CA2256694A1 - Combinatorial synthesis of carbohydrate libraries - Google Patents

Abstract

Disclosed are methods for synthesizing very large collections of diverse thiosaccharide derivatives optionally attached to a solid support. Also disclosed are libraries of diverse thiosaccharide derivatives.

Description

COMBINATORIAL SY~THESIS OF CARBOHYDRATE LIBRARIES

BACKGROUND OF THE INVENTION
Field of the Invention S This invention is directed to methods for synthesi7in~ very iarge coll~ctionc Of diverse thiosaccharide derivatives optionally attached to a solid support. This invention is further directed to a library of diverse thios~ec~ride derivatives.
References The following publications, patents and patent applications are cited in this application as superscript numbers:

International Patent Application Publication No. WO 93/06121.

2 U.S. Patent No. 5,143,854, issued September 1, 1992.

3 Hol, W. G. J., et al., "Structure and Function of E. coli Heat-Labile Enterotoxin and Cholera Toxin B Pent~mer", Bacterial Toxins and Virulence Factors in Disease, Ed. by J. Moss et al., Marcel Deldcer, Inc. (1995).

4 Spangler, B. D., "Structure and Function of Cholera Toxin and Related Escherichia coli Heat-Labile Enterotoxin", Microbiological Reviews, 56(4):622-647 (1992).
Williams (ed.), Synthesis of Optically Active o~-Amino Acids, Pergarnon Press (1989).
6 Evans et al., J. Amer. Chem. Soc., 112:4011-4030 (1990).

7 PU et al., J. Amer. Chem. Soc., 56:128CL1283 (1991).
8 Williams et al., J. Amer. Chem. Soc., 113:9276-9286 (1991).
s 9 Ratcliffe, et al., U.S. Patent No. 5,079,353.
'~ J. Defaye, et al., "Thiooligosa~çh~rides: Their Synthesis and ~elcti~l~c with Enzymes" in Studies in Natural Products Chemistry, Vol. 8, pp.
315-357, Elsevier Sci~n~es Publishers (1991).
" Kagen et al., Synlett, 1990, 643-650.
12 E. Hasegawa, K. Ishiyama, T. Horaguchi, T. Shimim, J. Org.
Chem. 1991, 56, 1631-1635.
3 H. Paulsen, K. Eberstein, W. Koebemick, Tetrahedron Letters, 4S-50, 4377-4380.
14 J.M. Kerr, S.C. Banville and R.N. Zuckermann, J. Am. Chem. Soc., 5:2529 (1993).
V. Nikolaiev, A. Stierandova, V. Krchnak, B. Se~ m~nn~ K.S. Iam, S.E. Salmon and M. Lebl, Pept. Res., 6:161 (1993) 6 M.C. Needels, D.G. Jones, E.M. Tate, G.L. ~einkPl, L.M.
Kochersperger, W.J. Dower, RW. Barrett and M.A. Gallop, Proc.
Natl. Acchl. Sci., USA, 90:10700 (1993) 17 M.H.J. Ohlmeyer, R.N. Swanson, L.W. Dillard, J.C. Reader, G.
Asouline, R. Kobayashi, M. Wigler and W.C. StiIl, Proc. Natl. Acad.
Sci. USA, ~2:10922 (1993) 18 U.S. Patent No. 4,137,401, issued January 30, 1979, tO R.

Lemieux et al.
9 H. H. Westal et al., "Methods of Enzymology," 34(b), 64 (1974).
T. Muk~iyama et al., Tetr~edron Letters, ~6, 5907-5908 (1968).
21 Svennerholm, A-M. et al., Current Microbiology, 1:19-23 (1978).

All of the above publications, patents and patent appli~tiol-s are herein incol~oldted by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually in(lic~ted to be incorporated by reference in its entirety.

State of the Art Compounds having biological activity can be identifiP~d by scr~ning diver_e collections of compounds (i.e., libraries of compounds) produced through either molecular biological or synthetic chernic~l techniques. Such screening metho~s 10 include methods wherein each member of the library is tagged with a unique identifi~r tag to facilitate identification of compounds having biological activity' or where the library comprises a plurality of compounds synthesi7e~ at spe~ific locations on the surface of a solid substrate wherein a receptor is apl,lop~iately labeled to identify binding to the compound, e.g., fluorescent or r~-lio~ctive labels. Correlation of the 15 labelled receptor bound to the substrate with its location on the substrate identifiP-c the binding compound.2 Central to these methods is the screenhlg of a multiplicity of co..l~unds in thelibrary and the ability to identify the structures of the compounds which have arequisite biological activity. Preferably, in order to facilitate synthesis and identific~tion, the compounds in the library are typically formed on solid SU~ swherein the compound is covalently ~tt~ehed to the support via a cleavable or non-cleavable linking arm. In this regard, libraries of diverse compounds are plc~a~ed and then screened to identify "lead compounds" having good binding affinity to the receptor.
Pharm~euti~l drug discovery relies heavily on studies of structure-activity relationships wherein the structure of "lead compounds" is typically altered to determine the effect of the alteration on activity. Alteration of the structure of the lead compounds permits evaluation of the effect of the structural alteration on activity.
Thus libraries of compounds derived from a lead compound can be created by including derivatives of the lead compound and repeating the screening procedures.

- W 0 98/22487 PCTtCA97ioo866 Ideally, the compounds are synthPci7ed in situ on the solid support so that the support can be tagged to identify the synthetic steps employed and/or the derivatiw incorporated onto the support. However, relatively simple synthetic metho l~ to produce a diverse collection of such derivatives on the ~.lp~lL~ are often not 5 available.
One particular class of compounds which would be useful for inclucion in screening libraries is thios~r~h~ride derivatives. It is well known that certain toxins and organisms bind to oligos~crh~ride recel)tol~ on host cells as an initial step in the pathological development of various disease conditions.3 For example, heat-labile 10 enterotoxin ("LT"), secreted by certain enterotoxigenic strains of Escherichia coli, and cholera toxin ("CT"), produced by Vibrio cholerae, are known to bind to g~nglis)~ e GMI. a glycosphingolipid situated in the outer leaflet of the host cell membrane and which has a char~cteri~tic pentasaccharide structure, i.e., Gal(,~l ~3)GalNAc(,B1~4){NeuAc(cY2 3)}-15 Gal(,B1~4)Glc, on its surface.3 LT has been i~lentified as one of the causative agentsof bacterial-induced traveller's diarrhea4 and CT is known to be the causative agent of the severe diarrheal disease, cholera.4 Additionally, many virulent org~nism~ (e.g., bacteria, virus, fungi, and the like) including enterovirulent org~nicm~ bind to cell surface receptors as part of the 20 disease process. For example, bacteria such as Vibrio cholerae and en~erotoAigenic strains of Escherichia coli can directly bind to cell surface r~cepLo,~ forming a colony at the point of ~tt~rhm~nt. Such binding is detrimental because it perrnits eApl..,3S~;
toxin to immeAi~t~ly interact with the cell surface.
Accordingly, in order to develop new pharm~reuti~i drugs to treat various 25 disease conditions, it would be highly desirable to be able to generate very large libraries of diverse thiosaccharide derivatives.

SIJMMARY OF THE INVENTION
This invention is directed to general synthetic m.otho ~s for gen~rating very large libraries of diverse thiosarch~ride derivatives optionally ~tt~ched to a solid support. The thio~cch~ride derivative libraries provided by this invention are S synthesi~eci by reacting a thios~cch~ride with a Mich~l acceptor or an a-halocarbonyl compound to provide for a thiosacch~ride carbonyl compound. The carbonyl group of the thio~cch~ride carbonyl compound can optionally be reduced to provide for a plurality of alcohol and/or amine thio~rch~ride derivatives. In one embo lime-nt, the alcohol and/or amine group of the thiosacch~ri~e derivative is10 further derivatized to provide for a plurality of thiosaçch~ride derivatives.In one embodiment of this invention, the thiosaccharide derivatives are covalently ~tt~che~ to a solid support. Solid SuppOll~ containing such thio~s~cch~ri~
derivatives preferably comprise a linking arm which links the solid support to the thioc~cch~ride derivative. The linking arm can be either cleavable or non-cleavable 15 and when cleavable, can be used to prepare a library of either solid phase or soluble thios~ch~ri~ie derivatives. The library of thiosacch~ride derivatives, whether soluble or insoluble, can be screened to isolate individual compounds that possess some desired biological activity. In a preferred embo~iment each compound in the library is unique.
Accordingly, in one of its method ~Cpectc~ this invention is directed to a method for synthesizing a thios~ch~ride derivative, which method comprises:
(a) providing a thios~cch~ride;
(b) providing at least a stoichiometric amount of a coupling reagent c~lectpd from the group concictin~ of Michael acceptors and ~-halocarbonyl co.npoul~ds; and (c) contacting the thioc~cch~ride and the coupling reagent under conditi~nc which provide for a thiosaccharide carbonyl compound.
In another of its method aspects, this invention is directed to a method for syntheci7ing a thio~crh~ride derivative on a solid support, which method compAses:
(a) providing a thiosacchaAde;

(b) providing at least a stoichiometric amount of a coupling reagent s~l~ted from Michael acceptors and cY-halocarbonyl compounds wherein either the thio.cacc.h~ride or the coupling reagent is covalently ~tt~r-hed to a solid sllppo~l, and (c) contacting the thios~cçh~ride and the coupling reagent under conciitiQnc S which provide for a thio~rch~ride carbonyl compound covalently ~tt~ched to a solid support.
In preferred embo~imentc of this invention, each of the above mPtho~35 for synthesi7ing a thios~r.ch~nde derivative further comprises reducing the carbony} group of the thio~cch~ride carbonyl compound to form a group sele~.ted from hydroxy and 10 amino derivatives. Optionally, the hydroxy or amino group can be further derivatized to form a group selected from esters, substituted amines, ~midçs, carb~m~tes, ureas, thiourea, thioesters and thiocarb~m~tçs.
In still another of its method aspects, this invention is directed to a method for preparing a thio~rcharide derivative library produced by syntheci7ine on each of a 15 plurality of solid suppolls a single compound wherein each compound compr ~s a thio~.ch~ride derivative, which library is synthesi7ed in a p-OCe5S comprising:
a) apportioning solid ~uppolLs among a plurality of reaction vessels which supports comprise a reactive functional group covalently bound thereto which group is capable of covalently binding a thiosaccharide at a position other than the thiol group;
b) contacting the supports in each reaction vessel with a unique thio~r.ch~n~e under conditions wherein the thiosaccharide is covalently attached to the solid supports through the reactive functional group;
c) pooling the ~.lp?olls;
d) apportioning the S~l~ Ol~ from (c) above among a plurality of reaction --25 vessels; and e) contacting the supports in each reaction vessel from (d) above with a unique coupling reagent selected from the group concicting of Michael açc~tc,r~ and a-halocarbonyl compounds under conditions which provide for a thios~cch~ride carbonyl compound covalently bound to said support.
And, in yet another of its method ~Cpectc~ this invention is directed to a method for preparing a thiosaccharide derivative library produced by syntheci7in~ on - wog8n2487 PCT/CA97100866 each of a plurality of solid supports a single compound wherein each con~pound comprises a thios~ch~ridc dcrivative, which library is synthesi7~d in a plOCCS5 comprising:
a) apportioning solid supports arnong a plurality of reaction vessels which S S~ S comprise.a reactive functional group covalently bound thereto which group is capable of covalently binding a coupling reagent;
b) contacting the supports in each reaction vessel with a unique coupling reagent sel~cted from the group conci.cting of Michael acceptols and a-halocarbonyl com~ounds under conditions wherein the coupling reagent is covalently ~tt~che~ to the 10 solid supports through the reactive functional group;
c) pooling the supports;
d) apportioning the supports from (c) above among a plurality of reaction vessels; and e) contacting the supports in each reaction vessel from (d) above with a 15 unique thic s~ch~ride under conditions which provide for a thioc~cch~ride carbonyl compound covalently bound to said support.
In prt:felled embo~iimçn~ of this invention, each of the above metho~3c for prepa~ing a thiosaccharide derivative library exemplified in procedures (a) through (e) further comprises: (f) pooling the supports from procedure (e);
20 (g) apportioning the supports from (f) above among a plurality of reaction ves~ls; and (h) reJu~ing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives. Still further, such methods optionally include the further steps of: (i) pooling the s.lp~lls from procedure (h) above; (j) apportioning the S~lppGlls from (i) above among a plurality of reaction 25 vessels; and (k) derivatizing the hydroxyl or amine groups to form a fun~ion~l group selected from esters, substituted ~mineS, amides, carb~m~tes, ureas, thioureas, thioesters and thiocarb~m~es.
The methods described above can be used to create a library of diverse thios~cch~ride derivatives. Accordingly, in one its composition ~Cpectc~ this invention 30 is directed to a library of diverse thiosaccharide derivatives comprising a plurality of solid supports having a plurality of covalently bound thios~rh~rides derivatives, wo s8l22487 pcTlcAs7loo866 wherein the thios~cch~ride derivative bound to each of said sU~p(JllSiS S~lbst~nt~ y homogeneous and further wherein the thios~cch~ride derivative bound on one support is different from the thios~rch~ride derivatives bound on the other Su~ and further wherein said thios~rch~ride derivative is l~ ~nted by the formula (1):
s Saccharide Y ~R4 R' R2 15 wherein Rl is s~Pl~Pcted from the group consi.cting of hydrogen, alkyl, su~ ul~d alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~l~P-nyl, heteroaryl, hel~ clic, thio~lkt xyalkyl and a linlcing arm covalently linking the compound of formula I' to the S~pOl~, 20. R2 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~lk-Pnyl, heteroaryl, het~ clic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to the ~u~>poll;
R3 is sel~te~ from the group collcicting of hydrogen, alkyl, substitut~P l aIlyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~lkPnyl, hetef~yl, het~,oc~clic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to the support;
or Rl and R2, or R' and R3, or R2 and R3, or Rl, R2 and R3 can be joined, together with the carbon atoms to which Rl and/or R2 and/or R3 are ~tt~che~ to form a cycloalkyl, cycloalkenyl or heterocyclic ring;
R4 is selected from the group concicting of-XR5, -XC(W)R6, - W098/22487 - PCTtCA97/00866 -XC(W)X'R7 and -C(W)XR8; wherein W is select~P~ from the group CQ. cicting of oxygen, sulfur and NH; and X and X' are each independently SPlP~ct~P~ from the group consisting of oxygen, sulfur and -NR9-, wherein R9 is sPlected from the group concicting of hydrogen and alkyl; or when R4 is -XR5 and R5 is not hydrogen, X can also be SPIPctP~ from the group Concictin& of -S(O)- and -SO2-;
R5 is sele~tP~ from the group consisting of hydrogen, alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thio~lkoxyalkyl and a linking arm covalently linking the compound of formula I' to the support, and when X is -NR9-, then R9 together with X can form an amino acid; or R5 and R~, or R5 and R2, or R5 and R3 can be joined, together with X of the -XR5 group and thecarbon atoms to which R' and/or R2 and/or R3 are ~tt~- hP~l, to form a heterocyclic ring;
R6 is selected from the group conciC~ing of alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, hetèrocyclic, thio~lkoxyalkyl and a linking arm covalently linking the compound of formula I' to the support; or R6 and R', or R6 and R2, or R6 and R3 can be joined, together with the -XC(W)- moiety of the -XC(W)R6 group and the carbon atoms to which R' and/or R2 and/or R3 are ~tt~hPd, to form a heterocyclic ring;
R7 is selected from the group con~i~ting of alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~lkPnyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to the sLIp~lL-, or R7 and R', or R~
and R2, or R7 and R3 can be joined, together with the -XC(W)X'- moiety of the -XC(W)X'R7 group and the carbon atoms to which Rl and/or R2 and/or R3 are ~tt~hP~, to form a heterocyclic ring;
R8 is selected from the group consisting of alkyl, alkenyl, alkaryl, alkoxyallyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thio~lkoxyalkyl and a linking arm covalently linking the compound of formula I' to the support; or R8 and R', or R8 and R2, or R8 and R3 can be joined, together with the -C(W)X- moiety of the -C(W)XR8 group and the carbon atoms to which Rl, R2 and/or R3 are ~s~chp~ to forma heterocyclic ring;

Y is selected from the group consisting of sulfur, -S(O)- and -S(O)2-;
n is an integer equal to 0 or 1; and pharm~ceuti~lly ~rc~pt~hle salts ll.e,~or, wherein the saccharide is selected from the group concictin~ of a monos~cçh~nde, an oligos~çh~ride, monos~cch~ricle-Z- and oligoc~e~h~rid~Z-, 5 wherein Z is a linking arm covalently linking the compound of formula I to the solid support;
with the proviso that only one of Rl, R2, R3, R4, R6, R', R8 and Z is linked to the solid support.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a preferred reaction scheme for synthç~i7ing a library of diverse thios~cch~ride derivatives using an ~"B-unsatuldted carbonyl co-,-you- d, i.e., cyclohept-2-en- 1 -one.
Figure 2 illustrates a preferred reaction scheme for synthe~i7ing a library of diverse thiosaccharide derivatives using an a-halocarbonyl compound, i.e, 2-chlorocycll he~none.

DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to libraries of diverse thio~cch~ndç derivatives optionally ~tt~hPcl to a solid support and to methods for genel~Ling such libraries.
However, prior to describing this invention in further detail, the following terrns will first be dçfinf?.i Definitions "Acyl" refers to the groups alkyl-C(O)-, aryl-C(O)-, and he~.~.a yl-C(O)-where alkyl, aryl and heteroaryl are as defined herein.
"Acylamino" refers to the group -C(O)NRR where each R is in-:lepçnd~nt1y hydrogen or alkyl.
"Acyloxy" refers to the groups alkyl-C(O)O-, aryl-C(O)O-, he~ro~yl-C(O)~, and heterocyclic-C(O)O- where alkyl, aryl, heteloalyl and heterocyclic are as defined herein.

"Alkaryl" refers to -alkylene-aryl groups preferably having from 1 to 8 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
"Alkoxy'' refers to the group alkyl-O-. Such alkoxy groups include~ by way of 5 example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, ter~-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
"Alkoxyalkyl" refers to the group -alkylene-O-alkyl which incl~ldes by way of example, methoxymethyl (CH30CH2-), methoxyethyl (CH3-O-CH2CH2-) and the like.
"Alkenyl" refers to alkenyl groups preferably having from 2 to 8 carbon atoms 10 and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation. Such alkenyl groups include ethenyl (-CH=CH2), n-propenyl (i.e., allyl) (-CH2CH=CH2), iso-propenyl (-C(CH3)=CH2), and the like.
"Alkyl" refers to monovalent alkyl groups preferably having from 1 to 8 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by 15 groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.
"Substituted alkyl" refers to a branched or straight chain alkyl group of from 1to 8 carbon atoms having from 1 to 3 s~lbstit~le~ts se~e~t~ from the group con~i~t~
of hydroxy, acyl, acylamino, acyloxy, alkoxy, alkenyl, alkynyl, amino, ~minoacyl, 20 aryl, aryloxy, carboxy, carboxyalkyl, cyano, cycloalkyl, guanidino, halo, hetelu~yl, heterocyclic, nitro, thiol, thioaryloxy, thioheteroaryloxy, and the like. Prefe~led substituents include hydroxy and amino.
"Alkylene" or "alkyldiyl" refers to divalent alkylene groups preferably having from 1 to 8 carbon atoms and more preferably 1 to 6 carbon atoms. This term is --25 exemplified by groups such as methylene (-CH2-), ethylene (-CH2CHr), the propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-) and the like.
"Alkynyl" refers to alkynyl groups preferably having from 2 to 8 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation. Such alkynyl groups include ethynyl ~-C 5 CH), 30 p~opalgyl (-CH2C--CH) and the like.

CA 022~6694 1998-11-24 "Amino acidN refers to any of the naturally occurring amino acids, as well as synthetic analogs and derivatives thereof. c~-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxy group, a hydrogen atom, and a riictinctive group referred to as a "side chain". The side chains of naturally occurring amino S acids are well kno~n in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in ~l~nine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), alkaryl (e.g., as in phenyl~l~nine and tryptQphan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in 10 hicti~in~). One of skill in the art will appreciate that the term "amino acid" can also include ,l~ , and ~-amino acids, and the like. Unnatural amino acids are also known in the art, as set forth in, for example, Williams3, Evans et al.4, Pu et al.5, Williams et al.6, and all references cited therein. Stereoisomers (e.g., D-arnino acids) of the twenty conventional amino acids, unnatural amino acids such as ~
15 disubstituted amino acids and other unconventional amino acids may also be suitable co..l~nents for compounds of the present invention. Examples of unconven~ign~l amino acids include: 4-hydroxyproline, 3-methylhi~ti~ine, S-hydroxylysine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
"~minoacyl" refers to the group -NRC(O)R where each R is indepen~e-ntly hydrogen or alkyl.
The term "amino derivative(s)" refers to a primary, secondary or tertiary amine compound produced by reductive amination of a thioc~ch~ride carbonyl compound in the presence of ammonia or an amine, including amino acids and derivatives thereof.
"Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple conden~ed rings (e.g., naphthyl or anthryl). ~lef~lled aryls include phenyl, naphthyl and the like.
Unless otherwise constrained by the definition for the aryl s~lbstituent, such aryl groups can optionally be substituted with from 1- to 3 substituents selPct~ from the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano, halo, nitro, heteroaryl, trihalomethyl and the like. Preferred subs~ituentc include alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and thio~lkoxy.
"Aryloxy" refers to the group aryl-O- where the aryl group is as defined herein including optiona}ly substituted aryl groups as also defined herein.
"Carboxy" refers to the group -COOH.
"Carboxyalkyl" refers to the group -C(O)O-alkyl where alkyl is as defined herein.
The term "coupling reagent" refers to Michael acceptola and a-haloc~lonyl com~2ounds. "Michael acc~pto-s" refers to ~,~-unsaturated carbonyl compounds 10 having the general formula (II):
o Il ~
R'-CH=C-C-R2 II

wherein R', R2 and R3 are as defined herein; or R'CH=CR2-C(O)XR8, wherein R', R2, R8 and X are as defined herein. Such Michael accepto~a include, by way of 20 example, ~"B-unsaturated aldehydes, ~,~-unsaturated ketones, a"B-unsaturated esters, a"~-unsaturated thioesters, ~ -unsaturated amides and the like. "cr-Halocarbonylcompounds" refers to compounds having the general formula: W-CHRI-C(O)R2 wherein Rl and R2 are as defined herein, and W is chloro, bromo or iodo. Such a-halocarbonyl compounds include, by way of example, a-chloroaldehydes, a-25 bromoaldehydes, a-iodoaldehydes, a-chloroketones, a-bromol~Ptones, a-ic~ ton~S
and the like.
"Cycloalkyl" refers to cyclic alkyl groups or cyclic alkyl rings of from 3 to 8 carbon atoms having a single cyclic ring or mnltiple conden~A rings which can beoptionally substituted with from 1 to 3 substituents sPle~ted from the group c~n~i~tin~
30 of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkylene, alkoxy, alkenyl, allynyl, amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano, halo, nitro, heteroaryl, trihalomethyl and the like. Preferred substituents include alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and thioalkoxy. Such cycloalkyl groups CA 022~6694 1998-11-24 W0 98/22487 pcTlcAs7loo866 include, by way of example, single ring structures such as cyclopropyl, cyclobut~
cyclopentyl, cyclooctyl, l-mothylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures such as ~A~ nt~nyl and the like, and spiro compounds. Examples of suitable cycloalkyl rings include single 5 ring structures such as cyclopentane, cycloheY~ne, cycloheptane, cyclooct~n~, and the like, or multiple ring structures such as bicyclo[2.2. l]heptane, bicyclo[3.2. l]octane, and the like. Preferred cycloalkyl rings include cyclopentane, cyclohPY~nP, cycloheptane and bicyclo[3.2. l]octane.
"Cycloalkenyl" refers to cyclic alkenyl groups or cyclic alkenyl rings of from 10 4 to 8 carbon atoms having a single cyclic ring and at least one pcint of internal unsaturation which can be optionally substituted with from 1 to 3 substihlentc SPlPct~p~d from the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkylene, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano, halo, nitro, h~elualyl, trihalomethyl and the like. Plere.led 15 substituents include alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and thio~lkoxy Examples of suitable cycloalkenyl groups include, for in~t~nce, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like. Such cyclo~lk~nyl rings include, by way of example, cyclopentene, cyclohexene, and the like.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and preferably is 20 either chloro or bromo.
"a-Halocarbonyl compound" refers to a compound having the general forrnula:
Q-CHRI-C(O)R2 wherein R' and R2 are as defined herein, and Q is chloro, bromo oriodo. Such a-halocarbonyl co,l~poullds include, by way of example, a-chloroaldehydes, a-bromo~ldehydes, ~x-iodoaldehydes, a-chloroketones, a-25 bromoketones, a-iodoketones and the like.
"Heteroaryl" refers to a monovalent aromatic carbocyclic group of from 2 to 8 carbon atoms and 1 to 4 heteroatoms s~l~cted from oxygen, nitrogen and sulfur within the ring.
Unless otherwise constrained by the definition for the heteloal~l subst~ ent, 30 such heteroaryl groups can be optionally substituted with 1 to 3 substituents sol~t~d from the group consisting of alkyl, substituted alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thio~lkoxy, thioaryloxy and the like. Such he~lo~yl groups can have a single ring (e.g., pyridyl or furyl) or multiple con~enced rings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or unsaturated group having a single ring or multiple condenced rings, from 1 to 8 carbon atoms and from 1 to 4 hetero atoms sel~cted from nitrogen, sulfur or oxygen within the ring. For the purposes of this application, the terrn "heterocycle" or "heterocyclic" does not include carbohydrate rings (i.e. mono- or oligos~çch~nde~).
Unless otherwise constrained by the definition for the heterocyclic substit~ent such heterocyclic groups can be optionally substituted with 1 to 3 substituents sel~tçd from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heteroaryl groups can have a single ring (e.g., pyrrolidinyl, piperidinyl, morpholinyl or tetrahydrofuranyl) or multiple condence~ rings (e.g., indolinyl).
Fy~mples of nitrogen heterocycles and heleroa,yls inclu~e, but are not ~imited to, pyrrole, imi~7ole, pyra_ole, pyridine, pyra_ine, pyrimi(~ine, pyri~7ine, indoli7ine, isoindole, indole, inda_ole, purine, quinolizine, isoquinoline, qllinoline, ph~h~l~7ine, naphthylpyridine, quinoxaline, quina_oline, cinnoline, pteridine, carbazole, carboline, phen~nthridine, acridine, phen~nthroline, isothi~701e, ph~n~7ine, isoxazole, phenoxa_ine, phenothia_ine, imidazolidine, imidazoline, piperidine, pipera_ine, indoline and the like.
~ich~el acce~to-" refers to an a"~-unsaturated carbonyl col"~und having the general formula (II):
O
R'-CH = C-C-R2 II

wherein Rl, R2 and R3 are as defined herein; or R'CH=CR2-C(O)XR8, wherein R', R2, R8 and X are as defined herein. Such Michael accepto,~ include, by way of example, cY,l~-unsaturated aldehydes, a,~-unsaturated ketones, a,l~-unsaturated esters, a"~-unsaturated thioesters, ~"B-unsaturated amides and the like.
"Thioalkoxyalkyl~ refers to the group -alkylene-S-alkyl which includes by way of example, thiomethoxymethyl (CH3SCH2-), thiomethoxyethyl (CH3-S-CH2CH2-) and S the like.
"Thiol" refers to the group -SH.
"Thioalkoxy" refers to the group -S-alkyl wherein the alkyl group is as defined herein.
"Thioaryloxy" refers to the group aryl-S- wherein the aryl group is as defined herein, including optionally substituted aryl groups as also defined herein.
"Thioheteroaryloxy" refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein, including optionally substituted heteroaryl groups as also defined herein.
The term "thios~cch~ride" refers to a monosaccharide or oligo~cch~ride having 2 to about 8 saccharide units wherein at least one, and preferably 1 or 2, of the hydroxyl groups of the ca~ch~ride is replaced with a thiol group. Preferably, the thiosaccharide is an animal c~ch~ride. The term "animal sacchariden refers to a saccharide which is naturally e~p~ssed by one or more ~nim~ls, such as m~mm~l$, birds or fish. Preferably, the animal saccharide is a m~mrn~ n ~rch~ride In particular, preferred rn~mm~]i~n saccharides include D-galactose, D-glucose, D-mannose, D-xylose, D-glucuronic acid, N-acetyl-D-glucos~mine, N-acetyl-D-g~ to~mine, sialyic acid, iduronic acid, L-fucose, and the like. Included within the definition of this terrn are acylated, phosphorylated and s--lf~t~d derivatives of animal saccharides~
The term ''thiosaccharide carbonyl compound" refers to a compound having the formula (III):

Saccharide--y ~ 111 wherein R', R2, R3, n and saccharide are as defined herein.
Ihe term "substrate" or "solid support" refers to a material having a rigid or semi-rigid surface which contains or can be derivatized to contain reactive functionality which covalently links a compound to the surface thereof. Such 5 materials are well known in the art and include, by way of example, silicon ~ioYide supports containing reactive Si-O~ groups, polyacrylamide supports, polystyrene supports, polyethyleneglycol ~up~olls, and the like. Such ~uppolls will plefe.dbly take the form of small beads, pellets, disks, or other conventional forms, although other forms may be used. In some embodiments, at least one surface of the substrate 10 will be substantially flat.
In one embodiment, the activated ketone compound is covalently ~tt~ e~
directly to the solid support or is ~ttache~ to the support via a linking arm. ~ inkin~
arms are well known in the art and include, by way of example only, conventim-~llinking arms such as those comprising ester, amide, carb~m~te~ ether, thio ether, 15 urea, amine groups and the like. The linking arm can also be a covalent bond. The linking arm can be cleavable or non-cleavable.
"Cleavable linking arms" refer to linking arms wherein at least one of the covalent bonds of the linking arm which ~tt~hes the compound to the solid support can be readily broken by specific chemical reactions thereby providing for co",~unds 20 comprising activated ketone groups free of the solid support ("soluble compoundsn).
The chemical reactions employed to break the covalent bond of the linking arm are s~lP~ted so as to be specific for bond breakage thereby preventing unintended reactions occurring elsewhere on the compound. The cleavable linking arm is s~1Pxt~d relative to the synthesis of the compounds to be forrned on the solid support 25 so as to prevent premature cleavage of this compound from the solid support as well as not to interfere with any of the procedures employed during compound synthesis on the support. Suitable cleavable linking arms are well known in the art.
A particularly preferred linking arm is illustrated in the formula:

(saccharide)-NH-(CH2)m-NHC(O)NH-(support) CA 022~6694 1998-11-24 wherein m is an integer of from 2 to about 10. Preferably, m is 6.

"Non-cleavable linking arms" refer to linking arms wherein the covalent bond(s) linking the activated ketone compound to the solid support can only be 5 cleaved under conditions which chemically alters ~Inintende~ parts of the structure of the compound ~tt~r~ed thereto.
The term "subst~nti~lly homogeneous" refers to collections of molecules wherein at least 80%, preferably at least about 90% and more preferably at leastabout 95~ of the molecules are a single compound or stereoisomers thereof.
The term "stereoisomer" refers to a chemic~l compound having the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain iden~ic~l chemi~l moieties are at different orientations in space and, therefore, when pure, have the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that 15 is so slight that it is un~et~t~hle with present instrumentation. The compounds described herein may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. A11 stereoisomers are included within the scope of the invention.
When chiral centers are found in the thios~cr~ride derivatives of this 20 invention, it is to be understood that this invention encompasses all possible stereoisomers. For example, when n is 0 in formula I, the carbon atoms to which R~
and R2 are attached may have an R,R or R,S or S,R or S,S configuration. Simil~rly, when n is 1, the carbon atoms to which Rl, R2 and R3 are ~tt~rhed may have an R,R,R or S,R,R or R,S,R or R,R,S or S,S,R or S,R,S or R,S,S or S,S,S
25 configuration.
The term "removable protecting group" or "protecting group" refers to any group which when bound to a functionality such as hydroxyl, amino, or carboxyl groups prevents reactions from occurring at these functional groups and which ~protecting group can be removed by conventional chemical or enzymatic steps to 30 reestablish the functional group. The particular removable protecLing group employed is not critical.

Wo 98/22487 PCr/cAs7/00866 The term "toxin" refers to a compound produced by an organism which causes or initi~tps the developln~nt of ~ noxious, poisonous or deleterious effect in a host presented with the toxin. Such deleterious con-lition~ may include fever, nausea, diarrhea, weight loss, neurologic disorders, renal disorders, hemorrhage, and the like.
S As used herein, the term "toxin" includes bacterial toxins, such as cholera toxin, heat-liable and heat-stable toxins of E. coli, toxins A and B of Clostridium difficile, aerolysins, hemolysins, and the like; toxins produced by protozoa, such as Giardia;
toxins produced by fungi; and the like. Included within this term are exotoxins, i.e., toxins secreted by an organism as an extracellular product, and enteroto"ins, i.e., 10 toxins present in the gut of an organism.
The terms "heat-labile enterotoxin" or "LT" refer to an enterotoxin of enterotoxigenic E. coli which initi~t~c traveller's diarrhea and related cQr-lition~. This toxin has a lectin-like activity.
The term "traveller's diarrhea" refers to diarrhea of sudden onset, often 15 accompanied by abdominal cramps, vomiting and fever that occurs sporadically in traveller's, usually during the first week of a trip. This diarrhea is most commonly caused by enterotoxigenic E. coli.
The term "cholera" refers to an acute epidemic infectious disease caused by Vibrio cholerae, wherein a soluble toxin elaborated in the inte5tin~1 tract by the Vibrio 20 alters the permeability of the mucosa, causing a profuse watery diarrhea, extreme loss of fluid and electrolytes, and a state of dehydration and collapse, but no grossmorphologic change in the intestinal mucosa.
The terms "cholera toxin" or "CT" refer to an ente~ Lin of V. cholerae which initi~t~s cholera and related contlit;ons. This toxin has a lectin-like activity.
The phrase "inhibit(s) the binding of a toxin to its receptor" means that a compound inhibits the binding of a toxin to its recep~or by at least 20%. For example, useful binding inhibition assays may measure inhibition of binding to ganglioside GD,b or ganglioside GMI~ neutralization of cytotoxic activity, or the like.
Such binding is reported herein as percent toxin activity rem~ining so that those 30 compounds which result in about 80% or less toxin activity ~ ining under the bioassay conditions dicclose~ herein are deemed to inhibit the binding of a toxin to its receptor.
The phrase "inhibit(s) the binding of heat-labile enterotoxin (LT~ andlor cholera toxin (C~ to an LT and/or CT r~plor" means that a compound inhibits ~e 5 binding of LT andlor CT to an LT and/or CT receptor by at least 20 %.
The phrase "inhibit(s) the binding of an organism to its cell surface r~pt~l-means that a çompound inhibits the binding of an organism, such as a bacterium, a virus, a protozoan, a fungus, and the like, to its cell surface receptor. For e~c~mple, for org~nicms such as Vibro cholera or enterotoxigenic strains of E. coli, a co".po~
10 is said to inhibit binding of an organism to a cell surface leceplor if it reduces binding of a bacteri~l surface adhesion antigen, such as CFA I pili, by at least 10%.
The term "pharm~reutir~lly acceptable salt" refers to pharmaceuti~lly acceptable salts of a compound of formula I which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of 15 example only, sodium, potassium, calcium, m~gnecium, ammonium, tetraalkylammonium, and the like; and when the mol~ le contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydroblolnide, tartrate, mesylate, ~cet~te, m~ tP, oxalate and the like.
For purpose of this application, all sugars are referenced using conven~ionql 20 three letter nomenclature. All sugars are assumed to be in the D-form unless otherwise noted, except for fucose, which is in the L-forrn. Further, all sugars are in the pyranose forrn.

General Synthetic Procedures 25 1. Method for Synthesizin~ Thiosaccharide Derivatives In one aspect, the methods of this invention involve the novel ~ ition of a thio~cch~ride to a coupling reagent selected from the group concictin~ of Michael reagents and ~-halocarbonyl co~pollnds.
Specific~lly, the thios~rch~ride derivatives of this invention are typically 30 prepared by reaction of a suitably protected thios~rrh~ride interrn~i~te with an a"B-unsaturated carbonyl compound or an a-halocarbonyl compound to provide for a thiosarch~ride carbonyl compound. The carbonyl group of the thios~cch~ride carbonyl compound is then optionally reduced to provide for a plurality of alcohol and/or amine thiosaccharide derivatives. In one embodiment, the alcohol and/or amine group of the thios~cc~ride derivative is further derivatized to provide for a 5 plurality of thiosaccharide derivatives.
The a"B-unsaturated carbonyl compounds employed in prepa~ g the thios~ch~ride derivatives of this invention preferably have the general formula (II):
o Rl-CH=C-C-R2 II

wherein R', R2 and R3 are as defined above; or RlCH=CR2-C(O)XR8, wherein Rl, R2, R8 and X are as defined above. These compounds are either commercially available or can be prepared from commercially available materials using art recognized procedures. For example, such compounds can be pr~ed via a Wittig reaction from an aldehyde, R'CHO, and a ~-carbonyl phosphorane, such as (Ph)3PC(R3)C(O)R2.
Preferred ~Y"~-unsaturated carbonyl compounds for use in this invention include, by way of example, cyclopent-2-en-1-one, 4,4-dimethylcyclopent-2-en-1-one, cyclohex-2-en-1-one, 4,4-dimethylcyclohex-2-en-1-one, 6,6-dimethylcyclohex-2-en-1-one, cyclohept-en-1-one, and 3-methylene-2-norbornanone.
The a-halocarbonyl compounds employed in preparing the thios~h~ e derivatives of this invention preferably have the general formula: W-CHR'-C(O)R2wherein R' and R2 are as defined above, and W is chloro, bromo or iodo. Such compounds are either commercially available or can be prepa-~d from commerciallyavailable materials using art recognized procedures. Preferred ~-halocarbonyl compounds for use in this invention include, by way of example, 2-chlorocyclopentanone and 2-chlorocyclohexanone. Alternatively, carbonyl co..,poul,ds having a leaving group other than a halogen in the a-position may be employed.
Suitable leaving groups include, by way of illustration, various sulfonic ester groups, CA 022~6694 1998-11-24 - W O 98n2487 PC~r/CA97/00866 such as tosylate, mesylate, brosylate and nosylate groups and the like, and fluc..;n3 sulfonic ester groups, such as triflate, nonaflate and tresylate groups and the like.
The sugars employed in this invention are any thiol containing saccharides or oligosaçch~ndP-s wherein the thiol substitution is at any position of the thioMc~h~ri~.
For example, thio!actose having a thiol (-SH) group at the 1, 2, 3, 6, 2', 3', 4' or 6' can be used. Methods for chemically modifying saccharides to introduce suitable substitution are well known in the art as illustrated in Ratcliffe, et al.9 and l~ere.~ces cited therein as well as by Defaye'~. For example, 1-thiosaccharides can be p-epa~d by reacting the saccharide with an acylating agent to convert all of the hydroxyl groups to acyl groups. The 1-acyl group is then selectively converted to the 1-thioacetyl group by reaction with an excess of thiolacetic acid. Hydrolysis thenprovides for the 1-thiosacch~ride.
Alternatively, selective protection of the hydroxyl groups of the saccharide provides for one or more free hydroxyl groups which can be converted into appro~liate leaving groups, such as mesyl or halo groups, by conventional çh~Pmis~ry well known in the art. Such leaving groups can then be displaced to afford the corresponding thiol groups. See, for example, International Patent Application Serial No. PCT/CA92/00242. Specifically, a mesyl group is selectively introduced at one of the hydroxyl groups and then reacted with a thioacetyl group (for eY~mple pot~ccillm thio~cet~tP~) to provide for the corresponding thio~r-et~t~ derivative. Tr~tmçnt of this compound with a-mild base provides for the collcs~nding thio group.
The resulting thiosaccharide is then reacted with a coupling reagent ~Ple~ted from the group consisting of Michael acceptors and ~-halocarbonyl compounds.
~ypically, this reaction is conductP~ by contacting the thiosa(~ch~ride with at least one equivalent, preferably 1 to 1.2 equivalents, of the coupling reagent in an inert diluent, such as dichloromPth~nP, at a temperature of from about -40~C to about 50~C for about l to about 6 hours to afford a thiosaccharide carbonyl compound. In a preferred embodiment, when the thiosaccharide reagent is ~tt~chPfl to a solid support, the coupling reagent is preferable used in excess to maximize the yield of the resulting thiosaccharide carbonyl compound. Alternatively, when the the coupling reagent is CA 022~6694 1998-11-24 - WO 98/22487 ' PCT/CA97/00866 ?,tt~che~ to a solid support, the thios~ch~ride is preferably used in excess relative to the coupling reagent.
The carbonyl group of the thiosaccharide carbonyl compound can then be optionally reduced using a re~ucin~ agent to provide for an alcohol derivative.
S Preferably, this reduction is conducted by con~cting the thio~rch~ride carbonyl compound with sodium borohydride, preferably about 1.2 to about 2.0 equivalents of sodium borohydride based on the carbonyl compound. Generally, this reaction is conducted in an inert diluent, such as tetrahydrofuran, isopropanol and mixture thereof, at a temperature of about 25~C to about 30~C for about 0.5 to about 3.0hours, to afford the alcohol derivative.
Alternatively, the carbonyl group of the thiosaccharide carbonyl compound can be reductively ~min~t~Pd to provide for an amine derivative. In this reaction, the thiosaccharide carbonyl compound is contacted with an excess of ammonium acetateand at least one equivalent of sodium cyanoborohydride based on the carbonyl compound. This reaction is typically con-lucted in an inert diluent, such as mPth~n- l, tetrahydrofuran and mixtures thereof, at a temperature of about 25~C to about 30~C
for about 1 to about 72 hours.
The thiosaccharide carbonyl compound can also be reductively ~min~t~ in the presence of a primary or secondary amine to provide for amine derivatives.
Preferably the amine used in the reductive amination is an amino acid or a derivative thereof, such as amino acid esters. Typically, this reaction is conducted by collt~ctin~
the thiosaccharide carbonyl compound with a molar excess of an amino acid ester,such as the methyl ester or the te~-butyl ester, preferably with 10 equivalents based on the carbonyl compound, in the presence of at least one molar equivalent, preferably about 1.0 to about 1.2 equivalents, of sodium cyanoborohydride.
Typically, this reaction is conducted in an essenti~l]y anhydrous inert diluent, such as acetonillile, at a ter,peldture of about 25~C to about 30~C for about 1 to about 72 hours. Subsequently, the ester group of the amino acid can be cle~aved using standard conditions to provide the col~es~onding carboxylic acid.
In a preferred embodiment, the alcohol and/or amine derivatives ~re~ cd as described above are further derivatized to form a group sPlerte~ from esters, CA 022~6694 1998-11-24 - W 0 98/22487 PCT/CAg7tO0866 substituted amines, amides, carb~m~tes, ureas, thioureas, thioesters and thiocarb~m~t~s. Methods for derivatizing alcohols and/or amines to provide for such functional groups are well known to those skilled in the art. For example, alcohols and arnines can be reacted with acyl halides to form esters and amides, respectively.
Amines can also be reductively alkylated to form substituted ~mines. Similarly, alcohols and amines can be reacted with isocyantes, among other reagents, to afford carbarnates and ureas, respectively. Conditions for such reactions are well r~ni7~d in the art.
Preferred embodiments of this invention are illustrated in Figures 1 and 2.
Figure 1 illustrates the synthesis of various 1-thiogalactose derivatives from cyclohept-2-en-1-one. Figure 2 illustrates the synthesis of various 1-thiogalactose from 2-chlorocyclohexanone. It will be readily apparent to those of ordinary shll in the art that the synthetic procedure illustrated in Figures 1 and 2 and following reaction conditions can be modified by selecting the applo~liate starting materials and reagents to allow the preparation of a plurality of 1-thiogalactose derivatives.
As shown in Figure 1, D-galactose is perlauroylated by cont~ctin~ D-~ rt~se with at least S equivalents, and preferably 10 equivalents, of lauroyl chlonde This reaction is generally conducted in an inert diluent, such pentane, hexane, dichloromethane and the like, using a tertiary amine such as pyridine or triethylamine to neutralize the bydrochloric acid generated during the reaction. Preferably, acatalytic amount of 4-(N,N-dimethylamino)pyridine is added to the reaction mixture to fa.~ilit~e this reaction. Typically, this reaction is conducted at a l~",l)el~ture of from about -78~C to about 30~C for about 0.5 to about 96 hours to afford 1,2,3,4,6-penta-O-lauroyl-a-D-galactopyranose, 1, in approximately 70% yield from D-~al~r-to~
Compound 1 is then converted into 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thi~-D-galactopyranose, 2, by reaction of I with an excess of thiolacetic acid. In one embodiment, this reaction is conducted in the presence of an excess of boron trifluoride etherate, preferably using about 15 to 20 equivalents of boron trifluoride etherate based on 1, in an inert diluent, such as dichloromethane and the like.
Typically, this reaction is conducted initially at about 0~C and then at about 20~C to about 30~C for about 0.5 to about 48 hours.

CA 022~6694 1998-11-24 In another embodiment, compound 2 can be prep~ed from 1 by collt~ct;n~ 1 with at least one equivalent, preferably 1 to 1.2 equivalents, of benzylamine toselectively remove the 1-lauroyl group. This reaction is typically conducted at about 25~C to about 30~C for about l to about 96 hours to provide for 2,3,4,6-tetra-O-5 lauroyl-(c~"B)-galactopyranoside. This intermediate is then converted into an O-(2,3,4,6-tetra-O-lauroyl-(~Y"~)-galactopyranosyl) trichloroacetimid~te interme~ te by contacting the tetralauroyl compound with an excess of trichloroacetonitrile, preferably about 10 equivalents, and about 0.8 to about 1.0 equivalents, of 1,8-diaza~bicyclo[5.4.0]undec-7-ene (DBU) in an inert diluent, such as dichlorometh~n~.
10 The resulting O-trichloroacetidate intermeAi~te is then contacted with an excess of thiolacetic acid in an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C for about 1 to about 96 hours to provide for 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-,B-D-galactopyranose, 2.
In still another embodiment, compound 2 can be prepared by contacting compound 1 with about 1.5 to about 2.0 equivalents of thiolacetic acid and about 0.5 equivalents of trimethylsilyl trifluoromethanesulfonate based on 1 in an inert diluent, such as dichloromethane and the like. Typically, this reaction is conducted initially at about 0~C and then at about 20~C to about 30~C for about 0.5 to about 72 hours.
This method is especiaIly preferred since it provides the highest yield of compound 2 20 and produces no detectable traces of the coll~sponding a-isomer.
If desired, however, the ~-isomer, i.e., 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranose, can be readily prepared by contacting compound 1 with an excess, preferably about 20 equivalents, of thioacetic acid in the presence of about 1.0 to l.1 equivalents of tin (IV) chloride in an inert diluent, such toluene, at ambient 25 temperature for about 0.5 to about 2 hours. Alternatively, tre~tment of compound 1 with an excess, preferably about 3 to about 6 equivalents, of thioacetic acid in the presence of about 2.0 to 3.0 equivalents of trimethylsilyl trifluorometh~nes~llfonate in an inert diluent, such dichloromethane, at ambient temperature for about 12 to about 48 hours affords 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-cY-D-galactopyranose.

.

CA 022~6694 1998-11-24 The Michael addition of compound 2 to cyclohept-2-en-1-one then affords cycloheptanon-3-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranoside, 3. Thisreaction is typically conducted by contacting 2 with at least one equivalent, ~ fe,ably 1.0 to 1.2 equivalents, of cyclohep-2-en-1-one in the presence of a molar excess of a S dialkylamine, such as diethylamine.
Without being limited by any theory, it is believed that the dialkylamine first reacts with the thio~cetyl of compound 2 thereby forming in situ the thiol derivative of compound 2 which then reacts under basic conditions generated by the dialkylamine with a Michael adduct.
Typically, this reaction is conducted in an inert diluent, such as dichloromethane, at a temperature of from about -40~C to about 50~C for about 1 to about 6 hours.
The carbonyl group of compound 3 can then reduced using a reducin~ agent to provide for 3-hydroxycycloheptyl 2,3,4,~tetra-O-lauroyl-1-thio-~B-D-15 galactopyranoside, 4. Preferably, this reduction is con~ucte~ by cont~rting 3 withsodium borohydride, preferably about 1.2 to about 2.0 equivalents of sodiumborohydride based on 3. Generally, this reaction is conducted in an inert diluent, such as tetrahydrofuran, isopropanol and mixture thereof, at a temperature of about 25~C to about 30~C for about 0.5 to about 3.0 hours. The resulting alcohol, 4, is 20 readily purified by solid-phase extraction on Cl8 silica gel using pentane as an eluent.
Removal of the lauroyl groups from alcohol 4 is then accomplished by treating 4 with an excess of sodium methoxide in methanol and an inert diluent, such as dichloromethane, at about 25~C to about 30~C for about 1 to about 24 hours.
Neutralization of the reaction mixture with Amberlite IR-50S (H+~ resin then provides 25 for 3-hydroxycycloheptyl 1-thio-,B-galactopyranoside, A5.
Alternatively, compound 3 can be reductively ~min~t~A to provide for 3-aminocycloheptyl 2,3,4,6-tetra-O-lauroyl-l-thio-,B-D-galactopyr~nosi~e, 5. In one embodiment of this reaction, compound 3 is contacted with an excess of ammonium acetate and at least one equivalent of sodium cyanoborohydride based on 3. This 30 reaction is typically conducted in an inert diluent, such as met~nol, tetrahydrorul~n and mixtures thereof, at a temperature of about 25~C to about 30~C for about 1 to about 72 hours.
In another preferred embo~iment, the reductive amination reaction is accomplished by cont~rting compound 3 with an excess of ammonium acetate and an 5 excess of trimethyl orthoformate based on 3, in an inert ~iluent, such as 1,2-dichloroethane at a temperature of about 25~C to about 30~C for about 12 to about 72 hours to form an imine intermeAi~tP. The imine interrnedi~t~ is generally not j~l~tJ~
but is contacted in situ with an excess of sodium borohydride, preferably about 1.2 to about 1.5 equiva}ents of sodium borohydride, based on 3. The resulting amino 10 compound 5 is then readily purified by solid-phase extraction on C18 silica gel using pentane as an eluent.
Optionally, the amine group formed by reductive amination can be acylated with conventional acylating agents under conventional conditions. The acylating agent is generally of the formula L-C(O)R6 where L is a leaving group such as a halide, an 15 activated ester, and the like.
The lauroyl groups are removed from compound 5 by cont~tin~ 5 with an excess of sodium methoxide in methanol and an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C for about 1 to about 24 hours.
Neutralization of the reaction mixture with Amberlite IR-50S (H+) resin then provides 20 for 3-aminocycloheptyl 1-thio-,B-galactopyranoside, B5.
In one example, the primary amine group of compound B5 can optionally be acylated by contacting B5 with an excess of acetic anhydride in meth~nQl containing a trace of water. Generally, this reaction is conducted at about 25~C to about 30~C for about 2 to about 24 hours to provide for 3-~rePmidocycloheptyl l-thio-~B-25 galactopyranoside, CS.
Alternatively, the primary amine group of 5 can be acylated with phthalicanhydride before removal of the lauroyl groups to provide for 3-(2-carboxybenzamido)cycloheptyl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranoside, 6. This reaction is typically conducted by contacting compound 5 with at least one 30 molar equivalent, preferably with an excess of phthalic anhydride. Preferably, this reaction is conducted in dry pyridine containing a catalytic amount of 4-(N,N-CA 022~6694 1998-11-24 WO 98/22487 PCTtCA97/00866 dimethylamino)pyridine. The reaction is typically conducted at about 25~C to about 30~C for about 12 to about 48 hours to provide for compound, 6. Removal of the lauroyl groups from 6 is then ~ccomplished by treating 6 with sodium methoxide in methanol and an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C
for about 1 to about 24 hours. Neutralization of the reaction mixture with ~mberlit~
IR-SOS (H+) resin then provides for 3-(2-carboxyben7~midQ)cycloheptyl l-thio-~-D-galactopyranoside, DS.
As shown in Figure 1, compound 3 can also be reductively ~min~tçd with an amino acid ester to provide for interm~Ai~t~s 7 or 8. Specifically, compound 3 is 10 contacted with a molar excess of ,~-alanine tert-butyl ester, preferably with 10 equivalents based on 3, in the presence of at least one molar equivalent, preferably about 1.0 to about 1.2 equivalents, of sodium cyanoborohydride. Typically, this reaction is conducted in an essentially anhydrous inert diluent, such as acetonitrile, at a temperature of about 25~C to about 30~C for about 1 to about 72 hours. The 15 resulting intermeAi~te 7 is readily purified by solid-phase extraction on C18 silica gel using pentane as the eluent.
The tert-butyl ester group of compound 7 is readily hydrolyzed to the co~ onding carboxylic acid by treating 7 with an excess of trifluoroacetic acid in an inert diluent such as dichlorometh~ne. This reaction is typically conducted at about 20 25~C to about 30~C for about 1 to about 10 hours. The lauroyl groups of the rçslllting carboxylic acid intermçdi~te are then removed using sodium methoxide in methanol as described above to provide for N,B-[l-(l-thio-,B-D-galactopyranosyl)cyclohept-3-yl]-~ nine7 F5.
In a similar manner, compound 3 can be reductively ~ n~te~ using other 25 amino acid esters, such as glycine tert-butyl ester, L-leucine ter~-butyl ester, L-histitline methyl ester, L-tryptophan methyl ester, and L-arginine methyl ester, to provide for intermediate 8. In those cases where the amino acid ester employed is a tert-butyl ester, the tert-butyl ester is cleaved as described above using trifluoroacetic acid to afford N~-tl-(l-thio-,B-D-galactopyranosyl)cyclohept-3-yl]-glycine, E5, and 30 No~-[ 1-(1-thio-B-D-galactopyranosyl)cyclohept-3-yl]-L-leucine, G5. Alternatively, in CA 022~6694 1998-11-24 those cases where an amino acid methyl ester is employed, the lauroyl groups of interm~Ai~te 8 are preferably removed before cleaving the methyl ester by tre~trn~-nt of 8 with sodium methoxide in methanol as described above. Subsequently, the methyl ester of the amino acid moiety is cleaved to the corresponding carboxylic acid 5 by tre~tm~nt with an excess of aqueous lithium hydroxide for about 0.5 to about 2 hours. Neutralization of the reaction mixture with Amberlite IR-SOS (H+) resin then provides for NcY-[l-(1-thio-1~-D-galactopyranosyl)cyclohept-3-yl]-L-hictilline, H5, Ncr-[l-(1-thio-,~-D-galactopyranosyl)cyclohept-3-yl~-L-tryptophan, I5, and Nol-[1-(1-thio-,B-D-galactopyranosyl)cyclohept-3-yl]-L-arginine, J5.
Additionally, if desired, the hydroxyl group of alcohol derivatives, such as compound 4, can be converted into a leaving group, such as the mesylate, tosylate, etc., and displaced with various nucleophiles. For example, treatment of an alcohol derivative with an excess, preferably about 1.1 to about 1.5 equivalents, of meth~nesulfonyl chloride in pyridine and an inert diluent, such as THF, affords the corresponding mesylate. The mesylate group can then be ~ pl~ced with, for example, sodium azide to provide the corresponding azido derivative. This reaction is typically conducted by contacting the mesylate compound with an excess, preferably about 5 to about 50 equivalents of sodium azide in an inert diluent, such as N,N-dimethylformamide, THF and mixtures thereof, at a temperature of from about 50~Cto about 100~C for about 1 to about 6 hours. Preferably, a crown ether, such as 18-crown-6, is added to the reaction mixture to promote the displ~cement re?~ction The azido derivative can then be reduced with a reduçing agent to afford the cGl-esponding primary amine, i.e., a compound such as 5. Preferably, this reaction is conducted by contacting the azido compound with about 1.0 to about 1.1 equivalents of sodium borohydride and about 2.0 to about 2.2 equivalents of nickel chloride (NiCl2) in an inert diluent, such as ethanol, isopropanol, or mixtures thereof, at a temperature of from about 0~C to about 50~C for about 0.5 to about 6 hours.Removal of the lauroyl protecting groups can then be accomplished using the procedures described above.
Additionally, the primary amine group of amino compounds such as 5 can be further derivatized by reductive alkylation to afford a secondary amine. Typically, CA 022~6694 1998-11-24 W0 98/22487 - PCTtCA97/00866 this reaction is conducted by contacting the primary amine with an excess, preferably about 2 to about 500 equivalents of an aldehyde or a kefone in the presence of at least one equivalent, preferably about 1.0 to about 10 equivalents, of a reduçin~ agent, such as sodium triacetoxyborohydride. This reaction is typically conduct~ in an inert S diluent, such as dichlorometh~ne, methanol, or mixtures thereof, at a te.~ dture of about 0~C to about 50~C for about 10 to about 48 hours. In a prefel.~d embo~ en~the ketone employed in this reaction is a cyclic ketone including, by way of e~mp'~, cyclobutanones, such as 3,3-dimethylcyclobutan-1-one; cyclopentanones, such as 3,3-dimethylcyclopentan-1-one; cyclohexanones and cycloheptanones.
The lauroyl groups of the resulting secondary amine are then removed by contacting the lauroyl-protected compound with an excess of sodium methoxide in mPth~nol and an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C
for about 1 to about 24 hours. Neutralization of the reaction mixture with Amberlite IR-SOS (H+) resin then provides the desired secondary amine compound.
As noted above, Figure 2 illustrates the synthesis of various 1-thio~ rtrse derivatives using an a-halocarbonyl carbonyl compound, i.e., 2-chlorocycloh~T~n~As shown in Figure 2, 1-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-,B-D-galactopyranose, 2, prepared as described above, reacts with 2-chlorocyclohexanone to give cyclohexanon-2-yl 2,3,4,6-tetra-0-lauroyl-1-thio-~-D-galactopyranoside, 9. This reaction is typically conducted by cont~cting 2 with at least one equivalent, preferably 1.0 to 1.2 equivalents, of 2-chlorocyclohexanone in the presence of an excess of a dialkylamine, such as diethylamine. Typically, this reaction is conducted in an inert diluent, such as dichloromPth~ne, at a te~-pe,dture of from about -40~C to about 50~C
for about 1 to about 6 hours to afford compound 9.
Compound 9 can then be reacted using the same reagents and condi~ions described above for compound 3 to afford various l-thiogalactose derivatives.
Specifically, compound 9 is reduced with sodium borohydride to provide 10 which,after removal of the lauroyl groups, affords 2-hydroxycyclohexyl l-thio-,B-D-galactopyranoside, A2.
Altematively, compound 9 is reductively ~min~t~d with ammonium acetate and sodium cyanoborohydride to provide for interme~ te 11 which, upon removal of the wo s8l22487 PCT/CA97/00866 lauroyl groups, affords 2-aminocyclohexyl l-thio-,B-D-galactopyranoside, B2.
Compound B2 can then be acylated with acetic anhydride to give 2-~et~midocyclohexyl 1-thio-B-D-galactopyranoside, C2. Alternatively, interrneAi~
11 can be acylated with phthalic anhydride to provide for interme~ te 12 which S affords 2-(2-carboxyben7~mitlocyclohexyl l-thio-~-D-galactopyr~noside, D2, by removal of the lauroyl groups using the conditions described above.
Additionally, compound 9 can be reductively ~min~ted using an ,B-alanine tert-butyl estèr to provide for intermediate 13 which then affords NB-tl-(l-thio-~-D-galactopyranosyl)cyclohex-2-yl]-~-alanine, F2, upon deprotection. Alternatively,10 compound 9 can be reductive ~min~tecl with other amino acid esters, such as glycine tert-butyl ester, L-leucine tert-butyl ester, L-hi~tidine methyl ester, L-tr~lophan methyl ester, and L-arginine methyl ester, to provide intermedi~t~ 14 which uponde~loteclion, affords NcY-[l-(1-thio-~-D-galactopyranosyl)cyclohex-2-yl]-glycine E2, N~-[1-(1-thio-,~-D-galactopyranosyl)cyclohex-2-yl]-L-leucineG2, Na-tl-(l-thio-,B-D-15 galactopyranosyl)cyclohex-2-yl]-L-histidine H2, Nc~-[1-(1-thio-,~-D-galactopyranosyl)cyclohex-2-yl]-L-tryptophan ~2, and Na-[1-~1-thio-,~-D-galactopyranosyl)cyclohex-2-yl]-L-arginine J2.
Optionally, the s~cch~ride derivatives of formula I wherein Y is a sulfide linking group (-S-) can be oxidized using conventional reagents and con~litions to 20 provide the corresponding sulfoxide (Y = -S(O)-) and sulfone (Y = -SO2-) derivatives. Suitable reagents for oxidizing a sulfide compound to a sulfoxide include, by way of example, hydrogen peroxide, peracids such as 3-chlolopeloxybenzoic acid (MCPBA), sodium periodate, sodium chlorite, sodium hypochlorite, calcium hypochlorite, tert-butyl hypochlorite and the like. Chiral25 oxidizing reagents (optically active reagents) may also be employed to provide chi~al sulfoxides. Such optically active reagents are well known in the art and include, for example, the reagents described in Kagen et al.'l and references cited therein.
The oxidation reaction is typically conducted by contacting the s~ch~ri(le derivative with about 0.95 to about 1.1 equivalents of the oxidizing reagent in an inert 30 diluent, such as dichloromethane, at a temperature ranging from about 0~C to about 50~C for about 1 to about 48 hours. The resulsing sulfoxide can then be further oxidized to the cor.. sponding sulfone by cont~Gting the sulfoxide with at least one additional equivalent of an o~ in~ reagent, such as hydrogen peroxide, MCPBA, potassium perm~n~n~tÇ and the like. Alternatively, the sulfone can be ~
S directly by cQnt~ting the sulfide with at least two equivalents, and preferably an excess, of the oxidi7ing reagent.
In a similar manner, the ~ch~rlde of formula I, wherein R4 is -XR5, X is sulfur and R5 is a defined substituent other than hydrogen, can be oxidized to afford the co~,es~onding sulfoxide (X = -S(0)-) and sulfone (X = -S02-) derivatives.
Additionally, if desired, the hydroxyl groups of the saccharide moiety may be readily acylated, sulfonylated- or phosphorylated using art recognized procedures and reagents to provide compounds of formula I wherein at least one of the hydroxyl groups of the saccharide is -O-SO2-OH, -C(O)RI~, -P(O)(ORIl)2 or pharn-~ceutir~lly acceptable salts thereof, where Rl~ and Rll are as defined above. Such acylation15 re~ctions may occur as an initial step of the synthesis (i.e., using an acyl halide, such as lauroyl chloride, as described above~ or as a post-synthetic transformation of compounds of formula I using, for eY~mple, acyl h~lides, anhydrides, halophosph~ -s sulfur trioxide, and the like.
For example, a de-blocked hydroxyl group can be sulfonylated by treating the 20 hydroxy-containing compound with an excess, preferably about 1.1 to about 1.2equivalents, of a pyridine:sulfur trioxide complex in an inert diluent, such as N,N-dimethylform~mide, at ambient te,l.p~l~ture for about 1 to about 24 hours. Typically, the resnlting sulfate (i.e., -O-SO2-OH) is isolated as its salt by tre~tnlent with, for example, a Na+ resin in an inert ~iluent, such as m~th~nol. Further reaction --25 conditions suitable for forming s-llf~tes and phosphates can be found, for eY~mrl~, in U.S. Patent No. 5,580,858'2.
The methods illustrated in Figures 1 and 2 were con~ucted in a solution phase.
Surprisingly, these methods can also be con~ucted on the solid phase using reaction ~con-litions similar to those described above for the solution phase. When conduc~d 30 on the solid phase, one of the reagents employed is attached to a solid support via a cleavable or non-cleavable linking arm. Such linking arms are well known in the art as well as their ~tt~chment to either the thios~cçh~ride or the coupling reagent.
Either of the reagents can be ~tt~hed to the solid support without criticality provided that the attachment does not alter the reactivity of the reagent. For example, S a linking arm may be covalently ~tt~ched to any position of the thio~ ch~ride other than the thiol group. Such ~tt~rhm~nts are preferably made through, for example, an ester or ether linkage to one the hydroxyl group of the thiosAc~h~ride. A pref~
linking arm is derived from succinic acid.
By way of example, 1-dithioethyl-~B-D-galactopyranoside is readily ~tt~hed to a trityl chloride resin having about 0.80 to about 1.00 mmol/g of active chlorine by contacting the resin with about 0.75 to about 2.0 equivalents of l-dithioethyl-~B-D-galactopyranoside in pyridine containing a catalytic amount of 4-(N,N-dimethylamino)pyridine at a telllpelature ranging from about 25~C to about 100~C for about 12 to 48 hours. A free thiol group at the 1-position of the covalently bound 15 galactose is then generated by treating the resin with dithiothreitol (C'l~l~nd's reagent) and triethylamine in an inert diluent, such as methanol, for about 6 to 24 hours at ambient ten-pe,dture. The resulting l-thio-~B-D-galactopyranoside is then reacted as described above to afford a 1-thiogalactose derivative of formula I covalently ~tt~Çh~d to the solid support resin. If desired, the 1-thiog~l~ctQse derivative can be cleaved 20 from the solid support resin by cont~cting the resin with an excess of trifluoroacetic acid and triisopropylsilane in an inert diluent, such as dichlorometh~ne, at ambient te---pe,dture.
Similarly, a linking arm can be covalently ~t~hed to any position of the coupling reagent provided that the point of ~t~-hment does not interfere with the 25 Michael addition of the thios~cch~ride to the a"B-unsaturated carbonyl group or with the displacement of the halide from the ~-halocarbonyl compound by the thiosacch~ride Accordingly, the linking arm is preferably ~tt~rh~d to the coupling reagent through any one of substituents R'-Ra via a covalent bond. Such linkage can be through, for example, an ester, ether, amine, amide, or urea functional group and 30 the like.

- W098/22487 PCT/CAs7/00866 By way of example, a carboxylic acid moiety can be covalently ~tt~hed to an ~min~ted solid support using convention~l coupling procedures and reagents.
Typically, such a coupling reaction will be conducte~d using well-known co.~.l;n~
reagents such as carbo~liimides, BOP reagent (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphonate) and the like. Suitable carbo~iimides in~ de, by way of example, dicyclohexylcarbo~iimide (IDCC), diisopropylcarbodiimide~ 1-(3-dimethylaminopropyl)-3-ethylcarbo liimi-~e (EDC) and the like. Preferably, a well-known coupling promoter, such as N-hydroxysucrinimi~e, 1-hydroxybenzotriazole and the like, is also employed in the reaction IlliX~ c; to f~ilit~te the coupling reaction.
The coupling reaction is typically conducted by contacting the solid support with an excess, preferably about 1.1 to about 10 or more equivalents, of the carboxylic acid-cont~ining compound (based on the number of equivalents of arnino groups present on the solid support) and at least one equivalent, preferably about 1.5 to about 3.0 equivalents, of the coupling reagent (based on the carboxylic acid groups) in an inert diluent, such N,N-dimethylformamide and the like. If desired, least one equivalent, preferably about 1.5 to about 3.0 equivalents (based on the l-thiog~l~r~se derivative), of a coupling promoter such as l-hydroxybenzotliazole may also be used in the reaction. Generally, the coupling reaction is conducted at a le~ ture ranging from about 0~C to about 50~C for about 24 to about 100 hours. Upon completion of the reaction, the solid support is preferably co~t~cte~ with excess acetic anhydride in methanol at a temperature ranging from about 0~C to about 40~C for about 12 to about 24 hours to cap any unreacted amino groups present on the solid support. The yield of inco.yGld~ion of a ~hio~-ch~ride onto the solid s~lppoll can be determined using well-established procedures such as those described, for eY~mple~ by M. Dubois et al.~3.

2. Method for Preparing A Thiosaccharide Derivative Library In another aspect, the methods of this invention provide for a thioc~ch~ride derivative library. Such libraries are produced by synthe-~i7in~ on each of a plurality of solid supports a single compound wherein each compound comprises a thios~ch~ride derivative.
The thio~l~ch~ride derivative libraries provided by this invention are syn~hesi7çd by first apportioning solid supports among a plurality of reaction vessels.
5 Such S~lppOll~ comprise a reactive functional group capable of covalently binding to the solid support. The function~l group is one that is capable of covalently binding a thios~ch~ride at a position other than the thiol group. Suitable functional groups include, by way of example, alcohols, amines, isocyanates, carboxylic acid groups, esters and the like. In one embo~iment this is accomplished by selectively blocking 10 the thiol group with a removable blocking group which, after coupling of the thiosaccharide to the solid support, is removed thereby freeing the thiol group for further reaction.
The supports in each reaction vessel are then cont~-t~d with a unique thio~( ch~ride under conditions wherein the thio~ ch~ride is covalently ~t ~-hed to 15 the solid supports through the reactive filncti~n~l group. This reaction is typically con~lucted by contacting the solid support with at least one equivalent, pf~feldbly 1 to S equivalents, of the thiosaccharide based on the functional groups on the solidsupport.
After ~tt~hing the thiosaccharide to the solid support, the su~ s are then 20 pooled and the pooled supports are then apportioned among a plurality of reaction vessels.
The supports having a thio~cch~ride covalently ~tt~chçd thereto are then contacted in each reaction vessel with a unique coupling reagent sP-l~ct~ from the group consi~tin~ of Michael accepto~ and ~-halocarbonyl compounds to provide for a 25 thio~h~ride carbonyl compound which covalently bound to the ~.lppoll. This reaction is preferably conducted as described above.
The thios~cçh~ride carbonyl compound is then reduced as described above to provide for an alcohol and/or an amine derivative. Optionally, the hydroxy or amino group of these compounds can be further derivatized as described above to form a30 group selected from esters, substituted ~minçs, ~ çs, carb~m~tPs, ureas, thioesters and thiocarb~m~t~s In an alternative embo~liment the thiosaccharide derivative libraries provide bythis invention are syntheci7ed by first apportioning solid suppo~Ls among a plurality of reaction vessels wherein such S~pOlls comprise a reactive filnrtion~l group covalently bound to the solid support such that the funrtio~ group one that is capable of covalently binding a coupling reagent. Such functional groups include, by way ofexample, alcohols, ~minPs~ isocyanates, carboxylic acid groups, esters and the like.
The supports in each reaction vessel is then cont~-tPd with a unique coupling reagent selP~cted from the group concicting of Michael acceptfjl~ and c~-halocarbonyl compounds under conditions wherein the coupling reagent is covalently ~tt~rhed to the 10 solid supports through the reactive functional group. Typically, this reaction is conducted by contacting the solid support with at least one equivalent of the coupling reagent, preferably with about 1 to about 5 equivalents, based on the funrtio groups on the solid support.
After ~tt~hing the coup}ing reagent to the solid support, the s~lppoll~ are then15 pooled and the pooled supports are then apportioned among a plurality of reaction vessels.
The supports having a coupling reagent covalently ~ttach-P~ thereto are then contacted in each reaction vessel with a unique thio~rçh~ride to provide for a thios~cch~ride carbonyl compound which is covalently bound to the suppoll. This 20 reaction is preferably contiucte~ as described above. The thiosacch~ride carbonyl compounds can then be reduced to provide for a plurality of alcohol and/or aminederivatives. As above, these alcohol and/or amine derivatives can optionally be further derivatized to provide for a group SPl~Pc~f ~ from esters, substituted ~min ~mides, carb~m~tes, ureas, thioesters, and thiocarb~m~tes.
In a preferred embo~iment~ an identifier tag is employed in the metho~s of this invention. The identifier tag has a recognizable feature that is, for eY~mp'e, microscopically or otherwise distinguishable in shape, size, mass, charge, or color.
This recognizable feature may arise from the optical, çh~.mic~l, electronic, or m~gmPtic properties of the tag, or from some combination of such pr~.Lies. In 30 essence, the tag serves to label a molecule and to encode information ~er;phf able at the level of one (or a few) molecules or solid supports. By using i~PntifiPr tags to CA 022=,6694 1998-11-24 track the synthesis pathway that each rnember of a chemic-~l library has taken, one can deduce the structure of ~ny chemi~l in the library by reading the id~ntifier tag.
The identifier tags identify each reagent or other reaction step that an individual library member or solid support has experienced and record the step in the 5 synthesis series in which each reagent was added or other ch~mic~l reaction performed. The tags may be ~tt~ched immedi~tely before, during, or after the reagent addition or other reaction, as convenient and compatible with the type of idpntifi~or tag, modes of ~t~ hment, and chemistry of activated ketone or other molecular synthesis. The i~lentifi~r tag can be ~ccoci~ted with the thioc~cch~ride derivatives 10 through a variety of m~rh~ni~m~, either directly, through a linking mol~nle, or through a solid support upon which the thiosaccharide derivative is synthesi7~d. In the latter mode, one could also attach the tag to another solid support that, in turn, is bound to the solid support upon which the thio~ch~-ide derivative is syn~h~i7~d The identifier tag is added when the solid supports that have undergone a specific 15 reagent addition or other chemic~l reaction step are physically together and so can be tagged as a group, i.e., prior to the next pooling step. P~felr~d iclentifi~- tags include, by way of example, peptides'4 l5 oligonucleotidesl6 and halocarbon derivatives'7.

20 3. Screening of Thiosaccharide Derivative Libraries The libraries of thiosa~c~l~ride derivatives (e.g., compounds of forrnula I) maybe screened for biological activity. Generally the library to be screen is e.~l)osed to a biological substance, usually a protein such as a r~ceptor, enzyme, ~ .llbld~ c binding protein or antibody, and the presence or ~hsence of an interaction between the 25 thiosaccharide derivative and the biological substance is deterrnined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by ~t~ching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. The degree of binding 30 affinity may be determined by quantitating the amount or intensity of the bound label.

, Thus, various lead compounds may be select~ by identifying which compounds bind the particular biological substance most effectively.
In a preferred embo~limPnt, bead-based libraries are scr~n~d by assays in which each different molecule in the library is assayed for its ability to bind to a S receptor of interest. The recepto~ is contacted with the library of thio~r~h~n~e derivatives, forming a bound member between the receptor and any thioc~-rh~nde derivative in the library able to bind the l~ceptor under the assay con~itinnc. The bound thiosaccharide derivative is then identified by eY~min~tion of the tag ~C~i~t~d with that thio~ch~ride derivative. The receptor to which the library is e ~pos~
10 under binding conditions can be a mixture of receptors, each of which is ~soci~fd with an idçntifi~r tag specifying the receptor type, and consequently two tags are ex~mine~ after the binding assay. Specific beads can be isolated in a receptor screening by a number of means, including infinite dilution, micromanipulation, or preferably, flow cytometry (e.g., fluorescçnce activated cell sorting (FACS)). By 15 adopting cell-sized solid SUppOltS or beads, one can use flow cylolllc~ly for high sensitivity receptor binding analysis and facile bead manipulation.
Thiosaccharide derivatives can be synthe~i7çd on beads and cleaved prior to assay. Cleavage of the thiosaccharide derivatives from the beads may be accomplished cleavable linker arms which are cleaved using conventional methods. In 20 either event, the thiosaccharide derivatives of interest are cleaved from the beads but remain cont~ined within the colllp~lment along with the bead and the identifiP,~ tag(s).
Soluble tagged thiosacch~ e derivatives can also be screened using an immobilized receptor. After cont~cting the tagged thiosa~ch~ride deAvatives with the immobilized receptor and washing away non-spe~ific~lly bound mQIt-rlJl~, bound, 25 tagged thiosaccharide derivatives are released from the receptor by any of a wide variety of methods. The tags are optionally amplified and then ex~min~ and ~ ed to identify the structure of the molecules that bind spe~ific~lly to the ~ tor. A
tagged thiosaccharide derivative in solution can be assayed using a l~cep~or immobilized by attachment to a bead, for example, by a co~ lition assay with a 30 fluorescently labeled ligand. One may recover the beads bearing immobilized receptors and sort the beads using FACS to identify positives (~iminished fluo~ n~e caused by the library molecule competing with the labeled ligand). The ~c~ qt~d identifier tag is then amplified and decoded.
Preferably, the libraries described herein will contain at least about 2 compounds, more preferably at least about 102 compounds, still more ~,ef~..bly from S about 102 to about 101~ compounds and even still more preferably from about lO3 to about 106 compounds.
Of particular interest is the identification of thios~ch~ride derivatives which block binding of a toxin, such as heat-labile en~eiuto~in or cholera toxin, the toxin's f~ceplor either in vitro or in vivo, and compounds which inhibit binding of or~nismS
(e.g., bacteria, virus, fungi, and the like), including enterovirulent organism such as ~brio cholerae and enterotoxigenic strains of Escherichia coli, to their cell surface receptors.
The following synthetic and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees CPI~;IIS.

EXAMPLES
In the e-~mples below, the following abbreviations have the following me~ning~. If an abbreviation is not defined, it has its generally accepted rn~nine.
A = angstroms bd = broad doublet bs = broad singlet d = doublet dd = doublet of doublets DMAP = dimethylaminopyridine eq. = equivalents g = grams L = liter m = multiplet meq = milliequivalent mg = milligram mL = milliliter mmol = millimol N = normal q = quartet quint. = quintet - WO 98n2487 PCTICA97100866 s = singlet t = triplet TFA = - trlfluoroacetic acid THF = tetrahydrofuran TLC = thin layer chromatography ~L = microliter IH-Nmr spectra were recorded with a Brueker AM-360 spe.;LIonleter and MALDI-TOF mass spectra were recorded with a HP G2020A (LD-TOF) insL,u.ll.,.~t.
10 Optical rotations were measured with a Perkin-Elmer 241 pol~rimeter ~oactiorls were monitored by TLC on Silica Gel FG254 (E. Merck, Darrnstadt, Ge~ any).

Example A
Solid-Phase Extraction of Lauroylated Intermediates As indicated in the following examples, certain lauroylated reaction intermediates were purifled by solid-phase extraction. In this purification procedure, the reaction mixture is concentrated, re-dissolved in meth~nol, and applied onto C18 silica (Waters Prep C18, 125 A, 1 g per 20 mg lauroylated carbohydrate). The C18silica is then washed with mPth~nol (10 mLt g C18 silica) and the product is eluted 20 with pentane (10 mL/ g C18 silica). For L-arginine cont~ining colll~unds, thereaction mixture is concentrated, re-dissolved in 70% mPth~nol and applied onto C18 silica. The C18 silica is then washed with 70% methanol and the product is eluted with meth~nol. The res~ltin~ product contains no residual reagents as deteln~ined'by TLC, 'H-nmr, or MALDI-TOF mass speclroscopy.
Example B
Synthesis of 1.2~3~4~6-Penta-O-lauroyl-~Y-D-galactopvranose 1 To a suspension of galactose (3.78 g, 21.0 mmol), pyridine (50 mL), and 4-30 dimethylaminopyridine (cat.) in pentane (150 mL) under argon ~tmosphPre, was added lauroyl chloride (50 mL, 210 mmol) at -78~C. The mixture was allowed to reach ambient l~ul~eldture~ The resulting white slurry slowly dissolved and a fine precipit~tP of pyridinium hydrochloride formed~ After 40 h, the pyridinium WO 98/22487 I'CT/CA97/00866 hydrochloride was filtered off and the pentane solution was concentrated. Columnchromaeography (SiO2, pentane/EtOAc 9:1) gave 1 (16.0 g, 70% yield), ta~]D25 +39~
(c 0.9, CHCI3). 'H-Nmr data (CHC13): ~ 6.39 (d, lH, J 2.4 Hz, H-l), 5.51 (br s, lH, H-4), 5.35 (m, 2H, H-2 and H-3), 4.32 (br t, lH, J 6.6 Hz, H-5), 4.08 (d, 2H, J
6.6 Hz, H-6a and H-6b), 2.39, 2.38, 2.30, 2.26 (4 t, 2H each, J 7.5 Hz, -CH2C~),2.21 (m, 2H, -CH2CO-), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C66H,22O~:
C, 72.2; H, 11.3. Found: C, 72.6; H, 11.5.

Example C
Synthesis of l-S-Acetyl-2.3.4.6-tetra-O-lauroyl-l-thio-B-D-~alactopyranose (2) Method 1: To compound 1 (from Fy~mple B, 1 g, 0.91 mmol) and thiollr~
acid (0.4 mL, 9.1 mmol) in dry dichlorometh~ne (5 mL) under argon at 0~C, was added boron trifluoride etherate (1.7 mL, 13.6 mmol). The cold-bath was removed after 10 min and after 24 h the mixture was diluted with dichlororneth~ne, washed with saturated sodium bicarbonate, dried over sodium sulfate, and conc~ntrated.
Column chromatography (SiO2, pentane/Et20/EtOAc 9:1:1) gave 2 (0.60 g, 70%
yield).

Method 2: To compound 1 (from Example B, 276.5 mg, 0.253 mmol) in dry tetrahydrofuran (2.0 mL) under argon, was added benzylamine (27.9 ~L, 0.255 mmol). The mixture was concen~rated after 70 h. The residue was dissolved in drydichloromethane (4.0 mL) under argon and then trichloroacetonitrile (250 ~LL, 2.5 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (30 ~L, 0.2 mmol) were added. The mixture was concentrated after 3 h and the residue was flashed through a short column (SiO2, pentane/EtOAc 19:1), then concentrated. To the residue in dry dichloromethane (3.5 mL) under argon, was added thiolacetic acid (1 mL). After 96 h, the reaction mixture was concentrated and the residue was purified by column ~ chromatography (SiO2, pentane, EtOAc 19: 1) to give 2 (90 mg, 37% yield), [~Y}D25 21~ (c 1, CHCl3). 'H-Nmr data (CHCl3): ~ 5.47 (d, lH, J 3.4 Hz, H-4), 5.32 (t, lH, J 10.0 Hz, H-2), 5.25 (d, lH, J 10.0 Hz, H-l), 5.12 (dd, lH, J 3.4 and 10.0 Hz, H-~ .... . . . ....

3), 4.08 (m, 3H, H-S, H-6a and H-6b), 2.14-2.43 (m, 8H, -CH2CO-), 2.37 (s, 3H, -SAc), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C56HI02Olos C, 69.5; H, 10.6; S, 3.3. Found: C, 69.4; H, 10.8; S, 3.5.

S Method 3: To compound 1 (20.0 g, 18.2 mmol) and thio~ tic acid (5.0 mL,1.9 eq.) in dry dichlorome~h~ne (300 mL) under argon, was added trimethy}silyl trifluorom~th~nesulfonate (5.0 mL, 0.5 eq.) at 0~C. The cold-bath was imm~i~ly removed and after 48 h the mixture was diluted with dichlorometh~ne, washed withsaluldted sodium hydrogen carbonate, dried (Na2SO4), and concentrated. Column chromatography (SiO2, pentane/EtOAc 20:1) gave 2 (13.7 g, 77%), [~]D25 ~21~ (c 1, CHCI3). IH-Nmr data (CHCl3): ~ 5.47 (d, lH, J 3.4 Hz, H-4), 5.32 (t, lH, J 10.0 Hz, H-2), 5.25 (d, lH, J 10.0 Hz, H-1), 5.12 (dd, lH, J 3.4 and 10.0 Hz, H-3), 4.08 (m, 3H, H-5, H-6a and H-6b), 2.14-2.43 (m, 8H, -CH2CO-), 2.37 (s, 3H, -SAc), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C56H,02OloS: C, 69.5; H, 10.6;
S, 3.3. Found: C, 69.4; H, 10.8; S, 3.5.

Fy~mplç C' Synthesis of 1 -S-Acetyl-2.3.4.6-tetra-O-laurovl-l-thio-cY-D-galactopyranose Method 1: To compound 1 (20.0 g, 18.2 mmol) and thioacetic acid (27.0 mL, 20 eq.) in dry toluene (80 mL) under argon was added tin (IV) chloride (21.3 mL)dropwise at room temperature. The reaction was monitored by Tlc carefully. After 1 h, 600 mL of lM aqueous HCl was added to the vigorously stirred mixture and the res--lting mixture was filtered through Celite to remove the emulsion of tin salts. The mixture was diluted with pentane (800 mL), washed with water (2 x 400 mL), saturated sodium hydrogen carbonate (300 mL) and water (300 mL), dried with Na2SO4 and concçntrated. The residue was purified by column chromatog~dphy threetimes (SiO2, pentane/EtOAc 20:1, 30:1, 40:1) to give 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-a-D-galactopyranose (3.65 g, 21 %). 'H-Nmr data (CHCl3): ~ 6.26 (d, lH, J 5.5 Hz, H-1), 5.47 (dd, lH, J 11.0 Hz, 5.5 Hz, H-2), 5.46 (d, lH, J 3.5 Hz, W O 98~2487 PCTICA97/00866 H-4), 5.04 (dd, lH, J 11.0 Hz, 3.5 Hz, H-3), 4.17 (t, lH, J 6.5 Hz, H-5), 4.06 (d, 2H, J 6.~ Hz, H-6a and H-6b), 2.38 (t, ~H, J 7.0 Hz, -COCH2-), 2.40 (s, 3H, -SAc), 0.87 (t, 15H, J 7.0 Hz, -CH3).

S Method 2: To compound 1 (25.0 g, 22.9 mmol) and thio~cetic acid (8.5 mL, 114.5 mmol) in dry dichlorometh~ne (100 mL) under argon, was added trimethylsilyl trifluororneth~nesulfonate (5.6 mL, 45.8 mmol) at room t~ ture. After 20 h, the mixture was diluted with dichloro-neth~ne (600 mL), washed with saturated sodiumhydrogen carbonate (250 mL) and water (2 x 200 mL), dried with Na2SO4 and concentrated. The residue was purified by column chromatography three times (SiQ2, pentane/EtOAc 20:1, 30:1, 40:1) to give 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thi~a-D-galactopyranose (1.59 g, 7.2%).

Example D
General Procedure for Michael Additions and c~!-Halocarbonyl Substitutions To compound 2 (1 mmol) and an electrophile (1.2 mmol) in dry dichlorometh~ne (~ mL) under argon, was added Et2NH (4 mL). After 1-3 h, the mixture was concentrated and the residue was purified by column chromatography on SiO2 by eluting with pentane/EtOAc. The products were characterized with 'H-nmr spectroscopy and MALDI-TOF mass spectroscopy.

Example E
General Procedure for Reduction to Alcohols To the product from Example D (100 ~mol) in dry tetrahydlorul~n (2.0 mL) and isopropanol (0.7 mL) under argon atmosphere, was added NaBH4 (150 ~mol).
After 0.5-3 h, the mixture was concentrated (acetic acid (about 40 ~L) was addedprior to concentration in some cases) and the residue was purified according to the solid-phase extraction procedure of Example A. The product alcohols were characterized with 'H-nmr spectroscopy and MALDI-TOF mass spect~vsco~y.

Example F
General Procedure for Reductive Amination to a Primary Amine Method 1: To the product from Example D (ioo ~mol) and ~mmonillm acetate (75 mg, 1 mmol) in dry methanol (2.3 mL) and tetrahydrofuran (0.9 mL) under S argon, was added NaCNBH3 (100 ~mol). After 1-72 h, the mixture was con~ t~d and the residue puAfied according to the solid-phase extraction procedure of Example A. The product amines were char~cter 7e~ with 'H-nmr syecLIosco~y and MALDI-TOF mass spectroscopy.

Method 2: The product from Example D (200 mg, 0.198 mmol) and dry NH40Ac (30 mg, 0.4 mmol) were stirred in dry MeOH (6 mL), dry 1,2-dichloroethane (6 mL), and trimethyl orthoformate (1 mL~ under argon for 24 h (un~l TLC analysis showed that most of the starting material was con~umed). NaBH4 (10 mg, 0.26 mmol) was added and after 1 h the mixture was concentrated. The residue15 was purified according to the solid-phase extraction procedure of Example A to provide the primary amine (containing traces of the co~ onding alcohol). This mixture was dissolved in pentane/EtOAc (1:1) and applied onto a Waters Sep-Pak Plus Longbody SiO2 cartridge. The cartridge was washed with pentane/EtOAc (1:1, 20 mL) (to remove the co~ ,uonding alcohol), followed by elution with toluene/EtOH
20 (9:1, 30 mL) to afford the primary amine.

Example G
General Procedure for Acylation of Primary Amines with Phthalic Anhydride The O-lauroylated primary amine from Example F (100 ~mol), phthalic anhydride (2.7 mmol), and 4-(N,N-dimethylamino)pyridine (catalytic) were dissolved in dry pyridine. The mixture was concentrated after 12-48 h and the residue purified according to the solid-phase extraction procedure of Example A. The product 2-carboxyben7~mides were characterized with 'H-nmr spect~uscopy and MALDI-TOF
30 mass spectroscopy.

.

Example H
General Procedure for Reductive Amination with Amino Acids To the product from Example D (100 ~mol) and an amino acid te~t-butyl ester 5 hydrochloride or methyl ester hydrochloride (l mmol) in dry MeCN (2.25 mL) andTHF (0.75 mL), was added NaCNBH3 (100 ~mol). After 1-72 h, the ~ ul~ was c~nc~ntrated and the residue was purified according to the solid-phase eYtrveti~n procedure of Example A. The product N-alkylated amino acids were chata.;l~,iz~d with IH-nmr spectroscopy and MALDI-TOF mass spectroscopy.
Example I
General Procedure for Deblocking of Alcohols To the lauroylated alcohol from Example E (100 ~mol) in dry meth-s-nQl (7.1 mL) and dichlorornethsne (1.4 mL) under argon ~tmosphçre, was added ml;!l.sn~
sodium methoxide (1 M, 50 ~L). After 1-24 h, the mixture was neutralized with Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in water and applied onto a column of C18 silica (Waters Prep C18, 125 A, s g). TheC18 silica was washed with water (50 mL), and the product was then eluted with 70%
methanol (50 mL). The resulting alco,hDls were characterized with lH-nmr spectroscopy and MALDI-TOF mass spectroscopy.

Example J
General Procedure for Deblocking of Primary Amines To the O-lauroylated primary amine from FY~mple F (100 ,umol) in dry meth~nol (7.1 mL) and dichloromethane (1.4 mL) under argon, was added methsn~
sodium methoxide (1 M, 50 ~LL). After 1-24 h, the mixture was neutralized with Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in dichloromethane/methanol 2:1 and applied to a Waters SepPak Plus Longbody SiO2 cartridge. The cartridge was washed with dichloromethane/me~h~nol (2:1) and thenthe product was eluted with dichloromethane/methanol/water (5:5:1) (20 mL) and concentrated. The residue was dissolved in water and applied onto a column of C18 ... ..... .. ..... ...... . .. .

WO 98t22487 PCT/CA97/00866 silica (Waters Prep C18, 125 A, s g). The C18 silica was washed with water (50 mL) and then the product was eluted with mçth~nol (50 mL). The res~ltin~
amines were characterized with lH-nmr sl,ecl,oscopy and MALDI-TOF mass spec~l~,s~opy.

Example K
General Procedure for N-Acetylation of Primary Amines To the primary amine from Fl~mple J (100 ~mol) in moist meth~nol (4.4 mL) was added acetic anhydride (0.4 mL). The mixture was col-centrated after 2-24 h, re-dissolved in water and applied to a column of C18 silica (Waters Prep C18, 125 A, S
g). The C18 silica was washed with water (50 mL) and then the product was elutedwith meth~nol (50 mL). The r~s-llting ~et~mides were characterized with 'H-nmr speclroscopy and MALDI-TOF mass ~e~ osco~y.
Example L
General Procedure for Deblocking of 2-Carboxybenzamides To the O-lauroylated 2-carboxybenzamide from Example G (100 ~mol) in dry meth~nol (7.1 mL) and dichlororneth~ne (1.4 mL) under argon, was added meth~n5 sodium methoxide (1 M, 50 ~L). After 1-24 h, the mixture was neutralized with Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in dichlorometh~n~/meth~nol (8:1) and applied to a Waters SepPak Plus Longbody SiO2cartridge. The cartridge was washed with dichlorometh~n~/m~th~-lol (8:1) and then the product was eluted with dichlorometh~ne/rneth~nol/water (65:35:5) (20 mL) and concen~ ted. The residue was dissolved in water and applied to a column of C18 silica (Waters Prep C18, 125 ~, 5 g~. The C18 silica was washed with water (50 mL), and then the product was eluted with methanol (50 mL). The res~ in~ 2-carbox~,l,e~,,..-.i~es were characterized with 'H-nmr ~peclrosco~y and MALDI-TOF30 mass speclfosC~l)Y-E,xample M
General Procedure for Deblocking of N-Alkylated Glycine, ~-Alanine. and L-Leucine Com~ounds The N-alkylated amino acid ten-butyl ester from Exarnple H (100 ~mol) was treated with trifluoroacetic acid (3.5 mL) in dry dichlorometh~ne (3.5 mL) for 1-10 h.
n-Propyl acetate (8 mL) and toluene (16 mL) were added and the mixture was concentrated, then co-concentrated twice with toluene. To the residue in dry methanol (7.1 mL) and dichlorometh~ne (1.1 mL) under an argon ~tmosph~ore was added methanolic sodium methoxide (1 M, 200 ~L). After 1-24 h, the l,li~lure was10 neutralized with Amberlite IR-SOS (H+) resin, filtered and concentrated. The residue was dissolved in dichloromethane/methanol (9:1) and applied to a Waters SepPak Plus Longbody SiO2 cartridge. The cartridge was washed with dichlorometh~ne/mPth~nol (9:1) and then the product was eluted with dichlorome~h~ne/methanol/water (65:35:5) (20 mL) and concentrated. The residue was dissolved in water and applied to a 15 column of C18 silica (Waters Prep C18, 125 A, s g). The C18 silica was washedwith water (50 mL) and then the product was eluted with 70% nleth~nol (50 mL).
The resulting N-alkylated glycine, ~-alanine, and L-leucine compounds were characterized with 'H-nmr spectroscopy and MALDI-TOF mass a~ecLloscop~r.

Example N
General Procedure for Deblocking of N-Alkylated L-Histidine and L-Tryptophan Compounds To the N-alkylated amino acid methyl ester from Example H (100 ~Lmol) in dry methanol (7.3 rnL) and dichlorometh~ne (1.1 mL) under an argon atm~hcr~ was 25 added methanolic sodium methoxide (1 M, 50 ~L). After 1-24 h, the .ni~lu~ wasneutralized with Amberlite IR-50S (H+) resin, filtered and concPntrated. The residue was dissolved in 70% methanol and applied to a column of C18 silica (Waters PrepC18, 125 A, s g) and then the product was eluted with 70% mPth~nol (50 mL). To the residue in water (3.7 mL) was added aqueous lithium hydroxide (lM, 0.3 mL).
30 After 0.5-2 h, the mixture was neutralized with Amberlite IR-SOS (H+) resin, filtered and concent~ated. The residue was dissolved in dichloromethane/methanol (9:1) and . _. . . ,,, ~ . .

applied to a Waters SepPak Plus Longbody SiO~ cartridge. The cartridge was washed with dichloromethane/methanol (9:1) and then the product was eluted with dichlorometh~n~/meth~nol/water (65:35:5) (20 mL) and conc~ntrated. The residue was dissolved in water and applied to a column of C18 silica (Waters Prep C18, 125 s A, 5 g). The C18 silica was washed with water (50 mL), and the product was eluted with 70% methanol (50 mL). The resulting N-alkylated L-histidine and L-tr~p~ophan compounds were characterized with IH-nmr spectroscopy and MALDI-TOF mass spectroscopy.

Example O
General Procedure for Deblocking of N-Alkylated L-Arginine Compounds To the l~-alkylated arginine methyl ester from Example H (100 ~mol) in dry meth~nQI (7.3 mL) and dichloromethane (1.1 mL) under an argon ~tmosph~re was added m~th~nolic sodium methoxide (lM, 50 ~L). After 1-24 h, the mixture was neutralized with Amberlite IR-SOS (H~) resin, filtered and concei,t.dted. The residue was dissolved in 70% methanol and applied to a column of C18 silica and then theproduct was eluted with 70% methanol (50 mL). To the residue in water (3.7 mL) wa then added aqueous lithium hydroxide (lM, 0.3 mL). After 0.5-2 h, the mixture20 was neutralized with Amberlite IR-SOs (H+) resin, filtered and concentrated. The residue was dissolved in water and applied to column of C18 silica (Waters Prep C18, 125 A, s g). The C18 silica was washed with water (50 mL) and then the product was eluted with 50% methanol (50 mL). The res~lting N-alkylated L-arginine cG...l~ou~ds were characterized with 'H-nmr s~ecLloscopy and MALDI-TOF mass spe~ osco~.
Example P
General Procedure for the P~el)aldlion of Mesylates To the alcohol from Example D (0.3 mmol) in dry tetrahydrofuran (2 mL) and 30 dry pyridine (4 mL) under an argon atmosphere was added meth~n~sl-lfonyl chloride (0.5 mL). After 12-24 h, the mixture was washed with O.5M HCI and extracted with pentane. The pentane extracts were concen~rated and the residue was purified on C18-silica to afford the mesylate derivative.

Example Q
S General Procedure for the Preparation of Azido Compounds To the mesylate from FY~mple P (0.2 mmol) in dry DMF (8 mL) and dry THF
(3 mL) under an argon atmosphere at 60~C was added sodium azide (5 mmol) and 18-crown-6 (180 mg). After 2 hours, the reaction mixture was concentrated and the 10 residue was purified on C18-silica. In some cases, the product was re-chromatographed with silica gel using pentane/EtOAc (9:1) as the eluant to afford the azido derivative.

Example R
General Procedure for Reduction of Azido Groups to Primary Amines To a solution of the azido compound from Fy~mple S (15 ~lmol) in dry isopro~anol (1 mL) and dry ethanol (1 mL) under an argon atmosphere, was added NaBH4 (15 ~mol) and NiCl2 (30 ~mol). ARer 1 hour, the reaction mixture was 20 neutralized with acetic acid (1 drop), concentrated and purified on C18-silica to afford the primary amine.

Example S
General P,ocedule for Reductive Alkylation of Primary Amines To the primary amine from Example F or S (6.8 ~mol) in dry m~th~nol (1 mL) and dry dichlororne~h~n~ (1 mL) under an argon atmosphere was added an aldehyde or ketone (3.4 mmol) and sodium triacetoxyborohydride (47 llmol). After 24~8 hours, toluene (1 mL) was added and the mixture was concentrated and the residue30 purified on C18-silica gel.

Fy~mple T
- General Procedure for Reductive Amination To the product from Example D (0.1 mmol) and a primary amine (0.45 mmol) in dry dichloromethane (2 mL), meth~nol (2 mL) and triethylorthoformate (1 mL) 5 under argon, was added NaCNBH3 (1 mmol). After 24 h, the mixture was concentrated and dissolved in toluene (1 mL) and purified on C18-silica gel (5 g).

Example U
General Procedure for Deblocking of Secondary Amines To the O-lauroylated secondary amine from Example S or T (100 ~mol) in dry methanol (7.1 mL) and dichloromethane (1.4 mL) under argon, was added meth~ns~
sodium methoxide (lM, 50 ~L). After 1-24 h, the mixture was neutralized with Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in dichloromethane/methanol 2:1 and applied to a Waters SepPak Plus Longbody SiO2 cartridge. The cartridge was washed with dichlorometh~nP/meth~ncl (2:1) and thenthe product was eluted with dichlorometh~ne/mPth~nol/water (5:5:1) (20 mL) and concent-ated. The residue was dissolved in water and applied onto a column of C18 silica (Waters Prep C18, 125 A, 5 g). The C18 silica was washed with water (50 mL) and then the product was eluted with methanol (50 mL). The res--lting secondary amines were characterized with lH-nmr spectroscopy and MALDI-TOF
mass spectroscopy.

Example Al Sylllll~is of 2-Hy~l-ox~-;yclopent-1-yl 1-Thi~B-D-galactopyr~no~
The title compound was prepared according to procedures D, E and I above using 2-chlorocyclopentanone as the electrophile. Mass spectra data was as follows:
M (calcd.): 280.34; M (found): 304.9 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.44 (H-l), 4.42, 4.38, and 4.35.

Example A2 Synthesis of 2-HydroA~c~clohex-1-yl l-Thi~-D-ga}actopyranoside The title compound was plcpar~d according to procedures D, E and I above 5 using 2-chlorocyclohe~nQlle as the electrophile. Mass spectra data was as follows:
M (calcd.): 294.34; M (found): 318.8 ~M+Na+). Select~d nmr data was as follows:
H-nmr (CD30D): ~ 4.55 (H-l), 4.43, 4.39, and 4.34.

Example A3 Synthesis of 3-Hydroxy-l-phenylbut-1-yl 1-Thio-,15-D-~ ctopyr~n~cj~
The title compound was prepared according to procedures Dt E and I above using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 345.43; M (found): 368.0 (M+Na+). Selected nmr data was as follows:
15IH-nmr (CD30D): ~ 4.45 (H-l), 4.43, 4.31, and 4.25.

Example A4 Synthesis of (3-Hydroxynorborn-2-yl)methyl 1-Thio-,B-D-~ ctopyranoside 20The title compound was prepared according to procedures D, E and I above using 3-methylene-2-norbornanone as the electrophile. Mass spectra data was as follows: M (calcd.): 320.41; M (found): 344.6 (M+Na+). Sel~c~ed nmr data was as follows: 'H-nmr (CD30D~: ~ 4.30 (H-1) and 4.29.

25Exarnple A5 Synthesis of 3-Hydroxycyclohept-1-yl l-Thio-,B-D-~ ctopyranoside - The title compound was plepa ed according to procedures D, E and I above using cyclohept-en-l-one as the electrophile. Mass spectra data was as follows: M
30(calcd.): 308.40; M (found): 332.1 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): â 4.394 (H-1), 4.389, and 4.381.

. . ..

Exarnple AS' _ Synthesis of 3-Hydrox~clQhept-l-yl 1-Thi~a-D-~ ctopyranoside The title compound was ~l~ paled according to procedures D, E and I above S using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl- l-thio-a-D-galactopyranose (from Example C' above) and cyclohépt-en-l-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 308.40; M (found): 331.3 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 5.44 (d, J 5.8 Hz, H-1) and 5.45 (d, J 5.8 Hz, H-1).

Example A6 Synthesis of 2,2-Dimethyl-~hydrox~c~clopent-l-yl 1-Thio-~B-D-g~ topyr~
The title compound was prepared according to procedures D, E and I above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 308.40; M (found): 332.1 (M+Na+). Sel~t~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.34 (H-1), 4.315, 4.310, and 4.305.

Example A7 Synthesis of 3-Hydroxycyclopent-l-yl l-Thio-,B-D-g~lqctopyranoside The title compound was prepared according to procedures D, E and I above using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 280.34; M (found): 304.9 (M+Na+). Sele~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.36 (H-l), 4.355, and 4.34.
Example A8 Synthesis of ~Hydroxypent-2-yl l-Thi~,~-D-galactopyranoside The title compound was prepared according to procedures D, E and I above using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 282.35; M (found): 305.3 (M+Na+). Select~l nmr data was as follows:
lH-nmr (CD30D): ~ 4.42 (H-l), 4.41, and 4.39.

- Wo 98/22487 - pcTlcAs7loo866 Fy~nlple A9 Synthesis of 2,2-Dimethyl-5-hylllo~cyclohex-1-yl l-Thio-~-D-gs~l~rtoFyr~nos~,de The title compound was prepared according to procedures D, E and I above S using 4,4-dimethylcyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd ): 322,42; M (found): 346.6 (M+Na+). S~lPct~d nmr data was as follows: ~H-nmr (CD30D): ~ 4.34 (H-1), 4.33, and 4.32.

Example A10 Synthesis of 3-Hydroxycyclohex-1-yl l-Thi~,B-D-galactopyr~nos;~
The title compound was prepared according to procedures D, E and I above using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 294.37; M (found): 317.3 (M+Na+). S~le~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.422 (H-l), 4.417, and 4.38.

Example A11 Synthesis of 4,4-Dimethyl-3-hydroxycyclohex-1-yl l-Thio-,B-D-galactopyr~n~
The title compound was plc~ed according to procedures D, E and I above using 6,6-dimethylcyclohex-2-en-1-one as the electrophile.

Exarnple B1 Synthesis of 2-Aminocyclor~nt-1-yl l-Thio-,B-D-galactopyranoside The title compound was prepared according to procedures D, F and J above using 2-chlorocyclopentanone as the electrophile. Mass spectra data was as follows:
M (calcd.): 279.36; M (found): 276.3 (M+H+). .~elec~d nmr data was as follows:
IH-nmr (CD30D): ~ 4.46 (H-l), 4.45, 4.37 and 4.27.

, . . ~ ~ . .. ... . . . ~

WO 98n2487 PCTICA97/00866 Example B2 Synthesis of 2-Aminocyclohex-1-yl 1-Thio-,B-D-g~ topyranoside The title compound was prepared according to procedures D, F and J above 5 using 2-chlorocycloheY~nonP as the electrophile. Mass spe~tra data was as follows:
M (calcd.3: 293.38; M (found): 295.8 (M+H+), and 319.7 (M+Na+). Sel~t~ nmr data was as follows: IH-nmr (CD30D): ~ 4.48 (H-1), 4.44, 4.40 and 4.30.

Example B3 Synthesis of 3-Amino-1-phenyl~ut-1-yl 1-Thio-~-D-galactopyr~n~c;Ae The title compound was prepared according to procedures D, F and J above using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 344.45; M (found): 345.1 (M+H+). Sele~t~d nmr data was as follows:
15IH-nmr (CD30D): ~ 4.41 (H-1), 4.12, and 3.90.

Example B4 Synthesis of (3_~minrnQrborn-2-yl)methyl 1-Thio-,B-D-galactopyranoside 20The title compound was prepared according to procedures D, F and J above using 3-methylene-2-norbomanone as the electrophile. Mass spectra data was as follows: M (calcd.): 319.42; M (found): 321.6 (M+H+). Sele~ted nmr data was as follows: 'H-nmr (CD30D): ~ 4.42 (H-1), 4.41, 4.38, and 4.35.

25Example B5 Synthesis of 3-Aminocy~ lohept-1-yl 1-Thio-,l~-D-galactopyranoside The title compound was prepared according to procedures D, F and J above using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
30(calcd.): 307.41; M (found): 333.0 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.41 (H-1), 4.39, and 4.38.

Example B6 Synthesis of 2,2-Dimethyl-~minocyclopent-l-yl 1-Thio~ D-g~ ctopyranoside The title compound was pr~ed according to procedures D, F and J above S using 4,4-dimethylcyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd;): 307.41; M (found): 307.2 (M+H+). SelP~ted nmr data was as follows: ~H-nmr (CD30D): ~ 4.35 (H-1), 4.33, 4.32, and 4.30.

Example B6A
Synthesis of 2,2-Dimethyl-~(methylamino)-cyclopent-1-yl l-Thio-,B-D-~ rtopyr~n~5i~1~
The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and methylamine as ~eprimary amine. Mass spectra data was as follows: M (calcd.): 321.43; M (found):
322.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.325 (H-1), 4.315, 4.308, 4.304.

Example B6B
Synthesls of 2,2-Dimethyl~(isopropylamino)-cyclopent-1-yl 1-Thio-~B-D-~ ~topyr~nos;~le The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and isopropylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 349.48: M (found):
350.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 41460 (H- -1), 4.401, 4.400, 4.391.

... .

Example B6C
Synthesis of 2,2-Dimethyl-~(n-propylamino)-cyclopent-l-yl 1-Thio-,B-D-galactopyr~nr~sid~
The title compound was prepared according to procedures D, T and U above using 4,4-dimethyicyclopent-2-en-1-one as the electrophile and n-propylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 349.49; M (found):
350.5 (M+~+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.324 (H-1), 4.317, 4.310, 4.307.~0 Example B6D
Synthesis of 2,2-Dimethyl~((R)-sec-butylamino)-cyclopent-l-yl 1-Thio-,B-D-~ ctopyranoside The title compound was prepa~ed according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and (R)-(-)-sec-butylan~ine as the primary amine. Mass spectra data was as follows: M (calcd.): 364.52; M
(found): 364.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.328 (H-1), 4.319, 4.313, 4.311.
Example B6E
Synthesis of 2,2-Dimethyl-4((S)-sec-butylamino)-cyrlopent-1-yl 1-Thi~,B-D-g~l~ctopyranoside The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and (S)-(+)-sec-bu~lamine as the primary amine. Mass spectra data was as fol}ows: M (calcd.): 364.52; M
(found): 364.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.333 (H-l), 4.330, 4.300, 4.290.

Wo 98/22487 PCT/CAg7/00866 Example B6F
Synthesis of 2,2-Dimethyl~(pent-3-ylamino)-cyclopent-l-yl l-Thio-,li-D-~ ctopyrqncsi-le The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-pentylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 377.53; M (found):
376.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.333 (H-1), 4.329, 4.300, 4.290.
Example B6G
Synthesis of 2,2-Dimethyl-4(n-hexylamino)-cyclopent-l-yl l-Thio-,B-D-galactopyr~nosi~le The title compound was prepared according to ~ ced-nes D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and n-hexylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 391.57; M (found):
394.3 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.336 (H-1), 4.332, 4.303, 4.291.
Example B6H
Synthesis of 2,2-Dimethyl-~cyclobut-1-ylamino)-~clope--l-l-yl 1-Thio-~-D-~ ctopyranoside The title compound was plepa,cd according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and cyclobutyl amine as the primary arnine. Mass spectra data was as follows: M (calcd.): 361.50; M
(found): 361.6 (M+H+). Sçlçcted nmr data was as follows: 'H-nmr (CD30D):
4.315 (H-l), 4.300, 4.292, 4.290.

Example B6I
Synthesis of 2,2-Dimethyl-4(3,3-dimethylcyclobut-1-ylamino)-cyclopent-1-yl 1-Thi~B-D-galactopyranoside S The title compound was prepared according to procedures D, T and U aboveusing 4,4-dimethyIcyclopent-2-en-1-one as the electrophile and 3,3-dimethylcyclobut-1-ylamine as the primary amine. Mass spectra data was as follows: M (calcd.):
389.55; M (found): 392.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.324 (H-l), 4.311, 4.305, 4.294.
Example B6J
Synthesis of - _ 2,2-Dimethyl-4-(cyclopent-1-ylamino)-cyclopent-1-yl 1-Thio-,B-D-galactopyr~r~ ~s;d~
The title compound was prepared according to ~loced-lres D, T and U a'oove using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and cyclopentylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 375.52; M
(found): 376.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.322 (H-l), 4.310, 4.304, 4.295.
Example B6K
Synthesis of 2,2-Dimethyl-1 (cyclohex-1-ylamino)-cyclopent-1-yl 1-Thio-,B-D-galactopyranoside The title compound was p~epa ed according to procedures D, T and U a~ove using 4,4-dimethylcyclopent-2-en-1-one as the elec~ophile and cyclohexylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 389.55; M (found):
391.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.319 (H-1), 4.310, 4.307, 4.293.

Examp}e B6L
Synthesis of 2,2-Dirnethyl-4-(~methylcyclohex-1-yla nino)-cyclop~n~ yl l-Thi~,B-D-~ ctopyr~n~s;~le The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 4-methylcyclohex-1-ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 403.47;
M (found): 404.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.333 (H-1), 4.312, 4.300, 4.295.
Example B6Q
Synthesis of 2,2-Dimethyl-~(3-methylcyclopent-1-ylamino)-cyclopent-1-yl 1-Thio-,B-D-galactopyr~n~-si~,~
The title compound was prepared according to procedures D, T and U above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-methylcyclopent-1-ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 389.55;
M (found): 390.7 (M+H+). Sele~t~Pd nmr data was as follows: IH-nmr (CD30D):
4.383 (H-1), 4.325, 4.300, 4.292.
Example B6R
Synthesis of 2,2-Dimethyl-4(3,3-dilnethylcyrlorP.~t-l-ylamino)-cyclopent-l-yl 1-Thio-,B-D-~ rtopyranoside The title compound was p~ ed according to procedures D, T and U
above using 4,4-dimethylcyclopent-2-en- 1 -one as the electrophile and 3,3-dimethylcyclopent-l-ylamine as the primary amine. Mass spectra data was as follows:
M (calcd.): 4.295; M (found): 404.3 (M+H+). SelP~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.322 (H-l), 4.305, 4.300, 4.295.

Example B6T
Synthesis of 2,2-Dimethyl~(3-methylcyclohex-1-ylamino)-cyclopent-l-yl l-Thio-,B-D-galactopyr~n~;de S The title compound was plepar~d according to procedures D, T and U aboveusing 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-methylcyclohex-1-ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 403.57;
M (found): 404.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.326 (H-1), 4.313, 4.303, 4.294.
Example B7 Synthesis of 3-Aminocyclopent-l-yl l-Thio-~B-D-g~ topyr~r~os;de The title compound was prepared according to procedures D, F and J above 15 using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 279.35; M (found): n.a. Selected nmr data was as follows: lH-nmr (CD30D): ~ 4.46, 4.40, 4.38, and 4.34 (4 d, J 10 Hz), 3.88 (br s), 2.61, 2.27, 2.15, 1.82, and 1.64 (5 m).

Example B8 Synthesis of ~Aminopent-2-yl l-Thio-,B-D-galactopyr~n~s;-le The title compound was prepared according to procedures D, F and J above using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 281.37; M (found): 283.4 (M+H+). Selectçd nmr data was as follows~
nmr (CD30D): ~ 4.41 (H-1), 4.40, and 4.36.

Example B10 Synthesis of 3-Aminocyclohex-l-yl l-Thio-~-D-gPl~rSopyranoside The title compound was prepated according to procedure D, F and J above using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M

(calcd.): 293.38; M (found): 317.9 (M+Na+). Selected nmr data was as follows:
H-nmr (CD30D): ~ 4.54 (H-1), 4.52, 4.49, and 4.47.

Example Bll S Synthesis of 3-Amino-4,~dimethylcyclohex-1-yl 1-Thi~,B-D-gala~ to~ ~. snoside The title compound was prepared according to procedure D, F and J above using-6,6-dimethylcyclohex-2-en-1-one as the electrophile.

Example Cl Synthesis of 2-Ac~t~mi~10cyclopent-l-yl l-Thio-,B-D-g~l~cto~yranoside The title compound was prepared according to procedures D, F, J and K above using 2-chlorocyclopent-1-one as the electrophile. Mass spectra data was as foIlows:
M (calcd.): 321.39; M (found): 345.8 (M+Na+). Selected nmr data was as follows:
H-nmr (CD30D): ~ 4.53 (H-l), 4.44, 4.32, and 4.24.

Example C2 Synthesis of 2-Acet~mirlocyclohex-1-yl 1-Thio-,B-D-galactopyr~noside The title compound was prepared according to procedures D, F, J and K above using 2-chlorocyclohexan-1-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 335.42; M (found): 359.4 (M+Na+). Selected nmr data was as follows:
~H-nmr (CD30D): ~ 4.43 (H-1), 4.42, 4.32, and 4.29.
Example C3 Synthesis of 3-Ac~t~ ~1-phenylbut-1-yl 1-Thi~,B-D-galactopyr~r~osi,d~
The title compound was prepared according to proced~res D, F, J and K above using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 386,48; M (found): 408.3 (M+Na+). Selected nmr data was as follows:
- lH-nmr (CD30D): ~ 4.32 (H-1), 4.25, 3.83, and 3.79.

, Example C5 Synthesis of 3-Acet~midocyrlQhel)t-l-yl l-Thi~,B-D-g~l~rtopyranoside The title compound was prepared according to procedures D, F, J and K above 5 using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 349.42; M (found): 372.5 (M+Na+). .Sele~t~ nmr data was as follows:
'H-nmr (CD30D): ~ 4.403 (H-l), 4.397, 4.34, and 4.33.

Example C7 Synthesis of 3-Acetamidocyclopent-l-yl l-Thi~,B-D-galactopyr~nn~;de The title compound was ~lel a~cd according to procedu~es D, F, J and K above using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 321.39; M (found): 349.5 (M+Na+).
Example C8 Synthesis of ~Acet~midopent-2-yl l-Thio-,B-D-galactopyranoside The title compound was prepared according to procedures D, F, J and K above using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 323.40; M (found): 347.7 (M+Na+). Selected nmr data was as follows:
H-nmr(CD30D):~ 4.42 (H-l), 4.38, 4.37, and 4.35.

Example C10 Synthesis of ~-3-Acet~mi~ocyclohexyl l-Thi~-D-galactopyranoside The title compound was prepared according to procedures D, F, J and K above using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 335.42; M (found): 373.7 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D):~ 4.52 (H-l), 4.464, and 4.455.

Example C l l Synthesis of 3-Ac~qmido~,~dimethylcyclohexyl l-Thio-,B-D-g,q~ ctopyrqn~
The title compound was prepared according to procedures D, F, J and K above using 6,6-dimethylcyclohex-2-en-1-one as the electrophile.

Example Dl Synthesis of 2-(2-Carboxyben7~mi~lc)cyclopent-l-yl l-Thio-,B-D-galactopyrqn~ e The htle compound was prepared according to procedures D, F, G and L above using 2-chlorocyclopentan-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 427.47; M (found): 450.5 (M+H'). Sele~ted nmr data was as follows: 'H-nmr (CD30D): ~ 4.69 (H-l), 4.58, 4.27, and 4.22.

Example D2 Synthesis of 2-(2-Carboxyben7~mido)cyclohex-1-yl l-Thi~,B-D-galactopyrqn~ e The title compound was prepared according to procedures D, F, G and L above using 2-chlorocyclohexan-1-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 441.50; M (found): 465.9 (M+Na+). .Sele~tçd nmr data was as follows:'H-nmr (CD30D): ~ 4.54 (H-1), 4.52, 4.50, and 4.35.

Exarnple D3 Synthesis of 3-(2-CarboA~l,c~ m;~lQ)-1-phenylbut-1-yl l-Thio-~B-~g,qlqctopyranos;de The title compound was prepared according to procedures D, F, G and L above using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 492.56; M (found): 513.0 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.41 (H-l), 4.115, 4.110, and 3.90.

~ . , Example D4 S~ is of [3-(Carboxyben7~mi~o)norborn-2-yl]methyl l-Thio-,B-~g~ topyr~nnsi l~
The title compound was prepared according to procedures D, F, G and L above using 3-methylene-2-norbornanone as the electrophile. Mass spectra data was as follows: M (calcd.): 467.54; M (found): 492.9 (M+Na+). SelPcted nmr data was as follows: ~H-nmr (CD30D): ~ 4.39 (H-l), 4.34, 4.31, and 4.26.

Example D5 10- Synthesis of 3-(2-Carboxyben7~mido)cyclohept-l-yl l-Thio-,B-D-g~l~rtopyranoside The title compound was prepared according to procedures D, F, G and L above using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 453.52; M (found): 479.6 (M+Na+). Selected nmr data was as follows:
IH-nmr (CD30D): â 4.53 (H-l), 4.51, 4.42, and 4.40.

Example D8 Synthesis of 3-(2-Carboxyben7~mi~lo)pent-2-yl 1 Thio-,B-D-galactopyranoside The title compound was prepared according to procedures D, F, G and L above using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 429.48; M (found): 452.7 (M+Na+). Selected nmr data was as follows:
IH-nmr (CD30D): ~ 4.42 (H-l), 4.41, 4.40, and 4.35.

Example D9 Synthesis of 5-(2-Carboxyben~mi~o)-2,2-dimethylcyclohex-1-yl l-Thio-~B-D-galactopyr~n~s;~l~
The title compound was prepared according to procedures D, F, G and L above using 4,4-dimethylcyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 469.55, M (found): 492.4 (M+Na+).

Example D10 Synthesis of 3-(2-Carboxyben7~mi~1o)cyclohex-l-yl l-Thio-~B-D-g~lactopyr~nos;~l~
S The title compound was prepared according to procedures D, F, G and L above using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 441.50, M (found): n.a. Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.37 (H-l), 4.34, and 4.32.

Example El Synthesis of N~Y-[2-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]glycine The title compound was prepared according to procedures D, H and M above using 2-chlorocyclopentan-l-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 337.39; M (found):
359.8 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.44 (H-1), 4.41, 4.40, and 4.34.

Example E2 Synthesis of Ncr-[2-(1-Thio-,B-D-galactopyranosyl)cyclohex-l-yl]glycine The title compound was l)r~paled according to procedures D, H and M above using 2-chlorocyclohexan-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found):
353.5 (M+H+), 376.5 (M+Na+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.48 (H-1), 4.47, 4.36, and 4.29.

Example E3 Synthesis of N~r-[~Phenyl-4-(1-thio-,B-D-galactopyranosyl)but-2-yl]glycine The title compound was prepared according to procedures D, H and M above using 4-phenylbut-3-en-2-one as the electrophile and glycine tert-butyl ester as the ~ ........ . . . . .

- WO 98122487 PCTtCA97/00866 amino acid ester. Mass spectra data was as follows: M (calcd.): 401.48; M (found):
403.1 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.29 (H-l), 4.18, 3.92, and 3.91.

Exa nple E4 Synthesis of Na-[3-((1-Thi~-D-~Pl ~ ~topyranosyl)methyl)nor~orn-2-yl]glycine The title compound was pl~ared according to procedures D, H and M above using 3-methylene-2-norbornanone as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.~: 377.46; M
(found): 401.4 (M+Na+). Sele~tecl nmr data was as follows: 'H-nmr (CD30D):
4.42 (H-1), 4.40, 4.383, 4.377, and 4.35.

Example E5 Synthesis of NcY-[3-(l-Thio-,B-D-~ ctopyranosyl)cyclQhept-l-yl]glycine The title compound was prepared according to procedures D, H and M above using cyclohept-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found): 367.4 (M+H+), 389.9 (M+Na+). Se1e~ted nmr data was as follows: 'H-nmr (CD30D):
4.46 (H-1), 4.45, 4.42, and 4.38.

Example E5' Synthesis of Nlx-[3-(1-Thio-'-D-galactopyranosyl)cycl~hept-1-yl]glycine The title compound was prepa,ed according to procedures D, H and M above using l-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-~-D-galactopyranose (from Fy~mpl~ C' above), cyclohept-2-en-1-one as the electrophile and glycine tert-butyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
366.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 5.51 (d, J
5.5 Hz, H-l (major), 5.46 (d, J 5.5 Hz, H-l), 5.47 (d, J 5.5 Hz, H-l (minor)), 5.48 (d, J 5.5 Hz, H-l).

Example E6 Synthesis of Na~-[4,~Dunethyl-3~ thio-,B-D-~Io~topyranosyl) cyclopent-1-yl]glycine The title compound was prepared according to procedures D, H and M above using 4,4-dimethyicyclopent-2-en-1-one as the electrophile and glycine te~-butyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 365.44; M
(found): 368;0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.330 (H-1), 4.325, 4.320, and 4.30.
Example E7 Synthesis of N~-[3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]glycine The title compound was prepared according to procedures D, H and M above 15 using cyclopent-2-en-1-one as the electrophile and glycine te)t-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 337.39; M (found): 360.9 (M+Na+). Selectçd nmr data was as follows: 'H-nmr (CD30D): ~ 4.38 (H-l), 4.375, 4.36, and 4.35.

Example E8 Synthesis of Na-[~(l-Thio-,B-D-galactopyranosyl)pent-2-yl]glycine The title compound was prepared according to procedures D, H and M above using pent-3-en-2-one as the electrophile and glycine ~erl-butyl ester as the amino acid 25 ester. Mass spectra data was as follows: M (calcd.): 338.39; M (found): 363.9(M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.43, 4.42, 4.37 (H-l), and 4.36.

.. , . . ,, ,, .,, ...... ~.. _. . . ..

Example E9 Syl~lLe~ls of N~-[4,~Dimethyl-3-(1-thio-,B-D-galactopyranosyl)-cyclohex-l -yl]glycine 5The title compound was piepal~d according to procedures D, H and M above using 4,4-dimethyicyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 379.47; M(found): 380.6 (M+H+), 403.5 ~M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.38 (H-1), 4.36, 4.34, and 4.31.
Example E10 Synthesis of Na-[3-(l-Thio-,B-D-~,q-l~rtopyranosyl)cyclohex-l-yl]glycine The title compound was prel)ared according to procedures D, H and M above 15 using cyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found): 377.1 (M+Na+). Selecte~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.44, 4.40, and 4.36.

20Example Ell Synthesis of Nc~-[5-(1-Thio-,B-D-galactopyranosyl)-2,2-dimethylcyclohex-1-yl]glycine The title compound was prepared according to procedures D, H and M above 25using 6,6-dimethylcyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester.

Exarnple F1 Synthesis of 30N~-[2-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-,B qlqnine The title compound was prepared according to~procedures D, H and M above using 2-chlorocyclopentan-1-one as the electrophile and ,~-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M

(found): 372.9 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.54 (H-l), 4.52, 4.36, and 4.35.

Example F2 Synthesis of N~B-[2-(1-Thio-,B-D-galactopyranosyl)cyclohex l yl] ~ n;n~
The title compound was prepared according to procedures D, H and M above using 2-chlorocyclohexan-1-one as the electrophile and ,B-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
367.4 (M+H+), 389.9 (M+Na+), 412.0 (M+K+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.47 (H-1), 4.42, 4.41, and 4.33.

Example F3 Synthesis of N~B-[4-Phenyl-~(1-thio-~B-D-galactopyranosyl)but-2-yl]-~-alanine The title compound was prepared according to procedures D, H and M above using 4-phenylbut-3-en-2-one as the electrophile and ,B-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 415.50; M (found):
417.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.28 (H-l), 4.17, 3.97, and 3.96.

Example F4 Synthesis of N~B-r3-((l-Thio-~-D-galactopyranosyl)methyl)norbol ll-2-yl]-~ n;n~
The title compound was prepared according to procedures D, H and M above using 3-methylene-2-norbornanone as the electrophile and ~-alanine te~r-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 391.48; M
(found): 393.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.40 (H-l), 4.37, 4.34, and 4.33.

~ , Fy~mpl~ F5 ~ - Synthesis of N,B-[3-(1-Thio-,B-D-~ etopyranosyl)cyclQ~ept-l-yl]-,B~Iqn;rle The title compound was prepared according to procedures D, H and M above S using cyclohept-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 379.45; M (found):
381.7 (M+H+), 403.5 (M+Na+), 426.0 (M+K+). S~lected nmr data was as follows: ~H-nmr (CD30D): ~ 4.46 (H-1), and 4.38.

Example F6 Synthesis of N,B-[4,4-Dirnethyl-3~ thio-~-D-galactopyranosyl)-cyclopent-l-yl]-~ nin~
The title compound was prepared according to procedures D, H and M above 15 using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and ~-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 379.44;
M (found): 3B3.2 (M+H+). Sel~ted nmr data was as follows: 'H-nmr (CD30D): ô
4.34 (H-1), 4.33, 4.315, and 4.310.

Example F7 Synthesis of N~ 3-(l-Thio-~-D-ga~actopyranosyl)cyclopent-l-y~ n;n~
The title compound was prepared according to procedures D, H and M above using cyclopent-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the 25 arnino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found):
375.1 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.41 (H-1), and 4.40.

Example F8 S~lltl~is of [~(l-Thio-~B-D-~ ctopyranosyl)pent-2-yl~-,B-alal~ine The title compound was prepared according to procedures D, H and M above 5 using pent-3-en-2-one as the electrophile and ~-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 352.42; M (found): 356.0 (M+H+). SPlected nmr data was as follows: ~H-nmr (CD30D): ~ 4.49 (H-l), 4.440, and 4.435.

Example F9 Synthesis of N,B-[4,4-Dimethyl-3-(1-thio-~B-D-galactopyranosyl)-cyclohex-l-yl]-~ n~
The title compound was prepared according to procedures D, H and M above 15 using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 393.50;
M (found): 399.3 (M+H+), 419.5 (M+Na+), 442.4 (M+K+). Sele~ted nmr data was as follows: 'H-nmr (CD30D): ô 4.35 (H-l), 4.34, and 4.32.

Example F10 Synthesis of N~ 3-(l-Thio-~-D-gs~l~ctopyranosyl)cyclohex~l-yl]-~- ~lqn;n~
The title compound was prepared according to procedures D, H and M above using cyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the 25 amino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
367.0 (M+H+), 389.9 (M+Na+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.46 (H-l~, 4.44, 4.43, and 4.36.

.... .. ..... . .. .

- W 0 98/22487 rCT/CA97/00866 Example F11 Synthesis of N~B-[5-(1-Thio-~-D-~ topyranosyl)-2,2-dimethylcyclohex-1-yl]-,B ql~ e The title compound was p.epa~ed according to procedures D, H and M above using 6,~dimethyrcyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the amino acid ester.

Example Gl Synthesis of Na-[2-(1-Thio-,B-D-g~ ctopyranosyl)cyclopent-1-yl]-L,leucine The title compound was p~pa-ed according to procedures ~ H and M above using 2-chlorocyclopentan-1-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 393.50; M
(found): 396.4 (M+H+). Sele~t~d nmr data was as follows: IH-nmr (CD30D):
4.47 (H-1), 4.43, 4.36, and 4.34.

Example G2 Synthesis of N~-[2-(1-Thio-~B-D-galactopyranosyl)cyclohex 1 yl] L~leucin~
The title compound was prepared according to procedures D, H and M above using 2-chlorocyclohexan-1-one as the electrophile and L-leucine ten-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 407.53; M (found):
410.9, (M+H ' ), 435.5 (M+Na+). S~lect~d nmr data was as follows: ~H-nmr (CD30D): ~ 4.49 (H-1), 4.44, 4.41, and 4.37.

Example G3 Synthesis of N~-[~Phenyl-4,-(1-thi~B-D-galactopyranosyl)but-2-yl]-L,leuciDe The title compound was prepared according to procedures D, H and M above using 4-phenylbut-3-en-2-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 458.59; M (found):

- W098/22487 PCI'/CA97/00866 480.5 (M+Na+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.39 (H-l), 4.36, 4.29, and 4.21.

Example G5 S Synthesis of N~-[1-(1-Thio-~-D-galactopyranosyl)cyclohept-3-yl]-L,leucine The title compound was prepared according to procedures D, H and M above using cyclohept-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 421.55; M (found):
421.7 (M+H+), 448.0 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.44 (H-1), 4.43, and 4.36.

Example G6 Synthesis of Ncr-[4,4 Dimethyl-3-(1-thi~,B-D-galactopyranosyl)-cyclopent-l-yl]-L,leucine The title compound was ~-epared according to procedures D, H and M above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 421.55;
M (found): 422.3 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.320 (H-l) and 4.315.

Example G7 Synthesis of Nc~-t3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-L,Ieucine The title compound was l~lc~aled according to procedures D, H and M above using cyclopent-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 393.50; M (found):
393.6 (M+H+), 417.0 (M+Na+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.380 (H-l), 4.375, 4.370 and 4.367.

, .. ~ . . ..

- W O 98/22487 P ~/CA97/00866 Example G8 Synthesis of N~ ~(l-Thio-~-D-galactopyranosyl)pent-2-yl]-L~leucine The title compound was prepared according to procedures D, H and M above 5 using pent-3-en-2-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 395.51; M (found): 396.8 (M+H+), 419.1 (M+Na+), and 440.9 (M+K+). Sel~ct~ nmr data was as follows:
IH-nmr (CD30D): ~ 4.42 (H-l), 4.41, 4.405 and 4.40.

Example G9 - Synthesis of N~-[4,4-Dimethyl-3-(1-thio-l~-D-g~lactQpyranosyJ)-cyclohex-l-yl]-L,leucine The title compound was prepared according to procedures D, H and M above using 4,4-dimethylcyclohex-2-en-l-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 436.58;
M (found): 438.0 (M+H+), 461.4 (M+Na+). Sele~t~ nmr data was as follows: IH-nmr (CD30D): ~ 4.38 (H-l) and 4.34.

Example G10 Synthesis of N~r-[3-(1-Thio-~B-D-galactopyranosyl)cyclohex-l-yl]-L~leucine The title compound was prepared according to procedures D, H and M above using cyclohex-2-en-1-one as the electrophile and L-leucine ten~-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 407.53; M (found):
408.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.42, 4.40, and 4.33.

- WO 98t22487 PCT/CA97/00866 Example Hl -- - Synthesis of Na-[2-(1-Thio-~-D-galactopyranosyl)cyclopent-l-yl]-L,hi~;(l;ne The title compound was prepared according to procedures D, H and N above S using 2-chlorocyclopentan-1-one as the electrophile and L-his~i~line methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 417.48; M (found):
418.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô 4.45 (H-l), 4.41, 4.40, and 4.29.

Example H2 Synthesis of N~ 2-(1-Thio-,B-D-galactopyranosyl)cyclohex-l-yl]-L-hictillin~
The title compound was prepared according to procedures D, H and N above using 2-chlorocyclohexan-1-one as the electrophile and L-hicti-line methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 431.50; M (found):
433.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.52 (H-1), 4.45, 4.40, and 4.28.

Example H3 Synthesis of Na~-[~Phenyl-~(l-thio-~B-D-galactopyranosyl)but-2-yl]-L,histidine The title compound was prepared according to procedures D, H and N above using 4-phenylbut-3-en-2-one as the electrophile and L-hi~ti~ine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 481.56; M (found):
482.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.38 (H-l), 4.36, 4.23, and 4.16.

Example H5 Synthesis of Nlx-[3-(1-Thio-,~-D-galactopyranosyl)cyclohept-l-yl]-~hi~;din~
The title compound was prepared according to procedures D, H and N above using cyclohept-2-en-1-one as the electrophile and L-histidine methyl ester as the . , . . , .. .~ ~ ~ . . . ... ... .. .

.

arnino acid ester. Mass spectra data was as follows: M (calcd.): 445.54; M (found):
448.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.50 (H-l), 4.44, 4.41, and 4.32.

Example H6 Synthesis of Na-[4,~Dimethyl-3-(1-thio-~B-D-~ ctopyranosyl)-cyclopent-l-yl]-~hicti-lin~
The title compound was prepared according to procedures D, H and N above 10 using-4,4-dimethylcyclopent-2-en-1-oneas the electrophile and L-histidine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 445.54;
M (found): 447.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.33 (H-l), 4.32, 4.305, and 4.30.

1~ Example H7 Synthesis of N~ 3-(~ o-~-D-~ ctopyranosyl)cyclopent-l-yl]-L~h~ ;r~
The title compound was plepared according to procedures D, H and N above using cyclopent-2-en-1-one as the electrophile and L-hi~tidine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 418.48; M (found):
418.0 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.39 (H-l), 4.38, 4.36, and 4.32.

Example H8 Synthesis of Na-[~(l-Thio-,B-D-~ rtopyranosyl)pent-2-yl]-L~hi.c~ te The title compound was plcpared according to ~ ,cedures D, H and N above using pent-3-en-2-one as the electrophile and L-hi~ti~line methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 419.49; M (found): 420.2 (M+Ht). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.44 (H-l), 4.41, 4.40, and 4.36.

Example H9 Synthesis of N~-[4,~Dimethyl-3-(1-thio-,B-D-~ ctopyranosyl)-cyclohex-1-yl]-L~histidine The title compound was prepared according to procedures D, H and N above using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and L-hi~tidine ethyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 459.56; M(found): 462.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.364 (H-l), 4.357, and 4.34.
Example H10 Synthesis of Nc~-[3-(1-Thio-~B-D-galactopyranosyl)cyclohex-l-yl]-L ~.;ct;~l;np The title compound was prepared according to procedures D, H and N above using cyclohex-2-en-1-one as the electrophile and L-histi~line methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 431.51; M (found):
433.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.43 (H-l), 4.425, 4.39 and 4.35.

Example I1 Synthesis of N~-~2-(1-Thio-~B-D-galactopyranosyl)cyclopent-1-yl]-Irtryptophan The title compound was prepared according to procedures D, H and N above using 2-chlorocyclopentan-1-one as the electrophile and L-tryptophan methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 466.55; M
(found): 467.5 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.51 (H-1), 4.39, 4.28 and 4.27.

.... .. . . .. . ..

-78- _ Examp}e I2 Synthesis of N~-[2-(l-Thio-~-D-gpl~ctopyranosyl)cyclohex-l-yl]-L~tryptorhql~
The title compound was prepared according to procedures D, H and N above S using 2-chlorocyclohexan-1-one as the electrophile and L-try~.tophan methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 480.59; M
(found): 481.9 ~M+H+), 505.3 (M~Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.40, 4.24 and 4.09.

Example I3 Synthesis of N~-[4-Phenyl-~(l-thio-~-D-galactopyranosyl)but-2-yl]-L,tryptophan The titIe compound was prcyared according to procedures D, H and N above using 4-phenylbut-3-en-2-one as the electrophile and L-tryl)to~)han methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 531.64; M (found):
531.3 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.24 (H-l), 4.23, 4.14 and 4.09.

Example I5 Synthesis of Ncr-[3-(1-Thio-,B-D-galactopyranosyl)cyclohept-l-yl]-L,tryptophan The title compound was prepared according to procedures D, H and N above using cyclohept-2-en-1-one as the electrophile and L-tryptophan methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 494.60; M (found):
495.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.50 (H-l), 4.44, 4.41 and 4.32.

FY~mple I6 Synthesis of Na-[4,~Di nethyl-3-(1-thio-,~-D~ ctopyranosyl)-cyclopent-l-y~ tryptophan S The title compound was prepared according to procedures D, H and N above using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and L-tryl~tophan methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 494.60;
M (found): 4gS.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.26 (H-1), 4.22, 4.20 and 4.13.
Example I7 Synthesis of N~-[3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-L,tryptorhD~
The title compound was prepared according to procedures D, H and N above 15 using cyclopent-2-en-1-one as the electrophile and L-tryptophan methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 466.55; M (found):
467.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.33 (H-1), 4.32, 4.30 and 4.23.

Example I8 Synthesis of N~-[~(l-Thio-,B-D-gPlncto~yranosyl)pent-2-yl]-Irtryptorh~-~
The title compound was prepared according to procedures D, H and N above using pent-3-en-2-one as the electrophile and L-tryptophan methyl ester as the amino 25 acid ester. Mass spectra data was as follows: M (calcd.): 468.S7; M (found): 490.9 (M+Na+). S~ ted nmr data was as follows: 'H-nmr (CD30D): ~ 4.30 (H-l), 4.27, 4.22 and 4.09.

.. ... . .. . . ..

Lxample I9 Synthesis of N~Y-14,4-Dimethyl-3-(1-thi~,B-D-g~l~ctopyranosyl~-cyclohex-l-yl~-L,tryptophan 5The title compound was prepared according to procedures D, H and N above using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and L-try~lo~han methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 508.63;
M (found): 512.1 (M+H+). Selecte4 nmr data was as ~ollows: 'H-nmr (CD30D):
4.30 (H-l), 4.26, and 4.21.
Example I10 Synthesis of Na-[3-(1-Thio-,B-D-galactopyranosyl)cyclohex-1-yl]-L,tryptophan The title compound was prepared according to procedures D, H and N above 15 using cyclohex-2-en-1-one as the electrophile and L-tr)~tophan methyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 480.59; M (found):
483.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.36 (H-l), 4.35, 4.33, and 4.24.

20Example Jl Synthesis of N~-[2-(1-Thi~,B-D-galactopyranosyl)cyclopent-1-yl]-L,arginine The title compound was ~f~d according to procedures D, H and 0 above using 2-chlorocyclopentan-1-one as the electrophile and L-arginine methyl ester as the 25amino acid ester. Mass spectra data was as follows: M (calcd.): 436.52; M (found):
436.2 (M+H+). SelPctP~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.54 (H-l), 4.43, 4.41, and 4.28.

Example J2 ~ Synthesis of N~-[2~ Thio-~B-D-g~ topyranosyl)cyclobex-l-yl]-L,arginine - The title compound was prepared according to procedures D, H and O above using 2-chlorocycloheY~n-1-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 450.56; M (found):
453.5 (M+H+). Select~d nmr data was as follows: 'H-nmr (CD30D): ~ 4.47 (H-1), 4.45, 4.44, and 4.38.

Example J3 Synthesis of Na-[~pheny~ -Thio-~B-D-galactopyranosyl)but'-2-yl~-Irarginine The title compound was prepared according to procedures D, H and O above using 2-chlorocyclohexan-1-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 501.62; M (found):
503.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.32 (H-l), 4.31, and 4.30.

Example JS
Synthesis of N~r-[3-(1-Thio-,B-D-galactopyranosyl)cyclohept-1-yl]-I~arginine The title compound was prepared according to procedures D, H and O above using cyclohept-2-en-1-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58; M (found):
467.1 (M+H+). Sçlecte~ nmr data was as follows: IH-nmr (CD30D): ~ 4.48 (H-l), 4.46, and 4.43.

WO 98n2487 PCT/CA97/00866 Example J6 ~ - Synthesis of N~Y-[4,~Dimethyl-3-(1-thio-~-D-g~l~~topyranosyl)-cyclopent-l-yl]-L,arginine S The title compound was prepared according to procedures D, H and 0 above using 4,4-dimethylcyclopent-2-en-1-one as the e}ectrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58; M(found): 465.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.37 (H-1), 4.35, 4.34, and 4.30.
Example J7 Synthesis of N~r-t3-(1-Thio-,B-D-galactopyranosyl)cy~lo~Je~ll-l-yl]-L~arginine The title compound was prepared according to procedures D, H and 0 above using cyclopent-2-en-1-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 436.53; M (found):
437.6 (M+H+). Sele~tecl nmr data was as follows: 'H-nmr (CD30D): ~ 4.37 (H-1), 4.35, and 4.34.

Example J8 Synthesis of N~Y-[~(l-Thio-,B-D-galactopyranosyl)pent-2-yl]-L,arginine The title compound was prepared according to procedures D, H and 0 above using pent-3-en-2-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 438.54; M (found): 437.3 (M+H+). .Celecte~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.41, 4.39, and 4.38.

Example J9 Synthesis of Na-[4,~Dimethyl-3-(1-thio-,B-D-~ ~topyranosyl)-cyclohex-1-yl]-L,arginine The title compound was prepared according to procedures D, H and O above using 4,4-dimethylcyclohex-2-en-l-one as the electrophile and L-arginine methyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 478.60; M
(found): 479.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.43 (H-l), 4.41, 4.38, and 4.32.
Example J10 Synthe~sis of Na-~3-(1-T;hio-~B-D-galactopyranosyl)cyclohex-1-yl]-L,arginine The title compound was prepared according to procedures D, H and O above using cyclohex-2-en-l-one as the electrophile and L-arginine methyl ester as the an~ino acid ester. Mass spectra data was as follows: M (calcd.): 450.55; M (found): 451.8 (M+H+). .S~l~cted nmr data was as follows: 'H-nmr (CD30D): ~ 4.34 (H-l), 4.33, 4.32, and 4.29.

Example 1 Synthesis of the Individual Diastereomers of 2,2-Dimethyl-~(cyclobut-l-ylamino)-cyclopent-1-yl 1-Thio-,B-D-galactopyranoside This example illustrates the preparation of individual diastereomers of a compound of formula I.
Step A--Syu~ is of (1R,S)-2,2-Dimethylcyclopentan-4On-1-yl2,3,4,6-Tetra-O-lauroyl-1-thio-~B-D-galactopyr~noside: To l-S-acetyl-2,3,4,6-tetra-O-lauryl-- 1-thio-,B-D-galactopyranose (5 g, 5 mmol) (from Example C above) and 4,4-dimethyl-2-cyclopenten-1-one (500 mg, 4.45 mmol) in dry CH2Cl2 (10 mL) under argon, was added Et2NH (6 mL). After 3 h, the mixture was concentrated and purified by column chromatography (siO2, pentane/EtOAc, 9: l) to give the title co~ ound as a mixture of diastereomers (3.54 g, 66%).

Step B Separation of the Diastereomers of (lR,S)-2,2-Dimethylcyclopentan-4-on-1-yl 2,3,4,~Tetra-O-lauroyl-1-thio-~B-D-galactopyranoside: The two diastereomers from Step A (5 g, 4.8 mmol) were separated by column chromatography (SiO2, pentanelEtOAc, 9:1) to give (lS)-2,2-S dimethylcyclopentan-4-on-1-yl 2,3,4,6-tetra-0-lauroyl-1-thio-~-D-galactopyr~nosi~e (428.8 mg, 8%) and (lR)-2,2-dimethylcyclopentan-4-on-1-yl2,3,4,6-tetra-O-lauroyl-l-thio-,B-D-galactopyr~noci~e (373.8 mg, 6%) along with a mixture of unresolved compounds (2.74 g, 52%).
Step C -- Synthesis of (lS, 4RS)- and (lR, 4RS)-2,2-Dimethyl~
hydroxycyclopent-1-yl2,3,4,~Tetra-O-lauroyl-l-thio-,B-D-galactopyr~n~si-le: To each of the purified diastereomers from Step B (in separate reaction flasks) (320 mg, 0.3 mmol) in dry tetrahydrofuran (3 mL), methanol (0.5 mL) and isopropanol (2 mL) under argon atmosphere, was added NaBH4 (0.12 mmol). After 30 min, AcOH (1 drop) is added and the mixtures were concentrated and the residues dissolved MeOH
(2 mL) and added to a column of C-18 silica (5 g). The columns were washed with MeOH (50 mL) and products eluted pentane (50 mL) to give (lS, 41~S)-2,2-dime~yl-4-hydroxy-cyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-l-thio-,~-D-galactopyr~nosi~e (281 mg, 88%) and (lR, 4RS)-2,2-dimethyl-4-hydroxy-cyclopent-1-y 2,3,4,~tet~a-~
lauroyl-l-thio-,B-D-galactopyranoside (297 mg, 93%).
Step D--Synthesis of (lS, 4RS)- and (lR, 4RS)-2,2-Dimethyl 4 O-m_th~n~~-.lfonyloxycyclopent-1-yl 2,3,4,6-Tetra-O-lauroyl-l-thio-~B-D-galactopyr~nos;~ To each of the (lS, 4RS) and (lR, 4RS) mixtures from Step C
(in separate reaction flasks) (280 mg, 0.3 mmol) in dry tetrahydrofuran (2 mL) and dry pyridine (4 mL) under argon atmosphere, was added mPth~n~slllfonyl chloride (0.5 mL). After 12 h, the mixtures were washed with 0.5 M HCl and extracted withpen~ne After concentration, the residues were purified on C18-silica (5 g) as described in Step C to afford (lS, 4RS)-2,2-dimethyl-4-O-mPth~nesulfonyloxycyclopent-l-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranoside (281 mg, 88%) and (5) (lR, 4RS)-2,2-dimethyl-4-O-me~h~n~suIfonyloxycyclopent-l-yl 2,3,4,6-tetra-O-lauroyl-l-thio-,~-D-galactopyranoside (297 mg, 93 %) as white solids after pentane evaporation.
Step E--Synthesis of (lS, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,~
Dimethyl-4azidocyclopent-1-yl 2,3,4,6-Tetra-O-lauroyl-1-thio-,B-~
5 galactopyranoside: To the (IS, 4RS) and (lR, 4RS) mixtures from Step D (inseparate reaction flasks) (250 mg, 0.2 mmol) in dry DMF (8 mL) and dry THF (3 mL) under argon atmosphere at 60~C was adde~d NaN3 (340 mg, 5 mmol) and 18 crown-6 (180 mg). After 2 h, the mixtures were concentrated and purified on C18-silica (5 g) as described in Step C. Re-chromatography (SiO2, pentane/EtOAc, 9:1) 10 permitted the separation of diastereomers to give pure (lS, 4R)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-l-thio-~-D-galactopyr~nosi~le (163 mg, 65 5~); (lS, 4S)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,6-tetla-O-lauroyl-l-thio-,B-D-galactopyranoside (29 mg, 9%); (lR, 4S)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,~
tetra-O-lauroyl-l-thio-,B-D-galactopyranoside (68 mg, 28%); and (lR, 4R)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyr~nos;de (21 mg, 9%).
Step F ~ Synthesis of (lS, 4R)-, (lS, 4S)-, (lR, 4S)- and (IR, 4R)-2,~
Dimethyl-4aminocyclopent-1-yl 2,3,4,~Tetra-O-lauroyl-1-thio-~B-D-g;~l~ctopyranoside: To each of the four diastereomers of 2,2-dimethyl-4-azido-cyclopent-1-yl l-thio-,B-D-galactopyranoside from Step E (5 mg, 15 ~umol) in dryisopropanol (1 mL) and dry ethanol (1 mL) under argon atmosphere, was added NaBH4 (15 ~mol) and NiCl2 (30 ~Lmol). After 1 h, the mixtures were neutralized wi~
AcOH (1 drop), concentrated and purified on C18-silica (2 g) as described in Step C
to give (IS, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,2-dimethyl-4-aminocyclopent- -l-yl l-thio-,B-D-galactopyranoside (each S mg; quant.).
Step G--Synthesis of (1S, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,2- -Dimethyl-~(cyclobut-1-ylamino)cyclopent-1-yl 2,3,4,~Tctra-O-lauroyl-1-thio-,B-D-galactopyranoside: To each of four diastereomers of 2,2-dimethyl-4-amino-cyclopent-1-yl l-thio~ D-galactopyranoside from Step F (in separate reaction flasks) (2 mg, 6.8 ~Lmol) in dry methanol (1 mL) and dry dichlororn~th~ne (1 mL) under argon atmosphere, was added cyclobutanone~(250 ~L, 3.4 mmol) and sodium - Wo 98/22487 - pcTlcAs7loo866 triacetoxyborohydride (10 mg, 47 ~mol). After 24-48 h, toluene (1 mL) was added and the mixture was concentrated and the residue purified on C18-silica as d~s.;libed in Step C to give 2.1-2.4 mg (quant.) each of:
(lS, 4R)-2,2-dimethyl~-(cyclobut-1-ylamino)cyclopent-1-yl l-thio-,B-D-5galactopyr~noside (B6HA); M (calcd.): 361.50; M (found): 361.6 (M+H+); ~H-nmr (CD30D): ~ 4.292 (H-l);
(lS, 4S)-2,2-dimethyl-4-(cyclobut-1-ylamino)cyclopent-1-yl l-thio-,B-D-galactopyranoside (B6HB); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr (CD30D): ~ 4.315 (H-1);
10(lR, 4S)-2,2-dimethyl-4-(cyclobut- 1-ylamino)cyclopent-1 -yl 1-thio-,B-D-galactopyranoside (B6HC); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr (CD30D): ~ 4.300 (H-l);
(1 R, 4R)-2,2-dimethyl-4-(cyclobut- 1 -ylamino)cyclopent- 1 -yl 1 -thio-,B-D-galactopyr~noside (B6HD); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr 15(CD30D): ~ 4.290 (H-l).

Example 2 Synthesis of 3-Hydlo~-~c~clohex-1-yl 1-Thio-c~!-L,fucopyr~n~sid~
20The title compound was prepared according to procedures D, E and I above using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the thioc~ch~ ide and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 278.37; M (found~: 302.5 (M+Na+). .S~le~te~ nmr data was asfollows: IH-nmr (CD30D): ~ 5.43 and 5.38 (H-1).
Example 3 Synthesis of 3-Aminocyclohex-1-yl 1-Thio-~-Irfucopyranoside The title compound was prepared according to procedure D, F and J above 30using 1 -S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the thioc~ch~ride and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 277.38; M (found): 278.3 (M+H+). Sele~ted nmr data was as follows: 'H-nmr (CD30D): ~ 5.43, 5.42, 5.36, and 5.34 (H-l).

Example 4 Synthesis of 3-~c~t~mid~cyclohexyl 1-Thio-~-l,fucopyranoside The title compound was p~e~,ared according to procedures D, F, J and K above using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the thi~nch~ride and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 319.42; M (found): 342.2 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 5.43, 5.42, 5.38, and 5.37 (H-l).

Example 5 Synthesis of 3-(2-CarboxybPn7~mido)cyclohex-1-yl l-Thi~-L,fucopyr~nos;~l~
The title compound was prepared according to procedures D, F, G and L above using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-~-L-fucopyranose (2') as the t~hiosaccharide and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M (calcd.): 425.50, M (found): 448.7 (M+Na+). Sele~ted nmr data was as follows: IH-nmr (CD30D): ô 5.48, 5.47, 5.45, and 5.40 ffI-1).

Example 6 Synthesis of Nar-[3-(1-Th;o-~-~fucopyranosyl)cyclohex-l-yl]glycine l he title compound was prepared according to procedures D, H and M above using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-cY-L-fucopyranose (2') as the thios~rh~.;de and cyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 335.42; M~found): 336.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.48, 5.47, 5.39, and 5.36 (H-1).

Example 7 Synthesis of N,B-[3-(1-Thio-cY-L,fucopyranosyl)cyclohex-l-yl]~ lqni-~D
The title compound was prepared according to procedures D, H and M above using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the thiosacch~ride and cyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 349.45;
M (found): 350.0 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ô
5.48, 5.47, 5.39 and 5.38 (H-l).
Example 8 Synthesis of N~Y-[3-(1-Thio-a-L,fucopyranosyl)cyclohex-l-yl]-L,leucine The title compound was prepaled according to procedures D, H and M above using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the thiosaccharide and cyclohex-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 391.53;
M (found): 392.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ô
5.46, 5.40, and 5.35 (H-l).
Example 9 Synthesis of Na-[3-(1-Thio-~ fucopyranosyl)cyclohex-l-yl]-L,hic~i lin-~
The title compound was pl~pared according to procedures D, H and N above using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the thio~cch~ride and cyclohex-2-en-1-one as the electrophile and L-hic~i~line methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 415.51; M(found): 418.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.44, 5.38, and 5.35 (H-l).

Example 10 Synthesis of Na-[3-(1-Thio-a-L,fucopyranosyl)cyclohex-l-yl~-L,tryptophan The title compound was ~ ed according to procedures D, H and N above S using l-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-cY-L-fucopyranose (2') as the thio~ ~h~ride and cyclohex-2-en-1-one as the electrophi}e and L-tr~ opha~l methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58;
M (found): 466.7 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.35, 5.32, 5.27, and 5.22 (H-1).
Example 11 - Synthesis of N~-~3-(1-Thio-~-L,fucopyranosyl)cyclohex-l-yll-L,arginine The title compound was prepared according to procedures D, H and O above using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-a-L-fucopyranose (2') as the thios~ch~ride and cyclohex-2-en-1-one as the electrophile and L-arginine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 434.56; M(found): 435.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.433, 5.427, 5.38 and 5.32 (H-1).
~xample 12 Synthesis of Nc~-[3-(5-AcePmi~1O-3,5-dideoxy-2-thio-D-glycero-cY-D-galacto-2-nonulopyronosyl)cyclohex-1-yl]-L,hicti l;ne The title compound was prepared according to procedures D, H and N abo~e using methyl-5-ace~mido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thi~D-glycero-a-D-glacto-2-nonulopyranosonate'2 as the thio~ch~ride and cyclohex-2-en-1-one as the electrophile and L-histi~ine methyl ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 415.51; M (found): 418.0 (M+H+).
Selected nmr data was as follows: 'H-nmr (CD30D): ~ 5.44, 5.38, and 5.35 (H-1).

Example 13 A~t~hment of [3-(Carboxyben7~m;d~)norborn-2-yllmethyl l-l'hi~,~-D-g~lqrtopyr~ e to a Solid Support STo [3-(carboxyben7~mido)norborn-2-yl]methyl l-thio-,~-D-galaclop~ Q~i~e (2.1 mg, 4.5 ~mor, from Example D4 above), silyl ~min~tP~ Chromosorb P (449 mg, prepared as described in U.S. Patent No. 4,137,401l8 and Westal et al.'9), and hydroxybenzotriazole (1.3 mg, 9.4 ~mol) in DMF (1 mL, dried over 4A molecular sieves), was added diisopropylcarbodiimide (1.4 ~L, 9.0 ~mol). The beads were 10 filtered off after 75 hours, washed with water, DMF, MeOH, and CHtCl2. To theres~llting beads in MeOH (1.5 mL) was added acetic anhydride (0.5 mL) and after 16.5 hours, the beads were filtered and washed with water, DMF, MeOH, CH2Cl2, and pentane. Fine particles were removed by suspending the beads in MeOH and decanting the supernatant repeatedly. Drying under high-vacuum gave 433 mg of a 15 product having [3-(carboxybçn7~mido)norborn-2-yl]methyl 1-thio-,B-D-galactopyranoside covalently ~tt~r~led to the Chromasorb P by formation of an amide linkage between amine group of the chromasorb P and the carboxy group of the 1-thiogalactose derivative as shown in formula III below. Phenol/H2SO4 assay using the procedure described in M. Dubois et al.'3 showed an incorporation yield of 4.0 20 ~mol/g.

~ ~ ~ Ch.~ b~
t HO ~ s ~

Example 14 Att~rhmPnt of a Thios~c~h~ride to a Solid Support To a solution of 1,2:3,4-di-0-isopropylidene-D-galactopyranose (1 eq.) in pyridine at room temperature is added succinic anhydride (1.2 eq.). The reaction is S stirred overnight then concentrated in vacuo to give 1,2:3,4-di-0-isopropylidene-~
(3-carboxy)propanoyl-D-galactopyranose. To the residue is added 80% aqueous acetic acid to remove the isopropylidene groups. When this reaction is complete, the reaction mixture is concentrated in vaCuo and to the residue is added excess 1:1 acetic anhydride/pyridine to afford 1,2,3,4-0-acetyl-6-0-(3-carboxy)propanoyl-D-galactopyranose. To this compound is then added excess thiolacetic acid in dry dichloromethane under argon at 0~C and boron trifluoride etherate. The cold-bath is removed after 10 min and after 24 h the mixture is diluted with dichlorometh~ne,washed with saturated sodium bicarbonate, dried over sodium sulfate, and concentrated to afford l-S-acetyl-2,3,4-tri-0-acetyl-6-0-(3-carboxy)~lu~anoyl-1-thio-~-D-galactopyranose. To this compound is added ~min7~ted Merrifield resin and a carbodiimide coupling reagent to afford the 0,S-protected galactûpyranose coupled to the resin through the 6-0-(3-carboxy)propanoyl group.

Example 15 Solid-Phase Synthesis of 1-Thio~ ctose Derivatives The example illustrates the solid-phase synthesis of 1-thiog~l~rt( se derivatives of formula I.
Step A Synthesis of 1-Di~hioe~hyl-2,3,4,~tetra-aacetyl-galactopyranoside:
1-Thio-2,3,4,6-tetra-0-acetyl-galactopyr~noside (500 mg, 1.37 mmol) and diethyl-N-ethyl-sulfenylhydr~70rlic~rboxylate (360 mg, 2.0 mmol) (plep~ed as described in T.
Mukaiyama20) are dissolved in dichlorornPth~ne (14 mL) and stirred at room ~."pelature. After 10 min, the solution is concentrated and column chromatoE~a~hy (siO2, hexane/ethylacetate 2:1) yields 1-dithioethyl-2,3,4,6-tetra-0-acetyl-galactopyranoside (580 mg, quant) as a white solid (R~ 0.27 in hPY~ne~/ethyl acetate (2: 1)).

'H-NMR (360 MHz, CHC13): ~ 1.30 (dd, 3 H, J = 7.4 Hz, CH3), 1.96, 2.02, 2.03, 2.13 (4 s, 12 H, 4 CH3CO), 2.79 (ddd, 2 H, J = 7.4 Hz, J--7.4 Hz, J = 1.3 Hz, CH2), 3.94 (ddd, 1 ~I, J45 = 1.0 Hz, J5.6. = 6.6 Hz, J5.6b = 7.6 Hz, 5-H), 4.10 (ddd, 2 H, 61-H, 6b-H), 4.51 (d, 1 H, J,.2 = 10.0 Hz, l-H), 5.05 (dd, 1 H, J2.3 = 10.0S Hz, J34 = 3.3 Hzj 3-H)), 5.38 (dd, 1 H, J~.2 = 10.0 Hz, J33= 10.0 Hz, 2-H), 5.40 (dd, 1 H, J3.4 = 3.3 Hz, J45 = 1.0 Hz, 4-H); m/z calcd. for C,6H2409S2 (M+Na) 447.1, found 447Ø
Step B--Synthesis of l-Dithioethyl-,~-D-galactopyranoside: l-Dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside from Step A (500 mg, 1.18 mmol) was dissolved in dry methanol (10 mL) and treated with methanolic sodium methoxide (1 M, 150 ~L). After 2 h, the solution was neutralized with Amberlite lR-120 (H+) resin, filtered and concentrated to give l-dithioethyl-6-,~-D-galactopyranoside as a white solid (300 mg, quant).
Step C--Coupling of l-Dithioethyl-,l5-D-galactopyr~nnc;~le to a Resin: 1-Dithioet~yl-6-,~-D-galactopyranoside (200 mg, 780 ~mol) was dissolved in dry pyridine (8 mL). Trityl chloride-resin (1 g, 950 ~mol trityl chloride resin, loading 0.95 mmol/g of active chlorine, polymer matrix: copolystyrene-l % DVB, 200~00 mesh, Novabiochem) and DMAP (5 mg) were added and the mixture was heated for 24 h at 60~C. The resin was filtered off, and washed succes~ively with meth~nnl,tetrahydrofuran, dichlorom~th~ne and diethyl ether (10 mL each) to afford 1-~ithioethyl-~-D-galactopyranoside covalently linked to the trityl resin through the hydroxyl group in the 6-position.
Step D--G~neration of the Free Thiol on the Resin: The resin from Step C
(50 mg) is swollen in dry tetrahydrofuran (1.5 mL). Dry meth~nol (300 ~L), dithiothreitol (74 mg) and triethylamine (180 ~L) are added and the mixture is shaken for 10 hours at room temperature. The resin is filtered off and washed succes~ively with methanol, tetrahydrofuran, dichloromethane and diethyl ether (10 mLleach). IR
(of intact beads): 2565 cm~l (SH stretch).
Step E Michael Addition Re~ct;Qn: The resin from Step D (50 mg) was swollen in dry N,N-dimethylformamide (1 rnL) and then cyclohept-2-en-1-one (70 ~1, 63 ~mol) was added and the mixture was shaken at room temperature. After 2 hours, the resin was filtered off and washed succescively with me~h~nol~ tetrahydrofuran, dichlorometh~ne and ~iethyl eSher (10 mL each).
Step F ~ Reductive ~n;n~tion with an Amino Acid: The resin from Step E
(50 mg) was swollen in dichlororneth~n~ (1 mL). Glycine ten-butyl ester hydrochloride (75.mg, 447 ~mol), sodium sulfate (100 mg), sodium triacetoxyborohydride (63 mg, 297 llmol) and acetic acid (lO ILL) were added at room temperature under argon atmosphere and the mixture shaken for 24 hours. The resin was then filtered off and washed successively with water, methanol, tetrahydrofuran and dichloromethane.
Step G -- Cleavage from the l-Thio~lact~se Derivative from the Resin and Deblocking of the Amino Acid Ester: The resin from Step F (50 mg) was shaken with trifluoroacetic acid (1 mL) and triisopropylsilane (20 ,uL) in dichlorol,.e~ ne (2 mL) at room temperature. After 3 hours, the resin was removed by filtration and washed with dichlorometh~ne (10 mL). After adding toluene (10 mL), the solution was concentrated, then co-evaporated twice with toluene. The residue was dissolved in water (1 mL) and applied onto two C,g-Sep-Pak-cartridges (Waters Sep-Pak Plus).
The C18 silica was washed with water (4 mL) and the final product was eluted with 20% meth~nol and concentrated. After freeze drying from 5 mL of water, Na-[3-(1-thio-,B-D-galactopyranosyl)cyclohept-1-yl]glycine was obtained as a white powder (4.8 mg). The diastereomers ratio was 10:10:8:6 as determined by 'H-NMR.
'H-NMR (360 MHz, CD30D, anomeric protons): ~ 4.36 (d, J, 2 = 9.6 Hz), 4.40 (d, Jl2 = 9.5 Hz), 4.44 (d, J~2 = 9.1 Hz), 4.45 (d, J, 2 = 9.2 Hz); m/z calcd. for C,5H27NO7S (M+H), 366.2, found 366.1.

Example 16 Inhibition of Heat-Labile Enterotoxin Binding to GDlb In this example, 1-thiog~lactose derivatives of forrnula I above were tested fortheir ability to inhibit the binding of heat-labile enterotoxin from E. coli to g~nglios~de GDlb- This bioassay was conduct~l using the procedure described by A.-M.
Svennerholm2' except that ganglioside GDlb was used instead of ganglioside GMI- The compounds of Examples A1, A2, A4-A7, A10, A11, B1, B2, B4-B7, B10, B11, C2, WO 98~2487 PCT/CA97/00866 CS, C7, C10, Cll, D2, D4, D5, El, E2, E4, E10, Ell, Fl, F2, F5, F7, F10, ~11, G2, G5, I2, I5, and J7 were tested in this bioassay. All of the compounds testedinhibited binding of heat-labile entcfotoAin to ganglioside GDIb by at least 20~, except for the compounds of Examples A2, A5, A7, C10, D2 and G2, which did not inhibit S binding by at least 20% at the concentration employed in the assay.

Example 17 Inhibition of Cholera Toxin Binding to GDlb In this example, l-thiog~l~etose derivatives of formula I above were tested for 10 their ability to inhibit the binding of cholera toxin to ganglioside GD,b. This bioassay was conducted using the following mo~ifi~tion of the procedure described by A.-M.
Svennerholm2l.
On day 1, microtiter plates (C96 Maxisorp) were coated with 100 ~LL of 1 mg/mL GDlb (disialoganglioside GDlb, MW = 2127, Fluka) in PBS per well and 15 incub~ted overnight at 37~C.
On day 2, the s~mples to be tested were diluted in BSA-Tween-PBS (0.1% BSA
and 0.05% Tween-20 in PBS; Sigma). A total of 500 ~L of each solution was plep~ so that each point could be measured in quadruplicate. A conce~
curve of 10, 20 and 30 ng/mL of CTB5-HRP (CT-B5 conjugated to HRP, Sigma, 20 lyophilized, diluted in Tween-PBS) was prepared. For the inhibition experiment~, 20 ng/mL of CTB5-HRP was used. The samples were then incub~t~d for 2 hours at room lem~ldture. After incub~tion~ the plates were emptied and un~tt~~h~d ganglioside was removed by washing the plates 2 times with 200 ~LL PBS per well.Additi'~ binding sites on the plastic surface were then bloc~ed by in~;uh~l;n~ the 25 plates with 200 ,uL of 1% BSA in PBS per well for 30 minutes at 37~C. The plates were then emptied and l-n~tt~hed BSA was removed by washing the plates 3 times with 200 ~L of 0.05% Tween 20-PBS per well. Samples (100 ~L) were added to 4 different wells and incubated for 30 minutes at room le,llp~;ldture. The plates were emptied and unat~che~ BSA was removed by washing the plates 3 times with 200 ~L
30 of 0.05% Tween 20-PBS per well.

W0 98/22487 PCT/CA97tO0866 _95_ A substrate solution was freshly piepar~d for each ELISA. Each sol~tion contained 10 mg of o-phenylene~ mine (Sigma), S mL of 0. lM sodium citrate (filter sterile or autoclaved), 5 mL of 0. lM citric acid (filter sterile or autoclaved) and 4 mL
of 30% H2O2. (Gloves should be worn since ~phenyl~n~Ai~mine is carcinogenic).
S The substrate solution (100 ~LL) was then added to each well and inc~ t~ for 30 minutes at room temperature. After incub~tion, the OD450 was recorded. Under theco~litions of the assay, D-galactose had an IC50 of 30 mM.
The compounds of Examples Al-A10, Bl-B6, B~A-B6L, B6Q, B6T, B7-B8, B10 C1-C3, C5, C7, C8, C10, Dl-D5, D8, El-E9, Fl-F10, G2, G3, G5-G10, H2, H3, H5-H10, I1-I3, I5-I10, J1-J3 and J5-J10 were tested in this bioassay. All of the compounds tested inhibited binding of cholera toxin to g~ iosi~e_GD,b by at least 20%, except for the compounds of Examples Al, A3, A4, A~A8, A10, Bl, B3, B4, B10, Cl, C3, C8, D3, _5, E8, E9, Fl, F5-F7, F9, F10, G3, G7-G10, H2, H5, H8-H10, I2, I8-I10, J5-J10, which did not inhibit binding by at least 20% at the 15 concentration employed in the assay (i.e., 1 mg/mL).

_xample 18 Neutralization of the Cytotonic Activity of CT and LT
In this example, the solid support material of Fy~mp]e 13 was tested for its 20 ability to neutralize the cytotonic activity of CT and LT. The cytotonic activity of CT
and LT was measured by the use of Chinese h~mster ovary cells (CHO) that were maintained in Hams F12 media supplemented with 10% fetal bovine serum (FBS) in an ~tmosphere of 5% CO2 at 37~C. Toxin samples were diluted 1:5 in Hams media and filter sterilized through 0.22 micron syringe filters. Samples were then serial 5-25 fold diluted in media and 100 ~L of each dilution was added to wells with co~fll~entmonolayers of CHO cells and incub~ for 24 h at 37~C (under 5% CO2). Each sample was analyzed two times. Cytotonic effects were readily visible after 24 hincub?,tion by comparing wells with controls that do not contain toxin. After 24 h, the cells were fixed with 95 % methanol and stained with Geimsa stain. Toxin 30 containing samples from neutralization experiments were treated in an analogous fashion except that the percent neutralization was determined by co".p~ing the . . -- . ., ~ , .

endpoint dilutions of ~mples with and without the solid support m~teri~l of F~ rl,o 13. ~
A solution cont~ininE purified CT or LT (2, 10 or 20 ~g in 1 mL PBS) was added to the solid support material of Example 13 (20 mg) in 1.5 mL mi.;ruc~ . ;fuge S tubes and incub~ted at room te~ ture for 1 h on an end-over rotator. After inc~lb~tion, the solid support m~tPri~l was allowed to settle to the bottom of the tubes and the supernatants were carefully removed. The supernatants were added to CHO
cells and the cytotonic endpoint determined after incub~tion for 24 h as desrri~e~
above. The extent of reduction in the endpoint in the presence of the solid S.
10 material was determined by comparing with controls in which solid support nl~tf~r was not added.
Results showed that the solid support material of F~mp1~ 13 neutralized more than 90% of CT and LT activity, regardless of toxin concentration, i.e., less than 10% toxin activity rem~inçd.
Example 19 Inhibition of ColQr~i7~t;on Factor Antigens (CFA pili) Binding to Glycophorin In this example, l-thiog~l~et- se derivatives of formula I above were tested for20 their ability to inhibit CFA pili binding to glycophorin. Bacterial surface ~ll~si~n antigens such as CFA pili are a virulence factor e,-~less~d by certain enteric pathogens, including enterotoxigenic E. coli. These pili are inlpo~ factors in bacterial ~tt~c~lm~nt to cell surface ~ecepto~. Accordingly, inhi~ition of CFA pili binding is a useful test to determine whether a coml~o~ d will inhibit the binding of a 25 pathogenic microorganism to cell surface lecep~ol~.
Binding assays were done by coating microtitre wells with 50 ~L of glyc~ph~
(10 ~g/mL) in PBS for 2 h at 37~C. The solution was removed by aspiration and replaced with 100 ~LL of 1% BSA in PBS cont~ining 0.05% Tween 20 (PBST) and incllb~t~d at 37~C for an additional 1 h. The microtitre wells were washed three30 times with 200 ~L of PBST and then replaced with biotinylated CFA I (5 ~g/mL) in 50 ~L of PBS containing 0.05% BSA. After incub~ing for 2 h at 37~C the binding W 0 98/22487 PCTtC A97/00866 reaction was stopped by aspirating the solutions and the plate was washed with PBST
- (3 X 200 ~L). Avidin-peroxidase (50 ~L of a 1/3000 dilution of a 1 mg/mL solution in PBST containing 0.05 % BSA) was added and the plates were incub~t~d for an additional 1 h. After washing the wells as described above, 100 ~L of the s~bst~te S solution (0.42 mM tetramethylben7idine (TMB) in 0.1 M sodium citrate buffer, pH
6.0, containing 0.5 ~LM urea peroxide) was added and the plates were inruh~te~ for 10 min at ambient temperature and the enzyme reaction stopped by adding 50 ~LL of 2N H2SO4. Binding assays were done in triplicate and background binding was measured in wells coated with BSA only.
Binding inhibition assays were done using oligo~rc-h~ride analogs at a concentration of 1 mg/mL in PBS. Inhibitors were preincub~ed with biotinyla~d CFA I pili (5 /l/mL) for 1 h at 37~C prior to adding to glycophorin-coated microtitre wells as outlined above. o-Nitrophenyl-,B-D-g~l~ctose was utilized as a control inhibitor for these experiments.
The l-thiogalactose derivatives of Examples Al-A10, Bl-B8, B10, Cl-C3, C5, C7, C8, C10, Dl-D5, D8, D10, El-E10, F1-F10, G1-G3, G5-G10, H1-H3, H5-H10, I1-I3, I5-I10, J1-J3 and J5-J10 were tested. Of these compounds, the results showed that the compounds of Examples B2, B5, H2, H3, H5, H6, H7, H8, H9, H10, I1, I2 and J9 inhibited CFA I pili binding to glycophorin, with the ~mollnt of inhibition 20 ranging from 13 to 71%. The compounds having a histidine or a tryptophan (Group H and I) moiety were particularly good inhibitors in this experiment.
From the foregoing desc~ tion, various mo~lification~ and changes in the col,lposiLion and method will occur to those skilled in the art. All such mo~ifi~tions coming within the scope of the appended claims are intended to be included therein.

Claims (21)

Claims:

1. A method for synthesizing a thiosaccharide derivative, which method comprises:
(a) providing a thiosaccharide;
(b) providing at least a stoichiometric amount of a coupling reagent selected from the group consisting of Michael acceptors and .alpha.-halocarbonyl compounds; and (c) contacting the thiosaccharide and the coupling reagent under conditions which provide for a thiosaccharide carbonyl compound.

2. The method of Claim 1, which method further comprises the step of:
(d) reducing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives.

3. A method for synthesizing a thiosaccharide derivative on a solid support, which method comprises:
(a) providing a thiosaccharide;
(b) providing at least a stoichiometric amount of a coupling reagent selected from Michael acceptors and .alpha.-halocarbonyl compounds wherein either the thiosaccharide or the coupling reagent is covalently attached to a solid support; and (c) contacting the thiosaccharide and the coupling reagent under conditions which provide for a thiosaccharide carbonyl compound covalently attached to a solid support.

4. The method of Claim 1, which method further comprises the step of:
(d) reducing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives.

5. A method for preparing a thiosaccharide derivative library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises a thiosaccharide derivative, which library is synthesized in a process comprising:

a) apportioning solid supports among a plurality of reaction vessels which supports comprise a reactive functional group covalently bound thereto which group is capable of covalently binding a thiosaccharide at a position other than the thiol group;

b) contacting the supports in each reaction vessel with a unique thiosaccharide under conditions wherein the thiosaccharide is covalently attached to the solid supports through the reactive functional group;
c) pooling the supports;
d) apportioning the supports from (c) above among a plurality of reaction vessels; and e) contacting the supports in each reaction vessel from (d) above with a unique coupling reagent selected from the group consisting of Michael acceptors and .alpha.-halocarbonyl compounds under conditions which provide for a thiosaccharide carbonyl compound covalently bound to said support.

6. The method of Claim 5, which method further comprises the steps of:
(f) pooling the supports from procedure (e);
(g) apportioning the supports from (f) above among a plurality of reaction vessels; and (h) reducing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives.

7. The method of Claim 6, which method further comprises the steps of:
(i) pooling the supports from procedure (h) above;
(j) apportioning the supports from (i) above among a plurality of reaction vessels; and (k) derivatizing the hydroxyl or amine groups to form a functional group selected from esters, substituted amines, amides, carbamates, ureas, thioureas, thioesters and thiocarbamates.

8. A method for preparing a thiosaccharide derivative library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises a thiosaccharide derivative, which library is synthesized in a process comprising:
a) apportioning solid supports among a plurality of reaction vessels which supports comprise a reactive functional group covalently bound thereto which group is capable of covalently binding a coupling reagent;
b) contacting the supports in each reaction vessel with a unique coupling reagent selected from the group consisting of Michael acceptors and .alpha.-halocarbonyl compounds under conditions wherein the coupling reagent is covalently attached to the solid supports through the reactive functional group;
c) pooling the supports;
d) apportioning the supports from (c) above among a plurality of reaction vessels; and e) contacting the supports in each reaction vessel from (d) above with a unique thiosaccharide under conditions which provide for a thiosaccharide carbonyl compound covalently bound to said support.

9. The method of Claim 8, which method further comprises the steps of:
(f) pooling the supports from procedure (e);
(g) apportioning the supports from (f) above among a plurality of reaction vessels; and (h) reducing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives.

10. The method of Claim 9, which method further comprises the steps of:
(i) pooling the supports from procedure (h) above;
(j) apportioning the supports from (i) above among a plurality of reaction vessels; and (k) derivatizing the hydroxyl or amine groups to form a functional group selected from esters, amides, carbamates, ureas, thioureas, thioesters and thiocarbamates.

11. A library of diverse thiosaccharide derivatives comprising a plurality of solid supports having a plurality of covalently bound thiosaccharides derivatives, wherein the thiosaccharide derivative bound to each of said supports is substantially homogeneous and further wherein the thiosaccharide derivative bound on one support is different from the thiosaccharide derivatives bound on the other supports andfurther wherein said thiosaccharide derivative is presented by the formula (I):

wherein R1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl,alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support;
R2 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroalyl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support;
R3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl,alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support;
or R1 and R2, or R1 and R3, or R2 and R3, or R1, R2 and R3 can be joined, together with the carbon atoms to which R1 and/or R2 and/or R3 are attached to form a cycloalkyl, cycloalkenyl or heterocyclic ring;
R4 is selected from the group consisting of -XR5, -XC(W)R6, -XC(W)X'R7 and -C(W)XR8; wherein W is selected from the group consisting of oxygen, sulfur and NH; and X and X' are each independently selected from the group consisting of oxygen, sulfur and -NR9-, wherein R9 is selected from the group consisting of hydrogen and alkyl; or when R4 is -XR5 and R5 is not hydrogen, X can also be selected from the group consisting of -S(O)- and -SO2-;
R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support, and when X is -NR9-, then R9 together with X can form an amino acid; or R5 and R1, or R5 and R2, or R5 and R3 can be joined, together with X of the -XR5 group and thecarbon atoms to which R1 and/or R2 and/or R3 are attached, to form a heterocyclic ring;
R6 is selected from the group consisting of alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support; or R6 and R1, or R6 and R2, or R6 and R3 can be joined, together with the -XC(W)- moiety of the -XC(W)R6 group and the carbon atoms to which R1 and/or R2 and/or R3 are attached, to form a heterocyclic ring;
R7 is selected from the group consisting of alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support; or R7 and R1, or R7 and R2, or R7 and R3 can be joined, together with the -XC(W)X'- moiety of the -XC(W)X'R7 group and the carbon atoms to which R1 and/or R2 and/or R3 are attached, to form a heterocyclic ring;
R8 is selected from the group consisting of alkyl, alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I to the support; or R8 and R1, or R8 and R2, or R8 and R3 can be joined, together with the -C(W)X- moiety of the -C(W)XR8 group and the carbon atoms to which R1, R2 and/or R3 are attached, to form a heterocyclic ring;
Y is selected from the group consisting of sulfur, -S(O)- and -S(0)2-;
n is an integer equal to 0 or 1; and pharmaceutically acceptable salts thereof;
wherein the saccharide is selected from the group consisting of a monosaccharide, an oligosaccharide, monosaccharide-Z- and oligosaccharide-Z-, wherein Z is a linking arm covalently linking the compound of formula I to the solid support;
with the proviso that only one of R1, R2, R3, R4, R6, R7, R8 and Z is linked to the solid support.

12. The library of Claim 11 wherein the compound of formula I is an .alpha.-anomer.

13. The library of Claim 11 wherein the compound of formula I is a .beta.-anomer.

14. The library of Claim 11 wherein, when n is 0, R1 and R2 are joined, together with the carbon to which they are attached, to form a cycloalkyl ring having 5 to 7 carbon atoms optionally substituted with 1 to 3 alkyl groups.

15. The library of Claim 14 wherein R1 and R2 are joined, together with the carbon to which they are attached, to form a cyclopentane or cyclohexane ring.

16. The library of Claim 11 wherein, when n is 1, R1 and R2 are joined, together with the carbon atoms to which R1, R2 and R3 are attached, to form a cycloalkyl ring having 5 to 7 carbon atoms optionally substituted with 1 to 3 alkyl groups.

17. The library of Claim 16 wherein R1 and R2 are joined, together with the carbon atoms to which R1, R2 and R3 are attached, to form a cycloheptane, dimethylcyclopentane, cyclohexane, dimethylcyclohexane or cycloheptane ring.

18. The library of Claim 16 wherein R4 is -XR5, where X is -NH- and R5 is cycloalkyl.

19. The library of Claim 11 wherein, when n is 1, R2 and R3 are joined, together with the carbon atoms to which they are attached, to form a norbornene ring.

20. The library of Claim 11 wherein R4 is -XR5, where X and R5 form an amino group, a hydroxy group or an amino acid selected from the group consisting of glycine, .beta.-alanine, leucine, histidine tryptophan and arginine.

21. The library of Claim 11 wherein R4 is -XC(O)R6, where X is -NH- and R6 is methyl or 2-carboxyphenyl.