EP0938492A1 - Synthese combinatoire de banques d'hydrates de carbone - Google Patents

Synthese combinatoire de banques d'hydrates de carbone

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Publication number
EP0938492A1
EP0938492A1 EP97913040A EP97913040A EP0938492A1 EP 0938492 A1 EP0938492 A1 EP 0938492A1 EP 97913040 A EP97913040 A EP 97913040A EP 97913040 A EP97913040 A EP 97913040A EP 0938492 A1 EP0938492 A1 EP 0938492A1
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EP
European Patent Office
Prior art keywords
group
thiosaccharide
compound
supports
follows
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP97913040A
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German (de)
English (en)
Inventor
Ole Hindsgaul
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synsorb Biotech Inc
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Synsorb Biotech Inc
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Publication date
Priority claimed from US08/751,510 external-priority patent/US6063769A/en
Priority claimed from US08/751,231 external-priority patent/US5780603A/en
Application filed by Synsorb Biotech Inc filed Critical Synsorb Biotech Inc
Publication of EP0938492A1 publication Critical patent/EP0938492A1/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/08—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium
    • C07H5/10—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium to sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00731—Saccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof

Definitions

  • This invention is directed to methods for synthesizing very large collections of diverse thiosaccharide derivatives optionally attached to a solid support. This invention is further directed to a library of diverse thiosaccharide derivatives.
  • Compounds having biological activity can be identified by screening diverse collections of compounds (i.e., libraries of compounds) produced through either molecular biological or synthetic chemical techniques. Such screening methods include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having biological activity 1 or where the library comprises a plurality of compounds synthesized at specific locations on the surface of a solid substrate wherein a receptor is appropriately labeled to identify binding to the compound, e.g., fluorescent or radioactive labels. Correlation of the labelled receptor bound to the substrate with its location on the substrate identifies the binding compound. 2
  • the compounds in the library are typically formed on solid supports wherein the compound is covalently attached to the support via a cleavable or non- cleavable linking arm.
  • libraries of diverse compounds are prepared and then screened to identify "lead compounds" having good binding affinity to the receptor.
  • Pharmaceutical 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.
  • libraries of compounds derived from a lead compound can be created by including derivatives of the lead compound and repeating the screening procedures.
  • the compounds are synthesized in situ on the solid support so that the support can be tagged to identify the synthetic steps employed and/or the derivative incorporated onto the support.
  • relatively simple synthetic methods to produce a diverse collection of such derivatives on the supports are often not available.
  • thiosaccharide derivatives One particular class of compounds which would be useful for inclusion in screening libraries is thiosaccharide derivatives. It is well known that certain toxins and organisms bind to oligosaccharide receptors on host cells as an initial step in the pathological development of various disease conditions.
  • LT heat-labile enterotoxin
  • CT cholera toxin
  • virulent organisms e.g., bacteria, virus, fungi, and the like
  • enterovirulent organisms bind to cell surface receptors as part of the disease process.
  • bacteria such as Vibrio cholerae and enterotoxigenic strains of Escherichia coli can directly bind to cell surface receptors forming a colony at the point of attachment. Such binding is detrimental because it permits expressed toxin to immediately interact with the cell surface.
  • This invention is directed to general synthetic methods for generating very large libraries of diverse thiosaccharide derivatives optionally attached to a solid support.
  • the thiosaccharide derivative libraries provided by this invention are synthesized by reacting a thiosaccharide with a Michael acceptor or an ⁇ -halocarbonyl compound to provide for a thiosaccharide carbonyl compound.
  • the carbonyl group of the thiosaccharide carbonyl compound can optionally be reduced to provide for a plurality of alcohol and/or amine thiosaccharide derivatives.
  • the alcohol and/or amine group of the thiosaccharide derivative is further derivatized to provide for a plurality of thiosaccharide derivatives.
  • the thiosaccharide derivatives are covalently attached to a solid support.
  • Solid supports containing such thiosaccharide derivatives preferably comprise a linking arm which links the solid support to the thiosaccharide derivative.
  • the linking arm can be either cleavable or non-cleavable and when cleavable, can be used to prepare a library of either solid phase or soluble thiosaccharide derivatives.
  • the library of thiosaccharide derivatives, whether soluble or insoluble can be screened to isolate individual compounds that possess some desired biological activity. In a preferred embodiment, each compound in the library is unique. Accordingly, in one of its method aspects, this invention is directed to a method for synthesizing a thiosaccharide derivative, which method comprises:
  • this invention is directed to a method for synthesizing a thiosaccharide derivative on a solid support, which method comprises:
  • each of the above methods for synthesizing a thiosaccharide derivative further comprises reducing the carbonyl group of the thiosaccharide carbonyl compound to form a group selected from hydroxy and amino derivatives.
  • the hydroxy or amino group can be further derivatized to form a group selected from esters, substituted amines, amides, carbamates, ureas, thiourea, thioesters and thiocarbamates.
  • this invention is directed to 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
  • this invention is directed to 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 ⁇ -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.
  • each of the above methods for preparing a thiosaccharide derivative library exemplified in procedures (a) through (e) further comprises: (f) pooling the supports from procedure (e); (g) apportioning the supports from (f) above among a plurality of reaction vessels; and
  • such methods optionally include the further 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.
  • this invention is directed to 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 and further wherein said thiosaccharide derivative is represented by the formula (I):
  • R 1 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; 20.
  • R 2 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;
  • R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, 25 alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to the support; or R 1 and R 2 , or R 1 and R 3 , or R 2 and R 3 , or R 1 , R 2 and R 3 can be joined, together with the carbon atoms to which R 1 and/or R 2 and/or R 3 are attached, to form 30 a cycloalkyl, cycloalkenyl or heterocyclic ring;
  • R 4 is selected from the group consisting of -XR 5 , -XC(W)R 6 , -XC(W)X'R 7 and -C(W)XR 8 ; 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 -NR 9 -, wherein R 9 is selected from the group consisting of hydrogen and alkyl; or when R 4 is -XR 5 and R s is not hydrogen, X can also be selected from the group consisting of -S(O)- and -SO 2 -;
  • R 5 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 -NR 9 -, then R 9 together with X can form an amino acid; or R 5 and R 1 , or R 5 and R 2 , or R 5 and R 3 can be joined, together with X of the -XR 5 group and the carbon atoms to which R 1 and/or R 2 and/or R 3 are attached, to form a heterocyclic ring;
  • R 6 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 R 6 and R 1 , or R 6 and R 2 , or R 6 and R 3 can be joined, together with the -XC(W)- moiety of the - XC(W)R 6 group and the carbon atoms to which R 1 and/or R 2 and/or R 3 are attached, to form a heterocyclic ring; R 7 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
  • R 8 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 R 8 and R 1 , or R 8 and R 2 , or R 8 and R 3 can be joined, together with the -C(W)X- moiety of the - C(W)XR 8 group and the carbon atoms to which R 1 , R 2 and/or R 3 are attached, to form a 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 pharmaceutically acceptable salts thereof; wherein the saccharide is selected from the group consisting of a monosaccharide, an oligosaccharide, monosaccharide
  • Figure 1 illustrates a preferred reaction scheme for synthesizing a library of diverse thiosaccharide derivatives using an ⁇ ,jS-unsaturated carbonyl compound, i.e., cyclohept-2-en- 1 -one.
  • Figure 2 illustrates a preferred reaction scheme for synthesizing a library of diverse thiosaccharide derivatives using an ⁇ -halocarbonyl compound, i.e, 2- chlorocyclohexanone.
  • This invention is directed to libraries of diverse thiosaccharide derivatives optionally attached to a solid support and to methods for generating such libraries.
  • the following terms will first be defined.
  • Acyl refers to the groups alkyl-C(O)-, aryl-C(O)-, and heteroaryl-C(O)- where alkyl, aryl and heteroaryl are as defined herein.
  • Acylamino refers to the group -C(O)NRR where each R is independently hydrogen or alkyl.
  • Alkyloxy refers to the groups alkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- where alkyl, aryl, heteroaryl 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 example, methoxy, ethoxy, n-propoxy, w ⁇ -propoxy, n-butoxy, r -butoxy, sec- butoxy, n-pentoxy, /i-hexoxy, 1,2-dimethylbutoxy, and the like.
  • Alkoxyalkyl refers to the group -alkylene-O-alkyl which includes by way of example, methoxymethyl (CH 3 OCH 2 -), methoxyethyl (CH 3 -O-CH 2 CH 2 -) and the like.
  • Alkenyl refers to alkenyl 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 alkenyl unsaturation.
  • 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 groups such as methyl, ethyl, n-propyl, tr ⁇ -propyl, rt-butyl, ⁇ -butyl, «-hexyl, and the like.
  • Substituted alkyl refers to a branched or straight chain alkyl group of from 1 to 8 carbon atoms having from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acylamino, acyloxy, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano, cycloalkyl, guanidino, halo, heteroaryl, heterocyclic, nitro, thiol, thioaryloxy, thioheteroaryloxy, and the like.
  • Preferred 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 exemplified by groups such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), the propylene isomers (e.g., -CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -) 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 ⁇ CH), propargyl (-CH 2 C ⁇ CH) and the like.
  • Amino acid refers to any of the naturally occurring amino acids, as well as synthetic analogs and derivatives thereof. ⁇ -Amino acids comprise a carbon atom to which is bonded an amino group, a carboxy group, a hydrogen atom, and a distinctive group referred to as a "side chain".
  • side chains of naturally occurring amino acids include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in alanine, 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 phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in histidine).
  • hydrogen e.g., as in glycine
  • alkyl e.g., as in alanine, valine, leucine, isoleucine, proline
  • substituted alkyl e.g., as in threonine, serine,
  • amino acid can also include ⁇ -, y-, ⁇ -, and ⁇ -amino acids, and the like.
  • Unnatural amino acids are also known in the art, as set forth in, for example, Williams 3 , Evans et al. 4 , Pu et al. 5 , Williams et al. 6 , and all references cited therein.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as ⁇ , ⁇ - disubstituted amino acids and other unconventional amino acids may also be suitable components for compounds of the present invention.
  • unconventional amino acids include: 4-hydroxyproline, 3-methylhistidine, 5-hydroxylysine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • aminoacyl refers to the group -NRC(O)R where each R is independently hydrogen or alkyl.
  • amino derivative(s) refers to a primary, secondary or tertiary amine compound produced by reductive amination of a thiosaccharide 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 condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
  • such aryl groups can optionally be substituted with from 1 to 3 substituents selected 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.
  • substituents include alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and thioalkoxy.
  • Aryloxy refers to the group aryl-O- where the aryl group is as defined herein including optionally 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.
  • Coupled reagent refers to Michael acceptors and ⁇ -halocarbonyl compounds.
  • Michael acceptors refers to ⁇ , ⁇ -unsaturated carbonyl compounds having the general formula (II):
  • Michael acceptors include, by way of example, ⁇ , ⁇ -unsaturated aldehydes, ⁇ ,/3-unsaturated ketones, ⁇ ,/3-unsaturated esters, ⁇ ,/3-unsaturated thioesters, ⁇ ,/3-unsaturated amides and the like.
  • ⁇ -Halocarbonyl compounds refers to compounds having the general formula: W-CHR 1 -C(O)R 2 wherein R 1 and R 2 are as defined herein, and W is chloro, bromo or iodo.
  • ⁇ - halocarbonyl compounds include, by way of example, ⁇ -chloroaldehydes, ⁇ - bromoaldehydes, ⁇ -iodoaldehydes, ⁇ -chloroketones, ⁇ -bromoketones, ⁇ -iodoketones 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 multiple condensed rings which can be optionally substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkylene, alkoxy, alkenyl, alkynyl, 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 include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2- methylcyclooctyl, and the like, or multiple ring structures such as adamantanyl and the like, and spiro compounds.
  • Suitable cycloalkyl rings include single ring structures such as cyclopentane, cyclohexane, cycloheptane, cyclooctane, and the like, or multiple ring structures such as bicyclo[2.2. l]heptane, bicyclo[3.2.1]octane, and the like.
  • Preferred cycloalkyl rings include cyclopentane, cyclohexane, cycloheptane and bicyclo[3.2.1]octane.
  • Cycloalkenyl refers to cyclic alkenyl groups or cyclic alkenyl rings of from 4 to 8 carbon atoms having a single cyclic ring and at least one point of internal unsaturation which can be optionally substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkylene, alkoxy, alkenyl, alkynyl, 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.
  • suitable cycloalkenyl groups include, for instance, cyclobut- 2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
  • Such cycloalkenyl rings include, by way of example, cyclopentene, cyclohexene, and the like.
  • Halo or halogen refers to fluoro, chloro, bromo and iodo and preferably is either chloro or bromo.
  • ⁇ -Halocarbonyl compound refers to a compound having the general formula: Q-CHR 1 -C(O)R 2 wherein R 1 and R 2 are as defined herein, and Q is chloro, bromo or iodo.
  • ⁇ -halocarbonyl compounds include, by way of example, ⁇ - chloroaldehydes, ⁇ -bromoaldehydes, ⁇ -iodoaldehydes, ⁇ -chloroketones, ⁇ - bromoketones, ⁇ -iodoketones and the like.
  • Heteroaryl refers to a monovalent aromatic carbocyclic group of from 2 to 8 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring.
  • heteroaryl groups can be optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like.
  • Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed 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 condensed rings, from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen within the ring.
  • heterocycle or “heterocyclic” does not include carbohydrate rings (i.e. mono- or oligosaccharides).
  • heterocyclic groups can be optionally substituted with 1 to 3 substituents selected 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 condensed rings (e.g., indolinyl).
  • nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline and the like.
  • Mathael acceptor refers to an ⁇ ,0-unsaturated carbonyl compound having the general formula (II): O
  • Michael acceptors include, by way of example, ⁇ ,/3-unsaturated aldehydes, ⁇ ,/3-unsaturated ketones, ⁇ , ⁇ -unsaturated esters, ⁇ ,/3-unsaturated thioesters, ⁇ ,/3-unsaturated amides and the like.
  • Thioalkoxyalkyl refers to the group -alkylene-S-alkyl which includes by way of example, thiomethoxymethyl (CH 3 SCH 2 -), thiomethoxyethyl (CH 3 -S-CH 2 CH 2 -) and 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.
  • thiosaccharide refers to a monosaccharide or oligosaccharide having 2 to about 8 saccharide units wherein at least one, and preferably 1 or 2, of the hydroxyl groups of the saccharide is replaced with a thiol group.
  • the thiosaccharide is an animal saccharide.
  • animal saccharide refers to a saccharide which is naturally expressed by one or more animals, such as mammals, birds or fish.
  • the animal saccharide is a mammalian saccharide.
  • preferred mammalian saccharides include D-galactose, D-glucose, D- mannose, D-xylose, D-glucuronic acid, N-acetyl-D-glucosamine, N-acetyl-D- galactosamine, sialyic acid, iduronic acid, L-fucose, and the like. Included within the definition of this term are acylated, phosphorylated and sulfated derivatives of animal saccharides.
  • thiosaccharide carbonyl compound refers to a compound having the formula (III):
  • 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.
  • materials are well known in the art and include, by way of example, silicon dioxide supports containing reactive Si-OH groups, polyacrylamide supports, polystyrene supports, polyethyleneglycol supports, and the like.
  • Such supports will preferably take the form of small beads, pellets, disks, or other conventional forms, although other _forms may be used.
  • at least one surface of the substrate will be substantially flat.
  • the activated ketone compound is covalently attached directly to the solid support or is attached to the support via a linking arm.
  • Linking arms are well known in the art and include, by way of example only, conventional linking arms such as those comprising ester, amide, carbamate, ether, thio ether, 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 attaches the compound to the solid support can be readily broken by specific chemical reactions thereby providing for compounds comprising activated ketone groups free of the solid support ("soluble compounds").
  • the chemical reactions employed to break the covalent bond of the linking arm are selected so as to be specific for bond breakage thereby preventing unintended reactions occurring elsewhere on the compound.
  • the cleavable linking arm is selected relative to the synthesis of the compounds to be formed on the solid support 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:
  • 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 cleaved under conditions which chemically alters unintended parts of the structure of the compound attached thereto.
  • substantially homogeneous refers to collections of molecules wherein at least 80%, preferably at least about 90% and more preferably at least about 95% of the molecules are a single compound or stereoisomers thereof.
  • stereoisomer refers to a chemical compound having the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical 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 is so slight that it is undetectable with present instrumentation.
  • the compounds described herein may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.
  • the carbon atoms to which R 1 and R 2 are attached may have an R,R or R,S or S,R or S,S configuration.
  • the carbon atoms to which R 1 , R 2 and R 3 are attached 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 configuration.
  • 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 reestablish the functional group.
  • the particular removable protecting group employed is not critical.
  • toxin refers to a compound produced by an organism which causes or initiates the development of a noxious, poisonous or deleterious effect in a host presented with the toxin. Such deleterious conditions may include fever, nausea, diarrhea, weight loss, neurologic disorders, renal disorders, hemorrhage, and the like.
  • 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 enterotoxins, i.e., toxins present in the gut of an organism.
  • heat-labile enterotoxin or "LT” refer to an enterotoxin of enterotoxigenic E. coli which initiates traveller's diarrhea and related conditions. This toxin has a lectin-like activity.
  • travelingler's diarrhea refers to diarrhea of sudden onset, often 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.
  • cholera refers to an acute epidemic infectious disease caused by Vibrio cholerae, wherein a soluble toxin elaborated in the intestinal tract by the Vibrio 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 gross morphologic change in the intestinal mucosa.
  • cholera toxin or "CT” refer to an enterotoxin of V. cholerae which initiates cholera and related conditions. This toxin has a lectin-like activity.
  • inhibitor(s) the binding of a toxin to its receptor means that a compound inhibits the binding of a toxin to its receptor by at least 20%.
  • useful binding inhibition assays may measure inhibition of binding to ganglioside G Dlb or ganglioside G M1 , neutralization of cytotoxic activity, or the like. Such binding is reported herein as percent toxin activity remaining so that those compounds which result in about 80% or less toxin activity remaining under the bioassay conditions disclosed herein are deemed to inhibit the binding of a toxin to its receptor.
  • inhibitor(s) the binding of heat-labile enterotoxin (LT) and/or cholera toxin (CT) to an LT and/or CT receptor means that a compound inhibits the binding of LT and/or CT to an LT and/or CT receptor by at least 20 %.
  • inhibitor(s) the binding of an organism to its cell surface receptor means that a compound inhibits the binding of an organism, such as a bacterium, a virus, a protozoan, a fungus, and the like, to its cell surface receptor.
  • an organism such as a bacterium, a virus, a protozoan, a fungus, and the like
  • a compound is said to inhibit binding of an organism to a cell surface receptor if it reduces binding of a bacterial surface adhesion antigen, such as CFA I pili, by at least 10%.
  • pharmaceutically acceptable salt refers to pharmaceutically 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 example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • the methods of this invention involve the novel addition of a thiosaccharide to a coupling reagent selected from the group consisting of Michael reagents and ⁇ -halocarbonyl compounds.
  • the thiosaccharide derivatives of this invention are typically prepared by reaction of a suitably protected thiosaccharide intermediate with an , ⁇ - unsaturated carbonyl compound or an ⁇ -halocarbonyl compound to provide for a thiosaccharide carbonyl compound.
  • the carbonyl group of the thiosaccharide carbonyl compound is then optionally reduced to provide for a plurality of alcohol and/or amine thiosaccharide derivatives.
  • the alcohol and/or amine group of the thiosaccharide derivative is further derivatized to provide for a plurality of thiosaccharide derivatives.
  • the ⁇ ,0-unsaturated carbonyl compounds employed in preparing the thiosaccharide derivatives of this invention preferably have the general formula (II):
  • These compounds are either commercially available or can be prepared from commercially available materials using art recognized procedures. For example, such compounds can be prepared via a Wittig reaction from an aldehyde, R'CHO, and a /3-carbonyl phosphorane, such as (Ph) 3 PC(R 3 )C(O)R 2 .
  • Preferred ⁇ ,/3-unsaturated carbonyl compounds for use in this invention include, by way of example, cyclopent-2-en-l-one, 4,4-dimethylcyclopent-2-en-l-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 ⁇ -halocarbonyl compounds employed in preparing the thiosaccharide derivatives of this invention preferably have the general formula: W-CHR 1 -C(O)R 2 wherein R 1 and R 2 are as defined above, and W is chloro, bromo or iodo.
  • W is chloro, bromo or iodo.
  • Preferred ⁇ -halocarbonyl compounds for use in this invention include, by way of example, 2- chlorocyclopentanone and 2-chlorocyclohexanone.
  • carbonyl compounds having a leaving group other than a halogen in the ⁇ -position may be employed.
  • Suitable leaving groups include, by way of illustration, various sulfonic ester groups, such as tosylate, mesylate, brosylate and nosylate groups and the like, and fluorinated 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 oligosaccharides wherein the thiol substitution is at any position of the thiosaccharide.
  • thiolactose 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 references cited therein as well as by Defaye 10 .
  • 1-thiosaccharides can be prepared 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 then provides for the 1 -thiosaccharide.
  • selective protection of the hydroxyl groups of the saccharide provides for one or more free hydroxyl groups which can be converted into appropriate leaving groups, such as mesyl or halo groups, by conventional chemistry well known in the art. Such leaving groups can then be displaced to afford the corresponding thiol groups.
  • appropriate leaving groups such as mesyl or halo groups
  • Such leaving groups can then be displaced to afford the corresponding thiol groups.
  • a mesyl group is selectively introduced at one of the hydroxyl groups and then reacted with a thioacetyl group (for example potassium thioacetate) to provide for the corresponding thioacetate derivative. Treatment of this compound with a mild base provides for the corresponding thio group.
  • the resulting thiosaccharide is then reacted with a coupling reagent selected from the group consisting of Michael acceptors and ⁇ -halocarbonyl compounds.
  • a coupling reagent selected from the group consisting of Michael acceptors and ⁇ -halocarbonyl compounds.
  • this reaction is conducted by contacting the thiosaccharide with at least one equivalent, preferably 1 to 1.2 equivalents, of the coupling reagent 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 to afford a thiosaccharide carbonyl compound.
  • the coupling reagent when the thiosaccharide reagent is attached to a solid support, the coupling reagent is preferable used in excess to maximize the yield of the resulting thiosaccharide carbonyl compound.
  • the thiosaccharide when the the coupling reagent is attached to a solid support, the thiosaccharide 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 reducing agent to provide for an alcohol derivative.
  • a reducing agent to provide for an alcohol derivative.
  • this reduction is conducted by contacting the thiosaccharide carbonyl compound with sodium borohydride, preferably about 1.2 to about 2.0 equivalents of sodium borohydride based on the carbonyl compound.
  • 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, to afford the alcohol derivative.
  • the carbonyl group of the thiosaccharide carbonyl compound can be reductively aminated to provide for an amine derivative.
  • the thiosaccharide carbonyl compound is contacted with an excess of ammonium acetate and at least one equivalent of sodium cyanoborohydride based on the carbonyl compound.
  • This reaction is typically conducted in an inert diluent, such as methanol, 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 aminated in the presence of a primary or secondary amine to provide for amine derivatives.
  • the amine used in the reductive amination is an amino acid or a derivative thereof, such as amino acid esters.
  • this reaction is conducted by contacting the thiosaccharide carbonyl compound with a molar excess of an amino acid ester, such as the methyl ester or the tert-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.
  • 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.
  • an essentially anhydrous inert diluent such as acetonitrile
  • the ester group of the amino acid can be cleaved using standard conditions to provide the corresponding carboxylic acid.
  • the alcohol and/or amine derivatives prepared as described above are further derivatized to form a group selected from esters, substituted amines, amides, carbamates, ureas, thioureas, thioesters and thiocarbamates.
  • alcohols and amines can be reacted with acyl halides to form esters and amides, respectively.
  • Amines can also be reductively alkylated to form substituted amines.
  • alcohols and amines can be reacted with isocyantes, among other reagents, to afford carbamates and ureas, respectively. Conditions for such reactions are well recognized in the art.
  • Figures 1 and 2 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-l-one.
  • Figure 2 illustrates the synthesis of various 1-thiogalactose from 2- chlorocyclohexanone. It will be readily apparent to those of ordinary skill in the art that the synthetic procedure illustrated in Figures 1 and 2 and following reaction conditions can be modified by selecting the appropriate starting materials and reagents to allow the preparation of a plurality of 1-thiogalactose derivatives.
  • D-galactose is perlauroylated by contacting D-galactose with at least 5 equivalents, and preferably 10 equivalents, of lauroyl chloride.
  • 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 hydrochloric acid generated during the reaction.
  • a catalytic amount of 4-(N,N-dimethylamino)pyridine is added to the reaction mixture to facilitate this reaction.
  • this reaction is conducted at a temperature 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- ⁇ -D-galactopyranose, 1, in approximately 70% yield from D-galactose.
  • Compound 1 is then converted into l-S-acetyl-2,3,4,6-tetra-0-lauroyl-l-thio-j8-
  • D-galactopyranose 2, by reaction of 1 with an excess of thiolacetic acid.
  • 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.
  • 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.
  • compound 2 can be prepared from 1 by contacting 1 with at least one equivalent, preferably 1 to 1.2 equivalents, of benzylamine to selectively remove the 1-lauroyl group.
  • This reaction is typically conducted at about 25°C to about 30°C for about 1 to about 96 hours to provide for 2,3,4,6-tetra-O- lauroyl-( ⁇ ,j3)-galactopyranoside.
  • This intermediate is then converted into an O- (2,3,4,6-tetra-Olauroyl-(a,0)-galactopyranosyl) trichloroacetimidate intermediate 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- diazabicyclo[5.4.0]undec-7-ene (DBU) in an inert diluent, such as dichloromethane.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • the resulting O-trichloroacetidate intermediate is then contacted with an excess of thiolacetic acid in an inert diluent, such as dichloromethane, at about 25 °C to about 30°C for about 1 to about 96 hours to provide for l-S-acetyl-2,3,4,6-tetra-0-lauroyl- 1-thio- ⁇ -D-galactopyranose, 2.
  • an inert diluent such as dichloromethane
  • 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 especially preferred since it provides the highest yield of compound 2 and produces no detectable traces of the corresponding ⁇ -isomer.
  • the ⁇ -isomer i.e., l-S-acetyl-2,3,4,6-tetra-O-lauroyl-l- thio- ⁇ -D-galactopyranose
  • compound 1 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 1.1 equivalents of tin (IV) chloride in an inert diluent, such toluene, at ambient temperature for about 0.5 to about 2 hours.
  • dialkylamine first reacts with the thioacetyl 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.
  • 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 reducing agent to provide for 3-hydroxycycloheptyl 2,3,4,6-tetra-0-lauroyl-l-thio-/3-D- galactopyranoside, 4.
  • this reduction is conducted by contacting 3 with sodium borohydride, preferably about 1.2 to about 2.0 equivalents of sodium borohydride based on 3.
  • 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 readily purified by solid-phase extraction on C18 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 for 3-hydroxycycloheptyl l-thio- ⁇ -galactopyranoside, A5.
  • compound 3 can be reductively aminated to provide for 3- aminocycloheptyl 2,3,4,6-tetra-( -lauroyl-l-thio-/3-D-galactopyranoside, 5.
  • compound 3 is contacted with an excess of ammonium acetate and at least one equivalent of sodium cyanoborohydride based on 3.
  • This reaction is typically conducted in an inert diluent, such as methanol, tetrahydrofuran and mixtures thereof, at a temperature of about 25 °C to about 30 °C for about 1 to about 72 hours.
  • the reductive amination reaction is accomplished by contacting compound 3 with an excess of ammonium acetate and an excess of trimethyl orthoformate based on 3, in an inert diluent, 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 intermediate.
  • the imine intermediate is generally not isolated but is contacted in situ with an excess of sodium borohydride, preferably about 1.2 to about 1.5 equivalents of sodium borohydride, based on 3.
  • the resulting amino compound 5 is then readily purified by solid-phase extraction on C18 silica gel using pentane as an eluent.
  • 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)R 6 where L is a leaving group such as a halide, an activated ester, and the like.
  • the lauroyl groups are removed from compound 5 by contacting 5 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.
  • an inert diluent such as dichloromethane
  • the primary amine group of compound B5 can optionally be acylated by contacting B5 with an excess of acetic anhydride in methanol 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-acetamidocycloheptyl 1-thio- ⁇ - galactopyranoside, C5.
  • the primary amine group of 5 can be acylated with phthalic anhydride before removal of the lauroyl groups to provide for 3-(2- carboxybenzamido)cycloheptyl 2,3,4,6-tetra-0-lauroyl-l-thio-j8-D-galactopyranoside, 6.
  • This reaction is typically conducted by contacting compound 5 with at least one molar equivalent, preferably with an excess of phthalic anhydride.
  • this reaction is conducted in dry pyridine containing a catalytic amount of 4-(N,N- 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 accomplished by treating 6 with 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.
  • an inert diluent such as dichloromethane
  • Neutralization of the reaction mixture with Amberlite IR-50S (H + ) resin then provides for 3-(2-carboxybenzamido)cycloheptyl l-thio-3-D- galactopyranoside, D5.
  • compound 3 can also be reductively aminated with an amino acid ester to provide for intermediates 7 or 8.
  • compound 3 is contacted with a molar excess of 0-alanine t -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.
  • 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 resulting intermediate 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 corresponding carboxylic acid by treating 7 with an excess of trifluoroacetic acid in an inert diluent such as dichloromethane. This reaction is typically conducted at about 25 °C to about 30°C for about 1 to about 10 hours.
  • the lauroyl groups of the resulting carboxylic acid intermediate are then removed using sodium methoxide in methanol as described above to provide for N ⁇ -[l-(l-thio-jS-D- galactopyranosyl)cyclohept-3-yl]-j8-alanine, F5.
  • compound 3 can be reductively aminated using other amino acid esters, such as glycine tert-butyl ester, L-leucine tert-butyl ester, L- histidine methyl ester, L-tryptophan methyl ester, and L-arginine methyl ester, to provide for intermediate 8.
  • amino acid esters such as glycine tert-butyl ester, L-leucine tert-butyl ester, L- histidine methyl ester, L-tryptophan methyl ester, and L-arginine methyl ester
  • the tert-butyl ester is cleaved as described above using trifluoroacetic acid to afford N ⁇ -[l-(l-thio-j8-D-galactopyranosyl)cyclohept-3-yl]-glycine, E5, and N ⁇ -[l-(l-thio- ⁇ -D-galactopyranosyl)cyclohept-3-yl]-L-leucine, G5.
  • the lauroyl groups of intermediate 8 are preferably removed before cleaving the methyl ester by treatment 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 by treatment with an excess of aqueous lithium hydroxide for about 0.5 to about 2 hours.
  • the hydroxyl group of alcohol derivatives can be converted into a leaving group, such as the mesylate, tosylate, etc., and displaced with various nucleophiles.
  • a leaving group such as the mesylate, tosylate, etc.
  • treatment of an alcohol derivative with an excess, preferably about 1.1 to about 1.5 equivalents, of methanesulfonyl chloride in pyridine and an inert diluent, such as THF affords the corresponding mesylate.
  • the mesylate group can then be displaced 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°C to about 100°C for about 1 to about 6 hours.
  • an inert diluent such as N,N- dimethylformamide, THF and mixtures thereof
  • a crown ether such as 18- crown-6, is added to the reaction mixture to promote the displacement reaction.
  • the azido derivative can then be reduced with a reducing agent to afford the corresponding primary amine, i.e., a compound such as 5.
  • a reducing agent i.e., a compound such as 5.
  • 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 ( ⁇ iCl 2 ) 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.
  • the primary amine group of amino compounds such as 5 can be further derivatized by reductive alkylation to afford a secondary amine.
  • this reaction is conducted by contacting the primary amine with an excess, preferably about 2 to about 500 equivalents of an aldehyde or a ketone in the presence of at least one equivalent, preferably about 1.0 to about 10 equivalents, of a reducing agent, such as sodium triacetoxyborohydride.
  • This reaction is typically conducted in an inert diluent, such as dichloromethane, methanol, or mixtures thereof, at a temperature of about 0°C to about 50°C for about 10 to about 48 hours.
  • the ketone employed in this reaction is a cyclic ketone including, by way of example, cyclobutanones, such as 3,3-dimethylcyclobutan-l-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 methanol and an inert diluent, such as dichloromethane, at about 25 °C to about 30°C for about 1 to about 24 hours.
  • Figure 2 illustrates the synthesis of various 1-thiogalactose derivatives using an ⁇ -halocarbonyl carbonyl compound, i.e., 2-chlorocyclohexanone.
  • This reaction is typically conducted by contacting 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.
  • 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 to afford compound 9.
  • compound 9 can then be reacted using the same reagents and conditions described above for compound 3 to afford various 1-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-j3-D- galactopyranoside, A2.
  • compound 9 is reductively aminated with ammonium acetate and sodium cyanoborohydride to provide for intermediate 11 which, upon removal of the lauroyl groups, affords 2-aminocyclohexyl 1-thio- ⁇ -D-galactopyranoside, B2.
  • Compound B2 can then be acylated with acetic anhydride to give 2- acetamidocyclohexyl 1-thio-jS-D-galactopyranoside, C2.
  • intermediate 11 can be acylated with phthalic anhydride to provide for intermediate 12 which affords 2-(2-carboxybenzamidocyclohexyl l-thio-j8-D-galactopyranoside, D2, by removal of the lauroyl groups using the conditions described above.
  • compound 9 can be reductively aminated using an jS-alanine tert- butyl ester to provide for intermediate 13 which then affords N/3-[l-(l-thio-/3-D- galactopyranosyl)cyclohex-2-yl]-
  • compound 9 can be reductive aminated with other amino acid esters, such as glycine tert-butyl ester, L-leucine tert-butyl ester, L-histidine methyl ester, L-tryptophan methyl ester, and L-arginine methyl ester, to provide intermediate 14 which upon deprotection, affords N ⁇ -[l-(l-thio- ⁇ -D-galactopyranosyl)cyclohex-2-yl]-glycine E2, N ⁇ -[ 1 -(1 -thio-/?-D-galactopyranosyl)cyclohex-2-yl]-L-leucine G2 , N ⁇ -[ 1 -( 1 -thio-/3-D- galactopyranosyl)cyclohex-2-yl]-L-histidine H2, N ⁇ -[l-(l-thio-/?-D- galactopyranosyl)cyclool
  • Suitable reagents for oxidizing a sulfide compound to a sulfoxide include, by way of example, hydrogen peroxide, peracids such as 3- chloroperoxybenzoic acid (MCPBA), sodium periodate, sodium chlorite, sodium hypochlorite, calcium hypochlorite, tert-butyl hypochlorite and the like.
  • Chiral oxidizing reagents may also be employed to provide chiral sulfoxides.
  • optically active reagents are well known in the art and include, for example, the reagents described in Kagen et al. 11 and references cited therein.
  • the oxidation reaction is typically conducted by contacting the saccharide derivative with about 0.95 to about 1.1 equivalents of the oxidizing reagent in an inert diluent, such as dichloromethane, at a temperature ranging from about 0°C to about 50°C for about 1 to about 48 hours.
  • the resulting sulfoxide can then be further oxidized to the corresponding sulfone by contacting the sulfoxide with at least one additional equivalent of an oxidizing reagent, such as hydrogen peroxide, MCPBA, potassium permanganate and the like.
  • the sulfone can be prepared directly by contacting the sulfide with at least two equivalents, and preferably an excess, of the oxidizing reagent.
  • 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-SO 2 -OH, -C(O)R 10 , -P(O)(OR u ) 2 or pharmaceutically acceptable salts thereof, where R 10 and R 11 are as defined above.
  • Such acylation reactions 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 example, acyl halides, anhydrides, halophosphates, sulfur trioxide, and the like.
  • a de-blocked hydroxyl group can be sulfonylated by treating the hydroxy-containing compound with an excess, preferably about 1.1 to about 1.2 equivalents, of a pyridine: sulfur trioxide complex in an inert diluent, such as N,N- dimethylformamide, at ambient temperature for about 1 to about 24 hours.
  • an inert diluent such as N,N- dimethylformamide
  • the resulting sulfate i.e., -O-SO 2 -OH
  • the resulting sulfate is isolated as its salt by treatment with, for example, a Na + resin in an inert diluent, such as methanol.
  • Further reaction conditions suitable for forming sulfates and phosphates can be found, for example, in U.S. Patent No. 5,580,858 12 .
  • Figures 1 and 2 were conducted in a solution phase. Surprisingly, these methods can also be conducted on the solid phase using reaction conditions similar to those described above for the solution 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 attachment to either the thiosaccharide or the coupling reagent.
  • a linking arm may. be covalently attached to any position of the thiosaccharide other than the thiol group. Such attachments are preferably made through, for example, an ester or ether linkage to one the hydroxyl group of the thiosaccharide.
  • a preferred linking arm is derived from succinic acid.
  • l-dithioethyl-/3-D-galactopyranoside is readily attached 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-/3-D- galactopyranoside in pyridine containing a catalytic amount of 4-(N,N- dimethylamino)pyridine at a temperature 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 galactose is then generated by treating the resin with dithiothreitol (Cleland's reagent) and triethylamine in an inert diluent, such as methanol, for about 6 to 24 hours at ambient temperature.
  • the resulting 1-thio-jS-D-galactopyranoside is then reacted as described above to afford a 1-thiogalactose derivative of formula I covalently attached to the solid support resin.
  • the 1-thiogalactose derivative can be cleaved from the solid support resin by contacting the resin with an excess of trifluoroacetic acid and triisopropylsilane in an inert diluent, such as dichloromethane, at ambient temperature.
  • an inert diluent such as dichloromethane
  • a linking arm can be covalently attached to any position of the coupling reagent provided that the point of attachment does not interfere with the Michael addition of the thiosaccharide to the ⁇ , 3-unsaturated carbonyl group or with the displacement of the halide from the ⁇ -halocarbonyl compound by the thiosaccharide.
  • the linking arm is preferably attached to the coupling reagent through any one of substituents R ⁇ R 8 via a covalent bond.
  • Such linkage can be through, for example, an ester, ether, amine, amide, or urea functional group and the like.
  • a carboxylic acid moiety can be covalently attached to an aminated solid support using conventional coupling procedures and reagents.
  • a coupling reaction will be conducted using well-known coupling reagents such as carbodiimides, BOP reagent (benzotriazol-1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphonate) and the like.
  • Suitable carbodiimides include, by way of example, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and the like.
  • DCC dicyclohexylcarbodiimide
  • EDC diisopropylcarbodiimide
  • EDC l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
  • 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-containing compound (based on the number of equivalents of amino 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 1-thiogalactose derivative), of a coupling promoter such as 1-hydroxybenzotriazole may also be used in the reaction.
  • a coupling promoter such as 1-hydroxybenzotriazole
  • the coupling reaction is conducted at a temperature ranging from about 0°C to about 50°C for about 24 to about 100 hours.
  • the solid support is preferably contacted 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 incorporation of a thiosaccharide onto the solid support can be determined using well-established procedures such as those described, for example, by M. Dubois et al. 13 .
  • the methods of this invention provide for a thiosaccharide derivative library.
  • Such libraries are produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises a thiosaccharide derivative.
  • the thiosaccharide derivative libraries provided by this invention are synthesized by first apportioning solid supports among a plurality of reaction vessels.
  • Such supports comprise a reactive functional group capable of covalently binding to the solid support.
  • the functional group is one that is capable of covalently binding a thiosaccharide 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 embodiment, this is accomplished by selectively blocking 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.
  • each reaction vessel is then contacted with a unique thiosaccharide under conditions wherein the thiosaccharide is covalently attached to the solid supports through the reactive functional group.
  • This reaction is typically conducted by contacting the solid support with at least one equivalent, preferably 1 to
  • the supports After attaching the thiosaccharide to the solid support, the supports are then pooled and the pooled supports are then apportioned among a plurality of reaction vessels.
  • the supports having a thiosaccharide covalently attached thereto are then contacted in each reaction vessel with a unique coupling reagent selected from the group consisting of Michael acceptors and ⁇ -halocarbonyl compounds to provide for a thiosaccharide carbonyl compound which covalently bound to the support.
  • This reaction is preferably conducted as described above.
  • the thiosaccharide carbonyl compound is then reduced as described above to provide for an alcohol and/or an amine derivative.
  • the hydroxy or amino group of these compounds can be further derivatized as described above to form a group selected from esters, substituted amines, amides, carbamates, ureas, thioesters and thiocarbamates.
  • the thiosaccharide derivative libraries provide by this invention are synthesized by first apportioning solid supports among a plurality of reaction vessels wherein such supports comprise a reactive functional group covalently bound to the solid support such that the functional group one that is capable of covalently binding a coupling reagent.
  • Such functional groups include, by way of example, alcohols, amines, isocyanates, carboxylic acid groups, esters and the like.
  • the supports in each reaction vessel is then contacted with a unique coupling reagent selected from the group consisting of Michael acceptors and ⁇ -halocarbonyl compounds under conditions wherein the coupling reagent is covalently attached to the solid supports through the reactive functional group.
  • 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 functional groups on the solid support.
  • the supports After attaching the coupling reagent to the solid support, the supports are then pooled and the pooled supports are then apportioned among a plurality of reaction vessels.
  • the supports having a coupling reagent covalently attached thereto are then contacted in each reaction vessel with a unique thiosaccharide to provide for a thiosaccharide carbonyl compound which is covalently bound to the support.
  • This reaction is preferably conducted as described above.
  • the thiosaccharide carbonyl compounds can then be reduced to provide for a plurality of alcohol and/or amine derivatives.
  • these alcohol and/or amine derivatives can optionally be further derivatized to provide for a group selected from esters, substituted amines, amides, carbamates, ureas, thioesters, and thiocarbamates.
  • an identifier tag is employed in the methods of this invention.
  • the identifier tag has a recognizable feature that is, for example, microscopically or otherwise distinguishable in shape, size, mass, charge, or color. This recognizable feature may arise from the optical, chemical, electronic, or magnetic properties of the tag, or from some combination of such properties. In essence, the tag serves to label a molecule and to encode information decipherable at the level of one (or a few) molecules or solid supports. By using identifier tags to track the synthesis pathway that each member of a chemical library has taken, one can deduce the structure of any chemical in the library by reading the identifier 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 synthesis series in which each reagent was added or other chemical reaction performed.
  • the tags may be attached immediately before, during, or after the reagent addition or other reaction, as convenient and compatible with the type of identifier tag, modes of attachment, and chemistry of activated ketone or other molecular synthesis.
  • the identifier tag can be associated with the thiosaccharide derivatives through a variety of mechanisms, either directly, through a linking molecule, or through a solid support upon which the thiosaccharide derivative is synthesized.
  • identifier tag is added when the solid supports that have undergone a specific reagent addition or other chemical reaction step are physically together and so can be tagged as a group, i.e., prior to the next pooling step.
  • Preferred identifier tags include, by way of example, peptides 14,15 oligonucleotides 16 and halocarbon derivatives 17 .
  • the libraries of thiosaccharide derivatives may be screened for biological activity.
  • the library to be screen is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody, and the presence or absence of an interaction between the thiosaccharide derivative and the biological substance is determined.
  • a biological substance usually a protein such as a receptor, enzyme, membrane binding protein or antibody
  • the presence or absence of an interaction between the thiosaccharide derivative and the biological substance is determined.
  • this will comprise determining whether the biological substance is bound to one or more of the members of the library.
  • binding may be determined by attaching 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 affinity may be determined by quantitating the amount or intensity of the bound label.
  • various lead compounds may be selected by identifying which compounds bind the particular biological substance most effectively.
  • bead-based libraries are screened by assays in which each different molecule in the library is assayed for its ability to bind to a receptor of interest.
  • the receptor is contacted with the library of thiosaccharide derivatives, forming a bound member between the receptor and any thiosaccharide derivative in the library able to bind the receptor under the assay conditions.
  • the bound thiosaccharide derivative is then identified by examination of the tag associated with that thiosaccharide derivative.
  • the receptor to which the library is exposed under binding conditions can be a mixture of receptors, each of which is associated with an identifier tag specifying the receptor type, and consequently two tags are examined 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. , fluorescence activated cell sorting (FACS)).
  • flow cytometry e.g. , fluorescence activated cell sorting (FACS)
  • FACS fluorescence activated cell sorting
  • Thiosaccharide derivatives can be synthesized 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 either event, the thiosaccharide derivatives of interest are cleaved from the beads but remain contained within the compartment along with the bead and the identifier tag(s).
  • Soluble tagged thiosaccharide derivatives can also be screened using an immobilized receptor. After contacting the tagged thiosaccharide derivatives with the immobilized receptor and washing away non-specifically bound molecules, bound, tagged thiosaccharide derivatives are released from the receptor by any of a wide variety of methods.
  • the tags are optionally amplified and then examined and decoded to identify the structure of the molecules that bind specifically to the receptor.
  • a tagged thiosaccharide derivative in solution can be assayed using a receptor immobilized by attachment to a bead, for example, by a competition assay with a fluorescently labeled ligand.
  • One may recover the beads bearing immobilized receptors and sort the beads using FACS to identify positives (diminished fluorescence caused by the library molecule competing with the labeled ligand).
  • the associated identifier tag is then amplified and decoded.
  • the libraries described herein will contain at least about 2 compounds, more preferably at least about 10 2 compounds, still more preferably from about 10 2 to about 10 10 compounds and even still more preferably from about 10 3 to about 10 6 compounds.
  • thiosaccharide derivatives which block binding of a toxin, such as heat-labile enterotoxin or cholera toxin, the toxin's receptor either in vitro or in vivo, and compounds which inhibit binding of organisms (e.g. , bacteria, virus, fungi, and the like), including enterovirulent organism such as Vibrio cholerae and enterotoxigenic strains of Escherichia coli, to their cell surface receptors.
  • ⁇ -Nmr spectra were recorded with a Brueker AM-360 spectrometer and MALDI-TOF mass spectra were recorded with a HP G2020A (LD-TOF) instrument.
  • Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Reactions were monitored by TLC on Silica Gel FG254 (E. Merck, Darmstadt, Germany).
  • Example E General Procedure for Reduction to Alcohols To the product from Example D (100 ⁇ mol) in dry tetrahydrofuran (2.0 mL) and isopropanol (0.7 mL) under argon atmosphere, was added NaBH 4 (150 ⁇ mol). After 0.5-3 h, the mixture was concentrated (acetic acid (about 40 ⁇ L) was added prior 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 1 H-nmr spectroscopy and MALDI-TOF mass spectroscopy.
  • Example F General Procedure for Reductive Amination to a Primary Amine Method 1: To the product from Example D (100 ⁇ mol) and ammonium acetate (75 mg, 1 mmol) in dry methanol (2.3 mL) and tetrahydrofuran (0.9 mL) under argon, was added NaCNBH 3 (100 ⁇ mol). After 1-72 h, the mixture was concentrated and the residue purified according to the solid-phase extraction procedure of Example A. The product amines were characterized with 'H-nmr spectroscopy and MALDI- TOF mass spectroscopy.
  • the cartridge was washed with pentane/EtOAc (1:1, 20 mL) (to remove the conesponding alcohol), followed by elution with toluene/EtOH (9: 1, 30 mL) to afford the primary amine.
  • Example I General Procedure for Deblocking of Alcohols To the lauroylated alcohol from Example E (100 ⁇ mol) in dry methanol (7.1 mL) and dichloromethane (1.4 mL) under argon atmosphere, was added methanolic sodium methoxide (1 M, 50 ⁇ L). After 1-24 h, the mixture was neutralized with
  • Example J General Procedure for Deblocking of Primary Amines
  • methanolic sodium methoxide (1 M, 50 ⁇ L).
  • 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 SiOi cartridge.
  • the cartridge was washed with dichloromethane/methanol (2:1) and then the 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 silica (Waters Prep C18, 125 A, 5 g). The C 18 silica was washed with water (50 mL) and then the product was eluted with methanol (50 mL). The resulting primary amines were characterized with 1 H-nmr spectroscopy and MALDI-TOF mass spectroscopy.
  • Example J To the primary amine from Example J (100 ⁇ mol) in moist methanol (4.4 mL) was added acetic anhydride (0.4 mL). The mixture was concentrated after 2-24 h, re- dissolved in water and applied to 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 resulting acetamides were characterized with ⁇ -nmr spectroscopy and MALDI-TOF mass spectroscopy.
  • N-alkylated glycine, ⁇ -alanine, and L-leucine compounds were characterized with 'H-nmr spectroscopy and MALDI-TOF mass spectroscopy.
  • Example 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), methanol (2 mL) and triethylorthoformate (1 mL) under argon, was added NaCNBH 3 (1 mmol). After 24 h, the mixture was concentrated and dissolved in toluene (1 mL) and purified on C18-silica gel (5 g).
  • the cartridge was washed with dichloromethane/methanol (2: 1) and then the 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 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 resulting secondary amines were characterized with IH-nmr spectroscopy and MALDI-TOF mass spectroscopy.
  • the title compound was prepared according to procedures D, H and M above using 6,6-dimethylcyclohex-2-en-l-one as the electrophile and glycine tert-butyl ester as the amino acid ester.
  • This example illustrates the preparation of individual diastereomers of a compound of formula I.
  • Tetra-O-lauroyl-l-thio-/3-D-galactopyranoside To l-S-acetyl-2,3,4,6-tetra-0-lauryl- l-thio-/3-D-galactopyranose (5 g, 5 mmol) (from Example C above) and 4,4-dimethyl-
  • Step B Separation of the Diastereomers of (lR,S)-2,2- Dimethylcyclopentan-4-on-l-yl 2,3,4,6-Tetra-0-lauroyl-l-thio-/3-D- galactopyranoside:
  • the two diastereomers from Step A (5 g, 4.8 mmol) were separated by column chromatography (SiO 2 , pentane/EtOAc, 9: 1) to give (lS)-2,2- dimethylcyclopentan-4-on- 1 -yl 2 , 3 ,4 , 6-tetra-O-lauroyl- 1 -thio-/3-D-galactopyranoside (428.8 mg, 8%) and (lR)-2,2-dimethylcyclopentan-4-on-l-yl 2,3,4,6-tetra-0-lauroyl- l-thio-/3-D-galactopyranoside (373.8 mg, 6%) along with
  • Step C Synthesis of (IS, 4RS)- and (IR, 4RS)-2,2-Dimethyl-4- hydroxycyclopent-1-yl 2,3,4,6-Tetra-0-lauroyI-l-thio-3-D-gaIactopyranoside: 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 NaBH 4 (0.12 mmol).
  • Step D Synthesis of (IS, 4RS)- and (IR, 4RS)-2,2-Dimethyl-4-0- methanesulfonyloxycyclopent-1-yl 2,3,4, 6-Tetra-O-lauroyl-l-thio- ?-D- galactopyranoside: To each of the (IS, 4RS) and (IR, 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 methanesulfonyl chloride (0.5 mL).
  • Step E Synthesis of (IS, 4R)-, (IS, 4S)-, (IR, 4S)- and (IR, 4R)-2,2- DimethyI-4-azidocyclopent-l-yl 2,3,4,6-Tetra-0-lauroyl-l-thio-i3-D- galactopyranoside: To the (IS, 4RS) and (IR, 4RS) mixtures from Step D (in separate reaction flasks) (250 mg, 0.2 mmol) in dry DMF (8 mL) and dry THF (3 mL) under argon atmosphere at 60 °C was added NaN 3 (340 mg, 5 mmol) and 18 crown-6 (180 mg).
  • Step F Synthesis of (IS, 4R)-, (IS, 4S)-, (IR, 4S)- and (IR, 4R)-2,2- Dimethyl-4-aminocyclopent-l-yl 2,3,4,6-Tetra- ⁇ 9-lauroyl-l-thio-/3-D- galactopyranoside: To each of the four diastereomers of 2,2-dimethyl-4-azido- cyclopent-1-yl 1-thio- ⁇ -D-galactopyranoside from Step E (5 mg, 15 ⁇ mol) in dry isopropanol (1 mL) and dry ethanol (1 mL) under argon atmosphere, was added NaBH 4 (15 ⁇ mol) and NiCl 2 (30 ⁇ mol).
  • Step G Synthesis of (IS, 4R)-, (IS, 4S)-, (IR, 4S)- and (IR, 4R)-2,2- Dimethyl-4-(cycIobut-l-ylamino)cyclopent-l-yI2,3,4,6-Tetra-0-Iauroyl-l-thio- ⁇ -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 ⁇ mol) in dry methanol (1 mL) and dry dichloromethane (1 mL) under argon atmosphere, was added cyclobutanone (250 ⁇ L, 3.4 mmol) and sodium triacetoxyborohydride (10 mg, 47 ⁇ mol). After 24-48 h, toluene (1 mL) was added and the mixture was concentrated and
  • the example illustrates the solid-phase synthesis of 1-thiogalactose derivatives of formula I.
  • Step A Synthesis of l-DithioethyI-2,3,4,6-tetra-0-acetyl-galactopyranoside: l-Thio-2,3,4,6-tetra- >-acetyl-galactopyranoside (500 mg, 1.37 mmol) and diethyl-N- ethyl-sulfenylhydrazodicarboxylate (360 mg, 2.0 mmol) (prepared as described in T. Mukaiyama 20 ) are dissolved in dichloromethane (14 mL) and stined at room temperature.
  • Step B Synthesis of l-Dithioethyl-ZJ-D-galactopyranoside: 1-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 1R-120 (H + ) resin, filtered and concentrated to give l-dithioethyl- ⁇ - ⁇ -D-galactopyranoside as a white solid (300 mg, quant).
  • Step C Coupling of l-Dithioethyl- ⁇ -D-galactopyranoside to a Resin: 1- Dithioethyl-6-jS-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-1 % DVB, 200-400 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 successively with methanol, tetrahydrofuran, dichloromethane and diethyl ether (10 mL each) to afford 1- dithioethyl-jS-D-galactopyranoside covalently linked to the trityl resin through the hydroxyl group in the 6-position.
  • Step D Generation of the Free Thiol on the Resin:
  • the resin from Step C (50 mg) is swollen in dry tetrahydrofuran (1.5 mL). Dry methanol (300 ⁇ L), dithiothreitol (74 mg) and trieth ylamine (180 ⁇ L) are added and the mixture is shaken for 10 hours at room temperature. The resin is filtered off and washed successively with methanol, tetrahydrofuran, dichloromethane and diethyl ether (10 mL/each). IR (of intact beads): 2565 cm "1 (SH stretch).
  • Step E Michael Addition Reaction: The resin from Step D (50 mg) was swollen in dry N,N-dimethylformamide (1 mL) and then cyclohept-2-en-l-one (70 ⁇ l, 63 ⁇ mol) was added and the mixture was shaken at room temperature. After 2 hours, the resin was filtered off and washed successively with methanol, tetrahydrofuran, dichloromethane and diethyl ether (10 mL each).
  • Step F Reductive Amination with an Amino Acid:
  • the resin from Step E (50 mg) was swollen in dichloromethane (1 mL).
  • Glycine tert-butyl ester hydrochloride (75. mg, 447 ⁇ mol), sodium sulfate (100 mg), sodium triacetoxyborohydride (63 mg, 297 ⁇ mol) and acetic acid (10 ⁇ L) 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 1-Thiogalactose Derivative from the Resin and
  • the C I8 silica was washed with water (4 mL) and the final product was eluted with 20% methanol and concentrated. After freeze drying from 5 mL of water, N ⁇ -[3-(l- thio-/3-D-galactopyranosyl)cyclohept-l-yl]glycine was obtained as a white powder (4.8 mg). The diastereomers ratio was 10: 10:8:6 as determined by 'H- ⁇ MR.
  • Example 17 Inhibition of Cholera Toxin Binding to G Dlb
  • 1-thiogalactose derivatives of formula I above were tested for their ability to inhibit the binding of cholera toxin to ganglioside G D ⁇ b .
  • This bioassay was conducted using the following modification of the procedure described by A.-M. Svennerholm 21 .
  • Each solution contained 10 mg of ⁇ -phenylenediamine (Sigma), 5 mL of 0.1M sodium citrate (filter sterile or autoclaved), 5 mL of 0.1M citric acid (filter sterile or autoclaved) and 4 mL of 30% H 2 O 2 . (Gloves should be worn since ⁇ -phenylenediamine is carcinogenic).
  • the substrate solution 100 ⁇ L was then added to each well and incubated for 30 minutes at room temperature. After incubation, the OD 450 was recorded. Under the conditions of the assay, D-galactose had an IC 50 of 30 mM.
  • All of the compounds tested inhibited binding of cholera toxin to ganglioside G Dlb by at least 20%, except for the compounds of Examples Al, A3, A4, A6-A8, A10, Bl, B3, B4, B10, CI, C3, C8, D3, E5, E8, E9, FI, F5-F7, F9, F10, G3, G7-G10, H2, H5, H8- H10, 12, 18-110, J5-J10, which did not inhibit binding by at least 20% at the concentration employed in the assay (i.e., 1 mg/mL).
  • the solid support material of Example 13 was tested for its ability to neutralize the cytotonic activity of CT and LT.
  • the cytotonic activity of CT and LT was measured by the use of Chinese hamster ovary cells (CHO) that were maintained in Hams F12 media supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO 2 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- fold diluted in media and 100 ⁇ L of each dilution was added to wells with confluent monolayers of CHO cells and incubated for 24 h at 37° C (under 5% COj). Each sample was analyzed two times.
  • a solution containing 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 microcentrifuge tubes and incubated at room temperature for 1 h on an end-over rotator. After incubation, the solid support material 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 incubation for 24 h as described above. The extent of reduction in the endpoint in the presence of the solid support material was determined by comparing with controls in which solid support material was not added.
  • 1-thiogalactose derivatives of formula I above were tested for their ability to inhibit CFA pili binding to glycophorin.
  • Bacterial surface adhesion antigens such as CFA pili are a virulence factor expressed by certain enteric pathogens, including enterotoxigenic E. coli. These pili are important factors in bacterial attachment to cell surface receptors. Accordingly, inhibition of CFA pili binding is a useful test to determine whether a compound will inhibit the binding of a pathogenic microorganism to cell surface receptors.
  • Binding assays were done by coating microtitre wells with 50 ⁇ L of glycophorin
  • Inhibitors were preincubated with biotinylated CFA I pili (5 ⁇ /mL) for 1 h at 37°C prior to adding to glycophorin-coated microtitre wells as outlined above. ⁇ -Nitrophenyl-j3-D-galactose was utilized as a control inhibitor for these experiments.

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Abstract

L'invention concerne des procédés de synthèse de collections très importantes de divers dérivés thiosaccharides, éventuellement fixés sur un support solide, et elle concerne également des banques de divers dérivés thiosaccharides.
EP97913040A 1996-11-15 1997-11-14 Synthese combinatoire de banques d'hydrates de carbone Ceased EP0938492A1 (fr)

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Application Number Priority Date Filing Date Title
US08/751,510 US6063769A (en) 1996-11-14 1996-11-15 1-thiogalactose derivatives
US751510 1996-11-15
US751231 1996-11-15
US08/751,231 US5780603A (en) 1996-11-15 1996-11-15 Combinatorial synthesis of carbohydrate libraries
PCT/CA1997/000866 WO1998022487A1 (fr) 1996-11-15 1997-11-14 Synthese combinatoire de banques d'hydrates de carbone

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ATE478083T1 (de) 1999-03-05 2010-09-15 Massachusetts Inst Technology Linker zur synthese von oligosacchariden auf festträgern
AU1075701A (en) 1999-10-08 2001-04-23 Protogene Laboratories, Inc. Method and apparatus for performing large numbers of reactions using array assembly
JP2006151948A (ja) * 2004-11-04 2006-06-15 Institute Of Physical & Chemical Research 糖鎖合成方法および糖鎖自動合成装置
KR101417135B1 (ko) 2005-11-22 2014-07-08 콘드롤드 케미컬즈, 인크. 옥시코돈 및 다른 조성물에서 오염물질인 마이클 수용체수준을 감소시키는 방법

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JPH0532689A (ja) * 1991-08-01 1993-02-09 Nippon Fine Chem Co Ltd 膜タンパク質の可溶化剤
ES2097925T3 (es) * 1991-09-18 1997-04-16 Affymax Tech Nv Metodo para sintetizar diversas colecciones de oligomeros.
JPH08511234A (ja) * 1993-02-23 1996-11-26 ザ トラスティーズ オブ プリンストン ユニヴァーシティー グリコシド結合の溶液及び固相形成
US5571902A (en) * 1993-07-29 1996-11-05 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
JP3429036B2 (ja) * 1993-09-30 2003-07-22 生化学工業株式会社 エンドトキシンの特異的測定剤
US5484773A (en) * 1994-02-14 1996-01-16 Alberta Research Council Treatment of antibiotic associated diarrhea

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Title
See references of WO9822487A1 *

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JP2001505558A (ja) 2001-04-24
AU736031B2 (en) 2001-07-26
WO1998022487A1 (fr) 1998-05-28
CA2256694A1 (fr) 1998-05-28
NZ332975A (en) 2001-06-29
AU5044098A (en) 1998-06-10

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