[go: up one dir, main page]

WO1999061583A2 - Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction - Google Patents

Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction Download PDF

Info

Publication number
WO1999061583A2
WO1999061583A2 PCT/US1999/012032 US9912032W WO9961583A2 WO 1999061583 A2 WO1999061583 A2 WO 1999061583A2 US 9912032 W US9912032 W US 9912032W WO 9961583 A2 WO9961583 A2 WO 9961583A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
group
methyl
compound
heterocyclic
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
PCT/US1999/012032
Other languages
French (fr)
Other versions
WO1999061583A3 (en
Inventor
Michael J. Sofia
Rakesh K. Jain
Andrew Vaughan
David M. Gange
Manuka Ghosh
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.)
Aeolus Pharmaceuticals Inc
Original Assignee
Incara Pharmaceuticals Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Incara Pharmaceuticals Corp filed Critical Incara Pharmaceuticals Corp
Priority to AU43227/99A priority Critical patent/AU4322799A/en
Publication of WO1999061583A2 publication Critical patent/WO1999061583A2/en
Publication of WO1999061583A3 publication Critical patent/WO1999061583A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • 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

Definitions

  • the present invention relates to the construction of carbohydrate- based "scaffold" molecules and combinatorial libraries. Such compounds and libraries can be used to screen for novel ligands capable of binding to therapeutically relevant biomolecular targets of interest.
  • Primary screening libraries are useful for the identification of new classes of drugs when little is known about the kinds of ligands that bind to particular receptors on a biological target or when it is desired to identify new compounds that bind similarly to known pharmacophores. Since little structural information is typically available upon which to base design of a library, the probability of identifying an active compound from a primary screening library is related to the number of compounds that can be constructed and screened. Hence, the strategy for designing a primary screening library should permit the creation of a large amount of structural variation within a given molecular system, and should provide access to a large diversity of structures of interest.
  • a "scaffold" molecular system is an advantageous approach to the construction of primary screening libraries.
  • a particular molecular system serves as a template upon which various chemical or biological appendages are attached to define the library.
  • the conformational rigidity and high degree of functionalization of carbohydrates suggests that these molecules may be ideal templates for the construction of primary screening libraries.
  • Previous approaches to the use of carbohydrates in the construction of a set of compounds are conveniently characterized as being directed towards the construction of oligosaccharide mimetics or monosaccharide peptidomimetics.
  • An example of the first approach is a random glycosylation method for producing oligosaccharide, e.g., trisaccharide, libraries [Kanie, 0. et al . , (1995)].
  • a recent variation on the synthesis of oligosaccharides, which entails linking carbohydrate molecules via nucleotide or peptide bonds [Nicolaou, K. et al., (1995); Suhara, Y. et al .
  • a further refinement to this approach for constructing carbohydrate scaffolds involves the use of a "sugar amino acid” as a building block for the construction of so-called “peptidomimetics” [von Roedern, E., et al . , (1996)].
  • This latter approach involves coupling one or more amino acids to a carboxylic acid group provided on the carbohydrate ring. Amino acids are also attached to an amino group provided on the carbohydrate ring.
  • This approach is limited to carbohydrate scaffolds bearing two functional groups which may be elaborated.
  • a largely non-peptidyl approach in which the libraries comprise disaccharides , trisaccharides and glycoconjugates of amino acids is the subject of PCT Publication No. 95/03315.
  • the present invention is directed to a compound of structure
  • X is 0 or S; Z is 0 or NH; Ri is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl ; Y is COOH, C00R 2 , CH 2 OR 3 , CH 3 , or CH(s)Y2 ⁇ 3-s) where Y 2 is F, Cl, Br or I , and s is 0, 1, or 2 or Y and one of ZR 4 and OR5 are linked to form a 6-membered cyclic acetal; R 2 is alkyl, aryl or aralkyl; R 3 , R 4 and R 5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1; m is 0 or 1; and n is 1 or 2; provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH;
  • This invention is also directed to a library of compounds, each having the structure
  • X is O or S
  • Ai is a residue of an ⁇ -amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 ⁇ -amino acids and attached through a terminal amino, RiO, RiS, Ri, RiNH or RiN-alkyl
  • a 2 is a residue of an ⁇ -amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 ⁇ -amino acids and attached through a terminal carboxyl, R2SO2, R 2 NHCO, R 2 OP (0) (0R 6 ) , R 2 P(0)(OR 6 ) or R 2 , or A 2 , A 3 and N combine to form a nitrogen heterocycle
  • a 3 is hydrogen when A3 is not combined with A 2 and N;
  • a 4 is OR4, NHR4, CH2OR 4 or CH 3 ;
  • a 5 is 0, NH or N-alkyl; p, q and r are independently 0 or
  • Ri, R 2 and R 3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl , heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl;
  • R4, R5, ⁇ and R 7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl;
  • m is 0 or 1; and n is 1 or 2; provided that: when n is 1, then m is 0; when A 5 is NH or N-alkyl, then L 3 is not NHP(O) (OR 7 ) ; when L is a single bond, CH 2 or carbonyl
  • the invention is further directed to a method of making the library of compounds by the steps of: (a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and an azido group; (b) performing, in any order, steps of: (i) allowing the free carboxyl group of the monosaccharide to react to produce a substituent Ai; (ii) reducing the azido group to an amino group and allowing the amino group to react with a compound capable of reacting with the amino group to produce a substituent A2; (iii) allowing a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent Brief Description of the Drawings
  • Figure 1 illustrates the preparation of compounds bearing the Guanidinium grou .
  • Figure 2 illustrates a library of compounds comprising the Guanidinium group, labeled Library BI-1.
  • Figure 3 illustrates a library of compounds comprising the Guanidinium group, labeled Library BI-2.
  • alkyl refers to an acyclic or non-aromatic cyclic group having from one to twenty carbon atoms connected by single or multiple bonds.
  • An alkyl group may be substituted by one or more of halo, hydroxyl, protected hydroxyl, amino, nitro, cyano , alkoxy, aryloxy, aralkyloxy, COOH, aroyloxy, alkylamino, dialkyla ino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl , aroyl, CONH 2 , CONH-alkyl or CON(alkyl) 2 , COO-aralkyl, COO-aryl or COO- alkyl .
  • aryl refers to a group derived from a non- heterocyclic aromatic compound having from six to twenty carbon atoms and from one to four rings which may be fused or connected by single bonds.
  • An aryl group may be substituted by one or more of alkyl, aralkyl, heterocyclic, halo, hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, aroyl, COO-alkyl, COO-aralkyl, COO-aryl, C0NH 2 , CONH- alkyl or CON (alkyl) 2 .
  • aralkyl refers to an alkyl group substituted by an aryl group.
  • heterocyclic refers to a group derived from a heterocyclic compound having from one to four rings, which may be fused or connected by single bonds; said compound having from three to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur or phosphorus.
  • a heterocyclic group may be substituted by one or more of alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl , aroyl, COO-alkyl, COO- aralkyl, COO-aryl, CONH 2 , CONH-alkyl or CON (alkyl) 2 •
  • alkoxy, " aryloxy” and “aralkyloxy” refer to groups derived from bonding an oxygen atom to an alkyl, aryl or aralkyl group, respectively.
  • alkanoyl refers to groups derived from bonding a carbonyl to an alkyl, aryl or aralkyl group, respectively.
  • protected hydroxyl refers to a hydroxyl group bonded to a group which is easily removable under basic conditions, including, e.g., acetyl, benzoyl, levulinoyl, chloroacetyl, pivaloyl, p-nitrobenzoyl and tert-butyl-diphenylsilyl, to generate a free hydroxyl grou .
  • the desired three-point motif is achieved by a scaffold design that incorporates a carboxylic acid moiety, a free or protected hydroxyl group and an azido group. This functional group triad affords the chemoselectivity necessary for rapid combinatorial synthesis allowing the maximum amount of molecular diversity while minimizing the number of solid phase synthetic steps.
  • the monosaccharide scaffold compounds of the present invention have the structure
  • X is O or S; Z is 0 or NH; Ri is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; Y is COOH, COOR 2 , CH2OR3, CH 3 or CHis) 2 ⁇ 3-s) where Y 2 is F, Cl , Br or I , and s is 0, 1, or 2 or Y and one of ZR 4 and OR 5 are linked to form a 6-membered cyclic acetal; R is alkyl, aryl or aralkyl; R 3 , R 4 and R 5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1; m is 0 or 1 ; and n is 1 or 2 ; provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH, then R is not hydrogen;
  • the monosaccharide scaffold compound is a five- or six- membered ring having one oxygen atom in the ring, and bearing one free or protected hydroxyl group, one free carboxylic acid group and one azido group.
  • the azido group may be attached directly to a monosaccharide ring carbon, or may be attached through a CH 2 substituent.
  • the hydroxyl or protected hydroxyl and the carboxylic acid group may be attached to monosaccharide ring carbons or to any substituent attached to the ring.
  • the scaffold compound may have both a free hydroxyl group and one or more protected hydroxyl groups, but has no more than one free hydroxyl group.
  • Suitable protecting groups for the hydroxyl group are those which are easily removable under basic conditions to generate a free hydroxyl group, including, e.g., acetyl, benzoyl, levulinoyl, chloroacetyl, pivaloyl, p-nitrobenzoyl and tert-butyl-diphenylsilyl.
  • a monosaccharide scaffold compound bearing a free hydroxyl, a carboxylic acid and an azido group is functionalized at the three reactive sites, i.e., the hydroxyl, carboxylic acid and azido group, to produce the library of compounds of this invention.
  • Suitable scaffold compounds are those disclosed in this invention, as well as other monosaccharides bearing the three reactive sites described hereinabove. Examples of other suitable monosaccharide scaffolds are compounds disclosed in copending application Serial No. 08/975,229, filed November 21, 1997, including compound (XI), having the structure
  • Each compound in the library has the structure
  • X is 0 or S;
  • Ai is a residue of an ⁇ -amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 ⁇ -amino acids and attached through a terminal amino,
  • RiO, RiS, Ri, RxNH or RiN-alkyl is a residue of an ⁇ -amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 ⁇ -amino acids and attached through a terminal carboxyl, R 2 S0 2 , R 2 NHCO, R 2 OP (O) (OR 6 ) , R 2 P(0)(OR 6 ) or R 2 , or A 2 , A 3 and N combine to form a nitrogen heterocycle;
  • a 3 is hydrogen when A 3 is not combined with A 2 and N;
  • a 4 is OR 4 , NHR 4 , CH 2 OR or CH 3 ;
  • a s is O, NH or N-alkyl; p, q and r are independently 0 or 1; Yi and Y 2 are independently O or CH 2 ; each of Li and L 2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or
  • Ri, R 2 and R 3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl;
  • R 4 , R5, e and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl;
  • m is 0 or 1; and n is 1 or 2; provided that: when n is 1, then m is 0; when A 5 is NH or N-alkyl, then L 3 is not NHP(O) (0R 7 ) ; when L 3 is a single bond, CH 2 or carbony
  • Difunctional groups Y1L1 and L 2 Y 2 in the library compounds when present, may be derived from scaffold compounds in which the carboxylic acid or hydroxyl groups, respectively, are not attached directly to a monosaccharide ring carbon, but instead are substituted on a group Y.Li or L2Y2 attached to a monosaccharide ring carbon.
  • the group Y 1 L 1 may also be derived from reaction of a difunctional molecule, e.g., a dicarboxylic acid in which Yx is 0 and Li is difunctional alkanoyl, with the carbonyl carbon of the difunctional alkanoyl attached to Yi, and another carbon connected to the COAi group, with a hydroxyl group directly substituted on a monosaccharide ring atom.
  • a difunctional molecule e.g., a dicarboxylic acid in which Yx is 0 and Li is difunctional alkanoyl
  • the group R 3 L 3 A5 is derived from reaction of the hydroxyl group of the scaffold molecule.
  • a 5 is oxygen.
  • As is NH or N-alkyl.
  • Suitable amino acids are the natural or unnatural amino acids described above.
  • the natural amino acids can be obtained commercially. Some unnatural amino acids can also be obtained commercially, however, it is frequently desired to prepare them, preferably in enantiomeric excess, from commercially available starting materials.
  • a general approach to unnatural amino acids has been described recently [Petasis, N. et al . , JACS, 119:445 (1997)].
  • Natural, nonnatural, and modified amino acids can be linked through their C-terminii to an amine-substituted saccharide in the presence of a carbodiimide or acid anhydride. Alternatively, they can be linked through their N-terminii to a carboxyl-substituted saccharide in the same way.
  • An ester linkage to the monosaccharide scaffold molecule is formed straightforwardly by reacting a carboxylic acid group on the monosaccharide scaffold with an alcohol, e.g., one provided by a ligand molecule linked to a solid support.
  • an ester linkage can be formed by reacting the free hydroxyl of the monosaccharide scaffold with a carboxylic acid ligand under standard esterification conditions.
  • An ester linkage can also be formed by reacting the monosaccharide scaffold with an acid anhydride or acyl halide.
  • the ester can also be prepared by an MCC reaction, such as the Passerini reaction, which entails reacting a carboxylic acid with an aldehyde and an isonitrile in a one-step synthesis.
  • the carboxylic acid component is conveniently provided by the monosaccharide .
  • An amido linkage between the amino group on the monosaccharide scaffold and a compound bearing a carboxylic acid group, or between the carboxylic acid group on the monosaccharide scaffold and a compound bearing an amino group is conveniently formed by reacting an amino group with a carboxylic acid group in the presence of dicyclohexylcarbodiimide (DCC) or an anhydride.
  • DCC dicyclohexylcarbodiimide
  • a secondary amino linkage can be formed by reacting the amino group on the monosaccharide scaffold with an aldehyde or a ketone under reductive alkylation conditions.
  • Preferred aldehydes are n- butyraldehyde and substituted alkyl compounds such as 3- methylthiopropionaldehyde; cycloaliphatic aldehydes such as cyclohexanecarboxaldehyde; aryl aldehydes such as benzaldehyde, 4- nitrobenzaldehyde and pyridine-2-carboxaldehyde; heteroatom- substituted cycloaliphatic aldehydes such as N-formylmorpholine; and alkarylaldehydes such as 2-phenylpropionaldehyde.
  • cycloaliphatic aldehydes such as cyclohexanecarboxaldehyde
  • aryl aldehydes such as benzaldehyde, 4- nitrobenzaldehyde and pyridine-2-carboxaldehyde
  • heteroatom- substituted cycloaliphatic aldehydes such as N-formylmorph
  • a sulfonamido linkage can be formed by reacting the amine with a sulfonyl halide, e.g., sulfonyl chloride.
  • a sulfonyl halide e.g., sulfonyl chloride.
  • Particularly preferred are substituted and unsubstituted aryl sulfonyl chlorides, such as 1- naphthalenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, p- toluenesulfonyl chloride, and N-acetylsulfanilyl chloride.
  • a urea linkage can be formed by reacting the amine with an isocyanate, e.g., phenyl isocyanate.
  • an isocyanate e.g., phenyl isocyanate.
  • aromatic, halogenated aromatic, and halogenated aliphatic isocyanates are particularly preferred.
  • a guanidine can be formed by reacting the sugar azides with isothiocyanates and triphenylphosphine, followed by the addition of a primary. or secondary amine, e.g., phenylisothiocyanate, triphenylphosphine, and methyl amine to give the phenyl, methyl guanidine attached to the sugar.
  • a primary. or secondary amine e.g., phenylisothiocyanate, triphenylphosphine, and methyl amine
  • a number of alcoholic reactions are available for connecting a free hydroxyl of the monosaccharide with a desired organic moiety.
  • an alkyl ether linkage is formed by alkylating a free hydroxyl group on the sugar molecule, e.g., by reacting the sugar with an alkyl halide in the presence of a base.
  • An aryl ether can be formed by reacting a monosaccharide bearing a free hydroxyl group with the desired phenol under Mitsunobu reaction conditions [Mitsunobu, 0., et al . , JACS, 94:679 (1972)].
  • a carbamate linkage is formed by reacting a free hydroxyl group of the sugar with an isocyanate.
  • a urea linkage is formed by reacting a free amino group of the sugar with an isocyanate.
  • a carbonate linkage can be formed by reacting an ester of a haloformate, e.g., a chloroformate ester, with the free hydroxyl of the monosaccharide in the presence of a base.
  • a phosphonate or phosphate linkage to the monosaccharide can be formed by reacting it with a phosphonic acid ester or phosphoramidite followed by oxidation to give the phosphonate or phosphate.
  • a phosphonic acid ester or phosphoramidite followed by oxidation to give the phosphonate or phosphate.
  • exemplary phosphoramidite reagents include chloro-methoxy-N,N-diisopropyl phosphoramidite or chloro-cyanoethoxy-N,N-diisopropyl phosphoramidite.
  • the library compounds are preferably hexoses with Ai being a residue of an ⁇ -amino acid or a peptide residue comprising residues of from two to ten ⁇ -amino acids. It is also preferred that r is 0, A 5 is 0 and L 3 is a carbamoyl group derived from an isocyanate. It is further preferred that q is 0 and A 2 is R 2 .
  • the present invention is also directed to a method for preparing the library of compounds described hereinabove.
  • the method comprises the steps of:
  • the protecting group is removed just prior to allowing the hydroxyl to react to form the substituent R3L3A5.
  • the leaving group G is any group which can easily be displaced by the free hydroxyl group. Suitable leaving groups include, but are not limited to halo, arylsulfonyloxy, alkylsulfonyloxy, alkanoyloxy, aroyloxy, hydroxy, alkoxy and aryloxy.
  • R 3 M is an isocyanate, R3NCO which reacts with the free hydroxyl group to form a carbamate, L 3 0 in which W is 0 and Z is NH.
  • a 5 is NH or N-alkyl
  • the hydroxyl group of the scaffold molecule is activated by conversion into a leaving group (e.g., tosylate, triflate, mesylate or halide) or by means of an activating reagent (e.g., a Mitsonobu reagent, PPI13-CCI4) , followed by displacement of the activated hydroxyl by a nucleophilic primary or secondary amine.
  • a leaving group e.g., tosylate, triflate, mesylate or halide
  • an activating reagent e.g., a Mitsonobu reagent, PPI13-CCI4
  • the carboxyl group of the monosaccharide reacts with a free or polymer-bound group having a terminal amino group. More preferably, the carboxyl group reacts with a terminal amino group of an amino acid or a peptide to form the amide linkage COAi. It is most preferable that the amino acid or peptide is bound to a polymer support through its terminal carboxyl group, and reacts with the carboxyl group on the scaffold molecule through its terminal amino group to form the amide linkage COAi .
  • the scaffold may be linked to a polymeric support through reaction of the amino group on the scaffold with a reactive group on the support, e.g., a carboxylic acid, carboxylic acid anhydride, isocyanate, aldehyde or sulfonyl halide.
  • a reactive group on the support e.g., a carboxylic acid, carboxylic acid anhydride, isocyanate, aldehyde or sulfonyl halide.
  • the azido group of the monosaccharide is reduced to an amino group and then allowed to react with a carboxylic acid to form an amide linkage AN in which A 2 is R 2 , which is alkanoyl, aroyl, aralkanoyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl.
  • the azido group is not reduced to an amino group in step (ii) , but is instead allowed to enter into a direct reaction with a suitable reactive compound.
  • the azido group may enter into a cycloaddition reaction with a suitable dienophile to produce a heterocyclic substituent A2A3N.
  • An azido group can react with an enolate to produce a triazole, as described in Colotta et al . , J. Med. Chem., 1990, 33, 2646; and Smalley et al . , Synthesis, 1990, 8, 654.
  • Azido groups also react with acetylenes to produce triazoles, as described in Radchenko et al . , Zh. Org. Khi . , 1991, 27, 1463; and Menyhart et al . , J. Carbohydr . Chem., 1990, 9, 253.
  • Another example of direct reactions of azido groups is the reaction of the azido group as a nucleophile in a Michael addition (Matsuda et al . , J. Med. Chem. 1991, 34, 999) or in reaction with an isocyanate (Delacotte et al . , J. Chem. Res., Synop . , 1991, 3, 64) or a chiral dichloroborane (Brown et al . , J. Org. Chem., 1991, 56, 1170).
  • step (i) is performed first, followed by steps (ii) and (iii) in that order.
  • the scaffold is attached to a polymeric support through a group on the support which has a terminal amino group and that the polymeric support is a solid support, i.e., one which is insoluble in the solvents used in the method.
  • Other polymeric supports include polymers which have a composition and molecular weight that renders them soluble in some solvents, allowing a reaction to be performed in solution, with subsequent precipitation of the bound product.
  • polymeric supports is the commercially available polyethylene glycols.
  • Suitable solid supports for use in the present invention include most synthetic polymer resins, preferably in the form of sheets, beads, or resins, such as polystyrene, polyolefins, polymethyl methacrylates , and the like, derivatives thereof and copolymers thereof. Polymers having varying degrees of crosslinking are also useful.
  • a preferred solid support is a Merrifield resin, which is a 1% divinylbenzene copolymer of polystyrene or TentagelTM, which is a polyethylene glycol-grafted polystyrene resin available from Novabiochem (La Jolla, CA) .
  • suitable polymer supports are insoluble in most organic solvents but swellable in some.
  • solid supports may be comprised of glass, ceramic, or metallic substances and their surfaces. It is important that any solid support contain functional groups that can participate in the instant reactions, so that the molecular residues of choice may be bound or attached to the surface of the solid support. Such functional groups will generally involve halides, unsaturated groups, carboxylic acids, hydroxyls, amines, esters, thiols, siloxy, aza, oxo and the like.
  • linker groups may be used.
  • Such linkers are well known in the art and may include, but are not limited to, polyamino, polycarboxylic , polyester, polyhalo, polyhydroxy, polyunsaturated groups, or combinations thereof.
  • the linker is preferably labile under a given set of conditions that do not adversely affect the compounds attached to the library or the reagents used in their preparation or manipulation. More preferably, the linker is acid labile or is photolabile.
  • Desirable linkers include a halotrityl moiety, a Rink amine linked polystyrene (Novabiochem) linking the scaffold molecule to the solid support, or an alpha-halo, alpha-methylphenacyl moiety.
  • the linkers may be used to covalently bind the scaffold molecules to the solid support.
  • covalent attachment may be through amine, ether, thioether, ester, thioester, amide, acetamide, phosphate, phosphonate, phosphinate, sulfonate or sulfate bonds.
  • Customized resin linkers e.g., those supporting an amino acid, can be obtained from Novabiochem.
  • the library compounds may be prepared using a "safety-catch" linker.
  • This type of linker is used to bond the scaffold to the resin, but unlike conventional linkers that are removed by hydrolysis, the linker is removed by displacement with a nucleophile. The nucleophile becomes part of one of the three functional groups on the library compound, allowing greater diversity of functional groups in the library.
  • Use and preparation of safety-catch linkers are described in Backes and Ellman, J. Am. Chem. Soc . , 1994, Vol. 116, p. 11171; and Backes et al., J. Am. Chem. Soc, 1996, Vol. 118, p. 3055.
  • the linker when the scaffold is attached via the carboxylic acid to an amino acid or peptide bound to a solid support via a safety-catch linker, the linker may be displaced with an amine or thiol compound to further functionalize the amino acid or peptide substituent, thereby producing the final Ai substituent.
  • a procedure for library preparation using a resin bearing a safety-catch linker is given in Example 14.
  • the linker when the scaffold is attached directly to the support via the carboxylic acid and the linker, the linker may be displaced with an amine or thiol compound which becomes the Ai substituent.
  • the compounds can be prepared using a mix and split strategy with directed sorting using the IRORI AccuTag®-100 radiofrequency tagged solid phase synthesis system or in an arrayed parallel synthesis using automated robotic methods. For instance, a TecanTM
  • the library compounds are typically used without purification with the product of a given preparation being characterized by standard techniques such as liquid chromatography and mass spectrometry. Quantitative analysis of the products is conveniently performed by preparing daughter multi-well plates from a mother plate, with one of the daughter plates being dedicated to the analytical studies. A suitable threshold for the screening studies is >85% purity of the scaffold product.
  • Various purification techniques can be employed, however, if so desired in order to increase the level of sample purity. These purification techniques include flash chromatography, high-performance liquid chromatography (HPLC) , solution phase "covalent scavenger” strategies, polymer-supported quenching, and resin capture, to name a few.
  • Compounds in the library of this invention can be screened for biological activity using routine methods well known to those skilled in the art, and described hereinafter in Example 15.
  • the compounds can be screened for anti-infective activity against viral, bacterial, or fungal agents.
  • Representative targets include strains of Staphylococcus and Streptococcus bacteria.
  • the activities of the compounds can be screened by contacting each compound with the biological target under conditions generally found to promote growth of the target. Observations are then made over a several hour or day period to determine whether proliferation of the target has been inhibited. Signs of inhibition are indicative of the compound having a positive activity against the target. For example, an observation that the growth rate of a microbe has ceased or diminished is an indication that the compound has anti-microbial activity. Screening may also be performed by directly assaying for peptidoglycan synthesis in the microbes.
  • a library of 1920 compounds was prepared from scaffold molecule (XI) .
  • the free hydroxyl group was allowed to react with an isocyanate RiNCO in which Ri is cyclohexyl, 3- ( trifluoromethyl ) phenyl , 4- (trifluoromethyloxy) phenyl, 3 , 5-bis (trifluoromethyl) phenyl , 2,4- difluorophenyl, and 3- (phenyloxy) phenyl .
  • an arylthio group i.e., X is S and R 5 is aryl
  • a suitable reagent for accomplishing this transformation is a mercury (II) salt.
  • Library compounds are preferably prepared on a solid support and then removed from the solid support by cleaving their covalent attachments thereto .
  • 6-Tetra-0-acetyl-3-azido-3-deoxy-D-glucopyranose (2) is prepared from 1 , 2-5 , 6-di-O-isopropylidene- ⁇ -D-allofuranose (1) following the procedure disclosed in copending application Serial No. 08/975,229, filed November 21, 1997.
  • pTSA p-toluenesulfonic acid
  • anisaldehyde dimethyl acetal 50mL
  • Methyl 2-0-benzoyl-4 6-0-benzylidene-3-0-methoxycarbonylmethyl- ⁇ -D- glucopyranoside (30) and Methyl 3-0-benzoyl-4 , 6-0-benzylidene-2-0- methoxycarbonylmethyl- ⁇ -D-Glucopyranoside (31)
  • NBS N-bromosuccinimide
  • reaction mixture is diluted with ethyl acetate and washed with water, aqueous HCl solution, dried over Na 2 S0 4 and concentrated in vacuo. It is purified on a silica gel column by using a solvent gradient consisting of hexane-ethyl acetate (9:1- ⁇ 2:3) to give (33) (13.5g,84%); ⁇ NMR (CDCl 3 ) : 8 8.04-7.44 (m,5H,ArH), 5.45 (t,lH,H-3), 5.10 (d,lH,H-l), 4.30-4.15 (m,2H,0CH 2 ), 3.73-3.68 (dd,lH,H-2), 3.62 & 3.47 (each s,6H,2xOMe) ; 13 C NMR: ⁇ 98.18 (C-l), 78.31(0-2), 76.30(0 5), 70.79(03), 70.58(OCH 2 ), 68.55(04
  • a mixture of (53) (2.4g, 12mmol ) and Bu 2 Sn0 (3.9g, 15.7mmol) in toluene (60ml) is heated for four hours at reflux temperature with azeotropic distillation of water.
  • the solution is concentrated to 40ml, then Bu 4 NI (2.6g, 7mmol ) and BrCH 2 C00Me (5.7ml, 60mmol) are added to the reaction mixture. It is heated under reflux for four hours and then evaporated to dryness to give a crude mixture of lactone and alkylated products.
  • This crude mixture is passed through small silica gel column using hexane-ethyl acetate (4:l->2:3) as the eluent.
  • reaction mixture is allowed to warm to room temperature over two hours, then diluted with ethyl acetate (250mL) and washed with ice-cold brine (800mL) .
  • the aqueous layer is extracted once with a further portion of ethyl acetate (250mL) , then the combined organic layers are washed with 3N citric acid (2 x 500mL) and brine (lOOmL) , then dried over Na 2 S0 4 and evaporated to give compound (56) as a yellow brown oil (49.3g;
  • reaction mixture is stirred overnight at room temperature then diluted with water (600mL) . After stirring for about 20 minutes, the reaction mixture is extracted with ethyl acetate (2 x 500mL) . The combined organic phases are washed with saturated sodium bicarbonate solution (2 x 400mL) , 2N HCl (2 x 300mL) and brine (200mL) , then dried over Na 2 S0 4 and evaporated to give compound (59) as a brown oil (62.5g; 166mmol); Rf (50% ethyl acetate-hexane): 0.45.
  • reaction mixture is diluted with dichloromethane (300mL) and washed with water (300mL) , saturated sodium bicarbonate solution (3 x 600mL) , 2N hydrochloric acid (2 x 500mL) , water (600mL) and brine (2 x 500mL) , then dried over Na 2 S0 4 and evaporated to a yellow solid (72 g) .
  • the crude product containing both the alpha and beta isomers is recrystallized from ethyl acetate/hexane to give compound (60)
  • the mixture is treated with acetic anhydride (17mL; 165mmol) , pyridine (80mL) and 4-dimethylamino pyridine (70mg) and the reaction mixture is stirred overnight at room temperature. TLC in 10% ethyl acetate-hexane showed no starting material.
  • the reaction mixture is poured into water (500mL) and extracted with ethyl acetate (200mL) .
  • the organic phase is washed with water (2 x 500mL) , saturated sodium bicarbonate solution (2 x 500mL) , 3N citric acid solution (2 x 500mL) then water (2 x 500mL) and dried over sodium sulfate.
  • Example 12 Methyl 6-azido-4-0-carboxymethyl-6-deoxy-3-0-methyl- ⁇ -D- glucopyranoside (XII) Methyl 2-0-benzoyl-4 , 6-0-benzylidene-3-0-methyl- ⁇ -D-glucopyranoside (67)
  • Methyl 2-0-benzoyl-4 6-0-benzylidene-3-deoxy- ⁇ -D-glucopyranoside (75).
  • a suspension of methyl 2-0-benzoyl-4 , 6-0-benzylidene-a-D- glucopyranoside [Kim, et.al., J. Org. Chem., 50, 1751, (1985)] (1.9g) in toluene (30ml) is added thiocarbonyldiimidazole (1.8g). The reaction mixture is heated under reflux for about two hours .
  • the solvent is then evaporated and the residue is dissolved in ethyl acetate and washed with cold aqueous 10% HCl, aqueous NaHC0 3 solution, saline, dried over Na2 ⁇ 0 4 , and concentrated under vacuum.
  • the residue is taken up in toluene (30ml) and AIBN (0.2g) and Bu 3 SnH (2ml) are added.
  • the reaction mixture is heated under reflux for about one hour and the solvent is removed under vacuum.
  • the residue is dissolved in acetonitrile and washed with hexane.
  • the mixture is cooled in an ice / water bath and ethanol is added to consume any unreacted acid chloride.
  • the solvent is removed under vacuum and the residue is taken up in ethyl acetate and washed with water, cold aqueous NaHC0 3 solution, dried over Na 2 S0 4 , and concentrated under vacuum.
  • Example 18 Methyl 3-azido-2-0-carboxymethyl-6-fluoro-3 , 6-dideoxy- ⁇ - D-glucopyranoside (XVIII).
  • Methyl 2-0-benzoyl-3 4-0-isopropylidene 6-O-p-toluenesulf onyl- ⁇ -D- galactopyranoside (88) .
  • the libraries outlined within were synthesized using Irori microcanisters (available from Irori Quantum Microchemistry (LaJolla, CA) with radio frequency tagging and an Accutag 100 software system.
  • Irori microcanisters available from Irori Quantum Microchemistry (LaJolla, CA) with radio frequency tagging and an Accutag 100 software system.
  • This approach allows for the synthesis of large numbers of compounds through the use of directed sorting.
  • the canisters are sorted into the proper flasks for the upcoming reaction. After these steps, and during all intervening washes, the cans are handled in a single container. In most cases, approximately 1 mL of solvent is used for each canister present in a reaction flask. Agitation of large numbers of cans (>500) is performed through mechanical stirring at the low speed. This approach is used for all washes and for the non-combinatorial reactions.
  • Proper loading of the second amino acid is quantified by photometric FMOC analysis and complete removal of the FMOC group is accomplished by treating the canisters with 20% piperidine in DMF for about 30 minutes. Subsequently, the canisters are rinsed four times with DMF.
  • the sugar is coupled to the dipeptide by treating the canisters with the appropriate scaffold carboxylic acid in the presence of equimolar HATU and DIPEA.
  • the canisters are stirred overnight and proper coupling is ensured by LC-MS analysis of the glycopeptide .
  • the canisters are washed with DMF and THF prior to the addition of trimethylphosphine for reduction of the azido group.
  • the reaction is allowed to proceed for at least six hours, at which time the absence of the azido group is checked by diffuse reflectance IR.
  • the canisters After washing with THF and then DMF, the canisters are sorted and treated with a coupling solution of the carboxylic acid, HATU and DIPEA. The canisters are rinsed after overnight shaking by washing with DMF and THF. Complete amide formation is ensured by LC-MS analysis of the products.
  • the canisters are sorted and then treated with the appropriate isocyanate in the presence of triethylamine .
  • the canisters are then washed with THF and then DMF. All of the canisters are treated with piperidine for about 30 minutes to remove any byproducts formed due to incorporation of more than one isocyanate molecule per scaffold molecule. After this treatment the cans are washed again with DMF and then THF. Complete formation of the mono-carbamate is checked by LC-MS.
  • the cans After washing the canisters four times with DMF and two times with DCM, the cans are sorted and then treated with 0.3 M of the appropriate isothiocyanate in tetrahydrofuran (THF) .
  • THF tetrahydrofuran
  • the canisters are shaken for about 30 minutes, 0.3M triphenyl phospine is then added.
  • the canisters are agitated overnight at room temperature to form the carbodiimides .
  • the canisters are then washed four times with anhydrous THF, twice with DCM and sorted. Each batch of cans is treated with the appropriate primary or secondary amine and agitated overnight at room temperature. After this treatment, the cans are washed again with THF and then with DCM. Complete formation of the guanidines is then checked by LC-MS.
  • Treatment of the protected scaffolds with lithium hydroxide at this stage provides the free alcohol at the four position of the sugar. This is performed by stirring the canisters in the presence of 0.05M lithium hydroxide in 1:1 THF/methanol for four hours.
  • thiophenyl group is present at the anomeric position, it is quickly removed using an 8mg/mL solution of mercury (II) trifluoroacetate in THF with 1% v/v water added. The reaction is conducted at 60°C with mechanical stirring for 15 minutes and complete lactol formation is checked by LC-MS.
  • the cans are scanned on the Irori scanning station and placed in a 5L round bottom flask. After washing the cans twice with 1.5L N, N- dimethylformamide (DMF), they are treated with 1.6L 20% piperidine for about 30 minutes. The cans are then washed four times with 1.5L DMF.
  • N, N- dimethylformamide DMF
  • the cans are sorted into five 2L flasks (384 cans/flask) and each batch is then reacted with the appropriate FMOOAmino acid, 0- (7-azabenzotriazol-l-yl) -1 , 1 , 3 , 3- tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamme (DIPEA) in 400mL of DMF (see Table 2 for exact reagent amounts and concentrations) .
  • the cans are then shaken at 110 rpm for about 16 hours.
  • Each set of 384 cans is washed twice with 400mL of DMF.
  • the cans are then combined and washed four times with 1.5L of DMF before being treated with 1.5L of 20% piperidine in DMF for about 30 minutes.
  • XI 0.011M phenyl 3-azido-3-deoxy-4-0-Me-l- ⁇ '- ⁇ -D- glucopyranosiduronic acid
  • the cans After washing four times with 1.7L of DMF and twice with 1.7L of THF, the cans are swelled in a combination of 722.5mL of distilled THF, 765mL of ethanol and 170mL of water. To this cocktail is added 42.5mL of 1.0M trimethylphosphine in THF (0.025M). The mixture is then stirred overnight at room temperature. After washing the cans six times with 1.7L of THF, the cans are washed twice with 1.7L of DMF and then sorted into eight 2L flasks (240 cans per flask) in preparation for the acylation reactions.
  • reaction mixtures are then shaken at 120rpm for 18 hours at room temperature. Each flask is washed twice with 250mL of DMF. The cans are then combined and washed four times with 1.7L of DMF and twice with 1.7L of THF.
  • the cans are sorted into six 2L flasks (320 cans/ flask) . Each flask is evacuated, purged with argon twice, and then washed twice with 400mL of THF. Flask 6 is then washed with an additional 2 x 400mL of DMF. THF (300mL) is added to flasks 1,2,3,4 and 5, while 300mL of DMF is added to flask 6. The appropriate volume of isocyanate and triethylamine are added to flasks 1-5 while the appropriate volume of isocyanate and mass of copper chloride are added to flask 6 (see Table 4) .
  • reaction flasks are then shaken overnight at 110 rpm. Each reaction flask is washed twice with 400mL of THF. All the cans are then combined and washed four times with 1.7L of THF. The cans are then treated with 1.7L of 20% piperidine in DMF for about 30 minutes before being washed six times with 1.7L of THF. The cans are manually sorted and half of them are treated with .019M mercury (II) trifluoroacetate (8g) in 1.0L THF with 1% water added (lOmL). The reaction mixture is stirred mechanically for 15 minutes at 60°C. The cans are then washed six times with 1.7L of THF and four times with 1.7L of DCM.
  • II .019M mercury trifluoroacetate
  • the cans are archived and individually cleaved in Irori Accucleave cleavage stations by adding 3mL of 20% trifluoroacetic acid in DCM to each tube. After shaking the stations at 250rpm for 90 minutes, the products are transferred to 48-well microtiter plates by vacuum filtration.
  • Radio frequency tags are inserted into microkans in twenty 96-well microtiter plates.
  • 30g of rink amide resin is swelled in lOOOmL 5:2 dichloromethane (DCM) / tetrahydrofuran (THF) and a 500 ⁇ L slurry was dispensed into each canister (15mg/can) using the TECAN mini-prep- dispensing robot.
  • the solvent is drained and the canisters are capped.
  • the cans are washed four times with 1.7L low amine DMF and then washed twice with DCM and dried.
  • the cans are scanned and sorted into six bins using the Irori Aufco ⁇ OrtTM-10K.
  • Each batch of 320 cans is transferred into a 2L reaction vessel and coupled with the first amino acid using the appropriate Fmoc-amino acid, 0- (7- azabenzotriazole-1-yl ) -1 , 1 , 3 , 3 , -tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in 400mL low amine DMF (see table one for the exact reagent amounts and concentrations) .
  • Fmoc-amino acid 0- (7- azabenzotriazole-1-yl ) -1 , 1 , 3 , 3
  • DIPEA diisopropylethylamine
  • the cans are shaken on an orbital shaker at 110-120 rpm overnight at room temperature. Each batch of cans is washed four times with DMF and then the complete coupling of the first amino acid is analyzed by two methods. The first tests for free amine using Kaiser test, where the resin beads turn blue if positive or remain brown if negative. The second test of the amino acid coupling quantifies Fmoc loading photometrically on the resin 1 .
  • the cans are washed four times with DMF, then analyzed for complete coupling of the second amino acid, followed by treatment with 20% piperidine in DMF for 30 minutes. After washing the cans with DMF and then washing with DCM, the cans are dried and sorted and then
  • Table 3 Reagents for coupling of the third amino acid (.025M Fmoc- Amino acid, .025M HATU and .025M DIPEA) in 450ml of DMF).
  • cans in vessel 1 are treated with 400mL acetic anhydride: pyridine: THF (1:1:2).
  • All the canisters are combined and washed four times with 1.8L THF and then added into a solution of 722.5mL distilled THF, 765mL ethanol and 170mL water. To this mixture is added 42.5mL of 0. IM trimethylphosphine in THF (.025M). The mixtures was agitated overnight at room temperature and then washed six times with 1.8mL THF. Following this, the cans are washed twice with DCM and dried. Some resin is removed from two cans and checked for the absence of azide by diffuse reflectance IR.
  • All the cans are sorted into 4 bins for the urea formation combinatorial step.
  • Each set of 480 cans is treated with 0.4M of the appropriate isocyanate, 0.05M triethylamine in 450mL of anhydrous THF (see table 5 for exact amounts) .
  • All the canisters are left shaking overnight and then the canisters in each batch are washed four times with 500mL THF. Resin is removed from a single canister from each batch and the cleaved products are analyzed for complete formation of the ureas by LC-MS.
  • the canisters are combined, archived and individually cleaved in the Irori Accucleave 96-well cleavage stations by addition of 2mL of 20 TFA, 2% triethylsilane in DCM into each well. After shaking the cleavage stations for about 90 minutes on the orbital shaker at 250 rpm, the products were drained into 48-well microtiter plates by vacuum filtration. A second rinse is performed by adding lmL of the acidic cleave cocktail into each well, shake the cleavage station for 20-30 minutes and drain the wells as described before.
  • the cans are treated with a .02M solution of the required sugar scaffold in DMF, in the presence of .02M O- (7-azabenzotriazol-l-yl) -1 , 1 , 3 , 3- tetramethyluronium hexafluorophosphate (HATU) and .02M DIPEA. Shaking is conducted for about 12 hours to ensure completion of this reaction step.
  • the cans are then washed four times with NMP and four times with THF before sorting the cans into IRORI cleavage stations for final cleavage.
  • This final step is generally conducted by adding 2.5ml of a .15M solution of the nucleophile (amine, ammonia, hydroxide or thiol) in THF to each can separately.
  • Volatile nucleophiles are generally used so that purification is possible through simple evaporation of the solvent and the nucleophile to leave the product in a microtiter plate.
  • the evaporation process is conducted in a Savant Speedvac® as described for the other procedures .
  • Bacteria All organisms are grown in a universal rich media to minimize media effects on the inhibition assay. All bacteria are demonstrated to grow in Brain Heart Infusion (BHI) media (Difco, Detroit, MI) supplemented with 0.1% Casamino Acids (CAA) (Difco).
  • BHI Brain Heart Infusion
  • CAA Casamino Acids
  • the following organisms are used in primary screening: Enterococcus faecium (ATCC 49624) Enterococcus faecalis (ATCC 29212) Staphylococcus aureu ⁇ (ATCC 29213) Staphylococcus epidermidis (ATCC 12228) Streptococcus pneumonia (ATCC 49150) Escherichia coli (ATCC 25922) Acinetobacter ani tra tus (ATCC 43498)
  • the cells are diluted 100 fold in BHI/CAA containing 0.7% agar maintained at 50°C to a cell density of approximately 5 X 10 colony forming units (CFU) per mL.
  • the agar slurry is poured into an 86 mm X 128 mm assay plate (Nunc) , which has the dimensions of a 96-well plate, and allowed to solidify for at least 30 minutes.
  • Streptococcus strains are diluted in BHI/CAA media without agar and 200 ⁇ l aliquoted to each well of a 96-well assay plate .
  • test Compounds are solubilized in sterile 20% DMSO/water to a concentration of approximately l-5mg/ml, aseptically aliquoted among several sterile "daughter” plates and frozen at - 20°C. Daughter plates are thawed at room temperature or 37°C just prior to assay.
  • a sterilized 96-well replicating device (Boekel) is inserted into the daughter plate and used to deliver the test compound to either a 96-well plate containing Streptococcus, or an agar plate imbedded with bacteria.
  • the replicator pierces the agar and is removed vertically to prevent damage to the agar surface.
  • the appearance of zones of inhibition is monitored after 15 to 24 hr. growth at 37°C.
  • Streptococcus inhibition is monitored by no observable turbidity in the wells of the 96-well plate after 24-48 hr . growth .
  • a control plate containing dilutions of antibiotic standards is run at the time of each assay with each organism.
  • the control antibiotics are Ampicillin, Vancomycin and Moenomycin.
  • Control samples are aliquoted in duplicate in a 96-well array. Each antibiotic is tested at eight serial two fold dilutions.
  • Antibiotic concentrations vary from lOmg/ml to O.OOlmg/ml.
  • MIC Assay Putative actives in the Lawn assay are further screened to determine the minimum inhibitory concentrations (MIC) of each compound for each organism affected. Test compounds are serially diluted in 20% DMSO/water and added to 96-well plates in a volume of 5 ⁇ l . Each bacterium, grown as described above and diluted in broth without agar, is added to the diluted compound in a volume of 200 ⁇ l . The range of concentrations used for each compound in the MIC assay is based on the potency implied by the size of the zone of inhibition in the lawn assay. Each compound is tested at five serial dilutions, ranging anywhere from 1:40 up to the maximum dilution necessary to alleviate the antimicrobial effect. The effect of the test compound on bacterial growth is measured after 18 hrs of growth at 37°C by determining the turbidity of the medium at 600 nm or by visual inspection. The MIC is defined as the lowest concentration of compound necessary to completely inhibit bacterial growth.
  • the peptidoglycan polymerization assay is adapted from that described by Mirelman, et al .
  • E. coli . (ATCC #23226) are permeabilized with ether according to Mirelman, et al.(1976), and
  • Polymerization assays are conducted in 96-well filter-bottom plates (Millipore GF/C - cat. # MAFC NOB 10).
  • a Tecan Genesis 150 robot is programmed for all subsequent liquid handling steps.
  • each well contains: 50 mM Tris - HCl (pH 8.3); 50 mM NH 4 C1 ; 20 mM MgS0 4 .7 H 2 0; 10 mM ATP (disodium salt); 0.5 mM ⁇ -mercaptoethanol; 0.15 mM D-aspartic acid; O.OOlmM UDP-N-acetyl [ 14 C-] -D-glucosamine (DuPont/N.E.N.
  • Novel test compounds are solubilized in 10% DMSO/water and screened at a final assay concentration of 10 ⁇ g/ml. With the exception of radiolabeled and isolated native pentapeptide, all remaining biochemicals are purchased from Sigma Chemical or Fisher Scientific.
  • Assay buffer (10 ⁇ L) , ATP (20 ⁇ L) , UDP pentapeptide (10 ⁇ L) and 14 C- UDP-GlcNAc (20 ⁇ L) are added to all wells, followed by either test compound, reference standard or buffer vehicle (20 ⁇ L) .
  • the reactions are then started by adding 20 ⁇ L aliquots of bacterial protein prepared in assay buffer into each well. Plates are covered, mixed for 30 sec., then incubated at 37°C for about 120 minutes. Ice cold 20% TCA (100 ⁇ L) is added to each well, the plates are gently mixed (60 sec), then refrigerated (4°C) for about 30 minutes to assure precipitation of all peptidoglycan.
  • the plates are placed under vacuum filtration on a Millipore manifold, filtered, and washed 3-4 times with 200 ⁇ L/well of 10% TCA. Optiphase scintillation cocktail (30 ⁇ L/well) is added, then the plates are incubated overnight prior to counting in a Wallac Microbeta. Percent inhibition of incorporation of 14 C-label into peptidoglycan is computed from control (total incorporation) and background (blank) wells containing 300 ⁇ g/ml of vancomycin or 100 ⁇ g/ml of the library compound, which completely inhibit incorporation of radiolabel . All wells are arrayed in duplicates, which usually vary by ⁇ 20%. Concentration-response curves for reference standards are arrayed on each plate as positive controls.
  • Radio frequency tars are inserted into microkans in 11 microtiter plates.
  • Into each microkan is dispensed 15mg of rink amide resin as a slurry in DCM: THF (4:1) .
  • the solvent is drained and the canisters are capped.
  • the microkans are washed twice with 1 liter of DMF and then treated with 1 liter of 20% piperidine for about 30 minutes.
  • the microkans are then washed with 1 liter of low-amine DMF.
  • the microkans are sorted into four flasks with the aid of the IRORI accutag rf tag reader.
  • the microkans in flasks 2-4 are treated with the appropriate amino acid, as shown in Table 1, along with HATU and DIPEA in 250 ml of DMF.
  • the microkans are shaken at 110 rpm on an orbital shaker overnight.
  • Each set of microkans was washed with 2 x 200 ml of DMF before combining all of the microkans and washing with 4 x 500 ml of DMF.
  • the microkans are then treated with 20% piperidine in DMF for about 30 minutes.
  • After washing with 5 x 1000 ml of DMF the microkans were treated with 0.0166M of (XII) (4.83g, 2.23 equiv) , 0.0166M HATU (6.3g) and 0.0166 DIPEA (2.89ml) in 1000ml DMF.
  • the mixture is shaken overnight at room temperature. After shaking overnight the microkans were washed with 4 x 1000ml of DMF and 4 x 1000ml of anhydrous THF.
  • the microkans were then dried under air for four hours .
  • the microkans are sorted into five containers. Flask one is set aside. To each flask is added 200ml of anhydrous DMF. The microkans are shaken for about 10 minutes. The solvent is removed and another 200ml of anhydrous DMF is added to each flask. The appropriate volume of isocyanate, along with 200mg of CuCl is then added to each flask (Table 2) .
  • microkans are sorted into five flasks for the next reaction sequence. Flasks 1-4 are treated with 0.5M of the appropriate isothiocyanate (Table 3) for 30 minutes and then the triphenylphosphine is added to each flask. The flasks are shaken for about four hours .
  • Flask five is treated with 20ml of . IM trimethylphosphine in 9:9:2 THF/EtOH/H 2 0 for about five hours. The microkans in flask five are then washed with four portions of THF and two portions of DMF, and then 20ml of 0.5M 1 pyrazole carboxamide hydrochloride (1.6g), 0.5M DIPEA (1.74ml) in DMF are added. The flask is shaken overnight
  • the flasks are individually washed twice with anhydrous THF and then combined and washed four times with anhydrous THF. After drying the microkans are sorted into 12 flasks and the appropriate amine (Table 4) is added to each flask as a solution in THF.
  • Flasks 1-4 are washed once separately and then six times together with THF. Flask five is washed four times with DMF and twice with THF and then the microkans are combined with the rest. All of the microkans are then washed four times with THF and four times with DCM.
  • the products are cleaved off the resin in IRORI Accucleave stations using 20% TFA in DCM with 1% triethylsilane added. Each vessel receives 2.5ml of cleavage solution.
  • the products are isolated by draining the cleavage stations into 48 well microtiter plates and evaporating the plates to dryness in a centrifugal evaporator.
  • Hirschmann, R. et al . "NONPEPTIDYL PEPTIDOMIMETICS WITH A ⁇ -D- GLUCOSE SCAFFOLDING. A PARTIAL SOMATOSTATIN AGAINST BEARING A CLOSE STRUCTURAL RELATIONSHIP TO A POTENT, SELECTIVE SUBSTANCE P ANTAGONIST", JACS, Vol. 114, pp. 9217-9218, (1992).
  • Hirschmann, R. et al . "DE NOVO DESIGN AND SYNTHESIS OF SOMATOSTATIN NON-PEPTIDE PEPTIDOMIMETICS UTILIZING ⁇ -D-GLUCOSE AS A NOVEL SCAFFOLDING", JACS, Vol. 115, pp. 12550-12568, (1993).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)

Abstract

A compound of structure (I) wherein X is O or S; Z is O or NH; Y is COOH, COOR2, CH2OR3, CH3, or CH(s)Y2(3-s) where Y2 is F, Cl, Br or I, and s is 0, 1, or 2 or Y and one of ZR4 and OR5 are linked to form a 6-membered cyclic acetal; p is O or 1; m is 0 or 1; n is 1 or 2. A library of compounds of structure (II) wherein X is O or S; A1 is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino, R1O, R1S, R1, R1NH or R1N-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2SO2, R2NHCO, R2OP(O) (OR6), R2P(O) (OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is OR4, NHR4, CH2OR4 or CH3; A5 is O, NH or N-alkyl; p, q and r are independently 0 or 1; Y1 and Y2 are independently O or CH2; each of L1 and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH2, carbonyl, OP(O) (OR7), NHP(O) (OR7), P(O) (OR7) or (III) wherein W is O, NH, N-alkyl or S, and Z is NH, O or S; m is 0 or 1; and n is 1 or 2.

Description

CARBOHYDRATE-BASED SCAFFOLD COMPOUNDS, COMBINATORIAL LIBRARIES AND METHODS FOR THEIR CONSTRUCTION
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to the construction of carbohydrate- based "scaffold" molecules and combinatorial libraries. Such compounds and libraries can be used to screen for novel ligands capable of binding to therapeutically relevant biomolecular targets of interest.
Related Background Art
Primary screening libraries are useful for the identification of new classes of drugs when little is known about the kinds of ligands that bind to particular receptors on a biological target or when it is desired to identify new compounds that bind similarly to known pharmacophores. Since little structural information is typically available upon which to base design of a library, the probability of identifying an active compound from a primary screening library is related to the number of compounds that can be constructed and screened. Hence, the strategy for designing a primary screening library should permit the creation of a large amount of structural variation within a given molecular system, and should provide access to a large diversity of structures of interest.
A "scaffold" molecular system is an advantageous approach to the construction of primary screening libraries. In a scaffold-based library, a particular molecular system serves as a template upon which various chemical or biological appendages are attached to define the library. Moreover, the conformational rigidity and high degree of functionalization of carbohydrates suggests that these molecules may be ideal templates for the construction of primary screening libraries.
Previous approaches to the use of carbohydrates in the construction of a set of compounds are conveniently characterized as being directed towards the construction of oligosaccharide mimetics or monosaccharide peptidomimetics. An example of the first approach is a random glycosylation method for producing oligosaccharide, e.g., trisaccharide, libraries [Kanie, 0. et al . , (1995)]. A recent variation on the synthesis of oligosaccharides, which entails linking carbohydrate molecules via nucleotide or peptide bonds [Nicolaou, K. et al., (1995); Suhara, Y. et al . , (1996)], also promises to permit construction of oligosaccharide mimetics on a solid phase support [Mueller, C, et al . , (1995); PCT Publication 96/27379]. Libraries of C-disaccharides and C-trisaccharides have also been described [Armstrong, R. et al . (1994); Sutherlin, D. et al . (1996)].
An approach to the synthesis of compounds using a carbohydrate template utilizes a monosaccharide backbone as a scaffold upon which various desired ligands (functional groups) are attached. This approach is frequently used in the study of structures analogous to target peptides, commonly referred to as peptidomimetics [see, Hirschmann, R. , et al . , (1996); Hirschmann, R. et al . , (1992); Hirschmann, R. et al . (1993)]. An exemplary approach along this line, which entails coupling an allyl group to the anomeric carbon atom of a carbohydrate molecule and constructing a C-glycoside conjugate, is described in PCT Publication No. 96/36627.
A further refinement to this approach for constructing carbohydrate scaffolds involves the use of a "sugar amino acid" as a building block for the construction of so-called "peptidomimetics" [von Roedern, E., et al . , (1996)]. This latter approach involves coupling one or more amino acids to a carboxylic acid group provided on the carbohydrate ring. Amino acids are also attached to an amino group provided on the carbohydrate ring. This approach is limited to carbohydrate scaffolds bearing two functional groups which may be elaborated. A largely non-peptidyl approach in which the libraries comprise disaccharides , trisaccharides and glycoconjugates of amino acids is the subject of PCT Publication No. 95/03315.
A significant recent development in this area has been the application of the Ugi multi-component condensation (MCC) reaction [Ugi, I., (1982)] to the construction of C-glycoside glycopeptide libraries. Along these lines, a disaccharide C-glycoside derived from neomycin B has been used to produce a library of C-glycoside peptides [Park, W. et al . , (1996)]. The Ugi reaction has also been carried out on a solid phase amine resin to produce acyl amino amides upon cleavage [Sutherlin, D. et al . , (1996)]. The latter reaction employs a monosaccharide aldehyde in the formation of C-glycosylated amino acid analogs. However, the library compounds of this reference consist of sugars functionalized at only one position. Multi- component condensation strategies for the construction of combinatorial chemical libraries have been reviewed [Armstrong, R. , et al. , (1996) ] .
In the context of constructing a large number of diverse chemical structures based on a carbohydrate template, it is desired to display a rich set of functionalities from the rigid backbone of a monosaccharide unit. Such an approach should provide a large number of compounds that afford a highly variegated three-dimensional arrangement of chemical structures. Indeed, a carbohydrate-based approach to the construction of a large number of related compounds is expected to span a greater and unique diversity space than is available to other templates. For reasons of efficiency and speed in the construction of these compounds, it is also desired that the products can be synthesized combinatorially, at least in part by automated techniques. This concept is referred to herein as being directed to the construction of a carbohydrate-based universal pharmacophore mapping library.
SUMMARY OF THE INVENTION
The present invention is directed to a compound of structure
Figure imgf000005_0001
wherein X is 0 or S; Z is 0 or NH; Ri is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl ; Y is COOH, C00R2, CH2OR3, CH3, or CH(s)Y2<3-s) where Y2 is F, Cl, Br or I , and s is 0, 1, or 2 or Y and one of ZR4 and OR5 are linked to form a 6-membered cyclic acetal; R2 is alkyl, aryl or aralkyl; R3, R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1; m is 0 or 1; and n is 1 or 2; provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH, then R4 is not hydrogen; when n is 1, then m is 0; when p is 1, then Y is not CH2OR3 or CH3; when Y is not COOH, then exactly one of Ri, R , 3 and R5 is substituted by exactly one COOH; when Y is COOH, then none of Ri, R2, R3, R4 and R5 is substituted by COOH; when one of R3, R4 and R5 is hydrogen, then two of R3, R4 and R5 are not hydrogen and none of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent; when one of Ri, R2, R3, R and R5 bears a hydroxyl substituent, then four of Ri, R2, R3, R and R5 do not bear a hydroxyl substituent and none of R3, R and R5 is hydrogen; and when none of OR3, OR4 and OR5 is hydroxyl or a protected hydroxyl group, then one or more of Ri, R2, R3, and R5 bears a hydroxyl substituent or a protected hydroxyl substituent.
This invention is also directed to a library of compounds, each having the structure
Figure imgf000006_0001
wherein X is O or S; Ai is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino, RiO, RiS, Ri, RiNH or RiN-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2SO2, R2NHCO, R2OP (0) (0R6) , R2P(0)(OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is OR4, NHR4, CH2OR4 or CH3; A5 is 0, NH or N-alkyl; p, q and r are independently 0 or 1; Yi and Y2 are independently 0 or CH2; each of Li and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH2, carbonyl, 0P(0)(0R7), NHP(0)(0R7), P(0) (0R7) or
W ll_
wherein is 0, NH, N-alkyl or S, and Z is NH, 0 or S ; Ri, R2 and R3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl , heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl; R4, R5, δ and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl; m is 0 or 1; and n is 1 or 2; provided that: when n is 1, then m is 0; when A5 is NH or N-alkyl, then L3 is not NHP(O) (OR7) ; when L is a single bond, CH2 or carbonyl, r is 0 and A5 is 0, then R3 is not aryl having fewer than 8 carbon atoms or aralkyl having fewer than 8 carbon atoms or alkyl having fewer than 3 carbon atoms; and when L3 is carbonyl, r is 0 and A5 is NH, then R3 is not alkyl having fewer than 3 carbon atoms.
The invention is further directed to a method of making the library of compounds by the steps of: (a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and an azido group; (b) performing, in any order, steps of: (i) allowing the free carboxyl group of the monosaccharide to react to produce a substituent Ai; (ii) reducing the azido group to an amino group and allowing the amino group to react with a compound capable of reacting with the amino group to produce a substituent A2; (iii) allowing a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent Brief Description of the Drawings
Figure 1 illustrates the preparation of compounds bearing the Guanidinium grou .
Figure 2 illustrates a library of compounds comprising the Guanidinium group, labeled Library BI-1.
Figure 3 illustrates a library of compounds comprising the Guanidinium group, labeled Library BI-2.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "alkyl" refers to an acyclic or non-aromatic cyclic group having from one to twenty carbon atoms connected by single or multiple bonds. An alkyl group may be substituted by one or more of halo, hydroxyl, protected hydroxyl, amino, nitro, cyano , alkoxy, aryloxy, aralkyloxy, COOH, aroyloxy, alkylamino, dialkyla ino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl , aroyl, CONH2, CONH-alkyl or CON(alkyl) 2, COO-aralkyl, COO-aryl or COO- alkyl . The term "aryl" refers to a group derived from a non- heterocyclic aromatic compound having from six to twenty carbon atoms and from one to four rings which may be fused or connected by single bonds. An aryl group may be substituted by one or more of alkyl, aralkyl, heterocyclic, halo, hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, aroyl, COO-alkyl, COO-aralkyl, COO-aryl, C0NH2, CONH- alkyl or CON (alkyl) 2. The term "aralkyl" refers to an alkyl group substituted by an aryl group. The term "heterocyclic" refers to a group derived from a heterocyclic compound having from one to four rings, which may be fused or connected by single bonds; said compound having from three to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur or phosphorus. A heterocyclic group may be substituted by one or more of alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl , aroyl, COO-alkyl, COO- aralkyl, COO-aryl, CONH2, CONH-alkyl or CON (alkyl) 2 • The terms "alkoxy, " aryloxy" and "aralkyloxy" refer to groups derived from bonding an oxygen atom to an alkyl, aryl or aralkyl group, respectively. The terms "alkanoyl," "aroyl" and "aralkanoyl" refer to groups derived from bonding a carbonyl to an alkyl, aryl or aralkyl group, respectively. The term "protected hydroxyl" refers to a hydroxyl group bonded to a group which is easily removable under basic conditions, including, e.g., acetyl, benzoyl, levulinoyl, chloroacetyl, pivaloyl, p-nitrobenzoyl and tert-butyl-diphenylsilyl, to generate a free hydroxyl grou .
To investigate the potential of carbohydrates for the preparation of universal pharmacophore mapping libraries, three sites of diversification are desirable in each scaffold to provide the minimal requirements needed for pharmacophoric chiral molecular recognition. The desired three-point motif is achieved by a scaffold design that incorporates a carboxylic acid moiety, a free or protected hydroxyl group and an azido group. This functional group triad affords the chemoselectivity necessary for rapid combinatorial synthesis allowing the maximum amount of molecular diversity while minimizing the number of solid phase synthetic steps.
The monosaccharide scaffold compounds of the present invention have the structure
Figure imgf000009_0001
wherein X is O or S; Z is 0 or NH; Ri is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; Y is COOH, COOR2 , CH2OR3, CH3 or CHis) 2<3-s) where Y2 is F, Cl , Br or I , and s is 0, 1, or 2 or Y and one of ZR4 and OR5 are linked to form a 6-membered cyclic acetal; R is alkyl, aryl or aralkyl; R3, R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1; m is 0 or 1 ; and n is 1 or 2 ; provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH, then R is not hydrogen; when n is 1, then m is 0; when p is 1, then Y is not CH2OR3 or CH3; when Y is not COOH, then exactly one of Ri, R2, R3, R4 and R5 is substituted by exactly one COOH; when Y is COOH, then none of Ri, R2, R , R4 and R5 is substituted by COOH; when one of R3, R4 and R5 is hydrogen, then two of R3, R and R5 are not hydrogen and none of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent; when one of Ri, R2, R3, 4 and R5 bears a hydroxyl substituent, then four of Ri, R2, R3, R4 and R5 do not bear a hydroxyl substituent and none of R3, R and R5 is hydrogen; and when none of OR3, OR4 and OR5 is hydroxyl or a protected hydroxyl group, then one or more of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent or a protected hydroxyl substituent.
Accordingly, the monosaccharide scaffold compound is a five- or six- membered ring having one oxygen atom in the ring, and bearing one free or protected hydroxyl group, one free carboxylic acid group and one azido group. The azido group may be attached directly to a monosaccharide ring carbon, or may be attached through a CH2 substituent. The hydroxyl or protected hydroxyl and the carboxylic acid group may be attached to monosaccharide ring carbons or to any substituent attached to the ring. The scaffold compound may have both a free hydroxyl group and one or more protected hydroxyl groups, but has no more than one free hydroxyl group. It is preferred that the scaffold compound has a six-membered ring, i.e., n is 2. It is further preferred that m=n-l, i.e., that R50 is present when the scaffold compound has a six-membered ring.
Suitable protecting groups for the hydroxyl group are those which are easily removable under basic conditions to generate a free hydroxyl group, including, e.g., acetyl, benzoyl, levulinoyl, chloroacetyl, pivaloyl, p-nitrobenzoyl and tert-butyl-diphenylsilyl.
The most preferred scaffold compounds are shown below in Scheme 1, with details of their preparation given in the Examples.
Figure imgf000011_0001
(I) (II)
Figure imgf000011_0002
(III) (IV)
Figure imgf000011_0003
Figure imgf000012_0001
(XVI) (XVII)
Figure imgf000012_0002
(XLX)
(xvm)
Figure imgf000012_0003
(XX) A monosaccharide scaffold compound bearing a free hydroxyl, a carboxylic acid and an azido group is functionalized at the three reactive sites, i.e., the hydroxyl, carboxylic acid and azido group, to produce the library of compounds of this invention. Suitable scaffold compounds are those disclosed in this invention, as well as other monosaccharides bearing the three reactive sites described hereinabove. Examples of other suitable monosaccharide scaffolds are compounds disclosed in copending application Serial No. 08/975,229, filed November 21, 1997, including compound (XI), having the structure
Figure imgf000013_0001
(XI) whose preparation is described in detail in Example 11 .
Each compound in the library has the structure
Figure imgf000013_0002
wherein X is 0 or S; Ai is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino,
RiO, RiS, Ri, RxNH or RiN-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2S02, R2NHCO, R2OP (O) (OR6) , R2P(0)(OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is OR4, NHR4, CH2OR or CH3; As is O, NH or N-alkyl; p, q and r are independently 0 or 1; Yi and Y2 are independently O or CH2; each of Li and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH2, carbonyl, OP(0)(OR7), NHP(0)(OR7), P(0) (0R7) or
W ll_
wherein is 0, NH, N-alkyl or S, and Z is NH, 0 or S; Ri, R2 and R3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl; R4, R5, e and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl; m is 0 or 1; and n is 1 or 2; provided that: when n is 1, then m is 0; when A5 is NH or N-alkyl, then L3 is not NHP(O) (0R7) ; when L3 is a single bond, CH2 or carbonyl, r is 0 and A5 is O, then R3 is not aryl having fewer than 8 carbon atoms or aralkyl having fewer than 8 carbon atoms or alkyl having fewer than 3 carbon atoms; and when L3 is carbonyl, r is 0 and As is NH, then R3 is not alkyl having fewer than 3 carbon atoms .
Difunctional groups Y1L1 and L2Y2 in the library compounds, when present, may be derived from scaffold compounds in which the carboxylic acid or hydroxyl groups, respectively, are not attached directly to a monosaccharide ring carbon, but instead are substituted on a group Y.Li or L2Y2 attached to a monosaccharide ring carbon. The group Y1L1 may also be derived from reaction of a difunctional molecule, e.g., a dicarboxylic acid in which Yx is 0 and Li is difunctional alkanoyl, with the carbonyl carbon of the difunctional alkanoyl attached to Yi, and another carbon connected to the COAi group, with a hydroxyl group directly substituted on a monosaccharide ring atom.
The group R3L3A5 is derived from reaction of the hydroxyl group of the scaffold molecule. When the hydroxyl has reacted as a nucleophile to displace a leaving group, or to add to an unsaturated compound such as an isocyanate, A5 is oxygen. Alternatively, when the hydroxyl has been displaced by an amino group, then As is NH or N-alkyl.
Suitable amino acids are the natural or unnatural amino acids described above. The natural amino acids can be obtained commercially. Some unnatural amino acids can also be obtained commercially, however, it is frequently desired to prepare them, preferably in enantiomeric excess, from commercially available starting materials. A general approach to unnatural amino acids has been described recently [Petasis, N. et al . , JACS, 119:445 (1997)]. Natural, nonnatural, and modified amino acids can be linked through their C-terminii to an amine-substituted saccharide in the presence of a carbodiimide or acid anhydride. Alternatively, they can be linked through their N-terminii to a carboxyl-substituted saccharide in the same way.
An ester linkage to the monosaccharide scaffold molecule is formed straightforwardly by reacting a carboxylic acid group on the monosaccharide scaffold with an alcohol, e.g., one provided by a ligand molecule linked to a solid support. Alternatively, an ester linkage can be formed by reacting the free hydroxyl of the monosaccharide scaffold with a carboxylic acid ligand under standard esterification conditions. An ester linkage can also be formed by reacting the monosaccharide scaffold with an acid anhydride or acyl halide. The ester can also be prepared by an MCC reaction, such as the Passerini reaction, which entails reacting a carboxylic acid with an aldehyde and an isonitrile in a one-step synthesis. The carboxylic acid component is conveniently provided by the monosaccharide .
An amido linkage between the amino group on the monosaccharide scaffold and a compound bearing a carboxylic acid group, or between the carboxylic acid group on the monosaccharide scaffold and a compound bearing an amino group, is conveniently formed by reacting an amino group with a carboxylic acid group in the presence of dicyclohexylcarbodiimide (DCC) or an anhydride. A secondary amino linkage can be formed by reacting the amino group on the monosaccharide scaffold with an aldehyde or a ketone under reductive alkylation conditions. Preferred aldehydes are n- butyraldehyde and substituted alkyl compounds such as 3- methylthiopropionaldehyde; cycloaliphatic aldehydes such as cyclohexanecarboxaldehyde; aryl aldehydes such as benzaldehyde, 4- nitrobenzaldehyde and pyridine-2-carboxaldehyde; heteroatom- substituted cycloaliphatic aldehydes such as N-formylmorpholine; and alkarylaldehydes such as 2-phenylpropionaldehyde.
A sulfonamido linkage can be formed by reacting the amine with a sulfonyl halide, e.g., sulfonyl chloride. Particularly preferred are substituted and unsubstituted aryl sulfonyl chlorides, such as 1- naphthalenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, p- toluenesulfonyl chloride, and N-acetylsulfanilyl chloride.
A urea linkage can be formed by reacting the amine with an isocyanate, e.g., phenyl isocyanate. Particularly preferred are aromatic, halogenated aromatic, and halogenated aliphatic isocyanates .
A guanidine can be formed by reacting the sugar azides with isothiocyanates and triphenylphosphine, followed by the addition of a primary. or secondary amine, e.g., phenylisothiocyanate, triphenylphosphine, and methyl amine to give the phenyl, methyl guanidine attached to the sugar.
A number of alcoholic reactions are available for connecting a free hydroxyl of the monosaccharide with a desired organic moiety. For instance, an alkyl ether linkage is formed by alkylating a free hydroxyl group on the sugar molecule, e.g., by reacting the sugar with an alkyl halide in the presence of a base. An aryl ether can be formed by reacting a monosaccharide bearing a free hydroxyl group with the desired phenol under Mitsunobu reaction conditions [Mitsunobu, 0., et al . , JACS, 94:679 (1972)]. A carbamate linkage is formed by reacting a free hydroxyl group of the sugar with an isocyanate. Likewise, a urea linkage is formed by reacting a free amino group of the sugar with an isocyanate. A carbonate linkage can be formed by reacting an ester of a haloformate, e.g., a chloroformate ester, with the free hydroxyl of the monosaccharide in the presence of a base.
A phosphonate or phosphate linkage to the monosaccharide can be formed by reacting it with a phosphonic acid ester or phosphoramidite followed by oxidation to give the phosphonate or phosphate. [See, e.g., Campbell, D. , et al . , JACS, 117, 5381-5382 (1995); Hebert, N. , et al., Tett.Lett. , 35: 9509-9512 (1994); Beaucage, S., et al . , Tett.Lett. , 22: 1859-1862 (1981)]. Exemplary phosphoramidite reagents include chloro-methoxy-N,N-diisopropyl phosphoramidite or chloro-cyanoethoxy-N,N-diisopropyl phosphoramidite.
The library compounds are preferably hexoses with Ai being a residue of an α-amino acid or a peptide residue comprising residues of from two to ten α-amino acids. It is also preferred that r is 0, A5 is 0 and L3 is a carbamoyl group derived from an isocyanate. It is further preferred that q is 0 and A2 is R2.
The present invention is also directed to a method for preparing the library of compounds described hereinabove. The method comprises the steps of:
(a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and an azido group;
(b) performing, in any order, steps of: (i) allowing the free carboxyl group of the monosaccharide to react to produce a substituent Ai;
(ii) reducing the azido group to an amino group and allowing the amino group to react with a compound capable of reacting with the amino group to produce a substituent A ; (iii) allowing a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent R3L3A5.
When the hydroxyl group is protected, the protecting group is removed just prior to allowing the hydroxyl to react to form the substituent R3L3A5.
In one embodiment of the invention, A5 is 0 and the free hydroxyl group reacts with a compound R3M to form a substituent R3L3O; wherein M is N=C=0, N=C=N-alkyl, N=C=S, OP(O) (0R7)G, NHP(O) (OR7)G, P(O) (OR7)G, OCOG, SCOG, COG, G or CH2G, wherein G is a leaving group.
The leaving group G is any group which can easily be displaced by the free hydroxyl group. Suitable leaving groups include, but are not limited to halo, arylsulfonyloxy, alkylsulfonyloxy, alkanoyloxy, aroyloxy, hydroxy, alkoxy and aryloxy. Preferably R3M is an isocyanate, R3NCO which reacts with the free hydroxyl group to form a carbamate, L30 in which W is 0 and Z is NH.
In another embodiment of the invention, A5 is NH or N-alkyl, and the hydroxyl group of the scaffold molecule is activated by conversion into a leaving group (e.g., tosylate, triflate, mesylate or halide) or by means of an activating reagent (e.g., a Mitsonobu reagent, PPI13-CCI4) , followed by displacement of the activated hydroxyl by a nucleophilic primary or secondary amine.
Preferably, the carboxyl group of the monosaccharide reacts with a free or polymer-bound group having a terminal amino group. More preferably, the carboxyl group reacts with a terminal amino group of an amino acid or a peptide to form the amide linkage COAi. It is most preferable that the amino acid or peptide is bound to a polymer support through its terminal carboxyl group, and reacts with the carboxyl group on the scaffold molecule through its terminal amino group to form the amide linkage COAi . Alternatively, the scaffold may be linked to a polymeric support through reaction of the amino group on the scaffold with a reactive group on the support, e.g., a carboxylic acid, carboxylic acid anhydride, isocyanate, aldehyde or sulfonyl halide.
Preferably, the azido group of the monosaccharide is reduced to an amino group and then allowed to react with a carboxylic acid to form an amide linkage AN in which A2 is R2, which is alkanoyl, aroyl, aralkanoyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl.
In a variation of the method for preparing the library of compounds, the azido group is not reduced to an amino group in step (ii) , but is instead allowed to enter into a direct reaction with a suitable reactive compound. For example, the azido group may enter into a cycloaddition reaction with a suitable dienophile to produce a heterocyclic substituent A2A3N. An azido group can react with an enolate to produce a triazole, as described in Colotta et al . , J. Med. Chem., 1990, 33, 2646; and Smalley et al . , Synthesis, 1990, 8, 654. Azido groups also react with acetylenes to produce triazoles, as described in Radchenko et al . , Zh. Org. Khi . , 1991, 27, 1463; and Menyhart et al . , J. Carbohydr . Chem., 1990, 9, 253. Another example of direct reactions of azido groups is the reaction of the azido group as a nucleophile in a Michael addition (Matsuda et al . , J. Med. Chem. 1991, 34, 999) or in reaction with an isocyanate (Delacotte et al . , J. Chem. Res., Synop . , 1991, 3, 64) or a chiral dichloroborane (Brown et al . , J. Org. Chem., 1991, 56, 1170).
In a preferred embodiment of the invention, step (i) is performed first, followed by steps (ii) and (iii) in that order. It is also preferred that the scaffold is attached to a polymeric support through a group on the support which has a terminal amino group and that the polymeric support is a solid support, i.e., one which is insoluble in the solvents used in the method. Other polymeric supports include polymers which have a composition and molecular weight that renders them soluble in some solvents, allowing a reaction to be performed in solution, with subsequent precipitation of the bound product. One example of such polymeric supports is the commercially available polyethylene glycols. Suitable solid supports for use in the present invention include most synthetic polymer resins, preferably in the form of sheets, beads, or resins, such as polystyrene, polyolefins, polymethyl methacrylates , and the like, derivatives thereof and copolymers thereof. Polymers having varying degrees of crosslinking are also useful. A preferred solid support is a Merrifield resin, which is a 1% divinylbenzene copolymer of polystyrene or Tentagel™, which is a polyethylene glycol-grafted polystyrene resin available from Novabiochem (La Jolla, CA) . Generally, suitable polymer supports are insoluble in most organic solvents but swellable in some. Still other solid supports may be comprised of glass, ceramic, or metallic substances and their surfaces. It is important that any solid support contain functional groups that can participate in the instant reactions, so that the molecular residues of choice may be bound or attached to the surface of the solid support. Such functional groups will generally involve halides, unsaturated groups, carboxylic acids, hydroxyls, amines, esters, thiols, siloxy, aza, oxo and the like.
To facilitate coupling and later release of a library compound synthesized on the solid phase, linker groups may be used. Such linkers are well known in the art and may include, but are not limited to, polyamino, polycarboxylic , polyester, polyhalo, polyhydroxy, polyunsaturated groups, or combinations thereof. The linker is preferably labile under a given set of conditions that do not adversely affect the compounds attached to the library or the reagents used in their preparation or manipulation. More preferably, the linker is acid labile or is photolabile. Desirable linkers include a halotrityl moiety, a Rink amine linked polystyrene (Novabiochem) linking the scaffold molecule to the solid support, or an alpha-halo, alpha-methylphenacyl moiety. The linkers may be used to covalently bind the scaffold molecules to the solid support. Typically, covalent attachment may be through amine, ether, thioether, ester, thioester, amide, acetamide, phosphate, phosphonate, phosphinate, sulfonate or sulfate bonds. Customized resin linkers, e.g., those supporting an amino acid, can be obtained from Novabiochem. In another embodiment of the method of this invention, the library compounds may be prepared using a "safety-catch" linker. This type of linker is used to bond the scaffold to the resin, but unlike conventional linkers that are removed by hydrolysis, the linker is removed by displacement with a nucleophile. The nucleophile becomes part of one of the three functional groups on the library compound, allowing greater diversity of functional groups in the library. Use and preparation of safety-catch linkers are described in Backes and Ellman, J. Am. Chem. Soc . , 1994, Vol. 116, p. 11171; and Backes et al., J. Am. Chem. Soc, 1996, Vol. 118, p. 3055. For example, when the scaffold is attached via the carboxylic acid to an amino acid or peptide bound to a solid support via a safety-catch linker, the linker may be displaced with an amine or thiol compound to further functionalize the amino acid or peptide substituent, thereby producing the final Ai substituent. A procedure for library preparation using a resin bearing a safety-catch linker is given in Example 14. Alternatively, when the scaffold is attached directly to the support via the carboxylic acid and the linker, the linker may be displaced with an amine or thiol compound which becomes the Ai substituent.
The compounds can be prepared using a mix and split strategy with directed sorting using the IRORI AccuTag®-100 radiofrequency tagged solid phase synthesis system or in an arrayed parallel synthesis using automated robotic methods. For instance, a Tecan™
(Switzerland) Genesis liquid handling system, a Tecan™ resin dispenser, and a Savant centrifugal evaporator can be used. Use of the IRORI AccuTag®-100 system is preferred.
The library compounds are typically used without purification with the product of a given preparation being characterized by standard techniques such as liquid chromatography and mass spectrometry. Quantitative analysis of the products is conveniently performed by preparing daughter multi-well plates from a mother plate, with one of the daughter plates being dedicated to the analytical studies. A suitable threshold for the screening studies is >85% purity of the scaffold product. Various purification techniques can be employed, however, if so desired in order to increase the level of sample purity. These purification techniques include flash chromatography, high-performance liquid chromatography (HPLC) , solution phase "covalent scavenger" strategies, polymer-supported quenching, and resin capture, to name a few.
Compounds in the library of this invention can be screened for biological activity using routine methods well known to those skilled in the art, and described hereinafter in Example 15. For example, the compounds can be screened for anti-infective activity against viral, bacterial, or fungal agents. Representative targets include strains of Staphylococcus and Streptococcus bacteria.
The activities of the compounds can be screened by contacting each compound with the biological target under conditions generally found to promote growth of the target. Observations are then made over a several hour or day period to determine whether proliferation of the target has been inhibited. Signs of inhibition are indicative of the compound having a positive activity against the target. For example, an observation that the growth rate of a microbe has ceased or diminished is an indication that the compound has anti-microbial activity. Screening may also be performed by directly assaying for peptidoglycan synthesis in the microbes.
A library of 1920 compounds was prepared from scaffold molecule (XI) . The free hydroxyl group was allowed to react with an isocyanate RiNCO in which Ri is cyclohexyl, 3- ( trifluoromethyl ) phenyl , 4- (trifluoromethyloxy) phenyl, 3 , 5-bis (trifluoromethyl) phenyl , 2,4- difluorophenyl, and 3- (phenyloxy) phenyl . The amino group produced by reduction of the azido group reacted with carboxylic acids R2C02H in which R is 3- (2-thienyl) -1-propyl , 1-undecyl, 5- (1 , 3-benzodioxole, 5- (1, 2 , 3-benzotriazole) , 2- (5-nitrofuran) , 4- (n-octyloxy) phenyl , 4- acetamidophenyl , and 1- (1-butenyl ) . The free carboxylic acid substituent reacted with internal amino acids Gly, Nle, Lys , His and Ser; and with terminal amino acids Ala, Leu, His and Met. Details of the library preparation are provided in Examples 12 and 13.
Further modification of the library compounds may be carried out after the three reactive sites are functionalized. For example, an arylthio group, i.e., X is S and R5 is aryl, at the anomeric position is removed by converting the XR5 group to a hydroxyl group. A suitable reagent for accomplishing this transformation is a mercury (II) salt.
Library compounds are preferably prepared on a solid support and then removed from the solid support by cleaving their covalent attachments thereto .
The following Examples illustrate, but do not limit the invention.
EXAMPLES
EXAMPLE 1: Methyl 3-azido-3-deoxy-4-0-methyl-β-D-glucopyranosiduronic acid (I)
1, 2 , 4 , 6-Tetra-0-acetyl-3-azido-3-deoxy-D-glucopyranose (2) is prepared from 1 , 2-5 , 6-di-O-isopropylidene-α-D-allofuranose (1) following the procedure disclosed in copending application Serial No. 08/975,229, filed November 21, 1997.
Methyl 3-azido-3-deoxy-β-D-glucopyranoside (5)
To a stirred solution of (2) (42g, 112. δmmol ) and BiBr3 (3g,6.7mmol) in CH2C12 (400mL) is added Me3SiBr (62mL, 470mmol ) under Argon atmosphere. After four hours at room temperature, the solution is washed with cold water, cold aqueous NaHC03 solution, dried over Na2SU4 and concentrated under reduced pressure to give crude bromide (3); Hϊ NMR (CDCI3) : 8 6.62 (d, J=4Hz , 1H, H-l ) , 5.04 (t,lH,H-4), 4.69- 4.65 (dd,lH,H-2), 2.18, 2.15 and 2.10 (each s,9H,3x0Ac). A suspension of Ag20 (60g), CaS0 (60g) in CH2C12 (500mL) and CH3OH (llOmL) is stirred for one half hour under protection of light and moisture. A solution of above bromide in CH2C12 (lOOmL) is then added dropwise and stirring is continued for an additional one hour. The mixture is filtered through Celite, the solids are thoroughly washed with CH2CI2, and the organic layer is washed with aqueous NaHC03 solution, dried over Na2S04 and concentrated under reduced pressure. A solution of this residue in MeOH (500mL) is treated with IM MeONa (pH~ll) for four hours at room temperature. The base is neutralized with Amberlite IR-120 (H+) cation-exchange resin, filter and concentrate in vacuo . The residue is purified on a silica gel column by using 10-15% MeOH in CH2C12 as eluent to give (5) (23g, 87%); H NMR (CD3OD) : 5 4.25 (d, J=7.5Hz , 1H, H-l ) , 3.89-3.86 (dd,lH,H-4), 3.71- 3.67 (dd,lH,H-2), 3.55 (s,3H,OMe), 3.22-3.17 (dd,lH,H-3); 13C NMR: δ 105.23(C-1), 78.32(C-5), 73.66(C-3), 70.99(C-2), 70.14(C-4), 62.28(C- 6) , 57.34 (OMe) .
Methyl 3-azido-4, 6-0-p-methoxybenzylidene-3-deoxy-β-D-glucopyranoside (6)
To a stirred solution of (5) (23g, 105mmol) in DMF (230mL) are added p-toluenesulfonic acid (pTSA) (2.3g) and anisaldehyde dimethyl acetal (50mL) . The stirring is continued for four hours at room temperature. The acid is neutralized with Et3N, and solution is concentrated under reduced pressure. The residue is purified on a silica gel column by using hexane-ethyl acetate (4:l- 1:1) as eluent to give (6) (24g, 68%); XH NMR (CDC13) : δ 7.41 (d,2H,ArH), 6.90 (d,2H,ArH), 5.52 (s,lH, acetal H) , 4.37-4.30 (m, 2H, H-1&H-4 ) , 3.79 & 3.56 (each s, 6H,2xOMe) , 3.43-3.38 (dd,lH,H-3).
Methyl 2-0-acetyl-3-azido-4 , 6-0-p-methoxybenzylidene-3-deoxy-β-D- glucopyranoside (7)
To an ice cooled solution of (6) (22.8g, 60.2mmol) in pyridine (200mL) containing DMAP(0.8g) is added acetic anhydride (lOOmL) and stirring is continued for four hours at room temperature. The solvent is evaporated under reduced pressure, the last traces being removed by coevaporation with toluene. The residue is dissolved in ethyl acetate and washed with aqueous NaHCθ3 solution and water, dried over Na2S04 and concentrated in vacuo . The residue gave a white amorphous solid (23.5g,94%) from ether-hexane ; XH NMR (CDC13) : δ 7.43 (d,2H,arom. ) , 6.91 (d, 2H, arom. ) , 5.56 (s , 1H, acetal H) , 4.91-4.86 (dd,lH,H-2), 4.44 (d,lH,H-l), 4.40-4.35 (dd,lH,H-4), 3.82& 3.51 (each s,6H,2xOMe), 2.16 (s,3H,OAc); 13C NMR: δ 102.34 (acetal C) , 101.45(C- 1), 79.29(C-5), 71.56(C-3), 68.51(C-2), 67.23(C-4), 63.14(C-6), 57.10 & 55.28(2xOMe) , 20.80(OAc).
Methyl 2-0-acetyl-3-azido-6-0-p-methoxybenzyl-3-deoxy-β-D- glucopyranoside ( 8 )
To a cold (0°C,bath) stirred mixture of (7) (6.8g, 17.9mmol), NaBH3CN (4.7g, 74.6mmol) and powdered 3A molecular sieves (lOg) in dry DMF (80mL) is added, dropwise, a trifluoroacetic acid (TFA) (10.8mL; 140mmol) solution in DMF (25mL) and stirring is continued at room temperature for 48 hours. The mixture is diluted with ethyl acetate and solids are filtered through Celite and thoroughly wash with ethyl acetate. The combined filtrate is washed with aqueous NaHCU3 solution and water, dried over Na2S04 and concentrated under reduced pressure. The residue is purified on a column of silica gel and is eluted with hexane-ethyl acetate (4:1-^ 2: 3) . The fractions corresponding to the product are concentrated to give (8) (6.6g,96%); !H NMR (CDCI3) : δ 7.32 (d,2H,ArH), 6.81 (d,2H,ArH), 4.82-4.76
(dd,lH,H-2) , 4.56-4.44 (m,2H,OCH2) , 4.30 (d,lH,H-l) , 3.78 & 3.44 (2xOMe) , 2.11(s,3H,OAc) ; 13C NMR: δ 109.61 (acetal C) , 101.6KC-1) , 74.27 (C-5) , 73.37(C-3) , 71.47(C-2) , 71.10 (OCH2) , 69.76 (C-6) , 56.65 & 55.17 (OMe) , 20.73 (OAc) .
Methyl 2-0-acetyl-3-azido-6-0-p-methoxybenzyl-3-deoxy-4-0-methyl-β- D-glucopyranoside ( 9 )
A mixture of (8) ( 6g, 15.7mmol) , Ag20 (12g) and CH3I (lOmL) in dry DMF (lOOmL) is stirred for 16 hours at room temperature. The mixture is diluted with ethyl acetate and solids are removed by filtration (celite bed) and thoroughly washed with ethyl acetate. The combined filtrate is washed with aqueous Na2S203 solution and water, dried over Na2S04 and evaporated under reduced pressure to give pure (9)
(6.1g,98%); XH NMR (CDCI3) : δ 7.27 (d,2H,ArH), 6.88 (d,2H,ArH), 4.82- 4.76 (dd,lH,H-2), 4.61-4.48 (m,2H,OCH2), 4.29 (d,lH,H-l), 3.79,3.50 & 3.47 (each s,9H,3xOMe) , 2.12 (s,3H,OAc); 13C NMR: 6 101.42 (C-l) , 78.29(C-4), 75.56(C-5), 72.93(C-3), 70.99(OCH2), 67.73(C-2), 66.34(C- 6), 60.23, 56.41 & 54.98 (3xOMe) , 20.58(OAc).
Methyl 2-0-acetyl-3-azido-3-deoxy-4-0-methyl-β-D-glucopyranoside (10)
To a solution of (9) (6g, 15.2mmol) in CH2C12 (150mL) saturated with water is added 2 , 3-dichloro-5 , 6-dicyano-l , 4-benzoquinone (DDQ) (6.6g, 29mmol) and stirring is continued for two hours. The organic layer is washed with cold aqueous NaHC03 solution, aqueous Na2S2θ3 solution, water, and dried over Na2S04 and concentrated in vacuo . It is purified on a silica gel column using 20-30% acetone in CH2C12 as eluent to give (10) (3.8g,91%); lK NMR (CDCI3) : δ 4.76 (dd,lH,H-2), 4.34 (d,lH,H-l), 3.58 & 3.46 (each s,6H,2xOMe), 2.12 (s,3H,OAc); 13C NMR: δ l69.26(CO), 101.53(C-1), 77.82(C-4), 75.90(C-5), 71.05(C-3), 66.09(C-2), 60.91 (OMe), 60.42(C-6), 56.73(OMe), 20.59(OAc).
Methyl 2-0-acetyl-3-azido-3-deoxy-4-0-methyl-β-D-glucopyranosiduronic acid (11)
A mixture of (10) (3.5g, 13mmol ) in acetone (12OmL) containing Jones reagent (22mL, 1.5eqv) is sonicated for one hour. Then, another batch of Jones reagent (6mL) is added to the reaction mixture and allow to sonicate for another one half hour. Isopropanol is added to decompose the excess of reagent. It is filtered through the Celite and solid is washed with an excess of acetone. The solvents are removed under reduced pressure and the residue is dissolved in ethyl acetate and washed with aqueous NaCl solution, dried over Na2S04 and concentrated in vacuo. The residue is purified on a silica gel column using 10- 12% MeOH in CH2C12 as eluent to give (11) (2.7g,73%); XH NMR (CD30D) : δ 4.76-4.70 (dd,lH,H-2), 4.54 (d,lH,H-l), 4.00 (d,lH,H-5), 3.77 (t,lH,H-4), 3.56 & 3.48 (each s,6H,2xOMe), 3.44 (t,lH,H-3), 2.12 (s,3H,OAc); 13C NMR: δ 102.99 (C-l ) , 81.64(C-4), 75.77(C-5), 72.44(C-3) 66.90(C-2), 60.71 & 57.37 (2xOMe) , 20.70(OAc).
Methyl 3-azido-3-deoxy-4-0-methyl-β-D-glucopyranosiduronic acid (I)
To a solution of (11) (2g) in MeOH (lOOmL) is added IM MeONa (pH -11) and the mixture is stirred at room temperature for 16 hours. The base is neutralized with IR-120 (H+) resin, filtered and concentrated under reduced pressure. It is purified on a silica gel column using a solvent gradient consisting of 10-15% MeOH in CH2C12 to give (I) (1.5g,88%); K NMR (CD3OD) : δ 4.32 (d,lH,H-l), 3.92 (d,lH,H-5), 3.56 & 3.55 (each s, 6H,2xOMe) , 3.46 (t,lH,H-4), 3.29 (t,lH,H-3), 3.26-3.20 (dd,lH,H-2) ; 13C NMR: δ l71.89(CO), 105.48(C-1), 81.5KC-4), 75.88(C- 5), 73.45(C-3), 69.57 (C-2), 60.61 & 57.59 (2xOMe) . ES-MS Calcd. For C8H13N3O16 (247); Found : 265 [M+NH4 ]+, 246[M-1].
EXAMPLE 2: Methyl 3-azido-4-Q-carboxymethyl-3-deoxy-2-0-methyl-β-D- glucopyranoside (II)
Methyl 3-azido-4 , 6-0-p-methoxybenzylidene-3-deoxy-2-0-methyl-β-D- glucopyranoside (12)
To a solution of (6) ( 6.8g, 20.2mmol) in DMF (70mL) is added NaH (1.6g,60% mineral-oil suspension, 2eqv) at 0°C . After one half hour, CH3I (1.84mL, 1.5eq. ) is added and stirring is continued for one hour at same temperature. The reaction mixture is diluted with ethyl acetate and washed with water and aqueous NaHC03 solution, dried over Na2Sθ and concentrated in vacuo. The residue is dissolved in CHC12 and addition of hexane gives amorphous (12) (6.7g,95%); XH NMR
(CDCI3) : δ 7.41 (d,2H,ArH) , 6.89 (d, 2H, arom. ) , 5.49 (s , 1H, acetal H) , 4.33 (d,lH,H-l), 3.79, 3.61 & 3.56 (each s,9H,3xOMe), 3.04-2.98 (dd,lH,H-3); 13C NMR: δ 104.94 (acetal C) , 101.4KC-1), 82.64(C-2), 79.05(C-5), 68.57(C-3), 66.83(C-4), 64.44(C-6), 60.83, 57.34 & 55.23 (3xOMe) . Methyl 3-azido-6-0-p-methoxybenzyl-3-deoxy-2-0-methyl-β-D- glucopyranoside (13)
To a cold (0°C bath) stirred mixture of (12) (6.3g, 17.9mmol), NaBH3CN (4.7g, 74.6mmol) and powdered 3A molecular sieves (lOg) in dry DMF
(80mL) are added, dropwise, a solution of TFA (10.8mL, 140mmol) in DMF (25mL) followed by processing as described for the preparation of (8) gives (13) (4g,63%) after silica gel column chromatography (solvent gradient consisting of 4 : l-> 1:1, hexane-ethyl acetate); 1H NMR (CDC13) : δ 7.26(d,2H,ArH) , 6.88 (d,2H,ArH), 4.57-4.46 (m,2H,OCH2), 4.23 (d,lH,H-l), 3.81, 3.59 & 3.54 (each s,9H,3xOMe), 3.00-2.95 (dd,lH,H-3); 13C NMR: δ 104.81 (C-l) , 81.78(C-2), 74.04(C-5), 73.26(C- 3), 70.83(C-6), 69.71 (OCH2) , 67.92(C-4), 60.34, 56.90 & 55.13 (3xOMe) .
Methyl 3-azido-3-deoxy-2-0-methyl-4-0-methoxycarbonylmethyl-β-D- glucopyranoside (15)
To a stirred solution of (13) (2.9g, 8.2mmol ) , Ag20 (lOg, 43.3mmol) , KI (3g,18mmol) and DMF (30mL) is added BrCH2COOMe (4mL,42mmol) and the mixture is stirred at room temperature for four hours. Processing as described for the preparation of (9) gives crude (14) which is utilized as such for the next step. Compound (14) on treatment with DDQ as described for the synthesis of (10) gives (15) (1.5g,60%) after silica gel column chromatography with 10-30% acetone in CH2C12; XH NMR (CDCI3) : δ 4.56-4.41 (m,2H,OCH2), 4.28 (d,lH,H-l), 3.80, 3.61 & 3.56 (each s,9H,3xOMe) , 3.07-2.98 (dd,lH,H-3); 13C NMR: δ 171.24 (CO), 104.25(C-1), 82.45(C-2), 76.46(C-4), 75.13(C-5), 68.61(C-3), 67.78(OCH2), 61.43(C-6), 60.28, 56.93 & 52.00 (3xOMe) .
Methyl 3-azido-4-0-carboxymethyl-3-deoxy-2-0-methyl-β-D- glucopyranoside (II)
To a solution of (15) (1.5g) in dioxane (75mL) is added 2N NaOH solution (8mL) at 0°C . It is allowed to stir at same temperature for one hour. The base is neutralized with IR-120 (Hτ) resin, filter and is concentrated under reduced pressure. The residue is purified on a silica gel column using a solvent gradient consisting of 10-25% MeOH in CH2CI2 to give (II) (1.3g,91%); H NMR (CD3OD) : δ 4.27 (d,lH,H-l), 3.86-3.72 (2xdd,2H, H-4&H-2), 3.54 & 3.49 (each s,6H,2x OMe) ,2.95-2.89 (dd,lH.H-3) ; 13C NMR: δ l73.95(CO), 105.47(C-1),
83.86(C-2), 78.06(C-4), 76.83(C-5), 69.87(OCH2), 69.20(C-3), 61.98(C-
6) , 60.63 & 57.18(2xOMe) .
ES-MS Calcd. for C10H17N3O7 (291 ) ; Found: 309 [M+NH ] +, 289 . 5 [M-1 ] .
EXAMPLE 3 : Methyl 3 -azido-2 -0-carboxymethyl-3 -deoxy-4-0-methyl-β-D- glucopyranoside ( III )
Methyl 3 -azido-4 , 6-0-p-methoxybenzylidene-3 -deoxy-2 -0- methoxycarbonylmethyl-β-D-glucopyranoside ( 16 )
To a stirred mixture of (6) (7g, 20.8mmol) , Ag20 (21g) , KI (7g) in DMF (70mL) is added BrCH2COOMe (llmL) and mixture is stirred at room temperature for two hours. Processing as described for the preparation of (9) provides (16) (6.8g,80%) after silica gel column chromatography (solvent gradient consisting of 4:1-^1:1, hexane-ethyl acetate); ^ NMR (CDCI3) : δ 7.43 (d,2H,ArH), 6.91 (d,2H,ArH), 5.51 (s,lH, acetal H) , 4.47-4.33 (m, 3H, H-I&OCH2) , 3.81, 3.79 & 3.51 (each s,9H,3xOMe), 3.25-3.19 (dd,lH,H-3); 13C NMR : δ 104.16 (acetal C) , 101.39(C-1), 82.09(C-2), 78.94(C-5), 69.56(C-3), 68.43 (0CH2) , 66.7KC-4), 63.85(C-6), 57.03, 55.21 & 51.78 (3xOMe) .
Methyl 3-azido-6-0-p-methoxybenzyl-3-deoxy-2-0-methoxycarbonylmethyl- β-D-Glucopyranoside (17)
Compound (16) ( 6.4g, 15.6mmol) is treated exactly as described for the preparation of (8) from (7) to give (17) (6.2g,97%) after silica gel column chromatography (solvent gradient consisting of 4:1~>2:3, hexane-ethyl acetate); Η NMR (CDCI3) : δ 7.24 (d, 2H, arom. ) , 6.87 (d,2H,ArH), 4.51-4.30 (m, 3H, H-l&OCH2) , 3.79, 3.76 & 3.46 (each s,9H,3xOMe), 3.16 (t,lH,H-3); 13C NMR: δ 103.63 (C-l ) , 81.13(C-2), 74.14(C-5), 73.19(C-3), 70.53(C-6), 69.42 & 69.03 (2xOCH2) , 67.32(C- 4), 56.66, 55.09 & 51.69 (3xOMe) .
Methyl 3-azido-3-deoxy-4-0-methyl-2-0-methoxycarbonylmethyl-β-D- glucopyranoside (19)
Methylation of (17) (5g, 12.2mmol) and followed by the removal of p- methoxybenzyl group is achieved in a manner analogous to that described for (8) (to give (10)) and the product mixture is purified on a silica gel column with 15-30% acetone in CH2C12 to afford (19)
(3.1g,83%); :H NMR (CDC13) : δ 4.43-4.29 (m, 3H, H-I&OCH2) , 3.77, 3.57 & 3.47 (each s,9H,3xOMe) ; 13C NMR: 170.36(CO), 103.69(C-1), 81.25(C-2), 77.60(C-4), 75.5KC-5), 69,09(C-3), 66.98(0CH2), 61.16(C-6), 60.44, 56.82 & 51.70(3xOMe) .
Methyl 3-azido-4-0-carboxymethyl-3-deoxy-2-0-methyl-β-D- glucopyranoside (III)
The ester hydrolysis of (19) (2.9g, 9.5mmol) in dioxane-2N NaOH is obtained in a manner analogous to that described for (15) (to give II) to afford (III) (2.6g,94%) ; XH NMR (CD3OD) : δ 4.39-4.35 (m,3H,H- I&OCH2) , 3.89-3.84 (dd,lH,H-4) , 3.75-3.70 (dd,lH,H-2) , 3.60 & 3.52 (each s,6H,2xOMe) , 3.18-3.12 (dd,lH,H-3) ; 13C NMR: δ 173.85 (CO) , 104.99(C-1) , 82.61 (C-2) , 79.26(C-4) , 77.36(C-5) , 69.94(OCH2) , 68.8KC-3) , 61.64(C-6) , 60.68 & 56.99 (2x0Me) .
ES-MS Calcd. For CιoHι7N3Oι7 (291) ; Found: 292[M+1]+, 309[M+NH4]+, 314[M+Na]+, 289.5 [M-l] .
EXAMPLE 4: Methyl 3-azido-2-0-carboxymethyl-3 -deoxy-4-O-methyl-β-D- galactopyranoside (IV)
Methyl 3-azido-4-0-benzoyl-6-0-p-methoxybenzyl-3-deoxy-2-0- methoxycarbonylmethy1-β-D-galactopyranoside (21) To a stirred solution of triflie anhydride (4mL, 23.8mmol ) in CH2C12 (lOOmL) at -15°C is added dropwise pyridine (3.8mL, 47mmol) followed by a solution of (17) (6g, 14.6mmol) in CH2C12 (lOOmL). After two hours at 0°C, the solution is washed with 10% aqueous Citric acid solution, aqueous NaHC03 solution, water, dried over Na2S04 and concentrated under reduced pressure. To a solution of this triflate 20 in the dry toluene (lOOmL) is added Bu4N+OBz" (3.3g, 1.5eqv) . The reaction is stirred at room temperature for one hour. The solvent is evaporated in vacuo and the residue is purified on a silica gel column by using hexane-ethyl acetate (4:1-^1:1) as eluent to give (21) (6.5g,86%); XH NMR (CDC13) : δ 8.06-6.74 (m,9H,ArH), 5.63 (d, J=3Hz,lH,H-4) , 4.24-4.33 (m, 5H, H-l&2xOCH2) , 3.84 (t,lH,H-2), 3.80- 3.76 (dd,lH,H-3), 3.74, 3.72 & 3.52 (each s,9H,3xOMe); 13C NMR: δ 104.15(C-1), 79.7KC-2), 73.22(C-5), 73.07(OCH2), 69.48(C-6), 68.4KC-3), 67.59 (OCH2) , 62.74(C-4), 56.93, 55.06 & 51.71 (3xOMe) .
Methyl 3-azido-4-0-benzoyl-3-deoxy-2-0-methoxycarbonylmethyl-β-D- galactopyranoside (22)
Compound (21) (5.6g, 9.7mmol) is converted to (22) (4.2g,95%) under similar reaction conditions as described for the preparation of (10) from (9); Η NMR (CDCl3) : δ 8.08-7.47 (m, 5H, arom. ) , 5.50 (d,lH,H-4), 4.48-4.45 (m,3H,H-l&OCH2) , 3.85-3.81 (dd,lH,H-2), 3.78 (s,3H,OMe); 13C NMR: δ 104.27 (C-l), 79.66(C-2), 74.14(C-5), 69.44 & 69.19 (2xOCH2) , 62.35(C-4), 60.24(C-6), 56.97 & 51.80 (2xOMe) .
Methyl 3-azido-6-0-benzoyl-3-deoxy-4-0-methyl-2-0- methoxycarbonylmethyl-β-D-Galactopyranoside (23 )
Treatment of a solution of (22) (4g,8.8mmol) in DMF (60mL) with Ag20 (8g) and CH3I (6mL) in a manner similar to that described for (8) to give (9) results in the migration of the benzoyl group to the C-6 position to give (23) (3.9g,94%) after silica gel column chromatography (9:1-^3:2), hexane-ethyl acetate as eluent); XH NMR (CDCI3) : δ 8.06-7.47 (m,5H,ArH), 4.55-4.40 (m,2H,OCH2) , 4.36 (d,lH,H- 1), 3.81 (t,lH,H-2), 3.76 & 3.62 (each s,6H,2x0Me), 3.53 (d,J=3Hz,lH,H-4) , 3.48 (s,3H,0Me); 13C NMR: δ 103.80 (C-l ) , 78.88(C-2), 77.23(C-5), 72.26(C-4), 69.21(C-3), 63.46(OCH2), 62.14(C-6), 61.50, 56.43 & 51.61(3xOMe) .
Methyl 3-azido-2-0-carboxymethyl-3-deoxy-4-0-methyl-β-D- galactopyranoside (IV)
Compound (23) (3.8g, 8. lmmol) is hydrolyzed exactly following the procedure described for the preparation of I from (11) , and the product mixture is purified in a column of silica gel with the solvent gradient consisting of 10-40% MeOH in CH2Cl2 to give (IV) (2.2g, 93%); lE NMR (CD3OD) : δ 4.37-4.35 (m, 3H, H-l&OCH2) , 3.74-3.71 (dd,lH,H-2), 3.64 (d, J=3Hz, lH,H-4 ) , 3.59 & 3.52 (each s , 6H, 2xOMe) ; 13C NMR: δ 105.50 (C-l) , 80.38(C-2), 78.82(C-5), 76.82 (C-3), 70.14(C-4), 65.22(OCH2), 61.79(OMe), 61.3KC-6), 56.98(OMe).
ES-MS Calcd. for C10H17N3O7 (291); Found: 309[M+NH4]+, 313.8[M+Na] + ,289.8[M-1] .
EXAMPLE 5: Methyl 3 -azido-2-0-carboxymethyl-3-deoxy-6-0-methyl-β-D- glucopyranoside (V)
Methyl 3-azido-4-0-methoxybenzyl-3-deoxy-2-0-methoxycarbonylmethyl-β- D-Glucopyranoside (24)
To a cold (0°C,bath) stirred mixture of (16) (1.8g, 4.4mmol ) , NaBH3CN (1.7g, 27mmol) and powdered 3A molecular sieves (5g) in dry acetonitrile (20mL) is added, dropwise, a Me3SiCl (3.5mL, 27.5mmol) solution in acetonitrile (20mL) and stirring is continued at room temperature for four hours. The mixture is diluted with ethyl acetate and wash with aqueous NaHC03 solution, water, 5% aqueous HCl solution, dry over Na2S04 and concentrate under reduced pressure. The residue is purified on a column of silica gel and is eluted with hexane-ethyl acetate (4:1- 1:4) to give 6-0-p-methoxybenzyl derivative (17) (0.4g,22%) and 4-0-methoxybenzyl compound (24) (0.8g,44%); :H NMR (CDCI3): δ 7.29 (d,2H,ArH), 6.87 (d,2H,ArH), 4.79- 4.31 (m,5H,H-l-i2x0CH2) , 3.78, 3.75 & 3.46 (each s,9H,3xOMe); 13C NMR: δ 103.60(01) , 81.28(02), 75.44(04), 75.19(C-5), 74.31 &
68.99 (2xOCH2) , 67.23(03), 61.06(06), 56.69, 55.01 & 51.40 (3xOMe) .
Methyl 3-azido-3-deoxy-6-0-methyl-2-0-methoxycarbonylmethyl-β-D- glucopyranoside (25)
Methylation of (24) (1.2g, 2.9mmol) and removal of p-methoxybenzyl group is performed in a manner similar to that described for (8) (to give (10)) and the residue is purified on a silica gel column with 10-20% acetone in CH2C12 to yield (25) (0.54g, 61%) ; XH NMR (CDC13) : δ 4.33-4.28 (m,3H,H-l&OCH2) , 3.72 (s,3H,0Me), 3.66-3.61 (dd,lH,H-2), 3.54-3.50 (dd,lH,H-4), 3.43 & 3.36 (each s,6H,2xOMe), 3.16-3.10 (dd,lH,H-3); 13C NMR: δ 170.46 (CO), 103.56 (C-l), 81.11 (C-2), 74.45(05), 71.70(06), 69.50(OCH2), 68.91(03), 67.39(04), 59.34, 56.58 & 51.63 (3xOMe) .
Methyl 3-azido-3-deoxy-6-0-methyl-2-0-carboxymethyl-β-D- glucopyranoside (V)
The hydrolysis of methyl ester in (25) ( 0.5g, 1.6mmol ) with dioxane-2N NaOH in a manner analogous to that described for (15) (to give II) provide (V) (0.45g,94%); *H NMR (CD3OD) : δ 4.26-4.20 (m, 3H, OCH2-iH-l) , 3.61-3.57 (dd,lH,H-2), 3.37 & 3.29(each s,6H,2xOMe), 3.01-2.95 (dd,lH,H-3); 13C NMR: δ 105.02 (C-l ) , 82.37(02), 76.99(05), 72.48(C- 6) ,
70.12 (C-3&OCH2) , 69.47(04), 59.59 & 57.06 (2xOMe) .
ES-MS Calcd. For C10Hi7N3O7 (291); Found: 291.8[M+1]+, 309[M+NH4]+,
313.8[M+Na]+, 289.8[M-1].
EXAMPLE 6: Methyl 3-azido-2-0-carboxymethyl-3-deoxy-6-0-methyl-β-D- Galactopyranoside (VI)
Methyl 3-azido-3-deoxy-2-0-methoxycarbonylmethyl-β-D- galactopyranoside (27) To a solution of triflate (20) ( 6g, 10.4mmol) in dry DMF (80mL) is added NaN02 (16g,20eqv) . The reaction is stirred at room temperature for four hours. It is diluted with ethyl acetate and washed with water, aqueous NaHC03 solution, and dried over Na2Sθ4 and concentrated in vacuo. This residue is dissolved in 5% TFA in CH2C12 (150mL) saturated with water and stir for two hours at room temperature. The solvent is removed under reduced pressure and coevaporated with toluene to remove traces of acid. It is purified on a silica gel column by using CH2Cl2-acetone (4:1->3:1) as eluent to give (27) (1.7g,40%); *H NMR (CDCl3) : δ 4.41-4.35 (m, 3H, OCH2&H-l) , 4.03
(d, J=3Hz,lH,H-4) , 3.78 (s,3H,OMe) , 3.62-3.58 (dd,lH,H-2) , 3.51 (s,3H,OMe) ; 13C MR: δ 104.25 (C-l ) , 79.03 (C-2 ) , 74.25 (C-5 ) , 69.21(OCH2) , 68.35(03) , 64.20(04) , 61.73(06) , 56.81 & 51.78 (2xOMe) .
Methyl 3-azido-3-deoxy-6-0-methyl-2-0-methoxycarbonylmethyl-β-D- galactopyranoside (28)
To a cold (0°C bath) stirred solution of (27) (1.45g, 5mmol) in CH2C12 (75mL) is added 2 , 6-di-tert-butyl-4-methyl pyridine (2g,9.8mmol) and trimethyloxonium tetrafluoroborate (1. lg, 7.5mmol) . Stirring is continued for 16 hours at ~5°C. It is washed with aqueous NaHCθ3 solution, dried over Na2S04 and concentrated under diminished pressure. The residue is applied to a column of silica gel and is eluted with a solvent gradient consisting of 10-15% acetone in CH2C12 to give (28) (1.3g,83%); :H NMR (CDC13) : δ 4.39-4.32 (m, 3H, 0CH2&H-1) , 3.98 (t,lH,H-2), 3.76, 3.48 & 3.39(each s , 9H, 3xOMe) ; 13C NMR: δ 104.14(01), 79.06(02), 73.15(05), 71.74(06), 69.28 (OCH2) , 68.59(03), 64.22(04), 59.43, 56.67 & 51.75 (3xOMe) .
Methyl 3-azido-2-0-carboxymethyl-3-deoxy-6-0-methyl-β-D- galactopyranoside (VI)
Compound (28) ( 1.2g, 3.9mmol) is transformed to title compound (VI) (lg,87%) under similar reaction conditions as described for the preparation of (II) from (15); H NMR (CD3OD) : δ 4.29-4.26 (m,3H,OCH2&H-l) , 3.82 (bs,lH,H-4) , 3.42 & 3.32 (each s, 6H, 2xOMe) ; 13C NMR: δ 105.55(01) , 80.07(02) , 75.11(05) , 72.56(06) , 70.07(OCH2) , 69.21(03) , 65.56
(C-4) , 59.44 & 56.95 (2xOMe) . ES-MS Calcd. For C10H17N3O7 (291) ; Found: 292[M+1]\ 309.3 [M+NH4]+, 314[M+Na]+, 289.8[M-1] .
EXAMPLE 7: Methyl 6-azido-2-0-carboxymethyl-6-deoxy-4-0-methyl-α-D- Glucopyranoside (VII)
Methyl 2-0-benzoyl-4 , 6-0-benzylidene-3-0-methoxycarbonylmethyl-α-D- glucopyranoside (30) and Methyl 3-0-benzoyl-4 , 6-0-benzylidene-2-0- methoxycarbonylmethyl-α-D-Glucopyranoside (31)
A mixture of methyl 4 , 6-0-benzylidene-α-D-glucopyranoside (E. Evans, Carbohydr. Res., 1972, 21, 473 ) (29.56g, 198.6 mmol) and Bu2SnO
(66g, 265mmol) in toluene (800mL) is heated for five hours at reflux temperature, with azeotropic distillation of water. The solution is concentrated to ~700mL, Bu4NI (50g, 135mmol ) and BrCH2COOMe (70mL, 740mmol) are added, and the mixture is heated (~130°C) under reflux for 16 hours. It is then evaporated to dryness to give a crude mixture of lactone and alkylated products. This crude mixture is passed through small silica gel using hexane-ethyl acetate (4:l->3:2) as eluent. The fractions corresponding to the products are concentrated and treated with methanolic MeONa (pH~10) for one half hour to convert lactone to methyl ester. It is neutralized with IR-120 (H+) resin, filtered and evaporated under reduced pressure. A solution of this crude material in pyridine (500mL) is treated with BzCl (lOOmL) for 16h at room temperature. The solvents are removed in vacuo and residue is taken in ethyl acetate. The organic layer is washed with aqueous citric acid, water, and aqueous NaHCθ3 solution, dried over Na2≤04 and concentrated under diminished pressure. It is purified on silica gel column using gradient consisting of hexane- ethyl acetate (4:1-^1:3) as eluent to give faster moving (30) (23g,25%); Η NMR (CDCI3) : δ 8.09-7.34 (m,10H,ArH), 5.54 (s,lH, acetal H) , 5.16-5.11 (dd,lH,H-2), 5.07 (d, J=4Hz , 1H, H-l) , 4.32-4.27 (dd,lH,H- 3), 4.15 (t,lH,H-4), 3.39 & 3.36 (each s, 6H,2x0Me); 13C NMR: δ 101.39 (acetal C) , 97.60(01), 81.65(03), 77.34(05), 72.90 (C-2), 69.62 (0CH ), 68.8KC-4), 61.92(C-6), 55.28 & 51.33 (2xOMe) ; and to also give slower moving (31) ( 19.5g, 22%) ; H NMR (CDCI3) : δ 8.07-7.26 (m, 10H,ArH), 5.84 (t,lH,H-3), 5.46 (s,lH, acetal H) , 5.14 (d, J=4Hz,lH,H-l) , 3.64 & 3.47(each s,6H,2xOMe); 13C NMR: δ 101.35 (acetal C) , 99.36(01), 79.62(02), 79.10(05), 71.25(03), 68.92(OCH2), 68.29(04), 62.32(06), 55.34 & 51.77 (2xOMe) .
Methyl 3-0-benzoyl-2-0-methoxycarbonylmethyl-α-D-glucopyranoside (32)
A solution of (31) (19g) in CH2C12 (400mL) , TFA (45mL) and H20 (5mL) is stirred for two hours at room temperature. The organic layer is washed with cold water, dried over Na2S04 and concentrated to dryness. It is purified on a silica gel column by using 5-10% MeOH in CH2C12 as eluent to give (32) (10.2g,67%); 'H NMR (CDCI3) : δ 8.06-7.44
(m,5H,ArH), 5.53 (t,lH,H-3), 5.10 (d,lH,H-l), 4.30-4.14 (m,2H,OCH2), 3.63 & 3.45 (each s,6H,2xOMe); 13C NMR (CDCI3) : δ 98.30 (C-l), 78.44(02), 75.96(05), 71.24(03), 69.73(OCH2), 68.34(04), 61.70(0 6) , 55.19 & 51.76 (2xOMe) .
Methyl 6-azido-6-deoxy-2-0-methoxycarbonylmethyl-α-D-glucopyranoside (33)
To a mixture of (32) (15g, 40.5mmol) , Ph3P (27g, 103mmol) in DMF (200mL) is added N-bromosuccinimide (NBS) (18.6g, 104mmol) at room temperature under an argon atmosphere. It is heated at 60°C for two hours and cooled to room temperature. Methanol (5mL) is added to the reaction mixture to decompose the excess of NBS. Sodium azide (21.6g, 332mmol) is added to the reaction mixture. It is heated at 0°C for two hours and cooled to room temperature. The reaction mixture is diluted with ethyl acetate and washed with water, aqueous HCl solution, dried over Na2S04 and concentrated in vacuo. It is purified on a silica gel column by using a solvent gradient consisting of hexane-ethyl acetate (9:1-^2:3) to give (33) (13.5g,84%); Η NMR (CDCl3) : 8 8.04-7.44 (m,5H,ArH), 5.45 (t,lH,H-3), 5.10 (d,lH,H-l), 4.30-4.15 (m,2H,0CH2), 3.73-3.68 (dd,lH,H-2), 3.62 & 3.47 (each s,6H,2xOMe) ; 13C NMR: δ 98.18 (C-l), 78.31(0-2), 76.30(0 5), 70.79(03), 70.58(OCH2), 68.55(04), 55.38 & 51.82 (2xOMe) , 51.11(06) .
Methyl 6-azido-3-0-benzoyl-6-deoxy-4-0-methyl-2-0- methoxycarbonylmethyl-α-D-Glucopyranoside (34)
Compound (33) ( 12g, 30.4mmol) is O-methylated under similar reaction conditions as described for the preparation of (9) from (8) to give (34) (8g,64%) after silica gel column chromatography (solvent gradient consisting of hexane-ethyl acetate, 9:1-^2:3); 1H NMR (CDC13) : δ 8.05-7.45 (m,5H,ArH), 5.73 (t,lH,H-3), 5.10 (d,lH,H-l), 4.28-4.08 (m,2H,OCH2), 3.72-3.67 (dd,lH,H-4), 3.64, 3.48 & 3.38(each s,9H,3xOMe) ; 1C NMR: δ 98.30(C-l), 78.70(02), 78.64(04), 73.77(C- 5), 69.53(03), 68.02(OCH2), 60.01, 55.37 & 51.72 (3xOMe) , 51.07(06).
Methyl 6-azido-6-deoxy-4-0-methyl-2-0-carboxymethyl-α-D- glucopyranoside (VII)
Compound (34) (7.5g, 18.3mmol) is de-O-benzoylated and hydrolyzed in a similar manner as described for the synthesis of I from (11) to give (VII) (3.9g,73%) after silica gel column chromatography using 10-30% MeOH in CH2C12 as eluent; XH NMR (CD3OD) : δ 4.15-3.98 (m,2H,OCH2), 4.94 (d,lH,H-l), 3.91 (t,lH,H-4), 3.53 & 3.42 (each s,6H,2xOMe), 3.14 (t,lH,H-3); I3C NMR: 5 98.27(01), 81.71(02), 81.02(04), 73.53(05), 71.50 (C-3), 70.04(OCH2), 60.98 & 55.55 (2xOMe) , 52.46(06). ES-MS Calcd. for C10H17N3O7 (291); Found: 309[M+NH4]+, 290[M-1].
EXAMPLE 8: Methyl 6-azido-6-deoxy-4-0-methyl-3-0-carboxymethyl-α-D- glucopyranoside (VIII)
Methyl 2-0-benzoyl-3-0-methoxycarbonylmethyl-α-D-glucopyranoside (35)
The removal of the benzylidene group from (30) (20g, 43.7mmol ) is accomplished under identical conditions as described for the preparation of (32) from (31) to give (35) (9.3g,58%) after silica gel column chromatography (5-10% MeOH in CH2C12 as eluent) ; 1H NMR (CDC13) : 5 8.06-7.48 (5,5H,ArH) , 5.02-4.96 (m, 3H, H-2&H-1 ) , 4.40-4.24 (m,2H,OCH2), 3.73 & 3.34(each s, 6H,2xOMe) ; 13C NMR: δ 96.96(01) , 82.24(03) , 74.14(05) , 71.30(02) , 69.84(OCH2) , 68.80 (C-4) , 62.43 (O 6) , 55.14 & 52.42 (2xOMe) .
Methyl 6-azido-2-0-benzoyl-6-deoxy-3-0-methoxycarbonylmethyl-α-D- glucopyranoside (36)
Compound (36) (from (35); 9.3g, 25. Immol) is obtained (4.5g,45%) by the same reaction sequence as described for the preparation of (33) from (32); 'H NMR (CDCI3) : δ 8.08-7.42 (m,5H,ArH), 5.13-5.01 (m,2H,H- 2&H-1), 4.42-4.26 (m, 2H, OCH2) , 3.84 (t,lH,H-3), 3.77 & 3.40 (each s,6H,2xOMe); 13C NMR: 5 96.80(01), 82.07(03), 73.92(05), 70.99(0 2), 70.02(OCH2), 68.72(C-4), 55.23 & 52.42 (2xOMe) , 51.37(06).
Methyl 6-azido-2-0-benzoyl-6-deoxy-4-0-methyl-3-0- methoxycarbonylmethyl-α-D-Glucopyranoside (37 )
Compound (36) (5g, 12.7mmol ) is O-methylated in a similar manner as described for the preparation of (9) from (8) to give (37) (5g,96%) after silica gel column chromatography (solvent gradient consisting of hexane-ethyl acetate, 9:1-»1:1); ^ NMR (CDCI3) : δ 8.08-7.38 (m,5H,ArH), 5.06-5.01 (m, 2H, H-2&H-1) , 4.42-4.31 (m,2H,OCH2), 3.95 (t,lH,H-4), 3.59, 3.46 & 3.36(each s,9H,3xOMe); 13C NMR: 5 96.73(C-1), 81.55(03), 79.79(04), 73.63(05), 70.23 (C-2), 69.86(OCH2), 60.93, 55.26 & 51.49 (3xOMe) , 51.07(06).
Methyl 6-azido-6-deoxy-4-0-methyl-3 -O-carboxymethyl-α-D- glucopyranoside (VIII)
Compound (37) (5g, 12.2mmol) provides (VIII) (3.1g,87%) under similar reaction conditions as mentioned in the preparation of (I) from (11) after silica gel column chromatography; 1H NMR (CDOD) : δ 4.70 (d,lH,H-l), 3.55 (s,3H,0Me), 3.50 (t,lH,H-4), 3.43 (s,3H,0Me), 3.21- 3.15 (dd,lH,H-3); 13C NMR: δ 100.87 (C-l ) , 84.80(C-3), 81.69(04), 72.97 (C-5), 71.66(C-2), 70.46 (0CH2) , 61.10 & 55.70 (2xOMe) , 52.35(06) .
ES-MS Calcd. for Cι0Hι7N3θ (291); Found: 292[M+1]+, 309.3 [M+NH4] +, 290[M-1] .
EXAMPLE 9: Carboxymethyl 2-acetamido-6-azido-4-0-benzyl-2 , 6-dideoxy- α-D-glucopyranoside (IX)
(a) Allyl 2-acetamido-2-deoxy-c.-D-glucopyranoside (38)
A mixture of 2-acetamido-2-deoxy-D-glucose (50 g, 226 mol) , allyl alcohol (500 mL, dried over molecular sieve), and BF3'Et20 (8 mL, 67.8 mmol) is refluxed for two hours. Allyl alcohol (100 mL, containing 5% of cone HCl) is then added to the above, and the reaction is heated under reflux for ten hours. The reaction mixture is concentrated under reduced pressure, and the resulting gummy residue is crystallized from Et0H-Et20 to afford compound (38) (37 g, 63%) as a light brown solid. τE NMR (300 MHz, DMS0-d6) δ 7.79 (d, J=6. OHz , 1H,NH) , 5.94-5.78 (m,lH,vinylic CH) , 5.28, 5.13 (2d, J=17.4 , 10.5Hz , 2H, vinylic CH2), 4.65 (d,J=3.3Hz,lH,H-l) , 4.07 (dd, J=13.8 , 3. OHz , IH, allylic CH2) , 4.87 (dd,J=14.1,4.8 Hz, IH, allylic CH2) , 3.72-3.54 (m, 2H, H-2 , H-6 , ) , 3.52-3.32 (m,3H,H-6,H-3,H-5) , 3.11 ( t , J=9. OHz , IH, H-4 ) , 1.82 (s,3H,NCOCH3) ; 13C NMR (75 MHz, DMSO-d6) δ 163.58, 134.69, 116.53, 95.99 (C-l), 72.99 (C-5), 70.92 (C-3), 70.63, 66.87 (C-4), 60.87 (C- 6), 53.79 (C-2), 22.65.
(b) Allyl 2-acetamido-4, 6-0-benzylidine-2-deoxy-α-D-glucopyranoside (39)
To a solution of compound (38) (26 g, 101.5 mmol) in anhydrous DMF (200 mL) is added benzaldehyde dimethylacetal (45 mL, 304.5 mmol) and pTSA (2 g) and the mixture is heated at 60°C under reduced pressure (ca. 10 mm/Hg) for constant removal of MeOH formed. After 3 hours, the reaction mixture is cooled, poured into saturated solution of NaHC03 (500 mL) to precipitate out the product as solid, filtered, and washed with Et0 to furnish compound (39) (30 g, 86%) as a white crystalline solid; λK NMR (300 MHz, DMS0-d6) δ 8.11 (d, J=6. OHz, 1H,NH) , 7.5-7.3 (m,5H,ArH), 5.98-5.83 (m, IH, vinylic CH) , 5.62 (s,lH,PhCH), 5.35, 5.18 (2d,J=17.4,10.5 Hz , 2H, vinylic CH2) , 5.30 (br Ξ,1H,3-OH) , 4.79 (d, J=3.0Hz,lH,H-l) , 4.22-4.15 (m, 2H, H-2 , allylic CH2) , 3.96 (dd, J=13.5,5.4 Hz , IH, allylic CH2) , 3.91-3.41 (m,5H,H-6, H-3 , H-5 , H-4) , 1.87 (s,3H,NCOCH3) ; 13C NMR (75 MHz, DMSO-d6) δ 169.74, 162.45, 137.79, 134.48, 129.56, 129.24, 128.95, 128.10, 126.47, 111.92,
100.98, 96.90 (C-l), 82.00 (C-5), 68.08, 67.64, 67.24, 62.83 (C-6), 54.43 (C-2), 22.62.
(c) Allyl 2-acetamido-3-0-acetyl-4, 6-0-benzylidine-2-deoxy- -D- glucopyranoside (40)
To a solution of compound (39) (29.3 g, 84.19 mmol) in anhydrous pyridine (200 mL) is added acetic anhydride (11.9 mL, 102 mmol) and DMAP (4 g) . The resulting mixture is stirred at room temperature for about two and a half hours under nitrogen, and then poured into ice- cold water. The precipitated white solid is filtered, washed repeatedly with water to remove residual pyridine, and redissolved in CH2CI2. The organic layer is dried over anhydrous NaS04, and then concentrated to obtain compound (40) (23 g, 72%) as a solid; XH NMR (300 MHz, CDCI3) δ 7.51-7.31 (m,5H,ArH), 6.02-5.98 (brs,lH,NH), 5.94- 5.81 (m,lH, vinylic CH) , 5.52 (s,lH,PhCH), 5.35, 5.22 (2d, J=10.2 , 12.0 Hz, 2H, vinylic CH2) , 5.31-5.25 (m,lH,H-3), 4.87 (d,J=3.0 Hz,lH,H-l), 4.36 (td, J=9.6,3.6 Hz,lH,H-2), 4.28 (dd, J=9.9 , 4.5 Hz,lH,H-6), 4.19 (dd,J=12.0,6.0 Hz,lH, allylic CH2,), 3.98 (dd, J=12.9,6.6 Hz, IH, allylic CH2) , 3.91 (dd, J=9.9 , 4.5 Hz,lH,H-6), 3.77 (t, J=10.2,lH,H-5) , 3.72 ( t , J=9.3Hz , IH, H-4 ) , 2.05 ( s , 3H, OCOCH3) , 1.96 (s,3H,NCOCH3) ; 13C NMR (75 MHz, CDCI3) δ 171.19, 169.99, 136.82, 133.07, 128.94, 128.05, 126.01, 118.15, 101.36, 96.91 (C-l), 78.85 (C-5), 70.11 (C-3), 68.65, 68.46 (C-4), 62.79 (C-6), 52.32 (C-2), 23.01, 20.76.
(d) Allyl 2-acetamido-3-0-acetyl-6-0-benzyl-2-deoxy-α-D- glucopyranoside (41)
To a suspension of compound (40) (4.9 g, 10 mmol), NaCNBH3 (60 mmol), 3A molecular sieves in anhydrous acetonitrile (200 mL) at 0°C is dropwise added chlorotrimethylsilane (TMSCl) (60 mmol), and then stirred for five hours at room temperature. The reaction mixture is filtered through a pad of celite, and poured into a saturated solution of NaHC03, and extracted three times with methylene chloride. The methylene chloride extract is dried over anhydrous Na2S04, and concentrated, and the residue is purified by column chromatography (EtOAc) to obtain compound (41) (3.0 g, 60%) as an oil; XH NMR (300 MHz, CD3OD) δ 7.78 (d,J=6.0 Hz,lH,NH), 7.42-7.21
(m,5H,ArH), 6.01-5.86 (m,lH, vinylic CH) , 5.43 (d,J=6.3 Hz,lH,C-4- OH) , 5.33, 5.17 (2d, J=17.1 , 10.5 Hz, 2H, vinylic CH2) , 4.51 (s , 2H, PhCH2) , 5.00 (dd,J=ll.l,9.3 Hz,lH,H-3), 4.69 (d,J=3.6 Hz,lH,H-l), 4.12 (dd,J=13.5,4.8 Hz,lH,H-2), 4.03-3.93 (m,2H, allylic CH,H-6), 3.73- 3.58 (m, 2H, allylic CH,H-6) , 3.46-3.32 (m, 2H, H-4 ,H-5 ) , 1.95
(s,3H,OCOCH3) , 1.80 (s, 3H,NCOCH3) ; 13C NMR (75 MHz, CD3OD) δ 173.36, 173.04, 138.99, 134.73, 129.17, 128.90, 128.59, 128.54, 118.20, 97.34 (C-l), 74.74 (C-3), 74.30 (C-5), 72.31, 69.84 (C-4), 69.59, 69.35 (C- 6), 52.87 (C-2), 22.56, 20.91.
(e) Allyl 2-acetamido-4-0-benzyl-2-deoxy-α-D-glucopyranoside (42)
To a suspension of LiAlH4 (250 mg, 6.56 mmol) in anhydrous CH2Cl2 (32 mL) and diethyl ether (90 mL) under argon is added the acetal (40) (2.0 g, 5.11 mmol) and A1C13 (875 mg, 6.56 mmol), and the reaction is stirred under reflux for about 20 hours. The reaction mixture is quenched with EtOAc (15 mL) , filtered through a pad of celite, and then concentrated. The residue is purified by column chromatography on silica-gel using 5% MeOH -CH2CI2 as eluent to obtain compound (42) (440 mg, 25%) as a white solid; XH NMR (300 MHz,CD3OD) δ 7.59-7.21 (m,5H,ArH), 6.11-5.82 (m,lH, vinylic CH) , 5.28, 5.15 (2d, J=17.1 , 9.6 Hz,2H, vinylic CH2) , 4.93, 4.83 (2d, J=10.8 , 11.0 Hz , 2H, PhCH2) , 4.64 (d, J=10.5 Hz,lH,H-l) , 4.18-3.93 (m, 2H, allylic CH2) , 3.89-3.58 (m,5H,H-2,H-3,H-5,H-6) , 3.43 ( t , J=9.0 Hz , IH, H-4) , 1.98 (s , 3H, OCOCH3) ; 13C NMR (75 MHz, CD3OD) δ 173.81, 140.07, 135.54, 130.05, 129.43, 129.16, 128.78, 127.65, 117.73, 97.70, 80.18 (C-5), 76.16 (C-3), 73.39, 73.20(04), 69.75, 69.24 (C-6), 62.44, 55.77 (C-2), 22.71.
(f) Allyl 2-acetamido-3-0-acetyl-4-0-benzyl-2-deoxy-α-D- glucopyranoside (43) To a cooled (-78°C) solution of compound (40) (10. Og, 25.5 mmol) in CH2C12 under argon is added a solution of H3B'NMe3 complex (7.4 g, 102 mmol) in toluene (50 mL) followed by Me2BBr (7.44 mL, 64 mmol) . After stirring for about 30 minutes, 200 mL of 0.5 M sodium phosphate buffer (pH 7.3) is added, and the solution is allowed to warm to room temperature. The organic layer is washed with saturated NaHC03 solution, water, dried (Na2S04),and evaporated. The residue is purified by chromatography on silica-gel using EtOAc to obtain compound (43) (9 g, 90%) as a white solid; ^ NMR (300 MHz, CD3OD) δ 7.41-7.22 (m,5H,ArH), 6.02-5.84 (m,lH, vinylic CH2) , 5.33, 5.20 (2d,J=17.1,10.5 Hz,2H, vinylic CH2) , 5.30 (m,lH,H-3), 4.81 (d,J=3.9 Hz,lH,H-l), 4.67, 4.60 (2d, J=ll .7 , 11.6 Hz , 2H, PhCH2) , 4.22-4.16 ( , allylic CH2,H-6) , 4.12-3.88 (m,lH, allylic CH2) , 3.86-3.68 (m,3H,H- 4,H-5,H-6), 1.92 (s,3H,OAc), 1.90 (s , 3H,NHCOCH3) ; 13C NMR (75 MHz, CD3OD) δ 173.35, 172.24, 139.49, 135.15, 129.36, 129.31, 128.90, 128.77, 128.66, 128.58, 118.15, 97.50 (C-l), 77.50 (C-3), 75.66 (C- 5), 74.51, 73.01 (C-4), 69.29, 61.75 (C-6), 53.34 (C-2), 22.44, 20.95.
(g) Allyl 2-acetamido-3-0-acetyl-4-0-benzyl-2-deoxy-6-0-tosyl-α: -D- glucopyranoside (44) To a solution of compound (43) (9.0 g, 23 mmol) in pyridine (100 mL) is added p-toluenesulfonyl chloride (pTsCl) (13.0 g, 63 mmol) and DMAP (3 g) . The reaction mixture is stirred at room temperature for six hours, poured into ice-water, and then extracted with methylene chloride. The organic layer is washed with brine, dried (Na2S0 ) , and evaporated. The residue is chromatographed on silica-gel using ethyl acetate as eluent to obtain compound (44) (8.0 g, 72%) as a gummy liquid; XH NMR (300 MHz, CDCl3) δ 7.35-7.28 (m,7H,ArH), 7.21-7.18 (m,2H,ArH), 5.88-5.72 (m,2H, vinylic CH,NH) , 5.27, 5.18 (2d, J=5.4,12.0Hz,2H, vinylic CH2) , 5.26-5.24 (m,lH,H-3), 4.73 (d,J=3.6 Hz,lH,H-l), 4.60, 4.52 (2d, J=10.8 , 11.1 Hz , 2H, PhCH2) , 4.28
(dd, J=4.2,10.8 Hz,H-6) , 4.22-4.04 (m, 4H, H-2 , H-6 , allylic CH2) , 3.93- 3.81 (m,2H, allylic CH2,H-5) , 3.63 (t,J=9.3 Hz,lH,H-4) , 2.44 (s,3H,ArMe) , 1.98 (s,3H,OAc) , 1.93 (s , 3H,NC0CH3) ; 13C NMR (75 MHz, CDCI3) δ 171.10, 169.90, 144.91, 137.16, 132.96, 132.59, 129.73, 128.39, 127.91, 127.87, 127.70, 118.10, 96.13 (C-l) , 75.11 (C-3) , 74.80 (C-5) , 73.44, 68.79 (C-4) , 68.97, 68.03 (C-6) , 51.88 (C-2) , 23.04, 21.54, 20.79. (h) Allyl 2-acetamido-3-0-acetyl-6-azido-4-0-benzyl-2, 6-dideoxy-α-D- glucopyranoside (45)
To a solution of compound (44) (7.0 g, 12.9 mmol) in DMF (50 mL) is added NaN3 (8.0g, 129 mmol), and the suspension is stirred at 90°C for three hours. The reaction mixture is poured into ice-water and extracted with methylene chloride. The combined organic layer is washed with water, brine, dried (Na2S04) , and evaporated. The residue is purified by column chromatography on silica-gel eluting with 50% ethyl acetate in hexane to obtain compound (45) (4.0 g, 75%) as a white solid; ^Η NMR (300 MHz, CDC13) 57.39-7.21 (m, 5H, ArH) , 5.97-5.79 (m,2H,NH, vinylic CH) , 5.31, 5.21 (2d, J=9.9 , 10.5 Hz,2H, vinylic CH2) , 5.26-5.25 (m,lH,H-3), 5.85 (d,J=3.9 Hz,lH,H-l), 4.65, 4.57 (2d, J=ll.1,11.1 Hz, 2H,PhCH2) , 4.28 (td, J=9.6 , 3.6 Hz , IH, H-2 ) , 4.18, 3.99 (2dd, J=12.6 , 5.1 , 12.9 , 6.0 Hz,2H, allylic CH2) , 3.87
(ddd, J=9.6,5.4,2.4 Hz,lH,H-5) , 3.64 (t , J=9.0 Hz , IH, H-4) , 3.48 (dd,J=12.9,2.1 Hz,lH,H-5) , 3.64 (t,J=9.0 Hz,lH,H-4), 3.48 (dd, J=12.9,2.1 Hz,H-6) , 3.36 (dd, J=13.2 , 5.4 Hz , IH, H-6) , 1.98 (s,3H,OAc) , 1.94 (s , 3H,NCOCH3) ; 13C NMR (75 MHz, CDCl3) δ 170.88, 169.77, 137.21, 132.93, 128.26, 127.78, 127.64, 127.57, 117.93,
96.02, 76.28, 74.70, 73.33, 70.23, 68.25, 51.86, 50.79, 22.83, 20.63.
(i) Carboxymethyl 2-acetamido-3-0-acetyl-6-azido-4-0-benzyl-2 , 6- dideoxy-α-D-glucopyranoside (48) To a solution of compound (45) (4 g, 9.6 mmol) in a mixture of CCl4 (20 mL) , CH3CN (20 mL) , and H20 (30 mL) is added NaI04 (8.4 g, 39.36 mmol). To this biphasic solution RuCl3.3H20 (43 mg, 2.2 mol%) is added and the resulting mixture is stirred vigorously for about two hours at room temperature. After dilution with 50 mL of CH2C12, the reaction mixture is filtered through a pad of celite, and the phases are separated. The aqueous layer is extracted twice with CH2C12. The combined organic layer is washed with water, brine, dried (Na2S04) and concentrated to obtain the mixture of aldehyde (47) and acid (48) . This crude mixture is chromatographed on silica-gel eluting with a gradient of MeOH in CH2Cl2 to obtain aldehyde (47) (2.7 g, 67%) and acid (48) (1 g, 25%) . Jones reagent (1.17M, lOmL, 8.54 mmol) is added to a solution of compound (47) (2.7 g, 5.97 mmol) in acetone (30 mL) and the mixture is subjected to sonication for about 30 minutes. After TLC analysis, excess reagent is quenched with 2-propanol (100 mL) , the chromate salt is filtered off through a pad of celite. The green solid residue obtained after concentration is purified by flash chromatography eluting with 20% methanol /methylene chloride to afford compound (48) as a white foamy solid (2.4 g, 86 %). The overall yield for the two step oxidation is 82%; XH NMR (300 MHz, CD3OD) δ 7.49-7.19 (m,5H,ArH), 5.32 (t,J=9.9 Hz,lH,H-3), 4.96 (br Ξ,1H,NH), 4.82 (br s,lH,H-l) , 4.60 (s , 2H, PhCH2) , 4.23-4.11 (m, 2H, H-2 , CH2C02) , 4.01-3.81 (m,2H,CH2C02,H-5) , 3.62 (t,J=9.3 Hz,lH,H-4), 3.53-2.28 (m,2H,H-6), 1.94 (s,3H,OAc), 1.91 (s , 3H,NCOCH3) ; 13C NMR (75 MHz, CD3OD) δ 173.45, 172.03, 139.25, 129.44, 128.95, 128.89, 98.81 (C-l), 78.17 (C-3), 75.77 (C-5), 74.73, 71.94 (C-4), 53.24 (C-6), 52.26 (C- 2), 22.59, 20.94. Fab-MS Cι9H2N408 434.8(M-1), 437.3 (MH+) .
(j) Carboxymethyl 2-acetamido-6-azido-4-0-benzyl-2 , 6-dideoxy-α-D- glucopyranoside (IX) To a stirred solution of (48) (3.4 g, 6.8 mmol) in anhydrous methanol (40 mL) is added sodium methoxide (365 mg, 6.8 mmol). The reaction mixture is stirred for about 45 minutes at room temperature by which time all the starting material has been consumed (TLC analysis) . Excess base is neutralized with amberlite-H resin, filtered and concentrated, and purified by silica-gel chromatography eluting with 25 % MeOH in CH2CI2 to afford compound (IX) as a brownish foamy solid (2.5 g, 83%); ^ NMR (300 MHz, CD3OD) δ 7.39-7.19 (m,5H,ArH), 4.79 (d, J=ll.lHz,lH,H-l) , 4.14 (d, J=15.9 Hz , IH, CH2C02) , 4.07 (d,J=10.8 Hz,lH,H-2), 3.93 (d, J=14.1 Hz , IH, CH2C02) , 3.90 (t , J=8.7 Hz , IH, H-3 ) , 3.82-3.72 (m,lH,H-5), 3.48-3.24 ( , 3H, H-6 , H-4 ) , 2.05 (s , 3H,NCOCH3) ; 13C NMR (75 MHz, CD3OD) δ 178.07, 174.02, 139.67, 129.31, 129.08, 128.74, 98.87 (C-l), 80.28 (C-5), 75.98 (C-3), 73.89, 71.99 (C-4), 67.53, 55.19 (C-6), 52.55 (C-2), 22.90; Fab-MS Cι7H22N407 392.8 (M-l), 395 (MH+) . EXAMPLE 10: Methyl 4-azido-4-deoxy-3-0-benzoyl-2-0-carboxymethyl-α-D- fucopyranoside (X)
Methyl 2 , 3-di-0-benzoyl-4 , 6-di-O-p-toluenesulfonyl-α-D- Glucopyranoside (49)
To a stirred solution of methyl 2 , 3-di-O-benzoyl-α-D-Glucopyranoside (40g, lOOmmol) in pyridine (500ml) are added p-toluenesulfonyl chloride (91g, 479mmol ) and N,N-dimethyl-4-amino pyridine (9g) at 0°C. The reaction mixture is allowed to stir at room temperature for 48 hours. Ethanol is added to decompose the excess of reagent. The solvent is removed under reduced pressure and residue is dissolved in CH2C12 and washed with cold water, 10% aqueous H2S04 solution, water, dried over Na2SU4 and concentrated in vacuo. The residue is purified on silica gel column by using 2-5% MeOH in CH2Cl2 as eluent to give
(49) (55g,78%); lH NMR (CDC13) : 5 7.90-6.95 (m, 18H, arom. ) , 5.95(t,lH,H- 3), 5.07-5.02 (m,2H,H-l&H-2) , 4.87(t, lH,H-4), 3.37 (s , 3H, OMe) , 2.47&2.15(each s , 6H, 2xAr-CH3) . ES-MS Calcd. For C35H34θι2S2 (710) ; Found: 728.3 (M+NH4) +, 769(M+CH3COO)~.
Methyl 2 , 3-di-0-benzoyl-6-deoxy-6-iodo-4-0-p-toluenesulfonyl-α-D- Glucopyranoside (50)
A mixture of (49) (7g, 9.8mmol ) , Nal (2.2g, 14.7mmol ) and 2- butanone (100ml) is stirred under reflux for 16 hours. The mixture is cooled to room temperature and filter. The filtrate is concentrated and a solution of residue in ethyl acetate is washed with 10% aqueous Na2S2θ3 solution and water, dried over Na2Sθ4 and concentrated. Treatment of the residue with hot ether gives pure amorphous (50) (5.5g,84%); XU NMR (CDC13) : δ 7.89- 6.92 (m,14H, arom. ) , 5.98 (t , IH, H-3 ) , 5.18-5.12 (m, 2H, H-1&H-2 ) , 4.81(t,lH, H-4), 3.74-3.72 (dd,lH,H-6) , 3.50 (s , 3H, OCH3) , 3.41- 3.34 (dd,lH,H-6' ) , 2.13 (s , 3H, Ar-CH3) ; 13C NMR: 5 165.52&164.86 (2 x CO), 144.79-127.22 (arom. C) , 96.60(01), 79.00(04), 71.91(05), 69.07(03), 68.69(02), 55.92(OCH3), 21.45 (Ar-CH3) , 4.17(-CH2I). ES-MS Calcd. For C28H2709SI (666); Found: 684 (M+NH4) +, 725(M+CH3COO)~.
Methyl 2 , 3-di-0-benzoyl-6-deoxy-4-0-p-toluenesulfonyl-α-D- Glucopyranoside (51)
A solution of iodide (50) (35g, 52.5mmol ) , Bu3SnH (32ml , 119mmol ) and a catalytic amount of azobisisobutyronitrile(3.4g) in toluene (125ml) is stirred under reflux for one hour. The solution is concentrated, followed by silica gel chromatographic purification of the residue with hexane-ethyl acetate (9:1-^2:3) give (51) (24g,84%); 1H NMR (CDC13) : δ 7.86-6.85 (m,14H, arom. ) , 5.91 ( t , IH, H-3 ) , 5.10- 5.04(m,2H,H-2&H-l) , 4.69 ( t , IH, H-4 ) , 3.36 (s , 3H, OCH3) 2.07 (s , 3H, Ar- CH3) , 1.43(d,J=6.3Hz,3H,C-CH3) ; 13C NMR : δ 165.67&164.96 (2 x CO), 144.36-127.10 (arom. C) , 96.44(C-1), 81.10(04), 72.33(05), 69.55(03), 65.39(02), 55.39(OCH3), 21.41 (Ar-CH3) , 17.51 (OCH3) . ES-MS Calcd. For C28H28O9S (540); Found: 558 (M+NH4) +.
Methyl 4-azido-2 , 3-di-0-benzoyl-4-deoxy-α-D-fucopyranoside (52)
A mixture of (51) (24g, 44.4mmol ) , NaN3 (12g, 184.6mmol ) and dry DMF (150ml) is stirred at 120°C for 16 hours. The mixture is cooled to room temperature and diluted with ethyl acetate. The resulting mixture is extracted with water, dried over Na2S04 and evaporated under reduced pressure. It is purified on a silica gel column by using (4:1-^3:1) hexane-ethyl acetate as an eluent to give (52) (14g,77%); XH NMR (CDCl3) : 5 8.04-7.34 (m, 10H, arom. ) , 5.92-5.87(dd,lH,H-3) , 5.58-5.53 (dd, IH, H-2 ) , 5.09 (d, IH, H-l) , 4.23- 4.20(m, lH,H-5), 4.09-4.07 (m, IH, H-4 ) , 3.38 (s , 3H, 0CH3) ,
1.34(d,3H,C-CH3) ; 13C NMR: δ 165.82&165.67 ( 2 x CO) , 133.44- 128.29(arom. C) , 97.28(01) , 70.55(05) , 68.93(03) , 64.73(02) , 64.27(04) , 55.43(OCH3) , 17.08 (OCH ) . ES-MS Calcd. For C2ιH2ιN306 (411) ; Found: 412.3 (M+l)+, 429.3 (M+NH )+, 470 (M+CH3C00) " . Methyl 4-azido-4-deoxy-α-D-fucopyranoside (53)
To a solution of (52) (14g) in MeOH (200ml) is added IM MeONa (25ml, pH~12) and the solution is stirred at room temperature for 16 hours. The base is neutralized with IR-120 (H+) resin, filtered and concentrated under reduced pressure. It is purified on a silica gel column by using a solvent gradient consisting of 5-10% MeOH in CH2Cl2 to give (53) (6.4g,92%); lK NMR (CDC13) : δ 4.73(d,lH,H-l) , 4.08-3.74(m,4H,H-3,H-5,H-3&H-2) , 3.39(s,3H, OCH3), 1.29(d,3H,OCH3); "c NMR: δ 99.42(01), 71.00(05),
69.27(03), 66.32(02), 64.97(04), 55.40(OCH3), 17.20 (OCH3) . ES-MS Calcd. For C17Hι3N304 (203); Found: 221 (M+NH4) +, 262 (M+CH3COO)~
Methyl 4-azido-2-0-benzoyl-4-deoxy-3-0-methoxycarbonylmethyl-α-D- fucopyranoside (54) and Methyl 4-azido-3-0-benzoyl-4-deoxy-2-0- methoxycarbonylmethyl-α-D-fucopyranoside ( 55 )
A mixture of (53) (2.4g, 12mmol ) and Bu2Sn0 (3.9g, 15.7mmol) in toluene (60ml) is heated for four hours at reflux temperature with azeotropic distillation of water. The solution is concentrated to 40ml, then Bu4NI (2.6g, 7mmol ) and BrCH2C00Me (5.7ml, 60mmol) are added to the reaction mixture. It is heated under reflux for four hours and then evaporated to dryness to give a crude mixture of lactone and alkylated products. This crude mixture is passed through small silica gel column using hexane-ethyl acetate (4:l->2:3) as the eluent. The fractions corresponding to the products are concentrated and treated with methanolic MeONa (pH~10) for lh to convert the lactone to a methyl ester. It is neutralized with IR-120 (H+) resin, filtered and evaporated under reduced pressure. A solution of this crude material in pyridine (30ml) is treated with BzCl (5ml) for four hours at room temperature. The solvents are removed in vacuo and the residue is dissolved in ethyl acetate and washed with 10% aqueous citric acid, water, aqueous NaHC03 solution, dried (Na2S0 ) and concentrated under reduced pressure. It is purified on a silica gel column using a solvent gradient consisting of 15-20% ethyl acetate in hexane to give (54) (0.4g,9%); XH NMR (CDC13) : δ 8.07-7.45(m,5H,arom. ) , 5.34-5.30 (dd, IH, H-2), 5.00 (d, IH, H-1 ) , 4.36-4.03 (m,5H,OCH2, H-4, H-3&H-5) , 3.68&3.31 (each s , 6H, 2xOCH3) , 1.31 (d,3H,C-CH3) .
Further elution with a solvent gradient consisting of 20-40% ethyl acetate in hexane provides (55) (1.4g,31%); XR NMR (CDC13) : δ 8.08-7.45(m,5H,arom. ) , 5.63-5.59 (dd, IH, H-3 ) , 5.05 (d, IH, H-1 ) , 4.26-3.95 (m,5H, H-4, H-2, H-5&OCH2) , 3.63&3.43 (each s , 6H, 2xOCH3) , 1.27 (d,3H,C-CH3) ; 13C NMR: δ 170.67&165.52 (2 x CO), 133.50-
128.55(arom. C) , 98.75(01), 75.49(02), 72.90(05), 69.02(OCH2), 64.84(03), 64.15(04), 55.45&51.81 (2xOCH3 ) , 17.03 (OCH3) . ES-MS Calcd. For C17H21N3O7 (379); Found: 397(M+NH4)\
Methyl 4-azido-3-0-benzoyl-2-0-carboxymethyl-4-deoxy-α-D- fucopyranoside (X)
A solution of (55) (0.5g, 1.32mmol) and Lil (0.85g, 6.3mmol in pyridine (20ml) is stirred for four hours at 120°C. The solvent is evaporated under reduced pressure and the residue is dissolved in CH2C12 and washed with saturated saline solution, dried (Na2S04) and concentrated under reduced pressure. The residue is purified by silica gel column chromatography using CH2Cl2-MeOH (9:1-^3:2) as the eluent to give (X) (0.26g,54%); XH NMR (CD3OD) : δ 8.12-7.48 (m,5H, arom. ) , 5.65-5.60 (dd, IH, H-3 ) , 5.03 (d, IH, H-1) , 4.14-4.12 (m,4H,H-4,H-5&OCH2) , 3.29 (s , 3H, 0CH3) , 1.26 (d, 3H, C-CH3) ; 13C NMR: δ l66.96(CO), 134.88-129.85 (arom. C) , 99.70(01), 75.70(02), 74.71(05), 72.77(OCH2), 66.17(03), 65.76(04), 55.35(OCH3), 17.37( CH3) . ES-MS Calcd. For Ci6Hι9N307 (365); Found: 383.3 (M+NH4) +, 364 (M-l)". EXAMPLE 11: Phenyl 3-azido-3-deoxy-4-Q-methyl-l-thio-β-D- glucopyranosiduronic acid (XI)
(a) Phenyl 2-0-acetyl-3-azido-3-deoxy-6-0-benzoyl-l-thio-β-D- glucopyranoside (64b) Trifluoromethanesulfonic anhydride (Tf20) (25mL; 41.9g; 148.7mmol) is added dropwise over 10 minutes to a stirred solution of 1, 2 : 5 , 6-di-0- isopropylidene-D-allofuranose (35g; 35.0mmol), commercially available from Pfanstiehl Laboratories, Inc. (Waukegan, IL) , in dry pyridine (150mL) under argon at -20°C. The reaction mixture is allowed to warm to room temperature over two hours, then diluted with ethyl acetate (250mL) and washed with ice-cold brine (800mL) . The aqueous layer is extracted once with a further portion of ethyl acetate (250mL) , then the combined organic layers are washed with 3N citric acid (2 x 500mL) and brine (lOOmL) , then dried over Na2S04 and evaporated to give compound (56) as a yellow brown oil (49.3g;
125mmol; 93%): Rf (30% ethyl acetate-hexane) : 0.39. XH NMR (300 MHz, CDC13) : δ 1.32, 1.36, 1.43, 1.56(12H, 4x s, isopropylidene, CH3, s); 3.88(1H, dd, J = 4.8 and 8.7 Hz); 4.07-4.19 (3H,m) ; 4.75(1H, t, J=5.7 Hz), 4.89(1H, dd, J=5.1 and 6.9 Hz), 5.82(1H, d, J=3.9 Hz, H-1). MS m/e 415 [M+Na]
Compound (56) (49.30g; 126mmol) and sodium azide (16.40g; 252mmol) are stirred in dry DMF (200mL) at 100°C for 30 min. The reaction mixture is cooled to room temperature, filtered, then diluted with water (200mL) and extracted with ethyl acetate (2 x 150mL) . The organic layers are washed with water (2 x 300mL) then brine (lOOmL) , dried over Na2S0 , and concentrated under reduced pressure to give compound (57) as a light yellow oil (37.3 g, quantitative yield); Rf (50% ethyl acetate-hexane): 0.64. 'Ή NMR (300 MHz, CDC13) : δ 1.32, 1.36, 1.43, 1.5K12H, 4 x s, isopropylidene CH3 s); 3.99(1H, dd) ;
4.08-4.25 (4H, m) ; 4.62(1H, d, J=3.3 Hz), 5.86(1H, d, J=3.3 Hz); MS m/e 308 [M+Na] .
Compound (57) (106g; 372mmol) is dissolved in a mixture of trifluoroacetic acid (50mL), dioxane (200mL) and 1.1 M aqueous HCl
(90mL) , and heated at 50-60 °C for 21 hours. The reaction mixture is neutralized with sodium hydroxide and concentrated to give a quantitative yield of compound (58) as a brown oil. XH NMR (300 MHz, CD3OD) : δ 3.1-3.75(10H, m) , 4.55(1/2H, d, H-1); 5.05(1/2H, d, H-1). MS FAB: m/e 228 [M+Na]. A stirred solution of compound (58) (38.6g; 180mmol) in pyridine containing a catalytic amount of 4-dimethylaminopyridine (DMAP) (0.5g) is treated dropwise with acetic anhydride (147g; 1.44mol). The reaction mixture is stirred overnight at room temperature then diluted with water (600mL) . After stirring for about 20 minutes, the reaction mixture is extracted with ethyl acetate (2 x 500mL) . The combined organic phases are washed with saturated sodium bicarbonate solution (2 x 400mL) , 2N HCl (2 x 300mL) and brine (200mL) , then dried over Na2S04 and evaporated to give compound (59) as a brown oil (62.5g; 166mmol); Rf (50% ethyl acetate-hexane): 0.45. XH NMR (300 MHz, CDCI3) : δ (2:1 mixture of β:α anomers at C-l) 2.04-2.11 (12H, 8s, 4x[02CCH3]); 3.66(1H, dd, J=9.0 and 9.9 Hz, H-3β) ; 3.76(1H, m, H-5β) ; 3.94(1H, t, J=10.2 Hz, H-3α) ; 3.98-4.23 (3H, m) ; 4.88-5.03 (2H, m) ; 5.64(2/3H, d, J=8.1 Hz, H-lβ) ; 6.26(1/3H, d, J=3.6 Hz, H-lα) . MS FAB: m/e 396 [M+Na]
BF3.Et20 (50mL; 40.6mmol) is added to a stirred solution of compound (59) (56. Og; 150mmol) and thiophenol (30ml; 28.7mmol) in dry dichloromethane (125mL) . The reaction mixture is heated under reflux for six hours then kept at room temperature for 16 hours. The reaction mixture is diluted with dichloromethane (300mL) and washed with water (300mL) , saturated sodium bicarbonate solution (3 x 600mL) , 2N hydrochloric acid (2 x 500mL) , water (600mL) and brine (2 x 500mL) , then dried over Na2S04 and evaporated to a yellow solid (72 g) . The crude product containing both the alpha and beta isomers is recrystallized from ethyl acetate/hexane to give compound (60)
(37.96g; 90mmol; 60%) as a white solid. mp 135°C. Rf (50% ethyl acetate-hexane): 0.53. X NMR (300 MHz, CDC13) : δ 2.07, 2.11 and 2.17(9H, 3xs, 3x 02CCH3) ; 3.66(2H, t and , Jt=9.9 Hz, H-3 and H-5), 4.17(2H, m, 2 x H-6); 4.65(1H, d, J=9.8 Hz, H-lβ); 4.91 and 4.93(2H, 2 x t, H-4 and H-2 ) , 7.27-7.52 (5H, m, Ar-H) . FAB MS: m/e 445 [M+Na] A stirred suspension of compound (60) (38. Og; 90mmol) in dry methanol (250mL) is treated with sodium methoxide (4.0g; 18.5mmol) at room temperature under argon. After two hours, the reaction mixture is adjusted to pH=7 with Dowex 50 (H+) resin, then filtered and the filtrate is evaporated to give compound (61) as a white solid (28. Og; quantitative yield), mp 159-160 °C . Rf (ethyl acetate): 0.44. XH NMR
(300 MHz, CD3OD) : δ 3.18-3.39 (4H, m) ; 3.66(1H, dd, J=10.5 and 5.4 Hz, H-6); 3.84(1H, d, J=10.5 and 2.0 Hz, H-6'); 4.64(1H, d, J=9.8 Hz, H- lβ) ; 7.27-7.33(3H, m, Ar-H) ; 7.53-7.56 (2H, d, Ar-H) ; MS m/e 320 [M+Na] .
Compound (61) (28g; 94mmol) and trimethyl orthobenzoate (39mL; 230mmol) are stirred in anhydrous acetonitrile (250mL) containing a catalytic amount of p-TSA (1.34 g) for 3 hours at room temperature. The solvent is evaporated and the residue is redissolved in fresh anhydrous acetonitrile (250mL) and stirred for a further two hours. Solid sodium bicarbonate is added (8.6g) and the crude compound (62) is concentrated under reduced pressure to a slurry. MS /e 438 [M+Na] and 384 [M-OMe] . The mixture is treated with acetic anhydride (17mL; 165mmol) , pyridine (80mL) and 4-dimethylamino pyridine (70mg) and the reaction mixture is stirred overnight at room temperature. TLC in 10% ethyl acetate-hexane showed no starting material. The reaction mixture is poured into water (500mL) and extracted with ethyl acetate (200mL) . The organic phase is washed with water (2 x 500mL) , saturated sodium bicarbonate solution (2 x 500mL) , 3N citric acid solution (2 x 500mL) then water (2 x 500mL) and dried over sodium sulfate. The solution is concentrated at 80°C to remove most of the remaining trimethyl orthobenzoate, giving compound (63) as a yellow gel (50. lg; quantitative yield). Rf (10% ethyl acetate- hexane): 0.31. Η NMR (300 MHz, CDC13) : δ 2.18(3H, s, 02CCH3) ;
3.09(3H, Ξ, -OCH3); 3.56(1H, dt , H-5); 3.75(1H, t, J=10.2 Hz, H-3); 4.00-4.10 (2H, m, 2 x H-6); 4.18(1H, t, J=10.2 Hz, H-4); 4.72(lH,d, J=10.0 Hz, H-1); 4.90(1H, t, J=10.0 Hz, H-2); 7.26-7.60 ( 10H, m, Ar- H) . A solution of compound (63) (50. lg; 94mmol) in acetonitrile (lOOmL) is treated with a 9:1 (v:v) mixture of trifluoroacetic acid and water (20mL) . The solution turns reddish and a white precipitate is formed after about one minute. The suspension is stirred for about two hours then filtered to remove the solid 6-0-benzoate product (12g) . A second crop of the 6-0-benzoate is obtained by concentration of the filtrate to 40mL and the remainder is evaporated to a yellow oil, containing primarily compound (64a) with a little of the 6-0-benzoate regioisomer (64b) and methyl benzoate. The crude product is purified by column chromatography (eluent: 20-50% ethyl acetate-hexane followed by 10% methanol dichloromethane) to give phenyl 2-0-acetyl- 3-azido-3-deoxy-4-0-benzoyl-l-thio-β-D-glucopyranoside (64a) : Rf (50% ethyl acetate-hexane): 0.33; λ NMR (300 MHz, CDC13) : δ 2.18 (3H, s, 02CCH3); 3.6-3.8 (3H, m, H-5, 2 x H-6); 3.89 (IH, t, J=9.8 Hz, H-3); 4.78 (IH, d, J=10.0 Hz, H-1); 4.98 (IH, t, J=9.8 Hz, H-2); 5.12 (IH, t, J=9.5 Hz, H-4); 7.31-7.35 (3H,m, -SAr-H) ; 7.44-7.51 (4H,m, 2 x- SPh-Ho, 2 x-Bz-Hm ); 7.61 (IH, t, Bz-Hp) ; 8.03 (2H, d, Bz-H0) . MS m/e 466 [M+Na] ; and phenyl 2-0-acetyl-3-azido-3-deoxy-6-0-benzoyl-l-thio- β-D-glucopyranoside (64b): Rf (50% ethyl acetate-hexane): 0.48. XH NMR (300 MHz, CDC13): δ 2.17 (3H, s, 02CCH3); 2.49 (IH, br s, OH);
3.47 (IH, t, J=9.5 Hz); 3.61 (IH, t, J=9.5 Hz); 3.67 (IH, m, H-5); 4.59 (IH, dd, J=2.2 and 12.2 Hz, H-6); 4.67 (IH, d, J=10.0 Hz, H-1); 4.68 (IH, dd, J=4.6 and 12.2 Hz, H-6); 5.36 (IH, t, J=9.8 Hz, H-4); 7.14-7.26 (3H,m, -SAr-H); 7.44-7.49 (4H,m, 2 x-SPh-HD, 2 x-Bz-Hm) ; 7.60 (IH, t, Bz-Hp); 8.03 (2H, d, Bz-H„) . MS m/e 466 [M+Na].
(b) Phenyl 3-azido-3-deoxy-4-0-methyl-l~thio-β-D-gluco- pyranosiduronic acid (XI) . To a solution of compound (64b) (16g; 0.037 mol) in anhydrous DMF (200mL) is added silver oxide (16 g) with stirring. Methyl iodide
(25mL; 0.401mol) is added to the resulting suspension and the mixture is stirred at room temperature for 24 hours. The reaction mixture is diluted with ethyl acetate (400mL) , filtered, washed with water, washed with brine, dried (Na2S04) and concentrated. The crude compound is purified by flash column chromatography eluting with 20% EtOAc/hexane to afford compound (65) (15g; 91%) as a white solid; p 75-77 °C; XH NMR (300 MHz, CDC13): δ 8.01 (d, J=8.1 Hz, 2H, ArH) , 7.5- 7.4(m, 5H, ArH), 7.3-7.1(m, 3H, ArH), 4.84(t, J=10.2 Hz, IH, H-2), 4.71(dd, J=12, 2.1 Hz, IH, H-6 pseudo equatorial), 4.64(d, J=9Hz, IH, H-1), 4.44 (dd, J=12 , 6 Hz, IH, H-6 pseudo axial), 3.71-3.53 (m, 2H, H- 3 and H-5, containing a singlet at 3.55, OMe), 3.21(t, J=9.3 Hz, IH, H-4), 2.18(s, 3H, OAc) . 13C NMR (75 MHz, CDCl3) : δ 169.15, 165.85, 133.15, 132.68, 132.65, 131.76, 129.56, 129.54, 129.52, 128.66, 128.35, 128.33, 127.94, 88.67, 78.46, 69.90, 67.84, 63.00, 60.66, 60.63, 20.75; FAB MS: m/z 480 (M+Na].
To a stirred solution of compound (65) (15g; 0.033mol) in anhydrous methanol (200mL) is added sodium methoxide (3.65g; 0.067mol). The reaction mixture is stirred for four hours at room temperature by which time all the starting material has been consumed (TLC analysis) . Excess base is neutralized with Amberlite-H+ resin, filtered and concentrated to afford compound (66) as a white solid in quantitative yield (mp 182-185°C) .
Jones reagent (0.7M; 36mL) is added to a solution of compound (66) (9.67g; 0.033mol) in acetone (150mL) and the mixture is sonicated. After about one hour, another portion (0.7M; 30mL) of Jones reagent is added and the sonication is continued for about 45 minutes. After TLC analysis, excess reagent is quenched with isopropanol (lOOmL) and the chromate salt is filtered off through a pad of Celite. The green solid residue obtained after concentration is purified by flash chromatography eluting with 5% methanol/methylene chloride to afford compound (XI) as a white foamy solid (mp 182-185 °C) . K NMR (300MHz, CDC13) : δ 7.21-7.75(m,5H, ArH), 4.58(d, J=9.3 Hz, IH, H-1), 3.93(d, J=9.3 Hz, H-5), 3.44-3.62(m, IH, H-3, containing a singlet at 3.54 - OMe), 3.26-3.42(m, 2H, H-2, 4); 13C NMR (75 MHz, CDC13) : δ 171.56,
132.91, 132.42, 130.80, 129.11, 128.48, 88.72, 79.23, 77.66, 70.61, 68.72, 60.50; FAB MS: calcd for Cι305SHι5N3 325. Found 324 (- ve mode), 343 (+ ve mode, M +18 (NH4) ) .
Example 12: Methyl 6-azido-4-0-carboxymethyl-6-deoxy-3-0-methyl-α-D- glucopyranoside (XII) Methyl 2-0-benzoyl-4 , 6-0-benzylidene-3-0-methyl-α-D-glucopyranoside (67)
To a stirred solution of methyl 2-0-benzoyl-4 , 6-0-benzylidene-a-D- glucopyranoside (lOg) in DCM is added 2,6-di tert butyl-4-methyl pyridine (11. lg) and trimethyloxonium tetrafluoroborate (6.1g). After processing as described for the preparation of (28) the 3-0-methyl compound (67) (8g, 77%) is obtained. The compound was purified by column chromatography on silica gel: :H NMR (CDC13) : δ 8.05-7.36 (m, 9H, arom), 5.59 (s, IH, acetal H) , 5.07-5.01 (m, 2H, H- 2 & H-1), 4.34-4.30 (dd, IH, H-4), 3.61 & 3.30 (s, 6H, 2 x OMe); 13C NMR: δ 101.35 (acetal C) , 97.78 (C-l), 81.86 (C-3), 77.73 (C-5), 73.65 (C-2), 68.90 (C-4), 62.27 (C-6), 61.05 & 55.32 (2 x OMe).
Methyl 2-0-benzoyl-3-0-methyl-α-D-glucopyranoside (68)
The product resulting from removal of 4 , 6-O-benzylidine group from (67) (15g) is obtained by treatment with 90% aq TFA in DCM under identical conditions as described for (32)to give (68) (lOg, 82%); :H NMR (CDC13) : δ 8.13-7.48 (m, 5H, arom), 5.07-5.06 (d, IH, H-1), 5.02- 4.97 (dd, IH, H-2), 3.63 & 3.39 (s, 6H, 2 x OMe); 13C NMR: δ 97.24 (C- 1), 81.28 (C-3), 74.12 (C-5), 70.91 (C-2), 69.92 (C-4), 61.85 (C-6), 61.16 & 55.25 (2 x OMe) .
Methyl 6-azido-2-0-benzoyl-6-deoxy-3-0-methyl-α-D-glucopyranoside (69)
Compound (68) (14g) is transformed under similar reaction conditions as described for the preparation of (33) by utilizing Ph3P/ NBS/ NaN3 in DMF to give (69) (12.5g, 83%); lH NMR: δ 8.09-7.45 (m, 5H, arom), 5.03-5.02 (d, IH, H-1), 5.;00-4.95 (dd, IH, H-2), 3.87-3.81 (m, IH, H-5), 3.75 (t, IH, H-4), 3.58 & 3.38 (s, 6H, 2x OMe); 13C NMR: δ 97.12 (C-l), 81.06 (C-3), 73.84 (C-5), 70.56 (C-2), 70.32 (C-4), 60.96 & 55.28 (2 x OMe), 51.28 (C-6). Methyl 6-azido-2-0-benzoyl-4-0-methoxycarbonylmethyl-6-deoxy- 3-O-methyl-α-D-glucopyranoside (70) .
Compound (69) (14g) is alkylated under similar reaction conditions as described for the preparation of (9) to give (70) (lOg, 59%) after silica gel column chromatography: XH NMR (CDC13) : δ 8.08 -7.43 (m, 5H, arom), 4.98-4.91 (m, 2H, H-2 & H-1), 4.41 (bs, 2H, OCH2) , 3.87 (t, IH, H-4), 3.75-3.67 (m, 4H, OMe & H-5), 3.61-3.59 (m, 2H, H-6), 3.52 (s, 3H, OMe), 3.44-3.40 (dd, IH, H-3), 3.35 (s, 3H, OMe); 13C NMR: δ 96.76 (C-l), 81.67 (C-3), 78.7 (C-4), 73.81 (C-5), 69.50 (O 2), 69.11 (OCH2), 60.57, 55.12, 51.58 (3 x OMe) , 51.24 (C-6).
Methyl 6-azido-4-0-carboxymethyl-6-deoxy-3-0-methyl-α-D- glucopyranoside (XII).
Compound (70) (9.5g) is de-O-benzoylated and hydrolyzed, as described for the synthesis of I, to give XII (5g, 74%) ; XH NMR (CD3OD) : δ 4.62- 4.61 (d, IH, H-1) , 4.31-4.30 (m, 2H, OCH2) , 3.75-3.69 (m, IH, H-5) , 3.63-3.57 (m, 4H, H-4 & OCH3) , 3.45 (t, IH, H-3) , 3.41 (s, 3H, OMe) , 3.29-3.27 (m, 2H, H-6) , 3.20 (t, IH, H-2) ; 13C NMR: d 101.21 (C-l) , 85.44 (C-3) , 80.33 (C-4) , 73.88 (C-5) , 71.27 (C-2) , 70.12 (OCH2) , 61.06 & 55.76 (2 x OMe) , 52.72 (C-6) ; ES-MS calcd. For C10H17N3O7 (291) : Found: 292 [M +1]+, 309.2 [M + NH4T, 314 [M + Na]+, 290 [M - 1]".
Example 13: Methyl 6-azido-3-0-benzoyl-4-0-carboxylmethyl-6-deoxy- 2-O-methyl-α-D-glucopyranoside (XIII)
Methyl 4, 6-0-benzylidene-3-0-benzoyl-2-0-methyl-α-D-glucopyranoside (71)
A solution of methyl 4 , 6-0-benzylidene-2-0-methyl-a-D-glucopyranoside [Grindley, et.al., J. Carbohydr. Chem. 15, 95-108, (1996)] (44g) in pyridine (400 ml) is treated with BzCl (90 ml) for five hours at room temperature. After processing, as described for the preparation of (30), compound (71) (54g, 91%) was obtained; ^Η NMR (CDCI3) : δ 8.10- 7.27 (m, 9H, arom.), 5.80 (t, IH, H-3), 5.50 (s, IH, acetal H) , 4.98- "4.96 (d, IH, H-1), 4.35-4.30 (dd, IH, H-4), 4.02-3.92 (m, IH, H-5), 3.82-3.72 ( , 2H, H-6), 3.62-3.58 (dd, IH, H-2), 3.47 & 3.44 (s, 6H, 2 x OMe); 13C NMR: d 101.19 (acetal C) , 98.17 (C-l), 79.89 (C-2), 79.41 (C-5), 71.27 (C-3), 68.77 (C-4), 62.23 (C-6), 59.11 & 55.16 (2 x OMe) .
Methyl 3-0-benzoyl-2-0-methyl-α-D-glucopyranoside (72)
Compound (71) (52g) is converted to (72) (28g, 69%) under reaction conditions as described for the preparation of (32): λK NMR (CDCl3) : δ 8.05-7.43 (m, 5H, arom.), 5.44 (t, IH, H-3), 4.92-4.91 (d, IH, H- 1), 3.44 & 3.43 (s, 6H, 2 x OMe); 13C NMR: δ 97.34 (C-l), 79.16 (C-2), 76.19 (C-5), 71.30 (C-3), 69.82 (C-4), 61.87 (C-6), 59.18 & 55.16 (2 x OMe) .
Methyl 6-azido-3-0-benzoyl-6-deoxy-2-0-methyl-a-D-glucopyranoside (73) . Compound (72) (26g) is reacted under reaction conditions as described for the preparation of (33) to give (73) (22.5g, 80%); XH NMR (CDCl3: δ 8.11-7.49 (m, 5H, arom.), 5.44 (t, IH, H-3), 5.00-4.99 (d, IH, H- 1), 4.10-4.03 (m, IH, H-5), 3.83-3.74 (m, 2H, H-6), 3.55-3.40 (m, 2H, H-4 & H-2); 13C NMR: δ 97.22 (C-l), 78.93 (C-2), 76.26 (C-5), 70.80 (C-3), 70.44 (C-4), 59.22 & 55.30 (2 x OMe) 51.13 (C-6).
Methyl 2-0-methyl-3-0-benzoyl-6-deoxy-6-azido-4-0-t- butoxycarbonylmethyl-α-D-glucopyranoside (74) .
To a solution of (73) (102g, 0.301mol) in anhydrous DMF (IL) is added successively silver oxide (348.8g, 1.505 mol, 5 eq. ) and tetrabutylammonium iodide (167g, 0.542mol, 1.5 eq. ) . The resulting suspension is cooled in a water/ ice bath and t-butyl bromoacetate (177.8 ml, 1.204 mol, 4 eq. ) is added dropwise over about 15 minutes. The mixture is stirred for about one half hour then warmed to room temperature and stirred for about 20 hours. The suspension is diluted with ethyl acetate (1.5L) . The solid was filtered off through Celite. The filtrate is washed with saturated NaHC03 solution (2 x 500 ml) . The yellow precipitate was filtered off through Celite. The filtrate is washed with saline. The organic phase is dried (Na2S0 ) and evaporated under reduced pressure to give a dark brown syrup, which is purified on a silica gel column (EtOAc : Hexane, 1 : 5 to 2:3) to give (74) (66g, 48%), a colorless syrup; XH NMR (CDC13) : δ 1.37 (s, 9H, t-Bu) , 3.38 (s, 3H, OMe), 3.43 (dd, IH, J=10, 3.6 Hz), 3.48 (s, 3H, OMe), 3.64 (t, IH, J=9.9Hz), 3.76-3.75 (m, 2H) , 4.10- 4.96 (m, 2H) , 4.94 (d, IH, J-3.3 Hz, H-1), 5.71 (t, IH, J=9.6 Hz), 7.63-7.46 (m, 3H, arom.), 8.08-8.12 (m, 2H, arom.); LC-MS: 469 (M + NH4) .
Methyl 2-0-methyl-3-0-benzoyl-6-deoxy-6-azido-4-0-carboxymethyl-α-D- glucopyranoside (XIII).
Compound (74) (21g, 47 mmol) is dissolved in DCM (75ml) and cooled in an ice bath. TFA (25ml, 386mmol, 8.2 eq. ) is added and the mixture is allowed to warm to room temperature and stirred for about four hours. The solvent is then removed under vacuum. Excess acid is removed by co-evaporation with toluene. The residue is dissolved in DCM (300ml) and washed with saline (3 x 70ml), dried (Na2S04) and concentrated. Precipitate was formed during concentration. The precipitate was collected and washed with DCM to give the product
(XIII) (lOg, 66%), a white crystalline solid; Η NMR (CD3OD) : δ 3.36 (s, 3H, OMe), 3.49 (s, 3H, OMe), 3.55-3.51 (m, IH) , 3.75-3.67 (m, 2H) , 3.91-3.86 (m, IH) , 4.21-4.07 (m, 2H) , 5.01 (d, IH, J=3.6 Hz), 5.59 (t, IH, J-9.9 Hz), 7.66-7.46 (m, 3H, arom.), 8.11-8.08 (m, 2H, arom.); 13C NMR: (CD3OD) : 52.56, 55.75, 58.94, 69.86, 71.29, 75.68, 79.51, 80.82, 98.10; LC-MS:413 [M + NH4]+), 394[M-H]".
Example 14: Methyl 6-azido-3 , 6-dideoxy-4-0-carboxymethyl-α-D- glucopyranoside (XIV)
Methyl 2-0-benzoyl-4 , 6-0-benzylidene-3-deoxy-α-D-glucopyranoside (75). To a suspension of methyl 2-0-benzoyl-4 , 6-0-benzylidene-a-D- glucopyranoside [Kim, et.al., J. Org. Chem., 50, 1751, (1985)] (1.9g) in toluene (30ml) is added thiocarbonyldiimidazole (1.8g). The reaction mixture is heated under reflux for about two hours . The solvent is then evaporated and the residue is dissolved in ethyl acetate and washed with cold aqueous 10% HCl, aqueous NaHC03 solution, saline, dried over Na2≤04, and concentrated under vacuum. The residue is taken up in toluene (30ml) and AIBN (0.2g) and Bu3SnH (2ml) are added. The reaction mixture is heated under reflux for about one hour and the solvent is removed under vacuum. The residue is dissolved in acetonitrile and washed with hexane. The acetonitrile layer is concentrated under vacuum and the residue is purified by chromatography on silica gel (Hexane :EtOAC, 9:1 to 3:1) to give the product (75) (1.2g, 66%); XH NMR (CDC13): δ 8.08-7.35 (m, 9H, arom.), 5.56 (s, IH, acetal H) , 5.16-5.10 (m, IH, H-2), 4.96-4.94 (d, IH, H-1), 4.32-4.27 (dd, IH, H-4), 3.49 (s, 3H, OMe), 2.38-2.32 (m, 2H, H-3); 13C NMR: δ 101.80 (acetal C) , 96.89 (C-l), 76.34 (C-5), 69.35 (C-2), 69.29 (C-4), 63.90 (C-6), 55.19 (OMe), 29.66 (C-3).
Methyl 2-0-benzoyl-3-deoxy-α-D-glucopyranoside (76) .
Compound (75) (14g) is hydrolyzed under similar reaction conditions as described for the preparation of (32) to give (76) (8g, 76%); H NMR (CDCI3) : δ 8.04-7.38 (m, 5H, arom.), 5.05-4.97 (m, IH, H-2), 4.92-4.91 (d IH, H-1), 3.41 (s, 3H, OMe), 2.34-2.26 (m, IH, H-3 eq. ) , 2.18-2.04 (m, IH, H-3 ax.); 13C NMR: δ 96.16 (C-l), 72.10 (C-5), 69.32 (C-2), 65.17 (C-4), 62.06 (C-6), 55.05 (OMe), 32.27 (C-3).
Methyl 6-azido-2-0-benzoyl-3 , 6-dideoxy-α-D-glucopyranoside (77) .
Compound (76) (1.96g) is treated with Ph3P/NBS/NaN3 in DMF under similar reaction conditions as described for the synthesis of (33) to give (77) (1.6g, 75%) after silica gel column chromatography; XH NMR (CDCI3) : δ 8.08-7.43 (m, 5H, arom.), 5.08-5.02 (m, IH, H-2), 4.96- 4.95 (d, IH, H-1), 3.57 (t, IH, H-4), 3.47 (s, 3H, OMe), 2.36-2.31 (m, IH, H-3 eq.), 2.16-2.04 (m, IH, H-3ax) ; 13C NMR: δ 96.05 (C-l), 71.74 (C-5), 69.01 (C-2), 65.90 (C-4), 55.09 (OMe), 51.27 (C-6), 32.49 (C-3) .
Methyl 6-azido-2-0-benzoyl-4-0-methoxycarbonylmethyl-3 , 6-dideoxy-α-D- glucopyranoside (78).
Compound (77) (1.5g) is treated with BrCH2COOMe/ Ag20 / Bu4NI in DMF under identical conditions as described for the preparation of (9) to give (78) (1.8g 65%) after silica gel column chromatography (EtOAc: Hexane, 1:9); λK NMR (CDC13): δ 8.09-7.44 (m, 5H, arom.), 5.04- 4.94 (m, 3H, H-2 & H-1), 4.25-4.14 (m, 2H, OCH2) , 3.49 (s, 3H, OMe); 13C NMR: δ 95.98 (C-l), 73.48 (C-4), 70.14 (C-5), 68.73 (C-2), 65.44 (OCH2) , 55.01 &51.20 (2 x OMe), 51.76 (C-6), 28.51 (C-3).
Methyl 6-azido-3 , 6-dideoxy-4-0-carboxymethyl-a-D-glucopyranoside (XIV) .
Compound (78) (1.2g) is transformed under similar reaction conditions as described for the preparation of (I), to give (XIV) (0.75g, 90%); Η NMR (CD3OD) : 5 4.59-4.58 (d, IH, H-1), 4.07-3.91 (m, 2H, 0CH2) ,
3.45 (s, 3H, OMe) , 2.28-2.24 (m, IH, H-3 eq. ) , 1.78-1.64 (m, IH, H-3 ax.) ; 13C NMR: δ 100.06 (C-l) , 75.11 (C-4) , 71.81 (C-5) , 68.01 (C-2) , 55.54 (OMe) , 52.74 (C-6) , 32.98 (C-3) ; ES-MS calc . for C9Hι5N306 (261) : Found: 262 [M+l]+, 279 [M+NH4]+, 284 [M+Na]+, 260 [M-l]~
Example 15: Methyl 2-azido-3-0-benzoyl-4-0-carboxymethyl-2-deoxy-
6-O-methyl-β-D-glucopyranoside (XV)
Methyl 2-azido-2-deoxy-β-D-glucopyranoside (79).
1 , 3 , 4 , 6-tetra-0-acetyl-2-azido-2-deoxy-D-glucopyranose is prepared by the procedure of Wong, et. al . (Tet. Lett. 37, 6029-6032, 1996). The compound is then reacted with Me3SiBr / BiBr3 in DCM followed by treatment with DCM / CH3OH / Ag20 under identical conditions as described for the preparation of (5) to give (79) in 90% yield; 1H NMR (CD3OD) : δ 4.21 (d, IH, H-1), 3.86-3.82 (m, IH, H-5), 3.70-3.64
(dd, IH, H-4), 3.52 (s, 3H, OMe), 3.32-3.24 (m, 3H, H-3& H-6), 3.16- 3.10 (dd, IH, H-2); 13C NMR : 5 103.93 (C-l), 77.54 (C-5), 76.09 (C-3), 71.21 (C-4), 67.78 (C-2), 62.28 (C-6), 57.26 (OMe).
Methyl 2-azido-3-0-benzoyl-2-deoxy-b-D-glucopyranoside (80).
To a stirred solution of (79) (54g) in DMF:DMP (2:1, 540ml) is added p-toluenesulfonic acid (6g) , and the mixture is stirred for about two hours. The acid is neutralized with triethylamine and the mixture is concentrated to dryness. The residue is taken up in ethyl acetate and washed with saturated aqueous NaHC03 solution, dried over Na2S04, and evaporated in vacuo. The residue is then taken up in pyridine (500ml) , and benzoyl chloride (100ml) is added. The mixture is stirred for about 16 hours at room temperature. The mixture is cooled in an ice / water bath and ethanol is added to consume any unreacted acid chloride. The solvent is removed under vacuum and the residue is taken up in ethyl acetate and washed with water, cold aqueous NaHC03 solution, dried over Na2S04, and concentrated under vacuum. The residue is treated with 90% aqueous TFA in DCM, as described for the preparation of (32), to give (80) (68g, 85% in 3 steps); XH NMR (CDCl3): δ 8.04-7.39 (m, 5H, arom.), 5.09 (t, IH, H-3), 4.36 (d, IH, H-1), 3.82-3.72 ( , 2H, H-5 & H-4), 3.55 (s, 3H, OMe), 3.51-3.45 (dd, IH, H-2); 13C NMR : δ 102.87 (C-l), 75.96 (C-5), 75.50 (C-3), 68.98 (C-4), 63.90 (C-2), 61.52 (C-6), 57.34 (OMe).
Methyl 2-azido-3-0-benzoyl-2-deoxy-6-0-methyl-β-D-glucopyranoside (81) .
Compound (80) (60g) in DCM (600ml) is reacted with Me3OBF4 / DTBMP under similar reaction conditions as described for the preparation of (28), to give (81) (40g, 64%), along with 4 , 6-di-O-methyl derivative (15%); XH NMR (CDC13): δ 8.05-7.42 (m, 5H, arom.), 5.14-5.08 (dd, IH, H-3), 4.33 (d, IH, H-1), 3.56 & 3.35 ( s, 6H, 2 x OMe), 3.54-3.48 (dd, IH, H-2); 13C NMR : δ 102.63 (C-l), 75.91 (C-5), 74.58 (C-3), 71.30 (C-6), 69.44 (C-4), 63.74 (C-2), 59.28 & 57.03 (2 x OMe). Methyl 2-azido-3-0-benzoyl-4-0-t-butoxycarbonylmethyl-2-deoxy- 6-O-methyl-b-D-glucopyranoside (82) .
Compound (81) (40 g) is transformed to (82) (37 g, 69%) under similar reaction conditions as described for the preparation of (73); """H NMR (CDC13) : 5 8.09 - 7.43 (m, 5H, arom.), 5.30 (t, IH, H-3), 4.35 - 4.30 (m, 3H, H-1 & OCH2) , 3.57 & 3.40 (s, 6H, 2 x OMe), 1.27 (bs, 9H, CMe3) ; 13C NMR: 5 102.73 (C-l), 81.27 (C-4), 77.42 (C-5), 74.16 (C-3), 70.25 (C-6), 70.06 (OCH2) 64.13 (C-2), 58.98 & 57.03 (2xOMe) , 27.58 (C-CH) .
Methyl 2-azido-3-0-benzoyl-4-0-carboxymethyl-2-deoxy-6-0-methyl-β-D- glucopranoside (XV) .
The tert-butyl ester of compound (82) (37 g) is hydrolysed with DCM- TFA (2:1) as described for the preparation of (XIII) to give (XV) (19.5 g, 60%); XH NMR (CD3OD) : 5 8.11 - 7.49 (m, 5H, arom.), 5.32 - 5.26 (dd, IH, H-2), 4.44 (d, IH, H-1), 4.18 - 4.04 (m, 2H, OCH2) , 3.56 & 3.39 (s, 6H, 2 x OMe), 13C NMR : 5 103.98 (C-l), 78.53 (C-4), 76.12 (C-5), 75.44 (C-3), 72.01 (C-6), 70.70 (OCH2), 65.68 (C-2),
59.50 & 57.43 (2 x OMe). ES-MS calcd. for C17H21N3O8 (395) found: 396 [M+l]+, 413 [M+NH4]+, 418.3 [M+Na]+, 394 [M-l]~.
Example 16: Methyl 2-azido-3-0-benzoyl-4-0-carboxymethyl-2-deoxy- 6-O-ethoxycarbonylmethyl-β-D-glucopranoside (XVI)
Methyl 2-azido-3-0-benzoyl-6-0-ethoxycarbonylmethyl-2-deoxy-β-D- glucopyranoside (83).
To an ice cold solution of (80) (6.5 g) in DCM (100 ml), ethyl diazoacetate (3.3 ml) is added under argon atmosphere. A solution of BF3-etherate (7.5 ml) in DCM (30 ml) is then added to the reaction mixture. The mixture is allowed to stir at room temperature for about 16 hours and then cold aqueous NaHC03 solution is added with stirring. The organic layer is separated out, dried under Na2S0 , and concentrated under reduced pressure. The residue is purified via silica gel column chromatography using hexane-ethyl acetate (9:1 to 1:1) to give (83) (3.1 g, 43%), along with the dialkylated derivative (10%); XH NMR (CDCl3): δ 8.09 - 7.43 (m, 5H, arom.), 5.18 (t, IH, H- 3), 4.35 (d, IH, H-1), 4.22 - 4.13 (m, 4H, 2 x OCH2) , 3.58 (s, 3H, OMe), 1.24 (m, 3H, CH2-CH3) ; 13C NMR: δ 103.19 (C-l), 75.08 (C-5), 74.71 (C-3), 69.45 (OCH2) , 68.69 (C-6), 67.62 (OCH2) , 64.22 (C-4), 61.45 (C-2), 57.27 (OMe), 14.02 ( Me) .
Methyl 2-azido-3-0-benzoyl-4-0-tert-butoxycarbonylmethyl- 6-0-ethoxycarbonylmethyl-2-deoxy-β-D-glucopranoside (84) .
Compound (83) (3.6 g) is alkylated with Ag20/BuNI/BrCH2C00Bu- 1 in DMF under similar reaction conditions as described for the synthesis of compound (73) to give (84) (2.0 g, 44%); 13C NMR (CDC13) : δ 102.86 (C-l), 81.40 (C-4), 74.60 (C-5), 74.46 (C-3), 70.26 (C-6), 69.60, 68.94, & 64.19 (3 x OCH2) , 60.69 (C-2), 57.12 (OMe), 27.75 (CMe) , 14.01 (CH2-CH3) .
Methyl 2-azido-3-0-benzoyl-4-0-carboxymethyl-2-deoxy- 6-O-ethoxycarbonylmethyl-β-D-glucopyranoside (XVI) .
The tert-butyl ester of compound (84) (2.0 g) is hydrolyzed with DCM- TFA as described for the preparation of (XIII) to give (XVI) (1.5 g, 84%); XH NMR (CD3OD) : δ 8.15-7.47 (m, 5H, arom.), 5.33 (t, IH, H-3), 4.49 (d, IH, H-1), 4.37-4.12 (m, 6H, 2 x OCH2) , 3.93 (t, IH, H-4), 3.59 (s, 3H, OMe), 1.29 (t, 3H, CH2-CH3) ; 13C NMR : δ 103.93 (C-l), 78.32 (C-4), 76.11 (C-5), 75.58 (C-3), 70.99, 70.28, & 65.63 (3 x OMe), 69.82 (C-6), 61.88 (C-2), 57.43 (OMe), 14.52 (CCH3) . ES-MS calcd. for C2oH25N3Oιι (467) found: 485 [M+NH4]+, 466 [M-l]".
Example 17: Methyl 4-azido-2-0-benzoyl-3-0-carboxymethyl-4-deoxy-α-D- fucopyranoside (XVII)
Compound (54) (4.1 g) is treated with LiI (9 g) in pyridine under similar reaction conditions as described for the preparation of (X) to give (XVII) (2.3 g, 58%) after silica gel column chromatography using DCM-MeOH (9:1 a 3:2) ; XH NMR (CD3OD) : δ 8.04 - 7.40 (m, 5H, arom.) , 5.19 - 5.14 (dd, IH, H-2) , 4.29 (d, IH, H-4) , 3.27 (s, 3H, OMe) , 1.25 (d, 3H, OMe) ; 13C NMR : δ 96.55 (C-l) , 77.96 (C-3) , 72.98 (C-5) , 66.42 (C-2) , 66.01 (C-4) , 55.69 (OMe) , 17.52 (OMe) . ES-MS calcd. for Cι6Hι9N307 (365) Found: 383 [M+NH ]+, 364 [M-l]".
Example 18: Methyl 3-azido-2-0-carboxymethyl-6-fluoro-3 , 6-dideoxy-β- D-glucopyranoside (XVIII).
Methyl 3-azido-2-0-methyoxycarbonylmethyl-4-0-benzoyl-3-deoxy-β-D- glucopyranoside (85)
Compound (17) (5.8 g) is benzoylated with pyridine-BzCl and followed by the removal of p-methoxybenzyl group with DDQ in a manner analogous to that described for the preparation of (10) to give (85) (3.29 g, 87%) ; XH NMR (CDC1 ) : δ 8.01 - 7.43 (m, 5H, arom.) , 4.98 (t, IH, H-4) , 4.42 (d, IH, H-1) , 4.36 - 4.35 (m, 2H, 0CH2) , 3.88 (t, IH, H-4) , 3.73 (s, 3H, OMe) , 3.69 - 3.48 ( , 3H, H-6 & H-5) , 3.47 (s, 3H, OMe) , 3.30 - 3.24 (dd, IH, H-3) ; 13C NMR: d 103.77 (C-l) , 81.41 (C-2) , 74.66 (C-5) , 69.26 (OCH2) , 69.21 (C-4) , 65.08 (C-3) , 61.05 (C-6) , 56.94 & 51.48 (2 x OMe) .
Methyl 3-azido-4-0-benzoyl-2-0-methoxycarbonylmethyl-3 , 6-dideoxy-6- fluoro-β-D-glucopyranoside (86)
To a solution of (85) (0.8 g) in DCM (20 ml), DAST (1 ml), is added at - 15° C. The reaction mixture is allowed to warm at room temperature and stirring is continued for about five hours at room temperature. The saturated aqueous NaHC03 is added to the reaction mixture very carefully. The organic layer is separated out, dried over Na2S04, and evaporated under reduced pressure. It is purified on silica gel column chromatography using hexane-ethyl acetate (9:1 a 3:1) to give (86) (0.3 g; 50%, on the basis of starting material reacted); H-NMR (CDCI3) : δ 8.02 - 7.43 (m, 5H, arom.), 4.99 (t, IH, H-4), 4.52 (dd, J=4Hz , IH, H-6), 4.42 (d, J=45.6 Hz, H-6, F) , 4.41 (d, IH, H-1), 4.37 (d, J=4Hz, IH, H-6), 3.82 (t, IH, H-2), 3.73 &
3.48 (s, 6H, 2 x OMe), 3.32-3.27 (dd, IH, H-3); 13C NMR: 5 103.61 (C- 1) , 81.33 (d, J=175 Hz, C-6) , 81.23 (C-2) , 73.18 (d, J=19 Hz, C-5) , 69.16 (OCH2) , 68.28 (J =7Hz, C-4) , 65.04 (C-3) , 56.93 & 51.79 (2 x OMe) .
Methyl 3-azido-2-0-carboxymethyl-3 , 6-dideoxy-6-fluoro-β-D- glucopyanoside (XVIII) .
Compound (86) (0.25 g) is de-O-benzoylated and hydrolyzed in a similar manner as described for the synthesis of (I) to give (XVIII) (0.13 g, 74%) ; 'H NMR (CD3OD) : 5 4.63 (d, IH, 6a) , 4.54 (d, J=47.7 Hz, H-6,F) , 4.40 (d, IH, H-1) , 4.26 - 4.09 (m, 2H, OCH2) , 3.53 (t, IH, H- 3) , 3.44 (s,3H,OMe) , 3.05 - 2.99 (dd, IH, H-3) ; 13C NMR : δ 104.98 (C- 1) , 82.85 (d, J=172 Hz, H-6) , 81.54 (C-2) , 76-59 (d, J=18 Hz, C-5) , 72.36 (OCH2) , 69.43 (C-4) , 69-28 (C-3) , 57.14 (OMe) . ES-MS Calcd. for C9Hι4N306F (279)
Found: 280[M+1]+, 297 [M+NH4]+, 278 [M-l]~.
Example 19: Methyl 2-azido-3-0-benzoyl-4-0-carboxymethyl-2 , 6-dideoxy- 6-f luoro-β-D-glucopyranoside (XIX)
Methyl 2-azido-3-0-benzoyl-3 , 6-dideoxy-6-f luoro-β-D-glucopyranoside (87) .
Compound (80) (1.3 g) is treated with DAST (2.2 ml) under similar reaction 'conditions as described for the preparation of (86) to give
(87) (0.34 g 26%) ; 1HNMR (CDC13) : δ 8.03 - 7.40 (m, 5H, arom.) , 5.49 - 5.48(dd, IH, H-3) , 4.55-4.52 (dd, IH, H-6) , 4.46 (d, J=46.5 Hz, H-6, F) , 4.39 - 4.37 (dd, IH, H-6) . 4.24 (d, IH, H-1) , 3.92 - 3.84 (m, IH, H-5) , 3.75 - 3.71 (dd, IH, H-4) , 3.60(t, IH, H-2) . 3.58(s, 3H, OMe) ; 13C NMR : δ 103.21 (C-l) , 81.15 (d, J=171 Hz, C-6) , 71.97 (d, J=23.4 Hz, C-5) , 70.93 (C-3) , 69.15 (d, J=6Hz, C-4) , 63.81 (C-2) , 57.38 (OMe) .
Compound (XIX) (0.12 g, 51%) is obtained from (87) by the same reaction sequence as described for the preparation of (XIII) from
(74) . 13C NMR (CD3OD) : d 104.32 (C-l) . 82.73 (J=170 Hz, C-6) , 79.38 (C-4) , 73.21 (d, J-22Hz, C-5) , 68.01 (C-3) , 64.50 (C-2) , 57.48 (OMe) ES-MS calc. for d6Hι8N307F (383) Found: 384 [M+l] +, 401-3 [M+NH ] + , 382 [M-l]~.
Example 20: Methyl 6-azido-4-0-carboxymethyl-6-deoxy-3-0-methyl-β-D- galactopyranoside (XX)
Methyl 2-0-benzoyl-3 , 4-0-isopropylidene 6-O-p-toluenesulf onyl-β-D- galactopyranoside (88) .
To a stirred solution of methyl 3 , 4-O-isopropylidene-b-D- galactopyranoside (23.4 g) [ Catelani, et. al . Carbohydr. Res. (1988), 182, 297] in pyridine (300 ml) is added p-toluenesulfonyl chloride (28.8 g) at 0° C. The reaction mixture is allowed to stir at room temperature for about 16 hours. Benzoyl chloride (29.2 ml) is then added to reaction mixture and stirring is continued for another five hours. Ethanol is added to decompose the excess of reagent. The solvent is removed under reduced pressure and residue is taken up in DCM and washed with cold water, 5% aqueous HCl, saturated aqueous NaHC03 solution, dried over Na2S04 and concentrated in vacuo. The residue on warming with ether gave pure amorphous (88) (29 g, 59%); XH NMR (CDC13) : δ 8.07 - 7.36 (m, 9H, arom.), 5.18 (t, IH, H-2), 4.41 (d, IH, H-1), 4.37 - 4.16 (M, 5H, H-3, H-4, H-5 & H-6) 3.44 (s, 3H, OMe), 2.47 (s, 3H, Ar-CH3) , 1.54 & 1.30 (each s, 6H, 2 x OMe); 13C NMR: δ 110.84 (acetal C), 101.20 (C-l), 76.58 (C-5), 72.97 (C-3), 72.89 (C-2), 70.65 (C-6), 68.58 (C-4), 56.71 (OMe), 27.39 & 26.17 (2x Me) 21.61 (Ar-Me) .
Methyl 6-azido-2-0-benzoyl-6-deoxy-β-D-galactopyranoside (89;
A mixture of (88) (27 g) , NaN3 (70 g) and dry DMF (250 ml) is stirred at 110°c for 30 hour. After processing as described for the preparation of (52) . The residue is treated with 90% aqueous TFA in DCM as reported for (32) to give (89) (17 g, 96%) after trituration with ether; XK NMR (CDC13) : δ 8.02 - 7.39 (m, 5H, arom.), 5.22 (t, IH, H-2), 4.47 (d, IH, H-1), 3.96 (d, IH, H-4), 3.86-3.77 (m, 2H, H-3 & H-5), 3.70 - 3.66 (dd, IH, H-6), 3.53 (s, 3H, OMe), 3.31 - 3.25 (dd, IH, H-6); 13C NMR: δ 101.84 (C-l), 74.47 (C-5), 73.42 (C-2), 72.40 (C- 3), 69.45 (C-4), 56.97 (OMe), 51.11 (C-6).
Methyl 6-azido-2-0-benzoyl-6-deoxy-3-0-methyl-β-D-galactopyranoside (90) .
A mixture of (89) (17g) and Bu2SnO (17 g) in methanol (300 ml) is heated for four hours at reflux temperature. The solvent is removed and coevaporated with toluene under reduced pressure. This residue is dissolved in DMF (200 ml) and CH3I (35 ml) is added to the reaction mixture. It is heated at 65° C for 1 hour and left stirring at room temperature for 16 hours. The solvent is evaporated under reduced pressure and the residue purified on silica gel column chromatography by using hexane-ethyl acetate (9:1 - 1:1) to give (90)
(15 g, 85%); XH NMR (CDC13) : δ 8.09 - 7.46 (m, 5H, arom.), 5.40 (t, IH, H-2), 4.51 (d, IH, H-1), 4.11 (d, IH, H-4), 3.89-3.82 (dd, IH, H- 5), 3.76 - 3.72 (dd, IH, H-6), 3.53 (t, IH, H-3), 3.51 (s, 3H, OMe), 3.41 (S, 3H, OMe), 3.38 - 3.34 (dd, IH, H-6); 13C NMR : δ 101.84 (C- 1), 80.96 (C-3), 74.11 (C-5), 71.06 (C-2), 65.96 (c-4), 57.87 d 56.66 (2 x OMe) , 51.02 (C-6) .
Methyl 6-azido-2-0-benzoyl-4-0-methoxycarbonylmethyl-6-deoxy-3-0- methyl-β-D-galactopyranoside (91) .
Compound (90) (14 g) is treated with BrCH2COOMe (30.4 ml), BuNI (35.6 g) , Ag2θ (92 g) in DMF (500 ml) under identical condition as described for the synthesis of (9) to give (91) (8.0 g, 77%, on the basis of starting material reacted along with the recovery of starting material, 5.5 g) . XH NMR (CDCl3) : δ 8.13-7.48 (m, 5H, arom.), 5.60 - 5.54 (m, IH, H-2), 4.58 - 4.30 (m, 3H, OCH2 & H-1), 4.07 (d, IH, H-1), 4.00 - 3.93 (dd, IH, H-6), 3.80, 3.78 (each s, 6H, 2xOMe) , 3.62 - 3.56 (dd, IH. H-6) 3.45 (s, 3H, OMe); 13C NMR: δ 101.89 (C-l), 82.81 (C-3), 74.17 (C-5). 73.72 (C-4), 71.44 (OCH2) , 68.24 (C- 2), 58.73, 56-36 & 51.56 (3 x OMe), 51.27 (C-6).
Methyl 6-azido-4-0-carboxymethyl-6-deoxy-3-0-methyl-β-D- galactopyranoside (XX) .
Compound (91) (5.5 g) is de-o-benzoylated and hydrolyzed in a similar manner as described for the synthesis of (I) to give (XX) (2.7 g, 69%); lH NMR (CD30D) :5 4.23 - 4.13 (m, 3H, H-1 & OCH2) , 3.90 (d, IH, H-4), 3.80-3.73 (dd, IH, H-2), 3.68-3.57 (m, 2H, H-5 & H-3), 3.49 & 3.47 (s, 6H, 2 x OMe), 3.37 - 3.32 (dd, IH, H-6), 3.22 - 3.18 (dd, IH, H-6); 13C NMR : 5 105.73 (C-l), 85.60 (C-3), 75.71 (C-5), 75.61 (C-4), 71.73 (C-2), 58.99 & 57.31 (2 x OMe), 52.81 (C-6). ES-MS Calcd. for C10H17N3O7 (291) .
Found: 292 [M+l]+, 309.2 [M+NH4]+, 314 [M+Na] + 290 [M-l ]" .
EXAMPLE 21: General Synthesis of Scaffold Library Compounds
(a) General Procedure
The libraries outlined within were synthesized using Irori microcanisters (available from Irori Quantum Microchemistry (LaJolla, CA) with radio frequency tagging and an Accutag 100 software system. This approach allows for the synthesis of large numbers of compounds through the use of directed sorting. At each combinatorial step the canisters are sorted into the proper flasks for the upcoming reaction. After these steps, and during all intervening washes, the cans are handled in a single container. In most cases, approximately 1 mL of solvent is used for each canister present in a reaction flask. Agitation of large numbers of cans (>500) is performed through mechanical stirring at the low speed. This approach is used for all washes and for the non-combinatorial reactions. When the canisters are sorted into separate flasks for a combinatorial step agitation is provided by orbital shaking at around 125rpm. For analysis of the reaction intermediates at each step, two methods are used. For the preliminary dipeptide formation, photometric FMOC determination is used to quantify the loading of the second amino acid. For the subsequent steps, cleavage of a small sample of the resin (3-5mg) is required. Cleavage reactions are conducted with 20% trifluoroacetic acid (TFA) in DCM for about 30 minutes. The resulting products are analyzed by direct infusion MS, and, if necessary, LC-MS.
(b) Resin Dispensing
Thirty milligrams of the appropriate Novasyn® TGT FMOC-amino acid resin (available from Novabiochem, San Diego, CA) is dispensed into each microcanister . The dispensing is performed on a TECAN® liquid handling platform (available from TECAN SLT Instruments, Research Triangle Park, NC) by slurrying the resin (30mg/mL) in 4:1 dichloromethane (DCM)/ tetrahydrofuran (THF) and dispensing lmL to each canister.
(c) Dipeptide Synthesis The canisters are washed twice with N, JV-dimethylformamide (DMF) and then treated with piperidine for 30 minutes. Subsequently, the canisters are washed with DMF and sorted for the addition of the second amino acid in the dipeptide. To each reaction flask is added DMF, the FMOC-amino acid, 0- (7-azabenzotriazol-l-yl ) -1 , 1 , 3 , 3- tetramethyluronium hexafluorophosphate (HATU) , and finally diisopropylethylamine (DIPEA) . After shaking the canisters overnight, the canisters are washed extensively with DMF. Proper loading of the second amino acid is quantified by photometric FMOC analysis and complete removal of the FMOC group is accomplished by treating the canisters with 20% piperidine in DMF for about 30 minutes. Subsequently, the canisters are rinsed four times with DMF.
(d) Coupling of the Sugar
The sugar is coupled to the dipeptide by treating the canisters with the appropriate scaffold carboxylic acid in the presence of equimolar HATU and DIPEA. The canisters are stirred overnight and proper coupling is ensured by LC-MS analysis of the glycopeptide . (e) Reduction of the Azido Group
The canisters are washed with DMF and THF prior to the addition of trimethylphosphine for reduction of the azido group. The reaction is allowed to proceed for at least six hours, at which time the absence of the azido group is checked by diffuse reflectance IR.
(f) Coupling of the Carboxylic Acid to the Amino Group
After washing with THF and then DMF, the canisters are sorted and treated with a coupling solution of the carboxylic acid, HATU and DIPEA. The canisters are rinsed after overnight shaking by washing with DMF and THF. Complete amide formation is ensured by LC-MS analysis of the products.
(g) Carbamate Formation
The canisters are sorted and then treated with the appropriate isocyanate in the presence of triethylamine . The canisters are then washed with THF and then DMF. All of the canisters are treated with piperidine for about 30 minutes to remove any byproducts formed due to incorporation of more than one isocyanate molecule per scaffold molecule. After this treatment the cans are washed again with DMF and then THF. Complete formation of the mono-carbamate is checked by LC-MS.
(h) Guanidine formation
After washing the canisters four times with DMF and two times with DCM, the cans are sorted and then treated with 0.3 M of the appropriate isothiocyanate in tetrahydrofuran (THF) . The canisters are shaken for about 30 minutes, 0.3M triphenyl phospine is then added. The canisters are agitated overnight at room temperature to form the carbodiimides . The canisters are then washed four times with anhydrous THF, twice with DCM and sorted. Each batch of cans is treated with the appropriate primary or secondary amine and agitated overnight at room temperature. After this treatment, the cans are washed again with THF and then with DCM. Complete formation of the guanidines is then checked by LC-MS.
(i) Urea formation The canisters are sorted and then treated with the appropriate isocyanate in the presence of triethyl amine. The canisters are shaken overnight and then the canisters in each batch are washed with THF and twice with DCM. Resin is removed from a single canister from each batch and analyzed for product formation by LC/MS.
(j) de-O-Benzoylation (if necessary)
Treatment of the protected scaffolds with lithium hydroxide at this stage provides the free alcohol at the four position of the sugar. This is performed by stirring the canisters in the presence of 0.05M lithium hydroxide in 1:1 THF/methanol for four hours.
(k) Anomeric Thiophenyl Hydrolysis
If a thiophenyl group is present at the anomeric position, it is quickly removed using an 8mg/mL solution of mercury (II) trifluoroacetate in THF with 1% v/v water added. The reaction is conducted at 60°C with mechanical stirring for 15 minutes and complete lactol formation is checked by LC-MS.
EXAMPLE 22 : Preparation of a Library Comprising 1920 Compounds
Ninety-six microkans containing radio-frequency tags are inserted into each of twenty 96-well microtiter plates. The appropriate mass of each resin (see Table 1) is swelled in 500 mL 4:1 dichloromethane (DCM)/ tetrahydrofuran (THF). One mL of each slurry is dispensed robotically into each well (30mg/can) .
Table 1: Resin Dispensing
Figure imgf000070_0001
The cans are scanned on the Irori scanning station and placed in a 5L round bottom flask. After washing the cans twice with 1.5L N, N- dimethylformamide (DMF), they are treated with 1.6L 20% piperidine for about 30 minutes. The cans are then washed four times with 1.5L DMF. For coupling the second amino acid, the cans are sorted into five 2L flasks (384 cans/flask) and each batch is then reacted with the appropriate FMOOAmino acid, 0- (7-azabenzotriazol-l-yl) -1 , 1 , 3 , 3- tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamme (DIPEA) in 400mL of DMF (see Table 2 for exact reagent amounts and concentrations) . The cans are then shaken at 110 rpm for about 16 hours.
Table 2 : Reagents for Coupling the Second Amino Acid (.025M FMOC- amino acid, .025M HATU & .025M DIPEA)
Figure imgf000071_0001
Each set of 384 cans is washed twice with 400mL of DMF. The cans are then combined and washed four times with 1.5L of DMF before being treated with 1.5L of 20% piperidine in DMF for about 30 minutes. After washing the cans four times with 1.5L of DMF, the cans are treated with 0.011M phenyl 3-azido-3-deoxy-4-0-Me-l-≤'-β-D- glucopyranosiduronic acid (XI) (MW=325, 5.99g, 1.6X/amine), 0.011M HATU (7.11g) and 0.011M DIPEA (3.26mL) in 1.7L DMF. The mixture is mechanically stirred overnight. After washing four times with 1.7L of DMF and twice with 1.7L of THF, the cans are swelled in a combination of 722.5mL of distilled THF, 765mL of ethanol and 170mL of water. To this cocktail is added 42.5mL of 1.0M trimethylphosphine in THF (0.025M). The mixture is then stirred overnight at room temperature. After washing the cans six times with 1.7L of THF, the cans are washed twice with 1.7L of DMF and then sorted into eight 2L flasks (240 cans per flask) in preparation for the acylation reactions.
To each flask is then added 250mL of DMF followed by the carboxylic acid, the HATU and the DIPEA. See Table 3 for exact amounts.
Table 3: Reagents for Amide Formation (0.025M acid, HATU & DIPEA)
Figure imgf000072_0001
The reaction mixtures are then shaken at 120rpm for 18 hours at room temperature. Each flask is washed twice with 250mL of DMF. The cans are then combined and washed four times with 1.7L of DMF and twice with 1.7L of THF.
The cans are sorted into six 2L flasks (320 cans/ flask) . Each flask is evacuated, purged with argon twice, and then washed twice with 400mL of THF. Flask 6 is then washed with an additional 2 x 400mL of DMF. THF (300mL) is added to flasks 1,2,3,4 and 5, while 300mL of DMF is added to flask 6. The appropriate volume of isocyanate and triethylamine are added to flasks 1-5 while the appropriate volume of isocyanate and mass of copper chloride are added to flask 6 (see Table 4) .
Table 4 : Reagents for Carbamate Formation (0.4M isocyanate, 0. IM triethylamine)
Figure imgf000073_0001
The reaction flasks are then shaken overnight at 110 rpm. Each reaction flask is washed twice with 400mL of THF. All the cans are then combined and washed four times with 1.7L of THF. The cans are then treated with 1.7L of 20% piperidine in DMF for about 30 minutes before being washed six times with 1.7L of THF. The cans are manually sorted and half of them are treated with .019M mercury (II) trifluoroacetate (8g) in 1.0L THF with 1% water added (lOmL). The reaction mixture is stirred mechanically for 15 minutes at 60°C. The cans are then washed six times with 1.7L of THF and four times with 1.7L of DCM. The cans are archived and individually cleaved in Irori Accucleave cleavage stations by adding 3mL of 20% trifluoroacetic acid in DCM to each tube. After shaking the stations at 250rpm for 90 minutes, the products are transferred to 48-well microtiter plates by vacuum filtration.
Preparation of a 1920 member Library
Radio frequency tags are inserted into microkans in twenty 96-well microtiter plates. 30g of rink amide resin is swelled in lOOOmL 5:2 dichloromethane (DCM) / tetrahydrofuran (THF) and a 500μL slurry was dispensed into each canister (15mg/can) using the TECAN mini-prep- dispensing robot. The solvent is drained and the canisters are capped. After washing the cans twice with 1.7L N, N- dimethylformamide DMF they are treated with 1.7L 20% piperidine for 30 minutes. The cans are washed four times with 1.7L low amine DMF and then washed twice with DCM and dried. The cans are scanned and sorted into six bins using the Irori Aufco≤Ort™-10K. Each batch of 320 cans is transferred into a 2L reaction vessel and coupled with the first amino acid using the appropriate Fmoc-amino acid, 0- (7- azabenzotriazole-1-yl ) -1 , 1 , 3 , 3 , -tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in 400mL low amine DMF (see table one for the exact reagent amounts and concentrations) .
Table 1: Reagents for coupling of the first amino acid (.025M Fmoc-Amino acid, .025M HATU and .025M DIPEA) in 450ml of DMF
Figure imgf000074_0001
Figure imgf000075_0001
The cans are shaken on an orbital shaker at 110-120 rpm overnight at room temperature. Each batch of cans is washed four times with DMF and then the complete coupling of the first amino acid is analyzed by two methods. The first tests for free amine using Kaiser test, where the resin beads turn blue if positive or remain brown if negative. The second test of the amino acid coupling quantifies Fmoc loading photometrically on the resin1.
After proper coupling of the first amino acid is confirmed, all the 1920 canisters are combined and treated with 20% piperidine in DMF for 30 minutes. The cans are washed four times with DMF, then washed twice with DCM and dried. After sorting the cans into four bins (480 cans/bin) , cans in each bin are transferred into a 2L reaction vessel containing 450mL of low amine and then coupled to the second amino acid as described above (see table 2 for the exact reagent amounts and concentrations) .
Table 2. Reagents for coupling of the second amino acid (.025M Fmoc-Amino acid, .025M HATU and .025M DIPEA) in 450ml of DMF).
Figure imgf000075_0002
The cans are washed four times with DMF, then analyzed for complete coupling of the second amino acid, followed by treatment with 20% piperidine in DMF for 30 minutes. After washing the cans with DMF and then washing with DCM, the cans are dried and sorted and then
Novabiochem catalog & peptide synthesis handbook, 1997-1998 coupled with the third amino acid as before (see reagent quantities " in table 3 ) .
Table 3: Reagents for coupling of the third amino acid (.025M Fmoc- Amino acid, .025M HATU and .025M DIPEA) in 450ml of DMF).
Figure imgf000076_0001
After washing the cans with DMF and checking for the complete coupling of the third amino acid, all the canisters are combined and the Fmoc is removed by treatment with 20% piperidine in DMF for about 30 minutes followed by washing four with DMF. The sugar scaffold carboxylic acid (0.013M, mw=394, 8.71g, 1.6eq. 22mmole) is coupled to the tripeptides using equimolar HATU (mw=394, 8.36g) and DIPEA (mw=129, .742g/mL, 3.82mL) in 1.7L of low amine DMF. The canisters are shaken overnight and then washed four times with DMF and then washed twice with DCM. A small portion of the resin was taken out of a canister to test for proper coupling of the sugar by both the Kaiser test and by LC-MS analysis of the glycopeptide .
The canister are sorted into 5 reaction vessels and canisters in batch 2-5 are treated with the appropriate isocyanate and triethylamine (0.01M, mw=101, 4.6g, d=726g/ml, 6.3mL) in 350mL of anhydrous THF (see table 4 for the exact amounts of the isocyanates) . To protect the 3 -OH from reacting with the isocyanates in the next urea formation combinatorial step, cans in vessel 1 are treated with 400mL acetic anhydride: pyridine: THF (1:1:2).
Table 4: Isocyanates for carbamate formation (.40M Isocyanate, .10M Et3N in THF, 350ml in each reaction)
Figure imgf000077_0001
After shaking the reaction vessels overnight at 110-120 rpm, cans in each vessel are washed four times with 400mL THF and washed twice with DMF. Cans in vessels 2-5 are then treated with 20% piperidine for about 30 minutes to remove any bis-carbamates and then washed four times with 400mL DMF. To check for complete formation of the carbamates, some resin is removed from a single can from each vessel and individually cleaved with 20% trifluoroacetic acid (TFA) , 2% triethylsilane in DCM. The solvent is evaporated and the products are analyzed by LC-MS.
All the canisters are combined and washed four times with 1.8L THF and then added into a solution of 722.5mL distilled THF, 765mL ethanol and 170mL water. To this mixture is added 42.5mL of 0. IM trimethylphosphine in THF (.025M). The mixtures was agitated overnight at room temperature and then washed six times with 1.8mL THF. Following this, the cans are washed twice with DCM and dried. Some resin is removed from two cans and checked for the absence of azide by diffuse reflectance IR.
All the cans are sorted into 4 bins for the urea formation combinatorial step. Each set of 480 cans is treated with 0.4M of the appropriate isocyanate, 0.05M triethylamine in 450mL of anhydrous THF (see table 5 for exact amounts) . All the canisters are left shaking overnight and then the canisters in each batch are washed four times with 500mL THF. Resin is removed from a single canister from each batch and the cleaved products are analyzed for complete formation of the ureas by LC-MS.
Table 5: Reagents for urea formation (0.4M isocyanate, 0.13M triethylamine, in 450mL THF)
Figure imgf000078_0001
To hydrolyze the 3-O-acetyl ester and the ethyl ester (incorporated with the ethyl 3-isocyanatoacetate) , all the canisters are combined and treated with . IM lithium hydroxide (mw=24, 6g) in THF: Methanol (2.5L 1:1) for three hours. Following ester hydrolysis, the cans are washed four times with THF and then washed four times with DCM. Some resin from two cans is taken out, cleaved and complete ester de- protection checked by LC-MS.
The canisters are combined, archived and individually cleaved in the Irori Accucleave 96-well cleavage stations by addition of 2mL of 20 TFA, 2% triethylsilane in DCM into each well. After shaking the cleavage stations for about 90 minutes on the orbital shaker at 250 rpm, the products were drained into 48-well microtiter plates by vacuum filtration. A second rinse is performed by adding lmL of the acidic cleave cocktail into each well, shake the cleavage station for 20-30 minutes and drain the wells as described before.
EXAMPLE 23: Library Preparation on a Safety-Catch Resin
1) Dispense 20mg of 4-Sulfamylbutyryl or 4-Sulfamylbenzoyl Safety- catch resin (available from Novabiochem USA) into each canister with the TECAN mini-prep dispensing robot. After capping the canisters, wash them twice with low-amine dimethylformamide (DMF). 2) Add the first amino acid to the resin by reacting the cans in a . IM solution of the Fmoc-protected amino acid in the presence of . IM benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (pyBOP) and .15M diisopropylethylamine (DIPEA) in DMF. Agitate the cans on an orbital shaker overnight at room temperature.
3) After washing the cans four times with DMF and four times with dichloromethane (DCM) , the loading of the first amino acid should be checked through photometric Fmoc-determination as described for the other scaffolds. 4) The cans are then treated with 20% piperidine in DMF for about 30 minutes before again washing the cans four times with DMF.
5) If necessary, additional amino acids can be coupled to the resin by repeating steps 2-4.
6) Once the complete peptide portion of the molecule has been formed and the terminal amine functionality deprotected, the cans are treated with a .02M solution of the required sugar scaffold in DMF, in the presence of .02M O- (7-azabenzotriazol-l-yl) -1 , 1 , 3 , 3- tetramethyluronium hexafluorophosphate (HATU) and .02M DIPEA. Shaking is conducted for about 12 hours to ensure completion of this reaction step.
7) The cans are then washed four times with DMF and five times with THF.
8) The sugar scaffolds are then elaborated in the same manner described hereinabove for the Rink Amide resin. 9) Once combinatorial elaboration has been completed but prior to the TFA cleavage step, the cans are washed four times with THF, once with 5% trifluoroacetic acid (TFA) in THF, four times with THF, and twice with 1-methyl-2-pyrrolidinone (NMP) .
10) The cans are then treated with a .5M solution of iodoacetonitrile in NMP in the presence of . IM DIPEA for about 24 hours at room temperature .
11) The cans are then washed four times with NMP and four times with THF before sorting the cans into IRORI cleavage stations for final cleavage. This final step is generally conducted by adding 2.5ml of a .15M solution of the nucleophile (amine, ammonia, hydroxide or thiol) in THF to each can separately.
12) Volatile nucleophiles are generally used so that purification is possible through simple evaporation of the solvent and the nucleophile to leave the product in a microtiter plate. The evaporation process is conducted in a Savant Speedvac® as described for the other procedures .
EXAMPLE 24: Procedures for Screening for Biological Activity
The following procedures for conducting an assay of bacterial inhibition by a library compound of the present invention can be performed.
Bacteria. All organisms are grown in a universal rich media to minimize media effects on the inhibition assay. All bacteria are demonstrated to grow in Brain Heart Infusion (BHI) media (Difco, Detroit, MI) supplemented with 0.1% Casamino Acids (CAA) (Difco). The following organisms are used in primary screening: Enterococcus faecium (ATCC 49624) Enterococcus faecalis (ATCC 29212) Staphylococcus aureuε (ATCC 29213) Staphylococcus epidermidis (ATCC 12228) Streptococcus pneumonia (ATCC 49150) Escherichia coli (ATCC 25922) Acinetobacter ani tra tus (ATCC 43498)
The bacteria are streaked for isolation from frozen glycerol stocks onto BHI/CAA plates containing 1.5% Bacto-agar (Difco). An isolated colony from each strain is used to inoculate 5mL of BHI/CAA media and allowed to grow overnight at 37°C with shaking. The exception is with Streptococcus strains, which are grown in a candle "jar at 37°C without shaking. After overnight growth, the organisms are diluted 1:100 and allowed to incubate until they reach early to mid-logarithmic growth (OD6oo = 0.5). The cells are diluted 100 fold in BHI/CAA containing 0.7% agar maintained at 50°C to a cell density of approximately 5 X 10 colony forming units (CFU) per mL. The agar slurry is poured into an 86 mm X 128 mm assay plate (Nunc) , which has the dimensions of a 96-well plate, and allowed to solidify for at least 30 minutes. Streptococcus strains are diluted in BHI/CAA media without agar and 200 μl aliquoted to each well of a 96-well assay plate .
Test Compounds. The test compounds are solubilized in sterile 20% DMSO/water to a concentration of approximately l-5mg/ml, aseptically aliquoted among several sterile "daughter" plates and frozen at - 20°C. Daughter plates are thawed at room temperature or 37°C just prior to assay.
Lawn Assay. A sterilized 96-well replicating device (Boekel) is inserted into the daughter plate and used to deliver the test compound to either a 96-well plate containing Streptococcus, or an agar plate imbedded with bacteria. The replicator pierces the agar and is removed vertically to prevent damage to the agar surface. The appearance of zones of inhibition is monitored after 15 to 24 hr. growth at 37°C. Similarly, Streptococcus inhibition is monitored by no observable turbidity in the wells of the 96-well plate after 24-48 hr . growth .
A control plate containing dilutions of antibiotic standards is run at the time of each assay with each organism. The control antibiotics are Ampicillin, Vancomycin and Moenomycin. Control samples are aliquoted in duplicate in a 96-well array. Each antibiotic is tested at eight serial two fold dilutions. Antibiotic concentrations vary from lOmg/ml to O.OOlmg/ml.
MIC Assay. Putative actives in the Lawn assay are further screened to determine the minimum inhibitory concentrations (MIC) of each compound for each organism affected. Test compounds are serially diluted in 20% DMSO/water and added to 96-well plates in a volume of 5 μl . Each bacterium, grown as described above and diluted in broth without agar, is added to the diluted compound in a volume of 200 μl . The range of concentrations used for each compound in the MIC assay is based on the potency implied by the size of the zone of inhibition in the lawn assay. Each compound is tested at five serial dilutions, ranging anywhere from 1:40 up to the maximum dilution necessary to alleviate the antimicrobial effect. The effect of the test compound on bacterial growth is measured after 18 hrs of growth at 37°C by determining the turbidity of the medium at 600 nm or by visual inspection. The MIC is defined as the lowest concentration of compound necessary to completely inhibit bacterial growth.
Peptidoglycan Synthesis Assay. The peptidoglycan polymerization assay is adapted from that described by Mirelman, et al .
[Biochemistry 15:1781-1790 (1976)] and modified by Allen, et al . [FEMS Microbiol. Lett. 98:109-116 (1992)]. E. coli . (ATCC #23226) are permeabilized with ether according to Mirelman, et al.(1976), and
Maas and Pelzer [Arch. Microbiol. , 130:301-306 (19 )], permitting exogenously added radiolabelled and non-radiolabelled cell wall precursors to penetrate the bacterial cell wall. Screening quantities of UDP muramyl-pentapeptide (UDP-N-acetylmuramyl-L-Ala-D- glu-meso-diaminopimelyl-D-ala-D-ala) are isolated by boiling from an aqueous extract of B . cereus (ATCC # 11778) according to published preparative (Kohlrausch and Holtje, FEMS Microbiol. Lett. 78:253-258 (1991) and analytical HPLC techniques (Kohlrausch, et al . , J . Gen . Microbiol. 153:1499-1506 (1989). Bacterial protein is determined by the method of Bradford [Anal. Biochem.. 72: 248 (1976)].
Polymerization assays are conducted in 96-well filter-bottom plates (Millipore GF/C - cat. # MAFC NOB 10). A Tecan Genesis 150 robot is programmed for all subsequent liquid handling steps. In a final assay volume of 100 μL, each well contains: 50 mM Tris - HCl (pH 8.3); 50 mM NH4C1 ; 20 mM MgS04.7 H20; 10 mM ATP (disodium salt); 0.5 mM β-mercaptoethanol; 0.15 mM D-aspartic acid; O.OOlmM UDP-N-acetyl [14C-] -D-glucosamine (DuPont/N.E.N. - 265 - 307 mCi/mmol); 0.05 mM UDP-MurNAc-pentapeptide, lOOug/ml tetracycline and 50ug/well ether- treated bacterial protein. Novel test compounds are solubilized in 10% DMSO/water and screened at a final assay concentration of 10 μg/ml. With the exception of radiolabeled and isolated native pentapeptide, all remaining biochemicals are purchased from Sigma Chemical or Fisher Scientific.
Assay buffer (10 μL) , ATP (20 μL) , UDP pentapeptide (10 μL) and 14C- UDP-GlcNAc (20 μL) are added to all wells, followed by either test compound, reference standard or buffer vehicle (20 μL) . The reactions are then started by adding 20 μL aliquots of bacterial protein prepared in assay buffer into each well. Plates are covered, mixed for 30 sec., then incubated at 37°C for about 120 minutes. Ice cold 20% TCA (100 μL) is added to each well, the plates are gently mixed (60 sec), then refrigerated (4°C) for about 30 minutes to assure precipitation of all peptidoglycan.
The plates are placed under vacuum filtration on a Millipore manifold, filtered, and washed 3-4 times with 200 μL/well of 10% TCA. Optiphase scintillation cocktail (30 μL/well) is added, then the plates are incubated overnight prior to counting in a Wallac Microbeta. Percent inhibition of incorporation of 14C-label into peptidoglycan is computed from control (total incorporation) and background (blank) wells containing 300 μg/ml of vancomycin or 100 μg/ml of the library compound, which completely inhibit incorporation of radiolabel . All wells are arrayed in duplicates, which usually vary by <20%. Concentration-response curves for reference standards are arrayed on each plate as positive controls.
Preparation of a 980 member library
Radio frequency tars are inserted into microkans in 11 microtiter plates. Into each microkan is dispensed 15mg of rink amide resin as a slurry in DCM: THF (4:1) . The solvent is drained and the canisters are capped. The microkans are washed twice with 1 liter of DMF and then treated with 1 liter of 20% piperidine for about 30 minutes. The microkans are then washed with 1 liter of low-amine DMF. The microkans are sorted into four flasks with the aid of the IRORI accutag rf tag reader. The microkans in flasks 2-4 are treated with the appropriate amino acid, as shown in Table 1, along with HATU and DIPEA in 250 ml of DMF. The microkans are shaken at 110 rpm on an orbital shaker overnight.
Table 1 :
Amino Acids : .025M in DMF, 250ml in each reaction
Figure imgf000084_0001
Each set of microkans was washed with 2 x 200 ml of DMF before combining all of the microkans and washing with 4 x 500 ml of DMF. The microkans are then treated with 20% piperidine in DMF for about 30 minutes. After washing with 5 x 1000 ml of DMF the microkans were treated with 0.0166M of (XII) (4.83g, 2.23 equiv) , 0.0166M HATU (6.3g) and 0.0166 DIPEA (2.89ml) in 1000ml DMF. The mixture is shaken overnight at room temperature. After shaking overnight the microkans were washed with 4 x 1000ml of DMF and 4 x 1000ml of anhydrous THF. The microkans were then dried under air for four hours .
The microkans are sorted into five containers. Flask one is set aside. To each flask is added 200ml of anhydrous DMF. The microkans are shaken for about 10 minutes. The solvent is removed and another 200ml of anhydrous DMF is added to each flask. The appropriate volume of isocyanate, along with 200mg of CuCl is then added to each flask (Table 2) .
Table 2
Isocyanates . IM Isocyanate, lmg/ml CuCl in DMF, 200ml / reaction
Figure imgf000085_0001
Shake flasks four and five for two hours at room temperature, then wash each flask twice with DMF and cool to -20 C overnight. Shake flasks two and three overnight at room temperature then wash twice with DMF. Then wash all microkans three times with DMF, once with piperidine, four times with DMF, and four times with THF. The microkans are then air dried for four hours.
The microkans are sorted into five flasks for the next reaction sequence. Flasks 1-4 are treated with 0.5M of the appropriate isothiocyanate (Table 3) for 30 minutes and then the triphenylphosphine is added to each flask. The flasks are shaken for about four hours .
Flask five is treated with 20ml of . IM trimethylphosphine in 9:9:2 THF/EtOH/H20 for about five hours. The microkans in flask five are then washed with four portions of THF and two portions of DMF, and then 20ml of 0.5M 1 pyrazole carboxamide hydrochloride (1.6g), 0.5M DIPEA (1.74ml) in DMF are added. The flask is shaken overnight
Table 3 Isothiocyanates , 5M Isothiocyanate in 250ml THF/ reaction
Figure imgf000086_0001
The flasks are individually washed twice with anhydrous THF and then combined and washed four times with anhydrous THF. After drying the microkans are sorted into 12 flasks and the appropriate amine (Table 4) is added to each flask as a solution in THF.
Table 4 Amines : .5M Amine in 80ml THF/ reaction
Figure imgf000087_0001
Flasks 1-4 are washed once separately and then six times together with THF. Flask five is washed four times with DMF and twice with THF and then the microkans are combined with the rest. All of the microkans are then washed four times with THF and four times with DCM.
The products are cleaved off the resin in IRORI Accucleave stations using 20% TFA in DCM with 1% triethylsilane added. Each vessel receives 2.5ml of cleavage solution. The products are isolated by draining the cleavage stations into 48 well microtiter plates and evaporating the plates to dryness in a centrifugal evaporator. REFERENCES CITED
Kanie, 0. et al . , "A STRATEGY OF ^RANDOM GLYCOSYLATION' FOR THE PRODUCTION OF OLIGOSACCHARIDE LIBRARIES", Angew. Chem. Int. Ed. Engl., Vol. 34, pp. 23/24, (1995).
Nicolaou, K. et al . , "CARBONUCLEOTOIDS AND CARBOPEPTOIDS : NEW CARBOHYDRATE OLIGOMERS", Tet . Lett., Vol. 36, No. 11, pp. 1775-1778, (1995) .
Suhara, Y. et al., "SYNTHESIS OF A NEW CARBOHYDRATE MIMETICS: "CARBOPEPTOID" CONTAINING A C-l CARBOXYLATE AND C-2 AMINO GROUP", Tet. Lett., Vol. 37, No. 10, pp. 1575-1578, (1996).
Mueller, C. et al . , "NOVEL OLIGOSACCHARIDE MIMETICS BY SOLID-PHASE SYNTHESIS," J. Chem. Soc, Chem. Comm. , 120(23), 2425-2426, (1995).
Nicolaou, K., PCT Publ. No. WO 96/27379.
Armstrong, R. , et al . , "STRATEGIES FOR THE SYNTHESIS OF C-
DISACCHARIDES CONTAINING D AND L SUGARS," Tet. Lett., 35(42): 7743- 7746, (1994) .
Sutherlin, D. et al., "THE SYNTHESIS OF C-TRISACCHARIDES EXPLOITING THE STEREOCHEMICAL DIVERSITY OF A CENTRAL SUGAR," JACS, 118: 9802- 9803 (1996).
Hirschmann, R. et al . , "SYNTHESIS OF POTENT CYCLIC HEXAPEPTIDE NK-1 ANTAGONISTS. USE OF A MINILIBRARY IN TRANSFORMING A PEPTIDAL SOMATOSTATIN RECEPTOR LIGAND INTO AN NK-1 RECEPTOR LIGAND VIA A
POLYVALENT PEPTIDOMIMETIC " , J. Med. Chem., Vol. 39, pp. 2441-2448, (1996) .
Hirschmann, R. et al . , "NONPEPTIDYL PEPTIDOMIMETICS WITH A β-D- GLUCOSE SCAFFOLDING. A PARTIAL SOMATOSTATIN AGAINST BEARING A CLOSE STRUCTURAL RELATIONSHIP TO A POTENT, SELECTIVE SUBSTANCE P ANTAGONIST", JACS, Vol. 114, pp. 9217-9218, (1992). Hirschmann, R. et al . , "DE NOVO DESIGN AND SYNTHESIS OF SOMATOSTATIN NON-PEPTIDE PEPTIDOMIMETICS UTILIZING β-D-GLUCOSE AS A NOVEL SCAFFOLDING", JACS, Vol. 115, pp. 12550-12568, (1993).
Anderson, M. et al . , PCT Publ. No. WO 96/36627.
von Roedern, E. et al . , "SYNTHESIS AND CONFORMATIONAL ANALYSIS OF LINEAR AND CYCLIC PEPTIDES CONTAINING SUGAR AMINO ACIDS", JACS, Vol. 118, pp. 10156-10167, (1996).
Sutherlin, D., et al . , "GENERATION OF C-GLYCOSIDE PEPTIDE LIGANDS FOR CELL SURFACE CARBOHYDRATE RECEPTORS USING A FOUR-COMPONENT CONDENSATION ON SOLID SUPPORT," J. Org. Chem., 61:8350-8354 (1996).
Park, W. , et al . , "RAPID COMBINATORIAL SYNTHESIS OF AMINOGLYCOSIDE ANTIBIOTIC MIMETICS: USE OF A POLYETHYLENE GLYCOL-LINKED AMINE AND A NEAMINE-DERIVED ALDEHYDE IN MULTIPLE COMPONENT CONDENSATION AS A STRATEGY FOR THE DISCOVERY OF NEW INHIBITORS OF THE HIV RNA REV RESPONSIVE ELEMENT," JACS, 118:10150-10155, (1996).
Armstrong, R., et al . , 'MULTIPLE-COMPONENT CONDENSATION STRATEGIES FOR COMBINATORIAL LIBRARY SYNTHESIS,' Ace . Chem. Res . , 29: 123-131 (1996) .
Campion, C. et al . , PCT Publ. No. WO 95/03315.
Ugi, I., Angew. Chem. Int ' 1. Ed. Engl . , 21: 810-819 (1982).
Carpino , L . , JACS , 115 : 4397 ( 1993 ) .
Mitsunobu, O., et al . , JACS, 94:679 (1972).
Synthesis, 12:1095 (1991); JACS, 107:7788 (1985); JACS, 107:7762 (1985); J. Chem. Soc, Chem. Comm. , 2425 (1995).
Petasis, N. et al . , JACS, 119:445 (1997). Campbell , D . , et al . , JACS , 117 , 5381 - 5382 ( 1995 ) . Hebert, N. , et al . , Tett.Lett., 35: 9509-9512 (1994). Beaucage, S., et al., Tett.Lett., 22: 1859-1862 (1981).
Merrifield, B., Science, 232: 342-347 (1986)

Claims

WHAT IS CLAIMED IS:
1. A compound of structure
Figure imgf000091_0001
wherein X is O or S; Z is 0 or NH; Ri is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; Y is COOH, C00R , CH2OR3, or CH3, or Y and one of ZR4 and OR5 are linked to form a 6-membered cyclic acetal; R2 is alkyl, aryl or aralkyl; R3, R and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1 ; m is 0 or 1 ; and n is 1 or 2;
provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH, then R is not hydrogen; when n is 1, then m is 0; when p is 1, then Y is not CH2OR3 or CH3; when Y is not COOH, then exactly one of Ri, R2, R3, R and R5 is substituted by exactly one COOH; when Y is COOH, then none of Ri, R2, R3, Rι and R5 is substituted by COOH; when one of R3, R4 and R5 is hydrogen, then two of R3, R and R5 are not hydrogen and none of Ri, R , R3, 4 and R5 bears a hydroxyl substituent; when one of Ri, R2, R3, R and R5 bears a hydroxyl substituent, then four of Ri, R2, R3, R and R5 do not bear a hydroxyl substituent and none of R3, R4 and R5 is hydrogen; and when none of OR3, OR and OR5 is hydroxyl or a protected hydroxyl group, then one or more of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent or a protected hydroxyl substituent.
2. The compound of claim 1 in which n is 2.
3. The compound of claim 2 in which Ri and R2 are independently alkyl or aralkyl, and R3, R and R5 are independently hydrogen, alkyl or aralkyl.
4. A compound which is methyl 3-azido-3-deoxy-4-0-methyl-β-D- glucopyranosiduronic acid.
5. A compound which is methyl 3-azido-4-0-carboxymethyl-3- deoxy-2-O-methyl-β-D-glucopyranoside.
6. A compound which is methyl 3-azido-2-0-carboxymethyl-3- deoxy-4-O-methyl-β-D-glucopyranoside.
7. A compound which is methyl 3-azido-2-0-carboxymethyl-3- deoxy-4-O-methyl-β-D-galactopyranoside.
8. A compound which is methyl 3-azido-2-0-carboxymethyl-3- deoxy-6-O-methyl-β-D-glucopyranoside.
9. A compound which is methyl 3-azido-2-0-carboxymethyl-3- deoxy-6-O-methyl-β-D-galactopyranoside.
10. A compound which is methyl 6-azido-2-0-carboxymethyl-6- deoxy-4-O-methyl-α-D-glucopyranoside .
11. A compound which is methyl 6-azido-6-deoxy-4-0-methyl-3-0- carboxymethyl-α-D-glucopyranoside.
12. A compound which is carboxymethyl 2-acetamido-6-azido-4-0- benzyl-2 , 6-dideoxy-α-D-glucopyranoside .
13. A compound which is methyl 4-azido-4-deoxy-3-0-benzoyl-2- O-carboxymethyl-α-D-fucopyranoside.
14. A library comprising a plurality of compounds, each of said compounds of structure
Figure imgf000093_0001
wherein X is O or S; Ai is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino, RiO, RiS, Ri, RjNH or RiN-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2S02, R2NHCO, ROP (0) (0R6) , R2P(0)(OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is 0R4, NHR , CH2OR4 or CH3; A is 0, NH or N-alkyl; p, q and r are independently 0 or 1 ; Yi and Y2 are independently O or CH2; each of Li and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH2, carbonyl, 0P(0) (0R7) , NHP(O) (0R7) , P(O) (0R7) or
W II _
wherein W is O, NH, N-alkyl or S, and Z is NH, 0 or S; Ri, R2 and R3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl; R4, R5, R6 and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl; is 0 or 1; and n is 1 or 2 ;
provided that: when n is 1, then m is 0; when A5 is NH or N-alkyl, then L3 is not NHP(O) (OR7) ; when L3 is a single bond, CH2 or carbonyl, r is 0 and A5 is O, then R3 is not aryl having fewer than 8 carbon atoms or aralkyl having fewer than 8 carbon atoms or alkyl having fewer than 3 carbon atoms; and when L3 is carbonyl, r is 0 and A5 is NH, then R3 is not alkyl having fewer than 3 carbon atoms.
15. The library of claim 14 in which n is 2.
16. The library of claim 15 in which Ai is a residue of an α- amino acid or a peptide residue comprising residues of from 2 to 10 α-amino acids.
17. The library of claim 16 in which r is 0.
The library of claim 17 in which L3 is
W
II
-c-
wherein W is 0 and Z is NH; and M is N=C=0.
19. The library of claim 18 in which q is 0.
20. The library of claim 19 in which A2 is R2.
21. The library of claim 20 in which R3 is alkyl or aryl
22. The library of claim 21 in which R is alkyl or aralkyl
23. The library of claim 22 in which R5 is hydrogen, alkyl, aryl , or aralkyl .
24. A method for preparing a solid-phase library comprising a plurality of compounds, each of said compounds of structure
Figure imgf000095_0001
wherein X is 0 or S; Ai is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino, RiO, RiS, Rx, RjNH or RiN-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2S02, R2NHCO, R2OP (0) (OR6) , R2P(0)(OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is 0R , NHR , CH2OR4 or CH3; A5 is 0, NH or N-alkyl; p, q and r are independently 0 or 1 ; Yi and Y2 are independently 0 or CH2; each of Li and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH , carbonyl, 0P(0)(0R7), NHP(0)(OR7), P(0) (0R7) or
W ll_
wherein W is 0, NH, N-alkyl or S, and Z is NH, O or S; Ri, R2 and R are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl; R4, R5, R6 and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl; m is 0 or 1; and n is 1 or 2 ; provided that: when n is 1, then m is 0; when A5 is NH or N-alkyl, then L3 is not NHP(O) (0R7) ; when L3 is a single bond, CH2 or carbonyl, r is 0 and A5 is 0, then R3 is not aryl having fewer than 8 carbon atoms or aralkyl having fewer than 8 carbon atoms or alkyl having fewer than 3 carbon atoms; and when L3 is carbonyl, r is 0 and A5 is NH, then R3 is not alkyl having fewer than 3 carbon atoms ;
said method comprising steps of:
(a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and an azido group;
(b) performing, in any order, steps of:
(i) allowing the free carboxyl group of the monosaccharide to react to produce a substituent Ai;
(ii) reducing the azido group to an amino group and allowing the amino group to react with a compound capable of reacting with the amino group to produce a substituent A2;
(iii) allowing a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent R3L3A5.
25. The method of claim 24 in which A5 is 0 and the free hydroxyl group reacts with a compound R3M to form a substituent R3L3O; wherein M is N=C=0, N=C=N-alkyl, N=C=S, 0P(0) (0R7)G, NHP(O) (OR7)G,
P(O) (0R7)G, OCOG, SCOG, COG, G or CH2G, wherein G is a leaving group.
26. The method of claim 25 in which step (i) is performed first .
27. The method of claim 26 in which step (iii) is performed last .
28. The method of claim 27 in which said free carboxyl group reacts with a group which is attached to a polymeric support; said group having a free terminus .
29. The method of claim 28 in which said free terminus is an amino group .
30. The method of claim 29 in which Ai is a peptide residue comprising residues of from 2 to 10 α-amino acids, said peptide residue being bonded to the polymeric support through a terminal carboxyl group .
31. The method of claim 30 in which n is 2.
32. The method of claim 31 in which the polymeric support is a solid support.
33. The method of claim 32 in which r is 0.
34. The method of claim 33 in which L3 is
W
—z—c—
wherein W is 0 and Z is NH; and M is N=C=0.
35. The method of claim 34 in which q is 0.
36. The method of claim 35 in which A2 is R2.
37. The method of claim 36 in which R3 is alkyl or aryl.
38. The method of claim 37 in which R is alkyl or aralkyl.
39. The method of claim 38 in which R5 is hydrogen, alkyl, aryl, or aralkyl.
40. The method of claim 39 in which X is S, R5 is aryl and XR5 is attached to the anomeric position.
41. The method of claim 40, further comprising a step of treating the compounds with a reagent effective to convert XR5 to hydroxyl .
42. The method of claim 41 in which the reagent is a mercury (II) salt.
43. The method of claim 24, further comprising a step of treating the compounds with a solution containing trifluoroacetic acid to remove the compounds from the solid support.
44. The method of claim 27 in which the scaffold is attached to a polymeric support via a safety-catch linker.
45. The method of claim 44 further comprising reacting the product of step (iii) with a nucleophile, thereby displacing the compounds from the polymeric support.
46. The method of claim 45 in which n is 2.
47. The method of claim 46 in which the polymeric support is a solid support.
48. The method of claim 44 in which a nucleophile capable of displacing the safety-catch linker and producing the substituent Ai displaces the compounds from the polymeric support.
49. The method of claim 48 in which n is 2.
50. The method of claim 49 in which the polymeric support is a solid support.
51. A method for preparing a solid-phase library comprising a plurality of compounds, each of said compounds of structure
Figure imgf000099_0001
wherein X is 0 or S; Ai is a residue of an α-amino acid attached through a terminal amino, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal amino, RiO, RiS, Ri, RiNH or RiN-alkyl; A2 is a residue of an α-amino acid attached through a terminal carboxyl, a peptide residue comprising residues of from 2 to 10 α-amino acids and attached through a terminal carboxyl, R2S02, R2NHC0, R2OP (O) (OR6) , R2P(0)(OR6) or R2, or A2, A3 and N combine to form a nitrogen heterocycle; A3 is hydrogen when A3 is not combined with A2 and N; A4 is OR4, NHR4, CH2OR or CH3; A5 is 0, NH or N-alkyl; p, q and r are independently 0 or 1; Yi and Y2 are independently 0 or CH2; each of Li and L2 is independently a difunctional alkyl, aryl, aralkyl, alkanoyl, aroyl or aralkanoyl group; L3 is a single bond, CH2, carbonyl, 0P(0) (0R7) , NHP(0) (OR ), P(0)(0R7) or
W ll_
wherein W is 0, NH, N-alkyl or S, and Z is NH, 0 or S; Ri, R2 and R3 are independently alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic-carbonyl; R , R5, R6 and R7 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl; m is 0 or 1; and n is 1 or 2; provided that: when n is 1, then m is 0; when A5 is NH or N-alkyl, then L3 is not NHP(O) (0R7) ; when L3 is a single bond, CH2 or carbonyl, r is 0 and A5 is O, then R3 is not aryl having fewer than 8 carbon atoms or aralkyl having fewer than 8 carbon atoms or alkyl having fewer than 3 carbon atoms; and when L3 is carbonyl, r is 0 and A5 is NH, then R3 is not alkyl having fewer than 3 carbon atoms ;
said method comprising steps of: (a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and an azido group; (b) performing, in any order, steps of:
(i) allowing the free carboxyl group of the monosaccharide to react to produce a substituent Ai; (ii) allowing the azido group to react with a compound capable of reacting with the azido group to produce a substituent A2A3N;
(iii) allowing a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent R3L3A5.
52. Use of the library of claim 14 for screening for biological activity.
53. The use of claim 52 in which a method of screening for activity against whole cells is employed, said method comprising steps of:
(a) combining the whole cells with individual compounds of the library to form separate mixtures thereof;
(b) maintaining the mixtures under conditions amenable to growth of the cells in the absence of the compounds; and
(c) observing any inhibition of growth of the cells in the presence of a library compound relative to a control sample.
54. The screening method of claim 53 in which the biological target is a strain of Streptococcus or Staphylococcus bacteria.
55. A compound of structure
Figure imgf000101_0001
wherein X is 0 or S; Z is O or NH; Rx is alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; Y is COOH, COOR2, CH2OR3, CH3 or CH(S)Y2(3-s) where Y2 is F, Cl, Br or I, and s is 0, 1, or 2 or Y and one of ZR4 and OR5 are linked to form a 6-membered cyclic acetal; R2 is alkyl, aryl or aralkyl; R3 R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl or aroyl; p is 0 or 1; m is 0 or 1; and n is 1 or 2;
provided that: when X is S, then XRi is not attached to the anomeric carbon; when Z is NH, then R4 is not hydrogen; when n is 1, then m is 0; when p is 1, then Y is not CH2OR3 or CH3; when Y is not COOH, then exactly one of Ri, R2, R3, R4 and R5 is substituted by exactly one COOH; when Y is COOH, then none of Ri, R2, R , R4 and R5 is substituted by COOH; when one of R3, R4 and R5 is hydrogen, then two of R3, R4 and R5 are not hydrogen and none of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent; when one of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent, then four of Ri, R2, R3, R and R5 do not bear a hydroxyl substituent and none of R3, R4 and R5 is hydrogen; and when none of OR3, 0R4 and 0R5 is hydroxyl or a protected hydroxyl group, then one or more of Ri, R2, R3, R4 and R5 bears a hydroxyl substituent or a protected hydroxyl substituent.
56. A method of preparing a solid-phase library comprising a plurality of compounds, each of said compounds having the structure:
Figure imgf000102_0001
in which the groups R, Ri, R and R3 can each independently be alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, hetercyclic-alkyl , heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl, except that Ri, R2 and R3 can each independently also be hydrogen, which method comprises:
(a) providing a monosaccharide bearing a free carboxylic acid, a free hydroxyl group at either position C-2 or C-3 and an azido group at position C-6 of the formula:
Figure imgf000102_0002
(b) converting the free carboxylic acid group to an amide;
(c) reducing the azido group to an amino group and allowing the amino group to react with a substituted isothiocyanate, RNCS, to provide a substituted thiourea group, -NHC(S)NHR, at position C-6;
(d) converting the thiourea group to a urea group and allowing the urea group to react with an amine to form a guanidinium group, -
NHC(NR)NR1R2, at position C-6;
(e) allowing the free hydroxyl group to react with a compound capable of reacting with said free hydroxyl group to form a substituent, R3NHC00-, at position C-2 or C-3.
PCT/US1999/012032 1998-05-28 1999-05-28 Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction Ceased WO1999061583A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU43227/99A AU4322799A (en) 1998-05-28 1999-05-28 Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8707298P 1998-05-28 1998-05-28
US60/087,072 1998-05-28

Publications (2)

Publication Number Publication Date
WO1999061583A2 true WO1999061583A2 (en) 1999-12-02
WO1999061583A3 WO1999061583A3 (en) 2000-04-06

Family

ID=22202951

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/012032 Ceased WO1999061583A2 (en) 1998-05-28 1999-05-28 Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction

Country Status (2)

Country Link
AU (1) AU4322799A (en)
WO (1) WO1999061583A2 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042067A1 (en) * 1999-01-12 2000-07-20 Princeton University Saccharides linked to compounds that bind cell-surface peptides or proteins
US6994966B2 (en) 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
WO2008069440A1 (en) * 2006-12-06 2008-06-12 Samchully Pharm. Co., Ltd. The preparation method of 2-de0xy-l-rib0se
CN102320920A (en) * 2011-07-08 2012-01-18 浙江工业大学 Method for removing benzyl protecting group of hydroxyl group
CN104140348A (en) * 2013-05-07 2014-11-12 中国药科大学 Method of removing p-methoxybenzyl type protection groups for carboxyl and hydroxyl
US9073960B2 (en) 2011-12-22 2015-07-07 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9243022B2 (en) 2012-12-21 2016-01-26 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9422323B2 (en) 2012-05-25 2016-08-23 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US9862743B2 (en) 2013-10-11 2018-01-09 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10485815B2 (en) 2012-03-21 2019-11-26 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
CN110691777A (en) * 2017-03-15 2020-01-14 兹洛有限公司 Macrocycles
USRE48171E1 (en) 2012-03-21 2020-08-25 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US11697666B2 (en) 2021-04-16 2023-07-11 Gilead Sciences, Inc. Methods of preparing carbanucleosides using amides
US11767337B2 (en) 2020-02-18 2023-09-26 Gilead Sciences, Inc. Antiviral compounds
US12030903B2 (en) 2020-02-18 2024-07-09 Gilead Sciences, Inc. Antiviral compounds
US12054507B2 (en) 2020-02-18 2024-08-06 Gilead Sciences, Inc. Antiviral compounds
US12116380B2 (en) 2021-08-18 2024-10-15 Gilead Sciences, Inc. Phospholipid compounds and methods of making and using the same

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2341397A (en) * 1996-03-21 1997-10-10 Princeton University Carbohydrate-based ligand library, assay and method

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042067A1 (en) * 1999-01-12 2000-07-20 Princeton University Saccharides linked to compounds that bind cell-surface peptides or proteins
US6994966B2 (en) 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US8114987B2 (en) 2006-12-06 2012-02-14 Samchully Pharm. Co. Ltd. Preparation method of 2-deoxy-L-ribose
WO2008069440A1 (en) * 2006-12-06 2008-06-12 Samchully Pharm. Co., Ltd. The preparation method of 2-de0xy-l-rib0se
KR100849979B1 (en) 2006-12-06 2008-08-01 주식회사 삼천리제약 The preparation method of 2-deoxy-L-ribose
GB2456999A (en) * 2006-12-06 2009-08-05 Samchully Pharm Co Ltd The preparation method of 2-deoxy-L-ribose
GB2456999B (en) * 2006-12-06 2011-04-27 Samchully Pharm Co Ltd Method for preparing 2-deoxy-L-ribose
CN102320920A (en) * 2011-07-08 2012-01-18 浙江工业大学 Method for removing benzyl protecting group of hydroxyl group
US11021509B2 (en) 2011-12-22 2021-06-01 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9073960B2 (en) 2011-12-22 2015-07-07 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10464965B2 (en) 2011-12-22 2019-11-05 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
USRE48171E1 (en) 2012-03-21 2020-08-25 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10485815B2 (en) 2012-03-21 2019-11-26 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10301347B2 (en) 2012-05-25 2019-05-28 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US9422323B2 (en) 2012-05-25 2016-08-23 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US10040814B2 (en) 2012-05-25 2018-08-07 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US10774106B2 (en) 2012-05-25 2020-09-15 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US10544184B2 (en) 2012-05-25 2020-01-28 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US9845336B2 (en) 2012-05-25 2017-12-19 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US9243022B2 (en) 2012-12-21 2016-01-26 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10793591B2 (en) 2012-12-21 2020-10-06 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10487104B2 (en) 2012-12-21 2019-11-26 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9249174B2 (en) 2012-12-21 2016-02-02 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US12173025B2 (en) 2012-12-21 2024-12-24 Janssen Pharmaceuticals, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10144755B2 (en) 2012-12-21 2018-12-04 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10683320B2 (en) 2012-12-21 2020-06-16 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US11485753B2 (en) 2012-12-21 2022-11-01 Janssen Pharmaceutica Nv Substituted nucleosides, nucleotides and analogs thereof
US10112966B2 (en) 2012-12-21 2018-10-30 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
CN104140348A (en) * 2013-05-07 2014-11-12 中国药科大学 Method of removing p-methoxybenzyl type protection groups for carboxyl and hydroxyl
US10370401B2 (en) 2013-10-11 2019-08-06 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US9862743B2 (en) 2013-10-11 2018-01-09 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
CN110691777B (en) * 2017-03-15 2024-03-01 诺和诺德牛津研究中心有限公司 Macrocycles
CN110691777A (en) * 2017-03-15 2020-01-14 兹洛有限公司 Macrocycles
US11767337B2 (en) 2020-02-18 2023-09-26 Gilead Sciences, Inc. Antiviral compounds
US12030903B2 (en) 2020-02-18 2024-07-09 Gilead Sciences, Inc. Antiviral compounds
US12054507B2 (en) 2020-02-18 2024-08-06 Gilead Sciences, Inc. Antiviral compounds
US12264173B2 (en) 2020-02-18 2025-04-01 Gilead Sciences, Inc. Antiviral compounds
US11697666B2 (en) 2021-04-16 2023-07-11 Gilead Sciences, Inc. Methods of preparing carbanucleosides using amides
US12116380B2 (en) 2021-08-18 2024-10-15 Gilead Sciences, Inc. Phospholipid compounds and methods of making and using the same

Also Published As

Publication number Publication date
WO1999061583A3 (en) 2000-04-06
AU4322799A (en) 1999-12-13

Similar Documents

Publication Publication Date Title
WO1999061583A2 (en) Carbohydrate-based scaffold compounds, combinatorial libraries and methods for their construction
Xie et al. Synthesis and applications of carbohydrate-derived macrocyclic compounds
AU697920B2 (en) Soluble combinatorial libraries
CN102256989B (en) The anionic conjugates of glycosylated bacterial metabolite
US6274716B1 (en) Combinatorial library of moenomycin analogs and methods of producing same
KR20100044849A (en) Lantibiotic-based compounds having antimicrobial activity
JP2002520422A (en) Glycopeptide antibiotics, combinatorial library of glycopeptide antibiotics and method for producing the same
US6111084A (en) Fuopeptide mimetics
EP0934076A4 (en)
EP0901629A1 (en) Carbohydrate-based ligand library, assay and method
AU2010234779A1 (en) Heparan sulfate synthesis
Au et al. Tuning the properties of a cyclic RGD-containing tetrapeptide through backbone fluorination
Schweizer et al. Synthesis of a galacto-configured C-ketoside-based γ-sugar-amino acid and its use in peptide coupling reactions
EP1147129A1 (en) Saccharides linked to compounds that bind cell-surface peptides or proteins
EP0998280A1 (en) Carbohydrate scaffold compounds and libraries
JP2005529910A (en) Disaccharides for drug discovery
US20030027980A1 (en) Process for preparing lipid ii
CA2925028A1 (en) Glycoclusters and their pharmaceutical use as antibacterials
EP1417223B1 (en) Process for preparing lipid ii
US20040028702A1 (en) Muramic acid derivative compounds
Rösch et al. Highly regioselective synthesis of a 3-O-sulfonated arabino Lewisa asparagine building block suitable for glycopeptide synthesis
Norgren et al. Glycosylated foldamers: synthesis of carbohydrate‐modified β3hSer and incorporation into β‐peptides
WO2000042052A1 (en) Substituted alpha-linked disaccharides
Janczuk Study of α-Gal epitope anti-α-Gal antibody binding using deoxy derivatives of α-Gal and the synthesis of biologically relevant molecules
Eniade Part A: Synthetic studies of C-linked analogs of N-acetylglucosamine-1-phosphate. Part B: Synthesis of C-linked analogs of antifreeze glycoproteins and their in vitro assessment

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase