WO1999061583A2 - Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction - Google Patents

Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction Download PDF

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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
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alkyl
group
methyl
compound
heterocyclic
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PCT/US1999/012032
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WO1999061583A3 (fr
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Michael J. Sofia
Rakesh K. Jain
Andrew Vaughan
David M. Gange
Manuka Ghosh
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Incara Pharmaceuticals Corp.
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Priority to AU43227/99A priority Critical patent/AU4322799A/en
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Publication of WO1999061583A3 publication Critical patent/WO1999061583A3/fr

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    • 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).

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Abstract

L'invention concerne un composé de structure (I), dans laquelle X représente O ou S; Z représente O ou NH; Y représente COOH, COOR2, CH2OR3, CH3, ou CH(s)Y2(3-s), Y2 désignant F, Cl, Br, ou I, et s étant égal à 0, à 1, ou à 2, ou Y, ZR4 ou OR5 sont liés pour former un acétal cyclique à 6 chaînons; p est égal à 0 ou à 1; m est égal à 0 ou à 1; et n est égal à 1 ou à 2. L'invention concerne également une banque de composés de structure (II), dans laquelle X représente O ou S; A1 désigne un reste d'un α-aminoacide relié par un amino terminal, un reste peptidique renfermant des restes d'environ 2 à 10 α-aminoacides et étant relié par un amino terminal, R1O, R1S, R1, R1NH, ou R1N-alkyle; A2 désigne un reste d'un α-aminoacide relié par un carboxyle terminal, un reste peptidique renfermant des restes d'environ 2 à 10 α-aminoacides et étant relié par un carboxyle terminal, R2SO2, R2NHCO, R2OP(O) (OR6), R2P(O) (OR6), ou R2, ou A2, A3, et N s'associent pour former un hétérocycle azoté; A3 représente hydrogène quand A3 n'est pas associé à A2 et à N; A4 représente OR4, NHR4, CH2OR4, ou CH3; A5 représente O, NH, ou N-alkyle; p, q, et r sont indépendamment égaux à 0 ou à 1; Y1 et Y2 désignent indépendamment O ou CH2; L1 et L2 représentent chacun un groupe alkyle, aryle, aralkyle, alkanoyle, aroyle, ou aralkanoyle dysfonctionnel; L3 symbolise une liaison simple, CH2, carbonyle, OP(O) (OR7), NHP(O) (OR7), P(O) (OR7), ou (III), dans laquelle W représente O, NH, N-alkyle ou S, et Z désigne NH, O, ou S; m est égal à 0 ou à 1; et n est égal à 1 ou à 2.
PCT/US1999/012032 1998-05-28 1999-05-28 Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction WO1999061583A2 (fr)

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US6994966B2 (en) 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
WO2008069440A1 (fr) * 2006-12-06 2008-06-12 Samchully Pharm. Co., Ltd. Procédé de préparation de 2-desoxy-l-ribose
CN102320920A (zh) * 2011-07-08 2012-01-18 浙江工业大学 一种脱除羟基的苄基类保护基的方法
CN104140348A (zh) * 2013-05-07 2014-11-12 中国药科大学 一种脱除羧基和羟基的对甲氧基苄基类保护基的方法
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 (zh) * 2017-03-15 2020-01-14 兹洛有限公司 大环化合物
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

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WO2000042067A1 (fr) * 1999-01-12 2000-07-20 Princeton University Saccharides portes par des composes se liant a des proteines ou des peptides cellulaires de surface
US6994966B2 (en) 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
WO2008069440A1 (fr) * 2006-12-06 2008-06-12 Samchully Pharm. Co., Ltd. Procédé de préparation de 2-desoxy-l-ribose
KR100849979B1 (ko) 2006-12-06 2008-08-01 주식회사 삼천리제약 2-데옥시-엘-리보오스의 제조방법
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
US8114987B2 (en) 2006-12-06 2012-02-14 Samchully Pharm. Co. Ltd. Preparation method of 2-deoxy-L-ribose
CN102320920A (zh) * 2011-07-08 2012-01-18 浙江工业大学 一种脱除羟基的苄基类保护基的方法
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
US10774106B2 (en) 2012-05-25 2020-09-15 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US9422323B2 (en) 2012-05-25 2016-08-23 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US9845336B2 (en) 2012-05-25 2017-12-19 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US10544184B2 (en) 2012-05-25 2020-01-28 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US10040814B2 (en) 2012-05-25 2018-08-07 Janssen Sciences Ireland Uc Uracyl spirooxetane nucleosides
US10301347B2 (en) 2012-05-25 2019-05-28 Janssen Sciences Ireland Unlimited Company Uracyl spirooxetane nucleosides
US10112966B2 (en) 2012-12-21 2018-10-30 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
US10144755B2 (en) 2012-12-21 2018-12-04 Alios 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
US11485753B2 (en) 2012-12-21 2022-11-01 Janssen Pharmaceutica Nv Substituted nucleosides, nucleotides and analogs thereof
US10793591B2 (en) 2012-12-21 2020-10-06 Janssen 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
US9249174B2 (en) 2012-12-21 2016-02-02 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
CN104140348A (zh) * 2013-05-07 2014-11-12 中国药科大学 一种脱除羧基和羟基的对甲氧基苄基类保护基的方法
US9862743B2 (en) 2013-10-11 2018-01-09 Alios Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
US10370401B2 (en) 2013-10-11 2019-08-06 Janssen Biopharma, Inc. Substituted nucleosides, nucleotides and analogs thereof
CN110691777A (zh) * 2017-03-15 2020-01-14 兹洛有限公司 大环化合物
CN110691777B (zh) * 2017-03-15 2024-03-01 诺和诺德牛津研究中心有限公司 大环化合物
US11767337B2 (en) 2020-02-18 2023-09-26 Gilead Sciences, Inc. Antiviral compounds
US11697666B2 (en) 2021-04-16 2023-07-11 Gilead Sciences, Inc. Methods of preparing carbanucleosides using amides

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