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
  • 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, including libraries thereof. 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, or scaffold, upon which various chemical or biological appendages are attached to define the library compounds.
• 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
• NP represents amino, protected amino or amino bound to a solid support
• p is 0 or 1
• X is COOH, COORi, CH 3 or CH 2 OR 2
• Y is CH 2 OR 3 , NHR 4 or 0R 4 , or Y and 0R 6 are linked to form a 6-membered cyclic acetal
• Z is 0, NH or S
• Ri is alkyl, aryl or aralkyl
• R 2 , 3 , ⁇ -i r R 5 and R 6 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl, aroyl or a hydroxyl protecting group
• m is 0 or 1
• n is 1 or 2; provided that: when Y is NHR 4 , then R 4 is not hydrogen; when Z is NH or S, then R 5 is not hydrogen; when n is 1, then m is 0; when p is 1, then X is not CH
• This invention is also directed to a library of compounds, each compound in the library having 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, RiNH or R ⁇ N-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, R 2 S0 2 , R 2 NHCO, R 2 OP (0) (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 is 0R 4 , NHR 4 , CH 2 OR 4 or CH 3 ;
• a 5 is 0, NH or N-alkyl;
• W is 0, NH, N-alkyl or S, and Z is NH, 0 or S
• 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 , R 5 , R 6 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
• 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,
• This invention is further directed to a method for making the library of compounds comprising steps of: (a) providing a monosaccharide bearing a free carboxyl group, a free or protected hydroxyl group and a protected amino 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 a free hydroxyl of the monosaccharide to react with a compound capable of reacting with said free hydroxyl group to form a substituent R 3 L 3 A 5 ; (iii) deprotecting the protected amino group to provide a free amino group and allowing the free amino group to react with a compound capable of reacting with the amino group to produce a substituent A 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, dialkylamino, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, aroyl, CONH 2 , CONH-alkyl or CON (alkyl) 2 , C00- 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, CONH 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
• 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 non-acidic conditions or non-basic conditions, e.g., homogeneous catalytic reduction or hydride reduction, to generate a free hydroxyl group. Examples of protecting groups removable by means of reduction are allyloxycarbonyl (alloc) , allyl and trichloroethyloxycarbonyl (troc) .
• 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 amino or protected amino 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
• NP represents amino, protected amino or amino bound to a solid support
• p is 0 or 1
• X is COOH, COORi, CH 3 or CH 2 OR 2
• Y is CH 2 OR 3 , NHR 4 or 0R 4 , or Y and OR ⁇ are linked to form a 6-membered cyclic acetal
• Z is 0, NH or S
• Ri is alkyl, aryl or aralkyl
• R 2 , R 3 , R 4 , R5 and Re are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aralkanoyl, aroyl or a hydroxyl protecting group
• m is 0 or 1
• n is 1 or 2; provided that: when Y is NHR 4 , then R 4 is not hydrogen; when Z is NH or S, then R5 is not hydrogen; when n is 1, then m is 0; when p is 1, then X is not CH 2 OR 2 or CH
• 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 amino or protected amino group.
• the amino or protected amino 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 amino group are those easily removed by treatment with acid or base, by catalytic reduction, or by exposure to light. Suitable protected amino groups do not include the azido group.
• Preferred protecting groups are the base-labile protecting groups, including, e.g., 9- fluorenylmethyloxycarbonyl (Fmoc) , trifluoroacetamido, phthalimido, tetrachlorophthalimido and allyloxycarbonyl . The most preferred protecting group is Fmoc.
• the monosaccharide scaffold compounds may be prepared from commercially available monosaccharides bearing an amino and a carboxyl group.
• suitable precursors obtained readily from commercially available sources are the 2-amino, 5- carboxylic acid derivatives of glucosamine, mannosamine, and galactosamine .
• the N-Fmoc-blocked derivatives of these particular regioisomers are illustrated below:
• 1 position of the monosaccharide can introduce a further variable in the construction of the library since both axial and equatorial positions are available.
• a library of 2400 compounds is available for a dipeptidyl library constructed from glucose, mannose, and galactose and the twenty natural amino acids, wherein the 6- position of the sugar has been oxidized to carboxyl and the 2- position is occupied by an amino group.
• non-natural regioisomers can also be constructed.
• a single carboxyl group can be provided at the 1, 3 or 4 position. Accordingly, 7200 compounds are possible in addition to the 2400 scaffold products mentioned previously.
• 9600 compound possibilities are available.
• N-blocked aminoglucuronic acid scaffold molecules which illustrate just a few of the regioisomers permitted on a glucose template, are depicted hereinbelow.
• the underlying functional amine group is readily apparent.
• the reactive carboxyl group can be attached directly to the hexose ring or through a linker to a hydroxyl group of the ring, such as via an acetic acid group or a bifunctional acid, e.g., oxalic acid or p-benzoic acid. Reactions that can be used to synthesize these scaffolds have been described previously. [See, e -9" Synthesis, 12:1095 (1991); JACS, 107:7788 (1985); JACS, 107:7762 (1985); J.Chem.Soc, Chem. Comm., 2425 (1995)]
• a ligand on the scaffold can be an N-derivatized urea or the product of an Ugi multi-component condensation (MCC) reaction.
• the Ugi condensation which involves the reaction of an amine, an aldehyde, a carboxylic acid and an isocyanide, can be used to attach a ligand to the monosaccharide scaffold compound.
• the amine component of the reaction can be provided by an amine linked to a solid support, and the monosaccharide can bear the carboxylic acid group.
• the monosaccharide can bear the amine group and the carboxylic acid group can be provided by another molecule.
• the product of the Ugi condensation is an ⁇ -acylamino amide coupled to the sugar.
• the monosaccharide scaffold has either of the structures _1 and 2_ shown below.
• scaffolds 1_ and 2_ The synthesis of scaffolds 1_ and 2_ is outlined in Schemes 1 and 2.
• Scaffold 1_ was prepared from D-glucosamine hydrochloride 3_ in eight steps, as shown in Scheme 1. Reaction of 3_ with benzylchloroformate in aqueous sodium carbonate gave the desired benzyloxycarbonyl (Cbz) protected glucosamine which on treatment with HCl/dioxane-MeOH gave the -methyl glucoside _. Protection of the C-4- and C-6 hydroxyls as the isopropylidene ketal followed by methylation at the C-3-hydroxyl gave the intermediate 5_.
• Scaffold 2_ was prepared from the commercially available ⁇ - methyl-D-glucoside *i- n seven steps, as shown in Scheme 2. Reaction of 1_ with excess sodium periodate and condensation of the resulting dialdehyde with nitromethane yielded the 3- nitrosugar 8_. Formation of the benzylidene acetal followed by acetylation at the C-2 hydroxyl gave intermediate 9_ . Simultaneous reduction of the nitro group and benzylidene removal followed by amino-group protection gave the Fmoc-amino diol IC. Selective oxidation of the C-6 hydroxyl group gave the glucuronic acid scaffold 2_. Details of the preparation are given in Example 2. Scheme 2
• the scaffold compounds are functionalized at the three reactive sites, i.e., the hydroxyl, carboxylic acid and amino or protected amino group, to produce a library of compounds.
• Each compound in the library has the structure
• 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, R 2 S0 2 , R 2 NHCO, R 2 OP(0) (OR 6 ) , R 2 P(0) (OR ⁇ ) 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 , CH 2 OR 4 or CH 3 ;
• W is 0, NH, N-alkyl or S, and Z is NH, 0 or S
• 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 , R 5 , Re 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
• 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
• YiLi 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 ⁇ L ⁇ or L 2 Y 2 attached to a monosaccharide ring carbon.
• the group Y ⁇ L ⁇ may also be derived from reaction of a difunctional molecule, e.g., a dicarboxylic acid in which Yi 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 Yi is 0 and Li is difunctional alkanoyl
• the group R 3 L 3 A 5 is derived from reaction of the hydroxyl group of the scaffold molecule.
• a 5 is oxygen.
• a 5 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.
• MCC reaction such as the Passerini reaction
• 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 .
• 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 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 i being a residue of an ⁇ -amino acid or a peptide residue comprising residues of from two to ten ⁇ -amino acids. It is 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 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
• R 3 NCO which reacts with the free hydroxyl group to form a carbamate
• 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, PPh 3 -CCl 4 ) , 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, PPh 3 -CCl 4
• 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 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 .
• 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 COA ⁇ . 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.
• a protected amino group of the monosaccharide is deprotected and then allowed to react with a carboxylic acid to form an amide linkage A 2 N in which A 2 is R 2 , which is alkanoyl, aroyl, aralkanoyl, heterocyclic-alkyl-carbonyl or heterocyclic- carbonyl .
• 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 free carboxylic acid group 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 7.
• 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 library compounds of this invention are preferably prepared in an arrayed parallel synthesis using automated robotic methods.
• TecanTM (Switzerland) Genesis liquid handling system
• TecanTM resin dispenser a TecanTM resin dispenser
• Savant centrifugal evaporator can be used.
• 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. Use of the IRORI AccuTag®- 100 system is preferred.
• the scaffold products are typically used without purification with the amount of product in a given preparation being quantified 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, 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 8.
• 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 based on scaffolds 1_ and 2_ was prepared as discrete compounds using the IRORI AccuTag ® -100 radiofrequency tagged solid phase synthesis system and the directed sorting split-pool method.
• the building blocks for the libraries are described in Scheme 6, and details of the library synthesis provided in Example 6.
• Scaffolds 1 and 2_ were each coupled to eight amino- acid functionalized trityl-Tentagel resins.
• Each scaffold-amino acid resin was then used to prepare a 48-member sub-library by reaction with six isocyanates and eight carboxylic acids.
• Several of the amino acid building blocks contained acid labile protecting groups. These protecting groups were removed concomitantly with cleavage of the final product from the solid support. In total, sixteen 48-member sub-libraries were prepared. Library analysis by LC/MS showed that 90% of the library products were produced in > 80% purity.
• AA is derived from the following amino acids: Phe, Ala, Val, Leu, He, His (Trt) , Asn(Trt), Gly-Val;
• Ri is derived from isocyanates RiNCO in which Ri is cyclohexyl, 3-acetylphenyl, 3- (trifluoromethyl) phenyl, 4-chloro-3- (trifluoromethyl) phenyl, 4- (trifluoromethyloxy) phenyl, 3,5- bis (trifluoromethyl) phenyl; and R 2 is derived from acids R 2 C0 2 H in which R 2 is 3- (2- thienyl) propyl, cyclohexyl, methyl, 3-tetrahydrofuryl, 4- biphenylyl, 2, 4-dimethoxyphenyl, 3-pyridyl, and phenyl.
• EXAMPLE 2 2-0-acetyl-3-deoxy-3-fluorenylmethyloxycarbonylamino- l- ⁇ -O-methyl-glucuronic acid (2) .
• EXAMPLE 3 Derivatization of Fmoc-leucine-Novasyn -TGT with sugar scaffold (1).
• scaffold 1 (10.5mL of a 0.133M solution in DMF, 1.4mmol), HATU (10.5mL of a 0.133M solution in DMF, 1.4mmol) and DIPEA (10.5mL of a 0.133M solution in DMF, 1.4mmol), and this suspension was shaken at room temperature for 20 hours.
• the resin was filtered and washed (4x) with DMF, (4x) with EtOAc, (4x) with CH 2 C1 2 then dried in vacuo over phosphorus pentoxide to give 2-Fmoc-aminoglucose-scaffold-linked solid support _12.
• a 200mg sample of resin JL2 was subjected to cleavage with 10% TFA/CH 2 C1 2 for -lhour.
• the cleavage mixture was filtered through a PrepsepTM solid phase extraction column and the resin washed with 10% TFA/CH 2 C1 2 .
• Co-evaporation of the filtrate with toluene under reduced pressure gave the desired product as a white solid (32mg) .
• the resin _1_2 (1.2g, 0.25mmol) was swelled in anhydrous DMF (25mL) , filtered and washed (lx) with anhydrous DMF. Following addition of isopropyl isocyanate (25mL of a 0.5M solution in anhydrous DMF) and a catalytic amount of Cu(I)Cl the resin was stirred at room temperature for 3 hours. The resin was filtered and washed (4x) DMF, (4x) EtOAc, (4x) CH 2 C1 2 and dried in vacuo over P 2 0 5 to give the desired solid support 14. 180mg of resin was cleaved with 10% TFA/CH 2 C1 2 as previously described to give 29mg as a white solid.
• the resin 1_4 (0.23g, 0.048mmol) was swelled in DMF (lOmL), filtered and washed (2x) with DMF then treated with 20% piperidine/DMF (lOmL) for 30 minutes.
• the resulting resin was filtered and washed (4x) with DMF then treated with 4- nitrobenzoic acid (3mL of a 0.03M solution in DMF), HATU (3mL of a 0.03M solution in DMF) and DIPEA (3mLof a 0.03M solution in DMF) and the resulting suspension was shaken at room temperature for 20 hours.
• EXAMPLE 5 Derivatization of Fmoc-Leucine-Novasyn TGT with scaffold (2) .
• scaffold 2_ (10.5mL of a 0.133M solution in DMF, 1.4mmol), HATU (10.5mL of a 0.133M solution in DMF, 1.4mmol) and DIPEA (10.5mL of a 0.133M solution in DMF, 1.4mmol) and this suspension was shaken at room temperature for 20 hours.
• the resin was filtered and washed (4x) with DMF, (4x) with EtOAc, (4x) with CH 2 C1 2 then dried in vacuo over P 2 Os to give the 3-Fmoc-aminoglucose scaffold ⁇ -linked solid support 13.
• Electrospray L.C - M.S. C 3 oH3 6 N 2 O ⁇ o requires 584, found 585 (+ve ion), 583 (-ve ion).
• the resin 1_6 (0.52g, O.llmmol) was swelled in DMF (15mL), filtered and washed (2x) with DMF then treated with 20% piperidine/DMF (15mL) for 30 minutes. The resin was filtered and washed (4x) with DMF then treated with 2, 4-dimethoxybenzoic acid (5mL of a 0.03M solution in DMF), HATU (5mL of a 0.03M solution in DMF) and DIPEA (5mL of a 0.03M solution in DMF) and the suspension was shaken at room temperature for 20 hours.
• the resin was filtered and washed (4x) DMF, (4x) THF, (4x) CH 2 C1 2 and dried in vacuo over P 2 0 5 to give the amide functionalized solid support. 180mg of resin was cleaved with 10% TFA/CH 2 C1 2 as previously described, to give 37mg of a white solid.
• the resin 1 (0.25g, 0.053mmol) was swelled in THF-MeOH (1:1)
• the library described in Scheme 6 was synthesized using the IRORI AccuTag ® -100 radiofrequency tagging solid phase synthesis system (available from Irori Quantum Microchemistry, LaJolla, CA) employing the "directed sorting" split-pool method.
• the 330 ⁇ L MicroKans® were used for the library synthesis.
• 30mg of scaffold functionalized resin (resin loading: 0.2 mmol/g) was placed into each MicroKan ® using a Tecan resin dispensing robot, All synthetic procedures for library synthesis were as described for the synthesis of compounds 5 and _18_. All reactions were performed in a narrow-neck flat-bottom reaction vessel and agitated on an orbital shaker.
• the products were obtained as discrete entities by sorting the MicroKans into individual test tubes and treating with the cleavage cocktail.
• the resulting product-containing solutions were transferred to 48 deep well microtiter plates, and the solvent was removed on a Savant Speed vac ® SC210A. Approximately, 3-4 mg of each library product was obtained.
• the analysis of library compounds was performed on an HPLC/MS system consisting of a Perkin-Elmer 200 HPLC, an Alltech 500 evaporative light scattering detector, and a Perkin-Elmer API 100 mass spectrometer.
• the cans are treated with a .02M solution of the required sugar scaffold in DMF, in the presence of .02M 0- (7-azabenzotriazol- 1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate (HATU) and .02M DIPEA. Shaking is conducted for 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.
• EXAMPLE 8 Procedures for Screening for Biological Activity.
• BHI Brain Heart Infusion
• CAA Casamino Acids
• Enterococcus faecium ATCC 49624.
• Enterococcus faecalis ATCC 29212
• Acinetobacter anitra 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 (OD 6 oo * 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 5 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 maximium 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.
• E. coli . (ATCC #23226) are permeabilized with ether according to Mirelman, et al.(1976), and Maas and Pelzer [Arch. Microbiol., 130:301-306
• 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.
• Assay buffer (10 ⁇ L) , ATP (20 ⁇ L) , UDP pentapeptide (10 ⁇ L) and 1 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 120 min. Ice cold 20% TCA (100 ⁇ L) is added to each well, the plates are gently mixed (60 sec), then refrigerated (4°C) for 30 min 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.

Landscapes

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

Applications Claiming Priority (3)

Publications (2)

Family

ID=21951894

Family Applications (1)

Country Status (4)

Families Citing this family (8)

Citations (2)

Family Cites Families (5)

• 1998
  • 1998-05-28 JP JP50089799A patent/JP2002502393A/ja active Pending
  • 1998-05-28 AU AU77000/98A patent/AU7700098A/en not_active Abandoned
  • 1998-05-28 WO PCT/US1998/010867 patent/WO1998053813A1/en not_active Application Discontinuation
  • 1998-05-28 EP EP19980924946 patent/EP0998280A4/de not_active Withdrawn

Patent Citations (2)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events