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WO1999038993A1 - Banque combinatoire de quinolones - Google Patents

Banque combinatoire de quinolones Download PDF

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Publication number
WO1999038993A1
WO1999038993A1 PCT/US1999/001738 US9901738W WO9938993A1 WO 1999038993 A1 WO1999038993 A1 WO 1999038993A1 US 9901738 W US9901738 W US 9901738W WO 9938993 A1 WO9938993 A1 WO 9938993A1
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WO
WIPO (PCT)
Prior art keywords
resin
linked
formula
quinolone
chosen
Prior art date
Application number
PCT/US1999/001738
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English (en)
Inventor
Roland Ellwood Dolle
Hongzhi Zhang
Christopher D'agostino
Christopher T. Louer
Original Assignee
Pharmacopeia, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacopeia, Inc. filed Critical Pharmacopeia, Inc.
Priority to AU23459/99A priority Critical patent/AU2345999A/en
Publication of WO1999038993A1 publication Critical patent/WO1999038993A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
    • C07D215/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3 with oxygen atoms in position 4
    • 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 synthesis of chemical compounds for biological assay, and more particularly, to the synthesis of combinatorial libraries of substituted quinolones.
  • the present invention relates to a combinatorial library of substituted quinolone compounds optionally encoded with tags.
  • the present invention also relates to the use of this library containing substituted quinolone compounds in assays to discover biologically active compounds.
  • the invention relates to a combinatorial chemical library for biological assay comprising a plurality of members of the Formula I:
  • T-L Q - -Z wherein: T * is a tag; L- is a first linker;
  • S is a solid support; wherein — is — ⁇ , wherein
  • L'- is a second linker
  • Z is a compound of formula R , wherein R 1 is chosen from the group consisting of C ⁇ . 2 o alkyl, aryl, arylalkyl, either aryl or heteroaryl fused to a 3- or 4-membered moiety to form a non-aromatic second ring, cycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, substituted alkyl, substituted aryl and substituted heteroaryl;
  • W is R 2 or R 3 NHCH 2 Y-, wherein R 2 is chosen from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, substituted alkenyl, substituted alkynyl, substituted aryl and substituted heteroaryl; R 3 is chosen from the group consisting of alkyl, alkoxyalkyl, arylalkyl, cycloalkylalkyl, heteroalkyl, heteroarylalkyl and heterocycloalkylalkyl; and
  • Y is chosen from the group consisting of aryl, heteroaryl, substituted aryl and substituted heteroaryl.
  • a preferred embodiment of the invention is a library comprising a plurality of members of Formula I wherein: R 1 is chosen from the group consisting of the amine residues of Table 1;
  • W is R 2 or R 3 NHCH 2 Y- wherein:
  • R" is chosen from the group consisting of the stannane residues of Table 2;
  • R 3 is chosen from the group consisting of the amine residues of Table 3;
  • Y is chosen from the group consisting of aryl, heteroaryl, substituted aryl and substituted heteroaryl.
  • R 1 is chosen from the group consisting of benzyl, butyl, 2-cyanoethyl, cyclopropylmethyl, cyclohexylmethyl, 2-dimethylaminoethyl, 3,3-dimethylbutyl, furfuryl, 3-(l-imidazoyl)ethyl, isobutyl, 2-methylbutyl, 4-methylphenethyl, 2-methylthioethyl, 2-(l-morpholinyl)ethyl, myrantyl, 1-naphthylenemethyl, piperonyl 2-phenylphenethyl, 2-( 1 -pyrrolidiny l)ethyl, 2-(2-aminoethyl)- 1 -pyrrolidinyl, 2-pyridylethyl, 3-pyridylmethyl,tetrahydrofurfuryl and 2-
  • R 2 is chosen from the group consisting of allyl, 1-ethoxyvinyl, 4-formylphenyl, 3-formyl-2-thienyl, 2-furanyl, phenyl, 2-phenylethynyl,thienyl and vinyl.
  • R 3 is chosen from the group consisting of butyl, benzyl, cyclohexylmethyl, 3-isopropoxypropyl, 2-methoxyethyl, phenethyl, 2-phenylphenethyl, piperonyl, 3-pyridylmethyl and 3-(l-pyrrolidi-2-one ⁇ propyl.
  • Y is chosen from the group consisting of m-substituted phenyl, p-substituted phenyl, and 3-substituted thienyl.
  • Another preferred embodiment of the invention is a combinatorial library comprising a plurality of members of Formula I wherein: (T'-L) is a compound of Formula II
  • n 3-12;
  • Ar is halophenyl
  • Ar is pentachlorophenyl.
  • -L' is an acid-cleavable linker.
  • Another preferred embodiment of the invention is a combinatorial library comprising a plurality of members of Formula I wherein:
  • -L' is selected from and ⁇ O-I ⁇ C °H
  • the compounds -Z of Formula I may be detached by acidic, oxidative, or other cleavage techniques.
  • Formula I is , acidic cleavage may be represented by:
  • R m represents a cleaved compound of Formula HI.
  • Another aspect of the invention is the use of the herein described combinatorial library in assays to discover biologically active compounds of Formula III.
  • another aspect of the invention is a method for identifying a compound having a desired characteristic which comprises testing a combinatorial library comprising a plurality of members of Formula I, either attached to, or detached from, the solid supports, in a biological assay which identifies compounds of Formula HI having the desired characteristic.
  • a further aspect of the invention is determining the structure of any compound identified as having the desired biological activity.
  • the chemical structures of compounds that are identified by biological assays as having a desired characteristic can be determined either by decoding the tags ( , T'-L- of Formula I) (Still et al., "Complex Combinatorial Chemical Libraries Encoded With Tags", WO 94/08051) or by deconvolution of the library (Smith et a Biomed. Chem. Lett. 4. 2821 (1994); Kurth et al., J. Ore. Chem. 59, 5862 (1994); Murphy et al., J. Am. Chem. Soc. 117, 7029 (1995); Campbell et al., J. Am. Chem. Soc. 118, 5381 (1995); and Erb et al., Proc. Natl. Acad. Sci. USA 91, 11422 (1994)).
  • a further embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid to a solid support via its carbonyl group to form a resin-linked ⁇ -keto ester;
  • An additional preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid to a solid support via its carbonyl group to form a resin-linked ⁇ -keto ester;
  • a preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: cleaving a resin-linked amino quinolone from the resin to provide an amino quinolone of Formula HI.
  • a further embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R l is chosen from the group consisting of C ⁇ . 2u alkyl, aryl, arylalkyl, either aryl or heteroaryl fused to a 3- or 4-membered moiety to form a non-aromatic second ring, cycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, substituted alkyl, substituted aryl and substituted heteroaryl; and
  • R 2 is chosen from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, substituted alkenyl, substituted alkynyl, substituted aryl and substituted heteroaryl.
  • Another preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R 1 is chosen from the group consisting of the amine residues of Table 1; and R 2 is chosen from the group consisting of the stannane residues of Table 2.
  • a further preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R 1 is chosen from the group consisting of benzyl, butyl, 2-cyanoethyl, cyclopropylmethyl, cyclohexylmethyl, 2-dimethylaminoethyl, 3,3-dimethylbutyl, furfuryl, 3-(l-imidazoyl)ethyl, isobutyl, 2-methylbutyl, 4-methylphenethyl, 2-methylthioethyl, 2-(l-morpholinyl)ethyl, myrantyl, 1-naphthylenemethyl, piperonyl 2-phenylphenethyl, 2-(l-pyrrolidinyl)ethyl, 2-(2-aminoethyl)-l-pyrrolidinyl, 2-pyridylethyl, 3-pyridylmethyl, tetrahydrofurfuryl, and 2-ti ienylmethyl; and
  • R is chosen from the group consisting of allyl, 1-ethoxy vinyl, 4-formylphenyl, 3-formyl-2-thienyl, 2-furanyl, phenyl, 2-phenylethynyl,thienyl and vinyl.
  • Yet another preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R 1 is chosen from the group consisting of C ⁇ . 2o alkyl, aryl, arylalkyl, either aryl or heteroaryl fused to a 3- or 4-membered moiety to form a non-aromatic second ring, cycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, substituted alkyl, substituted aryl and substituted heteroaryl;
  • R 2 is chosen from the group consisting of alkenyl, alkynyl, aryl, heteroaryl, substituted alkenyl, substituted alkynyl, substituted aryl and substituted heteroaryl;
  • R 3 is chosen from the group consisting of alkyl, alkoxyalkyl, arylalkyl, cycloalkylalkyl, heteroalkyl, heteroarylalkyl and heterocycloalkylalkyl;
  • Y is chosen from the group consisting of aryl, heteroaryl, substituted aryl and substituted heteroaryl.
  • Yet another preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R l is chosen from the group consisting of the amine residues of Table 1;
  • R 2 is chosen from the group consisting of the stannane residues of Table 2;
  • R 3 is chosen from the group consisting of the amine residues of Table 3; and
  • Y is chosen from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl.
  • Another preferred embodiment of the invention is a method of synthesizing a library comprising a plurality of members of Formula I which comprises: a. attaching an acyl Meldrum's acid of the formula:
  • R 1 is chosen from the group consisting of benzyl, butyl, 2-cyanoethyl, cyclopropylmethyl, cyclohexylmethyl, 2-dimethylaminoethyl, 3,3-dimethylbutyl, furfuryl, 3-(l-imidazoyl)ethyl, isobutyl, 2-methylbutyl, 4-methylphenethyl,
  • R 2 is chosen from the group consisting of allyl, 1 -ethoxyvinyl, 4-formylphenyl, 3-formyl-2-thienyl, 2-furanyl, phenyl, 2-phenylethynyl,thienyl and vinyl.
  • R 3 is chosen from the group consisting of butyl, benzyl, cyclohexylmethyl, 3-isopropoxypropyl, 2-methoxyethyl, phenethyl, 2-phenylphenethyl, piperonyl, 3-pyridylmethyl and 3-(l-pyrrolidi-2-one)propyl.
  • Y is chosen from the group consisting of m-substituted phenyl, p-substituted phenyl and 3-substituted thienyl.
  • An additional embodiment of each of the above inventions further comprises cleaving a resin-linked quinolone from said resin to provide a quinolone.
  • TMS trimethylsilyl
  • Alkoxy means alkoxy groups of from 1 to 8 carbon atoms of a straight- branched or cyclic configuration and combinations thereof. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy.cyclohexyloxy and the like.
  • Alkyl is intended to include linear or branched hydrocarbon structures and combinations thereof.
  • “Lower alkyl” means alkyl groups of from 1 to 12 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl, pentyl, hexyl, octyl and the like.
  • “Alkenyl” includes C 2 -C 8 hydrocarbons of a linear, branched or cyclic
  • alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, c-hexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, 2,4-hexadienyl and the like.
  • Alkynyl includes C 2 -C 8 hydrocarbons of a linear, branched, or cyclic
  • alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, 3-methyl-l -butynyl, 3,3-dimethyl-l-butynyl and the like.
  • Aryl means a 5- or 6-membered aromatic ring; a bicyclic 9- or 10-membered aromatic ring system; or a tricyclic 13- or 14-membered aromatic ring; each of which is optionally substituted with 1-3 groups selected from lower alkyl, substituted alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, methylenedioxy, alkoxyethoxy, cyano, acylamino, phenyl, benzyl, phenoxy, napthyloxy, aryloxy, benzyloxy .heteroaryl and heteroaryloxy.
  • phenyl, benzyl, phenoxy, benzyloxy.heteroaryl and heteroaryloxy substituents may be optionally substituted with 1-3 substituents selected from lower alkyl, alkenyl, alkoxy, alkynyl, benzyl, benzyloxy, carboxamido, cyano, formyl, halogen, heteroaryl, heteroaryloxy, hydroxy.nitro and phenyl.
  • “Arylalkyl” means an alkyl containing an aryl ring. For example: benzyl, phenethyl, 4-chlorobenzyl and the like.
  • Aryloxy means a phenoxy group where the aryl ring is optionally substituted with 1 to 2 groups selected from halo, alkoxy or alkyl.
  • Cycloalkyl includes cyclic hydrocarbon groups of from 3 to 12 carbon atoms.
  • Examples of “cycloalkyl” groups include c-propyl, c-butyl, c-pentyl, c-hexyl, 2-methylcyclopropyl, norbornyl.adamantyl and the like.
  • Cycloalkylalkyl means an alkyl substituted with a cycloalkyl functionality. Examples include cyclopropylmethyl, cyclohexylmethyl and myrantyl.
  • Halogen includes F, Cl, Br, and I, with F and Cl as the preferred groups.
  • Halophenyl means phenyl substituted by 1-5 halogen atoms.
  • Halophenyl includes pentachlorophenyl, pentafluorophenyl, and 2,4,6-trichlorophenyl.
  • Heterocycloalkyl means a cycloalkyl ringcontaining 0-2 heteroatoms selected from O, N, and S; where the methylene H atom may be optionally substituted with alkyl, alkoxy, formyl or halogen. Both methylene hydrogens on a particular carbon atom may be replaced with carbonyl.
  • Heteroaryl means a 5- or 6-membered heteroaromatic ring containing 0-2 heteroatoms selected from O r N, and S: or abicyclic 9- or 10-membered heteroaromatic ring system containing 0-2 heteroatoms selected from O, N, and S; where the methine H atom may be optionally substituted with alkyl, alkoxy, formyl or halogen.
  • the 5- to 10-membered aromatic heterocyclic rings include imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.
  • Heteroarylalkyl means an alkyl containing a heteroaryl ring. For example: pyridinylmethyl, pyrimidinylethyl and the like.
  • Substituted alkyl means an alkyl or branched alkyl optionally substituted with groups such as alkoxy, alkylsulfide, amino, cyano, formyl, halogen, hydroxy and nitro.
  • the term "combinatorial library” means a collection of molecules based upon a logical design and involving the selective combination of building blocks by means of simultaneous chemical reactions. Each species of molecule in the library is referred to as a member of the library.
  • the combinatorial library of the present invention represents a collection of molecules of sufficient number and diversity of design to afford a rich molecular population from which to identify biologically active members.
  • residue shall mean the portion of the reagent that is incorporated into the product molecule after the reaction between the reagent and die molecule designated as the starting material.
  • residues of primary amines R l NH 2
  • residues of trialkyl stannanes R 2 SnR 3
  • residues of primary amines R'NHi
  • R 1 chosen from the "amine residues” shall mean R 1 chosen from those residues as shown in Table I.
  • substituents of "amine residues” are R 1 as C ⁇ . 20 alkyl, aryl, arylalkyl, either aryl or heteroaryl fused to a 3- or 4-membered moiety to form a non-aromatic second ring, cycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, substituted alkyl, substituted aryl and substituted heteroaryl as substituents on amino groups.
  • R chosen from the “stannane residues” shall mean
  • R 2 chosen from those residues as shown in Table II.
  • substituents of "stannane residues” are R 2 as alkenyl, alkynyl, aryl, heteroaryl, substituted alkenyl, substituted alkynyl, substituted aryl and substituted heteroaryl. Specific examples are 31
  • allyl 1 -ethoxyvinyl, 4-formylphenyl, 3-formyl-2-thienyl, 2-furanyl, phenyl, 2- phenylethynyl, thienyl and vinyl.
  • R chosen from the "amine residues” shall mean R chosen from those residues as shown in Table HI.
  • substituents of "amine residues” are R 3 as alkyl, alkoxyalkyl, arylalkyl, cycloalkylalkyl, heteroalkyl, heteroarylalkyl and heterocycloalkylalkyl as substituents on amino groups.
  • the "reagent” shall mean the chemical entity shown in the reaction scheme or named and described in the specification.
  • the reagent is reacted with the molecule designated as the starting material.
  • reagents are the primary amines shown in Scheme 3 and Scheme 5, respectively, and the tributyl stannanes in Scheme 4.
  • the linker L' preferably is an acid-cleavable linker.
  • the compound Z is attached to the benzylic oxygen where the oxymethylene is para to the methylene oxygen attached to the support. If another substituent is present, for example methoxy, then this particular substituent is placed ortho to the oxymethylene group that is attached to the compound Z. 32
  • the identifiers or tags of this invention, T'-L of Formula I are chemical entities which possess several properties.
  • the identifiers are detachable from the solid supports, preferably by oxidative cleavage. They are individually differentiable, and preferably separable from one anotiier.
  • the identifiers must be stable under the synthetic conditions and capable of being detected at very low concentrations (i.e., IO "15 to IO "9 mole). Preferred identifiers are discerned with readily available technical equipment operated by someone with the capabilities of one skilled in analytical techniques.
  • the identifiers are relatively economical, and each is usually found attached to the solid supports at concentrations of at least 0.01 picomol, usually 0.1-10 pmol per bead after synthesis of the combinatorial library.
  • the tags may be structurally related or unrelated , e.g. a homologous series, repetitive functional groups, related member of the Periodic Chart, different isotopes, combinations thereof or the like. Distinguishing features may be the number of repetitive units, such as methylene groups in an alkyl moiety; alkyleneoxy groups in a polyalkyleneoxy moiety; halo groups in a polyhalo compound; ⁇ - and/or ⁇ -substituted ethylene groups
  • substituents may be alkyl, alkoxy, carboxy, amino, halo or the like; isotopes; etc.
  • Suitable tags and methods for their employment are described in US patent application 08/743,960 filed October 5, 1996, herein incorporated by reference.
  • TentaGelTM S-PHB (available from Rapp Polymere, Tubingen, Germany) is the hydroxyl-functionalized polyethylene glycol-grafted polystyrene resin.
  • the material upon which the syntheses of the present invention are performed are referred to as solid supports, beads and resins.
  • beads, pellets, disks, fibers, gels or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, etc.( i.e., material having a rigid or semi-rigid surface) and soluble supports such as low molecular weight, non- cross-linked polystyrene.
  • Optical Isomers Diastereomers - Geometric Isomers
  • Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers and other stereoisometric forms which may be defined in terms of absolute stereochemistry as (R> or (S)- or as (D)- or (L)- for amino acids.
  • the present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms.
  • Optically active (R)- and (S)- or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the library of the present invention is useful as a screening tool for discovering new lead structures by evaluation across an array of biological assays, including the discovery of selective inhibition patterns across isoenzymes.
  • the library 34 is useful as a screening tool for discovering new lead structures by evaluation across an array of biological assays, including the discovery of selective inhibition patterns across isoenzymes.
  • Fluorescence activated cell sorting is a screening method that can be used to detect compound activity.
  • a particularly useful method for identifying activities with respect to a large variety of enzymes and molecular targets is the lawn assay disclosed in US patent application 08/553,056, filed November 3, 1995.
  • a library of solid supports preferably beads, is screened for the ability of compounds on the supports to affect the activity of an enzyme.
  • supports containing the active compounds are quickly and easily located merely by viewing zones of inhibition in a matrix.
  • the solid supports are contacted with a colloidal matrix such as agarose.
  • the compounds are linked to the supports by a cleavable linker and released, e.g., by 35
  • the compounds contact enzyme contained in the matrix.
  • Substrate is contacted with the matrix and reacts with the enzyme. Conversion of substrate to product is measured by monitoring a photometric change in the substrate or in a coenzyme or cofactor involved in reaction.
  • the substrate can be fluorogenic, i.e., becoming fluorescent when converted to product.
  • compounds mat are active inhibitors of the enzyme reaction are detected as dark zones of inhibition. The less active, or inactive, compounds are contained in the lighter areas.
  • the lawn assay is used to determine compounds that bind to a target molecule and thereby affect a detectable signal generated by a labeled compound bound to the target molecule.
  • This assay allows screening of compounds that, e.g., act as agonists or antagonists of a receptor or that disrupt a protein: protein interaction. It also allows detection of binding to DNA. RNA or complex carbohydrates. For example, neurokinin receptor binds to a 36
  • NBD 7-nitrobenz-2-oxa-l,3-diazol-4-yl(NBD) - labeled peptide ligand.
  • the labeled ligand has the following formula: PhCO-2,4-diaminobutyric acid(gamma-NBD)-Ala-D-trp- Phe-D-pro-Pro-NH2.
  • NBD is a fluorophore and binding of the labeled ligand to the neurokinin receptor increases NBD's fluorescence. When a compound displaces the NBD-labeled ligand from the neurokinin receptor, fluorescence of the NBD fluorophore is reduced (G. Turcatti, H. Vogel, A.
  • a library of solid supports can be screened for compounds that bind to neurokinin receptor in a colloidal matrix using this method. Active compounds are found in zones of decreased fluorescence.
  • a radioligand tritium or 125 lodine-labeled
  • SPATM Scintillation Proximity Assay beads
  • FlashplatesTM DuPont NEN Research Products
  • the signal can be detected using x-ray film, or other commercially available film that is specifically designed to detect tritium dependent scintillations.
  • Compounds released into the matrix from the solid supports that bind to receptor and displace the radioligand reduce the scintillation signal, i.e., result in a zone of reduced scintillation.
  • the receptor used in the assay can be, e.g., membrane- bound, tethered to a solid phase or solubilized.
  • fluorogenic substrates that convert to fluorescent products, i.e., fluorogenic substrates.
  • fluorogenic substrates include fluorescein diacetate, which converts to fluorescein in me presence of an esterase, such as carbonic anhydrase.
  • fluorogenic substrates include 7-amino-trifluoromethyl coumarin (AFC), 4-trifluoromethylumbelliferyl
  • HFC 7-amino-4-methylcoumarin
  • MNA 4-methoxy-2-naphthylamine
  • a fluorescent substrate can be used that converts to a product having different excitation and emission characteristics. By using band-pass filters so that only the product is excited and detected, the substrate can be effectively screened out.
  • An example of such a fluorescent substrate is peptidylaminomethylcoumarin, which is converted by an appropriate protease such as thrombin, to free aminomethyl- coumarin. The free aminomethylcoumarin excites and emits at different wavelengths than does the peptidylaminomethylcoumarin (S. Kawabata et al, (1988) Eur. J. Biochem. 172, 17).
  • peptide substrate can be produced having two fluorophores at opposite ends, one absorbing the fluorescence of the other. The substrate therefore emits a negligible amount of light. Upon cleavage of the peptide by a suitable protease, the absorbing fluorophore is released and no longer quenches the other fluorophore, resulting in an increase in fluorescence.
  • DAB CYL 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • DAB CYL 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic acid
  • EDANS 4-(dimethylaminophenylazo)-benzoic
  • Fluorescence can be detected, e.g., using a field format fluorescence detection instrument such as FluorimagerTM from Molecular Dynamics. This type of fluorimeter is capable of determining fluorescence over a large area. It is also possible to detect fluorescence using a CCD camera and to transfer the image data to a computer. The image can be generated by illumination of the fluorophore with light of the wavelength that specifically excites it. Detection can be optimized by using a bandpass filter between the camera and the assay that is specific for the emission wavelength of the fluorophore.
  • a field format fluorescence detection instrument such as FluorimagerTM from Molecular Dynamics. This type of fluorimeter is capable of determining fluorescence over a large area. It is also possible to detect fluorescence using a CCD camera and to transfer the image data to a computer. The image can be generated by illumination of the fluorophore with light of the wavelength that specifically excites it. Detection can be optimized by using a bandpass filter between the camera and
  • an assay for compounds that affect a chromogenic substrate, p-nitrophenylphosphate is described in the examples. It is also possible, for example, to measure a change in absorbance.
  • NADP is a common cofactor in many enzymatic reactions. Absorbance changes as NADPH is converted to NADP by, for example, neutrophil NADPH oxidase (such as during an oxidative burst associated with an immune response). This change can be monitored to determine zones of inhibition for compounds that inhibit this and other enzymes that use NADP, NADPH, NAD and NADH as co-factors.
  • the sensitivity of assays that measure a change in absorbance is believed to be generally lower than those that measure a change in fluorescence.
  • detectable changes resulting from conversion of substrate to product include chemiluminescent changes and scintillation changes.
  • Scintillation changes can be detected as described above for receptor binding with the exception that a substrate is attached to the scintillant (i.e., to the bead or plate containing scintillant).
  • a radioactive reagent such as tritiated famesyl pyrophosphate, can be added to the substrate by an enzyme such as farnesyl protein transferase.
  • Transferase inhibitors prevent addition of the tritiated farnesyl pyrophosphate to the substrate, resulting in a reduction in detectable scintillations; i.e., transferase inhibitors are found in zones of reduced scintillation.
  • removal of the radioactive portion of a substrate attached to the scintillant such as by cleaving with a protease, releases the radiolabeled portion (i.e., moves it away from the scintillant).
  • protease inhibitors cause an increase in 40
  • the scintillation signal can be detected using x-ray film or film that is specifically designed to detect tritium dependent scintillations.
  • a labeled ligand For assaying binding to a target molecule, a labeled ligand provides a signal that indicates such binding.
  • the label is preferably a fluorescent moiety that alters its signal as a result of target molecule binding.
  • fluorescent moieties are NBD and 5-(dimethylamino)-l-naphthalenesulfonyl (Dansyl) chloride.
  • Colloidal matrices that are useful for the lawn assay include silica gel, agar, agarose, pectin, polyacrylamide, gelatin, starch, gellan gum, cross-linked dextrans (such as SephadexTM , available from Supelco, Bellefonte PA) and any other matrix that allows diffusion of compound from the solid supports in a limited region.
  • Low melting-temperature agarose is preferred, generally in an amount of 0.5-2.0%, wt./vol.
  • the colloidal matrix can be chosen to obtain a desired rate of diffusion. It is generally preferred to use a matrix that allows a high concentration of compounds to be easily obtained.
  • the solid supports are preferably embedded in a matrix containing the relevant enzyme. Following cleavage, compound diffuses from the support into the matrix and contacts the enzyme. Substrate is then added and, as it diffuses into the colloidal matrix, active compounds inhibit conversion to product. By following such a procedure, compounds to be screened are allowed to interact with enzyme before the 41
  • Solid supports can also be applied to the matrix's surface and the compounds allowed to diffuse into the matrix. This can be done, for example, by arraying the solid supports on the surface of a stretched sheet of plastic film (e.g., ParafilmTM, available from Aldrich Co., Milwaukee, WI), and then applying the sheet to the surface of the matrix.
  • a stretched sheet of plastic film e.g., ParafilmTM, available from Aldrich Co., Milwaukee, WI
  • one matrix can contain enzyme and beads and the other can contain substrate. Contacting the surfaces of the matrices with each other allows the substrate to come into contact with the enzyme. It is also possible to add a solution of substrate over the surface of a matrix containing enzyme and embedded supports. Adding solution is prefe ⁇ ed when, e.g., the substrate interferes with detection. Solution containing the substrate can be removed prior to determining the zones of activity. 42
  • a matrix contains the target molecule bound to the labeled ligand which emits a detectable signal indicating binding to the target molecule.
  • Compounds from the solid supports are diffused into d e matrix, preferably from embedded supports using photolysis.
  • labeled ligand can be diffused into the matrix from a second matrix (or liquid layer) after release of the compounds in the matrix. This allows the compounds to contact the receptor before interaction with the labeled ligand, which can be advantageous.
  • Solid supports may contain acid cleavable linkers, as further described below. These linkers can be cleaved in a gaseous acidic atmosphere before placing the supports on the matrix. The compounds, although cleaved, remain on the surface of the supports and diffuse into the matrix when the supports are placed on it. It is even possible to cleave the compounds prior to pouring low-melt liquid agarose over the solid supports. While some of the compounds will be washed away, sufficient compound can remain on the support's surface to result in a recognizable zone of activity.
  • the compounds are cleaved after the beads are embedded in the colloidal matrix
  • photolysis e.g., cleaving by exposure to UV light.
  • UV light By adjusting light exposure, it is possible to control the amount of compound that diffuses into the matrix. If more light is applied, by increasing intensity or duration, more cleavage results, in turn releasing more compound into the matrix.
  • the solid supports can be in a random arrangement, or in an ordered one.
  • a library of beads can be suspended in a solvent, such as ethanol, and deposited on the bottom of a Petri plate. After the solvent has completely evaporated, a layer of agarose containing the relevant enzyme or target molecule can be poured over the beads.
  • a layer of agarose containing the relevant enzyme or target molecule can be poured over the beads.
  • an ordered a ⁇ ay can be used to space beads apart and allow easier identification of those that are active.
  • beads are a ⁇ ayed on a rigid template such as a thin glass disk having tapered holes. The tapered holes are sized to allow only single beads to settle into them.
  • Beads are suspended in a solvent such as ethanol, and washed over the top of the template to fill each hole with one bead.
  • the beads can then be cleaved in the dry state and the template set down on the colloidal matrix. Capillary action wets the beads, facilitating diffusion of the cleaved compounds into the matrix. Zones of activity can be observed immediately below beads containing active compounds. It is possible to remove the template prior to detecting zones of activity if an image of the template on the matrix is made. This image can later be used to co ⁇ elate the zones of inhibition in the matrix with the positions of beads on the template.
  • Ordered a ⁇ ays also may be useful in identifying the compounds on supports that are associated with zones of activity. Specifically, the a ⁇ ay can be ordered so that 44
  • the identity of active compounds is determined using d e encoding system described above, which employs tags T' encoding the identities of the compounds on the solid supports.
  • the assay is preferably carried out so that there is slow diffusion of d e compound from the solid support following cleavage. This results in a high concentration of compound in the vicinity of me bead. Thus, very little compound is required to cause a distinct zone of activity. Most of the compound remains on the support for any subsequent assays that are required. Such further assays may be needed if more than one solid support is found in the zone of activity. It may then be necessary to retest the supports from the zone to determine which one releases the active compound. Reassaying may be required as a matter of course if many thousands of beads are screened at high density. Reassaying may also be desirable to test for selectivity, i.e. to determine which active compounds are inactive in a second assay that tests for a different property.
  • Reducing photolysis time reduces the amount of compound released from the support. As the concentration of the compounds is lowered, diose that are less active become more difficult to detect. As a result, the number of active compounds drops.
  • compounds that were detectable at the shortest elution times, i.e., that were most potent were also identified as most potent using conventional solution-phase screening. The activity of die inhibitors was found to co ⁇ elate with the size and duration of the zone of activity: the most potent compounds produced the largest zones for the longest time for any given amount of photolysis.
  • a second assay of the active compounds may be performed to choose those mat should be further evaluated.
  • the second assay can determine whether there is cross reactivity with other targets, i.e., a "selectivity screening".
  • a given library of compounds can be screened for activity against HTV protease, a member of the aspartyl protease family, using DABCYL-gAbu-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln- EDANS.
  • Compounds found active in the initial assay can be counterscreened against a second, different aspartyl protease, such as cathepsin D. Alternately, all compounds screened in the assay for activity against H1N protease could be simultaneously screened in the counter assay.
  • PI 6 is a known protein inhibitor of cyclin-dependent kinase-4 (Cdk-4).
  • Cdk-4, Cyclin Dl, P16, a fluorogenic substrate and a library of beads to be screened can be included in a layer of low-melt agarose.
  • die gel can be subjected to an electrophoretic separation.
  • Product migrates to the anode, where it is preferably trapped on an anode filter. The location of product on die filter indicates die position in the gel of compound that disrupts P16 inhibition of Cdk4.
  • an electrophoretic procedure is used to separate substrate from product to increase the sensitivity of the assay.
  • a substrate is used which changes charge when converted to product.
  • An example of such a substrate is the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly attached to a fluorophore, sold commercially as Pep-TagTM (Promega Corp.).
  • Protein kinase A (PKA) phosphorylates this substrate, which has net +1 charge, to form a phosphopeptide which has a net - 1 charge.
  • PKA Protein kinase A
  • a lawn assay is performed in which PKA is contacted in a colloidal matrix with substrate and a library of potential inhibitors.
  • An electrophoretic separation is then carried out across the width of (i.e., perpendicular to) the matrix.
  • the phosphopeptide i.e., product
  • the dephosphopeptide i.e., substrate
  • electroblotting can be achieved.
  • the phosphopeptide can be electroblotted to a suitable membrane such as an ImmobilonTM CD membrane.
  • the dephosphopeptide can be electrotransfe ⁇ ed to an appropriate paper such as WhatmanTM 3MM paper.
  • the substrate and product can be chosen so that one is neutral and one is charged. Application of die electrophoretic field will remove the charged moiety.
  • the resulting matrix will contain only the neutral moiety, thereby allowing detection of compounds that affect the conversion to product.
  • the position of die bead containing die active compound can be determined by fluorescent imaging of d e substrate or product using, e.g., photography or video imaging. This technique increases sensitivity of the lawn assay by separating fluorescent substrate from fluorescent product, concentrating the fluorescent image and by eliminating compounds from the matrix that might cause background signal.
  • Other protein kinases and phosphatases such as protein kinase C, cyclin dependent kinases, MAP kinases and inositol monophosphatase can also be used with appropriate substrates in this method.
  • a protease can also be screened by this method by using a substrate consisting of an appropriate peptide linked to a labeling moiety such as a fluorophore.
  • the peptide sequence is selected so mat the substrate and product will migrate differentially in an electric field.
  • Enzymes that can be used in the assay include, but are not limited to, the following: Acid Phosphatase
  • the lawn assay is performed in Petri plates using two layers of agarose, each about 1.5 mm thick.
  • the first layer contains TentaGel S-NHTM beads (Rapp Polymere, Tubingen, Germany) and enzyme.
  • the TentaGel S-NH>TM beads have compounds to be screened attached thereto by a photocleavable linker and chemical tags attached for identifying the compounds, prepared according to me ods described herein.
  • the beads are either placed on the Petri plate and agarose poured over them or beads and agarose are first mixed and then poured together onto the piate.
  • a second layer of agarose containing the fluorescein diacetate is contacted with the first layer to initiate the reaction.
  • Fluorescein diacetate is hydrolyzed to produce fluorescein as me reaction proceeds.
  • the plate then becomes significantly brighter except in the vicinity of beads that release inhibitors, thereby forming zones of inhibition. Beads at the center of these zones are removed with a hollow glass tube or a spatula and washed in kann anol/methylene chloride (1: 1) or with hot water (80°C) to remove most of the agarose. After a final rinse in methanol, beads are either retested in a separate assay using the methods described above to confirm activity or analyzed to determine the relevant compound structures by tag decoding.
  • Example 1 Assay of Two Known Inhibitors
  • Carbonic anhydrase inhibitors are useful in treating, e.g., glaucoma. Results were compared with ose obtained using a conventional solution phase assay.
  • Two aryl sulfonamide-containing compounds (compounds “A” and “B") were synthesized on TentaGelTM beads (Rapp Polymere) and assayed in the standard solution-phase assay and in the lawn assay.
  • Compounds containing aryl sulfonamide substituents are known to be potent inhibitors of carbonic anhydrase.
  • Ki's were determined to be 4 and 660 nM for compounds A and B respectively.
  • beads containing each compound were embedded in agarose in a series of Petri plates. The right side of each plate contained beads with compound A and the left side contained beads with compound B. Separate plates were i ⁇ adiated for 2.5, 5, 10, 20 and 30 minutes.
  • the more potent inhibitor of carbonic anhydrase (compound A) showed a clear zone of inhibition after only 2.5 minutes of photolysis.
  • the weaker inhibitor (compound B) caused only a weak zone of inhibition after five minutes of photolysis. Ten minutes of photolysis was required to obtain a distinct zone. The clearest zones of inhibition were observed at the shortest time after photolysis. Zones at five minutes after photolysis were all sharper man at 15 minutes after photolysis. At 30 minutes after photolysis, all zones were much less distinct; some zones (for compound B) had disappeared.
  • an assay for inhibitors of inositol monophosphate is carried out in the same manner as described above for carbonic anhydrase inhibitors wim die following substitutions: the buffer used is 20mM Tris, lmM EGTA, pH 7.8.
  • the agarose layer contains 1 mg/mL of recombinant human inositol monophosphate, purified from E. coli, and lOmM MgCl 2 .
  • the substrate is methylumbelliferyl phosphate (Sigma Chemical Company, St. Louis MO, M-8883), CSPD (Tropix, Bedford MA) or CDP- Star (Tropix).
  • CSPD and CDP-Star ® are chemiluminescent substrates.
  • the buffer used is 20mM Tris, lmM EGTA, pH 7.8.
  • the agarose layer contains 1 mg/mL of recombinant human inositol monophosphate, purified from E. coli, and l
  • Inositol monophosphate is believed to be the molecular
  • This test chromogenically assays compounds for their effect on the catalytic domain of human SHPTP1, a protein tyrosine phosphatase [Pei et al., (1993) PNAS 90, 1092] using p-nitrophenylphosphate as a substrate.
  • This enzyme is assayed as described above for carbonic anhydrase with the following substitutions.
  • the buffer used is lOOmM N, N-bis(2hydroxyethyl)glycine, pH 8.
  • the first (lower) agarose layer contains 0.5 mg/mL recombinant human SHPTP1 catalytic domain, purified fromE. Coli, and die substrate is 4-nitrophenylphosphate (Sigma Chemical Corp.). Enzyme activity co ⁇ esponds with the release of the 4-nitrophenolate anion ( ⁇ m 400nm,
  • the identifiers attached to the solid support associated with the bioactivity may be decoded to reveal the structural or synthetic history of the active compound (Still et al., Complex Combinatorial Libraries Encoded With Tags, WO 94/08501) or the structures may be determined by deconvolution.
  • the effectiveness of such a library as a screening tool is demonstrated by the example of screening encoded combinatorial libraries for carbonic anhydrase inhibition (Burbaum et al.. Proc. Nad. Acad. Sci. 92, 6027-6031 (1995)).
  • Bio assays for a wide variety of enzymes and molecular targets can identify activity among the entities of a combinatorial library.
  • the compounds of the present invention can be prepared according to die following methods.
  • the solid support upon which a compound is being synthesized is uniquels tagged to define the particular chemical event(s) occurring during that step.
  • the tagging is accomplished using identifiers such as those of Formula H which record the sequential events to which the support is exposed during the synthesis. Tagging thus provides a reaction history for the compound produced on each support.
  • the identifiers are used in combination with one another to form a binary or higher order encoding scheme permitting a relatively small number of identifiers to encode a relatively large number of reaction products. For example, when N identifiers are used in a binary code, up to 2 N - 1 different compounds and/or conditions can be encoded.
  • identifiers By associating each variable or combination of variables at each step of the synthesis with a combination of identifiers which uniquely defines the chosen variables such as reactant, reagent, reaction conditions or combinations of these, one can use identifiers to define the reaction history of each solid support.
  • one begins with at least 10 , desirably at least 10 7 , and generally not exceeding 10 10 solid supports.
  • the supports accordingly into as many containers.
  • the appropriate reagents and reaction conditions are applied to each container and the combination of identifiers which encode for each R 1 choice is applied.
  • the tagging may be done prior to, concomitantly with or following the reactions which comprise each choice.
  • a sample of the tagged support can be taken at any stage during the reaction sequence and analyzed to obtain information about the synthesized compound.
  • the supports are combined, mixed and again divided, this time into as many containers as predetermined for the number of R 2 choices for die second step in the synthesis. This procedure of dividing, reacting, tagging and remixing is repeated until the combinatorial synthesis is completed.
  • a batch of hydroxyl-functionalized PEG-grafted polystyrene beads such as TentaGel S-PHB TM 1 is coupled to acyl Meldrum's acid 2 through alcoholysis.
  • the acyl Meldrum's acid is prepared by the condensation of 4-bromo-2-fluorobenzoyl chloride with Meldrum's acid.
  • Coupling is achieved by reacting a suspension of 1 in toluene with 2. The suspension is shaken with heating, drained and washed in succession with toluene and DCM. The derivatized resin 3 so obtained is men dried overnight under vacuum.
  • N,N-dimethylformamide dimethyl acetal is attached to resin derivative 3 to form enamine 4. This is accomplished by adding N,N-dimethylformamide dimethyl acetal to a suspension of solid sample 3 in ethyl ether. The mixture is shaken overnight, washed with ether and DCM and then dried overnight under vacuum to give enamine 4 on resin. After the reaction, a small portion of enamine 4 may be cleaved off the resin with TFA and quantitated by HPLC.
  • the N,N-dimethyl amino moiety in resin derivative 4 is displaced with various primary amines (e.g., see Table 1) to generate compounds.
  • the displacement occurs through a substitution with other amines (i.e., R'NH ⁇ ) that results in the formation of new enamines.
  • R'NHa is added to a suspension of resin 4 in THF and d e mixture is shaken overnight. The mixture is drained, washed with THF as well as DCM and dried overnight under vacuum to yield resin 5. The procedure is repeated for each of the twenty-four amines.
  • the enamine 5 on resin is cyclized to produce a quinolone derivative 6.
  • the cyclization of each enamine involves the displacement of the aromatic fluoride and formation of a new carbon-nitrogen bond intramolecularly.
  • a THF solution of lithium bis(trimethylsilyl)amide is added to a suspension of resin 5 in THF. The mixture is shaken overnight, washed with THF and DCM and then dried under vacuum to give quinolone derivative 6. This is repeated for each of the twenty-four enamines.
  • a small portion of the product on resin may be cleaved off using TFA and quantitated by HPLC. 59
  • Identifiers are then added to each quinolone derivative.
  • Unique tagging of the quinolone bound supports is achieved by using combinations of identifiers encoded in a binary scheme (the identifiers are not shown in the schematics for die purpose of simplicity) for the all choices of R 1 .
  • the identifiers are attached by adding a solution of up to three identifiers in DCM (10% wt. ratio of each identifier to the solid support) to a batch of the supports suspended in DCM and shaking the mixture for an hour. A dilute solution of rhodium trifluoroacetate dimer in DCM is added and the mixture is then shaken overnight, washed in DCM and dried under vacuum.
  • the coding for each quinolone derivative is confirmed by GC analysis. A second cycle of tagging may be applied when it is necessary.
  • the twenty-four batches of tagged quinolone resin 6 are pooled, mixed by shaking in DCM and then divided into a pre-determined number of reaction vessels. At mis stage, since 9 stannane reagents are used in the subsequent combinatorial step, equal portions of resin are placed in 9 vessels.
  • the mixtures of tagged quinolone derivatives 6 are coupled with a stannane reagent, co ⁇ esponding to one of the 9 R 2 choices shown in Table 2, by carbon-carbon bond formation. This is accomplished by adding TPP and Pd 2 (dba) 3 , followed by die addition of each stannane, to the respective suspensions of resin 6 in DEE. The mixture is shaken overnight with heating, drained and washed with DEE, DCM, EtOH and again with DCM. Compound 7 is obtained after drying overnight under vacuum. This is repeated for all 9 vessels. After coupling, a small portion of each batch of resin may be cleaved off and quantitated by HPLC. 60
  • Identifiers are introduced to resin 7. Unique tagging of the supports in each of the 9 reaction vessels is achieved with combinations of identifiers encoded in a binary scheme for all 9 choices of R 2 .
  • the identifiers are attached by adding a solution of up to three identifiers in DCM (10% wt. ratio of each identifier to the solid support) to a batch of the supports suspended in DCM and shaking the mixture for an hour. A dilute solution of rhodium trifluoroacetate dimer in DCM is added and the mixture is then shaken overnight, washed in DCM and dried under vacuum. The coding for each aldehyde derivative is confirmed by GC analysis.
  • the 3 batches of tagged aldehyde resin 7 (only some R 2 reagents generate aldehyde products, i.e., stannanes #7, 8 and 9 in Table 2) are pooled, mixed by shaking in DCM and divided into a pre-determined number of reaction vessels. In this case, since 10 amine reagents are used in this third combinatorial step, equal portions of resin are placed in the 10 vessels.
  • the mixtures of tagged quinolone aldehyde 7 are coupled with an amine co ⁇ esponding to one of the 10 R 3 choices shown in Table 3 by a carbon-nitrogen bond formation.
  • the aldehydes 7 are treated with the solution of an amine in TMOF which is in the presence of sodium cyanoborohydride.
  • the mixture is shaken and drained.
  • the solid product is then washed with 1% HCl/MeOH, MeOH. 10% Hunig's base/DCM, MeOH, DMF, and DCM.
  • the washed product is finally dried under o l
  • n 3-12 and Ar is pentachlorophenyl were prepared in accordance with Scheme 6 and die following illustrative example as disclosed in U.S. patent application 08/743,960, filed 11/5/96.
  • the protocol for tagging resin in the present invention was taken from the same reference.
  • Step 2- The methyl ester product of step 1 (1.0 g, 1.7 mmol) was dissolved in 50 mL THF, followed by addition of 2 mL water and LiOH (1.2 g, 50 mmol). The mixture was sti ⁇ ed at 25°C for one hour then refluxed for five hours. After cooling to 25°C, the mixture was poured onto ethyl acetate (200mL) and washed widi 1 M HCl (3 x 50 mL), then saturated aqueous NaCl (1 x 50 mL) and dried over sodium sulfate. The solvent was removed and the crude acid azeotroped once with toluene.
  • Step 3- The crude material from step 2 was dissolved in 100 mL toluene. Thionyl chloride (10 mL, 1.63 g, 14 mmol) was added and die resulting mixture was refluxed for 90 minutes. The volume of the solution was reduced to approximately 30 mL by distillation, then the remaining toluene was removed by evaporation.
  • the crude acid chloride was dissolved in 20 mL dry DCM and cooled to -70°C under argon. A solution of approximately 10 mmol diazomethane in 50 mL anhydrous ether was added. The mixture was warmed to room temperature and sti ⁇ ed for 90 minutes. Argon was bubbled through the solution for ten minutes, then the solvents were removed by evaporation.
  • the crude material was purified by flash chromatography, eluting with 10-20% ethyl acetate in hexane.
  • the diazoketone (0.85 g, 1.4 mmol, 82% yield over three steps) was obtained as a pale yellow solid.
  • ALTERNATE Step 3- A 2.0 M solution of (trimethylsilyl)-diazomemane (5.7 mL, 11.4 mmol, 3.00 eq.) in hexanes was added to a solution of die acyl chloride (3.8 mmol, 1.00 eq.) and triethylamine (1.85 mL, 13.3 mmol, 3.5 eq.) in anhydrous THF/acetonitrile (1:1) at 0°C under argon. The resulting orange solution was sti ⁇ ed at 0°C for two hours, then at 25°C for 17 hours.
  • Step 1 2-methyl butylamine (Table 1. entry 3) is encoded "00011", which represents tagging this choice in the synthesis with the two 64
  • Step (l) Attachment of acyl Meldrum's acid to resin.
  • Step (2) Attachment of N.N-dimethylformamide dimethyl acetal.
  • resin 3 9 g, 2.52 mmol
  • anhydrous ethyl ether 100 mL
  • N,N-dimed ⁇ ylformamide dimethyl acetal 9 g, 75 mmol
  • the mixture was shaken under Argon at room temperature for 16 hours.
  • the resin was then drained, washed witii ether (2 x 50 mL) and DCM (2 x 50 mL) and dried overnight under vacuum to give resin 4.
  • a small portion of the resin (20 mg) was suspended in TFA (5 mL) and shaken for one hour to cleave the compound off the solid support.
  • the compound solution was collected, concentrated and analyzed by HPLC. A yield of 95% was obtained.
  • Step (3) Attachment of primary amines NH;R*. A portion of resin 4 was placed into each of twenty-four reaction vessels.
  • Step (4) Intramolecular cvclization. encoding and coupling with stannanes
  • Each tag reagent was dissolved in DCM (0.5 mL) and then added to the suspension. After agitating for one hour, rhodium trifluoroacetate dimer (0.2 mL of a 5 mg/mL solution in methylene chloride) was added, and the mixture was agitated at room temperature for an additional eight hours. The resin was then drained and washed with 5 mL portions of DCM (3x), MeOH (3x) and DCM (5x). The washed resin was dried overnight under vacuum. The tagging efficiency was determined by GC analysis (see Example 3).
  • each of the smaller portions was coupled separately, with each of the first six stannanes listed on Table 2 while each of die larger portions was coupled separately, with each of the bifunctional stannanes in Table 2 (reagent #'s: 7, 8 and 9).
  • a larger amount of resin 6 was used for die bifunctional stannanes since an additional synthetic step was performed after the Stille coupling. This would ensure that the final compound amounts in terms of the weight of resin for all the sub-libraries are the same.
  • a suspension of resin 6 (288.5 mg, 0.081 mmol) in DEE (5 mL) was degassed by Argon (bubbled for 15 minutes).
  • Step (5) Encoding and attachment of amines NH>R 3 .
  • rhodium trifluoroacetate dimer (0.2 mL of a 5 mg/mL solution in methylene chloride) was added and the mixture was agitated at room temperature for an additional 8 hours. The resin was then drained and washed with 5 mL portions of DCM (3x), MeOH (3x) and DCM (5x). The washed resin was dried overnight under vacuum. The tagging efficiency was determined by GC analysis (see Example 3).
  • the organic layer is removed by syringe and mixed with 1 ⁇ L of MSTFA.
  • tag solution (1 ⁇ L) is analyzed by GC with an ECD detector.
  • the GC analysis is performed with a Hewlett Packard 5890 plus .
  • TM gas chromatograph using a column injection method (5 , 0.32 mm retention gap connected to a 25 m, 0.2 mm crosslinked 5% phenylmethyl silicone column).
  • the ECD detector is maintained at 400°C and the
  • auxiliary gas He is set at 35 psi.
  • the identity of the library compound attached to the bead is ascertained based on the reagents utilized in the synthesis of the compound which are readily determined from the binary codes associated, respectively, with each of the identifiers for such reagents as characterized through the above procedure.
  • the binary codes for the identifiers are assigned to the various reagents (Tables 1 and 2) prior to initiating synthesis of the combinatorial library

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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Abstract

L'invention concerne une banque combinatoire comprenant une pluralité d'éléments représentés par la formule (a), dans laquelle (b) est un support solide et -Z- est un reste de composé. Dans cette banque renfermant des composés de quinolone, Z est représenté par la formule (c), dans laquelle R1 est un susbtituant amine primaire; W est sélectionné dans le groupe constitué de stannanes et d'amines. La banque combinatoire peut être éventuellement codée par des identificateurs T'-L, qui sont liés par covalence au support solide. L'invention concerne également un procédé de synthèse d'une telle banque, ainsi que son utilisation dans des analyses visant à découvrir des composés biologiquement actifs.
PCT/US1999/001738 1998-01-29 1999-01-28 Banque combinatoire de quinolones WO1999038993A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1367045A1 (fr) * 2002-05-31 2003-12-03 Personal Chemistry i Uppsala AB Produits intermédiaires, leur procédé de préparation et leur utilisation
US7019094B2 (en) 2002-05-31 2006-03-28 Biotage Ab Intermediate products, methods for their preparation and use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324483A (en) * 1992-10-08 1994-06-28 Warner-Lambert Company Apparatus for multiple simultaneous synthesis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324483A (en) * 1992-10-08 1994-06-28 Warner-Lambert Company Apparatus for multiple simultaneous synthesis
US5324483B1 (en) * 1992-10-08 1996-09-24 Warner Lambert Co Apparatus for multiple simultaneous synthesis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GALLOP M A, ET AL.: "APPLICATIONS OF COMBINATORIAL TECHNOLOGIES TO DRUG DISCOVERY 1. BACKGROUND AND PEPTIDE COMBINATORIAL LIBRARIES", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 37, no. 09, 29 April 1994 (1994-04-29), US, pages 1233 - 1251, XP002919220, ISSN: 0022-2623, DOI: 10.1021/jm00035a001 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1367045A1 (fr) * 2002-05-31 2003-12-03 Personal Chemistry i Uppsala AB Produits intermédiaires, leur procédé de préparation et leur utilisation
WO2003101933A1 (fr) * 2002-05-31 2003-12-11 Personal Chemistry I Uppsala Ab Produits intermediaires, procedes permettant de les preparer et leur utilisation
US7019094B2 (en) 2002-05-31 2006-03-28 Biotage Ab Intermediate products, methods for their preparation and use thereof

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