+

WO1997028168A1 - Oligomeres d'ester phosphoreux non nucleotidiques - Google Patents

Oligomeres d'ester phosphoreux non nucleotidiques Download PDF

Info

Publication number
WO1997028168A1
WO1997028168A1 PCT/US1997/001060 US9701060W WO9728168A1 WO 1997028168 A1 WO1997028168 A1 WO 1997028168A1 US 9701060 W US9701060 W US 9701060W WO 9728168 A1 WO9728168 A1 WO 9728168A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
diol
oligomer
group
functionality
Prior art date
Application number
PCT/US1997/001060
Other languages
English (en)
Inventor
Robert G. Gentles
Alan F. Cook
Morris J. Rudolph
Reza Fathi
Original Assignee
Pharmagenics, 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
Priority claimed from US08/595,264 external-priority patent/US6008398A/en
Application filed by Pharmagenics, Inc. filed Critical Pharmagenics, Inc.
Priority to JP09527718A priority Critical patent/JP2000506513A/ja
Priority to EP97933585A priority patent/EP0880532A4/fr
Priority to CA002244924A priority patent/CA2244924A1/fr
Priority to AU17532/97A priority patent/AU1753297A/en
Publication of WO1997028168A1 publication Critical patent/WO1997028168A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/141—Esters of phosphorous acids
    • C07F9/1411—Esters of phosphorous acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/141—Esters of phosphorous acids
    • C07F9/143—Esters of phosphorous acids with unsaturated acyclic alcohols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/141—Esters of phosphorous acids
    • C07F9/145—Esters of phosphorous acids with hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2416—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of cycloaliphatic alcohols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/242—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/572—Five-membered rings
    • C07F9/5728—Five-membered rings condensed with carbocyclic rings or carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/58—Pyridine rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/650952—Six-membered rings having the nitrogen atoms in the positions 1 and 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
    • C07F9/6539—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to the field of oligomeric molecules ("oligomers") containing non-nucleotide phosphorus esters, which are useful as mixtures, particularly in the form of combinatorial libraries, to screen for binding activity to biologically significant targets, including protein targets.
  • Esters with high affinity have value as drug or diagnostic candidates, and such combinatorial libraries thereof have value as products used to identify such candidates.
  • Non-nucleotide phosphorus esters have been incorporated into oligonucleotides as linker groups which connect two separate oligonucleotide sequences.
  • oligonucleotides containing a single non-nucleotide hexaethylene glycol phosphodiester have been described (Durand et al . , 1990).
  • the oligonucleotide regions of the molecule were base paired to form a duplex structure, and the non-nucleotide phosphorus ester functioned as a loop to connect the two strands.
  • oligonucleotides for example, several 1,3-propanediol phosphodiester groups have been incorporated into oligonucleotides (Richardson and Schepartz, 1991) to act as tethers of varying lengths which connect two separate oligonucleotide sequences.
  • the tethers enabled the oligonucleotide sequences to bind to non ⁇ contiguous regions of an RNA target.
  • Diethylene-glycol phosphodiester and deca-ethylene-glycol phosphodiester moieties have also been incorporated into oligonucleotides for similar purposes (Cload and Schepartz, 1991) .
  • Non-nucleotide phosphorus ester oligomers have been used for other functions.
  • oligomeric phosphorus esters possessing substituted alkyl substituents have been synthesized and used as fire retardants (Hardy and Jaffe 1980) .
  • Oligomeric phosphorus esters of phenolic compounds have also been described as fireproofing agents (Sase et al . , 1988) .
  • non-nucleotide compounds have been used in combination with nucleotides in a combinatorial manner to search for compounds which bind to biological targets.
  • Compounds possessing various backbone elements such as ethylene glycol phosphate, hydroxyproline phosphate, or PNA ("peptide nucleic acid") have been used, and a twelve residue pentamer combinatorial library containing substituted ethylene glycol phosphate residues in combination with nucleotide residues was reported (Ecker, 1994) .
  • Other groups have described the assembly of combinatorial libraries of oligomers made from a set of monomers consisting of a side chain and a uniform backbone element (Zuckermann et al . , 1994) .
  • the application of combinatorial libraries to drug discovery has recently been extensively reviewed (Gallop et al . , 1994, Gordon et al . , 1994) .
  • Combinatorial libraries of oligomers havingpotential for binding to proteins and other biological targets are valuable products for use in drug screening and other research by pharmaceutical and diagnostic industry members and by investigators in the biological and medical arts. They provide a convenient and rich source of candidates for
  • One aspect of the invention provides a phosphorus ester oligomer of monomeric units, which oligomer has the structure:
  • A can be the same or di ferent in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
  • B, and B 2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosph ⁇ monoester;
  • R can be the same or different in each monomeric unit, and in at least one of the non-nucleotide monomeric units, R, is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine- or pyrimidine-substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or aiicyclic hydrophobic functionality (iv) a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine- or pyrimidine-substituted 1,2-diol or a disubstituted heterocycle; (iii)
  • M diol attached to a ring substituted-anionic functionality and (v) a cationic moiety attached to a non-vicinal or aiicyclic diol, any of which can further include a detectable label; and n is at least one.
  • the hydrogen bond donor contains a hydroxyl, amide, imide or thiol group or is a basic compound.
  • the basic compound can contain an amine moiety or a substituted or unsubstituted thiazole.
  • Preferred hydrogen bond acceptors include an amine or ether moiety. Examples include the 5-substituted 2-hydroxymethyl-3-hydroxy- tetrahydrofurans and bis(hydroxyalkyl) -substituted heterocycles or substituted or unsubstituted theophyllines.
  • Preferred hydrophobic functionalities are selected from aromatic rings, alkanes, cycloalkanes and aromatic rings fused to alkanes.
  • Particularly preferred hydrophobic functionalities include those wherein R, is selected from a substituted alkane, a 3,3-disubstituted 3-amino-l,2- propanediol, a substituted or unsubstituted aiicyclic ring wherein the ring size is from 4-12, a 3-substituted indole, a substituted or unsubstituted hydroxyalkyl phenol and an aiicyclic dicarboxylic acid.
  • Preferred anionic functionalities include those wherein the anionic functionality is a mono-or dicarboxylic acid moiety.
  • moieties include tricyclononene dicarboxylic acids and cyclopentane acetic acids.
  • Preferred cationic functionalities include bis(hydroxyalkyl) - substituted nitrogen-containing heterocycles. More particularly preferred cationic functionalities include substituted alkanes and 3,3-disubstituted 3-amino-l f 2- propanediols.
  • Another preferrred embodiment of the above aspect is an oligomer wherein in at least one of the monomeric units R, is a heterocyclic, an aiicyclic or a polycyclic ring system, preferably containing an indole, thiazole, imidazole, pyridine, purine or pyrimidine ring.
  • the aiicyclic ring system preferably contains a cyclopentane or cyclooctane ring, and can be substituted with at least one carboxylic acid moiety.
  • the polycyclic ring system preferably contains a bicyclic or tricyclic ring or is a polycyclic arene.
  • the bicyclic ring system is preferably a bicyclic alkane, such as tricyclononene.
  • the polycyclic arene is preferably diphenyl bicyclooctane.
  • Another aspect of the invention provides a phosphorus ester oligomer of monomeric units, which oligomer has the structure:
  • A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
  • Bj and B 2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
  • R can be the same or different in each monomeric unit and in at least one of the monomeric units is independently selected from the group consisting of
  • an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety (iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety; (iv) an aiicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;
  • n is at least 1, preferably 2-20, and more preferably 2-6.
  • Another aspect of the invention provides a non- nucleotide monomeric unit having the structure:
  • X is a protecting group
  • X 2 is a branched or unbranched lower alkyl group or a substituted or unsubstituted alkoxy group,-
  • Y is a branched or unbranched lower alkyl group
  • R is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine- or pyrimidine-substituted 1,2-diol or a disubstituted
  • a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or aiicyclic hydrophobic functionality
  • a diol attached to a ring substituted anionic functionality and
  • a cationic moiety attached to a non-vicinal or aiicyclic diol, any of which can further include a detectable label.
  • Another aspect of the invention provides a non- nucleotide monomeric unit having the structure:
  • X is a protecting group
  • X 2 is a branched or unbranched lower alkyl group or a substituted or unsubstituted alkoxy group
  • Y is a branched or unbranched lower alkyl group
  • R is a condensation product of:
  • Another aspect of the invention provides a non- nucleotide monomeric unit having the structure.
  • X is a protecting group
  • R is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine- or pyrimidine-substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or aiicyclic hydrophobic functionality (iv) a diol attached to a ring-substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or aiicyclic diol, any of which can further include a detectable label.
  • Another aspect of the invention provides a non- nucleotide monomeric unit having the structure:
  • X is a protecting group
  • R is a condensation product of:
  • Another aspect of the invention provides a phosphorus ester oligomer of monomeric units, which oligomer has the structure:
  • A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
  • B] and B 2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
  • R can be the same or different in each monomeric unit, and is selected from the group of a nucleoside moiety and, in at least one monomeric unit, R ⁇ is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle; (iii) a non- vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or aiicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or aiicyclic diol, any of which can further include a detectable label; and n is at least one.
  • Another aspect of the invention provides a combinatorial mixture of the above oligomers.
  • Preferred embodiments of combinatorial mixtures of oligomers include those wherein A is 1-20, and particularly preferred embodiments include those wherein A is 1-6.
  • Preferred embodiments include combinatorial mixtures wherein the oligomers are labeled with 32 phosphorus, 35 sulfur, tritium, fluorescein or biotin.
  • a large, highly diverse set of structurally unrelated, derivatized monomers is prepared for incorporation into oligomeric libraries.
  • the libraries are synthesized using combinatorial techniques from subsets of available monomers to generate molecular ensembles from which can be selected those with maximum affinity for the target protein.
  • the number of monomers used in the library synthesis is chosen to exploit the full capability of the screening technology used to identify the best ligand.
  • the nature of the phosphorus linking group in these compounds can be varied at any position to create a greater diversity per unit length of the oligomer than would otherwise be achieved with one type of linkage, such as a phosphodiester or amide.
  • the flexibility of this linkage is also controllable by changing between freely rotatable phosphodiesters, and rotationally constrained phosphoramidates or other esters with bulky groups directly attached to phosphorus.
  • the gross physical characteristics of the molecules can be varied from anionic to neutral to cationic, with all intermediate cases represented.
  • the unique and advantageous aspects of the present invention lie principally in the highly variant set of monomers employed for the synthesis of combinatorial libraries, and the choice of the linking groups in the product oligomers.
  • the monomers fufill two roles: a structural function whereby the molecular geometry and flexibity of the oligomer are controlled, and secondly, they contain appropriate functional groups to bind to protein or other biological targets.
  • the monomers do not contain the sugar or sugar analog element that is found in most other types of oligomers. Instead, any molecule containing two hydroxyl functions may be considered a suitable monomer candidate.
  • control can be excercised over the degree of flexibility in these compounds.
  • constraints can be introduced by either using intrinsically inflexible monomers, or monomers in which the two hydroxy groups are vicinal and the resultant steric congestion limits certain bond rotations.
  • the importance of being able to modulate the conformational freedom of potential drug candidates in order to optimize their binding and selectivity profiles is a well understood principal in medicinal chemistry.
  • conformationally- constrained compounds are desirable as lead molecules in any rational drug design program.
  • the monomers In relation to the ability of the monomers to present appropiate binding groups to protein targets, essentially all common organic functional groups, when suitably protected, are compatible with the condensation chemistry that is employed for synthesizing the oligomers.
  • the monomers used for the synthesis of a library directed against a particular biological target are chosen to contribute to the affinity for the target.
  • the monomers can be subdivided into a range of different categories describing their principal mode of binding, examples being; hydrophobic, hydrogen bond donor, hydrogen bond acceptor, electrostatic cation, and electrostatic anion. Obviously, many monomers contain multiple binding elements and can be assigned to more than one category.
  • each phosphorus linking element connecting the monomer units can be independently modified to both introduce additional binding functionality, and also control the structural flexibility of the oligomers.
  • These linkages can exist either as phosphates, phosphorothioates or phosphoramidates, depending on the oxidation conditions employed for their synthesis.
  • Additional binding groups can be introduced into the oligomers by the oxidation of intermediate H-phosphonate linkages using a diverse range of primary and secondary, simple or highly functionalized amines, an obvious example being amino acids. By using secondary and or-branched amines, significant rotational constriction of the phosphorus linkage can be achieved, and this constitutes another mechanism for introducing conformational constrain .
  • a primary utility of oligomeric phosphate ester libraries is the screening of such mixtures for binding activity to particular biological targets, including but not limited to proteins, with the objective of identifying therapeutically important molecules.
  • the structures of the oligomers of the present invention that bind with high affinity to a biological target can be deduced using procedures such as that described in commonly assigned, copending U.S. patent application Serial No. 08/223,519, or by the use of encoded libraries similar to those described in Gallop et al . , 1994, or by other methods obvious to those skilled in the art.
  • Glysoaminoglycans are large sulfated oligosaccarides that bind to a range of important regulatory proteins. Work directed at the synthesis of mimetics of these compounds has focused on the preparation of structurally defined heparin oligosaccarides and sulfated dextrans (Lander 1994) .
  • Glycosaminoglycans are multiply charged, anionic, sulfated polysaccarides that are typically around 250 saccharide units in length. They are known to modulate a series of biochemical processes by binding to certain polycationic receptor sites on proteins, and play key roles in a conditions as diverse as cancer and Alzheimer's disease.
  • the interaction between the protein and the glycosaminoglycan is mostly electrostatic in nature, and consequently the dissociation constant for these interactions is comparatively large, typically in the range 10 '5 to 10 "8 M.
  • the oligomers of the present invention can be used in a method for detecting the presence or absence of a target molecule in a sample, or determining the number of such molecules in a sample.
  • the oligomer can be labeled, exposed to target, and the amount of labeled oligomer which is bound to the target is quantified by measurement of the signal derived from the label.
  • the label can be radioactive or non-radioactive. Radioactive labels include phosphorus-32, sulfur-35, tritium, and heavy metal isotopes which can be trapped by chelating groups attached to the oligomer.
  • Non- radioactive labels include, but are not limited to, biotin and its analogs, fluorescein, tetramethyl-rhodamine, substrates for enzymes such as alkaline phosphatase or horseradish peroxidase, haptens such as dinitrophenyl or digoxigenin and other labels known to those experienced in the art.
  • oligomers of the present invention can be used for diagnostics in a manner similar to methods employed for antibody-based diagnostics. The same labeling techniques may also be employed in the protocols for the identification of protein ligands, discussed above.
  • Figure 1 shows a method for introduction of a tritium label by reduction of a hydroxyacetophenone functionality.
  • Figure 2 shows a method for the introduction of glycolic amide groups at the terminus of a library (Structure 8) , including a method for the introduction of a radioactive label (Structure 10) .
  • Figure 3 shows the binding of a combinatorial library of non-nucleotide phosphorus ester oligomers to two protein targets.
  • Figure 4 shows the binding of a library of non-nucleotide phosphorus ester oligomers to thrombin.
  • Figure 5 shows the prolongation of clotting time for a library of non-nucleotide phosphorus ester oligomers as compared with a control in the absence of library.
  • Figure 6 shows the clotting time as a function of concentration of library of non-nucleotide phosphorus ester oligomers.
  • Preferred hydrogen bond donors are functionalities containing amine, amide, imide, alcohol and thiol moieties.
  • R is a 3- substituted dihydroxyalkyl indole (e.g., indolyl- dihydroxypropane) , or a bis(hydroxyalkyl) -substituted heterocycle ⁇ e. g. , 1,2-dihydroxyethylthiazole and pyridoxine) or a 2-amino-l,3-propandiol (e.g.; thiomicamine) .
  • R is a 3- substituted dihydroxyalkyl indole (e.g., indolyl- dihydroxypropane) , or a bis(hydroxyalkyl) -substituted heterocycle ⁇ e. g. , 1,2-dihydroxyethylthiazole and pyridoxine) or a 2-amino-l,3-propandiol (e.g.;
  • Preferred hydrogen bond acceptor compounds include those containing amine or ether moieties.
  • R is a purine or pyrimidine substituted 1,2-diol (e.g., theophylline) or a bis (hydroxyalkyl) -substituted heterocycle (e. g. , 1,2- dihydroxyethylthiazole and pyridine dimethanol) or a 5- substituted 2-hydroxymethyl 3-hydroxy-tetrahydrofuran (e.g., 1,2 dideoxy-D-ribose) .
  • R is a purine or pyrimidine substituted 1,2-diol (e.g., theophylline) or a bis (hydroxyalkyl) -substituted heterocycle (e. g. , 1,2- dihydroxyethylthiazole and pyridine dimethanol) or a 5- substituted 2-hydroxymethyl 3-hydroxy-tetrahydrofuran (e.g., 1,2 dideoxy-D-ribose)
  • Preferred hydrophobic groups are alkyl groups and aromatic rings. Examples of such compounds are those wherein R, is a substituted 1,3-dihydroxyalkane (e. g. 4- methoxyphenoxy-1,3-propanediol) , a substituted 1,2- dihydroxy alkene ⁇ e. g. ; 1,2-dihydroxy-3-butene) , a 3,3-
  • R is a substituted 1,3-dihydroxyalkane (e. g. 4- methoxyphenoxy-1,3-propanediol) , a substituted 1,2- dihydroxy alkene ⁇ e. g. ; 1,2-dihydroxy-3-butene) , a 3,3-
  • indolyl-dihydroxypropane or a substituted or unsubstituted hydroxyalkyl phenol (e. g. , tetralin and hydroxybenzyl alcohol) or a 1,2-dihydroxy aiicyclic, dicarboxylic acid (e. g. , tricyclononene dicarboxylic acid) .
  • hydroxyalkyl phenol e. g. , tetralin and hydroxybenzyl alcohol
  • 1,2-dihydroxy aiicyclic, dicarboxylic acid e. g. , tricyclononene dicarboxylic acid
  • Preferred electrostatically charged functionalities include anionic functionalities, such as carboxylic, sulfonic and phosphoric acid and tetrazole moieties.
  • anionic functionalities such as carboxylic, sulfonic and phosphoric acid and tetrazole moieties.
  • R is a dihydroxy-substituted carboxylic acid (e.g., cyclopentanediol acetic acid) or a 1,2-dihydroxy aiicyclic dicarboxylic acid (e. g. , tricyclononene dicarboxylic acid) .
  • Preferred electrostatically charged functionalities also include cationic functionalities.
  • R is a substituted 1,3- dihydroxyalkane (e. g. , 2-amino-1,3-propanediol, 1-phenyl- 2-amino-1,3-propanediol and thiomicamine) , a 3,3- disubstituted 3-amino-1,2-propanediol (e. g. , 3,3- diet ylaminopropanediol and 3-benzyl-3-methylamino-1,2- propanediol) or abis(hydroxyalkyl) -substitutedheterocycle (e. g. , 1,2-dihydroxyethyl-thiazole and pyridine dimethanol) .
  • R is a substituted 1,3- dihydroxyalkane (e. g. , 2-amino-1,3-propanediol, 1-phenyl- 2-amino-1
  • Preferred heterocyclic dihydroxy alcohols are those containing an indole, thiazole, imidazole, purine or pyrimidine ring structure.
  • R is a 3-substituted dihydroxyalkyl indole (e. g. , indolyl-dihydroxypropane) or a purine or pyrimidine substituted 1,2-diol (e. g.
  • Preferred aiicyclic dihydroxy alcohols are those containing a cyclopentane or cyclooctane ring structure, including those which are diols and which are substituted with at least one carboxylic acid moiety.
  • R is a substituted or unsubstituted aiicyclic diol wherein the ring size is from 4-12 (e. g. cyclooctanediol and cyclopentanediol) and those wherein R ! is a dihydroxy-substituted carboxylic acid (e. g. , cyclopentanediol acetic acid).
  • Preferred polycyclic dihydroxy alcohols are those containing a bicyclic or tricyclic ring structure, including alkanes such as a bicycloheptane, alkenes such as tricyclonene diol and polycyclic arenes such as diphenyl bicyclooctane diol.
  • alkanes such as a bicycloheptane
  • alkenes such as tricyclonene diol
  • polycyclic arenes such as diphenyl bicyclooctane diol.
  • R is a dihydroxy substituted polycyclic compound (e.g. tricyclononene diol dicarboxylic acid and pinanediol) .
  • non-nucleotide phosphorus oligomers where R, is a condensation product of an aliphatic acyclic hydrocarbon diol wherein the diol hydroxyl groups are non- vicinal or are substituted include those formed from monomers wherein R, is 1,3-propanediol or 2-amino-1,3- propanediol.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of a purine or pyrimidine substituted variant of the diols of (i) or of aliphatic acyclic hydrocarbon vicinal diols include those formed from monomers wherein R, is a purine substituted 1,2-diol (e.g. 2,3-dihydroxypropyl theophylline) .
  • Examples of non-nucleotide phosphorus oligomers where R, is a condensation product of an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety include those formed from monomers wherein R t is 3-diethylamino-1,3-propanediol.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of an aiicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent include those formed from monomers wherein R, is cyclopentanediol, cyclo-octanediol, and those formed from monomers wherein R, is a dihydroxy-substituted carboxylic acid (e. g. , cyclopentane diol acetic acid) or a 1,2-dihydroxy aiicyclic dicarboxylic acid (e. g. , tricyclononene diol dicarboxylic acid) .
  • R is a condensation product of an aiicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent
  • non-nucleotide phosphorus oligomers where Rj is a condensation product of a hydroxy or hydroxyalkyl substituted tetrahydrofuran include those formed from monomers wherein R, is 1,2-dideoxy-D-ribose.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of an indole substituted acyclic aliphatic diol include those formed from monomers wherein R, is indolyl-dihydroxypropane.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of an aromatic ring or ring system having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl include those formed from monomers wherein R, is 2,6-bis- hydroxymethyl-pyridine.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl include those
  • ⁇ 9C formed from monomers wherein R[ is 1,2,3,4-tetrahydro-l,5- dihydroxy-naphthalene.
  • non-nucleotide phosphorus oligomers where R, is a condensation product of a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline include those formed from monomers wherein R, is 2-amino-4- (1,2-dihydroxyethyl) -1,3- thiazole.
  • oligomers of the present invention that bind with high affinity to a protein target can be deduced using procedures including, but not limited to, those described in commonly assigned, copending U.S. patent application Serial No. 08/223,519, or by the use of encoded libraries similar to those described in Gallop et al . , 1994.
  • the libraries are typically synthesized from a diverse pool of about 20 to 30 of such monomer units which are chosen to ensure sufficient diversity with regard to chemical functionality, shape and physical properties, such as charge and hydrophobicity. However, larger or smaller numbers of such monomers can be employed.
  • these monomers are prepared as 4,4' -dimethoxytrityl (DMT) -protected phosphoramidites and condensed together using known techniques (see Andrus et al . , 1988) to produce oligomers with negative charges on the phosphorus backbone. Phosphodiester or phosphorothioate backbones can be produced by these methods.
  • DMT 4,4' -dimethoxytrityl
  • Phosphodiester or phosphorothioate backbones can be produced by these methods.
  • the monomers can be prepared as H-phosphonate derivatives which can be coupled together to produce oligomers using standard procedures (Milligen/Biosearch Inc., Novato CA. , H-phosphonate Synthesis Information) .
  • the oligomer H- phosphonate intermediates can be subsequently oxidized with iodine and water to produce phosphodiester or with a sulfur reagent to give phosphorothioate linkages.
  • Another aspect of the present invention is the introduction of additional diversity into the backbone of the oligomers by oxidation of the H-phosphonate intermediates with a diverse set of amines to produce phosphoramidate derivatives.
  • the amines can be selected so as to introduce aliphatic groups, aromatic groups, uncharged polar groups such as hydrogen bond donor or acceptor groups, negatively charged groups and/or positively charged groups.
  • the overall structural diversity of the oligomer can be greatly increased above that which can be achieved when the backbone is comprised exclusively of phosphodiester groups.
  • Examples of amines which can be reacted with oligomer H-phosphonate intermediates to produce oligomer phosphoramidates are as follows: 2- (2-aminoethyl)pyridine,- 1 - ( 2 - aminoethyl) piperidine,- 2- (3, 4-dimethyloxy- phenyl)ethylamine,- 4- (2-aminoethyl) -morpholine; 1- (3- aminopropyl) -4-methylpiperazine,- 1- (3-aminopropyl) -2- pyrrolidinone; 1- (3-aminopropyl) imidazole; l-(2- aminoethyl)pyrrolidine,- 3-aminopropionitrile,- 2- (2- aminoethyl) -1-methyl-pyrrolidine,- 4-fluorophenethylamine,- 4-bromophenethylamine,- aminomethy1-cyclopropane,- 3,3- diphenylpropyl
  • oligomers can be constructed that can be either anionic, neutral or cationic in character. Using this method, a highly diverse library of molecules with lower molecular weights can be produced.
  • Another aspect of the invention relates to the use of preferred labeling and protecting groups, for example as
  • B, and/or B 2 is a group containing at least one tritium atom for the purposes of detection. This approach to tritium labeling employs a
  • labeling monomer which introduces a reactive aldehyde or ketone group at a late stage of synthesis, followed by reduction with labeled borohydride to introduce the tritium label.
  • labeling monomer which introduces a reactive aldehyde or ketone group at a late stage of synthesis, followed by reduction with labeled borohydride to introduce the tritium label.
  • B, and B 2 is a phosphodiester attached to a tritium-labeled p- or m-
  • the phosphoramidite derivative 2 was prepared by a conventional procedure (Beaucage et al . ) and trial experiments with model reactions indicated that this could be coupled to support-bound thymidine and oxidized to produce the diester 3a. Other methods can be used to prepare 3a. For example, 1 can be converted into an H-phosphonate derivative which can be coupled to a support bound nucleoside using standard chemistry known to those in the art.
  • libraries of non-nucleotide phosphorus ester oligomers were prepared using a conventional thymidine derivatized solid support, which after cleavage from the support results in each member of the library having a thymidylate protecting group attached to its terminus at B 2 .
  • This thymidine group plays a role as a protecting group in stabilizing the libraries to degradation by the concentrated ammonia conditions required for cleavage from the support.
  • nucleosides In addition to thymidine, a wide variety of other nucleosides can be used as protecting groups for this purpose, in the absence of such a protecting group, compounds with flexible terminal groups might be expected to undergo degradation by attack of the terminal hydroxyl group on the neighboring phosphoryl group to produce a cyclic phosphate intermediate followed by elimination of the remainder of the molecule. Degradation of this type has been demonstrated with oligonucleotides which terminated in ethylene glycol units (Fontanel et al . ) .
  • Another embodiment provides glycolic amide protecting groups Bj and B 2 .
  • Methods have been developed for the synthesis of other protecting groups which impart stability to the libraries when subjected to alkaline degradation.
  • One approach was to prepare a support which, when treated with ammonia, generates an amide group, and is similar to a reported method for generating 3' -endcapped oligonucleotides (Hovinen et al . ) .
  • Amino-derivatized controlled pore glass was treated with O-dimethoxytrityl glycolic acid (5, Figure 2) to give the solid supported amide derivative 6, which was detritylated and coupled to a second molecule of 5 to give the diglycolic ester derivative 7.
  • This support was used as a replacement for a the thymidine derivatized support for the synthesis of libraries.
  • the glycolic acid derivatized support can be treated with a diamine such as ethylenediamine to produce a glycolic amide of structure 9, which can be used to introduce a radioactive label by reductive amination with formaldehyde and tritium labeled borohydride to give a labeled library such as 10, where T is a tritium atom.
  • a diamine such as ethylenediamine
  • a glycolic amide of structure 9 which can be used to introduce a radioactive label by reductive amination with formaldehyde and tritium labeled borohydride to give a labeled library such as 10, where T is a tritium atom.
  • the diversity in this type of oligomer is derived from both the monomer units as described in Examples 1-41, as well as the amines which are attached to the phosphorus atoms.
  • the library can be prepared by solid-phase synthesis on a solid support, using a pool and divide strategy employing techniques already reported (Furka et al., 1991) .
  • the libraries can be assembled from a monomer subset which is weighted to have
  • 3k a high likelihood of affinity for the binding site on the target as possible, thereby increasing the probability of identifying a suitable ligand.
  • the weighting of the subset can be achieved through the use of molecular modeling techniques. In the absence of such information, a monomer subset is chosen to be as diverse as is considered desirable. The number of monomers that may be employed for the synthesis of a library is limited by the screening technology used in identifying potential ligands.
  • the crude product (13.79 g) was purified by Normal Phase High Pressure Liquid Chromatography (NP-HPLC) eluting with a gradient of dichloromethane/hexane (10 to 0%) and then eluting with a gradient of dichloromethane/methanol (0 to 2%) .
  • NP-HPLC Normal Phase High Pressure Liquid Chromatography
  • This DMT derivative (3.25 g, 6.5 mmol) was dissolved in dry dichloromethane (20 mL) under nitrogen and treated with diisopropylethylamine (2.5 g, 19.5 mmol), and 2-cyanoethoxy- (N,N- diisopropylamino)chlorophosphine (2.3 g, 9.7 mmol). After stirring for 1 hour, the mixture was poured into 5% aqueous sodium bicarbonate (200 mL) . The mixture was extracted with ethyl acetate (3 x 200 mL) , the organic layers were washed with saturated aqueous sodium chloride
  • This DMT derivative (0.5 g, 1.1 mmol) was dissolved in dry dichloromethane (4 mL) under nitrogen and treated with diisopropylethylamine (0.43 g, 3.3 mmol), and 2-cyanoethoxy- (N.N- diisopropylaraino)chlorophosphine (0.39 g, 1.7 mmol). After stirring for 2.5 hours, dry methanol (0.5 mL) was added and the mixture was evaporated to dryness, dissolved in 5% aqueous sodium bicarbonate, and extracted with ethyl acetate (3 x 75 mL) .
  • This DMT derivative (2.4 g, 4.8 mmol) was dissolved in dry acetonitrile (5 mL) under nitrogen and treated with diisopropylamine (0.73 g, 7.2 mmol), tetrazole (340 mg, 4.8 mmol), and 2-cyanoethoxy- (N,N,N,N- tetraisopropylamino)phosphine (3.63 g, 12.5 mmol). After stirring for 16 hours at room temperature, the mixture was evaporated to dryness, dissolved in ethyl acetate (200 mL) and washed with 5% aqueous sodium bicarbonate (200 mL) .
  • This DMT derivative (3.3 g, 5.2 mmol) was dissolved in dry acetonitrile (65 mL) under nitrogen and treated with tetrazole (0.33 g, 4.6 mmol), diisopropy1amine (0.68 g, 6.7 mmol), and 2-cyanoethoxy- (N,N,N,N-tetraisopropylamino)phosphine (3.3 g, 10.9 mmol) .
  • This material (10.3 g, 55 mmol) in dry pyridine (200 mL) was treated with DMT chloride (16.7 g, 49 mmol), triethylamine (7.8 g, 77 mmol), and dimethylaminopyridine (335 mg, 2.7 mmol). Additional DMT chloride (3.35, 9.9 mmol), triethylamine (1.8 g, 18 mmol), and dimethylaminopyridine (70 mg, 0.6 mmol) was added after 18 hours and at 26 hours, and the reaction mixture was stirred at room temperature for a total of 90 hours.
  • This DMT derivative (4.4 g, 9 mmol) was dissolved in dry dichloromethane (20 mL) under nitrogen and treated with diisopropylethylamine (3.5 g, 27 mmol), and 2- cyanoethoxy- (N,N-diisopropylamino)chlorophosphine (3.2 g, 13.5 mmol) . After stirring for 45 minutes the mixture was poured into 5% aqueous sodium bicarbonate (200 mL) , and extracted with ethyl acetate (200 mL) . The organic layers were washed with saturated aqueous sodium chloride (200 mL) and dried over anhydrous sodium sulfate.
  • Dimethyl-exo-tricyclo[4.2.1.0 2,5 ] -3,7-diene-3,4- dicarboxylate (l g, 4.3 mmol) was added dropwise to a mixture of 88% formic acid (2.6 mL, 59 mmol) and 30% hydrogen peroxide (0.6 mL, 6 mmol) that had been cooled on an ice bath. The ice bath was removed and the mixture was stirred for 18 hours. The mixture was evaporated to dryness, co-evaporated with methanol, toluene, methanol, and dichloromethane.
  • This DMT derivative (1.2 g, 2.1 mmol) was dissolved in dry acetonitrile (20 mL) under nitrogen and treated with tetrazole (150 mg, 2.1 mmol), diisopropylamine (0.32 g, 3.15 mmol), and 2-cyanoethoxy- (N,N,N,N-tetraisopropylamino)phosphine (1.6 g, 5.3 mmol). After stirring for 21 hours the mixture was evaporated to dryness, dissolved in 5% aqueous sodium bicarbonate (125 mL) , and extracted with dichloromethane (2 x 100 mL) .
  • This DMT derivative (260 mg, 0.6 mmol) was dissolved in dry dichloromethane (5 mL) under nitrogen and treated with diisopropylethylamine (0.23 g, 1.8 mmol), and 2-cyanoethoxy- (N,N- diisopropylamino)chlorophosphine (0.21 g, 0.9 mmol). After stirring for 15 minutes the mixture was evaporated to dryness, dissolved in ethylacetate (75 mL) , washed with 5% aqueous sodium bicarbonate (100 mL) . The aqueous layer was extracted with ethylacetate (2 x 75 mL) .
  • This DMT derivative (4 g, 10.5 mmol) was dissolved in dry dichloromethane (25 mL) under nitrogen and treated with diisopropylethylamine (6.7 g, 52 mmol), and 2- cyanoethoxy- (N,N-diisopropylamino)chlorophosphine (3.2 g, 13.5 mmol) . After stirring for l hour, additional 2- cyanoethoxy- (N,N-diisopropylamino) -chlorophosphine (1.1 g, 4.2 mmol) was added and the mixture was stirred for 2 hours.
  • Dimethoxytrityl chloride (3.38 g, lOmmol) was added in portions to a stirred solution of the cyclopentanol (5 g, 49 mmol) in anhydrous pyridine (300mL) containing DMAP (20 mg) .
  • the mixture was left to stir at RT overnight after which the pyridine was removed under vacuum, and the residue adsorbed onto silica gel. This mixture was then applied to a flash silica gel column and the product eluted using 25% ethyl acetate in hexane.
  • Dimethoxytrityl chloride (3.76 g, 11.l mmol) was added to a stirred solution of cis-1,2- dihydroxycyclooctane (8 g, 55.5 mmol) in anhydrous pyridine (300 mL) containing DMAP (20 mg) .
  • the reaction was left to stir at RT under nitrogen overnight.
  • the reaction mixture was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column.
  • Dimethoxytrityl chloride (22.7 g, 67 mmol) was added in portions to a stirred solution of 7- (2,3- Dihydroxypropyl)theophylline (10 g, 61 mmol) in anhydrous pyridine (200 mL) containing DMAP (20 mg) .
  • the reaction was left to stir at RT under nitrogen overnight.
  • the reaction mixture was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column.
  • 4,4' -Dimethoxytrityl chloride (10.33 g, 30.5 mmol) was added in portions to a stirred solution of 1,2- butanediol (2.5 g, 27.7 mmol) in anhydrous pyridine (200 mL) containing DMAP (1.7 g) .
  • the mixture was stirred at RT under nitrogen overnight.
  • the solution was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column which was eluted using 10% ethyl acetate in hexane.
  • (+/-)-0 i - (4,4' -dimethoxytrityl) -1,2-dihydroxybutane as an almost colorless oil, (8.15 g, 75%).
  • 4,4' -Dimethoxytrityl chloride (9.4 g, 27.7 mmol) was added in portions to a stirred solution of freshly distilled 3,3 dimethyl-l,2-dihydroxybutanediol (3 g, 25.4 mmol.) in anhydrous pyridine (200 mL) containing DMAP (0.31 g) .
  • the mixture was stirred at RT under nitrogen overnight.
  • the solution was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel short path column.
  • 4,4' -Dimethoxytrityl chloride (7.42 g, 21.9 mmol) was added in portions to a stirred solution of 1,2- propanediol (1.52 g, 20.0 mmol) in anhydrous pyridine (200 mL) containing DMAP (0.28 g) .
  • the mixture was stirred at RT under nitrogen overnight.
  • the solution was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column.
  • Acetic anhydride (4.5 mL, 47.4 mmol) was added to a solution of the above aminothiazole (2.3 g, 14.4 mmol) in anhydrous pyridine (30 mL) and the solution stirred under nitrogen at room temperature overnight. The mixture was then evaporated under vacuum, and the residue dissolved in ethly acetate. This solution wa3s washed successively with 2M hydrochloric acid, saturated sodium bicarbonate solution, and then brine. The mixture was then dried over anhydrous sodium sulfate, filtered and the filtrate evaporated under reduced pressure.
  • N-acetyl-derivatized thiazole derivative from the above reaction (0.915 g, 2.7 mmol.) was added in a single portion to a stirred solution of N-acetyl- 2- amino-4- (1,2-dihydroxyethyl) -1,3-thiazole in anhydrous pyridine (20 mL) containing DMAP (20 mg) .
  • the resultant mixture was then left to stir at room temperature under nitrogen for 3 h, and was then evaporated under vacuum to give an orange colored oil.
  • This material was dissolved S in dichloromethane and adsorbed onto silica gel and then applied to a silica flash column.
  • Dimethoxytrityl chloride (11.35 g, 33.5 mmol) was added to a stirred solution of 1,5-dihydroxy-1,2,3,4- tetrahydronaphthalene (5 g, 30.5 mmol) in pyridine (100 mL) containing DMAP (200 mg) , and the resultant mixture was stirred for 2 h at room temperature under nitrogen. The reaction mixture was then poured into water and the product extracted with ethyl acetate. The extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate evaporated in vacuo.
  • the DMT derivatives from examples 1 to 21 are dissolved in dry dichloromethane and added a flask over 10 minutes containing imidazole (15 equivalents) , phosphorous trichloride (4.3 equivalents), triethylamine (29 equivalents) , and dry dichloromethane cooled in an ice bath.
  • the reaction mixture is stirred for 30 minutes at 0 °C, the ice bath is removed and the mixture is stirred for an additional 30 minutes. Water is added and this mixture was stirred for 10 minutes.
  • the layers are separated and the the aqueous layer is extracted with chloroform. The combined organic layers are evaporated and then co-evaporated twice with toluene.
  • the crude material is purified by column chromatography on silica gel eluting with a gradient of dichloromethane/methanol (0 to 30%) .
  • the purities of the fractions are monitored by thin layer chromatography (TLC) and fractions containing pure material are combined and evaporated to dryness.
  • TLC thin layer chromatography
  • the resulting material is dissolved in dichloromethane and washed with 0.1 M triethylammonium bicarbonate.
  • the aqueous layer is backwashed once with dichloromethane and the combined organic layers are evaporated to dryness to give the H-phosphonate monomer.
  • TLC Rf 0.12 (9:1 dichloromethane/methanol/O.1% triethylamine) .
  • TLC Rf 0.09 (9:1 dichloromethane/methanol/0.1% triethylamine) .
  • 3- (4,4' -dimethoxytrityl) -1-pheny1-1,3-propanediol (2.5 g, 5.5 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give 3- (4,4' -dimethoxytrityl) -l- phenyl-l,3-propanediol-H-phosphonate (2.3 g) as a yellow foam having the following structure:
  • TLC Rf 0.234 (95:5 dichloromethane/methanol/0.5% triethylamine) .
  • the oligomer was synthesized on a thymidine-derivatized solid support that had been initially derivatized with the chemical phosphorylation reagent (2-cyanoethoxy) -2- (2 ' -o-4, 4 ' - dimethoxytrityloxyethyl-sulfonyl) ethoxy-N,N' - d isopropylammophosphine.
  • the efficiency of the coupling was checked by monitoring of the trityl colors from each cycle, and was determined to be in excess of 95% throughout all of the coupling steps.
  • the library was synthesized on ten columns each loaded with 10 mg of 100 ⁇ m thymidine-derivatized controlled pore glass support that had been derivatized with the chemical phosphorylation reagent (2- cyanoethoxy) -2- (2' -0-4,4' -dimethoxytrityl- oxyethylsulfonyl)ethoxy-N,N' -diisopropyl-aminophosphine.
  • the efficiency of the coupling was checked by monitoring of the trityl colors from each synthesis, and was determined to be in excess of 90% throughout all of the coupling steps.
  • the resins were removed from the columns and pooled and divided using the isopycnic slurry method. The synthesis columns were then weighed to ensure an even division of the resin between the columns, the variation of which was determined to be less than 5% throughout the four pooling and dividing steps.
  • the oligomers in each of the ten columns were fluoresceinated using fluorescein 6-FAM amidite (Applied Biosystems, Foster City, CA) .
  • the resin from each column was then removed, and treated with concentrated ammonium hydroxide for 4 h at 55 "C.
  • the resultant suspensions were filtered and the ammonia removed from the filtrate by bubbling the solutions with nitrogen for 20 min.
  • the ten solutions were then lyophilyzed and the residues dissolved in water and purified over an OPC cartridge (Applied Biosystems) using the standard procedure as provided by the manufacturer.
  • the products from the purification were then analyzed by electrophoresis on a non-denaturing polyacrylamide gel.
  • Each of the ten products ran as a single band, with almost the same mobility as a pentameric deoxyoligonucleotide.
  • the library was then assembled by mixing all of these solutions and lyophilizing the resultant mixture.
  • Each column was initially reacted with the chemical phosphorylating reagent (2- cyanoethoxy) -2- (2' -0-4,4' - dimethoxytrityloxyethylsulfonyl)ethoxy-N, N' - diisopropylaminophosphine using the standard phosphoramidite procedure.
  • Each of the 10 monomers was then coupled to its specified column, monomer one to column one, monomer two to column two, etc. , up to monomer 10 to column 10. The efficiency of each coupling was checked by monitoring the trityl colors from each synthesis, and was determined to be in excess of 90% throughout all of the coupling steps.
  • the resins were then removed from the columns and pooled and divided using the isopycnic slurry method.
  • the synthesis columns were weighed to ensure an even division of the resin among the columns.
  • the non-nucleotide H-phosphonate linkages were converted to phosphoramidate linkages using the following oxidation procedure with each column being oxidized with one of the amines listed below:
  • a 10 % solution of each amine was prepared individually in carbon tetrachloride and the H- phosphonate linkages of the oligomers were then oxidized to phosphoramidates using the freshly prepared solution of the required amine for 10 minutes.
  • the amine solutions were then flushed from the columns and the supports were washed extensively with acetonitrile.
  • the supports were removed from the columns and pooled and divided as described above.
  • a second round of monomer addition followed by pooling, dividing and amine addition was carried out, followed by addition of thymidine-H- phosphonate at the last step to provide an attachment site for the 32 P label.
  • the supports were then removed from the columns and treated with concentrated ammonium hydroxide for 5.5 hours at 55°C.
  • the resultant suspensions were filtered and the ammonia removed from the filtrates by bubbling the solutions with nitrogen for 20 minutes.
  • the sixteen solutions then were lyophilized, redissolved in water and purified by reversed-phase HPLC using a gradient of 5-100% acetonitrile in TEAA, 0.1 M, pH 7.
  • the products from these purifications were combined and resultant mixture was lyophilized to give the oligomeric phosphoramidate library which was then labeled with 32 P by a conventional method using T4 polynucleotide kinase.
  • the 3' ,5' -dithiol oligomer was synthesized on an Applied Biosystems Model 394 DNA synthesizer on a 1 ⁇ mol scale using 0.02 M solution of iodine for all of the oxidation steps.
  • 1-O-Dimethoxytrityl-hexyl-disulfide-1' - [(2-cyanoethyl) -N,N-diisopropyl-phosphoramidite] (thiol modifier C6 S-S, Glen Research, Stirling, VA) was directly coupled to a nucleoside residue anchored to a controlled pore glass solid support (i.e.
  • the solution was concentrated to a small volume and the tritylated oligomer was isolated by HPLC on a preparative C 18 column using a gradient of acetonitrile in 0.1 M triethyl-ammonium acetate (TEAA) buffer (5-30% over 15 min then 30-55% over 30 min, then at 55% for 20 min) . Fractions eluting at 47-51 min were evaporated to dryness and redissolved in 0.1 M TEAA buffer (pH 8.0, 0.5 mL) .
  • TEAA triethyl-ammonium acetate
  • the area under the peak corresponding to starting material was measured versus time to determine the degradation rate. By this method the t 1/2 was determined to be greater than 1 month.
  • a 20- base oligodeoxynucleotide was used as a control, and under the same conditions, the t 1/2 for this oligonucleotide was determined to be 7 min.
  • the oligomeric library of Example 43 was screened against the target proteins IL-4 and IFN7 for high affinity ligands, using the COMPILE method as outlined in copending patent application "Determination and Identification of Active Compounds in a Compound Library," U. S. patent application serial number 08/223,519. Binding reactions consisting of 2.5 ⁇ M oligomeric library and 1 ⁇ M target protein as well as a protein-minus control (data not shown) were prepared in a buffer containing 150 mM NaCl, 3 mM MgCl 2 and 25 mM Tris- HC1 (pH 7.5).
  • M.Wt.cutoff 10,000. The M.Wt. cutoff of the filtration unit was selected to allow free passage of the oligomeric library members but not the target protein or the oligomeric molecules bound to it.
  • the centrifugation was adjusted to allow approximately 270 ⁇ L of the total reaction volume of 400 ⁇ L to flow through the membrane, resulting in the loss of approximately 67% of the unbound oligomeric library from the retained sample.
  • the volume of the retained sample was brought up to the original 400 ⁇ L with buffer (containing 20% of the original target concentration to compensate for losses/inactivation of the target during the experiment) and the selection was repeated.
  • the oligomeric library members are lost at approximately the same rate for both IL-4 and IFN7 (and for the protein-minus control - data not shown) over the first seven rounds of selection.
  • the IFN7 reaction begins to retain a progressively larger fraction of the library whereas the IL-4 reaction fails to show such increased proportion of retention.
  • TLC Rf 0.19 (99:1 dichloromethane/methanol/0.1% triethylamine) .
  • This label was introduced onto the terminal thymidine residue of a compound or library using ⁇ 3 P - labeled adenosine-5' -triphosphate as the donor source and bacteriophage T 4 polynucleotide kinase as the enzyme.
  • Conditions for labeling approximated those for labeling of an oligonucleotide (Sambrook et al . ) . Purification was achieved by preparative polyacrylamide gel electrophoresis.
  • the crude oil was partitioned between ethyl acetate (150 mL) and 5% aqueous sodium bicarbonate (150 mL) and the aqueous layer was extracted twice with ethyl acetate (100 mL) .
  • the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solid removed by filtration.
  • the solvents were removed by rotary evaporation and the crude yellow oil was adsorbed onto silica gel and applied to a silica gel flash column (16 x 10 cm) which was eluted from the column with hexane/ethyl acetate (5:1).
  • the labeling group is attached to a thymidine residue attached to a solid support.
  • Thymidine-5' - (m-acetylphenyl)phosphate (3a) was prepared
  • the reaction mixture was loaded onto a Sep-Pak * C18 plus cartridge previously primed with acetonitrile (10 mL) and water (10 mL) .
  • the cartridge was washed with water (5 x 10 mL) , then eluted with 6:4 methanol/water (v:v) (4 x 1 mL) followed by methanol (1 mL) and the methanolic fractions were evaporated to give the tritium labeled compound 4 (20 OD HJO , 73 nmol, 86 ⁇ Ci, 4% radiochemical yield) .
  • Specific activity 1.18 mCi/ ⁇ mol.
  • a library of non-nucleotide phosphorus ester oligomers was prepared and coupled to 3-acetylphenyl 2-cyanoethyl N,N-diisopropyl phosphoramidite (2) to produce a library 3b, possessing 3-acetylphenyl moiety at its terminus.
  • Sodium borohydride (0.024 mg, 0.63 ⁇ mol) in 0.01 N aqueous sodium hydroxide solution (200 gL of stock solution 2.4 mg/20 mL) was added and the mixture was allowed to react an additional 2 hours.
  • the reaction mixture was loaded onto a Sep-Pak ® C18 plus cartridge previously primed with acetonitrile (10 mL) and water (10 mL) .
  • the solid was washed with pyridine, dried under high vacuum and treated with acetic anhydride/pyridine/N-methylimidazole (1:5:1,7.0 mL, v/v) for 30 minutes at room temperature to acetylate unreacted amino groups.
  • Loading of the DMT-glycolic acid (5) according to the dimethoxytrityl cation assay was 48 umol/g.
  • the material obtained was washed with 5% dichloroacetic acid in dichloromethane (40 mL) on a glass filter funnel to remove the dimethoxytrityl group and then washed with CH2C1 2 and dried under vacuum.
  • a suspension of the dry solid support (2 g) in pyridine (2.5 mL) was treated with a solution of dimethoxytrityl-glycolic acid, (0.73g, 1.6 mmol), triisopropyl sulfonyl chloride (0-44 g, 1.44 mmol) and N-methyl imidazole (115 ⁇ l, 1.44 mmol) in 1.3 mL of pyridine (1.3 mL) and the mixture was shaken at room temperature overnight and then filtered through sintered glass funnel.
  • the support was treated with a mixture of acetic anhydride/pyridine/ N-methyl imidazole (1:5:1, 7.0 mL, v/v) for 30 minutes at room temperature.
  • the library is synthesized on 10 columns each loaded with 60 mg of modified solid support that had been derivatized with dimethoxytrityl-glycolic acid (50 umol/g) .
  • Each of the 10 monomer H-phosphonates is coupled to its specified support, monomer one to column one, monomer 2 to column 2, etc., up to monomer 10 to column 10.
  • the efficiency of each coupling can be checked by monitoring the intensity of the trityl cation released from each coupling step.
  • the supports are removed from the columns and pooled and divided using the isopycnic slurry method, with acetonitrile as the solvent. The synthesis columns are weighed to ensure an even division of the resin among the columns.
  • the non-nucleotide H-phosphonate linkages are converted to phosphoramidate linkages using the following oxidation procedure with each column being oxidized with one of the following amines: 2- (2-aminoethyl)pyridine, 1- (2-aminoethyl)piperidine, 2- (3,4-- dimethoxyphenyl)ethylamine, 4- (2-aminoethyl)morpholine, 1- (3-aminopropyl) -2-pyrolidinone,
  • a 10% solution of each amine is prepared individually in carbon tetrachloride and the H-phosphonate moieties are oxidized to phosphoramidate on the DNA synthesizer using a fresh solution of the required amine for 14 minutes.
  • the amine solutions are then flushed from the column and the supports are washed extensively with acetonitrile.
  • the supports are removed from the columns and pooled and divided as described above.
  • a second round of monomer addition followed by pooling, dividing and amine addition is carried out, followed by addition of the synthon required for introduction of the label (either fluorescein phosphoramidite, thymidine H-phosphonate or m-hydroxyacetophenone phosphoramidite) .
  • the supports are then removed from the columns and treated with concentrated ammonium hydroxide for 5.5 h at 55°C.
  • the resultant suspensions are filtered and the ammonia removed from the filtrates by bubbling nitrogen through the solutions for 20 minutes.
  • the sixteen solutions are then lyophilized, redissolved in water, purified by reversed-phase HPLC using a gradient of 5-100% acetonitrile in TEAA, 0.1 M, pH 7.0 and pooled to give the library.
  • a phosphoramidate library is prepared as synthesized above and after the last pool and divide step all 16 columns are opened and the solid supports are transfered to 16 screw capped glass tubes and suspended in ethylenediamine/CHjCN (500 ⁇ l, 2:1) for 2 hours at 55°C. After two hours the support is filtered and washed with absolute ethanol (2 X 500 ⁇ l) . The filtrates are combined, evaporated to dryness and purified by HPLC using a C4 reversed column to yield the desired sub-libraries.
  • Each sub-library is suspended in 0.1 M HEPES, pH 7.5 (1 mL) and treated with tritium labeled NaCNBH 3 (3 equiv, 0.57 mg) and formaldehyde (3 equiv, 0.27 mg) . After 3 hours at room temperature the reaction mixtures from each sub-library are loaded on Sep-Pak cartridges (C18, Waters). Each cartridge is washed initially with water (15 mL) and then with 50:50 H 2 0/CH 3 OH (5 mL) .
  • each sub-library is suspended in 25 mM Na 2 B 4 0 7 , pH 9.0 (1 mL) and initially treated with formaldehyde (10 equiv, 0.9 mg) for 30 minutes followed by NaBH 4 (3 equiv, 0.34 mg) . After 5 hours the reaction mixture is loaded on Sep-Pak cartridge as is described above and the desired material is eluted using 50:50 H 2 0/CH 3 OH (5 mL) .
  • the library is synthesized on 10 columns each loaded with 60 mg of modified solid support that had been derivatized with glycolic acid.
  • Each of the 10 monomers is coupled to its specified column, monomer one to column one, monomer 2 to column 2, etc. , up to monomer 10 to column 10.
  • the efficiency of each coupling is checked by monitoring the intensity of the trityl cation released from each coupling step to be in excess of 90 % throughout all of the coupling steps.
  • the resins are then removed from the columns and pooled and divided using the isopycnic slurry method, with acetonitrile as the solvent.
  • the synthesis columns are weighed to ensure an even division of the resin among the columns.
  • the nonnucleotide H-phosphonate linkages are converted to phosphoramidate linkage using the following oxidation procedure with each column being oxidized with one of the following aminoacid derivatives: L-methionine ethyl ester HC1, L-alanine methyl ester, L-valine ethyl ester HC1, L-phenylalanine methyl ester HC1, L-glutamic acid diethyl ester, L-tyrosine ethyl ester HC1.
  • a 12% solution of each aminoacid derivative is prepared in DMF/CC1 4 (1:1, 5 mL) and the solution is treated with 1 equiv of triethylamine, filtered and the solution is used directly for oxidation of the H-phosphonate moieties. Oxidation is carried out on the DNA synthesizer using a 30 minute reaction time.
  • the amino acid solutions are then flushed from the columns and the supports are washed extensively with acetonitrile. The supports are removed from the columns and pooled and divided as described above.
  • a second round of monomer addition followed by pooling, dividing and addition of amino acid derivative is carried out, followed by addition of the synthon required for introduction of the label (either fluorescein phosphoramidite, dimethoxytritylthymidine H-phosphonate or m-hydroxyacetophenone phosphoramidite) .
  • the supports are then removed from the columns and treated with ethylenediamine in acetonitrile (1-5 mL, 2:1) for 1 hour at 55°C.
  • the resultant suspensions are filtered and washed with ethanol (2 X 500 ⁇ l) .
  • the filtrates are combined, concentrated and then redissolved in water and purified by reversed-phase HPLC using a gradient of 5-- 100% acetonitrile in TEAA, 0.1 M, pH 7.0.
  • the purified fractions are combined to give a library with the structure as follows:
  • B is a labeling group
  • B 2 is a glycolic ethylenediamine amide
  • A is an N-linked amino acid ester
  • R, and R 2 are derived from diols as previously described.
  • the centrifugation was adjusted to allow approximately 360 ⁇ L of the total reaction volume of 400 ⁇ L to flow through the membrane, resulting in the loss of approximately 90% of the unbound oligomeric library from the retained sample.
  • the volume of the retained sample was brought up to the original 400 ⁇ L with buffer to which was added 20% of the original target concentration to compensate for losses/inactivation of the target during the experiment.
  • the library was labeled with 32 P using T4 kinase followed by purification of the labeled library by forced
  • ICO dialysis Approximately 38% of the 32 P counts were incorporated into the library. During subsequent rounds of selection the unincorporated counts flowed freely through the Microcon-10 membrane. To control for unincorporated counts retained, a "no library” sample was carried along with the library sample. The "no library” sample was identical to the library sample with the exception that no library was added to the reaction at the beginning of the selection.
  • the progressive increase in the fraction of compounds retained at each round is evidence of a subpopulation of the original library that binds to the target.
  • This subpopulation is enriched with each successive round of selection due to selective retention of compounds in the library that bind to the target protein. From the target protein concentration and the curve shown in Figure 3, it is possible to estimate that this subpopulation of thrombin-binding oligomeric species is between 0.001 and 0.01% of the original mixture, and that it binds an apparent Kd below 100-500 nM.
  • non-nucleotide phosphorus ester oligomers were prepared as previously described and tested for their ability to inhibit thrombin activity as measured by an extension of the time for thrombin-catalyzed fibrin clot formation.
  • the assay was performed as previously described by Bock et al .
  • Clotting reactions were initiated by the addition of human thrombin (100 uL in the same buffer preequilibrated to 37 °C for 1 min) to final concentrations of 2 mg/mL fibrinogen and 3 nM thrombin.
  • the first indication of the clot was taken as the clotting time. Clot formation was usually complete within 5 sec of the first appearence of the clot. As shown in Figure 5, the library substantially prolonged the thrombin induced clotting time as compared with control reactions without addition of library.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention porte sur un oligomère d'ester phosphoreux présentant la structure (I) dans laquelle: A peut être identique ou différent dans chaque unité monomérique, et chacun des A est choisi indépendamment parmi O, S, alkyle inférieur, alkylamino substitué ou non, arylamino et aminoalkyle substitués ou non; B1 et B2 peuvent être identiques ou différents, et chacun choisi indépendamment parmi H, alkyle inférieure, un groupe marqueur, un groupe protecteur, un phosphoramidate ou un phosphomonoester; R1 peut être identique ou différent dans chaque unité monomère, et au moins dans l'une des unités monomères non nucléotidiques, R1 est choisi indépendamment dans un groupe consistant dans les produits de condensation: (i) d'un diol non vicinal lié à une fonction de donneur de liaison hydrogène, (ii) d'un accepteur de liaison hydrogène choisi parmi un éther, un 1,2-diol à substitution purine ou pyrimidine, ou un hétérocycle disubstitué, (iii) d'un diol non vicinal lié à une fonction hydrophobe, ou d'un diol vicinal lié à une fonction hydrophobe aliphatique ou alicyclique, (iv) d'un diol lié à une fonction anionique à substitution de cycle, et (v) d'un fragment cationique lié à un diol non vicinal ou alicyclique, chacun d'eux pouvant de plus comporter un marqueur détectable; et n étant au moins 1. Les fragments R1 préférés comportent des produits de condensation de diols hétérocycliques, alicycliques ou polycycliques. L'invention porte également sur leurs monomères non nucléotidiques, sur des mélanges de bibliothèques combinatoires d'oligomères, et sur l'emploi d'oligomères comme composés se fixant sélectivement à des cibles.
PCT/US1997/001060 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques WO1997028168A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP09527718A JP2000506513A (ja) 1996-02-01 1997-01-22 非ヌクレオチドリンエステルオリゴマー
EP97933585A EP0880532A4 (fr) 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques
CA002244924A CA2244924A1 (fr) 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques
AU17532/97A AU1753297A (en) 1996-02-01 1997-01-22 Non-nucleotide phosphorous ester oligomers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/595,264 US6008398A (en) 1995-01-18 1996-02-01 Non-nucleotide phosphorus ester oligomers
US08/595,264 1996-02-01

Publications (1)

Publication Number Publication Date
WO1997028168A1 true WO1997028168A1 (fr) 1997-08-07

Family

ID=24382509

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/001060 WO1997028168A1 (fr) 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques

Country Status (4)

Country Link
EP (1) EP0880532A4 (fr)
JP (1) JP2000506513A (fr)
AU (1) AU1753297A (fr)
WO (1) WO1997028168A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034660A3 (fr) * 1999-11-09 2001-11-29 Stanford Res Inst Int Criblage et analyse radiographiques de polymeres, de specialites chimiques et de catalyseurs
US6602714B1 (en) 1999-11-09 2003-08-05 Sri International Viscosity and mass sensor for the high-throughput synthesis, screening and characterization of combinatorial libraries
US7033840B1 (en) 1999-11-09 2006-04-25 Sri International Reaction calorimeter and differential scanning calorimeter for the high-throughput synthesis, screening and characterization of combinatorial libraries
EP1819715A2 (fr) * 2004-11-08 2007-08-22 Neopharm, Inc. Synthese de produits analogues de la cardiolipine et leurs applications
WO2008141799A1 (fr) * 2007-05-24 2008-11-27 Roche Diagnostics Gmbh Oligophosphoramidates
WO2010060599A1 (fr) * 2008-11-27 2010-06-03 Roche Diagnostics Gmbh Synthèse dirigée de stéréoisomères d'oligophosphoramidates
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5493117B2 (ja) * 2006-02-15 2014-05-14 国立大学法人岐阜大学 オリゴヌクレオチド誘導体及びその利用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3772340A (en) * 1972-05-02 1973-11-13 Miles Lab Bis(coumarinyl)phosphates
US3891727A (en) * 1970-03-27 1975-06-24 Stauffer Chemical Co Fire retardant phosphorus oligomer compositions
US4093582A (en) * 1976-12-08 1978-06-06 General Electric Company Organo phosphorus ester plasticized polycarbonate composition
US4097560A (en) * 1977-06-29 1978-06-27 M & T Chemicals Inc. Novel phosphorus compounds and flame retardant compositions containing same
US5153347A (en) * 1991-01-31 1992-10-06 E. R. Squibb & Sons, Inc. Phosphonate, phosphinate derivatives useful as antihypertensive agents
US5183811A (en) * 1987-03-24 1993-02-02 Nippon Chemiphar Co., Ltd. Glycerol derivative and anti-hypertensive agent
US5234918A (en) * 1988-04-04 1993-08-10 American Cyanamid Company Certain phosphinyl-oxy-phenyl-methyl-pyridinium-hydroxide inner salts useful as antagonists of PAF
US5364564A (en) * 1993-02-04 1994-11-15 Noda Institute For Scientific Research Phosphatidyl chromanol derivative, the production method thereof, antioxidant and emulsifier

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004204A1 (fr) * 1991-08-23 1993-03-04 Isis Pharmaceuticals, Inc. Derandomisation synthetique de fragments oligomeres
US5637684A (en) * 1994-02-23 1997-06-10 Isis Pharmaceuticals, Inc. Phosphoramidate and phosphorothioamidate oligomeric compounds
US5919965A (en) * 1995-01-18 1999-07-06 Genzyme Corporation Non-nucleotide phosphorus ester oligomers

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3891727A (en) * 1970-03-27 1975-06-24 Stauffer Chemical Co Fire retardant phosphorus oligomer compositions
US3772340A (en) * 1972-05-02 1973-11-13 Miles Lab Bis(coumarinyl)phosphates
US4093582A (en) * 1976-12-08 1978-06-06 General Electric Company Organo phosphorus ester plasticized polycarbonate composition
US4097560A (en) * 1977-06-29 1978-06-27 M & T Chemicals Inc. Novel phosphorus compounds and flame retardant compositions containing same
US5183811A (en) * 1987-03-24 1993-02-02 Nippon Chemiphar Co., Ltd. Glycerol derivative and anti-hypertensive agent
US5234918A (en) * 1988-04-04 1993-08-10 American Cyanamid Company Certain phosphinyl-oxy-phenyl-methyl-pyridinium-hydroxide inner salts useful as antagonists of PAF
US5153347A (en) * 1991-01-31 1992-10-06 E. R. Squibb & Sons, Inc. Phosphonate, phosphinate derivatives useful as antihypertensive agents
US5364564A (en) * 1993-02-04 1994-11-15 Noda Institute For Scientific Research Phosphatidyl chromanol derivative, the production method thereof, antioxidant and emulsifier

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0880532A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034660A3 (fr) * 1999-11-09 2001-11-29 Stanford Res Inst Int Criblage et analyse radiographiques de polymeres, de specialites chimiques et de catalyseurs
US6602714B1 (en) 1999-11-09 2003-08-05 Sri International Viscosity and mass sensor for the high-throughput synthesis, screening and characterization of combinatorial libraries
US7033840B1 (en) 1999-11-09 2006-04-25 Sri International Reaction calorimeter and differential scanning calorimeter for the high-throughput synthesis, screening and characterization of combinatorial libraries
EP1819715A2 (fr) * 2004-11-08 2007-08-22 Neopharm, Inc. Synthese de produits analogues de la cardiolipine et leurs applications
EP1819715A4 (fr) * 2004-11-08 2010-02-17 Neopharm Inc Synthese de produits analogues de la cardiolipine et leurs applications
WO2008141799A1 (fr) * 2007-05-24 2008-11-27 Roche Diagnostics Gmbh Oligophosphoramidates
WO2010060599A1 (fr) * 2008-11-27 2010-06-03 Roche Diagnostics Gmbh Synthèse dirigée de stéréoisomères d'oligophosphoramidates
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates

Also Published As

Publication number Publication date
JP2000506513A (ja) 2000-05-30
AU1753297A (en) 1997-08-22
EP0880532A4 (fr) 1999-06-09
EP0880532A1 (fr) 1998-12-02

Similar Documents

Publication Publication Date Title
US6008398A (en) Non-nucleotide phosphorus ester oligomers
WO1996022297A9 (fr) Oligomeres d'ester de phosphore non nucleotidiques
CA2184005C (fr) Nouveaux composes oligomeres de phosphoramidate et de phosphorothiomidate
JP2710756B2 (ja) 新規ヌクレオシドホスホルアミダイトおよびその製造方法
Misiura et al. Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides
JPH09509408A (ja) ピロリジン含有モノマーおよびオリゴマー
US20030036066A1 (en) Linker phosphoramidites for oligonucleotide synthesis
CA2081320A1 (fr) Derives de phosphoramidite; preparation et utilisation de ces derives et incorporation de groupes decelables dans des oligonucleotides synthetiques
JPH0631305B2 (ja) ヌクレオシド誘導体
EP0266168A2 (fr) Compositions et méthodes de synthèse d'oligonucléotides dont l'extrémité 5' est phosphorylée
WO1997028168A1 (fr) Oligomeres d'ester phosphoreux non nucleotidiques
JP2004513629A (ja) オリゴヌクレオチドからユニバーサルリンカーを除去する方法
WO1999051547A2 (fr) Synthese en phase solide de composes organiques au moyen de reactifs de phosphitylation
CA2180867C (fr) Diols monomeres et oligomeres a liaison phosphate formes a partir de ces derniers
AU701299C (en) Non-nucleotide phosphorus ester oligomers
Fathi et al. Synthesis and properties of combinatorial libraries of phosphoramidates
US6096916A (en) Synthesis of D-3 phosphorylated phosphoinositides and analogues
Iyer et al. N-pent-4-enoyl (PNT) group as a universal nucleobase protector: Applications in the rapid and facile synthesis of oligonucleotides, analogs, and conjugates
US6184389B1 (en) Combinatorial libraries having aminodiol monomer subunits
Ma et al. The 9‐fluorenylmethyloxycarbonyl group as a 5′‐OH protection in oligonucleotide synthesis
US20030194741A1 (en) Combinatorial protecting group strategy for multifunctional molecules
CA2244924A1 (fr) Oligomeres d'ester phosphoreux non nucleotidiques
Lindell et al. The combinatorial synthesis of organophosphorus compounds
Hotoda et al. Pre− activation strategy for oligodeoxyribonudeotide synthesis using triaryloxydichloro− phosphoranes in the phosphotriester method
US20040203036A1 (en) Multimer polynucleotide synthesis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997933585

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2244924

Country of ref document: CA

Ref country code: CA

Ref document number: 2244924

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1997933585

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997933585

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载