EP0880532A1 - Oligomeres d'ester phosphoreux non nucleotidiques - Google Patents

Oligomeres d'ester phosphoreux non nucleotidiques

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Publication number
EP0880532A1
EP0880532A1 EP97933585A EP97933585A EP0880532A1 EP 0880532 A1 EP0880532 A1 EP 0880532A1 EP 97933585 A EP97933585 A EP 97933585A EP 97933585 A EP97933585 A EP 97933585A EP 0880532 A1 EP0880532 A1 EP 0880532A1
Authority
EP
European Patent Office
Prior art keywords
substituted
diol
oligomer
group
functionality
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97933585A
Other languages
German (de)
English (en)
Other versions
EP0880532A4 (fr
Inventor
Robert G. Gentles
Alan F. Cook
Morris J. Rudolph
Reza Fathi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genzyme Corp
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
Publication of EP0880532A1 publication Critical patent/EP0880532A1/fr
Publication of EP0880532A4 publication Critical patent/EP0880532A4/fr
Withdrawn legal-status Critical Current

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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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 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 phosphodiester linkages have been substituted with acetal groups (Matteucci et al . , 1993), and the regular deoxyribose element of the backbone has been replaced with a 5-3-ethano-bridged rigid analog, both with the objective of introducing conformational constraint.
  • the oligonucleotides bound with higher affinity to their cognate sequences than did conventional oligonucleotides (Leumann et al., 1993).
  • Combinatorial libraries of oligomers having potential 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 modulating biologically active agents, particularly proteins, through their binding potential. Thus these products constitute a pool of readily accessible and sophisticated oligomeric constructs and libraries that satisfies this market and thereby makes structures available to research programs which would not otherwise have access to the broad spectrum of candidates they provide.
  • One 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 1 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 1 can be the same or different in each monomeric unit, and in at least one of the non-nucleotide monomeric units, R 1 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 alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted-anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic 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 1 is selected from a substituted alkane, a 3,3-disubstituted 3-amino-1,2- propanediol, a substituted or unsubstituted alicyclic ring wherein the ring size is from 4-12, a 3-substituted indole, a substituted or unsubstituted hydroxyalkyl phenol and an alicyclic 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-1,2-propanediols.
  • Another preferrred embodiment of the above aspect is an oligomer wherein in at least one of the monomeric units R 1 is a heterocyclic, an alicyclic or a polycyclic ring system, preferably containing an indole, thiazole, imidazole, pyridine, purine or pyrimidine ring.
  • the alicyclic 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;
  • B 1 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 1 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
  • 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 1 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 1 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 alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic 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 1 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 1 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 1 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 alicyclic hydrophobic functionality (iv) a diol attached to a ring-substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label.
  • Another aspect of the invention provides a non-nucleotide monomeric unit having the structure: wherein
  • X is a protecting group
  • R 1 is a condensation product of:
  • Another aspect of the invention provides a phosphorus ester oligomer of monomeric units, which oligomer has the structure: wherein
  • 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 1 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 1 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 1 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 alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic 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 ⁇ -branched amines, significant rotational constriction of the phosphorus linkage can be achieved, and this constitutes another mechanism for introducing conformational constraint.
  • 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 1 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-1,3-propandiol (e.g.; thiomicamine).
  • Preferred hydrogen bond acceptor compounds include those containing amine or ether moieties.
  • R 1 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 1 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 1,2-dihydroxy-3-butene
  • a 3,3- disubstituted 3-amino-1,2-propanediol e.g., 3-benzyl-3-methylamino-1,2-propanediol and 3,3-diethylamino-1,2-propanediol
  • a 3-substituted dihydroxyalkyl indole e. g.
  • indolyl-dihydroxypropane or a substituted or unsubstituted hydroxyalkyl phenol (e. g., tetralin and hydroxybenzyl alcohol) or a 1,2-dihydroxy alicyclic, dicarboxylic acid (e. g., tricyclononene dicarboxylic acid).
  • hydroxyalkyl phenol e. g., tetralin and hydroxybenzyl alcohol
  • 1,2-dihydroxy alicyclic, 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 1 is a dihydroxy-substituted carboxylic acid (e.g., cyclopentanediol acetic acid) or a 1,2-dihydroxy alicyclic dicarboxylic acid (e. g., tricyclononene dicarboxylic acid).
  • Preferred electrostatically charged functionalities also include cationic functionalities.
  • R 1 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- diethylaminopropanediol and 3 -benzyl-3-methylamino-1,2-propanediol) or a bis(hydroxyalkyl)-substituted heterocycle (e. g., 1,2-dihydroxyethyl-thiazole and pyridine dimethanol).
  • R 1 is a substituted 1,3-dihydroxyalkane (e. g. , 2-amino-1,3-propanediol, 1-phenyl- 2-amino
  • Preferred heterocyclic dihydroxy alcohols are those containing an indole, thiazole, imidazole, purine or pyrimidine ring structure.
  • R 1 is a 3-substituted dihydroxyalkyl indole (e. g. , indolyl-dihydroxypropane) or a purine or pyrimidine substituted 1,2-diol (e. g.
  • 2,3-dihydroxypropyl-theophylline or a bis (hydroxyalkyl)- substituted heterocycle (e.g., thiazole and pyridine dimethanol) or a 5-substituted 2-hydroxymethyl 3-hydroxy-tetrahydrofuran (e.g., 1,2 dideoxy-D-ribose).
  • a bis (hydroxyalkyl)- substituted heterocycle e.g., thiazole and pyridine dimethanol
  • a 5-substituted 2-hydroxymethyl 3-hydroxy-tetrahydrofuran e.g., 1,2 dideoxy-D-ribose
  • Preferred alicyclic 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 1 is a substituted or unsubstituted alicyclic diol wherein the ring size is from 4-12 (e. g. cyclooctanediol and cyclopentanediol) and those wherein R 1 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 1 is a dihydroxy substituted polycyclic compound (e.g. tricyclononene diol dicarboxylic acid and pinanediol).
  • non-nucleotide phosphorus oligomers where R 1 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 1 is 1,3-propanediol or 2-amino-1,3-propanediol.
  • non-nucleotide phosphorus oligomers where R 1 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 1 is a purine substituted 1,2-diol (e.g. 2,3-dihydroxypropyl theophylline).
  • Examples of non-nucleotide phosphorus oligomers where R 1 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 1 is 3-diethylamino-1,3-propanediol.
  • non-nucleotide phosphorus oligomers where R 1 is a condensation product of an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent include those formed from monomers wherein R 1 is cyclopentanediol, cyclo-octanediol, and those formed from monomers wherein R 1 is a dihydroxy-substituted carboxylic acid (e. g., cyclopentane diol acetic acid) or a 1,2-dihydroxy alicyclic dicarboxylic acid (e. g. , tricyclononene diol dicarboxylic acid).
  • R 1 is a condensation product of an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent
  • R 1 is a condensation product of an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyal
  • non-nucleotide phosphorus oligomers where R 1 is a condensation product of a hydroxy or hydroxyalkyl substituted tetrahydrofuran include those formed from monomers wherein R 1 is 1,2-dideoxy-D-ribose.
  • non-nucleotide phosphorus oligomers where R 1 is a condensation product of an indole substituted acyclic aliphatic diol include those formed from monomers wherein R 1 is indolyl-dihydroxypropane.
  • non-nucleotide phosphorus oligomers where R 1 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 1 is 2,6-bis-hydroxymethyl-pyridine.
  • non-nucleotide phosphorus oligomers where R 1 is a condensation product of a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl include those formed from monomers wherein R 1 is 1,2,3,4-tetrahydro-1,5-dihydroxy-naphthalene.
  • non-nucleotide phosphorus oligomers where R 1 is a condensation product of a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline include those formed from monomers wherein R 1 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.
  • An example of a simple oligomeric phosphodiester compound of this type which has already been synthesized is as follows:
  • 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-dimethyloxyphenyl)ethylamine; 4-(2-aminoethyl)-morpholine; 1-(3-aminopropyl)-4-methylpiperazine; 1-(3-aminopropyl)-2-pyrrolidinone; 1-(3-aminopropyl)imidazole; 1-(2-aminoethyl)pyrrolidine; 3-aminopropionitrile; 2-(2-aminoethyl)-1-methyl-pyrrolidine; 4-fluorophenethylamine; 4-bromophenethylamine; aminomethyl-cyclopropane; 3,3-diphenylpropylamine; formylpiperazine; trifluoromethylphenyl
  • 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 1 and/or B 2 are 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.
  • B 1 and B 2 is a phosphodiester attached to a tritium-labeled p- or m- 1-hydroxy (C 1 -C 6 alkyl) phenyl.
  • An important consideration requires that the labeling group should also be stable to the alkaline conditions used to cleave the material from the solid support. Simple glycol groups with flexible backbones were therefore ruled out since Fontanel et al .
  • oligonucleotides terminated with glycol moieties undergo degradation in concentrated ammonia at 55°C, presumably by attack of the free hydroxyl group, on the adjacent phosphate group followed by cyclization and elimination of the terminal unit.
  • hydroxyacetophenone was selected as a commercially available starting material for the labeling monomer approach, since the possibility of attack of the hydroxyl group produced by reduction on the adjacent phosphorus atom was eliminated because of the rigidity of the benzene ring.
  • a comparison of reductions of the ketone group of both p-and m-hydroxyacetophenone indicated that the meta isomer was more easily reduced using sodium borohydride, this isomer was selected for further studies.
  • 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.
  • 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 B 1 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 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 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
  • the purities of the fractions were monitored by TLC and fractions containing pure material were combined and evaporated to dryness to give 5'-O-(4, 4'-dimethoxytrityl)-1,2-dideoxy-D-ribose-3'- O-(N,N- diisopropylamino-2-cyanoethyl)-phosphoramidite (13.8 g) as an oil having the following structure:
  • 1-(4,4'-dimethoxytrityl)-1,3-propanediol (6.8 g) was prepared by the method of Seela and Kaiser (1987). This DMT derivative (6.8 g, 18 mmol) was dissolved in dry dichloromethane (35 mL) under nitrogen and treated with diisopropylethylamine (9.3 g, 72 mmol), and 2-cyanoethoxy-(N,N-diisopropylamino)chlorophosphine (6.4 g, 27 mmol). After stirring for 30 minutes, the mixture was concentrated and 5% aqueous sodium bicarbonate (250 mL) was added.
  • dichloromethane 200 mL and poured into 5% aqueous sodium bicarbonate (250 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2 ⁇ 200 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (250 mL) and dried over anhydrous sodium sulfate. The solid was filtered off and the filtrate was evaporated to dryness and coevaporated with toluene, methanol, hexane, and
  • 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 (1 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.
  • dichloromethane to give crude product (3.3 g) which was purified by column chromatography on silica gel (150 g) eluting with a gradient of dichloromethane/methanol (0 to 2%). The purities of the fractions were monitored by TLC and fractions containing the desired material (520 mg) were combined, evaporated to dryness and repurified by NP-HPLC eluting with isocratic dichloromethane.
  • 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, 10mmol) was added in portions to a stirred solution of the
  • 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. The product was eluted using 30% ethyl acetate in hexane, and homogenous fractions
  • 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.
  • 4,4'-Dimethoxytrityl chloride (9.4 g, 27.7 mmol) was added in portions to a stirred solution of freshly distilled 3,3 dimethyl-1,2-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.
  • (+/-)-2-amino-4-(1,2-dihydroxyethyl)-1,3- thiazole product as a pale orange colored solid, (2.6 g, 32.5%) with the following structure:
  • 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-2-amino-4-(1,2-dihyroxyethyl)-1,3-thiazole as a tan colored powder (0.475 g, 69%).
  • 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 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 residue was then adsorbed onto silica gel, and the mixture applied to a silica gel short path column and the product eluted using 30% ethyl acetate in hexane.
  • 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
  • 3-(4,4'-dimethoxytrityl)-2-amino-1-phenyl-1,3-propanediol (8.0 g, 14.1 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)-2-amino-1-phenyl-1,3-propanediol-H-phosphonate (7.1 g) as a white foam having the following structure:
  • 3-(4,4'-dimethoxytrityl)-1-phenyl-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)-1-phenyl-1,3-propanediol-H-phosphonate (2.3 g) as a yellow foam having the following structure:
  • 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'-O-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 oC. 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
  • the library was then assembled by mixing all of these solutions and lyophilizing the resultant mixture.
  • synthesizers using a modified H-phosphonate cycle with an extended coupling time of 5 minutes for each monomer.
  • the library was synthesized on 10 columns each loaded with 60 mg of long chain alkylamine controlled pore glass (37-70 ⁇ m, 44 umol/g), that had been derivatized with
  • oligomeric phosphoramidate library which was then labeled with 32 P by a conventional method using T4 polynucleotide kinase.
  • 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
  • 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.
  • Example 43 The oligomeric library of Example 43 was screened against the target proteins IL-4 and IFN ⁇ for high affinity ligands, using the COMPILE method as outlined in copending patent application "Determination and
  • 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-HCl (pH 7.5). The reactions were equilibrated for 30 min at room temperature prior to centrifugation through
  • 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
  • fluorescence of the fluorescein tag incorporated into the library members is plotted as a function of the rounds of selection - i.e. the number of dialysis-based
  • 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.
  • 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
  • the labeling group is attached to a thymidine residue attached to a solid support.
  • Thymidine-5'-(m-acetylphenyl)phosphate (3a) was prepared on a DNA synthesizer using four 10 ⁇ mol cartridges with a modified 10 ⁇ mol phosphoramidite coupling cycle and 2 ⁇ 15 minute coupling times. A solution of 2 (0.2 M) in anhydrous acetonitrile was used as the coupling reagent. A portion of the solid support (25 ⁇ mol) was cleaved with 29% NH 4 OH to give crude 3a (559 OD 260 ).
  • This material was purified in 4 portions using a C4 reversed-phase HPLC column with a gradient of 5-35% acetonitrile in 0.1M TEAA pH 7 over 6 min and then to 45% acetonitrile over 20 min. Pure fractions were combined and evaporated to give pure 3a (424 OD 260 ) as white powder.
  • 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 ⁇ 10 mL), then eluted with 6:4 methanol/water (v:v) (4 ⁇ 1 mL) followed by methanol (1 mL) and the methanolic fractions were
  • non-nucleotide phosphorus ester oligomers in a screw-cap glass test tube was added sodium borotritiide (20 mCi, 0.29 ⁇ mol, American Radiolabeled Chemicals, Inc., St. Louis, specific activity 70 Ci/mmol) in 0.01 N aqueous sodium hydroxide solution (200 ⁇ L) and the mixture was allowed to react for 2 hours.
  • sodium borohydride (0.024 mg, 0.63 ⁇ mol) in 0.01 N aqueous sodium hydroxide
  • dimethoxytrityl-glycolic acid (0.73g, 1.6 mmol)
  • 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. After addition of all 10 monomers as previously described, followed by a second
  • 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,
  • a phosphoramidate library is prepared as
  • 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 O/CH 3 OH (5 mL).
  • the H 2 O/methanol solutions are collected and freeze dried to yield labeled libraries having stucture 10 which are further purified by HPLC using a C4 columm with 5-80% acetonitrile in TEAA, 0.1 M, pH 7.0 as the eluant.
  • the libraries can be reduced using sodium borohydride instead of sodium cyanoborohydride. In this case each sub-library is suspended in 25 mM
  • 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 %
  • 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.
  • 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 HCl, L-alanine methyl ester, L-valine ethyl ester HCl, L-phenylalanine methyl ester HCl, L-glutamic acid diethyl ester,
  • B 1 is a labeling group
  • B 2 is a glycolic
  • A is an N-linked amino acid ester
  • R 1 and R 2 are derived from diols as previously
  • 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.
  • 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.
  • This subpopulation is enriched with each
  • 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

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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.
EP97933585A 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques Withdrawn EP0880532A4 (fr)

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US08/595,264 US6008398A (en) 1995-01-18 1996-02-01 Non-nucleotide phosphorus ester oligomers
PCT/US1997/001060 WO1997028168A1 (fr) 1996-02-01 1997-01-22 Oligomeres d'ester phosphoreux non nucleotidiques
US595264 2000-06-15

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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
AU1591301A (en) 1999-11-09 2001-06-06 Sri International Workstation, apparatus, and methods for the high-throughput synthesis, screeningand characterization of combinatorial libraries
AU1476501A (en) * 1999-11-09 2001-06-06 Sri International Screening and analysis of polymers, specialty chemicals and catalysts using radiography
JP2008519058A (ja) * 2004-11-08 2008-06-05 ネオファーム、インコーポレイティッド カルジオリピン類似体の合成及びそれらの使用
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
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
EP3484908A4 (fr) 2016-07-15 2020-04-08 AM Chemicals Llc Supports solides non nucléosides et blocs de construction de phosphoramidite pour synthèse d'oligonucléotides

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WO1995023160A1 (fr) * 1994-02-23 1995-08-31 Isis Pharmaceuticals, Inc. Nouveaux composes oligomeres de phosphoramidate et de phosphorothiomidate
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