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  • C07D473/00—Heterocyclic compounds containing purine ring systems
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems
  • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
  • C07D473/32—Nitrogen atom
  • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
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  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
  • C07F7/1804—Compounds having Si-O-C linkages
• • 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 System
  • 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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  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
  • C12N15/1133—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
  • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
  • C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/323—Chemical structure of the sugar modified ring structure

Definitions

• This application is directed to oligonucleotide surrogate compounds and their intermediates and to their design, synthesis and use. More particularly this invention is directed to oligonucleotide surrogate compounds that include linked cyclobutyl rings having heterocyclic bases attached thereto. Such oligonucleotide surrogates are useful for therapeutics, diagnostics and as research reagents.
• Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single- stranded RNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted.
• Hybridization is the sequence specific hydrogen bonding via Watson-Crick base pairs of the heterocyclic bases of oligonucleotides to RNA or DNA. Such base pairs are said to be complementary to one another.
• Naturally-occurring events that provide for the disruption of the nucleic acid function are thought to be of two types.
• the first is hybridization arrest. This denotes the terminating event in which an oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid.
• Methyl phosphonate oligonucleotides Miller, P.S. and Ts'O, P.O. P. (1987) Anti-Cancer Drug Design , 2:117-128, and ⁇ -anomer oligonucleotides are the two most extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
• the relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex.
• the melting temperature (T ) a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coil (unhybridized) forms are present.
• T m is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T m . The higher the T m , the greater the strength of the binding of the strands. Non- Watson-Crick base pairing has a strong destabilizing effect on the T m .
• the second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H.
• a 2'-deoxyribo- furanosyl oligonucleotide hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA.
• Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
• oligonucleotides as antisense agents for diagnostics, research reagents and potential therapeutic purposes.
• This research has included the synthesis of oligonucleotides having various modification.
• modification have primarily been modifications of the phosphate links that connect the individual nucleosides of the oligonucleotide.
• Various phosphorothioates, phosphotriesters, phosphoramidates and alkyl phosphonates have been reported.
• Further research has been directed to replacement of the inter-nucleoside phosphates with other moieties such as carbamates, sulfonates, siloxanes and the formacetal group.
• conjugate groups are attached to the nucleosides of the oligonucleotide via linking groups.
• conjugates include fluorescent dyes, intercalating agents, proteins, cross-linking agents, chain-cleaving agents and other groups including biotin and cholesterol.
• heterocyclic bases of the nucleosides of an antisense oligonucleotide are necessary for the proper Watson/Crick binding of the antisense oligonucleotide to the target RNA or DNA, with the exception of cross- linking agents, little has been reported as to modification on the heterocyclic bases.
• Alpha nucleosides have be used to form oligonucleotides having "alpha” sugars incorporated therein. In a like manner 2'-O-methylribonucleotides also have been used as precursor building blocks for oligonucleotides.
• United States Patent No. 5,034,506 and PCT Patent Application PCT/US86/00544 suggest that the sugar portion of a nucleoside can be ring opened via oxidization and then ring closed by reactions with an amino or hydrazine group on an adjacent nucleoside. This links the nucleosides.
• a new ring is formed from the residue of the oxidized pentofuranose sugar ring of the nucleoside.
• PCT/US86/00544 also suggests that a linear amino acid based polymer might be used in place of a sugar-phosphate backbone to link heterocyclic bases together in an oligonucleotide-like linkage. Aside from these modifications, modification of the sugar moieties of the nucleosides of oligonucleotides is also little known.
• Cyclobut-A i.e. ( ⁇ )-9-[(1 ⁇ ,2 ⁇ ,3 ⁇ )-2,3-bis- (hydroxymethyl)-1-cyclobutyl]adenine
• cyclobut-G i.e. ( ⁇ )-9-[(1 ⁇ ,2 ⁇ ,3 ⁇ )-2,3-bis(hydroxymethyl)-1-cyclobutyl]- guanine
• a still further object is to provide research and diagnostic methods and materials for assaying bodily states in animals, especially diseased states.
• Another object is to provide therapeutic and research methods and materials for the treatment of diseases through modulation of the activity of DNA and RNA.
• oligonucleotide surrogates that are formed from a plurality of cyclobutyl moieties that are covalently joined by linking moieties; each of the cyclobutyl moieties has one of a purine or a pyrimidine heterocyclic base attached to it.
• the purine or pyrimidine heterocyclic base is a naturally-occurring or synthetic purin-9-yl, pyrimidin- 1-yl or pyrimidin-3-yl heterocyclic base.
• the purine or pyrimidine heterocyclic base is adenine, guanine, cytosine, thymidine, uracil, 5-methylcytosine, hypoxanthine or 2-aminoadenine.
• oligonucleotide surrogates the heterocyclic base is attached to each respective cyclobutyl moiety at the carbon-1 (C-1) position of said cyclobutyl moiety and the linking moieties connect to each respective cyclobutyl moiety at the carbon-3 (C-3) position thereof.
• a substituent group can be located on one of the carbon-2 (C-2) or the carbon- 4 (C-4) positions of at least one of the cyclobutyl moieties.
• Preferred substituents include halogen, C 1 -C 10 alkoxy, allyloxy, C 1 -C 1 0 alkyl or C 1 -C 1 0 alkylamine groups.
• the substituent group is positioned trans to the heterocyclic base.
• the linking moieties are 4 or 5 atoms chains that connect adjacent cyclobutyl moieties.
• each of the linking moieties preferably is of the structure L 1 -L 2 -L 3 , where L 1 and L 3 are CH 2 ; and L 2 is phosphodiester, phosphorothioate, phosphoramidate, phosphotriester, C 1 -C 6 alkyl phosphonate, phosphorodithioate, phosphonate, carbamate, sulfonate, C 1 -C 8 -dialkylsilyl or formacetal.
• each of the linking moieties is of the structure L 1 -L 2 - L 3 , where L 1 and L 3 are CH 2 and L 2 is phosphodiester or phosphorothioate.
• each of the linking moieties preferably is of the structure:
• L 4 and L 7 are CH 2 ;
• R 1 and R 2 are H, OH, SH, NH 2 , C 1 -C 10 alkyl, C 1 -C 1 0 substituted alkyl, C 1 -C 1 0 alkenyl, C 1 -C 1 0 aralkyl, C 1 -C 6 alkoxy, C 1 -C 6 thio- alkoxy, C 1 -C 6 alkylamino, C 7 -C 10 aralkylamino, C 1 - C 10 substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, polyalkylamino, halo, formyl, keto, benzoxy, carboxamido, thiocarboxamido, ester, thioester, carboxamidino, carbamyl, ureido, guanidino, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucle
• R 3 is H, OH, NH 2 , C 1 -C 6 alkyl, substituted lower alkyl, alkoxy, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, polyalkylamino, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligo nucleotide and a group for improving the pharmacodynamic properties of an oligonucleotide; and
• R 4 and R 5 independently, are C 1 -C 6 alkyl or alkoxy.
• a method for modulating the production or activity of a protein in an organism comprising contacting the organism with an oligonucleotide surrogate formulated in accordance with the forgoing considerations.
• an oligonucleotide surrogate is specifically hybridizable with at least a portion of a nucleic acid sequence, i.e. an RNA or DNA, coding for the protein.
• At least a portion of the oligonucleotide surrogate is formed from a plurality of linked cyclobutyl moieties, each moiety having an attached purine or pyrimidine heterocyclic base.
• a method of treating an organism having a disease characterized by the undesired production of a protein includes contacting the organism with an oligonucleotide surrogate also formulated in accordance with foregoing considerations.
• an oligonucleotide surrogate is specifically hybridizable with at least a portion of a nucleic acid sequence, i.e. an RNA or DNA sequence, coding for the protein whose production or activity is to modulated.
• At least a portion of the oligonucleotide surrogate is formed from a plurality of linked cyclobutyl moieties, wherein each moiety having an attached purine or pyrimidine heterocyclic base.
• composition containing as its active ingredient an effective amount of an oligonucleo- tide surrogate formed from a plurality of linked cyclobutyl moieties, each moiety having an attached purine or pyrimidine heterocyclic base; and a pharmaceutically acceptable diluent or carrier.
• a method of in vitro assaying a sequence specific nucleic acid i.e. an RNA or DNA
• a method of in vitro assaying a sequence specific nucleic acid comprising contacting an in vitro composition which includes the nucleic acid with an oligonucleotide surrogate that is specifically hybridizable with at least a portion of the nucleic acid.
• the oligonucleotide surrogate preferably is formulated in accordance with the foregoing considerations.
• at least a portion of the oligonucleotide surrogate compound is formed from a plurality of linked cyclobutyl moieties, each moiety having an attached purine or pyrimidine heterocyclic base.
• a process for the preparation of a compound formed from a plurality of linked cyclobutyl moieties wherein each moiety has an attached purine or pyrimidine heterocyclic base comprises the steps of: functionalizing the cyclobutyl moieties with a leaving group; displacing the leaving group on each of the cyclobutyl moieties with an independently selected purine or pyrimidine heterocyclic base; functionalizing each of the base-containing cyclobutyl moieties with a protecting group; further functionalizing the protected moieties with an activated linking group; and stepwise deprotecting and linking the heterocyclic-base-containing cyclobutyl moieties.
• Such processes can be augmented to include stepwise deprotection and linkage of the base-containing cyclobutyl moieties on a polymeric support.
• the base-containing cyclobutyl moieties are stepwise deprotected and linked together by: (a) deprotecting a first of the protected moieties; (b) linking a further of the protected moieties bearing an activated linking group with the deprotected moiety to form a linked structure; and (c) deprotecting the linked structure.
• Deprotecting and linking steps (b) and (c) preferably are repeated a plurality of times.
• an antiviral composition containing as its active ingredient an effect amount of a compound of structure (A) :
• B x is purin-9-yl, pyrimidin-1-yl or pyrimidin-3- yl; and a pharmaceutically acceptable diluent or carrier.
• a method of treating viral diseases in mammals comprising administering to the mammal a therapeutic amount of a composition containing as its active ingredient a compound of the structure (A).
• the viral disease is a herpes viral disease.
• nucleoside refers to a molecular species made up of a heterocyclic base and a sugar.
• the heterocyclic base typically is guanine, adenine, cytosine, thymine, or uracil.
• Other natural bases are known, as are a plenitude of synthetic or "modified” bases.
• the sugar is normally deoxyribose (DNA type nucleosides) or ribose (RNA type nucleosides).
• nucleoside has been used to refer to both naturally-occurring and synthetic species formed from naturally-occurring or synthetic heterocyclic base and sugar subunits.
• nucleotide refers to a nucleoside having a phosphate group esterified to one of its 2 ' , 3' or 5' sugar hydroxyl groups.
• the phosphate group normally is a monophosphate, a diphosphate or triphosphate.
• oligonucleotide normally refers to a plurality of joined monophosphate nucleotide units. These units are formed from naturally-occurring bases and pentofuranosyl sugars joined by native phosphodiester bonds. A homo-oligonucleotide is formed from nucleotide units having a common heterocyclic base.
• oligonucleotide analog has been used to refer to molecular species which are structurally similar to oligonucleotides but which have non-naturally-occurring portions. This term has been used to identify oligonucleotide-like molecules that have altered sugar moieties, altered base moieties, or altered inter-sugar linkages.
• oligonucleotide analog denotes structures having altered inter-sugar linkages such as phosphorothioate, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of native phosphodiester inter-nucleoside linkages; purine and pyrimidine heterocyclic bases other than guanine, adenine, cytosine, thymine or uracil; carbocyclic or acyclic sugars; sugars having other than the ⁇ pentofuranosyl configuration; or sugars having substituent groups at their 2' position or at one or more of the sugar hydrogen atoms.
• oligonucleosides Certain oligonucleotide analogs having non-phosphodiester bonds, i.e. an altered inter-sugar linkage, can be referred to as "oligonucleosides.”
• oligonucleo side thus refers to a plurality of joined nucleoside units joined by linking groups other than native phosphodiester linking groups.
• oligomers can be used to encompass oligonucleotides and oligonucleotide analogs.
• inter-sugar linkages of oligonucleotides and oligonucleotide analogs are from the 3' carbon of one nucleoside to the 5' carbon of a second nucleoside; however, the terms oligomer and oligonucleotide analog also have been used in reference to 2'- 5' linked oligonucleotides.
• Antisense therapy is the use of oligonucleotides or oligonucleotide analogs to bind with complementary strands of RNA or DNA. After binding, the oligonucleotide and the RNA or DNA strand are "duplexed" together in a manner analogous to native, double-stranded DNA.
• the oligonucleotide strand and the RNA or DNA strand can be considered complementary strands wherein the individual strands are positioned with respect to one another to allow Watson/Crick type hybridization of the heterocyclic bases of one strand to the heterocyclic bases of the opposing strand.
• Antisense therapeutics can be practiced in a plethora of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA- protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to antisense therapeutics and/or prophylactics. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, and all plant and all higher animal forms, including warm-blooded animals, can be treated by antisense therapy.
• each of the cells of multicellular eukaryotes includes both DNA-RNA transcription and RNA-protein translation as an integral part of their cellular activity
• antisense therapeutics and/or diagnos tics can also be practiced on such cellular populations.
• many of the ⁇ rganelles, e.g. mitochondria and chloroplasts, of eukaryotic cells also include transcription and translation mechanisms.
• single cells, cellular populations and organelles can also be included within the definition of organisms that are capable of being treated with antisense therapeutics or diagnostics.
• therapeutics is meant to include both the eradication of a disease state, killing of an organism, e.g. bacterial, protozoan or other infection, or control of erratic or harmful cellular growth or expression.
• Antisense therapy utilizing "oligonucleotide analogs" is exemplified in the disclosures of the following United States and PCT Patent Applications: Serial No. 463,358, filed January 11, 1990, entitled Compositions And Methods For Detecting And Modulating RNA Activity; Serial No. 566,836, filed August 13, 1990, entitled Novel Nucleoside Analogs; Serial No. 566,977, filed August 13, 1990, entitled Sugar Modified Oligonucleotides That Detect And Modulate Gene Expression; Serial No. 558,663, filed July 27, 1990, entitled Novel Polyamine Conjugated Oligonucleotides;, Serial No.
• This invention is directed to certain molecular species which are related to oligonucleotides but which do not included a sugar moiety.
• cyclobutane rings i.e. cyclobutyl moieties, having heterocyclic bases attached thereto are connected by linking moieties into oligonucleotide-like structures.
• Such structures while chemically different from oligonucleotides (or oligonucleotide analogs), are functionally similar.
• Such molecular species are therefore oligonucleotide surrogates. As oligonucleotide surrogates they serve as substitutes for oligonucleotides. We have found that they are capable of hydrogen bonding to complementary strands of DNA or RNA in the same manner as are oligonucleotides.
• a cyclobutane ring system may be considered as fixed when compared to a pentofuranose ring system.
• a pentofuranose ring system allows for rotation about intra-ring chemical bonds
• a cyclobutane ring system does not. Consequently, the pentofuranosyl ring system can "pucker"; a cyclobutane ring cannot.
• a cyclobutane ring system has a sufficient number of functional positions within the ring to allow for placement of a number of substituent functional groups.
• nucleoside chemistry utilizes unprimed numbers to identify the functional positions on the heterocyclic base portion of the nucleoside and primed numbers to identify the functional positions on the sugar portion of the nucleoside.
• ⁇ isomer of a ribofuranosyl ring is syn about the anomeric C-1 position is syn with respect to the C-5 carbon, while the ⁇ isomer is trans with respect to the C-5 carbon.
• IUPAC recommended nomenclature for the functional positions of cyclobutane differ from such standard nucleoside nomenclature. Utilizing IUPAC nomenclature, the substitution of position C-1 in the cyclobutane ring is always ⁇ . For the purposes of this invention, positional identification of the cyclobutane ring is made by reference to structure (B):
• the oligonucleotide surrogates of the invention are formed by linking together a plurality of cyclobutyl moieties via linking moieties.
• Each of the cyclobutyl moieties includes a covalently-bound purine or pyrimidine heterocyclic base.
• Each of the linking moieties covalently bond two adjacent cyclobutyl moieties. Linked together in this manner, the cyclobutyl moieties present their heterocyclic bases in spatial positions for hybridization with DNA or RNA strands.
• cyclobutyl-based oligonucleotide surrogates include a plurality of subunits. Each subunit includes a cyclobutane ring, a heterocyclic base, and a linking moiety for joining adjacent subunits.
• the oligonucleotide surrogates of the invention preferably comprise from about 3 to about 100 subunits. Preferably, oligonucleotide surrogates comprise greater than about 6 subunits, preferably from about 8 to about 60 subunits, even more preferably from about 10 to about 30 subunits.
• the heterocyclic base of each of the subunits can be a natural heterocyclic base or a synthetic heterocyclic base.
• the heterocyclic base is selected as a naturally-occurring or synthetic purin-9- yl, pyrimidin-1-yl or pyrimidin-3-yl heterocyclic base.
• Heterocyclic bases include but are not limited to adenine, guanine, cytosine, thymidine, uracil, 5-methylcytosine, hypoxanthine or 2-aminoadenine.
• heterocyclic bases include 2-aminopurine, 2,6-diaminopurine, 6-mercaptopurine, 2,6-dimercaptopurine, 3-deazapurine, 6-amino- 3-deazapurine, 6-amino-3-deaza-2-oxypurine, 2-amino-6- mercaptopurine, 5-methylcytosine, 4-amino-2-mercaptopyrimidine, 2,4-dimercaptopyrimidine, 5-fluorocytosine.
• linking moieties are selected to covalently link individual heterocyclic- base-containing cyclobutyl moieties together in an orientation wherein the heterocyclic bases are positioned in space in a conformation which allows hybridization with a complementary strand of DNA or RNA.
• the linking moieties are selected as 4 or 5 atoms chains.
• Such 4 and 5 atoms chains include the phosphodiester linkages of native DNA and RNA as well as the related synthetic phosphorothioate, phosphoramidate, alkyl phosphonate, phosphorodithioate and phosphotriester linkages of "oligonucleotide analogs.”
• Other linking moieties include phosphate, carbamate, sulfonate, C 1 -C 8 -dialkylsilyl or formacetal linkages.
• Further linkages include an -O-CH 2 - CH 2 -O- linkage and the novel linkages disclosed in United States Patent Applications Serial No. 566,836 and Serial No. 703,619, identified above.
• a preferred group of 5 atom linking moieties of the invention include linking moieties of the structure L 1 - L 2 -L 3 where L 1 and L 3 are CH 2 ; and L 2 is Phosphodiester, phosphorothioate, phosphoramidate, phosphotriester, C 1 -
• a particularly preferred group of such 5 atom linker is of the structure L 1 -L 2 -L 3 where L, and L 3 are CH 2 ; and L 2 is phosphodiester or phosphorothioate.
• linking moieties of the invention include linking moieties of the structure L.-
• R 1 and R 2 independently, are H, OH, SH, NH 2 ,
• R 3 is H, OH, NH 2 , C 1 -C 8 alkyl, substituted lower alkyl, alkoxy, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, polyalkylamino, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide and a group for improving the pharmacodynamic properties of an oligonucleotide; and
• R 4 and R 5 independently, are C 1 -C 6 alkyl or alkoxy.
• the cyclobutyl moieties of the invention can further include other substituent groups.
• substituent groups can be located on one or both of the C-2 or the C-4 position.
• Preferred substituent groups include halogen, C 1 -C 1 0 alkoxy, allyloxy, C 1 -C 1 0 alkyl or C 1 -C 1 0 alkylamine.
• the substituent group is positioned trans to said heterocyclic base.
• Certain preferred embodiments of the invention take advantage of the symmetry of 3 ,3-bis-hydroxymethylcyclobutane substituted at the C-1 position with a heterocyclic base, as in structure (C).
• 1-benzyloxy-3,3-bis-hydroxymethyl-cyclobutane, 1-thymidyl-3,3-bis-hydroxymethyl-cyclobutane and 1-adenyl-3,3-bis-hydroxymethyl-cyclobutane can be synthesized by direct introduction of the heterocyclic bases; uridyl-3,3-bis-hydroxymethyl-cyclobutane, 1-guanyl-3,3- bis-hydroxymethyl-cyclobutane and 1-cytidyl-3,3-bishydroxymethyl-cyclobutane are prepared by similar procedures, as are cyclobutanes substituted with other heterocyclic bases.
• the known 1-benzyloxy-3,3-bis-hydroxymethyl-cyclobutane is converted to the O 5 ,O 5' -isopropyli dene-ether of 1-hydroxy-3,3-bis-hydroxymethyl-cyclobutane.
• Various sulfonate esters were examined for the introduction of the heterocyclic base. In the series mesylate, tosylate, brosylate and nosylate, the best properties with respect to stability and reaction conditions for substitution were observed with the p-bromobenzenesulfonate.
• the desired heterocyclic-base-substituted compounds were purified by flash chromatography and were deprotected with hydrochloric acid in dioxane to yield corresponding 3,3-bis hydroxymethyl derivatives having an appropriate heterocyclic base at the C-1 position.
• oligonucleotide surrogate compounds were of a pseudo- ⁇ - and a pseudo- ⁇ -configuration.
• the designation "pseudo- ⁇ ” refers to an oligonucleotide surrogate of the invention formed from cyclobutane units having their heterocyclic base positioned cis to what would be the 5' position of a natural oligonucleotide; "pseudo- ⁇ ” refers to oligonucleotide surrogates where the heterocyclic base would be trans to that same 5' position.
• the pseudo- ⁇ and pseudo- ⁇ configurations are shown in structures (D) and (E). A "normal" terminal thymidine nucleotide has been included in the structures at the "3'" terminal ends to better illustrate the configurations of the cyclobutane based oligonucleotide surrogates of the invention.
• the cis and trans isomers of mono-hydroxymethyl substituted adenylcyclobutane were also prepared.
• a chromatographic separation of the cis and trans isomers of carboethoxy intermediates was utilized. The procedure was applied separately to both isomers of 1-benzyloxy-3-carbethoxycyclobutane.
• oligonucleotide surrogate compounds of the invention having phosphorothioate, phosphoramidate, phosphotriester, C 1 -C 6 alkyl phosphonate and phosphorodithioate groups
• the oligonucleotide synthetic methods based on phosphoramidate chemistry of the following, above-referenced United States and PCT Patent Applications: Serial No. 566,836, Serial No. 463,358, Serial No. 558,806, Serial No. 558,663, Serial No. 566,977 and Serial No. US91/00243.
• the 3-aldehydic intermediate can be blocked for use as an aldehyde or it can be further converted to a hydrazino compound.
• the 3-aldehydic intermediate is either then treated with 1,2-dianilinoethylene to afford a 3-diphenylimidazolidino-protected 3- aldehydo compound or the 3-aldehydic intermediate is treated with hydrazine hydrate and sodium cyanoborohydrate in acetonitrile to give the corresponding 3-hydrazino compound.
• guanyl or cytidyl or unprotected thymidyl and uridyl-3-monomethoxytrityl-3- hydroxymethyl-cyclobutane are converted to the corresponding 3-N-hydroxyphthalimide-3-monomethoxytrityl-3- hydroxymethyl-cyclobutanes by treatment of 3-monomethoxytrityl-3-hydroxymethyl-cyclobutane compounds with N- hydroxyphthalimide, triphenylphosphine, and diisopropylazodicarboxylate in dry DMF.
• N-hydroxyphthalimido compound is then converted to a corresponding 3-amino intermediate compound by treatment with methylhydrazine in dry CH 2 CI 2 under anhydrous conditions, as per the teaching of above-referenced Patent Application Serial No. 703,619.
• Oligonucleotide surrogates of the invention having sulfonate linkages are prepared by reacting protected 1- adenyl, guanyl or cytidyl or unprotected thymidyl and uracyl-3-monomethoxytrityl-3-hydroxymethyl-eyelobutaneas per the procedures of Musicki, B and Widlanski, T.S. (1990) J. Organic Chem . , 55:4231.
• Phosphoramidates linkages are formed as per the procedure of Gryaznov, S.M. and Sokolova, N.I. (1990) Tetrahedron Letters , 31:3205; formacetal linkages as per the procedure of Matteucci, M.
• the oligonucleotide surrogates of this invention can be used in diagnostics, therapeutics, and as research reagents and kits.
• the oligonucleotide surrogates are administered to an animal suffering from a disease modulated by a protein. It is preferred to administer to patients suspected of suffering from such a disease an amount of the oligonucleotide surrogate that is effective to reduce the symptoms of that disease.
• One skilled in the art may determine optimum dosages and treatment schedules for such treatment regimens.
• oligonucleotide surrogates in accordance with this invention internally such as orally, intravenously, or intramuscularly.
• Other forms of administration such as transdermally, topically, or intra-lesionally may also be useful.
• Inclusion in suppositories may also be useful.
• Use of the oligonucleotide surrogates of this invention in prophylaxis is also likely to be useful.
• Use of pharmacologically acceptable carriers is also preferred for some embodiments.
• reaction mixture was then transferred into a 2 1 flask containing silica gel (800 ml) and the solvent was removed under vacuum until a fine powder was obtained.
• This powder was added to a 5 cm Hyflo pad on a fritteglass and washed with ethyl acetate (400 ml fractions with TLC control). The fractions containing product were evaporated to give 55 g of crystalline 2 (79 % crude). After recrystallization from ethyl-acetate/hexane 43.2 g , 62 % of colourless crystals were obtained.
• H 2b + H 4b 1.76 (s: CH 3 thy ); 13 C (CD 3 OD at 50 MHz): d in PPM: 173.87: CO; 167.06: CO; 161.08: C 6thy ; 115.45: C 5thy ; 72.27: CH 2 O a ; 71.59: CH 2 O b ; 70.05: C 1 ; 69.97: C 1 ; 42.87: C 3 ; 42.66: C 3 ; 38.34: C 2 + C 4 ; 38.19: C 2 + C 4 ; 15.62: CH 3 thy; U.V. (water, 0.5 x 10 -4 mole/1): 1 max in nm (e max): 268 (9640).
• Methoxytritylchloride (481 mg, 1.56 mmoles) was added under argon in 100 mg portions every 2 hours to a solution of 17 (500 mg, 1.41 mmoles) in pyridine (5 ml) in the presence of dimethylaminopyridine (100 mg).
• fraction 1 100 mg, 8 % bis-methoxytrityl-derivative
• fraction 2 240 mg, 27 % methoxytrityl- ⁇ -derivative 22
• fraction 3 80 mg, 9 % methoxytrityl- ⁇ -derivative 23
• fraction 4 50 mg, 10 % unreacted diol 17.
• 71.78 C 1 ; 70.70: CH 2 , Bzl; 70.52: CH 2 , Bzl; 68.78: C 1 ;
• Neat oxalyl chloride (42.3 ml, 485 mmoles) was added slowly over 1 hour at 0° C to a solution of 27 cis + 27 trans (28.61g, 133.5 mmoles) in carbon tetrachloride (230 ml). The reaction started immediately with evolution of CO 2 . The mixture was stirred 1 hour at 0° C and 12 hours at room temperature.
• Lithium aluminum hydride (1.39 g, 36.36 mmoles) was stirred under argon in dimethoxyethane (50 ml).
• the 1:1 mixture of the acids 27 cis and 27 trans (5.19 g, 24.21 mmoles) in dimethoxyethane (10 ml) was added dropwise without cooling.
• H 8ad 7.65 (n: 4 H ar ); 7.37 (m: 6 H ar ); 5.22 (quint : H 1 ); 3.81 (s: CH 2 OSi); 2.87 (m: H 2a + H 4a ); 2.60 (m: H 2b + H 3 + H 4b ); 1.05 (s: CH 3 , tBu); U.V. (water, 0.5 x 10 -4 mole/l):
• a typical activated phosphoryl compound was prepared as follows: 1 ⁇ -(N-Benzoyl-adenyl)-3 ⁇ -hydroxymethyl-3 ⁇ - methoxytrityloxymethyl-cyclobutane 22 (0.25 mmole, 156.4 mg) was co-evaporated with pyridine to remove traces of water and phosphorylated with 2-chlorophenyl-di-(1- benzotriazolyl)-phosphate (1.00 ml, 0.25 M ) in THF at room temperature for 30 min (activated nucleotide) as per: Van Boom, J.H., Van der Marel, G.A., Van Boeckel, C.A.A., Wille, G. and Hoyng, C.
• the oligomer was cleaved from the carrier and the protecting groups removed by sequentially reacting the resin with 1 M tetramethylguanidinium 2-nitrobenzald- oximate in 200 ml 95% pyridine during 7 h at 60°C and 0.8 ml 33% aqueous ammonia for 24 h at 60°C
• the reaction mixture was extracted 3 times with diethylether (2 ml each) and the aqueous phase was applied to a Biogel P4 (50-100 mesh) column (3 x 26 cm) and the product eluted with water. Fractions were checked for correct size of the oligomer and homogeneity with polyacrylamide- or capillary-electrophoresis.
• a typical activated phosphoramidate is prepared as follows: 1 ⁇ -(N-Benzoyladenyl)-3 ⁇ -hydroxymethyl-3 ⁇ -methoxy- trityloxymethyl-cyclobutane 23 (1.89 mmol) is dissolved in anhydrous dichloromethane (13 ml) under an argon atmosphere, diisopropylethylamime (0.82 ml, 4.66 mmol) is added, and the reaction mixture cooled to ice temperature. Chloro(diisdpropylamino)- ⁇ -cyanoethoxyphosphine (0.88 ml, 4.03 mmol) is added to the reaction mixture and the reaction mixture is then allowed to warm to 20°C and stirred for 3 hours.
• Phosphoramidate oligonucleotide surrogates synthesis are performed on an Applied Biosystems 380 B or 394 DNA synthesizer following standard phosphoramidate protocols and cycles.
• the oligonucleotide subunits are as described above and all other reagents are as supplied by the manufacture. In those steps wherein phophoramidites subunits of the oligonucleotide surrogates of the invention are used, a longer coupling time (10-15 min) is employed.
• the oligonucleotide surrogates are normally synthesized in either a 10 ⁇ mol scale or a 3 x 1 ⁇ mol scale in the "Trityl-On" mode.
• Standard deprotection conditions (30% NH 4 OH, 55°C, 16 hr) are employed.
• HPLC is performed on a Waters 600E instrument equipped with a model 991 detector.
• the following reverse phase HPLC conditions are employed: Hamilton PRP-1 column (15 x 2.5 cm); solvent A: 50mm TEAA, pH 7.0; solvent B: 45mm TEAA with 80% CH 3 CN; flow rate: 1.5ml/min; gradient: 5% B for the first 5 minutes, linear (1%) increase in B every minute thereafter.
• the oligonucleotide surrogate is treated with the Beaucage reagent, i.e. 3H- 1,2-benzodithioate-3-one 1,1-dioxide, see Radhakrishnan, P.I., Egan, W., Regan, J.B. and Beaucage, S.L., (1990), J. Am . Chem . Soc , 112:1253 for conversion of the phosphordiester linkages to phosphorothioate linkages.
• the Beaucage reagent i.e. 3H- 1,2-benzodithioate-3-one 1,1-dioxide
• PROCEDURE 1 Hybridization Analysis.
• the relative ability of an oligonucleotide surrogate of the invention to bind to complementary nucleic acids can be compared by determining the melting temperature of a particular hybridization complex.
• the melting temperature (T m ) a characteristic physical property of double-stranded RNA or DNA, denotes the temperature in degrees centigrade at which 50% helical versus coil (unhybridized) forms are present. T is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T m .
• Non-Watson-Crick base pairing has a strong destabilizing effect on the T m . Consequently, absolute fidelity of base pairing is necessary to have optimal binding of an antisense oligonucleotide to its targeted RNA or DNA.
• thermodynamics of hybridization of oligonucleotide surrogates A. Evaluation of the thermodynamics of hybridization of oligonucleotide surrogates.
• RNA complements used were poly-rA and poly-rU.
• DNA complements used were poly-dA and (dT) 8 .
• oligonucleotide surrogates of the invention were hybridized against one another and the T m s determined. The oligonucleotide surrogates were added to either the RNA or DNA complement at stoichiometric concentrations and the absorbance (260 nm) hyperchromicity upon duplex to random coil transition was monitored using a Gilford Response II spectrophotometer.
• Tables 1 and 2 The results of the thermodynamic analysis is shown in Tables 1 and 2. In both tables both measured Txns and the percent hyperchromicity are shown. Table 1 shows results against other "normal" nucleic acid strands (that is ribofuranosyl or deoxy-ribofuranosyl based nucleic acid strands) and Table 2 shows results against complementary cyclobutane strands - that is against one another.
• pseudo ⁇ A refers to an 8 mer oligonucleotide surrogate of the invention formed from eight cyclobutane units wherein the adenine base is cis to the methoxytrityloxymethyl moiety, i.e. compound 22, 1- ⁇ (N-benzoyl-adenyl)-3 ⁇ -hydroxymethyl-3 ⁇ -methoxytrity- loxymethyl-cyclobutane.
• Pseudo ⁇ A refers to the corresponding 8 mer having the respective substituents of the cyclobutane ring trans to each other.
• pseudo ⁇ T and pseudo ⁇ T reference the corresponding 8 mer cis and trans thymine base oligonucleotide surrogates.
• oligonucleotide surrogates are incubated for various times, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamine-urea denaturing gels and subsequent autoradiography. Autoradiograms are quantitated by laser densitometry. Based upon the location of the modified linkage and the known length of the oligonucleotide surrogate, it is possible to determine the effect on nuclease degradation by the particular modification. For the cytoplasmic nucleases, an HL 60 cell line can be used.
• a post-mitochondrial supernatant is prepared by differential centrifugation and the labelled oligonucleotide surrogates are incubated in this supernatant for various times. Following the incubation, the oligonucleotide surrogates are assessed for degradation as outlined above for serum nucleolytic degradation. Autoradiography results are quantitated for comparison of the unmodified and the oligonucleotide surrogates of the invention. It is expected that the of oligonucleotide surrogates will be completely resistant to serum and cytoplasmic nucleases.
• oligonucleotides and oligonucleotide surrogates of the invention can be done to determine the exact effect of the modified linkage on degradation.
• the oligonucleotide surrogates are incubated in defined reaction buffers specific for various selected nucleases. Following treatment of the products with protease K, urea is added and analysis on 20% polyacrylamide gels containing urea is done. Gel products are visualized by staining with Stains All reagent (Sigma Chemical Co.). Laser densitometry is used to quantitate the extent of degradation.
• the effects of the modified linkage are determined for specific nucleases and compared with the results obtained from the serum and cytoplasmic systems. As with the serum and cytoplasmic nucleases, it is expected that the oligonucleotide surrogates of the invention will be completely resistant to endo- and exonucleases.
• PROCEDURE 3 5-Lipoxygenase Analysis, Therapeutics and Assays
• an animal suspected of having a disease characterized by excessive or abnormal supply of 5-lipoxygenase is treated by administering oligonucleotide surrogates of the invention.
• Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Such treatment is generally continued until either a cure is effected or a diminution in the diseased state is achieved. Long term treatment is likely for some diseases.
• oligonucleotide surrogates of this invention will also be useful as research reagents when used to cleave or otherwise modulate 5-lipoxygenase mRNA in crude cell lysates or in partially purified or wholly purified RNA preparations.
• This application of the invention is accomplished, for example, by lysing cells by standard methods, optimally extracting the RNA and then treating it with a composition at concentrations ranging, for instance, from about 100 to about 500 ng per 10 Mg of total RNA in a buffer consisting, for example, of 50 mm phosphate, pH ranging from about 4-10 at a temperature from about 30° to about 50° C
• a composition at concentrations ranging, for instance, from about 100 to about 500 ng per 10 Mg of total RNA in a buffer consisting, for example, of 50 mm phosphate, pH ranging from about 4-10 at a temperature from about 30° to about 50° C
• the cleaved 5-lipoxygenase RNA can be analyzed by agarose gel electrophoresis and hybridization with radiolabeled DNA probes or by other standard methods.
• oligonucleotide surrogates of this invention will also be useful in diagnostic applications, particularly for the determination of the expression of specific mRNA species in various tissues or the expression of abnormal or mutant RNA species.
• the oligonucleotide surrogates target a hypothetical abnormal mRNA by being designed complementary to the abnormal sequence, but would not hybridize to normal mRNA.
• Tissue samples can be homogenized, and RNA extracted by standard methods.
• the crude homogenate or extract can be treated for example to effect cleavage of the target RNA.
• the product can then be hybridized to a solid support which contains a bound oligonucleotide comple- mentary to a region on the 5' side of the cleavage site. Both the normal and abnormal 5' region of the mRNA would bind to the solid support.
• the 3' region of the abnormal RNA, which is cleaved, would not be bound to the support and therefore would be separated from the normal mRNA.
• Targeted mRNA species for modulation relates to 5- lipoxygenase; however, persons of ordinary skill in the art will appreciate that the present invention is not so limited and it is generally applicable.
• the inhibition or modulation of production of the enzyme 5-lipoxygenase is expected to have significant therapeutic benefits in the treatment of disease.
• an assay or series of assays is required.
• the cellular assays for 5-lipoxygenase preferably use the human promyelocytic leukemia cell line HL-60. These cells can be induced to differentiate into either a monocyte-like cell or neutrophil-like cell by various known agents. Treatment of the cells with 1.3% dimethyl sulfoxide, DMSO, is known to promote differentiation of the cells into neutrophils. It has now been found that basal HL-60 cells do not synthesize detectable levels of 5-lipoxygenase protein or secrete leukotrienes (a downstream product of 5-lipoxygenase). Differentiation of the cells with DMSO causes an appearance of 5-lipoxygenase protein and leukotriene biosynthesis 48 hours after addition of DMSO. Thus induction of 5-lipoxygenase protein synthesis can be utilized as a test system for analysis of antisense oligonucleotides surrogates which interfere with 5-lipoxygenase synthesis in these cells.
• a second test system for antisense oligonucleotides surrogates makes use of the fact that 5-lipoxygenase is a "suicide" enzyme in that it inactivates itself upon reacting with substrate.
• 5-lipoxygenase is a "suicide" enzyme in that it inactivates itself upon reacting with substrate.
• Treatment of differentiated HL- 60 or other cells expressing 5 lipoxygenase, with 10 ⁇ M A23187, a calcium ionophore promotes translocation of 5- lipoxygenase from the cytosol to the membrane with subsequent activation of the enzyme. Following activation and several rounds of catalysis, the enzyme becomes catalytically inactive.
• treatment of the cells with calcium ionophore inactivates endogenous 5-lipoxygenase.
• Oligonucleotide surrogates directed against 5-lipoxygenase can be tested for activity in two HL-60 model systems using the following quantitative assays. The assays are described from the most direct measurement of inhibition of 5-lipoxygenase protein synthesis in intact cells to more downstream events such as measurement of 5-lipoxygenase activity in intact cells.
• oligonucleotide surrogates can exert on intact cells and which can be easily be quantitated is specific inhibition of 5-lipoxygenase protein synthesis.
• cells can be labelled with 35 S-methionine (50 ⁇ Ci/mL) for 2 hours at 37° C to label newly synthesized protein.
• Cells are extracted to solubilize total cellular proteins and 5- lipoxygenase is immunoprecipitated with 5-lipoxygenase antibody followed by elution from protein A Sepharose beads.
• the immunoprecipitated proteins are resolved by SDS-polyacrylamide gel electrophoresis and exposed for autoradiography. The amount of immunoprecipitated 5- lipoxygenase is quantitated by scanning densitometry.
• a predicted result from these experiments would be as follows.
• the amount of 5-lipoxygenase protein immunoprecipitated from control cells would be normalized to 100%.
• Treatment of the cells with 1 ⁇ M, 10 ⁇ M, and 30 ⁇ M of effective oligonucleotide surrogates for 48 hours would reduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control, respectively.
• Measurement of 5-lipoxygenase enzyme activity in cellular homogenates could also be used to quantitate the amount of enzyme present which is capable of synthesizing leukotrienes.
• a radiometric assay has now been developed for quantitating 5-lipoxygenase enzyme activity in cell homogenates using reverse phase HPLC. Cells are broken by sonication in a buffer containing protease inhibitors and EDTA. The cell homogenate is centrifuged at 10,000 x g for 30 min and the supernatants analyzed for 5- lipoxygenase activity.
• Cytosolic proteins are incubated with 10 ⁇ M 14 C-arachidonic acid, 2mM ATP, 50 ⁇ M free calcium, 100 ⁇ g/ml phosphatidylcholine, and 50 mM bis- Tris buffer, pH 7.0, for 5 min at 37° C.
• the reactions are quenched by the addition of an equal volume of acetone and the fatty acids extracted with ethyl acetate.
• the substrate and reaction products are separated by reverse phase HPLC on a Novapak C18 column (Waters Inc., Millford, MA). Radioactive peaks are detected by a Beckman model 171 radiochromatography detector. The amount of arachidonic acid converted into di-HETE's and mono-HETE's is used as a measure of 5-lipoxygenase activity.
• a predicted result for treatment of DMSO differentiated HL-60 cells for 72 hours with effective oligonucleotide surrogates at 1 ⁇ M, 10 ⁇ M, and 30 ⁇ M would be as follows. Control cells oxidize 200 pmol arachidonic acid/ 5 min/ 10 6 cells. Cells treated with 1 ⁇ M, 10 ⁇ M, and 30 ⁇ M of an effective oligonucleotide surrogates would oxidize 195 pmol, 140 pmol, and 60 pmol of arachidonic acid/ 5 min/ 10 6 cells respectively.
• ELISA quantitative competitive enzyme linked immunosorbant assay
• Cell extracts (0.2% Triton X-100, 12,000 x g for 30 min.) or purified 5-lipoxygenase were incubated with a 1:4000 dilutxon of 5-lipoxygenase polyclonal antibody in a total volume of 100 ⁇ L in the microtiter wells for 90 min.
• the antibodies are prepared by immunizing rabbits with purified human recombinant 5-lipoxygenase.
• the wells are washed with TBS containing 0.05% tween 20 (TBST), then incubated with 100 ⁇ L of a 1:1000 dilution of peroxidase conjugated goat anti-rabbit IgG (Cappel Laboratories, Malvern, PA) for 60 min at 25° C
• the wells are washed with TBST and the amount of peroxidase labelled second antibody determined by development with tetramethylbenzidine.
• Predicted results from such an assay using a 30 mer oligonucleotide analog at 1 ⁇ M, 10 ⁇ M, and 30 ⁇ M would be 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10 6 cells, respectively with untreated cells containing about 34 ng 5-lipoxygenase.
• a net effect of inhibition of 5-lipoxygenase biosynthesis is a diminution in the quantities of leukotrienes released from stimulated cells.
• DMSO-differentiated HL- 60 cells release leukotriene B4 upon stimulation with the calcium ionophore A23187.
• Leukotriene B4 released into the cell medium can be quantitated by radioimmunoassay using commercially available diagnostic kits (New England Nuclear, Boston, MA).
• Leukotriene B4 production can be detected in HL-60 cells 43 hours following addition of DMSO to differentiate the cells into a neutrophil-like cell.
• Cells (2 x 10 5 cells/mL) will be treated with increasing concentrations of oligonucleotide surrogates for 48-72 hours in the presence of 1.3 % DMSO.
• the cells are washed and resuspended at a concentration of 2 x 10 6 cell/mL in Dulbecco's phosphate buffered saline containing 1% delipidated bovine serum albumin.
• Cells are stimulated with 10 ⁇ M calcium ionophore A23187 for 15 min and the quantity of LTB4 produced from 5 x 10 5 cell determined by radioimmunoassay as described by the manufacturer.
• Inhibition of the production of 5-lipoxygenase in the mouse can be demonstrated in accordance with the following protocol.
• Topical application of arachidonic acid results in the rapid production of leukotriene B 4 , leukotriene C 4 and prostaglandin E 2 in the skin followed by edema and cellular infiltration.
• Certain inhibitors of 5-lipoxygenase have been known to exhibit activity in this assay.
• 2 mg of arachidonic acid is applied to a mouse ear with the contralateral ear serving as a control.
• the polymorphonuclear cell infiltrate is assayed by myeloperoxidase activity in homogenates taken from a biopsy 1 hour following the administration of arachidonic acid.
• the edematous response is quantitated by measurement of ear thickness and wet weight of a punch biopsy. Measurement of leukotriene B 4 produced in biopsy specimens is performed as a direct measurement of 5-lipoxygenase activity in the tissue. Oligonucleotide surrogates will be applied topically to both ears 12 to 24 hours prior to administration of arachidonic acid to allow optimal activity of the compounds. Both ears are pretreated for 24 hours with either 0.1 ⁇ mol, 0.3 ⁇ mol, or 1.0 ⁇ mol of the oligonucleotide analog prior to challenge with arachidonic acid. Values are expressed as the mean for three animals per concentration.
• Inhibition of polymorphonuclear cell infiltration for 0.1 ⁇ mol, 0.3 ⁇ mol, and 1 ⁇ mol is expected to be about 10 %, 75 % and 92 % of control activity, respectively.
• Inhibition of edema is expected to be about 3 %, 58% and 90%, respectively while inhibition of leukotriene B 4 production would be expected to be about 15 %, 79% and 99%, respectively.
• heterocyclic base substituted cyclobutane compound of the invention were tested as to their antiviral activity. Both 1-adenyl-3,3-bis-hydroxymethylcyclobutane and 1-thymindyl-3,3-bis-hydroxymethyl-cyclobutane were tested against HSV-1 in human macrophages. Both of these compounds were tested up to 600 ⁇ g/ml without toxicity. In these tests 1-thymindyl-3,3-bis-hydroxymethyl-cyclobutane exhibited a MED 50 of 40 - 65 ⁇ g/ml and 1-adenyl-3,3-bis-hydroxymethyl-cyclobutane exhibited a MED 50 of 200 ⁇ g/ml. Both of these compounds showed no activity in a cellular HIV assay.

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