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WO2023097251A1 - Oligonucleotide-containing transcription factor targeting chimeras - Google Patents

Oligonucleotide-containing transcription factor targeting chimeras Download PDF

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Publication number
WO2023097251A1
WO2023097251A1 PCT/US2022/080390 US2022080390W WO2023097251A1 WO 2023097251 A1 WO2023097251 A1 WO 2023097251A1 US 2022080390 W US2022080390 W US 2022080390W WO 2023097251 A1 WO2023097251 A1 WO 2023097251A1
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compound
occurrence
group
independently
ona
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PCT/US2022/080390
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French (fr)
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Craig Crews
Kusal SAMARASINGHE
Ling Chu
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Yale University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)

Definitions

  • PROTACs are small molecule-based heterobifunctional molecules that recruit an E3 ligase complex to a protein of interest. By doing so, PROTACs induce the ubiquitination and degradation of the protein of interest via the proteasome. Although PROTACs have the potential to induce the degradation of numerous proteins, the identification of small molecule recruiting ligands for several classes of proteins is still challenging and time-consuming. Therefore, the development of alternative, proximity- inducing strategies would help to target disease-causing proteins, such as transcription factors.
  • TFs Transcription factors
  • Many diseases such as neurological disorders, autoimmunity, developmental syndromes, and many cancers result from abnormalities in the TF-controlled gene-regulatory circuitry within the diseased cell.
  • the TF c-Myc the most frequently amplified oncogene, has been extensively studied and established as a direct mediator of tumorigenesis in numerous cancers.
  • indirect approaches such as bromodomain protein inhibitors and PROTACs have been explored to control c-Myc levels, direct inhibition or degradation of c-Myc has been an as yet unrealized goal.
  • T-box transcription factor In contrast to c-Myc, T-box transcription factor (“brachyury”) is expressed only in a restricted set of cancer types and minimally expressed in normal cells.
  • Brachyury is a master developmental TF that plays a pivotal role during early embryonic development in vertebrates. Brachyury expression is limited only to embryonic developmental stages and, in general, expression levels are highly downregulated in adult tissues. In addition to its regulatory functions during development, brachyury expression in remnant notochord cells in adults has been shown to be a key oncogenic driver in the rare bone cancer, chordoma. Chromosomal aberrations such as chromosome 6 gains and partial polysomy have been identified as potential molecular mechanisms in brachyury-dependent chordoma tumors.
  • TFs such as NF-kB, STAT3/5, the androgen receptor (AR) and the estrogen receptor (ER) are other known oncogenic drivers that also rewire transcriptional circuitry in various cancer types.
  • AR androgen receptor
  • ER estrogen receptor
  • ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages
  • LNK is a chemical linker covalently bonding the ONA and the UBL
  • UBL is an E3 ubiquitin ligase ligand.
  • a compound of formula II, or a salt, enantiomer, diastereomer, or tautomer thereof is provided: wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is a chemical linker covalently bonding the ONA and the C ⁇ C ⁇ H.
  • Compounds of formula I are useful in preventing, treating, and/or ameliorating cancer in subjects, including human subjects.
  • a method of preventing, treating, and/or ameliorating cancer in a subject is provided. The method includes administering to the subject a therapeutically effective amount of at least one compound of formula I, which is optionally formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
  • FIG.1 is a schematic representation of oligoTRAFTAC-mediated transcription factor (TF) and E3 ligase recruitment, and proximity-dependent TF ubiquitination, in accordance with various embodiments.
  • FIGs.2A-2E illustrate show activity and aspects of oligoTRAFTACs, according to various embodiments. OligoTRAFTAC induces c-Myc degradation. (FIG.2A) The oligonucleotide selected for the c-Myc oligoTRAFTAC engages c-Myc.
  • FIG.2C OligoTRAFTAC-induced c-Myc degradation occurred via the proteasomal pathway.
  • HEK293T cells were treated with c-Myc-targeting oligoTRAFTAC with and without the neddylation inhibitor, MLN-4924 (1 mM), and then analyzed for c-Myc levels.
  • FIG.2D HEK293 cells were preincubated with and without 10 mM VHL ligand followed by OT7 transfection and analyzed for c-Myc levels.
  • FIG.2E Chemical structure of a c-Myc- targeting oligoTRAFTAC (OT7), according to various embodiments.
  • FIGs.3A-3E show brachyury-GFP degradation by oligoTRAFTACs.
  • FIG.3A Brachyury-targeting oligonucleotide used in the oligoTRAFTAC design engaged with brachyury-GFP.
  • BRCH biotinylated oligonucleotide
  • SCRM scrambled oligonucleotide
  • FIG.3D Washout experiment after 12 h of OT3 transfection. Cells were incubated continuously for 24 h in transfection medium or OT3 was aspirated after 12 h of transfection and fresh medium added to cells. Washout cells incubated for another 12 h and 24 h prior to harvesting.
  • FIGs.4A-4D show oligoTRAFTACs induce brachyury-GFP degradation via the proteasomal pathway.
  • FIG.4B OT3 induced brachyury degradation is VHL- dependent.
  • HEK293T cells were preincubated with and without 10 mM of VHL ligand for 1.5 h prior to OT3 transfection. After 20 h of transfection, cells lysates were prepared and analyzed for brachyury degradation.
  • FIGs.5A-5F show endogenous brachyury degradation by oligoTRAFTACs constructed with phosphorothioate backbone.
  • FIG.5C UM-Chor1 cells were transfected with 60 nM of OT17 and harvested at subsequent different time points as indicated.
  • FIG.5F OT3- and OT17-induced brachyury ubiquitination.
  • FIGs.6A-6D show microinjection of brachyury-targeting oligoTRAFTAC into zebrafish embryos: demonstration of in vivo activity. (FIG.
  • FIG.6A Schematic representation of OT17 and OT20 microinjection into zebrafish embryos.
  • FIGs.7A-7C show the oligonucleotide synthesis and click reaction. Oligonucleotide synthesis and click reaction.
  • FIG.7A Oligonucleotides were custom synthesized with a terminal alkyne either at 3' or 5' end of the oligo. Oligonucleotide sequences for both 3' and 5' alkyne targeting c-Myc (left panel) and brachyury (right panel).
  • FIG.7B Chemical structure of azido-VHL ligand and click reaction conditions.
  • FIG.7C Chemical structures of 3' modified oligonucleotides (OT17- with a phosphorothioate backbone) after the click reaction with azido-VHL ligand.
  • FIGs.8A-8G show oligoTRAFTAC mediated c-Myc degradation.
  • FIG.8B Varying concentrations of OT7 were transfected into HEK293T cells and lysed after 20 h. Cell lysates were separated and transferred to a PVDF membrane followed by immunoblotting with antibodies against c-Myc and GAPDH.
  • FIG.8F Degradation of E-box binding transcription factor, TCF3.
  • OT7 transfected cells were analyzed for c-Myc and TCF3.
  • FIGs.9A-9B show EMSA and brachyury-GFP degradation data.
  • FIG.9A shows click reaction mixtures with or without VHL ligand were loaded on to a 1.2% agarose gel and separated over 1 h at constant 120 mV.
  • FIG.9B shows that OT1 through 4 were transfected into HEK293T cells overexpressing brachyury-GFP and lysed after 30 h.
  • FIGs.10A-10B show time course and washout experiments for brachyury targeting OTs.
  • FIG.10A shows OT2 through 4 were transfected at 75 nM into HEK293T cells overexpressing brachyury-GFP and lysed after 12 h, 24 h, and 36 h. Lysates were probed for brachyury and GAPDH.
  • FIG.10B shows OT3 was transfected into HEK293T cells and washed out after 6 h and 12 h. Cell lysates were probed with antibodies against brachyury and GAPDH.
  • FIGs.11A-11C show brachyury degradation by oligoTRAFTACs in HEK293T and UM-Chor1 cells.
  • FIG.11C Proteasome dependent brachyury degradation in UM-Chor1 cells.
  • DETAILED DESCRIPTION OF THE INVENTION Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.
  • values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a range of "about 0.1% to about 5%” or "about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • substantially refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.
  • substantially free of can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less.
  • substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.
  • organic group refers to any carbon-containing functional group.
  • Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups.
  • an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group
  • a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester such as an alkyl and aryl sulfide group
  • sulfur-containing group such as an alkyl and aryl sulfide group
  • Non-limiting examples of organic groups include OR, OOR, OC(O)N(R) 2 , CN, CF 3 , OCF 3 , R, C(O), methylenedioxy, ethylenedioxy, N(R) 2 , SR, SOR, SO 2 R, SO 2 N(R) 2 , SO 3 R, C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R) 2 , OC(O)N(R) 2 , C(S)N(R) 2 , (CH 2 )0- 2 N(R)C(O)R, (CH 2 ) 0-2 N(R)N(R) 2 , N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R) 2 , N(R)SO 2 R,
  • substituted refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms.
  • the substitution can be direct substitution, whereby the hydrogen atom is replaced by a functional group or substituent, or an indirect substitution, whereby an intervening linker group replaces the hydrogen atom, and the substituent or functional group is bonded to the intervening linker group.
  • direct substitution is: RR-H ⁇ RR-Cl, wherein RR is an organic moiety/fragment/molecule.
  • a non-limiting example of indirect substitution is: RR-H ⁇ RR- (LL) zz -Cl, wherein RR is an organic moiety/fragment/molecule, LL is an intervening linker group, and 'zz' is an integer from 0 to 100 inclusive. When zz is 0, LL is absent, and direct substitution results.
  • (LL) zz can be lin -ear, branched, cyclic, acyclic, and combinations thereof. In certain embodiments, two consecutive LL's cannot be both O.
  • a halogen e.g., F, Cl, Br, and I
  • an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters
  • a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups
  • a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other hetero
  • Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R) 2 , CN, NO, NO 2 , ONO 2 , azido, CF 3 , OCF 3 , R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R) 2 , SR, SOR, SO 2 R, SO 2 N(R) 2 , SO 3 R, C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R) 2 , OC(O)N(R) 2 , C(S)N(R) 2 , (CH 2 ) 0-2 N(R)C(O)R, (CH 2 )N(R)N(R) 2
  • alkyl refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
  • Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • alkenyl refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms.
  • alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.
  • alkynyl refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms.
  • alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ⁇ CH, -C ⁇ C(CH 3 ), -C ⁇ C(CH 2 CH 3 ), -CH 2 C ⁇ CH, -CH 2 C ⁇ C(CH 3 ), and -CH 2 C ⁇ C(CH 2 CH 3 ) among others.
  • acyl refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom.
  • the carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like.
  • An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group.
  • An acyl group can include double or triple bonds within the meaning herein.
  • An acryloyl group is an example of an acyl group.
  • An acyl group can also include heteroatoms within the meaning herein.
  • a nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein.
  • Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like.
  • the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen
  • the group is termed a "haloacyl” group.
  • An example is a trifluoroacetyl group.
  • cycloalkyl refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.
  • the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7.
  • Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein.
  • Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • cycloalkenyl alone or in combination denotes a cyclic alkenyl group.
  • heterocycloalkyl refers to a cycloalkyl group as defined herein in which one or more carbon atoms in the ring are replaced by a heteroatom such as O, N, S, P, and the like, each of which may be substituted as described herein if an open valence is present, and each may be in any suitable stable oxidation state.
  • aryl refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups.
  • aryl groups contain about 6 to about 14 carbons in the ring portions of the groups.
  • Aryl groups can be unsubstituted or substituted, as defined herein.
  • substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.
  • aralkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.
  • Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • heterocyclyl refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S.
  • a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof.
  • heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members.
  • a heterocyclyl group designated as a C 2 -heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • the number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms.
  • a heterocyclyl ring can also include one or more double bonds.
  • a heteroaryl ring is an embodiment of a heterocyclyl group.
  • the phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups.
  • a dioxolanyl ring and a benzdioxolanyl ring system are both heterocyclyl groups within the meaning herein.
  • the phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein.
  • Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquino
  • heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.
  • heteroaryl refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members.
  • a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure.
  • a heteroaryl group designated as a C 2 -heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • the number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
  • a heterocyclyl ring designated C x-y can be any ring containing 'x' members up to 'y' members, including all intermediate integers between 'x' and 'y' and that contains one or more heteroatoms, as defined herein.
  • Heterocyclyl rings designated C x-y can also be polycyclic ring systems, such as bicyclic or tricyclic ring systems.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl,
  • Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein. Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydry
  • heterocyclylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
  • Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.
  • heteroarylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like.
  • Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like.
  • Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms.
  • an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.
  • amine refers to primary, secondary, and tertiary amines having, e.g., the formula N(group) 3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like.
  • Amines include but are not limited to R-NH 2 , for example, alkylamines, arylamines, alkylarylamines; R 2 NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R 3 N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like.
  • amine also includes ammonium ions as used herein.
  • amino group refers to a substituent of the form -NH 2 , - NHR, -NR 2 , -NR 3 + , wherein each R is independently selected, and protonated forms of each, except for -NR 3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
  • An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
  • alkylamino includes a monoalkylamino, dialkylamino, and trialkylamino group.
  • halo means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro.
  • haloalkyl examples include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.
  • epoxy-functional or "epoxy-substituted” as used herein refers to a functional group in which an oxygen atom, the epoxy substituent, is directly attached to two adjacent carbon atoms of a carbon chain or ring system.
  • epoxy-substituted functional groups include, but are not limited to, 2,3-epoxypropyl, 3,4-epoxybutyl, 4,5- epoxypentyl, 2,3-epoxypropoxy, epoxypropoxypropyl, 2-glycidoxyethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2-(glycidoxycarbonyl)propyl, 3-(3,4-epoxycylohexyl)propyl, 2-(3,4- epoxycyclohexyl)ethyl, 2-(2,3-epoxycylopentyl)ethyl, 2-(4-methyl-3,4- epoxycyclohexyl)propyl, 2-(3,4-epoxy-3-methylcylohexyl)-2-methylethyl, and 5,6- epoxyhexyl.
  • the term "monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.
  • the term "hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.
  • hydrocarbyl refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- C b )hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms.
  • (C1-C4)hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C 2 ), propyl (C 3 ), or butyl (C 4 ), and (C 0 -C b )hydrocarbyl means in certain embodiments there is no hydrocarbyl group.
  • the term "C 6-10 -5-6 membered heterobiaryl” means a C 6-10 aryl moiety covalently bonded through a single bond to a 5- or 6-membered heteroaryl moiety.
  • the C 6-10 aryl moiety and the 5-6-membered heteroaryl moiety can be any of the suitable aryl and heteroaryl groups described herein.
  • Non-limiting examples of a C 6-10 -5-6 membered heterobiaryl include When the C 6-10- 5-6 membered heterobiaryl is listed as a substituent (e.g., as an "R" group), the C 6-10- 5-6 membered heterobiaryl is bonded to the rest of the molecule through the C 6-10 moiety.
  • the term "5-6 membered- C 6-10 heterobiaryl" is the same as a C 6-10 -5- 6 membered heterobiaryl, except that when the 5-6 membered- C 6-10 heterobiaryl is listed as a substituent (e.g., as an "R” group), the 5-6 membered- C 6-10 heterobiaryl is bonded to the rest of the molecule through the 5-6-membered heteroaryl moiety.
  • the term "C 6-10 - C 6-10 biaryl” means a C 6-10 aryl moiety covalently bonded through a single bond to another C 6-10 aryl moiety.
  • the C 6-10 aryl moiety can be any of the suitable aryl groups described herein.
  • Non-limiting example of a C 6-10 - C 6-10 biaryl include biphenyl and binaphthyl.
  • solvent refers to a liquid that can dissolve a solid, liquid, or gas.
  • Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.
  • independently selected from refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise.
  • X 1 , X 2 , and X 3 are independently selected from noble gases” would include the scenario where, for example, X 1 , X 2 , and X 3 are all the same, where X 1 , X 2 , and X 3 are all different, where X 1 and X 2 are the same but X 3 is different, and other analogous permutations.
  • room temperature refers to a temperature of about 15 °C to 28 °C.
  • standard temperature and pressure refers to 20 °C and 101 kPa.
  • composition refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • the terms "effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • the term “efficacy” refers to the maximal effect (Emax) achieved within an assay.
  • pharmaceutically acceptable refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • pharmaceutically acceptable salt refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic,
  • Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • the term "pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injuri
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein.
  • Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • potency refers to the dose needed to produce half the maximal response (ED 50 ).
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • treatment is defined as the application or administration of a therapeutic agent, i.e., a compound or compounds as described herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, or the symptoms of a condition contemplated herein.
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • the following abbreviations for the commonly occurring nucleic acid bases are used.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • oligonucleotide refers to short polynucleotides, such as from two (2) to 50 nucleotides.
  • RNA sequence i.e., A, U, C, G
  • U replaces "T”
  • 3'-end refers to the terminal residue of a DNA or RNA strand that terminates at the hydroxyl group of the 3' carbon in a deoxyribose (DNA) or ribose (RNA).
  • the term "5'-end” refers to the terminal residue of a DNA or RNA strand that terminates at a hydroxyl group of the 5' carbon in a deoxyribose (DNA) or ribose (RNA).
  • a phosphate or phosphorothioate group can be attached to the hydroxyl group on the 5'-carbon.
  • the term "DNA” means deoxyribonucleic acid.
  • RNA means ribonucleic acid.
  • a compound of formula I or a salt, enantiomer, diastereomer, or tautomer thereof: wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonded to the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand.
  • ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages
  • LNK is a chemical linker covalently bonded to the ONA and the UBL
  • UBL is an E3 ubiquitin ligase ligand.
  • Oligonucleotide Suitable salts include any of the pharmaceutically acceptable salts described herein.
  • the ONA is bonded to the LNK at the 3'-end or the 5'-end.
  • the ONA is bonded to the LNK at the 3'-end.
  • the ONA is bonded to the LNK at the 5' end.
  • the ONA comprises deoxyribonucleotide(s) (DNA), ribonucleotide(s) (RNA), or any combinations thereof.
  • the ONA comprises deoxyribonucleotide(s) (DNA).
  • the ONA comprises a c-Myc-binding nucleotide sequence.
  • At least two nucleotides flank each end of the c-Myc-binding nucleotide sequence. In various embodiments, 2 to 20 nucleotides flank each end of the c- Myc-binding nucleotide sequence. The number of flanking nucleotides at each end of the c- Myc-binding nucleotide sequence can be the same or different, and the sequences can be the same, different, reverse sequences, complementary sequences, or reverse complementary sequences. In various embodiments, the c-Myc-binding nucleotide sequence is flanked on each side by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.
  • the c-Myc-binding nucleotide sequence comprises a sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% similar to 5'- CACGTGGTTGCCACGTG-3' (SEQ ID NO: 1). In various embodiments, the c-Myc-binding nucleotide sequence comprises the sequence 5'- CACGTGGTTGCCACGTG-3' (SEQ ID NO: 1)..
  • the c-Myc binding nucleotide sequence is 5'- TGGGAGCACGTGGTTGCCACGTGGTTGGG-3' (SEQ ID NO: 2) or 3'- GGGTTGGTGCACCGTTGGTGCACGAGGGT-5' (SEQ ID NO: 3).
  • the ONA comprises a brachyury-binding nucleotide sequence. In various embodiments, at least two nucleotides flank each end of the brachyury- binding nucleotide sequence. In various embodiments, 2 to 20 nucleotides flank each end of the brachyury-binding nucleotide sequence.
  • flanking nucleotides at each end of the brachyury-binding nucleotide sequence can be the same or different, and the sequences can be the same, different, reverse sequences, complementary sequences, or reverse complementary sequences.
  • the brachyury-binding nucleotide sequence is flanked on each side by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.
  • the brachyury-binding nucleotide sequence comprises a sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% similar to 5'- AATTTCACACCTAGGTGTGAAATT-3' (SEQ ID NO: 4). In various embodiments, the brachyury-binding nucleotide sequence comprises the sequence 5'- AATTTCACACCTAGGTGTGAAATT-3' (SEQ ID NO: 4).
  • the brachyury-binding nucleotide sequence is 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3' (SEQ ID NO: 5) or 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5' (SEQ ID NO: 6).
  • at least one of the phosphodiester groups in the internucleotide linkages comprising the ONA is replaced with a non-bridging phosphorothioate (PS) group, in which the sulfur atom in the PS group does not connect to a ribose or deoxyribose.
  • PS non-bridging phosphorothioate
  • phosphorothioate group has the structure: and can be present as a salt thereof. Suitable salts include any of the pharmaceutically acceptable salts described herein. In various embodiments, the salt is an sodium or potassium salt.
  • the terminal nucleotide in ONA at the 3'-end has the structure: wherein: Y is H or OH; and In various embodiments, the terminal nucleotide in ONA at the 5'-end has the structure: wherein: Y is H or OH; Z is O or S; and , , , , , The in the terminal nucleotide sugar, whether at the 3'-end or the 5'-end, designates the point of attachment of the rest of the nucleotides in ONA. The in NUC designates the point of attachment of the respective base to the ribose or deoxyribose.
  • A is phenylene or a C 5-18 heterocyclylene;
  • R is independently at each occurrence H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 6-10 aryl, C 5-10 heteroaryl, halogen, or combinations thereof;
  • R' is independently at each occurrence H, C 1-12
  • a given quantity of LL units can be used to form any suitable stable chemical structure, including branched, cyclic, and acyclic structures.
  • A is a heterocyclylene that contains one or more aliphatic carbons.
  • A is heteroarylene.
  • A is arylene.
  • suitable structures for –(LL) aa –A–(LL) bb — include:
  • suitable structures for –(LL) aa –A–(LL) bb — include:
  • A is a C5-18 heteroaryl, such as but not limited to a 1,2,3- triazolyl or a 1,2,4-triazolyl.
  • LNK-X1 any two X groups bonded to each other cannot be -O- such that (X) m does not contain a peroxide functionality.
  • X in LNK-X1 is independently at each occurrence -O- or -CH 2 -.
  • Contemplated herein are all combinations of m, X, and pp in LNK-X1.
  • LNK-X2 any two X groups bonded to each other cannot be -O- such that (X) m does not contain a peroxide functionality.
  • X in LNK-X2 is independently at each occurrence -O- or -CH 2 -.
  • Contemplated herein are all combinations of m, X, and pp in LNK-X2.
  • LNK has the structure: wherein pp is an integer from 1 to 10, X is CH 2 or O, k is an integer from 1 to 3; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • X is CH 2 .
  • LNK-1 in LNK-1, X is O. In various embodiments, in LNK-1, k is 1. In various embodiments, in LNK-1, k is 2. In various embodiments, in LNK-1, k is 3. Contemplated herein are all combinations of k, X, and pp in LNK-1. In various embodiments, LNK has the structure: wherein pp is an integer from 1 to 10, X is CH 2 or O, k is an integer from 1 to 3; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. In various embodiments, in LNK-2, X is CH 2 . In various embodiments, in LNK-2, X is O.
  • k is 1. In various embodiments, in LNK-2, k is 2. In various embodiments, in LNK-2, k is 3. Contemplated herein are all combinations of k, X, and pp in LNK-2.
  • aa is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
  • bb is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
  • zz is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
  • LNK-X1, LNK-X2, LNK-1, or LNK-2 When LNK-X1, LNK-X2, LNK-1, or LNK-2 is bonded the 3'-end, the O* atom connects directly to the ribose or deoxyribose 3' carbon.
  • LNK-X1, LNK-X2, LNK-1, or LNK-2 is bonded the 5'-end, the O* atom connects directly to the phosphorus atom in the 5'-phosphate in the ribose or deoxyribose.
  • pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 1. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 2.
  • pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 3. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 4. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 5. In various embodiments, in LNK-X1, LNK- X2, LNK-1, or LNK-2 pp is 6. In various embodiments, in LNK-X1, LNK-X2, LNK-1, or LNK-2 pp is 7. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 8.
  • pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 9. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 10. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 2, 3, or 5.
  • LNK comprises a group represented by a general structure selected from the group consisting of: -NR(CH 2 ) n -(lower alkyl)-, -NR(CH 2 ) n -(lower alkoxyl)- , -NR(CH 2 ) n -(lower alkoxyl)-OCH 2 -, -NR(CH 2 ) n -(lower alkoxyl)-(lower alkyl)-OCH 2 -, - NR(CH 2 ) n -(cycloalkyl)-(lower alkyl)-OCH 2 -, -NR(CH 2 ) n -(heterocycloalkyl)-, - NR(CH 2 CH 2 O) n -(lower alkyl)-O-CH 2 -, -NR(CH 2 CH 2 O) n -(heterocycloalkyl)-O-CH 2 -, -NR(CH 2 CH
  • LNK comprises a group represented by a general structure selected from the group consisting of: -N(R)-(CH 2 ) m -O(CH 2 ) n -O(CH 2 ) o -O(CH 2 ) p -O(CH 2 ) q -O(CH 2 ) r -OCH 2 -, -O-(CH 2 ) m -O(CH 2 ) n -O(CH 2 )o-O(CH 2 ) p -O(CH 2 ) q -O(CH 2 )r-OCH 2 -, -O-(CH 2 ) m -O(CH 2 ) n -O(CH 2 ) o -O(CH 2 ) p -O(CH 2 ) q -O(CH 2 )r-OCH 2 -, -O-(CH 2 ) m -O(CH 2 ) n -O(CH 2 )
  • the linker (L) is selected from the group consisting of:
  • LNK is selected from the group consisting of:
  • LNK is selected from the group consisting of:
  • the UBL is a ligand of an E3 ubiquitin ligase selected from the group consisting of von Hippel-Lindau (VHL), cereblon (CRBN), RING-type zinc-finger protein 114 (RNF114), cellular inhibitor of apoptosis (cIAP), mouse double minute 2 homologue (MDM2), damage-specific DNA binding protein 1 (DDB1)-CUL4 associated factor 16 (DCAF16), and Kelch-like ECH-associated protein 1 (KEAP1).
  • VHL von Hippel-Lindau
  • CRBN cereblon
  • RRF114 RING-type zinc-finger protein 114
  • cIAP cellular inhibitor of apoptosis
  • MDM2 mouse double minute 2 homologue
  • DDB1-CUL4 associated factor 16 DCAF16
  • KEAP1 Kelch-like ECH-associated protein 1
  • UBL is a ligand of VHL or CRBN.
  • any suitable E3 ubiquitin ligase recruiting ligand can be incorporated into the compounds of formula I.
  • suitable E3 ubiquitin ligase ligands and their syntheses can be found in Bricelj, A. et al., Front. Chem., 05 July 2021, DOI: 10.3389/fchem.2021.707317.
  • the UBL is a ligand having the structure: wherein R 1 is H or CH 3 ; and R 2 is selected from the group consisting of In the CRBN ligands, the attachment point shown in structures such as can be through any suitable atom/fragment as defined in LL herein.
  • the CRBN ligands can be attached via any of the following connectivities:
  • the UBL has the structure:
  • the compound of formula I has the structure of:
  • pp is an integer from 1 to 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • Modified Oligonucleotides a compound of formula II is provided: ONA–LNK–C ⁇ C-H (formula II), wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is a chemical linker connecting the ONA and the C ⁇ C-H. In various embodiments, LNK has any linker structure described elsewhere herein.
  • A is phenylene or a C5-18 heterocyclylene;
  • R is independently at each occurrence H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 6-10 aryl, C 5-10 heteroaryl, halogen, or combinations thereof;
  • R' is independently at each occurrence H,
  • Y in II-A is independently at each occurrence -O- or -CH 2 -; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • d in II-A is 2. In various embodiments, d in II-A is 3. In various embodiments, d in II-A is 4. In various embodiments, d in II-A is 5. In various embodiments, d in II-A is 6. In various embodiments, d in II-A is 7. In various embodiments, d in II-A is 8. In various embodiments, d in II-A is 9. In various embodiments, d in II-A is 10. In various embodiments, d in II-A is 11.
  • d in II-A is 12. In various embodiments, d in II-A is 13. In various embodiments, d in II-A is 14. In various embodiments, d in II-A is 15. In various embodiments, the compound of formula II has the structure: wherein pp is an integer from 1 to 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Degradation of c-Myc by oligoTRAFTACs Myc transcription factors are dysregulated in a range of cancers. Even though c-Myc has been targeted by indirect approaches, development of direct-targeting methods is hindered by the paucity of ligandable pockets and their highly disordered structure.
  • c- Myc binds to specific DNA sequences with a conserved E-box sequence (CACGTG)
  • CACGTG conserved E-box sequence
  • OT oligoTRAFTAC
  • a Myc binding consensus sequence (5' CACGTGGTTGCCACGTG 3') was taken from one of its target gene promoters. Additionally, a flanking sequence was included at both 3' and 5' end of the c-Myc- targeting oligonucleotide sequence to facilitate successful double strand formation of the recognition sequence while providing a flexibility for oligoTRAFTACs.
  • a c-Myc-targeting oligonucleotide sequence was synthesized with an orthogonal alkyne handle on either side as a reactive moiety to append an azide-containing VHL ligand (FIG.7A). Copper-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction was performed to synthesize two c-Myc targeting oligoTRAFTACs (OT7: VHL ligand at the 5' end and OT10: VHL ligand at the 3' end of the oligonucleotide) (FIG.7B, 7C). After 18 h at room temperature, the crude mixture was purified and analyzed by HPLC to verify reaction completion (FIG.8A).
  • CuAAC Copper-catalyzed alkyne-azide cycloaddition
  • the reaction was also monitored by electrophoretic mobility shift assay (EMSA) (FIG.8B).
  • ESA electrophoretic mobility shift assay
  • the single stranded oligoTRAFTACs were then purified by HPLC, dried and reconstituted in water.
  • an annealing reaction was performed in the presence of the reverse complementary oligonucleotide by heating to 95 0 C and slowly cooling to room temperature over a 1.5 h period.
  • a biotinylated version of the same c-Myc-targeting oligonucleotide was also synthesized to generate a double stranded oligonucleotide via the same annealing reaction conditions.
  • OT7 and OT10 induces c-Myc degradation via the proteasomal pathway.
  • the cell viability in response to the transfection of OT7 and OT12 was assessed.
  • the induction of OT7-mediated c-Myc degradation inhibited cell proliferation compared to scrambled control-transfected cells (FIG.8E).
  • OligoTRAFTAC-mediated brachyury degradation T-box transcription factor (brachyury) is a DNA-interacting TF that regulates gene expression during early embryonic development in vertebrates.
  • Brachyury is crucial for early mesoderm formation and plays a key role in notochord development where, as a homodimer, it recognizes and binds to a consensus palindromic DNA sequence.
  • a brachyury-binding DNA sequence AATTTCACACCTAGGTGTGAAATT
  • Oligonucleotides were synthesized with flanking bases and terminal alkyne moieties at either end (3' and 5') of the oligonucleotide (FIG.7A, right panel).
  • oligoTRAFTACs To generate brachyury-targeting oligoTRAFTACs, two azido-VHL ligands were synthesized with a long (5 PEG units) and a short (2 PEG units) linker (FIG.7C, FIG.9A). To test whether the oligonucleotide used in oligoTRAFTAC design could recruit brachyury, a streptavidin pull-down experiment was performed using a biotin-oligonucleotide.
  • brachyury-oligoTRAFTACs were transfected into brachyury-GFP-expressing HEK293T cells for 24 h, after which cells were collected and lysed, followed by western blotting. OligoTRAFTACs with 5' VHL-ligand and a longer linker (OT1) did not induce significant degradation of brachyury-GFP (FIG.9B), while its counterpart the shorter linker (OT2) successfully induced target degradation.
  • oligoTRAFTACs with 3' VHL-ligand both showed comparable, better degradation profiles relative to OT1 and OT2 (FIG.9B, last two panels).
  • OT3 and OT4 were transfected into HEK293T cells and lysed after 20 h.
  • the time course experiment suggested that OT3 can induce significant brachyury-GFP degradation at 12 h that is maintained up to 36 h post-transfection (FIG.3C and FIG.10A).
  • brachyury degradation was prominent even after the cells incubated in fresh medium for longer time (36 h), which is consistent with oligoTRAFTAC OT7 targeting c-Myc-targeting (FIG.3D, last panel).
  • brachyury levels at 36 h had increased relative to the 24 h period, indicating a progressive loss of brachyury degradability by OT3. This could be partially explained by the reduced stability of oligoTRAFTACs due to the oligonucleotide sensitivity towards intracellular nucleases, increased deubiquitinase (DUB) activity, or increased c-Myc resynthesis.
  • DAB deubiquitinase
  • oligoTRAFTAC-mediated brachyury recruitment and degradation is oligonucleotide-dependent
  • OT5 and OT6 scrambled- oligoTRAFTACs
  • VHL competition rescued oligoTRAFTAC-mediated brachyury degradation (FIG.4B), confirming that observed brachyury degradation is a result of VHL E3 ligase recruitment by OT3.
  • OT3-mediated brachyury degradation was evaluated in the presence of a neddylation inhibitor to further confirm that the intended mechanism is via the proteasomal pathway.
  • the neddylation inhibitor, MLN-4924 was pre-incubated with cells at 1 mM concentration. After 1.5 h, OT3 was transfected into the cell for 20 h following which the cell lysates were analyzed as indicated in FIG.4C.
  • OT3 Consistent with the brachyury-GFP degradation in HEK293T cells, 60 nM of OT3 induces ⁇ 70% brachyury degradation in UM- Chor1 cells 24 h post-transfection (FIG.11B). Although OT3 induced comparable levels of brachyury degradation in both cell lines, phosphodiester linkages within OT3 are susceptible to cleavage by both extra- and intracellular nucleases. Therefore, oligoTRAFTACs with a phosphorothioate (PS) backbone were synthesized. The addition of extra non-bridging sulfur atoms in the inter-nucleotide phosphate group has been shown to have both increased stability against nucleases and improved cell permeability.
  • PS phosphorothioate
  • brachyury-targeting oligoTRAFTAC OT17
  • UM-Chor1 chordoma cells
  • FIG.5A A similar degradation pattern was noticed in another chordoma cell line, JHC-7 (FIG.5B), although OT17 was slightly less potent in them, requiring 30 nM to induce brachyury knockdown comparable to UM-Chor1 cells.
  • oligoTRAFTAC-mediated brachyury ubiquitination resulting from induced proximity to VHL HA-ubiquitin was transfected into HEK293 cells overexpressing brachyury-GFP. After cells pre-incubated with proteasome inhibitor epoxomicin (500 nM), OT3 and OT17 were transfected and incubated for 12 hours.
  • brachyury overexpression in adult tissue is one of the key factors that leads to tumorigenesis in chordoma
  • brachyury is widely known for its essential biological activity in vertebrate notochord formation at early stages of embryonic development.
  • tail deformation in OT3-injected zebrafish embryos was examined relative to mock-injected embryos (FIG.6A).
  • OT3 could induce brachyury degradation in cells, OT3 did not induce tail deformation in zebrafish, possibly due to the sensitivity of its phosphodiester backbone to nucleases.
  • Described herein are coupled TF-binding short DNA sequences from target gene promoters with VHL ligands to create bifunctional molecules for the targeted degradation of those same TFs. Since the binding sequences already have been identified for many TFs, and since synthetic routes for oligonucleotide synthesis are well established and economical, these oligonucleotide sequences can be rapidly employed in oligoTRAFTAC design to use as a versatile tool for both basic discovery biology and therapy development.
  • the TRAFTACs described herein were synthesized by directly attaching a VHL ligand to c-Myc or brachyury-binding oligonucleotide sequences via click chemistry.
  • OligoTRAFTACs targeting c-Myc TF displayed a robust degradation. Addition of VHL ligand to either side of the oligonucleotide did not significantly alter its ability to induce c- Myc degradation. This can be partially attributed to the flexibility of oligoTRAFTACs provided by the extra flanking nucleotides between the VHL ligand and TF-recruiting oligonucleotide. Other oligoTRAFTACs used in this study induced the degradation of ectopically expressed brachyury-GFP and endogenous brachyury via the proteasomal pathway.
  • oligoTRAFTACs are not as kinetically efficient as conventional PROTACs
  • the transient binding nature of oligoTRAFTACs to both TF and VHL proteins suggests the probability of a similar catalytic degradation mechanism.
  • the compositions and methods described herein represent the first evidence of the degradability of untagged, endogenous brachyury in multiple chordoma cell lines.
  • OT3 containing a phosphodiester backbone
  • first generation TRAFTACs where oligonucleotides are partly protected by the ribonuclear complex, induced defective tails in zebrafish. Therefore, a potential reason for the failure of OT3 to induce the intended phenotype within the embryos may be their high sensitivity and exposure of oligoTRAFTACs to embryonic nucleases present at the early stages of development. Therefore, phosphodiester oligoTRAFTACs might not endure in vivo at the intracellular concentrations needed to achieve a significant brachyury degradation and the intended phenotype.
  • oligoTRAFTACs are programmable heterobifunctional molecules comprised of a TF-binding oligonucleotide sequence and a VHL binding ligand, which induce TF degradation in cells and displayed robust in vivo activity. Due to the simple and modular nature of their structure, oligoTRAFTACs can be rapidly designed for many non-ligandable DNA-binding TFs both for use as a chemical biology tool as well as a potential therapeutic strategy.
  • TFs such as TCF3
  • TCF3 also recognizes a similar E-box consensus sequence as c-Myc
  • the evaluation of TCF3 degradation indicated a sequence-dependent off-target activity of OT7 (FIG.8F).
  • NF-kB p65 protein levels in response to brachyury-targeting OT17.
  • p65 NF-kB
  • oligoTRAFTACs are programmable heterobifunctional molecules comprised of a TF-binding oligonucleotide sequence and a VHL binding ligand, which induce TF degradation in cells and displayed robust in vivo activity. Due to the simple and modular nature of their structure, oligoTRAFTACs can be rapidly designed for many non-ligandable DNA-binding TFs both for use as a chemical biology tool as well as a potential therapeutic strategy.
  • the compounds described herein can possess one or more stereocenters, and each stereocenter can exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers.
  • These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • the methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol.
  • the compounds described herein exist in unsolvated form.
  • the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • compounds described herein are prepared as prodrugs.
  • a "prodrug" refers to an agent that is converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compound(s) described herein are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway.
  • the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, 11 C, 13 C, 14 C, 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, and 35 S.
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes, such as 11 C, 18 F, 15 O and 13 N is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • the compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4 th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols.
  • Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed.
  • each protective group is removable by a different means.
  • Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
  • protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions.
  • Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
  • Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
  • carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc.
  • Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co- existing amino groups are blocked with fluoride labile silyl carbamates. Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts.
  • an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups.
  • Another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
  • blocking/protecting groups may be selected from: .
  • compositions containing the compound(s) described herein include a pharmaceutical composition comprising at least one compound as described herein and at least one pharmaceutically acceptable carrier.
  • the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • Methods of Treatment, Amelioration, and/or Prevention The disclosure includes a method of treating, ameliorating, and/or preventing cancer using the compounds of formula I.
  • Non-limiting examples of cancer include squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, renal cell carcinoma, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; multiple myeloma, sarcomas, including Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gli
  • the methods described herein include administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated in a pharmaceutical composition.
  • a therapeutically effective amount of at least one compound described herein present in a pharmaceutical composition is the only therapeutically active compound in a pharmaceutical composition.
  • the method further comprises administering to the subject an additional therapeutic agent that treats cancer.
  • administering the compound(s) described herein to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating a cancer in the subject.
  • the compound(s) described herein enhance(s) the activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect.
  • the compound(s) described herein and the therapeutic agent are co-administered to the subject.
  • the compound(s) described herein and the therapeutic agent are coformulated and co-administered to the subject.
  • the subject is a mammal. In other embodiments, the mammal is a human.
  • Combination Therapies The compounds useful within the methods described herein can be used in combination with one or more additional therapeutic agents useful for treating, ameliorating, and/or preventing cancer.
  • additional therapeutic agents may comprise compounds that are commercially available or synthetically accessible to those skilled in the art. These additional therapeutic agents are known to treat, ameliorate, prevent, and/or reduce the symptoms, of cancer.
  • the compounds described herein can be used in combination with radiation therapy.
  • the combination of administration of the compounds described herein and application of radiation therapy is more effective in treating, ameliorating, or preventing cancer than application of radiation therapy by itself.
  • the combination of administration of the compounds described herein and application of radiation therapy allows for use of lower amount of radiation therapy in treating the subject.
  • a synergistic effect is observed when a compound as described herein is administered with one or more additional therapeutic agents or compounds.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul.22:27-55).
  • Sigmoid-Emax equation Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453
  • Loewe additivity Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326
  • the median-effect equation Chou & Talalay, 1984, Adv. Enzyme Regul.22:27-55.
  • the corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
  • Administration/Dosage/Formulations The regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the onset of cancer. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions described herein to a patient may be carried out using known procedures, at dosages and for periods of time effective to treat cacner in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat cancer in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound.
  • the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • compositions described herein are administered to the patient in dosages that range from one to five times per day or more.
  • compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account.
  • the compound(s) described herein for administration may be in the range of from about 1 ⁇ g to about 10,000 mg, about 20 ⁇ g to about 9,500 mg, about 40 ⁇ g to about 9,000 mg, about 75 ⁇ g to about 8,500 mg, about 150 ⁇ g to about 7,500 mg, about 200 ⁇ g to about 7,000 mg, about 350 ⁇ g to about 6,000 mg, about 500 ⁇ g to about 5,000 mg, about 750 ⁇ g to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • a composition as described herein is a packaged pharmaceutical composition
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • other active agents e.g., other analgesic agents.
  • the compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • transdermal e.g., sublingual, lingual, (trans)buccal, (trans)urethral
  • vaginal e.g., trans- and perivaginally
  • intravesical, intrapulmonary, intraduodenal, intragastrical intrathecal
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compound(s) described herein can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose
  • fillers e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White, 32K18400).
  • OPADRYTM film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White, 32K18400).
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid.
  • Compositions as described herein can be prepared, packaged, or sold in a
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, dispersing agents, surface-active agents, disintegrating agents, binding agents, and lubricating agents.
  • Suitable dispersing agents include, but are not limited to, potato starch, sodium starch glycollate, poloxamer 407, or poloxamer 188.
  • One or more dispersing agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more dispersing agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Surface-active agents include cationic, anionic, or non-ionic surfactants, or combinations thereof.
  • Suitable surfactants include, but are not limited to, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, carbethopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridine chloride, didecyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, domiphen bromide, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, tetramethylammonium hydroxide, thonzonium bromide, stearalkonium chloride, octenidine dihydrochloride, olaflur, N-oleyl-1,3-propanediamine, 2-acrylamido-2-methylpropane sulfonic acid, alkylbenzen
  • One or more surfactants can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more surfactants can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable diluents include, but are not limited to, calcium carbonate, magnesium carbonate, magnesium oxide, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate, Cellactose ® 80 (75 % ⁇ - lactose monohydrate and 25 % cellulose powder), mannitol, pre-gelatinized starch, starch, sucrose, sodium chloride, talc, anhydrous lactose, and granulated lactose.
  • One or more diluents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more diluents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable granulating and disintegrating agents include, but are not limited to, sucrose, copovidone, corn starch, microcrystalline cellulose, methyl cellulose, sodium starch glycollate, pregelatinized starch, povidone, sodium carboxy methyl cellulose, sodium alginate, citric acid, croscarmellose sodium, cellulose, carboxymethylcellulose calcium, colloidal silicone dioxide, crosspovidone and alginic acid.
  • One or more granulating or disintegrating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more granulating or disintegrating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, anhydrous lactose, lactose monohydrate, hydroxypropyl methylcellulose, methylcellulose, povidone, polyacrylamides, sucrose, dextrose, maltose, gelatin, polyethylene glycol.
  • One or more binding agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more binding agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Suitable lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, hydrogenated castor oil, glyceryl monostearate, glyceryl behenate, mineral oil, polyethylene glycol, poloxamer 407, poloxamer 188, sodium laureth sulfate, sodium benzoate, stearic acid, sodium stearyl fumarate, silica, and talc.
  • One or more lubricating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form.
  • One or more lubricating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form.
  • Tablets can be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patent Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Tablets can also be enterically coated such that the coating begins to dissolve at a certain pH, such as at about pH 5.0 to about pH 7.5, thereby releasing a compound as described herein.
  • the coating can contain, for example, EUDRAGIT ® L, S, FS, and/or E polymers with acidic or alkaline groups to allow release of a compound as described herein in a particular location, including in any desired section(s) of the intestine.
  • the coating can also contain, for example, EUDRAGIT ® RL and/or RS polymers with cationic or neutral groups to allow for time controlled release of a compound as described herein by pH-independent swelling.
  • Parenteral Administration For parenteral administration, the compounds as described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as such as lauryl, stearyl, or oleyl alcohols, or similar alcohol.
  • Additional Administration Forms Additional dosage forms suitable for use with the compound(s) and compositions described herein include dosage forms as described in U.S.
  • Controlled Release Formulations and Drug Delivery Systems can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use with the method(s) described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the dosage forms to be used can be provided as slow or controlled- release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein can be readily selected for use with the pharmaceutical compositions described herein.
  • single unit dosage forms suitable for oral administration such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the compositions and dosage forms described herein.
  • controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance.
  • controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects.
  • Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time.
  • Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • the term "controlled-release component" is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient.
  • the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound described herein depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different.
  • a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the compound(s) described herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary.
  • the dosage or the frequency of administration, or both is reduced to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • the compounds described herein can be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED 50 .
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized. Examples Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.
  • DMEM fetal bovine serum
  • IMDM fetal bovine serum
  • RPMI fetal bovine serum
  • DMEM/F-12 fetal bovine serum
  • Primary antibodies for GAPDH (2118S), Vinculin (13901S), brachyury (81694S), HA-tag (3724S), protein A magnetic beads (73778S) and streptavidin magnetic beads (5947S) were purchased from Cell Signaling Technologies.
  • Primary antibody for c-Myc (sc-40) was purchased from Santa Cruz. Secondary rabbit (NA934) and mouse (NA931) antibodies were purchased from GE Health Care.
  • RNAiMAX (13778-150) transfecting reagent was purchased from Thermo Fisher Scientific.
  • MLN4924 (S7109) was purchased from Selleckchem. Copper (II) sulfate pentahydrate (209198) and L-Ascorbic acid (A4403) were purchased from Millipore Sigma, and THPTA purchased from Lumiprobe (H4050). All the oligonucleotide modifiers were purchased from Glen Research, 3' Alkyne (20-2992-41), 5' Alkyne (10-1992-90) and biotin (10-5950-90). All the oligonucleotides were custom synthesized by Yale Keck oligo synthesis facility.
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS heat inactivated fetal bovine serum
  • streptomycin 5 ⁇ g/mL
  • penicillin 95 U/mL penicillin 95 U/mL
  • OligoTRAFTAC transfection One day prior to oligoTRAFTACs transfection, cells (HEK293T: 0.7X10 6 /well, HeLa: 0.2 X10 6 /well, UM-Chor1: 0.2X10 6 /well and JHC7: 0.4X10 6 /well) were propagated into 6-well plates containing appropriate complete growth medium. Prior to transfection, complete medium was replaced with 1.75 mL of transfection medium (2%FBS, no Penstrep). Chimeric oligoTRAFTAC transfection was performed using RNAi-Max reagent according to the protocols provided by the manufacture.
  • oligoTRAFTACs concentrations of oligoTRAFTACs were calculated according to this volume (2 mL). Briefly, for 50 nM concentration, 4 ⁇ L from a 25 ⁇ M oligo TRAFTAC stock was added to a tube containing 125 ⁇ L of OPTIM-MEM and 12.5 ⁇ L of RNAi-Max reagent was added to a separate tube containing 125 ⁇ L of OPTIM-MEM (added ⁇ 4 ⁇ g of oligoTRAFTAC to 2 mL cell culture medium).
  • oligoTRAFTAC containing OPTI-MEM was then slowly added to the second tube with RNAi-MAX.
  • the solution in the tube was mixed well by pipetting up and down several times. After incubating for 10 minutes at room temperature, 250 ⁇ L of oligoTRAFTAC:RNAi-MAX complex was added drop wise onto cells containing the transfection medium. Transfection medium was mixed well before transferring the 6-well plate into the incubator. After appropriate time, cells were either harvested or transfection medium containing oligoTRAFTAC:RNAi-MAX complex was replaced with fresh medium and incubated for desired time point prior to harvesting.
  • MLN-4924 and VHL ligand competition assay were pre-incubated in the transfection medium (1.75 mL) for 1 h prior to transfection of oligoTRAFTACs.
  • Cell lysates were prepared by incubating cells in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF) on ice for 30 minutes and cell lysate was clarified by centrifugation at high speed (15000 rpm) for 20 minutes. Clear supernatant was collected for further experiments.
  • RIPA lysis buffer 25 mM Tris pH 7.6, 150 mM NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF
  • the program was set with a flow rate of 5 mL/min for 150 minutes, and a gradient of ACN increasing from 0-80%.
  • Annealing Reaction FPLC purified single stranded oligo conjugated to VHL (oligo-VHL) ligand and its reverse complement oligo were dissolved in ultra-pure water.
  • Single stranded oligo-VHL and single stranded reverse complement oligos were mixed 1:1 molar ratio (final concentrations of TRAFTACs were set to 25 ⁇ M) in 1X annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl and 1 mM EDTA) and incubated for 10 minutes in a water bath at 95 degrees Celsius.
  • Double stranded oligoTRAFTACs were mixed by gently vortexing and aliquoted and stored at -20 degrees Celsius.
  • Reverse complementary sequences OT3/17'- 5'CCCAATTTCACACCTAGGTGTGAAATTGGA3', OT7/10- 5' AACCACGTGGCAACCACGTGCTC 3'.
  • Western Blotting Protein concentration in all the cell lysates were measured by BCA protein assay kit and equal amounts from each lysate were mixed with 4X loading dye and boiled for 5 minutes followed by 2 minutes centrifugation prior to loading into SDS-PAGE gel.
  • EMSA Electrophoretic Mobility Shift Assay
  • transfected cells were split into three 10 cm cell culture dishes and incubated for 24 h prior to transfection of oligoTRAFTACs.
  • Epoxomicin (1 ⁇ M) was preincubated with cells for 1 h and oligoTRAFTACs (mock, OT3 and OT17) were then transfected using RNAi-MAX in 5 mL/dish of transfection medium.
  • RNAi-MAX 5 mL/dish of transfection medium.
  • cells were harvested and lysed using immunoprecipitation buffer (25 mM Tris pH 7.4, 150 mM NaCl, 0.4% NP40, 5% glycerol, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF).
  • lysate from each sample was incubated with brachyury antibody at 4 o C for 4 h.
  • Protein A agarose beads (30 ⁇ L) were added to antibody, lysates mixture and rock at 4 o C for ⁇ 18 h. Beads were washed with 1X TBS for three times with 5-minute incubation during each wash. Immunoprecipitated proteins were eluted by boiling agarose beads in 2X loading buffer (containing 10% ß-ME) for 8 minutes and centrifuged at high speed for 5 minutes prior to the loading into SDS-PAGE gel followed by western blot analysis. Cell viability assay Cells were split and subjected to oligoTRAFTAC transfection as described herein.
  • CellTiter-Glo reagent was prepared according to the manufactures recommendation and mixed with cell culture medium with 1:1 ratio. CellTiter-Glo reagent (100 ⁇ L) was added to each well and incubated for 10 minutes before taking the reading. Zebrafish Husbandry and Microinjection Tüpfel-longfin zebrafish were raised according to standard protocols approved by the Institutional Animal Care and Use Committee. Experiments were performed before sex determination in zebrafish 1 . Embryos were injected at the one cell stage with 180 picoliters of a 25 ⁇ M oligoTRAFTAC solution.
  • OTs 1-20 can have a linker LNK of formula LNK-X1, LNK- X2, LNK-1, or LNK-2.
  • the linker PEG number (n) which represents the number of ethylene glycol (-CH 2 CH 2 O-) units, is the same as variable pp in formulas LNK-X1, LNK-X2, LNK- 1, and LNK-2. Table 2. Oligonucleotide sequence, modification and modification site of the oligonucleotides used for biotin pull-down experiments.
  • Embodiment 1 provides a compound of formula I, or a salt, enantiomer, diastereomer, or tautomer thereof: wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonding the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand.
  • Embodiment 2 provides the compound of embodiment 1, wherein the 3'-end or the 5'- end of the ONA is covalently bonded to the LNK.
  • Embodiment 3 provides the compound of any one of embodiments 1-2, wherein at least one internucleotide linkage comprises a phosphorothioate.
  • Embodiment 4 provides the compound of any one of embodiments 1-3, wherein all internucleotide linkages are independently phosphorothioates.
  • Embodiment 5 provides the compound of any one of embodiments 1-4, wherein the ONA comprises at least one deoxyribonucleotide and/or ribonucleotide.
  • Embodiment 6 provides the compound of any one of embodiments 1-5, wherein the ONA comprises a c-Myc-binding nucleotide sequence.
  • Embodiment 7 provides the compound of any one of embodiments 1-6, wherein at least two nucleotides flank each end of the c-Myc-binding nucleotide sequence.
  • Embodiment 8 provides the compound of any one of embodiments 1-7, wherein the c- Myc-binding nucleotide sequence comprises 5'-CACGTGGTTGCCACGTG-3'.
  • Embodiment 9 provides the compound of any one of embodiments 1-8, wherein the ONA comprises a brachyury-binding nucleotide sequence.
  • Embodiment 10 provides the compound of any one of embodiments 1-9, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence.
  • Embodiment 11 provides the compound of any one of embodiments 1-10, wherein the brachyury-binding nucleotide sequence comprises 5'- AATTTCACACCTAGGTGTGAAATT-3'.
  • Embodiment 15 provides the compound of any one of embodiments 1-14, wherein A is phenylene or a C 5-18 heteroarylene.
  • Embodiment 16 provides the compound of any one of embodiments 1-15, wherein A Embodiment 17 provides the compound of any one of embodiments 1-16, wherein LNK is –(LL) aa –A–(LL) bb –.
  • Embodiment 20 provides the compound of any one of embodiments 1-19, wherein LNK-X1 has the structure wherein X is CH 2 or O; and k is 1, 2, or 3.
  • Embodiment 22 provides the compound of any one of embodiments 1-21, wherein LNK-X2 has the structure wherein X is CH 2 or O; and k is 1, 2, or 3.
  • Embodiment 23 provides the compound of any one of embodiments 1-22, wherein LNK has the structure: , wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • Embodiment 24 provides the compound of any one of embodiments 1-23, wherein pp is 2, 3, or 5.
  • Embodiment 25 provides the compound of any one of embodiments 1-24, wherein pp is 2, 3, or 5.
  • Embodiment 26 provides the compound of any one of embodiments 1-25, wherein the UBL is a ligand of an E3 ubiquitin ligase selected from the group consisting of von Hippel- Lindau (VHL), cereblon (CRBN), RING-type zinc-finger protein 114 (RNF114), cellular inhibitor of apoptosis (cIAP), mouse double minute 2 homologue (MDM2), damage-specific DNA binding protein 1 (DDB1)-CUL4 associated factor 16 (DCAF16), and Kelch-like ECH- associated protein 1 (KEAP1).
  • Embodiment 27 provides the compound of any one of embodiments 1-26, wherein the UBL is a ligand of VHL or CRBN.
  • Embodiment 28 provides the compound of any one of embodiments 1-27, wherein the UBL is a ligand having the structure:
  • Embodiment 29 provides the compound of any one of embodiments 1-28, wherein the UBL has the structure: .
  • Embodiment 30 provides the compound of any one of embodiments 1-29, wherein the compound is selected from the group consisting of: , , wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • Embodiment 31 provides the compound of any one of embodiments 1-30, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'.
  • Embodiment 33 provides the compound of embodiment 32, wherein the terminal nucleotide at the 3'-end of ONA has the structure: , wherein: zz is an integer from 1 to 100; Y is H or OH; and Embodiment 34 provides the compound of any one of embodiments 32-33, wherein the terminal nucleotide at the 5'-end of ONA has the structure: , wherein: zz is an integer from 1 to 100; Y is H or OH; Z is O or S; and Embodiment 35 provides the compound of any one of embodiments 32-34, wherein the ONA comprises at least one deoxyribonucleotide or ribonucleotide, or a combination thereof.
  • Embodiment 36 provides the compound of any one of embodiments 32-35, wherein the ONA comprises a c-Myc-binding nucleotide sequence.
  • Embodiment 37 provides the compound of any one of embodiments 32-36, wherein at least two nucleotides flank each end of the c-Myc-binding nucleotide sequence.
  • Embodiment 38 provides the compound of any one of embodiments 32-37, wherein the c-Myc-binding nucleotide sequence comprises 5'- CACGTGGTTGCCACGTG-3'.
  • Embodiment 39 provides the compound of any one of embodiments 32-38, wherein the ONA comprises a brachyury-binding nucleotide sequence.
  • Embodiment 40 provides the compound of any one of embodiments 32-39, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence.
  • Embodiment 41 provides the compound of any one of embodiments 32-40, wherein the brachyury-binding nucleotide sequence comprises 5'- AATTTCACACCTAGGTGTGAAATT-3'.
  • Embodiment 42 provides the compound of any one of embodiments 32-41, having the structure: wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
  • Embodiment 43 provides the compound of any one of embodiments 32-42, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'.
  • Embodiment 44 provides a pharmaceutical composition comprising the compound of any one of embodiments 1-31, and a pharmaceutically acceptable carrier, additive, and/or excipient.
  • Embodiment 45 provides a method of preventing, treating, and/or ameliorating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of at least one compound of any one of embodiments 1-31, which is optionally formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
  • Embodiment 46 provides the method of embodiment 45, wherein the cancer is at least one selected from the group consisting of squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, renal cell carcinoma, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemia; benign or malignant lymphoma; benign or malignant melanoma; myeloproliferative disease; multiple myeloma, sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell tumor, meningioma, meningeal sarcoma, neurofibrom
  • Embodiment 47 provides the method of any one of embodiments 45-46, wherein: the benign or malignant lymphoma comprises Burkitt's lymphoma and/or Non-Hodgkin's lymphoma; and the sarcoma comprises Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelioma, and/or synovial sarcoma.
  • the benign or malignant lymphoma comprises Burkitt's lymphoma and/or Non-Hodgkin's lymphoma
  • the sarcoma comprises Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelioma, and/or synovial sarcoma.
  • Embodiment 48 provides the method of any one of embodiments 45-47, wherein the compound and/or composition is administered by a route selected from the group consisting of oral, transdermal, transmucosal, (intra)nasal, (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra- arterial, intravenous, intrabronchial, inhalation, and topical.
  • Embodiment 49 provides the method of any one of embodiments 45-48, wherein the subject is a mammal.
  • Embodiment 50 provides the method of any one of embodiments 45-49, wherein the subject is human.

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Abstract

Provided herein are compounds containing a TF-binding oligonucleotide and an E3 ligase-recruiting ligand ("oligoTRAFTAC"). These oligoTRAFTACs induce the degradation of oncogenic transcription factors (TFs), c-Myc and brachyury. These compounds demonstrate a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway, which is useful in the treatment of a large variety of cancers.

Description

TITLE OF THE INVENTION Oligonucleotide-Containing Transcription Factor Targeting Chimeras CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application No. 63/282,298, entitled "OLIGONUCLEOTIDE-CONTAINING TRANSCRIPTION FACTOR TARGETING CHIMERAS," filed November 23, 2021, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under CA197589 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM This invention contains one or more sequences in a computer readable format in an accompanying text file titled "047162-7309WO1 Sequence Listing_ST26," which was created on November 22, 2022 and is 16 KB in size, the contents of which are incorporated herein by reference in their entirety. BACKGROUND Protein degradation is an integral component of cellular homeostasis, supporting the healthy environment within the cell. The ubiquitin-proteasomal pathway is one of the key mechanisms by which unwanted or defective proteins are degraded. Targeted protein degradation by PROteolysis TArgeting Chimeras, or PROTACs, has stimulated the development of novel strategies to induce protein degradation, for both discovery biology and therapeutics. PROTACs are small molecule-based heterobifunctional molecules that recruit an E3 ligase complex to a protein of interest. By doing so, PROTACs induce the ubiquitination and degradation of the protein of interest via the proteasome. Although PROTACs have the potential to induce the degradation of numerous proteins, the identification of small molecule recruiting ligands for several classes of proteins is still challenging and time-consuming. Therefore, the development of alternative, proximity- inducing strategies would help to target disease-causing proteins, such as transcription factors. Transcription factors (TFs) control gene expression by binding to specific DNA elements within a given gene promoter or distal enhancer regions. Many diseases such as neurological disorders, autoimmunity, developmental syndromes, and many cancers result from abnormalities in the TF-controlled gene-regulatory circuitry within the diseased cell. For instance, the TF c-Myc, the most frequently amplified oncogene, has been extensively studied and established as a direct mediator of tumorigenesis in numerous cancers. Although indirect approaches such as bromodomain protein inhibitors and PROTACs have been explored to control c-Myc levels, direct inhibition or degradation of c-Myc has been an as yet unrealized goal. In contrast to c-Myc, T-box transcription factor ("brachyury") is expressed only in a restricted set of cancer types and minimally expressed in normal cells. Brachyury is a master developmental TF that plays a pivotal role during early embryonic development in vertebrates. Brachyury expression is limited only to embryonic developmental stages and, in general, expression levels are highly downregulated in adult tissues. In addition to its regulatory functions during development, brachyury expression in remnant notochord cells in adults has been shown to be a key oncogenic driver in the rare bone cancer, chordoma. Chromosomal aberrations such as chromosome 6 gains and partial polysomy have been identified as potential molecular mechanisms in brachyury-dependent chordoma tumors. Other TFs such as NF-kB, STAT3/5, the androgen receptor (AR) and the estrogen receptor (ER) are other known oncogenic drivers that also rewire transcriptional circuitry in various cancer types. Although hundreds of TFs have been identified as key players in human diseases, the lack of direct therapeutic strategies is a bottleneck for accessing these difficult- to-drug targets. Although several TFs, such as STAT3, AR, and ER, have been successfully degraded by PROTACs, a plethora of disease-relevant yet traditionally "undruggable" TFs remain unaddressed. The present disclosure addresses and solves this unmet need. BRIEF SUMMARY OF THE INVENTION In various aspects, a compound of formula I, or a salt, enantiomer, diastereomer, or tautomer thereof is provided: wherein:
Figure imgf000003_0001
ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonding the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand. In various aspects, a compound of formula II, or a salt, enantiomer, diastereomer, or tautomer thereof is provided:
Figure imgf000004_0001
wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is a chemical linker covalently bonding the ONA and the C ≡C ≡H. Compounds of formula I are useful in preventing, treating, and/or ameliorating cancer in subjects, including human subjects. In various aspects, a method of preventing, treating, and/or ameliorating cancer in a subject is provided. The method includes administering to the subject a therapeutically effective amount of at least one compound of formula I, which is optionally formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. Compounds of formula II are useful, in various aspects, for synthesizing compounds of formula I. BRIEF DESCRIPTION OF THE FIGURES The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application. FIG.1 is a schematic representation of oligoTRAFTAC-mediated transcription factor (TF) and E3 ligase recruitment, and proximity-dependent TF ubiquitination, in accordance with various embodiments. FIGs.2A-2E illustrate show activity and aspects of oligoTRAFTACs, according to various embodiments. OligoTRAFTAC induces c-Myc degradation. (FIG.2A) The oligonucleotide selected for the c-Myc oligoTRAFTAC engages c-Myc. HeLa cell lysates were incubated with biotinylated oligonucleotide, or its scrambled sequence, followed by capture with streptavidin agarose and probing for c-Myc. (n = 2). (FIG.2B) Dose response of OT7-mediated c-Myc knockdown in HeLa cells. (n = 2) (FIG.2C) OligoTRAFTAC-induced c-Myc degradation occurred via the proteasomal pathway. HEK293T cells were treated with c-Myc-targeting oligoTRAFTAC with and without the neddylation inhibitor, MLN-4924 (1 mM), and then analyzed for c-Myc levels. (n = 2, *p < 0.05) (FIG.2D) HEK293 cells were preincubated with and without 10 mM VHL ligand followed by OT7 transfection and analyzed for c-Myc levels. (n = 2, *p < 0.05) (FIG.2E) Chemical structure of a c-Myc- targeting oligoTRAFTAC (OT7), according to various embodiments. FIGs.3A-3E show brachyury-GFP degradation by oligoTRAFTACs. (FIG.3A) Brachyury-targeting oligonucleotide used in the oligoTRAFTAC design engaged with brachyury-GFP. Brachyury targeting biotinylated oligonucleotide (BRCH) or its scrambled oligonucleotide (SCRM) incubated with cell lysate and captured by streptavidin agarose beads. (n = 2) (FIG.3B) Two oligoTRAFTACs with 30 VHL ligand modifications, OT3 (5 PEG unit linker) and OT4 (2 PEG unit linker) were transfected into HEK293T cells and brachyury-GFP levels were analyzed in lysates prepared after 20 h. (n = 2) (FIG.3C) OT3 induced brachyury-GFP degradation as early as 12 h in HEK293T cells. (n = 2) (FIG.3D) Washout experiment after 12 h of OT3 transfection. Cells were incubated continuously for 24 h in transfection medium or OT3 was aspirated after 12 h of transfection and fresh medium added to cells. Washout cells incubated for another 12 h and 24 h prior to harvesting. (n = 2) (FIG.3E) Chemical structure of brachyury-targeting oligoTRAFTAC (OT3). OT3 includes a phosphodiester backbone. FIGs.4A-4D show oligoTRAFTACs induce brachyury-GFP degradation via the proteasomal pathway. (FIG.4A) HEK293T cells expressing brachyury-GFP were transfected with 75 nM each of OT3 and its scrambled OT6, and lysates were probed for brachyury levels. (n = 2, **p < 0.005) (FIG.4B) OT3 induced brachyury degradation is VHL- dependent. HEK293T cells were preincubated with and without 10 mM of VHL ligand for 1.5 h prior to OT3 transfection. After 20 h of transfection, cells lysates were prepared and analyzed for brachyury degradation. (n = 3, ****p < 0.0001) (FIG.4C) OT3 induces brachyury degradation via the proteasomal pathway: neddylation inhibitor MLN-4924 was preincubated with cells prior to OT3 transfection. After 20 h of transfection of OT3, cells were harvested and analyzed for brachyury levels. (n = 2, **p < 0.01) (FIG.4D) Brachyury- GFP downregulation was monitored by GFP fluorescence in cells in the presence or absence of MLN-4924. (n = 2). FIGs.5A-5F show endogenous brachyury degradation by oligoTRAFTACs constructed with phosphorothioate backbone. (FIG.5A) Increasing concentrations of OT17 were transfected into UM-Chor1 cells and harvested after 24 h, subjected to lysis and analyzed for brachyury downregulation. Brachyury levels were normalized to loading control and presented as a bar graph. (n = 2, **p < 0.01) (FIG.5B) JHC-7 cells were transfected with OT17 and probe for brachyury levels (n = 2, ****p < 0.0001). (FIG.5C) UM-Chor1 cells were transfected with 60 nM of OT17 and harvested at subsequent different time points as indicated. (n = 3, ****p < 0.0001) (FIG.5D) Washout experiment: transfection medium was removed after 12 h of OT17 transfection and UM-Chor1 cells were incubated for another 12 h or 24 h in fresh complete cell culture medium. (n = 2, ***p < 0.001, ****p < 0.0001) (FIG. 5E) OT17-mediated brachyury degradation is oligonucleotide sequence dependent. UM- Chor1 cells were transfected with OT17 and scrambled OT20, cells were lysed and analyzed as shown. (n = 2, ***p < 0.001, ****p < 0.0001) (FIG.5F) OT3- and OT17-induced brachyury ubiquitination. HEK293T cells that overexpress brachyury-GFP were transfected with HA-ubiquitin, followed by the second transfection with OT3 or OT17. After 12 h, cell lysates were subjected to immunoprecipitation using brachyury antibody, and the eluates blotted for the indicated proteins. (n = 3). FIGs.6A-6D show microinjection of brachyury-targeting oligoTRAFTAC into zebrafish embryos: demonstration of in vivo activity. (FIG. 6A) Schematic representation of OT17 and OT20 microinjection into zebrafish embryos. (FIG.6B) Quantitation of the defective embryos in mock, OT17 and OT20 injected groups. Mock, OT17 and OT20 (180 picoliters from 25 mM of oligoTRAFTACs, or mock equivalent) were microinjected into embryos (number of embryos in each group for three independent experiments; mock-47, 50, 43; OT17-49, 52, 61; OT20-75, 74, 45). After 48 h, the number of defective tails in each group was recorded and presented as percentage in a bar graph. (n = 3, **p < 0.001) (FIG. 6C) Images of representative zebrafish from the cognate treatment groups. Pictures were captured after 48 h post microinjection of mock, OT17 and OT20. Scale bar 500 mm. (n = 3) (FIG.6D) Brachyury levels in zebrafish embryos after OT17 (180 picoliters from 25 mM of oligoTRAFTACs, or mock equivalent) injection. Embryos were collected at 8–10 somite stage, subjected to lysis, and probed for brachyury levels. (n = 3, ****P < 0.0001). FIGs.7A-7C show the oligonucleotide synthesis and click reaction. Oligonucleotide synthesis and click reaction. FIG.7A) Oligonucleotides were custom synthesized with a terminal alkyne either at 3' or 5' end of the oligo. Oligonucleotide sequences for both 3' and 5' alkyne targeting c-Myc (left panel) and brachyury (right panel). FIG.7B) Chemical structure of azido-VHL ligand and click reaction conditions. FIG.7C) Chemical structures of 3' modified oligonucleotides (OT17- with a phosphorothioate backbone) after the click reaction with azido-VHL ligand. FIGs.8A-8G show oligoTRAFTAC mediated c-Myc degradation. FIG.8A) Electrophoretic mobility shift assay data for the click reaction. Before and after click reaction, oligonucleotides were separated from a 1.2 % agarose gel for 1 h at constant 120 mV. VHL ligand reacted oligonucleotides were shifted compared to the unreacted oligo. (n=2) FIG.8B) Varying concentrations of OT7 were transfected into HEK293T cells and lysed after 20 h. Cell lysates were separated and transferred to a PVDF membrane followed by immunoblotting with antibodies against c-Myc and GAPDH. (n=2) FIG.8C) After 60 nM of OT7 transfection to HEK293T cells, cell culture media was replaced with fresh medium and continued the incubation for total of 20 h prior to lysis. Lysates were probed for c-Myc and GAPDH. (n=2) FIG.8D) Similarly, HEK293T cells were subjected to a washout experiment 12 h posttransfection and lysed after 24 h and 36 h. (n=2) FIG.8E) HeLa cells were seeded into 96-well plates transfected increasing concentrations of OT7 and OT12. After 48 h, cell viability was monitored using CellTiter-Glo reagent. FIG.8F) Degradation of E-box binding transcription factor, TCF3. OT7 transfected cells were analyzed for c-Myc and TCF3. FIG.8G) Analysis of brachyury and p65 degradation by OT17. (n=2) FIGs.9A-9B show EMSA and brachyury-GFP degradation data. FIG.9A shows click reaction mixtures with or without VHL ligand were loaded on to a 1.2% agarose gel and separated over 1 h at constant 120 mV. FIG.9B shows that OT1 through 4 were transfected into HEK293T cells overexpressing brachyury-GFP and lysed after 30 h. Cell lysates were subjected to SDS-PAGE and western blotting followed by probing with antibodies against brachyury and GAPDH. FIGs.10A-10B show time course and washout experiments for brachyury targeting OTs. FIG.10A shows OT2 through 4 were transfected at 75 nM into HEK293T cells overexpressing brachyury-GFP and lysed after 12 h, 24 h, and 36 h. Lysates were probed for brachyury and GAPDH. FIG.10B shows OT3 was transfected into HEK293T cells and washed out after 6 h and 12 h. Cell lysates were probed with antibodies against brachyury and GAPDH. FIGs.11A-11C show brachyury degradation by oligoTRAFTACs in HEK293T and UM-Chor1 cells. FIG.11A) Brachyury-GFP degradation by OTs is sequence dependent. OT3, OT4, and their scrambled OTs (OT5 and OT6) were transfected and lysed after 20 h. Cell lysates were subjected to SDS-PAGE and western blotting. Blots were probed with brachyury and GAPDH antibodies. Quantitation of western blot bands is shown on the right. (n=2, **p<0.005) FIG.11B) Increasing concentration of OT3 were transfected into UM- Chor1 cells and degradation was evaluated after 24 h. (n=2, **p<0.005) FIG.11C) Proteasome dependent brachyury degradation in UM-Chor1 cells. The proteasome inhibitors, MG-132 and Epoxomicin were pre-incubated with cells prior to OT17 transfection and assessed brachyury levels after 24 h. (n=2, *p<0.05). DETAILED DESCRIPTION OF THE INVENTION Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter. Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise. In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. Definitions The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of" as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of" can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%. The term "organic group" as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0- 2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1-C100)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted. The term "substituted" as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The substitution can be direct substitution, whereby the hydrogen atom is replaced by a functional group or substituent, or an indirect substitution, whereby an intervening linker group replaces the hydrogen atom, and the substituent or functional group is bonded to the intervening linker group. A non-limiting example of direct substitution is: RR-H → RR-Cl, wherein RR is an organic moiety/fragment/molecule. A non-limiting example of indirect substitution is: RR-H → RR- (LL)zz-Cl, wherein RR is an organic moiety/fragment/molecule, LL is an intervening linker group, and 'zz' is an integer from 0 to 100 inclusive. When zz is 0, LL is absent, and direct substitution results. The intervening linker group LL is at each occurrence independently selected from the group consisting of -H, -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡ CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, -C(=NR)-, and combinations thereof. (LL)zz can be lin -ear, branched, cyclic, acyclic, and combinations thereof. In certain embodiments, two consecutive LL's cannot be both O. The term "functional group" or "substituent" as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1-C100)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl. The term "alkyl" as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others. The term "alkynyl" as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ≡CH, -C ≡C(CH3), -C ≡C(CH2CH3), -CH2C ≡CH, -CH2C ≡C(CH3), and -CH2C ≡C(CH2CH3) among others. The term "acyl" as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group. The term "cycloalkyl" as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination denotes a cyclic alkenyl group. The term "heterocycloalkyl" as used herein refers to a cycloalkyl group as defined herein in which one or more carbon atoms in the ring are replaced by a heteroatom such as O, N, S, P, and the like, each of which may be substituted as described herein if an open valence is present, and each may be in any suitable stable oxidation state. The term "aryl" as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof. The term "aralkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. The term "heterocyclyl" as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. The term heterocyclyl includes rings where a CH2 group in the ring is replaced by one or more C=O groups, such as found in cyclic ketones, lactones, and lactams. Examples of heterocyclyl groups containing a C=O group include, but are not limited to, β- propiolactam, γ-butyrolactam, δ-valerolactam, and ε-caprolactam, as well as the corresponding lactones. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein. The term "heteroaryl" as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring designated Cx-y can be any ring containing 'x' members up to 'y' members, including all intermediate integers between 'x' and 'y' and that contains one or more heteroatoms, as defined herein. In a ring designated Cx-y, all non- heteroatom members are carbon. Heterocyclyl rings designated Cx-y can also be polycyclic ring systems, such as bicyclic or tricyclic ring systems. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein. Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like. The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl. The term "heteroarylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein. The term "alkoxy" as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith. The term "amine" as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein. The term "amino group" as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR3 +, wherein each R is independently selected, and protonated forms of each, except for -NR3 +, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group. The terms "halo," "halogen," or "halide" group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "haloalkyl" group, as used herein, includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like. The terms "epoxy-functional" or "epoxy-substituted" as used herein refers to a functional group in which an oxygen atom, the epoxy substituent, is directly attached to two adjacent carbon atoms of a carbon chain or ring system. Examples of epoxy-substituted functional groups include, but are not limited to, 2,3-epoxypropyl, 3,4-epoxybutyl, 4,5- epoxypentyl, 2,3-epoxypropoxy, epoxypropoxypropyl, 2-glycidoxyethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2-(glycidoxycarbonyl)propyl, 3-(3,4-epoxycylohexyl)propyl, 2-(3,4- epoxycyclohexyl)ethyl, 2-(2,3-epoxycylopentyl)ethyl, 2-(4-methyl-3,4- epoxycyclohexyl)propyl, 2-(3,4-epoxy-3-methylcylohexyl)-2-methylethyl, and 5,6- epoxyhexyl. The term "monovalent" as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. The term "hydrocarbon" or "hydrocarbyl" as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups. As used herein, the term "hydrocarbyl" refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (C1-C4)hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group. As used herein, the term "C6-10-5-6 membered heterobiaryl" means a C6-10 aryl moiety covalently bonded through a single bond to a 5- or 6-membered heteroaryl moiety. The C6-10 aryl moiety and the 5-6-membered heteroaryl moiety can be any of the suitable aryl and heteroaryl groups described herein. Non-limiting examples of a C6-10-5-6 membered heterobiaryl include
Figure imgf000017_0001
When the C6-10-5-6 membered heterobiaryl is listed as a substituent (e.g., as an "R" group), the C6-10-5-6 membered heterobiaryl is bonded to the rest of the molecule through the C6-10 moiety. As used herein, the term "5-6 membered- C6-10 heterobiaryl " is the same as a C6-10-5- 6 membered heterobiaryl, except that when the 5-6 membered- C6-10 heterobiaryl is listed as a substituent (e.g., as an "R" group), the 5-6 membered- C6-10 heterobiaryl is bonded to the rest of the molecule through the 5-6-membered heteroaryl moiety. As used herein, the term "C6-10- C6-10 biaryl" means a C6-10 aryl moiety covalently bonded through a single bond to another C6-10 aryl moiety. The C6-10 aryl moiety can be any of the suitable aryl groups described herein. Non-limiting example of a C6-10- C6-10 biaryl include biphenyl and binaphthyl. The term "solvent" as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids. The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X1, X2, and X3 are independently selected from noble gases" would include the scenario where, for example, X1, X2, and X3 are all the same, where X1, X2, and X3 are all different, where X1 and X2 are the same but X3 is different, and other analogous permutations. The term "room temperature" as used herein refers to a temperature of about 15 °C to 28 °C. The term "standard temperature and pressure" as used herein refers to 20 °C and 101 kPa. As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration. A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health. As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. As used herein, the term "efficacy" refers to the maximal effect (Emax) achieved within an assay. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. The terms "patient," "subject," or "individual" are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human. As used herein, the term "potency" refers to the dose needed to produce half the maximal response (ED50). A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound or compounds as described herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, or the symptoms of a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Homologous" as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means. The term "oligonucleotide" refers to short polynucleotides, such as from two (2) to 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which "U" replaces "T." As used herein, the term "3'-end" refers to the terminal residue of a DNA or RNA strand that terminates at the hydroxyl group of the 3' carbon in a deoxyribose (DNA) or ribose (RNA). As used herein, the term "5'-end" refers to the terminal residue of a DNA or RNA strand that terminates at a hydroxyl group of the 5' carbon in a deoxyribose (DNA) or ribose (RNA). A phosphate or phosphorothioate group can be attached to the hydroxyl group on the 5'-carbon. As used herein, the term "DNA" means deoxyribonucleic acid. As used herein, the term "RNA" means ribonucleic acid. Preparation of Compounds Compounds of formula I or formula II, or otherwise described herein, can be prepared by the general schemes described herein, using the synthetic method known by those skilled in the art. The following examples illustrate non-limiting embodiments of the compound(s) described herein and their preparation. In various embodiments, a compound of formula I, or a salt, enantiomer, diastereomer, or tautomer thereof:
Figure imgf000022_0001
wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonded to the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand. A schematic non-limiting diagram of the compound of formula I and, without being bound by theory, one mode of its action, is depicted in FIG.1. Oligonucleotide (ONA) Suitable salts include any of the pharmaceutically acceptable salts described herein. In various embodiments, the ONA is bonded to the LNK at the 3'-end or the 5'-end. In various embodiments, the ONA is bonded to the LNK at the 3'-end. In various embodiments, the ONA is bonded to the LNK at the 5' end. In various embodiments, the ONA comprises deoxyribonucleotide(s) (DNA), ribonucleotide(s) (RNA), or any combinations thereof. In various embodiments, the ONA comprises deoxyribonucleotide(s) (DNA). In various embodiments, the ONA comprises a c-Myc-binding nucleotide sequence. In various embodiments, at least two nucleotides flank each end of the c-Myc-binding nucleotide sequence. In various embodiments, 2 to 20 nucleotides flank each end of the c- Myc-binding nucleotide sequence. The number of flanking nucleotides at each end of the c- Myc-binding nucleotide sequence can be the same or different, and the sequences can be the same, different, reverse sequences, complementary sequences, or reverse complementary sequences. In various embodiments, the c-Myc-binding nucleotide sequence is flanked on each side by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In one embodiments, the c-Myc-binding nucleotide sequence comprises a sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% similar to 5'- CACGTGGTTGCCACGTG-3' (SEQ ID NO: 1). In various embodiments, the c-Myc-binding nucleotide sequence comprises the sequence 5'- CACGTGGTTGCCACGTG-3' (SEQ ID NO: 1).. In various embodiments, the c-Myc binding nucleotide sequence is 5'- TGGGAGCACGTGGTTGCCACGTGGTTGGG-3' (SEQ ID NO: 2) or 3'- GGGTTGGTGCACCGTTGGTGCACGAGGGT-5' (SEQ ID NO: 3). In various embodiments, the ONA comprises a brachyury-binding nucleotide sequence. In various embodiments, at least two nucleotides flank each end of the brachyury- binding nucleotide sequence. In various embodiments, 2 to 20 nucleotides flank each end of the brachyury-binding nucleotide sequence. The number of flanking nucleotides at each end of the brachyury-binding nucleotide sequence can be the same or different, and the sequences can be the same, different, reverse sequences, complementary sequences, or reverse complementary sequences. In various embodiments, the brachyury-binding nucleotide sequence is flanked on each side by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In various embodiments, the brachyury-binding nucleotide sequence comprises a sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% similar to 5'- AATTTCACACCTAGGTGTGAAATT-3' (SEQ ID NO: 4). In various embodiments, the brachyury-binding nucleotide sequence comprises the sequence 5'- AATTTCACACCTAGGTGTGAAATT-3' (SEQ ID NO: 4). In various embodiments, the brachyury-binding nucleotide sequence is 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3' (SEQ ID NO: 5) or 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5' (SEQ ID NO: 6). In various embodiments, at least one of the phosphodiester groups in the internucleotide linkages comprising the ONA is replaced with a non-bridging phosphorothioate (PS) group, in which the sulfur atom in the PS group does not connect to a ribose or deoxyribose. In various embodiments, all of the phosphate groups in the internucleotide linkages in the ONA are replaced with non-bridging phosphorothioate (PS) groups. A phosphorothioate group has the structure:
Figure imgf000024_0001
and can be present as a salt thereof. Suitable salts include any of the pharmaceutically acceptable salts described herein. In various embodiments, the salt is an sodium or potassium salt. In various embodiments, the terminal nucleotide in ONA at the 3'-end has the structure:
Figure imgf000024_0002
wherein: Y is H or OH; and
Figure imgf000024_0003
In various embodiments, the terminal nucleotide in ONA at the 5'-end has the structure:
Figure imgf000024_0004
wherein: Y is H or OH; Z is O or S; and
Figure imgf000025_0001
, , , , The
Figure imgf000025_0002
in the terminal nucleotide sugar, whether at the 3'-end or the 5'-end, designates the point of attachment of the rest of the nucleotides in ONA. The
Figure imgf000025_0003
in NUC designates the point of attachment of the respective base to the ribose or deoxyribose. In various embodiments, the LNK fragment is covalently bonded to the terminal nucleotide in ONA at the phosphorus atom in the 5'-phosphate, at an oxygen on the 5'-phosphate, at 3'- carbon in the ribose or deoxyribose through a -O-,-NR'-, -S-, -NRC(=O)-, -C(=O)NR'-, or - CR'2- group, wherein R' is H or C1-4 alkyl. Linker (LNK) In various embodiments, LNK has the structure: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-. In various embodiments, LNK has the structure: (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡ - ,CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100. In various embodiments, LNK has the structure: (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100. In various embodiments, LNK has the structure: (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡ - CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof. A given quantity of LL units can be used to form any suitable stable chemical structure, including branched, cyclic, and acyclic structures. In various embodiments, A is a heterocyclylene that contains one or more aliphatic carbons. In various embodiments, A is heteroarylene. In various embodiments, A is arylene. When LNK has the structure –(LL)aa–A–(LL)bb–, the LL units can be attached to any open valence(s) in A. For example, and without limitation, suitable structures for –(LL)aa–A–(LL)bb– include:
Figure imgf000027_0001
For example, and without limitation, suitable structures for –(LL)aa–A–(LL)bb– include:
Figure imgf000027_0002
In various embodiments, A is a C5-18 heteroaryl, such as but not limited to a 1,2,3- triazolyl or a 1,2,4-triazolyl. In various embodiments, A is
Figure imgf000028_0001
In various embodiments, LNK is –(LL)aa–A–(LL)bb–, wherein at each occurrence of LL is independently selected from the group consisting of -O-, -OR, -CH2-, -CHR'-, -C(=O)-, and -N(R)-. In various embodiments, LNK is –(LL)aa–A–(LL)bb–, wherein each occurrence of LL is at each occurrence independently selected from the group consisting of -O-, -N(R)-, - CR'R'-, -C(=O)-, and -C(=NR)-, wherein R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl, wherein R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, and (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; In various embodiments, LNK has the structure:
Figure imgf000028_0002
wherein at each occurrence X is independently selected from the group consisting of -O-, - CH2-, -CHR'-, and -C(=O)-, pp is an integer from 1 to 10, and m is an integer from 2 to 10. In the structure LNK-X1, any two X groups bonded to each other cannot be -O- such that (X)m does not contain a peroxide functionality. In various embodiments, X in LNK-X1 is independently at each occurrence -O- or -CH2-. Contemplated herein are all combinations of m, X, and pp in LNK-X1. In various embodiments, LNK has the structure:
Figure imgf000028_0003
wherein at each occurrence X is independently selected from the group consisting of -O-, - CH2-, -CHR'-, and -C(=O)-, pp is an integer from 1 to 10, and m is an integer from 2 to 10. In the structure LNK-X2, any two X groups bonded to each other cannot be -O- such that (X)m does not contain a peroxide functionality. In various embodiments, X in LNK-X2 is independently at each occurrence -O- or -CH2-. Contemplated herein are all combinations of m, X, and pp in LNK-X2. In various embodiments, LNK has the structure:
Figure imgf000029_0001
wherein pp is an integer from 1 to 10, X is CH2 or O, k is an integer from 1 to 3; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. In various embodiments, in LNK-1, X is CH2. In various embodiments, in LNK-1, X is O. In various embodiments, in LNK-1, k is 1. In various embodiments, in LNK-1, k is 2. In various embodiments, in LNK-1, k is 3. Contemplated herein are all combinations of k, X, and pp in LNK-1. In various embodiments, LNK has the structure:
Figure imgf000029_0002
wherein pp is an integer from 1 to 10, X is CH2 or O, k is an integer from 1 to 3; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. In various embodiments, in LNK-2, X is CH2. In various embodiments, in LNK-2, X is O. In various embodiments, in LNK-2, k is 1. In various embodiments, in LNK-2, k is 2. In various embodiments, in LNK-2, k is 3. Contemplated herein are all combinations of k, X, and pp in LNK-2. In various embodiments, aa is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In various embodiments, bb is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In various embodiments, zz is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. When LNK-X1, LNK-X2, LNK-1, or LNK-2 is bonded the 3'-end, the O* atom connects directly to the ribose or deoxyribose 3' carbon. When LNK-X1, LNK-X2, LNK-1, or LNK-2 is bonded the 5'-end, the O* atom connects directly to the phosphorus atom in the 5'-phosphate in the ribose or deoxyribose. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 1. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 2. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 3. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 4. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 5. In various embodiments, in LNK-X1, LNK- X2, LNK-1, or LNK-2 pp is 6. In various embodiments, in LNK-X1, LNK-X2, LNK-1, or LNK-2 pp is 7. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 8. In various embodiments, in LNK-X1, LNK-X2, LNK-1, or LNK-2 pp is 9. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 10. In various embodiments, pp in LNK-X1, LNK-X2, LNK-1, or LNK-2 is 2, 3, or 5. In certain embodiments, LNK comprises a group represented by a general structure selected from the group consisting of: -NR(CH2)n-(lower alkyl)-, -NR(CH2)n-(lower alkoxyl)- , -NR(CH2)n-(lower alkoxyl)-OCH2-, -NR(CH2)n-(lower alkoxyl)-(lower alkyl)-OCH2-, - NR(CH2)n-(cycloalkyl)-(lower alkyl)-OCH2-, -NR(CH2)n-(heterocycloalkyl)-, - NR(CH2CH2O)n-(lower alkyl)-O-CH2-, -NR(CH2CH2O)n-(heterocycloalkyl)-O-CH2-, - NR(CH2CH2O)n-Aryl-O-CH2-, -NR(CH2CH2O)n-(heteroaryl)-O-CH2-, -NR(CH2CH2O)n- (cycloalkyl)-O-(heteroaryl)-O-CH2-, -NR(CH2CH2O)n-(cycloalkyl)-O-Aryl-O-CH2-, - NR(CH2CH2O)n-(lower alkyl)-NH-Aryl-O-CH2-, -NR(CH2CH2O)n-(lower alkyl)-O-Aryl- CH2, -NR(CH2CH2O)n-cycloalkyl-O-Aryl-, -NR(CH2CH2O)n-cycloalkyl-O-(heteroaryl)l-, - NR(CH2CH2)n-(cycloalkyl)-O-(heterocycle)-CH2, -NR(CH2CH2)n-(heterocycle)- (heterocycle)-CH2, -N(R1R2)-(heterocycle)-CH2; wherein: each n of each linker can be independently 0 to 10; each R of the linker can be independently H or lower alkyl; each R1 and R2 of each linker can independently form a ring with the connecting N. Lower alkyl groups include linear or branched C1-4 alkyl groups. In certain embodiments, LNK comprises a group represented by a general structure selected from the group consisting of: -N(R)-(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-OCH2-, -O-(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-OCH2-, -O-(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-O-; -N(R)-(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-O-; -(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-O-; -(CH2)m-O(CH2)n-O(CH2)o-O(CH2)p-O(CH2)q-O(CH2)r-OCH2-;
Figure imgf000031_0001
Figure imgf000032_0001
each m, n, o, p, q, and r of each linker are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein, if n = 0, there is no N-O or O-O bond; each R of each linker is independently H, methyl, or ethyl; each X of each linker is independently H or F;
Figure imgf000032_0002
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
In any aspect or embodiment described herein, the linker (L) is selected from the group consisting of:
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
wherein each occurrence of m and n is independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6. In various embodiments, LNK is selected from the group consisting of:
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
- 58 - 9.2
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000068_0002
wherein each occurrence of m, n, o, p, q, and r is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In various embodiments, LNK is selected from the group consisting of:
Figure imgf000068_0003
;
Figure imgf000069_0001
;
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
;
Figure imgf000074_0001
;
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
E3 Ubiquitin Ligase-Recruiting Ligand (UBL) In various embodiments, the UBL is a ligand of an E3 ubiquitin ligase selected from the group consisting of von Hippel-Lindau (VHL), cereblon (CRBN), RING-type zinc-finger protein 114 (RNF114), cellular inhibitor of apoptosis (cIAP), mouse double minute 2 homologue (MDM2), damage-specific DNA binding protein 1 (DDB1)-CUL4 associated factor 16 (DCAF16), and Kelch-like ECH-associated protein 1 (KEAP1). In various embodiments, UBL is a ligand of VHL or CRBN. Any suitable E3 ubiquitin ligase recruiting ligand can be incorporated into the compounds of formula I. For example, suitable E3 ubiquitin ligase ligands and their syntheses can be found in Bricelj, A. et al., Front. Chem., 05 July 2021, DOI: 10.3389/fchem.2021.707317. In various embodiments, the UBL is a ligand having the structure:
Figure imgf000078_0001
wherein R1 is H or CH3; and R2 is selected from the group consisting of
Figure imgf000078_0002
In the CRBN ligands, the attachment point shown in structures such as
Figure imgf000078_0003
can be through any suitable atom/fragment as defined in LL herein. For example, and the without limitation, the CRBN ligands can be attached via any of the following connectivities:
Figure imgf000079_0001
In various embodiments, the UBL has the structure:
Figure imgf000079_0002
In various embodiments, the compound of formula I has the structure of:
Figure imgf000079_0003
Figure imgf000080_0001
wherein pp is an integer from 1 to 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Modified Oligonucleotides In various embodiments, a compound of formula II is provided: ONA–LNK–C ≡C-H (formula II), wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is a chemical linker connecting the ONA and the C ≡C-H. In various embodiments, LNK has any linker structure described elsewhere herein. In various embodiments, LNK has the structure: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-. In various embodiments, LNK has the structure: (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡- CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100. In various embodiments, LNK has the structure: (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100. In various embodiments, LNK has the structure: (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡ ^ ^-CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof. In various embodiments, the terminal nucleotide at the 3'-end of ONA has the structure:
Figure imgf000082_0001
, wherein: Y is H or OH; and
Figure imgf000082_0002
In various embodiments, the terminal nucleotide at the 5'-end of ONA has the structure:
Figure imgf000083_0001
, wherein: Y is H or OH Z is O or S; and
Figure imgf000083_0002
In various embodiments, the compound of formula II has the structure:
Figure imgf000083_0003
wherein at each occurrence Y is independently selected from the group consisting of - O-, -CH2-, -CHR'-, and -C(=O)-, and d is an integer from 2 to 15. In the structure II-A, any two Y groups cannot be -O- such that (Y)d does not contain a peroxide functionality. In various embodiments, Y in II-A is independently at each occurrence -O- or -CH2-; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. In various embodiments, d in II-A is 2. In various embodiments, d in II-A is 3. In various embodiments, d in II-A is 4. In various embodiments, d in II-A is 5. In various embodiments, d in II-A is 6. In various embodiments, d in II-A is 7. In various embodiments, d in II-A is 8. In various embodiments, d in II-A is 9. In various embodiments, d in II-A is 10. In various embodiments, d in II-A is 11. In various embodiments, d in II-A is 12. In various embodiments, d in II-A is 13. In various embodiments, d in II-A is 14. In various embodiments, d in II-A is 15. In various embodiments, the compound of formula II has the structure:
Figure imgf000084_0001
wherein pp is an integer from 1 to 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Degradation of c-Myc by oligoTRAFTACs Myc transcription factors are dysregulated in a range of cancers. Even though c-Myc has been targeted by indirect approaches, development of direct-targeting methods is hindered by the paucity of ligandable pockets and their highly disordered structure. Since c- Myc binds to specific DNA sequences with a conserved E-box sequence (CACGTG), we sought to incorporate a c-Myc binding consensus DNA-sequence into the oligoTRAFTAC (OT) design. A Myc binding consensus sequence (5' CACGTGGTTGCCACGTG 3') was taken from one of its target gene promoters. Additionally, a flanking sequence was included at both 3' and 5' end of the c-Myc- targeting oligonucleotide sequence to facilitate successful double strand formation of the recognition sequence while providing a flexibility for oligoTRAFTACs. A c-Myc-targeting oligonucleotide sequence was synthesized with an orthogonal alkyne handle on either side as a reactive moiety to append an azide-containing VHL ligand (FIG.7A). Copper-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction was performed to synthesize two c-Myc targeting oligoTRAFTACs (OT7: VHL ligand at the 5' end and OT10: VHL ligand at the 3' end of the oligonucleotide) (FIG.7B, 7C). After 18 h at room temperature, the crude mixture was purified and analyzed by HPLC to verify reaction completion (FIG.8A). Furthermore, the reaction was also monitored by electrophoretic mobility shift assay (EMSA) (FIG.8B). The single stranded oligoTRAFTACs were then purified by HPLC, dried and reconstituted in water. To generate double stranded oligoTRAFTACs, an annealing reaction was performed in the presence of the reverse complementary oligonucleotide by heating to 950C and slowly cooling to room temperature over a 1.5 h period. A biotinylated version of the same c-Myc-targeting oligonucleotide was also synthesized to generate a double stranded oligonucleotide via the same annealing reaction conditions. A biotin-pulldown experiment was conducted with the c-Myc-targeting biotin- oligonucleotide and scrambled control to confirm that the selected oligonucleotide sequence binds c-Myc. After incubation of biotin probes with HeLa cell lysate and their capture using streptavidin agarose, the eluates were probed with c-Myc antibody (FIG.2A). The data indicated successful c-Myc engagement with the biotin-oligonucleotide probe compared to its scrambled version, thereby predicting c-Myc recruitment by the oligoTRAFTAC. c-Myc degradation was tested upon transfection of increasing concentration of OT7 into HeLa cells. After 20 h post transfection, western blot analysis of the cell lysates indicated that OT7 can induce significant c-Myc degradation at 50 nM (FIG.2B). A similar pattern was observed in HEK293T cells in response to OT7 transfection (FIG.8C). To test the kinetics of oligoTRAFTAC-mediated c-Myc degradation we performed a post-transfection washout experiment. After 6 h or 12 h post-transfection, cell culture medium was replaced with fresh medium and incubated for a total of 20 h. When cells were transfected for 6 h prior to washout, we observed very low c-Myc degradation levels relative to the 12 h transfection (FIG.8D). Furthermore, the degradation was maintained for 24 h (total of 36 h) post-washout indicating a possible catalytic behavior of oligoTRAFTACs (FIG.8E). After confirming that c-Myc is susceptible to oligoTRAFTAC-mediated degradation, whether the observed degradation occurs through the proteasomal pathway was studied. To address this, neddylation inhibition was evaluated, which disrupts cullin RING E3 ligase function, affects the activity of the c-Myc-targeting oligoTRAFTACs. When cells were pre- incubated with MLN-4924, c-Myc degradation was significantly diminished, demonstrating that OT7 and OT10-mediated c-Myc degradation is neddylation-dependent and occurs via the proteasomal pathway (FIG.2C). Next, a VHL ligand competition experiment was performed to confirm that oligoTRAFTAC-mediated c-Myc degradation is VHL-dependent. Cells were preincubated in the presence or absence of excess VHL ligand for 1.5 h before transfecting oligoTRAFTACs for 20 h. The observed c-Myc levels in the VHL ligand-incubated cells were maintained relative to the cells that were not incubated with VHL ligand (FIG.2D). Overall, the data indicated that OT7 and OT10 induces c-Myc degradation via the proteasomal pathway. To test the effect of c-Myc degradation on cell proliferation, the cell viability in response to the transfection of OT7 and OT12 was assessed. As the data indicate, the induction of OT7-mediated c-Myc degradation inhibited cell proliferation compared to scrambled control-transfected cells (FIG.8E). OligoTRAFTAC-mediated brachyury degradation T-box transcription factor (brachyury) is a DNA-interacting TF that regulates gene expression during early embryonic development in vertebrates. Brachyury is crucial for early mesoderm formation and plays a key role in notochord development where, as a homodimer, it recognizes and binds to a consensus palindromic DNA sequence. To investigate brachyury degradation by oligoTRAFTACs, a brachyury-binding DNA sequence (AATTTCACACCTAGGTGTGAAATT) was adapted as the brachyury-recruiting element in our new oligoTRAFTACs design. Oligonucleotides were synthesized with flanking bases and terminal alkyne moieties at either end (3' and 5') of the oligonucleotide (FIG.7A, right panel). To generate brachyury-targeting oligoTRAFTACs, two azido-VHL ligands were synthesized with a long (5 PEG units) and a short (2 PEG units) linker (FIG.7C, FIG.9A). To test whether the oligonucleotide used in oligoTRAFTAC design could recruit brachyury, a streptavidin pull-down experiment was performed using a biotin-oligonucleotide. After incubating the brachyury targeting biotin-oligonucleotide or its scrambled version for 1.5 h with the lysate of HEK293T cells that stably express a brachyury-GFP fusion, streptavidin beads were added to cell lysates and incubated for another 16 h at 40C to capture the binary complex. After several washings, eluted fractions were analyzed by western blotting. Biotin pulldown data indicated that the oligonucleotide used in brachyury-targeting oligoTRAFTAC design is engaged with its target protein (FIG.3A). In contrast, scrambled oligonucleotide failed to bind brachyury-GFP, indicating sequence-specific brachyury recruitment. To test whether a brachyury-targeting oligoTRAFTAC induces degradation, brachyury-oligoTRAFTACs were transfected into brachyury-GFP-expressing HEK293T cells for 24 h, after which cells were collected and lysed, followed by western blotting. OligoTRAFTACs with 5' VHL-ligand and a longer linker (OT1) did not induce significant degradation of brachyury-GFP (FIG.9B), while its counterpart the shorter linker (OT2) successfully induced target degradation. However, oligoTRAFTACs with 3' VHL-ligand (OT3 and OT4) both showed comparable, better degradation profiles relative to OT1 and OT2 (FIG.9B, last two panels). Next, different concentrations of OT3 and OT4 were transfected into HEK293T cells and lysed after 20 h. Data indicated that both OT3 and OT4 could induce significant brachyury-GFP degradation at 50 nM concentration within 20 h of transfection (FIG.3B). Furthermore, the time course experiment suggested that OT3 can induce significant brachyury-GFP degradation at 12 h that is maintained up to 36 h post-transfection (FIG.3C and FIG.10A). A washout experiment was performed to determine the minimum incubation time to induce a noticeable brachyury degradation by OT3. To address this question, we transfected 75 nM of OT3 into HEK293T cells expressing brachyury-GFP followed by replacement of the transfection medium after 6 h and 12 h with fresh cell culture medium (FIG.10B). Degradation data indicated that OT3 incubated for at least 12 h induces significant brachyury degradation comparable to continuous 24 h incubation, whereas OT3 incubation for 6 h did not induce significant brachyury degradation (FIG.3D, center panel). Surprisingly and unexpectedly, brachyury degradation was prominent even after the cells incubated in fresh medium for longer time (36 h), which is consistent with oligoTRAFTAC OT7 targeting c-Myc-targeting (FIG.3D, last panel). However, brachyury levels at 36 h had increased relative to the 24 h period, indicating a progressive loss of brachyury degradability by OT3. This could be partially explained by the reduced stability of oligoTRAFTACs due to the oligonucleotide sensitivity towards intracellular nucleases, increased deubiquitinase (DUB) activity, or increased c-Myc resynthesis. To test whether the oligoTRAFTAC-mediated brachyury recruitment and degradation is oligonucleotide-dependent, a scrambled- oligoTRAFTACs (OT5 and OT6) was synthesized and tested for brachyury degradation in cells. Consistently, OT3 induced a robust brachyury-GFP degradation, whereas neither OT5 nor OT6 induced significant brachyury degradation (FIG.4A, FIGs. 11A-11B). A VHL ligand competition experiment was performed to confirm brachyury degradation is dependent on VHL. In this experiment, cells were pretreated with 100-fold excess VHL ligand prior to OT3 treatment. VHL competition rescued oligoTRAFTAC-mediated brachyury degradation (FIG.4B), confirming that observed brachyury degradation is a result of VHL E3 ligase recruitment by OT3. Furthermore, OT3-mediated brachyury degradation was evaluated in the presence of a neddylation inhibitor to further confirm that the intended mechanism is via the proteasomal pathway. Similar to the VHL competition experiment, the neddylation inhibitor, MLN-4924 was pre-incubated with cells at 1 mM concentration. After 1.5 h, OT3 was transfected into the cell for 20 h following which the cell lysates were analyzed as indicated in FIG.4C. The data indicated that OT3 could not induce brachyury degradation in the presence of MLN- 4924, showing that neddylation is crucial for the mechanism of action (FIG.4C). Furthermore, analysis of GFP-fluorescence confirmed brachyury degradation only in the absence of MLN-4924 (FIG.4D). Overall, these data support the fact that OT3-induced brachyury degradation is mediated through the proteasomal pathway and is dependent on the oligonucleotide sequence of OT3. OligoTRAFTACs induce brachyury degradation in chordoma cells Increasing OT3 concentrations were transfected into UM-Chor1 cells followed by western blotting to determine brachyury degradation. Consistent with the brachyury-GFP degradation in HEK293T cells, 60 nM of OT3 induces ~70% brachyury degradation in UM- Chor1 cells 24 h post-transfection (FIG.11B). Although OT3 induced comparable levels of brachyury degradation in both cell lines, phosphodiester linkages within OT3 are susceptible to cleavage by both extra- and intracellular nucleases. Therefore, oligoTRAFTACs with a phosphorothioate (PS) backbone were synthesized. The addition of extra non-bridging sulfur atoms in the inter-nucleotide phosphate group has been shown to have both increased stability against nucleases and improved cell permeability. A brachyury-targeting oligoTRAFTAC, OT17, was synthesized by incorporating PS bonds throughout the oligo sequence. Increasing OT17 concentrations were transfected into chordoma cells (UM-Chor1) for 24 h following which the cells were lysed and probed for brachyury levels -- western blotting showed that OT17 induced significant degradation of endogenous brachyury even at 15 nM (FIG.5A). A similar degradation pattern was noticed in another chordoma cell line, JHC-7 (FIG.5B), although OT17 was slightly less potent in them, requiring 30 nM to induce brachyury knockdown comparable to UM-Chor1 cells. Time course experiment showed that OT17 induced a modest degradation at 8 h, which increased to ~ 60% knockdown 16 h post-transfection (FIG.5C) although washout experiment indicated that incubation of transfection complex for 12 h is sufficient to induce significant brachyury degradation (FIG.5D). Furthermore, this same experiment displayed persistent degradation for 24 hours post washout (total of 36 h of post-transfection), suggesting an increased stability and possible catalytic mechanism of OT17. A scrambled oligoTRAFTAC with a PS backbone throughout the oligonucleotide sequence (OT20) was also synthesized and tested. Consistently, side-by-side comparison of brachyury levels UM- Chor1 in cells transfected with OT17 and OT20 indicated sequence-specific brachyury degradation (FIG.5E). To monitor oligoTRAFTAC-mediated brachyury ubiquitination resulting from induced proximity to VHL, HA-ubiquitin was transfected into HEK293 cells overexpressing brachyury-GFP. After cells pre-incubated with proteasome inhibitor epoxomicin (500 nM), OT3 and OT17 were transfected and incubated for 12 hours. Subsequent immunoprecipitation data indicated that OT3 and OT17 could induce ubiquitination of brachyury-GFP, confirming that degradation by oligoTRAFTAC follows a ubiquitination event mediated by recruited VHL (FIG.5F). Furthermore, the proteasome inhibition experiment indicated that OT17-mediated brachyury degradation occurred via the proteasome (FIG.11C). In vivo activity of oligoTRAFTACs: OT17-mediated developmental defects in zebrafish tail formation After confirming that brachyury-targeting oligoTRAFTACs induce efficient endogenous brachyury degradation in cells, we next evaluated their activity in animals using zebrafish as a model organism. Although brachyury overexpression in adult tissue is one of the key factors that leads to tumorigenesis in chordoma, brachyury is widely known for its essential biological activity in vertebrate notochord formation at early stages of embryonic development. To test for oligoTRAFTAC in vivo activity, tail deformation in OT3-injected zebrafish embryos was examined relative to mock-injected embryos (FIG.6A). Interestingly, although OT3 could induce brachyury degradation in cells, OT3 did not induce tail deformation in zebrafish, possibly due to the sensitivity of its phosphodiester backbone to nucleases. However, microinjection of OT17, in which PS linkages provide stability against both exonucleases and endonucleases, induced tail deformation in ~70% of injected embryos (FIG.6B, 6C) whereas <5% mock and scrambled OT20 injected embryos had defective tails. This result illustrates how the observed brachyury-mutant/null phenotype is sequence- dependent, and demonstrates in vivo oligoTRAFTAC activity in zebrafish. The majority of transcription factors, key mediators of gene expression, are considered undruggable. In this study, we developed a method for oligonucleotide-dependent TF recruitment and degradation by the proteasomal pathway. Described herein are coupled TF-binding short DNA sequences from target gene promoters with VHL ligands to create bifunctional molecules for the targeted degradation of those same TFs. Since the binding sequences already have been identified for many TFs, and since synthetic routes for oligonucleotide synthesis are well established and economical, these oligonucleotide sequences can be rapidly employed in oligoTRAFTAC design to use as a versatile tool for both basic discovery biology and therapy development. The TRAFTACs described herein were synthesized by directly attaching a VHL ligand to c-Myc or brachyury-binding oligonucleotide sequences via click chemistry. OligoTRAFTACs targeting c-Myc TF displayed a robust degradation. Addition of VHL ligand to either side of the oligonucleotide did not significantly alter its ability to induce c- Myc degradation. This can be partially attributed to the flexibility of oligoTRAFTACs provided by the extra flanking nucleotides between the VHL ligand and TF-recruiting oligonucleotide. Other oligoTRAFTACs used in this study induced the degradation of ectopically expressed brachyury-GFP and endogenous brachyury via the proteasomal pathway. Although washout experiments indicated that oligoTRAFTACs are not as kinetically efficient as conventional PROTACs, the transient binding nature of oligoTRAFTACs to both TF and VHL proteins suggests the probability of a similar catalytic degradation mechanism. Moreover, the compositions and methods described herein represent the first evidence of the degradability of untagged, endogenous brachyury in multiple chordoma cell lines. In the first attempt to test oligoTRAFTAC in vivo activity, OT3 (containing a phosphodiester backbone) could not induce the intended defective tail phenotype in zebrafish. It is noteworthy that first generation TRAFTACs, where oligonucleotides are partly protected by the ribonuclear complex, induced defective tails in zebrafish. Therefore, a potential reason for the failure of OT3 to induce the intended phenotype within the embryos may be their high sensitivity and exposure of oligoTRAFTACs to embryonic nucleases present at the early stages of development. Therefore, phosphodiester oligoTRAFTACs might not endure in vivo at the intracellular concentrations needed to achieve a significant brachyury degradation and the intended phenotype. The microinjection of phosphorothioate backbone-containing OT17 resulted in a significantly high occurrence rate of the defective tail phenotype compared to the mock or scrambled oligoTRAFTAC (OT20), confirming the increased stability and resistance of OT17 to nucleases. Overall, oligoTRAFTACs are programmable heterobifunctional molecules comprised of a TF-binding oligonucleotide sequence and a VHL binding ligand, which induce TF degradation in cells and displayed robust in vivo activity. Due to the simple and modular nature of their structure, oligoTRAFTACs can be rapidly designed for many non-ligandable DNA-binding TFs both for use as a chemical biology tool as well as a potential therapeutic strategy. It has been shown that multiple TFs recognize and bind to similar DNA sequences.43 Since TFs, such as TCF3, also recognizes a similar E-box consensus sequence as c-Myc, the evaluation of TCF3 degradation indicated a sequence-dependent off-target activity of OT7 (FIG.8F). To address the sequence specificity of oligoTRAFTACs, we tested NF-kB (p65) protein levels in response to brachyury-targeting OT17. We observed a consistent brachyury degradation in OT17-treated cells. However, since p65 does not recognize brachyury-binding DNA sequence, OT17 did not induce p65 degradation. Therefore, the data suggest a sequence-dependent brachyury degradation by OT17 (FIG.8G). Furthermore, in the current study, we report for the first time the use of PS modified oligonucleotides in oligoTRAFTAC design to improve its in vivo stability and demonstrated oligoTRAFTAC activity in zebrafish. Without being bound by theory, due to the faster degradation profile and catalytic nature, oligoTRAFTACs can exhibit improved therapeutic benefits than other oligonucleotide- based strategies such as RNAi. Overall, oligoTRAFTACs are programmable heterobifunctional molecules comprised of a TF-binding oligonucleotide sequence and a VHL binding ligand, which induce TF degradation in cells and displayed robust in vivo activity. Due to the simple and modular nature of their structure, oligoTRAFTACs can be rapidly designed for many non-ligandable DNA-binding TFs both for use as a chemical biology tool as well as a potential therapeutic strategy. The compounds described herein can possess one or more stereocenters, and each stereocenter can exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography. The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound(s) described herein, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form. In certain embodiments, the compound(s) described herein can exist as tautomers. All tautomers are included within the scope of the compounds presented herein. In certain embodiments, compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, sites on, for example, the aromatic ring portion of compound(s) described herein are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group. Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels. The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein. Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein. In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In other embodiments, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal. In certain embodiments, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable. In certain embodiments, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co- existing amino groups are blocked with fluoride labile silyl carbamates. Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react. Typically blocking/protecting groups may be selected from:
Figure imgf000094_0001
. Other protecting groups, plus a detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosure. Compositions The compositions containing the compound(s) described herein include a pharmaceutical composition comprising at least one compound as described herein and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Methods of Treatment, Amelioration, and/or Prevention The disclosure includes a method of treating, ameliorating, and/or preventing cancer using the compounds of formula I. Non-limiting examples of cancer include squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, renal cell carcinoma, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; multiple myeloma, sarcomas, including Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor, and teratocarcinomas. The methods described herein include administering to the subject a therapeutically effective amount of at least one compound described herein, which is optionally formulated in a pharmaceutical composition. In various embodiments, a therapeutically effective amount of at least one compound described herein present in a pharmaceutical composition is the only therapeutically active compound in a pharmaceutical composition. In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent that treats cancer. In certain embodiments, administering the compound(s) described herein to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating a cancer in the subject. For example, in certain embodiments, the compound(s) described herein enhance(s) the activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect. In certain embodiments, the compound(s) described herein and the therapeutic agent are co-administered to the subject. In other embodiments, the compound(s) described herein and the therapeutic agent are coformulated and co-administered to the subject. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human. Combination Therapies The compounds useful within the methods described herein can be used in combination with one or more additional therapeutic agents useful for treating, ameliorating, and/or preventing cancer. These additional therapeutic agents may comprise compounds that are commercially available or synthetically accessible to those skilled in the art. These additional therapeutic agents are known to treat, ameliorate, prevent, and/or reduce the symptoms, of cancer. In certain embodiments, the compounds described herein can be used in combination with radiation therapy. In other embodiments, the combination of administration of the compounds described herein and application of radiation therapy is more effective in treating, ameliorating, or preventing cancer than application of radiation therapy by itself. In yet other embodiments, the combination of administration of the compounds described herein and application of radiation therapy allows for use of lower amount of radiation therapy in treating the subject. In various embodiments, a synergistic effect is observed when a compound as described herein is administered with one or more additional therapeutic agents or compounds. A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul.22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. Administration/Dosage/Formulations The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of cancer. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions described herein to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat cacner in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat cancer in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation. Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound. In certain embodiments, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account. The compound(s) described herein for administration may be in the range of from about 1 µg to about 10,000 mg, about 20 µg to about 9,500 mg, about 40 µg to about 9,000 mg, about 75 µg to about 8,500 mg, about 150 µg to about 7,500 mg, about 200 µg to about 7,000 mg, about 350 µg to about 6,000 mg, about 500 µg to about 5,000 mg, about 750 µg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween. In some embodiments, the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. In certain embodiments, a composition as described herein is a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of cancer in a patient. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein. Oral Administration For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent. For oral administration, the compound(s) described herein can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid). Compositions as described herein can be prepared, packaged, or sold in a formulation suitable for oral or buccal administration. A tablet that includes a compound as described herein can, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, dispersing agents, surface-active agents, disintegrating agents, binding agents, and lubricating agents. Suitable dispersing agents include, but are not limited to, potato starch, sodium starch glycollate, poloxamer 407, or poloxamer 188. One or more dispersing agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more dispersing agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Surface-active agents (surfactants) include cationic, anionic, or non-ionic surfactants, or combinations thereof. Suitable surfactants include, but are not limited to, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, carbethopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridine chloride, didecyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, domiphen bromide, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, tetramethylammonium hydroxide, thonzonium bromide, stearalkonium chloride, octenidine dihydrochloride, olaflur, N-oleyl-1,3-propanediamine, 2-acrylamido-2-methylpropane sulfonic acid, alkylbenzene sulfonates, ammonium lauryl sulfate, ammonium perfluorononanoate, docusate, disodium cocoamphodiacetate, magnesium laureth sulfate, perfluorobutanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate, sodium laurate, sodium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium nonanoyloxybenzenesulfonate, sodium pareth sulfate, sodium stearate, sodium sulfosuccinate esters, cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide diethanolamine, cocamide monoethanolamine, decyl glucoside, decyl polyglucose, glycerol monostearate, octylphenoxypolyethoxyethanol CA-630, isoceteth-20, lauryl glucoside, octylphenoxypolyethoxyethanol P-40, Nonoxynol-9, Nonoxynols, nonyl phenoxypolyethoxylethanol (NP-40), octaethylene glycol monododecyl ether, N-octyl beta- D-thioglucopyranoside, octyl glucoside, oleyl alcohol, PEG-10 sunflower glycerides, pentaethylene glycol monododecyl ether, polidocanol, poloxamer, poloxamer 407, polyethoxylated tallow amine, polyglycerol polyricinoleate, polysorbate, polysorbate 20, polysorbate 80, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, and Tween 80. One or more surfactants can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more surfactants can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Suitable diluents include, but are not limited to, calcium carbonate, magnesium carbonate, magnesium oxide, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate, Cellactose ® 80 (75 % ^- lactose monohydrate and 25 % cellulose powder), mannitol, pre-gelatinized starch, starch, sucrose, sodium chloride, talc, anhydrous lactose, and granulated lactose. One or more diluents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more diluents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Suitable granulating and disintegrating agents include, but are not limited to, sucrose, copovidone, corn starch, microcrystalline cellulose, methyl cellulose, sodium starch glycollate, pregelatinized starch, povidone, sodium carboxy methyl cellulose, sodium alginate, citric acid, croscarmellose sodium, cellulose, carboxymethylcellulose calcium, colloidal silicone dioxide, crosspovidone and alginic acid. One or more granulating or disintegrating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more granulating or disintegrating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Suitable binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, anhydrous lactose, lactose monohydrate, hydroxypropyl methylcellulose, methylcellulose, povidone, polyacrylamides, sucrose, dextrose, maltose, gelatin, polyethylene glycol. One or more binding agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more binding agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Suitable lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, hydrogenated castor oil, glyceryl monostearate, glyceryl behenate, mineral oil, polyethylene glycol, poloxamer 407, poloxamer 188, sodium laureth sulfate, sodium benzoate, stearic acid, sodium stearyl fumarate, silica, and talc. One or more lubricating agents can each be individually present in the composition in an amount of about 0.01% w/w to about 90% w/w relative to weight of the dosage form. One or more lubricating agents can each be individually present in the composition in an amount of at least, greater than, or less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% w/w relative to weight of the dosage form. Tablets can be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patent Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Tablets can also be enterically coated such that the coating begins to dissolve at a certain pH, such as at about pH 5.0 to about pH 7.5, thereby releasing a compound as described herein. The coating can contain, for example, EUDRAGIT ® L, S, FS, and/or E polymers with acidic or alkaline groups to allow release of a compound as described herein in a particular location, including in any desired section(s) of the intestine. The coating can also contain, for example, EUDRAGIT ® RL and/or RS polymers with cationic or neutral groups to allow for time controlled release of a compound as described herein by pH-independent swelling. Parenteral Administration For parenteral administration, the compounds as described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used. Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as such as lauryl, stearyl, or oleyl alcohols, or similar alcohol. Additional Administration Forms Additional dosage forms suitable for use with the compound(s) and compositions described herein include dosage forms as described in U.S. Patents Nos.6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms suitable for use with the compound(s) and compositions described herein also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757. Controlled Release Formulations and Drug Delivery Systems In certain embodiments, the formulations described herein can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use with the method(s) described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In some cases, the dosage forms to be used can be provided as slow or controlled- release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions described herein. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the compositions and dosage forms described herein. Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects. Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient. In one embodiment, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. In one embodiment, the compound(s) described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Dosing The therapeutically effective amount or dose of a compound described herein depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors. A suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the compound(s) described herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection. The compounds described herein can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized. Examples Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein. Materials, Reagents, and Methods All cell culture media and fetal bovine serum (DMEM, IMDM, RPMI, DMEM/F-12) were purchased from Gibco unless otherwise specified. Primary antibodies for GAPDH (2118S), Vinculin (13901S), brachyury (81694S), HA-tag (3724S), protein A magnetic beads (73778S) and streptavidin magnetic beads (5947S) were purchased from Cell Signaling Technologies. Primary antibody for c-Myc (sc-40) was purchased from Santa Cruz. Secondary rabbit (NA934) and mouse (NA931) antibodies were purchased from GE Health Care. RNAiMAX (13778-150) transfecting reagent was purchased from Thermo Fisher Scientific. MLN4924 (S7109) was purchased from Selleckchem. Copper (II) sulfate pentahydrate (209198) and L-Ascorbic acid (A4403) were purchased from Millipore Sigma, and THPTA purchased from Lumiprobe (H4050). All the oligonucleotide modifiers were purchased from Glen Research, 3' Alkyne (20-2992-41), 5' Alkyne (10-1992-90) and biotin (10-5950-90). All the oligonucleotides were custom synthesized by Yale Keck oligo synthesis facility. General Biology Methods Cell Culture Human embryonic kidney cells HEK293T cells and HeLa cells were grown in Dulbecco's Modified Eagles Medium (DMEM) containing 10% heat inactivated fetal bovine serum (FBS), streptomycin (5 ^g/mL) and 5 U/mL penicillin 95 U/mL). All cell lines were maintained and cell culture experiments were carried out in humidified incubators at 37 degrees and 5% CO2 supplementation. OligoTRAFTAC transfection One day prior to oligoTRAFTACs transfection, cells (HEK293T: 0.7X106/well, HeLa: 0.2 X106/well, UM-Chor1: 0.2X106/well and JHC7: 0.4X106/well) were propagated into 6-well plates containing appropriate complete growth medium. Prior to transfection, complete medium was replaced with 1.75 mL of transfection medium (2%FBS, no Penstrep). Chimeric oligoTRAFTAC transfection was performed using RNAi-Max reagent according to the protocols provided by the manufacture. All transfections were carried out in 6-well plates with 2 mL of media and concentrations of oligoTRAFTACs were calculated according to this volume (2 mL). Briefly, for 50 nM concentration, 4 μL from a 25 μM oligo TRAFTAC stock was added to a tube containing 125 μL of OPTIM-MEM and 12.5 μL of RNAi-Max reagent was added to a separate tube containing 125 μL of OPTIM-MEM (added ~4 μg of oligoTRAFTAC to 2 mL cell culture medium). Two tubes were incubated for 5 minutes at room temperature and oligoTRAFTAC containing OPTI-MEM was then slowly added to the second tube with RNAi-MAX. The solution in the tube was mixed well by pipetting up and down several times. After incubating for 10 minutes at room temperature, 250 μL of oligoTRAFTAC:RNAi-MAX complex was added drop wise onto cells containing the transfection medium. Transfection medium was mixed well before transferring the 6-well plate into the incubator. After appropriate time, cells were either harvested or transfection medium containing oligoTRAFTAC:RNAi-MAX complex was replaced with fresh medium and incubated for desired time point prior to harvesting. For MLN-4924 and VHL ligand competition assay, these molecules were pre-incubated in the transfection medium (1.75 mL) for 1 h prior to transfection of oligoTRAFTACs. Cell lysates were prepared by incubating cells in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF) on ice for 30 minutes and cell lysate was clarified by centrifugation at high speed (15000 rpm) for 20 minutes. Clear supernatant was collected for further experiments. For cell proliferation/viability assays, cells were split (HeLa: 0.1 x105/well, UM-Chor1: 0.7x104/well and JHC7: 0.1 x105/well) into white, clear bottom 96-well plates. Transfection was carried out as described above, except volumes, i.e., 175 μL of transfection medium per one well, 0.5 μL of RNAi-MAX/well and 25 μL of OPTIMEM per well. Click Reaction Alkyne-modified oligonucleotides were dissolved in ultra-pure water at 500 μM concentrations and azide-modified VHL Ligands were dissolved in DMSO at 10 mM. Right before the click reaction, fresh stock solutions of Cu (II) sulfate pentahydrate (CuSO4 ·5H2O) (50 mM in water), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA: 100 mM in DMSO) and sodium ascorbate (100 mM in water) were made. The click reaction was carried out in 50% DMSO solution. First, alkyne modified oligonucleotide (250 μL) and azide-modified VHL Ligand (1:5 molar ratio) mixed in tube 1. Then, Cu (II) sulfate pentahydrate and THPTA was mixed first, followed by the addition of sodium ascorbate to be final molar ratio of 1:2:2. A 37-fold molar excess of Cu-THPTA complex was added to tube 1 and water and DMSO were added to get the final reaction mixture with 50% DMSO. Click reaction mixture was mixed thoroughly and flushed with inert gas (N2) for 1 minute. The reaction mixture was then incubated at room temperature for ~ 16 h. Click reaction product was purified by reverse phase high-performance liquid chromatography (HPLC) using a C18 column. HPLC method used for oligo purification (Buffer A-5% acetonitrile, 4.25% triethylamine acetate (TEAA) in water; Buffer B- 100% acetonitrile (ACN). The program was set with a flow rate of 5 mL/min for 150 minutes, and a gradient of ACN increasing from 0-80%. Annealing Reaction FPLC purified single stranded oligo conjugated to VHL (oligo-VHL) ligand and its reverse complement oligo were dissolved in ultra-pure water. Single stranded oligo-VHL and single stranded reverse complement oligos were mixed 1:1 molar ratio (final concentrations of TRAFTACs were set to 25 µM) in 1X annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl and 1 mM EDTA) and incubated for 10 minutes in a water bath at 95 degrees Celsius. Then, the hot-plate was turned off and the samples were left in the water bath and let cool down to room temperature over 2 h. Double stranded oligoTRAFTACs were mixed by gently vortexing and aliquoted and stored at -20 degrees Celsius. Reverse complementary sequences: OT3/17'- 5'CCCAATTTCACACCTAGGTGTGAAATTGGA3', OT7/10- 5' AACCACGTGGCAACCACGTGCTC 3'. Western Blotting Protein concentration in all the cell lysates were measured by BCA protein assay kit and equal amounts from each lysate were mixed with 4X loading dye and boiled for 5 minutes followed by 2 minutes centrifugation prior to loading into SDS-PAGE gel. Next proteins on the SDS-PAGE gel were transferred to a PVDF membrane by western blotting and the membrane was blocked with 5% milk in TBST (0.05%Tween 20) for 1 h. Primary antibodies (all Cell Signaling antibodies were diluted 1:1000, c-Myc 1:150) were prepared in TBST with 5% milk and membranes were incubated overnight at 4 ºC. On the following day, membrane was washes for 15 minutes (Incubate for three times, 5 minutes each) and appropriate secondary antibodies (1:5000) were prepared in TBST with 5% milk and incubated with the membrane for 1h at room temperature (RT). Membrane was washed for 30 minutes with TBST (incubate for three times, 10 minutes each) prior to imaging. EMSA (Electrophoretic Mobility Shift Assay) Click reaction mixtures and unreacted alkyne-modified oligonucleotides were separated in a 1.2 % agarose gel for 1 h at constant 120 mV and DNA bands were captured by the ChemiDoc system (BioRad) using SYBR safe mode. Biotin pull down Cells (HEK293 or HeLa) were grown in three T-175 flasks for 2 days. When cells reach >90% confluency, cells were harvested and washed once with 1X PBS. Cells were pooled together and resuspended in 1.5 mL immunoprecipitation buffer (25 mM Tris pH 7.4, 150 mM NaCl, 0.4% NP40, 5% glycerol, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF). Cells were then incubated for 30 minutes on ice prior to centrifugation at high speed (15,000 rpm) for 20 minutes. Equal amounts/volumes (~1 mg) of clarified lysate were transferred to individual tubes and incubated with biotinylated double stranded oligonucleotides for 2 h at RT. Pre-washed 30 μL of streptavidin agarose beads were transferred to each tube and incubated overnight at 40C. Beads were then washed with 1X TBS for 15 minutes (three times, 5-minute incubation each time). Bound proteins were eluted with 2X loading buffer by boiling for 8 minutes. Boiled samples were centrifuged at high speed for 5 minutes and supernatant was loaded onto SDS-PAGE gel followed by western blotting. Immunoprecipitation and ubiquitination assay A HA-tagged ubiquitin plasmid (4 ug) was transfected into HEK293 cells overexpressing brachyury-GFP in a 10 cm dish. On the following day, transfected cells were split into three 10 cm cell culture dishes and incubated for 24 h prior to transfection of oligoTRAFTACs. Epoxomicin (1 µM) was preincubated with cells for 1 h and oligoTRAFTACs (mock, OT3 and OT17) were then transfected using RNAi-MAX in 5 mL/dish of transfection medium. After 12 h post transfection, cells were harvested and lysed using immunoprecipitation buffer (25 mM Tris pH 7.4, 150 mM NaCl, 0.4% NP40, 5% glycerol, 1X protease inhibitor cocktail from Roche and 1 mM of PMSF). Approximately 1.5 mg of lysate from each sample was incubated with brachyury antibody at 4 oC for 4 h. Protein A agarose beads (30 μL) were added to antibody, lysates mixture and rock at 4 oC for ~ 18 h. Beads were washed with 1X TBS for three times with 5-minute incubation during each wash. Immunoprecipitated proteins were eluted by boiling agarose beads in 2X loading buffer (containing 10% ß-ME) for 8 minutes and centrifuged at high speed for 5 minutes prior to the loading into SDS-PAGE gel followed by western blot analysis. Cell viability assay Cells were split and subjected to oligoTRAFTAC transfection as described herein. Following the transfection, cells were incubated for the appropriate number of days before recording the luminescence reading from the plate reader. CellTiter-Glo reagent was prepared according to the manufactures recommendation and mixed with cell culture medium with 1:1 ratio. CellTiter-Glo reagent (100 µL) was added to each well and incubated for 10 minutes before taking the reading. Zebrafish Husbandry and Microinjection Tüpfel-longfin zebrafish were raised according to standard protocols approved by the Institutional Animal Care and Use Committee. Experiments were performed before sex determination in zebrafish 1 . Embryos were injected at the one cell stage with 180 picoliters of a 25 µM oligoTRAFTAC solution. They were raised for 48 hours at 28.6 °C and then scored for the presence of tail defects. While injected embryos showed severe, moderate, and mild tail defects, only those with severe and moderate tail defects were considered for quantitation 2. For western blot analysis, approximately 20 embryos were collected at 8-10 somite stage and deyolked using deyolking buffer (without Calcium: 55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3). Deyolked embryos were centrifuged at 1000 rcf for 30 seconds and the pellet was isolated and washed once with 1 mL of wash buffer (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris/Cl pH 8.5). After centrifuging at 1000 rcf for 30 seconds, the pellet was lysed in 20 µL of 2X SDS-loading buffer with 10% BME before loading the SDS-PAGE gel. General Chemistry Methods Chemical shifts are reported in δ ppm referenced to an internal CDCl3 (δ 7.26 ppm) for 1H NMR, CDCl3 (δ 77.00) for 13C NMR.
Figure imgf000113_0001
Synthesis of LC-HO-2100 To a solution of (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[[4-(4- methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide hydrochloride (1.0 eq., 20 mg) in DMF (1 mL) was added (2,5-dioxopyrrolidin-1-yl) 3-[2-(2-azidoethoxy)ethoxy]propanoate (1.2 eq.), and TEA (5.0 eq.). The mixture was stirred for 1 hour at room temperature. Upon completion, the mixture was diluted with H2O (5 mL), extracted with EtOAc (3X5 mL), dried over Na2SO4, concentrated under vacuum and purified by prep-TLC. The title compound (23 mg, 71% yield) was obtained as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 8.67 (s, 1H), 7.42 (t, J = 6.0 Hz, 1H), 7.36 – 7.32 (m, 4H), 6.99 (d, J = 8.4 Hz, 1H), 4.70 – 4.67 (m, 1H), 4.54 – 4.51 (m, 1H), 4.47 – 4.46 (m, 1H), 4.11 (d, J = 11.4 Hz, 1H), 3.73 – 3.69 (m, 3H), 3.64 (s, 3H), 3.65 – 3.62 (m, 3H), 3.59 (dd, J = 10.8, 3.6 Hz, 1H), 3.37 – 3.35 (m, 2H), 2.50 (s, 3H), 2.53 – 2.47 (m, 3H), 2.13 – 2.09 (m, 3H), 0.93 (s, 9H).13C NMR (150 MHz, CDCl3) δ 172.07, 171.71, 170.76, 150.28, 148.40, 138.10, 131.55, 130.87, 129.45, 128.06, 70.45, 70.42, 70.02, 69.97, 67.13, 58.37, 57.67, 56.58, 50.52, 43.17, 36.65, 35.82, 34.78, 26.35, 16.02.
Figure imgf000114_0001
Synthesis of LC-HO-2113 To a solution of (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[[4-(4- methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide hydrochloride (1.0 eq., 20 mg) in DMF (1 mL) was added (2,5-dioxopyrrolidin-1-yl)3-[2-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.2 eq.), and TEA (5.0 eq.). The mixture was stirred for 1 hour at room temperature. Upon completion, the mixture was diluted with H2O (5 mL), extracted with EtOAc (3X5 mL), dried over Na2SO4, concentrated under vacuum and purified by prep-TLC. The title compound (19 mg, 57% yield) was obtained as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H), 7.47 (s, 1H), 7.34 – 7.33 (m, 4H), 7.04 – 7.03 (m, 1H), 4.71 – 4.67 (m, 1H), 4.55 – 4.50 (m, 1H), 4.49 – 4.44 (m, 2H), 4.33 – 4.29 (m, 1H), 4.05 (d, J = 10.8 Hz, 1H), 3.69– 3.66 (m, 3H), 3.65 – 3.58 (m, 17H), 3.36 – 3.35 (m, 2H), 2.49 – 2.48 (m, 4H), 2.48 – 2.38 (m, 4H), 2.13 – 2.07 (m, 1H), 0.92 (s, 9H).13C NMR (150 MHz, CDCl3) δ 172.01, 171.59, 170.91, 150.25, 148.34, 138.16, 131.55, 130.76, 129.39, 127.99, 70.59, 70.54, 70.50, 70.44, 70.41, 70.37, 70.32, 69.97, 69.95, 67.04, 58.45, 57.65, 56.62, 50.59, 43.08, 36.61, 36.03, 34.87, 26.33, 15.99. Oligonucleotide sequences Table 1: Oligonucleotide sequence, modification site, and linker lengths of oligoTRAFTACs (OTs). In various embodiments, OTs 1-20 can have a linker LNK of formula LNK-X1, LNK- X2, LNK-1, or LNK-2. The linker PEG number (n), which represents the number of ethylene glycol (-CH2CH2O-) units, is the same as variable pp in formulas LNK-X1, LNK-X2, LNK- 1, and LNK-2.
Figure imgf000115_0001
Figure imgf000116_0001
Table 2. Oligonucleotide sequence, modification and modification site of the oligonucleotides used for biotin pull-down experiments.
Figure imgf000116_0002
The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application. Enumerated Embodiments The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance: Embodiment 1 provides a compound of formula I, or a salt, enantiomer, diastereomer, or tautomer thereof:
Figure imgf000117_0001
wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonding the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand. Embodiment 2 provides the compound of embodiment 1, wherein the 3'-end or the 5'- end of the ONA is covalently bonded to the LNK. Embodiment 3 provides the compound of any one of embodiments 1-2, wherein at least one internucleotide linkage comprises a phosphorothioate. Embodiment 4 provides the compound of any one of embodiments 1-3, wherein all internucleotide linkages are independently phosphorothioates. Embodiment 5 provides the compound of any one of embodiments 1-4, wherein the ONA comprises at least one deoxyribonucleotide and/or ribonucleotide. Embodiment 6 provides the compound of any one of embodiments 1-5, wherein the ONA comprises a c-Myc-binding nucleotide sequence. Embodiment 7 provides the compound of any one of embodiments 1-6, wherein at least two nucleotides flank each end of the c-Myc-binding nucleotide sequence. Embodiment 8 provides the compound of any one of embodiments 1-7, wherein the c- Myc-binding nucleotide sequence comprises 5'-CACGTGGTTGCCACGTG-3'. Embodiment 9 provides the compound of any one of embodiments 1-8, wherein the ONA comprises a brachyury-binding nucleotide sequence. Embodiment 10 provides the compound of any one of embodiments 1-9, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence. Embodiment 11 provides the compound of any one of embodiments 1-10, wherein the brachyury-binding nucleotide sequence comprises 5'- AATTTCACACCTAGGTGTGAAATT-3'. Embodiment 12 provides the compound of any one of embodiments 1-11, wherein the 3'-end of the ONA is covalently bonded to the LNK and wherein the terminal nucleotide at the 3'-end in ONA has the structure:
Figure imgf000118_0001
, wherein: Y is H or OH; and
Figure imgf000118_0002
Embodiment 13 provides the compound of any one of embodiments 1-12, wherein the 5'-end of the ONA is covalently bonded to the LNK and wherein the terminal nucleotide at the 5'-end in ONA has the structure:
Figure imgf000118_0003
, wherein: Y is H or OH; Z is O or S; and
Figure imgf000118_0004
Embodiment 14 provides the compound of any one of embodiments 1-13, wherein LNK has the structure: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-; (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡, -CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100; (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100; or (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡, -CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof. Embodiment 15 provides the compound of any one of embodiments 1-14, wherein A is phenylene or a C5-18 heteroarylene. Embodiment 16 provides the compound of any one of embodiments 1-15, wherein A
Figure imgf000120_0001
Embodiment 17 provides the compound of any one of embodiments 1-16, wherein LNK is –(LL)aa–A–(LL)bb–. Embodiment 18 provides the compound of any one of embodiments 1-17, wherein each occurrence of LL is independently selected from the group consisting of -O-, -N(R)-, - CR'R'-, -C(=O)-, and -C(=NR)-, with the provisos that (a) –(LL)zz– does not comprise -O-O-, and (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to - O- or -N(R)-. Embodiment 19 provides the compound of any one of embodiments 1-18, wherein LNK has the structure:
Figure imgf000121_0001
wherein at each occurrence X is independently selected from the group consisting of -O-, -CH2-, -CHR'-, and -C(=O)-; pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Embodiment 20 provides the compound of any one of embodiments 1-19, wherein LNK-X1 has the structure
Figure imgf000121_0002
wherein X is CH2 or O; and k is 1, 2, or 3. Embodiment 21 provides the compound of any one of embodiments 1-20, wherein LNK has the structure:
Figure imgf000121_0003
wherein at each occurrence X is independently selected from the group consisting of -O-, -CH2-, -CHR'-, and -C(=O)-; pp is an integer from 1 to 10, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Embodiment 22 provides the compound of any one of embodiments 1-21, wherein LNK-X2 has the structure
Figure imgf000122_0001
wherein X is CH2 or O; and k is 1, 2, or 3. Embodiment 23 provides the compound of any one of embodiments 1-22, wherein LNK has the structure:
Figure imgf000122_0002
, wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Embodiment 24 provides the compound of any one of embodiments 1-23, wherein pp is 2, 3, or 5. Embodiment 25 provides the compound of any one of embodiments 1-24, wherein pp is 2, 3, or 5. Embodiment 26 provides the compound of any one of embodiments 1-25, wherein the UBL is a ligand of an E3 ubiquitin ligase selected from the group consisting of von Hippel- Lindau (VHL), cereblon (CRBN), RING-type zinc-finger protein 114 (RNF114), cellular inhibitor of apoptosis (cIAP), mouse double minute 2 homologue (MDM2), damage-specific DNA binding protein 1 (DDB1)-CUL4 associated factor 16 (DCAF16), and Kelch-like ECH- associated protein 1 (KEAP1). Embodiment 27 provides the compound of any one of embodiments 1-26, wherein the UBL is a ligand of VHL or CRBN. Embodiment 28 provides the compound of any one of embodiments 1-27, wherein the UBL is a ligand having the structure:
Figure imgf000122_0003
Figure imgf000123_0001
, wherein R1 is H or CH3; and R2 is selected from the group consisting of
Figure imgf000123_0002
Embodiment 29 provides the compound of any one of embodiments 1-28, wherein the UBL has the structure:
Figure imgf000123_0003
. Embodiment 30 provides the compound of any one of embodiments 1-29, wherein the compound is selected from the group consisting of:
Figure imgf000123_0004
, ,
Figure imgf000124_0001
wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Embodiment 31 provides the compound of any one of embodiments 1-30, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'. Embodiment 32 provides a compound of formula II, or a salt, enantiomer, diastereomer, or tautomer thereof:
Figure imgf000124_0002
wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is selected from: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-; (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡, -CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100; (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100; or (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡, -CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof. Embodiment 33 provides the compound of embodiment 32, wherein the terminal nucleotide at the 3'-end of ONA has the structure:
Figure imgf000126_0001
, wherein: zz is an integer from 1 to 100; Y is H or OH; and
Figure imgf000127_0001
Embodiment 34 provides the compound of any one of embodiments 32-33, wherein the terminal nucleotide at the 5'-end of ONA has the structure:
Figure imgf000127_0002
, wherein: zz is an integer from 1 to 100; Y is H or OH; Z is O or S; and
Figure imgf000127_0003
Embodiment 35 provides the compound of any one of embodiments 32-34, wherein the ONA comprises at least one deoxyribonucleotide or ribonucleotide, or a combination thereof. Embodiment 36 provides the compound of any one of embodiments 32-35, wherein the ONA comprises a c-Myc-binding nucleotide sequence. Embodiment 37 provides the compound of any one of embodiments 32-36, wherein at least two nucleotides flank each end of the c-Myc-binding nucleotide sequence. Embodiment 38 provides the compound of any one of embodiments 32-37, wherein the c-Myc-binding nucleotide sequence comprises 5'- CACGTGGTTGCCACGTG-3'. Embodiment 39 provides the compound of any one of embodiments 32-38, wherein the ONA comprises a brachyury-binding nucleotide sequence. Embodiment 40 provides the compound of any one of embodiments 32-39, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence. Embodiment 41 provides the compound of any one of embodiments 32-40, wherein the brachyury-binding nucleotide sequence comprises 5'- AATTTCACACCTAGGTGTGAAATT-3'. Embodiment 42 provides the compound of any one of embodiments 32-41, having the structure:
Figure imgf000128_0001
wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA. Embodiment 43 provides the compound of any one of embodiments 32-42, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'. Embodiment 44 provides a pharmaceutical composition comprising the compound of any one of embodiments 1-31, and a pharmaceutically acceptable carrier, additive, and/or excipient. Embodiment 45 provides a method of preventing, treating, and/or ameliorating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of at least one compound of any one of embodiments 1-31, which is optionally formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. Embodiment 46 provides the method of embodiment 45, wherein the cancer is at least one selected from the group consisting of squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, renal cell carcinoma, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemia; benign or malignant lymphoma; benign or malignant melanoma; myeloproliferative disease; multiple myeloma, sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell tumor, meningioma, meningeal sarcoma, neurofibroma, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor, and teratocarcinoma. Embodiment 47 provides the method of any one of embodiments 45-46, wherein: the benign or malignant lymphoma comprises Burkitt's lymphoma and/or Non-Hodgkin's lymphoma; and the sarcoma comprises Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelioma, and/or synovial sarcoma. Embodiment 48 provides the method of any one of embodiments 45-47, wherein the compound and/or composition is administered by a route selected from the group consisting of oral, transdermal, transmucosal, (intra)nasal, (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra- arterial, intravenous, intrabronchial, inhalation, and topical. Embodiment 49 provides the method of any one of embodiments 45-48, wherein the subject is a mammal. Embodiment 50 provides the method of any one of embodiments 45-49, wherein the subject is human.

Claims

CLAIMS What is claimed is: 1. A compound of formula I, or a salt, enantiomer, diastereomer, or tautomer thereof:
Figure imgf000130_0001
wherein: ONA is an oligonucleotide having a 3'-end and a 5'-end and comprising one or more phosphodiester or phosphorothioate internucleotide linkages; LNK is a chemical linker covalently bonding the ONA and the UBL; and UBL is an E3 ubiquitin ligase ligand.
2. The compound of claim 1, wherein the 3'-end or the 5'-end of the ONA is covalently bonded to the LNK.
3. The compound of claim 2, wherein at least one internucleotide linkage comprises a phosphorothioate.
4. The compound of claim 3, wherein all internucleotide linkages are independently phosphorothioates.
5. The compound of claim 1, wherein the ONA comprises at least one deoxyribonucleotide and/or ribonucleotide.
6. The compound of claim 5, wherein the ONA comprises a c-Myc-binding nucleotide sequence.
7. The compound of claim 6, wherein at least two nucleotides flank each end of the c- Myc-binding nucleotide sequence.
8. The compound of claim 6, wherein the c-Myc-binding nucleotide sequence comprises 5'-CACGTGGTTGCCACGTG-3'.
9. The compound of claim 1, wherein the ONA comprises a brachyury-binding nucleotide sequence.
10. The compound of claim 9, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence.
11. The compound of claim 10, wherein the brachyury-binding nucleotide sequence comprises 5'-AATTTCACACCTAGGTGTGAAATT-3'.
12. The compound of claim 1, wherein the 3'-end of the ONA is covalently bonded to the LNK and wherein the terminal nucleotide at the 3'-end in ONA has the structure:
Figure imgf000131_0001
, wherein: Y is H or OH; and
Figure imgf000131_0002
13. The compound of claim 1, wherein the 5'-end of the ONA is covalently bonded to the LNK and wherein the terminal nucleotide at the 5'-end in ONA has the structure:
Figure imgf000131_0003
, wherein: Y is H or OH; Z is O or S; and
Figure imgf000132_0001
14. The compound of claim 1, wherein LNK has the structure: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-; (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡, -CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100; (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100; or (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡, -CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof.
15. The compound of claim 14, wherein A is phenylene or a C5-18 heteroarylene.
16. The compound of claim 14, wherein A is
Figure imgf000134_0003
17. The compound of claim 14, wherein LNK is –(LL)aa–A–(LL)bb–.
18. The compound of claim 17, wherein each occurrence of LL is independently selected from the group consisting of -O-, -N(R)-, -CR'R'-, -C(=O)-, and -C(=NR)-, with the provisos that (a) –(LL)zz– does not comprise -O-O-, and (b) any -N(R)-O- or -N(R)-N(R)- within – (LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-.
19. The compound of claim 18, wherein LNK has the structure:
Figure imgf000134_0002
wherein at each occurrence X is independently selected from the group consisting of -O-, -CH2-, -CHR'-, and -C(=O)-; pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
20. The compound of claim 19, wherein LNK-X1 has the structure
Figure imgf000134_0001
wherein X is CH2 or O; and k is 1, 2, or 3.
21. The compound of claim 18, wherein LNK has the structure:
Figure imgf000135_0001
wherein at each occurrence X is independently selected from the group consisting of -O-, -CH2-, -CHR'-, and -C(=O)-; pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
22. The compound of claim 21, wherein LNK-X2 has the structure
Figure imgf000135_0002
wherein X is CH2 or O; and k is 1, 2, or 3.
23. The compound of claim 18, wherein LNK has the structure:
Figure imgf000135_0003
wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
24. The compound of claim 19, wherein pp is 2, 3, or 5.
25. The compound of claim 21, wherein pp is 2, 3, or 5.
26. The compound of claim 1, wherein the UBL is a ligand of an E3 ubiquitin ligase selected from the group consisting of von Hippel-Lindau (VHL), cereblon (CRBN), RING- type zinc-finger protein 114 (RNF114), cellular inhibitor of apoptosis (cIAP), mouse double minute 2 homologue (MDM2), damage-specific DNA binding protein 1 (DDB1)-CUL4 associated factor 16 (DCAF16), and Kelch-like ECH-associated protein 1 (KEAP1).
27. The compound of claim 26, wherein the UBL is a ligand of VHL or CRBN.
28. The compound of claim 26, wherein the UBL is a ligand having the structure:
Figure imgf000136_0001
, wherein R1 is H or CH3; and R2 is selected from the group consisting of
Figure imgf000136_0002
29. The compound of claim 1, wherein the UBL has the structure:
Figure imgf000137_0002
.
30. The compound of claim 1, which is selected from the group consisting of:
Figure imgf000137_0001
wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
31. The compound of claim 30, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'.
32. A compound of formula II, or a salt, enantiomer, diastereomer, or tautomer thereof: wherein:
Figure imgf000138_0001
ONA is an oligonucleotide having a 3'-end and a 5'-end; LNK is selected from: (i) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, - C ≡C-, -CR'R'-, -C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)zz– is not further bound to -O- or -N(R)-; (ii) –(LL)zz–, wherein: LL is at each occurrence independently selected from the group consisting of -O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, - NR2, -CR=, -C ≡, -CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, and - C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; zz is an integer from 1 to 100; (iii) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -S-, -S(=O)-, -S(=O)2-, -N(R)-, -CR'=CR'-, -C ≡C-, -CR'R'-, - C(=O)-, and -C(=NR)-; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, phenyl, or C5-10 heteroaryl; R' is independently at each occurrence H, halogen, hydroxyl, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkenoxy, C2-12 alkynyl, C2-12 alkynoxy, phenyl, or C5-10 heteroaryl; with the provisos that (a) –(LL)zz– does not comprise -O-O-, -S-O-, or -S(=O)-O-; (b) any -N(R)-O- or -N(R)-N(R)- within –(LL)aa– and –(LL)bb– is not further bound to -O- or -N(R)-; A is phenylene or a C5-18 heterocyclylene; aa is an integer from 1 to 100; bb is an integer from 1 to 100; or (iv) –(LL)aa–A–(LL)bb–, wherein: LL is at each occurrence independently selected from the group consisting of - O-, -OR, -S-, -S(=O)-, -S(=O)2-, -SR, -N(R)-, -NR2, -CR'=, -C ≡, -CH2- , -CHR'-, -C(R')2-, -CH3, -C(=O)-, and -C(=NR)-; A is phenylene or a C5-18 heterocyclylene; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof; aa is an integer from 1 to 100; bb is an integer from 1 to 100; R is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, halogen, or combinations thereof; and R' is independently at each occurrence H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C6-10 aryl, C5-10 heteroaryl, or combinations thereof.
33. The compound of claim 32, wherein the terminal nucleotide at the 3'-end of ONA has the structure:
Figure imgf000140_0001
, wherein: zz is an integer from 1 to 100; Y is H or OH; and
Figure imgf000140_0002
34. The compound of claim 32, wherein the terminal nucleotide at the 5'-end of ONA has the structure:
Figure imgf000140_0003
, wherein: zz is an integer from 1 to 100; Y is H or OH; Z is O or S; and
Figure imgf000141_0001
35. The compound of claim 32, wherein the ONA comprises at least one deoxyribonucleotide or ribonucleotide, or a combination thereof.
36. The compound of claim 32, wherein the ONA comprises a c-Myc-binding nucleotide sequence.
37. The compound of claim 36, wherein at least two nucleotides flank each end of the c- Myc-binding nucleotide sequence.
38. The compound of claim 36, wherein the c-Myc-binding nucleotide sequence comprises 5'- CACGTGGTTGCCACGTG-3'.
39. The compound of claim 32, wherein the ONA comprises a brachyury-binding nucleotide sequence.
40. The compound of claim 39, wherein at least two nucleotides flank each end of the brachyury-binding nucleotide sequence.
41. The compound of claim 39, wherein the brachyury-binding nucleotide sequence comprises 5'-AATTTCACACCTAGGTGTGAAATT-3'.
42. The compound of claim 32, having the structure:
Figure imgf000141_0002
wherein pp is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the atom marked with * is covalently bonded to the 3'-end or the 5'-end of the ONA.
43. The compound of claim 32, wherein the ONA has a sequence selected from the group consisting of: 5'-TGGGAGCACGTGGTTGCCACGTGGTTGGG-3', 3'-GGGTTGGTGCACCGTTGGTGCACGAGGGT-5', 5'-CTTTCCAATTTCACACCTAGGTGTGAAATTGGGGAC-3', and 3'-CAGGGGTTAAAGTGTGGATCCACACTTTAACCTTTC-5'.
44. A pharmaceutical composition comprising the compound of claim 1, and a pharmaceutically acceptable carrier, additive, and/or excipient.
45. A method of preventing, treating, and/or ameliorating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of at least one compound of claim 1, which is optionally formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
46. The method of claim 45, wherein the cancer is at least one selected from the group consisting of squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, renal cell carcinoma, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemia; benign or malignant lymphoma; benign or malignant melanoma; myeloproliferative disease; multiple myeloma, sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell tumor, meningioma, meningeal sarcoma, neurofibroma, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor, and teratocarcinoma.
47. The method of claim 45, wherein: the benign or malignant lymphoma comprises Burkitt's lymphoma and/or Non- Hodgkin's lymphoma; and the sarcoma comprises Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelioma, and/or synovial sarcoma.
48. The method of claim 45, wherein the compound and/or composition is administered by a route selected from the group consisting of oral, transdermal, transmucosal, (intra)nasal, (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical.
49. The method of claim 45, wherein the subject is a mammal.
50. The method of claim 49, wherein the subject is human.
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