Compounds for cleaving nucleic acids

Abstract

The present invention relates to novel compounds of formula (I) as defined herein, which are suitable for non-enzymatic cleavage of target nucleic acids. The present invention also relates to pharmaceutical compositions comprising these compounds and to the use of these compounds, for example, in epigenomics and epitranscriptomics mapping, as well as in therapy, for example, antimicrobial and / or antiviral therapy.

Description

[Technical Field]

[0001] The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under European Research Council grant agreement no. 676832.

[0002] The present invention relates to novel compounds suitable for the non-enzymatic cleavage of target nucleic acids. The invention also relates to the use of these compounds in, for example, RNA structure mapping, as well as in therapy, for example, antimicrobial and / or antiviral therapy. [Background technology]

[0003] Targeted degradation of nucleic acids is part of the cutting edge of drug discovery. The destruction of ribonucleic acid (RNA) strands in living systems is crucial for effective biological function in the organism. Because it is critical for a wide range of functions, RNA is an important target for disrupting disease. Established methods for targeted degradation utilize cellular cofactors that complicate their promising deployment. Additionally, the specificity of this approach limits flexibility in the compounds that can be used.

[0004] In 2019, respiratory illnesses changed the way we lived. The virus that caused the disease, known as COVID-19 or SARS-CoV-2, suddenly brought to light the need for developing drugs that could target and disrupt viral machinery, something we had been aware of. Beyond covalent modifications, the secondary and tertiary structures of RNA complexes also have pathological effects and can therefore be used as a focus for treatment. The genome of SARS-CoV-2 contains four putative G-quadruplex sites [Zhao, C. et al.].

[0005] (2020) describe small molecule "click degraders" that can be covalently attached to RNA species via click chemistry and then cleave the bound RNA molecule. The authors demonstrate that N in the RNA sequence 6 -methyladenosine (m6 We describe a methylated CLICK degradation sequencing method (meCLICK-Seq) to identify the presence of A). The method hijacks RNA methyltransferase to introduce an alkyne moiety into RNA in place of a methyl group. A subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction incorporates the click degrader molecule, resulting in RNA cleavage. The method identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in introns and intergenic regions.

[0006] Because Click degrader molecules are covalently incorporated into target RNA, they can only be used to degrade RNA species that can be edited to contain a suitable Click-reactive group (typically an alkyne). Furthermore, the required RNA editing limits the use of the technology to therapeutic applications.

[0007] Furthermore, while the original biomimetic cleavage group (warhead) imidazole used in the meClick-seq method has proven sufficient to provide a proof-of-concept for the strategy, its simplicity inherently lacks the tunability for efficacy and DMPK properties aimed at human treatment. Furthermore, differences in topology within each new RNA binder when complexed can lead to target-specific variations in degrader efficacy. Therefore, there is a need to generate new degrader molecules suitable for pharmaceutical applications.

[0008] The present invention was made with the above in mind. Summary of the Invention

[0009] The present invention relates to the discovery that bifunctional compounds, also referred to herein as degrading agents, can be used as catalytic agents that non-covalently bind to target nucleic acid molecules and cleave them. The degrading agents disclosed herein bind to target nucleic acids through non-covalent interactions. Surprisingly, the inventors have found that non-covalent binding is sufficient to enable selective decomposition of target nucleic acids. Thus, the degrading agents do not require any chemical modification of the target nucleic acid, for example, the introduction of click-reactive groups into the target nucleic acid.

[0010] Selective cleavage of target nucleic acid molecules using the degradative agents described herein can be useful in RNA structure mapping, as well as in therapy, such as anti-cancer, anti-bacterial and anti-viral therapy.

[0011] In one aspect, the present invention provides a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof.

[0012] In another aspect, the present invention provides a pharmaceutical composition comprising a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

[0013] In another aspect, the present invention provides a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, for use as a medicament.

[0014] In another aspect, the present invention provides a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a disease or condition in which degradation of a target oligonucleotide is beneficial.

[0015] In another aspect, the present invention provides a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a proliferative disorder (e.g., cancer) or a bacterial or viral infection.

[0016] In another aspect, the present invention provides the use of a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of a disease or condition in which degradation of a target oligonucleotide is beneficial.

[0017] In another aspect, the present invention provides the use of a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of a proliferative disorder (e.g., cancer) or a bacterial or viral infection.

[0018] In another aspect, the present invention provides a method of treating a disease or condition in which degradation of a targeted oligonucleotide is beneficial, the method comprising administering a therapeutically effective dose of a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof.

[0019] In another aspect, the present invention provides a method of treating a proliferative disorder (e.g., cancer) or a bacterial or viral infection, comprising administering a therapeutically effective dose of a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof.

[0020] In another aspect, the present invention provides a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, for use in epigenetics and epitranscriptomics analysis / mapping.

[0021] In another aspect, the present invention provides the use of a bifunctional compound as defined herein, or a salt or solvate thereof, for epigenetics and epitranscriptomics analysis / mapping.

[0022] In another aspect, the present invention provides a method for cleaving a target nucleic acid molecule, comprising: contacting a target nucleic acid molecule with a bifunctional compound of the invention such that the compound non-covalently binds to the target nucleic acid molecule; allowing the compound to cleave the target nucleic acid molecule bound thereto; The present invention provides a method comprising:

[0023] In another aspect, the present invention provides a method for identifying secondary or tertiary structure within a target nucleic acid molecule, comprising: providing first and second populations of nucleic acid molecules, each population comprising a target nucleic acid molecule; introducing a bifunctional compound of the present invention to a first population of nucleic acid molecules; allowing a bifunctional compound of the invention to cleave target nucleic acid molecules present in the first population; identifying nucleic acid molecules present in the first population in reduced amounts relative to the second population; The present invention provides a method comprising:

[0024] The present invention further provides a method of synthesizing a bifunctional compound as defined herein, or a pharmaceutically acceptable salt thereof.

[0025] Preferred, preferred, and optional features of any one of the particular embodiments of the present invention are also preferred, preferred, and optional features of any other embodiment.

[0026] Detailed Description of the Invention definition Unless otherwise stated, the following terms used in the specification and claims have the following meanings set forth below.

[0027] It should be recognized that references to "treating" or "treatment" include prevention, even alleviation, of documented symptoms of a condition. Thus, "treating" or "treatment" of a condition, disorder, or condition includes (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder, or condition occurring in a person who may be affected by or susceptible to the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the occurrence of the disease or its recurrence (in the case of maintenance treatment) or the occurrence of at least one clinical or subclinical symptom thereof; or (3) palliating or attenuating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof.

[0028] "Therapeutically effective amount" means the amount of a bifunctional compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the mammal being treated.

[0029] As used herein, the term "alkyl" includes both straight-chain and branched-chain alkyl groups and their analogs. References to individual alkyl groups, such as "propyl," are specific for the straight-chain version only, and references to individual branched-chain alkyl groups, such as "isopropyl," are specific for the branched-chain version only. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl, and t-butyl. Similar rules apply to other radicals, for example, "phenyl(1-6C)alkyl" includes phenyl(1-4C)alkyl, benzyl, 1-phenylethyl, and 2-phenylethyl.

[0030] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to any group having m to n carbon atoms.

[0031] The term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms have been replaced with a heteroatom, such as N, O, and S. A heteroalkyl group may be a 1-6C heteroalkyl group, such as a 1-4C, 1-3C, or 1-2C heteroalkyl group. In this context, the prefix (e.g., 1-6C) indicates the number of atoms in the heteroalkyl backbone, whether carbon atoms or heteroatoms. A heteroalkyl group may be linear or branched.

[0032] An "alkylene" group is an alkyl group that is positioned between and serves to connect two other chemical groups. Thus, "(1-6C)alkylene" means a linear saturated divalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated divalent hydrocarbon radical of 3 to 6 carbon atoms, e.g., methylene, ethylene, propylene, 2-methylpropylene, pentylene, and the like.

[0033] "(3-8C)cycloalkyl" means a hydrocarbon ring containing 3 to 8 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or bicyclo[2.2.1]heptyl.

[0034] "(3-8C)cycloalkyl-(1-6C)alkylene" means a (3-8C)cycloalkyl group covalently linked to a (1-6C)alkylene group, both of which are defined herein.

[0035] The term "halo" or "halogeno" refers to fluoro, chloro, bromo and iodo.

[0036] The terms "heterocyclyl," "heterocyclic," or "heterocycle" refer to a non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic heterocyclic ring system. The term heterocyclyl includes both monovalent and divalent species. Monocyclic heterocyclic rings contain about 3 to 12 (preferably 3 to 7) ring atoms with 1 to 5 (preferably 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, or sulfur in the ring. Bicyclic heterocyclic rings contain 7 to 17 member atoms, preferably 7 to 12 member atoms, in the ring. Bicyclic heterocyclic rings contain about 7 to about 17 ring atoms, preferably 7 to 12 ring atoms. Bicyclic heterocyclic rings can be fused, spiro, or bridged ring systems.

[0037] Examples of heterocyclic groups include cyclic ethers such as oxiranyl, oxetanyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers. Nitrogen-containing heterocycles include, for example, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrotriazinyl, tetrahydropyrazolyl, and the like. Typical sulfur-containing heterocycles include tetrahydrothienyl, dihydro-1,3-dithiol, tetrahydro-2H-thiopyran, and hexahydrothiepin. Other heterocycles include dihydrooxathiolyl, tetrahydrooxazolyl, tetrahydrooxadiazolyl, tetrahydrodioxazolyl, tetrahydrooxathiazolyl, hexahydrotriazinyl, tetrahydrooxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. Sulfur-containing heterocycles also include oxidized sulfur heterocycles containing SO or SO groups. Examples include the sulfoxide and sulfone forms of tetrahydrothienyl and thiomorpholinyl, such as tetrahydrothienyl 1,1-dioxide and thiomorpholinyl 1,1-dioxide. Suitable values ​​for heterocyclyl groups bearing one or two oxo (=O) or thioxo (=S) substituents are, for example, 2-oxopyrrolidinyl, 2-thioxopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioxoimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl, or 2,6-dioxopiperidinyl. Particular heterocyclyl groups are saturated monocyclic 3- to 7-membered heterocyclyl containing 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur, such as azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1,1-dioxide, thiomorpholinyl, thiomorpholinyl 1,1-dioxide, piperidinyl, homopiperidinyl, piperazinyl, or homopiperazinyl. As will be appreciated by those skilled in the art, any heterocycle may be attached to another group through any suitable atom, for example, a carbon or nitrogen atom.However, references herein to piperidino or morpholino refer to the piperidin-1-yl or morpholin-4-yl ring attached via the ring nitrogen.

[0038] "Bridged ring system" means a ring system in which two rings share more than two atoms. See, for example, Advanced Organic Chemistry, by Jerry March, 4 th Edition, Wiley Interscience, pages 131-133, 1992. Examples of bridged heterocyclyl ring systems include aza-bicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane, and quinuclidine.

[0039] "Heterocyclyl(1-6C)alkyl" means a heterocyclyl group covalently linked to a (1-6C)alkylene group, both of which are defined herein.

[0040] The term "heteroaryl" or "heteroaromatic" refers to an aromatic monocyclic, bicyclic, or polycyclic ring incorporating one or more (e.g., 1 to 4, particularly 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, or sulfur. The term heteroaryl includes both monovalent and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing 5 to 12 ring members, more typically 5 to 10 ring members. Heteroaryl groups can be, for example, 5- or 6-membered monocyclic rings or 9- or 10-membered bicyclic rings, e.g., fused 5- and 6-membered rings or bicyclic structures formed from two fused 6-membered rings. Each ring may contain up to about four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Typically, heteroaryl rings will contain up to three heteroatoms, more typically up to two, e.g., a single heteroatom. In one embodiment, a heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl ring can be basic, as in the case of an imidazole or pyridine, or essentially non-basic, as in the case of an indole or pyrrole nitrogen. Generally, the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents on the ring, will be fewer than five.

[0041] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, and isoxazolyl. These include noryl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzoisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl, 1H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, and imidazo[1,2-b][1,2,4]triazinyl. "Heteroaryl" also includes partially aromatic bicyclic or polycyclic ring systems in which at least one ring is aromatic and one or more other rings are non-aromatic saturated or partially saturated, provided that at least one ring contains one or more heteroatoms selected from nitrogen, oxygen, or sulfur. Examples of partially aromatic heteroaryl groups include, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1,2,3,4-tetrahydroquinolinyl, dihydrobenzothienyl, dihydrobenzofuranyl, 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1,3]dioxolyl, 2,2-dioxo-1,3-dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indolinyl, 1,2,3,4-tetrahydro-1,8-naphthyridinyl, 1,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl, and 3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazinyl.

[0042] Examples of 5-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl groups.

[0043] Examples of 6-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and triazinyl.

[0044] Bicyclic heteroaryl groups include, for example: a benzene ring fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms; a pyridine ring fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms; a pyrimidine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrrole ring fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms; a pyrazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrazine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an imidazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an oxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isoxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a thiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isothiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a thiophene ring fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms; a furan ring fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms; a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2, or 3 ring heteroatoms; and A cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2, or 3 ring heteroatoms It may be a group selected from:

[0045] Particular examples of bicyclic heteroaryl groups containing a 6-membered ring fused to a 5-membered ring include, but are not limited to, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl, and pyrazolopyridinyl groups.

[0046] Specific examples of bicyclic heteroaryl groups containing two fused 6-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolidinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinyl groups.

[0047] "Heteroaryl(1-6C)alkyl" means a heteroaryl group covalently linked to a (1-6C)alkylene group, both of which are defined herein. Examples of heteroaralkyl groups include pyridin-3-ylmethyl, 3-(benzofuran-2-yl)propyl, and the like.

[0048] The term "aryl" refers to a cyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, and the like. In certain embodiments, aryl is phenyl.

[0049] The term "aryl(1-6C)alkyl" means an aryl group covalently linked to a (1-6C)alkylene group, both of which are defined herein. Examples of aryl-(1-6C)alkyl groups include benzyl, phenylethyl, and the like.

[0050] This specification also uses some composite terms to describe groups containing more than one functional group. Such terms will be understood by those skilled in the art. For example, heterocyclyl(m-nC)alkyl includes (m-nC)alkyl substituted with heterocyclyl.

[0051] The term "optionally substituted" refers to groups, structures, or molecules that are substituted and those that are not substituted. 1 The term "one / any CH, CH, CH group or heteroatom (i.e., NH) within the group is optionally substituted" preferably refers to R 1 This means that (any) one of the hydrogen radicals of the group is replaced by the associated defined group.

[0052] Where an optional substituent is selected from "one or more" groups, it is to be understood that this definition includes all substituents being selected from one of the specified group, or substituents being selected from two or more of the specified group.

[0053] The phrase "compounds of the invention" refers generally and specifically to compounds disclosed herein (e.g., compounds of Formula (I), (II), (III), (IV), or (V), (VI) or (VII)).

[0054] The present invention will now be described with reference to the figures listed below. [Brief explanation of the drawings]

[0055] [Figure 1]Schematic diagram of the mechanism of action of the degraders. Figure 1 illustrates the use of degraders in a coronavirus pseudoknot degradation strategy. The pseudoknot degraders bind to and then directly degrade the coronavirus region containing the pseudoknot, without the need for other drugs. [Figure 2] Graphs showing that rG4 degraders cleave rG4-containing oligomers and the SARS-CoV-2 genome in vitro. (a) Shows the effect of rG4 degraders on rG4-competent oligomers. The degraders cleave oligomers under conditions that promote rG4 formation. n=3. (b) Shows the effect of rG4 degraders on oligomers that do not form rG4s. No degradation was observed. n=3. (c) Shows nanopore sequencing data showing extensive degradation of the SARS-CoV-2 genome at its ORF1b upon treatment with the rG4 degrader PDS-deg6(9A). *p<0.05, **p<0.01, ***p<0.005, ns=not significant. [Figure 3] Figure 1 shows preliminary in vitro findings of the anti-SARS-CoV-2 activity of G4-resolving agents. (a) Inhibition of plaque-forming units (PFU) in samples treated with 50 μM PDS-deg4 (9B), PDS-deg6 (9A), and PDS-DegALK (8). (b) PCR measurements of viral RNA. Results demonstrate inhibition of viral replication by 5 μM and 50 μM PDS-deg6 (9A). As shown, PDS-deg6 (9A) appears to inhibit viral growth to a greater extent than PDS-deg4 (9B). (c) Cell viability after 24 hours of incubation with increasing concentrations of G4-resolving agents is shown. None of the compounds exhibited cytotoxicity up to 50 μM. [Figure 4]Graphs showing the in vivo anti-SARS-CoV-2 activity of G4-resolvers. (a) As observed in vehicle (0.1% DMSO in water)-treated animals (gray square symbols), mice administered PDS-deg4(9B) (purple, triangle symbols) showed a 10% loss of body weight on day 1 post-infection. Body weight stabilized between days 1 and 3, then decreased again, reaching the 75% threshold on day 5. As a control, uninfected mice treated with vehicle (0.1% DMSO in water) (circle symbols) showed no weight loss. (b) Quantification of lung viral load by plaque assay on day 5 showed a reduced load in animals treated with PDS-deg4(9B) (purple, right) compared with the vehicle control group (gray, left). *p<0.01. [Figure 5] Figure 1 shows that the MTDB-degrader (16a) cleaves coronavirus pseudoknots in vitro. (a) The synthetic design of MTDB-deg (16a) is shown. (b) The structure of the control molecule, TDB-deg (16b), which features a weak pseudoknot binder and an imidazole cleavage moiety, is shown. (c) LC-MS data showing degradation of the pseudoknot in the presence of the degrader relative to the control. n=3. (d) A gel image demonstrating the activity of the pseudoknot degrader is shown. (e) LCMS data showing that the pseudoknot degrader becomes less effective when one of the stems of the pseudoknot is mutated to disrupt the pseudoknot secondary structure. n=3. (f) A gel image showing degradation of native RNA extracted from SARS-CoV-2 by MTDB-deg (16a) relative to the control. ns - not significant. [Figure 6]Direct RNA nanopore sequencing reveals genomic loci resolved by MTDB-deg. (a) Distribution and abundance of aligned reads flanking pseudoknot regions for SARS-CoV-2 RNA treated with control (0.1% DMSO in water) or MTDB-deg, based on alignments in minimap2. b, Distribution and abundance of aligned reads mapping exclusively to the S sgRNA region for SARS-CoV-2 RNA treated with control or MTDB-deg, based on alignments in minimap2. [Figure 7] Graph showing that treatment with MTDB-deg has no effect on subgenomic SARS-CoV-2 RNA. Distribution and abundance of aligned reads mapping exclusively to the indicated sgRNA regions for SARS-CoV-2 RNA treated with control (0.1% DMSO in water) or MTDB-deg, based on alignments in minimap2. [Figure 8]Figure 1 shows that MTDB degraders inhibit SARS-CoV-2 replication in cells. (a) and (b) show the percentage inhibition of viral replication normalized to the vehicle control (dashed line) after incubation with increasing concentrations of the pseudoknot degrader (MTDB-deg(16a)) and control molecules (MTDB and TDB-deg(16b)). Viral replication was assessed 24 hours after infection (multiplicity of infection (MOI) of 0.05) based on E gene and pseudoknot region RNA levels. The antiviral activity of MTDB-deg(16a) was observed both before (a) and after (b) infection with SARS-CoV-2 at an MOI of 0.05. Means ± SD of triplicates are shown, with differences between means indicated with p<0.01. *, p<0.05; **, p<0.01; two-tailed paired t-test. (c) The IC50 of the pseudoknot disrupter MTDB-deg (16a) is lower when the drug is added postinfection. (d) Photographs of cell monolayers after 4 days of incubation with supernatants from virus cultures treated with 6 mM MTDB-deg (16a), MTDB, and TDB-deg (16b) for 24 hours. Treatment with 6 mM MTDB-deg (16a) for 24 hours showed a reduction in the number of viral plaques compared to vehicle controls, both when added before or after infection. The control molecule MTDB only showed a reduction in the number of viral plaques when added before infection, whereas TDB-deg (16b) showed no reduction. (e) Cell viability assay demonstrating that none of the compounds exhibited cytotoxicity in VeroCCL81 cells after 24 hours. (f) shows the percentage of viral replication relative to the vehicle control 24 hours after removal of the lytic agent-containing medium. Treatment with MTDB-deg(16a) for 24 hours impaired the virus's ability to recover from drug exposure. [Figure 9]Graphs showing dose-response curves for MTDB-deg, MTDB, and TDB-deg. (a) and (b) show the 50% inhibitory concentration (IC50) values ​​for the pseudoknot-dissolving agent MTDB-deg (16a) before and after infection. Control molecules (MTDB and TDB-deg (16b)) did not inhibit viral replication, and therefore, IC50 values ​​could not be determined. (c) is a dose-response curve including a high concentration of 18 μM, which shows an elevated IC50 (as determined by PCR targeting the E gene). [Figure 10] Figure 1 shows virus recovery and virucidal activity after exposure to MTDB-degraders. (a) The ability of virus to recover after 24 hours of incubation with MTDB-deg (16a) and the control molecules MTDB and TDB-deg (16b), as determined by qPCR targeting the pseudoknot region. Viral recovery was impaired in samples treated with MTDB-deg (16b), but not in samples treated with the control molecules MTDB and TDB-deg (16b). (b) Virucidal activity was assessed by incubating 1000 PFU of SARS-CoV-2 with 6 μM of compound for 1 hour at 37°C, followed by determining residual virus infectivity by plaque assay. MTDB-deg(16a), MTDB, and TDB-deg(16b) did not exhibit virucidal activity against cell-free virions, suggesting that the antiviral activity of MTDB-deg(16a) is mediated by inhibiting viral replication in host cells rather than by inactivating cell-free virions. [Figure 11] Agarose gel analysis of ribosome degradation assay. Left: Ethyl linker, no degradation observed. Center left: Diethylene glycol linker, degradation observed at 15 mM concentration. Center right: Hexaethylene glycol linker, no degradation observed. Right: Control chloramphenicol, no degradation observed. B = Blank (no degradation agent), Degradation agent concentrations: 1a / 1b = 15 mM, 2a / 2b = 7.5 mM, 3a / 3b = 3.75 mM, 4a / 4b = 1.88 mM, 5a / 5b = 0.94 mM, 6a / 6b = 0.47 mM. [Figure 12] In vivo activity of MTDB-degraders against SARS-CoV-2 infection in K18-hACE2 mice. (a) Eight- to 12-week-old female K18-hACE2-transgenic mice were intranasally infected with 10 plaque-forming units (PFU) of SARS-CoV-2 and treated intranasally 1 hour before and 3 hours after infection with MTDB-deg 16a (25 mg / kg) (n = 6), MTDB (10 mg / kg; the maximum dose that could be administered was solubility-limited) (n = 3), TDB-deg 16b (25 mg / kg) (n = 5), and vehicle control (n = 6). (b) Administration of MTDB-deg 16a results in a reduction in lung viral load in SARS-CoV-2-infected K18-hACE2 mice. No differences in lung viral load were observed between vehicle control and mice treated with MTDB and TDB-deg 16b. Mean ± SD is shown; *p<0.05; unpaired t-test. (c) Western blot analysis of phospho-p38 from lung extracts of transgenic K18-hACE2 mice treated with three doses of 10 mg / kg vehicle (V1, V2) or MTDB-deg 16a (D1, D2) 1 hour before infection and on days 1 and 2 post-infection (n=2). [Figure 13] Figure 1 is a graph of the evaluation of cleaving group / warhead efficacy using an in vitro assay. n=3, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant. [Figure 14] Graph comparing the potency of RNA degrader warheads after incubating degrader-functionalized RNA at 37° C. for 4 hours. (a) Degrader-cleavable groups compared to a warhead-free linker (Degrader 2) using a one-tailed t-test. (b) Degrader-cleavable groups compared to imidazole degrader 1 using a two-tailed t-test. n=3, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant. [Figure 15]Graph comparing the non-covalent PDS-based RNA degraders PDS-deg6 and PDS-Amimi after incubation with RNA for 4 hours at 37° C. Statistical significance calculated using a one-tailed t-test. n=3, ***p<0.001, **p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0056] Bifunctional Compounds of the Present Invention The present invention relates to the discovery that bifunctional compounds, also known as degrading agents herein, can be used as catalytic agents to non-covalently bind to and cleave target nucleic acid molecules.The degrading agents disclosed herein bind to target nucleic acids through non-covalent interactions.Therefore, degrading agents do not require the introduction of any reactive groups into target nucleic acids, which would be required if covalent interactions were desired.Selective cleavage of target nucleic acid molecules using the degrading agents described herein can be useful in epigenetics and epitranscriptomics analysis, bifunctional mapping, and even in therapy, such as anti-cancer, antibacterial, and antiviral therapy.

[0057] In one aspect, the present invention provides a bifunctional compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof: CLB (I) [In the formula, C is a cleaving group as defined herein; L is a linker; B is a non-covalent bonding group. Regarding.

[0058] Cutting group C The cleaving group may be any suitable group that is capable of reacting with a target nucleic acid molecule and causing the nucleic acid molecule to be cleaved.

[0059] Without wishing to be bound by any particular theory, the cleaving group may act by abstracting a proton from the 2'OH position of the target nucleic acid molecule, and / or the cleaving group may complex with copper to induce copper-mediated nucleic acid degradation (Li, Zhong-Rui, et al. Nat Chem 11.10(2019):880-889; Wong, K, et al. Can J Biochem 52.11(1974):950-958; Subramaniam, Siddharth, et al. F1000 Research 4(2015)).

[0060] The basicity of a group can be quantitatively assessed using the pKa of the associated conjugate acid: the basicity of a basic group [C] is determined by the pKa of the conjugate acid [CH]. + The pKa of the conjugate acid can be evaluated using a pKa of 5.5 or higher. The pKa of the conjugate acid may be known or can be determined using standard techniques such as acid-base titration. While not wishing to be bound by theory, the inventors believe that basic residues with conjugate acids having pKa values ​​above a certain threshold, e.g., 5.5 or higher, 6.0 or higher, 6.2 or higher, 6.5 or higher, or 6.8 or higher, can deprotonate the hydroxyl group at the 2' position of the ribose sugar to enable cleavage of the phosphodiester backbone within the target nucleic acid. Preferably, the cleaving group has a pKa in the range of 5.5 to 9, more preferably 6 to 9, and most preferably 6.2 to 8.6.

[0061] Alternatively and / or additionally, the cleaving group may be a group known to form a chelate complex with copper that is capable of inducing copper-mediated nucleic acid degradation.

[0062] Suitably, the cleaving group comprises a basic nitrogen atom or hydroxy group that either (i) has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) is a nitrogen atom or hydroxy group that is capable of forming a chelate complex with copper (and thereby inducing copper-mediated nucleic acid degradation).

[0063] Suitably, the basic nitrogen atom or hydroxy group is not sterically hindered, for example, by a substituent (e.g., alkyl substituent) present on the carbon atom directly bonded to the nitrogen or COH group. In one embodiment of the present invention, the carbon atom adjacent to the basic N atom or C-OH group having a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6 is unsubstituted.

[0064] Preferably, the cleaving group is (i) an imidazole (1,3-diazole) group optionally substituted with one, two or three (1-6C) alkyl groups, which may be the same or different; or (ii) a nucleic acid cleaving group of formula Z: [ka] Instead, where: [ka] indicates the point of attachment to L; Ring A is absent or is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C) alkyl, (1-4C) haloalkyl, (1-4C) hydroxyalkyl, OR c , C(O)R c , C(O)OR c ,OC(O)R c , C(O)N(R d )R c , N(R d )C(O)R c , S(O) y R c (where y is 0, 1, or 2), SO2N(R d )R c , N(R d )SO2R c , or NR c R dand R is a nitrogen-containing heteroaryl or heterocyclic ring optionally further substituted with one or more substituents selected from c and R d is selected from hydrogen or (1-4C) alkyl; The integer a1 is 0, 1, 2 or 3; Each occurrence of Ra and Rb is independently selected from hydrogen or (1-2C)alkyl; R1 and R2 are each independently selected from hydrogen, (1-6C)alkyl, heterocyclic, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, each of which is halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR e , C(O)R e , C(O)OR e ,OC(O)R e , C(O)N(R f )R e , N(R f )C(O)R e , S(O) y R e (where y is 0, 1, or 2), SO2N(R f )R e , N(R f )SO2R e , or NR e R f and R e and R f is selected from hydrogen or (1-4C) alkyl, or R1 and R2 are joined together with the atoms to which they are attached to form a 4- to 6-membered heterocycle or a 5- or 6-membered heteroaryl, and any 4- to 6-membered heterocycle or 5- or 6-membered heteroaryl may be selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C) alkyl, (1-4C) haloalkyl, (1-4C) hydroxyalkyl, OR g , C(O)R g, C(O)OR g ,OC(O)R g , C(O)N(R h )R g , N(R h )C(O)R g , S(O) y R g (where y is 0, 1, or 2), SO2N(R h )R g , N(R h )SO2R g , or NR g R h and R g and R h is selected from hydrogen or (1-4C) alkyl; When Ring A is absent, R1 and R2 are each independently selected from hydrogen, heterocyclic ring, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, each of which is selected from halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR e , C(O)R e , C(O)OR e ,OC(O)R e , C(O)N(R f )R e , N(R f )C(O)R e , S(O) y R e (where y is 0, 1, or 2), SO2N(R f )R e , N(R f )SO2R e , or NR e R f and R e and R f is selected from hydrogen or (1-4C) alkyl, with the proviso that R1 and R2 cannot both be hydrogen.

[0065] Preferably, the cleaving group is (i) an imidazole (1,3-diazole) group optionally substituted with one, two or three (1-6C) alkyl groups, which may be the same or different; or (ii) a nucleic acid cleaving group of formula Z: [ka] Instead, where: [ka] indicates the point of attachment to L; Ring A is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C) alkyl, (1-4C) haloalkyl, (1-4C) hydroxyalkyl, OR c , C(O)R c , C(O)OR c ,OC(O)R c , C(O)N(R d )R c , N(R d )C(O)R c , S(O) y R c (where y is 0, 1, or 2), SO2N(R d )R c , N(R d )SO2R c , or NR c R d and R is a nitrogen-containing heteroaryl or heterocyclic ring optionally further substituted with one or more substituents selected from c and R d is selected from hydrogen or (1-4C) alkyl; The integer a1 is 0, 1, 2 or 3; Each occurrence of Ra and Rb is independently selected from hydrogen or (1-2C)alkyl; R1 and R2 are each independently selected from hydrogen, (1-6C) alkyl, (3-6C) cycloalkyl, or (3-6C) cycloalkyl-(1-3C) alkyl, each of which is halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR e , C(O)R e , C(O)OR e ,OC(O)R e , C(O)N(R f )R e , N(R f )C(O)R e , S(O) y R e (where y is 0, 1, or 2), SO2N(R f )R e , N(R f )SO2R e , or NR e R f and R e and R f is selected from hydrogen or (1-4C) alkyl.

[0066] Particular compounds of the present invention include, for example, compounds of formula (I) or any subformula thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the cleaving group C, and any associated substituents, have any of the meanings defined herein above or in any of paragraphs (1) to (12) herein below: (1) The cleaving group C is (i) any N- or C-OH-containing moiety such that at least one N- or C-OH group has a pKa within the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; (ii) any N- or OH-containing moiety capable of chelating with a metal, e.g., copper or zinc, at physiological pH Although selected from; However, the cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above provided that it is not; (2) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-6C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1 )C(O)-, -N(R XC2 )C(O)N(R XC1 )-, -N(R XC1 )C(O)O-, -OC(O)N(R XC1 )-, -S(O)2N(R XC1 ), -N(R XC1 )SO2-, -C(O)N(R XC1 )SO2- or -SO2N(R XC1 )C(O)-; R XC1 and R XC2 are each independently hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkylene, -(CH2) m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-4; L2 is absent or (1-6C) alkylene; R C is selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl or aryl is one or more R Asubstituted by one or more R B optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) (1-6C)hydroxyalkyl; (iii) (3-6C)hydroxycycloalkyl; (iv) NR A1 R A2 ; (v) -(1-6C)alkylene-NR A1 R A2 ; (vi) -(3-6C)cycloalkylene-NR A1 R A2 ; (vii) -(CH2) m2 -R A3 ; (where m2 is 0 to 6) is a group selected from: where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R A3 is one or more OH or NR A1 R A2 optionally substituted with a substituent and one or more R B cycloalkyl or aryl optionally further substituted by substituents; or one or more OH or NR A1 R A2 Or R B heteroaryl or heterocyclyl optionally substituted by substituents; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2] t-C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )R BA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R BB )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; where t is 0, 1, 2, or 3; R BA is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl; The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (2A) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-6C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1)C(O)-, -N(R XC2 )C(O)N(R XC1 )-, -N(R XC1 )C(O)O-, -OC(O)N(R XC1 )-, -S(O)2N(R XC1 ), -N(R XC1 )SO2-, -C(O)N(R XC1 )SO2- or -SO2N(R XC1 )C(O)-; R XC1 and R XC2 are each independently hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkylene, -(CH2) m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-4; L2 is absent or (1-6C) alkylene; R C is selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl or aryl is one or more R A substituted by one or more R B optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) (1-6C)hydroxyalkyl; (iii) (3-6C)hydroxycycloalkyl; (iv) -(3-6C)cycloalkylene-NR A1 R A2 ; (v) -(CH2) m2 -R A3 (where m2 is 0 to 6) is a group selected from where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R A3 is one or more OH or NR A1 R A2 optionally substituted with a substituent and one or more R B cycloalkyl or aryl optionally further substituted by substituents; or one or more OH or NR A1 R A2 Or R B heteroaryl or heterocyclyl optionally substituted by substituents; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2] t -C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )R BA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R BB )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; where t is 0, 1, 2, or 3; R BA is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl; L1, X C and L2 does not exist, and R C is a nitrogen-containing heteroaryl or heterocyclic ring, A is replaced by The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (3) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-2C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1 )C(O)-, -S(O)2N(R XC1 ), or -N(R XC1 )SO2-; R XC1 is hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl (1-2C) alkylene, -(CH2) m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-2; L2 is absent or (1-2C) alkylene; R C is selected from hydrogen, (1-4C) alkyl, (3-6C) cycloalkyl, phenyl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and phenyl are each independently one or more RA substituted by one or more R B optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) NR A1 R A2 ; (iii) -(1-6C)alkylene-NR A1 R A2 is a group selected from R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2] t -C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )R BA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R BB )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; where t is 0, 1, 2, or 3; RBA is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl, The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (3A) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-2C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1 )C(O)-, -S(O)2N(R XC1 ), or -N(R XC1 )SO2-; R XC1 is hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl (1-2C) alkylene, -(CH2) m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-2; L2 is absent or (1-2C) alkylene; R C is selected from hydrogen, (1-4C) alkyl, (3-6C) cycloalkyl, phenyl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and phenyl are each independently one or more R A substituted by one or more RB optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) (1-6C)hydroxyalkyl; (iii) (3-6C)hydroxycycloalkyl; (iv) -(3-6C)cycloalkylene-NR A1 R A2 is a group selected from where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2] t -C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )R BA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R BB )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; where t is 0, 1, 2, or 3; R BAis hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl; L1, X C and L2 does not exist, and R C is a nitrogen-containing heteroaryl or heterocyclic ring, A is replaced by The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (4) The cleaving group C has the formula: -X C -R C is the basis of X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)N(R XC1 )- or -N(R XC1 )C(O)-; R XC1 is hydrogen or (1-6C) alkyl or -(CH2) m1 -heteroaryl, wherein m1 is 0-2; R C is selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and phenyl are each independently one or more R A substituted by one or more R B optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) NR A1 R A2 ; (iii) -(1-6C)alkylene-NR A1 R A2 ; is a group selected from where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B are halo, nitro, cyano, R BA -[CH2] t -OR BA , or -[CH2] t -C(O)OR BA is selected from where t is 0, 1, 2, or 3; R BA is (1-4C) alkyl; The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (4A) The cleaving group C has the formula: -X C -R C is the basis of X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)N(R XC1 )- or -N(R XC1 )C(O)-; R XC1 is hydrogen or (1-6C) alkyl or -(CH2) m1 -heteroaryl, wherein m1 is 0-2; R C is selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl or heterocyclyl; where alkyl or phenyl is one or more R A substituted by one or more R B optionally further substituted by substituents; A heteroaryl or heterocyclic ring may be formed by one or more R A and / or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) (1-6C) hydroxyalkyl is a group selected from R B are halo, nitro, cyano, R BA -[CH2] t -OR BA , or -[CH2] t -C(O)OR BA is selected from t is 0, 1, 2 or 3; R BA is (1-4C) alkyl; X C does not exist and R C is a nitrogen-containing heteroaryl or heterocyclic ring, A is replaced by The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (5) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-6C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1 )C(O)-, -N(R XC2 )C(O)N(R XC1 )-, -N(R XC1 )C(O)O-, -OC(O)N(R XC1 )-, -S(O)2N(R XC1 ), -N(R XC1 )SO2-, -C(O)N(R XC1 )SO2- or -SO2N(R XC1 )C(O)-; R XC1 and R XC2 are each independently hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkylene, -(CH2) m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-4; L2 is absent or (1-6C) alkylene; R C is selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and aryl are each represented by one or more R A substituted by one or more R B optionally further substituted by substituents; If the heteroaryl or heterocyclic ring does not contain one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may contain one or more R A substituted by one or more R B If the heteroaryl or heterocyclic ring is optionally substituted with a substituent, or contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may also contain one or more R A or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) (1-6C)hydroxyalkyl; (iii) (3-6C)hydroxycycloalkyl; (iv) NR A1 R A2 ; (v) -(1-6C)alkylene-NR A1 R A2 ; (vi) -(3-6C)cycloalkylene-NR A1 R A2 ; (vii) -(CH2) m2 -R A3 ; (where m2 is 0 to 6) is a group selected from where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R A3 is one or more OH or NR A1 R A2 optionally substituted with a substituent and one or more R B cycloalkyl or aryl optionally further substituted by substituents; or one or more OH or NR A1 R A2 Or R B heteroaryl or heterocyclyl optionally substituted by substituents; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2] t -C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )RBA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R Bb )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; t is 0, 1, 2 or 3; R BA is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl; The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (6) The cleaving group C has the formula: -L1-X C -L2-R C is the basis of L1 is absent or (1-2C) alkylene; X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R XC1 )-, -N(R XC1 )C(O)-, -S(O)2N(R XC1 ), or -N(R XC1 )SO2-; R XC1 is hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl (1-2C) alkylene, -(CH2)m1 -aryl, -(CH2) m1 -heteroaryl or -(CH2) m1 -heterocyclic rings, m1 is 0-2; L2 is absent or (1-2C) alkylene; R C is selected from hydrogen, (1-4C) alkyl, (3-6C) cycloalkyl, phenyl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and phenyl are each independently one or more R A substituted by one or more R B optionally further substituted by substituents; If the heteroaryl or heterocyclic ring does not contain one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may contain one or more R A substituted by one or more R B If the heteroaryl or heterocyclic ring is optionally substituted with a substituent, or contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may also contain one or more R A or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) NR A1 R A2 ; (iii) -(1-6C)alkylene-NR A1 R A2 is a group selected from R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA , -[CH2]t -C(O)R BA , -[CH2] t -C(O)OR BA , -[CH2] t -OC(O)R BA , -[CH2] t -C(O)N(R BB )R BA , -[CH2] t -N(R BB )C(O)R BA , -[CH2] t -S(O) p R BA (wherein p is 0, 1 or 2), -[CH2] t -SO2N(R Bb )R BA , or -[CH2] t -N(R BB )SO2R BA Selected from; where t is 0, 1, 2, or 3; R BA is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R BB is hydrogen or (1-2C) alkyl; The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (7) The cleaving group C has the formula: -X C -R C is the basis of X C is absent or -O-, -S-, -SO-, -SO2-, -N(R XC1 )-, -C(O)-, -C(O)N(R XC1 )- or -N(R XC1 )C(O)-; R XC1is hydrogen or (1-6C) alkyl or -(CH2) m1 -heteroaryl, wherein m1 is 0-2; R C is selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl or heterocyclyl; where alkyl, cycloalkyl, and phenyl are each independently one or more R A substituted by one or more R B optionally further substituted by substituents; If the heteroaryl or heterocyclic ring does not contain one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may contain one or more R A substituted by one or more R B If the heteroaryl or heterocyclic ring is optionally substituted with a substituent, or contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, the ring may also contain one or more R A or R B optionally substituted by substituents; R A teeth, (i) -OH; (ii) NR A1 R A2 ; (iii) -(1-6C)alkylene-NR A1 R A2 ; is a group selected from where R A1 and R A2 are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B are halo, nitro, cyano, R BA , -[CH2] t -OR BA or -[CH2] t -C(O)OR BA is selected from t is 0, 1, 2 or 3; R BA is (1-4C) alkyl; The cleaving group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above isn't it; (8) The cleaving group C is a group of the formula: [ka] Selected from; (9) The cleaving group C is a group of the formula: [ka] Selected from; (10) The cleaving group C is a group of the formula: [ka] Selected from; (11) The cleaving group C is a group of the formula: [ka] Selected from; (12) The cleaving group C is a group of the formula: [ka] is.

[0067] In one embodiment of the present invention, the cleaving group C is as defined in paragraph (1) above.

[0068] In one embodiment of the present invention, the cleaving group C is as defined in paragraph (2) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (2A) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (3) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (3A) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (4) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (4A) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (5) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (6) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (7) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (8) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (9) above. In one embodiment of the present invention, the cleaving group C is as defined in paragraph (10) above. In one embodiment of the invention, the cleaving group C is as defined above in paragraph (11). In one embodiment of the invention, the cleaving group C is as defined above in paragraph (12).

[0069] As shown above, when non-covalently bound to the target nucleic acid molecule through a linker and a binding group, the cleavage group C is proximal to the target nucleic acid and reacts with the target nucleic acid molecule to cleave one or more phosphodiester bonds, thereby causing the degradation of the target nucleic acid molecule.For example, the cleavage group C of the bound degrading agent can abstract a proton from the 2'OH position on the nucleic acid molecule, causing the cleavage of the phosphodiester bond in the target nucleic acid molecule.In addition, the cleavage group C can form a copper complex, which cleaves the phosphodiester bond in the target nucleic acid molecule.

[0070] Preferably, R C or R AOne of the substituents contains a basic nitrogen atom or hydroxy group that either (i) has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) is a nitrogen atom or hydroxy group that can form a chelate complex with copper (and thereby induce copper-mediated nucleic acid degradation).

[0071] Preferably, R C is alkyl, cycloalkyl, or aryl / phenyl, R A is a substituent that contains a basic nitrogen atom or a hydroxy group that either (i) has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) is a nitrogen atom or a hydroxy group that can form a chelate complex with copper (and thereby induce copper-mediated nucleic acid degradation).

[0072] Preferably, R C is heteroaryl or heterocyclyl, either the heteroaryl or heterocyclyl group contains a basic nitrogen atom that either (i) has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) is a nitrogen atom that can form a chelate complex with copper (and thereby induce copper-mediated nucleic acid degradation), or the heteroaryl or heterocyclyl contains a basic nitrogen atom or a hydroxy group that either (i) has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) is a nitrogen atom or hydroxy group that can form a chelate complex with copper (and thereby induce copper-mediated nucleic acid degradation). A It is substituted by a substituent.

[0073] Linker (-L-) The linker L of the degrading agent comprises a group for connecting (i.e., covalently linking) the cleavable group (C) to the non-covalent linking group (B). Suitable linkers are well known in the art.

[0074] Typically, the linker comprises a divalent group in which one of the free valences forms part of a single bond to the cleaving group (C) and the remaining free valence forms part of a single bond to the non-covalent linking group (B).

[0075] Preferably, the linker is a stable linker, i.e., the linker comprises a group that is not substantially cleaved or degraded in vivo. A stable linker is typically non-reactive at physiological pH and is not substantially degraded by enzymatic action in vivo.

[0076] Typically, the linker is a flexible linker, i.e., the linker allows the cleaving group (C) and the linking group (B) to move with respect to each other with a high degree of freedom.

[0077] Typical linkers include groups selected from alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene. Mixed linkers containing different groups in the covalent bond are acceptable, such as alkylene-arylene (aralkylene) and heteroalkylene-arylene.

[0078] An alkylene (alkanediyl) group is a divalent saturated hydrocarbon group in which two free valencies each form part of a single bond to an adjacent atom. The alkylene group may be a (1-6C) alkylene group, e.g., a 1-4C, 1-3C, or 1-2C alkylene group. In this context, the prefix (e.g., 1-6C) indicates the number of atoms in the hydrocarbon backbone. The alkylene group may be linear or branched. Examples of linear alkylene groups include methanediyl (methylene bridge), ethane-1,2-diyl (ethylene bridge), propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, and hexane-1,6-diyl. Examples of branched alkylene groups include ethane-1,1-diyl and propane-1,2-diyl.

[0079] A heteroalkylene group is an alkylene group in which one or more carbon atoms have been replaced with a heteroatom, such as N, O, or S. The heteroalkylene group may be a 1-6C heteroalkylene group, such as a 1-4C, 1-3C, or 1-2C heteroalkylene group. In this context, the prefix (e.g., 1-6C) indicates the number of atoms in the heteroalkylene backbone, whether carbon atoms or heteroatoms. The heteroalkylene group may be linear or branched.

[0080] Examples of linear heteroalkylene groups include those derived from oxymethylene (e.g., polyoxymethylene, POM), ethylene glycol (e.g., polyethylene glycol, PEG), ethyleneimine (e.g., linear polyethyleneimine, PEI; polyaziridine), and tetramethylene glycol (e.g., polytetramethylene glycol, PTMEG; polytetrahydrofuran). Examples of branched heteroalkylene groups include those derived from propylene glycol (e.g., polypropylene glycol, PPG). When a nitrogen atom is present in a heteroalkylene group, the nitrogen atom may be unsubstituted (NH) or optionally substituted with an alkyl group, e.g., a (1-4C) alkyl group. When a sulfur atom is present in a heteroalkyl group, the sulfur atom may be S, S(O), or S(O).

[0081] A cycloalkylene group is a divalent saturated hydrocarbon group containing a ring in which all of the ring atoms are carbon atoms and the two free valences each form part of a single bond to an adjacent atom. The cycloalkylene group may be a (5-6C) cycloalkylene group. In this context, the prefix (e.g., 5-6C) indicates the number or range of ring atoms. The cycloalkylene group may be monocyclic. Examples of monocyclic cycloalkylene groups include 1,3-cyclopentylene and 1,4-cyclohexylene.

[0082] A heterocycloalkylene (heterocyclene) group is a cycloalkylene group in which one or more carbon atoms are replaced with heteroatoms, such as N, O, and S, or one or more carbon atoms have an oxo substituent (=O). The heterocycloalkylene group may be a C5-6 heterocycloalkylene group. In this context, the prefix (e.g., 5-6C) indicates the number or range of ring atoms, whether carbon atoms or heteroatoms. The heterocycloalkylene group may be monocyclic. If a nitrogen atom is present in a heteroalkylene group, the nitrogen atom may be unsubstituted (NH) or optionally substituted with an alkyl group, such as a 1-4C alkyl group. If a sulfur atom is present in a heteroalkyl group, the sulfur atom may be S, S(O), or S(O).

[0083] An arylene group is a divalent hydrocarbon group containing an aromatic ring in which all of the ring atoms are carbon atoms and the two free valencies each form part of a single bond to an adjacent atom. The arylene group may be a 6-10C arylene group. In this context, the prefix (e.g., 6-10C) indicates the number or range of ring atoms. The arylene group may be monocyclic or may contain two or more rings. An example of a monocyclic arylene group includes 1,4-phenylene. An example of a bicyclic arylene group includes 2,6-naphthylene.

[0084] Heteroarylene groups are arylene groups containing an aromatic ring in which one or more ring atoms are heteroatoms, such as N, O, and S, or one or more carbon atoms have an oxo substituent (=O). Heteroarylene groups may be 6-10C heteroarylene groups. In this context, the prefix (e.g., 6-10C) indicates the number or range of ring atoms, whether carbon atoms or heteroatoms. Heteroarylene groups may be monocyclic or may contain two or more rings. Examples of monocyclic heteroarylene groups include pyrrolylene and pyridylene.

[0085] Preferred linkers include groups selected from alkylene and heteroalkylene. More preferred linkers include heteroalkylene groups. Even more preferred linkers include alkylene ether groups. Most preferred linkers include ethylene oxide groups (e.g., derived from polyethylene glycol, PEG).

[0086] Particular compounds of the present invention include, for example, compounds of formula (I) or any subformula thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the linker group L, and any associated substituents, have any of the meanings defined herein above or in any of paragraphs (13) to (34) herein below: (13) The linker has the formula (L-Ia) or (L-Ib): [ka] is or comprises a group represented by L 1 is a covalent bond or a (1-6C) alkylene group or a (1-6C) heteroalkylene; L 2 is a (1-6C) alkylene group or a (1-6C) heteroalkylene group; L 3 is a (1-6C) alkylene group; n is 0 to 8; * is the point of attachment to the non-covalent group (-B); ** is the point of attachment to the cleavage group (-C); (14) L 1 a linker as defined in paragraph (13), wherein is a covalent bond or methylene; (15) L 3 a linker as defined in paragraph (13) or (14), wherein is (1-4C) alkylene; (16) L 3 a linker as defined in paragraph (13) or (15), wherein is ethylene; (17) A linker as defined in paragraphs (13) to (16), wherein n is 0 to 5 or 2 to 5; (18) L 2 is selected from -CH2-X-, -CH2-CH2-X-, -CH2-CH2-CH2-X-, or -CH2-CH2-CH2-CH2-X-, where X is -O- or -NH-; (19) L 2 is selected from -CH2-X-, -CH2-CH2-X-, or -CH2-CH2-CH2-X-, where X is -O- or -NH-; (20) L 2 is selected from -CH2-X- or -CH2-CH2-X-, where X is -O- or -NH-; (21) L 2 is selected from -CH2-O-, -CH2-NH-, ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-) and tetramethylene oxide (-CH2CH2CH2CH2O-); (22) L 2 is ethylene oxide; (-CH2CH2O-) a linker as defined in paragraphs (13) to (21); (23) The linker is represented by formula (L-IIa) or (L-IIb): [ka] is or comprises a group represented by L 1 , L 3 ,n, * and ** is as described for formula (LI) in paragraph (13) above, and L 1 is optional and is as defined in paragraph (14), and L 3is optionally as defined in paragraph (15) or (16), and n is optionally as defined in paragraph (17); (24) The linker has the formula (L-IIIa) or (L-IIIb): [ka] is or comprises a group represented by L 4 is a (1-6C) alkylene group; L 5 is a (1-6C) alkylene group or a (1-6C) heteroalkylene group; L 6 is a covalent bond or a (1-2C) alkylene group; m is 1 to 8; * is the point of attachment to the non-covalent group (-B); ** is the point of attachment to the cleavage group (-C); (25) L 6 is a covalent bond or methylene; (26) L 4 a linker as defined in paragraph (24) or (25), wherein is (1-4C) alkylene; (27) L 4 a linker as defined in paragraphs (24) to (26), wherein (28) A linker as defined in paragraphs (24) to (27), wherein m is 0 to 5 or 2 to 5; (29) L 5 is selected from -CH2-X-, -CH2-CH2-X-, -CH2-CH2-CH2-X-, or -CH2-CH2-CH2-CH2-X-, where X is -O- or -NH-; (30) L 5is selected from -CH2-X-, -CH2-CH2-X-, or -CH2-CH2-CH2-X-, where X is -O- or -NH-; (31) L 5 is selected from -CH2-X- or -CH2-CH2-X-, where X is -O- or -NH-; (32) L 5 is selected from -CH2-O-, -CH2-NH-, ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-) and tetramethylene oxide (-CH2CH2CH2CH2O-); (33) L 5 a linker as defined in paragraphs (24) to (32), wherein is ethylene oxide (—CH2CH2O—); (34) The linker is represented by formula (L-IV) or (LV): [ka] is or comprises a group represented by L 4 , L 6 ,m, * and ** is as described for formula (L-III) in paragraph (24) above, and L 4 is optionally as defined in paragraph (26) or (27), and L 6 is optional and is as defined in paragraph (25), and m is optional and is as defined in paragraph (28).

[0087] Further preferred linkers are of the formula: [ka] and L 1 , L 2 , L 3 ,n, * ,** , L 4 , L 5 , L 6 , and m are each as defined above; X L is selected from —O—, —S—, —SO—, —SO2—, —NH—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NH— or —NHC(O)—, piperidine, piperazine or triazole.

[0088] Preferably, L 1 is as defined in paragraph (14) above.

[0089] Preferably, L 3 is as defined in paragraph (15) or (16) above.

[0090] Preferably, n is as defined in paragraph (17) above.

[0091] Preferably, L 2 is as defined in any one of paragraphs (18) to (22) above.

[0092] Preferably, L 1 is as defined in paragraph (14) above, and L 2 is as defined in paragraph (18) above, and L 3 is as defined in paragraph (15) above, and n is as defined in paragraph (17) above.

[0093] Preferably, L 1 is as defined in paragraph (14) above, and L 2 is as defined in paragraph (19) above, and L 3 is as defined in paragraph (16) above, and n is as defined in paragraph (17) above.

[0094] Preferably, L 1is as defined in paragraph (14) above, and L 2 is as defined in paragraph (20) above, and L 3 is as defined in paragraph (16) above, and n is as defined in paragraph (17) above.

[0095] Preferably, L 1 is as defined in paragraph (14) above, and L 2 is as defined in paragraph (21) above, and L 3 is as defined in paragraph (16) above, and n is as defined in paragraph (17) above.

[0096] Preferably, L 1 is as defined in paragraph (14) above, and L 2 is as defined in paragraph (22) above, and L 3 is as defined in paragraph (16) above, and n is as defined in paragraph (17) above.

[0097] Preferably, L 6 is as defined in paragraph (25) above.

[0098] Preferably, L 4 is as defined in paragraph (26) or (27) above.

[0099] Preferably, m is as defined in paragraph (28) above.

[0100] Preferably, L 5 is as defined in either one of paragraphs (26) or (27) above.

[0101] Preferably, L 6 is as defined in paragraph (22) above, and L 4 is as defined in paragraph (24) above, and L 5is as defined in paragraph (26) above, and m is as defined in paragraph (25) above.

[0102] Preferably, L 6 is as defined in paragraph (22) above, and L 4 is as defined in paragraph (24) above, and L 5 is as defined in paragraph (27) above, and m is as defined in paragraph (25) above.

[0103] Suitable (1-2C) alkylene groups include methylene (methanediyl), ethylene (ethane-1,2-diyl).

[0104] Suitable (1-6C) alkylene groups include methylene (methanediyl), ethylene (ethane-1,2-diyl), propylene (propane-1,3-diyl), butylene (butane-1,4-diyl), pentylene (pentane-1,5-diyl) and hexylene (hexane-1,6-diyl).

[0105] Suitable (1-6C)heteroalkylene groups include alkylene ether groups, for example ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-) and tetramethylene oxide (-CH2CH2CH2CH2O-).

[0106] Preferably, L 2 is ethylene oxide. Preferably, L 4 is (1-4C) alkylene. Most preferably, L 3 is ethylene.

[0107] Preferably, L 6 is methylene or ethylene. Preferably, m is 2 to 5. Preferably, L 5 is ethylene oxide.

[0108] In one embodiment, m or n is 4-8, 5-7, or 6.

[0109] Non-covalent group (-B) The binding group of the degrading agent comprises a group capable of binding to a target nucleic acid molecule. The binding group binds to the target nucleic acid molecule by a non-covalent bond.

[0110] Some small molecule ligands are known to bind non-covalently to nucleic acids, and therefore can form the basis of non-covalent binding groups.Any small molecule that can bind to nucleic acids can be used as a non-covalent binding group.Such compounds can bind to, for example, MYC or MALAT-1.Preferably, small molecule compounds can bind to secondary or tertiary structures within target nucleic acids.In one embodiment, non-covalent binding groups can target transcribed SNVs and indels (for example, rs4430796 SNP on HNF1B is associated with ovarian and prostate cancer; rs28897672 SNV on BRCA1 is associated with ovarian cancer; rs80359351 deletion on BRCA2 is associated with breast and ovarian cancer; all of these genetic changes are reflected in their respective mRNAs, and therefore can potentially be targeted by the degradation agents of the present invention).

[0111] Additionally, non-covalent binding group B may be an oligonucleotide, nanobody, antibody, or antibody fragment capable of binding to a target nucleic acid sequence. Binding of oligonucleotide binding group B to a target nucleic acid sequence allows for targeted cleavage / degradation of the target nucleic acid molecule. The target nucleic acid sequence may be any desired nucleic acid sequence, including sequences associated with specific medical conditions, such as sequences associated with cancer, nucleotide repeat disorders (e.g., Huntington's, Fragile X, Myotonic Dystrophy Type 1), and sequences of mRNA encoding unstructured proteins (e.g., IAPP in type II diabetes).

[0112] In some embodiments, the non-covalent binding group has a molecular weight of 1,000 kDa or less, for example, the non-covalent binding group has a molecular weight of 800 kDa or less.

[0113] In certain embodiments, the non-covalent binding group binds to a secondary or tertiary structure within the target nucleic acid. Suitable secondary or tertiary structures include quadruplexes, pseudoknots, triplexes, tetraloops, step-loops, and hairpin loops. Preferably, the non-covalent binding group binds to quadruplexes or pseudoknots.

[0114] Preferably, in such embodiments, the non-covalent binding group selectively binds to secondary or tertiary structures within the target nucleic acid. In such cases, the non-covalent binding group preferentially binds to secondary or tertiary structures within the target nucleic acid compared to linear or unstructured nucleic acids. Preferably, the non-covalent binding group selectively binds to quadruplexes or pseudoknots.

[0115] Suitably, the non-covalent binding group selectively binds to ribonucleic acid (RNA) and is therefore sometimes known as a non-covalent RNA binding group.

[0116] Non-covalent binding groups can bind to target nucleic acids via electrostatic interactions, such as ionic interactions, hydrogen bonds, and halogen bonds; van der Waals interactions, such as permanent dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions; and pi interactions, such as pi-pi interactions, pi-cation interactions, and polar-pi interactions.

[0117] The non-covalent binding group may be based on the following small nucleic acid binding molecules:

[0118] [Table 1]

[0119] The non-covalent bonding group may be attached to the linker at any suitable position. Typically, the non-covalent bonding group is attached to the linker via a heteroatom (such as O or NH) or adjacent to a carbonyl group (C=O).

[0120] Suitably, the linking group is selected from formula (BI), (B-II), (B-III) or (B-IV).

[0121] Particular compounds of the present invention include, for example, compounds of formula (I) or any subformula thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the non-covalently binding group B, and any associated substituents, have any of the meanings defined herein above or in any of paragraphs (35) to (39) herein below: (35) The binding group B is selected from an oligonucleotide, a nanobody, an antibody, an antibody fragment or a small molecule capable of binding to a target nucleic acid or one of the above formulae (BI), (B-II), (B-III) or (B-IV); (36) The linking group B is a group of formula (BI) above; (37) The linking group B is a group of the above formula (B-II); (38) The linking group B is a group of the above formula (B-III); (39) The linking group B is the group of formula (B-IV) above.

[0122] Specific Embodiments In one particular embodiment: C is as defined in paragraph (1) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0123] In one particular embodiment: C is as defined in paragraph (2) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0124] In one particular embodiment: C is as defined in paragraph (2A) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0125] In one particular embodiment: C is as defined in paragraph (3) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0126] In one particular embodiment: C is as defined in paragraph (3A) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0127] In one particular embodiment: C is as defined in paragraph (4) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0128] In one particular embodiment: C is as defined in paragraph (4A) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0129] In one particular embodiment: C is as defined in paragraph (5) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0130] In one particular embodiment: C is as defined in paragraph (6) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0131] In one particular embodiment: C is as defined in paragraph (7) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0132] In one particular embodiment: C is as defined in paragraph (8) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0133] In one particular embodiment: C is as defined in paragraph (9) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0134] In one particular embodiment: C is as defined in paragraph (10) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0135] In one particular embodiment: C is as defined in paragraph (11) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0136] In one particular embodiment: C is as defined in paragraph (12) above; L is as defined in any one of paragraphs (13) to (34) above; B is as defined in paragraph (35) above.

[0137] In one particular embodiment, B is a pyridostatin linking group, i.e., the compound has formula (II) shown below: [ka] have wherein L and C are each as defined above.

[0138] In one particular embodiment, B is an MTBD linking group, i.e., the compound has formula (III) shown below: [ka] have wherein L, X and C are each as defined above.

[0139] In one particular embodiment, B is a chloramphenicol linking group, i.e., the compound has formula (IV) shown below: [ka] have wherein L and C are each as defined above.

[0140] In one particular embodiment, B is a lincomycin linking group, i.e., the compound has formula (V) shown below: [ka] have wherein L and C are each as defined above.

[0141] Preferably, in compounds of formula (II), (III), (IV) or (V): Cleavage group C is as defined in any one of paragraphs (1) to (12) above (including 2A, 3A, and 4A); L is as defined in any one of paragraphs (13) to (34) above.

[0142] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (1) above; L is as defined in any one of paragraphs (13) to (34) above.

[0143] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (2) above; L is as defined in any one of paragraphs (13) to (34) above.

[0144] In certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (2A) above; L is as defined in any one of paragraphs (13) to (34) above.

[0145] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (3) above; L is as defined in any one of paragraphs (13) to (34) above.

[0146] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (4A) above; L is as defined in any one of paragraphs (13) to (34) above.

[0147] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (4) above; L is as defined in any one of paragraphs (13) to (34) above.

[0148] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (4A) above; L is as defined in any one of paragraphs (13) to (34) above.

[0149] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (5) above; L is as defined in any one of paragraphs (13) to (34) above.

[0150] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (6) above; L is as defined in any one of paragraphs (13) to (34) above.

[0151] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (7) above; L is as defined in any one of paragraphs (13) to (34) above.

[0152] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (8) above; L is as defined in any one of paragraphs (13) to (34) above.

[0153] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (9) above; L is as defined in any one of paragraphs (13) to (34) above.

[0154] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (10) above; L is as defined in any one of paragraphs (13) to (34) above.

[0155] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (11) above; L is as defined in any one of paragraphs (13) to (34) above.

[0156] For certain compounds of formula (II), (III), (IV) or (V): the cleaving group C is as defined in paragraph (12) above; L is as defined in any one of paragraphs (13) to (34) above.

[0157] Particular compounds of the present invention are compounds of formula VI shown below: [ka] or a pharmaceutically acceptable salt thereof wherein C is as defined hereinbefore, or [ka] [Selected from one of the following]

[0158] Dynamic characteristics The interaction between degrading agent and target nucleic acid can be quantified using dissociation constant (kD).The dissociation constant between degrading agent and nucleic acid containing a given non-covalent group can be known or can be determined using standard techniques such as surface plasmon resonance (SPR), for example, Biacore (Santos, et al., 2021).A suitable system for measuring dissociation constant includes Biacore T200.

[0159] Typically, a degrading agent binds to a target nucleic acid with a dissociation constant (kD) of 10,000 nM or less, as determined by SPR or the like. Preferably, a degrading agent binds to a target nucleic acid with a kD of 1,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.

[0160] As mentioned above, the non-covalent binding group of the degrading agent typically binds to the secondary or tertiary structure in the target nucleic acid.Therefore, the degrading agent typically binds to the secondary or tertiary structure with a dissociation constant (kD) of 10,000nM or less, as determined by SPR or the like.Preferably, the degrading agent binds to the secondary or tertiary structure with a kD of 1,000nM or less, more preferably 500nM or less, even more preferably 200nM or less, and most preferably 100nM or less.

[0161] In some embodiments, the degrading agent binds to the quadruplex with a dissociation constant (kD) of 10,000 nM or less, as determined by, for example, SPR. In such cases, the degrading agent preferably binds to the quadruplex with a kD of 1,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.

[0162] In some embodiments, the degrading agent binds to the pseudoknot with a dissociation constant (kD) of 10,000 nM or less, as determined by, for example, SPR. In such cases, the degrading agent preferably binds to the pseudoknot with a kD of 1,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.

[0163] As noted above, the non-covalent binding group of the degrading agent preferably selectively binds to secondary or tertiary structures within the target nucleic acid. Binding selectivity can be quantified using the ratio of the dissociation constant for binding to a given secondary or tertiary structure compared to the dissociation constant for binding to a linear or unstructured nucleic acid, such as a linear or unstructured RNA. Typically, a comparative linear or unstructured nucleic acid is prepared by mutating one or more residues within the desired secondary or tertiary structure so that it no longer forms a secondary or tertiary structure, while maintaining the rest of the sequence. For example, the selectivity of binding to an RNA G-quadruplex can be estimated by using a comparative RNA in which one or more GGG motifs are changed to AUC motifs.

[0164] Typically, the binding selectivity between a given secondary or tertiary structure and linear or unstructured nucleic acid is 5: 1 or greater. Preferably, the selectivity between a given secondary or tertiary structure and linear or unstructured nucleic acid is 10: 1 or greater, more preferably 20: 1 or greater, even more preferably 50: 1 or greater, and most preferably 100: 1 or greater.

[0165] In one embodiment, the binding selectivity between the quadruplex and linear or unstructured nucleic acid is 5: 1 or greater. Preferably, the selectivity between the quadruplex and linear or unstructured nucleic acid is 10: 1 or greater, more preferably 20: 1 or greater, even more preferably 50: 1 or greater, nM or less, and most preferably 100: 1 or greater.

[0166] In one embodiment, the binding selectivity between the quadruplex and linear or unstructured nucleic acid is 5: 1 or greater. Preferably, the selectivity between the quadruplex and linear or unstructured nucleic acid is 10: 1 or greater, more preferably 20: 1 or greater, even more preferably 50: 1 or greater, nM or less, and most preferably 100: 1 or greater.

[0167] Salts and solvates The degrading agents of the present invention may be provided in free base form.

[0168] The degrading agent of the present invention may be provided in the form of a salt, preferably a pharmaceutically acceptable salt.

[0169] In some embodiments, the degrading agents disclosed herein may be provided in protonated form as salts with suitable counter anions.

[0170] Suitable counterions include both organic and inorganic anions. An example of an inorganic anion is chloride (Cl). - ), bromide (Br - ), iodide (I -), sulfate (SO4), sulfite (SO3), nitrate (NO3), nitrite (NO2), phosphate (PO4), and phosphite (PO3), including those derived from inorganic acids. Examples of organic anions include 2-acetoxybenzoate, acetate, ascorbate, aspartate, benzoate, camphorsulfonate, cinnamate, citrate, edetate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glutamate, glycolate, hydroxymaleate, carboxylate, lactate, laurate, lactate, maleate, malate, methanesulfonate, oleate, oxalate, palmitate, phenylacetate, phenylsulfonate, propionate, pyruvate, salicylate, stearate, succinate, sulfanilate, tartarate, toluenesulfonate, and valerate. Examples of suitable polymeric organic anions include those derived from tannic acid and carboxymethylcellulose.

[0171] In some embodiments, the degrading agents disclosed herein may be provided in deprotonated form as a salt with a suitable counter cation.

[0172] Suitable counterions include both inorganic and organic cations. Examples of suitable inorganic cations are alkali metal ions, e.g., Na + and K. + , alkaline earth metal cations, e.g., Ca 2+ and Mg 2+ , as well as other cations, e.g., NH4 + or Al 3+ Examples of suitable organic cations include substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 +Examples of substituted ammonium ions include ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + is.

[0173] The disintegrating agent of the present invention may be provided in the form of a solvate (a complex of a solute (e.g., a compound, a salt of a compound) and a solvent). Examples of solvates include hydrates, such as monohydrates, dihydrates, and trihydrates.

[0174] The disintegrants of the present invention may be provided in a desolvated form, for example in a dehydrated form.

[0175] Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the spatial arrangement of their atoms are called "isomers." Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers." When a resolving agent of formula (I) has an asymmetric center, for example, if it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric center and are described by the Cahn-Ingold-Prelog R and S ordering rules or by the way the molecule rotates the plane of polarized light and can be designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers, respectively). Chiral compounds can exist as either individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture."

[0176] The compounds of the present invention may have one or more asymmetric centers, and therefore, such compounds can be produced as individual (R)- or (S)-stereoisomers or mixtures thereof. Unless otherwise indicated, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and their racemic or other mixtures. Methods for determining stereochemistry and separating stereoisomers are well known in the art, for example, by synthesis from optically active starting materials or by resolution of racemates (see the discussion in Chapter 4 of "Advanced Organic Chemistry," 4th edition, J. March, John Wiley and Sons, New York, 2001). Some of the compounds of the present invention may have geometric isomeric centers (E- and Z-isomers). It should be understood that the present invention encompasses all optical, diastereomeric, and geometric isomers and mixtures thereof that possess antiproliferative activity.

[0177] The present invention also includes compounds of the invention as defined herein that contain one or more isotopic substitutions. For example, H is 1 H, 2 H(D), and 3 H(T) may be in any isotopic form; C may be in any isotopic form, including 12 C. 13 C, and 14 C may be any isotopic form, including 16 O, and 18 It may be any isotopic form containing O.

[0178] It should also be understood that certain compounds of the present invention may exist in solvated as well as unsolvated forms, such as, for example, hydrated forms, and it should be understood that the present invention encompasses all such solvated forms that possess antiproliferative activity.

[0179] It is also to be understood that certain compounds of the present invention may exist in polymorphic forms, and that the invention encompasses all such forms which possess antiproliferative activity.

[0180] The compounds of the present invention may exist in several different tautomeric forms, and a reference to a compound of the present invention includes all such forms. For the avoidance of doubt, even if the decomposing agent of the present invention may exist in one of several tautomeric forms and only one is specifically described or shown, all others are encompassed by the present invention. Examples of tautomeric forms include keto, enol, and enolate forms, such as in the following tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, and nitro / acinitro.

[0181] [ka]

[0182] The compounds of the present invention containing an amine functional group may also form N-oxides. Reference herein to the decomposing agent of the present invention containing an amine functional group also includes N-oxides. When the decomposing agent of the present invention contains several amine functional groups, one or more nitrogen atoms may be oxidized to form N-oxides. Specific examples of N-oxides are the N-oxides of tertiary amines or nitrogen atoms of nitrogen-containing heterocycles. N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid). For example, see Advanced Organic Chemistry, by Jerry March, 4 thEdition, Wiley Interscience, pages 1977. More specifically, N-oxides can be prepared by the procedure of L.W. Deady (Syn. Comm. 1977, 7, 509-514), in which an amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA) in an inert solvent such as dichloromethane.

[0183] The compounds of the present invention may be administered in the form of prodrugs that are degraded in the human or animal body to release the degrading agents of the present invention. Prodrugs can be used to modify the physical and / or pharmacokinetic properties of the degrading agents of the present invention. A prodrug can be formed when the compounds of the present invention contain a suitable group or substituent to which a property-modifying group can be attached. Examples of prodrugs include in vivo cleavable ester derivatives that can be formed at a carboxy or hydroxy group in the degrading agents of the present invention, and in vivo cleavable amide derivatives that can be formed at a carboxy or amino group in the compounds of the present invention.

[0184] Therefore, the present invention includes the compounds of the present invention as defined hereinbefore when they are made available by organic synthesis and when they are made available in the human or animal body by a method of cleaving the prodrug. Thus, the present invention includes compounds of the present invention produced by means of organic synthesis, as well as such compounds produced in the human or animal body by metabolism of precursor compounds, i.e., the degraders of the present invention may be synthetically produced compounds or metabolically produced compounds.

[0185] Suitable pharmaceutically acceptable prodrugs of the degraders of the present invention are those that are based on sound medical judgment and are suitable for administration to the human or animal body without undesirable pharmacological activity and without undue toxicity.

[0186] Various forms of prodrugs are described, for example, in the following documents: a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H.Bundgaard,Chapter 5“Design and Application of Pro-drugs”, by H.Bundgaard p.113-191(1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1~38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N.Kakeya,et al.,Chem.Pharm.Bull.,32,692(1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, ACCSymposium Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0187] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention having a carboxy group are, for example, in vivo cleavable esters thereof. In vivo cleavable esters of the degrading agents of the present invention containing a carboxy group are, for example, pharmaceutically acceptable esters that are cleaved in the human or animal body to produce the parent acid. Suitable pharmaceutically acceptable esters at carboxy include C 1~6Alkyl esters, such as methyl, ethyl and tert-butyl, C 1~6 Alkoxymethyl esters, e.g., methoxymethyl esters, C 1~6 Alkanoyloxymethyl esters, such as pivaloyloxymethyl ester, 3-phthalidyl ester, C 3~8 Cycloalkylcarbonyloxy-C 1~6 Alkyl esters such as cyclopentylcarbonyloxymethyl and 1-cyclohexylcarbonyloxyethyl ester, 2-oxo-1,3-dioxolenylmethyl esters such as 5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl ester and C 1~6 Alkoxycarbonyloxy-C 1~6 Alkyl esters include, for example, methoxycarbonyloxymethyl and 1-methoxycarbonyloxyethyl esters.

[0188] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention having a hydroxy group are, for example, their in vivo cleavable esters or ethers. The in vivo cleavable esters or ethers of the degrading agents of the present invention containing a hydroxy group are, for example, pharmaceutically acceptable esters or ethers that are cleaved in the human or animal body to generate the parent hydroxy compound. Suitable pharmaceutically acceptable ester-forming groups at the hydroxy group include inorganic esters such as phosphate esters (including phosphoramido cyclic esters). Further suitable pharmaceutically acceptable ester-forming groups at the hydroxy group are C 1~10 Alkanoyl groups, such as acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, C 1~10 Alkoxycarbonyl groups, such as ethoxycarbonyl, N,N-(C 1~6 ) 2 carbamoyl, 2-dialkylaminoacetyl, and 2-carboxyacetyl groups. Examples of ring substituents on the phenylacetyl and benzoyl groups are aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl, and 4-(C 1~4Suitable pharmaceutically-acceptable ether-forming groups at a hydroxy group include α-acyloxyalkyl groups, for example, acetoxymethyl and pivaloyloxymethyl groups.

[0189] Suitable pharmaceutically acceptable prodrugs of the degraders of the invention bearing a carboxy group include, for example, their in vivo cleavable amides, e.g., amines, e.g., ammonia, C 1~4 Alkylamines, such as methylamine (C 1~4 alkyl)2amines, such as dimethylamine, N-ethyl-N-methylamine or diethylamine, C 1~4 Alkoxy-C 2~4 Alkylamines, such as 2-methoxyethylamine, phenyl-C 1~4 amides formed with alkylamines, such as benzylamine, and amino acids, such as glycine or its esters.

[0190] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention having an amino group are, for example, in vivo cleavable amide derivatives thereof. Suitable pharmaceutically acceptable amides from an amino group include, for example, C 1~10 Alkanoyl groups include amides formed with alkanoyl groups such as acetyl, benzoyl, phenylacetyl, and substituted benzoyl and phenylacetyl groups. Examples of ring substituents on the phenylacetyl and benzoyl groups are aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl, and 4-(C 1~4 alkyl)piperazin-1-ylmethyl.

[0191] The in vivo action of the degraders of the present invention may be exerted in part by one or more metabolic products formed in the human or animal body following administration of the degraders of Formula I. As described herein above, the in vivo action of the degraders of the present invention may also be exerted by metabolism of a precursor compound (prodrug).

[0192] The present invention may relate to any compound, or particular group of compounds, defined herein by any preferred or suitable feature or otherwise with respect to a particular embodiment, but the present invention may also relate to any compound, or particular group of compounds, that specifically excludes said any preferred or suitable feature or particular embodiment.

[0193] synthesis The compounds of the present invention can be prepared by any suitable technique known in the art. Specific processes for preparing these compounds are further illustrated in the accompanying examples.

[0194] In the descriptions of synthetic methods described herein, and in any reference synthetic methods used to prepare starting materials, it should be understood that all proposed reaction conditions, including the choice of solvents, reaction atmospheres, reaction temperatures, experimental times, and work-up procedures, can be selected by one of ordinary skill in the art.

[0195] It is understood by one skilled in the art of organic synthesis that the functions present on various portions of the molecule must be compatible with the reagents and reaction conditions employed.

[0196] It will be appreciated that during the synthesis of the compounds of the invention in the processes defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituents to prevent undesired reactions. The skilled chemist will know when such protection is necessary and how such protecting groups are put in place and how they may be later removed.

[0197] For examples of protecting groups, see one of the many general texts on the subject, such as 'Protective Groups in Organic Synthesis' by Theodora Green (publisher: John Wiley & Sons). Protecting groups can be removed by any convenient method described in the literature or known to the skilled chemist to be suitable for removing the protecting group in question, such method being chosen so as to effect removal of the protecting group with minimal disturbance of groups elsewhere in the molecule.

[0198] Thus, if reactants include groups such as amino, carboxy or hydroxy, it may be desirable to protect the group in some of the reactions mentioned herein.

[0199] For example, suitable protecting groups for amino or alkylamino groups include, for example, acyl groups, such as alkanoyl groups (e.g., acetyl), alkoxycarbonyl groups (e.g., methoxycarbonyl, ethoxycarbonyl, or t-butoxycarbonyl), arylmethoxycarbonyl groups (e.g., benzyloxycarbonyl), or aroyl groups (e.g., benzoyl). The deprotection conditions for the above-mentioned protecting groups necessarily vary with the choice of protecting group. Thus, for example, acyl groups, such as alkanoyl or alkoxycarbonyl groups, or aroyl groups can be removed by hydrolysis with a suitable base, such as an alkali metal hydroxide, e.g., lithium or sodium hydroxide. Alternatively, acyl groups, such as tert-butoxycarbonyl groups, can be removed by treatment with a suitable acid, e.g., hydrochloric acid, sulfuric acid, or phosphoric acid, or trifluoroacetic acid, and arylmethoxycarbonyl groups, such as benzyloxycarbonyl groups, can be removed by hydrogenation over a catalyst, e.g., palladium on carbon, or by treatment with a Lewis acid, e.g., boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or hydrazine.

[0200] Suitable protecting groups for hydroxy groups include, for example, acyl groups, e.g., alkanoyl groups such as acetyl, aroyl groups such as benzoyl, or arylmethyl groups such as benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, acyl groups such as alkanoyl or aroyl groups can be removed by hydrolysis with a suitable base, e.g., an alkali metal hydroxide, e.g., lithium or sodium hydroxide, or ammonia. Alternatively, arylmethyl groups such as benzyl groups can be removed by hydrogenation over a catalyst, e.g., palladium on carbon.

[0201] Suitable protecting groups for carboxy groups are, for example, esterifying groups such as methyl or ethyl groups which may be removed by hydrolysis with a base such as, for example, sodium hydroxide, or a t-butyl group which may be removed by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or a benzyl group which may be removed by hydrogenation over a catalyst such as, for example, palladium-on-carbon.

[0202] Resins can also be used as protecting groups.

[0203] The methods used to synthesize the decomposing agents of the present invention will vary depending on the nature of any substituents associated therewith. Suitable processes for their preparation are further illustrated in the accompanying examples.

[0204] Once the decomposition agent of the present invention has been synthesized by any one of the processes defined herein, the process may then additionally comprise the steps of: (i) optionally removing any protecting groups present; (ii) optionally converting a compound of the invention into another compound of the invention; (iii) optionally forming a pharmaceutically acceptable salt, hydrate or solvate thereof; and / or (iv) optionally forming a prodrug thereof It may further include:

[0205] An example of (ii) above is where a decomposing agent of the present invention is synthesised and then one or more of the groups can be further reacted to change the nature of the group to give an alternative compound of the present invention.

[0206] The resulting compounds of the invention can be isolated and purified using techniques well known in the art.

[0207] Methods for cleaving target nucleic acids The present invention provides a method for cleaving a target nucleic acid molecule, the method comprising: The target nucleic acid molecule is treated with a degrading agent of formula (I) as defined herein: CLB (I) wherein -C is a cleaving group, -L- is a linker, and -B is a non-covalent linking group, so that the degradative agent non-covalently binds to the target nucleic acid molecule; allowing the degradative agent to cleave the target nucleic acid molecule bound thereto; Includes:

[0208] Particular embodiments of the degrading agents of formula (I) are presented above.

[0209] In some embodiments, the target nucleic acid molecule can be contacted with the degradative agent in solution.

[0210] More preferably, the target nucleic acid molecule can be contacted with the degradative agent within a cell (i.e., intracellularly). The cell can be in vitro or can be an isolated cell, for example, an isolated cell line or a cell isolated from an individual (e.g., from a tissue sample such as a biopsy).

[0211] Suitable cells may include mammalian, preferably human, cells. The cells may include somatic and germ cells, may be at any stage of development, and may include fully or partially differentiated cells or undifferentiated or pluripotent cells, including stem cells, such as adult or somatic stem cells, fetal stem cells, or embryonic stem cells. For example, the cells may include nerve cells, including neurons and glial cells, contractile muscle cells, smooth muscle cells, hepatocytes, hormone-synthesizing cells, sebocytes, pancreatic islet cells, adrenal cortical cells, fibroblasts, keratinocytes, endothelial and urinary tract cells, bone cells, and chondrocytes. In some embodiments, the cells may be associated with a pathology, such as cancer cells, such as carcinomas, sarcomas, lymphomas, blastomas, or germline tumor cells, and cells containing the genotype of a genetic disorder, such as Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome, or Marfan syndrome.

[0212] The target nucleic acid molecule may be an endogenous nucleic acid present in a cell. The degradative agent may be an exogenous molecule. The method may include introducing the degradative agent into a cell and allowing it to bind to the target nucleic acid molecule.

[0213] The target nucleic acid molecule may be a DNA or RNA molecule. Suitable target RNA molecules may include mRNA and long non-coding RNA (lncRNA). RNA molecules may include introns and intergenic regions.

[0214] The target nucleic acid molecule may comprise a secondary or tertiary structure. Suitable secondary and tertiary structures include a quadruplex, a pseudoknot, a tetraloop, a step-loop, and a hairpin loop. Preferably, the target nucleic acid molecule comprises a quadruplex or a pseudoknot.

[0215] For example, a method for cleaving a target nucleic acid containing a secondary or tertiary structure includes: The target nucleic acid molecule is treated with a degrading agent of formula (I) as defined herein: CLB (I) wherein -C is a cleavage group, -L- is a linker, and -B is a non-covalent group that interacts with secondary or tertiary structure to non-covalently bind the degradative agent to the target nucleic acid molecule; allowing the degradative agent to cleave the target nucleic acid molecule bound thereto; may include:

[0216] In one particular embodiment, the secondary or tertiary structure is a quadruplex. In such a case, the method comprises: The target nucleic acid molecule comprising the quadruplex is treated with a degradation agent of formula (I) as defined herein: CLB (I) wherein -C is a cleavage group, -L- is a linker, and -B is a non-covalent group that interacts with the quadruplex to non-covalently bind the degradation agent to the target nucleic acid molecule; allowing the degradative agent to cleave the target nucleic acid molecule bound thereto; may include:

[0217] In one particular embodiment, the secondary or tertiary structure is a pseudoknot. In such a case, the method comprises: A target nucleic acid molecule containing a pseudoknot is treated with a degradation agent of formula (I) as defined herein: CLB (I) wherein -C is a cleavage group, -L- is a linker, and -B is a non-covalent group that interacts with the pseudoknot, to non-covalently bind the degradative agent to the target nucleic acid molecule; allowing the degradative agent to cleave the target nucleic acid molecule bound thereto; may include:

[0218] When non-covalently bound to a target nucleic acid molecule, the degradative agent cleaves the target nucleic acid. Without wishing to be bound by any particular theory, it is understood that the degradative agent cleaves one or more phosphodiester bonds in the target nucleic acid.

[0219] When non-covalently bound to a target nucleic acid molecule, the cleaving group can abstract a proton from the 2'OH of a nucleotide in the target nucleic acid. Cleavage of the phosphodiester backbone can occur by intramolecular attack on the phosphate group at the 3' position.

[0220] When non-covalently bound to a target nucleic acid molecule, the cleaving group may bind to one or more transition metals (e.g., copper), and the degrading agent may cleave the target nucleic acid by copper-mediated nucleolysis.

[0221] Binding of the degradative agent to the target nucleic acid can proceed via an intermediate species, i.e., the target nucleic acid molecule is converted to a target nucleic acid molecule of the formula: CLB~NA wherein -C is a cleavage group as defined herein, -L- is a linker as defined herein, -B is a non-covalent group as defined herein, ~ is a non-covalent interaction, and NA is the target nucleic acid; and allowing a degrading agent to cleave the target nucleic acid molecule.

[0222] Particular embodiments of the degrading agents of formula (I) are presented above.

[0223] Methods for identifying secondary or tertiary structure After selectively cleaving the target nucleic acid molecule with a degradative agent as described above, the method may include identifying the target nucleic acid molecule, which may be useful, for example, in mapping sites containing secondary or tertiary structure within the nucleic acid.

[0224] The method may include determining the abundance or amount of one or more nucleic acid molecules in a population of nucleic acids. A decrease in the abundance or amount of the nucleic acid molecule in the population compared to a control indicates that the nucleic acid molecule is a target nucleic acid molecule selectively cleaved by a degradative agent. A suitable control may be a population of nucleic acids that has not been treated with a degradative agent.

[0225] Accordingly, the present invention provides a method for identifying secondary or tertiary structure within a target nucleic acid molecule, comprising: providing first and second populations of nucleic acid molecules, each population comprising a target nucleic acid molecule; a first population of nucleic acid molecules, the first population of nucleic acid molecules being degraded by a degrading agent of formula (I): CLB (I) wherein -C is a cleaving group as defined herein, -L- is a linker as defined herein, and -B is a non-covalent linking group as defined herein, so that the degradative agent is non-covalently attached to the target nucleic acid molecule; allowing a degradative agent to cleave target nucleic acid molecules present in a first population; and identifying nucleic acid molecules present in the first population in reduced amounts relative to a second population. The present invention provides a method comprising:

[0226] Particular embodiments of the decomposition agents of formula (I) are presented above.

[0227] The non-covalent binding group can bind to a secondary or tertiary structure in the target nucleic acid molecule. Suitable secondary or tertiary structures include quadruplexes, pseudoknots, tetraloops, step-loops and hairpin loops. Preferably, the secondary or tertiary structure is a quadruplex or pseudoknot.

[0228] The first and second populations of nucleic acid molecules may independently be isolated (ex vivo) populations of nucleic acid molecules. Alternatively, one or more populations of nucleic acid molecules may be present within a cell.

[0229] Said method may comprise extracting total nucleic acid from cell, such as total DNA or total RNA.Nucleic acid may be further analyzed, for example, to determine the abundance or amount of one or more nucleic acid molecules.For example, the total nucleic acid extracted may be sequenced, and sequence readings may be analyzed.

[0230] Suitable methods for determining the abundance or quantity of nucleic acid molecules in cells are well known in the art and include RT-qPCR, RNA-sequencing (RNA-seq), next-generation sequencing (NGS), nanopore sequencing, and other sequencing technologies, such as Sanger sequencing, Tracking Indels by Composition (TIDE) (Brinkman et al. Nucleic Acids Res. 2014 Dec 16;42(22):e168), and PCR analysis. In some embodiments, the method may include extracting nucleic acid molecules from cells, sequencing the extracted nucleic acid molecules, and determining the number of sequence reads (i.e., read counts) for each extracted nucleic acid molecule to determine the abundance or quantity of each nucleic acid molecule in the cells. In some embodiments, the raw read counts may be normalized and expressed as RPKM (reads per kilobase of exon model per million reads) or FPKM (fragments per kilobase of exon model per million mapped reads). Suitable methods for sequencing and sequence analysis are well established in the art.

[0231] Use in medicines Selective cleavage of a target nucleic acid molecule by the degradative agents described above can alter the downstream effects of the target nucleic acid molecule, which can be useful, for example, in the treatment or prevention of diseases mediated by the target nucleic acid molecule.

[0232] Thus, the present invention provides a degrading agent of formula (I) for use in a method of treating the human or animal body by therapy, for example for use in a method of treating a disorder (e.g., a disease).

[0233] Another aspect of the present invention relates to a method of treating, e.g., a disorder (e.g., a disease), comprising administering a therapeutically effective amount of a degrading agent of formula (I) to a subject in need of treatment.

[0234] Another aspect of the present invention relates to the use of a degrading agent of formula (I) in the manufacture of a medicament for use in treating a disorder (e.g., a disease). Typically, the medicament comprises a degrading agent of formula (I).

[0235] Disorders to be treated (i) Proliferative disorders (e.g., cancer) According to a further aspect of the present invention there is provided a method of inhibiting cell proliferation in vitro or in vivo, comprising contacting a cell with an effective amount of a degrader of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0236] According to a further aspect of the present invention there is provided a method of treating a proliferative disorder in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a degrader of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0237] According to a further aspect of the present invention there is provided a method of treating cancer in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a degrader of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0238] According to a further aspect of the present invention there is provided a degrader of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein, for use in the treatment of a proliferative condition.

[0239] According to a further aspect of the present invention, there is provided a degrading agent of formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein, for use in the treatment of cancer. In a particular embodiment, the cancer is a human cancer.

[0240] According to a further aspect of the present invention there is provided the use of a disintegrator of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof in the manufacture of a medicament for treating a proliferative condition.

[0241] According to a further aspect of the present invention, there is provided the use of a disintegrant of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof in the manufacture of a medicament for treating cancer. Suitably, the cancer is a human cancer.

[0242] Optionally, the compound or pharmaceutical composition is administered in combination with one or more additional anti-proliferative agents (eg, checkpoint inhibitors and / or cytotoxic agents).

[0243] The term "proliferative disorder" is used herein to refer to unwanted or uncontrolled cell proliferation of unwanted, excessive, or abnormal cells, e.g., neoplastic or hyperplastic growth, whether in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, premalignant and malignant cell proliferation, including malignant neoplasms and tumors, cancer, leukemia, psoriasis, bone disease, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. Any type of cell can be treated, including, but not limited to, lung, colon, breast, ovary, prostate, liver, pancreas, brain, and skin.

[0244] If the proliferative disorder is cancer, the cancer is optionally selected from adenoid cystic carcinoma, adrenal carcinoma, amyloidosis, anal cancer, ataxia telangiectasia, atypical nevus syndrome, basal cell carcinoma, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brain tumor, breast cancer (male and female), carcinoid tumor, cervical cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), islet cell tumor, juvenile polyposis syndrome, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adult leukemia, childhood leukemia, chronic lymphocytic leukemia (CLL), Selected from chronic myeloid leukemia (CML), liver cancer, lobular carcinoma, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, malignant glioma, melanoma, meningioma, multiple myeloma, myelodysplastic syndrome (MDS), nasopharyngeal carcinoma, neuroendocrine tumor, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor, parathyroid cancer, penile cancer, peritoneal cancer, Peutz-Jeghers syndrome, pituitary tumor, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma, salivary gland cancer, sarcoma, Kaposi's sarcoma, skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine (endometrial) cancer, vaginal cancer, or Wilms' tumor.

[0245] In one embodiment, the decomposition agent of formula I can target the SNV and indel transcribed in various cancers.For example, the rs4430796 SNP on HNF1B is associated with ovarian and prostate cancer; the rs28897672 SNV on BRCA1 is associated with ovarian cancer; and the rs80359351 deletion on BRCA2 is associated with breast cancer and ovarian cancer.All of these genetic changes are reflected in their respective mRNAs, and therefore can potentially be the target of the decomposition agent of the present invention.

[0246] Particular cancers of interest include Ewing's sarcoma (e.g., by targeting ESWR1 with a pyridostatin (PDS) non-covalent binding group) and lung, breast, cervical and colon cancer (by targeting MALAT1 with a MALAT1-binding non-covalent binding group).

[0247] (ii) Bacterial and viral infections In one embodiment (eg, of use in methods of therapy, use in the manufacture of medicaments, methods of treatment), the treatment is treatment of a bacterial or viral infection.

[0248] Preferably, the viral infection is an RNA virus (for example, a virus whose viral genome comprises single-stranded or double-stranded RNA). Many pathogenic viruses utilize -1 ribosomal frameshifting as a mechanism for accurate protein translation, and this phenomenon is made possible by secondary RNA structures such as stem-loops and pseudoknots. Therefore, by targeting these secondary RNA structures with the degrading agent of formula (I), viral RNA can be cleaved and inactivated, thereby treating viral infection.

[0249] Examples of RNA viruses include (+)ssRNA viruses, such as coronaviruses, picornaviruses, and togaviruses; (-)ssRNA viruses, such as orthomyxoviruses and rhabdoviruses; and dsRNA viruses, such as reoviruses.

[0250] Preferably, the virus is a (+)ssRNA virus, more preferably a coronavirus. Examples of coronaviruses include alphacoronaviruses, such as transmissible gastroenteritis virus, feline coronavirus, and canine coronavirus; betacoronaviruses, such as Middle East respiratory syndrome-related coronavirus (MERS-CoV), murine coronavirus (M-CoV), and severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2); gammacoronaviruses, such as avian coronaviruses; and deltacoronaviruses, such as brown-eared coronavirus HKU11 and porcine coronavirus HKU15.

[0251] Bacterial infection can be infection with Gram-negative or Gram-positive bacteria. Both types of bacteria contain bacterial ribosomes, which are riboenzymes that contain both protein and RNA units. Therefore, by targeting the RNA unit with the degrading agent of formula (I), bacterial ribosomes can be cleaved and inactivated, thereby treating bacterial infection.

[0252] Examples of medically relevant Gram-negative bacteria include Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, and Pseudomonas aeruginosa (which are primarily associated with respiratory problems); Escherichia coli and Enterobacter cloacae (which are primarily associated with urinary tract problems); and Helicobacter pylori and Salmonella enterica (which are primarily associated with gastrointestinal problems), and Neisseria meningitidis (which is primarily associated with meningitis).

[0253] Thus, in one embodiment, the Gram-negative bacterial species is selected from the group consisting of E. coli, E. cloacae, H. pylori, S. enterica, H. influenzae, K. pneumoniae, L. pneumoniae, L. pneumophila, P. aeruginosa and N. meningitidis.

[0254] Examples of medically relevant Gram-positive bacteria include actinomyces, bacillus, clostridium, corynebacterium (e.g., Corynebacterium diphtheriae), enterococcus, erysipelothrix, listerial (e.g., Listeria monocytogenes), nocardia, staphylococcal, and streptococcal (e.g., Staphylococcus aureus).

[0255] Thus, in one embodiment, the Gram-negative bacterial genus is selected from the group consisting of Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Erysipelothricus, Listeria nocardia, Staphylococcus, and Streptococcus.

[0256] In one embodiment (eg, of use in a method of therapy, of use in the manufacture of a medicament, of a method of treatment), the treatment is treatment of a respiratory infection, a urinary tract infection, or gastroenteritis.

[0257] Other conditions The degrading agents of the present invention can also be used to degrade other nucleic acid sequences associated with other pathologies. For example, the degrading agents of the present invention can be used to treat mRNA sequences encoding nucleotide repeat disorders (e.g., Huntington's disease, Fragile X, Myotonic Dystrophy Type 1) and unstructured proteins (e.g., IAPP in type II diabetes).

[0258] Patients to be treated In one embodiment (eg, of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is administered to a subject in need of treatment.

[0259] The subject (patient) in need of treatment may be a chordate, vertebrate, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), or any animal. , equine (e.g., horse), porcine (e.g., pig), ovine (e.g., sheep), bovine (e.g., cow), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0260] The subject in need of treatment may be an adult or a juvenile.

[0261] Preferably, the subject in need of treatment is a human, more preferably an adult.

[0262] Alternatively, the subject in need of treatment is a non-human animal used in laboratory research. Preferably, the non-human animal is a rodent (e.g., guinea pig, hamster, rat, mouse).

[0263] Administration route In one embodiment (e.g., for use in methods of therapy, for use in the manufacture of medicaments, for methods of treatment), the treatment is administered by any convenient route of administration, whether systemic / peripheral or local (i.e., at the desired site of action).

[0264] Routes of administration may be oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by patches, plasters, etc.); transmucosal (including, e.g., by patches, plasters, etc.); intranasal (e.g., by nasal spray); ophthalmic (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy, e.g., using an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by vaginal suppository); parenteral, by injection, including, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intrathecal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; or by, e.g., subcutaneous or intramuscular implantation of a depot or reservoir.

[0265] formulation In one embodiment (e.g., for use in a method of treatment, for use in the manufacture of a medicament, for a method of treatment), the degrading agent of Formula (I) is administered alone. Typically, however, it is preferred to provide the degrading agent in a pharmaceutical formulation (e.g., a composition, preparation, medicament) that includes at least one degrading agent as described herein together with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, additives, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. The formulation may further include other active agents, such as other therapeutic or prophylactic agents.

[0266] Thus, the present invention further provides pharmaceutical compositions and methods of making pharmaceutical compositions comprising mixing at least one disintegrant described herein together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

[0267] The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of an appropriate subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0268] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 25 2005.

[0269] The formulation can be prepared by any method known in the art of pharmacy. Such methods include the step of bringing the disintegrating agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the formulation is prepared by uniformly mixing the disintegrating agent with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0270] The formulations may be prepared to provide immediate or sustained release; immediate, delayed, timed, or sustained release; or a combination thereof.

[0271] The formulation may suitably be in the form of a liquid, solution (e.g., aqueous, non-aqueous), suspension (e.g., aqueous, non-aqueous), emulsion (e.g., oil-in-water, water-in-oil), elixir, syrup, electuary, mouthwash, drops, tablet (e.g., including coated tablets), granules, powder, lozenge, pastille, capsule (e.g., including hard and soft gelatin capsules), cachet, pill, ampoule, bolus, suppository, pessary, tincture, gel, paste, ointment, cream, lotion, oil, foam, spray, mist, or aerosol.

[0272] Formulations may suitably be provided as patches, adhesives, plasters, bandages, dressings, or the like impregnated with one or more compounds and, optionally, one or more other pharmaceutically acceptable ingredients including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.

[0273] The disintegrant may be dissolved, suspended, or mixed with one or more other pharmaceutically acceptable components. For example, the compound may be applied to a liposome or other microparticle designed to target the compound to a blood component or one or more organs.

[0274] Dosage In one embodiment (e.g., of use in methods of therapy, use in the manufacture of medicaments, methods of treatment), the treatment comprises administering a therapeutically effective amount of a degrading agent of formula (I) to a subject in need of treatment.

[0275] Those skilled in the art will understand that the appropriate dosage of the degrading agent and composition comprising the degrading agent described herein may vary from patient to patient. Determining the optimal dosage generally requires balancing the level of therapeutic effect with any risk or harmful side effects. The selected dosage level will depend on various factors, including, but not limited to, the activity of the specific degrading agent, the route of administration, the time of administration, the excretion rate of the degrading agent, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the disorder, and the patient's species, sex, age, weight, condition, general health, and previous medical history. The amount and route of administration of the degrading agent will ultimately be at the discretion of the physician, veterinarian, or clinician, but generally, the dosage will be selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or toxic side effects.

[0276] Administration can be carried out in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment.Methods of determining the most effective means and dosage of administration are well known to those skilled in the art and will vary with the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated.Single or multiple administrations can be carried out, with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

[0277] In general, a suitable dose of the disintegrant ranges from about 10 μg to about 250 mg (more typically, from about 100 μg to about 25 mg) per kilogram of subject body weight per day.

[0278] Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

[0279] Other priorities Any and all compatible combinations of the embodiments described above are expressly disclosed herein just as if each and every combination were individually and expressly listed.

[0280] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0281] As used herein, "and / or" should be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be construed as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually presented herein.

[0282] Unless the context dictates otherwise, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described. [Example]

[0283] Certain aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the figures described above.

[0284] [Example 1] Studies demonstrating the concept of noncovalent nucleic acid degradation using model degraders containing imidazole cleavage groups General Experimental Protocol In vitro pseudoknot oligolysis RNA oligos (20 μM) were added to a pH 7.5 HEPES (20 mM) buffer supplemented with KCl (50 mM) and EDTA (10 mM). The mixture was incubated at 37° C. for 30 minutes. MTDB-deg 16a, MTDB, or TDB-deg 16b (1 mM) was then added. The reaction mixture was incubated at 37° C. for 3 hours and then maintained at 4° C. The reaction mixture was analyzed by LC-MS or gel electrophoresis.

[0285] LC-MS analysis of oligonucleotides. Oligonucleotides were analyzed by LC-MS according to the method of Mikutis et al., 2020.

[0286] Oligomers were analyzed using a Xevo G2-S TOF mass spectrometer connected to an Acquity UPLC system using an Acquity UPLC BEH C18 1.7 μm column. The system utilizes electrospray ionization (ESI). Two mobile phases were used: 16.3 mM TEA, 400 mM HFIP in HO and 16.3 mM TEA, 400 mM HFIP in 80:20 v / v MeCN and HO at a flow rate of 0.200 mL / min. Calibration curves for RNA species were based on either A260 or the intensity of the specified m / z signal minus the intensity of the signal. Integrated peak intensities were calculated using a native module in the KNIME software platform (33). Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled in MassLynx software (v.4.1, Waters) according to the manufacturer's instructions. To obtain the negative ion series described, oligomeric peaks in the chromatograms were selected for integration and further analysis.

[0287] RNA degradation gel electrophoresis Gel electrophoresis was performed according to the method of Mikutis et al., 2020.

[0288] In vitro RNA degradation reactions were performed as described above. The quenched reaction mixture was mixed with loading buffer (95% formamide, 0.025% SDS, 0.025% bromophenol blue (BPB), 0.025% xylene cyanol FF, 0.025% ethidium bromide, 0.5 mM EDTA) in a 1:1 ratio, heated at 70°C for 5 minutes, and cooled to 0°C. PAGE was performed on Novex™ TBEUrea Gels containing 15% polyacrylamide in 1x TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 180V for 60 minutes. Gel staining was performed in 1x TBE buffer using SYBR Green II RNA Gel Stain (Invitrogen). The stained RNA was visualized using a ChemiDoc MP (Bio-Rad, United Kingdom).

[0289] Virus stock The SARS-CoV-2 stock used to infect Vero CCL-81 cells was derived from passage 4 SARS-CoV-2 isolated from a Portuguese patient (internal reference: 606_IMM ID_5452) containing approximately 1.7 × 10 6 PFU / mL were established after 4 days in Vero CCL-81 cultures. Stock titers were calculated by plaque assay. Briefly, approximately 8 x 10 5CCL-81 cells were seeded in 6-well plates at 1000 μL / well and grown to confluence for 24 hours. The medium was removed, and 500 μL of 10-fold serial dilutions of virus-containing supernatant were adsorbed in duplicate for 1 hour at 37°C. The plate was manually rocked to redistribute the inoculum every 15 minutes. The cells were overlaid with 1.25% carboxymethylcellulose (CMC) supplemented with DMEM and incubated at 37°C for 4 days. After incubation, the overlaid CMC was removed, and the cells were fixed with 4% formaldehyde / PBS and stained with 0.1% toluidine blue. After inactivation by fixation, the plates were sealed with parafilm, sterilized, and then removed from the BSC and BSL3. Viral plaques were counted to determine the infectious titer (PFU (plaque-forming units) / mL).

[0290] Viral infection of Vero CCL-81 Vero CCL-81 cells at 80% confluence were incubated with the SARS-CoV-2 inoculum for 1 hour at 37°C. After incubation, the inoculum was removed and DMEM medium supplemented with 2.5% FCS was added for 24 hours or until samples were taken.

[0291] Gel electrophoresis analysis 500 ng of SARS-CoV-2 RNA was incubated with or without 100 μM MTDB-deg 16a in 1x HEPES buffer for 2 hours at 37°C with gentle agitation. Samples were then analyzed on a 1.5% agarose gel.

[0292] Nanopore sequencing 500 ng of SARS-CoV-2 RNA was incubated with or without 100 μM MTDB-deg 16a in 1x HEPES buffer for 2 hours at 37°C with gentle agitation. Samples were then prepared for sequencing according to the manufacturer's protocol for Direct RNA Sequencing (SQK-RNA002, ONT). The prepared libraries were loaded onto a FLO-MIN106D flow cell (ONT) and sequenced on a MinION Mk1C device (ONT).

[0293] The genome sequence and genome annotation (NC_045512.2) of the SARS-CoV-2 Wuhan-hu1 strain (GenBank: MN908947.3) were downloaded from the NCBI database. Sequence reads were aligned to the Wuhan-hu1 genome using minimap2 (Li et al., 2018) with the parameters "-ax splice -N32 -un -k13". CIGAR strings from the alignment were processed using a customized script. Reads were flagged as leaders if the splice junction within the read started within the first 60–120 bp of the genome. Reads were assigned to individual transcripts if they covered >90% of the annotated transcripts or if >90% of the read sequence was within the transcript range.

[0294] Drug assay to determine 50% inhibitory concentration Increasing concentrations of MTDB-deg 16a (ranging from 0.07 to 25 μM) were tested to determine the 50% inhibitory concentration (IC 50) was determined. Vehicle (HO) controls and control molecules were included in parallel. Cells were seeded into 96-well plates at approximately 40% confluency 24 hours before infection. MTDB-deg16a, MTDB, or TDB-deg16b were added either 1 hour before or 1 hour after infection. SARS-CoV-2 cryopreserved stocks were thawed at room temperature and used to infect cells at a multiplicity of infection (MOI) of 0.05. Inhibition of viral growth was measured by harvesting cells 24 hours post-infection. Viral growth was assessed by measuring viral load using PCR targeting the E gene and pseudoknot region.

[0295] Detection of viral plaque-forming units by plaque assay Approximately 8 x 10 5 CCL-81 cells per well were seeded in 6-well plates and grown for 24 hours to 80% confluence. Supernatants from compound-treated cultures were diluted in DMEM medium supplemented with 2.5% FCS and added to pre-seeded wells of the 6-well plates. The plates were then manually rocked to redistribute the inoculum every 15 minutes. The cells were overlaid with 1.25% CMC supplemented with DMEM and incubated at 37°C for 4 days. After incubation, the overlaid CMC was removed, and the cells were fixed with 4% formaldehyde / PBS and stained with 0.1% toluidine blue. After fixation inactivation, the plates were sealed with parafilm, sterilized, and then removed from the BSC and BSL3. Viral plaques were counted to determine the infectious titer (PFU (plaque-forming units) / mL).

[0296] Quantification of viral load by PCR The cell pellet was collected in 300 μL of lysis buffer. Viral RNA was extracted using the NZY Viral RNA Isolation Kit (NZYtech), and cDNA was synthesized using the NZY First-Strand cDNA Synthesis Kit (NZYtech) according to the manufacturer's instructions. Quantitative RT-PCR (RT-qPCR) was then performed using PowerUp SYBR Green Master Mix (BIO-RAD) configured with an Applied Biosystems RT-PCR 7500Fast instrument using the default SYBR Green program.

[0297] The primers used to detect SARS-CoV-2 were: E gene: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (forward), 5'-ATATTGCAGCAGTACGCACACA-3' (reverse); N gene: 5'-GACCCCAAAATCAGCGAAAT-3' (forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (reverse); Pseudoknot: 5'-CCGCGAACCCATGCTTCAGTCA-3' (forward), 5'-CACGGTGTAAGACGGGCTGCAC-3' (reverse); 18S: 5'-GTAACCCGTTGAACCCCATT-3' (forward), 5'-CCATCCAATCGGTAGTAGCG-3' (reverse) It was.

[0298] Viral recovery assay Two sets of samples were prepared for recovery assays, in which 80% confluent cells were infected with a SARS-CoV-2 cryopreserved stock at an MOI of 0.05 for 2 hours. The inoculum was then removed, and the infected cells were incubated with 6 μM MTDB-deg 16a, MTDB, and TDB-deg 16b for 24 hours at 37°C and 5% CO2. After 24 hours, in one set of samples, cells were harvested in lysis buffer for PCR analysis of viral growth. In the other set of samples, the compounds in the supernatant were removed and replaced with drug-free medium and incubated for an additional 24 hours at 37°C and 5% CO2. After 24 hours of incubation (corresponding to the 48-hour time point), cells were harvested in lysis buffer, and viral growth was measured by PCR targeting the E gene and pseudoknot region. The percentage of viral recovery was normalized to the vehicle control.

[0299] Cytotoxicity assay To determine if a compound is toxic to cells, add 1 x 10 cells per well. 4 Vero E6 cells were seeded in 96-well plates. After 24 hours, the cells were incubated with increasing concentrations of MTDB-deg 16a, MTDB, or TDB-deg 16b (ranging from 0.05 μM to 25 μM). Cell viability after 24 hours of compound incubation was assessed using the CellTiter Blue viability assay (Promega) according to the manufacturer's protocol. Briefly, Cell Titer Blue stock solution was diluted 1:20. A volume of 80 μL of diluted Cell Titer Blue was added to each well and incubated at 37°C for 2 hours.

[0300] Dynamic Light Scattering (DLS) Stock solutions (10 mM) of each screening molecule were prepared in solvent-free DMSO and serially diluted in water to final concentrations of 25 or 12.5 μM. Data were collected on a Zetasizer Nano S (Malvern) at 25°C.

[0301] Antiviral activity against animal models of SARS-CoV-2 infection This study used 10- to 12-week-old specific pathogen-free hemizygous mice for Tg(K18-ACE2)2Prlmn (strain B6.Cg-Tg(K18-ACE2)2Prlmn / J, the Jackson laboratory strain 034860). Mice were cultured at 1 × 10 in 50 μl of PBS. 4 Mice were infected intranasally with PFU of SARS-CoV-2. Compounds were administered intranasally 1 hour before and 3 hours after infection. Mice were treated with either vehicle (n = 6), 25 mg / kg MTDB-degrader 16a (n = 6), 10 mg / kg MTDB (n = 3), or 25 mg / kg TDB-degrader 16b (n = 5). Five days after SARS-CoV-2 infection, animals were humanely euthanized, and the left lung was harvested for virus quantification by plaque assay, and the right lung was harvested for histopathological analysis.

[0302] Western blot analysis. For in vitro experiments, samples were treated with vehicle (HO) or MTDB-deg 16a (6 mM) for 24 hours. Cells were then lysed using whole cell lysis buffer (50 mM Tris-HCl pH = 8.0, 450 mM NaCl, 0.1% NP-40, 1 mM EDTA) supplemented with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). For in vivo experiments, whole left lungs from mice were homogenized in 3 mL of DMEM, and 750 μL was transferred to an equal volume of whole cell lysis buffer supplemented as described above. Protein concentration was assessed using Bradford Assays (BioRad). Prior to loading, samples were supplemented with LDS Loading Buffer (Life Technologies) and Sample Reducing Agent (Life Technologies). 40 μg of protein was separated on an SDS-PAGE gel and blotted onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare). Western blot experiments were performed using the following antibodies: anti-beta-actin (Abcam, ab8224), anti-phospho-MAPKAPK-2 (Thr334) (27B7) (Cell Signalling, 3007), anti-phospho-p38 MAPK (Thr180 / Tyr182) (D3F9) XP® (Cell Signalling, 4511), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719), and goat anti-rabbit HRP (Abcam, ab6721).

[0303] Synthesis of azido-imidazole

[0304] [ka]

[0305] Scheme 1: Synthesis of azido-imidazoles Synthesis of hexaethylene glycol di(p-toluenesulfonate) (1) Hexaethylene glycol (1.0 mmol) was dissolved in DCM (10 mL), and p-toluenesulfonyl chloride (2.2 mmol) and KOH (10 mmol) were added at 0° C. The reaction mixture was stirred at room temperature for 6 hours, then filtered and washed with water. After drying over MgSO4, the solvent was evaporated under reduced pressure. No further purification was required. Yield: 95% (colorless oil). 1 H NMR (400MHz, CDCl): δ H 7.78 (d, 4H), 7.33 (d, 4H), 4.14 (t, 4H), 3.67 (br tr, 4H, 3.60 (br s, 8H), 3.57 (br s, 8H), 2.43 (br s, 6H). MS: C 26 H 39 O 11 m / z of S2: 591.19.

[0306] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0307] Synthesis of hexaethylene glycol p-toluenesulfonate azide (2a) Hexaethylene glycol di(p-toluenesulfonate) 1 (1.0 mmol) was dissolved in DMF (10 mL) and sodium azide (1.0 mmol) was added. The reaction mixture was stirred at 60° C. for 6 hours, then cooled to room temperature and stirred overnight. The mixture was washed with brine and dried over MgSO4. Toluene was added to remove DMF, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (EtOAc:hexane, 1:1). Yield: 54% (colorless oil). 1 H NMR (400 MHz, CDCl3) δ H 7.82 (d, 2H), 7.36 (d, 2H), 4.18 (t, 2H), 3.59 - 3.73 (20H, PEG), 3.41 (t, 2H), 2.47 (s, 3H). MS: C 19 H31 m / z of N3NaO8S: 484.2.

[0308] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0309] Synthesis of tetraethylene glycol p-toluenesulfonate azide (2b) Tetraethylene glycol di(p-toluenesulfonate) (2.7 g, 5.4 mmol) was dissolved in anhydrous DMF (10 mL). Sodium azide (355 mg, 5.4 mmol) was added, and the mixture was placed under N2 and stirred at 55 °C for 18 h. The solvent was removed in vacuo, and the product was purified by flash column chromatography (3:1 petroleum ether:AcOEt to 1:1 petroleum ether:AcOEt). The product was obtained as a colorless oil (798 mg, 2.1 mmol, 39%). 1 H NMR (400 MHz, CDCl3) δ 7.82 (d, 2H), 7.37 (d, 2H), 4.19 (t, 1H), 3.60 - 3.73 (12H, PEG), 3.40 (t, 2H), 2.47 (s, 3H). MS: C 15 H 23 N3NaO6S m / z 396.1207.

[0310] Synthesis of diethylene glycol p-toluenesulfonate azide (2c) Diethylene glycol di(p-toluenesulfonate) (1.0 mmol) was dissolved in DMF (10 mL) and sodium azide (1.0 mmol) was added. The reaction mixture was stirred at 60° C. for 6 hours, then cooled to room temperature and stirred overnight. The mixture was washed with brine and dried over MgSO4. Toluene was added to remove DMF, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (EtOAc:hexane, 1:1). Yield: 59% (colorless oil). 1 H NMR (400 MHz, CDCl3) δH 7.80 (d, 2H), 7.35 (d, 2H), 4.17 (t, 2), 3.70 (t, 2H), 3.61 (t, 2H), 3.32 (t, 2H), 2.45 (s, 3H). MS: C 11 H 15 m / z of N3NaO4S: 308.068.

[0311] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0312] Synthesis of hexaethylene glycol imidazolate azide (3a) Imidazole (1.0 mmol) was dissolved in anhydrous DMF (15 mL) under inert conditions, and sodium hydride (60% dispersion in mineral oil, 1.2 mmol) was added. After stirring for 30 minutes at 0°C, 2a (1.0 mmol) was added. The reaction mixture was stirred at 60°C overnight, and after cooling to room temperature, the mixture was quenched with water (20 mL). It was then extracted with EtOAc and DCM, dried over MgSO4, and the solvent was evaporated under reduced pressure to give the crude product. The crude product was then purified by column chromatography (EtOAc:MeOH, 3:1). Yield: 35% (colorless oil). 1 H NMR (400 MHz, CDCl3) δ H MS: C 15 H 28 N5O5 m / z 358.21.

[0313] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0314] Synthesis of tetraethylene glycol imidazolate azide (3b) Imidazole (18 mg, 0.27 mmol) and NaH (60% dispersion in mineral oil, 12 mg, 0.27 mmol) were suspended in anhydrous DMF (1 mL) at 0 °C. The mixture was placed under a N atmosphere, warmed to room temperature, and stirred for 30 min. 2b (100 mg, 0.27 mmol) was dissolved in anhydrous DMF (1 mL), and the resulting solution was added to the first mixture, which was then stirred at 55 °C for 20 h. The solvent was then removed in vacuo, and the resulting residue was purified by flash chromatography (dry loading, gradient from EtOAc to 9:1 EtOAc:MeOH). The product was obtained as a colorless oil (54 mg, 0.20 mmol, 74%). 1 H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.05 (s, 1H), 7.05 (s, 1H), 4.12 (t, 2H), 3.75 (t, 2H), 3.60 - 3.71 (10H, PEG), 3.39 (t, 2H). 13 C NMR (100 MHz, CDCl3) δ C 137.6, 129.2, 119.4, 70.5-70.7 (multiple PEG peaks), 70.0, 50.7, 47.1. MS: C 11 H 19 N5O3 m / z 270.1582.

[0315] Synthesis of diethylene glycol imidazolate azide (3c) Imidazole (1.0 mmol) was dissolved in anhydrous DMF (15 mL) under inert conditions, and sodium hydride (60% dispersion in mineral oil, 1.2 mmol) was added. After stirring for 30 minutes at 0°C, 2c (1.0 mmol) was added. The reaction mixture was stirred at 60°C overnight and then cooled to room temperature. The mixture was quenched with water (20 mL). After extraction with EtOAc and DCM, drying over MgSO4, and evaporation of the solvent under reduced pressure, the crude product was obtained. The crude product was then purified by column chromatography (EtOAc:MeOH, 3:1). Yield: 38% (colorless oil). 1H NMR (400 MHz, CDCl3) δ H 7.53 (s, 1H), 7.06 (s, 1H), 6.99 (s, 1H), 4.14 (t, 2H), 3.75 (t, 2H), 3.60 (t, 2H), 3.36 (t, 2H). MS: C 11 H 19 N5O3 m / z 182.1

[0316] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0317] Synthesis of azido-ethylimidazole (4)

[0318] [ka]

[0319] Scheme 2: Synthesis of ethyl azido-imidazole 4 Hydroxyethylimidazole (1.0 mmol) was dissolved in DCM (10 mL) at 0° C., and KOH (10 mmol) and p-toluenesulfonyl chloride (1.2 mmol) were added. The reaction mixture was stirred at room temperature for 6 hours, then filtered, and the solvent was removed under reduced pressure. The crude was dissolved in DMF, and sodium azide (1.0 mmol) was added. It was stirred at 60° C. for 6 hours, then cooled to room temperature, and stirred overnight. Toluene was added, and the solvent was removed under reduced pressure. Purification was carried out by column chromatography (EtOAC:MeOH, 3:1). Yield: 26% (white solid). 1 H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.09 (s, 1H), 6.96 (s, 1H), 4.09 (t, J = 5.7 Hz, 1H), 3.62 (t, J = 5.7 Hz, 1H).

[0320] Synthesis of pyridostatin decomposers

[0321] [ka]

[0322] Scheme 3: Synthesis of pyridostatin derivatives. Synthesis of chelidamic acid dimethyl ester (5) Chelidamic acid hydrate (2.0 g, 11 mmol) was suspended in 20 mL of MeOH. Thionyl chloride (500 μL, 6.9 mmol) was added dropwise to the suspension at −10° C. A color change from white to brown was observed. The solution was warmed to room temperature and stirred overnight. The brown solution was refluxed for 2 h, and the solvent was removed in vacuo. The crude brown product was then recrystallized from EtOH to give chelidamic acid dimethyl ester 5 (864 mg, 3.9 mmol, 36%) as a beige solid. 1 H NMR (400 MHz, DMSO) δ 11.77 (br s, 1H), 7.61 (s, 2H), 3.88 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 165.97, 164.88, 149.37, 115.33, 52.68. HRMS (ES) C9H 10 NO5([M+H] + ) Calculated m / z: 212.0559, found 212.0567.

[0323] Synthesis of propargylchelidamic acid (6) Chelidamic acid dimethyl ester 5 (0.82 g, 3.8 mmol), propargyl alcohol (0.33 mL, 5.7 mmol), and polymer-bound triphenylphosphine (3.47 g, 1.5 mmol loading / g, 5.2 mmol) were suspended in 55 mL of freshly distilled THF. The solution was degassed using freeze-pump-thaw cycles, cooled to 0 °C, and DIAD (1.0 mL, 5.1 mmol) was added dropwise under argon. The solution was warmed to room temperature and stirred for 3 days. The solution was filtered, and the solvent was removed in vacuo. Chelidamic acid dimethyl ester was obtained by column chromatography (50% EtOAc, 50% petroleum ether). It was then dissolved in 50 mL of MeOH, followed by the addition of 50 mL of aqueous NaOH (0.33 g, 7.5 mmol). The resulting mixture was stirred for 5 min, and deprotection was confirmed by TLC. The organic solvent was removed in vacuo. The mixture was acidified by the addition of 5% formic acid, followed by extraction with 3 x 100 mL of EtOAc. The organic layer was then dried over MgSO, filtered, and the solvent removed in vacuo to give propargyl chelidamic acid 6 (0.17 g, 0.77 mmol, 20%) as an off-white solid. 1 H NMR (400 MHz, MeOD) δ 7.93 (s, 2H), 5.02 (d, J = 2.4 Hz, 2H), 3.15 (t, J = 2.5 Hz, 1H). 13 C NMR (100 MHz, MeOD) δ 168.12, 166.98, 150.54, 150.43, 115.73, 78.97, 77.90, 77.88, 57.74, 57.68. HRMS (ES) C 10 H8NO5([M+H] + ) Calculated m / z: 222.0397, found 222.0391.

[0324] Synthesis of O-(ethyl-2-N-boc-amine)-2-aminoquinolinone (7) 2-Aminoquinolinone (1.0 g, 6.2 mmol), N-boc ethanolamine (1.4 mL, 9.1 mmol), and triphenylphosphine (3.3 g, 13 mmol) were dissolved in 10 mL of freshly distilled THF. The solution was degassed using freeze-pump-thaw cycles, cooled to 0 °C, and DIAD (1.8 mL, 9.2 mmol) was added dropwise under argon. The solution was warmed to room temperature and stirred for 3 days. The solvent was then removed in vacuo. The product was purified by column chromatography using a gradient from 100% EtOAc to 90% EtOAc, 10% MeOH. The solvent was removed in vacuo to give an off-white solid 7 (552 mg, 1.82 mmol, 29%). 1 H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 8.0, 1.0 Hz, 2H), 7.60 (dd, J = 8.4, 1.2 Hz, 2H), 7.55 (ddd, J = 8.5, 6.7, 1.6 Hz, 2H), 7.23 (ddd, J = 8.1, 6.6, 1.3 Hz, 2H), 6.04 (s, 1H), 5.01 (br s, 1H), 4.69 (br s, 2H), 4.18 (t, J = 5.1 Hz, 4H), 3.68 (q, J = 5.5 Hz, 4H), 1.46 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ 162.32, 158.13, 155.90, 148.55, 130.25, 125.63, 121.97, 121.60, 117.52, 90.09, 79.83, 67.52, 39.82, 28.38. HRMS (ES) C 16 H 22 N3O3([M+H] + ) Calculated m / z: 304.1661, found 304.1649.

[0325] Synthesis of the alkyne pyridostatin (8) Propargyl chelidamic acid 6 (0.12 g, 0.54 mol) was dissolved in 1.2 mL of DCM. Ghosez reagent (170 μL, 1.3 mmol) was then added dropwise at 0° C. The orange solution was then stirred at room temperature for 2 h. Chlorination was confirmed by TLC. Triethylamine (0.18 mL, 1.3 mmol) was added dropwise at 0° C. The solution was then stirred at room temperature for 1 h. 7 (0.37 g, 1.2 mmol) was suspended in 1.2 mL of DCM and then added dropwise to the mixture. The mixture turned reddish-brown and was stirred overnight under argon. The crude protected product 8a (not shown) was precipitated from warm MeCN as a red solid. The red solid 8a was then dissolved in DCM. A 2:1 mixture of DCM:TFA was added to acidify the solution and remove the N-boc protection. The solvent was removed in vacuo and the product was purified by HPLC (gradient from 100% HO, 0.1% FA to 100% MeCN, 0.1% FA). Lyophilization gave alkyne-pyridostatin 8 (51 mg, 86 μmol, 16%) as an off-white solid. HRMS(ES):C 32 H 30 N7O5([M+H] + ) Calculated m / z: 592.2308, found 592.2327.

[0326] Synthesis of pyridostatin degraders (9A-9C) Alkyne-pyridostatin 8 (15 mg, 25 μmol) was dissolved in 2.5 mL of a 2:1 mixture of HO:tBuOH. A solution of copper sulfate pentahydrate (250 μL, 100 mM, 25 μmol) was added, followed by a solution of sodium ascorbate (1.3 mL, 100 mM, 130 μmol). The cloudy yellow solution was degassed and stirred for 10 min. A solution of the appropriate azido-imidazole (3a, 3b, or 3-azidopropionic acid) (3.8 mL, 10 mM) was then added. The solution was stirred under argon for 2 h. The organic solvent was removed in vacuo. The product was then purified by HPLC (gradient from 100% HO, 0.1% FA to 100% MeCN, 0.1% FA). The product was obtained as a white or off-white solid.

[0327] 9A (PDS-deg6). Yield 48% (11.3 mg, 12 μmol). HRMS (ES): Calculated for C47H52N12O10 ([M+H]+) m / z: 949.4321, found 949.4344.

[0328] 9B (PDS-deg4). Yield 69% (14.8 mg, 17 μmol). HRMS (ES): Calculated for C43H49N12O8 ([M+H]+) m / z: 861.3796, found 861.376.

[0329] 9C (PDS-CBX). Yield 28% (4.9 mg, 6.9 μmol). HRMS (ES): Calculated for C35H34N10O7 ([M+H]+) m / z: 707.2690, found: 707.2684.

[0330] Synthesis of pseudoknot-resolving agents

[0331] [ka]

[0332] Scheme 4: Synthesis of pseudoknot-resolving agents Synthesis of compound 11a 2-Methylthiazole-4-carbaldehyde (10.0 g, 78.6 mmol, 1.0 equiv.) in DCM (100 mL) was added to compound 10 (16.5 g, 82.6 mmol, 16.2 mL, 1.1 equiv.) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 3 hours. NaBH(OAc)3 (25.0 g, 118 mmol, 1.5 equiv.) was added to the mixture and stirred for 10 hours. The residue was poured into water (50 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (3×20 mL). The combined organic phase was dried over anhydrous Na2SO4 and filtered; the solvent was removed in vacuo. The product was purified by column chromatography (gradient, petroleum ether to petroleum ether / ethyl acetate 10 / 1) to give compound 11a (13.5 g, 43.4 mmol, 55% yield) as a yellow oil. LCMS [+scan]: m / z C 20 H 26 Calculated N3O3S value 388.2; measured value 388.1.

[0333] Synthesis of compound 12a TFA (40.0 g, 351 mmol, 26 mL, 8.4 equiv.) was added to compound 11a (13.0 g, 41.7 mmol, 1.0 equiv.) in DCM (130 mL) at 25 °C under N. The mixture was stirred for 12 h. The solvent was removed in vacuo to give the TFA salt of compound 12a (23.0 g, crude) as a red oil. LCMS [+scan]: m / z C 10 H 18 Calculated N3S value 212.1; measured value 212.0.

[0334] Synthesis of MTDB (compound 13a) To a solution of compound 12a (20.0 g, 45.5 mmol, 1.0 equiv) in DCM (200 mL) was added TEA (9.21 g, 91.0 mmol, 12.7 mL, 2.0 equiv) at 20 °C under N2. Then, ethyl 2-isocyanatobenzoate (8.70 g, 45.5 mmol, 1.0 equiv) was added to the mixture at 0 °C. The mixture was stirred at 20 °C for 12 h. The solvent was removed in vacuo. The residue was purified by column chromatography (gradient, petroleum ether / ethyl acetate 100 / 1 to 20 / 1) to give MTDB (compound 13a 5.62 g, 14.0 mmol, 31% yield) as an off-white solid. 1H NMR (400 MHz, CD3OD): δ 8.42 (br d, J = 8.4 Hz, 1 H), 8.08 (br d, J = 8.0 Hz, 1 H), 7.62 (s, 1 H), 7.52 - 7.59 (m, 1 H), 7.09 (br t, J = 7.6 Hz, 1 H), 4.34 - 4.46 (m, 4 H), 3.93 (br s, 2 H), 3.77 (br t, J = 6.0 Hz, 2 H), 3.49 (br s, 4 H), 3.28 - 3.31 (m, 1 H), 2.74 (s, 3 H), 2.31 (br d, J = 4.8 Hz, 2 H), 1.43 (t, J = 7.2 Hz, 3H). LCMS [+scan]: m / z C 20 H 27 Calculated N4O3S value: 403.2; Measured value: 403.1.

[0335] Synthesis of compound 14a To a mixture of MTDB (compound 13a 5.60 g, 13.9 mmol, 1.0 equiv.) in EtOH (120 mL) and HO (30 mL), LiOH monohydrate (2.34 g, 55.7 mmol, 4.0 equiv.) was added at 25 °C under N 2 . The mixture was stirred at 25 °C for 12 h. The mixture was adjusted to pH 6 with 1 M HCl, and the aqueous phase was extracted with ethyl acetate (3 × 40 mL); the organic phase was then dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give compound 14a (2.60 g, 6.94 mmol, 50%) as a yellow solid. LCMS [+scan]: m / z C 18 H 23 Calculated N4O3S 375.1; Measured 375.1.

[0336] Synthesis of compound 15a To a mixture of compound 14a (2.60 g, 6.94 mmol, 1.0 equiv.) and propargylamine (1.15 g, 20.8 mmol, 1.33 mL, 3.0 equiv.) in DMF (200 mL), DIEA (4.49 g, 34.7 mmol, 6.05 mL, 5.00 equiv.) was added at 25 °C under N2. The mixture was added to T3P (4.42 g, 13.9 mmol, 4.13 mL, 2.0 equiv.) and stirred at 50 °C for 12 h. The mixture was poured into water (200 mL), and the aqueous phase was extracted with ethyl acetate (3 × 70 mL); the organic phase was then washed with brine (60 mL); the organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography (gradient, petroleum ether / ethyl acetate 100 / 1 to ethyl acetate) to give 15a (1.20 g, 2.80 mmol, 40% yield). 1 H NMR (400 MHz, CDCl3): δ 10.54 (br s, 1 H), 8.46 (d, J = 8.2 Hz, 1 H), 7.43 - 7.51 (m, 2 H), 7.11 - 7.11 (m, 1 H), 6.99 (q, J = 7.6 Hz, 2 H), 6.49 (br s, 1 H), 4.22 (dd, J = 5.2, 2.6 Hz, 2 H), 3.72 - 3.83 (m, 4 H), 3.64 - 3.69 (m, 2 H), 2.77 - 2.96 (m, 4 H), 2.72 (s, 3 H), 2.32 (t, J = 2.6 Hz, 1 H), 2.04 (br d, J = 15.2 Hz, 2 H). LCMS [+scan]: m / z C 21 H 26 Calculated N5O2S value 412.2; measured value 412.0.

[0337] Synthesis of MTDB-deg (compound 16a) A mixture of azido-imidazole 3a (200 mg, 280 μmol, 1.0 equiv.), compound 15a (115 mg, 280 μmol, 1.0 equiv.), and CuSO (22.3 mg, 140 μmol, 21.5 uL, 0.5 equiv.) in DCM (5 mL), MeOH (5 mL), and HO (5 mL) was stirred at 20 °C for 0.5 h; then, NaAsc (11.1 mg, 55.9 μmol, 0.2 equiv.) was added to the mixture and stirred at 20 °C for 4.5 h. The mixture was diluted with HO (10 mL) and then extracted with DCM (3 × 10 mL). The combined organic phase was dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by HPLC (column: Phenomenex Gemini-NX 80 × 40 mm × 3 μm; mobile phase: [water (10 mM NH4HCO3) - ACN]; B%: 15%–35%, 8 min) to give MTDB-deg 16a (28.0 mg, 34.9 μmol, 13% yield) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1 H), 9.29 (br t, J = 5.2 Hz, 1 H), 8.37 (d, J = 8.4 Hz, 1 H), 7.96 (s, 1 H), 7.74 (br d, J = 7.2 Hz, 1 H), 7.63 (br s, 1 H), 7.43 (t, J = 7.60 Hz, 1 H), 7.15 - 7.31 (m, 2 H), 6.99 (t, J = 7.6 Hz, 1 H), 6.89 (br s, 1 H), 4.46 - 4.53 (m, 4 H), 4.10 (br t, J = 5.2 Hz, 2 H), 3.77 - 3.81 (m, 2 H), 3.71 (br s, 2 H), 3.66 (br t, J = 5.07 Hz, 2 H), 3.44 - 3.57 (m, 20 H), 3.34 (s, 25 H), 2.73 (br s, 1 H), 2.62 (br s, 6 H), 1.84 (br s, 2 H). HRMS [+scan]: m / z C 36 H 53 N 10Calculated O7S value: 769.3819; measured value: 769.3830.

[0338] Synthesis of control decomposers for pseudoknot experiments

[0339] [ka]

[0340] Scheme 5: Synthesis of pseudoknot-control decomposer (TBD-deg) Synthesis of compound 11b To thiophene-3-carbaldehyde (2.00 g, 17.8 mmol, 1.63 mL, 1.0 equiv) in DCM (80 mL) was added compound 10 (3.93 g, 19.6 mmol, 3.85 mL, 1.1 equiv) at 20 °C under N2. The mixture was stirred at 20 °C for 3 h. Then, NaBH(OAc)3 (5.67 g, 26.8 mmol, 1.5 equiv) was added to the mixture at 0 °C and stirred at 20 °C for 10 h. The reaction mixture was quenched by the addition of water (60 mL) and extracted with DCM (2 × 20 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient petroleum ether / ethyl acetate=100 / 1 to 20 / 1) to give compound 11b (1.70 g, 5.73 mmol, 32% yield) as a red oil. 1 H NMR (400 MHz, CDCl): δ 7.21 - 7.31 (m, 1 H), 7.03 - 7.15 (m, 2 H), 3.64 (s, 2 H), 3.37 - 3.54 (m, 4 H), 2.55 - 2.68 (m, 4 H), 1.81 (br dd, J = 10.8, 4.9 Hz, 2 H), 1.40 - 1.52 (m, 9 H).

[0341] Synthesis of compound 12b To a solution of compound 11b (1.70 g, 5.73 mmol, 1.0 equiv) in DCM (20 mL) was added TFA (6.16 g, 54.0 mmol, 4.00 mL, 9.4 equiv) at 20° C. under N. The mixture was stirred at 20° C. for 8 h. The solvent was removed in vacuo to give the TFA salt of compound 12b (3.00 g, crude) as a red oil, which was used in the next step without further purification.

[0342] Synthesis of TDB (compound 13b) To a mixture of compound 12b (3.00 g, 9.67 mmol, 1.0 equiv.) in DCM (40 mL) was added TEA (1.96 g, 19.3 mmol, 2.69 mL, 2.0 equiv.) at 20 °C under N2. Then, ethyl 2-isocyanatobenzoate (1.85 g, 9.67 mmol, 1.0 equiv.) was added to the mixture at 0 °C. The mixture was stirred at 20 °C for 12 h. The solvent was removed in vacuo. The residue was purified by column chromatography (gradient, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to give TDB 13b (3.30 g, 8.35 mmol, 86% yield) as an off-white solid. 1 H NMR (400 MHz, CDCl3): δ 10.59 (s, 1 H), 8.52 (d, J = 8.4 Hz, 1 H), 7.94 (dd, J = 8.0, 1.53 Hz, 1 H), 7.42 (t, J = 7.6 Hz, 1 H), 7.14 - 7.24 (m, 1 H), 6.96 - 7.07 (m, 2 H), 6.88 (t, J = 7.2 Hz, 1 H), 4.28 (q, J = 7.2 Hz, 2 H), 3.55 - 3.67 (m, 6 H), 2.70 (br s, 2 H), 2.53 - 2.65 (m, 2 H), 1.91 (br s, 2 H), 1.33 (t, J = 7.2 Hz, 3 H). LCMS [+scan]: m / z C 20 H 26 Calculated N3O3S value: 388.2; Measured value: 388.1.

[0343] Synthesis of compound 14b To a mixture of TDB21 (100 mg, 0.26 mmol, 1.0 equiv) in EtOH (1.2 mL) and HO (1.2 mL) was added LiOH monohydrate (65 g, 1.5 mmol, 6.0 equiv) at 25 °C under N. The mixture was stirred at 25 °C for 16 h, after which time additional LiOH monohydrate (130 g, 3.1 mmol, 12.0 equiv) was added. After 2 h, the mixture was adjusted to pH 6 with 1 M HCl, and the aqueous phase was extracted with DCM (3 × 10 mL); the organic phase was then dried over anhydrous MgSO, filtered, and concentrated in vacuo to give compound 14b (70 mg, 0.19 mmol, 76%) as a yellow oily solid. 1 H NMR (400 MHz, CD3OD): δ H 8.41 (d, J = 8.5 Hz, 1 H), 8.08 (d, J = 8.5 Hz, 1 H), 7.76 (br s, 1 H), 7.64 (m, 1 H), 7.53 (t, J = 7.5 Hz, 1 H), 7.32 (d, J = 4.5 Hz, 1 H), 7.07 (t, J = 7.5 Hz, 1 H), 4.46 (br s, 2 H), 3.95 (m, 2 H), 3.75 (tr, J = 6.0 Hz, 2 H), 3.47 (m, 4 H), 2.34 (m, 2 H). 13 C NMR (100 MHz, CD3OD) δ C 170.9, 155.4, 142.7, 133.7, 131.2, 129.7, 129.3, 128.8, 127.6, 121.2, 119.0, 115.3, 55.2, 54.9, 53.3, 44.5, 40.2, 24.0. HRMS [+scan]: m / z C 18 H 22 Calculated N3O3S value: 360.1382; Measured value: 360.1386.

[0344] Synthesis of compound 15b To a mixture of compound 14b (70 mg, 195 μmol, 1.0 equiv.) and propargylamine (11.2 mg, 200 μmol, 13.1 μL, 1.0 equiv.) in DMF (3 mL) was added TEA (75.5 mg, 74.6 μmol, 104 μL, 4.0 equiv.) at 25 °C under N 2 . The mixture was added to 50% T3P (238 mg, 370 μmol, 1.9 equiv.) in DMF and stirred at 25 °C for 16 h. The solvent was removed in vacuo, and the title compound was purified by column chromatography (gradient, DCM to DCM:MeOH 9:1). To remove residual DMF, the compound was dissolved in DCM (10 mL) and washed with HO (10 mL) and 1% aqueous NaOH (10 mL). The organic phase was dried over anhydrous MgSO4 and the solvent was removed in vacuo to give 15b (26 mg, 66 μmol, 34% yield, 4:1 ratio). 1 H NMR (400 MHz, CDCl3, reported for the major diastereomer): δ 10.42 (br s, 1 H), 8.35 (d, J = 8.9 Hz, 1 H), 7.36 - 7.41 (m, 2 H), 7.26 (m, 1 H), 7.11 (m, 1 H), 7.06 (m, 1 H), 6.87 - 6.97 (m, 2 H), 6.49 (br s, 1 H), 4.16 (dd, J = 5.2, 2.5 Hz, 2 H), 3.59 - 3.71 (m, 6 H), 2.75 (br s, 2 H), 2.65 (t, J = 5.5 Hz, 2 H), 2.27 (t, J = 2.5 Hz, 1 H). 1.96 (br s, 2 H). 13 C NMR (100 MHz, CDCl3) δ C 169.4, 155.4, 141.5, 140.0, 132.5, 128.4, 126.8, 125.5, 122.7, 121.1, 120.9, 118.9, 79.2, 71.8, 57.4, 55.0, 46.0, 29.6. HRMS [+scan]: m / z C 36 H 52 Calculated value for N9O7S: 397.1698; Found: 397.1716.

[0345] Synthesis of TDB-deg (compound 16b) Compound 15b (9.9 mg, 25 μmol, 1.0 equiv.) was dissolved in a mixture of HO (1.7 mL) and tBuOH (0.8 mL). Aqueous CuSO (250 μL, 100 mM, 25 μmol, 1.0 equiv.) was added, followed by aqueous NaAsc (1.3 mL, 100 mM, 130 μmol, 5.2 equiv.). The resulting cloudy yellow mixture was placed under an argon atmosphere; then, an aqueous solution of azido-imidazole 3a (3.8 mL, 10 mM, 38 μmol, 1.5 equiv.) was added. The reaction was stirred at room temperature for 1 h, after which the reaction mixture turned clear yellow. The reaction was quenched with EDTA disodium dihydrate (9.3 mg, 25 μmol, 1 equiv.), the organic solvent was removed in vacuo, and the mixture was purified by HPLC. The product-containing fractions were lyophilized to give TDB-deg (15b) as a yellow-brown oily solid (10.2 mg, 14 μmol, 54% yield). HRMS [+ scan]: m / z C 36 H 52 Calculated value for N9O7S: 754.3710; Found: 754.3698.

[0346] Synthesis of a chloramphenicol decomposer

[0347] [ka]

[0348] Scheme 6: Synthesis of chloramphenicol-PEG-imidazole degrader Synthesis of chloramphenicol-6PEG-imidazole degrader (18a) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred for 30 min at 0 °C. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred for 24 h at room temperature. After filtration, the crude mixture was used directly for the copper-click reaction. Azido-imidazole 3a (1.0 mmol), CuSO (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred for another 24 h at room temperature. After adding toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 10% (yellow oil). 1 H NMR (400 MHz, MeOD) δ 8.29 (s, 1H), 8.14 (d, J = 8.8 Hz, 2H), 7.79 (s, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H), 7.22 (s, 1H), 5.23 (d, J = 2.6 Hz, 1H), 4.61 - 4.56 (m, 2H), 4.34 (s, 1H), 4.22 (t, J = 4.9 Hz, 2H), 3.89 - 3.82 (m, 2H), 3.79 - 3.68 (m, 2H), 3.62 - 3.50 (m, 18H). MS: C 27 H 39 N7O 10 m / z: 621.3.

[0349] Synthesis of chloramphenicol-2PEG-imidazole degrader (18b) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred for 30 min at 0 °C. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred for 24 h at room temperature. After filtration, the crude mixture was used directly for the copper-click reaction. Azido-imidazole 3c (1.0 mmol), CuSO (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred for another 24 h at room temperature. After the addition of toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 10% (white solid). 1 H NMR (400 MHz, MeOD) δ 8.19 (s, 1H), 8.16 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.55 (s, 1H), 7.00 (s, 1H), 6.88 (s, 1H), 5.25 (d, J = 2.8 Hz, 1H), 4.60 (dd, J = 5.5, 4.5 Hz, 2H), 4.38 (ddd, J = 7.0, 5.9, 2.9 Hz, 1H), 4.14 (dd, J = 5.5, 4.4 Hz, 2H), 3.89 (dd, J = 11.0, 7.0 Hz, 1H), 3.84 (t, J = 5.1 Hz, 2H), 3.75 (dd, J = 10.9, 5.9 Hz, 1H), 3.71 (t, J = 4.9 Hz, 2H). 19 H 23 N7O6 m / z: 445.2.

[0350] Synthesis of chloramphenicol-ethyl-imidazole decomposer (18c) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred for 30 min at 0 °C. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred for 24 h at room temperature. After filtration, the crude mixture was used directly for the copper-click reaction. Azido-imidazole 4 (1.0 mmol), CuSO (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred for another 24 h at room temperature. After adding toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 12% (white solid). 1 H NMR (400 MHz, MeOD) δ 8.14 (d, J = 8.8 Hz, 2H), 8.03 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.43 (s, 1H), 6.97 (s, 1H), 6.93 (s, 1H), 5.21 (d, J = 2.7 Hz, 1H), 4.82 (dd, J = 6.8, 5.0 Hz, 2H), 4.58 (dd, J = 6.7, 4.9 Hz, 2H), 4.31 (ddd, J = 7.2, 5.9, 2.8 Hz, 1H), 3.84 (dd, J = 10.9, 7.2 Hz, 1H), 3.69 (dd, J = 10.9, 5.9 Hz, 1H). MS: C 17 H 19 N7O5 m / z: 401,1.

[0351] G-quadruplex targeting and RNA degradation Two degraders were rationally designed to target RNA G-quadruplexes (rG4s) by combining the known G4 binder, pyridostatin, with azido-imidazoles of different lengths (9A, 9B). The copper-induced azide-alkyne cycloaddition (CuAAC) used to combine the two components tolerates a wide range of substrates, resulting in triazoles, amide bioisosteres, and moieties that are well tolerated in biological systems. We utilized the same strategy to synthesize pyridostatin derivatives known to selectively bind RNA G-quadruplexes but not CBX-PDS-DNA. The two rG4 degraders and binding controls were tested in vitro and in cell systems.

[0352] In vitro degradation of rG4 oligomers To demonstrate that our degraders could selectively degrade G4 structures, we tested their activity in the presence of various oligomers and cations. We incubated our degraders with either RNA oligomers corresponding to the rG4 structure in the 5'UTR of NRAS mRNA, or a mutant version thereof that cannot form rG4s. In addition, we tested our molecules against K + (promoter of rG4 formation) and Li + (known to block rG4 formation). In rG4-competent oligomers, degradation occurs at the K + observed only in the presence of Li + This suggests that it is not sufficient for oligomers to have G-rich sequences to be targeted by these molecules; they must also form G-quadruplexes to be degraded. + or Li +No degradation was observed with disordered rG4-incompetent NRAS sequences containing either PDS or CBX-PDS, again demonstrating that these degraders act specifically on rG4 structures (Figure 2b). Furthermore, no degradation was observed with CBX-PDS, a control molecule containing the rG4 binder moiety PDS but no degrader, demonstrating that binding alone is insufficient to achieve degradation; the degrader moiety is required (Figure 2a, b). Interestingly, we observed that PDS-deg6 (9A), a degrader containing a linker of six PEG subunits, degraded RNA more quickly than PDS-deg4 (9B), which contains four PEG subunits. This may be a result of the extended reach of the longer linker. Collectively, these experiments demonstrate that our degraders specifically cleave rG4 species but not unfolded RNA regions.

[0353] In vitro degradation of SARS-CoV-2 genomic material To provide evidence that the rG4 degrader can degrade the SARS-CoV-2 genome and gain insight into the mechanism of degradation, we extracted viral RNA from SARS-CoV-2-infected VERO cells, treated it with PDS-deg6(9A), and then analyzed it by direct RNA sequencing. Because the SARS-CoV-2 genome has been shown to contain multiple putative rG4 sites (Zhao et al., 2021) and to be densely packed, most of which are therefore in close proximity to a single rG4 (Ziv et al., 2020), we expected that our degrader would induce widespread damage. Indeed, we observed significant degradation across many regions of the genome, indicating that our degrader was effective in damaging SARS-CoV-2 genetic material (Figure 2c). This phenomenon suggests that our degrading agent should be able to degrade and therefore inactivate viral RNA within cells.

[0354] Antiviral activity of rG4 degraders in vitro To test the antiviral activity of G4-resolving agents in vitro, cells were incubated with 0.5 μM, 5 μM, and 50 μM G4-resolving agents (PDS-deg6(9A), PDS-deg4(9B), and PDS-Alk(8): control molecules without resolving agents) and 5 μM chloroquine as a control 1 h before infection (Figures 3a and 3b). Inhibition of viral growth was measured by harvesting both the supernatant and cells 24 h postinfection. Viral growth was assessed by measuring viral load by plaque assay (in the supernatant) and PCR (in the cells). Cell viability after 24 h of incubation with increasing concentrations of G4-resolving agents (ranging from 0.05 μM to 50 μM) was assessed using a conventional cell viability kit (e.g., CellTiter Blue assay) according to the manufacturer's protocol.

[0355] We observed that G4 degraders successfully inhibited viral replication at 5 μM and 50 μM (Figure 3). PCR results showed that PDS-deg4 did not inhibit viral replication compared to the DMSO control, whereas PDS-deg6(9A) inhibited 70% of viral replication at 5 μM (Figure 3b). Importantly, none of the compounds exhibited cytotoxicity up to 50 μM (Figure 3c).

[0356] Antiviral activity of rG4 degraders in vivo To evaluate the in vivo antiviral activity of G4 degraders, transgenic K18hACE2 mice (expressing hACE2 protein) were intranasally administered PDS-deg4 (9B) and PDS-deg6 (9A) at 25 mg / kg, 40 min before infection and again at 3 and 18 h postinfection (Figure 4). Mice were intranasally infected with SARS-CoV-2 (2.5–5 × 10 in 50 μl of PBS). 4PFU / mouse on day 0), body weight, morbidity and mortality (found dead or euthanized at the end of life), and clinical signs of infection were monitored daily. On day 5, all mice were sacrificed and the left lung was collected for viral load quantification by plaque assay. The right lung, heart, liver, kidney, and spleen were harvested for histopathological analysis.

[0357] The results showed that administration of PDS-deg6(9A) at 25 mg / kg was toxic, and these treated mice had to be sacrificed on day 0. Organs were collected for histopathological analysis. Mice administered PDS-deg4(9B) showed a 10% loss of body weight on day 1 postinfection (Fig. 4a). However, body weight stabilized between days 1 and 3 and then decreased again at the same rate as vehicle controls. Animals treated with PDS-deg4(9B) showed a significant reduction in lung viral load (Fig. 4b).

[0358] This pilot showed that administration of the G4 disruptor resulted in a reduction in viral load in the lungs of SARS-CoV-2-infected k18hACE2 mice.

[0359] Pseudoknot targeting and RNA degradation To target RNA pseudoknots, we rationally designed a noncovalent degrader molecule, MTDB-deg (16a), by combining the known pseudoknot binder MTDB with azido-imidazole 3a (Figure 5a). MTDB contains an ethyl ester moiety, which was exchanged for an amide to increase stability and serve as a handle for degrader attachment. We used azido-imidazole 3a with a linker composed of six PEG subunits, which we previously found to be more effective than its shorter counterpart for RNA degradation of alkynyl-tagged RNA (Mikutis et al., 2020). We chose CuAAC as the reaction for coupling the binder and degrader fragments because it is robust, easy to perform, highly modular, and allows us to vary the structure of the two fragments without altering the coupling step.

[0360] Selective disassembly of the three-stem coronavirus pseudoknot To validate our strategy, we tested our pseudoknot-resolving agent against RNA 69-er, which has a sequence corresponding to and is therefore predicted to form a coronavirus pseudoknot. We incubated 69-er with either MTDB-deg (16a) or one of two control molecules: MTDB, a parent binding molecule that cannot be resolved, or TDB-deg (16b), a resolving agent derived from 2-(4-(thiophen-3-ylmethyl)-[1,4]diazepane-1-carbonyl]-amino)-benzoic acid ethyl ester (TDB), which is closely related to MTDB but has low binding affinity for pseudoknots (Fig. 5b) (Park et al., 2011). After 3 hours of incubation with MTDB-deg(16a), the RNA pseudoknot was significantly degraded, remaining only 23% intact relative to the untreated sample, whereas TDB-deg(16b) showed ineffective degradation and MTDB showed no degradation (Figure 5c, d). To demonstrate that these molecules specifically bind and degrade pseudoknots, we performed the same experiment with oligos that resemble pseudoknots but contain a heavily disordered third stem that prevents the pseudoknot from forming properly. As expected, neither of the molecules affected oligo stability (Figure 5e). Thus, we demonstrated that MTDB-deg(16a) simultaneously binds and degrades pseudoknots efficiently and selectively.

[0361] To demonstrate that MTDB-deg(16a) is functional and can cleave full-length coronavirus RNA, we incubated MTDB-deg(16a) and the controls MTDB and TDB-deg(16b) with RNA extracted from SARS-CoV-2. The viral RNA was then analyzed on an agarose gel. We observed degradation only in the lane corresponding to MTDB-deg(16a) (Figure 5f). Furthermore, we confirmed that MTDB-deg(16a) indeed degraded the native coronavirus pseudoknot, whereas the two control molecules did not, highlighting the specificity of our approach.

[0362] Specificity of coronavirus pseudoknot resolution by MTDB-deg To further demonstrate that MTDB-deg(16a) cleaves viral RNA and obtain a more precise picture of where cleavage occurs, we analyzed the cleaved genomic RNA (gRNA) by direct RNA Nanopore sequencing. As expected, the region surrounding the pseudoknot was most affected (Figure 6a). Interestingly, the pseudoknot flanks were more degraded than the pseudoknot itself. Indeed, a study of the SARS-CoV-2 RNA interactome found that the region surrounding the frameshift element formed extensive short- and long-range interactions with neighboring ORF1a and, in particular, ORF1b; the proximity of these elements to the pseudoknot likely allowed MTDB-deg(16a) to cleave them effectively (Figure 6a) (Ziv et al., 2020). Interestingly, the only other structural element affected by the molecule was the S gene, which was shown to form a long-range interaction with ORF1b (Ziv et al., 2020) and is therefore predicted to be within the reach of the degrader (Figure 6b). Impressively, none of the other subgenomic regions were affected, providing strong evidence for the specificity of MTDB-deg(16a) (Figure 7). The above results provide strong proof of principle that MTDB-deg(16a) is a fully functional and selective degrader of the SARS-CoV-2 pseudoknot and its direct RNA-RNA interactome (Ziv et al., 2020).

[0363] Efficacy and specificity of pseudoknot resolution in SARS-CoV-2-infected cells Having demonstrated the efficacy of MTDB-deg(16a) against coronavirus pseudoknots in vitro, we investigated whether it could degrade the SARS-CoV-2 genome in infected cells and thus prevent viral replication. We performed an in vitro drug assay measuring SARS-CoV-2 replication in Vero CCL-81 cells. We observed that low micromolar concentrations of MTDB-deg(16a) exhibited significant antiviral activity (Figures 8a-c), accompanied by a significant reduction in coronavirus RNA in cells treated before (Figure 8a; Figure 9a) or after (Figure 8b; Figure 9b) infection. These results were supported by results from a plaque assay (Figure 8d). Importantly, the control molecules MTDB and TDB-deg(16b) did not exhibit antiviral activity, even though MTDB is known to disrupt frameshifting in SARS-CoV-2 (Kelly et al., 2020). Additionally, we found that none of the compounds were cytotoxic to host cells, indicating that the observed effects on viral replication were the result of the compounds' specific antiviral activity (Figure 8e). Curiously, the degraders were less active at concentrations higher than 6 μM (Figure 9c), but no colloidal aggregation, which could justify these readouts, was observed in the dynamic light scattering screen. Furthermore, the virus's ability to recover from 24 h of drug exposure was impaired in MTDB-deg(16a)-treated samples but not in samples treated with the control molecules MTDB and TDB-deg(16b) (Figure 8f; Figure 10a). Finally, no virucidal effect was observed when cell-free virus was incubated with MTDB-deg (16a), MTDB, or TDB-deg (16b), suggesting that the antiviral activity of MTDB-deg (16a) is mediated by direct inhibition of viral replication in host cells (Figure 10b). Overall, the antiviral drug assays demonstrate that MTDB-degrader (16a) is an effective antiviral drug against SARS-CoV-2 and is specific for the coronavirus 3-stem pseudoknot with irreversible effects.

[0364] Antiviral activity of pseudoknot disintegrators in vivo We determined the in vivo antiviral activity of MTDB-deg 16a using a SARS-CoV-2 mouse model of infection (transgenic K18-hACE2 mice) (Figure 12a). Animals administered MTDB-deg 16a (at 25 mg / kg) showed a significant reduction in lung viral load compared to the vehicle control group by plaque assay (Figure 12b). In addition, we investigated the in vivo antiviral potential of either MTDB-deg 16a or vehicle treatment using proteins extracted from the lungs of K18-hACE2 transgenic mice on days 3 or 6 post-infection. Encouragingly, we observed that at both time points of infection, the MTDB-deg 16a-treated cohort showed a significant reduction in the phosphorylation levels of p38, an important biomarker of SARS-CoV-2 infection and replication (Figure 12c).

[0365] Targeting the bacterial ribosome Three degraders were rationally designed to target bacterial ribosomes by combining the known ribosomal RNA degrader chloramphenicol with azido-imidazoles of different lengths (18a, 18b, 18c). Propargylic acid was peptide-coupled to (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol using HATU and DIPEA, followed by a coupling step using copper click chemistry to generate the chloramphenicol binding moiety. The copper-induced azide-alkyne cycloaddition (CuAAC) used to combine the two components tolerates a large number of substrates and yields triazoles, amide bioisosteres, and moieties that are well tolerated in biological systems. The three ribosomal degraders and binding controls were tested in vitro and in cell systems.

[0366] In vitro degradation of Escherichia coli ribozymes. In an in vitro assay, degradation activity was measured by targeting E. coli ribosomes. 200 μM ribozyme was incubated with different concentrations of degrading agent ranging from 15 mM to 0.47 mM for 18 hours at 37°C. The activity was assessed by agarose gel analysis.

[0367] The best results were obtained using the PEG-2 linker (18b), where ribosomal degradation could be observed at a concentration of 15 mM (Figure 11).

[0368] [Example 2] Studies to demonstrate the nucleolytic activity of new cleavage groups Targeted degradation is part of the cutting edge of drug discovery. The destruction of ribonucleic acid (RNA) strands in living systems is crucial for effective biological function in the organism. Because it is critical for a wide range of functions, RNA is an important target for disrupting disease. Established methods for targeted degradation utilize cellular cofactors that complicate their successful deployment. Additionally, the specificity of this approach limits flexibility in the compounds that can be used.

[0369] We recently described the development of a strategy to utilize small molecules to degrade specific RNAs [Mikutis, S. et al.]. An azide ("warhead") attached to a basic degrading agent cleavage group was shown to degrade RNA propargylated at adenosine bases by hijacking a methyltransferase enzyme. A copper-promoted "click" reaction allowed the degrading agent to bind to the methylated RNA transcript, which then underwent proximity-driven cleavage at a nearby site. Two mechanisms were found to be operative. The first, we speculated, was base-mediated deprotonation of the 2'-O position on the ribose scaffold, as determined by in vitro pH control. The second mechanism, based on chelation control, we hypothesized was mediated by a transition metal. These findings suggested that our strategy could constitute a powerful general platform for degrading a wide range of RNA targets.

[0370] In 2019, respiratory illnesses changed the way we lived. The virus that caused that disease, COVID-19, known as SARS-CoV-2, suddenly brought to light the need for developing drugs that could target and disrupt viral machinery, something we had recognized. Beyond covalent modifications, the secondary and tertiary structures of RNA complexes also have pathological effects and can therefore be used as a focus for treatment. Following our initial reports of chemical degraders, as shown in Example 1 herein, we developed a noncovalent strategy to target G-quadruplexes, tertiary structures implicated in numerous diseases. The SARS-CoV-2 genome contains four putative G-quadruplex sites [Zhao, C., et al.].

[0371] Although the original biomimetic cleavage group (warhead) we selected for the meClick-seq method, imidazole, proved sufficient to prove the basis of our strategy, its simplicity inherently lacks tunability for efficacy and DMPK properties aimed at human treatment. Furthermore, differences in topology within each new RNA binder when complexed could lead to target-specific variations in degrader efficacy. Therefore, we sought to generate a new library of click degraders to broaden the pharmaceutical potential of our technology.

[0372] result A library of chemical degraders is disclosed herein. Guided by theoretical predictions of basicity, efforts were directed toward degraders with pKa values ​​in the range of 6.2 to 8.6, which approximate the physiological pH of 7.4. Such pKa values ​​would allow the base to be significantly deprotonated under physiological conditions and thus maintain its basicity (Scheme A). We also investigated degrader cleavage groups with the ability to strongly bind transition metals (cleavage groups 5 and 7). A wide range of degraders were evaluated (cleavage groups 2, 9, 11, and 14) to both obtain enhanced potency and to verify our mechanistic hypothesis.

[0373] [ka]

[0374] Scheme A. Library of RNA degrader cleavage groups investigated in this study. Numbers designate the pKa values ​​of specific functional groups ranging from 0 to 16, as predicted in silico using the MarvinView module under KNIME 3.6.1, part of the ChemAxon / Infocom Marvin Extensions 3.6.0 package. All degraders were prepared as warhead-hexaethyleneglycol-azide constructs.

[0375] To assess the efficacy of the degraders, in vitro assays were performed that focused on the direct functionalization of RNA oligonucleotides with degrader warheads.

[0376] Briefly, alkyne-tagged RNA oligonucleotides in HEPES pH 7.4 buffer are reacted with the degrader warhead-hexaethyleneglycol-azide construct using the CuAAC (copper-catalyzed azide-alkyne cycloaddition) reaction. The mixture is then incubated at 37 °C for 0-4 h, followed by quenching the copper and storing the reaction mixture at 4 °C. The mixture is then analyzed by LC-MS (liquid chromatography-mass spectrometry). The mass spectrometry signal corresponding to the oligonucleotide-degrader construct is then integrated and various time points are compared to the signal at t = 0 to estimate degradation (Figure 2).

[0377] As a counterassay to assess the specificity of the degradant, a non-alkyne-functionalized oligomer is treated with the degradant and CuAAC components under identical conditions. This oligonucleotide cannot be covalently functionalized with azide, and therefore any observed degradation in this case would result from nonspecific interactions between the degradant warhead and the RNA oligonucleotide, whereas in cases where no such degradation is observed, the degradation of the covalently functionalized oligonucleotide must be the specific result of induced proximity.

[0378] [ka]

[0379] Scheme B. Scheme outlining an in vitro assay for evaluating the efficacy of degradant warheads. CuAAC is performed on alkyne-functionalized oligonucleotides using a degradant warhead-hexaethyleneglycol-azide construct to introduce degradant cleavage groups into the oligonucleotides, attached via triazole and hexaethyleneglycol linkages. The functionalized oligonucleotides are analyzed immediately to obtain a t=0 reading, or the mixtures are analyzed after incubation at 37° C. for 2 or 4 hours to assess degradation at these time points. Analysis is performed by liquid chromatography-mass spectrometry.

[0380] The degrader cleavage groups tested exhibited a range of activities, providing insight into the design of RNA degrader cleavage groups (Figure 13). We found that degraders with minimal cleavage groups were still able to perform degradation (2, 11). The most likely explanation is the linker's ability to bind copper and bring it into proximity with the nucleic acid, as the absence of degradation with the CTRL strand indeed suggests that this effect is proximity-induced. Curiously, a warhead predicted to be positively charged under the conditions of the assay (11) induced more significant degradation than the uncharged minimal warhead (2). This may be the case due to the positively charged amino group interacting with the negatively charged phosphodiester backbone, thus tethering the linker in proximity and presumably facilitating hydrogen bonding. Note that cleavage groups 9 and 10 have very similar structures, but 10 is a significantly more effective degrader. Without wishing to be bound by any particular theory, it is hypothesized that this may be explained by the fact that 10 has a predicted pKa value closer to 7.4 (8.31), and 9 is more basic (predicted pKa 9.43), and therefore would be fully protonated and unable to act as a base under these conditions. Interestingly, all cleavage groups except 4 and 5 did not induce any damage in the CTRL strand, suggesting that they selectively degrade RNA only when brought into close proximity to the RNA. The phenanthroline-based degrader warhead 5 is a well-known nucleic acid intercalator, which explains its ability to degrade RNA in a nonselective manner [Sigman, DS, et al.].

[0381] The efficacy of the 15 degradant cleavage groups was then compared with a warhead-free linker (Degradant 2) and with the imidazole-based degradant 1 (Figure 14). Interestingly, we found that three of the degradant cleavage groups were less effective degradants than the linker itself, suggesting that these moieties do not promote degradation but rather act as steric blockers, preventing access of the copper-bound linker to the RNA oligonucleotide. Conversely, we found that two of the degradants were significantly more potent than the imidazole degradant 1. One of these degradants, the phenanthroline degradant 5, was also found to degrade control RNA strands, although, as previously discussed, to a significantly lower degree than the functionalized strands. However, degradant warhead 6 did not exhibit nonspecific cleavage and exclusively degraded the covalently functionalized RNA. We found that eight additional degradants exhibited intermediate potency. They were more potent than the linker itself, but less potent than the imidazole head, which is not surprising given the widespread presence of imidazole in natural RNA degradation systems.

[0382] A library of novel, rationally designed RNA degrader cleaving groups was prepared and their efficacy was compared in in vitro RNA degradation assays with the previously described imidazole warhead 1 and with a warhead-free hexaethylene glycol linker. These degraders demonstrated a wide range of potency. The principles described herein can also be used to discover new, potentially superior RNA degrader cleaving groups.

[0383] experiment Direct Functionalization of Degradant Warheads in Vitro - Degradant Effectiveness Assessment Reactions CuSO4 (final concentration 1.0 mM), THPTA (3.0 mM), degradant warhead-hexaethyleneglycol-azide construct (2.0 mM), and RNA oligo (200 μM) were added to a pH 7.5 buffer supplemented with 10 mM MgCl2 and 100 mM KCl. CuAAC was initiated by adding NaAsc (50 mM). The reaction mixture was then incubated at 37°C for 10 minutes. The reaction was quenched immediately or after an additional 2 or 4 hours of incubation at 37°C. After quenching, the reaction mixture was analyzed using LC-MS. The MS signal corresponding to the initial degradant-functionalized RNA concentration was estimated from the reaction quenched immediately after 10 minutes of functionalization by integrating the three or four m / z intensities corresponding to the appropriate RNA species. The MS signal corresponding to 2 or 4 hours of degradation was compared to the t=0 signal.

[0384] chemical synthesis

[0385] [ka]

[0386] 17-Hydroxy-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate Hexaethylene glycol (3.0 g, 11 mmol) was dissolved in DCM and cooled to 0 °C, followed by the addition of p-toluenesulfonyl chloride (2.2 g, 12 mmol) and TEA (2.1 g, 2.9 mL, 21 mmol). The solution was stirred for 3 h at 0 °C and for 30 min at room temperature and quenched with HO (20 mL). The organic solvent and volatiles were removed in vacuo, followed by silica column purification (dry loading, gradient from 5% MeOH in EtOAc to 10% MeOH in EtOAc). The product was obtained as a colorless oil (1.72 g, 3.9 mmol, 37%). 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 4.15 (t, J = 4.4 Hz, 2H), 3. 72 - 3.58 (m, 22H), 2.52 (s, 1 H), 2.44 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ144.89, 133.19, 129.94, 128.10, 72.70, 70.84, 70.73, 70.68, 70.64, 70.41, 69.39, 68.81, 61.84, 21.75. HRMS m / z: m / z [C 19 H 32 O 9 S] + Calculated value: 437.1854; measured value: 437.1845.

[0387] 17-Azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (1)

[0388] [ka]

[0389] 17-Hydroxy-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (524 mg, 1.2 mmol) was dissolved in anhydrous DMF (3 mL). Sodium azide (112 mg, 1.7 mmol) was added, and the mixture was placed under N2 and stirred at 55 °C for 18 h. The solvent was removed in vacuo. To remove traces of DMF, the residue was co-evaporated successively with portions of toluene. The resulting residue was dissolved in DCM and filtered. The resulting residue was purified by column chromatography (gradient, 9:1 DCM:MeOH). The product was obtained as a pale yellow oil (200 mg, 0.64 mmol, 54%). 1 H NMR (400 MHz, CDCl3) δ 3.72 - 3.59 (m, 22H), 3.38 (t, J = 5.1 Hz, 2H), 2.54 (s, 1H).13 C NMR (101 MHz, CDCl3) δ 72.75, 70.78, 70.75, 70.68, 70.64, 70.41, 70.15, 61.85, 50.82. HRMS m / z: m / z [C 12 H 25 N3O6Na] + Calculated value: 330.1652; measured value: 330.1641.

[0390] 1-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)-1H-imidazole (2)

[0391] [ka]

[0392] Imidazole (44 mg, 0.65 mmol) and NaH (60% dispersion in mineral oil, 26 mg, 0.65 mmol) were suspended in anhydrous DMF (2 mL) at 0 °C. The mixture was placed under a N atmosphere, warmed to room temperature, and stirred for 30 min. 4 (250 mg, 0.54 mmol) was dissolved in anhydrous DMF (3 mL), and the resulting solution was added to the first mixture, which was then stirred at 55 °C for 20 h. The solvent was then removed in vacuo, and the resulting residue was purified by flash chromatography (dry loading, gradient from EtOAc to 9:1 EtOAc:MeOH). The product was obtained as a colorless oil (154 mg, 0.43 mmol, 80%). 1 H NMR (400 MHz, CDCl3) δ H 7.52 (s, 1H), 7.02 (s, 1H), 6.98 (s, 1H), 4.09 (t, 2H), 3.72 (t, 2H), 3.55 - 3.78 (18H, PEG), 3.36 (t, 2H). 13 C NMR (100 MHz, CDCl3) δ C137.6, 129.3, 119.4, 70.6-70.7 (multiple PEG peaks), 70.0, 50.7, 47.0. HRMS m / z: [M+H] + : m / z [C 15 H 28 N5O5] + Calculated value: 358.2090; measured value: 358.2084.

[0393] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)-pyridine (3)

[0394] [ka]

[0395] 4-Hydroxypyridine (77.4 mg, 810 mmol), hexaethylene glycol azide (200 mg, 650 μmol), and triphenylphosphine (214 mg, 810 μmol) were dissolved in anhydrous THF, degassed by freeze-pump-thaw (3 cycles), and placed at 0 °C under an argon atmosphere. DIAD (165 mg, 810 μmol) was then added, and the mixture was warmed to room temperature and stirred overnight. The reaction was then quenched by the addition of a 3 g / L solution of NH4Cl (20 mL), and the resulting mixture was washed with DCM (3 × 10 mL). 1% NaOH solution (10 mL) was added to the aqueous fraction, and the product was extracted with DCM (9 × 20 mL). The organic solvent was removed, and the resulting yellow oil was purified on a silica column (gradient: AcOEt to AcOEt containing 10% MeOH and 1% TEA). The title compound was obtained as a yellow oil (82 mg, 0.21 mmol, 33%). 1 H NMR (400MHz, CDCl3): δ 7.34 (d, 2H), 6.33 (d, 2H), 3.90 (tr, 2H), 3.74 (tr, 2H), 3.55-3.66 (m, 18H), 3.36 (tr, 2H), 2.43 (br s, 6H); 13 C NMR (100 MHz, CDCl): δ C179.0, 140.3, 118.5, 70.8, 70.5-70.7 (multiple PEG peaks), 70.1, 70.0, 56.6, 50.7; HRMS m / z: [M+H] + : m / z [C 17 H 28 N4O6] + Calculated m / z 385.2082; found 385.2079.

[0396] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)-2-methylpyridine (4)

[0397] [ka]

[0398] A mixture of 2-methylpyridin-4-ol (10.9 mg, 0.10 mmol, 1.0 equiv.), 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (47.3 g, 0.10 mmol, 1.0 equiv.), and KCO (26.6 mg, 0.20 mmol, 2.0 equiv.) in acetonitrile (2 mL) was stirred under nitrogen in a sealed tube at 85 °C for 16 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on silica (eluted with 80% acetone / hexanes) to afford the title compound as a colorless oil (28.1 mg, 0.071 mmol, 71%). 1 H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 5.8 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 5.8, 2.4 Hz, 1H), 4.15-4.13 (m, 2H), 3.86-3.84 (m, 2H), 3.71-3.69 (m, 2H), 3.67-3.64 (m, 16H), 3.37 (t, J = 5.1 Hz, 2H), 2.49 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 165.3, 160.1, 150.4, 109.6, 107.8, 71.0, 70.8, 70.8, 70.8, 70.7, 70.7, 70.7, 70.2, 69.5, 67.2. HRMS m / z: [M+H] + [C 18 H 31 N4O6] + Calculated value: 399.2238, Measured value: 399.2243.

[0399] 20-Azido-N-(1,10-phenanthrolin-5-yl)-3,6,9,12,15,18-hexaoxaicosanamide (5)

[0400] [ka]

[0401] Hexaethylene glycol azide (48.7 mg, 320 μmol) was dissolved in THF (1.5 mL) and placed under an argon atmosphere at 0° C. NaH (60% in mineral oil, 6.3 mg, 320 μmol) was then added, and the mixture was stirred for 30 minutes at 0° C., followed by 30 minutes at room temperature. 2-Bromo-N-(1,10-phenanthrolin-5-yl)acetamide (49.7 mg, 160 μmol) was then added, and the mixture was stirred overnight at room temperature. The solvent was then removed in vacuo; the resulting red oil was subjected to HPLC purification and lyophilization. The title compound was obtained as a red oil (18.3 mg, 33.7 μmol, 17%). 1H NMR (400MHz, CDCl3): δ 8.92 (br d, 1H), 8.87 (br d, 1H), 8.20 (d, 2H), 8.15 (d, 2H), 7.65 (m, 2H), 7.60 (m, 2H), 4.36 (br s, 2H), 3.92 (m, 2H), 3.82 (m, 2H), 3.71 (m, 2H), 3.68 (m, 2H), 3.59 (m, 2H), 3.52 (m, 2H), 3.43 (m, 4H), 3.32-3.38 (m, 10H). 13 C NMR (100 MHz, DO): δ C 172.4, 150.1, 150.0, 144.6, 143.2, 137.0, 131.9, 129.4, 127.8, 124.7, 121.1, 123.7, 122.6, 70.8, 69.9, 69.9, 69.7, 69.6, 69.3-69.5 (multiple PEG peaks), 69.1, 50.1. HRMS m / z: [M+H] + [C 26 H 34 N6O7] + Calculated m / z 543.2562; found 543.2560.

[0402] 1-(1-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)-1H-imidazol-2-yl)-N,N-dimethylmethanamine (6)

[0403] [ka]

[0404] To a solution of 1-(1H-imidazol-2-yl)-N,N-dimethylmethanamine (24.4 mg, 0.195 mmol, 1.2 equiv.) and 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (75.0 mg, 0.160 mmol, 1.0 equiv.) in DMF (2 mL) was added NaH (5.1 mg, 0.211 mmol, 1.3 equiv.) at 0 °C, then warmed to room temperature and stirred at 55 °C for 14 h. Subsequently, the reaction mixture was cooled and carefully quenched with HO (25 mL) at 0 °C. The aqueous layer was then extracted with EA (25 mL × 3). The combined organic layers were dried over MgSO and concentrated under reduced pressure. The crude residue was purified on basic alumina eluting with a gradient of 0% to 30% MeOH / EtOAc to afford the title compound as a colorless oil (35.1 mg, 0.085 mmol, 53%). 1 H NMR (500 MHz, CDCl3) δ 6.98 (s, 1H), 6.89 (s, 1H), 4.20 (t, J = 5.4 Hz, 2H), 3.87 (s, 2H), 3.72 (t, J = 5.1 Hz, 2H), 3.67-3.58 (m, 12H), 3.56-3.55 (m, 6H), 3.72 (t, J = 5.1 Hz, 1H), 3.51 (s, 2H), 3.37 (t, J = 4.9 Hz, 2H), 2.18 (s, 6H); 13 C NMR (126 MHz, CDCl3) δ 145.4, 127.1, 121.1, 70.8, 70.8, 70.8, 70.8, 70.7, 70.7, 70.6, 70.1, 56.3, 50.8, 45.9, 45.4. HRMS m / z: [M+H] + [C 18 H 35 N6O5] + Calculated value: 415.2663, Measured value: 415.2662.

[0405] N-((1H-imidazol-2-yl)methyl)-2-(ethylthio)-N-(2-(ethylthio)ethyl)ethan-1-amine

[0406] [ka]

[0407] 1H-Imidazole-2-carbaldehyde (3 mg, 3.67 mmol, 1.1 equiv.) and bis(2-(ethylthio)ethyl)amine (646 mg, 3.34 mmol, 1.0 equiv.) were mixed in THF (12 mL) at room temperature under N2. Glacial AcOH (200 mL, 3.34 mmol, 1 equiv.) was added, followed by sodium triacetoxyborohydride (1.06 g, 5.01 mmol, 1.5 equiv.), and the mixture was stirred at 20 °C for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO3 (50 mL), and the product was extracted with EtOAc. The combined organic phases were dried over MgSO4 and concentrated. The residue was purified on basic alumina eluting with a gradient of 0% to 70% EtOAc / petroleum ether (40-60 b.p.) to afford the title compound as a brown gum (320 mg, 1.17 mmol, 35%). 1 H NMR (700 MHz, CDCl3) δ 6.98 (s, 2H), 3.80 (s, 2H), 2.76 (t, J = 6.7 Hz, 4H), 2.63 (t, J = 7.6 Hz, 4H), 2.49 (q, J = 7.4 Hz, 4H), 1.22 (t, J = 7.4 Hz, 6H); 13 C NMR (126 MHz, CDCl3) δ 145.0, 121.4, 53.9, 51.7, 29.3, 26.2, 15.0. HRMS m / z: [M+H] + [C 12 H 24 N3S2] + Calculated value: 274.1406, Measured value: 274.1409.

[0408] N-((1-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)-1H-imidazol-2-yl)methyl)-2-(ethylthio)-N-(2-(ethylthio)ethyl)ethan-1-amine (7)

[0409] [ka]

[0410] To a solution of N-((1H-imidazol-2-yl)methyl)-2-(ethylthio)-N-(2-(ethylthio)ethyl)ethan-1-amine (78 mg, 0.283 mmol, 1.2 equiv) in anhydrous DMF (2 mL) was added NaH (12 mg, 0.295 mmol, 1.25 equiv), and the resulting suspension was stirred for 30 min at 0 °C. Then, 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (109 mg, 0.236 mmol, 1.0 equiv) in 1 mL of anhydrous DMF was added at 0 °C. The mixture was warmed to room temperature and stirred at 55 °C for 20 h. Subsequently, the reaction mixture was cooled and carefully quenched with saturated NaHCO solution (25 mL) at 0 °C. The mixture was then extracted with diethyl ether (3 × 25 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude residue was purified on silica equilibrated with 1% NEt3 / EtOAc. The title product was eluted with a gradient of 0% to 8% MeOH / EtOAc to give a yellow oil (99 mg, 0.176 mmol, 74%). 1 H NMR (700 MHz, CDCl3) δ 7.07 (s, 1H), 6.96 (s, 1H), 4.37 (t, J = 4.5 Hz, 2H), 3.87 (s, 2H), 3.76 (t, J = 5.2 Hz, 2H), 3.68-3.63 (m, 12H), 3.62-3.57 (m, 6H), 3.38 (t, J = 5.1 Hz, 2H), 2.72 (t, J = 7.2 Hz, 4H), 2.57 (t, J = 7.2 Hz, 4H), 2.47 (q, J = 7.4 Hz, 4H), 1.21 (t, J = 7.4 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 145.0, 127.2, 121.4, 71.0, 70.9, 70.8, 70.8, 70.8, 70.8, 70.7, 70.2, 53.9, 51.7, 50.8, 46.1, 29.3, 26.2, 15.0. HRMS m / z: [M+H] + [C 24 H 47 N6O5S2] + Calculated value: 563.3044, Measured value: 563.3045.

[0411] 4-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)morpholine (8)

[0412] [ka]

[0413] A 4 mL reaction vial was charged with 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (46.4 mg, 0.10 mmol, 1.0 equiv.) and KCO (26.6 mg, 0.20 mmol, 2.0 equiv.), sealed, and placed under nitrogen. Anhydrous acetonitrile (2 mL) was then added, followed by morpholine (12.9 mL, 0.15 mmol, 1.5 equiv.). The sealed tube was then heated at 50 °C for 18 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on basic alumina (eluted with 70% ethyl acetate in hexanes) to afford the title compound as a colorless oil (15.5 mg, 0.041 mmol, 41%). 1 H NMR (500 MHz, CDCl3) δ 3.68-3.63 (m, 16H), 3.62-3.60 (m, 2H), 3.57 (t, J = 5.9 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 2.50 (t, J = 5.9 Hz, 2H), 2.25 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 70.8, 70.8, 70.8, 70.7, 70.7, 70.5, 70.2, 69.5, 58.9, 50.8, 46.0. HRMS m / z: [M+H] + [C 16 H 33 N4O6] + Calculated values: 377.2395, 377.2399.

[0414] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (9)

[0415] [ka]

[0416] A 4 mL reaction vial was charged with 4-mercaptophenol (18.9 mg, 0.15 mmol, 1.0 equiv) and NaHCO (30.0 mg, 0.30 mmol, 2.0 equiv) and placed under nitrogen. Anhydrous acetonitrile (1 mL) was then added, and the mixture was stirred at 25 °C for 1 h. A solution of 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (71.9 g, 0.156 mmol, 1.04 equiv) in anhydrous acetonitrile (2 mL) was added, and the sealed tube was heated at 50 °C for 16 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on silica (eluted with 1% MeOH / CH2Cl2) to give the title compound as a colorless oil (39.9 mg, 0.096 mmol, 64%). 1 H NMR (500 MHz, CDCl3) δ 7.35-7.32 (m, 2H), 6.81-6.78 (m, 2H), 3.68-3.55 (m, 20H), 3.38 (t, J = 5.2 Hz, 2H), 2.97 (t, J = 6.8 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 155.8, 134.1, 116.3, 70.9, 70.9, 70.8, 70.7, 70.7, 70.6, 70.5, 70.3, 70.2, 50.8, 35.3. HRMS m / z: [MH] - [C 18 H 28 N3O6S] - Calculated value: 414.1704, Measured value: 414.1705.

[0417] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)sulfinyl)phenol (10)

[0418] [ka]

[0419] To a solution of 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (28 mg, 0.066 mmol, 1 equiv.) in 1:1 HO / EtOH (4 mL) was added oxone (25 mg, 0.165 mmol, 2.5 equiv.). The resulting suspension was stirred at 20 °C for 14 h. Distilled water was added (50 mL) and the pH was adjusted to 5.0 with 1 M HCl. The aqueous solution was extracted with CHCl (4 × 50 mL) and the combined organic phases were dried over anhydrous MgSO and concentrated. The residue was purified on silica eluting with 9% methanol in dichloromethane to give the title compound as a colorless oil (26.2 mg, 92%, 0.61 mmol). 1 H NMR (500 MHz, CDCl3) δ 7.50 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 3.84-3.80 (m, 1H), 3.69-3.52 (m, 19H), 3.37 (t, J = 5.1 Hz, 2H), 3.13-3.08 (m, 1H), 3.01-2.96 (m, 1H); 13C NMR (126 MHz, CDCl3) δ 160.1, 132.6, 126.6, 116.7, 70.8, 70.7, 70.7, 70.7, 70.7, 70.6, 70.1, 64.1, 57.4, 50.7. HRMS m / z: [MH] - [C 18 H 28 N3O7S] - Calculated value: 430.1653, Measured value: 430.1652.

[0420] 17-Azido-N,N-dimethyl-3,6,9,12,15-pentaoxaheptadecan-1-amine (11)

[0421] [ka]

[0422] A 4 mL reaction vial was charged with 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (46.4 mg, 0.10 mmol, 1.0 equiv.) and KCO (26.6 mg, 0.20 mmol, 2.0 equiv.), sealed, and placed under nitrogen. Anhydrous acetonitrile (2 mL) was then added, followed by a 2 M solution of diethylamine in methanol (75 mL, 0.15 mmol, 1.5 equiv.). The sealed tube was then heated at 50 °C for 18 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on basic alumina (eluting with ethyl acetate) to afford the title compound as a colorless oil (28.1 mg, 0.084 mmol, 84%). 1 H NMR (500 MHz, CDCl3) δ 3.68-3.63 (m, 16H), 3.62-3.60 (m, 2H), 3.57 (t, J = 5.9 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 2.50 (t, J = 5.9 Hz, 2H), 2.25 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 70.8, 70.8, 70.8, 70.7, 70.7, 70.5, 70.2, 69.5, 58.9, 50.8, 46.0. HRMS m / z: [M+H] + [C 14 H 31 N4O5] + Calculated value: 335.2289, Measured value: 335.2285.

[0423] 17-Azido-N-(2,4,6-trimethylpyridin-3-yl)-3,6,9,12,15-pentaoxaheptadecanamide (12)

[0424] [ka]

[0425] To a solution of 17-azido-3,6,9,12,15-pentaoxaheptadecanoic acid (30 mg, 0.09 mmol, 1 equiv.) in 1 mL of anhydrous CHCl was added dropwise HATU (38 mg, 0.1 mmol, 1.1 equiv.), followed by triethylamine (20 mg, 0.2 mmol, 2.2 equiv.). The solution was cooled to 0 °C under a nitrogen atmosphere and incubated for 30 min. Then, a solution of the 1,2-substance (13.6 mg, 0.11 mmol, 1.1 equiv.) in 1 mL of anhydrous CHCl was added. The reaction was warmed to 20 °C and stirred for 14 h. The resulting mixture was diluted with CHCl and washed with saturated sodium carbonate solution (3 × 20 mL). The combined organic phase was dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by chromatography eluting with 5% methanol in ethyl acetate to give the title compound as a yellowish oil (5 mg, 11%, 0.011 mmol). 1H NMR (500 MHz, CDCl3) δ 9.10 (br. s, 1H), 7.17 (s, 1H), 4.21 (s, 2H), 3.83-3.81 (m, 2H), 3.74-3.72 (m, 2H), 3.67-3.66 (m, 2H), 3.62 (m, 2H), 3.60-3.57 (m, 4H), 3.56-3.54 (m, 2H), 3.51-3.50 (m, 2H), 3.43-3.41 (m, 2H), 3.37-3.35 (m, 2H), 2.68 (s, 3H), 2.64 (s, 3H), 2.37 (s, 3H); HRMS m / z: [M+H] + [C 20 H 34 N5O6] + Calculated value: 440.2504, Measured value: 440.2510.

[0426] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)-6-methoxyquinoline (13)

[0427] [ka]

[0428] A mixture of 6-methoxyquinolin-4-ol (35.0 mg, 0.20 mmol, 1.0 equiv.), 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (92.3 g, 0.20 mmol, 1.0 equiv.), and KCO (55.3 mg, 0.40 mmol, 2.0 equiv.) in acetonitrile (2 mL) was stirred under nitrogen in a sealed tube at 85 °C for 16 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on silica (eluting with 50% acetone / hexanes) to give the title compound as a colorless oil (67.5 mg, 0.145 mmol, 73%). 1H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 5.5 Hz, 1H), 8.08 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 2.9 Hz, 1H), 7.39 (dd, J = 9.2, 2.9 Hz, 1H), 6.82 (d, J = 5.5 Hz, 1H), 4.42 (dd, J = 5.6, 4.2 Hz, 2H), 4.03-4.02 (m, 2H), 3.79-3.77 (m, 2H), 3.70-3.68 (m, 2H), 3.66-3.63 (m, 14H), 3.37 (t, J = 5.0 Hz, 2H); 13 C NMR (126 MHz, CDCl3) δ 161.7, 157.8, 147.3, 140.8, 129.0, 123.0, 122.1, 101.1, 100.1, 71.0, 70.7, 70.7, 70.7, 70.6, 70.6, 70.0, 69.3, 68.4, 55.7, 50.7. HRMS m / z: [M+H] + [C 22 H 32 N4O7] + Calculated value: 465.2344, Measured value: 465.2353.

[0429] N-(6-aminopyridin-2-yl)-17-azido-3,6,9,12,15-pentaoxaheptadecanamide (14)

[0430] [ka]

[0431] To a solution of 17-azido-3,6,9,12,15-pentaoxaheptadecanoic acid (126 mg, 0.39 mmol, 1 equiv.) in 1 mL of anhydrous CHCl was added dropwise HATU (224 mg, 0.59 mmol, 1.5 equiv.), followed by triethylamine (99 mg, 0.23 mmol, 2.5 equiv.). The solution was cooled to 0° C. under a nitrogen atmosphere and incubated for 30 minutes, after which a solution of 1,2-diaminopyridine (193 mg, 1.8 mmol, 4.5 equiv.) in anhydrous CHCl / DMF was added. The reaction was warmed to 20° C. and stirred for 14 hours. The resulting mixture was diluted with CHCl and washed with saturated sodium carbonate solution (3×20 mL). The combined organic phase was dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by chromatography eluting with ethyl acetate to give the title compound as a yellowish oil (100 mg, 81%, 0.24 mmol). 1 H NMR (500 MHz, CDCl3) δ 8.84 (s, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 6.25 (dd, J = 7.9, 0.7 Hz, 1H), 4.43 (s, 2H), 4.10 (s, 2H), 3.77-3.74 (m, 4H), 3.72-3.70 (m, 4H), 3.69-3.65 (m, 10H), 3.39 (t, J = 5.1 Hz, 2H). 13 C NMR (126 MHz, CDCl3) δ 168.6, 157.5, 149.4, 140.0, 104.6, 103.5, 71.5, 70.9, 70.7, 70.7, 70.7, 70.4, 70.1, 50.8. HRMS m / z: [M+H] + [C 17 H 29 N6O6] + Calculated value: 413.2143, Measured value: 413.2147.

[0432] 17-Azido-N-(2-(dimethylamino)ethyl)-3,6,9,12,15-pentaoxaheptadecanamide (15)

[0433] [ka]

[0434] To a solution of 17-azido-3,6,9,12,15-pentaoxaheptadecanoic acid (63 mg, 0.20 mmol, 1.25 equiv.), HATU (119 mg, 0.31 mmol, 2 equiv.) and triethylamine (55 mL, 0.39 mmol, 2.5 equiv.) in anhydrous dichloromethane stirred at 0 °C under a nitrogen atmosphere was added N 1 ,N 1 -Dimethylethane-1,2-diamine (17 mL, 0.16 mmol, 1.0 equiv) was added dropwise. The resulting mixture was stirred at 0 °C for 30 min, then warmed to 20 °C and stirred for an additional 14 h. The solvent was removed in vacuo, and the residue was redissolved in EtOAc (50 mL) and transferred to a separatory funnel. Saturated aqueous NaCO (10 mL) and HO (40 mL) were added, the funnel was shaken, and the layers were separated. The aqueous layer was further extracted with EtOAc (2 × 50 mL), and the combined organic phases were dried over anhydrous MgSO and concentrated. The residue was purified on basic alumina (eluting with a gradient of 0% to 10% MeOH / CH Cl ) to afford the title compound as a colorless oil (25.0 mg, 0.064 mmol, 40%). 1 H NMR (500 MHz, CDCl3) δ 7.15 (br. s, 1H), 3.98 (s, 2H), 3.67-3.64 (m, 18H), 3.37 (q, J = 6.0 Hz, 4H), 2.44 (t, J = 6.4 Hz, 2H), 2.25 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 170.0, 71.1, 70.8, 70.8, 70.7, 70.7, 70.7, 70.7, 70.4, 70.1, 58.2, 50.8, 45.4, 36.5. HRMS m / z: [M+H] + [C 16 H 34 N5O6] +Calculated value: 392.2504, Measured value: 392.2506.

[0435] 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)sulfonyl)phenol (16)

[0436] [ka]

[0437] To a solution of 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (12.4 mg, 0.030 mmol, 1 equiv.) in MeOH (2 mL) was added ammonium molybdate hydrate (1.7 mg, 0.006 mmol, 5 mol%) and 30 wt. % aqueous hydrogen peroxide (12 mL, 0.12 mmol, 4 equiv.). The resulting suspension was stirred at 20° C. for 2 h. Ethyl acetate (5 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The residue was purified on silica eluting with 2.5% methanol in dichloromethane to give the title compound as a colorless oil (6.8 mg, 51%, 0.015 mmol). δ 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 3.81-3.78 (m, 2H), 3.75-3.69 (m, 6H), 3.66-3.61 (m, 4H), 3.53-3.51 (m, 2H), 3.38-3.33 (m, 10H). HRMS m / z: [MH] - [C 18 H 28 N3O8S] - Calculated value: 446.1603, Measured value: 446.1602.

[0438] 2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (17)

[0439] [ka]

[0440] A 4 mL reaction vial was charged with 2-mercaptophenol (30.2 μL, 0.30 mmol, 1.0 equiv) and NaHCO (60.0 mg, 0.60 mmol, 2.0 equiv) and placed under nitrogen. Anhydrous acetonitrile (1 mL) was then added, and the mixture was stirred at 25° C. for 1 h. A solution of 17-azido-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (143.8 g, 0.312 mmol, 1.04 equiv) in anhydrous acetonitrile (2 mL) was added, and the sealed tube was heated at 50° C. for 16 h. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The resulting residue was purified on silica (eluting with 15% acetone / hexanes) to give the title compound as a colorless oil (14.6 mg, 0.035 mmol, 12%). 1 H NMR (500 MHz, CDCl3) δ 7.48 (dd, J = 7.6, 1.7 Hz, 1H), 7.28-7.24 (m, overlapping with solvent peak, 1H), 6.92 (dd, J = 8.2, 1.3 Hz), 6.84 (td, J = 7.6, 1.3 Hz), 7.68-7.62 (m, 18H), 3.54 (t, J = 6.0 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H). HRMS m / z: [M-H] - [C 18 H 28 N3O6S] - Calculated value: 414.1704, Measured value: 414.1699.

[0441] 2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)sulfinyl)phenol (18)

[0442] [ka]

[0443] To a solution of 2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (25.5 mg, 0.061 mmol, 1 equiv.) in 1:1 HO / EtOH (4 mL) was added oxone (23.3 mg, 0.153 mmol, 2.5 equiv.). The resulting suspension was stirred at 20 °C for 14 h. Distilled water was added (50 mL) and the pH was adjusted to 5.0 using 1 M HCl. The aqueous solution was extracted with CHCl (4 × 50 mL) and the combined organic phases were dried over anhydrous MgSO and concentrated. The residue was purified on silica eluting with 2% methanol in dichloromethane to give the title compound as a colorless oil (22.3 mg, 85%, 0.052 mmol). 1 H NMR (500 MHz, CDCl3) δ 7.37-7.33 (m, 1H), 7.16 (dd, J = 7.56, 1.4 Hz), 6.93-6.90 (m, 2H), 3.95-3.90 (m, 1H), 3.75-3.71 (m, 1H), 3.66-3.60 (m, 18H), 3.48-3.43 (m, 1H), 3.37 (t, J = 5.0 Hz, 2H), 3.23-3.18 (m, 1H); 13 C NMR (126 MHz, CDCl3) δ 133.1, 125.8, 121.9, 119.9, 119.4, 70.9, 70.8, 70.8, 70.7, 70.7, 70.7, 70.5, 70.1, 64.1, 55.7, 50.8. HRMS m / z: [MH] - [C 18 H 28 N3O7S] - Calculated value: 430.1653, Measured value: 430.1650.

[0444] 2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)sulfonyl)phenol (19)

[0445] [ka]

[0446] To a solution of 2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)thio)phenol (19.5 mg, 0.047 mmol, 1 equiv.) in MeOH (2 mL) was added ammonium molybdate hydrate (2.7 mg, 0.002 mmol, 5 mol%) and 30 wt. % aqueous hydrogen peroxide (19 μL, 0.19 mmol, 4 equiv.). The resulting suspension was stirred at 20° C. for 2 h. Ethyl acetate (5 mL) was added, and the mixture was filtered through Celite and concentrated in vacuo. The residue was purified on silica eluting with 2.5% methanol in dichloromethane to give the title compound as a colorless oil (14 mg, 67%, 0.031 mmol). δ 1 H NMR (500 MHz, CDCl3) δ 8.93 (br. s, 1H), 7.66 (dd, J = 7.6, 1.7 Hz, 1H), 7.52-7.49 (m, 1H), 7.03-6.99 (m, 2H), 3.85 (t, J = 6.0 Hz, 2H), 3.67-3.63 (m, 10H), 3.61-3.59 (m, 2H), 3.55-3.53 (m, 2H), 3.52-3.48 (m, 4H), 3.46-3.44 (m, 2H), 3.37 (t, J = 5.1 Hz, 2H); 13 C NMR (126 MHz, CDCl3) δ 156.6, 136.4, 129.5, 122.6, 120.5, 118.9, 70.8, 70.8, 70.8, 70.8, 70.7, 70.7, 70.7, 70.4, 70.2, 64.5, 56.9, 50.8. HRMS m / z: [MH] - [C 18 H 28 N3O8S] - Calculated value: 446.1603, Measured value: 446.1600.

[0447] [Example 3] Synthesis and evaluation of PDS-Amimi Following the procedures outlined in Example 1, the compound PDS-Amimi shown below was synthesized and its activity as a degrader was evaluated along with PDS-deg6 (prepared as described in Example 1) and the non-degrader control CBX-PDS (prepared as described in Example 1).

[0448] Synthesis of PDS-AmImi. N2,N6-bis(4-(2-aminoethoxy)quinolin-2-yl)-4-(prop-2-yn-1-yloxy)pyridine-2,6-dicarboxamide (12.4 mg, 20.7 μmol) was dissolved in a 2:1 mixture of HO:tBuOH (2.1 mL). A solution of copper sulfate pentahydrate (207 μL, 100 mM, 20.7 μmol) was added, followed by a solution of sodium ascorbate (1.07 mL, 100 mM, 107 μmol). The cloudy yellow solution was placed under argon and stirred for 10 minutes. A solution of 1-(1-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)-1H-imidazol-2-yl)-N,N-dimethylmethanamine (6) (2.9 mL, 10 mM, 29 μmol) was then added. The reaction mixture was stirred at 25° C. for 2 hours. The solvent was then removed in vacuo. The product was then purified by HPLC (gradient from 100% HO, 0.1% FA to 100% MeCN, 0.1% FA). The title compound was obtained as a beige solid (7.2 mg, 7.2 μmol, 35%). HRMS m / z: [M+H] + :[C 50 H 64 N 13 O 10 ] + Calculated value: 1006.4899, measured value: 1006.4896.

[0449] [ka]

[0450] Assay Protocol Protocol: G4-forming RNA oligomer (final concentration 200 μM, sequence 5'-UGUGGGAGGGGCGGGUCUGGGUGC-3') was added to pH 7.5 HEPES (20 mM) buffer supplemented with KCl (100 mM), MgCl (10 mM). The mixture was heated at 95°C for 5 minutes and then kept on ice for 30 minutes. CuSO (final concentration 200 μM), THPTA (700 μM), and NaAsc (50 mM) were then added along with CBX-PDS, PDS-deg6, or PDS-AmImi (200 μM). The reaction mixture was incubated at 37°C for 4 hours, quenched with EDTA (final concentration 12 mM), and then maintained at 4°C. The reaction mixture was analyzed by LC-MS.

[0451] The results are shown in FIG.

[0452] It was observed that PDS-Amimi was a more potent degrader than PDS-deg6.

[0453] References Several publications are cited above in order to more fully describe and disclose the present invention and the state of the art to which it pertains. Full citations for these references are provided below. Each of these references is incorporated herein in its entirety. Bobbin, et al., “RNA Interference (RNAi)-Based Therapeutics: Delivering on the Promise?”, Annual Review of Pharmacology and Toxicology, 2016, Vol. 56, pp. 103-122. Cox, et al., “RNA editing with CRISPR-Cas13”, Science, 2017, Vol. 358, pp. 1019-1027. Gasiunas, et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria”, Proc. Natl. Acad. Sci. U.S.A., 2012, Vol. 109, E2579-E2586. Jinek, et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science, 2012, Vol. 337, pp. 816-821. Kelly et al., “Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2)”, J. Biol. Chem., 2020, Vol. 295, pp. 10741-10748. Li. “Minimap2: pairwise alignment for nucleotide sequences”, Bioinformatics, 2018, Vol. 34, pp. 3094-3100. Mikutis et al., “meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation”, ACS Cent. Sci., 2020, Vol. 6, pp. 2196-2208. Park et al., “Identification of RNA Pseudoknot-Binding Ligand That Inhibits the -1 Ribosomal Frameshifting of SARS-Coronavirus by Structure-Based Virtual Screening”, J. Am. Chem. Soc., 2011, Vol. 133, pp. 10094-10100. Santos et al., “G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex / Ligand Interactions”, Pharmaceuticals, 2021, Vol. 14, No. 769 Sigman, D.S., et al., Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline . cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem, 1979. 254(24): p. 12269-72. Tzelepis, et al., “A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia”, Cell Reports, 2016, Vol. 17, pp. 1193-1205. Zamore, et al., “RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals”, Cell, 2000, Vol. 101, pp. 25-33. Zhao, C.; Qin, G.; Niu, J.; Wang, Z.; Wang, C.; Ren, J.; Qu, X., “Targeting RNA G-Quadruplex in SARS-CoV-2: A Promising Therapeutic Target for COVID-19?”, Angew. Chem. Int. Ed., 2021, 60 (1), 432-438. Ziv, et al., “The Short- and Long-Range RNA-RNA Interactome of SARS-CoV-2.” Mol. Cell., 2020, Vol. 80, pp. 1067-1077.

Claims

1. A bifunctional compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof: C-L-B (I) [In the formula, C is a cleaving group as defined herein; L is a linker; B is a non-covalent bonding group, however, C is (i) an imidazole (1,3-diazole) group optionally substituted with one, two or three (1-6C) alkyl groups, which may be the same or different; or (ii) a nucleic acid cleaving group of formula Z: 【Chemistry 1】 provided that it is not where: 【Chemistry 2】 indicates the point of attachment to L; Ring A is absent or is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C) alkyl, (1-4C) haloalkyl, (1-4C) hydroxyalkyl, OR ｃ , C(O)R ｃ , C(O)OR ｃ , O.C.(O.)R ｃ , C(O)N(R ｄ ) R ｃ , N(R ｄ ) C(O)R ｃ , S(O) ｙ R ｃ (wherein y is 0, 1 or 2), SO ２ N (R ｄ ) R ｃ , N(R ｄ ) SO ２ R ｃ , or NR ｃ R ｄ and R is a nitrogen-containing heteroaryl or heterocyclic ring optionally further substituted with one or more substituents selected from ｃ and R ｄ is selected from hydrogen or (1-4C) alkyl; The integer a1 is 0, 1, 2 or 3; Each occurrence of Ra and Rb is independently selected from hydrogen or (1-2C) alkyl; R １ and R ２ are each independently selected from hydrogen, (1-6C)alkyl, heterocyclic ring, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, each of which is selected from halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR ｅ , C(O)R ｅ , C(O)OR ｅ , O.C.(O.)R ｅ , C(O)N(R ｆ ) R ｅ , N(R ｆ ) C(O)R ｅ , S(O) ｙ R ｅ (wherein y is 0, 1 or 2), SO ２ N (R ｆ ) R ｅ , N(R ｆ ) SO ２ R ｅ , or NR ｅ R ｆ and R is optionally substituted with one or more substituents selected from ｅ and R ｆ is selected from hydrogen or (1-4C) alkyl, or R １ and R ２ are linked together with the atom to which they are attached to form a 4- to 6-membered heterocycle or a 5- or 6-membered heteroaryl, and any 4- to 6-membered heterocycle or 5- or 6-membered heteroaryl may be selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)hydroxyalkyl, OR ｇ , C(O)R ｇ , C(O)OR ｇ , O.C.(O.)R ｇ , C(O)N(R ｈ ) R ｇ , N(R ｈ ) C(O)R ｇ , S(O) ｙ R ｇ (wherein y is 0, 1 or 2), SO ２ N (R ｈ ) R ｇ , N(R ｈ ) SO ２ R ｇ , or NR ｇ R ｈ and R is optionally substituted with one or more substituents selected from ｇ and R ｈ is selected from hydrogen or (1-4C) alkyl; When ring A is absent, R １ and R ２ are each independently selected from hydrogen, heterocyclic ring, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, each of which is selected from halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR ｅ , C(O)R ｅ , C(O)OR ｅ , O.C.(O.)R ｅ , C(O)N(R ｆ ) R ｅ , N(R ｆ ) C(O)R ｅ , S(O) ｙ R ｅ (wherein y is 0, 1 or 2), SO ２ N (R ｆ ) R ｅ , N(R ｆ ) SO ２ R ｅ , or NR ｅ R ｆ and R is optionally substituted with one or more substituents selected from ｅ and R ｆ is selected from hydrogen or (1-4C) alkyl, with the proviso that R １ and R ２ cannot both be hydrogen.

2. C is, (i) having a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) a nitrogen atom or a hydroxy group capable of forming a chelate complex with a metal (e.g., copper or zinc); 2. The bifunctional compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, which contains a basic nitrogen atom or a hydroxy group, wherein the basic nitrogen atom is either

3. The cleavage group C is (i) any N- or C-OH-containing moiety, such that at least one N- or C-OH group has a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; (ii) any N- or OH-containing moiety capable of chelating copper at physiological pH 3. The bifunctional compound of formula (I) according to claim 1 or claim 2, or a pharmaceutically acceptable salt or solvate thereof, selected from:

4. The cleaving group C has the formula: -L １ -X Ｃ -L ２ -R Ｃ is the basis of L １ is absent or is (1-2C)alkylene; X Ｃ is absent or is —O—, —S—, —SO—, —SO ２ -, -N(R ＸＣ１ )-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R ＸＣ１ ) -, -N(R ＸＣ１ )C(O)-,-S(O) ２ N (R ＸＣ１ ), or -N(R ＸＣ１ ) SO ２ - selected from; R ＸＣ１ is hydrogen or (1-6C) alkyl, (3-6C) cycloalkyl, (3-6C) cycloalkyl(1-2C) alkylene, -(CH ２ ) ｍ１ -aryl, -(CH ２ ) ｍ１ -heteroaryl or -(CH ２ ) ｍ１ - heterocyclic rings, m1 being 0-2; L ２ is absent or is (1-2C)alkylene; R Ｃ is selected from hydrogen, (1-4C) alkyl, (3-6C) cycloalkyl, phenyl, heteroaryl or heterocyclyl; Alkyl, cycloalkyl, phenyl may be one or more R Ａ substituted by one or more R Ｂ optionally further substituted by substituents; The heteroaryl or heterocyclic ring may be one or more R Ａ and / or R Ｂ optionally substituted by substituents; R Ａ but, (i) —OH; ()) !2 Ａ１ ( Ａ２ 4 (iii) -(1-6C)alkylene-NR Ａ１ R Ａ２ is a group selected from R Ａ１ and R Ａ２ are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R Ｂ But halo, nitro, cyano, R ＢＡ , -[CH ２ ] ｔ -OR ＢＡ , -[CH ２ ] ｔ -C(O)R ＢＡ , -[CH ２ ] ｔ -C(O)OR ＢＡ , -[CH ２ ] ｔ -OC(O)R ＢＡ , -[CH ２ ] ｔ -C(O)N(R ＢＢ ) R ＢＡ , -[CH ２ ] ｔ -N(R ＢＢ ) C(O)R ＢＡ , -[CH ２ ] ｔ -S(O) ｐ R ＢＡ (wherein p is 0, 1 or 2), -[CH ２ ] ｔ -SO ２ N (R ＢＢ ) R ＢＡ , or −[CH ２ ] ｔ -N(R ＢＢ ) SO ２ R ＢＡ Selected from: t is 0, 1, 2 or 3; R ＢＡ is hydrogen or (1-4C)alkyl optionally substituted with halo, hydroxy, amino or cyano; R ＢＢ is hydrogen or (1-2C) alkyl, The cleavage group is a) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or b) a group of formula Z as defined above 4. A bifunctional compound of formula (I) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, which is not:

5. The cleaving group C has the formula: -X Ｃ -R Ｃ is the basis of X Ｃ is not present or -O-, -S-, -SO-, -SO ２ -, -N(R ＸＣ１ )-, -C(O)-, -C(O)N(R ＸＣ１ )- or -N(R ＸＣ１ )C(O)—; R ＸＣ１ is hydrogen or (1-6C) alkyl or —(CH ２ ) ｍ１ -heteroaryl, wherein m1 is 0-2; R Ｃ is selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl, or heterocyclyl; The alkyl, cycloalkyl, and phenyl may be one or more R Ａ substituted by one or more R Ｂ optionally further substituted by substituents; The heteroaryl or heterocyclic ring may be one or more R Ａ and / or R Ｂ optionally substituted by substituents; R Ａ but, (iv) —OH; (v) NR Ａ１ R Ａ２ ; (vi) -(1-6C)alkylene-NR Ａ１ R Ａ２ is a group selected from R Ａ１ and R Ａ２ are each independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R Ｂ But halo, nitro, cyano, R ＢＡ , -[CH ２ ] ｔ -OR ＢＡ , or −[CH ２ ] ｔ -C(O)R ＢＡ Selected from: t is 0, 1, 2 or 3; R ＢＡ is (1-4C) alkyl; The cleavage group is c) an imidazole (1,3-diazole) group optionally substituted with 1, 2 or 3 (1-6C) alkyl groups, which may be the same or different; or d) a group of formula Z as defined above 5. The bifunctional compound of formula (I) according to any one of claims 1 to 4, which is not: or a pharmaceutically acceptable salt or solvate thereof.

6. The cleaving group C is a group of the following formula: 【Transformation 3】 【change】 【change】 6. The bifunctional compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, selected from:

7. The linker group L is selected from the group of formula (LI), (L-II), (L-III) or (L-IV) shown below: 【Chemistry 4】 is selected from L １ is a covalent bond or a (1-6C)alkylene group or a (1-6C)heteroalkylene; L ２ is a (1-6C) alkylene group or a (1-6C) heteroalkylene group; L ３ is a (1-6C) alkylene group; n is 0 to 8; L ４ is a (1-6C) alkylene group; L ５ is a (1-6C) alkylene group or a (1-6C) heteroalkylene group; L ６ is a covalent bond or a (1-2C)alkylene group; m is 1 to 8; ＊ is the point of attachment to the non-covalent bonding group (-B); ＊＊ 7. The bifunctional compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, wherein: is the point of attachment to said cleavage group (-C).

8. L １ is a covalent bond or methylene, and L ６ 8. The bifunctional compound of formula (I) according to claim 7, or a pharmaceutically acceptable salt or solvate thereof, wherein is a covalent bond or methylene.

9. L ３ is (1-4C) alkylene, and L ４ The bifunctional compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof according to claim 7 or 8, wherein is (1-4C) alkylene.

10. L ３ and L ４ 10. The bifunctional compound of formula (I) according to any one of claims 7 to 9, or a pharmaceutically acceptable salt or solvate thereof, wherein is ethylene.

11. 11. The bifunctional compound of formula (I) according to any one of claims 7 to 10, wherein m and n are 2 to 5, or a pharmaceutically acceptable salt or solvate thereof.

12. L ２ and L ５ is ethylene oxide (-CH ２ CH ２ O-), propylene oxide (-CH ２ CH ２ CH ２ O-) and tetramethylene oxide (-CH ２ CH ２ CH ２ CH ２ 12. The bifunctional compound of formula (I) according to any one of claims 7 to 11, or a pharmaceutically acceptable salt or solvate thereof, selected from:

13. L ２ and L ５ is ethylene oxide (-CH ２ CH ２ 13. The bifunctional compound of formula (I) according to any one of claims 7 to 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R is R, ...

14. B is selected from an oligonucleotide, a nanobody, an antibody, an antibody fragment or a small molecule capable of binding to a nucleic acid; or B is a group of formula (B-I), (B-II), (B-III) or (B-IV) shown below: 【Transformation 5】 and 14. The bifunctional compound of formula (I) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, wherein X is O or NH.

15. When the compound of formula (I) is a compound of formula (II), (III), (IV) or (V) shown below: 【Transformation 6】 【change】 is selected from L and C are each as defined in any one of claims 1 to 14; 15. The bifunctional compound of formula (I) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, wherein X is NH or O.

16. 16. A pharmaceutical composition comprising the bifunctional compound of any one of claims 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

17. 17. A bifunctional compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 16, for use as a medicament.

18. 17. A bifunctional compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 16, for use in the treatment of a proliferative disorder (e.g., cancer) or a bacterial or viral infection.

19. 17. A method for treating a proliferative disorder (e.g., cancer) or a bacterial or viral infection, comprising administering a therapeutically effective dose of a bifunctional compound described in any one of claims 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition described in claim 16.

20. 17. Use of a bifunctional compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 16, for epigenetics and epitranscriptomics analysis / mapping.

21. 1. A method for cleaving a target nucleic acid molecule, comprising: contacting the target nucleic acid molecule with the bifunctional compound of any one of claims 1 to 15, or a salt or solvate thereof, so that the compound non-covalently binds to the target nucleic acid molecule; allowing the compound to cleave the target nucleic acid molecule bound thereto; A method comprising:

22. 1. A method for identifying secondary or tertiary structure within a target nucleic acid molecule, comprising: providing first and second populations of nucleic acid molecules, each population comprising said target nucleic acid molecule; introducing into said first population of nucleic acid molecules a bifunctional compound according to any one of claims 1 to 15, or a salt or solvate thereof; allowing a bifunctional compound of the present invention to cleave the target nucleic acid molecules present in the first population; identifying nucleic acid molecules present in the first population in reduced amounts relative to the second population; A method comprising:

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