Compounds for nucleic acid cleavage

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 invention also relates to pharmaceutical compositions comprising these compounds, and to the use of these compounds in, for example, epigenomic and epitranscriptome mapping, and in therapy, for example, antimicrobial and / or antiviral therapy.

Description

[0001] The project that facilitated the application has been funded by the EU Horizon 2020 research and innovation program under grant agreement number 676832 granted by the European Research Council. Technical Field

[0002] This invention relates to novel compounds suitable for non-enzymatic cleavage of target nucleic acids. The invention also relates to the use of these compounds in, for example, RNA structural mapping, and in therapeutic applications, such as antimicrobial and / or antiviral therapy. Background Technology

[0003] Targeted degradation of nucleic acids has become a frontier in drug development. The disruption of ribonucleic acid (RNA) chains in living systems is crucial for effective biological functions within organisms. RNA is essential for a wide range of functions and represents a significant target for disease disruption. Existing targeted degradation methods utilize cellular cofactors, complicating their potential applications. Furthermore, the specificity of these methods limits the compounds that can be used.

[0004] In 2019, a respiratory disease changed our lifestyles. The virus that caused this disease was named COVID-19 or SARS-CoV-2, which spurred an urgent need to develop drugs that could target and destroy the virus's mechanisms. In addition to covalent modifications, the secondary and tertiary structures of RNA complexes can also have pathological effects and thus can be a focus of treatment. The SARS-CoV-2 genome contains four putative G-quadruplex sites [Zhao, C., et al.].

[0005] Mikutis et al. (2020) described a small-molecule "click degrader" that can be chemically covalently linked to RNA species via click, and then the linked RNA molecule can be cleaved. The authors described a method for identifying N in RNA sequences. 6 -Methyladenosine (m 6 A) The existing methylation click degradation sequencing method (meCLICK-Seq). This method hijacks RNA methyltransferases to introduce an alkyne moiety onto RNA instead of a methyl group. A subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction incorporates the click degrading agent molecule, leading to RNA cleavage. This method identifies methylated transcripts, determines RNA methyltransferase specificity, and reliably locates modification sites in introns and intergenic regions.

[0006] Because click degradative molecules are covalently incorporated into the target RNA, they can only be used to degrade RNA types that can be edited to contain suitable click-reactive groups (usually alkynes). Furthermore, the required RNA editing limits the therapeutic applications of this technology.

[0007] Furthermore, while the biomimetic cleavage group (warhead) imidazole initially used in the meClick-seq method has proven sufficient to validate the strategy's basis, its simplicity inherently limits its ability to be optimized for efficacy and pharmacokinetic (DMPK) properties in human therapeutic applications. Moreover, the degradative efficiency can vary target-specifically due to the different topological structures of each novel RNA conjugate upon recombination. Therefore, there is a need to develop novel degradative molecules suitable for pharmaceutical applications.

[0008] This invention was designed with the foregoing in mind. Summary of the Invention

[0009] This invention relates to the discovery that bifunctional compounds (also referred to herein as degrading agents) can be used as catalysts for the non-covalent binding and cleavage of target nucleic acid molecules. 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 selectively degrade the target nucleic acid. Therefore, the degrading agents do not require any chemical modification of the target nucleic acid (e.g., incorporation of click-reactive groups into the target nucleic acid).

[0010] The selective cleavage of target nucleic acid molecules using the degrading agents described herein can be used for RNA structural mapping and therapeutic applications (e.g., anticancer, antibacterial, and antiviral therapies).

[0011] On the one hand, the present invention provides bifunctional compounds as defined herein, or pharmaceutically acceptable salts or solvates thereof.

[0012] On the other hand, 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] On the other hand, the present invention provides bifunctional compounds as defined herein or pharmaceutically acceptable salts or solvates thereof for use as medicines.

[0014] On the other hand, the present invention provides a bifunctional compound as defined herein or a pharmaceutically acceptable salt or solvate thereof for treating a disease or condition in which degradation of the target oligonucleotide is beneficial.

[0015] On the other hand, the present invention provides bifunctional compounds as defined herein, or pharmaceutically acceptable salts or solvates thereof, for the treatment of proliferative conditions (e.g., cancer) or bacterial or viral infections.

[0016] On the other hand, the present invention provides the use of a bifunctional compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating a disease or condition in which degradation of the target oligonucleotide is beneficial.

[0017] On the other hand, the present invention provides the use of bifunctional compounds as defined herein, or pharmaceutically acceptable salts or solvates thereof, in the preparation of medicaments for treating proliferative diseases (e.g., cancer) or bacterial or viral infections.

[0018] On the other hand, the present invention provides a method for treating a disease or condition in which degradation of the target 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] On the other hand, the present invention provides a method for treating proliferative diseases (e.g., cancer) or bacterial or viral infections, the method comprising administering a therapeutically effective dose of a bifunctional compound as defined herein or a pharmaceutically acceptable salt or solvate thereof.

[0020] On the other hand, the present invention provides bifunctional compounds as defined herein or pharmaceutically acceptable salts or solvates thereof for use in epigenetic and epitranscriptome analysis / mapping.

[0021] On the other hand, the present invention provides the use of bifunctional compounds or their salts or solvates as defined herein for epigenetic and epitranscriptome analysis / mapping.

[0022] On the other hand, the present invention provides a method for cleaving target nucleic acid molecules, the method comprising:

[0023] Contacting the target nucleic acid molecule with the bifunctional compound of the present invention, such that the compound non-covalently binds to the target nucleic acid molecule; and

[0024] The compound is allowed to cleave the target nucleic acid molecule it binds to.

[0025] On the other hand, the present invention provides a method for identifying secondary or tertiary structures in target nucleic acid molecules, the method comprising:

[0026] First and second populations of nucleic acid molecules are provided, each population containing the target nucleic acid molecule;

[0027] The bifunctional compound of the present invention is introduced into the first group of nucleic acid molecules;

[0028] The bifunctional compounds of this invention are permitted to cleave target nucleic acid molecules present in the first population; and

[0029] Identify nucleic acid molecules present in the first group in reduced amounts relative to the second group.

[0030] The present invention further provides a method for synthesizing bifunctional compounds or pharmaceutically acceptable salts as defined herein.

[0031] Preferred, suitable, and optional features of any particular aspect of the invention are also preferred, suitable, and optional features of any other aspect.

[0032] Detailed description of the invention

[0033] definition

[0034] Unless otherwise stated, the following terms used in the specification and claims have the following meanings.

[0035] It should be understood that references to “treatment” or “management” include prevention and relief of established symptoms of a condition. Therefore, “treatment” or “management” of a state, condition or illness includes: (1) preventing or delaying the development of clinical symptoms of the state, condition or illness in a person who may have or is susceptible to the state, condition or illness but has not yet experienced or exhibited clinical or subclinical symptoms of the state, condition or illness; (2) suppressing the state, condition or illness, i.e., preventing, reducing or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms; or (3) alleviating or reducing the disease, i.e. causing the disappearance of the state, condition or illness or at least one of its clinical or subclinical symptoms.

[0036] "Therapeutic effective dose" refers to the amount of a bifunctional compound that is sufficient to treat a disease when administered to a mammal. The "therapeutic effective dose" will vary depending on the compound, the disease and its severity, and the age and weight of the mammal being treated.

[0037] In this specification, the term "alkyl" includes straight-chain and branched alkyl groups and their analogues. When referring to a single alkyl group such as "propyl," the straight-chain form is used only; when referring to a single branched alkyl group such as "isopropyl," the branched form is used only. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl, and tert-butyl. Similar convention applies to other groups; for example, "phenyl(1-6C)alkyl" includes phenyl(1-4C)alkyl, benzyl, 1-phenylethyl, and 2-phenylethyl.

[0038] The terms “(m-nC)” or “(m-nC) group” used alone or as a prefix refer to any group having m to n carbon atoms.

[0039] The term "heteroalkyl" is an alkyl group in which one or more carbon atoms are replaced by heteroatoms (e.g., N, O, and S). Heteroalkyl can be 1-6C heteroalkyl, such as 1-4C, 1-3C, or 1-2C heteroalkyl. In this document, the prefix (e.g., 1-6C) indicates the number of atoms in the heteroalkyl backbone, whether carbon atoms or heteroatoms. Heteroalkyl can be straight-chain or branched.

[0040] An "alkylene" group is an alkyl group located between two other chemical groups and used to connect them. Therefore, "(1-6C)alkylene" refers to a straight-chain saturated divalent hydrocarbon group with 1-6 carbon atoms or a branched saturated divalent hydrocarbon group with 3-6 carbon atoms, such as methylene, ethylene, propylene, 2-methylpropylene, pentylene, etc.

[0041] “(3-8C)cycloalkyl” refers to a hydrocarbon ring containing 3-8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or bicyclic [2.2.1]heptyl.

[0042] "(3-8C)cycloalkyl-(1-6C)alkylene" refers to a (3-8C)cycloalkyl group covalently attached to a (1-6C)alkylene group, both as defined herein.

[0043] The term "halogen" or "halogenated" refers to fluorine, chlorine, bromine, and iodine.

[0044] The terms "heterocyclic group," "heterocyclic," or "heterocycle" refer to a non-aromatic saturated or partially saturated monocyclic or fused, bridged, or spirobicyclic heterocyclic ring system. The term heterocyclic group includes both monovalent and divalent substances. A monocyclic heterocycle contains about 3 to 12 (suitably 3 to 7) ring atoms, with 1 to 5 (suitably 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, or sulfur. A bicyclic heterocycle contains 7 to 17 member atoms, suitable to 7 to 12 member atoms. Bicyclic heterocycles can be fused, spirocyclic, or bridged ring systems.

[0045] Examples of heterocyclic groups include cyclic ethers, such as ethylene oxide, oxetyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers. Nitrogen-containing heterocycles include, for example, oxetyl, pyrrolidinyl, piperidinyl, piperazineyl, tetrahydrotriazineyl, and tetrahydropyrazolyl. Typical sulfur-containing heterocycles include tetrahydrothiopheneyl, dihydro-1,3-dithiol, tetrahydro-2H-thiaran, and hexahydrothiophene. Other heterocycles include dihydrooxothioyl, tetrahydrooxazolyl, tetrahydrooxadiazolyl, tetrahydrodioxazolyl, tetrahydrooxothiazolyl, hexahydrotriazineyl, tetrahydrooxazineyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. For sulfur-containing heterocycles, sulfur oxide heterocycles containing SO or SO2 groups are also included. Examples include the sulfoxide and sulfone forms of tetrahydrothiophene and thiomorpholinoyl groups, such as tetrahydrothiophene 1,1-dioxide and thiomorpholinoyl 1,1-dioxide. Suitable values ​​for heterocyclic groups with one or two oxo (=O) or thio (=S) substituents are, for example, 2-oxopyrrolidinyl, 2-thiopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl, or 2,6-dioxopiperidinyl. Specific heterocyclic groups are saturated monocyclic 3-7 membered heterocyclic groups containing one, two, or three heteroatoms selected from nitrogen, oxygen, or sulfur, such as azo-butyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrothiophenyl 1,1-dioxide, thiomorpholinyl, thiomorpholinyl 1,1-dioxide, piperidinyl, homopiperidinyl, piperazinyl, or homopiperazinyl. As those skilled in the art will understand, any heterocycle can be attached to another group by any suitable atom, for example, by a carbon or nitrogen atom. However, the piperidinyl or morpholinyl groups mentioned herein refer to piperidin-1-yl or morpholino-4-yl rings linked by a cyclic nitrogen atom.

[0046] A “bridged ring system” refers to a ring system in which two rings share two or more atoms, see, for example, Jerry March’s *Advanced Organic Chemistry*, 4th ed., Wiley Interscience, pp. 131-133, 1992. Examples of bridged heterocyclic ring systems include azabicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, azabicyclo[2.2.2]octane, azabicyclo[3.2.1]octane, and quinine rings.

[0047] "Heterocyclic (1-6C)alkyl" refers to a heterocyclic group covalently attached to a (1-6C)alkylene group, both as defined herein.

[0048] The term "heteroaryl" or "heteroaromatic" refers to an aromatic monocyclic, bicyclic, or polycyclic compound containing one or more (e.g., 1-4, particularly 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, or sulfur. The term heteroaryl includes both monovalent and divalent compounds. Examples of heteroaryls are monocyclic and bicyclic groups containing 5-12 ring members, more typically 5-10 ring members. Heteroaryls can be, for example, 5- or 6-membered monocyclic or 9- or 10-membered bicyclic, such as a bicyclic structure formed by fused 5-membered and 6-membered rings or two fused 6-membered rings. Each ring may contain up to about 4 heteroatoms, typically selected from nitrogen, sulfur, and oxygen. Typically, a heteroaryl ring will contain up to 3 heteroatoms, more typically up to 2, such as a single heteroatom. In one embodiment, the heteroaryl ring contains at least one cyclic nitrogen atom. The nitrogen atom in the heteroaryl ring can be basic, as in the case of imidazole or pyridine, or substantially non-basic, as in the case of indole or pyrrole nitrogen. Typically, the number of basic nitrogen atoms present in a heteroaryl group (including any amino substituents on the ring) will be less than five.

[0049] Examples of heteroaryl groups include furanyl, pyrrolyl, thiophenyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothiophenyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, benzothiazolyl, indolyl, purinyl, benzofuranyl, quinolinyl, isoquinolinyl, and quinazolinyl. The terms "heteroaryl" also include partially aromatic bicyclic or polycyclic ring systems, wherein at least one ring is an aromatic ring and one or more other rings are non-aromatic saturated or partially saturated rings, provided that at least one ring contains one or more heteroatoms selected from nitrogen, oxygen, or sulfur. Examples of some aromatic heteroaryl groups include, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1,2,3,4-tetrahydroquinolinyl, dihydrobenzothiopheneyl, dihydrobenzofuranyl, 2,3-dihydro-benzo[1,4]dioxolyl, benzo[1,3]dioxolyl, 2,2-dioxo-1,3-dihydro-2-benzothiopheneyl, 4,5,6,7-tetrahydrobenzofuranyl, indololinyl, 1,2,3,4-tetrahydro-1,8-naphthidyl, 1,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl, and 3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazinyl.

[0050] Examples of five-membered heteroaryl groups include, but are not limited to, pyrrole, furanyl, thiophene, imidazolyl, furazolidone, oxazolyl, oxadiazolyl, oxtriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl.

[0051] Examples of six-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridinyl, pyrimidinyl, and triazinyl.

[0052] The bicyclic heteroaryl group can be, for example, selected from the following groups:

[0053] A benzene ring fused with a 5- or 6-membered ring containing 1, 2, or 3 heteroatoms;

[0054] A pyridine ring fused with a 5- or 6-membered ring containing 1, 2, or 3 heteroatoms;

[0055] A pyrimidine ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0056] Pyrrole rings fused with 5- or 6-membered rings containing 1, 2, or 3 heteroatoms;

[0057] A pyrazole ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0058] A pyrazine ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0059] An imidazole ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0060] Oxazole rings fused with 5- or 6-membered rings containing 1 or 2 heteroatoms;

[0061] An isoxazole ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0062] A thiazole ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0063] An isothiazole ring fused with a 5- or 6-membered ring containing 1 or 2 heteroatoms;

[0064] Thiophene rings fused with 5- or 6-membered rings containing 1, 2, or 3 heteroatoms;

[0065] Furan rings fused with 5- or 6-membered rings containing 1, 2, or 3 heteroatoms;

[0066] Cyclohexyl rings fused with 5- or 6-membered heteroaromatic rings containing 1, 2, or 3 cyclic heteroatoms; and

[0067] A cyclopentyl ring fused with a 5- or 6-membered heteroaromatic ring containing 1, 2, or 3 cyclic heteroatoms.

[0068] Specific examples of bicyclic heteroaryl groups containing a six-membered ring fused with a five-membered ring include, but are not limited to, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indololinyl, isoindololinyl, purine (e.g., adenine, guanine), indazoleyl, benzodioxanepentyl, and pyrazolopyridyl.

[0069] Specific examples of bicyclic heteroaryl groups containing two fused six-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxazinyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridinylpyridinyl, quinoxolinyl, quinazolinyl, terpineyl, phthalazinyl, naphthidyl, and pteridylyl.

[0070] "Heteroaryl(1-6C)alkyl" refers to a heteroaryl group covalently linked to a (1-6C)alkylene group, both as defined herein. Examples of heteroarylalkyl groups include pyridin-3-ylmethyl, 3-(benzofuran-2-yl)propyl, etc.

[0071] 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 compounds. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In a particular embodiment, the aryl group is phenyl.

[0072] The term "aryl(1-6C)alkyl" refers to an aryl group covalently linked to a (1-6C)alkylene group, both as defined herein. Examples of aryl-(1-6C)alkyl groups include benzyl, phenethyl, etc.

[0073] This specification also uses several compound terms to describe groups containing more than one functional group. Those skilled in the art will understand such terms. For example, heterocyclic (m-nC)alkyl includes (m-nC)alkyl groups substituted with heterocyclic groups.

[0074] The term "optionally substituted" refers to substituted and unsubstituted groups, structures, or molecules. The term "wherein R..." 1 "One or any CH, CH2, CH3 group or heteroatom (i.e., NH) may be substituted" appropriately represents R. 1 Any hydrogen group in the group is replaced by the relevant specified group.

[0075] When the optional substituents are selected from "one or more" groups, it should be understood that this definition includes all substituents selected from a particular group or substituents selected from two or more particular groups.

[0076] The phrase “compounds of the present invention” refers to those compounds disclosed herein, including both general and specific ones (e.g., compounds of formula (I), (II), (III), (IV) or (V)). Attached Figure Description

[0077] The invention is described with reference to the accompanying drawings listed below.

[0078] Figure 1: Schematic diagram of the action mode of the degradation agent. Figure 1 This study demonstrates the application of a degrading agent in a strategy for degrading coronavirus pseudoknots. The pseudoknot-degrading agent binds to and then directly degrades the coronavirus region containing the pseudoknot, without the need for other reagents.

[0079] Figure 2 The rG4 degrader cleaved rG4-containing oligomers and the SARS-CoV-2 genome in vitro. (a) shows the effect of the rG4 degrader on rG4-competent oligomers. The degrader cleaved the oligomers under conditions that promoted rG4 formation. n=3. (b) shows the effect of the rG4 degrader on oligomers that did not form rG4. No degradation was observed. n=3. (c) shows nanopore sequencing data indicating that the SARS-CoV-2 genome was extensively degraded when treated with the rG4 degrader PDS-deg6(9A) for its ORF1b. *p<0.05,**p<0.01,***p<0.005, no significant difference in ns.

[0080] Figure 3 Preliminary in vitro findings of the anti-SARS-CoV-2 activity of G4 degraders. (a) Inhibition of plaque-forming units (PFU) on samples treated with PDS-deg4 (9B), PDS-deg6 (9A), and PDS-DegALK (8) at 50 μM is shown. (b) PCR measurements of viral RNA are presented. Results showed that PDS-deg6 (9A) inhibited viral replication at both 5 μM and 50 μM. As shown, PDS-deg6 (9A) appeared to inhibit viral growth to a greater extent than PDS-deg4 (9B). (c) Cell viability is shown after 24 hours of incubation with increased concentrations of G4 degraders. None of the compounds showed cytotoxicity up to 50 μM.

[0081] Figure 4 In vivo anti-SARS-CoV-2 activity of G4-degrading agent. (a) Mice administered PDS-deg4(9B) (purple, triangle marker) showed a 10% reduction in body weight on day 1 post-infection, which stabilized between day 1 and day 3, then decreased again, reaching a 75% threshold on day 5, as observed in animals treated with the medium (0.1% DMSO aqueous solution) (grey square marker). Uninfected mice treated with the medium (0.1% DMSO aqueous solution) as a control (circle marker) did not experience a decrease in body weight. (b) Quantification of lung viral load by plaque assay on day 5 showed a reduced viral load in animals treated with PDS-deg4(9B) (purple, right) compared to the medium control group (grey, left). *p<0.01.

[0082] Figure 5 MTDB-degrader (16a) cleaves coronavirus pseudoknots in vitro. (a) shows the synthetic design of MTDB-deg (16a). (b) shows the structure of the control molecule TDB-deg (16b), characterized by a weak pseudoknot conjugate and an imidazole cleavage moiety. (c) is LC-MS data showing the degradation of pseudoknots relative to the control in the presence of the degrader, n=3. (d) shows a gel photograph confirming the activity of the pseudoknot degrader. (e) is LC-MS data showing that the pseudoknot degrader loses its efficiency when one of the pseudoknot stems is mutated to disrupt the pseudoknot secondary structure, n=3. (f) is a gel photograph showing that native RNA extracted from SARS-CoV-2 was degraded by MTDB-deg (16a) compared to the control. ns – no significant difference.

[0083] Figure 6 Direct RNA nanopore sequencing revealed genomic sites degraded by MTDB-deg. (a) Based on minimap2 alignments, the distribution and abundance of alignment reads flanking the pseudoknot region of SARS-CoV-2 RNA treated with control (0.1% DMSO aqueous solution) or MTDB-deg are shown. (b) Based on minimap2 alignments, the distribution and abundance of alignment reads specifically mapped to the S sgRNA region in SARS-CoV-2 RNA treated with control or MTDB-deg are shown.

[0084] Figure 7 Treatment with MTDB-deg had no effect on subgenomic SARS-CoV-2 RNA. Based on the comparison with minimap2, the distribution and abundance of alignment reads specifically mapped to the specified sgRNA regions in SARS-CoV-2 RNA treated with either control (0.1% DMSO aqueous solution) or MTDB-deg are shown.

[0085] Figure 8MTDB degrader inhibits SARS-CoV-2 replication in cells. (a) and (b) show the percentage inhibition of viral replication relative to the vector control (dashed line) normalized after incubation with increased concentrations of the pseudoknot degrader (MTDB-deg(16a)) and control molecules (MTDB and TDB-deg(16b)). Viral replication was assessed based on E gene and pseudoknot RNA levels 24 hours after infection (MOI 0.05). Antiviral activity of MTDB-deg(16a) was observed both before infection (a) and after infection (b) (where SARS-CoV-2 MOI was 0.05). Mean ± SD of three replicates are shown, and differences between means of p < 0.01 are noted. *, p < 0.05; **, p < 0.01; paired two-tailed t-test. (c) shows the lower 50% inhibitory concentration (IC50) of the pseudoknot degrader MTDB-deg(16a) when the drug was added post-infection. (d) A photograph of a cell monolayer after 4 days of incubation with supernatant from a viral culture treated for 24 hours with 6 mM MTDB-deg (16a), MTDB, and TDB-deg (16b). Treatment with 6 mM MTDB-deg (16a) for 24 hours showed a reduction in viral plaque number compared to the vector control, whether added before or after infection. When added before infection, the control molecule MTDB showed only a reduction in viral plaque number, while TDB-deg (16b) showed no reduction. (e) Cell viability assays demonstrating that none of the compounds exhibited cytotoxicity in VeroCCL81 cells after 24 hours. (f) Percentage of viral replication relative to the vector control 24 hours after removal of the medium containing the degrading agent. Treatment with MTDB-deg (16a) for 24 hours impaired the virus's ability to recover from drug exposure.

[0086] Figure 9 Dose-response curves for MTDB-deg, MTDB, and TDB-deg. (a) and (b) show the 50% inhibitory concentration (IC50) values ​​of the pseudoknot degrader MTDB-deg (16a) before and after infection. The control molecules (MTDB and TDB-deg (16b)) did not inhibit viral replication, so IC50 values ​​could not be determined. (c) is the dose-response curve (determined by PCR targeting the E gene), including higher concentrations of 18 μM showing an increased IC50.

[0087] Figure 10Viral recovery and antiviral activity after exposure to MTDB degraders. (a) The ability of the virus to recover after 24 hours of incubation with MTDB-deg (16a) and control molecules MTDB and TDb-deg (16b) was determined by qPCR targeting the pseudoknot region. Viral recovery was impaired in samples treated with MTDB-deg (16b), but not in samples treated with control molecules MTDB and TDB-deg (16b). (b) Antiviral activity was assessed by incubating 1000 PFU of SARS-CoV-2 with the compound at 6 μM for 1 hour at 37 °C, followed by determination of residual viral infectivity by plaque assay. MTDB-deg (16a), MTDB, and TDB-deg (16b) had no antiviral activity against cell-free virions, indicating that the antiviral activity of MTDB-deg (16a) is mediated by inhibiting viral replication in host cells rather than by inactivating cell-free virions.

[0088] Figure 11 Agarose gel analysis of ribosome degradation assay. Left panel: Ethyl linker, no visible degradation. Left center: Diethylene glycol linker, degradation visible at 15 mM concentration. Center right: Hexaethylene glycol linker, no visible degradation. Right panel: Control chloramphenicol, no degradation. 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.

[0089] Figure 12 In vivo activity of MTDB degraders against SARS-CoV-2 infection in K18-hACE2 mice. (a) Using 10 4Female K18-hACE2 transgenic mice aged 8 to 12 weeks were intranasally infected with SARS-CoV-2 plaque-forming units (PFUs) and treated 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 can be administered given limited solubility) (n=3), TDB-deg 16b (25 mg / kg) (n=5), and a vector control (n=6). (b) Administration of MTDB-deg 16a resulted in a decrease in viral load in the lungs of SARS-CoV-2-infected K18-hACE2 mice. No difference in viral load in the lungs was observed between the vector 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 phosphorylated p38 in lung extracts from transgenic K18-hACE2 mice treated with three doses of 10 mg / kg mediator (V1, V2) or MTDB-deg 16a (D1, D2) 1 hour before infection and 1 and 2 days after infection (n=2).

[0090] Figure 13 In vitro assays were used to evaluate the cleavage group / projectile effectiveness. n=3, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns showed no significant difference.

[0091] Figure 14 After incubating RNA functionalized with the degrading agent at 37°C for 4 hours, the efficacy of the RNA degrading agent warheads was compared. (a) The cleavage groups of the degrading agent were compared with those without a warhead adapter (degrading agent 2) using a one-sided t-test. (b) The cleavage groups of the degrading agent were compared with those of imidazole degrading agent 1 using a two-sided t-test. n=3,****p<0.0001,***p<0.001,**p<0.01,*p<0.05,ns showed no significant difference.

[0092] Figure 15 Comparison of PDS-based non-covalent RNA degraders PDS-deg6 and PDS-Amimi with RNA after incubation at 37°C for 4 hours. Statistical significance was calculated using a one-sided t-test. n = 3, ***p < 0.001, **p < 0.01. Detailed Implementation

[0093] The bifunctional compound of the present invention

[0094] This invention relates to the discovery that bifunctional compounds (also referred to herein as degrading agents) can be used as catalysts for non-covalent binding and cleavage of target nucleic acid molecules. The degrading agents disclosed herein bind to target nucleic acids through non-covalent interactions. Therefore, conventional methods require the incorporation of reactive groups into the target nucleic acid molecule to achieve covalent interactions, while this degrading agent eliminates this requirement. The selective cleavage of target nucleic acid molecules using the degrading agents described herein can be used for epigenetic and epitranscriptomic analysis, bifunctional mapping, and therapeutic applications (e.g., anticancer, antibacterial, and antiviral therapies).

[0095] On one hand, the present invention relates to bifunctional compounds of formula (I) or pharmaceutically acceptable salts or solvates thereof:

[0096] CLB (I)

[0097] in:

[0098] C is the cleavage group defined in this article;

[0099] L is a connector; and

[0100] B is a non-covalently bonded group.

[0101] Cleavage group C

[0102] The cleavage group can be any suitable group that can react with the target nucleic acid molecule and cause the nucleic acid molecule to be cleaved.

[0103] Without being bound by any particular theory, the cleavage group can function by abstracting a proton from the 2'OH position of the target nucleic acid molecule and / or can form a 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)).

[0104] The basicity of a functional group can be quantitatively assessed using the pKa of the relevant conjugate acid. That is, the conjugate acid [CH] can be used. +The pKa is used to assess the basicity of the basic group [C]. The pKa of the conjugate acid can be known or determined using standard techniques such as acid-base titration. Not wishing to be bound by theory, the inventors believe that basic residues of conjugate acids with pKa values ​​above a certain threshold, such as pKa of 5.5 or greater, 6.0 or greater, 6.2 or greater, 6.5 or greater, or 6.8 or greater, can deprotonate the hydroxyl group at the 2' position of the ribose to allow cleavage of the phosphodiester backbone within the target nucleic acid. Suitably, the pKa of the cleavage group is in the range of 5.5 to 9, more suitably 6 to 9, and most suitably 6.2 to 8.6.

[0105] Alternatively, and / or furthermore, the cleavage group may be a group known to form a chelate complex with copper, which is capable of inducing copper-mediated nucleic acid degradation.

[0106] Suitablely, the cleavage group comprises a basic nitrogen atom or a hydroxyl group, which: (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 hydroxyl group that is capable of forming a chelate complex with copper (and thus capable of inducing copper-mediated nucleic acid degradation).

[0107] Suitably, the basic nitrogen atom or hydroxyl group is not sterically hindered by substituents (e.g., alkyl substituents) present, for example, on the carbon atom directly attached to the nitrogen or COH group. In one embodiment of the invention, the carbon atom adjacent to the basic N atom or C-OH group with a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6 is not substituted.

[0108] Appropriately, the cleavage group is not:

[0109] (i) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0110] (ii) Nucleic acid cleavage group of formula Z:

[0111]

[0112] in:

[0113] Indicates the connection point with L;

[0114] Ring A is absent or contains a nitrogen-containing heteroaryl or heterocyclic ring, optionally further composed of one or more elements selected from halogen, 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 cC(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 Substituents of R; where R c and R d Selected from hydrogen or (1-4C) alkyl;

[0115] The integer a1 is 0, 1, 2, or 3;

[0116] Ra and Rb are each independently selected from hydrogen or (1-2C) alkyl groups each time they appear;

[0117] 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, and each is optionally selected by one or more elements selected from halogen, 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

[0118] (where y is 0, 1, or 2), SO2N(R) f )R e 、N(R f SO2R e or NR e R f Substituents of the substituents;

[0119] Where R e and R f Selected from hydrogen or (1-4C) alkyl, or

[0120] R1 and R2 are linked such that they, together with the atoms to which they are attached, form a 4-6 membered heterocycle or a 5- or 6-membered heteroaryl group, wherein any 4- or 6 membered heterocycle or 5- or 6-membered heteroaryl group is optionally composed of one or more elements selected from halogen, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, (1-4C)alkyl, (1-4C) haloalkyl, etc.

[0121] (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 ,

[0122] 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 Substituents of R, wherein R g and R h Selected from hydrogen or (1-4C) alkyl;

[0123] When ring A is absent, R1 and R2 are each independently selected from hydrogen, heterocycle, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, and each is optionally selected by one or more elements selected from halogen, 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 ,

[0124] 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 Substituents of R, wherein R e and Rf Selected from hydrogen or (1-4C) alkyl, provided that R1 and R2 cannot both be hydrogen.

[0125] Appropriately, the cleavage group is not:

[0126] (i) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0127] (ii) Nucleic acid cleavage group of formula Z:

[0128]

[0129] in:

[0130] Indicates the connection point with L;

[0131] Ring A is a nitrogen-containing heteroaryl or heterocyclic ring, optionally further composed of one or more elements selected from halogen, 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 Substituents of R; where R c and R d Selected from hydrogen or (1-4C) alkyl;

[0132] The integer a1 is 0, 1, 2, or 3;

[0133] Ra and Rb are each independently selected from hydrogen or (1-2C) alkyl groups each time they appear;

[0134] R1 and R2 are each independently selected from hydrogen, (1-6C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, and each is optionally selected by one or more elements selected from halogen, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, OR e C(O)R e C(O)ORe 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 Substituents of R; where R e and R f Selected from hydrogen or (1-4C) alkyl.

[0135] Specific compounds of the present invention include, for example, compounds of formula (I) or any subform thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the cleavage group C and any associated substituents each have any meaning as defined in any of paragraphs (1) to (12) above or below:

[0136] (1) The cleavage group C is selected from:

[0137] (i) Any part containing N or C-OH, wherein 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;

[0138] (ii) Any N- or OH-containing moiety capable of chelating a metal (e.g., copper or zinc) at physiological pH; provided that the cleavage group is not:

[0139] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0140] b) The groups of formula Z as defined above;

[0141] (2) The cleavage group C is a group of the following formula:

[0142] -L1-X C -L2-R C

[0143] in:

[0144] L1 is absent or is a (1-6C) alkylene group;

[0145] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1-, -C(O)-, -C(O)O-,

[0146] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 )C(O)-、-N(R XC2 )C(O)N(R XC1 )-、

[0147] -N(R XC1 )C(O)O-、-OC(O)N(R XC1 )-、-S(O)2N(R XC1 ), -N(R XC1 SO2-

[0148] -C(O)N(R XC1 SO2- or -SO2N(R) XC1 )C(O)-; and where R XC1 and R XC2 Each is independently selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene,

[0149] -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 4;

[0150] L2 is absent or is a (1-6C) alkylene group;

[0151] R C Selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups;

[0152] in:

[0153] Alkyl, cycloalkyl, or aryl groups are bound by one or more R groups. A Substituents, and optionally one or more R B Substituents further substitute;

[0154] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution;

[0155] R A It is selected from the following groups:

[0156] (i)-OH;

[0157] (ii)(1-6C)hydroxyalkyl;

[0158] (iii) (3-6C) hydroxycycloalkyl;

[0159] (iv)NR A1 R A2 ;

[0160] (v)-(1-6C)alkylene-NR A1 R A2 ;

[0161] (vi)-(3-6C)cycloalkylene-NR A1 R A2 ;

[0162] (vii)-(CH2) m2 -R A3 Where m2 is between 0 and 6;

[0163] in:

[0164] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0165] R A3 It is optional to be subjected to one or more OH or NR. A1 R A2 Substituents are cycloalkyl or aryl groups, and optionally are one or more R groups. B Substituents may be further substituted; or optionally replaced by one or more OH groups or NR groups. A1 R A2 or R B Substituents: heteroaryl or heterocyclic groups;

[0166] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA -[CH2] t -C(O)R BA ,

[0167] -[CH2] t -C(O)OR BA -[CH2] t -OC(O)R BA -[CH2] t -C(O)N(R BB )R BA ,

[0168] -[CH2] t -N(R BB )C(O)R BA -[CH2]t -S(O) p R BA (where p is 0, 1, or 2)

[0169] -[CH2] t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ;

[0170] Where t is 0, 1, 2 or 3;

[0171] R BA It is hydrogen or an (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino, or cyano groups; and

[0172] R BB It is hydrogen or (1-2C) alkyl;

[0173] The cleavage group is not:

[0174] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0175] b) The groups of formula Z as defined above;

[0176] (2A) The cleavage group C is a group of the following formula:

[0177] -L1-X C -L2-R C

[0178] in:

[0179] L1 is absent or is a (1-6C) alkylene group;

[0180] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)O-,

[0181] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 )C(O)-、-N(R XC2 )C(O)N(R XC1 )-、

[0182] -N(R XC1 )C(O)O-、-OC(O)N(R XC1 )-、-S(O)2N(R XC1 ), -N(RXC1 SO2-

[0183] -C(O)N(R XC1 SO2- or -SO2N(R) XC1 )C(O)-; and where R XC1 and R XC2 Each is independently selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene,

[0184] -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, wherein m1 is 0 to 4; L2 is absent or is (1-6C) alkylene;

[0185] R C Selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups;

[0186] in:

[0187] Alkyl, cycloalkyl, or aryl groups are bound by one or more R groups. A Substituents, and optionally one or more R B Substituents further substitute;

[0188] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution; R A It is selected from the following groups:

[0189] (i)-OH;

[0190] (ii)(1-6C)hydroxyalkyl;

[0191] (iii) (3-6C) hydroxycycloalkyl;

[0192] (iv)-(3-6C)cycloalkylene-NR A1 R A2 ;

[0193] (v)-(CH2) m2 -R A3 Where m2 is between 0 and 6;

[0194] in:

[0195] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0196] RA3 It is optional to be subjected to one or more OH or NR. A1 R A2 Substituents are cycloalkyl or aryl groups, and optionally are one or more R groups. B Substituents may be further substituted; or optionally replaced by one or more OH groups or NR groups. A1 R A2 or R B Substituents: heteroaryl or heterocyclic groups;

[0197] R B Selected from halogen, 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 (where p is 0, 1, or 2), -[CH2) t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ;

[0198] Where t is 0, 1, 2 or 3;

[0199] R BA It is hydrogen or (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino or cyano groups;

[0200] and

[0201] R BB It is hydrogen or (1-2C) alkyl;

[0202] Where L1, X C L2 does not exist, and R C When it is a nitrogen-containing heteroaryl or heterocyclic ring, it is reacted with the above-mentioned R A Replace; and

[0203] The cleavage group is not:

[0204] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0205] b) The groups of formula Z as defined above;

[0206] (3) The cleavage group C is a group of the following formula:

[0207] -L1-X C -L2-R C

[0208] in:

[0209] L1 is absent or is a (1-2C) alkylene group;

[0210] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)O-,

[0211] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 C(O)-、-S(O)2N(R) XC1 ) or -N(R XC1 SO2-;

[0212] And R XC1 Selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene, -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 2;

[0213] L2 is absent or is a (1-2C) alkylene group;

[0214] R C Selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, phenyl, heteroaryl or heterocyclic;

[0215] in:

[0216] Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute;

[0217] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution;

[0218] R A It is selected from the following groups:

[0219] (i)-OH;

[0220] (ii)NR A1 R A2 ;

[0221] (iii)-(1-6C)alkylene-NR A1 R A2 ;

[0222] in:

[0223] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0224] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA -[CH2] t -C(O)R BA ,

[0225] -[CH2] t -C(O)OR BA -[CH2] t -OC(O)R BA -[CH2] t -C(O)N(R BB )R BA ,

[0226] -[CH2] t -N(R BB )C(O)R BA -[CH2] t -S(O) p R BA (where p is 0, 1, or 2)

[0227] -[CH2] t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ;

[0228] Where t is 0, 1, 2 or 3;

[0229] R BAIt is hydrogen or an (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino, or cyano groups; and

[0230] R BB It is hydrogen or (1-2C) alkyl;

[0231] The cleavage group is not:

[0232] (a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0233] (b) The groups of formula Z as defined above;

[0234] (3A) The cleavage group C is a group of the following formula:

[0235] -L1-X C -L2-R C

[0236] in:

[0237] L1 is absent or is a (1-2C) alkylene group;

[0238] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)O-,

[0239] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 C(O)-、-S(O)2N(R) XC1 ) or -N(R XC1 SO2-; and where R XC1 Selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)

[0240] Alkylene, -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 2;

[0241] L2 is absent or is a (1-2C) alkylene group; R C Selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, phenyl, heteroaryl or heterocyclic;

[0242] in:

[0243] Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more RB Substituents further substitute;

[0244] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution;

[0245] R A It is selected from the following groups:

[0246] (i)-OH;

[0247] (ii)(1-6C)hydroxyalkyl;

[0248] (iii) (3-6C) hydroxycycloalkyl;

[0249] (iv)-(3-6C)cycloalkylene-NR A1 R A2 ;

[0250] in:

[0251] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0252] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA -[CH2] t -C(O)R BA ,

[0253] -[CH2] t -C(O)OR BA -[CH2] t -OC(O)R BA -[CH2] t -C(O)N(R BB )R BA ,

[0254] -[CH2] t -N(R BB )C(O)R BA -[CH2] t -S(O) p R BA (where p is 0, 1, or 2)

[0255] -[CH2] t -SO2N(R BB )R BA Or -[CH2] t -N(RBB SO2R BA ;

[0256] Where t is 0, 1, 2 or 3;

[0257] R BA It is hydrogen or an (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino, or cyano groups; and

[0258] R BB It is hydrogen or (1-2C) alkyl;

[0259] Where L1, X C L2 does not exist, and R C When it is a nitrogen-containing heteroaryl or heterocyclic ring, it is reacted with the above-mentioned R A Replace; and

[0260] The cleavage group is not:

[0261] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0262] b) The groups of formula Z as defined above;

[0263] (4) The cleavage group C is a group of the following formula:

[0264] -X C -R C

[0265] in:

[0266] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 )-、-C(O)-、

[0267] -C(O)N(R XC1 - or -N(R) XC1 )C(O)-; and where R XC1 Selected from hydrogen, (1-6C)alkyl, or -(CH2). m1 -heteroaryl, where m1 is 0 to 2;

[0268] R C Selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl, or heterocyclic groups;

[0269] in:

[0270] Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute;

[0271] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution;

[0272] R A It is selected from the following groups:

[0273] (i)-OH;

[0274] (ii)NR A1 R A2 ;

[0275] (iii)-(1-6C)alkylene-NR A1 R A2 ;

[0276] in:

[0277] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0278] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA Or -[CH2] t -C(O)OR BA Where t is 0, 1, 2, or 3; and

[0279] R BA It is a (1-4C) alkyl group;

[0280] The cleavage group is not:

[0281] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0282] b) The groups of formula Z as defined above;

[0283] (4A) The cleavage group C is a group of the following formula:

[0284] -X C -R C

[0285] in:

[0286] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-,

[0287] -C(O)N(RXC1 - or -N(R) XC1 )C(O)-; and where R XC1 Selected from hydrogen or (1-6C)alkyl

[0288] Or -(CH2) m1 -heteroaryl, where m1 is 0 to 2;

[0289] R C Selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl, or heterocyclic groups;

[0290] in:

[0291] Alkyl or phenyl groups are affected by one or more R groups. A Substituents, and optionally one or more R B Substituents further substitute;

[0292] Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution;

[0293] R A It is selected from the following groups:

[0294] (i)-OH;

[0295] (ii)(1-6C)hydroxyalkyl;

[0296] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA Or -[CH2] t -C(O)OR BA Where t is 0, 1, 2, or 3; and

[0297] R BA It is a (1-4C) alkyl group;

[0298] Where X C It does not exist, and R C When it is a nitrogen-containing heteroaryl or heterocyclic ring, it is reacted with the above-mentioned R A Replace; and

[0299] The cleavage group is not:

[0300] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or b) a group of formula Z as defined above;

[0301] (5) The cleavage group C is a group of the following formula:

[0302] -L1-X C -L2-R C

[0303] in:

[0304] L1 is absent or is a (1-6C) alkylene group;

[0305] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)O-,

[0306] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 )C(O)-、-N(R XC2 )C(O)N(R XC1 )-、

[0307] -N(R XC1 )C(O)O-、-OC(O)N(R XC1 )-、-S(O)2N(R XC1 ), -N(R XC1 SO2-

[0308] -C(O)N(R XC1 SO2- or -SO2N(R) XC1 )C(O)-; and where R XC1 and R XC2 Each is independently selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene,

[0309] -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 4;

[0310] L2 is absent or is a (1-6C) alkylene group;

[0311] R C Selected from hydrogen, (1-6C)alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups;

[0312] in:

[0313] Alkyl, cycloalkyl, aryl are bound by one or more R A Substituents, and optionally one or more R B Substituents further substitute;

[0314] 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, then the ring is bounded by one or more R atoms. A Substituents, and optionally one or more R groups. B Substituent substitution, or if the heteroaryl or heterocyclic ring contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, then the ring is optionally substituted with one or more R atoms. A or R B Substituent substitution;

[0315] R A It is selected from the following groups:

[0316] (i)-OH;

[0317] (ii)(1-6C)hydroxyalkyl;

[0318] (iii) (3-6C) hydroxycycloalkyl;

[0319] (iv)NR A1 R A2 ;

[0320] (v)-(1-6C)alkylene-NR A1 R A2 ;

[0321] (vi)-(3-6C)cycloalkylene-NR A1 R A2 ;

[0322] (vii)-(CH2) m2 -R A3 Where m2 is between 0 and 6;

[0323] in:

[0324] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0325] R A3 It is optional to be subjected to one or more OH or NR. A1 R A2 Substituents are cycloalkyl or aryl groups, and optionally are one or more R groups. B Substituents may be further substituted; or optionally replaced by one or more OH groups or NR groups. A1 R A2 or R B Substituents: heteroaryl or heterocyclic groups;

[0326] R B Selected from halogen, nitro, cyano, RBA -[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 (where p is 0, 1, or 2), -[CH2) t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ;

[0327] Where t is 0, 1, 2 or 3;

[0328] R BA It is hydrogen or (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino or cyano groups;

[0329] and

[0330] R BB It is hydrogen or (1-2C) alkyl;

[0331] The cleavage group is not:

[0332] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0333] b) The groups of formula Z as defined above;

[0334] (6) The cleavage group C is a group of the following formula:

[0335] -L1-X C -L2-R C

[0336] in:

[0337] L1 is absent or is a (1-2C) alkylene group;

[0338] X CIt does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)O-,

[0339] -OC(O)-、-C(O)N(R XC1 )-、-N(R XC1 C(O)-、-S(O)2N(R) XC1 ) or -N(R XC1 SO2-;

[0340] And R XC1 Selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene, -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 2;

[0341] L2 is absent or is a (1-2C) alkylene group;

[0342] R C Selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, phenyl, heteroaryl or heterocyclic;

[0343] in:

[0344] Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute;

[0345] 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, then the ring is bounded by one or more R atoms. A Substituents, and optionally one or more R groups. B Substituent substitution, or if the heteroaryl or heterocyclic ring contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, then the ring is optionally substituted with one or more R atoms. A or R B Substituent substitution;

[0346] R A It is selected from the following groups:

[0347] (i)-OH;

[0348] (ii)NR A1 R A2 ;

[0349] (iii)-(1-6C)alkylene-NR A1 R A2 ;

[0350] in:

[0351] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0352] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA -[CH2] t -C(O)R BA ,

[0353] -[CH2] t -C(O)OR BA -[CH2] t -OC(O)R BA -[CH2] t -C(O)N(R BB )R BA ,

[0354] -[CH2] t -N(R BB )C(O)R BA -[CH2] t -S(O) p R BA (where p is 0, 1, or 2)

[0355] -[CH2] t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ;

[0356] Where t is 0, 1, 2 or 3;

[0357] R BA It is hydrogen or (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino or cyano groups;

[0358] and

[0359] R BB It is hydrogen or (1-2C) alkyl;

[0360] The cleavage group is not:

[0361] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0362] b) The groups of formula Z as defined above;

[0363] (7) The cleavage group C is a group of the following formula:

[0364] -X C -R C

[0365] in:

[0366] X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 )-、-C(O)-、

[0367] -C(O)N(R XC1 - or -N(R) XC1 )C(O)-; and where R XC1 Selected from hydrogen, (1-6C)alkyl, or -(CH2). m1 -heteroaryl, where m1 is 0 to 2;

[0368] R C Selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl, or heterocyclic groups;

[0369] in:

[0370] Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute;

[0371] 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, then the ring is bounded by one or more R atoms. A Substituents, and optionally one or more R groups. B Substituent substitution, or if the heteroaryl or heterocyclic ring contains one or more N atoms with a pKa of 5.5 to 9, or 6 to 9, or 6.2 to 8.6, then the ring is optionally substituted with one or more R atoms. A or R B Substituent substitution;

[0372] R A It is selected from the following groups:

[0373] (i)-OH;

[0374] (ii)NR A1 R A2 ;

[0375] (iii)-(1-6C)alkylene-NR A1 R A2 ;

[0376] in:

[0377] R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl;

[0378] R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA Or -[CH2] t -C(O)OR BA Where t is 0, 1, 2, or 3; and

[0379] R BA It is a (1-4C) alkyl group;

[0380] The cleavage group is not:

[0381] a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or

[0382] b) The groups of formula Z as defined above;

[0383] (8) The cleavage group C is selected from the following groups:

[0384]

[0385]

[0386] (9) The cleavage group C is selected from the following groups:

[0387]

[0388] (10) The cleavage group C is selected from the following groups:

[0389]

[0390] (11) The cleavage group C is selected from the following groups:

[0391] (12) The cleavage group C is a group of the following formula:

[0392]

[0393] In one embodiment of the invention, the cleavage group C is as defined in paragraph (1) above.

[0394] In one embodiment of the invention, the cleavage group C is as defined in paragraph (2) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (2A) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (3) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (3A) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (4) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (4A) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (5) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (6) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (7) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (8) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (9) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (10) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (11) above. In one embodiment of the invention, the cleavage group C is as defined in paragraph (12) above.

[0395] As described above, when non-covalently bound to a target nucleic acid molecule via a linker and binding group, the cleaving group C approaches and reacts with the target nucleic acid molecule to cleave one or more phosphodiester bonds, thereby leading to the degradation of the target nucleic acid molecule. For example, the cleaving group C of the bound degrading agent can abstract a proton from the 2'OH position on the nucleic acid molecule, resulting in the cleavage of the phosphodiester bonds in the target nucleic acid molecule. Furthermore, the cleaving group C can form a copper complex, which cleaves the phosphodiester bonds in the target nucleic acid molecule.

[0396] Appropriately, R C or R A One of the substituents contains a basic nitrogen atom or a hydroxyl group, which: (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 hydroxyl group that is capable of forming a chelate complex with copper (and thus capable of inducing copper-mediated nucleic acid degradation).

[0397] Appropriately, when R C When it is an alkyl, cycloalkyl, or aryl / phenyl group, then R AIt is a substituent containing a basic nitrogen atom or a hydroxyl group, which: (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 hydroxyl group that is capable of forming a chelate complex with copper (and thus capable of inducing copper-mediated nucleic acid degradation).

[0398] Appropriately, when R C When it is a heteroaryl or heterocyclic group, the heteroaryl or heterocyclic group contains a basic nitrogen atom, which (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 capable of forming a chelate complex with copper (and thus capable of inducing copper-mediated nucleic acid degradation), or the heteroaryl or heterocyclic group is surrounded by an R containing a basic nitrogen atom or a hydroxyl group. A The substituents are substituted, which (i) have a pKa in the range of 5.5 to 9, or 6 to 9, or 6.2 to 8.6; and / or (ii) are nitrogen atoms or hydroxyl groups that are capable of forming chelate complexes with copper (and thus capable of inducing copper-mediated nucleic acid degradation).

[0399] Connector (-L-)

[0400] The connector L of the degrading agent contains a group for attaching (i.e., covalently attaching) the cleaving group (C) to the non-covalently bound group (B). Suitable connectors are well known in the art.

[0401] Typically, the linker contains divalent groups, one of which is a free valence forming part of a single bond of the cleaving group (C), and the remaining free valence forming part of a single bond of the non-covalently bonded group (B).

[0402] Specifically, a linker is a stable linker. That is, a linker contains groups that are essentially not cleaved or degraded in vivo. Stable linkers are generally unreactive at physiological pH and are essentially not degraded by enzymes in vivo.

[0403] Typically, the joint is a flexible joint. That is, the joint allows the cleaving group (C) and the binding group (B) to move relative to each other with a large degree of freedom.

[0404] Typical connectors include groups selected from alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene. Mixed connectors containing different covalently linked groups are permissible, such as alkylene-arylene (arylene) and heteroalkylene-arylene.

[0405] Alkylenes (alkyldiyl) are divalent saturated hydrocarbon groups in which the two free valences each form part of a single bond with an adjacent atom. Alkylenes can be (1-6C) alkylenes, such as 1-4C, 1-3C, or 1-2C alkylenes. In this document, the prefix (e.g., 1-6C) indicates the number of atoms in the hydrocarbon backbone. Alkylenes can be straight-chain or branched. Examples of linear alkylenes 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 alkylenes include ethane-1,1-diyl and propane-1,2-diyl.

[0406] A heteroalkylene is an alkylene in which one or more carbon atoms are substituted with heteroatoms such as N, O, and S. Heteroalkylenes can be 1-6C heteroalkylenes, such as 1-4C, 1-3C, or 1-2C heteroalkylenes. In this document, the prefix (e.g., 1-6C) indicates the number of atoms (both carbon and heteroatoms) in the heteroalkylene backbone. Heteroalkylenes can be straight-chain or branched.

[0407] Examples of linear heteroalkylene compounds include those derived from formaldehyde (e.g., polyoxymethylene, POM), ethylene glycol (e.g., polyethylene glycol, PEG), ethyleneimine (e.g., linear polyethyleneimine, PEI; polyaziridinium), and tetramethylene glycol (e.g., polytetramethylene glycol, PTMEG; polytetrahydrofuran). Examples of branched heteroalkylene compounds include those derived from propylene glycol (e.g., polypropylene glycol PPG). When a nitrogen atom is present in a heteroalkylene compound, it may be unsubstituted (NH) or optionally substituted with an alkyl group (e.g., (1-4C)alkyl). When a sulfur atom is present in a heteroalkyl compound, it may be S, S(O), or S(O)₂.

[0408] A cycloalkylene group is a divalent saturated hydrocarbon group containing a ring, wherein all ring atoms are carbon atoms, and each of the two free valences forms part of a single bond with an adjacent atom. Cycloalkylene groups can be (5-6C)cycloalkylene groups. In this document, the prefix (e.g., 5-6C) indicates the number or range of ring atoms. Cycloalkylene groups can be monocyclic. Examples of monocyclic cycloalkylene groups include 1,3-cyclopentane and 1,4-cyclohexane.

[0409] Heterocyclic alkylenes (heterocyclic alkenes) are cycloalkylenes in which one or more carbon atoms are substituted with heteroatoms (e.g., N, O, and S), or where one or more carbon atoms have an oxosubstituent (=O). Heterocyclic alkylenes can be C5-6 heterocyclic alkylenes. In this document, the prefix (e.g., 5-6C) indicates the number or range of ring atoms, whether carbon or heteroatoms. Heterocyclic alkylenes can be monocyclic. When a nitrogen atom is present in a heteroalkylene, it can be unsubstituted (NH) or optionally substituted with an alkyl group (e.g., 1-4C alkyl). When a sulfur atom is present in a heteroalkylene, it can be S, S(O), or S(O)₂.

[0410] A arylene is a divalent hydrocarbon group comprising an aromatic ring, wherein all ring atoms are carbon atoms, and each of the two free valences forms part of a single bond with an adjacent atom. Alene groups can be 6-10C arylene groups. In this document, the prefix (e.g., 6-10C) indicates the number or range of ring atoms. Alene groups can be monocyclic, or they can contain two or more rings. Examples of monocyclic arylene groups include 1,4-phenylene. Examples of bicyclic arylene groups include 2,6-naphthylene.

[0411] A heteroarylene is an arylene containing an aromatic ring in which one or more ring atoms are heteroatoms, such as N, O, and S, or where one or more carbon atoms have an oxygen substituent (=O). Heteroarylenes can be 6-10C heteroarylenes. In this document, the prefix (e.g., 6-10C) indicates the number or range of ring atoms (whether carbon or heteroatoms). Heteroarylenes can be monocyclic, or they can contain two or more rings. Examples of monocyclic heteroarylenes include pyrrolemethyl and pyridinylmethyl.

[0412] Suitable linkers include groups selected from alkylene and heteroalkylene groups. More suitable linkers include heteroalkylene groups. Even more suitable linkers include alkylene ether groups. The most suitable linkers include ethylene oxide groups (e.g., derived from polyethylene glycol, PEG).

[0413] Specific compounds of the present invention include, for example, compounds of formula (I) or any subform thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the linker group L and any associated substituents each have any meaning as defined in any of paragraphs (13) to (34) above or below:

[0414] (13) The connector is or contains a group represented by formula (L-Ia) or (L-Ib):

[0415]

[0416] in:

[0417] L1 It is a covalent bond or a (1-6C) alkylene or a (1-6C) heteroalkylene;

[0418] L 2 It is (1-6C) alkylene or (1-6C) heteroalkylene;

[0419] L 3 It is a (1-6C) alkylene;

[0420] n is between 0 and 8;

[0421] * indicates the junction with a non-covalently bonded group (-B); and

[0422] ** is the connection point with the cleavage group (-C).

[0423] (14) The joint defined in paragraph (13), where L 1 It is a covalent bond or a methylene group;

[0424] (15) The joint defined in paragraph (13) or (14), where L 3 It is a (1-4C) alkylene;

[0425] (16) The joint defined in paragraph (13) or (15), where L 3 It is ethylene;

[0426] (17) The joints defined in paragraphs (13) through (16), where n is 0 to 5 or 2 to 5;

[0427] (18) The joints defined in paragraphs (13) through (17), where L 2 Selected from -CH2-X-, -CH2-CH2-X-,

[0428] -CH2-CH2-CH2-X- or -CH2-CH2-CH2-CH2-X-, where X is -O- or -NH-;

[0429] (19) The joints defined in paragraphs (13) through (18), where L 2 Selected from -CH2-X-, -CH2-CH2-X- or -CH2-CH2-CH2-X-, where X is -O- or -NH-;

[0430] (20) The joints defined in paragraphs (13) through (19), where L 2 Selected from -CH2-X- or -CH2-CH2-X-, where X is -O- or -NH-;

[0431] (21) The joints defined in paragraphs (13) through (20), where L 2Selected from -CH2-O-, -CH2-NH-, ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-), and tetramethylene oxide (-CH2CH2CH2CH2O-);

[0432] (22) The joints defined in paragraphs (13) through (21), where L 2 It is ethylene oxide (-CH2CH2O-);

[0433] (23) The connector is or contains a group represented by formula (L-IIa) or (L-IIb):

[0434]

[0435]

[0436] Where L 1 L 3 , n, * and ** are as described in paragraph (13) above for equation (LI), and L 1 Choose any of the options as defined in paragraph (14), L 3 Choose from the options defined in paragraphs (15) or (16), and choose from the options defined in paragraph (17).

[0437] Defined in [the document / reference].

[0438] (24) The connector is or contains a group represented by formula (L-IIIa) or (L-IIIb):

[0439]

[0440] in:

[0441] L 4 It is a (1-6C) alkylene;

[0442] L 5 It is (1-6C) alkylene or (1-6C) heteroalkylene;

[0443] L 6 It is a covalent bond or a (1-2C) alkylene bond;

[0444] m is 1 to 8;

[0445] * indicates the junction with a non-covalently bonded group (-B); and

[0446] ** is the connection point with the cleavage group (-C).

[0447] (25) The joint defined in paragraph (24), where L 6 It is a covalent bond or a methylene group;

[0448] (26) The joint defined in paragraph (24) or (25), where L 4 It is a (1-4C) alkylene;

[0449] (27) The joints defined in paragraphs (24) through (26), where L 4 It is ethylene;

[0450] (28) The joints defined in paragraphs (24) to (27), where m is 0 to 5 or 2 to 5;

[0451] (29) The joints defined in paragraphs (24) to (28), where L 5 Selected from -CH2-X-, -CH2-CH2-X-, -CH2-CH2-CH2-X- or -CH2-CH2-CH2-CH2-X-, where X is -O- or -NH-;

[0452] (30) The joints defined in paragraphs (24) to (29), where L 5 Selected from -CH2-X-, -CH2-CH2-X- or -CH2-CH2-CH2-X-, where X is -O- or -NH-;

[0453] (31) The joints defined in paragraphs (24) through (30), where L 5 Selected from -CH2-X- or -CH2-CH2-X-, where X is -O- or -NH-;

[0454] (32) The joints defined in paragraphs (24) through (31), where L 5 Selected from -CH2-O-, -CH2-NH-, ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-), and tetramethylene oxide (-CH2CH2CH2CH2O-);

[0455] (33) The joints defined in paragraphs (24) through (32), where L 5 It is ethylene oxide (-CH2CH2O-);

[0456] (34) The connector is or contains a group represented by formula (L-IV) or (LV):

[0457]

[0458]

[0459] Where L 4 L 6 、m、* and ** are as described in paragraph (24) above for equation (L-III), and L 4Choose L as defined in paragraphs (26) or (27). 6 Choose as defined in paragraph (25), and choose as defined in paragraph (28).

[0460] Other suitable connectors have the following characteristics:

[0461]

[0462] in:

[0463] L 1 L 2 L 3 n, *, **, L 4 L 5 L 6 and m are each defined as above; and

[0464] X L Selected from -O-, -S-, -SO-, -SO2-, -NH-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-, piperidine, piperazine or triazole.

[0465] Appropriately, L 1 As defined in paragraph (14) above.

[0466] Appropriately, L 3 As defined in paragraphs (15) or (16) above.

[0467] Appropriately, n is as defined in paragraph (17) above.

[0468] Appropriately, L 2 As defined in any of the paragraphs (18) to (22) above.

[0469] Appropriately, L 1 As defined in paragraph (14) above, L 2 As defined in paragraph (18) above, L 3 As defined in paragraph (15) above, n is as defined in paragraph (17) above.

[0470] Appropriately, L 1 As defined in paragraph (14) above, L 2 As defined in paragraph (19) above, L 3 As defined in paragraph (16) above, n is as defined in paragraph (17) above.

[0471] Appropriately, L 1 As defined in paragraph (14) above, L 2 As defined in paragraph (20) above, L3 As defined in paragraph (16) above, n is as defined in paragraph (17) above.

[0472] Appropriately, L 1 As defined in paragraph (14) above, L 2 As defined in paragraph (21) above, L 3 As defined in paragraph (16) above, n is as defined in paragraph (17) above.

[0473] Appropriately, L 1 As defined in paragraph (14) above, L 2 As defined in paragraph (22) above, L 3 As defined in paragraph (16) above, n is as defined in paragraph (17) above.

[0474] Appropriately, L 6 As defined in paragraph (25) above.

[0475] Appropriately, L 4 As defined in paragraphs (26) or (27) above.

[0476] Appropriately, m is as defined in paragraph (28) above.

[0477] Appropriately, L 5 As defined in either paragraph (26) or (27) above.

[0478] Appropriately, L 6 As defined in paragraph (22) above, L 4 As defined in paragraph (24) above, L 5 As defined in paragraph (26) above, m is as defined in paragraph (25) above.

[0479] Appropriately, L 6 As defined in paragraph (22) above, L 4 As defined in paragraph (24) above, L 5 As defined in paragraph (27) above, m is as defined in paragraph (25) above.

[0480] Suitable (1-2C) alkylenes include methylene (methanediyl) and ethylene (ethane-1,2-diyl).

[0481] Suitable (1-6C) alkylenes include methylene (methanediyl), ethylene (ethane-1,2-diyl), propylene (propane-1,3-diyl), butylene (but-1,4-diyl), pentylene (pent-1,5-diyl), and hexylene (hex-1,6-diyl).

[0482] Suitable (1-6C) heteroalkylene groups include alkylene ether groups, such as ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-), and tetramethylene oxide (-CH2CH2CH2CH2O-).

[0483] Appropriately, L 2 It is ethylene oxide. Appropriately, L 4 It is a (1-4C) alkylene group. Most preferably, L 3 It is ethylene.

[0484] Appropriately, L 6 It is methylene or ethylene. Suitable, m is 2 to 5. Suitable, L 5 It is ethylene oxide.

[0485] In one implementation, m or n is 4 to 8, 5 to 7, or 6.

[0486] Non-covalently bonded group (-B)

[0487] The binding groups of the degrading agent include groups capable of binding to target nucleic acid molecules. These binding groups bind to the target nucleic acid molecules via non-covalent bonds.

[0488] Certain small molecule ligands are known to bind nonvalently to nucleic acids, thus forming the basis for the nonvalent binding group. Any small molecule capable of binding nucleic acids can be used as a nonvalent binding group. For example, these compounds can bind to MYC or MALAT-1. Suitablely, the small molecule compound can bind to secondary or tertiary structures within the target nucleic acid. In one embodiment, the nonvalent binding group can target transcribed SNVs and insertion / deletion mutations (e.g., 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 these genetic changes are reflected in their respective mRNAs and are therefore potential targets for the degraders of this invention).

[0489] Furthermore, the non-covalent binding group B can be an oligonucleotide, nanobody, antibody, or antibody fragment capable of binding to the target nucleic acid sequence. The binding of the oligonucleotide binding group B to the target nucleic acid sequence enables targeted cleavage / degradation of the target nucleic acid molecule. The target nucleic acid sequence can be any desired nucleic acid sequence, including sequences associated with specific medical conditions, such as those related to cancer, nucleotide duplication disorders (e.g., Huntington's disease, fragile X chromosome, type 1 myotonic dystrophy), and mRNA sequences encoding non-structural proteins (IAPP in type 2 diabetes).

[0490] In some embodiments, the non-covalently bonded group has a molecular weight of 1,000 kDa or less. For example, the non-covalently bonded group has a molecular weight of 800 kDa or less.

[0491] In some implementations, a non-covalently binding group binds to a secondary or tertiary structure within the target nucleic acid. Suitable secondary or tertiary structures include tetra-strands, pseudo-junctions, triple-strands, tetra-rings, step rings, and hairpin rings. Suitablely, the non-covalently binding group binds to tetra-strands or pseudo-junctions.

[0492] Suitablely, in such an implementation, the non-covalent binding group selectively binds to secondary or tertiary structures in the target nucleic acid. In this case, the non-covalent binding group preferentially binds to secondary or tertiary structures in the target nucleic acid compared to linear or unstructured nucleic acids. Suitablely, the non-covalent binding group selectively binds to quadruplexes or pseudoknots.

[0493] Appropriately, non-covalent binding groups selectively bind to ribonucleic acid (RNA). Therefore, non-covalent binding groups can be called non-covalent RNA binding groups.

[0494] Non-covalent binding groups can bind to target nucleic acids through electrostatic interactions (e.g., ionic interactions, hydrogen bonds, and halogen bonds), van der Waals interactions (e.g., permanent dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions), and π-effects (e.g., π-π interactions, π-cation interactions, and polar-π interactions).

[0495] Non-covalent binding groups can be based on the following small nucleic acid binding molecules:

[0496]

[0497] Non-covalent groups can be attached to the linker at any suitable position. Typically, non-covalent groups are attached to the linker via heteroatoms (such as O or NH) or adjacent to a carbonyl group (C=O).

[0498] Suitablely, the binding group is selected from formula (BI), (B-II), (B-III) or (B-IV).

[0499] Specific compounds of the present invention include, for example, compounds of formula (I) or any subform thereof, or pharmaceutically acceptable salts and / or solvates thereof, wherein, unless otherwise stated, the nonvalently bound group B and any associated substituents have any meaning as defined in any of paragraphs (35) to (39) above or below:

[0500] (35) The binding group B is selected from oligonucleotides, nanobodies, antibodies, antibody fragments or small molecules that can bind to target nucleic acids, or one of the above formulas (BI), (B-II), (B-III) or (B-IV);

[0501] (36) The binding group B is a group of the above formula (BI);

[0502] (37) The binding group B is a group of the above formula (B-II);

[0503] (38) The binding group B is a group of the above formula (B-III);

[0504] (39) The binding group B is a group of the above formula (B-IV).

[0505] Specific implementation plan

[0506] In a specific implementation:

[0507] C is as defined in paragraph (1) above;

[0508] L is as defined in any of paragraphs (13) to (34) above; and B is as defined in paragraph (35) above.

[0509] In a specific implementation:

[0510] C is as defined in paragraph (2) above;

[0511] L is as defined in any of paragraphs (13) to (34) above; and B is as defined in paragraph (35) above.

[0512] In a specific implementation:

[0513] C is as defined in paragraph (2A) above;

[0514] L is as defined in any of paragraphs (13) to (34) above; and B is as defined in paragraph (35) above.

[0515] In a specific implementation:

[0516] C is as defined in paragraph (3) above;

[0517] L is as defined in any of paragraphs (13) to (34) above; and B is as defined in paragraph (35) above.

[0518] In a specific implementation:

[0519] C is as defined in paragraph (3A) above;

[0520] L is as defined in any of paragraphs (13) to (34) above; and B is as defined in paragraph (35) above.

[0521] In a specific implementation:

[0522] C is as defined in paragraph (4) above;

[0523] L is defined as in any of the paragraphs (13) to (34) above; and

[0524] B is as defined in paragraph (35) above.

[0525] In a specific implementation:

[0526] C is as defined in paragraph (4A) above;

[0527] L is defined as in any of the paragraphs (13) to (34) above; and

[0528] B is as defined in paragraph (35) above.

[0529] In a specific implementation:

[0530] C is as defined in paragraph (5) above;

[0531] L is defined as in any of the paragraphs (13) to (34) above; and

[0532] B is as defined in paragraph (35) above.

[0533] In a specific implementation:

[0534] C is as defined in paragraph (6) above;

[0535] L is defined as in any of the paragraphs (13) to (34) above; and

[0536] B is as defined in paragraph (35) above.

[0537] In a specific implementation:

[0538] C is as defined in paragraph (7) above;

[0539] L is defined as in any of the paragraphs (13) to (34) above; and

[0540] B is as defined in paragraph (35) above.

[0541] In a specific implementation:

[0542] C is as defined in paragraph (8) above;

[0543] L is defined as in any of the paragraphs (13) to (34) above; and

[0544] B is as defined in paragraph (35) above.

[0545] In a specific implementation:

[0546] C is as defined in paragraph (9) above;

[0547] L is defined as in any of the paragraphs (13) to (34) above; and

[0548] B is as defined in paragraph (35) above.

[0549] In a specific implementation:

[0550] C is as defined in paragraph (10) above;

[0551] L is defined as in any of the paragraphs (13) to (34) above; and

[0552] B is as defined in paragraph (35) above.

[0553] In a specific implementation:

[0554] C is as defined in paragraph (11) above;

[0555] L is defined as in any of the paragraphs (13) to (34) above; and

[0556] B is as defined in paragraph (35) above.

[0557] In a specific implementation:

[0558] C is as defined in paragraph (12) above;

[0559] L is defined as in any of the paragraphs (13) to (34) above; and

[0560] B is as defined in paragraph (35) above.

[0561] In a particular embodiment, B is a pyridine statidine binding group, meaning the compound has the following formula (II):

[0562]

[0563] L and C are defined as above.

[0564] In a particular embodiment, B is an MTBD-binding group, meaning the compound has the following formula (III):

[0565]

[0566] L, X, and C are each defined as above.

[0567] In a particular embodiment, B is a chloramphenicol binding group, meaning the compound has the following formula (IV):

[0568]

[0570] L and C are defined as above.

[0571] In a particular embodiment, B is a lincomycin binding group, meaning the compound has the following formula (V):

[0572]

[0573] L and C are defined as above.

[0574] Suitable, in compounds of formula (II), (III), (IV) or (V):

[0575] The cleavage group C is as defined in any of paragraphs (1) to (12) above (including 2A, 3A, and 4A); and

[0576] L is defined as in any of the paragraphs (13) to (34) above.

[0577] In specific compounds of formula (II), (III), (IV), or (V):

[0578] The cleavage group C is as defined in paragraph (1) above; and

[0579] L is defined as in any of the paragraphs (13) to (34) above.

[0580] In specific compounds of formula (II), (III), (IV), or (V):

[0581] The cleavage group C is as defined in paragraph (2) above; and

[0582] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0583] The cleavage group C is as defined in paragraph (2A) above; and

[0584] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0585] The cleavage group C is as defined in paragraph (3) above; and

[0586] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0587] The cleavage group C is as defined in paragraph (4A) above; and

[0588] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0589] The cleavage group C is as defined in paragraph (4) above; and

[0590] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0591] The cleavage group C is as defined in paragraph (4A) above; and

[0592] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0593] The cleavage group C is as defined in paragraph (5) above; and

[0594] L is as defined in any of paragraphs (13) to (34) above. In specific compounds of formula (II), (III), (IV) or (V):

[0595] The cleavage group C is as defined in paragraph (6) above; and

[0596] L is defined as in any of the paragraphs (13) to (34) above.

[0597] In specific compounds of formula (II), (III), (IV), or (V):

[0598] The cleavage group C is as defined in paragraph (7) above; and

[0599] L is defined as in any of the paragraphs (13) to (34) above.

[0600] In specific compounds of formula (II), (III), (IV), or (V):

[0601] The cleavage group C is as defined in paragraph (8) above; and

[0602] L is defined as in any of the paragraphs (13) to (34) above.

[0603] In specific compounds of formula (II), (III), (IV), or (V):

[0604] The cleavage group C is as defined in paragraph (9) above; and

[0605] L is defined as in any of the paragraphs (13) to (34) above.

[0606] In specific compounds of formula (II), (III), (IV), or (V):

[0607] The cleavage group C is as defined in paragraph (10) above; and

[0608] L is defined as in any of the paragraphs (13) to (34) above.

[0609] In specific compounds of formula (II), (III), (IV), or (V):

[0610] The cleavage group C is as defined in paragraph (11) above; and

[0611] L is defined as in any of the paragraphs (13) to (34) above.

[0612] In specific compounds of formula (II), (III), (IV), or (V):

[0613] The cleavage group C is as defined in paragraph (12) above; and

[0614] L is defined as in any of the paragraphs (13) to (34) above.

[0615] Specific compounds of the present invention include compounds of formula VI as shown below, or pharmaceutically acceptable salts thereof:

[0616]

[0617] Where C is as defined above, or is selected from one of the following:

[0618]

[0619] Dynamic characteristics

[0620] The interaction between the degrader and the target nucleic acid can be quantified using the dissociation constant (kD). The dissociation constant between the degrader containing a given non-covalently binding group and the nucleic acid can be known or determined using standard techniques such as surface plasmon resonance (SPR), for example, Biacore (Santos et al., 2021). Suitable systems for measuring the dissociation constant include the Biacore T200.

[0621] Typically, the degrading agent binds to the target nucleic acid with a dissociation constant (kD) of 10,000 nM or less, as determined, for example, by SPR. Suitably, the degrading agent binds to the 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.

[0622] As described above, the non-covalent binding groups of the degrading agent typically bind to secondary or tertiary structures in the target nucleic acid. Therefore, the degrading agent typically binds to secondary or tertiary structures with a dissociation constant (kD) of 10,000 nM or less, as determined by SPR. Suitablely, the degrading agent binds to secondary or tertiary structures with a kD of 1,000 nM or less, more suitablely 500 nM or less, even more suitablely 200 nM or less, and most suitablely 100 nM or less.

[0623] In some embodiments, the degrading agent binds to the tetrachain with a dissociation constant (kD) of 10,000 nM or less, as determined by SPR. In this case, the degrading agent suitably binds to the tetrachain with a kD of 1,000 nM or less, more suitably 500 nM or less, even more suitably 200 nM or less, and most suitably 100 nM or less.

[0624] In some embodiments, the degrading agent binds to the pseudojunction with a dissociation constant (kD) of 10,000 nM or less, as determined by SPR. In this case, the degrading agent suitably binds to the pseudojunction with a kD of 1,000 nM or less, more suitably 500 nM or less, even more suitably 200 nM or less, and most suitably 100 nM or less.

[0625] As described above, the non-covalent binding groups of the degrading agent selectively bind to secondary or tertiary structures in the target nucleic acid. Binding selectivity can be quantified using the ratio between the dissociation constant for binding a given compressed secondary or tertiary structure and the dissociation constant for binding a linear or unstructured nucleic acid (e.g., linear or unstructured RNA). Typically, a comparative linear or unstructured nucleic acid is prepared by mutating one or more residues in the secondary or tertiary structure of interest, such that the secondary or tertiary structure no longer forms while preserving the remainder of the sequence. For example, the selectivity for binding to the RNA G quadruplex can be assessed using comparative RNA in which one or more GGG motifs are replaced with AUC motifs.

[0626] Typically, the binding selectivity between a given secondary or tertiary structure and a linear or unstructured nucleic acid is 5:1 or higher. Suitablely, the selectivity between a given secondary or tertiary structure and a linear or unstructured nucleic acid is 10:1 or higher, more suitablely 20:1 or higher, even more suitablely 50:1 or higher, and most suitablely 100:1 or higher.

[0627] In one implementation, the binding selectivity between the quadruplex and the linear or unstructured nucleic acid is 5:1 or higher. Suitablely, the selectivity between the quadruplex and the linear or unstructured nucleic acid is 10:1 or higher, more suitablely 20:1 or higher, even more suitablely 50:1 or higher, and most suitablely 100:1 or higher.

[0628] In one implementation, the binding selectivity between the quadruplex and the linear or unstructured nucleic acid is 5:1 or higher. Suitablely, the selectivity between the quadruplex and the linear or unstructured nucleic acid is 10:1 or higher, more suitablely 20:1 or higher, even more suitablely 50:1 or higher nM or less, and most suitablely 100:1 or higher.

[0629] Salts and solvates

[0630] The degradation agent of the present invention can be provided in the form of free alkali.

[0631] The degrading agent of the present invention can be provided in the form of a salt, preferably a pharmaceutically acceptable salt.

[0632] In some implementations, the degrading agents disclosed herein can be provided as protonated salts together with suitable counter anions.

[0633] Suitable counterions include both organic and inorganic anions. Examples of inorganic anions include those derived from inorganic acids, including chloride (Cl-), bromide (Br-), iodide (I-), sulfate (SO4-), sulfite (SO3-), nitrate (NO3-), nitrite (NO2-), phosphate (PO4-), and phosphite (PO3-). Examples of organic anions include 2-acetoxybenzoate, acetate, ascorbate, aspartate, benzoate, camphor sulfonate, cinnamate, citrate, ethylenediaminetetraacetate, ethanesulfonate, ethanesulfonate, formate, fumarate, gluconate, glutamate, glycolate, hydroxymalate, carboxylic acid ester, lactate, laurate, lactate, maleate, malate, methanesulfonate, oleate, oxalate, palmitate, phenylacetate, benzenesulfonate, propionate, pyruvate, salicylate, stearate, succinate, sulfanilate, tartrate, toluenesulfonate, and valerate. Suitable examples of polymeric organic anions include those derived from tannins and carboxymethyl cellulose.

[0634] In some implementations, the degrading agents disclosed herein can be provided as deprotonated salts together with suitable countercations.

[0635] Suitable counterions include both inorganic and organic cations. Examples of suitable inorganic cations include alkali metal ions such as Na+.+ and K + Alkaline earth metal cations such as Ca 2+ and Mg 2+ and other cations such as NH4 + Or Al 3+ Suitable examples of organic cations include substituted ammonium ions (e.g., NH3R). + NH2R2 + NHR3 + NR4 + Examples of substituted ammonium ions include those derived from 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. A common example of a quaternary ammonium ion is N(CH3)4. + .

[0636] The degrading agents of the present invention can be provided in the form of solvates (complexes of solute (e.g., compounds, salts of compounds) and solvents). Examples of solvates include hydrates, such as monohydrates, dihydrates, and trihydrates.

[0637] The degradation agent of the present invention can be provided in a desolventized form, such as a dehydrated form.

[0638] Compounds with the same molecular formula but different atomic bonding properties, sequences, or spatial arrangements are called "isomers." Isomers with different spatial atomic arrangements are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," while those that are non-overlapping mirror images of each other are called "enantiomers." When the degrading agent of formula (I) has an asymmetric center, for example, 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 described by the R- and S-order rules of the Cahn-Ingold-Prelog, or by the way in which the molecule rotates the plane of polarization and is designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture."

[0639] The compounds of this invention may have one or more asymmetric centers; therefore, such compounds may be produced as individual (R) or (S) stereoisomers or mixtures thereof. Unless otherwise stated, the description or naming of particular compounds in the specification and claims is intended to include their individual enantiomers and mixtures thereof, racemic or other forms. Methods for determining stereochemistry and isolating stereoisomers are well known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry," 4th edition, J. March, John Wiley and Sons, New York, 2001), for example, by synthesis from optically active raw materials or by resolving racemic forms. Some compounds of this invention may have geometric isomer centers (E and Z isomers). It should be understood that this invention includes all optical isomers, diastereomers, and geometric isomers and mixtures thereof possessing antiproliferative activity.

[0640] This invention also includes compounds of the invention as defined herein, which contain one or more isotopic substitutions. For example, H can be any isotopic form, including... 1 H, 2 H(D) and 3 H(T); C can be any isotopic form, including 12 C 13 C and 14 C; and O can be any isotopic form, including 16 O and 18 O etc.

[0641] It should also be understood that some of the compounds of this invention can exist in solvated and non-solvated forms, such as hydrated forms. It should be understood that this invention includes all such solvated forms possessing antiproliferative activity.

[0642] It should also be understood that some of the compounds of the present invention may exhibit polymorphism, and the present invention includes all of these forms having antiproliferative activity.

[0643] The compounds of the present invention can exist in many different tautomeric forms, and all such forms are included when referring to the compounds of the present invention. For the avoidance of doubt, when the degrading agents of the present invention can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are included in the present invention. Examples of tautomeric forms include ketone-, enol-, and enolide-forms, such as the following tautomeric pairs: ketone / enol (as shown below), imine / enamine, amide / imino alcohol, amidine / amidinium, nitroso / oxime, thionone / enthiol, and nitro / acid-nitro.

[0644]

[0645] Compounds of the present invention containing amine functional groups may also form nitrogen oxides. The degrading agents of the present invention containing amine functional groups mentioned herein also include nitrogen oxides. When the degrading agents of the present invention contain several amine functional groups, one or more nitrogen atoms can be oxidized to form N-oxides. Specific examples of N-oxides are 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 (e.g., hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid)), see, for example, Jerry March's *Advanced Organic Chemistry*, 4th edition, Wiley Interscience, pages. More specifically, N-oxides can be prepared by the procedure of LWDeady (Syn. Comm. 1977, 7, 509-514), wherein an amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA) in, for example, an inert solvent (e.g., dichloromethane).

[0646] The compounds of the present invention can be administered as prodrugs, which decompose in the human or animal body to release the degrading agents of the present invention. Prodrugs can be used to modify the physical properties and / or pharmacokinetic properties of the degrading agents of the present invention. Prodrugs can be formed when the compounds of the present invention contain suitable groups or substituents, and the modifying groups can be attached to these groups or substituents. Examples of prodrugs include in vivo cleavable ester derivatives that can be formed from the carboxyl or hydroxyl groups of the degrading agents of the present invention, and in vivo cleavable amide derivatives that can be formed from the carboxyl or amino groups of the compounds of the present invention.

[0647] Therefore, this invention includes those compounds of the invention as defined above, when obtained by organic synthesis, and when obtained in humans or animals by cleavage of their prodrugs. Thus, this invention includes those compounds of the invention produced by organic synthesis methods, and such compounds produced in humans or animals by the metabolism of precursor compounds, i.e., the degradative agents of the invention, which can be synthetically produced compounds or metabolically produced compounds.

[0648] The suitable pharmaceutically acceptable prodrug of the degradation agent of the present invention is a drug based on reasonable medical judgment that is suitable for administration to humans or animals without undesirable pharmacological activity and excessive toxicity.

[0649] Various forms of prodrugs have been described, for example in the following documents:

[0650] a) Methods in Enzymology ,Vol. 42 , p.309-396, edited by K. Widder, et al. (Academic Press, 1985);

[0651] b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);

[0652] 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);

[0653] d)H.Bundgaard, Advanced Drug Delivery Reviews , 8 ,1-38(1992);

[0654] e)H.Bundgaard, et al., Journal of Pharmaceutical Sciences , 77 ,285(1988);

[0655] f)N.Kakeya, et al., Chem.Pharm.Bull. , 32 ,692(1984);

[0656] g)T.Higuchi and V.Stella, "Pro-Drugs as Novel Delivery Systems", ACSSymposium Series, Volume 14; and

[0657] h) E. Roche (editor), "Bioreversible Carriers in Drug Design", Pergamon Press, 1987.

[0658] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention containing a carboxyl group are, for example, esters that are cleavable in vivo. Cheavable esters of the degrading agents of the present invention containing a carboxyl group are, for example, pharmaceutically acceptable esters that, upon cleavage in humans or animals, produce a parent acid. Suitable pharmaceutically acceptable esters containing a carboxyl group include C... 1-6 Alkyl esters, such as methyl, ethyl, and tert-butyl, C 1-6 Alkoxymethyl esters, such as methoxymethyl esters, C 1-6 Alkyloxymethyl esters, such as neopentyloxymethyl ester and 3-phthalidyl ester, C3-8 Cycloalkylcarbonyloxy-C 1-6 Alkyl esters, such as cyclopentylcarbonyloxymethyl ester and 1-cyclohexylcarbonyloxyethyl ester, 2-oxo-1,3-dioxolenylmethyl ester, such as 5-methyl-2-oxo-1,3-dioxolenyl-4-yl ester, and C 1-6 Alkoxycarbonyloxy-C 1-6 Alkyl esters, such as methoxycarbonyloxymethyl ester and 1-methoxycarbonyloxyethyl ester.

[0659] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention containing a hydroxyl group are, for example, esters or ethers that are cleavable in vivo. Cleavable esters or ethers of the degrading agents of the present invention containing a hydroxyl group are, for example, pharmaceutically acceptable esters or ethers that, upon cleavage in a human or animal body, produce a parent hydroxyl compound. Suitable pharmaceutically acceptable esterifying groups of the hydroxyl group include inorganic esters, such as phosphate esters (including cyclic phosphoramide esters). Other suitable pharmaceutically acceptable esterifying groups of the hydroxyl group include C... 1-10 Alkyl 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. Examples of cyclic substituents on the phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(C 1-4 Alkyl)piperazine-1-ylmethyl. Suitable pharmaceutically acceptable etherifying groups of the hydroxyl group include α-acyloxyalkyl, such as acetoxymethyl and neopentyloxymethyl.

[0660] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention having a carboxyl group are, for example, amides that are cleavable in vivo, such as amides formed with amines, such as ammonia, C1-4 alkylamines such as methylamine, (C1-4 alkyl)2amines such as dimethylamine, N-ethyl-N-methylamine or diethylamine, C1-4 alkoxy-C 2-4 Alkylamines, such as 2-methoxyethylamine, phenyl-C 1-4 Alkylamines, such as benzylamine, and amino acids, such as glycine or its esters.

[0661] Suitable pharmaceutically acceptable prodrugs of the degrading agents of the present invention having an amino group are, for example, amide derivatives that are cleavable in vivo. Suitable pharmaceutically acceptable amides of the amino group include, for example, those derived from C 1-10 Amides formed from alkanoyl groups, wherein the C 1-10Alkyl groups include acetyl, benzoyl, phenylacetyl, and substituted benzoyl and phenylacetyl groups. Examples of cyclic substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(C 1-4 Alkyl)piperazine-1-ylmethyl.

[0662] The in vivo action of the degrading agent of the present invention can be partially exerted through one or more metabolites formed in the human or animal body after administration of the degrading agent of Formula I. As described above, the in vivo action of the degrading agent of the present invention can also be exerted through the metabolism of the precursor compound (prodrug).

[0663] While the invention may relate to any compound or particular group of compounds as defined herein by optional, preferred or suitable features or by particular embodiments, the invention may also relate to any compound or particular group of compounds that explicitly excludes the optional, preferred or suitable features or particular embodiments.

[0664] synthesis

[0665] The compounds of the present invention can be prepared by any suitable technique known in the art. Specific methods for preparing these compounds are further described in the appended examples.

[0666] In the description of the synthetic methods described herein and in any reference synthetic methods used to prepare the raw materials, it should be understood that those skilled in the art can choose all the proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, experimental duration, and post-treatment procedures.

[0667] Those skilled in the field of organic synthesis can understand that the functionality present in different parts of a molecule must be adapted to the reagents and reaction conditions used.

[0668] It should be understood that during the synthesis of the compounds of this invention using the methods defined herein, or during the synthesis of certain starting materials, it may be necessary to protect certain substituents to prevent them from undergoing undesirable reactions. A skilled chemist will understand when such protection is needed and how to place such protecting groups in the appropriate positions before removing them.

[0669] For examples of protecting groups, see one of the many general texts on the subject, such as Theodora Green's *Protective Groups in Organic Synthesis* (published by John Wiley & Sons). Protecting groups can be removed by any convenient method described in the literature or known to a skilled chemist suitable for removing the protecting group in question, chosen so as to achieve the removal of the protecting group with minimal interference to other groups in the molecule.

[0670] Therefore, if the reactants include groups such as amino, carboxyl, or hydroxyl, it may be necessary to protect these groups in some of the reactions mentioned herein.

[0671] For example, suitable protecting groups for amino or alkylamino groups are, for example, acyl groups, such as acetyl groups; alkoxycarbonyl groups, such as methoxycarbonyl, ethoxycarbonyl, or tert-butyloxycarbonyl groups; arylmethoxycarbonyl groups, such as benzyloxycarbonyl groups; or aromatic acyl groups, such as benzoyl groups. The deprotection conditions for these protecting groups necessarily vary depending on the choice of protecting group. Thus, for example, acyl groups such as alkanoyl, alkoxycarbonyl, or aromatic acyl groups can be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide such as lithium hydroxide or sodium hydroxide. Alternatively, acyl groups such as tert-butyloxycarbonyl groups can be removed, for example, by treatment with a suitable acid such as hydrochloric acid, sulfuric acid, or phosphoric acid or trifluoroacetic acid; and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups can be removed, for example, by hydrogenation on a catalyst such as a carbon-supported palladium, or by treatment with a Lewis acid such as tri(trifluoroacetic acid)borane. Suitable alternative protecting groups for primary amino groups are, for example, phthalyl groups, which can be removed by treatment with an alkylamine, such as dimethylaminopropylamine, or with hydrazine.

[0672] Suitable protecting groups for the hydroxyl group are, for example, acyl groups, such as alkanoyl groups like acetyl, aromatic acyl groups like benzoyl, or arylmethyl groups like benzyl. The deprotection conditions for these protecting groups will necessarily vary depending on the choice of protecting group. Therefore, for example, acyl groups like alkanoyl or aromatic acyl groups can be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide like lithium hydroxide, sodium hydroxide, or ammonia. Alternatively, arylmethyl groups like benzyl can be removed, for example, by hydrogenation on a catalyst such as a carbon-supported palladium.

[0673] Suitable protecting groups for the carboxyl group are, for example, esterification groups, such as methyl or ethyl groups that can be removed by hydrolysis with a base such as sodium hydroxide, or tert-butyl groups that can be removed by treatment with an acid such as an organic acid such as trifluoroacetic acid, or benzyl groups that can be removed by hydrogenation on a catalyst such as a carbon-supported palladium.

[0674] Resins can also be used as protecting groups.

[0675] The method used to synthesize the degrading agents of the present invention will vary depending on the nature of any substituents associated with them. Suitable methods for preparing them are further described in the accompanying examples.

[0676] Once the degrading agent of the present invention has been synthesized by any of the methods defined herein, the method may further include the following additional steps:

[0677] (i) Optionally remove any existing protecting groups;

[0678] (ii) Optionally, the compounds of the present invention may be converted into another compound of the present invention;

[0679] (iii) Optionally forming a pharmaceutically acceptable salt, hydrate, or solvate; and / or

[0680] (iv) Optionally form its prodrug.

[0681] An example of (ii) above is that when the degrading agent of the present invention is synthesized, one or more groups can then be further reacted to change the properties of the group and provide an alternative compound of the present invention.

[0682] The compounds obtained in this invention can be separated and purified using techniques well known in the art.

[0683] Methods for cleaving target nucleic acids

[0684] This invention provides a method for cleaving target nucleic acid molecules. The method includes:

[0685] Contact the target nucleic acid molecule with the degrading agent of formula (I):

[0686] CLB(I)

[0687] As defined in this paper (where -C is a cleavage group, -L- is a linker, and -B is a non-covalently binding group), the degradative agent non-covalently binds to the target nucleic acid molecule; and

[0688] The degrading agent is allowed to cleave the target nucleic acid molecule it binds to.

[0689] The specific implementation of the degrading agent of formula (I) is as described above.

[0690] In some implementations, the target nucleic acid molecules can come into contact with a degrading agent in solution.

[0691] More appropriately, the target nucleic acid molecule can come into contact with an intracellular (i.e., intracellular) degrading agent. The cell can be in vitro and can be an isolated cell, such as an isolated cell line or a cell isolated from an individual (from a tissue sample, such as a biopsy).

[0692] Suitable cells may include mammalian cells, preferably human cells. Cells may include somatic cells and germline cells, and may be at any stage of development, including fully or partially differentiated cells or undifferentiated or pluripotent cells, including stem cells such as adult stem cells or somatic stem cells, fetal stem cells or embryonic stem cells. For example, cells may include nerve cells, including neurons and glial cells, contractile muscle cells, smooth muscle cells, hepatocytes, hormone-synthesizing cells, sebaceous gland cells, pancreatic islet cells, adrenal cortical cells, fibroblasts, keratinocytes, endothelial cells and urothelial cells, osteoblasts and chondrocytes. In some embodiments, cells may be associated with disease conditions, such as cancer cells, such as carcinoma, sarcoma, lymphoma, blastoma, or germline tumor cells, and cells with genotypes of hereditary diseases, such as Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome, or Marfan syndrome.

[0693] The target nucleic acid molecule can be an endogenous nucleic acid present in the cell. The degrading agent can be an exogenous molecule. One approach may include introducing the degrading agent into the cell and causing it to bind to the target nucleic acid molecule.

[0694] Target nucleic acid molecules can be DNA or RNA molecules. Suitable target RNA molecules can include mRNA and long non-coding RNA (lncRNA). RNA molecules can contain introns and intergenic regions.

[0695] The target nucleic acid molecule may contain secondary or tertiary structures. Suitable secondary and tertiary structures include quadruplexes, pseudoknots, tetraloops, step rings, and hairpin rings. Preferably, the target nucleic acid molecule contains a quadruplex or a pseudoknot.

[0696] For example, methods for cleaving target nucleic acids containing secondary or tertiary structures may include:

[0697] Contact the target nucleic acid molecule with the degrading agent of formula (I):

[0698] CLB(I)

[0699] As defined herein, -C is a cleavage group, -L- is a linker, and -B is a non-covalently binding group that interacts with secondary or tertiary structures to non-covalently bind the degradative agent to the target nucleic acid molecule; and

[0700] The degrading agent is allowed to cleave the target nucleic acid molecule it binds to.

[0701] In one specific implementation, the secondary or tertiary structure is a quadruple chain. In this case, the method may include:

[0702] Contact the target nucleic acid molecule containing the tetrastrand with the degrading agent of formula (I):

[0703] CLB (I)

[0704] As defined in this paper, -C is a cleavage group, -L- is a linker, and -B is a non-covalent binding group that interacts with the tetramer to non-covalently bind the degrading agent to the target nucleic acid molecule;

[0705] and

[0706] The degrading agent is allowed to cleave the target nucleic acid molecule it binds to.

[0707] In one particular implementation, the secondary or tertiary structure is a pseudo-knot. In this case, the method may include:

[0708] Contact the target nucleic acid molecule containing the pseudoknot with the degrading agent of formula (I):

[0709] CLB(I)

[0710] As defined herein, -C is a cleavage group, -L- is a linker, and -B is a non-covalently binding group that interacts with the pseudojunction to non-covalently bind the degradative agent to the target nucleic acid molecule; and

[0711] The degrading agent is allowed to cleave the target nucleic acid molecule it binds to.

[0712] When non-covalently bound to the target nucleic acid molecule, the degrading agent cleaves the target nucleic acid. Without being bound by any particular theory, the degrading agent is understood to cleave one or more phosphodiester bonds in the target nucleic acid.

[0713] When non-covalently bound to a target nucleic acid molecule, the cleavage group can abstract a proton from the 2'OH group of the nucleotide in the target nucleic acid. Cleavage of the phosphodiester backbone can occur through intramolecular attack on the 3' phosphate group.

[0714] When non-covalently bound to the target nucleic acid molecule, the cleavage group can bind to one or more transition metals (e.g., copper). The degrading agent can cleave the target nucleic acid through copper-mediated nucleic acid degradation.

[0715] The binding of the degrading agent to the target nucleic acid can occur via an intermediate. That is, the target nucleic acid molecule can be cleaved, as described herein, by a method including binding the target nucleic acid molecule to the degrading agent, to produce an intermediate having the following formula:

[0716] CLB~NA

[0717] Where -C is the cleavage group as defined in this paper, -L- is the linker as defined in this paper, -B is the non-covalent binding group as defined in this paper, ~ is the non-covalent interaction, NA is the target nucleic acid, and allows the degrading agent to cleave the target nucleic acid molecule.

[0718] The specific implementation of the degrading agent of formula (I) is as described above.

[0719] Methods for identifying secondary or tertiary structures

[0720] After selectively cleaving the target nucleic acid molecule using a degrading agent as described above, one approach may include identifying the target nucleic acid molecule. This can be used, for example, to map sites in nucleic acids that contain secondary or tertiary structures.

[0721] The method may also include determining the abundance or amount of one or more nucleic acid molecules in a nucleic acid population. A decrease in the abundance or amount of nucleic acid molecules in the population relative to a control indicates that the nucleic acid molecule is a target nucleic acid molecule that has been selectively cleaved by the degrading agent. A suitable control could be a nucleic acid population that has not been treated with the degrading agent.

[0722] Therefore, the present invention provides a method for identifying secondary or tertiary structures in target nucleic acid molecules, the method comprising:

[0723] First and second populations of nucleic acid molecules are provided, each population containing the target nucleic acid molecule;

[0724] A degrading agent of formula (I) is introduced into the first group of nucleic acid molecules: CLB(I), wherein -C is a cleavage group as defined herein, -L- is a linker as defined herein, and -B is a non-covalent binding group as defined herein, such that the degrading agent non-covalently binds to the target nucleic acid molecule;

[0725] Allowing the degrading agent to cleave target nucleic acid molecules present in the first group; and identifying nucleic acid molecules present in the first group in a reduced amount relative to the second group.

[0726] The specific implementation of the degrading agent of formula (I) is as described above.

[0727] Non-covalent binding groups can bind to secondary or tertiary structures in target nucleic acid molecules. Suitable secondary or tertiary structures include tetra-strands, pseudo-junctions, tetra-rings, step rings, and hairpin rings. Preferably, the secondary or tertiary structure is a tetra-strand or a pseudo-junction.

[0728] The first and second nucleic acid molecule populations can be independently isolated (ex vivo) nucleic acid molecule populations. Alternatively, one or more nucleic acid molecule populations can be present within the cell.

[0729] This method may include extracting total nucleic acids (e.g., total DNA or total RNA) from cells. The nucleic acids can be further analyzed, for example, to determine the abundance or quantity of one or more nucleic acid molecules. For instance, the extracted total nucleic acids can be sequenced, and the sequence reads can be analyzed.

[0730] Appropriate 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 count) for each extracted nucleic acid molecule to determine the abundance or quantity of each nucleic acid molecule in the cell. In some embodiments, the raw read count may be normalized and expressed as RPKM (reads per thousand bases per million exon models) or FPKM (fragments per thousand bases per million exon models). Suitable sequencing and sequence analysis methods have been established in the art.

[0731] Applications in medicine

[0732] The selective cleavage of target nucleic acid molecules by the aforementioned degrading agents can alter the downstream effects of the target nucleic acid molecules. For example, this can be used to treat or prevent diseases mediated by target nucleic acid molecules.

[0733] Therefore, the present invention provides a degrading agent as shown in formula (I) for use in a method of treating a human or animal body by means of a therapy, for example, a method of treating a condition (e.g., a specific disease).

[0734] Another aspect of the invention relates to a treatment method, such as a method of treating a symptom (e.g., a disease), comprising administering a therapeutically effective amount of a degrading agent of formula (I) to a subject requiring treatment.

[0735] Another aspect of the invention relates to the use of the degrading agent of formula (I) in the preparation of a medicament for treating a condition (e.g., a disease). Typically, the medicament comprises a degrading agent of formula (I).

[0736] Treatment of conditions (i) proliferative conditions (e.g., cancer)

[0737] According to another aspect of the invention, a method for inhibiting cell proliferation in vitro or in vivo is provided, the method comprising contacting cells with an effective amount of a degrading agent of formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0738] According to another aspect of the invention, a method for treating proliferative diseases in a patient requiring such treatment is provided, the method comprising administering to the patient a therapeutically effective amount of a degrading agent of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0739] According to another aspect of the invention, a method of treating cancer in a patient requiring such treatment is provided, the method comprising administering to the patient a therapeutically effective amount of a degrading agent of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein.

[0740] According to another aspect of the invention, a degrading agent of formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as defined herein, is provided for the treatment of proliferative conditions.

[0741] According to another aspect of the invention, a degrading agent of formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, or a pharmaceutical composition thereof, is provided for the treatment of cancer. In a specific embodiment, the cancer is a human cancer.

[0742] According to another aspect of the invention, the use of a degrading agent of formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in the preparation of a medicament for treating proliferative conditions is provided.

[0743] According to another aspect of the invention, the use of a degrading agent of formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate, or solvation thereof, in the preparation of a medicament for treating cancer is provided. Suitably, the cancer is a human cancer.

[0744] Optionally, the compound or pharmaceutical composition may be administered in combination with one or more other antiproliferative agents (e.g., checkpoint inhibitors and / or cytotoxic agents).

[0745] As used herein, the term "proliferative disorder" refers to unwanted or uncontrolled cell proliferation, such as tumors or proliferative growths, whether in vitro or in vivo, involving an unwanted excess or abnormal number of cells. Examples of proliferative conditions include, but are not limited to, pre-malignant and malignant cell proliferation, including but not limited to malignant growths and tumors, cancer, leukemia, psoriasis, bone diseases, fibroproliferative disorders (e.g., connective tissue), and atherosclerosis. It can involve any type of cell, including but not limited to those from the lungs, colon, breast, ovary, prostate, liver, pancreas, brain, and skin.

[0746] When the proliferative disorder is cancer, the cancer may be selected from adenoid cystic carcinoma, adrenal carcinoma, amyloidosis, anal cancer, ataxia-telangiectasia, atypical hydatidiform mole, basal cell carcinoma, cholangiocarcinoma, Birt-Hogg-Dube 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 lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult leukemia, childhood leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), liver cancer, lobular carcinoma, non-small cell lung cancer, small cell lung cancer, Hodgkin 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 carcinoma, 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 nephroblastoma.

[0747] In one embodiment, the degrader of Formula I can target SNVs and insertion / deletion mutations 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 and ovarian cancer. All these genetic changes are reflected in their respective mRNAs, thus the degrader of the present invention has potential targeting capabilities.

[0748] Specific cancers of interest include Ewing sarcoma (by targeting ESWR1, for example with a non-covalently bound group of pyridostazine (PDS)) and lung, breast, cervical, and colon cancers (by targeting MALAT1 with a non-covalently bound group of MALAT1).

[0749] (ii) Bacterial and viral infections

[0750] In one implementation (e.g., for a treatment method, for the manufacture of a drug for a treatment method), the treatment is a treatment for a bacterial or viral infection.

[0751] Specifically, viral infection refers to RNA virus infection (e.g., viruses whose genomes contain single-stranded or double-stranded RNA). Many pathogenic viruses utilize -1 ribosome frameshifting as a mechanism for correct protein translation, a phenomenon facilitated by secondary RNA structures such as stem loops and pseudoknots. Therefore, targeting these secondary RNA structures with a degrading agent of formula (I) can cleave and inactivate viral RNA, and treat viral infections.

[0752] Examples of RNA viruses include (+)ssRNA viruses, such as coronaviruses, picornaviruses, and tunica albugineas; (-)ssRNA viruses, such as orthomyxoviruses and rhabdoviruses; and dsRNA viruses, such as reoviruses.

[0753] Preferably, the virus is a (+)ssRNA virus, more preferably a coronavirus. Examples of coronaviruses include alpha coronaviruses, such as porcine transmissible gastroenteritis virus, feline coronavirus, and canine coronavirus; beta coronaviruses, such as Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), mouse coronavirus (M-CoV), and severe acute respiratory syndrome-associated coronavirus (SARS-CoV, novel coronavirus); gamma coronaviruses, such as avian coronavirus; and delta coronaviruses, such as white-rumped swift coronavirus HKU11 and porcine coronavirus HKU15.

[0754] Bacterial infections can be caused by Gram-negative or Gram-positive bacteria. Both types of bacteria contain bacterial ribosomes, which are ribozymes containing protein and RNA units. Therefore, targeting the RNA units with a degrading agent of formula (I) can cleave and inactivate bacterial ribosomes, thus treating the bacterial infection.

[0755] Examples of medically relevant Gram-negative bacteria include Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, and Pseudomonas aeruginosa (primarily associated with respiratory problems); Escherichia coli and Enterobacter cloacae (primarily associated with urinary tract problems); Helicobacter pylori and Salmonella enterica (primarily associated with gastrointestinal problems); and Neisseria meningitidis (primarily associated with meningitis).

[0756] Therefore, in one implementation, the Gram-negative bacterial species are selected from the group consisting of Escherichia coli, Enterobacter cloacae, Helicobacter pylori, Streptococcus enterica, Haemophilus influenzae, Klebsiella pneumoniae, Lactobacillus pneumoniae, Pseudomonas aeruginosa, and Neisseria meningitidis.

[0757] Examples of medically relevant Gram-positive bacteria include actinomycetes, Bacillus, Clostridium, Corynebacterium (e.g., Corynebacterium diphtheriae), Enterococcus, Erysipelothrix rhusiopathiae, Listeria (e.g., Listeria monocytogenes), Nocardia, Staphylococcus, and Streptococcus (e.g., Staphylococcus aureus).

[0758] Therefore, in one embodiment, the Gram-negative bacteria genus is selected from the group consisting of Actinomycetes, Bacillus, Clostridium, Corynebacterium, Enterococcus, Erysipelothrix, Listeria, Staphylococcus, and Streptococcus.

[0759] In one implementation (e.g., for a treatment method, for the manufacture of a drug for a treatment method), the treatment is for a respiratory infection, a urinary tract infection, or gastroenteritis.

[0760] Other conditions

[0761] The degrading agents of this invention can also be used to degrade other nucleic acid sequences associated with other disease states. For example, the degrading agents of this invention can be used to treat nucleotide duplication disorders (e.g., Huntington's disease, Fragile X syndrome, myotonic dystrophy type 1) and mRNA sequences encoding non-structural proteins (e.g., IAPP in type 2 diabetes).

[0762] Patients receiving treatment

[0763] In one implementation (e.g., for a treatment method, for the manufacture of a drug for a treatment method), a subject in need of treatment is treated.

[0764] The subjects (patients) requiring treatment may be chordates, vertebrates, mammals, placental mammals, marsupials (e.g., kangaroos, wombats), rodents (e.g., guinea pigs, hamsters, rats, mice), rodents (e.g., mice), lagomorphs (e.g., rabbits), birds (e.g., birds), canids (e.g., dogs), felines (e.g., cats), equines (e.g., horses), pigs (e.g., pigs), sheep (e.g., sheep), cattle (e.g., cows), primates, apes (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or humans.

[0765] Subjects requiring treatment can be adults or adolescents.

[0766] Preferably, the subject requiring treatment is a human, more preferably an adult.

[0767] Alternatively, the subjects requiring treatment are non-human animals used for laboratory research. Preferably, the non-human animals are rodents (e.g., guinea pigs, hamsters, rats, mice).

[0768] Application route

[0769] In one implementation (e.g., for a treatment method, for the manufacture of a drug for a treatment method), treatment is administered via any convenient route of administration, whether systemic / peripheral or local (i.e., at the site of desired action).

[0770] Routes of administration may include oral (e.g., by ingestion); buccal; sublingual; transdermal (including, for example, by patches, plaster, etc.); transmucosal (including, for example, by patches, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or blowing therapy, such as through the mouth or nose); rectal (e.g., by suppositories or enemas); vaginal (e.g., by vaginal suppositories); parenteral, such as by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrasheath, intraspinal, intracapsular, intraorbital, intraperitoneal, intratracheal, subcutaneous, intra-articular, subarachnoid, and intrasternal; and by implantation of a reservoir or depot, for example, subcutaneously or intramuscularly.

[0771] preparation

[0772] In one embodiment (e.g., for a treatment method, for the manufacture of a drug for a treatment method), the degrading agent of formula (I) is administered alone. However, generally, it is preferred to include the degrading agent in a pharmaceutical formulation (e.g., a composition, a formulation, a drug) comprising at least one degrading agent described herein, and one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. The formulation may further comprise other active agents, such as other therapeutic or preventative agents.

[0773] Therefore, the present invention further provides pharmaceutical compositions and methods for preparing pharmaceutical compositions, comprising mixing at least one degrading agent described herein with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. If formulated into discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

[0774] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that, to a reasonable medical judgment, are suitable for contact with the tissues of a subject in question (e.g., a human) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc., must also be "acceptable" in the sense of compatibility with other components of the formulation.

[0775] Suitable carriers, diluents, excipients, etc., can be found in standard pharmacy textbooks, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 25 2005.

[0776] This formulation can be prepared by any method known in the pharmaceutical industry. This method involves combining a degrading agent with a carrier that constitutes one or more auxiliary components. Typically, the formulation is prepared by uniformly mixing the degrading agent with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0777] The formulation can be prepared to provide rapid or slow release; immediate release, delayed release, timed release or sustained release; or a combination of the above release methods.

[0778] The formulation may suitably be 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, granule, mouthwash, drops, tablet (including, for example, coated tablets), granule, powder, lozenge, tablet, capsule (including, for example, hard and soft gelatin capsules), capsule, pill, ampoule, large granule, suppository, vaginal suppository, tincture, gel, paste, ointment, cream, lotion, oil, foam, spray, aerosol or powder mist.

[0779] The formulation may be suitably provided in the form of patches, adhesive tapes, bandages, dressings, etc., impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration enhancers, permeation improvers, and absorption promoters. The formulation may also be suitably provided in the form of a reservoir or depot.

[0780] The degrading agent can be dissolved, suspended, or mixed in one or more other pharmaceutically acceptable components. The compound can be present in liposomes or other microparticles, and is designed to target the compound, for example, blood components or one or more organs.

[0781] dose

[0782] In one implementation (e.g., for a treatment method, for the manufacture of a drug for a treatment method), the treatment comprises administering a therapeutically effective amount of the degrading agent of formula (I) to a subject requiring treatment.

[0783] Those skilled in the art will understand that the appropriate dosage of the degrading agents described herein and compositions containing said degrading agents may vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the particular degrading agent, route of administration, time of administration, rate of excretion of the degrading agent, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health status, and medical history. The amount and route of administration of the degrading agent will ultimately be determined by a physician, veterinarian, or clinician, although a dosage will generally be chosen to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harm or harmful side effects.

[0784] It can be administered continuously or intermittently (e.g., divided doses at appropriate intervals) throughout the treatment. Methods for determining the most effective administration method and dosage are well known to those skilled in the art and will vary depending on the formulation used for treatment, the therapeutic purpose, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed based on the dosage level and pattern chosen by the treating physician, veterinarian, or clinician.

[0785] Typically, the appropriate dose of the degrading agent is in the range of about 10 μg to about 250 mg per kilogram of body weight per day for the subject (more typically about 100 μg to about 25 mg).

[0786] When the compound is a salt, ester, amide, prodrug, etc., the dosage is calculated based on the parent compound, so the actual weight used increases proportionally.

[0787] Other preferences

[0788] Each compatible combination of the implementation schemes described above is explicitly disclosed herein as if each combination were described separately and explicitly.

[0789] In view of this disclosure, various further aspects and embodiments of the invention will be apparent to those skilled in the art.

[0790] "And / or" is considered herein to be a specific disclosure of each of two particular features or parts, having or not having the other. For example, "A and / or B" will be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were described separately herein.

[0791] Unless the context otherwise requires, the description and definition of the above features are not limited to any particular aspect or embodiment of the invention, but are equally applicable to all aspects and embodiments described.

[0792] Example

[0793] Some aspects and embodiments of the invention will now be described by way of examples and with reference to the accompanying drawings. Example Example 1:

[0794] Research using model degraders containing imidazole cleavage groups to demonstrate the concept of non-covalent nucleic acid degradation

[0795] General experimental protocol

[0796] In vitro pseudo-knot oligomer degradation reaction

[0797] RNA oligomers (20 μM) were added to HEPES (20 mM) buffer at pH 7.5 supplemented with KCl (50 mM) and EDTA (10 mM). The mixture was incubated at 37 °C for 30 min. Then MTDB-deg 16a, MTDB, or TDB-deg 16b (1 mM) were added. The reaction mixture was incubated at 37 °C for 3 h, then maintained at 4 °C. The reaction mixture was analyzed by LC-MS or gel electrophoresis.

[0798] LC-MS analysis of oligonucleotides

[0799] Oligonucleotides were analyzed by LC-MS according to the method of Mikutis et al., 2020.

[0800] Oligomers were analyzed using an Acquity UPLC system equipped with an Acquity UPLC BEH C18 1.7 μm column coupled to a Xevo G2-S TOF mass spectrometer. The system utilized electrospray ionization (ESI). Two mobile phases were used: 16.3 mM TEA, 400 mM HFIP (in H2O) and 16.3 mM TEA, 400 mM HFIP (in 80:20 v / v MeCN and H2O), at a flow rate of 0.200 mL / min. Calibration curves for RNA substances were based on the intensity of the A260 or specific negative m / z signal. Integrated peak intensities were calculated using the built-in module of the KNIME software platform (33). Total mass spectra were reconstructed from ion series using the MaxEnt algorithm pre-installed on MassLynx software (version 4.1 from Waters), according to the manufacturer's instructions. To obtain the aforementioned negative ion series, oligomer peaks in the chromatograms were selected for integration and further analysis.

[0801] RNA degradation gel electrophoresis

[0802] Gel electrophoresis was performed according to the method described by Mikutis et al., 2020.

[0803] In vitro RNA degradation was 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 blue FF, 0.025% ethidium bromide, 0.5 mM EDTA) at a 1:1 ratio, heated at 70°C for 5 minutes, and then cooled to 0°C. PAGE was performed at 180V on a Novex™ TBEUrea gel containing 15% polyacrylamide for 60 minutes in 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). Gel staining was performed using SYBR Green II RNA gel staining agent (Invitrogen) in 1×TBE buffer. The stained RNA was observed using a ChemiDoc MP (Bio-Rad, UK).

[0804] Virus stock solution

[0805] The SARS-CoV-2 stock solution used to infect Vero CCL-81 cells was prepared from the fourth generation of SARS-CoV-2 isolated from a Portuguese patient (Internal Reference: 606_IMM ID_5452) after culturing in Vero CCL-81 medium for 4 days, with a concentration of approximately 1.7 × 10⁻⁶. 6 PFU / mL. The stock solution titer was calculated using plaque assay. In short, approximately 8 × 10⁻⁶ PFU / mL. 5 CCL-81 cells / well were seeded in 6-well plates and allowed to grow to confluence for 24 hours. The culture medium was removed, and two 10-fold serial dilutions of 500 μL containing viral supernatant were incubated at 37°C for 1 hour. The plates were manually shaken every 15 minutes to redistribute the inoculum. The cells were covered with supplemented DMEM containing 1.25% carboxymethyl cellulose (CMC) and incubated at 37°C for 4 days. After incubation, the CMC covering 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 paraffin film and sterilized before being removed from BSC and BSL3. Viral plaques were counted to determine the infectious titer (PFU (plaque-forming units) / mL). Viral infection of Vero CCL-81.

[0806] Vero CCL-81 cells with 80% confluence were incubated with SARS-CoV-2 inoculum at 37°C for 1 hour. After incubation, the inoculum was removed, and DMEM medium supplemented with 2.5% FCS was added and incubated for 24 hours, or until samples were harvested.

[0807] Gel electrophoresis analysis

[0808] 500 ng of SARS-CoV-2 RNA, with or without 100 μM MTDB-deg 16a, was incubated in 1xHEPES buffer at 37°C with gentle stirring for 2 hours. The sample was then analyzed on a 1.5% agarose gel.

[0809] Nanopore sequencing

[0810] 500 ng of SARS-CoV-2 RNA was incubated with or without 100 μM MTDB-deg 16a in 1xHEPES buffer at 37°C with gentle stirring for 2 hours. Samples were then prepared for sequencing according to the manufacturer's direct RNA sequencing protocol (SQK-RNA002, ONT). The prepared library was loaded into a FLO-MIN106D flow cell (ONT) and sequenced on a MinION Mk1C device (ONT).

[0811] The genome sequence (GenBank: MN908947.3) and genome annotation (NC_045512.2) of the Wuhan-hu1 strain of SARS-CoV-2 were downloaded from the NCBI database. Sequence reads were aligned to the Wuhan-hu1 genome using minimap2 (Li et al., 2018) with the parameter "-ax splice-N32-un-k13". The CIGAR strings of the alignment results were processed using a custom script. Readings were marked as leaders if the splice point within a read began in the first 60-120 bp of the genome. Readings were assigned to individual transcripts if they covered more than 90% of the annotated transcripts or if the read sequence was located within a transcript. A drug assay was used to determine the 50% inhibitory concentration.

[0812] Increasing concentrations of MTDB-deg 16a (range from 0.07 to 25 μM) were tested to determine the 50% inhibitory concentration (IC50). 50 Parallel assays included a mediator (H2O) control and a control molecule. Cells were seeded in 96-well plates at approximately 40% confluence 24 hours before infection. MTDB-deg16a, MTDB, or TDB-deg 16b was added 1 hour before or 1 hour after infection. Frozen SARS-CoV-2 stock solutions were thawed at room temperature and used to infect cells at 0.05 multiples of infection (MOI). Inhibition of viral growth was measured by harvesting cells 24 hours after infection. Viral load was assessed by PCR targeting the E gene and pseudoknot region.

[0813] Viral plaque-forming units were detected by plaque assay.

[0814] Approximately 8×10 5CCL-81 cells / well were seeded in 6-well plates and allowed to grow for 24 hours to 80% confluence. The supernatant of the compound-treated culture was diluted in DMEM supplemented with 2.5% FCS and added to the pre-seeded wells of the 6-well plates, and incubated at 37°C for 1 hour. The plates were manually shaken every 15 minutes to redistribute the inoculum. The cells were covered with supplemented DMEM containing 1.25% CMC and incubated at 37°C for 4 days. After incubation, the CMC covering 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 paraffin film and sterilized before being removed from BSC and BSL3. Viral plaques were counted to determine the infectious titer (PFU (plaque-forming units) / mL).

[0815] Quantitative PCR of viral load

[0816] Cell pellet was harvested into 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) on an AppliedBiosystems RT-PCR 7500Fast instrument using the default SYBR green program.

[0817] The primers used to detect SARS-CoV-2 are:

[0818] E gene:

[0819] 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (positive),

[0820] 5'-ATATTGCAGCAGTACGCACACA-3' (reverse);

[0821] N gene:

[0822] 5'-GACCCCAAAATCAGCGAAAT-3' (positive)

[0823] 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (reverse);

[0824] False knot:

[0825] 5'-CCGCGAACCCATGCTTCAGTCA-3' (positive)

[0826] 5'-CACGGTGTAAGACGGGCTGCAC-3' (reverse);

[0827] 18S:

[0828] 5'-GTAACCCGTTGAACCCCATT-3' (positive)

[0829] 5'-CCATCCAATCGGTAGTAGCG-3' (reverse).

[0830] Virus recovery test

[0831] Two sets of samples were prepared for the recovery assay. Cells at 80% confluence were infected with cryopreserved SARS-CoV-2 stock solution at 0.05 MOI for 2 hours. The inoculum was then removed, and the infected cells were incubated with MTDB-deg 16a, MTDB, and TDB-deg 16b at 6 μM, 37°C, and 5% CO2 for 24 hours. After 24 hours, in one set of samples, cells were harvested into lysis buffer for PCR analysis of viral growth. In the other set of samples, the supernatant was removed, replaced with drug-free medium, and incubated again at 37°C and 5% CO2 for 24 hours. After 24 hours of incubation (corresponding to the 48-hour time point), cells were harvested into lysis buffer, and viral growth was measured by PCR targeting the E gene and pseudoknot region. The percentage of viral recovery was normalized relative to the vector control.

[0832] Cytotoxicity assay

[0833] To determine whether the compound is toxic to cells, 1 × 10⁻⁶ ppm was used 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). After 24 hours of incubation with the compounds, cell viability was assessed using the CellTiter Blue viability assay (Promega) according to the manufacturer's protocol. Briefly, the CellTiter Blue stock solution was diluted 1:20. 80 μL of the diluted CellTiter Blue was added to each well and incubated at 37°C for 2 hours.

[0834] Dynamic light scattering (DLS)

[0835] Stock solutions (10 mM) of each screening molecule were prepared in pure DMSO and then serially diluted in water to a final concentration of 25 or 12.5 μM. Data were collected at 25 °C on a Zetasizer Nano S (Malvern).

[0836] Antiviral activity in animal models of SARS-CoV-2 infection

[0837] 10-12 week old specific pathogen-free Tg(K18-ACE2)2Prlmn (strain B6.Cg-Tg(K18-ACE2)2Prlmn / J, Jackson laboratory strain 034860) hemizygous mice were used in this study. Mice were intranasally infected with 1×10⁻⁶ ppm of PBS. 4 PFU for SARS-CoV-2. The compound was administered intranasally 1 hour before and 3 hours after infection. Mice were treated with the medium (n=6), 25 mg / kg of MTDB-degrading agent 16a (n=6), 10 mg / kg of MTDB (n=3), or 25 mg / kg of TDB-degrading agent 16b (n=5). On day 5 post-SARS-CoV-2 infection, animals were humanely euthanized, and the left lung was harvested for viral quantification by plaque assay, and the right lung was harvested for histopathological analysis.

[0838] Western blot analysis

[0839] For in vitro experiments, samples were treated with a medium (H2O) or MTDB-deg 16a (6mM) for 24 hours. Cells were then lysed with whole-cell lysis buffer (50mM Tris-HCl pH 8.0, 450mM NaCl, 0.1% NP-40, 1mM EDTA) supplemented with 1mM DTT, a protease inhibitor (Sigma), and a phosphatase inhibitor (Sigma). For in vivo experiments, the entire left lung of a mouse was homogenized in 3 mL of DMEM, and 750 μL was transferred to an equal volume of whole-cell lysis buffer (supplemented as above). Protein concentration was assessed using Bradford Assays (BioRad). Samples were replenished with LDS loading buffer (Lifetechnologies) and sample reducing agent (Life Technologies) before loading. 40 μg of protein was isolated on an SDS-PAGE gel and imprinted onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare). The following antibodies were used for Western blotting experiments: anti-β actin (Abcam, ab8224), anti-phosphorylated MAPKAPK-2 (Thr334) (27B7) (CellSignalling, 3007), and anti-phosphorylated p38 MAPK (Thr180 / Tyr182) (D3F9). (Cell Signalling, 4511), goat anti-IgG H&L (HRP) (Abcam, ab205719) and goat anti-rabbit HRP (Abcam, ab6721).

[0840] Synthesis of azide-imidazolium

[0841]

[0842] Scheme 1: Synthesis of azide-imidazolium

[0843] Synthesis of hexaethylene glycol bis(p-toluenesulfonate) (1)

[0844] 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 with MgSO4, the solvent was evaporated under reduced pressure. No further purification was required. Yield: 95% (colorless oil).

[0845] 1 H NMR (400MHz, CDCl3): 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 S2's m / z: 591.19.

[0846] The physical and spectroscopic data are consistent with those described in the literature (Mikutis et al., 2020).

[0847] Synthesis of hexaethylene glycol p-toluenesulfonyl azide (2a)

[0848] Hexaethylene glycol bis(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 magnesium sulfate. To remove DMF, toluene was added and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:hexane, 1:1). Yield: 54% (colorless oil).

[0849] 1 H NMR (400MHz, 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 The m / z of N3NaO8S is 484.2.

[0850] The physical and spectroscopic data are consistent with those described in the literature (Mikutis et al., 2020).

[0851] Synthesis of tetraethylene glycol p-toluenesulfonyl azide (2b)

[0852] Tetraethylene glycol bis(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 hours. The solvent was removed under vacuum, and the product was purified by rapid column chromatography (from 3:1 petroleum ether:ethyl acetate to 1:1 petroleum ether:ethyl acetate). The obtained product was a colorless oil (798 mg, 2.1 mmol, 39%).

[0853] 1 H NMR (400MHz, CDCl3) δ7.82(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 The m / z of N3NaO6S is 396.1207. Synthesis of diethylene glycol p-toluenesulfonyl azide (2c).

[0854] Diethylene glycol bis(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 magnesium sulfate. To remove DMF, toluene was added and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:hexane, 1:1). Yield: 59% (colorless oil).

[0855] 1 H NMR (400MHz, CDCl3)δ H 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 The m / z of N3NaO4S is 308.068.

[0856] The physical and spectroscopic data are consistent with those described in the literature (Mikutis et al., 2020).

[0857] Synthesis of hexaethylene glycol imidazole ester azide (3a)

[0858] 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 at 0 °C for 30 min, 2a (1.0 mmol) was added. The reaction mixture was stirred overnight at 60 °C, cooled to room temperature, and quenched with water (20 mL). The mixture was then extracted with EtOAc and DCM, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give the crude product. The crude product was then purified by column chromatography (ethyl acetate:methanol, 3:1). Yield: 35% (colorless oil).

[0859] 1 H NMR (400MHz, 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).MS:C 15 H 28 The m / z of N5O5 is 358.21.

[0860] The physical and spectroscopic data are consistent with those described in the literature (Mikutis et al., 2020).

[0861] Synthesis of tetraethylene glycol imidazole ester azide compound (3b)

[0862] 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 N2 atmosphere, heated 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. The mixture was then stirred at 55 °C for 20 h. The solvent was then removed under vacuum, and the resulting residue was purified by rapid chromatography (dry loading, gradient EtOAC to 9:1 EtOAC:MeOH). The product was a colorless oil (54 mg, 0.20 mmol, 74%).

[0863] 1 H NMR (400MHz, CDCl3) δ7.55(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). 13C NMR (100MHz, 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 The m / z of N5O3 is 270.1582.

[0864] Synthesis of diethylene glycol imidazole ester azide compound (3c)

[0865] 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 at 0 °C for 30 min, 2 c (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, the product was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give the crude product. The crude product was then purified by column chromatography (ethyl acetate:methanol, 3:1). Yield: 38% (colorless oil).

[0866] 1 H NMR (400MHz, 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 The m / z of N5O3 is 182.1.

[0867] The physical and spectroscopic data are consistent with those described in the literature (Mikutis et al., 2020).

[0868] Synthesis of azidoethylimidazolium (4)

[0869]

[0870] Scheme 2: Synthesis of azidoethylimidazolium 4

[0871] 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 product was dissolved in DMF, and sodium azide (1.0 mmol) was added. The mixture 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 performed by column chromatography (ethyl acetate:methanol, 3:1). Yield: 26% (white solid).

[0872] 1 H NMR (400MHz, CDCl3) δ7.51 (s, 1H), 7.09 (s, 1H), 6.96 (s, 1H), 4.09 (t, J = 5.7Hz, 1H), 3.62 (t, J = 5.7Hz, 1H).

[0873] Synthesis of Pyridostatin Degrading Agent

[0874]

[0875] Option 3: Synthesis of pyridine statidine derivatives

[0876] Synthesis of dimethyl chebulin (5)

[0877] Chelidonine hydrate (2.0 g, 11 mmol) was suspended in 20 mL of methanol. 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 heated to room temperature and stirred overnight. The brown solution was refluxed for 2 hours, and the solvent was removed under vacuum. The brown crude product was then recrystallized from EtOH to give a beige solid, dimethyl chelidonine 5 (864 mg, 3.9 mmol, 36%).

[0878] 1 H NMR (400MHz, DMSO) δ11.77(br s,1H),7.61(s,2H),3.88(s,6H). 13 CNMR (101MHz, DMSO) δ 165.97, 164.88, 149.37, 115.33, 52.68. HRMS (ES) calculated as C9H 10 NO5([M+H] + m / z: 212.0559, actual measurement is 212.0567.

[0879] Synthesis of propargyl chelidonine (6)

[0880] Dimethyl leucine 5 (0.82 g, 3.8 mmol), propargyl alcohol (0.33 mL, 5.7 mmol), and polymerically 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 and cooled to 0 °C using a freeze-pump-thaw cycle, 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 under vacuum. Dimethyl leucine was obtained by column chromatography (50% EtOAc, 50% petroleum ether). It was then dissolved in 50 mL of methanol, followed by the addition of 50 mL of aqueous sodium hydroxide (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 under vacuum. The mixture was acidified with 5% formic acid and then extracted with 3 × 100 mL of ethyl acetate. The organic layer was then dried over magnesium sulfate, filtered, and the solvent was removed under vacuum. This produced a grayish-white solid, propargyl chebuline 6 (0.17 g, 0.77 mmol, 20%).

[0881] 1 H NMR (400MHz, MeOD) δ7.93 (s, 2H), 5.02 (d, J = 2.4Hz, 2H), 3.15 (t, J = 2.5Hz, 1H). 13 C NMR (100MHz, MeOD) δ 168.12, 166.98, 150.54, 150.43, 115.73, 78.97, 77.90, 77.88, 57.74, 57.68. HRMS (ES) calculated as C 10 H8NO5([M+H) + m / z:222.0397, actual measurement is 222.0391.

[0882] Synthesis of O-(ethyl-2-N-boc-amine)-2-aminoquinolinone (7)

[0883] 2-Aminoquinone (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 and cooled to 0 °C using a freeze-pump-thaw cycle, 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 under vacuum. The product was purified by gradient column chromatography from 100% EtOAc to 90% EtOAc, 10% MeOH. The solvent was removed under vacuum to give a grayish-white solid 7 (552 mg, 1.82 mmol, 29%).

[0884] 1H NMR (400MHz, CDCl3) δ7.98 (dd, J=8.0, 1.0Hz, 2H), 7.60 (dd, J=8.4, 1.2Hz, 2H), 7.55 ( ddd,J=8.5,6.7,1.6Hz,2H),7.23(ddd,J=8.1,6.6,1.3Hz,2H),6.04(s,1H),5.01(br s,1H),4.69(br s,2H),4.18(t,J=5.1Hz,4H),3.68(q,J=5.5Hz,4H),1.46(s,9H). 13 C NMR (100MHz, 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) calculated as C 16 H 22 N3O3([M+H)) + m / z: 304.1661, actual measurement is 304.1649.

[0885] Synthesis of alkyne-pyridinesstatin (8)

[0886] Propylcaryophylline 6 (0.12 g, 0.54 mol) was dissolved in 1.2 mL of DCM. Then, Ghosez's reagent (170 μL, 1.3 mmol) was added dropwise at 0 °C, and the orange solution was stirred at room temperature for 2 hours. 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 hour. 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 brownish-red and was stirred overnight under argon. The crude protected product 8a (not shown) precipitated from hot 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 N-boc protection. The solvent was removed under vacuum, and the product was purified by HPLC (gradient 100% H₂O, 0.1% FA to 100% MeCN, 0.1% FA). Freeze-drying yielded a grayish-white solid, alkyne-pyridostatin 8 (51 mg, 86 μmol, 16%).

[0887] HRMS(ES) is calculated as C 32 H 30 N7O5([M+H)) + m / z: 592.2308, measured value: 592.2327. Synthesis of pyridine statadin degrading agents (9A to 9C).

[0888] Alkyne-pyridostatin 8 (15 mg, 25 μmol) was dissolved in 2.5 mL of a 2:1 mixture of H₂O and tert-butanol. Copper sulfate pentahydrate solution (250 μL, 100 mM, 25 μmol) was added, followed by sodium ascorbate solution (1.3 mL, 100 mM, 130 μmol). The turbid yellow solution was degassed and stirred for 10 min. Then, a suitable solution of azido-imidazolium (3a, 3b, or 3-azidopropionic acid) (3.8 mL, 10 mM) was added. The solution was stirred under argon for 2 h. The organic solvent was removed under vacuum. The product was then purified by HPLC (gradient from 100% H₂O, 0.1% FA to 100% MeCN, 0.1% FA). The obtained product was a white or off-white solid.

[0889] 9A (PDS-deg6). 48% yield (11.3 mg, 12 μmol). HRMS (ES) calculated as C47H52N12O10([M+H]+) m / z: 949.4321, measured as 949.4344.

[0890] 9B (PDS-deg4). 69% yield (14.8 mg, 17 μmol). HRMS (ES) calculated as C43H49N12O8([M+H]+) m / z: 861.3796, measured as 861.376.

[0891] 9C(PDS-CBX). 28% yield (4.9 mg, 6.9 μmol). HRMS(ES) calculated as C35H34N10O7([M+H]+) m / z: 707.2690, measured: 707.2684.

[0892] Synthesis of pseudoknot degradation agents

[0893]

[0894] Option 4: Synthesis of pseudo-knot degrading agent

[0895] Synthesis of compound 11a

[0896] A solution of 100 mL of DCM containing 2-methylthiazol-4-carboxaldehyde (10.0 g, 78.6 mmol, 1.0 equivalent) was added in a single batch to compound 10 (16.5 g, 82.6 mmol, 16.2 mL, 1.1 equivalent) at 25 °C under N2. The mixture was stirred at 25 °C for 3 h. NaBH(OAc)3 (25.0 g, 118 mmol, 1.5 equivalent) was added to the mixture and stirred for 10 h. The residue was poured into water (50 mL) and stirred for 10 min. The aqueous phase was extracted with DCM (3 × 20 mL). The combined organic phases were dried over anhydrous Na2SO4 and filtered; the solvent was removed under vacuum. 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.

[0897] LCMS [+scan]: Calculated as m / z C 20 H 26 N3O3S 388.2; observed 388.1.

[0898] Synthesis of compound 12a

[0899] TFA (40.0 g, 351 mmol, 26 mL, 8.4 equivalents) was added to a DCM (130 mL) solution of compound 11a (13.0 g, 41.7 mmol, 1.0 equivalents) at 25 °C under N2. The mixture was stirred for 12 hours. The solvent was removed under vacuum to give the TFA salt of compound 12a (23.0 g, crude product) as a red oil.

[0900] LCMS [+scan]: Calculated as m / z C 10 H 18 N3S212.1; 212.0 was observed.

[0901] Synthesis of MTDB (compound 13a)

[0902] TEA (9.21 g, 91.0 mmol, 12.7 mL, 2.0 equivalent) was added to a DCM (200 mL) solution of compound 12a (20.0 g, 45.5 mmol, 1.0 equivalent) at 20 °C under N2. Then, ethyl 2-isocyanobenzoate (8.70 g, 45.5 mmol, 1.0 equivalent) was added to the mixture at 0 °C. The mixture was stirred at 20 °C for 12 hours. The solvent was removed under vacuum. 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 a grayish-white solid.

[0903] 1 ¹H NMR (400MHz, CD3OD): δ 8.42 (br d, J = 8.4Hz, 1H), 8.08 (br d, J = 8.0Hz, 1H), 7.62 (s, 1H), 7.52-7.59 (m, 1H), 7.09 (br t, J = 7.6Hz, 1H), 4.34-4.46 (m, 4H), 3.93 (br s, 2H), 3.77 (br t, J = 6.0Hz, 2H), 3.49 (br s, 4H), 3.28-3.31 (m, 1H), 2.74 (s, 3H), 2.31 (brd, J = 4.8Hz, 2H), 1.43 (t, J = 7.2Hz, 3H). LCMS [+scan]: calculated as m / z C 20 H 27 N4O3S 403.2; observed 403.1.

[0904] Synthesis of compound 14a

[0905] LiOH monohydrate (2.34 g, 55.7 mmol, 4.0 equivalent) was added to a mixture of MTDB (compound 13a 5.60 g, 13.9 mmol, 1.0 equivalent) in ethanol (120 mL) and H2O (30 mL) at 25 °C under N2. The mixture was stirred at 25 °C for 12 hours. The pH of the mixture was adjusted to 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 Na2SO4, filtered, and concentrated under vacuum to give compound 14a (2.60 g, 6.94 mmol, 50%) as a yellow solid.

[0906] LCMS [+scan]: Calculated as m / z C 18 H 23 N4O3S 375.1; observed at 375.1.

[0907] Synthesis of compound 15a

[0908] DIEA (4.49 g, 34.7 mmol, 6.05 mL, 5.00 equivalent) was added to a mixture of compound 14a (2.60 g, 6.94 mmol, 1.0 equivalent) and propargylamine (1.15 g, 20.8 mmol, 1.33 mL, 3.0 equivalent) in DMF (200 mL) at 25 °C under N2. T3P (4.42 g, 13.9 mmol, 4.13 mL, 2.0 equivalent) was added to the mixture and the mixture was stirred at 50 °C for 12 hours. 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 under vacuum. 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).

[0909] 1 ¹H NMR (400MHz, CDCl₃): δ 10.54 (br s, 1H), 8.46 (d, J = 8.2Hz, 1H), 7.43–7.51 (m, 2H), 7.11–7.11 (m, 1H), 6.99 (q, J = 7.6Hz, 2H), 6.49 (br s, 1H), 4.22 (dd, J = 5.2, 2.6Hz, 2H), 3.72–3.83 (m, 4H), 3.64–3.69 (m, 2H), 2.77–2.96 (m, 4H), 2.72 (s, 3H), 2.32 (t, J = 2.6Hz, 1H), 2.04 (br d, J = 15.2Hz, 2H). LCMS [+scan]: calculated as m / z C 21 H 26 N5O2S 412.2; observed 412.0.

[0910] Synthesis of MTDB-deg (compound 16a)

[0911] A mixture of azide-imidazolium 3a (200 mg, 280 μmol, 1.0 equivalent), compound 15a (115 mg, 280 μmol, 1.0 equivalent), and copper sulfate (22.3 mg, 140 μmol, 21.5 μL, 0.5 equivalent) in DCM (5 mL), methanol (5 mL), and H₂O (5 mL) was stirred at 20 °C for 0.5 h. Then, NaAsc (11.1 mg, 55.9 μmol, 0.2 equivalent) was added to the mixture, and the mixture was stirred at 20 °C for 4.5 h. The mixture was diluted with H₂O (10 mL) and then extracted with DCM (3 × 10 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by HPLC (column: Phenomenex Gemini-NX80×40mm×3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 15%-35%, 8 min) to give MTDB-deg16a (28.0 mg, 34.9 μmol, 13% yield) as a pale yellow oil.

[0912] 1 H NMR (400MHz, DMSO-d6): δ11.03(s,1H),9.29(br t,J=5.2Hz,1H),8.37(d,J=8.4Hz,1H),7.96(s,1H),7.74(br d,J=7.2Hz,1H),7.63(br s,1H),7.43(t,J=7.60Hz,1H),7.15-7.31(m,2H),6.99(t,J=7.6Hz,1H),6.89(br s,1H),4.46-4.53(m,4H),4.10(brt,J=5.2Hz,2H),3.77-3.81(m,2H),3.71(br s,2H),3.66(br t, J = 5.07 Hz, 2H), 3.44-3.57 (m, 20H), 3.34 (s, 25H), 2.73 (br s, 1H), 2.62 (br s, 6H), 1.84 (br s, 2H). HRMS [+scan]: calculated as m / z C 36 H 53 N 10 O7S769.3819; observed at 769.3830.

[0913] Synthesis of a control degradation agent for pseudoknot experiments

[0914]

[0915] Option 5: Synthesis of pseudoknot control degrader (TBD-deg)

[0916] Synthesis of compound 11b

[0917] Compound 10 (3.93 g, 19.6 mmol, 3.85 mL, 1.1 equivalent) was added to a solution of thiophene-3-carboxaldehyde (2.00 g, 17.8 mmol, 1.63 mL, 1.0 equivalent) in DCM (80 mL) under N2 at 20 °C. The mixture was stirred at 20 °C for 3 h. Then, NaBH(OAc)3 (5.67 g, 26.8 mmol, 1.5 equivalent) was added to the mixture at 0 °C, and the mixture was stirred at 20 °C for 10 h. The reaction mixture was quenched by adding 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 under vacuum. 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.

[0918] 1 H NMR (400MHz, CDCl3): δ7.21-7.31(m,1H),7.03-7.15(m,2H),3.64(s,2H),3.37-3.54(m,4H),2.55-2.68(m,4H),1.81(br dd,J=10.8,4.9Hz,2H),1.40-1.52(m,9H).

[0919] Synthesis of compound 12b

[0920] TFA (6.16 g, 54.0 mmol, 4.00 mL, 9.4 equivalents) was added to a DCM (20 mL) solution of compound 11b (1.70 g, 5.73 mmol, 1.0 equivalent) under N2 at 20 °C. The mixture was stirred at 20 °C for 8 hours. The solvent was removed under vacuum to obtain the TFA salt of compound 12b (3.00 g, crude product), which was a red oil and could be used in the next step without further purification.

[0921] Synthesis of TDB (compound 13b)

[0922] TEA (1.96 g, 19.3 mmol, 2.69 mL, 2.0 equivalent) was added to a mixture of compound 12b (3.00 g, 9.67 mmol, 1.0 equivalent) in DCM (40 mL) at 20 °C under N2. Then, ethyl 2-isocyanobenzoate (1.85 g, 9.67 mmol, 1.0 equivalent) was added to the mixture at 0 °C. The mixture was stirred at 20 °C for 12 hours. The solvent was removed under vacuum. 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 a grayish-white solid.

[0923] 1 ¹H NMR (400MHz, CDCl₃): δ 10.59 (s, 1H), 8.52 (d, J = 8.4Hz, 1H), 7.94 (dd, J = 8.0, 1.53Hz, 1H), 7.42 (t, J = 7.6Hz, 1H), 7.14–7.24 (m, 1H), 6.96–7.07 (m, 2H), 6.88 (t, J = 7.2Hz, 1H), 4.28 (q, J = 7.2Hz, 2H), 3.55–3.67 (m, 6H), 2.70 (br s, 2H), 2.53–2.65 (m, 2H), 1.91 (br s, 2H), 1.33 (t, J = 7.2Hz, 3H). LCMS [+scan]: calculated as m / z C 20 H 26 N3O3S 388.2; observed 388.1.

[0924] Synthesis of compound 14b

[0925] Under N2 at 25°C, LiOH monohydrate (65 g, 1.5 mmol, 6.0 equivalent) was added to a mixture of TDB 21 (100 mg, 0.26 mmol, 1.0 equivalent) in ethanol (1.2 mL) and H2O (1.2 mL). The mixture was stirred at 25°C for 16 hours, after which another LiOH monohydrate (130 g, 3.1 mmol, 12.0 equivalent) was added. After 2 hours, 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 magnesium sulfate, filtered, and concentrated under vacuum to give compound 14b (70 mg, 0.19 mmol, 76%) as a yellow oily solid.

[0926] 1 H NMR (400MHz, CD3OD): δ H8.41(d,J=8.5Hz,1H),8.08(d,J=8.5Hz,1H),7.76(br s,1H),7.64(m,1H),7.53(t,J=7.5Hz,1H),7.32(d,J=4.5Hz,1H),7.07(t,J=7.5Hz,1H),4.46(br s,2H),3.95(m,2H),3.75(tr,J=6.0Hz,2H),3.47(m,4H),2.34(m,2H). 13 CNMR (100MHz, 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]: calculated as m / zC 18 H 22 N3O3S 360.1382; observed 360.1386.

[0927] Synthesis of compound 15b

[0928] TEA (75.5 mg, 74.6 μmol, 104 μL, 4.0 equivalent) was added to a mixture of compound 14b (70 mg, 195 μmol, 1.0 equivalent) and propargylamine (11.2 mg, 200 μmol, 13.1 μL, 1.0 equivalent) in DMF (3 mL) at 25 °C under N2. 50% T3P (238 mg, 370 μmol, 1.9 equivalent) in DMF was added to the mixture, and the mixture was stirred at 25 °C for 16 h. The solvent was removed under vacuum, and the title compound was purified on a column (gradient DCM to DCM:MeOH 9:1). To remove residual DMF, the compound was dissolved in DCM (10 mL) and washed with H2O (10 mL) and 1% NaOH aqueous solution (10 mL). The organic phase was dried with anhydrous magnesium sulfate and the solvent was removed under vacuum to give 15b (26 mg, 66 μmol, 34% yield, ratio 4:1).

[0929] 1¹H NMR (400 MHz, CDCl₃, reporting major diastereomers): δ 10.42 (br s, 1H), 8.35 (d, J = 8.9 Hz, 1H), 7.36–7.41 (m, 2H), 7.26 (m, 1H), 7.11 (m, 1H), 7.06 (m, 1H), 6.87–6.97 (m, 2H), 6.49 (br s, 1H), 4.16 (dd, J = 5.2, 2.5 Hz, 2H), 3.59–3.71 (m, 6H), 2.75 (br s, 2H), 2.65 (t, J = 5.5 Hz, 2H), 2.27 (t, J = 2.5 Hz, 1H), 1.96 (br s, 2H). 13 C NMR (100MHz, 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]: calculated as m / z C 36 H 52 N9O7S 397.1698; observed 397.1716.

[0930] Synthesis of TDB-deg (compound 16b)

[0931] Compound 15b (9.9 mg, 25 μmol, 1.0 equivalent) was dissolved in a mixture of H₂O (1.7 mL) and tert-butanol (0.8 mL). An aqueous solution of copper sulfate (250 μL, 100 mM, 25 μmol, 1.0 equivalent) was added, followed by an aqueous solution of NaAsc (1.3 mL, 100 mM, 130 μmol, 5.2 equivalent). The resulting turbid yellow mixture was placed under an argon atmosphere; then an aqueous solution of azide-imidazolium 3a (3.8 mL, 10 mM, 38 μmol, 1.5 equivalent) was added. The reaction was stirred at room temperature for 1 hour, after which the reaction mixture turned a clear yellow. The reaction was quenched with disodium ethylenediaminetetraacetate dihydrate (9.3 mg, 25 μmol, 1 equivalent), the organic solvent was removed under vacuum, and the mixture was purified by HPLC. The fraction containing the product was freeze-dried to give TDB-deg(15b) in the form of a yellowish-brown oily solid (10.2 mg, 14 μmol, 54% yield).

[0932] HRMS [+scan]: Calculated as m / z C 36 H 52 N9O7S 754.3710; observed 754.3698.

[0933] Synthesis of chloramphenicol degrading agents

[0934]

[0935] Option 6: Synthesis of Chloramphenicol-PEG-Imidazole Degrading Agent

[0936] Synthesis of chloramphenicol-6PEG-imidazole degrader (18a)

[0937] Under inert conditions, propynic acid (1.1 mmol) was dissolved in DMF (20 mL) and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred at 0 °C for 30 min. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly for the copper click reaction. Azide-imidazolium 3a (1.0 mmol), CuSO4 (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred at room temperature for another 24 h. After adding toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 10% (yellow oil).

[0938] 1 H NMR (400MHz, MeOD) δ8.29(s,1H),8.14(d,J=8.8Hz,2H),7.79(s,1H),7.67(d,J=8.7Hz,2H),7.44(s,1H),7.22(s,1H),5.23(d,J= 2.6Hz,1H),4.61–4.56(m,2H),4.34(s,1H),4.22(t,J=4.9Hz,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.

[0939] Synthesis of Chloramphenicol-2PEG-Imidazole Degrader (18b)

[0940] Under inert conditions, propynic acid (1.1 mmol) was dissolved in DMF (20 mL) and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred at 0 °C for 30 min. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly for the copper click reaction. Azide-imidazolium 3c (1.0 mmol), CuSO4 (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred at room temperature for another 24 h. After adding toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 10% (white solid).

[0941] 1 H NMR (400MHz, MeOD) δ8.19(s,1H),8.16(d,J=8.8Hz,2H),7.69(d,J=8.6Hz,2H),7.55( s,1H),7.00(s,1H),6.88(s,1H),5.25(d,J=2.8Hz,1H),4.60(dd,J=5.5,4.5Hz,2H),4 .38(ddd,J=7.0,5.9,2.9Hz,1H),4.14(dd,J=5.5,4.4Hz,2H),3.89(dd,J=11.0,7.0Hz ,1H),3.84(t,J=5.1Hz,2H),3.75(dd,J=10.9,5.9Hz,1H),3.71(t,J=4.9Hz,2H).MS:C 19 H 23 The m / z of N7O6 is 445.2.

[0942] Synthesis of Chloramphenicol-Ethyl-Imidazole Degrading Agent (18c)

[0943] Under inert conditions, propynic acid (1.1 mmol) was dissolved in DMF (20 mL) and cooled to 0 °C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added, and the reaction mixture was stirred at 0 °C for 30 min. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added, and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly for the copper click reaction. Azide-imidazolium 4 (1.0 mmol), CuSO4 (5 mol%), and sodium ascorbate (0.2 mmol) were added, and the reaction mixture was stirred at room temperature for another 24 h. After adding toluene, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC. Yield: 12% (white solid).

[0944] 1 H NMR (400MHz, MeOD) δ8.14(d,J=8.8Hz,2H),8.03(s,1H),7.64(d,J=8.3Hz,2H),7.43(s,1H),6.97(s,1H),6.93(s,1H),5.21(d,J=2.7Hz,1H),4.82(d d,J=6.8,5.0Hz,2H),4.58(dd,J=6.7,4.9Hz,2H),4.31(ddd,J=7.2,5.9,2.8Hz,1H),3.84(dd,J=10.9,7.2Hz,1H),3.69(dd,J=10.9,5.9Hz,1H).MS:C 17 H 19 The m / z of N7O5 is 401.1.

[0945] G-quadruplex targeting and RNA degradation

[0946] Two degraders targeting RNA G-quadruplex (rG4) were rationally designed by conjugating the known G4 conjugate pyridostatine with azido-imidazolium of varying lengths (9A, 9B). Copper-induced azido-acetylene cycloaddition (CuAAC) was used to link the two components, providing tolerance to a wide range of substrates and generating a triazole—a bioisosteric moiety of an amide with good tolerance in biological systems. We used the same strategy to synthesize CBX-PDS—a pyridostatine derivative known to selectively bind RNA G-quadruplex (but not DNA G-quadruplex). Both rG4 degraders and a binding control were tested in vitro and in cellular systems.

[0947] In vitro degradation of rG4 oligomers

[0948] To demonstrate that our degrading agents can selectively degrade the G4 structure, we examined their activity in the presence of different oligomers and cations. We incubated our degrading agents with RNA oligomers corresponding to the rG4 structure in the NRAS mRNA 5'UTR or with mutants that cannot form rG4. Furthermore, we incubated them with K... + (rG4 formation promoter) and Li + Our molecules were tested in the presence of (a known inhibitor of rG4 formation). For rG4-active oligomers, only in K + (instead of Li) + Degradation was observed in the presence of ) Figure 2 a) This indicates that it is not enough for oligomers to simply possess G-rich sequences that are targeted by these molecules; the oligomers must form G-quadruplexes to be degraded. Remarkably, using K... + Or Li + No degradation was observed in the perturbed rG4 inactive NRAS sequence, further demonstrating that these degradative agents specifically act on the rG4 structure. Figure 2 b). Furthermore, no degradation was observed with CBX-PDS (a control molecule containing the rG4 conjugate moiety of PDS but without the degrading agent), indicating that conjugation alone is insufficient, and the degrading agent moiety is necessary for degradation. Figure 2 (a, b). Interestingly, we observed that PDS-deg6(9A), a degrader with a linker having 6 PEG subunits, degraded RNA faster than PDS-deg4(9B), which has 4 PEG subunits. This is likely due to the elongation effect of the longer linker. In summary, these experiments demonstrate that our degrader specifically cleaves rG4 material but not unfolded RNA regions.

[0949] In vitro degradation of SARS-CoV-2 genomic materials

[0950] To provide evidence that rG4 degraders can degrade the SARS-CoV-2 genome and to gain a deeper understanding of the degradation mechanism, 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. Since the SARS-CoV-2 genome has several putative rG4 sites (Zhao et al., 2021) and exhibits tight packing, with most sites adjacent to rG4 (Ziv et al., 2020), we expected our degraders to induce widespread damage. Indeed, we observed substantial degradation across many regions of the genome, indicating that our degraders are effective in disrupting the genetic material of SARS-CoV-2. Figure 2c). This phenomenon suggests that our degrading agent should be able to degrade and thus inactivate viral RNA within the cell.

[0951] In vitro antiviral activity of rG4 degraders

[0952] To test the antiviral activity of G4 degraders in vitro, cells were incubated one hour before infection with 0.5 μM, 5 μM, and 50 μM of G4 degraders (PDS-deg6(9A), PDS-deg4(9B), and PDS-Alk(8): control molecules without degraders) and 5 μM chloroquine as a control. Figure 3 (a and 3b) Inhibition of viral growth was measured by harvesting supernatant and cells 24 hours post-infection. Viral growth was assessed by plaque assay (in supernatant) and PCR (in cells). Cell viability was assessed after 24 hours of incubation with increased concentrations of G4-degrading agent (ranging from 0.05 μM to 50 μM) using conventional cell viability kits (e.g., CellTiter Blue assay), according to the manufacturer's protocol.

[0953] We observed that the G4 degrading agent successfully inhibited viral growth at 5 μm and 50 μm. Figure 3 PCR results showed that, compared with the DMSO control, PDS-deg4 appeared to have no inhibitory effect on viral replication, while PDS-deg6(9A) inhibited viral replication by 70% at 5 μM. Figure 3 b). Importantly, none of the compounds showed cytotoxicity exceeding 50 μM. Figure 3 c).

[0954] In vivo antiviral activity of rG4 degraders

[0955] To evaluate the in vivo antiviral activity of the G4 degrader, transgenic K18hACE2 mice (expressing hACE2 protein) were intranasally administered 25 mg / kg of PDS-deg4 (9B) and PDS-deg6 (9A) at 40 minutes before infection and again at 3 hours and 18 hours after infection. Figure 4 Mice were intranasally infected with SARS-CoV-2 (on day 0, 2.5-5 × 10⁻⁶ mg / L PBS). 4 PFU / mouse was used to monitor body weight, morbidity, and mortality (in extreme cases, death or euthanasia was performed), as well as clinical signs of infection, daily. On day 5, all mice were sacrificed, and the left lung was collected for viral load quantification using plaque assay. The right lung, heart, liver, kidney, and spleen were harvested for histopathological analysis.

[0956] Results showed that administration of PDS-deg6(9A) at a dose of 25 mg / kg was toxic to mice, 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% reduction in body weight on day 1 post-infection. Figure 4 a). However, body weight remained stable between day 1 and day 3, then decreased again at the same rate as the vector control group. Animals treated with PDS-deg4(9B) showed a significant reduction in pulmonary viral load ( Figure 4 b).

[0957] This experiment showed that administration of the G4 degrading agent led to a reduction in viral load in the lungs of SARS-CoV-2-infected k18hACE2 mice.

[0958] Targeting of pseudoknots and degradation of RNA

[0959] By binding the known pseudoknot conjugate MTDB with azide-imidazolium 3a, the non-covalent degradation molecule MTDB-deg(16a) was rationally designed to target RNA pseudoknots. Figure 5 a) MTDB contains an ethyl ester moiety, which has been replaced with an amide to increase stability and serve as a handle for the attachment of the degradative agent. We used azide-imidazolium 3a with a linker consisting of 6 PEG subunits, which we previously found to be more efficient at RNA degradation of alkyne-tagged RNA than its shorter counterparts (Mikutis et al., 2020). We chose CuAAC as the reaction medium for the coupling binder and degradative agent fragments because of its advantages: high stability, ease of operation, high modularity, and the ability to adjust the structure of both fragments without altering the coupling steps.

[0960] Selective degradation of the pseudobulb of the tristalk coronavirus

[0961] To validate our strategy, we tested our pseudoknot degrader against RNA 69-er, which has a sequence corresponding to the coronavirus pseudoknot and is therefore predicted to form it. We incubated 69-er with MTDB-deg (16a) or one of two control molecules (the non-degradable parent binding molecule MTDB) or TDB-deg (16b) (a degrading molecule derived from ethyl 2-(4-(thiophene-3-ylmethyl)-[1,4]diaza-1-carbonyl]-amino)benzoate (TDB), the latter being closely associated with MTDB but having a lower affinity for pseudoknot binding. Figure 5b)(Park et al., 2011). After 3 hours of incubation with MTDB-deg(16a), RNA pseudoknots were significantly degraded, with only 23% remaining intact compared to the untreated sample, while ineffective degradation was observed with TDB-deg(16b), and no degradation was observed with MTDB. Figure 5 (c, d). To illustrate the specific binding and degradation of pseudoknots by these molecules, we performed the same experiment with an oligomer similar to the pseudoknot, but containing a severely disrupted third stem that prevented proper pseudoknot formation. As expected, no molecules affected the stability of this oligomer. Figure 5 e). Therefore, MTDB-deg(16a) shows both efficient and selective binding and degradation of pseudoknots.

[0962] To demonstrate that MTDB-deg(16a) is functional and capable of cleaving full-length coronavirus RNA, we incubated MTDB-deg(16a) and controls MTDB and TDB-deg(16b) with RNA extracted from SARS-CoV-2. Viral RNA was then analyzed on agarose gels. We observed degradation only in the lanes corresponding to MTDB-deg(16a). Figure 5 f) further confirmed that MTDB-deg(16a) did indeed degrade the natural coronavirus pseudoknot, while the two control molecules did not, highlighting the specificity of the method.

[0963] MTDB-deg specificity in degrading coronavirus pseudonodules

[0964] To further demonstrate that MTDB-deg(16a) cleaves viral RNA and obtain a more precise image of the cleavage location, we analyzed the cleaved genomic RNA (gRNA) using direct RNA nanopore sequencing. As expected, the region around the pseudoknot was most affected. Figure 6 a). Interestingly, the degradation level in the regions flanking the pseudoknot was higher than that in the pseudoknot itself. In fact, a study on the SARS-CoV-2 RNA interactome found that the regions around the frameshift elements formed extensive short-range and long-range interactions with neighboring ORF1a, especially ORF1b; it is likely that the proximity of these elements to the pseudoknot allowed MTDB-deg(16a) to effectively cleave them. Figure 6 a)(Ziv, et al., 2020). Interestingly, the only other structural element affected by this molecule is the S gene, which shows a long-range interaction with ORF1b (Ziv, et al., 2020), and is therefore expected to be within the range of the degrading agent ( Figure 6 b). Notably, no 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 interaction group (Ziv, et al., 2020).

[0965] Efficiency and specificity of pseudojunction degradation in SARS-CoV-2 infected cells

[0966] 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, thereby preventing viral replication. We performed an in vitro drug assay in which we measured SARS-CoV-2 replication in Vero CCL-81 cells. We observed that low micromolar concentrations of MTDB-deg(16a) exhibited significant antiviral activity. Figure 8 ac), before infection ( Figure 8 a; Figure 9 a) or after infection ( Figure 8 b; Figure 9 In cells treated with b), coronavirus RNA levels were significantly reduced. These results are supported by plaque assay results. Figure 8 d). Importantly, the control molecules MTDB and TDB-deg(16b) did not exhibit antiviral activity, although MTDB is known to disrupt the frameshift of SARS-CoV-2 (Kelly et al., 2020). Furthermore, we found that none of the compounds exhibited cytotoxicity against host cells, suggesting that the observed effects on viral replication are a result of the compound's specific antiviral activity. Figure 8 e). Oddly enough, the degrading agent exhibits lower activity at concentrations above 6 μM. Figure 9 c), although the absence of colloidal aggregation proves that these readings were observed on a dynamic light scattering screen. Furthermore, the ability of the virus to recover from 24-hour drug exposure was impaired in samples treated with MTDB-deg (16a), but not in samples treated with the control molecules MTDB and TDB-deg (16b). Figure 8 f; Figure 10 a). Finally, no antiviral effect was observed when cell-free virus was incubated with MTDB-deg(16a), MTDB, or TDB-deg(16b), indicating that the antiviral activity of MTDB-deg(16a) is mediated by directly inhibiting viral replication in host cells. Figure 10(b) In summary, the antiviral drug assays indicate that MTDB degrader (16a) is an effective antiviral agent against SARS-CoV-2 virus and has an irreversible effect specifically against coronavirus tristalk pseudoknot.

[0967] In vivo antiviral activity of pseudoknot degradation agents

[0968] An infected SARS-CoV-2 mouse model (transgenic K18-hACE2 mouse) was used to determine the in vivo antiviral activity of MTDB-deg 16a. Figure 12 a). Plaque assays showed that animals administered MTDB-deg 16a (25 mg / kg) exhibited a significantly lower pulmonary viral load compared to the vector control group. Figure 12 b). Furthermore, we investigated the in vivo antiviral potential of MTDB-deg 16a or the vector treatment using proteins extracted from the lungs of K18-hACE2 transgenic mice on day 3 or 6 post-infection. Reassuringly, we observed a strong reduction in p38 phosphorylation levels in the MTDB-deg 16a-treated group at both time points of infection. Figure 12 c) p38 is an important biomarker for SARS-CoV-2 infection and replication.

[0969] Targeting of bacterial ribosomes

[0970] Three degraders targeting bacterial ribosomes were rationally designed by conjugating the known ribosomal RNA conjugate chloramphenicol with azido-imidazolium of varying lengths (18a, 18b, 18c). The chloramphenicol binding site was generated by peptide coupling of propargyl acid with (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol using HATU and DIPEA, followed by a copper click chemistry coupling step. Copper-induced azido-acetylation cycloaddition (CuAAC) was used to link the two components, was tolerant to a wide range of substrates, and produced a triazole—a bioisosteric amide—with good tolerance in biological systems. The three ribosomal degraders and one binding control were tested in vitro and in cellular systems.

[0971] In vitro degradation of Escherichia coli ribozymes

[0972] Degradation activity was measured in vitro using E. coli ribosomes as the target. 200 μM ribozyme was incubated with different concentrations of the degrading agent ranging from 15 mM to 0.47 mM at 37 °C for 18 hours. Evaluation was performed by agarose gel electrophoresis.

[0973] Optimal results were obtained using the PEG-2 linker (18b), where ribosome degradation was observed at a concentration of 15 mM. Figure 11).

[0974] Exemplary Example 2:

[0975] Studies demonstrating the nucleic acid degradation activity of the new cleavage groups

[0976] Targeted degradation has become a frontier in drug development. The disruption of ribonucleic acid (RNA) chains in living systems is crucial for effective biological functions within organisms. Essential for a wide range of functions, RNA represents a significant target for disease disruption. Existing targeted degradation methods utilize cellular cofactors, complicating their potential applications. Furthermore, the specificity of these methods limits the compounds that can be used.

[0977] We recently described the development of a strategy for degrading specific RNAs using small molecules [Mikutis, S., et al.]. Azides attached to the cleavage group (“warhead”) of a basic degrading agent showed degradation of propynyl-modified RNA at the adenine base by hijacking methyltransferases. This degrading agent was covalently coupled to methylated RNA transcripts via a copper-promoted “click” chemistry, followed by specific cleavage at a nearby site mediated by spatial proximity. Two mechanisms were found to be at work. The first, determined by in vitro pH control, we suspect is base-mediated deprotonation at the 2'-O position on the ribose scaffold. The second mechanism, we propose, is transition metal-mediated, based on chelate control. These findings suggest that our strategy could constitute a robust and universal platform for degrading a wide range of RNA targets.

[0978] In 2019, a respiratory disease altered our lifestyles. The virus that caused this disease, known as COVID-19 or SARS-CoV-2, spurred an urgent need to develop drugs that can target and destroy viral mechanisms. In addition to covalent modifications, the secondary and tertiary structures of RNA complexes can also have pathological effects and thus serve as a therapeutic focus. Following our initial report on chemical degraders, as illustrated in Exemplary Example 1 of this paper, we developed a non-covalent strategy targeting the G-quadruplex: a tertiary structure associated with multiple diseases. The SARS-CoV-2 genome contains four putative G-quadruplex sites [Zhao, C., et al.].

[0979] Although the imidazole biomimetic cleavage group (warhead) we initially chose for the meClick-seq method was sufficient to validate the strategy's feasibility, its structural simplicity inherently limits its efficacy and pharmacokinetic (DMPK) properties, making it difficult to meet human therapeutic needs. Furthermore, due to the different topological structures of each new RNA conjugate upon recombination, degradation efficiency may vary from target to target. Therefore, we are attempting to establish a novel library of click-degrading agents to expand the pharmaceutical potential of our technology.

[0980] result

[0981] This paper discloses a library of chemical degrading agents. Guided by alkalinity theory predictions, the research team focused on designing degrading agents with pKa values ​​close to the physiological pH of 7.4 (ranging from 6.2 to 8.6). This pKa would allow for significant deprotonation of bases under physiological conditions, thus preserving their fundamental characteristics (Scheme A). We also investigated degrading agents with strong transition metal binding capabilities (cleaving groups 5 and 7). A wide range of degrading agents were evaluated to provide enhanced potency and to explore our mechanistic hypotheses (cleaving groups 2, 9, 11, and 14).

[0982]

[0983] Scheme A. A library of RNA degrading agents with cleavage groups constructed in this study. The numbers specify the pKa values ​​of specific functional groups in the range of 0-16, as predicted by computer using the MarvinView module in a KNIME 3.6.1 environment (part of the ChemAxon / Infocom Marvin Extensions 3.6.0 package). All degrading agents were prepared as warhead-hexaethylene glycol-azide structures.

[0984] To evaluate the efficacy of the degrading agent, an in vitro assay was performed, characterized by the direct functionalization of RNA oligonucleotides with the degrading agent warhead.

[0985] In summary, alkyne-labeled RNA oligonucleotides in HEPES pH 7.4 buffer were reacted with the degrading agent warhead-hexaethylene glycol-azide compound construct using a CuAAC (copper-catalyzed cycloaddition of alkyne) reaction. The mixture was then incubated at 37°C for 0–4 h, followed by copper quenching, and the reaction mixture was stored at 4°C. The mixture was then analyzed by LC-MS (liquid chromatography-mass spectrometry). The mass spectrometric signals corresponding to the oligonucleotide-degrading compound construct were then integrated, and the signals at different time points were compared with those at t=0 to assess the degradation. Figure 2 ).

[0986] As a reverse assay to assess the specificity of the degrading agent, non-alkyne-functionalized oligomers were treated with the degrading agent and CuAAC component under the same conditions. This oligonucleotide cannot be covalently functionalized with azide; therefore, all degradation observed in this case stemmed from non-specific interactions between the degrading agent warhead and the RNA oligonucleotide. In contrast, the degradation of covalently functionalized oligonucleotides, where no such degradation was observed, must have been a specific result of induced proximity.

[0987]

[0988] Protocol B. Overview of the in vitro assay protocol for assessing the efficiency of the degradation agent warhead. CuAAC is performed on alkyne-functionalized oligonucleotides, using a degradation agent warhead-hexaethylene glycol-azide construct to mount degradation agent cleavage groups onto the oligonucleotides via triazole and hexaethylene glycol bonds. The functionalized oligonucleotides are analyzed either immediately to obtain a t=0 reading, or the mixture is incubated at 37°C for 2 or 4 hours to assess degradation at these time points. Analysis is performed by liquid chromatography-mass spectrometry.

[0989] The tested degradative cleavage groups exhibited different activities, providing insights into the design of RNA degradative cleavage groups. Figure 13 We found that even the degradative agents with the smallest cleavage groups could induce degradation (2, 11). The most likely explanation is the ability of the linker to bind copper and bring it to the vicinity of the nucleic acid, as the lack of degradation on the CTRL chain does suggest that this effect is proximity-induced. Surprisingly, the positively charged warhead (11) predicted to induce more significant degradation under the assay conditions than the minimally charged uncharged warhead (2). This could be due to the interaction of the positively charged amino group with the negatively charged phosphodiester backbone, thus anchoring the linker to its vicinity, perhaps facilitating hydrogen bonding. Note that cleavage groups 9 and 10 have very similar structures, but 10 is a more effective degradative agent. Without being bound by any particular theory, we hypothesize that this phenomenon can be explained by the fact that the compound predicted a pKa value close to 7.4 (8.31), while 9 is more basic (predicted pKa 9.43), and therefore is fully protonated under these conditions and cannot function as a base. Interestingly, except for 4 and 5, none of the cleaving groups induced any damage on the CTRL strand, suggesting that they selectively degrade RNA only when brought near it. The phenanthroline-based degrader Warhead 5 is a well-known nucleic acid interceptor, which explains its ability to degrade RNA in a non-selective manner [Sigman, DS, et al.].

[0990] The efficiency of the 15 degrading agents in cleaving groups was then compared with that of the headless connector (degrading agent 2) and the imidazole-based degrading agent 1. Figure 14Interestingly, we found that three of the degrading agents exhibited lower degradation efficiency for the cleavage group than for the linker itself; this likely means that these parts do not promote degradation and act as steric blockers, preventing copper-bound linkers from approaching RNA oligonucleotides. Conversely, we found two of these degrading agents to be significantly more effective than imidazole degrading agent 1. We also found that one of these degrading agents, phenanthroline degrading agent 5, also degraded the control RNA strand, albeit to a much lesser extent than the functionalized strand, as previously described. However, degrading agent warhead 6 did not exhibit non-specific cleavage and specifically degraded covalently functionalized RNA. We found that eight other degrading agents exhibited moderate potency—they were more effective than the linker itself but less effective than the imidazole head, which is not surprising given the prevalence of imidazole in natural RNA degradation systems.

[0991] Libraries of novel, rationally designed RNA degrading cleavage groups have been prepared, and their efficiencies have been compared with those of previously described imidazole warhead 1 and warheadless hexaethylene glycol linkers in in vitro RNA degradation assays. These degraders exhibit broad potency. The principles described herein can be used to discover new, potential advanced RNA degrading cleavage groups.

[0992] Experimental Section

[0993] In vitro degrading agent direct functionalization of warhead - degrading agent efficiency evaluation reaction

[0994] CuSO4 (final concentration 1.0 mM), THPTA (3.0 mM), the degrading agent warhead-hexaethylene glycol-azide construct (2.0 mM), and RNA oligomer (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 min. The reaction was quenched immediately or after incubation at 37 °C for 2 or 4 hours. After quenching, the reaction mixture was analyzed using LC-MS. The MS signal corresponding to the initial degrading agent functionalization concentration was estimated from the reaction quenched immediately after 10 min functionalization by integrating an intensity of 3 or 4 m / z corresponding to the appropriate RNA species. The MS signal corresponding to degradation at 2 or 4 hours was compared with the t=0 signal.

[0995] Chemical synthesis

[0996]

[0997] 17-Hydroxy-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate

[0998] Hexaethylene glycol (3.0 g, 11 mmol) was dissolved in DCM and cooled to 0 °C, then p-toluenesulfonyl chloride (2.2 g, 12 mmol) and TEA (2.1 g, 2.9 mL, 21 mmol) were added. The solution was stirred at 0 °C for 3 hours, then at room temperature for 30 minutes, and quenched with H₂O (20 mL). The organic solvent and volatiles were removed under vacuum, followed by silica gel column purification (dry loading, gradient from EtOAc containing 5% methanol to EtOAc containing 10% methanol). The resulting product was a colorless oil (1.72 g, 3.9 mmol, 37%).

[0999] 1 H NMR (400MHz, CDCl3) δ7.79(d,J=7.9Hz,2H),7.33(d,J=7.9Hz,2H),4.15(t,J=4.4Hz,2H),3.72-3.58(m,22H),2.52(s,1H),2.44(s,3H). 13 C NMR (101MHz, CDCl3) δ 144.89, 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: Calculated m / z is [C 19 H 32 O 9 S] + 437.1854; observed 437.1845.

[1000] 17-Azide-3,6,9,12,15-pentaheptadecane-1-ol (1)

[1001]

[1002] 17-Hydroxy-3,6,9,12,15-pentaheptadecyl-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 hours. The solvent was removed under vacuum. To remove trace amounts of DMF, the residue was continuously co-evaporated with a portion of toluene. The resulting residue was dissolved in DCM and filtered. The resulting residue was purified on a column (gradient, 9:1 DCM:MeOH). The product was a pale yellow oil (200 mg, 0.64 mmol, 54%).

[1003] 1H NMR (400MHz, CDCl3) δ3.72–3.59 (m, 22H), 3.38 (t, J = 5.1Hz, 2H), 2.54 (s, 1H). 13 C NMR (101MHz, CDCl3) δ 72.75, 70.78, 70.75, 70.68, 70.64, 70.41, 70.15, 61.85, 50.82. HRMS m / z: Calculated m / z is [C 12 H 25 N3O6Na] + 330.1652; observed 330.1641.

[1004] 1-(17-azido-3,6,9,12,15-pentaheptadecyl)-1H-imidazolium (2)

[1005]

[1006] 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 nitrogen atmosphere, heated to room temperature, and stirred for 30 min. Imidazole (250 mg, 0.54 mmol) was dissolved in anhydrous DMF (3 mL), and the resulting solution was added to the first mixture. The mixture was then stirred at 55 °C for 20 h. The solvent was then removed under vacuum, and the residue was purified by rapid chromatography (dry loading, gradient EtOAC to 9:1 EtOAC:MeOH). The product was a colorless oil (154 mg, 0.43 mmol, 80%).

[1007] 1 H NMR (400MHz, 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 (100MHz, CDCl3)δ C 137.6, 129.3, 119.4, 70.6-70.7 (multiple PEG peaks), 70.0, 50.7, 47.0. HRMS m / z: [M+H] + : Calculate m / z as [C 15 H 28 N5O5] + 358.2090; observed at 358.2084.

[1008] 4-((17-azido-3,6,9,12,15-pentaheptadecyl)oxy)-pyridine(3)

[1009]

[1010] 4-Hydroxypyridine (77.4 mg, 810 mmol), hexaethylene glycol azide (200 mg, 650 μmol), and triphenylphosphine (214 mg, 810 μmol) were dissolved in dry THF, degassed by freeze-suction-thaw (3 cycles), and placed at 0 °C under an argon atmosphere. DIAD (165 mg, 810 μmol) was then added, and the mixture was heated to room temperature and stirred overnight. The reaction was then quenched by adding 3 g / L NH₄Cl solution (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). After removing the organic solvent, the resulting yellow oil was purified on a silica gel column (gradient, AcOEt to AcOEt containing 10% MeOH and 1% TEA). The yellow oily title compound was obtained (82 mg, 0.21 mmol, 33%).

[1011] 1 H NMR (400MHz, CDCl3): δ7.34(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 (100MHz, CDCl3): δ C 179.0, 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] + : Calculate m / z as [C 17 H 28 N4O6] + The calculated m / z is 385.2082; observed m / z is 385.2079. 4-((17-azido-3,6,9,12,15-pentaheptadecyl)oxy)-2-methylpyridine (4)

[1012]

[1013] A mixture of 2-methylpyridin-4-ol (10.9 mg, 0.10 mmol, 1.0 equivalent), 17-azido-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate (47.3 g, 0.10 mmol, 1.0 equivalent), and K₂CO₃ (26.6 mg, 0.20 mmol, 2.0 equivalent) in acetonitrile (2 mL) was stirred in a sealed tube at 85 °C for 16 hours under nitrogen. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through diatomaceous earth and concentrated under vacuum. The resulting residue was purified on silica (eluting with 80% acetone / hexane) to give a colorless oily title compound (28.1 mg, 0.071 mmol, 71%).

[1014] 1 H NMR (400MHz, CDCl3) δ8.29(d,J=5.8Hz,1H),6.68(d,J=2.4Hz,1H),6.64(dd,J=5.8,2.4Hz,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=5.1Hz,2H),2.49(s,3H); 13 C NMR (126MHz, CDCl3) δ165.3,160.1,150.4,109.6,107.8,71.0,70.8,70.8,70.8,70.7,70.7,70.7,70.2,69.5,67.2.HRMS m / z:[M+H] + Calculated as [C 18 H 31 N4O6] + 399.2238, actual measurement: 399.2243.

[1015] 20-Azide-N-(1,10-phenanthroline-5-yl)-3,6,9,12,15,18-hexaoxanetetracarbamate (5)

[1016]

[1017] Hexaethylene glycol azide (48.7 mg, 320 μmol) was dissolved in THF (1.5 mL) and placed under an argon atmosphere at 0 °C. Then, NaH (60% in mineral oil, 6.3 mg, 320 μmol) was added, and the mixture was stirred at 0 °C for 30 minutes, followed by stirring at room temperature for 30 minutes. Then, 2-bromo-N-(1,10-phenanthroline-5-yl)acetamide (49.7 mg, 160 μmol) was added, and the mixture was stirred overnight at room temperature. The solvent was then removed under vacuum; the resulting red oil was purified by HPLC and lyophilized. The red oily title compound (18.3 mg, 33.7 μmol, 17%) was obtained.

[1018] 1 H NMR (400MHz, CDCl3): δ8.92(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 (100MHz, D2O): 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] + Calculated as [C 17 H 28 N4O6] + The calculated m / z is 543.2562; the observed value is 543.2560.

[1019] 1-(1-(17-azido-3,6,9,12,15-pentaheptadecyl)-1H-imidazol-2-yl)-N,N-dimethylmethylamine (6)

[1020] At 0 °C, NaH (5.1 mg, 0.211 mmol, 1.3 equivalent) was added to a solution of 1-(1H-imidazol-2-yl)-N,N-dimethylmethylamine (24.4 mg, 0.195 mmol, 1.2 equivalent) and 17-azido-3,6,9,12,15-pentaheptadecyl 4-methylbenzenesulfonate (75.0 mg, 0.160 mmol, 1.0 equivalent) in DMF (2 mL), and the mixture was then heated to room temperature and stirred at 55 °C for 14 hours. The reaction mixture was then cooled and carefully quenched with H₂O (25 mL) at 0 °C. The aqueous layer was then extracted with EA (25 mL × 3). The combined organic layers were dried over Mg₂SO₄ and concentrated under reduced pressure. The crude residue was purified on alkaline alumina and eluted with a 0% to 30% gradient of MeOH / EtOAc to give the title compound (35.1 mg, 0.085 mmol, 53%) as a colorless oil.

[1021] 1 H NMR (500MHz, CDCl3) δ6.98(s,1H),6.89(s,1H),4.20(t,J=5.4Hz,2H),3.87(s,2H),3.72(t,J=5.1Hz,2H),3. 67-3.58(m,12H),3.56-3.55(m,6H),3.72(t,J=5.1Hz,1H),3.51(s,2H),3.37(t,J=4.9Hz,2H),2.18(s,6H); 13 C NMR (126MHz, CDCl3) δ145.4,127.1,121.1,70.8,70.8,70.8,70.8,70.7,70.7,70.6,70.1,56.3,50.8,45.9,45.4.HRMS m / z:[M+H] + Calculated as [C 18 H 35 N6O5] + 415.2663, actual measurement: 415.2662.

[1022] N-((1H-imidazol-2-yl)methyl)-2-(ethio)-N-(2-(ethio)ethyl)ethyl-1-amine

[1023]

[1024] 1H-imidazol-2-carboxaldehyde (3 mg, 3.67 mmol, 1.1 equivalents) and bis(2-(ethylthio)ethyl)amine (646 mg, 3.34 mmol, 1.0 equivalents) were mixed in THF (12 mL) at room temperature under N2. Glacial acetic acid (200 mL, 3.34 mmol, 1 equivalent) was added, followed by sodium triacetoxyborohydride (1.06 g, 5.01 mmol, 1.5 equivalents), and the mixture was stirred at 20 °C for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO3 solution (50 mL), and the product was extracted with EtOAc. The combined organic phases were dried over magnesium sulfate and concentrated. The residue was purified on basic alumina and eluted with a 0% to 70% gradient of EtOAc / petroleum ether (40–60 b.p.) to give the title compound as a brown gel (320 mg, 1.17 mmol, 35%).

[1025] 1 H NMR (700MHz, CDCl3) δ6.98 (s, 2H), 3.80 (s, 2H), 2.76 (t, J = 6.7Hz, 4H), 2.63 (t, J = 7.6Hz, 4H), 2.49 (q, J = 7.4Hz, 4H), 1.22 (t, J = 7.4Hz, 6H); 13 CNMR(126MHz, CDCl3)δ145.0,121.4,53.9,51.7,29.3,26.2,15.0.HRMS m / z:[M+H] + Calculated as [C 12 H 24 N3S2] + 274.1406, actual measurement: 274.1409.

[1026] N-((1-(17-azido-3,6,9,12,15-pentaheptadecyl)-1H-imidazol-2-yl)methyl)-2-(ethylthio)-N-(2-(ethylthio)ethyl)ethyl-1-amine(7)

[1027]

[1028] To a solution of N-((1H-imidazol-2-yl)methyl)-2-(ethylthio)-N-(2-(ethylthio)ethyl)ethyl-1-amine (78 mg, 0.283 mmol, 1.2 equivalents) in anhydrous DMF (2 mL), NaH (12 mg, 0.295 mmol, 1.25 equivalents) was added, and the resulting suspension was stirred at 0 °C for 30 min. Then, 1 mL of 17-azido-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate (109 mg, 0.236 mmol, 1.0 equivalents) in anhydrous DMF was added at 0 °C. The mixture was heated to room temperature and stirred at 55 °C for 20 h. The reaction mixture was then cooled and carefully quenched at 0 °C with a saturated NaHCO3 solution (25 mL). 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 and equilibrated with 1% NEt3 / EtOAc. The title product was eluted with a gradient of 0% to 8% methanol / ethyl acetate to give a yellow oil (99 mg, 0.176 mmol, 74%).

[1029] 1 H NMR (700MHz, CDCl3) δ7.07(s,1H),6.96(s,1H),4.37(t,J=4.5Hz,2H),3.87(s,2H),3.76(t,J=5.2Hz,2H),3.68-3.63(m,12H),3. 62-3.57(m,6H),3.38(t,J=5.1Hz,2H),2.72(t,J=7.2Hz,4H),2.57(t,J=7.2Hz,4H),2.47(q,J=7.4Hz,4H),1.21(t,J=7.4Hz,6H); 13 C NMR (126MHz, CDCl3) δ145.0,127.2,121.4,71.0,70.9,70.8,70.8,70.8,70.8,70.7,70.2,53.9,51.7,50.8,46.1,29.3,26.2,15.0.HRMS m / z:[M+H] + Calculated as [C 24 H 47 N6O5S2] + 563.3044, actual measurement: 563.3045. 4-(17-azido-3,6,9,12,15-pentaheptadecyl)morpholine (8)

[1030]

[1031] 17-Azide-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate (46.4 mg, 0.10 mmol, 1.0 equivalent) and K₂CO₃ (26.6 mg, 0.20 mmol, 2.0 equivalent) were added to a 4 mL reaction flask, sealed, and placed under nitrogen atmosphere. Anhydrous acetonitrile (2 mL) was then added, followed by morpholine (12.9 mL, 0.15 mmol, 1.5 equivalent). The sealed tube was then heated at 50 °C for 18 hours. The reaction was cooled, and ethyl acetate (20 mL) was added. The mixture was filtered through diatomaceous earth and concentrated under vacuum. The resulting residue was purified on alkaline alumina (eluted with a hexane solution of 70% ethyl acetate) to give a colorless oily title compound (15.5 mg, 0.041 mmol, 41%).

[1032] 1 H NMR (500MHz, CDCl3) δ3.68-3.63(m,16H),3.62-3.60(m,2H),3.57(t,J=5.9Hz,2H),3.38(t,J=5.1Hz,2H),2.50(t,J=5.9Hz,2H),2.25(s,6H). 13 CNMR(126MHz, CDCl3)δ70.8,70.8,70.8,70.7,70.7,70.5,70.2,69.5,58.9,50.8,46.0.HRMS m / z:[M+H] + Calculated as [C 16 H 33 N4O6] + 377.2395, 377.2399.

[1033] 4-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol(9)

[1034]

[1035] 4-Mercaptophenol (18.9 mg, 0.15 mmol, 1.0 equivalent) and sodium bicarbonate (30.0 mg, 0.30 mmol, 2.0 equivalent) were added to a 4 mL reaction flask and placed under nitrogen atmosphere. Anhydrous acetonitrile (1 mL) was then added and the mixture was stirred at 25 °C for 1 hour. A dry acetonitrile solution (2 mL) of 17-azido-3,6,9,12,15-pentaheptadecyl 4-methylbenzenesulfonate (71.9 g, 0.156 mmol, 1.04 equivalent) was added, and the sealed tube was heated to 50 °C and maintained for 16 hours. The reaction was cooled, ethyl acetate (20 mL) was added, the mixture was filtered through diatomaceous earth, and concentrated under vacuum. The resulting residue was purified on silica (eluting with 1% MeOH / CH2Cl2) to yield a colorless oily title compound (39.9 mg, 0.096 mmol, 64%).

[1036] 1 H NMR (500MHz, CDCl3) δ7.35-7.32(m,2H),6.81-6.78(m,2H),3.68-3.55(m,20H),3.38(t,J=5.2Hz,2H),2.97(t,J=6.8Hz,2H); 13 C NMR (126MHz, CDCl3) δ155.8,134.1,116.3,70.9,70.9,70.8,70.7,70.7,70.6,70.5,70.3,70.2,50.8,35.3.HRMS m / z:[MH] - Calculated as [C 18 H 28 N3O6S] - 414.1704, actual measurement: 414.1705.

[1037] 4-((17-azido-3,6,9,12,15-pentaheptadecyl)sulfinyl)phenol(10)

[1038]

[1039] Potassium peroxide (oxone) (25 mg, 0.165 mmol, 2.5 equivalences) was added to a solution of 4-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol (28 mg, 0.066 mmol, 1 equivalent) in 1:1 H₂O / ethanol (4 mL). The resulting suspension was stirred at 20 °C for 14 h. Distilled water (50 mL) was added and the pH was adjusted to 5.0 with 1 M HCl. The aqueous solution was extracted with CHCl₃ (4 × 50 mL), and the combined organic phases were dried over anhydrous magnesium sulfate and concentrated. The residue was purified on silica and eluted with 9% methanol dissolved in dichloromethane to give the title compound (26.2 mg, 92%, 0.61 mmol) as a colorless oil.

[1040] 1 H NMR(500MHz, CDCl3)δ7.50(d,J=8.7Hz,2H),6.97(d,J=8.7Hz,2H),3.84-3.80(m,1H ),3.69-3.52(m,19H),3.37(t,J=5.1Hz,2H),3.13-3.08(m,1H),3.01-2.96(m,1H); 13 C NMR (126MHz, CDCl3) δ160.1,132.6,126.6,116.7,70.8,70.7,70.7,70.7,70.7,70.6,70.1,64.1,57.4,50.7.HRMS m / z:[MH] - Calculated as [C 18 H 28 N3O7S] - 430.1653, actual measurement: 430.1652.

[1041] 17-Azide-N,N-Dimethyl-3,6,9,12,15-pentaheptadecane-1-amine (11)

[1042]

[1043] 17-Azide-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate (46.4 mg, 0.10 mmol, 1.0 equivalent) and K₂CO₃ (26.6 mg, 0.20 mmol, 2.0 equivalent) were added to a 4 mL reaction flask, sealed, and placed under nitrogen atmosphere. Anhydrous acetonitrile (2 mL) was then added, followed by a 2 M solution of diethylamine in methanol (75 mL, 0.15 mmol, 1.5 equivalent). The sealed tube was then heated at 50 °C for 18 hours. The reaction was cooled, and ethyl acetate (20 mL) was added. The mixture was filtered through diatomaceous earth and concentrated under vacuum. The resulting residue was purified on alkaline alumina (eluting with ethyl acetate) to give a colorless oily title compound (28.1 mg, 0.084 mmol, 84%).

[1044] 1 H NMR (500MHz, CDCl3) δ3.68-3.63(m,16H),3.62-3.60(m,2H),3.57(t,J=5.9Hz,2H),3.38(t,J=5.1Hz,2H),2.50(t,J=5.9Hz,2H),2.25(s,6H). 13 CNMR(126MHz, CDCl3)δ70.8,70.8,70.8,70.7,70.7,70.5,70.2,69.5,58.9,50.8,46.0.HRMS m / z:[M+H] + Calculated as [C 14 H 31 N4O5] + 335.2289, actual measurement: 335.2285.

[1045] 17-Azide-N-(2,4,6-trimethylpyridin-3-yl)-3,6,9,12,15-pentaheptadecanoamide (12)

[1046]

[1047] To a solution of 17-azido-3,6,9,12,15-pentaheptadecanoic acid (30 mg, 0.09 mmol, 1 equivalent) in 1 mL of anhydrous CH₂Cl₂, HATU (38 mg, 0.1 mmol, 1.1 equivalent) was added, followed by the dropwise addition of triethylamine (20 mg, 0.2 mmol, 2.2 equivalent). The solution was cooled to 0 °C under a nitrogen atmosphere and incubated for 30 min, followed by the addition of a solution of 1,2-thing (13.6 mg, 0.11 mmol, 1.1 equivalent) in 1 mL of anhydrous CH₂Cl₂. The reaction mixture was heated to 20 °C and stirred for 14 h. The resulting mixture was diluted with CH₂Cl₂ and washed with saturated sodium carbonate solution (3 × 20 mL). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified chromatographically by elution with 5% methanol in ethyl acetate to give the title compound as a yellow oil (5 mg, 11%, 0.011 mmol).

[1048] 1 H NMR(500MHz, CDCl3)δ9.10(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. HRMS m / z:[M+H] + Calculated as [C 20 H 34 N5O6] + 440.2504, actual measurement: 440.2510.

[1049] 4-((17-azido-3,6,9,12,15-pentaheptadecyl)oxy)-6-methoxyquinoline(13)

[1050]

[1051] The compound, consisting of 6-methoxyquinoline-4-ol (35.0 mg, 0.20 mmol, 1.0 equivalent), 17-azido-3,6,9,12,15-pentaheptadecyl-4-methylbenzenesulfonate (92.3 g, 0.20 mmol, 1.0 equivalent), and K₂CO₃ (55.3 mg, 0.40 mmol, 2.0 equivalent) in acetonitrile (2 mL), was stirred in a sealed tube at 85 °C for 16 h under nitrogen. The reaction was cooled, ethyl acetate (20 mL) was added, and the mixture was filtered through diatomaceous earth and concentrated under vacuum. The resulting residue was purified on silica (eluting with 50% acetone / hexane) to give the title compound (67.5 mg, 0.145 mmol, 73%) as a colorless oil.

[1052] 1 H NMR (500MHz, CDCl3) δ8.62(d,J=5.5Hz,1H),8.08(d,J=9.2Hz,1H),7.48(d,J=2.9Hz,1H),7.39(dd,J=9.2,2.9Hz,1H),6.82(d,J=5.5Hz ,1H),4.42(dd,J=5.6,4.2Hz,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=5.0Hz,2H); 13 C NMR (126MHz, CDCl3) δ161.7,157.8,147.3,140.8,129.0,123.0,122.1,101.1,100. 1,71.0,70.7,70.7,70.7,70.6,70.6,70.0,69.3,68.4,55.7,50.7.HRMSm / z:[M+H] + Calculated as [C 22 H 32 N4O7] + 465.2344, actual measurement: 465.2353.

[1053] N-(6-aminopyridin-2-yl)-17-azido-3,6,9,12,15-pentaheptadecanoamide (14)

[1054]

[1055] To a solution of 17-azido-3,6,9,12,15-pentaheptadecanoic acid (126 mg, 0.39 mmol, 1 equivalent) in 1 mL of anhydrous CH₂Cl₂, HATU (224 mg, 0.59 mmol, 1.5 equivalent) was added, followed by the dropwise addition of triethylamine (99 mg, 0.23 mmol, 2.5 equivalent). The solution was cooled to 0 °C under a nitrogen atmosphere and incubated for 30 min, then a dry CH₂Cl₂ / DMF solution of 1,2-diaminopyridine (193 mg, 1.8 mmol, 4.5 equivalent) was added. The reaction mixture was heated to 20 °C and stirred for 14 h. The resulting mixture was diluted with CH₂Cl₂ and washed with saturated sodium carbonate solution (3 × 20 mL). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by chromatography and eluted with ethyl acetate to give the title compound as a yellow oil (100 mg, 81%, 0.24 mmol).

[1056] 1 H NMR (500MHz, CDCl3) δ8.84(s,1H),7.53(d,J=7.9Hz,1H),7.44(t,J=7.9Hz,1H),6.25(dd,J=7.9,0.7Hz,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=5.1Hz,2H). 13 C NMR (126MHz, CDCl3) δ168.6,157.5,149.4,140.0,104.6,103.5,71.5,70.9,70.7,70.7,70.7,70.4,70.1,50.8.HRMS m / z:[M+H] + Calculated as [C 17 H 29 N6O6] + 413.2143, actual measurement: 413.2147.

[1057] 17-Azide-N-(2-(dimethylamino)ethyl)-3,6,9,12,15-pentaheptadecanoamide (15)

[1058]

[1059] Under a nitrogen atmosphere at 0°C, N2 was added dropwise to a stirred solution of 17-azido-3,6,9,12,15-pentaheptadecanoic acid (63 mg, 0.20 mmol, 1.25 equivalents), HATU (119 mg, 0.31 mmol, 2 equivalents), and triethylamine (55 mL, 0.39 mmol, 2.5 equivalents) in anhydrous dichloromethane. 1 N 1 -Dimethylethane-1,2-diamine (17 mL, 0.16 mmol, 1.0 equivalent). The resulting mixture was stirred at 0 °C for 30 min, then heated to 20 °C and stirred for another 14 h. The solvent was removed under vacuum, and the residue was redissolved in ethyl acetate (50 mL) and transferred to a separatory funnel. A saturated aqueous solution of sodium bicarbonate (10 mL) and H₂O (40 mL) were added, and the funnel was shaken to separate the layers. The aqueous layer was further extracted with ethyl acetate (2 × 50 mL), and the combined organic phases were dried over anhydrous magnesium sulfate and concentrated. The residue was purified on basic alumina (eluted with a gradient of 0% to 10% methanol / CH₂Cl₂) to give the title compound (25.0 mg, 0.064 mmol, 40%) as a colorless oil. 1 H NMR (500MHz, CDCl3) δ7.15 (br.s, 1H), 3.98 (s, 2H), 3.67-3.64 (m, 18H), 3.37 (q, J = 6.0Hz, 4H), 2.44 (t, J = 6.4Hz, 2H), 2.25 (s, 6H). 13 CNMR(126MHz, CDCl3)δ170.0,71.1,70.8,70.8,70.7,70.7,70.7,70.7,70.4,70.1,58.2,50.8,45.4,36.5.HRMS m / z:[M+H] + Calculated as [C 16 H 34 N5O6] + 392.2504, actual measurement: 392.2506.

[1060] 4-((17-azido-3,6,9,12,15-pentaheptadecyl)sulfonyl)phenol(16)

[1061]

[1062] To a solution of 4-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol (12.4 mg, 0.030 mmol, 1 equivalent) in methanol (2 mL), ammonium molybdate hydrate (1.7 mg, 0.006 mmol, 5 mol%) and 30% wt aqueous hydrogen peroxide solution (12 μL, 0.12 mmol, 4 equivalents) were added. The resulting suspension was stirred at 20 °C for 2 hours. Ethyl acetate (5 mL) was added, and the mixture was filtered through diatomaceous earth and concentrated under vacuum. The residue was purified on silica and eluted with 2.5% methanol dissolved in dichloromethane to give a colorless oily title compound (6.8 mg, 51%, 0.015 mmol).

[1063] δ 1 H NMR(400MHz, CDCl3) δ7.76(d,J=8.8Hz,2H),6.98(d,J=8.7Hz,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] - Calculated as [C 18 H 28 N3O8S] - 446.1603, actual measurement: 446.1602.

[1064] 2-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol(17)

[1065]

[1066] 2-Mercaptophenol (30.2 μL, 0.30 mmol, 1.0 equivalent) and sodium bicarbonate (60.0 mg, 0.60 mmol, 2.0 equivalent) were added to a 4 mL reaction flask and placed under nitrogen atmosphere. Anhydrous acetonitrile (1 mL) was then added and the mixture was stirred at 25 °C for 1 hour. A dry acetonitrile solution (2 mL) of 17-azido-3,6,9,12,15-pentaheptadecyl 4-methylbenzenesulfonate (143.8 g, 0.312 mmol, 1.04 equivalent) was added, and the sealed tube was heated to 50 °C and maintained for 16 hours. The reaction was cooled, ethyl acetate (20 mL) was added, the mixture was filtered through diatomaceous earth, and concentrated under vacuum. The resulting residue was purified on silica (eluting with 15% acetone / hexane) to give a colorless oily title compound (14.6 mg, 0.035 mmol, 12%).

[1067] 1H NMR (500MHz, CDCl3) δ7.48 (dd, J=7.6, 1.7Hz, 1H), 7.28-7.24 (m, overlapped with solvent peak,1H),6.92(dd,J=8.2,1.3Hz),6.84(td,J=7.6,1.3Hz),7.68-7.62(m,18H),3.54(t,J=6.0Hz,2H),3.38(t,J=5.1Hz,2H),2.89(t,J=6.0Hz,2H).HRMS m / z:[MH] - Calculated as [C 18 H 28 N3O6S] - 414.1704, actual measurement: 414.1699.

[1068] 2-((17-azido-3,6,9,12,15-pentaheptadecyl)sulfinyl)phenol(18)

[1069]

[1070] Potassium peroxide (23.3 mg, 0.153 mmol, 2.5 mmol) was added to a solution of 2-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol (25.5 mg, 0.061 mmol, 1 equivalent) in 1:1 H₂O / ethanol (4 mL). The resulting suspension was stirred at 20 °C for 14 h. Distilled water (50 mL) was added and the pH was adjusted to 5.0 with 1 M HCl. The aqueous solution was extracted with CHCl₃ (4 × 50 mL), and the combined organic phases were dried over anhydrous magnesium sulfate and concentrated. The residue was purified on silica and eluted with 2% methanol dissolved in dichloromethane to give the title compound (22.3 mg, 85%, 0.052 mmol) as a colorless oil.

[1071] 1 H NMR (500MHz, CDCl3) δ7.37-7.33(m,1H),7.16(dd,J=7.56,1.4Hz),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=5.0Hz,2H),3.23-3.18(m,1H); 13C NMR (126MHz, CDCl3) δ133.1,125.8,121.9,119.9,119.4,70.9,70.8,70.8,70.7,70.7,70.7,70.5,70.1,64.1,55.7,50.8.HRMS m / z:[MH] - Calculated as [C 18 H 28 N3O7S] - 430.1653, actual measurement: 430.1650.

[1072] 2-((17-azido-3,6,9,12,15-pentaheptadecyl)sulfonyl)phenol(19)

[1073]

[1074] To a solution of 2-((17-azido-3,6,9,12,15-pentaheptadecyl)thio)phenol (19.5 mg, 0.047 mmol, 1 equivalent) in methanol (2 mL), ammonium molybdate hydrate (2.7 mg, 0.002 mmol, 5 mol%) and 30% wt aqueous hydrogen peroxide solution (19 μL, 0.19 mmol, 4 equivalents) were added. The resulting suspension was stirred at 20 °C for 2 hours. Ethyl acetate (5 mL) was added, and the mixture was filtered through diatomaceous earth and concentrated under vacuum. The residue was purified on silica and eluted with 2.5% methanol dissolved in dichloromethane to give a colorless oily title compound (14 mg, 67%, 0.031 mmol).

[1075] δ 1 H NMR(500MHz, CDCl3)δ8.93(br.s,1H),7.66(dd,J=7.6,1.7Hz,1H),7.52-7.49(m,1H),7.03-6.99(m,2H),3.85(t,J=6.0Hz, 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=5.1Hz,2H); 13 C NMR (126MHz, CDCl3) δ156.6,136.4,129.5,122.6,120.5,118.9,70.8,70.8,70.8,70.8,70.7,70.7,70.7,70.4,70.2,64.5,56.9,50.8.HRMS m / z:[MH] - Calculated as [C18 H 28 N3O8S] - 446.1603, actual measurement: 446.1600.

[1076] Exemplary Example 3:

[1077] Synthesis and Evaluation of PDS-Amimi

[1078] Following the procedure outlined in Exemplary Example 1, the compound PDS-Amimi, as shown below, was synthesized, and its activity as a degrading agent was evaluated together with PDS-deg6 (prepared as described in Exemplary Example 1) and the non-degrading agent control CBX-PDS (prepared as described in Exemplary Example 1).

[1079] 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 H2O and tert-butanol (2.1 mL). Copper sulfate pentahydrate solution (207 μL, 100 mM, 20.7 μmol) was added, followed by sodium ascorbate solution (1.07 mL, 100 mM, 107 μmol). The turbid yellow solution was placed under argon and stirred for 10 minutes. Then, a solution of 1-(1-(17-azido-3,6,9,12,15-pentaheptadecyl)-1H-imidazol-2-yl)-N,N-dimethylmethylamine (6) (2.9 mL, 10 mM, 29 μmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. The solvent was then removed under vacuum. The product was then purified by HPLC (gradient from 100% H₂O, 0.1% FA to 100% MeCN, 0.1% FA). The title compound was given as a beige solid (7.2 mg, 7.2 μmol, 35%). HRMS m / z: [M+H] + Calculated as [C 50 H 64 N 13 O 10 ] + 1006.4899, actual measurement: 1006.4896.

[1080]

[1081] Measurement scheme

[1082] Protocol: The G4-forming RNA oligomer (final concentration 200 μM, sequence 5'-UGUGGGAGGGGCGGGUCUGGGUGC-3') was added to a pH 7.5 HEPES (20 mM) buffer supplemented with KCl (100 mM) and MgCl2 (10 mM). The mixture was heated at 95 °C for 5 min, then kept on ice for 30 min. CuSO4 (final concentration 200 μM), THPTA (700 μM), and NaAsc (50 mM), along with CBX-PDS, PDS-deg6, or PDS-AmImi (200 μM), were then added. The reaction mixture was incubated at 37 °C for 4 h, quenched with EDTA (final concentration 12 mM), and then maintained at 4 °C. The reaction mixture was analyzed by LC-MS.

[1083] The results are as follows Figure 15 As shown.

[1084] PDS-Amimi was observed to be a more effective degradative agent than PDS-deg6.

[1085] References

[1086] To more fully describe and disclose the present invention and the state of the art to which it pertains, numerous publications have been cited above. The full citations of these references are as follows. The entire contents of each of these references are incorporated herein by reference.

[1087] Bobbin, et al., "RNA Interference (RNAi)-Based Therapeutics: Delivering on the Promise?", Annual Review of Pharmacology and Toxicology, 2016, Vol.56, pp.103-122.

[1088] Cox, et al., "RNA editing with CRISPR-Cas13", Science, 2017, Vol.358, pp.1019-1027.

[1089] Gasiunas, et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria”, Proc. Sci. U.S.A., 2012, Vol. 109, E2579-E2586. Rangasamy, Murugesan

[1090] Jinek, et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science, 2012, Vol. 337, pp. 816-821.

[1091] 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.

[1092] Li. “Minimap2: pairwise alignment for nucleotide sequences”, Bioinformatics, 2018, Vol. 34, pp. 3094–3100.

[1093] 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.

[1094] Park et al.,“Identification of RNA Pseudoknot-Binding Ligand ThatInhibits the-1Ribosomal Frameshifting of SARS-Coronavirus by Structure-BasedVirtual Screening”,J.Am.Chem.Soc.,2011,Vol.133,pp.10094–10100.

[1095] Santos et al.,“G-Quadruplexes and Their Ligands:Biophysical Methodsto Unravel G-Quadruplex / Ligand Interactions”,Pharmaceuticals,2021,Vol.14,No.769

[1096] Sigman,D.S.,et al.,Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline.cuprous complex.Inhibition of Escherichia coli DNA polymeraseI.J Biol Chem,1979.254(24):p.12269-72.

[1097] Tzelepis,et al.,“A CRISPR Dropout Screen Identifies GeneticVulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia”,CellReports,2016,Vol.17,pp.1193-1205.

[1098] Zamore,et al.,“RNAi:Double-Stranded RNA Directs the ATP-DependentCleavageof mRNA at 21 to 23 Nucleotide Intervals”,Cell,2000,Vol.101,pp.25-33.

[1099] 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.

[1100] 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: CLB(I) in: C is the cleavage group defined in this article; L is a connector; and B is a non-covalently bonded group. The condition is that C is not: (i) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or (ii) Nucleic acid cleavage group of formula Z: in: Indicates the connection point with L; Ring A is absent or contains a nitrogen-containing heteroaryl or heterocyclic ring, optionally further composed of one or more elements selected from halogen, 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 Substituents of R; where R c and R d Selected from hydrogen or (1-4C) alkyl; The integer a1 is 0, 1, 2, or 3; Ra and Rb are each independently selected from hydrogen or (1-2C) alkyl groups each time they appear; 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, and each is optionally selected by one or more elements selected from halogen, 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 Substituents of R; where R e and R f Selected from hydrogen or (1-4C)alkyl, or R1 and R2 connected such that they together with the attached atoms form a 4-6 membered heterocycle or a 5- or 6-membered heteroaryl group, wherein any 4- or 6 membered heterocycle or 5- or 6-membered heteroaryl group is optionally surrounded by one or more atoms selected from halogen, trifluoromethyl, trifluoromethoxy, cyano, hydroxy, (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 Substituents of R, wherein R g and R h Selected from hydrogen or (1-4C) alkyl; When ring A is absent, R1 and R2 are each independently selected from hydrogen, heterocycle, heterocyclic-(1-3C)alkyl, heteroaryl, heteroaryl-(1-3C)alkyl, (3-6C)cycloalkyl, or (3-6C)cycloalkyl-(1-3C)alkyl, and each is optionally selected by one or more elements selected from halogen, 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 Substituents of R, wherein R e and R f Selected from hydrogen or (1-4C) alkyl, provided that R1 and R2 cannot both be hydrogen.

2. The bifunctional compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein C comprises a basic nitrogen atom or a hydroxyl group, wherein: (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) is a nitrogen atom or hydroxyl group that is capable of forming a chelate complex with a metal (e.g., copper or zinc).

3. The bifunctional compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the cleavage group C is selected from: (i) Any part containing N or C-OH, wherein 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 fraction of copper that can chelate at physiological pH.

4. A bifunctional compound of formula (I) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein the cleavage group C is a group of the following formula: -L1-X C -L2-R C in: L1 is absent or is a (1-2C) alkylene group; X C It does not exist or is selected from -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-; and where R XC1 Selected from hydrogen or (1-6C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkylene, -(CH2) m1 -Aryl, -(CH2) m1 -Heteroaryl or -(CH2) m1 - Heterocyclic ring, where m1 is 0 to 2; L2 is absent or is a (1-2C) alkylene group; R C Selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, phenyl, heteroaryl or heterocyclic; in: Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute; Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution; R A It is selected from the following groups: (i)-OH; (ii) NR A1 R A2 4 (iii)-(1-6C)alkylene-NR A1 R A2 ; in: R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B Selected from halogen, 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 (where p is 0, 1, or 2), -[CH2) t -SO2N(R BB )R BA Or -[CH2] t -N(R BB SO2R BA ; Where t is 0, 1, 2 or 3; R BA It is hydrogen or an (1-4C) alkyl group optionally substituted with halogen, hydroxyl, amino, or cyano groups; and R BB It is hydrogen or (1-2C) alkyl; The cleavage group mentioned above is not: (a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or (b) The groups of formula Z as defined above.

5. A bifunctional compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, wherein the cleavage group C is a group of the following formula: -X C -R C in: X C It does not exist or is selected from -O-, -S-, -SO-, -SO2-, -N(R) XC1 -, -C(O)-, -C(O)N(R) XC1 - or -N(R) XC1 )C(O)-; and where R XC1 Selected from hydrogen, (1-6C)alkyl, or -(CH2). m1 -heteroaryl, where m1 is 0 to 2; R C Selected from hydrogen, (1-4C)alkyl, phenyl, heteroaryl, or heterocyclic groups; in: Alkyl, cycloalkyl, phenyl are affected by one or more R A Substituents, and optionally one or more R B Substituents further substitute; Heteroaryl or heterocyclic rings are optionally enclosed by one or more R A and / or R B Substituent substitution; R A It is selected from the following groups: (i)-OH; (ii) NR A1 R A2 4 (iii)-(1-6C)alkylene-NR A1 R A2 ; in: R A1 and R A2 Each is independently selected from hydrogen, (1-6C)alkyl, or (1-6C)heteroalkyl; R B Selected from halogen, nitro, cyano, R BA -[CH2] t -OR BA Or -[CH2] t -C(O)OR BA Where t is 0, 1, 2, or 3; and R BA It is a (1-4C) alkyl group; The cleavage group mentioned above is not: (a) an imidazole (1,3-diazole) group, optionally substituted with one, two, or three identical or different (1-6C) alkyl groups; or (b) The groups of formula Z as defined above.

6. A bifunctional compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, wherein the cleavage group C is a group selected from the following formula: (i) (ii) (iii) or (iv) 7. A bifunctional compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker group L is selected from groups of formula (L1), (L-II), (L-III), or (L-IV): in: L 1 It is a covalent bond or a (1-6C) alkylene or a (1-6C) heteroalkylene; L 2 It is (1-6C) alkylene or (1-6C) heteroalkylene; L 3 It is a (1-6C) alkylene; n is between 0 and 8; L 4 It is a (1-6C) alkylene; L 5 It is (1-6C) alkylene or (1-6C) heteroalkylene; L 6 It is a covalent bond or a (1-2C) alkylene bond; m is 1 to 8; * indicates the junction with a non-covalently bonded group (-B); and ** is the connection point with the cleavage group (-C).

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

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

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 L 3 and L 4 It is ethylene.

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

12. The bifunctional compound of formula (I) according to any one of claims 7 to 11, or a pharmaceutically acceptable salt or solvate thereof, wherein L 2 and L 5 It is selected from ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-) and tetramethylene oxide (-CH2CH2CH2CH2O-).

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 L 2 and L 5 It is ethylene oxide (-CH2CH2O-).

14. A bifunctional compound of formula (I) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, wherein B is selected from oligonucleotides, nanobodies, antibodies, antibody fragments, or small molecules capable of binding to nucleic acids; or B is a group of formula (B-I), (B-II), (B-III), or (B-IV) as shown below: in: X is O or NH.

15. A bifunctional compound of formula (I) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound of formula (I) is selected from groups of formulas (II), (III), (IV) or (V) shown below: Wherein L and C are each as defined in any one of claims 1 to 14 above, and X is NH or O.

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

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

18. The bifunctional compound of any one of claims 1 to 15 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 16, for the treatment of proliferative disorders (e.g., cancer) or bacterial or viral infections.

19. A method of treating a proliferative disease (e.g., cancer) or a bacterial or viral infection, the method comprising administering a therapeutically effective dose 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.

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

21. A method for cleaving a target nucleic acid molecule, the method comprising: The target nucleic acid molecule is contacted with a bifunctional compound or its salt or solvate according to any one of claims 1 to 15, such that the compound non-covalently binds to the target nucleic acid molecule; and The compound is allowed to cleave the target nucleic acid molecule it binds to.

22. A method for identifying secondary or tertiary structures in a target nucleic acid molecule, the method comprising: First and second populations of nucleic acid molecules are provided, each population containing the target nucleic acid molecule; Introduce a bifunctional compound or its salt or solvate according to any one of claims 1 to 15 into the first group of nucleic acid molecules; The bifunctional compounds of this invention are allowed to cleave target nucleic acid molecules present in the first population; and Identify nucleic acid molecules present in the first group in reduced amounts relative to the second group.

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