WO2023246935A1 - Target-specific nucleic acid molecule having quinoline modification - Google Patents

Abstract

The present invention relates to the field of biomedicines. Specifically, the present invention relates to a target-specific nucleic acid molecule having a quinoline modification, the quinoline modification being capable of improving the binding affinity between the nucleic acid molecule and a target.

Description

Target-specific nucleic acid molecules comprising quinoline modifications Technical field

The invention relates to the field of biomedicine. Specifically, it relates to a target-specific nucleic acid molecule comprising a quinoline modification that can increase the binding affinity of the nucleic acid molecule to a target.

Background of the invention

Nucleic acid aptamers (Aptamers) are single-stranded DNA or RNA that can form a complementary structure with the target. They have specificity and affinity comparable to monoclonal antibody drugs. They are also non-immunogenic, easy to produce, low cost, and thermally stable. Good to wait for the advantage.

Osteosclerostin is a negative regulator of bone formation and is an ideal target for the development of drugs that promote bone formation and treat osteoporosis and other related diseases. In addition, studies have found that osteosclerostin also plays an important regulatory role in the occurrence and development of diseases such as osteogenesis imperfecta, hypophosphatemic rickets, and triple-negative breast cancer, and is therefore an important target for the treatment of these diseases. Sclerostin monoclonal antibody Romosozumab can promote bone formation. Drug regulatory agencies in the United States, Europe and Asia have approved osteosclerostin monoclonal antibody for marketing. The indication is postmenopausal patients with osteoporosis who are at high risk of fracture. Due to the serious clinical cardiovascular risks of osteosclerostin monoclonal antibody, the US drug regulatory agency FDA requires patients to jointly evaluate the benefits and cardiovascular risks of osteogenic treatment with their doctors before using it, and it can only be used for one year; European drugs The regulatory agency EMA has further strictly restricted the clinical use of sclerostin monoclonal antibody in patients with high risk of osteoporotic fractures who must not have a history of cardiovascular disease.

Sclerostin protein has three structural regions, namely loop1, loop2 and loop3. Among them, osteosclerostin monoclonal antibody mainly acts on loop2. Studies have found that loop2 is involved in both bone formation inhibition and cardiovascular protection, while osteosclerostin loop3 is only involved in bone formation inhibition but not cardiovascular protection. Therefore, the nucleic acid aptamer-APC001, which specifically targets osteosclerostin loop3, was screened through SELEX technology, and the aptamer was verified to have the ability to promote bone anabolism in the treatment of osteoporosis models and osteogenesis imperfecta models. Therapeutic potential without increasing cardiovascular disease risk. This therapeutic aptamer received Osteogenesis Imperfecta orphan drug certification from the U.S. FDA in 2019 (FDA, DRU-2019-6966).

Improving the affinity of aptamers for targets can help improve the druggability bottleneck of low efficacy of aptamer drugs. Currently, the affinity of aptamer APC001 for osteosclerostin (KD=39.9nM) is lower than that of osteosclerostin monoclonal antibody (KD=0.12nM). Therefore, in order to further improve the aptamer's ability to inhibit osteosclerostin (even beyond that of monoclonal antibodies), it is necessary to take effective measures to increase its affinity for the target osteosclerostin.

Studies have shown that hydrophobic modifications on aptamers can improve the affinity of aptamers to targets. Developing new aptamer modification methods and expanding the chemical diversity of aptamers are of great significance for the further clinical application of aptamers.

Brief description of the invention

The present invention at least provides the following embodiments:

Embodiment 1. A modified target-specific nucleic acid molecule comprising one or more quinoline-modified nucleotides, wherein the modified target-specific nucleic acid molecule is compared to a corresponding control nucleic acid molecule. Target binding affinity is increased.

Embodiment 2. The modified target-specific nucleic acid molecule of embodiment 1, wherein said nucleic acid molecule is selected from the group consisting of an aptamer (aptamer), an antisense nucleic acid (antisense RNA), dsRNA (eg, siRNA, shRNA).

Embodiment 3. The modified target-specific nucleic acid molecule of embodiment 1 or 2, wherein the modified target-specific nucleic acid molecule has a binding affinity to a target that is increased by at least about 10% compared to a corresponding control nucleic acid molecule. At least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more; or all The binding affinity of the modified target-specific nucleic acid molecule to the target is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about the affinity of the unmodified corresponding nucleic acid molecule. About 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 16 times, at least about 20 times, at least about 25 times, at least about 30 times, at least About 40 times, at least about 50 times, at least about 75 times, at least about 100 times or more.

Embodiment 4. The modified target-specific nucleic acid molecule of any one of embodiments 1-3, wherein the modified target-specific nucleic acid molecule has an affinity for the target of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM, preferably less than 5 nM or less KD (dissociation constant).

Embodiment 5. The modified target-specific nucleic acid molecule of any one of embodiments 1-4, wherein the quinoline modification is quinolin-2-yl substitution, quinolin-3-yl substitution, quinolin-4 - base substitution, quinolin-5-yl substitution, quinolin-6-yl substitution, quinolin-7-yl substitution or quinolin-8-yl substitution, preferably, the quinoline modification is quinolin-5- base substitution.

Embodiment 6. The modified target-specific nucleic acid molecule of any one of embodiments 1-5, wherein the quinoline-modified nucleotide comprises a substituent group R selected from ag:

Preferably,

Embodiment 7. The modified target-specific nucleic acid molecule of any one of embodiments 1-6, wherein the quinoline-modified nucleotide comprises the base moiety of Formula I:

in

Preferably,

Embodiment 8. The modified target-specific nucleic acid molecule of any one of embodiments 1-7, wherein the quinoline-modified nucleotide comprises the following structure of Formula II:

in

Preferably,

Embodiment 9. The modified target-specific nucleic acid molecule of any one of embodiments 1-8, wherein one or more naturally occurring nucleotides in the target-specific nucleic acid molecule are modified with the quinoline Nucleotide substitutions.

Embodiment 10. The modified target-specific nucleic acid molecule of any one of embodiments 1-9, wherein the target-specific nucleic acid molecule may further comprise one or more modifications that extend its half-life in vivo.

Embodiment 11. The modified target-specific nucleic acid molecule of any one of embodiments 1-10, wherein the modified target-specific nucleic acid molecule is a modified osteosclerostin-specific aptamer.

Embodiment 12. The modified target-specific nucleic acid molecule of embodiment 11, wherein the modified osteosclerostin-specific aptamer is derived from the osteosclerostin-specific aptamer of SEQ ID NO: 2.

Embodiment 13. The modified target-specific nucleic acid molecule of embodiment 12, wherein the modified osteosclerostin-specific aptamer is at one or more positions selected from the group consisting of corresponding to SEQ ID NO:2 The nucleotides are replaced by the quinoline modified nucleotides: position 1, position 2, position 3, position 4, position 5, position 6, position 8, position 11, position 13 , 15th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th and 39th 40 people.

Embodiment 14. The modified target-specific nucleic acid molecule of embodiment 12 or 13, wherein the modified osteosclerostin-specific nucleic acid molecule is at position 6, position 8, position corresponding to SEQ ID NO: 2 One or more nucleotides at position 11, 13, 32 and/or 34 are substituted with the quinoline modified nucleotide.

Embodiment 15. The modified target-specific nucleic acid molecule of any one of embodiments 12-14, wherein the modified osteosclerostin-specific nucleic acid molecule comprises a nucleotide selected from the group consisting of SEQ ID NOs: 3-23 Sequence, where x is the quinoline modified nucleotide.

Embodiment 16. The modified target-specific nucleic acid molecule of any one of embodiments 12-15, wherein the modified osteosclerostin-specific nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NO: 8, 10, 11, 16 and the nucleotide sequence of 18, wherein x is the quinoline-modified nucleotide.

Embodiment 17. The modified target-specific nucleic acid molecule of any one of embodiments 12-16, wherein the quinoline-modified nucleotide comprises the structure of Formula II:

in,

Embodiment 18. A composition comprising the modified target-specific nucleic acid molecule of any one of embodiments 1-17, and optionally a pharmaceutically or physiologically acceptable carrier.

Embodiment 19. A method of increasing the binding affinity of a target-specific nucleic acid molecule to a target, the method comprising replacing one or more of the target-specific nucleic acid molecules with one or more quinoline-modified nucleotides. Naturally occurring nucleotides.

Embodiment 20. The method of Embodiment 19, wherein the quinoline modification is quinolin-2-yl substitution, quinolin-3-yl substitution, quinolin-4-yl substitution, quinolin-5-yl substitution, quinolin-5-yl substitution, quinolin-6-yl substitution, quinolin-7-yl substitution or quinolin-8-yl substitution. Preferably, the quinoline modification is quinolin-5-yl substitution.

Embodiment 21. The method of embodiment 19 or 20, wherein the quinoline modified nucleotide comprises a substituent group R selected from the following ag:

Preferably,

Embodiment 22. The method of any one of embodiments 19-21, wherein the quinoline modified nucleotide comprises the base moiety of Formula I:

in

Preferably,

Embodiment 23. The method of any one of embodiments 19-22, wherein the quinoline modified nucleotide comprises the following structure of Formula II:

in

Preferably,

Embodiment 24. The method of any one of embodiments 19-23, said one or more quinoline modified nucleotide substitutions being introduced during chemical synthesis of said target-specific nucleic acid molecule.

Embodiment 25. The method of embodiment 24, wherein a modified phosphoramidite monomer selected from the group consisting of 2a-2g is added during chemical synthesis of the target-specific nucleic acid molecule:

Embodiment 26. A modified phosphoramidite monomer selected from the following 2a-2g, which is used to increase the binding affinity of a target-specific nucleic acid molecule to a target:

Brief description of the drawings

Figure 1. Measurement results of the binding ability of osteosclerostin aptamers modified with different subtypes of quinoline at key sites and targets. The highlighted parts in the sequence are key sites. The horizontal axis in the heat map represents the key sites modified with quinoline, and the vertical axis represents different subtypes of quinoline. The binding ability was tested using the ELONA (Enzyme Ligated OligoNucleotide Assay) method.

Figure 2. Results of measuring the affinity of quinoline-modified aptamers for targets using Biolayer interferometry (BLI). a) Binding and dissociation curves of interaction between each aptamer and target. b) The equilibrium dissociation constant KD value calculated by the final fitting.

Detailed description of the invention

Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which meanings will be understood by those skilled in the art. Reference, for example, standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin, "Genes VIII"; and Roitt et al., "Immunology" (8th ed.), as well as general prior art references herein; in addition, unless otherwise indicated, all references not specifically recited Methods, steps, techniques and operations can and have been performed in a manner known per se, which manner will be understood by a person skilled in the art. Reference is also made to, for example, standard manuals, the general prior art mentioned above and other references cited therein.

I. Modified target-specific nucleic acid molecules with increased binding affinity

In one aspect, the invention provides a modified target-specific nucleic acid molecule comprising one or more quinoline-modified nucleotides, wherein the modified target-specific nucleic acid molecule has a higher The binding affinity of the nucleic acid molecule to the target is increased. In some embodiments, the corresponding control nucleic acid molecule refers to a target-specific nucleic acid molecule that does not comprise the one or more quinoline-modified nucleotides.

As used herein, a "target-specific nucleic acid molecule" refers to a nucleic acid molecule that is capable of specifically binding to a particular target. The targets include but are not limited to proteins, nucleic acids, small molecule compounds, etc. The nucleic acid molecules include but are not limited to aptamers, antisense nucleic acids (antisense RNA), dsRNA (such as siRNA, shRNA), preferably aptamers.

In some embodiments, the modified target-specific nucleic acid molecule has an increased binding affinity to the target by at least about 10%, at least about 20%, at least about 30%, at least about 40% compared to a corresponding control nucleic acid molecule. , at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more. In some embodiments, the modified target-specific nucleic acid molecule binds the target with an affinity that is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times greater than the affinity of the corresponding unmodified nucleic acid molecule. , at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 16 times, at least about 20 times, at least about 25 times , at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times or more. The binding affinity can be measured, for example, by enzyme-linked oligonucleotide assay (ELONA) or by biofilm interferometry (BLI).

In some embodiments, the modified target-specific nucleic acid molecule has a KD for the target of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM, preferably less than 5 nM or less. (dissociation constant). The KD is measured, for example, by enzyme-linked oligonucleotide assay (ELONA) or by biofilm interferometry (BLI).

In some embodiments, the quinoline modification is quinolin-2-yl substitution, quinolin-3-yl substitution, quinolin-4-yl substitution, quinolin-5-yl substitution, quinolin-6-yl substitution Substituted, quinolin-7-yl substituted or quinolin-8-yl substituted. In some preferred embodiments, the quinoline modification is a quinolin-5-yl substitution. The substitution position numbers on quinoline are as follows:

In some embodiments, the quinoline modified nucleotide comprises a substituent group R selected from the following ag:

In some preferred embodiments,

In some embodiments, the quinoline modified nucleotide contains the substituent group R in the base portion thereof. In some embodiments, the quinoline modified nucleotide is derived from uridine, such as deoxyuridine.

In some embodiments, the quinoline modified nucleotide includes the base moiety of Formula I:

in

In some preferred embodiments,

In some embodiments, the quinoline modified nucleotides comprise the structure of Formula II:

in

In some preferred embodiments,

In some embodiments, one or more nucleotides (natural nucleotides) in the target-specific nucleic acid molecule are replaced with the quinoline-modified nucleotides.

In some embodiments, the target-specific nucleic acid molecule may also comprise one or more modifications that extend its half-life in vivo.

The modifications that extend its half-life in vivo include, for example, 3' and 5' modifications, such as 3' and 5' capping. In some embodiments, the aptamer is capped at the 3' end with inverted deoxythymidine, i.e., 3' inverted deoxythymidine (3'idT) modification.

The modification to extend its half-life in vivo may also include replacing one or more naturally occurring nucleotides with modified nucleotides. For example, the modified nucleotides for extending half-life include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and/or 2'propenoxy modified nucleosides Acid (that is, the hydroxyl group at the 2' position of ribose is replaced by fluorine, methoxyethyl, methoxy or propyleneoxy, etc.). The modified nucleotides for extending half-life may also include C-5 modified pyrimidines. The term "C-5 modified pyrimidine" refers to a pyrimidine with a modification at the C-5 position. C-5 modified pyrimidines can enhance the nuclease resistance of oligonucleotides and are known in the art. For example, see International Patent Application WO 2011/130195 and its cited documents.

Modifications for extending half-life also include modifications between nucleotides, such as nucleotides with uncharged bonds (e.g., methylphosphonate, phosphotriester, phosphate amine ester, carbamate, etc.) Inter-modifications and inter-nucleotide modifications with charged bonds (e.g. phosphorothioates, phosphorodithioates, etc.), inter-nucleotide modifications with intercalating agents (e.g. acridine, psoralen, etc.), Internucleotide modifications containing chelating agents (such as metals, radioactive metals, boron, oxidizing metals, etc.), internucleotide modifications containing alkylating agents, and nucleosides with modified bonds (such as alpha anomeric nucleic acids, etc.) Inter-acid modification.

The modification for extending half-life may also include pegylation modification (PEG modification).

In another aspect, the invention also provides a composition, such as a pharmaceutical composition, comprising a modified target-specific nucleic acid molecule of the invention. In some embodiments, the composition further includes a pharmaceutically or physiologically acceptable carrier or excipient. The composition may, for example, be used to detect the target, or to diagnose and/or treat a disease associated with the target.

In another aspect, the invention provides a method for increasing the binding affinity of a target-specific nucleic acid molecule to a target, the method comprising replacing the nucleotides in the target-specific nucleic acid molecule with one or more quinoline-modified nucleotides. One or more naturally occurring nucleotides. The quinoline modified nucleotide is as defined above.

In some embodiments, the nucleotide substitutions are introduced during chemical synthesis (eg, solid phase) of the target-specific nucleic acid molecule.

In some embodiments, a modified phosphoramidite monomer selected from 2a-2g below is added during chemical synthesis of the target-specific nucleic acid molecule:

In another aspect, the present invention also provides modified phosphoramidite monomers selected from the following 2a-2g, which are used to improve the binding affinity of target-specific nucleic acid molecules to targets:

II. Modified osteosclerostin-specific nucleic acid molecules with increased binding affinity

In some embodiments of the invention, the target-specific nucleic acid molecule is an osteosclerostin-specific nucleic acid molecule. Accordingly, the present invention also provides modified osteosclerostin-specific nucleic acid molecules comprising one or more quinoline-modified nucleotides as described above.

In some embodiments, the target is sclerostin, such as human sclerostin. An exemplary human osteosclerostin contains the amino acid sequence:

In some embodiments, the target-specific nucleic acid molecule is an aptamer that specifically binds osteosclerostin (modified osteosclerostin-specific aptamer). In some embodiments, the aptamer (unmodified) that specifically binds osteosclerostin comprises the nucleotide sequence:

5'-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3'(SEQ ID NO:2).

In some embodiments, the modified osteosclerostin-specific aptamer is a nucleoside modified by the quinoline at one or more positions of a nucleotide corresponding to SEQ ID NO: 2 selected from: Acid substitution: 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 11th, 13th, 15th, 29th, 30th , 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th and 40th.

In some preferred embodiments, the modified osteosclerostin-specific nucleic acid molecule is at position 6, position 8, position 11, position 13, position 32 and/or corresponding to SEQ ID NO:2 The nucleotide at position 34 is substituted with the quinoline modified nucleotide.

In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises a nucleotide sequence selected from:

5’-xGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:3),

5’-CxGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:4),

5’-CGxGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:5),

5’-CGGxGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:6),

5’-CGGGxTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:7),

5’-CGGGGxGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:8),

5’-CGGGGTGxGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:9),

5’-CGGGGTGTGGxTTCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:10),

5’-CGGGGTGTGGGTxCGTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:11),

5’-CGGGGTGTGGGTTCxTCGTTAGCTTGATTTGGCAGCTGCC-3’(SEQ ID NO:12),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATxTGGCAGCTGCC-3’(SEQ ID NO:13),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTxGGCAGCTGCC-3’(SEQ ID NO:14),

5'-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTxGCAGCTGCC-3'(SEQ ID NO:15),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGxCAGCTGCC-3’(SEQ ID NO:16),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGxAGCTGCC-3’(SEQ ID NO:17),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCxGCTGCC-3’(SEQ ID NO:18),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAxCTGCC-3’(SEQ ID NO:19),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGxTGCC-3’(SEQ ID NO:20),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCxGCC-3’(SEQ ID NO:21),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTxCC-3’(SEQ ID NO:22),

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGxC-3’ (SEQ ID NO: 23), and

5’-CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCx-3’(SEQ ID NO:24),

Where x is the quinoline modified nucleotide.

In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 8, wherein x is the quinoline modified nucleotide. In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 10, wherein x is the quinoline modified nucleotide. In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 11, wherein x is the quinoline modified nucleotide. In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 16, wherein x is the quinoline modified nucleotide. In some embodiments, the modified osteosclerostin-specific nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 18, wherein x is the quinoline modified nucleotide.

In some preferred embodiments, the quinoline modified nucleotides comprise the structure of Formula II:

in,

In some embodiments, the binding affinity of the modified osteosclerostin-specific nucleic acid molecule of the present invention to osteosclerosis is the affinity of the unmodified osteosclerostin-specific nucleic acid molecule shown in SEQ ID NO: 2 At least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, At least about 15 times, at least about 16 times or more. The binding affinity can be measured, for example, by enzyme-linked oligonucleotide assay (ELONA) or by biofilm interferometry (BLI).

In some embodiments, the modified osteosclerostin-specific nucleic acid molecule has a KD (dissociation constant) for osteosclerosis of less than 10 nM or even less than 5 nM. The KD is measured, for example, by enzyme-linked oligonucleotide assay (ELONA) or by biofilm interferometry (BLI).

In another aspect, the present invention also provides a composition comprising the modified osteosclerostin-specific nucleic acid molecule of the present invention, and optionally a pharmaceutically or physiologically acceptable carrier. The composition is used, for example, for detecting osteosclerostin, or for diagnosing and/or treating osteosclerostin-related diseases.

In another aspect, the invention also provides a method of treating a disease by a modified osteosclerostin-specific nucleic acid molecule of the invention, the method comprising administering to a subject in need thereof a therapeutically effective amount of a modified osteosclerostin of the invention Specific nucleic acid molecules.

The disease is, for example, an osteosclerostin-related disease, such as an osteosclerostin-mediated disease.

As used herein, "osteosclerostin-related disease" includes conditions in which bone mineral density (BMD) is abnormally and/or pathologically low relative to healthy subjects. Diseases characterized by low BMD and/or bone fragility include, but are not limited to: primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (bone necrosis), fractures and implant healing (dental implants and hip implants), bone loss due to other conditions (eg, associated with HIV infection, cancer, and arthritis). Other "osteosclerostin-related diseases" include, but are not limited to: hypophosphatemic rickets, rheumatoid arthritis, osteoarthritis, arthritis, and osteolytic lesions.

As used herein, "osteosclerostin-related disease" includes osteosclerosis-related cancers, such as myeloma (eg, multiple myeloma with osteolytic lesions), breast cancer (eg, triple-negative breast cancer), colon cancer, Melanoma, hepatocellular carcinoma, epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and any metastases thereto.

"Osteosclerin-related diseases" may also include renal and cardiovascular diseases caused by at least expression of osteosclerostin in the kidneys and in the cardiovascular system. Such conditions include, but are not limited to, renal diseases such as: glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, and Glomerular damage associated with systemic diseases such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasia, sickle cell disease, and chronic inflammation), renal tubules Diseases (e.g., acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary sponge kidney disease, medullary cystic disease, nephrogenic diabetes, and tubular acidosis), tubulointerstitial disease (e.g., pyelonephritis , drug- and toxin-induced tubulointerstitial nephritis, hypercalcaemic nephropathy, and hypokalemic nephropathy), acute and rapidly progressive renal failure, chronic renal failure, renal tuberculosis stones, gout, vascular disease (eg, hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic nephropathy, diffuse cortical necrosis, and renal infarction), or tumors (eg, renal cell carcinoma and nephroblastoma) tumor).

The osteosclerostin-related diseases also include, but are not limited to, cardiovascular diseases such as: ischemic heart disease (eg, angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, cor pulmonale, heart disease, Valvular disease (eg, rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic stenosis), congenital heart disease (eg, occlusive lesions of valves and blood vessels, atrial or ventricular septal defects , and persistent ductus arteriosus), or cardiomyopathies (eg, myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

As used herein, "treating" a subject suffering from a disease means that the subject's symptoms are partially or completely alleviated, or remain unchanged following treatment. Treatment therefore includes prevention, treatment and/or cure. Prevention means preventing underlying disease and/or preventing symptoms from worsening or disease progression.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material or composition containing a compound that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or condition. As used herein, "therapeutic effect" means an effect resulting from treatment of a subject that alters, generally ameliorates, or ameliorates the symptoms of a disease or disease condition, or cures a disease or disease condition.

Dosage regimens utilizing the nucleic acid molecules are selected based on a variety of factors, including, for example, the type, species, age, weight, gender, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the The patient's kidney and liver function; and the specific nucleic acid molecule or salt thereof used. The ordinarily skilled physician can readily determine and prescribe the effective amount of the composition required to prevent, combat, or inhibit the progression of the disorder.

Example

Example 1, synthetic routes of compounds 1a to 1g and 2a to 2g

1) The structures of different quinoline subtype modified nucleosides (1a～1g) are as follows:

2) Compounds 1a to 1g react with phosphorus reagents to obtain different quinoline subtype modified phosphoramidite monomers (2a to 2g), which can be used as raw materials for solid phase synthesis. The structures of 2a to 2g are as follows:

3) Synthetic routes of 1a～1g and 2a～2g:

Experimental part:

Synthesis of 1a ~ 1g: Add 5'-DMT-5-iododeoxyuracil (2.0g, 3.0mmol), aminomethylquinoline (0.95g, 6.0mmol) and tetrakis(triphenylphosphine) to a 100mL autoclave )Palladium (35mg, Pb 10%). Then 20 mL of anhydrous THF was added to dissolve the above reactants, and Et3N (2.1 mL, 15 mmol) was added. Cover the kettle lid and open the pressure reducing valve of the carbon monoxide cylinder. Replace the remaining space with carbon monoxide three times, and then fill it with carbon monoxide under a pressure of 0.30-0.40MPa. Place the reaction kettle in an oil bath at 60-70°C and stir for 3-4 days. During this process, if the pressure in the kettle drops below 0.30MPa, carbon monoxide gas needs to be added to maintain the pressure between 0.30-0.40MPa. Place the kettle in ice water for 20 minutes and then release the pressure in a fume hood. Open the kettle, pour the reaction solution into 100 ml of ice water, and extract three times with 50 ml of ethyl acetate. The organic phase was dried over anhydrous _Na2SO4 and concentrated _in vacuo. Next, the final product is purified using neutral silica, and the eluent is n-hexane/ethyl acetate = 1/2. After concentration and evaporation to dryness, a viscous solid product was obtained (yield: 1a: 66%, 1b: 26%, 1c: 65%, 1d: 72%, 1e: 60%, 1f: 68%, 1g: 53%).

The NMR data are as follows:

1a: ¹ H NMR (400MHz, DMSO) δ11.98 (s, 1H), 9.18 (t, J = 5.8Hz, 1H), 8.89 (d, J = 2.8Hz, 1H), 8.58 (d, J = 8.5 Hz,1H),8.54(s,1H),7.96(d,J＝8.4Hz,1H),7.73–7.64(m,1H),7.56(d,J＝7.0Hz,1H),7.49– 7.34(m,3H),7.23(tt,J＝14.4,7.4Hz,7H),6.87(d,J＝8.7Hz,4H),6.08(t,J＝6.3Hz,1H),5.35(d,J ＝4.3Hz,1H),5.07–4.89(m,2H),4.11(s,1H),3.97(d,J＝4.0Hz,1H),3.68(d,J＝3.7Hz,6H),3.20(d ,J＝15.7Hz,2H),2.39–2.14(m,2H). ¹³ C NMR(101MHz,DMSO)δ163.7,161.9,158.5,150.7,149.9,148.5,146.2,145.3,136.0,135.8,132.4,130.3, 130.2,129.5,129.1,128.3,128.1,127.1,126.4,121.8,113.7,105.4,86.7,86.5,86.3,70.9,64.1,55.4,39.93.

1b: ¹ H NMR (400MHz, DMSO) δ12.02(s,1H),9.27(t,J＝6.0Hz,1H),8.86(dd,J＝4.1,1.6Hz,1H),8.54(s,1H ),8.21(d,J＝7.8Hz,1H),7.97(d,J＝8.7Hz,1H),7.81(s,1H),7.70(dd,J＝8.7,1.7Hz,1H),7.48(dd ,J＝8.3,4.2Hz,1H),7.38(d,J＝7.5Hz,2H),7.27(dd,J＝8.7,4.3Hz,6H),7.13(t,J＝7.3Hz,1H),6.87 (dd,J＝8.8,1.8Hz,4H),6.11(t,J＝6.4Hz,1H),5.36(d,J＝4.1Hz,1H),4.83–4.59(m,2H),4.11(s, 1H),4.01–3.92(m,1H),3.68(s,6H),3.22(s,2H),2.37–2.18(m,2H). ¹³ C NMR(101MHz,DMSO)δ163.7,162.2,158.5,150.7 ,149.9,147.5,146.2,145.4,138.1,136.2,136.0,135.8,130.3,130.2,129.9,129.5,128.3,128.2,128.1,127.1,126.0,122.1,113.7,105.6,86 .6,86.5,86.3,70.9,64.1 ,55.4,42.6.1c: ¹ H NMR(400MHz,DMSO)δ12.01(s,1H),9.29(t,J＝6.1Hz,1H),8.88(dd,J＝4.2,1.7Hz,1H), 8.52(s,1H),8.33(d,J＝8.2Hz,1H),7.92(d,J＝8.7Hz,2H),7.55(dd,J＝8.4,1.5Hz,1H),7.49(dd,J ＝8.3,4.2Hz,1H),7.37(d,J＝7.5Hz,2H),7.33–7.21(m,6H),7.15(t,J＝7.3Hz,1H),6.87(d,J＝8.9Hz ,4H),6.10(t,J＝6.4Hz,1H),5.36(d,J＝4.6Hz,1H),4.81–4.64(m,2H),4.12(dt,J＝8.9,4.4Hz,1H) ¹³ C NMR (101MHz, DMSO) δ163.7,162.2,158.5,151.2,149.9,146.2,145.3,141.4,139.6,136.2,136.0,135.8,130.3,130.2,128.6,128.3,128.1,127.3,127. 1,126.8,121.6,113.7, 105.6,86.5,86.4,86.2,70.8,64.1,55.4,42.7.

1d: ¹ H NMR (400MHz, DMSO) δ12.04 (s, 1H), 9.29 (t, J = 5.9Hz, 1H), 8.77 (d, J = 4.4Hz, 1H), 8.54 (s, 1H), 8.19(d,J＝8.3Hz,1H),8.06(d,J＝8.4Hz,1H),7.77(t,J＝7.6Hz,1H),7.61(t,J＝7.5Hz,1H),7.37( d,J＝7.7Hz,2H),7.32(d,J＝4.3Hz,1H),7.26(dd,J＝11.1,6.7Hz,6H),7.16(t,J＝7.2Hz,1H),6.85( d,J＝8.5Hz,4H),6.10(t,J＝6.3Hz,1H),5.36(d,J＝4.4Hz,1H),5.09–4.94(m,2H),4.11(d,J＝4.3 ¹³ C NMR (101MHz, DMSO) δ163.7,162.3,158.5,150.8,149.9,148.1,146.3,145.3,144.9,135.9,135.8,130.3,130.2,130.1,129.8,128.3,128.1,127.1,126.4, 124.0,119.4,113.7,105.4 ,86.7,86.5,86.3,70.9,64.1,55.4,39.7.

1e: ¹ H NMR (400MHz, DMSO) δ11.99 (s, 1H), 9.28 (t, J = 6.1Hz, 1H), 8.89 (t, J = 3.1Hz, 1H), 8.50 (s, 1H), 8.17(s,1H),8.01(d,J＝8.4Hz,1H),7.85(d,J＝8.1Hz,1H),7.75–7.69(m,1H),7.60–7.54(m,1H),7.36 (d,J＝7.5Hz,2H),7.28–7.22(m,6H),7.12(t,J＝7.3Hz,1H),6.86(dd,J＝8.9,1.8Hz,4H),6.08(t, J＝6.4Hz,1H),5.37(d,J＝4.5Hz,1H),4.78–4.63(m,2H),4.18–4.05(m,1H),3.95(dd,J＝8.5,4.3Hz,1H ), 3.69 (s, 6H), 3.18 (t, J = 4.7Hz, 2H), 2.33–2.17 (m, 2H). ¹³ C NMR (101MHz, DMSO) δ 163.6, 162.3, 158.5, 151.4, 149.9, 147.2, 146.2,145.3,135.9,135.8,134.1,133.0,130.2,129.6,129.1,128.3,128.1,127.9,127.2,127.0,113.7,105.6,86.6,86.4,86.2,70.9,64.1,55 .4,40.5.

1f: ¹ H NMR (400MHz, DMSO) δ12.01 (s, 1H), 9.65 (t, J = 5.4Hz, 1H), 8.52 (s, 1H), 8.30 (d, J = 8.5Hz, 1H), 7.97(t,J＝7.8Hz,2H),7.81–7.72(m,1H),7.59(dd,J＝11.5,4.5Hz,1H),7.48(d,J＝8.5Hz,1H),7.37(d ,J＝7.4Hz,2H),7.31–7.23(m,6H),7.15(t,J＝7.3Hz,1H),6.87(d,J＝8.9Hz,4H),6.11(t,J＝6.4Hz ,1H),5.36(d,J＝4.6Hz,1H),4.79(d,J＝5.4Hz,2H),4.13(dt,J＝8.9,4.3Hz,1H),3.96(q,J＝4.3Hz ,1H),3.69(d,J＝2.1Hz,6H),3.20(d,J＝4.3Hz,2H),2.36–2.19(m,2H). ¹³ C NMR(101MHz, DMSO)δ163.6,162.1,158.6,158.5,150.0,146.1,145.3,137.1,136.0,135.8,130.3,130.2,128.9,128.4,128.3,128.1,127.4,127.1,126.7,120.2, 113.7,105.7,86.5,86.4, 86.2,70.8,64.1,55.4,45.3.

1g: ¹ H NMR (400MHz, DMSO) δ11.93 (s, 1H), 9.42 (t, J = 6.1Hz, 1H), 8.98 (dd, J = 4.2, 1.7Hz, 1H), 8.48 (s, 1H ),8.39(dt,J＝8.6,4.3Hz,1H),7.90(d,J＝8.1Hz,1H),7.64(d,J＝7.0Hz,1H),7.59(dd,J＝8.3,4.2Hz ,1H),7.56–7.50(m,1H),7.36(d,J＝7.5Hz,2H),7.30–7.22(m,6H),7.17(t,J＝7.2Hz,1H),6.87(d, J＝8.8Hz,4H),6.09(t,J＝6.4Hz,1H),5.34(d,J＝4.6Hz,1H),5.08(d,J＝6.0Hz,2H),4.17–4.08(m, 1H),3.94(q,J＝4.4Hz,1H),3.68(d,J＝3.7Hz,6H),3.19(d,J＝4.4Hz,2H),2.35–2.11(m,2H). ¹³ C NMR (101MHz, DMSO) δ163.6,161.9,158.5,150.3,149.9,146.2,146.0,145.4,136.9,135.9,135.8,130.3,130.1,128.4,128.3,128.1,127.8,127.1,126. 7,122.0,113.7,105.8, 86.4,86.3,86.2,70.8,64.1,55.4.

Synthetic route of 2a～2g: Under nitrogen protection and ice water bath conditions, add tetrazole to 50mL 3.3mmol 1a～1g (1a is 2.2g, 1b-1g is 2.4g) anhydrous dichloromethane solution (0.69g, 9.8mmol) and the phosphorus reagent 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite (2.9g, 9.8mmol). The solution was then moved to room temperature and stirred for 2 hours, and the solution was concentrated in vacuo. The residue was then purified by flash chromatography (n-hexane/ethyl acetate = 1/1) to obtain the desired product 2a-2g as a viscous and light yellow solid (yield: 2a: 70%, 2b: 72%, 2c:48%, 2d:70%, 2e:58%, 2f:79%, 2g:52%).

2a: ¹ H NMR (400MHz, DMSO) δ11.93 (s, 1H), 9.18 (d, J = 5.8Hz, 1H), 8.89 (s, 1H), 8.63–8.52 (m, 2H), 7.95 (d ,J＝8.4Hz,1H),7.73–7.63(m,1H),7.56(d,J＝7.0Hz,1H),7.47–7.35(m,3H),7.30–7.23(m,6H),7.18( dd,J＝7.6,5.9Hz,1H),6.86(d,J＝7.9Hz,4H),6.13–6.04(m,1H),5.05–4.91(m,2H),4.40–4.29(m,1H) ,4.10(dd,J＝15.9,4.1Hz,1H),3.70–3.67(m,6H),3.63–3.37(m,4H),3.23(d,J＝3.8Hz,2H),2.75(t,J ＝5.9Hz,1H),2.64(t,J＝5.4Hz,1H),2.47–2.35(m,2H),1.10(dd,J＝15.1,7.7Hz,9H),0.96(d,J＝6.7Hz ,3H). ¹³ C NMR (101MHz, DMSO) δ163.7, 161.9, 158.5, 150.7, 149.8, 148.5, 146.7, 146.5, 145.2, 136.0, 135.9, 135.8, 135.7, 132.4, 130.2, 129.5, 129.2, 128.3, 128.1,127.1,126.4,121.8,119.4,119.2,113.7,105.5,87.1,86.3,85.6,73.2,63.7,59.0,58.8,55.4,43.1,43.0,24.7,20.2. ³¹ P NMR (162MHz, DMSO) δ147. 6,147.3.

2b: ¹ H NMR (400MHz, DMSO) δ11.93 (s, 1H), 9.27 (s, 1H), 8.85 (d, J = 2.9Hz, 1H), 8.57 (d, J = 7.0Hz, 1H), 8.21(t,J＝8.2Hz,1H),7.96(d,J＝8.6Hz,1H),7.80(s,1H),7.70(d,J＝8.6Hz,1H),7.47(dd,J＝7.4 ,3.2Hz,1H),7.37(d,J＝7.3Hz,2H),7.25(t,J＝6.3Hz,6H),7.16–7.11(m,1H),6.86(d,J＝7.7Hz,4H ),6.15–6.04(m,1H),4.70(s,2H),4.34(s,1H),4.15–4.05(m,1H),3.68(s,6H),3.61–3.46(m,4H), 3.24(s,2H),2.75(t,J＝5.8Hz,1H),2.64(d,J＝6.1Hz,1H),2.47–2.38(m,2H),1.10(dd,J＝14.7,7.5Hz ,9H),0.96(d,J＝6.7Hz,3H). ¹³ C NMR(101MHz,DMSO)δ163.7,162.2,158.5,150.7,149.8,147.5,146.6,146.4,145.2,138.1,136.2,135.9,135.8, 135.7,130.2,130.1,129.8,129.5,128.3,128.2,128.1,127.1,126.0,122.1,119.4,119.2,113.7,105.6,87.0,86.4,85.6,73.4,63.7,59.0,58. 7,55.4,43.1,42.6, 24.7,20.2. ³¹ P NMR (162MHz, DMSO) δ147.7,147.4.

2c: ¹ H NMR (400MHz, DMSO) δ11.92 (s, 1H), 9.29 (t, J = 5.5Hz, 1H), 8.88 (d, J = 2.7Hz, 1H), 8.55 (d, J = 5.6 Hz,1H),8.33(d,J＝8.2Hz,1H),7.92(d,J＝9.9Hz,2H),7.55(d,J＝8.4Hz,1H),7.49(dd,J＝8.2,4.2 Hz,1H),7.41–7.33(m,2H),7.26(s,6H),7.15(t,J＝7.2Hz,1H),6.86(dd,J＝8.6,3.0Hz,4H), 6.16–6.04(m,1H),4.73(d,J＝5.5Hz,2H),4.36(d,J＝4.5Hz,1H),4.09(dd,J＝9.2,4.2Hz,1H),3.69(d ,J＝1.4Hz,6H),3.63–3.39(m,4H),3.26(dd,J＝12.2,7.2Hz,2H),2.75(t,J＝5.8Hz,1H),2.64(t,J＝ 5.5Hz, 1H), 2.42 (dd, J＝12.8, 5.9Hz, 2H), 1.10 (dd, J＝14.3, 7.4Hz, 9H), 0.96 (d, J＝6.7Hz, 3H). ¹³ C NMR ( 101MHz, DMSO) δ163.7,162.2,158.5,151.2,149.9,148.3,146.6,146.5,145.2,141.4,136.2,135.9,135.8,135.7,130.2,130.1,128.6,128.3,128.1, 127.3,127.1,126.8,121.6, 119.4,119.2,113.7,105.6,87.0,86.3,85.5,73.5,63.8,59.0,58.8,55.4,43.1,42.7,24..7,20.2. ³¹ P NMR (162MHz, DMSO) δ147.7,147.4.

2d: ¹ H NMR (400MHz, DMSO) δ11.94 (s, 1H), 9.28 (d, J = 4.3Hz, 1H), 8.77 (t, J = 4.7Hz, 1H), 8.57 (d, J = 6.1 Hz,1H),8.19(d,J＝8.4Hz,1H),8.06(d,J＝8.4Hz,1H),7.77(dd,J＝8.2,7.1Hz,1H),7.65–7.56(m,1H ),7.40–7.35(m,2H),7.33(t,J＝4.2Hz,1H),7.26(dt,J＝9.0,5.0Hz,6H),7.17(dd,J＝7.7,5.9Hz,1H) ,6.85(dd,J＝8.9,2.5Hz,4H),6.15–6.06(m,1H),5.02(s,2H),4.41–4.30(m,1H),4.14–4.07(m,1H),3.67 –3.63(m,6H),3.61–3.46(m,4H),3.23(d,J＝4.4Hz,2H),2.75(t,J＝5.9Hz,1H),2.64(t,J＝6.1Hz, 1H), 2.43 (dd, J＝13.1, 7.6Hz, 2H), 1.13–1.07 (m, 9H), 0.96 (d, J＝6.7Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 163.7, 162.3, 158.5,150.8,149.9,148.1,146.7,146.6,145.2,144.9,135.9,135.8,135.7,130.2,130.1,129.8,128.3,128.1,127.1,126.4,124.0,119.4,119 .2,113.7,105.5,87.1,86.4, 85.6,73.4,63.8,59.0,58.8,55.4,43.1,43.0,24.7,20.2. ³¹ P NMR (162MHz, DMSO) δ147.6,147.3.

2e: ¹ H NMR (400MHz, DMSO) δ11.93 (s, 1H), 9.30 (t, J = 5.6Hz, 1H), 8.89 (s, 1H), 8.54 (d, J = 6.9Hz, 1H), 8.17(s,1H),8.01(d,J＝8.4Hz,1H),7.85(t,J＝7.7Hz,1H),7.72(t,J＝7.6Hz,1H),7.57(t,J＝7.3 Hz,1H),7.40–7.33(m,2H),7.25(td,J＝6.4,2.9Hz,6H),7.13(t,J＝7.2Hz,1H),6.91–6.81(m,4H),6.13 –6.02(m,1H),4.70(d,J＝5.6Hz,2H),4.41–4.30(m,1H),4.14–4.07(m,1H),3.69(s,6H),3.62–3.46(m ,4H),3.22(d,J＝4.5Hz,2H),2.75(t,J＝5.9Hz,1H),2.64(dd,J＝6.1,4.7Hz,1H),2.48–2.35(m,2H) ,1.10(dd,J＝15.3,7.7Hz,9H),0.96(d,J＝6.7Hz,3H). ¹³ C NMR(101MHz,DMSO)δ163.6,162.3,158.5,151.4,149.9,147.2,146.6,146.4 ,145.2,135.9,135.8,135.7,134.1,133.0,130.2,129.6,129.1,128.3,128.1,127.9,127.2,119.4,119.2,113.7,105.6,87.0,86.3,85.6,73.4, 63.7,58.8,55.4,43.1 ,43.0,24.7,20.2. ³¹ P NMR (162MHz, DMSO) δ147.6,147.3.

2f: ¹ H NMR (400MHz, DMSO) δ12.01 (s, 1H), 9.66 (t, J = 5.1Hz, 1H), 8.56 (d, J = 6.0Hz, 1H), 8.31 (dd, J = 8.5 ,2.8Hz,1H),8.00–7.93(m,2H),7.76(dd,J＝11.3,4.2Hz,1H),7.58(t,J＝7.5Hz,1H),7.48(dd,J＝8.5, 2.5Hz,1H),7.37(dd,J＝7.5,3.1Hz,2H),7.29–7.23(m,6H),7.15(t,J＝7.2Hz,1H),6.86(dd,J＝8.9,3.0 Hz,4H),6.16–6.05(m,1H),4.83–4.76(m,2H),4.38(dd,J＝10.1,4.8Hz,1H),4.14–4.07(m,1H),3.69(d, J＝2.4Hz,6H),3.63–3.46(m,4H),3.29–3.18(m,2H),2.76(t,J＝5.9Hz,1H),2.68–2.61(m,1H),2.48–2.37 (m,2H),1.13–1.07(m,9H),0.96(d,J＝6.7Hz,3H). ¹³ C NMR(101MHz,DMSO)δ163.6,162.1,158.6,149.9,147.3,146.5,146.3,145.2 ,137.1,135.9,135.8,135.7,130.2,130.1,128.9,128.3,128.1,127.4,127.1,126.7,120.2,119.4,119.2,113.7,105.8,86.9,86.4,85.5,73.1, 63.5,59.0,55.4,45.3 ,43.1,24.8,20.2. ³¹ P NMR (162MHz, DMSO) δ147.6,147.3.

2g: ¹ H NMR (400MHz, DMSO) δ11.93 (s, 1H), 9.42 (t, J = 5.7Hz, 1H), 8.98 (d, J = 2.7Hz, 1H), 8.51 (d, J = 5.7 Hz,1H),8.40(d,J＝7.0Hz,1H),7.91(d,J＝8.1Hz,1H),7.68–7.56(m,2H),7.53(td,J＝7.7,2.8Hz,1H ),7.40–7.32(m,2H),7.31–7.21(m,6H),7.20–7.13(m,1H),6.86(dd,J＝8.8,3.5Hz,4H),6.14–6.03(m,1H ),5.08(d,J＝5.8Hz,2H),4.41–4.29(m,1H),4.13–4.05(m,1H),3.71–3.66(m,6H),3.62–3.38(m,4H), 3.24(dd,J＝25.5,5.9Hz,2H),2.75(t,J＝5.9Hz,1H),2.66–2.61(m,1H),2.46–2.33(m,2H),1.09(dd,J＝ 16.0,7.1Hz,9H),0.95(d,J＝6.7Hz,3H). ¹³ C NMR(101MHz,DMSO) δ163.6,161.1,158.5,150.3,149.9,146.4,146.2,145.2,136.9,135.9,135.8,135.7,130.2,130.1,128.4,128.3,128.1,127.8,127.1,126.7,12 2.0,119.4,119.2,113.7,105.9, 86.9,86.4,85.5,72.9,63.6,59.0,55.4,43.1,43.0,24.7,20.2. ³¹ P NMR (162MHz, DMSO) δ147.7,147.3.

Example 2. Modification of nucleic acid aptamer APC001 by quinoline to increase its target binding affinity

In previous work, the nucleic acid aptamer APC001, which specifically targets osteosclerostin, was screened through protein-SELEX technology. The specific sequence is: CGGGGTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCTGCC. Through chemical biology research, 22 key sites for aptamer-target binding were screened (respectively C1, G2, G3, G4, G5, T6, T8, G11, T13, G15, T29, T30 , G31, G32, C33, A34, G35, C36, T37, G38, C39, C40).

The present invention uses chemical modification technology to modify different subtypes of quinolines at these key sites. First, different quinolines are coupled to nucleosides to obtain compounds 1a-1g, and then reacted with phosphorus reagents to construct different quinoline isoforms modified phosphoramidite monomers 2a-2g. Finally, DNA solid-phase synthesis technology is used to obtain compounds in different Aptamers with quinoline modification at key sites.

Specifically, the phosphoramidite monomers 2a to 2g were inserted into the 22 key sites of the osteosclerostin aptamer through standard DNA solid-phase synthesis technology. The sequence and insertion position are shown in Table 1 below:

Table 1. Quinoline modified adapter sequences (x=a, b, c, d, e, f, g)

ELONA method to compare the binding ability of each quinoline-modified aptamer to the target osteosclerostin

Prepare the following buffer in advance:

ELONA B&W buffer: PBS, 1mM MgCl2, 0.05% Tween 20;

ELONA assay blocking buffer:PBS,0.1% Tween 20and 1%BSA;

ELONA assay washing buffer: PBS, 1mM MgCl2, 0.1% Tween 20 and 0.1% BSA;

ELONA assay Strep-HRP binding buffer: Strep-HRP 1:10000 diluted into ELONA assay washing buffer;

ELONA assay reaction stopping buffer:2M H2SO4.

Add 160ng of target protein to 96 wells containing 100μl SELEX B&W buffer and incubate overnight at 4°C. The 96-well plate was then blocked with Blocking buffer for 1 hour at room temperature, and then washed 4 times with SELEX B&W buffer. Add terminally biotinylated aptamers at 1 μM in 100 μL SELEX B&W buffer to each well and incubate at room temperature for 45 minutes while continuing to shake gently. Next, wash 4 times with SELEX B&W buffer to remove non-specific and very weak binding. Add 100 μl Strap-HRP (1:10000 dilution) to each well, incubate for 30 minutes, and wash 4 times with washing buffer. Add 50 μl TMB to each well and incubate for 20 minutes. _The reaction was stopped by adding 50 μl 2M _H2SO4 . Measure the absorbance at 450 nm with a microplate reader.

The results are shown in Figure 1. The obtained quinoline-modified osteosclerostin nucleic acid aptamer generally has improved target-binding ability compared with the natural APC001. Among them, after modification of 5-quinoline (d) at positions 6 and 11 of the aptamer (i.e. aptamers APC001-d6 and APC001-d11), the improvement effect is most significant (nearly 16 times).

Biolayer interferometry (BLI) determines the affinity between quinoline-modified aptamers and targets

Biolayer interferometry (BLI) was used to determine the affinity of the six sequences with the highest binding ability in Figure 1, namely APC001-d6, APC001-d8, APC001-d11, APC001-d13, APC001-d32, and APC001-d34.

The instrument used to analyze the interaction between quinoline-modified aptamers and osteosclerostin was Octet 96/96e system (ForteBio), and all operations were performed at room temperature. The program running procedure is as follows:

Baseline: 1×PBS buffer (10mM phosphate buffer, 2.7mM KCl and 137mM NaCl, pH 7.4), 120s, 1000rpm;

Loading:100nM 5-biotinated aptamer in 1×PBST buffer(1×PBS+0.02%Tween20, pH 7.4),600s,500rpm;

Baseline 2:1×PBST buffer,300s,500rpm;

Association: sclerostin (20nM, 16nM, 12nM, 8nM, 4nM, 0nM) in 1×PBST buffer, 600s, 500rpm;

Dissociation:1×PBST buffer,600s,500rpm;

Regenration:5M NaCl,30s,500rpm;

Neutralization: 1×PBS buffer, 60s, 500rpm.

Finally, the equilibrium dissociation constant KD value was calculated using the analysis software ForteBio Data analysis 11.0 with a 1:1 binding mode fitting.

The results are shown in Figure 2. The quinoline structure in the present invention greatly improves the affinity between the aptamer and the target.

Claims (26)

1. A modified target-specific nucleic acid molecule comprising one or more quinoline-modified nucleotides, wherein the modified target-specific nucleic acid molecule has a binding affinity to a target compared to a corresponding control nucleic acid molecule improve.
2. The modified target-specific nucleic acid molecule of claim 1, wherein the nucleic acid molecule is selected from the group consisting of aptamers, antisense nucleic acids (antisense RNA), dsRNA (such as siRNA, shRNA).
3. The modified target-specific nucleic acid molecule of claim 1 or 2, wherein the modified target-specific nucleic acid molecule has a binding affinity to a target that is increased by at least about 10%, at least about 20% compared to a corresponding control nucleic acid molecule. , at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more; or the modified The target-specific nucleic acid molecule has a binding affinity to the target that is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, the affinity of the unmodified corresponding nucleic acid molecule. At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 16 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, At least about 50 times, at least about 75 times, at least about 100 times or more.
4. The modified target-specific nucleic acid molecule of any one of claims 1-3, wherein the modified target-specific nucleic acid molecule has an effect on the target of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM, preferably less than 5 nM or less KD (dissociation constant).
5. The modified target-specific nucleic acid molecule of any one of claims 1-4, wherein the quinoline modification is quinolin-2-yl substitution, quinolin-3-yl substitution, quinolin-4-yl substitution, Quinolin-5-yl substitution, quinolin-6-yl substitution, quinolin-7-yl substitution or quinolin-8-yl substitution. Preferably, the quinoline modification is quinolin-5-yl substitution.
6. The modified target-specific nucleic acid molecule of any one of claims 1-5, wherein the quinoline-modified nucleotide comprises a substituent group R selected from the following ag:
   

   Preferably,
7. The modified target-specific nucleic acid molecule of any one of claims 1-6, wherein the quinoline-modified nucleotide comprises the base moiety of Formula I:

   in
   

   Preferably,
8. The modified target-specific nucleic acid molecule of any one of claims 1-7, wherein the quinoline-modified nucleotide comprises the following structure of Formula II:
   

   in
   

   Preferably,
9. The modified target-specific nucleic acid molecule of any one of claims 1-8, wherein one or more naturally occurring nucleotides in the target-specific nucleic acid molecule are replaced by the quinoline-modified nucleotides .
10. The modified target-specific nucleic acid molecule of any one of claims 1-9, wherein the target-specific nucleic acid molecule may further comprise one or more modifications that extend its half-life in vivo.
11. The modified target-specific nucleic acid molecule of any one of claims 1-10, wherein the modified target-specific nucleic acid molecule is a modified osteosclerostin-specific aptamer.
12. The modified target-specific nucleic acid molecule of claim 11, wherein the modified osteosclerostin-specific aptamer is derived from the osteosclerostin-specific aptamer of SEQ ID NO: 2.
13. The modified target-specific nucleic acid molecule of claim 12, wherein the modified osteosclerostin-specific aptamer is substituted at a nucleotide corresponding to one or more positions selected from SEQ ID NO: 2 The quinoline modified nucleotide substitutions: 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 11th, 13th, 15th , 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th and 40th.
14. The modified target-specific nucleic acid molecule of claim 12 or 13, wherein the modified osteosclerostin-specific nucleic acid molecule is at position 6, position 8, position 11, and position corresponding to SEQ ID NO: 2 One or more nucleotides at position 13, 32 and/or 34 are substituted by the quinoline modified nucleotide.
15. The modified target-specific nucleic acid molecule of any one of claims 12-14, wherein the modified osteosclerostin-specific nucleic acid molecule comprises a nucleotide sequence selected from SEQ ID NOs: 3-24, wherein x It is the quinoline modified nucleotide.
16. The modified target-specific nucleic acid molecule of any one of claims 12-15, wherein the modified osteosclerostin-specific nucleic acid molecule comprises a core selected from the group consisting of SEQ ID NOs: 8, 10, 11, 16 and 18 A nucleotide sequence, where x is the quinoline-modified nucleotide.
17. The modified target-specific nucleic acid molecule of any one of claims 12-16, wherein the quinoline-modified nucleotide comprises the structure of Formula II:
    

    in,
18. A composition comprising the modified target-specific nucleic acid molecule of any one of claims 1-17, and optionally a pharmaceutically or physiologically acceptable carrier.
19. A method of increasing the binding affinity of a target-specific nucleic acid molecule to a target, the method comprising replacing one or more naturally occurring nuclei in the target-specific nucleic acid molecule with one or more quinoline-modified nucleotides glycosides.
20. The method of claim 19, wherein the quinoline modification is quinolin-2-yl substitution, quinolin-3-yl substitution, quinolin-4-yl substitution, quinolin-5-yl substitution, quinolin-6-yl substitution base substitution, quinolin-7-yl substitution or quinolin-8-yl substitution. Preferably, the quinoline modification is quinolin-5-yl substitution.
21. The method of claim 19 or 20, wherein the quinoline modified nucleotide comprises a substituent group R selected from the following ag:
    

    Preferably,
22. The method of any one of claims 19-21, wherein the quinoline modified nucleotide comprises the base moiety of Formula I:
    

    in
    

    Preferably,
23. The method of any one of claims 19-22, wherein the quinoline modified nucleotide comprises the following structure of Formula II:
    

    in
    

    Preferably,
24. The method of any one of claims 19-23, said one or more quinoline modified nucleotide substitutions being introduced during chemical synthesis of said target-specific nucleic acid molecule.
25. The method of claim 24, wherein modified phosphoramidite monomers selected from the following 2a-2g are added during chemical synthesis of the target-specific nucleic acid molecules:
    
26. A modified phosphoramidite monomer selected from the following 2a-2g, which is used to increase the binding affinity of target-specific nucleic acid molecules to the target: