Oligonucleotide compound, medicine and application thereof

Abstract

The invention provides an oligonucleotide compound, a medicine and application of the oligonucleotide compound, the oligonucleotide compound comprises at least one carbocyclic nucleoside shown in the formula (I), the nucleic acid base sequence of the oligonucleotide compound is shown in SEQ ID NO.1, Base is a heterocyclic base, and # imgabs 1 # represents a connection site. The oligonucleotide compound has excellent structural stability, specificity and biological activity, can reduce the expression level of HBV RNA, HBV DNA and related protein, and is beneficial to prevention and treatment of HBV and / or HDV related diseases or symptoms.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to oligonucleotide compounds, drugs and their applications. Background Art

[0002] Chronic hepatitis B (CHB) is caused by infection with the hepatitis B virus (HBV). Its global impact is well-documented and represents an urgent public health issue. The World Health Organization (WHO) has compiled statistics on the disease, finding that as many as 250 million people worldwide, representing approximately 3.5% of the world's population, suffer from CHB.

[0003] Hepatitis D virus (HDV) is a defective virus that requires the assistance of HBV for replication, antigen expression, and infection of hepatocytes. Compared with other hepatitis virus infections, HDV infection is more likely to progress to chronic hepatitis, increasing the risk of cirrhosis, liver decompensation, hepatocellular carcinoma (HCC), and liver-related mortality. It is a serious viral liver infection. Chronic hepatitis D is a severe viral hepatitis caused by co-infection of HDV with HBV or infection with HDV in addition to HBV infection. HBV-infected patients with co-hepatitis D often experience more rapid disease progression and a poorer prognosis.

[0004] Given the severity of the HBV epidemic, despite the continuous development of new drugs and the improvement of antiviral efficacy in recent years, achieving a complete cure for chronic hepatitis B (CHB) remains a clinical challenge. While interferon has demonstrated some success in the treatment of HBV, it is associated with significant negative side effects, such as low response rates and numerous adverse reactions. Nucleoside (acid) analogs (NAs) for HBV treatment require long treatment courses, with limited sustained efficacy and a potential risk of developing drug-resistant viral mutations. With advances in clinical technology, combined immunotherapy with NAs has become the preferred treatment option for CHB. While no new cure is currently available, most studies have found that the sequential or combined use of NAs and pegylated interferon (PEG-IFN), two drugs with different mechanisms of action, can improve therapeutic efficacy. The 2019 guidelines also recommend that PEG-IFN-α be combined with NAs for optimal CHB treatment in select patients. While the combined use of NAs and PEG-IFN offers therapeutic advantages, achieving HBsAg negativity remains difficult, and clinical cure rates remain low (less than 7%). Therefore, there is a need in the art to discover and develop new anti-HBV viral therapies.

[0005] Antisense technology is emerging as an effective means of reducing the expression of specific gene products. Unlike nucleoside therapy, antisense therapy can directly target transcripts for HBV DNA, thereby reducing serum hepatitis B E antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) levels. Because HBV infection produces multiple overlapping transcripts, including pregenomic transcripts (pg RNA), a single antisense oligonucleotide has the potential to reduce HBV DNA in addition to HBeAg and HBsAg, offering new insights into the treatment of HBV and HDV. Summary of the Invention

[0006] In view of this, the present application provides an oligonucleotide compound, a drug and its application to prevent and treat HBV and / or HDV-related diseases or conditions.

[0007] In a first aspect, the present application provides an oligonucleotide compound, comprising at least one carbocyclic nucleoside represented by formula (I), wherein the nucleic acid base sequence of the oligonucleotide compound is as shown in SEQ ID NO.1,

[0008]

[0009] Among them, Base is a heterocyclic base, Indicates the junction site.

[0010] In a second aspect, the present application provides a drug comprising the oligonucleotide compound described in the first aspect or a pharmaceutically acceptable salt thereof.

[0011] In a third aspect, the present application provides the use of the oligonucleotide compound or a pharmaceutically acceptable salt thereof described in the first aspect, or the drug described in the second aspect, in the preparation of a drug for preventing and / or treating HBV and / or HDV-related diseases or conditions.

[0012] The oligonucleotide compounds provided in the present application can specifically target the transcripts of HBV antigens and reduce the levels of serum HBeAg, HBsAg and HBV DNA. At the same time, the carbocyclic nucleosides can improve the nuclease resistance and thermal stability of the oligonucleotide compounds, and can also increase their base pairing affinity, promote binding with complementary mRNA, improve specificity and targeting ability, enhance their biological activity and therapeutic effects, and are beneficial to the prevention and treatment of HBV and / or HDV-related diseases or conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0014] Figure 1 This is the HBV DNA inhibition rate fitting curve of Example 6.

[0015] Figure 2 This is the HBsAg inhibition rate fitting curve of Example 6.

[0016] Figure 3 This is the HBeAg inhibition rate fitting curve of Example 6.

[0017] Figure 4 This is the HBV RNA inhibition rate fitting curve of Example 6.

[0018] Figure 5 This is the HBV DNA inhibition rate fitting curve of Example 7.

[0019] Figure 6 This is the HBsAg inhibition rate fitting curve of Example 7.

[0020] Figure 7 This is the HBeAg inhibition rate fitting curve of Example 7.

[0021] Figure 8 This is the HBV RNA inhibition rate fitting curve of Example 7.

[0022] Figure 9 This is the HBV DNA inhibition rate fitting curve of Example 8.

[0023] Figure 10 This is the HBsAg inhibition rate fitting curve of Example 8.

[0024] Figure 11 This is the HBeAg inhibition rate fitting curve of Example 8.

[0025] Figure 12 This is the HBV RNA inhibition rate fitting curve of Example 8.

[0026] Figure 13 is the HBV DNA content in the plasma of the C57BL / 6-HBV transgenic model mice in Example 9.

[0027] Figure 14 is the HBsAg content in the plasma of the C57BL / 6-HBV transgenic model mice in Example 9.

[0028] Figure 15 is the HBeAg content in the plasma of the C57BL / 6-HBV transgenic model mice in Example 9. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0030] Unless otherwise indicated, the following terms have the meanings described below. Any undefined terms have their art-recognized meanings.

[0031] An "oligonucleotide compound" is composed of 13-24 linked nucleosides, covalently linked by phosphate groups such as phosphate, phosphorothioate, aminoalkylphosphotriester, and alkyl phosphate to form a linear polymer compound.

[0032] When an oligonucleotide compound is represented by a nucleic acid base sequence such as "CGCTGATTTG", unless otherwise indicated, it should be understood that the sequence is in 5'→3' order from left to right, and "A" represents adenine, "G" represents guanine, "C" represents cytosine, "T" represents thymine (T), and "U" represents uracil (U).

[0033] "Nucleoside" is a compound composed of a ribose (or deoxyribose) and a nucleobase (purine or pyrimidine) connected by a glycosidic bond, and is the basic building block of nucleic acids; wherein, nucleosides can include natural nucleosides in the 2'-deoxy and 2'-hydroxy forms, and can also include modified nucleosides having modified base moieties and / or modified sugar moieties.

[0034] "Base" refers to the conventional DNA and RNA bases (uracil, thymine, adenine, guanine, and cytosine) and also includes modified bases such as 5-methylcytosine.

[0035] "5-methylcytosine" means cytosine modified with a methyl group attached to position 5. 5-Methylcytosine is a modified nucleic acid base.

[0036] "cLNA" or "carbocyclic nucleoside" refers to a carbocyclic nucleoside comprising the structure of formula (I) herein.

[0037] "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0038] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'-MOE modified sugar moiety.

[0039] "2'-substituted nucleoside" means a nucleoside comprising a substituent other than H or OH at the 2' position of the furanosyl ring.

[0040] "Phosphorothioate linkage" or "PS linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom.

[0041] "Gapmer" means a chimeric antisense compound having an internal region of multiple nucleosides that supports RNase H cleavage and positioned between external regions of one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the nucleosides comprising the external regions. The internal region may be referred to as a "spacer" and the external regions may be referred to as "wings."

[0042] "Lipophilic group" refers to a group derived from any fat-soluble molecule, such as fats, oils, waxes, terpenes, sterols, fat-soluble vitamins (such as A, D, E and K), monoglycerides, diglycerides, triglycerides, fatty acids, hopanoids and phospholipids.

[0043] "Linker" refers to an organic moiety that connects two parts of a compound.

[0044] "Pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of compounds that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects thereto.

[0045] "Pharmaceutically acceptable carrier or diluent" refers to a medium or diluent that does not interfere with the structure or function of the oligonucleotide, and is an additive other than the oligonucleotide administered to an animal or human. Some of these carriers enable the pharmaceutical composition to be formulated into oral preparations such as tablets, capsules, liquids, suspensions, and some of these carriers enable the pharmaceutical composition to be formulated into injectable preparations for injection or infusion. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution or physiological saline.

[0046] "Agent" or "therapeutic agent" means an active substance that provides a therapeutic benefit when administered to an animal or human. "First agent" means a therapeutic compound described herein. For example, the first agent may be an antisense oligonucleotide targeting HBV and / or HDV. "Second agent" means another pharmaceutically acceptable active ingredient, such as a second therapeutic compound (e.g., a second antisense oligonucleotide targeting HBV and / or HDV) and at least one non-HBV and / or HDV therapeutic compound.

[0047] "Prevention" refers to completely or nearly completely preventing a disease or condition (e.g., infection, ischemia or reperfusion injury) from occurring, for example, when the patient or subject is susceptible to or at risk for the disease or condition; prevention can also include inhibition, i.e., stopping the development of the condition.

[0048] "Treating" refers to: 1) inhibiting the disease; e.g., inhibiting the disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptomology of the disease, condition or disorder (i.e., arresting further development of the pathology and / or symptomology); or 2) ameliorating the disease; e.g., ameliorating the disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptomology of the disease, condition or disorder (i.e., reversing the pathology and / or symptomology).

[0049] "Animal" refers to human and non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees.

[0050] "Administering" means providing an agent to an animal or individual and includes, but is not limited to, administration by a medical professional and self-administration.

[0051] "Simultaneous administration" refers to the co-administration of two pharmaceutical agents in any manner such that the pharmacological effects of both agents manifest simultaneously in the patient. Simultaneous administration does not require that the two agents be in a single pharmaceutical composition, in the same dosage form, or administered by the same route of administration. The effects of the two agents themselves do not need to manifest simultaneously. These effects need only overlap for a period of time and do not need to be co-prolonged.

[0052] "HBV" means mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses geographical genotypes of hepatitis B virus, particularly human hepatitis B virus, and variant strains of geographical genotypes of hepatitis B virus.

[0053] “HBV antigen” means any hepatitis B virus antigen or protein, including core protein, such as “hepatitis B core antigen” or “HBcAg” or “HBcAG” and “hepatitis B E antigen” or “HBeAg” or “HBeAG”, and envelope protein, such as “HBV surface antigen” or “HBsAg” or “HBsAG”.

[0054] "HBV RNA" means any messenger RNA expressed by the hepatitis B virus.

[0055] "HBV nucleic acid" or "HBV DNA" means any nucleic acid encoding HBV. For example, in certain embodiments, HBV nucleic acid includes, but is not limited to, any viral DNA sequence encoding the HBV genome or portion thereof, any RNA sequence transcribed from viral DNA, including any RNA sequence encoding an HBV protein.

[0056] "HBV protein" means any protein secreted by the hepatitis B virus. The term encompasses various HBV antigens, including core proteins, such as "hepatitis E antigen," "HBeAg," or "HBeAG," and envelope proteins, such as "HBV surface antigen," "HBsAg," or "HBsAG."

[0057] "Hepatitis B-related disease or symptom" or "HBV-related disease or symptom" means any disease, biological symptom, medical symptom, or event caused by, associated with, related to, or attributable to hepatitis B infection, exposure, or disease. The term hepatitis B-related symptom includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, transplantation associated with liver disease, and, in conjunction with a positive test for the presence of hepatitis B virus, hepatitis B virus antigens, or a positive test for the presence of antibodies specific for hepatitis B virus antigens, symptoms that may include any or all of the following: flu-like symptoms, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver area of the body, clay-colored or gray stools, general itching, and dark urine.

[0058] "HDV (hepatitis D virus)" refers to hepatitis D virus, which is a defective virus.

[0059] "Induce," "inhibit," "enhance," "elevate," "increase," "reduce," "reduce" or similar terms generally refer to a quantitative difference between two states. The term may refer to a statistically significant difference between two states. For example, "effectively inhibiting the activity or expression of HBV" means that the activity level or expression level of HBV in a treated sample is quantitatively different from the activity level or expression level of HBV in untreated cells, and may be statistically significant. The term is applicable to, for example, expression levels and activity levels. "Inhibit HBV" means reducing the level or expression of HBV RNA, DNA and / or protein. In certain embodiments, HBV is inhibited in the presence of an antisense compound targeting HBV (including antisense oligonucleotides targeting HBV) compared to the expression level of HBV RNA, HBV DNA and / or protein in the absence of an HBV antisense compound (such as an antisense oligonucleotide). In certain embodiments, HBV RNA levels are reduced. In certain embodiments, HBV DNA levels are reduced. In certain embodiments, HBV protein levels are reduced. In certain embodiments, HBV antigen levels are reduced. In certain embodiments, HBV s antigen (HBsAg) levels are reduced. In certain embodiments, HBV e antigen (HBeAg) levels are reduced. The reduction can occur in a time-dependent manner and / or in a dose-dependent manner.

[0060] In the chemical formula provided herein, DMTr is 4,4'-dimethoxytriphenylmethyl, Bn is benzyl, Ms is methylsulfonyl, Bz is benzoyl, Me is methyl, Ac is acetate, and (S) and (R) represent S configuration and R configuration.

[0061] The present application provides an oligonucleotide compound, which includes at least one carbocyclic nucleoside represented by formula (I), and the nucleic acid base sequence of the oligonucleotide compound is shown in SEQ ID NO.1.

[0062]

[0063] Among them, Base is a heterocyclic base, Indicates the junction site.

[0064] The oligonucleotide compounds of the present application can target HBV DNA transcripts. At the same time, the carbocyclic nucleosides can improve the nuclease resistance and thermal stability of the overall structure, and can also increase the base pairing affinity of the oligonucleotide compounds, improve specificity, promote the binding of the oligonucleotide compounds to complementary mRNA, improve the efficacy and safety of the oligonucleotide compounds, and help reduce serum HBeAg, HBsAg and HBV DNA levels, thereby preventing and treating HBV-related diseases or conditions; HDV infection requires the presence of HBV, and HDV relies on HBV-derived HBsAg for packaging and viral transmission in the liver. HBV and HDV use HBsAg together. The oligonucleotide compounds of the present application can also effectively knock down HBV transcripts, reduce the production of HBsAg, and exert antiviral effects on HDV, thereby preventing and treating HDV-related diseases or conditions, and providing a new treatment method for the treatment of hepatitis B and hepatitis D.

[0065] The oligonucleotide compound of the present application may include at least one carbocyclic nucleoside represented by formula (I). For example, the oligonucleotide compound contains one carbocyclic nucleoside represented by formula (I); for another example, the oligonucleotide compound has multiple (e.g., two, three, four, etc.) carbocyclic nucleosides represented by formula (I).

[0066] It is understood that the heterocyclic base in formula (I) is any base in the nucleic acid base sequence of the oligonucleotide compound. In one embodiment of the present application, the heterocyclic base can be selected from at least one of adenine, guanine, cytosine, 5-methylcytosine, thymine and uracil.

[0067] In one embodiment of the present application, the oligonucleotide compound is composed of a 5' wing segment, a spacer segment and a 3' wing segment, and the base of the 5' wing segment includes GCAGA, the base of the spacer segment includes GGTGAAGCGA, and the base of the 3' wing segment includes AGTGC. In certain embodiments, the nucleotides in the 5' wing segment and the 3' wing segment are ribonucleotides, and the nucleotides in the spacer segment are deoxyribonucleotides. In certain embodiments, the nucleosides in the 5' wing segment and the 3' wing segment have 2'-O-methoxyethyl modifications. It is understandable that the nucleosides in the 5' wing segment and the 3' wing segment can partially have 2'-O-methoxyethyl modifications, or all have 2'-O-methoxyethyl modifications. In certain embodiments, adjacent nucleosides in the oligonucleotide compound are connected by thiophosphates. In certain embodiments, all cytosines in the oligonucleotide compound are 5-methylcytosine.

[0068] In one embodiment of the present application, the 5' wing segment and / or the 3' wing segment contain carbocyclic nucleosides to enhance the structural stability and targeting of the oligonucleotide compound.

[0069] In one embodiment of the present application, the oligonucleotide compound further comprises a lipophilic group. The lipophilic group can improve the oligonucleotide compound's ability to deliver to liver cells, enhance its cell permeability, enhance the efficiency of intracellular drug delivery, and improve the distribution characteristics of the oligonucleotide compound within the cell, thereby enabling more rapid, direct, and efficient targeting of HBV antigen transcripts. Furthermore, the oligonucleotide compound containing a carbocyclic nucleoside and a lipophilic group has the advantages of good stability, strong anti-HBV activity in vitro and in vivo, and long-lasting efficacy. It can also act on HDV, facilitating the prevention and improvement of HBV- and HDV-related diseases or conditions, and providing a new treatment for hepatitis B and hepatitis D.

[0070] In one embodiment of the present application, the lipophilic group is located at at least one of the 5' and 3' ends of the oligonucleotide compound. In other words, the lipophilic group can be located only at the 5' end of the oligonucleotide compound, or only at the 3' end of the oligonucleotide compound, or at both the 5' and 3' ends of the oligonucleotide compound; that is, the lipophilic group is located at at least one of the 5' and 3' ends of the oligonucleotide compound's nucleic acid base sequence, which can also be understood as the lipophilic group being located at one or both ends of the oligonucleotide compound's molecular chain. Conjugating a lipophilic group can enhance the penetration and delivery effect of the oligonucleotide compound while not affecting the targeting effect of the oligonucleotide compound.

[0071] In one embodiment of the present application, the lipophilic group is derived from a lipophilic compound having 4-32 carbon atoms, so that the cell penetration ability of the oligonucleotide compound can be improved. Specifically, the number of carbon atoms of the lipophilic compound (i.e., the number of carbon atoms of the lipophilic group) can be, but is not limited to, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32, etc. In some embodiments, the lipophilic compound can be selected from at least one of alkanes, alkanoyl compounds, carboxyl compounds, retinyl compounds, cholesteryl olefins, cholesterol, dihydrotestosterone, hexadecyl glycerol (such as 1,3-di-O (hexadecyl) glycerol, etc.), menthol, borneol, dimethoxytrityl compounds, geranyloxyhexyl compounds and phenoxazine. Exemplarily, the alkane can be C4-C 32 Alkanes, i.e. lipophilic groups are C4-C 32 Exemplarily, the alkanoyl compound may be C4-C 32 Alkanoyl compounds, i.e. lipophilic groups are C4-C 32 For example, the carboxyl compound can be selected from at least one of cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, and bile acid.

[0072] In one embodiment of the present application, the oligonucleotide compound further comprises a linker, the linker is connected to the lipophilic group, and the linker comprises a biodegradable group. In some embodiments, the biodegradable group can be selected from at least one of phosphate and thiophosphate. In some embodiments, the linker is a biodegradable group. That is, the linker contains only biodegradable groups. In some embodiments, the linker can also include a linking group, that is, the linker includes a biodegradable group and a linking group, and the linking group connects the biodegradable group and the lipophilic group. Wherein, the linking group can be covalently coupled to the lipophilic group. In some embodiments, the linking group is selected from at least one of the groups shown in formula (II-1) to formula (II-5),

[0073]

[0074] In one embodiment of the present application, the combination of the linker and the lipophilic group is selected from at least one of the groups represented by formula (III-1) to formula (III-4).

[0075]

[0076] The present application also provides a drug comprising the oligonucleotide compound of any of the aforementioned embodiments or a pharmaceutically acceptable salt thereof. The drug exhibits excellent structural stability, specific targeting, and biological activity, and can reduce the expression of HBV RNA, HBV DNA, and related proteins, thereby facilitating the prevention and treatment of HBV- and HDV-related diseases or conditions.

[0077] In one embodiment of the present application, the pharmaceutically acceptable salt of the oligonucleotide compound may be at least one of a sodium salt and a potassium salt.

[0078] In one embodiment of the present application, the drug may further include a pharmaceutically acceptable carrier or diluent, which facilitates the administration of the drug.

[0079] In one embodiment of the present application, the oligonucleotide compound or its pharmaceutically acceptable salt constitutes a medicine as a single active ingredient or with other pharmaceutically acceptable active ingredients. That is to say, the oligonucleotide compound or its pharmaceutically acceptable salt is the only active ingredient in the medicine, or the oligonucleotide compound or its pharmaceutically acceptable salt and other pharmaceutically acceptable active ingredients are used as the active ingredient in the medicine. Other pharmaceutically acceptable active ingredients are the second agent, which can be but not limited to anti-HBV agents, chemotherapeutics, antibiotics, analgesics, anti-inflammatory agents, antifungals, antiparasitics, antinausea agents, antidiarrheals, immunomodulators and other antiviral agents. The second agent can be used to treat hepatitis B, cancer, bacteria, pain, inflammation, fungal infection and / or parasitic infection, alcoholism, substance abuse, diabetes, or can be used to treat other viral infections such as human immunodeficiency virus (HIV), hepatitis C virus (HCV) etc. In some embodiments, the anti-HBV agent can be, but is not limited to, selected from interferons (including but not limited to interferon α2b, interferon α2a, interferon α-1a, pegylated interferon α2a, pegylated interferon α2b), nucleoside (acid) analogs (including but not limited to entecavir; tenofovir disoproxil fumarate; tenofovir alafenamide fumarate; amitenofovir), other anti-HBV small nucleic acid drugs, Toll-like receptor (TLR) agonists, capsid inhibitors, HBV therapeutic vaccines, HBV preventive vaccines, neutralizing antibodies, HBV monoclonal or polyclonal antibody therapeutics, etc. In some embodiments, other anti-HBV small nucleic acid drugs can be selected from, but are not limited to, antisense oligonucleotides (ASOs), small interfering RNA (siRNA), aptamer RNA, microRNA (miRNA), circular RNA (circRNA), small activating RNA (saRNA) and CpG oligonucleotides (CpG oligonucleotide), CRISPR-Cas nucleic acids, ribozymes, transfer RNA (tRNA), antibody nucleic acid conjugates (ARCs), nucleic acid aptamer-drug conjugates (ADCs), etc.

[0080] The present application also provides the use of the oligonucleotide compound or pharmaceutically acceptable salt thereof in any of the above embodiments, or the drug in any of the above embodiments in the preparation of a drug for preventing and / or treating HBV and / or HDV-related diseases or conditions, which is beneficial to the prevention and treatment of HBV and / or HDV-related diseases or conditions.

[0081] In one embodiment of the present application, the drug may further include a second agent. The oligonucleotide compound or a pharmaceutically acceptable salt thereof and the second agent may be administered simultaneously in a single formulation or separately in different formulations, and the administration of the oligonucleotide compound or a pharmaceutically acceptable salt thereof and the second agent may be performed concurrently or sequentially.

[0082] The present application also provides a method for prevention and / or treatment, comprising administering to a subject an oligonucleotide compound or a pharmaceutically acceptable salt thereof in any of the above embodiments, or a drug in any of the above embodiments, thereby preventing and / or treating HBV and / or HDV-related diseases or conditions.

[0083] In one embodiment of the present application, the drug includes a second agent, and the treatment method further includes administering to the subject an oligonucleotide compound or a pharmaceutically acceptable salt thereof in any of the above embodiments and the second agent. The oligonucleotide compound or a pharmaceutically acceptable salt thereof and the second agent may be administered simultaneously or separately.

[0084] The oligonucleotide compound provided herein exhibits high HBV inhibitory activity. In one embodiment of the present invention, the oligonucleotide compound may include at least one of Compounds 1 to 12 shown in Table 1.

[0085] Table 1 Oligonucleotide compounds

[0086] Compound Sequence (5'-3') Positive control GCAGAggtgaagcgaAGTGC Compound 1 <![CDATA[GC cLNA AGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 2 <![CDATA[GC cLNA AGAggtgaagcga cLNA AT cLNA TGC]]> Compound 3 <![CDATA[ cLNA GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 4 <![CDATA[X s cLNA GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 5 <![CDATA[ cLNA GCAGAggtgaagcga cLNA AGT cLNA GC]]> Compound 6 <![CDATA[ cLNA GCAGAggtgaagcgaAG cLNA T cLNA GC]]> Compound 7 <![CDATA[GC cLNA A cLNA GAggtgaagcgaA cLNA GT cLNA GC]]> Compound 8 <![CDATA[ cLNA GC cLNA AGAggtgaagcgaAG cLNA T cLNA GC]]> Compound 9 <![CDATA[GCAGAggtgaagcga cLNA AGT cLNA GC]]> Compound 10 <![CDATA[GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 11 <![CDATA[X o GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 12 <![CDATA[X o GCAGAggtgaagcga cLNA AGT cLNA GC]]>

[0087] Among them, each nucleoside is connected by a phosphorothioate (PS) bond, and each cytosine is 5-methylcytosine; lowercase "a, g, c, t" represents deoxyribonucleoside, and uppercase "A, G, C, T" represents 2'-MOE modified nucleoside; cLNA A" represents a carbocyclic nucleoside with base A, cLNA G" represents a carbocyclic nucleoside with G as the base, cLNA T" represents a carbocyclic nucleoside with T as the base; "X o " represents the following structure: “X s " represents the following structure:

[0088] The effects of the oligonucleotide compounds provided by the present application are described in more detail through the following examples, but the following examples are only used to illustrate the compounds described herein and are not intended to limit the present application.

[0089] Example 1: cLNA G Synthesis of phosphoramidite monomer (Compound I-1-7)

[0090]

[0091] (1) Synthesis of compound 19

[0092]

[0093] Under nitrogen atmosphere, compound 18 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), O-6-benzylguanine (G Bn , 33.8 g, 0.14 mol) was suspended in 240 mL of dry N,N-dimethylformamide (DMF) and heated to 90°C for 3 h. The reaction was monitored by liquid chromatography-mass spectrometry (LCMS) upon completion. The temperature was then lowered, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate (v:v) in a ratio of 1:2 to 1:3 as the eluent to obtain 23.3 g of an oily substance, with a yield of 56.8%. [M+1] + =582.2; 1HNMR(400MHz,Chloroform-d)δ7.64(s,1H),7.57–7.47(m,2H),7.46–7.29(m ,13H),5.58(s,2H),4.94(d,J=11.4Hz,1H),4.88(s,2H),4.70(dd,J=10.8,3.0Hz,2H), 4.57(dd,J=8.4,5.6Hz,1H),4.52(s,2H),4.15(d,J=5.6Hz,1H),3.85(d,J=11.3Hz,1H) ,3.66(d,J=11.3Hz,1H),3.41(s,2H),2.38(dd,J=13.3,8.7Hz,1H),2.09–1.96(m,2H).

[0094] (2) Synthesis of Compound 20

[0095]

[0096] Under a nitrogen atmosphere, compound 19 (23.2 g, 0.04 mol) was dissolved in 230 mL of dry pyridine (Py.). The temperature was lowered to 0°C, and trimethylsilyl chloride (TMSCl, 26.1 g, 0.24 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature (rt) for 2 h. The temperature was lowered to 0°C, and isobutyryl chloride (iPrCOCl, 4.7 g, 0.044 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 2 h. 30 mL of concentrated aqueous ammonia (NH4OH) was added dropwise, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Water was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 to 1:2 ratio of petroleum ether to ethyl acetate as the eluent to give 26.3 g of an oily liquid in a 97% yield. [M+1] + =652.3.

[0097] (3) Synthesis of Compound 21

[0098]

[0099] Under a nitrogen atmosphere, compound 20 (25.5 g, 0.039 mol) was dissolved in 200 mL of dry pyridine (Py), cooled to 0°C, and a solution of methanesulfonic anhydride (Ms2O, 8.7 g, 0.051 mol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. LCMS monitored the reaction until the product content reached a maximum. Water was added to quench the reaction, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate (v:v) as the eluent to obtain 19.1 g of an oily substance with a yield of 56.8%. 4.5 g of the raw material was recovered. [M+1] + =730.3.

[0100] (4) Synthesis of Compound 22

[0101]

[0102] Under a nitrogen atmosphere, 60% sodium hydride (NaH, 3.5 g, 87.9 mmol) was carefully added to 210 mL of dry tetrahydrofuran (THF) and stirred for 10 min. The temperature was lowered to 0°C, and a solution of compound 21 (21.4 g, 29.3 mmol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. LCMS monitored the reaction completion, quenched with acetic acid, and saturated sodium bicarbonate solution was added. The mixture was extracted twice with ethyl acetate. The organic phases were combined, washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate (v:v) as the eluent to afford 15.8 g of an oily substance with a yield of 85%. [M+1] + =634.3.

[0103] (5) Synthesis of Compound I-1-3

[0104]

[0105] Under a nitrogen atmosphere, compound 22 (5 g, 7.9 mmol) was dissolved in 50 mL of dichloromethane (DCM), cooled to -78°C, and 71 mL of a 1 M solution of boron tribromide (BBr3) in dichloromethane was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40°C and stirred for 2 h. The reaction was monitored by LCMS. Methanol was added dropwise to quench the reaction, and the mixture was allowed to warm to room temperature and concentrated. The crude product was purified by column chromatography using a 10:1 ratio of dichloromethane to methanol (v:v) as the eluent to afford 2.5 g of a white solid in an 87.2% yield. [M+1] +=364.1;1H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.36–7.21(m,1H),5.17(d,J=4.2Hz,1H),4.59(t,J=5.2Hz,1H),4.44(dd,J=9.7,5.4Hz,1H),4.03(d,J=4.4Hz,1 H),3.96(s,1H),3.78–3.68(m,2H),2.79(p,J=6.8Hz,1H),2.23(ddd,J=26 .1,14.8,10.2Hz,2H),1.99(dd,J=10.2,6.6Hz,1H),1.12(d,J=6.8Hz,5H).

[0106] (6) Synthesis of Compound 24

[0107]

[0108] Under a nitrogen atmosphere, compound I-1-3 (3.1 g, 9.1 mmol) and 4,4'-bismethoxytrityl chloride (DMTrCl, 6.8 g, 20.2 mmol) were dissolved in 30 mL of dry dichloromethane (DCM). The temperature was lowered to 0°C, and N,N-diisopropylethylamine (DIEA, 5.89 g, 45.5 mmol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature (rt) and stirred for 2 h. The reaction was monitored by LCMS for completion, and the mixture was concentrated. The crude product was purified by column chromatography using a solvent ratio of dichloromethane:methanol:triethylamine (v:v:v) = 30:1:0.1% to give 4.6 g of a light yellow solid, a yield of 76%. [M+1] + =666.3;1H NMR(400MHz,Chloroform-d)δ12.10(s,1H),9.40(s,1H),7.75(s,1H),7.46–7.40(m,2H),7.34–7.25 (m,5H),7.23–7.17(m,2H),6.83(ddd,J=9.0,4.2,2.0Hz,4H),4.66(s,1H),4.48(t,J=7.6Hz,1H),4. 27(s,1H),3.95(d,J=7.1Hz,1H),3.82(s,1H),3.78(s,6H),3.68(d,J=7.0Hz,1H),3.46(d,J=9.6Hz, 1H), 3.27 (d, J = 9.6Hz, 1H), 2.53 (p, J = 6.9Hz, 1H), 2.32 (d, J = 7.6Hz, 2H), 1.19 (dd, J = 9.7, 6.8Hz, 6H).

[0109] (7) Synthesis of Compound I-1-7

[0110]

[0111] Under a nitrogen atmosphere, compound 24 (1.0 g, 1.5 mmol) and 4,5-dicyanoimidazole (DCl, 443 mg, 3.75 mmol) were dissolved in 10 mL of dry dichloromethane (DCM). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (PN2, 1.1 g, 3.75 mmol) was added dropwise at room temperature (rt), and stirring was continued for 2 h. LCMS monitored the reaction completion, followed by the addition of dichloromethane and 0.1 mL of triethylamine. The organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) (v:v) = 7:3, yielding 660 mg of a white solid with a yield of 51%. [M+1] + =866.4; 31 PNMR:147.27,147.17.

[0112] Example 2: cLNA A Synthesis of phosphoramidite monomer (Compound I-1-8)

[0113]

[0114] (1) Synthesis of compound 25

[0115]

[0116] Under a nitrogen atmosphere, compound 18 (26 g, 0.076 mol), adenine (A, 20.5 g, 0.15 mol), and potassium carbonate (K2CO3, 31.5 g, 0.23) were suspended in 260 mL of dry dimethyl sulfoxide (DMSO) and heated to 120°C for 4 h. Upon completion of the reaction, the temperature was lowered, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 20:1 ratio of dichloromethane to methanol (v:v) as the eluent to afford 29.2 g of a white solid in an 80.4% yield. [M+1] +=476.2;1H NMR(400MHz,DMSO-d6)δ8.14(s,1H),8.10(s,1H),7.43–7.24(m,10H),7.16(s, 2H),5.19(d,J=6.2Hz,1H),4.84(d,J=11.8Hz,1H),4.81–4.71(m,2H),4.61–4. 53(m,3H),4.50(t,J=4.9Hz,1H),3.90(d,J=4.3Hz,1H),3.65–3.56(m,2H),3.5 4(d,J=4.6Hz,2H), 2.10(dd,J=13.5,8.6Hz,1H), 1.82(dd,J=13.4,9.2Hz,1H).

[0117] (2) Synthesis of Compound 26

[0118]

[0119] Under a nitrogen atmosphere, compound 25 (22 g, 0.046 mol) was dissolved in 200 mL of dry pyridine (Py.), cooled to 0°C, and trimethylsilyl chloride (TMSCl, 25 g, 0.231 mol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 h. Benzoyl chloride (BzCl, 13.5 g, 0.097 mol) was slowly added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 2 h. The reaction was monitored for completion by LCMS. The mixture was cooled to 0°C, 35 mL of concentrated aqueous ammonia (NH4OH) was added, and the mixture was slowly warmed to room temperature and stirred for 1 h. The reaction was monitored for completion by LCMS, water was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using 100% ethyl acetate as the eluent to obtain 20 g of the product, with a yield of 73.7%. [M+1] + =560.3.

[0120] (3) Synthesis of Compound 27

[0121]

[0122] Under a nitrogen atmosphere, compound 26 (20 g, 0.034 mol) and methanesulfonic anhydride (Ms2O, 7.2 g, 0.041 mol) were dissolved in 200 mL of dry tetrahydrofuran, cooled to -10°C, and triethylamine (TEA, 10.4 g, 0.103 mol) was slowly added dropwise. After the addition was complete, the temperature was raised to 0°C and the reaction was allowed to proceed for 2 h. LCMS confirmed the substantial reaction of the starting material. Saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate (v:v) as the eluent to afford 17 g of a white solid in a 65% yield. [M+1] + =658.2.

[0123] (4) Synthesis of Compound 28

[0124]

[0125] Under a nitrogen atmosphere, carefully add 60% sodium hydride (NaH, 1.87 g, 77.7 mmol) to 170 mL of dry N,N-dimethylformamide (DMF). Stir for 10 minutes, cool to 0°C, and slowly add a solution of compound 27 (17 g, 25.9 mmol) in DMF dropwise. After addition is complete, continue stirring for 2 hours. LCMS monitors the reaction for completion. Add saturated ammonium chloride solution, extract twice with ethyl acetate, combine the organic phases, wash sequentially with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by column chromatography using 100% ethyl acetate as the eluent to obtain 12.3 g of a white solid, with a yield of 85%. [M+1] + =562.2.

[0126] (5) Synthesis of Compound I-1-4

[0127]

[0128] Under a nitrogen atmosphere, compound 28 (5.3 g, 10.7 mmol) was dissolved in 50 mL of dry dichloromethane (DCM) solution, cooled to -78°C, and 64 mL of a 1 M dichloromethane solution of boron trichloride (BCl3) was slowly added dropwise. After the addition was complete, the temperature was raised to -20°C and stirred overnight. LCMS monitored the reaction completion, and methanol was added to quench the reaction. After concentration under reduced pressure, ethyl acetate was added to the mixture, and the mixture was filtered to obtain 3.4 g of a white solid, the hydrochloride salt of compound I-1-4. [M+1] +=382.2;1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),8.71(s,1H),8.55(s,1H),8.11–8.02(m,2H),7.71–7.63( m,1H),7.56(dd,J=8.3,7.0Hz,2H),7.35(d,J=6.1Hz,5H),7.32–7.23(m,5H),4.79–4.70(m,1H) ,4.66–4.59(m,1H),4.55(d,J=4.7Hz,2H),4.52(d,J=3.1Hz,1H),4.29(s,1H),3.85(d,J=6.7Hz ,1H),3.80(d,J=9.5Hz,1H),3.73(d,J=6.5Hz,1H),3.65(d,J=9.5Hz,1H),2.44(d,J=9.1Hz,2H).

[0129] (6) Synthesis of Compound 30

[0130]

[0131] Under a nitrogen atmosphere, compound I-1-4 (1 g, 2.6 mmol) and 4,4'-bismethoxytrityl chloride (DMTrCl, 1.9 g, 5.72 mmol) were dissolved in dry dichloromethane (DCM). The temperature was lowered to 0°C, and N,N-diisopropylethylamine (DIEA, 1.3 g, 13 mmol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 hours. LCMS monitored the reaction completion, and the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography using a solvent ratio of dichloromethane:ethyl acetate:triethylamine (v:v:v) = 1:1:0.1% to obtain 1.3 g of a light yellow solid, with a yield of 72.2%. [M+1] +=684.3;1H NMR (400MHz, DMSO-d6) δ11.16(s,1H),8.76(s,1H),8.62(s,1H),8.05(d,J=7.7Hz,2H),7.65(t,J=7.4Hz ,1H),7.56(t,J=7.6Hz,2H),7.41(d,J=7.8Hz,2H),7.33(t,J=7.6Hz,2H),7.30–7.21(m,5H),6.96–6.87( m,4H),5.20(d,J=4.6Hz,1H),4.75(dd,J=9.8,5.5Hz,1H),4.36(d,J=4.7Hz,1H),4.11(s,1H),3.81(d,J= 6.5Hz,1H),3.75(s,6H),3.40(d,J=9.1Hz,1H),3.21–3.16(m,2H),2.57–2.52(m,1H),2.46-2.39(m,1H).

[0132] (7) Synthesis of Compound I-1-8

[0133]

[0134] Under a nitrogen atmosphere, compound 30 (800 mg, 1.17 mmol) and 4,5-dicyanoimidazole (DCl, 345 mg, 2.92 mmol) were dissolved in 10 mL of dry dichloromethane (DCM). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (PN2, 880 mg, 2.92 mmol) was added dropwise at room temperature (rt), and stirring was continued for 2 h. LCMS monitored the reaction completion, followed by the addition of dichloromethane and 0.1 mL of triethylamine. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using a high-pressure preparative liquid chromatography column using a C18 column and a mobile phase of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) (v:v) = 8:2, yielding 550 mg of a white solid with a yield of 53%. [M+1] + =884.4; 31 PNMR:148.40,148.31.

[0135] Example 3: C 16 Synthesis of phosphoramidite monomer (Compound 33)

[0136]

[0137] (1) Synthesis of compound 31

[0138]

[0139] Under nitrogen atmosphere, palmitic acid (20 g, 78.1 mmol), (3R, 5S)-5-(hydroxymethyl)pyrrolidin-3-ol hydrochloride (12 g, 78.1 mmol), HATU (C 10 H 15 OF6N6P (44.5 g, 117.1 mmol) was suspended in 300 mL of dry N,N-dimethylformamide (DMF), cooled to 0°C, and N,N-diisopropylethylamine (DIEA, 35.3 g, 273.4 mmol) was slowly added dropwise. After the addition was complete, stirring was continued at room temperature for 3 hours. The mixture was poured into 300 mL of water, and the pH was adjusted to approximately 8. After stirring for 1 hour, the mixture was filtered and washed with water to obtain a crude product. 140 mL of acetonitrile solution was added, the mixture was heated and slurried, filtered, and the filter cake was dried to obtain 22 g of an off-white solid with a yield of 80%. [M+1] + =356; 1HNMR (400MHZ, DMSO-d6) δ4.28 (q, J=4.5Hz, 1H), 4.04-3.93 (m, 1H), 3.45 (dq, J=14.7, 5.0Hz, 2H), 3.31-3.18 (m, 2H), 2.31-2.12(m,2H),1.92(dt,J=13.0,5.7Hz,1H),1.83-1.75(m,1H),1.48(p,J=6.7Hz,2H),1.24(m,24H),0.93-0.81(m,3H).

[0140] (2) Synthesis of compound 32

[0141]

[0142] Under a nitrogen atmosphere, compound 31 (10 g, 28.1 mmol) and 4-dimethylaminopyridine (0.69 g, 5.6 mmol) were dissolved in 70 mL of pyridine. 4,4'-bis(methoxytrityl) chloride (DMTrCl, 14.3 g, 42.1 mmol) was added portionwise at 0°C. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, TLC (dichloromethane:methanol (v:v) = 30:1) was used to determine the reaction's completion. Ethyl acetate and water were then added, and the layers were separated. The aqueous phase was extracted once more with ethyl acetate. The combined organic phases were washed with saturated brine, dried, filtered, and concentrated. The crude product was purified by column chromatography over 200-300 mesh neutral alumina using a dichloromethane:ethyl acetate (v:v) ratio of 15:1 to 10:1 as eluent. 1‰ triethylamine was added to the eluent to yield 13.3 g of an oily substance, with a yield of 72%. [M+1] + =658.

[0143] (3) Synthesis of compound 33

[0144]

[0145] Under a nitrogen atmosphere, compound 32 (12 g, 18.3 mmol), N-methylimidazole (1.5 g, 18.3 mmol), and tetrazole (2.56 g, 36.6 mmol) were dissolved in 120 mL of dichloromethane. The temperature was lowered to 0°C and bis(diisopropylamino)(2-cyanoethoxy)phosphine (16.5 g, 54.9 mmol) was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature. LCMS monitored the reaction. After completion, the organic phase was washed sequentially with saturated sodium bicarbonate solution and saturated brine, then dried. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column and acetonitrile (containing 0.0035% diisopropylamine) as the mobile phase to obtain 8.7 g of an oil, with a yield of 56%. [M+1] + =858; 31 PNMR: 148.1, 147.8, 147.6, 147.3.

[0146] Example 4: cLNA Synthesis of T phosphoramidite monomer (compound I-1-5)

[0147]

[0148] (1) Synthesis of compound 34

[0149]

[0150] Under a nitrogen atmosphere, compound 18 (22 g, 0.065 mol), thymine (T, 32.8 g, 0.26 mol), and potassium carbonate (K2CO3, 26.9 g, 0.195 mol) were suspended in 220 mL of dry dimethyl sulfoxide (DMSO), heated to 140°C, and stirred for 3 h. After completion of the reaction as determined by LCMS, the mixture was cooled to room temperature, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol (v:v) ratio of 50:1 to 20:1 as the eluent to afford 24.4 g of a white solid in an 81% yield. [M+1] +=467.2; 1HNMR(400MHz, DMSO-d6)δ11.19(s,1H),7.55–7.16(m,11H),5.09( d,J=6.5Hz,1H),4.81(d,J=11.7Hz,1H),4.71(q,J=9.7Hz,1H),4.59–4.45( m,4H),4.39(dt,J=9.7,5.8Hz,1H),3.80(d,J=4.9Hz,1H),3.61–3.39(m,4H ),1.93–1.82(m,1H),1.68(d,J=1.1Hz,3H),1.35(dd,J=13.5,10.1Hz,1H).

[0151] (2) Synthesis of compound 35

[0152]

[0153] Under a nitrogen atmosphere, compound 34 (17.3 g, 0.037 mol) and triethylamine (TEA, 15 g, 0.148 mol) were dissolved in 170 mL of dry tetrahydrofuran (THF). The temperature was lowered to -10°C, and a solution of methanesulfonic anhydride (Ms2O, 7.7 g, 0.044 mol) in THF was slowly added dropwise. After the addition was complete, the reaction was continued at -10°C for 30 min. After completion of the reaction, as determined by LCMS, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 100:1 ratio of dichloromethane to methanol (v:v) as the eluent to afford 14.5 g of a white foamy solid in a yield of 72.5%. [M+1] + =545.2.

[0154] (3) Synthesis of compound 36

[0155]

[0156] Under a nitrogen atmosphere, carefully add 60% sodium hydride (NaH, 3.2 g, 0.081 mol) to 100 mL of dry N,N-dimethylformamide (DMF). Cool to 0°C, and slowly add a solution of compound 35 (14 g, 0.027 mol) in DMF dropwise until addition is complete. Continue the reaction for 1 hour. LCMS confirms the reaction is complete, then add saturated ammonium chloride solution and extract twice with ethyl acetate. The combined organic phases are washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography using a 40:1 ratio of dichloromethane to methanol (v:v) as the eluent to afford 8.5 g of an oily liquid, yielding 74%. [M+1] + =449.2.

[0157] (4) Synthesis of Compound I-1-1

[0158]

[0159] Compound 36 (4 g, 8.9 mmol) was dissolved in 40 mL of anhydrous methanol (MeOH). 50 μL of acetic acid (AcOH) and 400 mg of 10% palladium on carbon (Pd / C, 60% aqueous content) were added. The atmosphere was replaced with hydrogen (H2) three times and stirred at room temperature overnight. The reaction was monitored by LCMS. The mixture was filtered and concentrated to yield 2.2 g of a white solid, a 90% yield. [M+1] + =269.2;1H NMR (400MHz, DMSO-d6) δ11.27(s,1H),7.43(d,J=1.3Hz,1H),5.76(s,2H),4.23(dd,J=9.7,5.6Hz,1H),3.91(d,J=14 .6Hz,2H),3.70–3.61(m,2H),3.53–3.43(m,3H),2.05(dd,J=13.6,9.7Hz,1H),1.81–1.78(m,3H),1.78–1.72(m,1H).

[0160] (5) Synthesis of Compound 37

[0161]

[0162] Under a nitrogen atmosphere, compound I-1-1 (1.6 g, 6.0 mmol) and 4,4'-bismethoxytrityl chloride (DMTrCl, 4.65 g, 13.8 mmol) were dissolved in 30 mL of dry dichloromethane. The temperature was lowered to 0°C and N,N-diisopropylethylamine (DIEA, 2.3 g, 18 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 hours. LCMS monitored the reaction completion. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 40:1 ratio of dichloromethane to methanol (v:v) as the eluent to afford 2.8 g of a pale yellow solid in an 86% yield. [M+1] +=571.2;1H NMR(400MHz, DMSO-d6)δ11.26(s,1H),7.42(d,J=1.4Hz,1H),7.38–7.28(m,4H),7.22( ddt,J=6.9,4.8,2.2Hz,5H),6.92–6.86(m,4H),5.10(d,J=4.6Hz,1H),4.30(dd,J=9.9 ,5.8Hz,1H),3.96(d,J=4.7Hz,1H),3.91(s,1H),3.74(s,6H),3.67(s,2H),3.29(d,J= 9.0Hz, 1H), 3.12 (d, J = 9.0Hz, 1H), 2.30 (dd, J = 13.5, 9.8Hz, 1H), 1.78 (d, J = 1.1Hz, 4H).

[0163] (6) Synthesis of Compound I-1-5

[0164]

[0165] Under a nitrogen atmosphere, compound 37 (700 mg, 1.2 mmol) and 4,5-dicyanoimidazole (354 mg, 3 mmol) were dissolved in 10 mL of dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine ((iPr2N2)2POCH2CH2CN, 904 mg, 3 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. LCMS monitored the reaction completion, followed by the addition of dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) (v:v) = 8:2, yielding 560 mg of a white solid with a yield of 60%. [M+1] + =771.3; 31 PNMR:148.30,148.04.

[0166] Example 5: Synthesis and purification of oligonucleotide compounds 1-13 containing carbocyclic nucleoside modifications

[0167] Using an AKTA OligoPilotPlus 100 automated oligonucleotide synthesizer, the target oligonucleotide compounds were synthesized on a polystyrene solid support (PS resin) using a multi-step solid-phase process involving deprotection, coupling, capping, oxidation, and aminolysis according to the sequences of oligonucleotide compounds 1-12. All phosphoramidite monomers were dissolved in anhydrous acetonitrile (200 mM) and dried over molecular sieves (3A). 5-Ethylthio-1H-tetrazole (ETT, 350 mM in acetonitrile) was used as the activator solution. The coupling time between commercially available MOE monomers and DNA monomers was 12 min. cLNA A. cLNA T and cLNA G phosphoramidite monomer and C 16 The coupling time for the phosphoramidite monomers was 30 minutes, with the first monomer coupled twice. A 50 mM aqueous iodine solution was used to construct the phosphate bond; a 200 mM solution of hydrogenated yellow element in pyridine was used to introduce the phosphorothioate bond. Unreacted active groups were capped using a mixture of acetic anhydride, N-methylimidazole, pyridine, and acetonitrile (N-methylimidazole / acetonitrile (v:v) 1:4; acetic anhydride / pyridine / acetonitrile (v:v:v) 2:3:5). After the reaction, the solid support was cleaved in concentrated ammonia at 55°C for 16 hours, filtered, and the mother liquor concentrated to obtain the crude product.

[0168] The crude oligonucleotide analog was purified by ion-pair reversed-phase liquid chromatography using a NanoQ-15L 9.5 mL (8 × 190 mm) ion exchange column at a flow rate of 2 mL / min. Mobile phase A consisted of 10 mM sodium hydroxide solution and mobile phase B consisted of 10 mM sodium hydroxide and 2.0 M sodium chloride solution. Gradient elution was performed, and fractions meeting the quality requirements were combined and collected, ultrafiltered and concentrated using an ultrafiltration membrane package, and freeze-dried to obtain oligonucleotide compounds meeting the purity requirements.

[0169] Example 6: Antisense inhibition of HBV by the tested oligonucleotide compounds in HepG2.2.15 cells

[0170] An antisense oligonucleotide compound targeting human HBV modified with a carbocyclic nucleoside was designed, and a sequence without the carbocyclic nucleoside modification was used as a control sequence. The sequence modification is shown in Table 1. This compound was used as a positive control and its inhibitory effect on HBV activity in vitro was tested in HepG2.2.15 cells.

[0171] method:

[0172] On day 0, the test compound was serially diluted with RNase-free water to eight concentrations (30.000 nM, 7.500 nM, 1.875 nM, 0.469 nM, 0.117 nM, 0.029 nM, 0.007 nM, and 0.002 nM). HepG2.2.15 cells cultured in a culture flask were washed with Dulbecco's phosphate buffered saline (DPBS) and then digested with trypsin. The cells were collected and counted, and the cells were adjusted to an appropriate density. 2.25 × 10 cells were added to each well. 4 The cells were seeded into 96-well plates at a density of 100 cells / well. At the same time as the cells were seeded, the compounds were transfected into the cells using Lipofectamine RNAiMAX reagent.

[0173] On day 3, fresh culture medium was replaced.

[0174] On day 6, cell supernatants were collected and assayed for HBV DNA by real-time quantitative PCR (qPCR), and HBeAg and HBsAg by enzyme-linked immunosorbent assay (ELISA). Finally, cells were harvested, RNA was extracted, and total intracellular HBV RNA was measured by real-time quantitative reverse transcription PCR (RT-qPCR).

[0175] The test compounds were transfected into HepG2.2.15 cells, and the anti-HBV activity of the test compounds was evaluated by detecting HBV DNA, HBeAg, HBsAg in the cell supernatant and intracellular HBV RNA.

[0176] The test compound was tested at 8 concentration points and 2 replicate wells were tested in parallel.

[0177] Statistics:

[0178] Inhibition percentage (%) = (1-(DNA copy number or HBsAg content or HBeAg content in the sample / DNA copy number or HBsAg content or HBeAg content in the nuclease-free water control)) × 100;

[0179] ΔCt = HBV RNA average Ct value - GAPDH (glyceraldehyde-3-phosphate dehydrogenase) average Ct value;

[0180] ΔΔCt = ΔCt (sample) - ΔCt (nuclease-free water control);

[0181] Relative amount of target gene mRNA = 2 ^-ΔΔCt ;

[0182] Inhibition rate (%) = (relative amount of nuclease-free water control - relative amount of sample) / relative amount of nuclease-free water control × 100

[0183] Cell viability (%) = (test well reading - average value of culture medium control) / (average value of nuclease-free water control - average value of culture medium control) × 100;

[0184] GraphPad Prism software was used to calculate the IC of the compounds. 50 (half inhibitory concentration) and CC 50 (50% toxic concentration) value, and the curve fitting method was log(inhibition) vs.response--Variable slope.

[0185] result:

[0186] The results are shown in Table 2. Figure 1 、 Figure 2 、 Figure 3 and Figure 4 , Figure 1 is the fitting curve of HBV DNA inhibition rate, Figure 2 is the fitting curve of HBsAg inhibition rate, Figure 3 is the HBeAg inhibition rate fitting curve, Figure 4 is the fitting curve of HBV RNA inhibition rate; it can be seen that the positive control, compound 1, compound 2, and compound 3 inhibit HBV DNA IC 50 The IC values of the four compounds for inhibiting HBsAg were 8.03nM, 13.60nM, 14.26nM and 10.39nM respectively. 50 The inhibitory concentrations of compounds 1 to 3 were 29.21 nM, 26.74 nM, 23.39 nM, and 19.59 nM, respectively. Compared with the positive control, compounds 1 to 3 were more effective in inhibiting HBsAg. The IC values of the positive control and compound 1 in inhibiting HBeAg were 29.21 nM, 26.74 nM, 23.39 nM, and 19.59 nM, respectively. 50 The inhibitory activity of other compounds against HBeAg and HBsAg was less than 50% at the highest concentration. Compared with the positive control and other compounds, compound 1 was more effective in inhibiting HBeAg. The inhibitory activity of all compounds against HBV RNA was less than 50% at the highest concentration tested (30 nM). 50 >30nM.

[0187] Table 2 Anti-HBV activity of the tested compounds

[0188]

[0189]

[0190] Example 7: Antisense inhibition of HBV by the test oligonucleotide compounds in HepG2.2.15 cells

[0191] An antisense oligonucleotide compound targeting human HBV modified with a carbocyclic nucleoside was designed. The sequence modification is shown in Table 1. Its inhibitory effect on HBV activity in vitro was tested in HepG2.2.15 cells.

[0192] Method: Same as Example 6. The difference is that the concentration of the test compound is different from that in Example 1, and the test compound is diluted into 8 concentrations (270.000 nM, 90.000 nM, 30.000 nM, 10.000 nM, 3.333 nM, 1.111 nM, 0.3704 nM, 0.1235 nM).

[0193] Data statistics: Same as Example 6.

[0194] result:

[0195] The results are shown in Table 3. Figure 5 、 Figure 6 、 Figure 7 and Figure 8 As shown in Table 3, NA means not detected. Figure 5 is the fitting curve of HBV DNA inhibition rate, Figure 6 is the fitting curve of HBsAg inhibition rate, Figure 7 is the HBeAg inhibition rate fitting curve, Figure 8 is the fitting curve of HBV RNA inhibition rate; it can be seen that all the tested compounds, compound 3 to compound 8, have inhibitory activity against HBV.

[0196] Table 3 Anti-HBV activity of the tested compounds

[0197]

[0198] Example 8: Antisense inhibition of HBV by the test oligonucleotide compounds in HepG2.2.15 cells

[0199] An antisense oligonucleotide compound targeting human HBV modified with a carbocyclic nucleoside was designed, and a sequence without the carbocyclic nucleoside modification was used as a control sequence. The sequence modification is shown in Table 1. This compound was used as a positive control and its inhibitory effect on HBV activity in vitro was tested in HepG2.2.15 cells.

[0200] Method: Same as Example 7.

[0201] Data statistics: Same as Example 6.

[0202] result:

[0203] The results are shown in Table 4. Figure 9 、 Figure 10 、 Figure 11 and Figure 12 As shown in Table 4, NA means not detected. Figure 9is the fitting curve of HBVDNA inhibition rate, Figure 10 is the fitting curve of HBsAg inhibition rate, Figure 11 is the HBeAg inhibition rate fitting curve, Figure 12 is the fitting curve of HBV RNA inhibition rate; it can be seen that compounds 9-12 have inhibitory activity against HBV.

[0204] Table 4 Anti-HBV activity of the tested compounds

[0205]

[0206] Example 9: Anti-HBV activity of the tested oligonucleotide compounds in C57BL / 6-HBV transgenic model mice

[0207] An antisense oligonucleotide compound targeting human HBV containing a carbocyclic nucleoside modification was designed, and a sequence without the carbocyclic nucleoside modification was used as a control sequence. The sequence modifications are shown in Table 1. This compound served as a positive control and its inhibitory effect on HBV activity in vivo was tested in C57BL / 6-HBV transgenic model mice.

[0208] Methods: Six-week-old female C57BL / 6-HBV transgenic mice were acclimated for 6 days. The day of the first administration was designated as day 0, the day before administration as day -1, the day after administration as day 1, and so on. Blood was collected from the submandibular venous plexus on day -3 before administration. The collected blood samples were anticoagulated with K2-EDTA (dipotassium ethylenediaminetetraacetic acid) and centrifuged at 7000 g / min at 4°C for 10 minutes. Approximately 30 μL of plasma was collected for the determination of HBV DNA, HBsAg, and HBeAg levels in plasma. HBV DNA levels were quantified by qPCR, and HBsAg and HBeAg levels were quantified by ELISA. All mice were grouped according to their plasma HBV DNA, HBsAg, and HBeAg levels and body weight on day -3. Mice with low HBV DNA, HBsAg, and HBeAg levels, as well as those with low body weight, were removed from the experiment. The 24 selected mice were evenly distributed into groups, 3 mice per group, and no statistical differences were found in the HBVDNA, HBsAg, HBeAg levels and body weight among the groups on day -3 (P>0.05).

[0209] Each experimental group received a subcutaneous injection of either saline or the compounds listed in Table 1. Except for the saline group, all other groups received a single dose of 45 mg / kJ. On days 7, 14, and 21 after administration, blood was collected from the submandibular venous plexus of the mice. The collected blood samples were anticoagulated with K2-EDTA and centrifuged at 7000 g / min at 4°C for 10 minutes. Approximately 30 μL of plasma was collected for the analysis of HBV DNA, HBsAg, and HBeAg levels in the plasma. On day 21, all mice were euthanized by CO2 inhalation. Quantitative PCR was used to determine HBV DNA levels in the mouse plasma, and ELISA was used to determine HBsAg and HBeAg levels in the mouse plasma.

[0210] Statistics:

[0211] ΔLog[HBsAg (IU / mL)]=D (n) Log[HBsAg(IU / mL)]-D (-3) Log[HBsAg(IU / mL)], n=7, 14, 21;

[0212] ΔLog[HBeAg (IU / mL)]=D (n) Log[HBeAg(IU / mL)]-D (-3) Log[HBeAg(IU / mL)], n=7, 14, 21;

[0213] ΔLog[HBVDNA(copy / μL)]=D (n) Log[HBV DNA(copy / μL)]-D (-3) Log[HBVDNA(copy / μL)], n=7, 14, 21.

[0214] result:

[0215] The changes in the log values of HBV DNA, HBsAg, and HBeAg levels in C57BL / 6-HBV transgenic mice on days 7, 14, and 21 after administration compared with those before administration (day -3) and their trend graphs are shown in Table 5. Figure 13 、 Figure 14 and Figure 15The data are presented as the reduction in Log values compared to before administration (day -3), including: ΔLog[HBsAg(IU / mL)], ΔLog[HBeAg(PEIU / mL)], and ΔLog[HBVDNA(copy / μL)]. It can be seen that compared with the normal saline group, the Log[HBsAg(IU / mL)], Log[HBeAg(PEIU / mL)], and Log[HBV DNA(copy / μL)] of the other groups of test compounds were significantly reduced on days 7, 14, and 21 after administration. At the same time, compared with the positive control, the Log[HBsAg(IU / mL)], Log[HBeAg(PEIU / mL)], and Log[HBVDNA(copy / μL)] of the compound 3, compound 5, compound 11, and compound 12 groups were significantly reduced, especially compound 11 and compound 12.

[0216] Table 5 Anti-HBV activity of the test compounds in transgenic mice

[0217]

[0218] Note: “Δ” indicates the decrease compared with the value before administration; Log value refers to the logarithm with 10 as the base.

[0219] From the above, it can be seen that the oligonucleotide compounds provided in the present application can reduce the expression levels of HBV RNA, HBV DNA and related proteins, have excellent anti-HBV activity, and can be used in the prevention and treatment of HBV- and HDV-related diseases or conditions.

[0220] The above describes the specific implementation methods in detail, but the present invention is not limited to the above specific implementation methods. Under the guidance of this application, technical personnel in this field can also make various forms of changes without departing from the scope of protection of this application, which all fall within the scope of protection of the present invention.

Claims

1. An oligonucleotide compound, characterized in that The oligonucleotide compound comprises at least one carbocyclic nucleoside represented by formula (I), and the nucleic acid base sequence of the oligonucleotide compound is shown in SEQ ID NO.

1. Among them, Base is a heterocyclic base, Indicates the attachment site.

2. The oligonucleotide compound according to claim 1, wherein The oligonucleotide compound further comprises a lipophilic group, and the lipophilic group is located at at least one of the 5' end and the 3' end of the oligonucleotide compound.

3. The oligonucleotide compound according to claim 2, wherein The lipophilic group is derived from a lipophilic compound having 4 to 32 carbon atoms; The lipophilic compound is selected from at least one of alkanes, alkanoyl compounds, carboxyl compounds, retinyl compounds, cholesteryl olefins, cholesterol, dihydrotestosterone, hexadecyl glycerol, menthol, borneol, dimethoxytrityl compounds, geranyloxyhexyl compounds and phenoxazine.

4. The oligonucleotide compound according to claim 2, wherein The oligonucleotide compound further comprises a linker, which is connected to the lipophilic group, and the linker comprises a biocleavable group.

5. The oligonucleotide compound according to claim 4, wherein The linker further comprises a linking group, which connects the biocleavable group and the lipophilic group; The connecting group is selected from at least one of the groups represented by formula (II-1) to formula (II-5), 6. The oligonucleotide compound according to claim 5, wherein The combination of the linker and the lipophilic group is selected from at least one of the groups represented by formula (III-1) to formula (III-4), 7. The oligonucleotide compound according to claim 1, wherein The oligonucleotide compound consists of a 5'-wing segment, a spacer segment and a 3'-wing segment, wherein the bases of the 5'-wing segment include GCAGA, the bases of the spacer segment include GGTGAAGCGA, and the bases of the 3'-wing segment include AGTGC, wherein all cytosines are 5-methylcytosine; the nucleosides in the 5'-wing segment and the 3'-wing segment have 2'-O-methoxyethyl modifications, the 5'-wing segment and / or the 3'-wing segment contain the carbocyclic nucleoside, wherein adjacent nucleosides are connected by phosphorothioate bonds.

8. A drug, characterized in that The invention comprises the oligonucleotide compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. The drug according to claim 8, wherein The oligonucleotide compound or its pharmaceutically acceptable salt constitutes the drug as a single active ingredient or together with other pharmaceutically acceptable active ingredients.

10. Use of the oligonucleotide compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the medicament according to claim 8 or 9, in the preparation of a medicament for preventing and / or treating HBV and / or HDV-related diseases or conditions.

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