Oligonucleotide compounds, medicaments and uses thereof

Abstract

The application provides an oligonucleotide compound, a drug and application thereof, the oligonucleotide compound comprising at least one carbon ring nucleoside shown in formula (I), and the nucleic acid base sequence of the oligonucleotide compound is shown in SEQ ID NO. 1, wherein Base is a heterocyclic base, indicating 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 proteins, and is beneficial to the prevention and treatment of HBV and / or HDV related diseases or disorders.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an oligonucleotide compound, a drug and application thereof. BACKGROUND

[0002] Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. The harm of this disease to the world has been widely recognized, and it is a public health problem that needs to be solved urgently. The World Health Organization (WHO) has conducted a data statistics on this disease. There are 250 million people worldwide suffering from CHB, accounting for about 3.5% of the world's total population.

[0003] Hepatitis D virus (HDV) is a defective virus, and its replication, antigen expression and infection of hepatocytes all require the assistance of HBV. Compared with other hepatitis virus infections, HDV infection is more likely to progress to chronic hepatitis, increasing the risk of liver cirrhosis, liver decompensation, hepatocellular carcinoma (HCC) and liver disease-related death, and is a serious viral infection of the liver. Chronic hepatitis D is a serious viral hepatitis caused by co-infection of HDV and HBV or infection of HDV on the basis of HBV infection. HBV infection combined with hepatitis D often progresses faster and has a poorer prognosis.

[0004] Due to the severity of HBV infection, although new drugs have been developed in recent years and antiviral drugs have been continuously improved, the complete cure of CHB is still one of the clinical problems. Although interferon has certain effect in the treatment of HBV, the low response rate, many adverse reactions and other negative effects are great, and the treatment of HBV with nucleos(t)ide analogues (NAs) has a long course and insufficient lasting effect, and may be accompanied by a high risk of viral drug resistance mutation. With the progress of clinical technology, the combination therapy of immunotherapy and NAs drugs has become the preferred solution for the current treatment of CHB. In the stage where there is no new drug for radical cure, most studies have found that the sequential / combined application of NAs and pegylated interferon (Peg-IFN) drugs with different mechanisms of action can improve the treatment effect. The 2019 version of the guidelines also points out that the combination of Peg-IFN-alpha can achieve clinical cure for the advantage population of NAs treated CHB patients. Although the combination of NAs and Peg-IFN has more treatment advantages, it is still difficult to achieve HBsAg negative conversion, and the clinical cure rate is still very low (not more than 7%). Therefore, there is a need to discover and develop new anti-HBV viral therapies in the field.

[0005] Antisense technology is becoming an effective means for reducing the expression of specific gene products, and antisense therapy differs from nucleoside therapy in that antisense therapy can directly target the transcript for HBV DNA and thereby reduce serum hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) levels. Due to the multiple overlapping transcripts produced upon HBV infection, including the pregenomic transcript (pg RNA), a single antisense oligonucleotide also has the opportunity to reduce HBV DNA in addition to both HBeAg and HBsAg, providing a new approach to the treatment of HBV, HDV. SUMMARY

[0006] In view of this, the present application provides an oligonucleotide compound, a medicament and applications thereof for preventing and treating HBV and / or HDV related diseases or disorders.

[0007] In a first aspect, the present application provides an oligonucleotide compound, wherein the oligonucleotide compound comprises at least one carbocyclic nucleoside of formula (I), and the nucleobase sequence of the oligonucleotide compound is represented by SEQ ID NO. 1,

[0008]

[0009] wherein Base is a heterocyclic base, represents a linking site.

[0010] In a second aspect, the present application provides a medicament comprising the oligonucleotide compound or a pharmaceutically acceptable salt thereof according to the first aspect.

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

[0012] The oligonucleotide compound provided by the present application can specifically target the transcript of HBV DNA, reduce the levels of serum HBeAg, HBsAg and HBV DNA, and the carbocyclic nucleoside can improve the nuclease resistance and thermal stability of the oligonucleotide compound, and also increase the base pairing affinity, promote the binding to the complementary mRNA, improve the specificity and targeting ability, enhance the biological activity and therapeutic effect, and is conducive to the prevention and treatment of HBV and / or HDV related diseases or disorders. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to describe the technical solutions of the embodiments of the present application or the prior art more clearly, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. The specific embodiments described herein are only used to explain the present application and not used to limit the present application.

[0014] Figure 1 Fitting curve for HBV DNA inhibition rate of Example 6.

[0015] Figure 2 Fitting curve for HBsAg inhibition rate of Example 6.

[0016] Figure 3 Fitting curve for HBeAg inhibition rate of Example 6.

[0017] Figure 4 Fitting curve for HBV RNA inhibition rate of Example 6.

[0018] Figure 5 Fitting curve for HBV DNA inhibition rate of Example 7.

[0019] Figure 6 Fitting curve for HBsAg inhibition rate of Example 7.

[0020] Figure 7 Fitting curve for HBeAg inhibition rate of Example 7.

[0021] Figure 8 Fitting curve for HBV RNA inhibition rate of Example 7.

[0022] Figure 9 Fitting curve for HBV DNA inhibition rate of Example 8.

[0023] Figure 10 Fitting curve for HBsAg inhibition rate of Example 8.

[0024] Figure 11 Fitting curve for HBeAg inhibition rate of Example 8.

[0025] Figure 12 Fitting curve for HBV RNA inhibition rate of Example 8.

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

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

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

[0029] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

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

[0031] An "oligonucleotide compound" is a linear polymeric compound consisting of 13-24 linked nucleosides, formed by covalent linkages through phosphoester, phosphorothioester, aminoalkylphosphotriester, alkylphosphonate or the like phosphoester linkage.

[0032] When an oligonucleotide compound is represented by a nucleic acid base sequence such as "CGCTGATTTG", unless otherwise indicated, the sequence is understood to be in the 5' to 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] A "nucleoside" is a compound consisting of a ribose (or deoxyribose) and a nucleobase (purine or pyrimidine) linked by a glycosidic bond, which is the basic constituent unit of nucleic acids; wherein the nucleoside can include natural nucleosides in 2'-deoxy and 2'-hydroxyl forms, and can also include modified nucleosides having a modified base moiety and / or a modified sugar moiety.

[0034] A "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 a cytosine modified with a methyl group attached to the 5 position. 5-methylcytosine is a modified nucleic acid base.

[0036] "cLNA", "carbocyclic nucleoside" refers to a carbocyclic nucleoside containing 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’-0-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 support cleavage by ribonuclease H and flanked between external regions of one or more nucleosides, wherein the nucleosides making up the internal region are chemically different from the nucleosides making up the external regions. The internal region can be referred to as a “gap” and the external regions can be referred to as “wing” regions.

[0042] “Lipophilic group” refers to a group derived from any lipid-soluble molecule, such as a fat, oil, wax, terpene, sterol, lipid-soluble vitamin (e.g., A, D, E, and K), monoglyceride, diglyceride, triglyceride, fatty acid, hopanoid, and phospholipid.

[0043] “Linker” refers to an organic moiety that links two portions of a compound.

[0044] “Pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of a compound, which retains the desired biological activity of the parent compound, and does not impart undesired toxicological effects to the recipient.

[0045] “Pharmaceutically acceptable carrier or diluent” means a medium or diluent that does not interfere with the effectiveness of the oligonucleotide, additional to the oligonucleotide, to which it is administered to an animal or human, certain such carriers enable the pharmaceutical composition to be formulated e.g., into a tablet, capsule, liquid, suspension, etc. for oral administration, certain such carriers enable the pharmaceutical composition to be formulated into an injection or infusion injection formulation, e.g., the pharmaceutically acceptable carrier can be a sterile aqueous solution, physiological saline.

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

[0047] "Prevention" refers to the complete or nearly complete inhibition of the occurrence of a disease or condition, e.g., an infection, ischemic or reperfusion injury, e.g., when a patient or subject is at risk of developing the condition. Prevention can also include inhibition, i.e., the prevention of the development of the condition.

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

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

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

[0051] "Simultaneous administration" means the co-administration of two agents in any manner such that the pharmacological effects of both are manifested simultaneously in the patient. Simultaneous administration does not require that the two agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of the two agents need not be manifested simultaneously in nature. The effects need only overlap in time without necessarily being coextensive.

[0052] "HBV" means a mammalian hepatitis B virus, including a human hepatitis B virus. The term encompasses hepatitis B viruses, particularly geographic genotypes of human hepatitis B virus, as well as variant strains of the geographic genotypes of hepatitis B virus.

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

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

[0055] "HBV nucleic acid" or "HBV DNA" means any nucleic acid encoding a HBV. For example, in certain embodiments, a HBV nucleic acid includes, without limitation, any viral DNA sequence encoding a HBV genome or portion thereof, any RNA sequence transcribed from the viral DNA, including any RNA sequence encoding a 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” or “HBsAg” or “HBsAG.”

[0057] “Hepatitis B related disease or condition” or “HBV related disease or condition” means any disease, biological condition, medical condition, or event resulting from, associated with, related to, or attributable to hepatitis B infection, exposure, or disease. The term hepatitis B related condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, transplantation related to liver disease, and having symptoms that can include any or all of the following when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for the presence of antibodies specific for hepatitis B virus antigen: flu-like symptoms, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the body's liver region, clay-colored or gray-colored stool, generalized itching, and dark-colored urine.

[0058] “HDV (hepatitis D virus)” means a delta hepatitis virus, which is a defective virus.

[0059] "Induction," "inhibition," "enhancement," "elevation," "increase," "decrease," or similar terms generally refer to quantitative differences between two states. The terms can refer to differences between two states that are statistically significant. For example, "an amount effective to inhibit activity or expression of HBV" means that the level of activity or expression of HBV in a treated sample is quantitatively different, and can be statistically significant, from the level of activity or expression of HBV in untreated cells. The terms apply to, for example, expression levels and activity levels. "Inhibiting 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 (such as an antisense oligonucleotide) targeted to HBV, as compared to the level of expression of HBV RNA, HBV DNA, and / or protein in the absence of the antisense compound (such as an antisense oligonucleotide) targeted to HBV. In certain embodiments, the level of HBV RNA is reduced. In certain embodiments, the level of HBV DNA is reduced. In certain embodiments, the level of HBV protein is reduced. In certain embodiments, the level of HBV antigen is reduced. In certain embodiments, the level of HBV s antigen (HBsAg) is reduced. In certain embodiments, the level of HBV e antigen (HBeAg) is reduced. The reduction can occur in a time-dependent manner and / or in a dose-dependent manner.

[0060] In the chemical formula provided in the present application, DMTr is 4,4'-dimethoxytrityl, 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, wherein the oligonucleotide compound comprises at least one carbocyclic nucleoside represented by Formula (I), and the nucleobase sequence of the oligonucleotide compound is represented by SEQ ID NO. 1,

[0062]

[0063] wherein Base is a heterocyclic base, represents a linking site.

[0064] The oligonucleotide compound of the present application can target the transcript of HBV DNA, while the carbon ring nucleoside can improve the nuclease resistance and thermal stability of the overall structure, increase the base pairing affinity of the oligonucleotide compound, improve the specificity, promote the binding of the oligonucleotide compound to the complementary mRNA, and improve the efficacy and safety of the oligonucleotide compound, thereby reducing the serum HBeAg, HBsAg and HBV DNA levels, and preventing and treating HBV-related diseases or disorders; HDV infection requires the presence of HBV, and HDV relies on HBV-derived HBsAg for packaging and viral transmission in the liver, and HBV and HDV share HBsAg, so the oligonucleotide compound of the present application can also effectively knock down the HBV DNA transcript, reduce the production of HBsAg, and exert antiviral efficacy on HDV, thereby preventing and treating HDV-related diseases or disorders, and providing a new treatment method for hepatitis B and hepatitis D.

[0065] The oligonucleotide compound of the present application can include at least one carbon ring nucleoside represented by formula (I). For example, the oligonucleotide compound contains one carbon ring nucleoside represented by formula (I); or for example, the oligonucleotide compound has multiple (such as two, three, four, etc.) carbon ring nucleosides represented by formula (I).

[0066] It can be understood that the heterocyclic base in formula (I) is any one base in the nucleic acid base sequence of the oligonucleotide compound. In an 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 an embodiment of the present application, the oligonucleotide compound is composed of a 5' wing segment, a spacer segment and a 3' wing segment, 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. In some 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 some embodiments, the nucleosides in the 5' wing segment and the 3' wing segment have 2'-O-methoxyethyl modification. It can be understood that the nucleosides in the 5' wing segment and the 3' wing segment can have partial 2'-O-methoxyethyl modification or complete 2'-O-methoxyethyl modification. In some embodiments, adjacent nucleosides in the oligonucleotide compound are connected by phosphorothioate. In some embodiments, all cytosines in the oligonucleotide compound are 5-methylcytosines.

[0068] In an embodiment of the present application, the 5' wing segment and / or the 3' wing segment contains a carbon ring nucleoside, which improves the structural stability and targeting of the oligonucleotide compound.

[0069] In an embodiment of the present application, the oligonucleotide compound further comprises a lipophilic group. The lipophilic group can improve the ability of the oligonucleotide compound to deliver to liver cells, increase its cell permeability, enhance the efficiency of intracellular drug delivery, improve the distribution characteristics of the oligonucleotide compound in cells, and thus more quickly, directly and efficiently target the transcripts of HBV DNA; at the same time, the oligonucleotide compound containing a carbocyclic nucleoside and a lipophilic group has the advantages of good stability, strong anti-HBV activity in vivo and in vitro, and long drug efficacy duration, can act on HDV at the same time, and is beneficial to the prevention and improvement of HBV and HDV related diseases or conditions, thereby providing a new treatment method for hepatitis B and hepatitis D.

[0070] In an embodiment of the present application, the lipophilic group is located at at least one of the 5' end and the 3' end of the oligonucleotide compound. That is, 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' end and the 3' end of the oligonucleotide compound; that is, the lipophilic group is located at at least one of the 5' end and the 3' end of the nucleobase sequence of the oligonucleotide compound, and can also be understood as the lipophilic group being located at one end or both ends of the molecular chain of the oligonucleotide compound. Conjugating the lipophilic group can improve the penetration and delivery effect of the oligonucleotide compound, while not affecting the targeting effect of the oligonucleotide compound.

[0071] In an embodiment of the present application, the lipophilic group is derived from a lipophilic compound having a carbon atom number of 4-32, which can improve the cell penetration ability of the oligonucleotide compound. Specifically, the carbon atom number of the lipophilic compound (i.e., the carbon atom number 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 alkane, alkanoyl compound, carboxyl compound, retinyl compound, cholesteric alkane, cholesterols, dihydrotestosterone, hexadecyl glycerol (such as 1,3-di-O(hexadecyl)glycerol, etc.), menthol, borneol, dimethoxytrityl compound, geranyloxyhexyl compound and phenoxazine. For example, the alkane can be C4-C32alkane, i.e., the lipophilic group is C4-C32alkyl. For example, the alkanoyl compound can be C4-C32alkanoyl compound, i.e., the lipophilic group is C4-C32alkanoyl. For example, the carboxyl compound can be selected from at least one of cholic acid, adamantane acetic acid, 1-pyrene butyric acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, and bile acid. 32 32 32 32

[0072] ​​​​In an 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 bio-cleavable group. In some embodiments, the bio-cleavable group can be selected from at least one of a phosphate and a phosphorothioate. In some embodiments, the linker is the bio-cleavable group. That is, the linker only contains the bio-cleavable group. In some embodiments, the linker can further comprise a linking group. That is, the linker comprises the bio-cleavable group and the linking group, and the linking group connects the bio-cleavable group and the lipophilic group. In some embodiments, 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 represented by formula (II-1) to formula (II-5),

[0073]

[0074] In an 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 further provides a medicament comprising the oligonucleotide compound or the pharmaceutically acceptable salt thereof according to any one of the embodiments described above. The medicament has excellent structural stability, specific targeting and biological activity, and can reduce the expression level of HBV RNA, HBV DNA and related proteins, and is beneficial for the prevention and treatment of HBV, HDV related diseases or conditions.

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

[0078] In an embodiment of the present application, the medicament can further comprise a pharmaceutically acceptable carrier or diluent, which is beneficial for the administration of the medicament.

[0079] In an embodiment of the present application, the oligonucleotide compound or a pharmaceutically acceptable salt thereof is used as a single active ingredient or with other pharmaceutically acceptable active ingredients in a medicament. That is, the oligonucleotide compound or a pharmaceutically acceptable salt thereof is the only active ingredient in the medicament, or the oligonucleotide compound or a pharmaceutically acceptable salt thereof and other pharmaceutically acceptable active ingredients are active ingredients in the medicament. The other pharmaceutically acceptable active ingredients are second agents, which can be, but are not limited to, selected from the group consisting of anti-HBV agents, chemotherapeutic agents, antibiotics, analgesics, anti-inflammatory agents, antifungal agents, antiparasitic agents, anti-nausea agents, anti-diarrhea agents, immunomodulatory agents, and other antiviral agents. The second agents 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 agents can be, but are not limited to, selected from the group consisting of interferons (including but not limited to interferon alpha 2b, interferon alpha 2a, interferon alpha-1a, pegylated interferon alpha 2a, pegylated interferon alpha 2b), nucleos(t)ide analogs (including but not limited to entecavir; tenofovir disoproxil fumarate; tenofovir alafenamide; amdoxovir), other anti-HBV small nucleic acid drugs, Toll-like receptor (TLR) agonists, capsid inhibitors, HBV therapeutic vaccines, HBV prophylactic vaccines, neutralizing antibodies, HBV monoclonal or polyclonal antibody therapeutics, etc. In some embodiments, the other anti-HBV small nucleic acid drugs can be, but are not limited to, selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), aptamer RNAs, microRNAs (miRNAs), circular RNAs (circRNAs), small activating RNAs (saRNAs), and CpG oligonucleotides, CRISPR-Cas nucleic acids, ribozymes, transfer RNAs (tRNAs), antibody nucleic acid conjugate drugs (AOCs), nucleic acid aptamer-drug conjugates (ADCs), etc.

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

[0081] In an embodiment of the present application, the medicament can further comprise a second agent. The oligonucleotide compound or a pharmaceutically acceptable salt thereof and the second agent can be administered simultaneously in a single preparation, or administered separately in different preparations, and the administration of the oligonucleotide compound or a pharmaceutically acceptable salt thereof and the second agent is performed concurrently or sequentially.

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

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

[0084] The oligonucleotide compounds provided in this application exhibit high HBV inhibitory activity. In one embodiment of this application, the oligonucleotide compounds 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 GC cLNA AGAggtgaagcgaA cLNA GT cLNA GC Compound 2 GC cLNA AG Aggtgaagcga cLNA AG cLNA TGC Compound 3 <![CDATA[ cLNA GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 4 X s cLNA GCAGAggtgaagcgaA cLNA GT cLNA GC]] Compound 5 cLNA GCAGAggtgaagcga cLNA AGT cLNA GC]] ​ Compound 6 cLNA GCAGAggtgaagcgaAG cLNA T cLNA GC]] ​ Compound 7 GC cLNA A cLNA GAggtgaagcgaA cLNA GT cLNA GC Compound 8 <![CDATA[ cLNA GC cLNA AGAggtgaagcgaAG cLNA T cLNA GC]]> Compound 9 GCAGAggtgaagcga cLNA AGT cLNA GC]] Compound 10 GCAGAggtgaagcgaA cLNA GT cLNA GC]] Compound 11 X o GCAGAggtgaagcgaA cLNA GT cLNA GC]]> Compound 12 X o GCAGAggtgaagcga cLNA AGT cLNA GC]]>

[0087] In this system, each nucleoside is linked by a phosphate thioester (PS) bond, and each cytosine is 5-methylcytosine; lowercase "a, g, c, t" represent deoxyribonucleosides, and uppercase "A, G, C, T" represent 2'-MOE-modified nucleosides; cLNA "A" indicates a carbocyclic nucleoside with the base A. cLNA "G" indicates a carbocyclic nucleoside with the base G. cLNA "T" indicates a carbocyclic nucleotide with a base of T; "X" indicates a carbocyclic nucleotide with a base of T o The structure is as follows: “X s The structure is as follows:

[0088] The effects of the oligonucleotide compounds provided in this application are illustrated in more detail by the following examples, but the following examples are only for illustrating the compounds described herein and are not intended to limit this application.

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

[0090]

[0091] (1) Synthesis of compound 19

[0092]

[0093] Under a nitrogen atmosphere, compound 18 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), and O-6-benzylguanine (G Bn 33.8 g (0.14 mol) was suspended in 240 mL of dry N,N-dimethylformamide (DMF), and the mixture was heated to 90 °C and reacted for 3 h. The reaction was monitored by liquid chromatography-mass spectrometry (LCMS) until completion. The mixture was then cooled, water was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice successively 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) = 1:2–1:3, yielding 23.3 g of 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.), cooled to 0 °C, and trimethylchlorosilane (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 mixture was then cooled 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 another 2 h. 30 mL of concentrated ammonia (NH4OH) was added dropwise, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. 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) = 1:1 to 1:2, yielding 26.3 g of an oily liquid, with a yield of 97%. [M+1] + =652.3.

[0097] (3) Synthesis of compound 21

[0098]

[0099] Compound 20 (25.5 g, 0.039 mol) was dissolved in 200 mL dry pyridine (Py) under nitrogen atmosphere, and cooled to 0 °C. Methylsulfonic anhydride (Ms20, 8.7 g, 0.051 mol) in tetrahydrofuran was added dropwise slowly. After the addition was completed, the mixture was stirred for another 2 h. The reaction was monitored by LCMS until the product content was the highest. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate twice. The organic phase was combined and washed with saturated brine twice, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with petroleum ether: ethyl acetate (v:v) = 1:1 as eluent to give 19.1 g of oil with a yield of 56.8%. The recovered starting material was 4.5 g. [M+1] + = 730.3.

[0100] (4) Synthesis of compound 22

[0101]

[0102] Compound 21 (21.4 g, 29.3 mmol) was dissolved in 210 mL dry tetrahydrofuran (THF) under nitrogen atmosphere, and stirred for 10 min. Compound 22 (5 g, 7.9 mmol) was dissolved in 50 mL dichloromethane (DCM) under nitrogen atmosphere, and cooled to -78 °C. 71 mL of 1 M boron tribromide (BBr3) in dichloromethane was added dropwise slowly. After the addition was completed, the mixture was stirred for another 2 h. The reaction was monitored by LCMS until the product content was the highest. Methanol was added to quench the reaction, and the mixture was warmed to room temperature and concentrated. The crude product was purified by column chromatography with dichloromethane:methanol (v:v) = 10:1 as eluent to give 2.5 g of white solid with a yield of 87.2%. [M+1] + = 634.3.

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

[0104]

[0105] Compound 22 (5 g, 7.9 mmol) was dissolved in 50 mL dichloromethane (DCM) under nitrogen atmosphere, and cooled to -78 °C. 71 mL of 1 M boron tribromide (BBr3) in dichloromethane was added dropwise slowly. After the addition was completed, the mixture was stirred for another 2 h. The reaction was monitored by LCMS until the product content was the highest. Methanol was added to quench the reaction, and the mixture was warmed to room temperature and concentrated. The crude product was purified by column chromatography with dichloromethane:methanol (v:v) = 10:1 as eluent to give 2.5 g of white solid with a yield of 87.2%. [M+1] += 364.1; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.36 - 7.21 (m, 1H), 5.17 (d, J = 4.2 Hz, 1H), 4.59 (t, J = 5.2 Hz, 1H), 4.44 (dd, J = 9.7, 5.4 Hz, 1H), 4.03 (d, J = 4.4 Hz, 1H), 3.96 (s, 1H), 3.78 - 3.68 (m, 2H), 2.79 (p, J = 6.8 Hz, 1H), 2.23 (ddd, J = 26.1, 14.8, 10.2 Hz, 2H), 1.99 (dd, J = 10.2, 6.6 Hz, 1H), 1.12 (d, J = 6.8 Hz, 5H).

[0106] (6) Synthesis of compound 24

[0107]

[0108] Compound I-1-3 (3.1 g, 9.1 mmol), 4,4’-dimethoxytrityl chloride (DMTrCl, 6.8 g, 20.2 mmol) were dissolved in 30 mL dry dichloromethane (DCM) under nitrogen atmosphere, and N,N-diisopropylethylamine (DIEA, 5.89 g, 45.5 mmol) was added slowly dropwise at 0 °C. After the addition was completed, the reaction was stirred at room temperature (r.t.) for 2 h. LCMS monitoring showed that the reaction was completed. The crude product was concentrated and purified by column chromatography with dichloromethane:methanol:triethylamine (v:v:v) = 30:1:0.1% as eluent to give 4.6 g of light yellow solid with a yield of 76%. [M+1] + = 666.3; 1H NMR (400 MHz, Chloroform-d) δ 12.10 (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, 2.0 Hz, 4H), 4.66 (s, 1H), 4.48 (t, J = 7.6 Hz, 1H), 4.27 (s, 1H), 3.95 (d, J = 7.1 Hz, 1H), 3.82 (s, 1H), 3.78 (s, 6H), 3.68 (d, J = 7.0 Hz, 1H), 3.46 (d, J = 9.6 Hz, 1H), 3.27 (d, J = 9.6 Hz, 1H), 2.53 (p, J = 6.9 Hz, 1H), 2.32 (d, J = 7.6 Hz, 2H), 1.19 (dd, J = 9.7, 6.8 Hz, 6H).

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

[0110]

[0111] Compound 24 (1.0 g, 1.5 mmol), 4,5-dicyanoimidazole (DCI, 443 mg, 3.75 mmol) were dissolved in 10 mL dry dichloromethane (DCM) under nitrogen atmosphere, bis(diisopropylamino)(2-cyanoethoxy)phosphine (PN2, 1.1 g, 3.75 mmol) was added dropwise at room temperature (r.t.) and stirring was continued for 2 h. LCMS monitoring showed the reaction was completed, dichloromethane and 0.1 mL triethylamine were added, the organic phase was washed with saturated brine twice, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by high pressure preparative liquid chromatography, the column was C18 and the mobile phase was acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) (v:v) = 7:3, 660 mg white solid was obtained, yield 51%. [M+1] + = 866.4; 31 PNMR: 147.27, 147.17.

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

[0113]

[0114] (1) Synthesis of compound 25

[0115]

[0116] Compound 18 (26 g, 0.076 mol), adenine (A, 20.5 g, 0.15 mol), potassium carbonate (K2CO3, 31.5 g, 0.23) were suspended in 260 mL dry dimethyl sulfoxide (DMSO) under nitrogen atmosphere, the temperature was raised to 120 °C and the reaction was continued for 4 h. LCMS monitoring showed the reaction was completed, the temperature was lowered, water was added, ethyl acetate was extracted twice, the organic phase was combined and washed with saturated brine twice, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography, the eluent was dichloromethane:methanol (v:v) = 20:1, 29.2 g white solid was obtained, yield 80.4%. [M+1] += 476.2; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.10 (s, 1H), 7.43 - 7.24 (m, 10H), 7.16 (s, 2H), 5.19 (d, J = 6.2 Hz, 1H), 4.84 (d, J = 11.8 Hz, 1H), 4.81 - 4.71 (m, 2H), 4.61 - 4.53 (m, 3H), 4.50 (t, J = 4.9 Hz, 1H), 3.90 (d, J = 4.3 Hz, 1H), 3.65 - 3.56 (m, 2H), 3.54 (d, J = 4.6 Hz, 2H), 2.10 (dd, J = 13.5, 8.6 Hz, 1H), 1.82 (dd, J = 13.4, 9.2 Hz, 1H).

[0117] (2) Synthesis of compound 26

[0118]

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

[0120] (3) Synthesis of compound 27

[0121]

[0122] Compound 26 (20 g, 0.034 mol), methylsulfonic anhydride (Ms20, 7.2 g, 0.041 mol) were dissolved in 200 mL dry tetrahydrofuran under nitrogen atmosphere, cooled to -10 °C, triethylamine (TEA, 10.4 g, 0.103 mol) was added slowly dropwise, after the addition was completed, the temperature was raised to 0 °C and the reaction was carried out for 2 h. LCMS monitoring showed that the raw material was substantially completely reacted, saturated sodium bicarbonate aqueous solution was added, extracted with ethyl acetate twice, the organic phase was combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was petroleum ether: ethyl acetate (v:v) = 1:1, to obtain 17 g of white solid, with a yield of 65%. [M+1] + = 658.2.

[0123] (4) Synthesis of compound 28

[0124]

[0125] Under nitrogen atmosphere, 60% sodium hydride (NaH, 1.87 g, 77.7 mmol) was carefully added to 170 mL of dry N,N-dimethylformamide (DMF), stirred for 10 min, cooled to 0 °C, and a solution of compound 27 (17 g, 25.9 mmol) in N,N-dimethylformamide was slowly added dropwise. After the addition was completed, stirring was continued for 2 h. LCMS monitoring showed that the reaction was complete, saturated ammonium chloride solution was added, extracted with ethyl acetate twice, the organic phase was combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography, with the eluent being 100% ethyl acetate, to obtain 12.3 g of white solid, with a yield of 85%. [M+1] + = 562.2.

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

[0127]

[0128] Compound 28 (5.3 g, 10.7 mmol) was dissolved in 50 mL of dry dichloromethane (DCM) under nitrogen atmosphere, cooled to -78 °C, and 64 mL of 1M boron trichloride (BC13) in dichloromethane was slowly added dropwise. After the addition was completed, the temperature was raised to -20 °C and stirring was continued overnight. LCMS monitoring showed that the reaction was complete, methanol was added to quench, concentrated under reduced pressure, and then the slurry was prepared with ethyl acetate, filtered, to obtain 3.4 g of white solid, which was a hydrochloride form of compound I-1-4. [M+1] += 382.2; 1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.71 (s, 11H), 8.55 (s, 1H), 8.11 - 8.02 (m, 2H), 7.71 - 7.63 (m, 1H), 7.56 (dd, J = 8.3, 7.0 Hz, 2H), 7.35 (d, J = 6.1 Hz, 5H), 7.32 - 7.23 (m, 5H), 4.79 - 4.70 (m, 1H), 4.66 - 4.59 (m, 1H), 4.55 (d, J = 4.7 Hz, 2H), 4.52 (d, J = 3.1 Hz, 1H), 4.29 (s, 1H), 3.85 (d, J = 6.7 Hz, 1H), 3.80 (d, J = 9.5 Hz, 1H), 3.73 (d, J = 6.5 Hz, 1H), 3.65 (d, J = 9.5 Hz, 1H), 2.44 (d, J = 9.1 Hz, 2H).

[0129] (6) Synthesis of compound 30

[0130]

[0131] Compound I-1-4 (1 g, 2.6 mmol), 4,4'-dimethoxytrityl chloride (DMTrCl, 1.9 g, 5.72 mmol) were dissolved in dry dichloromethane (DCM) under nitrogen atmosphere, cooled to 0 °C, slowly added N,N-diisopropylethylamine (DIEA, 1.3 g, 13 mmol), after the addition was completed, warmed to room temperature and stirred for 2 h. LCMS monitoring reaction was completed, concentrated under reduced pressure, the crude product was purified by column chromatography, eluent dichloromethane: ethyl acetate: triethylamine (v:v:v) = 1:1:0.1%, 1.3 g of light yellow solid was obtained, yield 72.2%. [M+1] += 684.3; 1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.76 (s, 1H), 8.62 (s, 1H), 8.05 (d, J = 7.7 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.30 - 7.21 (m, 5H), 6.96 - 6.87 (m, 4H), 5.20 (d, J = 4.6 Hz, 1H), 4.75 (dd, J = 9.8, 5.5 Hz, 1H), 4.36 (d, J = 4.7 Hz, 1H), 4.11 (s, 1H), 3.81 (d, J = 6.5 Hz, 1H), 3.75 (s, 6H), 3.40 (d, J = 9.1 Hz, 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] Compound 30 (800 mg, 1.17 mmol), 4,5-dicyanoimidazole (DCI, 345 mg, 2.92 mmol) were dissolved in 10 mL dry dichloromethane (DCM) under nitrogen atmosphere, bis(diisopropylamino)(2-cyanoethoxy) phosphine (PN2, 880 mg, 2.92 mmol) was added dropwise at room temperature (r.t.) and stirring was continued for 2 h. The reaction was monitored by LCMS and was complete. Dichloromethane and 0.1 mL triethylamine were added and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude was separated by high pressure preparative liquid chromatography using C18 column and mobile phase acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) (v:v) = 8:2, to give 550 mg of white solid with 53% yield. [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] Pivaloyl chloride (3.0 mL, 25.0 mmol) was added dropwise to a stirred solution of compound 1 (5.0 g, 25.0 mmol) and triethylamine (3.5 mL, 25.0 mmol) in 50 mL of dichloromethane at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 1 h and then warmed to room temperature and stirred for another 2 h. The reaction mixture was washed with water (3 x 50 mL) and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a crude product which was purified by column chromatography using 200-300 mesh neutral alumina and eluting with dichloromethane: ethyl acetate (v:v) = 15:1 to 10:1 to obtain compound 2 as a white solid (5.0 g, 75% yield). [M+1] 10 H 15 OF6N6P, 44.5 g, 117.1 mmol) was suspended in 300 mL of dry N,N-dimethylformamide (DMF) and cooled to 0 °C. N,N-diisopropylethylamine (DIEA, 35.3 g, 273.4) was added slowly dropwise. After the addition was complete, the stirring was continued at room temperature for 3 h. The reaction mixture was poured into 300 mL of water and the pH was adjusted to about 8. After stirring for 1 h, the mixture was filtered and washed with water to obtain a crude product. The crude product was dissolved in 140 mL of acetonitrile and heated to obtain a slurry. The slurry was filtered and the filter cake was dried to obtain 22 g of a white solid (80% yield). + = 356;1H NMR (400 MHz, DMSO-d6) δ 4.28 (q, J = 4.5 Hz, 1H), 4.04 - 3.93 (m, 1H), 3.45 (dq, J = 14.7, 5.0 Hz, 2H), 3.31 - 3.18 (m, 2H), 2.31 - 2.12 (m, 2H), 1.92 (dt, J = 13.0, 5.7 Hz, 1H), 1.83 - 1.75 (m, 1H), 1.48 (p, J = 6.7 Hz, 2H), 1.24 (m, 24H), 0.93 - 0.81 (m, 3H).

[0140] (2) Synthesis of compound 32

[0141]

[0142] Compound 31 (10 g, 28.1 mmol) and 4-dimethylaminopyridine (0.69 g, 5.6 mmol) were dissolved in 70 mL of pyridine under nitrogen atmosphere. 4,4'-Dimethoxytrityl chloride (DMTrCl, 14.3 g, 42.1 mmol) was added portionwise at 0 °C. After the addition was complete, the reaction was continued at room temperature for 2 h. After the reaction was completed as determined by TLC (dichloromethane:methanol (v:v) = 30:1), ethyl acetate and water were added and the mixture was 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 using 200-300 mesh neutral alumina and eluting with dichloromethane: ethyl acetate (v:v) = 15:1 to 10:1 with 1% triethylamine in the eluent to obtain 13.3 g of an oil (72% yield). [M+1] + = 658.

[0143] (3) Synthesis of compound 33

[0144]

[0145] Compound 32 (12 g, 18.3 mmol), N-methylimidazole (1.5 g, 18.3 mmol), tetrazole (2.56 g, 36.6 mmol) were dissolved in 120 mL of dichloromethane under nitrogen atmosphere, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (16.5 g, 54.9 mmol) was added dropwise at 0°C. After the addition was completed, the reaction was carried out at room temperature. After the reaction was completed as monitored by LCMS, the organic phase was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried, and the crude product was separated by high-pressure preparative liquid chromatography using a C18 column and a mobile phase of acetonitrile (containing 0.0035% diisopropylamine). This resulted in 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] 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) under a nitrogen atmosphere, and stirred at 140°C for 3 h. After the reaction was completed as monitored by LCMS, the reaction was cooled to room temperature, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using dichloromethane:methanol (v:v) = 50:1 to 20:1 as the eluent. This resulted in 24.4 g of a white solid, with a yield of 81%. [M+1] += 467.2; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 7.55 - 7.16 (m, 11H), 5.09 (d, J = 6.5 Hz, 1H), 4.81 (d, J = 11.7 Hz, 1H), 4.71 (q, J = 9.7 Hz, 1H), 4.59 - 4.45 (m, 4H), 4.39 (dt, J = 9.7, 5.8 Hz, 1H), 3.80 (d, J = 4.9 Hz, 1H), 3.61 - 3.39 (m, 4H), 1.93 - 1.82 (m, 1H), 1.68 (d, J = 1.1 Hz, 3H), 1.35 (dd, J = 13.5, 10.1 Hz, 1H).

[0151] (2) Synthesis of compound 35

[0152]

[0153] Compound 34 (17.3 g, 0.037 mol), triethylamine (TEA, 15 g, 0.148 mol) were dissolved in 170 mL dry tetrahydrofuran (THF) under nitrogen atmosphere, and methylsulfonic anhydride (Ms20, 7.7 g, 0.044 mol) was slowly added dropwise in tetrahydrofuran solution at -10 °C. After the addition was completed, the reaction was continued at -10 °C for 30 min. After the reaction was completed by LCMS detection, saturated aqueous sodium bicarbonate solution was added, and extracted twice with ethyl acetate. The organic phase was combined, washed with saturated brine solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with dichloromethane:methanol (v:v) = 100:1 as eluent to give 14.5 g of white foamy solid with a yield of 72.5%. [M+1] + = 545.2.

[0154] (3) Synthesis of compound 36

[0155]

[0156] Under nitrogen atmosphere, 60% sodium hydride (NaH, 3.2 g, 0.081 mol) was carefully added in 100 mL dry N,N-dimethylformamide (DMF) at 0 °C, and compound 35 (14 g, 0.027 mol) was slowly added dropwise in N,N-dimethylformamide solution. After the addition was completed, the reaction was continued for 1 h. After the reaction was completed by LCMS detection, saturated ammonium chloride solution was added, and extracted twice with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride solution twice, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with dichloromethane:methanol (v:v) = 40:1 as eluent to give 8.5 g of oily liquid with a yield of 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 μΐ of acetic acid (AcOH) and 400 mg of 10% palladium on carbon (Pd / C, 60% water) were added, and hydrogen gas (H2) was replaced three times, and stirred at room temperature overnight. The reaction was monitored by LCMS, filtered, concentrated to give 2.2 g of white solid, yield 90%. [M+1] + = 269.2; 1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.43 (d, J = 1.3 Hz, 1H), 5.76 (s, 2H), 4.23 (dd, J = 9.7, 5.6 Hz, 1H), 3.91 (d, J = 14.6 Hz, 2H), 3.70 - 3.61 (m, 2H), 3.53 - 3.43 (m, 3H), 2.05 (dd, J = 13.6, 9.7 Hz, 1H), 1.81 - 1.78 (m, 3H), 1.78 - 1.72 (m, 1H).

[0160] (5) Synthesis of compound 37

[0161]

[0162] Compound I-1-1 (1.6 g, 6.0 mmol), 4,4'-dimethoxytrityl chloride (DMTrCl, 4.65 g, 13.8 mmol) were dissolved in 30 mL of dry dichloromethane, and N,N-diisopropylethylamine (DIEA, 2.3 g, 18 mmol) was slowly added dropwise at 0°C, and stirred at room temperature for 2 h after the addition was completed. The reaction was monitored by LCMS, and the organic phase was washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, eluent dichloromethane:methanol (v:v) = 40:1, to give 2.8 g of light yellow solid, yield 86%. [M+1] += 571.2; 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 7.42 (d, J = 1.4 Hz, 1H), 7.38 - 7.28 (m, 4H), 7.22 (ddt, J = 6.9, 4.8, 2.2 Hz, 5H), 6.92 - 6.86 (m, 4H), 5.10 (d, J = 4.6 Hz, 1H), 4.30 (dd, J = 9.9, 5.8 Hz, 1H), 3.96 (d, J = 4.7 Hz, 1H), 3.91 (s, 1H), 3.74 (s, 6H), 3.67 (s, 2H), 3.29 (d, J = 9.0 Hz, 1H), 3.12 (d, J = 9.0 Hz, 1H), 2.30 (dd, J = 13.5, 9.8 Hz, 1H), 1.78 (d, J = 1.1 Hz, 4H).

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

[0164]

[0165] Compound 37 (700 mg, 1.2 mmol), 4,5-dicyanoimidazole (354 mg, 3 mmol) were dissolved in 10 mL dry dichloromethane under nitrogen atmosphere, bis(diisopropylamino)(2-cyanoethoxy)phosphine ((iPr2N2)2POCH2CH2CN, 904 mg, 3 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. The reaction was monitored by LCMS, dichloromethane was added, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by high pressure preparative liquid chromatography using a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) (v:v) = 8:2, to give 560 mg of 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 compound 1-12 containing a carbocyclic nucleoside modification

[0167] The target oligonucleotide compounds were synthesized on a solid support made of polystyrene (PS resin) according to the sequences of oligonucleotide compounds 1-12 using AKTA Oligo Pilot Plus 100 oligonucleotide automatic synthesizer. All phosphoramidite monomers were dissolved in anhydrous acetonitrile (200 mM) and dried with molecular sieves (3A). 5-ethylthio-1H-tetrazole (ETT, 350 mM in acetonitrile) was used as an activator solution, and the coupling time of commercially available MOE monomers and DNA monomers was 12 min, cLNA A、 cLNA T and cLNA G phosphoramidite monomers, and C 16 phosphoramidite monomers was 30 min, and the first monomer was coupled twice. Iodine aqueous solution (50 mM) was used for the construction of phosphate bonds; for the introduction of phosphorothioate bonds, hydrazine (200 mM in pyridine) was used. For unreacted active groups, a mixture of acetic anhydride, N-methylimidazole, pyridine and acetonitrile (N-methylimidazole / acetonitrile (v:v) was 1:4; acetic anhydride / pyridine / acetonitrile (v:v:v) was 2:3:5) was used as a capping reagent. After the reaction, the solid support was cleaved from the concentrated ammonia solution at 55°C for 16 h, filtered, and the mother liquor was concentrated to obtain the crude product.

[0168] The obtained oligonucleotide analogue crude product was purified by ion pair reversed-phase liquid chromatography, the chromatographic column was NanoQ-15L9.5 mL (8 x 190 mm) ion exchange column, the flow rate was 2 mL / min, the mobile phase A was 10 mM sodium hydroxide solution, the mobile phase B was 10 mM sodium hydroxide and 2.0 M sodium chloride solution, gradient elution was performed, the fractions with purity meeting the quality requirements were combined and collected, and ultrafiltration membrane bag was used for ultrafiltration concentration and freeze-drying to obtain the oligonucleotide compound with required purity.

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

[0170] An antisense oligonucleotide compound targeting human HBV containing a carbocyclic nucleoside modification was designed, and a sequence without a carbocyclic nucleoside modification was used as a control sequence, the sequence modification is shown in Table 1, and the compound was a positive control. The in vitro inhibition of HBV activity in HepG2.2.15 cells was tested.

[0171] Method:

[0172] On day 0, the test compound was diluted with RNase free water in 8 concentrations (30.000 nM, 7.500 nM, 1.875 nM, 0.469 nM, 0.117 nM, 0.029 nM, 0.007 nM, 0.002 nM). The HepG2.2.15 cells in culture flasks were washed with Dulbecco's Phosphate Buffered Saline (DPBS) and trypsinized. The cells were counted and adjusted to the appropriate density. The cells were seeded into 96-well plates at a density of 2.25 x 10 4 cells per well; at the same time, the compounds were transfected into the cells with Lipofectamine RNAiMAX reagent.

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

[0174] On day 6, the cell supernatant was collected, the content of HBV DNA in the supernatant was detected by real-time quantitative PCR (qPCR), and the content of HBeAg and HBsAg in the supernatant was detected by enzyme-linked immunoassay (ELISA). Finally, the cells were collected, RNA was extracted, and the total HBV RNA in the cells was detected by real-time quantitative reverse transcription PCR (RT-qPCR).

[0175] The test compound was transfected into HepG2.2.15 cells, and the cell supernatant HBV DNA, HBeAg, HBsAg, and intracellular HBV RNA were detected to evaluate the anti-HBV activity of the test compound.

[0176] The test compound was diluted in 8 concentrations, and 2 replicate wells were determined in parallel.

[0177] Data statistics:

[0178] The percentage of inhibition (%) = (1 - (the DNA copy number or the content of HBsAg or HBeAg in the sample / the DNA copy number or the content of HBsAg or HBeAg in the RNase free water control)) x 100;

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

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

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

[0182] The inhibition rate (%) = (the relative amount of the RNase free water control - the relative amount of the sample) / the relative amount of the RNase free water control x 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] Calculating the IC50 of compounds using GraphPad Prism software 50 (half-maximal inhibitory concentration) and CC 50 The curve fitting method for the (half-maximum toxicity concentration) value is 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 The curve showing the HBV DNA inhibition rate is shown. Figure 2 The curve fitted to the HBsAg inhibition rate. Figure 3 The curve fitted to the HBeAg inhibition rate. Figure 4 The curves showing the HBV RNA inhibition rate are as follows: It can be seen that the positive control, compound 1, compound 2, and compound 3 inhibit HBV DNA by an IC50 value. 50 The concentrations were 8.03 nM, 13.60 nM, 14.26 nM, and 10.39 nM, respectively; the IC50 values ​​of the four compounds inhibiting HBsAg were 8.03 nM, 13.60 nM, 14.26 nM, and 10.39 nM, respectively; 50 The concentrations of HBeAg inhibiting compounds 1 through 3 were 29.21 nM, 26.74 nM, 23.39 nM, and 19.59 nM, respectively, indicating that compounds 1 through 3 were more effective at inhibiting HBsAg than the positive control. The IC50 values ​​for HBeAg inhibition by the positive control and compound 1 were 29.21 nM, 26.74 nM, 23.39 nM, and 19.59 nM, respectively. 50 The concentrations were 25.65 nM and 17.88 nM, respectively. Other compounds showed less than 50% inhibitory activity against HBeAg and HBsAg at the highest concentration. Compared to the positive control and other compounds, compound 1 was more effective in inhibiting HBeAg. All compounds showed less than 50% inhibitory activity against HBV RNA at the highest tested concentration (30 nM), with an IC50 value of [missing value]. 50 >30nM.

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

[0188]

[0189]

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

[0191] Design of antisense oligonucleotide compounds targeting human HBV with carbon ring nucleoside modification, sequence modification as shown in Table 1, test their inhibitory effect on HBV activity in vitro in HepG2.2.15 cells.

[0192] Method: same as Example 6. The difference is that the concentration of the test compound is different from Example 6, and the test compound is diluted at 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] Results:

[0195] The results are shown in Table 3, Figure 5 , Figure 6 , Figure 7 and Figure 8 , Table 3, NA means not detected, Figure 5 HBV DNA inhibition rate fitting curve, Figure 6 HBsAg inhibition rate fitting curve, Figure 7 HBeAg inhibition rate fitting curve, Figure 8 HBV RNA inhibition rate fitting curve; it can be seen that all the test compounds, compounds 3-8, have inhibitory activity on HBV.

[0196] Table 3 Anti-HBV activity of test compounds

[0197]

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

[0199] Design of antisense oligonucleotide compounds targeting human HBV with carbon ring nucleoside modification, and use the sequence without carbon ring nucleoside modification as control sequence, sequence modification as shown in Table 1, the compound is a positive control, test their inhibitory effect on HBV activity in vitro in HepG2.2.15 cells.

[0200] Method: same as Example 7.

[0201] Data statistics: same as Example 6.

[0202] Results:

[0203] The results are shown in Table 4, Figure 9 , Figure 10 , Figure 11 and Figure 12 , Table 4, NA means not detected, Figure 9The curve was fitted for HBV DNA inhibition rate, Figure 10 The curve was fitted for HBsAg inhibition rate, Figure 11 The curve was fitted for HBeAg inhibition rate, Figure 12 The curve was fitted for HBV RNA inhibition rate; it can be seen that compounds 9-12 all have inhibitory activity against HBV.

[0204] Table 4 Anti-HBV activity of test compounds

[0205]

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

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

[0208] Methods: 6-week-old female C57BL / 6-HBV transgenic mice were adaptively fed for 6 days. The first day of administration was designated as day 0, the day before administration was day -1, the day after administration was day 1, and so on. All mice were submandibular venous plexus blood collected 3 days before administration, and the collected blood samples were anticoagulated with K2-EDTA (potassium ethylenediaminetetraacetate), centrifuged at 4°C, 7000g / min for 10 min and the plasma was collected ~30μL for detection of HBV DNA, HBsAg and HBeAg levels in the plasma; qPCR method was used to quantitatively detect the level of HBV DNA in the plasma of mice, ELISA method was used to quantitatively detect the level of HBsAg and HBeAg in the plasma of mice. All mice were grouped according to the levels of HBV DNA, HBsAg and HBeAg in the plasma on day -3 and body weight. Mice with lower levels of HBV DNA, HBsAg and HBeAg and lighter body weight were removed from the experiment. The selected 24 mice were evenly distributed in each group, 3 mice per group, and the mice in each group had no statistical difference in HBV DNA, HBsAg, HBeAg levels and body weight on day -3 (P>0.05).

[0209] Each test group was subcutaneously injected with the drug (injection of normal saline or the compound in Table 1), and each group of mice was administered a dose of 45 mpk, single administration. On the 7th, 14th, and 21st day after administration, the mice were submandibular venous plexus blood samples were collected, the collected blood samples were anticoagulated with K2-EDTA, centrifuged at 4°C, 7000g / min for 10 min, and the plasma ~30μL was collected for detection of the levels of HBV DNA, HBsAg, and HBeAg in the plasma; on the 21st day, all mice were euthanized by CO2 inhalation. The qPCR method was used to quantitatively detect the level of HBV DNA in the plasma of mice, and the ELISA method was used to quantitatively detect the levels of HBsAg and HBeAg in the plasma of mice.

[0210] Data 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[HBV DNA (copy / μL)] = D (n) Log[HBV DNA (copy / μL)] - D (-3) Log[HBV DNA (copy / μL)], n = 7, 14, 21.

[0214] Results:

[0215] The changes in the Log values of the levels of HBV DNA, HBsAg, and HBeAg and their trend charts compared to those before administration (on the 3rd day) of C57BL / 6-HBV transgenic mice on the 7th, 14th, and 21st day after administration are shown in Table 5, Figure 13 , Figure 14 and Figure 15The data are presented as the reduction in Log values compared to pre-dose (day -3), including: ΔLog[HBsAg (IU / mL)], ΔLog[HBeAg (PEIU / mL)], ΔLog[HBV DNA (copy / μL)]. It can be seen that compared to the normal saline group, the Log[HBsAg (IU / mL)], Log[HBeAg (PEIU / mL)], Log[HBV DNA (copy / μL)] of each of the other groups of test compounds were significantly reduced on day 7, day 14, day 21 after administration, and compared to the positive control, the Log[HBsAg (IU / mL)], Log[HBeAg (PEIU / mL)], Log[HBV DNA (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 test compounds in transgenic mice

[0217]

[0218] Note: "Δ" represents the reduction value compared to pre-dose; Log values refer to logarithmic values with base 10.

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

[0220] The above has described the specific embodiments in detail, but the present application is not limited to the above specific embodiments, and those skilled in the art can make various modifications under the inspiration of the present application without departing from the scope of the present application, and these all belong to the protection scope of the present application.

Claims

1. An oligonucleotide compound, characterized in that, the oligonucleotide compound is compound 4, compound 11 or compound 12, wherein the sequence of the compound 4 is X s cLNA GCAGAggtgaagcgaA cLNA GT cLNA GC, The sequence of the compound 11 is X o GCAGAggtgaagcgaA cLNA GT cLNA GC, The sequence of the compound 12 is X o GCAGAggtgaagcga cLNA AGT cLNA GC, each internucleosidic linkage is by a phosphorothioate linkage, each cytosine is a 5-methylcytosine, a, g, c, t represent deoxyribonucleosides, A, G, C, T represent 2’-MOE modified nucleosides, cLNA A represents a carbocyclic nucleoside with heterocyclic base A, cLNA G represents a carbocyclic nucleoside with heterocyclic base G, X o For , X s For , the carbocyclic nucleoside is shown in formula (I), (I), Base is a heterocyclic base, represents a point of attachment.

2. A medicament, characterized by comprising: The oligonucleotide compound or the pharmaceutically acceptable salt thereof of claim 1.

3. The medicament according to claim 2, wherein The oligonucleotide compound or the pharmaceutically acceptable salt thereof as a single active ingredient or with other pharmaceutically acceptable active ingredients constitutes the medicine.

4. The medicament according to claim 3, wherein The other pharmaceutically acceptable active ingredients are selected from anti-HBV agents, which are selected from interferons, other anti-HBV small nucleic acid drugs, TLR agonists, capsid inhibitors, HBV therapeutic vaccines, HBV preventive vaccines, neutralizing antibodies, HBV monoclonal or polyclonal antibody therapeutic agents.

5. The medicament according to claim 2, wherein The pharmaceutically acceptable salt of the oligonucleotide compound includes at least one of sodium salt and potassium salt.

6. The medicament according to claim 2, wherein The medicine further includes a pharmaceutically acceptable carrier or diluent.

7. Use of the oligonucleotide compound or the pharmaceutically acceptable salt thereof of claim 1, or the medicine of any one of claims 2-6 in the preparation of a medicine for preventing and / or treating HBV and / or HDV related diseases or disorders.

Images