Cyclic peptide liver-targeting compounds, pharmaceutical compositions and their uses

Abstract

The present invention belongs to the field of biological medicine, and specifically provides a cyclic peptide-based liver-targeting compound, a pharmaceutical composition and their uses. The cyclic peptide-based liver-targeting compound has the structure shown in Formula I. This compound has a strong liver-targeting ability and can significantly enhance the endocytosis in liver cells.

Description

Technical Field

[0001] The present invention provides a liver-targeting compound, particularly a liver-targeting compound containing a cyclic peptide. The present invention also relates to a pharmaceutical composition containing the cyclic peptide compound and the pharmaceutical and therapeutic uses of the compound. Background Art

[0002] The liver is the largest metabolic organ in the human body and is involved in the synthesis and metabolism of various chemical substances in the body. Liver-targeted drug delivery is a particularly attractive strategy for treating metabolic diseases, cardiovascular diseases, and other liver diseases. Asialoglycoprotein receptor (ASGPR) is an endocytic receptor protein specifically expressed in large amounts in liver tissue. ASGPR specifically recognizes and binds glycoproteins or molecules with D-galactose (Gal) or N-acetylgalactosamine (GalNAc) at the end in a calcium-dependent manner, and starts a cycle from the cell membrane to the intracellular and then back to the cell membrane surface with a cycle of about 15 minutes. The recyclable function of GalNAc-induced ASGPR receptor provides an ideal pathway for the liver tissue-targeted delivery of drug molecules or imaging reagents.

[0003] The lysosomal escape of small nucleic acid drugs conjugated to the GalNAc conjugate delivery system is very low (less than 0.1%), resulting in high clinical dosing and high prices of nucleic acid drugs. It is very important to develop a new liver-targeted delivery system with high bioavailability and / or high efficiency and low toxicity of GalNAc ligands. Summary of the Invention

[0004] One aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0005] (I)

[0006] wherein n is an integer between 1 and 5, and m is an integer between 0 and 5;

[0007] R1 is H, an amino protecting group or -L 1 -GalNAc; wherein L 1 is independently -C(O)(CH2)p- or -C(O)(CH2)q(OCH2CH2)t-, where p is an integer between 2 and 10, q is an integer between 1 and 4, and t is an integer between 1 and 4;

[0008] Z is selected from H, an amino protecting group, a small molecule drug moiety, a fluorescent dye moiety, and an oligonucleotide moiety.

[0009] Another aspect of the present invention provides a pharmaceutical composition comprising the compound provided by the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0010] Another aspect of the present invention provides the use of the compound provided by the present invention or a pharmaceutically acceptable salt thereof in the preparation of an RNAi drug for treating diseases.

[0011] The compound provided by the present invention has comparable or significantly enhanced endocytosis in cells compared to the targeting reagent L96 used in the already marketed RNAi drugs. When used in RNAi drugs, it can significantly improve the drug efficacy or reduce the drug dosage or toxicity, and has broad application prospects. Detailed implementation mode

[0012] Definitions

[0013] As used herein, the terms "RNAi drug" or "RNAi agent" refer to a reagent comprising an RNA molecule that can down-regulate the expression of a target gene through the RNA interference mechanism when introduced into cells. The terms "RNAi drug of the present invention", "RNAi drug described herein" or similar expressions include modified or unmodified oligonucleotides linked to a targeting moiety (such as L96 or some targeting compounds provided by the present invention). In a preferred embodiment, the oligonucleotides used in the present invention are selected from siRNA, shRNA, miRNA and ASO. RNAi (RNA interference) refers to the process by which nucleic acid molecules induce the cleavage and degradation of target RNA molecules (such as mRNA molecules) in a sequence-specific manner, for example, through the RNA-induced silencing complex (RISC) pathway. RNAi agents in this article include siRNA, shRNA and DNA / RNA hybrid molecules, which are sometimes collectively referred to as double-stranded RNA (dsRNA) in this article, and which comprise two antiparallel continuous nucleotide chains that are sufficiently complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, usually through hydrogen bonds between complementary bases in the two polynucleotides (such as Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds or reverse Hoogsteen hydrogen bonds). "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that form base pairs through hybridization, thereby forming a double strand between the two polynucleotide chains.

[0014] "siRNA" refers to a nucleic acid that forms double-stranded RNA, which has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNA is usually about 15 to about 30 base pairs in length, most commonly about 19 to 25 base pairs in length, such as 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.

[0015] Antisense oligonucleotides (ASOs) are single-stranded DNA or RNA that are complementary to a selected sequence. In the case of antisense DNA, it can be used to target specific complementary (coding or non-coding) RNAs. If binding occurs, this hybrid can be degraded by ribonuclease H1 (RNAase H1). A typical example of an ASO is a "gapmer", which has an internal "gap segment" flanked by two external "wing segments", where the gap segment consists of multiple nucleotides that support nuclease H cleavage, and each wing segment consists of one or more nucleotides that are chemically different from the nucleotides within the gap segment. For example, the 5' and 3' wing segments of a "gapmer" consist of nucleotides modified with 2'-O-methoxyethyl (2'-MOE), the gap segment consists of deoxyribonucleotides, and optionally the linkages between all nucleotides are phosphorothioate bonds.

[0016] The term "therapeutically effective amount" refers to an amount of a compound or composition of the present invention that is effective in producing some desired therapeutic effect in at least one subset of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0017] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0018] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting a compound of the present invention from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the patient.

[0019] The term "treatment" encompasses treatment as well as cure. The patients receiving this treatment are typically any animals in need, including primates (especially humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0020] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids, and the inorganic acids include, but are not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, etc. The pharmaceutically acceptable acids include organic acids, and the organic acids include, but are not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, acid hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (such as glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts.

[0021] Liver-targeting compounds and their pharmaceutically acceptable salts

[0022] One aspect of the present invention provides a liver-targeting compound and its pharmaceutically acceptable salt.

[0023] The present invention provides a compound of formula (I) or its pharmaceutically acceptable salt:

[0024] (I)

[0025] Wherein n is an integer between 1 and 5, for example, n is 1, 2, 3, 4 or 5, and m is an integer between 0 and 5, for example, m is 0, 1, 2, 3, 4 or 5;

[0026] R1 is H, an amino protecting group or -L 1 -GalNAc; wherein -L 1- independently -C(O)(CH2)p- or -C(O)(CH2)q(OCH2CH2)t-, where p is an integer between 2 and 10, such as p is 2, 3, 4, 5, 6, 7, 8, 9 or 10, q is an integer between 1 and 4, such as q is 1, 2, 3 or 4, and t is an integer between 1 and 4, such as t is 1, 2, 3 or 4;

[0027] Z is selected from H, an amino protecting group, a small molecule drug moiety, a fluorescent dye moiety and an oligonucleotide moiety.

[0028] In some embodiments, the present invention provides a compound having the structure of formula (II) or a pharmaceutically acceptable salt thereof,

[0029] (II)

[0030] where Z and -L 1 - are as defined in formula (I).

[0031] In some embodiments, the present invention provides a compound having the structure of formula (IIa) or a pharmaceutically acceptable salt thereof,

[0032] (IIa),

[0033] where Z and -L 1 - are as defined in formula (I).

[0034] In some embodiments, the present invention provides a compound having the structure of formula (IIb) or a pharmaceutically acceptable salt thereof,

[0035] (IIb),

[0036] where Z and -L 1 - are as defined in formula (I).

[0037] In some embodiments, the present invention provides a compound having the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof,

[0038] (IIIa)

[0039] where Z is as defined in formula (I).

[0040] In some embodiments, the present invention provides a compound having the structure of formula (IIIb) or a pharmaceutically acceptable salt thereof,

[0041] (IIIb)

[0042] where Z is as defined in formula (I).

[0043] In some embodiments, Z in the above structural formulas I, II, IIa, IIb, IIIa and IIIb is H.

[0044] In some embodiments, Z in the above structural formulas I, II, IIa, IIb, IIIa and IIIb is an amino protecting group.

[0045] In preferred embodiments, the amino protecting group is selected from benzyloxycarbonyl, tert-butoxycarbonyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl, 2,4-dimethoxybenzyl, p-methoxybenzyl and benzyl. In more preferred embodiments, the amino protecting group is benzyloxycarbonyl. In more preferred embodiments, the amino protecting group is tert-butoxycarbonyl. In more preferred embodiments, the amino protecting group is trifluoroacetyl.

[0046] In some embodiments, Z in the above structural formulas I, II, IIa, IIb, IIIa and IIIb is a fluorescent dye.

[0047] In preferred embodiments, the fluorescent dye is selected from 5FAM, FITC, Cy3, Cy5, Cy5.5 and Cy7. In more preferred embodiments, the fluorescent dye is 5FAM. In more preferred embodiments, the fluorescent dye is FITC.

[0048] In some embodiments, Z in the above structural formulas I, II, IIa, IIb, IIIa and IIIb is an oligonucleotide.

[0049] In preferred embodiments, the oligonucleotide is ASO, mRNA or siRNA. In more preferred embodiments, the oligonucleotide is ASO. In more preferred embodiments, the oligonucleotide is siRNA.

[0050] In the present invention, in embodiments where Z in the structural formulas I, II, IIa, IIb, IIIa and IIIb is siRNA or ASO, the compound is also referred to as an RNAi agent.

[0051] In some embodiments, each nucleotide of the sense strand and the antisense strand of the oligonucleotide portion of the RNAi agent of the present invention is modified. For example, the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), unlocked nucleic acids (UNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, and combinations thereof.

[0052] In some embodiments, both the sense strand and the antisense strand of the oligonucleotide portion of the RNAi agent of the present invention are modified, and the modification methods are selected from one of STC (Alnylam Pharmaceuticals, Inc., USA), ESC (Alnylam Pharmaceuticals, Inc., USA), Advanced ESC (Alnylam Pharmaceuticals, Inc., USA), ESC+ (Alnylam Pharmaceuticals, Inc., USA), AD1-3 (Arrowhead Research Corporation, USA), AD5 (Arrowhead Research Corporation, USA), and GalXC (Dicerna Pharmaceuticals, Inc., USA) (see, for example, Hu et al., Therapeutic siRNA: State of the Art. Signal Transduct Target Ther. 2020; 5(1):101 (Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020; 5(1):101), published on June 19, 2020).

[0053] The oligonucleotide portion of the RNAi agent may include a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are fully base-paired at the molecular termini and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the oligonucleotide portion of the RNAi agent includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the oligonucleotide portion of the RNAi agent includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand.

[0054] Specifically, for example, in one embodiment, the oligonucleotide portion of the RNAi agent includes (i) a sense strand that is 19 nucleotides in length, and (ii) an antisense strand that is 21 nucleotides in length, and the two strands form a double-stranded region having a length equal to the length of the sense strand. In another embodiment, the oligonucleotide portion of the RNAi agent comprises (i) a sense strand that is 21 nucleotides in length, and (ii) an antisense strand that is 23 nucleotides in length, and the two strands form a double-stranded region having a length equal to the length of the sense strand.

[0055] In some specific embodiments, the present invention provides a compound of one of the following structural formulas or a pharmaceutically acceptable salt thereof:

[0056] (MCPP-17-001-I)

[0057] (MCPP-17-001-II)

[0058] (MCPP-16-001-I)

[0059] (MCPP-17-001-I-A)

[0060] (MCPP-17-001-I-C)

[0061] (MCPP-17-001-II-A)

[0062] (MCPP-17-001-II-C)

[0063] (MCPP-16-001-I-A)

[0064] (MCPP-16-001-I-C)

[0065] (MCPP-17-001)

[0066] (MCPP-16-001)

[0067] (MCPP-17-001-I-siRNA)

[0068] (MCPP-16-001-I-siRNA)

[0069] Among them, Cbz represents benzyloxycarbonyl.

[0070] Pharmaceutical composition

[0071] Another aspect of the present invention provides a pharmaceutical composition or formulation, which comprises any of the compounds described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, excipient or diluent. In view of clinical applications, the pharmaceutical composition and formulation will be prepared in a form suitable for the intended application. Generally, this will require preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0072] The ingredients and methods for formulating a pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the type and severity of the disease or disorder to be treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral administration forms include intravenous, intra-arterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery carrier. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery.

[0073] In some embodiments, the pharmaceutical composition comprises a therapeutically or prophylactically effective amount of the compound described herein or a pharmaceutically acceptable salt thereof. The effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof can be from about 0.01 mg per kg body weight to about 100 mg per kg body weight, and can be administered daily, weekly, monthly or at longer intervals. Accurately determining the specific effective dosage and dosing frequency may be based on several factors, including the patient's body size, age and general condition, the type of disease to be treated (such as myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the specific compound or a pharmaceutically acceptable salt thereof used, and the route of administration.

[0074] Administration of the pharmaceutical composition of the present invention can be effected by any common route, so long as the target tissue is accessible by that route. These routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, buccal, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or by delivery through the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0075] Liposomal formulations are particularly suitable for topical administration and have several advantages over other formulations. Such advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a compound or its pharmaceutically acceptable salt into the skin. In some embodiments, liposomes are used to deliver a compound or its pharmaceutically acceptable salt to epidermal cells and also enhance the penetration of the compound or its pharmaceutically acceptable salt into dermal tissues such as within the skin. Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these formulations are sterile and to the extent that they are fluid, easy to inject. The formulations should be stable under the conditions of production and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvent or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and vegetable oils. For example, fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0076] Sterile injectable solutions can be prepared by incorporating the appropriate amount of the active compound with any other ingredients (such as those enumerated above) into a solvent and then filtering sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile dispersion medium containing the desired other ingredients, for example, as described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and freeze-drying techniques which yield a powder of the active ingredient and any additional desired ingredients from their previously sterile filtered solutions.

[0077] The compositions of the present invention can generally be formulated in neutral form or in salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed from free amino groups) derived from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric oxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the compounds of the present invention or their pharmaceutically acceptable salts are formulated as sodium salts.

[0078] For example, for parenteral administration in the form of an aqueous solution, the solution is typically suitably buffered, and the liquid diluent is first made isotonic with, for example, sufficient saline or glucose. Such aqueous solutions can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 mL of isotonic sodium chloride solution and added to 1000 mL of subcutaneous infusion fluid, or injected at the proposed infusion site. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local market regulatory authorities. In certain embodiments, the pharmaceutical composition of the present invention comprises the sterile saline solution described herein and the compound or its pharmaceutically acceptable salt or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises the compound or its pharmaceutically acceptable salt described herein and sterile water (e.g., water for injection) or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises the compound or its pharmaceutically acceptable salt described herein and phosphate-buffered saline (PBS) or consists thereof.

[0079] In some embodiments, the pharmaceutical composition of the present invention is packaged with or stored within a dosing device. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, auto-injectors, infusion pumps, in vivo syringes, and injection pens. Devices for aerosol or powder formulations include, but are not limited to, inhalers, insufflators, nebulizers, etc. Accordingly, the present invention includes dosing devices containing the pharmaceutical composition of the present invention for treating or preventing one or more of the diseases or disorders described herein.

[0080] Methods of Treatment and Uses

[0081] The present invention provides a method for reducing or inhibiting the expression of a specific gene in a cell (such as a hepatocyte) by contacting the cell with any one of the compounds described herein or a pharmaceutically acceptable salt thereof. The cell can be in vitro or in vivo. Gene expression can be evaluated by measuring the amount or level of the transcript or translated protein of the corresponding gene. The reduction in the expression of the corresponding gene in the cell or animal treated with the compound or a pharmaceutically acceptable salt thereof of the present invention can be determined relative to the expression of the corresponding gene in the cell and animal not treated with the compound or a pharmaceutically acceptable salt thereof or treated with a control compound or a pharmaceutically acceptable salt thereof. For example, in some embodiments, the reduction in the expression of the corresponding gene is evaluated by (a) measuring the amount or level of the mRNA of the corresponding gene in hepatocytes treated with the compound or a pharmaceutically acceptable salt thereof of the present invention, (b) measuring the amount or level of the mRNA of the corresponding gene in hepatocytes treated with a control compound or a pharmaceutically acceptable salt thereof (such as a compound or a pharmaceutically acceptable salt thereof directed against an RNA molecule not expressed in hepatocytes or a compound or a pharmaceutically acceptable salt thereof having a nonsense or scrambled sequence) or no compound or a pharmaceutically acceptable salt thereof, and (c) comparing the level of the mRNA of the corresponding gene measured in the treated cells in (a) with the level of the mRNA of the corresponding gene in the control cells in (b). Before the comparison, the levels of the mRNA of the corresponding gene in the treated cells and the control cells can be normalized to the RNA level of a control gene (such as 18S ribosomal RNA or a housekeeping gene). The level of the mRNA of the corresponding gene can be measured by a variety of methods, including Northern blot analysis, nuclease protection analysis, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, etc.

[0082] In some embodiments, the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for preventing or treating one or more diseases. In some embodiments, the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in preventing or treating one or more diseases. In some embodiments, the present invention provides a method for preventing or treating one or more diseases, the method comprising administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0083] In some embodiments of the present invention, the target genes of the oligonucleotides can be selected from: ANGPTL3 gene, KHK gene, DGAT2 gene, PCSK9 gene, apo(a) gene, apoB gene, complement C3 gene, complement C5 gene, complement D gene, complement F gene, SAA, TTR, RSV, PDGF β gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden, activated protein C, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, p73 mutant gene, mutations in the p21 (WAF1 / CIP1) gene, mutations in the p27 (KIP1) gene, mutations in the PPM 1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, mutations in the p53 tumor suppressor gene, and combinations of any two of the above.

[0084] In certain embodiments, the expression of the target gene in hepatocytes is reduced by at least 40%, at least 45% or at least 50% by the compounds of the present invention or their pharmaceutically acceptable salts. In some embodiments, the expression of the target gene in hepatocytes is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85% by the compounds of the present invention or their pharmaceutically acceptable salts. In other embodiments, the expression of the target gene in hepatocytes is reduced by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by the RNAi agents of the present invention. The percentage reduction in target gene expression can be measured by any of the methods described herein and other methods known in the art.

[0085] In certain embodiments, the present invention provides a method for reducing the expression of a target gene in a patient in need thereof, comprising administering to the patient any of the compounds described herein or a pharmaceutically acceptable salt thereof. Preferably, the expression level of the target gene in the patient's hepatocytes is reduced after administering the compound or a pharmaceutically acceptable salt thereof, compared to the level of target gene expression in a patient who has not received the compound or a pharmaceutically acceptable salt thereof, or compared to the level of target gene expression in the patient before administering the compound or a pharmaceutically acceptable salt thereof. In some embodiments, after administering the compound or a pharmaceutically acceptable salt of the present invention, the expression of the target gene in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The percentage reduction in target gene expression can be measured by any of the methods described herein and other methods known in the art. In certain embodiments, the percentage reduction in target gene expression is determined by assessing the level of the target gene protein in the patient's serum or plasma according to the method described herein.

[0086] In the various embodiments described above in this section, the disease can be, for example, a metabolic disease, a cardiovascular disease, a rare disease, a complement-related disease or a liver disease.

[0087] In a preferred embodiment, the metabolic disease is, for example, metabolic associated steatohepatitis (MASH) / metabolic associated fatty liver disease (MAFLD), type II diabetes (T2DM) / insulin resistance (IR), obesity, familial hypercholesterolemia (FH), hypertriglyceridemia (HTG).

[0088] In a preferred embodiment, the cardiovascular disease is, for example, atherosclerotic cardiovascular disease (ASCVD), stroke, lipoprotein(a)-elevated related cardiovascular risk.

[0089] In a preferred embodiment, the rare disease is, for example, transthyretin amyloidosis (ATTR), hereditary angioedema (HAE), α1-antitrypsin deficiency (AATD), familial chylomicron syndrome (FCS).

[0090] In a preferred embodiment, the complement-related disease is, for example, paroxysmal nocturnal hemoglobinuria (PNH), IgA nephropathy (IgAN), C3 glomerulopathy (C3G), immune complex membranoproliferative glomerulonephritis (IC-MPGN), generalized myasthenia gravis (gMG), myelin-associated glycoprotein antibody-related peripheral neuropathy (MAG-PN), age-related macular degeneration (AMD), geographic atrophy of the macula (GA).

[0091] In a preferred embodiment, the liver diseases are selected from viral hepatitis, liver fibrosis, cirrhosis, and liver tumors. The viral hepatitis is, for example, hepatitis B virus infection or hepatitis C virus infection. The liver tumors are, for example, hepatocellular carcinoma (HCC) or metastatic liver cancer.

[0092] L96=

[0093] 。

[0094] Example 1. Synthesis of Compounds and Intermediates

[0095] 1. N-acetylgalactosamine (GalNAc) is a compound with a reactive group at the end, including compounds such as Gal-C5 and Gal-C4 containing a reactive carboxylic acid. They are synthesized according to the reported methods, and the specific information is shown in Table 1.

[0096] Table 1: Chemical Structures, Nomenclatures, and Synthesis Methods of Compounds with GalNAc at the End Containing Reactive Groups

[0097]

[0098] The abbreviations of chemical reagents involved in the examples and their corresponding Chinese names are shown in the following table.

[0099] 2. Synthesis of Key Intermediate Compounds

[0100] 2.1 Synthesis of Fmoc-A

[0101]

[0102] Step 1: Dissolve tert-butyl (4-aminobutyl) carbamate (9.40 g, 50 mmol, 1.0 equiv) in DCM (200 mL), and add TEA (20.9 mL, 150 mmol, 3.0 equiv). Cool the mixed solution to 0 o °C, and dropwise add ethyl 2,2,2-tribromopropionate (9.05 g, 50 mmol, 1.0 equiv). Raise the temperature of the mixed solution to 10 o °C, and stir the reaction for 48 hours. After the reaction is completed, concentrate the reaction solution to obtain the crude product A-1, which is directly used for the next step of the reaction.

[0103] LCMS: (ESI) m / z = 289.2 [M + H] + 。

[0104] Step 2: Dissolve A-1 (crude, ~50 mmol) in THF (200 mL), add Fmoc-OSu (16.9 g, 50 mmol, 1.0 equivalent) and aqueous NaHCO3 solution (100 mL), and stir the mixed solution at room temperature for 2 hours. After the reaction detection is completed, concentrate the reaction solution, extract it with ethyl acetate (500 mL), and wash the organic phase successively with saturated brine (50 mL), 0.5 N HCl, and saturated brine (50 mL). After drying the organic solution with anhydrous sodium sulfate, filter it, concentrate it, and purify the crude product by normal-phase silica gel column (330 g). The mobile phase is petroleum ether / ethyl acetate, and the volume ratio of the two solvents ranges from 100:0 to 40:60 to obtain 20 g of light yellow solid A-2. LCMS: (ESI) m / z = 411.1 [M + H-Boc] + 。

[0105] Step 3: Dissolve A-2 (20 g, ~39 mmol, 1.0 equivalent) in THF (200 mL), add 150 mL of aqueous NaOH solution (7.8 g, 195.8 mmol, 5.0 equivalents), and heat the reaction solution to 45 o °C and stir for 6 hours. Concentrate the reaction solution, wash the aqueous solution with tetraethylene glycol dimethyl ether (200 mL), slowly add dry ice (50 g) to the aqueous solution to adjust the pH to ~9, then add a THF solution (100 mL) of Fmoc-OSu (13.1 g, 39 mmol), and stir the reaction solution for 2 hours. After concentrating the reaction solution to remove THF, adjust the pH of the aqueous solution to 5 - 6 with 2 N HCl, and then extract it twice with ethyl acetate (500 mL). After combining the organic layers, wash them with saturated brine (2 × 200 mL) and dry them with anhydrous sodium sulfate. After filtering and concentrating the organic solution, purify it by preparative C18 chromatographic column to obtain compound Fmoc-A (12.5 g, yield: 51.9%) as a white solid. The mobile phase is acetonitrile / water (containing 10 mmol / L NH4HCO3), and the volume ratio of the two solvents ranges from 15:85 to 50:50. LCMS: (ESI) m / z = 383.2 [M + H-Boc] + 。

[0106] 2.2 Synthesis of Cbz-β-Ala-Sar-Sar-Sar-OH

[0107]

[0108] Step 1: Weigh - CTC resin (12 g, 1.29 mmol) into a solid - phase synthesis reactor and wash it with DCM (3 × 120 mL). The washed resin is swollen in DCM (120 mL). Under nitrogen protection, add Fmoc - Sar - OH (2.4 g, 7.7 mmol) and stir, then add DIPEA (7.7 mL, 45 mmol). Stir the reactor for 4 hours. Wash the resin with DCM (3×120 mL), add a mixture of MeOH / DCM (v:v = 1:1, 120 mL) for capping, and slowly stir the reactor for 2 hours. After removing the solution, wash the resin with DMF (3 × 120 mL) to obtain Fmoc - Sar - CTC resin.

[0109] Step 2: Add a 20% piperidine in DMF solution (120 mL) to the Fmoc - Sar - CTC resin to remove the Fmoc protection, twice for 20 minutes each time. Wash the resin with DMF (5 × 120 mL) to obtain Sar - CTC resin.

[0110] Step 3: Weigh Fmoc - Sar - OH (3.1 g, 10.0 mmol), HATU (3.80 g, 10.0 mmol) and HOAt (1.36 g, 10 mmol) and dissolve them in DMF (120 mL). Pour the mixture into the reactor of Sar - CTC resin, add DIPEA (5.1 mL, 30 mmol), and stir for 1 hour. Detect the reaction with ninhydrin. Stop the reaction when the result is negative. Wash the resin with DMF (6 × 120 mL) to obtain Fmoc - Sar - Sar - CTC resin.

[0111] Step 4: Remove the Fmoc protecting group from the Fmoc - Sar - Sar - CTC resin according to the method in Step 2 to obtain Sar - Sar - CTC resin.

[0112] Step 5: Condense Fmoc - Sar - OH with Sar - Sar - CTC resin according to the method in Step 3 to obtain Fmoc - Sar - Sar - Sar - CTC resin.

[0113] Step 6: Remove the Fmoc protecting group from the Fmoc - Sar - Sar - Sar - CTC resin according to the method in Step 2 to obtain Sar - Sar - Sar - CTC resin.

[0114] Step 7: Sar-Sar-Sar-CTC resin was condensed with Cbz-β-Ala-OH (4.46 g, 20.0 mmol) according to the method in Step 3. The resin was washed successively with DMF (3 × 120 mL), DCM (3 × 120 mL), and MTBE (3 × 120 mL) and dried under vacuum to obtain Cbz-β-Ala-Sar-Sar-Sar-CTC resin.

[0115] Step 8: Cold HFIP / DCM (1:1) solution (50 mL) was added to Cbz-β-Ala-Sar-Sar-Sar-CTC resin for cleavage. The filtrate was collected by suction filtration. The resin was washed with DCM (2 × 20 mL). The filtrates were combined and distilled under reduced pressure to obtain 3.2 g of light yellow solid Cbz-β-Ala-Sar-Sar-Sar-OH with a yield of 95.2%.

[0116] LCMS: (ESI) m / z = 437.0 [M+H] + ; 459.1 [M+Na] + 。

[0117] 2.3 Synthesis of Compound C

[0118]

[0119] Step 1: 6 - ((Benzyloxy)carbonyl)aminohexanoic acid (5.3 g, 20 mmol) was dissolved in DMF (20 mL). (6-Aminohexyl)carbamic acid tert-butyl ester (4.33 g, 20 mmol), HBTU (7.58 g, 20 mmol), and DIPEA (10.2 mL) were added. The mixed solution was stirred at room temperature for 4 hours. After the reaction was detected to be complete, 150 mL of water was added, and a white precipitate appeared. The solid was filtered, washed with water (2×10 mL), and dried to obtain white solid B-1 (9.2 g, yield: 99%) which was directly used in the next step. LCMS: (ESI) m / z = 464.3 [M+H] + 。

[0120] Step 2: B-1 obtained in the previous step was dissolved in DCM (20 mL) and cooled to -5 o °C. TFA (20 mL) was added, and the mixed solution was stirred at -5 o °C for 4 hours. After the reaction was detected to be complete, the reaction solution was concentrated to obtain light yellow solid compound B (9.2 g, yield: 99%) which was directly used in the next step. LCMS: (ESI) m / z = 364.1 [M+H] + 。

[0121] Step 3: Fmoc-D-Glu-O(tBu) (7.8 g, 18.4 mmol) was dissolved in DMF (40 ml), HBTU (7.0 g, 18.4 mmol) and DIPEA (9.5 mL, 50.8 mmol) were added, and the mixed solution was cooled to 0 o °C. A solution of compound B (8.8 g, 18.4 mmol) in DMF (10 mL) was slowly added, and the temperature was raised to room temperature and stirred for 2 hours. After the reaction detection was completed, 200 mL of water was added to the mixed solution, a white precipitate appeared, the solid was filtered, washed with water (2×10 mL), and dried to obtain a white solid C-1 (14.0 g, yield: 99%), which was directly used in the next step of the reaction. LCMS: (ESI) m / z = 771.5 [M+H] + ; 793.5 [M+Na] + .

[0122] Step 4: C-1 obtained in the previous step was dissolved in DCM (20 mL), cooled to -5 o °C, TFA (20 mL), TIS (5 mL) and water (1 mL) were added, and the mixed solution was raised to 5 o °C and stirred for 4 hours. After the reaction detection was completed, the reaction solution was concentrated to obtain a pale yellow solid compound C (9.0 g, yield: 99%), which was directly used in the next step of the reaction. LCMS: (ESI) m / z = 715.4[M+H] + ; 737.3 [M+Na] + .

[0123] 3. Synthesis of cyclic pentapeptide backbone structure

[0124] 3.1 Synthesis of MCPP-17-001

[0125]

[0126] Step 1: Weigh -CTC-RS resin (16 g, 1.25 mmol / g) into a solid-phase synthesis reactor and wash it with DCM (3 × 160 mL). The washed resin is swollen in DCM (160 mL). Under nitrogen protection, add Fmoc-Lys(Boc)-OH (4.7 g, 10.0 mmol) and stir, then add DIPEA (10.2 mL, 60.0 mmol). Stir the reactor for 6 hours. Wash the resin with DCM (3 × 160 mL), add a mixture of MeOH / DCM (v:v = 1:1, 160 mL) for capping, and slowly stir the reactor for 2 hours. After removing the solution, wash the resin with DMF (3 × 120 mL) to obtain Fmoc-Lys(Boc)-CTC resin.

[0127] Step 2: Add a 20% piperidine in DMF solution (20% Py / DMF) (160 mL) to the Fmoc-Lys(Boc)-CTC resin to remove the Fmoc protecting group, twice for 20 minutes each time. Wash the resin with DMF (5 × 160 mL) to obtain Lys(Boc)-CTC resin.

[0128] Step 3: Weigh compound Fmoc-A (7.24 g, 15 mmol), HATU (5.70 g, 15.0 mmol) and HOAt (2.04 g, 15 mmol) and dissolve them in DMF (160 mL). Pour the mixture into the reactor of Lys(Boc)-CTC resin, add DIPEA (7.7 mL, 45 mmol), and stir for 2 hours. After detecting the completion of the reaction with ninhydrin, wash the resin with DMF (6 × 160 mL) to obtain Fmoc-A-Lys(Boc)-CTC resin.

[0129] Step 4: Add a 20% piperidine in DMF solution (20% Py / DMF) (160 mL) to the Fmoc-A-Lys(Boc)-CTC resin to remove the Fmoc protecting group, twice for 20 minutes each time. Wash the resin with DMF (5 × 160 mL) to obtain A-Lys(Boc)-CTC resin.

[0130] Step 5: Weigh Fmoc-βAla-OH (4.67 g, 15.0 mmol), HATU (5.70 g, 15.0 mmol) and HOAt (2.04 g, 15 mmol), dissolve them in DMF (160 mL). Pour the mixture into the reactor of A-Lys(Boc)-CTC resin, add DIPEA (7.7 mL, 45 mmol), and stir for 1 hour. After detecting the completion of the reaction with ninhydrin, wash the resin with DMF (6 × 160 mL) to obtain Fmoc-βAla-A-Lys(Boc)-CTC resin.

[0131] Step 6: Add a 20% piperidine in DMF solution (20% Py / DMF) (160 mL) to the Fmoc-βAla-A-Lys(Boc)-CTC resin to remove the Fmoc protecting group twice for 20 minutes each time. Wash the resin with DMF (5 × 160 mL) to obtain βAla-A-Lys(Boc)-CTC resin.

[0132] Step 7: Weigh Fmoc-Lys(Boc)-OH (7.02 g, 15.0 mmol), HATU (5.70 g, 15.0 mmol) and HOAt (2.04 g, 15 mmol), dissolve them in DMF (160 mL). Pour the mixture into the reactor of βAla-A-Lys(Boc)-CTC resin, add DIPEA (7.7 mL, 45 mmol), and stir for 40 minutes. After detecting the completion of the reaction with ninhydrin, wash the resin with DMF (6 × 160 mL) to obtain Fmoc-Lys(Boc)-βAla-A-Lys(Boc)-CTC resin.

[0133] Step 8: Add a 20% piperidine in DMF solution (20% Py / DMF) (160 mL) to the Fmoc-Lys(Boc)-βAla-A-Lys(Boc)-CTC resin to remove the Fmoc protecting group twice for 20 minutes each time. Wash the resin with DMF (5 × 160 mL) to obtain βAla-A-Lys(Boc)-CTC resin (D).

[0134] Step 9: Weigh Fmoc-Lys(Cbz)-OH (5.35 g, 10.66 mmol), HATU (4.05 g, 10.66 mmol) and HOAt (1.45 g, 10.66 mmol) and dissolve them in DMF (60 mL). Pour the mixture into a reactor containing βAla-A-Lys(Boc)-CTC resin (6.0 g, 3.68 mmol), add DIPEA (5.4 mL, 32 mmol), and stir for 2 hours. After detecting the completion of the reaction by ninhydrin, wash the resin with DMF (6 × 60 mL) to obtain Fmoc-Lys(Cbz)-βAla-A-Lys(Boc)-CTC resin.

[0135] Step 10: Add a 20% piperidine in DMF solution (20%Py / DMF) (40 mL) to the Fmoc-Lys(Cbz)-βAla-A-Lys(Boc)-CTC resin to remove the Fmoc protecting group twice, 20 minutes each time. Wash the resin with DMF (3 × 60 mL), DCM (3× 60 mL) and MTBE (3 × 60 mL) to obtain Lys(Cbz)-βAla-A-Lys(Boc)-CTC resin.

[0136] Step 11: Add the resin to a cold HFIP / DCM (1:1, 30 mL) solution for cleavage, filter with suction to collect the filtrate, wash the resin with DCM (2 × 10 mL), combine the filtrates and distill under reduced pressure to obtain 3.5 g of white solid MCPP-17-001-A, with a yield of 90.5%.

[0137] Step 12: Dissolve HBTU (2.44 g, 6.43 mmol) in DMF (70 mL), add DIPEA (2.2 mL, 12.78 mmol), and gradually add dropwise a DMF (70 mL) solution of MCPP-17-001-A (4.5 g, 4.29 mmol) to the mixture within 2 hours. Pour the reaction solution into 200 mL of water, precipitate the solid, filter and wash it with water (2 × 10 mL). Dry the solid to obtain a light yellow solid (5.2 g, 37% purity) which is directly used for the next step. LCMS: (ESI) m / z = 1055.3 [M+Na] + ;1032.40 [M+H] + ; 932.4 [M-100+H] + 。

[0138] Step 13: The solid from the previous step was dissolved in MeOH (70 mL), 10% Pd / C (40 mg) was added, and the mixture was stirred under hydrogen for 4 hours. After filtration, the filtrate was concentrated and purified by a C18 reverse silica gel column with the mobile phase of acetonitrile / water (containing 0.05% TFA), and the volume ratio of the two solvents was from 5:95 to 50:50. 898 mg of white solid MCPP-17-001-B was obtained with a two-step yield of 23%. LCMS: (ESI) m / z = 920.6 [M+Na] + ; 898.7 [M+H] + 。

[0139] Step 14: Cbz-β-Ala-Sar-Sar-Sar-OH (0.654 g, 1.5 mmol) was dissolved in DMF (15 mL), HATU (570 mg, 1.5 mmol) and DIPEA (0.51 mL, 3.0 mmol) were added, and the mixture was stirred at room temperature for 5 minutes. A solution of MCPP-17-001-B (898 mg, 1.0 mmol) in DMF (5 mL) was added to the mixture, and the mixture was stirred at room temperature for 2 hours. The reaction solution was quenched with water (100 mL) and extracted with ethyl acetate (2 × 100 mL). After combination, the extract was washed with saturated brine (2 × 20 mL), dried and concentrated to obtain a light yellow solid MCPP-17-001-C (1.3 g, 30.7% purity). LCMS: (ESI) m / z =559.2 [M-200+2H] / 2 + ; 1339.9 [M+Na] + 。

[0140] Step 15: The crude product obtained in the previous step (1.3 g, crude) was dissolved in DCM (10 mL), cooled to -5 o °C, TFA (10 mL) and TIS (0.5 mL) were added, and the mixed solution was stirred at -5 o °C for 2 hours. After the reaction was detected to be complete, the reaction solution was concentrated to 10 mL, cold MTBE (30 mL) and petroleum ether (10 mL) were added, and the resulting crude product was purified by preparative HPLC using an Xbridge C18 column (19 × 250 mm, 10 μm, 130 Å) with the mobile phase of acetonitrile / water (containing 0.05% TFA), and the volume ratio of the two solvents was from 5:95 to 35:65. 400 mg of white solid MCPP-17-001 was obtained with a two-step yield of 29.4%. LCMS: (ESI) m / z = 1016.7 [M+H] + ; 509.0 [M+2H] / 2 +。

[0141] 3.2 Synthesis of MCPP-17-002

[0142]

[0143] Step 1: Compound C (Cmd-C, 2.54 g, 3.55 mmol) was dissolved in DMF (40 mL). HATU (1.35 g, 3.55 mmol), HOAt (483 mg, 3.55 mmol) and DIPEA (1.8 mL, 10.7 mmol) were added successively. The mixture was added to resin D (4.0 g, 2.45 mmol), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed as detected by ninhydrin, the resin was washed with DMF (6 × 40 mL) to obtain resin E.

[0144] Step 2: Resin E was added to a 20% piperidine in DMF solution (20% Py / DMF) (40 mL) to remove the Fmoc protecting group, twice for 20 minutes each time. The resin was washed successively with DMF (3 × 40 mL), DCM (3 × 40 mL) and MTBE (3 × 40 mL), and dried in vacuo.

[0145] Step 3: The resin dried in the previous step was added to a cold DCM / HFIP (1 / 1, 30 mL) solution for cleavage. After cleavage, the filtrate was collected by suction filtration. The resin was washed with DCM (2 × 10 mL). The filtrates were combined and distilled under reduced pressure to obtain 2.5 g of white solid MCPP-17-002-A with a yield of 80.9%. LCMS: (ESI) m / z = 1263.8 [M+H] + ; 1285.8 [M+H] + ;582.0 [M-100+2H] / 2 + 。

[0146] Step 4: HBTU (1.52 g, 4.0 mmol) was dissolved in DMF (50 mL). DIPEA (1.02 mL, 6 mmol) was added. The mixture of MCPP-17-002-A (1.26 g, 1.0 mmol) in DMF (20 mL) was added dropwise to the mixture within 2 hours. The reaction solution was poured into 200 mL of water, and a solid precipitated. After filtration, it was washed with water (2 × 10 mL). The solid was dried to obtain a light yellow solid (0.75 g, 60.3% purity) which was directly used for the next step. LCMS: (ESI) m / z = 1266.6 [M+Na] +; 523.0[M - 200 + 2H] / 2 + 。

[0147] Step 5: The crude product obtained in the previous step (0.75 g, crude product) was dissolved in DCM (10 mL), cooled to -5 o °C, TFA (10 mL) and TIS (0.5 mL) were added, and the mixed solution was stirred at -5 o °C for 2 hours. After the reaction detection was completed, the reaction solution was concentrated to 5 mL, cold MTBE (50 mL) was added, the solid obtained by concentration was dissolved in 100 mL of water and freeze-dried to obtain 700 mg of a light-colored solid MCPP-17-002, with a yield of 80.9%. LCMS: (ESI) m / z = 944.7 [M + H] + ; 966.6 [M + Na] + ; 472.9 [M + 2H] / 2 + 。

[0148] 3.3 Synthesis of MCPP-16-001

[0149]

[0150] Step 1: Using CTC-RS resin (8 g, 1.25 mmol / g) and Fmoc-Lys(Boc)-OH (2.81 g, 6.0 mmol) as raw materials, according to the method of Step 1 of MCPP-17-001, Fmoc-Lys(Boc)-CTC resin was obtained.

[0151] Step 2: According to the method of Step 2 of MCPP-17-001, Lys(Boc)-CTC resin was obtained.

[0152] Step 3: According to the method of Step 3 of MCPP-17-001, Fmoc-A-Lys(Boc)-CTC resin was obtained.

[0153] Step 4: According to the method of Step 4 of MCPP-17-001, A-Lys(Boc)-CTC resin was obtained.

[0154] Step 5: Weigh Fmoc-Gly-OH (2.97 g, 10.0 mmol), HATU (3.80 g, 10.0 mmol) and HOAt (1.36 g, 10 mmol) and dissolve them in DMF (80 mL). Pour the mixture into the reactor of A-Lys(Boc)-CTC resin, add DIPEA (5.1 mL, 30 mmol), and stir for 1 hour. After the reaction is completed as detected by ninhydrin, wash the resin with DMF (6 × 80 mL) to obtain Fmoc-Gly-A-Lys(Boc)-CTC resin.

[0155] Step 6: Add the resin obtained in the previous step to a 20% piperidine in DMF solution (20% Py / DMF) (80 mL) to remove the Fmoc protecting group, twice for 20 minutes each time. Wash the resin with DMF (5 × 80 mL) to obtain Gly-A-Lys(Boc)-CTC resin.

[0156] Step 7: Weigh Fmoc-Lys(Boc)-OH (4.69 g, 10.0 mmol), HATU (5.70 g, 15.0 mmol) and HOAt (2.04 g, 15 mmol) and dissolve them in DMF (80 mL). Pour the mixture into the reactor of GLy-A-Lys(Boc)-CTC resin, add DIPEA (5.1 mL, 30 mmol), and stir for 90 minutes. After the reaction is completed as detected by ninhydrin, wash the resin with DMF (6 × 80 mL) to obtain Fmoc-Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0157] Step 8: Add the resin obtained in the previous step to a 20% piperidine in DMF solution (20% Py / DMF) (80 mL) to remove the Fmoc protecting group, twice for 20 minutes each time. Wash the resin with DMF (5 × 80 mL) to obtain Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0158] Step 9: Weigh Dde-Lys(Fmoc)-OH (4.88 g, 10.0 mmol), HATU (5.70 g, 15.0 mmol) and HOAt (2.04 g, 15 mmol), dissolve them in DMF (80 mL), pour the mixture into the reactor of Lys(Boc)-Gly-A-Lys(Boc)-CTC resin, add DIPEA (5.1 mL, 30 mmol), and stir for 2 hours. After detecting the completion of the reaction by ninhydrin, wash the resin with DMF (6 × 80 mL) to obtain Dde-Lys(Fmoc)-Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0159] Step 10: Add the resin obtained in the previous step to a 20% piperidine in DMF solution (20% Py / DMF) (80 mL) to remove the Fmoc protecting group, twice for 20 minutes each time. Wash the resin with DMF (5 × 80 mL) to obtain Dde-Lys(ε-NH2)-Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0160] Step 11: Dissolve Cbz-β-Ala-Sar-Sar-Sar-OH (4.36 g, 10 mmol) in DMF (80 mL), add HATU (3.80 mg, 10 mmol) and DIPEA (5.1 mL, 30 mmol), and stir at room temperature for 5 minutes. Add the mixture to the resin reactor and stir at room temperature for 2 hours. After detecting the completion of the reaction by ninhydrin, wash the resin with DMF (6 × 80 mL) to obtain Dde-Lys(Cbz-β-Ala-Sar-Sar-Sar-ε-NH)-Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0161] Step 12: Add the resin obtained in the previous step to a DMF (30 mL) solution containing 5% N2H4.H2O under nitrogen to remove the Dde protecting group, twice for 20 minutes each time. Wash the resin successively with DMF (3 × 80 mL), DCM (3 × 30 mL) and MTBE (3 × 30 mL), and dry it under vacuum to obtain α-NH2-Lys(Cbz-β-Ala-Sar-Sar-Sar-ε-NH)-Lys(Boc)-Gly-A-Lys(Boc)-CTC resin.

[0162] Step 13: The resin dried in the previous step was added to a cold DCM / HFIP (1 / 1, 100 mL) solution for cleavage. After cleavage, the filtrate was collected by suction filtration. The resin was washed with DCM (2 × 20 mL). The combined filtrates were distilled under reduced pressure to obtain 12.5 g of white solid MCPP-16-001-B. LCMS: (ESI) m / z = 1320.7 [M + H] + ; 611 [M-100+2H] / 2 + 。

[0163] Step 14: HBTU (999 mg, 2.63 mmol) was dissolved in DMF (80 mL), and DIPEA (0.9 mL, 5.28 mmol) was added. A solution of MCPP-16-001-B (1.74 g, 1.32 mmol) in DMF (20 mL) was added dropwise to the mixture within 2 hours. The mixture was stirred at room temperature for 1 hour. After the solution was concentrated, it was purified by preparative HPLC using an Xbridge C18 column (19 × 250 mm, 10 μm, 130 Å), with the mobile phase being acetonitrile / water (containing 10 mmol / L NH4HCO3), and the volume ratio of the two solvents changing from 30:70 to 50:50. 440 mg of white solid was obtained with a yield of 25.6%. LCMS: (ESI) m / z = 1302.6 [M+H] + ; 551.8 [M-200+2H] / 2 + 。

[0164] Step 15: The compound obtained in the previous step (440 mg, 0.337 mmol) was dissolved in DCM (4 mL) and cooled to -5 o °C. TFA (4 mL) was added, and the mixed solution was stirred at -5 o °C for 2 hours. After the reaction was detected to be complete, the reaction solution was added to cold MTBE (30 mL) and petroleum ether (10 mL) to precipitate the solid, which was washed with 50 mL of cold MTBE / petroleum ether mixture and dried to obtain 370 mg of light-colored solid MCPP-16-001 with a yield of 81.6%. LCMS: (ESI) m / z = 944.7 [M+H] + ; 966.6 [M+Na] + ; 472.9 [M+2H] / 2 + 。LCMS: (ESI) m / z = 1002.6 [M+H] + ; 1024.7 [M+Na] + ; 502.0[M+2H] / 2 + 。

[0165] 4. Synthesis of MCPP-17-001-I

[0166]

[0167] Step 1: Dissolve MCPP-17-001 (100 mg, 0.0736 mmol) in DMF (3 mL), add Gal-C4 (160 mg, 0.368 mmol, 5.0 equivalents), EDCI . HCl (71 mg, 0.368 mmol) and HOAt (50 mg, 0.368 mmol). Cool the mixture to 4 o °C, add DIPEA (126 μL, 0.736 mmol), and allow it to warm to room temperature and react for 4 hours. The reaction solution was directly purified by a reversed-phase preparative chromatography column to obtain MCPP-17-001-I-A (85 mg, yield: 51.0%) as a white solid. LCMS: (ESI) m / z = 1132.1 [M+2H] / 2 + ; 775.0 [M+3H] / 3 + .

[0168] HPLC preparative conditions: Preparative column: Xbridge C18, 19 × 250 mm, 10 μm, 130 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increase from 28%B to 38%B within 10 minutes.

[0169] Step 2: Sequentially add MCPP-17-001-I-A (25 mg, 0.011 mmol), potassium carbonate (10 mg) and Pd / C (10%, 5 mg) to a 50 mL round-bottom flask, and then add MeOH (3 mL). The reaction system was purged with H2 three times and stirred under hydrogen for 2 hours. The reaction solution was filtered and concentrated to obtain MCPP-17-001-I-C (18 mg, crude product) as a white solid. LCMS: (ESI) m / z = 672.5 [M+2H-408] / 2 + ; 770.4 [M+2H-204] / 2 + .

[0170] Step 3: Dissolve the crude product MCPP-17-001-I-C obtained in the previous step in a mixture of water (2 mL) and acetonitrile (0.5 mL) (H2O / ACN). Sequentially add 5FAM-OSu (8 mg, 0.015 mmol) and saturated NaHCO3 solution (0.5 mL). Stir the mixture at room temperature for 1 hour. Directly separate and purify the reaction solution using a preparative chromatographic column to obtain MCPP-17-001-I (11.8 mg, yield of two-step reaction: 50.9%) as an orange solid. LCMS: (ESI) m / z = 1076.6 [M+2Na] / 2 + 。

[0171] HPLC preparative conditions: Preparative column: Gemini C18, 21.2 × 250 mm, 10 μm, 110 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increase from 12%B to 22%B within 20 minutes.

[0172] HPLC purity: >99% (214 nm), RT = 11.368 min.

[0173] HPLC purity analysis conditions A: 1) Mobile phase: A: water (containing 0.05% TFA); B: acetonitrile; Gradient (0.05% TFA). 2) Mobile phase gradient: 3 minutes of B, increase from 5%B to 65%B within 20 minutes. 3) Flow rate: 1.0 mL / min. 4) Column: XBridge peptide BEH column C18, 4.6 × 150 mm, 3.5 μm, 130 Å. 5) Column temperature: 60°C.

[0174] 5. Synthesis of MCPP-17-001-II

[0175]

[0176] Step 1: Use MCPP-17-001 (100 mg, 0.0736 mmol) and Gal-C5 (165 mg, 0.368 mmol, 5.0 equivalents) as raw materials, and according to the method of Step 1 of MCPP-17-001-I, obtain white solid MCPP-17-001-II-A (110 mg, yield: 64.7%). LCMS: (ESI) m / z = 1152.8 [M+2H] / 2 + ; 769.0 [M+3H] / 3 + 。

[0177] Step 2: According to the method of Step 2 of MCPP-17-001-I, obtain white solid MCPP-17-001-II-C (crude product). LCMS: (ESI) m / z = 897.0 [M+2H] / 2 + ; 919.0 [M+2Na] / 2 + ; 795.3 [M+2H-204] / 2 + ; 693.5 [M+2H-408] / 2 + 。

[0178] Step 3: According to the method of Step 3 of MCPP-17-001-I, obtain MCPP-17-001-II (9.6 mg, yield of two-step reaction 40.6%) as an orange solid. LCMS: (ESI) m / z = 1097.5 [M+2Na] / 2 + 。

[0179] HPLC preparation conditions: Preparation column: Gemini C18, 21.2 × 250 mm, 10 μm, 110 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increase from 8%B to 18%B within 20 minutes.

[0180] HPLC purity: 95.3% (214 nm), RT = 11.7 min.

[0181] HPLC purity analysis condition A.

[0182] 6. Synthesis of MCPP-17-002-I

[0183]

[0184] Step 1: Dissolve MCPP-17-002 (100 mg, 0.0777 mmol) in DMF (3 mL), add Gal-C4 (168 mg, 0.389 mmol, 5.0 equivalents), EDCI . HCl (75 mg, 0.389 mmol) and HOAt (53 mg, 0.389 mmol). Cool the mixture to 4 oC, DIPEA (133 μL, 0.777 mmol) was added, and the reaction was allowed to proceed at room temperature for 4 h. The reaction solution was directly purified by reversed-phase preparative chromatography to obtain MCPP-17-002-I-A (70 mg, yield: 41.2%) as a white solid. LCMS: (ESI) m / z = 1096.2 [M+2H] / 2 + ; 731.0 [M+3H] / 3 + .

[0185] HPLC preparative conditions: Preparative column: Xbridge C18, 19 × 250 mm, 10 μm, 130 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increased from 34%B to 44%B within 10 minutes.

[0186] Step 2: In a 50 mL round-bottom flask, MCPP-17-002-I-A (25 mg, 0.0114 mmol), potassium carbonate (10 mg) and Pd / C (10%, 5 mg) were successively added, and then MeOH (3 mL) was added. The reaction system was purged with H2 three times and stirred under hydrogen for 2 h. The reaction solution was filtered and concentrated to obtain MCPP-17-002-I-C (crude product) directly used for the next step. LCMS: (ESI) m / z = 839.5 [M+2H] / 2 + ; 738.0 [M+2H-204] / 2 + ; 851.0 [M+H+Na] / 2 + ;636.5 [M+H+Na-406] / 2 + .

[0187] Step 3: The crude product MCPP-17-002-I-C obtained in the previous step was dissolved in a mixture of water (2 mL) and acetonitrile (0.5 mL) (H2O / ACN), 5FAM-OSu (8 mg, 0.015 mmmol) and saturated NaHCO3 solution (0.5 mL) were successively added. The mixture was stirred at room temperature for 1 h. The reaction solution was directly separated and purified by preparative chromatography to obtain MCPP-17-002-I (13.7 mg, two-step reaction yield 59.0%) as an orange solid. LCMS: (ESI) m / z = 1019.5 [M+2H] / 2 + ;1041.1 [M+2Na] / 2 + ; 917.3 [M-204+2H] / 2 + .

[0188] HPLC Preparation Conditions: Preparation Column: Gemini C18, 21.2 × 250 mm, 10 μm, 110 Å; Mobile Phase: A: Water (containing 10 mmol / L NH4HCO3); B: Acetonitrile; Gradient: From 12% B to 22% B within 20 minutes.

[0189] HPLC Purity: 98.47% (214 nm), RT = 12.74 min

[0190] HPLC Purity Analysis Condition A.

[0191] 7. Synthesis of MCPP - 17 - 002 - II

[0192]

[0193] Step 1: Using MCPP - 17 - 002 (100 mg, 0.0777 mmol) and Gal - C5 (174 mg, 0.389 mmol, 5.0 equivalents) as raw materials, following the method of Step 1 of MCPP - 17 - 002 - I, white solid MCPP - 17 - 002 - II - A (72 mg, Yield: 41.6%) was obtained. LCMS: (ESI) m / z = 1117.1 [M + 2H] / 2 + ; 745.1 [M + 3H] / 3 + 。

[0194] Step 2: Following the method of Step 2 of MCPP - 17 - 002 - I, white solid MCPP - 17 - 002 - II - C (crude product) was obtained. LCMS: (ESI) m / z = 860.5 [M + 2H] / 2 + ; 871.7.0 [M + 2Na] / 2 + 。

[0195] Step 3: Following the method of Step 3 of MCPP - 17 - 002 - I, MCPP - 17 - 002 - II (14.1 mg, overall two - step reaction yield 60.5%) was obtained as an orange - yellow solid. LCMS: (ESI) m / z = 1062.1 [M + 2Na] / 2 + ; 1039.6 [M + 2H] / 2 + 。

[0196] HPLC preparation conditions: Preparation column: Gemini C18, 21.2 × 250 mm, 10 μm, 110 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increased from 12% B to 22% B within 20 minutes.

[0197] HPLC purity: 97.64% (214 nm), RT = 12.96 min

[0198] HPLC purity analysis condition A.

[0199] 8. Synthesis of MCPP-16-001-I

[0201] Step 1: Dissolve MCPP-16-001 (60 mg, 0.0446 mmol) in DMF (2 mL), add Gal-C4 (97 mg, 0.223 mmol, 5.0 equiv.), EDCI . HCl (43 mg, 0.223 mmol) and HOAt (30 mg, 0.223 mmol). Cool the mixture to 4 o °C, add DIPEA (76 μL, 0.446 mmol), and let it rise to room temperature and react for 4 hours. The reaction solution was directly purified by a reversed-phase preparative chromatographic column to obtain MCPP-16-001-I-A (60 mg, yield: 59.6%) as a white solid. LCMS: (ESI) m / z = 1125.2 [M+2H] / 2 + ; 960.5 [M+2H-330] / 2 + ; 750.5 [M+3H] / 3 + .

[0202] HPLC preparation conditions: Preparation column: Xbridge C18, 19 × 250 mm, 10 μm, 130 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: increased from 5% B to 65% B within 10 minutes.

[0203] Step 2: Sequentially add MCPP-16-001-I-A (23 mg, 0.01 mmol), potassium carbonate (10 mg) and Pd / C (10%, 5 mg) into a 50 mL round-bottom flask, and then add MeOH (3 mL). The reaction system is purged with H2 three times and stirred under hydrogen for 2 hours. The reaction solution is filtered and concentrated to obtain a white solid MCPP-16-001-I-C (crude product). LCMS: (ESI) m / z = 868.5 [M+2H] / 2 + ; 767.0 [M+2H-204] / 2 + ; 665.5 [M+2H-408] / 2 + 。

[0204] Step 3: Dissolve the crude product MCPP-16-001-I-C obtained in the previous step in a mixture of water (2 mL) and acetonitrile (0.5 mL) (H2O / ACN), and sequentially add 5FAM-OSu (8 mg, 0.015 mmol) and saturated NaHCO3 solution (0.5 mL). The mixture is stirred at room temperature for 1 hour. The reaction solution is directly separated and purified by a preparative chromatographic column to obtain MCPP-16-001-I (12.0 mg, yield of two-step reaction 57.3%) as an orange solid. LCMS: (ESI) m / z = 1047.5 [M+2H] / 2 + ; 1076.6 [M+2Na] / 2 + 。

[0205] HPLC preparative conditions: Preparative column: Gemini C18, 21.2 × 250 mm, 10 μm, 110 Å; Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; Gradient: from 5%B to 65%B within 10 minutes.

[0206] HPLC purity: 95.19% (214 nm), RT = 11.32 min.

[0207] HPLC purity analysis condition A.

[0208] Example 2. Study on the endocytosis activity of GalNAc ligand in hepatocytes

[0209] Flow cytometry was used to evaluate the uptake efficiency of the test product labeled with 5FAM fluorescence in human hepatoma cell line (HepG2).

[0210] HepG2 cells were purchased from ATCC (USA) and cultured in DMEM medium (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (ExCell Bio, South America), 100 units / mL penicillin and 100 units / mL streptomycin in an incubator at 37 °C with 5% CO2.

[0211] On day 0, the HepG2 cell suspension was adjusted to an appropriate density (1.5E+05 / well) and seeded into 48-well plates. On day 1, the diluted fluorescently labeled test compounds were added. The test compounds were diluted to different concentrations, 40 nM, 200 nM, and 1000 nM. The GalNAc ligand (L96-5FAM) was used as a positive control, while 5FAM was used as a negative control. After incubating the test compounds with the cells for 24 hours, all the liquid was removed, and the cells were washed twice with PBS and then collected. After fixing the digested cells, the corresponding fluorescence in HepG2 cells was detected by flow cytometry (BD FACSCanto™ II, Becton Dickinson, USA). The mean fluorescence intensity (MFI) of FAM in the test compounds was analyzed and compared with the mean fluorescence intensity of the positive compound (L96-5FAM). The MFI ratio = [MFI of the test compound] / [MFI of L96-FAM].

[0212] Table 2. Ratio of MFI of hepatocyte endocytosis of the cyclic peptide-based GalNAc ligands of the present invention to MFI of the positive compound (FAM fluorescence pathway, 24-hour time point)

[0213]

[0214] It can be seen that the cyclic peptide-based GalNAc ligands MCPP-17-001-I, MCPP-17-001-II, and MCPP-16-001-I provided by the present invention have comparable or improved endocytosis compared to the positive compound. In particular, MCPP-17-001-I shows a significant improvement compared to the positive compound at all tested concentrations, being at least 1.5 times that of the positive compound at 1000 nM and 200 nM concentrations, and more than 1.7 times that of the positive compound at 1000 nM concentration.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (IIIa) or (IIIb): (IIIa) (IIIb) wherein Z is selected from H, an amino protecting group, a small molecule drug moiety, a fluorescent dye moiety, and an oligonucleotide moiety.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the amino protecting group is selected from benzyloxycarbonyl, tert-butoxycarbonyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, and benzyl.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the fluorescent dye moiety is selected from 5FAM, FITC, Cy3, Cy5, Cy5.5, and Cy7.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the oligonucleotide moiety is ASO, mRNA, or siRNA.

5. A compound or a pharmaceutically acceptable salt thereof selected from the following structural formulas, (MCPP-17-001-I) (MCPP-17-001-II) (MCPP-16-001-I) (MCPP-17-001-I-A) (MCPP-17-001-I-C) (MCPP-17-001-II-A) (MCPP-17-001-II-C) (MCPP-16-001-I-A) (MCPP-16-001-I-C) (MCPP-17-001-I-siRNA) (MCPP-16-001-I-siRNA) Among them, Cbz represents benzyloxycarbonyl.

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