Carrier peptide, conjugate comprising carrier peptide, composition, preparation method, and use

Abstract

A new carrier peptide and a conjugate comprising the carrier peptide. The length of the carrier peptide is 5-30 amino acid residues, wherein the carrier peptide comprises one or more amino acid sequences I, the length of each amino acid sequence I is independently 5-7 amino acid residues, and each amino acid sequence I is consistent with at least five contiguous amino acid residues set forth in SEQ ID NO: 1: RSLGDTG (SEQ ID NO:1), where each capital letter independently represents an amino acid residue, and each amino acid residue is independently a natural or modified amino acid residue. The carrier peptide can be covalently linked to a functional group to form a conjugate, thereby effectively reaching a target tissue or a target organ in a central nervous system, and exerting a therapeutic and / or diagnostic effect.

Description

A carrier peptide, a conjugate containing the carrier peptide, a composition, and a preparation method and use thereof Technical Field

[0001] The present disclosure relates to a carrier peptide, and also relates to a carrier peptide conjugate containing the carrier peptide, and compositions containing the conjugates, as well as preparation methods and uses thereof. Background Art

[0002] The central nervous system (CNS) is the main part of the nervous system, including the spinal cord located in the spinal canal and the brain located in the cranial cavity. It is responsible for controlling the autonomic nervous system to regulate basic physiological activities such as breathing, heartbeat, body temperature, internal organs, and activities. There are many diseases related to the central nervous system, including brain lesions, spinal cord lesions, etc., such as Parkinson's disease, multiple system atrophy, Alzheimer's disease, etc., and a major challenge in the development of drugs for the treatment of central nervous system-related diseases is the lack of a means to effectively administer drugs to target organs and target tissues in the central nervous system. Many drug molecules that have shown excellent pharmaceutical activity in preclinical pharmaceutical studies are difficult to effectively reach specific target organs or target tissues due to the lack of effective delivery vectors. Therefore, there is still a significant practical need in the art for delivery vectors that can effectively deliver drug molecules to specific target organs or target tissues, especially the central nervous system. Summary of the Invention

[0003] The present invention provides a carrier peptide that can be conjugated with a variety of functional groups to form a carrier peptide conjugate with high delivery efficiency, which can effectively deliver the functional groups to target organs or target tissues in the central nervous system.

[0004] In one aspect, the present disclosure provides a carrier peptide having a length of 5-30 amino acid residues, wherein the carrier peptide comprises one or more segments of amino acid sequence I, each segment of amino acid sequence I independently having a length of 5-7 amino acid residues, and each segment of amino acid sequence I being identical to at least 5 consecutive amino acid residues in the sequence shown in SEQ ID NO: 1:

[0005] RSLGDTG (SEQ ID NO: 1);

[0006] Each capital letter represents an amino acid residue, and each amino acid residue is independently a natural or modified amino acid residue.

[0007] On the other hand, the present disclosure also provides an active carrier peptide, which comprises one or more carrier peptide groups and one or more reactive groups, wherein the carrier peptide group is formed by removing one or more atoms from the carrier peptide provided by the present disclosure, and each of the reactive groups is covalently linked to an amino acid residue in the carrier peptide group, and the reactive group comprises one or more functional groups or protected functional groups, which are capable of undergoing a conjugation reaction, and the conjugation reaction refers to an addition reaction, coupling reaction or substitution reaction capable of forming a covalent connection.

[0008] On the other hand, the present disclosure also provides a carrier peptide conjugate, which contains one or more carrier peptide groups and one or more functional groups, wherein the carrier peptide group is formed by removing one or more atoms from the carrier peptide provided by the present disclosure, and each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group, a functional oligonucleotide group and a delivery aid group; each of the carrier peptide groups is connected to at least one of the functional groups or another carrier peptide group through a connecting group, and each functional group is connected to at least one of the carrier peptide groups or another functional group through a connecting group.

[0009] In yet another aspect, the present disclosure further provides use of the carrier peptide of the present disclosure in delivering functional molecules, wherein the functional molecules are selected from one or more of diagnostic agents, small molecule therapeutic agents, and functional oligonucleotides.

[0010] In yet another aspect, the present disclosure further provides a pharmaceutical composition comprising the carrier peptide conjugate of the present disclosure, one or more of its pharmaceutically acceptable salts, metabolites or prodrugs, and a pharmaceutically acceptable carrier.

[0011] In another aspect, the present disclosure also provides use of the conjugate of the present disclosure, one or more of its pharmaceutically acceptable salts, metabolites or prodrugs and / or the pharmaceutical composition of the present disclosure in the preparation of a medicament for diagnosing, treating and / or preventing diseases related to the central nervous system.

[0012] In yet another aspect, the present disclosure also provides a method for diagnosing, treating and / or preventing diseases related to the central nervous system, the method comprising administering to a subject an effective amount of the conjugate of the present disclosure, one or more of its pharmaceutically acceptable salts, metabolites or prodrugs, and / or the pharmaceutical composition of the present disclosure.

[0013] In addition, the present disclosure also provides a kit comprising the conjugate of the present disclosure, one or more of its pharmaceutically acceptable salts, metabolites or prodrugs, and / or the pharmaceutical composition of the present disclosure.

[0014] Incorporated by reference

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Beneficial effects

[0016] The carrier peptide provided by the present disclosure can be conjugated with a variety of functional groups to form carrier peptide conjugates with high delivery efficiency, and can effectively deliver a variety of functional groups to target organs or target tissues, such as the central nervous system.

[0017] When the functional groups contained in the carrier peptide conjugates provided by the present disclosure include small molecule therapeutic agent groups and / or functional oligonucleotide groups, the conjugates and / or pharmaceutical compositions containing the carrier peptides provided by the present disclosure can be efficiently targeted to the central nervous system, have high delivery efficiency in the central nervous system, exhibit high pharmaceutical activity, and have good stability. For example, when administered intracerebroventricularly, under the same dosage conditions, the carrier peptide siRNA conjugates disclosed by the present disclosure showed a high inhibition rate on the mRNA expression of the SOD1 gene in different regions of the mouse brain, wherein the inhibition rate in the cortex on the administration side was as high as 74.1%, which was 50.30% higher than the inhibition rate of siRNA without carrier peptide groups. The inhibition rates in the hippocampus and cerebellum on the administration side exceeded 50%, reaching 63.4% and 51.4%, respectively, which were 80.11% and 18.4% higher than the inhibition rates of siRNA without carrier peptide groups, respectively. Even in the contralateral brain region, the carrier peptide conjugate of the present disclosure demonstrated a high inhibition rate of SOD1 mRNA expression, with inhibition rates exceeding 40% in the contralateral cortex, contralateral hippocampus, and cerebellum, reaching 69.2%, 53.5%, and 41.4%, respectively. These inhibition rates were significantly higher than those of siRNA without a carrier peptide group, representing 63.15%, 123.85%, and 6.1%, respectively, demonstrating beneficial SOD1 mRNA inhibition activity. For another example, when administered intrathecally, under the same dosage conditions, the conjugate of the present disclosure showed a very high inhibition rate on SOD1 mRNA in different regions of the mouse brain, among which the inhibition rate in the striatum was as high as 89.4%, the inhibition rates in the right parietal cortex, hippocampus, thalamus and cerebellum all exceeded 75%, and the inhibition rates in the lumbar and thoracic spinal cords could reach more than 86%, indicating that the conjugate provided by the present disclosure connected with the carrier peptide of the present disclosure can specifically target different regions of the mouse brain and has a higher SOD1 mRNA inhibition rate than the reference siRNA A not containing the carrier peptide. For another example, when administered intrathecally, under the same dosage conditions, the conjugate of the present invention showed a very high inhibition rate on the mRNA expression of the APOE4 gene in different regions of the mouse brain, among which the inhibition rate in the lumbar and cervical segments of the spinal cord was as high as 95.3%, the inhibition rate in the thoracic segment of the spinal cord was as high as 94.7%, the inhibition rate in the myelin sheath was as high as 89.3%, the inhibition rate in the thalamus was as high as 87.6%, the inhibition rate in the hippocampus was as high as 80.7%, and the inhibition rates in the striatum and cerebellum were both over 70%, indicating that the conjugate provided by the present invention and connected with the carrier peptide of the present invention can specifically target different regions of the mouse brain and has a high APOE4 mRNA inhibition rate.

[0018] For another example, when the functional groups contained in the conjugates of the carrier peptides provided by the present disclosure include diagnostic agent groups, the conjugates and / or pharmaceutical compositions provided by the present disclosure can efficiently target the central nervous system and can display obvious and accurate diagnostic signals, thereby obtaining efficient and accurate diagnostic results.

[0019] For another example, when the functional group contained in the conjugate of the carrier peptide provided by the present disclosure includes a delivery aid group, the bioavailability can be further increased and the delivery efficiency can be improved. For example, the conjugate provided by the present disclosure, which is conjugated with a delivery aid group and an siRNA group, shows a high inhibition rate on APOE4 mRNA in different regions of the mouse brain, wherein the inhibition rate in the thoracic spinal cord is as high as 95.9%, the inhibition rate in the cervical spinal cord is as high as 97.4%, the inhibition rate in the striatum is as high as 80.0%, and the inhibition rate in the hippocampus is 71.7%, indicating that the conjugate of the carrier peptide provided by the present disclosure, which is simultaneously connected with a delivery aid group and an siRNA group, can specifically target different regions of the mouse brain, and still has a high APOE4 mRNA inhibition rate within a 29-day experimental period, which can further improve the delivery efficiency.

[0020] This shows that the carrier peptide provided by the present disclosure can efficiently deliver functional groups to the central nervous system, and the carrier peptide conjugate containing the carrier peptide group formed by the carrier peptide of the present disclosure can effectively treat and / or prevent diseases or symptoms related to the central nervous system in vivo, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a bar graph showing the inhibition rates of SOD1 mRNA in different regions of the mouse brain after administration of different drugs in Experimental Example 3, wherein the abscissa represents the blank group, the reference group, and the test group compounds, and the ordinate represents the relative expression levels of SOD1 mRNA in the cerebellum, thalamus, striatum, hippocampus, and parietal cortex. DETAILED DESCRIPTION

[0022] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0023] In the present disclosure, SOD1 mRNA refers to the mRNA with Genbank Accession No. NM_011434.2 or NM_000454.5; APOE4 mRNA refers to the mRNA with the sequence shown in Genbank Accession No. NM_000041.4; RPTOR mRNA refers to the sequence shown in Genbank Accession No. NM_020761.3.

[0024] definition

[0025] In the above and below of this article, a polypeptide sequence refers to a polypeptide sequence formed by a dehydration condensation reaction of multiple amino acid residues at the carboxyl position and the amino position. If not otherwise specified, the amino terminus of the polypeptide sequence refers to the end of the amino group in the polypeptide formed by condensation that does not undergo the above-mentioned dehydration condensation reaction, and the carboxyl terminus of the polypeptide sequence refers to the end of the carboxyl group in the polypeptide formed by condensation that does not undergo the above-mentioned dehydration condensation reaction. Unless otherwise specified, in the polypeptide sequence of the present invention, each amino acid residue is connected from left to right via a peptide bond (i.e., an amide bond) from the amino terminus (N terminus) to the carboxyl terminus (C terminus). Unless otherwise specified, in a peptide sequence represented by a sequence of uppercase letters of abbreviations, the connection between the amino acid residue represented by the letter on the left and the amino acid residue represented by the letter on the right is that the carboxyl group of the amino acid residue represented by the letter on the left forms a peptide bond with the amino group of the amino acid residue represented by the letter on the right. For example, in the polypeptide sequence LPILTPS, the connection relationship between the first letter L and the second letter P is that the carboxyl group of the leucine represented by L forms a peptide bond with the amino group of the proline represented by P.

[0026] Throughout this document, unless otherwise specified, each letter in a polypeptide sequence represents an amino acid monomer or amino acid residue. An amino acid monomer refers to an amino acid in which the active amino group or active carboxyl group is protected by a protecting group. An amino acid residue refers to a group formed by losing a hydrogen atom from the amino group of an amino acid and / or losing a hydroxyl group from the carboxyl group of an amino acid. For simplicity, depending on the context, when describing preparation methods, amino acids or corresponding capital letters refer to amino acid monomers, and when describing amino acid sequences, amino acids or corresponding capital letters refer to amino acid residues. Wherein, according to the context, G represents a glycine monomer or represents a glycine residue in a polypeptide sequence, A represents an alanine monomer or represents an alanine residue in a polypeptide sequence, V represents a valine monomer or represents a valine residue in a polypeptide sequence, L represents a leucine monomer or represents a leucine residue in a polypeptide sequence, I represents an isoleucine monomer or represents an isoleucine residue in a polypeptide sequence, P represents a proline monomer or represents a proline residue in a polypeptide sequence, F represents a phenylalanine monomer or represents a phenylalanine residue in a polypeptide sequence, Y represents a tyrosine monomer or represents a tyrosine residue in a polypeptide sequence, W represents a tryptophan monomer or represents a tryptophan residue in a polypeptide sequence, S represents a serine monomer or represents a serine residue in a polypeptide sequence. wherein the amino acid residue is a threonine monomer or a threonine residue in a polypeptide sequence, T represents a threonine monomer or a threonine residue in a polypeptide sequence, C represents a cysteine ​​monomer or a cysteine ​​residue in a polypeptide sequence, M represents a methionine monomer or a methionine residue in a polypeptide sequence, N represents an asparagine monomer or an asparagine residue in a polypeptide sequence, Q represents a glutamine monomer or a glutamine residue in a polypeptide sequence, D represents an aspartic acid monomer or an aspartic acid residue in a polypeptide sequence, E represents a glutamic acid monomer or a glutamic acid residue in a polypeptide sequence, K represents a lysine monomer or a lysine residue in a polypeptide sequence, R represents an arginine monomer or an arginine residue in a polypeptide sequence, and H represents a histidine monomer or a histidine residue in a polypeptide sequence.

[0027] Various protecting groups can be used in the present disclosure. In general, protecting groups make chemical functional groups insensitive to specific reaction conditions, and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, which are incorporated herein by reference. In some embodiments, protecting groups are stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, examples of protecting groups for amino groups in carrier peptides, active carrier peptides or carrier peptide groups herein include but are not limited to C1-C6 alkyl acyl, tert-butyloxycarbonyl (Boc), tert-butyl fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, examples of protecting groups for carboxyl groups in carrier peptides, active carrier peptides, or carrier peptide groups herein include, but are not limited to, amino, C1-C6 alkylamino, or C1-C6 alkoxy. In some embodiments, non-exclusive examples of hydroxyl protecting groups in nucleotide or oligonucleotide groups herein include dimethoxytrityl (DMTr), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups in nucleotide or oligonucleotide groups herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0028] In the above and below, unless otherwise specified, in the nucleotide sequence, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a thiophosphate group; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide. In some embodiments, P1 is VP, Ps, or P representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a thiophosphate-modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0029] In the above and below, the term "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by fluorine, and a "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by a non-fluorinated group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), and acyclic nucleotides. The term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0030] In the context of this article, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0031] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0032] As used above and below, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0033] In the above and below, particularly when describing the preparation method of double-stranded oligonucleotide, pharmaceutical composition or the carrier peptide conjugate containing double-stranded oligonucleotide, unless otherwise specified, described nucleoside monomer (nucleoside monomer) refers to, according to the kind and order of nucleotide in the double-stranded oligonucleotide or the carrier peptide conjugate of desire preparation, the modification used in phosphoramidite solid phase synthesis or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, RNA phosphoramidites is also referred to as Nucleoside phosphoramidites sometimes).Phosphoramidite solid phase synthesis is the method used in RNA synthesis well known to those skilled in the art.The nucleoside monomer used in the disclosure all can be commercially available.

[0034] It should be noted that, throughout this document, A in a polypeptide sequence represents alanine, while A in a nucleic acid molecule represents the purine base adenine; T in a polypeptide sequence represents threonine, while T in a nucleic acid molecule represents thymine; G in a polypeptide sequence represents glycine, while G in a nucleic acid molecule represents guanine; and C in a polypeptide sequence represents cysteine, while C in a nucleic acid molecule represents cytosine. Those skilled in the art are familiar with the different meanings of A, T, G, and C in polypeptide sequences and nucleic acid molecules, respectively, and should not cause confusion.

[0035] The "carrier peptide" described in the present disclosure refers to a polypeptide composed of a certain number of amino acid residues that can be connected to a functional group through a covalent bond or a linking group to form a conjugate, thereby achieving the delivery of the functional group. The "carrier peptide group" used in the present disclosure is a group formed by removing one or more atoms from the carrier peptide of the present disclosure. It will be understood by those skilled in the art that the removal of the one or more atoms will not destroy the delivery effect of the carrier peptide and is a necessary technical means to achieve the delivery of the functional group by the carrier peptide.

[0036] In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each of which has a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Further, "carrier peptide conjugate" refers to a compound formed by covalently linking one or more carrier peptides and a chemical moiety with a specific function. Carrier peptide conjugates should be understood as a general term for multiple carrier peptide conjugates or a specific one or more carrier peptide conjugates shown in a chemical formula, depending on the context. As used in the present disclosure, the "carrier peptide conjugate" comprises a carrier peptide group and a functional group portion, wherein the functional group refers to a chemical moiety formed by removing one or more atoms from a drug molecule. Within the scope of the present disclosure, each of the functional groups is independently selected from one of a diagnostic agent group formed by a diagnostic agent molecule, a therapeutic agent group formed by a small molecule therapeutic agent, an oligonucleotide group formed by a functional oligonucleotide, and a delivery aid group formed by a delivery aid. It will be understood by those skilled in the art that the removal of the above-mentioned one or more atoms will not destroy the activity or stability of the drug molecule. For example, when the functional group is an oligonucleotide group, the oligonucleotide group can be a chemical part formed by removing the hydrogen atom in the phosphate bond of the functional oligonucleotide molecule, or a chemical part formed by removing the hydrogen atom in the 5' hydroxyl group of the 5' terminal nucleotide of the sense chain or antisense chain of the functional oligonucleotide molecule, or a chemical part formed by removing the hydrogen atom in the 3' hydroxyl group of the 3' terminal nucleotide of the sense chain or antisense chain of the functional oligonucleotide molecule.

[0037] In the above or below, "substituted" or "substituted" groups include but are not limited to substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic, substituted heteroaliphatic, substituted acyl, substituted aryl or substituted heteroaryl. Wherein, unless otherwise specified, a "substituted" or "substituted" group refers to a group in which the hydrogen atoms in the group are replaced by one or more substituents. For example, "substituted alkoxy" refers to a group in which one or more hydrogen atoms in an alkoxy group are replaced by a substituent. It will be understood by those skilled in the art that various substituents may be contained in the compounds that can be used in the present disclosure, as long as the introduction of the substituent does not affect the function of the present disclosure and can achieve the purpose of the present disclosure, it can be used in the present disclosure. In some embodiments, the substituent is selected from the group consisting of the following groups: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 Alkylphenyl), -CN, -NO2, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 In some embodiments, the substituent is one of C1-C3 alkyl, C6-C8 aryl, -OC1-C3 alkyl, -OC1-C3 alkylphenyl, halogen, -OH, -NH2, -CN or -NO2. It will be understood by those skilled in the art that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically infeasible and / or inherently unstable.

[0038] The term "subject," as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any type of poultry.

[0039] As used herein, "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0040] In some embodiments, the carrier peptide conjugates of the present invention contain free carboxyl or amino groups. These carboxyl or amino groups can form salts with corresponding bases or acids. In some embodiments, the carrier peptide conjugates of the present invention also contain oligonucleotide groups. In some embodiments, the oligonucleotide group is an siRNA group. In some embodiments, the pharmaceutically acceptable salt of the carrier peptide conjugate containing the siRNA group is a sodium salt of the carrier peptide conjugate in which the hydroxyl hydrogen ions in all phosphate groups in the conjugate are replaced by sodium ions. In these carrier peptide conjugates containing oligonucleotide groups, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond. The non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge and can exist in the form of a hydroxyl or sulfhydryl group. The hydrogen ions in the hydroxyl or sulfhydryl group can also be partially or completely replaced by cations. Therefore, in some embodiments, the present disclosure also provides pharmaceutically acceptable salts of the carrier peptide conjugates. The pharmaceutically acceptable salt may be a salt formed by a carboxylate ion formed when a carboxyl group loses a hydrogen ion and a cation, or a salt formed by an organic ammonium cation formed when an amino group obtains a hydrogen ion and an anion, and / or a salt formed by a phosphate group or a thiophosphate group in an oligonucleotide group and a cation. The cation may be any cation, such as a metal cation, an ammonium ion NH4 + , one of the organic ammonium cations. In order to improve solubility, in some embodiments, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion can be K + and / or Na + , the cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. The anion can be any anion, such as one or more of a chloride ion and an organic acid ion. In some embodiments, the anion can be, for example, a chloride ion or an acetate anion. Therefore, the carrier peptide conjugate described in the present disclosure can exist at least partially in the form of a salt. In some embodiments, in the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure, the carrier peptide group does not contain a free carboxyl group or amino group, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphodiester bond in the oligonucleotide group is at least partially bound to a sodium ion, and the pharmaceutically acceptable salt of the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure refers to a water-soluble salt of the conjugate, such as one of an alkali metal salt or an ammonium salt. In some embodiments, the pharmaceutically acceptable salt of the carrier peptide conjugate refers to a sodium salt or a partial sodium salt of the conjugate.

[0041] Unless otherwise specified, in the context, when referring solely to a carrier peptide conjugate comprising an oligonucleotide as described in the applications or methods provided by the present disclosure, including but not limited to a carrier peptide conjugate represented by any structural formula as described in the applications or methods provided by the present disclosure, the pharmaceutically acceptable salt of the conjugate is also referred to according to the context.

[0042] The "functional oligonucleotide" as used herein refers to an oligonucleotide, including single-stranded or double-stranded oligonucleotides, that has the function of upregulating or downregulating the level of a target mRNA expressed by a target gene. Functional single-stranded oligonucleotides include single-stranded ribonucleic acid (ssRNA), single-stranded antisense oligonucleotides (ASOs), or microRNAs (miRNAs); functional double-stranded oligonucleotides include small activating RNAs (saRNAs) that can upregulate the level of a target mRNA expressed by a target gene, or small interfering RNAs (siRNAs) that can downregulate the level of a target mRNA expressed by a target gene.

[0043] Carrier peptides disclosed herein

[0044] In one aspect, the present disclosure provides a carrier peptide having a length of 5-30 amino acid residues, wherein the carrier peptide contains one or more segments of amino acid sequence I, each segment of amino acid sequence I having a length of 5-7 amino acid residues, and each segment of amino acid sequence I is identical to at least 5 consecutive amino acid residues in the sequence shown in SEQ ID NO: 1:

[0045] RSLGDTG (SEQ ID NO: 1);

[0046] Each capital letter independently represents an amino acid residue, and each amino acid residue is independently a natural or modified amino acid residue.

[0047] The carrier peptide disclosed herein can exist in a linear or cyclic form. The inventors of the present invention have found that for the sequence shown in SEQ ID NO: 1, when its two ends are truncated or extended, some truncated or extended sequences can still retain the delivery activity of the carrier peptide, for example, when the amino acid sequence I is a sequence of 5-6 amino acid residues formed by truncating 1 or 2 amino acid residues at both ends of the sequence shown in SEQ ID NO: 1, or an amino acid sequence of 8-20 amino acid residues formed by connecting one end and / or both ends of the sequence shown in SEQ ID NO: 1 and more extended amino acid residues, the carrier peptide still has excellent delivery activity, and the extended amino acid residues can be selected from natural or modified amino acid residues. It is well known to those skilled in the art that such truncated or extended amino acid sequence I will not significantly affect the delivery activity of the carrier peptide. In some embodiments, the length of the carrier peptide is 5-20 amino acid residues. In some implementations, the length of the carrier peptide is 7-20, 7-15, 7-12 or 7-10 amino acid residues. In some embodiments, the carrier peptide further comprises other amino acid residues in addition to the amino acid sequence I. In some embodiments, the carrier peptide comprises a peptide portion consisting of the amino acid sequence I. In some embodiments, the carrier peptide consists only of one or more amino acid sequences I.

[0048] In some embodiments, the carrier peptide contains 1-2 segments of amino acid sequence I, and the length of the carrier peptide is 5-20 amino acid residues.

[0049] In some embodiments, the composition of the carrier peptide can be expressed as (M A N A ) n0 Z A , where each M A and Z A Same or different, respectively, refers to an amino acid sequence consisting of 0-3 amino acid residues, each N A Independently refers to the amino acid sequence I, n0 is an integer from 1 to 3, and two adjacent amino acid sequences are covalently linked by peptide bonds. In some embodiments, the composition of the carrier peptide can be expressed as M A N A Z A 、M A N A M A N A Z A or M A N A M A N A M A N A ZA .

[0050] In some embodiments, each M A and / or Z A In some embodiments, each M A and Z A The same or different, each independently represents an amino acid residue. A and Z A All represent the same amino acid residue. In some embodiments, each M A and Z A All are cysteine ​​residues.

[0051] In some embodiments, each amino acid sequence I independently has a sequence as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 144-148:

[0052] RSLGDTG (SEQ ID NO: 1);

[0053] RSLGD (SEQ ID NO: 144);

[0054] RSLGDT (SEQ ID NO: 145);

[0055] LGDTG (SEQ ID NO: 146);

[0056] SLGDTG (SEQ ID NO: 147);

[0057] SLGDT (SEQ ID NO: 148).

[0058] In some embodiments, each amino acid sequence I has the sequence shown in SEQ ID NO:1.

[0059] In some embodiments, the carrier peptide has a sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 144-148, SEQ ID NO: 152, or SEQ ID NO: 153:

[0060] RSLGDTG (SEQ ID NO: 1);

[0061] RSLGD (SEQ ID NO: 144);

[0062] RSLGDT (SEQ ID NO: 145);

[0063] LGDTG (SEQ ID NO: 146);

[0064] SLGDTG (SEQ ID NO: 147);

[0065] SLGDT (SEQ ID NO: 148);

[0066] KRSLGDTG (SEQ ID NO: 152);

[0067] CRSLGDTGCRSLGDTGC (SEQ ID NO: 153).

[0068] In some embodiments, the carrier peptide disclosed herein consists of only one nucleotide sequence I. In some embodiments, the carrier peptide disclosed herein has a sequence as shown in SEQ ID NO: 1.

[0069] In some embodiments, the carrier peptide described in the present disclosure exists in a cyclic form (hereinafter referred to as "cyclic peptide"). In some embodiments, the amino terminus and the carboxyl terminus of the carrier peptide are further connected to a cyclizing group L, respectively. c1 and L c2 , the L c1 and L c2 The carrier peptide is connected by covalent bonds to form a cyclic peptide. c1 and L c2 By forming an amide bond, a thioether bond or a disulfide bond. In some embodiments, the L c1 and L c2 are all cysteine ​​residues, and the two cysteine ​​residues are connected by a disulfide bond through a sulfhydryl group. In some embodiments, the side chains of any non-adjacent amino acid residues in the carrier peptide are connected to each other, so that the carrier peptide forms a cyclic peptide. In some embodiments, the multiple amino acid residues whose side chains are connected to each other are multiple amino acid residues in amino acid sequence I. In some embodiments, the number of amino acid residues whose side chains are connected to each other is 2-3. In some embodiments, the two amino acid residues whose side chains are connected to each other are separated by 2-5 amino acid residues.

[0070] The multiple amino acid residues connected to each other in the carrier peptide are multiple amino acid residues in amino acid sequence 1; the number of the amino acid residues connected to each other is 2-3. In some embodiments, the interval between two amino acid residues connected to each other in the carrier peptide is 2-5 amino acid residues.

[0071] In some embodiments, the side chains of the multiple amino acid residues connected to each other by side chains are connected by amide bonds, thioether bonds, disulfide bonds or linking groups. In some embodiments, the multiple amino acid residues connected to each other by side chains can be connected by amide bonds or by linking groups to form a cyclic peptide. In some embodiments, the multiple amino acid residues connected to each other by side chains undergo amidation reaction between the amino groups or carboxyl groups contained in the side chains to form amide bonds, thereby forming a cyclic peptide structure. Alternatively, in some embodiments, each of the linking groups is independently selected from C4-C 12 In some embodiments, each of the linking groups is independently selected from C6-C 10 In some embodiments, at least one or each of the linking groups is a 4-octenylene group.

[0072] In some embodiments, the carrier peptide has an amino acid sequence as shown in SEQ ID NO: 152: KRSLGDTG (SEQ ID NO: 152),

[0073] Here, the side chains of the lysine residue represented by K and the aspartic acid residue represented by D are linked to each other.

[0074] In some embodiments, the carrier peptide disclosed herein has a structure as shown in formula (21):

[0075] Wherein, the amino terminal amino group of the carrier peptide is optionally protected by an amino protecting group; the carboxyl terminal carboxyl group is optionally protected by a carboxyl protecting group. In some embodiments, the amino protecting group is a C1-C3 alkyl acyl group, optionally an acetyl group. In some embodiments, the carboxyl protecting group is an amino group or a C1-C3 alkylamino group, optionally an amino group. In some embodiments, the carrier peptide disclosed herein is a carrier peptide obtained by reacting the amino terminal amino group in the structure of formula (21) to form an acetamide group and reacting the carboxyl group to form an amide group.

[0076] In some embodiments, the carrier peptide has a structure as shown in the following formula (22):

[0077] Wherein, the two amino acid residues whose side chains are interconnected are amino acid residues formed by replacing the α hydrogen atom of the glycine residue with a methyl group; and the amino terminal amino group of the carrier peptide is optionally protected by an amino protecting group; and the carboxyl terminal carboxyl group is optionally protected by a carboxyl protecting group. In some embodiments, the amino protecting group is a C1-C3 alkyl acyl group, optionally an acetyl group. In some embodiments, the carboxyl protecting group is an amino group or a C1-C3 alkylamino group, optionally an amino group. In some embodiments, the carrier peptide disclosed herein is a carrier peptide obtained by reacting the amino terminal amino group in the structure of formula (22) to form an acetamide group and reacting the carboxyl group to form an amide group.

[0078] To save costs and facilitate synthesis, in some embodiments, each amino acid residue in the carrier peptide is a natural amino acid residue. In some embodiments, to achieve better stability or delivery efficiency, at least one amino acid residue in the carrier peptide is a modified amino acid residue. In some embodiments, at least 30%, 50%, 70%, 80%, or 90% of the amino acid residues in the carrier peptide are modified amino acid residues. In some embodiments, all amino acid residues in the carrier peptide are modified amino acid residues.

[0079] In the context of the present disclosure, the "modified amino acid residues" include constructing the carrier peptides of the present disclosure by replacing natural L-amino acid residues with D-amino acid residues. Alternatively, natural L-amino acid residues are substituted with amino acid residue mimetics, which include structural analogs of amino acid residues, such as salts or esters of natural amino acid residues. In addition, the carboxyl terminus of the polypeptide can be a carboxyl group or an amide group obtained by amidation of a carboxyl group, or other groups generated by incorporating one of the above amino acid residue mimetics; the amino terminus of the polypeptide can be an amino group or an amide group obtained by amidation of an amino group, or other groups generated by incorporating one of the above amino acid residue mimetics. In addition, one or more natural peptide bonds in the above peptides can be replaced by any one of the peptide bond replacement groups, including but not limited to: sulfonamide, reverse amide, aminooxy-containing bonds, ester groups, alkyl ketone groups, α,α-difluoroketone groups, α-fluoroketone groups, and peptide-like bonds (N-alkylated glycylamide bonds). In addition, the modified amino acid residue can be an amino acid residue with a modified side chain, for example, one of a 4-fluorophenylalanine residue, a 4-isoleic acid residue, a 3-aminoproline residue, a 2-nitrotyrosine residue, or an N-alkylhistidine residue; or a β-branched amino acid residue or a β-branched amino acid residue mimetic having a chirality opposite to the natural chirality at the β-side chain carbon atom, such as an allo-threonine residue, an allo-isoleucine residue, and derivatives thereof. Representative modified amino acid residues are disclosed in Baran et al., Biochemistry, 2017, 56(30):3863-3873. and Mehta et al., Tetrahedron Letters, 2017, 58(14):1357-1372, each of which is incorporated herein by reference in its entirety. In some embodiments, at least one modified amino acid residue in the carrier peptide of the present disclosure is a D-amino acid residue. In some embodiments, all modified amino acid residues are D-amino acid residues.

[0080] In some embodiments, at least one amino acid residue is a modified amino acid residue. The modified amino acid residue is an amino acid residue with a modified side chain, wherein the modifying group is selected from C1-C8 hydrocarbon group, substituted C1-C8 hydrocarbon group, C3-C 10 Cycloalkyl, substituted C3-C 10 In some embodiments, the modifying group is selected from one or more of a C1-C3 hydrocarbon group. In some embodiments, the modifying group is one or more of a methyl group, an ethyl group, or a propyl group. In some embodiments, the modifying group is a methyl group. In some specific embodiments, the two amino acid residues connected to each other in the carrier peptide are amino acid residues whose side chains contain a methyl modifying group.

[0081] In some embodiments, the side chains of any three non-adjacent amino acid residues in the carrier peptide are connected to each other through a linker group, so that the carrier peptide forms a bicyclic peptide, and the linker group has a structure as shown in formula (231):

[0082] Among them, A L Selected from trivalent C1-C8 straight or branched alkyl, trivalent C3-C 10 Cycloalkyl, trivalent C3-C 10 Heterocyclic group, trivalent C6-C 10 Aryl and trivalent C5-C 10 One of the heteroaryl groups, B L1 、B L2 and B L3 Each is independently selected from one of C1-C8 alkylenecarbonyl, substituted C1-C8 alkylenecarbonyl, C1-C8 alkylene and substituted C1-C8 alkylene, represents the site of attachment to which a group is covalently attached.

[0083] In some embodiments, A L One selected from trivalent C3-C6 nitrogen-containing heterocyclic groups, B L1 、B L2 and B L3 Each is independently selected from one of C2-C4 alkylene carbonyl groups. L is a trivalent 1,3,5-triazine group, B L1 、B L2 and B L3 All are -CH2CH2CO-.

[0084] In some embodiments, the connecting structure has a structure shown in the following formula (232):

[0085] In some embodiments, each of the three amino acid residues in the carrier peptide is bound to the B L1 、B L2 or B L3 Covalently linked to form a bicyclic peptide. A and / or Z A In some embodiments, at least one or each of the three amino acid residues is M A or Z AIn some embodiments, each of the three amino acid residues is the same or different. In some embodiments, the three amino acid residues are all cysteine ​​residues, and each cysteine ​​residue is independently covalently linked to the linking group via an amino group, a carboxyl group, or a sulfhydryl group. In some embodiments, each cysteine ​​residue is independently covalently linked to the linking group via a sulfhydryl group.

[0086] In some embodiments, the carrier peptide has an amino acid sequence as shown in SEQ ID NO: 153: CRSLGDTGCRSLGDTGC (SEQ ID NO: 153), wherein the three cysteine ​​residues represented by C are independently connected to the B through the sulfhydryl group on the side chain. L1 、B L2 or B L3 connect.

[0087] In some embodiments, the bicyclic amino acid sequence has a structure as shown in the following formula (23):

[0088] Synthesis of carrier peptides disclosed herein

[0089] Those skilled in the art can prepare the carrier peptide of the present disclosure by any suitable technical means. In some embodiments, the carrier peptide of the present disclosure can be obtained by connecting amino acid monomers one by one according to the amino acid sequence of the disclosed carrier peptide under solid-phase synthesis conditions, starting from a solid-phase carrier, and the carrier peptide contains an amino acid sequence I, which is consistent with at least 5 consecutive amino acid sequences in the sequence shown in SEQ ID NO: 1: RSLGDTG (SEQ ID NO: 1); in the carrier peptide, each amino acid residue is independently a natural or modified amino acid residue. In some embodiments, the synthesis also includes appropriate separation and purification steps. Solid-phase polypeptide synthesis methods are conventional technical means in the field, and mature commercial custom synthesis services are already available. In some embodiments, the carrier peptide is obtained through commercial ordering.

[0090] Those skilled in the art can prepare the cyclic peptides of the present disclosure by any suitable technical means. In some embodiments, the cyclic peptide can be prepared by coupling cyclization of an uncyclized linear peptide. In some embodiments, the reaction conditions of the coupling cyclization include contacting the uncyclized linear peptide with a coupling agent in an organic solvent under the reaction conditions of the coupling cyclization, and separating to obtain the cyclic peptide. The organic solvent can be a commonly used solvent for cyclization reactions, such as DMF, DCM or DIC, and the amount of the organic solvent can be, for example, 20-100 L / mol. In some embodiments, the multiple amino acid residues whose side chains are linked to each other are linked via amide bonds, and the coupling cyclization reaction conditions are amidation reaction conditions. The coupling agent can be, for example, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the molar ratio of the coupling agent to the uncyclized linear peptide can be, for example, 1:1-3:1. In some embodiments, the multiple amino acid residues whose side chains are linked to each other are linked via carbon-carbon double bonds, and the coupling cyclization reaction conditions are olefinic condensation coupling reaction conditions. The coupling agent can be, for example, phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride, and the molar ratio of the coupling agent to the uncyclized linear peptide can be, for example, 1:1-3:1. In some embodiments, the multiple amino acid residues whose side chains are linked to each other are linked via disulfide bonds, and the coupling cyclization reaction conditions are sulfhydryl oxidative condensation reaction conditions. The coupling cyclization can be carried out, for example, at room temperature and normal pressure for 0.5-3 hours.

[0091] Active carrier peptide

[0092] On the other hand, the present disclosure also provides an active carrier peptide, which comprises one or more carrier peptide groups and one or more reactive groups, each of the carrier peptide groups being formed by removing one or more atoms from the carrier peptide of the present disclosure, each of the reactive groups being covalently linked to an amino acid residue in the carrier peptide group, and the reactive groups comprising one or more functional groups or protected functional groups capable of undergoing a conjugation reaction.

[0093] In some embodiments, the number of the reactive groups is 1-3. In some embodiments, in some embodiments, the active carrier peptide contains only 1 reactive group. In some embodiments, the active carrier peptide of the present disclosure consists of 1-3 carrier peptide groups and 1-3 reactive groups. In some embodiments, the active carrier peptide of the present disclosure consists of 1 carrier peptide group and 1 reactive group. The reactive group may contain one or more functional groups capable of undergoing conjugation reactions. In some embodiments, each reactive group independently contains 1-3 functional groups capable of undergoing conjugation reactions. In some embodiments, the active carrier peptide of the present disclosure consists of 1 carrier peptide group and 1 reactive group. In some embodiments, the reactive group has a structure shown in formula (501):

[0094] Among them, L R represents any linking group capable of connecting the functional group to the carrier peptide group, G F represents the functional group, n501 is an integer of 1-3, represents the site of attachment of a group to which it is covalently attached; or, L R It is a 2-7 valent straight chain or branched C1-C 25 or C3-C 15 Saturated hydrocarbon group, wherein one or more methylene groups (-CH2-, methylene) in the straight or branched saturated hydrocarbon group are optionally replaced by one or more connecting combinations selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenyl group, C2-C5 alkynyl group, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 Heteroarylene; and / or

[0095] If there is a methine group ( methanetriyl), wherein one or more methine groups are optionally replaced by one or more selected from the group consisting of: nitrogen atoms, trivalent C3-C 10 Cycloalkyl, trivalent C3-C 10 Heterocyclic group, trivalent C6-C 10 Aryl and trivalent C5-C 10 heteroaryl; and / or

[0096] The saturated hydrocarbon group may optionally have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

[0097] In the above and below, the word "optionally" means that the following event occurs or does not occur, or the described substance exists or does not exist, for example, "L R It is a 2-7 valent straight chain or branched C1-C 25 Saturated hydrocarbon group, wherein one or more methylene groups in the straight or branched saturated hydrocarbon group are optionally replaced by one or more selected from the group consisting of the following groups" means "L R It is a 2-7 valent straight chain or branched C1-C 25 Alkylene, or L R It is a 2-7 valent straight chain or branched C1-C 25 A group formed by replacing one or more methylene groups in a saturated hydrocarbon group by one or more groups selected from the group consisting of the following groups.

[0098] Those skilled in the art are aware of various reactive groups suitable for conjugating a carrier peptide to a functional molecule through a conjugation reaction. The conjugation reaction refers to a reaction in which two reactant molecules are connected to each other into a new molecule through the formation of a covalent bond after one or more atoms are removed from each other. In some embodiments, the conjugation reaction is one of an addition reaction, a coupling reaction, and a substitution reaction. The addition reaction can be, for example, an unsaturated bond oxidative addition, a Michael addition, an alkynyl-diazo cycloaddition, a Diels-Alder cycloaddition, or a ring-opening addition, the coupling reaction can be, for example, an acylation coupling, a phosphoramidite coupling, or a condensation coupling, and the substitution reaction can be, for example, an ester exchange reaction or a disulfide exchange reaction. It will be understood by those skilled in the art that the conjugation reaction disclosed herein can also be other types of reactions, as long as it can achieve the connection of the carrier peptide to the functional molecule.

[0099] Those skilled in the art will understand that, in some embodiments, the carrier peptide of the present invention does not need to be conjugated through the reactive group, but rather through the active amino group of the first amino acid residue at the amino terminus or the active carboxyl group of the first amino acid residue at the carboxyl terminus in the amino acid sequence of the carrier peptide, or the side chain of an amino acid residue having a functional group capable of undergoing a conjugation reaction and a functional molecule through a conjugation reaction. In some embodiments, in order to minimize interference between the carrier peptide group and the functional group, to fully exert the delivery effect and the diagnostic, therapeutic or preventive effect, and / or for efficient and convenient synthesis, the carrier peptide of the present invention is formed by connecting the reactive group to the active carrier peptide of the present invention, and then reacts with the functional molecule to form a carrier peptide conjugate capable of delivering the functional group to the central nervous system.

[0100] In some embodiments, the functional group capable of undergoing conjugation reaction is selected from one or more of an azido group, an alkynyl group, a dibenzocyclooctynyl group, a sulfhydryl group, an amino group, a hydroxyl group, a carboxyl group, an acyl halide group, an aldehyde group, a carbonate group, an aminooxy group, an active ester group, a disulfide group, an orthopyridyl disulfide group, a maleimide group, a toluenesulfonate group, a tetrazine group, a trans-cyclooctenyl group, a hydrazide group, and a phosphoramidite group. In some embodiments, for ease of synthesis, each of the functional groups is independently selected from one of an azido group, a sulfhydryl group, a maleimide group, a disulfide group, or an orthopyridyl disulfide group.

[0101] It will be understood by those skilled in the art that when the active carrier peptide of the present invention contains a variety of reactive groups, if the functional groups capable of undergoing conjugation reaction contained in the multiple reactive groups are functional groups that can react with the same functional molecule to form a covalent bond, for example, when it contains two functional groups that can react with acylation reaction active functional groups (such as carboxyl groups, activated ester groups or acyl chloride groups), such as amino groups and hydroxyl groups, in order to achieve selective reaction and obtain a carrier peptide conjugate of a specific structure, it is necessary to first protect the first functional group and allow the second functional group to undergo conjugation reaction to form a covalent bond, and after the reaction is completed, deprotect the first functional group. The protection and deprotection conditions are all conventional reaction conditions in the art. For ease of storage and transportation, in some embodiments, all reactive groups are protected reactive groups. In some embodiments, when necessary, a specific protecting group or all protecting groups are deprotected and used to form a carrier peptide conjugate by a conjugation reaction.

[0102] In some embodiments, in order to ensure the delivery activity of the carrier peptide disclosed herein, the reactive group is an amino acid residue, one end of the residue is connected to the first amino acid residue at the carboxyl end or amino end of the carrier peptide sequence by a peptide bond, and the other end of the residue contains the above-mentioned functional group capable of undergoing conjugation reaction. In this case, these amino acid residues are not considered to be part of the carrier peptide sequence disclosed herein. In this case, the functional group capable of undergoing conjugation reaction is the amino group, carboxyl group or functional group on the side chain of the amino acid residue, and the linking group L R is the portion remaining after removing the functional group from the amino acid residue. In some embodiments, the reactive group is a cysteine ​​residue or an azidohomoalanine residue. For example, when connected to the carboxyl terminus of a carrier peptide sequence, the cysteine ​​residue can be a group as shown in (L38) or (L39), in which case the functional group capable of undergoing a conjugation reaction is at least a sulfhydryl group; in some embodiments, the sulfhydryl group further forms an orthopyridyl disulfide group; the azidohomoalanine residue can be a group as shown in formula (L40), in which the functional group capable of undergoing a conjugation reaction is at least an azido group. In some embodiments, at least one or each reactive group comprises a maleimide group, which can undergo a Michael addition reaction with, for example, a sulfhydryl group to form a covalent thioether bond.

[0103] The reactive group can be attached to any amino acid residue in the carrier peptide. In some embodiments, for ease of synthesis, the reactive group is attached to the amino-terminal amino acid residue or the carboxyl-terminal amino acid residue of the carrier peptide. In some embodiments, all reactive groups are attached to the amino-terminal amino acid residue of the carrier peptide.

[0104] When attached to the amino terminus of the carrier peptide sequence, the cysteine ​​residue can be a group represented by a group represented by formula (L38) or (L39), and the azidohomoalanine residue can be a group represented by a group represented by formula (L40) or (L41). In some embodiments, the functional group is a maleimide group, and at least one or each of the reactive groups has a structure represented by formula (L401), (L402), or (L403):

[0105] in, It indicates the site at which a group is attached to the rest of the molecule;

[0106] n 38 、n 39 、n 40 、n 41 and n 401 Each independently represents an integer from 1 to 10;

[0107] R38 、R 40 and R 42 Each is independently a hydroxyl group, a protected hydroxyl group, an amino group, or a site for covalent attachment of a group;

[0108] R 39 、R 41 and R 43 Each is independently a hydrogen, an amino protecting group, or a site for covalent attachment of a group.

[0109] In some embodiments, for structural simplicity, n 38 、n 39 、n 40 、n 41 and n 401 are independently an integer from 1 to 5. In some embodiments, n 38 、n 39 、n 40 Both are 1, n 41 and n 401 All are 4. In some embodiments, R 38 and R 40 Each independently represents a hydroxyl group or an amino group, R 39 and R 41 Each is independently hydrogen or an amino protecting group. 42 and R 43 In some embodiments, R 42 Form an amide bond with the imino group in the linking group shown in formula (L38) or (L40); in some embodiments, R 43 It forms an amide bond with the carbonyl group in the linking group represented by formula (L39), (L41), (L401) or (L402).

[0110] In some embodiments, the reactive group is linked to the amino terminal amino acid residue of the carrier peptide of the present disclosure via an amide bond, and the reactive group has a structure as shown in formula (L404) or (L405):

[0111] In some embodiments, the carrier peptide group is formed by removing one or more atoms from the amino acid residue of the carrier peptide of the present invention, and the reactive group can be connected to any amino acid residue in the carrier peptide. In some embodiments, for ease of synthesis, the carrier peptide group is formed by removing one or more atoms from the amino-terminal and / or carboxyl-terminal amino acid residue of the carrier peptide of the present invention, and the reactive group is connected to the amino-terminal amino acid residue and / or carboxyl-terminal amino acid residue of the carrier peptide group. In some embodiments, all reactive groups are connected to the amino-terminal amino acid residue of the carrier peptide. In some embodiments, the active carrier peptide of the present invention comprises 1 carrier peptide group and 1 reactive group connected to the amino-terminal or carboxyl-terminal of the carrier peptide.

[0112] In some embodiments, the active carrier peptide disclosed herein has a structure represented by formula (930):

[0113] Preparation of active carrier peptides

[0114] In some embodiments, the active carrier peptide disclosed herein can be prepared by the following method: contacting the prepared carrier peptide with a reactive molecule under conditions that allow a conjugation reaction between the carrier peptide and the reactive molecule, and isolating the carrier peptide disclosed herein. The reactive molecule refers to a precursor molecule capable of obtaining the reactive group through a conjugation reaction.

[0115] In some embodiments, the conjugation reaction is one of an addition reaction, a coupling reaction, and a substitution reaction, and the conditions for the conjugation reaction between the carrier peptide and the reactive molecule are correspondingly one of an addition reaction condition, a coupling reaction condition, and a substitution reaction condition.

[0116] The amount of the precursor molecule of the reactive group to the carrier peptide can be any suitable amount, for example, a molar ratio of 1:10 to 10:1. The active carrier peptide of the present disclosure can be isolated using various suitable methods, for example, it can be obtained by separation and purification by column chromatography. The reactive molecule can be any of various molecules with dual reactive sites commonly used by those skilled in the art, as long as it can provide a reactive group after reaction with the carrier peptide.

[0117] In some embodiments, the reactive group is an amino acid residue, such as a cysteine ​​residue or an azidohomoalanine residue, and the active carrier peptide can be prepared by the following method: contacting the carrier peptide with an amino acid or amino acid analog having a corresponding modification under amino acid condensation coupling conditions, and isolating the active carrier peptide of the present disclosure. The amino acid analog can be, for example, cysteine, a cysteine ​​analog, azidohomoalanine, or an azidohomoalanine analog. In some embodiments, the amino acid condensation coupling conditions are acylation reaction conditions. In some embodiments, the acylation reaction conditions are dehydration condensation conditions, such as carrying out the dehydration condensation in an organic solvent, at normal pressure or pressure, at a heated temperature, in the presence of an acylating agent. The organic solvent can be, for example, at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol, and tetrahydrofuran. In some embodiments, the amount of organic solvent used relative to the carrier peptide of the present disclosure can be 2-6 L / mol, for example, 2.5-4.5 L / mol. In some embodiments, the temperature is 30-80° C., for example, 40-60° C. The acylating agent can be, for example, an activated ester or an acid, and the amount of the acylating agent used is sufficient to promote dehydration condensation. For example, the molar ratio of the acylating agent to the carrier peptide of the present disclosure is 0.1:1-10:1, for example, 0.5:1-5:1. The reaction time can be determined by monitoring the consumption of the reactants, for example, 0.5-20 hours, 1-10 hours, or 2-8 hours.

[0118] In some embodiments, the conditions for the amino acid condensation coupling are polypeptide solid phase synthesis reaction conditions. Peptide solid phase synthesis reaction conditions are known to those skilled in the art and can be easily obtained through commercial custom synthesis when the sequence is known and the amino acid monomers used are commercially available. In some embodiments, polypeptide solid phase synthesis reaction conditions are described in Atherton, E.; Sheppard, R.C. Solid Phase Peptide Synthesis: A Practical Approach. Oxford, England: IRL Press. 1989, which is incorporated herein by reference in its entirety.

[0119] In some embodiments, the solid phase peptide synthesis reaction conditions include: (1) condensation: contacting a resin containing amino or hydroxyl groups with an activated amino acid solution at room temperature and pressure for 1-5 hours, removing the solvent and washing to obtain a resin connected to one protected amino acid; (2) deprotection: contacting the resin connected to the protected amino acid with a deprotecting agent in the presence of an organic solvent, and separating to obtain the amino acid deprotected resin; (3) repeated connection: repeating the above condensation and deprotection steps on the obtained amino acid deprotected resin for a total of X0 connections, where X0 is the number of amino acids in the carrier peptide disclosed herein, to obtain a resin connected to a carrier peptide group; (4) cleavage, separation and purification: contacting the resin connected to the carrier peptide group with a cleavage solution for 1-5 hours, removing the solid and collecting the liquid phase, adding a precipitant to precipitate and collect the solid. When the carrier peptide molecule contains a disulfide bond, (5) an additional oxidative coupling step is also required: contacting the carrier peptide with an oxidant under conditions that can form a disulfide bond, and separating to obtain the carrier peptide disclosed herein.

[0120] The activated amino acid solution can be easily obtained by those skilled in the art through known methods. In some embodiments, the activated amino acid solution can be obtained by dissolving an amino group-protected amino acid monomer and a condensing agent in an organic solvent. Amino group-protected amino acid monomers are well known to those skilled in the art and can be easily prepared, for example, by commercially obtaining or by known methods. The molar ratio of the amino group-protected amino acid monomer to the amino acid terminus in the resin can be 1:1-10:1, for example, 1:1-5:1 or 1:1-3:1. The condensing agent can use a condensing agent commonly used in polypeptide synthesis processes. In some embodiments, the condensing agent is a carbodiimide-type condensing agent and / or an onium salt-type condensing agent. In some embodiments, the carbodiimide condensing agent is one or more of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl). In some embodiments, the onium salt type condensing agent is one or more of 1-hydroxybenzotriazole (HOBT), tetramethyluronium hexafluorophosphate (HBTU), and benzotriazole tetramethyltetrafluoroboric acid (TBTU). In some embodiments, the condensing agent uses HOBT and DIC. The molar ratio of the condensing agent to the amino acid monomer protected by the amino group can be 1:10-10:1, for example, 1:1-8:1, 2:1-6:1, or 3:1-5:1. The organic solvent can use solvents commonly used in the field of polypeptide solid phase. In some embodiments, the organic solvent is one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), and methanol (MeOH). Relative to the amino acid monomer protected by the amino group, the amount of the organic solvent can be 0.5-10L / mol, for example, 1-5L / mol or 2-4L / mol.

[0121] The conditions of the deprotection step are well known to those skilled in the art. In some embodiments, the deprotecting agent is piperidine and the organic solvent is DMF. In some embodiments, the amount of the deprotecting agent is sufficient to excessive, and the amount of the organic solvent relative to the deprotecting agent is 3:1-10:1 (v / v), for example 4:1 (i.e., 20% piperidine / DMF solution). The temperature and pressure of the deprotection step can be any temperature at which the deprotection reaction can occur, for example, at room temperature / normal pressure. The deprotection step is carried out for 10 min-1 h, for example 20-30 min.

[0122] The lysate can be a lysate commonly used in the field of polypeptide solid phase synthesis. In some embodiments, the lysate is a solution of TFA. In some embodiments, the lysate is a mixture of TFA, thioanisole, phenol, triisopropylsilane (TIS), and water. In some embodiments, the lysate is a lysate with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5. The amount of the lysate is in large excess relative to the carrier peptide, for example 2-10 L / mol, or 4-6 L / mol. The temperature and pressure of the cleavage step can be any temperature at which a cleavage reaction can occur, for example, at room temperature / normal pressure. The cleavage step is performed for 1-10 h, for example 2-4 h.

[0123] In the separation step, the reaction mixture can be added to a precipitation solvent to precipitate and separate the cleaved carrier peptide from the solution. In some embodiments, the precipitation solvent is a polar solvent, such as an ether solvent. In some embodiments, the precipitation solvent is one or more of diethyl ether and methyl tert-butyl ether (MTBE). The volume ratio of the precipitation solvent to the reaction mixture can be 2:1-10:1, for example 3:1-6:1.

[0124] The conditions for the oxidative coupling step are known to those skilled in the art. In some embodiments, the conditions enabling disulfide bond formation are, for example, carried out in a solvent for 2 min to 0.5 h, for example, 3 to 10 min. The solvent is a polar solvent, such as one or more of water, methanol, and ethanol. The reaction temperature and conditions for the oxidative coupling step are sufficient to allow the oxidative coupling to proceed adequately. In some embodiments, the oxidative coupling step is carried out at room temperature and pressure. In some embodiments, the solvent is a water / methanol mixture. The amount of solvent used can be an amount sufficient to form a solution with a carrier peptide concentration of 0.1 to 10 mM, for example, a solution with a concentration of 0.5 to 5 mM. In some embodiments, the oxidant is iodine. In some embodiments, the oxidant is in the form of a methanolic solution of iodine. The molar ratio of the oxidant used relative to the groups to be coupled is 1:1 to 1:1.5, for example, 1:1 to 1:1.2 or 1:1 to 1:1.05. In some embodiments, the oxidant is used in a slight excess. In some embodiments, a reducing agent is additionally added to remove excess oxidant. In some embodiments, the reducing agent is ascorbic acid.

[0125] The separation and purification step may include filtering to remove solids and removing the solvent. In some embodiments, the removal of the solvent is concentrated under reduced pressure. In some embodiments, the separation and purification also includes purification by column chromatography. In some embodiments, the column chromatography is reversed-phase preparative liquid chromatography. The conditions for the column chromatography purification are well known to those skilled in the art.

[0126] In some embodiments, the reactive group is a cysteine ​​residue as shown in formula (L38) or (L39), and the carrier peptide is a carrier peptide as shown in formula (107):

[0127] where n 107 =n38 or n39, pp represents the carrier peptide group;

[0128] In this case, the carrier peptide can be obtained by commercial order or prepared by the following method: under the reaction conditions of amino acid dehydration condensation, the cysteine ​​analogue shown in (110) and the naked carrier peptide are contacted. The reaction conditions of the amino acid dehydration condensation are as described above. In some embodiments, the active carrier peptide is obtained by commercial order.

[0129] Among them, R 107 Is -NH2 or -OH, n 107 =n38 or n39, R 110 is H, -S-Ph or -S-2-pyridyl.

[0130] In some embodiments, the reactive group in the active carrier peptide is obtained by combining one or more functional monomers containing functional groups capable of undergoing conjugation reactions with the carrier peptide under the conditions of a polypeptide solid phase synthesis method, wherein the functional monomer contains a functional group capable of undergoing conjugation reactions and a functional group capable of forming a peptide bond with the carrier peptide through a polypeptide solid phase synthesis method. In some embodiments, the functional monomer is well known to those skilled in the art and can be synthesized by those skilled in the art using known methods or commercially available. In some embodiments, the functional monomer includes but is not limited to a compound represented by formula (110A), (110B) or (110C):

[0131] In some embodiments, the active carrier peptide is composed of a carrier peptide group and a reactive group covalently linked, wherein the carrier peptide group has a polypeptide sequence as shown in SEQ ID NO: 1, and the reactive group has a structure as shown in formula (404). In this case, the active carrier peptide can be obtained by commercial order or prepared by the following method: under the reaction conditions of amino acid dehydration condensation, 6-azidolysine monomer is contacted with the active carrier peptide shown in formula (107); then, under the conditions of acylation condensation reaction, the amino group in the 6-azidolysine residue is converted into an amide group by acetylation for protection. The reaction conditions of the amino acid dehydration condensation and acylation condensation reactions are as described above and below.

[0132] In this case, the active carrier peptide can be obtained by commercial order or prepared by the following method: 6-azidolysine is contacted with the active carrier peptide represented by formula (107) under the reaction conditions of amino acid dehydration condensation; and then the amino group in the 6-azidolysine residue is converted into an amide group by acetylation under the conditions of acylation condensation reaction for protection. The reaction conditions of the amino acid dehydration condensation and acylation condensation reactions are as described above.

[0133] In some embodiments, the active carrier peptide is obtained by commercial order. In some embodiments, the amino group or carboxyl group in the carrier peptide sequence of the present disclosure is a protected amino group or carboxyl group. For example, in some embodiments, the amino group of the amino-terminal amino acid residue of the carrier peptide or the carboxyl group of the carboxyl-terminal amino acid residue is converted into an amide group by amidation for protection. In some embodiments, the 6-azidolysine monomer is 6-azidolysine (i.e., 2-amino-6-azidohexanoic acid). In some embodiments, the 6-azidolysine monomer is 6-azidolysine with protected amino groups. In some embodiments, the 6-azidolysine monomer is commercially available (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-azidohexanoic acid.

[0134] In some embodiments, the reactive group is a reactive group as shown in formula (L401) or formula (405), the active carrier peptide has a structure as shown in formula (L44) or (L44A), and the active carrier peptide can be commercially ordered through polypeptide solid-phase synthesis, or separated by reacting the carrier peptide with 6-(maleimido)hexanoic acid succinimide ester under acylation reaction conditions, or by first reacting the carrier peptide with N-fluorenylmethoxycarbonyl-alanine and then reacting with 6-(maleimido)hexanoic acid succinimide ester, and the conditions of the acylation reaction are as described above.

[0135] Wherein, PP represents the carrier peptide group, and the carrier peptide group is connected to the acyl group in formula (L401) or (L405) through an amide bond formed by the amino group at the N-terminus (amino terminus).

[0136] In some embodiments, the reactive group is a reactive group as shown in formula (L44), the active carrier peptide has a structure as shown in formula (L45), and the active carrier peptide can be commercially ordered through peptide solid phase synthesis, or isolated by reacting the carrier peptide with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (SMCC) under acylation reaction conditions. The acylation reaction conditions are as described above.

[0137] Wherein, PP represents the carrier peptide group, and the carrier peptide group is connected to the acyl group in formula L45 through the amino group at the N-terminus (amino terminus).

[0138] In some embodiments, the reactive group is an azidohomoalanine residue as shown in formula (L40), the carrier peptide has a structure as shown in formula (204), and the carrier peptide can be commercially ordered through polypeptide solid phase synthesis.

[0139] Wherein, PP represents the carrier peptide group, and n40 is an integer of 1-10.

[0140] In some embodiments, the reactive group is a reactive group as shown in formula (L44), the carrier peptide has a structure as shown in formula (L45), and the active carrier peptide can be commercially ordered through peptide solid-phase synthesis, or isolated by reacting the carrier peptide with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (SMCC) under acylation reaction conditions. The acylation reaction conditions are as described above.

[0141] Wherein, PP represents the carrier peptide group, and the carrier peptide group is connected to the acyl group via the amino group at the N-terminus.

[0142] In some embodiments, the active carrier peptide has a structure as shown in formula (930). In this case, the active carrier peptide can be obtained by commercial order or prepared by the following method: the carrier peptide shown in formula (21) is subjected to a peptide solid phase synthesis method and then connected to a cysteine ​​at the amino terminus, and then contacted with disulfide dipyridine in an organic solvent at room temperature and normal pressure for 0.2-1h to separate and obtain the active carrier peptide shown in formula (930). In some embodiments, the organic solvent is a mixed solution of methanol and water, and the amount of the organic solvent used relative to the carrier peptide is 1-20L / mol; the amount of the disulfide dipyridine used is 1:1-3:1, for example, 1.2:1-1.8:1.

[0143] Carrier peptide conjugates of the present disclosure

[0144] On the other hand, the present disclosure provides a carrier peptide conjugate, the carrier peptide conjugate contains one or more carrier peptide groups and one or more functional groups, each carrier peptide group is formed by removing one or more atoms from the carrier peptide provided by the present disclosure, and each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group, a functional oligonucleotide group, and a delivery aid group; each of the carrier peptide groups is connected to at least one of the functional groups or at least one other carrier peptide group through at least one linking group, and each functional group is connected to at least one of the carrier peptide groups or at least one other functional group through at least one linking group. In some embodiments, each of the linking groups is independently connected to the carboxyl group at the carboxyl end of the carrier peptide group, or to the amino group at the amino end of each carrier peptide group. In some embodiments, the carrier peptide conjugate of the present disclosure contains n carrier peptide groups and m functional groups, wherein n is an integer selected from 1-8 and m is an integer selected from 1-4.

[0145] In some embodiments, m is 1 in the carrier peptide conjugate, and n carrier peptide groups are connected to the functional group through an amino acid residue at the amino terminus (N-terminus) or the carboxyl terminus (C-terminus). In some embodiments, each of the carrier peptide groups is independently connected to the functional group through a connecting group. In some embodiments, each of the carrier peptide groups is connected to the functional group through one of the connecting groups. In order to simplify the synthesis process, in some embodiments, all of the carrier peptide groups are connected to the functional group through the connecting group. In some embodiments, all of the carrier peptide groups are connected to the functional group through the same connecting group. In some embodiments, the conjugate contains multiple connecting groups, wherein any 1, 2, 3, 4, 5 or 6 of the carrier peptide groups are connected to the functional group through one of the connecting groups, and the remaining carrier peptide ligands are connected to the functional group through one or more additional connecting groups.

[0146] In some embodiments, the linking group comprises a branching group, 1-6 branching moieties, and 1 conjugated linking group, wherein the branching group is respectively connected to the conjugated linking group and each of the branching moieties, the branching moieties are respectively connected to the branching group and the carrier peptide group, and the conjugated linking group is respectively connected to the branching group and the oligonucleotide group. In some embodiments, at least one of the linking groups is formed by the aforementioned reactive group. In some embodiments, the linking group has a structure shown in formula (101L), and the conjugate has a structure shown in formula (101):

[0147] Wherein, the linking group is composed of all L A , L B and L C Composition, L A is the branched part, L B To connect L C and Nu conjugated linker, L C is a branching group, Nu and each PP independently represent the functional group or the carrier peptide group, and at least one of Nu and all PP is the functional group, and at least one is the carrier peptide group; each k is independently an integer of 1-6, n 101 is an integer from 1 to 3; and the sum of all k is an integer from 1 to 8;

[0148] The branching group L C is a 2-7 valent, linear or branched C1-C 25 or C3-C 15Saturated hydrocarbon group, or one or more carbon atoms in the straight or branched saturated hydrocarbon group are replaced by one or more connecting combinations selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenyl group, C2-C5 alkynyl group, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 Heteroarylene; and / or

[0149] If there is a methine in the branched saturated hydrocarbon group, one or more methines are optionally replaced by one or more selected from the group consisting of the following groups: nitrogen atoms, trivalent C3-C 10 Cycloalkyl, trivalent C3-C 10 Heterocyclic group, trivalent C6-C 10 Aryl and trivalent C5-C 10 heteroaryl, and

[0150] wherein the saturated hydrocarbon group optionally has any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl);

[0151] The branched portion L A is a covalent bond, or C1-C 20 or C3-C 15 Alkylene, or one or more carbon atoms in the alkylene are replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenylene, C2-C5 alkynylene, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C10 wherein the alkylene group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH-SC1-C5 alkyl, -SC1-C5 alkylphenyl, -C1-C5 alkyl-SH, -OH, -SH, -NH2, -C1-C5 alkyl-NH2, -N(C1-C5 alkyl)(C1-C5 alkyl), -NH(C1-C5 alkyl), -N(C1-C5 alkyl)(C1-C5 alkylphenyl), -NH(C1-C5 alkylphenyl), nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH (C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

[0152] Conjugated linker L B The role of the functional group and the branch group L C Therefore, any linking group that can achieve the above connection and does not damage the corresponding diagnostic, therapeutic or preventive function of the functional group can achieve the purpose of the present invention. In some embodiments, the L B is one of the following linking groups: a phosphite diester group, a thiophosphite group, an amide group, an ester group, an oxygen atom, or a disulfide group. The phosphite group, thiophosphite group, ester group, or disulfide group has the structure shown in Formulas (L46) to (L49), respectively:

[0153] in, In some embodiments, LB is a phosphite group, a thiophosphite group, or a disulfide group.

[0154] In some embodiments, in the carrier peptide conjugates of the present disclosure, each L A is a covalent bond, or each L A Selected from the group consisting of groups (L4)-(L23) and their connected combinations:

[0155] Wherein, each j1 is an integer from 1 to 10;

[0156] Each R' is C1-C 10 alkyl;

[0157] Each Ra is a hydrogen atom, C1-C 10 Alkyl, or selected from the group consisting of groups (L24)-(L37):

[0158] Identifies the point at which a group is attached to the rest of a molecule.

[0159] In some embodiments, L A The length of L is 3-35 atoms, wherein the L A The length is L A Zhong and L C Directly connected atoms to L A In some embodiments, L A The length of L is 3-20 atoms. A The length of L is 4-15 atoms. In some embodiments, each L A is a combination of at least two of the groups (L4)-(L9), (L13), (L14), and (L18). A It is a connected combination of groups (L4), (L5), (L7), (L9), (L13), (L14), and (L18).

[0160] Branching group L C The role of the conjugated linker L B With one or more branched moieties L A Covalently linked, thereby achieving conjugation of one or more carrier peptide groups with the functional group, thereby enabling more efficient delivery of the functional group through the combined action of one or more carrier peptide groups. C It has a structure as shown in the formula -H1-H2-, wherein H1 is selected from the group consisting of groups (L4)-(L23) and their connected combinations; H2 is selected from any one of the groups shown in the formulas (B1)-(B5):

[0161] Wherein, the carbon atoms in formula (B1)-(B3) are directly connected with one or more L AConnection, the nitrogen atom is used to connect to H1 via an imino or amide bond;

[0162] In formula (B4), H2 is connected to each L through the nitrogen atom. A is linked via an imino or amide bond; and is linked to H1 via an imino or amide bond through one of the two oxygen atoms, and is linked to a hydrogen atom through the other oxygen atom;

[0163] In formula (B5), H2 is connected to H1 through one of the (k+1) nitrogen atoms via an imino or amide bond, and is connected to each L through the other k nitrogen atoms. A Connected via imino or amide bonds,

[0164] Indicates the site of covalent attachment of a group.

[0165] In some embodiments, H1 is selected from the group consisting of a combination of at least two of the groups (L4) to (L9), (L13), (L14), (L17), and (L18). In some embodiments, H1 is selected from the group consisting of a combination of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L17), and (L18). In some embodiments, the length of H1 is 5-20 atoms, and the length refers to the length of H1 from the group consisting of L4 to L9. B The number of atoms in the longest atomic chain between the site of attachment and the site of attachment to H2.

[0166] In some embodiments, L C It has the structure shown in formula (201a):

[0167] Among them, n 201 and m 201 are each independently an integer from 1 to 10, and n 201 With m 201 The sum is not greater than 10 or not greater than 5.

[0168] In this case, the linking group has a structure as shown in formula (201L):

[0169] The conjugate has a structure as shown in formula (201):

[0170] In some embodiments, each of k and n 101 are independently integers of 1-3, and the sum of all k is not greater than 6.

[0171] In some embodiments, in formulas (201a), (201L), and (201), each L A is a combination of at least two of the groups (L4)-(L9), (L13), (L14), (L17), and (L18). A The length of L is 3-35 atoms, 3-20 atoms, or 4-15 atoms, wherein the L A The length is L A Zhong and L C Directly connected atoms to L A The number of atoms in the longest chain of atoms formed by the atoms directly connected to PP is the number of atoms in the longest chain of atoms, and any cycloalkylene, arylene, heterocyclylene or heteroarylene group is calculated as one atom.

[0172] In some embodiments, L A It has the structure shown in formula (202):

[0173] Among them, n 202 、m 202 、p 202 ,q 202 are each independently an integer from 1 to 5, i 202 is an integer from 0 to 5, and the L A It is connected to PP in formula (201) through the imino group and is connected to the L through the methylene group. C .

[0174] In some embodiments, n 202 、m 202 、p 202 and q 202 Each independently is 2 or 3, i 202 3 or 4.

[0175] In some embodiments, m is 1, Nu represents the functional group, and each PP independently represents one of the carrier peptide groups.

[0176] In some embodiments, the conjugate has a structure as shown in formula (103):

[0177] Among them, R 107 is amino or hydroxyl; n 103 and m 103 Each independently represents an integer from 1 to 10; n 101 An integer from 1 to 3.

[0178] In this case, the linking group has a structure as shown in formula (103L):

[0179] In some embodiments, n 103 is an integer from 1 to 3, m 103 is an integer between 3 and 6, n 101 An integer from 1 to 3.

[0180] For ease of synthesis, in some embodiments, the conjugate has a structure as shown in formula (103), and n 103 is 1, m 103 is 4 or 6. In some embodiments, n 103 =1,m 103 =6.

[0181] In some embodiments, the conjugate has a structure as shown in formula (104):

[0182] where n 104 and m 104 Each independently represents an integer from 1 to 10; n 102 An integer from 1 to 3.

[0183] In this case, the linking group has a structure as shown in formula (104L):

[0184] In some embodiments, n 104 is an integer from 1 to 3, m 104 is an integer between 3 and 6, n 102 An integer from 1 to 3.

[0185] For ease of synthesis, in some embodiments, the conjugate has a structure as shown in formula (104), and n 104 is 1, m 104 is 4 or 6. In some embodiments, n 104 =1,m 104 =6.

[0186] In some embodiments, the linking group may also be a linking group as disclosed in WO2019128611A1, which is incorporated herein by reference in its entirety.

[0187] In the carrier peptide conjugates disclosed herein, each carrier peptide group can be linked to any position in the functional group, and each functional group can be linked to any amino acid residue in the carrier peptide group. In some embodiments, each functional group is linked to the amino acid residue at the carboxyl terminus or the amino terminus of the carrier peptide.

[0188] In some embodiments, at least one of the functional groups is a delivery aid group selected from C8-C30 One or more of an alkyl or alkenyl group, a cholesterol group, a lipid group, a palmitic acid group, and a cholic acid group. The conjugate of the present invention with a delivery aid group can be more compatible with the intracellular environment of the central nervous system, have better bioavailability, and / or enable the conjugate of the present invention to be more effectively delivered to relevant targeted cells.

[0189] In some embodiments, at least one of the functional groups is a diagnostic agent group, and each of the diagnostic agent groups is independently selected from at least one of a fluorescent group, a contrast agent group, and a group containing a radionuclide. By including the diagnostic agent group, the conjugates of the present disclosure can deliver the diagnostic agent group to relevant targeted central nervous system cells and / or tissues, thereby specifically, efficiently, and accurately diagnosing the progression status and / or symptom information of the relevant disease.

[0190] In some embodiments, at least one of the functional groups is a therapeutic group, and each of the therapeutic groups is selected from at least one of a cytotoxin group, an antibiotic group, an angiogenesis inhibitor, an antibody drug group, and a group containing a radioactive isotope. By comprising the disease therapeutic group, the conjugate of the present invention can specifically deliver the therapeutic group to the targeted central nervous system cells and / or tissues, thereby treating and / or alleviating the relevant disease process or symptoms through the action of the therapeutic group. For example, the cytotoxin group is specifically delivered to the relevant cells and / or tissues by the conjugate of the present invention, so that the targeted cells are specifically eliminated, thereby significantly reducing the number of targeted cells while reducing the side effects caused by the low targeting of the cytotoxin itself, thereby treating the relevant diseases, symptoms and / or disorders.

[0191] In some embodiments, at least one of the functional groups is an oligonucleotide group. The oligonucleotide group is capable of adjusting the level of target mRNA in cells in the central nervous system. By comprising the oligonucleotide group, the conjugate of the present disclosure can specifically deliver the oligonucleotide group to cells in the central nervous system, and then through the action of the oligonucleotide group, such as the RNA interference effect, the level of the relevant target mRNA in the cell is regulated, such as suppressing the expression of oncogenes, thereby treating and / or alleviating the relevant disease process or symptoms. In some embodiments, the functional oligonucleotide group is an siRNA group. The siRNA group refers to a chemical portion formed by the loss of one or more atoms or functional groups in an siRNA molecule.

[0192] In some embodiments, the functional group is a small molecule therapeutic agent group or a functional oligonucleotide group that has a therapeutic effect on central nervous system-related diseases or symptoms.

[0193] At this time, when there are multiple carrier peptide groups, the multiple carrier peptide groups can be connected to the same nucleotide in the functional oligonucleotide or respectively connected to different nucleotides. In order to reduce the influence of steric hindrance on the activity of the functional oligonucleotide, in some embodiments, each of the covalent bonds and / or linking groups connected with the carrier peptide group is connected to nucleotides at different positions in the functional oligonucleotide. In some embodiments, the nucleotides connected by each covalent bond and / or linking group connected with the carrier peptide group are adjacent nucleotides. In some embodiments, the nucleotides to which they are connected are spaced at least 1 nucleotide. In some embodiments, the nucleotides to which they are connected are spaced at least 3 nucleotides. In some embodiments, the nucleotides to which they are connected are spaced at least 5 nucleotides.

[0194] The carrier peptide group can be attached to any technically feasible position on the nucleotide, for example, it can be attached to the ribose ring of the nucleotide, and the attachment position can be the 2', 3' or 5' position of the ribose ring; alternatively, each carrier peptide group is attached to the base of the nucleotide. To simplify the synthesis process, in some embodiments, each carrier peptide group is attached to the ribose ring of the nucleotide, and the attachment position is the 3' or 5' position of the ribose ring.

[0195] In some embodiments, the functional oligonucleotide is a single-stranded oligonucleotide, such as a single-stranded ribonucleic acid (ssRNA) or a single-stranded antisense oligonucleotide (ASO). In this case, the carrier peptide group can be attached to any nucleotide in the single-stranded oligonucleotide using the above-mentioned connection method. In some embodiments, the carrier peptide group is attached to the first nucleotide at the 3' end or the first nucleotide at the 5' end of the single-stranded oligonucleotide.

[0196] In some embodiments, the functional oligonucleotide is a double-stranded oligonucleotide containing a sense strand and an antisense strand. Each of the carrier peptide groups is independently connected to the sense strand or the antisense strand. In some embodiments, part of the carrier peptide groups is connected to the sense strand, and part of the carrier peptide groups is connected to the antisense strand. In some embodiments, all of the carrier peptide groups are connected to the antisense strand. In some embodiments, all of the carrier peptide groups are connected to the sense strand.

[0197] In some embodiments, at least one of the carrier peptide groups is linked to the first nucleotide at the 3' end or the 5' end of the sense strand or antisense strand. In some embodiments, in the carrier peptide conjugate, the number n of carrier peptide groups is an integer from 1 to 3. The structure of the carrier peptide conjugate is as described above.

[0198] In some embodiments, n=1, and the carrier peptide group is connected to the first nucleotide at the 3' end or the 5' end of the sense strand or antisense strand. In this case, the carrier peptide conjugate of the present disclosure has a structure as shown in any one of Formulas (301) to (304):

[0199] Where 3' and 5' represent the first nucleotide at the 3' end or 5' end of the sense strand or antisense strand, respectively;

[0200] LK represents a linking group or a covalent bond;

[0201] PP represents a carrier peptide group, and the meanings of “3′”, “5′”, “LK” and “PP” used in the structural formulas hereinafter in this disclosure are the same as those herein.

[0202] In some embodiments, n=2, one carrier peptide group is linked to the first nucleotide at the 3' end or the 5' end of the sense strand, and the other carrier peptide group is linked to any one of the nucleotides at other positions in the sense strand, wherein the nucleotides at other positions refer to the nucleotides other than the first nucleotide at the 3' end or the 5' end of the sense strand. In this case, the carrier peptide conjugate of the present disclosure has a structure shown in Formula (305) or Formula (306):

[0203] Alternatively, one carrier peptide group is linked to the first nucleotide at the 3' end of the sense strand, and another carrier peptide group is linked to the first nucleotide at the 5' end of the sense strand. In this case, the carrier peptide conjugate of the present disclosure has a structure as shown in Formula (307):

[0204] In some embodiments, n=3, at least one of the carrier peptide groups is linked to the first nucleotide at the 3' end or the 5' end of the sense strand. In some embodiments, two of the carrier peptide groups are linked to the first nucleotide at the 3' end and the 5' end of the sense strand, respectively, and another carrier peptide group is linked to any one of the nucleotides at other positions in the sense strand. In this case, the carrier peptide conjugate of the present disclosure has a structure as shown in Formula (308):

[0205] Alternatively, in some embodiments, all carrier peptide groups are linked to the first nucleotide at the 3' end or the 5' end of the sense strand. In this case, the carrier peptide conjugate of the present disclosure has a structure as shown in Formula (309):

[0206] Where R IThe definition and selection range of are the same as those of the linking group above. For example, it can be a linking group as shown in formula (101L).

[0207] In some embodiments, the double-stranded oligonucleotide is an siRNA or shRNA, comprising a sense strand and an antisense strand, each comprising 15-25 nucleotides; each nucleotide being a modified or unmodified nucleotide. The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region; the nucleotide sequence II is at least partially reverse-complementary to a target mRNA, the target mRNA being mRNA expressed by a target gene in a target cell in a target tissue or organ, and the target cell being a cell having a surface receptor capable of binding to the carrier peptide.

[0208] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are both composed of 19 nucleotides, and the nucleotide sequence II is substantially reverse complementary, essentially reverse complementary, or completely reverse complementary to the nucleotide sequence I; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0209] In some embodiments, from the 5' end to the 3' end, at least nucleotides 2-19 of the nucleotide sequence II are completely reverse complementary to nucleotides 1-18 of the nucleotide sequence I. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or there is a base mismatch between the second nucleotide in the nucleotide sequence II from the 5' end to the 3' end and the second nucleotide in the nucleotide sequence I from the 3' end to the 5' end. By including this base mismatch, the target gene expression inhibition activity of the functional double-stranded oligonucleotide of the present disclosure can be further enhanced while maintaining low off-target effects.

[0210] In some embodiments, the sense strand and antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides long and the antisense strand being 19-26 nucleotides long. Thus, the length ratio of the sense strand and the antisense strand of the functional double-stranded oligonucleotide can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand in the functional double-stranded oligonucleotide is 19 / 21, 21 / 23, or 23 / 25.

[0211] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are 1-4 nucleotides in length, wherein nucleotide sequence III is linked to nucleotide sequence I, wherein nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and wherein nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. In some embodiments, nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary. In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide sequence IV is also determined.

[0212] In some embodiments, the sense strand and the antisense strand are of different lengths, and the antisense strand further comprises a nucleotide sequence V having a length of 1 to 3 nucleotides and attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In some embodiments, the sense strand further comprises a nucleotide sequence VI having a length of 1 to 3 nucleotides and attached to the 3' end of the sense strand to form a 3' overhang of the sense strand.

[0213] In some embodiments, the functional double-stranded oligonucleotide includes nucleotide sequence V, but does not include nucleotide sequence VI.Thus, the length ratio of the sense strand and the antisense strand can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26.In some embodiments, the functional double-stranded oligonucleotide includes nucleotide sequence V and VI.In some embodiments, the length of nucleotide sequence V is identical or different from the length of nucleotide sequence VI.Thus, the length ratio of the sense strand and the antisense strand can be (19-26): (19-26). In some embodiments, the length of the nucleotide sequence V and / or VI is 2 nucleotides, and thus, the length ratio of the sense strand to the antisense strand can be 19 / 21, 21 / 21, 21 / 23, 23 / 23, 23 / 25 or 25 / 25.

[0214] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save costs, in some embodiments, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); alternatively, to improve the affinity of the antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding position in the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand to the antisense strand is 19 / 21 or 21 / 23. In this case, the functional double-stranded oligonucleotide has better mRNA silencing activity.

[0215] Each nucleotide in the nucleotide sequence VI can be any nucleotide. To facilitate synthesis and save synthesis costs, in some embodiments, the nucleotide sequence VI is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); alternatively, to improve the affinity of the sense and antisense strands in the functional double-stranded oligonucleotide, the nucleotide sequence VI is identical to the nucleotides at the corresponding position of the target mRNA. Therefore, in some embodiments, the functional double-stranded oligonucleotide comprises nucleotide sequences V and VI, and the ratio of the length of the sense and antisense strands is 21 / 21 or 23 / 23. In this case, the functional double-stranded oligonucleotide has better mRNA silencing activity.

[0216] The nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to the 5' end of a nucleotide sequence of the target mRNA, wherein the nucleotide sequence of the target mRNA is substantially reverse complementary or completely reverse complementary to the nucleotide sequence II, or is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of the nucleotide sequence II and the nucleotide sequence IV.

[0217] As described above, the nucleotides in the functional double-stranded oligonucleotide are modified or unmodified nucleotides. In some embodiments, the nucleotides in the functional double-stranded oligonucleotide are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the functional double-stranded oligonucleotide are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the regulatory function of the functional double-stranded oligonucleotide on the target mRNA expressed by the target gene.

[0218] In some embodiments, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of the siRNA to inhibit gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.

[0219] In some embodiments, at least one nucleotide in the sense strand or antisense strand of the functional double-stranded oligonucleotide is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; that is, at least a portion of the phosphate group and / or ribose group in the phosphate-sugar backbone of at least one single strand in the sense strand and antisense strand is a phosphate group having a modified group and / or a ribose group having a modified group.

[0220] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide. The carrier peptide conjugates provided by the present disclosure have achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.

[0221] In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, the number of fluorinated modified nucleotides in the nucleotide sequence I is no more than 5, and from the 5' end to the 3' end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in the nucleotide sequence II is no more than 7, and the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.

[0222] In some embodiments, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 or positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.

[0223] In the context of the present disclosure, a "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (7). A "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is selected from one of a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0224] The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorinated groups are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0225] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (11):

[0226] Nucleotide analogs are groups that can replace nucleotides in nucleic acids but have a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0227] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

[0228] Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

[0229] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (—O-alkyl).

[0230] An isonucleotide is a compound formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide can be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18).

[0231] In the compounds of formula (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; and R is selected from H, OH, F or the non-fluorinated groups described above.

[0232] In some embodiments, the nucleotide analog is selected from one of an isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0233] In the above and below, “fluorinated nucleotides”, “2’-fluorinated nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by fluorine” and “nucleotides having a 2’-fluorinated ribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (7) formed by replacing the 2’-hydroxyl group of the nucleotide with fluorine; “methoxy-modified nucleotides”, “2’-methoxy-modified nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by a methoxy group” and “nucleotides having a 2’-methoxyribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (8) formed by replacing the 2’-hydroxyl group of the ribose group of the nucleotide with a methoxy group.

[0234] In some embodiments, the functional double-stranded oligonucleotide is a double-stranded oligonucleotide having the following modifications: in the direction from the 5' end to the 3' end, in the sense chain, the nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense chain are methoxy-modified nucleotides; in the antisense chain, the nucleotides at positions 2, 6, 14, and 16 or positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense chain are methoxy-modified nucleotides.

[0235] In some embodiments, the functional double-stranded oligonucleotide is a double-stranded oligonucleotide having the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence I in the sense strand of the functional double-stranded oligonucleotide are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxy-modified nucleotides; in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II in the antisense strand of the double-stranded oligonucleotide are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxy-modified nucleotides;

[0236] Alternatively, in the direction from the 5' end to the 3' end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxy-modified nucleotides; in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxy-modified nucleotides;

[0237] Alternatively, in the direction from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the sense chain are methoxy-modified nucleotides; in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense chain are methoxy-modified nucleotides.

[0238] Double-stranded oligonucleotides with these modifications can make it difficult for ribonucleases in the blood to cleave nucleic acids, thereby increasing their stability and making them more resistant to nuclease hydrolysis. Furthermore, these functional double-stranded oligonucleotides with these modifications still have a high regulatory function for target mRNA.

[0239] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the functional double-stranded oligonucleotide is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (1):

[0240] This modification can stabilize the double-stranded structure of double-stranded oligonucleotides and maintain high specificity and high affinity of base pairing.

[0241] In some embodiments, in the functional double-stranded oligonucleotide, the phosphorothioate linkage is present at at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0242] between the first and second nucleotides at the 5' end of the sense strand;

[0243] between the second and third nucleotides at the 5' end of the sense strand;

[0244] between the first and second nucleotides at the 3' end of the sense strand;

[0245] between the second and third nucleotides at the 3' end of the sense strand;

[0246] between the first and second nucleotides at the 5' end of the antisense strand;

[0247] between the second and third nucleotides at the 5' end of the antisense strand;

[0248] between the first and second nucleotides at the 3' end of the antisense strand; and

[0249] between the second and third nucleotides at the 3' end of the antisense strand.

[0250] In some embodiments, the 5'-terminal nucleotide of the antisense strand in the functional double-stranded oligonucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0251] Commonly used 5'-phosphate nucleotides or 5'-phosphate analogue-modified nucleotides are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have the following structure:

[0252] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analogue-modified nucleotides:

[0253] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0254] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a thiophosphate modified nucleotide as shown in formula (5).

[0255] In some embodiments, in the carrier peptide conjugates of the present disclosure, the functional double-stranded oligonucleotide is siRNA.

[0256] As previously described, the target cells of the carrier-peptide conjugates of the present disclosure are cells that have surface receptors capable of binding to the carrier peptides of the present disclosure. Therefore, the carrier-peptide conjugates of the present disclosure can be targeted to any target tissue or organ in a subject where such target cells are present. By using double-stranded oligonucleotides that target mRNAs expressed by different target genes, the function of regulating target genes expressed in these target tissues or organs can be achieved.

[0257] In some embodiments, the target tissue or target organ is selected from the brain or spinal cord. In some embodiments, the target tissue is the cerebral cortex or hippocampus. In some embodiments, the target gene is APP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR or TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B or RHO. In some embodiments, the target gene is SOD1, APOE4 or RPTOR.

[0258] The carrier peptide conjugates containing these siRNA groups showed significantly improved stability in plasma, low off-target effects, and high target mRNA silencing activity. Therefore, in some embodiments, the siRNA can be one of the siRNAs shown in Table 1.

[0259] Table 1 siRNA sequences in the carrier peptide conjugates disclosed herein

[0260] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a thiophosphate group; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide.

[0261] In some embodiments, the functional double-stranded oligonucleotide in the carrier peptide conjugate of the present disclosure is siRNA targeting SOD1 mRNA or siRNA targeting APOE4 mRNA.

[0262] Synthesis of carrier peptide conjugates of the present disclosure

[0263] Those skilled in the art can prepare the carrier peptide conjugates of the present disclosure by any suitable means. The carrier peptide conjugates contain one or more carrier peptide groups and one or more functional groups, wherein each carrier peptide group is formed by removing one or more atoms from the carrier peptide described in any one of claims 1 to 7, and each functional group is independently one of a diagnostic agent group, a small molecule therapeutic agent group, a functional oligonucleotide group, and a delivery aid group; each carrier peptide group is connected to at least one functional group or another carrier peptide group via a linker, and each functional group is connected to at least one carrier peptide group or another functional group via a linker.

[0264] In some embodiments, the method for synthesizing the carrier peptide conjugate of the present invention includes contacting the active carrier peptide provided by the present invention with one or more functional molecules containing functional groups capable of undergoing a conjugation reaction under conjugation reaction conditions, so that each carrier peptide is connected to one or more of the functional molecules or one or more additional carrier peptides, and each functional molecule is connected to one or more of the carrier peptides or one or more additional functional molecules through a covalent bond and / or a linking group.

[0265] In some embodiments, the method for synthesizing the carrier peptide conjugate of the present disclosure comprises contacting the active carrier peptide provided by the present disclosure with one or more functional molecules containing functional groups capable of undergoing a conjugation reaction under conjugation reaction conditions, or contacting a single active carrier peptide provided by the present disclosure with the same or different functional molecules containing functional groups capable of undergoing a conjugation reaction multiple times, so that the carrier peptide is linked to the one or more functional molecules via a covalent bond and / or a linking group. Alternatively, the method may further comprise contacting the conjugate molecule of the carrier peptide and functional molecule obtained in the above steps with one or more additional carrier peptides provided by the present disclosure under conditions capable of undergoing a conjugation reaction.

[0266] Those skilled in the art can use various suitable methods to separate and purify the carrier peptide conjugate disclosed herein, for example, separation and purification can be performed by column chromatography.

[0267] In some embodiments, the conditions of the conjugation reaction are conditions of a coupling reaction, specifically, in a solvent, under coupling reaction conditions, a functional molecule containing a functional group capable of undergoing a conjugation reaction is contacted with the active carrier peptide of the present disclosure, and the coupling reaction conditions can be condensation reaction conditions or thiol-disulfide exchange reaction conditions.

[0268] In some embodiments, the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry conditions. Accordingly, the functional groups capable of undergoing conjugation reaction contained in the active carrier peptide and the functional molecule are functional groups capable of undergoing acylation condensation or dehydration condensation, for example, one of the functional groups is an acyl halide group or a carboxyl group and the other is an amino group or a hydroxyl group, or one of the functional groups is an alkynyl group and the other is an azide group.

[0269] In some embodiments, the synthesis method of the carrier peptide conjugate disclosed herein includes separately synthesizing a functional molecule having a coupling reaction active group and the above-mentioned active carrier peptide disclosed herein, and contacting them with each other under the conditions of the coupling reaction to obtain the carrier peptide conjugate disclosed herein. It can be understood by those skilled in the art that for the carrier peptide conjugate disclosed herein containing different types of functional groups, different active groups and different coupling reaction conditions can be selected according to needs and conventional technical means in the art. Common methods for obtaining carrier peptide conjugates, i.e., polypeptide conjugates, are known to those skilled in the art, such as Y. Zhou et al., Carbohydrate Polymers (2021), https: / / doi.org / 10.1016 / j.carbpol.2021.118662; Ollivier, N. et al., Nat Protoc 10, 269–292 (2015), and Stanley Stein et al., Bioconjugate Chem. 1991, 2, 6, 464–465. The above-mentioned documents are each incorporated herein by reference in their entirety.

[0270] Each of the functional molecules is independently selected from one of a small molecule therapeutic agent, a functional oligonucleotide, a diagnostic agent molecule and a delivery aid. The diagnostic agent, small molecule therapeutic agent, functional oligonucleotide or delivery aid is a compound that can form a diagnostic agent group, a therapeutic agent group, an oligonucleotide group or a delivery aid group as described above. In some embodiments, at least one of the functional molecules is a delivery aid molecule selected from C8-C 30One or more of an alkane or alkene, a cholesterol molecule, a lipid molecule, a palmitic acid molecule, and a bile acid molecule. In some embodiments, at least one of the functional molecules is a diagnostic agent molecule, and each of the diagnostic agent molecules is independently selected from at least one of a fluorescent molecule, a contrast agent molecule, and a molecule containing a radionuclide. In some embodiments, at least one of the functional groups is a therapeutic agent molecule, and each of the therapeutic agent molecules is selected from at least one of a cytotoxin molecule, an antibiotic molecule, an angiogenesis inhibitor, an antibody drug molecule, and a molecule containing a radioisotope. In some embodiments, at least one of the functional groups is an oligonucleotide molecule. The oligonucleotide molecule can adjust the level of target mRNA in cells in the central nervous system.

[0271] In some embodiments, the functional group in the carrier peptide conjugate of the present disclosure is a double-stranded oligonucleotide group. In this case, the synthesis method of the carrier peptide conjugate of the present disclosure includes providing a sense chain and an antisense chain of a double-stranded oligonucleotide, annealing the sense chain and the antisense chain to obtain the double-stranded oligonucleotide, wherein the sense chain and the reaction chain respectively contain 15-25 nucleotides, each of which is a modified or unmodified nucleotide, at least one of the sense chain or antisense chain is a sense chain or antisense chain connected to the carrier peptide group, the total number of the carrier peptide groups connected to the sense chain or antisense chain is n, the carrier peptide group is formed by removing one or more atoms from the carrier peptide described in the present disclosure, each of the carrier peptide groups is connected to the sense chain and / or antisense chain by a covalent bond or by a linker, each of the carrier peptide groups is composed of 7-20 amino acid residues, wherein each amino acid residue is a modified or unmodified amino acid residue, and each of the carrier peptide groups is connected to the double-stranded oligonucleotide through the carboxyl end or the amino end. In some embodiments, the number of the carrier peptide groups, the position and manner of attachment to the sense strand and / or antisense strand are as described above.

[0272] In some embodiments, the synthesis method further comprises separating and purifying the double-stranded oligonucleotide.

[0273] In some embodiments, a single nucleotide chain connected with n carrier peptide groups can be prepared by the following method: in a solvent, under coupling reaction conditions, a single nucleotide chain having an active group R x1 The oligonucleotide single strand and the active group R x2 and a compound containing a carrier peptide group, and the reaction obtains an oligonucleotide single chain having n carrier peptide groups and an active group R x1 The oligonucleotide single strand and the active group R x2 The molar ratio of the compound of the carrier peptide group is 1:1-1:n. In some embodiments, the carrier peptide group is connected to the oligonucleotide single chain via a linker group, and the active group R x1and the active group R x2 is a group that can generate a linking group through a coupling reaction. In some embodiments, the active group R x2 and a carrier peptide group is a carrier peptide comprising a reactive group as described in the present disclosure. In some embodiments, the reactive group R x2 In some embodiments, the reactive group R x2 The compound containing a carrier peptide group is obtained by deprotecting the carrier peptide containing a protected reactive group as described in the present disclosure.

[0274] Those skilled in the art can obtain the active group R by various methods. x1 In some embodiments, the oligonucleotide having an active group R x1 The oligonucleotide single chain can be obtained by nucleic acid synthesis methods well known to those skilled in the art, such as phosphoramidite solid phase synthesis or phosphodiester / phosphotriester liquid phase synthesis. In some embodiments, the oligonucleotide having an active group R x1 The oligonucleotide single chain is obtained by phosphoramidite solid phase synthesis, which comprises connecting nucleoside monomers one by one according to the order of nucleotides in the oligonucleotide single chain under phosphoramidite solid phase synthesis conditions, wherein at least one nucleoside monomer has an active group R x1 The phosphoramidite solid phase synthesis method is well known to those skilled in the art, and its process and conditions are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, P17-P31, the entire contents of which are incorporated herein by reference.

[0275] The solvent can be any solvent that can achieve the coupling reaction. In some embodiments, the solvent is an organic solvent, such as at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol and tetrahydrofuran. In some embodiments, the amount of the solvent is relative to the amount of the active group R x1 For a single-stranded oligonucleotide, the molecular weight is 2-6 L / mol, for example, 2.5-4.5 L / mol.

[0276] In some embodiments, the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.

[0277] In some embodiments, the coupling reaction conditions are condensation reaction conditions, which are acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry reaction conditions, and the active group R x1 With active group R x2 In some embodiments, the condensation reaction conditions are the conditions of the acylation condensation reaction, and the active group R x1 and R x2 It is possible to undergo acylation condensation reaction to form R I In some embodiments, the condensation reaction conditions are dehydration condensation reaction conditions, and the active group R x1 and R x2 One of them is a group containing an acyl halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. In some embodiments, the condensation reaction conditions are click chemistry conditions, and the active group R x1 and R x2 One of the groups is a group containing an alkynyl group, and the other is a group containing an azide group.

[0278] In some embodiments, the active group R x1 The oligonucleotide single chain is formed by coupling the active group R x0 The oligonucleotide single chain is prepared by contacting with a cross-linking agent, wherein the cross-linking agent contains a click chemistry active group and an acylation group. X0 The click chemistry active group is connected to the single-stranded oligonucleotide by a coupling reaction with the acylation group to form a covalent connection.

[0279] In some embodiments, the active group R x1 It is an active group containing 1-3 click chemistry active groups at the end, and the click chemistry active group includes a terminal alkynyl group. In some embodiments, the acylated group is an active ester group, for example, it can be one of NHS ester group, imidate group and pentafluorophenyl ester group. Those skilled in the art can obtain the cross-linking agent by various methods. For example, when the acylated group is a pentafluorophenyl ester group and the click chemistry group includes a terminal alkynyl group, the cross-linking agent can be prepared as follows: Michael E., et al."Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8 (2015): 1451-1455, Scheme 1a (A) was used to prepare the oligonucleotide. In some embodiments, the active group R x0 In some embodiments, the coupling conditions are alkaline conditions. In some embodiments, the alkaline conditions are conditions in which a weak base aqueous solution exists, such as conditions in which a sodium bicarbonate aqueous solution exists. Those skilled in the art can obtain the active group R in various ways. x0 In some embodiments, the oligonucleotide having an active group R x0 The oligonucleotide single chain is prepared by using a phosphoramidite monomer containing an active group at the corresponding position during the synthesis of the oligonucleotide single chain. Those skilled in the art can obtain the phosphoramidite monomer containing an active group by various methods. In some embodiments, the active group R x0 It is amino, containing R x0 The phosphoramidite monomer can be commercially obtained or prepared by methods well known to those skilled in the art, for example, x0 The phosphoramidite monomer can be a commercially available 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, wherein the active group R x0 is an amino group, the active group R x0 The phosphoramidite monomer can be linked to a single-stranded oligonucleotide by a phosphoramidite solid phase synthesis method, and then the trifluoroacetyl protecting group can be removed by a deprotection reaction (such as aminolysis with concentrated ammonia) that can be easily achieved by those skilled in the art.

[0280] In some embodiments, the coupling reaction conditions are one of the sulfhydryl-disulfide exchange reactions, and the active group R x1 and R x2 In some embodiments, the above-mentioned active group R x1 R in the phosphoramidite monomer x1 With the protected R x1 ' form, the preparation method further comprises the step of: x1 The oligonucleotide single strand is contacted with a deprotection reagent to separate the oligonucleotide containing Rx1 In some embodiments, the R x1 'contains a disulfide bond leaving group, the deprotection reaction conditions are sulfhydryl-disulfide bond exchange reaction conditions, and the deprotection reagent is a disulfide bond activator. In some embodiments, the disulfide bond activator is disulfide dipyridine. Those skilled in the art can obtain the above-mentioned containing active group R by various methods. x1 or R x1 'phosphoramidite monomer, in some embodiments, the phosphoramidite monomer containing the active group R x1 or R x1 The phosphoramidite monomer is commercially available, for example, the phosphoramidite monomer shown in formula (105) can be obtained commercially.

[0281] where n 105 An integer from 1 to 10 ， m 105 =m 103 ;

[0282] Those skilled in the art can obtain the active group R in various ways. x2 In some embodiments, the coupling reaction conditions are sulfhydryl-disulfide exchange reaction conditions, and the active group R x2 Is an active group containing a thiol group, the active group R x2 In some embodiments, the coupling reaction conditions are click chemistry reaction conditions, and the active group R x2 It is a compound comprising an azide group and a carrier peptide group, wherein the active group R x2 Compounds containing peptides and carrier peptide groups are commercially available by order.

[0283] In some embodiments, the linking group is a linking group as shown in formula (103L). The carrier peptide conjugate of the present disclosure can be prepared by the following method: in a solvent, at room temperature and pressure, under sulfhydryl-disulfide exchange reaction conditions, a sulfhydryl exchange group as shown in formula (106), i.e., R x1 The oligonucleotide single strand and n oligonucleotides containing R as shown in formula (107) x2 The method is to contact the carrier peptide group with a compound containing the carrier peptide group, and separate and obtain a carrier peptide group-oligonucleotide single-stranded conjugate connected via a linker; the carrier peptide group-oligonucleotide single-stranded conjugate is annealed with another single strand of the carrier peptide conjugate of the present invention to form a double-stranded oligonucleotide, and separate and obtain the carrier peptide conjugate of the present invention.

[0284] where n 107=n 103 , pp represents the carrier peptide group;

[0285] Among them, Nu' represents a single-stranded oligonucleotide, m 106 =m 103 ; R 106 is a sulfhydryl exchanger residue. In some embodiments, R 106 C7-C 12 In some embodiments, R 106 It is 2-pyridyl.

[0286] The solvent and reaction conditions for the thiol-disulfide exchange reaction are commonly used in the art for thiol-disulfide exchange reactions, for example, in the presence of a 0.05-1 M aqueous ammonium acetate solution, and the reaction is carried out at room temperature and pressure for 2-10 hours, for example, 4-8 hours. The ratio of the solvent to the compound of formula (106) can be 100:1-2000:1 L / mol; the molar ratio of the compound of formula (107) to the compound of formula (106) can be 1:1-15:1, for example, 4:1-10:1.

[0287] Any suitable separation method can be used to separate the carrier peptide-oligonucleotide single-stranded conjugate from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then the carrier peptide-oligonucleotide single-stranded conjugate can be separated by chromatography. For example, the following chromatographic conditions can be used for separation: a C18 reverse phase chromatography column as the stationary phase, 100 mM TEAA (PH = 7.0-7.3): acetonitrile in a ratio of 5%-75% (V / V) as the mobile phase for gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the carrier peptide-oligonucleotide single-stranded conjugate, which can be directly used in subsequent reactions.

[0288] The carrier peptide represented by formula (107) can be obtained by the method described in the preparation of the carrier peptide above.

[0289] Those skilled in the art can obtain a single-stranded oligonucleotide having a thiol exchange group represented by formula (106) by various methods. In some embodiments, the compound represented by formula (106) can be prepared by the following method: in a solvent, under thiol-disulfide exchange reaction conditions, contacting the compound represented by formula (108) with a thiol-disulfide exchange agent represented by formula (111), and isolating the compound represented by formula (106).

[0290] Among them, m 106 、Nu'、R 106The definition and selection range of are the same as described above; the selection of the solvent and the conditions for the thiol-disulfide exchange reaction are the same as described above. The molar ratio of the compound of formula (111) to the compound of formula (108) is in large excess, for example, it can be 10:1-1000:1, and in some embodiments, it is 50:1-200:1.

[0291] Any suitable separation method can be used to separate the compound of formula (106) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then the compound of formula (106) can be separated by chromatography. For example, the separation can be performed using the following conditions: 20% (V / V) ethanol in water as the mobile phase, gel desalting purification in a purifier, and collecting the product eluate at a wavelength of 280 nm. In some embodiments, the solvent can be directly removed after filtration to obtain a crude product of the compound of formula (106), which can be directly used in subsequent reactions.

[0292] The thiol-disulfide exchange reagent represented by formula (111) can be prepared by methods well known to those skilled in the art. In some embodiments, the compound represented by formula (111) is commercially available 2-2'-disulfide dipyridine.

[0293] Those skilled in the art can obtain a single-stranded oligonucleotide having a thiol group as shown in formula (108) by various methods. In some embodiments, the single-stranded oligonucleotide as shown in formula (108) can be prepared by the following method: in an aqueous solution of a reducing agent, under reduction reaction conditions, contacting the single-stranded oligonucleotide as shown in formula (109) with a reducing agent, and separating to obtain the single-stranded oligonucleotide as shown in formula (108). In some embodiments, the reducing agent is a reducing agent that can reduce disulfide bonds to thiol groups. In some embodiments, the reducing agent is TCEP, and the reduction reaction conditions are to react in an aqueous solution of TCEP at room temperature and pressure.

[0294] Wherein, the definition and selection range of m106 and Nu' are the same as described above; the reducing agent and reduction reaction conditions are the reducing agents and reaction conditions commonly used in the field for reduction reactions. For example, in some embodiments, the reducing agent is TCEP. The reaction conditions are 1-5 hours at room temperature and pressure. In some embodiments, the reaction time is 2-3 hours, and the reducing agent is in large excess. In some embodiments, the "large excess" means that the mass ratio of the reducing agent to the oligonucleotide single chain can be 3:1-1:1; in some embodiments, the mass ratio of the reducing agent to the oligonucleotide single chain can be 1.5:1.

[0295] Any suitable separation method can be used to separate the compound of formula (108) from the reaction mixture. In some embodiments, the compound of formula (108) can be obtained by diluting the reaction solution with purified water and filtering, then repeating ultrafiltration and centrifugation until the ultrafiltration liquid conductivity is measured to be below 100 s, and collecting the product in the filter membrane.

[0296] Those skilled in the art can obtain the oligonucleotide single-stranded structure represented by formula (109) by various methods. In some embodiments, the oligonucleotide single-stranded structure represented by formula (109) is prepared by using a phosphoramidite monomer represented by formula (105) during solid-phase synthesis of the oligonucleotide single-stranded structure. The phosphoramidite monomer represented by formula (105) is readily available to those skilled in the art. In some embodiments, the phosphoramidite monomer represented by formula (105) is commercially available.

[0297] where n 105 An integer from 1 to 10 ， m 105 =m 103 ;

[0298] At the same time, modified nucleotide groups can also be introduced into the functional double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0299] Among them, R 107 Is -NH2 or -OH, n 107 =n 103 .

[0300] In some embodiments, in the carrier peptide conjugate of the present invention, each carrier peptide group is connected to a single chain in the double-stranded oligonucleotide through a linker group as shown in formula (201L). In this case, the carrier peptide conjugate of the present invention can be prepared by the following method: in a solvent, under the conditions of a click chemistry reaction, a single chain of an oligonucleotide containing a carbon-carbon triple bond is reacted with a click peptide containing a -N3 group through a click chemistry reaction, and the carrier peptide group is introduced into the single chain of the oligonucleotide containing the active group, and the carrier peptide-oligonucleotide single chain conjugate connected by the linker is obtained by separation, wherein the click peptide already has commercial customization services; the carrier peptide group-oligonucleotide single chain conjugate is annealed with the other single chain of the carrier peptide conjugate of the present invention to form a double-stranded oligonucleotide, and the carrier peptide conjugate of the present invention is obtained by separation. The click chemistry reaction conditions are the click chemistry reaction conditions commonly used in the art. In some embodiments, the click chemistry reaction conditions are to contact the click polypeptide with the single chain of the oligonucleotide under heating conditions and in the presence of a catalyst. In some embodiments, the catalyst is an aqueous solution containing a copper catalyst. In some embodiments, the catalyst is an aqueous solution of copper sulfate, and the molar ratio of copper sulfate to click polypeptide is 1:6-1:1. In some embodiments, the catalyst is a mixed solution of copper sulfate, TBTA and sodium ascorbate, wherein the molar ratio of copper sulfate to TBTA is 3:1-1:3, and the molar ratio of copper sulfate to sodium ascorbate is 1:5-1:10. Preferably, the molar ratio of copper sulfate to TBTA is 1:1, and the molar ratio of copper sulfate to sodium ascorbate is 1:7.5. In some embodiments, the heating conditions refer to reacting in a water bath at 30-60 degrees Celsius. In some embodiments, the heating conditions refer to reacting in a water bath at 30-50 degrees Celsius. In some embodiments, the molar ratio of the click polypeptide to the single-stranded oligonucleotide is 10:1-3:1, and in some embodiments, the molar ratio of the click polypeptide to the single-stranded oligonucleotide is 5:1-4:1. In some embodiments, the reaction endpoint is determined by monitoring the content of reactants and / or products in the reaction mixture by HPLC. In some embodiments, the click chemistry reaction conditions are as described in Zengmin Li; Tae Seok Seo; Jingyue Ju (2004). 1,3-Dipolar cycloaddition of azides with electron-deficient alkynes under mild conditions in water., 45(15), 3143–3146, the entire contents of which are incorporated herein by reference.

[0301] Any suitable separation method can be used to separate the carrier peptide-oligonucleotide single-stranded conjugate from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then the carrier peptide-oligonucleotide single-stranded conjugate can be separated by chromatography. For example, the following chromatographic conditions can be used for separation: a C18 reverse phase chromatography column as the stationary phase, 100 mM TEAA (PH = 7.0-7.3) and a mixed solvent of acetonitrile / isopropanol (V / V = 1:1) with a ratio of 5%-75% (V / V) as the mobile phase for gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the carrier peptide-oligonucleotide single-stranded conjugate, which can be directly used in subsequent reactions.

[0302] In some embodiments, the carrier peptide-oligonucleotide single-stranded conjugate is a conjugate as shown in formula (203), and the oligonucleotide single-stranded conjugate connected to the carrier peptide group can be prepared by the following method:

[0303] The definitions of Nu' and PP are the same as above.

[0304] Under the conditions of click chemistry reaction, the active group R x2 Click peptide and active group R as shown in formula (205) x1 The oligonucleotide single chain is contacted with the carrier peptide-oligonucleotide single chain conjugate shown in formula (203) is separated and obtained, wherein R x1 and R x2 The conditions of the click chemistry reaction are the same as those described above.

[0305] Those skilled in the art can obtain the compound containing an azide group and a carrier peptide group as shown in formula (204A) by various methods. In some embodiments, the compound as shown in formula (204A) is obtained by commercial order.

[0306] A person skilled in the art can obtain the oligonucleotide single chain as shown in formula (205) by various methods. For example, the oligonucleotide single chain as shown in formula (206) can be contacted with the active ester as shown in formula (207) under normal pressure and temperature under alkaline conditions to separate and obtain the oligonucleotide as shown in formula (205). In some embodiments, the alkaline conditions refer to the conditions in the presence of sodium bicarbonate aqueous solution. In some embodiments, the active ester as shown in formula (207) is in large excess compared to the oligonucleotide single chain as shown in formula (205); in some embodiments, the molar ratio of the active ester as shown in formula (207) to the oligonucleotide single chain as shown in formula (206) is 200:1-50:1; in some embodiments, the molar ratio of the active ester as shown in formula (207) to the oligonucleotide single chain as shown in formula (206) is 150:1-80:1.

[0307] Those skilled in the art can use any suitable separation method to separate the oligonucleotide single strand as represented by formula (205) from the reaction mixture. In some embodiments, the oligonucleotide single strand as represented by formula (205) can be obtained by adding a mixed solvent of acetonitrile and PBS to the reaction solution, centrifuging, adding a nucleic acid precipitation solution (for example, a commercially available 10% trichloroacetic acid aqueous solution) to the resulting supernatant, and separating the precipitate.

[0308] Those skilled in the art can obtain the active ester shown in formula (207) by various methods. In some embodiments, the active ester shown in formula (207) is obtained by using Michael E., et al."Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8(2015):1451-1455, the preparation method disclosed in Scheme 1a(A) was obtained, the only difference being that the initial reactant RCO(CH2)3COOH in Scheme 1a(A) was replaced by a cross-linker compound as shown in formula (208), the entire contents of which are incorporated herein by reference.

[0309] Those skilled in the art can obtain the compound shown in Formula (208) by various methods. In some embodiments, the compound shown in Formula (208) is obtained by the following method: in a solvent, under the hydrolysis conditions of the ester, hydrolyzing the ester shown in Formula (209), and separating and obtaining the compound shown in Formula (208). In some embodiments, the hydrolysis conditions of the ester refer to the presence of a catalyst in an alkaline aqueous solution. In some embodiments, the alkaline aqueous solution is an aqueous solution of sodium hydroxide, and the catalyst is ethanol, wherein the molar ratio of sodium hydroxide to the compound shown in Formula (209) is 7:1-3:1, the concentration of the sodium hydroxide aqueous solution is 2M-5M, and the volume ratio of ethanol to the sodium hydroxide aqueous solution is 7:1-3:1. Those skilled in the art can use various methods to separate the compound shown in Formula (208). In some embodiments, the pH value of the reaction solution can be adjusted to 5-6 using an acidic solution, and then separated by column chromatography to obtain the compound shown in Formula (208).

[0310] Those skilled in the art can obtain the compound represented by formula (209) by various methods. In some embodiments, the compound represented by formula (209) can be prepared by the following method: under condensation reaction conditions, the compound represented by formula (210) and the compound represented by formula (211) are contacted to obtain the compound represented by formula (209):

[0311] In some embodiments, the condensation reaction conditions are amidation reaction conditions, for example, conditions in which 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and N,N-diisopropylethylamine (DIEA) are present in an organic solvent. In some embodiments, the organic solvent is DMF. In some embodiments, the molar ratio of the compound represented by formula (210) to the compound represented by formula (211) is 1:2-1:5. In some embodiments, the molar ratio of the compound represented by formula (210) to 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine is 1:3-1:10, and the amount of DMF used and the amount of the compound represented by formula (210) are 7 ml / g-20 ml / g. Those skilled in the art can isolate the compound represented by formula (209) by various methods. In some embodiments, water and ethyl acetate can be added to the reaction solution to extract an organic phase, which is then washed with 10% citric acid, sodium bicarbonate, and a saturated aqueous salt solution, and the solvent is evaporated to obtain a compound as shown in formula (209). Those skilled in the art can obtain the compound as shown in formula (210) by various methods. In some embodiments, the compound as shown in formula (210) is prepared according to the method of Scheme 3 disclosed in WO2014025805A1. Those skilled in the art can obtain the compound as shown in formula (211) by various methods. In some embodiments, the compound as shown in formula (211) is obtained commercially.

[0312] Those skilled in the art can obtain the oligonucleotide single strand as shown in formula (206) by various methods, for example, by using a phosphoramidite monomer containing an active group at the corresponding position during the synthesis of the oligonucleotide single strand. In some embodiments, the phosphoramidite monomer containing an active group is a 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer. The phosphoramidite monomer is easily available to those skilled in the art, and in some embodiments, the phosphoramidite monomer is commercially available.

[0313] Furthermore, by forming the targeting peptide conjugates of the present disclosure, the present disclosure also provides a use of the carrier peptide of the present disclosure in delivering functional molecules, wherein the functional molecules are selected from one or more of diagnostic agents, small molecule therapeutic agents, functional oligonucleotides, and delivery aids. As previously mentioned, the targeting peptide conjugates of the present disclosure can rapidly, efficiently, and safely deliver functional molecules to the central nervous system in the form of functional groups, and upon reaching the target tissue or cells, release or not release the corresponding functional molecules, thereby exerting diagnostic, preventive, or therapeutic effects.

[0314] Pharmaceutical composition

[0315] In another aspect, the present disclosure provides a pharmaceutical composition comprising the carrier peptide conjugate as described above, or one or more of its pharmaceutically acceptable salts, metabolites or prodrugs, as well as an active ingredient and a pharmaceutically acceptable carrier.

[0316] The pharmaceutically acceptable carrier is one or more of various components conventionally used in the art, such as one or more of a solvent, a protective agent, an osmotic pressure regulator, and other pharmaceutically acceptable carriers.

[0317] For example, when the pharmaceutical composition is an injection, the pharmaceutically acceptable carrier is a solvent, such as deionized water, water for injection, ethanol, or a pH buffer. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0318] The amount of the solvent is adjusted according to the required solution concentration. Based on the functional groups in the carrier peptide conjugate, the concentration of the carrier peptide conjugate in the injection can be 0.01 mg / mL-20 mg / mL, 0.1 mg / mL-10 mg / mL, or 0.5 mg / mL-5 mg / mL.

[0319] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. The content of the protective agent may be 0.01-30% by weight based on the total weight of the pharmaceutical composition.

[0320] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles / kilogram (mOsm / kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage of the preparation made from the pharmaceutical composition during administration may be adjusted due to different administration methods.

[0321] In some embodiments, the pharmaceutical composition can be a liquid formulation, such as an injection, or a lyophilized powder injection, which is mixed with a liquid excipient to form a liquid formulation during administration. The liquid formulation can be administered, but is not limited to, by subcutaneous, intramuscular, intracerebroventricular, or intrathecal injection. The pharmaceutical composition can also be delivered, but is not limited to, via eye drops, nasal administration, oropharyngeal inhalation, spray administration, or the like. In some embodiments, the pharmaceutical composition is administered intravenously or intrathecally. In some embodiments, the pharmaceutical composition is injected intrathecally into the cerebrospinal fluid. The injection can be performed as a bolus injection or via a micropump implanted beneath the skin to provide regular and consistent delivery of the siRNA to the cerebrospinal fluid. In some embodiments, intrathecal administration is performed via a surgically implanted osmotic pump. In some embodiments, an osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration. Further details regarding this intrathecal delivery system can be found in PCT / US2015 / 013253, filed January 28, 2015, which is incorporated herein by reference in its entirety.

[0322] The other pharmaceutically acceptable carriers can be various conventionally used carriers. In some embodiments, the functional group is a double-stranded oligonucleotide group. In this case, the carrier includes but is not limited to magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate) phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

[0323] In some embodiments, the pharmaceutically acceptable carrier contains a physiologically acceptable compound that acts, for example, to stabilize the pharmaceutical composition or increase or decrease the absorption of the conjugate and / or pharmaceutical composition. The physiologically acceptable compound is selected from one or more of the following: carbohydrates, such as glucose, sucrose, and / or dextran; antioxidants, such as ascorbic acid and / or glutathione; chelating agents; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of any co-administered substances; excipients; stabilizers, and buffers. Detergents may also be used to stabilize the composition or increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound may also include one or more wetting agents, emulsifiers, dispersants, or preservatives specifically for preventing microbial growth or action. The physiologically acceptable compounds are known to those skilled in the art and will not be described in detail herein. It will be readily understood by those skilled in the art that the selection of a pharmaceutically acceptable carrier and a physiologically acceptable compound will depend, for example, on the route of administration and the specific physiochemical properties of any co-administered substances.

[0324] In some embodiments, the pharmaceutically acceptable carrier is sterile and generally free of undesirable substances. The pharmaceutical compositions of the present disclosure may further comprise pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, including but not limited to one or more of pH adjusters, buffers, and toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, or sodium lactate. The concentration of the conjugate of the present disclosure in the pharmaceutical composition can vary over a wide range and is primarily selected based on fluid volume, viscosity, body weight, etc. according to a specific administration method.

[0325] In some embodiments, there are no special requirements for the content of the carrier peptide conjugate and the pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of the carrier peptide conjugate to the pharmaceutically acceptable carrier can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).

[0326] Application of the carrier peptide conjugate and pharmaceutical composition disclosed herein

[0327] In some embodiments, the present disclosure further provides a use of a carrier peptide conjugate of the present disclosure, one or more of its pharmaceutically acceptable salts, metabolites, or prodrugs, and / or a pharmaceutical composition of the present disclosure in the preparation of a drug for treating and / or preventing a disease associated with the central nervous system. In some embodiments, the disease is a disease associated with mRNA expressed by a target gene in cells in the central nervous system.

[0328] In some embodiments, the present disclosure also provides a method for treating and / or preventing a disease associated with the central nervous system, the method comprising administering to a subject an effective amount of a carrier peptide conjugate of the present disclosure, a pharmaceutically acceptable salt, metabolite, or prodrug thereof, and / or a pharmaceutical composition of the present disclosure. In some embodiments, the disease is a disease associated with mRNA expression of a target gene in cells in the central nervous system.

[0329] In some embodiments, the target gene in the above uses or methods is APP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR, TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B or RHO. In some embodiments, the target gene is SOD1, APOE4 or RPTOR.

[0330] By administering the carrier peptide conjugates and / or pharmaceutical compositions provided by the present disclosure to a subject in need, the functional group can be targeted and delivered to relevant tissues and / or cells in the central nervous system, thereby exerting a diagnostic, therapeutic, and / or preventive effect through the functional group. For example, when the functional group is an oligonucleotide group, the purpose of preventing and / or treating pathological conditions or diseases caused by the expression of specific cell genes is achieved by regulating gene expression. Therefore, the carrier peptide compositions and / or pharmaceutical compositions provided by the present disclosure can be used to prevent and / or treat the pathological conditions or diseases, or to prepare drugs for preventing and / or treating the pathological conditions or diseases described herein.

[0331] In some embodiments, the functional group in the carrier peptide conjugate of the present disclosure is a diagnostic agent group. The present disclosure also provides a use of the carrier peptide conjugate and / or pharmaceutical composition of the present disclosure in the preparation of a method for diagnosing a disease related to the central nervous system. In some embodiments, the present disclosure also provides a method for diagnosing a disease related to the central nervous system, the method comprising administering an effective amount of the carrier peptide conjugate and / or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0332] As used herein, the term "administration" refers to placing a carrier peptide conjugate and / or pharmaceutical composition in a subject's body by a method or route that at least partially localizes the carrier peptide conjugate and / or pharmaceutical composition to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more carrier peptide conjugate and / or pharmaceutical composition being delivered to a specific site than to the entire body of the subject; whereas systemic administration results in delivery of the carrier peptide conjugate and / or pharmaceutical composition to substantially the entire body of the subject.

[0333] The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), intracerebroventricular administration, intrathecal administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration), intravitreal injection, and eye drop administration. The frequency of administration may be once or more daily, weekly, biweekly, three-weekly, monthly, two-monthly, three-monthly, six-monthly, or annually.

[0334] The dosage of the carrier peptide conjugates and / or pharmaceutical compositions disclosed herein can be conventional dosages in the art, which can be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining the LD50 (lethal dose that kills 50% of the population) and the ED50 (the dose that causes 50% of the maximum response in quantitative reactions and the dose that causes a positive response in 50% of the subjects in qualitative reactions). A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0335] When the method provided by the present disclosure is used to diagnose, treat and / or prevent central nervous system related diseases, the amount of the functional carrier peptide and the functional group in the carrier peptide conjugate and / or pharmaceutical composition provided is that those skilled in the art are easily determined according to the effect obtained by expectation. For example, in some embodiments, the functional group is a double-stranded oligonucleotide, and the functional double-stranded oligonucleotide amount in the carrier peptide conjugate provided is such amount: it is enough to reduce the expression of the target gene, and results in an extracellular concentration of 1pM to 1 μM, or 0.01nM to 100nM, or 0.05nM to 50nM or to about 5nM at the target cell surface. The amount required to reach this local concentration will vary with various factors, including delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, whether delivery is local or systemic, etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

[0336] Reagent test kit

[0337] The present disclosure provides a kit comprising an effective dose of the carrier peptide conjugate and / or pharmaceutical composition provided by the present disclosure.

[0338] In some embodiments, the kits described herein may provide the carrier peptide conjugate and / or pharmaceutical composition in one container. In some embodiments, the kits described herein may include a container for providing a pharmaceutically acceptable excipient. In some embodiments, the kits may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in a container other than the container in which the carrier peptide conjugate and / or pharmaceutical composition described herein is provided. In some embodiments, the kits may include instructions for mixing the carrier peptide conjugate and / or pharmaceutical composition with a pharmaceutically acceptable carrier and / or excipient or other ingredients (if any).

[0339] In the kits of the present disclosure, the carrier peptide conjugate and the pharmaceutically acceptable carrier and / or excipient, as well as the pharmaceutical composition and / or the pharmaceutically acceptable carrier and / or excipient can be provided in any form, such as liquid form, dry form and / or lyophilized form. In some embodiments, the carrier peptide conjugate and the pharmaceutically acceptable carrier and / or excipient, as well as the pharmaceutical composition and / or the optional pharmaceutically acceptable excipient are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure.

[0340] The present disclosure will be further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0341] Example

[0342] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory (1989)).

[0343] With respect to the amino acid sequences in the following examples, unless otherwise specified, the amino acids used in preparing the sequences and the amino acid residues in the sequences are all natural (unmodified) amino acids and natural amino acid residues.

[0344] Preparation Example 1 Synthesis of Conjugate 1 Provided by the Present Disclosure

[0345] (1-1) Preparation of single-stranded oligonucleotide S1

[0346] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 1 in Table 2 was synthesized by solid phase synthesis. The only difference was that, during the solid phase synthesis, before the first nucleoside monomer at the 3' end was connected and after the last nucleoside monomer at the 5' end was connected, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected. The oligonucleotide single chain was cut from the solid phase support to obtain the oligonucleotide single chain S1 (45.00 mg, 6.61 μmol) as shown in formula (1-1-1) (molecular weight: 6807.15, measured: 6807.91):

[0347] Where, Represents the sequence shown as SEQ ID NO:127.

[0348] 5'-UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm-3' (SEQ ID NO: 127).

[0349] (1-2) Synthesis of single-stranded oligonucleotide S2:

[0350] After dissolving S1 (45.00 mg, 6.61 μmol) prepared in (1-1) in 5.00 ml of purified water, TCEP aqueous solution (67.50 mg, 0.24 mmol, purchased from Bidex Pharmaceuticals, batch number: BD155793) was added to the resulting solution at a weight ratio of TCEP to S1 of 1.5:1. The mixture was mixed and allowed to react at room temperature for 2 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 14 mL of reaction solution. The reaction solution was transferred to a 15 mL, 3K specification ultrafiltration tube and centrifuged at 3900 rpm for 30 min. The ultrafiltration and centrifugation steps were repeated until the conductivity of the ultrafiltration liquid was measured to be below 100 s. The product on the filter membrane was collected to obtain the single-stranded oligonucleotide S2 (42.00 mg, 6.42 μmol, yield: 97.10%) (molecular weight: 6543.47, measured: 6542.57).

[0351] (1-3) Synthesis of single-stranded oligonucleotide S3:

[0352] S2 (42.00 mg, 6.61 μmol) prepared in step (1-2) was dissolved in 7.00 ml of purified water in a 15 ml centrifuge tube. After complete dissolution, Py-SS-Py (0.1515 g, 0.68 mmol, purchased from Aladdin, batch number: F2015072) was added to the centrifuge tube. The mixture was vortexed and reacted at room temperature for 6 hours. The reaction was detected using an Agilent 1260 HPLC instrument. After the reaction was complete, the reaction solution was diluted with 20% (v / v) ethanol aqueous solution, filtered, and purified by gel desalting using a purifier (purchased from Lisui Technology Co., Ltd.) using a mobile phase of 20% (v / v) ethanol aqueous solution and a collection wavelength of 280 nm. The eluate was collected and concentrated to obtain 41.62 mg (6.15 μmol, yield: 93.11%) of the product (molecular weight: 6761.76, measured: 6761.06).

[0353] (1-4) Synthesis of the positive chain in carrier peptide conjugate 1

[0354] Wherein, PP represents a carrier peptide group having a sequence as shown in SEQ ID NO: 1.

[0355] S3 (40.00 mg, 5.92 μmol) prepared in step (1-3) was dissolved in 8.00 ml of 0.1 M aqueous ammonium acetate solution (ammonium acetate was purchased from Aladdin, No. A112061). HS-P7 (custom-synthesized from Tanzhen Biotech) was added to the reaction solution, wherein HS-P7 is a commercially available compound RSLGDTG-Cys-NH2 containing a carrier peptide group and containing the sequence shown in SEQ ID NO: 1 (RSLGDTG) in the amino-terminal to carboxyl-terminal direction, wherein Cys represents a cysteine ​​group. After mixing, the reaction was allowed to react at room temperature for 6 hours. The reaction solution was diluted with an equal volume of purified water, filtered, and purified using an Agilent semi-preparative reverse phase column using a Kromasil 100-10-C18 column. 10um, 21.2*250mm; gradient elution with 100mM TEAA (PH=7.0-7.3): acetonitrile in a ratio of 5%-75% (V / V) was performed, and the product peak eluate was collected and concentrated to obtain 20.50mg (2.53μmol, yield: 41.19%) of the positive chain S4 (molecular weight: 8613.86, measured: 8612.76).

[0356] (1-5) Synthesis of Conjugate 1

[0357] in, Represents the siRNA group obtained by annealing the sense chain and the antisense chain.

[0358] The antisense strand in carrier peptide conjugate 1 was prepared according to the method described in Preparation Example 1 of WO2019105418 (A1), with the only difference being that the nucleoside monomers were sequentially linked according to the sequence shown in SEQ ID NO: 129 in Table 2; the sense strand prepared in steps (1-4) and the antisense strand prepared in steps (1-5) were dissolved in equimolar amounts using DEPC water, and then annealed to obtain conjugate 1 in Table 2. After the preparation was completed, the molecular weights of the sense and antisense strands were detected by LC-MS, respectively. The theoretical molecular weight of the sense strand was 8625.760 and the measured molecular weight was 8624.48, while the theoretical molecular weight of the antisense strand was 7096.788 and the measured molecular weight was 7095.82. The measured values ​​were consistent with the theoretical values, indicating that the obtained conjugate was a carrier peptide conjugate having the sequence corresponding to conjugate 1 in Table 2. Conjugate 1 is a double-stranded siRNA conjugate with one carrier peptide group conjugated to each end of the sense strand.

[0359] Table 2 Sequences of conjugates and siRNA

[0360] In Table 2, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m represents that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of letter f is a fluorinated-modified nucleotide; lowercase letter s represents that the two nucleotides to the left of letter s are connected by a thiophosphate group; the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; the capital letter combination -SS- represents a linking group, and the linking group has the structure shown in formula (103), wherein n 103 =1,n 101 =1,m 103 =6, R 107 is an amino group, the capital letter H represents a hydrogen atom; the capital letter combination -S'-S'- represents a linking group, and the linking group has a structure shown in formula (104), wherein n 104 =1,n 102 =1,m 104=6, capital H represents a hydrogen atom; P represents a carrier peptide group having a sequence as shown in SEQ ID NO: 1 from the N-terminal to the C-terminal direction, and the carboxyl group of the C-terminal amino acid residue in the sequence is protected by an amino group; P1 represents a carrier peptide group having a sequence as shown in SEQ ID NO: 149 from the N-terminal to the C-terminal direction, and the carboxyl group of the C-terminal amino acid residue in the sequence is protected by an amino group, and the carrier peptide group P2 is an amide group formed by the carboxyl group of the C-terminal amino acid residue of the carrier peptide having the sequence as shown in SEQ ID NO: 149, and a group formed by removing an amino hydrogen atom from the N-terminal amino acid residue; P2 represents a carrier peptide group having a sequence as shown in SEQ ID NO: 149 from the N-terminal to the C-terminal direction. The carrier peptide group of the sequence shown in NO:150, and the K and D in the sequence are connected by an amide bond formed by an amino group on the side chain and a carboxyl group through an amide reaction, so that the carrier peptide group forms a cyclic peptide, and the carboxyl group of the C-terminal amino acid residue is protected by an amino group. The carrier peptide group P2 is formed by forming an amide group with the carboxyl group of the C-terminal amino acid residue of the carrier peptide shown in formula (21), and a group formed by removing an amino hydrogen atom from the N-terminal amino acid residue; P3 represents a carrier peptide group having a sequence shown in SEQ ID NO:151 in the N-terminal to C-terminal direction, and S5 and S5 in the sequence are connected by a carbon-carbon double bond through a condensation reaction between the side chains, so that the carrier peptide group forms a cyclic peptide, and the carboxyl group of the C-terminal amino acid residue is protected by an amino group. The carrier peptide group P3 is formed by forming an amide group with the carboxyl group of the C-terminal amino acid residue of the carrier peptide shown in formula (22), and a group formed by removing an amino hydrogen atom from the N-terminal amino acid residue. P4 has the following structure as shown in formula (15-3-1). P5 has the following structure as shown in formula (17-3-1).

[0361] Preparation Example 2 Synthesis of Carrier Peptide Conjugate 2 Provided by the Present Disclosure

[0362] (2-1) Preparation of single-stranded oligonucleotide S1

[0363] According to step (1-1) in Preparation Example 1, single-stranded oligonucleotide S1 was prepared.

[0364] (2-2) Synthesis of single-stranded oligonucleotide S2:

[0365] According to steps (1-2) in Preparation Example 1, single-stranded oligonucleotide S2 was prepared.

[0366] (2-3) Synthesis of active carrier peptide 1:

[0367] Active carrier peptide 1 was synthesized according to the peptide solid phase synthesis method. The specific steps are as follows:

[0368] (2-3-1) Deprotection: 0.5 g of Rink amide MBHA resin (amino-protected resin, purchased from Gill Biochemicals) with a degree of substitution of 0.67 mmol / g was added with 10 mL of a 20 v / v% piperidine solution in DMF. The mixture was reacted at room temperature for 25 min to obtain an amino-deprotected resin.

[0369] (2-3-2) Coupling: 0.67 mmol of amino-protected amino acid G monomer: Fmoc-Gly-OH (Adamas brand, purchased from Shanghai Titan Technology Co., Ltd.) and 1.34 mmol of 1-hydroxybenzotriazole (HOBT) were dissolved in 20 mL of DMF. 1.34 mmol of N,N-diisopropylcarbodiimide (DIC) was added and reacted at room temperature for 5 min to obtain an activated amino acid solution.

[0370] The obtained activated amino acid solution was mixed with the amino-deprotected resin obtained in step (2-3-1), and the mixture was reacted at room temperature and normal pressure for 120 min. The solvent in the reaction solution was removed under reduced pressure, and the mixture was washed with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain a resin with an amino-protected amino acid residue connected thereto.

[0371] (2-3-3) Repeat the deprotection method in step (2-3-1) and the coupling method in step (2-3-2) on the resin to which the amino group-protected amino acid residue is attached, and sequentially couple the amino acid monomers corresponding to the remaining amino acid residues in the amino acid sequence shown in SEQ ID NO: 149 from the carboxyl terminus to the amino terminus to obtain a peptide resin to which the eight amino acid residues according to the amino acid sequence shown in SEQ ID NO: 149 are attached and the terminal amino group is protected:

[0372] CRSLGDTG (SEQ ID NO: 149).

[0373] (2-3-4) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5; after all monomers are connected, contact the peptide resin with terminal amino group protection with the cleavage solution at room temperature and normal pressure, with 5 mL of cleavage solution per 0.5 g of resin. React for 3 hours, filter, collect the filtrate, add it to 4 volumes of MTBE of the filtrate, and centrifuge to obtain a solid.

[0374] (2-3-5) Oxidative coupling: The solid obtained in step (2-3-4) was dissolved in a water / methanol mixture (water:methanol = 1:5 v / v). 1.00 mmol of disulfide dipyridine was added to the dissolved solid, and the mixture was shaken at room temperature for 0.5 h to obtain a product mixture of a peptide compound containing a disulfide bond.

[0375] (2-3-6) The product mixture was concentrated under reduced pressure to remove the solvent, and then purified by reverse-phase preparative column chromatography. The fractions containing the product were collected, concentrated to remove the solvent, and lyophilized to obtain active carrier peptide 1. Column chromatography conditions were as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2 × 250 mm; mobile phase: gradient elution, mobile phase A: 0.1% formic acid / water, mobile phase B: acetonitrile; gradient conditions: 0-6 min, 5%-95% B; 6-11 min, 95% B.

[0376] The obtained active carrier peptide 1 has a structure as shown in formula (920):

[0377] The obtained active carrier peptide 1 was diluted to a concentration of 0.2 mg / mL using ultrapure water (resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The theoretical molecular weight was 958.08, and the measured molecular weight was 957.38. The measured value was consistent with the theoretical value, indicating that the active carrier peptide 1 was obtained.

[0378] (2-4) Preparation of carrier peptide conjugate 2

[0379] (2-4-1) Preparation of single-chain oligonucleotide-carrier peptide 1 compound

[0380] Oligonucleotide single-strand S2 (40 mg, 1.0 eq) was dissolved in 3.9 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S2 solution. Active carrier peptide 1 (59 mg, 10.0 eq) was dissolved in 3.9 mL of DMF solution, and the obtained active carrier peptide 1 solution was added to the oligonucleotide single-strand S2 solution, mixed, and reacted at room temperature for 2 hours. The reaction solution was filtered through a 0.22 μm microporous filter membrane and then purified using the semi-preparative reverse phase purification described above. The fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 1 compound having a structure as shown in formula (2-4-1) (25 mg, yield 25 mg / 40 mg = 62.5%).

[0381] Molecular weight determination was performed on a liquid chromatography-mass spectrometry (LC-MS, Waters, model LCT Premier). The theoretical molecular weight was 8237.30, and the measured molecular weight was 8236.42. The LC-MS molecular weight was consistent with the theoretical value, indicating that the oligonucleotide single-chain-carrier peptide 1 compound with the structure shown in formula (2-4-1) was successfully obtained.

[0382] (2-4-2) Preparation of carrier peptide conjugate 2

[0383] The antisense strand in carrier peptide conjugate 2 was prepared according to the method described in Preparation Example 1 of WO2019105418 (A1), except that the nucleoside monomers were linked sequentially according to the sequence shown in SEQ ID NO: 129 in Table 2. The molecular weight of the antisense strand was determined by LC-MS, with a theoretical value of 7096.79 and a measured value of 7095.24, which was consistent with the theoretical value.

[0384] Equimolar amounts of the single-stranded oligonucleotide-carrier peptide 1 compound prepared in step (2-4-1) and the antisense strand prepared in step (2-4-2) were dissolved in DEPC water and then annealed to obtain Conjugate 2 shown in Table 2. Conjugate 2 is a double-stranded siRNA conjugate with a carrier peptide group attached to each end of the sense strand. It differs from Conjugate 1 in that the carrier peptide group is attached to the sense strand at the N-terminus.

[0385] Preparation Example 3 Synthesis of Carrier Peptide Conjugate 3 Provided by the Present Disclosure

[0386] (3-1) Preparation of single-stranded oligonucleotide S1

[0387] The single-stranded oligonucleotide S1 was prepared according to the steps (1-1) in Preparation Example 1.

[0388] (3-2) Synthesis of single-stranded oligonucleotide S2:

[0389] The single-stranded oligonucleotide S2 was prepared according to the steps (1-2) in Preparation Example 1.

[0390] (3-3) Synthesis of active carrier peptide 2:

[0391] Active carrier peptide 2 was synthesized according to the peptide solid phase synthesis method. The specific steps are as follows:

[0392] (3-3-1) Deprotection: 1.0 g of Rink amide MBHA resin with a degree of substitution of 0.32 mmol / g was added to 20 mL of a 20 v / v% piperidine solution in DMF. The mixture was reacted at room temperature for 25 min to obtain an amino-deprotected resin.

[0393] (3-3-2) Coupling: 0.67 mmol of amino-protected amino acid G monomer (Fmoc-Gly-OH) and 1.34 mmol of HOBT were dissolved in 20 mL of DMF, and 1.34 mmol of DIC was added. The mixture was reacted at room temperature for 5 minutes to obtain an activated amino acid solution.

[0394] The obtained activated amino acid solution was mixed with the amino-deprotected resin obtained in step (3-3-1), and the mixture was reacted at room temperature and normal pressure for 120 min. The solvent in the reaction solution was removed under reduced pressure, and the mixture was washed with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain a resin with an amino-protected amino acid residue connected thereto.

[0395] (3-3-3) Repeat the deprotection method in step (3-3-1) and the coupling method in step (3-3-2) on the resin to which the amino group-protected amino acid residue is attached, and sequentially couple the amino acid monomers corresponding to the remaining amino acids in the amino acid sequence shown in SEQ ID NO: 150 from the carboxyl terminus to the amino terminus to obtain a peptide resin to which the 9 amino acid residues according to the amino acid sequence shown in SEQ ID NO: 150 are attached and the terminal amino group is protected:

[0396] CKRSLGDTG (SEQ ID NO: 150).

[0397] (3-3-4) Deprotection and cyclization: 0.1 mmol of tetrakistriphenylphosphine palladium and 0.3 mmol of 5,5-dimethyl-1,3-cyclohexanedione were dissolved in 20 mL of DCM. The dissolved solution was poured into the peptide resin with protected terminal amino group obtained in step (3-3-3). Argon was introduced to the reaction for 1 hour. The solvent in the reaction solution was removed under reduced pressure. At room temperature and normal pressure, the mixture was washed with DCM, DMF, DCM, and DMF in sequence, 25 mL each time, to obtain a deprotected peptide resin.

[0398] 0.64 mmol HATU and 0.64 mmol DIEA were dissolved in 20 mL of DMF. The dissolved solution was added to the deprotected peptide resin obtained in the above step, and argon was introduced into the reaction solution for 3 hours. The solvent in the reaction solution was removed under reduced pressure. At room temperature and normal pressure, the solution was washed with DCM, DMF, DCM, and DMF in sequence, 25 mL each time, to obtain a cyclized peptide resin.

[0399] (3-3-5) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5. After all monomers are connected, the cyclized peptide resin obtained in the above step is contacted with the cleavage solution at room temperature and normal pressure. The amount of cleavage solution is 5 mL per 0.5 g of resin. The reaction is carried out for 3 hours, filtered, and the filtrate is collected and added to 4 volumes of MTBE of the filtrate. The solid is obtained by centrifugation.

[0400] (3-3-6) Activation: Dissolve the solid obtained in step (3-3-5) in a water / methanol mixture (water:methanol = 1:5 v / v), add 1.00 mmol of disulfide dipyridine to the dissolved solid, and shake the mixture at room temperature for 0.5 h to obtain a product mixture of a cyclic peptide compound containing a disulfide bond.

[0401] (3-3-7) The product mixture obtained in step (3-3-6) was concentrated under reduced pressure to remove the solvent, and then purified by reverse-phase preparative column chromatography. The fractions containing the product were collected, concentrated to remove the solvent, and lyophilized to obtain active carrier peptide 2. Column chromatography conditions were as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2 × 250 mm; mobile phase: gradient elution, mobile phase A: 0.1% formic acid / water, mobile phase B: acetonitrile; gradient conditions: 0-12 min, 5%-95% B; 12-20 min, 95% B.

[0402] The obtained active carrier peptide 2 has the structure shown in formula (930):

[0403] The obtained active carrier peptide 2 was diluted to a concentration of 0.2 mg / mL using ultrapure water (resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The theoretical molecular weight was 1068.24, and the measured molecular weight was 1067.46. The measured value was consistent with the theoretical value, indicating that the active carrier peptide 2 was obtained.

[0404] (3-4) Preparation of carrier peptide conjugate 3

[0405] (3-4-1) Preparation of single-chain oligonucleotide-carrier peptide 2 compound

[0406] Oligonucleotide single-strand S2 (10 mg, 1.0 eq) was dissolved in 1.0 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S2 solution. Active carrier peptide 2 (16 mg, 10.0 eq) was dissolved in 1.0 mL of DMF solution, and the obtained active carrier peptide 2 solution was added to the oligonucleotide single-strand S2 solution, mixed, and reacted at room temperature for 2 hours. The reaction solution was filtered through a 0.22 μm microporous filter membrane and then purified using the semi-preparative reverse phase purification described above. The fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 2 compound having a structure as shown in formula (3-4-1) (6.1 mg, yield 6.1 mg / 10 mg = 61.0%).

[0407] Molecular weight determination was performed on a liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The theoretical molecular weight was 8457.62, and the measured molecular weight was 8456.79. The LC-MS molecular weight was consistent with the theoretical value, indicating that the oligonucleotide single-chain-carrier peptide 2 compound with the structure shown in formula (3-4-1) was successfully obtained.

[0408] (3-4-2) Preparation of carrier peptide conjugate 3

[0409] The antisense strand in carrier peptide conjugate 3 was prepared according to the method described in Preparation Example 1 of WO2019105418 (A1), except that the nucleoside monomers were linked sequentially according to the sequence shown in SEQ ID NO: 129 in Table 2. The molecular weight of the antisense strand was determined by LC-MS, with a theoretical value of 7096.788 and a measured value of 7095.24, which was consistent with the theoretical value.

[0410] Equimolar amounts of the oligonucleotide single-stranded-carrier peptide 2 compound prepared in step (3-4-1) and the antisense strand prepared in step (3-4-2) were dissolved in DEPC water and then annealed to obtain Conjugate 3 shown in Table 2. Conjugate 3 is a double-stranded siRNA conjugate with one cyclic carrier peptide group attached to each end of the sense strand.

[0411] Preparation Example 4 Synthesis of Carrier Peptide Conjugate 4 Provided by the Present Disclosure

[0412] (4-1) Preparation of single-stranded oligonucleotide S1

[0413] The single-stranded oligonucleotide S1 was prepared according to the steps (1-1) in Preparation Example 1.

[0414] (4-2) Synthesis of single-stranded oligonucleotide S2:

[0415] The single-stranded oligonucleotide S2 was prepared according to the steps (1-2) in Preparation Example 1.

[0416] (4-3) Synthesis of active carrier peptide 3:

[0417] Active carrier peptide 3 was synthesized according to the peptide solid phase synthesis method. The specific steps are as follows:

[0418] (4-3-1) Deprotection: 1.0 g of Rink amide MBHA resin with a degree of substitution of 0.32 mmol / g was added to 20 mL of a 20 v / v% piperidine solution in DMF. The mixture was reacted at room temperature for 25 min to obtain an amino-deprotected resin.

[0419] (4-3-2) Coupling: 0.67 mmol of amino-protected amino acid G monomer (Fmoc-Gly-OH) and 1.34 mmol of HOBT were dissolved in 20 mL of DMF, and 1.34 mmol of DIC was added. The mixture was reacted at room temperature for 5 minutes to obtain an activated amino acid solution.

[0420] The obtained activated amino acid solution was mixed with the amino-deprotected resin obtained in step (4-3-1), and the mixture was reacted at room temperature and normal pressure for 120 min. The solvent in the reaction solution was removed under reduced pressure, and the mixture was washed with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain a resin with an amino-protected amino acid residue connected thereto.

[0421] (4-3-3) Repeat the deprotection method in step (4-3-1) and the coupling method in step (4-3-2) for the resin to which the amino group-protected amino acid residue is attached, and sequentially couple the remaining amino acid monomers or S5 monomer ((2R)-2-N-fluorenylmethoxycarbonylamino-2-methyl-6-heptenoic acid) in the direction from the carboxyl terminus to the amino terminus according to the sequence shown in SEQ ID NO: 151 to obtain a peptide resin to which the 9 amino acid residues according to the amino acid sequence shown in SEQ ID NO: 151 are attached and the terminal amino group is protected:

[0422] CS5RSLS5DTG (SEQ ID NO: 151).

[0423] (4-3-4) Cyclization: Dissolve 0.32 mmol of phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride in 20 mL of DMF, add the dissolved solution to the amino-terminally protected peptide resin obtained in step (4-3-3), and allow argon to flow through the reaction mixture for 2 hours. Remove the solvent from the reaction mixture under reduced pressure, and wash with DCM, DMF, DCM, and DMF, 25 mL each time, at room temperature and atmospheric pressure to obtain a stapled cyclic peptide resin.

[0424] (4-3-5) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5. The cyclized stapled cyclic peptide resin obtained in the above step is contacted with the cleavage solution at room temperature and normal pressure. The amount of cleavage solution is 5 mL per 0.5 g of resin. The reaction is carried out for 3 hours, filtered, and the filtrate is collected and added to 4 volumes of MTBE of the filtrate. The solid is obtained by centrifugation.

[0425] (4-3-6) Activation: Dissolve the solid obtained in step (4-3-5) in a water / methanol mixture (water:methanol = 1:5 v / v), add 1.00 mmol of disulfide dipyridine to the dissolved solid, and shake the mixture at room temperature for 0.5 h to obtain a product mixture of a stapled cyclic peptide compound containing a disulfide bond.

[0426] (4-3-7) The product mixture obtained in step (4-3-6) was concentrated under reduced pressure to remove the solvent, and then purified by reverse-phase preparative column chromatography. The fractions containing the product were collected, concentrated to remove the solvent, and lyophilized to obtain active carrier peptide 3. Column chromatography conditions were as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2 × 250 mm; mobile phase: gradient elution, mobile phase A: 0.1% formic acid / water, mobile phase B: acetonitrile; gradient conditions: 0-12 min, 5%-95% B; 12-20 min, 95% B.

[0427] The obtained active carrier peptide 3 has the structure shown in formula (940):

[0428] The obtained active carrier peptide 3 was diluted to a concentration of 0.2 mg / mL using ultrapure water (resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The theoretical molecular weight was 1151.37, and the measured molecular weight was 1150.53. The measured value was consistent with the theoretical value, indicating that the active carrier peptide 3 was obtained.

[0429] (4-4) Preparation of carrier peptide conjugate 4

[0430] (4-4-1) Preparation of single-chain oligonucleotide-carrier peptide 3 compound

[0431] Oligonucleotide single-strand S2 (10 mg, 1.0 eq) was dissolved in 1.0 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S2 solution. Active carrier peptide 3 (18 mg, 10.0 eq) was dissolved in 1.0 mL of DMF solution, and the obtained active carrier peptide 3 solution was added to the oligonucleotide single-strand S2 solution, mixed, and reacted at room temperature for 2 hours. The reaction solution was filtered through a 0.22 μm microporous filter membrane and then purified using the semi-preparative reverse phase purification described above. The fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 3 compound having a structure as shown in formula (4-4-1) (6.4 mg, yield 6.4 mg / 10 mg = 64.0%).

[0432] Molecular weight determination was performed on a liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The theoretical molecular weight was 8623.88, and the measured molecular weight was 8623.10. The LC-MS molecular weight was consistent with the theoretical value, indicating that the oligonucleotide single-chain-carrier peptide 3 compound with the structure shown in formula (4-4-1) was successfully obtained.

[0433] (4-4-2) Preparation of carrier peptide conjugate 4

[0434] The antisense strand in carrier peptide conjugate 4 was prepared according to the method described in Preparation Example 1 of WO2019105418 (A1), except that the nucleoside monomers were linked sequentially according to the sequence shown in SEQ ID NO: 129 in Table 2. The molecular weight of the antisense strand was determined by LC-MS, with a theoretical value of 7096.788 and a measured value of 7095.24, which was consistent with the theoretical value.

[0435] Equimolar amounts of the single-stranded oligonucleotide-carrier peptide 3 compound prepared in step (4-4-1) and the antisense strand prepared in step (4-4-2) were dissolved in DEPC water and then annealed to obtain Conjugate 4 shown in Table 2. Conjugate 4 is a double-stranded siRNA conjugate with a cyclic carrier peptide group attached to each end of the sense strand, wherein the two nucleotides of the carrier peptide are linked by an alkenylene group to form a cyclic structure.

[0436] Preparation Example 5 Synthesis of Carrier Peptide Conjugate 5 Provided by the Present Disclosure

[0437] (5-1) Preparation of single-stranded oligonucleotide S51

[0438] The single-stranded oligonucleotide S51 was prepared according to step (1-1) in Preparation Example 1, except that the nucleotide sequence shown in SEQ ID NO: 127 in Preparation Example 1 was replaced with the nucleotide sequence shown in SEQ ID NO: 154:

[0439] 5'-UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmsAmsAm-3' (SEQ ID NO: 154).

[0440] (5-2) Synthesis of single-stranded oligonucleotide S52:

[0441] According to the steps (1-2) in Preparation Example 1, single-stranded oligonucleotide S52 was prepared.

[0442] (5-3) Synthesis of active carrier peptide 1

[0443] Active carrier peptide 1 was prepared according to steps (2-3) in Preparation Example 2.

[0444] (5-4) Preparation of carrier peptide conjugate 5

[0445] (5-4-1) Preparation of single-chain oligonucleotide-carrier peptide 1 compound

[0446] Oligonucleotide single-chain-carrier peptide 1 was prepared according to step (2-4-1) of Preparation Example 2, except that oligonucleotide single-chain S52 (35 mg, 1.0 eq) was used instead of oligonucleotide single-chain S2. This step yielded 20.5 mg of oligonucleotide single-chain-carrier peptide 1 (yield 20.5 mg / 35 mg = 58.6%), with a theoretical molecular weight of 8920.60 and a detected molecular weight of 8919.10.

[0447] (5-4-2) Preparation of carrier peptide conjugate 5

[0448] The sequence of the siRNA antisense strand in carrier peptide conjugate 5 in Table 2 was synthesized by solid-phase synthesis according to the method described in Preparation Example 1 of WO2019105418 (A1), with the only difference being that the nucleoside monomers were linked sequentially according to the sequence shown in SEQ ID NO: 156 in Table 2. The molecular weight of the antisense strand was determined by LC-MS, with a theoretical value of 7851.18 and a measured value of 7850.39.

[0449] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 1 compound prepared in step (5-4-1) and the antisense chain prepared in step (5-4-2) were dissolved in DEPC water, and then annealed to obtain conjugate 5 in Table 2. Conjugate 5 has the same structure as conjugate 2, except that the 1st to 3rd nucleotides at the 3' end of the sense chain are linked by phosphorothioate groups, and the 5' nucleotide of the antisense chain is a 5'-vinyl phosphate (VP)-modified nucleotide.

[0450] Preparation Example 6 Synthesis of Carrier Peptide Conjugate 6 Provided by the Present Disclosure

[0451] (6-1) Preparation of single-stranded oligonucleotide S1

[0452] According to step (1-1) in Preparation Example 1, single-stranded oligonucleotide S1 was prepared.

[0453] (6-2) Synthesis of single-stranded oligonucleotide S2:

[0454] According to steps (1-2) in Preparation Example 1, single-stranded oligonucleotide S2 was prepared.

[0455] (6-3) Synthesis of active carrier peptide 2

[0456] Active carrier peptide 2 was prepared according to step (3-3) in Preparation Example 3.

[0457] (6-4) Preparation of carrier peptide conjugate 6

[0458] (6-4-1) Preparation of Single-chain Oligonucleotide-Carrier Peptide 2 Compound

[0459] According to the steps (3-4-1) in Preparation Example 3, the oligonucleotide single chain-carrier peptide 2 compound was prepared.

[0460] (6-4-2) Preparation of carrier peptide conjugate 6

[0461] The antisense chain in carrier peptide conjugate 6 was prepared according to step (5-4-2) in Preparation Example 5.

[0462] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 2 compound prepared in step (6-4-1) and the antisense chain prepared in step (6-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 6 shown in Table 2. After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 8459.633, and the measured molecular weight was 8457.11. The theoretical molecular weight of the antisense chain was 7172.779, and the measured molecular weight was 7172.01. The measured values ​​were consistent with the theoretical values. Conjugate 6 has the same structure as conjugate 3, except that the 5' nucleotide of the antisense chain is a 5'-vinyl phosphate (VP)-modified nucleotide.

[0463] Preparation Example 7 Synthesis of Carrier Peptide Conjugate 7 Provided by the Present Disclosure

[0464] (7-1) Preparation of single-stranded oligonucleotide S71

[0465] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 7 in Table 2 was synthesized by a solid phase synthesis method. The only difference was that, during the solid phase synthesis process, before connecting the first nucleoside monomer at the 3' end, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected, and the oligonucleotide single chain was cut from the solid phase support to obtain the oligonucleotide single chain S71 shown in formula (7-1-1).

[0466] Where, Represents the sequence shown as SEQ ID NO:175.

[0467] (7-2) Synthesis of single-stranded oligonucleotide S72:

[0468] After dissolving 45.00 mg of the single-stranded oligonucleotide S71 prepared in step (7-1) in 5.00 mL of purified water, a TCEP aqueous solution was added to the resulting solution at a weight ratio of 1:1 between TCEP and S71. Mix well and react at room temperature for 3 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 14 mL of reaction solution. The reaction solution was transferred to a 15 mL, 3K specification ultrafiltration tube and centrifuged at 3200 rpm for 30 minutes. The ultrafiltration and centrifugation steps were repeated until the conductivity of the ultrafiltration liquid was measured to be below 100 s. The product in the filter membrane was collected to obtain the single-stranded oligonucleotide S72 with a yield of more than 85%.

[0469] (7-3) Synthesis of active carrier peptide 1

[0470] Active carrier peptide 1 was prepared according to steps (2-3) in Preparation Example 2.

[0471] (7-4) Preparation of carrier peptide conjugate 7

[0472] (7-4-1) Preparation of Single-chain Oligonucleotide-Carrier Peptide 1 Compound

[0473] Dissolve 1.0eq of oligonucleotide single-strand S72 with 5mL of 0.1M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S72 solution. Dissolve 4.0eq of active carrier peptide 1 with 5mL of DMF solution, add the obtained active carrier peptide 1 solution to the oligonucleotide single-strand S72 solution, mix well, and react at room temperature for 3 hours. Add four times the volume of 1×PBS / acetone mixed solution (the volume ratio of 1×PBS and acetone is 1:14) to the obtained reaction solution, mix well, centrifuge at 3200rpm for 5min, take the precipitate after centrifugation and dissolve it in purified water, filter the reaction solution with a 0.45μm microporous filter membrane, and purify the reaction solution with the above-mentioned semi-preparative reverse phase purification. Collect the components containing the product and concentrate to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 1 compound having a structure shown in formula (7-4-1) with a yield of 66%.

[0474] (7-4-2) Preparation of carrier peptide conjugate 7

[0475] The antisense chain in carrier peptide conjugate 7 was prepared according to step (5-4-2) in Preparation Example 5.

[0476] Equimolar amounts of the oligonucleotide single-stranded peptide 1 compound prepared in step (7-4-1) and the antisense strand prepared in step (7-4-2) were dissolved in DEPC water and subsequently annealed to obtain Conjugate 7 (Table 2). After preparation, the molecular weights of the sense and antisense strands were determined by LC-MS. The theoretical molecular weight of the sense strand was 7195.188, and the measured molecular weight was 7193.58. The theoretical molecular weight of the antisense strand was 7172.779, and the measured molecular weight was 7172.02. The measured values ​​were consistent with the theoretical values. Conjugate 7 is a double-stranded siRNA conjugate with a carrier peptide group attached to the 3' end of the sense strand.

[0477] Preparation Example 8 Synthesis of Carrier Peptide Conjugate 8 Provided by the Present Disclosure

[0478] (8-1) Preparation of single-stranded oligonucleotide S1

[0479] According to step (1-1) in Preparation Example 1, single-stranded oligonucleotide S1 was prepared.

[0480] (8-2) Synthesis of single-stranded oligonucleotide S2:

[0481] According to steps (1-2) in Preparation Example 1, single-stranded oligonucleotide S2 was prepared.

[0482] (8-3) Synthesis of active carrier peptide 1

[0483] Active carrier peptide 1 was prepared according to steps (2-3) in Preparation Example 2.

[0484] (8-4) Preparation of carrier peptide conjugate 8

[0485] (8-4-1) Preparation of single-chain oligonucleotide-carrier peptide 1 compound

[0486] According to the steps (2-4-1) in Preparation Example 2, the oligonucleotide single chain-carrier peptide 1 compound was prepared.

[0487] (8-4-2) Preparation of carrier peptide conjugate 8

[0488] The antisense chain in carrier peptide conjugate 8 was prepared according to step (5-4-2) in Preparation Example 5.

[0489] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 1 compound prepared in step (8-4-1) and the antisense chain prepared in step (8-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 8 shown in Table 2. After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 8271.435, and the measured molecular weight was 8268.81. The theoretical molecular weight of the antisense chain was 7172.779, and the measured molecular weight was 7172.13. The measured values ​​were consistent with the theoretical values. Conjugate 8 has the same structure as conjugate 5, except that the 1st to 3rd nucleotides at the 3' end of the sense chain are linked by phosphate groups.

[0490] Preparation Example 9 Synthesis of Carrier Peptide Conjugate 9 Provided by the Present Disclosure

[0491] (9-1) Preparation of single-stranded oligonucleotide S1

[0492] According to step (7-1) of Preparation Example 7, single-stranded oligonucleotide S71 was prepared.

[0493] (9-2) Synthesis of single-stranded oligonucleotide S2:

[0494] According to step (7-2) of Preparation Example 7, single-stranded oligonucleotide S72 was prepared.

[0495] (9-3) Synthesis of active carrier peptide 2

[0496] Active carrier peptide 2 was prepared according to step (3-3) in Preparation Example 3.

[0497] (9-4) Preparation of carrier peptide conjugate 9

[0498] (9-4-1) Preparation of Single-chain Oligonucleotide-Carrier Peptide 2 Compound

[0499] Dissolve 1.0eq of oligonucleotide single-strand S72 with 5mL of 0.1M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S72 solution. Dissolve 4.0eq of active carrier peptide 2 with 5mL of DMF solution, add the obtained active carrier peptide 2 solution to the oligonucleotide single-strand S72 solution, mix well, and react at room temperature for 3 hours. Add four times the volume of 1×PBS / acetone mixed solution (the volume ratio of 1×PBS and acetone is 1:14) to the obtained reaction solution, mix well, centrifuge at 3200rpm for 5min, take the precipitate after centrifugation and dissolve it in purified water, filter the reaction solution with a 0.45μm microporous filter membrane, and purify the reaction solution with the above-mentioned semi-preparative reverse phase purification. Collect the components containing the product and concentrate to remove the solvent to obtain an oligonucleotide single-stranded-carrier peptide 2 compound having a structure shown in formula (9-4-1) with a yield of 60%.

[0500] (9-4-2) Preparation of carrier peptide conjugate 9

[0501] The antisense chain in carrier peptide conjugate 9 was prepared according to the steps (5-4-2) in Preparation Example 5.

[0502] Equimolar amounts of the oligonucleotide single-stranded-carrier peptide 2 compound prepared in step (9-4-1) and the antisense chain prepared in step (9-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 9 in Table 2. After preparation, the molecular weights of the sense and antisense chains were detected by LC-MS. The theoretical molecular weight of the sense chain was 7305.348 and the measured molecular weight was 7303.64, while the theoretical molecular weight of the antisense chain was 7172.779 and the measured molecular weight was 7172.04, which were consistent with the theoretical values. Conjugate 9 is a double-stranded siRNA conjugate with a cyclic carrier peptide group connected to the 3' end of the sense chain, wherein two non-adjacent nucleotides in the carrier peptide group form a cyclic connection through an amide bond between the side chains.

[0503] Preparation Example 10 Synthesis of the Carrier Peptide Conjugate 10 Provided by the Present Disclosure

[0504] (10-1) Preparation of single-stranded oligonucleotide S1

[0505] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 10 in Table 2 was synthesized by a solid phase synthesis method. The only difference was that during the solid phase synthesis, before connecting the first nucleoside monomer at the 3' end and after connecting the last nucleoside monomer at the 5' end, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected, and the oligonucleotide single chain was cut from the solid phase support to obtain an oligonucleotide single chain S101 having a structure shown in Formula (10-1-1).

[0506] Where, Represents the sequence shown as SEQ ID NO:161.

[0507] (10-2) Synthesis of single-stranded oligonucleotide S102:

[0508] After dissolving 45.00 mg of the single-stranded oligonucleotide S101 prepared in (10-1) in 5.00 mL of purified water, TCEP aqueous solution was added to the resulting solution at a weight ratio of 1:1 between TCEP and S101. Mix thoroughly and react at room temperature for 3 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 14 mL of reaction solution. The solution was transferred to a 15 mL, 3K ultrafiltration tube and centrifuged at 3200 rpm for 30 minutes. The ultrafiltration and centrifugation steps were repeated until the conductivity of the ultrafiltration liquid was measured to be below 100 s. The product on the filter membrane was collected to obtain the single-stranded oligonucleotide S102 with a yield of over 85%.

[0509] (10-3) Synthesis of active carrier peptide 2

[0510] Active carrier peptide 2 was prepared according to step (3-3) in Preparation Example 3.

[0511] (10-4) Preparation of carrier peptide conjugate 10

[0512] (10-4-1) Preparation of Single-chain Oligonucleotide-Carrier Peptide 2 Compound

[0513] 1.0 eq of oligonucleotide single-strand S102 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S102 solution. 8.0 eq of active carrier peptide 2 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 2 solution was added to the oligonucleotide single-strand S2 solution, mixed, and reacted at room temperature for 3 hours. Four times the volume of 1×PBS / acetone mixed solution (1×PBS and acetone volume ratio is 1:14) was added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by the above-mentioned semi-preparative reverse phase purification. The components containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 2 compound having a structure shown in formula (10-4-1) with a yield of 60%.

[0514] Where, Represents the sequence shown as SEQ ID NO:161.

[0515] (10-4-2) Preparation of carrier peptide conjugate 10

[0516] The antisense chain in carrier peptide conjugate 10 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0517] Equimolar amounts of the oligonucleotide single-stranded-carrier peptide 2 compound prepared in step (10-4-1) and the antisense strand prepared in step (10-4-2) were dissolved in DEPC water and subsequently annealed to obtain conjugate 10 in Table 2. After preparation, the molecular weights of the sense and antisense strands were determined by LC-MS. The theoretical molecular weight of the sense strand was 8586.751, and the measured molecular weight was 8583.81. The theoretical molecular weight of the antisense strand was 7069.687, and the measured molecular weight was 7068.97. The measured values ​​were consistent with the theoretical values. Conjugate 10 is a double-stranded siRNA conjugate with a cyclic carrier peptide group attached to each end of the sense strand, wherein two non-adjacent nucleotides in the carrier peptide group form a cyclic connection through an amide bond between the side chains.

[0518] Preparation Example 11 Synthesis of the Carrier Peptide Conjugate 11 Provided by the Present Disclosure

[0519] (11-1) Preparation of single-stranded oligonucleotide S111

[0520] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 11 in Table 2 was synthesized by a solid phase synthesis method, with the only difference being that, during the solid phase synthesis process, before connecting the first nucleoside monomer at the 3' end, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected, and the oligonucleotide single chain was cut from the solid phase support to obtain the oligonucleotide single chain S111 shown in formula (11-1-1).

[0521] Where, Represents the sequence shown as SEQ ID NO:161.

[0522] (11-2) Synthesis of single-stranded oligonucleotide S112:

[0523] After dissolving 45.00 mg of the single-stranded oligonucleotide S111 prepared in step (11-1) in 5.00 mL of purified water, a TCEP aqueous solution was added to the resulting solution at a weight ratio of 1:1 between TCEP and S111. Mix thoroughly and react at room temperature for 3 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 14 mL of reaction solution. The reaction solution was transferred to a 15 mL, 3K ultrafiltration tube and centrifuged at 3200 rpm for 30 minutes. The ultrafiltration and centrifugation steps were repeated until the conductivity of the ultrafiltration liquid was measured to be below 100 s. The product on the filter membrane was collected to obtain the single-stranded oligonucleotide S112 with a yield of over 85%.

[0524] (11-3) Synthesis of active carrier peptide 2

[0525] Active carrier peptide 2 was prepared according to step (3-3) in Preparation Example 3.

[0526] (11-4) Preparation of carrier peptide conjugate 11

[0527] (11-4-1) Preparation of single-chain oligonucleotide-carrier peptide 2 compound

[0528] 1.0eq of oligonucleotide single-strand S112 was dissolved in 5mL of 0.1M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S112 solution. 4.0eq of active carrier peptide 2 was dissolved in 5mL of DMF solution, and the obtained active carrier peptide 2 solution was added to the oligonucleotide single-strand S112 solution, mixed, and reacted at room temperature for 3 hours. Four times the volume of 1×PBS / acetone mixed solution (1×PBS and acetone volume ratio is 1:14) was added to the obtained reaction solution, mixed, and centrifuged at 3200rpm for 5min. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered with a 0.45μm microporous filter membrane. The reaction solution was purified by the above-mentioned semi-preparative reverse phase purification, and the components containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-stranded-carrier peptide 2 compound having a structure shown in formula (11-4-1) with a yield of 65%.

[0529] Where, Represents the sequence shown as SEQ ID NO:161.

[0530] (11-4-2) Preparation of carrier peptide conjugate 11

[0531] The antisense chain in carrier peptide conjugate 11 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0532] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 2 compound prepared in step (11-4-1) and the antisense chain prepared in step (11-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 11 (Table 2). After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 7432.466, and the measured molecular weight was 7430.70. The theoretical molecular weight of the antisense chain was 7069.687, and the measured molecular weight was 7068.93. The measured values ​​were consistent with the theoretical values. Conjugate 11 is an siRNA conjugate with the same structure as conjugate 9, but with a different siRNA sequence.

[0533] Preparation Example 12 Synthesis of the Carrier Peptide Conjugate 12 Provided by the Present Disclosure

[0534] (12-1) Preparation of single-stranded oligonucleotide S1

[0535] According to the step (10-1) of Preparation Example 10, single-stranded oligonucleotide S121 was prepared.

[0536] (12-2) Synthesis of single-stranded oligonucleotide S2:

[0537] According to step (10-2) of Preparation Example 10, single-stranded oligonucleotide S122 was prepared.

[0538] (12-3) Synthesis of active carrier peptide 1

[0539] Active carrier peptide 1 was prepared according to steps (2-3) in Preparation Example 2.

[0540] (12-4) Preparation of carrier peptide conjugate 12

[0541] (12-4-1) Preparation of single-chain oligonucleotide-carrier peptide 2 compound

[0542] 1.0 eq of single-stranded oligonucleotide S122 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain a single-stranded oligonucleotide S122 solution. 7.0eq of active carrier peptide 1 was dissolved in 5mL DMF solution, and the obtained active carrier peptide 1 solution was added to the oligonucleotide single-chain S122 solution, mixed, and reacted at room temperature for 4 hours. Four times the volume of 1×PBS / acetone mixed solution (the volume ratio of 1×PBS and acetone is 1:14) was added to the obtained reaction solution, mixed, and centrifuged at 3200rpm for 5min. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered with a 0.45μm microporous filter membrane. The reaction solution was purified by the above-mentioned semi-preparative reverse phase purification. The components containing the product were collected and concentrated to remove the solvent. The product was dissolved and diluted with 150mM sodium chloride, and centrifuged and ultrafiltered 3 times with a 3K ultrafiltration tube. After dilution with purified water, the sodium salt form of the oligonucleotide single-chain-carrier peptide 2 compound having the structure shown in formula (12-4-1) was obtained, with a yield of 46%.

[0543] Where, Represents the sequence shown as SEQ ID NO:161.

[0544] (12-4-2) Preparation of carrier peptide conjugate 12

[0545] The antisense chain in carrier peptide conjugate 12 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0546] Equimolar amounts of the oligonucleotide single-stranded carrier peptide 2 compound prepared in step (12-4-1) and the antisense strand prepared in step (12-4-2) were dissolved in DEPC water and subsequently annealed to obtain conjugate 12 (Table 2). After preparation, the molecular weights of the sense and antisense strands were determined by LC-MS. The theoretical molecular weight of the sense strand was 8366.431, and the measured molecular weight was 8360.23. The theoretical molecular weight of the antisense strand was 7069.687, and the measured molecular weight was 7066.03. The measured values ​​were consistent with the theoretical values. Conjugate 12 is an siRNA conjugate with the same structure as conjugate 10, but with a different carrier peptide group structure.

[0547] Preparation Example 13 Synthesis of Carrier Peptide Conjugate 13 Provided by the Present Disclosure

[0548] (13-1) Preparation of single-stranded oligonucleotide S1

[0549] According to the steps (11-1) in Preparation Example 11, single-stranded oligonucleotide S131 was prepared.

[0550] (13-2) Synthesis of single-stranded oligonucleotide S2:

[0551] According to the step (11-2) of Preparation Example 11, single-stranded oligonucleotide S132 was prepared.

[0552] (13-3) Synthesis of active carrier peptide 1

[0553] Active carrier peptide 1 was prepared according to steps (2-3) in Preparation Example 2.

[0554] (13-4) Preparation of carrier peptide conjugate 13

[0555] (13-4-1) Preparation of single-chain oligonucleotide-carrier peptide 2 compound

[0556] 1.0 eq of oligonucleotide single-chain S132 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-chain S132 solution. 4.0 eq of active carrier peptide 1 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 1 solution was added to the oligonucleotide single-chain S132 solution, mixed, and reacted at room temperature for 3.5 hours. Four volumes of 1×PBS / acetone mixed solution (1×PBS and acetone volume ratio is 1:14) were added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by the above-mentioned semi-preparative reverse phase purification. The fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-chain-carrier peptide 2 compound having a structure shown in formula (13-4-1) with a yield of 54%.

[0557] Where, Represents the sequence shown as SEQ ID NO:161.

[0558] (13-4-2) Preparation of carrier peptide conjugate 13

[0559] The antisense chain in carrier peptide conjugate 13 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0560] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 2 compound prepared in step (13-4-1) and the antisense chain prepared in step (13-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 13 (Table 2). After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 7322.306, and the measured molecular weight was 7320.57. The theoretical molecular weight of the antisense chain was 7069.687, and the measured molecular weight was 7069.11. The measured values ​​were consistent with the theoretical values. Conjugate 13 is a siRNA conjugate with the same structure as conjugate 11, differing only in the structure of the attached carrier peptide.

[0561] Preparation Example 14 Synthesis of Carrier Peptide Conjugate 14 Provided by the Present Disclosure

[0562] (14-1) Preparation of single-stranded oligonucleotide S51

[0563] According to step (1-1) in Preparation Example 5, single-stranded oligonucleotide S51 was prepared.

[0564] (14-2) Synthesis of single-stranded oligonucleotide S52:

[0565] According to step (5-2) of Preparation Example 5, single-stranded oligonucleotide S52 was prepared.

[0566] (14-3) Synthesis of active carrier peptide 2

[0567] Active carrier peptide 2 was prepared according to step (3-3) in Preparation Example 3.

[0568] (14-4) Preparation of carrier peptide conjugate 14

[0569] (14-4-1) Preparation of single-chain oligonucleotide-carrier peptide 2 compound

[0570] 1.0 eq of oligonucleotide single-strand S52 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S52 solution. 6.0 eq of active carrier peptide 2 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 2 solution was added to the oligonucleotide single-strand S52 solution, mixed, and reacted at room temperature for 3 hours. Four volumes of 1×PBS / acetonitrile mixed solution (1×PBS and acetonitrile volume ratio is 1:14) were added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by semi-preparative reverse phase purification, and the fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 2 compound having a structure shown in formula (14-4-1) with a yield of 72%.

[0571] Where, Represents the sequence shown as SEQ ID NO:154.

[0572] (14-4-2) Preparation of carrier peptide conjugate 14

[0573] The antisense chain in carrier peptide conjugate 14 was prepared according to step (5-4-2) in Preparation Example 5.

[0574] Equimolar amounts of the oligonucleotide single-stranded peptide 2 compound prepared in step (14-4-1) and the antisense strand prepared in step (14-4-2) were dissolved in DEPC water and subsequently annealed to obtain conjugate 14 (Table 2). After preparation, the molecular weights of the sense and antisense strands were determined by LC-MS. The theoretical molecular weight of the sense strand was 8489.739, and the measured molecular weight was 8489.24. The theoretical molecular weight of the antisense strand was 7172.779, and the measured molecular weight was 7172.46. The measured values ​​were consistent with the theoretical values. Conjugate 14 is an siRNA conjugate with the same structure as conjugate 10, but with a different siRNA duplex sequence.

[0575] Preparation Example 15 Synthesis of Carrier Peptide Conjugate 15 Provided by the Present Disclosure

[0576] (15-1) Preparation of single-stranded oligonucleotide S151

[0577] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 15 in Table 2 was synthesized by solid phase synthesis. The only difference was that during the solid phase synthesis, after the last nucleoside monomer at the 5' end was connected, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected, and the oligonucleotide single chain was cut from the solid phase support to obtain the oligonucleotide single chain S151 shown in formula (15-1-1):

[0578] Where, Represents the sequence shown as SEQ ID NO:163.

[0579] (15-2) Preparation of single-stranded oligonucleotide S152

[0580] According to step (11-2) in Preparation Example 11, single-stranded oligonucleotide S152 was prepared.

[0581] (15-3) Synthesis of active carrier peptide 4

[0582] Active carrier peptide 4 was synthesized according to the peptide solid phase synthesis method. The specific steps are as follows:

[0583] (15-3-1) Deprotection: 0.5 g of Rink amide MBHA resin (amino-protected resin, purchased from Gill Biochemicals) with a degree of substitution of 0.7 mmol / g was added with 10 mL of a 20 v / v% piperidine solution in DMF. The mixture was reacted at room temperature for 25 min to obtain the amino-deprotected resin.

[0584] (15-3-2) Coupling: 0.7 mmol of amino-protected amino acid G monomer: Fmoc-Gly-OH (Adamas brand, purchased from Shanghai Titan Technology Co., Ltd.), 1.4 mmol of 1-hydroxybenzotriazole (HOBT), and 1.05 mmol of peptide condensation reagent (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HATU) were dissolved in 20 mL of DMF, and 1.05 mmol of N,N-diisopropylethylamine (DIEA) was added. The mixture was reacted at room temperature for 5 minutes to obtain an activated amino acid solution.

[0585] The obtained activated amino acid solution was mixed with the amino-deprotected resin obtained in step (15-3-1), and the mixture was reacted under an argon atmosphere for 120 min. The solvent in the reaction solution was removed under reduced pressure, and the mixture was washed with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain a resin with an amino-protected amino acid residue connected thereto.

[0586] (15-3-3) Repeat the deprotection method in step (15-3-1) and the coupling method in step (15-3-2) on the resin to which the amino group-protected amino acid residue is attached, and sequentially couple the amino acid monomers corresponding to the remaining amino acid residues in the amino acid sequence shown in SEQ ID NO: 149 from the carboxyl terminus to the amino terminus to obtain a peptide resin to which the eight amino acid residues according to the amino acid sequence shown in SEQ ID NO: 149 are attached and the terminal amino group is protected:

[0587] CRSLGDTG (SEQ ID NO: 149).

[0588] The terminal amino protecting group Fmoc was removed, and a 20 v / v% acetic anhydride DCM solution was added and reacted at room temperature for 30 min to obtain an amino-deprotected peptide resin.

[0589] (15-3-4) Removal of OAll special protection: Take 0.7 mmol of tetrakistriphenylphosphine palladium and 2.1 mmol of bis(methylcyclohexanedione) and dissolve them in 20 mL of DCM. Add them to the peptide resin obtained in step (15-3-3) and react under argon atmosphere for 2 h. Remove the solvent in the reaction solution under reduced pressure. At room temperature and normal pressure, wash with DCM, DMF, DCM and DMF in sequence, 25 mL each time, to obtain the peptide resin with OAll special protection removed.

[0590] (15-3-5) Coupling: 0.7 mmol of hexadecylamine, 1.4 mmol of HOBT and 1.05 mmol of HATU were dissolved in 20 mL of DMF and added to the peptide resin obtained in step (15-3-4). 1.05 mmol of DIEA was then added and the mixture was reacted for 120 min at room temperature and atmospheric pressure under an argon atmosphere. The solvent in the reaction solution was removed under reduced pressure. The mixture was washed with DCM, DMF, DCM and DMF in sequence at room temperature and atmospheric pressure, 25 mL each time, to obtain a peptide resin coupled with hexadecylamine.

[0591] (15-3-6) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5; after all monomers are connected, contact the hexadecylamine-coupled peptide resin with the cleavage solution at room temperature and normal pressure. The amount of cleavage solution is 5 mL per 0.5 g of resin. The reaction is carried out for 3 hours, filtered, and the filtrate is collected and added to 4 volumes of MTBE of the filtrate. The solid is obtained by centrifugation.

[0592] (15-3-7) Oxidative coupling: The solid obtained in step (15-3-6) was dissolved in methanol solution, 0.7 mmol of disulfide dipyridine was added to the dissolved solid, and the reaction was shaken at room temperature for 0.5 h to obtain a product mixture of a peptide compound containing a disulfide bond.

[0593] (15-3-8) The product mixture was concentrated under reduced pressure to remove the solvent and then purified by reverse-phase preparative column chromatography. The fractions containing the product were collected, concentrated to remove the solvent, and lyophilized to obtain active carrier peptide 4. Column chromatography conditions were as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2 × 250 mm; mobile phase: gradient elution, mobile phase A: 0.1% formic acid / water, mobile phase B: 0.085% formic acid / acetonitrile; gradient conditions: B: 10%, 0-3 min; 10%-90%, 3-25 min; 90%, 25-30 min; 90%-10%, 30-31 min; 10%, 31-32 min.

[0594] The obtained active carrier peptide 4 has a structure as shown in formula (15-3-1):

[0595] (15-4) Preparation of carrier peptide conjugate 15

[0596] (15-4-1) Preparation of single-chain oligonucleotide-carrier peptide 4 compound

[0597] 1.0 eq of oligonucleotide single-strand S152 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-strand S152 solution. 5.0 eq of active carrier peptide 4 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 4 solution was added to the oligonucleotide single-strand S152 solution, mixed, and reacted at room temperature for 4 hours. Four times the volume of 1×PBS / acetonitrile mixed solution (1×PBS and acetonitrile volume ratio is 1:14) was added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by the above-mentioned semi-preparative reverse phase purification. The fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-strand-carrier peptide 4 compound having a structure shown in formula (15-4-1) with a yield of 52%.

[0598] (15-4-2) Preparation of carrier peptide conjugate 15

[0599] The antisense chain in carrier peptide conjugate 15 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0600] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 4 compound prepared in step (15-4-1) and the antisense chain prepared in step (15-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 15 in Table 2. After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 7576.868, and the measured molecular weight was 7572.91. The theoretical molecular weight of the antisense chain was 7069.687, and the measured molecular weight was 7066.01. The measured values ​​were consistent with the theoretical values. Conjugate 15 is an siRNA conjugate in which the 5' end of the sense chain is connected to a carrier peptide group, and the carrier peptide group is connected to a hexadecyl group as a delivery aid group.

[0601] Preparation Example 16 Synthesis of Carrier Peptide Conjugate 16 Provided by the Present Disclosure

[0602] (16-1) Preparation of single-stranded oligonucleotide S1

[0603] According to the method described in Preparation Example 1 of WO2019105418 (A1), the sequence of the siRNA sense chain in the carrier peptide conjugate 16 in Table 2 was synthesized by a solid phase synthesis method, except that, during the solid phase synthesis, before connecting the first nucleoside monomer at the 3' end, a phosphoramidite monomer containing a HO(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was additionally connected, and the oligonucleotide single chain was cut from the solid phase support to obtain the oligonucleotide single chain S161 shown in formula (16-1-1).

[0604] Where, Represents the sequence shown as SEQ ID NO:163.

[0605] (16-2) Synthesis of Single-Stranded Oligonucleotide S162

[0606] According to step (11-2) in Preparation Example 11, single-stranded oligonucleotide S162 was prepared.

[0607] (16-3) Synthesis of active carrier peptide 4

[0608] Active carrier peptide 4 was prepared according to step (15-3) in Preparation Example 15.

[0609] (16-4) Preparation of carrier peptide conjugate 16

[0610] (16-4-1) Preparation of single-chain oligonucleotide-carrier peptide 4 compound

[0611] 1.0 eq of single-stranded oligonucleotide S162 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain a single-stranded oligonucleotide S162 solution. 5.0 eq of active carrier peptide 4 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 4 solution was added to the single-stranded oligonucleotide S162 solution, mixed, and reacted at room temperature for 4 hours. Four volumes of 1×PBS / acetonitrile mixed solution (1×PBS and acetonitrile volume ratio is 1:14) were added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by semi-preparative reverse phase purification, and the fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-stranded-carrier peptide 4 compound having a structure shown in formula (16-4-1) with a yield of 90%.

[0612] (16-4-2) Preparation of carrier peptide conjugate 16

[0613] The antisense chain in carrier peptide conjugate 16 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0614] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 4 compound prepared in step (16-4-1) and the antisense chain prepared in step (16-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 16 in Table 2. After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 7576.868, and the measured molecular weight was 7573.04. The theoretical molecular weight of the antisense chain was 7069.687, and the measured molecular weight was 7066.03. The measured values ​​were consistent with the theoretical values. Conjugate 16 is an siRNA conjugate in which the 3' end of the sense chain is connected to a carrier peptide group, and the carrier peptide group is connected to a hexadecyl group as a delivery aid group.

[0615] Preparation Example 17 Synthesis of Carrier Peptide Conjugate 17 Provided by the Present Disclosure

[0616] (17-1) Preparation of single-stranded oligonucleotide S171

[0617] The single-stranded oligonucleotide S171 was prepared according to step (15-1) in Preparation Example 15.

[0618] (17-2) Preparation of single-stranded oligonucleotide S172

[0619] According to step (11-2) in Preparation Example 11, single-stranded oligonucleotide S172 was prepared.

[0620] (17-3) Synthesis of active carrier peptide 5

[0621] Active carrier peptide 5 was synthesized according to the peptide solid phase synthesis method. The specific steps are as follows:

[0622] (17-3-1) Deprotection: 0.5 g of Rink amide MBHA resin (amino-protected resin, purchased from Gill Biochemicals) with a degree of substitution of 0.7 mmol / g was added with 10 mL of a 20 v / v% piperidine solution in DMF. The mixture was reacted at room temperature for 25 min to obtain the amino-deprotected resin.

[0623] (17-3-2) Coupling: 0.7 mmol of amino-protected amino acid G monomer: Fmoc-Gly-OH (Adamas brand, purchased from Shanghai Titan Technology Co., Ltd.), 1.4 mmol of HOBT, and 1.05 mmol of HATU were dissolved in 20 mL of DMF. 1.05 mmol of DIEA was added and the mixture was reacted at room temperature for 5 min to obtain an activated amino acid solution.

[0624] The obtained activated amino acid solution was mixed with the amino-deprotected resin obtained in step (17-3-1), and the mixture was reacted under an argon atmosphere for 120 min. The solvent in the reaction solution was removed under reduced pressure, and the mixture was washed with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain a resin with an amino-protected amino acid residue connected thereto.

[0625] (17-3-3) For the resin obtained in step (17-3-2) connected to the amino acid residue with protected amino groups, repeat the deprotection method in step (17-3-1) and the coupling method in step (17-3-2), and according to the amino acid sequence shown in SEQ ID NO: 149, the amino acid monomers corresponding to the remaining amino acid residues in the sequence are coupled in sequence from the carboxyl terminus to the amino terminus to obtain 8 amino acid residues connected to the amino acid sequence shown in SEQ ID NO: 149, and after the last amino acid residue is connected, hexadecanoic acid is connected to obtain a peptide resin.

[0626] (17-3-4) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: thioanisole: phenol: TIS: water = 87.5:5:2.5:2.5:2.5; after all monomers are connected, the peptide resin obtained in step (17-3-3) is contacted with the cleavage solution at room temperature and normal pressure. The amount of cleavage solution is 5 mL per 0.5 g of resin. The reaction is carried out for 3 hours, filtered, and the filtrate is collected and added to 4 volumes of MTBE of the filtrate. The solid is obtained by centrifugation.

[0627] (17-3-5) Oxidative coupling: The solid obtained in step (17-3-4) was dissolved in methanol solution, 0.7 mmol of disulfide dipyridine was added to the dissolved solid, and the reaction was shaken at room temperature for 0.5 h to obtain a product mixture of a peptide compound containing a disulfide bond.

[0628] (17-3-6) The product mixture was concentrated under reduced pressure to remove the solvent and then purified by reverse-phase preparative column chromatography. The fractions containing the product were collected, concentrated to remove the solvent, and lyophilized to obtain active carrier peptide 5. Column chromatography conditions were as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2 × 250 mm; mobile phase: gradient elution, mobile phase A: 0.1% formic acid / water, mobile phase B: 0.085% formic acid / acetonitrile; gradient conditions: B: 10%, 0-3 min; 10%-90%, 3-25 min; 90%, 25-30 min; 90%-10%, 30-31 min; 10%, 31-32 min.

[0629] The obtained active carrier peptide 5 has a structure as shown in formula (17-3-1):

[0630] (17-4) Preparation of carrier peptide conjugate 17

[0631] (17-4-1) Preparation of single-chain oligonucleotide-carrier peptide 5 compound

[0632] 1.0 eq of oligonucleotide single-chain S172 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-chain S172 solution. 5.0 eq of active carrier peptide 5 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 5 solution was added to the oligonucleotide single-chain S172 solution, mixed, and reacted at room temperature for 4 hours. Four volumes of 1×PBS / acetonitrile mixed solution (1×PBS and acetonitrile volume ratio is 1:14) were added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by semi-preparative reverse phase purification, and the fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-chain-carrier peptide 5 compound having a structure shown in formula (17-4-1) with a yield of 77%.

[0633] (17-4-2) Preparation of carrier peptide conjugate 17

[0634] The antisense chain in carrier peptide conjugate 17 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0635] DEPC water was used to dissolve an equal amount of the oligonucleotide single-chain-carrier peptide 5 compound prepared in step (17-4-1) and the antisense chain prepared in step (17-4-2), and then annealed to obtain conjugate 17 in Table 2. After preparation, the molecular weights of the sense chain and antisense chain were detected by LC-MS. The theoretical molecular weight of the sense chain was 7549.798 and the measured molecular weight was 7545.94, while the theoretical molecular weight of the antisense chain was 7069.687 and the measured molecular weight was 7066.04, which were consistent with the theoretical values. Conjugate 17 is an siRNA conjugate in which the 5' end of the sense chain is connected to a carrier peptide group, and the carrier peptide group is connected to a hexadecyl group as a delivery aid group, wherein the connection position of the hexadecyl group on the carrier peptide group is different from that of conjugate 15.

[0636] Preparation Example 18 Synthesis of Carrier Peptide Conjugate 18 Provided by the Present Disclosure

[0637] (18-1) Preparation of single-stranded oligonucleotide S181

[0638] The single-stranded oligonucleotide S181 was prepared according to step (16-1) in Preparation Example 16.

[0639] (18-2) Synthesis of single-stranded oligonucleotide S182

[0640] According to step (11-2) of Preparation Example 11, single-stranded oligonucleotide S182 was prepared.

[0641] (18-3) Synthesis of active carrier peptide 5

[0642] Active carrier peptide 5 was prepared according to step (17-3) in Preparation Example 17.

[0643] (18-4) Preparation of carrier peptide conjugate 18

[0644] (18-4-1) Preparation of single-chain oligonucleotide-carrier peptide 5 compound

[0645] 1.0 eq of oligonucleotide single-chain S182 was dissolved in 5 mL of 0.1 M ammonium acetate aqueous solution to obtain an oligonucleotide single-chain S182 solution. 5.0 eq of active carrier peptide 5 was dissolved in 5 mL of DMF solution, and the obtained active carrier peptide 5 solution was added to the oligonucleotide single-chain S182 solution, mixed, and reacted at room temperature for 4 hours. Four volumes of 1×PBS / acetonitrile mixed solution (1×PBS and acetonitrile volume ratio is 1:14) were added to the obtained reaction solution, mixed, and centrifuged at 3200 rpm for 5 minutes. The precipitate after centrifugation was dissolved in purified water, and the reaction solution was filtered through a 0.45 μm microporous filter membrane. The reaction solution was purified by semi-preparative reverse phase purification, and the fractions containing the product were collected and concentrated to remove the solvent to obtain an oligonucleotide single-chain-carrier peptide 5 compound having a structure shown in formula (18-4-1) with a yield of 93%.

[0646] (18-4-2) Preparation of carrier peptide conjugate 18

[0647] The antisense chain in carrier peptide conjugate 18 was prepared according to step (5-4-2) in Preparation Example 5, except that the antisense chain had the sequence shown in SEQ ID NO:185.

[0648] Equimolar amounts of the oligonucleotide single-chain-carrier peptide 5 compound prepared in step (18-4-1) and the antisense chain prepared in step (18-4-2) were dissolved in DEPC water and then annealed to obtain conjugate 18 in Table 2. After preparation, the molecular weights of the sense and antisense chains were determined by LC-MS. The theoretical molecular weight of the sense chain was 7549.798, and the measured molecular weight was 7545.91. The theoretical molecular weight of the antisense chain was 7069.687, and the measured molecular weight was 7065.95. The measured values ​​were consistent with the theoretical values. Conjugate 18 is an siRNA conjugate in which the 3' end of the sense chain is connected to a carrier peptide group, and the carrier peptide group is connected to a hexadecyl group as a delivery aid group, wherein the hexadecyl group is connected to the carrier peptide group at a different position than in conjugate 16.

[0649] Comparative Preparation Example 1 Synthesis of Reference siRNA 1

[0650] The following reference siRNA1 NC was synthesized by solid-phase synthesis according to the method described in Preparation Example 1 of WO2019105418(A1), except that equimolar complementary sense strands (SEQ ID NO: 127) and antisense strands (SEQ ID NO: 129) were dissolved in DEPC water and subsequently annealed to obtain siRNA 1. siRNA 1 is an siRNA with the same sense and antisense strand sequences as conjugates 1-4, but without a carrier.

[0651] Comparative Preparation Example 2 Synthesis of Reference siRNA 2

[0652] The following reference siRNA2 NC was synthesized by solid-phase synthesis according to the method described in Preparation Example 1 of WO2019105418(A1), except that equimolar complementary sense strands (SEQ ID NO: 154) and antisense strands (SEQ ID NO: 156) were dissolved in DEPC water and subsequently annealed to yield siRNA 2. siRNA2 is an siRNA with the same sense and antisense strand sequences as conjugates 5-6, but without a carrier.

[0653] Experimental Example 1 Activity of the carrier peptide conjugate disclosed herein in mice

[0654] This experimental example investigated the inhibitory activity of the prepared conjugate 1 on SOD1 mRNA in mice, especially in the central nervous system.

[0655] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 25±1 g at purchase.

[0656] The detailed steps are as follows:

[0657] [1] Intracerebroventricular injection in mice:

[0658] For conjugate 1 and reference siRNA 1, the powdered conjugate was dissolved in PBS and diluted to an injection solution with a concentration of 20 μg / μL (based on the amount of siRNA) for use.

[0659] Fifteen mice were randomly divided into three groups, each containing five mice, numbered as a blank group, a test group, and a reference group. No medication was administered to the mice in the blank group. The mice in the test group were administered conjugate 1 at a dose of 10 μL / mouse, while the mice in the reference group were administered siRNA 1 at a dose of 10 μL / mouse. The specific steps were as follows: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia took effect, the drug was injected into the lateral ventricle using a microdosing pump (model: 78-8130, purchased from KDSCIENTIFIC) and a 25 μL microinjection needle (purchased from Hamilton). The injection time was 10 minutes, and the needle was left in place for 5 minutes after the injection, then slowly removed. The needle hole was then sealed with biological glue (purchased from Minnesota Mining and Manufacturing Co., Ltd.), the head skin was glued, and the mice were returned to the animal breeding room for continued breeding after they woke up. The injection day was recorded as the first day.

[0660] On the fifth day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the parietal cortex, hippocampus, cerebellum, medulla oblongata, thalamus, and striatum of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene SOD1.

[0661] [3] Detection

[0662] For each area of ​​the brain of each mouse, 6 samples were extracted, 3 from the administration side and 3 from the contralateral side. 1 μg of total RNA was taken from each sample and reverse transcription was performed using the Goldenstar reverse transcription kit. TM The reagents provided by RT6 cDNA Synthesis Kit (purchased from Yiyou Technology, product number 50000665) were selected from Goldenstar TM Oligo(dT) 17 As primers, 20 μl of reverse transcription reaction system was prepared according to the reverse transcription protocol in the kit instructions, and total RNA from cells in each well was reverse transcribed. Reverse transcription conditions were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50°C for 50 minutes, then at 85°C for 5 minutes, and finally at 4°C for 30 seconds. After the reaction was completed, 80 μl of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0663] For each reverse transcription reaction system, 5 μl of the above solution containing cDNA was taken as template and SYBR TMA 20 μl qPCR reaction system was prepared using the reagents provided in the Select Master Mix kit (purchased from Appliedbiosystems, Cat. No. 50000332). The PCR primer sequences for amplifying the target gene SOD1 and the internal reference gene GAPDH are shown in Table 3, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step protocol: a 95°C initial denaturation for 10 minutes, followed by a 95°C denaturation for 30 seconds, a 60°C annealing for 30 seconds, and a 72°C extension for 30 seconds. This denaturation, annealing, and extension cycle was repeated 40 times to obtain product W containing the amplified target gene SOD1 and the internal reference gene GAPDH. Product W was then incubated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The melting curves of the target gene SOD1 and the internal reference gene GAPDH in product W were collected by real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of the target gene SOD1 and the internal reference gene GAPDH.

[0664] Table 3 Primer information

[0665] The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene SOD1 in each test group. The calculation method is as follows:

[0666] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0667] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0668] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0669] ΔΔCt(control group) = ΔCt(control group) - ΔCt(average of control group)

[0670] Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) values ​​for each of the four samples.

[0671] The SOD1 mRNA expression level of the test group was normalized with the control group as the benchmark, and the SOD1 mRNA expression level of the blank control group was defined as 100%.

[0672] Relative expression level of SOD1 mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0673] SOD1 mRNA inhibition rate in the test group = (1 - relative expression level of SOD1 mRNA in the test group) × 100%. The inhibition rates of each siRNA conjugate on SOD1 mRNA in different brain regions are summarized in Table 4.

[0674] Table 4 Inhibition rate of the conjugate on SOD1 gene expression mRNA in the mouse central nervous system

[0675] As shown in Table 4, under the same dosage conditions, the conjugate 1 of the present invention showed a high inhibition rate on the mRNA expression of the SOD1 gene in different regions of the mouse brain. The inhibition rate in the cortex on the administration side was as high as 74.1%, which was 50.30% higher than the inhibition rate of the reference siRNA 1 without a carrier peptide group. The inhibition rates in the hippocampus and cerebellum on the administration side exceeded 50%, reaching 63.4% and 51.4%, respectively. The inhibition rates were 80.11% and 18.4% higher than the inhibition rates of siRNA 1 without a carrier peptide group, respectively.

[0676] Even in the contralateral brain region, the conjugate 1 of the present invention showed a high inhibition rate of SOD1 gene expression mRNA. The inhibition rates in the contralateral cortex, contralateral hippocampus and cerebellum were all higher than 40%, namely 69.2%, 53.5% and 41.4%, respectively. The inhibition rates were increased by 63.15%, 123.85% and 6.1% compared with the inhibition rates of siRNA1 without a carrier peptide group, respectively.

[0677] Experimental Example 2 Activity of the carrier peptide conjugate disclosed herein in mice

[0678] This experimental example investigated the inhibitory activity of Conjugate 2, Conjugate 3, and Conjugate 4 on the mRNA expression of the SOD1 gene in mice, particularly in the central nervous system.

[0679] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 26±1 g at purchase.

[0680] The detailed steps are as follows:

[0681] [1] Intracerebroventricular injection in mice:

[0682] For conjugate 2, conjugate 3, conjugate 4 and siRNA 1, the powdered conjugate was dissolved and diluted with PBS to prepare an injection solution with a concentration of 20 μg / μL (based on the amount of siRNA).

[0683] 30 mice were randomly divided into 5 groups, with 6 mice in each group, namely blank group, reference group, test group 1, test group 2 and test group 3. For the mice in the blank group, no drug was given. For the mice in the reference group, siRNA1 was administered at a dose of 10 μL / mouse. For the mice in test group 1, conjugate 2 was administered at a dose of 10 μL / mouse. For the mice in test group 2, conjugate 3 was administered at a dose of 10 μL / mouse. For the mice in test group 3, conjugate 4 was administered at a dose of 10 μL / mouse. The specific administration steps were: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia took effect, the drug was injected into the lateral ventricle using a microdosing pump (model: 78-8130, purchased from KDSCIENTIFIC) using a 25 μL microinjection needle (purchased from Hamilton). The injection time was 10 minutes. The needle was retained for 5 minutes after the injection, and then the needle was slowly removed. The needle hole was then sealed with biological glue (purchased from Minnesota Mining and Manufacturing Co., Ltd.), the head skin was glued, and the mouse was returned to the animal breeding room for continued breeding after it woke up. The injection day was recorded as the first day.

[0684] On the eighth day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the parietal cortex, hippocampus, cerebellum, medulla oblongata, thalamus, and striatum of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene SOD1.

[0685] According to the steps of Experimental Example 1, the inhibition rates of Conjugate 2, Conjugate 3 and Conjugate 4 on SOD1 mRNA in different brain regions were detected and calculated. The results are shown in Table 5.

[0686] Table 5 Inhibition rate of conjugates 2-4 on SOD1 gene expression mRNA in the mouse central nervous system

[0687] As shown in Table 5, under the same dosage conditions, conjugate 2 connected to a linear carrier peptide, conjugate 3 connected to a cyclic carrier peptide, and conjugate 4 connected to a stapled carrier peptide provided by the present disclosure all showed high inhibition rates on the mRNA expression of the SOD1 gene in different regions of the mouse brain, with the inhibition rate in the cortex reaching 62.04%, and the inhibition rate in the hippocampus reaching 59.41%, both of which were significantly higher than the reference siRNA 1 without a delivery carrier.

[0688] Experimental Example 3 Activity of the carrier peptide conjugate disclosed herein in mice

[0689] This experimental example investigated the inhibitory activity of conjugates 5 and 6 on SOD1 mRNA in mice, particularly in the central nervous system.

[0690] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 25±1 g at purchase.

[0691] The detailed steps are as follows:

[0692] [1] Intrathecal injection in mice:

[0693] For conjugate 5, conjugate 6 and siRNA 2, the powdered conjugate was dissolved in PBS and diluted to an injection solution with a concentration of 25 μg / μL (based on the amount of siRNA) for use.

[0694] 40 mice were randomly divided into 4 groups, with 10 mice in each group, numbered as blank group, reference group, test group one and test group two. For the mice in the blank group, no drug was given. For the mice in the reference group, siRNA2 was administered at a dose of 20 μL / mouse. For the mice in test group one, conjugate 5 was administered at a dose of 20 μL / mouse. For the mice in test group two, conjugate 6 was administered at a dose of 20 μL / mouse. The specific administration steps were: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia takes effect, an insulin injection needle is used to draw the drug from the junction of the mouse lumbar vertebrae L5 and L6, and the drug is injected into the subdural mater. After the injection, the needle is retained for 2 minutes, and then the needle is slowly withdrawn. After that, the needle hole is sealed with biological glue (purchased from Minnesota Mining Manufacturing Co., Ltd.), the skin is glued well, and the mouse is returned to the animal breeding room for continued breeding after it wakes up. The injection day is recorded as the first day.

[0695] On the 29th day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the right parietal cortex, hippocampus, cerebellum, medulla oblongata, thalamus, and striatum of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene SOD1.

[0696] According to the steps of Experimental Example 1, the inhibition rates of conjugates 5 and 6 on SOD1 mRNA in different brain regions were detected and calculated. The results are shown in Table 6.

[0697] Table 6 Inhibition rate of conjugate 5-6 on SOD1 mRNA in the mouse central nervous system

[0698] Figure 1 shows a bar graph of the SOD1 mRNA inhibition rates in different regions of the mouse brain after administration of conjugates 5 and 6 of the present disclosure and reference siRNA 2. As can be seen from Figure 1 and Table 6, under the same dosage conditions, conjugates 5 and 6 of the present disclosure showed high inhibition rates on SOD1 mRNA expression in different regions of the mouse brain, with inhibition rates as high as 89.4% in the striatum and exceeding 75% in the right parietal cortex, hippocampus, thalamus, and cerebellum. This indicates that the conjugates provided by the present disclosure, which are linked to the carrier peptide of the present disclosure, can specifically target different regions of the mouse brain and have higher SOD1 mRNA inhibition rates than the reference siRNA without the carrier peptide.

[0699] Experimental Example 4 Activity of the carrier peptide conjugate disclosed herein in mice

[0700] This experimental example investigated the inhibitory activity of conjugates 8, 9, and 14 on the mRNA expression of the SOD1 gene in mice, particularly in the central nervous system.

[0701] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 25±1 g at purchase.

[0702] The detailed steps are as follows:

[0703] [1] Intrathecal injection in mice:

[0704] For Conjugate 8, Conjugate 9, and Conjugate 14, the powdered conjugates were dissolved and diluted with PBS to prepare injection solutions with a concentration of 20 μg / μL (based on the amount of siRNA).

[0705] 40 mice were randomly divided into 4 groups, with 10 mice in each group, numbered as blank group, test group one, test group two and test group three. For the mice in the blank group, no drug was given. For the mice in test group one, conjugate 8 was administered at a dose of 20 μL / mouse. For the mice in test group two, conjugate 9 was administered at a dose of 20 μL / mouse. For the mice in test group three, conjugate 14 was administered at a dose of 20 μL / mouse. The specific drug administration steps were: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia takes effect, an insulin injection needle is used to draw the drug from the junction of the mouse lumbar vertebrae L5 and L6, and the drug is injected into the subdural mater. After the injection, the needle is retained for 2 minutes, and then the needle is slowly withdrawn. After that, the needle hole is sealed with biological glue (purchased from Minnesota Mining Manufacturing Co., Ltd.), the skin is glued, and the mouse is returned to the animal breeding room for continued breeding after it wakes up. The injection day is recorded as the first day.

[0706] On the 29th day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the lumbar spinal cord, thoracic spinal cord, cervical spinal cord, parietal cortex, hippocampus, and striatum of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene SOD1.

[0707] According to the steps of Experimental Example 1, the inhibition rates of Conjugate 8, Conjugate 9 and Conjugate 14 on SOD1 mRNA in different brain regions were detected and calculated. The results are shown in Table 7.

[0708] Table 7 Inhibition rate of conjugates 8, 9 and 14 on SOD1 mRNA in the mouse central nervous system

[0709] As can be seen from Table 7, under the same dosage conditions, conjugates 8, 9 and 14 of the present disclosure all showed very high inhibition rates on the mRNA expression of the SOD1 gene in different regions of the mouse brain. The inhibition rate in the lumbar spinal cord can reach more than 87%, and can even reach 90.2%; the inhibition rate in the thoracic spinal cord can reach more than 86%, and can even reach 91.6%; the inhibition rate in the cervical spinal cord can reach more than 68%, and can even reach 83.8%; the inhibition rate in the striatum can reach 69.5%; and the inhibition rate in the hippocampus can reach 55.6%, indicating that the conjugates provided by the present disclosure connected with the carrier peptide of the present disclosure can specifically target different regions of the mouse brain and still have a high SOD1 mRNA inhibition rate within the 29-day experimental period.

[0710] Experimental Example 5 Activity of the carrier peptide conjugate disclosed herein in mice

[0711] This experimental example investigated the inhibitory activity of conjugate 10, conjugate 11, conjugate 12, and conjugate 13 on APOE4 mRNA in mice, particularly in the central nervous system.

[0712] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 25±1 g at purchase.

[0713] The detailed steps are as follows:

[0714] [1] Intrathecal injection in mice:

[0715] For conjugate 10, conjugate 11, conjugate 12 and conjugate 13, the powdered conjugate was dissolved and diluted with PBS to prepare an injection solution with a concentration of 10 μg / μL (based on the amount of siRNA).

[0716] 50 mice were randomly divided into 5 groups, with 10 mice in each group, numbered as blank group, test group one, test group two, test group three and test group four. For the mice in the blank group, no medication was given. For the mice in test group one, conjugate 10 was administered at a dose of 20 μL / mouse. For the mice in test group two, conjugate 11 was administered at a dose of 20 μL / mouse. For the mice in test group three, conjugate 12 was administered at a dose of 20 μL / mouse. For the mice in test group four, conjugate 13 was administered at a dose of 20 μL / mouse. The specific administration steps were: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia took effect, the drug was injected into the mouse's lumbar vertebrae L5 and L6 using an insulin injection needle. The drug was injected subdurally. The needle was left in place for 2 minutes after injection and then slowly withdrawn. The needle hole was sealed with biological glue (purchased from Minnesota Mining and Manufacturing Co., Ltd.), the skin was glued shut, and the mouse was returned to the animal housing room for continued care after awakening. The day of injection was recorded as day one.

[0717] On the 29th day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the lumbar spinal cord, thoracic spinal cord, cervical spinal cord, parietal cortex, hippocampus, striatum, myelin sheath, cerebellum, and thalamus of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene APOE4.

[0718] According to the steps of Experimental Example 1, the inhibition rates of APOE4 mRNA in different brain regions for conjugates 10, 11, 12, and 13 were detected and calculated. The difference was that the PCR primer sequences used to amplify the target gene APOE4 and the internal reference gene GAPDH were shown in Table 8. The detection and calculation results are shown in Table 9.

[0719] Table 8 Primer information

[0720] Table 9 Inhibition rate of APOE4 mRNA in the central nervous system of mice by conjugates 10-13

[0721] As can be seen from Table 9, under the same dosage conditions, conjugates 10, 11, 12 and 13 of the present disclosure all showed very high inhibition rates on the mRNA expression of the APOE4 gene in different regions of the mouse brain, among which the inhibition rate in the lumbar segment of the spinal cord was as high as 95.3%, the inhibition rate in the cervical segment of the spinal cord was as high as 95.3%, the inhibition rate in the thoracic segment of the spinal cord was as high as 94.7%, the inhibition rate in the myelin sheath was as high as 89.3%, the inhibition rate in the thalamus was as high as 87.6%, the inhibition rate in the hippocampus was as high as 80.7%, and the inhibition rates in the striatum and cerebellum were both over 70%, indicating that the conjugates provided by the present disclosure connected with the carrier peptide of the present disclosure can specifically target different regions of the mouse brain, and still have a high APOE4 mRNA inhibition rate within the 29-day experimental period.

[0722] Experimental Example 6 Activity of the Carrier Peptide Conjugates of the Present Disclosure in Mice

[0723] This experimental example investigated the inhibitory activity of conjugate 15, conjugate 16, conjugate 17, and conjugate 18 on APOE4 mRNA in mice, particularly in the central nervous system.

[0724] The mice used in this experiment were purchased from Spafford. They were of ICR strain, SPF grade, male, and weighed 25±1 g at purchase.

[0725] The detailed steps are as follows:

[0726] [1] Intrathecal injection in mice:

[0727] For conjugates 15, 16, 17, and 18, the powdered conjugates were dissolved and diluted with PBS to prepare injection solutions with a concentration of 20 μg / μL (based on the amount of siRNA).

[0728] 50 mice were randomly divided into 5 groups, with 10 mice in each group, numbered as blank group, test group one, test group two, test group three and test group four. For the mice in the blank group, no drug was given. For the mice in test group one, conjugate 15 was administered at a dose of 20 μL / mouse. For the mice in test group two, conjugate 16 was administered at a dose of 20 μL / mouse. For the mice in test group three, conjugate 17 was administered at a dose of 20 μL / mouse. For the mice in test group four, conjugate 18 was administered at a dose of 20 μL / mouse. The specific administration steps were: each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg / kg body weight. After anesthesia took effect, the drug was injected into the mouse's lumbar vertebrae L5 and L6 using an insulin injection needle. The drug was injected subdurally. The needle was left in place for 2 minutes after injection and then slowly withdrawn. The needle hole was sealed with biological glue (purchased from Minnesota Mining and Manufacturing Co., Ltd.), the skin was glued shut, and the mouse was returned to the animal housing room for continued care after awakening. The day of injection was recorded as day one.

[0729] On the 29th day after the injection, each mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate at a dose of 400 mg / kg body weight, and the parietal cortex, hippocampus, striatum, thoracic spinal cord and cervical spinal cord of the mouse were extracted and stored in RNA later (No.: MFCD03453003, purchased from SIGMA). Subsequently, RNA was extracted using RNAVzol (purchased from Viglas Biotechnology (Beijing) Co., Ltd., Product No. N002) according to the method described in the instructions, and then q-PCR was performed to detect the expression of the target gene APOE4.

[0730] According to the steps of Experimental Example 1, the inhibition rates of APOE4 mRNA in different brain regions of conjugates 15, 16, 17, and 18 were detected and calculated. The difference was that the PCR primer sequences used to amplify the target gene APOE4 and the internal reference gene GAPDH were shown in Table 8. The detection and calculation results are shown in Table 10.

[0731] Table 10 Inhibition rate of APOE4 mRNA in the central nervous system of mice by conjugates 15-18

[0732] As can be seen from Table 11, under the conditions of the same dosage, the conjugates 15-18 provided by the present disclosure, which are simultaneously conjugated with a delivery aid group and a siRNA group, showed high inhibition rates on the mRNA expression of the APOE4 gene in different regions of the mouse brain, among which the inhibition rates in the thoracic segment of the spinal cord were as high as 95.9%, the inhibition rates in the cervical segment of the spinal cord were as high as 97.4%, the inhibition rates in the striatum were as high as 80.0%, and the inhibition rates in the hippocampus were as high as 71.7%, indicating that the conjugates provided by the present disclosure, which are simultaneously connected with a delivery aid group and a siRNA group, of the carrier peptide can specifically target different regions of the mouse brain, and still have a high APOE4 mRNA inhibition rate within the 29-day experimental period.

[0733] In summary, the carrier peptide disclosed in the present invention can effectively deliver siRNA to the spinal cord and brain areas, and can effectively achieve drug delivery to the central nervous system; the carrier peptide conjugate disclosed in the present invention comprising a carrier peptide group and a siRNA group can effectively reach different organs and tissues of the central nervous system, and enter the cells to efficiently exert RNAi effects; compared with siRNA that does not contain a delivery group, the carrier peptide conjugate disclosed in the present invention has a significantly increased inhibition rate of mRNA expressed by the target gene in the central nervous system of mice.

[0734] The specific details in the above embodiments are within the technical concept of the present disclosure, and a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0735] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0736] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A carrier peptide, the length of the carrier peptide being 5-30 amino acid residues, wherein, The carrier peptide contains one or more amino acid sequences I, and the length of each amino acid sequence I is independently 5 to 7 amino acid residues, and each amino acid sequence I is identical to at least 5 consecutive amino acid residues in the sequence shown in SEQ ID NO:1: RSLGDTG (SEQ ID NO:1); Wherein, each capital letter independently represents an amino acid residue, and each amino acid residue is independently a natural or modified amino acid residue.

2. The carrier peptide according to claim 1, wherein The carrier peptide contains 1 to 2 amino acid sequences I, and the length of the carrier peptide is 5 to 20 amino acid residues.

3. The carrier peptide according to claim 1, wherein The carrier peptide has the composition (M A N A ) n0 Z A , where each M A and Z A is the same as or different from each other and represents an amino acid sequence composed of 0 to 3 amino acid residues, each N A independently refers to the amino acid sequence I, and n0 is an integer from 1 to 3; or, M A and / or Z A represents an amino acid sequence composed of 1 to 3 amino acid residues; or, each M A and Z A is the same as or different from each other and each independently represents 1 amino acid residue; or each M A and Z A represents the same amino acid residue.

4. The carrier peptide according to claim 3, wherein The carrier peptide has an amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:144 - 148, SEQ ID NO:152 or SEQ ID NO:153: RSLGDTG (SEQ ID NO:1); RSLGD (SEQ ID NO:144); RSLGDT (SEQ ID NO:145); LGDTG (SEQ ID NO:146); SLGDTG (SEQ ID NO:147); SLGDT (SEQ ID NO:148); KRSLGDTG (SEQ ID NO:152); CRSLGDTGCRSLGDTGC (SEQ ID NO:153).

5. The carrier peptide according to any one of claims 1-4, wherein, The side chains of any non-adjacent multiple amino acid residues in the carrier peptide are connected to each other to form a cyclic peptide of the carrier peptide; alternatively, the multiple amino acid residues with side chains connected to each other are multiple amino acid residues in amino acid sequence I; alternatively, the number of amino acid residues with side chains connected to each other is 2-3; alternatively, there are 2-5 amino acid residues between the two amino acid residues with side chains connected to each other; alternatively, the side chains of the multiple amino acid residues with side chains connected to each other are connected by an amide bond, a thioether bond, a disulfide bond or a linking group; alternatively, each of the linking groups is independently selected from one of C4-C 12 linear alkylene groups; alternatively, each of the linking groups is independently selected from one of C6-C 10 alkenylene groups; alternatively, at least one or each of the linking groups is 4-octenylene.

6. The carrier peptide according to claim 5, wherein, The carrier peptide has an amino acid sequence shown in SEQ ID NO:152: KRSLGDTG (SEQ ID NO:152), wherein, the side chain of the lysine residue represented by K is connected to the side chain of the aspartic acid residue represented by D; Alternatively, the carrier peptide has a structure represented by the formula (21):

7. The carrier peptide according to claim 5, wherein The carrier peptide has a structure shown in the following formula (22):

8. The carrier peptide according to claim 3, wherein The side chains of any three non-adjacent amino acid residues in the carrier peptide are connected to each other through a linking group, so that the carrier peptide forms a cyclic peptide, and the linking group has the structure shown in formula (231): Among them, A L is selected from one of trivalent C1-C8 linear or branched alkyl groups, trivalent C3-C 10 cycloalkyl groups, trivalent C3-C 10 heterocyclic groups, trivalent C6-C 10 aryl groups, and trivalent C5-C 10 heteroaryl groups, B L1 , B L2 and B L3 are each independently selected from one of -C(O)C1-C8 alkyl groups, substituted -C(O)C1-C8 alkyl groups, C1-C8 alkylene groups, and substituted C1-C8 alkylene groups, represents the connection site of group covalent connection; Or, A L is selected from one of trivalent C3-C6 nitrogen-containing heterocyclic groups, and B L1 , B L2 and B L3 are each independently selected from one of -C(O)C2-C4 alkyl groups; or, A L is a trivalent 1,3,5-triazine group, and B L1 , B L2 and B L3 are all -CH2CH2CO-; Each of the three amino acid residues in the carrier peptide is covalently linked to the B L1 , B L2 or B L3 via an amino acid side chain, so that the carrier peptide forms a bicyclic peptide; alternatively, M A and / or Z A represents an amino acid sequence consisting of 1 to 3 amino acid residues, and at least one or each of the three amino acid residues is an amino acid residue in the amino acid sequence M A or Z A ; or each of the three amino acid residues is the same or different; or the three amino acid residues are all cysteine residues, and each cysteine is independently covalently linked to the linking group via an amino group, a carboxyl group or a mercapto group; Alternatively, the carrier peptide has an amino acid sequence shown in SEQ ID NO:153: CRSLGDTGCRSLGDTGC (SEQ ID NO:153), wherein the three cysteine residues represented by C are respectively linked through the sulfhydryl groups on the side chains to the B L1 , B L2 or B L3 ; Alternatively, the bicyclic amino acid sequence has a structure represented by the following formula (23):

9. The carrier peptide according to claim 1, wherein, Each amino acid residue is a natural amino acid residue.

10. The carrier peptide according to claim 1, wherein, At least one amino acid residue is a modified amino acid residue, and the modification refers to one or more of side chain modification, terminal group protection or chiral isomerism; or at least one or each of the side chain modifications is that a hydrogen atom or a functional group of the amino acid residue side chain is replaced by a modifying group, and each of the modifying groups is independently selected from a C1-C8 hydrocarbon group, a substituted C1-C8 hydrocarbon group, a C3-C 10 cycloalkyl group, a substituted C3-C 10 cycloalkyl group; or each of the modifying groups is independently selected from one of C1-C3 hydrocarbon groups; or each of the modifying groups is independently methyl, ethyl, propyl or isopropyl.

11. The carrier peptide according to claim 10, wherein, All amino acid residues are modified amino acid residues; or at least one of the modified amino acid residues is a D - amino acid residue.

12. An active carrier peptide, wherein, The active carrier peptide contains one or more carrier peptide groups and one or more reactive groups. Each carrier peptide group is formed by removing one or more atoms from the carrier peptide described in any one of claims 1 - 11. Each reactive group is covalently connected to the amino acid residue in the carrier peptide group. The reactive group contains one or more functional groups or protected functional groups, and this functional group can undergo a conjugation reaction; or the conjugation reaction refers to an addition reaction, a coupling reaction or a substitution reaction that can form a covalent connection.

13. The active carrier peptide according to claim 12, wherein, The number of the reactive groups is 1 to 3; or, the active carrier peptide only contains 1 reactive group.

14. The active carrier peptide according to claim 12 or 13, wherein, Each of the reactive groups independently contains 1-3 of the functional groups; alternatively, the reactive group has a structure represented by formula (501): wherein, L R represents any linking group capable of linking the functional group to the carrier peptide group, G F represents the functional group, and n501 is an integer from 1 to 3, represents the linking site for covalent linkage of the group; or, L R is a straight-chain or branched-chain C1-C with a valence of 2-7 25 or C3-C 15 saturated hydrocarbon group, and one or more methylene groups in the straight-chain or branched-chain saturated hydrocarbon group are optionally replaced by one or more linking combinations selected from the group consisting of the following groups: C(O), NH, O, S, CH═N, S(O)2, OP(O)2, C5-C8 sub-glycosyl group, C2-C5 sub-alkenyl group, C2-C5 sub-alkynyl group, C6-C 10 sub-aryl group, C3-C8 sub-heterocyclic group and C5-C 10 sub-heteroaryl group; and / or if there is a methine group in the branched-chain saturated hydrocarbon group, then one or more methine groups are optionally replaced by one or more selected from the group consisting of the following groups: nitrogen atom, trivalent C3-C 10 cycloalkyl group, trivalent C3-C 10 heterocyclic group, trivalent C6-C 10 aryl group and trivalent C5-C 10 heteroaryl group; and / or optionally, the saturated hydrocarbon group has a substituent selected from any one or more of the following groups: C1-C5 alkyl group, C6-C 10 aryl group, C5-C 10 heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH, -SC1-C5 alkyl group, nitro group, -C(O)O(C1-C5 alkyl group), -CON(C1-C5 alkyl group)(C1-C5 alkyl group), -CONH(C1-C5 alkyl group), -CONH2, -NHC(O)(C1-C5 alkyl group), -NHC(O)(phenyl group), -N(C1-C5 alkyl group)C(O)(C1-C5 alkyl group), -N(C1-C5 alkyl group)C(O)(phenyl group), -C(O)C1-C5 alkyl group, -C(O)C1-C5 alkylphenyl group, -OC(O)C1-C5 alkyl group, -SO2(C1-C5 alkyl group), -SO2(phenyl group), -SO2NH2, -SO2NH(C1-C5 alkyl group), -SO2NH(phenyl group), -NHSO2(C1-C5 alkyl group) and -NHSO2(phenyl group).

15. The active carrier peptide according to claim 12, wherein, Each of the functional groups is the same or different, and is independently selected from the group consisting of azide group, alkynyl group, dibenzocyclooctynyl group, mercapto group, amino group, hydroxyl group, carboxyl group, acyl halide group, aldehyde group, carbonate group, aminooxy group, active ester group, disulfide group, o - pyridyl disulfide group, maleimide group, tosylate group, tetrazine group, trans - cyclooctenyl group, hydrazide group and phosphoramidite group.

16. The active carrier peptide according to claim 15, wherein, Each of the functional groups is independently selected from the group consisting of an azide group, a thiol group, a hydroxyl group, an amino group, a maleimide group, a disulfide group, or an ortho-pyridyl disulfide group.

17. The active carrier peptide according to any one of claims 12-16, wherein, At least one or each of the reactive groups is a cysteine residue or an azidohomoalanine residue; alternatively, at least one or each of the reactive groups comprises a maleimide group.

18. The active carrier peptide according to claim 17, wherein, The cysteine residue is a group represented by formula (L38) or (L39); the azidohomoalanine residue is a group represented by formula (L40) or (L41); or, the reactive group has a structure represented by formula (L401), (L402) or (L403): Among them, Denotes the site where the group is linked to the rest of the molecule; n 38 、n 39 、n 40 、n 41 and n 401 are each independently an integer from 1 to 10; R 38 、R 40 and R 42 are each independently a hydroxyl group, a protected hydroxyl group, an amino group or a site covalently linked to a group; R 39 、R 41 and R 43 each independently represents hydrogen, an amino protecting group or a site covalently linked to a group; Optionally, the reactive group has a structure represented by formula (L404) or (L405):

19. The active carrier peptide according to any one of claims 12-18, wherein, At least one or each of the reactive groups is linked to an amino-terminal amino acid residue or a carboxyl-terminal amino acid residue of the carrier peptide group.

20. A carrier peptide conjugate, the carrier peptide conjugate containing one or more carrier peptide groups and one or more functional groups, wherein, Each carrier peptide group is formed by removing one or more atoms from the carrier peptide as described in any one of claims 1-11. Each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group, a functional oligonucleotide group, and a delivery aid group; each carrier peptide group is linked to at least one of the functional groups or at least one additional carrier peptide group through a linking group, and each functional group is linked to at least one of the carrier peptide groups or at least one additional functional group through a linking group.

21. The conjugate according to claim 20, wherein, Each carrier peptide group is independently linked to the linking group at the amino group at the amino terminus or the carboxyl group at the carboxyl terminus in the carrier peptide group, or each carrier peptide group is linked to the linking group at the amino terminus.

22. The conjugate according to claim 20 or 21, wherein, The conjugate contains n carrier peptide groups and m functional groups, where n is an integer selected from 1-8 and m is an integer selected from 1-4.

23. The conjugate according to claim 22, wherein, The conjugate has a structure represented by formula (101): wherein the linking group consists of all L A , L B and L C ; L A is the branched portion, L B is the conjugating linking group connecting L C and Nu; L C is the branching group; Nu and each PP independently represent the functional group or the carrier peptide group; at least one of Nu and all the PPs is the functional group and at least one is the carrier peptide group; each k is independently an integer from 1 to 6; n 101 is an integer from 1 to 3; and the sum of all k is an integer from 1 to 8; The branched group L C is a straight-chain or branched-chain C1-C with a valence of 2-7 25 or C3-C 15 saturated hydrocarbon group, or one or more methylene groups in the straight-chain or branched-chain saturated hydrocarbon group are optionally replaced by one or more linking combinations selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 sub-glycosyl group, C2-C5 sub-alkenyl group, C2-C5 sub-alkynyl group, C6-C 10 sub-aryl group, C3-C8 sub-heterocyclic group and C5-C 10 sub-heteroaryl group; and / or If there is a methylene group in the branched-chain saturated hydrocarbon group, one or more of the methylene groups are optionally replaced by one or more selected from the group consisting of: a nitrogen atom, a trivalent C3-C 10 cycloalkyl group, a trivalent C3-C 10 heterocyclic group, a trivalent C6-C 10 aryl group and a trivalent C5-C 10 heteroaryl group, and Among them, the saturated hydrocarbon group optionally has a substituent selected from any one or more of the groups consisting of: C1-C5 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl); The branched-chain portion L A is a covalent bond, or a C1-C 20 or C3-C 15 alkylene group, or one or more carbon atoms in the alkylene group are replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, a C5-C8 sub-glycosyl group, a C2-C5 sub-alkenyl group, a C2-C5 sub-alkynyl group, a C6-C 10 sub-aryl group, a C3-C8 sub-heterocyclic group and a C5-C 10 sub-heteroaryl group; wherein, the alkylene group may have any one or more substituents selected from the group consisting of: a C1-C5 alkyl group, a C6-C 10 aryl group, a C5-C 10 heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH-SC1-C5 alkyl group, -SC1-C5 alkylphenyl group, -C1-C5 alkyl-SH, -OH, -SH, -NH2, -C1-C5 alkyl-NH2, -N(C1-C5 alkyl)(C1-C5 alkyl), -NH(C1-C5 alkyl), -N(C1-C5 alkyl)(C1-C5 alkylphenyl), -NH(C1-C5 alkylphenyl), nitro group, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

24. The conjugate according to claim 23, wherein, The L B is one of the following linking groups: phosphite group, thiophosphite group, amide group, ester group, oxygen atom, dithio group; alternatively, the L B is a phosphite group, a thiophosphite group or a dithio group.

25. The conjugate according to claim 23 or 24, wherein 1) Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and combinations of their linkages; and / or 2) Each L C is -H1-H2-, where H1 is selected from the group consisting of the groups (L4)-(L23) and their linking combinations: In the formula, each j1 is an integer from 1 to 10; Each R’ is a C1-C 10 alkyl group; Each Ra is a hydrogen atom, a C1-C 10 alkyl group, or a group selected from the group consisting of the free radical groups (L24)-(L37): H2 is selected from any one of the groups represented by formulas (B1)-(B5), Among them, the carbon atoms in formulas (B1)-(B3) are directly connected to one or more L A connections, and the nitrogen atom is used to connect to H1 via an imino or amide bond; Each nitrogen atom in formula (B4) is used to connect to an L via an imino or amide bond A ; one oxygen atom is used to connect to H1 via an imino or amide bond, and the other oxygen atom forms a hydroxyl group with a hydrogen atom; One of the nitrogen atoms in formula (B5) is used to connect to H1 via an imino or amide bond, and each of the remaining nitrogen atoms is separately used to connect to an L via an imino or amide bond A connection Denotes the site of covalent linkage of the group.

26. The conjugate according to claim 25, wherein, H1 and / or each L A are independently selected from the group consisting of linking combinations of at least two of the groups (L4)-(L9), (L13), (L14), (L17) and (L18).

27. The conjugate according to claim 26, wherein, H1 and / or each L A is independently selected from the linking combinations of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L17) and (L18).

28. The conjugate according to claim 26 or 27, wherein, The length of H1 is 5 to 20 atoms, and the length refers to the number of atoms in the longest atomic chain in H1 from the site connected to L B to the site connected to H2.

29. The conjugate according to any one of claims 25-28, wherein, L C has the structure shown in formula (201a): where n 201 and m 201 are each independently an integer from 1 to 10, and the sum of n 201 and m 201 is not greater than 10 or not greater than 5.

30. The conjugate according to claim 29, wherein, The conjugate has a structure represented by formula (201):

31. The conjugate according to claim 30, wherein, each k and n 101 independently is an integer from 1 to 3, and the sum of all k's is not greater than 6.

32. The conjugate according to claim 25 or 30, wherein, Each L A is a linking combination of at least two of the groups (L4)-(L9), (L13), (L14), (L17), and (L18).

33. The conjugate according to claim 32, wherein, L A has a length of 3 to 35 atoms, 3 to 20 atoms, or 4 to 15 atoms, wherein the length of said L A refers to the number of chain-forming atoms in the longest atomic chain formed by the atoms directly connected to L A in L C and the atoms directly connected to PP in L A , and any cycloalkylidene, arylidene, heterocycloalkylidene or heteroarylidene is counted as one atom.

34. The conjugate according to claim 23 or 30, wherein, L A has the structure shown in formula (202): wherein, n 202 , m 202 , p 202 , q 202 are each independently an integer from 1 to 5, i 202 is an integer from 0 to 5, and the L A is connected to the PP through an imino group and is connected to the L C .

35. The conjugate according to claim 34, wherein, n 202 、m 202 、p 202 and q 202 Each independently is 2 or 3, i 202 Is 3 or 4.

36. The conjugate according to claim 35, wherein, In formula (202), n 202 , all m 202 , q 202 and i 202 The sum of them is not greater than 26, or not greater than 11, or not greater than 6.

37. The conjugate according to claim 23, the conjugate having the structure shown in formula (103): wherein R 107 is amino or hydroxyl; n 103 and m 103 are each independently an integer from 1 to 10; n 101 is an integer from 1 to 3; where n 104 and m 104 are each independently an integer from 1 to 10; n 102 is an integer from 1 to 3.

38. The conjugate according to claim 37, wherein, n 103 is an integer from 1 to 3, m 103 is an integer from 3 to 6, n 101 is an integer from 1 to 3; or, n 104 is an integer from 1 to 3, m 104 is an integer from 3 to 6, n 102 is an integer from 1 to 3.

39. The carrier peptide conjugate according to claim 19 or 22, wherein, At least one or each of the functional groups is a small molecule therapeutic agent group or a functional oligonucleotide group having a therapeutic effect on central nervous system-related diseases or symptoms.

40. The conjugate according to claim 39, wherein, The functional oligonucleotide is a single-stranded oligonucleotide.

41. The conjugate according to claim 39, wherein, The functional oligonucleotide is siRNA or shRNA. The siRNA or shRNA contains a sense strand and an antisense strand. The sense strand and the antisense strand each contain 15-25 nucleotides; each nucleotide is a modified or unmodified nucleotide.

42. The conjugate according to claim 41, wherein, All carrier peptide groups are linked to the sense strand.

43. The conjugate according to claim 42, wherein, At least one of the carrier peptide groups is linked to the first nucleotide at the 3'-end or the 5'-end of the sense strand.

44. The conjugate according to claim 43, wherein, At least one of the carrier peptide groups is linked to the first nucleotide at the 3'-end of the sense strand, and at least one of the carrier peptide groups is linked to the first nucleotide at the 5'-end of the sense strand.

45. The conjugate according to claim 42, wherein, The sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, the nucleotide sequence II is at least partially reverse complementary to the target mRNA, and the target mRNA is the mRNA expressed by the target gene in the target cells in the target tissue or target organ, and the target cells are cells having a receptor on the surface capable of binding to the carrier peptide.

46. The conjugate according to claim 45, wherein the target tissue or target organ is the brain or the spinal cord.

47. The conjugate according to claim 45, wherein, The target gene is one of APP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR, TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B or RHO.

48. The conjugate according to claim 20 or 23, wherein, The functional group is a diagnostic agent group.

49. The conjugate according to claim 48, wherein, The diagnostic agent group is a fluorescent group, a contrast agent group or a group containing a radionuclide.

50. Use of the carrier peptide according to claims 1-11 in delivering a functional molecule, wherein the functional molecule is selected from one or more of a diagnostic agent, a small molecule therapeutic agent, a functional oligonucleotide and a delivery aid.

51. A pharmaceutical composition comprising one or more of the conjugate according to any one of claims 20-47, its pharmaceutically acceptable salt, metabolite or prodrug, and a pharmaceutically acceptable carrier.

52. The pharmaceutical composition according to claim 51, wherein, The weight ratio of the pharmaceutically active molecule to the pharmaceutically acceptable carrier is 1:(1-100), or the weight ratio is 1:(1-50).

53. A pharmaceutical composition comprising the conjugate according to claim 48 or 49 and a pharmaceutically acceptable carrier.

54. The pharmaceutical composition according to claim 53, wherein, The weight ratio of the pharmaceutically active molecule to the pharmaceutically acceptable carrier is 1:(1-100), or the weight ratio is 1:(1-50).

55. Use of one or more of the conjugate according to any one of claims 20-47, its pharmaceutically acceptable salt, metabolite or prodrug, and / or the pharmaceutical composition according to claim 51 or 52 in the preparation of a drug for treating and / or preventing a disease related to the central nervous system.

56. The use according to claim 55, wherein The disease is a disease related to the mRNA of target gene expression in cells in the central nervous system.

57. The use according to claim 56, wherein, The target gene is one of APP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR, TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B or RHO.

58. A method for treating and / or preventing a disease related to the central nervous system, the method comprising administering to a subject in need an effective amount of one or more of the conjugate according to any one of claims 20-47, its pharmaceutically acceptable salt, metabolite or prodrug, and / or the pharmaceutical composition according to claim 51 or 52.

59. Use of the conjugate according to claim 48 or 49 and / or the pharmaceutical composition according to claim 53 or 54 in the preparation of a drug for diagnosing a disease related to the central nervous system.

60. A method for diagnosing a disease related to the central nervous system, the method comprising administering to a subject in need thereof an effective amount of one or more of the conjugate according to claim 48 or 49, a pharmaceutically acceptable salt, metabolite or prodrug thereof, and / or the pharmaceutical composition according to claim 53 or 54.

61. A kit comprising one or more of the conjugate according to any one of claims 20-49, a pharmaceutically acceptable salt, metabolite or prodrug thereof, and / or the pharmaceutical composition according to any one of claims 51-54.

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