CN116854753A - Compounds, conjugates, compositions and uses thereof - Google Patents

Abstract

The present disclosure relates to the field of pharmaceuticals, and in particular to a compound, conjugate, composition, and uses thereof. The compound has a structure shown in a formula (Ia). Conjugates provided by the present disclosure are formed by conjugating compounds provided by the present disclosure to oligonucleotides. The conjugates provided by the present disclosure are highly effective in targeting to the liver and targeting genes to the liverThe expression produces effective inhibition, can be used for treating and/or preventing liver diseases, has higher in vivo activity than a control vector, has more stable and lasting pharmacodynamic action, and is expected to have excellent safety and low animal level toxicity.

Description

Compounds, conjugates, compositions and uses thereof

Technical Field

The present disclosure relates to the field of medicine, in particular, the present disclosure relates to a compound, conjugate, composition and uses thereof.

Background

Small molecule nucleic acid drugs represented by small interfering RNAs (small interference RNA, siRNA), antisense oligonucleotides (antisense oligodeoxynucleotide, ASODN) and nucleic acid stimulatory motifs (CpG) are increasingly important in gene therapy, some of which have been approved by the FDA for marketing, and many more are currently in preclinical and clinical trials. Nucleic acid drugs refer to nucleic acid sequences that specifically target pathogenic genes or proteins by binding or cleavage, thereby inhibiting/promoting expression of certain genes/proteins, including all human normal genes that can replace defective genes, antisense nucleic acids that block gene expression, or single-stranded nucleic acids that promote triplex formation, etc., such as siRNA, DNA, microRNA or CpG, etc.

Delivery systems are one of the key technologies in the development of small nucleic acid drugs, and the most widely studied class of delivery systems for small nucleic acid delivery systems worldwide is currently targeted conjugated delivery technology. There remains a pressing need in the art to develop a new drug conjugate with higher efficacy of active drug delivery in vivo, lower toxicity, higher activity.

Disclosure of Invention

The present disclosure is directed to solving, at least in part, at least one of the technical problems existing in the prior art.

To this end, the present disclosure provides compounds, conjugates, compositions, and uses thereof. The conjugate provided by the present disclosure is capable of highly effectively targeting the liver and producing effective inhibition of expression of liver target genes, is useful for treating and/or preventing liver-derived diseases, has higher in vivo activity than control vectors, is more stable and durable in pharmacodynamic action, and is expected to have excellent safety and low animal level toxicity.

In a first aspect of the present disclosure, the present disclosure provides a compound having a structure represented by formula (Ia):

wherein R is ₁ Selected from H, hydroxy protecting groups or * Represents a ligation site for ligation of pharmaceutically active molecules;

each Z is independently selected from hydroxyl or mercapto;

R ₂ selected from H, reactive phosphorus groups orR _2b Selected from solid supports containing amino functions, R _2a A covalent linking group selected from the group consisting of a covalent linkage to the amino functional group;

n is selected from 0, 1, 2 or 3;

each j is independently selected from 1, 2 or 3;

each L is independently selected from substituted or unsubstituted C ₂ -C ₁₀ Alkylene orWherein each R _L Each independently selected from substituted or unsubstituted C ₁ -C ₅ Alkylene, each X is independently selected from O, S, NH or-NH-C (O) -, m is selected from 1, 2, 3, 4 or 5;

each R ₃ Each independently selected from H, substituted or unsubstituted C ₁ -C ₄ Alkylacyl or substituted or unsubstituted C ₅ -C ₇ Aryl acyl, R ₃ Each substituent group of (a) is independently selected from halogen, C ₁ -C ₂ An alkoxy group.

In some alternative embodiments of the present disclosure, each L is independently selected from substituted or unsubstituted C ₂ -C ₁₀ An alkylene group.

In some alternative embodiments of the present disclosure, each L is independentlySelected from the group consisting of substituted and unsubstituted C ₂ -C ₁₀ A linear alkylene group. For example: substituted or unsubstituted C ₂ -C ₉ Straight chain alkylene, substituted or unsubstituted C ₂ -C ₈ Straight chain alkylene, substituted or unsubstituted C ₂ -C ₇ Straight chain alkylene, substituted or unsubstituted C ₂ -C ₆ Straight chain alkylene, substituted or unsubstituted C ₂ -C ₅ Straight chain alkylene, substituted or unsubstituted C ₂ -C ₄ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₉ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₈ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₇ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₆ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₅ Straight chain alkylene, substituted or unsubstituted C ₃ -C ₄ Straight chain alkylene, substituted or unsubstituted C ₄ -C ₉ Straight chain alkylene, substituted or unsubstituted C ₄ -C ₈ Straight chain alkylene, substituted or unsubstituted C ₄ -C ₇ Straight chain alkylene, substituted or unsubstituted C ₄ -C ₆ Straight chain alkylene, substituted or unsubstituted C ₄ -C ₅ Straight chain alkylene, substituted or unsubstituted C ₄ A linear alkylene group.

In some alternative embodiments of the present disclosure, each L is independently selected from

In some embodiments of the disclosure, each L is selected from

In some embodiments of the present disclosure, j is selected from 1.

In some embodiments of the present disclosure, each Z is selected from hydroxyl.

In some alternative embodiments of the present disclosure, n is selected from 0, 1, or 2.

In some embodiments of the present disclosure, n is selected from 0.

In some embodiments of the disclosure, n is selected from 1.

In some embodiments of the disclosure, n is selected from 2.

In some alternative embodiments of the present disclosure, each R3 is independently selected from H, substituted or unsubstituted C ₁ -C ₄ Alkyl acyl group

(e.g., acetyl, propionyl, n-butyryl, isobutyryl, n-pentanoyl, isopentanoyl, 2-methylbutanoyl, pivaloyl/pivaloyl) or substituted or unsubstituted C ₅ -C ₇ Aryl acyl (e.g. benzoyl), R ₃ Each substituent group of (2) is independently selected from halogen (e.g., F).

In some alternative embodiments of the present disclosure, each R ₃ Each independently selected from H,

In some alternative embodiments of the present disclosure, each R ₃ Each independently selected from H or

In some embodiments of the disclosure, each R ₃ Are all selected from

In some embodiments of the disclosure, each R ₃ Are all selected from H.

In the disclosure, the hydroxyl protecting group may be various hydroxyl protecting groups, as long as the hydroxyl group can be protected, and the specific type is not limited. In some embodiments, the hydroxyl protecting group is stable under basic conditions, but can be removed under acidic conditions.

In some alternative embodiments of the present disclosure, hydroxyl protecting groups that may be used in the present disclosure include, but are not limited to, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and 9- (p-methoxyphenyl) xanthin-9-yl (Mox), trityl (Tr-yl), 4-methoxytrityl (MMTr-yl), 4 '-dimethoxytrityl (DMTr-yl) and 4,4',4 "-trimethoxytrityl (TMTr-yl).

In some alternative embodiments of the present disclosure, the hydroxyl protecting group is selected from trityl (Tr group), 4-methoxytrityl (MMTr group), 4 '-dimethoxytrityl (DMTr group) or 4,4',4 "-trimethoxytrityl (TMTr group).

In some embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4' -dimethoxytrityl (DMTr group).

In some alternative embodiments of the present disclosure,selected from-> * Representing a ligation site for ligation of pharmaceutically active molecules.

In some alternative embodiments of the present disclosure, the pharmaceutically active molecule is selected from a small molecule drug, an antibody, or an oligonucleotide.

In some alternative embodiments of the present disclosure, the oligonucleotide is selected from the group consisting of a single stranded oligonucleotide and a double stranded oligonucleotide.

In some alternative embodiments of the present disclosure, the single stranded oligonucleotide is selected from the group consisting of an antisense oligonucleotide, a nucleic acid aptamer, a ribozyme, a deoxyribozyme, a circular RNA, a sense strand of an siRNA, or an antisense strand of an siRNA.

In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide is selected from the group consisting of a small interfering RNA, a double-stranded RNA, a microrna, a small guide RNA, a small activating RNA, or a short hairpin RNA.

In some embodiments of the present disclosure, the pharmaceutically active molecule is selected from double-stranded oligonucleotides.

In some embodiments of the present disclosure, the pharmaceutically active molecule is selected from siRNA.

In some alternative embodiments of the present disclosure, the solid support is selected from an amino-functional group-containing resin (e.g., polystyrene, abbreviated PS) or an amino-functional group-containing controlled pore glass sphere (Controlled Pore Glass, abbreviated CPG).

In some embodiments of the disclosure, R _2a Selected from the group consisting of

In some embodiments of the disclosure, R _2b Selected from the group consisting ofSelected from resin or controllable pore size glass spheres;

in some embodiments of the present disclosure,selected from->

In some alternative embodiments of the present disclosure, each Z is independently selected from

In some embodiments of the present disclosure, each Z isIn some embodiments of the present disclosure, each Z is +.>In some embodiments of the present disclosure, each Z is +.>In some embodiments of the present disclosure, each Z is +.>

In some alternative embodiments of the present disclosure, eachEach independently selected from

In the disclosure, a "reactive phosphorus group" refers to a phosphorus-containing group that can be reacted with other compounds to remove.

In some alternative embodiments of the present disclosure, the reactive phosphorus group is selected from In some embodiments of the present disclosure, the reactive phosphorus group is selected fromIn some alternative embodiments of the present disclosure, the compound has a structural formula shown in formula (II):

wherein each R ₄ Each independently selected from H, C ₁ -C ₂ Alkyl or C ₁ -C ₂ An alkoxy group;

each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

R ₁ 、R ₂ 、n、j、Z、R ₃ as defined above.

In some embodiments of the disclosure, each R ₄ Are all selected from H.

In some embodiments of the present disclosure, each k is selected from 4.

In some alternative embodiments of the present disclosure, the compound has a structural formula shown in formula (III):

wherein R is ₁ 、R ₂ 、n、Z、R ₃ 、R ₄ K is as defined above.

In some selected embodiments of the present disclosure, the compound has a structural formula shown in formula (IV):

wherein R is ₁ 、R ₂ 、n、Z、R ₃ K is as defined above.

In some selected embodiments of the present disclosure, the compound is selected from any one of the following structures:

in a second aspect of the present disclosure, the present disclosure provides a conjugate having a structure represented by formula (Ib):

wherein Nu represents an oligonucleotide;

n' is selected from 1, 2, 3 or 4;

j. Z, L are as defined above.

In some embodiments of the disclosure, n' is selected from 1, 2, or 3.

In some embodiments of the disclosure, n' is selected from 3.

In some alternative embodiments of the present disclosure, the conjugate has a structural formula shown in formula (IIb):

wherein Nu, j, Z, n', R ₄ K is as defined above.

In some alternative embodiments of the present disclosure, the conjugate has a structural formula shown in formula (IIIb):

wherein Nu, Z, n', R ₄ K is as defined above.

In some alternative embodiments of the present disclosure, the conjugate has a structural formula shown in formula (IVb):

wherein Nu, Z, n', k are as defined previously.

In some embodiments of the present disclosure, the conjugate has a formula selected from the group consisting of

In a third aspect of the present disclosure, the present disclosure provides a composition comprising the conjugate of the second aspect.

In some alternative embodiments of the present disclosure, the composition further comprises at least one pharmaceutically acceptable carrier.

In a fourth aspect of the present disclosure, the present disclosure provides a use of any of the following in the manufacture of a medicament for the prevention and/or treatment of a disease:

(I) The compound of the first aspect; and/or

(II) the conjugate of the second aspect; and/or

(III) the composition of the third aspect.

In some alternative embodiments of the present disclosure, the disease is selected from a pathological condition or disease caused by abnormal expression of a target gene in liver cells. Illustratively, the target genes include, but are not limited to, at least one of Apoa, apoB, apoC, ANGPTL3, PCSK9, SOD1, FVII, p53, C3, C4, C5, AGT, CFB, USP, ASGR1, FTO, INHBE, HBV, and HCV.

In some embodiments of the disclosure, the target gene is selected from SOD1.

In a fifth aspect of the present disclosure, the present disclosure provides a method of reducing expression or activity of a target gene, the method comprising contacting a liver cell with any of:

(I) The conjugate of the second aspect; and/or

(II) the composition of the third aspect.

In some embodiments of the disclosure, the target gene includes, but is not limited to, at least one of Apoa, apoB, apoC, ANGPTL, PCSK9, SOD1, FVII, p53, C3, C4, C5, AGT, CFB, USP, ASGR1, FTO, INHBE, HBV, and HCV.

In some embodiments of the disclosure, the target gene is selected from SOD1.

Additional aspects and advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.

Advantageous effects

Conjugates provided by the present disclosure are formed by conjugating compounds provided by the present disclosure to oligonucleotides. The conjugates provided by the present disclosure are highly effective in targeting the liver and producing effective inhibition of expression of liver target genes, are useful for the treatment and/or prevention of liver-derived diseases, have higher in vivo activity than control vectors, have more stable and durable pharmacodynamic effects, and are expected to have excellent safety and low animal level toxicity.

Drawings

FIG. 1 is the inhibitory activity of a target gene of interest in mice after administration of the siRNA conjugates described in example 2.1;

FIG. 2 is the inhibitory activity of a target gene of interest in mice following administration of the siRNA conjugates described in example 2.2.

Detailed description of the preferred embodiments

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

Interpretation of the terms

In the context of the present disclosure, the term "alkyl" refers to a compound of the formula The alkanyl radical of (2) may be a straight-chain alkyl radical or a branched-chain alkyl radical. Wherein the term "C ₁ -C ₂ Alkyl "refers to an alkanyl radical having 1 to 2 carbon atoms, such as methyl or ethyl.

In the context of the present disclosure, the term "alkylene" refers to a compound of the general formulaThe chain alkylene group of (a) may be a straight chain alkylene group or a branched chain alkylene group. Wherein the term "C ₂ -C ₁₀ Alkylene "refers to an alkylene chain having 1 to 10 carbon atoms. />

In the context of the present disclosure, the term "NH" refers to an imino group of the formula

In the context of the present disclosure, the term "alkanoyl" has the formulaFor example "C ₁ -C ₅ Alkanoyl "may be acetyl, propionyl, n-butyryl or isobutyryl, and the like.

In the context of the present disclosure, the term "aroyl" has the structural formulaWherein Ar refers to an aryl group, ar refers in organic chemistry to any functional group or substituent derived from a simple aromatic ring. The simplest aryl group is Phenyl (Phenyl), which is derived from benzene.

In the context of the present disclosure, the term "alkoxy" has the formulaWherein "C ₁ -C ₂ Alkoxy "may be methoxy or ethoxy.

In the context of the present disclosure, "pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

In the context of the present disclosure, a "pharmaceutically acceptable acid addition salt" refers to a salt with an inorganic or organic acid that is capable of retaining the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formate, acetate, 2 dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

In the context of the present disclosure, a "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including natural substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (RNA), typically consisting of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as ribonucleic acid interference, ribonuclease-mediated target degradation, splice regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

TerminologyRepresenting the site of attachment of the group by covalent bonds.

In the structural formulae of the compounds of the present disclosure, the bond "-" represents an unspecified configuration. If chiral isomerism exists in the chemical structure, the bond "-" may be、/>Or at the same time contain->And->Two configurations. Although all of the above structural formulae are drawn as certain isomeric forms for simplicity, the present disclosure may include all isomers, for example: tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers.

In the structural formula of the compound disclosed in the present disclosure, a bond "=" represents an unspecified configuration. If cis-trans isomerism exists in the chemical structure, the bond "=" configuration may be E-type, Z-type, or both E and Z-containing configurations.

The following definitions as used herein should be applied unless otherwise indicated. For the purposes of this disclosure, chemical elements are consistent with CAS version of the periodic Table of the elements, and handbook of chemistry and physics, 75 th edition, 1994. In addition, general principles of organic chemistry may be referenced to the descriptions in "Organic Chemistry", thomas Sorrell, university Science Books, sausalato:1999, and "March's Advanced Organic Chemistry" by Michael b.smith and Jerry March, john Wiley & Sons, new york:2007, the entire contents of which are incorporated herein by reference. The articles "a," "an," and "the" are intended to include "at least one" or "one or more" unless the context clearly dictates otherwise or otherwise. Thus, as used herein, these articles refer to one or to more than one (i.e., to at least one) object. For example, "a component" refers to one or more components, i.e., more than one component is contemplated as being employed or used in embodiments of the described embodiments.

The term "comprising" is an open-ended expression, i.e., including what is indicated in the disclosure, but not excluding other aspects.

"stereoisomers" refer to compounds having the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

"chiral" is a molecule that has properties that do not overlap with its mirror image; and "achiral" refers to a molecule that may overlap with its mirror image.

"enantiomer" refers to two isomers of a compound that do not overlap but are in mirror image relationship to each other.

"diastereoisomers" refers to stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. The diastereomeric mixture may be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.

Stereochemical definitions and rules as used in the present disclosure generally follow s.p. parker, ed., mcGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, new York; and Eliel, e.and Wilen, s., "Stereochemistry of Organic Compounds", john Wiley & Sons, inc., new York,1994.

As described in the present disclosure, the compounds of the present disclosure may be optionally substituted with one or more substituents, such as the compounds of the general formula above, or as specific examples within the examples, subclasses, and classes of compounds encompassed by the present disclosure.

In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a specific substituent. Unless otherwise indicated, a substituted group may have a substituent substituted at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from a particular group, then the substituents may be the same or different substituted at each substitutable position.

The term "unsubstituted" means that the specified group does not carry a substituent.

The term "optionally substituted with … …" may be used interchangeably with the term "unsubstituted or substituted with … …," i.e., the structure is unsubstituted or substituted with one or more substituents described in this disclosure. Substituents described in this disclosure include, but are not limited to: D. f, cl, br, I, N3, CN, NO2, OH, SH, NH2, alkyl, haloalkyl, haloalkoxy, haloalkylamino, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like.

In addition, unless explicitly indicated otherwise, the descriptions used in this disclosure of the manner in which each … is independently "and" … is independently "and" … is independently "are to be construed broadly as meaning that particular items expressed between the same symbols in different groups do not affect each other, or that particular items expressed between the same symbols in the same groups do not affect each other. Taking R3 as an example, the specific options of R3 between the structural formula "C1-C50 alkylene optionally substituted with R3" and the structural formula "C (O) -NH-C1-50 alkylene optionally substituted with R3" are not affected by each other.

The term "small interfering RNA (Small interfering RNA; siRNA)" is a type of double-stranded RNA comprising a sense strand and an antisense strand, each strand being 17 to 30 nucleotides in length. siRNA mediates RNA transcript targeted cleavage of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into amino acids and conversion to proteins.

In the context of the present disclosure, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. "sense strand (or" follower strand) "means that it contains an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, e.g., the antisense strand is complete complementarity or at least partial complementarity to a target sequence. In the case of partial complementarity, the mismatch may be present within the interior or terminal region of the molecule, wherein the most tolerated mismatch is present within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 '-and/or 3' -end of the iRNA.

It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (RNA), typically consisting of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as ribonucleic acid interference, ribonuclease-mediated target degradation, splice regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

In the context of the present disclosure, an "antisense oligonucleotide (antisense oligonucleotides, ASO)" is a single stranded oligonucleotide molecule, typically consisting of 10 to 50 nucleotides. ASO enters cells and then is combined with complementary target mRNA through the base complementary pairing principle under the action of ribonuclease H1, so that the expression of target genes is inhibited.

In the context of the present disclosure, upper case A, U, G, C, T indicates the base composition of a nucleotide, and lower case m indicates that one nucleotide adjacent to the left of the letter m is a 2' -methoxy modified nucleotide, unless otherwise specified; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that there is a phosphorothioate linkage between two nucleotides adjacent to the letter s.

In the context of the present disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring, coloring agent, or combination thereof, all of which are known to those of skill in the art (as described in Remington's Pharmaceutical Sciences,18th Ed.Mack Printing Company,1990,pp.1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

In the context of the present disclosure, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the siRNA of the present disclosure, such as any adverse biological effect produced or interactions with any other component of the pharmaceutically acceptable composition that occur in a deleterious manner, their use is also contemplated by the present disclosure.

In the context of the present disclosure, "subject" refers to any animal, such as a mammal or a pouched animal. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., monkeys), mice, pigs, horses, donkeys, cattle, sheep, and any variety of poultry.

In the context of the present disclosure, "treatment," "alleviation," or "improvement" may be used interchangeably herein. These terms refer to methods of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Here, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

In the context of the present disclosure, "prevent" and "prevent" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," the conjugate, RNAi agent, or composition may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

In the context of the present disclosure, the ratio of reagents described in the various embodiments of the present disclosure are calculated as volume ratio (v/v) unless otherwise indicated.

Unless otherwise indicated, the starting materials and reagents used in the preparation of the compounds provided by the present disclosure were purchased from Beijing coupling technologies Inc. Details of some of the reagents used in the present disclosure are shown in table 1.

TABLE 1 details of the reagents

The reagent consumables (Table 2) and instrumentation (Table 3) used in the present disclosure were all derived from commercial products from the following manufacturers, unless otherwise specified.

Table 2 Primary reagent consumable

TABLE 3 Main instrumentation

Name of the name	Manufacturing factories
		Full-automatic nucleic acid extractor	Zhejiang Hanwei science and technology Limited liability company
High-speed refrigerated centrifuge	Eppendorf
		NANODROP OneC	Thermo Fisher Scientific
Gradient PCR amplification instrument	Eppendorf
		Fluorescent quantitative PCR instrument	ABI StepOne Plus
Gel imaging instrument	Shanghai Tianneng Life Science Co., Ltd.
		Electrophoresis apparatus	Beijing Liuyi Instrument Factory
Tissuelyser II type full-automatic tissue homogenate instrument	SHANGHAI JINGXIN INDUSTRIAL DEVELOPMENT Co.,Ltd.

Preparation example 1: synthesis of Compound CR01018

In this preparation, the synthetic route for compound CR01018 is as follows:

(1-1) Synthesis of Compound CR01018-2

Methanesulfonamide (1.28 g,13.5 mmol) was dissolved in a mixed solution of 60ml of t-butanol and 30ml of water at 25℃and AD-mix- β (21 g) was added, the reaction system was cooled to 0℃with an ice-water bath and compound CR01018-1 (2.85 g,13.5mmol, 4-vinylpiperidine-1-carboxylic acid t-butyl ester) was added at 0℃and the reaction system was warmed to 25℃and stirred at 25℃for 16 hours. After the completion of the reaction, the reaction system was cooled to 0℃with an ice-water bath and sodium thiosulfate pentahydrate (33.48 g,135 mmol) was added at 0℃and extracted three times with 100ml of ethyl acetate (3X 100 ml), the organic phases were combined, washed once with 50ml of saturated aqueous sodium chloride solution (1X 50 ml), dried over anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (elution gradient: dichloromethane/methanol=20/1, v/v) to give compound CR01018-2 (3.31 g, yield 96.6%) as a white solid.

(1-2) Synthesis of Compound CR01018-3

Compound CR01018-2 (3.31 g,13.5 mmol) was dissolved in 15ml of 1, 4-dioxane at 25℃and a solution of 1, 4-dioxane (15 mL,4 mol/L) of hydrogen chloride was added and the reaction stirred for 2 hours. After completion of the reaction, the reaction mixture was directly evaporated to dryness to give compound CR01018-3 (2.45 g, yield 100%) as a white solid.

(1-3) Synthesis of Compound CR01018-4

Compound GAL-5 (1.83 g,4.1 mmol) was dissolved in 10ml of N, N-Dimethylformamide (DMF), N, N-diisopropylethylamine (826.5 mg,6.4 mmol), benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (2.33 g,6.2 mmol) and compound CR01018-3 (2.45 g,13.5 mmol) were added and the reaction stirred for 3 hours.After the completion of the reaction, the reaction mixture was added to 100ml of saturated aqueous sodium hydrogencarbonate, followed by extraction three times (3X 50 ml) with 50ml of ethyl acetate, and the organic phases were combined, washed once with 50ml of saturated aqueous sodium chloride (1X 50 ml), dried over anhydrous sodium sulfate and filtered, and concentrated, followed by column chromatography reversed-phase purification (C18 column, elution gradient: water/acetonitrile=2/1, v/v) to give compound CR01018-4 (2.1 g, yield 90.6%) as a yellow solid. ESI-MS (m/z) =575.3 [ M+H ] ⁺ .

(1-4) Synthesis of Compound CR01018-5

Compound CR01018-4 (2.13 g,3.9 mmol) was dissolved in 20ml of pyridine at 25℃and the reaction system was cooled to 0℃with an ice-water bath and 4,4' -dimethoxytriphenylchloromethane (1.58 g,4.7 mmol) was added in portions at 0℃and reacted under stirring at 0℃for 1 hour, and the reaction mixture was quenched with methanol. After the completion of the reaction, the reaction mixture was directly evaporated to dryness and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=1/2, v/v) to give compound CR01018-5 (3.08 g, yield 90.3%) as a yellow solid. ESI-MS (m/z) =877.6 [ M+H ]] ⁺ .

(1-5) Synthesis of Compound CR01018

Compound CR01018-5 (800 mg,0.91 mmol) was dissolved in 8ml of methylene chloride at 25℃and 4, 5-dicyanoimidazole (87.0 mg,0.73 mmol), 2-cyanoethyl N, N, N ', N' -tetraisopropylphosphoramidite (306.3 mg,1.09 mmol) was added thereto, the mixture was replaced with nitrogen three times, and the reaction was stirred at 25℃for 1 hour. After completion of the reaction, the reaction mixture was washed twice with 10ml of saturated aqueous sodium hydrogencarbonate (2X 10 ml) and once with 20ml of saturated aqueous sodium chloride (1X 20 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=1/3, v/v) to give compound CR01018 (678 mg, yield 69.0%) as a white solid. ESI-MS (m/z) =1076.3 [ M+H ] ] ⁺ .

1H NMR(400MHz,DMSO-d6)δ7.79(d,J＝9.2Hz,1H),7.41(d,J＝7.6Hz,2H),7.35–7.18(m,7H),6.88(t,J＝8.0Hz,4H),5.21(d,J＝3.3Hz,1H),4.96(dd,J＝11.2,3.3Hz,1H),4.48(d,J＝8.5Hz,1H),4.02(s,3H),3.86(dt,J＝16.7,8.9Hz,3H),3.73(d,J＝2.7Hz,7H),3.71–3.45(m,4H),3.11(s,1H),2.76(t,J＝5.8Hz,1H),2.61–2.53(m,1H),2.23(s,2H),2.08(d,J＝9.0Hz,5H),1.99(s,3H),1.89(s,4H),1.75(s,3H),1.46(s,6H),1.13(t,J＝7.1Hz,9H),1.02(d,J＝6.7Hz,4H).

Preparation example 2: synthesis of Compound CR01018Z

In this preparation, the synthetic route for compound CR01018Z is as follows:

(2-1) Synthesis of Compound CR01018-6

Compound CR01018-5 (120 mg,0.14 mmol) was dissolved in 2ml of dichloromethane at 25℃and triethylamine (34.6 mg,0.35 mmol), 4-dimethylaminopyridine (1.67 mg,0.01 mmol) and succinic anhydride (20.5 mg,0.21 mmol) were added and the reaction stirred at 25℃for 16 hours. After the completion of the reaction, the reaction mixture was directly concentrated, and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=1/1, v/v) to give compound CR01018-6 (108 mg, yield 80.8%) as a yellow oil. ESI-MS (m/z) =977.3 [ M+H ]] ⁺ .

(2-2) Synthesis of Compound CR01018Z

Compound CR01018-6 (50 mg,0.05 mmol) was dissolved in 10ml acetonitrile at 25℃and benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate (24.2 mg,0.07 mmol), N, N-diisopropylethylamine (13.2 mg,0.1 mmol) and amino CPG (1.1 g,100-200 mesh, loading 80 umol/g) were added and reacted at 25℃with stirring for 16 hours. After the reaction, the reaction mixture was directly filtered, and the cake was washed twice with 50ml of methylene chloride (2X 50 ml), three times with 50ml of acetonitrile (3X 50 ml), once with 50ml of ethyl acetate (1X 50 ml), and then dried under vacuum.

Cap1 (5 ml), cap2 (0.56 ml) and 4-dimethylaminopyridine (6.25 mg) were added to the dried cake, and the reaction was stirred at 25℃for 5 hours. After the reaction, the reaction mixture was directly filtered, and the cake was washed three times with 50ml of acetonitrile (3X 50 ml) and then dried under vacuum to give compound CR01018Z (1.03 g, load of 30 umol/g).

Cap1 and Cap2 are capping reagents, cap1 is a pyridine/acetonitrile mixed solution of 20 volume percent of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; cap2 is a 20% by volume acetic anhydride in acetonitrile.

Preparation example 3: synthesis of reference Compound CR01014

In this preparation, compound CR01014 was synthesized as follows:

(3-1) Synthesis of Compound CR01014-3

Compound CR01014-1 (5.0 g, maleic anhydride), compound CR01014-2 (12.1 g, N- (methoxymethyl) -N- (trimethylsilyl) benzylamine) and trifluoroacetic acid (0.58 g) were dissolved in 35ml of dichloromethane, nitrogen was substituted 3 times, and the reaction was stirred at 25℃for 3 hours. After completion of the reaction, the reaction mixture was washed once with 10ml of purified water (1X 10 ml) and once with 10ml of saturated aqueous sodium chloride solution (1X 10 ml), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give compound CR01014-3 (11 g), which was directly subjected to the next reaction without purification. MS-ESI (m/z) =232.0 [ M+H ] ] ⁺ .

(3-2) Synthesis of Compound CR01014-4

Dissolving compound CR01014-3 (4.1 g) in 20ml tetrahydrofuran, displacing nitrogen for 3 times, cooling the reaction system to 0deg.C with ice-water bath, and dropwise adding LiAlH at 0deg.C ₄ Tetrahydrofuran (1.0 mol/L,17.7 ml), and the reaction was stirred at 0℃for 1 hour. After the completion of the reaction, 32ml of purified water was added dropwise to the reaction mixture, followed by 24ml of a 1.0mol/L aqueous sodium hydroxide solution, and the mixture was filtered to obtain a filtrate, which was evaporated under reduced pressure to obtain CR01014-4 (6.0 g), and the next reaction was directly carried out without purification. MS-ESI (m/z) =222.3 [ m+h] ⁺ .

(3-3) Synthesis of Compound CR01014-5

Compound CR01014-4 (5.0 g) was dissolved in 50ml of methanol, and wet palladium on carbon (0.5 g, load 10 mass%) and palladium on carbon hydroxide (0.5 g, load 10 mass%) were added, respectively, and hydrogen was replaced 3 times, and the reaction system was warmed to 40℃CAnd the reaction was stirred at 40℃for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was evaporated under reduced pressure to give Compound CR01014-5 (2.9 g). MS-ESI (m/z) =132.18 [ M+H ]] ⁺ .

(3-4) Synthesis of Compound CR01014-7

To 20ml of N, N-dimethylformamide were dissolved compound CR01014-5 (4.0 g), compound CR01014-6 (2.8 g, N-benzyloxycarbonyl-4-aminobutyric acid) and N, N-diisopropylethylamine (15.6 g), benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate (17.2 g) was added, and the mixture was stirred and reacted for 3 hours at 25℃with nitrogen substitution. After the reaction, the reaction mixture was concentrated directly, and purified by column chromatography in reverse phase (C18 column, elution gradient: acetonitrile/water=72/28, v/v) to give compound CR01014-7 (1.46 g, yield 35.6%). MS-ESI (m/z) =367.41 [ M+H ] ] ⁺ .

(3-5) Synthesis of Compound CR01014-8

Compound CR01014-7 (1.46 g) was dissolved in 10ml of methanol, and wet palladium on carbon (0.15 g,10 mass%) was added thereto, hydrogen was replaced 3 times, and the reaction was stirred at 25℃for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was evaporated under reduced pressure to give Compound CR01014-8 (1.06 g), which was then subjected to the next reaction without purification. MS-ESI (m/z) =233.28 [ M+H ]] ⁺ .

(3-6) Synthesis of Compound CR01014-9

Compound CR01014-8 (1.02 g) and compound GAL-5 (1.41 g) were dissolved in 14ml of N, N-dimethylformamide, and N, N-diisopropylethylamine (0.81 g) and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.8 g) were added thereto, followed by 3 times of nitrogen substitution, and the reaction was stirred at 25℃for 3 hours. After the reaction, the reaction mixture was concentrated directly, and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=2/1, v/v) to give compound CR01014-9 (1.28 g, yield 63.5%). MS-ESI (m/z) =648.68 [ M+H ]] ⁺ .

(3-7) Synthesis of Compound CR01014-10

Compound CR01014-9 (1.18 g) was dissolved in 12ml of pyridine, 4' -dimethoxytrityl chloride (0.98 g) was added in portions, nitrogen was substituted 3 times, and the reaction was stirred at 25℃for 3 hours. After the reaction is finished, the reaction liquid is directly concentrated, Column chromatography reversed phase purification (C18 column, elution gradient: water/acetonitrile=1/2, v/v) afforded compound CR01014-10 (1.0 g, yield 61.1%). MS-ESI (m/z) =951.0 [ M+H ]] ⁺ .

(3-8) Synthesis of Compound CR01014

Compound CR01014-10 (300 mg) was dissolved in 6ml of methylene chloride, and bis (diisopropylamino) (2-cyanoethoxy) phosphine (152 mg) and 4, 5-dicyanoimidazole (30 mg) were added in portions, followed by 3 times of nitrogen substitution and stirred at 25℃for 3 hours. After the reaction, the reaction mixture was concentrated directly, and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=1/3, v/v) to give compound CR01014 (270 mg, yield 74.4%). MS-ESI (m/z) =1151.27 [ M+H ]] ⁺ .

1H NMR(400MHz,DMSO-d6)δ:0.92–1.03(d,J＝6.7Hz,8H),1.04–1.13(d,J＝6.8Hz,7H),1.16–1.29(m,4H),1.38–1.53(dq,J＝7.2,14.6Hz,4H),1.55–1.68(dt,J＝9.0,15.1Hz,2H),1.75–1.82(s,3H),1.86–1.94(s,3H),1.97–2.07(s,6H),2.09–2.15(s,3H),2.15–2.29(m,2H),2.64–2.76(d,J＝5.5Hz,3H),2.99–3.16(dt,J＝6.7,14.4Hz,4H),3.20–3.30(d,J＝14.8Hz,1H),3.37–3.53(tt,J＝7.1,14.6Hz,6H),3.53–3.66(dt,J＝8.4,17.5Hz,3H),3.74–3.78(s,6H),4.00–4.09(s,3H),6.84–6.96(d,J＝8.3Hz,4H),7.19–7.28(t,J＝7.6Hz,5H),7.28–7.41(dt,J＝7.8,22.9Hz,4H),7.70–7.88(d,J＝5.9Hz,2H).

Preparation example 4: synthesis of reference Compound CR01014Z

In this preparation, the synthetic route for compound CR01014Z is as follows:

(4-1) Synthesis of Compound CR01014-11

Compound CR01014-10 (100 mg) was dissolved in 2ml of methylene chloride, succinic anhydride (15.7 mg), 4-dimethylaminopyridine (1.2 mg) and triethylamine (19.7 mg) were added thereto, the mixture was replaced with nitrogen 3 times, and the reaction was stirred at 25℃for 16 hours. After the reaction, the reaction mixture was concentrated directly, and purified by column chromatography in reverse phase (C18 column, elution gradient: water/acetonitrile=11, v/v) to give compound CR01014-11 (73 mg, yield 66.7%). MS-ESI (M/z) = [ M+H ] ] ⁺ .

(4-2) Synthesis of Compound CR01014Z

Compound CR01014-11 (50 mg), amino CPG (1.19 g), benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate (27 mg) and N, N-diisopropylethylamine (12 mg) were mixed and reacted for 16 hours on a shaker. After the reaction was completed, the reaction mixture was filtered, and the cake was washed once with 10ml of acetonitrile (1X 10 ml) and then dried under vacuum.

The dried cake, 4-dimethylaminopyridine (3 mg), cap1 (10 ml) and Cap2 (10 ml) were mixed and reacted for 6 hours on a shaker. After the reaction, the reaction mixture was filtered, and the cake was washed once with 10ml of acetonitrile (1X 10 ml) and then dried under vacuum to give compound CR01014Z (1.03 g, loading 20-30 umol/g).

Cap1 and Cap2 are capping reagents, cap1 is a pyridine/acetonitrile mixed solution of 20 volume percent of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; cap2 is a 20% by volume acetic anhydride in acetonitrile.

Reference compound L96-PS

Compound L96-PS was purchased from Kailin pharmaceutical group (Tianjin) Co., ltd, and the loading was 120.+ -.12. Mu. Mol/g (detection method: UV/HPLC).

The structural formula of the compound L96-PS is as follows:

wherein PS stands for Polystyrene (Polystyrene) resin solid support.

Preparation example 5: preparation of siRNA conjugates

(5-1) Synthesis of Sense Strand (SS)

By the method of phosphoramidite nucleic acid solid phase synthesis, the above-mentioned compound (i.e., CR01018Z, CR01014Z, L-PS) attached to a solid phase carrier is used to initiate a cycle, and nucleoside monomers are attached one by one in the 3'-5' direction according to the nucleotide sequence (the compounds CR01018 and CR01014 can be regarded as one nucleoside monomer, respectively). Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The synthesis conditions were given as follows:

nucleoside monomers were formulated as an acetonitrile solution of nucleoside monomers at a concentration of 0.1M.

The deprotection conditions are the same for each step. Conditions for deprotection reaction: the temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is dichloromethane solution (3 vol%) of dichloroacetic acid, and the molar ratio of the dichloroacetic acid to the 4,4' -dimethoxytrityl protecting group on the solid carrier is 5:1.

The conditions for each coupling reaction were identical. The conditions of the coupling reaction are: the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with the concentration of 0.5M, and the thioagent is an acetonitrile/pyridine mixed solution of hydrogenation Huang Yuansu with the concentration of 0.2mol/L (the volume ratio of acetonitrile to pyridine is 1:1).

The conditions for the capping reaction were the same for each step. The conditions of the capping reaction are: the temperature is 25 ℃; the reaction time was 2 minutes; the Cap reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, wherein Cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume percent, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is an acetonitrile solution of acetic anhydride with a annual attack rate of 20 volume percent; the molar ratio of the N-methylimidazole in the Cap1 capping reagent to the acetic anhydride in the Cap2 capping reagent to the nucleic acid sequence connected to the solid carrier is 1:1:1.

The conditions for each oxidation reaction are the same. The oxidation reaction conditions were: the temperature is 25 ℃; the reaction time was 3 seconds; iodine water with the concentration of 0.05M of the oxidizing reagent, wherein the molar ratio of iodine to the nucleic acid sequence connected to the solid carrier in the coupling reaction is 30:1; the oxidation reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9). The conditions of the vulcanization reaction are as follows: the temperature is 25 ℃; the reaction time was 360 seconds; a pyridine solution with a concentration of 0.2M hydrogenation Huang Yuansu of the thio reagent, wherein the molar ratio of the thio reagent to the nucleic acid sequence connected to the solid carrier in the coupling reaction is 4:1; the thio-reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9).

After the last nucleoside monomer is connected, the nucleic acid sequence connected on the solid phase carrier is sequentially cut, deprotected, purified and desalted, and then freeze-dried to obtain the sense strand, wherein:

the cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence to which the solid phase carrier was attached was added to aqueous ammonia having a concentration of 25% by mass, the amount of aqueous ammonia was 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the solvent was removed, and concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved with 0.4 ml/. Mu.mol of N-methylpyrrolidone, followed by the addition of 0.3 ml/. Mu.mol of triethylamine and 0.6 ml/. Mu.mol of triethylamine-tricofluoride, relative to the amount of single-stranded nucleic acid, and the 2' -O-TBDMS protection on ribose was removed.

Conditions for purification and desalination: purification of nucleic acids was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent 1 is 20mM sodium phosphate (pH=8.1), solvent is water/acetonitrile mixed solution (volume ratio of water and acetonitrile is 9:1); eluent 2 is 1.5M sodium chloride, 20mM sodium phosphate (pH=8.1), solvent is water/acetonitrile mixed solution (volume ratio of water and acetonitrile is 9:1); the elution gradient is eluent 1, eluent 2= (100:0) - (50:50). Collecting and combining product eluents, desalting by using a reverse chromatography purification column, wherein the desalting conditions comprise desalting by using a sephadex column, eluting with deionized water, wherein the filler is sephadex G25.

And (3) detection: purity detection using ion exchange chromatography (IEX-HPLC); and (3) detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the measured value and the theoretical value of the molecular weight, wherein if the measured value and the theoretical value are consistent, the obtained compound is conjugated at the 3' end of the siRNA sense strand.

During the synthesis of the sense strand, the following targeting ligands (vectors) were synthesized, respectively:

the structural formula of the two-cluster CR01018 (denoted as (CR 01018). Times.2) is:

the structural formula of the three-cluster CR01018 (denoted as (CR 01018). Times.3) is:

the structural formula of the two clusters CR01014 (denoted as (CR 01014) ×2) is:

the structural formula of three clusters CR01014 (noted as (CR 01014) ×3) is:

(5-2) Synthesis of Antisense Strand (AS)

Antisense strands were synthesized using a universal solid support. The conditions of deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting in the solid phase synthesis method of antisense strand are the same as those of step (5-1) for synthesizing sense strand.

And (3) detection: purity detection using ion exchange chromatography (IEX-HPLC); molecular weight detection is performed by using liquid chromatography-mass spectrometry (LC-MS), and the measured value and the theoretical value of the molecular weight are compared, and if the measured value and the theoretical value are consistent, the siRNA antisense strand is obtained.

(5-3) Synthesis of siRNA conjugates

The sense strand synthesized in step (5-1) and the antisense strand synthesized in step (5-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃, slowly cooled to room temperature and maintained at room temperature for 10 minutes, so that the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonds, thereby obtaining siRNA conjugates having the sense strand and the antisense strand shown in table 4.

Sense strand: umsUmsUmAmUfCfCfUmCmAmCmUmCmUmAmAm, as shown in SEQ ID NO:1 is shown in the specification;

antisense strand: umsUfsUmGlumGlumGlumGlumGlumGlumFUmAfAmAmAmAmAmsUmsGm, as shown in SEQ ID NO: 2.

When the carrier is three clusters CR01018, the structural formula of the siRNA conjugate is as follows:

when the carrier is three clusters CR01014, the structural formula of the siRNA conjugate is as follows:

when the carrier is L96, the structural formula of the siRNA conjugate is as follows:

wherein, the liquid crystal display device comprises a liquid crystal display device,representing the siRNA, the targeting ligand (carrier) is conjugated to the 3' end of the sense strand of the siRNA.

TABLE 4 sequence information for siRNA conjugates

Unless otherwise indicated, the base compositions and modifications described in the examples of the present disclosure are as follows: capital A, U, G, C, T indicates the base composition of the nucleotide, and lowercase m indicates that the nucleotide indicated by the preceding letter is a methoxy-modified nucleotide; the lower case letter f indicates that the nucleotide indicated by the preceding letter is a fluoro-modified nucleotide; lower case letter s indicates that phosphorothioate linkages are between the nucleotides indicated by the two letters before and after.

Unless otherwise indicated, the siRNA sequences used in the present disclosure were all assigned to the su Bei Xin biotechnology limited synthesis; the PCR primer synthesis used in the application is completed by Beijing qingke biological science and technology Co., ltd; experimental animals C57BL/6J mice used in the present disclosure were purchased from St Bei Fu (Beijing) biotechnology Co., ltd.

TABLE 5 detection results of siRNA conjugates

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As can be seen from the data in Table 5, the Sense Strand (SS) and the Antisense Strand (AS) are able to be attached to the ligand better and remain in higher purity.

General experiment

Experimental example 1 in vivo toxicity experiment of siRNA conjugate

C57BL/6J mice were randomly divided into 3 groups (i.e., RZ599001 experimental group, RZ899043 experimental group, RZ899027 experimental group), 2 mice per group, each half male and female, and 300mg/kg of siRNA conjugate of the mouse body weight (calculated as siRNA) was administered to each mice of each experimental group individually by subcutaneous injection, and continuous observation was performed for 14 days, without death of animals, without observation of clinical symptoms related to adverse drug reactions, and after the end of observation, the mice were subjected to general dissection, and no abnormality was found. Thus, the above results indicate that the siRNA conjugates of the present disclosure are safe and have low animal level toxicity.

Example 2 evaluation method of in vivo target Gene inhibitory Activity in mice

The 6-8 week old C57BL/6J mice were randomly grouped by body weight (females). The mice in each group were dosed in a single dose by abdominal subcutaneous injection based on body weight, and each siRNA conjugate was formulated with PBS solution as a corresponding concentration (calculated as siRNA) solution for dosing with a volume of 5ml/kg of body weight (calculated as siRNA) of the mice. The PBS control group was given 5ml/kg mouse body weight of PBS solution (without drug conjugate). Administration when astronomical was day 1 (noted D1), 5 mice were sacrificed for each group at a preset time after administration, e.g., day 8 (noted D8), day 15 (noted D15), and day 29 (noted D29). The sacrificed mice were each subjected to gross dissection and liver tissue of each sacrificed mouse was collected, and the liver tissue was cut into small pieces of about 2mm3 and preserved with RNA Later.

Taking liver tissue samples at different time points from the RNA later, crushing the liver tissue samples in a tissue lyser II type full-automatic tissue homogenizer for 60s, and extracting the total RNA by using a full-automatic nucleic acid extractor (purchased from Zhejiang Han Wei technology Co., ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Han Wei technology Co., ltd.) according to standard operation steps of total RNA extraction.

Using the above 1. Mu.g of total RNA, a reverse transcription kit (Promega Corp., reverse Transcription System, A3500) was used and Oligo (dT) 15 reverse transcription primer was selected, and a 20. Mu.L reverse transcription system was prepared according to the method described in the specification of the reverse transcription kit to complete the reverse transcription reaction. After completion of the reaction, 80. Mu.L of RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then, a real-time fluorescent quantitative PCR kit (ABI, SYBR was used ^TM Select Master Mix, catalyst number:4472908 Detecting the expression level of mRNA of the target gene in liver tissue. In the real-time fluorescent quantitative PCR method, a primer for a target gene and a primer for an internal reference gene are used to detect the target gene and the internal reference gene, respectively. 20. Mu.L Real-time PCR reaction systems were prepared for each PCR detection well according to the method described in the Real-time fluorescent quantitative PCR kit, each reaction system containing 5. Mu.L of the cDNA solution obtained by the above-mentioned reverse transcription reaction and 10. Mu.L of SYBR ^TM Select Master Mix, 0.5. Mu.L of 10. Mu.M upstream primer, 0.5. Mu.L of 10. Mu.M downstream primer, 4. Mu.L RNase-Free H2O. Placing the prepared reaction system in a real-time fluorescence quantitative PCR instrument (ABI company, stepOneGlus) ^TM ) The three-step method was used to perform Real-time PCR amplification by pre-denaturing at 95℃for 10min, then denaturing at 95℃for 30s, annealing at 60℃for 30s, and extending at 72℃for 30s, and repeating the denaturation, annealing, and extension steps for 40 cycles. In this real-time fluorescent quantification In the PCR method, the expression level and the inhibition rate of the target gene mRNA in each test group are calculated by adopting a delta Ct method, and the calculation method is as follows:

delta Ct (test group) =ct (test group target gene) -Ct (test group reference gene)

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Wherein, Δct (control group average) is the arithmetic average of Δct (control group) of each of 5 mice sacrificed at the same time point in the control group. Thus, each mouse of the test and control groups corresponds to one ΔΔct value.

And normalizing the mRNA expression level of the target gene of the test group by taking the control group as a reference, and defining the mRNA expression level of the target gene of the control group as 100%.

Relative expression level of target gene mRNA in test group = 2- ΔΔCt (test group) ×100%

Test group target gene mRNA expression inhibition ratio= (1-test group target gene mRNA relative expression level) ×100%

Unless otherwise indicated, in vivo activity assay data are all expressed as X+ -SD, and the assay data are plotted and analyzed using GraphPad prism 8.0 software.

Example 2.1 evaluation of in vivo Activity of three Cluster CR01018 vector conjugated superoxide dismutase 1 (SOD 1) target Gene

The experimental example adopts a method for evaluating the inhibition activity of a target gene in a mouse to evaluate the inhibition activity of an siRNA sequence RZ599001 of an L96 carrier conjugated with the 3 '-end of an siRNA sense strand and an siRNA sequence RZ899043 of a three-cluster CR01018 carrier conjugated with the 3' -end of the siRNA sense strand on the target gene SOD1 in the mouse. RZ899043 and RZ599001 have identical nucleic acid sequences and chemical modifications, differing only in the delivery vector.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 15 mice per group, and 3 total groups. Each group of mice was given PBS solution and the above siRNA conjugate by subcutaneous administration on the abdomen, wherein each mouse of the PBS control group was given a dose of 5mL/kg, and each mouse of the siRNA conjugate experimental group was given a dose of 3mg/kg (in terms of siRNA) in a dose volume of 5mL/kg. Dosing 5 mice per group were sacrificed when the day one (D1) was noted, on day 8 (D8), day 29 (D29) and day 54 (D54) after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

TABLE 6 primer sequence listing of this example

As shown in fig. 1 and table 4, the in vivo inhibition effect and the sustained effect of the siRNA conjugate RZ899043 conjugated to the three-cluster CR01018 on the target gene were equivalent to those of the siRNA conjugate RZ599001 conjugated to L96 and slightly better than those of the siRNA conjugate RZ599001 conjugated to L96.

TABLE 7 inhibitory Activity of target genes of interest in mice following administration of siRNA conjugates described in this example

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Example 2.2 evaluation of in vivo Activity of three Cluster CR01014 vector conjugated superoxide dismutase 1 (SOD 1) target Gene

The experimental example adopts a method for evaluating the inhibition activity of a target gene in a mouse to evaluate the inhibition activity of an siRNA sequence RZ599001 of an L96 carrier conjugated with the 3 '-end of an siRNA sense strand and an siRNA sequence RZ899027 of a three-cluster CR01014 carrier conjugated with the 3' -end of the siRNA sense strand on the target gene SOD1 in the mouse. RZ899043 and RZ899027 have identical nucleic acid sequences and chemical modifications, differing only in the delivery vector.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 20 mice per group, and 3 total groups. Each group of mice was given PBS solution and the above siRNA conjugate by subcutaneous administration on the abdomen, wherein each mouse of the PBS control group was given a dose of 5mL/kg of mouse body weight, and each mouse of the siRNA conjugate experimental group was given a dose of 3mg/kg (in terms of siRNA) in a dose volume of 5mL/kg of mouse body weight. Dosing when the day one (D1) was recorded, 5 mice per group were sacrificed on day 15 (D15), day 29 (D29), day 43 (D43) and day 57 (D57) after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

The primer sequences of this example are shown in Table 6 of example 2.1.

As shown in fig. 2 and table 5, the in vivo inhibition effect of the siRNA conjugate RZ899027 conjugated to the three-cluster CR01014 on the target gene was comparable to that of the siRNA conjugate RZ599001 conjugated to L96 at D15, and significantly weaker than that of the siRNA conjugate RZ599001 conjugated to L96 at D29, D43 and D57.

Table 8 inhibitory Activity of target genes of interest in mice following administration of siRNA conjugates described in this example

In combination with example 2.1 and example 2.2, the in vivo inhibition effect and the sustained effect of the siRNA conjugate RZ899043 conjugated with the three-cluster CR01018 on the target gene are obviously better than those of the siRNA conjugate RZ899027 conjugated with the three-cluster CR 01014.

It is to be understood that the above embodiments are merely exemplary embodiments employed to illustrate the principles of the present disclosure, however, the present disclosure is not limited thereto. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the disclosure, and are also considered to be within the scope of the disclosure.

Claims (14)

1. A compound having the structure of formula (Ia) or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof:

Wherein R is ₁ Selected from H, hydroxy protecting groups or* Represents a ligation site for ligation of pharmaceutically active molecules;

each Z is independently selected from hydroxyl or mercapto;

R ₂ selected from H, reactive phosphorus groups orR _2b Selected from solid supports containing amino functions, R _2a A covalent linking group selected from the group consisting of a covalent linkage to the amino functional group;

n is selected from 0, 1, 2 or 3;

each j is independently selected from 1, 2 or 3;

each L is independently selected from substituted or unsubstituted C ₂ -C ₁₀ Alkylene orWherein each R _L Each independently selected from substituted or unsubstituted C ₁ -C ₅ Alkylene, each X is independently selected from O, S, NH or-NH-C (O) -, m is selected from 1, 2, 3, 4 or 5;

each R ₃ Each independently selected from H, substituted or unsubstituted C ₁ -C ₄ Alkylacyl or substituted or unsubstituted C ₅ -C ₇ Aryl acyl, R ₃ Each substituent group of (a) is independently selected from halogen, C ₁ -C ₂ An alkoxy group.

2. The compound of claim 1, wherein each L is independently selected from substituted or unsubstituted C ₂ -C ₁₀ Straight chain alkylene；

Optionally, each L is independently selected from substituted or unsubstituted C ₂ -C ₆ A linear alkylene group;

optionally, each L is independently selected from substituted or unsubstituted C ₃ -C ₅ A linear alkylene group;

Optionally, each L is independently selected from substituted or unsubstituted C ₄ A linear alkylene group;

optionally, each L is independently selected from

Optionally, each L is selected from

Optionally, n is selected from 0, 1 or 2;

optionally, n is selected from 0;

optionally, n is selected from 1;

optionally, n is selected from 2;

optionally, each j is selected from 1;

optionally, each Z is selected from hydroxy;

optionally, each R ₃ Each independently selected from H,

Optionally, each R ₃ Each independently selected from H or

Optionally, each R ₃ Are all selected from

Optionally, each R ₃ Are all selected from H;

optionally, the hydroxyl protecting group is selected from trityl, 4-methoxytrityl, 4 '-dimethoxytrityl or 4,4',4 "-trimethoxytrityl;

optionally, the hydroxyl protecting group is selected from 4,4' -dimethoxytrityl;

optionally, the reactive phosphorus group is selected from

Optionally, the reactive phosphorus group is selected from

Optionally, the pharmaceutically active molecule is selected from a small molecule drug, an antibody or an oligonucleotide;

optionally, the oligonucleotide is selected from the group consisting of a single stranded oligonucleotide and a double stranded oligonucleotide;

optionally, the pharmaceutically active molecule is selected from double stranded oligonucleotides;

optionally, the pharmaceutically active molecule is selected from siRNA;

Optionally, the solid support is selected from an amino-functional group-containing resin or an amino-functional group-containing controllable pore size glass sphere;

optionally R _2a Selected from the group consisting of

Optionally R _2b Selected from the group consisting of Selected from resin or controllable pore size glass spheres;

optionally, the composition may be used in combination with,selected from->

3. The compound according to any one of claims 1-2, wherein the compound has a structural formula represented by formula (IIa):

wherein each R ₄ Each independently selected from H, C ₁ -C ₂ Alkyl or C ₁ -C ₂ An alkoxy group;

each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

optionally, each R ₄ Are all H;

optionally, each k is 4.

4. The compound according to any one of claims 1-2, wherein the compound has a structural formula shown in formula (III):

wherein each R ₄ Each independently selected from H, C ₁ -C ₂ Alkyl or C ₁ -C ₂ An alkoxy group;

each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

optionally, each R ₄ Are all H;

optionally, each k is 4.

5. The compound according to any one of claims 1-2, wherein the compound has a structural formula shown in formula (IV):

wherein each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

Optionally, each k is 4.

6. A compound according to claim 1, wherein the compound is selected from any one of the following structures:

7. a conjugate having a structure represented by formula (Ib) or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof:

wherein Nu represents an oligonucleotide;

n' is selected from 1, 2, 3 or 4;

j. z, L is as defined in any one of claims 1 to 6;

optionally, the oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide;

optionally, nu is selected from siRNA;

optionally, n' is selected from 3.

8. The conjugate of claim 7, wherein the conjugate has a structural formula of formula (IIb):

wherein each R ₄ Each independently selected from H, C ₁ -C ₂ Alkyl or C ₁ -C ₂ An alkoxy group;

each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

optionally, each R ₄ Are all H;

optionally, each k is 4.

9. The conjugate of claim 7, wherein the conjugate has a structural formula of formula (IIIb):

wherein each R ₄ Each independently selected from H, C ₁ -C ₂ Alkyl or C ₁ -C ₂ An alkoxy group;

each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

Optionally, each R ₄ Are all H;

optionally, each k is 4.

10. The conjugate of claim 7, wherein the conjugate has a structural formula of formula (IVb):

wherein each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

optionally, each k is 4.

11. The conjugate of claim 7, wherein the conjugate is selected from the group consisting of

12. A composition comprising the conjugate of any one of claims 7-11;

optionally, the composition further comprises at least one pharmaceutically acceptable carrier.

13. Use of any of the following for the manufacture of a medicament for the prevention and/or treatment of a disease:

(I) A compound according to any one of claims 1 to 6; and/or

(II) the conjugate of any one of claims 7-11; and/or

(III) the composition of claim 12;

optionally, the disease is selected from a pathological condition or disease caused by abnormal expression of a target gene in liver cells.

14. A method of reducing expression or activity of a target gene, the method comprising contacting a liver cell with any of:

(I) The conjugate of any one of claims 7-11; and/or

(II) the composition of claim 12.