CN116474107A - Conjugate, intermediate compound thereof and application - Google Patents

Conjugate, intermediate compound thereof and application Download PDF

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Publication number
CN116474107A
CN116474107A CN202211628857.9A CN202211628857A CN116474107A CN 116474107 A CN116474107 A CN 116474107A CN 202211628857 A CN202211628857 A CN 202211628857A CN 116474107 A CN116474107 A CN 116474107A
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alkylene
alkyl
group
conjugate
alpha
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李进
万金桥
张帅
杨琪
裴帅
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Hitgen Inc
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Hitgen Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides a drug conjugate, an intermediate compound thereof and application thereof in preparing drugs. The conjugate disclosed by the invention can specifically target the surface of a cell, so that the problem of in vivo delivery of a drug is effectively solved.

Description

Conjugate, intermediate compound thereof and application
Technical Field
The present invention relates to a drug conjugate and intermediate compounds thereof and use thereof in the preparation of a medicament.
Background
Drug delivery systems are one of the key technologies in modern drug development, especially for the field of nucleic acid pharmaceuticals. Means to efficiently deliver oligonucleotides, particularly double stranded siRNAs, to cells in vivo are becoming increasingly important and require specific targeting and substantial protection against extracellular environments, particularly serum proteins. One way to achieve specific targeting is to conjugate the targeting moiety to an RNAi duplex reagent. The targeting moiety aids in targeting the RNAi duplex to the desired target site and requires the design of an appropriate targeting moiety for the desired receptor site of the conjugated molecule that is taken up by the cell, e.g., by endocytosis.
There remains a need for more efficient receptor-specific ligand conjugates and methods of making the same for in vivo delivery of drugs such as oligonucleotides.
Disclosure of Invention
The present invention first provides a conjugate characterized in that: has a structure shown in formula I:
wherein, the liquid crystal display device comprises a liquid crystal display device,
nu is the active drug moiety;
m is selected from-O-, -NH-, -P (O) (OH) O-, -P (S) (OH) O-, or none;
L 1 selected from the group consisting of
L 11 Selected from-C 1～20 Alkylene-, -O-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
L 12 selected from-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
R a selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
R b selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
L 2 selected from-C (O) -C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-O-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 alkylene-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -, -C (O) -O-C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -orEach L 3 Independently selected from-C 1～10 Alkylene-, -O-, or none;
each n is independently selected from integers from 0 to 10;
each m is independently selected from integers from 0 to 5;
t is selected from
Each L 3 Each independently selected from a linking chain of 1 to 50 carbon atoms in length, wherein one or more methylene groups may be optionally replaced by one or more of the following groups: -C (O) -, -NH-, O, S, -S (O) -, -S (O) 2-, -P (O) (OH) -, -P (S) (OH) -, -ch=ch-, -c≡c-, -ch=n-, carbocyclyl, heterocyclyl; may each be further substituted with one or more of the following groups: c (C) 1～10 Alkyl, halogen substituted C 1～10 Alkyl, - (C) 0～10 Alkylene) -OH, - (C 0～10 Alkylene) -O- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -SH, - (C 0～10 Alkylene) -S- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -NH2, - (C 0～10 Alkylene) -NH (C) 1～10 Alkyl) - (C) 0～10 Alkylene) -N (C) 1～10 Alkyl) (C) 1～10 An alkyl group);
each G is independently selected from a ligand moiety capable of binding to a cell surface receptor.
In some embodiments of the invention, further,
L 1 selected from the group consisting of
In some embodiments of the invention, further,
L 2 selected from the group consisting of
In some embodiments of the invention, further,
L 3 selected from the group consisting of
In some embodiments of the invention, further, each G is independently selected from a sugar.
Still further, each G is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose, L-xylose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetyl galactosamine, N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-isobutyryl galactosamine, 2-amino-3-O) -1- [ (ethyl ] -2-carboxymethyl ] -2-D-glucopyranose, 4-dimethyl-6-D-deoxygalactopyranose, 4-D-methyl-2-D-deoxygalactopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-psicosonitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose.
Further, each G is
In some embodiments of the invention, the conjugate is selected from the following structures:
further, the Nu is a functional oligonucleotide.
Still further, the functional oligonucleotide is selected from one or more of a small interfering RNA, a microrna, an anti-microrna, a microrna antagonist, a microrna mimetic, a decoy oligonucleotide, an immunostimulatory substance, a G-quadrupole, an alternative splice, a single stranded RNA, an antisense nucleic acid, a nucleic acid aptamer, a stem loop RNA, an mRNA fragment, an activating RNA, or DNA.
Still further, the functional oligonucleotide is a single stranded oligonucleotide; wherein the remainder of the conjugate is attached to the 5 'end of the single stranded oligonucleotide or the remainder of the conjugate is attached to the 3' end of the single stranded oligonucleotide.
Still further, the functional oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand; wherein the remainder of the conjugate is attached to the 5 'end of the sense strand, or the remainder of the conjugate is attached to the 3' end of the sense strand, or the remainder of the conjugate is attached to the 5 'end of the antisense strand, or the remainder of the conjugate is attached to the 3' end of the antisense strand.
Further specifically, the Nu is siRNA.
Still more particularly, the siRNA is an siRNA comprising at least one modified nucleotide.
In some embodiments, the siRNA comprises a sense strand and an antisense strand; wherein the nucleotide sequence of the antisense strand is at least partially complementary to a target mRNA corresponding to a gene that is abnormally expressed in hepatocytes.
In some embodiments, the nucleotide sequence of the antisense strand is fully complementary to the target mRNA.
In some embodiments, the sense strand and the antisense strand are fully complementary.
In some embodiments, all nucleotides of the sense strand are modified nucleotides; in some embodiments, all nucleotides of the antisense strand are modified nucleotides.
In some embodiments, all nucleotides of the sense strand are 2 '-methoxy modified nucleotides or 2' -fluoro modified nucleotides; in some embodiments, all nucleotides of the antisense strand are 2 '-methoxy modified nucleotides or 2' -fluoro modified nucleotides.
In some embodiments, the 7, 9, 10, 11 nucleotides at the 5 'end of the sense strand are 2' -fluoro modified nucleotides; in some embodiments, the 2, 6, 14, 16 nucleotides at the 5 'end of the antisense strand are 2' -fluoro modified nucleotides. Preferably, the 7 th, 9 th, 10 th and 11 th nucleotides at the 5' end of the sense strand are 2' -fluoro modified nucleotides, and the rest are 2' -methoxy modified nucleotides; preferably, the 2, 6, 14, 16 nucleotides at the 5' end of the antisense strand are 2' -fluoro modified nucleotides, the remainder being 2' -methoxy modified nucleotides.
In some embodiments, the sense strand and the antisense strand comprise at least one phosphorothioate linkage between any two nucleotides; preferably, the 5' end of the sense strand contains two consecutive phosphorothioate linkages; preferably, the 5' end of the antisense strand contains two consecutive phosphorothioate linkages; preferably, the 3' -end of the antisense strand contains two consecutive phosphorothioate linkages.
In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length; in some embodiments, the antisense strand is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length. Preferably, the sense strand is 19 nucleotides in length and the antisense strand is 19 nucleotides in length. Preferably, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. Preferably, the sense strand is 25 nucleotides in length and the antisense strand is 27 nucleotides in length.
The invention also provides a compound which is characterized in that: has a structure shown in formula II:
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 is hydrogen, a protecting group or a solid support moiety;
m is selected from-O-, -NH-, -P (O) (OH) O-, -P (S) (OH) O-, or none;
L 1 selected from the group consisting of
L 11 Selected from-C 1～20 Alkylene-, -O-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
L 12 selected from-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
R 2 selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl group,A protecting group or a solid support moiety;
R b selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
L 2 selected from-C (O) -C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-O-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 alkylene-C (O) -, -C (O) -C 0～10 Alkylene-
(OCH 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -, -C (O) -O-C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -orEach L 3 Independently selected from-C 1～10 Alkylene-, -O-, or none;
each n is independently selected from integers from 0 to 10;
each m is independently selected from integers from 0 to 5;
t is selected from
Each L 3 Each independently selected from a linking chain of 1 to 50 carbon atoms in length, wherein one or more methylene groups may be optionally replaced by one or more of the following groups: -C (O) -, -NH-, O, S, -S (O) -, -S (O) 2-, -P (O) (OH) -, -P (S) (OH) -, -ch=ch-, -c≡c-, -ch=n-, carbocyclyl, heterocyclyl; may each be further substituted with one or more of the following groups: c (C) 1～10 Alkyl, halogen substituted C 1～10 Alkyl, - (C) 0～10 Alkylene) -OH, - (C 0～10 Alkylene) -O- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -SH, - (C 0～10 Alkylene) -S- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -NH2, - (C 0～10 Alkylene) -NH (C) 1～10 Alkyl) - (C) 0～10 Alkylene) -N (C) 1～10 Alkyl) (C) 1～10 An alkyl group);
each G is independently selected from a ligand moiety capable of binding to a cell surface receptor.
In some embodiments of the invention, further,
L 1 selected from the group consisting of
Further, R 1 As part of the solid support, R 2 Is DMTr.
In some embodiments of the invention, further,
L 2 selected from the group consisting of
In some embodiments of the invention, further,
L 3 selected from the group consisting of
In some embodiments of the invention, further, each G is independently selected from a sugar.
Still further, each G is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose, L-xylose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetyl galactosamine, N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-isobutyryl galactosamine, 2-amino-3-O) -1- [ (ethyl ] -2-carboxymethyl ] -2-D-glucopyranose, 4-dimethyl-6-D-deoxygalactopyranose, 4-D-methyl-2-D-deoxygalactopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-psicosonitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose.
Further, each G is
In some embodiments of the invention, the compound is selected from the following structures:
wherein R is 1 Is a solid support portion comprising CPG.
The invention also provides the application of any conjugate in preparing medicines for treating and/or preventing diseases caused by abnormal expression of specific genes in liver cells.
The invention also provides a method of inhibiting expression of a particular gene in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of any of the conjugates described above.
The invention also provides a preparation formed by the conjugate and pharmaceutically acceptable carriers, diluents and auxiliary materials thereof.
The conjugate disclosed by the invention can specifically target the surface of a cell, so that the problem of in vivo delivery of a drug is effectively solved. In particular to target the surface of hepatocytes. The conjugates disclosed herein have low toxicity and are highly stable in vivo.
Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
In the present invention, the group B9 represents the following structure:
in the structural formula of the invention, "Ac" represents acetyl.
In the present invention, "DMTr" means 4,4' -dimethoxytrityl.
In the present invention, "conjugation" refers to the attachment of two or more chemical moieties to each other by covalent attachment, unless otherwise specified; "conjugate" refers to a compound formed by covalent attachment between chemical moieties; an "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties to an siRNA.
In the present invention, unless otherwise specified, "partially complementary" means that the number of bases of complementary pairing between two nucleotide sequences involved exceeds 70%.
In the present invention, unless otherwise specified, capital C, G, U, A indicates the base composition of ribonucleotides; dC. dG, dT, dA represent the base composition of deoxyribonucleotides; the lower case letter m indicates that the adjacent nucleotide to the right of the letter m is a 2' -methoxy modified nucleotide; the identifier i2F represents that one nucleotide adjacent to the right side of the identifier i2F is a 2' -fluorine modified nucleotide; the identifier indicates that phosphorothioate linkages are between two nucleotides adjacent to the identifier.
In the present invention, unless otherwise specified, "complementary" or "reverse complementary" means that in a nucleic acid duplex molecule, bases of one strand are each paired with bases on the other strand in a complementary manner. That is, A/dA paired with U/dT and C/dC paired with G/dG, the two strands were considered complementary. Correspondingly, unless otherwise indicated, "mismatch" in the present invention means that the bases at corresponding positions in the nucleic acid duplex molecule do not exist in complementary form as a pair.
The compounds and derivatives provided in the present invention may be named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.
"substituted" means that a hydrogen atom in the molecule is replaced with a different atom or group; or the lone pair of electrons of an atom in the molecule is replaced by another atom or group.
The minimum and maximum values of the carbon atom content of the hydrocarbon groups are indicated by a prefix, e.g. prefix C a～b Alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1～6 Alkyl refers to alkyl groups containing 1 to 6 carbon atoms.
"alkyl" refers to a saturated hydrocarbon chain having the indicated number of member atoms. The alkyl group may be linear or branched. Representative branched alkyl groups have one, two or more branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. The alkyl group may also be part of other groups such as-O (C 1～6 Alkyl).
"alkylene" refers to a divalent saturated aliphatic hydrocarbon group having the indicated number of member atoms. C (C) a ～ b Alkylene refers to an alkylene group having a to b carbon atoms. Alkylene groups include branched and straight chain hydrocarbyl groups. For example, by the following structure:
the-C of the invention 0 ～ 4 The alkylene group may be C 0 Alkylene, C 1 Alkylene (e.g. -CH 2 -)、C 2 Alkylene (e.g. -CH 2 CH 2 -etc., C 3 Alkylene or C 4 Alkylene groupA base; c (C) 0 Alkylene means that the radicals are not present here and are attached in the form of chemical bonds, e.g.A-C 0 alkylene-B refers to A-B, i.e., the A group is directly linked to the B group by a chemical bond.
"alkenyl" means having at least 1 site of ethylenic unsaturation [ ]>C＝C<) A linear or branched hydrocarbyl group of (a). For example, C a-b Alkenyl refers to alkenyl groups having a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1, 3-butadienyl, and the like.
"alkynyl" refers to a straight or branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbyl groups having one triple bond and one double bond. For example, C 2-6 Alkynyl is intended to include ethynyl, propynyl, and the like.
"none" in the description of the radicals according to the invention is that the radical is not present here and the moiety to which it is attached is directly linked in the form of a chemical bond.
In the description of the radicals according to the inventionAre used to describe the positions of substitution of groups.
The structure of the '-P (O) (OH) O' -in the invention isThe structure of the "-P (S) (OH) O-" is +.>
In the present invention, the term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form, and physiologically compatible with the recipient.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Drawings
FIG. 1 is a diagram of compound b7 1 H NMR spectrum (DMSO-d 6);
FIG. 2 is a diagram of compound b7 1 H NMR spectrum (heavy water exchange);
FIG. 3 is a LCMS of compound b 8;
FIG. 4 is a LCMS of immobilized compound b 9;
FIG. 5 is a live image of mice tested for distribution of siRNA conjugates of test example 1;
FIG. 6 is a fluorescent quantitative graph of the distribution of siRNA conjugates detected in test example 1.
Detailed Description
The structure of the compounds was determined by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR shift (. Delta.) is given in units of 10-6 (ppm). NMR was performed using a nuclear magnetic resonance apparatus (Bruker Avance III 400 and Bruker Avance 300) with deuterated dimethyl sulfoxide (DMSO-d) 6 ) Deuterated chloroform (CDCl) 3 ) Deuterated methanol (CD) 3 OD), internal standard is Tetramethylsilane (TMS).
LC-MS was measured using Shimadzu LC-MS 2020 (ESI). HPLC was performed using a Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A). MPLC (medium pressure preparative chromatography) uses Gilson GX-281 reverse phase preparative chromatograph. The specification of the thin layer chromatography separation and purification product adopted by the smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate is 0.4 mm-0.5 mm. Column chromatography generally uses tobacco stand yellow sea silica gel 200-300 mesh silica gel as a carrier.
The known starting materials of the present invention may be synthesized using or according to methods known in the art, or may be purchased from An Naiji chemical, chengkoulochemical, shaoshan chemical technology, carbofuran technology, and the like. Materials and reagents used in the examples of the present invention are commercially available unless otherwise specified.
The reaction was carried out under nitrogen atmosphere without specific explanation in examples. The examples are not specifically described, and the solution refers to an aqueous solution. The temperature of the reaction was room temperature, unless otherwise specified in the examples. In the examples, M is mol/liter unless otherwise specified.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Synthesis of intermediate compound 9- (benzyloxy) -9-oxononanoic acid:
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to a 100mL round bottom flask was added THF (tetrahydrofuran, 18 mL), and then to the stirred solution was added the compound azelaic acid (3.0 g,15.9 mmol), DBU (1, 8-diazabicyclo undec-7-ene, 2.43g,15.9 mmol) and BnBr (2.7 g,15.9 mmol). The reaction was allowed to react at room temperature for 2 hours, and LCMS detected completion of the reaction, extracted with EA, and the organic phase was washed once with saturated brine. Preparation by MPLC gave 1.53g (34.5%).
Synthesis of intermediate compound C4:
in a 250mL round bottom flask, 9- (benzyloxy) -9-oxononanoic acid (1.06 g,3.8 mmol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (0.87 g,4.5 mmol), N-hydroxysuccinimide (0.48 g,4.17 mmol) and dichloromethane 20mL were added. After stirring the reaction at room temperature for 0.5 hours, compound c3 (1.5 g,3.8 mmol) was added and the reaction was completed after 4 hours by TLC monitoring. The reaction mixture was washed with 20mL of a saturated sodium hydrogencarbonate solution and 20mL of a saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by passing through a silica gel column (ethyl acetate: petroleum ether V: v=4:1) to obtain compound c4 (1.1 g, 48%).
Synthesis of intermediate compound C5:
into a 100mL round bottom flask was added THF (tetrahydrofuran, 30 mL), then to the stirred solution was added compound c4 (1.1 g,1.68 mmol), then NaHCO was added 3 (0.17 g,2.0 mmol) and 10% Pb/C (110 mg, based on 50% wet weight). The top was flushed with hydrogen (balloon), reacted at room temperature for 2 hours, LCMS checked for completion, filtered through celite, and concentrated to give 0.92g of compound c5 (97%).
EXAMPLE 1 Synthesis of immobilized Compound b9
Step 1, synthesis of Compound b3
To a dry three-necked flask was added b1 (5.94 g,17.6mmol,1.1 eq.) followed by THF (tetrahydrofuran, 60 mL). The resulting solution was stirred and N-methylmorpholine (3.88 g,38.4mmol,2.4 eq.) was added. The mixture was cooled to 0 ℃ with an ice bath. Isobutyl chloroformate (i-BuCOCl, 2.40g,16mmol,1.1 eq.) was added dropwise to the mixture over 10 minutes, b2 (4.73 g,16mmol,1.0 eq.) was added while maintaining a temperature below 4℃and the ice bath was removed and the reaction was allowed to warm to room temperature and stirred for 2 hours. LCMS detection showed no compound b2, addition of dilute hydrochloric acid to acidity, EA (ethyl acetate) extraction, collection of the organic phase, washing with saturated brine, drying over anhydrous sodium sulfate, and concentration. Preparation by MPLC gave 8.10g of compound b3 (87%).
Step 2 Synthesis of Compound b4
To a 500mL round bottom flask was added compound b3 (8.97 g,15.5 mmol), formic acid (180 mL) and stirred at 45℃for 1.5 h. LCMS showed complete reaction, the reaction was concentrated, acetonitrile was added, the reaction was concentrated again, and repeated three times to remove residual formic acid. Concentration gave 6.13g of compound b4 (96%).
Step 3 Synthesis of Compound b5
To a 100mL round bottom flask was added compound b4 (1.01 g,2.46 mmol), DMF (N, N-dimethylformamide, 36 mL), TBTU (O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate, 2.55g,7.94 mmol) and compound a4 (4.47 g,8.16 mmol) were added to the stirred solution, after dissolution of the TBTU, the flask was placed in an ice bath, then DIPEA (N, N-diisopropylethylamine, 2.22g,17.17 mmol) was slowly added dropwise and the ice bath was removed and stirred at room temperature for 4 hours. LCMS showed no b4 remaining. To the reaction solution was added a saturated ammonium chloride solution, which was extracted with DCM (dichloromethane), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated brine in this order. Preparation by MPLC gave 3.64g of compound b5 (89%).
Step 4, synthesis of Compound b6
To a 250mL round bottom flask was added methanol (110 mL), then compound b5 (4.2 g,2.53 mmol) was added to the stirred solution, followed by 10% Pb/C (420 mg based on 50% wet weight). The top was flushed with hydrogen (balloon) and reacted at room temperature for 2 hours, LCMS detected complete reaction, filtered through celite and concentrated to give 3.87g of compound b6 (93%).
Step 5 Synthesis of Compound b7
To a 100mL round bottom flask was added c5 (0.95 g,1.69mmol,1.1 eq.) and b6 (2.5 g,1.53mmol,1.0 eq.). The mixture was dissolved in dichloromethane (30 mL). TBTU (0.59 g,1.84mmol,1.2 eq.) was added via a separatory funnel to a solution stirred under nitrogen. At 20 minutes by syringeDIPEA (0.69 g,5.37mmol,3.5 eq.) was added dropwise over a clock, maintaining the reaction at less than 25 ℃. The mixture was stirred for 2 hours from the start of DIPEA addition and checked by HPLC. Analysis at 100 minutes showed complete reaction of the starting materials. After that, saturated NaCl solution and saturated NaHCO solution are used 3 Solution volume 1:1 (3X 60 mL) of the washing. Na for organic layer 2 SO 4 Dried, filtered and concentrated in vacuo to give the crude product, which is then purified by silica (CH 2 Cl 2 Meoh=20:1) to give compound b7 (2.56 g,80% yield, 96% purity) as a white foam solid, fig. 1 shows compound b7 1 H NMR spectrum (DMSO-d) 6 ) FIG. 2 shows compound b7 1 H NMR spectrum (heavy water exchange).
Step 6 Synthesis of Compound b8
To a 25mL round bottom flask was added dichloromethane (3 mL), to the stirred solution was added b7 (0.30 g,0.14 mmol), triethylamine (0.044 mg,0.43 mmol), c6 (0.035 g,0.35 mmol), 4-dimethylaminopyridine, stirred at room temperature for 2 hours, LCMS detected no starting material remaining, quenched with saturated solution of sodium bicarbonate, extracted with dichloromethane, brine wash the organic phase with sodium sulfate, dried under reduced pressure, concentrated under reduced pressure and purified through a silica column (MeOH: CH2 Cl2=1/10) to give 242mg of white powder b8 (99%). Fig. 3 shows LCMS diagram of compound b 8.
Step 7, synthesis of immobilized Compound b9
CPG solid was placed in a 10ml capped reagent bottle, washed once with dichloromethane and DMF sequentially, DMF (5 ml) was added to the bottle, b8 (0.24 g,0.11 mmol) and TBTU (0.070 g,0.22 mmol) were added to the slightly shaking bottle, and DIPEA (0.057 g,0.44mmol,3.5 eq.) was then added dropwise maintaining the reaction at less than 25 ℃. The mixture was stirred for 16 hours from the start of DIPEA addition. After the reaction, the mixture was washed 3 times with a solvent, 8mg of CPG solid was filtered by suction, and then the solid was dissolved in 1ml of ammonia water, reacted at 50℃for 1 hour, and the supernatant was taken for LCMS detection, which showed the formation of the target product. Then, the whole reaction solution was suction-filtered, and the solid was collected and dried by a freeze dryer to obtain 681mg of the immobilized compound b9. Fig. 4 shows LCMS of immobilized compound b9.
Example 2 preparation of siRNA conjugates
Synthesis using a solid phase phosphoramidite triester method, synthesis using a solid support oligonucleotide.
For the siRNA sequences of the invention, the sense strand was synthesized using the immobilization compound b9 as a solid support, and the antisense strand was synthesized using universal CPG.
The 48-channel synthesizer was used for sequence synthesis on a scale of 0.2. Mu. Mol. Phosphoramidite monomer was used at a concentration of 0.05M and activator was used at a concentration of 0.3M BTT.
Cleavage and deprotection of the sequence was performed in 1.5ml tubes, the first step using AMA and the second step using triethylamine hydrogen trifluoride to deprotect the two-position protecting group. For sequences containing all modifications of two positions, ammonolysis with ammonia is required. The sequence after cleavage and deprotection was precipitated using an acetone: ethanol (80:20) mixture and dissolved with RNase-free water. Each sequence was analyzed by LC-MS to determine sequence accuracy, quantified by a spectrophotometer, and purity determined by HPLC.
After HPLC purification, freeze-drying and quality inspection, the salts are exchanged by sodium acetate alcohol precipitation, desalted by a 3KD ultrafiltration tube, the sense strand and the antisense strand are quantitatively determined by a spectrophotometer after desalting, and the duplex is formed by mixing and annealing according to the ratio of 1:1. The sequence of the synthesized siRNA conjugate is as follows:
wherein the lowercase letter m indicates that one nucleotide adjacent to the right side of the letter m is a 2' -methoxy modified nucleotide; the identifier i2F represents that one nucleotide adjacent to the right side of the identifier i2F is a 2' -fluorine modified nucleotide; the identifier indicates that phosphorothioate linkages are between two nucleotides adjacent to the identifier; cy5.5 represents that the 5' -end of the antisense strand is linked to the fluorophore anthocyanin 5.5; the 5' -end of the sense strand of SIRNA1 and SIRNA2 is connected with a B9 group.
The effect of the conjugates of the invention is illustrated by the following test examples:
test example 1 inhibition of expression of ANGPTL3
This test example was used to examine the inhibition rate of the expression level of ANGPTL3 in liver tissue and the effect on total cholesterol, triglycerides, low density lipoprotein cholesterol in serum in normal C57BL6/J model mice (purchased from experimental animal technologies limited, beijing vernalia).
Normal C57BL6/J model mice of 6-8 weeks of age were randomly divided into 5 groups of 4 (male and female halves), and 2 groups of mice were treated with 1 x pbs, siRNA conjugate, respectively. The abdomen of each mouse was shaved and wiped clean with depilatory cream. The dose was calculated based on the body weight of the mice, and the neck was subcutaneously injected, and the dose of siRNA conjugate administered was 3mg/kg in a single dose, and the administration volume was 10mL/kg. The PSB group was given the same volume of PBS solution injected. Whole blood was taken from the canthus before and 72h after dosing, and two mice were sacrificed for each group 72h and 120h after dosing, respectively, and whole blood and liver tissue were collected.
Distribution of siRNA conjugates was examined before and 0.5, 3, 24, 48, 72, 96, 120h after SIRNA2 administration using IVIS (Perkinelmer, USA) with running imaging, and the results are shown in FIGS. 5 and 6.
The collected whole blood was centrifuged at 1000×g at 4 ℃ for 15min to obtain serum, and the serum was further tested for total cholesterol, triglyceride and low-density lipoprotein cholesterol content using a Dxc AU full-automatic biochemical analyzer (Beckman, usa), the test results of which are shown in table 1.
TABLE 1
Collected whole blood was centrifuged at 1000×g at 4 ℃ for 15min to obtain serum, and the serum and liver were further tested for ANGPTL3 protein content using a mouse ANGPTL3 elisas kit (SAB, usa), and the test results are shown in table 2, which were performed strictly according to the kit instructions.
TABLE 2
The test results show that the siRNA conjugate used in the invention has the expression inhibition rate of 78.87% to ANGPTL3 in serum in normal mice after 120 hours, and the expression inhibition rate of 55.05% to ANGPTL3 in liver after 120 hours. And the contents of total cholesterol, triglyceride and low-density lipoprotein cholesterol in blood are obviously reduced by inhibiting the expression of ANGPTL3, and the inhibition rates of the total cholesterol, the triglyceride and the low-density lipoprotein cholesterol are about 30%, 36% and 21% respectively at 120 hours. The siRNA conjugate can efficiently deliver siRNA drugs to target the liver and exert curative effects.
Project name Design B
State of generated
Creation date 2022-12-20
Last modified:2022-12-20
Sequence(s)
Sequence 1 "seq_1"
Features (e.g. a character)
Residues
acatatttga tcagtctttt t 21
Sequence 2 "seq_2"
Features (e.g. a character)
Residues
aaaaagactg atcaaatatg ttg 23
Sequence 3 "seq_3"
Features (e.g. a character)
Residues
aaaaagactg atcaaatatg ttg 23。

Claims (13)

1. A conjugate, characterized in that: has a structure shown in formula I:
wherein, the liquid crystal display device comprises a liquid crystal display device,
nu is the active drug moiety;
m is selected from-O-, -NH-, -P (O) (OH) O-, -P (S) (OH) O-, or none;
L 1 selected from the group consisting of
L 11 Selected from-C 1～20 Alkylene-, -O-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
L 12 selected from-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
R a selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
R b selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
L 2 selected from-C (O) -C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-O-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 alkylene-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -, -C (O) -O-C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -orEach L 3 Independently selected from-C 1～10 Alkylene-, -O-, or none;
each n is independently selected from integers from 0 to 10;
each m is independently selected from integers from 0 to 5;
t is selected from
Each L 3 Each independently selected from a linking chain of 1 to 50 carbon atoms in length, wherein one or more methylene groups may be optionally replaced by one or more of the following groups: -C (O) -, -NH-, O, S, -S (O) -, -S (O) 2-, -P (O) (OH) -, -P (S) (OH) -, -ch=ch-, -c≡c-, -ch=n-, carbocyclyl, heterocyclyl; may each be further substituted with one or more of the following groups: c (C) 1～10 Alkyl, halogen substituted C 1～10 Alkyl, - (C) 0～10 Alkylene) -OH, - (C 0～10 Alkylene) -O- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -SH, - (C 0～10 Alkylene) -S- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -NH2, - (C 0～10 Alkylene) -NH (C) 1～10 Alkyl) - (C) 0～10 Alkylene) -N (C) 1～10 Alkyl) (C) 1～10 An alkyl group);
each G is independently selected from a ligand moiety capable of binding to a cell surface receptor.
2. The conjugate according to claim 1, characterized in that:
L 1 selected from the group consisting of
L 2 Selected from the group consisting of
L 3 Selected from the group consisting of
3. The conjugate according to claim 1, characterized in that: each G is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucofuranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-glucopyranose alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamide-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose.
4. A conjugate according to claim 3, characterized in that: each G is
5. The conjugate according to claims 1-4, characterized in that: the conjugate is selected from the following structures:
6. the conjugate according to claim 1, characterized in that: the Nu is a functional oligonucleotide.
7. The conjugate of claim 6, wherein: the Nu is siRNA.
8. The conjugate of claim 7, wherein: the siRNA comprises a sense strand and an antisense strand; the remainder of the conjugate is attached to the 5' end of the sense strand.
9. A compound, characterized in that: has a structure shown in formula II:
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 is hydrogen, a protecting group or a solid support moiety;
m is selected from-O-, -NH-, -P (O) (OH) O-, -P (S) (OH) O-, or none;
L 1 selected from the group consisting of
L 11 Selected from-C 1～20 Alkylene-, -O-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
L 12 selected from-C 1～20 Alkylene-, -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 Alkylene-or absent;
R 2 selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl, protecting group or solid support moiety;
R b selected from hydrogen, -C 1～30 Alkyl, -C 2～30 Alkenyl or-C 2～30 Alkynyl;
L 2 selected from-C (O) -C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-C (O) -, -C (O) -O-C 1～20 alkylene-O-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 0～10 alkylene-C (O) -, -C (O) -C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -, -C (O) -O-C 0～10 Alkylene- (OCH) 2 CH 2 ) m -O-C 1～10 alkylene-O-C (O) -orEach L 3 Independently selected from-C 1～10 Alkylene-, -O-, or none;
each n is independently selected from integers from 0 to 10;
each m is independently selected from integers from 0 to 5;
t is selected from
Each L 3 Each independently selected from a linking chain of 1 to 50 carbon atoms in length, wherein one or more methylene groups may be optionally replaced by one or more of the following groups: -C (O) -, -NH-, O, S, -S (O) -, -S (O) 2-, -P (O) (OH) -, -P (S) (OH) -, -ch=ch-, -c≡c-, -ch=n-, carbocyclyl, heterocyclyl; may each be further substituted with one or more of the following groups: c (C) 1～10 Alkyl, halogen substituted C 1～10 Alkyl, - (C) 0～10 Alkylene) -OH, - (C 0～10 Alkylene) -O- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -SH, - (C 0～10 Alkylene) -S- (C 1～10 Alkyl) - (C) 0～10 Alkylene) -NH2, - (C 0～10 Alkylene) -NH (C) 1～10 Alkyl) - (C) 0～10 Alkylene) -N (C) 1～10 Alkyl) (C) 1～10 An alkyl group);
each G is independently selected from a ligand moiety capable of binding to a cell surface receptor.
10. A compound according to claim 16, wherein:
L 1 selected from the group consisting of
R 1 As part of the solid support, R 2 Is DMTr;
L 2 selected from the group consisting of
L 3 Selected from the group consisting of
11. A compound according to claim 10, characterized in that: the compound is selected from the following structures:
wherein R is 1 Is a solid support portion comprising CPG.
12. Use of a conjugate according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment and/or prophylaxis of a disease caused by abnormal expression of a specific gene in hepatocytes.
13. A method of inhibiting expression of a specific gene in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of the conjugate of any one of claims 1 to 8.
CN202211628857.9A 2021-12-24 2022-12-21 Conjugate, intermediate compound thereof and application Pending CN116474107A (en)

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