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CN120282953A - Ligands for delivery of SIRNA to the eye and central nervous system - Google Patents

Ligands for delivery of SIRNA to the eye and central nervous system Download PDF

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CN120282953A
CN120282953A CN202380076440.2A CN202380076440A CN120282953A CN 120282953 A CN120282953 A CN 120282953A CN 202380076440 A CN202380076440 A CN 202380076440A CN 120282953 A CN120282953 A CN 120282953A
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黄金宇
杨晓彦
邹昊
刘俊凯
方剑武
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Darui Biomedical Technology Shanghai Co ltd
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    • A—HUMAN NECESSITIES
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    • C07—ORGANIC CHEMISTRY
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Abstract

本发明提供式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体。所述式(I)化合物包含在核苷酸内部，或者在5′端和/或3′端，用于提高双链RNA穿过血脑屏障的能力。本发明还涉及包含式(I)化合物的寡核苷酸、双链RNA、载体、细胞、药物组合物和试剂盒。 The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. The compound of formula (I) is contained inside a nucleotide, or at the 5' end and/or the 3' end, and is used to improve the ability of double-stranded RNA to pass through the blood-brain barrier. The present invention also relates to oligonucleotides, double-stranded RNA, vectors, cells, pharmaceutical compositions and kits containing the compound of formula (I).

Description

Ligands for delivery of SIRNA to the eye and central nervous system
The present application claims priority from chinese application 202211347517.9 filed on 10/31 of 2022, which is incorporated herein by reference in its entirety.
Technical Field
The present invention is in the field of medicine, in particular to hydrophobic groups, such as R groups in formula (I), having the ability to enhance delivery of double stranded RNA across the blood brain barrier and/or the eye, and to compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which attach the hydrophobic groups to nucleotides.
Background
RNA interference is a phenomenon of efficient and specific degradation of target mRNA induced by double-stranded RNA (double-STRANDED RNA, DSRNA, also known as siRNA).
However, due to the presence of the blood brain barrier, it is difficult to deliver siRNA to the central nervous system to function in turn, which limits the use of siRNA. Some attempts in the art to deliver siRNA to the central nervous system have been made, for example WO2004094595A2 discloses the delivery of siRNA using a single lipid ligand (e.g. cholesterol or long chain alkane) at the end of the chain, WO2019217459A1 discloses the delivery of siRNA using a single lipid ligand inside the chain, WO2021092371A2 discloses a series of new lipid ligand structures.
There remains a need in the art to develop more hydrophobic groups to more effectively deliver siRNA to the eye and/or central nervous system.
Disclosure of Invention
In one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein each group is as defined below.
In another aspect, the invention provides an oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein each group is as defined below.
In another aspect, the invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence substantially complementary to the sense strand and a target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein each group is as defined below.
In another aspect, the hydrophobic groups provided herein (R groups in formula (I)) can be attached to the siRNA via existing linker structures, such as biodegradable linker structures.
In another aspect, the invention provides a vector comprising a nucleotide sequence encoding the aforementioned double stranded RNA.
In another aspect, the invention provides a cell comprising the aforementioned double stranded RNA or the aforementioned vector.
In another aspect, the invention provides a pharmaceutical composition comprising the aforementioned double stranded RNA, the aforementioned vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier or excipient.
In another aspect, the invention provides a kit comprising the aforementioned double stranded RNA, the aforementioned vector, or the aforementioned cell.
Drawings
FIG. 1 shows the decrease in the expression level of SOD1 in cervical spinal cord, thoracic spinal cord, cerebellum, brain stem, hippocampus and frontal cortex after injecting the siRNA of the present invention via intervertebral foramen puncture in SD rats.
FIG. 2 shows the decrease in the expression level of TTR gene in eyes after intravitreal injection of siRNA of the present invention in C57BL/6 mice.
Detailed Description
Definition of the definition
Chemical definition
The definition of specific functional groups and chemical terms is described in more detail below.
When numerical ranges are listed, it is intended to include each and every value and subrange within the range. For example, "C 1-6 alkyl" includes C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5 and C 5-6 alkyl.
"C 1-30 alkyl" refers to a straight or branched saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 5-25 alkyl, C 10-20 alkyl, C 1-20 alkyl, C 1-10 alkyl, and C 1-6 alkyl are preferred. Examples of C 1-6 alkyl groups include methyl (C 1), ethyl (C 2), n-propyl (C 3), Isopropyl (C 3), n-butyl (C 4), tert-butyl (C 4), sec-butyl (C 4), Isobutyl (C 4), n-pentyl (C 5), 3-pentyl (C 5), pentyl (C 5), Neopentyl (C 5), 3-methyl-2-butyl (C 5), tert-amyl (C 5) and n-hexyl (C 6). The term "C 1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., 1,2,3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include ：Me(-CH3)、Et(-CH2CH3)、iPr(-CH(CH3)2)、nPr(-CH2CH2CH3)、n-Bu(-CH2CH2CH2CH3) or i-Bu (-CH 2CH(CH3)2).
"C 2-30 alkenyl" refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-25 alkenyl, C 2-10 alkenyl, C 2-6 alkenyl, and C 2-4 alkenyl are preferred. Examples of C 2-6 alkenyl groups include vinyl (C 2), 1-propenyl (C 3), 2-propenyl (C 3), 1-butenyl (C 4), 2-butenyl (C 4), butadienyl (C 4), pentenyl (C 5), pentadienyl (C 5), hexenyl (C 6), and the like. The term "C 2-6 alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1,2,3, or 4) carbon atoms are replaced with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
"C 2-30 alkynyl" refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-25 alkynyl, C 2-10 alkynyl, C 2-6 alkynyl, and C 2-4 alkynyl are preferred. Examples of C 2-6 alkynyl include, but are not limited to, ethynyl (C 2), 1-propynyl (C 3), 2-propynyl (C 3), 1-butynyl (C 4), 2-butynyl (C 4), pentynyl (C 5), hexynyl (C 6), and the like. The term "C 2-6 alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1,2,3, or 4) carbon atoms are replaced with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
"C 1-10 Alkylene", "C 2-10 alkenylene" and "C 2-10 alkynylene" refer to the removal of C 1-10 alkyl, respectively, The divalent group formed by the other hydrogen of C 2-10 alkenyl and C 2-10 alkynyl groups, and may be substituted or unsubstituted. In some embodiments, C 2-8 alkylene, C 3-7 alkylene, C 4-6 alkylene, C 1-4 alkylene, C 2-4 alkylene and C 1-3 alkylene are preferred. Unsubstituted alkylene groups include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2CH2 -), propylene (-CH 2CH2CH2 -), butylene (-CH 2CH2CH2CH2 -), and, pentylene (-CH 2CH2CH2CH2CH2 -), hexylene (-CH 2CH2CH2CH2CH2CH2 -), and the like. Exemplary substituted alkylene groups, for example, alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH (CH 3)-、-C(CH3)2 -), substituted ethylene (-CH(CH3)CH2-、-CH2CH(CH3)-、-C(CH3)2CH2-、-CH2C(CH3)2-)、 substituted propylene (-CH(CH3)CH2CH2-、-CH2CH(CH3)CH2-、-CH2CH2CH(CH3)-、-C(CH3)2CH2CH2-、-CH2C(CH3)2CH2-、-CH2CH2C(CH3)2-),, and the like.
"C 0-10 alkylene" refers to a chemical bond and "C 1-10 alkylene" as described above.
"Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
Thus, "C 1-20 haloalkyl", "C 1-6 haloalkyl" and "C 1-4 haloalkyl" refer to "C 1-20 alkyl", "C 1-6 alkyl" and "C 1-4 alkyl" described above, respectively, substituted with one or more halo groups. In some embodiments, C 1-4 haloalkyl is particularly preferred, more preferably C 1-2 haloalkyl. Exemplary such haloalkyl groups include, but are not limited to ：-CF3、-CH2F、-CHF2、-CHFCH2F、-CH2CHF2、-CF2CF3、-CCl3、-CH2Cl、-CHCl2、2,2,2- trifluoro-1, 1-dimethyl-ethyl, and the like. The haloalkyl group may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or1 substituent.
The term "hydrophobic group" refers broadly to any chemical group having an affinity for lipids. One way of characterizing the hydrophobicity of a hydrophobic group is by the octanol-water partition coefficient log K ow, where K ow is the ratio of the concentration of chemical species in the octanol phase to its concentration in the aqueous phase at equilibrium for a two-phase system. Typically, the hydrophobic moiety has a log k ow of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. In particular, according to the invention, the hydrophobic moiety is an R group in a compound of formula I.
Alkyl, alkenyl, alkynyl, and the like as defined herein are optionally substituted groups.
Exemplary substituents on carbon atoms include, but are not limited to, halo 、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORaa、-ON(Rbb)2、-N(Rbb)2、-N(Rbb)3 +X-、-N(ORcc)Rbb、-SH、-SRaa、-SSRcc、-C(＝O)Raa、-CO2H、-CHO、-C(ORcc)2、-CO2Raa、-OC(＝O)Raa、-OCO2Raa、-C(＝O)N(Rbb)2、-OC(＝O)N(Rbb)2、-NRbbC(＝O)Raa、-NRbbCO2Raa、-NRbbC(＝O)N(Rbb)2、-C(＝NRbb)Raa、-C(＝NRbb)ORaa、-OC(＝NRbb)Raa、-OC(＝NRbb)ORaa、-C(＝NRbb)N(Rbb)2、-OC(＝NRbb)N(Rbb)2、-NRbbC(＝NRbb)N(Rbb)2、-C(＝O)NRbbSO2Raa、-NRbbSO2Raa、-SO2N(Rbb)2、-SO2Raa、-SO2ORaa、-OSO2Raa、-S(＝O)Raa、-OS(＝O)Raa、-Si(Raa)3、-OSi(Raa)3、-C(＝S)N(Rbb)2、-C(＝O)SRaa、-C(＝S)SRaa、-SC(＝S)SRaa、-SC(＝O)SRaa、-OC(＝O)SRaa、-SC(＝O)ORaa、-SC(＝O)Raa、-P(＝O)2Raa、-OP(＝O)2Raa、-P(＝O)(Raa)2、-OP(＝O)(Raa)2、-OP(＝O)(ORcc)2、-P(＝O)2N(Rbb)2、-OP(＝O)2N(Rbb)2、-P(＝O)(NRbb)2、-OP(＝O)(NRbb)2、-NRbbP(＝O)(ORcc)2、-NRbbP(＝O)(NRbb)2、-P(Rcc)2、-P(Rcc)3、-OP(Rcc)2、-OP(Rcc)3、-B(Raa)2、-B(ORcc)2、-BRaa(ORcc)、 alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3, 4, or 5R dd groups;
Or two geminal hydrogens on the carbon atom are substituted with a group ＝O、＝S、＝NN(Rbb)2、＝NNRbbC(＝O)Raa、＝NNRbbC(＝O)ORaa、＝NNRbbS(＝O)2Raa、＝NRbb or = NOR cc;
each of R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1,2, 3, 4, or 5R dd groups;
Each of R bb is independently selected from hydrogen 、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、 alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3, 4, or 5R dd groups;
Each of R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1,2, 3, 4, or 5R dd groups;
Each of R dd is independently selected from halogen 、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORee、-ON(Rff)2、-N(Rff)2,、-N(Rff)3 +X-、-N(ORee)Rff、-SH、-SRee、-SSRee、-C(＝O)Ree、-CO2H、-CO2Ree、-OC(＝O)Ree、-OCO2Ree、-C(＝O)N(Rff)2、-OC(＝O)N(Rff)2、-NRffC(＝O)Ree、-NRffCO2Ree、-NRffC(＝O)N(Rff)2、-C(＝NRff)ORee、-OC(＝NRff)Ree、-OC(＝NRff)ORee、-C(＝NRff)N(Rff)2、-OC(＝NRff)N(Rff)2、-NRffC(＝NRff)N(Rff)2、-NRffSO2Ree、-SO2N(Rff)2、-SO2Ree、-SO2ORee、-OSO2Ree、-S(＝O)Ree、-Si(Ree)3、-OSi(Ree)3、-C(＝S)N(Rff)2、-C(＝O)SRee、-C(＝S)SRee、-SC(＝S)SRee、-P(＝O)2Ree、-P(＝O)(Ree)2、-OP(＝O)(Ree)2、-OP(＝O)(ORee)2、 alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3, 4, or 5R gg groups, or two geminal R dd substituents may combine to form =o or =s;
Each of R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1,2, 3, 4, or 5R gg groups;
Each of R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1,2, 3, 4, or 5R gg groups;
R gg is each independently halogen, -CN, -NO 2、-N3、-SO2H、-SO3H、-OH、-OC1-6 alkyl, -ON (C 1-6 alkyl) 2、-N(C1-6 alkyl) 2、-N(C1-6 alkyl) 3 +X-、-NH(C1-6 alkyl) 2 +X-、-NH2(C1-6 alkyl) +X-、-NH3 +X-、-N(OC1-6 alkyl) (C 1-6 alkyl), -N (OH) (C 1-6 alkyl), -NH (OH), -SH, -SC 1-6 alkyl, -SS (C 1-6 alkyl), -C (=o) (C 1-6 alkyl), -CO 2H、-CO2(C1-6 alkyl), -OC (=o) (C 1-6 alkyl), -OCO 2(C1-6 alkyl), -C (=o) NH 2、-C(＝O)N(C1-6 alkyl) 2、-OC(＝O)NH(C1-6 alkyl, -NHC (=o) (C 1-6 alkyl), -N (C 1-6 alkyl) C (=o) (C 1-6 alkyl), -NHCO 2(C1-6 alkyl), -NHC (=o) N (C 1-6 alkyl) 2、-NHC(＝O)NH(C1-6 alkyl), -NHC (=o) NH 2、-C(＝NH)O(C1-6 alkyl), -OC (=nh) (C 1- 6 alkyl), -OC (=nh) OC 1-6 alkyl, -C (=nh) N (C 1-6 alkyl) 2、-C(＝NH)NH(C1-6 alkyl), -C (=nh) NH 2、-OC(＝NH)N(C1-6 alkyl) 2、-OC(NH)NH(C1-6 alkyl, -OC (NH) NH 2、-NHC(NH)N(C1-6 alkyl) 2、-NHC(＝NH)NH2、-NHSO2(C1-6 alkyl), -SO 2N(C1-6 alkyl) 2、-SO2NH(C1-6 alkyl, -SO 2NH2、-SO2C1-6 alkyl, -SO 2OC1-6 alkyl, -OSO 2C1-6 alkyl, -SOC 1-6 alkyl, -Si (C 1-6 alkyl) 3、-OSi(C1-6 alkyl) 3、-C(＝S)N(C1-6 alkyl) 2、C(＝S)NH(C1-6 alkyl), C (=s) NH 2、-C(＝O)S(C1-6 alkyl, -C (=s) SC 1-6 alkyl, -SC (=s) SC 1-6 alkyl, -P (=o) 2(C1-6 alkyl), -P (=o) (C 1- 6 alkyl) 2、-OP(＝O)(C1-6 alkyl) 2、-OP(＝O)(OC1-6 alkyl) 2、C1-6 alkyl, C 1-6 haloalkyl, C 2-C6 alkenyl, C 2-C6 alkynyl, C 3-C7 cycloalkyl, C 6-C10 aryl, C 3-C7 heterocyclyl, C 5-C10 heteroaryl, or two geminal R gg substituents may combine to form =o or =s, wherein X - is a counterion.
Exemplary substituents on the nitrogen atom include, but are not limited to, hydrogen 、-OH、-ORaa、-N(Rcc)2、-CN、-C(＝O)Raa、-C(＝O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(＝NRbb)Raa、-C(＝NRcc)ORaa、-C(＝NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(＝S)N(Rcc)2、-C(＝O)SRcc、-C(＝S)SRcc、-P(＝O)2Raa、-P(＝O)(Raa)2、-P(＝O)2N(Rcc)2、-P(＝O)(NRcc)2、 alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups attached to a nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R dd groups, and wherein R aa、Rbb、Rcc and R dd are as described above.
Other definitions
The term "siRNA" herein is a class of double stranded RNA molecules that can mediate silencing of target RNAs (e.g., mRNA, e.g., transcripts of genes encoding proteins) complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to a target RNA, and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNAs (e.g., tRNA's) and viral RNAs can be targeted.
The term "antisense strand" refers to a strand of an siRNA that comprises a region that is fully, substantially or complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is wholly, substantially or essentially complementary to a region that is the term antisense strand as defined herein.
The term "complementary region" refers to a region on the antisense strand that is fully, substantially or essentially complementary to a target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. Typically, the most tolerated mismatch is located in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotides of the 5 'and/or 3' end. The portion of the antisense strand that is most susceptible to mismatch is referred to as the "seed region". For example, in an siRNA comprising a strand of 19nt, the 19 th position (from 5 'to 3') may tolerate some mismatches.
The term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50℃or 70℃for 12-16 hours. "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides in terms of meeting the above requirements with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble base pairing or Hoogstein base pairing.
A polynucleotide that is "at least partially complementary," "substantially complementary," or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, if the sequence is substantially complementary to the non-interrupting portion of the PCSK 9-encoding mRNA, the polynucleotide is at least partially complementary to the PCSK9 mRNA. The terms "complementary," "fully complementary," "substantially complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense strand and the antisense strand of an siRNA agent, or between the antisense strand and a target sequence of an siRNA agent.
"Substantially complementary" refers to the extent to which the sense strand need only be complementary to the antisense strand in order to maintain the overall double-stranded character of the molecule. In other words, while perfect complementarity is often desired, in some cases, particularly in the antisense strand, one or more, e.g., 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands may still maintain the overall double stranded character of the molecule.
"ShRNA" refers to short hairpin RNA. shRNA comprises two short inverted repeats. shRNA cloned into shRNA expression vectors comprises two short inverted repeats, separated by a stem-loop (loop) sequence in the middle, constituting a hairpin structure, controlled by the polIII promoter. Then, 5-6T's were ligated as transcription terminators for RNA polymerase III.
"Nucleoside" is a compound consisting of a purine or pyrimidine base, and ribose or deoxyribose, and "nucleotide" is a compound consisting of three substances, purine or pyrimidine base, ribose or deoxyribose, and phosphate, and "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300, or 400 nucleotides, for example.
"Base" is the basic constituent unit of synthetic nucleosides, nucleotides and nucleic acids, the constituent elements of which contain nitrogen, also known as "nitrogenous bases". Herein, unless otherwise indicated, capital letters A, U, T, G and C represent the base composition of nucleotides, adenine, uracil, thymine, guanine and cytosine, respectively.
"Modification" of a nucleotide as described herein includes, but is not limited to, methoxy modification, fluoro modification, phosphorothioate linkage, or conventional protecting group protection, and the like. For example, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluoro, and the methoxy-modified nucleotide refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with methoxy.
"Modified nucleotides" herein include, but are not limited to, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing phosphorothioate groups, vinylphosphate modified nucleotides, locked nucleotides, 2' -amino-modified nucleotides, 2' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural bases containing nucleotides, and terminal nucleotides attached to cholesterol derivatives or dodecanoic didecanoyl amine groups, deoxyribonucleotides or conventional protecting group protection, and the like. For example, the 2 '-fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine. The 2 '-deoxy-modified nucleotide refers to a nucleotide formed by substituting a 2' -hydroxyl group of a ribosyl group with a methoxy group.
"Ligand moiety" refers to a chemical moiety conjugated to an siRNA that is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, cell or cell type, compartment (e.g., cell or organ compartment, tissue, organ or region of the body)) as compared to, for example, an siRNA in the absence of such a ligand.
"Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or in a nucleotide analog unit that can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or in another nucleotide analog, by nucleophilic attack reaction. Typically, such a reaction produces an ester internucleoside linkage linking the first nucleotide unit or the first nucleotide analogue unit to the second nucleotide unit or the second nucleotide analogue unit. The reactive phosphorus group may be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate ester mimics including, but not limited to, natural phosphates, phosphorothioates, phosphorodithioates, borane phosphates, borane phosphorothioates, phosphonates, halogen substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioates triesters, bisphosphates and triphosphates, preferably-P (OCH 2CH2CN)(N(iPr)2).
"Protecting group" refers to any atom or group of atoms that is added to a molecule to prevent undesired chemical reactions of existing groups in the molecule. "protecting groups" may be labile chemical moieties known in the art that serve to protect reactive groups, such as hydroxyl, amino, and thiol groups, from undesired or untimely reactions during chemical synthesis. The protecting groups are typically used selectively and/or orthogonally to the protecting site during the reaction of the other reactive site, and can then be removed to leave the unprotected group intact or available for further reaction.
Non-limiting lists of protecting groups include benzyl, substituted benzyl, alkylcarbonyl and alkoxycarbonyl (e.g., t-Butoxycarbonyl (BOC), acetyl or isobutyryl), aralkylcarbonyl and aralkoxycarbonyl (e.g., benzyloxycarbonyl), substituted methyl ethers (e.g., methoxymethyl ether), substituted diethyl ether, substituted benzyl ether, tetrahydropyranyl ether, silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2- (trimethylsilyl) ethoxy ] methyl or t-butyldiphenylsilyl), esters (e.g., benzoates), carbonates (e.g., methoxymethyl carbonate), sulfonates (e.g., tosylate or mesylate), acyclic ketals (e.g., dimethylacetal), acyclic ketals (e.g., 1, 3-dioxane, 1, 3-dioxolane and those described herein), acyclic acetals, cyclic hemiacetals, cyclic dithioketals (e.g., those described herein), cyclic dithioketals (e.g., 1, 3-dioxane, 3-dioxolane), methyl, trimethyl benzene (e.g., 1, 3-dimethyl sulfide, 4 '-trimethylbenzene, tri-4' -methyl), methyl (e.g., tri-4, 4 '-methoxy) and (e.g., methyl) tri-4, 4' -phenyl) methyl (e.g., tri-methyl), 4'' -trimethoxytrityl (TMTr), and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), beta-Methoxyethoxymethyl Ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, piv-onyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [ (4-methoxyphenyl) diphenylmethyl ] (MMT), dimethoxytrityl, [ bis- (4-methoxyphenyl) phenylmethyl (DMT), trimethylsilyl ether (TMS), t-butyldimethylsilyl ether (TBDMS), tri-isopropyl silyloxymethyl ether (TOM), tri-isopropyl silyl ether (TIPS), methyl ether, ethoxydiethyl Ether (EE) N, N-dimethyl formamidine and 2-Cyanoethyl (CE).
"Hydroxy protecting group" refers to a group that is capable of protecting a hydroxy group from chemical reaction and that can be removed under specific conditions to restore the hydroxy group. Mainly comprises a silane type protecting group, an acyl type protecting group or an ether type protecting group, preferably the following:
Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), C (CH 25, C (OH) or C4-C (OH) or more preferably C4-C (OH) 35O, 3, C (CH) or C4-O3, more preferably C (OH) 35O (CH) or C4-O (CH) 35.
The term "pharmaceutically acceptable salts" as used herein means those carboxylate salts, amino acid addition salts of the compounds of the invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including (if possible) zwitterionic forms of the compounds of the invention.
The present invention includes tautomers, which are functional group isomers that result from the rapid movement of an atom in a molecule at two positions. Compounds that exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.
The compounds of the invention may include one or more asymmetric centers and thus may exist in a variety of stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers may be separated from the mixtures by methods known to those skilled in the art, including chiral High Pressure Liquid Chromatography (HPLC) and formation and crystallization of chiral salts, or the preferred isomers may be prepared by asymmetric synthesis.
The invention also includes isotopically-labelled compounds (isotopically-variant) which are identical to those recited in formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F and 36 Cl, respectively. The compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs thereof, which contain the isotopes described above and/or other isotopes of other atoms, are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes (e.g., 3 H and 14 C) are introduced, are useful in drug and/or substrate tissue distribution assays. Tritium, i.e., 3 H, and carbon-14, i.e., 14 C isotopes are particularly preferred because they are easy to prepare and detect. Further, substitution with heavier isotopes, such as deuterium, i.e., 2 H, may be preferred in some circumstances because greater metabolic stability may afford therapeutic benefits such as increased in vivo half-life or reduced dosage requirements. Isotopically-labeled compounds of formula (I) of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and/or examples and preparations below by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent.
Compounds of the invention
The present invention relates in particular to compounds of formula (I), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
Wherein, the
L 1 and L 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group, or a solid support;
R s is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until fully deuterated;
m=0, 1, 2, 3,4, 5 or 6;
R is-C (O) -C 0-10 alkylene-L-R 1、-C(O)-C2-10 alkenylene-L-R 1 or-C (O) -C 2-10 alkynylene-L-R 1;
L is a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)- or-NHC (O) -CH 2-O-CH2CH(NH2) CH (OH) -;
R 1 is independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl, wherein non-adjacent 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms in the groups may be replaced by heteroatoms selected from O, S and N, or the-CH 2CH2 -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-substitution, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring;
Wherein the hydrogen atoms in the C 0-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 1-30 alkyl, C 2-30 alkenyl, and C 2-30 alkynyl groups may optionally be replaced by 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more halogens, C 1-6 alkyl, or C 1-6 haloalkyl, and which is optionally deuterated until completely deuterated.
The invention also relates to oligonucleotides comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
Wherein, the
Represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
L 2 represents H or a solid support, or represents a position linked to an adjacent nucleotide;
R s, m and R are as defined above.
The invention also relates to double stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof:
Wherein, the
Represents the position of ligation to an adjacent nucleotide;
L 2 represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
R s, m and R are as defined above.
L 1、L2
In one embodiment, L 1 is H, in another embodiment L 1 is a reactive phosphorus group, in another embodiment L 1 is a hydroxyl protecting group, and in another embodiment L 1 is a solid support.
In one embodiment, L 2 is H, in another embodiment L 2 is a reactive phosphorus group, in another embodiment L 2 is a hydroxyl protecting group, in another embodiment L 2 is a solid support, and in another embodiment L 2 represents the position of attachment to an adjacent nucleotide.
In a more particular embodiment, one of L 1 and L 2 is-C (O) CH 2CH2 C (O) OH or 4,4' -dimethoxytrityl, and in another more particular embodiment, one of L 1 and L 2 is-C (O) CH 2CH2 C (O) OH.
In one embodiment,Represents a group H, and in another embodiment,Represents a hydroxyl protecting group and in another embodiment,Indicating the position of the ligation to the adjacent nucleotide.
Rs
In one embodiment, R s is H, in another embodiment R s is D, in another embodiment R s is halogen, in another embodiment R s is C 1-6 alkyl, in another embodiment R s is C 1-6 haloalkyl, in another embodiment R s is C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until fully deuterated;
m
In one embodiment, m=0, in another embodiment, m=1, in another embodiment, m=2, in another embodiment, m=3, in another embodiment, m=4, in another embodiment, m=5, in another embodiment, m=6.
R
In one embodiment, R is-C (O) -C 0-10 alkylene-L-R 1; in another embodiment, R is-C (O) -C 2-10 alkenylene-L-R 1, and in another embodiment R is-C (O) -C 2-10 alkynylene-L-R 1.
In a more specific embodiment, R is-C (O) -L-R 1; in another more specific embodiment, R is-C (O) -C 2-8 alkylene-L-R 1; in another more specific embodiment, R is-C (O) -C 3-7 alkylene-L-R 1; in another more particular embodiment, R is-C (O) -C 4-6 alkylene-L-R 1, and in another more particular embodiment, R is-C (O) -C 1-3 alkylene-L-R 1.
L
In one embodiment, L is a bond; in another embodiment, L is-NHC (O) -; in another embodiment, L is-C (O) NH-; in another embodiment, L is-OC (O) -, in another embodiment L is-C (O) O-, in another embodiment L is-S-, in another embodiment L is-NHC (O) O-, in another embodiment L is-NHC (O) NH-, in another embodiment L is-OC (O) O-, in another embodiment L is-OC (O) NH-, in another embodiment L is-NHC (O) -CH (OR 1)CH2 O-, in another embodiment L is-C (O) NH-CH (OR 1)CH2 O-, in another embodiment L is-OC (O) -CH (OR 1)CH2 O-, in another embodiment L is-C (O) O-CH (OR 1)CH2 O-in another embodiment L is-NHC (O) -CH (R25) -, in another embodiment L is-CH (R35O) -, l is-OC (O) -CH (R 1) -; in another embodiment, L is-C (O) O-CH (R 1) -; in another embodiment, L is-CH (OR 1)CH2 O-; in another embodiment L is-O-CH (R 1)CH2 O-, in another embodiment L is-O-CH 2CH(R1) O-, in another embodiment L is-O-CH (CH (OH) CH 2 OH) -, in another embodiment L is-O-CH (CH (NH 2)CH2 OH) -, in another embodiment L is-O-CH (CH 2 OH) CH (OH) -, in another embodiment L is-NH-CH (CH 2 OH) CH (OH) -, in another embodiment L is-O-CH 2 CH (OH) -, in another embodiment L is-O-CH 2CH(NH2) CH (OH) -, in another embodiment L is-NHC (O) -CH 2-O-CH(CH(OH)CH2 OH) -, in another embodiment L is-NHC (O) -CH 2-O-CH(CH(NH2)CH2 OH) -, in another embodiment L is-NH-CH (CH) CH (OH) -, in another embodiment L is-CH (OH) -, l is-NHC (O) -CH 2-NH-CH(CH2 OH) CH (OH) -; in another embodiment, L is-NHC (O) -CH 2-O-CH2 CH (OH) CH (OH) -; in another embodiment, L is-NHC (O) -CH 2-O-CH2CH(NH2) CH (OH) -.
In a more specific embodiment, L is a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)- or-NHC (O) -CH 2-O-CH2CH(NH2) CH (OH) -; in another more specific embodiment, L is a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or-NHC (O) -CH 2-O-CH(CH(NH2)CH2 OH) -; in another more specific embodiment, L is a bond 、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or-NHC (O) -CH 2-O-CH(CH(NH2)CH2 OH) -; in another more specific embodiment, L is a bond, -NHC (O) -, -S-S-or-NHC (O) O-, and in another more specific embodiment L is-NHC (O) -.
In more specific embodiments, L is -NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O- OR-O-CH 2CH(R1) O-; in another more specific embodiment, L is-NHC (O) -CH (OR 1)CH2O-、-NHC(O)-CH(R1) -OR-CH (OR 1)CH2 O-; in another more specific embodiment, L is-NHC (O) -CH (OR 1)CH2 O-.
R1
In one embodiment, R 1 is C 1-30 alkyl; in another embodiment, R 1 is C 2-30 alkenyl; in another embodiment, R 1 is C 2-30 alkynyl; in another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms of the groups recited in R 1 that are not adjacent may be replaced by heteroatoms selected from O, S and N, in another embodiment the-CH 2CH2 -group in R 1 may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-, and in another embodiment the substituents on one or more carbon atoms in R 1 may be linked to form a saturated or unsaturated ring.
In more specific embodiments, R 1 is independently C 1-30 alkyl or C 2-30 alkenyl, wherein non-adjacent 1,2, 3, 4, 5, 6, 7, or 8 carbon atoms in the group may be replaced with a heteroatom selected from O, S and N, or the-CH 2CH2 -group may be replaced with-NHC (O) -or-C (O) NH-, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; in another more specific embodiment, R 1 is independently C 5-25 alkyl, C 10-25 alkenyl containing 1,2, 3, 4, 5 or 6 double bonds, C 5-25 alkyl wherein 1,2, 3, 4 or 5 carbon atoms are replaced with N heteroatoms and/or 1,2 or 3-CH 2CH2 -groups are replaced with-C (O) NH-, or C 5-25 alkyl wherein substituents on one or more carbon atoms are linked to form a steroidal ring, in another more specific embodiment R 1 is selected from the group consisting of:
c 6 alkyl, C 8 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, C 15 alkyl, C 16 alkyl, C 17 alkyl, C 21 alkyl,
Any one of the above embodiments or any combination thereof may be combined with any one of the other embodiments or any combination thereof. For example, any one of the technical schemes of L 1 or any combination thereof may be combined with any one of the technical schemes of L 2、Rs, m, R, L, R 1, etc., or any combination thereof. The invention is intended to include all such combinations, limited to the extent that they are not listed.
The invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the invention. The vector of the present invention is capable of amplifying or expressing the nucleotide encoding the siRNA of the present invention linked thereto.
For example, siRNA targeting the PCSK9 gene may be expressed from a transcription unit inserted into a DNA or RNA vector. Expression may be transient (hours to weeks) or continuous (weeks to months or more), depending on the particular construct and target tissue or cell type used. The siRNA encoding nucleotide can be incorporated into a linear construct, a circular body, or a viral vector. The nucleotides of the siRNA may be integrated into the cell genome for stable expression or may be expressed extrachromosomally for stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.
Viral vector systems comprising the coding sequence of the siRNA include, but are not limited to, (a) adenovirus vectors, (b) retrovirus vectors, (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vectors, (f) polyoma virus vectors, (g) papilloma virus vectors, (h) picornavirus vectors, (i) poxvirus vectors, and (j) helper-dependent adenoviruses or entero-free adenoviruses.
The invention also provides a cell comprising an siRNA or vector according to the invention, wherein the siRNA or vector according to the invention is capable of being transcribed in the cell.
The invention relates to the following technical scheme:
Technical scheme 1. A compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein, the
L 1 and L 2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group, or a solid support;
R s is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until fully deuterated;
m=0, 1, 2, 3,4, 5 or 6;
R is-C (O) -C 0-10 alkylene-L-R 1、-C(O)-C2-10 alkenylene-L-R 1 or-C (O) -C 2-10 alkynylene-L-R 1;
L is a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)- or-NHC (O) -CH 2-O-CH2CH(NH2) CH (OH) -;
R 1 is independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl, wherein non-adjacent 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms in the groups may be replaced by heteroatoms selected from O, S and N, or the-CH 2CH2 -group may be replaced by-OC (O) -, -C (O) O-, -NHC (O) -or-C (O) NH-substitution, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring;
Wherein the hydrogen atoms in the C 0-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 1-30 alkyl, C 2-30 alkenyl, and C 2-30 alkynyl groups may optionally be replaced by 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more halogens, C 1-6 alkyl, or C 1-6 haloalkyl, and which is optionally deuterated until completely deuterated.
A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R is-C (O) -C 0-10 alkylene-L-R 1, preferably-C (O) -L-R 1, preferably-C (O) -C 2-8 alkylene-L-R 1, more preferably-C (O) -C 3-7 alkylene-L-R 1, more preferably-C (O) -C 4-6 alkylene-L-R 1, more preferably-C (O) -C 1-3 alkylene-L-R 1.
A compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)- or-NHC (O) -CH 2-O-CH2CH(NH2) CH (OH) -, preferably a bond 、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or-NHC (O) -CH 2-O-CH(CH(NH2)CH2 OH) -, preferably a bond 、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or-NHC (O) -CH 2-O-CH(CH(NH2)CH2 OH) -, more preferably a bond, -NHC (O) -, -S-or-NHC (O) O-, more preferably-NHC (O) -.
A compound of formula (I) according to claim 1 OR 2, OR a pharmaceutically acceptable salt, tautomer OR stereoisomer thereof, wherein L is -NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-, preferably-NHC (O) -CH (OR 1)CH2O-、-NHC(O)-CH(R1) -OR-CH (OR 1)CH2 O-, more preferably-NHC (O) -CH (OR 1)CH2 O-.
A compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1 is independently C 1-30 alkyl or C 2-30 alkenyl, wherein non-adjacent 1,2, 3, 4, 5, 6, 7 or 8 carbon atoms in said groups may be replaced by heteroatoms selected from O, S and N, or the-CH 2CH2 -group may be replaced by-NHC (O) -or-C (O) NH-, or substituents on one or more carbon atoms may be attached to form a saturated or unsaturated ring, preferably R 1 is independently C 5-25 alkyl, C 10-25 alkenyl containing 1,2, 3, 4, 5 or 6 double bonds, C 5-25 alkyl wherein 1,2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and/or 1,2 or 3-CH 2CH2 -groups are replaced by-C (O) NH-, or a substituent on one or more carbon atoms may be attached to form a ring, preferably R 5-25 is selected from the group consisting of:
c 6 alkyl, C 8 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, C 15 alkyl, C 16 alkyl, C 17 alkyl, C 21 alkyl,
A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L 1 and L 2 are H.
A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein one of L 1 and L 2 is a reactive phosphorus group, preferably phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate or phosphate mimic, such as a natural phosphate, phosphorothioate, phosphorodithioate, borane phosphate, borane phosphorothioate, phosphonate, halogen substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, phosphorothioate diester, phosphorodithioate or triphosphate, preferably-P (OCH 2CH2CN)(N(iPr)2).
Technical solution a compound of formula (I) according to any one of claims 1-5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L 1 and L 2 are selected from protecting groups, preferably hydroxy protecting groups, such as Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxybenzyl (bm), methoxy (m, m-methoxy (m), 2-methoxy (m ' -methoxy), 2-methoxy (m-methoxy) methyl (m, m ' -methoxy) 2, m ' -methoxy (m, m ' -methoxy) 2, m ' -methoxy (m) 2, m ' -methoxy (m) 2m (m) methoxy (m) 2m ' -methoxy (m) methyl (m) 2, preferably-C (O) CH 2CH2 C (O) OH or 4,4' -dimethoxytrityl, more preferably-C (O) CH 2CH2 C (O) OH.
A compound of formula (I) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, selected from the following general formulae:
Wherein each group is as defined in claims 1-8.
A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound is selected from the group consisting of:
An oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein, the
Represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
L 2 represents H or a solid support, or represents a position linked to an adjacent nucleotide;
R s is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until fully deuterated;
m=0, 1, 2, 3,4, 5 or 6;
r is a hydrophobic group;
preferably, the method comprises the steps of,
Represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
L 2 represents H or a solid support, or represents a position linked to an adjacent nucleotide;
R s, m and R are as defined in any of claims 1 to 5.
The oligonucleotide of claim 12, claim 11, wherein the compound of formula (I') is selected from the group consisting of compounds of the following general formulas:
Wherein, the
Represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
L 2 represents H or a solid support, or represents a position linked to an adjacent nucleotide;
The other groups are defined in claims 1-5.
The oligonucleotide of claim 13, claim 11, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, wherein the compound is selected from the group consisting of:
Wherein the method comprises the steps of One represents H or a hydroxyl protecting group, or represents a position linked to an adjacent nucleotide, and the otherRepresents H or a solid support, or represents the position of attachment to an adjacent nucleotide.
Technical solution 14. The oligonucleotide of any one of claims 11-13, having 14 to 30 nucleotides.
Technical scheme 15 the oligonucleotide of any one of claims 11-14 comprising at the 5 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
Claim 16. The oligonucleotide of any one of claims 11-15, comprising at the 3 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
Claim 17 the oligonucleotide of any one of claims 11-16, which comprises a compound of formula (I ') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' and 3' ends, respectively.
Technical scheme 18 the oligonucleotide of any one of claims 11-17 comprising one or more compounds of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, within the oligonucleotide.
Claim 19. An oligonucleotide comprising two or more hydrophobic groups within, 5 'and/or 3' of the oligonucleotide, preferably the hydrophobic groups are as defined for the R groups in the compound of formula (I), preferably the hydrophobic groups are attached to the oligonucleotide by a linker, e.g.a biodegradable linker.
Technical solution 20. Double stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises one or more compounds of formula (I'), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:
Wherein, the
Represents H, or represents the position of attachment to an adjacent nucleotide;
L 2 represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
R s is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until fully deuterated;
m=0, 1, 2, 3,4, 5 or 6;
r is a hydrophobic group;
preferably, the method comprises the steps of,
Represents H, or represents the position of attachment to an adjacent nucleotide;
L 2 represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
R s, m and R are as defined in any of claims 1 to 5.
The double stranded RNA of claim 20, wherein the compound of formula (I') is selected from the group consisting of compounds of the following general formulae, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
Wherein, the
Represents H, or represents the position of attachment to an adjacent nucleotide;
L 2 represents H or a hydroxyl protecting group, or represents a position of attachment to an adjacent nucleotide;
R s, m and R are as defined in claims 1 to 5.
The double stranded RNA of claim 20, wherein said compound of formula (I') is selected from the group consisting of, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound is selected from the group consisting of:
Wherein the method comprises the steps of One of which represents H, or represents the position of attachment to an adjacent nucleotide, and the otherRepresents H or a hydroxyl protecting group, or a position linked to an adjacent nucleotide.
The double stranded RNA of any one of claims 20-22, wherein the sense strand comprises at the 5 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The double stranded RNA of any one of claims 20-23, wherein the sense strand comprises at the 3 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The double stranded RNA of any one of claims 20-24, wherein the sense strand comprises one compound of formula (I ') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' and 3' ends, respectively.
The double stranded RNA of any one of claims 20-25, wherein the sense strand comprises one or more compounds of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within an oligonucleotide.
The double stranded RNA of any one of claims 20-26, wherein the antisense strand comprises at the 5 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The double stranded RNA of any one of claims 20-27, wherein the antisense strand comprises at the 3 'end a compound of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.
The double stranded RNA of any one of claims 20-28, wherein the antisense strand comprises one compound of formula (I ') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, at the 5' and 3' ends, respectively.
The double stranded RNA of any one of claims 20-29, wherein the antisense strand comprises one or more compounds of formula (I') of any one of claims 11-13, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, within an oligonucleotide.
The double stranded RNA of any one of claims 20-30, wherein two or more compounds of formula (I') on the sense strand and/or the antisense strand, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, are separated by at least 5-30 nucleotides.
The double stranded RNA of any one of claims 20-31, wherein the double stranded RNA comprises two compounds of formula (I '), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at any two of the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand, preferably at the 5' end of the sense strand and the 3' end of the sense strand.
The double stranded RNA of any one of claims 20-32, wherein the double stranded RNA comprises three compounds of formula (I '), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at any three of the 5' end of the sense strand, the 3 'end of the sense strand, the 5' end of the antisense strand and the 3 'end of the antisense strand, preferably at the 5' end of the sense strand, the 3 'end of the sense strand and the 3' end of the antisense strand.
The double stranded RNA of any one of claims 20-33, wherein the double stranded RNA comprises four compounds of formula (I '), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand.
The double stranded RNA of any one of claims 20-33, further comprising a terminal phosphate protecting group or prodrug protecting group, preferably a vinyl phosphate group or a prodrug protecting group of formula (X), coupled to the 5' end of the antisense strand:
Wherein, the
X 1 is selected from OH or
R a is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl, optionally deuterated until fully deuterated;
R b and R c are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, said R b and R c optionally being substituted with D, C 6-10 aryl or 5-10 membered heteroaryl, until fully deuterated;
X 2 is a chemical bond to the first nucleotide at the 5' end of the antisense strand, preferably through a hydroxyl linkage;
X 3 is independently selected from O or S;
T is selected from
Each R T1 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2- 6 alkynyl, or GalNAc-containing chain, optionally deuterated until fully deuterated;
Each R T2 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl, optionally deuterated, up to fully deuterated;
Each R T3 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl, optionally deuterated, up to fully deuterated;
each R T4 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl, optionally deuterated, up to fully deuterated;
m is 0, 1, 2, 3,4 or 5;
n is 0, 1, 2, 3,4 or 5;
p is 0, 1, 2, 3,4 or 5;
X is selected from the group consisting of a bond, -O-, -S-, -C (O) O-, -OC (O) NR X1-、-NRX1C(O)O-、-NRX1 C (O) -, or-C (O) NR X1 -;
R X1 is selected from H, C 1-6 alkyl or C 1-6 haloalkyl, optionally deuterated until completely deuterated;
L is-Ar- (CH 2)1-6 -O-, wherein each CH 2 is optionally substituted by R# selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until completely deuterated;
ar in L is connected with X, and an oxygen atom is connected with a phosphorus atom;
Ar is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-14 membered heteroaryl, said C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl optionally substituted with 1,2,3, 4, or 5R;
R is selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, or C 2-6 alkynyl, optionally deuterated, up to complete deuteration;
Wherein P 1 is selected from protecting groups, preferably hydroxy protecting groups, such as Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, P-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), P-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-P-methoxytrityl (MEM), methoxymethyl (MOM), phenoxymethyl (BOM), 2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), P-methoxybenzyloxymethyl (PMM), 4' -dimethoxymethyl (Cbz), t-butoxycarbonyl (DMO) or (CH) of (OH) 32-C (OCH) or (DMO) 32, preferably-P (OCH 2CH2CN)(N(iPr)2) or-C (O) CH 2CH2 C (O) OH.
Technical solution 36. The double stranded RNA of any one of claims 20-35, selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA), is preferably used to inhibit a gene expressed in the eye.
The double stranded RNA of any one of claims 20-36, wherein the sense strand comprises one of the following nucleotide sequences:
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL6、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL8、
LL6s-CmsAmUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-dTdT-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs IbsIbsIbs-LL7、
LL6s-IbsIbsIbs-CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs-LL7,
Wherein LL6, LL7 and LL8 are each selected from the following compounds:
And wherein Represents the 3' carbon or corresponding position attached to the last nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group,Represents the 5' carbon or corresponding position attached to the next nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group, when the corresponding structure is located at a terminal position of the nucleic acid strand,Correspondingly, to a hydrogen, terminal modification, terminal protecting group or other terminal structure useful for nucleic acid strands.
The double-stranded RNA of claim 37, wherein the antisense strand comprises the nucleotide sequence of:
VPUmsUfsUmAmGmAfGmUfGfAmGmGmAmUfUmAfAmAmAmUmGmsAmsGm。
Technical scheme 39. Double stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence substantially complementary to the sense strand and target mRNA, wherein the sense strand and/or antisense strand comprises two or more hydrophobic groups at the internal, 5 'and/or 3' ends, preferably the hydrophobic groups are as defined for the R groups in the compound of formula (I), preferably the hydrophobic groups are attached to the sense strand and/or antisense strand by a linker, e.g.a biodegradable linker.
The vector of claim 40, which comprises a nucleotide sequence encoding the double stranded RNA of any one of the preceding claims 20-39.
Claim 41 a cell comprising the double stranded RNA of any one of claims 20-39 or the vector of claim 40.
A pharmaceutical composition comprising the double stranded RNA of any one of claims 20-39, the vector of claim 40, or the cell of claim 41, and optionally a pharmaceutically acceptable carrier or excipient.
A kit comprising the double stranded RNA of any one of claims 20-39, the vector of claim 40, or the cell of claim 41.
Technical solution 44 double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence substantially complementary to the sense strand and a target mRNA, wherein the 3' end of the sense strand is a compound of the formula:
And wherein Represents the 3' carbon or corresponding position attached to the last nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group,Represents the 5' carbon or corresponding position attached to the next nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group, when the corresponding structure is located at a terminal position of the nucleic acid strand,Correspondingly, to a hydrogen, terminal modification, terminal protecting group or other terminal structure useful for nucleic acid strands.
List of specific Compounds
The numbering and structure of the compounds of the invention in the oligonucleotides is as follows, wherein the sequence from the compounds is 5' - >3To the point ofAnd (5) connection.
Specifically, according to the order of 5'- >3', such as the corresponding structure being located at the intermediate position of the nucleic acid strand,Represents the 3' carbon or corresponding position attached to the last nucleotide or nucleotide analogue by a phosphate group, phosphorothioate group or other linking group,Represents the attachment to the 5' carbon or corresponding position of the next nucleotide or nucleotide analogue via a phosphate group, phosphorothioate group or other linking group, e.g.the corresponding structure is located at a terminal position of the nucleic acid strand,Correspondingly, to a hydrogen, terminal modification, terminal protecting group or other terminal structure useful for nucleic acid strands.
Synthetic examples
The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Abbreviations (abbreviations)
Example 1 preparation of intermediate Compounds example
Example 1.1 preparation of DL0066
Compound 1 was dissolved in DCM (20.0 mL) at room temperature, DCI (0.06 g,0.502 mmol), 5A molecular sieve and compound 2 (0.33 g,1.105 mmol) were added to the solution, nitrogen was displaced 3 times, and the reaction mixture was stirred at 25℃for 1 hour. The reaction solution was cooled to 0 ℃ in an ice salt bath, 6mL of saturated NaHCO 3 solution and 6mL of saturated brine solution were added to the reaction solution, then 50mL of DCM was added, the organic phase was further washed with a 1:1 mixed solution (20 mL x 3) of saturated NaHCO 3 and saturated brine, then the organic phase was dried over Na 2SO4 and spun-dried, and the obtained crude product was purified by column chromatography (petroleum ether/ethyl acetate=10/3 to 10/5) to give DL0066 (620 mg, yield 53.89% purity as a white solid) 96.00％).1H NMR(400MHz,DMSO-d6)δ7.25-7.36(m,4H),7.13-7.25(m,5H),7.01-7.03(m,1H),6.78-6.93(m,4H),5.30-5.32(m,1H),4.47-4.72(m,1H),4.24-4.34(m,1H),4.12-4.15(m,1H),3.69-3.71(m,8H),3.48-3.59(m,3H),3.35-3.47(m,1H),3.13-3.25(m,1H),2.86-3.06(m,3H),2.70-2.78(m,2H),2.09-2.31(m,5H),1.86-1.96(m,2H),1.70-1.85(m,3H),1.47-1.51(m,6H),1.22-1.42(m,11H),1.06-1.16(m,20H),0.93-0.95(m,4H),0.87-0.89(m,4H),0.82-0.85(m,7H),0.65(s,3H)
EXAMPLE 1.2 preparation of DL0067
Compound 1 (250 mg,0.380 mmol) was dissolved in DCM (4.00 mL), DIEA (0.377 mL,2.28 mmol), DMAP (11.6 mg,0.095 mmol) and Compound 2 (228 mg,2.28 mmol) were added. The reaction solution was stirred at 25 ℃ for 18 hours. LCMS detected the product with this mass response. TLC (PE/EA=3/1) was found to have a new spot, and the reaction mixture was concentrated directly and purified by a basic silica gel normal phase column (SiO 2, PE/EA=1/0 to 3/1) to give DL0067 (87.0 mg, yield 28.65%, purity as a yellow oil 94.84％).LCMS(ESI):m/z＝756.4[M-H]-;1H NMR(400MHz,CD3OD)δ7.36-7.38(m,2H),7.24-7.30(m,6H),7.16-7.22(m,1H),6.81-6.87(m,4H),5.38-5.49(m,1H),4.24-4.29(m,1H),3.81-3.89(m,1H),3.77(d,J＝2.0Hz,6H),3.67-3.70(m,1H),3.54-3.58(m,1H),3.08-3.25(m,1H),2.57-2.59(m,4H),2.30-2.39(m,2H),2.21-2.28(m,1H),2.07-2.16(m,1H),1.46-1.62(m,2H),1.22-1.35(m,24H),0.90(t,J＝6.4Hz,3H).
EXAMPLE 1.3 preparation of DL0082
1. Preparation of intermediate 3
Compound 1 (2.00 g,13.6 mmol) was dissolved in DCM (50.0 mL) at 25℃and DIEA (6.74 mL,40.8 mmol), HATU (7.75 g,20.4 mmol) and Compound 2 (3.48 g,13.6 mmol) were added sequentially and the reaction stirred at 25℃for 12h. LCMS showed product formation. TLC (DCM/meoh=10/1, pma) showed that compound 2 was not completely reacted and several new spots were formed. To this was added ethyl acetate (200 mL), the mixed liquid was washed three times with brine (15.0 mL. Times.3), and the organic phase was concentrated under reduced pressure to give the crude compound. Purification of the crude product by column chromatography TLC (DCM/meoh=10/1 to 5/1, pma) gave compound 3 as a yellow oil (1.15 g, yield 21.95％).1H NMR(400MHz,CD3OD)δ3.79-3.91(m,2H),3.75-3.78(m,1H),3.57-3.69(m,4H),3.47-3.56(m,2H),3.27-3.34(m,1H),2.29-2.50(m,2H),1.53-1.66(m,2H),1.27-1.37(m,24H),0.84-0.95(m,3H)
2. Preparation of intermediate 4
Compound 3 (1.15 g,2.98 mmol) was dissolved in pyridine (20.0 mL) at 25℃and nitrogen was replaced three times. A solution of DMTrCl (1.11 g,3.28 mmol) in DCM (10.0 mL) was then added thereto. The mixed liquid was stirred at 25 ℃ for 3 hours. LCMS showed product formation, starting material was not consumed completely. TLC (DCM/meoh=10/1, pma) showed complete consumption of starting material and formation of new spots. DCM (200 mL) was added thereto, and the mixture was washed three times (20.0 mL. Times.3) with saturated sodium bicarbonate solution (20.0 mL. Times.3) and saturated brine. The organic phase was concentrated under reduced pressure to give the crude compound. The crude product was purified by column chromatography TLC (PE/ea=3/1 to 1/1, pma) to give compound 4 (620 mg, yield 30.22%) as a yellow oil.
Preparation of DL0082
Compound 4 (420 mg,0.611 mmol) was dissolved in DCM (3.00 mL) at 25℃and DIEA (0.605 mL,3.66 mmol), DMAP (18.6 mg,0.153 mmol) and compound 5 (367 mg,3.66 mmol) were added to the above mixture. The reaction liquid was stirred at 25 ℃ for 3 hours. LCMS showed product formation, starting material was not consumed completely. DCM (50.0 mL) was added thereto and washed three times with saturated brine (50.0 mL. Times.3). The organic phase was concentrated under reduced pressure to give the crude compound. The crude product was purified by prep-HPLC (column: waters Xbridge BEH C18:150:25:5 um; mobile phase: TEAA-ACN; gradient: 55% -95%/16min; flow: 15 ml/min) to give compound DL0082 as a yellow oil (170 mg, yield 35.34%, purity 97.26%).
1H NMR(400MHz,CD3OD)δ7.43(d,J＝8.0Hz,2H),7.18-7.34(m,7H),6.82-6.89(m,4H),4.16-4.25(m,1H),3.92-4.13(m,2H),3.85(d,J＝5.20Hz,1H),3.78(s,6H),3.66-3.76(m,1H),3.53-3.64(m,2H),3.33-3.42(m,1H),3.28(s,1H),3.12-3.23(m,3H),2.48-2.65(m,4H),2.19-2.43(m,2H),1.52(s,2H),1.25-1.35(m,27H),0.86-0.93(m,3H)
EXAMPLE 1.4 preparation of DL0084
1. Preparation of Compound 3
Compound 1 (207 mg,0.37 mmol) was dissolved in DCM (5.00 mL), HATU (177 mg,0.46 mmol) and DIEA (0.15 mL,0.93 mmol) were added and the reaction stirred for half an hour at 25℃after which compound 2 (140 mg,0.31 mmol) was added and the reaction was reacted for 16 hours at 25 ℃. TLC (DCM/meoh=10/1) showed a new spot formation. To the reaction solution was added 10.0mL DCM,10.0mL H 2 O, and the solution was separated, and sodium hydrogencarbonate solution (10.0 ml x 1) was added to the organic phase, which was washed with saturated brine (10.0 ml x 1), dried over anhydrous sodium sulfate, filtered, and spun-dried to give crude product compound 3 (200 mg,0.20mmol, 65.10%) as a white solid.
1H NMR(400MHz,CD3OD)δ7.45(d,J＝8.0Hz,2H),7.16-7.38(m,7H),6.82-6.90(m,4H),4.39(d,J＝8.0Hz,1H),3.93-4.17(m,2H),3.80(s,7H),3.39-3.71(m,8H),3.10-3.31(m,2H),2.84-3.03(m,2H),1.46-1.70(m,4H),1.16-1.45(m,51H),0.91(t,J＝8.0Hz,6H)
Preparation of DL0084
Compound 3 was dissolved in DCM (7.0 mL), DIEA (0.20 mL,1.21 mmol), DMAP (9.9 mg,0.08 mmol) and compound 4 (121 mg,1.21 mmol) were added and the reaction was allowed to react at 25℃for 3 hours. LCMS showed MS with the desired product. The reaction mixture was dried by rotary evaporation, and the crude product was purified by prep-HPLC (column: 01-Waters Xbridge BEH C, 19X 150mm,5 μm; conditions: TEAA-ACN; begin B65-95; gradient time: 15 minutes; 100% B hold time: 2 minutes; flow rate: 15 ml/min) to give the product DL0084 as a yellow oil (80.0 mg, yield: 36.27%).
1H NMR(400MHz,CDCl3)δ7.35(d,J＝4.0Hz,2H),7.26-7.30(m,6H),7.09-7.25(m,1H),6.75(d,J＝8.0Hz,4H),4.20-4.31(m,1H),3.75-4.16(m,5H),3.71(s,6H),3.32-3.56(m,6H),3.05-3.29(m,4H),2.99(q,J＝8.0Hz,2H),2.50-2.60(m,4H),1.47(d,J＝8.0Hz,4H),1.18(s,52H),0.77-0.84(m,6H)
EXAMPLE 1.5 preparation of DL0127
1. Preparation of Compound 3
Compound 1 (1.00 g,3.90 mmol) was dissolved in DCM (20 mL), HATU (1.48 g,3.90 mmol) and DIEA (3.86 mL,23.3 mmol) were added and the reaction stirred for 30 min at 25℃after which compound 2 (0.68 g,4.68 mmol) was added and the reaction stirred for 16h at 25 ℃. LCMS showed ms value generation of product. TLC (PE/ea=1/1) showed new spot formation. To the reaction was added 20mL of water, and the reaction was extracted with DCM (15.0 mL. Times.2). The combined organic phases were washed with ammonium chloride solution (20.0 ml x 1), sodium bicarbonate solution (20.0 ml x 1), saturated brine (20.0 ml x 1), dried over anhydrous sodium sulfate, filtered and spun-dried to give the crude product. The crude product was purified by column chromatography (PE: ea=1/0-1/1) to give compound 3 (2.24 g,5.839mmol, 149.73%) as a pale yellow solid.
1H NMR(400MHz,CD3OD)δ3.65(s,3H),3.15-3.19(m,2H),2.32(t,J＝7.6Hz,2H),2.11-2.19(m,1H),1.59-1.68(m,2H),1.48-1.58(m,4H),1.37-1.40(m,2H),1.22-1.32(m,22H),0.89(t,J＝6.8Hz,6H)
2. Preparation of Compound 4
Compound 3 was dissolved in H 2 O (1.00 mL) and THF (4.00 mL), then LiOH (218 mg,5.21 mmol) was added and the reaction was allowed to react at 25℃for 16 hours. LCMS showed the disappearance of starting material and TLC (PE/ea=1/1) showed the formation of new spots. To the reaction was added 10.0mL of water, and the reaction was extracted with EtOAc (10.0 mL. Times.2). The aqueous phase was adjusted to pH6 with 3M HCl, the aqueous phase was extracted with EtOAc (10.0 mL x 2), and the combined organic phases were washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered and spun-dried to give compound 4 (220 mg,0.595mmol, 45.67%) as a white solid.
1H NMR(400MHz,CD3OD)δ3.18(t,J＝8.0Hz,2H),2.29(t,J＝8.0Hz,2H),2.15(m,1H),1.60-1.67(m,2H),1.48-1.59(m,4H),1.37-1.62(m,2H),1.17-1.35(m,22H),0.84-0.93(m,6H)
3. Preparation of Compound 6
Compound 4 (213 mg,0.57 mmol) was dissolved in DCM (5.00 mL), HATU (219 mg,0.57 mmol) and DIEA (0.22 mL,1.335 mmol) were added and the reaction stirred for 30 min at 25℃after which compound 5 (200 mg,0.44 mmol) was added and the reaction stirred for 16h at 25 ℃. TLC (DCM/meoh=10/1) showed a new spot formation. LCMS showed the disappearance of starting material, and the reaction was diluted with 10.0mL of DCM, and sodium bicarbonate solution (10.0 mL x 2) was added to the reaction, washed with saturated brine (10.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and spun-dried to give the crude product. The crude product was purified by column chromatography (DCM/meoh=1/0-10/1) to give the product compound as a yellow oil 6(300mg,0.374mmol,84.15％).1H NMR(400MHz,CD3OD)δ7.41-7.47(m,2H),7.20-7.36(m,7H),6.84-6.92(m,4H),3.85-4.06(m,2H),3.80(d,J＝4.0Hz,6H),3.72-3.77(m,1H),3.42-3.71(m,5H),3.15-3.27(m,4H),2.13-2.43(m,3H),1.46-1.67(m,6H),1.20-1.38(m,23H),0.84-0.93(m,6H)
Preparation of DL0127
Compound 6 (300 mg,0.374 mmol) was dissolved in DCM (5.0 mL), DIEA (0.371 mL,2.247 mmol), DMAP (18.30 mg,0.150 mmol) and compound 7 (224.84 mg,2.247 mmol) were added and the reaction was allowed to react at 25℃for 16 hours. LCMS showed MS with the desired product. The reaction was spin-dried and the crude product purified by prep-HPLC (column: waters Xbridge BEH C18100 x 25mm x 5um; conditions: TEAA-ACN; begin B55-95; gradient time: 15 min; 100% B hold time: 6 min; flow rate: 15 ml/min) to give the product DL0127 as a yellow oil (120 mg,0.133mmol, 35.56%).
1H NMR(400MHz,CD3OD)δ7.43(d,J＝8.0Hz,2H),7.18-7.34(m,7H),6.86(dd,J＝8.0,8.0Hz,4H),4.16-4.25(m,1H),3.92-4.14(m,2H),3.84-3.90(m,1H),3.78-3.82(m,6H),3.52-3.76(m,3H),3.34-3.46(m,1H),3.17-3.22(m,4H),2.49-2.62(m,4H),2.22-2.41(m,2H),2.10-2.17(m,1H),1.43-1.62(m,6H),1.25-1.35(m,24H),0.88(t,J＝8.0Hz,6H)
EXAMPLE 1.6 preparation of DL0133
Preparation of Compound 3
Compound 2 (1.66 g,8.26 mmol) was dissolved in DCM (50.0 mL) at 25℃and HATU (3.93 g,10.3 mmol) and DIEA (3.42 mL,20.7 mmol) were added sequentially, the reaction stirred at 25℃for 0.5 h, then Compound 1 (1.00 g,6.89 mmol) was added and the reaction stirred at 25℃for 14 h. LCMS showed mass values for the product. Thin layer chromatography (PE/ea=3/1) showed complete consumption of the reactants and a new point of formation. The reaction was diluted with DCM (50.0 mL), washed with saturated aqueous citric acid (10.0 mL x 3), and the organic phase was washed with saturated aqueous NaHCO 3 (20.0 mL x 3) followed by saturated aqueous saline (20.0 mL x 3), then the organic phase was dried over Na 2SO4 and the organic phase was dried by spinning to give the crude product. The crude product was purified by column chromatography (PE/ea=1/0-3/1) to give the compound as a white solid 3(1.50g).1H NMR(400MHz,CD3OD)δ3.65(s,3H),3.11-3.20(m,2H),2.33(t,J＝7.2Hz,2H),2.16(t,J＝7.6Hz,2H),1.57-1.68(m,4H),1.51(q,J＝7.2Hz,2H),1.25-1.40(m,19H),0.86-0.93(m,3H).
2 Preparation of Compound 4
Compound 3 (1.00 g,3.05 mmol) was dissolved in a mixed solvent of THF (5.00 mL) and H 2 O (2.50 mL) at 25℃and LiOH (25.9 mg,1.08 mmol) was added thereto, and the reaction was stirred at 25℃for 18 hours. Thin layer chromatography (PE/ea=3/1) showed complete consumption of starting material and a new point of formation. To the reaction mixture was added a 1M HCl (1.00 mL) solution to adjust acidity, followed by addition of methylene chloride (20.0 mL) and separation. The organic phase was dried over Na 2SO4 and the organic phase was dried by spinning to give compound 4 (900 mg, yield) as a white solid 94.04％).1H NMR(400MHz,CDCl3)δ3.26(q,J＝6.8Hz,2H),2.37(t,J＝7.2Hz,2H),2.13-2.20(m,2H),1.80-1.90(m,1H),1.58-1.71(m,4H),1.53(q,J＝7.2Hz,2H),1.36-1.43(m,2H),1.21-1.32(m,16H),0.84-0.92(m,3H).
Preparation of Compound 6
Compound 4 (167 mg, 0.284 mmol) was dissolved in DCM (10.0 mL) at 25℃and HATU (169 mg,0.445 mmol) and DIEA (0.074 mL,0.445 mmol) were added sequentially, the reaction stirred at 25℃for 0.5 h, then compound 5 (200 mg,0.445 mmol) was added and the reaction stirred at 25℃for 16 h. LCMS showed mass values for the product. Thin layer chromatography (DCM/meoh=10/1) showed a new point. The reaction was diluted with DCM (20.0 mL), washed with saturated aqueous citric acid (5.00 mL. Times.3), saturated aqueous NaHCO 3 (5.00 mL. Times.3) and saturated aqueous saline (5.00 mL. Times.3), then the organic phase was dried over anhydrous Na 2SO4 and the organic phase was dried by spinning to give the crude product. The crude product was purified by column chromatography (DCM/meoh=1/0 to 10/1) to give crude compound 6 (460 mg) .1H NMR(400MHz,CD3OD)δ7.42(dd,J＝7.6,2.0Hz,2H),7.17-7.33(m,6H),6.80-6.92(m,4H),3.89-3.98(m,1H),3.78(s,6H),3.36-3.75(m,8H),3.09-3.19(m,3H),2.11-2.40(m,4H),1.43-1.63(m,6H),1.28(s,18H),0.85-0.94(m,3H)
Preparation of 4 DL0133
Compound 6 (460 mg, 0.611 mmol) was dissolved in DCM (10.0 mL) at 25℃and DMAP (75.4 mg, 0.611 mmol), DIEA (0.102 mL, 0.611 mmol) and compound 7 (375 mg,3.71 mmol) were added in sequence and the reaction stirred at 25℃for 2h. LCMS showed mass values for the product. The reaction solution is spin-dried to obtain a crude product. Separating the crude product by prep-HPLC (chromatographic column: waters Xbridge BEH C18.250.50 mm.10 um; mobile phase: TEAA-ACN; B%:55% -81%,10min; flow rate: 15 ml/min) to obtain colorless oily liquid DL0133 (180 mg, yield 34.52%, purity) 98.01％).1H NMR(400MHz,CD3OD)δ7.43(d,J＝7.6Hz,2H),7.17-7.34(m,7H),6.86(dd,J＝7.8,5.9Hz,4H),4.18-4.26(m,1H),3.93-4.15(m,2H),3.87(d,J＝2.8Hz,1H),3.78(d,J＝1.6Hz,6H),3.33-3.69(m,4H),3.10-3.23(m,5H),2.51-2.62(m,4H),2.23-2.41(m,2H),2.15(q,J＝7.6Hz,2H),1.45-1.62(m,6H),1.21-1.39(m,22H),0.89(t,J＝6.8Hz,3H).
EXAMPLE 1.7 preparation of DL0134
Preparation of Compound 3
Compound 2 (1.537 mL,5.739 mmol) was dissolved in DCM (20.0 mL) at 25℃and HATU (3.27 g, 8.319 mmol) and DIEA (2.846 mL,17.218 mmol) were added sequentially and the combined liquid was stirred at 25℃for 0.5 h before compound 1 (1 g,6.887 mmol) was added to the reaction. The reaction liquid was stirred at 25 ℃ for 12 hours. Thin layer chromatography (PE/ea=2/1) showed complete reaction of starting material with new point formation. The reaction solution was diluted with methylene chloride (30 mL), and washed successively with a saturated citric acid solution (15 mLx 3), a saturated sodium bicarbonate solution (15 mLx 3) and a saturated sodium chloride solution (15 mLx 3). The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which is then purified by column chromatography (PE/ea=50/50 to 40/60). Yield 3 as white solid (630 mg, yield) 30.87％).1H NMR(400MHz,METHANOL-d4)δ3.65(s,3H),3.16(t,J＝7.03Hz,2H),2.33(t,J＝7.40Hz,2H),2.16(t,J＝7.40Hz,2H),1.45-1.66(m,6H),1.25-1.36(m,22H),0.85-0.95(m,3H)
2 Preparation of Compound 4
Compound 3 (630 mg,1.772 mmol) was dissolved in THF (10 mL) at 25℃and H 2 O (5 mL) and KOH (149.13 mg, 2.618 mmol) were added and reacted at 25℃for 12 hours. T1:thin layer chromatography (PE/ea=2/1) and t2:thin layer chromatography (DCM: meoh=10:1) showed the disappearance of starting material with new spot. The reaction solution was diluted with water (15 mL), pH was adjusted to 3 to 4 with 1moL/L hydrochloric acid, extraction was performed 3 times with ethyl acetate (15 mL X3), and the reaction solution was concentrated under reduced pressure to give a white solid product 4 (610 mg, yield) 100.80％).1H NMR(400MHz,METHANOL-d4)δ3.11-3.21(m,2H),2.29(t,J＝7.40Hz,2H),2.16(t,J＝7.40Hz,2H),1.43-1.68(m,6H),1.22-1.43(m,22H),0.83-0.96(m,3H)
Preparation of Compound 6
Compound 4 (227.92 mg,0.667 mmol) was dissolved in DCM (10 mL) at 25℃and HATU (317.18 mg,0.834 mmol) and DIEA (0.365 mL,2.224 mmol) were added and after 0.5h the reaction was continued for 12 h. Thin layer chromatography (DCM/meoh=10/1) showed complete reaction of starting material with a new point. The reaction mixture was diluted with dichloromethane (20 mL), washed three times with saturated brine (10 mL. Times.3), and the organic phase was concentrated under reduced pressure to give the crude compound. The crude product was purified by column chromatography (DCM/meoh=95/5-92/8). The product was obtained as a white solid 6(350mg,0.453mmol,81.41％).1H NMR(400MHz,METHANOL-d4)δ7.42(dd,J＝2.26,7.78Hz,2H),7.18-7.35(m,7H),6.78-6.94(m,4H),3.85-4.04(m,2H),3.78(d,J＝2.51Hz,6H),3.66-3.75(m,1H),3.44-3.63(m,4H),3.33-3.42(m,1H),3.06-3.22(m,4H),2.26-2.42(m,2H),2.15(q,J＝7.19Hz,2H),1.44-1.62(m,6H),1.28(s,22H),0.87-0.92(m,3H)
Preparation of DL0134
Compound 6 (350 mg, 0.4573 mmol)) was dissolved in DCM (10 mL) at 25℃and DMAP (13.83 mg,0.113 mmol), DIEA (0.449 mL,2.716 mmol) and compound 7 (271.83 mg,2.716 mmol) were added. The reaction solution was stirred at 25 ℃ for 12 hours. LCMS showed complete reaction of starting material with product formation. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Prep-HPLC (column: waters Xbridge BEH C100 x 25mm x 5um; mobile phase: TEAA-ACN; gradient: 55% -95%/15min; flow rate: 15 ml/min). Yield DL0134 (216 mg, 54.64% yield, purity) was obtained as a colourless oil 95.94％),1H NMR(400MHz,METHANOL-d4)δ7.41-7.47(m,2H),7.19-7.34(m,7H),6.81-6.91(m,4H),4.16-4.26(m,1H),3.84-4.14(m,3H),3.45-3.81(m,9H),3.35-3.43(m,1H),3.25-3.28(m,1H),3.11-3.23(m,8H),2.55-2.61(m,2H),2.48-2.54(m,2H),2.20-2.39(m,2H),2.11-2.18(m,2H),1.43-1.64(m,6H),1.21-1.37(m,30H),0.87-0.92(m,3H)
EXAMPLE 1.8 preparation of DL0135
Preparation of Compound 3
Compound 1 (1.00 g,3.90 mmol) was dissolved in DCM (20.0 mL) and HATU (1.78 g,4.68 mmol) and DIEA (3.87 mL,23.4 mmol) were added and the reaction was allowed to react at 25℃for 0.5 h. Compound 2 (0.92 g,5.07 mmol) was then added and the reaction was allowed to react at 25℃for 16.5 hours. Thin layer chromatography (PE/etoac=2/1) showed new spots to be generated. To the reaction was added DCM (25.0 mL) and water (20.0 mL), the reaction was partitioned, and the aqueous phase was extracted with DCM (15.0 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and spun-dried to give the crude product. The crude product was purified by column chromatography (PE/etoac=1/0 to 1/1) to give compound 3 (1.20 g, yield) as a white solid product 80.21％).1H NMR(400MHz,CD3OD)δ3.67(s,3H),3.13-3.22(m,2H),2.35(t,J＝7.6Hz,2H),2.18(t,J＝7.6Hz,2H),1.47-1.71(m,6H),1.24-1.43(m,28H),0.87-0.97(m,3H)
2 Preparation of Compound 4
Compound 3 (740 mg,1.93 mmol) was dissolved in THF (2.00 mL) and H 2 O (6.00 mL), liOH (324 mg,7.72 mmol) was added and the reaction was allowed to react at 20℃for 16H. Thin layer chromatography (PE/ea=3/1, pma) showed complete consumption of starting material with new point formation. The reaction liquid was adjusted to pH 5 with 1M HCl, the product was extracted with dimethyltetrahydrofuran (30.0 mL X3), and the organic phase was dried under reduced pressure to give compound 4 (650 mg, yield 91.17%) as a white solid.
Preparation of Compound 6
Compound 4 (267 mg,0.723 mmol) was dissolved in DCM (5.00 mL), HATU (275 mg,0.723 mmol) and DIEA (0.276 mL,1.67 mmol) were added, the reaction stirred at 25℃for 0.5 h, compound 5 (250 mg, 0.554 mmol) was added and the reaction stirred at 25℃for 16 h. Thin layer chromatography (DCM/meoh=10/1) showed a new spot formation. DCM (10.0 mL) was added to the reaction mixture to dilute the reaction mixture, sodium bicarbonate solution (10.0 mL X2) was added to the reaction mixture, and the mixture was washed with saturated brine (10.0 mL X2), dried over anhydrous sodium sulfate, filtered and spun-dried to give a crude product. The crude product was purified by column chromatography (DCM/meoh=1/0-10/1) to give compound 3 (390 mg, yield 87.54%) as a yellow oil.
Preparation of 4 DL0135
Compound 6 (390 mg,0.487 mmol) was dissolved in DCM (7.00 mL), DIEA (0.4813 mL,2.92 mmol), DMAP (23.8 mg,0.195 mmol) and compound 7 (292 mg,2.92 mmol) were added and the reaction was allowed to react at 25℃for 3 hours. LCMS showed MS with the desired product. The reaction mixture was spin-dried and the crude product purified by prep-HPLC (column: waters Xbridge BEH C18.25 mm 5um; conditions: TEAA-CAN; begin B55-95; gradient time: 15min; 100% B hold time: 6 min; flow rate: 15 ml/min) to give the product DL0135 (140 mg, 31.91% yield, purity) as a yellow oil 99.54％).1H NMR(400MHz,CD3OD)δ7.39-7.48(m,2H),7.17-7.36(m,7H),6.80-6.92(m,4H),4.18-4.26(m,1H),3.83-4.16(m,3H),3.78(m,6H),3.49-3.76(m,3H),3.33-3.45(m,1H),3.09-3.20(m,4H),2.50-2.64(m,4H),2.22-2.42(m,2H),2.15(q,J＝8.0Hz,2H),1.42-1.65(m,6H),1.24-1.32(m,28H),0.84-0.96(m,3H)
EXAMPLE 1.9 preparation of DL0136
Preparation of Compound 3
Compound 2 (1305.00 mg,4.587 mmol) was dissolved in DMF (50 mL) at 25℃and HATU (2616.43 mg,6.881 mmol) and DIEA (2.275 mL,13.762 mmol) were added sequentially and the mixed liquor was stirred at 25℃for 0.5 hours before compound 1 (1000 mg,5.505 mmol) was added to the reaction. The reaction liquid was stirred at 25 ℃ for 11.5 hours. Thin layer chromatography (PE/ea=2/1) showed complete reaction of starting material with new point formation. The reaction solution was diluted with ethyl acetate (50 mL), and washed successively with a saturated citric acid solution (30 mLx 3), a saturated sodium bicarbonate solution (30 mLx 3) and a saturated sodium chloride solution (30 mLx 3). The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which is then purified by column chromatography (PE/ea=70/30 to 60/40). Yield 3 as a white solid (1530 mg, yield) 81.02％).1H NMR(400MHz,METHANOL-d4)δ3.65(s,3H),3.15(br d,J＝6.38Hz,2H),2.33(br t,J＝7.25Hz,2H),2.09-2.24(m,2H),1.42-1.72(m,6H),1.29(br s,30H),0.90(br s,3H)
2 Preparation of Compound 4
Compound 3 (1.53 g, 3.719 mmol) was dissolved in THF (20 mL) at 25℃and H 2 O (10 mL) and LiOH (0.19 g,4.460 mmol) were added and the reaction stirred for 12 hours. Thin layer chromatography (PE/ea=2/1) showed the disappearance of starting material with new spot formation. The reaction solution was concentrated under reduced pressure, and dissolved in water (10 mL) and acetonitrile (5 mL) to freeze-dry. Yield 4 as white solid (1313 mg, yield) 87.54％).1H NMR(400MHz,METHANOL-d4)δ3.16(t,J＝6.94Hz,2H),2.29(t,J＝7.38Hz,2H),2.16(t,J＝7.50Hz,2H),1.45-1.63(m,6H),1.29(s,29H),0.90(br t,J＝6.75Hz,3H)
Preparation of Compound 6
Compound 4 (300 mg,0.754 mmol)) was dissolved in DCM (10 mL) at 25℃and HATU (406.40 mg,1.069 mmol) and DIEA (0.416 mL, 2.515mmol) were added and after 0.5 h reaction compound 5 (282.64 mg,0.629 mmol) was added and the reaction continued for 7.5 h. Thin layer chromatography (DCM/meoh=8/1) showed complete reaction of starting material with a new point. The reaction solution was diluted with methylene chloride (10 mL), and washed with a saturated citric acid solution (10 mLX a), a saturated sodium hydrogencarbonate solution (10 mLX a) and a saturated sodium chloride solution (10 mLX a) in this order. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (DCM/meoh=95/5 to 94/6). Yield 6 (358 mg, yield) as colorless oil 68.71％).1H NMR(400MHz,METHANOL-d4)δ7.38-7.48(m,2H),7.20-7.35(m,7H),6.79-6.91(m,4H),3.86-4.08(m,2H),3.78(d,J＝2.51Hz,6H),3.36-3.74(m,6H),3.06-3.24(m,4H),2.23-2.43(m,2H),2.15(q,J＝6.94Hz,2H),1.44-1.64(m,6H),1.28(s,30H),0.86-0.92(m,3H)
Preparation of 4 DL0136
Compound 6 (356 mg, 0.433 mmol) was dissolved in DCM (5 mL) at 25℃and DMAP (13.19 mg,0.108 mmol), DIEA (0.428 mL,2.591 mmol) and compound 7 (259.23 mg,2.591 mmol) were added. The reaction solution was stirred at 25 ℃ for 3 hours. Thin layer chromatography (DCM/meoh=10/1) showed complete reaction of starting material with a new point. The reaction solution was diluted with dichloromethane (10 mL), washed 3 times with saturated NaHCO 3 solution (10 mL x 3) and saturated NaCl solution (10 mL x 3) in this order, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM/meoh=95/5 to 90/10) to give the crude product. The crude product was purified by Prep-HPLC (column: waters Xbridge BEH C100 x 25mm x 5um; mobile phase: TEAA-ACN; gradient: 65% -95%/15min; flow rate: 15 ml/min). Yield DL0136 (163 mg, 40.63% yield, purity) was obtained as a colourless oil 97.57％),1H NMR(400MHz,METHANOL-d4)δ7.43(br d,J＝8.03Hz,2H),7.18-7.34(m,7H),6.82-6.91(m,4H),4.08-4.26(m,2H),3.84-4.04(m,3H),3.78(d,J＝2.01Hz,6H),3.54-3.65(m,2H),3.35-3.45(m,1H),3.13-3.22(m,4H),2.49-2.65(m,4H),2.23-2.44(m,2H),2.16(q,J＝7.53Hz,2H),1.45-1.62(m,6H),1.25-1.36(m,33H),0.85-0.94(m,3H)
EXAMPLE 1.10 preparation of DL0137
1. Preparation of Compound 3
Compound 2 (896 mg,2.54 mmol) was dissolved in DCM (15.0 mL) at 25℃and HATU (1205 mg,3.17 mmol), DIEA (1.05 mL,6.34 mmol) was added sequentially. After the mixed liquid was stirred at 25℃for 0.5 hours, compound 1 (950 mg,2.113 mmol) was added thereto. The mixed liquid was stirred at 25 ℃ for 3 hours. LCMS showed product formation. TLC (DCM/meoh=10/1) showed complete reaction of starting material. After methylene chloride (60.0 ml) was added to the reaction liquid, the mixture was washed three times with saturated brine (10.0 ml. Times.3), and the organic phase was dried over anhydrous sodium sulfate and then dried under reduced pressure to give a crude compound. The crude product was purified by column chromatography (PE/ea=5/1 to 1/1) to give compound 3 (1.3 g,1.66mmol, yield) as a colourless oil 78.37％).1H NMR(400MHz,CD3OD)δ7.78(d,J＝7.6Hz,2H),7.57-7.69(m,2H),7.34-7.45(m,4H),7.24-7.32(m,7H),7.18-7.21(m,1H),6.93-7.10(m,1H),6.79-6.89(m,4H),4.29-4.39(m,2H),4.17(s,1H),3.86-4.01(m,1H),3.42-3.82(m,12H),3.33-3.40(m,1H),2.99-3.26(m,5H),2.13-2.43(m,2H),1.39-1.62(m,4H)
2. Preparation of Compound 4
Compound 3 (650 mg, 0.8238 mmol) was dissolved in MeCN (6.00 mL) at 25℃and Et 2 NH (1.00 mL) was added thereto. The mixed liquid was stirred at 25 ℃ for 3 hours. LCMS and TL (PE: ea=1:1) showed complete reaction of starting material. The reaction liquid was directly concentrated under reduced pressure and dried to give compound 4 (450 mg,0.800mmol, yield 96.57%)
3. Preparation of Compound 6
Compound 5 (58.1 mg,0.171 mmol) was dissolved in DMF (5.00 mL) at 25℃and HATU (70.3 mg,0.185 mmol), DIEA (0.070 mL,0.427 mmol) was added in sequence, and after stirring for 0.5 h compound 4 (80.0 mg,0.142 mmol) was added thereto and the mixed liquor stirred at 25℃for 12h. TLC (DCM/meoh=10/1) showed a new spot formation. Ethyl acetate (60.0 mL) was added to the reaction mixture, which was then washed with a saturated sodium hydrogencarbonate solution (10.0 mL. Times.3) and a saturated brine (10.0 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate and dried under reduced pressure to give a crude compound. The crude product was purified by column chromatography TLC (DCM/meoh=10/1 to 5/1) to give compound 6 (120 mg, yield) as a yellow oil 95.34％).1H NMR(400MHz,CD3OD)δ7.39-7.46(m,2H),7.16-7.34(m,7H),6.81-6.91(m,4H),3.71-3.78(m,8H),3.43-3.70(m,5H),3.33-3.43(m,2H),3.18-3.23(m,4H),3.12-3.17(m,2H),2.11-2.42(m,4H),1.44-1.64(m,6H),1.32-1.40(m,6H),1.25-1.32(m,38H),0.85-0.93(m,3H)
Preparation of product DL0137
Compound 6 (120 mg,0.136 mmol) and compound 7 (81.4 mg, 0.803 mmol) were added to DCM (3.00 mL) at 25℃followed by DMAP (4.14 mg,0.034 mmol) and DIEA (0.134 mL,0.813 mmol). The mixed liquid was stirred at 25 ℃ for 12 hours. TLC (DCM/meoh=10/1, uv) showed complete reaction of starting material, formation of new spots. After methylene chloride (60.0 mL) was added to the reaction liquid, the mixture was washed with a saturated sodium hydrogencarbonate solution (10.0 mL. Times.3) and a saturated brine (10.0 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate and dried under reduced pressure to give a crude compound. Purifying the crude product by column chromatography (DCM/MeOH=10/1-8/1) to obtain crude colorless oily compound, and purifying the crude product by prep-HPLC (chromatographic column: waters Xbridge BEH C18.150.25 mm.5 um; mobile phase: TEAA-ACN; gradient: 65% -95%/15min; flow rate: 15 ml/min) to obtain white solid compound DL0137 (45.0 mg, yield 33.69%, purity) 95.06％).1H NMR(400MHz,CD3OD)δ7.43(d,J＝8.0Hz,2H),7.16-7.34(m,7H),6.84-6.87(m,4H),3.85-4.26(m,4H),3.67-3.78(m,7H),3.56-3.65(m,2H),3.33-3.46(m,1H),3.10-3.23(m,6H),2.48-2.63(m,4H),2.20-2.42(m,2H),2.16(q,J＝7.6Hz,2H),1.45-1.63(m,6H),1.14-1.44(m,44H),0.85-0.94(m,3H)
EXAMPLE 1.11 preparation of DL0138
Preparation of Compound 3
Compound 2 (1.47 mL,4.60 mmol) was dissolved in DCM (30.0 mL) at 25℃and HATU (2.10 g,5.52 mmol) and DIEA (2.28 mL,13.8 mmol) were added sequentially and stirred for 0.5 h, then compound 1 (1.00 g,5.52 mmol) was added and the reaction stirred for 16 h at 25 ℃. Thin layer chromatography (PE/ea=3/1) showed complete consumption of starting material and a new point of formation. Dichloromethane (30.0 mL) was added to the reaction solution to dilute, which was washed with saturated aqueous citric acid (10.0 mL x 3), saturated aqueous NaHCO 3 (10.0 mL x 3) and saturated aqueous NaCl (10.0 mL x 3), then the organic phase was dried over anhydrous Na 2SO4 and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE/ea=1/0 to 5/1) to give compound 3 (1.30 g, yield 68.95％).1H NMR(400MHz,CD3OD)δ5.35-5.41(m,2H),3.65(s,3H),3.16(t,J＝6.8Hz,2H),2.33(t,J＝7.2Hz,2H),2.16(t,J＝7.6Hz,2H),1.98(d,J＝4.8Hz,4H),1.56-1.68(m,4H),1.46-1.55(m,2H),1.27-1.40(m,22H),0.86-0.94(m,3H).
2 Preparation of Compound 4
Compound 3 (1.30 g,3.17 mmol) was dissolved in a mixed solvent of THF (5.00 mL) and H 2 O (2.50 mL) at 25℃and LiOH (0.150 g,6.35 mmol) was added thereto, and the reaction was stirred at 25℃for 18 hours. Thin layer chromatography (PE/ea=3/1) showed complete consumption of starting material and a new point of formation. To the reaction mixture was added a 1M HCl (10.0 mL) solution to adjust acidity, followed by addition of methylene chloride (20.0 mL) and separation. The organic phase was dried over Na 2SO4, and the organic phase was dried by spinning to give compound 4 (800 mg, yield) as a white solid 63.72％).1H NMR(400MHz,CDCl3)δ5.54-5.63(m,1H),5.38(dt,J＝4.4,2.4Hz,2H),3.26(q,J＝6.8Hz,2H),2.36(t,J＝7.2Hz,2H),2.12-2.20(m,2H),1.91-2.02(m,4H),1.47-1.72(m,6H),1.24-1.42(m,22H),0.84-0.93(m,3H).
Preparation of Compound 6
Compound 4 (255 mg, 0.640 mmol) was dissolved in DCM (9.00 mL) at 25℃and HATU (304 mg,0.80 mmol) and DIEA (0.529 mL,3.20 mmol) were added sequentially and stirred for 0.5 h, then compound 5 (240 mg, 0.284 mmol) was added and the reaction stirred at 25℃for 16h. Thin layer chromatography (DCM/meoh=10/1) showed complete consumption of starting material and a new point of formation. Dichloromethane (30.0 mL) was added to the reaction solution for dilution, which was washed with saturated aqueous NaHCO 3 (10.0 mL x 3) and saturated aqueous NaCl (10.0 mL x 3), then the organic phase was dried over anhydrous Na 2SO4 and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (DCM/meoh=1/0 to 10/1) to give compound 6 (300 mg, yield 67.92%) as a pale yellow oil.
Preparation of 4 DL0138
Compound 6 (300 mg, 0.803 mmol) was dissolved in DCM (10.0 mL) at 25℃and DIEA (0.360 mL,2.17 mmol), DMAP (17.7 mg,0.145 mmol) and compound 7 (218 mg,2.18 mmol) were added sequentially and the reaction stirred at 25℃for 2 hours. LCMS showed mass values for the product. The reaction solution is spin-dried to obtain a crude product. Separating the crude product with reversed phase column (chromatographic column: waters Xbridge BEH C18.25.5 μm; mobile phase: TEAA-CAN; B%:60% -95%,10min; flow rate: 15 ml/min) to obtain colorless oily liquid DL0138 (178 mg, yield 52.93%, purity) 97.55％).1H NMR(400MHz,CD3OD)δ7.41-7.48(m,2H),7.20-7.35(m,7H),6.84-6.93(m,4H),5.39(t,J＝3.6Hz,2H),3.93-4.26(m,3H),3.89(d,J＝3.6Hz,1H),3.80(d,J＝2.0Hz,6H),3.54-3.76(m,3H),3.35-3.48(m,1H),3.30(d,J＝5.2Hz,1H),3.13-3.24(m,10H),2.49-2.63(m,4H),2.23-2.42(m,2H),2.17(q,J＝7.6Hz,2H),1.98(d,J＝4.8Hz,4H),1.46-1.64(m,6H),1.24-1.39(m,32H),0.88-0.95(m,3H).
EXAMPLE 1.12 preparation of DL0139
Preparation of Compound 3
Compound 1 (200 mg, 0.719 mmol), compound 2 (647 mg,3.566 mmol), HOBt (144.55 mg,1.070 mmol), EDCI (205.06 mg,1.070 mmol), DIEA (0.943 mL, 5.704 mmol) were added sequentially to DCM (5.00 mL) and reacted at 25℃for 14 h. TLC (petroleum ether: ethyl acetate=2:1) showed disappearance of starting compound 1 with formation of new spots. The reaction solution was diluted with dichloromethane (50.0 mL), washed with citric acid (10 ml×3), sodium bicarbonate (10 ml×3), saturated sodium chloride (10 ml×3), and the organic phase was dried over anhydrous sodium sulfate, filtered and spun-dried to give a yellow solid product compound 3(250mg,0.613mmol,86.00％).1H NMR(400MHz,CDCl3)δ5.39-5.47(m,4H),3.65-3.70(m,3H),3.25(q,J＝6.8Hz,2H),2.65-2.70(m,2H),2.32(t,J＝7.2Hz,2H),2.15(t,J＝7.6Hz,2H),1.94-2.03(m,4H),1.60-1.70(m,4H),1.52(q,J＝7.2Hz,2H),1.26-1.38(m,16H),0.89(t,J＝6.8Hz,3H).
2 Preparation of Compound 4
Compound 3 (330 mg,0.810 mmol) was dissolved in THF (4.00 mL) and H 2 O (2.00 mL), liOH (67.9 mg,1.62 mmol) was added and reacted at 25℃for 14 hours. TLC (petroleum ether: ethyl acetate=1:1) showed disappearance of starting material and formation of new spots. The reaction was cooled to 0 ℃, ph=5 was adjusted with 1M HCl, the aqueous phase was extracted with dimethyltetrahydrofuran (20 ml x 3), the organic phase was dried over anhydrous sodium sulfate, filtered and spun-dried to give the product compound as a brown solid 4(350mg,0.889mmol,109.84％).1H NMR(400MHz,CD3OD)δ5.32-5.46(m,4H),3.16(t,J＝6.8Hz,2H),2.65(d,J＝4.0Hz,2H),2.29(t,J＝7.2Hz,2H),2.13-2.21(m,2H),1.96-2.03(m,4H),1.48-1.67(m,6H),1.26-1.38(m,16H),0.90(t,J＝6.8Hz,3H).
Preparation of Compound 6
Compound 4 (200 mg,0.508 mmol), HATU (263 mg,0.693 mmol), DIEA (0.229 mL,1.38 mmol) was added to DCM (3.00 mL), compound 5 (207 mg, 0.460 mmol) was added and reacted at 25℃for 30 min. LCMS showed 83% product formation. The reaction solution was diluted with dichloromethane (100 ml), washed with saturated aqueous sodium bicarbonate (30 ml x 3) and saturated aqueous sodium chloride (30 ml x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered and dried by spin to give compound 6 (600 mg,0.727 mmol) as a brown solid.
Preparation of 4 DL0139
Compound 6 (600 mg,0.727 mmol) and compound 7 (436 mg,4.36 mmol) were dissolved in DCM (6.00 mL), DIEA (0.721 mL,4.36 mmol) and DMAP (22.2 mg,0.182 mmol) were added, nitrogen was displaced three times, the reaction solution was reacted at 25℃for 1 hour, LCMS showed the starting material disappeared, 84.2% of the product was formed. The reaction solution was directly spin-dried to give a crude product, which was purified by Prep-HPLC (column: 01-Waters Xbridge BEH C, 19 x 150mm; mobile phase: TEAA-ACN; gradient: 55% -95%/15min; flow rate: 15 ml/min). The product was obtained as a colourless oil DL0139(200mg,0.216mmol,29.73％).MS:m/z＝1971.7(M+H)+;1H NMR(400MHz,CD3OD)δ7.38-7.46(m,2H),7.18-7.35(m,7H),6.81-6.90(m,4H),5.32-5.45(m,4H),4.16-4.26(m,1H),3.85-4.13(m,3H),3.66-3.81(m,7H),3.56-3.65(m,2H),3.35-3.46(m,1H),3.14-3.22(m,9H),2.63-2.68(m,2H),2.54-2.61(m,2H),2.46-2.53(m,2H),2.23-2.41(m,2H),2.10-2.19(m,2H),1.94-2.02(m,4H),1.45-1.61(m,6H),1.22-1.38(m,26H),0.85-0.93(m,3H).
EXAMPLE 1.13 preparation of DL0140
Preparation of Compound 3
Compound 1 (500 mg,1.52 mmol) and HATU (7512 mg,1.97 mmol) were dissolved in DCM (5.00 mL) and DIEA (1.50 mL,9.13 mmol) was added. The reaction was allowed to react at 40 degrees celsius for 0.5 hours. Compound 2 (106 mg,0.731 mmol) was then added. The reaction was allowed to react at 40 degrees celsius for 16 hours. TLC (PE/etoac=2/1) showed new spots to form. To the reaction was added 25mL of DCM and 20mL of water, the reaction was partitioned, and the aqueous phase was extracted with DCM (15 mL. Times.1). The combined organic phases were washed with saturated brine (20 ml x 1), dried over anhydrous sodium sulfate, filtered and spun-dried to give the crude product. The crude product was purified by column chromatography (PE/etoac=1/0-1/1) to give the compound 3(520mg,1.14mmol,74.97％).1H NMR(400MHz,CD3OD)δ5.29-5.47(m,12H),3.67(s,3H),3.13-3.23(m,2H),2.80-2.95(m,10H),2.31-2.45(m,4H),2.19-2.27(m,2H),2.06-2.16(m,2H),1.58-1.70(m,2H),1.46-1.57(m,2H),1.32-1.42(m,2H),0.99(t,J＝7.6Hz,3H)
2 Preparation of Compound 4
Compound 3 (520 mg,1.14 mmol) was dissolved in H 2 O (2 mL) and THF (5 mL) and LiOH (191 mg,4.56 mmol) was added. The reaction was allowed to react at 20 degrees celsius for 16 hours. TLC (PE/etoac=2/1) showed new spots to form. The reaction solution was dried by spin-drying. To the crude product was added 15mL of DCM and 15mL of water, the reaction was partitioned, and the aqueous phase was adjusted to pH 5 with 3M HCl. The aqueous phase was extracted with DCM (15 mL x 2). The organic phase was washed with saturated brine (20 mL. Times.1), dried over anhydrous sodium sulfate, filtered, and spin-dried to give the product compound as a colourless oil 4(420mg,0.951mmol,83.34％).1H NMR(400MHz,CD3OD)δ5.21-5.48(m,12H),3.16(t,J＝7.2Hz,2H),2.78-2.94(m,10H),2.34-2.43(m,2H),2.29(t,J＝7.2Hz,2H),2.19-2.24(m,2H),2.04-2.15(m,2H),1.57-1.67(m,2H),1.46-1.56(m,2H),1.31-1.42(m,2H),0.97(t,J＝7.6Hz,3H).
Preparation of Compound 6
Compound 4 (319 mg,0.72 mmol) was dissolved in DCM (5 mL), HATU (274 mg,0.72 mmol) and DIEA (0.27 mL,1.66 mmol) were added and the reaction stirred for half an hour at 25℃and compound 5 (250 mg, 0.554 mmol) was added and the reaction was reacted for 16 hours at 25 ℃. TLC (DCM/meoh=10/1) showed a new spot formation. To the reaction solution was added 10mL of DCM and 15mL of water, the reaction solution was partitioned, and the combined organic phases were added with sodium bicarbonate solution (10 mL x 2), washed with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, filtered and dried by spinning to give a crude product. The crude product was purified by column chromatography (DCM/meoh=1/0-10/1) to give compound 6 as a yellow oil.
Preparation of 4 DL0140
Compound 6 (300 mg,0.344 mmol) was dissolved in DCM (7 mL), DIEA (0.34 mL,2.06 mmol), DMAP (16.7 mg,0.13 mmol) and compound 7 (206 mg,2.06 mmol) were added and the reaction was allowed to react at 25℃for 3 hours. The reaction mixture was spin-dried and the crude product purified by prep-HPLC (column: waters Xbridge BEH C18.5.25 mm. 5um; conditions: TEAA-ACN; begin B55-95; gradient time: 15min; 100% B hold time: 6 min; flow rate: 15 ml/min) to give DL0140 (120 mg, yield 35.89% purity) as a yellow oil 97.34％).1H NMR(400MHz,CD3OD)δ7.43(d,J＝8.0Hz,2H),7.17-7.36(m,7H),6.81-6.92(m,4H),5.19-5.46(m,11H),4.16-4.29(m,1H),3.84-4.15(m,3H),3.76-3.80(m,6H),3.35-3.73(m,4H),3.07-3.18(m,6H),2.76-2.93(m,10H),2.50-2.64(m,4H),2.16-2.44(m,6H),2.03-2.13(m,2H),1.42-1.66(m,4H),1.32-1.38(m,6H),0.96(t,J＝8.0Hz,3H)
EXAMPLE 1.14 preparation of DL0142
Preparation of Compound 2
Compound 1 (2.00 g,10.9 mmol) was dissolved in THF (80.0 mL), TBAB (0.71 g,2.19 mmol), compound 1A (13.4 g,43.9 mmol) and KOH (3.08 g,54.8 mmol) were added and the reaction was allowed to proceed at 25℃for 16 hours. TLC (PE/ea=10/1) showed new spot formation. To the reaction was added 15.0mL of water, and the reaction was extracted with EtOAc (15.0 mL. Times.2). The combined organic phases were washed with saturated brine (15.0 ml x 1), dried over anhydrous sodium sulfate, filtered and spun-dried to give the crude product. The crude product was purified by column chromatography (PE/ea=1/0-10/1) to give the product as a colourless oil 2(5.5g,8.71mmol,79.40％).1H NMR(400MHz,CD3OD)δ7.14-7.40(m,5H),4.41-4.55(m,2H),3.28-3.64(m,9H),0.26-1.08(m,56H),-0.58-0.00(m,6H)
2 Preparation of Compound 3
Compound 2 (5.50 g,8.75 mmol) was dissolved in EtOH (70.0 mL) and Pd (OH) 2 (0.20 g,3.16 mmol) was added and the reaction was carried out at 50℃under H 2 (1 atm) pressure for 16 hours. TLC (PE/ea=10/1) showed new spot formation. Filtering and spin-drying to obtain a crude product. The crude product was purified by column chromatography (PE/etoac=1/0-10/1) to give the product as a white solid 3(2.45g,4.52mmol,51.9％).1H NMR(400MHz,CD3OD)δ3.51-3.64(m,5H),3.47(s,4H),1.51-1.62(m,4H),1.29(s,52H),0.90(t,J＝8.0Hz,6H)
Preparation of Compound 4
Compound 3 (2.45 g,4.52 mmol) was dissolved in DCM (15 mL) and DMF (30.0 mL) and Pyridinium Dichromate (PDC) (5.96 g,15.8 mmol) was added and the reaction was allowed to react at 25℃for 16 hours. TLC (PE/ea=3/1) showed new spot formation. To the reaction solution was added 40.0mL of H 2 O, followed by extraction with DCM (40.0 mL. Times.2), drying over anhydrous sodium sulfate, filtration and spin-drying to give the crude product. The crude product was purified by column chromatography (PE/etoac=1/0-5/1) to give the product as a white solid 4(1.1g,1.982mmol,43.77％).1H NMR(400MHz,CDCl3)δ4.02-4.11(m,1H),3.77-3.87(m,1H),3.59-3.77(m,3H),3.44-3.56(m,2H),1.53-1.61(m,4H),1.26(s,52H),0.86-0.95(m,6H)
Preparation of Compound 6
Compound 4 (661mg, 1.39 mmol) was dissolved in DCM (15.0 mL), HATU (292 mg,1.55 mmol), DIEA (1.18 mL,7.19 mmol) and compound 5 (28.7 mg,0.19 mmol) were added and the reaction was allowed to react at 25℃for 16h. TLC (PE/ea=3/1) showed new spot formation. Adding 15.0mL of DCM to dilute the reaction solution, adding citric acid monohydrate (15.0 mL x 1), sodium bicarbonate solution (15.0 mL x 1), washing with saturated saline (15.0 mL x 1), drying over anhydrous sodium sulfate, filtering, and spinning to obtain a crude product as a white solid 6(510mg,0.748mmol,62.39％).1H NMR(400MHz,CDCl3)δ3.89(dd,J＝4.0,5.6Hz,1H),3.76-3.81(m,1H),3.60-3.70(m,5H),3.39-3.56(m,3H),3.29(q,J＝8.0Hz,2H),2.33(t,J＝8.0Hz,2H),1.52-1.72(m,12H),1.27-1.38(m,50H),0.87-0.93(m,6H)
Preparation of Compound 7
Compound 6 (510 mg,0.748 mmol) was dissolved in THF (6.0 mL) and H 2 O (3.0 mL), liOH (62.7 mg,1.49 mmol) was added and the reaction was allowed to react at 25℃for 16 hours. TLC (PE/ea=3/1) showed new spot formation. The reaction solution was dried by spinning, 10.0mL of water was added, the pH of the aqueous phase was adjusted to 4 with 1M HCl, the aqueous phase was extracted with dimethyltetrahydrofuran (10.0 mL. Times.2), the combined organic phases were washed with saturated brine (10.0 mL. Times.1), dried over anhydrous sodium sulfate, filtered, and spun-dried to give a white solid crude product 7(460mg,0.55mmol,73.6％).1H NMR(400MHz,CD3OD)δ3.83-3.90(m,1H),3.36-3.65(m,6H),3.19-3.27(m,2H),2.29(t,J＝8.0Hz,2H),1.49-1.68(m,8H),1.23-1.37(m,54H),0.86-0.94(m,6H)
6 Preparation of Compound 9
Compound 7 (326 mg,0.489 mmol) was dissolved in DCM (5.0 mL), HATU (219 mg,0.57 mmol) and DIEA (0.22 mL,1.33 mmol) were added and the reaction was reacted at 25℃for 30min, then compound 8 (200 mg,0.44 mmol) was added and the reaction was reacted at 25℃for 16 h. TLC (DCM/meoh=10/1) showed a new spot formation. 10.0mL of H 2 O was added to the reaction solution, the reaction solution was extracted with DCM (10.0 mL. Times.2), sodium bicarbonate solution (10.0 mL. Times.1) was added to the reaction solution, washed with saturated brine (10.0 mL. Times.1), dried over anhydrous sodium sulfate, filtered, and spun-dried to give a crude product, which was purified by column chromatography (DCM/MeOH=1/0-5/1) to give a crude product as a white solid 9(194mg,0.17mmol,39.7％).1H NMR(400MHz,CDCl3)δ7.38-7.46(m,1H),7.27-7.35(m,7H),7.18(d,J＝8.0Hz,1H),6.84(d,J＝8.0Hz,4H),4.75-4.80(m,1H),3.87(s,2H),3.77-3.85(m,6H),3.69-3.76(m,3H),3.57-3.68(m,4H),3.37-3.56(m,5H),3.11-3.36(m,3H),2.72-2.81(m,1H),2.81(s,1H),2.18-2.45(m,2H),1.66-1.75(m,4H),1.43-1.51(m,4H),1.26(s,54H),0.86-0.92(m,6H).
Preparation of 7 DL0142
Compound 9 (390 mg,0.48 mmol) was dissolved in DCM (7.00 mL), DIEA (0.16 mL,0.98 mmol), DMAP (9.91 mg,0.08 mmol) and compound 10 (98.28 mg,0.982 mmol) were added and the reaction was allowed to react at 25℃for 3 hours. LCMS showed MS with the desired product. The reaction mixture was spin-dried and the crude product purified by prep-HPLC (column: waters Xbridge BEH C, 19 x 150mm; conditions: TEAA-ACN; begin B65-95; gradient time: 15min; 100% B hold time: 2 min; flow rate: 15 ml/min) to give a white solid product DL0142(106mg,0.08mmol,53.9％).1H NMR(400MHz,CDCl3)δ7.31-7.39(m,2H),7.21-7.27(m,5H),7.12-7.17(m,1H),6.89-6.96(m,1H),6.73-6.79(m,4H),4.29-4.40(m,1H),3.94-4.04(m,1H),3.77-3.92(m,3H),3.70-3.76(m,6H),3.67(d,J＝4.0Hz,1H),3.29-3.51(m,6H),3.04-3.28(m,5H),2.94(q,J＝8.0Hz,3H),2.47-2.59(m,4H),2.17-2.27(m,2H),1.43-1.61(m,8H),1.18-1.26(m,54H),0.78-0.85(m,6H)
EXAMPLE 1.15 preparation of DL0143
1. Preparation of Compound 3
Compound 2 (2.00 g,11.0 mmol) was dissolved in THF (60.0 mL) at 25℃and TBAB (0.71 g,2.20 mmol), compound 1 (10.5 mL,43.9 mmol) and KOH (3.08 g,54.9 mmol) were added sequentially to the above mixed liquid. The reaction liquid was stirred at 25 ℃ for 12 hours. Thin layer chromatography (PE/ea=10/1) showed complete reaction of starting material with new point formation. The reaction solution was diluted with EA (50.0 mL), washed with water (30.0 mL X3), saturated citric acid solution (30.0 mL X3), water (30.0 mL X3) and saturated NaCl solution (30.0 mL X3), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (PE/ea=97/3 to 96/4) to give compound 3 (5.21 g, yield 91.48％).1H NMR(400MHz,CD3OD)δ7.21-7.38(m,5H),4.53(s,2H),3.43-3.65(m,9H),1.49-1.62(m,4H),1.25-1.37(m,36H),0.87-0.93(m,6H)
2. Preparation of Compound 4
Compound 3 (2.77 g,5.34 mmol) was added to EtOH (30.0 mL), pd (OH) 2 (0.30 g,0.214 mmol) was added and the system gas was replaced 3 times with hydrogen (14.696 psi) and the reaction was carried out with heating in an oil bath at 50℃for 12 hours. Thin layer chromatography (PE/ea=10/1) showed the disappearance of starting material with new spot formation. The reaction solution was filtered through celite, and the organic phase was concentrated under reduced pressure to give crude product. Purifying the crude product by column chromatography (PE/EA=95/5-94/6) to obtain a colorless oily crude compound 4(2.20g).1H NMR(400MHz,CD3OD)δ3.42-3.62(m,9H),1.50-1.63(m,4H),1.27-1.39(m,36H),0.84-0.97(m,6H)
3. Preparation of Compound 5
Compound 4 (700 mg,1.63 mmol) was dissolved in DCM (10.0 mL) and DMF (5.00 mL) at 25℃and PDC (2.15 g,5.71 mmol) was added thereto. The mixed liquid was stirred at 25 ℃ for 12 hours. Thin layer chromatography (PE/ea=3/1) showed the disappearance of starting material and the creation of new spots. To the reaction liquid was added water (50.0 mL), the mixed liquid was extracted with ethyl acetate (100 mL X3), and the organic phase was concentrated under reduced pressure to dryness to obtain a crude product. The crude product was purified by column chromatography (PE/ea=5/1 to 3/1) to give compound 5 (320 mg, yield) as a white solid 44.27％).1H NMR(400MHz,CDCl3)δ4.05(dd,J＝3.2,5.2Hz,1H),3.78-3.83(m,1H),3.69-3.75(m,1H),3.64(t,J＝6.4Hz,2H),3.42-3.56(m,2H),1.52-1.70(m,4H),1.23-1.37(m,36H),0.89(t,J＝6.4Hz,6H)
4. Preparation of Compound 7
Compound 5 (320 mg,0.723 mmol) was added to DCM (5.00 mL) at 25℃followed by HATU (412 mg,1.08 mmol) and DIEA (0.178 mL,2.17 mmol). After the mixed liquid was stirred at 25℃for 0.5 hours, compound 6 (158 mg,0.867 mmol) was added thereto. The reaction liquid was stirred at 25 ℃ for 12 hours. TLC (PE/ea=3/1, pma) showed complete consumption of starting material and generation of new spots. To the reaction liquid was added dichloromethane (100 mL), and the mixture was washed three times with saturated sodium bicarbonate solution (15.0 mL of X3) and saturated brine (15.0 mL of X3) in this order. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude compound. The crude product was purified by column chromatography (PE/ea=3/1 to 1/1, pma) to give colorless liquid compound 7 (200 mg, yield) 48.55％).1H NMR(400MHz,CD3OD)δ3.86(dd,J＝3.2,5.2Hz,1H),3.64-3.70(m,4H),3.40-3.63(m,5H),3.20-3.24(m,2H),2.33(t,J＝7.6Hz,2H),1.47-1.69(m,8H),1.22-1.43(m,38H),0.86-0.94(m,6H)
5. Preparation of Compound 8
Compound 7 (200 mg,0.351 mmol) was dissolved in THF (4.00 mL) and H 2 O (2.00 mL) at 25℃to which LiOH (44.2 mg,1.05 mmol) was added. The mixed liquid was stirred at 25 ℃ for 12 hours. Thin layer chromatography (PE/ea=3/1, pma) showed complete consumption of starting material with new point formation. The reaction liquid was adjusted to pH 5 with 1M HCl, the product was extracted with dimethyltetrahydrofuran (30.0 mL X3), and the organic phase was suspended under reduced pressure to give the crude compound as a colorless oil 8(200mg).1H NMR(400MHz,CD3OD)δ3.83-3.89(m,1H),3.64-3.71(m,1H),3.39-3.63(m,5H),3.18-3.28(m,2H),2.29(t,J＝7.6Hz,2H),1.50-1.68(m,8H),1.24-1.40(m,38H),0.86-0.94(m,6H)
6. Preparation of Compound 10
Compound 8 (193 mg,0.347 mmol) was added to DCM (5.00 mL) at 25℃followed by HATU (165 mg,0.433 mmol) and DIEA (0.143 mL,0.867 mmol) added thereto. After the reaction solution was stirred at 25℃for 0.5 hours, compound 9 (130 mg,0.289 mmol) was added thereto, and stirring was continued for 12 hours. LCMS showed MS formation of the product. TLC (DCM/meoh=1/1) showed new spot formation. To the reaction liquid was added dichloromethane (100 mL), and the mixture was washed three times with saturated sodium bicarbonate solution (15.0 mL of X3) and saturated brine (15.0 mL of X3) in this order. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude compound. The crude product was purified by column chromatography (DCM/meoh=10/1 to 8/1, pma) to give compound 10 (180 mg, yield) as a colorless liquid 63.04％).1H NMR(400MHz,CD3OD)δ7.69-7.87(m,1H),7.43(d,J＝8.0Hz,1H),7.17-7.33(m,6H),7.07-7.15(m,1H),6.79-6.91(m,4H),3.83-4.05(m,2H),3.74-3.81(m,6H),3.63-3.73(m,3H),3.56-3.63(m,3H),3.40-3.56(m,6H),3.32-3.40(m,1H),3.15-3.28(m,4H),2.14-2.46(m,2H),1.46-1.68(m,8H),1.28(s,34H),0.84-0.94(m,6H)
7. Preparation of Compound DL0143
Compound 10 (180 mg,0.182 mmol) was dissolved in DCM (5.00 mL) at 25℃and compound 11 (109 mg,1.09 mmol), DIEA (0.181 mL,1.09 mmol) and DMAP (5.57 mg,0.046 mmol) were added sequentially. The reaction solution was stirred at 25 ℃ for 12 hours. LCMS showed product formation. The reaction liquid was directly dried under reduced pressure to give a crude product, which was isolated by preparative reverse phase column (column chromatography: 01-Waters Xbridge BEH C, 19 x 150mm; mobile phase: TEAA-ACN; gradient: 75% -95%/18 min; flow rate: 15 ml/min) to give a colorless oily compound DL0143 (110 mg, yield 55.49%, purity) 99.49％).1H NMR(400MHz,CD3OD)δ7.43(d,J＝7.6Hz,2H),7.18-7.33(m,7H),6.84-6.87(m,4H),4.09-4.28(m,2H),3.83-4.03(m,4H),3.78(d,J＝2.0Hz,7H),3.61-3.70(m,2H),3.41-3.61(m,7H),3.33-3.41(m,1H),3.10-3.26(m,4H),2.51-2.66(m,4H),2.18-2.44(m,2H),1.45-1.69(m,8H),1.20-1.43(m,41H),0.85-0.93(m,6H)
EXAMPLE 1.16 preparation of DL0144
1. Preparation of Compound 3
Compound 1 (1.00 g,2.59 mmol) was dissolved in DCM (16.0 mL) at 25deg.C, pyridine (Py) (0.418 mL,5.17 mmol) was added, cooled to 0deg.C, then Compound 2 (0.630 g,3.10 mmol) was added and the reaction stirred at 25deg.C for 2 hours. Thin layer chromatography (PE/ea=3/1, PE/ea=10/1) showed that the starting material was not reacted completely, with a new point. The reaction was diluted with DCM (50.0 mL), washed with saturated aqueous NaHCO 3 (20.0 mL. Times.3) and the organic phase was washed with saturated aqueous saline (20.0 mL. Times.3) and then the organic phase was dried over Na 2SO4 and the organic phase was dried by spinning to give the crude product. The crude product was purified by column chromatography (PE/ea=1/0-10/1) to give the compound as a white solid 3(800mg,55.94％).1H NMR(400MHz,CDCl3)δ8.22-8.34(m,2H),7.36-7.44(m,2H),5.44(d,J＝4.8Hz,1H),4.56-4.68(m,1H),2.43-2.55(m,2H),1.68-2.10(m,7H),1.44-1.54(m,4H),1.24-1.40(m,4H),0.95-1.24(m,14H),0.92(d,J＝6.4Hz,3H),0.87(dd,J＝6.57,1.6Hz,6H),0.69(s,3H).
2. Preparation of Compound 5
Compound 4 (200 mg,1.10 mmol) was dissolved in DCM (10.0 mL) at 25℃and TEA (0.650 mL,4.72 mmol) and compound 3 (433 mg,0.787 mmol) were added sequentially and the reaction stirred at 25℃for 16h. Thin layer chromatography (PE/ea=10/1, PE/ea=3/1) showed complete consumption of starting material and a new point of formation. The reaction was diluted with DCM (50.0 mL), washed with saturated aqueous NaHCO 3 (20.0 mL X3), the organic phase was further washed with saturated aqueous saline (20.0 mL X3), then the organic phase was dried over Na 2SO4 and the organic phase was dried by spinning to give the crude product. The crude product was purified by column chromatography (PE/ea=1/0-5/1) to give the crude compound as a white solid 5(380mg).1H NMR(400MHz,CDCl3)δ5.35-5.41(m,1H),4.42-4.54(m,1H),3.68(s,3H),3.17(q,J＝6.4Hz,2H),2.24-2.40(m,4H),1.92-2.05(m,2H),1.78-1.90(m,3H),1.65(dt,J＝15.2,7.2Hz,3H),1.46-1.54(m,6H),1.23-1.45(m,8H),0.97-1.22(m,13H),0.92(d,J＝6.4Hz,3H),0.87(dd,J＝6.4,1.6Hz,6H),0.68(s,3H).
3. Preparation of Compound 6
Compound 5 (380 mg,0.681 mmol) was dissolved in a mixed solvent of THF (5.00 mL) and H 2 O (2.50 mL) at 25℃and LiOH (32.6 mg,1.36 mmol) was added and the reaction was reacted at 25℃for 18 hours. Thin layer chromatography (PE/ea=3/1) showed complete consumption of starting material and a new point of formation. To the reaction solution was added a 1M HCl (10.0 mL) solution to adjust acidity, then DCM (20.0 mL) was added, the organic phase was collected by extraction, and dried over Na 2SO4, and spin-dried to give Compound 6 (230 mg, yield) as a white solid 62.09％).1H NMR(400MHz,CDCl3)δ5.36-5.40(m,1H),4.45-4.56(m,1H),3.18(q,J＝6.4Hz,2H),2.27-2.41(m,4H),1.96-2.05(m,2H),1.67(dt,J＝15.2,7.2Hz,3H),1.46-1.59(m,9H),1.29-1.44(m,8H),0.97-1.18(m,13H),0.92(d,J＝6.4Hz,3H),0.87(dd,J＝6.4,1.6Hz,6H),0.68(s,3H).
4. Preparation of Compound 8
Compound 6 (232 mg,0.427 mmol) was dissolved in DCM (10.0 mL) at 25℃and HATU (203 mg, 0.284 mmol) and DIEA (0.353 mL,2.14 mmol) were added sequentially, the reaction stirred at 25℃for 0.5 h, then compound 7 (160 mg,0.356 mmol) was added and the reaction stirred at 25℃for 16 h. LCMS showed mass values for the product. Thin layer chromatography (DCM/meoh=10/1) showed a new spot formation. The reaction was diluted with DCM (20.0 mL), washed with saturated aqueous citric acid (5.00 mL X3), saturated aqueous NaHCO 3 (5.00 mL X3) and saturated aqueous saline (5.00 mL X3), then the organic phase was dried over anhydrous Na 2SO4 and the organic phase was dried by spin to give the crude product. The crude product was purified by column chromatography (DCM/meoh=1/0-10/1) to give the crude compound as a colourless oil 8(500mg).1H NMR(400MHz,CDCl3)δ7.27-7.34(m,5H),7.15-7.21(m,4H),6.81-6.87(m,4H),5.35-5.41(m,1H),4.63-4.80(m,1H),4.31-4.52(m,1H),3.86-3.94(m,2H),3.79-3.83(m,6H),3.57-3.74(m,7H),3.47-3.55(m,1H),3.12-3.22(m,2H),2.24-2.41(m,4H),1.81-2.05(m,6H),1.45-1.59(m,11H),1.21-1.31(m,4H),1.08-1.17(m,6H),0.95(d,J＝5.2Hz,6H),0.90-0.94(m,3H),0.86-0.89(m,6H),0.66-0.71(m,3H).
Preparation of DL0144
Compound 8 (500 mg,0.308 mmol) was dissolved in DCM (10.0 mL) at 25℃and DIEA (0.305 mL,1.85 mmol), DMAP (15.0 mg,0.123 mmol) and compound 9 (185 mg,1.85 mmol) were added in sequence, and the reaction stirred at 25℃for 2 hours. LCMS showed mass values for the product. The reaction solution is spin-dried to obtain a crude product. Separating the crude product with reversed phase column (chromatographic column: 01-Waters Xbridge BEH C18.19X105 mm; mobile phase: TEAA-ACN; B%:75% -95%,15min; flow rate: 15 ml/min) to obtain white solid compound DL0144 (218 mg, yield 65.91%, purity) 91.17％).1H NMR(400MHz,CDCl3)δ7.37-7.46(m,2H),7.27-7.35(m,7H),6.83(d,J＝8.8Hz,4H),5.38(s,1H),4.33-4.72(m,2H),3.83-4.04(m,3H),3.79-3.81(m,6H),3.05-3.58(m,8H),2.96(q,J＝7.2Hz,5H),2.49-2.69(m,5H),2.23-2.40(m,5H),1.79-2.05(m,6H),1.42-1.62(m,11H),1.31-1.39(m,4H),1.26(t,J＝7.2Hz,7H),1.08-1.18(m,6H),0.97-1.07(m,6H),0.92(d,J＝6.4Hz,3H),0.87(dd,J＝6.4,1.6Hz,6H),0.68(s,3H).
EXAMPLE 1.17 preparation of DL0145
Preparation of Compound 3
Compound 2 (134 mg,0.667 mmol) was dissolved in DCM (10.0 mL) at 25℃and HATU (380 mg,1.00 mmol) and DIEA (0.331 mL,2.00 mmol) were added sequentially, and compound 1 (300 mg,0.667 mmol) was added after the mixed solution was stirred at 25℃for 0.5 h. The reaction liquid was stirred at 25 ℃ for 12 hours. Thin layer chromatography (DCM/meoh=10/1, pma) and (DCM/meoh=10/1, pma) showed complete reaction of the two starting materials with a new point. To this was added ethyl acetate (10.0 mL), and the mixed liquid was washed three times with brine (10 mL X3), and the organic phase was concentrated under reduced pressure to give a crude product compound. The crude product was purified by column chromatography (DCM/meoh=93/7-90/10) to give compound 3 (412 mg, yield) as a colourless oil 97.71％).1H NMR(400MHz,METHANOL-d4)δ7.39-7.51(m,2H),7.16-7.35(m,7H),6.86(dt,J＝2.89,5.83Hz,4H),3.79-4.04(m,2H),3.78(d,J＝1.25Hz,6H),3.41-3.76(m,5H),3.32-3.38(m,1H),3.18(br d,J＝6.27Hz,1H),2.94-3.14(m,1H),2.16-2.42(m,2H),1.52(br d,J＝6.02Hz,2H),1.19-1.39(m,18H),0.87-0.92(m,3H)
Preparation of 2 DL0145
Compound 3 (200 mg,0.317 mmol) was dissolved in DCM (3.00 mL) at 25℃and DIEA (0.314 mL,1.90 mmol), DMAP (9.67 mg,0.079 mmol) and Compound 4 (31.7 mg,0.317 mmol) were added to the above mixture. The reaction liquid was stirred at 25 ℃ for 3 hours. Thin layer chromatography (DCM/meoh=10/1) showed complete reaction of starting material with a new point. Liquid mass (LCMS) showed complete reaction of starting material. The reaction solution was diluted with dichloromethane (10.0 mL), washed 3 times with saturated NaHCO 3 solution (10.0 mL X3) and saturated NaCl solution (10.0 mL X3) in this order, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM/meoh=95/5 to 90/10) to give crude product. The crude product was purified by Prep-HPLC (column: waters Xbridge BEH C100 x 25mm x 5um; mobile phase: TEAA-ACN; gradient: 45% -95%/16min; flow: 15 ml/min) to give DL0145 (84.0 mg, 36.26% yield, purity) as a colorless oil 98.70％).1H NMR(400MHz,METHANOL-d4)δ7.43(br d,J＝7.53Hz,2H),7.18-7.36(m,7H),6.82-6.93(m,4H),4.16-4.27(m,1H),4.11(d,J＝5.52Hz,1H),3.97(br d,J＝11.29Hz,1H),3.85(br s,1H),3.78(s,6H),3.43-3.75(m,3H),3.34-3.43(m,1H),3.10-3.28(m,5H),2.55-2.64(m,2H),2.52(d,J＝6.02Hz,2H),2.33(br d,J＝12.30Hz,2H),1.52(br s,2H),1.18-1.38(m,21H),0.83-0.95(m,3H)
EXAMPLE 2 Synthesis of siRNA
The siRNA of the present invention is prepared using a solid phase phosphoramidite method well known in the art. Specific methods are described in, for example, PCT publication Nos. WO2016081444 and WO2019105419, and are briefly described below.
1. Preparation of siRNA with ligand not connected to 3' -end of sense strand
1.1 Synthesis of sense strand (SS strand)
By the solid-phase phosphoramidite synthesis method, blank CPG solid-phase carriers are used as initial circulation, and nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the sense strand. Each nucleoside monomer attached involved four steps of deprotection, coupling, capping, oxidation or thio reactions to synthesize oligonucleotides on a scale of 5 umol. The synthesis conditions were as follows:
The nucleoside monomer was provided in 0.05mol/L acetonitrile, the conditions for each step were the same, i.e., the temperature was 25 ℃, 3% trichloroacetic acid-dichloromethane solution was used for deprotection, 3 times, 0.25 mol/L5-Ethylthiotetrazole (ETT) -acetonitrile solution was used for coupling, 2 times for coupling, 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/v), 2 times for capping, 0.05mol/L of iodine in tetrahydrofuran/pyridine/water (70/20/10, v/v/v) for oxidation, 2 times for oxidation, 0.2mol/L phenylacetyl disulfide (PADS) in acetonitrile/3-methylpyridine (1/1, v/v) for coupling, and 2 times for thio.
1.2 Synthesis of antisense strand (AS strand)
By the solid-phase phosphoramidite synthesis method, blank CPG solid-phase carriers are used as initial circulation, and nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each of the linked nucleoside monomers involved four steps of deprotection, coupling, capping, oxidation or thio reactions, 5umol of the antisense strand was synthesized under identical conditions as the sense strand.
1.3 Purification and annealing of oligonucleotides
1.3.1 Ammonolysis
Adding the synthesized solid phase carrier (sense strand or antisense strand) into a 5mL centrifuge tube, adding 3% diethylamine/ammonia water (v/v), reacting for 16 hours (or 8 hours) in a constant temperature water bath at 35 DEG (or 55 DEG), filtering, washing the solid phase carrier with ethanol/water three times, 1mL each time, centrifuging and concentrating the filtrate, and purifying the crude product.
1.3.2 Purification
Methods of purification and desalination are well known to those skilled in the art. For example, the column can be packed with strong anionic packing, the sodium chloride-sodium hydroxide system is used for eluting and purifying, the product is collected and is managed, the gel packing purification column can be used for desalting, and the eluting system is pure water.
1.3.3 Annealing
According to the specification, mixing the sense strand (SS strand) and the antisense strand (AS strand) in a molar ratio (SS strand/AS strand=1/1.05), heating to 70-95 ℃ in a water bath, maintaining for 3-5min, naturally cooling to room temperature, and freeze-drying the system to obtain the product.
Test examples
Example 3 in vivo Activity detection (CNS delivery)
1. Single strand sequence information
In this context, the meanings of the abbreviations are as follows:
A. u, G and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide and cytosine ribonucleotide, respectively.
D indicates that the nucleotide adjacent to the right thereof is deoxyribonucleotide. For example dA, dT, dG and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide and cytosine deoxyribonucleotide, respectively.
I represents inosine ribonucleotides.
M represents that the nucleotide adjacent to the left side thereof is a 2' -OCH 3 modified nucleotide. For example, am, um, gm and Cm represent 2' -OCH 3 modified A, U, G and C.
F represents that the nucleotide adjacent to the left thereof is a 2' -F modified nucleotide. For example, af, uf, gf and Cf represent 2' -F modified A, U, G and C, respectively.
"S" means that two nucleotides adjacent to each other and/or the delivery vehicle are linked by phosphorothioate.
VP indicates that the nucleotide adjacent to the right is a vinyl phosphate modified nucleotide.
Ib represents an inverted abasic deoxyribonucleotide and may include the following three structures depending on the position/linkage thereof in the siRNA.
2. Double-stranded sequences used
3. Experimental method
Experimental animals are SD rats, male, for 6-8 weeks, 2-3 animals per group;
Experimental operation:
After the animals are anesthetized by isoflurane gas, placing an electric blanket with constant temperature, preparing skin on the back of buttocks, sterilizing by using iodophor and 75% alcohol, and applying eye ointment;
Accurately positioning the L3-L5 level, cutting the section of skin to expose the spinal column, puncturing through the intervertebral foramen, slowly pushing 30uL (0.9 mg,30 mg/mL) of corresponding compound after an obvious tail-flick animal (serving as a puncture success mark) is seen, leaving a needle for 15-30s after the injection is finished, and then suturing the skin;
Animal postoperative care, meloxicam and antibiotics via subcutaneous injection;
At 14 days post injection, all animals were euthanized, neck and chest spinal cords were harvested, brain tissue was harvested and cerebellum, brainstem, hippocampus, frontal cortex was isolated, and placed in RNAlater for proper preservation for subsequent SOD1mRNA extraction and QPCR detection.
Methods for mRNA extraction and QPCR detection are well known in the art, and the primer information used is as follows:
The residual inhibition was calculated by the following formula:
A2 -△△Ct value was calculated and converted to a percentage to obtain the residual inhibition. Delta ct= [ (Ct experimental group gene of interest-Ct experimental group internal reference) - (Ct control group gene of interest-Ct control group internal reference) ].
The target gene is SOD1, the internal reference is GAPDH, and the control group is injected with artificial cerebrospinal fluid (aCSF).
4. Experimental results
Residual inhibition data are shown in the following table (see also fig. 1), DR005713, DR005714, DR005715, DR005717, DR005718, DR005716, DR005735 all reduced SOD1 expression in the central nervous system. Wherein the knockdown effect of DR005716 and DR005735 is superior to other sequences.
Example 4 in vivo Activity detection (ocular delivery)
The sequence information used in this example is as follows:
Administration and separation of ocular tissues
C57BL/6 mice (males, 6-8 weeks) were randomly grouped and administered in a single dose by intravitreal injection to each eye at a dose of 2 μg and the siRNA conjugate was administered as a 5mg/mL solution (phosphate buffered saline as solvent), specifically, prior to the experiment, the siRNA conjugate was dissolved and sized to the desired solution concentration and volume with phosphate buffered saline and the administration volume of the siRNA conjugate was 1.5 μl/eye.
Eyeballs were harvested on day14 post-dose and isolated in three parts, ① retinal pigment epithelial cells (RPE) +choroid+sclera, ② retina, ③ cornea+iris+ciliary body, and the isolated samples were immediately frozen in liquid nitrogen and then stored at-80 ℃ for detection of mTTR mRNA.
RNA extraction and detection
Cellular RNA extraction was performed using a nucleic acid extractor (Oriental, hangzhou, auto-pure 96), reverse transcription with reference to PRIMESCRIPT TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B), fluorescent quantitative PCR reaction (ABI, quantStudio 3) with reference to TaqMan TM FAST ADVANCED MASTER Mix (ABI, 4444965) 20. Mu.L system, and detection of primers are shown in the following Table, according to the protocol of the high throughput tissue RNA extraction kit (well known medical science, FG 0412).
Data statistics
Calculating a value of 2 -△△Ct and converting the value into a percentage to obtain a residual inhibition rate;
delta ct= [ (Ct experimental group gene of interest-Ct experimental group internal reference) - (Ct control group gene of interest-Ct control group internal reference) ].
The target gene is mTTR, and the internal reference is mGAPDH.
The experimental results are shown in FIG. 2. The results show that DR005938, DR 007505 and DR007871 can reduce the expression level of TTR gene in eyes, and the reduction effect is equal to or better than that of positive sequence DR 005933.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (33)

式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体： A compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: 其中，in, L1和L2独立地选自H、反应性磷基团、羟基保护基或固相载体； L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support; Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代； Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated; m＝0、1、2、3、4、5或6；m = 0, 1, 2, 3, 4, 5 or 6; R为-C(O)-C0-10亚烷基-L-R1、-C(O)-C2-10亚烯基-L-R1或-C(O)-C2-10亚炔基-L-R1；R is -C(O)-C 0-10 alkylene-LR 1 , -C(O)-C 2-10 alkenylene-LR 1 or -C(O)-C 2-10 alkynylene-LR 1 ; L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-；L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR 1 )CH 2 O-, -C(O)NH-CH(OR 1 )CH 2 O-, -OC(O)-CH(OR 1 )CH 2 O-, -C(O)O-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, -C(O)NH-CH(R 1 )-, -OC(O)-CH(R 1 )-, -C(O)O-CH(R 1 )-, -CH(OR 1 )CH 2 O-, -O-CH(R 1 ) CH 2 O-, -O-CH 2 CH(R 1 )O-, -O-CH(CH(OH)CH 2 OH)-, -O-CH(CH(NH 2 )CH 2 OH)-, -O-CH(CH 2 OH)CH(OH)-, -NH-CH(CH 2 OH)CH(OH)-, -O-CH 2 CH(OH)CH(OH)-, -O-CH 2 CH(NH 2 )CH(OH)-, -NHC(O)-CH 2 -O-CH(CH(OH)CH 2 OH)-, -NHC(O)-CH 2 -O-CH(CH(NH 2 )CH 2 OH)-, -NHC(O)-CH 2 -O-CH(CH 2 OH)CH(OH)-, -NHC(O)-CH 2 -NH-CH(CH 2 OH)CH(OH)-, -NHC(O)-CH 2 -O-CH 2 CH(OH)CH(OH)- or -NHC(O)-CH 2 -O-CH 2 CH(NH 2 )CH(OH)-; R1独立地为C1-30烷基、C2-30烯基或C2-30炔基，其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；R 1 is independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; 其中C0-10亚烷基、C2-10亚烯基、C2-10亚炔基、C1-30烷基、C2-30烯基和C2-30炔基中的氢原子可以任选地被1、2、3、4、5、6、7、8、9、10或更多个卤素、C1-6烷基或C1-6卤代烷基替换，而且其任选地被氘代，直至完全氘代。wherein the hydrogen atoms in the C0-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C1-30 alkyl, C2-30 alkenyl and C2-30 alkynyl groups may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C1-6 alkyl or C1-6 haloalkyl groups, and are optionally deuterated until fully deuterated. 权利要求1的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，R为-C(O)-C0-10亚烷基-L-R1，优选-C(O)-L-R1，优选-C(O)-C2-8亚烷基-L-R1，更优选-C(O)-C3-7亚烷基-L-R1，更优选-C(O)-C4-6亚烷基-L-R1，更优选-C(O)-C1-3亚烷基-L-R1。The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R is -C(O)-C 0-10 alkylene-LR 1 , preferably -C(O)-LR 1 , preferably -C(O)-C 2-8 alkylene-LR 1 , more preferably -C(O)-C 3-7 alkylene-LR 1 , more preferably -C(O)-C 4-6 alkylene-LR 1 , more preferably -C(O)-C 1-3 alkylene-LR 1 . 权利要求1或2的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-，优选化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-，优选化学键、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-，更优选化学键、-NHC(O)-、-S-S-或-NHC(O)O-，更优选-NHC(O)-。 The compound of formula (I) of claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH 2 OH)-, -O-CH(CH(NH 2 )CH 2 OH)-, -O-CH(CH 2 OH)CH(OH)-, -NH-CH(CH 2 OH)CH(OH)-, -O-CH 2 CH( OH )CH(OH)-, -O-CH 2 CH(NH 2 )CH(OH)-, -NHC(O)-CH 2 -O-CH(CH(OH)CH 2 OH)-, -NHC(O)-CH 2 -O-CH(CH(NH 2 )CH 2 OH)-, -NHC(O)-CH 2 -O-CH(CH 2 OH)CH(OH)-, -NHC(O)-CH 2 -NH-CH(CH 2 OH)CH(OH)-, -NHC(O)-CH 2 -O-CH 2 CH(OH)CH(OH)- or -NHC(O)-CH 2 -O-CH 2 CH(NH 2 )CH(OH)-, preferably chemical bonds, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH 2 OH)-, -O-CH(CH(NH 2 )CH 2 Preferably , the amine group is a chemical bond, -NHC(O)-, -SS- , -NHC(O)O-, -O -CH(CH(OH)CH2OH)-, -O-CH(CH( NH2 ) CH2OH )-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, preferably a chemical bond, -NHC(O) - , -SS-, -NHC(O)O-, -O-CH(CH( OH ) CH2OH )-, -O - CH(CH(NH2)CH2OH)-, -NHC(O) -CH2-O -CH(CH(OH)CH2OH)-, or -NHC(O) -CH2 -O-CH(CH( NH2 ) CH2OH )-, more preferably a chemical bond, -NHC(O)-, -SS-, or -NHC(O)O-, and more preferably -NHC(O)-. 权利要求1或2的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，L为-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-，优选-NHC(O)-CH(OR1)CH2O-、-NHC(O)-CH(R1)-或-CH(OR1)CH2O-，更优选-NHC(O)-CH(OR1)CH2O-。The compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is -NHC(O)-CH(OR 1 )CH 2 O-, -C(O)NH-CH(OR 1 )CH 2 O-, -OC(O)-CH(OR 1 )CH 2 O-, -C(O)O-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, -C(O)NH-CH(R 1 )-, -OC(O)-CH(R 1 )-, -C(O)O-CH(R 1 )-, -CH(OR 1 )CH 2 O-, -O-CH(R 1 )CH 2 O-, -O-CH 2 CH(R 1 )O-, preferably -NHC(O)-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )- or -CH(OR 1 )CH 2 O-, more preferably -NHC(O)-CH(OR 1 )CH 2 O-. 权利要求1-4中任一项的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，R1独立地为C1-30烷基或C2-30烯基，其中所述基团中不相邻的1、2、3、4、5、6、7或8个碳原子可以被选自O、S和N的杂原子替换，或者-CH2CH2-基团可以被-NHC(O)-或-C(O)NH-替换，或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环；优选地，R1独立地为C5-25烷基、含有1、2、3、4、5或6个双键的C10-25烯基、其中1、2、3、4或5个碳原子被N杂原子替换和/或1、2或3个-CH2CH2-基团被-C(O)NH-替换的C5-25烷基、或者一个或多个碳原子上的取代基连接成甾体环的C5- 25烷基；优选地，R1选自以下基团：The compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1 is independently C 1-30 alkyl or C 2-30 alkenyl, wherein 1, 2, 3, 4, 5, 6, 7 or 8 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be connected to form a saturated or unsaturated ring; preferably, R 1 is independently C 5-25 alkyl, C 10-25 alkenyl containing 1, 2, 3, 4, 5 or 6 double bonds, C 10-25 alkenyl wherein 1, 2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and/or 1, 2 or 3 -CH 2 CH 2 - groups are replaced by -C(O)NH-; Preferably , R 1 is selected from the following groups: C6烷基、C8烷基、C11烷基、C12烷基、C13烷基、C15烷基、C16烷基、C17烷基、C21烷基、 C6 alkyl, C8 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C21 alkyl, 权利要求1-5中任一项的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，L1和L2之一为反应性磷基团，优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物，例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯，优选-P(OCH2CH2CN)(N(iPr)2)。A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein one of L 1 and L 2 is a reactive phosphorus group, preferably a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a thiophosphate, a dithiophosphate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a thiophosphate diester, a thiophosphate triester, a diphosphate or a triphosphate, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ). 权利要求1-5中任一项的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，L1和L2选自保护基，优选羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基，更优选-C(O)CH2CH2C(O)OH。The compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L 1 and L 2 are selected from protecting groups, preferably hydroxy protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxy Carbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl, preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH 2 CH 2 C(O)OH. 权利要求1-7中任一项的式(I)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其选自以下通式： A compound of formula (I) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, which is selected from the following general formula: 其中各基团如权利要求1-7所定义。The groups are as defined in claims 1-7. 权利要求1-8中任一项的化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，所述化合物选自以下： The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound is selected from the following: 寡核苷酸，其包含一个或多个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体： An oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: 其中，in, 表示H或羟基保护基，或表示与相邻核苷酸连接的位置； represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2表示H或固相载体，或表示与相邻核苷酸连接的位置；L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide; Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代； Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated; m＝0、1、2、3、4、5或6；m = 0, 1, 2, 3, 4, 5 or 6; R为疏水性基团；R is a hydrophobic group; 优选地， Preferably, 表示H或羟基保护基，或表示与相邻核苷酸连接的位置； represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2表示H或固相载体，或表示与相邻核苷酸连接的位置；L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide; Rs、m和R如权利要求1-5中任一项所定义。R s , m and R are as defined in any one of claims 1 -5. 权利要求10的寡核苷酸，其中所述式(I’)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体： The oligonucleotide of claim 10, wherein the compound of formula (I') is selected from the following compounds of the general formula, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: 其中，in, 表示H或羟基保护基，或表示与相邻核苷酸连接的位置； represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide; L2表示H或固相载体，或表示与相邻核苷酸连接的位置；L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide; 其他各基团如权利要求1-5所定义。The other groups are as defined in claims 1-5. 权利要求10的寡核苷酸，其中所述式(I’)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，所述化合物选自以下： The oligonucleotide of claim 10, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein the compound is selected from the following: 其中之一表示H或羟基保护基，或表示与相邻核苷酸连接的位置，并且另一个表示H或固相载体，或表示与相邻核苷酸连接的位置。in One of them represents H or a hydroxyl protecting group, or represents the position of attachment to the adjacent nucleotide, and the other represents H or a solid phase support, or represents the position of connection with the adjacent nucleotide. 权利要求10-12中任一项的寡核苷酸，其具有14至30个核苷酸。The oligonucleotide of any one of claims 10 to 12, having 14 to 30 nucleotides. 权利要求10-13中任一项的寡核苷酸，其在5’端包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。An oligonucleotide according to any one of claims 10 to 13, comprising at the 5' end a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 权利要求10-14中任一项的寡核苷酸，其在3’端包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。An oligonucleotide according to any one of claims 10 to 14, comprising at the 3' end a compound of formula (I') according to any one of claims 10 to 12, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 权利要求10-15中任一项的寡核苷酸，其在5’端和3’端分别包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。An oligonucleotide according to any one of claims 10 to 15, comprising a compound of formula (I') according to any one of claims 10 to 12 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 寡核苷酸，其在寡核苷酸内部、5’端和/或3’端包含两个或多个疏水性基团；优选地，所述疏水性基团如式(I)化合物中R基团所定义；优选地，所述疏水性基团通过接头，例如可生物降解的接头，连接至寡核苷酸上。An oligonucleotide comprising two or more hydrophobic groups within the oligonucleotide, at the 5' end and/or at the 3' end; preferably, the hydrophobic groups are defined as the R groups in the compound of formula (I); preferably, the hydrophobic groups are connected to the oligonucleotide via a linker, such as a biodegradable linker. 双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链包含一个或多个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体： A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: 其中， in, 表示H，或表示与相邻核苷酸连接的位置； represents H, or indicates the position of connection with the adjacent nucleotide; L2表示H或羟基保护基，或表示与相邻核苷酸连接的位置；L 2 represents H or a hydroxyl protecting group, or represents the position for connecting to the adjacent nucleotide; Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代； Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated; m＝0、1、2、3、4、5或6；m = 0, 1, 2, 3, 4, 5 or 6; R为疏水性基团；R is a hydrophobic group; 优选地，Preferably, 表示H，或表示与相邻核苷酸连接的位置； represents H, or indicates the position of connection with the adjacent nucleotide; L2表示H或羟基保护基，或表示与相邻核苷酸连接的位置；L 2 represents H or a hydroxyl protecting group, or represents the position for connecting to the adjacent nucleotide; Rs、m和R如权利要求1-5中任一项所定义。R s , m and R are as defined in any one of claims 1 -5. 权利要求18的双链RNA，其中所述式(I’)化合物选自以下通式化合物，或其药学上可接受的盐、互变异构体或立体异构体： The double-stranded RNA of claim 18, wherein the compound of formula (I') is selected from the following compounds of the general formula, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: 其中，in, 表示H，或表示与相邻核苷酸连接的位置； represents H, or indicates the position of connection with the adjacent nucleotide; L2表示H或羟基保护基，或表示与相邻核苷酸连接的位置；L 2 represents H or a hydroxyl protecting group, or represents the position for connecting to the adjacent nucleotide; Rs、m和R如权利要求1-5所定义。 Rs , m and R are as defined in claims 1-5. 权利要求18的双链RNA，其中所述式(I’)化合物选自以下化合物，或其药学上可接受的盐、互变异构体或立体异构体，其中，所述化合物选自以下： The double-stranded RNA of claim 18, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein the compound is selected from the following: 其中之一表示H，或表示与相邻核苷酸连接的位置，并且另一个表示H或羟基保护基，或与相邻核苷酸连接的位置。in One of them represents H, or represents the position of connection with the adjacent nucleotide, and the other indicates H or a hydroxyl protecting group, or the position of attachment to the adjacent nucleotide. 权利要求18-20中任一项的双链RNA，其中所述正义链在5’端包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。The double-stranded RNA of any one of claims 18 to 20, wherein the sense strand comprises a compound of formula (I') of any one of claims 10 to 12 at the 5' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 权利要求18-21中任一项的双链RNA，其中所述正义链在3’端包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。The double-stranded RNA of any one of claims 18 to 21, wherein the sense strand comprises a compound of formula (I') of any one of claims 10 to 12 at the 3' end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 权利要求18-22中任一项的双链RNA，其中所述正义链在5’端和3’端分别包含一个权利要求10-12中任一项的式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体。A double-stranded RNA according to any one of claims 18 to 22, wherein the sense strand comprises a compound of formula (I') according to any one of claims 10 to 12 at the 5' end and the 3' end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 权利要求18-23中任一项的双链RNA，其中所述正义链和/或反义链上的两个或多个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体之间间隔至少5-30个核苷酸。The double-stranded RNA of any one of claims 18 to 23, wherein two or more compounds of formula (I') on the sense strand and/or antisense strand, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof are separated by at least 5 to 30 nucleotides. 权利要求18-24中任一项的双链RNA，其中所述双链RNA包含两个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其位于以下位点中的任两个：正义链的5’端、正义链的3’ 端、反义链的5’端和反义链的3’端；优选位于正义链的5’端和正义链的3’端。A double-stranded RNA according to any one of claims 18 to 24, wherein the double-stranded RNA comprises two compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any two of the following sites: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand; preferably located at the 5' end of the sense strand and the 3' end of the sense strand. 权利要求18-25中任一项的双链RNA，其中所述双链RNA包含三个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其位于以下位点中的任三个：正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端；优选位于正义链的5’端、正义链的3’端和反义链的3’端。The double-stranded RNA of any one of claims 18 to 25, wherein the double-stranded RNA comprises three compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any three of the following sites: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand; preferably located at the 5' end of the sense strand, the 3' end of the sense strand and the 3' end of the antisense strand. 权利要求18-26中任一项的双链RNA，其中所述双链RNA包含四个式(I’)化合物，或其药学上可接受的盐、互变异构体或立体异构体，其位于以下位点：正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端。The double-stranded RNA of any one of claims 18 to 26, wherein the double-stranded RNA comprises four compounds of formula (I'), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, located at the following sites: the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the antisense strand. 权利要求18-26中任一项的双链RNA，其进一步在所述反义链的5’端偶联末端磷酸保护基团或前药保护基团，优选乙烯基磷酸酯基或式(X)所示的前药保护基团： The double-stranded RNA according to any one of claims 18 to 26, further comprising a terminal phosphate protecting group or a prodrug protecting group coupled to the 5' end of the antisense strand, preferably a vinyl phosphate group or a prodrug protecting group represented by formula (X): 其中，in, X1选自OH或 X1 is selected from OH or Ra选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；R a is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated; Rb和Rc独立地选自H、C1-6烷基或C1-6卤代烷基，所述Rb和Rc可任选地被D、C6-10芳基或5-10元杂芳基取代，直至完全氘代；R b and R c are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, and said R b and R c may be optionally substituted by D, C 6-10 aryl or 5-10 membered heteroaryl until fully deuterated; X2是与所述反义链的5’端的第一个核苷酸连接的化学键，优选通过羟基连接； X2 is a chemical bond connected to the first nucleotide at the 5' end of the antisense strand, preferably connected through a hydroxyl group; X3独立地选自O或S； X3 is independently selected from O or S; T选自 T is selected from 每个RT1独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C1-6卤代烷基、C2-6烯基、C2- 6炔基或包含GalNAc的链，其任选地被氘代，直至完全氘代；each R T1 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, or a chain comprising GalNAc, which is optionally deuterated, up to fully deuterated ; 每个RT2独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；Each R T2 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated; 每个RT3独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；Each R T3 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated; 每个RT4独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；Each R T4 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated; m为0、1、2、3、4或5；m is 0, 1, 2, 3, 4 or 5; n为0、1、2、3、4或5；n is 0, 1, 2, 3, 4 or 5; p为0、1、2、3、4或5；p is 0, 1, 2, 3, 4 or 5; X选自化学键、-O-、-S-、-C(O)-、-C(O)O-、-OC(O)-、-OC(O)NRX1-、-NRX1C(O)O-、-NRX1C(O)-或-C(O)NRX1-；X is selected from a chemical bond, -O-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O) NRX1- , -NRX1C (O)O-, -NRX1C (O)- or -C(O) NRX1- ; RX1选自H、C1-6烷基或C1-6卤代烷基，其任选地被氘代，直至完全氘代； RX1 is selected from H, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated; L为-Ar-(CH2)1-6-O-，其中每一个CH2可任选地被R#取代，R#选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；L is -Ar-(CH 2 ) 1-6 -O-, wherein each CH 2 may be optionally substituted by R#, R# is selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated; L中的Ar与X相连，氧原子与磷原子相连；Ar in L is connected to X, and the oxygen atom is connected to the phosphorus atom; Ar选自C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基，所述C3-10环烷基、3-10元杂环基、C6-10芳基或5-10元杂芳基可任选被1个、2个、3个、4个或5个R*取代；Ar is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, wherein the C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl may be optionally substituted with 1, 2, 3, 4 or 5 R*; R*选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基，其任选地被氘代，直至完全氘代；R* is selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated up to fully deuterated; 其中P1选自保护基，优选羟基保护基，例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、4,4'-二甲氧基三苯甲基、-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH，优选-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH。wherein P1 is selected from a protecting group, preferably a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Allo c), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4′-dimethoxytrityl, —P(OCH 2 CH 2 CN)(N(iPr) 2 ) or —C(O)CH 2 CH 2 C(O)OH, preferably —P(OCH 2 CH 2 CN)(N(iPr) 2 ) or —C(O)CH 2 CH 2 C(O)OH. 权利要求18-28中任一项的双链RNA，其选自小干扰RNA(siRNA)和短发夹RNA(shRNA)，优选用于抑制在眼部表达的基因。The double-stranded RNA according to any one of claims 18 to 28, which is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA), preferably for inhibiting a gene expressed in the eye. 双链RNA，其具有正义链和反义链，各链具有14至30个核苷酸，所述反义链包含与所述正义链和靶标mRNA充分互补的序列，其中所述正义链和/或反义链在内部、5’端和/或3’端包含两个或多个疏水性基团；优选地，所述疏水性基团如式(I)化合物中R基团所定义；优选地，所述疏水性基团通过接头，例如可生物降解的接头，连接至正义链和/或反义链上。A double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is fully complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises two or more hydrophobic groups at the interior, 5' end and/or 3' end; preferably, the hydrophobic group is as defined as the R group in the compound of formula (I); preferably, the hydrophobic group is connected to the sense strand and/or the antisense strand via a linker, such as a biodegradable linker. 细胞，其含有如权利要求18-30中任一项所述的双链RNA。A cell comprising the double-stranded RNA according to any one of claims 18 to 30. 药物组合物，其包含如权利要求18-30中任一项所述的双链RNA、或如权利要求31所述的细胞，以及任选的药学上可接受的载剂或赋形剂。A pharmaceutical composition comprising the double-stranded RNA according to any one of claims 18 to 30, or the cell according to claim 31, and optionally a pharmaceutically acceptable carrier or excipient. 试剂盒，其包含如权利要求18-30中任一项所述的双链RNA、或如权利要求31所述的细胞。 A kit comprising the double-stranded RNA according to any one of claims 18 to 30, or the cell according to claim 31.
CN202380076440.2A 2022-10-31 2023-10-30 Ligands for delivery of SIRNA to the eye and central nervous system Pending CN120282953A (en)

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