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TW202516005A - Sirna, conjugate which contain sirna, pharmaceutical composition and uses thereof - Google Patents

Sirna, conjugate which contain sirna, pharmaceutical composition and uses thereof Download PDF

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TW202516005A
TW202516005A TW113123276A TW113123276A TW202516005A TW 202516005 A TW202516005 A TW 202516005A TW 113123276 A TW113123276 A TW 113123276A TW 113123276 A TW113123276 A TW 113123276A TW 202516005 A TW202516005 A TW 202516005A
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管小陽
欣 曾
祝力
澤 劉
宗宏
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美商翰森生物有限責任公司
大陸商上海翰森生物醫藥科技有限公司
大陸商常州恆邦藥業有限公司 中國江蘇省常州市新北區遼河路1028號 郵編：213001
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Abstract

The invention relates to siRNA, e.g., double stranded ribonucleic acid (dsRNA), that interfere with target gene expression or inhibit its expression. The invention also relates to a conjugate or a pharmaceutical composition containing the siRNA. And the use of the siRNA, the conjugate and the pharmaceutical composition thereof in preparing a drug used for treating and/or preventing the disease or disorder.

Description

SIRNA，含有SIRNA的綴合物，醫藥組成物及其用途 SIRNA, conjugates containing SIRNA, pharmaceutical compositions and their uses

本揭露涉及一種能夠抑制補體成分C3基因表達的核酸、含有該核酸的醫藥組成物和siRNA綴合物，屬於核酸藥物領域。本揭露還涉及該核酸、醫藥組成物和siRNA綴合物的製備方法和用途。 The present disclosure relates to a nucleic acid capable of inhibiting the expression of complement component C3 gene, a pharmaceutical composition containing the nucleic acid, and a siRNA complex, belonging to the field of nucleic acid drugs. The present disclosure also relates to the preparation method and use of the nucleic acid, pharmaceutical composition, and siRNA complex.

補體途徑是宿主抵禦入侵病原體的先天免疫系統的一部分。補體主要由一系列以前體形式在血流中循環或存在於細胞膜上的蛋白質組成。補體的激活會導致級聯酶促反應，從而形成強效過敏毒素，引發一系列生理反應，包括趨化作用和細胞死亡。補體蛋白C3是補體級聯的紐帶。C3水解形成與細胞表面結合的高反應性C3b。這種結合導致順序形成多個複合物，從而觸發所謂的“替代途徑(AP)”的自動擴增，最終導致炎症反應，導致吞噬細胞吸引、調理作用，從而清除病原體、免疫複合物和細胞碎片。補體的不適當激活與許多疾病的傳播和/或引發有關，包括C3相關疾病(例如，C3腎小球病)、陣發性睡眠性血紅蛋白尿(PNH)、非典型溶血性尿毒癥綜合症(aHUS)、類風濕性關節炎、缺血再灌注損傷和神經退行性疾病。因此，能夠抑制補體系統的治療劑(包括抑制C3活性的治 療劑)可以被證明是有益的。目前，可用於治療補體成分C3相關疾病的療法有限，這些療法需要耗時且侵入性施用，成本高昂。因此，本領域需要為患有補體成分C3相關疾病的受試者提供替代療法。 The complement pathway is part of the host's innate immune system that defends against invading pathogens. Complements are primarily composed of a series of proteins that circulate in the bloodstream in precursor form or are present on the cell membrane. Activation of complements leads to a cascade of enzymatic reactions, resulting in the formation of potent allergic toxins, which trigger a series of physiological reactions, including chemotaxis and cell death. The complement protein C3 is the backbone of the complement cascade. C3 is hydrolyzed to form highly reactive C3b that binds to the cell surface. This binding leads to the sequential formation of multiple complexes, which triggers the auto-expansion of the so-called "alternative pathway (AP)", ultimately leading to an inflammatory response, resulting in the attraction and opsonization of phagocytes, thereby clearing pathogens, immune complexes and cell debris. Inappropriate activation of complement components has been implicated in the propagation and/or initiation of numerous diseases, including C3-related diseases (e.g., C3 glomerulopathy), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. Therefore, therapeutics that can inhibit the complement component system, including those that inhibit C3 activity, may prove beneficial. Currently, there are limited therapies available for the treatment of complement component C3-related diseases that require time-consuming and invasive administration and are costly. Therefore, there is a need in the art to provide alternative treatments for subjects suffering from complement component C3-related diseases.

基於RNA干擾(RNAi)機制的小干擾RNA(siRNA)，能夠以序列特異性的方式抑制或阻斷靶基因的表達，從而達到靶向抑制的特異性，達到治療疾病的目的。顯然，藉由調節C3的mRNA水平來降低C3基因的表達，是阻斷補體蛋白產生、維持正常免疫功能、防止異常免疫反應的有效途徑。 Small interfering RNA (siRNA) based on the RNA interference (RNAi) mechanism can inhibit or block the expression of target genes in a sequence-specific manner, thereby achieving the specificity of targeted inhibition and the purpose of treating diseases. Obviously, reducing the expression of the C3 gene by regulating the mRNA level of C3 is an effective way to block the production of complement proteins, maintain normal immune function, and prevent abnormal immune responses.

開發抑制C3基因表達的siRNA藥物的關鍵在於尋找合適的靶標選擇性siRNA序列，用於化學穩定性的合適修飾及其有效的遞送系統。由於在分泌到血液之前，C3表達主要侷限於肝細胞中，因此其是siRNA治療的理想靶標。 The key to developing siRNA drugs that inhibit C3 gene expression lies in finding the right target-selective siRNA sequence, suitable modification for chemical stability and its effective delivery system. Since C3 expression is mainly confined to liver cells before secretion into the blood, it is an ideal target for siRNA therapy.

本發明的目的在於提供一種用於抑制C3基因表達的siRNA、含有該siRNA作為藥物活性成分的醫藥組成物和siRNA綴合物、利用該siRNA、該醫藥組成物或該siRNA綴合物抑制C3基因表達的方法以及該siRNA、該醫藥組成物或該siRNA綴合物在治療和/或預防C3相關疾病中的用途。 The purpose of the present invention is to provide a siRNA for inhibiting C3 gene expression, a pharmaceutical composition and a siRNA complex containing the siRNA as a pharmaceutical active ingredient, a method for inhibiting C3 gene expression using the siRNA, the pharmaceutical composition or the siRNA complex, and the use of the siRNA, the pharmaceutical composition or the siRNA complex in the treatment and/or prevention of C3-related diseases.

即，本發明藉由提供以下技術方案來實現上述目的。 That is, the present invention achieves the above-mentioned purpose by providing the following technical solutions.

在一個方面，本發明提供了一種用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中dsRNA包含第一鏈和第二鏈，其中第一鏈序列包含至少15個連續核苷酸，其與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482- 2500或4698-4716中的任一核苷酸序列相差不超過3個核苷酸；並且第二鏈包含與第一鏈至少部分互補的核苷酸序列。 In one aspect, the present invention provides a siRNA agent having a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the dsRNA comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, which differs from any of the nucleotide sequences of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

在一個方面，第一鏈序列包含至少16個，較佳至少17個，更佳至少18個，最佳全部19個連續核苷酸，其與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過2個核苷酸，較佳相差不超過1個核苷酸，更佳沒有任何核苷酸差異。 In one aspect, the first strand sequence comprises at least 16, preferably at least 17, more preferably at least 18, and most preferably all 19 consecutive nucleotides, which differ from any of the nucleotide sequences of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 2 nucleotides, preferably by no more than 1 nucleotide, and more preferably by no more than 1 nucleotide.

在一個方面，本發明提供了一種用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中核酸包含第一鏈和第二鏈，其中第一鏈序列包含至少15個連續核苷酸的序列，其與序列SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188中的任一相差不超過3個核苷酸。 In one aspect, the present invention provides a siRNA agent having a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 consecutive nucleotides, which is identical to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 ,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110, Any of 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 differs by no more than 3 nucleotides.

在一個方面，siRNA劑是核酸，其中，第一鏈序列分別包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、 134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188的序列；以及視需要地，其中第二鏈序列分別包含SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189的序列。 In one aspect, the siRNA agent is a nucleic acid wherein the first strand sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 14, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second chain sequence comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 ,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,10 9, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, The sequence of 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

本發明的一個方面涉及一種siRNA試劑，其中第一鏈和第二鏈存在於核酸的單鏈上，第一鏈和第二鏈能夠相互雜交，從而形成具有雙鏈區的雙鏈核酸。 One aspect of the present invention relates to an siRNA reagent, wherein a first chain and a second chain are present on a single strand of nucleic acid, and the first chain and the second chain can hybridize with each other to form a double-stranded nucleic acid having a double-stranded region.

在一個方面，第一鏈和第二鏈形成15-25個核苷酸長的雙鏈區；較佳形成15-23個核苷酸長的雙鏈區；更佳形成15-19個核苷酸長的雙鏈區；進一步佳形成17-19個核苷酸長的雙鏈區；最佳形成15、16、17、18、19、20、21、22、23個核苷酸長的雙鏈區。 In one aspect, the first chain and the second chain form a double-chain region of 15-25 nucleotides in length; preferably, a double-chain region of 15-23 nucleotides in length; more preferably, a double-chain region of 15-19 nucleotides in length; further preferably, a double-chain region of 17-19 nucleotides in length; and most preferably, a double-chain region of 15, 16, 17, 18, 19, 20, 21, 22, 23 nucleotides in length.

在一個方面，核酸的第一鏈和第二鏈是分開的鏈。兩條分開的鏈較佳各為15-25個核苷酸長，更佳為17-25個核苷酸長。兩條鏈的長度可以相同或不同。第一鏈可以為17-25個核苷酸長，較佳地，其可以為18-24個核苷酸長，其可以為18、19、20、21、22、23或24個核苷酸長。最佳地，第一鏈為19個核苷酸長。第二鏈可以獨立地為17-25個核苷酸長，較佳地，其可以為18-24個 核苷酸長，其可以為18、19、20、21、22、23或24個核苷酸長。更佳地，第二鏈為18或19或20個核苷酸長，最佳地，其是19個核苷酸長。 In one aspect, the first and second strands of the nucleic acid are separate strands. The two separate strands are preferably 15-25 nucleotides long, more preferably 17-25 nucleotides long. The lengths of the two strands can be the same or different. The first strand can be 17-25 nucleotides long, preferably, it can be 18-24 nucleotides long, it can be 18, 19, 20, 21, 22, 23 or 24 nucleotides long. Optimally, the first strand is 19 nucleotides long. The second strand can independently be 17-25 nucleotides long, preferably, it can be 18-24 nucleotides long, it can be 18, 19, 20, 21, 22, 23 or 24 nucleotides long. More preferably, the second strand is 18 or 19 or 20 nucleotides long, optimally, it is 19 nucleotides long.

在一個方面，siRNA劑是核酸，其中，第一鏈序列具有分別由SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188表示的核苷酸序列；以及視需要地，其中第二鏈具有分別由SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189表示的核苷酸序列。 In one aspect, the siRNA agent is a nucleic acid wherein the first strand sequence has the sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, , 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second strand has a nucleotide sequence represented by SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 1 11, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 represent the nucleotide sequence.

在一個方面，第一鏈具有分別由SEQ ID NO：20、96、16、150、32、100、110、134、136表示的核苷酸序列。 In one aspect, the first chain has a nucleotide sequence represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively.

在一個方面，與第一鏈互補的第二鏈具有分別由SEQ ID NO：21、97、17、151、33、101、111、135、137表示的核苷酸序列。 In one aspect, the second chain complementary to the first chain has a nucleotide sequence represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, 137, respectively.

在一個方面，第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In one aspect, the first chain has a nucleotide sequence represented by SEQ ID NO: 20 and the second chain has a nucleotide sequence represented by SEQ ID NO: 21; or the first chain has a nucleotide sequence represented by SEQ ID NO: 96 and the second chain has a nucleotide sequence represented by SEQ ID NO: 97; or the first chain has a nucleotide sequence represented by SEQ ID NO: 16 and the second chain has a nucleotide sequence represented by SEQ ID NO: 17; or the first chain has a nucleotide sequence represented by SEQ ID NO: 150 and the second chain has a nucleotide sequence represented by SEQ ID NO: 151; or the first chain has a nucleotide sequence represented by SEQ ID NO: 32 and the second chain has a nucleotide sequence represented by SEQ ID NO: 33; or the first chain has a nucleotide sequence represented by SEQ ID NO: 100 and the second chain has a nucleotide sequence represented by SEQ ID NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 120 and the second chain has a nucleotide sequence represented by SEQ ID NO: 121. The nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在一個方面，siRNA劑包含至少一個修飾的核苷酸，較佳地，其中第一鏈的基本上全部核苷酸都是修飾的核苷酸，第二鏈的基本上全部核苷酸都是修飾的核苷酸；更佳地，其中第一鏈的全部核苷酸都是修飾的核苷酸，第二鏈的全部核苷酸都是修飾的核苷酸。 In one aspect, the siRNA agent comprises at least one modified nucleotide, preferably, wherein substantially all nucleotides of the first strand are modified nucleotides and substantially all nucleotides of the second strand are modified nucleotides; more preferably, wherein all nucleotides of the first strand are modified nucleotides and all nucleotides of the second strand are modified nucleotides.

如本文所用，“基本上全部核苷酸都是修飾的”是大部分但不全部是修飾的，包括不超過5、4、3、2或1個未修飾的核苷酸。 As used herein, "substantially all nucleotides are modified" means that most but not all nucleotides are modified, including no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

在一個方面，修飾的核苷酸選自3’末端脫氧胸腺嘧啶(dT)核苷酸、2'-OCF2H修飾的核苷酸、2'-甲氧基修飾的核苷酸、2’-氟修飾的核苷酸、包含5'-硫代磷酸酯基團的核苷酸和包含5'-(E)-乙烯基膦酸酯的核苷酸。 In one aspect, the modified nucleotides are selected from 3' terminal deoxythymidine (dT) nucleotides, 2'- OCF2H modified nucleotides, 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing a 5'-phosphorothioate group and nucleotides containing a 5'-(E)-vinylphosphonate.

在一個方面，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾，5’端起第2、4、6、8、12、14、16和18位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾，5’端起第1、3、5、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、6、8、14和16位核苷酸殘基的戊糖經2’-氟取代修飾，5’端起第1、3、5、7、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。5’端起第4、9、10和18位經2’-甲氧基修飾或2’-氟取代修飾。 In one aspect, all nucleotides of the first strand comprise the following pattern of modification: pentoses from the 5' end at nucleotide residues 7, 9, 10 and 11 are modified by 2'-fluorine substitution, pentoses from the 5' end at nucleotide residues 2, 4, 6, 8, 12, 14, 16 and 18 are modified by 2'-methoxy modification, and pentoses from the 5' end at nucleotide residues 1, 3, 5, 13, 15, 17 and 19 are modified by 2'-methoxy or 2'-fluorine substitution. All nucleotides of the second strand comprise the following pattern of modification: pentoses from the 5' end at nucleotide residues 2, 6, 8, 14 and 16 are modified by 2'-fluorine substitution, and pentoses from the 5' end at nucleotide residues 1, 3, 5, 7, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification. The 4th, 9th, 10th and 18th positions from the 5' end are modified by 2'-methoxy or 2'-fluorine substitution.

在一個方面，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第3、11和14位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第4、8和10位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾得到修飾。 In one aspect, all nucleotides of the first strand comprise the following pattern of modifications: the pentoses at the 5', 7', 8' and 9' nucleotide residues are modified by 2'-fluorine substitution; the pentoses at the 5', 2', 4', 6', 10', 12', 13', 15', 16', 17', 18' and 19' nucleotide residues are modified by 2'-methoxy; the pentoses at the 5', 3', 11' and 14' nucleotide residues are modified by 2'-methoxy or 2'-fluorine substitution. All nucleotides in the second strand contain the following pattern of modifications: the pentoses at the 5' end of the nucleotide residues at positions 2, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; the pentoses at the 5' end of the nucleotide residues at positions 4, 8 and 10 are modified by 2'-methoxy modification or 2'-fluorine substitution modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、5、6、8、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、6、8、9、14和16位核苷酸殘基的戊糖經2’-氟取代；5’端起第1、3、4、5、7、 10、11、12、13、15、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 7th, 9th, 10th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 6th, 8th, 9th, 14th and 16th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 15th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 3, 5, 7, 8, and 9 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 2, 4, 14, 16, and 18 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 8th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、 7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 5, 7, 8, 9 and 11 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 2, 4, 14, 16 and 18 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 8th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 4th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、 6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 3, 5, 7, 8, and 9 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 2, 10, 14, 16, and 18 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 3, 5, 7, 8, and 9 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 2, 8, 14, 16, and 18 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 5, 7, 8, 9, and 11 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses at the 5' end of the nucleotide residues at positions 2, 10, 14, 16, and 18 are modified by 2'-fluorine substitution; pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

在某些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、 6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In certain embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification. All nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 10th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一個方面，第一鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵。 In one aspect, the first strand comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end.

在某些實施方案中，第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 3' terminus; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在某些實施方案中，第一鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在某些實施方案中，第一鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵，和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在一個方面，第二鏈在5’端包含末端5’(E)-乙烯基膦酸酯核苷酸。 In one aspect, the second strand comprises a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end.

在本發明的說明書中，“相同或共同修飾”是指對任何核苷酸的相同修飾，是用基團，如甲基(2'-OMe)或氟基(2’-F)修飾的A、G、C或U。例如，2'-F-dU、2'-F-dA、2'-F-dC、2'-F-dG都被視為相同或共同修飾，2'-OMe-rU、2’-OMe-rA；2'-OMe-rC；2'-OMe-rG也是如此。相反，2’-F修飾與2’-OMe修飾相比是不同的修飾。 In the specification of the present invention, "same or common modification" refers to the same modification of any nucleotide, which is A, G, C or U modified with a group such as methyl (2'-OMe) or fluoro (2'-F). For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, 2'-F-dG are all considered the same or common modification, as are 2'-OMe-rU, 2'-OMe-rA; 2'-OMe-rC; 2'-OMe-rG. In contrast, 2'-F modification is a different modification than 2'-OMe modification.

較佳地，核酸的第一鏈和/或第二鏈的至少一個核苷酸是修飾的核苷酸，較佳是非自然存在的核苷酸，如較佳是2’-F或2’-OMe修飾的核苷酸。 Preferably, at least one nucleotide of the first strand and/or the second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, such as preferably a 2'-F or 2'-OMe modified nucleotide.

在某些實施方案中，siRNA劑還包含靶向肝組織的靶向配體。 In certain embodiments, the siRNA agent further comprises a targeting ligand that targets liver tissue.

在一個方面，靶向配體是GalNAc衍生物。 In one aspect, the targeting ligand is a GalNAc derivative.

在一個方面，靶向配體是GalNAc綴合物。 In one aspect, the targeting ligand is a GalNAc complex.

在一個方面，靶向配體綴合至siRNA劑的第一鏈的3’末端或5’末端。 In one aspect, the targeting ligand is conjugated to the 3' end or the 5' end of the first strand of the siRNA agent.

在某些實施方案中，靶向配體綴合至siRNA劑的第一鏈的3’末端。在某些實施方案中，靶向配體綴合至siRNA劑的第一鏈的5’末端。 In certain embodiments, the targeting ligand is conjugated to the 3' end of the first strand of the siRNA agent. In certain embodiments, the targeting ligand is conjugated to the 5' end of the first strand of the siRNA agent.

在一個方面，siRNA劑： In one aspect, a siRNA agent:

(i)包含與第一鏈的3’末端核苷酸或5’末端核苷酸綴合的靶向配體； (i) comprising a targeting ligand linked to the 3' terminal nucleotide or the 5' terminal nucleotide of the first chain;

(ii)在與靶向配體綴合的一個末端相對的第一鏈另一個末端的三個核苷酸之間有硫代磷酸酯鍵； (ii) There is a phosphorothioate bond between the three nucleotides at the other end of the first strand opposite to the end ligated to the targeting ligand;

(iii)在第二鏈的末端三個3’核苷酸和末端三個5’核苷酸之間具有硫代磷酸酯鍵；以及 (iii) having phosphorothioate bonds between the terminal three 3' nucleotides and the terminal three 5' nucleotides of the second strand; and

(iv)視需要地，第一鏈和/或第二鏈的核苷酸之間的所有剩餘鍵均為磷酸二酯鍵。 (iv) Optionally, all remaining bonds between nucleotides in the first strand and/or the second strand are phosphodiester bonds.

在某些實施方案中，靶向配體與第一鏈的3’末端綴合；第一鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵，第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵以及在3’末端包含兩個硫代磷酸酯核苷酸間鍵，視需要地，第一鏈和/或第二鏈的核苷酸之間的所有剩餘鍵均為磷酸二酯鍵。 In certain embodiments, the targeting ligand is conjugated to the 3' terminus of the first strand; the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus, the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and optionally, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

在某些實施方案中，靶向配體與第一鏈的5’末端綴合；第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵，第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵以及在3’末端包含兩個硫代磷酸酯核苷酸間鍵，視需要地，第一鏈和/或第二鏈的核苷酸之間的所有剩餘鍵均為磷酸二酯鍵。 In certain embodiments, the targeting ligand is conjugated to the 5' terminus of the first strand; the first strand comprises two phosphorothioate internucleotide bonds at the 3' terminus, the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and optionally, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

在一個方面，第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：208)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 In one aspect, the first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO：212)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO： 212)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO：213)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：214)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：214)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second chain has a nucleotide sequence represented by (E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：216)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO：221)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：222)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO： 222)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：223)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：223)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列； The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217);

其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵，“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分，“GN”是靶向配體。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond, "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end, and "GN" is a targeting ligand.

在一個方面，siRNA劑與配體綴合，如下圖所示：配體包括(i)一個或多個N-乙醯半乳糖胺(GalNAc)部分或其衍生物，和(ii)接頭，其中接頭將至少一個GalNAc部分或其衍生物與核酸綴合，和(iii)雙環基團連接GalNAc和siRNA，或(iv)四官能團，其中連接GalNAc部分，其中X是O或S。 In one aspect, the siRNA agent is conjugated to a ligand as shown in the following figure: the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid, and (iii) a bicyclic group connecting the GalNAc and the siRNA, or (iv) a tetrafunctional group, wherein the GalNAc moiety is connected, wherein X is O or S.

Figure 113123276-A0202-12-0018-1
Figure 113123276-A0202-12-0018-1

或者 or

Figure 113123276-A0202-12-0018-2
Figure 113123276-A0202-12-0018-2

在一個方面，圖中的每個接頭視需要地相同或不同。 In one aspect, each connector in the figure may be the same or different as desired.

在一個方面，圖中的接頭獨立地選自： In one aspect, the connectors in the figure are independently selected from:

(1)-(CH2)x-C(O)NH-(CH2)y-C(O)-；(2)-(CH2)x-C(O)NH-(CH2)y-NHC(O)-(CH2)z-C(O)-；(3)-NH-(CH2)y-C(O)-；(4)-C(O)-(CH2)z-C(O)-；x、y和z獨立地選自1-10；較佳地，x和y獨立地選自2-8，z選自1-6；更佳地，x和y獨立地選自3-6，z選自1-4。 (1) -(CH 2 )xC(O)NH-(CH 2 )yC(O)-; (2) -(CH 2 )xC(O)NH-(CH 2 )y-NHC(O)-(CH 2 )zC(O)-; (3) -NH-(CH 2 )yC(O)-; (4) -C(O)-(CH 2 )zC(O)-; x, y and z are independently selected from 1-10; preferably, x and y are independently selected from 2-8, and z is selected from 1-6; more preferably, x and y are independently selected from 3-6, and z is selected from 1-4.

在一個方面，siRNA劑與配體綴合，如下圖所示： In one aspect, the siRNA agent is conjugated to a ligand, as shown below:

Figure 113123276-A0202-12-0019-3
Figure 113123276-A0202-12-0019-3

其中X是O或S； Where X is O or S;

Figure 113123276-A0202-12-0019-84
是：
Figure 113123276-A0202-12-0019-84
yes:

Figure 113123276-A0202-12-0019-5
Figure 113123276-A0202-12-0019-5

該

Figure 113123276-A0202-12-0020-6
是： The
Figure 113123276-A0202-12-0020-6
yes:

Figure 113123276-A0202-12-0020-7
Figure 113123276-A0202-12-0020-7

Figure 113123276-A0202-12-0021-8
Figure 113123276-A0202-12-0021-8

Figure 113123276-A0202-12-0022-9
Figure 113123276-A0202-12-0022-9

在一個方面，siRNA劑與配體綴合，如下圖所示： In one aspect, the siRNA agent is conjugated to a ligand, as shown below:

Figure 113123276-A0202-12-0023-10
Figure 113123276-A0202-12-0023-10

其中X是O或S， Where X is O or S,

Figure 113123276-A0202-12-0023-12
是：
Figure 113123276-A0202-12-0023-12
yes:

Figure 113123276-A0202-12-0023-13
Figure 113123276-A0202-12-0023-13

Figure 113123276-A0202-12-0024-14
Figure 113123276-A0202-12-0024-14

Figure 113123276-A0202-12-0025-15
Figure 113123276-A0202-12-0025-15

Figure 113123276-A0202-12-0026-16
Figure 113123276-A0202-12-0026-16

在一個方面，siRNA劑與配體綴合，如下圖所示： In one aspect, the siRNA agent is conjugated to a ligand, as shown below:

Figure 113123276-A0202-12-0027-17
Figure 113123276-A0202-12-0027-17

四官能團選自： Four functional groups are selected from:

Figure 113123276-A0202-12-0027-18
Figure 113123276-A0202-12-0027-18

Figure 113123276-A0202-12-0028-19
Figure 113123276-A0202-12-0028-19

在某些實施方案中，GalNAc綴合物是一種或多種GalNAc衍生物，視需要地藉由接頭或載體與雙鏈RNA劑綴合，一種或多種GalNAc衍生物視需要地藉由接頭或載體與雙鏈RNAi劑綴合。 In certain embodiments, the GalNAc conjugate is one or more GalNAc derivatives, optionally conjugated to a double-stranded RNA agent via a linker or a carrier, and one or more GalNAc derivatives are optionally conjugated to a double-stranded RNAi agent via a linker or a carrier.

在某些實施方案中，siRNA劑與配體綴合，如下圖所示： In certain embodiments, the siRNA agent is conjugated to a ligand, as shown in the following figure:

Figure 113123276-A0202-12-0029-20
Figure 113123276-A0202-12-0029-20

Figure 113123276-A0202-12-0030-21
Figure 113123276-A0202-12-0030-21

Figure 113123276-A0202-12-0031-22
Figure 113123276-A0202-12-0031-22

Figure 113123276-A0202-12-0032-23
Figure 113123276-A0202-12-0032-23

Figure 113123276-A0202-12-0033-24
Figure 113123276-A0202-12-0033-24

Figure 113123276-A0202-12-0034-25
Figure 113123276-A0202-12-0034-25

Figure 113123276-A0202-12-0035-26
Figure 113123276-A0202-12-0035-26

Figure 113123276-A0202-12-0036-27
Figure 113123276-A0202-12-0036-27

Figure 113123276-A0202-12-0037-28
Figure 113123276-A0202-12-0037-28

Figure 113123276-A0202-12-0038-29
Figure 113123276-A0202-12-0038-29

Figure 113123276-A0202-12-0039-30
Figure 113123276-A0202-12-0039-30

Figure 113123276-A0202-12-0040-31
Figure 113123276-A0202-12-0040-31

Figure 113123276-A0202-12-0041-32
Figure 113123276-A0202-12-0041-32

其中X是O或S。 Where X is O or S.

在某些實施方案中，siRNA劑與配體綴合，如下圖所示： In certain embodiments, the siRNA agent is conjugated to a ligand, as shown in the following figure:

Figure 113123276-A0202-12-0041-35
Figure 113123276-A0202-12-0041-35

Figure 113123276-A0202-12-0041-37
Figure 113123276-A0202-12-0041-37

Figure 113123276-A0202-12-0042-38
Figure 113123276-A0202-12-0042-38

Figure 113123276-A0202-12-0042-39
Figure 113123276-A0202-12-0042-39

Figure 113123276-A0202-12-0042-40
Figure 113123276-A0202-12-0042-40

Figure 113123276-A0202-12-0042-41
Figure 113123276-A0202-12-0042-41

Figure 113123276-A0202-12-0043-42
Figure 113123276-A0202-12-0043-42

Figure 113123276-A0202-12-0043-43
Figure 113123276-A0202-12-0043-43

Figure 113123276-A0202-12-0043-44
Figure 113123276-A0202-12-0043-44

Figure 113123276-A0202-12-0043-45
Figure 113123276-A0202-12-0043-45

Figure 113123276-A0202-12-0044-46
Figure 113123276-A0202-12-0044-46

Figure 113123276-A0202-12-0044-47
Figure 113123276-A0202-12-0044-47

Figure 113123276-A0202-12-0044-48
Figure 113123276-A0202-12-0044-48

Figure 113123276-A0202-12-0044-49
Figure 113123276-A0202-12-0044-49

Figure 113123276-A0202-12-0044-50
Figure 113123276-A0202-12-0044-50

Figure 113123276-A0202-12-0045-51
Figure 113123276-A0202-12-0045-51

Figure 113123276-A0202-12-0045-52
Figure 113123276-A0202-12-0045-52

Figure 113123276-A0202-12-0045-53
Figure 113123276-A0202-12-0045-53

Figure 113123276-A0202-12-0045-54
Figure 113123276-A0202-12-0045-54

Figure 113123276-A0202-12-0045-55
Figure 113123276-A0202-12-0045-55

Figure 113123276-A0202-12-0046-56
Figure 113123276-A0202-12-0046-56

Figure 113123276-A0202-12-0046-57
Figure 113123276-A0202-12-0046-57

Figure 113123276-A0202-12-0046-58
Figure 113123276-A0202-12-0046-58

Figure 113123276-A0202-12-0046-59
Figure 113123276-A0202-12-0046-59

Figure 113123276-A0202-12-0046-60
Figure 113123276-A0202-12-0046-60

Figure 113123276-A0202-12-0046-61
Figure 113123276-A0202-12-0046-61

Figure 113123276-A0202-12-0047-62
Figure 113123276-A0202-12-0047-62

Figure 113123276-A0202-12-0047-63
Figure 113123276-A0202-12-0047-63

Figure 113123276-A0202-12-0047-64
Figure 113123276-A0202-12-0047-64

Figure 113123276-A0202-12-0047-65
Figure 113123276-A0202-12-0047-65

Figure 113123276-A0202-12-0047-66
Figure 113123276-A0202-12-0047-66

Figure 113123276-A0202-12-0048-67
Figure 113123276-A0202-12-0048-67

Figure 113123276-A0202-12-0048-68
Figure 113123276-A0202-12-0048-68

Figure 113123276-A0202-12-0048-69
Figure 113123276-A0202-12-0048-69

Figure 113123276-A0202-12-0048-70
Figure 113123276-A0202-12-0048-70

Figure 113123276-A0202-12-0049-71
Figure 113123276-A0202-12-0049-71

Figure 113123276-A0202-12-0049-72
Figure 113123276-A0202-12-0049-72

Figure 113123276-A0202-12-0049-73
Figure 113123276-A0202-12-0049-73

在較佳的實施方案中，siRNA劑與配體綴合，如下圖所示： In a preferred embodiment, the siRNA agent is conjugated to a ligand, as shown in the following figure:

Figure 113123276-A0202-12-0050-74
Figure 113123276-A0202-12-0050-74

Figure 113123276-A0202-12-0050-75
Figure 113123276-A0202-12-0050-75

在一個方面，第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：224)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 In one aspect, the first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO: 225); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID N：228)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID N: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：228)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO：229)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：230)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：230)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：232)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 232), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：234)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：234)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO： 237)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列； The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP- mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233);

其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵；“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond; "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end.

在一個方面，醫藥組成物包含本文揭露的siRNA劑，或其藥學上可接受的鹽或溶劑化物，以及溶劑(較佳水)和/或遞送載劑和/或生理學上可接受的賦形劑和/或藥學上可接受的載體和/或鹽和/或稀釋劑和/或緩衝劑和/或防腐劑。 In one aspect, the pharmaceutical composition comprises the siRNA agent disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent (preferably water) and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a pharmaceutically acceptable carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.

在一個方面，醫藥組成物包含本文揭露的siRNA劑和選自寡核苷酸、小分子、單株抗體、多株抗體和肽中的另一種治療劑。 In one aspect, the pharmaceutical composition comprises the siRNA agent disclosed herein and another therapeutic agent selected from oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

在一個方面，醫藥組成物包含本文揭露的試劑和藥學上可接受的載體。 In one aspect, a pharmaceutical composition comprises an agent disclosed herein and a pharmaceutically acceptable carrier.

本揭露的另一方面提供了一種抑制細胞(如神經細胞或眼細胞)中C3基因表達的方法，該方法包括：(a)將細胞與本揭露的雙鏈RNAi劑或本揭露的醫藥組成物接觸；以及(b)將步驟(a)中產生的細胞維持足夠的時間以獲得C3基因的mRNA轉錄物的降解，從而抑制細胞中C3基因的表達。 Another aspect of the present disclosure provides a method for inhibiting the expression of C3 gene in cells (such as nerve cells or eye cells), the method comprising: (a) contacting the cells with the double-chain RNAi agent disclosed herein or the pharmaceutical composition disclosed herein; and (b) maintaining the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the C3 gene, thereby inhibiting the expression of the C3 gene in the cells.

本揭露的另一方面提供了一種治療患有將受益於補體成分C3表達降低的疾病或病症的受試者的方法，該方法包括向受試者施用治療有效量的上述siRNA劑或上述醫藥組成物，從而治療該受試者。 Another aspect of the present disclosure provides a method for treating a subject suffering from a disease or condition that would benefit from reduced expression of complement component C3, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned siRNA agent or the above-mentioned pharmaceutical composition, thereby treating the subject.

要用本文揭露的核酸或組成物治療的疾病或病症是補體介導的疾病、病症或綜合症，較佳地，該疾病或病症是C3相關疾病或病症。 The disease or condition to be treated with the nucleic acid or composition disclosed herein is a complement-mediated disease, condition or syndrome, preferably, the disease or condition is a C3-related disease or condition.

要用本文揭露的核酸或組成物治療的疾病或病症較佳選自IgA腎病(IgAN)、C3腎小球病(C3G)、陣發性睡眠性血紅蛋白尿(PNH)、非典型溶血性尿毒癥綜合症(aHUS)、視神經脊髓炎(NMO)、多灶性運動神經病(MMN)、重症肌無力(MG)。 The diseases or conditions to be treated with the nucleic acid or composition disclosed herein are preferably selected from IgA nephropathy (IgAN), C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), and myasthenia gravis (MG).

本發明提供的siRNA可以序列特異性地介導對C3基因表達的抑制。該基因可以位於細胞內，例如，受試者的細胞內，如人的細胞內。不受理論的束縛，相信上述特性的組合或子組合以及這些iRNA中的特定靶位點或特定修飾賦予本發明的iRNA改善的療效、穩定性、效力、持久性和安全性。 The siRNA provided by the present invention can mediate sequence-specific inhibition of C3 gene expression. The gene can be located in a cell, for example, in a subject's cell, such as a human cell. Without being bound by theory, it is believed that the combination or subcombination of the above-mentioned properties and the specific target sites or specific modifications in these iRNAs give the iRNA of the present invention improved efficacy, stability, potency, durability and safety.

圖1顯示表2A中的siRNA對C3 mRNA表達水平的抑制效果的檢測結果。 Figure 1 shows the results of the test on the inhibitory effect of siRNA in Table 2A on the expression level of C3 mRNA.

圖2A顯示9個選擇的siRNA在HepG2細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 2A shows the dose-response curves of the C3 mRNA attenuation effects of nine selected siRNAs in HepG2 cells.

圖2B顯示9個選擇的siRNA在HepG2細胞中的C3 mRNA減弱效果的檢測結果，濃度為100nM和0.16nM。 Figure 2B shows the results of the detection of C3 mRNA attenuation effects of 9 selected siRNAs in HepG2 cells at concentrations of 100nM and 0.16nM.

圖3A顯示9個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的檢測結果，濃度為1000nM和1nM。 Figure 3A shows the results of the detection of C3 mRNA attenuation effects of 9 selected GalNAc-conjugated siRNAs in PHH cells at concentrations of 1000nM and 1nM.

圖3B顯示四個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 3B shows the dose-response curves of four selected GalNAc-conjugated siRNAs for attenuation of C3 mRNA in PHH cells.

圖4A顯示二十個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 4A shows the dose-response curves of twenty selected GalNAc-conjugated siRNAs for C3 mRNA attenuation in PHH cells.

圖4B顯示二十個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 4B shows the dose-response curves of twenty selected GalNAc-conjugated siRNAs for C3 mRNA attenuation in PHH cells.

圖4C顯示二十個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 4C shows the dose-response curves of twenty selected GalNAc-conjugated siRNAs for C3 mRNA attenuation in PHH cells.

圖4D顯示二十個選擇的GalNAc綴合的siRNA在PHH細胞中的C3 mRNA減弱效果的劑量反應曲線。 Figure 4D shows the dose-response curves of twenty selected GalNAc-conjugated siRNAs for C3 mRNA attenuation in PHH cells.

圖5A顯示兩個選擇的GalNAc綴合的siRNA在人C3轉基因小鼠(雄性)中血清人C3蛋白降低效果的檢測結果。 Figure 5A shows the results of the detection of the serum human C3 protein-lowering effect of two selected GalNAc-ligated siRNAs in human C3 transgenic mice (male).

圖5B顯示兩個選擇的GalNAc綴合的siRNA在人C3轉基因小鼠(雌性)中血清人C3蛋白降低效果的檢測結果。 Figure 5B shows the results of the detection of the serum human C3 protein-lowering effect of two selected GalNAc-ligated siRNAs in human C3 transgenic mice (female).

圖6顯示測試的綴合的siRNA降低人C3蛋白效果的檢測結果。 Figure 6 shows the results of the test combined siRNA to reduce the effect of human C3 protein.

圖7顯示測試的不同劑量的GalNAc綴合的siRNA降低人C3蛋白效果的檢測結果。 Figure 7 shows the results of the test of different doses of GalNAc-conjugated siRNA to reduce the effect of human C3 protein.

本發明藉由以下具體描述進一步說明。 The present invention is further described in detail below.

本發明提供一種siRNA劑，其影響RNA誘導沉默複合物(RISC)介導的補體成分C3(C3)基因的RNA轉錄物的切割。該基因可以位於細胞內，例如，受試者的細胞內，如人的細胞內。使用這些iRNA能夠靶向降解哺乳動物中相應基因(C3)的mRNA。 The present invention provides a siRNA agent that affects RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of complement component C3 (C3) gene. The gene can be located in a cell, for example, in a subject's cell, such as a human cell. The use of these siRNAs can target and degrade the mRNA of the corresponding gene (C3) in mammals.

本發明的siRNA劑被設計為靶向人補體成分C3(C3)基因，包括該基因在其他哺乳動物物種的C3直系同源物中保守的部分。本發明的siRNA劑具有改進的療效、穩定性、效力、持久性和安全性。 The siRNA agent of the present invention is designed to target the human complement component C3 (C3) gene, including the portion of the gene that is conserved in the C3 orthologs of other mammalian species. The siRNA agent of the present invention has improved efficacy, stability, potency, durability and safety.

I.核酸序列 I. Nucleic acid sequence

本發明的第一方面是用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中核酸包含第一鏈和第二鏈，其中第一鏈序列包含至少15個連續核苷酸，其與表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中的第一鏈的任一核苷酸序列相差不超過3個核苷酸。 The first aspect of the present invention is a siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the first strand in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F by no more than 3 nucleotides.

較佳地，第一鏈序列包含至少16個，更佳至少17個，還更佳至少18個以及最佳全部19個連續核苷酸，其與表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中所示的第一鏈序列中的任一的相差不超過3個核苷酸，較佳不超過2個核苷酸，更佳不超過1個核苷酸，最佳沒有任何核苷酸差異。 Preferably, the first strand sequence comprises at least 16, more preferably at least 17, still more preferably at least 18 and most preferably all 19 consecutive nucleotides, which differ from any of the first strand sequences shown in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, Table 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference.

本發明還提供了一種用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中該核酸包含第一鏈和第二鏈，其中第一鏈序列包含至少15個連續核苷酸，其與表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中的第一鏈的任一核苷酸序列相差不超過3個核苷；以及第二鏈序列包含至少15個連續核苷酸，其與表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中的第二鏈的任一核苷酸序列的相差不超過3個核苷酸。 The present invention also provides a siRNA agent having a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the first strand in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F by no more than 3 nucleotides; and the second strand sequence comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the second strand in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F by no more than 3 nucleotides.

較佳地，該第一鏈序列包含至少16個，更佳至少17個，仍更佳至少18個以及最佳全部19個連續核苷酸，其與表1、表2A、表3A、表3B、表 3C、表3D、表3E、表3F中任一中所示的任一第一鏈序列相差不超過3個核苷酸，較佳不超過2個核苷酸，更佳不超過1個核苷酸，最佳沒有任何核苷酸差異；第二鏈序列包含至少16個，更佳至少17個，仍更佳至少18個以及最佳全部19個連續核苷酸，其與表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中所示的任一第二鏈序列相差不超過3個核苷酸，較佳不超過2個核苷酸，更佳不超過1個核苷酸，最佳沒有任何核苷酸差異。 Preferably, the first chain sequence comprises at least 16, more preferably at least 17, still more preferably at least 18 and best all 19 consecutive nucleotides, which differ from any first chain sequence shown in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and best no nucleotide difference; the second chain sequence comprises at least 16, more preferably at least 17, still more preferably at least 18 and best all 19 consecutive nucleotides, which differ from any second chain sequence shown in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and best no nucleotide difference.

本發明還提供了一種用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中該核酸包含第一鏈和第二鏈，其中第一鏈序列包含選自表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中的第一鏈的任一核苷酸序列的核苷酸序列；以及第二鏈包含選自表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一中的第二鏈的任一核苷酸序列的核苷酸序列。 The present invention also provides a siRNA agent having a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a nucleotide sequence selected from any nucleotide sequence of the first strand in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F; and the second strand comprises a nucleotide sequence selected from any nucleotide sequence of the second strand in any of Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, and Table 3F.

應當理解，儘管例如表1中的序列未被描述為修飾或綴合的序列，但本發明的siRNA劑的dsRNA(例如本發明的dsRNA)可以包含表1、表2A、表3A、表3B、表3C、表3D、表3E、表3F中任一所示的任一序列，其可以是未修飾、未綴合的，或與其中該相比為不同的修飾或綴合。換句話說，本發明涵蓋未修飾的、未綴合的、修飾的或綴合的表2-7中的dsRNA，如本文所述。 It should be understood that although the sequences in Table 1, for example, are not described as modified or conjugated sequences, the dsRNA of the siRNA agent of the present invention (e.g., the dsRNA of the present invention) may include any sequence shown in Table 1, Table 2A, Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, Table 3F, which may be unmodified, unconjugated, or modified or conjugated differently than the sequence in Table 2-7. In other words, the present invention encompasses unmodified, unconjugated, modified, or conjugated dsRNAs in Tables 2-7, as described herein.

此外，本發明提供了用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中第一鏈序列包含與下列序列中的任一相差不超過3個核苷酸的至少15個連續核苷酸的序列：SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、 84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188；以及視需要地，其中第二鏈序列包含與下列序列中的任一相差不超過3個核苷酸的至少15個連續核苷酸的序列：SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189。 In addition, the present invention provides a siRNA agent having a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the first strand sequence comprises a sequence of at least 15 consecutive nucleotides that differs from any of the following sequences by no more than 3 nucleotides: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 ,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 , 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second strand sequence comprises a sequence of at least 15 consecutive nucleotides that differs by no more than 3 nucleotides from any of the following sequences: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 5 7, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 1 09, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

在一些實施方案中，第一鏈序列包含至少16個，更佳至少17個，還更佳至少18個以及最佳全部19個連續核苷酸，其與以下序列中的任一相差不超過3個核苷酸，較佳不超過2個核苷酸，更佳不超過1個核苷酸，以及最佳沒有任何核苷酸差異：SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188；以及第二鏈序列包含至少16個，更佳至少17個，還更佳 至少18個以及最佳全部19個連續核苷酸，其與以下序列中的任一相差不超過3個核苷酸，較佳不超過2個核苷酸，更佳不超過1個核苷酸，以及最佳沒有任何核苷酸差異：SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189。 In some embodiments, the first strand sequence comprises at least 16, preferably at least 17, even more preferably at least 18 and most preferably all 19 consecutive nucleotides, which differ from any of the following sequences by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide differences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 7 4, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 13 4, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and the second strand sequence comprises at least 16, preferably at least 17, still more preferably at least 18 and most preferably all 19 consecutive nucleotides, which differ from any of the following sequences by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 5 7, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 1 09, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

在一些實施方案中，siRNA劑是核酸，其中，第一鏈序列分別包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186、188的序列；以及視需要地，其中第二鏈序列分別包含SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123、125、127、129、131、133、135、137、 139、141、143、145、147、149、151、153、155、157、159、161、163、165、167、169、171、173、175、177、179、181、183、185、187、189的序列。 In some embodiments, the siRNA agent is a nucleic acid wherein the first strand sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 14, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second chain sequence comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 ,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109 ,111,113,115,117,119,121,123,125,127,129,131,133,135,137, The sequence of 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

II.修飾的SIRNA劑 II. Modified SIRNA Agents

本發明的第二個方面是針對多聚核酸分子的特定修飾模式，該多聚核酸分子是包含第一鏈和第二鏈的雙鏈RNA(dsRNA)。 The second aspect of the present invention is directed to a specific modification pattern of a polynucleic acid molecule, which is a double-stranded RNA (dsRNA) comprising a first strand and a second strand.

在一個方面，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈和第二鏈包含至少一個修飾的核苷酸，較佳地，其中第一鏈的基本上全部核苷酸都是修飾的核苷酸，第二鏈的基本上全部核苷酸都是修飾的核苷酸；更佳地，其中第一鏈的全部核苷酸都是修飾的核苷酸，第二鏈的全部核苷酸都是修飾的核苷酸。 In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand and the second strand comprise at least one modified nucleotide, preferably, wherein substantially all nucleotides of the first strand are modified nucleotides, and substantially all nucleotides of the second strand are modified nucleotides; more preferably, wherein all nucleotides of the first strand are modified nucleotides, and all nucleotides of the second strand are modified nucleotides.

如本文所用，“基本上全部核苷酸都是修飾的”是大部分但不全部都是修飾的，包括不超過5、4、3、2或1個未修飾的核苷酸。 As used herein, "substantially all nucleotides are modified" means that most but not all nucleotides are modified, including no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中，第一鏈的全部核苷酸包含如下模式的修飾：從5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾，5’端起第2、4、6、8、12、14、16和18位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第1、3、5、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、6、8、14和16位核苷酸殘基的戊糖經2’-氟取代修飾，5’端起第1、3、5、7、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。5’端起第4、9、10和18位經2’-甲氧基修飾或2’-氟取代修飾。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein all nucleotides of the first strand comprise the following pattern of modification: the pentoses of the 7th, 9th, 10th and 11th nucleotide residues from the 5' end are modified by 2'-fluorine substitution, and the pentoses of the 2nd, 4th, 6th, 8th, 12th, 14th, 16th and 18th nucleotide residues from the 5' end are modified by 2'-methoxy modification; The pentoses at the 1st, 3rd, 5th, 13th, 15th, 17th and 19th nucleotide residues from the 5’ end are modified by 2’-methoxy or 2’-fluorine substitution; all nucleotides in the second chain contain the following pattern of modification: the pentoses at the 2nd, 6th, 8th, 14th and 16th nucleotide residues from the 5’ end are modified by 2’-fluorine substitution, and the pentoses at the 1st, 3rd, 5th, 7th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues from the 5’ end are modified by 2’-methoxy. The 4th, 9th, 10th and 18th nucleotide residues from the 5’ end are modified by 2’-methoxy or 2’-fluorine substitution.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第3、11和14位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第4、8和10位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾得到修飾。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein all nucleotides of the first strand comprise the following pattern of modifications: the pentoses at the 5th, 7th, 8th and 9th nucleotide residues from the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 4th, 6th, 10th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification; the pentoses at the 3rd, 11th and 14th nucleotide residues from the 5' end are modified by 2'-methoxy modification; The pentose of the nucleotide residue is modified by 2’-methoxy or 2’-fluorine substitution; all nucleotides of the second chain contain the following pattern of modification: the pentose of the nucleotide residues at positions 2, 14, 16 and 18 from the 5’ end is modified by 2’-fluorine substitution; the pentose of the nucleotide residues at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17 and 19 from the 5’ end is modified by 2’-methoxy; the pentose of the nucleotide residues at positions 4, 8 and 10 from the 5’ end is modified by 2’-methoxy or 2’-fluorine substitution.

在一些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)，補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In some embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈的全部核苷酸含有如下 模式的修飾：5’端起第5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾，5’端起第1、2、4、6、10、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第3、11和14位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；5’端起第4、8和10位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾；其中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein all nucleotides of the first strand contain the following pattern of modifications: the pentoses at the 5th, 7th, 8th and 9th nucleotide residues from the 5' end are modified by 2'-fluorine substitution, the pentoses at the 1st, 2nd, 4th, 6th, 10th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxyl modification; the pentoses at the 3rd, 11th and 14th nucleotide residues from the 5' end are modified by 2'-methoxyl modification or 2'-fluorine substitution; the second strand All nucleotides of the double-stranded RNA (dsRNA) agent comprise the following pattern of modification: the pentoses at the 2nd, 14th, 16th and 18th nucleotide residues from the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 3rd, 5th, 6th, 7th, 9th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues from the 5' end are modified by 2'-methoxyl modification; the pentoses at the 4th, 8th and 10th nucleotide residues from the 5' end are modified by 2'-methoxyl modification or 2'-fluorine substitution; wherein the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a sequence of SEQ The first chain has a nucleotide sequence represented by SEQ ID NO: 20 and the second chain has a nucleotide sequence represented by SEQ ID NO: 21; or the first chain has a nucleotide sequence represented by SEQ ID NO: 96 and the second chain has a nucleotide sequence represented by SEQ ID NO: 97; or the first chain has a nucleotide sequence represented by SEQ ID NO: 16 and the second chain has a nucleotide sequence represented by SEQ ID NO: 17; or the first chain has a nucleotide sequence represented by SEQ ID NO: 150 and the second chain has a nucleotide sequence represented by SEQ ID NO: 151; or the first chain has a nucleotide sequence represented by SEQ ID NO: 32 and the second chain has a nucleotide sequence represented by SEQ ID NO: 33; or the first chain has a nucleotide sequence represented by SEQ ID NO: 100 and the second chain has a nucleotide sequence represented by SEQ ID NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 120 and the second chain has a nucleotide sequence represented by SEQ ID NO: 121. NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、5、6、8、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、6、8、9、14和16位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、7、10、11、12、13、15、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses from the 5' end of the 7th, 9th, 10th and 11th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end of the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses from the 5' end of the 2nd, 6th, 8th, 9th, 14th and 16th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end of the 1st, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 15th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses from the 5' end at the 3rd, 5th, 7th, 8th and 9th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 2nd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses from the 5' end at the 2nd, 4th, 14th, 16th and 18th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 8th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 4th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 8th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 4th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses from the 5' end at the 3rd, 5th, 7th, 8th and 9th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 2nd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses from the 5' end at the 2nd, 10th, 14th, 16th and 18th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses from the 5' end at the 3rd, 5th, 7th, 8th and 9th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 2nd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses from the 5' end at the 2nd, 8th, 14th, 16th and 18th nucleotide residues are modified by 2'-fluorine substitution; pentoses from the 5' end at the 1st, 3rd, 4th, 5th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 10th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在一些實施方案中，第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 In some embodiments, all nucleotides of the first chain contain the following pattern of modifications: pentoses starting from the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; all nucleotides of the second chain contain the following pattern of modifications: pentoses starting from the 2nd, 10th, 14th, 16th and 18th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; pentoses starting from the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification.

在某些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；其中，雙鏈RNA(dsRNA)劑的靶基因選自補體成分C3(C3)、補體成分C5(C5)，補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、 KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4；較佳地，是補體成分C3(C3)。在較佳的實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：150表示的核苷酸序列以及第二鏈具有由SEQ ID NO：151表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：32表示的核苷酸序列以及第二鏈具有由SEQ ID NO：33表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：100表示的核苷酸序列以及第二鏈具有由SEQ ID NO：101表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：110表示的核苷酸序列以及第二鏈具有由SEQ ID NO：111表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：134表示的核苷酸序列以及第二鏈具有由SEQ ID NO：135表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：136表示的核苷酸序列以及第二鏈具有由SEQ ID NO：137表示的核苷酸序列。 In certain embodiments, all nucleotides of the first strand of a double-stranded RNA (dsRNA) agent comprise the following pattern of modifications: the pentoses at the 5', 7', 8', 9' and 11' nucleotide residues are modified by 2'-fluorine substitution; the pentoses at the 1', 2', 3', 4', 6', 10', 12', 13', 14', 15', 16', 17', 18' and 19' nucleotide residues are modified by 2'-methoxy modification; and all nucleotides of the second strand comprise the following pattern of modifications: the pentoses at the 2', 8', 14', 16' and 18' nucleotide residues are modified by 2'-fluorine substitution; substitution modification; the pentose of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 at the 5' end is modified by 2'-methoxy modification; wherein the target gene of the double-stranded RNA (dsRNA) agent is selected from complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, A SGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG-1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SC AP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53, CD4, CD8, CD40, CD71, DUX4, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4; preferably, it is complement component C3 (C3). In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and a nucleotide sequence represented by SEQ ID NO: 21 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and a nucleotide sequence represented by SEQ ID NO: 97 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and a nucleotide sequence represented by SEQ ID NO: 17 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 150 and a nucleotide sequence represented by SEQ ID NO: 151 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 32 and a nucleotide sequence represented by SEQ ID NO: 33 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 100 and a nucleotide sequence represented by SEQ ID NO: 101 in the second strand; NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 134 and the second chain has a nucleotide sequence represented by SEQ ID NO: 135; or the first chain has a nucleotide sequence represented by SEQ ID NO: 136 and the second chain has a nucleotide sequence represented by SEQ ID NO: 137.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnxnNfnNfNfNfnxnnxnnnnn-3’；第二鏈包含5’-nNfnxnnnxnxnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnxnNfnNfNfnfnxnnxnnnnn-3'; the second strand comprises 5'-nNfnxnnnxnnnxnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluorine- or 2'-O-methyl-modified nucleotide.

在一些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)，補體因子B(CFB)、PCSK9、TTR、AGT、LPA、 Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In some embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RI G-1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP5 3. CD4, CD8, CD40, CD71, DUX4, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnxnNfnNfNfNfnxnnxnnnnn-3’；第二鏈包含5’-nNfnxnnnxnxnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸；其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：150表示的核苷酸序列以及第二鏈具有由SEQ ID NO：151表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：32表示的核苷酸序列以及第二鏈具有由SEQ ID NO：33表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：100表示的核苷酸序列以及第二鏈具有由SEQ ID NO：101表示的核苷酸序列；或者第 一鏈具有由SEQ ID NO：110表示的核苷酸序列以及第二鏈具有由SEQ ID NO：111表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：134表示的核苷酸序列以及第二鏈具有由SEQ ID NO：135表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：136表示的核苷酸序列以及第二鏈具有由SEQ ID NO：137表示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnxnNfnNfNfnfnxnnxnnnnn-3'; the second strand comprises 5'-nNfnxnnnxnnnxnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide and "x" represents a 2'-fluorine or 2'-O-methyl-modified nucleotide; wherein the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: NO:21; or the first chain has a nucleotide sequence represented by SEQ ID NO:96 and the second chain has a nucleotide sequence represented by SEQ ID NO:97; or the first chain has a nucleotide sequence represented by SEQ ID NO:16 and the second chain has a nucleotide sequence represented by SEQ ID NO:17; or the first chain has a nucleotide sequence represented by SEQ ID NO:150 and the second chain has a nucleotide sequence represented by SEQ ID NO:151; or the first chain has a nucleotide sequence represented by SEQ ID NO:32 and the second chain has a nucleotide sequence represented by SEQ ID NO:33; or the first chain has a nucleotide sequence represented by SEQ ID NO:100 and the second chain has a nucleotide sequence represented by SEQ ID NO:101; or the first chain has a nucleotide sequence represented by SEQ ID NO:110 and the second chain has a nucleotide sequence represented by SEQ ID NO:111; or the first chain has a nucleotide sequence represented by SEQ ID NO:152 and the second chain has a nucleotide sequence represented by SEQ ID NO:153; NO: 134 and the second chain has a nucleotide sequence represented by SEQ ID NO: 135; or the first chain has a nucleotide sequence represented by SEQ ID NO: 136 and the second chain has a nucleotide sequence represented by SEQ ID NO: 137.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnNfnNfnNfNfNfnnnnnnnnnn-3’；第二鏈包含5’-nNfnnnnnnnnNfnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfnnnnnnnnn-3'; the second strand comprises 5'-nNfnnnnnnnnNfnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnNfnNfnNfNfNfnnnnnnnnnn-3’；第二鏈包含5’-nNfnnnnnNfnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfnnnnnnnnn-3'; the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnNfnNfnNfNfNfnnnnnnnnnn-3’；第二鏈包含5’-nNfnNfnnnnnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfnnnnnnnnn-3'; the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnNfnnnnnnnn-3’；第二鏈包含5’-nNfnnnnnnnnNfnnnNfnNfnNfn- 3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnnnnnn-3'; the second strand comprises 5'-nNfnnnnnnnnNfnNfnNfnNfn- 3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnNfnnnnnnnn-3’；第二鏈包5’-nNfnnnnnNfnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnnnnnn-3'; the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnNfnnnnnnnn-3’；第二鏈包含5’-nNfnNfnnnnnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnnnnnn-3'; the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnnnnNfnnnnn-3’；第二鏈包5’-nNfnnnnnnnNfnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnnnnNfnnnnn-3'; the second strand comprises 5'-nNfnnnnnnnNfnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnnnnNfnnnnn-3’；第二鏈包含5’-nNfnnnnnNfnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnnnnNfnnnnn-3'; the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnnnnNfnnnnn-3’；第二鏈包含5’-nNfnNfnnnnnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnnnNfnnnnn-3'; the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-nnnnnnNfnNfNfNfnnnnnnnn-3’；第二鏈包含5’-nNfnnnNfnNfNfnnnnNfnNfnnn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnnnNfnNfNfNfnnnnnnnn-3'; the second strand comprises 5'-nNfnnnNfNfnnnNfnNfnnn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide.

在一些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)，補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In some embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，包括第一鏈和第二鏈，其中第一鏈包含5’-nnnnNfnNfNfNfnNfnnnnnnnn-3’；第二鏈包含5’-nNfnnnnnNfnnnnnNfnNfnNfn-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者 第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfNfnNfnnnnnn-3'; the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: 21; or the first strand has a nucleotide sequence represented by SEQ ID NO: NO:96 and the second chain has the nucleotide sequence represented by SEQ ID NO:97; or the first chain has the nucleotide sequence represented by SEQ ID NO:16 and the second chain has the nucleotide sequence represented by SEQ ID NO:17; or the first chain has the nucleotide sequence represented by SEQ ID NO:150 and the second chain has the nucleotide sequence represented by SEQ ID NO:151; or the first chain has the nucleotide sequence represented by SEQ ID NO:32 and the second chain has the nucleotide sequence represented by SEQ ID NO:33; or the first chain has the nucleotide sequence represented by SEQ ID NO:100 and the second chain has the nucleotide sequence represented by SEQ ID NO:101; or the first chain has the nucleotide sequence represented by SEQ ID NO:110 and the second chain has the nucleotide sequence represented by SEQ ID NO:111; or the first chain has the nucleotide sequence represented by SEQ ID NO:134 and the second chain has the nucleotide sequence represented by SEQ ID NO:135; or the first chain has the nucleotide sequence represented by SEQ ID NO:136 The nucleotide sequence shown in NO: 136 and the second chain have the nucleotide sequence shown in SEQ ID NO: 137.

在一些實施方案中，第一鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵。 In some embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end.

在某些實施方案中，第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 3' terminus; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在某些實施方案中，第一鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵；和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus; and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在某些實施方案中，第一鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵，和/或第二鏈在5'末端包含兩個硫代磷酸酯核苷酸間鍵和在3'末端包含兩個硫代磷酸酯核苷酸間鍵。 In certain embodiments, the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and/or the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus.

在一些實施方案中，siRNA劑還包含靶向肝組織的靶向配體。 In some embodiments, the siRNA agent also comprises a targeting ligand that targets liver tissue.

在一些實施方案中，靶向配體是GalNAc衍生物。在一個方面，靶向配體是GalNAc綴合物。 In some embodiments, the targeting ligand is a GalNAc derivative. In one aspect, the targeting ligand is a GalNAc complex.

在一些實施方案中，靶向配體綴合至siRNA劑的第一鏈的3’末端或5’末端。 In some embodiments, the targeting ligand is conjugated to the 3' end or 5' end of the first strand of the siRNA agent.

在某些實施方案中，靶向配體與第一鏈的3’末端綴合；第一鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵，第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵以及在3’末端包含兩個硫代磷酸酯核苷酸間鍵，視需要地，第一鏈和/或第二鏈的核苷酸之間的所有剩餘鍵均為磷酸二酯鍵。 In certain embodiments, the targeting ligand is conjugated to the 3' terminus of the first strand; the first strand comprises two phosphorothioate internucleotide bonds at the 5' terminus, the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and optionally, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

在某些實施方案中，靶向配體與第一鏈的5’末端綴合；第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵，第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵以及在3’末端包含兩個硫代磷酸酯核苷酸間鍵，視需要地，第一鏈和/或第二鏈的核苷酸之間的所有剩餘鍵均為磷酸二酯鍵。 In certain embodiments, the targeting ligand is conjugated to the 5' terminus of the first strand; the first strand comprises two phosphorothioate internucleotide bonds at the 3' terminus, the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus, and optionally, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*xnNfnNfNfNfnxnnxnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*xnNfnNfNfnfnxnnxnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nxnnnxnnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluorine or 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnxnNfnNfNfNfnxnnxnnn*n*n-3’；第二鏈包含5’- n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnxnNfnNfNfnfnxnnxnnn*n*n-3'; the second strand comprises 5'- n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluorine or 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在某些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)、補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In certain embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*NfnNfnNfNfNfnnnnnnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*NfnNfnNfNfnnnnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’- n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnn(GN)-3'; the second strand comprises 5'- n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnnnNfnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first chain and a second chain, wherein the first chain comprises 5'-n*n*nnNfnNfNfnnnNfnnnnn(GN)-3'; the second chain comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnNfnNfnNfNfNfnnnnnnnn*n*n-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnNfnNfnNfNfnnnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3’；第二鏈包含5’-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其 中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first chain and a second chain, wherein the first chain comprises 5'-(GN)nnnnNfnNfNfnnnnNfnnn*n*n-3'; the second chain comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3’；第二鏈包含5’-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, and wherein "GN" represents a targeting ligand.

在某些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)、補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In certain embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其 中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a sequence represented by SEQ ID NO:20 and the second chain has the nucleotide sequence represented by SEQ ID NO:21; or the first chain has the nucleotide sequence represented by SEQ ID NO:96 and the second chain has the nucleotide sequence represented by SEQ ID NO:97; or the first chain has the nucleotide sequence represented by SEQ ID NO:16 and the second chain has the nucleotide sequence represented by SEQ ID NO:17; or the first chain has the nucleotide sequence represented by SEQ ID NO:150 and the second chain has the nucleotide sequence represented by SEQ ID NO:151; or the first chain has the nucleotide sequence represented by SEQ ID NO:32 and the second chain has the nucleotide sequence represented by SEQ ID NO:33; or the first chain has the nucleotide sequence represented by SEQ ID NO:100 and the second chain has the nucleotide sequence represented by SEQ ID NO:101; or the first chain has the nucleotide sequence represented by SEQ ID NO:110 and the second chain has the nucleotide sequence represented by SEQ ID NO:111; or the first chain has the nucleotide sequence represented by SEQ ID NO:112. NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的 核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a sequence represented by SEQ ID NO: 20 and the second chain has a nucleotide sequence represented by SEQ ID NO: 21; or the first chain has a nucleotide sequence represented by SEQ ID NO: 96 and the second chain has a nucleotide sequence represented by SEQ ID NO: 97; or the first chain has a nucleotide sequence represented by SEQ ID NO: 16 and the second chain has a nucleotide sequence represented by SEQ ID NO: 17; or the first chain has a nucleotide sequence represented by SEQ ID NO: 150 and the second chain has a nucleotide sequence represented by SEQ ID NO: 151; or the first chain has a nucleotide sequence represented by SEQ ID NO: 32 and the second chain has a nucleotide sequence represented by SEQ ID NO: 33; or the first chain has a nucleotide sequence represented by SEQ ID NO: 100 and the second chain has a nucleotide sequence represented by SEQ ID NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 120 and the second chain has a nucleotide sequence represented by SEQ ID NO: 121. NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在一些實施方案中，第二鏈在5’端包含末端5’(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the second strand comprises a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*xnNfnNfNfNfnxnnxnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*xnNfnNfNfnfnxnnxnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nxnnnxnnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluorine or 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnxnNfnNfNfNfnxnnxnnn*n*n-3’；第二鏈包含5’-(E-vP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸，“n”代表2’-O-甲基修飾的核苷酸以及“x”代表2’-氟或2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnxnNfnNfNfnfnxnnxnnn*n*n-3'; the second strand comprises 5'-(E-vP)n*Nf*nxnnnxnnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluorine or 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在某些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)、補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In certain embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*NfnNfnNfNfNfnnnnnnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以 及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*NfnNfnNfNfnnnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnnnNfnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’- n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnnnNfnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnNfnNfnNfNfNfnnnnnnnn*n*n-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnNfnNfnNfNfnnnnnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3’；第二鏈包含5’-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnnnNfnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在一些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3’；第二鏈包含5’-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸。 In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluorine-modified nucleotide and wherein "n" represents a 2'-O-methyl-modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide.

在某些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)、補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In certain embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-n*n*nnNfnNfNfNfnNfnnnnnnnn(GN)-3’；第二鏈包含5’-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸；其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents Table 2'-Fluoro-modified nucleotides and wherein "n" represents a 2'-O-methyl modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond; wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide; wherein the target gene of the above double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a sequence of SEQ The first chain has a nucleotide sequence represented by SEQ ID NO: 20 and the second chain has a nucleotide sequence represented by SEQ ID NO: 21; or the first chain has a nucleotide sequence represented by SEQ ID NO: 96 and the second chain has a nucleotide sequence represented by SEQ ID NO: 97; or the first chain has a nucleotide sequence represented by SEQ ID NO: 16 and the second chain has a nucleotide sequence represented by SEQ ID NO: 17; or the first chain has a nucleotide sequence represented by SEQ ID NO: 150 and the second chain has a nucleotide sequence represented by SEQ ID NO: 151; or the first chain has a nucleotide sequence represented by SEQ ID NO: 32 and the second chain has a nucleotide sequence represented by SEQ ID NO: 33; or the first chain has a nucleotide sequence represented by SEQ ID NO: 100 and the second chain has a nucleotide sequence represented by SEQ ID NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 120 and the second chain has a nucleotide sequence represented by SEQ ID NO: 121. NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在某些實施方案中，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其包含第一鏈和第二鏈，其中第一鏈包含5’-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3’；第二鏈包含5’-(E- VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3’；其中，“Nf”代表2’-氟修飾的核苷酸以及其中“n”代表2’-O-甲基修飾的核苷酸；其中，“*”代表硫代磷酸酯核苷酸間鍵，其中“GN”代表靶向配體；其中“E-VP”代表(E)-乙烯基膦酸酯核苷酸；其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)；較佳地，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In certain embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-(E- VP)n*Nf*nnnnnNfnnnnnNfn*Nf*n-3'; wherein "Nf" wherein "n" represents a 2'-fluoro modified nucleotide and wherein "n" represents a 2'-O-methyl modified nucleotide; wherein "*" represents a phosphorothioate internucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents an (E)-vinylphosphonate nucleotide; wherein the target gene of the double-stranded RNA (dsRNA) agent is the complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has a sequence consisting of SEQ The first chain has a nucleotide sequence represented by SEQ ID NO: 20 and the second chain has a nucleotide sequence represented by SEQ ID NO: 21; or the first chain has a nucleotide sequence represented by SEQ ID NO: 96 and the second chain has a nucleotide sequence represented by SEQ ID NO: 97; or the first chain has a nucleotide sequence represented by SEQ ID NO: 16 and the second chain has a nucleotide sequence represented by SEQ ID NO: 17; or the first chain has a nucleotide sequence represented by SEQ ID NO: 150 and the second chain has a nucleotide sequence represented by SEQ ID NO: 151; or the first chain has a nucleotide sequence represented by SEQ ID NO: 32 and the second chain has a nucleotide sequence represented by SEQ ID NO: 33; or the first chain has a nucleotide sequence represented by SEQ ID NO: 100 and the second chain has a nucleotide sequence represented by SEQ ID NO: 101; or the first chain has a nucleotide sequence represented by SEQ ID NO: 110 and the second chain has a nucleotide sequence represented by SEQ ID NO: 111; or the first chain has a nucleotide sequence represented by SEQ ID NO: 120 and the second chain has a nucleotide sequence represented by SEQ ID NO: 121. NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在某些實施方案中，第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：208)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 In certain embodiments, the first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO：212)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO：212)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO： 213)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：214)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：214)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or

第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mU*fA*mAfUmGmCmAmCmAmCmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second chain has a nucleotide sequence represented by (E)-VP- mU*fA*mAfUmGmCmAmCmAmCmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or

第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：216)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO：221)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：222)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：222)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO： 223)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or

第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：223)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列； The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217);

其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵，“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分，“GN”是靶向配體。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond, "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end, and "GN" is a targeting ligand.

III.SIRNA綴合物 III. SIRNA complexes

本發明的第三方面，siRNA試劑與一個或多個靶向部分偶聯(couple)或綴合(conjugate)。在某些情況下，靶向部分的選擇基於其選擇性地或較佳地將本文所述的綴合物分子靶向所需細胞群、組織或器官的能力。在某些情況下，靶向部分靶向表達該靶向部分的相應結合伴侶(例如，相應的受體或配體)的細胞、組織或器官。例如，與N-乙醯半乳糖胺(GalNAc)綴合的多核苷酸分子可以靶向表達唾液酸糖蛋白(ASGP-R)的肝細胞。本領域已知用作靶向部分的任何合適的GalNAc分子都是可以考慮的。示例性GalNAc分子包括三分支GalNAc(例如，L96)。 In a third aspect of the invention, the siRNA reagent is coupled or conjugated to one or more targeting moieties. In some cases, the selection of the targeting moiety is based on its ability to selectively or preferably target the complex molecules described herein to a desired cell population, tissue or organ. In some cases, the targeting moiety targets cells, tissues or organs that express the corresponding binding partner (e.g., the corresponding receptor or ligand) of the targeting moiety. For example, a polynucleotide molecule conjugated to N-acetylgalactosamine (GalNAc) can target hepatocytes expressing asialoglycoprotein (ASGP-R). Any suitable GalNAc molecule known in the art for use as a targeting moiety is contemplated. Exemplary GalNAc molecules include three-branched GalNAc (e.g., L96).

在一個方面，本發明提供了能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其中siRNA劑與配體綴合。 In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, wherein the siRNA agent is conjugated to a ligand.

在一個方面，本發明提供了一種能夠抑制靶基因表達的雙鏈RNA(dsRNA)劑，其中siRNA劑與配體綴合，如下圖所示：配體包括(i)一個或多個N-乙醯半乳糖胺(GalNAc)部分或其衍生物，和(ii)接頭，其中接頭將至少一個GalNAc部分或其衍生物與核酸綴合，和(iii)雙環基團連接GalNAc和siRNA，或(iv)四官能團，其中連接GalNAc部分， In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, wherein the siRNA agent is conjugated to a ligand, as shown in the following figure: the ligand includes (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to a nucleic acid, and (iii) a bicyclic group connecting GalNAc and siRNA, or (iv) a tetrafunctional group, wherein the GalNAc moiety is connected,

Figure 113123276-A0202-12-0093-76
Figure 113123276-A0202-12-0093-76

其中X是O、S。 Where X is O, S.

在一個方面，

Figure 113123276-A0202-12-0094-77
是： In one respect,
Figure 113123276-A0202-12-0094-77
yes:

Figure 113123276-A0202-12-0094-80
Figure 113123276-A0202-12-0094-80

Figure 113123276-A0202-12-0094-82
是：
Figure 113123276-A0202-12-0094-82
yes:

Figure 113123276-A0202-12-0094-83
Figure 113123276-A0202-12-0094-83

Figure 113123276-A0202-12-0095-85
Figure 113123276-A0202-12-0095-85

Figure 113123276-A0202-12-0096-86
Figure 113123276-A0202-12-0096-86

Figure 113123276-A0202-12-0097-87
Figure 113123276-A0202-12-0097-87

在一個方面，siRNA劑與配體綴合，如下圖所示： In one aspect, the siRNA agent is conjugated to a ligand, as shown below:

Figure 113123276-A0202-12-0097-88
Figure 113123276-A0202-12-0097-88

Figure 113123276-A0202-12-0098-89
Figure 113123276-A0202-12-0098-89

四官能團選自： Four functional groups are selected from:

Figure 113123276-A0202-12-0098-90
Figure 113123276-A0202-12-0098-90

Figure 113123276-A0202-12-0099-91
Figure 113123276-A0202-12-0099-91

在一個方面，圖中的接頭獨立地選自： In one aspect, the connectors in the figure are independently selected from:

(1)-(CH2)x-C(O)NH-(CH2)y-C(O)-； (1)-(CH 2 )xC(O)NH-(CH 2 )yC(O)-;

(2)-(CH2)x-C(O)NH-(CH2)y-NHC(O)-(CH2)z-C(O)-； (2)-(CH 2 )xC(O)NH-(CH 2 )y-NHC(O)-(CH 2 )zC(O)-;

(3)-NH-(CH2)y-C(O)-； (3) -NH-(CH 2 )yC(O)-;

(4)-C(O)-(CH2)y-C(O)-； (4) -C(O)-(CH 2 )yC(O)-;

其中，圖中的每個接頭視需要地相同或不同；x、y和z獨立地選自1-10；較佳地，x和y獨立地選自2-8，z選自1-6；更佳地，x和y獨立地選自3-6，z選自1-4。 Wherein, each joint in the figure is the same or different as needed; x, y and z are independently selected from 1-10; preferably, x and y are independently selected from 2-8, and z is selected from 1-6; more preferably, x and y are independently selected from 3-6, and z is selected from 1-4.

在一個方面，siRNA劑與配體綴合，如下圖所示： In one aspect, the siRNA agent is conjugated to a ligand, as shown below:

Figure 113123276-A0202-12-0100-92
Figure 113123276-A0202-12-0100-92

Figure 113123276-A0202-12-0100-93
Figure 113123276-A0202-12-0100-93

Figure 113123276-A0202-12-0100-94
Figure 113123276-A0202-12-0100-94

Figure 113123276-A0202-12-0101-95
Figure 113123276-A0202-12-0101-95

Figure 113123276-A0202-12-0101-96
Figure 113123276-A0202-12-0101-96

Figure 113123276-A0202-12-0101-97
Figure 113123276-A0202-12-0101-97

Figure 113123276-A0202-12-0101-98
Figure 113123276-A0202-12-0101-98

Figure 113123276-A0202-12-0102-99
Figure 113123276-A0202-12-0102-99

Figure 113123276-A0202-12-0102-100
Figure 113123276-A0202-12-0102-100

Figure 113123276-A0202-12-0102-101
Figure 113123276-A0202-12-0102-101

Figure 113123276-A0202-12-0103-102
Figure 113123276-A0202-12-0103-102

Figure 113123276-A0202-12-0103-103
Figure 113123276-A0202-12-0103-103

Figure 113123276-A0202-12-0103-104
Figure 113123276-A0202-12-0103-104

Figure 113123276-A0202-12-0103-105
Figure 113123276-A0202-12-0103-105

Figure 113123276-A0202-12-0103-106
Figure 113123276-A0202-12-0103-106

Figure 113123276-A0202-12-0104-107
Figure 113123276-A0202-12-0104-107

Figure 113123276-A0202-12-0104-108
Figure 113123276-A0202-12-0104-108

Figure 113123276-A0202-12-0104-109
Figure 113123276-A0202-12-0104-109

Figure 113123276-A0202-12-0104-110
Figure 113123276-A0202-12-0104-110

Figure 113123276-A0202-12-0104-111
Figure 113123276-A0202-12-0104-111

Figure 113123276-A0202-12-0105-112
Figure 113123276-A0202-12-0105-112

Figure 113123276-A0202-12-0105-113
Figure 113123276-A0202-12-0105-113

Figure 113123276-A0202-12-0105-114
Figure 113123276-A0202-12-0105-114

Figure 113123276-A0202-12-0105-116
Figure 113123276-A0202-12-0105-116

Figure 113123276-A0202-12-0105-117
Figure 113123276-A0202-12-0105-117

Figure 113123276-A0202-12-0106-118
Figure 113123276-A0202-12-0106-118

Figure 113123276-A0202-12-0106-119
Figure 113123276-A0202-12-0106-119

Figure 113123276-A0202-12-0106-120
Figure 113123276-A0202-12-0106-120

Figure 113123276-A0202-12-0106-121
Figure 113123276-A0202-12-0106-121

Figure 113123276-A0202-12-0106-122
Figure 113123276-A0202-12-0106-122

Figure 113123276-A0202-12-0107-123
Figure 113123276-A0202-12-0107-123

Figure 113123276-A0202-12-0107-124
Figure 113123276-A0202-12-0107-124

Figure 113123276-A0202-12-0107-125
Figure 113123276-A0202-12-0107-125

Figure 113123276-A0202-12-0107-126
Figure 113123276-A0202-12-0107-126

Figure 113123276-A0202-12-0108-127
Figure 113123276-A0202-12-0108-127

Figure 113123276-A0202-12-0108-128
Figure 113123276-A0202-12-0108-128

Figure 113123276-A0202-12-0108-129
Figure 113123276-A0202-12-0108-129

Figure 113123276-A0202-12-0108-130
Figure 113123276-A0202-12-0108-130
GalNAc-38GalNAc-38

其中，X是O或S。 Where X is O or S.

在一些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)、補體成分C5(C5)，補體因子B(CFB)、PCSK9、TTR、AGT、LPA、Agtr1、ALK、VEGF、ANGPTL3、ANGPTL4、ANGPTL8、APOA、APOC3、ASGR1、CIDEB、COL1A1、COL3A1、CTGF、DGAT2、DMPK、DNAJC15/MCJ、DPP4、因子VIII、因子X、因子IX、因子XI、因子XII、GPR146、GPR75、GRB10/14、TLR7/8/RIG-1、HSD17B13、INHBE、ITGV6、KHK、KLK1、MASP2、MTARC1、MUC5B、NPC1L1、PNPLA3、ASGR1、SCAP、SERPINA1、SERPINF2、SREBF2、HMGCR、TGFB1、COX-2、TP53、CD4、CD8、CD40、CD71、DUX4、XDH、LDHA、ALDH2、DMD、EPHA2、KIF11、BCL2L12、APOA1、TRPV1、CASP2、KRAS、TMPRSS6、STAT3、PRDM14、PTGS2、CTGF或DDIT4。 In some embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15/MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10/14, TLR7/8/RIG -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

在某些實施方案中，上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)。 In certain embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3).

在某些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有包含至少15個連續核苷酸的序列，這些序列與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過3個核苷酸；並且第二鏈包含與第一鏈至少部分互補的核苷酸序列。 In certain embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a sequence comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

在一些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有分別由SEQ ID NO：20、96、16、150、32、100、110、134、136表示的核苷酸序列。 在一些實施方案中，與第一鏈互補的第二鏈具有分別由SEQ ID NO：21、97、17、151、33、101、111、135、137表示的核苷酸序列。 In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively. In some embodiments, the second strand complementary to the first strand has a nucleotide sequence represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, 137, respectively.

在一些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and a nucleotide sequence represented by SEQ ID NO: 21 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and a nucleotide sequence represented by SEQ ID NO: 97 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and a nucleotide sequence represented by SEQ ID NO: 17 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 150 and a nucleotide sequence represented by SEQ ID NO: 151 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 32 and a nucleotide sequence represented by SEQ ID NO: 33 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 100 and a nucleotide sequence represented by SEQ ID NO: 101 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 110 and a nucleotide sequence represented by SEQ ID NO: ID NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在一些實施方案中，雙鏈RNA(dsRNA)劑如下圖所示： In some embodiments, the double-stranded RNA (dsRNA) agent is as shown below:

Figure 113123276-A0202-12-0111-131
或者
Figure 113123276-A0202-12-0111-131
or

Figure 113123276-A0202-12-0111-132
Figure 113123276-A0202-12-0111-132

其中上述雙鏈RNA(dsRNA)劑的靶基因是補體成分C3(C3)。 The target gene of the double-stranded RNA (dsRNA) agent is the complement component C3 (C3).

在一些實施方案中，雙鏈RNA(dsRNA)劑如下圖所示： In some embodiments, the double-stranded RNA (dsRNA) agent is as shown below:

Figure 113123276-A0202-12-0111-133
Figure 113123276-A0202-12-0111-133

其中第一鏈具有包含至少15個連續核苷酸的序列，其與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、 589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過3個核苷酸；並且第二鏈包含至少部分與第一鏈互補的核苷酸序列。在一些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有分別由SEQ ID NO：20、96、16、150、32、100、110、134、136表示的核苷酸序列。在一些實施方案中，與第一鏈互補的第二鏈具有分別由SEQ ID NO：21、97、17、151、33、101、111、135和137表示的核苷酸序列。在較佳的實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 wherein the first strand has a sequence comprising at least 15 consecutive nucleotides, which differs from any of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively. In some embodiments, the second strand complementary to the first strand has a nucleotide sequence represented by SEQ ID NOs: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively. In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and a nucleotide sequence represented by SEQ ID NO: 21 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and a nucleotide sequence represented by SEQ ID NO: 97 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and a nucleotide sequence represented by SEQ ID NO: 17 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 150 and a nucleotide sequence represented by SEQ ID NO: 151 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 32 and a nucleotide sequence represented by SEQ ID NO: 33 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 100 and a nucleotide sequence represented by SEQ ID NO: 101 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 152 and a nucleotide sequence represented by SEQ ID NO: 153 in the second strand; NO: 110 and the second chain has the nucleotide sequence shown in SEQ ID NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在一些實施方案中，雙鏈RNA(dsRNA)劑如下圖所示： In some embodiments, the double-stranded RNA (dsRNA) agent is as shown below:

Figure 113123276-A0202-12-0113-134
Figure 113123276-A0202-12-0113-134

其中第一鏈具有包含至少15個連續核苷酸的序列，其與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過3個核苷酸；並且第二鏈包含與第一鏈至少部分互補的核苷酸序列。在一些實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有分別由SEQ ID NO：20、96、16、150、32、100、110、134、136表示的核苷酸序列。在一些實施方案中，與第一鏈互補的第二鏈具有分別由SEQ ID NO：21、97、17、151、33、101、111、135和137表示的核苷酸序列。在較佳的實施方案中，雙鏈RNA(dsRNA)劑的第一鏈具有由SEQ ID NO：20表示的核苷酸序列以及第二鏈具有由SEQ ID NO：21表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：96表示的核苷酸序列以及第二鏈具有由SEQ ID NO：97表示的核苷酸序列；或者第一鏈具有由SEQ ID NO：16表示的核苷酸序列以及第二鏈具有由SEQ ID NO：17表示的核苷酸序列；或第一鏈具有SEQ ID NO：150所示的核苷酸序列以及第二鏈具有SEQ ID NO：151所示的核苷酸序列；或第一鏈具有SEQ ID NO：32所示的核苷酸序列以及第二鏈具有SEQ ID NO：33所示的核苷酸序列；或第一鏈具有SEQ ID NO：100所示的核苷酸序列以及第二鏈具有SEQ ID NO：101所示的核苷酸序列；或第一鏈具有SEQ ID NO：110所示的核苷酸序列以及第二鏈具有SEQ ID NO：111所示的核苷酸 序列；或第一鏈具有SEQ ID NO：134所示的核苷酸序列以及第二鏈具有SEQ ID NO：135所示的核苷酸序列；或第一鏈具有SEQ ID NO：136所示的核苷酸序列以及第二鏈具有SEQ ID NO：137所示的核苷酸序列。 wherein the first strand has a sequence comprising at least 15 consecutive nucleotides, which differs from any of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively. In some embodiments, the second strand complementary to the first strand has a nucleotide sequence represented by SEQ ID NOs: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively. In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and a nucleotide sequence represented by SEQ ID NO: 21 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and a nucleotide sequence represented by SEQ ID NO: 97 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and a nucleotide sequence represented by SEQ ID NO: 17 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 150 and a nucleotide sequence represented by SEQ ID NO: 151 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 32 and a nucleotide sequence represented by SEQ ID NO: 33 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 100 and a nucleotide sequence represented by SEQ ID NO: 101 in the second strand; or the first strand has a nucleotide sequence represented by SEQ ID NO: 152 and a nucleotide sequence represented by SEQ ID NO: 153 in the second strand; NO: 110 and the second chain has the nucleotide sequence shown in SEQ ID NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

在某些實施方案中，第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：224)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 In certain embodiments, the first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID N：228)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID N: 228), and the second chain has a nucleotide sequence represented by (E)-VP- mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO: 227); or

第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：228)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO：229)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：230)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：230)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or

第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or

第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：232)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 232), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：234)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc- 7)(SEQ ID NO：234)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc- 7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or

第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO：237)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及第二鏈具有由(E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or

第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列； The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233);

其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵；“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond; "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end.

定義 Definition

在下面詳述本發明之前，將理解的是，本發明不限於本文中描述的特定方法、方案和試劑，因為它們可以不同。還將理解的是，本文所用的術語僅用於描述特定實施方案的目的，而不旨在限制本發明的範圍，本發明的範圍將僅由所附申請專利範圍限制。除非另有定義，否則本文所用的所有技術術語和科學術語具有與本發明所屬領域具有通常知識者通常理解的相同含義。 Before describing the present invention in detail below, it will be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It will also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will be limited only by the scope of the attached patent application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

對於說明書的解釋，將應用以下定義，並且在任何適當的情況下，單數形式使用的術語也可以包括複數形式，反之亦然。將理解的是，本文所用的術語僅用於描述具體實施方案的目的，而不旨在限制。 For the interpretation of the specification, the following definitions will apply, and where appropriate, terms used in the singular may also include the plural form, and vice versa. It will be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

如本文所用，術語“補體成分3”與術語“C3”互換使用，是指眾所周知的基因和多肽，在本領域中也稱為ARMD9、C3a過敏毒素、ASP、補體成分C3a、C3a、補體成分C3b、C3b、prepro-C3、醯化刺激蛋白切割產物、CPAMD1、補體C3、C3和含PZP樣α-2-巨球蛋白結構域的蛋白1、補體成分C3和AHUS5。 As used herein, the term "complement component 3" is used interchangeably with the term "C3" to refer to the well-known gene and polypeptide, which is also known in the art as ARMD9, C3a allergic toxin, ASP, complement component C3a, C3a, complement component C3b, C3b, prepro-C3, acetylation stimulating protein cleavage product, CPAMD1, complement C3, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, complement component C3 and AHUS5.

如本文所用，“靶序列”指在補體成分C3基因轉錄期間所形成的mRNA分子的核苷酸序列的連續部分，包括作為主要轉錄產物的RNA加工產物的mRNA。序列的靶部分將至少足夠長，以用作在補體成分C3基因轉錄期間形成的mRNA分子的核苷酸序列部分上或在其附近進行iRNA定向切割的底物。 As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a complement component C3 gene, including mRNA that is an RNA processing product of the primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage on or near the nucleotide sequence portion of an mRNA molecule formed during transcription of a complement component C3 gene.

術語“iRNA”、“RNAi劑”、“iRNA劑”、“RNA干擾劑”在本文中可互換使用，是指含有本文所定義的RNA的試劑，其藉由RNA誘導的沉默複合體(RISC)途徑介導RNA轉錄物的靶向切割。iRNA藉由稱為RNA干擾(RNAi)的過程指導mRNA的序列特異性降解。iRNA調節(例如抑制)細胞(例如受試者內的細胞，如哺乳動物受試者內的細胞)中C3基因的表達。 The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interferor" are used interchangeably herein and refer to an agent containing RNA as defined herein that mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). iRNA modulates (e.g., inhibits) expression of the C3 gene in a cell (e.g., a cell in a subject, such as a cell in a mammalian subject).

術語“mRNA(信使RNA)”是指作為體內蛋白質翻譯模板的RNA分子，其將基因編碼信息從DNA轉移到蛋白質產物。術語“RNA干擾”或“RNAi”是指生物體中基因表達的轉錄後調控現象。該現象是由單鏈或雙鏈RNA介導的靶mRNA特異性降解引起的。RNAi調控機制的詳細內容，請參見Biotech.Adv.2008,26(3)：202以及其它文獻的描述。 The term "mRNA (messenger RNA)" refers to an RNA molecule that serves as a template for protein translation in vivo, transferring gene coding information from DNA to protein products. The term "RNA interference" or "RNAi" refers to the post-transcriptional regulatory phenomenon of gene expression in an organism. The phenomenon is caused by the specific degradation of target mRNA mediated by single-stranded or double-stranded RNA. For details of the RNAi regulatory mechanism, please refer to Biotech.Adv.2008,26(3):202 and other literature descriptions.

在本發明中，除非另有說明，術語“小干擾RNA/小干擾的RNA(small interference/small interfering RNA)”或“siRNA”是指能夠序列特異性地誘導RNAi現象的RNA分子，由兩個長度為15-27個核苷酸的單鏈RNA組成，並且具有部分或完全互補的雙鏈結構。在本發明的siRNA中，互補雙鏈結構的長度可以為16-25、17-22或18-21個鹼基對。本發明所述的siRNA可以為由兩條長度為15-27個核苷酸的單鏈RNA組成的平端雙鏈RNA結構，也可以為在雙鏈結構的至少一端具有由1-3個連續核苷酸組成的3’突出端的結構。 In the present invention, unless otherwise specified, the term "small interference RNA/small interfering RNA" or "siRNA" refers to an RNA molecule capable of sequence-specifically inducing RNAi phenomenon, consisting of two single-stranded RNAs of 15-27 nucleotides in length, and having a partially or completely complementary double-stranded structure. In the siRNA of the present invention, the length of the complementary double-stranded structure can be 16-25, 17-22 or 18-21 base pairs. The siRNA of the present invention can be a blunt-ended double-stranded RNA structure consisting of two single-stranded RNAs of 15-27 nucleotides in length, or a structure having a 3' overhang consisting of 1-3 consecutive nucleotides at at least one end of the double-stranded structure.

在本發明中，除非另有說明，術語“第一單鏈”或“正義鏈”是指siRNA的兩條單鏈中的一條單鏈，其具有與siRNA在靶mRNA中的作用位點的核苷酸序列部分或全部相同的核苷酸序列；而術語“第二單鏈”或“反義鏈”是指siRNA的兩條單鏈中的另一條單鏈，其具有與siRNA在靶mRNA中的作用位點的核苷酸序列部分或全部互補的核苷酸序列。本發明中提到的siRNA的第一單鏈(或正義鏈)和相應的第二單鏈(或反義鏈)可以形成部分或完全互補的雙鏈結構。 In the present invention, unless otherwise specified, the term "first single strand" or "positive sense strand" refers to one of the two single strands of siRNA, which has a nucleotide sequence that is partially or completely identical to the nucleotide sequence of the siRNA's action site in the target mRNA; and the term "second single strand" or "antisense strand" refers to the other of the two single strands of siRNA, which has a nucleotide sequence that is partially or completely complementary to the nucleotide sequence of the siRNA's action site in the target mRNA. The first single strand (or positive sense strand) and the corresponding second single strand (or antisense strand) of the siRNA mentioned in the present invention can form a partially or completely complementary double strand structure.

本文所用的“互補”序列還可以包括非沃森-克裡克鹼基對和/或由非天然和修飾的核苷酸形成的鹼基對，或完全由非沃森-克裡克鹼基對和/或由非 天然和修飾的核苷酸形成的鹼基對形成，只要符合上述有關雜交能力的要求即可。這種非沃森-克裡克鹼基對包括但不限於G：U擺動或Hoogsteen鹼基配對。 The "complementary" sequence used herein may also include non-Watson-Crick base pairs and/or base pairs formed by non-natural and modified nucleotides, or be completely formed by non-Watson-Crick base pairs and/or base pairs formed by non-natural and modified nucleotides, as long as the above-mentioned requirements for hybridization ability are met. Such non-Watson-Crick base pairs include but are not limited to G:U swing or Hoogsteen base pairs.

本發明中，除非另有說明，術語“壓制/壓制的(suppress/suppressing)、抑制/抑制的(inhibit/inhibiting)”是指siRNA或其他小干擾核酸(siNA)抑制劑介導的mRNA降解從而導致靶基因表達顯著下調的情況。“顯著下調”是指靶基因表達與正常水平或治療前水平相比降低5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%或更多，或100%的情況。 In the present invention, unless otherwise specified, the terms "suppress/suppressing", "inhibit/inhibiting" refer to the situation where the target gene expression is significantly downregulated due to mRNA degradation mediated by siRNA or other small interfering nucleic acid (siNA) inhibitors. "Significant downregulation" refers to the situation where the target gene expression is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more, or 100% compared with the normal level or the level before treatment.

本文中使用的短語“使細胞與RNAi劑(如dsRNA)接觸”包括藉由任何可能的方式接觸細胞。使細胞與RNAi劑接觸包括使細胞在體外與RNAi劑接觸或使細胞在體內與RNAi劑接觸。接觸可以直接或間接進行。因此，例如，執行該方法的個體可使RNAi劑與細胞發生物理接觸，或者，可將RNAi劑置於允許或導致其隨後與細胞接觸的環境中。例如，可以藉由將細胞與RNAi劑一起孵育來實現體外細胞接觸。例如，可藉由將RNAi劑注射到細胞所在組織中或附近來實現體內細胞接觸，或者可藉由將RNAi劑注射到另一區域(例如中樞神經系統(CNS))來實現體內細胞接觸，視需要地藉由鞘內、玻璃體內或其他注射，或注射到血流或皮下空間，使得試劑隨後到達要接觸的細胞所在的組織。 As used herein, the phrase "contacting a cell with an RNAi agent (e.g., dsRNA)" includes contacting the cell by any possible means. Contacting the cell with an RNAi agent includes contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. The contacting can be done directly or indirectly. Thus, for example, an individual performing the method may bring the RNAi agent into physical contact with the cell, or the RNAi agent may be placed in an environment that allows or causes it to subsequently come into contact with the cell. For example, in vitro cell contact may be achieved by incubating the cell with the RNAi agent. For example, in vivo cell contact can be achieved by injecting the RNAi agent into or near the tissue where the cells are located, or in vivo cell contact can be achieved by injecting the RNAi agent into another area, such as the central nervous system (CNS), optionally by intrathecal, intravitreal or other injection, or injection into the bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located.

“G”、“C”、“A”和“U”通常分別代表含有鳥嘌呤、胞嘧啶、腺嘌呤和尿嘧啶作為鹼基的核苷酸。然而，應理解術語“核糖核苷酸”或“核苷酸”也可以指修飾的核苷酸，如下文進一步詳述，或替換取代部分。所屬技術領域中具有通常知識者充分瞭解，鳥嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可以被其他部分取代，而不會顯著改變包含帶有這種取代部分的核苷酸的寡核苷酸的鹼基配對特性。 "G", "C", "A" and "U" generally represent nucleotides containing guanine, cytosine, adenine and uracil as bases, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as further described below, or to replacement substitution moieties. It is well understood by those of ordinary skill in the art that guanine, cytosine, adenine and uracil may be substituted with other moieties without significantly altering the base pairing properties of an oligonucleotide containing nucleotides bearing such substitution moieties.

本發明對siRNA進行的化學修飾可以是選自以下的一種化學修飾、或選自以下的多種化學修飾的組合： The chemical modification of siRNA performed in the present invention can be one of the following chemical modifications, or a combination of the following chemical modifications:

1)RNA鏈主鏈結構中連接核苷酸殘基的磷酸二酯鍵的修飾； 1) Modification of the phosphodiester bonds connecting nucleotide residues in the main chain structure of the RNA chain;

2)RNA鏈主鏈結構中的核糖的修飾； 2) Modification of ribose in the main chain structure of RNA chain;

3)RNA的核苷酸殘基中的鹼基的修飾。 3) Modification of the bases in the nucleotide residues of RNA.

siRNA的自然存在的核苷間鍵是3’至5’磷酸二酯鍵。具有一個或多個修飾的(即非自然存在的)核苷間鍵的RNA鏈通常比具有自然存在的核苷間鍵的RNA鏈更易被選擇，因為其具有理想的特性，如增強的細胞攝取、增強的對靶核酸的親和性以及在核酸酶存在下的穩定性增加。 The naturally occurring internucleoside bond of siRNA is a 3' to 5' phosphodiester bond. RNA strands with one or more modified (i.e., non-naturally occurring) internucleoside bonds are often selected over RNA strands with naturally occurring internucleoside bonds because they possess desirable properties, such as enhanced cellular uptake, enhanced affinity for the target nucleic acid, and increased stability in the presence of nucleases.

具有修飾的核苷間鍵的寡核苷酸包括保留磷原子的核苷間鍵以及不具有磷原子的核苷間鍵。代表性的含磷的核苷間鍵包括但不限於磷酸二酯、磷酸三酯、甲基膦酸酯、胺基磷酸酯和硫代磷酸酯。 Oligonucleotides with modified nucleoside bonds include those that retain phosphorus atoms and those that do not. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphamidate, and phosphorothioate.

RNAi劑可以在正義鏈或反義鏈的5’端包含含磷基團。5’端含磷基團可以是5’端磷酸酯(5’-P)、5’端硫代磷酸酯(5’-PS)、5’端二硫代磷酸酯(5’-PS2)、5’端乙烯基膦酸酯(5’-VP)、5’端甲基膦酸酯(MePhos)或5’-脫氧-5’-C-丙二醯)。當5’端含磷基團是5’端乙烯基膦酸酯(5’-VP)時，5’-VP可以是5’-E-VP異構體磷酸酯、異構體(即，順式乙烯基磷酸酯)或其混合物。 The RNAi agent may contain a phosphorus-containing group at the 5' end of the positive or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl). When the 5'-terminal phosphorus-containing group is a 5'-terminal vinylphosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomeric phosphate, an isomer (i.e., cis-vinyl phosphate), or a mixture thereof.

在本發明中，RNAi劑包含一個或多個修飾的核苷間鍵。在某些實施方案中，修飾的核苷間鍵是硫代磷酸酯鍵。在某些實施方案中，RNAi劑在第二鏈中包含5’-E-VP。 In the present invention, the RNAi agent comprises one or more modified nucleoside interlinks. In certain embodiments, the modified nucleoside interlink is a phosphorothioate bond. In certain embodiments, the RNAi agent comprises 5'-E-VP in the second chain.

本發明中，核糖基團的修飾是指核糖基團上2’-羥基(2’-OH)的修飾。在核糖基團的2’-羥基位置引入某些取代基，如甲氧基或氟基，使得血清中 的核糖核酸酶不易消化核酸，從而提高核酸的穩定性，使核酸具有更強的抵抗核酸酶水解的能力。核苷酸的戊糖上的2’-羥基的修飾包括2’-氟修飾、2’-甲氧基修飾、2’-甲氧基乙氧基修飾、2’-2,4-二硝基苯酚修飾(2’-DNP修飾)、鎖核酸修飾(LNA修飾)、2’-胺基修飾、2’-脫氧修飾等。 In the present invention, the modification of the ribose group refers to the modification of the 2'-hydroxyl (2'-OH) on the ribose group. Certain substituents, such as methoxy or fluoro, are introduced at the 2'-hydroxyl position of the ribose group, so that the ribonuclease in the serum is not easy to digest the nucleic acid, thereby improving the stability of the nucleic acid and making the nucleic acid have a stronger ability to resist nuclease hydrolysis. The modification of the 2'-hydroxyl on the pentose of the nucleotide includes 2'-fluoro modification, 2'-methoxy modification, 2'-methoxyethoxy modification, 2'-2,4-dinitrophenol modification (2'-DNP modification), locked nucleic acid modification (LNA modification), 2'-amine modification, 2'-deoxy modification, etc.

本發明中，鹼基的修飾是指核苷酸基團上的鹼基的修飾。例如，5’-溴尿嘧啶修飾和5’-碘尿嘧啶修飾，在尿嘧啶的5位引入溴或碘，是鹼基的常見修飾方法。還可以使用修飾，如N3-甲基尿嘧啶修飾、2,6-二胺基嘌呤修飾。 In the present invention, the modification of the base refers to the modification of the base on the nucleotide group. For example, 5'-bromouracil modification and 5'-iodouracil modification, which introduce bromine or iodine at the 5th position of uracil, are common methods of modifying the base. Modifications such as N3-methyluracil modification and 2,6-diaminopurine modification can also be used.

在某些實施方案中，雙鏈RNAi劑還包含靶向肝組織的靶向配體。在某些實施方案中，靶向配體是GalNAc綴合物。在某些實施方案中，GalNAc綴合物是一種或多種GalNAc衍生物，視需要地藉由接頭或載體與雙鏈RNAi劑綴合，一種或多種GalNAc衍生物視需要地藉由接頭或載體與雙鏈RNAi劑綴合。 In some embodiments, the double-chain RNAi agent further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a GalNAc complex. In some embodiments, the GalNAc complex is one or more GalNAc derivatives, which are optionally conjugated to the double-chain RNAi agent via a linker or a carrier, and one or more GalNAc derivatives are optionally conjugated to the double-chain RNAi agent via a linker or a carrier.

在一些實施方案中，綴合物包括一種或多種GalNAc衍生物。GalNAc衍生物可以藉由接頭連接，例如二價或三價分支接頭。在一些實施方案中，GalNAc綴合物綴合至正義鏈的3’端。在一些實施方案中，GalNAc綴合物藉由接頭(例如本文所述的接頭)與iRNA劑(例如，與正義鏈的3’端)綴合。在一些實施方案中，GalNAc綴合物綴合至正義鏈的5’端。在一些實施方案中，GalNAc綴合物藉由接頭(例如本文所述的接頭)與iRNA劑(例如，與正義鏈的5’端)綴合。 In some embodiments, the conjugate includes one or more GalNAc derivatives. The GalNAc derivatives can be linked by a linker, such as a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the positive sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 3' end of the positive sense strand) by a linker (e.g., a linker described herein). In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the positive sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 5' end of the positive sense strand) by a linker (e.g., a linker described herein).

在本發明的某些實施方案中，GalNAc或GalNAc衍生物藉由單價接頭與本發明的iRNA劑連接。在一些實施方案中，GalNAc或GalNAc衍生物藉由二價接頭與本發明的iRNA劑連接。在本發明的其他實施方案中，GalNAc 或GalNAc衍生物藉由三價接頭與本發明的iRNA劑連接。在本發明的其他實施方案中，GalNAc或GalNAc衍生物藉由四價接頭與本發明的iRNA劑連接。 In certain embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a divalent linker. In other embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a trivalent linker. In other embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a tetravalent linker.

本發明的siRNA的一條RNA單鏈可以藉由固相或液相核酸合成法合成。這些方法包括四個加工步驟：1)寡核苷酸合成；2)脫保護；3)純化和分離；4)脫鹽。上述四個步驟的技術細節對於所屬技術領域中具有通常知識者來說是眾所周知的，因此在此不再詳細描述。 An RNA single strand of the siRNA of the present invention can be synthesized by solid phase or liquid phase nucleic acid synthesis methods. These methods include four processing steps: 1) oligonucleotide synthesis; 2) deprotection; 3) purification and separation; 4) desalination. The technical details of the above four steps are well known to those with ordinary knowledge in the relevant technical field, so they will not be described in detail here.

本發明的siRNA除了化學合成外，還可以藉由質粒和/或病毒載體表達獲得。例如，設計一段長度為50-90個核苷酸的DNA序列，在其兩端添加兩種不同的限制性酶切位點，例如BamHI和EcoRI酶切位點。設計的DNA編碼的RNA轉錄物的中間段序列可以形成環狀結構，而環在U型轉彎之後的兩端序列可以形成互補配對的雙鏈結構。藉由選殖技術，將設計的DNA插入到經相應限制性酶消化的表達載體中。將表達載體引入細胞，由設計的DNA序列產生的RNA轉錄物可以藉由細胞固有的siRNA加工機制加工成成熟的siRNA。從而，siRNA可以在細胞中短暫或穩定地表達。 In addition to chemical synthesis, the siRNA of the present invention can also be obtained by expression of plasmids and/or viral vectors. For example, a DNA sequence of 50-90 nucleotides in length is designed, and two different restriction enzyme sites, such as BamHI and EcoRI restriction sites, are added to its two ends. The middle segment sequence of the designed DNA-encoded RNA transcript can form a circular structure, and the two end sequences of the loop after the U-turn can form a complementary double-stranded structure. Through cloning technology, the designed DNA is inserted into the expression vector digested with the corresponding restriction enzyme. The expression vector is introduced into the cell, and the RNA transcript produced by the designed DNA sequence can be processed into mature siRNA by the cell's inherent siRNA processing mechanism. Thus, siRNA can be expressed transiently or stably in the cell.

除非另有定義，否則本文所用的所有技術術語和科學術語具有與本發明所屬領域具有通常知識者通常理解的相同含義。儘管在實施或測試本發明中的RNAi劑和方法時可以使用與本文所述相似或等效的方法和材料，但下文描述了合適的方法和材料。本文中提及的所有出版物、專利申請、專利和其他參考文獻均以引用的方式全文併入本文。如有衝突，以本說明書(包括定義)為準。此外，材料、方法和實施例僅用於說明目的，並不旨在限制。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by persons of ordinary skill in the art to which the present invention belongs. Although similar or equivalent methods and materials as those described herein may be used in practicing or testing the RNAi agents and methods of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

實施例Embodiment

本發明將結合下文的實施例進行說明。如果試劑和其他實驗材料的來源在本文中沒有具體說明，則可從任何分子生物學試劑供應商處獲得此類試劑和其他實驗材料，只要其質量/純度符合分子生物學應用的標準。 The present invention will be described in conjunction with the following embodiments. If the source of reagents and other experimental materials is not specifically described herein, such reagents and other experimental materials can be obtained from any molecular biology reagent supplier as long as their quality/purity meets the standards for molecular biology applications.

實施例1.人源C3基因的小干擾核苷酸的設計Example 1. Design of small interfering nucleotides of human C3 gene

19bp的核苷酸序列選自1-5231bp範圍內的人源C3基因的mRNA序列，其序列相對保守(Genbank中的登錄號為NM_000064，SEQ ID NO：1)。 The 19bp nucleotide sequence was selected from the mRNA sequence of the human C3 gene in the range of 1-5231bp, and its sequence is relatively conservative (the accession number in Genbank is NM_000064, SEQ ID NO: 1).

表1分別列出了每個siRNA的正義鏈和互補反義鏈的序列。具體而言，例如，siRNA C3-1的第一鏈(正義鏈)具有SEQ ID NO：2所示的序列，其與C3 mRNA序列中對應的靶位點序列相同；以及第二鏈(反義鏈)具有SEQ ID NO：3所示的序列，其與C3 mRNA序列中對應的靶位點序列互補。其他每個siRNA的兩條單鏈的序列以與siRNA C3-1相同的方式依次編號。 Table 1 lists the sequences of the positive sense strand and the complementary antisense strand of each siRNA. Specifically, for example, the first strand (positive sense strand) of siRNA C3-1 has the sequence shown in SEQ ID NO: 2, which is the same as the target site sequence corresponding to the C3 mRNA sequence; and the second strand (antisense strand) has the sequence shown in SEQ ID NO: 3, which is complementary to the target site sequence corresponding to the C3 mRNA sequence. The sequences of the two single strands of each other siRNA are numbered in sequence in the same manner as siRNA C3-1.

表1

Figure 113123276-A0202-12-0125-135
Table 1
Figure 113123276-A0202-12-0125-135

Figure 113123276-A0202-12-0126-136
Figure 113123276-A0202-12-0126-136

Figure 113123276-A0202-12-0127-137
Figure 113123276-A0202-12-0127-137

Figure 113123276-A0202-12-0128-138
Figure 113123276-A0202-12-0128-138

Figure 113123276-A0202-12-0129-139
Figure 113123276-A0202-12-0129-139

實施例2 siRNA對C3基因表達的抑制效果的驗證Example 2 Verification of the inhibitory effect of siRNA on C3 gene expression

表2A中設計的siRNA由Thermo Fisher Scientific Inc.或GenScript化學合成。互補的寡核糖核苷酸可以按照“Molecular Cloning：A Laboratory Manual”中描述的方法退火，形成雙鏈RNA。 The siRNAs designed in Table 2A were chemically synthesized by Thermo Fisher Scientific Inc. or GenScript. Complementary oligoribonucleotides can be annealed to form double-stranded RNAs as described in "Molecular Cloning: A Laboratory Manual".

表2A

Figure 113123276-A0202-12-0130-140
Table 2A
Figure 113123276-A0202-12-0130-140

Figure 113123276-A0202-12-0131-141
Figure 113123276-A0202-12-0131-141

Figure 113123276-A0202-12-0132-142
Figure 113123276-A0202-12-0132-142

Figure 113123276-A0202-12-0133-143
Figure 113123276-A0202-12-0133-143

Figure 113123276-A0202-12-0134-144
Figure 113123276-A0202-12-0134-144

(1)HepG2細胞培養物 (1) HepG2 cell culture

使用定制設計的沉默子siRNA文庫(C3)轉染HepG2細胞。RQ值標準化為空的RNAiMAX轉染的HepG2細胞。對每個樣品和/或探針進行三次測定。(n=3) HepG2 cells were transfected with a custom designed silencer siRNA library (C3). RQ values were normalized to empty RNAiMAX transfected HepG2 cells. Each sample and/or probe was assayed in triplicate. (n=3)

(2)HepG2細胞轉染的方法 (2) HepG2 cell transfection method

轉染前一天，將17,000個細胞/孔的HepG2親本細胞接種在96孔組織培養處理板中。使用LipofectamineTM RNAiMAX轉染試劑盒(目錄號#13778030)，使用每 孔20nM siRNA和0.3 RNAiMAX試劑製備siRNA轉染。轉染後72小時收穫細胞，並藉由RT-qPCR分析。從siRNA處理和未處理的細胞中分離出RNA。使用TaqMan®Gene Expression Cells-to-Ct試劑盒(#4399002)提取RNA。生成的cDNA用作qPCR反應模板，使用TaqMan®基因表達主混合物和特異於靶基因β-肌動蛋白(Hs01060665_g1)、人C3(Hs00163811_m1)的定制TaqMan®基因表達測定探針。β-肌動蛋白用作內源性對照。樣品在Quant StudioTM 12K Flex實時PCR系統上運行。使用比較CT方法分析數據，如表2B和圖1所示。 One day before transfection, 17,000 cells/well of HepG2 parental cells were seeded in 96-well tissue culture treated plates. siRNA transfections were prepared using Lipofectamine TM RNAiMAX Transfection Kit (Catalog #13778030) with 20 nM siRNA and 0.3 RNAiMAX reagent per well. Cells were harvested 72 hours after transfection and analyzed by RT-qPCR. RNA was isolated from siRNA treated and untreated cells. RNA was extracted using TaqMan® Gene Expression Cells-to-Ct Kit (#4399002). The generated cDNA was used as template for qPCR reactions using TaqMan® Gene Expression Master Mix and custom TaqMan® Gene Expression Assay probes specific for the target genes β-actin (Hs01060665_g1), human C3 (Hs00163811_m1). β-actin was used as an endogenous control. Samples were run on a Quant Studio ™ 12K Flex Real-Time PCR System. Data were analyzed using the comparative CT method as shown in Table 2B and Figure 1.

相對C3 mRNA(%)=2-(樣品△Ct(C3的Ct-管家基因的Ct)-模擬△Ct(C3的Ct-管家基因的Ct))×100% Relative C3 mRNA (%) = 2 - (sample △Ct (C3 Ct - housekeeping gene Ct) - simulation △Ct (C3 Ct - housekeeping gene Ct)) × 100%

結果表明，測試的示例性siRNA劑有效降低人C3信使RNA的水平。 The results showed that the tested exemplary siRNA agents effectively reduced the levels of human C3 messenger RNA.

表2B

Figure 113123276-A0202-12-0135-146
Table 2B
Figure 113123276-A0202-12-0135-146

Figure 113123276-A0202-12-0136-147
Figure 113123276-A0202-12-0136-147

Figure 113123276-A0202-12-0137-148
Figure 113123276-A0202-12-0137-148

實施例3.轉染0.128pM-100nM選擇的siRNA的HepG2細胞中的C3 mRNA減弱效果(劑量反應曲線實驗)。Example 3. C3 mRNA attenuation effect in HepG2 cells transfected with 0.128pM-100nM selected siRNA (dose response curve experiment).

在HepG2細胞中轉染0.128pM-100nM siRNA後，確定選擇的siRNA C3-8-dTdT、C3-10-dTdT、C3-16-dTdT、C3-48-dTdT、C3-50-dTdT、C3-55-dTdT、C3-67-dTdT、C3-68-dTdT和C3-75-dTdT的C3減弱效果。siRNA序列信息如表2A所示。結果如圖2A所示。轉染後，所有siRNA均顯示出劑量依賴性的C3 mRNA減弱。最有效的siRNA是C3-48-dTdT、C3-16-dTdT、C3-8-dTdT，濃度為100nM，如圖2B所示。 The C3 attenuation effects of the selected siRNAs C3-8-dTdT, C3-10-dTdT, C3-16-dTdT, C3-48-dTdT, C3-50-dTdT, C3-55-dTdT, C3-67-dTdT, C3-68-dTdT, and C3-75-dTdT were determined after transfection of 0.128pM-100nM siRNA in HepG2 cells. The siRNA sequence information is shown in Table 2A. The results are shown in Figure 2A. After transfection, all siRNAs showed dose-dependent attenuation of C3 mRNA. The most effective siRNAs were C3-48-dTdT, C3-16-dTdT, and C3-8-dTdT, with a concentration of 100 nM, as shown in Figure 2B.

HepG2細胞轉染的方法 HepG2 cell transfection method

將細胞以13,300個細胞/孔的密度接種在96孔透明F底TC處理板(VWR #734-2327)中。根據製造商的方案，使用LipofectamineTM RNAiMAX轉 染試劑(Invitrogen life technology)進行siRNA轉染。使用8種濃度的C3 siRNA進行劑量反應實驗，從100nM開始，稀釋5倍至0.128pM。模擬(mock)是未經siRNA處理的細胞。 Cells were seeded at 13,300 cells/well in 96-well clear F-bottom TC-treated plates (VWR #734-2327). siRNA transfection was performed using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen life technology) according to the manufacturer's protocol. Dose response experiments were performed using 8 concentrations of C3 siRNA, starting from 100 nM and diluted 5-fold to 0.128 pM. Mocks were cells that were not treated with siRNA.

轉染後72小時，根據製造商的方案，使用DynabeadsTM mRNA DIRECTTM純化試劑盒(Invitrogen life technology)提取RNA。簡而言之，從細胞中去除介質後，向細胞中添加100uL的裂解緩衝液。將細胞培養板放入振盪器中，在室溫下以300rpm的速度振盪30分鐘。然後添加Dynabeads以純化裂解物中的mRNA。RNA提取後，立即藉由SuperScript VILO cDNA合成試劑盒(Invitrogen life technology)進行逆轉錄。C3和TBP mRNA由Quantstudio 5實時PCR系統(Invitrogen life technology)確定，使用TaqMan qPCR引子、C3-FAM(測定ID：Hs00163811_m1)和TBP-VIC(測定ID：Hs00427620_m1)。將給定C3 siRNA的活性表示為相對於未用siRNA處理的細胞中歸一化至TBP mRNA的C3 mRNA(模擬)，在處理細胞中歸一化至TBP mRNA的剩餘C3 mRNA的百分比。用GraphPad Prism 9版使用四參數邏輯模型製作劑量反應曲線。圖2A和圖2B中顯示的數據來自三個生物學重複。圖2B中的數據來自圖2A中的0.16nM和100nM數據。 72 hours after transfection, RNA was extracted using Dynabeads TM mRNA DIRECT TM Purification Kit (Invitrogen life technology) according to the manufacturer's protocol. Briefly, after removing the medium from the cells, 100uL of lysis buffer was added to the cells. The cell culture plate was placed in a shaker and shaken at 300rpm for 30 minutes at room temperature. Dynabeads were then added to purify the mRNA in the lysate. After RNA extraction, reverse transcription was immediately performed by SuperScript VILO cDNA Synthesis Kit (Invitrogen life technology). C3 and TBP mRNA were determined by Quantstudio 5 real-time PCR system (Invitrogen life technology) using TaqMan qPCR primers, C3-FAM (Assay ID: Hs00163811_m1) and TBP-VIC (Assay ID: Hs00427620_m1). The activity of a given C3 siRNA was expressed as the percentage of remaining C3 mRNA normalized to TBP mRNA in treated cells relative to C3 mRNA normalized to TBP mRNA in cells not treated with siRNA (mock). Dose response curves were prepared using a four-parameter logic model with GraphPad Prism version 9. The data shown in Figures 2A and 2B are from three biological replicates. The data in Figure 2B are derived from the 0.16 nM and 100 nM data in Figure 2A.

實施例4.siRNA的化學修飾Example 4. Chemical modification of siRNA

為了增強小干擾核苷酸的穩定性並改善藥物施用的肝靶向性，對人源C3基因的小干擾核苷酸C3-8、C3-10、C3-16、C3-48、C3-50、C3-55、C3-67、C3-68和C3-75進行化學修飾設計，設計原理如下：第一單鏈或第二單鏈的5’端的第1號核苷酸開始依次編號核苷酸。 In order to enhance the stability of small interfering nucleotides and improve the liver targeting of drug administration, chemical modification design was performed on small interfering nucleotides C3-8, C3-10, C3-16, C3-48, C3-50, C3-55, C3-67, C3-68 and C3-75 of the human C3 gene. The design principle is as follows: the nucleotides are numbered sequentially starting from the first nucleotide at the 5' end of the first single chain or the second single chain.

第一單鏈(正義鏈)的修飾模式： Modification mode of the first single chain (justice chain):

SS7：第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾；第1、2、3、4、5、6、8、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 SS7: The pentoses at nucleotide residues 7, 9, 10, and 11 are modified by 2'-fluorine substitution; the pentoses at nucleotide residues 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification.

SS1：第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 SS1: The pentoses at the nucleotide residues at positions 3, 5, 7, 8, and 9 are modified by 2'-fluorine substitution; the pentoses at the nucleotide residues at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2'-methoxy modification.

SS2：第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 SS2: The pentoses at the 5th, 7th, 8th, 9th and 11th nucleotide residues are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxyl modification.

SS3：第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 SS3: The pentoses at the 5th, 7th, 8th, 9th and 14th nucleotide residues are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues are modified by 2'-methoxyl modification.

第二單鏈(反義鏈)的修飾模式： Modification pattern of the second single chain (antisense chain):

AS8：第2、6、8、9、14和16位核苷酸殘基的戊糖經2’-氟取代修飾；第1、3、4、5、7、10、11、12、13、15、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 AS8: The pentoses at nucleotide residues 2, 6, 8, 9, 14, and 16 are modified by 2'-fluorine substitution; the pentoses at nucleotide residues 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, and 19 are modified by 2'-methoxyl modification.

AS4：第2、10、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；第1、3、4、5、6、7、8、9、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 AS4: The pentoses at nucleotide residues 2, 10, 14, 16, and 18 are modified by 2'-fluorine substitution; the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

AS5：第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 AS5: The pentoses at nucleotide residues 2, 8, 14, 16, and 18 are modified by 2'-fluorine substitution; the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

AS6：第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 AS6: The pentoses at nucleotide residues 2, 4, 14, 16, and 18 are modified by 2'-fluorine substitution; the pentoses at nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2'-methoxy modification.

siRNA包含SS7/AS8、SS1/AS4、SS1/AS5、SS1/AS6、SS2/AS4、SS2/AS5、SS2/AS6、SS3/AS4、SS3/AS5或SS3/AS6的修飾模式。 The siRNA contains modification patterns of SS7/AS8, SS1/AS4, SS1/AS5, SS1/AS6, SS2/AS4, SS2/AS5, SS2/AS6, SS3/AS4, SS3/AS5, or SS3/AS6.

在第二單鏈(反義鏈)5'端和3'端的最後3個核苷酸之間引入了硫代磷酸酯(表3A、表3B、表3C、表3D、表3E、表3F中的*表示硫代磷酸酯)。在第一單鏈(正義鏈)5'端或3'端的最後3個核苷酸之間也引入了硫代磷酸酯。(E)-乙烯基膦酸酯(表3A、表3B、表3C、表3D、表3E、表3F中的“(E)-VP”表示(E)-乙烯基膦酸酯)用於第二單鏈(反義鏈)的5'端。 Phosphorothioate was introduced between the last three nucleotides at the 5' end and 3' end of the second single strand (antisense strand) (* in Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, Table 3F indicates phosphorothioate). Phosphorothioate was also introduced between the last three nucleotides at the 5' end or 3' end of the first single strand (positive strand). (E)-vinylphosphonate ("(E)-VP" in Table 3A, Table 3B, Table 3C, Table 3D, Table 3E, Table 3F indicates (E)-vinylphosphonate) was used at the 5' end of the second single strand (antisense strand).

表3A、表3B和表3C中的“GN”是包含N-乙醯半乳糖胺(GalNAc)的靶向配體。 "GN" in Table 3A, Table 3B, and Table 3C is a targeting ligand containing N-acetylgalactosamine (GalNAc).

修飾的siRNA C3-144至C3-152包含修飾模式SS7/AS8。修飾的siRNA C3-144至C3-152的序列信息如表3A所示。修飾的siRNA C3-104至C3-113包含修飾模式SS1/AS6、SS2/AS5、SS2/AS6、SS3/AS5、SS3/AS6。修飾的siRNA C3-104至C3-113的序列信息如表3B所示。修飾的siRNA C3-114至C3-123包含修飾模式SS1/AS6、SS2/AS5、SS2/AS6、SS3/AS5、SS3/AS6。修飾的siRNA C3-114至C3-123的序列信息如表3C所示。 Modified siRNAs C3-144 to C3-152 contain modification patterns SS7/AS8. The sequence information of modified siRNAs C3-144 to C3-152 is shown in Table 3A. Modified siRNAs C3-104 to C3-113 contain modification patterns SS1/AS6, SS2/AS5, SS2/AS6, SS3/AS5, SS3/AS6. The sequence information of modified siRNAs C3-104 to C3-113 is shown in Table 3B. Modified siRNAs C3-114 to C3-123 contain modification patterns SS1/AS6, SS2/AS5, SS2/AS6, SS3/AS5, SS3/AS6. The sequence information of modified siRNAs C3-114 to C3-123 is shown in Table 3C.

表3A

Figure 113123276-A0202-12-0141-149
Table 3A
Figure 113123276-A0202-12-0141-149

表3B

Figure 113123276-A0202-12-0142-150
Table 3B
Figure 113123276-A0202-12-0142-150

Figure 113123276-A0202-12-0143-151
Figure 113123276-A0202-12-0143-151

表3C

Figure 113123276-A0202-12-0143-152
Table 3C
Figure 113123276-A0202-12-0143-152

Figure 113123276-A0202-12-0144-153
Figure 113123276-A0202-12-0144-153

具體來說，GalNAc-1、GalNAc-3和GalNAc-7與dsRNA綴合，用於體外和體內篩選。siRNA劑與GalNAc-1、GalNAc-3和GalNAc-7綴合，如下圖所示： Specifically, GalNAc-1, GalNAc-3, and GalNAc-7 are conjugated to dsRNA for in vitro and in vivo screening. siRNA agents are conjugated to GalNAc-1, GalNAc-3, and GalNAc-7, as shown in the figure below:

Figure 113123276-A0202-12-0144-155
Figure 113123276-A0202-12-0144-155

Figure 113123276-A0202-12-0145-156
Figure 113123276-A0202-12-0145-156

Figure 113123276-A0202-12-0145-157
Figure 113123276-A0202-12-0145-157

修飾的siRNA C3-95至C3-103包含修飾模式SS7/AS8。修飾的siRNA C3-95至C3-103的序列信息如表3D所示。修飾的siRNA C3-124至C3-133包含修飾模式SS1/AS6、SS2/AS5、SS2/AS6、SS3/AS5、SS3/AS6。修飾的siRNA C3-124至C3-133的序列信息如表3E所示。修飾的siRNA C3-134至C3-143包含修飾模式SS1/AS6、SS2/AS5、SS2/AS6、SS3/AS5、SS3/AS6。修飾的siRNA C3-134至C3-143的序列信息如表3F所示。 Modified siRNAs C3-95 to C3-103 contain modification patterns SS7/AS8. The sequence information of modified siRNAs C3-95 to C3-103 is shown in Table 3D. Modified siRNAs C3-124 to C3-133 contain modification patterns SS1/AS6, SS2/AS5, SS2/AS6, SS3/AS5, SS3/AS6. The sequence information of modified siRNAs C3-124 to C3-133 is shown in Table 3E. Modified siRNAs C3-134 to C3-143 contain modification patterns SS1/AS6, SS2/AS5, SS2/AS6, SS3/AS5, SS3/AS6. The sequence information of modified siRNAs C3-134 to C3-143 is shown in Table 3F.

表3D

Figure 113123276-A0202-12-0146-158
Table 3D
Figure 113123276-A0202-12-0146-158

表3E

Figure 113123276-A0202-12-0147-159
Table 3E
Figure 113123276-A0202-12-0147-159

Figure 113123276-A0202-12-0148-160
Figure 113123276-A0202-12-0148-160

表3F

Figure 113123276-A0202-12-0148-161
Table 3F
Figure 113123276-A0202-12-0148-161

Figure 113123276-A0202-12-0149-162
Figure 113123276-A0202-12-0149-162

化學修飾的小干擾核苷酸由藥明康德(Wuxi AppTec)合成。靶向配體(GalNAc)可以作為單條(三分支)或以1+1+1三價的形式分步綴合到寡核苷酸的3'端或5'端。要實現三分支GalNAc綴合或1+1+1組裝，應當分別合成GalNAc醯胺化物和/或GalNAc固體載體。然後將GalNAc醯胺化物和/或GalNAc固體載體用作構建塊用於固相寡核苷酸合成。 Chemically modified siRNAs were synthesized by Wuxi AppTec. The targeting ligand (GalNAc) can be conjugated to the 3' or 5' end of the oligonucleotide as a single strand (three-branched) or in a 1+1+1 trivalent form in a stepwise manner. To achieve three-branched GalNAc conjugation or 1+1+1 assembly, GalNAc amides and/or GalNAc solid supports should be synthesized separately. The GalNAc amides and/or GalNAc solid supports are then used as building blocks for solid phase oligonucleotide synthesis.

GalNAc綴合的亞磷醯胺： GalNAc-conjugated phosphoramidites:

所有GalNAc綴合的亞磷醯胺的合成遵循在實施例5中描述的方法。 The synthesis of all GalNAc-conjugated phosphamides followed the method described in Example 5.

GalNAc綴合的琥珀酸酯和上樣到固體載體上： GalNAc-conjugated succinate and loading onto solid support:

GalNAc綴合的琥珀酸酯的合成和上樣至固體載體遵循在實施例5中描述的方法。 The synthesis and loading of GalNAc-conjugated succinate onto the solid support followed the method described in Example 5.

siRNA製備的一般方法 General methods for siRNA preparation

1.上樣：在5mL注射器中GalNAc-或核苷預裝的CPG(0.19g，500A，~10μmol)。 1. Loading: GalNAc- or nucleoside-preloaded CPG (0.19 g, 500 A, ~10 μmol) in a 5 mL syringe.

2.洗滌：乙腈(2.0mL，0.3分鐘，重複兩次，室溫)。 2. Washing: acetonitrile (2.0 mL, 0.3 min, repeated twice, room temperature).

3.去三苯甲基化：3%三氯乙酸的二氯甲烷溶液(2.0mL，0.7分鐘，在室溫中重複四次)。 3. Detritylation: 3% trichloroacetic acid in dichloromethane (2.0 mL, 0.7 min, repeated four times at room temperature).

4.洗滌：乙腈(2.0mL，0.3分鐘，重複兩次，室溫)。 4. Washing: acetonitrile (2.0 mL, 0.3 min, repeated twice, room temperature).

5.偶聯：0.067M的反應單體在乙腈(1.0mL)中的溶液和0.30M 5-(苄基硫基)-1H-四唑(BTT)在乙腈(1.0mL)中的溶液作為活化劑(7.0分鐘，在室溫中重複三次) 5. Coupling: 0.067M solution of the reaction monomer in acetonitrile (1.0mL) and 0.30M solution of 5-(benzylthio)-1H-tetrazole (BTT) in acetonitrile (1.0mL) as an activator (7.0 minutes, repeated three times at room temperature)

6.硫化或氧化：N,N-二甲基-N'-(3-硫代-3H-1,2,4-二唑-5-基)甲脒(DDTT，4.80M，在吡啶/乙腈=2/1，2.0mL中，1分鐘，兩次，在室溫中)或0.05M I2在吡啶/H2O=80/20(v/v)中的溶液(2.0mL，1分鐘，兩次，在室溫中)。 6. Sulfurization or oxidation: N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-oxadiazole-5-yl)formamidine (DDTT, 4.80M, in pyridine/acetonitrile = 2/1, 2.0mL, 1 minute, twice, at room temperature) or 0.05M I2 in pyridine/H2O = 80/20 (v/v) solution (2.0mL, 1 minute, twice, at room temperature).

7.封端：1-甲基咪唑(NMI)/乙腈=15/85(v/v)(2.0mL)和乙酸酐/乙腈=20/80(v/v)(0.9mL，1分鐘，一次，在室溫中)。 7. End-capping: 1-methylimidazole (NMI)/acetonitrile = 15/85 (v/v) (2.0 mL) and acetic anhydride/acetonitrile = 20/80 (v/v) (0.9 mL, 1 minute, once, at room temperature).

8.洗滌：乙腈(2.6mL，0.3分鐘，重複兩次，室溫)。 8. Washing: acetonitrile (2.6 mL, 0.3 min, repeated twice, room temperature).

9.合成程序自動循環19次。 9. The synthesis process automatically cycles 19 times.

10.將固體載體浸入10% DEA中20分鐘。將固體載體懸浮在NH3．H2O(5mL)中，在48mL密封管中在40℃中攪拌16小時。使反應混合物冷卻至25℃。然後過濾固體載體，將水相在真空中濃縮得到黃色溶液。 10. Immerse the solid support in 10% DEA for 20 minutes. Suspend the solid support in NH3.H2O (5 mL) and stir at 40°C in a 48 mL sealed tube for 16 hours. Cool the reaction mixture to 25°C. Then filter the solid support and concentrate the aqueous phase in vacuo to obtain a yellow solution.

11.向濾液(10mL)中加入40mL乙醇，然後接著加入0.3mL NaCl(3M)。離心管在-20℃靜置20分鐘。然後將管離心。棄去上清液並收集剩餘的固體。 11. Add 40 mL of ethanol to the filtrate (10 mL), followed by 0.3 mL of NaCl (3 M). Incubate the tube at -20°C for 20 minutes. Then centrifuge the tube. Discard the supernatant and collect the remaining solid.

12.白色固體藉由製備型HPLC純化(管柱：O-C18 150*40mm*10μm；流動相：[0.1M TEAB-ACN]；B%：14%-24%，30分鐘)。 12. The white solid was purified by preparative HPLC (column: O-C18 150*40mm*10μm; mobile phase: [0.1M TEAB-ACN]; B%: 14%-24%, 30 minutes).

13.凍乾後得到期望的為白色固體的化合物。 13. After freeze-drying, the desired compound is obtained as a white solid.

退火步驟： Annealing step:

1.計算雙鏈體的莫耳值(例如，2mg的雙鏈體，莫耳值=2mg/雙鏈體M.W.，游離酸)，這是單鏈莫耳值。 1. Calculate the molar value of the dimer (e.g., 2 mg of dimer, molar value = 2 mg/M.W. of dimer, free acid), which is the molar value of the single chain.

2.在25℃中，分別將正義鏈和反義鏈溶解在超純水(無DNase/RNase，無菌)中。寡聚物的濃度藉由紫外-可見光(UV-vis)定量。 2. Dissolve the positive and antisense chains separately in ultrapure water (DNase/RNase-free, sterile) at 25°C. The concentration of the oligomers is quantified by UV-vis.

3.以1：1的莫耳比混合兩種溶液。 3. Mix the two solutions in a molar ratio of 1:1.

4.將混合樣品在室溫下放置10分鐘。藉由HPLC測試以監測配比是否合適。 4. Place the mixed sample at room temperature for 10 minutes. Use HPLC test to monitor whether the ratio is appropriate.

5.將溶液分裝到試管中並凍乾以得到siRNA樣品。 5. Dispense the solution into test tubes and freeze-dry to obtain siRNA samples.

實施例5：GalNAc配體的化學合成。Example 5: Chemical synthesis of GalNAc ligand.

Figure 113123276-A0202-12-0151-164
Figure 113123276-A0202-12-0151-164

化合物1-2：Compound 1-2:

將化合物1-1(5g，12.84mmol)溶於無水1,2-二氯乙烷(30mL)中，在0℃下攪拌，在10分鐘內逐滴加入TMSOTf(3.43g，15.41mmol，2.78mL)，繼續在室溫下攪拌過夜。用冷的飽和NaHCO3溶液(200mL)淬滅反應混合物，分離有機層。用二氯甲烷(60mLx2)萃取產物；合併的有機層用水洗滌，用無水Na2SO4乾 燥，減壓蒸發至乾，得到化合物1-2(4.23g，產率為99%)，為黃色油狀物，無需進一步純化即可使用。 Compound 1-1 (5 g, 12.84 mmol) was dissolved in anhydrous 1,2-dichloroethane (30 mL), stirred at 0 ° C, TMSOTf (3.43 g, 15.41 mmol, 2.78 mL) was added dropwise over 10 minutes, and stirring was continued at room temperature overnight. The reaction mixture was quenched with cold saturated NaHCO 3 solution (200 mL), and the organic layer was separated. The product was extracted with dichloromethane (60 mL x 2); the combined organic layers were washed with water, dried over anhydrous Na 2 SO 4 , and evaporated to dryness under reduced pressure to obtain compound 1-2 (4.23 g, yield 99%), which was a yellow oil and used without further purification.

C14H19NO8質量計算為：329.1；實測：330.1[M+H]+，ESI。 Mass calculated for C 14 H 19 NO 8 : 329.1; found: 330.1 [M+H] + , ESI.

化合物1-3：Compound 1-3:

將化合物1-2(4.23g，12.85mmol)溶於無水1,2-二氯乙烷(20mL)中，與4Å分子篩(4.7g)在室溫下攪拌5分鐘。加入5-己烯-1-醇(1.42g，14.13mmol)，繼續攪拌30分鐘。在0℃下逐滴加入TMSOTf(1.43g，6.42mmol，1.16mL)，在室溫下繼續攪拌2小時。用冷的飽和NaHCO3溶液(100mL)淬滅反應混合物，分離有機層。用二氯甲烷(60mLx2)萃取產物；合併的有機層用水洗滌，用無水Na2SO4乾燥，減壓蒸發至乾，得到化合物1-3(5.5g，產率為99%)，為黃色油狀物，無需進一步純化即可使用。 Compound 1-2 (4.23 g, 12.85 mmol) was dissolved in anhydrous 1,2-dichloroethane (20 mL) and stirred with 4Å molecular sieve (4.7 g) at room temperature for 5 minutes. 5-Hexen-1-ol (1.42 g, 14.13 mmol) was added and stirring was continued for 30 minutes. TMSOTf (1.43 g, 6.42 mmol, 1.16 mL) was added dropwise at 0 ° C and stirring was continued at room temperature for 2 hours. The reaction mixture was quenched with cold saturated NaHCO 3 solution (100 mL) and the organic layer was separated. The product was extracted with dichloromethane (60 mL x 2); the combined organic layers were washed with water, dried over anhydrous Na 2 SO 4 , and evaporated to dryness under reduced pressure to obtain compound 1-3 (5.5 g, yield 99%) as a yellow oil, which was used without further purification.

C20H31NO9質量計算為：429.2；實測：430.2[M+H]+，ESI。 Mass calculated for C 20 H 31 NO 9 : 429.2; found: 430.2 [M+H] + , ESI.

化合物1-4：Compound 1-4:

向化合物1-3(5.5g，12.81mmol)在DCM(35mL)和MeCN(35mL)中的溶液中加入4.0mol當量的(偏)高碘酸鈉(10.96g，51.24mmol)在水(45.5mL)中的溶液。將混合物在冰浴中冷卻至0℃並攪拌15分鐘。將三水合氯化釕(110.5mg，423μmol)加入冷的反應混合物中。將反應混合物在室溫下攪拌4小時。用水(90mL)稀釋反應混合物，加入固體NaHCO3將pH調節至7.5。除去DCM層，用DCM(30mLx2)洗滌水層，棄去有機萃取物。加入檸檬酸將水層的pH調節至3，將羧酸1-4萃取到DCM(50mLx3)中。將有機層與飽和鹽水(50mLx1)一起攪拌，然後逐滴加入Na2S2O3溶液(50mLx1)，直到深綠色有機相變成淡黃色。分離各 層，有機層經無水Na2SO4乾燥，減壓蒸發，得到化合物1-4，為白色固體(2.3g，產率為40%)。無需進一步純化即可使用。 To a solution of compound 1-3 (5.5 g, 12.81 mmol) in DCM (35 mL) and MeCN (35 mL) was added 4.0 mol equivalents of sodium (meta) periodate (10.96 g, 51.24 mmol) in water (45.5 mL). The mixture was cooled to 0 ° C in an ice bath and stirred for 15 minutes. Ruthenium chloride trihydrate (110.5 mg, 423 μmol) was added to the cold reaction mixture. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water (90 mL) and solid NaHCO 3 was added to adjust the pH to 7.5. The DCM layer was removed, the aqueous layer was washed with DCM (30 mL x 2), and the organic extract was discarded. The pH of the aqueous layer was adjusted to 3 by adding citric acid, and the carboxylic acid 1-4 was extracted into DCM (50 mL x 3). The organic layer was stirred with saturated brine (50 mL x 1), and then Na 2 S 2 O 3 solution (50 mL x 1) was added dropwise until the dark green organic phase turned light yellow. The layers were separated, and the organic layer was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to obtain compound 1-4 as a white solid (2.3 g, 40% yield). It was used without further purification.

C19H29NO11質量計算為：447.2；實測：448.2[M+H]+，ESI。 Mass calculated for C 19 H 29 NO 11 : 447.2; found: 448.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.81(d,J=9.2Hz,1H),5.84-5.73(m,1H),5.21(d,J=3.3Hz,1H),4.98-4.93(m,3H),4.48(d,J=8.5Hz,1H),4.04-4.00(m,3H),3.88-3.83(m,1H),3.73-3.68(m,1H),3.40-3.36(m,1H),2.10(s,3H),2.03-2.02(m,2H),2.00(s,3H),1.89(s,3H),1.76(s,3H),1.43-1.32(m,4H). 1 H NMR(400MHz, DMSO-d6)δ 7.81(d, J =9.2Hz,1H),5.84-5.73(m,1H),5.21(d, J =3.3Hz,1H),4.98-4.93(m,3H),4.48(d, J =8.5Hz,1H),4.04-4.00(m,3H),3.88-3.83(m,1H),3.73-3.68(m,1H),3.40-3.36(m,1H), 2.10(s,3H),2.03-2.02(m,2H),2.00(s,3H),1.89(s,3H),1.76(s,3H),1.43-1.32(m,4H).

化合物1-6：Compounds 1-6:

向化合物1-5(1.16g，6.17mmol)和酸1-4(2.3g，5.14mmol)在DMF(30mL)中的溶液中加入HBTU(2.92g，7.71mmol)和DIPEA(1.99g，15.42mmol，2.69mL)。將反應在室溫下攪拌43小時，並用水稀釋(150mL)。用乙酸乙酯(60mLx3)萃取混合物。用水(100mLx3)和鹽水(100mL)連續洗滌合併的有機層。經無水Na2SO4乾燥後，減壓蒸發溶劑，得到化合物1-6，為黃色油狀物(4.3g，粗產物)，無需進一步純化即可使用。 To a solution of compound 1-5 (1.16 g, 6.17 mmol) and acid 1-4 (2.3 g, 5.14 mmol) in DMF (30 mL) was added HBTU (2.92 g, 7.71 mmol) and DIPEA (1.99 g, 15.42 mmol, 2.69 mL). The reaction was stirred at room temperature for 43 hours and diluted with water (150 mL). The mixture was extracted with ethyl acetate (60 mL x 3). The combined organic layers were washed successively with water (100 mL x 3) and brine (100 mL). After drying over anhydrous Na 2 SO 4 , the solvent was evaporated under reduced pressure to give compound 1-6 as a yellow oil (4.3 g, crude product) which was used without further purification.

C29H48N2O12質量計算為：616.3；實測：617.4[M+H]+，ESI。 Mass calculated for C 29 H 48 N 2 O 12 : 616.3; found: 617.4 [M+H] + , ESI.

化合物1-7：Compound 1-7:

將化合物1-6(4.23g，假設6.86mmol)加入到甲酸(30mL)中，室溫下攪拌混合物過夜。LC-MS監測反應完成。減壓蒸發混合物，藉由矽膠層析純化(DCM：MeOH=10：1)，得到化合物1-7，為黃色油狀物(2.88g，2步產率75%)。 Compound 1-6 (4.23 g, assumed 6.86 mmol) was added to formic acid (30 mL), and the mixture was stirred at room temperature overnight. LC-MS monitored the completion of the reaction. The mixture was evaporated under reduced pressure and purified by silica gel chromatography (DCM: MeOH = 10: 1) to obtain compound 1-7 as a yellow oil (2.88 g, 2-step yield 75%).

C25H40N2O12質量計算為：560.3；實測：561.3[M+H]+，ESI。 Mass calculated for C 25 H 40 N 2 O 12 : 560.3; found: 561.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ12.02(s,1H),7.83(d,J=9.2Hz,1H),7.25(t,J=5.6Hz,1H),5.21(d,J=3.2Hz,1H),4.96(dd,J=11.2,3.6Hz,1H), 4.47(d,J=8.4Hz,1H),4.04-4.00(m,3H),3.90-3.83(m,1H),3.73-3.68(m,1H),3.42-3.36(m,1H),3.02-2.97(m,1H),2.18(t,J=7.2Hz,2H),2.10(s,3H),2.02(t,J=7.2Hz,2H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.54-1.41(m,7H),1.27-1.21(m,3H). 1 H NMR (400MHz, DMSO-d6) δ12.02(s,1H),7.83(d, J =9.2Hz,1H),7.25(t, J =5.6Hz,1H),5.21(d, J =3.2Hz,1H),4.96(dd, J =11.2,3.6Hz,1H), 4.47(d, J =8.4Hz,1H),4.04-4.00(m,3H),3.90-3.83(m,1H),3.73-3.68(m,1H),3.42-3.36(m,1H),3.02-2.97(m,1H),2.18(t, J =7.2Hz,2H),2.10(s,3H),2.02(t, J =7.2Hz,2H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.54-1.41(m,7H),1.27-1.21(m,3H).

化合物2-1：Compound 2-1:

Figure 113123276-A0202-12-0154-165
Figure 113123276-A0202-12-0154-165

將2,2-雙(溴甲基)丙烷-1,3-二醇(270g，1.03mol)、苯甲醛(114.86g，1.08mol)、TsOH(17.74g，340.55mmol)的混合物在甲苯(1L)中回流6小時。將混合物冷卻並用EA(1L)萃取，用NaHCO3溶液洗滌，然後用鹽水洗滌，用Na2SO4乾燥。減壓除去溶劑，殘餘物用MeOH再結晶以得到所需產物2-1(247g，產率為68%)。 A mixture of 2,2-bis(bromomethyl)propane-1,3-diol (270 g, 1.03 mol), benzaldehyde (114.86 g, 1.08 mol), and TsOH (17.74 g, 340.55 mmol) was refluxed in toluene (1 L) for 6 hours. The mixture was cooled and extracted with EA (1 L), washed with NaHCO 3 solution, then with brine, and dried over Na 2 SO 4. The solvent was removed under reduced pressure, and the residue was recrystallized with MeOH to obtain the desired product 2-1 (247 g, 68% yield).

1H NMR(400MHz,DMSO-d6)δ 7.47-7.41(m,2H),7.40-7.36(m,3H),5.48(s,1H),4.09-3.93(m,6H),3.46(s,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.47-7.41(m,2H),7.40-7.36(m,3H),5.48(s,1H),4.09-3.93(m,6H),3.46(s,2H).

化合物2-2：Compound 2-2:

向t-BuOK(137.27g，1.43mol)在無水DMF(700mL)中的懸浮液中逐滴加入丙二酸二異丙酯(268.85g，1.43mol)(溫度保持在70℃以下)，然後加入化合物2-1(250g，714.19mmol)。將所得反應混合物在140℃下加熱6小時。冷卻後，加入飽和NH4Cl溶液(1.5L)，並用己烷(500mLx3)萃取混合物。將合併的有機萃取物用硫酸鈉乾燥並在真空中濃縮。藉由過濾將固體產物與液體殘餘物分離，用己烷(100mLx2)洗滌並乾燥，得到純產物2-2，為白色固體(194g，產率為68%)。 To a suspension of t-BuOK (137.27 g, 1.43 mol) in anhydrous DMF (700 mL) was added diisopropyl malonate (268.85 g, 1.43 mol) dropwise (temperature maintained below 70°C), followed by compound 2-1 (250 g, 714.19 mmol). The resulting reaction mixture was heated at 140°C for 6 hours. After cooling, saturated NH 4 Cl solution (1.5 L) was added, and the mixture was extracted with hexane (500 mL x 3). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The solid product was separated from the liquid residue by filtration, washed with hexane (100 mL x 2) and dried to give the pure product 2-2 as a white solid (194 g, 68% yield).

C21H28O6質量計算為：376.2；實測：377.2[M+H]+，ESI。 Mass calculated for C 21 H 28 O 6 : 376.2; found: 377.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.45-7.31(m,5H),5.45(s,1H),5.02-4.93(m,2H),3.96(d,J=11.2Hz,2H),3.77(d,J=11.1Hz,2H),2.55(s,2H),2.13(s,2H),1.23-1.15(m,12H). 1 H NMR(400MHz, DMSO-d6)δ 7.45-7.31(m,5H),5.45(s,1H),5.02-4.93(m,2H),3.96(d, J =11.2Hz,2H),3.77(d, J =11.1Hz,2H),2.55(s,2H),2.13(s,2H),1.23-1.15(m,12H).

化合物2-3：Compound 2-3:

向化合物2-2(155g，411.7mmol)的MeOH(750mL)溶液中加入10% Pd/C(31g)，所得懸浮液在5個大氣壓的H2下，在環境溫度下攪拌氫化48小時。過濾催化劑並真空除去溶劑，得到化合物2-3，為無色油狀物(118g，產率為99%)，無需進一步純化即可用於下一步。 10% Pd/C (31 g) was added to a solution of compound 2-2 (155 g, 411.7 mmol) in MeOH (750 mL), and the resulting suspension was stirred and hydrogenated under 5 atmospheres of H2 at ambient temperature for 48 hours. The catalyst was filtered and the solvent was removed in vacuo to obtain compound 2-3 as a colorless oil (118 g, 99% yield), which was used in the next step without further purification.

C21H28O6質量計算為：288.2；實測：289.4[M+H]+，ESI。 Mass calculated for C 21 H 28 O 6 : 288.2; found: 289.4 [M+H] + , ESI.

化合物2-4：Compound 2-4:

向化合物2-3(140g，485.5mmol)的二氯甲烷(840mL)溶液中加入甲磺醯氯(155.7g，1.36mol)。將所得混合物冷卻至-30℃，然後逐滴加入三乙胺(323.9g，3.2mol)。加入完成後，將反應混合物升溫至環境溫度並攪拌12小時，然後用水(1000mL)、10%檸檬酸水溶液(1000mL)和鹽水(1000mL)洗滌。有機相經硫酸鈉乾燥並減壓蒸發以得到化合物2-4(125g，產率為58%)。 Methanesulfonyl chloride (155.7 g, 1.36 mol) was added to a solution of compound 2-3 (140 g, 485.5 mmol) in dichloromethane (840 mL). The resulting mixture was cooled to -30 °C, and triethylamine (323.9 g, 3.2 mol) was then added dropwise. After the addition was complete, the reaction mixture was warmed to ambient temperature and stirred for 12 hours, then washed with water (1000 mL), 10% aqueous citric acid solution (1000 mL) and brine (1000 mL). The organic phase was dried over sodium sulfate and evaporated under reduced pressure to obtain compound 2-4 (125 g, 58% yield).

C21H28O6質量計算為：444.1；實測：445.2[M+H]+，ESI。 Mass calculated for C 21 H 28 O 6 : 444.1; found: 445.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 5.02-4.93(m,2H),4.20(s,4H),3.23(s,6H),2.45(s,4H),1.22-1.14(m,12H). 1 H NMR(400MHz, DMSO-d6)δ 5.02-4.93(m,2H),4.20(s,4H),3.23(s,6H),2.45(s,4H),1.22-1.14(m,12H).

化合物2-5：Compound 2-5:

將化合物2-4(63g，141.73mmol)、碳酸鉀(100.88g，729.89mmol)和對甲苯磺醯胺(25.48g，148.81mmol)在DMSO(300mL)中的溶液在85℃加熱12小時。冷卻後，加入水(300mL)，用EtOAc(600mL)萃取混合物。將合併的有機相用10%檸檬酸水溶液(600mL)和鹽水(600mL)洗滌，用硫酸鈉乾燥並真空蒸發，然後用異丙醚再結晶，得到化合物2-5(48g，產率為80%)。 A solution of compound 2-4 (63 g, 141.73 mmol), potassium carbonate (100.88 g, 729.89 mmol) and p-toluenesulfonamide (25.48 g, 148.81 mmol) in DMSO (300 mL) was heated at 85 ° C for 12 hours. After cooling, water (300 mL) was added and the mixture was extracted with EtOAc (600 mL). The combined organic phase was washed with 10% citric acid aqueous solution (600 mL) and brine (600 mL), dried over sodium sulfate and evaporated in vacuo, and then recrystallized with isopropyl ether to obtain compound 2-5 (48 g, yield 80%).

C21H28O6計算為423.2；實測：424.2[M+H]+，ESI。 Calculated for C 21 H 28 O 6 : 423.2; found: 424.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.67(d,J=8.2Hz,2H),7.45(d,J=8.0Hz,2H),4.45(t,J=5.4Hz,2H),3.60(s,4H),3.14(d,J=5.7Hz,4H),2.43(s,3H),1.67(s,4H). 1 H NMR(400MHz, DMSO-d6)δ 7.67(d, J =8.2Hz,2H),7.45(d, J =8.0Hz,2H),4.45(t, J =5.4Hz,2H),3.60(s,4H),3.14(d, J =5.7Hz,4H),2.43(s,3H),1.67(s,4H).

化合物2-6：Compound 2-6:

在-20℃下，向化合物2-5(58.7g，138.60mmol)在THF(100mL)中的溶液中加入硼氫化鋰(2M)在THF(263.82mL)中的溶液。將所得混合物在室溫下攪拌16小時。將混合物緩慢加入冰水(1L)中。用檸檬酸水溶液調節pH至7。混合物用EA(500mL)萃取，用無水硫酸鈉乾燥，減壓蒸發以得到化合物2-6(40g，產率為93%)。 To a solution of compound 2-5 (58.7 g, 138.60 mmol) in THF (100 mL) was added a solution of lithium borohydride (2 M) in THF (263.82 mL) at -20°C. The resulting mixture was stirred at room temperature for 16 hours. The mixture was slowly added to ice water (1 L). The pH was adjusted to 7 with aqueous citric acid solution. The mixture was extracted with EA (500 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give compound 2-6 (40 g, yield 93%).

C21H28O6質量計算為：311.1；實測：312.1[M+H]+，ESI。 Mass calculated for C 21 H 28 O 6 : 311.1; found: 312.1 [M+H] + , ESI.

1H NMR(400MHz,DMSO-D6)δ 7.67(d,J=8.2Hz,2H),7.45(d,J=8.0Hz,2H),4.45(t,J=5.4Hz,2H),3.60(s,4H),3.14(d,J=5.7Hz,4H),2.43(s,3H),1.67(s,4H). 1 H NMR(400MHz, DMSO-D6)δ 7.67(d, J =8.2Hz,2H),7.45(d, J =8.0Hz,2H),4.45(t, J =5.4Hz,2H),3.60(s,4H),3.14(d, J =5.7Hz,4H),2.43(s,3H),1.67(s,4H).

化合物2-7：Compound 2-7:

將化合物2-6(5g，16.1mmol)和Mg(粉末，3.47g，144.6mmol)混合在MeOH(無水，60mL)中並在室溫下攪拌過夜。LC-MS顯示完全轉化，並加入水(30mL)。形成白色沉澱。過濾混合物，用HCl(4M，水溶液)將濾液調節至pH 6，濃縮至乾以得到化合物2-7(2.55g，粗產物)，為黃色固體。粗產物無需進一步純化即可使用。 Compound 2-6 (5 g, 16.1 mmol) and Mg (powder, 3.47 g, 144.6 mmol) were mixed in MeOH (anhydrous, 60 mL) and stirred at room temperature overnight. LC-MS showed complete conversion, and water (30 mL) was added. A white precipitate was formed. The mixture was filtered, and the filtrate was adjusted to pH 6 with HCl (4 M, aqueous solution) and concentrated to dryness to give compound 2-7 (2.55 g, crude product) as a yellow solid. The crude product was used without further purification.

C8H15NO2質量計算為：157.1；實測：158.1[M+H]+，ESI。 Mass calculated for C 8 H 15 NO 2 : 157.1; found: 158.1 [M+H] + , ESI.

化合物2-8：Compound 2-8:

將化合物2-7(2.55g，假定為16.2mmol)溶於二噁烷(20mL)中，在0℃下緩慢加入FmocCl(4.60g，17.8mmol)，然後加入Na2CO3(飽和的，20mL)。將反應在30℃下攪拌3小時。LC-MS顯示完全轉化。用EA(20mLx3)萃取反應混合物。濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOH=97：3)以得到白色固體2-8(2.75g，2步產率44.7%)。 Compound 2-7 (2.55 g, assumed to be 16.2 mmol) was dissolved in dioxane (20 mL), and FmocCl (4.60 g, 17.8 mmol) was slowly added at 0°C, followed by Na 2 CO 3 (saturated, 20 mL). The reaction was stirred at 30°C for 3 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mLx3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 97:3) to give a white solid 2-8 (2.75 g, 2-step yield 44.7%).

C23H25NO4質量計算為：379.2；實測：380.2[M+H]+，ESI。 Mass calculated for C 23 H 25 NO 4 : 379.2; found: 380.2 [M+H] + , ESI.

化合物2-9：Compound 2-9:

在燒瓶中混合化合物2-8(2.7g，7.1mmol)和吡啶(20mL)，然後分批加入DMTr-Cl(2.4g，7.1mmol)。將反應在室溫下攪拌4小時。LC-MS顯示完全轉化。用EA(20mLx3)萃取反應混合物。濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOH=97：3)以得到黃色固體2-9(2.8g，產率為58%)。 Compound 2-8 (2.7 g, 7.1 mmol) and pyridine (20 mL) were mixed in a flask, and then DMTr-Cl (2.4 g, 7.1 mmol) was added in batches. The reaction was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 97: 3) to give a yellow solid 2-9 (2.8 g, yield 58%).

C44H43NO6質量計算為：681.3；實測：682.3[M+H]+，ESI。 Mass calculated for C 44 H 43 NO 6 : 681.3; found: 682.3 [M+H] + , ESI.

化合物2-10： Compound 2-10 :

將化合物2-9(2.3g，6.8mmol)、哌啶(5mL)和MeOH(無水，45mL)在燒瓶中混合，並在30℃下攪拌3小時。LC-MS顯示完全轉化。濃縮反應混合物，藉由矽膠快速管柱層析純化(DCM：MeOH=95：5)以得到黃色固體2-10(0.5g，產率為33%)。 Compound 2-9 (2.3 g, 6.8 mmol), piperidine (5 mL) and MeOH (anhydrous, 45 mL) were mixed in a flask and stirred at 30 °C for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 95: 5) to give a yellow solid 2-10 (0.5 g, yield 33%).

C29H33NO4質量計算為：459.2；實測：460.2[M+H]+，ESI。 Mass calculated for C 29 H 33 NO 4 : 459.2; found: 460.2 [M+H] + , ESI.

化合物2-11： Compound 2-11 :

將化合物1-7(325mg，0.58mmol)、HOBT(105mg，0.77mmol)和EDCI(150mg，0.78mmol)溶於DCM(6mL)中，在室溫下攪拌15分鐘，然後冷卻至0℃。加入DIPEA(211mg，1.6mmol)，然後加入化合物2-9(300mg，0.65mmol)，在室溫下攪拌4小時。LC-MS顯示完全轉化。加入NaHCO3(飽和的10mL溶液)。用DCM(5mLx3)萃取反應混合物。濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOH=95：5)以得到白色固體粗化合物2-11(350mg，210nm處純度88%，產率為46%)。製備型HPLC純化：粗化合物2-7(1.8g，210nm處純度為88%)經製備型HPLC(C-18管柱，水/ACN，10%-80% ACN)純化，以得到化合物2-11(650mg，回收率42%)。C54H71N3O15質量計算為：1001.5；實測：1024.5[M+Na]+，ESI。 Compound 1-7 (325 mg, 0.58 mmol), HOBT (105 mg, 0.77 mmol) and EDCI (150 mg, 0.78 mmol) were dissolved in DCM (6 mL), stirred at room temperature for 15 minutes, and then cooled to 0°C. DIPEA (211 mg, 1.6 mmol) was added, followed by compound 2-9 (300 mg, 0.65 mmol), and stirred at room temperature for 4 hours. LC-MS showed complete conversion. NaHCO 3 (saturated 10 mL solution) was added. The reaction mixture was extracted with DCM (5 mL×3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 95: 5) to obtain crude white solid compound 2-11 (350 mg, purity at 210 nm 88%, yield 46%). Preparative HPLC purification: Crude compound 2-7 (1.8 g, purity at 210 nm 88%) was purified by preparative HPLC (C-18 column, water/ACN, 10%-80% ACN) to obtain compound 2-11 (650 mg, recovery rate 42%). C 54 H 71 N 3 O 15 mass calculated: 1001.5; found: 1024.5 [M+Na] + , ESI.

1H NMR(400MHz,DMSO)δ 7.82(d,J=9.2Hz,1H),7.75-7.69(br,1H),7.40-7.35(m,2H),7.37-7.21(m,7H),6.95-6.88(m,4H),5.21(d,J=3.3Hz,1H),5.00-4.90(m,1H),4.66-4.63(m,1H),4.48(d,J=8.4Hz,1H),4.08-4.00(br,4H),3.91-3.83(m,1H),3.73(s,6H),3.72-3.66(m,3H),3.51(s,1H),3.43-3.37(m,3H), 3.03-2.96(m,2H),2.91(s,2H),2.10(s,3H),2.00-1.76(m,17H),1.49-1.30(m,8H),1.23-1.19(m,2H). 1 H NMR (400MHz, DMSO) δ 7.82 (d, J =9.2Hz, 1H), 7.75-7.69 (br, 1H), 7.40-7.35 (m, 2H), 7.37-7.21 (m, 7H), 6.95-6.88 (m, 4H), 5.21 (d, J =3.3Hz,1H),5.00-4.90(m,1H),4.66-4.63(m,1H),4.48(d, J =8.4Hz,1H),4.08-4.00(br,4H),3.91-3.83(m,1H),3.73(s,6H),3.72-3.66(m,3H),3.51(s,1H),3.43-3.37(m,3H), 3.03-2.96(m,2H),2.91(s,2H),2.10(s,3H),2.00-1.76(m,17H),1.49-1.30(m,8H),1.23-1.19(m,2H).

Figure 113123276-A0202-12-0159-166
Figure 113123276-A0202-12-0159-166

化合物2-12： Compound 2-12 :

在室溫中向化合物2-11(300.0mg，0.30mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(32.0mg，0.27mmol)和2-氰乙基N,N,N’,N’-四異丙基磷二醯胺(108mg，0.36mmol)，攪拌1小時。LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱：C18矽膠；流動相：CH3CN/H2O=1/1，20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物2-12，為白色固體(177mg，產率為49%)。 To a solution of compound 2-11 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (32.0 mg, 0.27 mmol) and 2-cyanoethyl N , N , N' , N' -tetraisopropylphosphodiamidate (108 mg, 0.36 mmol) at room temperature and stirred for 1 hour. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3 ), and dried over Na2SO4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1, increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 2-12 was produced as a white solid (177 mg, yield 49%).

C63H88N5O16P質量計算為：1201.60；實測：1202.6[M+H]+，ESI。 Mass calculated for C 63 H 88 N 5 O 16 P: 1201.60; found: 1202.6 [M+H] + , ESI.

1HNMR(600MHz,CD3CN)δ 7.37-7.34(m,2H),7.24-7.20(m,6H),7.14-7.12(m,1H),6.79-6.77(m,4H),6.57-6.54(m,1H),6.44-6.43(m,1H),5.20(d,J=6.0Hz,1H),4.93-4.91(m,1H),4.44(d,J=6.0Hz,1H),4.04-3.83(m,5H),3.73-3.39(m,16H),3.04-2.91(m,4H),2.52-2.50(m,2H),2.03-1.75(m,24H),1.53-1.33(m,9H),1.21-1.04(m,15H). 1 HNMR (600MHz, CD 3 CN) δ 7.37-7.34(m,2H),7.24-7.20(m,6H),7.14-7.12(m,1H),6.79-6.77(m,4H),6.57-6.54(m,1H),6.44-6.43(m,1H),5.20(d, J =6.0Hz,1H),4.93-4.91(m,1H),4.44(d, J =6.0Hz,1H),4.04-3.83(m,5H),3.73-3.39(m,16H),3.04-2.91(m,4H),2.5 2-2.50(m,2H),2.03-1.75(m,24H),1.53-1.33(m,9H),1.21-1.04(m,15H).

31PNMR(242MHz,CD3CN)δ 147.37,147.30. 31 PNMR(242MHz,CD 3 CN)δ 147.37,147.30.

化合物2-13： Compound 2-13 :

向化合物2-11(80mg，0.080mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(5mg，0.04mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗。將反應混合物用DCM(10mL)稀釋，用H2O(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物2-13，為白色固體(85mg，產率為97%)。 To a solution of compound 2-11 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL) was added DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H 2 O (3 mL×4), and then with brine (3 mL×4), and the organic layer was concentrated to give compound 2-13 as a white solid (85 mg, 97% yield).

C58H75N3O18質量計算為：1101.50；實測：1100.4[M-H]-，ESI。 Mass calculated for C 58 H 75 N 3 O 18 : 1101.50; found: 1100.4 [MH] - , ESI.

1HNMR(600MHz,DMSO-d6)δ 12.22(s,1H),7.84-7.81(m,1H),7.71-7.69(m,1H),7.37-7.16(m,9H),6.92-6.89(m,4H),5.21(d,J=6.0Hz,1H),4.97-4.95(m,1H),4.48(d,J=12.0Hz,1H),4.07-4.01(m,6H),3.89-3.84(m,1H),3.73-3.65(m,9H),3.49(s,1H),3.41-3.38(m,1H),3.01-2.95(m,4H),2.45-2.44(m,4H),2.09-1.76(m,20H),1.48-1.19(m,10H). 1 HNMR(600MHz,DMSO-d6)δ 12.22(s,1H),7.84-7.81(m,1H),7.71-7.69(m,1H),7.37-7.16(m,9H),6.92-6.89(m,4H),5.21(d, J =6.0Hz,1H),4.97-4.95(m,1H),4.48(d, J =12.0Hz,1H),4.07-4.01(m,6H),3.89-3.84(m,1H),3.73-3.65(m,9H),3.49(s,1H),3.41- 3.38(m,1H),3.01-2.95(m,4H),2.45-2.44(m,4H),2.09-1.76(m,20H),1.48-1.19(m,10H).

固體載體2-14：Solid carrier 2-14:

天然胺基-LCAA-CPG(上樣值：75μmol/g，1000Å)用ACN(100mLx2)、DMF(100mLx2)和DCM(100mLx2)洗滌，然後在高真空下乾燥過夜。向含有琥珀酸酯2-13(85mg，0.077mmol)和HBTU(53mg，0.14mmol)的無水DMF(1.5mL)的溶液中，加入DIPEA(30mg，0.23mmol)，室溫振盪反應混合物10分鐘，然後加入天然胺基-LCAA-CPG(250mg，上樣75μmol/g)，室溫振盪懸浮液20小時，然後過濾，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，得到固體載體(260mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將固體載體上未反應的胺封端，用DMF(20mLx5)、CAN(20mLx5)、DCM(20mLx5) 洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體2-14(260mg)。為了計算上樣，取6.5mg乾燥的上樣CPG，加入25mL含3% DCA的DCM溶液。振盪溶液，測量500nm處的紫外吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式：上樣(μmol/g)=(添加的DCA總體積(mL))*(500nm處的Abs值)*1000)/(76*(取的CPGmg數)) Native amine-LCAA-CPG (loading value: 75 μmol/g, 1000 Å) was washed with ACN (100 mL x 2), DMF (100 mL x 2) and DCM (100 mL x 2), and then dried under high vacuum overnight. To a solution of succinate 2-13 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL), DIPEA (30 mg, 0.23 mmol) was added, and the reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-LCAA-CPG (250 mg, loading 75 μmol/g) was added, and the suspension was shaken at room temperature for 20 hours, and then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spot in the eluent at 254 nm. The solid support was vacuum dried for 2 hours to obtain a solid support (260 mg). Stir with Ac2O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the solid support, and wash with DMF (20mLx5), CAN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 2-14 (260mg). In order to calculate the loading, 6.5mg of the dried loading CPG was taken and 25mL of DCM solution containing 3% DCA was added. The solution was shaken and the UV absorbance at 500nm was measured. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula: Load (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 500nm) * 1000) / (76 * (number of mg of CPG taken))

添加的DCA總體積(mL)=25mL Total volume of DCA added (mL) = 25mL

500nm處的Abs值=0.7721 Abs value at 500nm = 0.7721

取的CPG mg數=6.5mg The amount of CPG mg taken = 6.5mg

上樣(μmol/g)=((25)*(0.7721)*1000)/(76*(6.5))=39μmol/g。 Loading (μmol/g) = ((25)*(0.7721)*1000)/(76*(6.5)) = 39μmol/g.

方法2：Method 2:

Figure 113123276-A0202-12-0161-273
Figure 113123276-A0202-12-0161-273

化合物2-2：Compound 2-2:

將化合物2-1(11g，26.0mmol)溶解在250mL燒瓶中的THF(無水，100mL)中。在0℃下分批加LiAlH4(1.97g，52mmol)，歷時15分鐘。然後將反應在室溫下攪拌4小時。LC-MS顯示完全轉化。將反應冷卻至0℃，緩慢加入水(2mL)，然後加入NaOH(10%，2mL)，然後加入水(6mL)。過濾混合物，將濾液濃縮至乾以得到化合物2-2(6.5g，產率為80%)，為白色固體。粗產物無需進一步純化即可使用。 Compound 2-1 (11 g, 26.0 mmol) was dissolved in THF (anhydrous, 100 mL) in a 250 mL flask. LiAlH 4 (1.97 g, 52 mmol) was added in portions at 0°C for 15 minutes. The reaction was then stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction was cooled to 0°C, water (2 mL) was added slowly, followed by NaOH (10%, 2 mL), and then water (6 mL). The mixture was filtered and the filtrate was concentrated to dryness to give compound 2-2 (6.5 g, 80% yield) as a white solid. The crude product was used without further purification.

C15H21NO4S為：311.1；實測：312.1[M+H]+，ESI。 C 15 H 21 NO 4 S: 311.1; found: 312.1 [M+H] + , ESI.

1H NMR(400MHz,DMSO)δ 7.67(d,J=8.0Hz,2H),7.48(d,J=8.0Hz,2H),4.46(t,J=5.4Hz,2H),3.60(s,4H),3.31(d,J=5.6Hz,4H),2.43(s,3H),1.67(s,4H). 1 H NMR (400MHz, DMSO) δ 7.67 (d, J =8.0Hz, 2H), 7.48 (d, J = 8.0Hz, 2H), 4.46 (t, J = 5.4Hz, 2H), 3.60 (s, 4H), 3.31 (d, J =5.6Hz,4H),2.43(s,3H),1.67(s,4H).

化合物2-3：Compound 2-3:

將化合物2-2(5g，16.1mmol)和Mg(粉末，3.47g，144.6mmol)混合在MeOH(無水，60mL)中並在室溫下攪拌過夜。LC-MS顯示完全轉化，並加入水(30mL)。形成白色沉澱。過濾混合物，用HCl(4M水溶液)將濾液調節至pH 6，濃縮至乾以得到化合物2-3(2.55g，粗產物)，為黃色固體。粗產物無需進一步純化即可使用。 Compound 2-2 (5 g, 16.1 mmol) and Mg (powder, 3.47 g, 144.6 mmol) were mixed in MeOH (anhydrous, 60 mL) and stirred at room temperature overnight. LC-MS showed complete conversion, and water (30 mL) was added. A white precipitate was formed. The mixture was filtered, and the filtrate was adjusted to pH 6 with HCl (4 M aqueous solution) and concentrated to dryness to give compound 2-3 (2.55 g, crude product) as a yellow solid. The crude product was used without further purification.

C8H15NO2質量計算為：157.1；實測：158.1[M+H]+，ESI。 Mass calculated for C 8 H 15 NO 2 : 157.1; found: 158.1 [M+H] + , ESI.

化合物2-4：Compound 2-4:

將化合物2-3(2.55g，假定為16.2mmol)溶於二噁烷(20mL)中，在0℃下緩慢加入FmocCl(4.60g，17.8mmol)，然後加入Na2CO3(飽和的，20mL)。將反應在30℃下攪拌3小時。LC-MS顯示完全轉化。用EA(20mLx3)萃取反應混合物。 濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOH=97：3)以得到白色固體2-4(2.75g，2步產率44.7%)。 Compound 2-3 (2.55 g, assumed to be 16.2 mmol) was dissolved in dioxane (20 mL), and FmocCl (4.60 g, 17.8 mmol) was slowly added at 0°C, followed by Na 2 CO 3 (saturated, 20 mL). The reaction was stirred at 30°C for 3 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mLx3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 97: 3) to give a white solid 2-4 (2.75 g, 2-step yield 44.7%).

C23H25NO4質量計算為：379.2；實測：380.2[M+H]+，ESI。 Mass calculated for C 23 H 25 NO 4 : 379.2; found: 380.2 [M+H] + , ESI.

化合物2-5：Compound 2-5:

在燒瓶中混合化合物2-4(2.7g，7.1mmol)和吡啶(20mL)，然後分批加入DMTr-Cl(2.4g，7.1mmol)。將反應在室溫下攪拌4小時。LC-MS顯示完全轉化。用EA(20mLx3)萃取反應混合物。濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOh=97：3)以得到黃色固體2-5(2.8g，產率為58%)。 Compound 2-4 (2.7 g, 7.1 mmol) and pyridine (20 mL) were mixed in a flask, and then DMTr-Cl (2.4 g, 7.1 mmol) was added in batches. The reaction was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOh = 97: 3) to give a yellow solid 2-5 (2.8 g, yield 58%).

C44H43NO6質量計算為：681.3；實測：682.3[M+H]+，ESI。 Mass calculated for C 44 H 43 NO 6 : 681.3; found: 682.3 [M+H] + , ESI.

化合物2-6：Compound 2-6:

將化合物2-5(2.3g，6.8mmol)、哌啶(5mL)和MeOH(無水，45mL)在燒瓶中混合，並在30℃下攪拌3小時。LC-MS顯示完全轉化。濃縮反應混合物，藉由矽膠快速管柱層析純化(DCM：MeOH=95：5)以得到黃色固體2-6(0.5g，產率為33%)。 Compound 2-5 (2.3 g, 6.8 mmol), piperidine (5 mL) and MeOH (anhydrous, 45 mL) were mixed in a flask and stirred at 30 °C for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 95: 5) to give a yellow solid 2-6 (0.5 g, yield 33%).

C29H33NO4質量計算為：459.2；實測：460.2[M+H]+，ESI。 Mass calculated for C 29 H 33 NO 4 : 459.2; found: 460.2 [M+H] + , ESI.

化合物2-7：Compound 2-7:

將化合物1-7(325mg，0.58mmol)、HOBT(105mg，0.77mmol)和EDCI(150mg，0.78mmol)溶於DCM(6mL)中，在室溫下攪拌15分鐘，然後冷卻至0℃。加入DIPEA(211mg，1.6mmol)，然後加入化合物2-6(300mg，0.65mmol)，在室溫下攪拌4小時。LC-MS顯示完全轉化。加入NaHCO3(飽和的10mL溶液)。用DCM(5mLx3)萃取反應混合物。濃縮有機相，藉由矽膠快速管柱層析純化(DCM：MeOH=95：5)以得到白色固體粗化合物2-7(350mg，210nm處純度88%， 產率為46%)。製備型HPLC純化：粗化合物2-7(1.8g，210nm處純度為88%)經製備型HPLC(C-18管柱，水/ACN，10%-80% ACN)純化，以得到化合物2-7(650mg，210nm處純度為96%，回收率為42%)。C54H71N3O15質量計算為：1001.5；實測：1024.5[M+Na]+，ESI。 Compound 1-7 (325 mg, 0.58 mmol), HOBT (105 mg, 0.77 mmol) and EDCI (150 mg, 0.78 mmol) were dissolved in DCM (6 mL), stirred at room temperature for 15 minutes, and then cooled to 0°C. DIPEA (211 mg, 1.6 mmol) was added, followed by compound 2-6 (300 mg, 0.65 mmol), and stirred at room temperature for 4 hours. LC-MS showed complete conversion. NaHCO 3 (saturated 10 mL solution) was added. The reaction mixture was extracted with DCM (5 mL×3). The organic phase was concentrated and purified by silica gel flash column chromatography (DCM: MeOH = 95: 5) to give a crude white solid compound 2-7 (350 mg, purity at 210 nm 88%, yield 46%). Preparative HPLC purification: Crude compound 2-7 (1.8 g, purity at 210 nm 88%) was purified by preparative HPLC (C-18 column, water/ACN, 10%-80% ACN) to give compound 2-7 (650 mg, purity at 210 nm 96%, recovery rate 42%). C 54 H 71 N 3 O 15 mass calculated: 1001.5; found: 1024.5 [M+Na] + , ESI.

1H NMR(400MHz,DMSO)δ 7.82(d,J=9.2Hz,1H),7.75-7.69(br,1H),7.40-7.35(m,2H),7.37-7.21(m,7H),6.95-6.88(m,4H),5.21(d,J=3.3Hz,1H),5.00-4.90(m,1H),4.66-4.63(m,1H),4.48(d,J=8.4Hz,1H),4.08-4.00(br,4H),3.91-3.83(m,1H),3.73(s,6H),3.72-3.66(m,3H),3.51(s,1H),3.43-3.37(m,3H),3.03-2.96(m,2H),2.91(s,2H),2.10(s,3H),2.00-1.76(m,17H),1.49-1.30(m,8H),1.23-1.19(m,2H). 1 H NMR (400MHz, DMSO) δ 7.82 (d, J =9.2Hz, 1H), 7.75-7.69 (br, 1H), 7.40-7.35 (m, 2H), 7.37-7.21 (m, 7H), 6.95-6.88 (m, 4H), 5.21 (d, J =3.3Hz,1H),5.00-4.90(m,1H),4.66-4.63(m,1H),4.48(d, J =8.4Hz,1H),4.08-4.00(br,4H),3.91-3.83(m,1H),3.73(s,6H),3.72-3.66(m,3H),3.51(s,1H),3.43-3.37( m,3H),3.03-2.96(m,2H),2.91(s,2H),2.10(s,3H),2.00-1.76(m,17H),1.49-1.30(m,8H),1.23-1.19(m,2H).

化合物2-8： Compound 2-8 :

室溫下向化合物2-7(300.0mg，0.30mmol)在無水DCM(3.0mL)中的溶液中加入DCI(32.0mg，0.27mmol)和CEP[N(iPr)2]2(108mg，0.36mmol)，攪拌1小時，LCMS顯示SM已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物使用以下條件藉由MPLC純化(管柱：C18矽膠；流動相：CH3CN/H2O=1/1，20分鐘內增加至CH3CN/H2O=1/0；沖提的產物以CH3CN/H2O=3/2收集；檢測器：UV 254nm)。產生了化合物2-8，為白色固體(177mg，0.15mmol，97.0%純度，產率為49%)。 To a solution of compound 2-7 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL) at room temperature, DCI (32.0 mg, 0.27 mmol) and CEP[N(iPr) 2 ] 2 (108 mg, 0.36 mmol) were added, stirred for 1 hour, LCMS showed that SM was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL×3), and dried over Na 2 SO 4 . The solution was then concentrated under reduced pressure, and the residue was purified by MPLC using the following conditions (column: C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1, increased to CH 3 CN/H 2 O=1/0 within 20 minutes; the eluted product was collected with CH 3 CN/H 2 O=3/2; detector: UV 254 nm). Compound 2-8 was produced as a white solid (177 mg, 0.15 mmol, 97.0% purity, 49% yield).

C63H88N5O16P質量計算為：1201.60；實測：1202.6[M+H]+，ESI。 Mass calculated for C 63 H 88 N 5 O 16 P: 1201.60; found: 1202.6 [M+H] + , ESI.

1H-NMR(600MHz,CD3CN)：δ=7.37-7.34(m,2H),7.24-7.20(m,6H),7.14-7.12(m,1H),6.79-6.77(m,4H),6.57-6.54(m,1H),6.44-6.43(m,1H),5.20-5.19(d,J=6.0Hz,1H),4.93-4.91(m,1H),4.44-4.43(d,J=6.0Hz,1H),4.04-3.83 (m,5H),3.73-3.39(m,16H),3.04-2.91(m,4H),2.52-2.50(m,2H),2.03-1.75(m,24H),1.53-1.33(m,9H),1.21-1.04(m,15H). 1 H-NMR (600MHz, CD 3 CN): δ=7.37-7.34(m,2H),7.24-7.20(m,6H),7.14-7.12(m,1H),6.79-6.77(m,4H),6.57-6.54(m,1H),6 .44-6.43(m,1H),5.20-5.19(d,J=6.0Hz,1H),4.93-4.91(m,1H),4.44-4.43(d,J=6.0Hz,1H),4.04-3.83 (m,5H),3.73-3.39(m,16H),3.04-2.91(m,4H),2.52-2.50(m,2H),2.03-1.75(m,24H),1.53-1.33(m,9H),1.21-1.04(m,15H).

31PNMR(242MHz,CD3CN)δ 147.37,147.29. 31 PNMR(242MHz,CD 3 CN)δ 147.37,147.29.

化合物2-9： Compound 2-9 :

向化合物2-7(80mg，0.080mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(5mg，0.04mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗。將反應混合物用DCM(10mL)稀釋，用H2O(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物2-9，為白色固體(85mg，0.077mmol，純度97%，產率為97%)，C58H75N3O18質量計算為：1101.50；實測：1100.4[M-H]-，ESI。 To a solution of compound 2-7 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL) was added DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H 2 O (3 mL×4), and then with brine (3 mL×4). The organic layer was concentrated to give compound 2-9 as a white solid (85 mg, 0.077 mmol, purity 97%, yield 97%), mass calculated for C 58 H 75 N 3 O 18 : 1101.50; found: 1100.4 [MH] - , ESI.

1H-NMR(600MHz,DMSO-d6)：δ=12.22(s,1H),7.84-7.81(m,1H),7.71-7.69(m,1H),7.37-7.16(m,9H),6.92-6.89(m,4H),5.21-5.20(d,J=6.0Hz,1H),4.97-4.95(m,1H),4.49-4.47(d,J=12.0Hz,1H),4.07-4.01(m,6H),3.89-3.84(m,1H),3.73-3.65(m,9H),3.49(s,1H),3.41-3.38(m,1H),3.01-2.95(m,4H),2.45-2.44(m,4H),2.09-1.76(m,20H),1.48-1.19(m,10H). 1 H-NMR (600MHz, DMSO-d6): δ=12.22(s,1H),7.84-7.81(m,1H),7.71-7.69(m,1H),7.37-7.1 6(m,9H),6.92-6.89(m,4H),5.21-5.20(d,J=6.0Hz,1H),4.97-4.95(m,1H),4.49-4.47(d,J =12.0Hz,1H),4.07-4.01(m,6H),3.89-3.84(m,1H),3.73-3.65(m,9H),3.49(s,1H),3.41- 3.38(m,1H),3.01-2.95(m,4H),2.45-2.44(m,4H),2.09-1.76(m,20H),1.48-1.19(m,10H).

固體載體2-10：Solid carrier 2-10:

天然胺基-LCAA-CPG(上樣值：75μmol/g，1000Å)用ACN(100mLx2)、DMF(100mLx2)和DCM(100mLx2)洗滌，然後在高真空下乾燥過夜。 Native amine-LCAA-CPG (loading value: 75 μmol/g, 1000Å) was washed with ACN (100 mLx2), DMF (100 mLx2) and DCM (100 mLx2), and then dried under high vacuum overnight.

向琥珀酸酯2-9(85mg，0.077mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)的溶液中加入DIPEA(30mg，0.23mmol)，室溫振盪反應混合物10分鐘，然後加入天然胺基-LCAA-CPG(250mg，上樣75μmol/g)，室溫振盪懸浮液20小時，然後過濾，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，得 到固體載體(260mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將固體載體上未反應的胺封端，用DMF(20mLx5)、CAN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體2-10(260mg)。為了計算上樣，取6.5mg乾燥的上樣CPG，加入25mL含有3% DCA的DCM溶液。振盪溶液，測量500nm處的紫外吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式：上樣(μmol/g)=(添加的DCA總體積(mL))*(500nm處的Abs值)*1000)/(76*(取的CPG mg數)) To a solution of succinate 2-9 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (30 mg, 0.23 mmol), the reaction mixture was shaken at room temperature for 10 minutes, then natural amino-LCAA-CPG (250 mg, loading 75 μmol/g) was added, the suspension was shaken at room temperature for 20 hours, then filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), DCM (20 mL x 5) until TLC showed no spot in the eluent at 254 nm. The solid support was vacuum dried for 2 hours to obtain a solid support (260 mg). Stir with Ac2O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the solid support, and wash with DMF (20mLx5), CAN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support is vacuum dried for 15 hours to obtain solid support 2-10 (260mg). In order to calculate the loading, take 6.5mg of the dried loading CPG and add 25mL of DCM solution containing 3% DCA. Vibrate the solution and measure the UV absorbance at 500nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula: Loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 500nm) * 1000) / (76 * (mg of CPG taken))

添加的DCA總體積(mL)=25mL Total volume of DCA added (mL) = 25mL

500nm處的Abs值=0.7721 Abs value at 500nm = 0.7721

取的CPG mg數=6.5mg The amount of CPG mg taken = 6.5mg

上樣(μmol/g)=((25)*(0.7721)*1000)/(76*(6.5))=39μmol/g。 Loading (μmol/g) = ((25)*(0.7721)*1000)/(76*(6.5)) = 39μmol/g.

Figure 113123276-A0202-12-0166-274
Figure 113123276-A0202-12-0166-274

化合物3-2：Compound 3-2:

向化合物3-1(40g，211.36mmol)的NH3/MeOH(7M，192mL)溶液中逐滴添加2-氰基乙酸乙酯(47.82g，422.71mmol)，並將混合物攪拌過夜。LC-MS顯示完全轉化，將反應混過濾。將濾餅在PE(100mL)中研磨，過濾。乾燥濾餅以得到為固體的化合物3-2(41g，產率為44%)。 To a solution of compound 3-1 (40 g, 211.36 mmol) in NH 3 /MeOH (7M, 192 mL) was added ethyl 2-cyanoacetate (47.82 g, 422.71 mmol) dropwise, and the mixture was stirred overnight. LC-MS showed complete conversion, and the reaction mixture was filtered. The filter cake was triturated in PE (100 mL) and filtered. The filter cake was dried to give compound 3-2 as a solid (41 g, 44% yield).

C18H18N4O2質量計算為：322.1；實測：323.1[M+H]+，ESI。 Calculated mass of C18H18N4O2: 322.1; Found: 323.1[M+H] + , ESI.

化合物3-3：Compound 3-3:

將化合物3-2(80g，248.17mmol)溶於硫酸溶液(濃H2SO4/水=1：1，v/v，160mL)，並在120℃下攪拌5小時。LC-MS顯示完全轉化，藉由添加NaOH(30%水溶液)將反應混合物調節至pH 9-10，並藉由添加1M HCl溶液重新調節至pH 4-5。然後過濾反應物，將濾液濃縮至乾以得到粗化合物3-3(72g，95%產率)。粗產物無需進一步純化即可直接使用。 Compound 3-2 (80 g, 248.17 mmol) was dissolved in sulfuric acid solution (conc. H 2 SO 4 /water=1:1, v/v, 160 mL) and stirred at 120° C. for 5 hours. LC-MS showed complete conversion, and the reaction mixture was adjusted to pH 9-10 by adding NaOH (30% aqueous solution), and readjusted to pH 4-5 by adding 1M HCl solution. The reaction was then filtered, and the filtrate was concentrated to dryness to give crude compound 3-3 (72 g, 95% yield). The crude product was used directly without further purification.

C16H21NO4質量計算為：291.2；實測：292.3[M+H]+，ESI。 The mass of C16H21NO4 was calculated as: 291.2; found as: 292.3 [M+H] + , ESI.

化合物3-4：Compound 3-4:

將化合物3-3(72.3g粗產物)在MeOH(1600mL)和H2SO4(160mL，濃H2SO4/水=1：1，v/v)中在85℃下攪拌4小時。LC-MS顯示完全轉化。將反應混合物過濾並濃縮至乾，並藉由矽膠管柱(0-25% EA在PE中)純化以得到化合物3-4(62.5g，2步產率78%)。 Compound 3-3 (72.3 g crude product) was stirred in MeOH (1600 mL) and H 2 SO 4 (160 mL, concentrated H 2 SO 4 /water = 1:1, v/v) at 85° C. for 4 hours. LC-MS showed complete conversion. The reaction mixture was filtered and concentrated to dryness, and purified by silica gel column (0-25% EA in PE) to give compound 3-4 (62.5 g, 2-step yield 78%).

C18H25NO4質量計算為：319.2；實測：320.3[M+H]+，ESI。 Mass calculated for C 18 H 25 NO 4 : 319.2; found: 320.3 [M+H] + , ESI.

化合物3-5：Compound 3-5:

在0℃下向化合物3-4(40g，125.2mmol)的THF(295mL)溶液中分批加入LiAlH4(14.3g，375.1mmol)，攪拌1小時。LC-MS顯示完全轉化，向反應混合物 中加入乙酸乙酯(100mL)，加入10% NaOH(水溶液)調節pH至9-10。然後過濾混合物並用乙酸乙酯(50mL X 2)洗滌。合併濾液濃縮至乾以得到粗化合物3-5(23g，83%產率)。粗產物無需進一步純化即可用於下一步驟。 LiAlH 4 (14.3 g, 375.1 mmol) was added in portions to a solution of compound 3-4 (40 g, 125.2 mmol) in THF (295 mL) at 0°C and stirred for 1 hour. LC-MS showed complete conversion, ethyl acetate (100 mL) was added to the reaction mixture, and 10% NaOH (aqueous solution) was added to adjust the pH to 9-10. The mixture was then filtered and washed with ethyl acetate (50 mL x 2). The combined filtrate was concentrated to dryness to give crude compound 3-5 (23 g, 83% yield). The crude product was used in the next step without further purification.

C16H25NO2質量計算為：263.2；實測：264.3[M+H]+，ESI。 The mass of C16H25NO2 was calculated as: 263.2; found as: 264.3 [M+H] + , ESI.

化合物3-6：Compound 3-6:

將化合物3-5(20g，75.94mmol)、Boc2O(16.57g，75.94mmol)和Pd/C(10%碳載鈀，濕，~55%水)置於MeOH(150mL)中，室溫下在1個大氣壓的H2下攪拌過夜。LC-MS顯示完全轉化，將反應混合物過濾並濃縮以得到油狀化合物3-6(20g，96%產率)，無需進一步純化即可使用。 Compound 3-5 (20 g, 75.94 mmol), Boc 2 O (16.57 g, 75.94 mmol) and Pd/C (10% palladium on carbon, wet, ~55% water) were placed in MeOH (150 mL) and stirred under 1 atmosphere of H 2 at room temperature overnight. LC-MS showed complete conversion, and the reaction mixture was filtered and concentrated to give compound 3-6 (20 g, 96% yield) as an oil, which was used without further purification.

C14H27NO4質量計算為273.2；實測：274.1[M+H]+，ESI。 Mass calculated for C 14 H 27 NO 4 : 273.2; found: 274.1 [M+H] + , ESI.

化合物3-7：Compound 3-7:

向化合物3-6(58g，212.17mmol)在三乙胺(85.88g，848.67mmol，118.37mI)和DCM(1.16L)中的溶液中加入MsCl(72.91g，636.51mmol)。將反應在室溫攪拌過夜。LC-MS顯示完全轉化(藉由EtOH猝滅和二醚檢測)。將反應混合物用DCM(1L)稀釋，用10%檸檬酸洗滌，然後用NaHCO3(飽和水溶液)洗滌，並乾燥以得到化合物3-7，為油狀物(87g，產率為95%)，無需進一步純化，直接用於下一步。 To a solution of compound 3-6 (58 g, 212.17 mmol) in triethylamine (85.88 g, 848.67 mmol, 118.37 ml) and DCM (1.16 L) was added MsCl (72.91 g, 636.51 mmol). The reaction was stirred at room temperature overnight. LC-MS showed complete conversion (by EtOH quench and diether detection). The reaction mixture was diluted with DCM (1 L), washed with 10% citric acid, then washed with NaHCO 3 (saturated aqueous solution), and dried to give compound 3-7 as an oil (87 g, 95% yield), which was used directly in the next step without further purification.

C16H31NO8S2質量計算為：429.2；實測：430.3[M+H]+，ESI。 Mass calculated for C 16 H 31 NO 8 S 2 : 429.2; found: 430.3 [M+H] + , ESI.

化合物3-8：Compound 3-8:

在氮氣中，將NaH(60%礦物油，3.72g，93.12mmol)懸浮於無水DMF(60mL)中，加入丙二酸二異丙酯(8.76g，46.56mmol)。將反應混合物在室溫下攪拌30分鐘，形成澄清溶液。加入KI(1.55g，9.31mmol)，然後加入化合物3-7(20g， 46.56mmol)。將反應混合物在70℃下攪拌30分鐘，然後在140℃下再攪拌30分鐘。LC-MS顯示完全轉化。將反應冷卻至室溫，用乙酸乙酯(300mL)稀釋，用檸檬酸(10%水溶液，200mL x 2)和NaHCO3(飽和水溶液，200mL)洗滌。有機相濃縮並藉由矽膠管柱(0-10% EA在PE中的溶液)純化以得到化合物3-8，為無色油狀物(3.55g，產率為18%)。 NaH (60% mineral oil, 3.72 g, 93.12 mmol) was suspended in anhydrous DMF (60 mL) under nitrogen and diisopropyl malonate (8.76 g, 46.56 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes to form a clear solution. KI (1.55 g, 9.31 mmol) was added, followed by compound 3-7 (20 g, 46.56 mmol). The reaction mixture was stirred at 70 °C for 30 minutes and then at 140 °C for another 30 minutes. LC-MS showed complete conversion. The reaction was cooled to room temperature, diluted with ethyl acetate (300 mL), and washed with citric acid (10% aqueous solution, 200 mL x 2) and NaHCO 3 (saturated aqueous solution, 200 mL). The organic phase was concentrated and purified by silica gel column (0-10% EA in PE) to give compound 3-8 as a colorless oil (3.55 g, 18% yield).

C23H39NO6質量計算為：425.3；實測：426.2[M+H]+，ESI。 Mass calculated for C 23 H 39 NO 6 : 425.3; found: 426.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.96-4.90(m,2H),3.27(br,4H),1.86(t,J=10.8Hz,4H),1.38(br,13H),1.28(s,4H),1.17-1.16(m,12H). 1 H NMR(400MHz, DMSO-d6)δ 4.96-4.90(m,2H),3.27(br,4H),1.86(t, J =10.8Hz,4H),1.38(br,13H),1.28(s,4H),1.17-1.16(m,12H).

化合物3-9：Compound 3-9:

在0℃下向化合物3-8(7.5g，17.62mmol)中加入LiBH4(172mL，1M，在THF中)，將混合物攪拌過夜。LC-MS顯示完全轉化。向反應混合物中加入乙酸乙酯(10mL)，然後加入NaOH(10%水溶液，20mL)並過濾。濾液用水(200mL)稀釋，用乙酸乙酯(50mL X3)萃取，濃縮以得到化合物3-9(6.35g，產率為98%)，為無色油狀物，無需進一步純化即可直接用於下一步。 LiBH4 (172 mL, 1 M in THF) was added to compound 3-8 (7.5 g, 17.62 mmol) at 0°C, and the mixture was stirred overnight. LC-MS showed complete conversion. Ethyl acetate (10 mL) was added to the reaction mixture, followed by NaOH (10% aqueous solution, 20 mL) and filtered. The filtrate was diluted with water (200 mL), extracted with ethyl acetate (50 mL x 3), and concentrated to give compound 3-9 (6.35 g, yield 98%) as a colorless oil, which was used directly in the next step without further purification.

C17H31NO4質量計算為：313.2；實測：314.2[M+H]+，ESI。 Mass calculated for C 17 H 31 NO 4 : 313.2; found: 314.2 [M+H] + , ESI.

化合物3-10：Compound 3-10:

將化合物3-9(6.35g，20.26mmol)與HCl(63.5mL，4M在二噁烷中)混合，在室溫下攪拌3小時。LC-MS顯示完全轉化。將反應混合物濃縮至乾並與甲苯共沸乾燥，得到化合物3-10(7g，產率為99%)，無需進一步純化即可直接用於下一步。 Compound 3-9 (6.35 g, 20.26 mmol) was mixed with HCl (63.5 mL, 4 M in dioxane) and stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to dryness and azeotropically dried with toluene to give compound 3-10 (7 g, yield 99%), which was used directly in the next step without further purification.

C12H23NO2質量計算為(游離鹼基)：213.2；實測：214.2[M+H]+，ESI。 Mass calculated for C 12 H 23 NO 2 (free base): 213.2; found: 214.2 [M+H] + , ESI.

1H NMR(400MHz,D2O)δ 3.39(br,4H),3.18(t,J=11.6Hz,4H),1.59(t,J=12.0Hz,4H),1.36-1.33(m,4H),1.25(t,J=12.4Hz,4H). 1 H NMR (400MHz, D 2 O) δ 3.39 (br, 4H), 3.18 (t, J =11.6Hz, 4H), 1.59 (t, J =12.0Hz, 4H), 1.36-1.33 (m, 4H), 1.25 (t, J =12.4Hz, 4H).

化合物3-11：Compound 3-11:

將化合物3-10(5g，20.02mmol)、DIEA(6.47g，50.04mmol，8.72mL)、HOBT(3.25g，24.02mmol)、EDCI(4.60g，24.02mmol)在DCM(150mL)中的溶液，0℃攪拌30分鐘，然後加入Fmoc-6-胺基己酸(5.66g，16.01mmol)，室溫攪拌混合物1小時。LC-MS顯示完全轉化。將反應混合物用另外的DCM(100mL)稀釋，用檸檬酸(10%水溶液，200mL)、NaHCO3(飽和水溶液，200mL)和水(200mL)洗滌，用Na2SO4乾燥，濃縮得到粗產物，其用矽膠管柱(0-10% EA在PE中的溶液)純化以得到呈膠狀的化合物3-11(3.65g，LC-MS純度為92%，產率為33%)。 A solution of compound 3-10 (5 g, 20.02 mmol), DIEA (6.47 g, 50.04 mmol, 8.72 mL), HOBT (3.25 g, 24.02 mmol), and EDCI (4.60 g, 24.02 mmol) in DCM (150 mL) was stirred at 0° C. for 30 minutes, and then Fmoc-6-aminohexanoic acid (5.66 g, 16.01 mmol) was added and the mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was diluted with additional DCM (100 mL), washed with citric acid (10% aqueous solution, 200 mL), NaHCO3 (saturated aqueous solution, 200 mL) and water (200 mL), dried over Na2SO4 , concentrated to give a crude product, which was purified with a silica gel column (0-10% EA in PE) to give compound 3-11 (3.65 g, 92% purity by LC-MS, 33% yield) as a gel.

C33H44N2O5質量計算為：548.3；實測：549.3[M+H]+，ESI。 Mass calculated for C 33 H 44 N 2 O 5 : 548.3; found: 549.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.88(d,J=7.5Hz,2H),7.84(d,J=7.5Hz,2H),7.41(t,J=7.3Hz,2H),7.34(t,J=7.3Hz,2H),6.65(t,J=5.3Hz,1H),6.28(s,2H),4.36-4.18(m,3H),3.36(dt,J=19.9,5.2Hz,4H),3.24(s,4H),2.92-2.87(m,2H),2.24(t,J=7.4Hz,2H),1.49-1.26(m,18H). 1 H NMR (400MHz, DMSO-d6)δ 7.88(d, J =7.5Hz,2H),7.84(d, J =7.5Hz,2H),7.41(t, J =7.3Hz,2H),7.34(t, J =7.3Hz,2H),6.65(t, J =5.3Hz,1H),6.28(s,2H),4.36-4.18(m,3H),3.36(dt, J =19.9,5.2Hz,4H),3.24(s,4H),2.92-2.87(m,2H),2.24(t, J =7.4Hz,2H),1.49-1.26(m,18H).

化合物3-12：Compound 3-12:

將化合物3-11(2.55g，4.65mmol)、4，4'-(氯(苯基)亞甲基)雙(甲氧基苯)(1.57g，4.65mmol)、DIEA(600.6mg，4.65mmol)和DMAP(56.8mg，0.47mmol)在DCM(51mL)中的溶液在室溫下攪拌過夜。LC-MS顯示部分轉化。然後將反應混合物濃縮並藉由矽膠管柱純化(10-40% EA在PE中的溶液以沖提雜質，然後用5-10% MeOH和0.1% NH3‧H2O在DCM中的溶液以沖提雜質)以得到化合物3-12(2.75g，產率為69%)。 A solution of compound 3-11 (2.55 g, 4.65 mmol), 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (1.57 g, 4.65 mmol), DIEA (600.6 mg, 4.65 mmol) and DMAP (56.8 mg, 0.47 mmol) in DCM (51 mL) was stirred at room temperature overnight. LC-MS showed partial conversion. The reaction mixture was then concentrated and purified by silica gel column (10-40% EA in PE to wash out impurities, then 5-10% MeOH and 0.1% NH 3 ‧H 2 O in DCM to wash out impurities) to give compound 3-12 (2.75 g, 69% yield).

1H NMR(400MHz,DMSO-d6)δ 7.88-7.87(m,2H),7.69-7.68(m,2H),7.41-7.37(m,4H),7.33-7.25(m,9H),6.89-6.80(m,5H),4.41(t,J=5.0Hz,1H),4.28(d,J=6.8Hz,2H),4.20(t,J=6.8Hz,1H),3.72(s,8H),3.40-3.39(m,2H),3.32-3.27(m,4H),2.96(q,J=6.3Hz,2H),2.22(t,J=7.9Hz,2H),1.48-0.86(m,18H). 1 H NMR(400MHz, DMSO-d6)δ 7.88-7.87(m,2H),7.69-7.68(m,2H),7.41-7.37(m,4H),7.33-7.25(m,9H),6.89-6.80(m,5H),4.41(t, J =5.0Hz,1H),4.28(d, J =6.8Hz,2H),4.20(t, J =6.8Hz,1H),3.72(s,8H),3.40-3.39(m,2H),3.32-3.27(m,4H),2.96(q, J =6.3Hz,2H),2.22(t, J =7.9Hz,2H),1.48-0.86(m,18H).

化合物3-13：Compound 3-13:

向化合物3-12(1.7g，2.00mmol)在MeOH(51mL)的溶液中加入哌啶(5.1mL)，室溫下攪拌混合物3小時。LC-MS顯示完全轉化。濃縮反應混合物，用二甲苯共沸乾燥。殘餘物經矽膠管柱純化(0-10% MeOH與0.1% NH3‧H2O在DCM中的溶液)以得到呈膠狀的化合物3-13(1.1g，產率為87%)。 To a solution of compound 3-12 (1.7 g, 2.00 mmol) in MeOH (51 mL) was added piperidine (5.1 mL), and the mixture was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and azeotropically dried with xylene. The residue was purified by silica gel column (0-10% MeOH and 0.1% NH 3 ‧H 2 O in DCM) to give compound 3-13 (1.1 g, yield 87%) as a gel.

C39H52N2O5質量計算為：628.4；實測：629.4[M+H]+，ESI。 Mass calculated for C 39 H 52 N 2 O 5 : 628.4; found: 629.4 [M+H] + , ESI.

化合物3-14：Compound 3-14:

將化合物3-13(0.94g，1.49mmol)、DIEA(482.98mg，3.74mmol)、HOBT(242.38mg，1.79mmol)、EDCI(343.87mg，1.79mmol)在DCM(29mL)中的溶液在0℃下攪拌，然後加入化合物1-4(936.36mg，2.09mmol)，在室溫下攪拌混合物1小時。LC-MS顯示完全轉化。將反應混合物用另外的DCM(20mL)稀釋，用NaHCO3(飽和水溶液，50mL)和水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物3-14(700mg，產率為44%)。 A solution of compound 3-13 (0.94 g, 1.49 mmol), DIEA (482.98 mg, 3.74 mmol), HOBT (242.38 mg, 1.79 mmol), EDCI (343.87 mg, 1.79 mmol) in DCM (29 mL) was stirred at 0°C, and then compound 1-4 (936.36 mg, 2.09 mmol) was added, and the mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was diluted with additional DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 3-14 (700 mg, 44% yield).

C58H79N3O15質量計算為：1057.6；實測：1080.5[M+Na]+，ESI。 Mass calculated for C 58 H 79 N 3 O 15 : 1057.6; found: 1080.5 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.82(d,J=9.2Hz,1H),7.69(t,J=5.5Hz,1H),7.40-7.38(m,2H),7.32-7.19(m,7H),6.89-6.87(m,4H),5.21(d,J=3.3Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.48(d,J=8.5Hz,1H),4.40(t,J=5.0Hz,1H),4.02(s,3H),3.91-3.83(m,1H),3.73-3.68(m,7H),3.43-3.39(m,3H),3.32- 3.27(m,2H),2.99(q,J=6.6Hz,2H),2.87(s,2H),2.22(t,J=7.3Hz,2H),2.10(s,3H),2.03(t,J=7.0Hz,2H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.53-1.03(m,20H),0.90-0.83(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.82(d, J =9.2Hz,1H),7.69(t, J =5.5Hz,1H),7.40-7.38(m,2H),7.32-7.19(m,7H),6.89-6.87(m,4H),5.21(d, J =3.3Hz,1H),4.97(dd, J =11.2,3.4Hz,1H),4.48(d, J =8.5Hz,1H),4.40(t, J =5.0Hz,1H),4.02(s,3H),3.91-3.83(m,1H),3.73-3.68(m,7H),3.43-3.39(m,3H),3.32- 3.27(m,2H),2.99(q, J =6.6Hz,2H),2.87(s,2H),2.22(t, J =7.3Hz,2H),2.10(s,3H),2.03(t, J =7.0Hz,2H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.53-1.03(m,20H),0.90-0.83(m,2H).

化合物3-15： Compound 3-15 :

在室溫中向化合物3-14(670.0mg，0.61mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(96.0mg，0.81mmol)和2-氰乙基N,N,N’,N’-四異丙基磷二醯胺(324mg，1.08mmol)，攪拌1小時。LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱：C18矽膠；流動相：CH3CN/H2O=1/1，20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物3-15，為白色固體(600mg，產率為83%)。 To a solution of compound 3-14 (670.0 mg, 0.61 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N , N , N' , N' -tetraisopropylphosphodiamidate (324 mg, 1.08 mmol) at room temperature and stirred for 1 hour. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3 ), and dried over Na2SO4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1, increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 3-15 was produced as a white solid (600 mg, yield 83%).

C67H96N5O16P質量計算為：1257.66；實測：1204.5[M-CH2CH2CN]-，ESI。 Mass calculated for C 67 H 96 N 5 O 16 P: 1257.66; found: 1204.5 [M-CH2CH2CN] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.81-7.67(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d,J=6.0Hz,1H),4.98-4.94(m,1H),4.49(d,J=6.0Hz,1H),4.04-3.83(m,4H),3.73-3.32(m,15H),3.01-2.89(m,4H),2.73-2.70(m,2H),2.24-2.20(m,2H),2.11-1.76(m,15H),1.49-0.91(m,34H). 1 HNMR(600MHz, DMSO- d 6 )δ 7.81-7.67(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d, J =6.0Hz,1H),4.98-4.94(m,1H),4.49(d, J =6.0Hz,1H),4.04-3.83(m,4H),3.73-3.32(m,15H),3.01-2.89(m,4H),2.7 3-2.70(m,2H),2.24-2.20(m,2H),2.11-1.76(m,15H),1.49-0.91(m,34H).

31PNMR(242MHz,DMSO-d 6)δ 146.21,146.19. 31 PNMR(242MHz,DMSO- d 6 )δ 146.21,146.19.

Figure 113123276-A0202-12-0173-275
Figure 113123276-A0202-12-0173-275

化合物4-2：Compound 4-2:

在0℃下將碳酸鉀(8.10g，58.61mmol)加入MeOH(80mL)、H2O(60mL)、甲醛(3.52g，117.22mmol)和化合物4-1(25g，117.22mmol)的溶液中。將反應混合物在0℃中攪拌過夜。將反應混合物濃縮至其體積的70%，用乙酸乙酯萃取殘餘物三次。有機相用水，鹽水洗滌，經無水硫酸鈉乾燥，然後濃縮，得到粗產物4-2(27g，產率為95%)，其無需進一步純化即可用於下一步。 Potassium carbonate (8.10 g, 58.61 mmol) was added to a solution of MeOH (80 mL), H 2 O (60 mL), formaldehyde (3.52 g, 117.22 mmol) and compound 4-1 (25 g, 117.22 mmol) at 0°C. The reaction mixture was stirred at 0°C overnight. The reaction mixture was concentrated to 70% of its volume, and the residue was extracted three times with ethyl acetate. The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, and then concentrated to obtain a crude product 4-2 (27 g, yield 95%), which was used in the next step without further purification.

C12H21NO4質量計算為：243.2；實測：244.1[M+H]+，ESI。 Mass calculated for C 12 H 21 NO 4 : 243.2; found: 244.1 [M+H] + , ESI.

化合物4-3：Compound 4-3:

將化合物4-2(35g，143.86mmol)在甲醇(28ml)中的溶液，在0℃下約5分鐘內加入硼氫化鈉(11.97g，316.48mmol)。將反應混合物在0℃下攪拌，然後升溫至室溫並攪拌5小時。用飽和氯化銨溶液猝滅反應，然後濃縮，用乙酸乙酯(EA)萃取三次，用鹽水洗滌，然後用EA：PE=1：3再結晶以得到化合物4-3(19.6g，產率為54%)。 To a solution of compound 4-2 (35 g, 143.86 mmol) in methanol (28 ml), sodium borohydride (11.97 g, 316.48 mmol) was added at 0°C for about 5 minutes. The reaction mixture was stirred at 0°C, then warmed to room temperature and stirred for 5 hours. The reaction was quenched with a saturated ammonium chloride solution, then concentrated, extracted three times with ethyl acetate (EA), washed with brine, and then recrystallized with EA:PE=1:3 to obtain compound 4-3 (19.6 g, yield 54%).

C12H23NO4質量計算為：245.2；實測：146.1[M+H-Boc]+，ESI。 Mass calculated for C 12 H 23 NO 4 : 245.2; found: 146.1 [M+H-Boc] + , ESI.

化合物4-4：Compound 4-4:

將化合物4-3(30g，122.29mmol)、三乙胺(30.94g，305.73mmol，42.64mL)和DCM(300mL)混合，然後將混合物冷卻至-30℃，然後緩慢加入MsCl(35.02g，305.73mmol)。室溫下攪拌1.5小時後，用400mL水萃取反應混合物，用100mL的10%檸檬酸水溶液洗滌，無水硫酸鈉乾燥。濃縮後，殘餘物經矽膠管柱純化以得到化合物4-4，為淡黃色油狀液體(40g，產率為81%)。 Compound 4-3 (30 g, 122.29 mmol), triethylamine (30.94 g, 305.73 mmol, 42.64 mL) and DCM (300 mL) were mixed, and then the mixture was cooled to -30 °C, and then MsCl (35.02 g, 305.73 mmol) was slowly added. After stirring at room temperature for 1.5 hours, the reaction mixture was extracted with 400 mL of water, washed with 100 mL of 10% citric acid aqueous solution, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by silica gel column to obtain compound 4-4 as a light yellow oily liquid (40 g, yield 81%).

C14H27NO8S2質量計算為：401.1；實測：302.1[M+H-Boc]+，ESI。 Mass calculated for C 14 H 27 NO 8 S 2 : 401.1; found: 302.1 [M+H-Boc] + , ESI.

化合物4-5：Compound 4-5:

在氮氣下向t-BuONa(9.57g，99.63mmol)在DMA(50mL)中的混合物中緩慢加入丙二酸二異丙酯(14.06g，74.72mmol)。攪拌20分鐘後，加入化合物4-4(20g，49.81mmol)、KI(4.13g，24.9lmmol)。將反應混合物加熱至140℃並攪拌過夜。藉由加入飽和氯化銨猝滅反應並用乙酸乙酯萃取。濃縮後，殘餘物藉由矽膠管柱(PE：EA)純化以得到化合物4-5(6g，產率為30%)。 To a mixture of t-BuONa (9.57 g, 99.63 mmol) in DMA (50 mL) was slowly added diisopropyl malonate (14.06 g, 74.72 mmol) under nitrogen. After stirring for 20 minutes, compound 4-4 (20 g, 49.81 mmol) and KI (4.13 g, 24.91 mmol) were added. The reaction mixture was heated to 140 ° C and stirred overnight. The reaction was quenched by adding saturated ammonium chloride and extracted with ethyl acetate. After concentration, the residue was purified by silica gel column (PE: EA) to obtain compound 4-5 (6 g, yield 30%).

C21H35NO6質量計算為：397.3；實測：398.2[M+H]+，ESI。 Mass calculated for C 21 H 35 NO 6 : 397.3; found: 398.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO)δ 4.98-4.90(m,2H),3.22-3.20(m,4H),2.25(s,4H),1.42(t,J=5.6Hz,4H),1.38(s,9H),1.17(d,J=6.0Hz,12H). 1 H NMR(400MHz, DMSO)δ 4.98-4.90(m,2H),3.22-3.20(m,4H),2.25(s,4H),1.42(t, J =5.6Hz,4H),1.38(s,9H),1.17(d, J =6.0Hz,12H).

化合物4-6：Compounds 4-6:

在0℃下，向化合物4-5(9.75g，24.53mmol)在無水THF(200mL)中的溶液中加入硼氫化鋰(10.69g，490.56mmol)。將反應混合物緩慢升溫至室溫並攪拌過夜。然後將乙酸乙酯(200mL)加入到反應混合物中，用水(150mL x3)萃取，然後用飽和氯化鈉洗滌，用無水硫酸鈉乾燥，濃縮得到粗產物4-6(6.52g，產率為93%)，無需進一步純化即可用於下一步。 Lithium borohydride (10.69 g, 490.56 mmol) was added to a solution of compound 4-5 (9.75 g, 24.53 mmol) in anhydrous THF (200 mL) at 0°C. The reaction mixture was slowly warmed to room temperature and stirred overnight. Ethyl acetate (200 mL) was then added to the reaction mixture, extracted with water (150 mL x 3), then washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a crude product 4-6 (6.52 g, 93% yield), which was used in the next step without further purification.

C15H27NO4質量計算為：285.2；實測：186.2[M+H-Boc]+，ESI。 Mass calculated for C 15 H 27 NO 4 : 285.2; found: 186.2 [M+H-Boc] + , ESI.

化合物4-7：Compound 4-7:

在0℃下向產物4-6(7.52g，26.35mmol)在DCM(100mL)中的溶液中加入HCl(4M在1,4-二噁烷中，21.52mL)，歷時15分鐘。將反應混合物升溫至室溫並攪拌3小時。將反應混合物過濾，濾餅用二氯甲烷(x3)洗滌以得到粗產物4-7(3.7g，產率為76%)。 To a solution of product 4-6 (7.52 g, 26.35 mmol) in DCM (100 mL) was added HCl (4M in 1,4-dioxane, 21.52 mL) at 0°C over 15 minutes. The reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction mixture was filtered and the filter cake was washed with dichloromethane (x3) to give the crude product 4-7 (3.7 g, 76% yield).

C10H19NO2計算為(游離鹼基)：185.1m/z；實測：186.2[M+H]+，ESI。 Calculated for C 10 H 19 NO 2 (free base): 185.1 m/z; found: 186.2 [M+H] + , ESI.

化合物4-8：Compound 4-8:

室溫攪拌Fmoc-6-胺基己酸(1.91g，5.41mmol)、1-羥基苯并三唑(HOBT,609.41mg，4.51mmol)、EDCI(864.59mg，4.51mmol)、DIEA(582.89mg，4.51mmol，785.56μL)在DCM(10mL)溶液中的混合物30分鐘，然後加入化合物4-7(1g，4.51mmol)在DCM中的溶液，攪拌混合物4小時。反應混合物用飽和碳酸氫鈉溶液、鹽水洗滌，無水硫酸鈉乾燥。濃縮後，殘餘物經矽膠管柱純化以得到化合物4-8(1.35g，產率為58%)。 A mixture of Fmoc-6-aminohexanoic acid (1.91 g, 5.41 mmol), 1-hydroxybenzotriazole (HOBT, 609.41 mg, 4.51 mmol), EDCI (864.59 mg, 4.51 mmol), and DIEA (582.89 mg, 4.51 mmol, 785.56 μL) in DCM (10 mL) was stirred at room temperature for 30 minutes, and then a solution of compound 4-7 (1 g, 4.51 mmol) in DCM was added and the mixture was stirred for 4 hours. The reaction mixture was washed with saturated sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by silica gel column to obtain compound 4-8 (1.35 g, yield 58%).

C31H40N2O5質量計算為：520.3；實測：521.2[M+H]+，ESI。 Mass calculated for C 31 H 40 N 2 O 5 : 520.3; found: 521.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO)δ 7.88(d,J=7.5Hz,2H),7.68(d,J=7.4Hz,2H),7.39(d,J=7.5Hz,4H),7.34-7.18(m,10H),6.89(d,J=8.8Hz,4H),4.64(t,J=4.7Hz,1H),4.28(d,J=6.8Hz,2H),4.20(t,J=6.8Hz,1H),3.73(s,6H),3.44(d,J=5.0Hz,2H),3.30-3.10(m,4H),3.01(s,2H),3.00-2.90(m,2H),2.21(t,J=7.2Hz,2H),1.56(d,J=11.9Hz,2H),1.46-1.30(m,8H),1.21-1.00(m,4H). 1 H NMR(400MHz, DMSO)δ 7.88(d, J =7.5Hz,2H),7.68(d, J =7.4Hz,2H),7.39(d, J =7.5Hz,4H),7.34-7.18(m,10H),6.89(d, J =8.8Hz,4H),4.64(t, J =4.7Hz,1H),4.28(d, J =6.8Hz,2H),4.20(t, J =6.8Hz,1H),3.73(s,6H),3.44(d, J =5.0Hz,2H),3.30-3.10(m,4H),3.01(s,2H),3.00-2.90(m,2H),2.21(t, J =7.2Hz,2H),1.56(d, J =11.9Hz,2H),1.46-1.30(m,8H),1.21-1.00(m,4H).

化合物4-9：Compound 4-9:

將DMTr-Cl(488.07mg，1.44mmol)和化合物4-8(0.75g，1.44mmol)在吡啶(6ml)溶液中的混合物室溫下攪拌過夜。將反應混合物在真空中濃縮。將殘餘物重新溶解在含有1%吡啶的乙酸乙酯中，並與氧化鋁一起乾燥，用於在氧化鋁上進行管柱層析純化(0-10% MeOH，0.1% TEA在DCM中)得到化合物4-9(0.39g，產率為32.9%)。 A mixture of DMTr-Cl (488.07 mg, 1.44 mmol) and compound 4-8 (0.75 g, 1.44 mmol) in pyridine (6 ml) solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was redissolved in ethyl acetate containing 1% pyridine and dried with alumina for column chromatography on alumina (0-10% MeOH, 0.1% TEA in DCM) to give compound 4-9 (0.39 g, 32.9% yield).

1H NMR(400MHz,DMSO)δ 7.39(d,J=7.3Hz,2H),7.34-7.18(m,7H),6.89(d,J=8.8Hz,4H),4.63(s,1H),3.73(s,6H),3.43(s,2H),3.30-3.18(m,6H),3.05(s,2H),2.22(t,J=7.4Hz,2H),1.57(d,J=12.1Hz,2H),1.47-1.08(m,12H). 1 H NMR (400MHz, DMSO) δ 7.39 (d, J =7.3Hz, 2H), 7.34-7.18 (m, 7H), 6.89 (d, J =8.8Hz,4H),4.63(s,1H),3.73(s,6H),3.43(s,2H),3.30-3.18(m,6H),3.05(s,2H),2.22(t, J =7.4Hz,2H),1.57(d, J =12.1Hz,2H),1.47-1.08(m,12H).

化合物4-10：Compound 4-10:

將化合物4-9(1.4g，1.70mmol)溶解於MeOH(40mL)中，然後添加哌啶(4ml)。將混合物在室溫下攪拌過夜。將反應混合物在真空中濃縮。藉由與二甲苯共沸乾燥進一步除去哌啶殘餘物。然後，將殘餘物藉由矽膠管柱(DCM：MeOH=10：1，甲醇含有10%氨水)純化得到化合物4-10(1g，產率為97%)。 Compound 4-9 (1.4 g, 1.70 mmol) was dissolved in MeOH (40 mL), and then piperidine (4 ml) was added. The mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. Piperidine residues were further removed by azeotropic drying with xylene. Then, the residue was purified by silica gel column (DCM: MeOH = 10: 1, methanol containing 10% ammonia water) to obtain compound 4-10 (1 g, yield 97%).

C37H48N2O5質量計算為：600.4；實測：601.4[M+H]+，ESI。 Mass calculated for C 37 H 48 N 2 O 5 : 600.4; found: 601.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.40-7.39(m,2H),7.33-7.21(m,7H),6.89(d,J=8.8Hz,4H),4.63(s,1H),3.73(s,6H),3.44-3.16(m,8H),3.01-3.00(m,2H),2.24-2.21(m,2H),1.57(d,J=12.2Hz,2H),1.45-1.33(m,7H),1.27-1.21(m,4H),1.09(s,1H). 1 H NMR(400MHz,DMSO-d6)δ 7.40-7.39(m,2H),7.33-7.21(m,7H),6.89(d, J =8.8Hz,4H),4.63(s,1H),3.73(s,6H),3.44-3.16(m,8H),3.01-3.00(m,2H),2.24-2.21(m,2H),1.57(d, J =12.2Hz,2H),1.45-1.33(m,7H),1.27-1.21(m,4H),1.09(s,1H).

化合物4-11：Compound 4-11:

將化合物1-4(959.23mg，2.14mmol)、HOBt(289.68mg，2.14mmol)、EDCI(410.98mg，2.14mmol)、DIEA(395.82mg，3.06mmol，533.45μL)在DCM(10mL)溶液中的混合物攪拌20分鐘，然後將其加入化合物4-10(920mg，1.53mmol) 在DCM(5mL)中的溶液中，室溫攪拌過夜。加入水淬滅反應，分離有機層並濃縮。藉由製備型HPLC純化殘餘物以得到化合物4-11(1g，產率為63%)。 A mixture of compound 1-4 (959.23 mg, 2.14 mmol), HOBt (289.68 mg, 2.14 mmol), EDCI (410.98 mg, 2.14 mmol), DIEA (395.82 mg, 3.06 mmol, 533.45 μL) in DCM (10 mL) solution was stirred for 20 minutes, and then added to a solution of compound 4-10 (920 mg, 1.53 mmol) in DCM (5 mL) and stirred at room temperature overnight. Water was added to quench the reaction, and the organic layer was separated and concentrated. The residue was purified by preparative HPLC to give compound 4-11 (1 g, yield 63%).

C56H75N3O15質量計算為：1029.5；實測：1052.3[M+Na]+，ESI。 Mass calculated for C 56 H 75 N 3 O 15 : 1029.5; found: 1052.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.82(d,J=9.2Hz,1H),7.79(t,J=5.0Hz,1H),7.40-7.19(m,9H),6.90-6.88(m,2H),5.21(d,J=3.2Hz,1H),4.97(dd,J=11.2,3.3Hz,1H),4.62(t,J=4.9Hz,1H),4.48(d,J=8.4Hz,1H),4.02(s,3H),3.87(dd,J=20.4,9.6Hz,1H),3.97-3.67(m,7H),3.45-3.38(m,3H),3.30-3.13(m,4H),3.05-2.96(m,4H),2.22(t,J=7.3Hz,2H),2.10(s,3H),2.04-1.99(m,5H),1.89(s,3H),1.77(s,3H),1.59-1.21(m,18H). 1 H NMR(400MHz, DMSO-d6)δ 7.82(d, J =9.2Hz,1H),7.79(t, J =5.0Hz,1H),7.40-7.19(m,9H),6.90-6.88(m,2H),5.21(d, J =3.2Hz,1H),4.97(dd, J =11.2,3.3Hz,1H),4.62(t, J =4.9Hz,1H),4.48(d, J =8.4Hz,1H),4.02(s,3H),3.87(dd, J =20.4,9.6Hz,1H),3.97-3.67(m,7H),3.45-3.38(m,3H),3.30-3.13(m,4H),3.05-2.96(m,4H),2.22(t, J =7.3Hz,2H),2.10(s,3H),2.04-1.99(m,5H),1.89(s,3H),1.77(s,3H),1.59-1.21(m,18H).

Figure 113123276-A0202-12-0177-276
Figure 113123276-A0202-12-0177-276

化合物4-12：Compound 4-12:

在室溫中向化合物4-11(750.0mg，0.75mmol)在無水DCM(7.0mL)中的溶液中加入4,5-二氰基咪唑(80.0mg，0.67mmol)和2-氰乙基N,N,N’,N’-四異丙基亞磷二醯胺(270mg，0.90mmol)，攪拌1小時，LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了4-12，為白色固體(500mg，產率為53%)。 To a solution of compound 4-11 (750.0 mg, 0.75 mmol) in anhydrous DCM (7.0 mL) was added 4,5-dicyanoimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (270 mg, 0.90 mmol) at room temperature, stirred for 1 hour, LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na 2 SO 4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). 4-12 was obtained as a white solid (500 mg, yield 53%).

C65H92N5O16P質量計算為：1229.63；實測：1129.0[M-N(CH(CH3)2)2]+，ESI。 Mass calculated for C 65 H 92 N 5 O 16 P: 1229.63; found: 1129.0 [MN(CH(CH 3 ) 2 ) 2 ] + , ESI.

1HNMR(600MHz,DMSO-d 6)：δ 7.44-7.18(m,9H),6.85-6.83(m,4H),6.52-6.44(m,2H),5.26(d,J=6.0Hz,1H),4.99-4.96(m,1H),4.49(d,J=6.0Hz,1H),4.13-3.89(m,4H),3.79-3.55(m,13H),3.44-3.48(m,1H),3.35-3.07(m,8H),2.58-2.56(m,2H),2.24-1.89(m,16H),1.62-1.11(m,31H). 1 HNMR (600MHz, DMSO- d 6 ): δ 7.44-7.18(m,9H),6.85-6.83(m,4H),6.52-6.44(m,2H),5.26(d, J =6.0Hz,1H),4.99-4.96(m,1H),4.49(d, J =6.0Hz,1H),4.13-3.89(m,4H),3.79-3.55(m,13H),3.44-3.48(m,1H),3.3 5-3.07(m,8H),2.58-2.56(m,2H),2.24-1.89(m,16H),1.62-1.11(m,31H).

31PNMR(242MHz,DMSO-d 6)δ 147.17,147.16. 31 PNMR(242MHz,DMSO- d 6 )δ 147.17,147.16.

化合物4-13：Compound 4-13:

向化合物4-11(80mg，0.080mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(5mg，0.04mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗，然後將反應混合物用DCM稀釋，用水(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物4-13，為白色固體(85mg，產率為97%)。 To a solution of compound 4-11 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL) was added DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, LCMS showed that the starting material was completely consumed, then the reaction mixture was diluted with DCM, washed with water (3 mL x 4), then with brine (3 mL x 4), and the organic layer was concentrated to give compound 4-13 as a white solid (85 mg, 97% yield).

C60H79N3O18質量計算為：1129.54；實測：1128.6[M-H]-，ESI。 Mass calculated for C 60 H 79 N 3 O 18 : 1129.54; found: 1128.6 [MH] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 12.22(s,1H),7.84-7.81(m,1H),7.71-7.69(m,1H),7.37-7.16(m,9H),6.92-6.89(m,4H),5.21(d,J=6.0Hz,1H),4.97-4.95(m,1H),4.48(d,J=12.0Hz,1H),4.12-4.01(m,3H),3.89-3.84(m,1H),3.73-3.65(m,7H),3.41-3.38(m,2H),3.28-3.15(m,4H),3.15-2.98(m,4H),2.46-2.44(m,4H),2.22-2.20(m,2H),2.09-1.76(m,14H),1.65-1.60(m,2H),1.48-1.06(m,19H). 1 HNMR (600MHz, DMSO- d 6 ) δ 12.22 (s, 1H), 7.84-7.81 (m, 1H), 7.71-7.69 (m, 1H), 7.37-7.16 (m, 9H), 6.92-6.89 (m, 4H), 5.21 (d, J =6.0Hz,1H),4.97-4.95(m,1H),4.48(d, J =12.0Hz,1H),4.12-4.01(m,3H),3.89-3.84(m,1H),3.73-3.65(m,7H),3.41-3.38(m,2H),3.28-3.15(m,4H),3.1 5-2.98(m,4H),2.46-2.44(m,4H),2.22-2.20(m,2H),2.09-1.76(m,14H),1.65-1.60(m,2H),1.48-1.06(m,19H).

化合物4-14：Compound 4-14:

向化合物4-13(85mg，0.077mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)中的溶液加入DIPEA(30mg，0.23mmol)。將反應混合物室溫振盪 10分鐘，然後加入天然胺基-lcaa-CPG(250mg，上樣75μmol/g)，室溫振盪混懸液20小時，然後過濾，用DMF(20mLx5)、ACN(20mL x5)、DCM(20mLx5)洗滌，直到TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時以得到固體載體(260mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將載體上未反應的胺封端，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時以得到固體載體4-14(260mg)。為了計算上樣，取5.97mg乾燥的上樣CPG，加入25mL的3% DCA在DCM中的溶液。振盪溶液，測量500nm處的紫外吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式： To a solution of compound 4-13 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (30 mg, 0.23 mmol). The reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-lcaa-CPG (250 mg, loading 75 μmol/g) was added, and the suspension was shaken at room temperature for 20 hours, and then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spot in the eluent at 254 nm. The solid support was dried in vacuo for 2 hours to obtain a solid support (260 mg). Stir with Ac 2 O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the support, and wash with DMF (20mLx5), ACN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 4-14 (260mg). To calculate the loading, take 5.97mg of dry loading CPG and add 25mL of 3% DCA solution in DCM. Vibrate the solution and measure the UV absorbance at 500nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula:

上樣(μmol/g)=(添加的DCA總體積(mL))*(500nm處的Abs值)*1000)/(76*(取的CPG mg數)) Sample loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 500nm) * 1000) / (76 * (mg of CPG taken))

添加的DCA總體積(mL)=25mL Total volume of DCA added (mL) = 25mL

500nm處的Abs值=0.6689 Abs value at 500nm = 0.6689

取的CPG的Mg數=5.97mg The Mg content of CPG taken = 5.97mg

上樣(μmol/g)=((25)*(0.6689)*1000)/76*(5.97)=36.85μmol/g。 Loading (μmol/g) = ((25)*(0.6689)*1000)/76*(5.97) = 36.85μmol/g.

Figure 113123276-A0202-12-0180-277
Figure 113123276-A0202-12-0180-277

化合物5-2：Compound 5-2:

在0℃、氮氣下，向甲氧基甲基(三苯基)氯化磷(12.89g，37.61mmol)和KOtBu(4.69g，41.79mmol)在THF(150mL)中的溶液中加入化合物5-1(10g，41.78mmol)。將反應混合物在25℃中攪拌過夜。TLC顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應調節至pH6，並用乙酸乙酯(EA)萃取。有機相濃縮並藉由快速管柱層析(0-10% EA在PE中的溶液)純化以得到呈油狀的化合物5-2(2.92g，產率為37%)。 To a solution of methoxymethyl (triphenyl) phosphonium chloride (12.89 g, 37.61 mmol) and KOtBu (4.69 g, 41.79 mmol) in THF (150 mL) at 0°C under nitrogen was added compound 5-1 (10 g, 41.78 mmol). The reaction mixture was stirred overnight at 25°C. TLC showed complete conversion. The reaction was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with ethyl acetate (EA). The organic phase was concentrated and purified by flash column chromatography (0-10% EA in PE) to give compound 5-2 (2.92 g, 37% yield) as an oil.

C15H25NO3質量計算為：267.2；實測：268.2[M+H]+，ESI。 Mass calculated for C 15 H 25 NO 3 : 267.2; found: 268.2 [M+H] + , ESI.

化合物5-3：Compound 5-3:

向化合物5-2(8.2g，30.67mmol)在MeCN(220mL)中的混合物中加入2,2,2-三氯乙酸(6.31g，38.64mmol)在水(70mL)中的溶液，將混合物攪拌過夜。藉由TLC完全轉化。然後用NaHCO3(飽和水溶液)將pH調節至7-8以淬滅反應，並 用乙酸乙酯萃取。濃縮有機相以得到呈油狀的化合物5-3(7.8g)。粗產物無需進一步純化即可直接用於下一步驟。 To a mixture of compound 5-2 (8.2 g, 30.67 mmol) in MeCN (220 mL) was added a solution of 2,2,2-trichloroacetic acid (6.31 g, 38.64 mmol) in water (70 mL), and the mixture was stirred overnight. Complete conversion by TLC. The reaction was then quenched by adjusting the pH to 7-8 with NaHCO 3 (saturated aqueous solution), and extracted with ethyl acetate. The organic phase was concentrated to give compound 5-3 (7.8 g) as an oil. The crude product was used directly in the next step without further purification.

C14H23NO3質量計算為：253.2；實測：154.2[M+H-Boc]+，ESI。 Mass calculated for C 14 H 23 NO 3 : 253.2; found: 154.2 [M+H-Boc] + , ESI.

化合物5-4：Compound 5-4:

向化合物5-3(1.35g，5.33mmol)和NaOH(21.32mg，532.89μmol，29.38μL)在MeOH(30mL)中的混合物中加入甲醛溶液(454.47mg，15.13mmol，37%水溶液，0.91g)。將反應混合物在25℃攪拌36小時。濃縮反應混合物以除去大部分甲醇，用乙酸乙酯萃取。濃縮有機相並藉由管柱純化(0-5% MeOH在DCM中的溶液)以得到呈固體的化合物5-4(1.5g，2步產率為96%)。 To a mixture of compound 5-3 (1.35 g, 5.33 mmol) and NaOH (21.32 mg, 532.89 μmol, 29.38 μL) in MeOH (30 mL) was added formaldehyde solution (454.47 mg, 15.13 mmol, 37% aqueous solution, 0.91 g). The reaction mixture was stirred at 25 ° C for 36 hours. The reaction mixture was concentrated to remove most of the methanol and extracted with ethyl acetate. The organic phase was concentrated and purified by column (0-5% MeOH in DCM) to give compound 5-4 as a solid (1.5 g, 96% yield for 2 steps).

C15H25NO4質量計算為：283.2；實測：184.2[M+H-Boc]+，ESI。 Mass calculated for C 15 H 25 NO 4 : 283.2; found: 184.2 [M+H-Boc] + , ESI.

化合物5-5：Compound 5-5:

向化合物5-4(2.95g，10.41mmol)在MeOH(90mL)中的溶液中加入硼氫化鈉(866.50mg，22.90mmol)。將反應混合物攪拌1小時。LC-MS顯示完全轉化。用檸檬酸(10%水溶液)將反應混合物調節至pH6，然後濃縮至乾，殘餘物用DCM萃取並過濾。濃縮濾液以得到化合物5-5(2.95g，產率為73%)，無需進一步純化，直接用於下一步。 To a solution of compound 5-4 (2.95 g, 10.41 mmol) in MeOH (90 mL) was added sodium borohydride (866.50 mg, 22.90 mmol). The reaction mixture was stirred for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 with citric acid (10% aqueous solution), then concentrated to dryness, and the residue was extracted with DCM and filtered. The filtrate was concentrated to give compound 5-5 (2.95 g, 73% yield), which was used directly in the next step without further purification.

C15H27NO4質量計算為：285.2；實測：271.2[M+ACN+H-tBu]+，ESI。 Mass calculated for C 15 H 27 NO 4 : 285.2; found: 271.2 [M+ACN+H-tBu] + , ESI.

化合物5-6：Compound 5-6:

向化合物5-5(4g，14.02mmol)中加入HCl(4M在二噁烷中的溶液，63.5mL)。將反應混合物攪拌3小時。LC-MS顯示完全轉化。將反應混合物濃縮以得到化合物5-6(3.5g)，無需進一步純化，直接用於下一步。 To compound 5-5 (4 g, 14.02 mmol) was added HCl (4 M solution in dioxane, 63.5 mL). The reaction mixture was stirred for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to give compound 5-6 (3.5 g), which was used directly in the next step without further purification.

C10H19NO2質量計算為(游離鹼基)：185.1；實測：186.1[M+H]+，ESI。 Mass calculated for C 10 H 19 NO 2 (free base): 185.1; found: 186.1 [M+H] + , ESI.

化合物5-7：Compound 5-7:

將Fmoc-6-胺基己酸(3.19g，9.02mmol)、DIEA(2.91g，22.55mmol，3.93mL)、HOBT(1.46g，10.82mmol)、EDCI(2.08g，10.82mmol)在DCM(12mL)中的溶液在0℃下攪拌30分鐘，然後加入化合物5-6(2g，9.02mmol)。將反應混合物在室溫攪拌過夜。LC-MS顯示完全轉化。用DCM(12mL)稀釋反應混合物，並用檸檬酸(10%水溶液，20mL)和水洗滌。有機相濃縮並藉由矽膠管柱(0-5% MeOH在DCM中的溶液)純化以得到化合物5-7(3.5g，產率為34%)。 A solution of Fmoc-6-aminohexanoic acid (3.19 g, 9.02 mmol), DIEA (2.91 g, 22.55 mmol, 3.93 mL), HOBT (1.46 g, 10.82 mmol), EDCI (2.08 g, 10.82 mmol) in DCM (12 mL) was stirred at 0 ° C for 30 minutes, and then compound 5-6 (2 g, 9.02 mmol) was added. The reaction mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (12 mL) and washed with citric acid (10% aqueous solution, 20 mL) and water. The organic phase was concentrated and purified by silica gel column (0-5% MeOH in DCM) to give compound 5-7 (3.5 g, 34% yield).

C31H40N2O5質量計算為：520.3；實測：521.2[M+H]+，ESI。 Mass calculated for C 31 H 40 N 2 O 5 : 520.3; found: 521.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,5H),4.32-4.28(m,4H),4.20(t,J=6.6Hz,1H),3.69(s,2H),3.43(s,2H),3.22(t,J=5.6Hz,4H),2.02-1.98(m,3H),1.55-1.52(t,J=6.6Hz,4H),2.26-2.23(m,4H),1.24-1.16(m,8H). 1 H NMR (400MHz, DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,5H),4.32-4.28(m,4H),4.20(t, J =6.6Hz,1H),3.69(s,2H),3.43(s,2H),3.22(t, J =5.6Hz,4H),2.02-1.98(m,3H),1.55-1.52(t, J =6.6Hz,4H),2.26-2.23(m,4H),1.24-1.16(m,8H).

化合物5-8：Compound 5-8:

向化合物5-7(2.2g，4.23mmol)、DMAP(51.6mg，0.423mmol)和DIEA(1.09g，8.46mmol)在DCM(66mL)中的溶液中加入DMTrCl(1.43g，4.23mmol)。將反應混合物在室溫下攪拌過夜。LC-MS顯示部分轉化。反應混合物藉由氧化鋁墊過濾，並用DCM(50mL)洗滌。將濾液濃縮並藉由矽膠管柱純化(10-40% EA與0.1%氨水在PE中的溶液，然後0-10% MeOH與0.1%氨水在DCM中的溶液)以得到化合物5-8(3.0g，產率為86%)。 To a solution of compound 5-7 (2.2 g, 4.23 mmol), DMAP (51.6 mg, 0.423 mmol) and DIEA (1.09 g, 8.46 mmol) in DCM (66 mL) was added DMTrCl (1.43 g, 4.23 mmol). The reaction mixture was stirred at room temperature overnight. LC-MS showed partial conversion. The reaction mixture was filtered through an alumina pad and washed with DCM (50 mL). The filtrate was concentrated and purified by a silica gel column (10-40% EA with 0.1% ammonia in PE, then 0-10% MeOH with 0.1% ammonia in DCM) to give compound 5-8 (3.0 g, 86% yield).

C52H58N2O7質量計算為：822.4；實測：845.3[M+Na]+，ESI。 Mass calculated for C 52 H 58 N 2 O 7 : 822.4; found: 845.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.67(m,4H),7.42-7.39(m,4H),7.41-7.24(m,14H),6.89-6.87(m,4H),4.44(t,J=7.6Hz,1H),4.24-4.19(m,1H),4.06-4.01(m,1H),3.74-3.73(m,6H),3.65-3.59(m,2H),3.42-3.39(m,3H),3.32(br,1H),3.00-2.83(m,4H),1.99-1.96(m,4H),1.45-1.16(m,16H). 1 H NMR(400MHz, DMSO-d6)δ 7.90-7.67(m,4H),7.42-7.39(m,4H),7.41-7.24(m,14H),6.89-6.87(m,4H),4.44(t, J =7.6Hz,1H),4.24-4.19(m,1H),4.06-4.01(m,1H),3.74-3.73(m,6H),3.65-3.59(m,2H),3 .42-3.39(m,3H),3.32(br,1H),3.00-2.83(m,4H),1.99-1.96(m,4H),1.45-1.16(m,16H).

化合物5-9：Compound 5-9:

向化合物5-8(3g，3.65mmol)在MeOH(90mL)中的溶液中加入哌啶(9mL)。將反應混合物在室溫攪拌過夜。LC-MS顯示完全轉化。濃縮反應混合物，然後用二甲苯共沸蒸餾。殘餘物藉由矽膠管柱層析純化(0-10% MeOH與1% TEA在DCM中的溶液)以得到化合物5-9(1.1g，產率為91%)。 To a solution of compound 5-8 (3 g, 3.65 mmol) in MeOH (90 mL) was added piperidine (9 mL). The reaction mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was concentrated and then azeotropically distilled with xylene. The residue was purified by silica gel column chromatography (0-10% MeOH and 1% TEA in DCM) to give compound 5-9 (1.1 g, 91% yield).

C37H48N2O5質量計算為：600.4；實測：601.3[M+H]+，ESI。 Mass calculated for C 37 H 48 N 2 O 5 : 600.4; found: 601.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.40-7.21(m,9H),6.89(d,J=7.2Hz,4H),3.74(s,6H),3.67-3.61(m,2H),3.39-3.33(m,2H),2.86-2.62(m,4H),1.99(t,J=7.4Hz,2H),1.51-1.21(m,16H). 1 H NMR(400MHz,DMSO-d6)δ 7.40-7.21(m,9H),6.89(d, J =7.2Hz,4H),3.74(s,6H),3.67-3.61(m,2H),3.39-3.33(m,2H),2.86-2.62(m,4H),1.99(t, J =7.4Hz,2H),1.51-1.21(m,16H).

化合物5-10：Compound 5-10:

將化合物1-4(2.09g，4.66mmol)、DIEA(1.08g，8.32mmol，1.45mL)、HOBT(539.77mg，3.99mmol)和EDCI(765.80mg，3.99mmol)在DCM(60mL)中的溶液在0℃攪拌30分鐘，然後加入化合物5-9(2g，3.33mmol)。將反應混合物攪拌過夜。將反應混合物濃縮，殘餘物藉由製備型HPLC(C18，水/ACN)純化以得到化合物5-10(1.0g，產率為15%)。 A solution of compound 1-4 (2.09 g, 4.66 mmol), DIEA (1.08 g, 8.32 mmol, 1.45 mL), HOBT (539.77 mg, 3.99 mmol) and EDCI (765.80 mg, 3.99 mmol) in DCM (60 mL) was stirred at 0° C. for 30 minutes, and then compound 5-9 (2 g, 3.33 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was concentrated and the residue was purified by preparative HPLC (C18, water/ACN) to give compound 5-10 (1.0 g, 15% yield).

C56H75N3O15質量計算為：1029.5；實測：1052.3[M+Na]+，ESI。 Mass calculated for C 56 H 75 N 3 O 15 : 1029.5; found: 1052.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.83(d,J=9.2Hz,1H),7.71(t,J=5.2Hz,1H),7.40-7.38(m,2H),7.32-7.19(m,5H),6.90-9.87(m,2H),5.22(d,J=3.4Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.49(d,J=8.5Hz,1H),4.42(t,J=5.0 Hz,1H),4.03(s,2H),3.91-3.84(m,2H),3.74(s,6H),3.67-3.61(m,1H),3.44-3.37(m,4H),3.00(dd,J=12.7,6.6Hz,2H),2.85(d,J=9.6Hz,2H),2.11(s,3H),2.05-1.96(m,7H),1.90(s,3H),1.78(s,3H),1.52-1.18(m,18H). 1 H NMR(400MHz, DMSO-d6)δ 7.83(d, J =9.2Hz,1H),7.71(t, J =5.2Hz,1H),7.40-7.38(m,2H),7.32-7.19(m,5H),6.90-9.87(m,2H),5.22(d, J =3.4Hz,1H),4.97(dd, J =11.2,3.4Hz,1H),4.49(d, J =8.5Hz,1H),4.42(t, J =5.0 Hz,1H),4.03(s,2H),3.91-3.84(m,2H),3.74(s,6H),3.67-3.61(m,1H),3.44-3.37(m,4H),3.00(dd, J =12.7,6.6Hz,2H),2.85(d, J =9.6Hz,2H),2.11(s,3H),2.05-1.96(m,7H),1.90(s,3H),1.78(s,3H),1.52-1.18(m,18H).

化合物5-11：Compound 5-11:

在室溫中向化合物5-10(690.0mg，0.66mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(96.0mg，0.81mmol)和2-氰乙基N,N,N,,N’-四異丙基亞磷二醯胺(324mg，1.08mmol)，攪拌1小時，LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物5-11，為白色固體(460mg，產率為56%)。 To a solution of compound 5-10 (690.0 mg, 0.66 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N,,N'-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) at room temperature, stirred for 1 hour, LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3 ), and dried over Na2SO4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 5-11 was produced as a white solid (460 mg, yield 56%).

C65H92N5O16P質量計算為：1229.63；實測：1230.3[M+H]+，ESI。 Mass calculated for C 65 H 92 N 5 O 16 P: 1229.63; found: 1230.3 [M+H] + , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.81-7.67(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d,J=6.0Hz,1H),4.98-4.94(m,1H),4.49(d,J=6.0Hz,1H),4.04-3.83(m,4H),3.73-3.32(m,18H),3.01-2.83(m,4H),2.72-2.69(m,2H),2.10-1.76(m,16H),1.52-1.04(m,30H). 1 HNMR(600MHz, DMSO- d 6 )δ 7.81-7.67(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d, J =6.0Hz,1H),4.98-4.94(m,1H),4.49(d, J =6.0Hz,1H),4.04-3.83(m,4H),3.73-3.32(m,18H),3.01-2.83(m,4H),2.72-2.69(m,2H),2.10-1.76(m,16H),1.52-1.04(m,30H).

31PNMR(242MHz,DMSO-d 6)δ 146.33,146.26. 31 PNMR(242MHz,DMSO -d6 ) δ 146.33,146.26.

Figure 113123276-A0202-12-0185-278
Figure 113123276-A0202-12-0185-278

化合物6-2：Compound 6-2:

將化合物6-1(20g,99.88mmol)溶解於THF(200mL)中。將NaH(4.79g，119.86mmol，純度60%)加入到反應混合物中，在0℃下攪拌30分鐘。將2,3-二溴丙烯(20.96g，104.88mmol)緩慢加入到反應混合物中。並將反應在室溫攪拌2小時。將反應混合物用飽和NH4Cl溶液(100mL)淬滅，用EA(200mLX3)萃取，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由矽膠快速管柱層析純化以得到化合物6-2，為無色油狀物(31g，產率為97%)。 Compound 6-1 (20 g, 99.88 mmol) was dissolved in THF (200 mL). NaH (4.79 g, 119.86 mmol, purity 60%) was added to the reaction mixture and stirred at 0°C for 30 minutes. 2,3-Dibromopropylene (20.96 g, 104.88 mmol) was slowly added to the reaction mixture. The reaction was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NH 4 Cl solution (100 mL), extracted with EA (200 mL×3), washed with brine (100 mL), dried with Na 2 SO 4 , concentrated in vacuo and purified by silica gel flash column chromatography to obtain compound 6-2 as a colorless oil (31 g, yield 97%).

C13H19BrO4質量計算為：318.0；實測：319.0[M+H]+，ESI。 Mass calculated for C 13 H 19 BrO 4 : 318.0; found: 319.0 [M+H] + , ESI.

1H NMR(400MHz,CDCl3)δ 5.71-5.58(m,3H),5.14-5.10(m,2H),4.25-4.13(m,4H),3.14(s,2H),2.7(d,J=7.2Hz,2H),1.26(t,J=6.8Hz,6H). 1 H NMR (400MHz, CDCl 3 )δ 5.71-5.58(m,3H),5.14-5.10(m,2H),4.25-4.13(m,4H),3.14(s,2H),2.7(d, J =7.2Hz,2H),1.26(t, J =6.8Hz,6H).

化合物6-3：Compound 6-3:

將化合物6-2(31g，97.12mmol)、Pd(OAc)2(2.18g，9.71mmol)、PPh3(5.09g，19.42mmol)和AgOAc(19.45g，116.54mmol)溶解於CAN(600mL)中。將反應混合物在N2氣氛下在85℃下攪拌3小時。將反應冷卻至室溫並過濾、真空濃縮並藉由矽膠快速管柱層析純化以得到化合物6-3，為無色油狀物(21g，產率為91%)。C13H18O4質量計算為：238.1；實測：239.2[M+H]+，ESI。 Compound 6-2 (31 g, 97.12 mmol), Pd(OAc) 2 (2.18 g, 9.71 mmol), PPh 3 (5.09 g, 19.42 mmol) and AgOAc (19.45 g, 116.54 mmol) were dissolved in ACN (600 mL). The reaction mixture was stirred at 85 °C for 3 hours under N 2 atmosphere. The reaction was cooled to room temperature and filtered, concentrated in vacuo and purified by silica gel flash column chromatography to give compound 6-3 as a colorless oil (21 g, yield 91%). Mass calculated for C 13 H 18 O 4 : 238.1; found: 239.2 [M+H] + , ESI.

1H NMR(400Mhz,CDCl3)δ 5.39(t,J=2Hz,2H),4.95(t,J=1.6Hz,2H),4.18(q,J=7.2Hz,4H),3.03(t,J=7.2Hz,4H),1.24(t,J=7.2Hz,6H). 1 H NMR (400Mhz, CDCl3) δ 5.39 (t, J =2Hz, 2H), 4.95 (t, J =1.6Hz, 2H), 4.18 (q, J =7.2Hz, 4H), 3.03 (t, J =7.2Hz, 4H), 1.24 (t, J =7.2Hz, 6H).

化合物6-4：Compound 6-4:

將化合物6-3(21g，88.13mmol)溶於DCM(300mL)中並冷卻至-78℃。將Br2(14.08g，88.13mmol，4.51mL)溶於DCM(100mL)中並在1小時內加入到反應混合物中。將混合物在-78℃中攪拌1小時。將反應混合物用飽和Na2SO3溶液(100mL)淬滅，用DCM(200mLX3)萃取，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由矽膠快速管柱層析純化以得到化合物6-4，為淺黃色油狀物(28g，產率為80%)。 Compound 6-3 (21 g, 88.13 mmol) was dissolved in DCM (300 mL) and cooled to -78 °C. Br 2 (14.08 g, 88.13 mmol, 4.51 mL) was dissolved in DCM (100 mL) and added to the reaction mixture over 1 hour. The mixture was stirred at -78 °C for 1 hour. The reaction mixture was quenched with saturated Na 2 SO 3 solution (100 mL), extracted with DCM (200 mL×3), washed with brine (100 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by silica gel flash column chromatography to give compound 6-4 as a light yellow oil (28 g, 80% yield).

C13H18Br2O4質量計算為：396.0；實測：397.0[M+H]+，ESI。 Mass calculated for C 13 H 18 Br 2 O 4 : 396.0; found: 397.0 [M+H] + , ESI.

1H NMR(400MHz,CDCl3)δ：4.21(q,J=7.2Hz,4H),4.00(s,4H),3.19(s,4H),1.26(t,J=7.2Hz,6H). 1 H NMR (400MHz, CDCl3) δ: 4.21 (q, J =7.2Hz, 4H), 4.00 (s, 4H), 3.19 (s, 4H), 1.26 (t, J =7.2Hz, 6H).

化合物6-5：Compound 6-5:

將4-甲基苯磺醯胺(12.05g，70.35mmol)溶解於DMF(260mL)中。在0℃下將NaH(6.19g，154.77mmol)加入到反應混合物中。將反應混合物在0℃攪拌30分鐘。將化合物6-4(28g，70.35mmol)在DMF(50mL)中的溶液緩慢加入到反應混合物中。將混合物在室溫攪拌1小時。將反應混合物用飽和NH4Cl溶液(500mL)淬滅，用EA(200mLx3)萃取，用水(500mL x3)、鹽水(300mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由矽膠快速管柱層析純化以得到化合物6-5，為無色固體(22g，產率為76%)。 4-Methylbenzenesulfonamide (12.05 g, 70.35 mmol) was dissolved in DMF (260 mL). NaH (6.19 g, 154.77 mmol) was added to the reaction mixture at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. A solution of compound 6-4 (28 g, 70.35 mmol) in DMF (50 mL) was slowly added to the reaction mixture. The mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with saturated NH 4 Cl solution (500 mL), extracted with EA (200 mL×3), washed with water (500 mL×3), brine (300 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by silica gel flash column chromatography to give compound 6-5 as a colorless solid (22 g, yield 76%).

C20H25NO6S質量計算為：407.1；實測：408.2[M+H]+，ESI。 Mass calculated for C 20 H 25 NO 6 S: 407.1; found: 408.2 [M+H] + , ESI.

1H NMR(400MHz,CDCl3)δ 7.70-7.68(m,2H),7.32-7.30(m,2H),4.17(q,J=7.2Hz,4H),3.96(s,4H),2.89(s,4H),2.42(s,3H),1.22(t,J=7.2Hz,6H). 1 H NMR(400MHz, CDCl3)δ 7.70-7.68(m,2H),7.32-7.30(m,2H),4.17(q, J =7.2Hz,4H),3.96(s,4H),2.89(s,4H),2.42(s,3H),1.22(t, J =7.2Hz,6H).

化合物6-6：Compound 6-6:

將化合物6-5(22g，54.05 mmol)溶解於THF(300mL)中。在0℃下將LiBH4(11.8g，540.5mmol)加入到反應混合物中。將反應在室溫攪拌1小時。將反應混合物用水(100mL)淬滅，用EA(200mLx3)萃取，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由矽膠快速管柱層析純化以得到化合物6-6，為白色固體(14.8g，產率為89%)。 Compound 6-5 (22 g, 54.05 mmol) was dissolved in THF (300 mL). LiBH4 (11.8 g, 540.5 mmol) was added to the reaction mixture at 0°C. The reaction was stirred at room temperature for 1 hour. The reaction mixture was quenched with water (100 mL), extracted with EA (200 mL x 3), washed with brine (100 mL ), dried over Na2SO4 , concentrated in vacuo and purified by silica gel flash column chromatography to give compound 6-6 as a white solid (14.8 g, 89% yield).

C16H21NO4S質量計算為：323.1；實測：324.1[M+H]+，ESI。 Mass calculated for C 16 H 21 NO 4 S: 323.1; found: 324.1 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.70-7.68(m,2H),7.43-7.41(m,2H),4.56(t,J=5.2Hz,2H),3.26(d,J=4.8Hz,4H),2.84(s,4H),2.39(s,3H),1.95(s,4H). 1 H NMR(400MHz, DMSO-d6)δ 7.70-7.68(m,2H),7.43-7.41(m,2H),4.56(t, J =5.2Hz,2H),3.26(d, J =4.8Hz,4H),2.84(s,4H),2.39(s,3H),1.95(s,4H).

化合物6-7：Compound 6-7:

將化合物6-6(4.1g，12.68mmol)和DIEA(4.92g，3.83mmol)溶於DCM(20mL)中。在0℃下將DMTrCl(4.30g，12.68mmol)在DCM(80mL)中的溶液緩慢加入到反應混合物中。將反應在室溫攪拌1小時。將反應混合物用飽和NaHCO3溶液(20mL)淬滅，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-3% MeOH)在Al2O3上純化以得到化合物6-7，為白色泡沫狀固體(6.39g，產率為80%)。 Compound 6-6 (4.1 g, 12.68 mmol) and DIEA (4.92 g, 3.83 mmol) were dissolved in DCM (20 mL). A solution of DMTrCl (4.30 g, 12.68 mmol) in DCM (80 mL) was slowly added to the reaction mixture at 0 °C. The reaction was stirred at room temperature for 1 hour. The reaction mixture was quenched with saturated NaHCO 3 solution (20 mL), extracted with DCM (50 mLx2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-3% MeOH) on Al 2 O 3 to give compound 6-7 as a white foamy solid (6.39 g, 80% yield).

C37H39NO6S質量計算為：625.25；實測：648.25[M+Na]+，ESI。 Mass calculated for C 37 H 39 NO 6 S: 625.25; found: 648.25 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.68(d,J=8.0Hz,2H),7.41(d,J=8.0Hz,2H),7.33(d,J=4.0Hz,2H),7.31-7.18(m,7H),6.85(d,J=8.0Hz,4H),4.68(t,J=4.8Hz,1H),3.83(s,4H),3.72(s,6H),3.37(d,J=2.0Hz,2H),2.94(s,2H),2.39(s,3H),2.07-2.03(m,2H),1.92-1.89(m,2H). 1 H NMR (400MHz, DMSO- d 6 ) δ 7.68 (d, J =8.0Hz, 2H), 7.41 (d, J =8.0Hz, 2H), 7.33 (d, J =4.0Hz, 2H), 7.31-7.18 (m, 7H), 6.85 (d, J =8.0Hz, 4H), 4.68 (t, J =4.8Hz,1H),3.83(s,4H),3.72(s,6H),3.37(d, J =2.0Hz,2H),2.94(s,2H),2.39(s,3H),2.07-2.03(m,2H),1.92-1.89(m,2H).

化合物6-8：Compound 6-8:

將化合物6-7(6.0g，9.59mmol)溶解於MeOH(300mL)中，然後在0℃下將Mg(18g)添加到反應混合物中。將混合物在室溫攪拌過夜。將反應混合物用H2O淬滅，用DCM萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-15%MeOH)在Al2O3上純化以得到化合物6-8，為白色泡沫狀固體(1.2g，產率為26.6%)。 Compound 6-7 (6.0 g, 9.59 mmol) was dissolved in MeOH (300 mL), and then Mg (18 g) was added to the reaction mixture at 0°C. The mixture was stirred at room temperature overnight. The reaction mixture was quenched with H2O , extracted with DCM, washed with brine (50 mL), dried over Na2SO4 , concentrated in vacuo and purified by flash column chromatography (0-15% MeOH) on Al2O3 to give compound 6-8 as a white foamy solid (1.2 g, 26.6% yield).

C30H33NO4質量計算為：471.24；實測：472.20[M+H]+，ESI。 Mass calculated for C 30 H 33 NO 4 : 471.24; found: 472.20 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.37(d,J=8.0Hz,2H),7.32-7.28(m,2H),7.25-7.19(m,5H),6.89-6.87(m,4H),4.76(s,1H),3.83(s,1H),3.73(s,6H),3.47(s,3H),3.45(s,3H),3.00(s,2H),2.13-2.09(m,2H),1.97-1.93(m,2H). 1 H NMR (400MHz, DMSO- d 6 ) δ 7.37 (d, J =8.0Hz,2H),7.32-7.28(m,2H),7.25-7.19(m,5H),6.89-6.87(m,4H),4.76(s,1H),3.83(s,1 H),3.73(s,6H),3.47(s,3H),3.45(s,3H),3.00(s,2H),2.13-2.09(m,2H),1.97-1.93(m,2H).

化合物6-9：Compound 6-9:

將化合物6-8(2.4g，5.09mmol)和Fmoc-6-胺基己酸(1.80g，5.09mmol)溶於DCM(60mL)，然後加入DIEA(1.97g，15.27mmol)、EDCI(1.95g，10.18mmol)和HOBT(1.38g，10.18mmol)到混合物中。將反應在室溫攪拌2小時。將反應混合物用飽和NaHCO3溶液(200mL)淬滅，用DCM(60mLx3)萃取，用鹽水(60mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-10% MeOH)在Al2O3上純化以得到化合物6-9，為白色泡沫狀固體(3.1g，產率為75%)。 Compound 6-8 (2.4 g, 5.09 mmol) and Fmoc-6-aminohexanoic acid (1.80 g, 5.09 mmol) were dissolved in DCM (60 mL), and then DIEA (1.97 g, 15.27 mmol), EDCI (1.95 g, 10.18 mmol) and HOBT (1.38 g, 10.18 mmol) were added to the mixture. The reaction was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (200 mL), extracted with DCM (60 mL×3), washed with brine (60 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on Al 2 O 3 to give compound 6-9 as a white foamy solid (3.1 g, 75% yield).

1H NMR(400MHz,DMSO-d 6)δ 7.88(d,J=7.4Hz,2H),7.68(d,J=7.4Hz,2H),7.43-7.37(m,4H),7.34-7.28(m,5H),7.26-7.21(m,5H),6.88(d,J=8.9Hz,4H),4.75(t,J=4.8Hz,1H),4.30-4.28(m,2H),4.22-4.19(m,1H),4.11-3.91(m,2H),3.91-3.85(m,2H),3.73(s,6H),3.49-3.48(d,J=4.6Hz,2H),3.02-2.95(m,4H),2.20-2.14(m,4H),2.00-1.97(m,2H),1.52-1.46(m,2H),1.44-1.36(m,2H),1.24-1.29(m,2H). 1 H NMR (400MHz, DMSO- d 6 ) δ 7.88 (d, J =7.4Hz, 2H), 7.68 (d, J = 7.4Hz, 2H), 7.43-7.37 (m, 4H), 7.34-7.28 (m, 5H), 7.26-7.21 (m, 5H), 6.88 (d, J =8.9Hz,4H),4.75(t, J =4.8Hz,1H),4.30-4.28(m,2H),4.22-4.19(m,1H),4.11-3.91(m,2H),3.91-3.85(m,2H),3.73(s,6H),3.49-3.48(d, J =4.6Hz,2H),3.02-2.95(m,4H),2.20-2.14(m,4H),2.00-1.97(m,2H),1.52-1.46(m,2H),1.44-1.36(m,2H),1.24-1.29(m,2H).

化合物6-10：Compound 6-10:

將化合物6-9(3.1g，3.84mmol)溶解於MeOH(60mL)中，然後添加哌啶(6ml)到混合物中。將反應在室溫攪拌8小時。將反應混合物用水淬滅，用DCM(100mLx3)萃取，用鹽水(60mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-20% MeOH)在Al2O3上純化以得到化合物6-10，為白色泡沫狀固體(1.45g，產率為64%)。 Compound 6-9 (3.1 g, 3.84 mmol) was dissolved in MeOH (60 mL), and then piperidine (6 ml) was added to the mixture. The reaction was stirred at room temperature for 8 hours. The reaction mixture was quenched with water, extracted with DCM (100 mL x 3), washed with brine (60 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-20% MeOH) on Al 2 O 3 to give compound 6-10 as a white foamy solid (1.45 g, 64% yield).

C36H44N2O5質量計算為：584.33；實測：585.4[M+H]+，ESI。 The mass of C36H44N2O5 was calculated as: 584.33; found: 585.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.39-7.39(m,2H),7.37-7.28(m,2H),7.25-7.19(m,5H),6.88(d,J=8.8Hz,2H),4.76(s,1H),4.04(m,2H),3.86(m,2H),3.78(s,6H),3.49(s,2H),3.02(s,2H),2.55(m,2H),2.22-2.14(m,4H),2.01-1.97(m,2H),1.53-1.46(m,2H),1.37-1.25(m,5H). 1 H NMR (400MHz, DMSO- d 6 ) δ 7.39-7.39(m,2H),7.37-7.28(m,2H),7.25-7.19(m,5H),6.88(d, J =8.8Hz,2H),4.76(s,1H),4.04(m,2H),3.86(m,2H),3.78(s,6H),3.49(s,2H),3.02(s,2H) ,2.55(m,2H),2.22-2.14(m,4H),2.01-1.97(m,2H),1.53-1.46(m,2H),1.37-1.25(m,5H).

化合物6-11：Compound 6-11:

將化合物6-10(1.5g，2.57mmol)和化合物1-4(1.15g，2.57mmol)溶於DCM(60mL)，然後加入DIEA(1.66g，12.83mmol)、EDCI(1.10g，6.41mmol)和HOBT(519.92mg，3.85mmol)到混合物中。將反應在室溫攪拌2小時。將反應混合物用飽和NaHCO3溶液(200mL)淬滅，用DCM(60mLx3)萃取，用鹽水(60mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-10% MeOH)在Al2O3上純化以得到化合物6-11，為白色泡沫狀固體(2g，產率為76%)。 Compound 6-10 (1.5 g, 2.57 mmol) and compound 1-4 (1.15 g, 2.57 mmol) were dissolved in DCM (60 mL), and then DIEA (1.66 g, 12.83 mmol), EDCI (1.10 g, 6.41 mmol) and HOBT (519.92 mg, 3.85 mmol) were added to the mixture. The reaction was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (200 mL), extracted with DCM (60 mL×3), washed with brine (60 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on Al 2 O 3 to give compound 6-11 as a white foamy solid (2 g, 76% yield).

C55H71N3O15質量計算為：1013.49；實測：1014.4[M+H]+，ESI。 Mass calculated for C 55 H 71 N 3 O 15 : 1013.49; found: 1014.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.82(d,J=9.2Hz,1H),7.72(t,J=5.6Hz,1H),7.39-7.37(m,2H),7.32-7.28(m,2H),7.26-7.19(m,5H),6.90-6.87(m,4H),5.22(d,J=3.4Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.50-4.48(m,1H),4.03-4.00(m,5H),3.91-3.84(m,3H),3.74(s,7H),3.49(d,J=4.6Hz,1H),3.43-3.38(m,1H),3.02-2.98(m,4H),2.21-2.15(m,4H),2.10-2.08(m,3H),2.05-2.00(m,7H),1.89(s,3H),1.78(s,3H),1.52-1.35(m,9H),1.27-1.24(m,3H). 1 H NMR (400MHz, DMSO- d 6 ) δ 7.82 (d, J = 9.2 Hz, 1H), 7.72 (t, J =5.6Hz,1H),7.39-7.37(m,2H),7.32-7.28(m,2H),7.26-7.19(m,5H),6.90-6.87(m,4H),5.22(d, J =3.4Hz,1H),4.97(dd, J =11.2,3.4Hz,1H),4.50-4.48(m,1H),4.03-4.00(m,5H),3.91-3.84(m,3H),3.74(s,7H),3.49(d, J =4.6Hz,1H),3.43-3.38(m,1H),3.02-2.98(m,4H),2.21-2.15(m,4H),2.10-2.08(m,3 H),2.05-2.00(m,7H),1.89(s,3H),1.78(s,3H),1.52-1.35(m,9H),1.27-1.24(m,3H).

化合物6-12：Compound 6-12:

在室溫中向化合物6-11(790.0mg，0.78mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(96.0mg，0.81mmol)和2-氰乙基N,N,N’,N’-四異丙基亞磷二醯胺(324mg，1.08mmol)，攪拌1小時，LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物6-12，為白色固體(680mg，產率為71%)。 To a solution of compound 6-11 (790.0 mg, 0.78 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) at room temperature, stirred for 1 hour, LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na 2 SO 4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 6-12 was produced as a white solid (680 mg, yield 71%).

C64H88N5O16P質量計算為：1213.60；實測：1159.5[M-CH2CH2CN]-，ESI。 Mass calculated for C 64 H 88 N 5 O 16 P: 1213.60; found: 1159.5 [M-CH 2 CH 2 CN] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.82-7.68(m,2H),7.38-7.19(m,9),6.90-6.86(m,4H),5.21(d,J=6.0Hz,1H),4.98-4.94(m,1H),4.48(d,J=12.0Hz,1H),4.04-3.99(m,4H),3.90-3.37(m,16H),3.09-2.87(m,4H),2.71-2.68(m,2H),2.19-1.76(m,20H),1.52-1.05(m,22H). 1 HNMR(600MHz, DMSO- d 6 )δ 7.82-7.68(m,2H),7.38-7.19(m,9),6.90-6.86(m,4H),5.21(d, J =6.0Hz,1H),4.98-4.94(m,1H),4.48(d, J =12.0Hz,1H),4.04-3.99(m,4H),3.90-3.37(m,16H),3.09-2.87(m,4H),2.71-2.68(m,2H),2.19-1.76(m,20H),1.52-1.05(m,22H).

31PNMR(242MHz,DMSO-d 6)δ 146.51,146.47. 31 PNMR(242MHz,DMSO -d6 ) δ 146.51,146.47.

Figure 113123276-A0202-12-0191-279
Figure 113123276-A0202-12-0191-279

化合物7-2：Compound 7-2:

在0℃下向甲氧基甲基(三苯基)氯化磷(54.78g，159.80mmol)在THF(600mL)中的溶液中加入KOtBu(19.92g，177.55mmol)並攪拌0.5小時。然後在0℃下加入化合物7-1(20g，88.78mmol)，並攪拌過夜，使反應升溫至室溫。LC-MS顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，過濾並濃縮至乾。殘餘物藉由矽膠管柱(0-5% EA在PE中的溶液)純化，以得到呈油狀的化合物7-2(15.5g，產率為68%)。 To a solution of methoxymethyl (triphenyl) phosphonium chloride (54.78 g, 159.80 mmol) in THF (600 mL) was added KOtBu (19.92 g, 177.55 mmol) at 0°C and stirred for 0.5 hours. Compound 7-1 (20 g, 88.78 mmol) was then added at 0°C and stirred overnight, allowing the reaction to warm to room temperature. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered and concentrated to dryness. The residue was purified by silica gel column (0-5% EA in PE) to give compound 7-2 (15.5 g, 68% yield) as an oil.

C14H23NO3質量計算為：253.2；實測：239.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 14 H 23 NO 3 : 253.2; found: 239.1 [M-tBu+ACN+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 5.99(pent,J=2.1Hz,1H),3.48(s,3H),3.44-3.36(m,2H),2.99-2.91(m,2H),2.59(br,2H),2.44-2.36(m,2H),2.04-1.97(m,2H),1.38(s,9H). 1H NMR(400MHz,DMSO-d6)δ 5.99(pent,J=2.1Hz,1H),3.48(s,3H),3.44-3.36(m,2H),2.99-2.91(m,2H),2.59(br,2H),2.44-2.36(m,2H),2.04-1.97(m,2H),1.38(s,9H).

化合物7-3：Compound 7-3:

向化合物7-2(5g，19.74mmol)在MeCN(150mL)中的溶液中加入2,2,2-三氯乙酸(4.06g，24.87mmol，在50mL的水中)。將反應混合物在室溫攪拌過夜。TLC顯示完全轉化。藉由加入NaHCO3(飽和水溶液)將反應混合物調節至pH7，並用EA萃取。濃縮有機相以得到粗化合物7-3，為油狀物(4.7g，產率為99%)，無需進一步純化即可直接用於下一步。 To a solution of compound 7-2 (5 g, 19.74 mmol) in MeCN (150 mL) was added 2,2,2-trichloroacetic acid (4.06 g, 24.87 mmol in 50 mL of water). The reaction mixture was stirred at room temperature overnight. TLC showed complete conversion. The reaction mixture was adjusted to pH 7 by adding NaHCO 3 (saturated aqueous solution) and extracted with EA. The organic phase was concentrated to give crude compound 7-3 as an oil (4.7 g, yield 99%), which was used directly in the next step without further purification.

C13H21NO3質量計算為：239.2；實測：225.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 13 H 21 NO 3 : 239.2; found: 225.1 [M-tBu+ACN+H] + , ESI.

化合物7-4：Compound 7-4:

向化合物7-3(4.7g，19.64mmol)和K2CO3(542.87mg，3.93mmol)在MeOH(150mL)中的混合物中加入福爾馬林(15.9g，37%甲醛水溶液，55.78mmol)。將反應混合物在室溫攪拌過夜。TLC顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，並用EA萃取。有機相濃縮並藉由矽膠管柱(0-3% MeOH在DCM中的溶液)純化以得到呈油狀的化合物7-4(5.2g，產率為98%)。 To a mixture of compound 7-3 (4.7 g, 19.64 mmol) and K 2 CO 3 (542.87 mg, 3.93 mmol) in MeOH (150 mL) was added formalin (15.9 g, 37% aqueous formaldehyde, 55.78 mmol). The reaction mixture was stirred at room temperature overnight. TLC showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with EA. The organic phase was concentrated and purified by silica gel column (0-3% MeOH in DCM) to give compound 7-4 (5.2 g, 98% yield) as an oil.

C14H23NO4質量計算為：269.2；實測：170.1[M-Boc+H]+，ESI。 Mass calculated for C 14 H 23 NO 4 : 269.2; found: 170.1 [M-Boc+H] + , ESI.

化合物7-5：Compound 7-5:

向化合物7-4(17g，63.12mmol)在MeOH(190mL)中的溶液中加入NaBH4(5.25g，138.86mmol)。將反應混合物在室溫下攪拌1小時。LC-MS顯示 完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，過濾，並用DCM萃取。有機相濃縮並藉由矽膠管柱(0-5% MeOH在DCM中的溶液)純化以得到化合物7-5(14.5g，產率為84%)。 To a solution of compound 7-4 (17 g, 63.12 mmol) in MeOH (190 mL) was added NaBH 4 (5.25 g, 138.86 mmol). The reaction mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and extracted with DCM. The organic phase was concentrated and purified by silica gel column (0-5% MeOH in DCM) to give compound 7-5 (14.5 g, 84% yield).

C14H25NO4質量計算為：271.2；實測：172.1[M-Boc+H]+，ESI。 Mass calculated for C 14 H 25 NO 4 : 271.2; found: 172.1 [M-Boc+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.53(t,J=5.3Hz),3.37-3.32(m,2H),3.27(d,J=5.3Hz,2H),3.19(d,J=5.3Hz,2H),3.07(dd,J=10.78,3.5Hz,2H),1.60(dd,J=13.4,7.9Hz,2H)1.39(s,9H),1.27(dd,J=13.5,6.3Hz,2H). 1H NMR(400MHz,DMSO-d6)δ 4.53(t,J=5.3Hz),3.37-3.32(m,2H),3.27(d,J=5.3Hz,2H),3.19(d,J=5.3Hz,2H),3.07(dd, J=10.78,3.5Hz,2H),1.60(dd,J=13.4,7.9Hz,2H)1.39(s,9H),1.27(dd,J=13.5,6.3Hz,2H).

化合物7-6：Compound 7-6:

在裝有化合物7-5(14.5g，53.44mmol)的反應瓶中加入HCl在二噁烷中的溶液(4M，140mL)。將反應混合物在室溫下攪拌1.5小時。LC-MS顯示完全轉化。將反應混合物濃縮以得到粗化合物7-6(11g)，其無需進一步純化即可直接用於下一步。 A solution of HCl in dioxane (4M, 140 mL) was added to a reaction bottle containing compound 7-5 (14.5 g, 53.44 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to give crude compound 7-6 (11 g), which was used directly in the next step without further purification.

C9H17NO2質量計算為(游離鹼基)：171.1；實測：172.1[M-Boc+H]+，ESI。 Mass calculated for C 9 H 17 NO 2 (free base): 171.1; found: 172.1 [M-Boc+H] + , ESI.

化合物7-7：Compound 7-7:

將Fmoc-6-胺基己酸(18.38g，52.00mmol)、DIEA(16.80g，130.00mmol，22.64mL)、HOBT(8.43g，62.40mmol)和EDCI(11.96g，62.40mmol)在DCM(300mL)中的混合物在0℃攪拌30分鐘，然後加入化合物7-6(10.8g，52.00mmol)。將反應混合物攪拌過夜，允許其升溫至室溫。LC-MS顯示完全轉化。用DCM(300mL)稀釋反應混合物並用NaHCO3(飽和水溶液)和水洗滌。有機相濃縮並藉由矽膠管柱(0-10% MeOH在DCM中的溶液)純化以得到呈膠狀的化合物7-7(19.6g，產率為74%)。 A mixture of Fmoc-6-aminohexanoic acid (18.38 g, 52.00 mmol), DIEA (16.80 g, 130.00 mmol, 22.64 mL), HOBT (8.43 g, 62.40 mmol) and EDCI (11.96 g, 62.40 mmol) in DCM (300 mL) was stirred at 0°C for 30 minutes, and then compound 7-6 (10.8 g, 52.00 mmol) was added. The reaction mixture was stirred overnight and allowed to warm to room temperature. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (300 mL) and washed with NaHCO 3 (saturated aqueous solution) and water. The organic phase was concentrated and purified by silica gel column (0-10% MeOH in DCM) to give compound 7-7 (19.6 g, 74% yield) as a gel.

C30H38N2O5質量計算為：506.3；實測：507.3[M+H]+，ESI。 Mass calculated for C 30 H 38 N 2 O 5 : 506.3; found: 507.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.33(m,5H),4.55(s,2H),4.29(d,J=6.8Hz,2H),4.23-4.18(m,1H),3.28-3.21(m,6H),2.97-2.96(m,2H),2.61-2.53(m,2H),2.17(t,J=7.6Hz,2H),1.63-1.23(m,12H). 1 H NMR(400MHz, DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.33(m,5H),4.55(s,2H),4.29(d, J =6.8Hz,2H),4.23-4.18(m,1H),3.28-3.21(m,6H),2.97-2.96(m,2H),2.61-2.53(m,2H),2.17(t,J=7.6Hz,2H),1.63-1.23(m,12H).

化合物7-8：Compound 7-8:

在0℃中向化合物7-7(19g，37.50mmol)、N，N-二甲基吡啶-4-胺(458.17mg，3.75mmol)和DIEA(7.27g，56.25mmol，9.80mL)在DCM(590mL)中的溶液中分批加入DMTrCl(13.98g，41.25mmol)。將反應混合物攪拌過夜。LC-MS顯示部分轉化，形成了2種異構體和副產物二-DMTr。將反應混合物濃縮並藉由矽膠管柱純化(20-100% EA，含有0.2% TEA的PE溶液)以得到化合物7-8a和7-8b，為內型和外型異構體的混合物(17.9g，產率為47%)。 To a solution of compound 7-7 (19 g, 37.50 mmol), N,N-dimethylpyridin-4-amine (458.17 mg, 3.75 mmol) and DIEA (7.27 g, 56.25 mmol, 9.80 mL) in DCM (590 mL) was added DMTrCl (13.98 g, 41.25 mmol) in portions at 0°C. The reaction mixture was stirred overnight. LC-MS showed partial conversion, with the formation of 2 isomers and byproduct di-DMTr. The reaction mixture was concentrated and purified by silica gel column (20-100% EA, PE solution containing 0.2% TEA) to give compounds 7-8a and 7-8b as a mixture of endo and exo isomers (17.9 g, 47% yield).

藉由製備型HPLC(C18管柱，水/ACN)分離異構體以得到化合物7-8a(外型異構體，7.5g)和化合物7-8b(內型異構體，5.7g)。 The isomers were separated by preparative HPLC (C18 column, water/ACN) to give compound 7-8a (exo-isomer, 7.5 g) and compound 7-8b (endo-isomer, 5.7 g).

7-8a：7-8a:

C51H56N2O7質量計算為：808.4；實測：832.3[M+Na]+，ESI。 Mass calculated for C 51 H 56 N 2 O 7 : 808.4; found: 832.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.88(m,2H),7.69-7.67(m,2H),7.43-8.18(m,13H),6.88-6.85(m,4H),4.62(br,1H),4.37-4.28(m,2H),4.20(t,J=6.7Hz,1H),3.73(s,6H),3.47(dd,J=9.9,8.2Hz,1H),3.28(br,2H),3.11(td,J=12.0,4.3Hz,2H),2.99-2.91(m,4H),2.66-2.54(m,2H),2.19-2.06(m,2H),1.67(pent,J=6.8Hz,2H),1.51-1.17(m,10H). 1 H NMR (400 MHz, DMSO-d6)δ 7.90-7.88(m,2H),7.69-7.67(m,2H),7.43-8.18(m,13H),6.88-6.85(m,4H),4. 62(br,1H),4.37-4.28(m,2H),4.20(t,J=6.7Hz,1H),3.73(s,6H),3.47(dd,J=9. 9,8.2Hz,1H),3.28(br,2H),3.11(td,J=12.0,4.3Hz,2H),2.99-2.91(m,4H),2.6 6-2.54(m,2H),2.19-2.06(m,2H),1.67(pent,J=6.8Hz,2H),1.51-1.17(m,10H).

7-8b：7-8b:

C51H56N2O7質量計算為：808.4；實測：832.3[M+Na]+，ESI。 Mass calculated for C 51 H 56 N 2 O 7 : 808.4; found: 832.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.84(m,4H),7.44-7.20(m,13H),6.91-6.89(m,4H),6.66(br,1H),6.29(s,2H),3.74(s,6H),3.36-3.31(m,3H),3.22-3.12(m,3H),2.90(dd,J=12.7,6.5Hz,2H),2.86(s,2H),2.44-2.29(m,2H),2.15(t,J=7.1Hz,2H),1.64-1.18(m,12H). 1H NMR(400MHz,DMSO-d6)δ 7.90-7.84(m,4H),7.44-7.20(m,13H),6.91-6.89(m,4H),6.66(br,1H), 6.29(s,2H),3.74(s,6H),3.36-3.31(m,3H),3.22-3.12(m,3H),2.90(dd, J =12.7,6.5Hz,2H),2.86(s,2H),2.44-2.29(m,2H),2.15(t, J =7.1Hz,2H),1.64-1.18(m,12H).

Figure 113123276-A0202-12-0195-280
Figure 113123276-A0202-12-0195-280

化合物7-9a：Compound 7-9a:

將化合物7-8a(6.5g，8.03mmol)在MeOH(200mL)和哌啶(20mL)中的溶液，室溫下攪拌過夜，LC-MS顯示完全轉化。將反應混合物濃縮，並將殘餘物藉由矽膠管柱純化(20-30% MeOH，含有1% TEA的DCM溶液)以得到化合物7-9a(4.4g，產率為94%)。 A solution of compound 7-8a (6.5 g, 8.03 mmol) in MeOH (200 mL) and piperidine (20 mL) was stirred at room temperature overnight, and LC-MS showed complete conversion. The reaction mixture was concentrated, and the residue was purified by silica gel column (20-30% MeOH, DCM solution containing 1% TEA) to give compound 7-9a (4.4 g, yield 94%).

C36H46N2O5質量計算為：586.3；實測：587.3[M+H]+，ESI。 Mass calculated for C 36 H 46 N 2 O 5 : 586.3; found: 587.3 [M+H] + , ESI.

化合物7-10a：Compound 7-10a:

將化合物1-4(4.70g，10.50mmol)、DIEA(2.42g，18.75mmol，3.27mL)、HOBT(1.22g，9.00mmol)和EDCI(1.73g，9.00mmol)在DCM(132mL)中的溶液在0℃攪拌30分鐘，然後加入化合物7-9a(4.4g，7.50mmol)。將反應混合物攪拌過夜。LC-MS顯示完全轉化。將反應混合物用DCM(150mL)稀釋，用水洗滌。有機相濃縮並藉由矽膠管柱(含有1% TEA的0-10% MeOH在DCM中的溶液)純化以得到化合物7-10a(3.8g，產率為51%)。 A solution of compound 1-4 (4.70 g, 10.50 mmol), DIEA (2.42 g, 18.75 mmol, 3.27 mL), HOBT (1.22 g, 9.00 mmol) and EDCI (1.73 g, 9.00 mmol) in DCM (132 mL) was stirred at 0 °C for 30 minutes, and then compound 7-9a (4.4 g, 7.50 mmol) was added. The reaction mixture was stirred overnight. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column (0-10% MeOH in DCM containing 1% TEA) to give compound 7-10a (3.8 g, 51% yield).

C55H73N3O15質量計算為：1015.5；實測：1039.1[M+Na]+，ESI。 Mass calculated for C 55 H 73 N 3 O 15 : 1015.5; found: 1039.1 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.85(d,J=9.2Hz,1H),7.74(t,J=5.3Hz,1H),7.38-7.36(m,2H),7.31-7.27(m,2H),7.24-7.19(m,5H),6.88-6.86(m,4H),5.22(d,J=2.8Hz,1H),4.97(dd,J=11.2,2.8Hz,1H),4.65(t,J=4.8Hz,1H),4.49(d,J=8.4Hz,1H),4.03(s,3H),3.88(dd,J=19.5,9.1Hz,1H),3.73-3.70(m,7H),3.50-3.39(m,2H),3.34-3.31(m,1H),3.27(d,J=4.5Hz,2H),3.17(d,J=5.2Hz,1H),3.15-3.09(m,2H),3.00(dd,J=12.4,6.3Hz,2H),2.94(br,1H),2.67-2.53(m,2H),2.19-2.08(m,5H),2.04(t,J=6.9Hz,2H),2.00(s,3H),1.89(s,3H),1.78(s,3H),1.70-1.63(m,2H),1.53-1.16(m,14H). 1 H NMR (400MHz, DMSO-d6) δ 7.85 (d, J =9.2Hz, 1H), 7.74 (t, J =5.3Hz,1H),7.38-7.36(m,2H),7.31-7.27(m,2H),7.24-7.19(m,5H),6.88-6.86(m,4H),5.22(d, J =2.8Hz,1H),4.97(dd, J =11.2,2.8Hz,1H),4.65(t, J =4.8Hz,1H),4.49(d, J =8.4Hz,1H),4.03(s,3H),3.88(dd, J =19.5,9.1Hz,1H),3.73-3.70(m,7H),3.50-3.39(m,2H),3.34-3.31(m,1H),3.27(d, J =4.5Hz,2H),3.17(d, J =5.2Hz,1H),3.15-3.09(m,2H),3.00(dd, J =12.4,6.3Hz,2H),2.94(br,1H),2.67-2.53(m,2H),2.19-2.08(m,5H),2.04(t, J =6.9Hz,2H),2.00(s,3H),1.89(s,3H),1.78(s,3H),1.70-1.63(m,2H),1.53-1.16(m,14H).

化合物7-11a：Compound 7-11a:

在室溫中向化合物7-10a(680.0mg，0.67mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(96.0mg，0.81mmol)和2-氰乙基N,N,N’,N’-四異丙基亞磷二醯胺(324mg，1.08mmol)，攪拌1小時，LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。這產生了7-11a，為白色固體(390mg，產率為47%)。 To a solution of compound 7-10a (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) at room temperature, stirred for 1 hour, LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na 2 SO 4 . The solution was then concentrated under reduced pressure and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 in 20 minutes; detector: UV 254 nm). This gave 7-11a as a white solid (390 mg, 47% yield).

C64H90N5O16P質量計算為：1215.61；實測：1161.3[M-CH2CH2CN]-，ESI。 Mass calculated for C 64 H 90 N 5 O16 P : 1215.61; found: 1161.3 [M-CH 2 CH 2 CN] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.81-7.69(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d,J=6.0Hz,1H),4.98-4.94(m,1H),4.49(d,J=6.0Hz,1H),4.02-3.83(m,4H),3.73-3.32(m,18H),3.01-2.97(m,6H),2.71-2.68(m,4H),2.12-1.99(m,18H),1.49-1.04(m,24H). 1 HNMR(600MHz,DMSO- d 6 )δ 7.81-7.69(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d, J =6.0Hz,1H),4.98-4.94(m,1H),4.49(d, J =6.0Hz,1H),4.02-3.83(m,4H),3.73-3.32(m,18H),3.01-2.97(m,6H),2.71-2.68(m,4H),2.12-1.99(m,18H),1.49-1.04(m,24H).

31PNMR(242MHz，DMSO-d 6)δ 146.70. 31 PNMR (242MHz, DMSO- d 6 ) δ 146.70.

Figure 113123276-A0202-12-0197-281
Figure 113123276-A0202-12-0197-281

化合物7-9b：Compound 7-9b:

將化合物7-8b(5.70g，7.05mmol)在MeOH(200mL)和哌啶(20mL)中的溶液在室溫下攪拌過夜。LC-MS顯示完全轉化。將反應混合物濃縮並藉由矽膠管柱純化(10-20% EA，含有1% TEA的DCM溶液)以得到化合物7-9b(4.1g，產率為99%)。 A solution of compound 7-8b (5.70 g, 7.05 mmol) in MeOH (200 mL) and piperidine (20 mL) was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by silica gel column (10-20% EA, DCM solution containing 1% TEA) to give compound 7-9b (4.1 g, yield 99%).

C36H46N2O5質量計算為：586.3；實測：587.3[M+H]+，ESI。 Mass calculated for C 36 H 46 N 2 O 5 : 586.3; found: 587.3 [M+H] + , ESI.

化合物7-10b：Compound 7-10b:

將化合物1-4(4.38g，9.78mmol)、DIEA(2.26g，17.47mmol，3.04mL)、HOBT(1.13g，8.39mmol)和EDCI(1.61g，8.39mmol)在DCM(120mL)中的溶液在0℃攪拌30分鐘，然後加入化合物7-9b(4.10g，6.99mmol)。將反應混合物攪拌過夜。LC-MS顯示完全轉化。將反應混合物用DCM(150mL)稀釋，用水洗滌。有機相濃縮並藉由矽膠管柱(含有1% TEA的0-10% MeOH在DCM中的溶液)純化以得到粗產物4.4g，藉由製備型HPLC(C18，水/ACN)進一步純化以得到化合物7-10b(2.0g，產率為28%)。 A solution of compound 1-4 (4.38 g, 9.78 mmol), DIEA (2.26 g, 17.47 mmol, 3.04 mL), HOBT (1.13 g, 8.39 mmol) and EDCI (1.61 g, 8.39 mmol) in DCM (120 mL) was stirred at 0 °C for 30 minutes, and then compound 7-9b (4.10 g, 6.99 mmol) was added. The reaction mixture was stirred overnight. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column (0-10% MeOH in DCM containing 1% TEA) to give a crude product 4.4 g, which was further purified by preparative HPLC (C18, water/ACN) to give compound 7-10b (2.0 g, 28% yield).

C55H73N3O15質量計算為：1015.5；實測：1039.1[M+Na]+，ESI。 Mass calculated for C 55 H 73 N 3 O 15 : 1015.5; found: 1039.1 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84(d,J=9.2Hz,1H),7.72(t,J=5.5Hz,1H),7.40-7.38(m,2H),7.33-7.29(m,2H),7.28-7.20(m,5H),6.91-6.88(m, 4H),5.22(d,J=3.3Hz,1H),4.97(dd,J=11.2,3.3Hz,1H),4.66(t,J=4.9Hz,1H),4.49(d,J=8.5Hz,1H),4.03(s,3H),3.88(dd,J=20.2,8.8Hz,1H),3.74-3.69(m,7H),3.48-3.38(m,4H),3.34-3.31(m,1H),3.21(dd,J=10.4,4.2Hz,1H),3.14(dd,J=12.0,4.6Hz,1H),3.00(dd,J=12.7,6.6Hz,2H),2.86(s,2H),2.46-2.27(m,2H),2.16(t,J=6.7Hz,2H),2.11(s,3H),2.04(t,J=7.0Hz,2H),2.00(s,3H),1.89(s,3H),1.78(s,3H),1.63-1.12(m,14H). 1 H NMR(400MHz, DMSO-d6)δ 7.84(d,J=9.2Hz,1H),7.72(t,J=5.5Hz,1H),7.40-7.38(m,2H),7.33-7.29(m,2H),7.28-7.20(m,5H),6.91-6.88(m, 4H),5.22(d,J=3.3Hz,1H),4.97(dd,J=11.2,3.3Hz,1H),4.66(t,J=4.9Hz,1H),4.49(d,J=8.5Hz,1H),4.03(s ,3H),3.88(dd,J=20.2,8.8Hz,1H),3.74-3.69(m,7H),3.48-3.38(m,4H),3.34-3.31(m,1H),3.21(dd,J=10.4, 4.2Hz,1H),3.14(dd,J=12.0,4.6Hz,1H),3.00(dd,J=12.7,6.6Hz,2H),2.86(s,2H),2.46-2.27(m,2H),2.16(t ,J=6.7Hz,2H),2.11(s,3H),2.04(t,J=7.0Hz,2H),2.00(s,3H),1.89(s,3H),1.78(s,3H),1.63-1.12(m,14H).

化合物7-11b：Compound 7-11b:

在室溫中向化合物7-10b(680.0mg，0.67mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(96.0mg，0.81mmol)和2-氰乙基N,N,N’,N’-四異丙基磷二醯胺(324mg，1.08mmol)，攪拌1小時。LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物7-11b，為白色固體(390mg，產率為47%)。 To a solution of compound 7-10b (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N , N , N' , N' -tetraisopropylphosphodiamidamide (324 mg, 1.08 mmol) at room temperature and stirred for 1 hour. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3 ), and dried over Na2SO4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 7-11b was produced as a white solid (390 mg, yield 47%).

C64H90N5O16P質量計算為：1215.61；實測：1161.6[M-CH2CH2CN]-，ESI。 Mass calculated for C 64 H 90 N 5 O 16 P: 1215.61; found: 1161.6 [M-CH 2 CH 2 CN] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.81-7.69(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d,J=6.0Hz,1H),4.98-4.94(m,1H),4.49(d,J=6.0Hz,1H),4.02-3.83(m,4H),3.73-3.32(m,18H),3.01-2.97(m,6H),2.71-2.68(m,4H),2.12-1.99(m,18H),1.49-1.04(m,24H). 1 HNMR(600MHz,DMSO- d 6 )δ 7.81-7.69(m,2H),7.40-7.19(m,9H),6.88-6.86(m,4H),5.21(d, J =6.0Hz,1H),4.98-4.94(m,1H),4.49(d, J =6.0Hz,1H),4.02-3.83(m,4H),3.73-3.32(m,18H),3.01-2.97(m,6H),2.71-2.68(m,4H),2.12-1.99(m,18H),1.49-1.04(m,24H).

31PNMR(242MHz，DMSO-d 6)δ 146.22. 31 PNMR(242MHz, DMSO- d 6 )δ 146.22.

Figure 113123276-A0202-12-0199-282
Figure 113123276-A0202-12-0199-282

化合物8-2：Compound 8-2:

在0℃下向甲氧基甲基(三苯基)氯化磷(17.71g，51.67mmol)在THF(120mL)中的溶液中加入KOtBu(9.66g，86.12mmol)並攪拌0.5小時。然後在0℃下加入化合物8-1(9.7g，43.06mmol)，並攪拌過夜，使反應升溫至室溫。LC-MS顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，過濾並濃縮至乾。殘餘物藉由矽膠管柱(0-5% EA在PE中的溶液)純化，以得到呈油狀的化合物8-2(6.0g，產率為55%)。 To a solution of methoxymethyl (triphenyl) phosphonium chloride (17.71 g, 51.67 mmol) in THF (120 mL) was added KOtBu (9.66 g, 86.12 mmol) at 0°C and stirred for 0.5 hours. Compound 8-1 (9.7 g, 43.06 mmol) was then added at 0°C and stirred overnight, allowing the reaction to warm to room temperature. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered and concentrated to dryness. The residue was purified by silica gel column (0-5% EA in PE) to give compound 8-2 (6.0 g, 55% yield) as an oil.

1H NMR(400MHz,DMSO-d6)δ 6.01(s,1H),3.48(s,3H),3.43-3.33(m,2H),2.82-2.74(m,2H),2.35-2.26(m,2H),1.90-1.79(m,4H),1.38(s,9H). 1 H NMR (400MHz, DMSO-d6)δ 6.01(s,1H),3.48(s,3H),3.43-3.33(m,2H),2.82-2.74(m,2H),2.35-2.26(m,2H),1.90-1.79(m,4H),1.38(s,9H).

化合物8-3：Compound 8-3:

向化合物8-2(5.85g，23.09mmol)在THF(50mL)中的溶液中加入鹽酸(3.28g，90.00mmol，2N，45mL)。將反應混合物在室溫下攪拌3小時。TLC顯示完全轉化。藉由加入NaHCO3(飽和水溶液)將反應混合物調節至pH7，並用EA萃取。濃縮有機相得到粗化合物8-3，為油狀物(5.5g)，無需進一步純化即可直接用於下一步。 To a solution of compound 8-2 (5.85 g, 23.09 mmol) in THF (50 mL) was added hydrochloric acid (3.28 g, 90.00 mmol, 2N, 45 mL). The reaction mixture was stirred at room temperature for 3 hours. TLC showed complete conversion. The reaction mixture was adjusted to pH 7 by adding NaHCO 3 (saturated aqueous solution) and extracted with EA. The organic phase was concentrated to give crude compound 8-3 as an oil (5.5 g), which was used directly in the next step without further purification.

1H NMR(400MHz,DMSO-d6)δ 9.62(pent,J=1.2Hz,1H),3.42-3.33(m,3H),2.82-2.72(m,2H),2.03-1.93(m,1H),1.92-1.87(m,2H),1.75-1.62(m,1H),1.51-1.44(m,1H),1.38(s,9H). 1H NMR(400MHz,DMSO-d6)δ 9.62(pent, J =1.2Hz,1H),3.42-3.33(m,3H),2.82-2.72(m,2H),2.03-1.93(m,1H),1.92-1.87(m,2H),1.75-1.62(m,1H),1.51-1.44(m,1H),1.38(s,9H).

化合物8-4：Compound 8-4:

向化合物8-3(5.25g，21.94mmol)和K2CO3(660.39mg，4.39mmol)在MeOH(30mL)中的混合物中加入福爾馬林(6.59g，37%水溶液，219.38mmol)。將反應混合物在室溫攪拌過夜。TLC顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，並用EA萃取。濃縮有機相得到化合物8-4(6.7g)，其無需進一步純化即可直接用於下一步。 To a mixture of compound 8-3 (5.25 g, 21.94 mmol) and K 2 CO 3 (660.39 mg, 4.39 mmol) in MeOH (30 mL) was added formalin (6.59 g, 37% aqueous solution, 219.38 mmol). The reaction mixture was stirred at room temperature overnight. TLC showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with EA. The organic phase was concentrated to give compound 8-4 (6.7 g), which was used directly in the next step without further purification.

化合物8-5：Compound 8-5:

向化合物8-4(6.3g，23.39mmol)在MeOH(50mL)中的溶液中加入NaBH4(2.65g，70.17mmol)。將反應混合物在室溫下攪拌1小時。LC-MS顯示完全轉化。藉由加入檸檬酸(10%水溶液)將反應混合物調節至pH6，過濾，並用DCM萃取。有機相濃縮並藉由矽膠管柱(0-5% MeOH在DCM中的溶液)純化以得到化合物8-5(1.4g，產率為22%)。 To a solution of compound 8-4 (6.3 g, 23.39 mmol) in MeOH (50 mL) was added NaBH 4 (2.65 g, 70.17 mmol). The reaction mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and extracted with DCM. The organic phase was concentrated and purified by silica gel column (0-5% MeOH in DCM) to give compound 8-5 (1.4 g, 22% yield).

C14H25NO4質量計算為：271.18；實測：257.24[M-tBu+CH3CN+H]+，ESI。 Mass calculated for C 14 H 25 NO 4 : 271.18; found: 257.24 [M-tBu+CH 3 CN+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.16-7.55(m,1H),4.52-4.48(m,2H),3.36-3.27(m,5H),2.74-2.50(m,2H),1.98-1.84(m 2H),1.54-1.50(m 2H),1.49(s,9H),1.05-0.97(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.16-7.55(m,1H),4.52-4.48(m,2H),3.36-3.27(m,5H),2.74-2.50(m,2H),1.98-1.84(m 2H),1.54-1.50(m 2H),1.49(s,9H),1.05-0.97(m,2H).

化合物8-6：Compound 8-6:

將化合物8-5(1.35g，4.97mmol)溶解於DCM(5mL)中，室溫下向混合物中加入HCl/二噁烷(4M，10mL)，室溫下攪拌2小時。將反應混合物在真空中濃縮以得到粗產物8-6(1.3g)，為油狀物，無需進一步純化即可用於下一步。 Compound 8-5 (1.35 g, 4.97 mmol) was dissolved in DCM (5 mL), and HCl/dioxane (4 M, 10 mL) was added to the mixture at room temperature, and stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to give the crude product 8-6 (1.3 g) as an oil, which was used in the next step without further purification.

C9H17NO2質量計算為：171.1；實測：172.2[M+H]+，ESI。 Mass calculated for C 9 H 17 NO 2 : 171.1; found: 172.2 [M+H] + , ESI.

化合物8-7：Compound 8-7:

將Z-6-胺基己酸(1.99g，7.51mmol)、HATU(4.72g，12.52mmol)和DIPEA(2.43g，18.78mmol，3.27mL)溶解於DCM(20mL)中。將反應攪拌20分鐘，然後加入化合物8-6(1.3g，6.26mmol)，再攪拌2小時。反應混合物用H2O(20mL)淬滅，用飽和NaHCO3水溶液(30mL)洗滌，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(DCM：MeOH=20：1)在矽膠管柱上純化，以得到化合物8-7，為白色泡沫狀固體(1.5g，3.58mmol，產率為57%)。C23H34N2O5質量計算為：418.3；實測：419.3[M+H]+，ESI。 Z-6-aminohexanoic acid (1.99 g, 7.51 mmol), HATU (4.72 g, 12.52 mmol) and DIPEA (2.43 g, 18.78 mmol, 3.27 mL) were dissolved in DCM (20 mL). The reaction was stirred for 20 minutes, then compound 8-6 (1.3 g, 6.26 mmol) was added and stirred for another 2 hours. The reaction mixture was quenched with H 2 O (20 mL), washed with saturated aqueous NaHCO 3 solution (30 mL), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (DCM: MeOH = 20: 1) on a silica gel column to give compound 8-7 as a white foamy solid (1.5 g, 3.58 mmol, yield 57%). Mass calculated for C 23 H 34 N 2 O 5 : 418.3; found: 419.3 [M+H] + , ESI.

化合物8-8：Compound 8-8:

將化合物8-7(1.2g，2.87mmol)溶解於DCM(20mL)中。加入DMTrCl(971.49mg，2.87mmol)和DIPEA(741.12mg，5.73mmol，998.81μL)。將反應混合物在室溫下攪拌2小時。然後用水淬滅反應，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌。收集有機相，乾燥，真空濃縮，並藉由快速管柱層析(DCM：MeOH：TEA=20：1：0.5%)在Al2O3上純化以得到化合物8-8，為白色泡沫狀固體(850mg，產率為41%)。 Compound 8-7 (1.2 g, 2.87 mmol) was dissolved in DCM (20 mL). DMTrCl (971.49 mg, 2.87 mmol) and DIPEA (741.12 mg, 5.73 mmol, 998.81 μL) were added. The reaction mixture was stirred at room temperature for 2 hours. The reaction was then quenched with water, extracted with DCM (50 mL x 2), and washed with brine (50 mL). The organic phase was collected, dried, concentrated in vacuo, and purified by flash column chromatography (DCM: MeOH: TEA = 20: 1: 0.5%) on Al 2 O 3 to obtain compound 8-8 as a white foamy solid (850 mg, 41% yield).

C44H52N2O7質量計算為：720.4；實測：721.4[M+H]+，ESI。 Mass calculated for C 44 H 52 N 2 O 7 : 720.4; found: 721.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.41-7.36(m,3H),7.35-7.29(m,6H),7.27-7.20(m,5H),6.89(d,J=8.9Hz,4H),5.00(s,2H),4.67(t,J=4.9Hz,1H), 3.75(s,6H),3.55-3.47(m,2H),3.39(d,J=4.5Hz,2H),3.00-2.95(m,4H),2.92-2.84(m,1H),2.62(q,J=11Hz,1H),2.16-2.10(m,2H),1.95-1.70(m,2H),1.62-1.58(m,1H),1.51-1.35(m,5H),1.27-1.23(m,3H),1.12-1.00(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.41-7.36(m,3H),7.35-7.29(m,6H),7.27-7.20(m,5H),6.89(d, J =8.9Hz,4H),5.00(s,2H),4.67(t, J =4.9Hz,1H), 3.75(s,6H),3.55-3.47(m,2H),3.39(d, J =4.5Hz,2H),3.00-2.95(m,4H),2.92-2.84(m,1H),2.62(q, J =11Hz,1H),2.16-2.10(m,2H),1.95-1.70(m,2H),1.62-1.58(m,1H),1.51-1.35(m,5H),1.27-1.23(m,3H),1.12-1.00(m,2H).

化合物8-9：Compounds 8-9:

將化合物8-8(0.8g，1.11mmol)溶解於MeOH(10mL)中。加入Pd/C(80mg)。將反應混合在氫氣氛圍下，室溫下攪拌3小時。將反應混合物過濾並在真空中濃縮以得到粗產物8-9，為白色泡沫狀固體(550mg，產率為84%)，其無需進一步純化即可用於下一步。 Compound 8-8 (0.8 g, 1.11 mmol) was dissolved in MeOH (10 mL). Pd/C (80 mg) was added. The reaction mixture was stirred under hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered and concentrated in vacuo to give the crude product 8-9 as a white foamy solid (550 mg, 84% yield), which was used in the next step without further purification.

C36H46N2O5質量計算為：586.3；實測：587.3[M+H]+，ESI。 Mass calculated for C 36 H 46 N 2 O 5 : 586.3; found: 587.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.40-7.38(m,2H),7.31(t,J=7.7Hz,2H),7.29-7.18(m,5H),6.89(d,J=8.9Hz,4H),3.74(s,6H),3.54-3.43(m,2H),3.34-3.21(m,2H),2.96(s,2H),2.94-2.85(m,2H),2.67-2.55(m,1H),2.17-2.10(m,2H),1.99-1.74(m,2H),1.63-1.58(m,1H),1.53-1.42(m,3H),1.36-1.24(m,5H),1.16-1.10(m,1H),1.05-0.98(m,1H). 1 H NMR(400MHz, DMSO-d6)δ 7.40-7.38(m,2H),7.31(t, J =7.7Hz,2H),7.29-7.18(m,5H),6.89(d, J =8.9Hz,4H),3.74(s,6H),3.54-3.43(m,2H),3.34-3.21(m,2H),2.96(s,2H),2.94-2.85(m,2H),2.67-2.55(m,1H),2.17-2. 10(m,2H),1.99-1.74(m,2H),1.63-1.58(m,1H),1.53-1.42(m,3H),1.36-1.24(m,5H),1.16-1.10(m,1H),1.05-0.98(m,1H).

Figure 113123276-A0202-12-0202-283
Figure 113123276-A0202-12-0202-283

化合物9-2：Compound 9-2:

將甲氧基甲基(三苯基)氯化磷(116.8g，340.72mmol)溶於THF(1200mL)中。在0℃下將KOtBu(46.74g，416.56mmol)加入混合物中。將反應混合物在0℃下再攪拌30分鐘。將化合物9-1(40g，189.34mmol)加入混合物中。將混合物加熱至室溫並在室溫攪拌過夜。用飽和檸檬酸淬滅反應並將pH調節至6-7。用EA(500mL x3)萃取反應混合物，用鹽水(200mL)洗滌，用Na2SO4乾燥，真空濃縮，藉由矽膠快速管柱層析(10-30% EA在PE中的溶液)純化，得到化合物9-2，為油狀物(24g，產率為52%)。 Methoxymethyl (triphenyl) phosphonium chloride (116.8 g, 340.72 mmol) was dissolved in THF (1200 mL). KOtBu (46.74 g, 416.56 mmol) was added to the mixture at 0°C. The reaction mixture was stirred for another 30 minutes at 0°C. Compound 9-1 (40 g, 189.34 mmol) was added to the mixture. The mixture was heated to room temperature and stirred at room temperature overnight. The reaction was quenched with saturated citric acid and the pH was adjusted to 6-7. The reaction mixture was extracted with EA (500 mL x 3), washed with brine (200 mL), dried over Na 2 SO 4 , concentrated in vacuo, and purified by silica gel flash column chromatography (10-30% EA in PE) to give compound 9-2 as an oil (24 g, yield 52%).

C13H21NO3質量計算為：239.3；實測：225.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 13 H 21 NO 3 : 239.3; found: 225.1 [M-tBu+ACN+H] + , ESI.

1H NmR(400MHz,DMSO-d6)δ 5.88(t,J=2.4Hz,1H),3.83(s,4H),3.47(s,3H),2.79-2.74(m,4H),1.41(s,9H). 1 H NmR(400MHz,DMSO-d6)δ 5.88(t, J =2.4Hz,1H),3.83(s,4H),3.47(s,3H),2.79-2.74(m,4H),1.41(s,9H).

化合物9-3：Compound 9-3:

將化合物9-2(15g，62.68mmol)溶解於ACN(450mL)中。將2,2,2-三氯乙酸(12.90g，78.98mmol)在水中(150mL)的溶液加入到反應混合物中。將反應在室溫攪拌過夜，然後用飽和NaHCO3淬滅反應並將pH調節至7-8。用EA(200mLx3)萃取混合物，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮以得到粗產物9-3，為油狀物(13g，產率為92%)，無需進一步純化即可用於下一步。 Compound 9-2 (15 g, 62.68 mmol) was dissolved in ACN (450 mL). A solution of 2,2,2-trichloroacetic acid (12.90 g, 78.98 mmol) in water (150 mL) was added to the reaction mixture. The reaction was stirred at room temperature overnight, then quenched with saturated NaHCO 3 and the pH was adjusted to 7-8. The mixture was extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na 2 SO 4 , and concentrated in vacuo to give the crude product 9-3 as an oil (13 g, 92% yield), which was used in the next step without further purification.

C12H19NO3質量計算為：225.3；實測：211.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 12 H 19 NO 3 : 225.3; found: 211.1 [M-tBu+ACN+H] + , ESI.

化合物9-4：Compound 9-4:

將化合物9-3(13g，57.71mmol)溶解於MeOH(400mL)中。向混合物中加入K2CO3(1.60g，11.54mmol)和甲醛(4.92g，163.88mmol，在水中的溶液)。將反 應在室溫攪拌過夜，然後用飽和檸檬酸淬滅反應並將pH調節至6-7。將混合物在真空中濃縮以除去MeOH。用EA(200mLx3)萃取殘餘物，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮以得到粗產物9-4(14g，產率為95%)。 Compound 9-3 (13 g, 57.71 mmol) was dissolved in MeOH (400 mL). K 2 CO 3 (1.60 g, 11.54 mmol) and formaldehyde (4.92 g, 163.88 mmol, solution in water) were added to the mixture. The reaction was stirred at room temperature overnight, then quenched with saturated citric acid and the pH was adjusted to 6-7. The mixture was concentrated in vacuo to remove MeOH. The residue was extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na 2 SO 4 , and concentrated in vacuo to give crude product 9-4 (14 g, 95% yield).

C13H21NO4質量計算為：255.3；實測：241.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 13 H 21 NO 4 : 255.3; found: 241.1 [M-tBu+ACN+H] + , ESI.

化合物9-5：Compound 9-5:

將化合物9-4(14g，54.84mmol)溶解於MeOH(420mL)中。分批加入NaBH4(4.56g，120.64mmol)。將反應在室溫攪拌1小時，然後用水將反應淬滅。用飽和檸檬酸調節PH至6-7。將混合物在真空下濃縮以除去MeOH，然後用DCM(200mLx3)萃取殘餘物，用鹽水(100mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-5% MeOH)在矽膠上純化，以得到化合物9-5(7.05g，產率為50%)。 Compound 9-4 (14 g, 54.84 mmol) was dissolved in MeOH (420 mL). NaBH4 (4.56 g, 120.64 mmol) was added in batches. The reaction was stirred at room temperature for 1 hour and then quenched with water. The pH was adjusted to 6-7 with saturated citric acid. The mixture was concentrated under vacuum to remove MeOH, and the residue was extracted with DCM (200 mL x 3 ), washed with brine (100 mL), dried with Na2SO4 , concentrated in vacuum and purified on silica gel by flash column chromatography (0-5% MeOH) to give compound 9-5 (7.05 g, 50% yield).

C13H23NO4質量計算為：257.3；實測：243.1[M-tBu+ACN+H]+，ESI。 Mass calculated for C 13 H 23 NO 4 : 257.3; found: 243.1 [M-tBu+ACN+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.51-4.11(m,2H),3.83-3.75(m,4H),3.25-3.17(m,4H),2.01-1.91(m,4H),1.36(s,9H). 1 H NMR(400MHz, DMSO-d6)δ 4.51-4.11(m,2H),3.83-3.75(m,4H),3.25-3.17(m,4H),2.01-1.91(m,4H),1.36(s,9H).

化合物9-6：Compound 9-6:

將化合物9-5(13.1g，50.91mmol)溶解於HCl在1,4-二噁烷(4M，131mL)的溶液中。將反應混合物在室溫攪拌1小時，然後在真空下濃縮以得到粗產物9-6(9.8g)，無需進一步純化即可用於下一步。 Compound 9-5 (13.1 g, 50.91 mmol) was dissolved in a solution of HCl in 1,4-dioxane (4 M, 131 mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under vacuum to give the crude product 9-6 (9.8 g), which was used in the next step without further purification.

C8H13NO2Cl質量計算為：157.2；實測：158.2[M+H]+，ESI。 Mass calculated for C 8 H 13 NO 2 Cl: 157.2; found: 158.2 [M+H] + , ESI.

化合物9-7：Compound 9-7:

將化合物9-6(9.8g，50.60mmol)溶解於DCM(300mL)中。將DIEA(22.89g，177.10mmol，30.85mL)、HOBT(8.20g，60.72mmol)、EDCI(11.64g，60.72mmol)和Fmoc-6-己酸(17.88g，50.60mmol)加入反應混合物中。將反應在室溫攪拌過夜，然後用水淬滅反應，用10%檸檬酸(50mL)、飽和NaHCO3溶液(50mL)、鹽水(50mL)洗滌，Na2SO4乾燥，真空濃縮，快速管柱層析(0-10% MeOH)在矽膠上純化，以得到化合物9-7(6.7g，兩步產率為25%)。 Compound 9-6 (9.8 g, 50.60 mmol) was dissolved in DCM (300 mL). DIEA (22.89 g, 177.10 mmol, 30.85 mL), HOBT (8.20 g, 60.72 mmol), EDCI (11.64 g, 60.72 mmol) and Fmoc-6-hexanoic acid (17.88 g, 50.60 mmol) were added to the reaction mixture. The reaction was stirred at room temperature overnight, then quenched with water, washed with 10% citric acid (50 mL), saturated NaHCO 3 solution (50 mL), brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo, and purified by flash column chromatography (0-10% MeOH) on silica gel to give compound 9-7 (6.7 g, two-step yield 25%).

C30H36N2O5質量計算為：492.3；實測：493.3[M+H]+，ESI。 Mass calculated for C 30 H 36 N 2 O 5 : 492.3; found: 493.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,4H),6.76(s,1H),6.68-6.66(m,1H),6.29(s,2H),4.01(s,2H),3.74(s,2H),3.69(s,2H),3.27(s,4H),2.92-2.87(m,2H),1.98-1.91(m,4H),1.46-1.18(m,6H). 1H NMR(400MHz,DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,4H),6.76(s,1H),6.68-6.66(m,1H),6.29(s,2H),4.01(s,2H), 3.74(s,2H),3.69(s,2H),3.27(s,4H),2.92-2.87(m,2H),1.98-1.91(m,4H),1.46-1.18(m,6H).

化合物9-8：Compound 9-8:

將化合物9-7(6.7g，13.60mmol)、DMAP(166.15mg，1.36mmol)和DIEA(2.64g，20.40mmol，3.18mL)溶於DCM(180mL)。將DMTrCl(5.52g，14.96mmol)在0℃分批加入到反應混合物中。將反應在室溫攪拌過夜，然後用水淬滅反應。用DCM(50mLx2)萃取混合物，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(50-100% EA)在Al2O3上純化以得到化合物9-8(4.3g，產率為40%)。 Compound 9-7 (6.7 g, 13.60 mmol), DMAP (166.15 mg, 1.36 mmol) and DIEA (2.64 g, 20.40 mmol, 3.18 mL) were dissolved in DCM (180 mL). DMTrCl (5.52 g, 14.96 mmol) was added to the reaction mixture in portions at 0 °C. The reaction was stirred at room temperature overnight and then quenched with water. The mixture was extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (50-100% EA) on Al 2 O 3 to give compound 9-8 (4.3 g, 40% yield).

C51H56N2O8質量計算為：824.4；實測：848.3[M+Na]+，ESI。 Mass calculated for C 51 H 56 N 2 O 8 : 824.4; found: 848.3 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,4H),7.75-7.71(m,6H),6.90-6.88(m,6H),6.67-6.65(m,1H),6.29(s,2H),3.96(s,1H),3.74(s,9H),3.69-3.67(m,2H),3.53(s,1H),3.39-3.37(m,2H),2.92-2.89(m,4H),2.03-1.79(m,8H),1.46-1.18(m,6H). 1H NMR(400MHz,DMSO-d6)δ 7.91-7.68(m,4H),7.44-7.28(m,4H),7.75-7.71(m,6H),6.90-6.88(m,6H),6.67-6.65(m,1H),6.29(s,2H),3.96(s,1H), 3.74(s,9H),3.69-3.67(m,2H),3.53(s,1H),3.39-3.37(m,2H),2.92-2.89(m,4H),2.03-1.79(m,8H),1.46-1.18(m,6H).

化合物9-9：Compound 9-9:

將化合物9-8(4.2g，5.09mmol)溶解於MeOH(126mL)中，然後添加哌啶(12.6ml)到混合物中。將反應混合物在室溫攪拌過夜，然後將反應混合物在真空濃縮，藉由快速管柱層析(0-10% MeOH)在Al2O3上純化以得到化合物9-9，為白色泡沫(3g，產率為97%)。 Compound 9-8 (4.2 g, 5.09 mmol) was dissolved in MeOH (126 mL), and then piperidine (12.6 ml) was added to the mixture. The reaction mixture was stirred at room temperature overnight, and then the reaction mixture was concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on Al 2 O 3 to give compound 9-9 as a white foam (3 g, yield 97%).

C36H46N2O6質量計算為：602.3；實測：603.4[M+H]+，ESI。 Mass calculated for C 36 H 46 N 2 O 6 : 602.3; found: 603.4 [M+H] + , ESI.

化合物9-10：Compounds 9-10:

將化合物1-4(3.2g，7.16mmol)和化合物9-9(3g，5.11mmol)溶於DCM(130mL)中。將DIEA(1.65g，12.78mmol，2.17mL)、HOBT(807.01mg，6.13mmol)和EDCI(1.18g，6.13mmol)加入到混合物中。反應在室溫攪拌過夜，然後將反應用水(40mL)淬滅，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由HPLC純化以得到化合物9-10(610mg，產率為12%)。 Compound 1-4 (3.2 g, 7.16 mmol) and compound 9-9 (3 g, 5.11 mmol) were dissolved in DCM (130 mL). DIEA (1.65 g, 12.78 mmol, 2.17 mL), HOBT (807.01 mg, 6.13 mmol) and EDCI (1.18 g, 6.13 mmol) were added to the mixture. The reaction was stirred at room temperature overnight, then the reaction was quenched with water (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by HPLC to give compound 9-10 (610 mg, 12% yield).

C55H73N3O16質量計算為：1031.5；實測：1054.5[M+Na]+，ESI。 Mass calculated for C 55 H 73 N 3 O 16 : 1031.5; found: 1054.5 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.82-7.73(m,2H),7.24-6.88(m,12H),5.22(s,1H),4.97(d,J=10.8Hz,1H),4.64-4.48(m,2H),4.05-3.85(m,4H),3.71-3.69(m,12H),3.52(s,1H),3.39-3.38(m,1H),3.04-2.91(m,4H),2.11-1.78(m,19H),1.46-1.18(m,14H). 1 H NMR(400MHz,DMSO-d6)δ 7.82-7.73(m,2H),7.24-6.88(m,12H),5.22(s,1H),4.97(d, J =10.8Hz,1H),4.64-4.48(m,2H),4.05-3.85(m,4H),3.71-3.69(m,12H),3.52(s,1 H),3.39-3.38(m,1H),3.04-2.91(m,4H),2.11-1.78(m,19H),1.46-1.18(m,14H).

Figure 113123276-A0202-12-0206-284
Figure 113123276-A0202-12-0206-284

化合物9-11：Compound 9-11:

在室溫中向化合物9-10(1.0g，0.97mmol)在無水DCM(3.0mL)中的溶液中加入4,5-二氰基咪唑(32.0mg，0.27mmol)和2-氰乙基N,N,N’,N’-四異丙基磷二醯胺(108mg，0.36mmol)，攪拌1小時。LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱：C18矽膠；流動相：CH3CN/H2O=1/1，20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物9-11，為白色固體(680mg，產率為56%)。 To a solution of compound 9-10 (1.0 g, 0.97 mmol) in anhydrous DCM (3.0 mL) was added 4,5-dicyanoimidazole (32.0 mg, 0.27 mmol) and 2-cyanoethyl N , N , N' , N' -tetraisopropylphosphodiamidamide (108 mg, 0.36 mmol) at room temperature and stirred for 1 hour. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3 ), and dried over Na2SO4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1, increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 9-11 was produced as a white solid (680 mg, yield 56%).

C64H90N5O17P質量計算為：1231.61；實測：1177.1[M-CH2CH2CN]-，ESI。 Mass calculated for C 64 H 90 N 5 O 17 P: 1231.61; found: 1177.1 [M-CH2CH2CN] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.81-7.67(m,2H),7.26-7.23(m,6H),6.88-6.86(m,6H),5.22(d,J=6.0Hz,1H),4.98-4.95(m,1H),4.48(d,J=12.0Hz,1H),4.04-3.96(m,4H),3.89-3.84(m,1H),3.73-3.38(m,20H),3.01-2.93(m,4H),2.73-2.71(m,2H),2.10-1.75(m,20H),1.51-1.07(m,22H). 1 HNMR(600MHz,DMSO- d 6 )δ 7.81-7.67(m,2H),7.26-7.23(m,6H),6.88-6.86(m,6H),5.22(d, J =6.0Hz,1H),4.98-4.95(m,1H),4.48(d, J =12.0Hz,1H),4.04-3.96(m,4H),3.89-3.84(m,1H),3.73-3.38(m,20H),3. 01-2.93(m,4H),2.73-2.71(m,2H),2.10-1.75(m,20H),1.51-1.07(m,22H).

31PNMR(242MHz,DMSO-d 6)δ 146.82,146.70. 31 PNMR(242MHz,DMSO -d6 ) δ 146.82,146.70.

化合物9-12：Compounds 9-12:

向化合物9-10(100mg，0.097mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(5mg，0.04mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗。將反應混合物用DCM(10mL)稀釋，用H2O(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物9-12，為白色固體(85mg，產率為90%)。 To a solution of compound 9-10 (100 mg, 0.097 mmol) in anhydrous DCM (1.0 mL) was added DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H 2 O (3 mL×4), and then with brine (3 mL×4), and the organic layer was concentrated to give compound 9-12 as a white solid (85 mg, 90% yield).

C59H77N3O19質量計算為：1131.52；實測：1130.2[M-H]-，ESI。 Mass calculated for C 59 H 77 N 3 O 19 : 1131.52; found: 1130.2 [MH] - , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.87-7.72(m,2H),7.24-7.21(m,6H),6.90-6.87(m,6H),5.21(d,J=6.0Hz,1H),4.98-4.95(m,1H),4.49(d,J=12.0Hz,1H),4.06-3.98(m,6H),3.90-3.83(m,1H),3.73-3.50(m,13H),3.02-2.94(m,4H),2.45-2.44(m,4H),2.11-1.76(m,20H),1.48-1.18(m,12H). 1 HNMR(600MHz,DMSO- d 6 )δ 7.87-7.72(m,2H),7.24-7.21(m,6H),6.90-6.87(m,6H),5.21(d, J =6.0Hz,1H),4.98-4.95(m,1H),4.49(d, J =12.0Hz,1H),4.06-3.98(m,6H),3.90-3.83(m,1H),3.73-3.50(m,13H),3. 02-2.94(m,4H),2.45-2.44(m,4H),2.11-1.76(m,20H),1.48-1.18(m,12H).

固體載體9-13：Solid carrier 9-13:

天然胺基-LCAA-CPG(上樣值：75μmol/g，1000Å)用ACN(100mLx2)、DMF(100mLx2)和DCM(100mLx2)洗滌，然後在高真空下乾燥過夜。 Native amine-LCAA-CPG (loading value: 75 μmol/g, 1000Å) was washed with ACN (100 mLx2), DMF (100 mLx2) and DCM (100 mLx2), and then dried under high vacuum overnight.

向琥珀酸酯9-12(85mg，0.075mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)的溶液中加入DIPEA(30mg，0.23mmol)，在室溫下振盪反應混合物10分鐘，然後加入天然胺基-LCAA-CPG(300mg，上樣75μmol/g)，室溫振盪懸浮液20小時，然後過濾，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，以得到固體載體(300mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將固體載體上未反應的胺封端，用DMF(20mLx5)、CAN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體9-13(300mg)。為了計算上樣，取5.00mg乾燥的上樣CPG，加入25mL的3% DCA在DCM中的溶液。振盪溶液，測量482nm處的UV吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式： To a solution of succinate 9-12 (85 mg, 0.075 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (30 mg, 0.23 mmol), the reaction mixture was shaken at room temperature for 10 minutes, then natural amino-LCAA-CPG (300 mg, loading 75 μmol/g) was added, the suspension was shaken at room temperature for 20 hours, then filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), DCM (20 mL x 5) until TLC showed no spot in the eluent at 254 nm. The solid support was dried in vacuo for 2 hours to obtain a solid support (300 mg). Stir with Ac2O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the solid support, and wash with DMF (20mLx5), CAN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 9-13 (300mg). To calculate the loading, take 5.00mg of the dried loading CPG and add 25mL of 3% DCA solution in DCM. Vibrate the solution and measure the UV absorbance at 482nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula:

上樣(μmol/g)=(添加的DCA總體積(mL))*(482nm處的Abs值)*1000)/(78.3*(取的CPG mg數)) Sample loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 482nm) * 1000) / (78.3 * (mg of CPG taken))

添加的DCA總體積(mL)=25mL Total volume of DCA added (mL) = 25mL

482nm處的Abs值=0.577 Abs value at 482nm = 0.577

取的CPG的Mg數=5.00mg The Mg content of CPG taken = 5.00mg

上樣(μmol/g)=((25)*(0.577)*1000)/78.3*(5.00)=36.8μmol/g。 Sample loading (μmol/g) = ((25)*(0.577)*1000)/78.3*(5.00) = 36.8μmol/g.

Figure 113123276-A0202-12-0209-285
Figure 113123276-A0202-12-0209-285

化合物10-2：Compound 10-2:

將化合物10-1(0.6g，0.299mmol)、DIEA(96.63mg，0.748mmol，130.23μL)、HOBT(48.49mg，0.359mmol)和EDCI(68.80mg，0.359mmol)混合於DCM(18mL)中，室溫攪拌30分鐘，然後加入化合物2-10(412.34mg，0.897mmol)，室溫攪拌1小時。LC-MS顯示完全轉化。將反應混合物濃縮，經製備型HPLC(C18管柱，沖提液A：水，沖提液B：ACN)純化，以得到化合物10-2，為白色固體(580mg，產率為79%)。 Compound 10-1 (0.6 g, 0.299 mmol), DIEA (96.63 mg, 0.748 mmol, 130.23 μL), HOBT (48.49 mg, 0.359 mmol) and EDCI (68.80 mg, 0.359 mmol) were mixed in DCM (18 mL), stirred at room temperature for 30 minutes, and then compound 2-10 (412.34 mg, 0.897 mmol) was added and stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by preparative HPLC (C18 column, eluent A: water, eluent B: ACN) to obtain compound 10-2 as a white solid (580 mg, yield 79%).

C120H179N11O42質量計算為：2446.22；實測：1247.61[M+2Na]2+，ESI。 Mass calculated for C 120 H 179 N 11 O 42 : 2446.22; found: 1247.61 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.86-7.82(m,6H),7.75(t,J=5.5Hz,3H),7.39-7.29(m,4H),7.26-7.19(m,5H),7.00(br,1H),6.89(dd,J=8.9,3.7Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,3H),4.66-4.64(m,1H),4.78(d,J=8.4Hz,3H),4.04-3.96(m,10H),3.90-3.83(m,3H),3.73-3.67(m,11H),3.55-3.50(m,13H),3.43-3.37(m,5H),3.02(pent,J=5.8Hz,12H),2.90(s,2H),2.27(t,J=6.2Hz,6H),2.10(s,9H)2.07-2.00(m,10H),1.99(s,9H),1.96-1.92(m,2H),1.89(s,9H),1.85-1.80(m,2H),1.77(s,9H),1.54-1.37(m,22H),1.22-1.20(m,12H). 1 H NMR(400MHz, DMSO-d6)δ 7.86-7.82(m,6H),7.75(t, J =5.5Hz,3H),7.39-7.29(m,4H),7.26-7.19(m,5H),7.00(br,1H),6.89(dd, J =8.9,3.7Hz,4H),5.21(d, J =3.4Hz,3H),4.96(dd, J =11.2,3.4Hz,3H),4.66-4.64(m,1H),4.78(d, J =8.4Hz,3H),4.04-3.96(m,10H),3.90-3.83(m,3H),3.73-3.67(m,11H),3.55-3.50(m,13H),3.43-3.37(m,5H),3.02(pent, J =5.8Hz,12H),2.90(s,2H),2.27(t, J =6.2Hz,6H),2.10(s,9H)2.07-2.00(m,10H),1.99(s,9H),1.96-1.92(m,2H),1.8 9(s,9H),1.85-1.80(m,2H),1.77(s,9H),1.54-1.37(m,22H),1.22-1.20(m,12H).

化合物10-3：Compound 10-3:

向化合物10-2(150mg，0.06mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(14mg，0.12mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗。將反應混合物用DCM稀釋，用水(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物10-3(130mg，產率為97%)，為白色固體。 To a solution of compound 10-2 (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL) was added DMAP (14 mg, 0.12 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM, washed with water (3 mL x 4), then with brine (3 mL x 4), and the organic layer was concentrated to give compound 10-3 (130 mg, 97% yield) as a white solid.

C124H183N11O45質量計算為：2546.24；實測：1273.5[M+2H]2+，ESI。 Mass calculated for C 124 H 183 N 11 O 45 : 2546.24; found: 1273.5 [M+2H] 2+ , ESI.

1HNMR(600MHz，DMSO-d 6)：7.84-7.73(m,9H),7.37-7.21(m,9H),6.98-6.89(m,5H),5.21(d,J=6.0Hz,3H),4.97-4.94(m,3H),4.48(d,J=12.0Hz,3H),4.07-3.66(m,26H),3.55-3.37(m,17H),3.04-2.95(m,14H),2.45(d,J=6.0Hz,4H),2.29-2.25(m,6H),2.11-1.72(m,50H),1.51-1.12(m,38H). 1 HNMR (600MHz, DMSO- d 6 ): 7.84-7.73(m,9H),7.37-7.21(m,9H),6.98-6.89(m,5H),5.21(d, J =6.0Hz,3H),4.97-4.94(m,3H),4.48(d, J =12.0Hz,3H),4.07-3.66(m,26H),3.55-3.37(m,17H),3.04-2.95(m,14H),2.45(d, J =6.0Hz,4H),2.29-2.25(m,6H),2.11-1.72(m,50H),1.51-1.12(m,38H).

化合物10-4：Compound 10-4:

向化合物10-3(130mg，0.05mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)中的溶液加入DIPEA(30mg，0.23mmol)。將反應混合物室溫振盪10分鐘，然後加入天然胺基-lcaa-CPG(300mg，上樣75μmol/g)，室溫振盪混懸液20小時，然後過濾，用DMF(20mLx5)、ACN(20mL x5)、DCM(20mLx5)洗滌，直到TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，以得到固體載體(300mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將載體上未反應的胺封端，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體10-4(300mg)。為了計算上樣，取5.19mg乾燥的上樣CPG，加入20mL的3% DCA在DCM中的溶液。振盪溶液，測量500nm處的UV吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式： To a solution of compound 10-3 (130 mg, 0.05 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (30 mg, 0.23 mmol). The reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-lcaa-CPG (300 mg, loading 75 μmol/g) was added, and the suspension was shaken at room temperature for 20 hours, and then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed that the eluent had no spots at 254 nm. The solid support was dried in vacuo for 2 hours to obtain a solid support (300 mg). Stir with Ac 2 O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amines on the support, and wash with DMF (20mLx5), ACN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 10-4 (300mg). To calculate the loading, take 5.19mg of the dried loading CPG and add 20mL of 3% DCA solution in DCM. Vibrate the solution and measure the UV absorbance at 500nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula:

上樣(μmol/g)=(添加的DCA總體積(mL))*(500nm處的Abs值)*1000)/(76*(取的CPG mg數)) Sample loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 500nm) * 1000) / (76 * (mg of CPG taken))

添加的DCA總體積(mL)=20mL Total volume of DCA added (mL) = 20mL

500nm處的Abs值=0.335 Abs value at 500nm = 0.335

取的CPG的Mg數=5.19mg The Mg content of CPG taken = 5.19mg

上樣(μmol/g)=((20)*(0.335)*1000)/76*(5.19)=17μmol/g。 Sample loading (μmol/g) = ((20)*(0.335)*1000)/76*(5.19) = 17μmol/g.

Figure 113123276-A0202-12-0212-286
Figure 113123276-A0202-12-0212-286

化合物11-1：Compound 11-1:

向化合物2-10(1.27g，3.59mmol)在DCM(15mL)中的溶液中加入TEA(495.41mg，4.90mmol)和HATU(1.35g，3.59mmol)。攪拌15分鐘後，將Fmoc-6-己酸(1.5g，3.26mmol)加入反應混合物中，持續攪拌4小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用DCM/MeOH=20/1沖提以得到600mg的化合物11-1(產率為23.2%)。 To a solution of compound 2-10 (1.27 g, 3.59 mmol) in DCM (15 mL) was added TEA (495.41 mg, 4.90 mmol) and HATU (1.35 g, 3.59 mmol). After stirring for 15 minutes, Fmoc-6-hexanoic acid (1.5 g, 3.26 mmol) was added to the reaction mixture and stirring was continued for 4 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH = 20/1 to obtain 600 mg of compound 11-1 (yield 23.2%).

1H NMR(400MHz,DMSO-d6)δ 7.87(d,J=7.6Hz,2H),7.68(d,J=7.4Hz,2H),7.44-7.35(m,4H),7.35-7.28(m,4H),7.27-7.17(m,6H),6.88(d,J= 7.6Hz,4H),4.64(td,J=5.04,1.6Hz,1H),4.28(d,J=6.9Hz,2H),4.20(t,J=6.8Hz,1H),3.96(s,1H),3.73(d,J=3.8Hz,6H),3.66(d,J=10.4Hz,2H),3.50(s,1H),3.39(t,J=5.9Hz,2H),2.96(t,J=6.6Hz,2H),2.91(s,2H),2.06-1.74(m,6H),1.47-1.31(m,4H),1.26-1.13(m,2H). 1 H NMR (400MHz, DMSO-d6)δ 7.87(d, J =7.6Hz,2H),7.68(d, J =7.4Hz,2H),7.44-7.35(m,4H),7.35-7.28(m,4H),7.27-7.17(m,6H),6.88(d, J = 7.6Hz,4H),4.64(td, J =5.04,1.6Hz,1H),4.28(d, J =6.9Hz,2H),4.20(t, J =6.8Hz,1H),3.96(s,1H),3.73(d, J =3.8Hz,6H),3.66(d, J =10.4Hz,2H),3.50(s,1H),3.39(t, J =5.9Hz,2H),2.96(t, J =6.6Hz,2H),2.91(s,2H),2.06-1.74(m,6H),1.47-1.31(m,4H),1.26-1.13(m,2H).

化合物11-2：Compound 11-2:

將哌啶(0.4mL)加入化合物11-1(600mg，0.75mmol)在MeOH(4mL)中的溶液中。攪拌過夜後，真空濃縮混合物。殘餘物藉由管柱層析純化，用DCM/MeOH/NH3OH=100/10/1沖提以得到360mg的化合物11-2(產率為82%)。 Piperidine (0.4 mL) was added to a solution of compound 11-1 (600 mg, 0.75 mmol) in MeOH (4 mL). After stirring overnight, the mixture was concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH/NH 3 OH=100/10/1 to obtain 360 mg of compound 11-2 (yield 82%).

C35H44N2O5質量計算為：572.3；實測：573.4[M+H]+，ESI。 Mass calculated for C 35 H 44 N 2 O 5 : 572.3; found: 573.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.27-7.21(m,4H),6.91-6.85(m,9H),3.97(s,1H),3.73(s,6H),3.68(s,2H),3.47(d,J=6.5Hz,2H),3.16(s,2H),2.90(s,2H),2.66-2.62(m,2H),2.54-2.51(m,2H),1.49-1.17(m,12H). 1H NMR(400MHz,DMSO-d6)δ 7.27-7.21(m,4H),6.91-6.85(m,9H),3.97(s,1H),3.73(s,6H),3.68(s,2H),3.47(d, J =6.5Hz,2H),3.16(s,2H),2.90(s,2H),2.66-2.62(m,2H),2.54-2.51(m,2H),1.49-1.17(m,12H).

化合物11-4：Compound 11-4:

將化合物11-3(10g,84.65mmol)、4-溴吡啶(14.71g，93.11mmol)、Pd(PPh3)2Cl2(4g，5.70mmol)和t-BuOK(19.00g，169.30mmol)在1,4-二噁烷(200mL)中的混合物溶液用氮氣吹掃脫氣。然後將混合物在60℃下加熱6小時。濃縮混合物。然後，殘餘物藉由管柱層析純化，用DCM/MeOH/NH4OH=95/5/1沖提以得到8g的化合物11-4，為黃色的固體(產率為48%)。 A mixture solution of compound 11-3 (10 g, 84.65 mmol), 4-bromopyridine (14.71 g, 93.11 mmol), Pd(PPh 3 ) 2 Cl 2 (4 g, 5.70 mmol) and t-BuOK (19.00 g, 169.30 mmol) in 1,4-dioxane (200 mL) was degassed by purging with nitrogen. The mixture was then heated at 60° C. for 6 hours. The mixture was concentrated. The residue was then purified by column chromatography and extracted with DCM/MeOH/NH 4 OH=95/5/1 to obtain 8 g of compound 11-4 as a yellow solid (yield 48%).

C12H9N3質量計算為：195.1；實測：196.2[M+H]+，ESI。 Mass calculated for C 12 H 9 N 3 : 195.1; found: 196.2 [M+H] + , ESI.

化合物11-5：Compound 11-5:

將化合物11-4(10g，51.22mmol)溶於三級丁醇並升溫至50℃，然後加入KOH(574.84mg，10.24mmol)和MeOH(5mL)。然後向反應混合物中緩慢加入丙 烯酸三級丁酯(7.22g，56.35mmol)，攪拌5小時。將混合物真空濃縮。將殘餘物在乙酸乙酯(250mL)和水(150mL)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用PE/EA=1/2沖提以得到11g的化合物11-5(產率為66%)。 Compound 11-4 (10 g, 51.22 mmol) was dissolved in tert-butyl alcohol and heated to 50 ° C, then KOH (574.84 mg, 10.24 mmol) and MeOH (5 mL) were added. Then tert-butyl acrylate (7.22 g, 56.35 mmol) was slowly added to the reaction mixture and stirred for 5 hours. The mixture was concentrated in vacuo. The residue was distributed between ethyl acetate (250 mL) and water (150 mL), the ethyl acetate extract was washed with brine (100 ml), dried with sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with PE/EA = 1/2 to obtain 11 g of compound 11-5 (yield 66%).

C19H21N3O2質量計算為：323.2；實測：324.2[M+H]+，ESI。 Mass calculated for C 19 H 21 N 3 O 2 : 323.2; found: 324.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.65(d,J=5.6Hz,4H),7.50(d,J=6.0Hz,4H),2.82(t,J=7.8Hz,2H),2.23(t,J=7.8Hz,2H),1.35(s,9H). 1 H NMR (400MHz, DMSO-d6) δ 8.65 (d, J =5.6Hz, 4H), 7.50 (d, J =6.0Hz, 4H), 2.82 (t, J =7.8Hz, 2H), 2.23 (t, J =7.8Hz, 2H), 1.35 (s, 9H).

化合物11-6：Compound 11-6:

將六水合氯化鈷(1.47g，6.18mmol)加入化合物11-5(1g，3.09mmol)在MeOH(20mL)中的溶液中。然後，在-6℃下將硼氫化鈉(1.17g，30.92mmol)緩慢加入到反應混合物中。將混合物升溫至室溫並攪拌5小時。將混合物真空濃縮。殘餘物溶於EA，藉由矽藻土床過濾，並用MeOH洗滌。濃縮濾液以得到0.7g的化合物11-6(產率為69%)。 Cobalt chloride hexahydrate (1.47 g, 6.18 mmol) was added to a solution of compound 11-5 (1 g, 3.09 mmol) in MeOH (20 mL). Then, sodium borohydride (1.17 g, 30.92 mmol) was slowly added to the reaction mixture at -6 °C. The mixture was warmed to room temperature and stirred for 5 hours. The mixture was concentrated in vacuo. The residue was dissolved in EA, filtered through a diatomaceous earth bed, and washed with MeOH. The filtrate was concentrated to give 0.7 g of compound 11-6 (yield 69%).

C19H25N3O2質量計算為：327.2；實測：328.3[M+H]+，ESI。 Mass calculated for C 19 H 25 N 3 O 2 : 327.2; found: 328.3 [M+H] + , ESI.

化合物11-7：Compound 11-7:

將化合物11-6(2.12g，9.16mmol)溶解於DCM(40mL)中。然後，向該溶液中加入TEA(925.42mg，9.16mmol)和HATU(3.46g，9.16mmol)。攪拌20分鐘後，將Boc-6-胺基己酸(3g，9.16mmol)加入到反應混合物中，並將混合物攪拌5小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用DCM/MeOH=9/1沖提以得到2.5g的化合物11-7，為黃色的油狀(產率為71%)。 Compound 11-6 (2.12 g, 9.16 mmol) was dissolved in DCM (40 mL). Then, TEA (925.42 mg, 9.16 mmol) and HATU (3.46 g, 9.16 mmol) were added to the solution. After stirring for 20 minutes, Boc-6-aminohexanoic acid (3 g, 9.16 mmol) was added to the reaction mixture, and the mixture was stirred for 5 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried with sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH = 9/1 to obtain 2.5 g of compound 11-7 as a yellow oil (yield of 71%).

C30H44N4O5質量計算為：540.3；實測：541.4[M+H]+，ESI。 Mass calculated for C 30 H 44 N 4 O 5 : 540.3; found: 541.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.48(d,J=5.9Hz,4H),7.45(t,J=6.6Hz,1H),7.14(d,J=6.1Hz,4H),6.73(t,J=5.4Hz,1H),3.88(d,J=6.1Hz,2H),2.85(q,J=6.6Hz,2H),2.31(t,J=8.0Hz,2H),1.94(q,J=8.0Hz,4H),1.36(s,9H),1.32(s,9H),1.31-1.23(m,4H),1.1-0.9(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 8.48(d, J =5.9Hz,4H),7.45(t, J =6.6Hz,1H),7.14(d, J =6.1Hz,4H),6.73(t, J =5.4Hz,1H),3.88(d, J =6.1Hz,2H),2.85(q, J =6.6Hz,2H),2.31(t, J =8.0Hz,2H),1.94(q, J =8.0Hz,4H),1.36(s,9H),1.32(s,9H),1.31-1.23(m,4H),1.1-0.9(m,2H).

化合物11-8：Compound 11-8:

將氧化鉑(2.2g，9.69mmol)加入到化合物11-7(4.5g，8.32mmol)在AcOH(15ml)的溶液中。混合物在~0.4MPa下氫化56小時。催化劑藉由矽藻土床過濾並用MeOH洗滌。混合物真空濃縮。藉由添加濃NaHCO3水溶液將pH調節至7，然後真空乾燥。將殘餘物溶解在THF中，然後過濾混合物，減壓蒸發濾液以得到4g的化合物11-8，為無色油狀物，產率為87%。 Platinum oxide (2.2 g, 9.69 mmol) was added to a solution of compound 11-7 (4.5 g, 8.32 mmol) in AcOH (15 ml). The mixture was hydrogenated at ~0.4 MPa for 56 hours. The catalyst was filtered through a diatomaceous earth bed and washed with MeOH. The mixture was concentrated in vacuo. The pH was adjusted to 7 by adding concentrated aqueous NaHCO 3 solution and then dried in vacuo. The residue was dissolved in THF, and the mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain 4 g of compound 11-8 as a colorless oil with a yield of 87%.

C30H56N4O5質量計算為：552.4；實測：553.4[M+H]+，ESI。 Mass calculated for C 30 H 56 N 4 O 5 : 552.4; found: 553.4 [M+H] + , ESI.

化合物11-9：Compound 11-9:

將TEA(402.72mg，3.98mmol)和HATU(1.50g，3.98mmol)加入到Boc-6-胺基己酸(920.49mg，3.98mmol)在DCM(10mL)的溶液中。攪拌溶液20分鐘後，將化合物11-8(1g，1.18mmol)加入到反應混合物中並繼續攪拌5小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用DCM/MeOH=15/1沖提以得到0.4g的化合物11-9，為白色固體(產率為22%)。 TEA (402.72 mg, 3.98 mmol) and HATU (1.50 g, 3.98 mmol) were added to a solution of Boc-6-aminohexanoic acid (920.49 mg, 3.98 mmol) in DCM (10 mL). After stirring the solution for 20 minutes, compound 11-8 (1 g, 1.18 mmol) was added to the reaction mixture and stirring was continued for 5 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH=15/1 to obtain 0.4 g of compound 11-9 as a white solid (yield 22%).

C52H94N6O11質量計算為：978.7；實測：979.8[M+H]+，ESI。 Mass calculated for C 52 H 94 N 6 O 11 : 978.7; found: 979.8 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.41(t,J=6.0Hz,1H),6.78-6.70(m,3H),3.88(d,J=11.4Hz,2H),3.02(d,J=6.5Hz,6H),2.92-2.82(m,8H),2.25(t, J=7.4Hz,4H),2.15(t,J=8.1Hz,2H),2.16(t,J=7.2Hz,2H),1.70-1.50(m,6H),1.49-1.38(m,10H),1.38(s,9H),1.36(s,27H),1.34-1.15(m,14H). 1 H NMR (400MHz, DMSO-d6)δ 7.41(t, J =6.0Hz,1H),6.78-6.70(m,3H),3.88(d, J =11.4Hz,2H),3.02(d, J =6.5Hz,6H),2.92-2.82(m,8H),2.25(t, J =7.4Hz,4H),2.15(t, J =8.1Hz,2H),2.16(t, J =7.2Hz,2H),1.70-1.50(m,6H),1.49-1.38(m,10H),1.38(s,9H),1.36(s,27H),1.34-1.15(m,14H).

化合物11-10：Compound 11-10:

將TFA(2mL)加入化合物11-9(1.4g，1.43mmol)在DCM(6mL)中的溶液中，然後在室溫下攪拌混合物4小時。將混合物真空濃縮以得到850mg的化合物11-10，為無色油狀物，產率為95%。 TFA (2 mL) was added to a solution of compound 11-9 (1.4 g, 1.43 mmol) in DCM (6 mL), and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated in vacuo to give 850 mg of compound 11-10 as a colorless oil with a yield of 95%.

C33H62N6O5質量計算為：622.5；實測：623.6[M+H]+，ESI。 Mass calculated for C 33 H 62 N 6 O 5 : 622.5; found: 623.6 [M+H] + , ESI.

化合物11-11：Compound 11-11:

將TEA(584.84mg，5.78mmol)和HATU(1.64g，4.33mmol)加入到化合物1-4(2.13g，4.77mmol)在DMF(10mL)中的溶液中。攪拌20分鐘後，將化合物11-10(0.9g，1.44mmol)加入到反應混合物中並繼續攪拌5小時。混合物經反相管柱(C18管柱)純化，得到1.3g的化合物11-11，為白色固體，產率為47%。 TEA (584.84 mg, 5.78 mmol) and HATU (1.64 g, 4.33 mmol) were added to a solution of compound 1-4 (2.13 g, 4.77 mmol) in DMF (10 mL). After stirring for 20 minutes, compound 11-10 (0.9 g, 1.44 mmol) was added to the reaction mixture and continued to stir for 5 hours. The mixture was purified by a reverse phase column (C18 column) to obtain 1.3 g of compound 11-11 as a white solid with a yield of 47%.

C90H143N9O35質量計算為：1909.9；實測：956.0[M+2H]2+，ESI。 Mass calculated for C 90 H 143 N 9 O 35 : 1909.9; found: 956.0 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 12.0(br,1H),7.83(d,J=9.2Hz,3H),7.70(t,J=5.5Hz,3H),7.50-7.40(m,1H),5.20(d,J=3.4Hz,3H),4.96(dd,J=11.24,3.4Hz,3H),4.50-4.40(m,5H),4.08-3.96(m,9H),3.92-3.82(m,5H),3.76-3.66(m,3H),3.46-3.36(m,4H),3.15-2.75(m,11H),2.40-2.30(m,2H),2.25(t,J=7.3Hz,4H),2.10(s,9H),2.08-2.00(m,8H),1.99(s,9H),1.98(s,9H),1.77(s,9H),1.60-1.08(m,42H). 1 H NMR(400MHz, DMSO-d6)δ 12.0(br,1H),7.83(d, J =9.2Hz,3H),7.70(t, J =5.5Hz,3H),7.50-7.40(m,1H),5.20(d, J =3.4Hz,3H),4.96(dd, J =11.24,3.4Hz,3H),4.50-4.40(m,5H),4.08-3.96(m,9H),3.92-3.82(m,5H),3.7 6-3.66(m,3H),3.46-3.36(m,4H),3.15-2.75(m,11H),2.40-2.30(m,2H),2.25(t, J =7.3Hz,4H),2.10(s,9H),2.08-2.00(m,8H),1.99(s,9H),1.98(s,9H),1.77(s,9H),1.60-1.08(m,42H).

化合物11-12：Compound 11-12:

將化合物11-11(1.4g，0.73mmol)溶解於DCM(15mL)中，然後將HOBT(148.47mg，1.10mmol)、EDCI(210.10mg，1.10mmol)和DIEA(236.69mg，1.83mmol)加入該溶液中。攪拌15分鐘後，將化合物11-2(503.47mg，0.88mmol) 加入到反應混合物中並繼續攪拌4小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫酸鈉乾燥並真空濃縮。將殘餘物經反相管柱(C18管柱)純化，得到0.8g的化合物11-12，為白色粉末，產率為44%。 Compound 11-11 (1.4 g, 0.73 mmol) was dissolved in DCM (15 mL), and then HOBT (148.47 mg, 1.10 mmol), EDCI (210.10 mg, 1.10 mmol) and DIEA (236.69 mg, 1.83 mmol) were added to the solution. After stirring for 15 minutes, compound 11-2 (503.47 mg, 0.88 mmol) was added to the reaction mixture and stirring was continued for 4 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by reverse phase column (C18 column) to obtain 0.8 g of compound 11-12 as white powder with a yield of 44%.

C125H185N11O39質量計算為：2464.3；實測：1255.6[M+2Na]2+，ESI。 Mass calculated for C 125 H 185 N 11 O 39 : 2464.3; found: 1255.6 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.86-7.76(m,4H),7.71(t,J=5.3Hz,3H),7.45(s,1H),7.41-7.35(m,2H),7.34-7.28(m,2H),7.27-7.18(m,5H),6.93-6.86(m,4H),5.27-5.15(d,J=3.3Hz,3H),5.02-4.90(dd,J=11.2,3.4Hz,3H),4.69-4.60(m,1H),4.57-4.33(m,5H),4.11-3.98(m,9H),3.97(s,1H),3.93-3.81(m,5H),3.73(s,6H),3.72-3.62(m,5H),3.51(s,1H),3.46-3.36(m,5H),3.14-2.73(m,14H),2.43-2.29(m,2H),2.29-2.19(m,4H),2.01(s,9H),2.06-1.97(m,19H),1.96-1.91(m,3H),1.87(s,9H),1.86-1.80(m,2H),1.77(s,9H),1.70-1.55(m,5H),1.54-1.13(m,44H). 1 H NMR(400MHz,DMSO-d6)δ 7.86-7.76(m,4H),7.71(t, J =5.3Hz,3H),7.45(s,1H),7.41-7.35(m,2H),7.34-7.28(m,2H),7.27-7.18(m,5H),6.93-6.86(m,4H),5.27-5.15(d, J =3.3Hz,3H),5.02-4.90(dd, J =11.2,3.4Hz,3H),4.69-4.60(m,1H),4.57-4.33(m,5H),4.11-3.98(m,9H),3.97(s,1H),3. 93-3.81(m,5H),3.73(s,6H),3.72-3.62(m,5H),3.51(s,1H),3.46-3.36(m,5H),3.14-2.73 (m,14H),2.43-2.29(m,2H),2.29-2.19(m,4H),2.01(s,9H),2.06-1.97(m,19H),1.96-1.91 (m,3H),1.87(s,9H),1.86-1.80(m,2H),1.77(s,9H),1.70-1.55(m,5H),1.54-1.13(m,44H).

Figure 113123276-A0202-12-0218-287
Figure 113123276-A0202-12-0218-287

化合物11-13：Compounds 11-13:

在室溫中向化合物11-12(2.0g，0.8mmol)在無水DCM(7.0mL)中的溶液中加入4,5-二氰基咪唑(80.0mg，0.67mmol)和2-氰乙基N,N,N’,N’-四異丙基亞磷二醯胺(270mg，0.90mmol)，攪拌1小時，LCMS顯示起始材料已完全消耗。用水(100mL)淬滅溶液，用鹽水(300mLx3)洗滌，並用Na2SO4乾燥。然後將溶液減壓濃縮，殘餘物藉由以下條件藉由快速製備型HPLC純化(管柱，C18矽膠；流動相：CH3CN/H2O=1/1在20分鐘內增加至CH3CN/H2O=1/0；檢測器：UV 254nm)。產生了化合物11-13，為白色固體(1.0g，產率為45%)。 To a solution of compound 11-12 (2.0 g, 0.8 mmol) in anhydrous DCM (7.0 mL) was added 4,5-dicyanoimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (270 mg, 0.90 mmol) at room temperature, stirred for 1 hour, LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na 2 SO 4 . The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH 3 CN/H 2 O=1/1 increased to CH 3 CN/H 2 O=1/0 within 20 minutes; detector: UV 254 nm). Compound 11-13 was produced as a white solid (1.0 g, yield 45%).

C134H202N13O40P質量計算為：2664.39；實測：1132.5[M-DMTr-N(CH(CH3)2)2+4H]2+，ESI。 Mass calculated for C 134 H 202 N 13 O 40 P: 2664.39; found: 1132.5 [M-DMTr-N(CH(CH 3 ) 2 ) 2 +4H] 2+ , ESI.

1HNMR(600MHz,DMSO-d 6)δ 7.82-7.69(m,6H),7.44-7.19(m,9H),6.90-6.87(m,4H),5.21(d,J=6.0Hz,3H),4.98-4.94(m,3H),4.49-4.44(m,5H),4.04-3.37(m,35H),3.02-2.71(m,15H),2.33-1.75(m,56H),1.64-1.07(m,57H). 1 HNMR(600MHz, DMSO- d 6 )δ 7.82-7.69(m,6H),7.44-7.19(m,9H),6.90-6.87(m,4H),5.21(d, J =6.0Hz,3H),4.98-4.94(m,3H),4.49-4.44(m,5H),4.04-3.37(m,35H),3.02-2.71(m,15H),2.33-1.75(m,56H),1.64-1.07(m,57H).

31PNMR(242MHz,DMSO-d 6)δ 146.79,146.67. 31 PNMR(242MHz,DMSO- d 6 )δ 146.79,146.67.

Figure 113123276-A0202-12-0219-288
Figure 113123276-A0202-12-0219-288

化合物11-14：Compounds 11-14:

向化合物11-12(150mg，0.06mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(5mg，0.04mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)，將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗， 將反應混合物用DCM稀釋，用水(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層，以得到化合物11-14(135mg，產率為97%)，為白色固體。 To a solution of compound 11-12 (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL) were added DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), and then succinic anhydride (20 mg, 0.2 mmol), and the reaction mixture was stirred at room temperature for 3 hours. LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM, washed with water (3 mL x 4), and then with brine (3 mL x 4), and the organic layer was concentrated to give compound 11-14 (135 mg, yield 97%) as a white solid.

C129H189N11O42質量計算為：2564.30；實測：1132.5[M-DMTr+2H]2+，ESI。 Mass calculated for C 129 H 189 N 11 O 42 : 2564.30; found: 1132.5 [M-DMTr+2H] 2+ , ESI.

1H NMR(600MHz,DMSO-d 6)：7.84-7.71(m,6H),7.45-7.21(m,10H),6.92-6.89(m,4H),5.21(d,J=6.0Hz,3H),4.98-4.94(m,3H),4.49-4.44(m,5H),4.07-3.37(m,36H),3.16-2.84(m,14H),2.44-1.77(m,62H),1.45-1.21(m,50H). 1 H NMR (600MHz, DMSO- d 6 ): 7.84-7.71 (m, 6H), 7.45-7.21 (m, 10H), 6.92-6.89 (m, 4H), 5.21 (d, J =6.0Hz,3H),4.98-4.94(m,3H),4.49-4.44(m,5H),4.07-3.37(m,36H),3.16-2.84(m,14H),2.44-1.77(m,62H),1.45-1.21(m,50H).

化合物11-15：Compounds 11-15:

向化合物11-14(135mg，0.05mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)中的溶液加入DIPEA(30mg，0.23mmol)。將反應混合物室溫振盪10分鐘，然後加入天然胺基-lcaa-CPG(300mg，上樣75μmol/g)，室溫振盪混懸液20小時，然後過濾，用DMF(20mLx5)、ACN(20mL x5)、DCM(20mLx5)洗滌，直到TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，以得到固體載體(300mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將載體上未反應的胺封端，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體11-15(300mg)。為了計算上樣，取5.00mg乾燥的上樣CPG，加入20mL的3% DCA在DCM中的溶液。振盪溶液，測量500nm處的UV吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式： To a solution of compound 11-14 (135 mg, 0.05 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (30 mg, 0.23 mmol). The reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-lcaa-CPG (300 mg, loading 75 μmol/g) was added, and the suspension was shaken at room temperature for 20 hours, and then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed that the eluent had no spots at 254 nm. The solid support was dried in vacuo for 2 hours to obtain a solid support (300 mg). Stir with Ac 2 O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the support, and wash with DMF (20mLx5), ACN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 11-15 (300mg). To calculate the loading, take 5.00mg of the dried loading CPG and add 20mL of 3% DCA solution in DCM. Vibrate the solution and measure the UV absorbance at 500nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula:

上樣(μmol/g)=(添加的DCA總體積(mL))*(500nm處的Abs值)*1000)/(76*(取的CPG mg數)) Sample loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 500nm) * 1000) / (76 * (mg of CPG taken))

添加的DCA總體積(mL)=20mL Total volume of DCA added (mL) = 20mL

500nm處的Abs值=0.464 Abs value at 500nm = 0.464

取的CPG的Mg數=5.0mg The Mg content of CPG taken = 5.0mg

上樣(μmol/g)=((20)*(0.464)*1000)/76*(5.0)=24μmol/g。 Loading (μmol/g) = ((20)*(0.464)*1000)/76*(5.0) = 24μmol/g.

Figure 113123276-A0202-12-0221-289
Figure 113123276-A0202-12-0221-289

化合物12-1：Compound 12-1:

將化合物3-4(1.1g，5.16mmol)溶解於H2O(10mL)和THF(20mL)中。加入K2CO3(1.43g，10.31mmol)和氯甲酸苄酯(967mg，5.67mmol)。將反應混合物在室溫攪拌3小時。然後真空濃縮，藉由矽膠快速管柱層析(PE/EA=5/1)純化，得到化合物12-1，為白色泡沫固體(1.5g，產率為84%)。 Compound 3-4 (1.1 g, 5.16 mmol) was dissolved in H 2 O (10 mL) and THF (20 mL). K 2 CO 3 (1.43 g, 10.31 mmol) and benzyl chloroformate (967 mg, 5.67 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. Then it was concentrated in vacuo and purified by silica gel flash column chromatography (PE/EA=5/1) to obtain compound 12-1 as a white foam solid (1.5 g, yield 84%).

C20H29NO4質量計算為：347.2；實測：348.3[M+H]+，ESI。 Mass calculated for C 20 H 29 NO 4 : 347.2; found: 348.3 [M+H] + , ESI.

化合物12-2：Compound 12-2:

將化合物12-1(1.52g，4.37mmol)、DMAP(147.10mg，1.20mmol)和DIEA(3.11g，24.08mmol，4.19mL)溶於DCM(20mL)。在0℃下將DMTrCl(1.48g，4.37mmol)在DCM(20mL)中的溶液緩慢加入到反應混合物中。將反應在室溫攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-3% MeOH)在Al2O3上純化以得到化合物12-4，為白色泡沫狀固體(1.9g，產率為67%)。 Compound 12-1 (1.52 g, 4.37 mmol), DMAP (147.10 mg, 1.20 mmol) and DIEA (3.11 g, 24.08 mmol, 4.19 mL) were dissolved in DCM (20 mL). A solution of DMTrCl (1.48 g, 4.37 mmol) in DCM (20 mL) was slowly added to the reaction mixture at 0° C. The reaction was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (50 mL×2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-3% MeOH) on Al 2 O 3 to give compound 12-4 as a white foamy solid (1.9 g, 67% yield).

C41H47NO6質量計算為：649.34；實測：650.34[M+H]+，ESI。 Mass calculated for C 41 H 47 NO 6 : 649.34; found: 650.34 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.41-7.19(m,15H),6.90-6.87(m,3H),5.06-5.05(m,2H),4.42-4.40(m,1H),3.76(s,6H),3.40-3.38(m,6H),2.88(s,2H),1.39-1.11(m,12H). 1H NMR(400MHz,DMSO-d6)δ 7.41-7.19(m,15H),6.90-6.87(m,3H),5.06-5.05(m,2H),4.42-4.40(m ,1H),3.76(s,6H),3.40-3.38(m,6H),2.88(s,2H),1.39-1.11(m,12H).

化合物12-3：Compound 12-3:

將化合物12-2(0.7g，1.08mmol)溶解於MeOH(30mL)中，然後添加Pd/C(140mg)到溶液中。將反應混合物在氫氣氛圍下在室溫攪拌3小時。將反應混合物過濾並在真空中濃縮以得到粗化合物12-3，為白色泡沫固體(520mg)，無需進一步純化即可用於下一步。 Compound 12-2 (0.7 g, 1.08 mmol) was dissolved in MeOH (30 mL), and then Pd/C (140 mg) was added to the solution. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated in vacuo to give crude compound 12-3 as a white foam solid (520 mg), which was used in the next step without further purification.

C33H41NO4質量計算為：515.30；實測：516.30[M+H]+，ESI。 Mass calculated for C 33 H 41 NO 4 : 515.30; found: 516.30 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.38(d,J=8.8Hz,2H),7.32-7.20(m,7H),6.89-6.87(m,4H),4.51-4.49(m,1H),3.73(s,6H),2.89-2.86(m,8H),1.52-1.51(m,2H),1.19-1.12(m,7H),0.91-0.88(m,2H). 1 H NMR (400MHz, DMSO-d 6 ) δ 7.38 (d, J =8.8Hz,2H),7.32-7.20(m,7H),6.89-6.87(m,4H),4.51-4.49(m,1H),3.73(s,6 H),2.89-2.86(m,8H),1.52-1.51(m,2H),1.19-1.12(m,7H),0.91-0.88(m,2H).

化合物12-4：Compound 12-4:

化合物10-1(1.56g，0.756mmol)溶解於DCM(20mL)中。在0℃下將DIEA(300.74mg，2.33mmol)、EDCI(297.39mg，1.55mmol)和HOBT(209.62mg，1.55mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物12-3(400mg，0.756mmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL) 洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-8% MeOH)在Al2O3上純化，以得到化合物12-4，為白色泡沫狀固體(1g，產率為51%)。 Compound 10-1 (1.56 g, 0.756 mmol) was dissolved in DCM (20 mL). DIEA (300.74 mg, 2.33 mmol), EDCI (297.39 mg, 1.55 mmol) and HOBT (209.62 mg, 1.55 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 12-3 (400 mg, 0.756 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-8% MeOH) on Al 2 O 3 to give compound 12-4 as a white foamy solid (1 g, 51% yield).

C124H187N11O42質量計算為：2503.28；實測：1101.6[M-DMTr+2H+]2+，ESI。 Mass calculated for C 124 H 187 N 11 O 42 : 2503.28; found: 1101.6 [M-DMTr+2H + ] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.85-7.81(m,6H),7.73(t,J=5.6Hz,3H),7.39(d,J=7.32Hz,2H),7.32-7.19(m,8H),6.99(s,1H),6.88(d,J=8.9Hz,4H),5.21-5.20(m,3H),4.98-4.94(m,3H),4.48(d,J=8.44Hz,3H),4.42(t,J=5.0Hz,1H),4.05-4.00(m,9H),3.90-3.83(m,3H),3.73-3.67(m,9H),3.55-3.52(m,11H),3.43-3.38(m,6H),3.22-3.21(m,6H),3.06-3.00(m,11H),2.29-2.24(m,5H),2.22(t,J=7.4Hz,2H),2.10(s,8H),2.05(t,J=6.96Hz,8H),1.99-1.98(m,9H),1.89(s,8H),1.77(s,8H),1.52-1.44(m,22H),1.30-1.22(m,24H). 1 H NMR (400MHz, DMSO-d 6 )δ 7.85-7.81(m,6H),7.73(t, J =5.6Hz,3H),7.39(d, J =7.32Hz,2H),7.32-7.19(m,8H),6.99(s,1H),6.88(d, J =8.9Hz,4H),5.21-5.20(m,3H),4.98-4.94(m,3H),4.48(d, J =8.44Hz,3H),4.42(t, J =5.0Hz,1H),4.05-4.00(m,9H),3.90-3.83(m,3H),3.73-3.67(m,9H),3.55-3.52(m,11H ),3.43-3.38(m,6H),3.22-3.21(m,6H),3.06-3.00(m,11H),2.29-2.24(m,5H),2.22(t, J =7.4Hz,2H),2.10(s,8H),2.05(t, J =6.96Hz,8H),1.99-1.98(m,9H),1.89(s,8H),1.77(s,8H),1.52-1.44(m,22H),1.30-1.22(m,24H).

Figure 113123276-A0202-12-0223-290
Figure 113123276-A0202-12-0223-290

化合物13-1：Compound 13-1:

將化合物4-7(1.3g，7.01mmol)溶解於THF(30mL)和H2O(15mL)中。將K2CO3(2.91g，21.05mmol)加入到該溶液中。在0℃下將氯甲酸苄酯(1.32g，7.72mmol)加入到反應混合物中。將混合物在室溫攪拌3小時。用EA(50mLX2)萃取反應混合物，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮，藉由矽膠快速管 柱層析(0-5% MeOH)純化，得到化合物13-1，為白色泡沫固體(1.95g，產率為87%)。 Compound 4-7 (1.3 g, 7.01 mmol) was dissolved in THF (30 mL) and H 2 O (15 mL). K 2 CO 3 (2.91 g, 21.05 mmol) was added to the solution. Benzyl chloroformate (1.32 g, 7.72 mmol) was added to the reaction mixture at 0°C. The mixture was stirred at room temperature for 3 hours. The reaction mixture was extracted with EA (50 mL×2), washed with brine (50 mL), dried with Na 2 SO 4 , concentrated in vacuo, and purified by silica gel flash column chromatography (0-5% MeOH) to obtain compound 13-1 as a white foam solid (1.95 g, yield 87%).

C18H25NO4質量計算為：319.2；實測：320.2[M+H]+，ESI。 Mass calculated for C 18 H 25 NO 4 : 319.2; found: 320.2 [M+H] + , ESI.

化合物13-2：Compound 13-2:

將化合物13-1(2g，6.26mmol)和DIEA(2.43g，18.79mmol，4.19mL)溶於DCM(60mL)中。在0℃下將DMTrCl(2.02g，5.95mmol)加入到反應混合物中。將反應在0℃攪拌1小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(50mLX2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(PE/EA=0-40%)在Al2O3上純化，以得到化合物13-2，為白色泡沫狀固體(2.6g，產率為66%)。 Compound 13-1 (2 g, 6.26 mmol) and DIEA (2.43 g, 18.79 mmol, 4.19 mL) were dissolved in DCM (60 mL). DMTrCl (2.02 g, 5.95 mmol) was added to the reaction mixture at 0 °C. The reaction was stirred at 0 °C for 1 hour. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (50 mL×2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified on Al 2 O 3 by flash column chromatography (PE/EA=0-40%) to give compound 13-2 as a white foamy solid (2.6 g, 66% yield).

化合物13-3：Compound 13-3:

將化合物13-2(600mg，0.96mmol)溶解於MeOH(20mL)中，然後將Pd/C添加到反應混合物中。將反應混合物在氫氣氛圍下在室溫攪拌3小時。將反應混合物過濾並在真空中濃縮以得到粗產物13-3，為白色泡沫狀固體(470mg，粗產物)，其無需進一步純化即可用於下一步。 Compound 13-2 (600 mg, 0.96 mmol) was dissolved in MeOH (20 mL), and then Pd/C was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated in vacuo to give the crude product 13-3 as a white foamy solid (470 mg, crude product), which was used in the next step without further purification.

C31H37NO4質量計算為：487.3；實測：488.3[M+H]+，ESI。 Mass calculated for C 31 H 37 NO 4 : 487.3; found: 488.3 [M+H] + , ESI.

化合物13-4：Compound 13-4:

將化合物10-1(1.23g，0.61mmol)溶解於DCM(20mL)中。在0℃下將DIEA(239mg，1.85mmol，0.32mL)、EDCI(236mg，1.23mmol)和HOBT(166mg，1.23mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物13-3(300mg，0.61mmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL) 洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-8% MeOH)在Al2O3上純化以得到化合物13-4，為白色泡沫狀固體(1g，產率為66%)。 Compound 10-1 (1.23 g, 0.61 mmol) was dissolved in DCM (20 mL). DIEA (239 mg, 1.85 mmol, 0.32 mL), EDCI (236 mg, 1.23 mmol) and HOBT (166 mg, 1.23 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 13-3 (300 mg, 0.61 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-8% MeOH) on Al 2 O 3 to give compound 13-4 as a white foamy solid (1 g, 66% yield).

1H NMR(400MHz,DMSO-d6)δ 7.85-7.82(m,6H),7.75-7.73(m,3H),7.40-7.38(m,2H),733-7.29(m,2H),7.27-7.25(m,4H),7.23-7.19(m,1H),6.98(s,1H),6.90-6.88(m,4H),5.21(d,J=3.2Hz,3H),4.97(dd,J=3.2,11.2Hz,3H),4.63-4.61(m,1H),4.48(d,J=8.4Hz,3H),4.04-4.00(m,9H),3.91-3.83(m,3H),3.73(s,6H),3.71-3.67(m,3H),3.55-3.52(m,12H),3.44-3.37(m,5H),3.27-3.15(m,5H),3.06-3.01(m,14H),2.27(t,J=12.8Hz,6H),2.21(d,J=14.8Hz,2H),2.10(s,9H),2.04(t,J=6.8Hz,8H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.58-1.33(m,30H),1.29-1.23(m,12H). 1H NMR(400MHz,DMSO-d6)δ 7.85-7.82(m,6H),7.75-7.73(m,3H),7.40-7.38(m,2H),733-7.29(m,2H),7 .27-7.25(m,4H),7.23-7.19(m,1H),6.98(s,1H),6.90-6.88(m,4H),5.21(d, J =3.2Hz,3H),4.97(dd, J =3.2,11.2Hz,3H),4.63-4.61(m,1H),4.48(d, J =8.4Hz,3H),4.04-4.00(m,9H),3.91-3.83(m,3H),3.73(s,6H),3.71-3.67(m,3H),3 .55-3.52(m,12H),3.44-3.37(m,5H),3.27-3.15(m,5H),3.06-3.01(m,14H),2.27(t, J =12.8Hz,6H),2.21(d, J =14.8Hz,2H),2.10(s,9H),2.04(t, J =6.8Hz,8H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.58-1.33(m,30H),1.29-1.23(m,12H).

Figure 113123276-A0202-12-0225-291
Figure 113123276-A0202-12-0225-291

化合物14-1：Compound 14-1:

將化合物5-6(3.6g，19.27mmol)溶解於THF(30mL)和H2O(15mL)中。將K2CO3(5.33g，38.54mmol)加入到該溶液中。在0℃下將氯甲酸苄酯(3.62g，21.20mmol)加入到反應混合物中。將混合物在室溫攪拌2小時。用EA(50mLx2)萃取反應混合物，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮，藉由矽膠快速管柱層析(0-2% MeOH)純化，得到化合物14-1，為白色泡沫固體(5g，產率為81%)。 Compound 5-6 (3.6 g, 19.27 mmol) was dissolved in THF (30 mL) and H 2 O (15 mL). K 2 CO 3 (5.33 g, 38.54 mmol) was added to the solution. Benzyl chloroformate (3.62 g, 21.20 mmol) was added to the reaction mixture at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EA (50 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo, and purified by silica gel flash column chromatography (0-2% MeOH) to give compound 14-1 as a white foam solid (5 g, yield 81%).

C18H25NO4質量計算為：319.2；實測：320.2[M+H]+，ESI。 Mass calculated for C 18 H 25 NO 4 : 319.2; found: 320.2 [M+H] + , ESI.

1H NNR(400MHz,DMSO)δ 7.39-7.28(m,5H),5.01(s,2H),4.27(t,J=5.4Hz,2H),3.61-3.50(m,4H),3.22(d,J=5.3Hz,4H),1.55(t,J=6.1Hz,4H),1.25-1.22(m,8H). 1 H NNR(400MHz,DMSO)δ 7.39-7.28(m,5H),5.01(s,2H),4.27(t, J =5.4Hz,2H),3.61-3.50(m,4H),3.22(d, J =5.3Hz,4H),1.55(t, J =6.1Hz,4H),1.25-1.22(m,8H).

化合物14-2：Compound 14-2:

將化合物14-1(3.5g，10.96mmol)和DIEA(2.12g，16.44mmol)溶於DCM(60mL)中。在0℃下將DMTrCl(3.34g，9.86mmol)加入到反應混合物中。將反應在0℃攪拌3小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(50mLX2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(PE/EA=0-60%)在Al2O3上純化，以得到化合物14-2，為白色泡沫狀固體(5.5g，產率為81%)。 Compound 14-1 (3.5 g, 10.96 mmol) and DIEA (2.12 g, 16.44 mmol) were dissolved in DCM (60 mL). DMTrCl (3.34 g, 9.86 mmol) was added to the reaction mixture at 0 °C. The reaction was stirred at 0 °C for 3 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (50 mL×2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified on Al 2 O 3 by flash column chromatography (PE/EA=0-60%) to give compound 14-2 as a white foamy solid (5.5 g, 81% yield).

1H NMR(400MHz,DMSO-d6)δ 7.39-7.18(m,14H),6.88(d,J=8.8Hz,4H),5.00(s,2H),4.39(t,J=4.9Hz,1H),3.73(s,6H),3.58-3.35(m,6H),2.83(s,2H),1.29-1.15(m,8H). 1 H NMR(400MHz, DMSO-d6)δ 7.39-7.18(m,14H),6.88(d, J =8.8Hz,4H),5.00(s,2H),4.39(t, J =4.9Hz,1H),3.73(s,6H),3.58-3.35(m,6H),2.83(s,2H),1.29-1.15(m,8H).

化合物14-3：Compound 14-3:

將化合物14-2(254mg，408.52μmol)溶解於MeOH(10mL)中，然後將Pd/C添加到反應混合物中。將反應混合物在氫氣氛圍下在室溫攪拌2小時。將反應混合物過濾並在真空中濃縮以得到粗產物14-3，為白色泡沫狀固體(199mg，粗產物)，其無需進一步純化即可用於下一步。 Compound 14-2 (254 mg, 408.52 μmol) was dissolved in MeOH (10 mL), and then Pd/C was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated in vacuo to give the crude product 14-3 as a white foamy solid (199 mg, crude product), which was used in the next step without further purification.

C31H37NO4質量計算為：487.3；實測：488.3[M+H]+，ESI。 Mass calculated for C 31 H 37 NO 4 : 487.3; found: 488.3 [M+H] + , ESI.

化合物14-4：Compound 14-4:

將化合物10-1(820mg，408.51μmol)溶解於DCM(20mL)中。在0℃下將DIEA(158mg，1.23mmol)、EDCI(157mg，817μmol)和HOBT(110mg，817μmol) 加入到混合物中。將反應在0℃攪拌30分鐘。將化合物14-3(199mg，408.51μmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-6% MeOH)在Al2O3上純化以得到化合物14-4，為白色泡沫狀固體(540mg，產率為53%)。 Compound 10-1 (820 mg, 408.51 μmol) was dissolved in DCM (20 mL). DIEA (158 mg, 1.23 mmol), EDCI (157 mg, 817 μmol) and HOBT (110 mg, 817 μmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 14-3 (199 mg, 408.51 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-6% MeOH) on Al 2 O 3 to give compound 14-4 as a white foamy solid (540 mg, 53% yield).

1H NMR(400MHz,DMSO-d6)δ 7.89-7.80(m,6H),7.75(t,J=5.5Hz,3H),7.37(d,J=7.3Hz,2H),7.30(t,J=7.7Hz,2H),7.23-7.17(m,5H),6.99(s,1H),6.87(d,J=8.4Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=10.8,3.4Hz,3H),4.47(d,J=8.5Hz,3H),4.41(t,J=4.9Hz,1H),4.04-3.98(m,9H),3.91-3.82(m,3H),3.74-3.48(m,23H),3.43-3.36(m,6H),3.06-2.98(m,12H),2.83(d,J=9.4Hz,2H),2.27(t,J=6.3Hz,6H),2.09(s,9H),2.03(t,J=7.0Hz,8H),2.00-1.95(m,12H),2.01(s,9H),1.89(s,9H),1.77(s,9H),1.54-1.37(m,22H),1.31-1.14(m,20H). 1 H NMR(400MHz, DMSO-d6)δ 7.89-7.80(m,6H),7.75(t, J =5.5Hz,3H),7.37(d, J =7.3Hz,2H),7.30(t, J =7.7Hz,2H),7.23-7.17(m,5H),6.99(s,1H),6.87(d, J =8.4Hz,4H),5.21(d, J =3.4Hz,3H),4.96(dd, J =10.8,3.4Hz,3H),4.47(d, J =8.5Hz,3H),4.41(t, J =4.9Hz,1H),4.04-3.98(m,9H),3.91-3.82(m,3H),3.74-3.48(m,23H),3.43-3.36(m,6H),3.06-2.98(m,12H),2.83(d, J =9.4Hz,2H),2.27(t, J =6.3Hz,6H),2.09(s,9H),2.03(t, J =7.0Hz,8H),2.00-1.95(m,12H),2.01(s,9H),1.89(s,9H),1.77(s,9H),1.54-1.37(m,22H),1.31-1.14(m,20H).

Figure 113123276-A0202-12-0227-292
Figure 113123276-A0202-12-0227-292

化合物15-1：Compound 15-1:

將化合物7-6(3.8g，18.30mmol)溶解於THF(30mL)和H2O(15mL)中。將K2CO3(5.06g，36.59mmol)加入到反應混合物中。在0℃下將氯甲酸苄酯(3.43g，20.13mmol)加入到反應混合物中。將混合物在室溫攪拌2小時。用EA(50mL x2) 萃取反應混合物，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮，以得到粗產物15-1，為白色泡沫(3.46g，粗產物)。 Compound 7-6 (3.8 g, 18.30 mmol) was dissolved in THF (30 mL) and H 2 O (15 mL). K 2 CO 3 (5.06 g, 36.59 mmol) was added to the reaction mixture. Benzyl chloroformate (3.43 g, 20.13 mmol) was added to the reaction mixture at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EA (50 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , and concentrated in vacuo to give the crude product 15-1 as a white foam (3.46 g, crude product).

C17H23NO4質量計算為：305.2；實測：306.2[M+H]+，ESI。 Mass calculated for C 17 H 23 NO 4 : 305.2; found: 306.2 [M+H] + , ESI.

化合物15-2：Compound 15-2:

將化合物15-1(3.07g，10.06mmol)和DIEA(1.95g，15.09mmol)溶於DCM(60mL)中。在0℃下將DMTrCl(3.41g，10.06mmol)加入到反應混合物中。將反應在0℃攪拌3小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(PE/EA=0-60%)在Al2O3和製備型HPLC上純化，以得到化合物15-2a，為白色泡沫狀固體(1.04g，產率為34%)和化合物15-2b(1.78g，產率為58%)。 Compound 15-1 (3.07 g, 10.06 mmol) and DIEA (1.95 g, 15.09 mmol) were dissolved in DCM (60 mL). DMTrCl (3.41 g, 10.06 mmol) was added to the reaction mixture at 0° C. The reaction was stirred at 0° C. for 3 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (50 mL×2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (PE/EA=0-60%) on Al 2 O 3 and preparative HPLC to give compound 15-2a as a white foamy solid (1.04 g, yield 34%) and compound 15-2b (1.78 g, yield 58%).

15-2a： 15-2a :

1H NMR(400MHz,DMSO-d6)δ 7.38-7.36(m,1H),7.36-7.31(m,5H),7.31-7.20(m,7H),7.20-7.15(m,1H),6.85(d,J=8.9Hz,4H),5.02(d,J=6.1Hz,2H),6.98(s,1H),4.61(t,J=5.0Hz,1H),3.71(s,6H),3.46-3.35(m,2H),3.27(d,J=5.0Hz,2H),3.13-3.05(m,2H),2.93(s,2H),2.64-2.55(m,2H),1.72-1.62(m,2H),1.30-1.21(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.38-7.36(m,1H),7.36-7.31(m,5H),7.31-7.20(m,7H),7.20-7.15(m,1H),6.85(d, J =8.9Hz,4H),5.02(d, J =6.1Hz,2H),6.98(s,1H),4.61(t, J =5.0Hz,1H),3.71(s,6H),3.46-3.35(m,2H),3.27(d, J =5.0Hz,2H),3.13-3.05(m,2H),2.93(s,2H),2.64-2.55(m,2H),1.72-1.62(m,2H),1.30-1.21(m,2H).

15-2b： 15-2b :

1H NMR(400MHz,DMSO-d6)δ 7.40-7.27(m,9H),7.27-7.22(m,4H),7.22-7.16(m,1H),6.89(d,J=8.9Hz,4H),5.03(s,2H),6.98(s,1H),4.64(t,J=5.1Hz,1H),3.73(s,6H),3.43-3.34(m,4H),3.19-3.09(m,2H),3.13-3.05(m,2H),2.84(s,2H),2.41-2.31(m,2H),1.62-1.54(m,2H),1.38-1.30(m,2H). 1 H NMR (400MHz, DMSO-d6)δ 7.40-7.27(m,9H),7.27-7.22(m,4H),7.22-7.16(m,1H),6.89(d, J =8.9Hz,4H),5.03(s,2H),6.98(s,1H),4.64(t, J =5.1Hz,1H),3.73(s,6H),3.43-3.34(m,4H),3.19-3.09(m,2H),3.13-3.05( m,2H),2.84(s,2H),2.41-2.31(m,2H),1.62-1.54(m,2H),1.38-1.30(m,2H).

化合物15-3：Compound 15-3:

將化合物15-2a(210mg，350.48μmol)溶解於MeOH(10mL)中，然後將Pd/C添加到溶液中。將反應混合物在氫氣氛圍下在室溫攪拌2小時。將反應混合物過濾並在真空中濃縮以得到粗產物15-3a，為白色泡沫狀固體(166mg，粗產物)，其無需進一步純化即可用於下一步。 Compound 15-2a (210 mg, 350.48 μmol) was dissolved in MeOH (10 mL), and then Pd/C was added to the solution. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated in vacuo to give the crude product 15-3a as a white foamy solid (166 mg, crude product), which was used in the next step without further purification.

C30H35NO4質量計算為：473.3；實測：474.3[M+H]+，ESI。 Mass calculated for C 30 H 35 NO 4 : 473.3; found: 474.3 [M+H] + , ESI.

將化合物15-2b(610mg，1.00mmol)溶解於MeOH(10mL)中，然後將Pd/C添加到溶液中。將反應混合物在氫氣氛圍下在室溫攪拌2小時。將反應混合物過濾並在真空中濃縮以得到粗產物15-3b，為白色泡沫狀固體(475mg，粗產物)，其無需進一步純化即可用於下一步。 Compound 15-2b (610 mg, 1.00 mmol) was dissolved in MeOH (10 mL), and then Pd/C was added to the solution. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated in vacuo to give the crude product 15-3b as a white foamy solid (475 mg, crude product), which was used in the next step without further purification.

化合物15-4：Compound 15-4:

將化合物10-1(700mg，348.92μmol)溶解於DCM(20mL)中。在0℃下將DIEA(135mg，1.05mmol)、EDCI(134mg，698μmol)和HOBT(94mg，698μmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物15-3a(199mg，408.51μmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-6% MeOH)在Al2O3上純化以得到化合物15-4a，為白色泡沫狀固體(550mg，產率為64%)。 Compound 10-1 (700 mg, 348.92 μmol) was dissolved in DCM (20 mL). DIEA (135 mg, 1.05 mmol), EDCI (134 mg, 698 μmol) and HOBT (94 mg, 698 μmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 15-3a (199 mg, 408.51 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-6% MeOH) on Al 2 O 3 to give compound 15-4a as a white foamy solid (550 mg, 64% yield).

1H NMR(400MHz,DMSO-d6)δ 7.88-7.80(m,6H),7.75(t,J=5.4Hz,3H),7.38-7.34(m,2H),7.28(t,J=7.6Hz,2H),7.24-7.17(m,5H),7.00(s,1H),6.87(d,J=8.9Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.3,3.4Hz,3H),4.64-4.60(m,1H),4.47(d,J=8.5Hz,3H),4.05-3.98(m,9H),3.92-3.83(m,3H),3.76-3.65(m,9H),3.62-3.45(m,13H),3.45-3.37(m,6H),3.28-3.23(m,2H),3.17-3.09(m, 2H),3.07-2.98(m,12H),2.97-2.89(m,2H),2.69-2.53(m,2H),2.27(t,J=6.2Hz,6H),2.16-2.07(m,11H),2.06-1.96(m,17H),1.88(s,9H),1.78(s,9H),1.57-1.36(m,22H),1.26-1.15(m,14H). 1 H NMR(400MHz, DMSO-d6)δ 7.88-7.80(m,6H),7.75(t, J =5.4Hz,3H),7.38-7.34(m,2H),7.28(t, J =7.6Hz,2H),7.24-7.17(m,5H),7.00(s,1H),6.87(d, J =8.9Hz,4H),5.21(d, J =3.4Hz,3H),4.96(dd, J =11.3,3.4Hz,3H),4.64-4.60(m,1H),4.47(d, J =8.5Hz,3H),4.05-3.98(m,9H),3.92-3.83(m,3H),3.76-3.65(m,9H),3 .62-3.45(m,13H),3.45-3.37(m,6H),3.28-3.23(m,2H),3.17-3.09(m, 2H),3.07-2.98(m,12H),2.97-2.89(m,2H),2.69-2.53(m,2H),2.27(t, J =6.2Hz,6H),2.16-2.07(m,11H),2.06-1.96(m,17H),1.88(s,9H),1.78(s,9H),1.57-1.36(m,22H),1.26-1.15(m,14H).

將化合物10-1(1g，500.29μmol)溶解於DCM(20mL)中。在0℃下將DIEA(194mg，1.50mmol)、EDCI(192mg，1.00mmol)和HOBT(135mg，1.00mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物15-3b(238mg，502.53μmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-6% MeOH)在Al2O3上純化，以得到化合物15-4b，為白色泡沫狀固體(550mg，產率為44%)。 Compound 10-1 (1 g, 500.29 μmol) was dissolved in DCM (20 mL). DIEA (194 mg, 1.50 mmol), EDCI (192 mg, 1.00 mmol) and HOBT (135 mg, 1.00 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 15-3b (238 mg, 502.53 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-6% MeOH) on Al 2 O 3 to give compound 15-4b as a white foamy solid (550 mg, 44% yield).

1H NMR(400MHz,DMSO-d6)δ 7.87-7.80(m,4H),7.74(t,J=5.6Hz,3H),7.40-7.36(m,2H),7.31(t,J=7.7Hz,2H),7.27-7.18(m,5H),6.99(s,1H),6.91-6.87(m,4H),5.22(d,J=3.4Hz,3H),4.96(dd,J=11.3,3.4Hz,3H),4.66-4.62(m,1H),4.48(d,J=8.4Hz,3H),4.05-3.98(m,9H),3.92-3.83(m,3H),3.75-3.67(m,9H),3.57-3.47(m,11H),3.46-3.36(m,6H),3.34-3.27(m,4H),3.23-3.09(m,2H),3.07-2.98(m,12H),2.85(s,2H),2.46-2.31(m,2H),2.30-2.24(m,6H),2.18-2.12(m,2H),2.09(s,9H),2.06-2.01(m,8H),1.99(s,9H),1.88(s,9H),1.77(s,9H),1.55-1.37(m,22H),1.33-1.14(m,14H). 1 H NMR(400MHz, DMSO-d6)δ 7.87-7.80(m,4H),7.74(t, J =5.6Hz,3H),7.40-7.36(m,2H),7.31(t, J =7.7Hz,2H),7.27-7.18(m,5H),6.99(s,1H),6.91-6.87(m,4H),5.22(d, J =3.4Hz,3H),4.96(dd, J =11.3,3.4Hz,3H),4.66-4.62(m,1H),4.48(d, J =8.4Hz,3H),4.05-3.98(m,9H),3.92-3.83(m,3H),3.75-3.67(m,9H),3.57-3.47(m,11 H),3.46-3.36(m,6H),3.34-3.27(m,4H),3.23-3.09(m,2H),3.07-2.98(m,12H),2.85(s ,2H),2.46-2.31(m,2H),2.30-2.24(m,6H),2.18-2.12(m,2H),2.09(s,9H),2.06-2.01 (m,8H),1.99(s,9H),1.88(s,9H),1.77(s,9H),1.55-1.37(m,22H),1.33-1.14(m,14H).

Figure 113123276-A0202-12-0230-293
Figure 113123276-A0202-12-0230-293

化合物16-1：Compound 16-1:

將化合物6-8(190mg，0.403mmol)和化合物10-1(809mg，0.403mmol)溶於DCM(10mL)，然後加入DIEA(156.35mg，1.21mmol)、EDCI(154.61mg，0.81mmol)和HOBT(108.97mg，0.81mmol)到混合物中。將反應在室溫攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-10% MeOH)在Al2O3上純化以得到化合物16-1，為白色泡沫狀固體(772mg，產率為77%)。 Compound 6-8 (190 mg, 0.403 mmol) and compound 10-1 (809 mg, 0.403 mmol) were dissolved in DCM (10 mL), and then DIEA (156.35 mg, 1.21 mmol), EDCI (154.61 mg, 0.81 mmol) and HOBT (108.97 mg, 0.81 mmol) were added to the mixture. The reaction was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on Al 2 O 3 to give compound 16-1 as a white foamy solid (772 mg, 77% yield).

C121H179N11O42質量計算為：2458.22；實測：1079.2[M-DMTr+2H]2+，ESI。 Mass calculated for C 121 H 179 N 11 O 42 : 2458.22; found: 1079.2 [M-DMTr+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.85-7.82(m,6H),7.75-7.72(m,3H),7.38-7.36(m,2H),7.29-7.27(m,2H),7.25-7.22(m,5H),6.98(s,1H),6.87(d,J=8.0Hz,4H),5.21(d,J=4.0Hz,3H),4.97(dd,J=3.2,8.0Hz,3H),4.80-4.70(m,1H),4.48(d,J=8.4Hz,3H),4.02-4.01(m,11H),3.88-3.86(m,5H),3.73-3.69(m,9H),3.55-3.52(m,12H),3.46-3.36(m,5H),3.17(d,J=5.2Hz,1H),3.04-3.00(m,14H),2.29-2.26(m,10H),2.10-2.07(m,8H),2.06-2.02(m,19H),1.99(s,9H),1.77(s,9H),1.52-1.46(m,22H),1.45-1.22(m,12H). 1H NMR(400MHz,DMSO - d6 )δ 7.85-7.82(m,6H),7.75-7.72(m,3H),7.38-7.36(m,2H),7.29-7.27(m,2H),7.25-7.22(m,5H),6.98(s,1H),6.87(d, J =8.0Hz,4H),5.21(d, J =4.0Hz,3H),4.97(dd, J =3.2,8.0Hz,3H),4.80-4.70(m,1H),4.48(d, J =8.4Hz,3H),4.02-4.01(m,11H),3.88-3.86(m,5H),3.73-3.69(m,9H),3.55-3.52(m,12H),3.46-3.36(m,5H),3.17(d, J =5.2Hz,1H),3.04-3.00(m,14H),2.29-2.26(m,10H),2.10-2.07(m,8H),2.06-2 .02(m,19H),1.99(s,9H),1.77(s,9H),1.52-1.46(m,22H),1.45-1.22(m,12H).

Figure 113123276-A0202-12-0232-294
Figure 113123276-A0202-12-0232-294

化合物17-1：Compound 17-1:

將化合物11-11(1.5g，0.78mmol)、DIEA(302.32mg，2.35mmol)、HOBT(212.11mg，1.57mmol)和EDCI(300.92mg，1.57mmol)在0℃下溶於DCM(15mL)，然後加入化合物3-12(493.56mg，0.78mmol)，將混合物在室溫攪拌1小時。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用NaHCO3(飽和水溶液，50mL)和水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物17-1(1.14g，產率為58%)。 Compound 11-11 (1.5 g, 0.78 mmol), DIEA (302.32 mg, 2.35 mmol), HOBT (212.11 mg, 1.57 mmol) and EDCI (300.92 mg, 1.57 mmol) were dissolved in DCM (15 mL) at 0°C, and then compound 3-12 (493.56 mg, 0.78 mmol) was added, and the mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 17-1 (1.14 g, yield 58%).

C129H193N11O39質量計算為：2520.35，實測：1109.35(M-DMTr+2H)2+，ESI。 Mass calculated for C 129 H 193 N 11 O 39 : 2520.35, found: 1109.35 (M-DMTr+2H) 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.83(d,J=9.2Hz,4H),7.73(t,J=9.2Hz,3H),7.47-7.39(m,3H),7.33-7.19(m,7H),6.88(d,J=8.92Hz,4H),5.21 (d,J=3.3Hz,3H),4.98-4.94(m,3H),4.49-4.42(m,5H),4.02(s,9H),3.91-3.86(m,5H),3.73-3.68(m,9H),3.43-3.39(m,5H),3.34-3.30(m,3H),3.02-2.99(m,10H),2.87(s,4H),2.34-2.33(m,2H),2.25-2.21(m,6H),2.10(s,9H),2.10-2.08(m,8H),2.0(s,9H),1.99(s,3H),1.89(s,9H),1.77(s,9H),1.45-1.24(m,64H). 1 H NMR (400MHz, DMSO-d6) δ 7.83 (d, J =9.2Hz, 4H), 7.73 (t, J =9.2Hz, 3H), 7.47-7.39 (m, 3H), 7.33-7.19 (m, 7H), 6.88 (d, J =8.92Hz, 4H), 5.21 (d, J =3.3Hz,3H),4.98-4.94(m,3H),4.49-4.42(m,5H),4.02(s,9H),3.91-3.86(m, 5H),3.73-3.68(m,9H),3.43-3.39(m,5H),3.34-3.30(m,3H),3.02-2.99(m,10 H),2.87(s,4H),2.34-2.33(m,2H),2.25-2.21(m,6H),2.10(s,9H),2.10-2.08 (m,8H),2.0(s,9H),1.99(s,3H),1.89(s,9H),1.77(s,9H),1.45-1.24(m,64H).

Figure 113123276-A0202-12-0233-295
Figure 113123276-A0202-12-0233-295

化合物18-1：Compound 18-1:

0℃下向化合物11-11(1.01g，0.45mmol)在DCM(20mL)中的溶液中加入DIEA(193.61mg，1.85mmol，0.32mL)、EDCI(191.45mg，0.99mmol)和HOBT(134.95mg，0.99mmol)。將反應在0℃攪拌30分鐘。將化合物4-10(300mg，0.49mmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。將反應混合物用飽和NaHCO3溶液(40mL)淬滅，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-8% MeOH)在Al2O3上純化，以得到化合物18-1，為白色泡沫狀固體(500mg，產率為46%)。 To a solution of compound 11-11 (1.01 g, 0.45 mmol) in DCM (20 mL) was added DIEA (193.61 mg, 1.85 mmol, 0.32 mL), EDCI (191.45 mg, 0.99 mmol) and HOBT (134.95 mg, 0.99 mmol) at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 4-10 (300 mg, 0.49 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-8% MeOH) on Al 2 O 3 to give compound 18-1 as a white foamy solid (500 mg, 46% yield).

C127H189N11O39質量計算為：2493.31；實測：1096.7[M-DMTr+2H]2+，ESI。 Mass calculated for C 127 H 189 N 11 O 39 : 2493.31; found: 1096.7 [M-DMTr+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84-7.78(m,2H),7.72(t,J=5.4Hz,3H),7.45(br,1H),7.40-7.39(m,2H),7.33-7.30(m,2H),7.28-7.20(m,5H),5.22(d,J=3.4Hz,3H),4.99-4.95(m,3H),4.64-4.62(m,3H),4.49(d,J=8.4Hz,2H),4.03(s,9H),3.91-3.84(m,5H),3.74(s,6H),3.72-3.68(m,3H),3.45-3.41(m,2H),3.40-3.34(m,4H),3.28-3.16(m,4H),3.01-2.99(m,11H),2.85-2.67(m,4H),2.34-2.32(m,2H),2.27-2.22(m,6H),2.07-2.00(m,17H),1.89(s,9H),1.78(s,9H),1.64-1.56(m,4H),1.55-1.34(m,36H),1.28-1.21(m,16H). 1 H NMR(400MHz,DMSO-d6)δ 7.84-7.78(m,2H),7.72(t, J =5.4Hz,3H),7.45(br,1H),7.40-7.39(m,2H),7.33-7.30(m,2H),7.28-7.20(m,5H),5.22(d, J =3.4Hz,3H),4.99-4.95(m,3H),4.64-4.62(m,3H),4.49(d, J =8.4Hz,2H),4.03(s,9H),3.91-3.84(m,5H),3.74(s,6H),3.72-3.68(m,3H),3 .45-3.41(m,2H),3.40-3.34(m,4H),3.28-3.16(m,4H),3.01-2.99(m,11H),2.8 5-2.67(m,4H),2.34-2.32(m,2H),2.27-2.22(m,6H),2.07-2.00(m,17H),1.89( s,9H),1.78(s,9H),1.64-1.56(m,4H),1.55-1.34(m,36H),1.28-1.21(m,16H).

Figure 113123276-A0202-12-0234-296
Figure 113123276-A0202-12-0234-296

化合物19-1：Compound 19-1:

將化合物11-11(1.59g,832.24μmol)(3.2g,7.16mmol)和化合物5-9(500mg,832.24μmol)溶解於DCM(50mL)。將DIEA(215.12mg，1.66mmol，289.92μL)、 EDCI(239.31mg，1.25mmol)和HOBT(112.45mg，832.24μmol)加入到混合物中。將反應在室溫攪拌過夜。將反應混合物用水(40mL)淬滅，用DCM(50mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由HPLC純化以得到化合物19-1(840mg，產率為36%)。 Compound 11-11 (1.59 g, 832.24 μmol) (3.2 g, 7.16 mmol) and compound 5-9 (500 mg, 832.24 μmol) were dissolved in DCM (50 mL). DIEA (215.12 mg, 1.66 mmol, 289.92 μL), EDCI (239.31 mg, 1.25 mmol) and HOBT (112.45 mg, 832.24 μmol) were added to the mixture. The reaction was stirred at room temperature overnight. The reaction mixture was quenched with water (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by HPLC to give compound 19-1 (840 mg, 36% yield).

C127H189N11O39質量計算為：2492.3；實測：1258.4[M+2Na]2+，ESI。 Mass calculated for C 127 H 189 N 11 O 39 : 2492.3; found: 1258.4 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.85-7.71(m,7H),7.49(s,1H),7.37-7.20(m,9H),6.82-6.87(m,J=8.8Hz,4H),5.22(d,J=3.2Hz,3H),4.97(d,J=11.6Hz,3H),4.49-4.46(m,6H),4.05(s,9H),3.91-3.85(m,5H),3.77-3.62(m,11H),3.41(s,6H),3.01-2.71(m,14H),2.32-2.25(m,6H),2.11-1.78(m,48H),1.62-1.34(m,37H),1.26-1.21(m,20H). 1 H NMR(400MHz, DMSO-d6)δ 7.85-7.71(m,7H),7.49(s,1H),7.37-7.20(m,9H),6.82-6.87(m, J =8.8Hz,4H),5.22(d, J =3.2Hz,3H),4.97(d, J =11.6Hz,3H),4.49-4.46(m,6H),4.05(s,9H),3.91-3.85(m,5H),3.77-3.62(m,11H),3.41(s,6H), 3.01-2.71(m,14H),2.32-2.25(m,6H),2.11-1.78(m,48H),1.62-1.34(m,37H),1.26-1.21(m,20H).

Figure 113123276-A0202-12-0235-297
Figure 113123276-A0202-12-0235-297

化合物20-1a：Compound 20-1a:

將化合物11-11(554mg，289.73μmol)溶解於DCM(20mL)中。在0℃下將DIEA(112mg，869.18μmol)、EDCI(222mg，1.16mmol)和HOBT(157mg，1.16mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物7-9a(170mg，289.73μmol)加入反應混合物中，並將混合物在室溫攪拌2小時。用飽和NaHCO3溶液(40mL)猝滅反應，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾 燥，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物20-1a，為白色泡沫狀固體(640mg，產率為89%)。 Compound 11-11 (554 mg, 289.73 μmol) was dissolved in DCM (20 mL). DIEA (112 mg, 869.18 μmol), EDCI (222 mg, 1.16 mmol) and HOBT (157 mg, 1.16 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 7-9a (170 mg, 289.73 μmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 20-1a as a white foamy solid (640 mg, yield 89%).

1H NMR(400MHz,DMSO-d6)δ 7.86-7.76(m,4H),7.72(t,J=5.4Hz,3H),7.49-7.42(m,1H),7.38(d,J=8.6Hz,2H),7.31(t,J=7.7Hz,2H),7.27-7.19(m,5H),6.88(d,J=8.9Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,3H),4.65(t,J=5.0Hz,1H),4.50-4.41(m,5H),4.05-3.99(m,9H),3.91-3.82(m,5H),3.76-3.66(m,9H),3.49-3.36(m,6H),3.32-3.28(m,1H),3.23-3.09(m,2H),3.08-2.91(m,10H),2.84(s,2H),2.44-2.21(m,8H),2.20-1.92(m,30H),1.88(s,9H),1.77(s,9H),1.70-1.08(m,54H). 1 H NMR(400MHz, DMSO-d6)δ 7.86-7.76(m,4H),7.72(t, J =5.4Hz,3H),7.49-7.42(m,1H),7.38(d, J =8.6Hz,2H),7.31(t, J =7.7Hz,2H),7.27-7.19(m,5H),6.88(d, J =8.9Hz,4H),5.21(d, J =3.4Hz,3H),4.96(dd, J =11.2,3.4Hz,3H),4.65(t, J =5.0Hz,1H),4.50-4.41(m,5H),4.05-3.99(m,9H),3.91-3.82(m,5H),3.76-3.66(m,9H),3.49-3.36(m,6H),3.32-3.28(m,1H),3.23 -3.09(m,2H),3.08-2.91(m,10H),2.84(s,2H),2.44-2.21(m,8H),2.20-1.92(m,30H),1.88(s,9H),1.77(s,9H),1.70-1.08(m,54H).

化合物20-1b：Compound 20-1b:

將化合物11-11(1.3g，681.71μmol)溶解於DCM(20mL)中。在0℃下，將DIEA(176mg，1.36mmol)、EDCI(261mg，1.36mmol)和HOBT(138mg，1.02mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物7-9b(400mg，681.71μmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。 Compound 11-11 (1.3 g, 681.71 μmol) was dissolved in DCM (20 mL). DIEA (176 mg, 1.36 mmol), EDCI (261 mg, 1.36 mmol) and HOBT (138 mg, 1.02 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 7-9b (400 mg, 681.71 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours.

用飽和NaHCO3溶液(40mL)猝滅反應混合物，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物20-1b，為白色泡沫狀固體(610mg，產率為36%)。 The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 20-1b as a white foamy solid (610 mg, yield 36%).

1H NMR(400MHz,DMSO-d6)δ：7.87-7.76(m,4H),7.72(t,J=5.3Hz,3H),7.46(s,1H),7.35(d,J=7.4Hz,2H),7.28(t,J=7.6Hz,2H),7.25-7.18(m,5H),6.86(d,J=8.9Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,3H),4.62(t,J=5.0Hz,1H),4.50-4.38(m,5H),4.07-3.98(m,9H),3.92-3.82(m,5H),3.75-3.66(m,9H),3.51-3.36(m,6H),3.29-3.23(m,2H),3.15-3.07(m,2H),3.01-2.94(m,10H),2.93-2.88(m,2H),2.87-2.78(m,2H),2.68-2.60(m,1H),2.37-2.23(m,2H), 2.29-2.19(m,4H),2.13-2.08(m,10H),2.07-1.98(m,18H),1.88(s,9H),1.77(s,9H),1.71-1.58(m,6H),1.53-1.13(m,48H). 1 H NMR (400MHz, DMSO-d6) δ: 7.87-7.76 (m, 4H), 7.72 (t, J =5.3Hz, 3H), 7.46 (s, 1H), 7.35 (d, J =7.4Hz, 2H), 7.28 (t, J =7.6Hz,2H),7.25-7.18(m,5H),6.86(d, J =8.9Hz,4H),5.21(d, J =3.4Hz,3H),4.96(dd, J =11.2,3.4Hz,3H),4.62(t, J =5.0Hz,1H),4.50-4.38(m,5H),4.07-3.98(m,9H),3.92-3.82(m,5H),3.75-3.66(m,9H),3.51-3.36(m,6H),3.29-3.23(m ,2H),3.15-3.07(m,2H),3.01-2.94(m,10H),2.93-2.88(m,2H),2.87-2.78(m,2H),2.68-2.60(m,1H),2.37-2.23(m,2H), 2.29-2.19(m,4H),2.13-2.08(m,10H),2.07-1.98(m,18H),1.88(s,9H),1.77(s,9H),1.71-1.58(m,6H),1.53-1.13(m,48H).

Figure 113123276-A0202-12-0237-298
Figure 113123276-A0202-12-0237-298

化合物21-1：Compound 21-1:

將化合物11-11(1.76g，920.31μmol)、HOBt(248.70mg，1.84mmol)、EDCI(529.27mg，2.76mmol)溶於DCM(20mL)中。攪拌混合物30分鐘，然後加入化合物8-9(540mg，920.31μmol)。繼續攪拌反應混合物2小時，然後將其濃縮後經製備型HPLC純化以得到化合物21-1(1.2g，產率為53%)。 Compound 11-11 (1.76 g, 920.31 μmol), HOBt (248.70 mg, 1.84 mmol), and EDCI (529.27 mg, 2.76 mmol) were dissolved in DCM (20 mL). The mixture was stirred for 30 minutes, and then compound 8-9 (540 mg, 920.31 μmol) was added. The reaction mixture was stirred for 2 hours, and then concentrated and purified by preparative HPLC to obtain compound 21-1 (1.2 g, yield 53%).

C126H187N11O39質量計算為：2478.3；實測：1089.6[M-DMTr+2H]2+，ESI。 Mass calculated for C 126 H 187 N 11 O 39 : 2478.3; found: 1089.6 [M-DMTr+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84-7.80(m,4H),7.72(t,J=5.4Hz,3H),7.39(s,1H),7.40-7.37(m,2H),7.31(t,J=7.7Hz,2H),7.27-7.20(m,5H),6.98(d,J=8.9Hz,4H),5.22(d,J=3.4Hz,3H),4.97(dd,J=3.4,7.8Hz,3H),4.67 (t,J=4.8Hz,1H),4.48(d,J=8.5Hz,3H),4.47-4.42(m,2H),4.05-3.98(m,9H),3.92-3.83(m,5H),3.73(s,6H),3.72-3.67(m,3H),3.55-3.45(m,2H),3.44-3.35(m,5H),3.05-2.93(m,11H),2.91-2.80(m,3H),2.65-2.55(m,2H),2.38-2.20(m,9H),2.10(s,9H),2.08-2.01(m,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.69-1.52(m,6H),1.47-0.98(m,48H). 1 H NMR(400MHz, DMSO-d6)δ 7.84-7.80(m,4H),7.72(t, J =5.4Hz,3H),7.39(s,1H),7.40-7.37(m,2H),7.31(t, J =7.7Hz,2H),7.27-7.20(m,5H),6.98(d, J =8.9Hz,4H),5.22(d, J =3.4Hz,3H),4.97(dd, J =3.4,7.8Hz,3H),4.67 (t, J =4.8Hz,1H),4.48(d, J =8.5Hz,3H),4.47-4.42(m,2H),4.05-3.98(m,9H),3.92-3.83(m,5H),3.73(s,6H) ,3.72-3.67(m,3H),3.55-3.45(m,2H),3.44-3.35(m,5H),3.05-2.93(m,11H),2.9 1-2.80(m,3H),2.65-2.55(m,2H),2.38-2.20(m,9H),2.10(s,9H),2.08-2.01(m,9 H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.69-1.52(m,6H),1.47-0.98(m,48H).

Figure 113123276-A0202-12-0238-299
Figure 113123276-A0202-12-0238-299

化合物22-1：Compound 22-1:

將化合物6-10(410mg，0.71mmol)和化合物11-11(1.34g，0.71mmol)溶於DCM(60mL)，然後加入DIEA(453.09mg，3.51mmol)、EDCI(299.57mg，1.75mmol)和HOBT(236.85mg，1.75mmol)到該溶液中。將反應混合物在室溫下攪拌2小時。將反應用飽和NaHCO3溶液(100mL)淬滅，用DCM(60mLx3)萃取，用鹽水(60mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-20% MeOH)在Al2O3上純化以得到化合物22-1，為白色泡沫狀固體(1.2g，產率為69%)。 Compound 6-10 (410 mg, 0.71 mmol) and compound 11-11 (1.34 g, 0.71 mmol) were dissolved in DCM (60 mL), and DIEA (453.09 mg, 3.51 mmol), EDCI (299.57 mg, 1.75 mmol) and HOBT (236.85 mg, 1.75 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated NaHCO 3 solution (100 mL), extracted with DCM (60 mL×3), washed with brine (60 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-20% MeOH) on Al 2 O 3 to give compound 22-1 as a white foamy solid (1.2 g, 69% yield).

C126H185N11O39質量計算為：2476.28；實測：1088.2[M-DMTr+2H]2+，ESI。 Mass calculated for C 126 H 185 N 11 O 39 : 2476.28; found: 1088.2 [M-DMTr+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d 6)δ 7.85-7.81(m,3H),7.75-7.72(m,3H),7.47(s,H),7.39-7.37(m,2H),7.32-7.25(m,2H),7.24-7.19(m,5H),6.88(d,J=8.84Hz,4H),5.22(d,J=3.4Hz,2H),4.97(d,J=11.2Hz,3H),4.75(t,J=4.4Hz,1H),4.50-4.48(m,5H),4.03(s,11H),3.94-3.80(m,7H),3.77-3.66(m,9H),3.50-3.41(m,6H),3.02-3.00(m,12H),2.92-2.79(m,2H),2.43-2.30(m,2H),2.29-2.22(m,2H),2.21-2.19(m,3H),2.11-2.08(m,11H),2.05-2.02(m,8H),2.00(s,9H),1.90(s,9H),1.78(s,9H),1.62-1.23(m,48H). 1H NMR(400MHz,DMSO - d6 )δ 7.85-7.81(m,3H),7.75-7.72(m,3H),7.47(s,H),7.39-7.37(m,2H),7.32-7.25(m,2H),7.24-7.19(m,5H),6.88(d, J =8.84Hz,4H),5.22(d, J =3.4Hz,2H),4.97(d, J =11.2Hz,3H),4.75(t, J =4.4Hz,1H),4.50-4.48(m,5H),4.03(s,11H),3.94-3.80(m,7H),3.77-3. 66(m,9H),3.50-3.41(m,6H),3.02-3.00(m,12H),2.92-2.79(m,2H),2.43- 2.30(m,2H),2.29-2.22(m,2H),2.21-2.19(m,3H),2.11-2.08(m,11H),2.0 5-2.02(m,8H),2.00(s,9H),1.90(s,9H),1.78(s,9H),1.62-1.23(m,48H).

Figure 113123276-A0202-12-0239-300
Figure 113123276-A0202-12-0239-300

化合物23-1：Compound 23-1:

N-Boc-4-哌啶酮(5g，25.09mmol)、丙二腈(2.49g，37.64mmol)、AcNH2(3.87g，50.19mmol)和AcOH(4.52g，75.28mmol)在甲苯(50mL)中的溶液在110℃下攪拌2小時。LC-MS顯示完全轉化。用水(50mL)洗滌反應混合物，用Na2SO4乾燥，濃縮並藉由矽膠管柱(0-10% EA在PE中的溶液)純化以得到呈膠狀的化合物23-1(6.18g，產率為98%)。 A solution of N- Boc-4-piperidone (5 g, 25.09 mmol), malononitrile (2.49 g, 37.64 mmol), AcNH2 (3.87 g, 50.19 mmol) and AcOH (4.52 g, 75.28 mmol) in toluene (50 mL) was stirred at 110 °C for 2 hours. LC-MS showed complete conversion. The reaction mixture was washed with water ( 50 mL), dried over Na2SO4 , concentrated and purified by silica gel column (0-10% EA in PE) to give compound 23-1 (6.18 g, 98% yield) as a gel.

C13H17O2N3質量計算為：247.1；實測：246.2[M-H]-，ESI。 Mass calculated for C 13 H 17 O 2 N 3 : 247.1; found: 246.2 [MH] - , ESI.

1H NMR(400MHz,DMSO-d6)δ 3.53(t,J=11.6Hz,4H),2.69(d,J=11.6Hz,4H),7.41(t,J=7.3Hz,2H),1.427(s,9H). 1 H NMR (400MHz, DMSO-d6) δ 3.53 (t, J =11.6Hz, 4H), 2.69 (d, J =11.6Hz, 4H), 7.41 (t, J =7.3Hz, 2H), 1.427 (s, 9H).

化合物23-2：Compound 23-2:

將化合物23-1(7.6g，30.73mmol)在MeOH(76mL)的溶液中在0℃下攪拌。向攪拌溶液中分批加入NaBH4(1.16g，30.73mmol)，攪拌20分鐘。混合物真空濃縮。將殘餘物在乙酸乙酯(250mL)和水(150mL)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用PE/EA=1/5沖提以得到呈膠狀的化合物23-2(4.6g，產率為60%)。 Compound 23-1 (7.6 g, 30.73 mmol) was stirred in a solution of MeOH (76 mL) at 0°C. NaBH 4 (1.16 g, 30.73 mmol) was added in batches to the stirred solution and stirred for 20 minutes. The mixture was concentrated in vacuo. The residue was partitioned between ethyl acetate (250 mL) and water (150 mL), the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with PE/EA = 1/5 to obtain compound 23-2 (4.6 g, 60% yield) in a gelatinous state.

C13H19O2N3質量計算為：249.2；實測：248.3[M-H]-，ESI。 Mass calculated for C 13 H 19 O 2 N 3 : 249.2; found: 248.3 [MH] - , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.04-4.01(m,2H),2.75(s,2H),2.31-2.27(m,1H),1.82-1.78(m,2H),1.04(s,9H),1.27-1.16(m,3H). 1 H NMR (400MHz, DMSO-d6)δ 4.04-4.01(m,2H),2.75(s,2H),2.31-2.27(m,1H),1.82-1.78(m,2H),1.04(s,9H),1.27-1.16(m,3H).

化合物23-3：Compound 23-3:

將化合物23-2(25g，100.28mmol)、3-溴丙酸三級丁酯(41.93g，200.56mmol)、t-BuOK(22.50g，200.56mmol)和KI(33.29g，200.56mmol)在DMSO(250mL)中的混合物在60℃下攪拌過夜。LC-MS顯示完全轉化。混合物真空濃縮。將殘餘物在乙酸乙酯(100mL×3)和水(150mL×3)之間分配，用鹽水(100ml)洗滌乙酸乙 酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用PE/EA=10/1沖提以得到呈膠狀的化合物23-3(32.87g，產率為87%)。 A mixture of compound 23-2 (25 g, 100.28 mmol), 3-bromopropionic acid tributyl ester (41.93 g, 200.56 mmol), t-BuOK (22.50 g, 200.56 mmol) and KI (33.29 g, 200.56 mmol) in DMSO (250 mL) was stirred at 60 ° C overnight. LC-MS showed complete conversion. The mixture was concentrated in vacuo. The residue was partitioned between ethyl acetate (100 mL×3) and water (150 mL×3), and the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and eluted with PE/EA = 10/1 to obtain compound 23-3 (32.87 g, yield 87%) as a gel.

C20H31O4N3質量計算為：377.2；實測：319.4[M-Boc+MeCN+H]+，ESI。 Mass calculated for C 20 H 31 O 4 N 3 : 377.2; found: 319.4 [M-Boc+MeCN+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.06(d,J=11.2Hz,2H),2.75(s,2H),2.55-2.53(m,1H),2.35-2.31(m,3H),1.89(d,J=12.4Hz,2H),1.43(s,9H),1.41(s,9H),1.27-1.19(m,3H). 1 H NMR(400MHz, DMSO-d6)δ 4.06(d, J =11.2Hz,2H),2.75(s,2H),2.55-2.53(m,1H),2.35-2.31(m,3H),1.89(d, J =12.4Hz,2H),1.43(s,9H),1.41(s,9H),1.27-1.19(m,3H).

化合物23-4：Compound 23-4:

將六水合氯化鈷(40.34g，169.55mmol)加入化合物23-3(16g，42.39mmol)在MeOH(400mL)中的溶液中，然後，在-20℃下將硼氫化鈉(32.07g，847.73mmol)緩慢加入到反應混合物中，攪拌1小時。然後，將混合物在室溫下攪拌1小時。將反應混合物濃縮至乾，用DMF共沸乾燥，以得到粗化合物23-4，其無需進一步純化即可直接用於下一步。 Cobalt chloride hexahydrate (40.34 g, 169.55 mmol) was added to a solution of compound 23-3 (16 g, 42.39 mmol) in MeOH (400 mL), and then sodium borohydride (32.07 g, 847.73 mmol) was slowly added to the reaction mixture at -20°C and stirred for 1 hour. Then, the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness and azeotropically dried with DMF to obtain crude compound 23-4 , which was directly used in the next step without further purification.

C20H39O4N3質量計算為：385.5；實測：386.5[M+H]+，ESI。 Mass calculated for C 20 H 39 O 4 N 3 : 385.5; found: 386.5 [M+H] + , ESI.

化合物23-5：Compound 23-5:

將Z-6-胺基己酸(27.53g，103.75mmol)、TEA(12.6g，124.50mmol)、HATU(46.97g，124.50mmol)在DMF(240mL)中的溶液在室溫攪拌30分鐘，將化合物23-4(16g，41.50mmol)加入，並在室溫攪拌過夜。LC-MS顯示完全轉化。將水(50mL)和EA(50mL)加入到反應混合物。將混合物過濾，將過濾物在乙酸乙酯(100mL×3)和水(150mL×3)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由製備型HPLC(C18管柱，ACN/水)純化，以得到呈膠狀的化合物23-5(8.3g，產率為23%)。 A solution of Z-6-aminohexanoic acid (27.53 g, 103.75 mmol), TEA (12.6 g, 124.50 mmol), HATU (46.97 g, 124.50 mmol) in DMF (240 mL) was stirred at room temperature for 30 minutes, compound 23-4 (16 g, 41.50 mmol) was added, and stirred at room temperature overnight. LC-MS showed complete conversion. Water (50 mL) and EA (50 mL) were added to the reaction mixture. The mixture was filtered, and the filtrate was partitioned between ethyl acetate (100 mL×3) and water (150 mL×3), and the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC (C18 column, ACN/water) to give compound 23-5 (8.3 g, 23% yield) as a gel.

C48H72O11N4質量計算為：879.5；實測：880.1[M+H]+，ESI。 Mass calculated for C 48 H 72 O 11 N 4 : 879.5; found: 880.1 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.73(t,J=12.4Hz,2H),7.38-7.30(m,10H),7.23(t,J=10.8Hz,2H),5.00(s,4H),4.06-3.60(m,4H),3.00-2.97(m,8H),2.23-2.19(m,4H),1.99(s,2H),1.58-1.48(m,6H),1.42-1.38(m,25H),1.25-1.23(m,4H). 1 H NMR(400MHz,DMSO-d6)δ 7.73(t, J =12.4Hz,2H),7.38-7.30(m,10H),7.23(t, J =10.8Hz,2H),5.00(s,4H),4.06-3.60(m,4H),3.00-2.97(m,8H),2.23-2.19( m,4H),1.99(s,2H),1.58-1.48(m,6H),1.42-1.38(m,25H),1.25-1.23(m,4H).

化合物23-6：Compound 23-6:

在氫氣氣氛下，將化合物23-5(3.8g，4.31mmol)、NH3.H2O(3.5mL)和Pd/C(760mg)在MeOH(38mL)中的混合物在室溫下攪拌2小時。LC-MS顯示完全轉化。過濾混合物，將濾液在真空中濃縮，以得到粗化合物23-6，其無需進一步純化即可直接用於下一步。 Under hydrogen atmosphere, a mixture of compound 23-5 (3.8 g, 4.31 mmol), NH 3 .H 2 O (3.5 mL) and Pd/C (760 mg) in MeOH (38 mL) was stirred at room temperature for 2 hours. LC-MS showed complete conversion. The mixture was filtered and the filtrate was concentrated in vacuo to give crude compound 23-6 , which was used directly in the next step without further purification.

C32H60N4O7質量計算為：611.8；實測：612.8[M+H]+，ESI。 Mass calculated for C 32 H 60 N 4 O 7 : 611.8; found: 612.8 [M+H] + , ESI.

化合物23-7：Compound 23-7:

將化合物1-4(2.46g，5.49mmol)、TEA(757.89mg，7.49mmol)和HATU(2.35mg，6.24mmol)在DCM(15mL)中的混合物在室溫下攪拌30分鐘。將化合物23-6(1.53g，2.5mmol)加入，並在室溫攪拌混合物過夜。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，然後用水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物23-7(3.14g，產率為85%)。 A mixture of compound 1-4 (2.46 g, 5.49 mmol), TEA (757.89 mg, 7.49 mmol) and HATU (2.35 mg, 6.24 mmol) in DCM (15 mL) was stirred at room temperature for 30 minutes. Compound 23-6 (1.53 g, 2.5 mmol) was added, and the mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), then washed with water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 23-7 (3.14 g, 85% yield).

1H NMR(400MHz,DMSO-d6)δ 7.82(d,J=9.2Hz,2H),7.71(s,4H),5.52(d,J=0.4Hz,2H),4.99-4.95(m,2H),4.48(d,J=8.8Hz,2H),4.03(s,8H),3.95-3.84(m,2H),3.72-3.70(m,2H),3.44-3.38(m,2H),3.01-2.99(m,8H),2.23-2.19(m,2H),2.11-2.07(m,10H),2.05-2.00(m,10H),1.90(s,6H),1.78(s,6H),1.58-1.49(m,14H),1.39-1.38(m,25H),1.24-1.23(m,8H). 1 H NMR(400MHz, DMSO-d6)δ 7.82(d, J =9.2Hz,2H),7.71(s,4H),5.52(d, J =0.4Hz,2H),4.99-4.95(m,2H),4.48(d, J =8.8Hz,2H),4.03(s,8H),3.95-3.84(m,2H),3.72-3.70(m,2H),3.44-3.38(m,2H),3.01-2.99(m,8H),2.23-2.19(m,2H),2. 11-2.07(m,10H),2.05-2.00(m,10H),1.90(s,6H),1.78(s,6H),1.58-1.49(m,14H),1.39-1.38(m,25H),1.24-1.23(m,8H).

化合物23-8：Compound 23-8:

將化合物23-7(3.0g，2.04mmol)和TFA(12mL)在DCM(30mL)中的溶液在室溫下攪拌4小時。LC-MS顯示完全轉化。真空濃縮溶液，然後加入TEA進行鹼化。化合物23-8無需進一步純化即可直接用於下一步。 A solution of compound 23-7 (3.0 g, 2.04 mmol) and TFA (12 mL) in DCM (30 mL) was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The solution was concentrated in vacuo and then TEA was added for alkalization. Compound 23-8 was used directly in the next step without further purification.

C61H99O24N7質量計算為：1313.4；實測：1314.4[M+H]+，ESI。 Mass calculated for C 61 H 99 O 24 N 7 : 1313.4; found: 1314.4 [M+H] + , ESI.

化合物23-9：Compound 23-9:

將化合物1-7(2.68g，2.04mmol)、TEA(412.62.89mg，4.08mmol)和HATU(769.19mg，2.04mmol)在DCM(30mL)中的溶液在室溫攪拌4小時。然後，將該溶液加入到化合物23-8(2.68g，2.04mmol)，並在室溫攪拌過夜。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物23-9(1.76g，產率為47%)。 A solution of compound 1-7 (2.68 g, 2.04 mmol), TEA (412.62.89 mg, 4.08 mmol) and HATU (769.19 mg, 2.04 mmol) in DCM (30 mL) was stirred at room temperature for 4 hours. Then, the solution was added to compound 23-8 (2.68 g, 2.04 mmol) and stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 23-9 (1.76 g, 47% yield).

C86H137O35N9質量計算為：1857.0；實測：929.0[M+2H]2+，ESI。 Mass calculated for C 86 H 137 O 35 N 9 : 1857.0; found: 929.0 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.94(d,J=9.2Hz,2H),7.88-7.79(m,6H),5.21(d,J=3.2Hz,3H),4.99-4.95(m,3H),4.51-4.48(m,4H),4.05-4.00(m,9H),3.92-3.84(m,4H),3.73-3.37(m,3H),2.99(s,10H),2.81(t,J=23.2Hz,1H),2.48-2.44(m,2H),2.34-2.16(m,6H),2.11-2.09(m,13H),2.05-2.00(m,16H),1.89(s,9H),1.78(s,9H),1.49-1.35(m,31H),1.24-1.23(m,6H). 1 H NMR(400MHz,DMSO-d6)δ 7.94(d, J =9.2Hz,2H),7.88-7.79(m,6H),5.21(d, J =3.2Hz,3H),4.99-4.95(m,3H),4.51-4.48(m,4H),4.05-4.00(m,9H),3.92-3.84(m,4H),3.73-3.37(m,3H),2.99(s,10H),2.81(t, J =23.2Hz,1H),2.48-2.44(m,2H),2.34-2.16(m,6H),2.11-2.09(m,13H),2.05- 2.00(m,16H),1.89(s,9H),1.78(s,9H),1.49-1.35(m,31H),1.24-1.23(m,6H).

化合物23-10：Compound 23-10:

將化合物23-9(1.76g，0.94mmol)、DIEA(367.47mg，2.84mmol)、HOBT(256.12mg，1.90mmol)和EDCI(363.37mg，1.90mmol)在DCM(20mL)中的混合物在0℃攪拌1小時，然後加入化合物11-2(542.80mg，0.94mmol)。將混合物在室溫攪拌過夜。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20 mL)稀釋，用NaHCO3(飽和水溶液，50mL)和水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物23-10(1.07g，產率為47%)。 A mixture of compound 23-9 (1.76 g, 0.94 mmol), DIEA (367.47 mg, 2.84 mmol), HOBT (256.12 mg, 1.90 mmol) and EDCI (363.37 mg, 1.90 mmol) in DCM (20 mL) was stirred at 0 ° C for 1 hour, and then compound 11-2 (542.80 mg, 0.94 mmol) was added. The mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 23-10 (1.07 g, yield 47%).

1H NMR(400MHz,DMSO-d6)δ 7.83(d,J=9.2Hz,3H),7.76-7.65(m,6H),7.40-7.30(m,4H),7.26-7.22(m,5H),6.92-6.89(m,4H),5.22(d,J=0.8Hz,3H),4.99-4.95(m,3H),4.65(d,J=10.4Hz,1H),4.49(d,J=8.4Hz,4H),4.03-3.98(m,10H),3.91-3.89(m,4H),3.74-3.67(m,11H),3.51(s,1H),3.41-3.39(m,5H),3.01-2.92(m,14H),2.27-2.25(m,14H),2.05-2.02(m,7H),2.00-1.94(m,12H),1.78(s,9H),1.49-1.37(m,33H),1.24-1.22(m,11H). 1 H NMR (400MHz, DMSO-d6) δ 7.83 (d, J =9.2Hz, 3H), 7.76-7.65 (m, 6H), 7.40-7.30 (m, 4H), 7.26-7.22 (m, 5H), 6.92-6.89 (m, 4H), 5.22 (d, J =0.8Hz,3H),4.99-4.95(m,3H),4.65(d, J =10.4Hz,1H),4.49(d, J =8.4Hz,4H),4.03-3.98(m,10H),3.91-3.89(m,4H),3.74-3.67(m,11H),3.51(s,1H),3.41-3.39(m,5H),3.01-2.92(m, 14H),2.27-2.25(m,14H),2.05-2.02(m,7H),2.00-1.94(m,12H),1.78(s,9H),1.49-1.37(m,33H),1.24-1.22(m,11H).

Figure 113123276-A0202-12-0244-301
Figure 113123276-A0202-12-0244-301

化合物24-1：Compound 24-1:

將4-吡啶基乙腈(5g，42.32mmol)、N-Cbz-4-碘哌啶(21.91g，63.49mmol)和KOH(4.75g，84.64mmol)溶解於DMF(50mL)中。將反應混合物在室溫攪拌過夜。LC-MS顯示完全轉化。將殘餘物在乙酸乙酯(100mL×2)和水(80mL×3)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘 物藉由管柱層析純化，用DCM/MeOH=20/1沖提以得到化合物24-1，為淺棕色油狀物(11.3g，產率為80%)。 4-Pyridylacetonitrile (5 g, 42.32 mmol), N-Cbz-4-iodopiperidine (21.91 g, 63.49 mmol) and KOH (4.75 g, 84.64 mmol) were dissolved in DMF (50 mL). The reaction mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The residue was partitioned between ethyl acetate (100 mL×2) and water (80 mL×3), the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH=20/1 to give compound 24-1 as a light brown oil (11.3 g, 80% yield).

C20H21N3O2質量計算為：335.4；實測：336.2[M+H]+，ESI。 Mass calculated for C 20 H 21 N 3 O 2 : 335.4; found: 336.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.62-8.61(m,2H),7.40-7.31(m,7H),5.06(s,2H),4.35(d,J=7.2Hz,1H),4.34-4.00(m,2H),2.75(s,2H),2.12-2.06(m,1H),1.64-1.53(m,2H),1.24-1.14(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 8.62-8.61(m,2H),7.40-7.31(m,7H),5.06(s,2H),4.35(d, J =7.2Hz,1H),4.34-4.00(m,2H),2.75(s,2H),2.12-2.06(m,1H),1.64-1.53(m,2H),1.24-1.14(m,2H).

化合物24-2：Compound 24-2:

在N2氣氛下，將KI(2.97g，17.89mmol)和NaH(858.6mg，35.78mmol)在無水THF(10mL)中的溶液在0℃下冷卻10分鐘。加入化合物24-1(3g，8.94mmol)在無水THF(15mL)中的溶液。之後，將混合物攪拌30分鐘。然後，加入N-(氯甲基)胺基甲酸苄酯(2.14g，10.73mmol)在無水THF(5mL)中的溶液，在N2氣氛下在室溫攪拌過夜。用水淬滅混合物，用乙酸乙酯(50mLx3)萃取混合物。用鹽水(60ml)洗滌合併的有機層，用無水MgSO4乾燥並真空濃縮。殘餘物藉由管柱層析純化，用二氯甲烷/甲醇4：1沖提以得到化合物24-2，為黃色油狀物(1.8g，產率為40%)。 Under N2 atmosphere, a solution of KI (2.97 g, 17.89 mmol) and NaH (858.6 mg, 35.78 mmol) in anhydrous THF (10 mL) was cooled at 0 ° C for 10 minutes. A solution of compound 24-1 (3 g, 8.94 mmol) in anhydrous THF (15 mL) was added. After that, the mixture was stirred for 30 minutes. Then, a solution of N- (chloromethyl)benzylcarbamate (2.14 g, 10.73 mmol) in anhydrous THF (5 mL) was added and stirred at room temperature overnight under N2 atmosphere. The mixture was quenched with water and extracted with ethyl acetate (50 mLx3). The combined organic layers were washed with brine (60 ml), dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by column chromatography and extracted with dichloromethane/methanol 4:1 to give compound 24-2 as a yellow oil (1.8 g, yield 40%).

C29H30N4O4質量計算為：498.2；實測：499.2[M+H]+，ESI。 Mass calculated for C 29 H 30 N 4 O 4 : 498.2; found: 499.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO)δ 8.61(d,J=5.2Hz,2H),7.52-7.57(m,1H),7.44-7.22(m,12H),5.06(s,2H),5.03-4.91(m,2H),4.14(d,J=13.6Hz,1H),3.97-3.89(m,2H),3.73-3.68(m,1H),2.89-2.68(m,2H),2.39-2.34(t,J=11.6Hz,1H),2.07(d,J=11.8Hz,2H),1.31-1.21(m,1H),1.18-1.12(m,1H),1.04-0.94(m,1H). 1 H NMR (400MHz, DMSO) δ 8.61 (d, J =5.2Hz, 2H), 7.52-7.57 (m, 1H), 7.44-7.22 (m, 12H), 5.06 (s, 2H), 5.03-4.91 (m, 2H), 4.14 (d, J =13.6Hz,1H),3.97-3.89(m,2H),3.73-3.68(m,1H),2.89-2.68(m,2H),2.39-2.34(t, J =11.6Hz,1H),2.07(d, J =11.8Hz,2H),1.31-1.21(m,1H),1.18-1.12(m,1H),1.04-0.94(m,1H).

化合物24-3：Compound 24-3:

將CoCl2.6H2O(2.97g，17.89mmol)和化合物24-2(5g，10.03mmol)在無水MeOH(50mL)中的溶液在N2氣氛下在-50℃冷卻20分鐘。在-50℃下逐滴加入LiBH4在THF(50mmol，2M,25mL)中的溶液，並攪拌3小時，然後在-50℃下逐滴加入無水MeOH(50mL)。將混合物升溫至室溫，並在N2氣氛下攪拌過夜。混合物真空濃縮。將殘餘物在二氯甲烷(250mL)和水(150mL)之間分配，用鹽水(100mL)洗滌二氯甲烷萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用DCM/MeOH=15/1沖提以得到化合物24-3，為淺黃色固體(1.8g，產率為40%)。 A solution of CoCl 2 .6H 2 O (2.97 g, 17.89 mmol) and compound 24-2 (5 g, 10.03 mmol) in anhydrous MeOH (50 mL) was cooled at -50 °C for 20 min under N 2 atmosphere. A solution of LiBH 4 in THF (50 mmol, 2M, 25 mL) was added dropwise at -50 °C and stirred for 3 hours, and then anhydrous MeOH (50 mL) was added dropwise at -50 °C. The mixture was warmed to room temperature and stirred overnight under N 2 atmosphere. The mixture was concentrated in vacuo. The residue was partitioned between dichloromethane (250 mL) and water (150 mL), and the dichloromethane extract was washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH = 15/1 to give compound 24-3 as a light yellow solid (1.8 g, yield 40%).

C29H34N4O4質量計算為：502.2；實測：503.2[M+H]+，ESI。 Mass calculated for C 29 H 34 N 4 O 4 : 502.2; found: 503.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.48(d,J=5.2Hz,2H),7.50-7.47(m,1H),7.37-7.25(m,12H),5.02(s,2H),4.99(s,2H),3.96(d,J=12.4Hz,2H),3.69-3.58(m,2H),3.16(d,J=14Hz,1H),2.94(d,J=13.2Hz,1H),2.63(s,2H),1.93(s,2H),1.77-1.68(m,2H),1.26(d,J=8.8Hz,1H),0.86-0.77(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 8.48(d, J =5.2Hz,2H),7.50-7.47(m,1H),7.37-7.25(m,12H),5.02(s,2H),4.99(s,2H),3.96(d, J =12.4Hz,2H),3.69-3.58(m,2H),3.16(d, J =14Hz,1H),2.94(d, J =13.2Hz,1H),2.63(s,2H),1.93(s,2H),1.77-1.68(m,2H),1.26(d, J =8.8Hz,1H),0.86-0.77(m,2H).

化合物24-4：Compound 24-4:

將化合物24-3(2.1g，4.18mmol)、5-(三級丁氧基)-5-側氧戊酸(943.7mg，5.01mmol)、HATU(3.15g，8.36mmol)和Et3N(1.27g，12.53mmol)溶解於DCM(20mL)中。將反應混合物在室溫下攪拌2小時。然後，用CH2Cl2(50mL)稀釋反應混合物。用水(50mLx2)洗滌有機層，用硫酸鈉乾燥，真空濃縮。殘餘物藉由管柱層析純化，用DCM/MeOH=20/1沖提以得到化合物24-4，為淺黃色固體(2.23g，產率為81%)。 Compound 24-3 (2.1 g, 4.18 mmol), 5-(t-butyloxy)-5-oxopentanoic acid (943.7 mg, 5.01 mmol), HATU (3.15 g, 8.36 mmol) and Et 3 N (1.27 g, 12.53 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 hours. Then, the reaction mixture was diluted with CH 2 Cl 2 (50 mL). The organic layer was washed with water (50 mL x 2), dried over sodium sulfate, and concentrated in vacuo. The residue was purified by column chromatography and extracted with DCM/MeOH = 20/1 to obtain compound 24-4 as a light yellow solid (2.23 g, yield 81%).

C38H48N4O7質量計算為：672.3；實測：673.3[M+H]+，ESI。 Mass calculated for C 38 H 48 N 4 O 7 : 672.3; found: 673.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.48(d,J=5.2Hz,2H),7.83-7.79(m,1H),7.36-7.21(m,13H),5.01(s,2H),4.98(s,2H),3.96(d,J=12.4Hz,2H),3.65- 3.52(m,4H),2.68-2.62(m,2H),2.12-2.09(m,4H),1.77-1.69(m,5H),1.35(s,9H),0.76-0.64(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 8.48(d, J =5.2Hz,2H),7.83-7.79(m,1H),7.36-7.21(m,13H),5.01(s,2H),4.98(s,2H),3.96(d, J =12.4Hz,2H),3.65- 3.52(m,4H),2.68-2.62(m,2H),2.12-2.09(m,4H),1.77-1.69(m,5H),1.35(s,9H),0.76-0.64(m,2H).

化合物24-5：Compound 24-5:

將PtO2(1.21g，5.36mmol)和化合物24-4(1.8g，2.67mmol)在HOAc(10mL)中的溶液在H2氣氛下在室溫下攪拌過夜。然後，過濾混合物，將溶劑在真空中蒸發，以得到粗產物24-5，其直接用於下一步。 A solution of PtO 2 (1.21 g, 5.36 mmol) and compound 24-4 (1.8 g, 2.67 mmol) in HOAc (10 mL) was stirred under H 2 atmosphere at room temperature overnight. Then, the mixture was filtered and the solvent was evaporated in vacuo to give the crude product 24-5 , which was used directly in the next step.

C22H42N4O3質量計算為：410.3；實測：411.3[M+H]+，ESI。 Mass calculated for C 22 H 42 N 4 O 3 : 410.3; found: 411.3 [M+H] + , ESI.

化合物24-6：Compound 24-6:

將化合物1-7(4.76g，8.52mmol)、TEA(1.48g，14.6mmol)、HATU(2.94g，7.8mmol)在DCM(15mL)中的混合物在室溫下攪拌30分鐘，然後加入化合物24-5(1g，2.44mmol)，並在室溫攪拌2小時。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物24-6，為白色固體(1g，產率為20%)。 A mixture of compound 1-7 (4.76 g, 8.52 mmol), TEA (1.48 g, 14.6 mmol), HATU (2.94 g, 7.8 mmol) in DCM (15 mL) was stirred at room temperature for 30 minutes, then compound 24-5 (1 g, 2.44 mmol) was added and stirred at room temperature for 2 hours. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 24-6 as a white solid (1 g, yield 20%).

C97H156N10O36質量計算為：2037.07；實測：1019.1[M+2H]2+，ESI。 Mass calculated for C 97 H 156 N 10 O 36 : 2037.07; found: 1019.1 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.80(d,J=9.2Hz,3H),7.73-7.63(m,6H),5.22(d,J=3.2Hz,3H),4.97(dd,J=11.2,3.2Hz,3H),4.50-4.47(m,5H),4.08-4.01(m,9H),3.91-3.86(m,5H),3.84-3.70(m,3H),3.43-3.40(m,3H),3.07-3.05(m,3H),2.81(d,J=9.2Hz,2H),2.68-2.67(m,1H),2.34-2.23(m,12H),2.18(s,9H),2.16-2.11(m,5H),2.03(s,9H),1.90(s,9H),1.78(s,9H),1.72-1.67(m,14H),1.54-1.48(m,18H),1.43-1.37(m,13H),1.28-1.18(m,12H). 1 H NMR (400MHz, DMSO-d6) δ 7.80 (d, J =9.2Hz, 3H), 7.73-7.63 (m, 6H), 5.22 (d, J = 3.2Hz, 3H), 4.97 (dd, J =11.2,3.2Hz,3H),4.50-4.47(m,5H),4.08-4.01(m,9H),3.91-3.86(m,5H),3.84-3.70(m,3H),3.43-3.40(m,3H),3.07-3.05(m,3H),2.81(d, J =9.2Hz,2H),2.68-2.67(m,1H),2.34-2.23(m,12H),2.18(s,9H),2.16-2.11(m,5H),2.03(s,9H),1.90 (s,9H),1.78(s,9H),1.72-1.67(m,14H),1.54-1.48(m,18H),1.43-1.37(m,13H),1.28-1.18(m,12H).

化合物24-7：Compound 24-7:

將化合物24-6(1.0g，0.49mmol)和TFA(12mL)在DCM(30mL)中的溶液在室溫下攪拌4小時。LC-MS顯示完全轉化。真空濃縮溶液，然後加入TEA進行鹼化。粗化合物24-7無需進一步純化直接用於下一步。 A solution of compound 24-6 (1.0 g, 0.49 mmol) and TFA (12 mL) in DCM (30 mL) was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The solution was concentrated in vacuo and then TEA was added for alkalization. The crude compound 24-7 was used directly in the next step without further purification.

C93H148N10O36質量計算為：1981.0；實測：991.4[M+2H]2+，ESI。 Mass calculated for C 93 H 148 N 10 O 36 : 1981.0; found: 991.4 [M+2H] 2+ , ESI.

化合物24-8：Compound 24-8:

將化合物24-7(400mg，0.21mmol)、TEA(61.26mg，0.61mmol)、HATU(252.26mg，0.40mmol)在DCM(5mL)中的混合物，室溫攪拌1小時，然後加入化合物11-2(115.16mg，0.20mmol)，室溫攪拌過夜。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(5mL)稀釋，用水(20mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物24-8，為白色固體(200mg，產率為39%)。 A mixture of compound 24-7 (400 mg, 0.21 mmol), TEA (61.26 mg, 0.61 mmol), HATU (252.26 mg, 0.40 mmol) in DCM (5 mL) was stirred at room temperature for 1 hour, and then compound 11-2 (115.16 mg, 0.20 mmol) was added and stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (5 mL), washed with water (20 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 24-8 as a white solid (200 mg, 39% yield).

1H NMR(400MHz,DMSO-d6)δ 7.83(d,J=9.2Hz,3H),7.76-7.66(m,5H),7.40-7.30(m,4H),7.26-7.22(m,5H),6.92-6.88(m,4H),5.22(d,J=3.2Hz,3H),4.99-4.95(m,3H),4.66(t,J=5.6Hz,1H),4.48(d,J=8.4Hz,4H),4.43-4.42(m,1H),4.05-3.97(m,9H),3.91-3.86(m,5H),3.74-3.62(m,11H),3.51(s,1H),3.43-3.38(m,5H),3.06-2.92(m,13H),2.86-2.82(m,2H),2.34-2.22(m,6H),2.11(s,9H),2.08-2.06(m,3H),2.05-2.02(m,8H),2.00(s,9H),1.96-1.94(m,3H),1.89(s,9H),1.85-1.82(m,3H),1.78(s,9H),1.72-1.62(m,5H),1.52-1.42(m,18H),1.39-1.33(m,8H),1.28-1.22(m,9H),0.95(t,J=8.4Hz,1H). 1 H NMR (400MHz, DMSO-d6) δ 7.83 (d, J =9.2Hz, 3H), 7.76-7.66 (m, 5H), 7.40-7.30 (m, 4H), 7.26-7.22 (m, 5H), 6.92-6.88 (m, 4H), 5.22 (d, J =3.2Hz,3H),4.99-4.95(m,3H),4.66(t, J =5.6Hz,1H),4.48(d, J =8.4Hz,4H),4.43-4.42(m,1H),4.05-3.97(m,9H),3.91-3.86(m,5H),3.74-3.62(m,11H),3.51(s, 1H),3.43-3.38(m,5H),3.06-2.92(m,13H),2.86-2.82(m,2H),2.34-2.22(m,6H),2.11(s,9H),2.0 8-2.06(m,3H),2.05-2.02(m,8H),2.00(s,9H),1.96-1.94(m,3H),1.89(s,9H),1.85-1.82(m,3H), 1.78(s,9H),1.72-1.62(m,5H),1.52-1.42(m,18H),1.39-1.33(m,8H),1.28-1.22(m,9H),0.95(t, J =8.4Hz,1H).

Figure 113123276-A0202-12-0249-302
Figure 113123276-A0202-12-0249-302

化合物25-1：Compound 25-1:

將N-Boc-哌啶-4-甲醛(50g，234.44mmol)、丙二腈(18.59g，281.33mmol)和二異丙胺(11.86g，117.21mmol)在H2O(200mL)和EtOH(800mL)中的溶液在室溫攪拌2小時。LC-MS顯示完全轉化。用水(50mL)洗滌反應混合物，用Na2SO4乾燥，濃縮並藉由矽膠管柱(0-50% EA在PE中的溶液)純化，得到化合物25-1(30.00g，產率為46%)。 A solution of N-Boc-piperidine-4-carbaldehyde (50 g, 234.44 mmol), malononitrile (18.59 g, 281.33 mmol) and diisopropylamine (11.86 g, 117.21 mmol) in H 2 O (200 mL) and EtOH (800 mL) was stirred at room temperature for 2 hours. LC-MS showed complete conversion. The reaction mixture was washed with water (50 mL), dried over Na 2 SO 4 , concentrated and purified by silica gel column (0-50% EA in PE) to give compound 25-1 (30.00 g, 46% yield).

C14H19N3O2質量計算為：261.1；實測：260.2[M-H]-，ESI。 Mass calculated for C 14 H 19 N 3 O 2 : 261.1; found: 260.2 [MH] - , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84(d,J=10.2Hz,1H),3.97-3.89(m,2H),2.94-2.72(m,3H),1.69-1.59(m,2H),1.45-1.34(m,13H). 1 H NMR (400MHz, DMSO-d6) δ 7.84 (d, J =10.2Hz, 1H), 3.97-3.89 (m, 2H), 2.94-2.72 (m, 3H), 1.69-1.59 (m, 2H), 1.45-1.34 (m, 13H).

化合物25-2：Compound 25-2:

將化合物25-1(30g，114.80mmol)在MeOH(450mL)中的溶液在-5℃下攪拌。向溶液中分批加入NaBH4(4.34g，114.80mmol)，攪拌3小時。混合物真空濃縮。 將殘餘物在乙酸乙酯(250mL)和水(150mL)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用PE/EA=3/1沖提得到化合物25-2(19.4g，產率為64%)。 A solution of compound 25-1 (30 g, 114.80 mmol) in MeOH (450 mL) was stirred at -5 °C. NaBH 4 (4.34 g, 114.80 mmol) was added to the solution in batches and stirred for 3 hours. The mixture was concentrated in vacuo. The residue was partitioned between ethyl acetate (250 mL) and water (150 mL), the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with PE/EA = 3/1 to give compound 25-2 (19.4 g, yield 64%).

C14H21N3O2質量計算為：263.2；實測：262.3[M-H]-，ESI。 Mass calculated for C 14 H 21 N 3 O 2 : 263.2; found: 262.3 [MH] - , ESI.

1H NMR(400MHz,DMSO-d6)δ 4.87(t,J=7.6Hz,1H),3.95-3.87(m,2H),2.82-2.61(m,2H),1.98(t,J=7.2Hz,2H),1.70-1.56(m,3H),1.42-1.35(m,9H),1.11-0.98(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 4.87(t, J =7.6Hz,1H),3.95-3.87(m,2H),2.82-2.61(m,2H),1.98(t, J =7.2Hz,2H),1.70-1.56(m,3H),1.42-1.35(m,9H),1.11-0.98(m,2H).

化合物25-3：Compound 25-3:

將化合物25-2(15g，56.96mmol)、3-溴丙酸三級丁酯(17.86g，85.44mmol)、t-BuOK(8.31g，74.05mmol)和KI(9.46g，56.96mmol)在DMSO(250mL)中的混合物在室溫下攪拌3小時。LC-MS顯示完全轉化。將混合物在乙酸乙酯(100mL×3)和水(150mL×3)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由管柱層析純化，用PE/EA=7/1沖提得到化合物25-3(17.4g，產率為94%)。 A mixture of compound 25-2 (15 g, 56.96 mmol), 3-bromopropionic acid tributyl ester (17.86 g, 85.44 mmol), t-BuOK (8.31 g, 74.05 mmol) and KI (9.46 g, 56.96 mmol) in DMSO (250 mL) was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The mixture was partitioned between ethyl acetate (100 mL×3) and water (150 mL×3), the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography and extracted with PE/EA=7/1 to give compound 25-3 (17.4 g, yield 94%).

C21H33N3O4質量計算為：391.2；實測：333.3[M-Boc+MeCN+H]+，ESI。 Mass calculated for C 21 H 33 N 3 O 4 : 391.2; found: 333.3 [M-Boc+MeCN+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 3.96-3.87(m,2H),2.76(s,2H),2.55-2.50(m,2H),2.36-2.31(m,2H),2.05(d,J=6.2Hz,2H),1.85-1.74(m,3H),1.42(s,9H),1.39(s,9H),1.21-1.08(m,2H). 1 H NMR(400MHz,DMSO-d6)δ 3.96-3.87(m,2H),2.76(s,2H),2.55-2.50(m,2H),2.36-2.31(m,2H),2.05(d, J =6.2Hz,2H),1.85-1.74(m,3H),1.42(s,9H),1.39(s,9H),1.21-1.08(m,2H).

化合物25-4：Compound 25-4:

將六水合氯化鈷(29.17g，122.60mmol)加入化合物25-3(12g，30.65mmol)在MeOH(350mL)中的溶液中，然後在-20℃下將硼氫化鈉(23.19g，613.02mmol) 緩慢加入到反應混合物，攪拌1小時。然後，將混合物在室溫下攪拌1小時。將反應混合物濃縮至乾，用DMF共沸乾燥。粗化合物25-4無需進一步純化直接用於下一步。 Cobalt chloride hexahydrate (29.17 g, 122.60 mmol) was added to a solution of compound 25-3 (12 g, 30.65 mmol) in MeOH (350 mL), and then sodium borohydride (23.19 g, 613.02 mmol) was slowly added to the reaction mixture at -20 °C and stirred for 1 hour. Then, the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness and azeotropically dried with DMF. The crude compound 25-4 was used directly in the next step without further purification.

C21H41N3O4質量計算為：399.3；實測：400.4[M+H]+，ESI。 Mass calculated for C 21 H 41 N 3 O 4 : 399.3; found: 400.4 [M+H] + , ESI.

化合物25-5：Compound 25-5:

將Z-6-胺基己酸(17.52g，66.04mmol)、TEA(9.10g，90.05mmol)和HATU(28.31g，75.04mmol)在DMF(240mL)中的溶液在室溫攪拌30分鐘，將化合物25-4(12g，30.03mmol)加入，並在室溫攪拌過夜。LC-MS顯示完全轉化。將水(50mL)和EA(50mL)加入到反應混合物。將混合物過濾，並將濾液在乙酸乙酯(100mL×3)和水(150mL×3)之間分配，用鹽水(100ml)洗滌乙酸乙酯萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由製備型HPLC(C18管柱，ACN/水)純化，得到化合物25-5(4.08g，產率為14%)。 A solution of Z-6-aminohexanoic acid (17.52 g, 66.04 mmol), TEA (9.10 g, 90.05 mmol) and HATU (28.31 g, 75.04 mmol) in DMF (240 mL) was stirred at room temperature for 30 minutes, compound 25-4 (12 g, 30.03 mmol) was added, and stirred at room temperature overnight. LC-MS showed complete conversion. Water (50 mL) and EA (50 mL) were added to the reaction mixture. The mixture was filtered, and the filtrate was partitioned between ethyl acetate (100 mL×3) and water (150 mL×3), and the ethyl acetate extract was washed with brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC (C18 column, ACN/water) to give compound 25-5 (4.08 g, yield 14%).

C49H75N5O10質量計算為：893.6；實測：894.7[M+H]+，ESI。 Mass calculated for C 49 H 75 N 5 O 10 : 893.6; found: 894.7 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.68(t,J=6.1Hz,2H),7.38-7.27(m,10H),7.22(t,J=5.5Hz,2H),4.99(s,4H),3.89-3.79(m,2H),3.00-2.93(m,4H),2.92-2.85(m,3H),2.73-2.56(m,2H),2.23-2.16(m,2H),2.13-2.06(m,4H),1.61-1.42(m,9H),1.42-1.34(m,24H),1.28-1.18(m,4H),1.05-0.99(m,2H). 1 H NMR(400MHz,DMSO-d6)δ 7.68(t, J =6.1Hz,2H),7.38-7.27(m,10H),7.22(t, J =5.5Hz,2H),4.99(s,4H),3.89-3.79(m,2H),3.00-2.93(m,4H),2.92-2.85(m,3H),2.73-2.56(m,2H),2.23- 2.16(m,2H),2.13-2.06(m,4H),1.61-1.42(m,9H),1.42-1.34(m,24H),1.28-1.18(m,4H),1.05-0.99(m,2H).

化合物25-6：Compound 25-6:

在氫氣氣氛下，將化合物25-5(3.0g，3.36mmol)、NH3.H2O(15mL)和Pd/C(1.20g)在MeOH(60mL)中的混合物在室溫下攪拌4小時。LC-MS顯示完全轉化。過濾混合物，將濾液在真空中濃縮，粗化合物25-6無需進一步純化直接用於下一步。 Under hydrogen atmosphere, a mixture of compound 25-5 (3.0 g, 3.36 mmol), NH 3 .H 2 O (15 mL) and Pd/C (1.20 g) in MeOH (60 mL) was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The mixture was filtered, the filtrate was concentrated in vacuo, and the crude compound 25-6 was used directly in the next step without further purification.

C33H63N5O6質量計算為：625.5；實測：626.5[M+H]+，ESI。 Mass calculated for C 33 H 63 N 5 O 6 : 625.5; found: 626.5 [M+H] + , ESI.

化合物25-7：Compound 25-7:

將化合物1-4(3.30g，7.38mmol)、TEA(1.02g，10.07mmol)和HATU(3.04g，8.05mmol)在DCM(15mL)中的混合物在室溫下攪拌30分鐘。將化合物25-6(2.10g，3.36mmol)加入，並在室溫攪拌過夜。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化得到化合物25-7(3.14g，產率為86%)。 A mixture of compound 1-4 (3.30 g, 7.38 mmol), TEA (1.02 g, 10.07 mmol) and HATU (3.04 g, 8.05 mmol) in DCM (15 mL) was stirred at room temperature for 30 minutes. Compound 25-6 (2.10 g, 3.36 mmol) was added and stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 25-7 (3.14 g, 86% yield).

1H NMR(400MHz,DMSO-d6)δ 7.82(d,J=9.3Hz,2H),7.74-7.65(m,4H),5.21(d,J=3.4Hz,2H),4.99-4.93(m,2H),4.48(d,J=8.5Hz,2H),4.05-3.98(m,6H),3.91-3.80(m,4H),3.74-3.67(m,2H),3.44-3.36(m,2H),3.03-2.95(m,4H),2.91-2.86(m,3H),2.73-2.60(m,2H),2.24-2.17(m,2H),2.13-2.07(m,10H),2.05-1.98(m,10H),1.89(s,6H),1.71(s,6H),1.61-1.32(m,41H),1.27-1.19(m,4H),1.04-0.99(m,2H). 1 H NMR(400MHz, DMSO-d6)δ 7.82(d, J =9.3Hz,2H),7.74-7.65(m,4H),5.21(d, J =3.4Hz,2H),4.99-4.93(m,2H),4.48(d, J =8.5Hz,2H),4.05-3.98(m,6H),3.91-3.80(m,4H),3.74-3.67(m,2H),3. 44-3.36(m,2H),3.03-2.95(m,4H),2.91-2.86(m,3H),2.73-2.60(m,2H) ,2.24-2.17(m,2H),2.13-2.07(m,10H),2.05-1.98(m,10H),1.89(s,6H) ,1.71(s,6H),1.61-1.32(m,41H),1.27-1.19(m,4H),1.04-0.99(m,2H).

化合物25-8：Compound 25-8:

將化合物25-7(1.0g，673.53μmol)和TFA(10mL)在DCM(20mL)中的溶液在室溫下攪拌4小時。LC-MS顯示完全轉化。真空濃縮溶液，然後加入TEA進行鹼化。粗化合物25-8無需進一步純化直接用於下一步。 A solution of compound 25-7 (1.0 g, 673.53 μmol) and TFA (10 mL) in DCM (20 mL) was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The solution was concentrated in vacuo and then TEA was added for alkalization. The crude compound 25-8 was used directly in the next step without further purification.

C62H101N7O24質量計算為：1327.7；實測：665.0[M+2H]2+，ESI。 Mass calculated for C 62 H 101 N 7 O 24 : 1327.7; found: 665.0 [M+2H] 2+ , ESI.

化合物25-9：Compound 25-9:

將化合物1-7(1.43g，2.56mmol)、TEA(777mg，7.68mmol)和HATU(1.06g，2.82mmol)在DCM(30mL)中的混合物在室溫下攪拌30分鐘。然後，將該溶液加入到化合物22-8(3.4g，2.56mmol)，並在室溫攪拌過夜。LC-MS顯示完全 轉化。將反應混合物藉由加入DCM(20mL)稀釋，用水(50mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物25-9(2.5g，產率為49%)。 A mixture of compound 1-7 (1.43 g, 2.56 mmol), TEA (777 mg, 7.68 mmol) and HATU (1.06 g, 2.82 mmol) in DCM (30 mL) was stirred at room temperature for 30 minutes. Then, the solution was added to compound 22-8 (3.4 g, 2.56 mmol) and stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with water (50 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 25-9 (2.5 g, 49% yield).

C87H139N9O35質量計算為：1869.9；實測：936.3[M+2H]2+，ESI。 Mass calculated for C 87 H 139 N 9 O 35 : 1869.9; found: 936.3 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.96-7.90(m,2H),7.87-7.75(m,5H),7.71(s,1H),5.21(d,J=3.4Hz,3H),4.99-4.94(m,3H),4.52-4.47(m,3H),4.32-4.25(m,1H),4.05-3.98(m,9H),3.92-3.82(m,3H),3.80-3.67(m,5H),3.52-3.42(m,4H),3.03-2.91(m,10H),2.89-2.80(m,2H),2.24(t,J=7.5Hz,2H),2.20-2.13(m,2H),2.13-2.07(m,13H),2.05-1.96(m,15H),1.89(s,9H),1.77(s,9H),1.68-1.55(m,4H),1.54-1.32(m,26H),1.28-1.18(m,6H),1.04-0.98(m,2H). 1 H NMR(400MHz,DMSO-d6)δ 7.96-7.90(m,2H),7.87-7.75(m,5H),7.71(s,1H),5.21(d, J =3.4Hz,3H),4.99-4.94(m,3H),4.52-4.47(m,3H),4.32-4.25(m,1H),4.05-3.98(m,9H),3.92-3 .82(m,3H),3.80-3.67(m,5H),3.52-3.42(m,4H),3.03-2.91(m,10H),2.89-2.80(m,2H),2.24(t, J =7.5Hz,2H),2.20-2.13(m,2H),2.13-2.07(m,13H),2.05-1.96(m,15H),1.89(s,9H),1. 77(s,9H),1.68-1.55(m,4H),1.54-1.32(m,26H),1.28-1.18(m,6H),1.04-0.98(m,2H).

化合物25-10：Compound 25-10:

將化合物25-9(750mg，400.84μmol)、DIEA(155mg，1.20mmol)、HOBT(217.12mg，1.60mmol)和EDCI(307.36mg，1.06mmol)在DCM(20mL)中的混合物攪拌30分鐘，加入化合物11-2(229.57mg，400.84μmol)，室溫攪拌3小時。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用NaHCO3(飽和水溶液，30mL)和水(30mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物25-10(500mg，產率為51%)。 A mixture of compound 25-9 (750 mg, 400.84 μmol), DIEA (155 mg, 1.20 mmol), HOBT (217.12 mg, 1.60 mmol) and EDCI (307.36 mg, 1.06 mmol) in DCM (20 mL) was stirred for 30 minutes, compound 11-2 (229.57 mg, 400.84 μmol) was added, and stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 30 mL) and water (30 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 25-10 (500 mg, yield 51%).

1H NMR(400MHz,DMSO-d6)δ 7.83(d,J=9.2Hz,3H),7.75-7.65(m,6H),7.41-7.34(m,2H),7.31(t,J=7.1Hz,2H),7.27-7.18(m,5H),6.93-6.84(m,4H),5.22(d,J=3.0Hz,3H),5.00-4.90(m,3H),4.71-4.56(m,1H),4.48(d,J=8.4Hz,3H),4.28(d,J=10.7Hz,1H),4.11-3.93(m,10H),3.92-3.82(m,3H),3.77-3.63(m,12H),3.51(s,1H),3.45-3.36(m,6H),3.05-2.79(m,15H),2.28-2.19(m,2H),2.12-2.08(m,12H),2.06-1.98(m,17H),1.96-1.92(m,3H),1.89(s,9H),1.85-1.80(m,2H),1.77(s,9H),1.60-1.00(m,47H). 1 H NMR(400MHz, DMSO-d6)δ 7.83(d, J =9.2Hz,3H),7.75-7.65(m,6H),7.41-7.34(m,2H),7.31(t, J =7.1Hz,2H),7.27-7.18(m,5H),6.93-6.84(m,4H),5.22(d, J =3.0Hz,3H),5.00-4.90(m,3H),4.71-4.56(m,1H),4.48(d, J =8.4Hz,3H),4.28(d, J =10.7Hz,1H),4.11-3.93(m,10H),3.92-3.82(m,3H),3.77-3.63(m,12H),3.51(s,1H),3.45-3.36(m,6H),3.05-2.79(m,15H),2.28-2. 19(m,2H),2.12-2.08(m,12H),2.06-1.98(m,17H),1.96-1.92(m,3H),1.89(s,9H),1.85-1.80(m,2H),1.77(s,9H),1.60-1.00(m,47H).

Figure 113123276-A0202-12-0254-303
Figure 113123276-A0202-12-0254-303

化合物26-1：Compound 26-1:

將5-(苄氧基)-5-側氧戊酸(0.3g，1.35mmol)、化合物11-2(773.14mg，1.35mmol)、DIEA(523.40mg，4.05mmol)、EDC(517.56mg，2.70mmol)、HOBT(364.81mg，2.70mmol)在DCM(20mL)中的混合物在室溫攪拌3小時。LC-MS顯示完全轉化。反應混合物藉由加入DCM(20mL)稀釋，用NaHCO3(飽和水溶液，150mL)和水(150mL)洗滌，濃縮並藉由管柱層析純化，用DCM/MeOH=20：1沖提，以得到化合物26-1(480mg，產率為45%)。 A mixture of 5-(benzyloxy)-5-hydroxypentanoic acid (0.3 g, 1.35 mmol), compound 11-2 (773.14 mg, 1.35 mmol), DIEA (523.40 mg, 4.05 mmol), EDC (517.56 mg, 2.70 mmol), HOBT (364.81 mg, 2.70 mmol) in DCM (20 mL) was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 150 mL) and water (150 mL), concentrated and purified by column chromatography, and extracted with DCM/MeOH=20:1 to obtain compound 26-1 (480 mg, yield 45%).

C47H56N2O8質量計算為：776.4；實測：799.3[M+Na]+，ESI。 Mass calculated for C 47 H 56 N 2 O 8 : 776.4; found: 799.3 [M+Na] + , ESI.

化合物26-2：Compound 26-2:

化合物26-1(0.48g，0.629mmol)和Pd/C(48mg)在MeOH(20mL)中的混合物在室溫下在H2下攪拌3小時。LC-MS顯示完全轉化。將反應混合物濃縮並藉由 管柱層析純化，用DCM/MeOH=10：1沖提以得到化合物26-2(280mg，產率為65%)。 A mixture of compound 26-1 (0.48 g, 0.629 mmol) and Pd/C (48 mg) in MeOH (20 mL) was stirred at room temperature under H 2 for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by column chromatography, and extracted with DCM/MeOH=10:1 to give compound 26-2 (280 mg, yield 65%).

C47H56N2O8質量計算為：686.3；實測：725.3[M+K]+,ESI C 47 H 56 N 2 O 8 mass calculated: 686.3; found: 725.3 [M+K] + , ESI

1H NMR(400MHz,DMSO-d6)δ 7.79-7.75(m,1H),7.40-7.35(m,2H),7.34-7.30(m,2H),7.27-7.20(m,5H),6.92-6.89(m,4H),3.98(s,1H),3.68(d,J=6.4Hz,2H),3.51(s,1H),3.40-3.38(m,2H),3.00(t,J=6.4Hz,2H),2.93-2.92(m,2H),2.19(t,J=7.4Hz,2H),2.09-2.05(m,2H),2.03-1.88(m,4H),1.85-1.80(m,2H),1.71-1.68(m,2H),1.42-1.33(m,4H),1.24-1.18(m,4H). 1 H NMR (400MHz, DMSO-d6)δ 7.79-7.75(m,1H),7.40-7.35(m,2H),7.34-7.30(m,2H),7.27-7.20(m,5H),6.92-6.89(m,4H),3.98(s,1H),3.68(d, J =6.4Hz,2H),3.51(s,1H),3.40-3.38(m,2H),3.00(t, J =6.4Hz,2H),2.93-2.92(m,2H),2.19(t, J =7.4Hz,2H),2.09-2.05(m,2H),2.03-1.88(m,4H),1.85-1.80(m,2H),1.71-1.68(m,2H),1.42-1.33(m,4H),1.24-1.18(m,4H).

化合物26-3：Compound 26-3:

化合物26-2(3.1g，6.17mmol)和Pd/C(2.97g)在無水MeOH(40mL)中的混合物在室溫下在H2下攪拌過夜。過濾混合物，將濾液減壓濃縮，以得到粗化合物26-3，為白色固體(1.4g，產率為97%)，其無需純化即使用。 A mixture of compound 26-2 (3.1 g, 6.17 mmol) and Pd/C (2.97 g) in anhydrous MeOH (40 mL) was stirred at room temperature under H 2 overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure to give crude compound 26-3 as a white solid (1.4 g, yield 97%), which was used without purification.

化合物26-4：Compound 26-4:

在N2氣氛下，將化合物26-3(1.50g，6.40mmol)、化合物1-7(11.12g，19.84mmol)、EDCI(7.34g，38.41mmol)、HOBT(5.19g，38.41mmol)、DIEA(7.45g，57.61mmol)在無水DCM(50mL)中的混合物在室溫攪拌3小時。將混合物真空濃縮，然後用二氯甲烷(100mLx3)萃取，用鹽水(100mL)洗滌，用硫酸鈉乾燥並真空濃縮。殘餘物藉由製備型HPLC(C18管柱，ACN/水)純化，以得到化合物26-4，為淺黃色固體(6g，產率為50%)。 Under N2 atmosphere, a mixture of compound 26-3 (1.50 g, 6.40 mmol), compound 1-7 (11.12 g, 19.84 mmol), EDCI (7.34 g, 38.41 mmol), HOBT (5.19 g, 38.41 mmol), DIEA (7.45 g, 57.61 mmol) in anhydrous DCM (50 mL) was stirred at room temperature for 3 hours. The mixture was concentrated in vacuo, then extracted with dichloromethane (100 mL x 3), washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC (C18 column, ACN/water) to obtain compound 26-4 as a light yellow solid (6 g, 50% yield).

C88H136N10O33質量計算為：1860.9；實測：931.5[M+2H]2+，ESI。 Mass calculated for C 88 H 136 N 10 O 33 : 1860.9; found: 931.5 [M+2H] 2+ , ESI.

化合物26-5：Compound 26-5:

化合物26-4(1.01g，0.53mmol)和PtO2(0.243g，1.07mmol)在AcOH(20mL)中的混合物在H2下攪拌過夜。將反應混合物過濾並在真空中濃縮。然後，殘餘物藉由製備型HPLC(C18管柱，ACN/水)純化，以得到化合物26-5，為白色固體(640mg，產率為64%)。 A mixture of compound 26-4 (1.01 g, 0.53 mmol) and PtO 2 (0.243 g, 1.07 mmol) in AcOH (20 mL) was stirred under H 2 overnight. The reaction mixture was filtered and concentrated in vacuo. The residue was then purified by preparative HPLC (C18 column, ACN/water) to give compound 26-5 as a white solid (640 mg, yield 64%).

C88H142N10O33質量計算為：1867.0；實測：934.5[M+2H]2+，ESI。 Mass calculated for C 88 H 142 N 10 O 33 : 1867.0; found: 934.5 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.86-7.84(m,3H),7.75-7.73(m,3H),7.68-7.61(m,2H),5.22(d,J=3.3Hz,3H),4.97(q,J=3.3Hz,3H),4.50-4.43(m,4H),4.03(s,9H),3.91-3.84(m,4H),3.75-3.70(m,3H),3.43-3.38(m,11H),3.06-2.99(m,12H),2.83-2.77(m,1H),2.46-2.42(m,2H),2.27-2.23(m,4H),2.11-2.09(m,13H),2.07-2.00(m,16H),1.90(s,9H),1.78(s,9H),1.68-1.62(m,5H),1.49-1.46(m,22H),1.39-1.36(m,9H),1.25-1.20(m,9H). 1 H NMR(400MHz, DMSO-d6)δ 7.86-7.84(m,3H),7.75-7.73(m,3H),7.68-7.61(m,2H),5.22(d, J =3.3Hz,3H),4.97(q, J =3.3Hz,3H),4.50-4.43(m,4H),4.03(s,9H),3.91-3.84(m,4H),3.75-3.70(m,3H ),3.43-3.38(m,11H),3.06-2.99(m,12H),2.83-2.77(m,1H),2.46-2.42(m,2H), 2.27-2.23(m,4H),2.11-2.09(m,13H),2.07-2.00(m,16H),1.90(s,9H),1.78(s, 9H),1.68-1.62(m,5H),1.49-1.46(m,22H),1.39-1.36(m,9H),1.25-1.20(m,9H).

化合物26-6：Compound 26-6:

將化合物26-5(0.543g，0.29mmol)、化合物26-2(200mg，2.12mmol)、DIEA(112mg，0.87mmol)、EDC(111mg，0.58mmol)和HOBT(80mg，0.58mmol)在DCM(20mL)中的混合物在室溫攪拌3小時。LC-MS顯示完全轉化。將反應混合物藉由加入DCM(20mL)稀釋，用NaHCO3(飽和水溶液，150mL)和水(150mL)洗滌，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物26-6(0.15g，產率為20%)。 A mixture of compound 26-5 (0.543 g, 0.29 mmol), compound 26-2 (200 mg, 2.12 mmol), DIEA (112 mg, 0.87 mmol), EDC (111 mg, 0.58 mmol) and HOBT (80 mg, 0.58 mmol) in DCM (20 mL) was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO 3 (saturated aqueous solution, 150 mL) and water (150 mL), concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 26-6 (0.15 g, yield 20%).

C121H179O39N11質量計算為：3411.7；實測：1117.2[M-DMTr+2H]2+，ESI。 Mass calculated for C121H179O39N11: 3411.7; found: 1117.2 [M-DMTr+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84(d,J=9.3Hz,3H),7.78-7.72(m,5H),7.65(s,2H),7.40-7.37(m,2H),7.32(t,J=7.1Hz,2H),7.26-7.20(m,6H), 6.92-6.89(m,4H),5.22(d,J=3.4Hz,2H),4.98-4.95(m,2H),4.65(t,J=5.0Hz,1H),4.48(d,J=8.4Hz,2H),4.45-4.42(m,2H),4.05-4.03(m,9H),3.98(s,2H),3.91-3.84(m,5H),3.74(s,7H),3.71-3.67(m,5H),3.51(s,1H),3.43-3.39(m,6H),3.07-2.97(m,13H),2.92(s,2H),2.81-2.78(m,2H),2.34-2.22(m,8H),2.13-2.07(m,16H),2.05-1.96(m,20H),1.89(s,11H),1.79-1.77(m,10H),1.70-1.62(m,7H),1.47-1.41(m,23H),1.40-1.35(m,14H),1.24-1.22(m,14H). 1 H NMR(400MHz, DMSO-d6)δ 7.84(d, J =9.3Hz,3H),7.78-7.72(m,5H),7.65(s,2H),7.40-7.37(m,2H),7.32(t, J =7.1Hz,2H),7.26-7.20(m,6H), 6.92-6.89(m,4H),5.22(d, J =3.4Hz,2H),4.98-4.95(m,2H),4.65(t, J =5.0Hz,1H),4.48(d, J =8.4Hz,2H),4.45-4.42(m,2H),4.05-4.03(m,9H),3.98(s,2H),3.91-3.84(m,5H),3.74(s,7 H),3.71-3.67(m,5H),3.51(s,1H),3.43-3.39(m,6H),3.07-2.97(m,13H),2.92(s,2H),2.81- 2.78(m,2H),2.34-2.22(m,8H),2.13-2.07(m,16H),2.05-1.96(m,20H),1.89(s,11H),1.79- 1.77(m,10H),1.70-1.62(m,7H),1.47-1.41(m,23H),1.40-1.35(m,14H),1.24-1.22(m,14H).

Figure 113123276-A0202-12-0257-304
Figure 113123276-A0202-12-0257-304

化合物27-1：Compound 27-1:

將化合物11-6(15g，45.81mmol)、Boc-8-胺基辛酸(13.07g，50.39mmol)、Et3N(6.95g，68.72mmol)和HATU(19.01g，50.39mmol)的混合物在室溫攪拌1小時。將矽藻土(15g)、水(300mL)和EA(400mL)添加到混合物中。攪拌30分鐘後過濾反應物。用EA(400mL)萃取濾液。用水(500mLx2)和鹽水(300mL)洗滌有機相。用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(0-10% MeOH)在矽膠上純化，以得到化合物27-1(15g，產率為57%)。 A mixture of compound 11-6 (15 g, 45.81 mmol), Boc-8-aminocaprylic acid (13.07 g, 50.39 mmol), Et3N (6.95 g, 68.72 mmol) and HATU (19.01 g, 50.39 mmol) was stirred at room temperature for 1 hour. Celite (15 g), water (300 mL) and EA (400 mL) were added to the mixture. After stirring for 30 minutes, the reaction was filtered. The filtrate was extracted with EA (400 mL). The organic phase was washed with water (500 mL x 2) and brine (300 mL). Dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on silica gel to give compound 27-1 (15 g, 57% yield).

C32H48N4O5質量計算為：568.3；實測：569.4[M+H]+，ESI。 Mass calculated for C 32 H 48 N 4 O 5 : 568.3; found: 569.4 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 8.49-8.48(m,4H),7.46-6.77(m,6H),3.89(t,J=6.4Hz,2H),2.91-2.86(m,2H),2.33-2.29(m,2H),1.98-1.92(m,4H),1.38(s,22H),1.32-1.24(m,6H). 1 H NMR(400MHz,DMSO-d6)δ 8.49-8.48(m,4H),7.46-6.77(m,6H),3.89(t, J =6.4Hz,2H),2.91-2.86(m,2H),2.33-2.29(m,2H),1.98-1.92(m,4H),1.38(s,22H),1.32-1.24(m,6H).

化合物27-2：Compound 27-2:

在化合物27-1(14.5g，25.49mmol)在AcOH(120mL)中的溶液中加入PtO2(8g，35.23mmol)。將反應在0.4Mpa氫氣氣氛下攪拌88小時，倒出反應混合物的清液，真空濃縮。用飽和NaHCO3調節pH為7-8。將水相在真空中濃縮。向殘餘物中加入THF(300mL)，並攪拌30分鐘。將混合物過濾並在真空中濃縮以得到粗化合物27-2(15.32g)。C32H60N4O5質量計算為：580.5；實測：581.6[M+H]+，ESI。 PtO2 (8 g, 35.23 mmol) was added to a solution of compound 27-1 (14.5 g, 25.49 mmol) in AcOH (120 mL). The reaction was stirred under 0.4 MPa hydrogen atmosphere for 88 hours, the clear solution of the reaction mixture was poured out and concentrated in vacuo. The pH was adjusted to 7-8 with saturated NaHCO3 . The aqueous phase was concentrated in vacuo. THF ( 300 mL) was added to the residue and stirred for 30 minutes. The mixture was filtered and concentrated in vacuo to give crude compound 27-2 (15.32 g). C32H60N4O5 mass calculated: 580.5; found: 581.6 [M+H] + , ESI.

化合物27-3：Compound 27-3:

將Boc-6-胺基己酸(12.80g，55.38mmol)、HATU(23.87g，63.29mmol)和TEA(8.0g，79.11mmol)在DCM(60mL)中的混合物在室溫攪拌1小時。將化合物27-2(15.32g，26.37mmol)在DCM(60mL)中的溶液加入到反應混合物中，室溫攪拌1.5小時。用水(200mL)淬滅反應，用DCM(200mL×3)萃取。將合併的有機相用鹽水(200mL)洗滌，用Na2SO4乾燥，過濾，真空濃縮，藉由快速管柱層析(0-10% MeOH)在矽膠上純化，以得到化合物27-3(23g，產率為75%)。 A mixture of Boc-6-aminohexanoic acid (12.80 g, 55.38 mmol), HATU (23.87 g, 63.29 mmol) and TEA (8.0 g, 79.11 mmol) in DCM (60 mL) was stirred at room temperature for 1 hour. A solution of compound 27-2 (15.32 g, 26.37 mmol) in DCM (60 mL) was added to the reaction mixture and stirred at room temperature for 1.5 hours. The reaction was quenched with water (200 mL) and extracted with DCM (200 mL×3). The combined organic phases were washed with brine (200 mL), dried over Na 2 SO 4 , filtered, concentrated in vacuo, and purified by flash column chromatography (0-10% MeOH) on silica gel to give compound 27-3 (23 g, 75% yield).

C54H98N6O11質量計算為：1006.7；實測：907.7[M+H-Boc]+，ESI。 Mass calculated for C 54 H 98 N 6 O 11 : 1006.7; found: 907.7 [M+H-Boc] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.44-6.76(m,4H),4.46(d,J=11.6Hz,2H),3.89(d,J=12.0Hz,2H),3.06-2.86(m,10H),2.40-2.23(m,12H),1.70-1.16(m,68H). 1 H NMR (400MHz, DMSO-d6) δ 7.44-6.76 (m, 4H), 4.46 (d, J =11.6Hz, 2H), 3.89 (d, J =12.0Hz,2H),3.06-2.86(m,10H),2.40-2.23(m,12H),1.70-1.16(m,68H).

化合物27-4：Compound 27-4:

將化合物27-3(10g，9.93mmol)溶解於DCM(54mL)中，然後添加TFA(59mL)到混合物中。將反應在30℃中攪拌3小時。將反應在真空中濃縮，而沒有進一步純化以得到化合物27-4(6.64g)。 Compound 27-3 (10 g, 9.93 mmol) was dissolved in DCM (54 mL), and then TFA (59 mL) was added to the mixture. The reaction was stirred at 30 °C for 3 hours. The reaction was concentrated in vacuo without further purification to give compound 27-4 (6.64 g).

C35H66N6O5質量計算為：650.5；實測：651.6[M+H]+，ESI。 Mass calculated for C 35 H 66 N 6 O 5 : 650.5; found: 651.6 [M+H] + , ESI.

化合物27-5：Compound 27-5:

將化合物1-4(13.32g，29.77mmol)、HATU(13.10g，34.73mmol)和TEA(5.02g，49.62mmol，6.92mL)在DCM(50mL)中的混合物在室溫攪拌2小時。將化合物27-4添加到混合物中。將反應在室溫下繼續攪拌4小時。反應在真空濃縮，並藉由製備型HPLC純化，以得到化合物27-5(13g，產率為67%)。 A mixture of compound 1-4 (13.32 g, 29.77 mmol), HATU (13.10 g, 34.73 mmol) and TEA (5.02 g, 49.62 mmol, 6.92 mL) in DCM (50 mL) was stirred at room temperature for 2 hours. Compound 27-4 was added to the mixture. The reaction was continued to stir at room temperature for 4 hours. The reaction was concentrated under vacuum and purified by preparative HPLC to give compound 27-5 (13 g, 67% yield).

C92H147N9O35質量計算為：1938.0；實測：970.1[M+2H]2+，ESI。 Mass calculated for C 92 H 147 N 9 O 35 : 1938.0; found: 970.1 [M+2H] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 12.01(s,1H),7.86-7.50(m,7H),5.22-4.96(m,6H),4.50-4.48(m,4H),4.03-3.69(m,17H),3.41-3.32(m,6H),3.01-2.60(m,8H),2.46-2.21(m,4H),2.15-1.78(m,42H),1.68-1.23(m,46H),0.95(t,J=7.6Hz,6H). 1H NMR(400MHz,DMSO-d6)δ 12.01(s,1H),7.86-7.50(m,7H),5.22-4.96(m,6H),4.50-4.48(m,4H),4.03-3.69(m,17H),3.41-3 .32(m,6H),3.01-2.60(m,8H),2.46-2.21(m,4H),2.15-1.78(m,42H),1.68-1.23(m,46H),0.95(t, J =7.6Hz,6H).

化合物27-6：Compound 27-6:

將化合物27-5(2.75g，1.42mmol)、DIEA(383.61mg，2.97mmol，517.00μL)、EDCI(426.76mg，2.23mmol)和HOBT(200.53mg，1.48mmol)在DCM(30mL)中的混合物在室溫攪拌30分鐘。將化合物11-2(0.85g，1.48mmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。用水(50mL)淬滅反應，用DCM(50mL×2)萃取。合併的有機相在真空濃縮，並藉由製備型HPLC純化，以得到化合物27-6(1.2g，產率為32%)。 A mixture of compound 27-5 (2.75 g, 1.42 mmol), DIEA (383.61 mg, 2.97 mmol, 517.00 μL), EDCI (426.76 mg, 2.23 mmol) and HOBT (200.53 mg, 1.48 mmol) in DCM (30 mL) was stirred at room temperature for 30 minutes. Compound 11-2 (0.85 g, 1.48 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with water (50 mL) and extracted with DCM (50 mL×2). The combined organic phases were concentrated in vacuo and purified by preparative HPLC to obtain compound 27-6 (1.2 g, 32% yield).

C127H189O39N11質量計算為：2493.9；實測：1270.0[M+2Na]2+，ESI。 Mass calculated for C 127 H 189 O 39 N 11 : 2493.9; found: 1270.0 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.84-7.72(m,7H),7.44-7.23(m,10H),6.92-6.88(m,4H),5.22(d,J=3.6Hz,3H),4.99-4.95(m,3H),4.66-4.45(m,6H),4.03-3.69(m,26H),3.41-3.36(m,6H),3.01-2.86(m,14H),2.46-2.21(m,6H),2.15-1.78(m,52H),1.68-1.23(m,52H). 1 H NMR(400MHz, DMSO-d6)δ 7.84-7.72(m,7H),7.44-7.23(m,10H),6.92-6.88(m,4H),5.22(d, J =3.6Hz,3H),4.99-4.95(m,3H),4.66-4.45(m,6H),4.03-3.69(m,26H),3.41-3.36(m ,6H),3.01-2.86(m,14H),2.46-2.21(m,6H),2.15-1.78(m,52H),1.68-1.23(m,52H).

Figure 113123276-A0202-12-0260-305
Figure 113123276-A0202-12-0260-305

化合物28-1：Compound 28-1:

將8-(Fmoc-胺基)辛酸(1.27g，3.59mmol)、TEA(495.41mg，4.90mmol)和HATU(1.35g，3.59mmol)在DCM(15mL)中的混合物攪拌20分鐘，然後將化合物2-10(1.5g，3.26mmol)加入到反應混合物中，並繼續攪拌4小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫 酸鈉乾燥，真空濃縮，經快速管柱層析(DCM/MeOH=0-6%)在Al2O3上純化，以得到化合物28-1，為白色泡沫狀固體(600mg，產率為23%)。 A mixture of 8-(Fmoc-amino)octanoic acid (1.27 g, 3.59 mmol), TEA (495.41 mg, 4.90 mmol) and HATU (1.35 g, 3.59 mmol) in DCM (15 mL) was stirred for 20 minutes, and then compound 2-10 (1.5 g, 3.26 mmol) was added to the reaction mixture and stirring was continued for 4 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate, concentrated in vacuo, and purified by flash column chromatography (DCM/MeOH = 0-6%) on Al 2 O 3 to obtain compound 28-1 as a white foamy solid (600 mg, 23% yield).

1H NMR(400MHz,DMSO-d6)δ 7.87(d,J=7.6Hz,2H),7.68(d,J=7.4Hz,2H),7.44-7.35(m,4H),7.34-7.28(m,4H),7.27-7.17(m,6H),6.89(d,J=7.6Hz,4H),4.64(td,J=5.0,1.6Hz,1H),4.28(d,J=6.9Hz,2H),4.20(t,J=6.8Hz,1H),3.96(s,1H),3.73(d,J=3.8Hz,6H),3.67(d,J=10.4Hz,2H),3.50(s,1H),3.39(t,J=5.9Hz,2H),2.96(t,J=6.6Hz,2H),2.91(s,2H),2.06-1.74(m,6H),1.47-1.31(m,4H),1.26-1.13(m,6H). 1 H NMR(400MHz, DMSO-d6)δ 7.87(d, J =7.6Hz,2H),7.68(d, J =7.4Hz,2H),7.44-7.35(m,4H),7.34-7.28(m,4H),7.27-7.17(m,6H),6.89(d, J =7.6Hz,4H),4.64(td, J =5.0,1.6Hz,1H),4.28(d, J =6.9Hz,2H),4.20(t, J =6.8Hz,1H),3.96(s,1H),3.73(d, J =3.8Hz,6H),3.67(d, J =10.4Hz,2H),3.50(s,1H),3.39(t, J =5.9Hz,2H),2.96(t, J =6.6Hz,2H),2.91(s,2H),2.06-1.74(m,6H),1.47-1.31(m,4H),1.26-1.13(m,6H).

化合物28-2：Compound 28-2:

將哌啶(0.4mL)加入化合物28-1(600mg，0.75mmol)在MeOH(4mL)中的溶液中。攪拌過夜後，真空濃縮混合物，以得到粗化合物28-2，為白色固體(600mg)，其無需進一步純化即使用。 Piperidine (0.4 mL) was added to a solution of compound 28-1 (600 mg, 0.75 mmol) in MeOH (4 mL). After stirring overnight, the mixture was concentrated in vacuo to give crude compound 28-2 as a white solid (600 mg), which was used without further purification.

C35H44N2O5質量計算為：600.4；實測：601.4[M+H]+，ESI。 Mass calculated for C 35 H 44 N 2 O 5 : 600.4; found: 601.4 [M+H] + , ESI.

化合物28-3：Compound 28-3:

將化合物11-11(1.75g，915.47μmol)溶解於DCM(30mL)中。在0℃下將DIEA(355mg，2.75mmol)、EDCI(351mg，1.83mmol)和HOBT(247mg，1.83mmol)加入到混合物中。將反應在0℃攪拌30分鐘。將化合物28-2(550mg，915.47μmol)加入反應混合物中。將反應混合物在室溫下攪拌2小時。用飽和NaHCO3溶液(40mL)猝滅反應混合物，用DCM(30mLx2)萃取，用鹽水(30mL)洗滌，用Na2SO4乾燥，濃縮並藉由製備型HPLC(C18管柱，ACN/水)純化以得到化合物28-3，為白色泡沫狀固體(1.1g，產率為48%)。 Compound 11-11 (1.75 g, 915.47 μmol) was dissolved in DCM (30 mL). DIEA (355 mg, 2.75 mmol), EDCI (351 mg, 1.83 mmol) and HOBT (247 mg, 1.83 mmol) were added to the mixture at 0°C. The reaction was stirred at 0°C for 30 minutes. Compound 28-2 (550 mg, 915.47 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO 3 solution (40 mL), extracted with DCM (30 mL×2), washed with brine (30 mL), dried over Na 2 SO 4 , concentrated and purified by preparative HPLC (C18 column, ACN/water) to give compound 28-3 as a white foamy solid (1.1 g, yield 48%).

1H NMR(400MHz,DMSO-d6)δ 7.86-7.76(m,4H),7.73(t,J=5.4Hz,3H),7.46(s,1H),7.40-7.35(m,2H),7.31(t,J=7.6Hz,2H),7.27-7.18(m,5H),6.93-6.85(m,4H),5.21(d,J=3.4Hz,3H),4.95(dd,J=11.2,3.4Hz,3H),4.65(t,J=5.1Hz,1H),4.50-4.40(m,5H),4.10-3.94(m,10H),3.93-3.80(m,5H),3.70-3.64(m,11H),3.50(s,1H),3.45-3.36(m,5H),3.08-3.75(m,14H),2.39-2.20(m,6H),2.15-1.92(m,31H),1.92-1.73(m,21H),1.67-1.13(m,52H). 1 H NMR(400MHz, DMSO-d6)δ 7.86-7.76(m,4H),7.73(t, J =5.4Hz,3H),7.46(s,1H),7.40-7.35(m,2H),7.31(t, J =7.6Hz,2H),7.27-7.18(m,5H),6.93-6.85(m,4H),5.21(d, J =3.4Hz,3H),4.95(dd, J =11.2,3.4Hz,3H),4.65(t, J =5.1Hz,1H),4.50-4.40(m,5H),4.10-3.94(m,10H),3.93-3.80(m,5H),3.70-3.64(m,11H),3.50(s,1H),3.45-3 .36(m,5H),3.08-3.75(m,14H),2.39-2.20(m,6H),2.15-1.92(m,31H),1.92-1.73(m,21H),1.67-1.13(m,52H).

Figure 113123276-A0202-12-0262-306
Figure 113123276-A0202-12-0262-306

化合物29-1：Compound 29-1:

將化合物27-5(2.74g，1.41mmol)、DIEA(365.70mg，2.83mmol，492.86μL)、EDCI(406.83mg，2.12mmol)和HOBT(191.17mg，1.41mmol)在DCM(30mL)中的混合物在室溫攪拌30分鐘。將化合物28-2(0.85g，1.41mmol)加入混合物中。將反應在室溫下繼續攪拌2小時。用水(50mL)淬滅反應，用DCM(50mL×2)萃取。合併的有機相在真空濃縮，並藉由製備型HPLC純化，以得到化合物29-1(1.1g，產率為29%)。 A mixture of compound 27-5 (2.74 g, 1.41 mmol), DIEA (365.70 mg, 2.83 mmol, 492.86 μL), EDCI (406.83 mg, 2.12 mmol) and HOBT (191.17 mg, 1.41 mmol) in DCM (30 mL) was stirred at room temperature for 30 minutes. Compound 28-2 (0.85 g, 1.41 mmol) was added to the mixture. The reaction was stirred for 2 hours at room temperature. The reaction was quenched with water (50 mL) and extracted with DCM (50 mL×2). The combined organic phases were concentrated in vacuo and purified by preparative HPLC to give compound 29-1 (1.1 g, 29% yield).

C129H193O39N11質量計算為：2520.4；實測：1284.0[M+2Na]2+，ESI。 Mass calculated for C 129 H 193 O 39 N 11 : 2520.4; found: 1284.0 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ：7.84-7.72(m,7H),7.44-7.23(m,10H),6.92-6.88(m,4H),5.22(d,J=3.6Hz,3H),4.99-4.95(m,3H),4.66-4.45(m,6H),4.03-3.69(m,26H),3.41-3.36(m,6H),3.01-2.86(m,14H),2.46-2.21(m,6H),2.15-1.78(m,52H),1.68-1.23(m,56H). 1 H NMR (400MHz, DMSO-d6) δ: 7.84-7.72(m,7H),7.44-7.23(m,10H),6.92-6.88(m,4H),5.22(d, J =3.6Hz,3H),4.99-4.95(m,3H),4.66-4.45(m,6H),4.03-3.69(m,26H),3.41-3.36(m ,6H),3.01-2.86(m,14H),2.46-2.21(m,6H),2.15-1.78(m,52H),1.68-1.23(m,56H).

Figure 113123276-A0202-12-0263-307
Figure 113123276-A0202-12-0263-307

化合物30-1：Compound 30-1:

將苄胺(25g，233.31mmol)、聚甲醛(42.03g，1.40mol)和K2CO3(32.24g，233.31mmol)在MeOH(30mL)中的混合物在室溫攪拌過夜。 A mixture of benzylamine (25 g, 233.31 mmol), polyoxymethylene (42.03 g, 1.40 mol) and K2CO3 ( 32.24 g, 233.31 mmol) in MeOH (30 mL) was stirred at room temperature overnight.

將反應混合物過濾並在真空中濃縮。向殘餘物中加入PE：EA=1：1(100mL)，然後將混合物過濾並在真空下濃縮以得到粗化合物30-1，為無色油狀物(45.56g)。 The reaction mixture was filtered and concentrated in vacuo. PE:EA=1:1 (100 mL) was added to the residue, and then the mixture was filtered and concentrated in vacuo to give crude compound 30-1 as a colorless oil (45.56 g).

1H NMR(400MHz,DMSO-d6)δ：7.33-7.27(m,5H),4.27(s,4H),4.04(s,2H),3.30(s,6H). 1 H NMR (400MHz, DMSO-d6) δ: 7.33-7.27 (m, 5H), 4.27 (s, 4H), 4.04 (s, 2H), 3.30 (s, 6H).

化合物30-2：Compound 30-2:

將滴液漏斗中的3-((苄氧基)甲基)環丁酮(23.38g，122.92mmol)、化合物30-1(24g，122.92mmol)在MeCN(200mL)中的混合物以及另一個滴液漏斗中的氯三甲基矽烷(26.70g，245.84mmol)在MeCN(200mL)中的溶液逐滴混合到乙腈(200mL)中，在40℃下攪拌3小時。將反應在20℃中攪拌過夜。用飽和NaHCO3將pH調節至7-8，並在真空下除去ACN。用EA(200mLx3)萃取水層。將合併的有機相在真空下濃縮。藉由C18反相管柱和快速管柱層析純化殘餘物以得到30-2(1.5g，4%產率)。 A mixture of 3-((benzyloxy)methyl)cyclobutanone (23.38 g, 122.92 mmol), compound 30-1 (24 g, 122.92 mmol) in MeCN (200 mL) in a dropping funnel and a solution of chlorotrimethylsilane (26.70 g, 245.84 mmol) in MeCN (200 mL) in another dropping funnel were mixed dropwise into acetonitrile (200 mL) and stirred at 40 ° C for 3 hours. The reaction was stirred at 20 ° C overnight. The pH was adjusted to 7-8 with saturated NaHCO 3 , and ACN was removed under vacuum. The aqueous layer was extracted with EA (200 mL×3). The combined organic phases were concentrated under vacuum. The residue was purified by C18 reverse phase column and flash column chromatography to give 30-2 (1.5 g, 4% yield).

C21H23O2N質量計算為：321.2；實測：322.1[M+H]+，ESI。 Mass calculated for C 21 H 23 O 2 N: 321.2; found: 322.1 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.35-7.24(m,10H),4.48(s,2H),3.61(s,2H),3.40(d,J=6.8Hz,2H),3.33-3.08(m,4H),2.88(d,J=2.0Hz,2H),2.24(t,J=6.8Hz,1H). 1 H NMR(400MHz, DMSO-d6)δ 7.35-7.24(m,10H),4.48(s,2H),3.61(s,2H),3.40(d, J =6.8Hz,2H),3.33-3.08(m,4H),2.88(d, J =2.0Hz,2H),2.24(t, J =6.8Hz,1H).

化合物30-3：Compound 30-3:

將甲氧基甲基(三苯基)氯化磷(2.88g，8.40mmol)溶於THF(30mL)中。在0℃下將KOtBu(1.05g，9.33mmol)加入混合物中。將反應混合物在0℃下再攪拌30分鐘。將化合物30-2(1.5g，4.67mmol)加入混合物中。將混合物加熱至室溫並在室溫攪拌過夜。用飽和檸檬酸淬滅反應混合物，並將pH調節至6-7。將用EA(50mLx3)萃取反應混合物，用鹽水(20mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(10-30% MeOH在DCM中的溶液)在矽膠上純化以得到呈油狀的化合物30-3(1.5g，產率為92%)。 Methoxymethyl (triphenyl) phosphonium chloride (2.88 g, 8.40 mmol) was dissolved in THF (30 mL). KOtBu (1.05 g, 9.33 mmol) was added to the mixture at 0°C. The reaction mixture was stirred at 0°C for another 30 minutes. Compound 30-2 (1.5 g, 4.67 mmol) was added to the mixture. The mixture was heated to room temperature and stirred at room temperature overnight. The reaction mixture was quenched with saturated citric acid and the pH was adjusted to 6-7. The reaction mixture was extracted with EA (50 mL x 3), washed with brine (20 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by flash column chromatography (10-30% MeOH in DCM) on silica gel to give compound 30-3 (1.5 g, 92% yield) as an oil.

C23H27O2N質量計算為：349.20；實測：350.2[M+H]+，ESI。 C23H27O2N mass calculated: 349.20; found: 350.2[M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.37-7.26(m,10H),6.02(s,1H),4.47(s,2H),3.85(s,2H),3.54(d,J=7.6Hz,2H),3.46(s,3H),3.13-3.09(m,4H),2.88(d,J=2Hz,2H),2.24(t,J=6.8Hz,1H). 1 H NMR(400MHz,DMSO-d6)δ 7.37-7.26(m,10H),6.02(s,1H),4.47(s,2H),3.85(s,2H),3.54(d, J =7.6Hz,2H),3.46(s,3H),3.13-3.09(m,4H),2.88(d, J =2Hz,2H),2.24(t, J =6.8Hz,1H).

化合物30-4：Compound 30-4:

將2,2,2-三氯乙酸(883.64mg，5.41mmol)在水中(15mL)的溶液加入到化合物30-3(1.5g，4.29mmol)在ACN(45mL)中的溶液。將反應在室溫攪拌過夜。然後用飽和NaHCO3淬滅反應並將pH調節至7-8。用EA(20mLx3)萃取反應混合物，用鹽水(10mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由快速管柱層析(10-30% EA在PE中的溶液)在矽膠上純化以得到呈油狀的化合物30-4(600mg，產率為42%)。 A solution of 2,2,2-trichloroacetic acid (883.64 mg, 5.41 mmol) in water (15 mL) was added to a solution of compound 30-3 (1.5 g, 4.29 mmol) in ACN (45 mL). The reaction was stirred at room temperature overnight. The reaction was then quenched with saturated NaHCO 3 and the pH was adjusted to 7-8. The reaction mixture was extracted with EA (20 mL×3), washed with brine (10 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified on silica gel by flash column chromatography (10-30% EA in PE) to give compound 30-4 (600 mg, 42% yield) as an oil.

C22H25O2N質量計算為：335.25；實測：336.2[M+H]+，ESI。 Mass calculated for C 22 H 25 O 2 N: 335.25; found: 336.2 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 9.90(s,1H),7.37-7.26(m,10H),4.41(s,2H),3.85(s,2H),3.44(d,J=7.6Hz,2H),3.03-2.89(m,4H),2.86(d,J=4Hz,1H),2.69(s,2H),2.04(t,J=6.8Hz,1H). 1 H NMR(400MHz, DMSO-d6)δ 9.90(s,1H),7.37-7.26(m,10H),4.41(s,2H),3.85(s,2H),3.44(d, J =7.6Hz,2H),3.03-2.89(m,4H),2.86(d, J =4Hz,1H),2.69(s,2H),2.04(t, J =6.8Hz,1H).

化合物30-5：Compound 30-5:

將化合物30-4(600mg，1.79mmol)溶解於MeOH(18mL)中，然後分批緩慢加入NaBH4(148.88mg，3.94mmol)。將反應在室溫下攪拌1小時。用水將反應淬滅。用飽和檸檬酸調節PH至6-7。將反應在真空中濃縮以除去MeOH。用DCM(20mLx3)萃取殘餘物，用鹽水(10mL)洗滌，用Na2SO4乾燥，真空濃縮，以得到化合物30-5(600mg)。 Compound 30-4 (600 mg, 1.79 mmol) was dissolved in MeOH (18 mL), and then NaBH4 (148.88 mg, 3.94 mmol) was slowly added in portions. The reaction was stirred at room temperature for 1 hour. The reaction was quenched with water. The pH was adjusted to 6-7 with saturated citric acid. The reaction was concentrated in vacuo to remove MeOH. The residue was extracted with DCM (20 mL x 3), washed with brine (10 mL ), dried over Na2SO4 , and concentrated in vacuo to obtain compound 30-5 (600 mg).

C22H27NO2質量計算為：337.15；實測：338.2[M+H]+，ESI。 The mass of C22H27NO2 was calculated as: 337.15; found as: 338.2[M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.37-7.26(m,10H),4.47(s,2H),3.90(s,2H),3.69(d,J=7.6Hz,2H),3.62-3.59(m,4H),3.11(d,J=10.8Hz,2H),2.61-2.56(m,2H),2.19-2.04(m,2H). 1H NMR(400MHz,DMSO-d6)δ 7.37-7.26(m,10H),4.47(s,2H),3.90(s,2H),3.69(d, J =7.6Hz,2H),3.62-3.59(m,4H),3.11(d, J =10.8Hz,2H),2.61-2.56(m,2H),2.19-2.04(m,2H).

化合物30-6：Compound 30-6:

將化合物30-5(200mg，592.67μmol)、Pd/C(100.00mg，823.38μmol)在MeOH(6mL)中的混合物在0.4MPa H2下攪拌88小時。將混合物過濾並在真空中濃縮，藉由製備型HPLC純化，以得到化合物30-6(70mg，產率為46%)。 A mixture of compound 30-5 (200 mg, 592.67 μmol) and Pd/C (100.00 mg, 823.38 μmol) in MeOH (6 mL) was stirred under 0.4 MPa H2 for 88 hours. The mixture was filtered and concentrated in vacuo, and purified by preparative HPLC to give compound 30-6 (70 mg, 46% yield).

C13H23NO2質量計算為：257.2；實測：158.2[M-Boc+H]+，ESI。 The mass of C13H23NO2 was calculated to be 257.2; found to be 158.2 [M-Boc+H] + , ESI.

1H NMR(400MHz,CDCl3)δ 3.96(d,J=8.0Hz,4H),3.70-3.68(m,4H),3.25-3.17(m,4H),2.36-2.30(m,2H),2.00-1.95(m,2H),1.49(s,9H). 1H NMR (400MHz, CDCl3) δ 3.96 (d, J =8.0Hz, 4H), 3.70-3.68 (m, 4H), 3.25-3.17 (m, 4H), 2.36-2.30 (m, 2H), 2.00-1.95 (m, 2H), 1.49 (s, 9H).

化合物30-7：Compound 30-7:

將化合物30-6(1g，3.89mmol)溶解於HCl/1,4-二噁烷(4M,10mL)中。將反應混合物在室溫攪拌3小時。將反應混合物在真空中濃縮以得到粗產物30-7(610mg)，無需進一步純化即可用於下一步。 Compound 30-6 (1 g, 3.89 mmol) was dissolved in HCl/1,4-dioxane (4 M, 10 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated in vacuo to give the crude product 30-7 (610 mg), which was used in the next step without further purification.

C8H15NO2質量計算為：157.2；實測：158.1[M+H]+，ESI。 The mass of C8H15NO2 was calculated to be 157.2; found to be 158.1 [M+H] + , ESI.

化合物30-8：Compound 30-8:

將DIEA(1.25g，9.70mmol，1.69mL)、HOBT(629.15mg，4.66mmol)、EDCI(892.59mg，4.66mmol)、Fmoc-6-胺基己酸(1.37g，3.88mmol)在DCM(30mL)中的混合物在0℃攪拌30分鐘。將化合物30-7(610mg，3.88mmol)加入反應混合物中，在室溫攪拌過夜。用水淬滅反應，用10%檸檬酸(10mL)和飽和NaHCO3溶液(10mL)和鹽水(10mL)洗滌，Na2SO4乾燥，真空濃縮，快速管柱層析(0-10% MeOH)在矽膠上純化，以得到化合物30-8(1.55g，產率為81%)。 A mixture of DIEA (1.25 g, 9.70 mmol, 1.69 mL), HOBT (629.15 mg, 4.66 mmol), EDCI (892.59 mg, 4.66 mmol), and Fmoc-6-aminohexanoic acid (1.37 g, 3.88 mmol) in DCM (30 mL) was stirred at 0° C. for 30 minutes. Compound 30-7 (610 mg, 3.88 mmol) was added to the reaction mixture and stirred at room temperature overnight. The reaction was quenched with water, washed with 10% citric acid (10 mL) and saturated NaHCO 3 solution (10 mL) and brine (10 mL), dried over Na 2 SO 4 , concentrated in vacuo, and purified by flash column chromatography (0-10% MeOH) on silica gel to give compound 30-8 (1.55 g, yield 81%).

C29H36N2O5質量計算為：492.3；實測：493.3[M+H]+，ESI。 Mass calculated for C 29 H 36 N 2 O 5 : 492.3; found: 493.3 [M+H] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.25(m,5H),4.56(t,J=5.2Hz,2H),4.31-4.20(m,3H),3.70-3.67(m,4H),3.55(s,2H),3.31(s,4H),2.99-2.95(m,2H),2.29-2.21(m,2H),1.71(t,J=6.0Hz,2H),1.53-1.24(m,6H). 1 H NMR(400MHz,DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.25(m,5H),4.56(t, J =5.2Hz,2H),4.31-4.20(m,3H),3.70-3.67(m,4H),3.55(s,2H),3.31(s,4H),2 .99-2.95(m,2H),2.29-2.21(m,2H),1.71(t,J=6.0Hz,2H),1.53-1.24(m,6H).

化合物30-9：Compound 30-9:

將化合物30-8(1.55g，3.15mmol)、DMAP(38.44mg，314.65μmol)、DIEA(610.00mg，4.72mmol，822.10μL)溶於DCM(50mL)。在0℃下分批將DMTrCl(1.17g，3.46mmol)加入到反應混合物中。將反應在室溫攪拌過夜。然後將反應混合物在真空濃縮，藉由快速管柱層析(20-50% EA在PE中的溶液)在Al2O3上純化以得到化合物30-9(1g，產率為40%)。 Compound 30-8 (1.55 g, 3.15 mmol), DMAP (38.44 mg, 314.65 μmol), DIEA (610.00 mg, 4.72 mmol, 822.10 μL) were dissolved in DCM (50 mL). DMTrCl (1.17 g, 3.46 mmol) was added to the reaction mixture in portions at 0°C. The reaction was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo and purified by flash column chromatography (20-50% EA in PE) on Al 2 O 3 to give compound 30-9 (1 g, 40% yield).

C50H54N2O7質量計算為：794.4；實測：817.4[M+Na]+，ESI。 Mass calculated for C 50 H 54 N 2 O 7 : 794.4; found: 817.4 [M+Na] + , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.25(m,14H),6.90(d,J=8.8Hz,4H),4.56(t,J=5.2Hz,1H),4.31-4.20(m,3H),3.74(s, 6H),3.70-3.67(m,4H),3.55(s,2H),3.31(s,4H),2.99-2.95(m,2H),2.29-2.21(m,2H),1.89-1.67(m,2H),1.53-1.24(m,6H). 1 H NMR(400MHz, DMSO-d6)δ 7.90-7.68(m,4H),7.44-7.25(m,14H),6.90(d, J =8.8Hz,4H),4.56(t, J =5.2Hz,1H),4.31-4.20(m,3H),3.74(s, 6H),3.70-3.67(m,4H),3.55(s,2H),3.31(s,4H),2.99-2.95(m,2H),2.29-2.21(m,2H),1.89-1.67(m,2H),1.53-1.24(m,6H).

化合物30-10：Compound 30-10:

將化合物30-9(700mg，880.54μmol)溶解於MeOH(14mL)中，然後添加哌啶(1.4mL)到混合物中。將反應混合物在室溫攪拌過夜。將反應混合物在真空濃縮，藉由快速管柱層析(0-10% MeOH)在Al2O3上純化以得到化合物30-10，為白色泡沫狀固體(300mg，產率為59%)。 Compound 30-9 (700 mg, 880.54 μmol) was dissolved in MeOH (14 mL), and then piperidine (1.4 mL) was added to the mixture. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (0-10% MeOH) on Al 2 O 3 to give compound 30-10 as a white foamy solid (300 mg, 59% yield).

C35H44N2O5質量計算為：572.3；實測：595.3[M+Na]+，ESI。 Mass calculated for C 35 H 44 N 2 O 5 : 572.3; found: 595.3 [M+Na] + , ESI.

化合物30-11：Compound 30-11:

將化合物11-11(1.00g，523.80μmol)、DIEA(169.25mg，1.31mmol，228.09μL)、HOBT(84.93mg，628.57μmol)和EDCI(120.50mg，628.57μmol)在DCM(50mL)中的混合物在0℃攪拌30分鐘。將化合物30-10(300mg，523.80μmol)加入混合物中。將反應在室溫攪拌過夜。將反應混合物用水(20mL)淬滅，用DCM(25mLx2)萃取，用鹽水(50mL)洗滌，用Na2SO4乾燥，真空濃縮並藉由HPLC純化以得到化合物30-11(595mg，產率為46%)。 A mixture of compound 11-11 (1.00 g, 523.80 μmol), DIEA (169.25 mg, 1.31 mmol, 228.09 μL), HOBT (84.93 mg, 628.57 μmol) and EDCI (120.50 mg, 628.57 μmol) in DCM (50 mL) was stirred at 0 °C for 30 minutes. Compound 30-10 (300 mg, 523.80 μmol) was added to the mixture. The reaction was stirred at room temperature overnight. The reaction mixture was quenched with water (20 mL), extracted with DCM (25 mL x 2), washed with brine (50 mL), dried over Na 2 SO 4 , concentrated in vacuo and purified by HPLC to give compound 30-11 (595 mg, 46% yield).

C125H185N11O39質量計算為：2464.3；實測：1255.2[M+2Na]2+，ESI。 Mass calculated for C 125 H 185 N 11 O 39 : 2464.3; found: 1255.2 [M+2Na] 2+ , ESI.

1H NMR(400MHz,DMSO-d6)δ 7.83-7.69(m,7H),7.43(s,1H),7.37-7.20(m,9H),6.91-6.89(m,J=8.8Hz,4H),5.22(d,J=3.2Hz,3H),4.99-4.95(m,3H),4.49-4.46(m,6H),4.03(s,9H),3.91-3.85(m,5H),3.74(s,6H),3.74-3.26(m,9H),3.01-2.99(m,10H),2.90-2.87(m,2H),2.27-2.20(m,9H),2.12(s,11H),2.06-2.02(m,8H),2.02(s,11H),1.89(s,11H),1.78(s,9H),1.62-1.21(m,52H). 1 H NMR(400MHz, DMSO-d6)δ 7.83-7.69(m,7H),7.43(s,1H),7.37-7.20(m,9H),6.91-6.89(m, J =8.8Hz,4H),5.22(d, J =3.2Hz,3H),4.99-4.95(m,3H),4.49-4.46(m,6H),4.03(s,9H),3.91-3.85(m,5H),3.74(s,6H),3.74-3.26(m,9H),3.01-2.99(m,10H) ,2.90-2.87(m,2H),2.27-2.20(m,9H),2.12(s,11H),2.06-2.02(m,8H),2.02(s,11H),1.89(s,11H),1.78(s,9H),1.62-1.21(m,52H).

Figure 113123276-A0202-12-0269-308
Figure 113123276-A0202-12-0269-308

化合物31-1：Compound 31-1:

將化合物11-11(3.17g，1.66mmol)、HOBT(448.34mg，3.32mmol)、EDCI(954.11mg，4.98mmol)和DIEA(857.66mg，6.64mmol)在DCM(20mL)中的混合物攪拌15分鐘，然後化合物9-9(1g，1.66mmol)加入到反應混合物中，繼續攪拌4小時。將混合物在DCM(50mL)和水(35mL)之間分配，用鹽水(100mL)洗滌DCM萃取物，用硫酸鈉乾燥並真空濃縮。殘餘物藉由反相管柱(C18管柱)純化，以提供化合物31-1(2.25g，產率為54%)。 A mixture of compound 11-11 (3.17 g, 1.66 mmol), HOBT (448.34 mg, 3.32 mmol), EDCI (954.11 mg, 4.98 mmol) and DIEA (857.66 mg, 6.64 mmol) in DCM (20 mL) was stirred for 15 minutes, and then compound 9-9 (1 g, 1.66 mmol) was added to the reaction mixture, and stirring was continued for 4 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by a reverse phase column (C18 column) to provide compound 31-1 (2.25 g, 54% yield).

1H NMR(400MHz,DMSO-d6)δ 7.85-7.78(m,4H),7.72(t,J=5.4Hz,3H),7.45(s,1H),7.27-7.20(m,6H),6.91-6.85(m,6H),5.21(d,J=3.4Hz,3H),4.99-4.94(m,3H),4.64(t,J=5.1Hz,1H),4.50-4.41(m,5H),4.05-3.95(m,10H),3.91-3.83(m,5H),3.75-3.65(m,14H),3.51(s,1H),3.44-3.31(m,5H),3.06-2.94(m,10H),2.90(s,2H),2.89-2.78(m,2H),2.41-2.30(m,2H),2.28-2.21(m,4H),2.10(s,9H),2.08-2.00(m,10H),1.99(s,11H),1.96-1.92(m,2H),1.89(s,9H),1.86-1.80(m,2H),1.77(s,9H),1.66-1.31(m,35H),1.30-1.13(m,13H). 1 H NMR(400MHz, DMSO-d6)δ 7.85-7.78(m,4H),7.72(t, J =5.4Hz,3H),7.45(s,1H),7.27-7.20(m,6H),6.91-6.85(m,6H),5.21(d, J =3.4Hz,3H),4.99-4.94(m,3H),4.64(t, J =5.1Hz,1H),4.50-4.41(m,5H),4.05-3.95(m,10H),3.91-3.83(m,5H),3.75-3.65(m,14H), 3.51(s,1H),3.44-3.31(m,5H),3.06-2.94(m,10H),2.90(s,2H),2.89-2.78(m,2H),2.41-2 .30(m,2H),2.28-2.21(m,4H),2.10(s,9H),2.08-2.00(m,10H),1.99(s,11H),1.96-1.92(m ,2H),1.89(s,9H),1.86-1.80(m,2H),1.77(s,9H),1.66-1.31(m,35H),1.30-1.13(m,13H).

化合物31-2： Compound 31-2 :

向化合物31-1(150mg，0.06mmol)在無水DCM(1.0mL)中的溶液中加入DMAP(14mg，0.12mmol)和TEA(24mg，0.24mmol)，然後加入琥珀酸酐(20mg，0.2mmol)。將反應混合物在室溫攪拌3小時，LCMS顯示起始材料已完全消耗。將反應混合物用DCM(10mL)稀釋，用H2O(3mLx4)洗滌，然後用鹽水(3mLx4)洗滌，濃縮有機層以得到化合物31-2，為白色固體(130mg，產率為97%)。 To a solution of compound 31-1 (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL) was added DMAP (14 mg, 0.12 mmol) and TEA (24 mg, 0.24 mmol), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H 2 O (3 mL×4), and then with brine (3 mL×4), and the organic layer was concentrated to give compound 31-2 as a white solid (130 mg, 97% yield).

C130H191N11O43質量計算為：2594.31；實測：1297.4[M+2H]2+，ESI。 Mass calculated for C 130 H 191 N 11 O 43 : 2594.31; found: 1297.4 [M+2H] 2+ , ESI.

1HNMR(600MHz,DMSO-d6)δ 7.92-7.69(m,7H),7.50-7.41(m,1H),7.31-7.12(m,9H),6.92-6.89(m,6H),5.21(d,J=6.0Hz,3H),4.98-4.95(m,3H),4.52-4.41(m,5H),4.07-4.01(m,12H),3.90-3.83(m,5H),3.80-3.64(m,14H),3.49-3.25(m,17H),3.05-2.84(m,14H),2.47-2.23(m,8H),2.12-1.77(m,52H)1.61-1.21(m,46H). 1 HNMR(600MHz,DMSO-d6)δ 7.92-7.69(m,7H),7.50-7.41(m,1H),7.31-7.12(m,9H),6.92-6.89(m,6H),5.21(d, J =6.0Hz,3H),4.98-4.95(m,3H),4.52-4.41(m,5H),4.07-4.01(m,12H),3.90-3.83(m,5H),3.80-3.64(m, 14H),3.49-3.25(m,17H),3.05-2.84(m,14H),2.47-2.23(m,8H),2.12-1.77(m,52H)1.61-1.21(m,46H).

固體載體31-3：Solid carrier 31-3:

天然胺基-LCAA-CPG(上樣值：75μmol/g，1000Å)用ACN(100mLx2)、DMF(100mLx2)和DCM(100mLx2)洗滌，然後在高真空下乾燥過夜。 Native amine-LCAA-CPG (loading value: 75 μmol/g, 1000Å) was washed with ACN (100 mLx2), DMF (100 mLx2) and DCM (100 mLx2), and then dried under high vacuum overnight.

向琥珀酸酯31-2(130mg，0.05mmol)和HBTU(53mg，0.14mmol)在無水DMF(1.5mL)中的溶液中，加入DIPEA(30mg，0.23mmol)，在室溫下振盪反應混合物10分鐘，然後加入天然胺基-LCAA-CPG(300mg，上樣75μmol/g)，室溫振盪懸浮液20小時，然後過濾，用DMF(20mLx5)、ACN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥2小時，得到固體載體(300mg)。與Ac2O/吡啶/N-甲基咪唑(90μL/1.0mL/80μL)在室溫攪拌1小時，將固體載體上未反應的胺封端，用DMF(20mLx5)、CAN(20mLx5)、DCM(20mLx5)洗滌，直至TLC顯示沖提液在254nm處無斑點。固體載體真空乾燥15小時，以得到固體載體31-3(300mg)。為了計算上樣，取5.3mg乾燥的上樣CPG，加入20mL的3% DCA在DCM中的溶液。振盪溶液，測量482nm處的紫外吸光度。確保吸光度值低於1.0單位，以確保沒有信號飽和。然後應用以下公式： To a solution of succinate 31-2 (130 mg, 0.05 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL), DIPEA (30 mg, 0.23 mmol) was added, the reaction mixture was shaken at room temperature for 10 minutes, then natural amino-LCAA-CPG (300 mg, loading 75 μmol/g) was added, the suspension was shaken at room temperature for 20 hours, then filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), DCM (20 mL x 5) until TLC showed no spot in the eluent at 254 nm. The solid support was dried in vacuo for 2 hours to obtain a solid support (300 mg). Stir with Ac2O/pyridine/N-methylimidazole (90μL/1.0mL/80μL) at room temperature for 1 hour to cap the unreacted amine on the solid support, and wash with DMF (20mLx5), CAN (20mLx5), and DCM (20mLx5) until TLC shows that the eluent has no spots at 254nm. The solid support was vacuum dried for 15 hours to obtain solid support 31-3 (300mg). To calculate the loading, take 5.3mg of the dried loading CPG and add 20mL of 3% DCA solution in DCM. Vibrate the solution and measure the UV absorbance at 482nm. Make sure the absorbance value is less than 1.0 unit to ensure that there is no signal saturation. Then apply the following formula:

上樣(μmol/g)=(添加的DCA總體積(mL))*(482nm處的Abs值)*1000)/(78.3*(取的CPG mg數)) Sample loading (μmol/g) = (total volume of DCA added (mL)) * (Abs value at 482nm) * 1000) / (78.3 * (mg of CPG taken))

添加的DCA總體積(mL)=20mL Total volume of DCA added (mL) = 20mL

482nm處的Abs值=0.681 Abs value at 482nm = 0.681

取的CPG的Mg數=5.3mg The Mg content of CPG taken = 5.3mg

上樣(μmol/g)=((20)*(0.681)*1000)/78.3*(5.3)=32.82μmol/g。 Sample loading (μmol/g) = ((20)*(0.681)*1000)/78.3*(5.3) = 32.82μmol/g.

如前所述，GalNAc可以以單條或分步(1+1+1)的方式與寡核苷酸綴合。 As mentioned above, GalNAc can be conjugated to oligonucleotides in a single or stepwise (1+1+1) manner.

Figure 113123276-A0202-12-0272-309
Figure 113123276-A0202-12-0272-309

要實現1+1+1組裝，需要合成單-GalNAc醯胺和單-GalNAc固體載體。 To achieve 1+1+1 assembly, it is necessary to synthesize mono-GalNAc amide and mono-GalNAc solid support.

具體方法如下： The specific method is as follows:

Figure 113123276-A0202-12-0273-310
Figure 113123276-A0202-12-0273-310

Figure 113123276-A0202-12-0274-311
Figure 113123276-A0202-12-0274-311

Figure 113123276-A0202-12-0275-312
Figure 113123276-A0202-12-0275-312

實施例6用0.01-100nM(GalNAc-1)綴合的siRNA處理的原代人肝細胞(PHH)中C3 mRNA減弱的效果(劑量反應曲線實驗)。Example 6 The effect of C3 mRNA attenuation in primary human hepatocytes (PHH) treated with 0.01-100 nM (GalNAc-1)-conjugated siRNA (dose response curve experiment).

我們藉由在PHH細胞中自由攝取1nM和1000nM，檢測了使用(GalNAc-1)綴合的siRNA--具有(GalNAc-1)的C3-95、C3-96、C3-97、C3-98、C3-99、C3-100、C3-101、C3-102和C3-103，對C3 mRNA減弱的效果。 We examined the effect of (GalNAc-1)-conjugated siRNAs, C3-95, C3-96, C3-97, C3-98, C3-99, C3-100, C3-101, C3-102, and C3-103 with (GalNAc-1), on C3 mRNA attenuation by free uptake of 1 nM and 1000 nM in PHH cells.

所有(GalNAc-1)綴合的siRNA均能夠減弱C3 mRNA，在PHH中自由攝取濃度為1000nM的siRNA。如圖3A和表4所示，效果最好的四種siRNA是C3-98、C3-96、C3-103和C3-95，濃度為1000nM。然後，以劑量反應形式分析了這四種(GalNAc-1)綴合的siRNA的C3減弱效果，在PHH細胞中自由攝取 的濃度從0.01nM到100nM，如圖3B所示，劑量反應曲線中C3-95、C3-96、C3-98和C3-103的IC50值分別為1.6、0.6、0.3和1.0。管家基因TBP的mRNA水平作為這些實驗的內部對照。 All (GalNAc-1)-conjugated siRNAs were able to attenuate C3 mRNA, with a free uptake concentration of 1000 nM in PHH. As shown in Figure 3A and Table 4, the four most effective siRNAs were C3-98, C3-96, C3-103, and C3-95 at a concentration of 1000 nM. Then, the C3 attenuation effects of these four (GalNAc-1)-conjugated siRNAs were analyzed in a dose-response format, with free uptake concentrations ranging from 0.01 nM to 100 nM in PHH cells. As shown in Figure 3B, the IC 50 values of C3-95, C3-96, C3-98, and C3-103 in the dose-response curve were 1.6, 0.6, 0.3, and 1.0, respectively. The mRNA level of the housekeeping gene TBP served as an internal control in these experiments.

表4.在原代人肝細胞中進行的體外研究，其表明測試的(GalNAc-1)綴合的siRNA的C3 mRNA減弱效果

Figure 113123276-A0202-12-0276-313
Table 4. In vitro studies in primary human hepatocytes demonstrating the C3 mRNA attenuation effect of the tested (GalNAc-1)-conjugated siRNAs
Figure 113123276-A0202-12-0276-313

PHH細胞自由攝取的方法 Method for free uptake of PHH cells

可接種的人冷凍保存的原代肝細胞購自Gibco(目錄號HMCPUS，批號：Hu8115)。將細胞從液氮中取出後立即在37℃中解凍，然後在完全融化前轉移到預溫的解凍介質(Gibco，CM7000)中。將細胞在室溫中以100g離心10分鐘，去除解凍的介質，添加接種介質，即補充有肝細胞接種補充包(Gibco，CM3000)的William Medium E(Gibco，A1217601)，以7.6×104個細胞/100ul介質/孔的密度在I型膠原蛋白包被的96孔板(Gibco，A1142803)中將細胞接種。在37℃孵育4小時後，將接種介質更換為孵育介質(補充有肝細胞維持補充包(Gibco，CM4000)的Williams Medium E(Gibco，A1217601))，其中含有(GalNAc-1)綴合的siRNA，藉 由3倍稀釋從100nM降到0.01nM(圖3B)，或濃度為1nM和1000nM(圖3A)。對數據進行了兩次生物重複，如圖3A及圖3B所示。在(GalNAc-1)綴合的siRNA添加後48小時收穫細胞。藉由RT-qPCR測定C3 mRNA，方法與HepG2細胞轉染實驗相同。C3 mRNA水平歸一化至TBP mRNA。用GraphPad Prism 9.3.1版使用四參數邏輯模型製作劑量反應曲線。劑量反應曲線中C3-95、C3-96、C3-98和C3-103的IC50值分別為1.6、0.6、0.3和1.0。 Vaccinable human cryopreserved primary hepatocytes were purchased from Gibco (Catalog No. HMCPUS, Lot No. Hu8115). Cells were thawed at 37°C immediately after being taken out of liquid nitrogen and then transferred to pre-warmed thawing medium (Gibco, CM7000) before complete thawing. The cells were centrifuged at 100 g for 10 minutes at room temperature, the thawed medium was removed, and the inoculation medium, namely William Medium E (Gibco, A1217601) supplemented with a hepatocyte inoculation supplement pack (Gibco, CM3000), was added, and the cells were inoculated at a density of 7.6×10 4 cells/100 ul medium/well in a type I collagen-coated 96-well plate (Gibco, A1142803). After incubation at 37°C for 4 hours, the inoculation medium was replaced with incubation medium (Williams Medium E (Gibco, A1217601) supplemented with Hepatocyte Maintenance Supplement Pack (Gibco, CM4000)) containing (GalNAc-1)-conjugated siRNA at concentrations of 1 nM and 1000 nM (Figure 3A) by 3-fold dilution from 100 nM to 0.01 nM (Figure 3B), or at concentrations of 1 nM and 1000 nM (Figure 3A). The data were performed in two biological replicates, as shown in Figures 3A and 3B. Cells were harvested 48 hours after the addition of (GalNAc-1)-conjugated siRNA. C3 mRNA was measured by RT-qPCR using the same method as the HepG2 cell transfection experiment. C3 mRNA levels were normalized to TBP mRNA. Dose response curves were prepared using a four-parameter logic model using GraphPad Prism version 9.3.1. The IC 50 values of C3-95, C3-96, C3-98, and C3-103 in the dose response curves were 1.6, 0.6, 0.3, and 1.0, respectively.

實施例7.0.1-300nM(GalNAc-3或GalNAc-7)綴合的siRNA處理的原代人肝細胞(PHH)中C3 mRNA減弱的效果(劑量反應曲線實驗)。Example 7. Effect of C3 mRNA attenuation in primary human hepatocytes (PHH) treated with 0.1-300 nM (GalNAc-3 or GalNAc-7) conjugated siRNA (dose response curve experiment).

藉由劑量反應曲線分析了使用表5中所示的GalNAc siRNA綴合物孵育後，C3 mRNA的表達。結果顯示在表5和圖4A、圖4B、圖4C、圖4D中。所有GalNAc綴合的siRNA對C3 mRNA水平均表現出劑量反應效應。表5結果顯示，所有GalNAc綴合的siRNA在100nM時的最大抑制率均接近或超過70%。綴合的siRNA C3-125、C3-126、C3-127、C3-128、C3-134、C3-136、C3-138、C3-141和C3-143在100nM時的最大抑制率均接近或超過80%。 The expression of C3 mRNA after incubation with the GalNAc siRNA conjugates shown in Table 5 was analyzed by dose-response curves. The results are shown in Table 5 and Figures 4A, 4B, 4C, and 4D. All GalNAc-conjugated siRNAs showed a dose-response effect on C3 mRNA levels. The results in Table 5 show that the maximum inhibition rate of all GalNAc-conjugated siRNAs at 100nM is close to or exceeds 70%. The maximum inhibition rate of conjugated siRNAs C3-125, C3-126, C3-127, C3-128, C3-134, C3-136, C3-138, C3-141, and C3-143 at 100nM is close to or exceeds 80%.

PHH細胞自由攝取的方法 Method for free uptake of PHH cells

可接種的人冷凍保存的原代肝細胞購自XenoTech(目錄號HPCH10+，批號：2010146)。將細胞從液氮中取出後立即在37℃中解凍，然後將移細胞沉澱導入預溫的解凍介質(XenoTech，K8000 OptiThaw肝細胞介質)。細胞在室溫下以100g離心10分鐘，除去解凍介質，加入接種介質(XenoTech，K8200 Optiplate肝細胞介質)以重新懸浮細胞。然後，將細胞接種到膠原蛋白I包被的96孔板(Gibco，A1142803)中，密度為1.0x105個細胞/100ul介質/孔。在37℃孵育4小時後，將接種介質更換為孵育介質(XenoTech，K8300 OptiCulture肝細胞介質)，其中含有 GalNAc綴合的siRNA，藉由3倍稀釋從300nM降到0.1nM。在圖4所示的數據中，用siRNA處理細胞48小時，進行了兩次生物重複。藉由RT-qPCR測定C3 mRNA，方法與HepG2細胞轉染實驗相同。C3 mRNA水平歸一化至TBP mRNA。用GraphPad Prism 9.3.1版使用四參數邏輯模型製作劑量反應曲線。 Human cryopreserved primary hepatocytes ready for inoculation were purchased from XenoTech (Catalog No. HPCH10+, Lot No. 2010146). Cells were immediately thawed at 37°C after being removed from liquid nitrogen, and the cell pellet was then transferred into pre-warmed thawing medium (XenoTech, K8000 OptiThaw Hepatocyte Medium). Cells were centrifuged at 100 g for 10 minutes at room temperature, the thawing medium was removed, and inoculation medium (XenoTech, K8200 Optiplate Hepatocyte Medium) was added to resuspend the cells. The cells were then seeded into collagen I-coated 96-well plates (Gibco, A1142803) at a density of 1.0x10 5 cells/100ul medium/well. After incubation at 37°C for 4 hours, the seeding medium was replaced with incubation medium (XenoTech, K8300 OptiCulture Hepatocyte Medium) containing GalNAc-conjugated siRNA, which was reduced from 300nM to 0.1nM by 3-fold dilution. In the data shown in Figure 4, cells were treated with siRNA for 48 hours in two biological replicates. C3 mRNA was measured by RT-qPCR, using the same method as the HepG2 cell transfection experiment. C3 mRNA levels were normalized to TBP mRNA. Dose-response curves were prepared using GraphPad Prism version 9.3.1 using a four-parameter logic model.

表5.在原代人肝細胞中進行的體外研究，其表明測試的(GalNAc-3或GalNAc-7)綴合的siRNA的C3 mRNA減弱效果

Figure 113123276-A0202-12-0278-314
Table 5. In vitro studies in primary human hepatocytes demonstrating the C3 mRNA attenuation effect of the tested (GalNAc-3 or GalNAc-7) conjugated siRNAs
Figure 113123276-A0202-12-0278-314

實施例8.測試的GalNAc-綴合的siRNA在小鼠中的體內減弱效果Example 8. Testing the in vivo attenuation effect of GalNAc-conjugated siRNA in mice

B6-hC3小鼠(種系編號：T05196)，9週齡，獲自中國南京的GemPharmatech，該小鼠轉基因表達人C3基因。動物繁殖和飼養、siRNA給藥、血清收集和hC3 ELISA測定均在GemPharmatech進行。根據給藥前(D-3，基線)血清中人C3的量，針對雄性和雌性小鼠將小鼠隨機分為3組。在體內研究的第0天，小鼠接受單次皮下劑量5mg/kgsiRNA(溶於鹽水中)或僅有鹽水。我們分別選擇了兩種siRNA，C3-138和C3-139綴合的GalNAc-7和GalNAc-3用於給藥。研究期間監測小鼠的生存能力、體重和行為。在給藥前(D-3)、第8天(D8)、第14天(D14)採集血清樣本。使用市售的人C3 ELISA試劑盒分析血清樣本。分析根據製造商的方案進行，計算血清人C3蛋白水平相對於相應基線水平。結果顯示在表6、圖5A和圖5B中。數據顯示小鼠血清樣本的相對人C3蛋白水平(以百分比表示)，這些血清樣本分別在給藥前D-3、以及在給藥5mg/kg鹽水、C3-138和C3-139的GalNAc-siRNA後D8、D14採集。siRNA大大降低了D8和D14天的雄性和雌性小鼠血清中的人C3蛋白水平。接受鹽水處理的動物在D8時，雄性和雌性小鼠的人C3蛋白水平相對於基線降低了約30%，但在D14時人C3蛋白水平幾乎恢復到基線。體內研究的數據顯示，藉由向小鼠皮下給藥，測試的GalNAc綴合的siRNA能顯著降低循環中人C3蛋白的水平。 B6-hC3 mice (strain number: T05196), 9 weeks old, were obtained from GemPharmatech, Nanjing, China, and were transgenic for expressing the human C3 gene. Animal breeding and maintenance, siRNA administration, serum collection, and hC3 ELISA assays were all performed at GemPharmatech. Mice were randomly divided into 3 groups for male and female mice based on the amount of human C3 in the serum before administration (D-3, baseline). On day 0 of the in vivo study, mice received a single subcutaneous dose of 5 mg/kg siRNA (dissolved in saline) or saline only. We selected two siRNAs, C3-138 and C3-139 conjugated to GalNAc-7 and GalNAc-3, respectively, for administration. The viability, weight, and behavior of the mice were monitored during the study. Serum samples were collected before dosing (D-3), on day 8 (D8), and on day 14 (D14). Serum samples were analyzed using a commercially available human C3 ELISA kit. The analysis was performed according to the manufacturer's protocol, and serum human C3 protein levels were calculated relative to the corresponding baseline levels. The results are shown in Table 6, Figure 5A and Figure 5B. The data show the relative human C3 protein levels (expressed as percentages) of mouse serum samples, which were collected on D-3 before dosing, and on D8 and D14 after dosing with 5 mg/kg saline, C3-138 and C3-139 GalNAc-siRNA. siRNA significantly reduced the human C3 protein levels in the serum of male and female mice on days D8 and D14. In animals treated with saline, human C3 protein levels in male and female mice decreased by about 30% relative to baseline at D8, but human C3 protein levels almost returned to baseline at D14. Data from in vivo studies showed that the tested GalNAc-conjugated siRNA could significantly reduce circulating human C3 protein levels by subcutaneous administration to mice.

表6.在人C3基因轉基因小鼠中的體內研究，其表明測試的綴合的siRNA降低人C3蛋白的效果

Figure 113123276-A0202-12-0280-315
Table 6. In vivo studies in human C3 gene transgenic mice showing the efficacy of the tested conjugated siRNAs in reducing human C3 protein
Figure 113123276-A0202-12-0280-315

實施例9.測試的GalNAc-綴合的siRNA在小鼠中的體內減弱效果Example 9. Testing the in vivo attenuation effect of GalNAc-conjugated siRNA in mice

雄性人源化的C3小鼠(C57BL/6JSmoc-C3em1(hC3)/Smoc，目錄號NM-HU-200079)，7-8週齡，購自上海模式生物中心有限公司，使用前隔離一週。動物繁殖和飼養、siRNA給藥、血清收集和hC3 ELISA測定均在上海模式生物中心有限公司進行。根據給藥前(D-5，基線)血清中人C3的量，將小鼠隨機分為14組，每組6隻小鼠。在體內研究的第0天，小鼠接受單次皮下劑量3mg/kg siRNA(溶於鹽水中)或僅有鹽水。研究期間監測小鼠的生存能力、體重和行為。在給藥前(D-5)、給藥後第7天(D7)、第14天(D14)、第21天(D21)、第28天(D28)和第35天(D35)採集血清樣本。使用市售的人C3 ELISA試劑盒分析血清樣本。分析根據製造商的方案測定，計算血清人C3蛋白水平相對於相應基線水平。 Male humanized C3 mice (C57BL/6JSmoc-C3em1(hC3)/Smoc, catalog number NM-HU-200079), 7-8 weeks old, were purchased from Shanghai Model Organism Center Co., Ltd. and isolated for one week before use. Animal breeding and maintenance, siRNA administration, serum collection and hC3 ELISA assay were all performed at Shanghai Model Organism Center Co., Ltd. Mice were randomly divided into 14 groups of 6 mice each based on the amount of human C3 in serum before administration (D-5, baseline). On day 0 of the in vivo study, mice received a single subcutaneous dose of 3 mg/kg siRNA (dissolved in saline) or saline only. The viability, weight and behavior of the mice were monitored during the study. Serum samples were collected before dosing (D-5), 7 days (D7), 14 days (D14), 21 days (D21), 28 days (D28), and 35 days (D35) after dosing. Serum samples were analyzed using a commercially available human C3 ELISA kit. The analysis was determined according to the manufacturer's protocol, and serum human C3 protein levels were calculated relative to the corresponding baseline levels.

結果如表7和圖6所示。數據顯示人C3蛋白水平與基線(D-5)相比相對降低(百分比)。siRNA降低了小鼠血清中的人C3蛋白水平，最大減弱範圍在D21至D28。鹽水處理的動物在處理期間顯示出人C3蛋白水平降低，但在D35時恢復到基線。體內研究的數據顯示，藉由給小鼠皮下給藥，測試的GalNAc 綴合的siRNA顯著降低循環中人C3蛋白的水平。C3-127和C3-130均達到80%的最大減弱效率，因此被選中進行進一步評價。 The results are shown in Table 7 and Figure 6. The data show a relative decrease (percentage) in human C3 protein levels compared to baseline (D-5). siRNA reduced human C3 protein levels in mouse serum, with maximum reduction ranging from D21 to D28. Animals treated with saline showed a decrease in human C3 protein levels during the treatment period, but returned to baseline at D35. Data from in vivo studies showed that the tested GalNAc conjugated siRNAs significantly reduced circulating human C3 protein levels by subcutaneous administration to mice. C3-127 and C3-130 both achieved a maximum reduction efficiency of 80% and were therefore selected for further evaluation.

表7.在人C3基因轉基因小鼠中的體內研究，其表明測試的綴合的siRNA降低人C3蛋白的效果

Figure 113123276-A0202-12-0281-316
Table 7. In vivo studies in human C3 gene transgenic mice showing the efficacy of the tested conjugated siRNA in reducing human C3 protein
Figure 113123276-A0202-12-0281-316

實施例10.不同劑量的測試的GalNAc-綴合的siRNA在小鼠中的體內減弱效果Example 10. In vivo attenuation effect of different doses of tested GalNAc-conjugated siRNA in mice

在實施例9中，在人源化的C3小鼠模型(SMOC)中測試了C3-127和C3-130。兩種siRNA在3mpk時均表現出顯著的減弱效果。為了進一步分析減弱效果和持續時間，在表達人C3的小鼠中測試了不同劑量的這些siRNA。雌性B6-hC3小鼠(種系編號：T05196)，8週齡，獲自中國南京的GemPharmatech，該小鼠轉基因表達人C3基因。動物繁殖和飼養、siRNA給藥、血清收集和hC3ELISA測定均在GemPharmatech進行。根據給藥前(D-3，基線)血清中人C3水平，針對C3-127和C3-130，將小鼠隨機分為5組。在體內研究的第0天，小鼠 接受單次皮下劑量1mg/kg、3mg/kg、5mg/kg或10mg/kg siRNA(溶於鹽水中)或僅有鹽水。研究期間監測小鼠的生存能力、體重和行為。在給藥前(D-3)、第7天(D7)、第14天(D14)、第21天(D21)、第28天(D28)和第35天(D35)採集血清樣本。使用市售的人C3 ELISA試劑盒分析血清樣本。分析根據製造商的方案進行，計算血清人C3蛋白水平相對於相應基線水平。結果如表8、圖7所示。數據顯示，分別在給藥前D-3，以及在多次給藥C3-127和C3-130的GalNAc-siRNA後D7、D14、D21、D28、D35採集的小鼠血清樣本中，相對人C3蛋白水平(以百分比表示)。siRNA大大降低了在D7、D14和D21時，在小鼠血清中的人C3蛋白水平。數據證實，C3-127和C3-130有效降低不同小鼠模型中的C3蛋白水平。兩種siRNA的減弱效果均可持續至給藥後D35，表明siRNA在減弱老年小鼠的循環C3蛋白水平方面具有很強的持久性。 In Example 9, C3-127 and C3-130 were tested in a humanized C3 mouse model (SMOC). Both siRNAs showed significant attenuation effects at 3 mpk. To further analyze the attenuation effect and duration, different doses of these siRNAs were tested in mice expressing human C3. Female B6-hC3 mice (strain number: T05196), 8 weeks old, were obtained from GemPharmatech in Nanjing, China, which were transgenic for expressing the human C3 gene. Animal breeding and husbandry, siRNA administration, serum collection and hC3 ELISA assays were all performed at GemPharmatech. Based on the level of human C3 in serum before administration (D-3, baseline), mice were randomly divided into 5 groups for C3-127 and C3-130. On day 0 of the in vivo study, mice received a single subcutaneous dose of 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10 mg/kg siRNA (dissolved in saline) or saline alone. The viability, weight, and behavior of the mice were monitored during the study. Serum samples were collected before dosing (D-3), on day 7 (D7), day 14 (D14), day 21 (D21), day 28 (D28), and day 35 (D35). Serum samples were analyzed using a commercially available human C3 ELISA kit. The analysis was performed according to the manufacturer's protocol, and serum human C3 protein levels were calculated relative to the corresponding baseline levels. The results are shown in Table 8 and Figure 7. The data showed the relative human C3 protein levels (expressed as a percentage) in mouse serum samples collected at D-3 before administration and at D7, D14, D21, D28, and D35 after multiple administrations of GalNAc-siRNA of C3-127 and C3-130. siRNA significantly reduced the human C3 protein levels in mouse serum at D7, D14, and D21. The data confirmed that C3-127 and C3-130 effectively reduced C3 protein levels in different mouse models. The attenuation effects of both siRNAs lasted until D35 after administration, indicating that siRNA has a strong persistence in attenuating circulating C3 protein levels in aged mice.

表8.在人C3基因轉基因小鼠中的體內研究，其顯示不同劑量的測試的GalNAc綴合的siRNA降低人C3蛋白的效果

Figure 113123276-A0202-12-0283-317
Table 8. In vivo studies in human C3 gene transgenic mice showing the effect of different doses of the tested GalNAc-conjugated siRNA on reducing human C3 protein
Figure 113123276-A0202-12-0283-317

TW202516005A_113123276_SEQL.xmlTW202516005A_113123276_SEQL.xml

Claims (24)

一種用於抑制補體成分C3(C3)表達的具有雙鏈核糖核酸(dsRNA)的siRNA劑，其中該dsRNA包含第一鏈和第二鏈，其中該第一鏈序列包含至少15個連續核苷酸，該連續核苷酸與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過3個核苷酸；以及該第二鏈包含與所述第一鏈至少部分互補的核苷酸序列。 A siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the dsRNA comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, and the consecutive nucleotides differ from any of the nucleotide sequences of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand. 如請求項1所述的siRNA劑，其中該第一鏈序列包含至少16個，較佳至少17個，更佳至少18個以及最佳全部19個連續核苷酸，該連續核苷酸與SEQ ID NO：1的核苷酸3123-3141、5019-5037、2304-2322、494-512、2643-2661、2244-2262、589-607、2482-2500或4698-4716中的任一核苷酸序列相差不超過2個核苷酸，較佳相差不超過1個核苷酸，以及更佳沒有任何核苷酸差異。 The siRNA agent as described in claim 1, wherein the first strand sequence comprises at least 16, preferably at least 17, more preferably at least 18 and most preferably all 19 consecutive nucleotides, and the consecutive nucleotides differ from any of the nucleotide sequences of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 2 nucleotides, preferably by no more than 1 nucleotide, and more preferably without any nucleotide difference. 如請求項1或2所述的siRNA劑，其中該第一鏈和該第二鏈形成15-25個核苷酸長的雙鏈區；較佳形成15-23個核苷酸長的雙鏈區；更佳形成15-19個核苷酸長的雙鏈區；進一步佳形成17-19個核苷酸長的雙鏈區；最較佳形成15、16、17、18、19、20、21、22、23個核苷酸長的雙鏈區。 The siRNA agent as described in claim 1 or 2, wherein the first chain and the second chain form a double-chain region of 15-25 nucleotides in length; preferably a double-chain region of 15-23 nucleotides in length; more preferably a double-chain region of 15-19 nucleotides in length; further preferably a double-chain region of 17-19 nucleotides in length; most preferably a double-chain region of 15, 16, 17, 18, 19, 20, 21, 22, 23 nucleotides in length. 如請求項1至3中任一項所述的siRNA劑，其中該第一鏈具有分別由SEQ ID NO：20、96、16、150、32、100、110、134、136表示的核苷酸序列；和/或與該第一鏈互補的所述第二鏈具有分別由SEQ ID NO：21、97、17、151、33、101、111、135、137表示的核苷酸序列。 The siRNA agent as described in any one of claims 1 to 3, wherein the first chain has a nucleotide sequence represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively; and/or the second chain complementary to the first chain has a nucleotide sequence represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, 137, respectively. 如請求項1至4中任一項所述的siRNA劑，其中該第一鏈具有SEQ ID NO：20所示的核苷酸序列以及該第二鏈具有SEQ ID NO：21所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：96所示的核苷酸序列以及該第二鏈具有SEQ ID NO：97所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：16所示的核苷酸序列以及該第二鏈具有SEQ ID NO：17所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：150所示的核苷酸序列以及該第二鏈具有SEQ ID NO：151所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：32所示的核苷酸序列以及該第二鏈具有SEQ ID NO：33所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：100所示的核苷酸序列以及該第二鏈具有SEQ ID NO：101所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：110所示的核苷酸序列以及該第二鏈具有SEQ ID NO：111所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：134所示的核苷酸序列以及該第二鏈具有SEQ ID NO：135所示的核苷酸序列；或者該第一鏈具有SEQ ID NO：136所示的核苷酸序列以及該第二鏈具有SEQ ID NO：137所示的核苷酸序列。 The siRNA agent as described in any one of claims 1 to 4, wherein the first chain has the nucleotide sequence shown in SEQ ID NO: 20 and the second chain has the nucleotide sequence shown in SEQ ID NO: 21; or the first chain has the nucleotide sequence shown in SEQ ID NO: 96 and the second chain has the nucleotide sequence shown in SEQ ID NO: 97; or the first chain has the nucleotide sequence shown in SEQ ID NO: 16 and the second chain has the nucleotide sequence shown in SEQ ID NO: 17; or the first chain has the nucleotide sequence shown in SEQ ID NO: 150 and the second chain has the nucleotide sequence shown in SEQ ID NO: 151; or the first chain has the nucleotide sequence shown in SEQ ID NO: 32 and the second chain has the nucleotide sequence shown in SEQ ID NO: 33; or the first chain has the nucleotide sequence shown in SEQ ID NO: 100 and the second chain has the nucleotide sequence shown in SEQ ID NO: 101; or the first chain has the nucleotide sequence shown in SEQ ID NO: 110 and the second chain has the nucleotide sequence shown in SEQ ID NO: NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137. 如請求項1至5中任一項所述的siRNA劑，其中該siRNA劑包含至少一個修飾的核苷酸；較佳地，其中沒有全部修飾並且包括不超過5、4、3、2或1個未修飾的核苷酸；更佳地，其中該第一鏈的全部核苷酸都是修飾的核苷酸，該第二鏈的全部核苷酸都是修飾的核苷酸。 The siRNA agent as described in any one of claims 1 to 5, wherein the siRNA agent comprises at least one modified nucleotide; preferably, wherein none of the nucleotides are modified and comprises no more than 5, 4, 3, 2 or 1 unmodified nucleotides; more preferably, wherein all the nucleotides of the first chain are modified nucleotides and all the nucleotides of the second chain are modified nucleotides. 如請求項6所述的siRNA劑，其中至少一個修飾的核苷酸選自3’末端脫氧胸腺嘧啶(dT)核苷酸、2'-甲氧基修飾的核苷酸、2’-氟修飾的核苷酸、2'-OCF2H修飾的核苷酸、包含5'-硫代磷酸酯基團的核苷酸和包含5'-(E)-乙烯基膦酸酯的核苷酸。 The siRNA agent of claim 6, wherein at least one modified nucleotide is selected from 3' terminal deoxythymine (dT) nucleotides, 2'-methoxy modified nucleotides, 2'-fluorine modified nucleotides, 2'-OCF 2 H modified nucleotides, nucleotides containing 5'-phosphorothioate groups and nucleotides containing 5'-(E)-vinylphosphonate. 如請求項6或7所述的siRNA劑，其中， The siRNA agent as described in claim 6 or 7, wherein, (i)該第一鏈的全部核苷酸包含如下模式的修飾： (i) All nucleotides in the first chain contain the following pattern of modifications: 5’端起第5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾； The pentose sugars at the 5’ end of the nucleotide residues at positions 5, 7, 8 and 9 are modified by 2’-fluorine substitution; 5’端起第1、2、4、6、10、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； The pentoses at the 1st, 2nd, 4th, 6th, 10th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; 5’端起第3、11和14位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾； The pentose sugars at the 5' end of the nucleotide residues at positions 3, 11 and 14 are modified by 2'-methoxy or 2'-fluorine substitution; 該第二鏈的全部核苷酸包含如下模式的修飾： All nucleotides in the second chain contain the following pattern of modifications: 5’端起第2、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾； The pentose sugars at the 5' end of the 2nd, 14th, 16th and 18th nucleotide residues are modified by 2'-fluorine substitution; 5’端起第1、3、5、6、7、9、11、12、13、15、17和19位核苷酸殘基的戊 糖經2’-甲氧基修飾得到修飾； The pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; 5’端起第4、8和10位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾；或者 The pentose sugars at the 5' end of the nucleotide residues at positions 4, 8 and 10 are 2'-methoxy-modified or 2'-fluorinated; or (ii)該第一鏈的全部核苷酸包含如下模式的修飾： (ii) All nucleotides in the first chain contain the following pattern of modifications: 5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾； The pentose sugars at the 7th, 9th, 10th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; 5’端起第2、4、6、8、12、14、16和18位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； The pentoses at the 5' end of the nucleotide residues at positions 2, 4, 6, 8, 12, 14, 16 and 18 are modified by 2'-methoxy modification; 5’端起第1、3、5、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾或2’-氟取代修飾； The pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 13, 15, 17 and 19 are modified by 2'-methoxy or 2'-fluorine substitution; 該第二鏈的全部核苷酸包含如下模式的修飾： All nucleotides in the second chain contain the following pattern of modifications: 5’端起第2、6、8、14和16位核苷酸殘基的戊糖經2’-氟取代修飾； The pentose sugars at the 5' end of the 2nd, 6th, 8th, 14th and 16th nucleotide residues are modified by 2'-fluorine substitution; 5’端起第1、3、5、7、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； The pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 7, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; 5’端起第4、9、10和18位經2’-甲氧基修飾或2’-氟取代修飾。 The 4th, 9th, 10th and 18th positions from the 5' end are modified by 2'-methoxy or 2'-fluorine substitution. 如請求項8所述的siRNA劑，其中， The siRNA agent as described in claim 8, wherein, (i)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (i) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；或者 All nucleotides of the second chain contain the following pattern of modification: the pentoses at the 5' end of the nucleotide residues at positions 2, 8, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; or (ii)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第3、5、7、8和9位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、4、6、10、11、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (ii) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 5' end of the nucleotide residues at positions 3, 5, 7, 8 and 9 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸都包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；或者 All nucleotides of the second chain contain the following pattern of modifications: the pentoses at the 5' end of the nucleotide residues at positions 2, 4, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; or (iii)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (iii) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 5th, 7th, 8th, 9th and 11th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；或者 All nucleotides of the second chain contain the following pattern of modification: the pentoses at the 5' end of the nucleotide residues at positions 2, 4, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; or (iv)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (iv) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、8、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、6、7、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；或者 All nucleotides of the second chain contain the following pattern of modification: the pentoses at the 5' end of the nucleotide residues at positions 2, 8, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; or (v)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第5、7、8、9和14位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、6、10、11、12、13、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (v) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 5th, 7th, 8th, 9th and 14th nucleotide residues at the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues at the 5' end are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、4、14、16和18位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、5、6、7、8、9、10、11、12、13、15、17和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾；或者 All nucleotides of the second chain contain the following pattern of modification: the pentoses at the 5' end of the nucleotide residues at positions 2, 4, 14, 16 and 18 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 are modified by 2'-methoxy modification; or (vi)該第一鏈的全部核苷酸包含如下模式的修飾：5’端起第7、9、10和11位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、2、3、4、5、6、8、12、13、14、15、16、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾； (vi) All nucleotides of the first chain contain the following pattern of modification: the pentoses at the 7th, 9th, 10th and 11th nucleotide residues from the 5' end are modified by 2'-fluorine substitution; the pentoses at the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification; 該第二鏈的全部核苷酸包含如下模式的修飾：5’端起第2、6、8、9、14和16位核苷酸殘基的戊糖經2’-氟取代修飾；5’端起第1、3、4、5、7、10、11、12、13、15、17、18和19位核苷酸殘基的戊糖經2’-甲氧基修飾得到修飾。 All nucleotides of the second chain contain the following pattern of modifications: the pentoses at the 5' end of the nucleotide residues at positions 2, 6, 8, 9, 14 and 16 are modified by 2'-fluorine substitution; the pentoses at the 5' end of the nucleotide residues at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 are modified by 2'-methoxy modification. 如請求項1至9中任一項所述的siRNA劑，其中第二鏈在5’端包含末端5’(E)-乙烯基膦酸酯核苷酸。 An siRNA agent as described in any one of claims 1 to 9, wherein the second strand comprises a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end. 如請求項1至10中任一項所述的siRNA劑，其中， The siRNA agent as described in any one of claims 1 to 10, wherein, 該第一鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵；和/或該第二鏈在5’末端和/或3’末端包含兩個硫代磷酸酯核苷酸間鍵； The first chain comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end; and/or the second chain comprises two phosphorothioate internucleotide bonds at the 5' end and/or the 3' end; 較佳地， Preferably, (i)該第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵； (i) the first strand comprises two phosphorothioate internucleotide bonds at the 3' end; 以及該第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵並且在3’末端包含兩個硫代磷酸酯核苷酸間鍵；或者 and the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus; or (ii)該第一鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵； (ii) the first strand contains two phosphorothioate internucleotide bonds at the 5' end; 以及該第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵並且在3’末端包含兩個硫代磷酸酯核苷酸間鍵。 and the second strand comprises two phosphorothioate internucleotide bonds at the 5' terminus and two phosphorothioate internucleotide bonds at the 3' terminus. 如請求項1至11中任一項所述的siRNA劑，其還包含靶向配體；較佳地，該靶向配體包含N-乙醯半乳糖胺(GalNAc)綴合物或其衍生物。 The siRNA agent as described in any one of claims 1 to 11 further comprises a targeting ligand; preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) conjugate or a derivative thereof. 如請求項12所述的siRNA劑，其中該靶向配體與該siRNA劑的第一鏈的3'末端或5'末端綴合。 The siRNA agent as described in claim 12, wherein the targeting ligand is conjugated to the 3' end or 5' end of the first chain of the siRNA agent. 如請求項12或13所述的siRNA劑，其中， The siRNA agent as described in claim 12 or 13, wherein, 該靶向配體與該第一鏈的5'末端綴合； The targeting ligand is conjugated to the 5' end of the first chain; 該第一鏈在3’末端包含兩個硫代磷酸酯核苷酸間鍵； The first strand contains two phosphorothioate internucleotide bonds at the 3' end; 該第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵並且在3’末端包含兩個硫代磷酸酯核苷酸間鍵； The second strand comprises two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide bonds at the 3' end; 視需要地，該第一鏈和/或該第二鏈的核苷酸之間的全部剩餘鍵均為磷酸二酯鍵； Optionally, all remaining bonds between nucleotides in the first chain and/or the second chain are phosphodiester bonds; 或者 or 該靶向配體與該第一鏈的3'末端綴合； The targeting ligand is conjugated to the 3' end of the first chain; 該第一鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵； The first strand contains two phosphorothioate internucleotide bonds at the 5' end; 該第二鏈在5’末端包含兩個硫代磷酸酯核苷酸間鍵並且在3’末端包含兩個硫代磷酸酯核苷酸間鍵； The second strand comprises two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide bonds at the 3' end; 視需要地，該第一鏈和/或該第二鏈的核苷酸之間的全部剩餘鍵均為磷酸二酯鍵。 Optionally, all remaining bonds between nucleotides of the first chain and/or the second chain are phosphodiester bonds. 如請求項12至14中任一項所述的siRNA劑，其中， The siRNA agent as described in any one of claims 12 to 14, wherein, 該第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：214)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or 該第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：208)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN(SEQ ID NO：210)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO：212)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN(SEQ ID NO：212)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO：213)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO： 214)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：211)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or 該第一鏈具有由GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：215)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：209)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or 該第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：216)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列以及該第二鏈具有由(E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218) and the second chain has a nucleotide sequence represented by (E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN(SEQ ID NO：218)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN(SEQ ID NO：220)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or 該第一鏈具有由GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO：221)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or 該第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：222)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or 該第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：222)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or 該第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：223)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：219)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or 該第一鏈具有由GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：223)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：217)表示的核苷酸序列； The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217); 其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵，“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分，“GN”是靶向配體。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond, "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end, and "GN" is a targeting ligand. 如請求項12至15中任一項所述的siRNA劑，其中該siRNA劑與配體綴合，如下圖所示：該配體包括(i)一個或多個N-乙醯半乳糖胺(GalNAc)部分或其衍生物，和(ii)接頭，其中該接頭將至少一個GalNAc部分或其衍生物與核酸綴合，和(iii)雙環基團連接GalNAc和siRNA，或(iv)四官能團，其中連接該GalNAc部分， A siRNA agent as described in any one of claims 12 to 15, wherein the siRNA agent is conjugated to a ligand, as shown in the following figure: the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to a nucleic acid, and (iii) a bicyclic group connecting GalNAc and siRNA, or (iv) a tetrafunctional group, wherein the GalNAc moiety is connected,
Figure 113123276-A0202-13-0012-318
Figure 113123276-A0202-13-0012-318
 其中X是O、S。 Where X is O, S. 如請求項16所述的siRNA劑，其中該siRNA劑與該配體綴合，如下圖所示： The siRNA agent as described in claim 16, wherein the siRNA agent is conjugated with the ligand, as shown in the following figure:
Figure 113123276-A0202-13-0013-321
Figure 113123276-A0202-13-0013-321
 其中X是O或S； Where X is O or S; 該
Figure 113123276-A0202-13-0013-319
是： The
Figure 113123276-A0202-13-0013-319
yes:
Figure 113123276-A0202-13-0013-322
Figure 113123276-A0202-13-0013-322
 該
Figure 113123276-A0202-13-0013-320
是： The
Figure 113123276-A0202-13-0013-320
yes:
Figure 113123276-A0202-13-0014-323
Figure 113123276-A0202-13-0014-323
Figure 113123276-A0202-13-0015-324
Figure 113123276-A0202-13-0015-324
Figure 113123276-A0202-13-0016-325
Figure 113123276-A0202-13-0016-325
 或 or
Figure 113123276-A0202-13-0017-327
Figure 113123276-A0202-13-0017-327
 其中X是O或S； Where X is O or S; 該
Figure 113123276-A0202-13-0017-326
是 The
Figure 113123276-A0202-13-0017-326
yes
Figure 113123276-A0202-13-0017-328
Figure 113123276-A0202-13-0017-328
Figure 113123276-A0202-13-0018-329
Figure 113123276-A0202-13-0018-329
Figure 113123276-A0202-13-0019-330
Figure 113123276-A0202-13-0019-330
Figure 113123276-A0202-13-0020-331
Figure 113123276-A0202-13-0020-331
 如請求項16或17所述的siRNA劑，其中該圖中的接頭獨立地選自： The siRNA agent as described in claim 16 or 17, wherein the linkers in the figure are independently selected from: (1)-(CH2)x-C(O)NH-(CH2)y-C(O)-； (1)-(CH 2 )xC(O)NH-(CH 2 )yC(O)-; (2)-(CH2)x-C(O)NH-(CH2)y-NHC(O)-(CH2)z-C(O)-； (2)-(CH 2 )xC(O)NH-(CH 2 )y-NHC(O)-(CH 2 )zC(O)-; (3)-NH-(CH2)y-C(O)-； (3) -NH-(CH 2 )yC(O)-; (4)-C(O)-(CH2)y-C(O)-； (4) -C(O)-(CH 2 )yC(O)-; 其中，該圖中的每個接頭視需要地相同或不同；該x、y和z獨立地選自1-10；較佳地，該x和y獨立地選自2-8，z選自1-6；更佳地，該x和y獨立地選自3-6，z選自1-4。 Wherein, each joint in the figure is the same or different as needed; x, y and z are independently selected from 1-10; preferably, x and y are independently selected from 2-8, and z is selected from 1-6; more preferably, x and y are independently selected from 3-6, and z is selected from 1-4. 如請求項12至18中任一項所述的siRNA劑，其中該siRNA劑與該配體綴合，如下圖所示： The siRNA agent as described in any one of claim 12 to 18, wherein the siRNA agent is conjugated with the ligand, as shown in the following figure:
Figure 113123276-A0202-13-0021-332
Figure 113123276-A0202-13-0021-332
Figure 113123276-A0202-13-0022-333
Figure 113123276-A0202-13-0022-333
Figure 113123276-A0202-13-0023-334
Figure 113123276-A0202-13-0023-334
Figure 113123276-A0202-13-0024-335
Figure 113123276-A0202-13-0024-335
Figure 113123276-A0202-13-0025-336
Figure 113123276-A0202-13-0025-336
Figure 113123276-A0202-13-0026-337
Figure 113123276-A0202-13-0026-337
Figure 113123276-A0202-13-0027-338
Figure 113123276-A0202-13-0027-338
Figure 113123276-A0202-13-0028-339
Figure 113123276-A0202-13-0028-339
Figure 113123276-A0202-13-0029-340
Figure 113123276-A0202-13-0029-340
Figure 113123276-A0202-13-0030-341
Figure 113123276-A0202-13-0030-341
Figure 113123276-A0202-13-0031-342
Figure 113123276-A0202-13-0031-342
Figure 113123276-A0202-13-0032-343
Figure 113123276-A0202-13-0032-343
Figure 113123276-A0202-13-0033-344
Figure 113123276-A0202-13-0033-344
 該X是O或S。 The X is O or S. 如請求項12至19中任一項所述的siRNA劑，其中， An siRNA agent as described in any one of claims 12 to 19, wherein, 該第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO：230)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or 該第一鏈具有由mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：224)表示的核苷酸序列以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：226)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID N：228)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID N: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO: 227); or 該第一鏈具有由mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7)(SEQ ID NO：228)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由(GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA(SEQ ID NO：229)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA(SEQ ID NO： 230)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：227)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or 該第一鏈具有由(GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA(SEQ ID NO：231)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG(SEQ ID NO：225)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or 該第一鏈具有由mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：232)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 232), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：234)表示的核苷酸序列，以及該第二鏈具有由(E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP- mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：234)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 235); or 該第一鏈具有由mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO：236)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7)(SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO: 233); or 該第一鏈具有由(GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU(SEQ ID NO：237)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or 該第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or 該第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU(SEQ ID NO：238)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or 該第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：235)表示的核苷酸序列；或者 The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or 該第一鏈具有由(GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU(SEQ ID NO：239)表示的核苷酸序列，以及該第二鏈具有由(E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG(SEQ ID NO：233)表示的核苷酸序列； The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233); 其中mA、mC、mG和mU分別代表2'-O-甲基腺苷、胞苷、鳥苷和尿苷；fA、fC、gG和fU分別代表2'-氟腺苷、胞苷、鳥苷或尿苷；“*”是硫代磷酸酯核苷酸間鍵；“(E)-VP”是5'端的(E)-乙烯基膦酸酯部分。 Wherein mA, mC, mG and mU represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; fA, fC, gG and fU represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; "*" is a phosphorothioate intermolecular bond; "(E)-VP" is the (E)-vinylphosphonate moiety at the 5 ' end. 一種醫藥組成物，其包含如請求項1至20中任一項所述的siRNA劑和藥學上可接受的載體。 A pharmaceutical composition comprising an siRNA agent as described in any one of claims 1 to 20 and a pharmaceutically acceptable carrier. 一種抑制細胞中補體成分C3基因表達的方法，該方法包括： A method for inhibiting the expression of complement component C3 gene in cells, the method comprising: (a)將該細胞與如請求項1至20中任一項所述的siRNA試劑或如請求項21所述的醫藥組成物接觸；以及 (a) contacting the cell with an siRNA reagent as described in any one of claims 1 to 20 or a pharmaceutical composition as described in claim 21; and (b)將步驟(a)中產生的該細胞維持一段足以獲得補體成分C3基因的mRNA轉錄物降解的時間，從而抑制該補體成分C3基因在該細胞中的表達。 (b) maintaining the cell produced in step (a) for a period of time sufficient to cause the mRNA transcript of the complement component C3 gene to degrade, thereby inhibiting the expression of the complement component C3 gene in the cell. 一種治療患有將受益於補體成分C3表達降低的疾病或病症的受試者的方法，該方法包括向該受試者施用治療有效量的如請求項1至20中任一項所述的siRNA劑或如請求項21所述的醫藥組成物，從而治療該受試者。 A method for treating a subject suffering from a disease or condition that would benefit from reduced expression of complement component C3, the method comprising administering to the subject a therapeutically effective amount of an siRNA agent as described in any one of claims 1 to 20 or a pharmaceutical composition as described in claim 21, thereby treating the subject. 如請求項23所述的方法，其中該疾病或病症是補體介導的疾病、病症或綜合症，較佳該疾病或病症是C3相關疾病或病症，更佳地，該疾病或病症選自IgA腎病(IgAN)、C3腎小球病(C3G)、陣發性睡眠性血紅蛋白尿(PNH)、非典型溶血性尿毒癥綜合症(aHUS)、視神經脊髓炎(NMO)、多灶性運動神經病(MMN)和重症肌無力(MG)。 The method of claim 23, wherein the disease or condition is a complement-mediated disease, condition or syndrome, preferably the disease or condition is a C3-related disease or condition, and more preferably, the disease or condition is selected from IgA nephropathy (IgAN), C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN) and myasthenia gravis (MG).
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