Novel therapeutic delivery moieties and uses thereof

Abstract

To provide novel compounds comprising novel delivery moieties for delivery of oligonucleotides, which are useful in the treatment of disease, suitably diseases of the liver.SOLUTION: A compound including Formula I and R is provided, wherein R includes an oligonucleotide, and R may be conjugated to Formula I via a linker.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a method for the delivery of oligonucleotides useful in the treatment of diseases, preferably diseases of the liver. The present invention relates to novel compounds comprising novel delivery moieties for [Background technology]

[0002] Compounds, including oligonucleotides, have been shown to be effective against many different types of genes involved in gene dysregulation. It allows targeting genes in a sequence-specific manner for personalized treatment of disease. Compounds containing oligonucleotides may vary depending on the specific type of oligonucleotide used. These have different mechanisms of action depending on the type of drug. For example, antisense oligonucleotides igonucleotide (ASO) and small interfering RNA (siRNA) RNA interference (RNAi) compounds, including In contrast, other oligonucleotide-containing compounds can induce short chain activity. Using oligonucleotides such as short activating RNA (saRNA), By delivering the oligonucleotide to the desired tissue in the patient, Gene expression can be downregulated, upregulated, or corrected.

[0003] N-acetylgalactosamine is used to target the asialoglycoprotein receptor on hepatocytes. oligonucleotides using delivery moieties containing N-acetylgalactosamine (GalNAc) Delivery of oxidases is one mode for delivery to the desired tissue. Examples containing GalNAc A potential compound targets the ALAS1 gene to treat acute hepatic porphyria. The drug is givosiran, an FDA-approved siRNA. Summary of the Invention [Problem to be solved by the invention]

[0004] Despite the existence of such commercially available compounds, the delivery of oligonucleotides to the liver or other tissues has not been reported. Alternative or improved moieties for delivery of peptides, and delivery moieties and therapeutic siRNAs There remains a need to provide improved therapeutic compounds comprising one or more oligonucleotides. It exists as such. [Means for solving the problem]

[0005] More specifically, compounds are provided that include a delivery moiety and one or more oligonucleotides. A need exists for such compounds to have improved tissue exposure, preferably improved liver exposure. improved liver-to-kidney exposure ratio; improved knockdown; improved durability response; improved pharmacokinetic profile, fewer off-target effects, improved toxicity improved activity profile, improved safety profile, and / or improved synthetic process; For example, but not limited to, synthetic processes with fewer steps, fewer decomposition products and processes for producing compounds with improved safety or efficacy profiles. a synthetic process for producing a compound having improved yields, or a process for producing an improved yield, or any combination thereof. This indicates one or more of the combinations. DETAILED DESCRIPTION OF THE INVENTION

[0006] In one aspect, the present invention provides a delivery moiety of formula I:

[0007] [ka]

[0008] The compounds herein containing Formula I may be modified or added to one or more atoms within Formula I, or The compound may have deletions or may contain additional moieties, for example, one or more of the moieties in Formula I The alkyl chain may be lengthened or shortened, or the compound comprising Formula I may contain one or more oligomers. The compounds of the present invention, including Formula I, typically comprise, for example, 3 The asialoglycoprotein receptor (Asialoglycoprotein rec) binds to two GalNAc moieties. one or more N-acetylgalactosamines (GalNAc or ASPGR), One or more agonists are introduced into cells that have receptors for the N-GalNAc or galnac moiety. Thus, compounds comprising Formula I herein are useful for delivering oligonucleotides. preferentially binds to hepatocytes expressing ASPGR, thereby inhibiting compound penetration into hepatocytes. ASPGR is also present on adipose tissue, so it can be used to promote To achieve this, compounds of Formula I can be used to deliver oligonucleotides to adipocytes expressing ASPGR. can be achieved.

[0009] In one embodiment, a compound comprising Formula I, wherein R comprises two bonded hydrogen molecules. In another embodiment, the compound of Formula I, wherein R comprises a methyl group. In one embodiment, the compound of formula I is a compound of formula I, wherein R comprises a protecting group. , R is a compound comprising one or more oligonucleotides. A delivery moiety comprising Formula I binds to the extracellular receptor ASPGR and delivers the compound to cells, including liver tissue. By allowing the entry of one or more oligonucleotides into the liver, delivered to liver tissue.

[0010] Delivery moieties comprising Formula I can be used to deliver oligonucleotides for diagnostic or therapeutic purposes. The one or more oligonucleotides may be DNA or RNA nucleotides, or may comprise DNA or RNA nucleosides, or any combination thereof; It may contain one or more, or all, modified nucleotides or modified linkages.

[0011] The oligonucleotides herein target specific DNA fragments in cells to regulate gene expression. A or RNA sequence that is designed to target, i.e., bind or anneal to, In one embodiment, the compound comprises Formula I, wherein R is a compound that decreases expression of a target transcript. In a further embodiment, the compound comprising Formula I, R is: The oligonucleotides used to reduce expression of target transcripts further reduce protein expression. In another embodiment, the decrease in expression of the target transcript or target protein is about 99% or less. 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 percent. In further embodiments, the decrease in expression is about 3 weeks, about 1 month, about 1.5 months, about 2 months, about 3 months lasts for about 1 month, about 4 months, about 5 months, or about 6 months.

[0012] Those skilled in the art will appreciate that one or more mismatches may occur between the oligonucleotide and the target nucleotide sequence. It is recognized that a protein can exist in a nucleotide sequence and still function to regulate gene expression. In one embodiment, the oligonucleotide has a target sequence and a nucleotide sequence similar to the target sequence. 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 8 1, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, or 70 percent The oligonucleotide may also have 1 to 1 nucleotides at either the 5' or 3' end. may have overhangs of 0, 1-5, or 1-3, or 3, 2, or 1 residue The 5' or 3' terminus may be further modified, for example, but not limited to, with an abasic residue or a phosphate group. Suitable modifications are known in the art.

[0013] Those skilled in the art will appreciate that 2'-modifications on the sugar residues, preferably the ribose, increase their stability and half-life. These modifications include a 2' OH group in place of the 2' OH group of the unmodified sugar. These modifications include, but are not limited to, fluoro or 2' methoxy modifications. It has been shown that changes in the backbone of oligonucleotides can also increase their stability and half-life. These backbone modifications involve the conversion of phosphodiester bonds to phosphorothioates. orothioate, PS) linkages.

[0014] Thus, as used herein, an oligonucleotide (or interchangeably referred to herein) As used herein, a polymer or oligomer (as used in means a chain of two nucleotide or nucleoside residues, modified or unmodified bases and / or may contain modified or unmodified bonds. Nucleotide residues may be linked by phosphodiester bonds or The linkage may be by a modified bond (in the absence of a phosphate, the residue is typically (As is known, they are called nucleosides). The nucleotide or nucleoside residue is a pyrimidine. One or more atoms in a diamine or purine ring, or one or more atoms in a sugar residue, or a ring-sugar The modification may be at one or more atoms of the bond between the bases. The modification may also be at the 5' end of the oligonucleotide chain. It may be at the 1' or 3' end, and unless otherwise clear from the context, it is referred to herein as an oligonucleotide. It is called leotide.

[0015] In certain embodiments, the one or more oligonucleotides are small interfering RNA (siRNA). A) Small activating RNA (saRNA) , microRNA (miRNA), short hairpin RNA (short hairpin RNA) shRNA, single guide RNA (sgRNA), or antisense In a preferred embodiment, one or more oligonucleotides In another preferred embodiment, the oligonucleotide comprises one or more siRNA. is an siRNA comprising a sense strand and an antisense strand.

[0016] In some embodiments, R is conjugated to Formula I via a linker. Suitable linkers are known in the art. In one embodiment, the linker is an alkyl chain. , preferably C 1~10 In a further embodiment, the linker comprises Linker 1, Formula II: In another embodiment, the linker comprises piperidine. In an embodiment, the linker is shown below as Linker 2, Formula III.

[0017] [ka]

[0018] In one embodiment, linker 1 (Formula II), connection point A, or linker 2 (Formula III), connection point B, Attachment point C is conjugated to Formula I. In one embodiment, Linker 1, attachment point A is , is conjugated to Formula I, and the connection point B is conjugated to R. In embodiments, Linker 2, attachment point C, is conjugated to Formula I, and attachment point D is In one embodiment, linker 1, connection point A is conjugated to formula I. The attachment point B is conjugated to a phosphate group that is conjugated to R. In one embodiment, linker 2, connection point C, is conjugated to formula I The attachment point D is conjugated to a phosphate group that is conjugated to R. There are.

[0019] Those skilled in the art will appreciate that the linker may be on the 5' or 3' end of the oligonucleotide. or to one of the internal nucleotide or nucleoside bases. Those skilled in the art will also appreciate that the linker may be attached to the 5' or 3' end of the oligonucleotide. Those skilled in the art will also recognize that the hydroxyl group may be conjugated via a linker. a delivery moiety to the 5' end of the oligonucleotide, e.g., comprising Formula I, The placement of the delivery moiety may result in potential inefficient loading, or RI, of Ago2 loading. It will be appreciated that other obstacles to SC complex activity may need to be overcome, for example: Formula I linked or directly conjugated to siRNA comprising a sense strand and an antisense strand For a delivery moiety comprising Ago, the placement of the delivery moiety at the 5' end of the antisense strand is 2 This can cause difficulties with loading and may prevent efficient knockdown. In a preferred embodiment, the one or more oligonucleotides comprise a sense strand and an antisense strand. The delivery moiety comprising Formula I is present at the 3' end of the sense strand. In embodiments, a delivery moiety comprising Formula I is conjugated to the 3' end of the sense strand via a linker. In still further embodiments, the linker is a ring structure, preferably a piperidine ring. In still further embodiments, the linker comprises Linker 2.

[0020] Thus, in one embodiment, the ribose of at least one nucleotide is 2' fluoro. The present invention includes one or more oligonucleotides modified with a 2' or 2' methoxy group. In another embodiment, one or more oligonucleotides have one or more modifications or substitutions. In a further embodiment, the amino acid has one or more substituted phosphodiester linkages. In yet a further embodiment, the one or more oligonucleotides contains at least one nucleotide modified with a 2' fluoro or 2' methoxy group , the backbone has one or more modified or substituted phosphodiester linkages, preferably PS linkages.

[0021] In other embodiments of the compounds disclosed herein, the one or more oligonucleotides are In a further embodiment, the siRNA comprises a sense strand and an antisense strand. In another embodiment, the sense and antisense strands are each 15 to 40 nucleotides in length. The sense and antisense strands are annealed and, optionally, one or more 5' or 3' nucleotides are inserted. overhangs, one or more 5' or 3' blunt ends, or a combination of both. nothing.

[0022] In another embodiment of the compounds disclosed herein, the 5' or 3' terminus is further modified. In a further embodiment, the 5' end of the antisense strand is optionally phosphorylated. In one embodiment, the 5' terminal nucleotide or nucleoside of the antisense strand is 5' Contains vinyl phosphonate modifications.

[0023] The compounds herein, comprising Formula I and one or more oligonucleotides, are administered to the liver or adipose tissue. In one embodiment, the compounds are useful in the therapy or treatment of diseases involving and a pharmaceutical composition for administering a compound comprising Formula I and one or more nucleotides for the treatment of cancer. One embodiment is a compound comprising Formula I and one or more oligonucleotides for use in therapy. A further embodiment is a compound comprising a compound or pharmaceutical composition thereof, wherein the therapy is for a disease of the liver. An alternative embodiment is directed to the detection of genes involved in adipose tissue, e.g., in adipocytes. Another embodiment is for diseases involving gene dysregulation. A compound of formula I and one or more oligonucleotides, preferably administered in an effective amount, a compound containing benzodiazepine or any of the aforementioned pharmaceutical compositions for the treatment of liver disease. Another embodiment is a method of treating liver disease using the compounds disclosed herein, preferably Preferably, the compound of formula I and one or more oligonucleotides for use in the manufacture of a medicament for the treatment of A compound containing a nucleotide or a pharmaceutical composition thereof.

[0024] In another embodiment, the compound comprises Formula IV:

[0025] [ka] where Z is a solid support, resin, or bead.

[0026] The pharmaceutical compositions disclosed herein may be administered in the presence of a compound or other component of a composition or formulation. It contains one or more carriers, diluents, and excipients that are compatible and not harmful to the patient. Examples of compositions and processes for their preparation are given in "Remington: The Science nce and Practice of Pharmacy”, Loyd, V., et al. al. Eds., 22 nd Ed.,Mack Publishing Co.,201 It can be found in 2.

[0027] As used herein, the term "effective amount" refers to an amount that is effective to treat a disorder or disease. Refers to the effective amount.

[0028] As used herein, a "region of complementarity" refers to a region of complementarity that, under appropriate hybridization conditions, Hybridization between two sequences of nucleotides (e.g., in phosphate buffer, in cells, etc.) A nucleic acid (e.g., d) that is sufficiently complementary to the antiparallel nucleotide sequence to allow for synthesis. In some embodiments, the present invention provides a method for the preparation of a nucleotide sequence of an oligonucleotide. The oligonucleotides in this specification are targeting sequences that have a region complementary to an mRNA target sequence. Contains columns.

[0029] Delivery moieties comprising Formula I can be made by the following non-limiting synthetic steps and schemes: can be done.

[0030] Certain abbreviations are defined as follows: "1,2-DCE" is 1,2-dichloroethene; "DCM" refers to dichloromethane, and "DIEA" refers to N,N-diisopropyl "DMF" refers to N,N-dimethylformamide, and "DM "AP" refers to 4-dimethylaminopyridine, and "DMTCl" refers to 4,4'-dimethoxamine. "DPP4" refers to dipeptidyl peptidase 4 (EDP4). "C" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "E "tOAc" refers to ethyl acetate, and "GalNAc" refers to N-acetylgalactosamine. "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3- Triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate "HBTU" is O-(benzotriazol-1-yl)-N,N,N',N'-tetrazole "HOBt" refers to tetramethyluronium hexafluorophosphate, and "HOBt" refers to 1-hydroxybenzoate. "HPRT" refers to hypoxanthine-guanine phosphodiesterase (HMPS) "IPA" refers to isopropanol and isopropyl "LDHA" refers to lactate dehydrogenase-A, and "MeCN" refers to "MeOH" refers to methanol and methyl alcohol, and " "MWCO" refers to molecular weight cut-off and "NHS" refers to N-hydroxysuccinimide "OD" refers to optical density, "PBS" refers to phosphate buffered saline, "PhSiH3" refers to phenylsilane, and "PTS" refers to the portable endotoxin test system. "siRNA" refers to small interfering ribonucleic acid, and "TEA" refers to triethyl ester. "TFA" refers to trifluoroacetic acid, and "THF" refers to tetrahydrofuran. "TLC" refers to thin line chromatography, and "TMP" refers to 2,2,6 ,6-tetramethylpiperidine.

[0031] [ka]

[0032] Step A of Scheme 1 involves the reaction of trifluoromethanesulfonic acid in a solvent such as 1,2-DCE. Step B shows the cyclization of compound (1) using trimethylsilyl to obtain compound (2). The reaction was carried out using trimethylsilyl trifluoromethanesulfonate in a solvent such as 1,2-DCE. Addition of hex-5-en-1-ol to compound (2) using the method of In step C, a catalyst such as ruthenium(III) chloride is used together with sodium periodate. The method for oxidizing compound (3) to give compound (4) using a suitable oxidizing agent is shown.

[0033] [ka]

[0034] Step A of Scheme 2 involves the reaction of HBT with a suitable base such as DIEA in a solvent such as DMF. Compound (5) and tert-butyl N-[2-[2-(tert -butoxycarbonylamino)ethylamino]ethyl]carbamate and amide coupling Step B shows that N is reacted with HCl in a THF and MeOH solvent system to give compound (6). Compound (6) is hydrolyzed using a base such as aqueous OH to give compound (7). Step C shows that HAT is reacted with a suitable base such as DIEA in a solvent such as DMF. Compound (7) was amide-coupled with allyl 11-aminoundecanoate hydrochloride using U. Step D shows that the compound (8) is obtained by cleaving the hydroxyl group with TFA in a solvent such as DCM. This shows that compound (8) is deprotected with an acid to obtain compound (9). Compound (9) and compound (4) were amide-coupled using EDC and HOBt in a solvent of Step F shows the formation of compound (10) by tetracyclization in a solvent such as DCM. Compound (10) was deprotected by bis(triphenylphosphine)palladium and PhSiH3. Step F shows the use of EDC in a solvent such as DCM to obtain compound (11). The compound (11) is coupled with NHS to give the compound (12). .

[0035] [ka]

[0036] Steps A to C of Scheme 3 are essentially similar to Steps C to E of Scheme 2, Starting from compound (7), compounds (13), (14) and (15) are obtained. Compound (15) is hydrogenated using palladium on carbon in a solvent such as eOH to give compound (1 6). Step E is essentially similar to the preparation of Step G in Scheme 2, Compound (17) is obtained.

[0037] [ka]

[0038] Steps A through I of Scheme 4 are essentially similar processes to those shown in Schemes 2 and 3. This consists of a series of amide couplings and deprotections using to obtain compound (27).

[0039] [ka]

[0040] Steps A-C of Scheme 5 are essentially similar to steps G-I of Scheme 4. Starting from compound (24), compound (30) is obtained.

[0041] [ka]

[0042] Step A of Scheme 6 involves the reaction of DMT with a suitable base such as DIEA in a solvent such as DCM. Step B shows the protection of compound (31) using Cl to obtain compound (32). Compound (32) was synthesized using HBTU and HOBt with TMP in a solvent such as DCM. Amide coupling with peridin-4-ylmethanol to give compound (33) In step C, compound (33) was deprotected with 20% piperidine in DMF to give compound ( 34) is obtained.

[0043] [ka]

[0044] Step A of Scheme 7 is essentially similar to Step A of Scheme 2, and involves the preparation of compound (16). The compound (35) is obtained by coupling the compound (34) with the compound (35). Add succinic anhydride to compound (35) along with a basic system of TEA and DMAP in a suitable solvent. Step C shows the formation of compound (36) by reacting MeCN and DCM. In a solvent system such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylbenzoate Resin using a base such as methyluronium hexafluorophosphate and DIEA Compound (36) is loaded onto the column to give compound (37).

[0045] Preparation 1 (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pi Methyl lano[3,2-d]oxazol-5-ylacetate

[0046] [ka]

[0047] (5-acetamido-3,4,6-triacetoxy- in 1,2-DCE (46 mL) To a solution of methyl tetrahydropyran-2-ylacetate (9.00 g, 23.1 mmol) Trimethylsilyl trifluoromethanesulfonate (6.5 mL, 35 mmol) was added. The mixture is heated to 50°C and stirred for 18 hours. After this time, the mixture is diluted with DCM (200 mL), and dilute with 200 mL of saturated aqueous NaHCO3 and 20 mL of saturated aqueous sodium chloride. 0 mL, dried over sodium sulfate, filtered and concentrated in vacuo. The product was purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM. Purify by HPLC to give the title compound (6.434 g, 84%). ES / MS m / z 330(M+H).

[0048] Preparation 2 (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydro (2-(2-pyran-2-yl))acetic acid methyl ester

[0049] [ka]

[0050] (6,7-diacetoxy-2-methyl-5, ... ,7a-Tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetic acid methyl To a solution of hexa-5-en-1-ol (30.43 g, 92.42 mmol), 22.2 mL, 185 mmol), followed by activated powdered 4 Å molecular sieves (15.6 g) is added. The suspension is stirred at ambient temperature for 30 minutes, then trimethylsilyl trifluoride is added. Add 19 mL (101.9 mmol) of methylparaben and allow the mixture to stand at ambient temperature. The mixture is stirred at room temperature for 18 hours. After this time, the solution is filtered through diatomaceous earth and concentrated in vacuo. The fraction was purified by silica gel flash chromatography eluting with 30-100% EtOAc / hexane. Purify by chromatography to give the title compound (34.76 g, 86%). m / z 430.4 (M+H).

[0051] Preparation 3 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrofuran] Dropyran-2-yl]oxypentanoic acid

[0052] [ka]

[0053] (5-acetamido-3,4- Diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate methyl (34.76 g, 80.93 mmol) is cooled to 0°C. (22.4 g, 104.7 mmol) is added and stirring is continued at 0° C. for 10 min. After this time, ruthenium(III) chloride (270 mg, 1.3 mmol) was added and the mixture Stir while warming to ambient temperature. After stirring for 2 hours, add additional sodium periodate (6 6 g, 308.4 mmol) is added and stirring is continued for 18 hours. After this time, the mixture is mixed with 3: Extract with 1 part of CHCl:IPA (2 × 500 mL) and add saturated aqueous sodium chloride (1 L ), dried over sodium sulfate, filtered and concentrated in vacuo. Silica gel flash chromatography eluting with 0-40% MeOH / DCM Purify to give the title compound (29.75 g, 82%). ES / MS m / z 4 48.4(M+H).

[0054] Preparation 4 Benzyl 6-aminohexanoate hydrochloride

[0055] [ka]

[0056] 6-Aminohexanoic acid (5.00 g, 38.1 mmol) suspended in THF (38 mL) To this was added benzyl alcohol (47 mL, 453.7 mmol) and the mixture was cooled to 0°C. Thionyl chloride (8.6 mL, 120 mmol) is added dropwise and the mixture is allowed to warm to ambient temperature. The mixture was stirred for 18 hours while heating. After this time, ether (166 mL) was added and the reaction vessel was heated to - Transfer to a 20°C freezer for 1 hour. After this time, the solid precipitate was collected by filtration to give the title compound ( 8.57 g, 81%) was obtained. ES / MS m / z 222 (M+H).

[0057] Preparation 5 Benzyl 11-aminoundecanoate hydrochloride

[0058] [ka]

[0059] The title compound was prepared from 11-aminoundecanoic acid in a manner essentially similar to that of Preparation 4. ES / MS m / z 292.2 (M+H).

[0060] Preparation 6 Allyl 11-aminoundecanoate hydrochloride

[0061] [ka]

[0062] 11-aminoundecanoic acid (9.00 g, 44.7 mL) in allyl alcohol (42 mL) Add thionyl chloride (6.5 mL, 89. 4 mmol) is added and the mixture is stirred for 18 hours while warming to ambient temperature. The mixture is concentrated in vacuo and ether (200 mL) is added to the residue to give a white suspension. The mixture was stirred at ambient temperature for 10 minutes and the solid precipitate was collected by filtration to give the product (12. 0g, 97%). ES / MS m / z 242.2 (M+H).

[0063] Preparation 7 (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H -Fluoren-9-ylmethoxycarbonylamino)propanoic acid

[0064] [ka]

[0065] (2S)-2-(9H-fluoren-9-ylmethoxy) Stirring of (carbonylamino)-3-hydroxy-propanoic acid (40 g, 0.122 mol) To the solution is added DIEA (64 mL, 0.366 mol) at 0° C. under an inert atmosphere. To this, a solution of DMTCl (49.6 g, 0.146 mol) in DCM (200 mL) The mixture is allowed to warm to ambient temperature and stirred for 16 hours. After this time, the reaction mixture The mixture is diluted with water (12.5 vol) and extracted with DCM (25 vol). The organic layer is washed with anhydrous sulfuric acid. The crude product is then diluted with 10% EtO, dried over sodium hydroxide, filtered and concentrated in vacuo. Washing with Ac / hexane (12.5 vol) and drying under vacuum gave the title compound in a light brown Obtained as a solid (62 g, crude). This material was used in the next step without further purification. TLC: 5% MeOH / CH2Cl2 (R f :0.5)UV, 254nM.

[0066] Preparation 8 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl) 2-[4-(hydroxymethyl)-1-piperidinyl]-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl Diethyl]-2-oxo-ethyl]carbamate

[0067] [ka]

[0068] (2S)-3-[bis(4-methoxyphenyl)-phenyl] in DCM (750 mL) -Methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid To a stirred solution of HBTU (78.3 g, 0.206 mol ), HOBt (27.9 g, 0.206 mol), and piperidin-4-ylmethanol (15.4 g, 0.134 mol), followed by TMP (15 mL, 0.113 mol). The resulting reaction mixture was allowed to warm to ambient temperature and was stirred for 4 hours. After this time, the reaction mixture was diluted with water (8 vol) and extracted with DCM (15 vol). The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue is eluted with 20-40% EtOAc / hexane and 1% MeOH / DCM. Purification by silica gel flash chromatography gave the title compound (40 g, 2 steps) and get 52%). 1 H NMR(DMSO-d6)δ7.88(br d, J=7.5 Hz, 2H), 7.79-7.59(m, 3H), 7.45-7.12(m, 13H), 6.92-6.76(m, 4H), 4.79-4.44(m, 2H), 4.32(br d, J=11.4Hz, 2H), 4.20(br s, 2H), 3.71(s, 6H), 3.21(br s, 4H), 2.99-2.79(m, 1H), 2.69(br s, 2H), 1.81-1.43(m, 3H), 1.08-0.73(m, 2H).

[0069] Preparation 9 (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy] -1-[4-(hydroxymethyl)-1-piperidyl]propan-1-one

[0070] [ka]

[0071] A solution of 20% piperidine in DMF (400 mL) was heated at 0°C under an inert atmosphere for 9H- Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)- Phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]- 2-oxo-ethyl]carbamate (40 g, 0.055 mol) was added slowly. The resulting reaction mixture is stirred at ambient temperature for 1 hour, after which the mixture is diluted with water (15 vol). Dilute and extract with EtOAc (30 vol). Dry the organic layer with anhydrous sodium sulfate. The resulting residue was purified by elution with 1-8% MeOH / DCM. The title compound was purified by silica gel flash chromatography using (13 g, 47%). ES / MS m / z 1009.5 (2M+H).

[0072] Preparation 10 Methyl(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl] Amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate

[0073] [ka]

[0074] S-4-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentyl benzoic acid (7.00 g, 26.8 mmol) and HOBt (4.16 g, 30.8 mmol) In a flask containing (179 mL) DMF and (2-(1H-benzotriazole) -1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( Add DIEA (14 mL, 80.3 mmol) to the reaction mixture. is added and the mixture is stirred at ambient temperature for 5 minutes. 2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate (8 Add 0.94 g (29.5 mmol) in one portion and continue stirring at ambient temperature. Stir for 18 hours. After stirring, the mixture was diluted with EtOAc (400 mL) and added water (2×400 mL) and saturated Wash with aqueous sodium chloride (400 mL), dry over sodium sulfate, filter, and Concentrate in air. Elute the resulting residue with 40-100% EtOAc / hexanes. Purification by silica gel flash chromatography gave the title compound (13.01 g , 89%). ES / MS m / z 547.40 (M+H).

[0075] Preparation 11 (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino ]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid

[0076] [ka]

[0077] In a flask, add methyl(2S)-5-[bis[2-(tert-butoxycarbonylamine] 2-(tert-butoxycarbonylamino)-5-oxopentylamino)-2-(tert-butoxycarbonylamino)-5-oxopentylamino thanoate (13.01 g, 23.8 mmol), THF (120 mL), and MeO Add 120 mL of 1N NaOH (71 mL, 71 mmol) and mix. The mixture was stirred at ambient temperature. After 1 h, the mixture was concentrated in vacuo and redissolved in water (300 mL). Add 5N HCl (12 mL) to bring the pH to 4. The mixture is diluted with DCM (3 × 3 The combined organic layer was washed with saturated aqueous sodium chloride (1 L) and Dry over sodium hydroxide, filter, and concentrate to give the title compound (12.41 g, 98%). ES / MS m / z 531.60(MH).

[0078] Preparation 12 Allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino )Ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentane [Tanoyl]amino]undecanoate

[0079] [ka]

[0080] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino ]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (500m g, 0.94 mmol) and allyl 11-aminoundecanoate hydrochloride (31 3 mg, 1.13 mmol) in a flask, DMF (6.25 mL) and (1-[ Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine Dinium 3-oxide hexafluorophosphate (428 mg, 1.12 mmol) After adding DIEA (0.5 mL, 3 mmol), the mixture was stirred at ambient temperature for 18 h. After this time, the mixture is diluted with EtOAc (200 mL) and water (3 x 200 mL) Wash with saturated aqueous sodium chloride (200 mL) and dry with sodium sulfate. The resulting residue was diluted with 40-100% EtOAc / Hexane Purification by silica gel flash chromatography eluting with hexane gave the title compound ( Yield 687mg, 97%). 1 H NMR (DMSO-d6) δ 7.78-7.64 ( m, 1H), 6.98-6.7(m, 2H), 5.96-5.84(m, 1H), 5.3 1-5.25(m, 1H), 5.23-5.17(m, 1H), 4.56-4.50(m , 2H), 3.88-3.67(m, 1H), 3.30-3.19(m, 4H), 3.1 1-2.91(m, 6H), 2.35-2.12(m, 4H), 1.88-1.65(m , 2H), 1.58-1.47(m, 2H), 1.46-1.30(m, 30H), 1. 30-1.18(m, 12H).

[0081] Preparation 13 Allyl(S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5- Oxopentanamido)undecanoate

[0082] [ka]

[0083] Allyl 11-[[(2S)-5-[bis[2-(tert-butyl) [tert-butoxycarbonylamino]ethyl]amino]-2-(tert-butoxycarbonylamino)ethyl]amino 5-Oxo-pentanoylaminoundecanoate (687mg, 0.91m To a solution of 1.5 mol of TFA (15 mL) was added. The mixture was stirred at room temperature. After a period of time, the mixture is concentrated in vacuo. The residue is dissolved in MeOH and applied to an ion exchange cartridge. The cartridge was eluted with MeOH (150 mL), followed by 7N NH3 / M Elute with eOH (150 mL). Concentrate the basic fractions in vacuo to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0084] Preparation 14 Allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino] -5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxy) Dimethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino ]-5-oxo-pentanoyl]amino]undecanoate

[0085] [ka]

[0086] In a flask, add 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) tetrahydropyran-2-yl]oxypentanoic acid (489 mg, 1.09 mmol ) and allyl S-11-(2-amino-5-(bis(2-aminoethyl)amino)-5- Add 150 mg (0.33 mmol) of oxopentanamido) undecanoate. DCM (3.35 mL) was added, followed by 1-hydroxybenzotriazole monohydrate ( 164 mg, 1.07 mmol) and 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride (206 mg, 1.07 mmol) is added. The mixture is heated to ambient temperature. After this time, the solution was diluted with EtOAc (100 mL) and saturated NaHC1 O3 (2 x 100 mL), saturated aqueous NH4Cl (100 mL), and saturated sodium chloride The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel filtration eluting with 0-10% MeOH / DCM. Purify by rush chromatography to give the title compound (424 mg, 74%) . ES / MS m / z 872.80(M+2H) / 2.

[0087] Preparation 15 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-( Acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5- [bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) (I)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5 -Oxo-pentanoyl]amino]undecanoic acid

[0088] [ka]

[0089] Allyl 11-[[(2S)-2-[5-[3-acetamido-4-yl]amino]-4 in DCM (2 mL) ,5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy Pentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetamido] Oxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl a amino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate (35 4 mg, 0.20 mmol) was added to a solution of tetrakis(triphenylphosphine)palladium. (29 mg, 0.02 mmol), followed by PhSiH3 (51 μL, 0.41 mmol) The mixture is stirred at ambient temperature for 2 hours, after which saturated aqueous NaHCO3 (1 Dilute with 100 mL of water. Add 15 mL of 1N NaOH to bring the pH to approximately 10. The solution was washed with DCM (3 x 100 mL), then concentrated HCl (5 mL), then 5N H Acidify with aqueous Cl (15 mL). Extract the aqueous layer with DCM (100 mL) and the organic layer Dry over sodium sulfate, filter, and concentrate in vacuo. Purify by silica gel flash chromatography eluting with MeOH / DCM The title compound (151 mg, 44%) was obtained. ES / MS m / z 852.60 (M +2H) / 2.

[0090] Preparation 16 (2,5-dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[3-a Cetoamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2 -yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4 ,5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy Pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undeca Noate

[0091] [ka]

[0092] In a reaction vial, add 11-[[(2S)-2-[5-[3-acetamido-4,5-di Acetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentano ylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6 -(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]e ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid (50 mg, 0.0 3 mmol), N-hydroxysuccinimide (5 mg, 0.04 mmol), and 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8 mg, 0.04 DCM (0.3 mL) is added and the mixture is stirred at ambient temperature. After 18 h, the mixture was loaded directly onto a silica gel cartridge and the crude mixture was diluted with 0–10% Purification by silica gel flash chromatography eluting with MeOH / DCM gave The title compound (49 mg, 93%) was obtained. ES / MS m / z 901.40 (M+2H ) / 2.

[0093] Preparation 17 Benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino )Ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentane [Tanoyl]amino]hexanoate

[0094] [ka]

[0095] The title compound was synthesized by the reaction of (2S)-5-[bis[2-(tert-butoxycarbonylamino) Ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-penta Preparation 10 is essentially similar to the method of 10 from benzoic acid and benzyl 6-aminohexanoate hydrochloride. Prepared in the manner of: ES / MS m / z 736.40 (M+H).

[0096] Preparation 18 Benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]- 5-Oxo-pentanoyl]amino]hexanoate tris(trifluoroacetic acid)

[0097] [ka]

[0098] Benzyl 6-[[(2S)-5-[bis[2-(tert- butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)ethyl]amino [amino]-5-oxo-pentanoyl]amino]hexanoate (15.47g, 21.0 To a solution of 2 mmol of TFA (16 mL, 210.2 mmol) was added TFA (16 mL, 210.2 mmol). Stir at ambient temperature for 24 h. After this time, add additional TFA (16 mL, 210.2 mmol). is added and stirring is continued for a further 2 hours. After this time, the mixture is concentrated in vacuo. The distillate was azeotroped with toluene (2 x 30 mL). The resulting oil was heated in a vacuum oven at 40 °C for 4 hours. The title compound (28.08 g, 58% purity considering residual toluene, 9 9+%). ES / MS m / z 436.40 (M+H). The compound was dissolved in 70 mL of D Dissolve in MF to make a 0.3 M solution, which will be used in the next step.

[0099] Preparation 19 Benzyl 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino] -5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxy) Dimethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino ]-5-oxo-pentanoyl]amino]hexanoate

[0100] [ka]

[0101] The title compound was synthesized by the method of 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) 6-[[(2S) ethyl)tetrahydropyran-2-yl]oxypentanoic acid and benzyl 6-[[(2S) -2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl] [amino]hexanoate tristrifluoroacetic acid in a manner essentially similar to that of Preparation 10. Prepared in the same manner as above. ES / MS m / z 862(M+2H) / 2.

[0102] Preparation 20 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (ethoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[ Bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl )tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- Oxopentanoylaminohexanoic acid

[0103] [ka]

[0104] Palladium on carbon (1.90 g, 0.89 mmol, 5% by weight, 50% wet) was placed in a round-bottom flask. Place in a scope and evacuate and refill with nitrogen three times. 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (ethoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[ Bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl )tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- Oxo-pentanoyl]amino]hexanoate (15.41 g, 8.94 mmol) The solution is added via syringe. The flask is evacuated and refilled with 1 atm of hydrogen and mixed. The mixture is stirred at ambient temperature under 1 atm of hydrogen for 18 hours. After this time, the mixture is passed through diatomaceous earth. Filter and concentrate the filtrate in vacuo to give the title compound (13.85 g, 95%). MS m / z 817.2(M+2H) / 2.

[0105] Preparation 21 (2,5-dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[3-acetate Amido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4, 5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxype pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]hexanoe Route

[0106] [ka]

[0107] The title compound was synthesized by the reaction of 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetamido Oxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl a amino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (ethoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl] Methods and essence of preparation 16 from amino-5-oxo-pentanoyl aminohexanoic acid Prepared in a similar manner. ES / MS m / z 866.20(M+2H) / 2.

[0108] Preparation 22 Benzyl(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl ]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid to

[0109] [ka]

[0110] The title compound was synthesized by the reaction of tert-butyl N-[2-[2-(tert-butoxycarbonyl) (4S)-5-benzyloxy-4-( Method 12 of preparation from tert-butoxycarbonylamino)-5-oxo-pentanoic acid Prepared in essentially the same manner as in Example 1. ES / MS m / z 623.6 (M+H).

[0111] Preparation 23 Benzyl(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo So-pentanoate tris(trifluoroacetic acid) salt

[0112] [ka]

[0113] The title compound was synthesized by the reaction of benzyl(2S)-5-[bis[2-(tert-butoxycarbonyl Amino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo Prepared from the -pentanoate in a manner essentially similar to the method of Preparation 18. ES / MS m / z 323.2 (M+H).

[0114] Preparation 24 Benzyl(2S)-5-[bis[2-[5-(tert-butoxycarbonylamino) pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino] amino)pentanoylamino]-5-oxo-pentanoate

[0115] [ka]

[0116] The title compound was synthesized by the reaction of 5-(tert-butoxycarbonylamino)pentanoic acid and benzyl (2S)-2-Amino-5-[bis(2-aminoethyl)amino]-5-oxopenta Prepared from benzoate tris(trifluoroacetate) in a manner essentially similar to that of Preparation 10. ES / MS m / z 920.6 (M+H).

[0117] Preparation 25 Benzyl(2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5- Aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(tris(trimethylamino)ethyl)amino)-5-oxo-pentanoate tris(trimethylamino)ethyl Trifluoroacetic acid salt

[0118] [ka]

[0119] The title compound was synthesized by the reaction of benzyl(2S)-5-[bis[2-[5-(tert-butoxycarbonyl) 2-[5-(tert-butylamino)pentanoylamino]ethyl]amino]- ... Preparation 1 from [(dicarbonylamino)pentanoylamino]-5-oxo-pentanoate Prepared in a manner essentially similar to that of 8. ES / MS m / z 620.4 (M+H) .

[0120] Preparation 26 Benzyl(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6 -(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pe 2-[5-[3-acetamido-4,5-diazomethane]-5-[bis[ ... acetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl 5-oxo-pentanoylamino-ethylamino-5-oxopentanoate

[0121] [ka]

[0122] The title compound was synthesized by the method of 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) Benzyl (2S)-2-(tetrahydropyran-2-yl)oxypentanoic acid and benzyl (2S)-2-( 5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino) Prepared from ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetate) salt Prepared in a manner essentially similar to that of Preparation 10. ES / MS m / z 954.80 (M +2H) / 2.

[0123] Preparation 27 (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (Tetrahydropyran-2-yl)oxypentanoylamino]pentanoyl 3-acetamido-4,5-diacetoxy]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy]-5-[ ... -6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino ]Pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid

[0124] [ka]

[0125] A round-bottom flask was charged with palladium on carbon (467 mg, 0.22 mmol, 5% by weight, 50% wet). The flask was evacuated and refilled with nitrogen three times. Di(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (ethoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentano ylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 5-oxo-pentanoate (4.19g) A solution of 2.20 mmol) is added via syringe, followed by 3 drops of acetic acid. The flask was evacuated and backfilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 2 hours, the mixture is filtered through diatomaceous earth and the filtrate is concentrated in vacuo to give the title compound. The compound (3.99 g, 99+%) was obtained. ES / MS m / z 909.6 (M+2H) / 2.

[0126] Preparation 28 Benzyl 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetamido Oxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl a amino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4, 5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxype pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl ]amino]hexanoate

[0127] [ka]

[0128] The title compound was synthesized by the reaction of (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and From benzyl 6-aminohexanoate hydrochloride in a manner essentially similar to that of Preparation 10 Prepare. ES / MS m / z 1011.6(M+2H) / 2.

[0129] Preparation 29 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6 -(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pe 2-[5-[3-acetamido-4,5-diazomethane]-5-[bis[ ... acetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl 5-oxopentanoylamino]ethylamino]-5-oxopentanoylamino]ethylamino ]hexanoic acid

[0130] [ka]

[0131] Add palladium on carbon (24 mg, 0.01 mmol, 5% by weight, 50% wet) to a round-bottom flask. ), evacuate the flask and backfill with nitrogen. 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (Acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentane 5-[bis[2-[5-[3-acetamido-4,5-diacetamido]- ...]-2-[5-[3-acetamido]-2-[5-[3 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl Amino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino] A solution of hexanoate (222 mg, 0.11 mmol) was added via syringe, followed by Add 3 drops of acetic acid. The flask is evacuated and refilled with 1 atm of hydrogen. The mixture was stirred at ambient temperature under 1 atm of hydrogen. After 5 h, the flask was purged with nitrogen and the mixture was Filter through diatomaceous earth. Concentrate the filtrate in vacuo to give the title compound (180 mg, 85%). . ES / MS m / z 966.2(M+2H) / 2.

[0132] Preparation 30 (2,5-dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[5-[3 -Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran -2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5 -[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrofuran] Hydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]a 5-oxo-pentanoylaminohexanoate

[0133] [ka]

[0134] The title compound was synthesized by the reaction of 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-di Acetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentano 2-[5-[(2-ylamino)pentanoylamino]-5-[bis[2-[5-[(3-acetamide -4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]o 5-Oxopentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoylamino Prepared from [amino]hexanoic acid in a manner essentially similar to that of Preparation 16. S / MS m / z 1014.6(M+2H) / 2.

[0135] Preparation 31 Benzyl 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetamido 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl Amino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4 ,5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy Pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl 1,2-diamino]undecanoate

[0136] [ka]

[0137] The title compound was synthesized by the reaction of (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and From benzyl 11-aminodecanoate hydrochloride in a manner essentially similar to that of Preparation 10 Prepare. ES / MS m / z 1046.6(M+2H) / 2.

[0138] Preparation 32 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino] pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-di Acetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentano ylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino Undecanoic acid

[0139] [ka]

[0140] Add palladium on carbon (35 mg, 0.02 mmol, 5% by weight, 50% wet) to a round-bottom flask. ) is added and the flask is evacuated and refilled with nitrogen three times. 2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) )tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]- 5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (Tetrahydropyran-2-yl)oxypentanoylamino]pentanoyl [ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate ( A solution of 285 mg of 80% pure HCl (0.11 mmol) is added via syringe. The mixture was evacuated and backfilled with 1 atm of hydrogen, and then stirred under 1 atm of hydrogen at ambient temperature. After stirring for 3 hours, the flask is purged with nitrogen and the mixture is filtered through diatomaceous earth. The solution is concentrated to give the title compound (213 mg, purity 79%, 77%). / z 1001.20(M+2H) / 2.

[0141] Preparation 33 (2,5-dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[5-[ 3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyridine 5-[bis[2-[ 5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrofuran [(2-hydroxypentanoylamino)pentanoylamino]ethyl] Amino]-5-oxo-pentanoyl]amino]undecanoate

[0142] [ka]

[0143] The title compound was synthesized by the reaction of 11-[[(2S)-2-[5-[5-[3-acetamido-4,5- Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypenta 5-[bis[2-[5-[3-acetamido]pentanoylamino]-5-[bis[2-[5 ... 4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl] Oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoylamino Prepared from [methylamino]undecanoic acid in a manner essentially similar to that of Preparation 16 ES / MS m / z 1050(M+2H) / 2

[0144] Preparation 34 [5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido -4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]o xypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl) (phenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperazin-2-yl Lysyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo 2-[5-[3-acetamido-4,5-diacetoxy-6-( Acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl ]amino]ethylamino]-5-oxo-pentoxy]-3,4-diacetoxytetrahydrochloride Methyl dropyran-2-ylacetate

[0145] [ka]

[0146] The title compound was synthesized by the reaction of 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetamido Oxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl a amino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (ethoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl] Amino]-5-oxo-pentanoyl]amino]hexanoic acid and (2S)-2-amino- 3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxy

[0073] From [methyl]-1-piperidyl]propan-1-one, essentially similar to the method of Preparation 10 Prepared in the manner described above. ES / MS m / z 1059.2(M-2H) / 2.

[0147] Preparation 35 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido- 4,5-Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy Cypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetamido] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoyl amino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino] -3-[bis(4-methoxyphenyl)-phenyl-methoxy]propanoyl]-4-pi Peridyl]methoxy]-4-oxobutanoic acid

[0148] [ka]

[0149] [5-acetamido-6-[5-[2-[[(4S)-4-[ 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrochloride ropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[ [bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydrogen Oxymethyl)-1-piperidyl]-2-oxo-ethyl]amino]-6-oxo-hexyl yl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamido-4,5 -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen thanoylamino]ethyl]amino]ethylamino]-5-oxo-pentoxy]-3,4 -diacetoxy-tetrahydropyran-2-yl]methyl acetate (1.194 g, 0.56 A solution of succinic anhydride (113 mg, 1.13 mmol), TEA (0. Add 4 mL of HCl (3 mmol) and DMAP (213 mg, 1.69 mmol). The mixture was stirred at ambient temperature for 1 h, after which the mixture was diluted with saturated NH4Cl (200 mL). Extract with DCM (3 x 200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers are combined, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM. The resulting product was purified by filtration and dried in a vacuum oven at 40°C for 3 hours. The title compound (1.081 g, 86%) was obtained. ES / MS m / z 1109.60 (M- 2H) / 2.

[0150] Preparation 36 Resin Loading

[0151] [ka]

[0152] 4-[[1-[(2S)-2-[6-[ in MeCN (6 mL) and DCM (1 mL) [(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxy Methyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2 -[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrofuran] Hydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo- pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)- Phenyl-methoxy]propanoyl]-4-piperidyl]methoxy]-4-oxo-butyric acid (1.00 g, 0.61 mmol) solution is transferred to a resin loading cartridge. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronate ammonium hexafluorophosphate (386 mg, 0.97 mmol) and DIEA (0 0.25 mL, 0.48 mmol) is added and the cartridge is shaken for 5 minutes at ambient temperature. This was followed by 1000Å LCAA controlled pore glass resin (5.39 g, 90 μmol / g rho). Ding (purchased from ChemGenes) was added and the mixture was shaken at ambient temperature for 18 hours. After this, the cartridge was drained by suction and the resin was washed with DCM (10 mL) for 10 minutes. Drain the cartridge and repeat the wash and drain steps with 10% M Repeat with eOH / DCM (10 mL) and EtO (10 mL). After draining, add acetic anhydride ( A solution of 6.4 mL of pyridine (20 mL) and TEA (0.22 mL) was added and the resulting mixture was stirred at 4°C for 1 hour. Shake the cartridge for 2 hours. After this time, drain the cartridge and repeat the wash and drain procedure described above. , DCM (10 mL), 10% MeOH / DCM (10 mL) and diethyl ether ( Repeat with 10 mL of PEG. After draining, dry the resin under vacuum for 30 minutes. Resin loading is measured using a standard trityl assay. The calculated value was 7 μmol / g.

[0153] Preparation 37 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]ethyl]amino]-5-oxo-pentanoyl] Benzyl aminoethoxyacetate

[0154] [ka]

[0155] The title compound was synthesized by the reaction of (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and From 2-(2-aminoethoxy)benzyl acetate hydrochloride, essentially the same procedure as in Preparation 10 Prepared in the manner described above. ES / MS m / z 1005.2 (M+2H / 2).

[0156] Preparation 38 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]ethyl]amino]-5-oxo-pentanoyl] Amino]ethoxy]acetic acid

[0157] [ka]

[0158] 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]ethyl]amino]-5-oxo-pentanoyl] Benzyl aminoethoxyacetate (0.120 mmol, 240 mg) was dissolved in MeOH (12 Mix with 5% Pd / C (1.17 mmol, 124 mg) in 0.0 mL of ethanol. Hydrogenated on a shaker (ambient temperature, 10 psi) for 48 minutes, filtered through diatomaceous earth, and concentrated in vacuo. Concentrate to give the title compound as a grey solid (187 mg, 82%). ES / MS m / z 960.0(M+2H / 2).

[0159] Preparation 39 (2,3,5,6-tetrafluorophenyl)2-[2-[[(2S)-2-[5-[ 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrochloride 5-[bis[ 2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) )tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]e ethyl]amino]5-oxo-pentanoyl]amino]ethoxy]acetic acid

[0160] [ka]

[0161] 2-[2-[[(2S)-2-[5-[5-[3-acetate in DCM (3.0 mL) Mido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl ]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[ 3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyridine pentanoylamino]pentanoylamino]ethyl]amino]-5 -Oxo-pentanoyl]amino]ethoxy]acetic acid (0.096 mmol, 184 mg) and DIEA (0.765 mmol, 140 μL), (2,3,5,6-tetrafluoroethylene) (2,2,2-trifluorophenyl)acetic acid (0.383 mmol, 100 mg) The mixture was stirred at ambient temperature for 16 hours. The reaction mixture was diluted with 0% to 50% Me Purify directly by silica gel flash chromatography eluting with OH / DCM , to give the title compound as a tan solid (197 mg, 99%). ES / MS m / z 1 034.0(M+2H / 2).

[0162] preparation 40 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5 -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen 2-[5-[3-acetamino]pentanoylamino]-5-bis[ ... Mido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl ]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoylamino benzyl ethoxy]ethoxy]ethoxy]ethoxy]acetate

[0163] [ka]

[0164] The title compound was synthesized by the reaction of (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy] 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamine 2-[5-[3-acetamido-4,5-amino]pentanoylamino]-5-[bis[2-[5 ... -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen pentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and From 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]benzyl acetate hydrochloride , prepared in a manner essentially similar to that of Preparation 10. ES / MS m / z 1049.0 (M+2H / 2).

[0165] Preparation 41 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5 -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen 2-[5-[3-acetamino]pentanoylamino]-5-bis[ ... Mido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl ]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoylamino Aminoethoxyethoxyethoxyethoxyacetic acid

[0166] [ka]

[0167] 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5 -Diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypen 2-[5-[3-acetamino]pentanoylamino]-5-bis[ ... Mido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl ]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoylamino benzyl ethoxy]ethoxy]ethoxy]acetate (0.118mmol) 1, 247 mg) was dissolved in 5% Pd / C (1.17 mmol) in MeOH (12.0 mL). The mixture was mixed with 124 mg of PEG-400 (124 mg of PEG-400). The mixture was shaken on a Parr shaker (ambient temperature, 10 psi) for 1 hour. Hydrogenate, filter through diatomaceous earth, and concentrate in vacuo to obtain the title compound as a grey solid (22 7 mg, 96%). ES / MS m / z 1004.0(M+2H / 2).

[0168] Preparation 42 (2,3,5,6-tetrafluorophenyl)2-[2-[2-[2-[[(2S)- 2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) )tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]- 5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetamido) (Tetrahydropyran-2-yl)oxypentanoylamino]pentanoyl 5-oxopentanoylamino]ethylamino]5-oxopentanoylamino]ethoxy]ethoxy ]ethoxy]acetic acid

[0169] [ka]

[0170] 2-[2-[2-[2-[[(2S)-2-[5-[5-[ 3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyridine 5-[bis[2-[ 5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydrofuran [(2-hydroxypentanoylamino)pentanoylamino]ethyl] Amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid ( 0.111 mmol, 222 mg) and DIEA (0.883 mmol, 154 μL) , (2,3,5,6-tetrafluorophenyl) 2,2,2-trifluoroacetic acid (0.4 43mmol, 116mg) is added dropwise to the mixture. The mixture is stirred at ambient temperature for 16 hours. The reaction mixture was purified by silica gel flash chromatography eluting with 0% to 50% MeOH / DCM. The title compound was purified directly by chromatography to give the title compound as a tan solid (174 mg, 73%). ES / MS m / z 1078.2 (M+2H / 2).

[0171] Example 1 Conjugation Protocol For the synthesis of GalNAc-conjugated sense strands, a 3'C6-NH2 functional group was used. The sense strand is first synthesized using standard phosphoramidite chemistry. Stock solution of ligand-NHS ester (10 mmol / L in acetonitrile; 1 equiv. Prepare the solution by dissolving borate buffer (10% v / v; 20x) in an Eppendorf tube. The oligonucleotide C6-NH2 was added to the sense strand, followed by GalNAc ligand (5 equiv. The mixture is shaken at ambient temperature for 16 hours. After this time, the mixture is diluted with 15 mL of The mixture was transferred to a fluororesin tube, ammonium hydroxide (28% by weight) was added, and the mixture was allowed to stand at ambient temperature. The mixture is shaken at RT for 2 hours. The ammonia is then removed in vacuo. The residue is purified by ion exchange chromatography. Purify by chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH PO4, Solvent B: 15% MeCN / 20 mM NaH2PO4, 1 M NaBr; 8 m 35-55% B over 5 CV at 1 L / min, column temperature 60 °C. Pool the desired fractions. Desalt by Eppendorf centrifugation or spin filtration using desalting columns. Afterwards, the material is collected and the OD and volume are measured to obtain the concentration.

[0172] Alternatively, GalNAc ligands can be immobilized on microporous polystyrene resin or controlled pore glass. Stabilized and established solid-phase oligomerization using 5'-CE(β-cyanoethyl) phosphoramidite Conjugation was performed on the sense strand by synthesis using nucleotide synthesis methods. This is done at the 5' position.

[0173] Alternatively, the GalNAc ligand can be converted to a suitable phosphoramidite and then subjected to standard phospho- It is delivered to the 5' position of the sense strand using lamidite chemistry.

[0174] Example 2: Annealing To generate siRNA duplexes of sense and antisense strands, the following procedure is carried out: Falcon tubes containing oligonucleotide sense strand-GalNAc conjugates Add the corresponding antisense oligonucleotide (1 equivalent) to the mixture and vortex for 10 seconds. After rinsing, the solution was spin-filtered through a 100K MWCO Amicon filter unit. The filtrate is collected and concentrated in vacuo in a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2 µm filter, and the OD and volume were measured. and obtain the concentration.

[0175] Endosafe®-nexgen PTS device for Limulus amebocyte lysate The lysate is used to perform an endotoxin test.

[0176] [Table 1]

[0177] Example 3: General Procedure for Oligo Synthesis Using GalNAc-Functionalized CPG Oligo synthesis is performed on a Mermade 12 instrument using phosphoramidite chemistry. The on-sense strand was synthesized from a pre-functionalized GalNAc solid support, and the anti-sense strand was synthesized from a pre-functionalized GalNAc solid support. The first nucleotide of the oligo sequence is synthesized using a standard support preloaded with The oligo was cleaved and deprotected using concentrated ammonium hydroxide solution (28% by weight) and Purify by ion exchange chromatography using the conditions of: Desalting, Annealing , and endotoxin testing is performed as described above.

[0178] [Table 2]

[0179] Important note: [Phos] indicates the phosphorylated 5' residue, and m indicates the nucleotide sequence immediately following m. indicates the 2'O-Me group on the ribose of the nucleotide / nucleoside base, and f is immediately after f represents the 2' fluoro group on the ribose on the nucleotide / nucleoside base described in * indicates a modified linkage that is phosphorothioate (PS).

[0180] Example 4

[0181] [Table 3]

[0182] Example 5

[0183] [Table 4]

[0184] Example 6: Biological Assays GalNAc-LDHA siRNA conjugate gene knockdown in vivo Rating animal All animals were housed in a temperature-controlled facility (24°C) with a 12-h / 12-h light / dark cycle. Animal protocols were approved by Eli Lilly and Company IA. Approved by the CUC. Male C57BL / 6 mice, approximately 8 weeks old (Envigo). Animals are weighed and randomized by weight into treatment groups of 6 animals per group. Treat with either PBS or siRNA conjugate via intravenous injection. 14 days after administration , the animals are sacrificed and liver tissue is rapidly dissected and flash-frozen in liquid nitrogen.

[0185] RNA isolation and real-time quantitative RT-PCR Total RNA was purified using TRIzol reagent (Ambion) and PureLink Pro 96 Total RNA was isolated from liver samples using a total RNA purification kit (Invitrogen). High-Capacity cDNA Reverse PCR using a single RNA fragment e Transcription kit (Applied Biosystems) was used. cDNA was synthesized using the Quantitative Real-Time PCR kit (Applied Biosystems). stems QuantStudio 7 Flex Real-Time PCR System The CT value was calculated using a RPLP0 (Mm01 974474_gH, Applied Biosystems) and LD Relative expression of HA (Mm03646738_gI, Applied Biosystems) Expression is calculated by the ΔΔC method. Relative expression is normalized to vehicle-treated animals. Calculate the fold change by:

[0186] [Table 5]

[0187] [Table 6]

[0188] GalNAc-LDHA siRNA 24-hour tissue exposure screening animal All animals were housed in a temperature-controlled facility (24°C) with a 12-h / 12-h light / dark cycle. Animal protocols were approved by Eli Lilly and Company. The study was approved by the IACUC. Male C57BL / 6 mice (Envig) were approximately 8 weeks old. o) are weighed and randomized by weight into treatment groups of 5 animals per group. Mice are treated with either PBS vehicle or siRNA conjugate via subcutaneous injection. 24 hours after administration, the animals were sacrificed, plasma samples were collected by cardiac puncture bleeding, and liver / kidney tissues were analyzed. Tissues are rapidly dissected and flash frozen in liquid nitrogen. Tissue concentrations of siRNA and metabolites are determined by Anion-exchange high-performance liquid chromatography coupled with PNA hybridization and fluorescence detection Determined by roughness analysis.

[0189] [Table 7]

[0190] [Table 8]

[0191] Example 7: In vitro knockdown of mouse genes Freshly isolated mouse primary hepatocytes (MPH) were cultured at 1000 x g / well. The siRNA conjugates were plated onto Corning plates at 15kJ / well and added to the plates in PBS. Dose-response experiments were performed in primary mouse hepatocytes at 1000, 333, 1 11, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM The procedure is carried out using a final GalNAc-conjugated duplex concentration of 0.05%.

[0192] Pre-RNA was prepared using the Quick-RNA 96 kit from Zymo Research. RNA is isolated directly from the transfected cells. The final purified and eluted RNA is Fast A from Invitrogen, used immediately or stored frozen. Advanced RT Master Mix and QuantStudio 7 Fle x Real-Time PCR System (Life Technologies ) to synthesize cDNA from purified RNA, and incubate at 37°C for 30 min and 95°C for 5 min. Incubate for 1 minute and hold at 4°C. cDNA is then used to analyze the DNA in QuantStudio 7 Flex Real-Time PCR System (Life Techno Real-time PCR using RT-PCR (RT-PCR) was performed with the following parameters: 2 min at 50°C; for 10 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Do this:.

[0193] The following mouse target genes were identified: HPRT, LDHA, and DPP4 (Life Technologies The results of the RT-PCR assay for the α-glucanase (α-glucanase) and IC50 calculations are shown in Table 8. Knockdown levels represent relative knockdown compared to vehicle alone and are shown in mice. Further normalization to Rplp0 (Life Technologies) was performed between samples. Compare IC using a four-parameter fit model using XLFit.50 Calculate These results demonstrate the effective knockdown of the delivery moieties disclosed herein. with 80-97% knockdown of three different genes.

[0194] [Table 9]

Claims

1. A compound comprising Formula I conjugated to R, 【Chemistry 1】 wherein R may be conjugated to Formula I via a linker, and R is an anti the sense strand, the antisense strand comprising a region complementary to a gene of interest, A compound wherein the antisense strand is 15 to 40 nucleotides in length.

2. 2. The compound of claim 1, wherein Formula I is conjugated to R via a linker.

3. The linker comprises a linker of Formula II having attachment points A and B, or comprises Formula III having connection points C and D, 【Chemistry 2】 The compound of claim 2, wherein junction A or junction C is conjugated to formula I. 。

4. The linker further comprises a phosphate group at connection point B or connection point D, and the phosphate group is 4. The compound of claim 2 or 3, which is conjugated.

5. the linker comprises a linker of Formula III having attachment points C and D; 【Transformation 3】 5. The compound according to claim 2, wherein the connecting point C is conjugated to formula I. compound.

6. The region of complementarity to the gene of interest is at least 15 contiguous nucleotides in length. The compound according to any one of claims 1 to 5.

7. R further comprises a sense strand, and the sense strand and the antisense strand are at least 15 The compound of claim 6, which forms a complementary region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31

8. 8. Formula I is conjugated to the sense strand or the antisense strand. The compound described in

9. The compound of claim 7 or 8, wherein Formula I is conjugated to the antisense strand. Compound.

10. 9. The compound of claim 7 or 8, wherein Formula I is conjugated to the sense strand.

11. 10. The method of claim 1, wherein Formula I is conjugated to the 3' terminal nucleotide of the sense strand.

1. The compound according to claim 0.

12. The sense strand and the antisense strand have a complementary region of at least 18 nucleotides in length. The compound according to any one of claims 7 to 11, which forms

13. The antisense strand forms a complementary region of at least 15 nucleotides with the sense strand. The compound according to any one of claims 7 to 12,

14. the sense strand and the antisense strand are each independently 18 to 23 nucleotides in length; The compound according to any one of claims 7 to 13.

15. the sense strand and the antisense strand are each independently 21 to 23 nucleotides in length; The compound according to any one of claims 7 to 14.

16. The antisense strand comprises a 3' overhang of two nucleotides.

16. The compound according to any one of claims 15.

17. 10. The method of claim 9, wherein the sense strand or the antisense strand comprises one or more modified nucleotides.

17. The compound according to any one of claims 7 to 16.

18. The one or more modified nucleotides may be a 2' fluoro-modified nucleotide, a 2'-O-methyl 2'-O-alkyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, or O-methoxyethyl-modified nucleotides The compound according to any one of claims 7 to 17, which is a modified nucleotide.

19. the sense strand comprises one or more modified nucleotides, 19. The compound of any one of claims 7 to 18, wherein each of the nucleotides is a modified nucleotide.

20. the antisense strand comprises one or more modified nucleotides, 20. The method according to any one of claims 7 to 19, wherein each of the nucleotides is a modified nucleotide. The compounds listed above.

21. the sense strand or the antisense strand comprises one or more modified internucleotide linkages; The compound according to any one of claims 7 to 20.

22. 8. The method of claim 7, wherein the one or more modified internucleotide linkages are phosphorothioate linkages.

22. The compound according to any one of claims 1 to 21.

23. The 5' nucleotide of the antisense strand is phosphorylated or phosphate analogue. The compound according to any one of claims 7 to 22, comprising

24. 24. The method of claim 7, wherein the 5' nucleotide of the antisense strand contains a vinyl phosphate group. The compound according to any one of claims 1 to 4.

25. Any one of claims 7 to 24, wherein the sense strand and the antisense strand are siRNA.

3. The compound according to claim 1.

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