Liver-targeted three-antenna GalNAc connexon precursor and preparation method thereof

Abstract

The invention relates to a GalNAc connexon precursor and a preparation method thereof, in particular to a preparation method of a liver targeting three-antenna GalNAc connexon precursor with a structure shown in a formula 13. The method provided by the invention has the advantages of few steps, simple purification, high yield and mild reaction conditions, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis. Specifically, this invention relates to a liver-targeting three-antennae GalNAc linker precursor and its preparation method. Background Technology

[0002] With the development of biotechnology, nucleic acid drugs have ushered in a new era of innovative drugs following chemical drugs. Currently, the research and development of nucleic acid drugs mainly focuses on two aspects: first, modifying and optimizing the nucleic acid structure; and second, employing drug delivery systems. Both aspects aim to enable effective targeted accumulation and cellular uptake of drugs, lysosomal escape, and improved in vivo drug performance. GalNAc, a classic liver-targeting delivery ligand, has already seen four marketed drugs based on it: Givosiran, Lumasilan, Inclisiran, and Vutrisiran, with more than a dozen other nucleic acid drugs currently undergoing clinical trials. This demonstrates the significant application value of GalNAc ligands as conjugates for RNA drugs.

[0003] To accelerate the development of RNA drugs, a literature review revealed that the synthesis method of this compound is cumbersome, difficult to purify, involves repeated deprotection and reprotection, complex post-processing, a long route, poor atom economy, and is unsuitable for industrialization. Patents CN1104717982, CN11527207, CN102006890, CN111050806, CN115028670A, and WO2012037254 all employ the method of first condensing monoprotected propylenediamine and then splicing it with an aminogalactose fragment. This process prolongs the reaction route, and both the intermediate itself and its purification methods have shortcomings.

[0004] The patent CN11527207 describes a complex synthetic route for compound 11 that involves 14 steps, with several steps having a yield of approximately 55%. Furthermore, it employs preparative liquid-phase purification, which is costly and difficult to produce.

[0005] The reaction route in CN104717982 is quite long, with 17 steps. The synthesis of compound 12 even uses a three-step continuous feeding method, and all intermediates need to be post-processed before continuous feeding. The reaction time is too long. When synthesizing compound 26B, the trifluoroacetate of the starting material will produce impurities, resulting in low yield and difficulty in purification. The synthesis of the sugar chain part has a low yield and a long route.

[0006] As can be seen, the methods used in the above literature are largely the same. Although the starting materials differ, the reaction steps are all lengthy, the post-processing is cumbersome, and they all require a strategy of repeatedly adding and removing protecting groups. The purification methods for intermediates are limited, and many require column purification. This increases the number of reaction steps and is clearly not atom-economical in the selection of starting materials.

[0007] Therefore, there is an urgent need in the field to develop a method for preparing liver-targeting tri-antenna GalNAc linker precursors that is short in route, low in cost, simple in post-processing, and convenient in purification. Summary of the Invention

[0008] One objective of this invention is to provide a method for preparing liver-targeting GalNAc linker precursors that is low in cost, has a short synthetic route, and is simple to purify.

[0009] In a first aspect of the present invention, a method for preparing a compound of formula 12 is provided, comprising the following steps:

[0010]

[0011] (1) Under the protection of an inert gas and in the presence of a condensing agent, compound 8 and compound 8-1 undergo a condensation reaction in the first solvent to obtain compound 9.

[0012] (2) Under acidic conditions, compound 9 undergoes a hydrolysis reaction to give compound 10;

[0013] (3) Under the protection of an inert gas and in the presence of a condensing agent, compound 10 and compound 5 undergo a condensation reaction in the first solvent to obtain compound 11.

[0014] (4) In the second solvent, the compound of formula 11 undergoes a reduction reaction to obtain the compound of formula 12;

[0015] Where n is an integer between 8 and 15; preferably, n is 10.

[0016] In another preferred embodiment, the compound of formula 8 is obtained by reacting the compound of formula 6 with the compound of formula 7.

[0017]

[0018] In another preferred embodiment, step (1) includes one or more features selected from the group consisting of:

[0019] (a) The first solvent is selected from the group consisting of: C 1-6 Hydrocarbon solvents, amide solvents, or combinations thereof; preferably, dichloromethane, N,N-dimethylformamide, or combinations thereof;

[0020] (b) The condensing agent is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), HBTU, DIPEA, CDI, DIPEA, or combinations thereof;

[0021] (c) The molar ratio of the compound of formula 8 to the compound of formula 8-1 is 1:(1-1.5), preferably 1:(1-1.3), and more preferably 1:1.15; and / or

[0022] (d) The mass-to-volume ratio (g / mL) of the compound of Formula 8 to the first solvent is 1:(10-20), preferably 1:(10-15).

[0023] In another preferred embodiment, in step (1), the molar ratio of the compound of formula 8 to the condensing agent is 1:(1-10); more preferably, the molar ratio of the compound of formula 8 to the condensing agent is 1:(1-5).

[0024] In another preferred embodiment, in step (1), the molar ratio of the compound of formula 8 to HOBt is 1:(1.2-2); more preferably 1:1.5; the molar ratio of the compound of formula 8 to EDCI is 1:(1.0-1.5); more preferably 1:(1.2-1.3); and / or, the molar ratio of the compound of formula 8 to DIEA is 1:(2.5-4); more preferably 1:(3-3.5).

[0025] In another preferred embodiment, in step (1), the reaction time is 6-10 h, more preferably 6-8 h.

[0026] In another preferred embodiment, in step (1), the reaction temperature is 10-45°C; more preferably 15-30°C.

[0027] In another preferred embodiment, step (3) includes one or more of the following features:

[0028] (3-1) The first solvent is selected from the group consisting of: C 1-6 Hydrocarbon solvents, amide solvents, or combinations thereof; preferably, dichloromethane, N,N-dimethylformamide, or combinations thereof;

[0029] (3-2) The condensing agent is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), HBTU, DIPEA, CDI, DIPEA, or a combination thereof;

[0030] (3-3) The molar ratio of compound 10 to compound 5 is 1:(3-6); preferably 1:(3-4); and / or

[0031] The mass-to-volume ratio (g / mL) of compound 10 of formula (3-4) to the first solvent is 1:(15-30); preferably 1:(20-25).

[0032] In another preferred embodiment, in step (3), the molar ratio of the compound of formula 5 to the condensing agent is 1:(1-15).

[0033] In another preferred embodiment, in step (3), the molar ratio of compound 5 to HOBt is 1:(1.2-2); more preferably 1:(1.2-1.5); the molar ratio of compound 5 to EDCI is 1:(1.0-1.5); more preferably 1:(1.0-1.2); and / or the molar ratio of compound 5 to DIEA is 1:(2.5-4); more preferably 1:(2.5-3).

[0034] In another preferred embodiment, the reaction time in step (3) is 10-12 h.

[0035] In another preferred embodiment, in step (3), the reaction temperature is 15-40°C, more preferably 15-30°C.

[0036] In another preferred embodiment, step (3) further includes purification; preferably column purification.

[0037] In another preferred embodiment, in step (3), the preparation method of the compound of formula 5 is as follows:

[0038]

[0039] (3-a) In the presence of an inert gas, in a third solvent, and in the presence of a hydrolyzing agent, compound 3-1 and compound 3-2 undergo a ring-opening reaction. The product is purified by slurrying with a C2-6 ether solvent to obtain compound 4.

[0040] (3-b) In the fourth solvent, compound 4 undergoes a salt-forming reaction to give compound 5;

[0041] The fourth solvent is selected from the group consisting of C1-C6 alcohol solvents.

[0042] In another preferred embodiment, the hydrolysant is selected from trimethylsilyl trifluoromethanesulfonate and diethylsilyl trifluoromethanesulfonate.

[0043] In another preferred embodiment, the C2-6 ether solvent is selected from the group consisting of methyl tert-butyl ether, diethyl ether, or combinations thereof; preferably, methyl tert-butyl ether.

[0044] In another preferred embodiment, step (3-b) includes the following steps:

[0045] In the fourth solvent, under a hydrogen atmosphere and in the presence of palladium on carbon, compound 4 undergoes reduction and salt formation reactions, and after filtration and concentration, compound 5 is obtained.

[0046] In another preferred embodiment, in step (3-a), the molar ratio of the compound of formula 3-1 to the compound of formula 3-2 is 1:(1±0.3); more preferably 1:1; and / or

[0047] The molar ratio of the compound of formula 3-1 to the hydrolysant is (5-10):1; preferably (5-7):1.

[0048] In another preferred embodiment, the compound of formula 3-1 was prepared by the method reported in document CN11527207A, which is incorporated herein by reference:

[0049]

[0050] In another preferred embodiment, step (4) includes one or more of the following features:

[0051] (i) The second solvent is selected from the group consisting of C1-C6 alcohol solvents, C1-C6 hydrocarbon solvents, or combinations thereof; preferably methanol, dichloromethane, or combinations thereof;

[0052] (ii) The reduction reaction is carried out in a hydrogen atmosphere in the presence of 10% palladium on carbon.

[0053] In another preferred embodiment, the reaction time in step (4) is 3-5 hours.

[0054] In another preferred embodiment, in step (4), the reaction temperature is 15-40°C; more preferably 20-30°C.

[0055] In another preferred embodiment, step (4) further includes post-processing: filtration and concentration.

[0056] In another preferred embodiment, in step (2), the acid is formic acid, trifluoroacetic acid, acetic acid, hydrochloric acid, sulfuric acid, or a combination thereof; preferably formic acid, trifluoroacetic acid, or a combination thereof.

[0057] In another preferred embodiment, step (2) further includes a purification step: after the reaction is complete, the compound of formula 10 is recrystallized and purified in a fifth solvent, wherein the fifth solvent is selected from the group consisting of: C 1-6 Ester solvents, C 1-6 Hydrocarbon solvents, or combinations thereof; preferably ethyl acetate / n-hexane = 1:1.

[0058] In another preferred embodiment, in step (2), the reaction time is 6-10 h, more preferably 6-8 h.

[0059] In another preferred embodiment, in step (2), the reaction temperature is 15-40°C, more preferably 20-30°C.

[0060] In a second aspect of the invention, the use of the compound of formula 12 is provided for preparing a liver-targeting tri-antennae GalNAc linker; comprising the following steps:

[0061]

[0062] (M1) In a first solvent, in the presence of a condensing agent, compound 12 reacts with compound 12-1 to give compound 13.

[0063] (M2) The reaction of compound 13 with succinic anhydride of compound 13-1 yields the tri-antennae GalNAc linker shown in formula 14;

[0064] Where n is an integer between 8 and 15; preferably, n is 10.

[0065] The compound of formula 12 is prepared by the method described in the first aspect of the present invention.

[0066] In another preferred embodiment, in step (M1), the molar ratio of the compound of formula 12 to the compound of formula 12-1 is 1:(1-1.8), more preferably 1:(1-1.3); and / or

[0067] The molar ratio of the compound of Formula 12 to the condensing agent is 1:(1-1.8), preferably 1:(1-1.3).

[0068] In another preferred embodiment, in step (M2), the reaction system further includes a base (e.g., triethylamine) and 4-dimethylaminopyridine.

[0069] In another preferred embodiment, the molar ratio of compound 13 to compound 13-1 is 1:(2-3);

[0070] The molar ratio of the compound of Formula 13 to the base is 1:(5-10), preferably 1:(6-7); and / or

[0071] The molar ratio of compound 13 to 4-dimethylaminopyridine is 1:(1.5-3), preferably 1:(1.8-2.2).

[0072] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0073] Through extensive and in-depth research, the inventors have, for the first time, provided a novel liver-targeting tri-antenna GalNAc linker precursor and its preparation method. The preparation route of the liver-targeting tri-antenna GalNAc linker precursor of this invention is short, low-cost, and the purification method is simple, making it suitable for industrial production. Furthermore, the obtained product has high purity and high yield. Based on these findings, the inventors completed this invention.

[0074] the term

[0075] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0076] As used herein, unless otherwise specified, solvents or solutions are added by pouring directly, adding at a constant rate, or adding slowly dropwise.

[0077] The term “room temperature” or “normal temperature” refers to a temperature of 4-40°C, preferably 25±5°C.

[0078] In this invention, in the reaction system of each reaction step, each substance (except the solvent) can participate in the reaction independently in any suitable ratio, such as 1:(0.1-10), 1:(0.2-5), 1:(0.5-2), 1:(0.8-1.5) or 1:(1.0-1.2).

[0079] As used in this article, the abbreviations and chemical names of the reagents are as follows:

[0080] DCC: N,N'-Dicyclohexylcarboimide

[0081] DIC: N,N'-Diisopropylcarbodiimide

[0082] EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0083] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate

[0084] HOBt: 1-Hydroxybenzotriazole

[0085] DIEA: N,N-Diisopropylethylamine

[0086] Preparation method of liver-targeting three-antennae GalNAc linker precursor

[0087] N-acetylgalactosamine (GalNAc) is a ligand that binds to the desialization glycoprotein receptor (ASGPR) on the liver surface. The desialization glycoprotein receptor is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, the high-affinity ligand GalNAc for ASGPR has been used as a targeting molecule, achieving some progress in liver-targeted delivery of nucleic acid drugs. The liver-targeting triangular GalNAc linker precursor is a classic liver-targeting delivery ligand, exhibiting a much higher affinity for ASGPR than biangular and monoangular GalNAc.

[0088] In existing technologies, the preparation methods for liver-targeting tri-antenna GalNAc linkers are lengthy, have high production and purification costs, and low yields.

[0089]

[0090] To address the shortcomings of existing technologies, this invention provides a novel method for preparing a liver-targeting tri-antenna GalNAc linker precursor (compound of formula 12). The method described has high yield, high product purity, mild reaction conditions, simple post-processing, and few steps.

[0091] The liver-targeting tri-antenna GalNAc linker precursor (compound of formula 12) of the present invention can be prepared by the following method; however, the conditions of the method, such as reactants, solvents, bases, acids, amounts of compounds used, reaction temperatures, reaction times, and material ratios, are not limited to the explanations below.

[0092] Typically, the preparation process of the compounds of the present invention is as follows, wherein the raw materials and reagents used can be purchased commercially unless otherwise specified.

[0093] Specifically, this invention provides a novel synthetic route for a key intermediate 12, a precursor of the GalNAc linker. Using readily available 2-amino-2-deoxy-D-galactose hydrochloride as a starting material, intermediate 5 is constructed through a four-step reaction involving full acetylation, cyclization, ring-opening, and hydrogenation. This avoids the need for additional protecting groups in existing routes, improves reaction yield, simplifies purification methods, and avoids heavy metal catalysis, thus enhancing production safety and saving costs.

[0094]

[0095] Specifically,

[0096] For example, in an inert solvent, in the presence of trimethylsilyl trifluoromethanesulfonate, compound 2 reacts at room temperature to produce compound 3-1;

[0097] In an inert solvent, in the presence of trimethylsilyl trifluoromethanesulfonate, compound 3-1 undergoes a ring-opening reaction to produce compound 4. After the reaction is complete, the reaction solution is washed with saturated sodium bicarbonate solution, the organic phase is washed with brine, dried with anhydrous sodium sulfate, filtered and concentrated, and the residue is stirred with an ether solvent to crystallize, thus obtaining the purified compound 4.

[0098] In a C1-6 alcohol solvent, in the presence of 10% palladium on carbon and hydrogen, compound 4 undergoes a hydrogenation reaction to give compound 5.

[0099] The reaction temperature is 10-50℃, preferably carried out at room temperature, such as 15-40℃.

[0100] Using tris(hydroxymethyl)aminomethane as a starting material, intermediate 10 was constructed through a three-step reaction involving Michael addition, amide condensation, and acid hydrolysis. This modified the protecting group reported in the original literature, shortening the route and simplifying the operation for subsequent synthesis.

[0101]

[0102] GalNAc linker precursor intermediate 13 was constructed through a four-step reaction involving amide condensation, hydrogenation, amide condensation, and esterification of intermediates 5 and 10. The overall reaction operation is simple, the route is short, the purification method is simple, and the yield is high.

[0103]

[0104] In the above steps, the reaction solvent, reaction temperature, reaction time, etc., can be selected according to the specific reactants.

[0105] It should be understood that the raw material compounds of the present invention (e.g., compounds of formula 8, formula 3-1, and formula 3-2) can be prepared by the above-described preparation method, or by methods known in the prior art, or may be commercially available products. The above reaction also includes a post-treatment purification step, the specific steps of which are conventionally selected according to the reaction conditions.

[0106] For example, the reaction times in this application can be selected as needed, such as 1-48h, 2-24h, 2-12h, 2-8h, 2-5h.

[0107] Each reaction can be carried out under inert gas or air conditions, preferably under inert gas protection. Preferably, the inert gas is a commonly used inert gas in the art, including but not limited to: nitrogen, argon, helium, or combinations thereof.

[0108] Unless otherwise specified, the reaction solvents for each reaction in this application may be conventionally selected according to the reaction type, including but not limited to: hydrocarbon solvents, halogenated hydrocarbon solvents, aromatic hydrocarbon solvents, sulfone solvents, sulfoxide solvents, alcohol solvents, ether solvents, amide solvents, ester solvents, or combinations thereof; preferably, dichloromethane, ethyl acetate, N,N-dimethylformamide, or combinations thereof.

[0109] The compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art, such combinations can be easily performed by those skilled in the art.

[0110] Compared with the prior art, the main advantages of the present invention include:

[0111] (1) The preparation route of liver-targeting tri-antenna GalNAc linker precursor is short, low-cost, and simple to purify, making it suitable for industrial production.

[0112] (2) The liver-targeting tri-antenna GalNAc linker precursor prepared by the method of the present invention has high purity and high yield.

[0113] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed according to the conditions described in the conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0115] Example 1: Synthesis of Compound 3-1

[0116]

[0117] Under a nitrogen atmosphere, compound 2 (50 g, 128 mmol) was dispersed in 250 mL of 1,2-dichloroethane with stirring. Trimethylsilyl trifluoromethanesulfonate (34.29 g, 154 mmol) was slowly added dropwise. The reaction was carried out at room temperature, and the mixture was stirred for 10-20 minutes until it was completely dissolved. After the reaction was complete, the reaction solution was added dropwise to 216 mL of an aqueous solution of sodium bicarbonate (32.39 g, 386 mmol). After stirring for 30 minutes, the mixture was separated. The organic phase was washed once with saturated brine (216 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give a yellow oily substance 3-1 (44.06 g, quantitative). 1 H NMR (600MHz, DMSO-d6) δ6.05(d,J=7.0Hz,1H),5.24(dd,J=3.9,2.9Hz,1H),4.88(dd,J=6.8,3.9Hz,1H),4.26(ddd,J=7.5,4.9,2.9Hz,1 H),4.11(dd,J=11.6,7.3Hz,1H),4.07–4.01(m,1H),3.95(tq,J=7.0,1.3Hz,1H),2.07(s,3H),2.02–1.98(m,6H),1.95(d,J=1.4Hz,3H).

[0118] Example 2: Synthesis of Compound 4

[0119]

[0120] Under a nitrogen atmosphere, intermediate 3-1 (15.80 g, 48 mmol) was dissolved in dichloromethane (150 mL) with stirring. Intermediate 3-2 (8.87 g, 48 mmol) was added and dispersed with stirring. Trimethylsilyl trifluoromethanesulfonate (2.13 g, 9.60 mmol) was slowly added dropwise. As the reaction proceeded, the solids in the reaction system dissolved. After the reaction was complete, the reaction solution was added dropwise to an aqueous solution of sodium bicarbonate (1.21 g, 14.39 mmol) (150 mL). After stirring for 30 minutes, the mixture was separated. The organic phase was washed once with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness, yielding a yellow oily substance. This oily substance was then added... Methyl tert-butyl ether (300mL) Stirring to crystallize, yielding a pale yellowish-white solid 4 (25.11g, yield 82%, purity 93.05%). 1 H NMR(400MHz, DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.72(t,J=5.7Hz,1H),7.40–7.27(m,5H),7.21(t,J=5.8Hz,1H),5 .21(d,J=3.4Hz,1H),5.00(s,2H),4.96(dd,J=11.2,3.4Hz,1H),4.48(d,J=8.5Hz,1H),4.06–3.97(m,3H),3.87(d t,J=11.3,8.8Hz,1H),3.70(dt,J=10.7,5.6Hz,1H),3.45–3.36(m,1H),3.01(dq,J=15.7,6.7Hz,4H),2.10(s,3H) ,2.03(t,J=7.0Hz,2H),1.99(d,J=2.8Hz,3H),1.89(s,3H),1.77(s,3H),1.57–1.38(m,6H).HRMS(ESI)m / z:Calcd for C 30 H 43 N3O 12 [M+H] + :638.28,found638.29.

[0121] Example 3: Synthesis of Compound 5

[0122]

[0123] Intermediate 4 (10 g, 15.69 mmol) was dissolved in methanol (100 mL), and 10% palladium on carbon (1 g) and 1,1,2-trichloroethane (2.31 g, 17.26 mmol) were added. Hydrogen gas was introduced, and the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered through diatomaceous earth, and the reaction solution was evaporated to dryness to give a white foamy solid 5 (8.15 g, yield 96%).1 H NMR (400MHz, DMSO-d6+D2O) δ5.19(d,J=3.5Hz,1H),4.94(dd,J=11.2,3.4Hz,1H),4.47(d,J=8.5Hz,1H ),4.00(h,J=4.2Hz,3H),3.86(dd,J=11.2,8.4Hz,1H),3.70(dd,J=9.9,5.9Hz,1H),3.43–3.38(m,1H) ,3.15(s,8H),3.08(t,J=6.8Hz,2H),2.78–2.68(m,2H),2.08(s,3H),2.05(d,J=7.4Hz,2H),1.98(s,3 H),1.87(d,J=3.8Hz,3H),1.77(s,3H),1.65(p,J=7.0Hz,2H),1.52–1.39(m,4H).HRMS(ESI)m / z:Calcd for C 22 H 38 ClN3O 10 [M-HCl+H] + :504.22,found504.25.

[0124] Example 4: Synthesis of Compound 9

[0125]

[0126] Under a nitrogen atmosphere, intermediate 8 (10.88 g, 21.53 mmol), monobenzyl dodecanoate (7.97 g, 24.76 mmol), 1-hydroxybenzotriazole HOBt (4.36 g, 32.29 mmol), N,N-dimethylformamide (11 mL), and dichloromethane (110 mL) were added to the reaction flask and stirred until dissolved. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI (5.43 g, 27.34 mmol) was added in portions. After stirring for 30 minutes, N,N-diisopropylethylamine DIEA (8.34 g, 64.59 mmol) was added dropwise at 10 °C. The reaction was carried out at room temperature. After the reaction was completed, the reaction solution was evaporated to dryness, and water (110 mL) was added. After stirring for 30 minutes, the aqueous layer was poured off after standing. Ethyl acetate (110 mL) was added to dissolve the aqueous layer. The solution was washed successively with water (110 mL), 10% citric acid aqueous solution (110 mL), saturated sodium bicarbonate solution (55 mL), and saturated saline solution (55 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain oily substance 9 (18.22 g, quantitative, purity 92.39%). It was not purified and directly added to the next step. 1H NMR (400MHz, DMSO-d6) δ7.40–7.29(m,5H),6.86(s,1H),5.08(s,2H),3.54(q,J=7.4Hz,12H),2.44–2.31(m,8H),2.23(dt,J=3 8.3,7.5Hz,2H),2.03(t,J=7.3Hz,2H),1.52(d,J=7.0Hz,2H),1.39(d,J=1.4Hz,27H),1.24–1.20(m,12H).MS(ESI)m / z:Calcd for C 19 H 28 O4[M+H] + :808.51,found 808.52.

[0127] Example 5: Synthesis of Compound 10

[0128]

[0129] At room temperature, formic acid (182 mL) was added to oily substance 9 (18.22 g, 22.56 mmol), and the mixture was stirred overnight. After the reaction was complete, the reaction solution was evaporated to dryness, co-evaporated with toluene (20 mL * 2), and then ethyl acetate and n-hexane (1:1, 140 mL) were slowly added. A white solid precipitated, which was filtered, washed with ethyl acetate and n-hexane (1:1, 24 mL), and dried to give white solid 10 (11.57 g, yield 84%, purity 98.72%). 1 H NMR (400MHz, DMSO-d6) δ12.15(s,3H),7.43–7.27(m,5H),6.90(s,1H),5.07(s,2H),3.58–3.52(m,12H),2.41(t,J=6.4Hz,6H),2.3 3(t,J=7.4Hz,2H),2.03(t,J=7.3Hz,2H),1.52(q,J=7.1Hz,2H),1.42(t,J=7.1Hz,2H),1.22(d,J=7.2Hz,12H).MS(ESI)m / z:Calcd for C 19 H 28 O4[M+H] + :640.73, found 640.33.

[0130] Example 6: Synthesis of Compound 11

[0131]

[0132] Under a nitrogen atmosphere, intermediate 10 (1.2 g, 1.9 mmol), intermediate 5 (3.69 g, 7.3 mmol), 1-hydroxybenzotriazole (1.27 g, 9.4 mmol), N,N-dimethylformamide (2.4 mL), and dichloromethane (24 mL) were added to the reaction flask and stirred until dissolved. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.49 g, 7.5 mmol) was added in portions. After stirring for 30 minutes, N,N-diisopropylethylamine (2.42 g, 18.8 mmol) was added dropwise at 10 °C. The reaction was carried out at room temperature. After the reaction was completed, the reaction solution was evaporated to dryness, and water (20 mL) was added. After stirring for 30 minutes, the aqueous layer was poured off after standing. Ethyl acetate (40 mL) was added to dissolve the aqueous layer. The solution was washed successively with water (20 mL), 10% citric acid aqueous solution (40 mL), saturated sodium bicarbonate solution (20 mL), and saturated saline solution (20 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a yellow foamy solid. Finally, the solid was purified by silica gel column chromatography (elution with a gradient of 5% to 15% methanol / dichloromethane). Evaporation to dryness gave a white foamy solid 11 (3.39 g, yield 86.21%, purity 98.44%). 1 H NMR(400MHz, DMSO-d6+D2O)δ8.03–7.76(m,9H),7.43–7.30(m,5H),7.07(s,1H),5.22(d,J=3.4Hz,3H),5.08(s,2H),4. 96(dd,J=11.2,3.4Hz,3H),4.50(d,J=8.5Hz,3H),4.10–3.97(m,9H),3.90(dt,J=11.1,8.8Hz,3H),3.74–3.67(m,3H), 3.55(dd,J=11.4,5.0Hz,12H),3.43(dt,J=9.6,6.0Hz,3H),3.05(p,J=6.0Hz,12H),2.38–2.26(m,8H),2.11(s,9H),2. 06(q,J=5.9Hz,8H),2.00(s,9H),1.90(s,9H),1.80(s,9H),1.59–1.38(m,22H),1.26–1.18(m,12H).MS(ESI)m / z:Calcd for C 98 H 154 N 10 O 39 [M+H] + :2097.34,found 2097.04.

[0133] Example 7: Synthesis of Compound 12

[0134]

[0135] Intermediate 11 (10 g, 4.8 mmol) was dissolved in methanol (100 mL), 10% palladium on carbon (1 g) was added, hydrogen gas was introduced, and the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered through diatomaceous earth and the reaction solution was evaporated to dryness to obtain a white foamy solid 12 (9.19 g, yield 96%, purity 96.04%). 1 H NMR (400MHz, DMSO-d6) δ7.77(dd,J=37.8,7.0Hz,9H),6.98(s,1H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,3 H),4.48(d,J=8.4Hz,3H),4.02(q,J=3.9Hz,9H),3.87(dt,J=11.3,8.8Hz,3H),3.74–3.65(m,3H),3.53(d,J=7.6Hz, 12H),3.42–3.39(m,3H),3.03(p,J=6.1Hz,12H),2.27(t,J=6.4Hz,6H),2.17(t,J=7.4Hz,2H),2.10(s,9H),2.04(t, J=7.1Hz,8H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.54–1.39(m,22H),1.23(d,J=5.0Hz,12H).MS(ESI)m / z:Calcd for C 91 H 148 N 10 O 39 [M+H] + :2006.99,found2006.99.

[0136] The above compounds of formulas 8 to 12 were obtained through a total of 4 steps, with an overall yield of 69.51%.

[0137] Example 8: Synthesis of Compound 13

[0138] Under a nitrogen atmosphere, intermediate 12 (2.1 g, 1.1 mmol) was added to the reaction flask.

[0139] (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidine-3-ol (Formula 12-1) (0.53 g, 1.3 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (0.48 g, 1.3 mmol), N,N-dimethylformamide (2 mL), and dichloromethane (21 mL) were stirred until dissolved. After stirring for 30 minutes, N,N-diisopropylethylamine (0.42 g, 3.2 mmol) was added dropwise at 10 °C. After the addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete, dichloromethane (10 mL) was added for dilution. The mixture was washed with saturated sodium bicarbonate solution (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a yellow solid 13 (2.61 g). This solid was not purified and was directly added to the next step. 1 H NMR(600MHz,DMSO-d6)δ7.89–7.79(m,6H),7.74(t,J=5.7Hz,3H),7.36–7.26(m,4H ),7.20(ddd,J=12.5,8.5,3.2Hz,5H),6.99(d,J=3.2Hz,1H),6.88(td,J=8.3,3.4H z,4H),5.22(d,J=3.4Hz,3H),4.99–4.95(m,3H),4.49(d,J=8.4Hz,3H),4.40(q,J= 5.0Hz,1H),4.19–4.12(m,1H),4.07–3.99(m,J=4.4,3.5Hz,9H),3.88(dt,J=11.1,8 .8Hz,3H),3.74(s,6H),3.70(dd,J=10.8,4.9Hz,3H),3.54(dd,J=11.7,5.2Hz,12H ),3.41(dt,J=9.4,6.2Hz,3H),3.17(dd,J=9.0,5.1Hz,1H),3.04(p,J=6.6Hz,12H) ,2.28(t,J=6.4Hz,6H),2.21(t,J=7.5Hz,2H),2.11(s,9H),2.08(s,3H),2.05(t,J =7.3Hz,8H),2.00(s,9H),1.90(s,9H),1.78(s,9H),1.48(dq,J=31.5,7.3Hz,22H),

[0140] 1.23(d,J=18.6Hz,12H).MS(ESI)m / z:Calcd for C 117 H 175 N 11 O 42

[0141] [M+Na] + :2430.19,found 2430.19.[M / 2+Na] + :1226.59,found1226.59.

[0142] Example 9: Synthesis of Compound 14

[0143] Under a nitrogen atmosphere, intermediate 13 (2.61 g, 0.6 mmol), succinic anhydride (0.16 g, 1.6 mmol), triethylamine (0.47 g, 4.7 mmol), 4-dimethylaminopyridine (0.15 g, 1.2 mmol), and dichloromethane (15 mL) were added to a reaction flask, and the reaction was carried out at room temperature. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate (10 mL) and saturated brine (10 mL), the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give a yellowish-white solid 14 (2.18 g, two-step yield 81%). 1 H NMR (600MHz, DMSO-d6) δ7.87–7.78(m,6H),7.74(t,J=5.7Hz,3H),7.31(dq,J=15.4,7.3Hz ,4H),7.20(td,J=8.4,4.4Hz,5H),6.99(d,J=3.2Hz,1H),6.93–6.81(m,4H),5.37(t,J=4.7 Hz,1H),5.22(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,3H),4.49(d,J=8.4Hz,3H),4.20(t ,J=7.0Hz,1H),4.03(h,J=4.3Hz,9H),3.88(dt,J=11.2,8.8Hz,3H),3.82–3.76(m,1H),3.7 4(s,6H),3.72–3.63(m,3H),3.54(dd,J=11.8,5.2Hz,12H),3.41(dt,J=9.6,6.3Hz,3H),3. 26–3.20(m,1H),3.04(p,J=6.7Hz,12H),2.50–2.48(m,2H),2.28(t,J=6.4Hz,6H),2.24–2. 19(m,2H),2.11(s,9H),2.08(s,4H),2.05(t,J=7.3Hz,8H),2.00(s,9H),1.90(s,9H),1.78 (s,9H),1.48(ddt,J=37.9,14.1,6.7Hz,22H),1.23(d,J=20.5Hz,12H).MS(ESI)m / z:Calcd for C 121 H179 N 11 O 45 [M+Na] + :2530.21,found 2530.20.

[0144] In Example 4 of this invention, a compound of formula 8-1 with a benzyl protecting group was used. Compared with similar compounds with other protecting groups in the prior art, it showed excellent purification effect in the preparation of compound 14.

[0145] discuss

[0146] One improvement of this invention is that the hydrochloride form of Formula 5 compound is synthesized to replace other salt forms (e.g., trifluoroacetate) or non-salt forms of Formula 5 compound in the prior art for subsequent reactions. Compared to the trifluoroacetate or non-salt forms of Formula 5 compound, the hydrochloride form of Formula 5 compound simplifies the subsequent purification process and reduces the generation of impurities. pole Earth improved reaction yield and product purity. .

[0147] Specifically, the applicant provided the following comparative experimental data:

[0148]

[0149] In Example 2 of the present invention, after the reaction was completed, methyl tert-butyl ether was used for pulping and purification to obtain a high-yield and high-purity compound of Formula 4. Compared with the cumbersome column purification in the prior art, the purification steps were simplified, and a high-purity product was obtained at the same time.

[0150] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing a compound of formula 12, characterized in that, Includes the following steps: (1) Under the protection of an inert gas and in the presence of a condensing agent, compound 8 and compound 8-1 undergo a condensation reaction in the first solvent to obtain compound 9. (2) Under acidic conditions, compound 9 undergoes a hydrolysis reaction to give compound 10; (3) Under the protection of an inert gas and in the presence of a condensing agent, compound 10 and compound 5 undergo a condensation reaction in the first solvent to obtain compound 11. (4) In the second solvent, the compound of formula 11 undergoes a reduction reaction to obtain the compound of formula 12; Where n is an integer between 8 and 15; preferably, n is 10.

2. The preparation method according to claim 1, characterized in that, Step (1) includes one or more features selected from the following group: (a) The first solvent is selected from the group consisting of: C 1-6 Hydrocarbon solvents, amide solvents, or combinations thereof; preferably, dichloromethane, N,N-dimethylformamide, or combinations thereof; (b) The condensing agent is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), HBTU, DIPEA, CDI, DIPEA, or combinations thereof; (c) The molar ratio of the compound of formula 8 to the compound of formula 8-1 is 1:(1-1.5), preferably 1:(1-1.3), and more preferably 1:1.15; and / or (d) The mass-to-volume ratio (g / mL) of the compound of Formula 8 to the first solvent is 1:(10-20), preferably 1:(10-15).

3. The preparation method according to claim 1, characterized in that, Step (3) includes one or more of the following features: (3-1) The first solvent is selected from the group consisting of: C 1-6 Hydrocarbon solvents, amide solvents, or combinations thereof; preferably, dichloromethane, N,N-dimethylformamide, or combinations thereof; (3-2) The condensing agent is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), HBTU, DIPEA, CDI, DIPEA, or a combination thereof; (3-3) The molar ratio of compound 10 to compound 5 is 1:(3-6); preferably 1:(3-4); and / or The mass-to-volume ratio (g / mL) of compound 10 of formula (3-4) to the first solvent is 1:(15-30); preferably 1:(20-25).

4. The preparation method according to claim 1, characterized in that, In step (3), the preparation method of the compound of formula 5 is as follows: (3-a) In the presence of an inert gas, in a third solvent, and in the presence of a hydrolyzing agent, compound 3-1 and compound 3-2 undergo a ring-opening reaction. The product is purified by slurrying with a C2-6 ether solvent to obtain compound 4. (3-b) In the fourth solvent, compound 4 undergoes a salt-forming reaction to give compound 5; The fourth solvent is selected from C1-C6 alcohol solvents.

5. The preparation method according to claim 4, characterized in that, Step (3-b) includes the following steps: In the fourth solvent, under a hydrogen atmosphere and in the presence of palladium on carbon, compound 4 undergoes reduction and salt formation reactions, and after filtration and concentration, compound 5 is obtained.

6. The preparation method according to claim 4, characterized in that, In step (3-a), the molar ratio of the compound of formula 3-1 to the compound of formula 3-2 is 1:(1±0.3); preferably 1:1; and / or The molar ratio of the compound of formula 3-1 to the hydrolysant is (5-10):1; preferably (5-7):

1.

7. The preparation method according to claim 1, characterized in that, Step (4) includes one or more of the following features: (i) The second solvent is selected from the group consisting of C1-C6 alcohol solvents, C1-C6 hydrocarbon solvents, or combinations thereof; preferably methanol, dichloromethane, or combinations thereof; (ii) The reduction reaction is carried out in a hydrogen atmosphere in the presence of 10% palladium on carbon.

8. The preparation method according to claim 1, characterized in that, In step (2), the acid is formic acid, trifluoroacetic acid, acetic acid, hydrochloric acid, sulfuric acid, or a combination thereof; preferably formic acid, trifluoroacetic acid, or a combination thereof.

9. The preparation method according to claim 1, characterized in that, Step (2) also includes a purification step: after the reaction is complete, the compound of formula 10 is recrystallized and purified in a fifth solvent, wherein the fifth solvent is selected from the group consisting of: C 1-6 Ester solvents, C 1-6 Hydrocarbon solvents, or combinations thereof; preferably ethyl acetate / n-hexane = 1:

1.

10. Use of the compound of formula 12, characterized in that, The preparation of a liver-targeting tri-antennae GalNAc linker includes the following steps: (M1) In a first solvent, in the presence of a condensing agent, compound 12 reacts with compound 12-1 to give compound 13. (M2) The reaction of compound 13 with succinic anhydride of compound 13-1 yields the tri-antennae GalNAc linker shown in formula 14; Where n is an integer between 8 and 15; preferably, n is 10. The compound of formula 12 is prepared by the method described in claim 1.

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