CN116444589B

CN116444589B - GalNAc compounds, their thio-oligonucleotide conjugates and coupling methods

Info

Publication number: CN116444589B
Application number: CN202310705604.5A
Authority: CN
Inventors: 宋更申; 王杰; 黄泽傲; 谢豪; 马儒杰
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: HANGZHOU TIANLONG PHARMACEUTICAL CO Ltd
Priority date: 2023-06-15
Filing date: 2023-06-15
Publication date: 2023-09-19
Anticipated expiration: 2043-06-15
Also published as: CN116444589A

Abstract

The present invention provides a novel GalNAc compound, which is a compound of formula (I), and a thio oligonucleotide conjugate and a coupling method thereof. The compound of the formula (I) can be directly coupled with the thioated oligonucleotide to obtain the conjugate by adopting a liquid phase synthesis method, and the method has mild reaction conditions and high yield.

Description

GalNAc compounds, their thio-oligonucleotide conjugates and coupling methods

Technical Field

The invention belongs to the field of biological medicine, and relates to a novel GalNAc compound, a thio oligonucleotide conjugate thereof and a coupling method.

Background

N-acetylgalactosamine (GalNAc) is a ligand that binds to hepatic surface asialoglycoprotein receptor (ASGPR). The asialoglycoprotein receptor is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, high affinity ligand GalNAc of asialoglycoprotein receptor has been used as targeting molecule in nucleic acid medicineCertain advances have been made in the liver targeted delivery of (c). For example, alnicom corporation (Alnylam pharmaceuticals, inc.) reported that siRNA based on GalNAc conjugation technology exerted gene silencing activity in mice (j. Am. chem. Soc. 2014, 136, 16958). Conjugates of GalNAc and siRNA are reported herein to exhibit good delivery activity in both in vivo and in vitro assays. Single dose ED by in vivo experiments in subcutaneously administered mice ₅₀ Determined to be 1 mg/kg, the single injection dose was less than 1 mL. In long-term dosing trials, stable interfering activity of up to 9 months was obtained once weekly subcutaneous injections.

Currently, conventional methods for conjugation of GalNAc to oligonucleotides are solid phase synthesis methods. One method is to attach a GalNAc compound to a CPG (Controlled Pore Glass, controllable microporous glass) column and achieve attachment of the GalNAc compound to the oligonucleotide by solid phase synthesis. The other method is to prepare the GalNAc compound into phosphoramidite monomer first and realize the connection of the GalNAc compound and any position of the oligonucleotide through solid phase synthesis.

The conventional method has the following defects: 1) The coupling efficiency of solid phase synthesis may be reduced due to the larger steric hindrance of the GalNAc compound, and even the coupling is difficult, so that the overall yield is greatly reduced. 2) The GalNAc phosphoramidite monomer has long synthetic route and great difficulty.

Current solid phase methods have certain limitations on the groups that are reacted, typically linking GalNAc compounds to the oxygen atom of the phosphodiester bond in the nucleotide, and therefore the type of conjugation is relatively limited. For a thioated oligonucleotide, if a GalNAc compound is prepared as a corresponding phosphoramidite monomer according to a conventional solid phase synthesis method, and the GalNAc compound is required to be linked to a phosphoramidite group through a thioether bond, it is difficult to perform with a conventional method for the following reasons:

1) The preparation of phosphoramidite monomers by linking GalNAc compounds to phosphoramidite groups via thioether linkages has not been reported for similar compounds to date, and it is presumed that this technique is difficult to implement.

2) Although some simple synthetic-like methods are described in the related patent and literature, such as US5218088 mentioning that the methyl sulfide group is attached to the phosphoryl group, WO2021178885 mentioning that the 3 'or 5' oxygen atom is replaced by a sulfur atom to be attached to the phosphoryl group, toshiki, tanaka et al mentioning that the phenylmercapto group is attached to the phosphoryl group (Tetrahedron Letters, 1986, 27 (2): 199-202.), the above methods involve small molecules of very simple chemical structure and stable chemical properties.

3) The nucleotide structure is complex, contains groups such as base, ribose (or deoxyribose), phosphate and the like, has huge chemical properties different from simple small molecules, and has not been reported about conjugation of GalNAc compounds with oligonucleotides through thioether bonds.

Thus, there remains a need in the art to develop methods of preparing conjugates with thio-oligonucleotides.

Disclosure of Invention

In order to overcome one or more of the problems of the prior art described above, the present invention devised and synthesized a series of novel GalNAc compounds which can be efficiently conjugated with a thio oligonucleotide by a liquid phase synthesis method.

In one aspect, the invention provides a compound of formula (I):

wherein,,

X ₁ is- (CH) ₂ ) _a -or- (CH) ₂ CH ₂ O) _a CH ₂ -, a is an integer from 1 to 5;

X ₂ is- (CH) ₂ ) _b -, b is an integer from 1 to 6;

X ₃ is-NHC (O) -or-C (O) NH-;

X ₄ is- (CH) ₂ ) _c -, c is an integer from 1 to 5;

Y ₁ 0 or 1;

Y ₂ 0, 1 or 2;

Y ₃ 1, 2 or 3;

r is Cl、Br、I、Or->。

For example, X ₁ Is- (CH) ₂ ) ₄ -、-(CH ₂ ) ₂ -or- (CH) ₂ CH ₂ O) ₃ CH ₂ -。

For example, X ₂ Is- (CH) ₂ ) ₃ -、-(CH ₂ ) -or- (CH) ₂ ) ₅ -。

For example, X ₄ Is- (CH) ₂ ) -or- (CH) ₂ ) ₂ -。

For example, the compound of formula (I) is a compound YK-GAL-001, YK-GAL-002, YK-GAL-003, YK-GAL-004, YK-GAL-005, YK-GAL-006, YK-GAL-007, YK-GAL-008, YK-GAL-009, or YK-GAL-010 having the following structure:

，

。

preferably, the compound of formula (I) or a pharmaceutically acceptable salt thereof is capable of binding to an asialoglycoprotein receptor (ASGPR).

In another aspect, the present invention provides a method of coupling a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, to a thio oligonucleotide comprising the steps of:

1) Reacting a thioate oligonucleotide with a compound of formula (I) or a pharmaceutically acceptable salt thereof in an organic solvent;

2) Concentrating the solvent after the reaction is finished to obtain a residue; and

3) Purifying the residue to obtain the product.

The organic solvent is, for example, a polar aprotic solvent, preferably one or more of dimethyl sulfoxide, N-dimethylformamide and tetrahydrofuran, more preferably dimethyl sulfoxide.

The thio oligonucleotide is, for example, an oligonucleotide in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom.

The molar ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the thioated oligonucleotide is, for example, from about 5:1 to about 30:1, preferably from about 7.5:1 to about 20:1, more preferably about 10:1.

The reaction temperature of step 1) is, for example, about 20 to 60 ℃, preferably about 25 to 55 ℃, more preferably about 50 ℃.

The reaction time of step 1) is, for example, about 1 to 15 hours, preferably about 5 to 10 hours, more preferably about 8 hours.

The thio oligonucleotide is, for example, a small interfering nucleotide, DNA, microrna, small activating RNA, small guide RNA, transfer RNA, antisense nucleotide or aptamer, preferably an antisense nucleotide or small interfering nucleotide.

In another aspect, the invention provides a conjugate comprising a thio oligonucleotide and a GalNAc moiety, wherein the GalNAc moiety is

，

X ₁ 、X ₂ 、X ₃ 、X ₄ 、Y ₁ 、Y ₂ 、Y ₃ As defined above for the compounds of formula (I).

Preferably, the thio oligonucleotide modulates the expression of a target gene.

For example, the thioate oligonucleotide and the GalNAc moiety are linked by a bond or a cleavable linker.

For example, the thioate oligonucleotide and the GalNAc moiety are linked by a phosphorothioate linkage.

For example, the conjugate has the following structural formula:

，

Another aspect of the invention provides a conjugate as described above for use as a medicament.

In another aspect the invention provides the use of a conjugate as described above in the manufacture of a medicament.

In another aspect, the invention provides a pharmaceutical composition comprising the conjugate described above and at least one pharmaceutically acceptable excipient.

Detailed Description

The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The invention includes additional embodiments resulting from such combinations.

All publications and patents mentioned in this specification are herein incorporated by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with a use or term used in the present invention, the use or term of the present invention controls.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.

Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative properties such as dosages set forth in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range.

As used in this application, the use of the terms "comprising," "including," or "containing," and the like, are intended to cover an element listed as a means that it is followed by such term and equivalents thereof, without excluding unrecited elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "pharmaceutically acceptable" in the present application means: the compound or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.

The term "pharmaceutically acceptable salt" refers to the phase of a compound of the inventionNon-toxic, inorganic or organic acid addition salts. See, for example, s.m. Berge et al, "Pharmaceutical Salts",J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, and the like. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid may be used to form pharmaceutically acceptable salts with the compounds of formula (I).

The thioated oligonucleotides of the present application include single stranded oligonucleotides (e.g., antisense nucleotides, ASO for short) and double stranded oligonucleotides (e.g., small interfering nucleotides, siRNA for short).

In one embodiment, the thioate oligonucleotide is selected from the group consisting of small interfering nucleotides, DNA, micrornas (mirnas), small activating RNAs (sarnas), small guide RNAs (sgrnas), transfer RNAs (trnas), antisense nucleotides or aptamers, preferably the thioate oligonucleotide is an antisense nucleotide or a small interfering nucleotide.

Oligonucleotides in the present application include natural oligonucleotides and chemically modified oligonucleotides. Chemical modifications herein include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. Chemical modification of oligonucleotides does not include cases where there is only a difference in nucleobase sequence. Natural herein refers to the situation where naturally occurring RNA or DNA corresponds.

Natural oligonucleotides are difficult to enter cells, are easily degraded by intracellular nucleases, and have poor effects. By chemical modification of the oligonucleotide, its properties can be improved and its bioavailability increased. Among the many modified oligonucleotides, the most representative are thio-oligonucleotides in which one of the thio-modes is the substitution of one of the non-bridging oxygen atoms in the phosphodiester bond by a sulfur atom, e.g 。

The thio oligonucleotide may be commercially available or may be prepared by conventional solid phase synthesis methods. As vulcanizing agent, hydrogenated Huang Yuansu can be used. For example, thio oligonucleotides can be synthesized with reference to CN1479745A and CN113150041 a.

The conjugation of the compound of formula (I) to the thioate oligonucleotide according to the application is obtained by a hydrogen leaving reaction of the "R" group of the compound of formula (I) with SH on the phosphorothioate in the thioate oligonucleotide.

The prior studies indicate that the reaction yield of a compound with a thiol in a phosphorothioate is greatly affected by the structure of the compound and the group attached to the thiol in the phosphorothioate (Bioorganic Chemistry, 2009, 37 (4): 101-105; advanced synthesis & analysis, 2018, 360 (10): 1913-1918). Although the oligonucleotide has very large molecular weight, complex three-dimensional structure and complex GalNAc compound structure, the application couples the designed GalNAc compound with the thio oligonucleotide, and the yield is more than 65 percent and up to more than 85 percent.

In one aspect, the application provides a compound of formula (I):

wherein,,

X ₁ is- (CH) ₂ ) _a -or- (CH) ₂ CH ₂ O) _a CH ₂ -a is 1, 2, 3, 4 or 5;

X ₂ is- (CH) ₂ ) _b -b is 1, 2, 3, 4, 5 or 6;

X ₃ is-NHC (O) -or-C (O) NH-;

X ₄ Is- (CH) ₂ ) _c -c is 1, 2, 3, 4 or 5;

Y ₁ 0 or 1;

Y ₂ 0, 1 or 2;

Y ₃ 1, 2 or 3, preferably 3;

r is Cl, br, I,Or->。

For example, X ₂ Is- (CH) ₂ ) ₃ -、-(CH ₂ ) -or- (CH) ₂ ) ₅ -。

For example, X ₄ Is- (CH) ₂ ) -or- (CH) ₂ ) ₂ -。

，

。

3) Purifying the residue to obtain the product.

The thio oligonucleotide may be commercially available or prepared by solid phase chemical synthesis methods.

In a preferred embodiment, the thio oligonucleotide comprises 7-30 nucleotides. Conjugates made by such thio-oligonucleotides would be of greater therapeutic value.

In another aspect, the invention provides a conjugate comprising a thioate oligonucleotide and a GalNAc moiety, wherein the GalNAc moiety is a compound of formula (I) above minus an "R" group, i.e

Wherein

X ₁ 、X ₂ 、X ₃ 、X ₄ 、Y ₁ 、Y ₂ 、Y ₃ As defined above.

Preferably, the thio oligonucleotide modulates the expression of a target gene.

For example, the thioate oligonucleotide and the GalNAc moiety are linked by a bond or a cleavable linker. The linkages herein may include, but are not limited to, phosphate linkages and phosphorothioate linkages.

The cleavable linker as used in the present application refers to a linker which is cleaved by intracellular metabolism after internalization, for example by hydrolysis, reduction or enzymatic reaction. Suitable linkers include, but are not limited to, acid labile linkers, hydrolytically labile linkers, enzymatically cleavable linkers, and reduction labile linkers. The acid labile linker may be referred to as an acid labile linker in ADC drugs (Mylotarg, besponsas, trodelvys).

For example, the conjugate has the following structural formula:

wherein

X ₁ 、X ₂ 、X ₃ 、X ₄ 、Y ₁ 、Y ₂ 、Y ₃ As defined above.

Another aspect of the application provides a conjugate as described above for use as a medicament.

In another aspect the application provides the use of a conjugate as described above in the manufacture of a medicament.

In another aspect, the application provides a pharmaceutical composition comprising the conjugate described above and at least one pharmaceutically acceptable excipient.

Excipients include, but are not limited to, dispersing media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening agents, emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington's The Science and Practice of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).

The beneficial effects of the application are at least as follows:

1. a brand new GalNAc compound is designed, and compared with the GalNAc compound in the prior art, the chemical structure of the GalNAc compound is significantly different.

2. The GalNAc compound designed by the application has high synthesis yield, wherein the final step yield of some representative compounds can reach more than 70%, and the total yield can reach 65%.

3. The synthesized GalNAc compound can be efficiently coupled with a thio oligonucleotide. For example, YK-GAL-004, YK-GAL-009 and YK-GAL-010, coupled with the thio oligonucleotide in yields of 88.1%, 87.8% and 85.0%, respectively.

4. According to the application, the thioated oligonucleotide is synthesized by a solid phase method, and the GalNAc compound is coupled with the thioated oligonucleotide by a liquid phase method, so that the total yield of the synthesized GalNAc thioated oligonucleotide conjugate can reach more than 60%, and is improved by more than 10 times compared with the total yield of the solid phase method in the prior art.

1) The prior art conventional methods for preparing GalNAc thioate oligonucleotide conjugates, synthesizing thioate oligonucleotides and coupling GalNAc compounds to thioate oligonucleotides all use solid phase methods. This method is limited by the structure of the thioate oligonucleotide and the coupling site, and yields are low when the GalNAc compound is sterically hindered.

2) According to the application, the 20-base thioated oligonucleotide is synthesized by a solid phase method, and then the GalNAc compound is coupled with the thioated oligonucleotide by a liquid phase method, so that the GalNAc compound is not required to be prepared into a corresponding phosphoramidite monomer, the GalNAc compound can be directly coupled with the thioated oligonucleotide, the total yield of the synthesized GalNAc thioated oligonucleotide conjugate can reach more than 60%, and can be improved by more than 10 times compared with the conventional solid phase method.

Examples

The application is further described below with reference to examples. The present application is not limited to the following examples. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present application fall within the protection scope of the present application. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. In the specific examples of the present application, all the raw materials used are commercially available. All temperatures are given in degrees celsius unless otherwise indicated. The technical features of the various embodiments of the present application may be combined with each other as long as they do not collide with each other.

Example 1: synthesis of GalNAc compound

The following abbreviations represent the following reagents, respectively: DMF: n, N-dimethylformamide; THF: tetrahydrofuran; HBTU: o-benzotriazol-tetramethylurea hexafluorophosphate; DIEA: n, N-diisopropylethylamine; DMSO: dimethyl sulfoxide; TMSOTF: trisilyl triflate; HOBt: 1-hydroxybenzotriazole; t (T) ₃ P: 1-propylphosphoric acid cyclic anhydride

Synthesis of YK-GAL-001

The synthetic route is as follows:

step 1: synthesis of G1-3

G1-1 (53.46G, 0.20 mol) was dissolved in N, N-dimethylformamide (750 mL), O-benzotriazol-tetramethylurea hexafluorophosphate (83.44G, 0.22 mol) and N, N-diisopropylethylamine (64.63G, 0.50 mol) were added, and stirred at 25℃for 30 minutes. G1-2 (69.70G, 0.40 mol) was added thereto and stirred at 25℃for 15 hours. Ethyl acetate was added to the reaction solution, and the mixture was washed 3 times with a saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 67.63. 67.63 g as a yellow oil in 79.8% yield. MS 324.3 [ M-Boc+H] ⁺ 。

Step 2: synthesis of G1-4

G1-3 (67.60G, 0.16 mol) was dissolved in methylene chloride (300 mL), trifluoroacetic acid (300 mL, 4.05 mol) was added, and 1 h was stirred at 25 ℃. The reaction mixture was concentrated under reduced pressure at 45-50℃to remove the solvent, and a yellow oil was obtained as 66.65% g in 99.0% yield. MS 324.2 [ M+H ] ] ⁺ 。

Step 3: synthesis of G1-6

G1-5 (50.00G, 0.13 mol) was added to dichloroethane (500 mL) and stirred at 25℃for 30 min. TMSOTF (44.45 g, 0.20 mol) was added and stirred at 25℃for 15h. The reaction was slowly poured into a mixture of sodium bicarbonate (33.60 g, 0.40 mol) and ice water (1L), stirring 1 h. Extracting with dichloromethane for 3 times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure at 40-45deg.CThe solvent was removed to give 41.85 g as a pale yellow oil in 98.9% yield. MS 330.2 [ M+H ]] ⁺ 。

Step 4: synthesis of G1-8

G1-6 (41.05G, 0.12 mol) and G1-7 (37.50G, 0.18 mol) were added to dichloroethane (500 mL), dissolved with stirring, 3A molecular sieves (48.20G) were added and 1 h was stirred at 25 ℃. TMSOTF (13.33, g, 0.06 mol) was added and the reaction stirred at 25℃for 15h. The reaction was filtered through celite, and the filtrate was slowly poured into a mixture of sodium bicarbonate (10.08 g, 0.12 mol) and ice water (1L) and stirred 1 h. The mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate, concentrated at 40-45℃under reduced pressure to remove the solvent, affording 65.66 g as a yellow oil in 98.0% yield. ¹ HNMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 9.2 Hz, 1H), 7.37-7.32 (m, 5H), 5.21 (d, J = 3.3 Hz, 1H), 4.99-4.92 (m, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.09-4.02 (m, 5H), 3.91-3.84 (m, 1H), 3.74-3.68 (m, 1H), 3.45-3.39 (m, 1H), 2.36 (t, J = 7.2 Hz, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.74 (s, 3H), 1.58-1.47 (m, 4H)。 MS: 560.4 [M + Na] ⁺ 。

Step 5: synthesis of G1-9

G1-8 (65.00G, 0.12 mol), ammonium formate (26.49G, 0.42 mol) and palladium on charcoal (6.50G) were added to ethanol (500 mL), heated, and refluxed for 2 hours. Palladium on charcoal was removed by filtration, and the filtrate was concentrated under reduced pressure at 40-45 ℃ to remove the solvent, affording 53.21 g as a colorless oil in 98.4% yield. ¹ HNMR (400 MHz, DMSO-d6) δ 7.84 (d, J = 9.2 Hz, 1H), 5.21 (d, J = 3.3 Hz, 1H), 4.98-4.94 (m, 1H), 4.49 (d, J = 8.5 Hz, 1H), 4.04-3.99 (m, 5H), 3.90-3.84 (m, 1H), 3.44-3.40 (m, 1H), 2.16-2.12 (m, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.88 (s, 3H), 1.77 (s, 3H), 1.52-1.44 (m, 4H)。MS: 448.2 [M + H] ⁺ 。

Step 6: synthesis of G1-10

G1-9 (20.00G, 44.7 mmol) was dissolved in N, N-dimethylformamide (240 mL), and O-benzotriazol-tetramethylurea hexafluorophosphate (18.66G, 49.2 mmol), 1-hydroxybenzotriazole (6.65G, 49.2 mmol) and N, N-diisopropylethylamine (20.68G, 0.16 were addedmol), stirred for 30 min, and finally G1-4 (22.53G, 53.6 mmol) was added, stirred at 25℃for 24. 24h. The reaction solution was concentrated under reduced pressure at 55-60℃to remove the solvent, dissolved in methylene chloride, washed with saturated sodium hydrogencarbonate solution and water, respectively, and the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45-50℃to remove the solvent, to give a yellow oily substance. Column chromatography purification (MeOH/DCM) gave 25.55. 25.55 g as a brown oil in 76.0% yield. MS 753.4 [ M+H ]] ⁺ 。

Step 7: synthesis of G1-11

G1-10 (25.00G, 33.2 mmol), ammonium formate (14.75G, 0.23 mol) and palladium on charcoal (6.25G) were added to ethanol (1L), heated, and refluxed for 1 hour. Palladium on charcoal was removed by filtration, and the filtrate was concentrated under reduced pressure at 40-45 ℃ to remove the solvent, affording 18.25. 18.25 g as a white oil in 89.0% yield. MS 619.3 [ M+H ]] ⁺ 。

Step 8: synthesis of YK-GAL-001

G1-12 (0.50G, 3.6 mmol) was dissolved in ethyl acetate (25 mL), 1-propylphosphoric acid cyclic anhydride (1.52G, 4.8 mmol) and N, N-diisopropylethylamine (1.87 mL,12.0 mmol) were added, stirred for 5min, G1-11 (1.49G, 2.4 mmol) was added, and stirred at 25℃for 24h. Ethyl acetate was added to the reaction solution, which was washed with water for 3 times, and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 1.33. 1.33 g as an oil, yield 74.7%. MS 739.0 [ M+H ] ] ⁺ 。

Synthesis of YK-GAL-002

The synthetic route is as follows:

g1-9 (600 mg, 1.3 mmol) was dissolved in N, N-dimethylformamide (12 mL), O-benzotriazol-tetramethylurea hexafluorophosphate (552 mg, 1.5 mmol) and N, N-diisopropylethylamine (698 mg,5.4 mmol) were added, stirred at room temperature for 5min, G2-1 (405 mg, 2.0 mmol) was added, and stirred at 25℃for 24h. Ethyl acetate was added to the reaction solution, the mixture was washed 3 times with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give a pale yellow oily substance629 mg, yield 86.0%. MS 554.3 [ M+H ]] ⁺ 。

Synthesis of YK-GAL-003

The synthetic route is as follows:

step 1: synthesis of G3-1

G1-9 (1.0G, 2.2 mmol) was dissolved in N, N-dimethylformamide (20 mL), O-benzotriazol-tetramethylurea hexafluorophosphate (0.92G, 2.4 mmol) and N, N-diisopropylethylamine (0.72G, 5.6 mmol) were added, stirred at room temperature for 5min, and G1-2 (0.57G, 3.3 mmol) was added thereto and stirred at 25℃for 20h. Ethyl acetate was added to the reaction solution, which was washed 3 times with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 1.24. 1.24 g as a yellow oil, yield 91.9%. MS 604.3 [ M+H ] ] ⁺ 。

Step 2: synthesis of G3-2

G3-1 (1.24G, 2.1 mmol) was dissolved in dichloromethane (17 mL), trifluoroacetic acid (4.27 mL, 56.89 mmol) was added and stirred at 25℃for 0.5h. The reaction mixture was concentrated under reduced pressure at 45-50℃to remove the solvent, whereby 1.21. 1.21 g as a yellow oil was obtained in 98.4% yield. MS 504.2 [ M+H ]] ⁺ 。

Step 3: synthesis of YK-GAL-003

G1-12 (0.25G, 1.8 mmol) was dissolved in ethyl acetate (12 mL), 1-propylphosphoric acid cyclic anhydride (0.75G, 2.4 mmol) and N, N-diisopropylethylamine (0.76G, 5.9 mmol) were added, stirred at room temperature for 5min, and finally G3-2 (0.59G, 1.2 mmol) was added and stirred at 25℃for 20h. Ethyl acetate was added to the reaction solution, which was washed with water for 3 times, and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 0.59. 0.59 g as a yellow oil, with a yield of 80.8%. MS 624.3 [ M+H ]] ⁺ 。

Synthesis of YK-GAL-004

The synthetic route is as follows:

step 1: synthesis of G4-2

G4-1 (10.0G, 0.11 mol) was dissolved in tetrahydrofuran/water (370 mL, 1:1), to which was added potassium carbonate (37.96G, 0.27 mol), stirred to dissolve, a tetrahydrofuran solution (15 mL) of di-tert-butyl dicarbonate (35.93G, 0.16 mol) was slowly added dropwise, and stirred at 25℃for 15h. The mixture was separated, the aqueous phase was extracted 3 times with tetrahydrofuran and the organic phases were combined. The organic phase was washed with water and saturated sodium chloride solution, respectively, and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 19.2. 19.2 g as a white solid in 91.4% yield. MS 214.2 [ M+Na ] ⁺ 。

Step 2: synthesis of G4-4

G4-2 (19.12G, 0.10 mol) was dissolved in DMSO (55 mL), 5M sodium hydroxide solution (2.2 mL) was added thereto, stirred at 25℃for 2 hours, and G4-3 (28.20G, 0.22 mol) was added thereto, and stirred at 25℃for 15 hours. The reaction solution was added to ice water of 8 times by weight, extracted 3 times with methylene chloride, and the organic phases were combined and washed with water and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give 24.58g of a white oil. The yield thereof was found to be 54.9%. MS 414.3 [ M-56 (tert-butyl) +Na] ⁺ 。

Step 3: synthesis of G4-5

G4-4 (7.10G, 15.9 mol) was dissolved in methylene chloride (28 mL), and a solution of 4M hydrochloric acid/1, 4-dioxane (110 mL) was added thereto and stirred at 25℃for 20 hours. The reaction mixture was concentrated under reduced pressure at 45-50℃to remove the solvent, whereby 3.53. 3.53 g as a yellow oil was obtained in 94.6% yield.

Step 4: synthesis of G4-6

G4-5 (1.87G, 7.9 mmol) was dissolved in 10% sodium carbonate solution (110 mL), cooled to 0-5℃and a tetrahydrofuran solution (12.3 mL) of Cbz-Cl (1.14 mL) was added dropwise thereto and stirred at 25℃for 4 hours. The reaction solution was adjusted to pH 2-3 with 10% citric acid solution, extracted 3 times with dichloromethane, and the organic phases were combined and washed with water and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50deg.C to give a yellow oil 1.75 g, yield 59.5%. MS 304.2 [ M-2CO ₂ + Na] ⁺ 。

Step 5: synthesis of G4-7

Starting with G4-6 (1.75G, 4.7 mmol) and G1-2 (3.28G, 18.8 mmol), G1-3 was synthesized in the same manner as described above to give 2.56: 2.56G as a yellow oil in 79.3% yield. MS 582.1 [ M-Boc+H] ⁺ 。

Step 6: synthesis of G4-8

Using G4-7 (2.56G, 3.8 mmol) as a starting material, the procedure was followed to synthesize G1-4, giving 2.46G of a yellow oil in 96.9% yield. MS 504.4 [ M+Na ]] ⁺ 。

Step 7: synthesis of G4-9

Using G1-9 (1.34G, 3.0 mmol) and G4-8 (2.46G, 3.6 mmol) as raw materials, according to the method for synthesizing G1-10, a white sticky material 3.14G was obtained, yield 78.1%. MS 671.0 [ M+2H ]] ²⁺ 。

Step 8: synthesis of G4-10

Using G4-9 (3.14G, 2.3 mmol) as a starting material, the procedure was followed to synthesize G1-11 to give a white dope, 2.45G, in 88.2% yield. MS 604.1 [ M+2 ] 2H] ²⁺ 。

Step 9: synthesis of YK-GAL-004

Using G4-10 (2.45G, 2.0 mmol) and G1-12 (0.42G, 3.0 mmol) as raw materials, according to the method for synthesizing YK-GAL-001, a white solid was obtained in 2.01G, yield 75.5%. MS 664.2 [ M+H ]] ²⁺ 。

Synthesis of YK-GAL-005

The synthetic route is as follows:

step 1: synthesis of G5-2

Starting with G5-1 (3.25G, 6.9 mmol) and G1-2 (4.56G, 26.2 mmol), the procedure was followed to give G1-3 as a crude yellow oil which was purified on a silica gel column (dichloromethane/methanol) to give a pale yellow oil, 6.12G, in 94.4% yield. ¹ HNMR (400MHz, DMSO-d6) δ 7.79 (t, J = 5.5 Hz, 1H), 7.37-7.28 (m, 5H), 7.64 (s, 3H), 4.98 (s, 2H), 3.54 (t, J = 6.2 Hz, 6H), 3.48 (s, 6H), 3.05-3.00 (m, 6H), 2.93-2.88 (m, 6H), 2.27 (t, J = 6.2 Hz, 6H), 1.52-1.41 (m, 6H), 1.36 (s, 30H)。MS: 804.5 [M – 100（Boc）+ H] ⁺ 。

Step 2: synthesis of G5-3

Starting with G5-2 (5.29G, 5.6 mmol), G1-4 was synthesized to yield a yellow oil, 5.23G, in 100% yield. ¹ H-NMR (400 HZ; DMSO): δ 8.10 (t, J = 5.10 HZ, 3H), 8.05 (t, J = 5.60 HZ, 3H), 7.32-7.33 (m, 3H), 4.98 (s, 2H), 3.97-4.02 (m, 3H), 3.64 (t, J = 5.75 HZ, 3H), 3.57 (t, J = 6.20 HZ, 6H), 3.49 (s, 5H), 3.45 (s, 3H), 2.67 (s, 1H), 2.37 (t, J = 6.17 HZ, 3H), 2.31 (t, J = 6.21 HZ, 5H), 1.65-1.73 (m, 9H), 1.14 (t, J = 7.18 HZ, 4H)。MS: 640.8 [M + H] ⁺ 。

Step 3: synthesis of G5-4

G1-9 (9.66G, 21.6 mmol) and G5-3 (5.00G, 5.4 mmol) were used as starting materials and the procedure for G1-10 was followed to give crude products. The crude product was purified by column on silica gel (dichloromethane/methanol) to give 7.74. 7.74 g as a pale yellow oil in 74.7% yield. MS 1928.9 [ M+H ]] ⁺ 。

Step 4: synthesis of G5-5

G5-4 (7.74, G, 4.0 mmol) was used as a starting material, and the crude product was obtained by the method for synthesizing G1-11. The crude product was purified by Buchi Pure C-815 Flash 300 g C18 column in reverse phase to give 5.98: 5.98 g as an off-white solid in 83.0% yield. ¹ HNMR (400 MHz, DMSO-d6) δ 7.95-7.60 (m, 11H), 5.21 (d, J = 3.4 Hz, 1H), 4.98-4.95 (m, 1H), 4.74-4.68 (m, 1H), 4.48 (d, J = 8.4 Hz, 1H), 4.39-4.32 (m, 1H), 4.28-4.20 (m, 2H), 4.15-4.08 (m, 2H), 3.99-3.93 (m, 2H), 3.90-3.83 (m, 2H), 3.70-3.63 (m, 8H), 3.55 (t, J = 6.2 Hz, 9H), 3.16 (s, 7H), 3.06-3.00 (m, 14H)。MS: 1795.5 [M + H] ⁺ 。

Step 5: synthesis of YK-GAL-005

Starting from G5-5 (1.00G, 0.6 mmol) and G1-12 (0.13G, 0.9 mmol) according to the following formulaThe procedure for the synthesis of YK-GAL-001 was followed to give 0.78. 0.78 g as a white solid in 72.9% yield. MS 958.2 [ M+2H ]] ²⁺ 。

Synthesis of YK-GAL-006

The synthetic route is as follows:

step 1: synthesis of G6-2

G5-1 (2.36G, 5.0 mmol) and G6-1 (3.34G, 16.5 mmol) were dissolved in N, N-dimethylformamide (30 mL), O-benzotriazole-tetramethylurea hexafluorophosphate (6.83G, 18.0 mmol) and 1-hydroxybenzotriazole (41 mg, 0.3 mmol) were added, N-diisopropylethylamine (2.13G, 16.5 mmol) was slowly added dropwise under ice-bath conditions, and stirred at room temperature for 8h. Under ice bath conditions, 40 mL water was added to quench the reaction, followed by 30 mL methyl tert-butyl ether. The solution was separated and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined and washed 1 time with 40 mL saturated sodium bicarbonate solution, 3 more times with water and finally 1 time with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 45-50℃to give a pale yellow oil (4.12. 4.12 g, 4.0 mmol) in 80.3% yield. MS 1024.6 [ M+H ] ] ⁺ 。

Step 2: synthesis of G6-3

G6-2 (4.10G, 4.0 mmol) was used as a starting material and the procedure was followed to give G1-4 as a yellow oil in a yield of 4.01. 4.01G% and 98.8%. ¹ H-NMR (400 HZ; DMSO): δ 7.94 (s, 1H), 7.68-7.77 (m, 4H), 7.14-7.15 (m, 4H), 4.80 (s, 2H), 3.79-3.85 (m, 5H), 3.47 (t, J = 6.00 HZ, 4H), 3.39 (t, J = 6.00 HZ, 5H), 3.33 (s, 4H), 3.28 (s, 3H), 2.18 (t, J = 6.10 HZ, 3H), 2.12 (t, J = 6.10 HZ, 5H), 1.76 (s, 7H), 1.32-1.37 (m, 10H), 0.97 (t, J = 7.16 HZ, 8H)。MS: 724.6 [M + H] ⁺ 。

Step 3: synthesis of G6-4

Starting from G1-9 (1.79G, 4.0 mmol) and G6-3 (1.02G, 1.0 mmol), the procedure was followed to give G1-10 as a pale yellow oil(1.49. 1.49 g, 0.74 mmol) in 74.0% yield. MS 1007.0 [ M+2H ]] ²⁺ 。

Step 4: synthesis of G6-5

Starting with G6-4 (1.49G, 0.74 mmol), G1-11 was synthesized to give a white solid (1.17G, 0.62 mmol) in 84.2% yield. MS 939.6 [ M+2H ]] ²⁺ 。

Step 5: synthesis of YK-GAL-006

Using G6-5 (1.17G, 0.62 mmol) and G1-12 (0.13G, 0.93 mmol) as starting materials, the procedure was followed to synthesize YK-GAL-001 as a white solid (0.87G, 0.44 mmol) in 70.2% yield. MS 1000.1 [ M+2 ] 2H] ²⁺ 。

Synthesis of YK-GAL-007

The synthetic route is as follows:

step 1: synthesis of G7-2

G1-6 (1.51G, 4.6 mmol) and G7-1 (1.64G, 5.5 mmol) were dissolved in dichloroethane (25 mL), TMSOTF (0.47G, 2.3 mmol) was slowly added dropwise under ice-bath conditions and stirred at room temperature for 15h. The reaction was quenched by the addition of 0.5. 0.5 mL triethylamine, washed 1 with saturated sodium bicarbonate solution and 1 with saturated sodium chloride solution. The solution was separated, the aqueous phase was extracted 2 times with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45-50 ℃ to remove the solvent, to give a yellow oil (2.61 g, 4.2 mmol) in 90.6% yield. MS 649.1 [ M+Na ] ] ⁺ 。

Step 2: synthesis of G7-3

G1-9 was synthesized from G7-2 (0.56G, 0.89 mmol) to give a yellow oil (0.47G, 0.87 mmol) in 97.7% yield. MS 538.2 [ M+H ]] ⁺ 。

Step 3: synthesis of G7-4

Starting with G7-3 (0.47G, 0.87 mmol) and G5-3 (0.20G, 0.22 mmol), the procedure was followed to synthesize G1-10 to give a pale yellow oil (0.35G, 0.16 mmol) in 72.9% yield. MS: 1115.8 [M + 2 H] ²⁺ 。

Step 4: synthesis of G7-5

Using G7-4 (0.35G, 0.16 mmol) as a starting material, according to the method for synthesizing G1-11, a white bran-like solid (0.28G, 0.13 mmol) was obtained in a yield of 84.8%. MS 1049.0 [ M+2H ]] ²⁺ 。

Step 5: synthesis of YK-GAL-007

Using G7-5 (0.28G, 0.13 mmol) and G1-12 (27.8 mg, 0.20 mmol) as starting materials, the procedure was followed to synthesize YK-GAL-001 as a white solid (0.22G, 0.09 mmol) in 73.3% yield. MS 1109.4 [ M+2H ]] ²⁺ 。

Synthesis of YK-GAL-008

The synthetic route is as follows:

using G8-1 (0.28G, 3.0 mmol) and G1-11 (1.24G, 2.0 mmol) as starting materials, the procedure was followed to synthesize YK-GAL-001 as a white oil (1.11G, 1.6 mmol) in 79.9% yield. MS 696.3 [ M+H ]] ⁺ 。

Synthesis of YK-GAL-009

The synthetic route is as follows:

Starting with G9-1 (0.69G, 3.0 mmol) and G1-11 (1.24G, 2.0 mmol) according to the method for synthesizing YK-GAL-001, a pale yellow oil (1.25G, 1.5 mmol) was obtained. The yield thereof was found to be 75.3%. MS 832.0 [ M+H ]] ⁺ 。

Synthesis of YK-GAL-010

The synthetic route is as follows:

starting from G10-1 (0.21G, 3.0 mmol) and G1-11 (1.24G, 2.0 mmol) according to the following formulaThe procedure for the synthesis of YK-GAL-001 gave a yellow oil (1.18 g, 1.5 mmol). The yield thereof was found to be 72.8%. MS 809.9 [ M+H ]] ⁺ 。

The synthetic yields of each of the above GalNAc compounds are shown in Table 1:

TABLE 1 Synthesis yield of GalNAc Compounds

As can be seen from Table 1, the synthetic GalNAc compounds YK-GAL-001, YK-GAL-002, YK-GAL-003, YK-GAL-004, YK-GAL-005, YK-GAL-006, YK-GAL-007, YK-GAL-008, YK-GAL-009 and YK-GAL-010 designed by the application have higher yields in each step, and the highest yield is 100% of the synthetic YK-GAL-005 in step 2. The final step yield of each compound is above 70%. The overall yield is also higher, for example, the total yield of YK-GAL-002 reaches 64.8%.

Example 2: coupling of GalNAc Compounds with thio oligonucleotides (Oligo S)

2.1 Synthesis of thio oligonucleotide (Oligo S)

The sequence of the thio oligonucleotide (i.e., oligo S) according to this example was as follows:

5 'TCCTCCGGAGCCAGACTTC A3' (xrepresents a thio site)

The thio oligonucleotide is synthesized by adopting a solid phase method, and the specific experimental process is as follows:

(1) Reagent and monomer preparation

A100 nmol standard universal CPG column was used, commercial universal DNA monomers (dA, dT, dC, dG) were dissolved in ultra-dry anhydrous acetonitrile to prepare a solution with a concentration of about 0.1-0.2M, and 3A molecular sieves were added. An acetonitrile solution of 5-ethylthiotetrazole (5-BTT) is used as an activator (0.25M), a pyridine/water/THF solution of 0.05M iodine is used as an oxidant, a 3% trichloroacetic acid (TCA) dichloromethane solution is used as a deprotection agent, acetic anhydride/water is used as a capping agent A (CAPA), pyridine/N-methylimidazole/THF is used as a capping agent B (CAPB), and a pyridine solution of 0.2M hydrogenated Huang Yuansu is used as a vulcanizing agent and is filled into a reagent position designated in a 192 model P DNA/RNA automatic synthesizer.

(2) Crude product synthesis

The designated oligonucleotide sequence was entered and the synthesis procedure was set up, and after checking for errors, the synthesis of the circulating oligonucleotides was started. The universal DNA monomer coupling time is about 1 minute, with an oxo time of about 30-45 seconds and a thio time of about 2 minutes. After the cycle is completed, the solid phase synthesis of the thio oligonucleotide is completed.

(3) Deprotection of

After synthesis, the thio oligonucleotide needs to be detached from the CPG column and the protecting group on the monomer. The CPG column with the thio-oligonucleotide is taken out from the synthesizer and put into a gas phase ammonolysis instrument for ammonolysis at 95 ℃ for 2 h, and the temperature is reduced to below 60 ℃ after the ammonolysis is finished. The mixture was taken out and cooled to room temperature, and rinsed 1 time with 400. Mu.L of 90% acetonitrile solution. Centrifuging, washing with 400 μl of purified water for 1 time, and concentrating the solution at low temperature to obtain crude residue.

(4) Purification

The crude residue after deprotection was dissolved in purified water and purified by HPLC, the product peak solution was collected and the content was measured with a microplate reader to give about 14.3. 14.3 OD, which was converted to 78.2 nmol, i.e. 0.47 mg, yield 78.2%. The product peak solution was lyophilized and the lyophilized product was subjected to HPLC and MS detection. The purity was 98.0%. MS:863.9 [ M-7H ]] ^7- 。

2.2 coupling of GalNAc Compounds with thio oligonucleotides (Oligo S)

2.2.1 Coupling of YK-GAL-002 with a thio oligonucleotide

(1) Dimethyl sulfoxide (DMSO) as solvent

The synthetic route is as follows:

YK-GAL-002 (1.5. Mu. Mol) and the thio-oligonucleotide (150 nmol) were dissolved in dimethyl sulfoxide to maintain the thio-oligonucleotide concentration at about 200. Mu.M. Stirring at 50 ℃ for 8 hours, and detecting the thio-oligonucleotide by LC-MS basically without residue. The solvent was concentrated under vacuum to give a residue. The residue was dissolved in 0.3. 0.3 mL purified water and purified by HPLC to give a product of about 2 2.3 OD (about 0.80 mg,122.0 nmol), yield 81.3%. MS 814.8 [ M-8H ]] ^8- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, and thus the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-002 was 63.6%.

(2) N, N-Dimethylformamide (DMF) as solvent

YK-GAL-002 (1.5. Mu. Mol) and a thio oligonucleotide (150 nmol) were dissolved in N, N-dimethylformamide to give a product of about 11.2. 11.2 OD (about 0.40 mg,61.5 nmol) in a yield of 41.0% according to the method of (1). MS 814.7 [ M-8H ]] ^8- 。

(3) Tetrahydrofuran (THF) as solvent

YK-GAL-002 (1.5. Mu. Mol) and the thio oligonucleotide (150 nmol) were dissolved in tetrahydrofuran to give the product of about 16.6. 16.6 OD (about 0.60 mg,90.8 nmol) in a yield of 60.5% according to the method of (1). MS 814.7 [ M-8H ]] ^8- 。

The solvent and yield comparisons for each experimental reaction are shown in Table 2.

TABLE 2 reaction conditions and yields

As can be seen from the above experimental results, the coupling of YK-GAL-002 with the thioated oligonucleotide was carried out using dimethyl sulfoxide, N-dimethylformamide and tetrahydrofuran as solvents, and without the addition of other reagents, the GalNAc thioated oligonucleotide conjugate was obtained. The different reaction solvents have great influence on the yield, the yield can reach 81.3 percent by taking dimethyl sulfoxide as a solvent, the yield is only 41.0 percent by taking N, N-dimethylformamide as a solvent, the yield is 60.5 percent by taking tetrahydrofuran as a solvent, and the dimethyl sulfoxide with the highest yield can reach 2 times of the lowest N, N-dimethylformamide. The most preferred reaction solvent is dimethyl sulfoxide, which is used in the examples below.

The optimized results of other reaction parameters show that under the conditions that the reaction temperature is 20-60 ℃, the reaction time is 1-15 h, the feeding ratio (GalNAc compound: thio oligonucleotide) is 5:1-30:1 (molar ratio), the GalNAc compound YK-GAL-002 and the thio oligonucleotide (Oligo S) can be coupled, and the experimental results are similar to the above experiments.

2.2.2 Coupling of YK-GAL-004 with thio-oligonucleotides

The synthetic route is as follows:

using YK-GAL-004 (1.5. Mu. Mol) and thio oligonucleotide (150 nmol) as raw materials and DMSO as a reaction solvent, the coupling method of YK-GAL-002 and thio oligonucleotide gave a product of about 24.2. 24.2 OD (about 0.97 mg,132.2 nmol) in 88.1% yield. MS 1041.9 [ M-7H ]] ^7- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, and thus the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-004 was 68.9%.

2.2.3 Coupling of YK-GAL-005 with a thio oligonucleotide

The synthetic route is as follows:

using YK-GAL-005 (1.5. Mu. Mol) and a thio oligonucleotide (150 nmol) as raw materials and DMSO as a reaction solvent, the coupling method of YK-GAL-002 and the thio oligonucleotide gave a product of about 24.0. 24.0 OD (about 1.04 mg,131.3 nmol) in a yield of 87.5%. MS 985.0 [ M-8H ] ] ^8- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, and thus the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-005 was 68.4%.

2.2.4 Coupling of YK-GAL-008 with a thio oligonucleotide

The reaction route is as follows:

using YK-GAL-008 (1.5. Mu. Mol) and a thio oligonucleotide (150 nmol) as raw materials, the coupling method of YK-GAL-002 and the thio oligonucleotide gave a product of about 18.0. 18.0 OD (0.66 mg, 98.1 nmol) in a yield of 65.4%. MS 838.3 [ M-8H ]] ^8- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, so that the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-008 was 51.1%.

2.2.5 Coupling of YK-GAL-009 with a thio oligonucleotide

The reaction route is as follows:

using YK-GAL-009 (1.5. Mu. Mol) and the thio oligonucleotide (150 nmol) as raw materials and DMSO as a reaction solvent, the coupling procedure of YK-GAL-002 and the thio oligonucleotide gave a product of about 24.1. 24.1 OD (0.88 mg, 131.7 nmol) in 87.8% yield. MS 838.2 [ M-8H ]] ^8- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, so that the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-009 was 68.7%.

2.2.6 Coupling of YK-GAL-010 with a thio oligonucleotide

The reaction route is as follows:

Using YK-GAL-010 (1.5. Mu. Mol) and a thio oligonucleotide (150 nmol) as raw materials and DMSO as a reaction solvent, the coupling method of YK-GAL-002 and the thio oligonucleotide gave a product of about 23.4 OD (0.86 mg, 127.5 nmol) in 85.0% yield. MS 838.3 [ M-8H ]] ^8- 。

The yield of the synthesized thioate oligonucleotide was 78.2%, and thus the total yield of the synthesized GalNAc thioate oligonucleotide conjugate GalNAc-Oligo S-010 was 66.5%.

The structure of each GalNAc compound, the coupling yield with the thioated oligonucleotide, and the total yield of the synthesized GalNAc thioated oligonucleotide conjugate are listed in table 3.

TABLE 3 GalNAc Compound Structure, coupling yield with thio-oligonucleotide and Total yield

/>

As can be seen from Table 3, by the liquid phase synthesis method, the GalNAc compounds YK-GAL-002, YK-GAL-004, YK-GAL-005, YK-GAL-008, YK-GAL-009 and YK-GAL-010 of different structures were coupled with the thio oligonucleotide in yields of 65% -90%, wherein the highest yield was YK-GAL-009, namely GalNAc p-toluenesulfonate compound, up to 87.8%. The total yield of the synthesized GalNAc thio oligonucleotide conjugate is 50% -70%, wherein the highest yield is YK-GAL-004, and the total yield reaches 68.9%.

The three compounds of YK-GAL-002, YK-GAL-004 and YK-GAL-005 are GalNAc bromide, and the structural difference between the three compounds is that the YK-GAL-002 has 1 connecting arm, the YK-GAL-004 and YK-GAL-005 have 2 connecting arms and 3 connecting arms respectively, the chemical structure is more complex, but the yield of conjugation with the thio oligonucleotide is not obviously different, and the three compounds reach more than 80 percent.

The prior studies indicate that the reaction yield of small molecule compounds with thiol groups in phosphorothioates is greatly affected by the structure of the compound and the groups attached to thiol groups in phosphorothioates (Bioorganic Chemistry, 2009, 37 (4): 101-105; advanced synthesis & analysis, 2018, 360 (10)). The chemical structures of the small molecular compounds and thiophosphoryl ester are very simple, but the reaction yield greatly fluctuates due to the influence of different groups, and the minimum is only about 30 percent. The phosphorothioate oligonucleotides used in this example are 20 base oligonucleotides, and the thiol-linked groups, including phosphodiester linkages, ribose, purine and pyrimidine, are very complex in chemical structure, and have a vast difference from phosphorothioate compounds reported in the literature. It is therefore impossible to deduce from the prior art whether or not the GalNAc compound designed according to the present application can react with a thio oligonucleotide having 20 bases and a very complex chemical structure, and how much can the reaction yield.

The inventor optimizes the reaction condition, so that the GalNAc compound designed by the application can be conjugated with the thioated oligonucleotide with 20 bases with high efficiency, the yield can reach more than 85%, the total yield of synthesized GalNAc thioated oligonucleotide conjugate can reach more than 60%, and unexpected technical effects are obtained.

Comparative example 1: coupling of YK-GAL-002 with a thio oligonucleotide (cf. The methods of the prior art)

The reaction route is as follows:

refer to the method in CN111484540A (description, page 26, [0158 ]]Paragraph) under the protection of nitrogen, the thiooligonucleotide (0.15 mmol), N, N-dimethylformamide (0.88, mL), diisopropylethylamine (23 mg,0.18 mmol), sodium iodide (22 mg,0.15 mmol) and YK-GAL-002 (99.6 mg,0.18 mmol) were added to a reaction flask in this order, and the reaction mixture was stirred at room temperature overnight and purified by column chromatography to give a product 342 mg in a yield of 34.9%. MS:814.5 [ M-8H ]] ^8- 。

From experimental results, the coupling yield of the GalNAc compound YK-GAL-002 designed by the application and the thio oligonucleotide with 20 bases is only 34.9% by adopting the prior art method, namely N, N-dimethylformamide is taken as a solvent, diisopropylethylamine and sodium iodide are added into a reaction system, while the coupling yield is 81.3% by adopting the method of the application in the embodiment 1, the 46.4% is improved, and the yield is remarkably improved.

Comparative example 2: solid phase synthesis of GalNAc oligonucleotide conjugates with shorter linker arms

The yield of GalNAc oligonucleotide conjugates with short linker arms and simple structure were synthesized by the solid phase method was examined by this experiment. GalNAc compound G1-9 has a short linking arm, which is- (CH) ₂ ) ₄ C (O) O-, and only 1 chain, the chemical structure is relatively simple. G1-9 is firstly connected with a solid support CPG, and then an oligonucleotide with 20 bases is synthesized by a solid phase methodThe oligonucleotide sequences of (2) were identical to the thio oligonucleotide used in example 2, except that no thio was present.

The specific experiment is as follows:

g1-9 is reacted with compound 1 to obtain compound 2, and then compound 2 is connected with CPG of solid support to obtain CPG-GalNAc compound 3.

1. Preparation of CPG-GalNAc Compounds

Step 1: synthesis of Compound 2

G1-9 (3.53G, 7.9 mmol), O-benzotriazol-tetramethylurea hexafluorophosphate (3.50G, 9.2 mmol) and N, N-diisopropylethylamine (4.16G, 32.2 mmol) were added to dichloromethane (200 mL), dissolved by stirring, and then compound 1 (3.00G, 7.2 mmol) was added thereto and reacted at 25℃for 2 hours by stirring. The solvent was removed by concentration under reduced pressure, and compound 2 (5.21 g, 86.0%) was obtained by separation and purification. MS 849.8 [ M+H ]] ⁺ 。

Step 2: preparation of CPG-GalNAc Compound 3

Compound 2 (0.58 g, 0.68 mmol), succinic anhydride (0.14 g, 1.36 mmol) and 4-dimethylaminopyridine (0.25 g, 2.05 mmol) were dissolved in dichloromethane (5 mL) and stirred at 25 ℃ for 20h. The reaction system was diluted with dichloromethane and washed with water and cold citric acid solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to remove the solvent to give succinate (0.32 g, 0.34 mmol) in 50.4% yield.

Succinic acid ester (0.32 g,0.34 mmol), O-benzotriazol-tetramethylurea hexafluorophosphate (0.13 g,0.34 mmol) and N, N-dimethylformamide (15 mL) were added to the reaction system, and dissolved with stirring. N, N-diisopropylethylamine (0.13 g,1.0 mmol) was then added and the mixture was shaken for 5min. Polystyrene scaffolds (5.0 g) were added and shaken at 25℃for 24h. The mixture was filtered, washed with dichloromethane, 10% methanol/dichloromethane and diethyl ether in this order, and dried in vacuo for 2h. And oscillating the filter material in 25% acetic anhydride/pyridine solution for 0.5h, filtering, washing sequentially with dichloromethane, 10% methanol/dichloromethane and diethyl ether, and vacuum drying the filter material for 2h to obtain CPG-GalNAc compound 3.

2. GalNAc oligonucleotide conjugate solid phase synthesis

The synthesized oligonucleotide sequence was identical to the thioated oligonucleotide used in example 2, except that no thioation was present. The GalNAc oligonucleotide conjugate was synthesized by loading an acetonitrile solution (0.1-0.2M) of the DNA monomers (A, T, C, G) and CPG-GalNAc compound 3 into a 192 synthesizer, debugging the apparatus and setting the synthesis procedure in which the first monomer was coupled to the CPG column for an activated coupling time of about 20 min. After the synthesis is completed, gas phase ammonolysis is carried out, and simple desalination and purification are carried out. And quantifying by using an enzyme-labeled instrument, wherein the yield is 32.0%. MS:918.9 [ M-7H ] ] ^7- 。

Experimental results:

comparative example 2 GalNAc oligonucleotide conjugate was synthesized by solid phase method with a total yield of only 13.9%. In example 2, the thioated oligonucleotide is synthesized by a solid phase method, and then the GalNAc compound is coupled with the thioated oligonucleotide by a liquid phase method, so that the total yield can reach 50-70%, which is 3.6-5.0 times that of comparative example 2.

1) Preparation of CPG-GalNAc Compounds

In the step 1, galNAc compound G1-9 is reacted with compound 1 to obtain compound 2, and the reaction yield in the step is 86.0%; in step 2, the yield of succinate was only 50.4%, and the subsequent yield of CPG was not accurately calculated, but even if the yield of CPG was calculated as 100%, the total yield of CPG-GalNAc compound 3 was only 43.3%.

2) GalNAc oligonucleotide conjugate solid phase synthesis

Starting with CPG-GalNAc compound 3, the synthesis of 20 base oligonucleotides was carried out by solid phase method in a yield of only 32.0%. The oligonucleotides are generally synthesized by solid phase methods (i.e., no GalNAc coupling on the starting nucleotide) in 99% yield per base, and thus 20 base oligonucleotides are synthesized in about 80% yield. From this, it was found that, in the present experiment, when the oligonucleotide was prepared by the solid phase method, the GalNAc compound was coupled to the starting nucleotide, which greatly affected the subsequent reaction, and the yield was greatly lowered, which was only 40% of that when the GalNAc coupling was not performed to the starting nucleotide.

Conclusion:

the solid phase method synthesizes GalNAc oligonucleotide conjugate with short connecting arm, and the total yield is only 13.9%. In the application, the solid phase method is used for synthesizing the thioated oligonucleotide, and the liquid phase method is used for coupling the GalNAc compound and the thioated oligonucleotide, so that the total yield can reach 50-70%, which is 3.6-5.0 times that of comparative example 2, and the yield is obviously improved.

Comparative example 3: solid phase synthesis of GalNAc oligonucleotide conjugates with longer linker arms

The effect of the length of the linker arm of the GalNAc compound on the yield of the GalNAc oligonucleotide conjugate synthesized by the solid phase method was examined. GalNAc compound G1-11 used in comparative example 3, wherein the linker was- (CH) ₂ ) ₄ C(O)NH(CH ₂ ) ₃ NHC(O)(CH ₂ ) ₂ O(CH ₂ ) ₂ NH-is longer in the connecting arm and more complicated in the structure than G1-9 in comparative example 2.

1. Preparation of CPG-GalNAc Compounds

The synthetic route is as follows:

step 1: synthesis of Compound 5

Starting from G1-11 (4.89G, 7.9 mmol) and compound 4 (4.45G, 7.2 mmol), compound 2 was synthesized to give a pale yellow oil (6.60G, 5.41 mmol) in 75.2% yield. MS 1219.7 [ M+H ]] ⁺ 。

Step 2: preparation of CPG-GalNAc Compound 6

Succinic acid ester (0.54 g,0.27 mmol) was prepared first using compound 5 (0.83 g, 0.68 mmol) as a starting material according to the method for preparing CPG-GalNAc compound 3, yield 40.5%, and CPG-GalNAc compound 6 was then obtained using succinic acid ester as a starting material.

2. GalNAc oligonucleotide conjugate solid phase synthesis

The synthesized oligonucleotide sequence was identical to the thioated oligonucleotide used in example 2, except that no thioation was present. DNA sheetA solution of the body (A, T, C, G) in acetonitrile (0.1-0.2M) and CPG-GalNAc compound 6 were charged into a 192 synthesizer, the apparatus was set up and the synthesis procedure was set up (wherein the first monomer was coupled to the CPG column for an active coupling time of about 20 min) to synthesize a GalNAc oligonucleotide conjugate. After the synthesis is completed, gas phase ammonolysis is carried out, and simple desalination and purification are carried out. And quantifying by an enzyme-labeled instrument, wherein the yield is 20.3%. MS 850.2 [ M-8H ]] ^8- 。

Experimental results:

1. as is clear from comparison of comparative example 2 with comparative example 3, the longer the connecting arm of the GalNAc compound is, the more complex the chemical structure is, and the lower the synthesis yield is

The yield of comparative example 3 was significantly reduced as compared with comparative example 2. The GalNAc compounds G1-11 used in comparative example 3 were longer in the linking arm and more complicated in the chemical structure than those of the G1-9 in comparative example 2. It is found that the yield of GalNAc oligonucleotide conjugates synthesized by the solid phase method is lower for GalNAc compounds having longer linker arms. The reaction yields of each step of comparative example 2 and comparative example 3 are shown in Table 4.

Table 4 comparative example 2 and comparative example 3 yield comparison

The method comprises the following steps:

Production yield of CPG-GalNAc compound: comparative example 2 step 1 yield 86.0%, comparative example 3 yield 75.2%, 10.8% lower than comparative example 2; step 2 of comparative example 2 gave a yield of succinate of 50.4%, 40.5% for comparative example 3 and 9.9% lower than for comparative example 2. The total yield of the two-step reaction was 43.3% for comparative example 2, 30.5% for comparative example 3 and 12.8% lower than comparative example 2.

Yield of synthetic GalNAc oligonucleotide conjugate: comparative example 2 was 32.0%, comparative example 3 was 20.3%, and the reduction was 11.7% compared to comparative example 2.

III, total yield: comparative example 2 was 13.9%, comparative example 3 was 6.2%, and 7.7% lower than comparative example 2. The total yield of comparative example 2 was 2 times or more that of comparative example 3.

As is clear from the comparison results, the longer the linker arm of the GalNAc compound is, the more complicated the structure is, and the lower the yield of the GalNAc oligonucleotide conjugate synthesized by the solid phase method is.

2. The method of the application synthesizes GalNAc thio oligonucleotide conjugate with longer connecting arm and more complex structure, and compared with the solid phase synthesis method, the yield is obviously improved, and the yield can be improved by at least more than 10 times

I. GalNAc compounds YK-GAL-008, YK-GAL-009 and YK-GAL-010 of the present application were chlorides, p-toluenesulfonate compounds and trifluoromethanesulfonate compounds prepared from G1-11 as raw materials in yields of 79.9%, 75.3% and 72.8% (examples 1, 8-10), respectively, and the total yields of GalNAc thio oligonucleotide conjugates synthesized from these 3 compounds reached 51.1%, 68.7% and 66.5% (examples 2,2.2.4-2.2.6), respectively. GalNAc compounds YK-GAL-001, YK-GAL-002, YK-GAL-003, YK-GAL-004, YK-GAL-005, YK-GAL-006, YK-GAL-007, YK-GAL-008, YK-GAL-009 and YK-GAL-010 of the present application have longer linking arms and more complex structures than G1-9 and G1-11, some of which have 2 or 3 chains. If the GalNAc oligonucleotide conjugate is synthesized by solid phase method, the total yield is expected to be lower than that of comparative example 3, i.e., lower than 6%.

YK-GAL-002 has 1 linker arm coupled to the thioate in a yield of 81.3% and the total yield of synthesized GalNAc thioate conjugate was 63.6% (example 2,2.2.1). The total yield was 10 times that of comparative example 3, solid phase synthesis.

YK-GAL-004 and YK-GAL-005 have 2 and 3 connecting arms, respectively, and the connecting arms are longer and the structure is more complicated compared with G1-9 and G1-11, but the coupling yield with the thioated oligonucleotide can reach 88.1% and 87.5% respectively by the method of the application, the total yield of the synthesized GalNAc thioated oligonucleotide conjugate is 68.9% and 68.4% respectively (examples 2,2.2.2 and 2.2.3), and the total yield is 11 times that of the solid phase synthesis method of comparative example 3, which is remarkably improved.

YK-GAL-001 and YK-GAL-003 have 1 connecting arm, YK-GAL-006 and YK-GAL-007 have 3 connecting arms, and the total yield of GalNAc thio oligonucleotide conjugates synthesized by the method of the application can be expected to be more than 60% and can be more than 10 times that of the solid phase synthesis method of comparative example 3.

Conclusion:

1. the solid phase method is used for synthesizing the GalNAc oligonucleotide conjugate, and the structure of the GalNAc compound, such as the length of a connecting arm, has great influence on the yield, and the longer the connecting arm is, the lower the yield is.

2. The GalNAc compound designed by the application has complex structure, and the total yield is expected to be lower than 6% when the GalNAc oligonucleotide conjugate is synthesized by a solid phase method.

3. By using the method, the coupling yield of the GalNAc compound and the thioated oligonucleotide can reach 85%, the total yield of the synthesized GalNAc thioated oligonucleotide conjugate can reach more than 60%, the total yield can reach more than 10 times of that of the solid phase synthesis method of comparative example 3, and the yield is obviously improved.

In summary, the present application designs and synthesizes a series of novel GalNAc compounds, and couples the GalNAc compounds with the thioated oligonucleotides by a liquid phase method to obtain GalNAc thioated oligonucleotide conjugates. The method can realize the fixed-point coupling of the thio oligonucleotide and the GalNAc compound, and has mild reaction conditions, simple experimental operation and high yield.

Claims

1. A compound of formula (I):

wherein,,

X ₂ is- (CH) ₂ ) _b -, b is an integer from 1 to 6;

X ₃ is-NHC (O) -or-C (O) NH-;

X ₄ is- (CH) ₂ ) -or- (CH) ₂ ) ₂ -；

Y ₁ 0 or 1;

Y ₂ 0, 1 or 2;

Y ₃ 1, 2 or 3;

r is Cl, br, I,

2. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X ₁ Is- (CH) ₂ ) ₄ -、-(CH ₂ ) ₂ -or- (CH) ₂ CH ₂ O) ₃ CH ₂ -。

3. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X ₂ Is- (CH) ₂ ) ₃ -、-(CH ₂ ) -or- (CH) ₂ ) ₅ -。

4. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound YK-GAL-001, YK-GAL-002, YK-GAL-003, YK-GAL-004, YK-GAL-005, YK-GAL-006, YK-GAL-007, YK-GAL-008, YK-GAL-009, or YK-GAL-010) having the structure:

5. a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, which is capable of binding to an asialoglycoprotein receptor (ASGPR).

6. A method of coupling a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of the preceding claims to a thioate oligonucleotide comprising the steps of:

3) Purifying the residue to obtain the product.

7. The method of claim 6, wherein the organic solvent is a polar aprotic solvent.

8. The method of claim 7, wherein the organic solvent is one or more of dimethyl sulfoxide, N-dimethylformamide, and tetrahydrofuran.

9. The method according to claim 8, wherein the organic solvent is dimethyl sulfoxide.

10. The method of any one of claims 6-9, wherein the thio oligonucleotide is an oligonucleotide in which one non-bridging oxygen atom in the phosphodiester linkage is replaced with a sulfur atom.

11. The method of any one of claims 6-9, wherein the molar ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the thioa oligonucleotide is from 5:1 to 30:1.

12. The method of claim 11, wherein the molar ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the thioated oligonucleotide is from 7.5:1 to 20:1.

13. The method of claim 12, wherein the molar ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the thioa oligonucleotide is 10:1.

14. The process according to any one of claims 6-9, wherein the reaction temperature of step 1) is 20 to 60 ℃.

15. The process of claim 14, wherein the reaction temperature of step 1) is 25 to 55 ℃.

16. The process of claim 15, wherein the reaction temperature of step 1) is 50 ℃.

17. The process according to any one of claims 6-9, wherein the reaction time of step 1) is 1 to 15 hours.

18. The method of any one of claims 6-9, wherein the thioate oligonucleotide is a small interfering nucleotide, DNA, microrna, small activating RNA, small guide RNA, transfer RNA, antisense nucleotide, or aptamer.

19. The method of claim 18, wherein the thio oligonucleotide is an antisense nucleotide or a small interfering nucleotide.

20. A conjugate comprising a thioate oligonucleotide and a GalNAc moiety, wherein the GalNAc moiety isWherein the method comprises the steps of

X ₁ 、X ₂ 、X ₃ 、X ₄ 、Y ₁ 、Y ₂ 、Y ₃ A compound of formula (I) as defined in any one of claims 1 to 5.

21. The conjugate of claim 20, wherein the thio oligonucleotide modulates expression of a target gene.

22. The conjugate of any one of claims 20-21, wherein the thio oligonucleotide and the GalNAc moiety are linked by a bond or a cleavable linker.

23. The conjugate of claim 22, wherein the thio oligonucleotide and the GalNAc moiety are linked by a phosphorothioate linkage.

24. The conjugate of claim 23, having the following structural formula:

wherein the method comprises the steps of

25. The conjugate according to any one of claims 20-24 for use as a medicament.

26. Use of a conjugate according to any one of claims 20-24 in the manufacture of a medicament.

27. A pharmaceutical composition comprising the conjugate of any one of claims 20-24 and at least one pharmaceutically acceptable excipient.