Novel galnac targeted delivery fragment and preparation and use thereof

Abstract

Disclosed in the present invention is a GalNAc delivery molecule as shown in formula (I), or a stereoisomer, enantiomer or pharmaceutically acceptable salt thereof, wherein the definition of each group is as described in the description.

Description

Novel GalNAc targeted delivery fragment and its preparation and application Technical Field

[0001] The present invention relates to the field of medicine. Specifically, the present invention provides a novel targeted delivery fragment of GalNAc and its preparation and application. Background Art

[0002] Hepatitis B virus (HBV) belongs to the Hepaciviridae family of small, enveloped, primarily hepatotropic viruses with a partially double-stranded DNA genome of approximately 3.2 kb. HBV infection remains a major public health problem. Despite the availability of safe and effective preventive HBV vaccines, an estimated 250 million people worldwide are chronically infected with HBV. Chronic HBV infection places patients at high risk for cirrhosis and liver cancer, and an estimated 686,000 people die annually from complications of HBV. (WHO Global Hepatitis Report 2017)

[0003] Current treatments are limited to two classes of drugs, namely nucleoside (nucleotide) analogs (lamivudine, adefovir, tenofovir, telbivudine and entecavir) and interferon alpha (INF-α, including non-PEGylated and PEGylated). Although both therapies can reduce HBV DNA and normalize liver enzymes, neither therapy can provide a high level of clinical cure, which is defined by the loss of HBV surface antigen (HBsAg) (with or without seroconversion). Interferon-based therapies are poorly tolerated and are only effective against certain viral genotypes. Although nucleoside (nucleotide) analogs often require extended or potentially lifelong treatment, and some have resistance, low efficacy and tolerability issues. Therefore, there is still a huge medical need to discover and develop effective and safe anti-HBV drugs with novel mechanisms of action to improve disease cure rates. (Marcellin, Patrick, et al. The Lancet 381.9865(2013):468-475; Tang, Lydia SY, et al. Jama 319.17(2018):1802-1813.)

[0004] GalNAc-mediated liver-targeted delivery of small interfering RNA (siRNA) drugs (GalNAc-siRNA), which inhibits target gene expression at the basic level of mRNA translation into protein, has recently been used to treat a variety of liver-related diseases. GalNAc-siRNA molecules offer unique advantages in the treatment of hepatitis B due to their potent efficacy, high liver-targeting specificity, and favorable safety profile. However, current investigational drugs are unable to effectively reduce HBsAg levels and achieve high clinical cure rates. Therefore, optimization of these molecules is needed, with a focus on structural modification of the GalNAc-containing targeted delivery fragment to improve delivery efficiency and druggability.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a GalNAc-siRNA molecule and a nucleic acid delivery pharmaceutical composition prepared using the same.

[0007] In a first aspect, the present invention provides a GalNAc delivery molecule represented by the following formula (I), or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof:

[0008] in,

[0009] R1 is selected from the group consisting of H, DMTr, phosphate, phosphorothioate, phosphorothioate-siRNA conjugate or phosphate-siRNA conjugate;

[0010] Linker is a divalent linking group;

[0011] r is 0, 1, 2, 3, 4, 5, or 6;

[0012] R2 is a GalNAc target head portion and has a structure as shown below:

[0013] wherein L1, L2 and L3 are each independently a divalent linking group, and R3, R4 and R5 are each independently a substituted or unsubstituted sugar group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule;

[0014] The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of: 1-8 Alkyl, C 2- 8-alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, C 3-8 Aryl, 5- to 7-membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C2-10 Ester group, amino group, C 1-8 Alkoxy, C 1-10 or two substituents located on adjacent ring atoms may together with the connected ring atoms form a group selected from the following group: a 5-7 membered carbon ring or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring.

[0015] In another preferred embodiment, the siRNA is selected from the following group: siRNA that inhibits the expression of hepatitis B virus (HBV) gene, siRNA that inhibits the expression of apolipoprotein C3 (APOC3) gene, siRNA that inhibits the expression of PCSK9 gene, siRNA that inhibits the expression of CCR4 gene, and siRNA that inhibits the expression of thyroxine transporter (TTR) gene.

[0016] In another preferred embodiment, the Linker has a structure selected from the following group:

[0017] C4-C20 alkyl, wherein n and m are each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0018] n1 and n2 are each independently selected from the group consisting of: 0 or 1;

[0019] W is selected from the group consisting of a single bond, a substituted or unsubstituted C 1-8 Alkylene, a substituted or unsubstituted 7-12 membered fused bicyclic ring, a substituted or unsubstituted 7-15 membered spirocyclic ring, a substituted or unsubstituted 5-15 membered bridged ring, a -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring, a -NH-substituted or unsubstituted 7-15 membered spirocyclic ring, a -NH-substituted or unsubstituted 5-15 membered bridged ring, a -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring-NH-, a -NH-substituted or unsubstituted 7-15 membered spirocyclic ring-NH-, a -NH-substituted or unsubstituted 5-15 membered bridged ring-NH-; wherein the ring atoms of the fused bicyclic ring, spirocyclic ring and bridged ring may optionally be carbon atoms or heteroatoms;

[0020] The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of: 1-8 Alkyl, C 2- 8-alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, C 1-8 Alkoxy, C 1-10Sulfonyl; or two substituents located on adjacent ring atoms may together with the connected ring atoms form a group selected from the following group: a 5-7 membered carbocyclic or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring;

[0021] Or W has a structure selected from the group consisting of:

[0022] wherein s1, s2, s, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2;

[0023] s7 is selected from the group consisting of 1, 2, 3, 4, 5, or 6;

[0024] Each of M1, M2, M3 and M4 is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR;

[0025] M5 and M6 are each independently selected from the group consisting of: (CHR) s6 、C(R)R、C(O)、(CHR) s6 -C(O), O, S, NR;

[0026] R is selected from the following group: H, halogen, methyl; or two R located on adjacent reducing atoms and the carbon atom to which they are connected together form a substituted or unsubstituted 4-8 membered carbocyclic or heterocyclic ring (including saturated, unsaturated or aromatic ring).

[0027] In another preferred embodiment, the W has a structure selected from the following group:

[0028] In another preferred embodiment, L1, L2 and L3 are each independently selected from the following structures:

[0029] wherein each u, v and w are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; u1 and u2 are independently selected from 0 or 1; Glu is an unmodified or modified 5-6 membered glycosyl group;

[0030] X and Y are each independently selected from the following groups: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group;

[0031] The cycloalkyl and heterocyclic groups may be monocyclic, condensed, bridged or spirocyclic; and the heterocyclic group may be saturated or partially unsaturated (but not aromatic).

[0032] In another preferred embodiment, R2 and the linker portion include at least one monocyclic, fused, bridged or spirocyclic structural unit.

[0033] In another preferred embodiment, R2 and the linker portion include at least one fused ring, bridged ring or spiro ring structural unit.

[0034] In another preferred embodiment, X and Y each independently have a structure selected from the following group:

[0035] -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, or a structure selected from the following group:

[0036] In another preferred embodiment, the sugar group has a structure shown in the following formula III:

[0037] Among them, the R 11 Selected from the group consisting of -NH(C2-C6 acyl), -NH(halogenated C2-C6 acyl), -NH(C2-C6 sulfonyl), -NH(halogenated C2-C6 sulfonyl);

[0038] R 12 、R 13 and R 14 Each is independently selected from the group consisting of H or C1-C6 acyl.

[0039] In another preferred embodiment, the R 11 Selected from the group consisting of: -NH(C2-C4 acyl); R 12 、R 13 and R 14 Each is independently selected from the group consisting of H or C1-C4 acyl.

[0040] In another preferred embodiment, the R 11 is -NHAc; R 12 、R 13 and R 14 Each is independently selected from the group consisting of H or Ac.

[0041] In another preferred embodiment, the GalNAc delivery molecule has a structure selected from the group consisting of:

[0042] wherein X, Y, Z and W are each independently selected from the following groups: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group;

[0043] R3, R4, R5 and R5' are each independently a substituted or unsubstituted sugar group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule;

[0044] The remaining groups are as defined above.

[0045] In another preferred embodiment, the GalNAc delivery molecule has a structure selected from the group consisting of:

[0046] The R1 group is as defined in claim 1.

[0047] In another preferred embodiment, the GalNAc delivery molecule has a structure selected from the group consisting of:

[0048] The second aspect of the present invention provides a pharmaceutical composition comprising: one or more of the compound of formula I as described in the first aspect of the present invention, its pharmaceutically acceptable salts, racemates, R-isomers, S-isomers or mixtures thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0049] The third aspect of the present invention provides a compound of formula I as described in the first aspect of the present invention, and the use of its pharmaceutically acceptable salt, racemate, R-isomer, S-isomer or mixture thereof for preparing a pharmaceutical composition for treating or preventing tumors or infections caused by viruses.

[0050] In a fourth aspect, the present invention provides the use of the compound of formula (I) as described in the first aspect of the present invention for preparing a medicament for treating and / or preventing a disease or condition selected from the following group: a disease or condition caused by hepatitis B virus, a disease or condition caused by abnormal expression of the ANGPTL3 gene, a disease or condition caused by abnormal expression of the TTR gene, a disease or condition caused by abnormal expression of hepatocyte genes, or other liver diseases or conditions.

[0051] In another preferred embodiment, the disease or condition caused by abnormal ANGPTL3 gene expression is cardiovascular disease and / or metabolic disease, including hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, obesity, diabetes and / or ischemic heart disease.

[0052] In another preferred embodiment, the disease or condition caused by abnormal TTR gene expression is selected from the following group: sensory neuropathy, including distal limb paresthesia and hypoesthesia; autonomic neuropathy, including gastrointestinal dysfunction such as gastric ulcer, or orthostatic hypotension; motor neuropathy; epileptic seizures; dementia; myelopathy; polyneuropathy; carpal tunnel syndrome; autonomic deficiency; cardiomyopathy; vitreous opacities; renal insufficiency; nephropathy; substantial decrease in mBMI; cranial nerve dysfunction and lattice corneal dystrophy.

[0053] In another preferred embodiment, the hepatocyte gene is selected from the following group: ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, CollA1, FVII, STAT3, p53, HBV, and HCV.

[0054] In another preferred embodiment, the other liver diseases or disorders are selected from the group consisting of hepatocellular carcinoma (HCC), liver metastasis, hepatoblastoma, diseases involving coagulation factors, complement-mediated inflammation or fibrosis, dyslipidemia and irregularities in glucose regulation.

[0055] The fifth aspect of the present invention provides a method for detecting ASGPR-GalNAc affinity based on primary monkey cells.

[0056] In a sixth aspect of the present invention, there is provided a GalNAc linker as shown in formula (Ia), or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof:

[0057] The moiety shown in formula (Ia) has a structure selected from the group consisting of:

[0058] in, It is the site connected to R1.

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. DETAILED DESCRIPTION

[0060] Based on extensive and in-depth research, the inventors have developed a novel GalNAc targeted drug delivery fragment with a novel structure. Drugs or pharmaceutical compositions composed of these GalNAc targeted drug delivery fragments exhibit excellent targeted delivery effects, therapeutic efficacy, and enhanced safety. Based on these findings, the inventors completed the present invention.

[0061] the term

[0062] In the present invention, the halogen is F, Cl, Br or I.

[0063] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0064] In the present invention, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

[0065] In the present invention, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy.

[0066] In the present invention, the term "C3-C7 cycloalkyl" refers to a cyclic alkyl group having 3 to 7 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The terms "C5-C6 cycloalkyl" and "C3-C6 cycloalkyl" have similar meanings.

[0067] In the present invention, the term "aromatic ring" or "aryl" has the same meaning, preferably "aryl" is "C6-C 12 Aryl" or "C6-C 10 Aryl. The term "C6-C 12 "Aryl" refers to an aromatic ring group with 6 to 12 carbon atoms and no heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C6-C 10 "Aryl" has a similar meaning.

[0068] In the present invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning and refer to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to herein include oxygen, sulfur, and nitrogen. Examples include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0069] In the present invention, the term "3-9 membered carbocyclic group" refers to a 3-9 membered cyclic group that is saturated or unsaturated (non-aromatic ring, including monocyclic, cyclic, spirocyclic, bridged, etc.), and its ring skeleton structure only includes carbon atoms, such as cyclopentyl, cyclohexyl, etc.

[0070] In the present invention, the term "3- to 9-membered heterocyclic group" refers to a saturated or unsaturated (non-aromatic ring, including monocyclic, fused, spirocyclic, bridged, etc.) 3- to 9-membered cyclic group containing 1 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen in the ring, such as dioxolanyl. The term "3- to 7-membered heterocyclic group" has a similar meaning.

[0071] In the present invention, the term "substituted" means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent is the substituent described above, or the substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, and the substituent may be the same or different at each position. A cyclic substituent, such as a heterocycloalkyl, may be connected to another ring, such as a cycloalkyl, to form a spirobicyclic system, for example, the two rings have a common carbon atom. It will be understood by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically feasible. The substituents are, for example (but not limited to): C 1-8 Alkyl, C 2-8 Alkenyl, C 2- 8 alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, alkoxy group, C 1-10 Sulfonyl, etc.

[0072] Pharmaceutical compositions containing active ingredients

[0073] The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention and a pharmaceutically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.01-99.99% by weight of the compound of the present invention per dose, more preferably 0.1-99.9% by weight of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0074] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0075] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration, or subcutaneous administration).

[0076] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0077] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0078] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0079] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0080] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0081] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0082] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (eg, anti-HBV agents).

[0083] When administered in combination, the pharmaceutical composition may further comprise one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds may be used simultaneously, separately, or sequentially with the compound of the present invention to prevent and / or treat the relevant disease or condition.

[0084] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0085] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0086] In the following examples, the structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and tetramethylsilane (TMS) was used as the internal standard.

[0087] SHIMADZU LC system (chromatographic column: CSH TM Prep-C18, 19*150mm, liquid handler LH-40, pump LC-20AP, detector SPD-20A, system controller CBM-20A, solvent system: acetonitrile and 0.05% trifluoroacetic acid aqueous solution).

[0088] LC / MS spectra of the compounds were obtained using LC / MS (Agilent Technologies 1200 Series). LC / MS conditions were as follows (run time 10 minutes):

[0089] Acidic conditions: A: 0.05% trifluoroacetic acid in water; B: 0.05% trifluoroacetic acid in acetonitrile;

[0090] Alkaline conditions: A: 0.05% NH3·H2O aqueous solution; B: acetonitrile

[0091] Neutral conditions: A: 10 mM NH4OAC in water; B: acetonitrile

[0092] Unless otherwise specified, in the following examples, intermediates and final compounds were purified by silica gel column chromatography or by using CSH TM Prep-C18 (5 μm, OBD TM 19*150mm) column or XBridgeTM Prep Phenyl (5μm, OBD TM 30*100 mm) was purified by preparative HPLC on a reverse phase column.

[0093] Silica gel column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0094] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0095] Thin layer chromatography (TLC) silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the silica gel plates used in thin layer chromatography detection products are 0.15mm~0.2mm, and the specifications used in thin layer chromatography separation and purification products are 0.4mm~0.5mm.

[0096] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0097] Abbreviations:

[0098] TMSOTf: trimethylsilyl trifluoromethanesulfonate; NaIO4: sodium periodate; RuCl3: ruthenium trichloride; BnBr: benzyl bromide; K2CO3: potassium carbonate; DIEPA (DIEA): N,N-diisopropylethylamine; TFA: trifluoroacetic acid; DCM: dichloromethane; TEA: triethylamine; HATU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; CbzCl: benzyl chloroformate; NaOH: sodium hydroxide; DMSO: dimethyl sulfoxide; HOBt: 1-hydroxybenzotriazole; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMF: N,N-dimethylformamide; tert-butyl acrylate: tert-butyl acrylate; Formic acid: formic acid; DMTr: 4,4'-dimethoxytrityl; Ac: acetyl; Boc: tert-butyloxycarbonyl; Bn: benzyl; Cbz: benzyloxycarbonyl

[0099] Preparation Example Preparation of Compound A1

[0100] Step 1: Preparation of compound A1-1

[0101] 4A molecular sieves (30 g) and TMSOTf (18.8 g, 84.8 mmol) were slowly added to a solution of Compound Int.1 (30 g, 77.1 mmol) in dichloromethane (300 mL). After the addition was complete, the reaction mixture was stirred at 55°C for 3 hours. The reaction mixture was then neutralized with triethylamine and poured into 500 mL of water. The mixture was diluted and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford Compound A1-1 (22.8 g) as a yellow oil. MS: 330.2 (M+H). + .

[0102] Step 2: Preparation of Compound A1-2

[0103] Under nitrogen, 4A molecular sieves (10 g) and compound Int.2 (3.7 g, 15.2 mmol) were slowly added to a solution of compound A1-1 (11 g, 30.4 mmol) in dichloroethane (100 ml). After the addition, the reaction mixture was stirred at 25°C for 30 minutes. TMSOTf (3.7 g, 15.2 mmol) was then slowly added to the reaction mixture with stirring at 0°C, and the reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was filtered, the filter cake washed with dichloromethane, the filtrate neutralized with sodium bicarbonate solution, and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified to yield compound A1-2 (7 g, 48.8%) as a yellow oil. MS: 430.2 (M+H). + .

[0104] Step 3: Preparation of Compound A1-3

[0105] Under nitrogen, a mixture of NaIO4 (13.0 g, 60.6 mmol) and compound A1-3 (6.5 g, 15.2 mmol) in dichloromethane (30 mL), acetonitrile (30 mL), and water (45 mL) was stirred at 0°C for 30 minutes. RuCl3 3H2O (95 mg, 0.45 mmol) was then slowly added to the reaction mixture while stirring at 0°C. The reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with 100 mL of water and the pH was adjusted to 7.5 with sodium bicarbonate. The mixture was then extracted with dichloromethane. The aqueous phase was adjusted to pH 3 with citric acid and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to yield compound A1-3 (3.2 g, 45%) as a white foamy solid. MS: 448.2 (M+H). + . 1 H NMR (400MHz, DMSO-d6) δ11.98(s,1H),7.81(d,J=9.2Hz,1H),5.21(d,J=3.2Hz,1H),4.96(dd,J=11.2,3.4Hz,1H),4.49(d,J=8.4Hz,1H),4.02(s, 3H),3.87(d,J=10.0Hz,1H),3.78–3.37(m,2H),2.20(t,J=6.8Hz,2H),2. 10(s,3H),2.00(s,3H),1.89(s,3H),1.77(s,3H),1.49(d,J=2.8Hz,4H).

[0106] Step 4: Preparation of Compound A1

[0107] Under nitrogen protection at 0°C, compound Int.3 (10.3 g, 36.8 mmol) was slowly added to a solution of compound A1-3 (11.0 g, 24.6 mmol) and DIEA (6.4 g, 49.2 mmol) in dichloromethane (100 mL). The reaction mixture was then stirred at 25°C for 3 hours. The reaction mixture was concentrated to obtain a crude product, which was then purified to obtain compound A1 (11.2 g, 74.2%) as a yellow oil. MS: 614.2 (M+H) + . 1 H NMR(400MHz,DMSO-d6)δ7.83(d,J=9.2Hz,1H),5.22(d,J=3.2Hz,1H),4.96(dd, J=11.2,3.4Hz,1H),4.51(d,J=8.4Hz,1H),4.03(s,3H),3.90(dd,J=20.0,9.0H z,1H),3.62(dtd,J=15.8,10.2,6.0Hz,2H),2.79(t,J=7.4Hz,2H),2.11(s,3H) ,2.00(s,3H),1.89(s,3H),1.76(s,3H),1.73–1.64(m,2H),1.63–1.54(m,2H).

[0108] Preparation Example Preparation of Compound A2

[0109] Step 1: Preparation of compound A2-1

[0110] Benzyl bromide (159 g, 0.927 mol) was slowly added to a reaction mixture of Compound Int.4 (150 g, 1.03 mol) and potassium carbonate (185 g, 1.34 mol) in DMF (750 ml) and 1,4-dioxane (750 ml) with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at 90°C for 16 hours. The reaction mixture was then diluted with 3000 ml of water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to yield Compound A2-1 (60 g) as a white solid. MS: 235.0 (MH) + .

[0111] Step 2: Preparation of Compound A2-3

[0112] A reaction mixture of compound A2-1 (59 g, 0.252 mol), compound Int.5 (25 g, 0.126 mol), DIPEA (49 g, 0.378 mol), and HATU (48 g, 0.126 mol) in dichloromethane (300 mL) was stirred at 25°C for 0.5 hours. The reaction mixture was then diluted with 300 mL of water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to obtain compound A2-3 (25 g) as a yellow oil. MS: 417.7 (M+H) + . 1 H NMR(400MHz,DMSO-d6)δ7.38–7.32(m,5H),5.08(s,2H),4.18(s,2H),3.95(s,2H),3.93(s,2H),3.67–3.5 7(m,1H),3.19–3.10(m,1H),2.35(t,J=7.2Hz,2H),2.00(t,J=7.2Hz,2H),1.56–1.42(m,4H),1.37(s,9H).

[0113] Step 3: Preparation of Compound A2-4

[0114] Trifluoroacetic acid (10 mL) was slowly added to a solution of compound A2-3 (3 g, 7.2 mmol) in dichloromethane (30 mL) at 0°C with stirring, and the reaction mixture was stirred at 25°C for 3 hours. The reaction mixture was then concentrated to give compound A2-4 (2.3 g). MS: 317.2 (M+H) + .

[0115] Step 4: Preparation of Compound A2-5

[0116] Under stirring conditions at room temperature, compound Int.3 (34 g, 0.121 mol) was slowly added to a reaction mixture of compound Int.4 (8 g, 55 mmol) and DIPEA (21 g, 0.165 mol) in dichloromethane (100 ml). After the addition, the reaction mixture was heated and stirred for 0.5 hour; then the reaction mixture was diluted with 100 ml of water and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain compound A2-5 (20 g) as a white solid. 1 H NMR (400MHz, CDCl3) δ2.81–2.68(m,4H),2.01–1.85(m,4H).

[0117] Step 5: Preparation of Compound A2

[0118] Compound A2-4 (2.3 g, 7.3 mmol) was slowly added to a reaction mixture of compound A2-5 (8.6 g, 18.2 mmol) and DIPEA (2.8 g, 21.9 mmol) in dichloromethane (100 mL) at 0°C with stirring. After the addition was complete, the reaction mixture was stirred at 0°C for 0.5 hours. The reaction mixture was then diluted with 100 mL of water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A2 (2.2 g) as a white solid. MS: 611.7 (M+H) + .

[0119] Preparation Example Preparation of Compound A3

[0120] Step 1: Preparation of compound A3-1

[0121] Tert-butyl acrylate (1060 g, 8260 mmol) and NaOH (5 M, 19.8 mL) were slowly added sequentially to a DMSO (100 mL) solution of Compound Int.6 (100 g, 825.5 mmol). After complete addition, the reaction mixture was stirred at 20°C for 28 hours. The reaction mixture was then extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford a crude product. The crude product was purified to afford Compound A3-1 (74 g, 17.7%) as a light yellow oil. MS: 506.4 (M+H). + .

[0122] Step 2: Preparation of compound A3-2

[0123] Under stirring conditions at 0 degrees, CbzCl (22.7 g, 133.6 mmol) was slowly added to a dichloromethane (100 ml) reaction solution of compound A3-1 (15 g, 29.7 mmol) and TEA (9.0 g, 89.1 mmol). After the addition, the reaction mixture was stirred at 20°C for 12 hours; the reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified to give compound A3-2 (18.8 g) as a yellow oil. 1 H NMR (400MHz, DMSO-d6) δ7.36–7.30 (m, 6H), 4.97 (s, 2H), 3.54 (t, J = 6.4Hz, 6H), 3.51 (s, 6H), 2.38 (t, J = 6.0Hz, 6H), 1.39 (s, 27H).

[0124] Step 3: Preparation of compound A3-3

[0125] A reaction mixture of compound A3-2 (18.8 g) and formic acid (200 mL) was stirred at 20°C for 12 hours. The reaction mixture was then concentrated to afford compound A3-3 (16.5 g) as a yellow oil. 1H NMR (400 MHz, DMSO) δ 12.12 (s, 3H), 7.40–7.32 (m, 5H), 5.17 (s, 1H), 4.99 (s, 2H), 3.57 (t, J = 6.4 Hz, 6H), 3.50 (s, 6H), 2.42 (t, J = 6.4 Hz, 6H).

[0126] Step 4: Preparation of Compound A3-4

[0127] Under nitrogen, HOBt (13.2 g, 97.6 mmol) and EDCI (18.7 g, 97.6 mmol) were slowly added to a DMF (120 mL) solution of Compound A3-3 (11.5 g, 24.4 mmol), N-tert-butyloxycarbonyl-1,3-propanediamine (17.0 g, 97.6 mmol), and DIEA (25 g, 193.6 mmol) with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to afford Compound A3-4 (16 g) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.37–7.28(m,5H),6.83(s,3H),5.55(s,1H),5.16(t,J=6.4Hz,3H),5.03(s,2H),3.68(t,J=6. 0Hz, 6H), 3.65 (s, 6H), 3.27–3.24 (m, 6H), 3.14–3.10 (m, 6H), 2.40 (t, J = 5.6Hz, 6H), 1.70–1.47 (m, 6H), 1.43 (s, 27H).

[0128] Step 5: Preparation of Compound A3-5

[0129] A reaction mixture of compound A3-4 (16 g) in dichloromethane (100 ml) and trifluoroacetic acid (20 ml) was stirred at 20°C for 12 hours; the reaction mixture was then concentrated to obtain a yellow oil compound A3-5 (20 g), which was used directly in the next reaction without purification.

[0130] Step 6: Preparation of Compound A3-6

[0131] Under stirring and nitrogen protection at 0 degrees, compound A1 (5 g) in dichloromethane (20 ml) was slowly added to the reaction mixture of compound A3-5 (5 g) and DIEA (2.8 g) in acetonitrile (20 ml). After the addition, the reaction mixture was stirred at room temperature for 12 hours; then the reaction mixture was diluted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain compound A3-6 (3.85 g) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ7.82-7.79(m,6H),7.71(t,J=5.6Hz,3H),7.37-7.27(m,5H),6.51(s,1H),5 .21(d,J=7.6Hz,3H),4.98-4.95(m,5H),4.48(d,J=7.6Hz,3H),4.04-4.00(m,9H),3.90-3.83(m,3H ),3.72-3.67(m,3H),3.56-3.48(m,12H),3.43-3.37(m,3H),3.05-2.99(m,12H),2.27(t,J=6.4Hz, 6H), 2.10 (s, 9H), 2.04 (t, J = 7.6Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.51–1.45 (m, 18H).

[0132] Step 7: Preparation of Compound A3

[0133] Compound A3-6 (4.5 g) was dissolved in methanol (25 ml), palladium carbon (1 g) was added to the reaction mixture under stirring, and then the reaction mixture was stirred under hydrogen pressure at room temperature for 1.5 hours; the reaction mixture was filtered and concentrated to obtain white solid compound A3 (4.2 g).

[0134] Preparation of Example Compound A4

[0135] Step 1: Preparation of compound A4-1

[0136] Under nitrogen, a reaction mixture of compound A3 (410 mg, 0.23 mmol), compound A2 (209 mg, 0.34 mmol), and TEA (70 mg, 0.69 mmol) in DMF (5 mL) was stirred at 30°C for 12 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound A4-1 (370 mg), which was used directly in the next step without purification. MS: 1110.8 (1 / 2M+H)+ .

[0137] Step 2: Preparation of compound A4-2

[0138] Compound A4-1 (370 mg) was dissolved in methanol (5 ml), and palladium carbon (300 mg) was added to the reaction mixture under stirring. The reaction mixture was then stirred under hydrogen pressure at room temperature for 1.5 hours. The reaction mixture was filtered and concentrated to obtain compound A4-2 (280 mg), which was used directly in the next reaction without purification.

[0139] Step 3: Preparation of compound A4-3

[0140] Under nitrogen protection and stirring at 0°C, compound Int.3 (72 mg, 0.26 mmol) was slowly added to a solution of compound A4-2 (280 mg, 0.13 mmol) and DIEA (50 mg, 0.39 mmol) in acetonitrile (5 mL). The reaction mixture was then stirred at 25°C for 12 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound A4-3 (280 mg). MS: 1148 (1 / 2M+H) + .

[0141] Step 4: Preparation of Compound A4

[0142] A reaction mixture of compound A4-3 (280 mg, 0.12 mmol), compound Int.7 (100 mg, 0.24 mmol), and DIEA (46 mg, 0.36 mmol) in acetonitrile (5 mL) was stirred at 20°C for 12 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was then separated and purified to yield compound A4 (80 mg). MS: 1115.1 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.90–7.78(m,6H),7.74(t,J=5.6Hz,3H),7.38–7.26(m,5H),7.26–7.13(m,5H),7.01(s,1H),6.88(ddd,J=9.0 ,6.6,1.9Hz,4H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,4H),4.48(d,J=8.5Hz,3H),4.24–4.11(m,6H),4.03(d,J=3.8Hz,9H), 3.93(dd,J=12.6,5.7Hz,5H),3.86(dd,J=11.2,8.8Hz,3H),3.77-3.65(m,10H),3.58-3.47(dd,J=11.1,4.5Hz,14H),3.03(dq,J=12.4, 6.5, 5.4Hz, 14H), 2.27 (t, J = 6.3Hz, 6H), 2.10 (s, 10H), 2.08–2.01 (m, 12H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.56–1.38 (m, 26H).

[0143] Preparation of Example Compound A5 and Compound A6

[0144] Step 1: Preparation of Compound A5

[0145] Synthesis of Compound A5: Compound A4 was prepared by using Compound Int.8 instead of Compound Int.5. MS: 1129.9 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.93–7.82(m,6H),7.77(s,3H),7.42(d,J=7.7Hz,1H),7.33(t,J=6.8Hz,5H),7.22(dd,J=10.4,7.7Hz,6H),7 .04(s,1H),6.90(dd,J=8.7,6.0Hz,6H),5.23(d,J=3.4Hz,3H),4.99(dd,J=11.2,3.4Hz,3H),4.50(d,J=8.5Hz,3H),4.42(s,1H),4.1 6(s,1H),4.04(s,8H),3.89(d,J=10.2Hz,3H),3.84–3.68(m,14H),3.55(d,J=7.2Hz,14H),3.48–3.39(m,7H),3.06(q,J=6.3Hz,15H) ,2.30(t,J=6.5Hz,10H),2.12(s,9H),2.06(t,J=7.3Hz,10H),2.02(s,9H),1.91(s,9H),1.79(s,9H),1.51(dt,J=19.5,9.9Hz,32H).

[0146] Step 2: Preparation of Compound A6

[0147] A reaction mixture of compound A5 (59 mg, 0.023 mmol) and 7M ammonia in methanol (6 mL) was stirred at 20°C for 40 hours. The reaction mixture was then concentrated to obtain a crude product, which was then separated and purified to obtain compound A6 (17.5 mg). MS: 941.1 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.88(t,J=5.7Hz,3H),7.78(t,J=5.6Hz,3H),7.66(d,J=9.0Hz,3H),7.37–7.29(m,4H),7.22(td,J=9.5,2.8 Hz,5H),7.06(s,1H),6.90(dt,J=8.9,3.4Hz,4H),5.01-4.90(m,3H),4.67–4.55(m,6H),4.51(d,J=4.3Hz,3H),4.42(d,J=4.4Hz,1H) ,4.24(d,J=8.4Hz,3H),4.20-4.14(m,1H),3.82(d,J=8.5Hz,2H),3.76(s,8H),3.74–3.70(m,5H),3.67(q,J=3.5Hz,4H),3.62–3.39( m,29H),3.32(dd,J=10.9,4.8Hz,6H),3.12-2.95(m,15H),2.39–2.21(m,10H),2.12-1.98(m,12H),1.82(s,9H),1.69–1.38(m,33H).

[0148] Preparation of Example Compound A7

[0149] Compound A7 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.9. MS: 954.6 (1 / 2[M-DMTr]+H) + . 1H NMR(400MHz,DMSO-d6)δ7.89(s,3H),7.78(s,3H),7.66(d,J=9.1Hz,3H),7.38–7.30(m,4H),7 .21(dd,J=8.9,2.6Hz,5H),7.06(s,1H),6.95–6.86(m,4H),5.02(d,J=4.0Hz,1H),4.96–4.89 (m,1H),4.66–4.55(m,6H),4.51(d,J=4.3Hz,3H),4.24(d,J=8.4Hz,2H),4.17(s,1H),3.76(s ,8H),3.72(d,J=9.6Hz,4H),3.66(d,J=4.1Hz,4H),3.54(td,J=11.8,11.3,5.4Hz,20H),3.48– 3.41(m,8H),3.38(s,7H),3.34–3.28(m,6H),3.05(q,J=6.2Hz,15H),2.30 (t,J=6.3Hz,12H),2.07(t,J=7.3Hz,9H),1.82(s,9H),1.59–1.31(m,36H).

[0150] Preparation of Example Compound A8 and Compound A9

[0151] Compound A8 was synthesized by referring to Compound A4 and replacing Compound Int.5 with Compound Int.10 to obtain Compound A8. MS: 1122.6 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.93–7.82(m,5H),7.78(t,J=5.6Hz,2H),7.38–7.29(m,4H),7.23(td,J=9.6,9.1,2.7Hz,5H),7.06(s,1H),6 .96–6.88(m,4H),5.25(d,J=3.4Hz,2H),5.06–4.91(m,3H),4.52(d,J=8.5Hz,2H),4.43(s,0H),4.32(s,-1H),4.18(s,1H),4.06(s,1 0H),3.97–3.85(m,3H),3.83–3.69(m,12H),3.65–3.52(m,14H),3.44(d,J=9.3Hz,6H),3.35(s,4H),3.20(s,1H),3.08(d,J=7.6Hz,1 4H),2.39–2.21(m,9H),2.14(s,10H),2.08(t,J=7.2Hz,12H),2.03(s,11H),1.93(s,9H),1.81(s,9H),1.52(dt,J=20.3,7.0Hz,26H).

[0152] Compound A9 was synthesized by referring to Compound A6 and replacing Compound A5 with Compound A8 to obtain Compound A9. MS: 933.01 (1 / 2[M-DMTr]+H) + . 1 H NMR(400MHz, DMSO-d6)δ7.88(s,2H),7.77(s,2H),7.65(d,J=9.0Hz,2H),7.37–7.29(m,4H),7.28–7.17(m, 5H),7.05(s,0H),6.90(d,J=7.5Hz,4H),4.66–4.55(m,6H),4.50(d,J=4.3Hz,3H),4.24(d,J=8.3Hz,3H),4 .08–3.95(m,1H),3.76(s,10H),3.74–3.63(m,11H),3.54(td,J=12.1,11.7,5.5Hz,23H),3.46(d,J=9.5Hz ,8H),3.31(s,6H),3.06(s,16H),2.37–2.21(m,9H),2.13–1.96(m,14H),1.82(s,9H),1.58–1.39(m,29H).

[0153] Preparation of Example Compound A10

[0154] Step 1: Preparation of compound A10-1

[0155] Saturated sodium bicarbonate solution (30 mL) and CbzCl (5.4 g, 32 mmol) were added to a solution of tert-butyl (3-aminopropyl)carbamate (5 g, 29 mmol) in dichloromethane (10 mL) with stirring at room temperature. After the addition, the reaction mixture was stirred at 20°C for 1 hour. The reaction mixture was then extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield benzyl tert-butylpropane-1,3-diyldicarbamate (7 g) as a white solid. MS: 309.4 (M+H). + .

[0156] Trifluoroacetic acid (10 mL) was slowly added to a solution of benzyl tert-butylpropane-1,3-diyldicarbamate (3 g, 9.7 mmol) in dichloromethane (10 mL) at 0°C with stirring. The reaction mixture was stirred at 25°C for 3 hours. The reaction mixture was then concentrated to obtain compound A10-1 (5 g) as a trifluoroacetate salt. MS: 209.2 (M+H) + .

[0157] Step 2: Preparation of compound A10-2

[0158] TEA (5.8 g, 58 mmol) and glutaric anhydride (1.25 g, 12.5 mmol) were slowly added to a dichloromethane (10 mL) reaction solution of the trifluoroacetate salt of Compound A10-1 (5 g, 9.6 mmol) at 20°C with stirring. After the addition, the reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield Compound A10-2 (2.5 g) as a white solid. MS: 323.2 (M+H). + .

[0159] Step 3: Preparation of compound A10-3

[0160] TEA (2.35 g, 23.3 mmol) and intermediate Int.3 (2.6 g, 9.3 mmol) were slowly added to a dichloromethane (10 mL) solution of compound A10-2 (2.5 g, 7.76 mmol) under stirring at 20°C. After the addition was complete, the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A10-3 (2.5 g) as a white solid. MS: 489.2 (M+H)+ .

[0161] Step 4: Preparation of Compound A10-4

[0162] Under nitrogen, a reaction mixture of Compound A3 (1 g, 0.558 mmol), Compound A10-3 (410 mg, 0.839 mmol), and TEA (0.17 g, 1.67 mmol) in DMF (5 mL) was stirred at 35°C for 16 hours. The reaction mixture was then diluted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford a crude product. The crude product was purified to afford Compound A10-4 (0.9 g) as a white solid. MS: 1096.7 (1 / 2 M+H) + .

[0163] Step 5: Preparation of Compound A10-5

[0164] Compound A10-4 (900 mg, 0.43 mmol) was dissolved in methanol (20 mL). Palladium on carbon (600 mg) was added to the reaction mixture with stirring. The reaction mixture was then stirred under hydrogen pressure at room temperature for 0.5 hours. The reaction mixture was filtered and concentrated to obtain compound A10-5 (750 mg), which was used directly in the next reaction without purification. MS: 982.8 (1 / 2M+H) + .

[0165] Step 6: Preparation of Compound A10-6

[0166] Under nitrogen protection and stirring at 20°C, TEA (93 mg, 0.93 mmol) and Cy5-NHS-ester (100 mg, 0.135 mmol) were slowly added to a reaction mixture of compound A10-5 (344 mg, 0.176 mmol) in DMF (2 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then diluted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, compound A10-6 (120 mg). MS: 1295.0 (1 / 2M+H). + .

[0167] Step 7: Preparation of Compound A10

[0168] A reaction mixture of compound A10-6 (120 mg, 0.046 mmol) and 7M ammonia in methanol (6 mL) was stirred at 20°C for 2 hours. The reaction mixture was then concentrated to obtain a crude product, which was then separated and purified to yield compound A10 (22.1 mg). MS: 1105.9 (1 / 2 [M] + H) + .1 H NMR (400MHz, DMSO-d6) δ8.33(t,J=13.2Hz,2H),7.79(s,2H),7.67(d,J=5.1Hz,2H),7.33(dd,J=8.3,2.7Hz,2H),6.59(t,J=12.3 Hz,1H),6.29(t,J=14.2Hz,2H),4.23(d,J=8.4Hz,3H),4.09(s,2H),3.73–3.66(m,6H),3.65(d,J=3.0Hz,3H),3.60(s,3H),3.54( d,J=2.9Hz,6H),3.49(dd,J=11.1,6.4Hz,11H),3.42(dd,J=10.5,3.1Hz,3H),3.38–3.33(m,3H),3.31(t,J=6.3Hz,3H),3.06–2.9 6(m,17H),2.28(t,J=6.1Hz,6H),2.05(t,J=7.2Hz,12H),1.81(s,9H),1.69(s,12H),1.56–1.37(m,24H),1.28(d,J=26.7Hz,4H).

[0169] Preparation of Example Compound A11

[0170] Compound A11 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.11. MS: 940.0 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.85(d,J=6.0Hz,2H),7.75(t,J=5.5Hz,2H),7.63(d,J=9.0Hz,2H),7.37-7.26(m,4 H),7.26–7.12(m,5H),7.03(s,1H),6.94–6.78(m,4H),5.02-4.86(m,1H),4.65–4.52(m,5H),4.48(d,J=4.3H z,2H),4.21(d,J=8.3Hz,2H),4.14(s,1H),3.83–3.60(m,19H),3.60-3.46(m,22H),3.45–3.38(m,5H),3.29 -3.13(m,9H),3.10-2.90(m,14H),2.35-2.19(m,10H),2.13-1.92(m,12H),1.79(s,9H),1.61-1.33(m,32H).

[0171] Preparation of Example Compound A12

[0172] Compound A12 was synthesized with reference to Compound A6, and Compound A12 was prepared by using Compound Int.12 instead of Compound Int.8. MS: 933.2 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.89(t,J=5.6Hz,3H),7.78(t,J=5.6Hz,3H),7.66(d,J=9.1Hz,3H),7.40–7.29(m,4H),7.2 2(dt,J=9.1,5.1Hz,5H),7.06(s,1H),6.94–6.86(m,4H),5.02(s,1H),4.67–4.57(m,6H),4.51(d,J=4.3Hz,3H),4.4 2(s,1H),4.24(d,J=8.4Hz,3H),4.15(d,J=10.6Hz,1H),4.06–3.92(m,2H),3.79–3.64(m,18H),3.61–3.41(m,26H) ,3.31(t,J=6.2Hz,2H),3.05(q,J=6.3Hz,14H),2.36–2.18(m,11H),2.12(s,15H),1.82(s,9H),1.60–1.37(m,28H).

[0173] Preparation of Example Compound A13

[0174] Compound A13 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.13 to obtain Compound A13. MS: 926.2 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.85 (s, 2H), 7.63 (d, J = 8.8Hz, 2H), 7.31 (s, 4H), 6.88 (s, 5H), 5.03-4.87 (m, 1H), 4 .58(dd,J=14.0,6.1Hz,6H),4.48(d,J=4.4Hz,3H),4.21(d,J=8.5Hz,2H),4.16-4.06(m,1H),3.96-3.80(m ,2H),3.73(s,10H),3.69(d,J=9.4Hz,4H),3.64(s,4H),3.57–3.45(m,20H),3.45-3.37(m,4H),3.30(d,J= 9.5Hz,10H),3.10-2.95(s,15H),2.46-2.36(m,1H),2.12-1.94(m,13H),1.79(s,9H),1.59–1.32(m,27H).

[0175] Preparation of Example Compound A14

[0176] Compound A14 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.14 to obtain Compound A14. MS: 941.2 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.08-8.00(m,1H),7.86(s,2H),7.75(t,J=5.7Hz,3H),7.63(d,J=9.1Hz,2H),7.34-7.26(m,4H),7.24 –7.14(m,5H),7.03(s,1H),6.92–6.83(m,4H),5.03-4.85(m,1H),4.65–4.53(m,6H),4.48(d,J=4.3Hz,3H),4.43-4.30(m,1H), 4.21(d,J=8.4Hz,4H),4.17-3.75(m,4H),3.73(s,8H),3.69(d,J=9.7Hz,5H),3.65(d,J=7.3Hz,4H),3.61–3.38(m,27H),3.32– 3.25(m,7H),3.21-3.14(m,1H),3.11–2.95(m,15H),2.38–2.14(m,9H),2.13-1.93(m,16H),1.79(s,10H),1.61–1.29(m,28H).

[0177] Preparation of Example Compound A15

[0178] Compound A15 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.15. MS: 940.1 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.85(t,J=5.6Hz,3H),7.75(t,J=5.6Hz,3H),7.63(d,J=9.1Hz,2H),7.37–7.26(m,4H),7.19(td,J=9.6, 9.1,3.0Hz,5H),7.04(s,1H),6.92–6.83(m,4H),5.05-4.85(m,1H),4.58(s,6H),4.49(s,3H),4.43-4.35(m,1H),4.21(d,J=8.4H z,3H),4.17-4.07(m,1H),3.73(s,9H),3.71-3.61(m,9H),3.61–3.46(m,23H),3.42-3.38(m,6H),3.31-3.23(m,6H),3.20-3.13 (m,1H),3.10-2.96(m,14H),2.28(t,J=6.4Hz,15H),2.04(t,J=7.3Hz,10H),1.95-1.84(m,3H),1.79(s,9H),1.60–1.36(m,28H).

[0179] Preparation of Example Compound A16

[0180] Synthesis of Compound A16 Referring to Compound A6, Compound A16 was prepared by using Compound Int.16 instead of Compound Int.8. MS: 971.3 (1 / 2[M-DMTr]+H) + . 1H NMR(400MHz, DMSO-d6)δ8.13-8.06(m,1H),7.75(t,J=5.4Hz,2H),7.63(d,J=9.0Hz,2H),7.35–7.13(m,14H),7.09-6.97(m, 1H),6.91–6.84(m,6H),5.04-4.87(m,1H),4.58(dd,J=14.3,6.0Hz,6H),4.48(d,J=4.3Hz,2H),4.40(s,1H),4.21(d,J=8.4 Hz,3H),4.15(s,1H),4.06-3.98(m,1H),3.76–3.71(m,11H),3.71–3.60(m,9H),3.59–3.34(m,29H),3.31-3.26(m,6H),3.2 1-3.16(m,1H),3.09-2.95(m,14H),2.38-2.22(m,2H),2.10(s,1H),2.04(t,J=7.3Hz,7H),1.79(s,9H),1.68–1.35(m,29H).

[0181] Preparation of Example Compound A17

[0182] Compound A17 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.17. MS: 940.1 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.86(t,J=5.7Hz,3H),7.75(t,J=5.6Hz,2H),7.63(d,J=9.1Hz,2H),7.58-7.51(m,1H),7.35–7.25(m,4H),7. 19(td,J=9.4,3.2Hz,5H),7.03(s,1H),6.92–6.82(m,4H),5.02-4.84(m,1H),4.66–4.53(m,6H),4.48(d,J=4.3Hz,3H),4.39(s,1H),4 .21(d,J=8.4Hz,3H),4.14(d,J=10.8Hz,2H),3.73(s,8H),3.69(d,J=9.6Hz,6H),3.65(d,J=7.2Hz,4H),3.59-3.46(m,21H),3.44-3.3 4(m,7H),3.32-3.25(m,5H),3.20-2.90(m,16H),2.26(q,J=11.5,8.9Hz,10H),2.16–1.96(m,15H),1.79(s,10H),1.60–1.27(m,30H).

[0183] Preparation of Example Compound A18 and Compound A19

[0184] Compound A18 was synthesized by referring to Compound A4 and replacing Compound Int.5 with Compound Int.18. MS: 1122.7 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.92–7.82(m,6H),7.77(t,J=5.7Hz,3H),7.36–7.31(m,4H),7.22(dd,J=10.3,7.4Hz,5H),7.04(s,1H),6.90 (dd,J=8.5,5.7Hz,4H),5.24(d,J=3.3Hz,3H),4.99(dd,J=11.3,3.5Hz,4H),4.51(d,J=8.4Hz,3H),4.17(s,1H),4.05(s,9H),3.90(q, J=9.3Hz,3H),3.76(s,12H),3.67–3.52(m,18H),3.46–3.41(m,3H),3.29(s,3H),3.19(d,J=10.5Hz,3H),3.06(q,J=6.2Hz,12H),2.30 (t,J=6.4Hz,6H),2.24(s,6H),2.13(s,9H),2.10–2.05(m,8H),2.02(s,9H),1.91(s,9H),1.80(s,9H),1.52(dd,J=15.6,9.2Hz,28H).

[0185] Compound A19 was synthesized by referring to Compound A6 and replacing Compound A5 with Compound A18 to obtain Compound A19. MS: 1106.6 (1 / 2 [M] + Na) + . 1 H NMR (400MHz, DMSO-d6) δ7.89(t,J=5.6Hz,3H),7.78(t,J=5.7Hz,3H),7.66(d,J=9.1Hz,3H),7.34(t,J=6.8Hz,4H),7.31–7.27(m, 1H),7.22(dt,J=9.1,5.0Hz,5H),6.91(td,J=7.4,3.7Hz,6H),5.03(d,J=4.0Hz,1H),4.66–4.57(m,6H),4.51(d,J=4.3Hz,3H),4. 25(d,J=8.4Hz,3H),3.77(s,9H),3.74–3.66(m,9H),3.55(dq,J=11.2,5.5Hz,21H),3.49–3.41(m,6H),3.32(t,J=6.1Hz,6H),3.1 9(d,J=10.4Hz,3H),3.06(t,J=6.2Hz,12H),2.35–2.20(m,12H),2.14–2.00(m,10H),1.83(s,9H),1.50(dt,J=29.8,7.5Hz,28H).

[0186] Preparation of Example Compound A20

[0187] Compound A20 was synthesized with reference to Compound A6, and Compound A4 was used instead of Compound A5 to prepare Compound A20. MS: 926.0 (1 / 2[M-DMTr]+H) + . 1 H NMR(400MHz,DMSO-d6)δ7.28(dd,J=6.5,4.3Hz,4H),7.24–7.12(m,5H),6.90–6.82(m,4H ),4.43-4.25,(m,1H),4.24–4.08(m,8H),3.97–3.86(m,4H),3.72–3.61(m,16H),3.55–3. 45(m,18H),3.40(dd,J=10.7,3.0Hz,4H),3.31(q,J=9.4,6.4Hz,7H),3.01(q,J=6.4Hz,13 H), 2.30–2.17 (m, 8H), 2.03 (t, J = 7.2Hz, 14H), 1.79 (d, J = 1.4Hz, 9H), 1.56–1.34 (m, 27H).

[0188] Preparation of Example Compound A21

[0189] Compound A21 was synthesized with reference to Compound A6, and Compound A21 was prepared by using Compound Int.21 instead of Compound Int.8. MS: 926.2 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ8.37–8.29(m,3H),7.84(t,J=5.6Hz,3H),7.74(t,J=5.7Hz,3H),7.62(d,J=9.0Hz,3H), 7.33–7.27(m,4H),7.22–7.15(m,5H),7.02(s,1H),6.90–6.84(m,4H),5.34-5.30(m,1H),5.00-4.86(m,1H), 4.61–4.53(m,6H),4.47(d,J=4.3Hz,3H),4.21(d,J=8.4Hz,3H),3.73(s,6H),3.69(d,J=9.5Hz,5H),3.64(q,J =3.8,3.3Hz,4H),3.58-3.46(m,20H),3.45-3.35(m,6H),3.45-3.35(m,6H),3.29(t,J=6.0Hz,5H),3.09-2.9 5(m,15H),2.27(t,J=6.4Hz,6H),2.21(d,J=7.2Hz,2H),2.08-1.96(m,15H),1.80(s,9H),1.55–1.38(m,28H).

[0190] Preparation of Example Compound A22 (L96-deAc)

[0191] Compound A22 was synthesized by referring to compound A6 and replacing compound A5-1 with compound A29-1 to obtain compound A22. MS: 864.7 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.88(t,J=5.7Hz,3H),7.78(t,J=5.7Hz,3H),7.66( d,J=9.0Hz,3H),7.36–7.29(m,4H),7.21(td,J=8.8,3.0Hz,5H),7.03(s,1H) ,6.91–6.87(m,4H),5.01(d,J=4.0Hz,1H),4.65–4.56(m,6H),4.51(d,J=4. 3Hz,3H),4.23(d,J=8.4Hz,3H),4.15(d,J=11.9Hz,1H),3.75(s,6H),3.73–3 .63(m,9H),3.54(td,J=11.7,11.3,5.6Hz,21H),3.44(ddd,J=9.9,6.2,2.9 Hz,3H),3.31(q,J=6.2,5.1Hz,4H),3.17(s,1H),3.05(p,J=6.4Hz,15H),2.3 0(t,J=6.4Hz,6H),2.22(t,J=7.4Hz,2H),2.05(q,J=11.3,9.2Hz,10H),1.82 (s, 9H), 1.52 (dd, J = 12.7, 6.6 Hz, 16H), 1.43 (t, J = 7.2 Hz, 6H), 1.23 (s, 12H).

[0192] Preparation of Example Compound A23

[0193] Compound A23 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.23. MS: 926.2 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.88(t,J=5.6Hz,3H),7.78(t,J=5.6Hz,3H),7.66(d,J=9.0Hz,3H),7.37– 7.30(m,4H),7.22(td,J=9.4,3.1Hz,5H),7.06(s,1H),6.96–6.86(m,4H),6.06(s,3H),4.52(s,1H ),4.42(s,1H),4.33–4.13(m,6H),3.81–3.63(m,20H),3.63–3.45(m,33H),3.43–3.30(m,18H),3. 12–2.98(m,15H),2.30(t,J=6.5Hz,11H),2.07(t,J=7.2Hz,11H),1.82(s,9H),1.58–1.38(m,28H).

[0194] Preparation of Example Compound A24

[0195] Compound A24 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.24 to obtain Compound A24. MS: 960.1 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.87(t,J=5.6Hz,3H),7.76(t,J=5.6Hz,2H),7.63(d,J=9.1Hz,2H),7.37–7.26(m,6H),7.25–7.14(m,5H),7.02(s,1H), 6.88(ddd,J=8.8,6.3,1.7Hz,4H),5.06-4.87(m,1H),4.64–4.53(m,6H) ,4.49(d,J=4.3Hz,3H),4.43-4.36(m,1H),4.21(d,J=8.4Hz,2H),4.16-

[0196] 4.08(m,1H),3.73(d,J=1.0Hz,8H),3.69(d,J=9.8Hz,4H),3.67-3.63(m, 4H),3.61–3.38(m,22H),3.32-3.23(m,5H),3.18-3.13(m,1H),3.06-2.94 (m,14H),2.27(t,J=6.4Hz,6H),2.22(d,J=8.0Hz,1H),2.12(s,2H),2.09 –1.93(m,16H),1.84(d,J=16.3Hz,8H),1.79(s,10H),1.56–1.31(m,30H).

[0197] Preparation of Example Compound A25

[0198] Compound A25 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.25. MS: 933.4 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.10–8.00(m,1H),7.89(t,J=5.6Hz,3H),7.78(t,J=5.7Hz,3H),7.66(d,J=9.1Hz,3H),7.40–7.31(m,4H),7 .30–7.14(m,5H),7.06(s,1H),6.96–6.86(m,4H),5.06–4.90(m,1H),4.66–4.57(m,6H),4.52(d,J=4.3Hz,3H),4.43(d,J=4.8Hz,1H) ,4.25(d,J=8.4Hz,3H),4.23–3.96(m,3H),3.87(s,1H),3.80–3.64(m,17H),3.65–3.42(m,23H),3.33(dt,J=12.4,6.2Hz,5H),3.20 (s,1H),3.17–2.95(m,13H),2.43(d,J=9.3Hz,2H),2.36–2.21(m,8H),2.06(q,J=8.8,7.9Hz,16H),1.83(s,9H),1.59–1.35(m,27H).

[0199] Preparation of Example Compound A26

[0200] Compound A26 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.26. MS: 933.4 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.07(dd,J=20.3,7.7Hz,1H),7.88(t,J=5.7Hz,3H),7.78(t,J=5.6Hz,3H),7.66(d,J=9.0Hz,3H), 7.39–7.29(m,4H),7.27–7.16(m,5H),7.05(s,1H),6.96–6.85(m,4H),5.05–4.90(m,1H),4.67–4.57(m,6H),4.51(d,J=4. 3Hz,3H),4.24(d,J=8.4Hz,4H),3.99(d,J=18.3Hz,1H),3.80–3.65(m,16H),3.62–3.42(m,24H),3.36–3.16(m,8H),3.13– 2.98(m,14H),2.37–2.20(m,9H),2.07(t,J=7.2Hz,12H),1.89(s,1H),1.82(s,9H),1.61–1.34(m,27H),0.78–0.50(m,3H).

[0201] Preparation of Example Compound A27

[0202] Compound A27 was synthesized by referring to compound A29 and replacing compound Int.5 with compound Int.18 to obtain compound A27. MS: 1094.9 (1 / 2 [M] + Na) + . 1H NMR (400MHz, DMSO-d6) δ7.63(d,J=9.0Hz,3H),7.33(t,J=7.3Hz,4H),7.21(dd,J=8.8,2.5Hz,5H),7.11(s,1H),6.92–6.87(m,4 H),5.00(d,J=4.0Hz,1H),4.63–4.55(m,6H),4.49(d,J=4.3Hz,3H),4.42(s,1H),4.25(d,J=8.4Hz,3H),4.16(s,1H),3.78–3.65 (m,22H),3.56(td,J=12.0,6.3Hz,25H),3.51–3.37(m,12H),3.31(t,J=6.1Hz,6H),3.20(d,J=11.8Hz,6H),3.00(d,J=10.1Hz,3 H),2.90(s,3H),2.75(d,J=39.9Hz,2H),2.47(s,6H),2.27–2.17(m,8H),2.08(s,2H),1.81(s,9H),1.50(s,16H),1.24(s,12H).

[0203] Preparation of Example Compound A28

[0204] Compound A28 was synthesized by referring to compound A29 and replacing compound Int.5 with compound Int.21 to obtain compound A28. MS: 1051.7 (1 / 2 [M] + H) + . 1H NMR (400MHz, DMSO-d6) δ8.44(s,3H),8.35(s,3H),7.64(d,J=9.0Hz,3H),7.34(dt,J=8.6,4.4Hz,4H),7.22(td,J=9.2,8.8,3.7Hz,5H ),7.01(s,1H),6.92–6.87(m,4H),5.01(d,J=3.8Hz,1H),4.63–4.56(m,6H),4.50(d,J=4.3Hz,3H),4.42(s,1H),4.23(d,J=8.4Hz,3H) ,4.15(d,J=15.1Hz,1H),3.73(d,J=24.0Hz,12H),3.67(t,J=3.9Hz,3H),3.54(q,J=5.8Hz,21H),3.46–3.42(m,3H),3.33–3.16(m,5H ), 3.01 (d, J = 7.5Hz, 1H), 2.27 (t, J = 6.6Hz, 6H), 2.17 (s, 21H), 2.03 (t, J = 6.9Hz, 10H), 1.82 (s, 9H), 1.52–1.40 (m, 16H), 1.24 (s, 12H).

[0205] Preparation of Example Compound A29

[0206] Synthesis of Compound A29-1 Referring to Compound A1, Compound A29-1 was prepared by using Compound Int.29 instead of Compound A1-3. MS: 509.1 (M+Na) + .

[0207] Compound A29-2 was synthesized with reference to compound A3, and compound A29-2 was prepared by using compound Int.5 instead of N-tert-butyloxycarbonyl-1,3-propylenediamine. MS: 934.1 (M / 2+H) + .

[0208] Compound A29 was synthesized with reference to compound A20 by using compound A29-2 instead of compound A3 and compound A29-1 instead of compound A2 to obtain compound A29. MS: 1051.7 (1 / 2 [M] + H) +. 1H NMR (400MHz, DMSO-d6) δ7.63(d,J=9.1Hz,3H),7.33(t,J=7.3Hz,4H),7.25–7.19(m,5H),7.08(s,1H),6.92–6.87(m,4H),5.00(d,J =4.1Hz,1H),4.62–4.55(m,6H),4.49(d,J=4.3Hz,3H),4.42(s,1H),4.30–4.22(m,15H),4.15(d,J=13.6Hz,1H),4.02–3.96(m,12H ),3.76(s,6H),3.72(d,J=9.0Hz,5H),3.67(s,3H),3.58–3.53(m,15H),3.53–3.48(m,3H),3.48–3.40(m,6H),3.31(t,J=6.3Hz,6H ), 3.19 (dd, J = 8.7, 5.1Hz, 1H), 3.01 (d, J = 7.7Hz, 2H), 2.25 (s, 8H), 2.02 (d, J = 6.4Hz, 9H), 1.82 (s, 9H), 1.47 (s, 16H), 1.25 (s, 12H).

[0209] Preparation of Example Compound A30

[0210] Compound A30 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.30. MS: 940.7 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.89(t,J=5.6Hz,3H),7.78(t,J=5.6Hz,3H),7.66(d,J=9.0Hz,3H),7.37–7.32(m,4H),7.22(dt,J=9.0 ,5.6Hz,5H),7.06(s,1H),6.94–6.88(m,4H),5.03(d,J=4.1Hz,1H),4.65–4.57(m,6H),4.51(d,J=4.3Hz,3H),4.43(s,1H),4.2 5(d,J=8.4Hz,3H),4.18(s,1H),3.79–3.65(m,18H),3.61–3.42(m,27H),3.32(t,J=6.2Hz,4H),3.07(p,J=6.1Hz,15H),2.35–2 .23(m,10H),2.14–2.02(m,12H),1.91(d,J=12.6Hz,1H),1.83(s,9H),1.56–1.44(m,26H),1.27(s,2H),0.59(d,J=96.6Hz,2H).

[0211] Preparation of Example Compound A31

[0212] Synthesis of Compound A31: Compound A20 was used as a reference, and Compound A31 was prepared by using Compound Int.31 instead of Compound Int.4. MS: 912.5 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.84(t,J=5.6Hz,3H),7.74(t,J=5.7Hz,3H),7.62(d,J=9.0Hz,3H),7.31(s,4H),7.19(dt,J=9.2,4.9Hz,5H),7. 11(s,1H),6.91–6.84(m,4H),5.34-5.30(m,1H),5.00-4.85(m,1H),4.63–4.51(m,6H),4.47(d,J=4.3Hz,3H),4.21(t,J=7.5Hz,6H),3.94 (d,J=11.2Hz,4H),3.73(s,6H),3.69(d,J=9.5Hz,4H),3.64(d,J=4.1Hz,4H),3.56-3.46(m,18H),3.44-3.35(m,5H),3.29(t,J=6.1Hz,4H ),3.20-3.13(m,1H),3.08-2.98(m,13H),2.27(t,J=6.5Hz,8H),2.24-2.13(m,4H),2.08-1.92(m,11H),1.80(s,9H),1.55–1.37(m,24H).

[0213] Preparation of Example Compound A32

[0214] Compound A32 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.32. MS: 1106.7 (1 / 2 [M] + Na) + . 1H NMR (400MHz, DMSO-d6) δ7.90(t,J=5.6Hz,3H),7.80(t,J=5.5Hz,3H),7.68(d,J=9.1Hz,3H),7.36–7.30(m,4H),7.21(dt,J=9.1,5.1Hz ,5H),7.07(s,1H),6.93–6.87(m,4H),5.07(d,J=3.9Hz,1H),4.67(t,J=5.6Hz,3H),4.61(d,J=6.3Hz,3H),4.52(d,J=4.3Hz,3H),4.23( d,J=8.4Hz,3H),4.16(s,1H),3.99(s,1H),3.71(d,J=35.9Hz,21H),3.55(d,J=8.7Hz,27H),3.42–3.29(m,15H),3.10–3.02(m,12H),2. 30(t,J=6.4Hz,6H),2.23(d,J=8.0Hz,3H),2.07(t,J=7.3Hz,10H),1.82(s,9H),1.49(dq,J=25.1,7.2Hz,25H),0.59(t,J=41.8Hz,1H).

[0215] Preparation of Example Compound A33

[0216] Compound A33 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.33. MS: 926.2 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.99(s,1H),7.88(t,J=5.6Hz,3H),7.77(t,J=5.6Hz,3H),7.65(d,J=9.2Hz,3H),7.39–7.30(m,4H),7.2 2(dt,J=9.1,5.1Hz,5H),7.04(s,1H),6.95–6.85(m,4H),5.01(d,J=4.8Hz,1H),4.65–4.56(m,6H),4.50(d,J=4.3Hz,4H),4.24( d,J=8.4Hz,4H),3.79–3.65(m,15H),3.55(tt,J=11.8,5.6Hz,21H),3.49–3.37(m,9H),3.32(t,J=6.2Hz,4H),3.16(d,J=5.4Hz, 1H),3.05(q,J=6.1Hz,14H),2.34–2.22(m,8H),2.07(t,J=7.3Hz,11H),1.87(d,J=9.9Hz,1H),1.83(s,9H),1.60–1.35(m,28H).

[0217] Preparation of Example Compound A34

[0218] Compound A34 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.34. MS: 940.4 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.88(t,J=5.6Hz,3H),7.77(t,J=5.5Hz,3H),7.65(d,J=9.1Hz,3H),7.33(q,J=8.4,7.7Hz,4H),7.2 8–7.17(m,5H),7.06(s,1H),6.90(dd,J=8.7,2.5Hz,4H),5.06–4.87(m,2H),4.66–4.57(m,7H),4.51(d,J=4.3Hz,3H),4.38( d,J=40.5Hz,2H),4.27–4.13(m,4H),3.80–3.64(m,18H),3.55(td,J=11.5,5.4Hz,20H),3.47–3.38(m,8H),3.34–3.17(m,6 H),3.12–2.96(m,14H),2.30(q,J=11.1,8.7Hz,10H),2.07(t,J=7.2Hz,11H),1.89(s,1H),1.83(s,9H),1.60–1.37(m,27H).

[0219] Preparation of Example Compound A35

[0220] Step 1: Preparation of compound A35-1

[0221] A solution of potassium carbonate (7.72 g, 55.9 mmol), 6-bromohexanoic acid (5.45 g, 27.9 mmol), and benzyl bromide (9.56 g, 55.9 mmol) in DMF (60 mL) was stirred at 20°C for 16 hours. The reaction mixture was then poured into ice water and diluted with petroleum ether for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A35-1 (7.22 g, 91%). MS: 285.2 & 287.2 (M+H) + .

[0222] Step 2: Preparation of compound A35-2

[0223] A solution of potassium carbonate (0.977 g, 7.07 mmol), intermediate Int.35 (0.8 g, 3.53 mmol), and compound A35-1 (1.21 g, 4.24 mmol) in DMF (30 mL) was stirred at 50°C for 1 hour. The reaction mixture was then poured into ice water and diluted with dichloromethane for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A35-2 (2.5 g, 99%). MS: 431.5 (M+H). + .

[0224] Step 3: Preparation of compound A35-3

[0225] A solution of trifluoroacetic acid (0.429 g, 3.76 mmol) and compound A35-2 (2.7 g, 3.76 mmol) in dichloromethane (30 mL) was stirred at 20°C for 0.5 hours. The reaction mixture was then concentrated to give the crude product, compound A35-3 (5.8 g, 97%). MS: 331.5 (M+H) + .

[0226] Step 4: Preparation of compound A35-4

[0227] A solution of potassium carbonate (5.01 g, 36.2 mmol), compound A35-3 (2.02 g, 3.62 mmol), and tert-butyl 6-bromohexanoate (2.6 g, 3.63 mmol) in DMF (30 mL) was stirred at 80°C for 2 hours. The reaction mixture was then poured into ice water and diluted with dichloromethane for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A35-4 (1.2 g, 33%). MS: 501.6 (M+H). + .

[0228] Step 5: Preparation of compound A35-5

[0229] A solution of trifluoroacetic acid (0.137 g, 1.198 mmol) and compound A35-4 (1.2 g, 1.198 mmol) in dichloromethane (30 mL) was stirred at 20°C for 2 hours. The reaction mixture was then concentrated to obtain a crude product, which was purified to give compound A35-5 (470 mg, 88%). MS: 445.4 (M+H) + .

[0230] Step 6: Preparation of Compound A35-6

[0231] Under nitrogen protection at 0°C, a solution of Intermediate Int.3 (850 mg, 3.04 mmol), DIEA (654 mg, 5.06 mmol), and Compound A35-5 (45 mg, 1.012 mmol) in dichloromethane (20 mL) was stirred at 25°C for 16 hours. The reaction mixture was concentrated to obtain a crude product, which was then purified to obtain Compound A35-6 (540 mg, 87%). MS: 611.5 (M+H) + .

[0232] Step 7: Preparation of Compound A35

[0233] Compound A35 was synthesized with reference to compound A6. Compound A35-6 was used instead of compound A5-1 to prepare compound A35. MS: 926.6 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.88(t,J=5.6Hz,3H),7.78(t,J=5.7Hz,3H),7.66(d,J=9.0Hz,3H),7.33(t,J=6.8Hz,4H),7.22(td,J =8.7,2.7Hz,5H),7.03(s,1H),6.96–6.84(m,4H),5.40-5.27(m,4H),5.06–4.83(m,8H),4.67–4.54(m,6H),4.51(d,J=4.3Hz,3 H),4.41(s,3H),4.32(s,2H),4.24(d,J=8.4Hz,3H),4.16(s,3H),3.82–3.63(m,13H),3.63–3.42(m,24H),3.32–3.14(m,4H), 3.12-2.92(m,13H),2.74-2.66(m,1H),2.43-2.15(m,11H),2.13-1.97(m,10H),1.82(s,9H),1.63(s,1H),1.58-1.29(m,38H).

[0234] Preparation of Example Compound A36

[0235] Compound A36 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.36. MS: 954.3 (1 / 2[M-DMTr]+H) + . 1H NMR(400MHz, Methanol-d4)δ7.40–7.33(m,2H),7.32–7.16(m,8H),6.86(t,J=9.0Hz,4H),4.35 (d,J=8.4Hz,3H),3.96–3.86(m,8H),3.83(d,J=3.3Hz,3H),3.79–3.70(m,16H),3.69-3.62(m, 13H),3.58(dd,J=10.7,3.3Hz,4H),3.55–3.46(m,9H),3.25–3.17(m,13H),2.46-2.36(m,10H) ,2.20(t,J=7.3Hz,10H),1.98(s,9H),1.73–1.53(m,30H),1.44-1.27(dt,J=28.2,14.6Hz,7H).

[0236] Preparation of Example Compound A37

[0237] Synthesis of Compound A37 Referring to Compound A29, Compound A37 was prepared by using Compound Int.16 instead of Compound Int.5. 1 H NMR (400MHz, Methanol-d4) δ8.14(d,J=7.9Hz,3H),7.35(d,J=7.5Hz,2H),7.28-7.13(m,13H),7.04(t,J=7.4Hz,3H),6.86–6.79( m,4H),4.55(d,J=12.6Hz,4H),4.37(d,J=8.4Hz,3H),4.12(s,6H),4.01(d,J=14.5Hz,3H),3.92(t,J=9.3Hz,6H),3.82(d,J=2.9Hz ,3H),3.78–3.67(m,26H),3.60–3.45(m,12H),3.17-3.05(m,1H),3.07(d,J=7.0Hz,1H),2.88-2.74(m,9H),2.46(s,6H),2.30(d, J=7.0Hz,1H),2.24–2.16(m,2H),2.05(d,J=10.7Hz,4H),1.97(s,9H),1.90-1.82(m,3H),1.80–1.57(m,24H),1.46–1.24(m,12H).

[0238] Preparation of Example Compound A38

[0239] Compound A38 was synthesized by referring to Compound A6 and replacing Compound Int.8 with Compound Int.38 to obtain Compound A38. MS: 947.7 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.85(t,J=5.6Hz,3H),7.74(t,J=5.6Hz,3H),7.62(d,J=9.0Hz,3H),7.30(q,J=8.2,7.7Hz,4H),7.20(t d,J=9.7,9.1,3.0Hz,5H),7.02(s,1H),6.92–6.84(m,4H),4.94(dd,J=35.3,4.2Hz,1H),4.64–4.52(m,6H),4.51–4.28(m,4H), 4.25–4.09(m,4H),3.89(d,J=19.2Hz,2H),3.78–3.60(m,19H),3.58-3.46(m,19H),3.45–3.36(m,8H),3.29(t,J=6.1Hz,6H),3 .03(p,J=6.1Hz,14H),2.34–2.18(m,10H),2.12–1.95(m,12H),1.80(s,9H),1.58–1.35(m,27H),0.83(dt,J=21.2,7.8Hz,3H).

[0240] Example: A galactosamine compound III-LA20-CPG attached to a solid support

[0241] Step 1: Preparation of compound III-LA20-1

[0242] Succinic anhydride (32 mg, 0.32 mmol) was slowly added to a reaction solution of Compound A4 (400 mg, 0.16 mmol) and DMAP (117 mg, 0.96 mmol) in acetonitrile (10 mL) with stirring at 20°C. After complete addition, the reaction mixture was stirred at 20°C for 12 hours. The reaction mixture was then diluted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude product, which was purified to afford Compound III-LA20-1 (150 mg) as a yellow solid. MS: 1165.1 (1 / 2[M-DMTr]+H) + .

[0243] Step 2: Preparation of aminogalactose compound III-LA20-CPG attached to a solid support

[0244] Compound III-LA20-1 (50 mg, 0.021 mmol) was added to acetonitrile (1.5 mL), followed by HATU (10 mg, 0.026 mmol) and DIEA (10 μL). lcaa-CPG (500°A, 125 mg) was then added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was washed with acetonitrile, and 0.5 mL of CapA (acetic anhydride:tetrahydrofuran = 1:9, v / v) and 0.5 mL of CapB (n-methylimidazole:pyridine:acetonitrile = 15:10:75, v / v / v) were added to the lcaa-CPG, followed by stirring at room temperature for another 1 hour. The mixture was then filtered and washed three times with acetonitrile. Lyophilization afforded the target product, Compound III-LA20-CPG (210 mg, white powder), attached to a solid phase support.

[0245] Example Synthesis of siRNA Conjugated with Galactosamine Clusters

[0246] The siRNA used for the test, siRNA targeting mouse TTR gene mRNA (Nucleic Acids Res. 2020 Dec 2; 48(21): 11827-11844) is as follows: an aminogalactose molecule cluster M is covalently linked to the 3' end of the SS chain,

[0247] SS chain (5'-3'):a·a·caguGuUCUugcucuauaaL

[0248] AS chain (5'-3'):u·U·auaGaGCaagaAcAcuguu·u·u

[0249] Capital letters and lowercase letters represent monomers modified with 2′-deoxy-2′-fluoro (2′-F) and 2′-O-methyl (2′-OMe), respectively. · represents a thio group, and L represents an N-acetylgalactosamine ligand.

[0250] The siRNA conjugates disclosed herein are synthesized by methods well known to those skilled in the art or by methods described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates.

[0251] Synthesize siRNA conjugated with aminogalactose clusters. The siRNA used in the experiment targets mouse TTR mRNA.

[0252] Table X. siRNA numbers and sequences for evaluation of targeting ligand activity

[0253] Among them, S-L96 was purchased from Jima Gene; C, G, A, U or the base composition of nucleotides, uppercase and lowercase letters represent monomers modified with 2′-deoxy-2′-fluoro (2′-F) and 2′-O-methyl (2′-OMe), respectively, · represents thio, and L represents N-acetylgalactosamine ligand.

[0254] After the siRNA conjugates of the present invention are prepared, they are lyophilized into solid powder using standard methods and stored for future use. When used, they can be redissolved in a solution of desired concentration using, for example, water for injection, physiological saline, phosphate buffer, or phosphate buffer.

[0255] (I) ASGPR-GalNAc™ affinity assay based on monkey primary cells

[0256] Principle: The test compound competes with GalNAc-Cy5 for binding to the ASGPR receptor on the surface of monkey primary cells (PCMH, purchased from Miaoshun Biotechnology Co., Ltd.). The fluorescence intensity of Cy5 detected by flow cytometry can reflect the affinity of the test compound to ASGPR. The weaker the Cy5 fluorescence intensity, the stronger the affinity of the test compound to ASGPR, and vice versa.

[0257] Experimental procedure: Monkey primary cells were plated at 40,000 / well in 96-well plates and incubated with GalNAc-Cy5 (final concentration 100 nM) and the test compound (final concentration starting from 1 μM, 10-fold dilution for initial screening) in a 37°C, 5% CO2 incubator for 4 hours. The cells were then harvested and the fluorescence intensity of Cy5 in the cells was analyzed by flow cytometry.

[0258] Conclusion: The results of affinity detection experiments show that the affinity of most of the compounds currently tested, such as A11, A14, A15, A16, A17, A19, A21, A23, A24, A25, A26, A27, A28, A31, A32, A33, A34, A35, A36, A37, and A38 to ASGPR shows activity comparable to or slightly better than that of the L96-DeAc molecule.

[0259] (II) Inhibition of mRNA expression in vivo by siRNA conjugated with aminogalactose clusters

[0260] C57 / BL6 mice aged 6 to 8 weeks were randomly divided into 3 groups (n=3), without any treatment, The mice were divided into a control group (S-L96), a positive control group (S-L96), and a drug-treated group (S-LA20). GalNac-siRNA was administered subcutaneously at a dose of 1 mg / kg. Blood was collected from the orbits before dosing (day 0) and 3 days after dosing (day 3) for serum separation. Mice were sacrificed on day 7, and liver samples were obtained. TTR levels in mouse serum and liver samples were measured using the Mouse Prealbumin ELISA Kit (cat# ALPCO(41-PALMS-E01)). The results are as follows:

[0261] Conclusion: The in vivo efficacy test data showed that S-LA20 exhibited excellent mTTR gene expression inhibition efficiency, and the activity level at 1 mpk was comparable to that of the control group S-L96.

[0262] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A GalNAc delivery molecule as shown in the following formula (I), or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof: in, R1 is selected from the group consisting of H, DMTr, phosphate, phosphorothioate, phosphorothioate-siRNA conjugate or phosphate-siRNA conjugate; Linker is a divalent linking group; r is 0, 1, 2, 3, 4, 5 or 6; R2 is a GalNAc target head portion and has a structure as shown below: Wherein, L1, L2 and L3 are each independently a divalent linking group, and R3, R4 and R5 are each independently a substituted or unsubstituted sugar group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule; The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the following group: 1-8 Alkyl, C 2- 8-Alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, C 3-8 Aryl, 5- to 7-membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, C 1-8 Alkoxy, C 1-10 or two substituents located on adjacent ring atoms may together with the connected ring atoms form a group selected from the following group: a 5-7 membered carbon ring or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring.

2. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The linker has a structure selected from the following group: C4-C20 alkyl, wherein n and m are each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; n1 and n2 are each independently selected from the following group: 0 or 1; W is selected from the group consisting of a single bond, a substituted or unsubstituted C 1-8 Alkylene, substituted or unsubstituted 7-12 membered fused bicyclic ring, substituted or unsubstituted 7-15 membered spirocyclic ring, substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring, -NH-substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring-NH-, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring-NH-, -NH-substituted or unsubstituted 5-15 membered bridged ring-NH-; wherein the ring atoms of the fused bicyclic ring, spirocyclic ring and bridged ring may be optionally carbon atoms or heteroatoms; The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the following group: 1-8 Alkyl, C 2- 8-Alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, C 1-8 Alkoxy, C 1-10 Sulfonyl or two substituents located on adjacent ring atoms may together with the connected ring atoms form a group selected from the following group: a 5-7 membered carbon ring or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring; Or W has a structure selected from the group consisting of: wherein s1, s2, s, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2; s7 is selected from the group consisting of 1, 2, 3, 4, 5 or 6; Each of M1, M2, M3 and M4 is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR; M5 and M6 are each independently selected from the following group: (CHR) s6 、C(R)R、C(O)、(CHR) s6 -C(O), O, S, NR; R is selected from the following group: H, halogen, methyl; or two R located on adjacent reducing atoms and the carbon atom to which they are connected together form a substituted or unsubstituted 4-8 membered carbocyclic or heterocyclic ring (including saturated, unsaturated or aromatic ring).

3. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: Said W has a structure selected from the following group:

4. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The L1, L2 and L3 are each independently selected from the following structures: wherein each u, v and w are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; u1 and u2 are independently selected from 0 or 1; Glu is an unmodified or modified 5-6-membered glycosyl; X and Y are each independently selected from the following group: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group; The cycloalkyl and heterocyclic groups may be monocyclic, condensed, bridged or spirocyclic; the heterocyclic group may be saturated or partially unsaturated (but not aromatic).

5. The GalNAc delivery molecule according to claim 4, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The X and Y each independently have a structure selected from the following group: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, or The structure of the bottom group:

6. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The sugar group has a structure shown in the following formula III: Among them, the R 11 Selected from the group consisting of -NH(C2-C6 acyl), -NH(halogenated C2-C6 acyl), -NH(C2-C6 sulfonyl), -NH(halogenated C2-C6 sulfonyl); R 12 , R 13 and R 14 Each is independently selected from the following group: H or C1-C6 acyl.

7. The GalNAc delivery molecule according to claim 6, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The R 11 Selected from the group consisting of: -NH(C2-C4 acyl); R 12 , R 13 and R 14 Each is independently selected from the following group: H or C1-C4 acyl.

8. The GalNAc delivery molecule according to claim 6, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The R 11 is -NHAc; R 12 , R 13 and R 14 Each is independently selected from the group consisting of H or Ac.

9. The GalNAc delivery molecule according to any one of claims 1 to 8, or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof, characterized in that: The GalNAc delivery molecule has a structure selected from the group consisting of: Wherein, X, Y, Z and W are each independently selected from the following groups: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group; R3, R4, R5 and R5' are each independently a substituted or unsubstituted sugar group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule; The remaining groups are as defined above.

10. The GalNAc delivery molecule according to any one of claims 1 to 8, or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof, characterized in that: The GalNAc delivery molecule has a structure selected from the group consisting of: The R1 group is as defined in claim 1.

11. The GalNAc delivery molecule according to any one of claims 1 to 8, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, characterized in that: The GalNAc delivery molecule has a structure selected from the group consisting of:

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: one or more of the compound of formula I according to any one of claims 1 to 11, its pharmaceutically acceptable salt, racemate, R-isomer, S-isomer or mixture thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

13. The use of a compound of formula I as claimed in any one of claims 1 to 11, its pharmaceutically acceptable salt, racemate, R-isomer, S-isomer or mixture thereof, characterized in that: Used for preparing a pharmaceutical composition for treating or preventing tumors or infections caused by viruses.

14. Use of a compound of formula (I) according to any one of claims 1 to 11 for preparing a medicament for treating and / or preventing a disease or condition selected from the group consisting of a disease or condition caused by hepatitis B virus, a disease or condition caused by abnormal expression of ANGPTL3 gene, a disease or condition caused by abnormal expression of TTR gene, a disease or condition caused by abnormal expression of hepatocyte genes, or other liver diseases or conditions.

15. A method for detecting ASGPR-GalNAc affinity based on monkey primary cells.

16. A GalNAc delivery molecule linker as shown in formula (Ia), or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof: The moiety shown in formula (Ia) has a structure selected from the group consisting of: in, It is the site connected to R1.

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