Novel GalNAc targeted delivery fragment and preparation and application thereof

Abstract

The invention discloses a GalNAc delivery molecule shown in the following formula (I), or a stereoisomer, an enantiomer or a pharmaceutically acceptable salt thereof, wherein the definition of each group is described in the specification.

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] Ax is a substituted or unsubstituted 4-12 membered saturated or partially unsaturated nitrogen-containing heterocyclic group, or a substituted or unsubstituted 4-12 membered nitrogen-containing heteroaryl group;

[0010] R1 is selected from the group consisting of H, DMTr, phosphate, phosphorothioate, phosphorothioate-siRNA conjugate, or phosphate-siRNA conjugate;

[0011] Linker is a divalent linking group;

[0012] Q is selected from the group consisting of a chemical bond, -NH-C(O)-, and -C(O)-;

[0013] r is 0, 1, 2, 3, 4, 5, or 6;

[0014] R2 is a GalNAc target head portion and has a structure selected from the group consisting of:

[0015] wherein L1 and L2 are each independently a trivalent linking group; L3 is 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;

[0016] 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 2-8Alkenyl, 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.

[0017] In another preferred embodiment, Having a structure selected from the group consisting of:

[0018] Wherein, R' is selected from the following group: H or C 1-8 alkyl;

[0019] p is 1, 2, 3, 4, 5 or 6.

[0020] 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.

[0021] In some embodiments, the linker has a structure selected from the following group:

[0022] C4-C20 alkylene,

[0023] wherein n, n3 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;

[0024] n1 and n2 are each independently selected from the group consisting of: 0 or 1;

[0025] Z is selected from the following groups: single bond, substituted or unsubstituted C 1-8Alkylene, 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;

[0026] 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 1-10 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 carbocyclic or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring;

[0027] Alternatively, Z has a structure selected from the group consisting of:

[0028] wherein s1, s2, s3, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2;

[0029] s7 is selected from the group consisting of 1, 2, 3, 4, 5, or 6;

[0030] Each of M1, M2, M3 and M4 is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR;

[0031] 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;

[0032] R is selected from the following group: H, halogen, methyl; or two R located on adjacent reducing sub-atoms and the carbon atom to which they are connected together constitute a substituted or unsubstituted C 3-8 Saturated or partially unsaturated carbon ring, 4-8 membered saturated or partially unsaturated heterocyclic ring, C 6-10aromatic ring or 5-12 membered heteroaromatic ring.

[0033] In some embodiments, Z has a structure selected from the group consisting of:

[0034] In some embodiments, L1, L2 and L3 are each independently a structure shown in the following formula:

[0035] The L3 is a structure shown in the following formula:

[0036] Among them, R 11 Selected from the following group: H or C 1-4 alkyl;

[0037] Each u, v and w is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; Glu is an unmodified or modified 5-6 membered glycosyl;

[0038] X1 is selected from the following group: substituted or unsubstituted C 4-10 Saturated or partially unsaturated carbocyclic group, substituted or unsubstituted 4-10 membered saturated or partially unsaturated heterocyclic group (preferably nitrogen-containing heterocyclic group), substituted or unsubstituted C 6-10 Phenyl, or substituted or unsubstituted 5-10 membered heteroaryl;

[0039] X2 is selected from the following groups: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, substituted or unsubstituted C 4-10 Saturated or partially unsaturated carbocyclic group, substituted or unsubstituted 4-10 membered saturated or partially unsaturated heterocyclic group (preferably nitrogen-containing heterocyclic group), substituted or unsubstituted C 6-10 Phenyl, or substituted or unsubstituted 5-10 membered heteroaryl;

[0040] The carbocyclic group and heterocyclic group may be a monocyclic ring, a condensed ring, a bridged ring or a spiro ring.

[0041] In some embodiments, the R2 has a structure as shown below:

[0042] In some embodiments, X1 is a substituted or unsubstituted structure selected from the following group:

[0043] wherein s1, s2, s, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2;

[0044] s7 is selected from the group consisting of 1, 2, 3, 4, 5, or 6;

[0045] Each of M1, M2, M3 and M4 is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR;

[0046] 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;

[0047] 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).

[0048] In another preferred embodiment, the X2 is a substituted or unsubstituted structure selected from the following group:

[0049] In some embodiments, the sugar group has a structure as shown in Formula III below:

[0050] Among them, the R 15 Selected from the group consisting of -NH(C2-C6 acyl), -NH(halogenated C2-C6 acyl), -NH(C2-C6 sulfonyl), -NH(halogenated C2-C6 sulfonyl);

[0051] R 12 、R 13 and R 14 Each is independently selected from the group consisting of H or C1-C6 acyl.

[0052] In some embodiments, the R 15 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.

[0053] In some embodiments, the R 15 is -NHAc; R 12 、R 13 and R 14 Each is independently selected from the group consisting of H or Ac.

[0054] In some embodiments, the GalNAc delivery molecule has a structure selected from the group consisting of:

[0055] In some embodiments, the GalNAc delivery molecule has a structure selected from the group consisting of:

[0056] In the second aspect of the present invention, a pharmaceutical composition is provided, characterized in that the pharmaceutical composition comprises: one or more of the compound of formula I according to the first aspect of the present invention, 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.

[0057] In some embodiments, the invention is used to prepare a pharmaceutical composition for treating or preventing tumors or infections caused by viruses.

[0058] In some embodiments, the invention is used to prepare a medicament for treating and / or preventing a disease or condition selected from the following groups: 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.

[0059] In a third aspect of the present invention, there is provided a GalNAc delivery molecule linker as shown in formula (Ia), or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof:

[0060] The part shown in formula (Ia) has a structure selected from the group consisting of

[0061] in, It is the site connected to R1.

[0062] 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

[0063] Based on extensive and in-depth research, the inventors have developed a novel GalNAc-based targeted drug delivery fragment. These GalNAc-based targeted drug delivery fragments offer excellent targeted delivery, therapeutic efficacy, and enhanced safety. Based on these findings, the inventors completed the present invention.

[0064] the term

[0065] In the present invention, the halogen is F, Cl, Br or I.

[0066] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0067] 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.

[0068] 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, butoxy and the like.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Unless otherwise stated, where substituents are designated by their conventional chemical formula (written from left to right), they equivalently include chemically identical substituents resulting from the structure written from right to left, e.g., -NH-C(O)- is intended to include -C(O)-NH-, and -COO- is intended to include -OCO-.

[0076] Pharmaceutical compositions containing active ingredients

[0077] 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.

[0078] "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 Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0079] 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).

[0080] 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.

[0081] 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.

[0082] 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.

[0083] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0084] 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.

[0085] 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.

[0086] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (eg, anti-HBV agents).

[0087] 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.

[0088] 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.

[0089] The present invention will be further described below in conjunction with 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 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.

[0090] 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.

[0091] 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).

[0092] 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):

[0093] Acidic conditions: A: 0.05% trifluoroacetic acid in water; B: 0.05% trifluoroacetic acid in acetonitrile;

[0094] Alkaline conditions: A: 0.05% NH3·H2O aqueous solution; B: acetonitrile

[0095] Neutral conditions: A: 10 mM NH4OAC in water; B: acetonitrile

[0096] 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.

[0097] Silica gel column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0098] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0099] 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.

[0100] 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.

[0101] Abbreviations:

[0102] 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

[0103] Preparation Example Preparation of Compound A1

[0104] Step 1: Preparation of compound A1-1

[0105] 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). + .

[0106] Step 2: Preparation of Compound A1-2

[0107] Under nitrogen, 4A molecular sieves (10 g) and compound Int.02 (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). + .

[0108] Step 3: Preparation of Compound A1-3

[0109] 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 under stirring at 0°C, and 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).

[0110] Step 4: Preparation of Compound A1

[0111] 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).

[0112] Preparation of Example Compound A2 (L96-deAc)

[0113] Step 1: Preparation of compound A2-1

[0114] Synthesis of Compound A2-1 Referring to Compound A1, Compound A2-1 was prepared by using Compound Int.2 instead of Compound A1-3. MS: 509.1 (M+Na) + .

[0115] Step 2: Preparation of compound A2-2

[0116] Under nitrogen, a reaction mixture of compound A3 (400 mg, 0.223 mmol), compound A2-1 (163 mg, 0.334 mmol), and DIEA (144 mg, 1.115 mmol) in acetonitrile (20 mL) was stirred at 20°C for 40 hours. The reaction mixture was then concentrated to obtain a crude product, which was purified to yield compound A2-2 (270 mg, 57.8%) as a white solid. MS: 1049.3 (1 / 2M+H) + .

[0117] Step 3: Preparation of Compound A2-3

[0118] Compound A2-2 (270 mg, 0.129 mmol) was dissolved in tetrahydrofuran (10 ml). Palladium on carbon (14 mg) was added to the reaction mixture under stirring. The reaction mixture was then stirred under hydrogen pressure at room temperature for 16 hours. The reaction mixture was filtered and concentrated to obtain compound A2-3 (210 mg, 81%). MS: 1003.6 (1 / 2M+H) + .

[0119] Step 4: Preparation of Compound A2-4

[0120] Under nitrogen protection and stirring at 0°C, compound Int.3 (56.8 mg, 0.209 mmol) was slowly added to a solution of compound A2-3 (210 mg, 0.105 mmol) and DIEA (67.6 mg, 0.523 mmol) in acetonitrile (10 mL). The reaction mixture was then stirred at 20°C for 15 minutes. 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 compound A2-4 (220 mg, 97%). MS: 1087.2 (1 / 2M+H) + .

[0121] Step 5: Preparation of Compound A2-5

[0122] A reaction mixture of compound A2-4 (220 mg, 0.101 mmol), compound Int.7 (63.7 mg, 0.152 mmol), and DIEA (66 mg, 0.506 mmol) in acetonitrile (10 mL) was stirred at 20°C for 15 minutes. 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 A2-5 (120 mg, 49.2%). MS: 1053.7 (1 / 2[M-DMTr]+H) + .

[0123] Step 6: Preparation of Compound A2

[0124] A reaction mixture of compound A2-5 (120 mg, 0.05 mmol) and 7M ammonia in methanol (6 mL) was stirred at 20°C for 4 hours. The reaction mixture was then concentrated to obtain a crude product, which was then separated and purified to yield compound A2 (30 mg, 29.7%). 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).

[0125] Preparation Example Preparation of Compound A3

[0126] Step 1: Preparation of compound A3-1

[0127] 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 obtain a crude product. The crude product was purified to yield compound A3-1 (74 g, 17.7%) as a light yellow oil. MS: 506.4 (M+H). + .

[0128] Step 2: Preparation of compound A3-2

[0129] 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).

[0130] Step 3: Preparation of compound A3-3

[0131] 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).

[0132] Step 4: Preparation of Compound A3-4

[0133] 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. 1H 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).

[0134] Step 5: Preparation of Compound A3-5

[0135] 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.

[0136] Step 6: Preparation of Compound A3-6

[0137] 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 give a crude product, which was purified to give 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).

[0138] Step 7: Preparation of Compound A3

[0139] 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).

[0140] Preparation Example Preparation of Compound A4

[0141] Compound A4 was synthesized with reference to compound A1, using compound A1-1 as the starting material to prepare compound A4. MS: 628.4 (M+H) + .

[0142] Preparation Example Preparation of Compound A7

[0143] The synthesis of compound A7-1 was carried out by referring to compound A8, and compound A7-1 was prepared by using compound A1-1 as the starting material. MS: 552.4 (M+H) + .

[0144] Compound A7 was synthesized by referring to compound A1 and replacing compound A1-3 with compound A7-1 to obtain compound A7. MS: 718.1 (M+H) + .

[0145] Preparation Example Preparation of Compound A8

[0146] Compound A8-1 was synthesized with reference to compound A1-2, and compound A8-1 was prepared by using compound A1-1 as a starting material. MS: 549.6 (M+H) + .

[0147] Compound A8 was synthesized by referring to compound A3 and replacing compound A3-6 with compound A8-1 to obtain compound A8. MS: 523.4 (M+H) + .

[0148] Preparation of Example Compound A12

[0149] Step 1: Preparation of compound A12-1

[0150] A solution of Intermediate Int.12 (3 g, 6.359 mmol), TEA (3.21 g, 31.7 mmol), HATU (7.24 g, 19.04 mmol), and Intermediate Int.2 (2.24 g, 7.62 mmol) in DMF (10 mL) was stirred at 20°C for 1 hour. The reaction mixture was then poured into ice water and diluted with dichloromethane. The organic phase was washed sequentially with 5% citric acid solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford Compound A12-1 (4.8 g, 97.5%) as a yellow oil. MS: 775.7 (M+H) + .

[0151] Step 2: Preparation of compound A12-2

[0152] A solution of trifluoroacetic acid (4.41 g, 38.7 mmol) and compound A12-1 (1 g, 1.29 mmol) in dichloromethane (10 mL) was stirred at 20°C for 1 hour. The reaction mixture was then poured into ice water and diluted with dichloromethane for extraction. The organic phase was washed sequentially with 5% citric acid solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to yield compound A12-2 (0.6 g, 98%) as a yellow oil. MS: 475.6 (M+H) + .

[0153] Step 3: Preparation of compound A12-3

[0154] A solution of TEA (6.4 g, 63.2 mmol), glutaric anhydride (4.33 g, 37.9 mmol), and compound A12-2 (5 g, 9.6 mmol) in dichloroethane (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was then concentrated to obtain a crude product, which was purified to give compound A12-3 (8.5 g, 99%) as a yellow oil. MS: 817.7 (M+H) + .

[0155] Step 4: Preparation of Compound A12-4: A solution of tert-butyl (3-aminopropyl)carbamate (3.07 g, 17.63 mmol), DIEA (1.898 g, 14.69 mmol), Compound A12-3 (4 g, 2.94 mmol), and HATU (4.47 g, 11.75 mmol) in DMF (10 mL) was stirred at 20°C for 40 hours. The reaction mixture was then concentrated to obtain a crude product, which was purified to afford Compound A12-4 (630 mg, 16.7%) as a yellow oil. MS: 1286.1 (M+H) + .

[0156] Step 5: Preparation of Compound A12-5

[0157] A solution of trifluoroacetic acid (1.676 g, 14.7 mmol) and compound A12-4 (630 mg, 0.49 mmol) in dichloromethane (10 mL) was stirred at 20°C for 40 hours. The reaction mixture was then concentrated to obtain a crude product, which was purified to give compound A12-5 (0.48 g, 99.9%) as a yellow oil. MS: 329.6 (M / 3+H) + .

[0158] Step 6: Preparation of Compound A12-6

[0159] A reaction mixture of compound A1 (1.195 g, 1.949 mmol), DIEA (630 mg, 4.87 mmol), and compound A12-5 (480 mg, 0.487 mmol) in dichloromethane (10 mL) was stirred at room temperature for 40 hours. The reaction mixture was then filtered and concentrated to obtain a crude product, which was purified to yield compound A12-6 (0.77 g, 69.5%) as a white solid. MS: 1138.0 (M / 2+H). + .

[0160] Step 7: Preparation of Compound A12-7

[0161] Compound A12-6 (600 mg, 0.264 mmol) was dissolved in tetrahydrofuran (20 mL). Palladium on carbon (28 mg) was added to the reaction mixture with stirring. The reaction mixture was then stirred under hydrogen pressure at room temperature for 40 hours. The reaction mixture was filtered and concentrated to obtain a crude product, which was purified to obtain Compound A12-7 (0.52 g, 90%) as a white solid. MS: 1092.9 (M / 2+H). + .

[0162] Step 8: Preparation of Compound A12-8

[0163] Under nitrogen protection at 0°C, compound Int.3 (133 mg, 0.476 mmol) was slowly added to a solution of compound A12-7 (520 mg, 0.238 mmol) and DIEA (154 mg, 1.19 mmol) in acetonitrile (10 mL). The reaction mixture was then stirred at 20°C for 0.25 hours. The reaction mixture was then diluted and extracted with dichloromethane. The organic phase was washed sequentially with 5% citric acid solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, compound A12-8 (560 mg, 100%), as a yellow oil. MS: 1175.9 (M / 2+H). + .

[0164] Step 9: Preparation of Compound A12-9

[0165] A reaction mixture of compound A12-8 (560 mg, 0.238 mmol), compound Int.7 (150 mg, 0.358 mmol), and DIEA (154 mg, 1.192 mmol) in acetonitrile (10 mL) was stirred at 20°C for 15 minutes. The reaction mixture was then concentrated to obtain a crude product, which was then isolated and purified to yield compound A12-9 (310 mg, 50.3%). MS: 1142.4 (1 / 2[M-DMTr]+H) + .

[0166] Step 10: Preparation of Compound A12

[0167] A reaction mixture of compound A12-9 (110 mg, 0.043 mmol) and 7M ammonia in methanol (10 mL) was stirred at 20°C for 4 hours. The reaction mixture was then concentrated to obtain a crude product, which was then separated and purified to yield compound A12 (45 mg, 47.9%) as a white solid. MS: 953.2 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.81(d,J=18.1Hz,6H),7.68(d,J=9.1Hz,3H),7.36-7.30(m,4H),7.22(ddd,J=8.8,6.7,2.6Hz,5H),6.90(ddd,J=8.9,6.0,2 .4Hz,4H),5.04(d,J=4.1Hz,1H),4.65(t,J=5.7Hz,3H),4.60(d,J=6.3Hz, 3H), 4.52 (d, J = 4.4Hz, 3H), 4.24 (d, J = 8.4Hz, 3H), 4.16 (d, J = 9.4Hz, 1H), 3. 77-3.66(m,16H),3.54(tt,J=11.1,5.3Hz,22H),3.34(dt,J=21.3,5.4Hz, 12H),3.19(dd,J=9.0,5.0Hz,1H),3.08-2.99(m,14H),2.35(s,8H),2.23(t ,J=7.5Hz,2H),2.13(s,6H),2.06(d,J=7.2Hz,6H),1.82(s,9H),1.76(d,J =9.0Hz, 6H), 1.53 (d, J = 11.9Hz, 16H), 1.45 (d, J = 7.1Hz, 6H), 1.26 (s, 12H).

[0168] Preparation of Example Compound A13

[0169] Step 1: Preparation of compound A13-1

[0170] Lithium hydroxide (3.13 g, 131 mmol) was slowly added to a mixture of 1-(tert-butyl)-2-methyl(S)-pyrrolidine-1,2-dicarboxylate (10 g, 43.6 mmol) in tetrahydrofuran (50 mL) and water (50 mL). The reaction mixture was then stirred and refluxed for 2 hours. The reaction mixture was concentrated and the pH was adjusted to 3 with dilute hydrochloric acid. The mixture was filtered, and the filter cake was washed with water and dried to yield Compound A13-1 (5.2 g, 55%) as a white solid. MS: 160.2 (M-56+H). + .

[0171] Step 2: Preparation of compound A13-2

[0172] Synthesis of Compound A13-2 With reference to Compound A12-8, Compound A13-2 was prepared by using Compound A13-1 instead of Compound A12-7.

[0173] Step 3: Preparation of compound A13-3

[0174] Compound A13-3 was synthesized by referring to compound A12-9 to obtain compound A13-3. MS: 386.2 (M-56+H) + .

[0175] Step 4: Preparation of Compound A13-6 Synthesis of Compound A13-6 Referring to Compound A13-3, Compound A13-6 was prepared by using Compound A13-3 instead of 1-(tert-butyl) 2-methyl (S)-pyrrolidine-1,2-dicarboxylate. MS: 598.2 (M-56+H) + &654.1(M+H) + .

[0176] Step 5: Preparation of Compound A13-7

[0177] Synthesis of Compound A13-7 Referring to Compound A13-1, Compound A13-7 was prepared by using Compound A13-6 instead of 1-(tert-butyl) 2-methyl (S)-pyrrolidine-1,2-dicarboxylate. MS: 584.2 (M-56+H) + .

[0178] Step 6: Preparation of Compound A13-8

[0179] A solution of trifluoroacetic acid (16.76 g, 147 mmol) and compound A13-7 (550 mg, 0.86 mmol) in dichloromethane (30 mL) was stirred at 20°C for 1 hour. The reaction mixture was then concentrated to give the crude product, compound A13-8 (270 mg, 97%). MS: 340.1 (M+H) + .

[0180] Step 7: Preparation of Compound A13-9

[0181] Under nitrogen protection at 0°C, EDCI (4.94 g, 25.8 mmol), benzyl alcohol (3.29 g, 30.5 mmol), and DMAP (286 mg, 2.343 mmol) were slowly added to a solution of 6-(tert-butyloxycarbonyl)amino)hexanoic acid (5.42 g, 23.43 mmol) in dichloromethane (100 ml). The reaction mixture was then stirred at 20°C for 16 hours. The reaction mixture was then quenched with saturated ammonium chloride solution 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 separated and purified to obtain Compound A13-9 as a yellow oil. MS: 322.1 (M+H) + .

[0182] Step 8: Preparation of Compound A13-11

[0183] The synthesis of compound A13-1 was carried out by referring to compound A3-6. Compound A13-9 was used instead of compound A3-4 to prepare compound A13-11. MS: 651.2 (M+H) + .

[0184] Step 9: Preparation of Compound A13-13

[0185] Compound A13-13 was synthesized with reference to compound A2-5. Compound A13-13 was prepared by using compound A13-11 instead of compound A2-2 and compound A13-8 instead of compound Int.7. MS: 984.2 (M / 2+H) + .

[0186] Step 10: Preparation of Compound A13-14

[0187] Synthesis of Compound A13-14 Compound A13-14 was prepared by referring to Compound A12-4. MS: 1121.1 (M+H) + .

[0188] Step 11: Preparation of Compound A13

[0189] Compound A13 was synthesized by referring to compound A12 and replacing compound A12-6 with compound A13-14 to obtain compound A13. MS: 936.6 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.06(s,1H),7.74(s,3H),7.64(d,J=9.0Hz,3H),7.38-7.29(m,4H),7.22(td,J=8.7,3.7Hz,5H ),6.94-6.85(m,4H),5.00(d,J=4.0Hz,1H),4.67-4.54(m,6H),4.49(d,J=4.2Hz,3H),4.44-4.11(m,10H),3.80-3.65( m,17H),3.63-3.40(m,13H),3.34-3.16(m,7H),3.03(d,J=7.0Hz,11H),2.24(d,J=9.6Hz,6H),2.05(q,J=9.7,8.5Hz,1 1H), 1.91 (d, J = 21.9Hz, 4H), 1.82 (s, 9H), 1.77 (d, J = 7.2Hz, 1H), 1.56-1.34 (m, 29H), 1.24 (s, 18H), 1.03-0.94 (m, 2H).

[0190] Preparation of Example Compound A15

[0191] Compound A15 was synthesized by referring to compound A13 and using appropriate starting materials to prepare compound A15. MS: 971.8 (1 / 2[M-DMTr]+H) + . 1H NMR (500MHz, DMSO-d6) δ7.83 (s, 1H), 7.72 (t, J = 5.7 Hz,3H),7.67(s,1H),7.62(d,J=9.1Hz,3H),7.58(s,1H),7.34-7.25(m,4H),7.24-7.1 4(m,5H),6.90-6.82(m,4H),4.99(d,J=4.0Hz,1H),4.91(d,J=4.3Hz,1H),4.59(t,J=5. 6Hz,3H),4.56(d,J=6.3Hz,3H),4.48(d,J=4.4Hz,3H),4.38(s,2H),4.21(d,J=8.4Hz,3 H),4.17-4.08(m,1H),4.04(s,1H),3.87(s,2H),3.73(d,J=0.6Hz,6H),3.72(s,1H),3. 71-3.65(m,6H),3.63(d,J=3.9Hz,3H),3.52(dddd,J=32.7,16.4,10.6,5.6Hz,8H),3. 41(ddd,J=10.3,6.4,3.2Hz,4H),3.28(t,J=6.3Hz,6H),3.26-3.12(m,8H),3.11-2.91( m,14H),2.79(s,1H),2.65-2.62(m,2H),2.39-2.34(m,2H),2.30-2.14(m,8H),2.03(t, J=7.4Hz,10H),1.97-1.81(m,4H),1.79(s,9H),1.71-1.61(m,2H),1.55-1.32(m,33H).

[0192] Preparation of Example Compound A21

[0193] Compound A21 was synthesized with reference to compound A15, using compound A13-7 as the starting material to obtain compound A21. MS: 788.1 (1 / 2[M-DMTr]+H) + . 1H NMR(400MHz, DMSO-d6)δ8.37-7.76(m,2H),7.60(d,J=8.7Hz,3H),7.43-7.23(m,4H),7.24-7.14(m,6H),6.92-6.81(m,4H),4.9 7-4.96(m,1H),4.89-4.88(m,1H),4.55(dd,J=18.2,5.9Hz,6H),4.45(d,J=4.1Hz,3H),4.38(s,2H),4.21(td,J=25.6,24.3,12 .9Hz,9H),3.73(s,8H),3.70-3.62(m,9H),3.52(tt,J=11.3,5.9Hz,8H),3.43(d,J=9.4Hz,8H),3.27(s,5H),2.98(d,J=7.6Hz, 4H),2.22-2.21(m,7H)2.04-1.85(m,9H),1.79(d,J=3.5Hz,9H),1.75(s,2H),1.46(d,J=7.8Hz,16H),1.25(d,J=21.4Hz,20H).

[0194] Preparation of Example Compound A22

[0195] Compound A22 was synthesized with reference to compound A15, and compound A22 was prepared by using compound A21-2 as the starting material. MS: 957.8 (1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ8.28-7.99(s,2H),7.70(s,4H),7.58(d,J=9.0Hz,3H),7.37-7.24(m,4H),7.24-7.11(m,5H),6.91-6.80(m,4H) ),4.95-4.86(m,1H),4.61-4.45(m,6H),4.43(d,J=4.3Hz,3H),4.40-4.23(m,5H),4.19(d,J=8.4Hz,4H),4.10(d,J=30.2Hz,2H),3.70 -3.69(m,7H),3.68-3.59(m,9H),3.56-3.36(m,12H),3.33(d,J=6.8Hz,3H),3.28-3.19(m,6H),3.15-3.12(m,1H),2.97-2.94(m,9H), 2.20-2.16(m,6H),1.99(t,J=7.4Hz,10H),1.92-1.88(m,5H),1.77(s,9H),1.73-1.68(m,2H),1.45-1.33(m,29H),1.22-1.19(m,24H).

[0196] Preparation of Example Compound A26

[0197] Compound A26 was synthesized with reference to compound A22, and compound A26 was prepared by using compound A21-2 as the starting material. MS: 923.3 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.49-8.26(m,1H),8.19-7.92(m,2H),7.76(s,1H),7.61(d,J=9.0 Hz,3H),7.33(t,J=7.4Hz,4H),7.21(dt,J=8.8,4.0Hz,5H),6.93-6.86(m,4H),4.98(d,J= 3.9Hz,1H),4.65-4.54(m,6H),4.50(d,J=4.3Hz,3H),4.31(d,J=8.4Hz,10H),3.85-3.70( m,13H),3.70-3.41(m,54H),3.35(s,21H),2.25-1.87(m,10H),1.83(s,9H),1.24(s,15H).

[0198] Example: A galactosamine compound III-LA15-CPG attached to a solid support

[0199] The aminogalactose compound attached to the solid support disclosed herein is 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 a method for preparing an aminogalactose compound attached to a solid support.

[0200] Compound III-LA15-1; MS: 1210.8 (1 / 2[M-DMTr]+H) + . 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=9.0Hz,3H),7.69(t,J=5.7Hz,3H),7.37-7.25(m,3H),7.19(ddd,J=8.9,5.6,3.2Hz,5H),6.87(ddd,J=9.0,4. 9,2.3Hz,3H),5.21(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,2H),4.49(d,J=8.4Hz,2H),4.02(q,J=4.1Hz,11H),3.87(dt,J=11.2,8.8Hz,6H),3 .73(s,17H),3.41(dt,J=11.2,6.0Hz,5H),3.22(s,5H),3.11-2.87(m,1 3H),2.53(d,J=7.1Hz,8H),2.45(d,J=9.5Hz,4H),2.32-2.14(m,8H),2. 10(s,10H),2.01(d,J=15.5Hz,19H),1.89(s,11H),1.77(s,9H),1.67(s ,1H),1.56-1.31(m,31H),1.24(d,J=9.7Hz,21H),0.96(t,J=7.1Hz,8H).

[0201] Example: A galactosamine compound III-LA13-CPG attached to a solid support

[0202] The synthesis of the aminogalactose compound III-LA13-CPG linked to a solid support was based on the synthesis of the aminogalactose compound III-LA15-CPG linked to a solid support.

[0203] Compound III-LA13-1; MS:1175.8(1 / 2[M-DMTr]+H) + . 1H NMR (400MHz, DMSO-d6) δ7.82(d,J=9.2Hz,2H),7.71(s,3H),7.36-7.25(m,4H),7.20(ddd,J=9.0,6.1,3.5Hz,5H),6.87(ddd,J=8.7,5. 4,2.4Hz,4H),5.21(d,J=3.4Hz,2H),4.97(dd,J=11.2,3.4Hz,2H),4.49(d,J=8.5Hz,2H),4.24(d,J=35.6Hz,4H),4.03(d,J=4.2Hz,9H ),3.87(dt,J=10.9,8.8Hz,3H),3.73(s,12H),3.12-2.92(m,11H),2.48(s,10H),2.45(d,J=7.7Hz,4H),2.22(d,J=7.9Hz,7H),2.10(s ,10H),2.02(d,J=6.9Hz,9H),1.99(s,11H),1.89(s,12H),1.77(s,11H),1.41(d,J=38.9Hz,29H),1.22(s,19H),0.95(t,J=7.1Hz,8H).

[0204] Example Synthesis of siRNA Conjugated with Galactosamine Clusters

[0205] 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,

[0206] SS chain (5'-3'):a·a·caguGuUCUugcucuauaaL

[0207] AS chain (5'-3'):u·U·auaGaGCaagaAcAcuguu·u·u

[0208] 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.

[0209] 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.

[0210] Synthesize siRNA conjugated with aminogalactose clusters. The siRNA used in the experiment targets mouse TTR mRNA.

[0211] Table X. siRNA numbers and sequences for evaluation of targeting ligand activity

[0212] 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.

[0213] 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.

[0214] (I) ASGPR-GalNAc™ affinity assay based on monkey primary cells

[0215] 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.

[0216] 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.

[0217] Conclusion: The affinity detection test results show that the affinity of most of the compounds tested so far, such as A13, A15, A21, A22 and A26, to ASGPR showed comparable or slightly better activity than that of the L96-DeAc molecule.

[0218] (II) Inhibition of mRNA expression in vivo by siRNA conjugated with aminogalactose clusters

[0219] 6-8 week old C57 / BL6 mice were randomly divided into 3 groups (3 mice in each group), which were either treated with any The mice were treated with a positive control group (S-L96), a positive control group (S-LA13, S-LA15), and a drug-treated group (S-LA13, S-LA15). GalNac-siRNA was administered subcutaneously at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Blood was collected from the orbits of the mice for serum separation before (day 0), 3 days after (day 3), 7 days after (day 7), 14 days after (day 14), and 21 days after (day 21). Mice were sacrificed on day 21, 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:

[0220] Table 1. Detection of TTR protein levels in mouse serum and liver samples

[0221] Conclusion: The in vivo efficacy test data showed that S-LA13 and S-LA15 exhibited excellent mTTR gene expression inhibition efficiency, and the activity levels at 0.3mpk, 1mpk and 3mpk were comparable to or slightly superior to the control group S-L96.

[0222] 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, for R1 is selected from the group consisting of H, DMTr, phosphate, phosphorothioate, phosphorothioate-siRNA conjugate, or phosphate-siRNA conjugate; Linker has a structure selected from the group consisting of C4-C20 alkylene, wherein n, n3 and m are each independently selected from the following group: 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; Z is selected from the following group: a single bond, a substituted or unsubstituted C 1-8 Alkylene; Q is selected from the group consisting of a chemical bond, -NH-C(O)-, -C(O)-; r is 0, 1, 2, 3, 4, 5 or 6; R2 is a GalNAc target head portion and has a structure selected from the group consisting of: Wherein, L1 and L2 are each independently a structure selected from the following group: L3 is a structure selected from the following group: Among them, R 11 Selected from the following group: H or C 1-4 alkyl; Each u, v and w is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; Glu is an unmodified or modified 5-6-membered sugar group; X1 is selected from the following group: substituted or unsubstituted C 4-10 a saturated or partially unsaturated carbocyclic group, a substituted or unsubstituted 4-10 membered saturated or partially unsaturated heterocyclic group (preferably a nitrogen-containing heterocyclic group), a substituted or unsubstituted C 6-10 Phenyl, or substituted or unsubstituted 5-10 membered heteroaryl; X2 is selected from the following groups: -NH-(C1-C6 alkyl)-NH-, -NH-(C3-C8 cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, substituted or unsubstituted C 4-10 a saturated or partially unsaturated carbocyclic group, a substituted or unsubstituted 4-10 membered saturated or partially unsaturated heterocyclic group (preferably a nitrogen-containing heterocyclic group), a substituted or unsubstituted C 6-10 Phenyl, or substituted or unsubstituted 5-10 membered heteroaryl; Wherein, the carbocyclic group and heterocyclic group may be a monocyclic ring, a condensed ring, a bridged ring or a spirocyclic ring. 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 group consisting of C4-C20 alkylene.

3. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: 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.

4. The GalNAc delivery molecule according to claim 1, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The L1 and L2 are each independently a structure as shown below: The L3 is a structure shown in the following formula: Wherein, the definitions of each group are as described in claim 1.

5. The GalNAc delivery molecule according to claim 4, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The R2 has a structure as shown below:

6. The GalNAc delivery molecule according to claim 4, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The X1 is a substituted or unsubstituted structure selected from the following group: Wherein, s1 and s2 are each independently selected from the following group: 0, 1 or 2; Each of M1 and M3 is independently selected from the group consisting of CHR, C(R)R, 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).

7. The GalNAc delivery molecule according to claim 4, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof, wherein: The X2 is a substituted or unsubstituted structure selected from the following group:

8. 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 15 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 independently selected from the group consisting of: H or C1-C6 acyl; Preferably, the R 15 Selected from the group consisting of: -NH(C2-C4 acyl); R 12 , R 13 and R 14 Each independently selected from the following group: H or C1-C4 acyl; More preferably, the R 15 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:

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:

11. 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 10, 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.

12. The use of a compound of formula I as claimed in any one of claims 1 to 10, 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.

13. Use of a compound of formula (I) according to any one of claims 1 to 10 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.

14. A GalNAc delivery molecule linker as shown in formula (Ia), or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof: The part 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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