Lipid compound for delivering therapeutic agent and composition containing same

Abstract

Disclosed in the present invention are a lipid compound for delivering a therapeutic agent and a composition containing same. The present invention provides a lipid compound represented by formula I or a pharmaceutically acceptable salt thereof. The lipid compound provided by the present invention, as a drug carrier, exhibits high encapsulation efficiency, high expression level, and good targeting property.

Description

Lipid compounds for delivering therapeutic agents and compositions containing them

[0001] This application claims priority to Chinese Patent Application No. 2024115427323, filed on October 31, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] The present invention specifically relates to a lipid compound for delivering a therapeutic agent and a composition containing the same. Background Technology

[0003] The efficient targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids has always been a challenge in medicine. In particular, delivering nucleic acids to cells is difficult due to their relative instability and low cell permeability. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutics and / or preventative agents (e.g., nucleic acids) to cells. Summary of the Invention

[0004] The technical problem this invention aims to solve is the limited variety of lipid compounds available for delivering therapeutic and / or preventative agents to cells in existing technologies. Therefore, this invention provides a lipid compound for delivering therapeutic agents and a composition containing the same. The lipid compound provided by this invention, as a drug carrier, has the advantages of high encapsulation efficiency, high expression levels, and good targeting.

[0005] This invention provides a lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0006] Where A is

[0007] m and n are independently 0 or 1, and m and n are not both 0 at the same time;

[0008] X can be CH2 or NH independently;

[0009] Y 1 and Y 2 Independently CH, CH2, N, or NH;

[0010] It can be a single bond or a double bond;

[0011] R 1 H and C independently 1-6 Alkyl or -OC 1-6 Alkyl, the C 1-6 Alkyl and -OC 1-6 Alkyl groups are independently and optionally surrounded by one or more R groups. a Replace, R a Each can be independently a halogen, amino group, oxo group (=O), or hydroxyl group;

[0012] H is an optional substituted linking group; the optional substituted linking group may optionally contain one or more biodegradable groups;

[0013] t is 0, 1, or 2;

[0014] K 1 and K 2 Independently alkyl, alkenyl, or ynyl, wherein the alkyl, alkenyl, and ynyl groups optionally contain one or more biodegradable groups; wherein the alkyl, alkenyl, and ynyl groups optionally containing one or more biodegradable groups are optionally independently denoted by one or more R groups. b replace;

[0015] R b Each can be independently an oxo group (=O), a cyano group, a hydroxyl group, or a C group. 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl; the C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 The aryl and 5-10 heteroaryl groups are independently and optionally separated by one or more R groups. b2 replace;

[0016] R b1 For H or C 1-6 alkyl;

[0017] R b2 It is either hydroxyl or amino;

[0018] And K 1 and K 2 At least one of them contains more than 11 carbon atoms;

[0019] The heteroatoms in the 3-10 membered heterocyclic alkyl and 5-10 membered heteroaryl groups are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently one, two or three.

[0020] And when A is In this case, the linking group is -(CH2). n1 -, n1 is 1, 2, 3, 4, 5 or 6.

[0021] In some embodiments, certain groups in the lipid compound of Formula I or its pharmaceutically acceptable salt have the following definitions, and the definitions of groups not mentioned are as described in any embodiment of the invention (hereinafter referred to as "in some embodiments").

[0022] In some embodiments, the linking group is a single bond or

[0023] Z is N, C, CH or CH2; n2 is an integer from 1 to 20;

[0024] The One or more methylene units are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently -NH-, -O-, -S-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -S(O2)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, C 3-10 Cycloalkyl, 3-10 heterocycloalkyl, C 6-10 Aryl or 5-10 heteroaryl; the one or more methylene units are independently and optionally replaced by biodegradable groups. Optionally by one or more R c Replace, R c Each independently is C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl, wherein C 1- 6-alkyl groups are optionally surrounded by one or more R c1 Replace; R c1 Each independently consists of hydroxyl and C. 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl;

[0025] The heteroatoms in the 3-10 membered heterocyclic alkyl groups and 5-10 membered heteroaryl groups are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0026] In some implementation schemes, K 1 and K 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl or C 2-30alkynyl group, the C 1-30 Alkyl, C 2-30 alkenyl and C 2-30 One or more methylene units in the alkynyl group are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently -NH-, -O-, -S-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -S(O)2-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, C 3-10 Cycloalkyl, 3-10 heterocycloalkyl, C 6-10 Aryl or 5-10 heteroaryl; the one or more methylene units are independently and optionally replaced by biodegradable groups. 1-30 Alkyl, C 2-30 alkenyl and C 2-30 The alkynyl group is independently and optionally surrounded by one or more R b replace;

[0027] R b Each can be independently an oxo group (=O), a cyano group, a hydroxyl group, or a C group. 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl; the C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 The aryl and 5-10 heteroaryl groups are independently and optionally separated by one or more R groups. b2 replace;

[0028] The heteroatoms in the 3-10 membered heterocyclic alkyl groups and 5-10 membered heteroaryl groups are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0029] In some implementation schemes, C 1-6 Alkyl, -OC 1-6 Alkyl, -C(O)C 1-6Alkyl and -NR b1 C(O)C 1-6 C in alkyl 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, such as ethyl.

[0030] In some implementations, the C 3-10 Each cycloalkyl group is independently C10. 3-6 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and again, cyclopropyl.

[0031] In some embodiments, the 3-10 membered heterocyclic alkyl groups are each independently a 3-6 membered heterocyclic alkyl group, such as azirrobutyl, azirropentyl or azirrohexyl, or azirropentyl.

[0032] In some implementations, the C 6-10 Each aryl group can be either phenyl or naphthyl, for example, phenyl.

[0033] In some implementations, X is independently NH.

[0034] In some implementation schemes, R 1 For H or C 1-6 Alkyl, the C 1-6 Alkyl groups are optionally surrounded by one or more R a Replace, R a Each is an independent hydroxyl group.

[0035] In some embodiments, the linking group is The One or more (e.g., one) methylene units are independently and optionally replaced by biodegradable groups; each of the biodegradable groups is independently NH, O, S, Or C 6-10 Aryl; wherein one or more (e.g., one) methylene unit is independently and optionally replaced by a biodegradable group. Optionally by one or more R c replace.

[0036] In some implementations, n2 is 1, 2, 3, 4, 5, 6, 7, or 8.

[0037] In some implementation schemes, R c Each independently is C 1-6 Alkyl; the C 1-6 Alkyl groups are optionally surrounded by one or more R c1 replace.

[0038] In some implementation schemes, Rc1 Each is an independent hydroxyl group.

[0039] In some embodiments, the linking group is

[0040] Each x1 and x2 is independently 1, 2, 3, or 4, and x3 is 0, 1, 2, 3, or 4.

[0041] Each V1 is independently a bond, NH, NR c O Or C 6-10 Aryl.

[0042] In some implementations, each V1 is independently a bond, NH, O, For example, O.

[0043] In some implementation schemes, K 1 and K 2 Independent for straight chain C 1-30 Alkyl, straight-chain C 2-30 alkenyl, straight-chain C 2-30 acetylinyl or

[0044] The straight chain C 1-30 Alkyl, straight-chain C 2-30 Alkenyl and straight-chain C 2-30 One or more (e.g., 1, 2, or 3) methylene units in the alkynyl group are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently O, NH, -S(O)2-, -S-, -C(O)-, C 3-10 cycloalkyl or C 6-10 aryl; a straight-chain C-terminal in which one or more (e.g., 1, 2, or 3) methylene units are independently and optionally replaced by biodegradable groups. 1-30 Alkyl, straight-chain C 2-30 Alkenyl and straight-chain C 2-30 The alkynyl group is independently and optionally surrounded by one or more R b replace;

[0045] f1 is 1, 2, 3, 4, 5, 6, 7 or 8; f2 and f3 are independently 0, 1, 2, 3, 4 or 5;

[0046] Z 1 It is a single bond or -O-;

[0047] Z 2 for

[0048] R 3 and R4 Independently for C 1-10 alkyl.

[0049] In some implementations, the linear C 2-30 The number of alkene bonds in the alkenyl group is one or two, and the straight-chain C 2-30 The number of alkyne bonds in an alkyne group is one or two.

[0050] In some implementation schemes, K 1 and K 2 Independently

[0051] Each r1 is an independent integer from 1 to 12, and r2 is an integer from 1 to 16, and the sum of r1 and r2 is ≤ 30;

[0052] r3 is an integer from 1 to 10, r4 is an integer from 0 to 6, r5 is an integer from 1 to 12, and the sum of r3, r4 and r5 is ≤ 30;

[0053] r6 is an independent integer from 1 to 6; and the sum of each r6 and r3 is ≤ 30;

[0054] G 1 C 3-10 cycloalkyl or C 6-10 Aryl;

[0055] G 2 For -CHR b -、 Or C 3-10 cycloalkyl;

[0056] R b Hydroxyl group, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl;

[0057] G 3 for

[0058] G 4 G 5 and G 6 Independently for O, -CHR b -, -CH2=CH2-, C 3-10 cycloalkyl, C 6-10 Aryl or a C 1-6 Alkyl-substituted C 6-10 Aryl;

[0059] f1 is 1, 2, 3, 4, 5, 6, 7 or 8; f2 and f3 are independently 0, 1, 2, 3, 4 or 5;

[0060] Z 1 It is a single bond or -O-;

[0061] Z 2 for

[0062] R 3 and R 4 Independently for C 1-10 alkyl.

[0063] In some implementation schemes, R b It is a hydroxyl group.

[0064] In some implementations, G 4 For O or C 6-10 Aryl.

[0065] In some implementation schemes, K 1 and K 2 In this context, each of the biodegradable groups is independently... C 3-10 cycloalkyl or C 6-10 Aryl.

[0066] In some implementation schemes, R b Each independently consists of hydroxyl and C. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl or C 6-10 Aryl.

[0067] In some embodiments, the lipid compound shown in Formula I is a compound shown in Formula II:

[0068] In some embodiments, the lipid compound shown in Formula I is a compound shown in Formula III-1:

[0069] In some embodiments, the lipid compound shown in Formula I is a compound shown in Formula III-2:

[0070] K1 and K2 are defined as follows:

[0071] Option 1: K 1 and K 2 Independently

[0072] Option 2: K 1 for

[0073] K2 for

[0074] In some implementation schemes, for

[0075] In some implementation schemes, for

[0076] In some implementation schemes, R 1 For H or

[0077] In some implementation schemes, A is

[0078] In some implementations, H is It can also be used for

[0079] In some implementation schemes, R 3 and R 4 Independently

[0080] In some implementation schemes, for

[0081] In some implementation schemes, for

[0082] In some implementation schemes, R b Each is independently hydroxyl, ethyl, n-propyl, n-butyl, cyclopropyl, Or phenyl.

[0083] In some embodiments, the biodegradable groups are each independently O, NH,

[0084] In some implementation schemes, K 1 and K 2 Independently It can also be used for

[0085] In some implementations, t is 0, K 1 for

[0086] In some implementations, t is 1. for It can also be used for

[0087] In some embodiments, the compound represented by Formula I is, for example, HI-19, HI-19-1-1, HI-19-1-2, HI-19-2-2, HI-19-2-3, HI-19-2-4, HI-19-3-1, HI-19-4-1, HI-19-5-1, HI-19-6-1, HI-19-7-1, HI-19-8-1, HI-19-9-1, HI-19-9-2, HI-19-10-1, HI-19-111, HI-19-112, HI-19-113, HI-19-114, HI-19-115, HI-19-116, HI-19-117, HI-19-118, HI-19-119. HI-19-120, HI-19-121, HI-19-122, HI-19-123, HI-19-124, HI-19-125, HI-19 -126, HI-19-127, HI-19-128, HI-19-129, HI-19-130, HI-19-131, HI-19-132, Compounds represented by HI-19-133, HI-19-138, HI-19-139, HI-19-140, HI-19-2-1, HI-19-134, HI-19-135, HI-19-136, HI-19-137, HI-19-R, HI-19-S, HI-19-162 or HI-621-2.

[0088] This invention provides the use of any of the compounds of Formula I or a pharmaceutically acceptable salt thereof in the preparation of lipid carriers.

[0089] In one embodiment, the lipid carrier is a lipid nanoparticle (LNP).

[0090] In some embodiments, the lipid carrier is used for the delivery of nucleic acid molecules (e.g., nucleic acid drugs) selected from one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), gene editing tools, self-replicating RNA (samRNA), microRNA (miRNA), circular RNA (circRNA), and messenger RNA (mRNA), such as messenger RNA (mRNA), or for example, firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, EGFP-Flag mRNA, mRNA sequences encoding human papillomavirus E7 antigen (e.g., having the sequence shown in SEQ ID NO:3), mRNA sequences encoding infectious disease antigens, or mRNA sequences encoding tumor antigens; for example, firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, EGFP-Flag mRNA, or mRNA sequences encoding human papillomavirus E7 antigen;

[0091] The liposomes can be used to deliver mRNA sequences encoding antigens for infectious diseases, including but not limited to respiratory diseases such as influenza, COVID-19, and respiratory syncytial virus (RSV); hemorrhagic fever viruses such as Ebola, Marburg, and dengue fever; vector-borne viruses such as chikungunya, West Nile virus, and Zika virus; and neurological diseases such as rabies, Japanese encephalitis, and poliomyelitis. In addition, they can be used to deliver chronic infections such as hepatitis B (HBV), hepatitis C (HCV), HIV, influenza, tuberculosis, herpes zoster (VZV), and tumor-associated viruses such as human papillomavirus (HPV).

[0092] The liposomes described herein can be used for tumor treatment, including but not limited to primary liver cancer, metastatic liver cancer, adrenocortical carcinoma, AIDS-related cancers (including AIDS-related lymphoma), anal cancer, basal cell carcinoma, cholangiocarcinoma, primary bone tumors, metastatic bone tumors, brain tumors (including brainstem gliomas, cerebellar astrocytomas, cerebral astrocytomas, malignant gliomas, ependymomas, blastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), metastatic brain tumors, breast cancer, bronchial adenoma / carcinoid tumors, Burkitt lymphoma, gastrointestinal cancers, cancers of unknown primary location, central nervous system lymphomas, cervical cancer, chronic myeloproliferative disorders, colon cancer, colorectal cancer, gastric cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymomas, esophageal cancer, extracranial germ cell tumors, gonadal germ cell tumors, ovarian germ cell tumors, and ocular tumors. Cancers including intraocular melanoma and retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, gliomas, pediatric brainstem gliomas, head and neck cancer, hematologic malignancies, adult and pediatric (primary) hepatocellular carcinoma, hypopharyngeal cancer, islet cell or pancreatic cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, adult and pediatric acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Warburg's macroglobulinemia, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary sites, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, and geranioid granulomatosis. Fungoides, myelodysplastic syndromes, myelodysplastic disorders, multiple myeloma, chronic myelodysplastic disorders, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, head and neck cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, potential low-grade ovarian tumors, pancreatic cancer, parathyroid cancer, penile cancer, jejunal chromocytoma, pineal blastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasmacytoma / multiple osteomas, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter cancer, transitional cell carcinoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, sezary syndrome Syndrome), skin cancer (including melanoma and non-melanoma skin), small bowel cancer, sclerodermal carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumors.Testicular cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor, pregnancy, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and nephroblastoma (Tilms' tumor), metastatic renal cell carcinoma. The liposomes can also be used for tumor neoantigen therapy.

[0093] The lipid carrier can be used for cellular peptide production, and the method of peptide production involves contacting cells with a lipid carrier composition comprising mRNA encoding the peptide of interest. After contact with the mRNA lipid carrier composition, the mRNA can be absorbed into the cell and translated to produce the peptide of interest.

[0094] The lipid carrier-mRNA compositions described above can be used to treat diseases, symptoms, or conditions. Specifically, these compositions can be used to treat diseases, symptoms, or conditions characterized by the absence or abnormal activity of proteins or peptides. For example, lipid nanoparticle compositions containing mRNA encoding absent or abnormal peptides can be administered or delivered to cells. The mRNA is subsequently translated to produce the peptide, thereby reducing or eliminating problems caused by the absence or abnormal activity of the peptide. Because translation can occur rapidly, these methods and compositions can be used to treat acute diseases, symptoms, or conditions such as sepsis, stroke, and myocardial infarction. Therapeutic and / or preventative agents included in the nanoparticle compositions can also alter the transcription rate of a given species, thereby affecting gene expression.

[0095] The composition of the lipid carrier and mRNA can also be used for protein replacement therapy to treat congenital diseases, metabolic diseases, genetic diseases, cardiovascular diseases, respiratory diseases, urinary diseases, etc., as well as in CAR-T and gene editing.

[0096] In some embodiments, the lipid carrier further includes one, two, three, four, five, or six of the following: ionizable lipids, cationic lipids, adjuvants, diluents, phospholipids, PEG lipids, and sterols.

[0097] The ionizable lipids (positively charged at low pH, uncharged at neutral or high pH) are conventional lipids in the art, characterized by a pKa range of 2-10. Lipid compounds of Formula I or their pharmaceutically acceptable salts, as described in any embodiment of this invention, also belong to the category of ionizable lipids. In some embodiments, the ionizable lipid molecule may be one or more. In some embodiments, the ionizable lipid is ALC0315.

[0098] Cationic lipid molecules refer to a class of positively charged lipid molecules. In some embodiments, the cationic lipid molecule is (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), methyl 4-(N,N-dimethylamino)butyrate (dilinyl)methyl ester (DLin-MC3-DMA), or dioleoylpropyltrimethylammonium chloride (DOTMA), such as (2,3-dioleoyl-propyl)-trimethylamine (DOTAP).

[0099] Adjuvants are commonly used components in vaccine products. They are substances that can non-specifically alter or enhance the body's specific immune response to antigens, playing a supportive role, and are now widely used in prophylactic and therapeutic vaccines. Common adjuvants include aluminum adjuvants or MPL, etc. In some embodiments, adjuvants refer to adjuvant lipids, which are lipid compounds prepared by incorporating molecules that act as adjuvants into lipid carriers conventional in the art. For example, the adjuvant is selected from any of the following compounds:

[0100] The diluent is a conventional diluent in the art. In some embodiments, the diluent is a citrate buffer, a phosphate buffer, or an acetate buffer; preferably an acetate buffer, such as an acetate buffer with a pH of 4.

[0101] In some embodiments, the diluent has a pH of 4-5.

[0102] The phospholipid is a conventional phospholipid in the art, preferably a neutral phospholipid. In some embodiments, it is an amphoteric accessory molecule that facilitates the fusion of lipid particles and cell membranes. The phospholipid may be a phospholipid molecule with a polar end and a nonpolar end of a fatty chain, such as distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearylphosphatidylethanolamine (DSPE), distearylphosphatidylglycerol (DSPG), DSPG-Na, disqualylphosphatidylcholine (DEPC), palmitoylphosphatidylglycerol (DPPG), DPPG-Na, dipalmitoylphosphatidic acid (DPPA), docosanoylphosphatidylcholine (DUPC), or palmitoylphosphatidylcholine (POPC); another example is distearylphosphatidylcholine (DSPC).

[0103] In some embodiments, the PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end. The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, such as PEG-DSPE, PEG-DMG, or ALC-0159; more preferably PEG-DMG-2000 or DSPE-PEG2000.

[0104] In some embodiments, the PEGylated lipid has a PEG portion size of 1000 Da to about 40 KDa, for example 1800-200 Da.

[0105] The sterol is a conventional sterol in the art, and in some embodiments, the sterol is an animal, plant, or fungal sterol. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, and α-tocopherol, for example, cholesterol.

[0106] In some embodiments, the lipid carrier comprises a compound of Formula I as described in any one of the present invention, or a pharmaceutically acceptable salt, phospholipid, sterol, or PEG lipid thereof.

[0107] In some embodiments, the lipid carrier further includes one or more of ionizable lipids, cationic lipids, and adjuvants, such as adjuvants.

[0108] In some embodiments, the lipid carrier includes a diluent.

[0109] In some embodiments, the lipid carrier comprises a compound of Formula I as described in any one of the present invention, or a pharmaceutically acceptable salt thereof, diluent, neutral phospholipid, sterol, or PEG lipid.

[0110] In some embodiments, the lipid carrier comprises a compound of Formula I as described in any one of the present invention, or a pharmaceutically acceptable salt thereof, a diluent, a neutral phospholipid, a sterol, and a PEG lipid.

[0111] In some embodiments, the lipid carrier comprises a compound of Formula I as described in any one of the present invention, or a pharmaceutically acceptable salt thereof, adjuvant, diluent, neutral phospholipid, sterol, and PEG lipid; preferably, the lipid carrier is composed of a compound of Formula I as described in any one of the present invention, or a pharmaceutically acceptable salt thereof, adjuvant, diluent, neutral phospholipid, and sterol.

[0112] In this invention, the molar percentage of each component of the lipid carrier is calculated as the sum of the molar amounts of the compound as shown in Formula I or its pharmaceutically acceptable salts, sterols, phospholipids and PEG lipids.

[0113] In some embodiments, the molar amount of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof accounts for 30% to 80% of the total molar amount, for example 50%.

[0114] In some embodiments, the molar percentage of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof is 20-80 mol%, for example 33 mol%-58 mol%, for example 40 mol%-52 mol%, 35-49 mol%, 48-52 mol%, or 40-44 mol%.

[0115] In some embodiments, the molar percentages of the compound as shown in Formula I or its pharmaceutically acceptable salts are 32 mol%, 34.52 mol%, 35.19 mol%, 35.81 mol%, 36.41 mol%, 37.59 mol%, 38 mol%, 38.79 mol%, 38.88 mol%, 39.3 mol%, 39.54 mol%, 40.79 mol%, 42.03 mol%, 42.57 mol%, 43.32 mol%, 43.9 mol%, 44.09 mol%, and 44.09 mol%. l%, 44.91mol%, 45mol%, 45.82mol%, 45.89mol%, 46.91mol%, 47.44mol%, 47.46mol%, 48.68mol%, 49.33mol%, 49.37mo 1%, 50.22mol%, 50.52mol%, 51.62mol%, 52.73mol%, 53.4mol%, 53.4mol%, 54.77mol%, 55mol%, 55.33mol% or 57.51mol%.

[0116] In some embodiments, the phospholipid accounts for 5% to 20% of the total molar amount, for example, 10%.

[0117] In some embodiments, the phospholipid has a molar percentage of 3 mol%-40 mol%, for example 3 mol%-35 mol%, for example 4 mol%-21 mol%, for example 5 mol%-18 mol%, and for example 5 mol%-15 mol%, 8-12 mol%.

[0118] In some implementations, the values ​​are 4.96 mol%, 5.11 mol%, 5.19 mol%, 5.52 mol%, 5.92 mol%, 6.44 mol%, 6.5 mol%, 6.72 mol%, 7.41 mol%, 8.67 mol%, 9.08 mol%, 9.27 mol%, 10.13 mol%, 10.59 mol%, 10.81 mol%, 11 mol%, 11.37 mol%, 11.63 mol%, 12.13 mol%, 12.66 mol%, 13.18 mol%, 14.07 mol%, 14.08 mol%, 14.8 mol%, or 15.08 mol%.

[0119] In some embodiments, the sterol accounts for 20% to 60% of the total molar amount, for example, 38.5%.

[0120] In some embodiments, the molar percentage of the sterol is 8 mol%-60 mol%, for example 10 mol%-60 mol%, for example 28 mol%-52 mol%, for example 35 mol%-52 mol%, and for example 40 mol%-52 mol%, 40-50 mol%.

[0121] In some embodiments, the molar percentage of the sterol is 35.07 mol%, 36.19 mol%, 37.63 mol%, 37.65 mol%, 38.58 mol%, 39.02 mol%, 39.23 mol%, 40.18 mol%, 40.32 mol%, 40.76 mol%, 41.31 mol%, 42.05 mol%, 42.33 mol%, 42.5 mol%, 42.94 mol%, 44.16 mol%, 45.08 mol%, 45.43 mol%, 46.86 mol%, 47.45 mol%, 47.94 mol%, 48.56 mol%, 49.7 mol%, 49.99 mol%, 51.11 mol%, or 51.79 mol%.

[0122] In some embodiments, the molar amount of the PEG lipid is 0.2% to 5% of the total molar amount, for example, 1.5%.

[0123] In some embodiments, the PEG lipid has a molar percentage of 0.1 mol% to 8 mol%, for example 0.1 mol% to 2.5 mol%, for example 1 mol% to 2.5 mol%, for example 1 mol% to 2 mol%.

[0124] In some embodiments, the molar percentage of the PEG lipid is 1.01, 1.04 mol%, 1.09 mol%, 1.13 mol%, 1.2 mol%, 1.21 mol%, 1.22 mol%, 1.33 mol%, 1.39 mol%, 1.39 mol%, 1.5 mol%, 1.51 mol%, 1.54 mol%, 1.55 mol%, 1.59 mol%, 1.6 mol%, 1.71 mol%, 1.75 mol%, 1.77 mol%, 1.81 mol%, 1.85 mol%, 1.94 mol%, 1.96 mol%, 1.97 mol%, or 2 mol%.

[0125] In some embodiments, the lipid carrier comprises 20-80 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof, 3-40 mol% of phospholipids, 8-60 mol% of sterols and 0.1-8 mol% of PEG lipids.

[0126] In some embodiments, the lipid carrier comprises 33-58 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof, 5-18 mol% of phospholipids, 35-52 mol% of sterols, and 1-2 mol% of PEG lipids.

[0127] In some embodiments, the lipid carrier comprises 35-49 mol% of a lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 5-18 mol% of phospholipids, 40-50 mol% of sterols, and 1-2 mol% of PEG lipids.

[0128] In some embodiments, the lipid carrier comprises 48-52 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof, 8-12 mol% of phospholipids, 36.5-40.5 mol% of sterols, and 1-2 mol% of PEG lipids.

[0129] In some embodiments, the lipid carrier comprises 40-44 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof, 4.5-8.5 mol% of phospholipids, 48-52 mol% of sterols, and 1-2 mol% of PEG lipids.

[0130] In some embodiments, the phospholipid is DPPC or DMPC, and the PEG lipid is ALC0159.

[0131] In some embodiments, the phospholipid is DEPC or DSPC, and the PEG lipid is DMG-PEG2000.

[0132] In some embodiments, the phospholipid is DMPC or DSPC, and the PEG lipid is DSPE-PEG2000.

[0133] In some embodiments, the lipid compound represented by Formula I is any of the following compounds: HI-19, HI-19, HI-19-2-1, HI-19-10-1, HI-19-129, HI-19-125, HI-19-9-2, HI-19-111, HI-19-112, HI-19-115, HI-19-129, or HI-19-135.

[0134] In some embodiments, the lipid carrier comprises 48-52 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof (preferably HI-19), 8-12 mol% of DSPC, 36.5-40.5% of cholesterol, and 1-2 mol% of DMG-PEG2000; preferably 49.5-50.5 mol% of a lipid compound of Formula I or a pharmaceutically acceptable salt thereof (preferably HI-19), 9.5-10.5 mol% of DSPC, 38-39% of cholesterol, and 1-2 mol% of DMG-PEG2000; preferably, the lipid carrier further comprises a diluent.

[0135] In some embodiments, the lipid carrier comprises 40-44 mol% of the compound as shown in Formula HI-19, 4.5-8.5 mol% of DEPC, 48-52% of cholesterol, and 1-2 mol% of DMG-PEG2000; preferably 41.5-42.5 mol% of the compound as shown in Formula HI-19, 6-7 mol% of DEPC, 49.5-50.5% of cholesterol, and 1-2 mol% of DMG-PEG2000.

[0136] In some embodiments, the lipid carrier comprises 43-47 mol% of the compound as shown in Formula HI-19, 9-13 mol% of DEPC, 40.5-45% of cholesterol, and 1-2 mol% of DMG-PEG2000; preferably 44.5-45.5 mol% of the compound as shown in Formula HI-19, 10.5-11.5 mol% of DEPC, 42-43% of cholesterol, and 1-2 mol% of DMG-PEG2000.

[0137] In some embodiments, the adjuvant in the lipid carrier is a percentage of 0.5% to 60%, for example 1% to 50%, or even 8% to 12%, of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0138] In some embodiments, the mass of the adjuvant is 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, or 50% of the mass of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, for example, 10%.

[0139] In some embodiments, the lipid carrier comprises 48-52 mol% of a compound of formula HI-19, 8-12 mol% of distearate phosphatidylcholine (DSPC), 36.5-40.5 mol% of cholesterol, 1-2 mol% of DMG-PEG2000, and an adjuvant, said adjuvant being a compound of formula HA-TLR-2-A, HA-1, HA-TLRAa, or HA-37, wherein the mass of said adjuvant is 8%-12% of the mass of the compound of formula I or a pharmaceutically acceptable salt thereof; preferably, the lipid carrier further comprises a diluent.

[0140] In some embodiments, the lipid carrier comprises 40-44 mol% of a compound as shown in Formula HI-19, 4.5-8.5 mol% of DEPC, 48-52% of cholesterol, 1-2 mol% of DMG-PEG2000, and a compound as shown in Formula HA-TLR-2-A, wherein the mass of the compound as shown in Formula HA-TLR-2-A is 8%-12% of the mass of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the lipid carrier is selected from Scheme 1 or Scheme 2:

[0142] Option 1: The lipid carrier is composed of a compound as shown in Formula HI-19, an acetate buffer at pH 4, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol.

[0143] The total molar amount is calculated as the sum of the molar amounts of the compound shown in formula HI-19, distearate phosphatidylcholine, cholesterol, and PEG-DMG-2000 lipid:

[0144] The molar amount of the compound as shown in formula HI-19 accounts for 50% of the total molar amount;

[0145] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0146] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0147] The PEG-DMG-2000 lipid accounts for 1.5% of the total molar amount;

[0148] Option 2: The lipid carrier is composed of a compound as shown in Formula HI-19, an adjuvant, an acetate buffer at pH 4, distearate phosphatidylcholine, PEG-DMG-2000, and cholesterol.

[0149] The total molar amount is calculated as the sum of the molar amounts of the compound shown in formula HI-19, distearate phosphatidylcholine, cholesterol, and PEG-DMG-2000 lipid:

[0150] The molar amount of the compound as shown in formula HI-19 accounts for 50% of the total molar amount;

[0151] The molar amount of the distearate phosphatidylcholine accounts for 10% of the total molar amount;

[0152] The molar amount of cholesterol accounts for 38.5% of the total molar amount;

[0153] The PEG-DMG-2000 lipid accounts for 1.5% of the total molar amount;

[0154] The mass percentage of the adjuvant to the mass of the compound as shown in Formula HI-19 is 10%.

[0155] The adjuvant is selected from compounds shown in formula HA-TLR-2-A, HA-1, HA-TLRAa or HA-37.

[0156] In some embodiments, the lipid carrier is selected from either scheme A or scheme B:

[0157] Option A: The lipid carrier consists of 50 mol% of the compound shown in Formula I, 10 mol% of phospholipids, 1.5 mol% of PEG lipids, and 38.5 mol% of cholesterol.

[0158] Preferably, the lipid carrier comprises 50 mol% of the compound represented by formula HI-19, 10 mol% of distearate phosphatidylcholine, 1.5 mol% of PEG-DMG-2000, and 38.5 mol% of cholesterol.

[0159] Preferably, the lipid carrier further includes a diluent;

[0160] Option B: The lipid carrier comprises 42 mol% of the compound of formula HI-19, 6.5 mol% of dierucic acid phosphatidylcholine (DEPC), 1.5 mol% of PEG-DMG-2000, and 50 mol% of cholesterol; or the lipid carrier comprises 45 mol% of the compound of formula HI-19, 11 mol% of dierucic acid phosphatidylcholine (DEPC), 1.5 mol% of PEG-DMG-2000, and 42.5 mol% of cholesterol.

[0161] Preferably, the lipid carrier further includes a diluent;

[0162] Option C: The lipid carrier consists of 50 mol% of a compound as shown in formula HI-19, an adjuvant, 10 mol% distearate phosphatidylcholine, 1.5 mol% PEG-DMG-2000, and 38.5 mol% cholesterol.

[0163] The mass percentage of the adjuvant to the mass of the compound as shown in Formula HI-19 is 10%.

[0164] The adjuvant is selected from compounds of the formula HA-1, HA-TLR-2-A, HA-TLRa, or HA-37:

[0165] Preferably, the lipid carrier further includes a diluent;

[0166] Scheme D: The lipid carrier consists of 42 mol% of the compound represented by formula HI-19, an adjuvant, 6.5 mol% of disorhodophosphatidylcholine (DEPC), 1.5 mol% of PEG-DMG-2000, and 50 mol% of cholesterol.

[0167] The adjuvant is a compound as shown in formula HA-TLR-2-A; the mass percentage of the adjuvant to the mass of the compound as shown in formula HI-19 is 10%;

[0168] Preferably, the lipid carrier further includes a diluent.

[0169] The present invention provides a liposomal drug (or lipid nanoparticle composition) comprising a nucleic acid molecule as described above and a lipid carrier as described in any of the aforementioned embodiments.

[0170] In some embodiments, the lipid carrier is a lipid nanoparticle (empty, unloaded lipid nanoparticles without nucleic acid molecules).

[0171] In some embodiments, the nucleic acid drug is firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, mRNA sequence encoding human papillomavirus E7 antigen, or EGFP-Flag mRNA.

[0172] In some embodiments, the liposomal drug wherein the lipocarrier is selected from scheme A, and the nucleic acid molecule is firefly luciferase (Fluc) mRNA or enhanced green fluorescent protein (eGFP) mRNA.

[0173] In some embodiments, in the liposomal drug, the lipid carrier is selected from scheme C, and the nucleic acid molecule is mRNA encoding the human papillomavirus E7 antigen.

[0174] In some embodiments, the liposomal drug wherein the lipocarrier is selected from scheme B, and the nucleic acid molecule is mRNA encoding luciferase (Luc).

[0175] In some embodiments, in the liposomal drug, the lipid carrier is selected from scheme D, and the nucleic acid molecule is mRNA encoding luciferase (Luc).

[0176] In some embodiments, the nitrogen-to-phosphorus ratio in the liposomal drug is (2-30):1. This nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in the lipid compound to the number of moles of phosphate groups in the RNA within the lipid carrier. In this application, the nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in the ionizable lipid (the compound shown in Formula I or a pharmaceutically acceptable salt thereof) to the number of moles of phosphate groups in the mRNA. Preferably, the nitrogen-to-phosphorus ratio is (2-20):1, more preferably (3-20):1, for example (3-16):1, and further for example (3-8):1, 3:1, 4:1, 5:1, 6:1, or 7:1.

[0177] In one embodiment, the average particle size of the liposomal drug is 40-170 nm, 40-150 nm, 60-150 nm, 80-130 nm, 100-130 nm, for example, 110 nm, 115 nm, 116 nm, 121 nm, 123 nm or 125 nm.

[0178] In one embodiment, the encapsulation rate of the liposomal drug is 80%-100%, 90%-100%, for example 94.20%, 94.22%, 95.25%, 95.56%, 95.93%, 96.24%, or 95.46%.

[0179] In one embodiment, the liposomal drug consists of firefly luciferase (Fluc) mRNA and a lipid carrier as described in Embodiment 1 above, with a nitrogen-to-phosphorus ratio of 3:1, 5:1, 6:1, or 7:1.

[0180] In one embodiment, the liposomal drug enhances green fluorescent protein (eGFP) mRNA and consists of a lipid carrier as described in embodiment 1 above, with a nitrogen-to-phosphorus ratio of 6:1.

[0181] In one embodiment, the liposomal drug consists of an mRNA sequence encoding the human papillomavirus E7 antigen and a lipid carrier as described in embodiment 2 above, with a nitrogen-to-phosphorus ratio of 6:1.

[0182] The present invention also provides a lipid carrier as described above.

[0183] In one embodiment, the liposomal drug contains a lipocarrier that encapsulates the nucleic acid as described above.

[0184] Unless otherwise specified, the terms used in this invention have the following meanings:

[0185] The descriptive phrase "...independently" used in this invention should be interpreted broadly, meaning that the described entities are independent of each other and can independently be the same or different specific functional groups. More specifically, the descriptive phrase "...independently" can mean that in different functional groups, the specific options expressed by the same symbols do not affect each other; or it can mean that in the same functional group, the specific options expressed by the same symbols do not affect each other.

[0186] In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0187] The term "optionally by one or more R" a "Replace" indicates that it was not replaced by R a Replaced and by one or more R a Replaces both scenarios.

[0188] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0189] The terms “substituted” or “replaced” refer to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the substituted compound is stable.

[0190] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the fact that the corresponding group is connected to other fragments or groups in the compound through this site.

[0191] In this article, the single dash "-" on both sides of the substituent indicates that the substituent is connected to the parent part by a chemical bond.

[0192] The term "multiple" refers to 2, 3, 4 or 5, preferably 2 or 3.

[0193] The term "pharmaceutically acceptable" means that the salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for patient use. The term "patient" preferably refers to a mammal, and more preferably a human.

[0194] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of this invention with a relatively non-toxic, pharmaceutically acceptable acid or base.

[0195] When any variable (e.g., R) f When a variable appears multiple times in the definition of a compound, the definition at each position is independent of the definitions at the other positions; their meanings are independent and do not affect each other. Therefore, if a group is surrounded by one, two, or three R... f Group substitution, meaning that the group can be replaced by up to 3 R groups. f Replace, the position R f Definition and other positions R f The definitions are independent of each other. Furthermore, combinations of substituents and / or variables are only permitted if the combination produces a stable compound.

[0196] The term "biodegradable group" refers to a group that promotes faster lipid metabolism in mammalian bodies. Biodegradable groups can be selected from, but are not limited to, -NH-, -O-, -S-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -S(O2)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, and -OP(S)(OH)-. Cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0197] The term "linking group" refers to a head group in a lipid compound used to link the lipid compound (e.g., a cationic or ionizable head group of the lipid compound; in this application, the head group refers to...). ) and long-chain hydrocarbon groups.

[0198] The term "oxo group" refers to the =O group (e.g., when connected to C or S, it is C=O, S=O / SO). 2 ) or O - (For example, together with N to form N) + O - ).

[0199] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0200] The term "alkyl" refers to a straight-chain or branched saturated alkyl group having a specified number of carbon atoms. For example, C 1-30 Alkyl, C 5-30 C 1-6 C 1-10 Alkyl groups, examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and straight-chain or branched C-aryl groups. 11 C12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 Alkyl groups and similar alkyl groups.

[0201] The term "alkenyl" refers to a straight-chain or branched alkene with a specified number of carbon atoms, containing one or more (e.g., one or two) carbon-carbon double bonds and no carbon-carbon triple bonds. These carbon-carbon double bonds can be internal or terminal. For example, C... 2-30 C 5-20 The alkenyl group.

[0202] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group having one or more (e.g., one or two) carbon-carbon triple bonds with a specified number of carbon atoms. These carbon-carbon triple bonds can be internal or terminal, such as the propynyl group with internal triple bonds. Or a propynyl group at the end of the triple bond Etc. For example, C2-C 30 Alkyne group, for example, C5-C 20 Alkyne group.

[0203] The term "cycloalkyl" refers to a saturated cyclic alkyl group having a specified number of ring carbon atoms, preferably a saturated cyclic alkyl group having 3-10 ring carbon atoms, more preferably 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0204] The term "heterocyclic alkyl" refers to a cyclic group having a specified number of ring atoms (e.g., 3-10), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatom (1, 2 or 3 of N, O, S and S(O2), preferably 1, 2 or 3 of N, O and S), and each ring is saturated.

[0205] "Aryl" refers to a polycyclic group consisting of an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) and having a conjugated π-electron system. "6-10 aryl" refers to an all-carbon aryl group containing 6-10 carbon atoms, such as phenyl and naphthyl; phenyl is preferred.

[0206] The term "heteroaryl" refers to a monocyclic or bicyclic aromatic group containing a specified number of ring atoms (e.g., 5 to 10, 5 to 6), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatom (1, 2 or 3 of N, O and S).

[0207] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0208] The reagents and raw materials used in this invention are all commercially available.

[0209] The positive and progressive effects of this invention are as follows: the lipid compounds provided by this invention, as drug carriers, have the advantages of high encapsulation efficiency, high expression level, and good targeting. Detailed Implementation

[0210] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0211] Example 1:

[0212] Step 1:

[0213] To a mixture of compound 1 (31 g, 140 mmol, 1.2 eq.) and compound 2 (30 g, 117 mmol, 1.0 eq.) in DCM (400 mL), EDCI (45 g, 234 mmol, 2.0 eq.), DMAP (5.7 g, 46.8 mmol, 0.4 eq.), and DIEA (60 g, 468 mmol, 4.0 eq.) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (PE:EA = 9 / 1) showed that the reaction was complete and new major spots were observed. The mixture was quenched with water (400 mL) and extracted with DCM (250 mL × 3). The combined organic layers were washed with saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% EA in PE) to give a clear oily compound 3 (42 g, 52.7%).

[0214] Step 2:

[0215] To a mixture of compound 1 (43 g, 191 mmol, 1.1 eq.) and compound 2A (30 g, 174 mmol, 1.0 eq.) in DCM (400 mL), EDCI (67 g, 348 mmol, 2.0 eq.), DMAP (8.5 g, 69.6 mmol, 0.4 eq.), and DIEA (90 g, 696 mmol, 4.0 eq.) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (PE:EA = 9 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (400 mL) and extracted with DCM (250 mL × 3). The combined organic layers were washed with saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% EA in PE) to give a clear oily compound 4 (42 g, 52.3%).

[0216] Step 3:

[0217] Compound 5 (39 g, 521 mmol, 30.0 eq.) was added to compound 4 (8.0 g, 17.4 mmol, 1.0 eq.) dissolved in ethanol (5.0 mL). The reaction mixture was stirred at 50 °C for 16 h under nitrogen protection. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–20% MeOH in DCM) to give a clear oily compound 6 (6.1 g, 77.1%).

[0218] Step 4:

[0219] To a mixture of compound 6 (4.3 g, 9.57 mmol, 1.0 eq.) and compound 3 (3.6 g, 9.57 mmol, 1.0 eq.) in acetonitrile (40 mL), K₂CO₃ (6.6 g, 47.7 mmol, 5.0 eq.), KI (3.2 g, 19.2 mmol, 2.0 eq.), and CPME (40 mL) were added. The reaction was stirred overnight at 90 °C under nitrogen protection. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (70 mL) and extracted with EA (80 mL × 3). The combined organic layers were washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a clear, oily compound 7 (3.7 g, 51.5%).

[0220] Step 5:

[0221] Under nitrogen protection, methanesulfonyl chloride (711 mg, 6.24 mmol, 1.25 eq.) was added to a mixture of compound 7 (3.7 g, 4.99 mmol, 1.0 eq.) and TEA (656 mg, 6.5 mmol, 1.3 eq.) in DCM (40 mL) at 0 °C. The reaction mixture was brought to room temperature and stirred for 16 h. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (50 mL) and extracted with DCM (50 mL × 3). The combined organic layers were washed successively with 5% NaHCO3 solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. A clear oily compound 8 (3.7 g, 96.4%) was given, which was used directly in the next reaction without further purification.

[0222] Step 6:

[0223] Compound 8 (3.7 g, 4.99 mmol, 1.0 eq.) was dissolved in DMF (40 mL) under nitrogen protection, and sodium azide (1.6 g, 25.0 mmol, 5.0 eq.) was added. The reaction mixture was stirred at 100 °C for 16 h. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. A yellow oily compound 9 (3.6 g, 93.0%) was given, which was used directly in the next reaction without further purification.

[0224] Step 7:

[0225] Pd / C (800 mg) was added to compound 9 (3.6 g) in ethanol (150 mL). The reaction mixture was stirred overnight at 25 °C under hydrogen atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to give compound 10 (3.0 g, 89.4%), a yellow oil.

[0226] LCMS:Rt:1.847min;MS m / z(ELSD):751.6[M+H] + ;

[0227] Step 8:

[0228] Compound 11 (5 g, 65.79 mmol, 1.0 eq.) was dissolved in water (50 mL), and the mixture was cooled to 0 °C and paraformaldehyde (11.39 g, 144.74 mmol, 2.2 eq.) was added. The reaction mixture was then stirred at 50 °C for 4 hours. The reaction mixture was directly concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography using 10% MeOH in DCM to give a milky white oily compound 12 (2.05 g, 22%).

[0229] LCMS:Rt:0.558min;MS m / z(ELSD):137.0[M+H] + ;

[0230] Step 2:

[0231] Compound 12 (109 mg, 0.79 mmol, 1.0 eq.) and compound 10 were mixed together and added to ethanol (5 mL), then stirred overnight at 70 °C under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and a new principal spot was formed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a colorless, transparent oily compound HI-19 (217.57 mg, 32%).

[0232] LCMS:Rt:1.538min;MS m / z(ELSD):851.9[M+H] + .

[0233] CAD: 91.69% purity.

[0234] 1H NMR (400MHz, CDCl3) δ6.40(s,2H),4.90–4.77(m,2H),4.23(s,4H),2.95–2.15(m,12H),1.76–1.18(m,66H),0.92–0.84(m,12H).

[0235] Example 2

[0236] Step 1:

[0237] Pd(dppf)Cl2 (1.606 g, 2.19 mmol, 0.1 eq.) and K2CO3 (9.079 g, 65.8 mmol, 3.0 eq.) were added to a mixture of compound 1 (5 g, 21.9 mmol, 1.0 eq.) and compound 2 (3.356 g, 32.9 mmol, 1.5 eq.) in 1,4-dioxane (50 mL) and water (5 mL). The mixture was stirred overnight at 100 °C under nitrogen protection. TLC (PE / EA = 20 / 1) showed that the reaction was complete and new major spots were observed. The mixture was extracted with ethyl acetate (200 mL × 3) and washed with water (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (0%–10% EA in PE) to give compound 3 (1.555 g, 34%) as a colorless oil.

[0238] LCMS:Rt:0.890min;MS m / z(ELSD):496.5[M+H] + .

[0239] 1H NMR (400MHz, CDCl3) δ7.24–7.13(m,4H),3.68(s,3H),3.66(s,2H),2.64–2.60(m,2H),1.58–1.50(m,2H),1.44–1.35(m,2H),0.94(t,J=7.3Hz,3H).

[0240] Step 2:

[0241] LAH (287 mg, 7.55 mol, 1.0 eq.) was added to a mixture of compound 1 (1.555 g, 7.55 mmol, 1.0 eq.) in THF (15 mL) under nitrogen protection at 0 °C. The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL) and treated with 1 N hydrochloric acid to adjust the pH to between 6 and 7. The mixture was then quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–20% EA in PE) to give a clear, oily compound 4 (1.17 g, 87%).

[0242] 1H NMR (400MHz, CDCl3) δ7.19–7.11(m,4H),3.83(t,J=7.0Hz,2H),2.91(t,J=7.0Hz,2H),2. 65–2.61(m,2H),1.60–1.52(m,2H),1.40(dq,J=14.5,7.3Hz,2H),0.94(t,J=7.3Hz,3H).

[0243] Step 3:

[0244] To a mixture of compound 4 (1.17 g, 6.57 mmol, 1.5 eq.) and compound 5 (973 mg, 4.38 mmol, 1.0 eq.) in DCM (20 mL), EDCI (1.674 g, 8.76 mmol, 2.0 eq.), DMAP (214 mg, 1.75 mmol, 0.4 eq.), and DIEA (2.261 g, 17.5 mmol, 4.0 eq.) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (PE:EA = 20 / 1) showed that the reaction was complete and new major spots were observed. The mixture was quenched with water (100 mL) and extracted with EA (80 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% EA in PE) to give a clear oily compound 6 (917 mg, 37%).

[0245] 1H NMR (400MHz, CDCl3) δ7.17–7.12(m,4H),4.26(t,J=7.4Hz,2H),3.40(t,J=6.8Hz,2H),2.96(t,J=7.4Hz,2H),2.66–2.62(m,2H),2.30( dd,J=10.1,4.9Hz,2H),1.83(dd,J=14.6,7.0Hz,2H),1.59–1.57(m,4H),1.45–1.38(m,4H),1.33–1.29(m,4H),0.95(t,J=7.3Hz,3H).

[0246] Step 4:

[0247] To a mixture of compound 7 (1.093 g, 2.4 mmol, 1.0 eq.) in CPME / MeCN (20 mL, v / v), KI (999 mg, 2.4 mmol, 1.0 eq.), K₂CO₃ (994 mg, 7.2 mmol, 3.0 eq.), and compound 6 (917 mg, 2.4 mmol, 1.0 eq.) were added. The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 8 (990 mg, 54%) as a yellow oil.

[0248] LCMS:Rt:1.455min; MS m / z(ELSD):759.9[M+H] + .

[0249] Step 5:

[0250] TEA (330 mg, 3.27 mmol, 2.5 eq.) and MSCl (300 mg, 2.61 mmol, 2.0 eq.) were added to a mixture of compound 8 (990 mg, 1.31 mmol, 1.0 eq.) in DCM (10 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (3 mL). Water (100 mL) was added, and the mixture was extracted with dichloromethane (100 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 9 (1.252 g, crude), which was a yellow oil.

[0251] LCMS:Rt:1.335min; MS m / z(ELSD):836.7[M+H] + .

[0252] Step 6:

[0253] To a mixture of compound 9 (1092 mg, 1.3 mmol, 1.0 eq.) in DMF (10 mL), NaN3 (170 mg, 2.6 mmol, 2.0 eq.) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (DCM:MeOH = 10 / 1) showed the reaction was complete, and a new major spot was observed. The mixture was quenched with water (150 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic layers were washed with brine (8 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 10 (845 mg, 83%) as a yellow solid. LCMS: Rt: 1.378 min; MS m / z (ELSD): 784.7 [M+H] + .

[0254] 1H NMR(400MHz, CDCl3)δ7.20–7.09(m,4H),4.86(p,J=6.2Hz,1H),4.25(t,J=7.4Hz, 2H),3.34(t,J=6.7Hz,2H),2.94(dd,J=22.1,15.1Hz,4H),2.68–2.60(m,2H),2.45 (dd,J=38.4,6.4Hz,6H),2.28(dd,J=14.2,7.1Hz,4H),1.79–1.68(m,2H),1.64–1. 39(m,16H),1.27(d,J=15.4Hz,34H),0.95(t,J=7.3Hz,3H),0.88(t,J=6.7Hz,6H).

[0255] Step 7:

[0256] Pd / C (300 mg) was added to a mixture of compound 10 (845 mg, 1.08 mmol, 1.0 eq.) in MeOH (10 mL) and THF (5 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 11 (587 mg, 74%) as a yellow oil.

[0257] LCMS:Rt:1.423min; MS m / z(ELSD):757.7[M+H] + .

[0258] 1 H NMR (400MHz, CDCl3) δ7.21–7.08(m,4H),4.86(p,J=6.3Hz,1H),4.25(t,J=7 .4Hz,2H),2.96(t,J=7.4Hz,2H),2.76(t,J=6.7Hz,2H),2.68–2.60(m,2H),2 .52–2.44(m,2H),2.43–2.36(m,4H),2.28(dd,J=14.4,7.2Hz,4H),1.65–1.3 7(m,19H),1.32–1.22(m,37H),0.95(t,J=7.3Hz,3H),0.88(t,J=6.8Hz,6H).

[0259] Step 8:

[0260] Compound 12 (161 mg, 0.71 mmol, 1.0 eq.) was added to a mixture of compound 11 (537 mg, 0.71 mmol, 1.0 eq.) in ethanol (0.5 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 hours. TLC (EA:THF = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily compound HI-19-10-1 (mg, %).

[0261] LCMS:Rt:1.260min;MS m / z(ELSD):857.7[M+H] + .

[0262] Example 3

[0263] Step 1:

[0264] Compound 1 (6 g, 26.2 mmol, 1 eq.), compound 2 (3.7 g, 31.4 mmol, 1.2 eq.), Pd(dppf)Cl2 (1.92 g, 2.62 mmol, 0.1 eq.), and potassium carbonate (11 g, 78.60 mmol, 3 eq.) were added sequentially to a solution of 1,4-dioxane (60 mL) and water (6 mL). The reaction was then heated to 100 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 30:1), indicating complete consumption of the starting materials. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated brine (40 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–5% EA in PE) to give a colorless, transparent oily product 3 (4.4 g, 76%).

[0265] 1 H NMR (400MHz, CDCl3) δ7.26–7.11(m,4H),3.68(s,3H),3.66(s,2H),2.66–2.59(m,2H),1.59–1.52(m,2H),1.39–1.29(m,4H),0.94–0.86(m,3H).

[0266] Step 2:

[0267] Lithium aluminum hydride (40 mL, 40 mmol, 1 M in THF) was added dropwise to a solution of compound 3 (4.4 g, 20 mmol, 1.0 eq.) in tetrahydrofuran (50 mL) at 0 °C. The reaction was then stirred at room temperature for two hours under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 30:1), indicating that the starting material was completely consumed. The reaction was quenched with sodium sulfate decahydrate, then filtered and the residue was washed with dichloromethane (30 mL). The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–20% EA in PE) to give a colorless oily product 4 (2.8 g, 73%).

[0268] 1 H NMR (400MHz, CDCl3) δ7.21–7.19(m,4H),3.83(t,J=6.8Hz,2H),2.91(t,J=6.8Hz,2 H),2.70–2.55(m,2H),1.59–1.52(m,1H),1.40–1.28(m,4H),0.90(t,J=6.8Hz,3H).

[0269] Step 3:

[0270] Compound 4 (2.795 g, 14.8 mmol), EDCI (5.7 g, 29.60 mmol), DMAP (767 mg, 5.92 mmol, 0.4 eq.), and DIEA (7.6 g, 59.19 mmol) were added sequentially to a solution of compound 5 (3.3 g, 14.8 mmol) in dichloromethane (30 mL). The reaction was then stirred overnight at room temperature under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 5:1), indicating complete consumption of the starting materials. The reaction was quenched with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–5% EA in PE) to give a yellow oily product 6 (2.69 g, 48%).

[0271] 1 H NMR (400MHz, CDCl3) δ7.20–7.07(m,4H),4.26(t,J=8.8,2H),3.55–3.37(m,2H),2.96(t,J=7.6Hz,2H),2.67–2. 59(m,2H),2.29(t,J=7.6Hz,2H),1.89–1.71(m,2H),1.66–1.52(m,4H),1.46–1.23(m,10H),0.97–0.81(m,3H).

[0272] Step 4:

[0273] Compound 7 (1 g, 2.20 mmol, 1 eq.), potassium iodide (1.1 g, 6.59 mmol, 3.8 eq.), and potassium carbonate (1.5 g, 10.99 mmol, 5 eq.), and compound 6 (1 g, 2.64 mmol, 1.2 eq.) were added to a solution of acetonitrile (15 mL) and cyclopentyl methyl ether (15 mL). The reaction was then heated to 90 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), indicating that the starting materials were completely consumed. The reaction mixture was diluted with ethyl acetate (80 mL) and washed with water (80 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 8 (1.077 g, 64%).

[0274] LCMS:Rt:1.515min;MS m / z(ELSD):772.9[M+H] + ;

[0275] Step 5:

[0276] Compound 8 (1.077 g, 1.40 mmol, 1.0 eq.) and triethylamine (352 mg, 3.49 mmol, 2.5 eq.) were dissolved in dichloromethane (10 mL). Methanesulfonyl chloride (321 mg, 2.99 mmol, 2 eq.) was slowly added dropwise to the solution at 0 °C. The reaction was then stirred at room temperature for two hours under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10:1), showing the disappearance of the starting material and the formation of a new spot. The reaction was quenched with water (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under pressure to give a yellow oily product 9 (1.1 g, 100%).

[0277] LCMS:Rt:1.338min; MS m / z(ELSD):790.7[M+H] + ;

[0278] Step 6:

[0279] Compound 9 (1.1 g, 1.29 mmol, 1.0 eq.) was dissolved in a solution of N,N-dimethylformamide (10 mL), and sodium azide (230 mg, 3.88 mmol, 3.0 eq.) was added. The reaction was heated to 100 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), showing that the starting material was completely consumed and a new spot was formed. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 10 (689 mg, 67%).

[0280] LCMS:Rt:1.737min; MS m / z(ELSD):797.9[M+H] + ;

[0281] Step 7:

[0282] Compound 10 (689 mg, 0.86 mmol) was dissolved in a solution of methanol (5 mL) and tetrahydrofuran (5 mL), and palladium on carbon (300 mg, 10% wt) was added. The reaction was stirred overnight at room temperature under hydrogen protection. The reaction was monitored by LCM to show that the starting material was consumed. The reaction mixture was filtered and concentrated to dryness under pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 11 (402 mg, 60%).

[0283] LCMS:Rt:2.278min; MS m / z(ELSD):771.8[M+H] + ;

[0284] Step 8:

[0285] Compound 11 (200 mg, 0.26 mmol, 1.0 eq) was added to compound 12 (47 mg, 0.26 mmol, 1.0 eq.) in ethanol (0.2 mL). The reaction was stirred overnight at 70 °C under argon protection. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give the target compound HI-19-111 (mg, 4.9%) as a yellow oil.

[0286] Example 4

[0287] Step 1:

[0288] Compound 1 (6.6 g, 28.82 mmol, 1 eq.), compound 2 (4 g, 34.39 mmol, 1.2 eq.), Pd(dppf)Cl 2 (2.1 g, 2.88 mmol, 0.1 eq.) and potassium carbonate (11.8 g, 86.46 mmol, 3 eq.) were added sequentially to a solution of 1,4-dioxane (70 mL) and water (7 mL). The reaction was then heated to 100 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 30:1), indicating that the starting materials were completely consumed. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–5% EA in PE) to give a colorless, transparent oily product 3 (4 g, 63%).

[0289] 1 H NMR (400MHz, CDCl3) δ7.35–7.19(m,2H),7.10–7.06(m,2H),3.69(s,3H),3.60(s,2 H),2.62–2.53(m,2H),1.67–1.51(m,2H),1.38–1.26(m,4H),0.89(t,J=6.8Hz,3H).

[0290] Step 2:

[0291] Lithium aluminum hydride (36 mL, 36.33 mmol, 1 M in THF) was added dropwise to a solution of compound 3 (4.0 g, 18.16 mmol, 1.0 eq.) in tetrahydrofuran (50 mL) at 0 °C. The reaction was then stirred at room temperature under nitrogen protection for two hours. The reaction was monitored by spot TLC (PE:EA = 30:1), indicating that the starting material was completely consumed. The reaction was quenched with sodium sulfate decahydrate, then filtered and the residue was washed with dichloromethane (30 mL). The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–20% EA in PE) to give a colorless oily product 4 (2.33 g, 67%).

[0292] 1H NMR (400MHz, CDCl3) δ7.24–7.18(m,1H),7.07–6.99(m,3H),3.83(t,J=6.8Hz,2H),2.83(t,J =6.8Hz,2H),2.62–2.50(m,2H),1.69–1.53(m,2H),1.40–1.25(m,4H),0.89(t,J=6.8Hz,3H).

[0293] Step 3:

[0294] Compound 4 (2.33 g, 12.11 mmol), EDCI (4.65 g, 24.22 mmol), DMAP (590 mg, 4.84 mmol, 0.4 eq.), and DIEA (6.3 g, 48.43 mmol) were added sequentially to a solution of compound 5 (2.7 g, 12.11 mmol) in dichloromethane (30 mL). The reaction was then stirred overnight at room temperature under nitrogen protection. The reaction was monitored by spot TLC (PE:EA = 5:1), indicating complete consumption of the starting materials. The reaction was quenched with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–5% EA in PE) to give a yellow oily product 6 (1.763 g, 38%).

[0295] 1 H NMR (400MHz, CDCl3) δ7.31–6.95(m,4H),4.28(t,J=7.2Hz,2H),3.57–3.37(m,2H),2.91(t,J=7.2Hz,2H),2.65–2. 48(m,2H),2.28(t,J=7.6Hz,2H),1.89–1.71(m,2H),1.65–1.52(m,4H),1.45–1.21(m,10H),0.89(t,J=7.2Hz,3H).

[0296] Step 4:

[0297] Compound 7 (1 g, 2.20 mmol, 1 eq.), potassium iodide (1.1 g, 6.59 mmol, 3.8 eq.), and potassium carbonate (1.5 g, 10.99 mmol, 5 eq.), and compound 6 (1 g, 2.64 mmol, 1.2 eq.) were added to a solution of acetonitrile (15 mL) and cyclopentyl methyl ether (15 mL). The reaction was then heated to 90 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), indicating that the starting materials were completely consumed. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with water (50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 8 (934 mg, 55%).

[0298] LCMS:Rt:1.374min;MS m / z(ELSD):772.8[M+H] + .

[0299] Step 5:

[0300] Compound 8 (937 mg, 1.21 mmol, 1.0 eq.) and triethylamine (305 mg, 3.02 mmol, 2.5 eq.) were dissolved in dichloromethane (10 mL). Methanesulfonyl chloride (278 mg, 2.42 mmol, 2 eq.) was slowly added dropwise to the solution at 0 °C. The reaction was then stirred at room temperature for two hours under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10:1), showing the disappearance of the starting material and the formation of a new spot. The reaction was quenched with water (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under pressure to give a yellow oily product 9 (1 g, 100%).

[0301] LCMS:Rt:1.338min; MS m / z(ELSD):790.7[M+H] + .

[0302] Step 6:

[0303] Compound 9 (1.0 g, 1.18 mmol, 1.0 eq.) was dissolved in a solution of N,N-dimethylformamide (10 mL), and sodium azide (229 mg, 3.53 mmol, 3.0 eq.) was added. The reaction was heated to 100 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), showing complete consumption of the starting material and formation of a new spot. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 10 (659 mg, 70%).

[0304] LCMS:Rt:1.457min; MS m / z(ELSD):797.6[M+H] + .

[0305] Step 7:

[0306] Compound 10 (659 mg, 0.83 mmol) was dissolved in a solution of methanol (5 mL) and tetrahydrofuran (5 mL), and palladium on carbon (300 mg, 10% wt) was added. The reaction was stirred overnight at room temperature under hydrogen protection. The reaction was monitored by LCM to show that the starting material was consumed. The reaction mixture was filtered and concentrated to dryness under pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 11 (466 mg, 73%).

[0307] LCMS:Rt:2.418min;MS m / z(ELSD):771.8[M+H] + .

[0308] Step 8:

[0309] Compound 11 (200 mg, 0.26 mmol, 1.0 eq) was added to compound 12 (47 mg, 0.26 mmol, 1.0 eq.) in ethanol (0.2 mL). The reaction was stirred overnight at 70 °C under argon protection. The reaction was monitored by spot TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give a yellow oily target compound HI-19-112 (mg, 4.9%).

[0310] Example 5

[0311] Step 1:

[0312] Compound 1 (6.18 g, 16.44 mmol, 1.0 eq.) was dissolved in ethanol (6 mL), and compound 2 (37 g, 493.48 mmol, 30.0 eq.) was added. The reaction was stirred overnight at 50 °C under nitrogen protection. TLC (DCM:MeOH = 10:1) showed that the reaction was complete and new spots were formed. The reaction mixture was diluted with EA (200 mL) and washed with saturated brine (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 3 (4.85 g, 79%) as a yellow oil.

[0313] LCMS:Rt:1.031min;MS m / z(ELSD):372.4[M-100+H] + ;

[0314] 1H NMR (400MHz, CDCl3) δ4.92–4.75(m,1H),3.90–3.72(m,2H),2.98–2.90(m,2H),2.71–2.60(m,2H),2 .28(t,J=7.5Hz,2H),1.80–1.70(m,2H),1.65-1.48(m,8H),1.20-1.10(m,20H),0.95–0.82(m,6H).

[0315] Step 2:

[0316] Compound 3 (1 g, 2.69 mmol, 1.0 eq.) was dissolved in CPME / MeCN (10 mL, 1:1, v / v), followed by the addition of KI (895 mg, 4.04 mmol, 2.0 eq.) and K₂CO₃ (1.86 g, 5.39 mmol, 5.0 eq.). The reaction was stirred overnight at 90 °C under nitrogen protection. TLC (DCM:MeOH = 15:1) showed that the reaction proceeded to completion and a new principal spot was formed. The reaction was quenched with water (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under pressure to give compound 5 (819 mg, 64%) as a yellow oil.

[0317] LCMS:Rt:1.069min; MS m / z(ELSD):498.4[M+H+23] + ;

[0318] 1H NMR(400MHz, CDCl3)δ7.29(dd,J=10.0,4.6Hz,2H),7.24–7.17(m,3H),4.89–4.76(m,1H),3.89–3.74(m,2H),2.90–2.71(m,6H),2 .64–2.48(m,2H),2.29(t,J=7.5Hz,2H),1.75(dt,J=10.8,5.4Hz,2H),1.65–1.48(m,8H),1.35–1.23(m,18H),0.94–0.80(m,6H).

[0319] Step 3:

[0320] Compound 5 (819 mg, 1.72 mmol, 1.0 eq.) was dissolved in dichloromethane (10 mL) and cooled to 0 °C. Triethylamine (435 mg, 4.31 mmol, 2.5 eq.) and MsCl (397 mg, 3.45 mmol, 2.0 eq.) were added dropwise. The reaction was then carried out at room temperature for 2 h. TLC (MeOH:DCM = 15:1) monitoring showed that the starting material was completely consumed and a new principal spot was formed. The reaction was quenched with water (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily crude compound 6 (953 mg).

[0321] LCMS:Rt:1.072min; MS m / z(ELSD):458.4[M+H] + ;

[0322] Step 4:

[0323] Compound 6 (953 mg, 1.72 mmol, 1.0 eq.) was dissolved in DMF (10 mL) and NaN3 (336 mg, 5.17 mmol, 3.0 eq.) was added. The reaction was carried out overnight at 100 °C under nitrogen protection with stirring. TLC (DCM:MeOH = 10:1) showed that the starting material was completely consumed and a new spot was formed. The reaction mixture was diluted with EA (200 mL), washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure until evaporated to dryness. The residue was purified by silica gel column chromatography to give compound 7 (748 mg, 86%) as a yellow oil.

[0324] LCMS:Rt:1.089min;MS m / z(ELSD):501.5[M+H] + .

[0325] 1H NMR (400MHz, CDCl3) δ7.33–7.27(m,2H),7.21(t,J=7.7Hz,3H),4.89–4.74(m,1H),2.98(dd,J=127. 8,98.9Hz,10H),2.29(t,J=7.5Hz,2H),1.66–1.47(m,10H),1.38–1.19(m,20H),0.90–0.84(m,6H).

[0326] Step 5:

[0327] Compound 7 (748 mg, 1.47 mmol, 1.0 eq.) was dissolved in MeOH / THF (10 mL, 1:1, v / v) and Pd / C (180 mg) was added. The mixture was stirred overnight at room temperature under hydrogen protection. TLC (DCM:MeOH = 10 / 1) was used to monitor complete consumption of the starting material. The reaction mixture was directly filtered and concentrated to dryness. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give compound 8 (450 mg, 63%) as a yellow oil.

[0328] LCMS:Rt:0.973min;MS m / z(ELSD):476.4[M+H] + .

[0329] Step 6:

[0330] Compound 8 (250 mg, 0.53 mmol, 1.0 eq.) was dissolved in EtOH (95%), and compound 7 (96 mg, 0.53 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature for 15 min, then heated to 70 °C and stirred overnight. TLC (DCM:MeOH = 10 / 1) showed complete consumption of the starting material. The reaction mixture was quenched with water (50 mL) and extracted with EA (50 mL × 3). The organic phase was washed with saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under pressure until dry. The residue was purified by silica gel column chromatography (50% EA / THF (5:1, v / v) in PE) to give a yellow oily compound HI-19-120 (g, 88%).

[0331] Example 6

[0332] Step 1:

[0333] Compound 5 (10 g, 131.57 mmol, 1.0 eq.) and formalin solution (22.78 g, 289.47 mmol, 2.02 eq.) were mixed together under nitrogen protection and stirred at 50 °C for 25 minutes, then kept in a crystallizer for 10–12 h. The syrupy liquid was kept at 0 °C for 1 hour, and the solidified material was ground into a white powder and dried under reduced pressure to obtain a white solid compound 6 (13.5 g, 75% purity).

[0334] 1H NMR spectrum (DMSO-d6), δ4.83(s,4H),5.48(s,2H),8.03(s,2H).

[0335] Step 2:

[0336] Compound 3 (970 mg, 3.08 mmol, 1.0 eq.) was dissolved in dichloromethane (10 mL), and compound 2 (665 mg, 4.64 mmol, 1.5 eq.), EDCI (1.18 g, 6.16 mmol, 2.0 eq.), DIEA (1.6 g, 12.32 mmol, 4.0 eq.), and DMAP (150 mg, 1.23 mmol, 0.4 eq.) were added. The reaction was stirred overnight at room temperature under nitrogen protection. TLC (EA:PE = 1:2) showed complete consumption of the starting material and the formation of a principal spot. The reaction mixture was quenched with water (100 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure until evaporated to dryness. The residue was purified by silica gel column chromatography to give compound 5 (960 mg, 70%) as a yellow solid.

[0337] LCMS:Rt:2.145min;MS m / z(ELSD):386.4[M+H-56] + .

[0338] 1H NMR (400MHz, CDCl3) δ4.47(s,1H),4.05(t,J=6.7Hz,2H),3.10(t,J=7.0Hz,2H),2.29( t,J=7.5Hz,2H),1.65–1.58(m,4H),1.44(s,9H),1.27(s,28H),0.88(t,J=6.9Hz,4H).

[0339] Step 3:

[0340] Compound 3 (400 mg, 0.91 mmol, 1.0 eq.) was dissolved in DCM (10 mL), cooled to 0 °C, and HCl (5 mL, HCl in dixoane) was added. The reaction was stirred at room temperature for 2 hours. TLC (MeOH:DCM = 15:1) showed that the reaction was complete and a new principal spot was formed. The reaction was quenched with saturated NaHCO3 (50 mL), and extracted with DCM (50 mL × 3). The organic phase was washed with saturated brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give compound 4 (295 mg, 95%) as a yellow solid.

[0341] LCMS:Rt:1.078min;MS m / z(ELSD):342.3[M+H] + .

[0342] 1H NMR (400MHz, CDCl3) δ4.05(t,J=6.7Hz,2H),2.71(t,J=7.1Hz,2H),2.29(t,J=7.5Hz,2H),2.11 (s,2H),1.68–1.54(m,4H),1.46(dd,J=13.9,7.0Hz,2H),1.27(s,26H),0.88(t,J=6.8Hz,3H).

[0343] Step 4:

[0344] Compound 4 (275 mg, 0.81 mmol, 1.0 eq.) was dissolved in EtOH (0.3 mL), and compound 6 (146 mg, 0.81 mmol, 1.0 eq. 75% purity) was added. The mixture was stirred at room temperature for 15 minutes, and then stirred overnight at 70 °C. . The reaction was monitored to be complete by TLC plate (DCM:MeOH = 10:1). The reaction mixture was diluted with ethyl acetate (50 mL) and then washed with brine (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (mobile phase: 0%–10% MeOH in DCM) to give a pale yellow oily compound HI-19-127 (mg, 86%).

[0345] Example 7

[0346] Step 1:

[0347] Compound 2 (2.8 g, 6.15 mmol, 1 eq.), potassium iodide (3.1 g, 18.46 mmol, 3.0 eq.), and potassium carbonate (4.3 g, 30.77 mmol, 5 eq.), and compound 1 (3.4 g, 7.38 mmol, 1.2 eq.) were added to a solution of acetonitrile (30 mL) and cyclopentyl methyl ether (30 mL). The reaction was then heated to 90 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), indicating that the starting materials were completely consumed. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with water (50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 3 (2.828 g, 55%).

[0348] LCMS:Rt:2.162min; MS m / z(ELSD):837.0[M+H] + .

[0349] Step 2:

[0350] Compound 3 (1.5 g, 1.79 mmol, 1.0 eq.) and triethylamine (453 mg, 4.49 mmol, 2.5 eq.) were dissolved in dichloromethane (20 mL). Methanesulfonyl chloride (413 mg, 3.59 mmol, 2 eq.) was slowly added dropwise to the solution at 0 °C. The reaction was then stirred at room temperature for two hours under nitrogen protection. The reaction was monitored by spot TLC (DCM:MeOH = 10:1), showing the disappearance of the starting material and the formation of a new spot. The reaction was quenched with water (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under pressure to give a yellow oily product 4 (548 mg, 33%).

[0351] LCMS:Rt:2.139min;MS m / z(ELSD):914.8[M+H] + .

[0352] Step 3:

[0353] Compound 4 (548 mg, 0.6 mmol, 1.0 eq.) was dissolved in a solution of N,N-dimethylformamide (7 mL), and sodium azide (110 mg, 1.80 mmol, 3.0 eq.) was added. The reaction was heated to 100 °C and stirred overnight under nitrogen protection. The reaction was monitored by spot TLC (DCM / MeOH = 10 / 1), showing that the starting material was completely consumed and a new spot was formed. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 5 (424 mg, 82%).

[0354] LCMS:Rt:2.256min;MS m / z(ELSD):861.9[M+H] + .

[0355] Step 4:

[0356] Compound 5 (424 mg, 0.49 mmol) was dissolved in a solution of methanol (5 mL) and tetrahydrofuran (5 mL), and palladium on carbon (150 mg, 10% wt) was added. The reaction was stirred overnight at room temperature under hydrogen protection. The reaction was monitored by LCMS, which showed that the starting material was consumed. The reaction mixture was filtered and concentrated to dryness under pressure. The residue was purified by silica gel column chromatography (0%–10% MeOH in DCM) to give a yellow oily product 6 (352 mg, 86%).

[0357] LCMS:Rt:2.074min;MS m / z(ELSD):836.0[M+H] + .

[0358] Step 5:

[0359] Compound 6 (252 mg, 0.3 mmol, 1.0 eq) was added to compound 7 (55 mg, 0.3 mmol, 1.0 eq.) in ethanol (0.2 mL), and the reaction was stirred overnight at 70 °C under argon protection. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new spots were formed. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH in DCM) to give a yellow oily target compound HI-19-129 (mg, 4.9%).

[0360] Example 8:

[0361] Step 1:

[0362] To a mixed solution of compound 1 (1 g, 2.31 mmol, 1.5 eq.) and compound 2 (2 g, 4.63 mmol, 1 eq.) in acetonitrile (10 mL) and cyclopentyl methyl ether (10 mL), potassium iodide (KI, 1.5 g, 9.26 mmol, 2.0 eq.) and potassium carbonate (K₂CO₃, 3.2 g, 23.14 mmol, 5.0 eq.) were added. The reaction mixture was stirred at 90 °C for 16 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent) to give compound 3 (1.53 g, 42% yield) as a pale yellow solid.

[0363] LCMS: Rt: 1.113min; MS m / z (ELSD): 486.9[M-55+H]+;

[0364] 1 H NMR (400MHz, CDCl3) δ4.98–4.73(m,1H),3.84–3.74(m,2H),3.66–3.50(m,4H),2.96–2.86(m,2H),2.80–2.70(m,2H),2.33-2. 28(m,2H),1.96–1.77(m,4H),1.72–1.60(m,4H),1.52–1.45(m,4H),1.42-1.34(m,2H),1.32-1.23(m,26H),0.91-0.85(m,6H).

[0365] Step 2:

[0366] To a solution of compound 4A (10 g, 5.28 mmol, 1.0 eq.) in dichloromethane (100 mL), compounds 4B (8.8 g, 5.28 mmol, 1.0 eq.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 19.5 g, 10.25 mmol, 2.0 eq.), 4-dimethylaminopyridine (DMAP, 2.5 g, 2.05 mmol, 0.4 eq.), and N,N-diisopropylethylamine (DIEA, 26.4 g, 20.51 mmol, 4.0 eq.) were added. The reaction mixture was stirred at 25 °C for 16 hours under nitrogen protection. Thin-layer chromatography (TLC, petroleum ether:ethyl acetate = 10:1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% ethyl acetate in petroleum ether) to give compound 4 (8.9 g, 49% yield) as a yellow oil.

[0367] 1 H NMR (400MHz, CDCl3) δ4.91–4.72(m,1H),3.47(dt,J=51.5,6.7Hz,2H),2.32(t,J=7.4Hz,2H),1.96–1.42(m,10H),1.26(s,12H),0.95–0.80(m,6H).

[0368] Step 3:

[0369] To a mixed solution of compound 3 (1.53 g, 3.15 mmol, 1.0 eq.) in acetonitrile (10 mL) and cyclopentyl methyl ether (10 mL), potassium iodide (KI, 1.0 g, 6.30 mmol, 2.0 eq.) and potassium carbonate (K₂CO₃, 2.17 g, 15.74 mmol, 5.0 eq.) were added. The reaction mixture was stirred at 90 °C for 16 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent) to give compound 5 (854 mg, 42% yield) as a pale yellow solid.

[0370] LCMS: Rt: 1.200min; MS m / z (ELSD): 755.1[M+H]+;

[0371] Step 4:

[0372] To a solution of compound 5 (1.397 g, 1.85 mmol) in dichloromethane (20 mL), triethylamine (TEA, 468 mg, 4.630 mmol) and methanesulfonyl chloride (MsCl, 423 mg, 3.704 mmol) were added, and the mixture was stirred at room temperature for 2 hours under argon protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (70 mL), and the mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with 5% sodium bicarbonate solution (70 mL) and saturated brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 6 (1.651 g, yield >100%), a colorless oil, which was used directly in the next reaction.

[0373] Step 5:

[0374] Sodium azide (NaN3, 387 mg, 5.95 mmol) was added to a solution of compound 6 (1.65 g, 1.99 mmol) in N,N-dimethylformamide (20 mL), and the mixture was stirred at 100 °C for 16 h under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with water (150 mL × 2) and saturated brine (150 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent at a volume fraction of 0%–10%) to give compound 7 (1.383 g, 87% yield) as a yellow oil.

[0375] LCMS:Rt:1.484min; MS m / z(ELSD):779.7[M+H] + ;

[0376] Step 6:

[0377] To a 20 mL ethanol solution of compound 7 (1.38 g, 1.78 mmol), palladium on carbon (Pd / C, 300 mg, 10% by mass) was added, and the mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere (30 psi). Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent) to give compound 8 (980 mg, 78% yield) as a colorless oil.

[0378] 1 H NMR (400MHz, CDCl3) δ4.90–4.75(m,2H),3.62–3.46(m,4H),2.98(dt,J=14.5,6.8Hz,4H),2.79–2.73(m,4H),2.31( td,J=7.4,4.8Hz,4H),1.94(dd,J=18.0,12.0Hz,4H),1.75–1.47(m,16H),1.40–1.24(m,40H),0.93–0.80(m,12H).

[0379] Step 7:

[0380] Compound 9 (135 mg, 0.6 mmol) was added to a 0.5 mL ethanol solution of compound 8 (450 mg, 0.6 mmol). The mixture was stirred at room temperature for 0.5 h, then stirred overnight at 70 °C. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched with water (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with saturated brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0%–50% volume fraction of ethyl acetate / tetrahydrofuran (5 / 3) in petroleum ether as eluent) and preparative high-performance liquid chromatography (pre-HPLC) to give compound HI-19-2-1 (35.03 mg, yield 6.8%) as a colorless oil.

[0381] LCMS:Rt:1.328min;MS m / z(ELSD):854.0[M+H] + ;

[0382] CAD:90.56% purity at ELSD; RT=25.596min.

[0383] 1 H NMR (400MHz, CDCl3) δ6.54(s,2H),4.88–4.79(m,2H),4.22(d,J=2.0Hz,4H),3.47(t,J=6.0Hz,4H),2.80(dd,J=16.7,9.3Hz,4 H),2.64–2.26(m,8H),1.80–1.75(m,2H),1.64(d,J=7.1Hz,10H),1.53–1.49(m,6H),1.33–1.23(m,42H),0.90–0.85(m,12H).

[0384] Example 9

[0385] Step 1:

[0386] A mixture of compound 1 (30 g, 180.7 mmol, 1.0 eq.) in DCM (30 mL) was added dropwise at -10 °C. A solution of compound 2 (20.6 g, 180.7 mmol, 1.0 eq.) and BF3·OEt2 (0.3 mL) were added dropwise. The reaction mixture was stirred at room temperature under N2 for 16 hours. TLC (ethyl acetate / petroleum ether = 1 / 5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (180 mL) and extracted with ethyl acetate (200 mL × 2). The combined organic layers were washed with brine (3 × 150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 20% ethyl acetate in petroleum ether to give a yellow oily compound 3 (8.3 g, 18%).

[0387] Step 2:

[0388] To a mixture of compound 3 (8.3 g, 32.9 mmol, 1.0 eq.) in ETOH (40 mL) and H₂O (40 mL), NaOH (1.581 g, 39.5 mmol, 1.2 eq.) was added. The reaction mixture was stirred at 50 °C for 3 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction mixture was quenched with water (100 mL). The aqueous phase was acidified with 1 M hydrochloric acid (10 mL) and extracted with ethyl acetate (3 x 120 mL). The combined organic compounds were dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness to give compound 4 (6.3 g, crude) as a yellow oil.

[0389] 1H NMR(400MHz, CDCl3)δ9.72–8.79(m,1H),4.14–4.11(m,2H),3.60–3.54(m,2H) ,3.44–3.39(m,2H),1.94–1.86(m,2H),1.70–1.64(m,2H),1.59–1.50(m,2H).

[0390] Step 3:

[0391] To a mixture of compound 4 (6.3 g, 28.1 mmol, 1.0 equivalent) and compound 5 (7.2 mg, 28.1 mmol / 1.0 equivalent) in DCM (100 mL), EDCI (10.744 g, 56.25 mmol, 2.0 equivalent), DMAP (1.373 g, 11.25 mmol, 0.4 equivalent), and DIEA (14.5 g, 112.5 mmol, 4.0 equivalent) were added. The reaction mixture was stirred under N2 at room temperature for 16 hours. TLC (ethyl acetate / petroleum ether = 1 / 20) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (130 mL × 3). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% ethyl acetate in petroleum ether to give a yellow oily compound 6 (2.8 g, 22%).

[0392] 1H NMR (400MHz, CDCl3) δ5.02–4.91(m,1H),4.04(s,2H),3.58–3.51(m,2H),3.46–3.38(m,2H),1. 96–1.83(m,2H),1.70–1.61(m,2H),1.58–1.53(m,4H),1.35–1.19(m,26H),0.90–0.86(m,6H).

[0393] Step 4:

[0394] To a mixture of compound 6 (1.82 g, 3.94 mmol, 1.0 eq.) in CPME / MeCN (20 mL, v / v), KI (654 mg, 3.94 mol, 1.0 eq.), K₂CO₃ (1.631 g, 11.8 mmol, 3.0 eq.), and compound 7 (2.742 g, 3.94 mmol, 1.0 eq.) were added. The reaction mixture was stirred at 90 °C under N₂ for 16 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with H₂O (100 mL) and extracted with EA (120 mL × 3). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% ethyl acetate in petroleum ether to give yellow oily compound 8 (1.969 g, 46%).

[0395] Step 5:

[0396] Compound 8 (1.969 g, 1.83 mmol, 1.0 equivalent) was added to a mixture in hydrochloric acid (15 mL). The reaction mixture was stirred at room temperature for 1 hour. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The pH was adjusted to approximately 6–7 by stepwise addition of aqueous sodium bicarbonate. The mixture was quenched with water (90 mL) and extracted with dichloromethane (100 mL × 3). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 9 (1.685 g, crude), a yellow oil.

[0397] LCMS:Rt:2.402min;MS m / z(ELSD):965.9[M+H] + ;

[0398] Step 6:

[0399] TEA (442 mg, 4.37 mmol, 2.5 equivalence) and MsCl (402 mg, 3.50 mmol, 2.0 equivalence) were added to a mixture of compound 9 (1.685 g, 1.75 mmol, 1.0 equivalence) in DCM (17 mL). The reaction mixture was stirred at 0 °C under Ar for 1 hour. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (120 mL × 3). The combined organic layers were washed with brine (2 × 70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 10 (1.959 g, crude) as a yellow oil.

[0400] LCMS:Rt:2.507min; MS m / z(ELSD):1043.5[M+H] + ;

[0401] Step 7:

[0402] To a mixture of compound 10 (1.959 g, 1.88 mmol, 1.0 equivalent) in DMF (19 mL), NaN3 (245 mg, 3.76 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (120 mL × 3). The combined organic layers were washed with brine (5 × 80 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 10% ethyl acetate to give a yellow oily compound 11 (1.24 g, 67%).

[0403] Step 8:

[0404] Pd / C (400 mg) was added to a mixture of compound 11 (1.24 g, 1.25 mmol, 1.0 eq.) in methanol (6 mL) and tetrahydrofuran (6 mL). The reaction mixture was stirred in H2 at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with a solution of 10% methanol in dichloromethane, to give compound 12 (761 mg, 63%) as a yellow oil.

[0405] LCMS:Rt:2.501min;MS m / z(ELSD):964.2[M+H] + ;

[0406] Step 9:

[0407] Compound 13 (99 mg, 0.73 mmol / 1.0 equivalent) was added to a mixture of compound 12 (700 mg, 0.73 mmol / 1.0 equivalent) in ethanol (0.7 mL, 95%). The reaction mixture was stirred at 70 °C under N2 for 16 hours. TLC (ethyl acetate:tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (60 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give compound 14 (94 mg, 12%) as a yellow oil.

[0408] LCMS:Rt:2.565min; MS m / z(ELSD):1064.1[M+H] + ;

[0409] Step 10:

[0410] To a mixture of compound 14 (94 mg, 0.088 mmol, 1.0 equivalent) in THF (1 mL), pyridine (0.1 mL) and pyridine hydrofluoride (0.1 mL) were added. The reaction mixture was stirred at 0 °C under N2 for 1 hour. The reaction was monitored by LCMS. The mixture was quenched with H2O (40 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether from 20% ethyl acetate, to give a white solid compound HI-19-2-3 (18.38 mg, 25%).

[0411] LCMS:Rt:1.439min;MS m / z(ELSD):826.0[M+H] + ;

[0412] CAD:84.54%purity.Rt:22.501min

[0413] 1 H NMR (400MHz, CDCl3) δ6.46(s,2H),4.95(s,1H),4.23(s,4H),4.04(s,2H),3.53(t,J=6.2Hz,3H ),2.79(s,8H),1.64(dd,J=13.7,6.4Hz,5H),1.43(s,5H),1.25(s,57H),0.87(d,J=7.0Hz,9H).

[0414] Example 10

[0415] Step 1:

[0416] Add the mixture of compound 1 (20.3 g, 271.3 mmol, 30.0 equivalents) and compound 2 (3.4 g, 9.04 mmol, 1.0 equivalents) to a mixture of ethanol (5 mL). Stir the mixture overnight at 50 °C under nitrogen protection. TLC (methanol / dichloromethane = 1 / 10) showed the reaction was complete and a new major spot was observed. Quench the mixture with water (100 mL) and extract with ethyl acetate (150 mL × 3). Wash the combined organic layers with brine (5 × 80 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Concentrate the mixture under reduced pressure. Purify the residue by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 3 (2.9 g, 86%) as a yellow oil.

[0417] LCMS:Rt:1.054min;MS m / z(ELSD):372.8[M+H] + ;

[0418] 1H NMR (400MHz, CDCl3) δ4.86–4.76(m,1H),3.85–3.77(m,2H),3.49–3.48(m,1H),2.91–2.84(m,2H),2.62–2. 58(m,2H),2.31–2.25(m,2H),1.73–1.66(m,2H),1.64–1.43(m,8H),1.38–1.14(m,19H),0.91–0.84(m,6H).

[0419] Step 2:

[0420] Compound 4 (1.246 g, 2.70 mmol, 1.0 equivalent), potassium carbonate (1.116 g, 8.09 mmol, 3.0 equivalent), and potassium iodide (448 mg, 2.70 mmol, 1.0 equivalent) were added to a mixture of compound 7 (1 g, 2.70 mmol, 1.0 equivalent) in cyclopentylmethyl ether / acetonitrile (20 mL, v / v). The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (120 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 5 (1.5 g, 74%) as a yellow oil.

[0421] LCMS:Rt:1.433min;MS m / z(ELSD):773.7[M+H]+ ;

[0422] 1H NMR (400MHz, CDCl3) δ7.14–7.09(m,4H),4.86(p,J=6.2Hz,1H),4.26(t,J=7.2H z,2H),3.81–3.78(m,2H),2.90(t,J=7.2Hz,2H),2.68–2.65(m,2H),2.59–2.55( m,2H),2.45–2.42(m,4H),2.28(td,J=7.5,2.4Hz,4H),1.71–1.68(m,2H),1.61 –1.58(m,6H),1.49(d,J=5.3Hz,9H),1.33–1.26(m,41H),0.88(q,J=6.7Hz,9H).

[0423] Step 3:

[0424] Triethylamine (503 mg, 4.97 mmol, 2.5 eq.) and methanesulfonyl chloride (456 mg, 3.98 mmol, 2.0 eq.) were added to a mixture of compound 5 (1.5 g, 1.99 mmol, 1.0 eq.) in dichloromethane (15 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N hydrochloric acid (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (120 mL × 3). The combined organic layers were washed with brine (2 × 70 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 9 (1.986 g, crude) as a yellow oil.

[0425] Step 4:

[0426] Sodium azide (311 mg, 4.77 mmol, 2.0 equivalent) was added to a mixture of compound 6 (1.986 g, 2.39 mmol, 1.0 equivalent) in DMF (20 mL). The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane:methanol = 10 / 1) showed the reaction was complete, and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine (6 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 7 (1.314 mg, 71%) as a yellow oil.

[0427] LCMS:Rt:1.591min; MS m / z(ELSD):780.5[M+H] + ;

[0428] 1H NMR (400MHz, CDCl3) δ5.00–4.90(m,1H),4.87–4.75(m,1H),4.04(s,2H),3.57–3.49(m,2H),3.43–3.32(m ,2H),2.77–2.36(m,4H),2.31–2.26(m,2H),1.96–1.38(m,21H),1.35–1.06(m,43H),0.93–0.83(m,12H).

[0429] Step 5:

[0430] Palladium on carbon (400 mg) was added to a mixture of compound 8 (1.314 g, 1.69 mmol, 1.0 eq.) in methanol (6 mL) and tetrahydrofuran (6 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 8 (724 mg, 57%) as a yellow oil.

[0431] LCMS:Rt:1.591min;MS m / z(ELSD):752.6[M+H] + ;

[0432] Step 6:

[0433] Compound 9 (60%) (121 mg, 0.53 mmol, 1.0 eq.) was added to a mixture of compound 8 (400 mg, 0.53 mmol, 1.0 eq.) in ethanol (0.5 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 h. TLC (ethyl acetate:tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (60 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 70% ethyl acetate:tetrahydrofuran (1:5) in petroleum ether, to give a yellow oily compound HI-19-2-4 (22.7 mg, 0.05%).

[0434] LCMS:Rt:1.305min; MS m / z(ELSD):854.1[M+H] + ;

[0435] CAD:90.04% purity at ELSD; RT=23.697min.

[0436] 1 H NMR (400MHz, CDCl3) δ4.74(m,5H),4.03(s,2H),3.54(s,3H),3.01(s,6H),2.29(s ,4H),1.64(s,6H),1.53(s,10H),1.31(d,J=42.5Hz,47H),0.88(t,J=6.3Hz,12H).

[0437] Example 11

[0438] Step 1:

[0439] A solution of compound 1 (3 g, 1.12 mmol) and compound 2 (2.8 g, 0.74 mmol) was stirred at 80 °C for 16 hours under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was purified directly by silica gel column chromatography (using 0%–10% methanol in dichloromethane as eluent) to give compound 3 (1.478 g, 21% yield) as a yellow oil.

[0440] LCMS:Rt:1.608min; MS m / z(ELSD):459.1[M+H] + ;

[0441] Step 2:

[0442] Imidazole (410 mg, 6.03 mmol) and tert-butyldiphenylchlorosilane (TBDPSCl, 1.24 g, 4.52 mmol) were added to a dichloromethane (30 mL) solution of compound 3 (1.378 g, 3.02 mmol), and the mixture was stirred at room temperature for 16 hours under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent at a volume fraction of 0%–10%) to give compound 4 (1.388 g, 76% yield) as a yellow oil.

[0443] LCMS:Rt:1.673min; MS m / z(ELSD):696.7[M+H] + ;

[0444] Step 3:

[0445] Compound 5 (1.69 g, 3.67 mmol), potassium carbonate (K₂CO₃, 2.1 g, 15.30 mmol), and potassium iodide (KI, 1.02 g, 6.12 mmol) were added to a solution of compound 4 (2.13 g, 3.06 mmol) in acetonitrile / cyclopentyl methyl ether (10 mL / 10 mL). The mixture was stirred at 90 °C for 16 hours under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent at a volume fraction of 0%–10%) to give compound 6 (1.388 g, 76% yield) as a yellow oil.

[0446] Step 4:

[0447] To a mixture of compound 6 (1.285 g, 1.19 mmol) and tetrahydrofuran (10 mL), 1 M hydrochloric acid (1 mL, aqueous solution) was added. The mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding water (30 mL), and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent at a volume fraction of 0%–10%) to give compound 7 (582 mg, 66% yield) as a yellow oil.

[0448] LCMS:Rt:1.381min;MS m / z(ELSD):962.9[M+H] + ;

[0449] Step 5:

[0450] Triethylamine (TEA, 127 mg, 1.25 mmol) and methanesulfonyl chloride (MsCl, 115 mg, 1 mmol) were added to a dichloromethane (10 mL) solution of compound 7 (482 mg, 0.5 mmol), and the mixture was stirred at room temperature for 2 hours under argon protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (30 mL) to the mixture, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phases were combined, washed with 5% sodium bicarbonate solution (30 mL) and saturated brine (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 8 (520 mg, 100% yield), a colorless oil, which was used directly in the next reaction.

[0451] Step 6:

[0452] Sodium azide (NaN3, 96 mg, 1.47 mmol) was added to a solution of compound 8 (614 mg, 0.59 mmol) in N,N-dimethylformamide (6 mL), and the mixture was stirred at 100 °C for 16 h under argon protection. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (20 mL) to the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with water (50 mL × 2) and saturated brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent at a volume fraction of 0%–10%) to give compound 9 (415 mg, 65% yield) as a brown oil.

[0453] LCMS:Rt:1.731min; MS m / z(ELSD):988.0[M+H] + ;

[0454] Step 7:

[0455] Palladium on carbon (Pd / C, 150 mg, 10% by mass) was added to a tetrahydrofuran / methanol (5 mL / 5 mL) solution of compound 9 (415 mg, 0.42 mmol), and the mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere (30 psi). Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0%–10% methanol in dichloromethane containing 0.05% ammonia as eluent) to give compound 10 (220 mg, 45% yield) as a colorless oil.

[0456] LCMS:Rt:2.408min;MS m / z(ELSD):963.3[M+H] + ;

[0457] Step 8:

[0458] Compound 11 (28 mg, 0.21 mmol) was added to a 0.2 mL ethanol solution of compound 10 (196 mg, 0.21 mmol). The mixture was stirred at room temperature for 0.5 h, then stirred overnight at 70 °C. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0%–50% (v / v) of ethyl acetate / tetrahydrofuran in petroleum ether) as eluent to give compound 12 (100 mg, 57% yield) as a yellow oil.

[0459] LCMS:Rt:2.744min;MS m / z(ELSD):1062.0[M+H] + ;

[0460] Step 9:

[0461] At 0 °C, pyridine (0.2 mL) and pyridine hydrogen fluoride (0.2 mL) were added to a mixture of compound 12 (130 mg, 0.12 mmol) and tetrahydrofuran (1 mL). The mixture was stirred at 45 °C for 16 hours under nitrogen protection. The reaction was quenched with water (20 mL), and the mixture was extracted with ethyl acetate (20 mL × 2). The combined organic phases were washed with saturated brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using ethyl acetate / tetrahydrofuran (5 / 3) in petroleum ether as eluent at a volume fraction of 0%–50%) and preparative high-performance liquid chromatography (pre-HPLC) to give compound HI-19-9-1 (13.94 mg, yield 17.1%) as a colorless oil.

[0462] LCMS:Rt:1.713min;MS m / z(ELSD):824.1[M+H] + ;

[0463] CAD: 80% purity. Rt: 26.940 min

[0464] 1H NMR (400MHz, CDCl3) δ6.55(s,2H),4.89–4.83(m,1H),4.25(s,4H),2.81(s,9H),2.28(t,J=7.5Hz ,2H),1.61(s,4H),1.50(d,J=5.2Hz,4H),1.35–1.32(m,5H),1.25(s,58H),0.87(d,J=6.9Hz,9H).

[0465] Example 12

[0466] Step 1:

[0467] A mixture of compound 1 (11 g, 52.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.66 g, 50.4 mmol), 4-dimethylaminopyridine (0.51 g, 5.2 mmol), and N,O-dimethylhydroxylamine (4.5 g, 46.2 mmol) in dichloromethane (200 mL) was stirred at room temperature for 16 hours. The mixture was diluted with 200 mL of dichloromethane, washed successively with water (70 mL), hydrochloric acid (0.5 N, 60 mL), sodium hydroxide (0.5 N, 60 mL), and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 3 (9 g, 73%) as a colorless oil.

[0468] Step 2:

[0469] Compound 4 (95 mL, 94.94 mmol, 2.5 eq. 1 M) was added to a tetrahydrofuran (90 mL) solution of compound 3 (9 g, 37.97 mmol, 1.0 eq.) at 0 °C under an argon atmosphere. The reaction mixture was stirred at room temperature for 4 h under an argon atmosphere. The reaction was quenched by adding an aqueous solution of ammonium chloride (100 mL) and extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 5 (7.4 g, 71%) as a white solid.

[0470] 1 H NMR (400MHz, CDCl3) δ3.47 (dt, J=51.0, 6.7Hz, 2H), 2.40 (dt, J=12.9, 7.4Hz, 4 H),1.93–1.75(m,2H),1.66–1.38(m,8H),1.25(s,16H),0.88(t,J=6.8Hz,3H).

[0471] Step 3:

[0472] Sodium borohydride (1.2 g, 31.99 mmol, 1.5 eq.) was added to a tetrahydrofuran / methanol (350 mL, 3 / 1) mixture of compound 5 (7.4 g, 21.33 mmol, 1.0 eq.) at 0 °C under an argon atmosphere. The reaction mixture was stirred at room temperature under an argon atmosphere for 2 h. The reaction was quenched by adding 1 M hydrochloric acid (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 6 (6.67 g, 91%) as a white solid.

[0473] 1 H NMR (400MHz, CDCl3) δ3.58(dd,J=10.4,7.3Hz,1H),3.56–3.36(m,2H),1.86(dd,J=24.2,17.2Hz,2H),1.56–1.16(m,28H),0.88(t,J=6.8Hz,3H).

[0474] Step 4:

[0475] Under a nitrogen atmosphere, tert-butyldimethylchlorosilane (3.9 g, 25.86 mmol, 1.5 eq.) and imidazole (2.34 g, 34.48 mmol, 2 eq.) were added to a solution of compound 6 (6 g, 17.24 mmol, 1.0 eq.) in dichloromethane (60 mL). The reaction mixture was stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 7 (3.48 mg, 39%) as a yellow oil.

[0476] 1 H NMR (400MHz, CDCl3) δ3.64–3.54(m,1H),3.50–3.34(m,2H),1.93–1.69(m,2H),1.43–1.16(m,28H),0.86–0.82(m,12H),-0.00(d,J=0.8Hz,6H).

[0477] Step 5:

[0478] Under a nitrogen atmosphere, potassium iodide (1.09 g, 6.59 mmol, 2.0 eq.), potassium carbonate (2.27 g, 16.48 mmol, 5.0 eq.), and compound 7 (2.28 g, 4.95 mmol, 1.5 eq.) were added to a methylcyclopentyl ether / acetonitrile (30 mL, 1 / 1) mixture of compound 8 (1.5 g, 3.30 mmol, 1.0 eq.). The reaction mixture was stirred at 90 °C for 16 h. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 9 (838 mg, 42%) as a yellow oil.

[0479] LCMS:Rt:2.466min;MS m / z(ELSD):839.2[M+H] + ;

[0480] Step 6:

[0481] Under an argon atmosphere, triethylamine (275 mg, 2.72 mmol, 2.0 eq.) and methanesulfonyl chloride (186 mg, 1.63 mmol, 1.5 eq.) were added to a solution of compound 9 (912 mg, 1.09 mmol, 1.0 eq.) in dichloromethane (15 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding water (30 mL) and extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 10 (996 mg, 100%) as a yellow oil.

[0482] LCMS:Rt:2.715min; MS m / z(ELSD):917.2[M+H] + ;

[0483] Step 7:

[0484] Under a nitrogen atmosphere, sodium azide (177 mg, 2.72 mmol, 2.5 eq.) was added to a solution of compound 10 (996 mg, 1.09 mmol, 1.0 eq.) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 100 °C for 16 hours. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with brine (5 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 11 (450 mg, 43%) as a yellow oil.

[0485] LCMS:Rt:1.422min; MS m / z(ELSD):864.3[M+H] + ;

[0486] Step 8:

[0487] Palladium on carbon (200 mg) was added to a mixed solution of compound 11 (450 mg, 0.51 mmol, 1.0 eq.) in ethanol (5 mL) and tetrahydrofuran (5 mL). The reaction mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The catalyst was removed by diatomaceous earth filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 12 (410 mg, 96%) as a yellow oil.

[0488] LCMS:Rt:2.283min;MS m / z(ELSD):838.3[M+H] + ;

[0489] Step 9:

[0490] Under a nitrogen atmosphere, compound 13 (49 mg, 0.36 mmol, 1.0 eq.) was added to a solution of compound 12 (300 mg, 0.36 mmol, 1.0 eq.) in 95% ethanol (0.3 mL). The reaction mixture was stirred at 70 °C for 16 hours. Thin-layer chromatography (ethyl acetate: tetrahydrofuran = 5 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (30 mL) to the mixture and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with a mixture of petroleum ether containing 70% ethyl acetate / tetrahydrofuran (1:5) to give compound 14 (200 mg, 59%) as a yellow oil.

[0491] 1H NMR (400MHz, CDCl3) δ6.35(s,2H),4.88–4.76(m,1H),4.18(s,4H),3.62–3.50(m,1H),2.71(t,J=7.0Hz,2H),2.40(t,J=7 .0Hz,2H),2.37–2.29(m,4H),2.25(t,J=7.5Hz,2H),1.63–1.15(m,70H),0.84(d,J=9.4Hz,18H),0.02(d,J=14.2Hz,6H).

[0492] Step 10:

[0493] Under a nitrogen atmosphere, tetrabutylammonium fluoride (2.5 mL, 1 M tetrahydrofuran solution) was added to a tetrahydrofuran (5 mL) solution of compound 14 (200 mg, 0.2 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction was quenched by adding water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography to give compound HI-19-9-2 (49.14 mg, 30%) as a colorless oil.

[0494] LCMS:Rt:1.585min; MS m / z(ELSD):824.1[M+H] + ;

[0495] CAD:85.83%purity.Rt:21.407min

[0496] 1 H NMR (400MHz, CDCl3) δ6.73(s,2H),4.97–4.79(m,1H),4.21(s,4H),3.59(s,1H),2.75(t,J=7.0Hz,2H),2.46(d,J=28.4 Hz, 6H), 2.28 (t, J = 7.4Hz, 2H), 1.64 (dd, J = 12.7, 6.8Hz, 4H), 1.51–1.41 (m, 12H), 1.26 (s, 55H), 0.88 (t, J = 6.5Hz, 9H).

[0497] Example 13

[0498] Step 1:

[0499] Pd(dppf)Cl2 (1.918 g, 2.62 mmol, 0.1 equivalent) and K2CO3 (10.9 g, 78.6 mmol, 3.0 equivalent) were added to a mixture of compound 1 (6 g, 26.2 mmol, 1.0 equivalent) and compound 2 (9.7 g, 39.3 mmol, 1.5 equivalent) in 1,4-dioxane (60 mL) and water (6 mL). The mixture was stirred overnight at 100 °C under nitrogen protection. TLC (ethyl acetate / petroleum ether = 1 / 20) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate (200 mL × 3) and washed with water (160 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 3 (4.387 g, 64%), a pale green oil.

[0500] 1H NMR (400MHz, CDCl3) δ7.22–7.13(m,4H),3.68(s,3H),3.66(s,2H),2.65–2.57(m,2H),1.63–1.18(m,12H),0.91–0.86(m,3H).

[0501] Step 2:

[0502] LAH (673 mg, 17.7 mmol, 1.0 eq.) was added to a mixture of compound 3 (4.387 g, 17.7 mmol, 1.0 eq.) in THF (50 mL) at 0 °C under N2. The mixture was stirred at room temperature for 3 hours. TLC (petroleum ether / ethyl acetate = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL) and treated with 1 N hydrochloric acid to adjust the pH to between 6 and 7. The mixture was quenched with water (150 mL) and extracted with EA (200 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 20% ethyl acetate to give compound 4 (3.7 g, 89%) as a yellow oil.

[0503] Step 3:

[0504] To a mixture of compound 4 (3.8 g, 19.8 mmol, 1.5 equivalences) and compound 5 (2.93 mg, 13.2 mmol, 1.0 equivalences) in DCM (50 mL), EDCI (5.041 g, 26.4 mmol, 2.0 equivalences), DMAP (644 mg, 5.28 mmol, 0.4 equivalences), and DIEA (6.809 g, 52.8 mmol, 4.0 equivalences) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (petroleum ether:ethyl acetate = 20 / 1) showed that the reaction was complete and new major spots were observed. The mixture was quenched with water (200 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic layers were washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% ethyl acetate in petroleum ether to give a clear oily compound 6 (3.978 g, 51%).

[0505] 1H NMR (400MHz, CDCl3) δ7.18–7.11(m,4H),4.28–4.23(m,2H),3.44–3.35(m,2H),2.98–2.93(m,2H),2.66–2. 60(m,2H),2.34–2.26(m,2H),1.90–1.80(m,2H),1.65–1.56(m,4H),1.44–1.27(m,14H),0.92–0.86(m,3H).

[0506] Step 4:

[0507] To a mixture of compound 7 (161 mg, 2.14 mmol, 1.0 eq.) in CPME (10 mL) and MeCN (10 mL), KI (356 mg, 2.14 mmol, 1.0 eq.), K₂CO₃ (888 mg, 6.43 mmol, 3.0 eq.), and compound 6 (2 g, 4.72 mmol, 1.0 eq.) were added. The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (110 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 8 (1.132 g, 31%) as a yellow oil. LCMS:Rt:1.183min;MS m / z(254nm):764.7[M+H] + ;

[0508] 1H NMR (400MHz, CDCl3) δ7.18–7.10(m,8H),4.29–4.21(m,4H),3.82–3.77(m,2H),2.99–2.93(m,4H),2.72–2.66(m,2H),2.66–2.60(m,4H) ,2.49–2.42(m,4H),2.32–2.27(m,4H),1.74–1.68(m,2H),1.64–1.54(m,8H),1.52–1.45(m,4H),1.40–1.25(m,29H),0.91–0.86(m,6H).

[0509] Step 5:

[0510] TEA (375 mg, 2.96 mmol, 2.5 equivalent) and MSCl (341 mg, 2.96 mmol, 2.0 equivalent) were added to a mixture of compound 8 (1.132 g, 1.48 mmol, 1.0 equivalent) in DCM (10 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (120 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 9 (1.246 g, crude) as a yellow oil.

[0511] LCMS:Rt:1.183min; MS m / z(ELSD):842.7[M+H] + ;

[0512] Step 6:

[0513] To a mixture of compound 9 (1.246 g, 1.48 mmol, 1.0 equivalent) in DMF (10 mL), NaN3 (192 mg, 2.96 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (110 mL) and extracted with EA (120 mL × 3). The combined organic layers were washed with brine (6 × 70 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 10 (805 mg, 69%) as a yellow oil.

[0514] LCMS:Rt:1.247min; MS m / z(ELSD):789.7[M+H] + ;

[0515] 1H NMR (400MHz, CDCl3) δ7.18–7.10(m,8H),4.29–4.22(m,4H),3.43–3.31(m,2H),2.99–2.91(m,4H),2.66–2.60(m,4H),2.56–2. 38(m,4H),2.32–2.27(m,4H),1.82–1.73(m,2H),1.70–1.60(m,8H),1.52–1.43(m,4H),1.41–1.25(m,30H),0.92–0.85(m,6H).

[0516] Step 7:

[0517] Pd / C (200 mg) was added to a mixture of compound 10 (805 mg, 1.02 mmol, 1.0 eq.) in MeOH (5 mL) and THF (5 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane, to give compound 11 (645 mg, 82%) as a yellow oil.

[0518] LCMS:Rt:1.136min;MS m / z(ELSD):764.1[M+H] + ;

[0519] Step 8:

[0520] Compound 12 (60%) (173 mg, 0.76 mmol, 1.0 eq.) was added to a mixture of compound 11 (580 mg, 0.76 mmol, 1.0 eq.) in ethanol (0.7 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 hours. TLC (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (50 mL) and extracted with ethyl acetate (70 mL × 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-115 (63 mg, 10%).

[0521] LCMS:Rt:1.159min; MS m / z(ELSD):864.1[M+H] + ;

[0522] CAD:88.33% purity at ELSD; RT=22.915min.

[0523] 1 H NMR(400MHz, CDCl3)δ7.15(dt,J=8.1,4.3Hz,8H),6.42(s,2H),4.25(t,J=7.4Hz,8H),3.15–2.38(m,1 6H), 2.30 (t, J=7.4Hz, 4H), 1.59 (dd, J=15.1, 7.3Hz, 14H), 1.38–1.26 (m, 28H), 0.88 (t, J=6.9Hz, 6H).

[0524] Example 14

[0525] Step 1:

[0526] Add the compound 1 (32.4 g, 431.03 mmol, 30.0 eq.) and compound 2 (5 g, 14.37 mmol, 1.0 eq.) to a mixture of ethanol (5 mL). Stir the mixture overnight at 50 °C under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. Quench the mixture with water (120 mL) and extract with ethyl acetate (130 mL × 3). Wash the combined organic layers with brine (3 × 80 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Concentrate the mixture under reduced pressure. Purify the residue by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 3 (2.1 g, 43%) as a yellow solid.

[0527] LCMS:Rt:0.920min;MS m / z(ELSD):344.3[M+H] + ;

[0528] 1H NMR (400MHz, CDCl3) δ4.08–4.02(m,2H),3.84–3.78(m,2H),2.90–2.85(m,2H),2.64–2.56(m,2H),2.33–2. 25(m,2H),1.73–1.66(m,2H),1.65–1.56(m,4H),1.51–1.42(m,2H),1.36–1.22(m,18H),0.91–0.85(m,3H).

[0529] Step 2:

[0530] Compound 4 (1.4 g, 3.49 mmol, 1.2 eq.), K₂CO₃ (1.2 g, 8.74 mmol, 3.0 eq.), and KI (484 mg, 2.91 mol, 1.0 eq.) were added to a mixture of compound 3 (1 g, 2.91 mmol, 1.0 eq.) in CPME (10 mL) and MeCN (10 mL). The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (110 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 5 (1.282 g, 67%) as a yellow oil.

[0531] LCMS:Rt:1.188min;MS m / z(ELSD):600.7[M+H] + ;

[0532] 1H NMR (400MHz, CDCl3) δ7.15–7.08(m,4H),4.29–4.23(m,2H),4.08–4.02(m,2H),3.82–3.77(m,2H),2.93–2.86(m,2H),2.72–2.66(m,2H),2.60–2. 54(m,2H),2.49–2.43(m,4H),2.31–2.26(m,4H),1.75–1.68(m,2H),1.65 –1.56(m,8H),1.54–1.45(m,4H),1.35–1.25(m,28H),0.91–0.86(m,6H).

[0533] Step 3:

[0534] TEA (491 mg, 4.86 mmol, 2.5 eq.) and MSCl (447 mg, 3.88 mmol, 2.0 eq.) were added to a mixture of compound 5 (1.282 g, 1.94 mmol, 1.0 eq.) in DCM (16 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (100 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 6 (1.692 g, crude) as a yellow oil.

[0535] LCMS:Rt:1.177min;MS m / z(ELSD):738.5[M+H] + ;

[0536] Step 4:

[0537] To a mixture of compound 6 (1.692 g, 2.29 mmol, 1.0 equivalent) in DMF (20 mL), NaN3 (299 mg, 4.59 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (110 mL) and extracted with ethyl acetate (110 mL × 3). The combined organic layers were washed with brine (6 × 90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 7 (981 mg, 62%) as a yellow oil.

[0538] LCMS:Rt:1.136min;MS m / z(254nm):685.6[M+H] + ;

[0539] 1H NMR (400MHz, CDCl3) δ7.15–7.09(m,4H),4.30–4.23(m,2H),4.08–4.02(m,2H),3.37–3.30(m,2H),2.92–2.86(m,2H),2.60–2.54(m,2H),2.50–2. 44(m,2H),2.41–2.33(m,4H),2.32–2.25(m,4H),1.74–1.66(m,2H),1.65 –1.56(m,8H),1.45–1.37(m,4H),1.35–1.25(m,28H),0.91–0.86(m,6H).

[0540] Step 5:

[0541] Pd / C (210 mg) was added to a mixture of compound 8 (981 mg, 1.43 mmol, 1.0 eq.) in methanol (5 mL) and tetrahydrofuran (5 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 8 (725 mg, 77%) as a yellow oil.

[0542] LCMS:Rt:1.107min;MS m / z(ELSD):659.7[M+H] + ;

[0543] Step 6:

[0544] Compound 9 (60%) (69 mg, 0.30 mmol, 1.0 equivalent) was added to a mixture of compound 8 (200 mg, 0.30 mmol, 1.0 equivalent) in ethanol (0.2 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 hours. TLC (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (50 mL) and extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-116 (15.36 mg, 7%).

[0545] LCMS:Rt:1.107min; MS m / z(ELSD):759.7[M+H] + ;

[0546] CAD:89.04% purity at ELSD; RT=21.266min.

[0547] 1 H NMR (400MHz, CDCl3) δ7.12(s,4H),6.48(s,2H),4.39–3.99(m,8H),3.11(s,2H),2.99(s,4H),2.93–2.83(m,4H),2.60–2.55(m,2H),2.29(t,J=7 .3Hz,4H),2.11(s,2H),1.77(s,4H),1.60(d,J=6.4Hz,6H),1.33(dd,J=11.8,8.2Hz,18H),1.26(d,J=6.0Hz,12H),0.88(dd,J=7.0,3.2Hz,6H).

[0548] Example 15

[0549] Step 1:

[0550] To a solution of compound 1 (1 g, 11.63 mmol, 1.0 eq.) in dichloromethane (20 mL), triethylamine (1.76 g, 17.44 mmol, 1.5 eq.) and methanesulfonyl chloride (1.6 g, 13.95 mmol, 1.2 eq.) were added. The reaction mixture was stirred at 25 °C for 2 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 2 (1.1 g, crude product) as a yellow solid.

[0551] Step 2:

[0552] To a mixed solution of compound 3 (1 g, 2.69 mmol, 1.0 eq.) in acetonitrile (10 mL) and cyclopentyl methyl ether (10 mL), potassium iodide (895 mg, 5.39 mmol, 2.0 eq.), compound 2 (663 mg, 4.04 mmol, 1.5 eq.), and potassium carbonate (1.86 g, 13.48 mmol, 5.0 eq.) were added. The reaction mixture was stirred at 90 °C for 16 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (80 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent to give compound 4 (936 mg, 78% yield) as a pale yellow solid.

[0553] LCMS: Rt: 1.060min; MS m / z (ELSD): 440.8[M-55+H]+;

[0554] Step 3:

[0555] To a solution of compound 4 (0.936 g, 2.13 mmol, 1.0 eq.) in dichloromethane (10 mL), triethylamine (0.431 g, 4.26 mmol, 2.0 eq.) and methanesulfonyl chloride (0.368 g, 3.19 mmol, 1.5 eq.) were added. The reaction mixture was stirred at 25 °C for 2 hours under nitrogen protection. The reaction mixture was quenched with water (80 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 5 (1.1 g, crude product) as a yellow oil.

[0556] LCMS: Rt: 1.130min; MS m / z (ELSD): 518.4[M-55+H]+;

[0557] Step 4:

[0558] Sodium azide (0.34 g, 5.33 mmol, 2.5 eq.) was added to a solution of compound 5 (1.1 g, 2.14 mmol, 1.0 eq.) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 100 °C for 16 hours under nitrogen protection. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (40 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent to give compound 6 (0.936 mg, 94% yield) as a pale yellow solid.

[0559] LCMS: Rt: 1.130min; MS m / z (ELSD): 465.9[M-55+H]+;

[0560] 1 H NMR (400MHz, CDCl3) δ4.87–4.77(m,1H),3.33(d,J=29.1Hz,2H),2.67–2.38(m,4H),2.33–2.24(m,2H),1.91–1.67(m,2H ),1.67–1.44(m,10H),1.38–1.16(m,20H),0.94–0.83(m,6H),0.73–0.60(m,1H),0.54–0.30(m,2H),0.08–0.02(m,2H).

[0561] Step 5:

[0562] Palladium on carbon (0.2 g) was added to a 10 mL ethanol solution of compound 6 (0.93 g, 2.02 mmol, 1.0 eq.). The reaction mixture was stirred at 25 °C for 16 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0-10 / 1, v / v) as eluent to give compound 7 (556 mg, 62% yield) as a pale yellow solid.

[0563] LCMS: Rt: 0.978min; MS m / z (ELSD): 439.5[M-55+H]+;

[0564] 1H NMR (400MHz, CDCl3) δ4.87–4.74(m,1H),2.83–2.72(m,2H),2.57–2.44(m,4H),2.44–2.37(m,2H),2.34–2.24(m,2H),2.06( s,2H),1.67–1.39(m,11H),1.37–1.20(m,21H),0.94–0.82(m,6H),0.68–0.59(m,1H),0.47–0.36(m,2H),0.06–0.01(m,2H).

[0565] Step 6:

[0566] Compound 8 (163 mg, 1.14 mmol, 1.0 eq.) was added to a 0.5 mL ethanol solution of compound 7 (500 mg, 1.14 mmol, 1.0 eq.). The reaction mixture was stirred at 70 °C for 16 hours under argon protection. The reaction mixture was concentrated under reduced pressure. The residue was separated by column chromatography (0%–10% methanol in dichloromethane solution) and preparative high-performance liquid chromatography to give HI-19-121 (178 mg, 28% yield) as a yellow oil.

[0567] LCMS:Rt:1.037min;MS m / z(ELSD):539.8[M+H] + ;

[0568] CAD:87.32%purity.Rt:16.159min

[0569] 1 H NMR(400MHz, CDCl3)δ6.55(s,2H),4.88–4.75(m,1H),4.23(d,J=4.0Hz,4H),2.81–2.74(m,2H),2.72–2.36(m,6H),2.32–2.27(m,2H),1.79–1.6 7(m,2H),1.66–1.57(m,3H),1.56–1.46(m,5H),1.36–1.22(m,20H),0.9 0–0.85(m,6H),0.70–0.60(m,1H),0.48–0.40(m,2H),0.06–0.03(m,2H).

[0570] Example 16

[0571] Step 1:

[0572] Sodium hydride (1.31 g, 32.7 mmol, 60% dispersed in mineral oil) and tetrahydrofuran (60 mL) were added to a round-bottom flask cooled to 0 °C, followed by dropwise addition of triethyl phosphonoacetate (5.6 mL, 28.2 mmol). After stirring at 0 °C for 10 min, a tetrahydrofuran solution (15 mL) of compound 1 (5.0 g, 29.4 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and then refluxed for 1 h. After cooling, the solution was diluted with distilled water and ethyl acetate (1:1, 50 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with 1 M sodium hydroxide solution, water, and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a 20:1 hexane / ethyl acetate (v / v) eluent to give compound 3 (4.8 g, 68% yield) as a colorless oil.

[0573] Step 2:

[0574] Cuprous bromide (574 mg, 4 mmol) and lithium chloride (340 mg, 8 mmol) were dissolved in tetrahydrofuran (50 mL), and the resulting solution was stirred at 0 °C for 10 min. Then, a tetrahydrofuran solution of compound 3 (4.8 g, 20 mmol) and trimethylchlorosilane (2.39 g, 22 mmol) was added dropwise. The mixture was stirred at 0 °C for 15 min under an argon atmosphere. Next, compound 4 (1.0 M) (48 mL, 48 mmol) was added dropwise, and the mixture was stirred at 0 °C for 2 h under an argon atmosphere. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed one main spot. The mixture was extracted with ethyl acetate (100 mL × 3) and an aqueous solution of ammonium chloride (300 mL), the organic phases were combined, washed with brine (150 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using petroleum ether with the proportion of ethyl acetate increasing from 0% to 10% as the eluent, to give compound 5 (2g, yield 35.4%), which was a light green oil.

[0575] LCMS:Rt:1.282min;MS m / z(ELSD):283.3[M+H] + ;

[0576] Step 3:

[0577] To a tetrahydrofuran (30 mL) solution of compound 5 (2 g, 7.092 mmol), lithium aluminum hydride (2.5 M) (5.67 mL, 14.184 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 16 hours under an argon atmosphere. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed a new main spot. The reaction was quenched by sodium sulfate decahydrate, followed by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using petroleum ether with the ethyl acetate ratio increased from 0% to 20% as eluent to give compound 6 (1.2 g, 70.5% yield) as a colorless oil.

[0578] 1 H NMR (400MHz, CDCl3) δ3.82–3.68(m,2H),1.75–1.55(m,2H),1.45–1.17(m,18H), 0.88(t,J=6.8Hz,3H),0.72–0.60(m,1H),0.55–0.40(m,3H),0.10–0.03(m,2H).

[0579] Step 4:

[0580] Triethylamine (1.578 g, 15.625 mmol) and methanesulfonyl chloride (1.43 g, 12.5 mmol) were added to a dichloromethane (30 mL) solution of compound 6 (1.5 g, 6.25 mmol). The mixture was stirred at room temperature for 2 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (70 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with 5% sodium bicarbonate solution (50 mL) and brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 7 (1.8 g, 90.5% yield), a colorless oil, which was used directly in the next reaction.

[0581] Step 5:

[0582] Compound 8 (1.91 g, 5.15 mmol), potassium carbonate (3.91 g), and potassium iodide (1.71 g) were added to a solution of compound 7 (1.8 g, 5.66 mmol) in acetonitrile / cyclopentyl methyl ether (20 mL / 20 mL). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as an eluent increasing from 0% to 10%, to give compound 9 (1.1 g, yield 32.7%) as a yellow oil.

[0583] LCMS:Rt:1.212min;MS m / z(ELSD):595.0[M+H] + ;

[0584] Step 6:

[0585] Triethylamine (468 mg, 4.630 mmol) and methanesulfonyl chloride (423 mg, 3.704 mmol) were added to a dichloromethane (15 mL) solution of compound 9 (1.1 g, 1.852 mmol), and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (70 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with 5% sodium bicarbonate solution (70 mL) and brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 10 (1.3 g, yield >100%) as a colorless oil, which was used directly in the next reaction.

[0586] Step 7:

[0587] Sodium azide (252 mg, 3.869 mmol) was added to a solution of compound 10 (1.3 g, 1.935 mmol) in N,N-dimethylformamide (20 mL), and the mixture was stirred at 100 °C for 16 h under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (150 mL × 2) and brine (150 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as eluent from 0% to 10%, to give compound 11 (900 mg, yield 75.2%) as a white solid.

[0588] LCMS:Rt:2.320min;MS m / z(ELSD):619.8[M+H] + ;

[0589] Step 8:

[0590] Palladium on carbon (300 mg, 10% wt%) was added to a tetrahydrofuran / methanol (10 mL / 10 mL) solution of compound 11. The mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere (30 psi). Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane solution of methanol (0.05% ammonia, increasing from 0% to 10%) as eluent to give compound 12 (0.8 g, 69.5% yield) as a colorless oil.

[0591] LCMS:Rt:1.258min;MS m / z(ELSD):593.9[M+H] + ;

[0592] Step 9:

[0593] Compound 13 (160 mg, 1.18 mmol) was added to a 0.7 mL ethanol solution of compound 12 (700 mg, 1.18 mmol). The mixture was stirred at room temperature for 0.5 h, then stirred overnight at 70 °C. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched with water (50 mL), extracted with ethyl acetate (30 mL × 2), and the combined organic phases were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using petroleum ether with an ethyl acetate / tetrahydrofuran (5 / 3) ratio increased from 0% to 50% as eluent) and preparative high-performance liquid chromatography (pre-HPLC) to give HI-19-122 (150.46 mg, yield 18.4%) as a colorless oil.

[0594] LCMS:Rt:1.183min;MS m / z(ELSD):693.9[M+H] + ;

[0595] CAD:95.99% purity at ELSD; RT=18.261min.

[0596] 1 H NMR (400MHz, CDCl3) δ6.66 (s, 2H), 4.84–4.77 (m, 1H), 4.27–4.13 (m, 4H), 2.74 (t, J = 7.2Hz, 2H), 2.60–2.36 (m, 6H), 2.2 8(t,J=7.6Hz,2H),1.67–1.47(m,10H),1.38–1.19(m,38H),0.90–0.83(m,9H),0.56–0.35(m,4H),0.08–-0.00(m,2H).

[0597] Example 17

[0598] Step 1:

[0599] Compound 2 (3 g, 8.62 mmol, 2.2 eq.), K₂CO₃ (1.623 g, 11.76 mmol, 3.0 eq.), and KI (651 mg, 3.92 mmol, 1.0 eq.) were added to a mixture of compound 1 (294 mg, 3.92 mmol, 1.0 eq.) in CPME (20 mL) and MeCN (20 mL). The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (methanol / dichloromethane = 1 / 10) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (120 mL × 3). The combined organic layers were washed with brine (90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 3 (1.7 g, 32%) as a yellow oil.

[0600] LCMS:Rt:1.241min; MS m / z(ELSD):614.7[M+H] + ;

[0601] 1H NMR (400MHz, CDCl3) δ4.09–4.02(m,4H),3.83–3.77(m,2H),2.76–2.67(m,2H),2.54–2.45(m,4H),2.33–2. 25(m,4H),1.77–1.70(m,2H),1.66–1.57(m,8H),1.56–1.46(m,4H),1.36–1.25(m,36H),0.91–0.86(m,6H).

[0602] Step 2:

[0603] TEA (702 mg, 6.94 mmol, 2.5 equivalence) and MSCl (639 mg, 5.56 mmol, 2.0 equivalence) were added to a mixture of compound 3 (1.7 g, 2.78 mmol, 1.0 equivalence) in DCM (20 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (110 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 4 (1.917 g, crude) as a yellow oil.

[0604] LCMS:Rt:1.340min; MS m / z(ELSD):691.1[M+H] + ;

[0605] Step 3:

[0606] To a mixture of compound 4 (1.917 g, 2.8 mmol, 1.0 equivalent) in DMF (20 mL), NaN3 (362 mg, 5.6 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (110 mL) and extracted with ethyl acetate (130 mL × 2). The combined organic layers were washed with brine (6 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give a yellow oily compound 9 (1.337 g, 76%).

[0607] LCMS:Rt:1.422min;MS m / z(ELSD):639.7[M+H] + ;

[0608] Step 4:

[0609] Pd / C (400 mg) was added to a mixture of compound 5 (1.337 g, 2.10 mmol, 1.0 eq.) in MeOH (10 mL) and THF (10 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 6 (696 mg, 54%) as a yellow oil.

[0610] LCMS:Rt:1.241min;MS m / z(ELSD):612.2[M+H] + ;

[0611] 1H NMR(400MHz, CDCl3)δ4.10–4.00(m,4H),2.84–2.74(m,2H),2.53–2.45(m,4H),2.42–2.37(m,4H), 2.32–2.26(m,4H),1.67–1.56(m,10H),1.46–1.39(m,4H),1.35–1.23(m,36H),0.92–0.84(m,6H).

[0612] Step 5:

[0613] Compound 7 (60%) (240 mg, 1.06 mmol, 1.0 eq.) was added to a mixture of compound 6 (646 mg, 1.06 mmol, 1.0 eq.) in ethanol (0.5 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 hours. TLC (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (60 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-125 (50.44 mg, 7%).

[0614] LCMS:Rt:1.148min;MS m / z(ELSD):711.9[M+H] + ;

[0615] CAD:91.08% purity at ELSD; RT=21.362min.

[0616] 1 H NMR (400MHz, CDCl3) δ6.48(s,2H),4.26(s,4H),4.06(t,J=6.8Hz,4H),2.82(s,6H),2.29(t,J=7. 4Hz, 4H), 1.99 (s, 2H), 1.62 (dd, J=13.6, 6.9Hz, 14H), 1.35–1.26 (m, 36H), 0.88 (t, J=6.8Hz, 6H).

[0617] Example 18

[0618] Step 1:

[0619] Under an argon atmosphere, potassium tert-butoxide (15.2 g, 135.86 mmol, 5 eq.) was added to a tetrahydrofuran (100 mL) solution of compound 1 (5 g, 27.17 mmol, 1.0 eq.). The mixture was stirred at 0 °C for 30 min. Then, a tetrahydrofuran (30 mL) solution of compound 2 (27.1 g, 67.93 mmol, 2.5 eq.) was added dropwise to the mixture. The mixture was stirred at room temperature for 2 h under an argon atmosphere. The reaction was quenched with water (150 mL) and extracted with ethyl acetate (150 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography to give compound 3 (4 g, 77%) as a yellow oil.

[0620] 1 H NMR (400MHz, CDCl3) δ5.02 (t, J=7.2Hz, 1H), 4.06 (q, J=7.1Hz, 2H), 2.49 (d, J= 15.2Hz,1H),2.11(t,J=10.3Hz,3H),1.99(t,J=12.7Hz,1H),1.88(dd,J=14.4 ,7.2Hz,3H),1.71(dd,J=23.4,12.5Hz,3H),1.26(dd,J=14.6,7.3Hz,2H),1.1 9(t,J=7.1Hz,3H),0.95(ddd,J=24.9,12.0,3.8Hz,2H),0.81(t,J=7.3Hz,3H).

[0621] Step 2:

[0622] Pd / C (1 g) was added to a mixture of compound 3 (4 g, 17.84 mmol, 1.0 eq.) in ETOH (40 mL). The reaction mixture was stirred in H2 at room temperature for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with 10% petroleum ether in ethyl acetate to give compound 4 (3.833 g, 95%) as a yellow oil.

[0623] Step 3:

[0624] At 0℃ in N 2Compound 4 (3.833 g, 16.95 mmol, 1.0 eq.) was added to a mixture in THF (40 mL) with LAH (644 mg, 16.95 mmol, 1.0 eq.). The mixture was stirred at room temperature for 3 hours. TLC (petroleum ether / ethyl acetate = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL) and treated with 1 N hydrochloric acid to adjust the pH to between 6 and 7. The mixture was quenched with H2O (150 mL) and extracted with EA (200 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 20% ethyl acetate to give compound 5 (2.335 g, 75%) as a yellow oil.

[0625] 1 H NMR (400MHz, CDCl3) δ3.68(t,J=6.6Hz,2H),1.80–1.54(m,4H),1.51–1.44(m,2H),1.39–1.22(m,7H),1.16(d,J=5.8Hz,2H),0.97–0.79(m,6H).

[0626] Step 4:

[0627] To a mixture of compound 6 (2.818 g, 12.7 mmol, 1.0 eq.) and compound 5 (2.335 g, 12.7 mmol, 1.0 eq.) in DCM (50 mL), EDCI (4.848 g, 25.4 mmol, 2.0 eq.), DMAP (620 mg, 5.08 mmol, 0.4 eq.), and DIEA (6.549 g, 50.8 mmol, 4.0 eq.) were added. The reaction mixture was stirred at room temperature under N2 for 16 h. TLC (PE:EA = 20 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (120 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% EA in PE to give a clear oily compound 7 (3.191 g, 65%).

[0628] 1H NMR (400MHz, CDCl3) δ4.14–4.06(m,2H),3.43–3.37(m,2H),2.32–2.24(m,2H),1.89–1.81(m,2H),1.77–1. 70(m,3H),1.67–1.55(m,3H),1.55–1.37(m,5H),1.35–1.21(m,10H),1.19–1.12(m,2H),0.98–0.81(m,6H).

[0629] Step 5:

[0630] Compound 8 (1.76 g, 3.87 mmol, 1.0 eq.) was stirred in CPME (15 mL) and MeCN (15 mL) at 90 °C under N2 for 16 h. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with H2O (110 mL) and extracted with EA (100 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give compound 9 (2.062 g, 70%) as a yellow oil.

[0631] LCMS:Rt:1.649min;MS m / z(ELSD):786.6[M+H] + ;

[0632] Step 6:

[0633] TEA (682 mg, 6.7 mmol, 2.5 eq.) and MSCl (621 mg, 5.4 mmol, 2.0 eq.) were added to a mixture of compound 9 (2.062 g, 2.7 mmol, 1.0 eq.) in DCM (20 mL). The reaction mixture was stirred at 0 °C under Ar for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (90 mL) and extracted with dichloromethane (120 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 10 (2.67 g, crude) as a yellow oil.

[0634] LCMS:Rt:1.509min; MS m / z(ELSD):843.0[M+H] + ;

[0635] Step 7:

[0636] To a mixture of compound 10 (2.67 g, 3.17 mmol, 1.0 equivalent) in DMF (20 mL), NaN3 (413 mg, 6.34 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (110 mL) and extracted with EA (120 mL × 3). The combined organic layers were washed with brine (6 × 70 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane to give a yellow oily compound 11 (1.456 mg, 58%).

[0637] LCMS:Rt:1.655min;MS m / z(ELSD):790.1[M+H] + ;

[0638] Step 8:

[0639] Pd / C (500 mg) was added to a mixture of compound 11 (1.456 g, 1.84 mmol, 1.0 eq.) in MeOH (8 mL) and THF (8 mL). The reaction mixture was stirred in H2 at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with a 10% methanol solution in dichloromethane, to give compound 12 (1.089 g, 78%) as a yellow oil.

[0640] LCMS:Rt:1.754min;MS m / z(ELSD):764.5[M+H] + ;

[0641] Step 9:

[0642] Compound 13 (60%) (149 mg, 0.66 mmol, 1.0 eq.) was added to a mixture of compound 12 (500 mg, 0.66 mmol, 1.0 eq.) in ethanol (0.5 mL, 95%). The reaction mixture was stirred at 70 °C under N2 for 16 hours. TLC (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (50 mL) and extracted with ethyl acetate (70 mL × 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-126 (mg, %).

[0643] LCMS:Rt:1.428min;MS m / z(ELSD):864.1[M+H] + ;

[0644] CAD:88.50% purity at ELSD; RT=24.181min.

[0645] 1 H NMR (400MHz, CDCl3) δ6.44(s,2H),4.90–4.82(m,1H),4.25(s,4H),4.10(dd,J=9.0,4.8Hz,2H),2.79(s,8H),2.29(td,J=7.4,3 .7Hz,4H),1.74(d,J=10.9Hz,4H),1.60(d,J=6.1Hz,8H),1.51(d,J=6.7Hz,8H),1.26(s,40H),1.16(s,3H),0.94–0.83(m,14H).

[0646] Example 19

[0647] Step 1:

[0648] Compound 2 (3 g, 21.13 mmol, 1.0 eq.), EDCI (8.07 g, 42.25 mmol, 2.0 eq.), DMAP (1.03 g, 8.45 mmol, 0.2 eq.), and DIEA (10.9 g, 84.51 mmol, 4.0 eq.) were added to a dichloromethane (80 mL) solution of compound 1. The reaction mixture was stirred at 25 °C for 16 h under nitrogen protection. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic layers were washed with brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (eluting from 0% to 10% ethyl acetate in petroleum ether) to give compound 3 (2.876 g, 23%) as a yellow oil.

[0649] 1 H NMR (400MHz, CDCl3) δ5.63(s,1H),5.54(s,1H),4.62(d,J=6.8Hz,2H),3.43(dd,J=31.9,25.0Hz,2H),2.31(t,J=7.5Hz ,2H),2.10(d,J=7.2Hz,2H),1.90–1.72(m,2H),1.63(dd,J=8.5,6.2Hz,2H),1.49–1.18(m,14H),0.88(t,J=6.9Hz,3H).

[0650] Step 2:

[0651] To a mixed solution of compound 4 (283 mg, 3.78 mmol, 1.0 eq.) in acetonitrile (20 mL) and methyl tert-butyl ether (20 mL), potassium iodide (1.25 g, 7.55 mmol, 2.0 eq.), compound 3 (2.876 g, 8.30 mmol, 2.2 eq.), and potassium carbonate (2.6 g, 18.87 mmol, 5.0 eq.) were added. The reaction mixture was stirred at 90 °C for 16 h under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol 1 / 0-10 / 1, v / v gradient elution) to give compound 5 (1.338 g, 52%) as a pale yellow oil.

[0652] LCMS:Rt:1.124min;MS m / z(ELSD):608.6[M+H] + ;

[0653] Step 3:

[0654] Triethylamine (556 mg, 5.5 mmol, 2.0 eq.) and methanesulfonyl chloride (506 mg, 4.4 mmol, 2 eq.) were added to a solution of compound 5 (1.338 g, 2.2 mmol, 1.0 eq.) in dichloromethane (15 mL). The reaction mixture was stirred at 25 °C for 2 h under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 6 (1.34 g, 88.8%) as a yellow oil.

[0655] LCMS:Rt:1.130min;MS m / z(ELSD):686.6[M+H] + ;

[0656] Step 4:

[0657] Sodium azide (358 mg, 5.5 mmol, 2.5 eq.) was added to a DMF (20 mL) solution of compound 6 (1.51 g, 2.2 mmol, 1.0 eq.). The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. After dilution with ethyl acetate (70 mL), the mixture was washed with water (40 mL × 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol 1 / 0-10 / 1, v / v gradient elution) to give compound 7 (703 mg, 41%) as a yellow oil.

[0658] LCMS: Rt: 1.130min; MS m / z (ELSD): 633.6[M+H]+;

[0659] Step 5:

[0660] Triphenylphosphine (541 mg, 2.09 mmol, 2 eq.) was added to a tetrahydrofuran / water (10 mL, 9 / 1) mixture of compound 7 (653 mg, 1.03 mmol, 1.0 eq.). The reaction mixture was stirred at 80 °C for 2 h. The reaction was quenched with water (25 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol 1 / 0–10 / 1, v / v gradient elution) to give compound 8 (326 mg, 51%) as a pale yellow oil.

[0661] LCMS: Rt: 1.054min; MS m / z (ELSD): 607.6[M+H]+;

[0662] Step 6:

[0663] Compound 9 (111 mg, 0.54 mmol, 1.0 eq.) was added to a 0.4 mL ethanol solution of compound 8 (326 mg, 0.54 mmol, 1.0 eq.). The mixture was stirred at 70 °C for 16 hours under argon protection. After concentration under reduced pressure, the residue was separated by column chromatography (0%–10% methanol gradient elution in dichloromethane) and preparative HPLC to give a yellow oily HI-19-130 (52.12 mg, 13.6%).

[0664] LCMS:Rt:1.280min;MS m / z(ELSD):708.0[M+H] + ;

[0665] CAD:95.54%purity.Rt:16.489min

[0666] 1 H NMR(400MHz, CDCl3)δ6.46(s,2H),5.68–5.60(m,2H),5.55–5.48(m,2H),4.62(d,J=6.8Hz,4H),4.24(s,4H),2 .91–2.27(m,12H),2.10(q,J=7.0Hz,4H),1.65–1.56(m,8H),1.37–1.25(m,30H),0.88(t,J=6.8Hz,6H).Example 20

[0667] Step 1:

[0668] At -20 °C, a mixture of compound 1 (5 g, 29.4 mmol, 1.0 eq.) in tetrahydrofuran (50 mL) was added dropwise. A solution of compound 2 (5.506 g, 38.2 mmol, 1.3 equivalence) was added dropwise. The mixture was stirred overnight at room temperature under nitrogen. TLC (ethyl acetate / petroleum ether = 1 / 10) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (150 mL) and extracted with ethyl acetate (170 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a solution of 10% ethyl acetate in petroleum ether, to give a yellow oily compound 3 (2.88 g, 46%).

[0669] 1H NMR (400MHz, CDCl3) δ2.89–2.81(m,1H),1.63–1.53(m,3H),1.51–1.36(m,2H) ,1.32–1.25(m,14H),0.93–0.85(m,4H),0.58–0.43(m,2H),0.31–0.17(m,2H).

[0670] Step 2:

[0671] To a mixture of compound 3 (2.886 g, 13.6 mmol, 1.0 eq.) and compound 4 (2.451 g, 13.6 mmol, 1.0 equivalence) in dichloromethane (50 mL), EDCI (5.201 g, 27.2 mmol, 2.0 equivalence), DMAP (665 mg, 5.45 mmol, 0.4 equivalence), and DIEA (7.025 g, 54.5 mmol, 4.0 equivalence) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (ethyl acetate / petroleum ether = 1 / 20) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (150 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% ethyl acetate in petroleum ether to give yellow oily compound 5 (1.773 g, 30%).

[0672] 1H NMR (400MHz, CDCl3) δ4.31–4.20(m,1H),3.45–3.37(m,2H),2.37–2.30(m,2H),1.95–1.86(m,2H),1.83–1.75(m,2H) ,1.68–1.60(m,2H),1.35–1.28(m,4H),1.31–1.22(m,12H),0.99–0.91(m,1H),0.90–0.85(m,3H),0.58–0.22(m,4H).

[0673] Step 3:

[0674] Compound 5 (1.773 g, 4.74 mmol, 1.0 equivalent), potassium carbonate (1.963 g, 14.2 mmol, 3.0 equivalent), and potassium iodide (787 mg, 4.74 mmol, 1.0 equivalent) were added to a mixture of compound 6 (1.759 g, 4.74 mmol, 1.0 equivalent) in cyclopentyl methyl ether / acetonitrile (20 mL, v / v). The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (methanol / dichloromethane = 1 / 10) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (120 mL) and extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with a 10% methanol-dichloromethane solution to give a yellow oily compound 7 (2.4 g, 76%).

[0675] LCMS:Rt:1.130min;MS m / z(ELSD):667.0[M+H] + ;

[0676] 1H NMR (400MHz, CDCl3) δ4.85–4.77(m,1H),4.30–4.22(m,1H),3.82–3.76(m,2H),2.72–2.63(m,2H),2.53–2.40(m,4H),2.37–2.25(m,4H), 1.73–1.68(m,2H),1.67–1.45(m,18H),1.35–1.30(m,8H),1.29–1.25(m,23H),0.99–0.91(m,1H),0.90–0.84(m,9H),0.58–0.22(m,4H).

[0677] Step 4:

[0678] TEA (909 mg, 9.0 mmol, 2.5 eq.) and MSCl (828 mg, 7.2 mmol, 2.0 eq.) were added to a mixture of compound 7 (2.4 g, 3.6 mmol, 1.0 eq.) in dichloromethane (25 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (100 mL) and extracted with dichloromethane (120 mL × 3). The combined organic layers were washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 9 (3.13 g, crude), a yellow oil.

[0679] LCMS:Rt:1.451min;MS m / z(ELSD):746.6[M+H] + ;

[0680] Step 5:

[0681] To a mixture of compound 6 (3.13 g, 4.21 mmol, 1.0 equivalent) in DMF (30 mL), NaN3 (547 mg, 8.41 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (120 mL) and extracted with ethyl acetate (150 mL × 2). The combined organic layers were washed with brine (6 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give a yellow oily compound 9 (1.75 mg, 60%).

[0682] LCMS:Rt:1.404min; MS m / z(ELSD):692.3[M+H] + ;

[0683] Step 6:

[0684] Pd / C (600 mg) was added to a mixture of compound 9 (1.75 g, 2.53 mmol, 1.0 eq.) in methanol (10 mL) and tetrahydrofuran (10 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 10 (865 mg, 51%) as a yellow oil.

[0685] LCMS:Rt:1.241min;MS m / z(ELSD):872.7[M+H] + ;

[0686] Step 7:

[0687] Compound 11 (103 mg, 0.75 mmol, 1.0 equivalent) was added to a mixture of compound 10 (500 mg, 0.75 mmol, 1.0 equivalent) in ethanol (0.5 mL, 95%). The reaction mixture was stirred under nitrogen at 70 °C for 16 hours. TLC (ethyl acetate:tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (60 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-131 (68.12 mg, 12%).

[0688] LCMS:Rt:1.177min; MS m / z(ELSD):766.0[M+H] + ;

[0689] CAD:91.93% purity at ELSD; RT=21.756 min.

[0690] 1 H NMR (400MHz, CDCl3) δ6.42(s,2H),4.84–4.78(m,1H),4.27(s,5H),2.82(s,6H),2.37(s,2H),2.29(t,J=7.4Hz,2H),1.87(s,2H),1.67–1.57(m ,12H),1.30(d,J=32.1Hz,39H),0.95(d,J=8.5Hz,1H),0.90–0.85(m,9 H),0.56(s,1H),0.44(d,J=5.1Hz,1H),0.34(s,1H),0.30–0.24(m,1H).

[0691] Example 21

[0692] Step 1:

[0693] To a mixture of compound 1 (1.7 g, 6.49 mmol, 1.0 equivalence) and compound 2 (1.441 mg, 6.49 kmol, 1.0 eq.) in dichloromethane (20 mL), EDCI (2.479 g, 12.98 mmol, 2.0 equivalence), DMAP (317 mg, 2.60 mmol, 0.4 equivalence), and DIEA (3.349 g, 25.95 mmol, 4.0 equivalence) were added. The reaction mixture was stirred under nitrogen at room temperature for 16 hours. TLC (ethyl acetate / petroleum ether = 1 / 20) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (90 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with petroleum ether in 10% ethyl acetate to give yellow oily compound 3 (1.1 g, 36%).

[0694] 1H NMR (400MHz, CDCl3) δ7.09–7.05(m,1H),7.00–6.94(m,2H),4.31–4.22(m,2H),3.44–3.37(m,2H),2.91–2.83(m,2H),2.60–2.53(m,4H) ,2.33–2.24(m,2H),1.88–1.80(m,2H),1.64–1.56(m,4H),1.56–1.51(m,2H),1.46–1.40(m,2H),1.39–1.29(m,12H),0.96–0.87(m,6H).

[0695] Step 2:

[0696] To a mixture of compound 4 (810 mg, 2.36 mmol, 1.0 eq.) in CPME / MeCN (20 mL, v / v), KI (392 mg, 2.36 mmol, 1.0 eq.), K₂CO₃ (978 mg, 7.08 mmol, 3.0 eq.), and compound 3 (1.1 g, 2.36 mmol / 1.0 eq.) were added. The reaction mixture was stirred at 90 °C under nitrogen for 16 h. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (120 mL × 3). The combined organic layers were washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 5 (947 mg, 55%) as a yellow oil.

[0697] LCMS:Rt:1.719min; MS m / z(ELSD):731.2[M+H] + ;

[0698] Step 3:

[0699] TEA (328 mg, 3.24 mmol, 2.5 equivalence) and MSCl (299 mg, 2.60 mmol, 2.0 equivalence) were added to a mixture of compound 5 (947 mg, 1.30 mmol, 1.0 equivalence) in dichloromethane (10 mL). The reaction mixture was stirred at 0 °C under argon for 1 hour. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1 N HCl (2 mL). The mixture was quenched with water (70 mL) and extracted with dichloromethane (90 mL × 3). The combined organic layers were washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give compound 6 (1.297 g, crude) as a yellow oil.

[0700] LCMS:Rt:1.503min;MS m / z(ELSD):808.7[M+H] + ;

[0701] Step 4:

[0702] To a mixture of compound 6 (1.297 g, 1.61 mmol, 1.0 equivalent) in DMF (12 mL), NaN3 (209 mg, 3.21 mmol, 2.0 equivalent) was added. The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (100 mL) and extracted with ethyl acetate (120 mL × 3). The combined organic layers were washed with brine (5 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 7 (897 mg, 74%) as a yellow oil.

[0703] LCMS:Rt:1.807min;MS m / z(ELSD):756.3[M+H] + ;

[0704] Step 5:

[0705] Pd / C (200 mg) was added to a mixture of compound 7 (897 mg, 1.19 mmol, 1.0 eq.) in methanol (5 mL) and tetrahydrofuran (5 mL). The reaction mixture was stirred under hydrogen at room temperature for 16 hours. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with 10% methanol in dichloromethane to give compound 8 (390 mg, 45%) as a yellow oil.

[0706] LCMS:Rt:1.048min;MS m / z(ELSD):729.7[M+H] + ;

[0707] 1H NMR (400MHz, CDCl3) δ7.09–7.04(m,1H),7.00–6.94(m,2H),4.28–4.22(m ,2H),4.07–4.02(m,2H),3.17–3.10(m,3H),2.90–2.80(m,6H),2.64–2.5 4(m,8H),2.32–2.26(m,4H),1.98–1.87(m,2H),1.64–1.51(m,14H),1.38 –1.33(m,10H),1.33–1.29(m,12H),1.28–1.25(m,9H),0.92–0.86(m,9H).

[0708] Step 6:

[0709] Compound 9 (116 mg, 0.51 mmol, 1.0 eq.) was added to a mixture of compound 8 (372 mg, 0.51 mmol, 1.0 eq.) in ethanol (0.5 mL, 95%). The reaction mixture was stirred at 70 °C under N2 for 16 hours. TLC (ethyl acetate:tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (60 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 70% ethyl acetate / tetrahydrofuran (1:5) to give a yellow oily compound HI-19-132 (39.37 mg, 0.09%).

[0710] LCMS:Rt:1.241min;MS m / z(ELSD):830.0[M+H] + ;

[0711] CAD:95.24% purity at ELSD; RT=23.427min.

[0712] 1 H NMR (400MHz, CDCl3) δ7.07(d,J=7.6Hz,1H),6.97(d,J=9.8Hz,2H),6.42(s,2H),4.32–4.23(m,6H),4.05(t,J=6.8Hz,2H),2.89(dd,J=24.0,16.7Hz ,8H),2.59–2.54(m,4H),2.29(t,J=7.4Hz,4H),2.10(s,2H),1.79–1.66( m,4H),1.60(d,J=6.4Hz,6H),1.31(d,J=32.2Hz,38H),0.93–0.88(m,9H).

[0713] Example 22

[0714] Step 1:

[0715] Compound 2 (1.64 g, 5.89 mmol), potassium carbonate (2.44 g, 17.67 mmol), and potassium iodide (978 mg, 5.89 mmol) were added to a solution of compound 1 (2.946 g, 6.47 mmol) in acetonitrile / cyclopentyl methyl ether (35 mL / 35 mL). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as an eluent increasing from 0% to 10%, to give compound 3 (3.1 g, 73.2% yield) as a yellow oil.

[0716] Step 2:

[0717] Triethylamine (1.159 g, 11.468 mmol) and methanesulfonyl chloride (1.05 g, 9.174 mmol) were added to a solution of compound 3 (3 g, 4.587 mmol) in dichloromethane (40 mL). The mixture was stirred at room temperature for 2 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (70 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with 5% sodium bicarbonate solution (70 mL) and brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 4 (3.2 g, 95.3% yield), a colorless oil, which was used directly in the next reaction.

[0718] Step 3:

[0719] Sodium azide (580 mg, 8.743 mmol) was added to a solution of compound 4 (3.2 g, 4.372 mmol) in N,N-dimethylformamide (40 mL), and the mixture was stirred at 100 °C for 16 h under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (150 mL × 2) and brine (150 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as eluent from 0% to 10%, to give compound 5 (2.0 g, yield 67.4%) as a white solid.

[0720] LCMS:Rt:1.363min; MS m / z(ELSD):680.3[M+H] + ;

[0721] Step 4:

[0722] Palladium on carbon (500 mg, 10% by weight) was added to a tetrahydrofuran / methanol (15 mL / 15 mL) solution of compound 5 (2 g, 2.95 mmol), and the mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere (30 psi). Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane solution of methanol (0.05% ammonia, increasing from 0% to 10%) as eluent to give compound 6 (1.8 g, 93.5% yield) as a pale yellow oil.

[0723] LCMS:Rt:1.142min;MS m / z(ELSD):653.9[M+H] + ;

[0724] Step 5:

[0725] Compound 7 (312 mg, 2.297 mmol) was added to a 2 mL ethanol solution of compound 6 (1.5 g, 2.297 mmol). The mixture was stirred at room temperature for 0.5 h, then stirred overnight at 70 °C. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched with water (50 mL) and extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using petroleum ether in an ethyl acetate / tetrahydrofuran (5 / 3) ratio increased from 0% to 50% as eluent, to give compound 8 (750 mg, yield 43.4%) as a colorless oil.

[0726] LCMS:Rt:1.118min;MS m / z(ELSD):753.6[M+H] + ;

[0727] 1 H NMR(400MHz, CDCl3)δ6.55(s,2H),4.89–4.82(m,1H),4.25–4.16(m,4H),2.75(t,J=7.0Hz,2H),2.49–2.24 (m,8H),2.23–2.16(m,2H),1.65–1.48(m,10H),1.46–1.37(m,15H),1.30–1.23(m,34H),0.89–0.85(m,6H).

[0728] Step 6:

[0729] A 3 mL solution of compound 8 (600 mg, 0.797 mmol) in 4.0 M hydrochloric acid / dioxane was stirred at room temperature for 3 hours. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was concentrated to give compound 9 (600 mg, yield >100%) as a colorless oil, which was used directly in the next reaction.

[0730] LCMS:Rt:1.093min; MS m / z(ELSD):697.5[M+H] + ;

[0731] Step 7:

[0732] Compound 10 (182 mg, 1.291 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 331 mg, 1.722 mmol), 4-dimethylaminopyridine (DMAP, 22 mg, 0.172 mmol), and N,N-diisopropylethylamine (DIEA, 445 mg, 3.44 mmol) were added to a dichloromethane (15 mL) solution of compound 9 (600 mg, 0.861 mmol). The mixture was stirred at room temperature for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched with water (30 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with brine (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (using methanol in dichloromethane as an eluent, increasing from 0% to 10%) and preparative high performance liquid chromatography (pre-HPLC) to obtain compound HI-19-134 (34.7 mg, yield 4.9%), which was a colorless oil.

[0733] LCMS:Rt:2.168min;MS m / z(ELSD):819.5[M+H] + ;

[0734] CAD:92.99%purity.Rt:18.610min

[0735] 1 H NMR (400MHz, CDCl3) δ6.47(s,2H),4.92–4.79(m,1H),4.66(t,J=2.2Hz,2H),4.21(s,4H),2.75(t,J=7.0Hz,2H),2. 56–2.24(m,10H),2.23–2.18(m,2H),1.67–1.58(m,6H),1.54–1.47(m,6H),1.40–1.21(m,46H),0.91–0.85(m,9H).

[0736] Example 23

[0737] Step 1:

[0738] To a solution of compound 1 (6.8 g, 44.84 mmol, 1.0 eq.) in dichloromethane (100 mL), compound 2 (10 g, 44.84 mmol, 1.0 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (17 g, 89.69 mmol, 2.0 eq.), 4-dimethylaminopyridine (2.2 g, 17.94 mmol, 0.4 eq.), and N,N-diisopropylethylamine (23 g, 179.37 mmol, 4.0 eq.) were added. The reaction mixture was stirred at 25 °C for 16 hours under nitrogen protection. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–10% ethyl acetate in petroleum ether solution) to give compound 3 (5.67 g, yield 33%), which was a yellow oil.

[0739] 1 H NMR (400MHz, CDCl3) δ4.28–4.17(m,2H),3.70–3.59(m,2H),3.50–3.34(m,4H),2.34(t,J= 8.0Hz,2H),1.91–1.78(m,2H),1.70–1.54(m,5H),1.48–1.26(m,12H),0.96–0.80(m,3H).

[0740] Step 2:

[0741] To a mixed solution of compound 4 (1 g, 2.20 mmol, 1.0 eq.) in acetonitrile (10 mL) and cyclopentyl methyl ether (10 mL), potassium iodide (730 mg, 3.30 mmol, 2.0 eq.), compound 3 (1.15 g, 3.30 mmol, 1.5 eq.), and potassium carbonate (1.2 g, 8.80 mmol, 5.0 eq.) were added. The reaction mixture was stirred at 90 °C for 16 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent to give compound 5 (1 g, 62% yield) as a pale yellow solid.

[0742] LCMS: Rt: 1.250min; MS m / z (ELSD): 727.4[M+H]+;

[0743] 1H NMR (400MHz, CDCl3) δ4.92–4.81(m,1H),4.25–4.18(m,2H),3.84–3.76(m,2H),3.67–3.57(m,2H),3.50–3.41(m,2H),2.7 5(s,2H),2.53(s,3H),2.37–2.23(m,4H),1.82–1.70(m,2H),1.59–1.52(m,12H),1.34–1.22(m,41H),0.91–0.84(m,9H).

[0744] Step 3:

[0745] To a solution of compound 5 (1 g, 1.38 mmol, 1.0 eq.) in dichloromethane (10 mL), triethylamine (0.348 g, 3.44 mmol, 2.0 eq.) and methanesulfonyl chloride (0.317 g, 2.75 mmol, 1.5 eq.) were added. The reaction mixture was stirred at 25 °C for 2 hours under nitrogen protection. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (2 × 40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 6 (1.1 g, crude product) as a yellow oil.

[0746] LCMS: Rt: 1.381min; MS m / z (ELSD): 804.9[M+H]+;

[0747] Step 4:

[0748] Sodium azide (0.18 g, 2.75 mmol, 2.0 eq.) was added to a solution of compound 6 (1.1 g, 2.14 mmol, 1.0 eq.) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. The mixture was diluted with ethyl acetate (100 mL) and washed with water (40 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent to give compound 7 (0.751 mg, 71% yield) as a pale yellow solid.

[0749] LCMS: Rt: 1.323min; MS m / z (ELSD): 752.4[M+H]+;

[0750] Step 5:

[0751] Palladium on carbon (0.2 g) was added to a 10 mL ethanol solution of compound 7 (0.75 g, 1.01 mmol, 1.0 eq.). The reaction mixture was stirred at 25 °C for 16 hours under a hydrogen atmosphere. The mixture was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (1 / 0–10 / 1, v / v) as eluent to give compound 8 (480 mg, 66% yield) as a pale yellow solid.

[0752] LCMS: Rt: 1.340min; MS m / z (ELSD): 726.0[M+H]+;

[0753] 1 H NMR(400MHz, CDCl3)δ4.93–4.80(m,1H),4.29–4.16(m,2H),3.69–3.53(m,2H),3.49–3.33(t,J=6.7Hz,2H), 3.36–3.30(t,J=6.6Hz,2H),2.53–2.21(m,9H),1.76–1.47(m,21H),1.45–1.15(m,47H),0.93–0.81(m,9H).

[0754] Step 6:

[0755] Compound 9 (81 mg, 0.59 mmol, 1.0 eq.) was added to a 0.5 mL ethanol solution of compound 8 (432 mg, 0.59 mmol, 1.0 eq.). The mixture was stirred at 70 °C for 16 hours under argon protection. The mixture was concentrated under reduced pressure. The residue was separated by column chromatography (0%–10% methanol in dichloromethane solution) and preparative high-performance liquid chromatography to give HI-19-135 (53.74 mg, 10% yield) as a yellow oil.

[0756] LCMS:Rt:1.223min;MS m / z(ELSD):826.2[M+H] + ;

[0757] CAD:90.8%purity.Rt:19.302min

[0758] 1H NMR(400MHz, CDCl3)δ6.53(s,2H),4.93–4.79(m,1H),4.30–4.15(m,6H),3.69–3.57(m,2H),3.5 1–3.42(m,2H),2.82–2.73(m,2H),2.64-2.44(m,8H),2.24-2.26(m,2H),1.69-1.50(m,7H),1.50 1.35(m,6H),1.36–1.22(m,45H),0.92–0.84(m,9H).

[0759] Example 24

[0760] Step 1:

[0761] To a mixture of compound 1 (6 g, 34.85 mmol, 1.0 eq.) and pyridine (40 mL), p-toluenesulfonyl chloride (7.28 g, 38.33 mmol, 1.1 eq.) was added. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. Thin-layer chromatography (ethyl acetate / petroleum ether = 1 / 10) showed that the reaction was complete and a new main spot was observed. The mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0–10 / 1) as eluent to give compound 3 (8.63 g, 76%).

[0762] 1 H NMR (400MHz, CDCl3) δ7.79(d,J=8.3Hz,2H),7.32(d,J=8.0Hz,2H),4.59–4.39(m,1H),2.44(s ,3H),1.67–1.59(m,2H),1.56–1.51(m,2H),1.30–1.14(m,12H),0.85(dt,J=14.9,7.2Hz,6H).

[0763] Step 2:

[0764] Acetyl potassium(III) sulfide (437 mg, 3.83 mmol, 2.5 eq.) was added to a mixture of compound 2 (500 mg, 1.53 mmol, 1.0 eq.) and N,N-dimethylformamide (5 mL). The mixture was stirred at 80 °C for 2 h under a nitrogen atmosphere. Thin-layer chromatography (ethyl acetate / petroleum ether = 1 / 20) showed that the reaction was complete and a new main spot was observed. The mixture was diluted with ethyl acetate (50 mL) and washed with water (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0-20 / 1) as eluent to give compound 3 (285 mg, 81%) as a yellow oil.

[0765] Step 3:

[0766] LAH (657 mg, 17.29 mmol, 1.0 eq.) was added to a mixture of compound 3 (3.98 g, 17.29 mmol, 1.0 eq.) in THF (40 mL) under nitrogen protection at 0 °C. The mixture was stirred at room temperature for 3 hours. TLC (petroleum ether / ethyl acetate = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL) and treated with 1 N hydrochloric acid to adjust the pH to between 6 and 7. The mixture was quenched with H2O (150 mL) and extracted with EA (200 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether solution of 20% ethyl acetate to give compound 4 (2.6 g, 89%) as a yellow oil. Step 4:

[0767] To a mixture of DCM (30 mL) of compounds 4 (2.6 g, 13.82 mmol, 1.0 equivalence) and 5 (3.069 g, 13.83 mmol, 1.0 wt), EDCI (5.279 g, 27.64 mmol, 2.0 equivalence), DMAP (675 mg, 5.53 mmol, 0.4 equivalence), and DIEA (7.131 g, 55.27 mmol, 4.0 equivalence) were added. The reaction mixture was stirred at room temperature under N2 for 16 h. TLC (PE:EA = 20 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with H2O (200 mL) and extracted with EA (200 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 10% EA in PE to give a clear oily compound 6 (3.4 g, 63%).

[0768] Step 5:

[0769] Potassium iodide (548 mg, 3.3 mmol, 1.0 eq.), potassium carbonate (1.365 g, 9.9 mmol, 3.0 eq.), and compound 6 (1.293 g, 3.3 mmol, 1.0 eq.) were added to a mixed solution of compound 7 (1.5 g, 3.3 mmol, 1.0 eq.) in methyl cyclopentyl ether (15 mL) and acetonitrile (15 mL). The reaction mixture was stirred at 90 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (110 mL) and extracted with ethyl acetate (100 mL × 3). The organic layers were combined, washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with dichloromethane containing 10% methanol to give compound 8 (1.5 g, 59%) as a yellow oil.

[0770] LCMS:Rt:1.754min;MS m / z(ELSD):769.5[M+H] + ;

[0771] Step 6:

[0772] Under an argon atmosphere, triethylamine (494 mg, 4.88 mmol, 2.5 eq.) and methanesulfonyl chloride (450 mg, 3.90 mmol, 2.0 eq.) were added to a dichloromethane (15 mL) solution of compound 8 (1.5 g, 1.95 mmol, 1.0 eq.). The reaction mixture was stirred at 0 °C for 1 hour. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding 1 N hydrochloric acid (2 mL). Water (90 mL) was then added, and the mixture was extracted with dichloromethane (120 mL × 3). The organic layers were combined, washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 9 (1.784 g, crude product) as a yellow oil.

[0773] LCMS:Rt:1.468min; MS m / z(ELSD):847.4[M+H] + ;

[0774] Step 7:

[0775] Sodium azide (274 mg, 4.2 mmol, 2.0 eq.) was added to a mixture of compound 9 (1.784 g, 2.1 mmol, 1.0 eq.) and N,N-dimethylformamide (20 mL). The reaction mixture was stirred at 100 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (110 mL) and extracted with ethyl acetate (120 mL × 3). The organic layers were combined, washed with brine (6 × 70 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane containing 10% methanol as the eluent to give compound 10 (841 mg, 50%) as a yellow oil.

[0776] LCMS:Rt:1.678min;MS m / z(ELSD):794.4[M+H] + ;

[0777] Step 8:

[0778] Palladium on carbon (250 mg) was added to a mixed solution of compound 10 (841 mg, 1.06 mmol, 1.0 eq.) in methanol (5 mL) and tetrahydrofuran (5 mL). The reaction mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The catalyst was removed by diatomaceous earth filtration. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 11 (505 mg, 62%) as a yellow oil.

[0779] LCMS:Rt:1.708min;MS m / z(ELSD):768.4[M+H] + ;

[0780] Step 9:

[0781] Compound 12 (82 mg, 0.6 mmol, 1.0 eq.) was added to a mixture of compound 11 (459 mg, 0.6 mmol, 1.0 eq.) and 95% ethanol (0.5 mL). The reaction mixture was stirred at 70 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (50 mL) and extracted with ethyl acetate (70 mL × 3). The organic layers were combined, washed with brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a mixture of petroleum ether containing 70% ethyl acetate / tetrahydrofuran (1:5), to give compound HI-19-136 (35.21 mg, 7%) as a yellow oil.

[0782] LCMS:Rt:1.521min;MS m / z(ELSD):867.9[M+H] + ;

[0783] CAD:93.03%purity.Rt:22.814min

[0784] 1 H NMR (400MHz, CDCl3) δ6.39(s,2H),4.86(t,J=6.1Hz,1H),4.26(s,4H),3.46(t,J=6.6Hz,1H),3.05–2.21( m,12H),1.62(dd,J=16.2,9.3Hz,10H),1.34–1.24(m,56H),0.92(d,J=7.4Hz,3H),0.88(t,J=6.8Hz,9H).

[0785] Example 25

[0786] Step 1:

[0787] Under an argon atmosphere, ammonium acetate (18.1 g, 235.29 mmol, 1.0 eq.) was added to a methanol (100 mL) mixture of compound 1 (5 g, 29.419 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour. Sodium cyanoborohydride (3.7 g, 235.29 mmol) was then added to the mixture. The mixture was stirred at room temperature for another 24 hours under an argon atmosphere. Thin-layer chromatography (petroleum ether / ethyl acetate = 5 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with brine (2 × 90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 2 (2.3 g, 45.7%) as a yellow oil.

[0788] LCMS:Rt:0.850min;MS m / z(ELSD):172.3[M+H] + ;

[0789] 1 H NMR (400MHz, DMSO) δ7.92(s,2H),2.96(p,J=6.3Hz,1H),1.62–1.45(m,4H),1.28(d,J=17.3Hz,12H),0.95–0.81(m,6H).

[0790] Step 2:

[0791] Under a nitrogen atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (5.14 g, 26.90 mmol, 2.0 eq.), 1-hydroxybenzotriazole (2.7 g, 20.18 mmol, 1.5 eq.), and N,N-diisopropylethylamine (5.2 g, 40.35 mmol, 3.0 eq.) were added to a mixture of compound 3 (2.99 g, 13.45 mmol, 1.0 eq.) and compound 2 (2.3 g, 13.45 mmol, 1.0 eq.) in dichloromethane (40 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (2 × 90 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 4 (3.4 g, 67%) as a yellow oil.

[0792] LCMS:Rt:1.404min;MS m / z(ELSD):378.1[M+3] + ;

[0793] Step 3:

[0794] Under a nitrogen atmosphere, potassium iodide (730 mg, 4.39 mmol, 2.0 eq.), potassium carbonate (1.5 g, 10.99 mmol, 5.0 eq.), and compound 4 (989 mg, 2.64 mmol, 1.2 eq.) were added to a mixture of methyl cyclopentyl ether (10 mL) and acetonitrile (10 mL) of compound 5 (1.0 g, 2.19 mmol, 1.0 eq.). The reaction mixture was stirred at 90 °C for 16 h. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The organic layers were combined, washed with brine (2 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with dichloromethane containing 10% methanol to give compound 6 (1.1 g, 67%) as a yellow oil.

[0795] LCMS:Rt:1.358min;MS m / z(ELSD):752.5[M+H] + ;

[0796] Step 4:

[0797] Under an argon atmosphere, triethylamine (370 mg, 3.66 mmol, 2.5 eq.) and methanesulfonyl chloride (337 mg, 2.93 mmol, 2.0 eq.) were added to a mixture of compound 6 (1.1 g, 1.46 mmol, 1.0 eq.) and dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 1 hour. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (50 mL) and extracted with dichloromethane (50 mL × 3). The organic layers were combined, washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 7 (1.21 g, 100%) as a yellow oil, which was used directly in the next reaction without further purification.

[0798] Step 5:

[0799] Under a nitrogen atmosphere, sodium azide (190 mg, 2.92 mmol, 2.0 eq.) was added to a mixture of compound 7 (1.21 g, 1.46 mmol, 1.0 eq.) and N,N-dimethylformamide (15 mL). The reaction mixture was stirred at 100 °C for 16 h. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic layers were combined, washed with brine (3 × 70 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 8 (1.05 g, 92.7%) as a yellow oil.

[0800] LCMS:Rt:1.305min;MS m / z(ELSD):777.4[M+H] + ;

[0801] Step 6:

[0802] Palladium on carbon (300 mg) was added to a methanol (20 mL) mixture of compound 8 (1.05 g, 1.35 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The catalyst was removed by diatomaceous earth filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane containing 10% methanol, to give compound 9 (589 mg, 63%) as a yellow oil.

[0803] Step 7:

[0804] Under a nitrogen atmosphere, compound 10 (89 mg, 0.65 mmol, 1.0 eq.) was added to a mixture of compound 9 (489 mg, 0.65 mmol, 1.0 eq.) and 95% ethanol (0.5 mL). The reaction mixture was stirred at 70 °C for 16 hours. Thin-layer chromatography (ethyl acetate / tetrahydrofuran = 5 / 1.5) showed that the reaction was complete and a new main spot was observed. The reaction was quenched by adding water (50 mL) and extracted with ethyl acetate (70 mL × 3). The organic layers were combined, washed with brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with a mixture of petroleum ether containing 70% ethyl acetate / tetrahydrofuran (1:5)) and preparative high-performance liquid chromatography to give compound HI-19-137 (103.78 mg, 18.8%) as a yellow oil.

[0805] LCMS:Rt:1.206min;MS m / z(ELSD):851.0[M+H] + ;

[0806] CAD:92.36%purity.Rt:19.926min

[0807] 1 H NMR (400MHz, CDCl3) δ6.52 (s, 2H), 5.16 (d, J = 9.2Hz, 1H), 4.86 (p, J = 6.2Hz, 1H),4.22(s,4H),3.91–3.80(m,1H),2.77(t,J=6.9Hz,2H),2.36(d,J=53.2 Hz,6H),2.28(t,J=7.5Hz,2H),2.19–2.14(m,2H),1.62(dd,J=13.9,7.0Hz, 6H), 1.51 (dd, J=11.4, 6.2Hz, 8H), 1.36–1.24 (m, 52H), 0.90–0.85 (m, 12H).

[0808] Example 26

[0809] Step 1:

[0810] To a mixture of compound 1 (30.0 g, 174.4 mmol, 1.0 eq.) and toluene (600 mL), vinyl acetate (67.5 g, 784.8 mmol, 4.5 eq.) and Novozymes lipase 435 (15.0 g, 63.0 mmol, 0.36 eq.) were added. The mixture was stirred overnight at room temperature under nitrogen protection. Thin-layer chromatography (TLC, petroleum ether / ethyl acetate = 20 / 1) showed that the reaction was complete and two new main spots were observed. The mixture was filtered through diatomaceous earth, and the filtrate was washed with water (200 mL) and saturated brine (2 × 200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (using 0-5% ethyl acetate in petroleum ether as eluent) to obtain compound 2A (12g, purity 40%) and compound 2B (19g, purity 50.9%), both of which were colorless oily substances.

[0811] 1H NMR1(2A)(400MHz, CDCl3)δ7.26(s,1H),3.52(dd,J=7.3,4.6Hz,1H),1.58–1.15(m,19H),0.91(dt,J=13.7,7.2Hz,6H),-0.00(s,1H).

[0812] 1H NMR2(2B)(400MHz, CDCl3)δ4.86–4.74(m,1H),2.04(s,3H),1.59–1.54(m,2H),1.26(s,14H),0.87(t,J=7.2Hz,6H).

[0813] Step 2:

[0814] Compound 2B (19 g, 88.79 mmol, 1.0 eq.) was dissolved in sodium methoxide / methanol solution (30%, 100 mL) and stirred at room temperature for 16 hours. Thin-layer chromatography (TLC, petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was concentrated to remove methanol, and water (100 mL) was added to the residue, followed by extraction with petroleum ether (100 mL × 3). The organic phases were combined, washed with water (100 mL) and saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether with 0%–5% ethyl acetate) to give compound 4 (16.7 g, 97% yield) as a pale yellow oil.

[0815] 1H NMR (400MHz, CDCl3) δ3.51 (dd, J=11.5, 4.6Hz, 1H), 1.51–1.44 (m, 2H), 1.43–1. 38(m,2H),1.28(d,J=2.9Hz,12H),0.94(t,J=7.5Hz,3H),0.88(t,J=6.8Hz,3H).

[0816] Step 3:

[0817] To a mixture of compound 4 (20.0 g, 116.28 mmol, 1.0 eq.) and compound 3 (28.5 g, 127.9 mmol, 1.1 eq.) in dichloromethane (500 mL), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 28.9 g, 151.2 mmol, 1.3 eq.) and 4-dimethylaminopyridine (DMAP, 2.9 g, 23.3 mmol, 0.2 eq.) was added. The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. Thin-layer chromatography (TLC, petroleum ether:ethyl acetate = 20 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was washed with water (300 mL) and saturated brine (2 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 3% ethyl acetate in petroleum ether as eluent) to give compound 5 (39 g, yield 89.2%), which was a transparent oil.

[0818] 1H NMR (400MHz, CDCl3) δ4.86–4.76(m,1H),3.39(t,J=6.8Hz,2H),2.28(t,J=7.5Hz,2H),1.91–1.79(m,2H),1.63(dd,J= 8.9,6.6Hz,2H),1.58–1.47(m,4H),1.46–1.39(m,2H),1.36–1.31(m,4H),1.25(t,J=7.1Hz,12H),0.91–0.82(m,6H).

[0819] Step 4:

[0820] To a mixture of compound 6 (16 g, 35.16 mmol, 1.0 eq.) and cyclopentyl methyl ether / acetonitrile (350 mL, 1 / 1 v), potassium iodide (KI, 11.7 g, 70.33 mmol, 2.0 eq.), potassium carbonate (K₂CO₃, 24.3 g, 175.8 mmol, 5.0 eq.), and compound 5 (19.8 g, 52.75 mmol, 1.5 eq.) were added. The reaction mixture was stirred at 90 °C for 24 h under nitrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0–5% methanol in dichloromethane as eluent) to give compound 7 (23 g, 87.5% yield) as a yellow oil.

[0821] 1H NMR (400MHz, CDCl3) δ4.89–4.77(m,2H),3.84–3.74(m,2H),2.72–2.63(m,2H),2.49–2.38(m,4H),2.28(td,J=7.5,3.9 Hz,4H),1.72–1.68(m,2H),1.65–1.56(m,6H),1.51(dd,J=12.9,6.0Hz,10H),1.35–1.21(m,48H),0.92–0.80(m,12H).

[0822] Step 5:

[0823] Triethylamine (TEA, 4.6 g, 45.9 mmol, 1.5 eq.) and methanesulfonyl chloride (MSCl, 4.2 g, 36.8 mmol, 1.2 eq.) were added to a mixture of compound 7 (23 g, 30.6 mmol, 1.0 eq.) and dichloromethane (350 mL). The reaction mixture was stirred at 0 °C for 1 hour under argon protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was washed with water (3 × 100 mL) and saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Concentration under reduced pressure gave compound 8 (21 g, crude product) as a yellow oil.

[0824] Step 6:

[0825] Sodium azide (NaN3, 5.32 g, 81.81 mmol, 3.0 eq.) was added to a mixture of compound 8 (21 g, 27.27 mmol, 1.0 eq.) and N,N-dimethylformamide (DMF, 200 mL). The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (250 mL) and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0–5% methanol in dichloromethane as eluent) to give compound 9 (19 g, 89.6% yield) as a yellow oil.

[0826] 1H NMR (400MHz, CDCl3) δ4.89–4.76(m,2H),3.32(t,J=6.8Hz,2H),2.45(t,J=7.0Hz,2H),2.40–2.32(m,4H),2.28(td,J=7.5,3.8Hz,4H),1. 70(dd,J=13.8,6.9Hz,2H),1.59(dd,J=14.4,4.6Hz,6H),1.54–1.48(m,6H),1.43–1.37(m,4H),1.32–1.20(m,48H),0.91–0.83(m,12H).

[0827] Step 7:

[0828] Palladium on carbon (Pd / C, 2.3 g) was added to a mixture of compound 9 (10 g, 12.87 mmol, 1.0 eq.) and ethanol (150 mL). The reaction mixture was stirred at room temperature for 16 hours under hydrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The catalyst was removed by diatomaceous earth filtration, and the residue was purified by silica gel column chromatography (using 0-10% methanol in dichloromethane as eluent) to give compound 10 (7.5 g, 77.3% yield) as a yellow oil.

[0829] LCMS:Rt:1.543min; MS m / z(ELSD):752.1[M+H] + ;

[0830] 1 H NMR (400MHz, CDCl3) δ4.89–4.75(m,2H),2.71(t,J=6.8Hz,2H),2.47–2.41(m,2H),2.39–2.33(m,4H),2.28(td,J=7. 5,3.9Hz,4H),1.61(d,J=3.8Hz,4H),1.57–1.48(m,10H),1.43–1.38(m,4H),1.32–1.23(m,48H),0.90–0.85(m,12H).

[0831] Step 8:

[0832] Compound 9 (181 mg, 1.33 mmol, 1.0 eq.) was added to a mixture of compound 10 (1.0 g, 1.33 mmol, 1.0 eq.) and ethanol (1.0 mL, 95%). The reaction mixture was stirred at room temperature for 0.5 h, and then heated to 70 °C and stirred for 16 h under nitrogen protection. Thin-layer chromatography (TLC, ethyl acetate:tetrahydrofuran = 3 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was concentrated to remove ethanol, and the residue was purified by silica gel column chromatography (using 40% ethyl acetate / tetrahydrofuran (3:1) in petroleum ether as eluent) to give a yellow oil (0.5 g), which was further purified by preparative high-performance liquid chromatography (Prep-HPLC) to give compound HI-19-R (199.4 mg, yield 17.6%) as a yellow oil.

[0833] LCMS:Rt:1.533min;MS m / z(ELSD):852.0[M+H] + ;

[0834] CAD:97.96% purity at ELSD; RT=8.518min in 25min.

[0835] 1 1H NMR (400MHz, CDCl3) δ6.69(s,2H),4.89–4.76(m,2H),4.20(d,J=1.7Hz,4H),2.73(t,J=7.1Hz,2H),2.45(t,J=6.9Hz,2H),2.42–2.32(m,4H),2.28(t d,J=7.5,4.1Hz,4H),1.62(dd,J=13.4,6.7Hz,6H),1.56–1.48(m,6H),1.4 3–1.37(m,4H),1.30(d,J=5.8Hz,12H),1.25(s,36H),0.89–0.84(m,12H).

[0836] Example 27

[0837] Step 1:

[0838] At 0 °C, 4-dimethylaminopyridine (DMAP, 2.298 g, 18.70 mmol, 0.2 eq.) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 26.759 g, 140.1 mmol, 1.5 eq.) were added to a mixture of compound 1 (25.0 g, 112.1 mmol, 1.2 eq.) and compound 1A (23.916 g, 93.4 mmol, 1.0 eq.) in dichloromethane (500 mL). The mixture was stirred overnight at room temperature under nitrogen protection. Thin-layer chromatography (TLC, petroleum ether / ethyl acetate = 20 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was washed with water (200 mL) and saturated brine (2 × 200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (using petroleum ether / ethyl acetate (1 / 0-20 / 1) as eluent) to give compound 3 (38.565 g, 90% yield) as a colorless oil.

[0839] 1H NMR (400MHz, CDCl3) δ4.86(p,J=6.4Hz,1H),3.40(t,J=6.8Hz,2H),2.28(t,J=7.6Hz,2H),1.90–1.80(m,2H),1.66 –1.58(m,2H),1.54-1.46(m,4H),1.46–1.40(m,2H),1.36–1.30(m,6H),1.28-1.22(m,22H),0.87(t,J=6.8Hz,6H).

[0840] Step 2:

[0841] Compound 1C (101.9 g, 1.36 mol, 25.0 eq.) was added to a mixture of compound 1 (25.0 g, 54.3 mmol, 1.0 eq.) and ethanol (15 mL). The mixture was stirred at 50 °C for 16 hours under nitrogen protection. Thin-layer chromatography (TLC, dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (200 mL) and extracted with ethyl acetate (150 mL × 3). The organic phases were combined, washed with saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using dichloromethane / methanol (1 / 0-20 / 1) as eluent) to give compound 4 (16.5 g, 67% yield) as a pale yellow oil.

[0842] 1H NMR (400MHz, CDCl3) δ4.90–4.82(m,1H),3.86–3.76(m,2H),2.90–2.85(m,2H),2.72(s,1H),2.60(t,J=7.1Hz,2H),2.27(t,J=7.5 Hz,2H),1.69(dt,J=10.9,5.4Hz,2H),1.63–1.57(m,2H),1.48(t,J=10.9Hz,6H),1.28(d,J=22.3Hz,30H),0.87(t,J=6.8Hz,6H).

[0843] Step 3:

[0844] To a mixture of compound 2 (55.0 g, 247.4 mmol, 1.15 eq.) and compound 2 (37 g, 215.1 mmol, 1.0 eq.) in dichloromethane (1000 mL), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 53.4 g, 279.6 mmol, 1.3 eq.) and 4-dimethylaminopyridine (DMAP, 5.3 g, 43.0 mmol, 0.2 eq.) was added. The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. Thin-layer chromatography (TLC, petroleum ether:ethyl acetate = 20 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was washed with water (400 mL) and saturated brine (2 × 400 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 3% ethyl acetate in petroleum ether as eluent) to give compound 3 (74 g, yield 91.5%), which was a transparent oil.

[0845] 1H NMR (400MHz, CDCl3) δ4.85–4.77(m,1H),3.40(t,J=6.8Hz,2H),2.29(t,J=7.5Hz,2H),1.89–1.80(m,2H),1.64(dd,J=14.7,7.3Hz,2H) ,1.55(t,J=3.6Hz,2H),1.53–1.49(m,2H),1.46–1.41(m,2H),1.34(dd,J=7.3,3.7Hz,4H),1.27(d,J=7.3Hz,12H),0.90–0.85(m,6H).

[0846] Step 4:

[0847] To a mixture of compound 4 (10.5 g, 23.08 mmol, 1.0 eq.) and cyclopentyl methyl ether / acetonitrile (250 mL, 1 / 1 v / v), potassium iodide (KI, 7.66 g, 46.15 mmol, 2.0 eq.), potassium carbonate (K₂CO₃, 15.9 g, 115.4 mmol, 5.0 eq.), and compound 3 (13.02 g, 34.62 mmol, 1.5 eq.) were added. The reaction mixture was stirred at 90 °C for 24 h under nitrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0–5% methanol in dichloromethane as eluent) to give compound 5 (11.5 g, 66.5% yield) as a yellow oil.

[0848] 1H NMR(400MHz, CDCl3) δ4.86(dd,J=12.5,6.2Hz,1H),4.80(dd,J=12.1,6.2Hz,1H),3.83–3.75(m,2H),2.72(s,2H),2.50(s,4H),2.28(td ,J=7.5,4.0Hz,4H),1.73(s,2H),1.61(s,4H),1.54–1.45(m,12H),1.31(s,10H),1.27(d,J=15.4Hz,38H),0.87(dt,J=7.4,3.8Hz,12H).

[0849] Step 5:

[0850] Triethylamine (TEA, 5.04 g, 49.93 mmol, 1.5 eq.) and methanesulfonyl chloride (MSCl, 4.55 g, 39.94 mmol, 1.2 eq.) were added to a mixture of compound 5 (25 g, 33.29 mmol, 1.0 eq.) and dichloromethane (300 mL). The reaction mixture was stirred at 0 °C for 1 hour under argon protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was washed with water (3 × 100 mL) and saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Concentration under reduced pressure gave compound 6 (21 g, crude product) as a yellow oil.

[0851] Step 6:

[0852] Sodium azide (NaN3, 5.32 g, 81.81 mmol, 3.0 eq.) was added to a mixture of compound 6 (21 g, 27.27 mmol, 1.0 eq.) and N,N-dimethylformamide (DMF, 200 mL). The reaction mixture was stirred at 100 °C for 16 h under nitrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The reaction was quenched with water (250 mL) and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using 0–5% methanol in dichloromethane as eluent) to give compound 7 (19 g, 89.6% yield) as a yellow oil.

[0853] LCMS:Rt:1.533min;MS m / z(ELSD):778.0[M+H] + ;

[0854] Step 7:

[0855] Palladium on carbon (Pd / C, 2.3 g) was added to a mixture of compound 7 (19 g, 24.45 mmol, 1.0 eq.) and ethanol (250 mL). The reaction mixture was stirred at room temperature for 16 hours under hydrogen protection. Thin-layer chromatography (TLC, dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new main spot was observed. The catalyst was removed by diatomaceous earth filtration, and the residue was purified by silica gel column chromatography (using 0–10% methanol in dichloromethane as eluent) to give compound 8 (15 g, 81.7% yield) as a yellow oil.

[0856] LCMS:Rt:1.541min;MS m / z(ELSD):752.1[M+H] + ;

[0857] 1 H NMR (400MHz, CDCl3) δ4.91–4.74(m,2H),2.73(t,J=6.8Hz,2H),2.49–2.43(m,2H),2.41–2.34(m,4H),2.28(td,J=7.5,3.9H z,4H),1.64–1.54(m,8H),1.54–1.48(m,6H),1.44–1.39(m,4H),1.30(d,J=6.2Hz,12H),1.25(s,37H),0.90–0.84(m,12H).

[0858] Step 8:

[0859] Compound 9 (181 mg, 1.33 mmol, 1.0 eq.) was added to a mixture of compound 8 (1.0 g, 1.33 mmol, 1.0 eq.) and ethanol (1.0 mL, 95%). The reaction mixture was stirred at room temperature for 0.5 h, and then heated to 70 °C and stirred for 16 h under nitrogen protection. Thin-layer chromatography (TLC, ethyl acetate:tetrahydrofuran = 3 / 1) showed that the reaction was complete and a new main spot was observed. The mixture was concentrated to remove ethanol, and the residue was purified by silica gel column chromatography (using 40% ethyl acetate / tetrahydrofuran (3:1) in petroleum ether as eluent) to give a yellow oil (0.5 g), which was further purified by preparative high-performance liquid chromatography (Prep-HPLC) to give compound HI-19-S (156.08 mg, yield 13.8%) as a yellow oil.

[0860] LCMS:Rt:1.264min;MS m / z(ELSD):857.7[M+H] + ;

[0861] CAD:95.21% purity at ELSD; RT=8.380min in 25min.

[0862] 1 1H NMR (400MHz, CDCl3) δ6.49(s,2H),4.96–4.75(m,2H),4.22(s,4H),2.78(t,J=6.7Hz,8H),2.28(td ,J=7.4,4.2Hz,4H),1.57(ddd,J=21.4,13.5,6.8Hz,20H),1.34–1.23(m,46H),0.92–0.82(m,12H).

[0863] Example 28

[0864] Step 1:

[0865] Compound 2 (1.515 g, 4.342 mmol), potassium carbonate (2.73 g, 19.535 mmol), and potassium iodide (1.31 g, 7.894 mmol) were added to a solution of compound 1 (1.8 g, 3.947 mmol) in acetonitrile / cyclopentyl methyl ether (15 mL / 15 mL). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as an eluent increasing from 0% to 10%, to give compound 3 (2.2 g, 77% yield) as a yellow oil.

[0866] LCMS:Rt:2.256min;MS m / z(ELSD):725.0[M+H] + ;

[0867] Step 2:

[0868] Triethylamine (768 mg, 7.598 mmol) and methanesulfonyl chloride (696 mg, 6.077 mmol) were added to a dichloromethane (30 mL) solution of compound 3 (2.2 g, 3.309 mmol), and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed a new main spot. The reaction was quenched by adding water (70 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with 5% sodium bicarbonate solution (70 mL) and brine (70 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 4 (2.5 g, 94.2% yield), a colorless oil, which was used directly in the next reaction.

[0869] Step 3:

[0870] Sodium azide (405 mg, 6.234 mmol) was added to a solution of compound 4 (2.5 g, 3.117 mmol) in N,N-dimethylformamide (40 mL), and the mixture was stirred at 100 °C for 16 h under an argon atmosphere. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched by adding water (70 mL) to the mixture, and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (150 mL × 2) and brine (150 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using methanol in dichloromethane as eluent from 0% to 10%, to give compound 5 (1.3 g, yield 55.7%) as a brown oil.

[0871] LCMS:Rt:1.358min;MS m / z(ELSD):750.0[M+H] + ;

[0872] Step 4:

[0873] Palladium on carbon (250 mg, 10% by weight) was added to a tetrahydrofuran / methanol (10 mL / 10 mL) solution of compound 5 (1 g, 1.33 mmol), and the mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere (30 psi). Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane solution of methanol (0.05% ammonia, increasing from 0% to 10%) as eluent to give compound 6 (0.6 g, 62.4% yield) as a colorless oil.

[0874] LCMS:Rt:1.818min;MS m / z(ELSD):723.6[M+H] + ;

[0875] Step 5:

[0876] Compound 7 (95 mg, 0.691 mmol) was added to a 0.5 mL ethanol solution of compound 6 (500 mg, 0.691 mmol). The mixture was stirred at room temperature for 0.5 h, then stirred overnight at 70 °C. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The reaction was quenched with water (50 mL) and extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using petroleum ether with an ethyl acetate / tetrahydrofuran (5 / 3) ratio increased from 0% to 50% as eluent) and preparative high-performance liquid chromatography (pre-HPLC) to give compound HI-162 (86.36 mg, yield 15.2%) as a colorless oil.

[0877] LCMS:Rt:1.352min;MS m / z(ELSD):823.9[M+H] + ;

[0878] CAD:93.45%purity.Rt:19.218min

[0879] 1 H NMR (400MHz, CDCl3) δ6.76(s,2H),4.89–4.81(m,1H),4.26–4.13(m,4H),4.04(t,J=6.8Hz,2H),2.73(t,J=7.2Hz,2H),2.42(t,J =7.2Hz,2H),2.39–2.31(m,4H),2.31–2.23(m,4H),1.65–1.55(m,8H),1.53–1.44(m,4H),1.41–1.21(m,52H),0.91–0.81(m,9H).

[0880] Example 29

[0881] Step 1:

[0882] Under nitrogen protection, compound 2 (2.4 g, 10.78 mmol), 4-dimethylaminopyridine (240 mg, 1.95 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.4 g, 12.74 mmol) were added fractionally to a solution of compound 1 (2.5 g, 9.8 mmol) in dichloromethane (50 mL) at 0 °C. The mixture was stirred at room temperature for 16 hours. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. Dichloromethane (30 mL) was added to the mixture, and the mixture was washed with water (50 mL) and brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% ethyl acetate in petroleum ether) to give compound 3 (4.4 g, 87.3% yield) as a colorless oil.

[0883] LCMS:Rt:1.082min;MS m / z(ELSD):455.9[M+H] +

[0884] 1 H NMR (400MHz, CDCl3) δ5.05(d,J=9.2Hz,1H),3.90(s,1H),3.39(t,J=6.8Hz,2H),2.15(t,J=7.5Hz,2H),1.84(dd,J =14.5,7.0Hz,2H),1.64(d,J=6.4Hz,2H),1.44(dd,J=12.9,6.2Hz,4H),1.35–1.22(m,30H),0.87(t,J=6.8Hz,6H).

[0885] Step 2:

[0886] Compound 4 (16 g, 213 mol, 20.0 eq.) was added to a 5 mL ethanol solution of compound 3 (4.4 g, 9.6 mmol, 1.0 eq.). The mixture was stirred at 50 °C for 16 h. Thin-layer chromatography (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was concentrated to remove ethanol. Water (50 mL) was added to the residue and extracted with ethyl acetate (30 mL × 3). The organic layers were combined, washed with water (40 mL) and brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 5 (3.2 g, 73.6% yield) as a white solid.

[0887] LCMS:Rt:1.082min;MS m / z(ELSD):455.9[M+H] +

[0888] 1H NMR (400MHz, CDCl3) δ5.27(d,J=9.0Hz,1H),3.89(s,1H),3.84–3.78(m,2H),2.93–2.85(m,2H),2.62(t,J=7.0Hz,2H),2. 16–2.12(m,2H),1.73–1.69(m,2H),1.64–1.60(m,2H),1.47(d,J=8.1Hz,4H),1.30–1.23(m,26H),0.87(t,J=6.8Hz,6H).

[0889] Step 3:

[0890] Potassium carbonate (1.5 g, 11 mmol, 1.0 eq.) and potassium iodide (730 mg, 4.4 mmol, 2.0 eq.) were added to a cyclopentyl methyl ether / acetonitrile (1 / 1, 20 mL) solution of compound 6 (1.0 g, 2.2 mmol, 1.0 eq.) and compound 5 (1 g, 2.66 mmol, 1.2 eq.). The mixture was stirred at 95 °C for 48 h under argon protection. LC-MS showed the formation of the target product. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 7 (1.2 g, 72.7% yield) as a yellow oil.

[0891] LCMS:Rt:1.245min;MS m / z(ELSD):752.1[M+H] +

[0892] Step 4:

[0893] Under nitrogen protection, methanesulfonyl chloride (320 mg, 2.8 mmol, 1.5 eq.) was added to a mixture of compound 7 (1.4 g, 1.86 mmol, 1.0 eq.) and triethylamine (376 mg, 3.72 mmol, 2.0 eq.) in dichloromethane (20 mL) at 0 °C. The mixture was allowed to return to room temperature and stirred for 16 hours. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. Dichloromethane (10 mL) was added to the mixture. The mixture was washed with water (10 mL) and brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give compound 8 (1.3 g, 92.9% yield) as a clear yellow oil, which was used directly in the next reaction without further purification.

[0894] Step 5:

[0895] Sodium azide (329 mg, 5.06 mmol, 3.0 eq.) was added to a solution of compound 8 (1.3 g, 1.69 mmol, 1.0 eq.) in N,N-dimethylformamide (15.0 mL). The mixture was stirred at 100 °C for 16 h under nitrogen protection. Thin-layer chromatography (dichloromethane:methanol = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched by adding water (20 mL) and extracted with ethyl acetate (15 mL × 3). The organic layers were combined, washed with brine (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 9 (1 g, 76.9% yield) as a yellow oil.

[0896] LCMS:Rt:1.318min;MS m / z(ELSD):777.0[M+H] + ;

[0897] 1H NMR (400MHz, CDCl3) δ5.13(d,J=8.9Hz,1H),4.85–4.76(m,1H),3.89(s,1H),3.37(s,2H),2.49(s,6H),2.28(t,J=7.5Hz,2H) ,2.17–2.11(m,2H),1.63(d,J=6.7Hz,6H),1.52(dd,J=12.2,5.1Hz,8H),1.34–1.19(m,52H),0.87(dd,J=10.9,3.9Hz,12H).

[0898] Step 6:

[0899] Palladium on carbon (300 mg) was added to a 15 mL ethanol solution of compound 9 (1.0 g, 1.29 mmol). The reaction mixture was stirred overnight at 25 °C under a hydrogen atmosphere. LC-MS showed that the reaction was complete. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (0%–5% methanol in dichloromethane) to give compound 10 (780 g, 80.4% yield) as a yellow oil.

[0900] LCMS:Rt:1.412min;MS m / z(ELSD):751.1[M+H] + ;

[0901] 1 H NMR (400MHz, CDCl3) δ5.33 (d, J = 9.2Hz, 1H), 4.85–4.76 (m, 1H), 3.88 (s, 1H), 2.96(t,J=6.1Hz,2H),2.63(t,J=6.4Hz,2H),2.51–2.43(m,4H),2.28(t,J=7 .5Hz,2H),2.15(t,J=7.5Hz,2H),1.80–1.71(m,2H),1.62(d,J=6.9Hz,4H),1 .47(dd,J=16.5,5.9Hz,8H),1.28(d,J=26.0Hz,54H),0.87(t,J=6.7Hz,12H).

[0902] Step 7:

[0903] Compound 11 (54.3 mg, 0.39 mmol) was added to a 0.3 mL solution of compound 10 (300 mg, 0.39 mmol) in ethanol. The mixture was stirred at room temperature for 30 min under nitrogen protection. The reaction was then heated to 70 °C and maintained for 16 h. LC-MS showed the formation of the target product. The mixture was concentrated to remove ethanol. The residue was purified by preparative high-performance liquid chromatography to give HI-621-2 (19.16 mg, 5.6% yield) as a colorless oil.

[0904] LCMS:Rt:1.225min;MS m / z(ELSD):851.1[M+H] + ;

[0905] CAD:88.78% purity at ELSD; RT=7.151min in 25min.

[0906] 1H NMR(400MHz, CDCl3)δ6.46(s,2H),5.15(d,J=9.0Hz,1H),4.84–4.78(m,1H),4.24(s,4H),3.90(s,1H),2.80(s,6H),2.29(t,J=7.4Hz ,2H),2.15(t,J=7.5Hz,2H),1.95–1.84(m,2H),1.51(dd,J=19.4,12.3Hz,12H),1.33(s,12H),1.25(s,42H),0.87(t,J=6.5Hz,12H).

[0907] Example 30: Preparation and efficacy testing of mRNA sequence formulations encapsulated in lipid nanoparticles (LNPs).

[0908] LNP nanoparticles can be prepared by rapidly mixing two fluid streams, namely an aqueous phase and an alcohol phase, using a rapid mixing method such as a microfluidic device. One of the two fluid streams contains an aqueous solution of mRNA, and the other is an ethanol organic phase containing dissolved lipid components.

[0909] Taking the ionizable lipids in the aforementioned examples as an example, 50 mol% ionizable lipids were mixed with 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG and dissolved in an ethanol solution. DSPC, cholesterol, and PEG-DMG-2000 were all purchased from Xiamen Sinobond Biotechnology Co., Ltd. The mRNA was dissolved in acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was either mRNA encoding luciferase (Luc) or eGFP mRNA encoding green fluorescent protein, wherein each uridine was replaced with N1-methylpseudouridine. The molar ratio of the ionizable lipid molecules to the bases in the mRNA solution was defined as the N / P ratio. In this example, the N / P ratio was 6:1. During mixing in a microfluidic device, the ratio of the aqueous phase to the ethanol phase was controlled at 3:1. After preparation by rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa) or tangential flow filtration (TFF) device. The LNP was aseptically filtered using a 0.22 μM filter and stored at 4°C for further use.

[0910] DLS characterization: The hydrodynamic diameter and polydispersity index (PDI) of the LNP were measured using Malvern particle size high-throughput dynamic light scattering (DLS) (Zetasizer Lab, Malvern Panalytical). The LNPs were diluted to appropriate concentrations with 1×PBS and analyzed, as shown in Table 1.

[0911] mRNA concentration and LNP encapsulation efficiency assay: The mRNA concentration was determined using the Qubit-iT RiboGreen kit according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring the mRNA concentration in uncleaved and cleaved LNPs, as detailed in Table 1.

[0912] Table 1

[0913] mRNA expression level assay:

[0914] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, the mice in the sample group were given mRNA-LNP by intramuscular injection or tail vein injection, respectively, at a dose of 0.25mg / kg. 3h, 6h and 24h after administration, 200μL of D-luciferin substrate (15mg / ml) was injected intraperitoneally into each group of mice. 10min after substrate injection, the bioluminescent signal of luciferase was acquired by mouse in vivo imaging spectrometry (IVIS). The signal intensity results are shown in Table 2.

[0915] Table 2

[0916] Example 31: Preparation of mRNA-LNP formulations with different N / P ratios

[0917] 50 mol% HI-19 lipids were mixed with 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG and dissolved in ethanol. DSPC, cholesterol, and PEG-DMG-2000 were purchased from Xiamen Sinobond Biotechnology Co., Ltd. The mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was the mRNA encoding luciferase (Luc), in which each uridine was replaced with N1-methylpseudouridine. The molar ratio of ionizable lipid molecules to bases in the mRNA solution was defined as the N / P ratio. In this example, the N / P ratios were 3:1, 5:1, and 7:1. During microfluidic mixing, the ratio of the aqueous phase to the ethanol phase was controlled at 3:1. After preparation by rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa) or tangential flow filtration (TFF) device. The LNPs were aseptically filtered using a 0.22 μM filter and stored at 4°C for further use. See Table 3 for details.

[0918] Table 3

[0919] Example 32: A549 cell transfection with mRNA-LNP formulations with different N / P ratios

[0920] One day before the transfection experiment, A549 cells were seeded in 96-well plates and incubated for 24 hours in an incubator (cells were purchased from Fenghui Biotechnology). Cell status was checked before transfection to avoid dead cells, and suitable areas were selected and marked with a marker. Each sample corresponded to three wells to ensure the reproducibility and accuracy of subsequent experimental data. 100 ng of Luciferase mRNA was administered to the samples, with the SM-102 sample serving as the control group. The ionizable lipid molecule used in the SM-102 control group was SM-102, with other lipid components including 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. The N / P ratio in the SM-102 control group was 6:1.

[0921] When administering samples, use 100 ng of Luciferase mRNA as the dosage. For transfection, first take 27 μL of culture medium, then add 3 μL of mRNA-LNP sample to the corresponding well. Simultaneously, add 10 μL of the 10-fold diluted sample to the other wells, gently pipetting while adding to ensure thorough mixing, taking care to avoid splashing to the bottom. After transfection, cap the plate and gently tap around the edges to prevent spillage. Finally, incubate the plate in an incubator for 24 hours.

[0922] The Beyotime Firefly Luciferase Reporter Gene Detection Kit was used for testing. The procedure was followed according to the product instructions. The results are shown in Table 4.

[0923] Table 4:

[0924] Experiments have shown that the ionizable lipid compound of the present invention can effectively deliver nucleic acid molecules, with a delivery efficiency several times higher than that of the existing molecule SM-102.

[0925] Example 33: Study of formulations containing adjuvant molecules

[0926] The molecular structure of the adjuvant is as follows:

[0927] The preparation method is the same as in Example 31.

[0928] One or more adjuvant lipid molecules were added to an LNP formulation based on 50 mol% HI-19, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. The amount of the additional adjuvant was 10% of the mass of the ionizable lipid HI-19.

[0929] The specific steps are as follows:

[0930] 20 mg / mL solutions of HA lipids, ionizable lipids (HI-19), DSPC, cholesterol, and PEG-DMG were prepared using ethanol solution. 50 mol% ionizable lipids were mixed with 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG, and additional HA lipids (HA lipids at 10% of the mass of ionizable lipids) were added, resulting in a final total mass of ionizable lipids, cofactor phospholipids, cholesterol, and PEG lipids of 10 mg / mL. DSPC, cholesterol, and PEG-DMG-2000 were purchased from Xiamen Sinobond Biotechnology Co., Ltd. mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was the mRNA encoding the human papillomavirus E7 antigen. The molar ratio of ionizable lipid molecules to bases in the mRNA solution was defined as the N / P ratio. In this example, the N / P ratio was 6:1. During mixing in a microfluidic device, the ratio of the aqueous phase to the ethanol phase was controlled at 3:1. After preparation via rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa) or tangential flow filtration (TFF) device. The LNP was then aseptically filtered using a 0.22 μM filter and stored at 4°C for further use.

[0931] The encapsulation efficiency of the five LNP-mRNA components was all above 90%. See Table 5 for details.

[0932] Table 5

[0933] Example 34

[0934] Preparation of enCureLip LNP: 42.03 mol% HI-19 lipid was mixed with 6.44 mol% DEPC, 49.99 mol% cholesterol, and 1.54 mol% PEG-DMG and dissolved in ethanol. DEPC was purchased from Avitol (Shanghai) Pharmaceutical Technology Co., Ltd. Cholesterol and PEG-DMG-2000 were purchased from Xiamen Sinobond Biotechnology Co., Ltd. mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was the mRNA encoding luciferase (Luc), in which uridines were replaced with N1-methylpseudouridines. The molar ratio of ionizable lipid molecules (HI-19 lipid) to bases in the mRNA solution was defined as the N / P ratio. In this example, the N / P ratios were 3:1, 4:1, 5:1, 6:1, and 7:1. During mixing in the microfluidic device, the ratio of the aqueous phase to the ethanol phase was controlled at 3:1. After preparation via rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa).

[0935] The encapsulation efficiency of different N / P formulations was tested according to the method in Example 30, as shown in Table 6. Under low N / P ratio conditions, the Hi-19 formulation exhibited a higher encapsulation efficiency compared to the classic SM-102 formulation.

[0936] Table 6

[0937] The encapsulation efficiency data for SM-102 comes from the article "Minimizing the ratio of ionizable lipid in lipid nanoparticles for in vivo base editing," National Science Review, Volume 11, Issue 6, June 2024, nwae135, https: / / doi.org / 10.1093 / nsr / nwae135.

[0938] Example 35: Testing the in vivo expression effect of the formulation with an N / P ratio of 6:1 in Example 34 (i.e., B5 formulation LNP).

[0939] The ionizable lipid molecule used in the SM-102 control group was SM-102, with other lipid components including 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG-2000. The N / P ratio in the SM-102 control group was 6:1. SM-102, DSPC, cholesterol, and PEG-DMG-2000 were all purchased from Xiamen Sinobond Biotechnology Co., Ltd. The mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was the mRNA encoding luciferase (Luc), in which uridines were replaced with N1-methylpseudouridines. During microfluidic mixing, the ratio of the aqueous phase to the alcohol phase was controlled at 3:1. After preparation via rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa). The LNP was aseptically filtered using a 0.22 μM filter and stored at 4°C for further use.

[0940] (1) Effect of tail vein injection on in vivo expression of luciferase in animals:

[0941] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, they were randomly divided into two groups of 4 mice each. The mice in both groups were injected intravenously with B5 formulation LNP and SM-102 LNP, respectively, at a dose of 0.25 mg / kg. At 6, 24, and 48 hours after intravenous injection, each group of mice was intraperitoneally injected with 200 μL of D-luciferin substrate (15 mg / ml). Ten minutes after substrate injection, the luciferase bioluminescence signal in the mice was acquired using an in vivo imaging system (IVIS). The results are shown in Table 7 (unit: P / S). Compared with the classic SM-102 formulation, the B5 formulation significantly improved the luciferase expression effect of this combination.

[0942] Table 7

[0943] (2) Effects of intramuscular administration of luciferase on in vivo expression in animals

[0944] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, they were randomly divided into two groups of 4 mice each. The two groups of mice were intramuscularly injected with B5 formulation LNP and SM-102 LNP, respectively, at a dose of 0.25mg / kg. 3h, 6h, 12h, 24h, 48h, 72h, and 96h after intramuscular administration, 200μL of D-luciferin substrate (15mg / ml) was injected intraperitoneally into each group of mice. Ten minutes after substrate injection, the bioluminescent signal of luciferase was acquired using a mouse in vivo imaging system (IVIS). The results are shown in Table 8 (unit: P / S). According to the results in Table 8, it can be concluded that, compared with SM-102, HI-19 lipid formulation B5 showed higher protein expression levels and longer protein expression time after intramuscular administration to mice.

[0945] Table 8

[0946] Example 36: Stability comparison of the five-component formulation LNP of HI-19 molecule B5-A at -20℃ with the classic BNT formulation LNP.

[0947] The ionizable lipid molecule used in the BNT control group was 47 mol% ALC-0315, and other lipid components were 10 mol% DSPC, 41.5 mol% cholesterol, and 1.5 mol% PEG-DMG-2000. The N / P ratio in the BNT control group was 6:1. DEPC was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd. ALC-0315, DSPC, cholesterol, and PEG-DMG-2000 were all purchased from Xiamen Sinobond Biotechnology Co., Ltd. The mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was the mRNA encoding luciferase (Luc), in which each uridine was replaced with N1-methylpseudouridine. During mixing in a microfluidic device, the ratio of the aqueous phase to the alcohol phase was controlled at 3:1. After preparation by rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer through an ultrafiltration tube (100 kDa). Stability studies were conducted by aseptically filtering LNPs using a 0.22 μM filter and storing them at -20 °C.

[0948] Organic phase preparation of the B5-A five-component formulation sample: 20 mg / mL solutions of HA-TLR-2-A lipid, HI-19, DEPC, cholesterol, and PEG-DMG were prepared using ethanol. 42.03 mol% ionizable lipid was mixed with 6.44 mol% DEPC, 49.99 mol% cholesterol, and 1.54 mol% PEG-DMG, with additional HA lipid (HA-TLR-2-A lipid mass equal to 10% of HI-19 mass) added, resulting in a final total mass of ionizable lipid, cofactor phospholipid, cholesterol, and PEG lipid of 10 mg / mL. The mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was encoding luciferase (Luc), with each uridine replaced by N1-methylpseuuridine (N / P = 6). During microfluidic mixing, the ratio of aqueous to ethanol phase was controlled at 3:1. After preparation via rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa). The B5-A LNP was aseptically filtered using a 0.22 μM filter and stored at -20°C for stability studies.

[0949] Organic phase preparation of the BNT-A five-component formulation sample: 20 mg / mL solutions of HA-TLR-2-A lipid, ALC-0315, DSPC, cholesterol, and PEG-DMG were prepared using ethanol. 47 mol% ionizable lipid was mixed with 10 mol% DEPC, 41.05 mol% cholesterol, and 1.5 mol% PEG-DMG, with additional HA lipid added (HA-TLR-2-A lipid mass equal to 10% of ALC-0315 mass), resulting in a final total mass of ionizable lipid, cofactor phospholipids, cholesterol, and PEG lipid of 10 mg / mL. The mRNA was dissolved in citrate or acetate buffer at pH 4.0 as the aqueous phase. The mRNA used was encoding luciferase (Luc), with each uridine replaced by N1-methylpseudouridine (N / P = 6). During microfluidic mixing, the ratio of aqueous to ethanol phase was controlled at 3:1. After preparation via rapid mixing, the ethanol content in the mRNA-LNP was reduced and dispersed into the target buffer using an ultrafiltration tube (100 kDa). The BNT-A LNP was then aseptically filtered using a 0.22 μM filter and stored at -20°C for stability studies.

[0950] As shown in Table 9 below, in the 9-month stability study, the B5 formulation showed no changes in particle size, PDI, and encapsulation efficiency, while the control group BNT formulation showed a continuous increase in particle size and PDI, and a continuous decrease in encapsulation efficiency. Similarly, the B5-A formulation showed no changes in particle size, PDI, and encapsulation efficiency, while the control group BNT-A formulation showed a continuous increase in particle size and PDI, and a continuous decrease in encapsulation efficiency.

[0951] Table 9

[0952] Example 37

[0953] Jurkat cells were loaded at 1×10 5 Cells were seeded at a density of cells / well in 24-well plates. LNP formulations encapsulating EGFP-Flag mRNA (using the classic SM-102LNP and B5 LNP formulations as in Example 35, with an N / P ratio of 6) were added to achieve final mRNA concentrations of 0.1 μg / mL, 0.5 μg / mL, and 1.0 μg / mL, respectively. A control group was also included, transfected with EGFP-Flag mRNA using Lipo3000 at the same concentration as described above. Except for the Blank group, each experiment had two replicates. After transfection, cells were cultured at 37°C and 5% CO2 for 24 hours. After culture, cells from each well were collected into 1.5 mL centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed twice with PBS, then resuspended in 200 μL of PBS and filtered through a 100-mesh filter to obtain a single-cell suspension. Finally, the transfection rate and fluorescence intensity of cells were detected using a CytExpert flow cytometer at a 525nm channel.

[0954] The B5 LNP formulation showed significantly higher transfection efficiency for Jurkat T cells at low doses of 0.1 μg / mL and 0.5 μg / mL compared to the classic SM-102 formulation.

[0955] Table 10

[0956] Example 38: Screening of phospholipids and PEGylated lipids in the lipid components of nanoparticle compositions

[0957] The ethanol organic phase was prepared according to the lipid composition in Table 11. The molar percentages of each lipid component were 50% ionizable lipids (HI-19), 10% phospholipids, 38.5% cholesterol, and 1.5% PEGylated lipids, with a total lipid concentration of 10 mg / mL. Luciferase mRNA was used as the target encapsulation sequence. An aqueous solution of luciferase mRNA was prepared using pH 4 acetate buffer, and the final concentration of the acetate buffer was adjusted to 20 mM. The mRNA concentration was determined based on a nitrogen-to-phosphorus ratio (N / P) of 6 between the organic and aqueous phases. The lipid nanoparticles were prepared using a microfluidic process. The organic and aqueous solutions were uniformly and rapidly mixed in a microfluidic device at a volume ratio of 1:3, with a total mixing flow rate of 20 mL / min. The initial microfluidically mixed sample was immediately diluted with 15 mM acetate buffer, and ultrafiltration was performed using RC membrane ultrafiltration centrifuge tubes (MWCO = 100 kDa) to remove ethanol from the sample. The sample was then diluted again with 10 mM Tris buffer (10% sucrose), ultrafiltration was performed, and the sample was concentrated to the target concentration of 0.1 mg / mL. The average particle size and polydispersity index of the sample were measured using a Malvern nanoparticle size potentiometer. The mRNA concentration and encapsulation efficiency in the sample were determined using a Ribogreen assay kit. The physicochemical properties of the final prepared sample are shown in Table 12.

[0958] Table 11. Lipid composition formulas of each sample in the study of phospholipids and PEGylated lipids.

[0959] Table 12 Characterization of physicochemical properties of different lipid samples

[0960] Example 39: In vivo pharmacodynamic expression of luciferase in animals of LNP samples with different combinations of phospholipids and ALC0159 in nanoparticle compositions (formulation samples 2-9 in Example 38).

[0961] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, they were randomly divided into 9 groups of 3 mice each. The mice were administered the drug via tail vein according to the grouping in Table 13, with a dosage of 10 μg / mouse. Six hours after the tail vein injection, each group of mice was intraperitoneally injected with 200 μL of D-luciferin substrate (15 mg / ml). Ten minutes after substrate injection, the bioluminescent signal of luciferase was acquired using a mouse in vivo imaging system (IVIS). The imaging results are shown in Table 14. As can be seen from the quantitative data of mouse luciferase bioluminescent signal in Table 14, except for the G7 group (using DPPG phospholipids), whose bioluminescent signal was weaker than the G1 control group, the bioluminescent signals of the other experimental groups were higher than the control group, with the strongest bioluminescent signals observed in G4 (DPPC) and G9 (DMPC). The results indicate that among the combinations using ALC0159 as the PEGylated lipid, DPPC and DMPC are most effective in enhancing luciferase expression.

[0962] The molar percentages of each lipid component in the formulation are 50% ionizable lipids, 10% phospholipids, 38.5% cholesterol, and 1.5% PEGylated lipids.

[0963] Table 13. Animal experimental dosing grouping information for different phospholipid-ALC0159 combinations.

[0964] Table 14 RNA expression levels in mice with different phospholipid and ALC0159 combinations.

[0965] Example 40: In vivo luciferase pharmacological expression in animals of LNP samples with different phospholipid combinations and DMG-PEG2000 in nanoparticle compositions (formulation samples 10-17 in Example 38).

[0966] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, they were randomly divided into 9 groups of 3 mice each. The mice were administered the drug via tail vein according to the grouping in Table 15, with a dosage of 10 μg / mouse. Six hours after the tail vein injection, each group of mice was intraperitoneally injected with 200 μL of D-luciferin substrate (15 mg / ml). Ten minutes after substrate injection, the bioluminescent signal of luciferase was acquired using a mouse in vivo imaging system (IVIS). The imaging results are shown in Table 16. According to the quantitative data of mouse luciferase bioluminescent signal in Table 16, the bioluminescent signal values ​​of group G6 (using DEPC) and group G2 (using DSPC) were closest to the control group. This result indicates that in the combination using DMG-PEG2000 as the PEGylated lipid, DEPC and DSPC can best improve the luciferase expression effect of this combination.

[0967] Table 15. Dosing grouping information for different phospholipid-DMG-PEG2000 combinations in animal experiments.

[0968] Table 16 RNA expression levels in mice with different phospholipid and DMG-PEG2000 combinations

[0969] Example 41: In vivo luciferase pharmacological expression in animals of LNP samples with different phospholipid combinations and DSPE-PEG2000 in nanoparticle compositions (formulation samples 18-25 in Example 38).

[0970] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used for the in vivo expression experiment of luciferase. After the mice passed the inspection and quarantine at the animal center and were fed for 3 days, they were randomly divided into 9 groups of 3 mice each. The mice were administered the drug via tail vein according to the grouping in Table 17, with a dosage of 10 μg / mouse. Six hours after the tail vein injection, each group of mice was intraperitoneally injected with 200 μL of D-luciferin substrate (15 mg / ml). Ten minutes after the substrate injection, the bioluminescence signal of luciferase was acquired using a mouse in vivo imaging system (IVIS). The imaging results are shown in Table 18. According to the quantitative data of mouse luciferase bioluminescence signal in Table 18, the bioluminescence signal values ​​of group G2 (using phospholipid DSPC) and group G9 (using phospholipid DMPC) were closest to the control group. This result indicates that in the combination using DSPE-PEG2000 as the PEGylated lipid, DMPC and DSPC can significantly improve the luciferase expression effect of this combination.

[0971] Table 17. Animal experimental dosing grouping information for different phospholipid-DSPE-PEG2000 compositions.

[0972] Table 18 RNA expression levels in mice with different phospholipid and DSPE-PEG2000 combinations

[0973] Example 42: Investigation of the molar percentage of each lipid component in the nanoparticle composition

[0974] To study the molar percentage of lipid components in the nanoparticle composition, the Design of Experiments (DOE), widely used in the pharmaceutical industry, was employed to design experiments and generate formulations for the molar percentage of lipid components. Four mixing factors were generated in the DOE software: ionizable lipids, phospholipids, structural lipids, and PEGylated lipids. In this example, HI-19 was selected as the ionizable lipid, DEPC as the phospholipid, cholesterol as the structural lipid, and DMG-PEG2000 as the PEGylated lipid. The screening ranges for the four mixing factors were set as follows: HI-19 (20-80 mol%), DEPC (3-35 mol%), cholesterol (10-60 mol%), and DMG-PEG2000 (0.1-5 mol%). A rapid and flexible space-filling design method was used to generate 28 lipid ratio formulations as shown in Table 19. Based on the lipid component ratios shown in the table and referring to the nanoparticle composition preparation method in Example 39, microparticle control technology and ultrafiltration centrifugation were used to prepare samples with different lipid ratios encapsulating luciferase mRNA. The average particle size and polydispersity index of the samples were measured using a Malvern nanoparticle size potentiometer, and the mRNA concentration and encapsulation efficiency in the samples were determined using a Ribogreen assay kit. The physicochemical properties of the final prepared samples are shown in Table 8.

[0975] Table 19 shows the results of DOE experimental design for four mixing factors with different molar percentages and their physicochemical properties.

[0976] Example 43: Investigation of the molar percentage of each lipid component in the nanoparticle composition

[0977] To study the molar percentage of lipid components in the nanoparticle composition, the Design of Experiments (DOE), widely used in the pharmaceutical industry, was employed to design experiments and generate formulations for the molar percentage of lipid components. Four mixing factors were generated in the DOE software: ionizable lipids, phospholipids, structural lipids, and PEGylated lipids. In this example, HI-19 was selected as the ionizable lipid, DEPC as the phospholipid, cholesterol as the structural lipid, and DMG-PEG2000 as the PEGylated lipid. The screening ranges for the four mixing factors were set as follows: HI-19 (30-55 mol%), DEPC (5-10 mol%), cholesterol (30-50 mol%), and DMG-PEG2000 (1-2 mol%). A rapid and flexible space-filling design method was used to generate 21 lipid ratio formulations as shown in Table 20. Based on the lipid component ratios shown in the table and referring to the nanoparticle composition preparation method in Example 39, samples with different lipid ratios encapsulating luciferase mRNA were prepared using microparticle control technology and ultrafiltration centrifugation. The average particle size and polydispersity index of the samples were measured using a Malvern nanoparticle size potentiometer, and the mRNA concentration and encapsulation efficiency in the samples were determined using a Ribogreen assay kit. The physicochemical properties of the final prepared samples are shown in Table 20.

[0978] Table 20 shows the results of DOE experimental design for four mixing factors with different molar percentages and their physicochemical properties.

[0979] Example 44: Evaluation of the effect of different ratios of nanoparticle compositions on luciferase expression in animals

[0980] In this embodiment, 7-week-old female BALB / c mice (18-22g) were used as experimental animals. After passing the inspection and quarantine at the animal center and being fed for 3 days, the mice were randomly divided into 14 groups of 3 mice each. They were administered the drugs according to the different formulation ratios (N / P = 6) shown in Table 21 via intramuscular injection at a dose of 5 μg / mouse. At 3, 6, 24, and 48 hours after administration, 200 μL of D-luciferin potassium salt (15 mg / ml) was injected intraperitoneally. Ten minutes after injection, the bioluminescence signal of each group was detected using an in vivo imaging system (IVIS). The imaging results are shown in Tables 22-23. According to the quantitative data of bioluminescence signals at different time points in Tables 22-23, the luciferase bioluminescence signal value of group G13 was higher than that of the other experimental groups. This result indicates that the lipid molar ratio of G13 is more conducive to increasing the expression level of luciferase in vivo using the HI-19 / DEPC / Chol / DMG-PEG2000 formulation.

[0981] Table 21 Animal Dosing Grouping Information for Nanoparticle Compositions with Different Ratios

[0982] Table 22 RNA expression levels in mice with different nanoparticle compositions.

[0983] Table 23 RNA expression levels in mice with different nanoparticle compositions.

[0984] Example of adjuvant molecule preparation:

[0985] Preparation Example 1:

[0986] Step 1:

[0987] Compound 1 (3 g, 12.35 mmol, 1.0 eq.) was dissolved in DCM (400 mL), followed by the addition of compound 2 (1.98 g, 12.35 mmol, 1.0 eq.) and TEA (3.74 g, 37.04 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 5:1) showed the reaction was complete, with a new major spot observed. The mixture was quenched with H2O (100 mL) and extracted with EA (100 mL × 3). The organic layers were combined, washed with saturated brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–30% EA in PE) to give compound 3 (4 g, 88.9%) as a yellow solid.

[0988] LCMS:Rt:2.063min;MS m / z(ELSD):367.2[M+H] + .

[0989] Step 2

[0990] Compound 1 (4 g, 10.93 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (40 mL), followed by the addition of Fe (1.84 g, 32.79 mmol, 3.0 eq.) and DIEA (1.77 g, 32.79 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 2:1) showed the reaction was complete, with a new major spot observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The organic layers were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (2.75 g, 74.9%) as a yellow solid.

[0991] LCMS:Rt:1.523min;MS m / z(ELSD):337.2[M+H] + ;

[0992] 1H NMR(400MHz, CDCl3)δ7.90(d,J=8.2Hz,1H),7.82(d,J=8.2Hz,1H),7.51–7.39 (m,2H),4.92(s,1H),4.29-3.65(m,3H),3.42(d,J=25.6Hz,4H),1.47(s,9H).

[0993] Step 3

[0994] A solution of compound 4 (2.75 g, 8.18 mmol, 1.0 eq.) and CH(OEt)3 (2.4 g, 16.37 mmol, 2.0 eq.) was stirred at 80 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed the reaction was complete, and a new major spot was observed. The mixture was quenched with H2O (100 mL) and extracted with EA (50 mL × 3). The organic layers were combined, washed with saturated brine (2 × 50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 5 (2.1 g, 74.2%) as a pale yellow solid.

[0995] LCMS:Rt:1.464min; MS m / z(ELSD):347.2[M+H] + ;

[0996] 1H NMR (400MHz, CDCl3) δ8.21-8,16m,2H),7.90(s,1H),7.76–7.63(m,2H),5.05(s,1H),4.83-4.75(m,2H),3.73-3.69(m,2H),1.46(s,9H).

[0997] Step 4:

[0998] A mixture of compound 5 (2.1 g, 6.25 mmol, 1.0 eq.) and NH3 (30 mL, NH3 in MeOH, 7 M.) was stirred at 140 °C for 48 h. The desired product was obtained by thin-layer chromatography (DCM:MeOH = 10 / 1). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 6 (900 mg, 45.5%) as a pale yellow solid.

[0999] LCMS:Rt:0.968 min; MS m / z(ELSD):328.2[M+H] + ;

[1000] Step 5:

[1001] Compound 6 (900 mg, 2.75 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and TFA (10 mL) was added at 0 °C and the reaction was carried out under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was quenched with TEA and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give compound 7 (610 mg, 96.4%) as a white solid.

[1002] LCMS:Rt:1.050min;MS m / z(ELSD):228.1[M+H] + ;

[1003] Step 6:

[1004] Compound 7 (210 mg crude, 0.93 mmol, 1.0 eq.) and compound 8 (553 mg, 1.20 mmol, 1.3 eq.) were dissolved in MeCN (10.0 mL), and K₂CO₃ (383 mg, 2.78 mmol, 3.0 eq.) and KI (154 mg, 0.93 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 h under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction mixture was quenched with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine (2 × 100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 9 (230 mg, 40.9%) as a yellow oil.

[1005] LCMS:Rt:1.301min;MS m / z(ELSD):608.5[M+H] + ;

[1006] Step 7:

[1007] Compound 9 (230 mg, 0.38 mmol, 1.0 eq.) and compound 10 (713 mg, 1.89 mmol, 5.0 eq.) were dissolved in MeCN (10.0 mL), and K₂CO₃ (157 mg, 1.14 mmol, 3.0 eq.) and KI (63 mg, 0.38 mmol, 1.0 eq.) were added. The mixture was stirred at 90 °C for 16 hours under an Ar atmosphere. TLC (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The mixture was quenched with H₂O (30 mL) and extracted with EA (30 mL × 3). The combined organic layers were washed with saturated brine (2 × 100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluted with 10% MeOH in DCM, to give a crude product as a yellow oil. The residue was purified by preparative high performance liquid chromatography (PreP-HPLC) to obtain HA-1 (33.85 mg, 4.9%) as a yellow oil.

[1008] LCMS: MS m / z (ELSD): 904.8 [M+H] + ;

[1009] 1H NMR (400MHz, CDCl3) δ8.02–7.89(m,3H),7.66–7.58(m,1H),7.49–7.42(m,1H),4.92–4.75(m,2H),4.56(t,J=6.0Hz,2H),2. 93(t,J=6.0Hz,2H),2.46–2.37(m,4H),2.32–2.22(m,4H),1.61–1.41(m,12H),1.33–1.15(m,53H),0.87(t,J=7.2Hz,12H).

[1010] Preparation Example 2:

[1011] Step 1:

[1012] A solution of compound 1 (1 g, 2.09 mmol, 1.0 eq.) was dissolved in DCM (10 mL), followed by the addition of TFA (10 mL). The mixture was stirred at 25 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated by hydration of NaHCO3 and the pH was adjusted to 8. The mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 2 (732 mg, 96%) as a white solid.

[1013] LCMS:Rt:1.311min;MS m / z(ELSD):379.1[M+H] + ;

[1014] Step 2:

[1015] A solution of compound 2 (260 mg, 0.69 mmol, 1.0 eq.) was dissolved in MeCN / CPME (2 mL, 1:1 v / v), followed by the addition of compound 3 (527 mg, 1.37 mmol, 2.0 eq.), KI (228 mg, 1.37 mmol, 2.0 eq.), and K₂CO₃ (475 mg, 3.44 mmol, 5.0 eq.). The mixture was stirred at 90 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H₂O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give HA-TLR-2-A (89 mg, 14%) as a yellow oil.

[1016] LCMS:Rt:1.305min; MS m / z(ELSD):915.6[M+H] + ;

[1017] CAD:97.55% purity at ELSD; RT=21.027min.

[1018] 1 H NMR (400MHz, DMSO) δ8.15-8.03(m,2H),7.64(t,J=7.6Hz,1H),7.52-7.45(m,2H),7.22(d, J=7.8Hz,1H),7.15(d,J=7.7Hz,1H),6.99(d,J=8.0Hz,1H),6.00(d,J=34.2Hz,2H),4.26( s,1H),4.02–3.88(m,4H),3.40(s,1H),2.99(d,J=6.7Hz,2H),2.95-2.85(m,2H),2.33-2. 15(m,6H),1.82-1.75(m,2H),1.59-1.35(m,12H),1.30–1.11(m,38H),0.89-0.75(m,9H).

[1019] Preparation Example 3:

[1020] Step 1:

[1021] A solution of compound 1 (4 g, 16.46 mmol, 1.0 eq.) was dissolved in DCM (40 mL), followed by the addition of compound 2 (3.88 g, 16.46 mmol, 1.0 eq.) and TEA (4.98 g, 49.38 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 1:1) showed that the reaction was complete and a new major spot was observed. The mixture was terminated with H2O (150 mL) and extracted with EA (100 mL × 3). The combined organic phases were washed with brine (2 × 100 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–70% EA in PE) to give compound 3 (5.645 g, 76.7%) as a yellow solid.

[1022] LCMS:Rt:1.867min; MS m / z(ELSD):443.2[M+H] + ;

[1023] Step 2:

[1024] A solution of compound 3 (5.645 g, 12.37 mmol, 1.0 eq.) was dissolved in EtOH / H₂O (60 mL, 5:1, v / v), followed by the addition of Fe (2.146 g, 38.31 mmol, 3.0 eq.) and NH₄Cl (2.07 g, 38.31 mmol, 3.0 eq.). The reaction mixture was stirred at 70 °C for 3 hours under a nitrogen atmosphere. Thin-layer chromatography (PE:EA = 1:3) showed that the reaction was complete and a new major spot was observed. The mixture was filtered, and the filter cake was washed with MeOH (150 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0%–60% EA in PE) to give compound 4 (3.698 g, 70.3%) as a yellow solid.

[1025] LCMS:Rt:1.727min;MS m / z(ELSD):413.2[M+H] + ;

[1026] Step 3: A solution of compound 4 (3 g, 7.28 mmol, 1.0 eq.) was dissolved in THF (50 mL), and then pyridine (2.88 g, 36.44 mmol, 5.0 eq.) and valeryl chloride (917 mg, 7.65 mmol, 1.05 eq.) were added at 0 °C under an argon atmosphere. The mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was terminated with H2O (100 mL) and extracted with EA (80 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with 10% MeOH in DCM to give compound 5 (3.6 g, 98%) as a pale yellow solid.

[1027] LCMS:Rt:1.702min;MS m / z(ELSD):497.2[M+H] + ;

[1028] 1H NMR (400MHz, CDCl3) δ7.93 (d, J=8.4Hz, 1H), 7.86 (d, J=8.4Hz, 1H), 7.61 (t, J= 7.6Hz,1H),7.38(t,J=7.6Hz,1H),7.29-7.15(m,4H),5.43(s,1H),4.90(s,1H ),4.73(d,J=4.9Hz,2H),4.32(d,J=5.6Hz,2H),2.41(t,J=7.6Hz,2H),1.75–1 .62(m,2H),1.46(s,9H),1.38(dt,J=14.2,7.2Hz,2H),0.93(t,J=7.3Hz,3H).

[1029] Step 4:

[1030] A solution of compound 5 (2.75 g, 8.18 mmol, 1.0 eq.) was dissolved in EtOH / H2O (20 mL), followed by the addition of K2CO3 (556 mg, 4.03 mmol, 2.0 eq.). The mixture was stirred at 55 °C for 16 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H2O (100 mL) and extracted with EA (80 mL × 3). The combined organic phases were washed with brine (2 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 6 (1.95 g, 73.6%) as a pale yellow solid.

[1031] LCMS:Rt:1.964min;MS m / z(ELSD):479.3[M+H] + ;

[1032] 1H NMR (400MHz, CDCl3) δ8.16(d,J=8.3Hz,1H),7.86(d,J=8.3Hz,1H),7.60(t,J=7.7Hz,1H),7.42(t,J=7.6Hz,1H),7.25(d,J=8.2Hz,2H),6.99( d,J=7.9Hz,2H),5.78(s,2H),4.84(s,1H),4.28(s,2H),3.07–2.88(m,2H),1.82(dd,J=15.4,7.8Hz,4H),1.43(s,9H),0.93(t,J=7.3Hz,3H).

[1033] Step 5:

[1034] A mixture of compound 6 (1.85 g, 3.87 mmol, 1.0 eq.) was placed in a reactor with NH3 (30 mL, NH3 in MeOH, 7 M) and stirred at 140 °C for 48 hours. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed the desired product. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 7 (880 mg, 49.7%) as a white solid.

[1035] LCMS: Rt: 1.237min; MS m / z (ELSD): 460.3M+H] + ;

[1036] 1H NMR (400MHz, CDCl3) δ7.87(d,J=8.1Hz,1H),7.70(d,J=7.9Hz,1H),7.48(t,J= 7.3Hz,1H),7.26(d,J=6.9Hz,2H),7.21(t,J=7.6Hz,1H),7.00(d,J=8.1Hz,2H ),5.73(s,2H),4.87(s,1H),4.29(d,J=5.2Hz,2H),3.25-3.05(m,2H),2.91–2 .83(m,2H),1.81(dt,J=15.4,7.6Hz,2H),1.43(s,9H),0.94(t,J=7.4Hz,3H).

[1037] Step 6:

[1038] A mixture of compound 6 (880 mg, 1.92 mmol, 1.0 eq.) was dissolved in DCM (10 mL), and then TFA (10 mL) was added at 0 °C under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was terminated with hydrated NaHCO3 and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound 8 (609 mg, 88.5%) as a white solid.

[1039] LCMS:Rt:1.050min;MS m / z(ELSD):228.1[M+H] + ;

[1040] 1H NMR (400MHz, CDCl3) δ7.93(d,J=8.3Hz,1H),7.71(d,J=8.2Hz,1H),7.51(t,J=7.3Hz,1H),7.32(d,J=8.2Hz,2H),7.23(d,J=8.2Hz,1 H),7.00(d,J=8.1Hz,2H),5.72(s,2H),3.86(s,2H),2.90–2.84(m,2H),1.86–1.78(m,2H),1.44-1.55(m,2H),0.94(t,J=7.3Hz,3H).

[1041] Step 7:

[1042] A solution of compound 8 (200 mg, 0.56 mmol, 1.0 eq.) was dissolved in EtOH (3 mL, 95%), followed by the addition of compound 9 (225 mg, 1.23 mmol, 2.2 eq.). The mixture was stirred at 80 °C for 48 hours under a nitrogen atmosphere. Thin-layer chromatography (DCM:MeOH = 10 / 1) showed that the reaction was complete, and a new major spot was observed. The reaction was terminated with H2O (50 mL) and extracted with EA (50 mL × 3). The combined organic phases were washed with brine (3 × 50 mL), dried, filtered through anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluting with 10% MeOH in DCM, to give compound HA-TLRa (50.84 mg, 11%) as a white solid.

[1043] LCMS:Rt:1.060min;MS m / z(ELSD):728.7[M+H] + ;

[1044] CAD:97.99% purity at ELSD; RT=31.007min.

[1045] 1 H NMR (400MHz, DMSO) δ7.77(d,J=8.2Hz,1H),7.57(d,J=8.2Hz,1H),7.31(t,J=7.6Hz,1H) ,7.26(d,J=7.6Hz,2H),7.05-6.95(m,3H),6.52(s,2H),5.83(s,2H),4.26(d,J=24.2Hz ,2H),3.55(d,J=8.2Hz,1H),3.46(d,J=14.0Hz,3H),2.89(t,J=7.6Hz,2H),2.31(d,J=8 .5Hz,2H),1.75-1.65(m,2H),1.39-1.31(m,2H),1.29-1.05(m,34H),0.87–0.81(m,9H).

[1046] Preparation Example 4:

[1047] Step 1:

[1048] Compound 2 (3.4 g, 26.19 mmol, 1.2 eq.), tetrakis(triphenylphosphine)palladium (1.26 g, 1.09 mmol, 0.05 eq.), and potassium carbonate (9.05 g, 65.48 mmol, 3.0 eq.) were added to a mixture of compound 1 (5 g, 21.83 mmol, 1.0 eq.) in toluene (50 mL) and water (10 mL). The reaction mixture was stirred at 100 °C for 16 h under a nitrogen atmosphere. Thin-layer chromatography (petroleum ether / ethyl acetate = 10 / 1) showed the formation of a new spot. The reaction was quenched with 100 mL of water and extracted with ethyl acetate (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0–10 / 1, v / v) as eluent to give compound 3 (2.12 g, 41% yield) as a colorless oil.

[1049] Step 2:

[1050] Under a nitrogen atmosphere, lithium aluminum hydride (8.53 mL, 21.34 mmol, 2.5 eq.) was added to a mixture of compound 3 (2 g, 8.53 mmol, 1.0 eq.) in tetrahydrofuran (20 mL) at 0 °C. The mixture was allowed to return to room temperature and stirred for 2 hours. Thin-layer chromatography (petroleum ether: ethyl acetate = 5 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with sodium sulfate decahydrate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography to give compound 4 (1.49 g, 84% yield) as a colorless oil.

[1051] Step 3:

[1052] Under a nitrogen atmosphere, triphenylphosphine (2.24 g, 8.55 mmol, 1.2 eq.) and N-bromosuccinimide (1.52 g, 8.55 mmol, 1.2 eq.) were added to a solution of compound 4 (1.47 g, 7.12 mmol, 1.0 eq.) in dichloromethane (20 mL), and the mixture was stirred at 25 °C for 16 hours. Thin-layer chromatography (petroleum ether: ethyl acetate = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 30 mL of water and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (1 / 0–10 / 1, v / v) as eluent to give compound 5 (599 mg, 31% yield) as a yellow oil.

[1053] 1 H NMR (400MHz, CDCl3) δ7.22(t,J=7.8Hz,1H),7.07(d,J=7.7Hz,1H),7.02(d,J=5.7Hz,2H),3.56(t,J=7.8Hz, 2H),3.14(t,J=7.8Hz,2H),2.73–2.47(m,2H),1.65–1.57(m,2H),1.40–1.26(m,6H),0.88(t,J=6.7Hz,3H).

[1054] Step 4:

[1055] Compound 5 (179 mg, 0.67 mmol, 1.5 eq.), potassium iodide (147 mg, 0.89 mmol, 2.0 eq.), and potassium carbonate (307 mg, 2.22 mmol, 5.0 eq.) were added to a mixture of compound 8 (220 mg, 0.44 mmol, 1.0 eq.) in acetonitrile (3 mL) and cyclopentyl methyl ether (3 mL). The mixture was stirred at 90 °C for 16 hours under an argon atmosphere. The mixture was quenched with 40 mL of water and extracted with ethyl acetate (40 mL × 3). The combined organic phases were washed with 40 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane containing 0%–10% methanol) and preparative high-performance liquid chromatography to give a yellow oil, HA-37 (79.97 mg, 28% yield).

[1056] LCMS:Rt:1.101min;MS m / z(ELSD):684.6[M+H] + ;

[1057] CAD:91.82%purity.Rt:13.033min

[1058] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=8.0Hz,1H),7.85(d,J=8.0Hz,1H),7.58-7.50(m,1H),7.44(s,1H),7.36- 7.30(m,1H),7.24-7.17(m,1H),7.04(d,J=8.0Hz,1H),6.98-6.92(m,2H),5.63(s,2H),4.47-4.42(m,2H), 4.09-4.02(m,2H),3.00-2.93(m,2H),2.79-2.71(m,2H),2.67-2.60(m,2H),2.60-2.53(m,2H),2.49-2.4 2(m,2H),2.30-2.23(m,2H),1.64-1.52(m,6H),1.34-1.24(m,20H),1.17-1.08(m,6H),0.90-0.85(m,6H).

[1059] Preparation Example 5:

[1060] Step 1: Compound 1 (1 g, 2.09 mmol, 1.0 eq, from HA-TLRa) was dissolved in dichloromethane (10 mL), and the mixture was cooled to 0 °C and trifluoroacetic acid (10 mL) was added dropwise. The reaction was then brought to room temperature and stirred for two hours. The reaction mixture was quenched with saturated sodium bicarbonate solution and pH = 8, and then evaporated to dryness. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give compound 2 (762 mg, 96%) as a white solid.

[1061] LCMS:Rt:1.053min; MS m / z(ELSD):379.2[M+H] + ;

[1062] Step 2:

[1063] Compound 2 (186 mg, 0.49 mmol, 1.0 eq.) was dissolved in ethanol (4 mL, 95%) and compound 3 (199 mg, 1.08 mmol, 2.2 eq.) was added. The mixture was then stirred at 80 °C for 48 hours under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 10 / 1), showing that the starting material was consumed and new products were formed. The reaction was quenched with water (50 mL), extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography with 10% MeOH in DCM to give a yellow oily product HA-TLRa-A (10.68 mg, 3%).

[1064] LCMS:Rt:1.130min;MS m / z(ELSD):747.6[M+H] + ;

[1065] CAD:89.91% purity at ELSD; RT=17.239min.

[1066] 1H NMR (400MHz, CDCl3) δ8.16(d,J=8.5Hz,1H),7.84(s,1H),7.59(t,J=7.6Hz,1H),7.39(dd,J =21.6,13.7Hz,3H),7.02(d,J=15.7Hz,2H),5.80(s,2H),3.66(d,J=11.6Hz,3H),3.50–3.36 (m,1H),3.12–2.83(m,2H),2.43(d,J=61.7Hz,3H),1.89–1.79(m,2H),1.46(dd,J=15.2,7. 6Hz, 4H), 1.31 (s, 4H), 1.25 (d, J = 4.6Hz, 34H), 0.93 (t, J = 7.4Hz, 3H), 0.89 (d, J = 6.5Hz, 6H).

[1067] Preparation example:

[1068] Step 1:

[1069] Under a nitrogen atmosphere, trifluoroacetic acid (3 mL) was added to a mixture of compound 1 (1 g, 2.326 mmol, 1.0 equivalent) in dichloromethane (15 mL) at 0 °C. The reaction mixture was allowed to return to room temperature and stirred for 2 hours. The reaction mixture was quenched with 1.0 M sodium hydroxide solution and the pH was adjusted to 8. The mixture was then concentrated under vacuum. The residue was purified by silica gel column chromatography using a dichloromethane solution containing 0.05% ammonia (increasing from 0% to 15%) as eluent to give compound 2 (750 mg, 97.6% yield) as a white solid.

[1070] LCMS:Rt:0.780min;MS m / z(ELSD):331.2[M+H] + ;

[1071] Step 2:

[1072] Compound 3 (420 mg, 2.273 mmol) was added to a 5 mL ethanol solution of compound 2 (250 mg, 0.758 mmol), and the mixture was stirred at 80 °C for 48 hours. Liquid chromatography-mass spectrometry (LC-MS) showed the formation of the target product (DP). The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using methanol in dichloromethane as eluent, increasing from 0% to 15%) and preparative high-performance liquid chromatography (pre-HPLC) to give HA-TLRb-A (123.81 mg, yield 23.3%) as a white solid.

[1073] LCMS:Rt:1.107min;MS m / z(ELSD):699.5[M+H] + ;

[1074] CAD:98.21% purity at ELSD; RT=14.383min.

[1075] 1 H NMR (400MHz, DMSO-d6) δ8.37–8.29(m,1H),8.10–8.02(m,1H),7.75–7.66(m,2H),4.69–4.51(m,2H),4.24(s,2H),3.37(s,2H),2.99(t,J=7.6H z,2H),2.35–2.26(m,2H),2.24–2.18(m,2H),1.92–1.78(m,4H),1.61– 1.43(m,4H),1.34–1.04(m,38H),1.00–0.94(m,3H),0.87–0.80(m,6H).

[1076] Luciferase (Luc) mRNA (SEQ ID NO:1):

[1077] eGFP mRNA (SEQ ID NO:2)

[1078] mRNA encoding human papillomavirus E7 antigen (SEQ ID NO:3):

[1079] EGFP-Flag mRNA (SEQ ID NO:4):

Claims

A lipid compound as shown in Formula I, or a pharmaceutically acceptable salt thereof. in, A is m and n are independently 0 or 1, and m and n are not both 0 at the same time; X is CH2 or NH; Y 1 and Y 2 Independently, it can be CH, CH2, N, or NH; It can be a single bond or a double bond; R 1 H and C independently 1-6 Alkyl or -OC 1-6 Alkyl, the C 1-6 Alkyl and -OC 1-6 Alkyl groups are independently and optionally surrounded by one or more R groups. a Replace, R a Each can be independently a halogen, amino, oxo, or hydroxyl group; H is an optional substituted linking group; the optional substituted linking group may optionally contain one or more biodegradable groups; t is 0, 1, or 2; K 1 and K 2 Independently alkyl, alkenyl, or ynyl, wherein the alkyl, alkenyl, and ynyl groups optionally contain one or more biodegradable groups; wherein the alkyl, alkenyl, and ynyl groups optionally containing one or more biodegradable groups are optionally independently denoted by one or more R groups. b replace; R b Each is independently an oxo group, cyano group, hydroxyl group, C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl; the C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 The aryl and 5-10 heteroaryl groups are independently and optionally separated by one or more R groups. b2 replace; R b1 For H or C 1-6 alkyl; R b2 It is either hydroxyl or amino; And K 1 and K 2 At least one of them contains more than 11 carbon atoms; The heteroatoms in the 3-10 membered heterocyclic alkyl and 5-10 membered heteroaryl groups are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently one, two or three. And when A is In this case, the linking group is -(CH2). n1 -, n1 is 1, 2, 3, 4, 5 or 6. The lipid compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, is characterized in that, It meets one or two of the following conditions: (1) The linking group is a single bond or Z is N, C, CH or CH2; n2 is an integer from 1 to 20; The One or more methylene units are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently -NH-, -O-, -S-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -S(O2)-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, C 3-10 Cycloalkyl, 3-10 heterocycloalkyl, C 6-10 Aryl or 5-10 heteroaryl; the one or more methylene units are independently and optionally replaced by biodegradable groups. Optionally by one or more R c Replace, R c Each independently is C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl; the C 1-6 Alkyl groups are optionally surrounded by one or more R c1 Replace; R c1 Each independently consists of hydroxyl and C. 1-6 Alkyl, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl; The heteroatoms in the 3-10 membered heterocyclic alkyl and 5-10 membered heteroaryl groups are independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently one, two or three. (2)K 1 and K 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 alkynyl group, the C 1-30 Alkyl, C 2-30 alkenyl and C 2-30 One or more methylene units in the alkynyl group are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently -NH-, -O-, -S-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -S(O)2-, -P(O)(OH)O-, -OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, C 3-10 Cycloalkyl, 3-10 heterocycloalkyl, C 6-10 Aryl or 5-10 heteroaryl; the one or more methylene units are independently and optionally replaced by biodegradable groups. 1-30 Alkyl, C 2-30 alkenyl and C 2-30 The alkynyl group is independently and optionally surrounded by one or more R groups. b replace; R b Each is independently an oxo group, cyano group, hydroxyl group, C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 Aryl or 5-10 heteroaryl; the C 1-6 Alkyl, C 2-6 alkenyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl, -NR b1 C(O)C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic alkyl, C 6-10 The aryl and 5-10 heteroaryl groups are independently and optionally separated by one or more R groups. b2 replace; The heteroatoms in the 3-10 membered heterocyclic alkyl groups and 5-10 membered heteroaryl groups are independently selected from one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three. The lipid compound of formula I as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, is characterized in that, It meets one or more of the following conditions: (1)C 1-6 Alkyl, -OC 1-6 Alkyl, -C(O)C 1-6 Alkyl and -NR b1 C(O)C 1-6 C in alkyl 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, for example, ethyl; (2) The C 3-10 Each cycloalkyl group is independently C10. 3-6 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and cyclopropyl, for example; (3) Each of the 3-10 membered heterocyclic alkyl groups is independently a 3-6 membered heterocyclic alkyl group, such as aziridine, aziridine pentyl or aziridine hexyl, and aziridine pentyl; (4) The C 6-10 Each aryl group can be either phenyl or naphthyl, for example, phenyl. The lipid compound of formula I as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, is characterized in that, It meets one or more of the following conditions: (1)R 1 For H or C 1-6 Alkyl, the C 1-6 Alkyl groups are optionally surrounded by one or more R a Replace, R a Each is independently a hydroxyl group; (2) The linking group is The One or more methylene units are independently and optionally replaced by biodegradable groups; each of the biodegradable groups is independently NH, O, S, Or C 6-10 Aryl; the one or more methylene units are independently and optionally replaced by biodegradable groups. Optionally by one or more R c Substitution; preferably, the linking group is Each x1 and x2 is independently 1, 2, 3, or 4, x3 is 0, 1, 2, 3, or 4, and each V1 is independently a bond, NH, or NR. c O Or C 6-10 Aryl; (3) n2 is 1, 2, 3, 4, 5, 6, 7 or 8; (4)R c Each independently is C 1-6 Alkyl; the C 1-6 Alkyl groups are optionally surrounded by one or more R c-1 replace; (5)R c-1 Each is independently a hydroxyl group; (6)K 1 and K 2 Independent for straight chain C 1-30 Alkyl, straight-chain C 2-30 Alkenyl, straight-chain C 2-30 acetylinyl or The straight chain C 1-30 Alkyl, straight-chain C 2-30 Alkenyl and straight-chain C 2-30 One or more methylene units in the alkynyl group are independently and optionally replaced by biodegradable groups; said biodegradable groups are each independently NH, -S(O)2-, -S-, -C(O)-, C 3-10 cycloalkyl or C 6-10 aryl; a straight-chain C whose one or more methylene units are independently and optionally replaced by biodegradable groups. 1-30 Alkyl, straight-chain C 2-30 Alkenyl and straight-chain C 2-30 The alkynyl group is independently and optionally surrounded by one or more R groups. b replace; f1 is 1, 2, 3, 4, 5, 6, 7 or 8; f2 and f3 are independently 0, 1, 2, 3, 4 or 5; Z 1 It is a single bond or -O-; Z 2 for R 3 and R 4 Independently for C 1-10 Alkyl group; preferably, each of the biodegradable groups is independently alkyl. C 3-10 cycloalkyl or C 6-10 Aryl; Preferably, K 1 and K 2 Independently Each r1 is an independent integer from 1 to 12, and r2 is an integer from 1 to 16, and the sum of r1 and r2 is ≤ 30; r3 is an integer from 1 to 10 independently, r4 is an integer from 0 to 6, r5 is an integer from 1 to 12, and the sum of r3, r4 and r5 is ≤ 30; r6 is an independent integer from 1 to 6; and the sum of each r6 and r3 is ≤ 30; G 1 C 3-10 cycloalkyl or C 6-10 Aryl; G 2 For -CHR b -、 Or C 3-10 cycloalkyl; R b Hydroxyl group, C 3-10 Cycloalkyl or 3-10 membered heterocyclic alkyl; G 3 for G 4 G 5 and G 6 Independent O, -CHR b -, -CH2=CH2-, C 3-10 cycloalkyl, C 6-10 Aryl or a C 1-6 Alkyl-substituted C 6-10 Aryl; f1 is 1, 2, 3, 4, 5, 6, 7 or 8; f2 and f3 are independently 0, 1, 2, 3, 4 or 5; Z 1 It is a single bond or -O-; Z 2 for R 3 and R 4 Independently for C 1-10 alkyl; (7)R b Each independently consists of hydroxyl and C. 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl or C 6-10 Aryl; Preferably, the lipid compound of Formula I satisfies one or more of the following conditions: (1) Each V1 is independently a bond, NH, O, (2)R b Each is independently hydroxyl, ethyl, n-propyl, n-butyl, cyclopropyl, Or phenyl; preferably hydroxyl; (3)R 3 and R 4 Independently Better, for The lipid compound of formula I as claimed in claim 4, characterized in that, The lipid compound represented by Formula I is selected from any of the following schemes: Option 1: The lipid compound shown in Formula I is a compound shown in Formula II: n2, Z, K 1 and K 2 The definition is as described in any one of claims 2-4; Option 2: The lipid compound shown in Formula I is a compound shown in Formula III-1: The lipid compound shown in Formula I is a compound shown in Formula III-2: K 1 and K 2 The definition is as described in any of the following schemes: Option A: K 1 and K 2 Independently Option B: K 1 for K 2 for The lipid compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, is characterized in that, It meets one or more of the following conditions: (1) for for Preferably, A is (2) H is (3)K 1 and K 2 Independently The lipid compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, is characterized in that, The compound shown in Formula I is any of the following compounds: A lipid nanoparticle composition, characterized in that, It includes lipid nanoparticle carriers and nucleic acid molecules, wherein the lipid nanoparticle carriers include lipid compounds as shown in Formula I as described in any one of claims 1-7, or pharmaceutically acceptable salts, phospholipids, sterols, and PEG lipids thereof. The lipid nanoparticle composition according to claim 8, characterized in that, It meets one or more of the following conditions: (1) The molar percentage of the lipid compound or its pharmaceutically acceptable salt as shown in Formula I is 20-80 mol%, preferably 33 mol%-58 mol%, more preferably 40 mol%-52 mol%, 35-49 mol%, 48-52 mol%, or 40-44 mol%. (2) The phospholipid is a neutral phospholipid, preferably a distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), palmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), distearyl phosphatidylethanolamine (DSPE), distearyl phosphatidylglycerol (DSPG), DSPG-Na, disqualyl phosphatidylcholine (DEPC), palmitoyl phosphatidylglycerol (DPPG), DPPG-Na, dipalmitoyl phosphatidic acid (DPPA), docosyl phosphatidylcholine (DUPC), or palmitoyl phosphatidylcholine (POPC). (3) The phospholipid has a molar percentage of 3 mol%-40 mol%, preferably 3 mol%-35 mol%, more preferably 4 mol%-21 mol%, further preferably 5 mol%-18 mol%, and even more preferably 5 mol%-15 mol% or 8-12 mol%. (4) The sterol is an animal, plant or fungal sterol, preferably selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid and α-tocopherol, more preferably cholesterol; (5) The molar percentage of the sterol is 8 mol%-60 mol%, preferably 28 mol%-52 mol%, more preferably 35 mol%-52 mol%, and even more preferably 40 mol%-52 mol% or 40-50 mol%. (6) The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, preferably PEG-DSPE, PEG-DMG or ALC-0159; more preferably PEG-DMG-2000 or DSPE-PEG2000. (7) The molar percentage of the PEG lipid is 0.1 mol%-8 mol%, preferably 0.1 mol%-5 mol%, more preferably 1 mol%-2 mol%; (8) The lipid nanoparticle carrier further includes one or more of ionizable lipids, cationic lipids, and adjuvants. Preferably, the ionizable lipid is ALC0315. The cationic lipid molecule is (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), methyl 4-(N,N-dimethylamino)butyrate (dilinyl)methyl ester (DLin-MC3-DMA), or dioleoylpropyltrimethylammonium chloride (DOTMA), for example (2,3-dioleoyl-propyl)-trimethylamine (DOTAP); The adjuvant is selected from any of the following compounds: More preferably, the lipid nanoparticle carrier includes an adjuvant, the mass of which is 0.5%-60% of the mass of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, preferably 1%-50%, more preferably 8-12%. (9) The lipid nanoparticle composition further includes a diluent, preferably a citrate buffer, a phosphate buffer or an acetate buffer; or the pH of the diluent is 4-5. The lipid nanoparticle composition according to claim 9, characterized in that, The lipid nanoparticle composition is selected from any of the following: Option 1: The lipid nanoparticle carrier comprises 20-80 mol% of the lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 3-40 mol% of phospholipids, 8-60 mol% of sterols and 0.1-8 mol% of PEG lipids; Option 2: The lipid nanoparticle carrier comprises 33-58 mol% of the lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 5-18 mol% of phospholipids, 35-52 mol% of sterols and 1-2 mol% of PEG lipids; Option 3: The lipid nanoparticle carrier comprises 48-52 mol% of a lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 8-12 mol% of phospholipids, 36.5-40.5 mol% of sterols and 1-2 mol% of PEG lipids; Option 4: The lipid nanoparticle carrier comprises 40-44 mol% of a lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 4.5-8.5 mol% of phospholipids, 48-52 mol% of sterols and 1-2 mol% of PEG lipids. The lipid nanoparticle composition according to claim 10, characterized in that, The phospholipids and PEG lipids are selected from any of the following: Option 1: The phospholipid is DPPC or DMPC, and the PEG lipid is ALC0159; Option 2: The phospholipid is DEPC or DSPC, and the PEG lipid is DMG-PEG2000; Option 3: The phospholipid is DMPC or DSPC, and the PEG lipid is DSPE-PEG2000. The lipid nanoparticle composition according to any one of claims 8-11, characterized in that, The nucleic acid molecule is selected from one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), gene editing tools, self-replicating RNA (samRNA), microRNA (miRNA), circular RNA (circRNA), and messenger RNA (mRNA), preferably messenger RNA (mRNA), more preferably firefly luciferase (Fluc) mRNA, enhanced green fluorescent protein (eGFP) mRNA, EGFP-Flag mRNA, mRNA sequence encoding human papillomavirus E7 antigen, mRNA sequence encoding infectious disease antigen, or mRNA sequence encoding tumor antigen; Preferably, the nitrogen-to-phosphorus ratio in the lipid nanoparticle composition is (2-30):1, more preferably (3-8):

1. The lipid nanoparticle composition according to claim 8, characterized in that, The lipid nanoparticle composition is selected from any of the following: Option 1: The lipid nanoparticle carrier comprises 48-52 mol% of a lipid compound as shown in Formula I or a pharmaceutically acceptable salt thereof, 8-12 mol% of DSPC, 36.5-40.5% of cholesterol, and 1-2 mol% of DMG-PEG2000; the nucleic acid molecule is firefly luciferase (Fluc) mRNA or enhanced green fluorescent protein (eGFP) mRNA; Option 2: The lipid nanoparticle carrier comprises 40-44 mol% of the compound shown in Formula HI-19, 4.5-8.5 mol% of DEPC, 48-52% of cholesterol, and 1-2 mol% of DMG-PEG2000; or, the lipid nanoparticle carrier comprises 43-47 mol% of the compound shown in Formula HI-19, 9-13 mol% of DEPC, 40.5-45% of cholesterol, and 1-2 mol% of DMG-PEG2000; the nucleic acid molecule is firefly luciferase (Fluc) mRNA; Option 3: The lipid nanoparticle carrier comprises 48-52 mol% of a compound as shown in Formula HI-19, 8-12 mol% of distearate phosphatidylcholine (DSPC), 36.5-40.5 mol% of cholesterol, 1-2 mol% of DMG-PEG2000, and an adjuvant, wherein the adjuvant is a compound as shown in Formula HA-TLR-2-A, HA-1, HA-TLRAa, or HA-37, and the mass percentage of the adjuvant to the mass of the compound as shown in Formula I or its pharmaceutically acceptable salt is 8%-12%; the nucleic acid molecule is mRNA encoding the human papillomavirus E7 antigen. Option 4: The lipid nanoparticle carrier comprises 40-44 mol% of the compound shown in Formula HI-19, 4.5-8.5 mol% of DEPC, 48-52% of cholesterol, 1-2 mol% of DMG-PEG2000 and the compound shown in Formula HA-TLR-2-A, wherein the mass percentage of the compound shown in Formula HA-TLR-2-A to the mass percentage of the compound shown in Formula I or a pharmaceutically acceptable salt thereof is 8%-12%, and the nucleic acid molecule is mRNA encoding luciferase (Luc). A pharmaceutical composition or vaccine comprising a lipid nanoparticle composition as described in any one of claims 1-13 and a pharmaceutically acceptable carrier. A lipid carrier comprising a compound of Formula I as described in any one of claims 1-7 or a pharmaceutically acceptable salt thereof, preferably, the lipid carrier being a lipid nanoparticle carrier as described in any one of claims 9-13.

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