Compositions and methods for modulating the immune response by activating α-protein kinase 1

Abstract

The present disclosure provides compositions and methods related to the activation of alpha-kinase 1 (ALPK1) to modulate immune responses, treat or prevent cancer, infection, inflammation and related diseases and disorders, and enhance immune responses to target antigens. The present disclosure provides heterocyclic compounds of formula (I), wherein A 1 , A 2 , L 1 , L 2 , L 3 , Z 1 , Z 2 , W 1 , W 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 (wherein x is as defined herein) and their uses in activating ALPK1, modulating immune responses, and treating diseases such as cancer.

Description

[Technical Field]

[0001]

[01] The present invention relates to compositions and methods for therapeutic purposes by activation of alpha protein kinase 1 (ALPK1). [Background technology]

[0002]

[02] Research into the mechanisms of inflammatory responses has identified various protein kinases that act as essential signaling components. Deficiencies in protein kinases are often associated with the pathogenesis of human inflammatory diseases, cancer, and diabetes.

[0003]

[03] α-kinases are a unique superfamily of protein kinases that show little sequence similarity to typical protein kinases. A total of six α-kinase members have been identified, including α-protein kinase 1 (ALPK1), ALPK2, ALPK3, elongation factor-2 kinase (eEF2K), and transient receptor potential cation channels M6 and M7 (TRPM6 and TRPM7) (Ryazanov AG et al., Curr Biol 1999 9(2):R43-45; Ryazanov AG et al., Proc Natl Acad Sci USA 1997 94(10):4884-4889).

[0004]

[04] ALPK1 was identified as a novel component of sucrose-isomerase (SI) vesicles containing rafts in epithelial cells (Heinet M et al., J. Biol. Chem. 2005 280(27):25637-43). It was shown that ALPK1 phosphorylates myosin 1 and plays an essential role in exocytotic transport to the apical plasma membrane. Transposon insertion homozygous inactivating mutations in ALPK1 in mice resulted in coordination disorders that could be rescued by overexpression of full-length ALPK1 (Chen M et al., BMC Neurosci. 2011 12:1).

[0005]

[05] Not all of the identified polymorphisms replicated in all populations, but several gene association studies have shown that ALPK1 is associated with the risk of gout (Wang SJ et al., J. Mol. Med. 2011 89:1241-51; Ko AM et al., J. Intl. Epidemiol. 2013 42:466-474; Chiba T et al., Human Cell 2015 28:1-4). Other gene-related studies have associated ALPK1 with a risk factor for chronic kidney disease, myocardial infarction, and diabetes (Yamada Y et al., J Med Genet 2013 50:410-418; Fujimaki T et al., Biomed Report 2014 2:127-131; Shimotaka S et al., Biomed Report 1 2013 940-44; Yamada Y et al., Biomed. Report 2015 DOI:10.3892 / br.2015.439).

[0006]

[06] Overexpression of ALPK1 in mice led to decreased testosterone levels and increased production of inflammatory cytokines IL-1β and TGF-β. This suggests that the balance between ALPK1 and testosterone may play a role in testosterone-mediated inhibition of inflammatory cytokines (Kuo TM et al., J Steroid Biochem Mol Biol 2015 154:150-158).

[0007]

[07] ALPK1 activation has also been suggested to play a role in cancer, including lung cancer, colorectal cancer, and breast cancer (Liao HF et al., Scientific Reports 2016 6:27350; Strietz J et al., Oncotarget 2016 1-16).

[0008]

[08] Recent studies have suggested the involvement of ALPK1 as a key regulator of innate immune responses activated by certain bacteria. For example, APLK1 has been suggested to be a major regulator of innate immunity against bacteria by promoting TIFA oligomer formation and interleukin-8 (IL-8) expression in response to infection with Shigella flexneri, Salmonella typhimurium, and Neisseria meningitides (Milivojevic M et al., PLoS Pathog 2017 13(2):e1006224). Zimmerman et al. have reported an ALPK1 and TIFA-dependent innate immune response triggered by the type IV secretory system of Helicobacter pylori (Zimmermann S et al., Cell Reports 2017 20(10):2384-95). Both studies suggest that the bacterial metabolite heptose-1,7-bisphosphate (HBP) activates TIFA-dependent innate immunity. [Overview of the project] [Problems that the invention aims to solve]

[0009]

[09] Many diseases, disorders, and conditions result from inflammation and various infections. Novel methods are needed to modulate inflammation in target tissues to treat such diseases, disorders, and conditions. This disclosure addresses this need. [Means for solving the problem]

[0010]

[10] The present invention is based in part on the discovery that several bacterial metabolites, including D-glycero-β-D-manno-heptose-1,7-bisphosphate (heptose-1,7-bisphosphate or "HBP") and D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and their derivatives represented by formulas IA, IB, or IC as described herein, induce ALPK1-dependent activation of downstream signaling, including increased expression of inflammatory cytokines such as IL-8 and TNFα. The biological activity of HMP-1bP, and its downstream product H1b-ADP-6L, particularly H1b-ADP, was unexpected given the currently known roles of ALPK1 and bacterial metabolites in activating innate immunity. This disclosure also provides evidence of antitumor activity of H1b-ADP and H1b-ADP derivatives, and reveals that co-administration of H1b-ADP with immune checkpoint inhibitors and immunomodulators (including anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA4 antibodies, and anti-CD4 antibodies) has a synergistic antitumor effect. This disclosure also shows that co-administration of H1b-ADP with immunomodulators (including interferon-alpha (INFα), interferon gene-stimulating factor ("STING") agonists, and TLR agonists (resquimod)) has a synergistic antitumor effect.

[0011]

[11] Accordingly, the present disclosure provides compositions and methods amenable to modulating the immune response through activation of ALPK1, treating cancer, enhancing the immune response against a target antigen, treating diseases or disorders suitable for treatment by activation of the NFkB, p38, and JNK cell signaling pathways, and treating or preventing diseases or disorders caused by infectious pathogens. In some embodiments, ALPK1 activation is achieved by administration of an ALPK1 agonist selected from HBP, HMP-1bP, H1b-ADP-6L, and H1b-ADP, preferably HMP-1bP, H1b-ADP-6L, and H1b-ADP, most preferably H1b-ADP-6L and H1b-ADP, or derivatives thereof represented by Formula IA, IB, or IC described herein. In some embodiments, the present disclosure provides a method of modulating an immune response in a subject, the method comprising administering to the subject a composition comprising any one of the ALPK1 agonists represented by Formula I, IA, IB, or IC described herein.

[0012]

[12] The present invention discloses novel heterocyclic compounds as ALPK1 agonists. The compounds are represented by formula (I)

[0013] [Chemical formula]

[0014] [Wherein, A 1 , 7 , 1 , 5 , 3 , 1 , 2 , 2 , 3 , 6 , 4 , 2 , A 2 , L 1 , L 2 , L 3 , Z 1 , Z 2 , W 1 , W 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7[This is defined herein]. Stereoisomers, tautomers, stable isotopes, prodrugs, and pharmaceutically acceptable salts of the compounds of formula I are also included within the scope of this disclosure. A 1 and A 2 These are independently O, S, and -C(R 8 R 9 )- Selected from, R 8 and R 9 A is independently selected from H, D, -OH, N3, -CN, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkanoyloxyl, and substituted or unsubstituted aralkyloxyl (optional substituents are 1-3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl and C1-C4 alkoxy), and A 1 Or A 2 At least one of them is -C(R 8 R 9 ) and A 1 In R 8 or R 9 is, A 2 In R 8 or R 9 It can be cyclized to form a C3-C6 cycloalkyl and a cycloheteralkyl having 3-9 ring members, with 1-3 heteroatoms selected from N, O, and S as ring members, each optionally substituted with 1-3 substituents independently selected from D, halogens, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. L 1 and L 2 These are independently selected from O, CH2, CHF, and CF2. L 3 These are O, S, CH2, or CH(OH), Z 1 and Z 2 These are independently selected from O and S, W 1 -C(R 10 R 11 )- and R10 and R 11 These are independently H, D, -OH, halogens, as well as C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4-haloalkoxyl, C1-C4 alkenyloxyl, aralkyloxyl and R 12 CO2-(R 12 R is selected from groups that are optionally substituted from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members, 10 and R 11 The optional substituents are 1 to 3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. W 2 These are H, or D, halogen, -OH, =O, C1-C3 alkoxyl, C1-C3 haloalkyl, C1-C3 haloalkoxyl, C1-C3 alkenyloxyl and R 12 CO2-(R 12 This refers to a C1-C3 alkyl group that is optionally substituted with 1-3 substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteroalkyl, C6-C10 aryl, and heteroaryl (which contains 5-10 ring atoms and has 1-3 heteroatoms selected from N, O, and S as ring members), R 1 This is a heteroaryl compound containing C6-C10 aryls, or 5-10 ring atoms, and having 1-4 heteroatoms selected from N, O, and S as ring members, R 1D, halogen, -OH, =O, CN, NH2, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamine, C1-C4 dialkylamine and (R 13 R 14 )NCO- is optionally substituted with 1 to 3 substituents selected from R 13 and R 14 These are independently selected from H, C1-C4 alkyl, C3-C6 cycloalkyl, cycloheteralkyl, C6-C10 aryl, C6-C10 aryl, and heteroaryl, which comprises 5-10 ring atoms and has 1-3 heteroatoms selected from N, O, and S as ring members. R 2 , R 3 and R 4 These are independently selected from H, D, halogens, C1-C4 alkyls and C1-C4 haloalkyls. R 5 , R 6 and R 7 H, D, halogens and -OH, R 12 CO2-(R 12 (Selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 4-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members), R 5 , R 6 and R 7 Any two of the adjacent groups can be cyclized to form a cycloheteralkyl group containing 5 to 9 ring members, each having 1 to 3 heteroatoms selected from N, O, and S, each optionally substituted with 1 to 3 substituents independently selected from D, halogens, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy.

[0015]

[13] In embodiments, the Disclosure provides a method for modulating an immune response in a subject requiring modulation of the immune response, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist represented by formula I, IA, IB, or IC as described herein, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant of said protein. In embodiments, the method for modulating the immune response is selected from activation of innate immunity and activation of adaptive immunity.

[0016]

[14] In embodiments, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof, including an ALPK1 agonist represented by formula I, IA, IB, or IC as described herein. In embodiments, the composition comprises an ALPK1 agonist selected from compounds represented by formula I, IA, IB, or IC as described herein, or an ALPK1 agonist selected from HBP, HMP-1bP, H1b-ADP-6L, and H1b-ADP, preferably HMP-1bP, H1b-ADP-6L, and H1b-ADP, most preferably H1b-ADP-6L and H1b-ADP. In embodiments, the ALPK1 agonist is H1b-ADP. In embodiments, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In embodiments, the ALPK1 agonist is compound 15. In embodiments, the cancer is selected from soft tissue sarcoma, breast cancer, head and neck cancer, melanoma, cervical cancer, bladder cancer, hematological malignancies, glioblastoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, kidney cancer, lung cancer, Merkel cell carcinoma, small intestine cancer, thyroid cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), gastric cancer, gastrointestinal stromal tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, liver cancer, leukemia, lymphoma, T-cell lymphoma, brain cancer, and multiple myeloma. In an embodiment, the cancer is selected from breast cancer, head and neck cancer, melanoma, kidney cancer, lung cancer, Merkel cell carcinoma, and lymphoma.

[0017]

[15] In embodiments, the present disclosure provides a method for enhancing an immune response to a target antigen in a subject, comprising the step of administering to a subject a composition comprising an ALPK1 agonist represented by formula I, IA, IB, or IC as described herein, a polynucleotide encoding an ALPK1 agonist, ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant of the protein, as a vaccine or immune adjuvant that works to enhance an immune response to a target antigen. In embodiments, the target antigens include adenovirus, coxsackie B virus, cytomegalovirus, eastern equine encephalitis virus, ebola virus, enterovirus, Epstein-Barr virus, Haemophilus influenzae type b (Hib), hepatitis C virus (HCV), herpes virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), hookworm, Marburg virus, norovirus, respiratory syncytial virus (RSV), rotavirus, and Salmonella typhi. It is an antigen of infectious pathogens selected from the group consisting of typhi, Staphylococcus aureus, Streptococcus pyogenes, varicella, West Nile virus, Yersinia pestis, and Zika virus.In embodiments, including ALPK1 agonists represented by formulas I, IA, IB, or IC as described herein, polynucleotides encoding ALPK1 agonists, ALPK1 or its constitutively active variants, or the ALPK1 protein or its constitutively active variants are used to treat anthrax, caries, Chagas disease, dengue fever, diphtheria, ehrlichiosis, hepatitis A or B, herpes, seasonal influenza, Japanese encephalitis, and leprosy. It acts as a vaccine adjuvant for vaccines in the treatment or prevention of meningococcal diseases, including senicitis, Lyme disease, malaria, measles, mumps, meningitis and sepsis; onchocerciasis (river blindness); pertussis; pneumococcal disease; polio; rabies; rubella; schistosomiasis; severe acute respiratory syndrome (SARS); herpes zoster; smallpox; syphilis; tetanus; tuberculosis; tularemia; tick-borne encephalitis virus; typhoid fever; trypanosomiasis; yellow fever; or visceral leishmaniasis.

[0018]

[16] In embodiments, the present disclosure provides a method for treating a disease or disorder that is amendable to treatment by activation of NFκB, p38, and JNK cell signaling pathways in cells of interest, comprising the step of administering to a target a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof. In embodiments, the disease or disorder is selected from tuberculosis, meningitis, pneumonia, ulcers, sepsis, rhinitis, asthma, allergies, COPD, inflammatory bowel disease, arthritis, obesity, radiation-induced inflammation, psoriasis, atopic dermatitis, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, systemic lupus, lupus erythematosus (SLE), autoimmune thyroiditis (Graves' disease), multiple sclerosis, ankylosing spondylitis, vesicular diseases, and diseases and disorders caused by hepatitis C virus (HCV), hepatitis B virus (HBV), or human immunodeficiency virus (HIV).

[0019]

[17] In embodiments, the Disclosure provides a method for treating or preventing a disease or disorder caused by an infectious pathogen selected from bacteria, viruses, or parasites in a subject requiring treatment or prevention of such a disease or disorder, comprising the step of administering to a subject a composition comprising one of the following: an ALPK1 agonist represented by formula I, IA, IB, or IC as described herein, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant of said protein. In embodiments, the infectious pathogen is a bacterium. In embodiments, the infectious pathogen is a virus. In embodiments, the infectious pathogen is a parasite. In embodiments, the bacterium is a Gram-negative bacterium or a Gram-positive bacterium. In this embodiment, the Gram-negative bacteria include Acinetobacter baumanii, Aggregatobacter actinomycetemcomitans, Bartonella bacilliformis, Bartonella henselae, Bartonella quintana, Bifidobacterium, Borrelia, Bortadella pertussis, Brucella sp, Burkholderia cepacis, Burkholderia psedomallei, and Campylobacter jejuni. jejuni), Cardiobacterium hominis, Campylobacter fetus, Chlamydia pneumonia, Chlamydia trachomatis, Clostridium difficileDifficile), Cyanobacteria, Eikennella corrodens, Enterobacter, Enterococcus faccium, Escherichia coli, Escherichia coli O157, Franceilla tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Haemophilus aphrophilus, Haemophilus ducreyi, Haemophilus parainfluenzae, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae Legionella bacteria, Legionella pneumophila serogroup 1, Leptospira, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus mirabilis, Proteus vulgaris, Proteus myxofaciens, Providencia rettgeri, Providencia alcalifaciens, Providencia stuartii, Pseudomonas aeruginosa Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas acidoboransacidovorans), Rickettsiae, Salmonella enterica, Salmonella typhi, Salmonella paratyphi types A and B typhus, Salmonella dublin, Salmonella arizonae, Salmonella choleraesuis, Serratia marcescens, Schigella dysenteriae, Schigella flexneri, Schigella boydii, Schigella sonnei, Treponema, Stenotrophomonas maltophilia The group is selected from the following: Maltophilia, Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio hollisae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia pestitis. In this embodiment, Gram-positive bacteria include Actinomycetes, Bacillus anthracis, Bacillus subtilis, Clostridium tetani, Clostridium perfingens, Clostridium botulinum, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix ruhsiopathiae, and Listeria monocytogenes.Mycobacterium monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma, Nocardia, Propionibacterium, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus aureus (VRSA), Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus pneumoniae In some embodiments, the virus is selected from the group consisting of pneumonia, Streptococcus pyogenes, and Streptococcus mutants. In this embodiment, the parasites include species of the genus Acanthamoeba, American trypanosomiasis, Balamuthia mandnillanis, and Babesia polymorphism.divergenes), Babesia bigemina, Babesia equi, Babesia microfti, Babesia duncani, Balantidium coli, lastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Diphyllobothrium latum, Leishmania amazonesis, Naegleria fowderi, Plasmodium falciparum, Plasmodium vivax The group is selected from the following: *Typha vivax*, *Plasmodium ovale curtisi*, *Plasmodium malariae*, *Rhinosporidium seeberi*, *Sarcocystis bovihominis*, *Sarcocystississimus suihominis*, *Toxoplasma gondii*, *Trichmonas vaginalis*, *Trypanosoma bruseyi*, *Trypanosoma cruzi*, and *Taenia polycephalus*.

[0020]

[18] In any embodiment of the above-described method, the method may further include a step of administering one or more additional therapeutic agents or immunomodulators, and combinations thereof, to a target. In embodiments, one or more additional therapeutic agents are selected from antimicrobial agents such as antibacterial agents, antiviral agents or antiparasitic agents, anticancer agents, or therapeutic agents for the treatment of tuberculosis, meningitis, pneumonia, ulcers, sepsis, rhinitis, asthma, allergies, COPD, inflammatory bowel disease, arthritis, obesity, radiation-induced inflammation, psoriasis, atopic dermatitis, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, systemic lupus, lupus erythematosus (SLE), autoimmune thyroiditis (Graves' disease), multiple sclerosis, ankylosing spondylitis, and bullous diseases.

[0021]

[19] In embodiments of a method for treating cancer, one or more additional therapeutic agents are immunomodulators. In embodiments, the immunomodulator is selected from one or more inhibitors or antagonists of immune checkpoint regulators, immunostimulatory molecules, and agonists of immune costimulatory molecules. In embodiments, the inhibitor or antagonist of immune checkpoint regulators is a PD-1 / PD-L1 inhibitor. In embodiments, the PD-1 / PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, and avelumab. In embodiments, the immunomodulator is selected from interferon-alpha (INFα), interferon gene-stimulating factor ("STING") agonists, TLR agonists (e.g., resquimod), and anti-OX40 (CD134) agonist antibodies. In embodiments, the agonist of the immunocostimulatory molecule is an anti-OX40 (CD134) agonist antibody. In embodiments, the cancer is selected from advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin lymphoma, liver cancer, gastric cancer, colon cancer, breast cancer, non-Hodgkin lymphoma, prostate cancer, head and neck cancer, thyroid cancer, brain cancer, acute myeloid leukemia (AML), Merkel cell carcinoma, multiple myeloma, cervical cancer, and sarcoma.

[0022]

[20] In embodiments, one or more additional immune modulators are inhibitors or antagonists of immune checkpoint modulators, or vaccines against immune checkpoint modulators. In embodiments, one or more additional immune modulators are agonists of immune checkpoint modulators, such as costimulatory molecules, e.g., agonists of OX40 (CD134). In embodiments, immune checkpoint regulators include programmed cell death 1 (PD-1) receptor (CD279), PD-1 ligand (e.g., PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), tumor necrosis factor receptor superfamily member 9 (or TNFRSF9, 4-1BB) and 4-1BB ligand, tumor necrosis factor receptor superfamily member 4 (or TNFRSF4, OX40) and OX40 ligand, glucocorticoid-inducible TNFR-associated protein (GITR), tumor necrosis factor receptor superfamily member 7 (or TNFRSF7, differentiation cluster 27, CD27), TNFRSF25 and TNF-like ligand 1A (TL1A), TNF receptor superfamily member 5 (or TNFRSF5, CD40) and CD40 ligand, herpesvirus entry-mediation factor (HVEM)-tumor necrosis factor ligand superfamily member Lymphocyte 14 (or TNFSF14, LIGHT)-lymphotoxin α (LTA), herpesvirus entry-mediating factor (HVEM)-B and T lymphocyte attenuator (BTLA)-CD160 (or TNFSF14), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and mucin domain-containing 3 (TIM3), sialic acid-binding immunoglobulin-like lectin (SIGLEC), inducible T cell costimulatory factor (ICOS) and ICOS ligator T cell activation inhibitor 1 containing V-set domain (VTCN1, or B7-H4), T cell activation inhibitor containing type V immunoglobulin domain (VISTA), human endogenous retrovirus type H long-terminal repeat-binding protein 2 (HHLA2)-transmembrane and immunoglobulin domain 2 (TMIGD2), butyrophyllin, natural killer cell receptor 2B4 (or NKR2B4),CD244) and B cell membrane protein (CD48), T cell immune receptor (TIGIT) and poliovirus receptor (PVR) family members containing an immunoglobulin (Ig) domain and an immunoreceptor-suppressive tyrosine motif domain, killer cell immunoglobulin-like receptor (KIR), immunoglobulin-like transcript (ILT) and leukocyte immunoglobulin-like receptor (LIR), member D of natural killer protein 2 (NKG2D) and member A of natural killer protein 2 (NKG2A), major histocompatibility complex (MHC) class I polypeptide-associated sequence A (MICA) and MHC class I polypeptide-associated sequence B (MI Selected from CB), natural killer cell receptor 2B4 (CD244), colony-stimulating factor 1 receptor (CSF1R), indoleamine 2,3-dioxygenase (IDO), transforming growth factor β (TGFβ), adenosine-ectonucleoside triphosphate diphosphohydrolase 1 (CD39)-5'-nucleotidase (CD73), CXC-motif chemokine receptor 4 (CXCR4) and CXC-motif chemokine ligand 12 (CXCL12), phosphatidylserine, signal regulatory protein α (SIRPA) and integrin-binding protein (CD47), vascular endothelial growth factor (VEGF), and neuropilin.

[0023]

[21] In embodiments, one or more additional immune modulators are vaccines.

[22] In embodiments of methods for treating cancer, the vaccine is a vaccine against a tumor antigen. In embodiments, the tumor antigen is selected from glycoprotein 100 (gp100), mucin 1 (MUC1), and melanoma-associated antigen 3 (MAGEA3).

[0024]

[23] In embodiments, one or more additional immune modulators are T cells, preferably chimeric antigen receptor T cells. In embodiments, one or more additional immune modulators are recombinant proteins, preferably recombinant proteins selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-7 (IL-7), IL-12, IL-15, IL-18, and IL-21.

[0025]

[24] In any embodiment of the above-described method, the composition may include an ALPK1 agonist represented by formula I, IA, IB, or IC as described herein, or an ALPK1 agonist selected from D-glycero-β-D-manno-heptose 1,7-bisphosphate (HBP) and its prodrugs, analogs, and derivatives. In an embodiment, the ALPK1 agonist is HBP. In an embodiment, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In an embodiment, the ALPK1 agonist is compound 15.

[0026]

[25] In embodiments, the ALPK1 agonist is a prodrug of HBP. In embodiments, the prodrug comprises a protecting group selected from the group consisting of carbonyloxymethyl, cyclosaligenyl, cyclic 1-aryl-1,3-propanyl esters, aryloxyphosphorumidates or phosphonoamides, and methylaryl haloalkylamdiates. In embodiments, the prodrug is a compound of formula 3a, 3b, 3c, 3d, or 3e. In embodiments, the prodrug is selected from the compounds in Table 1.

[0027]

[26] In any embodiment of the above-described method, the composition may include a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant of the said protein. In an embodiment, the composition comprises a polynucleotide encoding ALPK1 or a constitutively active variant thereof. In an embodiment, the composition is adapted for administration to a subject using a viral or nonviral gene delivery system. In an embodiment, the composition is adapted for administration to a subject using a viral gene delivery system. In an embodiment, the composition further comprises viral particles. In an embodiment, the composition is adapted for administration to a subject using a nonviral gene delivery system. In an embodiment, the composition further comprises one or more liposome particles, nanoparticles, minicircles, minivectors, and polymer carriers. In an embodiment, the nonviral gene delivery system comprises a gene editing technique. In an embodiment, the gene editing technique utilizes a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or CRISPR / Cas-9.

[0028]

[27] In embodiments, the present disclosure provides a method for treating a liver disease or disorder in a subject requiring treatment for such a disorder, comprising the step of administering a low dose of H1b-ADP or a derivative thereof to the subject. In embodiments, the low dose is in the range of 1 nanogram to 1 milligram per kilogram of body weight (1 ng / kg to 1 mg / kg), preferably 1 microgram to 100 micrograms per kilogram of body weight (1 μg / kg to 100 μg / kg). In embodiments, the liver disease or disorder is selected from liver cancer, non-alcoholic steatohepatitis (NASH), and diseases or disorders caused by infection with hepatitis C virus (HCV) or hepatitis B virus (HBV).

[0029]

[28] In embodiments, the present disclosure provides a method for treating cancer, comprising the step of administering to a subject requiring such treatment a composition comprising bacteria producing H1b-ADP or H1b-ADP-6L. In embodiments, the composition is administered by intratumoral injection.

[0030]

[29] In any embodiment of the above-described method, the subject may be a vertebrate. In the embodiment, the subject is a human.

[30] The disclosure also provides vaccine compositions or vaccine adjuvant compositions comprising an ALPK1 agonist, as well as pharmaceutical compositions comprising an ALPK1 agonist and a carrier. In embodiments of these compositions, the ALPK1 agonist is a compound represented by formula I, IA, IB, or IC as described herein, HBP, or a prodrug, analog, or derivative thereof. In embodiments, the ALPK1 agonist is selected from one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In embodiments, the ALPK1 agonist is compound 15. In embodiments, the ALPK1 agonist is a prodrug of HBP. In embodiments, the prodrug comprises a protecting group selected from the group consisting of carbonyloxymethyl, cyclosaligenyl, cyclic 1-aryl-1,3-propanyl ester, aryloxyphosphoramidate or phosphonoamidate, and methylaryl haloalkylamidate. In embodiments, the prodrug is a compound of formula 3a, 3b, 3c, 3d, or 3e. In embodiments, the prodrug is selected from the compounds in Table 1.

[0031]

[31] The disclosure also provides a method for selecting a compound capable of modulating an immune response in a mammalian subject, comprising the steps of contacting ALPK1 with the test compound in the presence of ATP and separately and simultaneously in the absence of ATP, and then performing an assay to detect ALPK1 phosphorylation and / or activation of one or more downstream targets of ALPK1 signaling. In embodiments, the step of contacting ALPK1 with the test compound is performed in a cell-free system or a cell system. In embodiments, assays for detecting ALPK1 phosphorylation and / or activation of one or more downstream targets of ALPK1 signaling include radiometric-based kinase assays, fluorescence-based kinase assays, time-resolved fluorescence energy transfer (TR-FRET)-based assays, alpha-technology-based assays, enzyme-conjugated immunosorbent assays, luminescence detection, mobility-shift-based kinase assays, Western-based kinase assays, and ligand-kinase binding assays.

[0032]

[32] According to any of the methods described herein, the ALPK1 agonist may be selected from the compounds represented by formula I, IA, IB, or IC as described herein, D-glycero-bD-manno-heptose-1,7-bisphosphate (HBP), D-glycero-bD-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L), and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), as well as prodrugs, analogs, and derivatives of any of the above molecules. In embodiments, the ALPK1 agonist is selected from HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is H1b-ADP or H1b-ADP-6L, and its derivatives as described herein. In the embodiment, the ALPK1 agonist is H1b-ADP. In the embodiment, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In the embodiment, the ALPK1 agonist is compound 15.

[0033]

[33] In embodiments, the Disclosure provides a vaccine composition or vaccine adjuvant composition comprising an ALPK1 agonist selected from compounds represented by formula I, IA, IB, or IC as described herein, HBP, HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is selected from HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is H1b-ADP or H1b-ADP-6L, and its derivatives as described herein. In embodiments, the ALPK1 agonist is H1b-ADP. In embodiments, the ALPK1 agonist is a compound represented by formula I, IA, IB, or IC as described herein. In embodiments, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In this embodiment, the ALPK1 agonist is compound 15.

[0034]

[34] In embodiments, the Disclosure provides pharmaceutical compositions comprising a compound represented by formula I, IA, IB, or IC as described herein, an ALPK1 agonist selected from HBP, HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is selected from HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is H1b-ADP or H1b-ADP-6L, and its derivatives as described herein. In embodiments, the ALPK1 agonist is H1b-ADP. In embodiments, the ALPK1 agonist is a compound represented by formula I, IA, IB, or IC as described herein. In embodiments, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In embodiments, the ALPK1 agonist is compound 15.

[0035]

[35] In embodiments, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, comprising the step of administering to the subject a composition comprising a compound represented by formula I, IA, IB, or IC as described herein, an ALPK1 agonist selected from the group consisting of HBP, HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is selected from HMP-1bP, H1b-ADP, and H1b-ADP-6L. In embodiments, the ALPK1 agonist is H1b-ADP or H1b-ADP-6L, and its derivatives as described herein. In embodiments, the ALPK1 agonist is H1b-ADP. In embodiments, the ALPK1 agonist is selected from any one of compounds 1-3, 9-17, 19, 20-22, and 26-32. In embodiments, the ALPK1 agonist is compound 15. In embodiments, the method further comprises the step of administering a PD-1 / PD-L1 inhibitor or an agonist of an immunocostimulatory molecule to a subject. In embodiments, the ALPK1 agonist is H1b-ADP, and the PD-1 / PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, and avelumab. In embodiments, the ALPK1 agonist is H1b-ADP, and the agonist of the immunocostimulatory molecule is an anti-OX40 (CD134) agonist antibody. According to the method described above, the subject may be a human subject, and the cancer may be one of the cancers described herein. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is refractory.

[0036]

[36] The present disclosure provides compositions for therapeutic use comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs and derivatives; or further provides compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0037]

[37] The Disclosure also provides compositions for use in methods of modulating an immune response in subjects requiring modulation of an immune response, comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs and derivatives; or compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0038]

[38] The Disclosure also provides compositions for use in methods of treating cancer in subjects requiring treatment for cancer, comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs and derivatives; or compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0039]

[39] The Disclosure also provides compositions for use in methods of enhancing an immune response in subjects requiring enhancement of an immune response, comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs and derivatives; or compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0040]

[40] The present disclosure is amendable to treat target cells by activating NFκB, p38, and JNK cell signaling pathways in target cells in which treatment is required for disease or disorder. The present invention also provides compositions for use in methods of treating a disease or disorder, comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L), and D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs, and derivatives; or compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0041]

[41] The present disclosure relates to compositions for use in the treatment or prevention of diseases or disorders caused by infectious pathogens selected from bacteria, viruses, or parasites in subjects requiring treatment or prevention of diseases or disorders caused by infectious pathogens selected from bacteria, viruses, or parasites, comprising D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), L-glycero- The present invention also provides compositions comprising D-manno-heptose-1β-ADP(H1b-ADP-6L) and D-glycero-D-manno-heptose-1β-ADP(H1b-ADP), and ALPK1 agonists selected from their prodrugs, analogs, and derivatives; or compositions comprising H1b-ADP-6L, H1b-ADP, or ALPK1 agonists selected from any one of the compounds described in claims 1 to 22 and their derivatives selected from any one of the compounds 1 to 33 in Table 1.

[0042]

[42] The Disclosure also provides a composition for use in a method of treating cancer in a subject requiring treatment for cancer, wherein the composition comprises an ALPK1 agonist selected from the group consisting of H1b-ADP-6L, H1b-ADP, or compounds described in any one of claims 1 to 22 and derivatives thereof selected from any one of compounds 1 to 33 in Table 1, wherein the method comprises a combination therapy of the ALPK1 agonist with an immunomodulator selected from one or more agonists of immune checkpoint regulators, immunostimulatory molecules, and immunocostimulatory molecules.

[0043]

[43] The Disclosure also provides compositions for use in methods of treating liver disease or disorder in subjects requiring treatment of liver disease or disorder, wherein the compositions comprise a low dose of H1b-ADP or a derivative thereof, and the liver disease or disorder is optionally selected from liver cancer, non-alcoholic steatohepatitis (NASH), and diseases or disorders caused by infection with hepatitis C virus (HCV) or hepatitis B virus (HBV).

[0044]

[44] The disclosure also provides compositions for use in methods of treating cancer, wherein the compositions comprise bacteria that produce H1b-ADP or H1b-ADP-6L, and which are optionally adapted for intratumoral injection. [Brief explanation of the drawing]

[0045] [Figure 1A]

[45] Figure (A) shows the protein sequence of ALPK1 isoform 1. [Figure 1B] Figure (B) shows the protein sequence of isoform 2. [Figure 2]

[46] Figure 2A is Figure (A) showing that IL-8 mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) HBP. Figure 2B is Figure (B) showing that TNFα mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) HBP. [Figure 3]

[47] Figure 3A is Figure (A) showing that IL-8 mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) HMP-1bP. Figure 3B is Figure (B) showing that TNFα mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) HMP-1bP. [Figure 4]

[48] ​​Figure 4A is Figure (A) showing that IL-8 mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) H1b-ADP. Figure 4B is Figure (B) showing that TNFα mRNA expression was increased in an ALPK1-dependent manner by (chemically synthesized) H1b-ADP. [Figure 5]

[49] Figure 5A is Figure (A) showing IL-8 mRNA expression induced by HBP, HMP-1bP, and H1b-ADP, respectively. Figure 5B is Figure (B) showing TNFα mRNA expression induced by HBP, HMP-1bP, and H1b-ADP, respectively. [Figure 6]

[50] This figure shows a thermal shift assay that reveals the binding of chemically synthesized HBP, HMP-1bP, and H1b-ADP to ALPK1 (only H1b-ADP binds). [Figure 7]

[51] This figure shows a cell-free kinase assay that reveals the phosphorylation of the ALPK1 substrate TIFA in the presence of chemically synthesized HBP, HMP-1bP, and H1b-ADP (TIFA is phosphorylated only in the presence of H1b-ADP). [Figure 8]

[52] This figure shows a cell-free kinase assay of ALPK1 autophosphorylation in the presence of (chemically synthesized) H1b-ADP. [Figure 9]

[53] This figure shows a cell-free kinase assay of ALPK1-dependent phosphorylation of IκB in the presence of (chemically synthesized) H1b-ADP. [Figure 10]

[54] This figure shows a cell-free kinase assay elucidating the phosphorylation of the ALPK1 substrate TIFA in the presence of chemically synthesized H1b-ADP and H1b-ADP-6L. [Figure 11]

[55] This figure shows that intratumoral injection of H1b-ADP inhibits tumor growth in a mouse CT26 xenograft model, but HMP-1bP does not. [Figure 12]

[56] This figure shows that intratumoral injection of H1b-ADP increases the expression of cytokines and PD-1 and PD-L1. [Figure 13]

[57] Figure 13A shows that intratumoral injection of H1b-ADP and anti-PD-1 antibody (RMP1-14) synergistically inhibits tumor growth in injected tumors of a mouse CT26 xenograft model (Figure A). Figure 13B shows that intratumoral injection of H1b-ADP and anti-PD-1 antibody (RMP1-14) synergistically inhibits tumor growth in distal tumors of a mouse CT26 xenograft model (Figure B). [Figure 14]

[58] This figure shows that intratumoral injection of H1b-ADP and anti-PD-1 antibody (OX40) synergistically inhibits tumor growth in a mouse CT26 xenograft model. [Figure 15]

[59] This figure shows the Western blotting analysis of phospho-TIFA after in vitro kinase reaction of ALPK1-dependent TIFA phosphorylation in the presence of HBP+HIda purified from HIDE mutant Escherichia coli (left) or HBP+HIda purified from wild-type Escherichia coli (right). [Figure 16]

[60] Figure 16A shows that intratumoral injection of H1b-ADP and anti-PD-L1 antibody synergistically inhibits tumor growth in injected tumors of a mouse CT26 xenograft model (Figure A). Figure 16B shows that intratumoral injection of H1b-ADP and anti-PD-L1 antibody synergistically inhibits tumor growth in distal tumors of a mouse CT26 xenograft model (Figure B). [Figure 17]

[61] Figure 17A shows that intratumoral injection of H1b-ADP and IFN-a synergistically inhibits tumor growth in injected tumors of a mouse CT26 xenograft model (Figure A). Figure 17B shows that intratumoral injection of H1b-ADP and IFN-a synergistically inhibits tumor growth in distal tumors of a mouse CT26 xenograft model (Figure B). [Figure 18]

[62] This figure shows that intratumoral injection of H1b-ADP and anti-CTLA-4 antibody synergistically inhibits tumor growth in a mouse CT26 xenograft model. Paired p-values ​​were determined by T-scan and are shown by the vertical lines on the right: *p<0.05, **p<0.01, ***p<0.001. [Figure 19]

[63] This figure shows that intratumoral injection of H1b-ADP and the STING agonist c-di-AM(PS)2 synergistically inhibits tumor growth in a mouse CT26 xenograft model. Paired p-values ​​were determined by T-scan and are shown by the vertical lines on the right: *p<0.05, **p<0.01, ***p<0.001. [Figure 20]

[64] This figure shows that intratumoral injection of H1b-ADP and anti-CD4 antibody synergistically inhibits tumor growth in a mouse CT26 xenograft model. [Figure 21]

[65] This figure shows that intratumoral injection of H1b-ADP and the TLR agonist resquimod synergistically inhibits tumor growth in a mouse CT26 xenograft model. [Figure 22]

[66] This figure shows that fetal bovine, human, and mouse serum reduce H1b-ADP activity when inducing IL8 secretion in HEK293 cells. [Figure 23]

[67] This figure shows that Na3VO4 protects H1b-ADP from degradation by fetal bovine serum. [Figure 24]

[68] This figure shows that Na3VO4 protects H1b-ADP from degradation by fetal bovine serum and retains its activity to induce IL8 secretion in HEK293 cells. [Figure 25]

[69] This figure shows that AMP protects H1b-ADP from degradation by fetal bovine serum and retains its activity to induce IL8 secretion in HEK293 cells. [Figure 26]

[70] This figure shows that compounds of formula I(1, 2, 9-12) activate IL8 secretion in HEK293 cells by activating ALPK1. HEK293 cells were cultured without FBS. [Figure 27]

[71] Figure 27A is a diagram (A) showing that compounds of formula I(3, 15, 19, 20) activate IL8 secretion in HEK293 without FBS by activating ALPK1. Figure 27B is a diagram (B) showing that compounds of formula I(3, 15, 19, 20) activate IL8 secretion in HEK293 using 10% FBS by activating ALPK1. Compound 15 is resistant to FBS degradation. [Figure 28]

[72] Figure 28A is a figure (A) showing that compounds of formula I (5, 13, 14, 17, 21, 22) activate ALPK1, as demonstrated by increased IL8 secretion in HEK293 cells without FBS. Figure 28B is a figure (B) showing that compounds of formula I (5, 13, 14, 17, 21, 22) activate ALPK1, as demonstrated by increased IL8 secretion in HEK293 cells using 10% FBS. [Figure 29]

[73] Figure 29A is a figure (A) showing that the compound of formula I(16, 26-32) activates ALPK1, as demonstrated by increased IL8 secretion in HEK293 cells without FBS. Figure 29B is a figure (B) showing that the compound of formula I(16, 26-32) activates ALPK1, as demonstrated by increased IL8 secretion in HEK293 cells using 10% FBS. [Figure 30]

[74] This figure shows that the compound of formula I(1,2) inhibits tumor growth in a CT26 syngeneic mouse tumor model. [Figure 31]

[75] This figure shows that mouse bone marrow-derived macrophages can be activated by very low concentrations of H1b-ADP. [Figure 32]

[76] Figure 32A shows that compound 1 activates the inflammatory response in liver tissue at a low dose of 2 nmole (1.2 μg). Figure 32B shows that compound 1 activates the inflammatory response in lung tissue at a dose of 200 nmole. [Figure 33]

[77] This is a schematic diagram showing the bacterial H1b-ADP biosynthesis pathway. [Modes for carrying out the invention]

[0046]

[78] The present disclosure provides compositions and methods relating to the therapeutic activation of ALPK1 using a suitable agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof. definition

[79] As used herein, the term “ALPK1” may refer to one of the two splice variants of the human ALPK1 gene, isoform 1 or isoform 2. Each isoform shares the same kinase domain. For reference, the human ALPK1 gene is identified by Entrez Gene ID 80216.

[0047]

[80] As used herein, the term “activation of ALPK1” means activation of ALPK1 kinase activity. In embodiments, the Disclosure provides a method for activating ALPK1 by providing an ALPK1 agonist which can be an ALPK1 activating ligand, such as HBP or its prodrug, analog, or derivative. Methods for producing synthetic HBP are known, for example, as described in Inuki S et al., Organic Letters 2017 19(12):3079-82. In embodiments, the ALPK1 agonist is selected from HMP-1bP and H1b-ADP, as well as their prodrugs, analogs, and derivatives. In embodiments, the ALPK1 agonist is H1b-ADP or its prodrug, analog, or derivative. In some embodiments, the Disclosure provides a method for activating ALPK1 by providing an ALPK1 agonist represented by formula I, IA, IB, or IC.

[0048]

[81] As used herein, the term “alkyl” refers to a linear or branched saturated aliphatic group having the indicated number of carbon atoms. Alkyl is C 1~2 , C1~3 , C 1~4 , C 1~5 , C 1~6 , C 1~7 , C 1~8 , C 1~9 , C 1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 and C 5~6 etc., and can contain any number of carbons. For example, C 1~6 Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbon atoms, such as heptyl, octyl, nonyl, decyl, etc., but are not limited thereto. The alkyl group may or may not be substituted. In some embodiments, the alkyl group is substituted with 1 to 2 substituents. Non-limiting examples of suitable substituents include halogen and hydroxyl.

[0049]

[82] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon having at least 2 carbon atoms and at least 1 double bond. Alkenyl is C2, C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 2~7 , C 2~8 , C 2~9 , C 2~10 , C3, C 3~4 , C 3~5 , C 3~6 , C4, C 4~5 , C 4~6 , C5, C 5~6The alkenyl group may contain any number of carbon atoms, including C6. The alkenyl group may have any number of double bonds, including but not limited to 1, 2, 3, 4, 5 or more. The alkenyl group may be substituted or unsubstituted.

[0050]

[83] As used herein, the term “alkylene” means a linear or branched saturated aliphatic group, i.e., a divalent hydrocarbon group, having the indicated number of carbon atoms and linking at least two other groups. The two parts linked to the alkylene may be linked to the same or different atoms of the alkylene group. For example, a linear alkylene may be a divalent group of -(CH2)n- (where n is 1, 2, 3, 4, 5, or 6). Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene. The alkylene group may be substituted or unsubstituted. In some embodiments, the alkylene group is substituted with one or two substituents. Non-limiting examples of preferred substituents include halogens and hydroxyls.

[0051]

[84] As used herein, the terms “alkoxy” or “alkoxyl” refer to an alkyl group having an oxygen atom connected to the alkyl group at the bonding site: alkyl-O-. With respect to alkyl groups, alkoxyl groups may have any preferred number of carbon atoms, such as C1 to C6. Examples of alkoxyl groups include methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. The alkoxy group may be substituted or unsubstituted.

[0052]

[85] As used herein, the terms “alkenyloxy” or “alkenyloxyl” refer to the alkenyl group as defined above, having an oxygen atom that connects the alkenyl group to a bonding site: alkenyl-O-. The alkenyloxyl group may have any number of carbon atoms, such as C1 to C6. The alkenyloxyl group may be further substituted with various substituents listed within the range. The alkenyloxyl group may be substituted or unsubstituted.

[0053]

[86] As used herein, the terms “alkylamine” or “alkylamino” refer to an alkyl group having a nitrogen atom connected to the alkyl group at the bonding site: alkyl-N-. With respect to alkyl groups, the alkoxyl group may have any preferred number of carbon atoms, such as C1-6.

[0054]

[87] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[88] As used herein, the term “haloalkyl” refers to the alkyl groups defined above, in which some or all of the hydrogen atoms are replaced by halogen atoms. With respect to alkyl groups, haloalkyl groups are C 1~6 It can have any suitable number of carbon atoms, such as trifluoromethyl and fluoromethyl. For example, examples of haloalkyls include trifluoromethyl and fluoromethyl.

[0055]

[89] As used herein, the terms “haloalkoxyl” or “haloalkoxy” refer to an alkoxyl group in which some or all of the hydrogen atoms are replaced by halogen atoms. With respect to alkyl groups, a haloalkoxy group is C 1~6 It can have any suitable number of carbon atoms, such as the following. The alkoxy group can be substituted with one, two, three, or more halogens.

[0056]

[90] As used herein, the term “alkanoyl” means an alkyl group having a carbonyl group attached to the alkyl group at the bonding site: alkyl-C(O)-. With respect to alkyl groups, the alkanoyloxyl group may have any preferred number of carbon atoms, such as C1 to C4. For example, alkanoyl groups include acetyl, propionyl, and butyryl.

[0057]

[91] As used herein, the term “alkanoyloxyl” refers to an alkanoyl group having an oxygen atom connected to the alkanoyl group at the bonding site: alkyl-C(O)-O-. With respect to the alkyl group, the alkanoyloxyl group may have any preferred number of carbon atoms, such as C1-C4. Examples of alkanoyloxyl groups include acetoxy, propionyloxy, and butyryloxy.

[0058]

[92] As used herein, the term “oxo” refers to an oxygen atom (=O) connected to a bond by a double bond.

[93] As used herein, the term “aryl” refers to an aromatic ring system having any preferred number of ring atoms and any preferred number of rings. An aryl group may include any preferred number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, and 6-10, 6-12, or 6-14 ring members. An aryl group may be a monocyclic aryl group that condenses to form a bicyclic or tricyclic group, or is linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl having a methylene linkage. Some aryl groups have 6-12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6-10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. The aryl group may be substituted or unsubstituted. In some embodiments, the aryl group is substituted with one or two substituents. As non-limiting examples, suitable substituents include halogens, hydroxyls, -NO2, C1-8 alkyls, and C1-8 alkoxys.

[0059]

[94] As used herein, the term “aralkyloxyl” refers to the aryl group as defined above, having an alkyl and an oxygen atom connected to the bond site of the aryl group: aryl-alkyl-O-. With respect to the alkyl group, the aralkyloxyl group may have any preferred number of carbon atoms, such as C1-4.

[0060]

[95] As used herein, the term “heteroaryl” refers to a monocyclic or fused bicyclic aromatic ring assembly comprising 5 to 12 ring atoms, of which 1 to 5 are heteroatoms such as N, O, or S. Additional heteroatoms, including but not limited to B, Al, Si, and P, may also be useful. Heteroatoms can also be oxidized, but are not limited to -S(O)- or -S(O)2-. Heteroaryl groups may contain any number of ring atoms, such as 3-6, 4-6, 5-6, 3-8, 4-8, 5-8, 6-8, 3-9, 3-10, 3-11, or 3-12 ring members. A heteroaryl group may contain any number of heteroatoms, such as 1, 2, 3, 4, or 5, or 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5. Heteroaryl groups can have 5-9 ring members and 1-4 heteroatoms, or 5-9 ring members and 1-3 heteroatoms, or 5-6 ring members and 1-4 heteroatoms, or 5-6 ring members and 1-3 heteroatoms. Examples of heteroaryl groups include pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), and purines. Heteroaryl groups can also be condensed with aromatic ring systems such as phenyl rings to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include bipyridine, which is a heteroaryl ring linked by a bond. The heteroaryl group may be substituted or unsubstituted.

[0061]

[96] As used herein, “cycloalkyl” refers to a saturated ring set containing 3 to 8 ring atoms or the indicated number of atoms. Cycloalkyl is C 3~6 , C 4~6 , C 5~6 , C 3~8 , C4~8 , C 5~8 , C 6~8 It can contain any number of carbon atoms. Examples of cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. The cycloalkyl group may be substituted or unsubstituted.

[0062]

[97] As used herein, “cycloheteroalkyl” refers to a saturated ring system having 3 to 12 ring members and 1 to 4 N, O, and S heteroatoms. Additional heteroatoms, including but not limited to B, Al, Si, and P, may also be useful. Heteroatoms can also be oxidized, but are not limited to -S(O)- or -S(O)2-. Heterocycloalkyl groups may contain any number of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any number of heteroatoms may be included in a heterocycloalkyl group, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Examples of heterocycloalkyl groups include aziridine, azetidine, pyrrolidine, piperidine, azepan, azocan, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3-, and 1,4-isomers), oxirane, tetrahydrofuran, oxane (tetrahydropyran), oxepan, thiolane (tetrahydrothiophene), thian (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, and morpholine. Heterocycloalkyl groups may be unsubstituted or substituted. For example, heterocycloalkyl groups include, in particular, C 1~6 It can be substituted with alkyl or oxo (=O) atoms.

[0063]

[98] Some of the compounds of the present invention have an asymmetric carbon atom (optical center) or a double bond. Racemates, diastereomers, geometric isomers, positional isomers and individual isomers (e.g., distinct enantiomers) are all intended to be included within the scope of the present invention. In some embodiments, the compounds of the present invention are specific enantiomers, anomers, or diastereomers that substantially do not contain other forms.

[0064]

[99] As used herein, “substantially not” means an amount of 10% or less of another form, preferably 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less of another form. In some embodiments, the isomers are stereoisomers. Detailed description of the embodiment

[0100] In some embodiments, the disclosure provides an ALPK1 agonist represented by formula (I), and / or its stereoisomers, tautomers, stable isotopes, prodrugs, or pharmaceutically acceptable salts.

[0065] [ka]

[0066] [In the formula, A 1 and A 2 These are independently O, S, and -C(R 8 R 9 )- Selected from, R 8 and R 9 A is independently selected from H, D, -OH, N3, -CN, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl and substituted or unsubstituted aralkyloxyl (optional substituents are 1 to 3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl and C1-C4 alkoxy), and A 1 Or A 2 At least one of them is -C(R 8 R9 ) and A 1 In R 8 or R 9 is, A 2 In R 8 or R 9 It can be cyclized to form a C3-C6 cycloalkyl and a cycloheteralkyl having 3-9 ring members, with 1-3 heteroatoms selected from N, O, and S as ring members, each optionally substituted with 1-3 substituents independently selected from D, halogens, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. L 1 and L 2 These are independently selected from O, CH2, CHF, and CF2. L 3 These are O, S, CH2, or CH(OH), Z 1 and Z 2 These are independently selected from O and S, W 1 -C(R 10 R 11 )- and R 10 and R 11 These are independently H, D, -OH, halogens, as well as C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4-haloalkoxyl, C1-C4 alkenyloxyl, aralkyloxyl and R 12 CO2-(R 12 R is selected from groups that are optionally substituted from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members, 10 and R 11The optional substituents are 1 to 3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. W 2 These are H, or D, halogen, -OH, =O, C1-C3 alkoxyl, C1-C3 haloalkyl, C1-C3 haloalkoxyl, C1-C3 alkenyloxyl and R 12 CO2-(R12 is a C1-C3 alkyl group that is optionally substituted with 1-3 substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteroalkyl, C6-C10 aryl, and heteroaryl, which contains 5-10 ring atoms and has 1-3 heteroatoms selected from N, O, and S as ring members), R 1 This is a heteroaryl compound containing C6-C10 aryls, or 5-10 ring atoms, and having 1-4 heteroatoms selected from N, O, and S as ring members, R 1 D, halogen, -OH, =O, CN, NH2, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamine, C1-C4 dialkylamine and (R 13 R 14 )NCO- is optionally substituted with 1 to 3 substituents selected from R 13 and R 14 These are independently selected from H, C1-C4 alkyl, C3-C6 cycloalkyl, cycloheteralkyl, C6-C10 aryl, C6-C10 aryl, and heteroaryl, which comprises 5-10 ring atoms and has 1-3 heteroatoms selected from N, O, and S as ring members. R 2 , R 3 and R 4 These are independently selected from H, D, halogens, C1-C4 alkyls and C1-C4 haloalkyls. R 5 , R 6and R 7 H, D, halogens and -OH, R 12 CO2-(R 12 (Selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteroalkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members), R 5 , R 6 and R 7 Any two of the adjacent groups can be cyclized to form a cycloheteralkyl group containing 5 to 9 ring members, with 1 to 3 heteroatoms selected from N, O, and S as ring members, each optionally substituted by 1 to 3 substituents independently selected from D, halogens, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy.

[0067]

[0101] In some embodiments, the compound of formula I is represented by the compound of formula IA and / or its stereoisomers, stable isotopes, prodrugs, or pharmaceutically acceptable salts.

[0068] [ka]

[0069] [In the formula, Y 1 and Y 2 The group is independently selected from H, D, -OH, N3, -CN, halogens, and optionally substituted groups selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl, and aralkyloxyl, where the optional substituents are 1 to 3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. R 1 ~R 7 、L 1 ~L 3 、Z 1 、Z 2 、W 1 およびW 2 are as defined above.

[0070]

[0102] In some embodiments, Y in the compound of formula IA 1 and Y 2 are independently selected from H, D, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, and C1-C4 alkenyloxyl, and R 1 -R 7 、L 1 -L 3 、Z 1 、Z 2 、W 1 およびW 2 are as defined above.

[0071]

[0103] In some embodiments, Y in the compound of formula IA 1 およびY 2 are independently selected from -OH, halogen, C1-C4 alkyl, and C1-C4 alkanoyloxyl, and R 1 ~R 7 、L 1 ~L 3 、Z 1 、Z 2 、W 1 およびW 2 are as defined above.

[0072]

[0104] In some embodiments, the compound of formula I or formula IA is also D-glycero-D-manno-heptose-1β-ADP (also referred to herein as H1b-ADP or H1b-D-ADP), the compound shown below

[0073] [ka]

[0074]

[0105] It also does not contain its diastereomer L-glycero-D-manno-heptose-1β-ADP (also known herein as H1b-ADP-6L or H1b-L-ADP).

[0075]

[0106] In some embodiments, the compound of formula I is represented by the compound of formula IB and / or its stereoisomers, stable isotopes, prodrugs, or pharmaceutically acceptable salts.

[0076] [ka]

[0077] [In the formula, n 1 and n 2 Each of these is an integer independently selected from the group consisting of 0 to 2. X 1 and X 2 The group is independently selected from H, D, -OH, N3, -CN, halogens, and optionally substituted groups selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl, and aralkyloxyl, where the optional substituents are 1 to 3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. R 1 ~R 7 , L 1 ~L 3 , Z 1 , Z 2 , W 1 and W 2 This is defined as described above.

[0078]

[0107] In some embodiments, n of formula IB1 and n 2 These values ​​are both 0.

[0108] In some embodiments, X of formula IB 1 and X 2 R is independently selected from H, D, C1-C4 alkoxyls and C1-C4 alkyls, 1 ~R 7 , L 1 ~L 3 , Z 1 , Z 2 , W 1 and W 2 This is defined as described above.

[0079]

[0109] In some embodiments, the compound of formula I is represented by the compound of formula IC and / or its stereoisomers, stable isotopes, prodrugs, or pharmaceutically acceptable salts.

[0080] [ka]

[0081] [In the formula, A 1 -C(R 10 R 11 )-, O or S, R 1 ~R 9 , L 1 ~L 3 , Z 1 , Z 2 , W 1 and W 2 This is defined as described above.

[0082]

[0110] In some embodiments, in formulas I, IA, IB, and IC, R 2 , R 3 , and R 4 These are H.

[0111] In some embodiments, in formulas I, IA, IB, and IC, R 5 , R 6 , and R7 is independently selected from the group consisting of -OH and C1-C4 alkanoyloxyl-.

[0083]

[0112] In some embodiments, in Formulas I, IA, IB, and IC, L 3 is O.

[0113] In some embodiments, in Formulas I, IA, IB, and IC, L 2 is O.

[0114] In some embodiments, in Formulas I, IA, IB, and IC, L 1 is O or S.

[0084]

[0115] In some embodiments, in Formulas I, IA, IB, and IC, W 1 is -C(R 10 R 11 )-, and R 10 and R 11 and R 11 are independently H, D, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4-haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl, R 12 CO2-(R 12 where R 12 is selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkanoyloxyl and C1-C4 alkenyloxyl). 12 is selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkanoyloxyl and C1-C4 alkenyloxyl).

[0085]

[0116] In some embodiments, in Formulas I, IA, IB, and IC, W 1 is -C(R 10 R 11 )-, and R 10 and R 11 and R 11 are independently selected from H, D, -OH, halogen and C1-C4 alkanoyloxyl. <00(00981>

[0117] In some embodiments, in Formulas I, IA, IB, and IC, W 2These include D, halogen, -OH, =O, C1-C3 alkoxyl, C1-C3 haloalkyl, C1-C3 haloalkoxyl, C1-C3 alkenyloxyl and R 12 CO2-(R 12 The C1-C3 alkyl group is optionally substituted with 1-3 substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 alkylamino groups.

[0087]

[0118] In some embodiments, in formulas I, IA, IB, and IC, W 2 D, halogen, -OH and R 12 CO2-(R 12 (is a C1-C3 alkyl group) is a C1-C3 alkyl group that is optionally substituted with 1-3 substituents independently selected from the C1-C3 alkyl group.

[0088]

[0119] In some embodiments, in formulas I, IA, IB, and IC, W 2 -OH and R 12 CO2-(R 12 It is a C1 alkyl group that is optionally substituted with one substituent selected from C1-C3 alkyl groups.

[0089]

[0120] In some embodiments, in formulas I, IA, IB, and IC, R 1 teeth,

[0090] [ka]

[0091] That is the case.

[0121] In some embodiments, in formulas I, IA, IB, and IC, R 1 teeth,

[0092] [ka]

[0093] That is the case.

[0122] In some embodiments, in formulas I, IA, IB, and IC, R 1 teeth,

[0094] [ka]

[0095] That is the case.

[0123] In some embodiments, the compound of formula I is

[0096] [ka]

[0097] [ka]

[0098] [ka]

[0099] and / or their stereoisomers, stable isotopes, prodrugs, or pharmaceutically acceptable salts.

[0124] In some embodiments, the compound of formula I is the compound described in the examples of this application.

[0100]

[0125] The compounds of this disclosure may be prepared using the general processes described in Schemes I, II, III, and IV, as well as the techniques described in the exemplary embodiments.

[0126] In embodiments, the Disclosure provides ALPK1 agonists in the form of small organic molecules such as D-glycero-β-D-manno-heptose-1,7-bisphosphate (heptose-1,7-bisphosphate or "HBP"), D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP), D-glycero-D-manno-heptose-1β-ADP (H1b-ADP), and L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L), as well as prodrugs, analogs, and derivatives thereof, or large biomolecules such as proteins (e.g., ALPK1 itself, or an antibody against ALPK1 or its Fc fragment that activates ALPK1 kinase activity) or polynucleotides (e.g., polynucleotides encoding ALPK1).

[0101]

[0127] In embodiments, the Disclosure provides a method for treating cancer by administering an ALPK1 agonist selected from HBP, HMP-1bP, H1b-ADP-6L, and H1b-ADP, preferably HMP-1bP, H1b-ADP-6L, and H1b-ADP, most preferably H1b-ADP-6L and H1b-ADP. In a further embodiment of the method for treating cancer, the Disclosure provides a combination therapy comprising the step of administering an ALPK1 agonist selected from H1b-ADP-6L and H1b-ADP and an immune checkpoint modulator selected from checkpoint inhibitors such as anti-PD-1 / PD-L1 antibodies and agonists of immune costimulatory molecules such as anti-OX40 (CD134) agonist antibodies. Without being bound by any particular theory, the inventors propose that H1b-ADP and similar molecules such as H1b-ADP-6L can promote the antigen-presenting function of tumor-infiltrating antigen-presenting cells (APCs), as well as tumor-specific T cell proliferation and differentiation. Furthermore, these molecules increase tumor-specific CD8 expression by increasing PD-L1 expression in tumor cells. + This can also enhance the recruitment of T cells to tumors.

[0102]

[0128] In other embodiments, the Disclosure provides a method for activating ALPK1 by administering ALPK1 to a target or introducing ALPK1 into cells, e.g., target cells or tissues, in the form of a recombinant protein or a polynucleotide encoding ALPK1, or in the form of a composition comprising a recombinant ALPK1 protein or a polynucleotide encoding it. The polynucleotide encoding ALPK1 is a polynucleotide that, when controlled by an appropriate regulatory sequence, e.g., a promoter sequence, is transcribed and translated into the ALPK1 protein. Such polynucleotides may include sequences derived from prokaryotic or eukaryotic DNA, or synthetic DNA sequences, and any combination thereof.

[0103]

[0129] Preferably, the administered or introduced ALPK1 is constitutively active ALPK1 (or the polynucleotide encoding it). The term “constitutively active” refers to an ALPK1 protein whose kinase activity is active in the absence of a ligand. In embodiments, constitutively active ALPK1 contains an activating mutation in its N-terminal domain that promotes ligand-independent oligomerization and kinase activation.

[0104]

[0130] The polynucleotide encoding ALPK1 can take the form of a nucleic acid vector or other vehicle suitable for gene transfer into living cells. Plasmids are a common type of nucleic acid vector, being extrachromosomal DNA molecules that can replicate independently of chromosomal DNA. Plasmids can be single-stranded or double-stranded and are often circular. Other useful vehicles include DNA or RNA minicircles and minivectors. Minicircles are formed by removing most of the bacterial DNA from a parent plasmid using site-directed recombination. The resulting circular DNA molecule contains the desired gene sequence to be transferred, e.g., the ALPK1 sequence, and only a small amount of bacterial DNA. Minivectors are similar, except they contain a short integration sequence. Other suitable non-viral DNA vectors for gene transfer are described, for example, Hardee et al., "Advances in Non-Viral DNA Vectors for Gene Therapy," Genes 2017 8:65.

[0105]

[0131] Other suitable nucleic acid vectors for ALPK1 gene transfer include viral vectors such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentiviral vectors.

[0106]

[0132] Nucleic acid vectors encoding ALPK1 can be introduced into target cells using preferred techniques, such as direct injection including viral delivery systems and gene guns, or nonviral delivery systems, including liposomes, nanoparticles, polymers, electroporation, cell compression, sonoporation, optical transfection, impale infection, and hydrodynamic delivery. Exemplary nonviral delivery systems and their uses are described, for example, by Jones et al., “Contemporary approaches for nonviral gene therapy,” Discov. Med. 2015;19:447-454.

[0107]

[0133] According to any embodiment of the methods described herein, ALPK1 may be administered in a suitable formulation, including, for example, viral particles, liposome particles, or nanoparticles, as a complex with a polymer carrier, including polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Liposome particles may be used to deliver ALPK1 in various forms, including DNA, RNA, and plasmids. In embodiments, ALPK1 polynucleotides may be administered as plasmid DNA in the absence of another particle or carrier.

[0108]

[0134] In embodiments, a polynucleotide encoding ALPK1 or an active variant thereof is inserted into a cell using gene editing techniques. Gene editing techniques include meganucleases, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), and CRISPR / Cas-9 based techniques.

[0109]

[0135] In embodiments, the present disclosure provides a method for modulating an immune response in a subject, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant of said protein.

[0110]

[0136] In embodiments, the Disclosure provides a method for enhancing an immune response to a target antigen in a subject, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof. In embodiments, the target antigen may be an antigen of an infectious pathogen, such as a bacterial antigen, a viral antigen, or a parasitic antigen. In embodiments, the antigen is a tumor antigen. According to any of these embodiments, the ALPK1 agonist, polynucleotide, or protein described herein may serve as an adjuvant to a vaccine composition for the treatment or prevention of a disease or disorder caused by an infectious pathogen, or for the treatment of cancer, or for the treatment of another disease or disorder that may be treated with the vaccine composition, including, for example, Alzheimer's disease. In embodiments, the antigen is selected from amyloid proteins in the treatment of Alzheimer's disease. In embodiments, the antigen is selected from glycoprotein 100 (gp100), mucin 1 (MUC1), and melanoma-associated antigen 3 (MAGEA3) in cancer treatment. In embodiments, the cancer is selected from breast cancer, ovarian cancer, or prostate cancer. In embodiments, the cancer is HTLV-1T lymphotropic leukemia.

[0111]

[0137] In embodiments, the cancer is melanoma, and the ALPK1 agonists, polynucleotides, or proteins described herein may serve as adjuvants to treatment with tarimozine-laharpalepbec (T-VEC) or may be used in combination therapy regimens with T-VEC.

[0112]

[0138] In embodiments for the treatment or prevention of infectious diseases, the ALPK1 agonists, polynucleotides, or proteins described herein can serve as adjuvants to vaccine compositions for the treatment or prevention of meningococcal diseases, including anthrax, caries, Chagas disease, dengue fever, diphtheria, ehrlichiosis, hepatitis A or B, herpes, seasonal influenza, Japanese encephalitis, leprosy, Lyme disease, malaria, measles, mumps, meningitis and sepsis, onchocerciasis (river blindness), pertussis, pneumococcal disease, polio, rabies, rubella, schistosomiasis, severe acute respiratory syndrome (SARS), herpes zoster, smallpox, syphilis, tetanus, tuberculosis, tularemia, tick-borne encephalitis virus, typhoid fever, trypanosomiasis, yellow fever, and visceral leishmaniasis.

[0113]

[0139] In embodiments for the treatment or prevention of infectious diseases, the ALPK1 agonists, polynucleotides, or proteins described herein can serve as adjuvants to vaccine compositions for the treatment or prevention of diseases or disorders caused by adenoviruses, coxsackie B viruses, cytomegaloviruses, Eastern equine encephalitis virus, Ebola virus, Enterovirus 71, Epstein-Barr virus, Haemophilus influenzae type b (Hib), Hepatitis C virus (HCV), herpesviruses, human immunodeficiency virus (HIV), human papillomavirus (HPV), hookworms, Marburg virus, norovirus, polynuclear respiratory virus (RSV), rotavirus, Salmonella typhi, Staphylococcus aureus, Streptococcus pyogenes, varicella, West Nile virus, Plague bacillus, and Zika virus.

[0114]

[0140] According to any of the embodiments described above, the method may include administering a vaccine composition or adjuvant comprising one of the following: an ALPK1 agonist, preferably selected from HBP, HMP-1bP, H1b-ADP-6L, and H1b-ADP; or an ALPK1 agonist selected from HMP-1bP, H1b-ADP-6L, and H1b-ADP, most preferably an ALPK1 agonist selected from H1b-ADP-6L and H1b-ADP; a polynucleotide encoding ALPK1 or a constitutively active variant thereof; or the ALPK1 protein or a constitutively active variant of said protein.

[0115]

[0141] In embodiments, the present disclosure provides a method for treating a disease or disorder that is amendable to treatment by activation of NFκB, p38, and JNK cell signaling pathways in target cells, comprising the step of administering to target a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof. In embodiments, the disease or disorder is caused by bacterial, viral, or parasitic infections, as described in more detail below, including, for example, diseases and disorders caused by hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). In embodiments, the disease or disorder is selected from tuberculosis, meningitis, pneumonia, ulcers, and sepsis. In embodiments, the disease or disorder is selected from rhinitis, asthma, allergy, COPD, inflammatory bowel disease, arthritis, obesity, radiation-induced inflammation, psoriasis, atopic dermatitis, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, systemic lupus, lupus erythematosus (SLE), autoimmune thyroiditis (Graves' disease), multiple sclerosis, ankylosing spondylitis, and bullous diseases. In embodiments, the disease or disorder is selected from actinic keratosis, ulcerative colitis, Crohn's disease, and alopecia areata.

[0116]

[0142] In embodiments, the present disclosure provides a method for treating or preventing a bacterial, viral, or parasitic infection in a subject requiring treatment or prevention of such infection, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof.

[0117]

[0143] In embodiments, the method is a method for treating or preventing a bacterial infection. In embodiments, the bacterial infection is caused by Gram-negative or Gram-positive bacteria. In this embodiment, the bacteria include Acinetobacter baumanii, Aggregatobacter actinomycetemcomitans, Bartonella basiliformis, Bartonella henselae, Bartonella quintana, Bifidobacterium, Borrelia, Bortadella pertussis, Brucella species, Burkholderia cepacis, Glandworm, Campylobacter jejuni, Cardiobacterium hominis, Campylobacter phytus, Chlamydia pneumoniae, and Chlamydia trachoma. Trachomatis, Clostridium difficile, cyanobacteria, Eikennella corrodens, Enterobacter species, Enterococcus faccium, Escherichia coli, Escherichia coli O157, Franceilla tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Haemophilus afrofilus, Chancroid, Parainfluenza bacillus, Helicobacter pylori, Kingella, Klebsiella pneumoniaePneumonia, Legionella, Legionella pneumophila serogroup 1, Leptospira, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus mirabilis, Proteus vulgaris, Proteus micsophaciens, Providencia rettgeri, Providencia alkalifaciens, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas pausimovilis, Ptida, fluorescent bacteria, Pseudomonas acidovorans, Rickettsia, Salmonella, Salmonella typhi, Salmonella paratyphi A, Salmonella B, Salmonella dublin, Salmonella Arizona, Salmonella choleresus, Shigella, Shigella dysenteriae, Shigella flexner It is a Gram-negative bacterium selected from the group consisting of flexneri, Schigella boydii, Schigella sonnei, Treponema species, Stenotrophomonas maltophilia, Vibrio cholerae, Vibrio mimicus, Vibrio arginoticus, Vibrio horrisae, Vibrio parahaemolyticus, Vibrio brunificus, and Yersinia pestitis.

[0118]

[0144] In embodiments, the bacteria include Actinomycetes, Bacillus anthracis, Bacillus subtilis, Clostridium perfingens, Clostridium botulinum, Clostridium tetani, Bacillus diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix ruhsiopathiae, Listeria monocytogenes, Mycoplasma, Nocardia, Propionibacterium, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus aureus (VRSA), Staphylococcus rugduensis, Staphylococcus saprophyticus, and Streptococcus pneumoniae. It is a Gram-positive bacterium selected from the group consisting of *Streptococcus pneumoniae*, *Streptococcus pyogenes*, and *Streptococcus mutans* (Streptococcus mutants).

[0119]

[0145] In one embodiment, the method is a method for treating or preventing a viral infection. In the embodiment, viral infections include adeno-associated virus, Aichi virus, Alpha virus, Arena virus, Arbovirus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Birnavirus, Bornavirus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Valicivirus, Cercopithecine herpesvirus, Chandipla virus, Chikungunya virus, and Cosavirus A. A) Cowpox virus, Coxsakievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Devenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, Flavivirus, GB virus / Hepatitis G virus, Hantan virusHepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Herpes simplex virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, 70, Human herpesvirus 1, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus (HIV), Human papillomavirus (HPV-6, HPV-11), Human spumaretrovirus, Human T lymphotropic virus T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, Kaposi's sarcoma (HHV-8), KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Vitoria marbugvirus, Langat virus, Lassa virus, LMC virus, Lordsdale virus, Louping ill virus, Lymphocytic choriomeningitis virusChoriomeningitis virus, Machupovirus, Marmath forest virus, Mayaro virus, MERS coronavirus, measles virus, Mengo encephalomycarditis virus, Merkel cell polyomavirus, molluscum contagiosum virus, parvovirus B19, Mokola virus, mumps virus, Murray valley encephalitis virus, New York virus, Nipha virus, Norwalk virus, O'nyong-hyong virus, Orf virus, Oropouche virus Viruses such as: Orthomyxovirus, Parainfluenza virus, Paramyxovaris, Parvovirus, Phchinde virus, Picornavirus, Poliovirus, Polyomavirus, Poxvirus, Punta toro phleboviris, Puumala virus, Rhabdovirus, Rabies virus, Reovirus, Rhinovirus, Polynuclear Respiratory Virus, Rift Valley Fever Virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, and Rubella virus.Viruses such as Sagiyama virus, Salivirus A, Sandfly fever sicillian virus, Sapporo virus, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne powassan virus, Togavirus, Torque virus, Toscana virus, Uukuniemi virus, Vaccinia virus, and Varicella-zoster virus. It is caused by viruses selected from the group consisting of (virus), Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitits virus, Western equine encephalitis virus, UU polyomavirus, West Nile virus, Yabasal tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus.

[0120]

[0146] In embodiments, the method is a method for treating or preventing parasitic infections. In embodiments, the parasitic infections include species of Acanthamoeba, American tryppanosomiasis, Balamuthia mandnillanis, Babesia divergenes, Babesia diaphylla, Babesia microfti, Babesia dankani, Balantidium colonis, species of Blastocystis, species of Cryptosporidium, Cyclospora caietanensis, Dientamoeba fragilis, Diphyllobothrium latum, Leishmania amazonesis, and Naegleria fowleri. It is caused by parasites selected from the group consisting of Plasmodium fowderi, Plasmodium vivax, Plasmodium tertianum, Plasmodium obare curtisii, Plasmodium quartanum, Rhinosporidium sebergii, Sarcocystis bovihominis, Sarcocystississimus suihominis, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brussei, Trypanosoma cruzi, and Tapeworms multicephalus.

[0121]

[0147] In embodiments, the Disclosure provides a method for treating cancer in a subject, comprising the step of administering to the subject a composition comprising one of the following: an ALPK1 agonist, a polynucleotide encoding ALPK1 or a constitutively active variant thereof, or the ALPK1 protein or a constitutively active variant thereof. In embodiments of the method for treating cancer, the ALPK1 agonist is selected from HBP, HMP-1bP, H1b-ADP-6L, and H1b-ADP, preferably from HMP-1bP, H1b-ADP-6L, and H1b-ADP, and most preferably from H1b-ADP-6L and H1b-ADP, as well as their prodrugs, analogs, and derivatives. In some embodiments of the method for treating cancer, the ALPK1 agonist is HMP-1bP, H1b-ADP-6L, or H1b-ADP, or its prodrug, analog, or derivative. In a further embodiment of the method for treating cancer, the ALPK1 agonist is H1b-ADP-6L or H1b-ADP, or a prodrug, analog or derivative thereof. In embodiments, cancer is selected from soft tissue sarcoma, breast cancer, head and neck cancer, melanoma, cervical cancer, bladder cancer, hematological malignancies, glioblastoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, kidney cancer, lung cancer, Merkel cell carcinoma, small intestine cancer, thyroid cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), gastric cancer, gastrointestinal stromal tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, liver cancer, leukemia, lymphoma, and T-cell lymphoma.

[0122]

[0148] In any embodiment of the methods described herein, an ALPK1 agonist, preferably selected from H1b-ADP-6L and H1b-ADP, may be administered in combination with one or more additional therapeutic agents or immunomodulators, including, for example, a vaccine or vaccine adjuvant. In embodiments, one or more additional therapeutic agents include, for example, programmed cell death 1 (PD-1) receptor (CD279), PD-1 ligand (e.g., PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), tumor necrosis factor receptor superfamily member 9 (or TNFRSF9, 4-1BB) and 4-1BB ligand, tumor necrosis factor receptor superfamily member 4 (or TNFRSF4, OX40) and OX40 ligand, glucocorticoid-inducible TNFR-associated protein (GITR), tumor necrosis factor receptor superfamily member 7 (or TNFRSF7, differentiation cluster 27, CD27), TNFRSF25 and TNF-like ligand 1A (TL1A), TNF receptor superfamily member 5 (or TNFRSF5, CD40) and CD40 ligand, herpesvirus entry-mediator (HVEM)-tumor necrosis factor ligand superfamily member 14 (or TN FSF14, LIGHT)-Lymphotoxin α (LTA), Herpesvirus Entry-Mediating Factor (HVEM)-B- and T Lymphocyte Attenuator (BTLA)-CD160 (or TNFSF14), Lymphocyte Activator Gene 3 (LAG3), T Cell Immunoglobulin and Mucin Domain-Containing Gene 3 (TIM3), Sialic Acid-Binding Immunoglobulin-like Lectin (SIGLEC), Inducible T Cell Costimulator (ICOS) and ICOS Ligand, B7-H3 (B7 Family, (or CD276), V-set domain-containing T cell activation inhibitor 1 (VTCN1, or B7-H4), V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA), human endogenous retrovirus H-type long-terminal repeat-binding protein 2 (HHLA2)-transmembrane and immunoglobulin domain-containing 2 (TMIGD2), butyrophyllin, natural killer cell receptor 2B4 (or NKR2B4, CD244) and B cell membrane protein (CD48),T cell immune receptors (TIGIT) and poliovirus receptor (PVR) family members containing an immunoglobulin (Ig) domain and an immunoreceptor-suppressive tyrosine motif domain, killer cell immunoglobulin-like receptors (KIRs), immunoglobulin-like transcripts (ILTs) and leukocyte immunoglobulin-like receptors (LIRs), natural killer protein 2 member D (NKG2D) and natural killer protein 2 member A (NKG2A), major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB), natural killer cell receptor 2B4 (CD244), and colony-stimulating factor 1 receptors. (CSF1R), indoleamine 2,3-dioxygenase (IDO), transforming growth factor β (TGFβ), adenosine ectonucleoside triphosphate diphosphohydrolase 1 (CD39)-5'-nucleotidase (CD73), CXC-motif chemokine receptor 4 (CXCR4) and CXC-motif chemokine ligand 12 (CXCL12), phosphatidylserine, signal regulatory protein α (SIRPA) and integrin-binding protein (CD47), vascular endothelial growth factor (VEGF), and neuropilin are inhibitors or antagonists of immune checkpoint molecules, or vaccines against immune checkpoint molecules.

[0123]

[0149] In any embodiment of the methods described herein, the ALPK1 agonist may be administered in combination with an agonist of an immunocostimulatory molecule, such as a checkpoint inhibitor or an anti-OX40 (CD134) agonist antibody. In embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor, such as an anti-PD1 antibody or an anti-PD-L1 antibody, and the ALPK1 agonist is selected from H1b-ADP-6L and H1b-ADP, as well as their prodrugs, analogs, and derivatives.

[0124]

[0150] In embodiments, the ALPK1 agonist may be administered in combination with one or more immune modulators. In embodiments, the immune modulator may be a vaccine. In embodiments, the vaccine is a vaccine against the infectious pathogens described above. In embodiments, the vaccine is a cancer vaccine. In embodiments, the cancer vaccine targets tumor antigens selected from glycoprotein 100 (gp100), mucin 1 (MUC1), and melanoma-associated antigen 3 (MAGEA3).

[0125]

[0151] In embodiments, one or more immunomodulators may be recombinant proteins, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-7 (IL-7), IL-12, IL-15, IL-18, or IL-21.

[0126]

[0152] In some embodiments of cancer treatment, ALPK1 agonists may be administered in combination with T-cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy.

[0153] In embodiments of methods for treating cancer, the ALPK1 agonist may be administered in combination with an agonist of an immunocostimulatory molecule, such as a PD-1 / PD-L1 inhibitor or an anti-OX40 (CD134) agonist antibody. In embodiments, the ALPK1 agonist administered in combination with a PD-1 / PD-L1 inhibitor or an immunocostimulatory molecule agonist is selected from H1b-ADP-6L and H1b-ADP. In embodiments, the ALPK1 agonist is H1b-ADP or its prodrug, analogue, or derivative. In embodiments, the cancer is selected from advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, liver cancer, gastric cancer, colon cancer, breast cancer, non-Hodgkin lymphoma, prostate cancer, head and neck cancer, thyroid cancer, brain cancer, acute myeloid leukemia (AML), Merkel cell carcinoma, multiple myeloma, cervical cancer, and sarcoma, and the method further comprises the step of administering a PD-1 / PD-L1 inhibitor or an agonist of an immunocostimulatory molecule to the target.

[0127]

[0154] In embodiments of methods for modulating an immune response, or for treating or preventing bacterial, viral, or parasitic infections, one or more additional therapeutic agents may be immune modulators, such as inhibitors or antagonists of immune checkpoint molecules. Such molecules generally act as major regulators of the immune system, for example, as co-stimulators of the immune response.

[0128]

[0155] In embodiments, the disclosure also provides vaccine compositions or vaccine adjuvants comprising an ALPK1 agonist. The vaccine compositions described herein may further comprise one or more adjuvants.

[0129]

[0156] In embodiments, the disclosure also provides pharmaceutical compositions comprising an ALPK1 agonist. In embodiments, the ALPK1 agonist may take the form of a small organic molecule such as HBP, or a large biomolecule such as a protein (e.g., ALPK1 itself, or an antibody against ALPK1 that activates ALPK1 kinase activity, or an Fc fragment thereof) or a polynucleotide (e.g., a polynucleotide encoding ALPK1), as discussed above. In embodiments, the ALPK1 agonist is selected from HMP-1bP and H1b-ADP, as well as their prodrugs, analogs, and derivatives. In embodiments, the ALPK1 agonist is H1b-ADP, or its prodrug, analog, or derivative.

[0130]

[0157] In embodiments, the disclosure also provides a method for selecting a compound that can modulate an immune response by measuring the effect of the test compound on ALPK1 autophosphorylation and / or activation of downstream targets of ALPK1 signaling, the method comprising the steps of contacting ALPK1 and the test compound in the presence of ATP and separately in the absence of ATP, and then performing an assay to detect ALPK1 autophosphorylation and / or activation of one or more downstream targets of ALPK1 signaling. In embodiments, the step of contacting ALPK1 and the test compound is performed in a cell-free system or a cell-based system.

[0131]

[0158] In the context of the methods described herein, the term “to treat” may mean the improvement or stabilization of one or more symptoms associated with the disease, disorder, or condition being treated. The term “to treat” may also encompass the management of the disease, disorder, or condition and refers to the beneficial effects that the subject derives from the treatment, but does not result in a cure of the underlying disease, disorder, or condition. In the context of this disclosure, the term “prevention” means preventing the recurrence, onset, progression, or onset of one or more symptoms of the disease, disorder, or condition.

[0132]

[0159] In embodiments in which a therapeutically effective amount of a compound or composition is administered to a target, the therapeutically effective amount is sufficient to achieve a desired therapeutic outcome, for example, improvement or stabilization of one or more symptoms of the disease, disorder, or condition being treated, or, in a preventive context, sufficient to achieve prevention of recurrence, onset, progression, or development of one or more symptoms of the disease, disorder, or condition.

[0133]

[0160] In this embodiment, the therapeutically effective dose is the amount required to achieve at least an equivalent therapeutic effect compared to standard treatment. An example of standard treatment is an FDA-approved drug used to treat the same disease, disorder, or condition.

[0134]

[0161] In the context of any of the methods described herein, the subject may be a non-human vertebrate, preferably a human. In other embodiments, the non-human vertebrate may be, for example, a dog, a cat, a rodent (e.g., a mouse, a rat, a rabbit), a horse, a cattle, a sheep, a goat, a chicken, a duck, or any other non-human vertebrate.

[0135]

[0162] In embodiments, human subjects are selected from adults, children, or elderly individuals, and these are terms understood by physicians, as defined, for example, by the U.S. Food and Drug Administration.

[0136]

[0163] In embodiments, this disclosure provides compositions comprising an ALPK1 agonist, or a polynucleotide encoding ALPK1, or a composition comprising an ALPK1 protein, and one or more excipients or carriers, preferably pharmaceutically acceptable excipients or carriers. As used herein, the term “pharmaceutically acceptable” means, within reasonable medical judgment, a compound, material, composition, carrier, and / or dosage form suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit-risk ratio. Excipients for preparing pharmaceutical compositions are generally known to be safe and non-toxic when administered to human or animal bodies. Examples of pharmaceutically acceptable excipients include, but are not limited to, sterile liquids, water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), oils, detergents, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, and suitable mixtures of any of the above. The specific excipients used in a composition depend on a variety of factors, including the chemical stability and solubility of the compound being formulated, as well as the intended route of administration.

[0137]

[0164] Pharmaceutical compositions may be supplied in bulk or as unit dosage forms. Formulating pharmaceutical compositions in unit dosage forms is particularly advantageous for ease of administration and uniformity of dosage. The term "unit dosage form" refers to a physically discontinuous unit suitable as a unit dose for the target to be treated. Each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. Unit dosage forms may be ampoules, vials, suppositories, sugar-coated tablets, tablets, capsules, IV bags, or single pumps for aerosol inhalation.

[0138]

[0165] In therapeutic applications, the dose may vary depending on the chemical and physical properties of the active compound, as well as the clinical characteristics of the subject, including, for example, age, weight, and comorbidities. Generally, the dose should be a therapeutically effective dose. An effective dose of a pharmaceutical composition is the amount that provides an objectively identifiable improvement, such as a reduction in the symptoms of a disorder, disease, or condition, as noted by a clinician or other qualified observer.

[0139]

[0166] The pharmaceutical composition may take any form suitable for administration via any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, percutaneous, transmucosal, rectal, etc.) (e.g., liquid, aerosol, solution, inhalant, mist, spray; or solid, powder, ointment, paste, cream, lotion, gel, patch, etc.). In embodiments, the pharmaceutical composition may take any form of dosage form acceptable for oral administration, including, but not limited to, capsules, tablets, buccal tablets, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions, or solutions. Capsules may contain excipients such as inert fillers and / or diluents, including starches (e.g., corn, potato, or tapioca starch), sugars, artificial sweeteners, powdered cellulose such as crystalline and microcrystalline cellulose, wheat flour, gelatin, and rubber. For tablets intended for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate may also be added.

[0140]

[0167] In embodiments, the pharmaceutical composition takes the form of a tablet. The tablet may contain a unit dose of the compound described herein together with an inert diluent or carrier such as a sugar or sugar alcohol, e.g., lactose, sucrose, sorbitol, or mannitol. The tablet may further contain a non-sugar-derived diluent such as sodium carbonate, calcium phosphate, or calcium carbonate, or cellulose or its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose), and starch such as corn starch. The tablet may further contain a binder and granulator such as polyvinylpyrrolidone, a disintegrant (e.g., a swelling crosslinked polymer such as crosslinked carboxymethylcellulose), a lubricant (e.g., stearate), a preservative (e.g., parabens), an antioxidant (e.g., butylated hydroxytoluene), a buffer (e.g., phosphoric acid or citrate buffer), and a foaming agent such as a citrate / bicarbonate mixture. The tablet may be a coated tablet. The coating may be a protective film coating (e.g., wax or varnish), or a coating designed to control the release of the active compound, such as a timed release (release of the active substance after a predetermined lag time following ingestion) or release at a specific location in the gastrointestinal tract. The latter can be achieved using enteric film coatings, such as those marketed under the trade name Eudragit®.

[0141]

[0168] Tablet formulations may be prepared by conventional compression, wet granulation, or dry granulation methods, and pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), suspending agents, or stabilizers may be used, including but not limited to magnesium stearate, stearic acid, talc, sodium lauryl sulfate, crystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, zanthang gum, sodium citrate, complex silicate, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dried starch, and powdered sugar. Preferred surface modifiers include nonionic and anionic surface modifiers. Representative examples of surface modifiers include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsified wax, sorbitan esters, colloidal silicon dioxide, phosphate, sodium dodecyl sulfate, aluminum magnesium silicate, and triethanolamine.

[0142]

[0169] In embodiments, the pharmaceutical composition takes the form of a hard or soft gelatin capsule. In this formulation, the compound of the present invention may take the form of a solid, semi-solid, or liquid.

[0143]

[0170] In embodiments, the pharmaceutical composition takes the form of a sterile aqueous solution or dispersion suitable for parenteral administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intrafocal, and intracranial injection or infusion techniques.

[0144]

[0171] In embodiments, the pharmaceutical composition takes the form of a sterile aqueous solution or dispersion suitable for administration by direct injection or by addition to a sterile infusion fluid for intravenous infusion, and comprises a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, or one or more vegetable oils. The solution or suspension may be prepared in water using a cosolvent or surfactant. Examples of suitable surfactants include polyethylene glycol (PEG) fatty acids and PEG fatty acid mono and diesters, PEG glycerol esters, alcohol-oil transesterification reaction products, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugars and their derivatives, polyethylene glycol alkylphenols, polyoxyethylene-polyoxypropylene (POE-POP) block copolymers, sorbitan fatty acid esters, ionic surfactants, lipid-soluble vitamins and their salts, water-soluble vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides. The dispersion system can also be prepared, for example, with glycerol in oil, liquid polyethylene glycol, or mixtures thereof.

[0145]

[0172] In embodiments, the compounds or compositions described herein may be administered as monotherapy or adjuvant therapy. In embodiments, the compounds or compositions described herein may be administered alone or in combination with one or more additional therapeutic agents (i.e., additional APIs), or as part of a therapeutic regimen, for example, including aspects of diet and exercise. In embodiments, the methods described herein include the administration of an ALPK1 agonist as a primary treatment. In other embodiments, the administration of an ALPK1 agonist is adjuvant therapy. In either case, the methods of the present invention intend the administration of an ALPK1 agonist in combination with one or more additional therapeutic agents and / or therapies for the treatment or prevention of the diseases, disorders, or conditions described herein. The terms “therapy” and “therapies” refer to any method, protocol, and / or agonist that may be used in the prevention, treatment, management, or improvement of a disease, disorder, or condition, or one or more of its symptoms.

[0146]

[0173] This disclosure also provides packaging and kits comprising pharmaceutical compositions used in the manner described herein. The kit may include one or more containers selected from the group consisting of bottles, vials, ampoules, blister packs, and syringes. The kit may further include one or more instructions for use, one or more syringes, one or more applicators, or a sterile solution suitable for reconstituting the compounds or compositions described herein. Preparation of the compound of formula I and the exemplary compound

[0174] Compound of formula I (wherein L 1Compound VI (where M is O) can be prepared by the general synthetic method exemplified in Scheme I. Compound II (where "PG" refers to a protecting group) can be obtained from Compound I (when M is OH) with a protected phosphorochloride under basic conditions or a suitable protected phosphate under Mitsunobu reaction conditions. Compound II can be obtained as a mixture of α and β isomers, which can be fractionated by silica gel chromatography. Compound III is obtained by deprotecting the β isomer of Compound II under H2 at 1–4 atmospheres catalyzed with Pd / C or PtO2. Compound V is obtained by coupling Compound IV with morpholine or another suitable base by DCC in a suitable solution such as t-BuOH / H2O. Compound VI is obtained by coupling Compound III and Compound V at room temperature for 24–72 hours using a suitable catalyst such as tetrazole in a suitable solvent such as pyridine.

[0147]

[0175]

[0148] [ka]

[0149]

[0176] Compound of formula I (wherein Z 2 (is S)(L 1 Compound XI) is O and can be synthesized as illustrated in Scheme II. Compound VIII ("PG" refers to the protecting group) can be obtained by reacting compound VII with a protected dialkyl phosphoramidite in a suitable solvent such as dichloromethane at a temperature of -10 to 25°C. Compound IX can be obtained by coupling VIII with III in a suitable solvent such as DMF under an inert gas system at a temperature below 25°C, and compound X can be obtained by in-situ oxidation of this compound with sulfur. The final compound XI can be obtained by deprotection of compound X.

[0150]

[0177]

[0151] [ka]

[0152]

[0178] Compound of formula I (wherein Z 2 (is S)(L 1 Compound XV, where is O, can be synthesized by alternative methods illustrated in Scheme III. Compound III can be activated by forming an imidazole salt in a suitable solvent such as DMF at 10–40°C under an inert gas system. A phosphate group is introduced to compound VII by reaction with phenoxyphosphonoyloxybenzene. Compound XIV can be obtained by oxidation with sulfur at 0–10°C. Compounds XII and XIV are coupled using a Lewis acid catalyst under mild conditions such as 0–40°C in a suitable solvent such as DMF under an inert gas system to obtain the final compound XV.

[0179]

[0153] [ka]

[0154]

[0180] Compound of formula I (wherein L 1 Compound XVIII (where M is CH2) can be prepared by the general synthetic method illustrated in Scheme IV. Compound XVI (where "PG" refers to the protecting group) is obtained by the Mitsunobu reaction of compound I (when M is OH) with a protected methyl diphosphate at 30-50°C for 2-4 hours. Compound XVI is subjected to a second Mitsunobu reaction with compound VI under similar conditions to obtain compound XVII. The final compound XVIII is obtained by the deprotection reaction of compound XVII.

[0155]

[0181]

[0156] [ka]

[0157]

[0182] Compound of formula I (wherein L 1Compound XXV (where M is CF2) can be prepared by the general synthesis method illustrated in Scheme V. Compound XIX (where "PG" refers to a protecting group) is converted to the protected difluoromethyl diphosphate compound XX by using N-fluorobenzenesulfonimide (NFSI) in a suitable solvent under basic NaH conditions, starting at a low reaction temperature of -20°C to 0°C. Compound XXI is obtained by selectively removing one of the protecting groups of compound XX. Compound VII is converted to compound XXII by converting the hydroxyl group to a leaving group such as OTs, OMs, or halogens. Compound XXIII is obtained by carrying out the Mitsunobu reaction of compound I (when M is OH) and compound XXI at 30 to 50°C for 2 to 4 hours. Compound XXIV is obtained by deprotection of compound XXIII. The final compound XXV is obtained by coupling compound XXIV and compound XXII in a suitable solvent such as CH3CN using the base Bu4N.

[0158] [ka]

[0159] Compounds of formula IC (wherein A 1Compound XXVII (where PG is the protecting group) can be prepared by the general synthetic method illustrated in Scheme VI. Compound XXVII (where "PG" refers to the protecting group) is obtained as a mixture of two isomers by reacting compound XXVI with a protected 2-hydroxyacetaldehyde similar to 2-oxoethylbenzoate in a suitable solvent. Alternatively, compound XXVII with the R configuration is obtained by reacting compound XXVI with 2-oxoethylbenzoate in a suitable solvent such as THF, in the presence of an organic base (e.g., triethylamine), phenyl acetate, subtilisine (STS) treated with a surfactant, and Carlsberg. Compound XXVII with the S configuration can be obtained by using CAL B instead of STS (Reference: Hu, L et al., Chem. Commun., 2013, 49, 10376-10378). Compound XXVIII is obtained by reacting compound XXVII with SnCl4 in a suitable solvent solution. Compound XXIX is obtained by deprotection of compound XXVIII. Phosphorination of compound XXIX can be carried out by reacting it with POCl3 and pyridine in a suitable solvent to obtain compound XXX. The remaining reaction to convert compound XXX to the final compound XXXII can be carried out by the same reaction procedure as described in Scheme I.

[0160] [ka]

[0161]

[0183] Table 1 lists example compounds prepared according to the procedures described herein.

[0162] [Table 1-1]

[0163] [Table 1-2]

[0164] [Table 1-3]

[0165] [Table 1-4]

[0166] [Table 1-5]

[0167] [Table 1-6]

[0168] Synthesis of representative compounds of formula (I):

[0184] All humidity-sensitive reactions were performed under Ar using the syringe-septum cap technique. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F 254 plates (Qindao, 0.25 mm thick). Using a Varian-400 spectrometer, 1 ¹H-NMR spectra were recorded, and the chemical shifts were reported as values ​​(ppm) relative to the residual protons of the internal tetramethylsilane or deuterated solvent. Using a Varian-400 spectrometer, 13 ¹³C-NMR spectra were recorded, and the chemical shifts were reported as δ values ​​(ppm) for residual protons of the internal tetramethylsilane or deuterated solvent. Using a Varian-400 spectrometer, 31 P-NMR spectra were recorded, and the chemical shift was reported as the δ value (ppm) relative to the external 85% phosphate. 1 The 1H-NMR spectrum is summarized as follows: chemical shift, multiplicity (br=broad, s=singular, d=double, t=tripular, q=quadular, m=multiplex), number of protons, and coupling constant. compound 1

[0185] (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0169] [ka]

[0170]

[0186] Step 1. Preparation of compound (2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-2-(hydroxymethyl)-4-((4-methoxybenzyl)oxy)tetrahydrofuran-3-ol

[0171] [ka]

[0172] A suspension of adenosine (40 g, 149.6 mmol) in DMF (500 mL) was cooled to -5°C. NaH (8.0 g, 200.0 mmol, 60% purity) was added to the mixture, and the mixture was stirred at -5°C for 1 hour. Then, PMB-Cl (23.0 mL, 168.8 mmol) was added dropwise to the mixture over 1 hour at that temperature. After the addition, the reaction mixture was stirred at 15°C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. H2O (50 mL) and EA (100 mL) were added to the residue, and the organic layer was separated. The organic layer was washed with brine (50 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (DCM / MeOH: 20 / 1 to 10 / 1) to obtain a mixture of the desired compound and isomers (27 g, yield: 46.1%) as a white solid. The mixture was used in the next step without further separation. 1H NMR (400MHz, DMSO-d6) δ 8.38 - 8.29 (m, 1H), 8.15 - 8.06 (m, 1H), 7.39 - 7.30 (m, 2H), 7.11 - 6.91 (m, 2H), 6.88 - 6.69 (m, 2H), 6.08 - 5.90 (m, 1H), 5.58 - 5.44 (m, 1H), 5.29 (d, J = 5.3 Hz, 1H), 4.71 - 4.50 (m, 2H), 4.40 - 3.99 (m, 3H), 3.76 - 3.68 (m, 3H), 3.68 - 3.63 (m, 1H), 3.60 - 3.47 (m, 1H).

[0187] Step 2. Preparation of compound (2R,3R,4R,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)-2-((trityloxy)methyl)tetrahydrofuran-3-ol

[0173] [ka]

[0174] To a solution of pyridine (20 mL) of the product and its isomer mixture from Step 1 above (10 g, 25.8 mmol), DMAP (2.5 g, 20.7 mmol) and TrtCl (16.4 g, 59.0 mmol) were added. The reaction mixture was then stirred at 80°C for 4 hours. HCl (1 N, 20 mL) and EA (50 mL) were added to the mixture, and the organic layer was separated. The organic layer was washed with HCl (1 N, 20 mL x 3 times) and brine (100 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (PE / EA: 20 / 1 to 1 / 1) to obtain the desired product and its isomer mixture (total 18 g, yield: 77.2%) as a white solid. This mixture was used in the next step without further separation. 1H NMR (400MHz, DMSO-d6) δ 8.40 - 8.27 (m, 1H), 7.86 - 7.77 (m, 1H), 7.57 - 7.46 (m, 1H), 7.37 - 7.31 (m, 11H), 7.30 - 7.16 (m, 22H), 6.88 - 6.74 (m, 2H), 6.16 - 5.91 (m, 1H), 5.70 - 5.30 (m, 1H), 5.01 - 4.44 (m, 1H), 4.53 - 4.23 (m, 1H), 4.19 - 4.08 (m, 1H), 3.72 - 3.66 (m, 3H), 3.30 - 3.07 (m, 2H).

[0188] Step 3. Preparation of compound (2R,4S,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)-2-((trityloxy)methyl)dihydrofuran-3(2H)-one

[0175] [ka]

[0176] A mixture of the product and its isomers from step 2 (2.6 g, 2.98 mmol) was dissolved in DCM (30 mL) and DMP (2.54 g, 5.99 mmol) and t-BuOH (503.9 mg, 6.80 mmol, 650.17 μL) were added. The mixture was stirred at 25°C for 4 hours. The reaction mixture was diluted with DCM (100 mL) and quenched with saturated Na2S2O3 / saturated NaHCO3 (1 / 1, 700 mL). The organic layer was separated, and the aqueous layer was extracted with DCM (100 mL x 3 times). The organic layers were combined, washed with brine (300 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated to obtain the residue. The desired product and isomers (2.79 g, crude) were obtained as a pale yellow solid. This was used in the next step without further purification. MS(ESI) m / z(M+H) + :870.4.

[0177]

[0189] Step 4. Preparation of compound (2R,3S,4R,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)-2-((trityloxy)methyl)tetrahydrofuran-3-ol

[0178] [ka]

[0179] A solution of NaBH4 (565.3 mg, 14.94 mmol) in CH3CO2H (25 mL) was stirred at 15°C for 10 minutes, and then added to the mixture of the product from step 3 and its isomer (2 g, 2.30 mmol). The mixture was stirred at 25°C for 20 hours. The reaction mixture was evaporated using EtOH (50 mL x 2 times), then partitioned with DCM (40 mL x 3 times) and H2O (50 mL). The organic layer was washed with saturated NaHCO3 (60 mL) and brine (60 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain the residue. The two isomer products were separated by flash silica gel chromatography (PE:EA = 1:0 to 2:1). The desired product (824 mg, yield: 40.8%) was obtained as a white solid. The isomer (203 mg, yield: 10%) was also obtained as a white solid. MS(ESI) m / z(M+H) + :872.4. Desired product: 1 H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.69 (s, 1H), 7.44 - 7.09 (m, 32H), 7.03 (s, 1H), 6.85 (d, J = 8.6 Hz, 2H), 5.73 (s, 1H), 4.63 (d, J = 11.2 Hz, 1H), 4.44 (d, J = 11.2 Hz, 1H), 4.32 (s, 1H), 4.28 - 4.16 (m, 2H), 3.78 (s, 3H), 3.56 - 3.44 (m, 2H).

[0190] Step 5.9 Preparation of 9-((2R,3S,4R,5R)-4-fluoro-3-((4-methoxybenzyl)oxy)-5-((trityloxy)methyl)tetrahydrofuran-2-yl)-N-trityl-9H-purine-6-amine

[0180] [ka]

[0181] To a 20 mL solution of the starting product (824 mg, 944.94 μmol) from step 4 above, pyridine (747.4 mg, 9.45 mmol, 762.70 μL) and DAST (913.9 mg, 5.67 mmol, 749.08 μL) were added. The mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with 20 mL of DCM, washed with saturated NaHCO3 (40 mL), water (40 mL), and brine (40 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 2:1). The desired product (218 mg, yield: 23.7%) was obtained as a colorless oil. MS(ESI) m / z(M+H) + :874.4 1 H NMR (400MHz, CDCl3) δ 7.91 (s, 1H), 7.80 (s, 1H), 7.42 - 7.15 (m, 30H), 7.09 (br d, J = 8.6 Hz, 2H), 6.99 - 6.93 (m, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.07 (d, J = 7.6 Hz, 1H), 5.18 - 4.89 (m, 2H), 4.60 - 4.48 (m, 2H), 4.48 - 4.36 (m, 1H), 3.75 (s, 3H), 3.48 (dd, J = 4.6, 10.5 Hz, 1H), 3.30 (dd, J = 4.2, 10.5 Hz, 1H).

[0191] Step 6. Preparation of (2R,3R,4R,5R)-2-(6-amino-9H-purine-9-yl)-4-fluoro-5-(hydroxymethyl)tetrahydrofuran-3-ol

[0182] [ka]

[0183]

[0192] To a stirred solution of the product from step 5 (1.2 g, 1.37 mmol) in CHCl3, TFA (0.51 mL, 5 equivalents) was added at room temperature. The solution was stirred at this temperature for 2 hours. The solution was concentrated under reduced pressure to obtain the desired product as an oily residue (360 mg, 1.34 mmol). This residue was used in the next step without further purification.

[0184]

[0193] Step 7. Preparation of (2R,3R,4S,5R)-2-(6-acetamido-9H-purine-9-yl)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl acetate

[0185] [ka]

[0186]

[0194] To a stirred solution of the product from step 6 (360 mg, 1.34 mmol) in pyridine (10 mL), DMAP (16 mg, 0.134 mmol) was added at room temperature. The solution was heated to 50 °C. At this temperature, TBDPSCl (734 mg, 2.68 mmol) was added, and the reaction mixture was stirred overnight at this temperature. LC-MS showed that no SM remained. The solution was added dropwise to Ac₂O (633 μL, 6.7 mmol). After stirring at this temperature for 5 hours, LC-MS showed that the desired compound had been formed. The reaction mixture was partitioned into DCM and water. The extracts were combined, washed with H₂O and brine, and dehydrated with Na₂SO₄. The filtrate was concentrated under reduced pressure to obtain the desired product as an oily residue (792 mg, 1.34 mmol). This was used in the next step without further purification.

[0187]

[0195] Step 8. Preparation of (2R,3R,4S,5R)-2-(6-acetamido-9H-purine-9-yl)-4-fluoro-5-(hydroxymethyl)tetrahydrofuran-3-yl acetate

[0188] [ka]

[0189]

[0196] To a stirred solution of the product from step 7 (792 mg, 1.34 mmol) in THF (10 mL), TBAF (1 M in THF, 2.00 mL, 2.00 mmol) was added at room temperature. After stirring overnight, the reaction mixture was quenched with saturated NH4Cl. The reaction mixture was partitioned into DCM and water. The extracts were combined, washed with brine, and dehydrated with Na2SO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This was purified by silica gel flash chromatography eluting with DCM / MeOH (20:1) to obtain the desired product (254 mg, 0.72 mmol) as a colorless oil.

[0190]

[0197] Step 9. Preparation of (2R,3R,4S,5R)-2-(6-acetamido-9H-purine-9-yl)-5-(2-(bis(benzyloxy)phosphoryl)ethyl)-4-fluorotetrahydrofuran-3-yl acetate)

[0191] [ka]

[0192]

[0198] In a 25 mL round-bottom flask, under a nitrogen atmosphere, the product from step 8 (254 mg, 0.72 mmol) and 1H-imidazole-4,5-dicarbonitride (170 mg, 1.44 mmol) were added. Dry DCM and MeCN were added (DCM:MeCN = 5:1, v / v). The resulting solution was cooled in an ice bath, and dibenzyl diisopropyl phosphoramidite (497 mg, 1.44 mmol) was added. After warming the reaction mixture to room temperature, it was stirred for another 1-2 hours. The reaction mixture was cooled again in an ice bath, and mCPBA (291 mg, 1.44 mmol) was added directly. After warming it to room temperature, saturated NaHCO3 (aqueous solution) was added to quench the reaction mixture and separate the organic phase. The aqueous phase was extracted twice with DCM. The extracts were combined, washed with H2O and brine, and dehydrated with Na2SO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This residue was purified by silica gel flash chromatography eluting with DCM / MeOH (30:1) to obtain the desired product (441 mg, 0.72 mmol).

[0193]

[0199] Step 10. Preparation of (2R,3R,4S,5R)-2-(6-acetamido-9H-purine-9-yl)-4-fluoro-5-((phosphonooxy)methyl)tetrahydrofuran-3-yl acetate

[0194] [ka]

[0195]

[0200] The mixture of the product from step 9 (441 mg, 0.72 mmol) and Pd / C (132 mg) in MeOH (4 mL) was stirred at room temperature under H2. After stirring overnight, the mixture was filtered through a 0.45 μm pore size Advantec PTFE membrane filter using MeOH. The filtrate was concentrated under reduced pressure to obtain the desired product (233 mg, 0.54 mmol). This was used in the next step without further purification.

[0196]

[0201] Step 11. Preparation of morphine DCC salt of ((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methylhydrogen morpholinophosphonate.

[0197] [ka]

[0198]

[0202] A solution of DCC (445 mg, 2.16 mmol) in t-butyl alcohol (5 mL) was added dropwise to a reflux solution of the product from step 10 (233 mg, 0.54 mmol) and purified morpholine (188 mg, 2.16 mmol) in a t-BuOH / H2O mixture (1:1, 10 mL). The addition was completed in approximately 3 hours, and the mixture was refluxed overnight until the TLC indicated completion of the reaction. The mixture was cooled to room temperature. The filtrate was evaporated until most of the t-BuOH was removed, and the remaining aqueous phase was extracted three times with ether. The clear aqueous solution was then evaporated to dryness by freeze-drying to obtain the desired product. This was used in the next step without further purification.

[0199]

[0203] Step 12. Preparation of (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-((diphenoxyphosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0200] [ka]

[0201] A solution of (2R,3R,4S,5S)-2-((R)-1,2-diacetoxyethyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyltriacetate (400 mg, 1 equivalent; Shinsuke Inuki et al., Org. Lett. 2017, 19:3079-3082; Alla Zamyatina et al., Carbohydrate Research, 2003, 338:2571-2589) and DMAP (265.1 mg, 2.17 mmol, 2.28 equivalents) in a solution of diphenyl phosphorochloride (600.7 mg, 2.35 equivalents) in a solution of DCM (10 mL) was added over 1 hour using a syringe. The reaction mixture was then stirred at 25°C for 2 hours. The starting material was detected by TLC (PE:EA = 2:1, 3 times) and some residue was retained. DMAP (1.2 g) was added, followed by the addition of a 15 mL solution of diphenyl phosphorochloride (0.6 g) in DCM, and the mixture was stirred at 25°C for 2 hours. The reaction mixture was diluted with DCM (20 mL) and washed with saturated NaHCO3 (30 mL) and brine (30 mL). The organic phase was concentrated to obtain the residue. The residue was purified by silica gel column chromatography (PE:EA = 10:1 to 1:1) to obtain both the isomer (α conformation, 70 mg, yield: 11.3%) and the desired product (β conformation, 400 mg, yield: 64.4%) as colorless oils. β conformation: 1 H NMR (400MHz, CDCl3) δ 7.42 - 7.12 (m, 10H), 5.70 - 5.61 (m, 1H), 5.44 (br d, J=1.2 Hz, 1H), 5.32 - 5.21 (m, 2H), 5.12 - 5.03 (m, 1H), 4.44 - 4.35 (m, 1H), 4.24 - 4.15 (m, 1H), 3.92 - 3.83 (m, 1H), 2.15 - 1.94 (m, 15H).α conformation: 1H NMR (400MHz, CDCl3) δ 7.42 - 7.30 (m, 4H), 7.29 - 7.15 (m, 6H), 5.85 (br d, J=6.4 Hz, 1H), 5.41 - 5.26 (m, 3H), 5.19 - 5.11 (m, 1H), 4.37 (dd, J=3.7, 12.0 Hz, 1H), 4.29 - 4.17 (m, 2H), 2.23 - 1.96 (m, 15H).

[0204] Step 13. Preparation of (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0202] [ka]

[0203]

[0205] A solution consisting of the product from step 12 (400 mg, 1 equivalent) in à (4 mL) and EtOH (4 mL) was mixed with PtO2 (69.60 mg, 0.5 equivalents) and stirred at 25°C for 16 hours under an H2 atmosphere at 1 atm. The mixture was filtered, and the filtrate was concentrated to obtain the residue. The desired product (300 mg, yield 97.81%) was obtained as a colorless oil. The product was of sufficient purity to be used directly in the next step. 1 H NMR (400MHz, methanol-d4) δ 5.52 - 5.44 (m, 2H), 5.25 - 5.18 (m, 3H), 4.44 (dd, J=3.4, 12.0 Hz, 1H), 4.27 (dd, J=7.2, 12.1 Hz, 1H), 4.01 - 3.95 (m, 1H), 2.15 (s, 3H), 2.10 - 2.02 (m, 9H), 1.98 - 1.94 (m, 3H).

[0206] Step 14.2 Preparation of (R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetatetriethylammonium salt

[0204] [ka]

[0205]

[0207] The solution of the product from step 13 (300 mg, 1 equivalent) and Et3N (0.2 mL, 2.40 equivalents) in MeOH (5 mL) was stirred at 25°C for 1.5 hours. The solvent was removed under reduced pressure to obtain the desired product of triethylammonium salt (340 mg, yield: 80.69%, containing 2Et3N) as a white solid. The product was used directly in the next step.

[0206]

[0208] Step 15. Preparation of (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0207] [ka]

[0208]

[0209] A mixture of the product from step 14 (200 mg, 1 equivalent) and the morphine DCC salt of the compound from step 9 (357.17 mg, 3 equivalents, DCC-morpholine) was dried with dried pyridine (5 mL x 3 times). The residue was then dissolved in pyridine (3 mL), 1H-tetrazole (99.68 mg, 5 equivalents) was added, and the mixture was stirred at 25°C for 32 hours. The reaction product was concentrated to obtain the residue. This residue was purified by silica gel column chromatography (CHCl3:MeOH:NH3,H2O:H2O=1:0:0:0 to 50:50:1:1) to obtain the crude product (300 mg). This was purified by preparative HPLC (column: Waters Xbridge 150*25 5μ, conditions: water (10mM NH4HCO3)-ACN, 3% to 33%), yielding the lower purity portion of the desired product (15 mg, yield: 3.9%, purity 61.6%) as a white solid, and the higher purity portion of the desired product (18 mg, yield: 7.16%, purity 94.1%) as a white solid. MS(ESI) m / z(M+H) + :832.4. 1 H NMR (400MHz, methanol-d4) δ 8.71 (s, 1H), 8.27 (s, 1H), 6.11 (d, J = 7.6 Hz, 1H), 5.61 - 5.56 (br. s, 2H), 5.34 (br d, J=4.2 Hz, 0.5H ), 5.25 - 5.15 (m, 3.5H), 4.61 - 4.50 (m, 1H), 4.45 - 4.41 (m, 1H), 4.31 - 4.21 (m, 3H), 3.95 - 3.90 (m, 1H), 2.13 (s, 3H), 2.08 - 2.02 (m, 6H), 1.99 (s, 3H), 1.91 (s, 3H). compound 2

[0210] Adenosine-3'-fluoro-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0209] [ka]

[0210]

[0211] Step 1. Preparation of adenosine-3'-fluoro-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0211] [ka]

[0212]

[0212] The compound (15.0 mg, 1 equivalent) produced in step 15 of the preparation of compound 1 described above was dissolved in 3 mL of solvent consisting of (TEAB (0.1 M):MeOH:Et3N = 4:3:0.05) and stirred at -28°C for 42 hours. The reaction mixture was then freeze-dried in a freeze-dryer to obtain a white solid. The obtained solid was sequentially purified by preparative HPLC (RP-C18, constant composition elution using triethylammonium acetate buffer (pH 6.8) / 2% acetonitrile) and G25 Sephadex chromatography eluting with distilled H2O to obtain the desired compound (6.1 mg, yield: 54.4%). MS (ESI) m / z (MH) - :619.8. compound 3

[0213] (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0213] [ka]

[0214]

[0214] Step 1. Preparation of compound 1-((2R,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-yl)-2-(trityloxy)ethane-1-ol

[0215] [ka]

[0216] TrtCl (19.6g, 70.4mmol) was added to a solution of compound 1-((2R,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-yl)ethane-1,2-diol (17.4g, 35.2mmol; Tiehai Li et al., (2014) Bioorg. Med. Chem. 22:1139-1147; Shinsuke Inuki et al., Org. Lett. (2017), 19:3079-3082), TEA (7.1g, 70.4mmol, 9.8mL), and DMAP (2.2g, 17.6mmol) in DCM (200mL). The mixture was stirred at 50°C for 20 hours. The reaction mixture was quenched with H2O (100mL) and then separated. The aqueous layer was extracted by DCM (60 mL x 2 times). The organic layers were combined, washed with brine (150 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 1:1). The desired compound (24.6 g, yield: 95%, purity 93%) was obtained as a pale yellow oil. MS(ESI) m / z(M+H) + :782.4.

[0217]

[0215] Step 2. Preparation of compound 1-((2S,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-yl)-2-(trityloxy)ethane-1-one

[0218] [ka]

[0219] The mixture of the product obtained from step 1 (24.6 g, 33.4 mmol), NMO (19.6 g, 166.9 mmol, 17.6 mL), and 4A molecular sieve (24 g, 33.4 mmol) in DCM (250 mL) was stirred at 25°C for 0.5 hours. Then, TPAP (1.17 g, 3.34 mmol) was added at 0°C. The mixture was stirred at 25°C for 4 hours. The mixture was filtered and washed with DCM (50 mL x 3 times). The filtrate was concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 4:1). The desired product (21.7 g, yield: 85.6%) was obtained as a light yellow oil. MS (ESI) m / z (M+H) + :757.3. 1 H NMR (400 MHz, CDCl3): δ7.45-7.25 (m, 30H), 4.72-4.52 (m, 6H), 4.20-4.07 (m, 4H), 3.99 (s, 2H), 3.68-3.67 (m, 1H), 3.22 (s, 3H).

[0216] Step 3. Preparation of (R)-1-((2R,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-yl)-2-(trityloxy)ethane-1-ol

[0220] [ka]

[0221] To a solution of the product obtained from step 2 above (21.7 g, 29.5 mmol) in THF (200 mL), Zn(BH4)2 (0.5 M, 66.7 mL) was added dropwise at 0°C for 0.5 hours. The reaction mixture was carefully quenched with H2O (50 mL). The organic layer was extracted with ethyl acetate (150 mL x 3 times). The organic layer was dehydrated with Na2SO4 and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 7:1). The desired compound (19.5 g, yield: 88.27%, purity 98.5%) was obtained as a colorless oil. MS(ESI) m / z(M+H) + :759.3.

[0222]

[0217] Step 4. Preparation of the compound (2S,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-2-((S)-1-fluoro-2-(trityloxy)ethyl)-6-methoxytetrahydro-2H-pyran

[0223] [ka]

[0224] To a mixture of the compound (9.5 g, 12.9 mmol) of the product from step 3 above in DCM (100 mL), DAST (10.4 g, 64.5 mmol, 8.5 mL) and pyridine (10.2 g, 128.9 mmol, 10.4 mL) were added at 0°C. The mixture was stirred at 25°C for 16 hours. The reaction product was carefully quenched with saturated NaHCO3 (100 mL). The mixture was extracted with DCM (100 mL x 3 times). The organic layers were combined, washed with 2N HCl (150 mL), dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 12:1). The desired product (4.2 g, yield: 44.1%) was obtained as a bright yellow oil. 1 H NMR (400MHz, CDCl3): δ 7.38-7.18 (m, 30H), 4.92-4.61 (m, 2H), 4.53-4.51 (m, 6H), 4.06-4.02 (m, 1H), 3.77-3.75 (m, 1H), 3.65-3.51 (m, 3H), 3.14-3.06 (m, 1H), 2.96 (s, 3H).

[0218] Step 5. Preparation of compound (S)-2-fluoro-2-((2S,3S,4S,5S,6S)-3,4,5-tris(benzyloxy)-6-methoxytetrahydro-2H-pyran-2-yl)ethane-1-ol

[0225] [ka]

[0226] To a 60 mL solution of the compound produced in step 4 above (5.8 g, 7.9 mmol), TFA (13.9 g, 121.6 mmol, 9 mL) was added. The mixture was stirred at 25°C for 1 hour. Saturated NaHCO3 (150 mL) was added to the mixture. The mixture was extracted with DCM (100 ml x 3 times). The organic layers were combined, dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 10:0 to 1:1). The desired compound (3.2 g, yield: 79.7%, purity 96.2%) was obtained as a colorless oil. MS(ESI) m / z(M+H) + :519.1. 1 H NMR (400MHz, CDCl3): δ 7.35-7.28 (m, 15H), 4.99-4.96 (m, 2H), 4.73-4.65 (m, 4H), 4.60 (s, 2H), 4.14-4.10 (m, 3H), 3.77-3.76 (m, 1H), 3.70 (m, 1H), 3.60-3.57 (m, 1H), 3.27 (s, 3H). 19 F NMR δ -207.84.

[0219] Step 6. Preparation of the compound (3S,4S,5S,6S)-6-((S)-2-acetoxy-1-fluoroethyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl acetate

[0227] [ka]

[0228] To a solution of the compound (3.2 g, 6.5 mmol) of the product from step 5 above in HOAc (15 mL) and Ac2O (15 mL), H2SO4 (2.8 g, 27.6 mmol, 1.5 mL, 98% purity) was added. The mixture was stirred at 25°C for 1 hour. The reaction product was quenched with methanol (15 mL) at 0°C. Most of the solvent was removed under vacuum. 30 mL of saturated NaHCO3 was added, and the mixture was extracted with ethyl acetate (50 mL x 3 times). The organic layers were combined, washed with brine (50 mL), dehydrated with Na2SO4, and concentrated under vacuum. The desired compound (3.9 g, crude) was obtained as a light yellow oil. This was used directly in the next step.

[0229]

[0220] Step 7. Preparation of compound (3S,4S,5S,6S)-6-((S)-2-acetoxy-1-fluoroethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-ylacetate

[0230] [ka]

[0231] Pd(OH)2 / C (0.6g, 20% purity) was added to a mixture of the compound (3.9g, 6.9 mmol) produced in step 6 above in methanol (20mL), THF (10mL), H2O (2mL), and HOAc (0.5mL) at 25°C. The mixture was stirred at 25°C for 32 hours under hydrogen (50psi). The mixture was filtered through Celite and washed with methanol (50mL x 3 times). The filtrate was collected and concentrated under vacuum. The desired compound (2.5g, crude) was obtained as a light yellow oil. This was used directly in the next step.

[0232]

[0221] Step 8. Preparation of the compound (3S,4S,5S,6S)-6-((S)-2-acetoxy-1-fluoroethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

[0233] [ka]

[0234] To a solution of the compound produced in step 7 above (2.5 g, 8.4 mmol) in pyridine (20 mL), Ac2O (4.3 g, 42.2 mmol, 4.0 mL) and DMAP (515.5 mg, 4.2 mmol) were added. The mixture was stirred at 25°C for 0.5 hours. The reaction product was quenched with methanol (15 mL). Most of the pyridine was removed under vacuum. 1N HCl (20 mL) was added to the residue. The residue was extracted with ethyl acetate (30 mL x 3 times). The organic layers were combined, washed with 2N HCl (30 mL), dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 10:1 to 3:2). The desired compound (1.6 g, yield: 44.6%) was obtained as a colorless oil. MS(ESI) m / z(M+H) + :445.0. 1 H NMR (400MHz, CDCl3): δ 6.07 (s, 1H), 5.54-5.49 (m, 1H), 5.34-5.31 (m, 1H), 5.24-5.22 (m, 1H), 4.70-4.56 (m, 1H), 4.38-4.24 (m, 2H), 3.98-3.89 (m, 1H), 2.16 (d, J = 6.4Hz, 6H), 2.06 (d, J = 6.0Hz, 6H), 1.99 (s, 3H).

[0222] Step 9. Preparation of compound (2S,3S,4S,5S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyltriacetate

[0235] [ka]

[0236] To a solution of the product compound from Step 8 (1.6 g, 3.8 mmol) in DMF (15 mL), hydrazine acetate (520.1 mg, 5.7 mmol) was added. The mixture was stirred at 25°C for 20 minutes. The reaction product was quenched with H₂O (15 mL). The mixture was extracted with ethyl acetate (20 mL x 3 times). The organic layers were combined, washed with H₂O (20 mL x 3 times), dehydrated with Na₂SO₄, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 10:1 to 1:1). The desired compound (860 mg, yield: 60.1%) was obtained as a colorless oil. 1 H NMR (400MHz, CDCl3): δ 5.52-5.47 (m, 1H), 5.42-5.39 (m, 1H), 5.26-5.25 (m, 2H), 4.75-4.60 (m, 1H), 4.39-4.31 (m, 2H), 4.14-4.05 (m, 1H), 2.15 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H), 1.99 (s, 3H).

[0223] Step 10. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-((diphenoxyphosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0237] [ka]

[0238] [Chloro(phenoxy)phosphoryl]oxybenzene (2.1 g, 7.7 mmol, 1.6 mL) in DCM (50 mL) was added dropwise to a solution of the product compound from step 9 (970 mg, 2.6 mmol) and DMAP (1.6 g, 12.8 mmol) in DCM (50 mL) at 25°C within 3.5 hours. The mixture was stirred at 25°C for 16 hours. The reaction product was quenched with saturated NaHCO3 (50 mL). The mixture was extracted with DCM (80 ml x 3 times). The organic layers were combined, dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (PE:EA = 10:1 to 3:2). The desired compound (1.21 g, yield: 77.5%, purity 100%) was obtained as a colorless oil. MS(ESI) m / z(M+H) + :658.1. 1 H NMR (400MHz, CDCl3) δ 7.35-7.13 (m, 10H), 5.54 (d, J = 6.8Hz, 1H), 5.50-5.46 (m, 2H), 5.07-5.04 (m, 1H), 4.72-4.57 (m, 1H), 4.30-4.26 (m, 1H), 4.23-4.19 (m, 1H), 3.74-3.65 (m, 1H), 2.10(s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H). 19 F NMR δ-205.5.

[0224] Step 11. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0239] [ka]

[0240] PtO2 (150 mg) was added to a mixture of the compound (600 mg, 979.6 μmol) produced in step 10 above in ethanol (10 mL) and ethyl acetate (10 mL). The mixture was stirred at 25°C for 20 hours under hydrogen (15 psi). The reaction mixture was filtered through Celite and washed with methanol (20 mL x 4 times). The filtrate was collected and concentrated under vacuum. The desired compound (450 mg, crude) was obtained as a white solid. The compound was used directly in the next step.

[0241]

[0225] Step 12. Preparation of compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetatetriethylamine salt

[0242] [ka]

[0243] The compound from step 11 (980 mg, 2.1 mmol) was dissolved in methanol (10 mL). TEA (646.3 mg, 6.4 mmol, 889 μL) was added to the mixture, and the mixture was stirred at 25°C for 0.5 hours. The mixture was concentrated under vacuum. The desired salt (950 mg, yield: 96.9%) was obtained as a bright yellow foam. The compound was used directly in the next step.

[0244]

[0226] Step 13. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0245] [ka]

[0246] The compound (300 mg, 651.8 μmol, TEA salt) and compound AMP-molfolide (4'-morpholine-N'N'-dicyclohexylcarboxamidinium salt) (693.9 mg, 977.6 μmol) from step 12 above were dehydrated twice with pyridine (4 mL). Then, 1H-tetrazole (228.3 mg, 3.3 mmol, 289.0 μL) was added, and the residue was dissolved in pyridine (5 mL). The mixture was stirred under nitrogen at 25°C for 40 hours. The mixture was concentrated under vacuum. The residue was dissolved in methanol (30 mL). The mixture was filtered, and the solid was discarded. The filtrate was concentrated under vacuum. The residue was purified by flash silica gel chromatography (DCM:methanol:NH3.H2O = 20:1:0.05 to 1:1:0.05) to obtain 240 mg of crude product as colorless oil. The crude compound was purified by preparative HPLC (neutral conditions, column: Waters Xbridge 150*25 5μ; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min). The desired compound (75.1 mg, yield: 14.5%, purity 99.2%) was obtained as a white solid. MS(ESI) m / z(M+H) + :790.1. 1 H NMR (400MHz, CD3OD) δ 8.60 (s, 1H), 8.19 (s, 1H), 6.08 (d, J = 6.8Hz, 1H), 5.57 - 5.55 (m, 2H), 5.36 - 5.21 (m, 2H), 4.74 - 4.72 (m, 1H), 4.64 - 4.37 (m, 4H), 4.23 - 4.22 (m, 3H), 3.86 - 3.78 (m, 1H), 2.12 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H). compound 4 Adenosine-5'-(L-glycero-β-D-manno-6-fluoroheptopyranosyl)diphosphate

[0247] [ka]

[0248]

[0227] Step 1. Preparation of the compound adenosine-5'-(L-glycero-β-D-manno-6-fluoro-heptopyranosyl)diphosphate The compound (24 mg, 30.4 μmol, 1 equivalent) of the product of step 13 in the preparation of compound 3 described above was dissolved in TEAB / MeOH / TEA (0.3 mL, v / v / v=1 / 1 / 1). The mixture was stirred at -28°C for 48 hours. The reaction product was diluted with CH3CN (2 mL) and lyophilized. The desired compound (15.3 mg, yield: 61.1%, 2Et3N) was obtained as a white solid. 1 H NMR (400MHz, D2O) δ 8.34 (s, 1H), 8.08-8.07 (m, 1H), 5.97-5.96 (m, 1H), 5.05 (d, J = 9.6Hz, 1H), 4.61-4.58 (m, 2H), 4.37-4.35 (m, 1H), 4.23-4.22 (m, 1H), 4.07-4.04 (m, 2H), 3.92-3.91(m, 1H), 3.83-3.60 (m, 3H), 3.53-3.50 (m, 1H), 3.25 (dd, J = 10.4Hz, 26.8Hz, 1H), 3.05-3.00 (m, 12H), 1.09 (t, J = 7.6Hz, 18H). compound 5

[0228] (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0249] [ka]

[0250]

[0229] Step 1. Preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyltriacetate

[0251] [ka]

[0252] AcOH (6.92 g, 115.28 mmol, 6.59 mL, 1.5 equivalents) was added to a solution of NH2NH2.H2O (5.60 mL, 115.28 mmol) in DMF (60 mL) at 0°C and stirred for 0.5 hours. (3S,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (30 g, 76.86 mmol) was added to the system and stirred at 25°C for 1.5 hours. The reaction mixture was diluted with H2O (200 mL) and extracted with ELISA (150 mL x 3 times). The organic layers were combined, washed with brine (150 mL x 3 times), concentrated, and the residue was obtained. The residue was purified by silica gel column chromatography (PE:EA = 1:0 to 1:1) to obtain the desired compound (26 g, yield: 97.1%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ 5.45 - 5.20 (m, 3H), 4.30 - 4.10 (m, 4H), 2.20 - 2.00 (m, 12H).

[0230] Step 2. Preparation of (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-((diphenoxyphosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0253] [ka]

[0254] A mixture of the product from Step 1 (864.4 mg, 2.48 mmol) and DMAP (3.03 g, 24.82 mmol) in DCM (10 mL) was added dropwise by adding a solution of diphenyl phosphorochloride (5 g, 18.61 mmol) in DCM (40 mL), and the mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with DCM (50 mL), washed with saturated NaHCO3 (50 mL) and brine (50 mL), and concentrated to obtain the residue. The residue was purified by silica gel column chromatography (PE:EA = 1:0 to 1:1) to obtain the α-conformation compound (750 mg, yield: 52.1%) and the desired β-conformation compound (380 mg, yield: 26.4%). Both were obtained as yellow oil. α-conformation compound: 1 H NMR (400MHz, CDCl3) δ 7.42 - 7.11 (m, 10H), 5.59 (dd, J = 1.1, 7.2 Hz, 1H), 5.48 (d, J = 2.9 Hz, 1H), 5.25 (t, J = 9.7 Hz, 1H), 5.07 (dd, J = Beta configuration compounds: 1 H NMR (400MHz, CDCl3) δ 7.43 - 7.14 (m, 11H), 5.87 (dd, J = 1.6, 6.7 Hz, 1H), 5.42 - 5.26 (m, 3H), 4.25 - 4.02 (m, 3H), 3.92 (dd, J = 2.1, 12.3 Hz, 1H), 2.16 (s, 3H), 2.08 - 1.94 (m, 9H).

[0231] Step 3. Preparation of the compound (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0255] [ka]

[0256] A mixture of the compound from step 2 (400 mg, 689.09 μmol) and PtO2 (15.65 mg, 68.91 μmol) in ethyl acetate (4 mL) and ethanol (4 mL) was stirred at 25°C for 16 hours under an H2 atmosphere (1 atm). The reaction mixture was filtered, and the filter cake was washed with ethyl acetate / ethanol (5 mL / 5 mL). The filtrate was concentrated to obtain the target compound (300 mg, crude) as a colorless oil. The crude product was used directly in the next step. 1 H NMR (400MHz, methanol-d4) δ 5.54 - 5.48 (m, 2H), 5.27 - 5.22 (m, 2H), 4.38 - 4.31 (m, 1H), 4.17 (dd, J = 2.5, 12.5 Hz, 1H), 3.97 - 3.90 (m, 1H), 2.19 (s, 3H), 2.08 (s, 3H), 2.07 - 2.05 (m, 3H), 1.98 (s, 3H).

[0232] Step 4. Preparation of (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate ditriethylammonium salt

[0257] [ka]

[0258] A mixture of the above product 3 compounds (300 mg, 700.47 μmol) and Et3N (0.2 mL, 1.40 mmol) in MeOH (10 mL) was stirred at 25°C for 2 hours. The solvent was removed to obtain triethylammonium salt (450 mg, crude, containing 2Et3N) as a colorless oil. The crude product was used directly in the next step.

[0259]

[0233] Step 5. Preparation of (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0260] [ka]

[0261]

[0234] The mixture of the product from step 4 above (56.44 mg, 135.57 μmol) and compound AMP-molfolide (4'-morpholine-N'N'-dicyclohexylcarboxamidinium salt) (50 mg, 70.4 μmol) was dried with dry pyridine (5 mL x 3 times). The mixture was then dissolved in pyridine (1 mL), 1H-tetrazole (16.66 mg, 237.84 μmol) was added, and the mixture was stirred at 25°C for 16 hours. The solvent was removed to obtain the residue. The compound was purified by preparative HPLC (column: Waters Xbridge 150*25 5μ, mobile phase: water (10mM NH4HCO3)-CAN, B%: 5% to 25%, gradient time (min): 7, 100% B hold time (min): 0.5, flow rate (mL / min): 25) to obtain the desired compound (5.5 mg, yield: 2.7%) as a white solid. MS(ESI) m / z(M+H) + :758.2. 1H NMR (400MHz, D2O) δ 8.43 (s, 1H), 8.16 (s, 1H), 6.05 (d, J=5.8 Hz, 1H), 5.43 (d, J=2.5 Hz, 1H), 5.37 (d, J=9.5 Hz, 1H), 5.06 - 4.96 (m, 2H), 4.64 - 4.58 (m, 2H), 4.42 - 4.37 (m, 1H), 4.31 - 4.25 (m, 1H), 4.19 (dd, J=3.1, 12.7 Hz, 1H), 4.14 - 4.07 (m, 2H), 3.94 (dd, J=2.0, 12.5 Hz, 1H), 3.58 (br d, J=9.0 Hz, 1H), 2.10 (s, 3H), 1.96 (d, J=10.3 Hz, 6H), 1.88 (s, 3H). compound 6

[0235] Adenosine 5'-(β-D-manno-heptopyranosyl)diphosphate

[0262] [ka]

[0263]

[0236] Step 1. Preparation of adenosine 5'-(β-D-manno-heptopyranosyl) diphosphate

[0264] [ka]

[0265]

[0237] The compound (2.5 mg, 3.30 μmol) produced in step 5 of the preparation of compound 5 described above was dissolved in 0.3 mL of solution (TEAB (0.1 M) / MeOH / TEA (13 / 14 / 1)) and stirred at -20°C for 4 days. The reaction product was freeze-dried to obtain the desired compound (0.9 mg, yield: 17.5%, Et3N salt) as a white solid. MS (ESI) m / z (MH) - :587.8. compound 7

[0238] (3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl hydrogen (((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonate

[0266] [ka]

[0267]

[0239] Step 1. Preparation of tetrabenzylmethylenebis(phosphonate)

[0268] [ka]

[0269] A mixture of dried phenylmethanol (6.2 g, 57.3 mmol, 6.0 mL) and dried pyridine (4.2 g, 52.5 mmol, 4.2 mL) was added over 30 minutes at 0°C using a syringe pump to a suspension of methylenebis(phosphonic acid dichloride) (3.45 g, 13.8 mmol) in dried toluene (10 mL). After the addition was complete, the reaction mixture was brought to 20°C and stirred for a further 3 hours. After the reaction was complete, the solid was removed by filtration and washed twice with toluene (2 times × 20 mL). The filtrate was washed twice with 2 M NaOH (2 times × 15 mL) and water (15 mL), dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product. This was purified using a silica gel column (PE:EA = 1:0 to 1:1) to obtain the desired compound (3g, yield: 40.5%, purity: 99.9%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.31 (s, 20H), 4.96 - 5.09 (m, 8H), 2.44 - 2.59 (m, 2H).

[0240] Step 2. Preparation of benzyl hydrogen ((bis(benzyloxy)phosphoryl)methyl)phosphonate

[0270] [ka]

[0271]

[0241] DABCO (627 mg, 5.59 mmol, 615 μL) was added to a solution of tetrabenzylmethylenebis(phosphonate) (product from step 1 above) (3 g, 5.59 mmol) in toluene (50 mL). The resulting mixture was stirred at 110 °C for 3 hours. Volatile substances were removed under vacuum, and the residue was treated by adding aqueous HCl (37%, 1.2 mL) dropwise. The mixture was extracted with siRNA (20 mL), the organic layer was dehydrated with Na₂SO₄, and evaporated under reduced pressure to obtain the desired compound (2.2 g, crude) as a yellow oil. The product was used directly in the next step. 1 H NMR (400 MHz, CDCl3) δ 7.27 - 7.36 (m, 15H), 4.99 - 5.10 (m, 6H), 2.51 - 2.64 (m, 2H)

[0242] Step 3. Preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(((benzyloxy)((bis(benzyloxy)phosphoryl)methyl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0272] [ka]

[0273]

[0243] PPh3 (360.0 mg, 1.4 mmol) and DEAD (239.5 mg, 1.4 mmol, 250 μL) were sequentially added to a solution of the product from step 2 (200 mg, 448.1 μmol) and (2R,3R,4S,5S)-2-(acetoxymethyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyltriacetate (225 mg, 574.9 μmol) in THF (5 mL). The resulting mixture was stirred at 40°C for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. The solution was purified by preparative HPLC (column: Boston Green ODS 150*30 5μ; mobile phase: [water (0.075% TFA)-ACN]; B%: 55%-75%, 9 min) to obtain the desired compound (130 mg, yield: 36.6%, purity 98.0%) as a white solid. MS(ESI) m / z(M+Na) + :799.1. 1 H NMR (400 MHz, CDCl3) δ 7.20 - 7.33 (m, 15H), 4.86 - 5.76 (m, 10H), 3.50 - 4.29 (m, 3H), 2.40 - 2.62 (m, 2H), 1.88 - 2.09 (m, 12H)

[0244] Step 4. Preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(((benzyloxy)(((benzyloxy)(hydroxy)phosphoryl)methyl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0274] [ka]

[0275]

[0245] DABCO (40.0 mg, 356.6 μmol, 39.2 μL) was added to the toluene (6 mL) solution of the product from step 3 (250 mg, 321.9 μmol). The resulting mixture was stirred at 120°C for 2 hours. After the reaction was complete, the solvent was removed under vacuum, the residue was dissolved in siRNA (20 mL), and washed with 1 N aqueous HCl (10 mL). The aqueous phase was extracted with siRNA (20 mL), the organic layers were combined, dehydrated with Na₂SO₄, filtered, and evaporated under reduced pressure to obtain the desired compound (220 mg, crude) as a yellow syrup. The product was used directly in the next step. MS(ESI) m / z(M+H) + :686.9

[0246] Step 5. Preparation of (2R,3R,4S,5R)-2-(6-(tritylamino)-9H-purine-9-yl)-5-((trityloxy)methyl)tetrahydrofuran-3,4-diol

[0276] [ka]

[0277]

[0247] To a 100 mL solution of adenine in pyridine, TrtCl (38.5 g, 138.0 mmol) and DMAP (5.9 g, 48.6 mmol) were added. The mixture was stirred at 80°C for 20 hours. The reaction mixture was concentrated to obtain the residue. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 0:1, EA:MeOH = 1:0 to 20:1) to obtain the desired compound (25.9 g, yield: 53.5%) as a white solid. MS(ESI) m / z(M+H) - :752.3. 1H NMR (400MHz, CDCl3) δ 8.08 (s, 1H), 8.00 (s, 1H), 7.44 - 7.03 (m, 30H), 6.67 (br s, 1H), 5.89 (d, J = 6.4 Hz, 1H), 4.78 (br t, J = 5.7 Hz, 1H), 4.44 (br s, 1H), 4.30 (br d, J = 4.4 Hz, 1H), 3.49 (dd, J = 3.4, 10.5 Hz, 1H), 3.18 (dd, J = 2.9, 10.8 Hz, 1H).

[0248] Step 6. Preparation of (2R,3R,4R,5R)-2-(6-(tritylamino)-9H-purine-9-yl)-5-((trityloxy)methyl)tetrahydrofuran-3,4-diyldiacetate

[0278] [ka]

[0279]

[0249] Ac2O (545.0 mg, 5.34 mmol, 500 μL) and DMAP (52 mg, 425.6 μmol) were added to a pyridine (5 mL) solution of the product from step 5 (1.6 g, 2.13 mmol). The resulting mixture was stirred at 15-20°C for 24 hours. After the reaction was complete, the reaction product was quenched by adding MeOH (2 mL). The mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (30 mL) and washed with 1N aqueous HCl (20 mL). The organic phase was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column (PE:EA = 1:0 to 1:1) to obtain the desired compound (1.43 g, yield: 77.0%, purity 95.8%) as a white foam. MS (ESI) m / z (M+H) + :836.4. 1H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.76 (s, 1H), 7.61 (s, 1H), 7.21 - 7.35 (m, 30H), 6.22 (d, J = 5.4 Hz, 1H), 6.12 - 6.17 (m, 1H), 5.68 (t, J = 5.4 Hz, 1H), 4.26 (q, J = 4.4 Hz, 1H), 3.28 (d, J = 4.2 Hz, 2H), 2.06 (s, 3H), 2.03 (s, 3H).

[0250] Step 7. Preparation of (2R,3R,4R,5R)-2-(hydroxymethyl)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-3,4-diyldiacetate

[0280] [ka]

[0281]

[0251] To a solution of the product from step 6 (1.9 g, 2.3 mmol) in HCl (76.5 mL), HCl / HCl (4 M, 8.50 mL) was added, and the reaction mixture was stirred at 15°C for 2 hours. After the reaction was complete, the pH was adjusted to 7 using Et3N, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (10 mL) and washed with saturated NaHCO3 (5 mL) and brine (5 mL). The organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was purified by silica gel chromatography (PE:EA = 1:0 to 1:1) to obtain the desired compound (735 mg, yield: 49.6%, purity 91%) as a white solid. MS(ESI) m / z(M+H) + :594.1. 1H NMR (400MHz, DMSO-d6): δ 8.50 (s, 1H), 7.93 (s, 1H), 7.66 (s, 1H), 7.39 - 7.16 (m, 9H), 6.21 (d, J = 6.8 Hz, 1H), 6.02 - 5.88 (m, 1H), 5.65 - 5.34 (m, 2H), 4.26 - 4.12 (m, 1H), 3.76 - 3.50 (m, 2H), 2.12 (s, 3H), 1.99 (s, 3H).

[0252] Step 8. Preparation of 2R,3R,4S,5S)-2-(acetoxymethyl)-6-(((benzyloxy)(((benzyloxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoryl)methyl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0282] [ka]

[0283]

[0253] PPh3 (221.7 mg, 845.1 μmol) and DEAD (145.6 mg, 836.1 μmol, 152.0 μL) were sequentially added to the solution of the product from step 4 (190 mg, 276.7 μmol) and the product from step 7 (171.0 mg, 288.1 μmol) in THF (3 mL). The resulting mixture was stirred at 40°C for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Boston Green ODS 150*30 5 μm; mobile phase: [water (0.075% TFA)-ACN]; B%: 70%-80%, 9 min) to obtain the desired compound (120 mg, yield: 28.2%, purity 82.0%) as a white solid. MS (ESI) m / z (M+H) + :1262.3.

[0284]

[0254] Step 9. Preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(((benzyloxy)(((benzyloxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoryl)methyl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0285] [ka]

[0286]

[0255] TFA (616.0 mg, 5.4 mmol, 400.0 μL) was added to the 1,4-dioxane (1.6 mL) solution of the product from step 8 (120 mg, 95.1 μmol). The mixture was stirred at 25°C for 3 hours. After the reaction was complete, the mixture was diluted with EA (30 mL), washed with saturated NaHCO3 (20 mL x 2 times), the organic phase was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired compound (110 mg, crude) as a yellow syrup. This was used directly in the next step. MS(ESI) m / z(M+H) + :1020.5

[0256] Step 10. Preparation of benzyl((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)(((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(benzyloxy)phosphoryl)methyl)phosphonate

[0287] [ka]

[0288]

[0257] The solution of the product from step 9 (30 mg, 29.4 μmol) in MeOH (1.4 mL), Et3N (0.6 mL), and H2O (0.2 mL) was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Boston Green ODS 150*30 5 μm; mobile phase: [water (0.075% TFA)-ACN]; B%: 24%-44%, 9 min) to obtain the desired compound (8 mg, yield: 35.4%, purity 99.9%) as a white solid. MS (ESI) m / z (MH) - :517.1 / 604.2. 1 H NMR (400 MHz, methanol-d4) δ 8.71 (br s, 1H), 8.24 (br s, 1H), 7.27 - 7.32 (m, 10H), 6.04 (d, J = 4.4 Hz, 1H), 5.26 - 5.31 (m, 1H), 4.99 - 5.16 (m, 6H), 4.91 - 4.92 (m, 1H), 4.49 - 4.64 (m, 1H), 4.37 - 4.44 (m, 1H), 4.26 - 4.36 (m, 1H), 4.16 - 4.26 (m, 2H), 3.60 - 3.94 (m, 3H), 2.56 - 2.76 (m, 2H).

[0258] Step 11. Preparation of (3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl hydrogen (((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonate

[0289] [ka]

[0290]

[0259] To a solution of the benzyl product (6 mg, 7.8 μmol) from step 10 above in MeOH (2 mL), dried Pd / C (10 mg, purity 10%) was added under N2. The suspension was degassed under vacuum and purged several times with H2. The mixture was stirred under hydrogen (15 psi) at 25°C for 2 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the desired compound (4 mg) as a white solid. MS (ESI) m / z (MH) - :337.0120 / 424.0460;MS(ESI) m / z(M+H) - :426.0591. 1 H NMR (400 MHz, D2O) δ 8.38 (s, 1H), 8.09 (s, 1H), 5.97 (d, J = 5.87 Hz, 1H), 5.14 (d, J = 1.00 Hz, 1H), 4.79 - 4.91 (m, 1H), 4.38 - 4.44 (m, 1H), 4.17 - 4.28 (m, 2H), 3.97 - 4.05 (m, 2H), 3.60 - 3.74 (m, 5H), 1.99 - 2.04 (m, 2H). compound 8

[0260] (3S,4S,5S,6R)-6-((R)-1,2-dihydroxyethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl hydrogen (((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonate

[0291] [ka]

[0292]

[0261] Step 1. Preparation of (3S,4S,5R,6R)-2-(((benzyloxy)((bis(benzyloxy)phosphoryl)methyl)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0293] [ka]

[0294]

[0262] PPh3 (540.0 mg, 2.1 mmol) and DEAD (367.9 mg, 2.1 mmol, 384.0 μL) were sequentially added to the product of step 2 of the preparation of compound 7 above (300 mg, 672.1 μmol) and a solution of (2R,3R,4S,5S)-2-((R)-1,2-diacetoxyethyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyltriacetate (300.0 mg, 713.7 μmol) in THF (10 mL). The resulting mixture was stirred at 40°C for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. The solution was purified by preparative HPLC (column: Boston Green ODS 150*30 5μ; mobile phase: [water (0.075% TFA)-ACN]; B%: 58%-74%, 8 min) to obtain the desired compound (188 mg, yield: 28.6%, purity: 87.0%) as a white solid. MS(ESI) m / z(M+Na) + :871.5

[0263] Step 2. Preparation of (3S,4S,5R,6R)-2-(((benzyloxy)(((benzyloxy)(hydroxy)phosphoryl)methyl)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0295] [ka]

[0296]

[0264] DABCO (36.4 mg, 324.0 μmol) was added to the toluene (6 mL) solution of the product from step 1 (250 mg, 294.6 μmol). The resulting mixture was stirred at 120°C for 2 hours. After the reaction was complete, the solvent was removed under vacuum, the residue was dissolved in siRNA (20 mL), and washed with 1 N aqueous HCl (10 mL). The aqueous phase was extracted with siRNA (20 mL), the organic layers were combined, dehydrated with Na₂SO₄, filtered, and evaporated under reduced pressure to obtain the desired compound (220 mg, crude) as a yellow syrup. The crude product was used directly in the next step. MS(ESI) m / z(M+H) + :759.5

[0265] Step 3. Preparation of 3S,4S,5R,6R)-2-(((benzyloxy)(((benzyloxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoryl)methyl)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0297] [ka]

[0298]

[0266] PPh3 (210.0 mg, 800.6 μmol) and DEAD (143.7 mg, 825.1 μmol, 150.0 μL) were sequentially added to the solution of the product from step 7 (172.2 mg, 290.0 μmol) and the product from step 2 (200 mg, 263.6 μmol) in THF (5 mL) of the above compound 7 preparation. The resulting mixture was stirred at 40°C for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Boston Green ODS 150*30 5 μm; mobile phase: [water (0.075% TFA)-ACN]; B%: 71%-85%, 9 min) to obtain the desired compound (117 mg, yield: 29.9%, purity 90.0%) as a white solid. MS(ESI) m / z(M+H)+ :1334.3

[0267] Step 4. Preparation of (3S,4S,5R,6R)-2-(((benzyloxy)(((benzyloxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoryl)methyl)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0299] [ka]

[0300]

[0268] TFA (1.23 g, 10.8 mmol, 800 μL) was added to the product from step 3 (113 mg, 84.7 μmol) in a solution of dioxane (1.2 mL). The mixture was stirred at 40°C for 1.5 hours. After the reaction was complete, the mixture was diluted with EA (30 mL), washed with saturated NaHCO3 (20 mL x 2 times), the organic phase was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired compound (110 mg, crude) as a white solid. The crude product was used directly in the next step. MS(ESI) m / z(M+H) + :1092.2

[0269] Step 5. Preparation of benzyl((3S,4S,5S,6R)-6-((R)-1,2-dihydroxyethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)(((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(benzyloxy)phosphoryl)methyl)phosphonate

[0301] [ka]

[0302]

[0270] The solution of the product from step 4 (105 mg, 96.2 μmol) in MeOH (3.5 mL), Et3N (0.5 mL), and H2O (0.5 mL) was stirred at 25°C for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Boston Green ODS 150*30 5 μm; mobile phase: [water (0.075% TFA)-ACN]; B%: 30%-50%, 7.5 min) to obtain the desired compound (16 mg, 20.0 μmol, yield: 20.7%, purity 99.4%) as a white solid (white colid). MS (ESI) m / z (MH) - :547.1 / 604.1

[0271] Step 6. Preparation of (3S,4S,5S,6R)-6-((R)-1,2-dihydroxyethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl hydrogen (((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonate

[0303] [ka]

[0304]

[0272] To a solution of the product from step 5 (14 mg, 17.6 μmol) in MeOH (2.5 mL), dried Pd / C (20 mg, 10% purity) was added under N2. The suspension was degassed under vacuum and purged several times with H2. The mixture was stirred at 25°C for 2 hours under H2 (15 psi). After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the desired compound (10 mg, 16.2 μmol) as a white solid. MS (ESI) m / z (MH) - :366.4 / 423.8 compound 9

[0273] 2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0305] [ka]

[0306]

[0274] Step 1. Preparation of (2R,3R,4R,5R)-5-(6-acetamido-9H-purine-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl acetate

[0307] [ka]

[0308]

[0275] A stirred solution of (2R,3R,4R,5R(2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol)-5-(6-amino-9H-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (389 mg, 1.44 mmol) in pyridine (3 mL) was mixed with DMAP (18 mg, 0.14 mmol) at room temperature. The solution was heated to 50 °C. At this temperature, TBDPSCl (594 mg, 2.16 mmol) was added, and the reaction mixture was stirred overnight at this temperature. LC-MS showed that no SM remained. The solution was added dropwise to Ac₂O (642 μL, 6.85 mmol). After stirring at this temperature for 5 hours, LC-MS showed that the desired compound had been formed. The reaction mixture was partitioned into DCM and water. The extracts were combined, washed with H2O and brine, and dehydrated with Na2SO4. The filtrate was concentrated under reduced pressure to obtain the desired compound (531 mg, 0.90 mmol) as a white foam. MS(ESI) m / z(M+H) + :592.

[0309]

[0276] Step 2. Preparation of (2R,3S,4S,5R)-5-(6-acetamido-9H-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl acetate

[0310] [ka]

[0311]

[0277] To a stirred solution of the compound produced in Step 1 (531 mg, 0.90 mmol) in THF (4 mL), TBAF (1 M in THF, 1.4 mL, 1.35 mmol) was added at room temperature. After stirring overnight, the reaction mixture was quenched with saturated NH4Cl. The reaction mixture was partitioned into DCM and water. The extracts were combined, washed with brine, and dehydrated with Na2SO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This was purified by silica gel flash chromatography eluting with DCM / MeOH (20:1) to obtain the desired compound as a white foam (96 mg, 0.27 mmol). MS(ESI) m / z(M+H) + :354.

[0312]

[0278] Step 3. Preparation of (2R,3R,4R,5R)-5-(6-acetamido-9H-purine-9-yl)-2-(((bis(benzyloxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl acetate

[0313] [ka]

[0314]

[0279] In a 25 mL round-bottom flask, under a nitrogen atmosphere, the compound of product from step 2 (96 mg, 0.27 mmol) and 1H-imidazole-4,5-dicarbonitride (64 mg, 0.54 mmol) were added. Dry DCM and MeCN were added (DCM:MeCN = 5:1, v / v). The resulting solution was cooled in an ice bath and dibenzyl diisopropyl phosphoramidite (188 mg, 0.54 mmol) was added. After warming the reaction mixture to room temperature, it was stirred for another 2 hours. The reaction mixture was cooled again in an ice bath and mCPBA (110 mg, 0.54 mmol) was added as is. After warming it to room temperature, LC-MS showed that the desired compound had formed as the main product. Saturated NaHCO3 (aqueous solution) was added to quench the reaction mixture and separate the organic phase. The aqueous phase was extracted twice with DCM. The extracts were combined, washed with H2O and brine, and dehydrated with Na2SO4. The filtrate was concentrated under reduced pressure to obtain the oil. This oil was purified by silica gel flash chromatography to obtain the desired compound (126 mg, 0.21 mmol). MS(ESI) m / z(M+H) + :612.

[0315]

[0280] Step 4. Preparation of (2R,3S,4S,5R)-5-(6-acetamido-9H-purine-9-yl)-4-fluoro-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl acetate

[0316] [ka]

[0317]

[0281] A mixture of the compound produced in step 3 (126 mg, 0.21 mmol) and Pd / C (132 mg) in MeOH (4 mL) was stirred at room temperature under H2. After stirring overnight, the mixture was filtered through MeOH using an Advantec PTFE membrane filter with a pore size of 0.45 μm. The filtrate was concentrated under reduced pressure to obtain the desired compound (90 mg, 0.21 mmol). MS(ESI) m / z(M+H) + :434.

[0318]

[0282] Step 5. Preparation of morphine DCC salt of (2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-4-fluoro-2-(((hydroxy(morpholino)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl acetate

[0319] [ka]

[0320]

[0283] A solution of DCC (173 mg, 0.84 mmol) in t-butyl alcohol (5 mL) was added dropwise to a reflux solution of the compound (90 mg, 0.21 mmol) of the product from step 4 above, in a mixture of t-BuOH / H2O (1:1) (10 mL) and purified morpholine (113 mg, 1.30 mmol). The addition was completed in 3 hours, and the mixture was refluxed overnight until TLC indicated completion of the reaction. The mixture was cooled to room temperature. The filtrate was evaporated until most of the t-BuOH was removed, and the remaining aqueous phase was extracted three times with ether. The clear aqueous solution was then evaporated to dryness by freeze-drying to obtain the desired compound as the DCC salt (133 mg, 90% yield). MS(ESI) m / z(M+H) + :419.

[0321]

[0284] Step 6. Preparation of (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0322] [ka]

[0323] A mixture of the compound produced in step 5 above (152 mg, 213 μmol) and the compound produced in step 14 of the preparation of compound 1 above (100 mg, 142 μmol) was azeotropically dehydrated using anhydrous pyridine (3 mL x 3 times). The solvent was then dissolved in pyridine (2 mL), and 2H-tetrazole (49.84 mg, 711.52 μmol) was added. The reaction mixture was stirred at 25°C for 3 days. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CHCl3:MeOH:NH3.H2O=1:0:0:0 to 50:50:1) to obtain the crude product (200 mg). The compound was purified by preparative HPLC (column: Waters Xbridge 150*25 5μ, water (10mM NH4HCO3)-ACN, 0% to 30%) to obtain the title compound (35 mg, yield 28.2%, purity 95.2%) as a bright yellow solid. MS(ESI) m / z(M+H) + :832.2. 1 H NMR (400MHz, methanol-d4) δ 8.60 (s, 1H), 8.29 (s, 1H), 6.36 - 6.26 (m, 1H), 5.62 - 5.52 (m, 2H), 5.38 - 5.33 (m, 0.5H), 5.24 - 5.21 (m, 0.5H), 5.20 - 5.15 (m, 3H), 4.72 - 4.63 (m, 1H), 4.46 - 4.38 (m, 2H), 4.32 - 4.20 (m, 3H), 3.94 - 3.89 (m, 1H), 2.11 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.90 (s, 3H). compound 10

[0285] Adenosine-2'-fluoro-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0324] [ka]

[0325]

[0286] Step 1. Preparation of adenosine-2'-fluoro-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0326] [ka]

[0327]

[0287] The compound (10 mg, 12.0 μmol) of the product of step 6 in the preparation of compound 9 above in 3 mL of solvent (TEAB (0.1 M, 16.00 mL), MeOH (12 mL), and Et3N (145 mg, 1.44 mmol, 200 μL) was stirred at -28 °C for 40 hours. After the reaction was complete, the mixture was freeze-dried in a freeze-dryer to obtain the desired compound (7 mg, yield: 38.5%, purity 54.5%, 2Et3N salt) as a bright yellow solid. MS (ESI) m / z (MH) - :619.8. 1 H NMR (400MHz, D2O) δ 8.28 (s, 1H), 8.10 (s, 1H), 6.30 - 6.24 (m, 1H), 5.36 - 5.30 (m, 1H), 5.23 - 5.17 (m, 1H), 5.06 - 5.00 (m, 1H), 4.56 - 4.51 (m, 3H), 4.47 - 4.42 (m, 1H), 4.2- 4.18 (m, 2H), 4.14 - 4.04 (m, 3H), 3.85 - 3.80 (m, 1H). compound 11

[0288] (2S,3S,4S,5R,6R)-2-(((((((3aR,4R,6R,6aR)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0328] [ka]

[0329]

[0289] Step 1. Preparation of ((3aS,4R,6R,6aS)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methanol

[0330] [ka]

[0331]

[0290] Adenosine (2.5 g, 9.36 mmol) was suspended in dry acetone (100 mL) containing p-toluenesulfonic acid monohydrate (8 g, 42.1 mmol). The mixture was then vigorously stirred, and trimethyl orthoformate (6.6 mL, 60.8 mmol) was added over 1 hour at ambient temperature to obtain a clear solution, after which a white solid formed. The mixture was stirred overnight. The pH of the mixture was adjusted to 8 using aqueous saturated potassium carbonate. The precipitate was removed by filtration, the filtrate was evaporated, and the residue was extracted with EA. The organic phases were combined, washed with aqueous saturated potassium carbonate and water, dried, and concentrated. The crude product was powdered (PE:EA = 10:1) to obtain the desired compound (2.6 g, 8.47 mmol). MS(ESI) m / z(M+H) + :308.

[0332]

[0291] Step 2. Preparation of (Z)-N'-(9-((3aS,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)-N,N-dimethylformimamide

[0333] [ka]

[0334]

[0292] To a stirred solution of the compound (1 g, 3.26 mmol) produced in step 1 above in DMF (2 mL), DMF-DMA (1.64 mL, 12.04 mmol) was added at room temperature. The solution was heated at 45 °C for 1 hour. LC-MS showed that the desired compound had been formed. The solvent was then removed by vacuum, the residue was dissolved in DCM, washed with brine, dehydrated with anhydrous Na2SO4, filtered, and evaporated to dryness. The dried product was purified by silica gel flash chromatography to obtain the desired compound (750 mg, 2.07 mmol). MS(ESI) m / z(M+H) + :363.

[0335]

[0293] Step 3. Preparation of dibenzyl(((3aS,4R,6R,6aS)-6-(6-(((Z)-(dimethylamino)methylene)amino)-9H-purine-9-yl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methyl)phosphate

[0336] [ka]

[0337]

[0294] To a stirred solution of the compound (400 mg, 1.10 mmol) produced in step 2 above in DCM / MeCN (6 mL), DCI (260 mg, 2.20 mmol), mCPBA (447 mg, 2.20 mmol), and dibenzyl diisopropyl phosphoramidite (760 mg, 2.20 mmol) were added at 0°C. The reaction mixture was then stirred overnight at room temperature. LC-MS indicated that the reaction was complete. The reaction mixture was concentrated, and the residue was purified by silica gel flash chromatography eluting with DCM / MeOH (30:1) to obtain the desired compound as colorless rubber (609 mg, 0.98 mmol). MS(ESI) m / z(M+H) + :623.

[0338]

[0295] Step 4. Preparation of ((3aS,4R,6R,6aS)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluoride[3,4-d][1,3]dioxol-4-yl)methyl dihydrogen phosphate

[0339] [ka]

[0340]

[0296] A mixture of the compound produced in step 3 above (609 mg, 0.98 mmol) and Pd(OH)2 (200 mg) in MeOH / H2O (10 mL) was stirred at room temperature under H2. After stirring overnight, the mixture was filtered through MeOH using an Advantec PTFE membrane filter with a pore size of 0.45 μm. The filtrate was concentrated under reduced pressure to obtain the desired compound (383 mg, 0.99 mmol). MS(ESI) m / z(M+H) + :388.

[0341]

[0297] Step 5. Preparation of morphine DCC salt of ((3aS,4R,6R,6aS)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluoride[3,4-d][1,3]dioxol-4-yl)methyl dihydrogen phosphate

[0342] [ka]

[0343]

[0298] A solution of DCC (825 mg, 4.00 mmol) in t-butyl alcohol (20 mL) was added dropwise to a reflux solution of the compound of the product from step 4 (383 mg, 0.99 mmol) in a mixture of t-BuOH / H2O (1:1) (20 mL) and purified morpholine (384 mg, 4.00 mmol). The addition was completed in approximately 3 hours, and the mixture was refluxed overnight until TLC indicated completion of the reaction. The mixture was cooled to room temperature. The filtrate was evaporated until most of the t-BuOH was removed, and the remaining aqueous phase was extracted three times with ether. The clear aqueous solution was then evaporated to dryness by freeze-drying to obtain the desired product. MS(ESI) m / z(M+H) + :457.

[0344]

[0299] Step 6. Preparation of (2S,3S,4S,5R,6R)-2-(((((((3aR,4R,6R,6aR)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0345] [ka]

[0346]

[0300] A mixture of the compound produced in step 14 of the preparation of compound 1 above (200 mg, 284.61 μmol, 2Et3N) and the compound produced in step 5 above (389.68 mg, 853.82 μmol) was dried with dried pyridine ("Py") (5 mL x 3 times). The residue was then dissolved in pyridine (5 mL), mixed with 1H-tetrazole (99.69 mg, 1.42 mmol), and stirred at 25°C for 72 hours. The solvent was removed to obtain the residue. This was purified by silica gel column chromatography (DCM:MeOH:NH3.H2O 1:0:0 to 30:50:1) to obtain the impurity product (200 mg). The compound was purified by preparative HPLC (column: Waters Xbridge 150*25 5μ, conditions: water (10mM NH4HCO3)-ACN, 3% to 33%) to obtain the desired compound (50 mg, yield: 19.6%) as a white solid. MS(ESI) m / z(M+H) + :870.3. 1 H NMR (400 MHz, methanol-d4) δ 8.59 (s, 1H), 8.21 (s, 1H), 6.22 (d, J=3.4 Hz, 1H), 5.58 - 5.52 (m, 2H), 5.27 (dd, J=3.3, 6.0 Hz, 1H), 5.22 - 5.14 (m, 4H), 4.53 (br s, 1H), 4.41 (dd, J=3.4, 12.0 Hz, 1H), 4.27 - 4.13 (m, 3H), 3.91 (dd, J=2.9, 9.8 Hz, 1H), 2.11 (s, 3H), 2.03 (d, J=3.7 Hz, 6H), 1.99 (s, 3H), 1.91 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H). compound 12

[0301] ((3aR,4R,6R,6aR)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methanol(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0347] [ka]

[0348]

[0302] Step 1. Preparation of ((3aR,4R,6R,6aR)-6-(6-amino-9H-purine-9-yl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methanol (D-glycero-β-D-mannoheptopyranosyl) diphosphate

[0349] [ka]

[0350]

[0303] The compound (10 mg, 11.50 μmol) of the product of step 6 in the preparation of compound 11 described above was dissolved in 2 mL of a mixed solvent consisting of TEAB (8 mL), MeOH (6 mL), and Et3N (0.1 mL). The resulting solution was stirred at -28 °C for 46 hours. The reaction product was freeze-dried in a freeze-dryer. The desired compound (9 mg, yield: 70.84%, 2.6Et3N salt) was obtained as a white solid. MS (ESI) m / z (MH) - :657.9. 1 H NMR (400 MHz, D2O) δ 8.28 (s, 1H), 8.09 (s, 1H), 6.13 (d, J = 3.2 Hz, 1H), 5.23 (br d, J = 3.4 Hz, 1H), 5.13 - 4.97 (m, 2H), 4.01 (br s, 2H), 3.92 - 3.78 (m, 1H), 3.66 - 3.42 (m, 4H), 3.33 - 3.23 (m, 1H), 1.52 (s, 3H), 1.29 (s, 3H). compound 13

[0304] (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0351] [ka]

[0352]

[0305] Step 1. Preparation of 9-((2R,3R,4R,5R)-3,4-dimethoxy-5-((trityloxy)methyl)tetrahydrofuran-2-yl)-N-trityl-9H-purine-6-amine

[0353] [ka]

[0354]

[0306] To a solution of the product (20 g, 18.62 mmol) from step 5 of the preparation of compound 7 above in DMF (100 mL), NaH (1.71 g, 42.83 mmol, 60%) was added and stirred at 0°C for 30 minutes, then CH3I (7.85 g, 55.31 mmol, 3.44 mL) was added at 0°C. The mixture was stirred at 25°C for 4 hours. The reaction mixture was quenched with H2O (200 mL) at 0°C, extracted with EA (100 mL x 3 times), the organic layers were combined, washed with brine (200 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by flash silica gel chromatography (PE:EA = 1:0 to 2:1). The desired compound (7.3 g, yield: 39.57%) was obtained as a white solid. MS (ESI) m / z (M+H) + :780.3.

[0355]

[0307] Step 2. Preparation of ((2R,3R,4R,5R)-3,4-dimethoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methanol

[0356] [ka]

[0357]

[0308] To a solution of the product from step 1 (3.81 g, 3.85 mmol) in HCl (126 mL), HCl / HCl (4 M, 14 mL) was added. The reaction mixture was stirred at 20°C for 0.5 hours. The pH was adjusted to 7 using Et3N (5 mL), and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (30 mL) and washed with saturated NaHCO3 (20 mL x 3 times) and brine (20 mL x 2 times). The organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column (PE:EA = 1:0 to 1:1, then PE:EA = 0:1) to obtain the desired compound (1.23 g, 2.25 mmol, yield 58.62%) as a white solid. MS (ESI) m / z (M+H) + :538.3.

[0358]

[0309] Step 3. Preparation of dibenzyl(((2R,3R,4R,5R)-3,4-dimethoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl)phosphate

[0359] [ka]

[0360]

[0310] The product from step 2 (2.03 g, 3.78 mmol) in DCM (40 mL) and CH3CN (8 mL) and 1H-imidazole-4,5-dicarbonitride (892 mg, 7.55 mmol, 2 equivalents) were mixed with dibenzyl diisopropyl phosphoramidite (2.61 g, 7.55 mmol, 2.53 mL) under nitrogen at 0°C. The mixture was stirred at 0°C for 5 minutes, then warmed to 25°C and stirred at 25°C for 1 hour. The mixture was cooled to 0°C, and m-CPBA (1.63 g, 7.55 mmol, 80% purity) was added in portions at 0°C. After addition, the mixture was stirred at 25°C for 0.25 hours. 35 mL of saturated NaHCO3 was added, and the mixture was extracted with DCM (40 mL x 3 times). The organic layers were combined, dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by silica gel chromatography (200-300 mesh, eluent: 20-55% ethyl acetate / petroleum ether gradient). The desired compound (2.54 g, yield: 79.92%) was obtained as colorless rubber. MS(ESI) m / z(M+H) + :798.2. 1 H NMR (400MHz, CDCl3) δ 8.00-7.98 (m, 2H), 7.33-7.25 (m, 25H), 6.97 (s, 1H), 6.03-6.02 (m, 1H), 5.07-5.01 (m, 4H), 4.51(t, J = 4.4Hz, 1H), 4.31-4.24 (m, 3H), 4.00-3.97 (m, 1H), 3.37 (s, 3H), 3.35 (s, 3H).

[0311] Step 4. Preparation of ((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methyldibenzyl phosphate

[0361] [ka]

[0362]

[0312] The product from step 3 (2.54 g, 3.18 mmol) was dissolved in dioxane (15 mL). TFA (5 mL, 67.53 mmol) was added, and the mixture was stirred at 40°C for 6 hours. Saturated NaHCO3 (approximately 50 mL) was added to the mixture until the pH became 8. The mixture was extracted with ethyl acetate (50 mL x 4 times). The organic layers were combined, dehydrated with Na2SO4, and concentrated under vacuum. The residue was purified by flash silica gel chromatography (ISCO®, 4 g SepaFlash® silica flash column, eluent: 0-10% methanol / ethyl acetate gradient, 35 mL / min). The desired compound (1.75, yield: 98.95%) was obtained as a colorless oil. MS (ESI) m / z (M+H) + :556.1. 1 H NMR (400MHz, CDCl3) δ 8.30 (s, 1H), 8.04 (s, 1H), 7.34-7.31 (m, 10H), 6.08-6.02 (m, 2H), 5.07-5.02 (m, 4H), 4.48-4.20 (m, 5H), 4.00-3.97 (m, 1H), 3.49 (s, 3H), 3.38 (s, 3H).

[0313] Step 5. Preparation of ((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methyl dihydrogen phosphate

[0363] [ka]

[0364]

[0314] The mixture of the product from step 4 (500 mg, 900.0 μmol) was dissolved in t-BuOH (20 mL) and H2O (20 mL), Pd / C (100 mg, 10% purity) and Pd(OH)2 (126 mg, 89.72 μmol, 10% purity) were added, and the mixture was stirred at 25°C for 16 hours under an H2 atmosphere (50 psi). The mixture was filtered, and the filtrate was concentrated to obtain the desired compound (400 mg, crude) as a colorless oil. 1H NMR (400MHz, CD3OD) δ 8.56 (s, 1H), 8.18 (s, 1H), 6.14 (d, J = 6.4 Hz, 1H), 4.60 - 4.50 (m, 1H), 4.35 - 4.25 (m, 1H), 4.20 - 4.10 (m, 1H), 4.09 - 3.95 (m, 2H), 3.49 (s, 3H), 3.39 (s, 3H).

[0315] Step 6. Preparation of ((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methylhydrogenmorpholinophosphonate

[0365] [ka]

[0366]

[0316] DCC (836 mg, 4.05 mmol) in t-BuOH (12 mL) was added dropwise to a reflux solution (110°C) of the product from step 5 (380 mg, 1.01 mmol) and morpholine (353 mg, 4.05 mmol) in H2O (12 mL) and t-BuOH (12 mL). The mixture was stirred at 110°C for 12 hours. The mixture was cooled to room temperature. The solid was removed by filtration. The filtrate was collected and the organic solvent was removed by vacuum. The remaining aqueous phase was collected and washed with MTBE (10 mL x 3 times). The aqueous phase was collected and concentrated by vacuum to obtain the desired compound (440 mg, crude) as a bright yellow viscous oil. This was used directly in the next step without further purification.

[0367]

[0317] Step 7. Preparation of (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0368] [ka]

[0369]

[0318] The product from step 14 (130 mg, 309.3 μmol) and the product from step 6 (390 mg, 878.3 μmol) of the above preparation of compound 1 were dried separately with pyridine (4 mL x 3 times). The residue was redissolved in pyridine (4 mL) and 1H-tetrazole (108 mg, 1.55 mmol) was added. The solution was stirred at 30°C for 12 hours. The solvent was removed by vacuum. The residue was redissolved in MeOH (10 mL). The solution was filtered. The filtrate was collected and concentrated. The residue was purified by column chromatography (DCM:(MeOH:NH3.H2O=50:1)=1:1) to obtain the crude product (80 mg). The solution was then re-purified by preparative HPLC (column: Waters Xbridge 150*25 5μ; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min) to obtain the desired compound (30 mg, yield 11.21%) as a white solid. MS(ESI) m / z(M+H) + :858.4. 1 H NMR (400MHz, CD3OD) δ 8.65 (s, 1H), 8.24 (s, 1H), 6.15 - 6.12 (m, 1H), 5.60 - 5.56 (m, 2H), 5.22 - 5.17 (m, 3H), 4.55 - 4.12 (m, 7H), 3.92 - 3.89 (m, 1H), 3.49 (s, 3H), 3.44 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.88 (s, 3H). compound 14

[0319] Adenosine-2'3'-dimethoxy-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0370] [ka]

[0371]

[0320] Step 1. Preparation of adenosine-2'3'-dimethoxy-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0372] [ka]

[0373]

[0321] The solution of the product (8.4 mg, 9.79 μmol) from step 7 of the preparation of compound 13 above in 4 mL of buffer (TEAB (12 mL): MeOH (9 mL): TEA (0.15 mL)) was maintained at -20°C for 24 hours. The solution was dried under lyophilization to obtain the desired compound (8 mg, yield: 65.85%) as a white viscous solid. MS (ESI) m / z (MH) + :645.9. compound 15

[0322] (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0374] [ka]

[0375]

[0323] Step 1. Preparation of compound ((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methanol

[0376] [ka]

[0377]

[0324] To a solution of the product from step 5 of the preparation of compound 7 above (37.4 g, 49.8 mmol) in acetone (100 mL) and 2,2-dimethoxypropane (51.8 g, 497 mmol, 61.0 mL), p-TsOH.H2O (11.4 g, 59.7 mmol) was added. The mixture was stirred at 25°C for 16 hours. After the reaction was complete, the mixture was cooled to 0°C and quenched with saturated NaHCO3 (300 mL). The reaction mixture was extracted with EA (200 mL x 3 times), the organic layers were combined, washed with brine (200 mL), dehydrated with Na2SO4, filtered, and concentrated to obtain the residue. This was purified by silica gel column (PE:EA = 1:0 to 2:3) to obtain the desired compound (9.96 g, yield: 35.57%) as a white solid. MS (ESI) m / z (M+H) + = 550.1. 1 H NMR (400MHz, DMSO-d6) δ 8.44 (s, 1H), 7.92 (s, 1H), 7.52 (s, 1H), 7.34 - 7.18 (m, 15H), 6.12 (d, J = 2.9 Hz, 1H), 5.34 (dd, J = 2.8, 6.2 Hz, 1H), 5.14 (t, J = 5.5 Hz, 1H), 4.93 (dd, J = 2.7, 6.1 Hz, 1H), 4.25 - 4.15 (m, 1H), 3.60 - 3.42 (m, 2H), 1.52 (s, 3H), 1.30 (s, 3H).

[0325] Step 2. Preparation of compound ((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofluoride[3,4-d][1,3]dioxol-4-yl)methylhydrogen phosphonate triethylamine salt

[0378] [ka]

[0379]

[0326] Phenoxyphosphonoyloxybenzene (3.41 g, 14.6 mmol) was added to the pyridine (20 mL) solution of the product from step 1 (2 g, 3.64 mmol). The resulting mixture was stirred at 25°C for 2 hours. Then, Et3N (2.21 g, 21.8 mmol, 3.04 mL) and H2O (786.9 mg, 43.7 mmol) were added. The resulting mixture was stirred at 25°C for 0.5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column (DCM:MeOH = 1:0 to 10:1, with 0.5% Et3N added) to obtain the desired compound (2 g, 2.55 mmol, yield 70.0%, purity 78%) as a yellow syrup. MS(ESI) m / z(M+H) + :614.1

[0327] Step 3. Preparation of O-(((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methyl)phosphorothioate triethylamine salt

[0380] [ka]

[0381]

[0328] To a solution of the product from step 2 (1.5 g, 2.40 mmol) in pyridine (6 mL) and Et3N (6 mL), TMSCl (2.4 mL, 19.1 mmol) was added dropwise over 15 minutes under an N2 atmosphere. The mixture was stirred at 0°C for 1 hour, and then S (730 mg, 22.7 mmol) was added. The mixture was stirred at 0°C for another 45 minutes. After the reaction was complete, the reactants were quenched with H2O (10 mL), and the mixture was concentrated under reduced pressure to obtain the crude product. The compound was purified by silica gel chromatography (DCM:MeOH = 20:1 to 10:1) and preparative HPLC (column: Boston Prime C18 150*30mm 5μm; mobile phase: [water (0.05% ammonium hydroxide (v / v))-ACN]; B%: 15%-45%, 9 min) to obtain the desired compound (700 mg, yield 44.4%, purity 86%) as a white solid. MS(ESI) m / z(M+H) + :646.1. 1 H NMR (400MHz, DMSO) δ 8.71 (s, 1H), 7.91 (s, 1H), 7.48 (s, 1H), 7.39 - 7.12 (m, 15H), 6.12 (d, J = 3.3 Hz, 1H), 5.28 (dd, J = 3.3, 5.8 Hz, 1H), 5.07 (d, J = 5.8 Hz, 1H), 4.39 (br s, 1H), 3.95 - 3.84 (m, 1H), 3.73 (td, J = 5.6, 10.9 Hz, 1H), 1.56 - 1.48 (s, 3H), 1.31 (s, 3H)

[0329] Step 4. Preparation of (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0382] [ka]

[0383]

[0330] CDI (945 mg, 5.83 mmol) was added to a solution of the compound (350 mg, 0.58 mmol) of the product of step 14 in the preparation of compound 1 above in anhydrous DMF (15 mL) under an N2 atmosphere. The resulting mixture was stirred at 25°C for 3 hours. After the reaction was complete, MeOH (0.2 mL) was added to quench the reaction product, and the mixture was concentrated under reduced pressure to obtain the crude desired product (1 g, crude). This was used as is in the next step.

[0384]

[0331] Step 5. Preparation of compound (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(((((((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0385] [ka]

[0386]

[0332] ZnCl2 (1 g, 7.34 mmol) was added to a solution of the product from step 4 (320 mg, 0.58 mmol) and the product from step 3 (500 mg, 0.67 mmol) in anhydrous DMF (15 mL) under an N2 atmosphere. The resulting mixture was stirred at 25°C for 16 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column chromatography (DCM:MeOH = 10:1, with 0.5% Et3N added) to obtain the desired compound (600 mg, crude) as a bright yellow solid. This was used directly in the next step without further purification. MS(ESI) m / z(M+H) + :1128.6.

[0387]

[0333] Step 6. Preparation of the compound (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0388] [ka]

[0389]

[0334] TFA (0.3 mL, 4.05 mmol) was added to a solution of the product compound (200 mg, crude) from step 5 above in H2O (2 mL). The mixture was stirred at 25°C for 0.5 hours. After the reaction was complete, the reaction product was adjusted to pH=7 by adding Et3N. The mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Waters Xbridge 150*25 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min) to obtain the desired compound (18.6 mg, purity 99%) as a white solid. MS (ESI) m / z (M+H) + :846.3. 1 H NMR (400MHz, methanol-d4) δ = 8.78 (s, 0.5H), 8.71 (s, 0.5H), 8.21 (s, 1H), 6.13 (dd, J = 2.0, 6.0 Hz, 1H), 5.76 - 5.60 (m, 2H), 5.26 - 5.16 (m, 3H), 4.74 - 4.68 (m, 1H), 4.53 - 4.42 (m, 2H), 4.37 - 4.21 (m, 4H), 4.01 - 3.89 (m, 1H), 2.17 (s, 3H), 2.10 - 2.06 (m, 6H), 2.04 (s, 1.5H), 2.02 (s, 1.5H), 1.96 (s, 3H) compound 16

[0335] Adenosine-5'-(D-glycero-β-D-manno-6-fluoro-heptopyranosyl)phosphoroyloxyphosphate

[0390] [ka]

[0391]

[0336] Step 1. Preparation of adenosine-5'-(D-glycero-β-D-manno-6-fluoro-heptopyranosyl)(hydroxyl)phosphoroyloxyphosphate

[0392] [ka]

[0393]

[0337] In the preparation of compound 15 described above, the compound (4 mg, 4.73 μmol) of the product of step 6 was mixed in MeOH / water / Et3N (7:3:1 ratio) (2 mL) and stirred at 25°C for 5 hours. After the reaction was complete, the mixture was concentrated and freeze-dried from the water to obtain the desired compound (3.2 mg, yield: 80.6%, 2Et3N salt) as a white solid. MS (ESI) m / z (MH) + :634.1. 1 H NMR (400MHz, D2O) δ 8.46 (s, 0.5H), 8.43 (s, 0.5 H), 8.08 (s, 0.5 H), 8.06 (s, 0.5H), 5.96 (dd, J = 5.9, 10.0 Hz, 1H), 5.38 - 5.28 (m, 0.5H), 5.13 - 5.03 (m, 0.5H), 4.45 - 4.31 (m, 2H), 4.23 (d, J = 11.0 Hz, 1H), 4.14 - 4.03 (m, 1H), 3.96 - 3.87 (m, 2H), 3.85 - 3.76 (m, 1H), 3.66 - 3.47 (m, 4H), 3.34 - 3.24 (m, 1H), 3.02 (q, J = 7.3 Hz, 12H), 1.09 (t, J = 7.3 Hz, 18H) compound 17

[0338] (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0394] [ka]

[0395]

[0339] Step 1. Preparation of the compounds (2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-2-(hydroxymethyl)-4-((4-methoxybenzyl)oxy)tetrahydrofuran-3-ol and (2R,3R,4S,5R)-2-(6-amino-9H-purine-9-yl)-5-(hydroxymethyl)-4-((4-methoxybenzyl)oxy)tetrahydrofuran-3-ol

[0396] [ka]

[0397]

[0340] A suspension of adenosine (25 g, 93.55 mmol) in DMF (900 mL) was cooled to -5°C. NaH (4.86 g, 121.61 mmol, 60% purity) was added to the solution. The mixture was stirred at -5°C for a further 1 hour. PMBCl (17.58 g, 112.26 mmol, 15.29 mL) was added dropwise to the suspension over a period of 1 hour. After the addition was complete, the reaction mixture was brought to 25°C and stirred for 16 hours. 40 mL of saturated NaHCO3 solution was added to the mixture at 0°C and stirred at room temperature for 10 minutes. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (elution with 0-2% MeOH in DCM). The compound (2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-2-(hydroxymethyl)-4-((4-methoxybenzyl)oxy)tetrahydrofuran-3-ol (22 g, 59.70 mmol, yield 63.8%, purity 96.37%) was obtained as a white solid. A mixture (12 g) of the two desired isomers was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ 8.29 (s, 1H), 8.06 (s, 1H), 7.35 (s, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 6.01 (d, J = 6.4 Hz, 1H), 5.47 (dd, J = 4.4, 7.2 Hz, 1H), 5.29 (d, J = 4.8 Hz, 1H), 4.65 - 4.42 (m, 2H), 4.39 - 4.20 (m, 2H), 4.00 (q, J = 2.8 Hz, 1H), 3.72 (s, 3H), 3.71-3.61 (m, 1H), 3.58 - 3.47 (m, 1H).

[0341] Step 2. Preparation of compound (2R,3R,4R,5R)-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)-2-((trityloxy)methyl)tetrahydrofuran-3-ol

[0398] [ka]

[0399]

[0342] The compound produced in step 1 (15.00 g, 38.72 mmol) was evaporated twice with pyridine (10 mL x 2 times) and dissolved in pyridine (300 mL). TrtCl (26.99 g, 96.80 mmol) and DMAP (3.78 g, 30.98 mmol) were added. The mixture was stirred under N2 at 80°C for 15 hours. The mixture was diluted with EA (800 mL) and washed with saturated NaHCO3 solution (200 mL x 2 times) and brine (200 mL x 2 times). The organic layer was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by flash column chromatography (elution in PE with 0-40% EA). The desired compound (22.6 mg, yield: 66.9%) was obtained as a white solid. MS(ESI) m / z(M+H) + =872.4

[0343] Step 3. Preparation of compound 9-((2R,3S,4S,5R)-4-fluoro-3-((4-methoxybenzyl)oxy)-5-((trityloxy)methyl)tetrahydrofuran-2-yl)-N-trityl-9H-purine-6-amine

[0400] [ka]

[0401]

[0344] To a solution of the compound produced in step 2 (5 g, 5.73 mmol) in DCM (50 mL), DAST (3.85 mL, 29.1 mmol) and pyridine (4.6 mL, 57.2 mmol) were added. The mixture was stirred at 20°C for 12 hours. The reaction product was quenched with saturated NaHCO3 (30 mL), and the organic layer was separated. The aqueous layer was extracted with DCM (50 mL x 2 times), the organic layers were combined, washed with HCl (50 mL) and brine (50 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA: 20 / 1 to 2 / 1) to obtain the desired compound (2.1 g, yield 42.0%) as a yellow solid. MS(ESI) m / z(M+H) + :874.4 1 H NMR (400MHz, DMSO-d6) δ 7.99 (s, 1H), 7.91 (s, 1H), 7.56 (s, 1H), 7.44 - 7.18(m, 32H), 6.84 (d, J = 8.8 Hz, 2H), 6.16 - 6.10 (m, 1H), 5.50 - 5.28 (m, 1H), 4.81 - 4.70 (m, 1H), 4.62 (s, 2H), 4.57 - 4.43 (m, 1H), 3.71 (s, 3H), 3.42 - 3.37 (m, 1H), 3.31 - 3.24 (m, 1H).

[0345] Step 4. Preparation of compound ((2R,3S,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methanol

[0402] [ka]

[0403]

[0346] The compound (2.7 g, 3.09 mmol) produced in step 3 above was mixed in HCl / dioxane (20 mL) and stirred at 28°C for 4 hours. The reaction mixture was neutralized with saturated NaHCO3 to pH=7, and then extracted with siRNA (50 mL x 3 times). The organic layers were combined, washed with brine (50 mL), dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. This residue was purified by silica gel column chromatography (PE:EA = 20:1 to 3:1). The desired compound (3 g, yield: 76.9%) was obtained as a yellow solid. MS(ESI) m / z(M+H) + = 631.2, 632.2. 1 H NMR (400MHz, DMSO-d6) δ 8.20 (s, 1 H), 7.94 (s, 1 H), 7.52 (s, 1 H), 7.37 - 7.18 (m, 17 H), 6.90 - 6.83 (m, 2 H), 6.12 - 6.05 (m, 1 H), 5.41 - 5.25 (m, 1 H) 5.10 - 5.05 (m, 1 H), 4.81 - 4.74 (m, 1 H), 4.70 - 4.62 (m, 2 H), 4.34 - 4.22 (m, 1 H) 3.81 - 3.73 (m, 1 H) 3.72 (s, 3 H).

[0347] Step 5. Preparation of the compound dibenzyl(((2R,3S,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl)phosphate

[0404] [ka]

[0405]

[0348] The compound of the product of step 4 above (1.5 g, 2.37 mmol) and 1H-imidazole-4,5-dicarbonitric acid (560 mg, 4.75 mmol) were mixed in CH2Cl2 (20 mL) and CH3CN (4 mL) and (BnO)2P-N(i-Pr)2 (1.64 g, 4.75 mmol) at 0 °C. The mixture was stirred at 0 °C for 10 minutes and then warmed to 25 °C. The resulting mixture was stirred for another hour and then cooled again to 0 °C. m-CPBA (1.02 g, 4.75 mmol, 80% purity) was added as is, and the reaction mixture was then warmed to 25 °C and stirred at 25 °C for 16 hours. The reaction mixture was diluted with DCM (20 mL) and washed with saturated NaHCO3 (30 mL x 2 times) and brine (30 mL). The organic phase was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 1:0 to 1:1). The desired compound (2.2 g, yield: 81.0%, purity 78%) was obtained as yellow oil. MS(ESI) m / z(M+H) + :892.3.

[0406]

[0349] Step 6. Preparation of the compounds ((2R,3R,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methyldibenzyl phosphate and ((2R,3S,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-((4-methoxybenzyl)oxy)tetrahydrofuran-2-yl)methyldibenzyl phosphate

[0407] [ka]

[0408]

[0350] The solution of the product compound from step 5 (2.2 g, 2.47 mmol) and TFA (562 mg, 4.93 mmol, 365 μL) in DCM (18 mL) was stirred at 25-30°C for 4 hours. The reaction mixture was adjusted to a pH of approximately 8-9 using saturated NaHCO3 (40 mL) and extracted with DCM (50 mL x 2 times). The organic phases were combined, washed with brine (50 mL), and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 1:0 to 0:1). A mixture of the two compounds (1.1 g, yield: 76.7%) was obtained as a yellow oil. This was used in further steps without purification. MS (ESI) m / z (M+H) + = 530.1, 650.1.

[0409]

[0351] Step 7. Preparation of compound ((2R,3R,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate

[0410] [ka]

[0411]

[0352] To a mixture of the compound (1.1 g, 1.69 mmol) of the product from step 6 above in t-BuOH (15 mL) and H2O (15 mL), Pd / C (0.2 g) and Pd(OH)2 (0.2 g) were added. The mixture was stirred at 25°C for 36 hours under a hydrogen atmosphere (50 psi). The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was used in the next step without further purification. The desired compound (0.45 g, crude) was obtained as a gray solid. 1H NMR (400MHz, DMSO-d6) δ 8.17 (s, 1H), 8.12 (s, 1H), 7.34 (br s, 2H), 5.96 (d, J = 2.4 Hz, 1H), 5.34 - 4.95 (m, 1H), 4.76 (br d, J = 15.6 Hz, 1H), 4.63 - 4.42 (m, 1H), 4.20 - 3.93 (m, 2H).

[0353] Step 8. ((2R,3R,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methylhydrogen morpholinophosphonate (4'-morpholine-N,N'-dicyclohexylcarboxamidinium salt)

[0412] [ka]

[0413]

[0354] DCC (354.50 mg, 1.72 mmol, 347.5 μL) in t-BuOH (4 mL) was added dropwise over 15 minutes to a reflux solution (110°C) of H2O (4 mL) and the compound of product from step 7 above (150 mg, 430 μmol) and morpholine (150 mg, 1.72 mmol) in t-BuOH (4 mL). The mixture was stirred under N2 at 100°C for 12 hours. The solution was filtered. The filtrate was collected and concentrated. The residue was diluted with H2O (30 mL) and washed with TBME (20 mL x 2 times). The aqueous phase was collected and concentrated under vacuum. The desired compound (290 mg, crude) was obtained as yellow oil. It was used directly in the next step without further purification. 1H NMR (400MHz, D2O) δ 8.11 (s, 1H), 8.05 (s, 1H), 6.01 - 5.98 (m, 1H), 5.22 - 5.07 (m, 1H), , 4.80 - 4.73 (m, 1H), 4.55 - 4.48 (m, 1H), 4.09 - 4.02 (m, 1H), 4.00 - 3.92 (m, 1H), 3.44 - 3.39 (m, 4H), 2.85 - 2.80 (m, 4H). 31 P NMR δ 7.5.

[0355] Step 9. Preparation of the compound (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0414] [ka]

[0415]

[0356] The compound produced in step 8 above (150 mg, 299.79 μmol) and the compound produced in step 14 in the preparation of compound 1 above (290 mg, 693.25 μmol) were dried with pyridine (3 mL x 3 times). The residue was redissolved in pyridine (5 mL) and 1H-tetrazole (105.01 mg, 1.50 mmol, 132.92 μL) was added. The solution was stirred at 30°C for 20 hours. Volatile substances were removed by vacuum. The residue was dissolved in MeOH (5 mL) and filtered. The filtrate was collected. The solution was purified by column chromatography (DCM: (MeOH:NH3.H2O=50:1)=1.2:1) to obtain the crude product (120 mg). The crude product was purified by preparative HPLC (column: Waters Xbridge 150*25 5μ; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 0%-30%, 12 min) to obtain the desired compound (46.9 mg, purity: 90.6%, yield: 18%) as a white solid. MS(ESI) m / z(M+H) + :832.2. 1 H NMR (400MHz, CD3OD) δ 8.25 (s, 1H), 8.19 (s, 1H), 6.12 (s, 1H), 5.60 - 5.50 (m, 2H), 5.25 - 5.09 (m, 4H), 4.80 - 4.71 (m, 1H), 4.69 - 4.62 (m, 1H), 4.47 - 4.31 (m, 3H), 4.27 - 4.18 (m, 1H), 3.92 - 3.88 (m, 1H), 2.12 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H).MS(ESI) m / z(M+H) + = 832.2. compound 18

[0357] 3'-(s)-fluoroadenosine-5'-(D-glycero-β-D-manno-heptopyranosyl)diphosphate

[0416] [ka]

[0417]

[0358] Step 1. Preparation of compound 3'-(s)-fluoroadenosine-5'-(D-glycero-β-D-manno-heptopyranosyl)diphosphate

[0418] [ka]

[0419]

[0359] The solution of the compound (6 mg, 7.22 μmol) produced in step 9 of the preparation of compound 17 above in a 4 mL solution (consisting of MeOH (9 mL), TEA (0.15 mL), and TEAB (12 mL)) was maintained at -20°C for 36 hours. The solution was lyophilized. The desired compound (4 mg, 6.44 μmol) was obtained as a white solid. MS (ESI) m / z (MH) + :620.2. 1 H NMR (400MHz, D2O) δ 8.13 (s, 1H), 8.05 (s, 1H), 6.03 - 5.99 (m, 1H), 5.22 - 5.21 (m, 1H), 5.08 - 4.96 (m, 3H), 4.27 - 4.10 (m, 2H), 4.00 - 3.95 (m, 1H), 3.90 - 3.85 (m, 1H), 3.82 - 3.75 (m, 1H), 3.56 - 3.38 (m, 3H), 3.26 - 3.17 (m, 1H), 2.97 - 2.86 (m, 15H), 1.12 - 0.97 (m, 23H). compound 19

[0360] (2S,3S,4S,5R,6R)-2-(((((((3aS,4S,6R,6aR)-6-(6-amino-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0420] [ka]

[0421]

[0361] Step 1. Preparation of N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide

[0422] [ka]

[0423]

[0362] N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purine-6-yl)benzamide (23.0 g, 61.9 mmol) and 2,2-dimethoxypropane (64.5 g, 619.3 mmol) were dissolved in acetone (400 mL). Then, p-TsOH.H2O (12.8 g, 74.3 mmol) was added. The reaction mixture was stirred at 25°C for 4 hours. After the reaction was complete, the reaction mixture was cooled to 0°C and quenched with saturated NaHCO3 solution (200 mL). The reaction mixture was diluted with ethyl acetate (250 mL), and the emulsion aqueous layer was extracted with ethyl acetate (250 mL x 2 times). The organic layers were combined, washed with brine (250 mL), dried, and concentrated under vacuum to obtain the desired compound (25.0 g, crude) as a bright yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (br, 1H), 8.74 (s, 1 H), 8.65 (s, 1 H), 8.03 - 7.99 (m, 2H), 7.64 - 7.60 (m, 1H), 7.54 - 7.50 (m, 2H), 6.25 - 6.24 (m, 1H), 5.41 (dd, J = 6.4Hz, 2.4 Hz, 1 H), 5.11 (t, J = 5.2 Hz,1 H), 4.99 - 4.97 (m, 1H), 4.26 - 4.23 (m, 1H), 3.56 - 3.50 (m, 2H), 1.54 (s, 3H), 1.32 (s, 3 H).

[0363] Step 2. Preparation of ((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purine-9-yl)-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methyl 4-methylbenzene sulfonate

[0424] [ka]

[0425]

[0364] TosCl (15.0 g, 79.0 mmol) in CH2Cl2 (50 mL) was added at 0°C to a solution of the compound obtained in step 1 above (25.0 g, 60.7 mmol), DMAP (1.4 g, 12.1 mmol), and TEA (12.3 g, 121.5 mmol) in CH2Cl2 (250 mL). The reaction mixture was stirred at 25°C for 6 hours. After the reaction was complete, the reaction mixture was cooled to 0°C and quenched with saturated NaHCO3 solution (200 mL). The reaction mixture was diluted with ethyl acetate (200 mL), and the emulsion aqueous layer was extracted with ethyl acetate (200 mL x 2 times). The organic layers were combined, washed with brine (200 mL), dried, and concentrated under vacuum to obtain the crude product. The solution was purified using a silica gel column (petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain the desired compound (35.0 g, yield: 76.7%, purity: 75.3%) as a white solid. MS(ESI) m / z(M+H) + :566.0

[0365] Step 3. Preparation of N-(9-((3aR,4R,6aS)-2,2-dimethyl-6-methylenetetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide

[0426] [ka]

[0427]

[0366] t-BuOK (15.7 g, 139 mmol) was added to a solution of the compound of product from step 2 (35.0 g, 46.6 mmol) in THF (400 mL). The resulting mixture was stirred at 25°C for 2 hours. The reaction mixture was added to aqueous NH4Cl (200 mL) and extracted with ethyl acetate (200 mL x 2 times). The organic phase was dried (Na2SO4) and concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain the desired compound (9.7 g, yield 52.9%) as a bright yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ11.24 (s, 1 H), 8.72 (s, 1 H), 8.58 (s, 1 H), 8.02 - 8.00 (m, 2H), 7.64 - 7.60 (m, 1H), 7.54 - 7.50 (m, 2H), 6.58 (s, 1H), 5.63 - 5.61 (m, 1H), 5.43 - 5.41 (m, 1H), 4.46 (s, 1H), 4.38 - 4.37 (m, 1H), 1.47 (s, 3 H), 1.35 (s, 3 H).

[0367] Step 4. Preparation of two isomers of compound N-(9-((3aR,4R,6R,6aS)-6-fluoro-6-(iodomethyl)-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide

[0428] [ka]

[0429] Subsequently, I2 (18.0 g, 71.1 mmol) was added to a solution of the compound of product from step 3 (7 g, 17.7 mmol) in CH3CN (300 mL) at -20°C. Then, a solution of AgF (2.26 g, 17.7 mmol) in CH3CN (300 mL) was added. The mixture was stirred at -20°C to -25°C for 16 hours. The reaction mixture was filtered, concentrated under reduced pressure, then ethyl acetate (300 mL) was added, washed with aqueous sodium bicarbonate (100 mL), the organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column (petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain a mixture of two isomers of the desired compound (4.00 g, yield: 40.4%, purity 97.1%) as a bright yellow solid. MS(ESI) m / z(M+H) + :540.0. 1 H NMR (400 MHz, DMSO-d6) δ11.25 (br, 1H), 8.78 - 8.75 (m, 1H), 8.64 - 8.52 (m, 1H), 8.03 - 8.01 (m, 2H), 7.66 - 7.61 (m, 1H), 7.55 - 7.51 (m, 2H), 6.66 - 6.53 (m, 1H), 5.88 - 5.86 (m, 0.5H), 5.44 - 5.37 (m, 1H), 5.29 - 5.26 (m, 0.5H), 3.65 - 3.48 (m, 2H), 1.55 (s, 1.5H), 1.52 (s, 1.5H),1.37 (s, 1.5H), 1.32 (s, 1.5H).

[0368] Step 5. Preparation of two isomers of N-(9-((3aR,4R,6S,6aS)-6-fluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide

[0430] [ka]

[0431]

[0369] TFA (3.80 g, 33.3 mmol) and tetra(n-butyl)ammonium hydroxide (5.19 g, 19.9 mmol) were added to a CH2Cl2 (80 mL) solution of the mixture of the two isomers obtained from step 4 above (3.70 g, 6.66 mmol), and then m-CPBA (6.76 g, 33.3 mmol, purity 85%) was added at 25°C. The mixture was stirred at 25°C for 16 hours. The reaction mixture was washed with saturated Na2SO3 solution (20 mL) and aqueous NaHCO3 solution (20 mL). The organic layer was dehydrated with anhydrous Na2SO4 and concentrated at low pressure. The crude product was purified by silica gel column (petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain the desired mixture of the two isomers (1.20 g, yield: 39.8%, purity 94.9%) as a bright yellow solid. MS(ESI) m / z(M+H) + :430.0. 1 H NMR (400 MHz, DMSO-d6) δ 11.23 (br. s, 1H), 8.79 - 8.73 (m, 1H), 8.62 (s, 0.3H), 8.54 - 8.48 (m, 0.7H), 8.05 - 7.98 (m, 2H), 7.65 - 7.59 (m, 1H), 7.56 - 7.49 (m, 2H), 6.63 (s, 0.3H), 6.48 (s, 0.7H), 5.81 - 5.75 (m, 1H), 5.41 - 5.32 (m, 1H), 5.21 - 5.14 (m, 1H), 3.80 - 3.54 (m, 2H), 1.52 (s, 1H), 1.50 (s, 2H), 1.36 (s, 2H), 1.32 (s, 1H).

[0370] Step 6. Preparation of two isomers of ((3aS,4S,6R,6aR)-6-(6-benzamido-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methyldibenzylphosphate

[0432] [ka]

[0433]

[0371] To a mixture of the two isomers obtained from step 5 above (850 mg, 1.98 mmol) in CH2Cl2 (50 mL) and CH3CN (17 mL) and 1H-imidazole-4,5-dicarbonitric acid (467 mg, 3.96 mmol), N-dibenzyloxyphosphanyl-N-isopropyl-propane-2-amine (1.37 g, 3.96 mmol) was added at 0°C. The reaction mixture was stirred for 10 minutes and then warmed to 25°C. The resulting mixture was stirred for another 2 hours and then cooled again to 0°C. m-CPBA (803 mg, 3.96 mmol, 85% purity) was added as is, and the reaction mixture was slowly warmed to 25°C and stirred for 2 hours. The reaction mixture was quenched with saturated NaHCO3 (50 mL) and the organic phase was separated. The aqueous phase was extracted with CH2Cl2 (50 mL x 2 times). The organic phases were combined, dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 0:1) to obtain the two desired isomers (1.10 g, yield: 73.2%, purity: 90.8%) as bright yellow solids. MS(ESI) m / z(M+H) + :690.1. H NMR (400 MHz, DMSO-d6) δ 11.25 (br. s, 1H), 8.75 (s, 0.7H), 8.64 (s, 0.3H), 8.61 (s, 0.3H), 8.54 (s, 0.7H), 8.01 (d, J = 8.4 Hz, 2H), 7.66 - 7.58 (m, 1H), 7.56 - 7.48 (m, 2H), 7.36 - 7.24 (m, 10H), 6.73 (s, 0.3H), 6.57 (s, 0.7H), 5.85 (d, J = 5.6 Hz, 0.7H), 5.51 - 5.42 (m, 0.3H), 5.41 - 5.37 (m, 0.3H), 5.30 (t, J = 6.0 Hz, 0.7H), 5.05 - 4.94 (m, 4H), 4.37 - 4.14 (m, 2H), 1.51 (s, 1H), 1.46 (s, 2H), 1.34 (s, 2H), 1.32 (s, 1H).

[0372] Step 7. Preparation of two isomers of ((3aS,4S,6R,6aR)-6-(6-amino-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methyldibenzylphosphate

[0434] [ka]

[0435]

[0373] The two isomers obtained from step 6 above (1.10 g, 1.45 mmol) were dissolved in NH3 / MeOH (20 mL, 7 M). The reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 0:1, then CH2Cl2:MeOH = 10:2) to obtain the desired isomer (710 mg, yield: 77.5%, purity 92.7%) as a bright yellow solid. MS(ESI) m / z(M+H) + = 586.1.

[0436]

[0374] Step 8. Preparation of two isomers of ((3aS,4S,6R,6aR)-6-(6-amino-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluor[3,4-d][1,3]dioxol-4-yl)methyl dihydrogen phosphate

[0437] [ka]

[0438]

[0375] A mixture of the two isomers obtained from step 7 above (600 mg, 1.02 mmol) in t-BuOH (20 mL) and H2O (20 mL) was mixed with Pd(OH)2 (300 mg, 427.23 μmol, 20%) and Pd / C (50 mg, 1.02 mmol, 10%), and the reaction mixture was then stirred at 25°C for 16 hours under an N2 atmosphere (45 psi). The mixture was filtered, and the filtrate was concentrated to obtain the desired isomer (200 mg, crude) as a bright yellow solid. 1 H NMR (400MHz, DMSO-d6) δ 8.26 - 8.17 (m, 2H), 6.59 - 6.46 (m, 1H), 5.68 (d, J = 8.0 Hz, 1H), 5.57 - 5.45 (m, J = 11.7 Hz, 1H), 5.37 - 5.30 (m, 1H), 4.21 - 4.06 (m, 2H), 1.80 - 1.52 (m, 3H), 1.49 - 1.35 (m, 3H).

[0376] Step 9. Preparation of ((3aS,4S,6R,6aR)-6-(6-amino-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methylhydrogen morpholinophosphonate DCC morpholine salt

[0439] [ka]

[0440]

[0377] A solution of DCC (407 mg, 1.97 mmol) in t-BuOH (10 mL) was added dropwise to a solution of H2O (10 mL) and t-BuOH (10 mL) containing the two isomers obtained from step 8 above (200 mg, 493 μmol) and morpholine (171 mg, 1.97 mmol) under 80-90°C. The solution was stirred under N2 at 80-90°C for 16 hours. The reaction mixture was cooled to room temperature, the solvent was removed, and the residue was obtained. The residue was dissolved in H2O (10 mL), extracted with TBME (10 mL x 2 times), and the aqueous phase was concentrated under reduced pressure to obtain the desired isomer (310 mg, crude) as a bright yellow solid. 1H NMR (400MHz, D2O) δ 8.41 - 8.17 (m, 2H), 6.70 - 6.50 (m, 1H), 5.81 - 5.68 (m, 1H), 5.62 - 5.46 (m, 1H), 5.42 - 5.28 (m, 1H), 4.34 - 4.09 (m, 1H), 4.07 - 4.00 (m, 1H), 3.88 - 3.74 (m, 5H), 3.73 - 3.55 (m, 2H), 3.51 - 3.22 (m, 8H), 3.07 - 3.02 (m, 1H), 2.96 - 2.91 (m, 1H), 2.87 - 2.78 (m, 2H), 1.90 (br s, 4H), 1.81 - 1.70 (m, 4H), 1.68 - 1.57 (m, 5H), 1.51 - 1.44 (m, 3H), 1.39 - 1.26 (m, 8H), 1.19 - 1.08 (m, 2H).

[0378] Step 10. Preparation of two isomers of (2S,3S,4S,5R,6R)-2-(((((((3aS,6R,6aR)-6-(6-amino-9H-purine-9-yl)-4-fluoro-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0441] [ka]

[0442]

[0379] The two isomers obtained from step 9 above (310 mg, 403 μmol) and the product from step 14 in the preparation of compound 1 above (226 mg, 322 μmol) were dehydrated with dried pyridine (10 mL x 3 times). The mixture was dissolved in pyridine (15 mL). 1H-tetrazole (94.2 mg, 1.35 mmol) was added and the mixture was stirred at 25°C for 72 hours. The solvent was removed to obtain the residue. The residue was purified by silica gel column chromatography (DCM:MeOH (containing 2% NH3, H2O) = 1:0 to 1:1) to obtain the crude desired product (170 mg, crude) as a white solid. The crude product (60 mg) was purified by preparative HPLC (column: Waters Xbridge 150*25 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 0%-35%, 10 min) to obtain isomer 1 (17 mg) and isomer 2 (7 mg). Isomer 1: 1 H NMR (400 MHz, D2O) δ 8.24 - 8.11 (m, 2 H), 6.46 (s, 1 H) ,5.41 (dd, J = 11.7, 6.6 Hz, 1 H), 5.35 - 5.23 (m, 2 H) ,5.19 (d, J = 6.4 Hz, 1 H), 5.09 - 4.85 (m, 3 H), 4.26 - 3.97 (m, 3 H), 3.91 (dd, J = 12.1, 7.2 Hz, 1 H),3.77 (br d, J = 9.8 Hz, 1 H), 1.98 (s, 3 H), 1.91 (s, 3 H), 1.89 (s, 3H), 1.82 (s, 3 H), 1.78(s, 3 H), 1.47 (s, 3 H), 1.25 (s, 3 H) Isomer 2: 1H NMR (400 MHz, D2O) δ 8.24 - 8.07 (m, 2 H), 6.50 - 6.36 (m, 1 H), 5.57 (d, J = 5.6 Hz, 1 H) ,5.47 - 5.24 (m, 2 H), 5.23 - 5.13 (m, 1 H), 5.09 - 4.85 (m, 4 H), 4.24 - 4.10 (m, 2 H), 4.03 (dd, J = 12.0, 7.1 Hz, 1 H), 3.80 (dd, J = 10.0, 2.7 Hz, 1 H), 1.98 (s, 3 H), 1.95 (s, 3 H), 1.93 (s, 3 H), 1.84 (s, 3 H), 1.77(s, 3 H), 1.44 (s, 3 H), 1.27 (s, 3 H) compound 20 (2S,3S,4S,5R,6R)-2-(((((((2S,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-2-fluoro-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0443] [ka]

[0444]

[0380] Step 1. Preparation of compound (2S,3S,4S,5R,6R)-2-(((((((3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-2-fluoro-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0445] [ka]

[0446]

[0381] Solutions of the two isomers (80.0 mg, 90.1 μmol) obtained from step 10 in the preparation of compound 19 in TFA (0.6 mL) and H2O (0.4 mL) were stirred at 25°C for 0.5 hours. The mixture was adjusted to pH=7 using Et3N and concentrated to obtain the crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge 150*25 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 0%-35%, 9 min) to obtain one isomer (10 mg, 11.8 μmol, yield 13.1%). MS(ESI) m / z(M+H) + :848.2 1 H NMR (400 MHz, methanol-d4) δ 8.37 (s, 1 H), 8.13 (s, 1 H) ,6.26 (br s, 1 H), 5.55 - 5.37 (m, 2 H), 5.08 (br s, 3 H), 4.86 - 4.70 (m, 1H), 4.44 (d, J = 5.8 Hz, 1 H) ,4.32 (br d, J = 12.0 Hz, 1 H), 4.23 - 4.04 (m, 3 H), 3.84 (br s, 1 H), 3.22 - 3.16 (m, 7 H), 2.01 (s, 3 H), 1.93 (s, 3H), 1.88 (s, 3H), 1.80 (s, 3 H), 1.72(s, 3 H). 19 Fδ-123.7, 31 P δ -13.22 and -15.21. compound 21 (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((S)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0447] [ka]

[0448] Step 1. Preparation of (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((S)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0449] [ka]

[0450]

[0382] A mixture of compound (2R,3R,4S,5S,6S)-2-((S)-1,2-diacetoxyethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate trimethylamine salt (200 mg, 399.72 μmol; Inuki et al., Org. Lett. 2017, 19, 3079-3082; Zamyatina et al., Carbohydrate Research (2003), 338:2571-2589) and compound AMP-molfolide (4'-morpholine-N,N'-dicyclohexylcarboxamidinium salt) (360 mg, 864.71 μmol) was dried with pyridine (5 mL x 3 times). The residue was then redissolved in pyridine (5 mL). ¹H-tetrazole (100 mg, 1.43 mmol) was added. The solution was stirred at 30°C for 24 hours. The solvent was removed by vacuum. The residue was dissolved in MeOH (5 mL). The solid was removed by filtration. The filtrate was collected and concentrated. The residue was purified by column chromatography (DCM: (MeOH:NH3.H2O 50:1) = 1:0~1:1.2) to obtain the crude product. This was re-purified by preparative HPLC (column: Waters Xbridge 150*25 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 0%-35%, 10 min) to obtain the desired compound (68 mg, yield: 20.24%) as a white solid. MS(ESI) m / z(M+H) + :830.2. 1 H NMR (400 MHz, CD3OD) δ 8.59 (s, 1H), 8.19 (s, 1H), 6.08 (d, J = 5.2 Hz, 1H), 5.62 - 5.57 (m, 1H), 5.55 - 5.47 (m, 1H), 5.28 - 5.22 (m, 1H), 5.19 - 5.12 (m, 2H), 4.65 - 4.57 (m, 1H), 4.49 - 4.39 (m, 2H), 4.33 - 4.15 (m, 4H), 3.94 - 3.84 (m, 1H), 2.14 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H). compound 22

[0383] Adenosine-5'-(L-glycero-β-D-mannoheptopyranosyl)diphosphate

[0451] [ka]

[0452]

[0384] Step 1. Preparation of adenosine-5'-(L-glycero-β-D-mannoheptopyranosyl) diphosphate

[0453] [ka]

[0454]

[0385] The compound (14.4 mg, 17.36 μmol) of the product of step 1 in the preparation of compound 21 above in 2 mL of solvent (consisting of 0.1 M TEAB (8 mL), MeOH (6 mL), and TEA (0.1 mL)) was stirred at -20°C for 56 hours. The solution was vacuum freeze-dried to obtain the trimethylamine salt of the desired compound (8 mg, yield: 30.51%) as a white viscous solid. MS (ESI) m / z (MH) + :617.9. 1 H NMR (400 MHz, D2O) δ 8.30 (s, 1H), 8.04 (s, 1H), 6.00 - 5.92 (m, 1H), 5.04 - 4.99 (m, 1H), 4.58 - 4.54 (m, 1H), 4.25 - 4.33 (m, 1H), 4.23 - 4.15 (m, 1H), 4.09 - 3.97 (m, 3H), 3.91 - 3.85 (m, 1H), 3.76 - 3.67 (m, 1H), 3.55 - 3.42 (m, 3H), 3.17 - 3.10 (m, 1H), 3.00 - 2.94 (m, 14H), 1.08 - 1.03 (m, 22H). compound 23

[0386] (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0455] [ka]

[0456]

[0387] Step 1. Preparation of ((2-cyanoethoxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphanyl)dipropylamine

[0457] [ka]

[0458]

[0388] 1H-tetrazole (71 mg, 1.01 mmol) in CH3CN (1.5 mL) was added dropwise to a solution of the compound from step 7 of the preparation of compound 7 above (300 mg, 0.51 mmol) in DCM (7.5 mL) and 3-bis(diisopropylamino)phosphanyloxypropanenitrile (304 mg, 1.01 mmol, 320 μL) under an N2 atmosphere at 0°C. The resulting mixture was stirred at 25°C for 2 hours. After the reaction was complete, the mixture was filtered and concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column (PE:EA = 1:0~1:1) to obtain the desired compound (80 mg, yield: 19.0%, purity 89%) as a colorless oil. MS (ESI) m / z (M+H) + :711.1 (mass after hydrolysis).

[0459]

[0389] Step 2. Preparation of (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((2-cyanoethoxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoroyl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0460] [ka]

[0461]

[0390] 4,5-dicyanoimidazole (24 mg, 203 μmol) was added under an N2 atmosphere to a solution in DMF (3 mL) of the product compound from step 1 (80 mg, 0.10 mmol) and the product compound from step 4 in the preparation of compound 5 (121 mg, 151 μmol). The resulting mixture was stirred at 25°C for 1 hour. Then, sulfur (5 mg, 151 μmol) was added. The resulting mixture was stirred at 25°C for another 0.5 hours. After the reaction was complete, the mixture was purified directly by preparative HPLC (column: Waters Xbridge 150*25 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 38%-64.25%, 7 min) to obtain the desired compound (23 mg, yield: 15.8%, purity 80%) as a white solid. MS(ESI) m / z(M+H)+: 1153.5.

[0462]

[0391] Step 3. Preparation of the compound (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((2-cyanoethoxy)(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)phosphoroyl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0463] [ka]

[0464]

[0392] TFA (616 mg, 5.40 mmol, 0.4 mL) was added to a solution of the compound of product from step 2 (5 mg, 4.34 μmol) in dioxane (0.6 mL). The resulting mixture was stirred at 40°C for 2 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (10 mL) and washed with saturated NaHCO3 (10 mL). The organic phase was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound G-3 (6 mg, crude) as a light yellow syrup. This was used directly in the next step. MS(ESI) m / z(M+H) + :909.9.

[0465]

[0393] Step 4. Preparation of compound (2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(((((((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0466] [ka]

[0467]

[0394] DBU (3.30 mg, 21.7 μmol, 3.3 μL) was added to the CH3CN (0.5 mL) solution of the compound from step 3 (5 mg, 4.34 μmol). The resulting mixture (reluting mixture) was stirred at 25°C for 0.5 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (10 mL) and washed with 1N HCl (10 mL). The organic phase was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired crude compound (7 mg) as a bright yellow syrup. This was purified by preparative HPLC (column: Boston Green ODS 150*30 5 μ; mobile phase: [water (0.075% TFA)-ACN]; B%: 30%-50%, 7.5 min) to obtain the desired compound. MS (ESI) m / z (M+H) + :858.5. compound 24

[0395] Adenosine-(5'-(mannose-pyranosyl)(hydroxy)phosphoroyloxyphosphate

[0468] [ka]

[0469]

[0396] Step 1. Preparation of adenosine-(5'-(mannose-pyranosyl)(hydroxy)phosphoroyloxyphosphate

[0470] [ka]

[0471]

[0397] The solution of the compound of product from step 4 in the preparation of compound 23, dissolved in a 7:3:1 ratio MeOH / water / Et3N solution, was stirred at 25°C for 10 hours. After the reaction was complete, the mixture was concentrated and freeze-dried from water to obtain the desired compound. compound 25

[0398] (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0472] [ka]

[0473]

[0399] Step 1. Preparation of (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0474] [ka]

[0475]

[0400] The mixture of the compound (220 mg, 439.70 μmol) produced in step 14 of the preparation of compound 1 above and AMP-molfolide (4'-morpholine-N,N'-dicyclohexylcarboxamidinium salt) (549.2 mg, 1.32 mmol) was dried with pyridine (5 mL x 3 times). The residue was then redissolved in pyridine (5 mL) and 1H-tetrazole (154.01 mg, 2.20 mmol) was added. The solution was stirred at 30°C for 48 hours. The solvent was removed by vacuum. The residue was dissolved in MeOH (10 mL). The solid was removed by filtration. The filtrate was collected and concentrated. The residue was purified by column chromatography (DCM:(MeOH:NH3.H2O=50:1)=1:0~1.2:1) to obtain the crude product (140 mg). The solution was then re-purified by preparative HPLC (column: Waters Xbridge 150*25 5μ; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 0%-35%, 10 min) to obtain the desired compound (50 mg, yield 13.6%) as a white solid. 1 H NMR (400MHz, methanol-d4) δ 8.58 (s, 1H), 8.18 (s, 1H), 6.07 (d, J = 5.2 Hz, 1H), 5.53 - 5.56 (br. s, 2H), 5.16-5.22 (m, 3H), 4.60 - 4.63 (m, 1H), 4.40 - 4.46 (m, 2H), 4.19 - 4.24 (m, 4H), 3.87 -3.89 (m, 1H), 2.13 (s, 3H), 2.04 2.03 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H), 1.92 (s, 3H).MS(ESI) m / z(M+H) + :830.4. compound 26

[0401] (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0476] [ka]

[0477]

[0402] Step 1. Preparation of compound ((2R,3R,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methanol

[0478] [ka]

[0479]

[0403] A mixture of the product compounds from Step 5 of Example 1 (4.1 g, 4.6 mmol) in dioxane (100 mL) was added dropwise with HCl-dioxane (4 M, 10 mL). The mixture was stirred at 26 °C for 30 minutes. After the reaction was complete, the mixture was diluted with EA (500 mL) and washed with saturated NaHCO3 (100 mL x 3 times) and brine (100 mL x 3 times). The organic layer was dehydrated with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (PE:EA = 1:0 to 1:1) to obtain the desired compound (1.8 g, yield: 60.7%) as a white solid. MS (ESI) m / z (M+H) + :646.1. 1H NMR (400MHz, DMSO-d6) δ 8.42 (s, 1H), 7.86 (s, 1H), 7.34 - 7.19 (m, 15H), 7.03 (d, J = 8.6 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.06 (d, J = 8.1 Hz, 1H), 5.58 - 5.52 (m, 1H), 5.46 - 5.22 (m, 1H), 4.98 - 4.89 (m, 1H), 4.57 - 4.26 (m, 4H), 3.70 (s, 3H), 3.63 - 3.60 (m, 2H).

[0404] Step 2. Preparation of the compound ((2R,3R,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methylhydrogen phosphonate

[0480] [ka]

[0481]

[0405] Phenoxyphosphonoyloxybenzene (1.65 mL, 8.6 mmol) was added to a solution of the compound produced in Step 1 (1.8 g, 2.8 mmol) in pyridine (6 mL). The mixture was stirred at 25°C for 2 hours. Then, TEA (1.45 g, 14.37 mmol, 2 mL) and H2O (515 μL, 28.5 mmol) were added to the mixture. The mixture was stirred at 25°C for another 0.5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1 to 10:1, with 0.5% Et3N added) to obtain the desired compound (2 g, yield: 90%) as a light yellow oil. MS (ESI) m / z (M+H) + :696.2.

[0482]

[0406] Step 3. Preparation of compound O-(((2R,3R,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl)O,O-dihydrogen phosphorothioate triethylamine salt

[0483] [ka]

[0484]

[0407] To a solution of the compound (2 g, 2.8 mmol) of the product from step 2 above in pyridine (5 mL) and Et3N (5 mL), TMSCl (1.82 mL, 14.3 mmol) was added dropwise over 15 minutes under an N2 atmosphere. The mixture was stirred at 0°C for 1 hour, and then sulfur (555 mg, 17.3 mmol) was added. The mixture was stirred at 0°C for another 45 minutes. After the reaction was complete, the reactants were quenched with H2O (10 mL), and the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel chromatography (DCM:MeOH = 20:1 to 10:1, with 0.5% Et3N added) to obtain the desired compound (900 mg, yield 43%) as a yellow syrup. MS(ESI) m / z(M+H) + :728.3. 1 H NMR (400MHz, DMSO-d6) δ 8.60 (s, 1H), 7.87 (s, 1H), 7.45 (s, 1H), 7.37 - 7.23 (m, 15H), 7.08 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 6.05 (d, J = 8.0 Hz, 1H), 5.60 - 5.35 (m, 1H), 5.14 - 4.96 (m, 1H), 4.63 - 4.34 (m, 3H), 4.11 - 3.82 (m, 2H), 3.69 (s, 3H), 3.12 - 2.89 (m, 12H), 1.19 (t, J=7.3 Hz, 18H).

[0408] Step 4. Preparation of the compound (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0485] [ka]

[0486]

[0409] CDI (943 mg, 5.8 mmol) was added to a solution of the compound of product from step 14 in Example 1 (350 mg, 581.8 μmol, Et3N) in anhydrous DMF (15 mL) under an N2 atmosphere. The resulting mixture was stirred at 25°C for 3 hours. After the reaction was complete, MeOH (0.28 mL) was added to quench the reaction product, and the mixture was concentrated under reduced pressure to obtain the desired compound (1 g, crude). This was then used directly in the next step.

[0487]

[0410] Step 5. Preparation of the compound (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(((((((2R,3R,4S,5R)-3-fluoro-4-((4-methoxybenzyl)oxy)-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioyl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0488] [ka]

[0489]

[0411] ZnCl2 (1.1 g, 8.2 mmol) was added to a solution of the product compound from step 4 (380 mg, 690.4 μmol) and the product compound from step 3 (580 mg, 699.7 μmol) in anhydrous DMF (10 mL) under an N2 atmosphere. The resulting mixture was stirred at 25°C for 16 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column chromatography (DCM:MeOH = 10:1, with 0.5% Et3N added) to obtain the desired compound (350 mg, yield: 40%) as a white solid. MS (ESI) m / z (M+H) + :1210.5

[0412] Step 6. Preparation of the compound (2S,3S,4S,5R,6R)-2-(((((((2R,3S,4S,5R)-5-(6-amino-9H-purine-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0490] [ka]

[0491]

[0413] A solution of the compound (350 mg, 289 μmol) of the product from step 5 above in DCM (1 mL) and TFA (0.2 mL, 2.7 mmol) was stirred at 25°C for 3 hours. After the reaction was complete, the mixture was adjusted to pH=7 by adding Et3N. The reaction product was concentrated under reduced pressure. The product was purified by preparative HPLC (water (10 mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min) to obtain the desired compound (70 mg, yield 39.9%) as a white solid. MS(ESI) m / z(M+H) + :848.2 1H NMR (400MHz, MeOD) δ 8.83 (s, 0.5H), 8.75 (s, 0.5H), 8.20 (s, 1H), 6.27 - 6.06 (m, 1H), 5.76 - 5.61 (m, 2H), 5.30 - 5.16 (m, 3H), 5.09 - 4.93 (m, 2H), 4.60 - 4.12 (m, 5H), 3.97 - 3.95 (m, 1H), 2.15 (s, 1.5H), 2.14 (s, 1.5H), 2.08 - 2.04 (m, 6H), 2.02 (s, 1.5H), 2.00 (s, 1.5H), 1.95 (s, 1.5H), 1.94 (s, 1.5H). compound 27

[0414] Adenosine-3'-fluoro-5'-(D-glycero-β-D-manno-heptopyranosyl)(hydroxy)phosphoroyloxyphosphate

[0492] [ka]

[0493]

[0415] Step 1. Adenosine-3'-fluoro-5'-(D-glycero-β-D-manno-heptopyranosyl)(hydroxy)phosphoroyloxyphosphate

[0494] [ka]

[0495] The compound (10 mg, 11.8 μmol) of the product from Step 6 in Example 26 was stirred at 20°C for 5 hours in a MeOH / water / Et3N (7:3:1, 1.1 mL) solution. After the reaction was complete, the mixture was concentrated and freeze-dried from the water to obtain the desired compound as trimethylamine salt (8 mg, 91.8% yield) as a white solid. MS (ESI) m / z (MH) - :636.0. 1H NMR (400MHz, D2O) δ 8.67 - 8.49 (m, 1H), 8.15 (s, 1H), 6.16 - 6.02 (m, 1H), 5.45 - 5.18 (m, 1H), 4.97 - 4.82 (m, 1H), 4.32 - 4.18 (m, 1H), 4.15 - 3.87 (m, 4H), 3.75 - 3.53 (m, 5H), 3.44 - 3.35 (m, 1H), 2.97 (q, J = 7.2 Hz, 12H), 1.12 (t, J=7.2 Hz, 18H). compound 28

[0416] (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0496] [ka]

[0497]

[0417] Step 1. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0498] [ka]

[0499]

[0418] To a mixture of the compounds produced in Step 12 of Example 3 (420 mg, 748.01 μmol, Et3N) in DMF (8 mL), CDI (1.2 g, 7.5 mmol) was added. The mixture was stirred at 25°C for 4 hours. After the reaction was complete, MeOH (0.3 mL) was added to quench the reaction product, and the mixture was concentrated under reduced pressure to obtain the desired compound (1.3 g, crude). This was used as is in the next step.

[0500]

[0419] Step 2. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(((((((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0501] [ka]

[0502]

[0420] ZnCl2 (1.2 g, 8.5 mmol) was added under an N2 atmosphere to a solution of the product compound from Step 1 (1.3 g, 2.6 mmol) and the product compound from Step 3 in Example 26 (530 mg, 709 μmol, Et3N) in anhydrous DMF (10 mL). The resulting mixture was stirred at 25°C for 16 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the crude product. This was purified by silica gel column chromatography (DCM:MeOH = 20:1, with 1% Et3N added) to obtain the desired compound (380 mg, yield: 47.5%) as a white solid. MS(ESI) m / z(M+H) + :1088.7.

[0503]

[0421] Step 3. Preparation of the compound (2S,3S,4S,5S,6S)-2-((S)-2-acetoxy-1-fluoroethyl)-6-(((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0504] [ka]

[0505]

[0422] TFA (0.6 mL, 8.10 mmol) was added to the H2O (0.4 mL) solution of the compound obtained in step 2 (370 mg, 340.1 μmol) above. The mixture was stirred at 25°C for 1.5 hours. After the reaction was complete, the reaction product was adjusted to pH=7 by adding Et3N. The mixture was concentrated under reduced pressure to obtain the crude product. This was purified by preparative HPLC (column: Waters Xbridge 150*25 5 μ; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min) to obtain the desired compound (44.5 mg, yield: 16.0%, purity 98.5%) as a white solid. MS (ESI) m / z (M+H) + :806.1. 1 H NMR (400MHz, CD3OD) δ 8.68 (s, 1H), 8.19 (s, 1H), 6.11 - 6.09 (m, 1H), 5.72 - 5.57 (m, 2H), 5.37 - 5.32 (m, 1H), 5.22 - 5.20 (m, 1H), 4.77 - 4.73 (m, 1H), 4.69 - 4.60 (m, 2H), 4.52 - 4.45 (m, 2H), 4.26 - 4.24 (m, 3H), 3.91-3.83 (m, 1H), 2.13(s, 3H), 2.03(s, 3H), 2.02(s, 3H), 1.92(s, 3H). compound 29

[0423] Adenosine-5'-(L-glycero-β-D-manno-6-fluoro-heptopyranosyl)(hydroxy)phosphorooyloxyphosphate

[0506] [ka]

[0507]

[0424] Step 1. Preparation of the compound adenosine-5'-(L-glycero-β-D-manno-6-fluoro-heptopyranosyl)(hydroxy)phosphorooyloxyphosphate

[0508] [ka]

[0509]

[0425] A solution of the compound (16.1 mg, 10.0 μmol) of the product from Step 3 in Example 28 in MeOH / water / Et3N (7:3:1, 2 mL) was stirred at 25°C for 3.5 hours. After the reaction was complete, the mixture was concentrated and freeze-dried from water to obtain the desired compound (14.3 mg, yield: 85.2%, 2Et3N) as a white amorphous solid. MS (ESI) m / z (MH) + :636.1. 1 H NMR (400MHz, D2O) δ 8.47 (s, 0.3H), 8.43 (s, 0.7H), 8.07 (s, 0.7H), 8.06 (s, 0.3H), 5.98 - 5.95 (m, 1H), 5.41 - 5.11(m, 1H), 4.86 - 4.73 (m, 1H), 4.39 - 4.37 (m, 1H), 4.26 - 4.24 (m, 1H), 4.12 - 4.10 (m, 2H), 4.00 - 3.94(m, 1H), 3.83 - 3.64 (m, 4H), 3.54 - 3.51(m, 1H), 3.33 - 3.21(m, 1H), 3.02 (q, J = 7.2Hz, 12H), 1.10 (t, J = 7.2Hz, 18H). compound 30

[0426] (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0510] [ka]

[0511]

[0427] Step 1. Preparation of the compound ((2R,3R,4R,5R)-3,4-dimethoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methylhydrogen phosphonate

[0512] [ka]

[0513]

[0428] PH(OPh)2 (1.35 g, 5.75 mmol) was added to a pyridine (10 mL) solution of the compound produced in Step 2 of Example 13 (1 g, 1.86 mmol). The resulting mixture was stirred at 25°C for 2 hours. Then, Et3N (1.33 mL, 9.52 mmol) and H2O (0.37 mL, 20.42 mmol) were added. The resulting mixture was stirred at 25°C for 0.5 hours. The solvent was removed to obtain the crude product. This was purified by silica gel chromatography (DCM:MeOH = 1:0 to 10:1, with 0.5% Et3N added) to obtain the desired compound (1.5 g, yield: 94.7%, Et3N) as a bright yellow oil. MS(ESI) m / z(M+H) + :602.1. Step 2. Preparation of compound O-(((2R,3R,4R,5R)-3,4-dimethoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl)O,S-dihydrogen phosphorothioate

[0514] [ka]

[0515]

[0429] TMSCl (2.00 mL, 15.7 mmol) was added to the solution of the compound (1.3 g, 1.85 mmol, Et3N) of the product from step 1 in pyridine (15 mL) and Et3N (15 mL). The mixture was stirred at 25°C for 0.5 hours, then sulfur (758 mg, 23.6 mmol) was added. The resulting mixture was stirred for another 1 hour. Then H2O (3.79 mL, 210 mmol) was added. The mixture was stirred for another 0.5 hours. The reaction product was filtered, and the filtrate was concentrated to obtain the crude product. This was purified by silica gel column chromatography (DCM:MeOH = 1:0 to 10:1, with 0.5% Et3N added). The desired compound (400 mg, yield: 33.1%, 2Et3N) was obtained as yellow oil. MS(ESI) m / z(M+H) + :634.1. 1 H NMR (400MHz, CDCl3) δ 8.37 (s, 1H), 8.00 (s, 1H), 7.40 - 7.35 (m, 2H), 7.34 - 7.27 (m, 8H), 7.23 - 7.15 (m, 5H), 6.14 - 6.05 (m, 1H), 4.49 - 4.42 (m, 1H), 4.37 - 4.28 (m, 2H), 4.24 - 4.10 (m, 2H), 3.45 - 3.41 (m, 6H), 3.03 (q, J = 7.2 Hz, 12H), 1.27 (t, J = 7.2 Hz, 18H).

[0430] Step 3. Preparation of the compound (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(((((((2R,3R,4R,5R)-3,4-dimethoxy-5-(6-(tritylamino)-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0516] [ka]

[0517]

[0431] ZnCl2 (516 mg, 3.79 mmol) was added to a solution of the product compound from Step 4 in Example 26 (191 mg, 293 μmol, Et3N) and the product compound from Step 2 (400 mg, 315 μmol) in DMF (10 mL). The mixture was stirred under an Ar atmosphere at 25°C for 24 hours. The solvent was removed to obtain the crude product. The residue was purified by silica gel chromatography column (DCM:MeOH = 1:0 to 10:1, with 1% Et3N added). The desired compound (400 mg, yield: 95.4%) was obtained as a colorless oil. MS (ESI) m / z (M+H) + :1116.5.

[0518]

[0432] Step 4. Preparation of the compound (2S,3S,4S,5R,6R)-2-(((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dimethoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorotioil)oxy)(hydroxy)phosphoryl)oxy)-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0519] [ka]

[0520]

[0433] The mixture of the compound produced in step 3 (400 mg, 358 μmol) and TFA (1 mL) in DCM (5 mL) was stirred at 25°C for 1 hour. After the reaction was complete, the mixture was adjusted to pH=7 using Et3N, the solvent was removed, and the residue was purified by preparative HPLC (Waters Xbridge 150*25 5 μ, water (10 mM NH4HCO3)-CH3CN, 0-30%) to obtain the desired compound (20 mg, yield: 6.4%) as a white solid. MS (ESI) m / z (M+H) + :874.2. 1 H NMR (400MHz, CD3OD) δ 8.77 (s, 0.4H), 8.71 (s, 0.6H), 8.17 (s, 1H), 6.18 - 6.12 (m, 1H), 5.76 - 5.59 (m, 2H), 5.21 - 5.13 (m, 3H), 4.61 - 4.53 (m, 2H), 4.45 - 4.32 (m, 3H), 4.27 - 4.20 (m, 2H), 3.95 - 3.90 (m, 1H), 3.51 (s, 3H), 3.42 (s, 1.4H), 3.41 (s, 1.6H), 2.11(s, 3H), 2.06 - 2.02 (m, 6H), 1.99 (s, 1.6H), 1.97 (s, 1.4H), 1.91 (s, 3H). compound 31

[0434] Adenosine-2'3'-dimethoxy-5'-(D-glycero-β-D-mannoheptopyranosyl)(hydroxy)phosphoroyloxyphosphate

[0521] [ka]

[0522]

[0435] Step 1. Preparation of the compound adenosine-2'3'-dimethoxy-5'-(D-glycero-β-D-mannoheptopyranosyl)(hydroxy)phosphoroyloxyphosphate

[0523] [ka]

[0524]

[0436] A mixture of the compound of the product from Step 4 in Example 30 (8 mg, 9.16 μmol) in MeOH (0.7 mL), H2O (0.3 mL), and Et3N (0.1 mL) was stirred at 15-20°C for 3 hours. The solution was then freeze-dried in a freeze-dryer. The desired compound (6 mg, yield: 76%, 2Et3N) was obtained as a white solid. MS (ESI) m / z (MH) - = 662.1. 1 H NMR (400MHz, D2O) δ 8.47 (s, 0.4H), 8.45 (s, 0.6H), 8.10 (s, 1H), 6.02 (d, J = 6.0 Hz, 1H), 5.13 - 5.05 (m, 1H), 4.50 - 4.42 (m, 2H), 4.40 - 4.36 (m, 1H), 4.28 - 4.19 (m, 1H), 4.20 - 4.10 (m, 2H), 4.00 - 3.92 (m, 1H), 3.85 - 3.80 (m, 1H), 3.59 - 3.55 (m, 2H), 3.52 - 3.41 (m, 2H), 3.36 (s, 3H), 3.27 (s, 3H), 3.02 (q, J = 7.3 Hz, 12H), 1.10 (t, J = 7.3 Hz, 18H). compound 32

[0437] (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(((((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-hydroxy-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0525] [ka]

[0526]

[0438] Step 1. Preparation of the compound ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-hydroxy-9H-purine-9-yl)tetrahydrofuran-2-yl)methylhydrogenmorpholinophosphonate

[0527] [ka]

[0528]

[0439] DCC (1.19 g, 5.74 mmol) in t-BuOH (6 mL) was added under N2 conditions to a reflux solution (110°C) of compound ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-hydroxy-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (500 mg, 1.44 mmol) and morpholine (500 mg, 5.74 mmol) in t-BuOH (6 mL) and H2O (6 mL). The solution was stirred under N2 conditions at 110°C for 12 hours. After mixing was complete, the solution was cooled to 20°C and the solid was removed by filtration. The filtrate was collected and the organic solvent was removed by vacuum. The residue was diluted with H2O (10 mL) and washed with TBME (20 mL x 3 times). The aqueous phase was recovered and concentrated in a vacuum to obtain a light yellow oil as the DCC salt (810 mg, crude) of the desired compound. This was used directly in the next step without further purification.

[0529]

[0440] Step 2. Preparation of compound (2R,3R,4S,5S,6S)-2-((R)-1,2-diacetoxyethyl)-6-(((((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-hydroxy-9H-purine-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate

[0530] [ka]

[0531]

[0441] The compound of the product from Step 1 (250 mg, 500 μmol) and the compound of the product from Step 14 in Example 14 (810 mg, 1.94 mmol) were dried with pyridine (5 mL x 3 times). The residue was dissolved in anhydrous pyridine (5 mL) and 1H-tetrazole (175 mg, 2.50 mmol) was added. The solution was stirred at 20°C for 12 hours. Then the solution was heated to 30°C and stirred for 12 hours. The solvent was removed by vacuum. The residue was dissolved in EtOH (20 mL). The solid was removed by filtration. The filtrate was collected and concentrated by vacuum. The residue was purified by silica gel column (DCM: (MeOH:NH3.H2O=50:1)=1:0 to 1:1.2) to obtain the crude product (80 mg). The solution was then re-purified by preparative HPLC (column: Waters Xbridge 150*25 5μ; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 0%-30%, 10 min) to obtain the desired compound (20 mg, yield: 4.82%) as a white solid. MS(ESI) m / z(M+H) + :831.4. 1 H NMR (400MHz, CD3OD) δ 8.53 (s, 1H), 8.06 (s, 1H), 6.07 (d, J = 5.4 Hz, 1H), 5.48 - 5.47 (m, 1H), 5.34 - 5.32 (m, 1H), 5.22 - 5.19 (m, 3H), 4.66 - 4.58 (m, 1H), 4.47 - 4.43 (m, 2H), 4.25 - 4.19 (m, 4H), 3.89 - 3.88 (m, 1H), 2.12 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.92 - 1.90 (m, 3H). compound 33

[0442] Inosine-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0532] [ka]

[0533]

[0443] Step 1. Preparation of the compound inosine-5'-(D-glycero-β-D-mannoheptopyranosyl)diphosphate

[0444] The compound of the product from step 2 in Example 32 above was stirred at -20°C for 2 days in 2 mL of solvent (consisting of 0.1 M TEAB (8 mL), MeOH (6 mL), and TEA (0.1 mL)). The solution was vacuum freeze-dried to obtain the desired compound as a trimethylamine salt. HBP Prodrug

[0445] The HBP shown in formula 1b below is highly hydrophilic, and therefore it is difficult for this molecule to permeate the cell membrane and reach the cytoplasmic protein ALPK1.

[0534] [ka]

[0535]

[0446] Accordingly, the present disclosure provides a variety of prodrugs of HBP adapted to enable permeation of the plasma membrane. In embodiments, the prodrug comprises one or more biounstable protecting groups in one or more phosphate moieties of HBP. In embodiments, one or more biounstable protecting groups are linked to one or more phosphate moieties of HBP via ester linkages. Exemplary protecting groups that may be thus linked include, for example, carbonyloxymethyl (e.g., POM, POC), cyclosaligenyl (e.g., cycloSal), cyclic 1-aryl-1,3-propanyl esters (e.g., HepDirect), aryloxyamino acid phosphoramidates or phosphonoamidates (e.g., ProTide), and methylarylhaloalkylamidates. Further examples include S-acyl-2-thioethyl (SATE), S-[(2-hydroxyethyl)sulfidyl]-2-thioethyl (DTE), alkyloxyalkyl (e.g., HDP, ODE), amino acid phosphoramidate or phosphonoamide monoesters, bis(amino acid) phosphoramidate or phosphonoamide, and di- or tri-phosphonates. Type I: Carbonyl oxymethyl

[0447] Carbonyloxymethyl is a class of phosphate protecting groups. In some embodiments, the carbonyloxymethyl protecting group has general formula 2a.

[0536] [ka]

[0537] [In the formula, R 1a and R 1b Each is independent of C 1~12 Alkyl or C 1~12 It is an alkoxy, and the wavy line indicates the bond point to the rest of the molecule. In some embodiments, R 1a and R 1b Each is independent of C 1~8 Alkyl or C 1~8It is an alkoxy.

[0538]

[0448] Without being bound by any particular theory, it is thought that a phosphate group protected by a carbonyloxymethyl moiety can be deprotected in vivo by a series of chemical transformations described in Scheme I below.

[0539] [ka]

[0540]

[0449] In some embodiments, the prodrug of HBP has formula 3a

[0541] [ka]

[0542] [In the formula, R 1a , R 1b , R 1c , and R 1d Each is independent of C 1~12 Alkyl or C 1~12 It is an alkoxy. In some embodiments, R 1a , R 1b , R 1c , and R 1d Each is independent of C 1~8 Alkyl or C 1~8 It is an alkoxy.

[0543] The carbonyloxymethyl prodrug of

[0450] HBP may be prepared using the methods described in Hwang, Y. and Cole, PA Organic Letters 2004, 6, 1555; Inuki, S. et al., Fujimoto, Y. Organic Letters 2017, 19, 3079, or similar publications. Type II: Cyclosaligenyl (cycloSal)

[0451] Cyclosaligenyl (cycloSal) is a class of phosphate protecting groups. In some embodiments, the cycloSal protecting group has general formula 2b.

[0544] [ka]

[0545] [In the formula, R 2 H, C 1~8 Alkyl or halogen, R 3 H, C 1~8 [It is an alkyl or halogen, where the subscript n is an integer from 1 to 3, and the tilde indicates a bond point to the rest of the molecule.] In some embodiments, R 2 is H or C 1~8 It is alkyl. In some embodiments, R 3 C 1~8 It is alkyl. In some embodiments, the subscript n is 1.

[0546]

[0452] Without being bound by any particular theory, a phosphate group protected by one or more cycloSal moieties is thought to be deprotected in vivo via one or more pathways described in Scheme II below.

[0547] [ka]

[0548]

[0453] In some embodiments, the prodrug of HBP has formula 3b

[0549] [ka]

[0550] [In the formula, R 2a and R 2b These are H and C, which are independent of each other. 1~8Alkyl or halogen, R 3a and R 3b These are H and C, which are independent of each other. 1~8 [An alkyl or halogen, where the subscripts n1 and n2 are independently integers from 1 to 3]. In some embodiments, R 2a and R 2b Each of these is independently H or C 1~8 It is alkyl. In some embodiments, R 3a and R 3b Each is independent of C 1~8 It is alkyl. In some embodiments, the subscripts n1 and n2 are each 1.

[0551]

[0454] CycloSal prodrugs of HBP may be prepared using the methods described in Spacilova, P. et al., ChemMedChem 2010, 5, 1386; Inuki, S. et al., Organic Letters 2017, 19, 3079, or similar. Type III: Cyclic 1-aryl-1,3-propanyl ester (HepDirect)

[0455] Cyclic 1-aryl-1,3-propanyl esters (HepDirects) are a class of phosphate protecting groups. In some embodiments, the HepDirect protecting group has general formula 2c

[0552] [ka]

[0553] [In the formula, R 4 [ is an aryl or 5- or 6-membered heteroaryl, where the heteroaryl group has 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S, and the dashed line indicates a bond point to the rest of the molecule]. In some embodiments, R 4 is an aryl or 6-membered heteroaryl. In some embodiments, R 4 It is phenyl or pyridyl.

[0554]

[0456] Without being bound by any particular theory, the phosphate group protected by the HepDirect moiety is thought to be deprotected in vivo via the pathway described in Scheme III below.

[0555] [ka]

[0556]

[0457] In some embodiments, the prodrug of HBP has formula 3c

[0557] [ka]

[0558] [In the formula, R 4a and R 4b Each is independently an aryl or a 5- or 6-membered heteroaryl, and the heteroaryl group has 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S. In some embodiments, R 4a and R 4b Each of these is independently an aryl or a 6-membered heteroaryl. In some embodiments, R 4a and R 4b These are independently phenyl or pyridyl.

[0559]

[0458] The HepDirect prodrug of HBP may be prepared using the methods described in Reddy, KR et al., Tetrahedron Letters 2005, 46, 4321; Inuki, S. et al., Organic Letters 2017, 19, 3079, or similar. Type IV: Aryloxy amino acid amide (Protide)

[0459] Aryloxy amino acid amides (Protide) are a class of phosphate protecting groups. In some embodiments, the protectide protecting group has general formula 2d

[0560] [ka]

[0561] [In the formula, R 5 and R 6 Each of these is independently H or C 1~8 It is alkyl, R 7 C 1~8 It is alkyl, R 8 [where R is an aryl group, and the wavy line indicates the bond point to the rest of the molecule.] In some embodiments, R 7 is methyl or isopropyl. In some embodiments, R 8 It is phenyl.

[0562]

[0460] Without being bound by any particular theory, the phosphate group protected by the Protect moiety is thought to be deprotected in vivo via the pathway described in Scheme IV below.

[0563] [ka]

[0564]

[0461] In some embodiments, the prodrug of HBP has formula 3d

[0565] [ka]

[0566] [In the formula, R 5a , R 5b , R 6a , and R 6 Each of these is independently H or C 1~8 It is alkyl, R7a and R 7b Each is independent of C 1~8 It is alkyl, R 8a and R 8b Each is independently an arrow. In some embodiments, R 7a and R 7b Each is independently methyl or isopropyl. In some embodiments, R 8a and R 8b Each of these is phenyl.

[0567]

[0462] The Protide prodrug of HBP may be prepared using the methods described in van Boom, JH et al., Tetrahedron 1975, 31, 2953; Inoue, J.-i. and Fujimoto, Y., Organic Letters 2017, 19, 3079, or similar. Type V: Methylarylhaloalkylamidate

[0463] Methylaryl haloalkylamidate is a class of phosphate protecting groups. In some embodiments, the methylaryl haloalkylamidate protecting group has general formula 2e.

[0568] [ka]

[0569] [In the formula, R 9 C 1~8 It is alkyl, X 1 C 3~5 It is alkylene, R 10 These are aryl, heteroaryl, and aryl C 1~4 Alkylene, or heteroaryl C 1~4 It is an alkylene, and the heteroaryl group is a 5 or 6-membered ring having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S. In some embodiments, R 9 C 1~4It is alkyl. In some embodiments, X 1 is a C4 alkylene. In some embodiments, R 10 is aryl or aryl C 1~4 It is an alkylene. In some embodiments, R 10 R is phenyl. In some embodiments, R 10 It is benzyl.

[0570]

[0464] Without being bound by any particular theory, it is thought that phosphate groups protected by a methylaryl haloalkylamdiate moiety can be deprotected in vivo through a series of chemical transformations described in Scheme V below. 9 and R 10 It should be understood that the bases defined for this are illustrative and not intended to be limiting.

[0571] [ka]

[0572]

[0465] In some embodiments, the prodrug of HBP has formula 3e

[0573] [ka]

[0574] [In the formula, R 9a and R 9b Each is independent of C 1~8 It is alkyl, X 1a and X 1b Each is independent of C 3~5 It is alkylene, R 10a and R 10b These are independently aryl, heteroaryl, and aryl C. 1~4 Alkylene, or heteroaryl C 1~4It is an alkylene, and the heteroaryl group is a 5 or 6-membered ring having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S. In some embodiments, R 9a and R 9b Each is independent of C 1~4 It is alkyl. In some embodiments, X 1a and X 1b Each of these is independently a C4 alkylene. In some embodiments, R 10a and R 10b Each is independently either aryl or aryl C 1~4 It is an alkylene. In some embodiments, R 10a and R 10b Each is independently phenyl. In some embodiments, R 10a and R 10b Each of them is independently benzyl.

[0575]

[0466] Methylaryl haloalkylamdiate prodrugs of HBP can be prepared using the methods described in Wu, W. et al., Journal of Medicinal Chemistry 2007, 50, 3743; Inoue, J.-i. and Fujimoto, Y., Organic Letters 2017, 19, 3079, or similar publications.

[0576]

[0467] Those skilled in the art will recognize that each of the two phosphate moieties of HBP can be independently protected with any of type I to type V protecting groups using the method described above. Thus, in some embodiments, the prodrug of HBP is represented by Formula 3

[0577] [ka]

[0578] [In the formula, Y 1 and Y 2Each of these is independently a phosphate, formula 2a, formula 2b, formula 2c, formula 2d, or formula 2e. However, Y 1 and Y 2 [Provided that neither of them is a phosphate]

[0579]

[0468] In some embodiments, the prodrug of HBP is one of the compounds listed in Table 1.

[0580] [Table 2-1]

[0581] [Table 2-2]

[0582] [Table 2-3]

[0583] [Table 2-4]

[0584] Synthesis of H1b-ADP and HMP-1bP

[0469] D-glycero-D-manno-heptose-1β-ADP ("H1b-ADP", compound IX) and D-glycero-D-manno-heptose-1β-P (HMP-1bP, compound VIII)

[0470] The synthesis of H1b-ADP proceeded from compound I, synthesized according to Inuki et al., Organic Letters (2017), 19:3079-3082. Compound IX, described below, was synthesized according to Zamyatina et al., Angewandte Chemie, Int'l Ed. (2000), 39(22):4150-4153. Synthesis scheme

[0585] [ka]

[0586]

[0471] All humidity-sensitive reactions were performed under Ar using the syringe-septum cap technique. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F 254 plates (Qindao, 0.25 mm thick). ¹H-NMR spectra were recorded using a Varian-400 spectrometer, and the chemical shift was reported as a value (ppm) relative to the residual proton of the internal tetramethylsilane or deuterated solvent. ¹³C-NMR spectra were recorded using a Varian-400 spectrometer, and the chemical shift was reported as a δ value (ppm) relative to the residual proton of the internal tetramethylsilane or deuterated solvent. ³¹P-NMR spectra were recorded using a Varian-400 spectrometer, and the chemical shift was reported as a δ value (ppm) relative to the external 85% phosphoric acid. The 1H-NMR spectrum is summarized as follows: chemical shift, multiplicity (br=broad, s=singleline, d=doubleline, t=tripleline, q=quadrupline, m=multiline), number of protons, and coupling constant.

[0587]

[0472] Step 1. Synthesis of Compound II:

[0588] [ka]

[0589]

[0473] Compound I (17.93 g, 23.65 mmol), TBAI (0.9 g, 2.365 mmol), and BnBr (7.1 mL, 59.14 mmol) in DMF (270 mL) were stirred, and NaH (60% oil dispersion, 2.4 g, 59.14 mmol) was added at 0°C. After stirring overnight, the reaction product was quenched with H2O. The entire mixture was extracted with PE / siRNA (1:9). The extract was washed with H2O and brine and dehydrated with MgSO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This was purified by silica gel flash chromatography using PE-siRNA (5:1) to obtain Compound III (6.3407 g, yield 32%) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ (ppm): 1 H NMR (CDCl3, 400 MHz) 1.04 (s,9H); 3.75~3.77(m,1H); 3.84~3.96(m,5H); 2.44~2.47(d,1H); 4.05~4.14(m, 3H); 4.56~4.86 (m,8H); 5.10~5.21(m,2H); 5.79~5.84(m,1H); 7.02~7.05(m,2H), 7.16~7.38(m, 24H); 7.60~7.67 (m,4H)

[0474] Step 2. Synthesis of Compound III:

[0590] [ka]

[0591]

[0475] A solution of Ir[(cod)(MePh2P)2]PF6 (210 mg, 253 mmol) in THF (35 mL) was stirred at room temperature under an H2 atmosphere at 1 atm until a bright yellow solution was produced, and then N2 was passed through the solution to remove any remaining hydrogen gas. The obtained Ir catalyst solution was added to a stirred solution of compound II (1.0741 g, 1.27 mmol) in THF (35 mL) at room temperature. After stirring at this temperature for 6 hours, H2O (22 mL) and I2 (650 mg, 2.56 mmol) were added to the stirred mixture at room temperature. After stirring at this temperature for 1 hour, the reaction product was quenched with saturated Na2S2O3. The entire mixture was extracted with siRNA. The extract was washed with saturated NaHCO3 and dehydrated with MgSO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This was purified by silica gel flash chromatography using PE-siRNA (1:1) to obtain compound III (0.68 g, yield 66.3%) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ (ppm): 1.03 (s, 9H), 3.72 (s, 1H); 3.93~4.05 (m, 6H); 4.23~4.45 (m, 1H); 4.55~4.80 (m, 7H); 5.13 (br, 1H); 7.02~7.07 (m, 2H); 7.21~7.37 (m, 24H); 7.62~7.68 (m, 4H).

[0476] Step 3. Synthesis of Compounds IV and V:

[0592] [ka]

[0593]

[0477] Compound III (680 mg, 0.842 mmol), dibenzyl phosphate (702 mg, 2.53 mmol), n-Bu3P (0.51 g, 2.53 mmol), and MS 5Å (500 mg) in CH2Cl2 (20 mL) were stirred, and Et3N (0.71 mL, 5.06 mmol) was added at room temperature. After stirring at this temperature for 30 minutes, DIAD (0.51 g, 2.53 mmol) was added at room temperature. After stirring overnight, the mixture was concentrated under reduced pressure to obtain an oily residue. The crude product was purified by silica gel flash chromatography using PE-Â(7:3) to obtain a mixture of compounds IV and V (0.966 g, 100%). This mixture was used directly in the next step.

[0594]

[0478] Step 4. Synthesis of Compounds VI and VII:

[0595] [ka]

[0596]

[0479] A mixture of compounds IV and V (0.966 g, 0.904 mmol) in THF (20 mL) was stirred, and TBAF (1 M in THF, 1.4 mL, 1.4 mmol) was added at room temperature. After stirring overnight, the reaction was quenched with saturated NH4Cl. The entire mixture was extracted with ethyl acetate. The extract was washed with saturated NaHCO3 and dehydrated with MgSO4. The filtrate was concentrated under reduced pressure to obtain an oily residue. This was purified by flash chromatography using petroleum / ethyl acetate (3:1) to obtain compound VI (169.7 mg, 22.6%) and compound VII (225 mg, 30%) as colorless oil. Compound VI: 1H NMR (CDCl3, 400 MHz) δ (ppm): 3.52~3.54 (m, 1H); 3.67~3.73 (m, 2H), 3.84~3.87 (dd, 1H); 3.97~3.99(m, 1H); 4.04~4.07 (m, 1H); 4.47~4.50 (m, 1H); 4.58~4.60 (m, 1H); 4.71~4.74 (m, 1H); 4.87~4.89 (m, 1H); 4.93~5.03 (m, 9H); 5.70~5.72 (dd, 1H); 7.19~7.33 (m, 30H). 31 P NMR (CDCl3, 400 MHz) δ -2.60. Compound VII: 1H NMR (CDCl3, 400 MHz) δ 3.56~3.59 (dd, 1H); 3.65~3.68 (m, 2H); 3.81~3.84 (m, 2H); 4.02~4.07 (m, 1H); 4.52~4.56 (m, 1H); 4.59~4.61 (m, 1H); 4.68~4.78 (m, 3H); 4.86~4.88 (m, 1H); 4.95~5.11 (m, 7H); 5.24~5.26 (d, 1H); 7.18~7.39 (m, 30H). 31 P NMR (CDCl3, 400 MHz) δ -2.50

[0480] ステップ5. Synthesis of compound VIII (D-glycero-D-manno-ヘプトース-1β-P):

[0597]

change

[0598]

[0481] A mixture of compound VI (105 mg, 0.126 mmol) and 20% (w / w) Pd(OH)2 / C (21 mg, 0.03 mmol) in 1,4-dioxane / H2O (5 mL, 4:1) was stirred at room temperature under H2 (1 atm) for 2 days. The mixture was filtered through an Advantec PTFE membrane filter with a pore size of 0.5 m using H2O. The filtrate was cooled to 0°C, TEA (53 μL, 0.378 mmol) was added, and the mixture was stirred at this temperature for 3 hours. The resulting mixture was freeze-dried to obtain compound VIII·2Et3N (74.3 mg, quantitatively) as a white solid.

[0599]

[0482] Step 6. Synthesis of compound IX (D-glycero-D-manno-heptose-1β-ADP):

[0600] [ka]

[0601]

[0483] Compound VIII (28.6 mg, 0.058 mmol) was dissolved in anhydrous pyridine and concentrated under vacuum. This azeotropic reaction was repeated three times to remove residual water. AMP-molholite (97 mg, 0.233 mmol) and 1H-tetrazole (32 mg, 0.453 mmol) were added to the dried compound VIII. Anhydrous pyridine (2 mL) was added, and the mixture was stirred under an N2 atmosphere for 78 hours. After concentration, the residue was precipitated and washed with ethyl acetate. The obtained solid was purified by preparative HPLC (RP-C18) (column: HSS T3 1.8 μm, 2.1*100 mm, column 40C) using 10 mm NH4COOCH3 in water (solvent A) and acetonitrile (solvent B) as mobile phases, eluten as a gradient at a flow rate of 0.4 ml / min over 3 minutes to 5%-85% (solvent B), then over 0.5 minutes to 85%-95% (solvent B), and held at 95% for 1 minute to obtain a mixture of compound IX and compound VIII. The mixture was purified again using a Sephadex G-15 column to obtain compound IX (3.5 mg, 10%) as a white solid. 1 1H NMR and 31The P NMR data was consistent with data reported in the literature.

[0602]

[0484] The present invention is further described and illustrated by the following non-limiting embodiments.

[0485] HBP is a metabolic intermediate in the bacterial ADP-heptose biosynthesis pathway. Depending on the bacterial strain, HBP is produced from D-glycero-D-manno-heptose-7-phosphate by the kinase domain of the enzyme HIdA or HIdE, and converted to D-glycero-β-D-manno-heptose-1-phosphate (HMP-1bP) by the bacterial enzyme GmhB. HMP-1bP is converted to D-glycero-D-manno-heptose-1β-ADP (H1b-ADP) by the bacterial enzyme HIdC, or in some bacterial cells, by the ADP-transferase domain of HIdE. Then, H1b-ADP is converted to L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) by HIdD (GmhD). See Figure 16 for a schematic diagram of the pathway and related enzymes.

[0603]

[0486] Other researchers have used genetic techniques to knock out various enzymes located both upstream and downstream of HBP in this biosynthetic pathway to elucidate the role of HBP in inducing the innate immune response. In particular, HBP is involved in infection-induced NFκB activation via ALPK1-TIFA-TRAF6. See, for example, Gaudet et al., Science 348:1251 2015; Milivojevic et al., PLOS Pathogens 13(2) e1006224 2017; and Zimmermann et al., Cell Reports 20:2384 2017. Guadet concluded that "disruption of the ADP-heptose pathway, located upstream of HBP, suppressed NF-κB activation in Escherichia coli or N. meningitidis," and therefore "the presence of HBP is the direct cause of NF-κB activation" (Guadet, p. 1252). Milivojevic used Salmonella typhimurium cells lacking the HIdE gene and showed that these cells, unable to synthesize HBP, could not induce IL-8 production in either infected or bystander cells, but cells lacking enzymes acting downstream of HBP, GmhB, or WaaC induced potent IL-8 expression (Milivojevic, p. 12).

[0604]

[0487] In light of previous research that identifies HBP as an important molecule for inducing innate immunity, we were surprised to find that, in addition to HBP, molecules such as HMP-1bP and H1b-ADP can also induce IL-8 and TNFα mRNA expression in cells in an ALPK1-dependent manner using chemically synthesized HBP, HMP-1bP, and H1b-ADP (Example 1). Furthermore, to our surprise, we found that H1b-ADP was far more potent than HMP-1bP in inducing cytokine expression in this assay. Unexpectedly, we also found that chemically synthesized HBP could not bind to ALPK1 in the thermal shift assay (Example 2), nor could it induce ALPK1 autophosphorylation (Example 3). Instead, we unexpectedly found that in these assays, only H1b-ADP could bind to ALPK1 and induce its autophosphorylation. In further experiments, the inventors found that H1b-ADP can activate ALPK1-dependent NFκB pathway signaling via IκB phosphorylation (Example 4). Furthermore, the inventors found that H1b-ADP-6L can also activate ALPK1-dependent phosphorylation of its downstream substrate TIFA (Example 5). Finally, the inventors found that in a mouse tumor model, H1b-ADP exhibits potent antitumor activity, while neither HBP nor HMP-1bP exhibits potent antitumor activity (Examples 7 and 8), and that this activity synergistically enhances the antitumor activity of both checkpoint inhibitors (anti-PD-1 antibodies) and agonists of immunocostimulatory molecules (anti-OX40 agonist antibodies).

[0605]

[0488] Overall, the data described herein demonstrate that, in addition to HBP, bacterial metabolites, namely HMP-1bP, H1b-ADP, and H1b-ADP-6L, can induce ALPK1-dependent signaling associated with the induction of innate immunity, and that at least one of these molecules, H1b-ADP, further possesses surprising and unexpected antitumor activity, both alone and in combination with other immunomodulators. This molecule is recognized as a TLR-9 agonist (US 20100016250, Nagata et al., Kyowa Hakko Kirin Co.) and, based on that evidence, has been proposed as being useful in treating allergies, tumors, and infections, and as an immunostimulant. However, these results are the first to demonstrate its activity in ALPK1-dependent signaling and the first to demonstrate its antitumor activity in an animal model. [Examples]

[0606] Example 1: Chemically synthesized HBP, HMP-1bP, and H1b-ADP each induce IL-8 and TNFα mRNA expression in 293HEK cells in an ALPK1-dependent manner.

[0489] To test whether HBP, HMP-1bP, and H1b-ADP can induce cytokine expression in an ALPK1-dependent manner, the inventors used small interfering RNA (siRNA) against ALPK1 to suspend ALPK1 expression in HEK293 cells (1 × 10⁻⁶ cells). 4Cells were seeded in 96-well plates and transfected with either control siRNA or siRNA against ALPK1 according to the manufacturer's protocol (Lipofectamine® RNAiMax®, Invitrogen 13778075). After 2 days of incubation, one of the following was added to the culture medium: (1) HBP (500 μM, 100 μM, 20 μM, Figures 2A-2B), (2) HMP-1bP (500 μM, 100 μM, Figures 3A-3B), or (3) H1b-ADP (100 nM, 20 nM, 4 nM, 0.8 nM, Figures 4A-4B). Cells were harvested after 4 hours. Total RNA was isolated (TRIzol®, ThermoFisher), and cDNA was synthesized (PrimeScript® RT reagent kit (Takara), amplified according to the manufacturer's protocol using QuantStudio® 7 Flex Real-Time PCR Systems (ThermoFisher) (AceQ® qPCR SYBR® Green Master Mix, Vazyme Biotech)). Both IL-8 and TNFα mRNA expression increased in an ALPK1-dependent manner, as evidenced by the decrease in the expression of both cytokines in the presence of siRNA against ALPK1. These results suggest that HBP, HMP-1bP, and H1b-ADP, respectively, activate IL-8 and TNFα gene expression via ALPK1.

[0607]

[0490] Surprisingly, H1b-ADP was significantly potent compared to the other molecules in this assay. As shown in Figure 5, both IL-8 and TNFα mRNA expression were induced by nanomolar concentration (10 nM) of H1b-ADP, requiring either 100 μM of HBP or HMP-1bP. This was surprising, because, as discussed above, previous reports from two groups have shown that HBP is responsible for IL-8 induction via the ALPK1-TIFA pathway, while its downstream metabolites, including HMP-1bP and H1b-ADP, are not (Gaudet et al., Science 348:1251 2015; Milivojevic et al., PLOS Pathogens 13(2) e1006224 2017).

[0608] Example 2: H1b-ADP binds to ALPK1, but neither HBP nor HMP-1bP binds to ALPK1.

[0491] Thermal shift assays are widely used to determine the binding of molecules to a target protein. The assay is based on the fact that when another molecule is bound to a protein, the amount of thermal energy required for the protein to denature increases. SYPRO Orange is a fluorescent compound used in the detection of thermal shift. SYPRO Orange binds to the hydrophobic surface of a protein, and water strongly quenches its fluorescence. When the protein is unfolded, the exposed hydrophobic surface binds to the dye, and fluorescence increases. When another molecule is bound to a protein, an increase in the temperature required to unfold the protein is observed.

[0609]

[0492] This assay system was used to determine whether chemically synthesized HBP, HMP-1bP, and H1b-ADP directly bind to ALPK1. Figure 6 shows the thermal shift of ALPK1 (mixed with 7 μM 1000 × SYPRO Orange) incubated in the absence or presence of 1 μM, 5 μM, 25 μM, or 125 μM of HBP, HMP-1bP, and H1b-ADP, respectively. Neither HBP nor HMP-1bP induced a thermal shift. Only H1b-ADP induced a shift greater than 1 degree at the three highest concentrations of 5 μM, 25 μM, and 125 μM. These results indicate that H1b-ADP can directly bind to ALPK1, but neither HBP nor HMP-1bP can.

[0610]

[0493] The inventors previously found that HBP binds to ALPK1 in this assay using HBP produced in vitro from its precursor D-glycero-D-manno-heptose-7-P (HMP) by enzymatic catalysis. In such studies, the enzyme used for in vitro production of HBP was the sugar kinase HIda purified from wild-type E. coli cells. As described below in Example 9, the inventors now believe that the purified HIda enzyme was contaminated with additional enzymes, possibly Gmhb and H1dE, that converted at least a portion of HBP to H1b-ADP in the previous assay.

[0611] Example 3: H1b-ADP induces ALPK1 autophosphorylation, but neither HBP nor HMP-1bP induces ALPK1 autophosphorylation.

[0494] ALPK1 autophosphorylation occurs upon activation. Therefore, the inventors then investigated whether the binding of H1b-ADP to ALPK1 was sufficient to induce ALPK1 autophosphorylation. Phosphorylation assays were performed according to standard protocols. Briefly, ALPK1 was incubated (20 μl, 25 mM HEPES, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 1.5 mM CaCl2, 10 mM MgCl2) with ATP and chemically synthesized HBP, HMP-1bP, or H1b-ADP (2 nM, 25°C, 1 hour), and then ALPK1 autophosphorylation was detected by denature gel electrophoresis and Western blotting using anti-phosphothreonine antibody (CST). As shown in Figure 7, ALPK1 phosphorylation was detected only in the presence of H1b-ADP (10 nM, 1 nM, and 0.2 nM), indicating that H1b-ADP induces ATP-dependent autophosphorylation and ALPK1 activation, while neither HBP nor HMP-1bP induces ATP-dependent autophosphorylation or ALPK1 activation. The concentration ranges of HBP and HMP-1bP used in this assay (1 nM, 10 nM, and 100 nM) were 10 times higher than those used for H1b-ADP.

[0612]

[0495] The inventors previously found in this assay that HBP can induce ALPK1 autophosphorylation using in vitro-produced HBP from its precursor D-glycero-D-manno-heptose-7-P (HMP) via enzymatic catalysis. In such studies, the enzyme used for in vitro production of HBP was the sugar kinase HIda purified from wild-type E. coli cells. As described later in Example 9, the inventors now believe that the purified HIda enzyme was contaminated with an additional enzyme, which converted at least a portion of the HBP to H1b-ADP in the previous assay.

[0613] Example 4: H1b-ADP induces ALPK1-dependent phosphorylation of IκB.

[0496] NFκB RelA(p65) is a transcription factor that must translocate from the cytoplasm to the nucleus, where it interacts with the promoter regions of numerous target genes to regulate their transcription. Target genes of NFκB RelA include inflammatory cytokines such as IL-8, TNFα, CXCL1, and CXCL3. For p65 to translocate to the nucleus, the cytoplasmic complex containing p65 must first be degraded. This process is triggered by the activation of IκB in conjunction with phosphorylation. Therefore, IκB phosphorylation can be used as a marker of NFκB activation.

[0614]

[0497] The inventors then tested whether H1b-ADP-induced autophosphorylation of ALPK1 is effective in activating NFκB, using phosphorylation of IκB as a marker of this activity. Phosphorylation assays were performed according to standard protocols, and phosphorylated proteins were detected by gel electrophoresis and Western blotting using an antibody that detects phosphorylated IκB. Briefly, the assays were performed in 20 μl volumes at 25°C in assay buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 1.5 mM CaCl2, 10 mM MgCl2) containing 2 nM ALPK1 and H1b-ADP (some amount) with or without ATP, or ALPK1 alone or H1b-ADP alone, both in the presence of ATP. After incubation for 1 hour, the reaction product was loaded onto a denature gel for protein electrophoresis, and IκB phosphorylation was detected by Western blotting using a p-IκB antibody (Abcam).

[0615]

[0498] As shown in Figure 9, IκB phosphorylation was detected only in the presence of ALPK1 and H1b-ADP in the presence of ATP. These results indicate that HBP-induced autophosphorylation of ALPK1 activates the NFκB pathway.

[0616] Example 5: H1b-ADP-6L induces ALPK1-dependent phosphorylation of TIFA. L-glycero-D-manno-heptose-1β-ADP (H1b-ADP-6L) is another bacterial metabolite in the same biosynthetic pathway as HBP, HMP-1bP, and H1b-ADP. It is formed from H1b-ADP by the action of the bacterial HIdD(GmhD) enzyme. The inventors investigated whether this molecule, which is structurally very similar to H1b-ADP, possesses ALPK1 biological activity.

[0617]

[0499] The inventors performed in vitro kinase assays using H1b-ADP and H1b-ADP-6L together with ALPK1 protein (2 nM) and TIFA protein (1.6 μM) in a kinase buffer containing 50 μM ATP. TIFA phosphorylation was analyzed by denature gel electrophoresis followed by Western blotting using an anti-phosphothreonine antibody. H1b-ADP or H1b-ADP-6L was added at concentrations of 2 nM, 0.4 nM, 80 pM, 16 pM, and 3 pM. As shown in Figure 10, H1b-ADP-6L activated ALPK1-dependent signaling in a manner similar to that of H1b-ADP.

[0618] Example 6: Intratumoral injection of H1b-ADP delays tumor growth and induces overexpression of inflammatory genes in tumor tissue, but HMP-1bP is not used.

[0500] The inventors tested the anticancer activity of H1b-ADP using a CT26 tumor xenograft model. Tumor cells (2 × 10 per 100 μL) 5 CT26 cells were subcutaneously inoculated into the right flank of BALB / c mice. On day 6, mice with tumor diameters reaching 3–5 mm were randomized and divided into three groups (n=8 each): control, HMP-1bP (580 μg), and H1b-ADP (1.2 μg). Injections were administered in a total volume of 20 μL on days 6, 8, 10, 12, and 14 after inoculation. (Formula: L × W) 2Tumor volume was calculated every two days from the caliper measurement of tumor diameter using the formula ) / 2 (where L is the longer measurement). As shown in Figure 11, in this model system, H1b-ADP reduced tumor growth, but HMP-1bP did not, indicating that H1b-ADP was able to induce an antitumor immune response that was effective in suppressing tumor growth.

[0619]

[0501] Next, the inventors investigated whether H1b-ADP caused an increase in inflammatory cytokines in tumor cells (2 × 10 per 100 μL). 5 CT26 cells were subcutaneously inoculated into the right flank of BALB / c mice. Seven days post-inoculation, 2.5 μg (n=2), 250 ng (n=2), and 50 ng (n=2) of H1b-ADP or control (n=2) were injected intratumorally (20 μL). Four hours post-injection, tumor tissue (tumor issue) was dissected and collected. Total RNA was isolated (TRIzol®, ThermoFisher), and cDNA was synthesized (PrimeScript® RT reagent kit (Takara), amplified according to the manufacturer's protocol using QuantStudio® 7 Flex Real-Time PCR Systems (ThermoFisher) (AceQ® qPCR SYBR® Green Master Mix, Vazyme Biotech).

[0620]

[0502] The results are shown in Figure 12. In this experiment, H1b-ADP injection increased the mRNA expression of inflammatory cytokines IL-1b, Tnfa, Ifnγ, and IL-6, as well as the chemokine Cxcl1, indicating that inflammation was activated in the tumor. H1b-ADP injection increased the mRNA expression of the cytotoxic T cell marker CD8, the helper T cell marker CD4, the regulatory T cell marker Foxp3, and the Th1 cell marker T-bet, indicating an increase in the number of cytotoxic T cells, helper T cells, regulatory T cells, and Th1 cells in H1b-ADP-injected tumors. Increased PD-1 and PD-L1 expression in H1b-ADP-injected tumors suggests that combining anti-PD-1 or anti-PD-L1 therapy with H1b-ADP injection may have a synergistic effect in delaying tumor growth.

[0621] Example 7: H1b-ADP and anti-PD-1 antibody work synergistically to inhibit tumor growth.

[0503] Antagonist antibodies targeting the B7 immunoglobulin superfamily molecules (CTLA-4, PD-1, and PD-L1) represent an immune checkpoint inhibitor approach that has demonstrated antitumor immunity and clinical efficacy in various types of cancer. However, many patients do not respond to monotherapy based on these antibodies, and many others experience relapse after treatment. Therefore, co-therapy is needed to address primary and secondary resistance to immune checkpoint inhibitor treatment.

[0622]

[0504] To test whether H1b-ADP can enhance the antitumor response mediated by checkpoint inhibitors, the inventors tested the effect of co-administration with an anti-PD1 antibody. Tumor cells (2 × 10 per 100 μL) 5CT26 cells were subcutaneously inoculated into the left and right abdomens of BALB / c mice. On day 7, mice with tumor diameters reaching 5 mm were randomized and divided into the following four groups (n=8 in each group): anti-PD1 antibody; rat IgG; H1b-ADP + rat IgG; H1b-ADP + anti-PD1 antibody. The inventors used RMP1-14 as the anti-PD1 antibody (10 mg / kg) and rat IgG 2a (2A3) as the control IgG. On days 6, 10, 12, and 17 after inoculation, anti-PD-1 antibody and control IgG were administered intraperitoneally in volumes of 200 μL each. On days 6, 8, 10, 12, and 15, H1b-ADP (6.2 μg) was injected intratumorally in volumes of 20 μL. Formula (L×W 2 Tumor volume was calculated every two days from the calipass measurements of tumor diameter using the formula ) / 2 (where L is the longer measurement). Results for injected tumors are shown in Figure 13A, and results for distal tumors are shown in Figure 13B. In this experiment, administration of H1b-ADP or anti-PD1 antibody alone significantly suppressed tumor growth to a similar degree. The combination of H1b-ADP and anti-PD1 antibody not only inhibited tumor growth but also led to the disappearance of some tumors. These results indicate that the combination of H1b-ADP and checkpoint inhibitors such as anti-PD1 antibodies is effective in suppressing growth and further inhibiting the viability of tumor cells in vivo.

[0623] Example 8: H1b-ADP and anti-OX40 agonist antibody work synergistically to inhibit tumor growth.

[0505] The inventors then conducted similar experiments using an anti-OX40(CD134) agonist antibody. OX40(CD134) is a tumor necrosis factor receptor superfamily costimulatory receptor molecule expressed by activated immune cells. As noted above, co-therapy is needed to address primary and secondary resistance to checkpoint inhibitor treatment, and one approach is to administer immune costimulatory factors such as anti-OX40 agonist antibodies.

[0624]

[0506] The experiment was carried out as described above, with the following modifications. On day 7, mice whose tumor diameter reached 5 mm were randomized and divided into the following four groups: anti-OX40 antibody; rat IgG; H1b-ADP + rat IgG; H1b-ADP + anti-OX40 antibody. The inventors used BE0031 as the anti-OX40 antibody and rat IgG 2a (2A3) as the control IgG. On days 7, 9, and 11 after inoculation, BE0031 (2 μg), rat IgG (2 μg), and / or H1b-ADP (6.2 μg) were administered intratumorally in a volume of 20 μL. The results are shown in Figure 14.

[0625] Example 9: HIda enzyme purified from wild-type E. coli appeared to be contaminated with other bacterial enzymes.

[0507] The inventors previously found that HBP can bind to ALPK1 using in vitro-produced HBP from its precursor D-glycero-D-manno-heptose-7-phosphate via enzymatic catalysis in a thermal shift assay. In such studies, the enzyme used for in vitro production of HBP was the sugar kinase HIdA, purified from wild-type E. coli cells transfected with a HIdA expression plasmid. E. coli cells do not express either the HIdA or HIdC enzymes, but instead express HIdE, a fusion protein containing a kinase domain and an ADP-transferase domain. These two domains of HIdE are homologous to the kinase domain of HIdA and the ADP-transferase domain of HIdC, respectively. In related studies using the same in vitro-produced HBP, the inventors demonstrated HBP activation of ALPK1 autophosphorylation and phosphorylation of IκB, which is located downstream of ALPK1.

[0626]

[0508] The inventors believe that the purified HIdA enzyme used in the experiment was contaminated with E. coli enzymes such as HIdE, which converts at least a portion of HBP to H1b-ADP. Previous reports on the structure-functional activity of the HIdA enzyme suggest that the kinase domains of the HIdA and HIdE enzymes can dimerize, leading to their co-purification. Lee TW et al., J. Med.Chem. 2013 56:1405-17. Therefore, it is possible that some E. coli HIdE was co-purified with the recombinant HIdA used in the inventors' previous study. The inventors further hypothesize that GmhB and HIdA also form a complex so that GmhB is also co-purified with recombinant HIdA. If such contamination by E. coli GmhB and HIdE occurred, at least a portion of the in vitro-produced HBP should have been converted to H1b-ADP in the previous study.

[0627]

[0509] To test this, the inventors performed an in vitro kinase reaction for ALPK1-dependent TIFA phosphorylation in the presence of HBP or HIdA purified from E. coli containing inactivated HIdE, or in the presence of HBP and HIdA purified from the same HIdE wild-type E. coli used in previous experiments. The results are shown in Figure 15. Phosphorylated TIFA (three concentrations, 1%, 0.2%, and 0.4%) was detected by denature ring gel electrophoresis followed by Western blotting analysis as described above. Phosphorylated TIFA was detected only in the assay using HIdA purified from wild-type E. coli, and not in the assay using HIdA purified from HldE mutant E. coli cells.

[0628]

[0510] To avoid abnormal results obtained using in vitro-produced HBP due to contaminated bacterial enzymes, the inventors utilized chemically synthesized sugar molecules in Examples 1-8 above and Examples 10-18 below.

[0629] Example 10: H1b-ADP and anti-PD-L1 antibody work synergistically to inhibit tumor growth.

[0511] The experiment was conducted as an anti-PD-1 combo experiment, with the following modifications. On day 7, mice whose tumor diameter reached 5 mm were randomized and divided into the following four groups: anti-PD-L1 antibody; rat IgG; H1b-ADP + rat IgG; H1b-ADP + anti-PD-L1 antibody. The inventors used BP0101 (BioxCell) as the anti-PD-L1 antibody and rat IgG 2a (2A3) as the control IgG. On days 7, 9, 11, and 15 after inoculation, anti-PD-L1 antibody (200 μg) and control IgG (200 μg) were administered intraperitoneally in a volume of 200 μL each. On days 7, 9, 11, 13, and 15 after inoculation, H1b-ADP (6.2 μg) was administered intratumorally in a volume of 20 μL. The results for the injected tumor are shown in Figure 16A, and the results for the distal tumor are shown in Figure 16B. These results indicate that the combination of H1b-ADP and checkpoint inhibitors such as anti-PD-L1 antibodies is effective in suppressing proliferation and further inhibiting the viability of tumor cells in vivo.

[0630] Example 11: H1b-ADP and IFNα work synergistically to inhibit tumor growth.

[0512] The experiment was conducted as an anti-PD-1 combo experiment, with the following modifications. On day 8, mice with tumor diameters reaching 5 mm were randomized and divided into the following four groups: INFα (752803, BioLegend); PBS; H1b-ADP; H1b-ADP + INFα. On days 8, 10, and 12 after inoculation, IFNα (0.1 μg) and H1b-ADP (6.2 μg) were administered intratumorally in 20 μL volumes each. Results for injected tumors are shown in Figure 17A, and results for distal tumors are shown in Figure 17B. These results indicate that the combination of H1b-ADP and interferon pathway or JAK-STAT pathway activators such as IFNα is effective in suppressing proliferation and further inhibiting the viability of tumor cells in vivo.

[0631] Example 12: H1b-ADP and anti-CTLA-4 antibody work synergistically to inhibit tumor growth.

[0513] The experiment was conducted as an anti-PD1 combo experiment, with the following modifications. On day 6, mice whose tumor diameter reached 5 mm were randomized and divided into the following four groups: anti-CTLA-4 antibody; rat IgG; H1b-ADP + rat IgG; H1b-ADP + anti-CTLA-4 antibody. The inventors used 9D9 (BioxCell) as the anti-CTLA-4 antibody and rat IgG 2b isotype (MPC-11 clone, BE0086, BioXCell) as the control IgG. On days 6 and 9 after inoculation, anti-CTLA-4 antibody (25 μg) and control IgG (25 μg) were administered intraperitoneally in volumes of 200 μL each. On days 6, 7, 9, and 11 after inoculation, H1b-ADP (6.2 μg) was administered intratumorally in volumes of 20 μL. The results are shown in Figure 18. These results indicate that combinations of H1b-ADP with checkpoint inhibitors such as anti-CTLA-4 antibodies or deletion-regulated T cells are effective in suppressing proliferation and even inhibiting tumor cell viability in vivo.

[0632] Example 13: H1b-ADP and STING agonists work synergistically to inhibit tumor growth.

[0514] The experiment was conducted as an anti-PD1 combo experiment, with the following modifications. On day 6, mice whose tumor diameter reached 5 mm were randomized and divided into the following four groups: c-di-AM(PS)2; PBS; H1b-ADP; H1b-ADP + c-di-AM(PS)2. The inventors used c-di-AM(PS)2 as a STING agonist. On days 6, 7, and 9 after inoculation, c-di-AM(PS)2 (1 μg) and H1b-ADP (6.2 μg) were administered intratumorally in volumes of 20 μL each. The results are shown in Figure 19. These results indicate that combinations of H1b-ADP with STING agonists such as c-di-AM(PS)2 and innate immune agonists are effective in suppressing proliferation and further inhibiting the viability of tumor cells in vivo.

[0633] Example 14: H1b-ADP and anti-CD4 antibody work synergistically to inhibit tumor growth.

[0515] The experiment was conducted as an anti-PD-1 combo experiment, with the following modifications. On day 6, mice with tumor diameters reaching 5 mm were randomized and divided into the following four groups: anti-CD4 antibody (GK1.5 clone, BE0003-1, BioXCell); rat IgG; H1b-ADP + rat IgG; H1b-ADP + anti-CD4 antibody. On days 3, 4, and 8 after inoculation, anti-CD4 antibody (200 μg) and control IgG (200 μg) were administered intraperitoneally in volumes of 200 μL each. On days 6, 8, 10, 12, and 14 after inoculation, H1b-ADP (6.2 μg) was administered intratumorally in volumes of 20 μL. The results are shown in Figure 20. These results indicate that the combination of H1b-ADP and CD4 or regulatory T cell deletion antibodies is effective in suppressing proliferation and further inhibiting tumor cell viability in vivo.

[0634] Example 15: H1b-ADP and TLR agonists work synergistically to inhibit tumor growth.

[0516] The experiment was conducted as an anti-PD-1 combo experiment, with the following modifications. On day 6, mice whose tumor diameter reached 5 mm were randomized and divided into the following four groups: Resquimod; PBS; H1b-ADP; H1b-ADP Resquimod. On days 6, 8, and 11 after inoculation, Resquimod (10 μg) and H1b-ADP (6.2 μg) were administered intratumorally in a volume of 20 μL. The results are shown in Figure 21. These results indicate that the combination of H1b-ADP and a TLR agonist is effective in suppressing proliferation and further inhibiting the viability of tumor cells in vivo. The results are shown in Figure 21.

[0635] Example 16: H1b-ADP can be degraded by serum phosphatases and protected by phosphatase inhibitors and AMP.

[0517] The activity of H1b-ADP, which activates ALPK1 in HEK293 cells, was significantly reduced when the cells were cultured with fetal bovine, human, or mouse serum (Figure 22), suggesting that components in animal serum can neutralize the activity of H1b-ADP. To test whether H1b-ADP is chemically converted to an inactive form, the inventors incubated H1b-ADP with FBS and analyzed the product using LC-MS. The inventors found that as the incubation time increased, the amount of H1b-ADP decreased, and a new substance with similar absorption intensity at UV·254nm increased. This substance was identified as AMP using a standard substance, suggesting that the POP phosphate anhydride bond in H1b-ADP is hydrolyzed by enzymes in the serum. FBS contains alkaline phosphatase at 110-352 μU / ml. Alkaline phosphatases are widely used dephosphorylation reagents capable of hydrolyzing phosphate esters in a variety of molecules, including alcohols, amines, pyrophosphates, and phenols. Phosphatases, such as alkaline phosphatases, can be responsible for the hydrolysis of H1b-A...

Claims

1. α-protein kinase 1 (ALPK1) agonists comprising the compound represented by formula (I), its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof. 【Chemistry 1】 [In the formula, A １ and A ２ are independently selected from O, S and -C(R ８ R ９ ), and R ８ and R ９ are independently selected from H, D, -OH, N ３ , -CN, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl and substituted or unsubstituted aralkyloxyl (the optional substituents are 1-3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl and C1-C4 alkoxy), and A １ or A ２ at least one of which is -C(R ８ R ９ ), and R １ in A ８ or R ９ is cyclized with R ２ in A ８ or R ９ to form C3-C6 cycloalkyl and cycloheteroalkyl having 3-9 ring members and having 1-3 heteroatoms selected from N, O and S as ring members, each being optionally substituted by 1-3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl and C1-C4 alkoxy, L １ and L ２ These are independently O, CH ２ CHF and CF ２ Selected from, L ３ is O, S, or CH ２ And, Z １ and Z ２ These are independently selected from O and S, W １ is -C(R １０ R １１ ) - and R １０ is a halogen, R １１ H, D, -OH, halogens, and C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4-haloalkoxyl, C1-C4 alkenyloxyl, aralkyloxyl and R １２ CO ２ - (R １２ R is selected from groups that are optionally substituted from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members, １１ The optional substituents are one to three substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy. W ２ is H or C1-C3 alkyl, and C1-C3 alkyl is D, halogen, -OH, =O, C1-C3 alkoxyl, C1-C3 haloalkyl, C1-C3 haloalkoxyl, C1-C3 alkenyloxyl and R １２ CO ２ - (R １２ (These are optionally substituted with 1 to 3 substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members.) R １ This is a heteroaryl having C6-C10 aryl or 5-10 ring atoms, and having 1-4 heteroatoms selected from N, O, and S as ring members, R １ D, halogen, -OH, =O, CN, NH ２ , C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamine, C1-C4 dialkylamine and (R １３ R １４ ) It is optionally substituted with 1 to 3 substituents selected from NCO-, R １３ and R １４ These are independently selected from H, C1-C4 alkyl, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members. R ２ , R ３ and R ４ These are independently selected from H, D, halogens, C1-C4 alkyls and C1-C4 haloalkyls. R ５ , R ６ And R7 is H, D, halogen and -OH, R １２ CO ２ - (R １２ (Selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkenyloxyl, C1-C4 alkylamino, C3-C6 cycloalkyl, cycloheteralkyl having 3-6 ring members and 1-3 heteroatoms selected from N, O, and S as ring members, C6-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-3 heteroatoms selected from N, O, and S as ring members), R ５ , R ６ and R ７ Any two adjacent groups can be cyclized to form a cycloheteralkyl group having 5 to 9 ring members, with 1 to 3 heteroatoms selected from N, O, and S as ring members, each optionally substituted with 1 to 3 substituents independently selected from D, halogens, -OH, =O, C1-C4 alkyl, and C1-C4 alkoxy groups.

2. Compounds of formula IA, their stereoisomers, or pharmaceutically acceptable salts. 【Chemistry 2】 [In the formula, Y １ and Y ２ These are independently H, D, -OH, and N ３ The group is selected from groups that are optionally substituted with -CN, halogens, and C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl, and aralkyloxyl, and the optional substituent is one to three substituents independently selected from D, halogen, -OH, -O, C1-C4 alkyl, and C1-C4 alkoxy. R １ ~R ７ , L １ ~L ３ Z １ Z ２ , W １ and W ２ This is as described in claim 1. The ALPK1 agonist according to claim 1, comprising the above.

3. a) Y １ and Y ２ These are independently selected from H, D, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl and C1-C4 alkenyloxyl, R １ ~R ７ , L １ ~L ３ Z １ Z ２ , W １ and W ２ This is either as described in claim 1, or b) Y １ and Y ２ These are independently selected from -OH, halogen, C1-C4 alkyl and C1-C4 alkanoyloxyl, R １ ~R ７ , L １ ~L ３ Z １ Z ２ , W １ and W ２ This is as described in claim 1, The ALPK1 agonist according to claim 2.

4. Compounds of formula IB, their stereoisomers, or pharmaceutically acceptable salts. 【Transformation 3】 [In the formula, n １ and n ２ Each of these is an integer independently selected from the group consisting of 0 to 2. X １ and X ２ are independently selected from H, D, -OH, N ３ , -CN, halogen, and optionally substituted groups selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4 haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl and aralkyloxyl, and the optional substituents are 1-3 substituents independently selected from D, halogen, -OH, =O, C1-C4 alkyl and C1-C4 alkoxy, R １ ~R ７ 、L １ ~L ３ 、Z １ 、Z ２ 、W １ およびW ２ are as described in claim 1 and, optionally, n １ およびn ２ are each 0] The ALPK1 agonist according to claim 1, comprising the above.

5. X １ and X ２ These are independently selected from H, D, and C1-C4 alkyl groups. R １ ~R ７ , L １ ~L ３ Z １ Z ２ , W １ and W ２ The ALPK1 agonist according to claim 4, as described in claim 1.

6. Compounds of formula IC, their stereoisomers, or pharmaceutically acceptable salts. 【Chemistry 4】 [In the formula, A １ is -C(R8R9)-, O or S, R １ ~R ９ , L １ ~L ３ Z １ Z ２ , W １ and W ２ This is as described in Equation I. The ALPK1 agonist according to claim 1, comprising the above.

7. R ２ , R ３ , and R ４ H is each and / or R ５ , R ６ , and R ７ The ALPK1 agonist according to any one of claims 1 to 6, wherein each is independently selected from the group consisting of -OH and C1-C4 alkanoyloxyl.

8. L ３ is O, and / or L ２ is O, and / or L １ However, it is O. An ALPK1 agonist according to any one of claims 1 to 7.

9. a) W １ However, -C(R １０ R １１ ) - and R １０ is a halogen, R １１ H, D, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 haloalkyl, C1-C4-haloalkoxyl, C1-C4 alkanoyloxyl, C1-C4 alkenyloxyl and R １２ CO ２ - (R １２ (Selected from C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkanoyloxyl and C1-C4 alkenyloxyl), or b) W １ However, -C(R １０ R １１ ) - and R １０ is a halogen, R １１ The elements are selected from H, D, -OH, halogens, and C1-C4 alkanoyloxyls. An ALPK1 agonist according to any one of claims 1 to 8.

10. W ２ but, a) D, halogens, -OH, =O and C1-C3 alkoxyls, C1-C3 haloalkyls, C1-C3 haloalkoxyls, C1-C3 alkenyloxyls and R １２ CO ２ - (R １２ (where C1-C alkyl, C1-C4 alkoxy, and C1-C4 alkylamino) is one to three substituents independently selected from the following: b) D, halogens, -OH and R １２ CO ２ - (R １２ (is C1-C3 alkyl) and one to three substituents independently selected from; or c) -OH and R １２ CO ２ - (R １２ (is a C1-C3 alkyl group) The ALPK1 agonist according to any one of claims 1 to 9, wherein the C1 to C3 alkyl group is optionally substituted.

11. R １ but, a) 【Transformation 5】 Select from, b) 【Transformation 6】 Select from, c) 【Transformation 7】 That is, An ALPK1 agonist according to any one of claims 1 to 10. 【Request Item 12】 【Chemistry 8】 The ALPK1 agonist according to claim 1, comprising a compound selected from, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

13. The ALPK1 agonist according to any one of claims 1 to 12, wherein the ALPK1 agonist binds to ALPK1.

14. The ALPK1 agonist i) Induce autophosphorylation of ALPK1; ii) Induce ALPK1-dependent phosphorylation of TIFA; and / or iii) Induces phosphorylation of IκB, An ALPK1 agonist according to any one of claims 1 to 13.

15. A pharmaceutical composition for activating ALPK1, comprising an ALPK1 agonist according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. An ALPK1 agonist according to any one of claims 1 to 14, for use in the treatment of a disease or disorder that can be treated by activating ALPK1.

17. a) For use as a drug that modulates the immune response, b) For use in methods of treating cancer, c) For use as a drug to enhance the immune response to a target antigen, d) For use in methods of treating diseases or disorders suitable for treatment by activation of NFκB, p38, and JNK cell signaling pathways in cells, or e) For use in methods of treating or preventing diseases or disorders caused by infectious pathogens selected from bacteria, viruses, or parasites The ALPK1 agonist according to claim 16.

18. An ALPK1 agonist according to claim 16 for use in a method for treating a liver disease or disorder in a subject requiring treatment for such a liver disease or disorder, wherein the method comprises the step of administering a low dose of the ALPK1 agonist to a subject, the low dose of the ALPK1 agonist corresponding to an ALPK1 agonist of 1 ng / kg to 1 mg / kg, wherein the liver disease or disorder is optionally selected from liver cancer, non-alcoholic steatohepatitis (NASH), and diseases or disorders caused by infection with hepatitis C virus (HCV) or hepatitis B virus (HBV).

19. The pharmaceutical composition according to claim 15, for use in the treatment of a disease or disorder that can be treated by activating ALPK1.

20. a) For use as a drug that modulates the immune response, b) For use in methods of treating cancer, c) For use as a drug to enhance the immune response to a target antigen, d) For use in methods of treating diseases or disorders suitable for treatment by activation of NFκB, p38, and JNK cell signaling pathways in cells, or e) For use in methods of treating or preventing diseases or disorders caused by infectious pathogens selected from bacteria, viruses, or parasites The pharmaceutical composition according to claim 19.

21. A pharmaceutical composition according to claim 19 for use in a method for treating liver disease or disorder in a subject requiring treatment for liver disease or disorder, wherein the method comprises the step of administering a low dose of the pharmaceutical composition to the subject, the low dose of the pharmaceutical composition corresponding to 1 ng / kg to 1 mg / kg of the ALPK1 agonist, and optionally, the liver disease or disorder being selected from liver cancer, non-alcoholic steatohepatitis (NASH), and diseases or disorders caused by infection with hepatitis C virus (HCV) or hepatitis B virus (HBV).

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