A water-soluble phosphoric acid derivative, preparation method, pharmaceutical composition and application thereof

Abstract

The present invention relates to a novel compound with TLR7 / 8 agonist activity, in particular to a phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine and its isomers, prodrugs, isotope derivatives, solvates, esters or salts, and its use in preparing vaccines, preventing and / or treating viral infections and tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a special type of phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine and a preparation method and a pharmaceutical composition thereof. The derivative produces a vaccine adjuvant effect by activating Toll-like receptor agonists (TLRs) TLR7 and 8 and can be used to prepare vaccines to prevent and / or treat viral infections and tumors in animals or humans. Background Art

[0002] Vaccines are a historic breakthrough in human medical history. In addition to protective antigens, effective vaccine systems usually require highly effective natural immune system activation components, which are called vaccine adjuvants. They can non-specifically change or enhance antigen-specific immune responses. Currently known adjuvants generally include aluminum salt adjuvants, small molecule peptides or glycoproteins, nucleic acids, etc. Among them, Toll-like receptor ligand molecules as immune adjuvants have made significant progress in clinical or preclinical trials.

[0003] Toll-like receptors (TLR1, 2, 3-13) are a very important class of receptors that specifically recognize pathogen-associated molecular patterns (PAMPs). They play an important role in both innate and acquired immunity and are an important bridge connecting innate and acquired immunity. These receptors are widely expressed on immune cells and epithelial cells. Toll-like receptor activation can activate NF-κB through myeloid differentiation protein 88 (MyD88) H or TRIF-dependent signaling pathways, thereby inducing the production of cytokines and chemokines, promoting the maturation of DC cells and the expression of co-stimulatory ligands on the surface of T cells, and enhancing innate and adaptive immune responses. Therefore, Toll-like receptor agonists are a class of vaccine adjuvants with important value.

[0004] TLRs agonists have been successful in the prevention and treatment of various diseases as vaccine adjuvants, including infectious diseases, tumors, influenza, etc. Live Mycobacterium bovis (BCG) vaccine has been approved for the prevention of tuberculosis. The main components of BCG are the bacterial cell wall skeleton and peptidoglycan. Studies have shown that these components rely on the activation of TLR2 and 4 signaling pathways to exert immune enhancement. TLR4 ligand MPLA can strongly induce the production of IgG2a Abs and induce TH-1 immune response, and is currently a widely used TLR ligand vaccine adjuvant. MPLA adsorbed onto aluminum salt preparations and combined with hepatitis B vaccine (Fendrix) was approved for marketing by the European Union as early as 2005. Dan H. Barouch's research group reported in Nature in 2016 and 2018 that TLR7 activation can be used as an important strategy for effective combined treatment of AIDS, and it has been verified in monkeys (Nature, 540, 284–287; Nature, 563, 360–364). Compared with the negative control group, the combined use of vaccine Ad26 / MVA and adjuvant TLR7 agonist GS-986 can reduce the median viral load by 1.74 log and prolong the time of viral rebound after ART termination by 2.5 times.

[0005] Although TLRs agonists have been shown to have significant vaccine adjuvant effects, the development of most drugs targeting TLRs targets has made limited progress due to side effects caused by insufficient efficacy or uncontrolled cytokine release. Taking TLR7 and 8 as an example, although the TLR7 / 8 agonist raquimod showed good clinical effects, the clinical trial has been terminated due to adverse side effects (NCT01676831). In Phase Ib clinical trials, the TLR8 agonist VTX-2337 showed the ability to enhance the antibody-dependent cellular cytotoxicity (ADCC) of cetuximab, but no encouraging clinical progress has been reported. These compounds are very difficult to use systematically due to poor efficacy or toxic side effects (such as lymphocytopenia, flu-like symptoms, etc.) (AIDS Res., 30, 457–467). In order to find effective and safe TLR7 / 8 activation schemes and increase the possibility of their combination with vaccines, it is necessary to develop new vaccine adjuvants. Inducing protective Th-1 immune responses based on TLRs activation is a very promising development area in vaccine design. This patent designs new ligand agonists around TLR7 / 8. Compared with other Toll-like receptor targets, these targets can be effectively activated by small molecule ligands, which is a great advantage for drug development. Summary of the invention

[0006] The present invention provides a special class of phosphoric acid derivatives based on pyridine [3,2-d] pyrimidine-2-amine. The derivatives have good water solubility, are convenient for drug preparation or chelated with other drugs to form a complex, and produce a vaccine adjuvant effect by activating Toll-like receptor agonists (TLRs) TLR7 and 8, so that they can be used to prepare vaccines, and prevent and / or treat viral infections and tumors in animals or humans.

[0007] The phosphoric acid derivatives based on pyridine [3,2-d] pyrimidine-2-amine provided by the present invention and their isomers, prodrugs, isotopic derivatives, solvates, esters, and salts have a structural formula as shown in general formula (I):

[0008]

[0009] Wherein, R1, R2, and R3 are independently selected from H, F, Cl, Br, I, substituted or unsubstituted alkyl, or substituted or unsubstituted cycloalkyl;

[0010] Furthermore, at least one of R1, R2, and R3 is:

[0011] wherein L1 is selected from -OCH2CH2-, -CH2CH2CH2- and -OCH2CH2O-;

[0012] L2 is absent or selected from -NR6-, -(CH2) m - and -NR6(CH2) m -;

[0013] Y is -(CH2) t -;

[0014] R4 and R5 are independently selected from H, F, Cl, Br, I, substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl;

[0015] m is selected from an integer between 1 and 10;

[0016] n is selected from an integer between 1 and 10;

[0017] L is selected from -(CR7R8) p -, -NR9-, -O-, -S-;

[0018] X is selected from -(CR 10 R 11 ) q -、-NR 12 -, -O-;

[0019] R6, R7, R8, R9, R 10 , R 11, R 12 , R 13 independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl;

[0020] R 14 is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; p is selected from an integer between 1 and 10;

[0021] q is an integer selected from 1 to 10;

[0022] t is an integer selected from 1 to 10.

[0023] Preferably, R1, R2, R3 are independently selected from H or C1-C3 alkyl, and at least one is:

[0024] More preferably, R1, R2, R3 are independently selected from H or methyl, and at least one is:

[0025] More preferably, R1, R2 are independently selected from H or methyl, and R3 is

[0026] Preferably, R4 and R5 are independently selected from H, F or C1-C3 alkyl.

[0027] More preferably, R4 and R5 are both H, or R4 and R5 are both F.

[0028] Preferably, R6 is H or a C1-C3 alkyl group, more preferably, R6 is H or a methyl group.

[0029] Preferably, m is 1, 2, 3, 4 or 5.

[0030] Preferably, n is 1, 2, 3, 4 or 5.

[0031] Preferably, Specifically, the group has the following structure:

[0032]

[0033] Preferably, R 13 It is H or C1-C3 alkyl.

[0034] Preferably, R 14 is a C1-C3 alkyl group, more preferably, R 14 It is methyl.

[0035] Preferably, t is 1, 2, 3, 4 or 5.

[0036] Preferably, the phosphoric acid derivatives based on pyridine [3,2-d] pyrimidine-2-amine and their isomers, prodrugs, isotopic derivatives, solvates, esters and salts provided by the present invention have the following structural formulas:

[0037]

[0038] Preferably, R7, R8, and R9 are independently selected from H and C1-C3 alkyl.

[0039] Preferably, R 10 , R 11 , R 12 Independently selected from H, C1-C3 alkyl.

[0040] p is 1, 2, 3, 4 or 5;

[0041] q is 1, 2, 3, 4 or 5.

[0042] Preferably, L is selected from -(CH2)-, (include ), -NH- or -O-;

[0043] Preferably, X is selected from -CH2-, -NH- or -O-.

[0044] The present invention also provides a phosphoric acid derivative based on pyridyl[3,2-d]pyrimidin-2-amine having the following N1 or N2 structural formula and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts:

[0045]

[0046] The present invention also provides a method for preparing a phosphoric acid derivative based on pyridine [3,2-d] pyrimidin-2-amine having the structural formula N1, comprising:

[0047] (1) Using diethyl (bromodifluoromethyl)phosphonate and 1,2-bis(2-iodoethoxy)ethane as starting materials, under the action of lithium diisopropylamide, compound 2 is prepared, and the reaction formula is:

[0048]

[0049] (2) Compound 2 is dissolved in acetonitrile, intermediate 3 and potassium carbonate are added, and heated to obtain compound 4. The reaction formula is:

[0050]

[0051] (3) Compound 4 was dissolved in dry dichloromethane and hydrolyzed under the action of TMSBr (trimethylsilane bromide) to obtain compound 5. The reaction formula is:

[0052]

[0053] The present invention also provides a method for preparing a phosphoric acid derivative based on pyridine [3,2-d] pyrimidin-2-amine having the N2 structural formula, comprising:

[0054] (1) Compound 6 was dissolved in dry DMF, and cesium carbonate and intermediate 2 were added in sequence. The mixture was reacted at 60° C. for 5 hours to obtain compound 7. The reaction formula is:

[0055]

[0056] (2) Compound 7 was dissolved in dry dichloromethane, and TMSBr was added successively, and the mixture was reacted at room temperature for 2 hours to obtain compound 8. The reaction formula is:

[0057]

[0058] The preparation methods of other phosphoric acid derivatives based on pyridine [3,2-d] pyrimidine-2-amine as shown in the general formula (I) of the present invention can be prepared by adopting the preparation method of phosphoric acid derivatives of pyridine [3,2-d] pyrimidine-2-amine with structural formula N1 or N2 as described above through conventional adjustments in the art.

[0059] In another aspect, the present invention provides the use of the pyridine [3,2-d] pyrimidine-2-amine-based phosphoric acid derivatives and their isomers, prodrugs, isotope derivatives, solvates, esters or salts as vaccine adjuvants.

[0060] In another aspect, the present invention provides the use of the phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts in the preparation of vaccines, wherein the phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts are used as vaccine adjuvants.

[0061] Another aspect of the present invention provides a pharmaceutical composition comprising the pyridinium [3,2-d] pyrimidine-2-amine-based phosphoric acid derivatives and isomers, prodrugs, isotopic derivatives, solvates, esters, salts thereof, and pharmaceutically acceptable excipients.

[0062] The pharmaceutically acceptable excipients of the present invention include, but are not limited to, sweeteners, diluents, stabilizers, emulsifiers, dispersants, preservatives, colorants, flavor enhancers, surfactants, wetting agents, disintegrants, suspending agents, isotonic agents, and solvents.

[0063] The pharmaceutical composition of the present invention can be prepared into tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, and gels.

[0064] The routes of administration of the pyridinium [3,2-d] pyrimidin-2-amine-based phosphoric acid derivatives and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof or pharmaceutical compositions thereof of the present invention include, but are not limited to, oral, rectal, transmucosal, topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intramuscular, subcutaneous, and intravenous administration.

[0065] The composition of the present invention also contains at least one other therapeutic agent, which is selected from chemotherapeutic agents, immunotherapy, anti-angiogenic agents, cytokines, hormones, polynucleotides, antibodies, inactivated pathogens, pathogen surface antigens, mRNA or DNA encoding pathogen surface antigens, peptides, whole cells, genetically modified whole cells, recombinant proteins or tumor-associated antigens expressed by recombinant viral vectors and immunologically active fragments.

[0066] The present invention also provides a vaccine, which includes an antigenic vaccine component or an anti-tumor vaccine component for infections, viruses and parasitic diseases, and a phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine and its isomers, prodrugs, isotope derivatives, solvates, esters or salts.

[0067] The antigenic vaccine components for infections, viruses and parasitic diseases described in the present invention are, for example, one or more of inactivated pathogens, pathogen surface antigens, and mRNA or DNA encoding pathogen surface antigens.

[0068] The anti-tumor vaccine components of the present invention are, for example, peptides, whole cells, genetically modified whole cells, recombinant proteins or tumor-associated antigens expressed by recombinant viral vectors.

[0069] The vaccine of the present invention can be a human vaccine or a veterinary vaccine.

[0070] The human vaccine described in the present invention is selected from the group consisting of tumor vaccines, diphtheria vaccines, pertussis vaccines, tetanus vaccines, polio vaccines, hepatitis A vaccines, hepatitis B vaccines, rabies vaccines, measles vaccines, rubella vaccines, influenza vaccines, mumps vaccines, varicella vaccines, rotavirus vaccines, smallpox vaccines, yellow fever vaccines, mite-type encephalitis vaccines, type B Haemophilus influenzae conjugate vaccines, typhoid vaccines, cholera vaccines, BCG vaccines, pneumococcal vaccines, coronavirus vaccines (including new coronavirus COVID-19 vaccines), norovirus vaccines, Ebola virus vaccines, dengue virus vaccines, Zika virus vaccines, coxsackie virus vaccines, and vaccines for meningitis caused by Neisseria meningitidis.

[0071] The tumor vaccine of the present invention can be used to treat melanoma, basal cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer (including small cell lung cancer and non-small cell cancer), leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, malignant carcinoid, bladder cancer, skin precancerous lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignant tumors, cervical cancer, endometrial cancer, glioblastoma, and adrenocortical carcinoma.

[0072] The veterinary vaccines of the present invention include poultry vaccines, pig vaccines or cattle vaccines. The poultry vaccines include but are not limited to avian influenza vaccines, Newcastle disease inactivated vaccines, chicken egg drop syndrome inactivated vaccines or combination vaccines of the above vaccines. The poultry vaccines include porcine foot-and-mouth disease inactivated vaccines, porcine circovirus vaccines, porcine pseudorabies inactivated vaccines, bivalent inactivated Streptococcus suis vaccines, inactivated Haemophilus parasuis vaccines, inactivated porcine reproductive and respiratory syndrome vaccines, inactivated Mycoplasma suis vaccines or combination vaccines of the above vaccines. The cattle vaccines include bovine foot-and-mouth disease inactivated vaccines, bovine Salmonella inactivated vaccines or combination vaccines of the above vaccines.

[0073] The antibodies of the present invention include, but are not limited to: PRO542, which is an anti-HIV gp120 antibody fused to CD4 (Progenics / Genzyme Transgenics); MDX-010 (Medarex, New Jersey), which is a humanized anti-CTLA-4 antibody; SYNAGIS (MedImmune, Maryland), which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody; HERCEPTIN (trastuzumab) (Genentech, California), which is a humanized anti-HER2 monoclonal antibody; Humanized anti-CD18 F(ab')2 (Genentech); CDP860, which is a humanized anti-CD18 F(ab')2 (Celltech, UK); Ostavir, which is a human anti-hepatitis B virus antibody (Protein Design Lab / Novartis); PROTOVIRTM, which is a humanized anti-CMV IgG1 antibody (Protein Design Lab / Novartis); MAK-195 (SEGARD), which is a murine anti-TNF-α F(ab')2 (Knoll Pharmaceuticals / BASF); llPharma / BASF); IC14, which is an anti-CD14 antibody (ICOS Pharmaceuticals (ICOS Pharm)); humanized anti-VEGF IgG1 antibody (Genentech); OVAREXM, which is a mouse anti-CA125 antibody (Altarex); PANOREXM, which is a mouse anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome / Centocor); BEC2, which is a mouse anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C2 25, which is a chimeric anti-EGFR IgG antibody (Immunoclonal Systems); VITAXIN™, which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution / MedImmune); Campath 1H / LDP-03, which is a humanized anti-CD52 IgG1 antibody (Leukosite); SmartM195, which is a humanized anti-CD33 IgG antibody (Protein Design Lab / Kanebo);RITUXAN™ (rituximab™), a chimeric anti-CD20 IgG1 antibody (IDEC Pharmaceuticals / Genentech, Roche / Zettyaku); LYMPHOCIDE™, a humanized anti-CD22 IgG antibody (Immunomedics); SmartID10, a humanized anti-HLA antibody (Protein Design Laboratories); ONCOLYM™ (Lym-1), a radiolabeled mouse anti-HLADR antibody (Techniclone); ABX-IL8, a human anti-IL8 antibody (Abgeni x)); anti-CD11a, a humanized IgG1 antibody (Genentech / Xoma); ICM3, a humanized anti-ICAM3 antibody (ICOS Pharmaceuticals); IDEC-114, a primatized anti-CD80 antibody (IDEC Pharmaceuticals / Mitsubishi); ZEVALIN™, a radiolabeled murine anti-CD20 antibody (IDEC / Schering AG); IDEC-131, a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151, a primatized anti-CD4 antibody (IDEC); IDEC-152, a primatized anti-CD4 antibody (IDEC); SMART anti-CD3, a humanized anti-CD3 IgG (Protein Design Laboratories); 5G1.1, a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7, a humanized anti-TNF-α antibody (CAT / BASF); CDP870, a humanized anti-TNF-α Fab fragment (Celtec); IDEC-151, a primatized anti-CD4 IgG1 antibody (IDEC Pharmaceuticals / SmithKline Beecham); MDX-CD4, a humanized anti- CD4 IgG antibody (Medarex / Eisai / Genmab); CDP571, a humanized anti-TNF-α IgG4 antibody (Celtech); LDP-02, a humanized anti-α4β7 antibody (Luxeter / Genentech); OrthoCloneOKT4A, a humanized anti-CD4 IgG antibody (OrthoBiotech); ANTOVATM, a humanized anti-CD40L IgG antibody (Biogen); ANTEGRENTM, a humanized anti-VLA-4 IgG antibody (Elan);MDX-33, a human anti-CD64 (FcγR) antibody (Medlex / Centeon); SCH55700, a humanized anti-IL-5 IgG4 antibody (Celtech / Schering); SB-240563 and SB-240683, humanized anti-IL-5 and anti-IL-4 antibodies, respectively (SmithKline Beecham); rhuMab-E25, a humanized anti-IgE IgG1 antibody (Genentech / Norvartis / Tanox Biosystems); ABX-CBL, a mouse anti-CD-147 IgM antibody (Abgenix); BTI-322, a rat anti-CD2 IgG antibody (Medical Immunology / BioTransplant); Odoclone / OKT3 (Orth Biosystems); oclone / OKT3), a mouse anti-CD3 IgG2a antibody (OrthoBiotech); SIMULECTTM, a chimeric anti-CD25 IgG1 antibody (Novartis); LDP-01, a humanized anti-β2-integrin IgG antibody (Luxet); anti-LFA-1, a mouse anti-CD18 F(ab′)2 (Pasteur-Merieux / Immunotech); CAT-152, a human anti-TGF-β2 antibody (Cambridge AbTech); CorsevinM, a chimeric anti-factor VII antibody (Santok); MDX-1106, a PD-1 antibody (Bristol-Myers Squibb); MDX-1105, a PDL1 antibody (Roche). ;

[0074] The anticancer agents described in the present invention include but are not limited to: acivicin, aclarubicin, acodazole hydrochloride, acronine, adolesin, aldesleukin, hexamethylmelamine, ambromycin, amenthranol acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, troline, azacitidine, azatepa, azomycin, batimastat, benzotepa, bicalutamide, bisantrene hydrochloride, binefad dimesylate, bisezole, bleomycin sulfate, buquina sodium, bropiridamine, busulfan, actinomycin C, caprotestosterone, carcetamide, carbetimer, carboplatin, carmustine, carrubicin hydrochloride, carzelesin, cedifingol, chlorambucil, cyromycin, cisplatin, cladribine, clinatol mesylate, cyclophosphamide, cytarabine glycosides, dacarbazine, actinomycin D, daunorubicin hydrochloride, decitabine, dextromaplatin, dezaguanine, dezaguanine mesylate, diazocone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drolostanone propionate, dazomicin, edatrexa, eflornithine hydrochloride, elsamitrucin, enloplatin, enprobamate, epiripridine, epirubicin hydrochloride, erbulazole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, chlorpheniramine, fadrozole hydrochloride, fazarabine, fenretinide, azauridine, fludarabine phosphate, fluorouracil, flucitabine, fosquatone, fostramoxine sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide Amine, Imofosine, Interleukin II (including recombinant interleukin II or rIL2), Interferon α2a, Interferon α-2b, Interferon α-n1, Interferon α-n3, Interferon β-Ia, Interferon γ-Ib, Iproplatin, Irinotecan hydrochloride, Lanreotide acetate, Letrozole, Leuprorelin acetate, Lirazo hydrochloride, Lometrexol sodium, Lomustine, Losoxantrone hydrochloride, Masoprofen, Maytansine, Nitrogen Mustard Hydrochloride, Progesterone acetate, Melengestrol acetate, Phenylalanine mustard, Menolil, Mercaptopurine, Methotrexate, Methotrexate sodium, Chlorpheniramine, Metodepa, Mibutamide, Mitokacin, Mitoclopramide, Mitoxantrone, Mitoxantrone, Mitomycin, Mitosep, Mitotane, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Noramycin, Omaplatin, Oxysulen, Paclitaxel, Pegaspargase, Pelimycin, Namustine, Pelimycin Sulfate, Perfosfamide, Pipobroman, Piposufan, Pyroxantrone Hydrochloride, Prucamycin, Promectin, Porfirol Sodium, Porfirol, Prednimustine, Procarbazine Hydrochloride, Puromycin, Puromycin Hydrochloride, Pyrazofuranoside, Liboadenosine, Roglulimide, Safingol, Safingol Hydrochloride, Semustine, Simtraqin, Sodium Phosacetyl Aspartate, Sparamycin, Spirogermanium Hydrochloride, Spiromustine, Spiroplatin, Streptozocin, Streptozocin, Sulfonyl Chloride, Tarimycin, Tecogalan Sodium, Tegafur, Tiloxantrone Hydrochloride, Temoporfin, Teniposide, Teroxilon, Testolactone, Thiamidopamine, Thioguanine, Thiotepa, Thiazofurine, Tirapazamine,Toremifene citrate, triptolon acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronide, triptorelin, tobradox hydrochloride, uramustine, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinposide sulfate, vinorelbine sulfate, vinorelbine tartrate, vinprodine sulfate, vinorelbine sulfate, vorozole, zeniplatin, jingsistatin, zorubicin hydrochloride. Other anticancer agents that can be used include, but are not limited to, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adolesin, aldesleukin, ALL-TK antagonists, hexamethylmelamine, aminostine, 2,4-dichlorophenoxyacetic acid (amidox), amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, anrelix, anti-dorsalization protein 1, antiandrogens, prostate cancer, antiestrogens, antineoplastons (antineoplasto n), antisense oligonucleotides, glycine aphidicolin, apoptosis gene regulators, apoptosis regulators, apurinic nucleic acids, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, amustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorin, benzoylstaurosporine, beta-lactam derivatives, β-alethine, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, diazinon spermine, binefad, bistratene A, bizolexin, breflate, bropiridamine, budotitan, butylthionine imide, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL2, capecitabine, carboxyamide-amino-triazole, carboxyamide triazole, CaRestM3, CARN700, cartilage Derived inhibitors, cardzelesin, casein kinase inhibitor (ICOS), castanospermine, bactericidal peptide B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, enclomiphene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, clenato, cryptophycin 8, cryptophycin A derivatives,Curacin A, cyclopentanthraquinone, cycloplatam, cypemycin, cytarabine sodium octadecyl phosphate, cytolytic factor, estradiol phosphate (cytostatin), dacximab, decitabine, dehydrogenated oxadiazine B, deslorelin, dexamethasone, right ifosfamide, right razoxane, right verapamil, diazadiquinone, dehydrogenated oxadiazine B, 3,4-dihydroxybenzohydroxamic acid (didox), diethylspermidine, dihydro 5-azacytidine, dihydropaclitaxel, 9-, dioxamycin, diphenylspiromustine, docetaxel, docosanol, dola Setron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, etiomustine, edilfosine, edrecolomab, eflornithine, elemene, etifur, epirubicin, etirostamide, estramustine analogs, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozoles, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, fluasterone, fludarabine, fluorodaunorunicin, fomemax, formestane, fostramexine, fostramustine, gadolinium texap hyrin), gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, cyclohexylbisacetamide, hyperanthrone, ibandronic acid, bilirubin, idoxifene, idemenone, imofosine, ilomastat, imidazoacridone, imiquimod, immunostimulatory peptides, insulin-like growth factor 1 receptor inhibitors, interferon agonists, interferons, interleukins, iodobenzylguanidine, iodoxorubicin, 4-sweet potato bitter alcohol, iropra, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide , kahalalide F, lamellarin-N triacetic acid, lanreotide, leinamycin, lelagostine, mushroom polysaccharide sulfate, leptolstatin, letrozole, leukemia inhibitory factor, interferon alpha, leuprolide + estrogen + progesterone, leuprolide, levamisole, lirazo, linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, guanidineethylphosphoserine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lutetiumtexaphyrin,Lysofylline, cytolytic peptide, metansine, mannostatin A, marimastat, maspin, mammary gland serine protease inhibitor, matrix lytic factor inhibitor, matrix metalloproteinase inhibitor, menolipin, thiobutanil, metirol, methionase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, milistostatin, mismatched double-stranded RNA, mitoxantrone, dibromodole, mitomycin analogs, mitonadamide, mitotoxin, fibroblast growth factor-saponin, mitoxantrone, mofarotin, morasporastatin, monoclonal antibody, human chorionic gonadotropin, monophosphoryl lipid A+ mycobacterial cell wall skeleton, mopidarol, multidrug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, Indian Ocean sponge (mycaperoxide) B, mycobacterial cell wall extract, myriaporone, N-acetyl danaline, N-substituted benzamide, nafarelin, nagrestip, naloxone + analgesic, napavin, naphterpin, natograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide regulator, nitrooxygen antioxidant, nitrullyn ), O6-benzylguanine, octreotide, okicenone, oligonucleotide, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, oxatron, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analog, paclitaxel derivative, palauamine, palmitoyl rhizoxin, pamidronate, panaxytriol, panomifen, parabactin, palzeptin, pegaspargase, perdecan, sodium pentothiosulfate, pentostatin, pentrozole, perflurane, perfosfamide, perillyl alcohol, phenazino mycin), phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piroxine, placenta A, placenta B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfibril sodium, porfibrin, chlorprednisolone, propyl diacridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurin, pyrazoline acridine, pyridoxalated hemoglobin polyoxyethylene conjugates,raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reteptine, etidronate rhenium Re186, rhizoxin, ribozymes, RII retinamide, rogulimide, rohitukine, romotide, roquimek, rubiginone B1, ruboxyl, safengo, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetics, semustine, senescence-derived inhibitor 1, sense oligonucleotides , signal transduction inhibitor, signal transduction regulator, single-chain antigen binding protein, sizoran, sobuzoxane, borca ​​sodium, sodium phenylacetate, solverol, somatomedin binding protein, sonarmin, fospartic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamide, matrix degrading inhibitor, sulfinosine, potent vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine , synthetic mucopolysaccharides, tamustine, tamoxifen methyl iodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thiocobalamin, thrombopoietin, thrombopoietin mimetics, thymofasin, thymopoietin receptor agonist, thymosin, thyroid stimulating hormone, tin ethyl ester, tirapazamine, dichlorocyclopentadienyl titanium, topsentin, torel Mifen, totipotent stem cell factor, translation inhibitors, retinoic acid, triacetyl uridine, triciribine, trimetrexate, triptorelin, tropisetron, torosterol, tyrosine kinase inhibitors, tyrphostin, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, ureakinase receptor antagonist, vapreotide, variolin B, vector system, erythrocyte gene therapy, vilaresol, veratrum, verdins, verteporfin, vinorelbine, vinxaltine, Vitaxin, vorozole, zanoteron, zeniplatin, benzyl retinol and statins.

[0075] The present invention also provides a method for improving the body's positive immune response, comprising: administering a therapeutically effective amount of the pyridine [3,2-d] pyrimidine-2-amine-based phosphoric acid derivative and its isomers, prodrugs, isotope derivatives, solvates, esters or salts or the composition of the present invention to a system or individual in need.

[0076] The present invention also provides a method for enhancing the effect of chemotherapy, comprising administering a therapeutically effective amount of a chemotherapeutic drug to a system or individual in need thereof, and simultaneously or subsequently administering a therapeutically effective amount of a pyridine [3,2-d] pyrimidine-2-amine-based phosphoric acid derivative and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts or the composition of the present invention.

[0077] The present invention also provides a method for improving immunotherapy, comprising: administering a therapeutically effective amount of a pyridine [3,2-d] pyrimidine-2-amine-based phosphoric acid derivative of formula (I) and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts or the composition of the present invention to a system or individual in need thereof, and simultaneously or subsequently introducing chimeric antigen receptor T cells (CAR-T) into the system or individual.

[0078] Another aspect of the present invention provides a method for treating or preventing a mammalian disease, comprising: administering a therapeutically effective amount of a pyridine [3,2-d] pyrimidine-2-amine-based phosphoric acid derivative and its isomers, prodrugs, isotopic derivatives, solvates, esters or salts or the composition of the present invention to a system or individual in need thereof.

[0079] Preferably, the mammal is a human or a pig.

[0080] Preferably, the disease is a disease or condition associated with TLRs activity. More preferably, the disease or condition associated with TLRs activity is a disease or condition associated with TLR7 and 8 activity, for example, the disease or condition associated with TLRs activity is selected from infectious diseases, immune diseases, viral infections or tumors.

[0081] Preferably, the immune disease is an autoimmune disease, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Wegener's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, Behcet's syndrome, etc.

[0082] Preferably, the viral infection includes but is not limited to infectious diseases caused by the following viruses: Ebola virus disease, anthrax, genital warts, simple warts, plantar warts, respiratory syncytial virus (RSV), hepatitis B, hepatitis C, dengue virus, herpes simplex virus (e.g., HSV-I, HSV-II, CMV or VZV), molluscum contagiosum, vaccinia, smallpox, lentivirus, human immunodeficiency virus (HIV), human papillomavirus (HPV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rhinovirus, enterovirus, adenovirus, influenza, parainfluenza, mumps virus, measles virus, papovavirus, flavivirus, retrovirus, coronavirus (e.g., novel coronavirus 2019-nCoV), arenavirus (e.g., LCM, Junin virus, Machupo virus, Guanarito virus and Lassa fever) and filovirus (e.g., Ebola virus or Marburg virus).

[0083] More preferably, the viral infection is an infectious disease caused by the following viruses: human immunodeficiency virus (HIV), hepatitis B (HBV), and African swine fever virus.

[0084] Preferably, the tumor includes but is not limited to: human sarcoma and cancer, such as fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, epithelial cancer, Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloblastic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroblastic leukemias); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's and non-Hodgkin's), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

[0085] More preferably, the tumor disease is: colon cancer, bladder cancer, melanoma, meningioma, lung cancer, or pancreatic cancer.

[0086] The term "TLR" described in the present invention refers to Toll like receptor.

[0087] The term "pharmaceutically acceptable" as used herein refers to materials that have no adverse biological or other effects, such as materials that can be incorporated into a pharmaceutical composition administered to a patient without causing any adverse biological effects or interacting in a harmful manner with any other component of the composition containing it. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it means that the carrier or excipient meets the required standards for toxicology and manufacturing testing or is included in the inactive ingredient guidelines established by the U.S. Food and Drug Administration.

[0088] The term "combination" or "pharmaceutical combination" as used herein refers to a product obtained by mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.

[0089] The term "TLR disease" or "disease or condition associated with TLR activity" as used herein refers to any disease state associated with toll-like receptors.

[0090] The room temperature of the present invention is 20-30°C.

[0091] The term "alkyl" of the present invention refers to a hydrocarbon chain radical that is straight or branched and does not contain an unsaturated bond, and the hydrocarbon chain radical is connected to the rest of the molecule by a single bond. Typical alkyl groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, etc. If the alkyl is substituted by a cycloalkyl, it is correspondingly a "cycloalkylalkyl", such as cyclopropylmethyl. If the alkyl is substituted by an aryl, it is correspondingly an "aralkyl", such as benzyl, diphenylmethyl or phenethyl. If the alkyl is substituted by a heterocyclic radical, it is correspondingly a "heterocyclic alkyl".

[0092] The term "cycloalkyl" as used herein refers to alicyclic hydrocarbons. Typical cycloalkyl groups contain 1 to 4 monocyclic and / or condensed rings and 3 to 18 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as cyclopropyl, cyclohexyl or adamantyl. DETAILED DESCRIPTION

[0093] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0094] The starting materials and reagents used in the present invention are all commercially available, and the suppliers include but are not limited to: Aldrich Chemical Company, Lancaster Synthesis Ltd. Among them, dimethylformamide, tetrahydrofuran and dioxane are all ultra-dry solvents purchased from J&K Company and stored in a glove box, dichloromethane and acetone are respectively taken from a solvent purification system, and all glassware used for water-sensitive reactions are first dried in an oven at 100°C. Unless otherwise specified, the raw materials and reagents used are used directly without further treatment.

[0095] 1 H NMR and 13 C NMR spectra were measured using a Bruker DRX 400 nuclear magnetic resonance spectrometer, and chemical shifts were expressed in ppm. Tetramethylsilane was used as an internal standard (0.00 ppm), and CDCl3 or DMSO-d6 was used as a solvent (or other solvents). 1HNMR representation method: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If coupling constants are provided, their units are Hz. Mass spectra were measured using a Finnigan Advantage mass spectrometer, and the ionization mode was ESI. The compounds were purified by column chromatography (silica gel GF254: 200-400 mesh). The purity of the compounds, unless otherwise specified, was determined using HPLC, and all refer to the isolated yield.

[0096] Example 1: Synthesis of Compound 2

[0097] Take diethyl (bromodifluoromethyl)phosphonate (265 mg, 1 mmol) and dissolve it in 5 mL of tetrahydrofuran. Place the reaction solution at -78 degrees Celsius, add a tetrahydrofuran solution of lithium diisopropylamide (containing 1 mmol LDA), react for 30 minutes, then dropwise add a tetrahydrofuran (2 mL) solution of 1,2-bis(2-iodoethoxy)ethane (400 mg, 1.1 mmol), store the reaction solution at -78°C and react for 12 hours. After the reaction is completed, slowly add ice water to quench the reaction. Add hydrochloric acid to adjust the solution to a clear color, extract three times with ethyl acetate, combine the organic layers, spin dry, and purify by column chromatography to obtain compound 2. LCMS (M+H + ) is: 431.2.

[0098]

[0099] Example 2: Synthesis of Compound 4

[0100] Compound 3 (350 mg, 1.0 mmol) was dissolved in 5 mL of dry acetonitrile, and intermediate 2 (473 mg, 1.1 mmol) and potassium carbonate (350 mg, 2.0 mmol) were added. The reaction solution was stirred at 75° C. for 12 hours, filtered, concentrated, and extracted with DCM / H2O. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography to obtain yellow solid compound 4. The reaction formula is:

[0101]

[0102] 1 H NMR(400MHz,Chloroform-d)δ7.80(d,J=8.6Hz,1H),7.53(t,J=5.7Hz,1H),7.39(dd ,J=14.7,8.2Hz,3H),7.22(d,J=7.9Hz,2H),6.02(s,2H),4.34–4.15(m,6H),3.85–3 .70(m,6H),3.69–3.58(m,6H),2.81(t,J=5.7Hz,2H),2.51–2.28(m,5H),1.80–1.68 (m,2H),1.50(dt,J=15.0,7.4Hz,2H),1.39(t,J=7.1Hz,6H),1.02(t,J=7.3Hz,3H). 13 CNMR(101MHz,Chloroform-d)δ160.02,157.30,156.7,138.28,130.22,129.39,129.15,128.93,126.88,70.37, 70.33,68.38,64.65,64.59,63.8,63.7,61.74,55.74,43.91,42.14,40.77,31.18,20.15,16.46,16.41,13.82.

[0103] Example 3: Synthesis of Compound 5

[0104] Compound 24 (326 mg, 0.5 mmol) was dissolved in 5 mL of dry dichloromethane, TMSBr (398 μL, 3 mmol) was added in an ice bath, and the mixture was heated to room temperature and stirred for 1 hour, then filtered and concentrated. White solid 5 was obtained by reverse phase HPLC with a yield of 45%. The reaction formula is:

[0105]

[0106] 1H NMR(400MHz, Deuterium Oxide)δ7.84(d,J=8.6Hz,1H),7.52(t,J=5.8Hz,1H),7.35(dd,J=14.7,8.2Hz,3H),7.20(d,J=7.9Hz,2H),4.34–4.15(s,2H),3.85–3.70(m ,6H),3.69–3.58(m,6H),2.78(t,J=5.8Hz,2H),2.46–2.20(m,5H),1.77–1.64(m,2H),1.50(dt,J=14.7,7.6Hz,2H),1.00(t,J=7.4Hz,3H). 13 C NMR (101MHz,Deuterium Oxide)δ160.57,157.34,156.83,138.58,130.41,129.33,129.21,128.93,126.92,70.35,70 .26,68.42,63.78,63.66,61.72,55.70,43.79,42.09,40.74,31.15,20.10,13.77.LCMS(M+H + ):597.3.

[0107] Example 4: Synthesis of Compound 6

[0108] Compound 6 (350 mg, 1.0 mmol) was dissolved in 5 mL of dry DMF, and intermediate 2 (473 mg, 1.1 mmol) and cesium carbonate (750 mg, 2.0 mmol) were added. The reaction solution was stirred at 75° C. for 12 hours, filtered, concentrated, and extracted with DCM / H2O. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography to obtain yellow solid compound 7 with a yield of 80%. The reaction formula is:

[0109]

[0110] 1H NMR(400MHz,Chloroform-d)δ7.81(d,J=8.6Hz,1H),7.52(t,J=5.8Hz,1H),7.42(d d,J=14.8,8.0Hz,3H),7.16(d,J=8.0Hz,2H),6.02(s,2H),4.31–3.75(m,4H),3.95– 3.40(m,8H),3.27–3.18(m,4H),3.01(t,J=6.0Hz,2H),2.41–2.12(m,4H),1.87–1.7 0(m,2H),1.49-1.37(M,2H),1.36(t,J=7.2Hz,6H),1.00(t,J=7.4Hz,3H).LCMS(M+H + ):626.4.

[0111] Example 4: Synthesis of Compound 8

[0112] Compound 7 (315 mg, 0.5 mmol) was dissolved in 5 mL of dry dichloromethane, TMSBr (398 μL, 3 mmol) was added in an ice bath, and the mixture was heated to room temperature and stirred for 1 hour, then filtered and concentrated. White solid 8 was obtained by reverse phase HPLC with a yield of 50%. The reaction formula is:

[0113]

[0114] 1 H NMR(400MHz, Deuterium Oxide)δ7.50(d,J=8.6Hz,1H),7.35(d,J=8.6Hz,1H),7.33–7.24(m,4H),3.83(s,2H),4.42–3.95(m,6H),3.85–3.6 0(m,6H),3.56(t,J=7.2Hz,2H),3.27–3.18(m,4H),1.75–1.62(m,2H),1.44(m,2H),0.99(t,J=7.4Hz,3H).LCMS(M+H + ):498.3.

[0115] Example 5: EC of small molecule agonists 50 Determination

[0116] Reagent: HEK-Blue TM hTLR7、HEK-Blue TMReagents required for hTLR8 and control HEK-Blue Null2k cell culture and detection: DMEM (4.5g / l glucose), bovine serum FBS, streptomycin (50μg / ml), penicillin (50U / ml) Blasticidin (10mg / ml), ZeocinTM (10mg / ml), Normocin (50mg / ml), HEK-BlueTMDetection. Basic culture medium: DMEM + 10% FBS + streptomycin + penicillin + Normocin (100μg / ml); Selective culture medium for resistance genes: DMEM + 10% FBS + streptomycin + penicillin + Normocin (100μg / ml) + Blasticidin (100μg / ml) and Zeocin TM (100 μg / ml).

[0117] Cell preparation: HEK-Blue TM hTLR7、HEK-Blue TM hTLR8 cell line was purchased from Invivogen. Take out the cells frozen in liquid nitrogen, quickly put them into a 37℃ water bath, shake them from time to time, and completely thaw them within 1 minute; transfer the cells to 15mL preheated basal culture medium and resuspend them; centrifuge at 1000r / min for 5min, discard the supernatant; resuspend in 1ml basal culture medium, transfer to a T25 culture flask, add 5ml culture medium, and culture in a 37℃ incubator; after two stable passages, add selective antibiotics for screening: HEK-Blue TM hTLR7 or HEK-Blue TM hTLR8: 100 μg / ml Zeocin+Blasticidin (30 μg / ml), HEK-Blue Null2-k: 100 μg / ml Zeocin; change the medium three times a week; when the cell volume reaches 70%-80%, add PBS and gently blow to make the cells fall off.

[0118] Activity detection steps: (1) Add 20 μL of the sample to be tested to each well of a 96-well plate (set up three replicates); (2) Add 20 μL of positive control (such as R848); Add 20 μL of negative control (such as ddH2O); (3) Remove the T75 culture flask from the incubator, discard the culture medium, add 5-10 mL of preheated PBS, and gently blow the cells; add 2-5 mL of PBS to the culture flask and return it to the incubator for 1-2 minutes, gently blow to scatter the cells; (4) Mix well, count the cells, and do not centrifuge. Use HEK-Blue TMResuspend the cells in the test solution to adjust the number of cells. Do not incubate for too long to avoid too dark background or false positives. The number of HEK-Blue hTLR cells is about 2.2×10 5 / mL, 180 μL (about 40,000 cells) was added to each well, and the amount of HEK-Blue Null2-k cells was about 2.8×10 5 / ml, 180 μL per well (about 50,000 cells); (5) incubate at 37°C for 6-16 h, and detect SEAP readings at 620-655 nm.

[0119] Effect % = (mean OD of drug group at each concentration - mean OD of H2O group) / (OD of positive drug group max Average value-average value of OD in H2O group)×100. GraphPad Prism5 software was used to fit the Lg [concentration]-effect curve to calculate the EC 50 .

[0120] Experimental results: Concentration for 50% of maximal effect (EC 50 ) refers to the concentration of small molecules that can cause 50% of the maximum agonist effect. The experimental results show that the half-maximal effect concentration of the positive control R848 is hTLR7 (75nM) and hTLR8 (480nM), while the agonist activity of the TLR agonist numbered N1 of the present invention is hTLR7 = (1026nM) and hTLR8 = (640nM), and the agonist activity of N2 is hTLR7 = (980nM) and hTLR8 = (750nM). EC 50 The smaller the value, the better the effect. In the table, the unit of agonist activity is nanomolar unless otherwise specified. Compounds N1 and N2 containing phosphate groups have good biological activity. The phosphate group can be coupled with aluminum adjuvant to serve as a vaccine adjuvant combination with increased potency.

[0121] Compound No. <![CDATA[hTLR7(EC 50 )]]> <![CDATA[hTLR8(EC 50 )]]> R848 (positive control) 75nM 480nM N1 1026nM 640nM N2 980nM 750nM

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0123] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0124] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A phosphoric acid derivative or a salt thereof based on pyridin[3,2-d]pyrimidin-2-amine, wherein the phosphoric acid derivative has the following structure: in, R1, R2 are independently selected from H or methyl, and R3 is L1 is selected from -OCH2CH2-, -CH2CH2CH2- and -OCH2CH2O-; L2 is absent or selected from -NR6-, -(CH2) m - and -NR6(CH2) m -; R6 is H or a C1-C3 alkyl group; R4 and R5 are independently selected from H, F or C1-C3 alkyl; m is 1, 2, 3, 4 or 5; n is 1, 2, 3, 4 or 5; L is selected from -CH2-, -NH- or -O-; X is selected from -CH2-, -NH- or -O-.

2. The pyridin[3,2-d]pyrimidin-2-amine-based phosphoric acid derivative or a salt thereof according to claim 1, wherein R4 and R5 are H at the same time, or R4 and R5 are F at the same time.

3. The pyridin[3,2-d]pyrimidin-2-amine-based phosphoric acid derivative or a salt thereof according to claim 1, wherein A group having the following structure:

4. A phosphoric acid derivative based on pyridin[3,2-d]pyrimidin-2-amine or a salt thereof, wherein: The phosphoric acid derivative based on pyridine [3,2-d] pyrimidine-2-amine has the following structure: or 5. Use of the pyridinium [3,2-d] pyrimidin-2-amine-based phosphoric acid derivative or its salt as claimed in any one of claims 1 to 4 as a vaccine adjuvant.

6. A pharmaceutical composition comprising the pyridinium [3, 2-d] pyrimidin-2-amine-based phosphoric acid derivative or a salt thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable excipient.

7. A vaccine comprising an antigenic vaccine component or an anti-tumor vaccine component against infections, viruses and parasitic diseases, and a phosphoric acid derivative or a salt thereof based on pyridinium [3,2-d] pyrimidine-2-amine according to any one of claims 1 to 4.

8. Use of the pyridinium [3, 2-d] pyrimidin-2-amine-based phosphoric acid derivative or its salt according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 6 in the preparation of a drug for enhancing a positive immune response of an organism.

9. Use of the pyridinium [3, 2-d] pyrimidin-2-amine-based phosphoric acid derivative or its salt according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 6 in the preparation of a drug for enhancing the effect of chemotherapy.

10. Use of a pyridinium [3,2-d] pyrimidine-2-amine-based phosphoric acid derivative or a salt thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 6 in the preparation of a medicament for treating or preventing a mammalian disease; the disease is a disease or condition associated with TLR7 and 8 activity.

11. The use according to claim 10, wherein the disease is selected from infectious diseases, immune diseases, viral infections or tumors.

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