CN115611945A - Water-soluble phosphoric acid derivative, preparation method thereof, pharmaceutical composition and application - Google Patents

Water-soluble phosphoric acid derivative, preparation method thereof, pharmaceutical composition and application Download PDF

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CN115611945A
CN115611945A CN202210816438.1A CN202210816438A CN115611945A CN 115611945 A CN115611945 A CN 115611945A CN 202210816438 A CN202210816438 A CN 202210816438A CN 115611945 A CN115611945 A CN 115611945A
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cancer
pyridine
phosphoric acid
amine
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廖学斌
王志松
张岩
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Beijing Yufan Biotechnology Co ltd
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    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
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Abstract

The invention relates to a novel compound with TLR7/8 agonistic activity, in particular to a phosphate derivative based on pyridine [3,2-d ] pyrimidine-2-amine, and an isomer, a prodrug, an isotope derivative, a solvate, an ester or a salt thereof, and application thereof in preparing vaccines, preventing and/or treating virus infection and tumors.

Description

Water-soluble phosphoric acid derivative, preparation method thereof, pharmaceutical composition and application
Technical Field
The invention belongs to the field of biological medicines, and relates to a special phosphate derivative based on pyridine [3,2-d ] pyrimidine-2-amine, a preparation method and a pharmaceutical composition thereof, wherein a vaccine adjuvant effect is generated by activating Toll-like receptor agonists (TLRs) TLR7 and TLR8, so that the phosphate derivative can be used for preparing a vaccine and preventing and/or treating virus infection and tumors in an animal or human body.
Background
Vaccines are a historical breakthrough in human medical history, and effective vaccine systems often require highly effective natural immune system-activating components, known as vaccine adjuvants (adjuvants), which are capable of non-specifically altering or enhancing antigen-specific immune responses, in addition to protective antigens. Currently known adjuvants typically include aluminum salt adjuvants, small molecule polypeptides or glycoproteins, nucleic acids, and the like. Among them, ligand molecules of Toll-like receptors have been greatly developed as immunoadjuvants in clinical or preclinical trials.
Toll-like receptors (TLR 1, TLR2, TLR 3-TLR 13) are important receptors for specifically recognizing pathogen-associated molecular patterns (PAMP), play important roles in both innate immunity and adaptive immunity and are important bridges for connecting innate immunity and adaptive immunity. These receptors are widely expressed on immune and epithelial cells. Toll-like receptor activation can activate NF-kB through a myeloid differentiation protein 88 (MyD 88) H or TRIF dependent signal pathway, further induce the production of cytokines and chemokines, promote the maturation of DC cells and the expression of co-stimulation ligands on the surfaces of T cells, and enhance innate and adaptive immune responses. Toll-like receptor agonists are therefore a class of vaccine adjuvants of great value.
TLRs agonists have been successful as vaccine adjuvants in the prevention and treatment of a variety of diseases, including infectious diseases, tumors, influenza, and the like. Live mycobacterium Bovis (BCG) vaccines are currently approved for the prevention of tuberculosis. The major components of BCG are the bacterial cell wall skeleton and peptidoglycan, and studies have shown that these components are dependent on activation of TLR2 and 4 signaling pathways to exert immunopotentiating effects. The TLR4 ligand MPLA can strongly cause the generation of IgG2a Abs and induce TH-1 immune response, and is a TLR ligand vaccine adjuvant which is widely used at present. Adsorption of MPLA to aluminium salt formulations in combination with hepatitis B vaccine (Fendrix) was approved by the European Union to be marketed as early as 2005. The Dan h bouuch topic group distribution in 2016 and 2018 reports TLR7 activation as an important strategy for effective combination therapy for aids in Nature, and was validated in monkeys (Nature, 540,284-287, nature,563, 360-364). Compared with a negative control group, the combination of the vaccine Ad26/MVA and the adjuvant TLR7 agonist GS-986 can reduce the median of the viral load by 1.74log and prolong the virus rebound time after ART termination by 2.5 times.
TLRs agonists, although proven to have significant vaccine adjuvant effects, have resulted in limited progress in the development of most drugs directed against TLRs targets due to side effects resulting from insufficient potency or uncontrolled cytokine release. Taking TLR7 and 8 as examples, the TLR7/8 agonist, resiquimod, showed good clinical efficacy, but clinical trials have been terminated due to adverse side effects (NCT 01676831). In phase Ib clinical trials, the TLR8 agonist VTX-2337 was shown to have the ability to enhance cetuximab antibody-dependent cellular cytotoxicity (ADCC), but no favorable clinical progress has yet been reported. These compounds are very difficult to use systemically due to poor efficacy or toxic side effects (e.g., lymphopenia, influenza-like symptoms, etc.) (AIDS res, 30, 457-467). In order to find an effective and safe TLR7/8 activation protocol and to increase the possibility of their combined administration with vaccines, it is essential to develop novel vaccine adjuvants. The induction of protective Th-1 type immune responses based on TLRs activation is a promising development field in vaccine design. The patent designs novel ligand agonists around TLR7/8, and compared with other Toll-like receptor targets, the targets can be effectively activated by small-molecule ligands, so that the novel ligand agonists have great advantages for drug development.
Disclosure of Invention
The invention provides a special phosphate derivative based on pyridine [3,2-d ] pyrimidine-2-amine, which has good water solubility, is convenient for a pharmaceutical preparation or is chelated with other drugs to form a compound, and can be used for preparing vaccines and preventing and/or treating virus infection and tumors in animals or human bodies by activating Toll-like receptor agonists (TLRs) TLR7 and TLR8 to generate a vaccine adjuvant effect.
The invention provides a pyridine [3,2-d ] pyrimidine-2-amine-based phosphoric acid derivative, and an isomer, a prodrug, an isotope derivative, a solvate, an ester and a salt thereof, wherein the structural formula of the derivative is shown as the general formula (I):
Figure BDA0003742604580000021
wherein R is 1 ,R 2 ,R 3 Independently selected from H, F, cl, br, I, substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl;
and, R 1 ,R 2 ,R 3 At least one of them is:
Figure BDA0003742604580000022
wherein L is 1 Is selected from-OCH 2 CH 2 -、-CH 2 CH 2 CH 2 -and-OCH 2 CH 2 O-；
L 2 Is absent or selected from-NR 6 -、-(CH 2 ) m -and-NR 6 (CH 2 ) m -；
Y is- (CH) 2 ) t -；
R 4 And R 5 Independently selected from H, F, cl, br, I, substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl;
m is an integer selected from 1 to 10;
n is selected from an integer between 1 and 10;
l is selected from- (CR) 7 R 8 ) p -、-NR 9 -、-O-、-S-；
X is selected from- (CR) 10 R 11 ) q -、-NR 12 -、-O-；
R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 Independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl;
R 14 selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; p is selected from an integer between 1 and 10;
q is an integer selected from 1 to 10;
t is selected from an integer between 1 and 10.
Preferably, R 1 ,R 2 ,R 3 Independently selected from H or C1-C3 alkyl, and at least one is:
Figure BDA0003742604580000031
more preferably, R 1 ,R 2 ,R 3 Independently selected from H or methyl, and at least one is:
Figure BDA0003742604580000032
more preferably, R 1 ,R 2 Independently selected from H or methyl, and R 3 Is composed of
Figure BDA0003742604580000033
Preferably, R 4 ，R 5 Independently selected from H, F or C1-C3 alkyl.
More preferably, R 4 And R 5 While being H, or R 4 And R 5 And is also F.
Preferably, R 6 Is H or C1-C3 alkyl, more preferably, R 6 Is H or methyl.
Preferably, m is 1,2,3, 4 or 5.
Preferably, n is 1,2,3, 4 or 5.
In a preferred embodiment of the method of the invention,
Figure BDA0003742604580000034
in particular a group of the structure:
Figure BDA0003742604580000035
preferably, R 13 Is H or C1-C3 alkyl.
Preferably, R 14 Is C1-C3 alkyl, more preferably, R 14 Is methyl.
Preferably, t is 1,2,3, 4 or 5.
Preferably, the pyridine [3,2-d ] pyrimidine-2-amine-based phosphoric acid derivatives provided by the invention have the following structural formula:
Figure BDA0003742604580000041
preferably, R 7 、R 8 、R 9 Independently selected from H and C1-C3 alkyl.
Preferably, R 10 、R 11 、R 12 Independently selected from H and C1-C3 alkyl.
p is 1,2,3, 4 or 5;
q is 1,2,3, 4 or 5.
Preferably, L is selected from- (CH) 2 )-、
Figure BDA0003742604580000042
(comprises
Figure BDA0003742604580000043
) -NH-or-O-;
preferably, X is selected from-CH 2 -, -NH-or-O-.
The present invention also provides pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivatives having the following structural formula N1 or N2 and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof:
Figure BDA0003742604580000044
the present invention also provides a method for preparing a pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivative having the structural formula of N1, comprising:
(1) Taking (bromodifluoromethyl) diethyl phosphonate and 1, 2-bis (2-iodoethoxy) ethane as starting materials, and preparing a compound 2 under the action of lithium diisopropylamide, wherein the reaction formula is as follows:
Figure BDA0003742604580000045
(2) Dissolving the compound 2 in acetonitrile, adding the intermediate 3 and potassium carbonate, and heating to obtain a compound 4, wherein the reaction formula is as follows:
Figure BDA0003742604580000051
(3) Dissolving the compound 4 in dry dichloromethane, and hydrolyzing under the action of TMSBr (trimethyl bromosilane) to obtain a compound 5, wherein the reaction formula is as follows:
Figure BDA0003742604580000052
the present invention also provides a method for preparing a pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivative having the structural formula of N2, comprising:
(1) Dissolving the compound 6 in dry DMF, sequentially adding cesium carbonate and the intermediate 2, and reacting at 60 ℃ for 5 hours to obtain a compound 7, wherein the reaction formula is as follows:
Figure BDA0003742604580000053
(2) Dissolving the compound 7 in dry dichloromethane, sequentially adding TMSBr, reacting at room temperature for 2 hours to obtain a compound 8, wherein the reaction formula is as follows:
Figure BDA0003742604580000054
the preparation method of other phosphate derivatives based on pyridine [3,2-d ] pyrimidine-2-amine shown in the general formula (I) can be prepared by adopting the preparation method of the phosphate derivatives of pyridine [3,2-d ] pyrimidine-2-amine with the structural formula of N1 or N2 through routine adjustment in the field.
In a further aspect, the invention provides the use of said pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivatives and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof as vaccine adjuvants.
The invention also provides application of the pyridine [3,2-d ] pyrimidine-2-amine-based phosphoric acid derivative and an isomer, a prodrug, an isotope derivative, a solvate, an ester or a salt thereof in preparation of vaccines, wherein the pyridine [3,2-d ] pyrimidine-2-amine-based phosphoric acid derivative and the isomer, the prodrug, the isotope derivative, the solvate, the ester or the salt thereof are used as vaccine adjuvants.
In another aspect, the present invention provides a pharmaceutical composition comprising the pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivative of the present invention and its isomers, prodrugs, isotopic derivatives, solvates, esters, salts, and pharmaceutically acceptable excipients.
The pharmaceutically acceptable auxiliary materials of the 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.
The pharmaceutical composition can be prepared into tablets, pills, capsules, powder, granules, paste, emulsion, suspending agents, solutions, suppositories, injections, inhalants and gels.
Routes of administration of the pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivatives of the present invention and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, transmucosal, topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intramuscular, subcutaneous, intravenous administration.
The compositions of the invention also contain at least one additional therapeutic agent selected from the group consisting of chemotherapeutic agents, immunotherapies, 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 based on expression from recombinant viral vectors, and immunologically active fragments.
The invention also provides a vaccine comprising antigenic or anti-tumor vaccine components against infections, viruses and parasitic diseases, and phosphoric acid derivatives based on pyridine [3,2-d ] pyrimidin-2-amine according to the invention and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof.
The antigenic vaccine components of the invention against infections, viruses and parasitic diseases are for example one or more of inactivated pathogens, pathogen surface antigens, mRNA or DNA encoding pathogen surface antigens.
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 based on expression from recombinant viral vectors.
The vaccine of the invention can be a human vaccine or a veterinary vaccine.
The human vaccine is selected from a vaccine selected from a tumor vaccine, a diphtheria vaccine, a pertussis vaccine, a tetanus vaccine, a polio vaccine, a hepatitis a vaccine, a hepatitis B vaccine, a rabies vaccine, a measles vaccine, a rubella vaccine, an influenza vaccine, a mumps vaccine, a varicella vaccine, a rotavirus vaccine, a smallpox vaccine, a yellow fever vaccine, a mite-type encephalitis vaccine, a B-type haemophilus influenzae conjugate vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a pneumococcal vaccine, a coronavirus vaccine (including a novel coronavirus COVID-19 vaccine), a norovirus vaccine, an ebola virus vaccine, a dengue virus vaccine, a Zika virus vaccine, a coxsackie virus vaccine and a meningitis vaccine caused by neisseria meningitidis.
The tumor vaccine of the invention can be used for treating 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, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, malignant carcinoid cancer, bladder cancer, skin precancerous lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignant tumor, cervical cancer, endometrial cancer, glioblastoma, and adrenal cortex cancer.
The veterinary vaccine comprises a poultry vaccine, a pig vaccine or a cattle vaccine. The poultry vaccine includes but is not limited to avian influenza vaccine, newcastle disease inactivated vaccine, egg drop syndrome inactivated vaccine or combined vaccine of the vaccines. The vaccine for the pig comprises a foot-and-mouth disease inactivated vaccine, a porcine circovirus vaccine, a porcine pseudorabies inactivated vaccine, a streptococcus suis bivalent inactivated vaccine, a haemophilus parasuis inactivated vaccine, a porcine reproductive and respiratory syndrome inactivated vaccine, a porcine mycoplasma inactivated vaccine or a combined vaccine of the vaccines. The bovine vaccine comprises a bovine foot-and-mouth disease inactivated vaccine, a bovine salmonella inactivated vaccine or a combined vaccine of the vaccines.
Antibodies of the invention include, but are not limited to: PRO542, which is an anti-hiv gp120 antibody fused to CD4 (Progenics/gen zymetransgenics); MDX-010 (Medarex, md., midlik, N.J., which is a humanized anti-CTLA-4 antibody; SYNAGIS (Medmanne, md.), which is a humanized anti-Respiratory Syncytial Virus (RSV) monoclonal antibody; HERCEPTIN (trastuzumab) (Gentatak, calif.) which is a humanized anti-HER 2 monoclonal antibody; humanized anti-CD 18F (ab ') 2 (Genentech.); CDP860 (humanized anti-CD 18F (ab') 2 (Genentech.)); ostavir, which is a human anti-hepatitis B virus antibody (protein design laboratory/Noval corporation) (ProteingelnDellab/Novartis)); PROTOTOVIRTM (human anti-humanized OSPG 1 antibody) (protein design laboratory/Noval), GAMAK-195 (Biokura, inc.); igG) (human anti-CTLA-IgG) (Marek, inc.; protex GmbH, inc.; protovorax, novax, inc.); protov., it is a chimeric anti-egfr igg antibody (immune clone systems); VITAXIN (VITAXIN) TM, which is a humanized anti- α V β 3 integrin antibody (applied molecular evolution/medical immunology corporation); karpas (Campath) 1H/LDP-03, a humanized anti-CD 52IgG1 antibody (Liukesait, leukosite); smartM195, a humanized anti-CD 33IgG antibody (protein design laboratory (ProteinDesignLab)/Carnbo (Kanebo)); RITUXANTM (rituximab TM), which is a chimeric anti-CD 20IgG1 antibody (IDEC pharmaceutical (IDEC pharm)/genetag, roche/jack (Zettyaku)); LYMPHOCIDETM, which is a humanized anti-CD 22IgG antibody (immunology corporation)); smartID10, which is a humanized anti-HLA antibody (protein design laboratory); oncolmtm (Lym-1), a radiolabeled murine anti-HLADR antibody (Techniclone); ABX-IL8, which is a human anti-IL 8 antibody (Anronex (Abgenix)); anti-CD 11a, a humanized IgG1 antibody (gene tag/thauma (Xoma)); ICM3, a humanized anti-ICAM 3 antibody (ICOS pharmaceuticals); IDEC-114, a primatized anti-CD 80 antibody (IDEC pharmaceuticals/Mitsubishi); ZEVALINTM, a radiolabeled murine anti-CD 20 antibody (IDEC/pioneer AG); IDEC-131, a humanized anti-CD 40L antibody (IDEC/Ware corporation (Eisai)); IDEC-151, a primatized anti-CD 4 antibody (IDEC); IDEC-152, a primatized anti-CD 23 antibody (Seikagaku, IDEC/SKK); SMART anti-CD 3, a humanized anti-CD 3IgG (protein design laboratory); 5G1.1, a humanized anti-complement factor 5 (C5) antibody (AlexionPharm); D2E7, which is a humanized anti-TNF- α antibody (CAT/Pasteur); CDP870, a humanized anti-TNF- α Fab fragment (selttack); IDEC-151, a primatized anti-CD 4IgG1 antibody (IDEC pharmaceuticals/Shikebichen (SmithKlineBeecham)); MDX-CD4, a human anti-CD 4IgG antibody (Medax/Wei/Gimby (Genmab)); CDP571, a humanized anti-TNF-. Alpha.IgG 4 antibody (Seltark, inc.); LDP-02, a humanized anti- α 4 β 7 antibody (Liuxett/Genetik); ortho cloneokt4A, a humanized anti-CD 4IgG antibody (OrthoBiotech); ANTOVATM, a humanized anti-CD 40LIgG antibody (Bayer Gene (Biogen)); ANTEGRENTM, a humanized anti-VLA-4 IgG antibody (Elan, iland); MDX-33, which is a human anti-CD 64 (Fc γ R) antibody (McDelex/Berlin (Centeon)); SCH55700, a humanized anti-IL-5 IgG4 antibody (Sertole/Xianling); SB-240563 and SB-240683, humanized anti-IL-5 and anti-IL-4 antibodies (SmithKlineBeecham), respectively); rhuMab-E25, a humanized anti-IgEIgG 1 antibody (genetat/noralis/tanohio biosystems) (TanoxBiosystems); ABX-CBL, a murine anti-CD 147IgM antibody (Angen Nix (Abgenix)); BTI-322, a rat anti-CD 2IgG antibody (BioTransplant, immunol/Biotransplant); ondolone/OKT 3 (Orthoclone/OKT 3), a murine anti-CD 3IgG2a antibody (orthobiostech); SIMULECTTM, a chimeric anti-CD 25IgG1 antibody (Norwa pharmaceutical); LDP-01, a humanized anti-beta 2-integrin IgG antibody (Liuxett Corp.); anti-LFA-1, murine anti-CD 18F (ab') 2 (PM (Pasteur-Merieux)/Immunotech); CAT-152, a human anti-TGF-. Beta.2 antibody (Cambridge antibody technology Inc. (Cambridge AbTech)); corsevin, a chimeric anti-factor VII antibody (santoch); MDX-1106, PD-1 antibody (bristol-myerssquibb); MDX-1105 is PDL1 antibody (Roche).
The anticancer agents described in the present invention include, but are not limited to: <xnotran> , , , , , , , , , , , , , , , , , , , , , , , , , , , , C, , , , , , , , , , , , , , , , , D, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , A , , , , , , , , , , , , , II ( II rIL 2), α 2a, </xnotran> <xnotran> α -2b, α -n1, α -n3, β -Ia, γ -Ib, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , </xnotran> Uramustine, urelpide, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinrosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorazole, zeniplatin, setastatin, zorubicin hydrochloride. Other anti-cancer agents that can be used include, but are not limited to: 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adenosylpentanol (adecanenol), aldeoxin, aldesleukin, ALL-TK antagonist, altretamine, amtemustine, 2,4 dichlorophenoxyacetic acid (amidox), amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, enriches, anti-dorsal chemoattractant protein 1, antiandrogen, prostate cancer, antiestrogens, antineoplastic ketones (antineoplaston), antisense oligonucleotides, glycine aphidicolin, apoptotic gene modulators, apoptosis modulators, depurination nucleic acids, ara-CDP-DL-PTBA, arginine deaminase, anilidine (asularnine), atamestane, amoxicillin, axistatin (axinstatin) 1, axistatin 2, axistatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivatives, palanol (balanol), batimastat, BCR/ABL antagonists, antisense oligonucleotides, glycine aphidicolin, apoptosis gene modulators, apurinic nucleic acids, ara-CDP-DL-PTBA, arginine deaminase, aniline acridide (asulamine), atamestane, amoxicillin, baccatin (baccatin), baccatin (balanol), batimastat, BCR/ABL antagonists, and the like benzo chlorin (benzodioxin), benzoyl staurosporine, beta lactam derivatives, beta-alexin (beta-alethine), clarithromycin (betacanthacin) B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, diazirine spermine, bisnefad, bistratene A, bizelesin, bevacride, bripirimidine, brookite, buthionine, calcipotriol, carboflavin C, camptothecin derivatives, canarypox IL2, capecitabine, carboxamide-amino-triazole, and pharmaceutically acceptable salts thereof, <xnotran> , caRestM3, CARN700, , , (ICOS), , B, , (chlorlns), , , - , , , , (collismycin) A, B, A4, , (conagenin), (crambescidin) 816, , (cryptophycin) 8, A , (curacin) A, (cyclopentanthraquinone), (cycloplatam), (cypemycin), , , (cytostatin), , , B, , , , , , , B, 3,4- (didox), , 5- , ,9-, (dioxamycin), , , , , , , , SA, , , , , , , , , , , , , , , </xnotran> Exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavudine (flavopiridol), fludrostine, fulululone (flusterone), fludarabine, fluorodaunorubicin hydrochloride (fluodanuroninin), fophenmetin, formestane, forskocin, fotemustine, gadolinium desporphyrin (gadolinium xanthophyrin), gallium nitrate, galotatabine, ganirelix, gelatinase inhibitor, gemcitabine, glutathione inhibitor, heusufam (hepsulfam), heregulin, cyclohexylbisacetamide, hypericum anthrone, ibandronic acid, flavobilin, idoxifene, idodronone, imofovir, ilomastat, imidazoloacridone, quimod, immunostimulatory factor 1 receptor inhibitor, kinin-like growth factor 1 receptor inhibitor, interferon agonist, interferon, interleukin iobenguanide, ioxygrim, 4-sweetpotato picrol, iloprazine, isosorafenib, isobrewagazole (isobengazole), isodigoxigenin (isohomohalichondrin) B, itasetron, desmediphamide (jasplaminolide), cahalalide (kahalalide) F, lamellarin (lamellarin) -NTAC, lanreotide, renomycin (leinamycin), leguminoxane, lentinan sulfate, letostratin (leptin), letrozole, leukemia inhibitory factor, leukocyte interferon-alpha, leuprorelin + estrogen + progesterone, leuprorelin, levamisole, liazole, a linear polyamine analog, a lipophilic glycopeptide, a lipophilic platinum compound, lisopronamide (lissilonamide) 7, lobaplatin, ethylphosphoserine, lomethamine, lonidamine, lovastatin, lotadine, loporin, loporixatin, loporine, lobin, solithromycin, solithrone, solithrotecan, solithromycin, solithrone, solithromycin, solithrob, lutetium deuteroporphyrin (lutetium texaphyrin), lexophyrine (lysofylline), lytic peptides, maytansine, minostatin (manostatin) a, marimastat, maxol, mammary serine protease inhibitor (maspin), matrix-dissolving factor inhibitor, matrix metalloproteinase inhibitor, methonuril, thiobartolidine, mettirelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirisperidone, mitoguazone, dibromodulcitol, mitomycin analogs, mitonaphthylamine, mitotoxin (mitotoxin) fibroblast growth factor-saporin, mitoxantrone, mofastine, morastine, monoclonal antibodies, human chorionic gonadotropin, monophosphoryl lipid a + mycobacterial cell wall matrix, mopidanol, multidrug resistance gene inhibitors, multiple tumor suppressor 1-based therapies, mustard anti-cancer agents, indian sponge (mycaperoxide) B, mycobacterial cell wall extract, miyapirone (myriapionone), N-acetyldinaline, N-substituted benzamides, nafarelin, naretipen (nagrinip), naloxone + analgesia, naphazin (napavin), napthalene glycol (naperpin), natostim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin (nisamycin), nitric oxide modulators, nitroxide antioxidants, nidocin (nitrulyn), O6-benzylguanine, octreotide, oxycodone (okicenone), oligonucelolone, ondansetron, dantron, and other drugs, olanexin (oracin), oral cytokine inducers, ormaplatin, oxaliplatin, oxainomycin (oxaauromycin), paclitaxel analogs, paclitaxel derivatives, pamolamine (palaamine), palmitoyl rhizomycin, pamidronic acid, panaxytriol, panomifene, paraferin (paraabacin), paclobutratin, pemetrexed, pedimicin, pentostatin (pentoxazole), perflurane, perfosfamide, perillyl alcohol, phenamycin (phenazinomocin), phenyl acetate (phenacylacetate), phosphatase inhibitors, picolinate (picolinil), pilocarpine hydrochloride, pirarubicin, pirtroxine, placentin (placitin) A, placentin B, plasminogen activator inhibitors, platinum complexes, platinum-triamine complexes, sodium-platinum complexes, prednisolone, prednisone, doxorubin, acridine J2, thromboxane inhibitors, porphinidin C, thrombospondin-based inhibitors, microalgae (microalga), protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, rhodopsin, pyrazoline acridine, pyridoxalated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras-GAP inhibitors, demethylated reteplatin, rhenium (Re 186) etidronate, rhizomycin, ribozymes, RII vecarboxamide (retinamide), roguamine, roxitue base (rohikitune), romotein, roquinacre, lubiginone (rubiginone) B1, lubiginone (Robinone) B1, and Lupinone (Robinitrone), luboxyl, safrano, escitaloprin (saintopin), sarCNU, saxophytol (sarcophytol) A, sargrastim, sdi1 mimetic, semustine, senescence-derived inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single-chain antigen binding protein, sizopyran, sobuconazole, sodium borocaate, sodium phenylacetate, solomon (solvol), growth regulator binding protein, sonamin, fomendonic acid, scamycin (spimycin) D, spiromustine, splenic pentapeptide (splenoptin), spongistatin (spongistatin) 1, squalamine, stem cell inhibitor, inhibitor of stem cell division, stilinamide (stiiamide), matrilysin inhibitor, semustine (supinosine), potent vasoactive intestinal peptide antagonist, sultid (sultina), sultam (sulindac), etc suramin, swainsonine, synthetic mucopolysaccharide, tamoxifen methyl iodide, taurolistin, tazarotene, tegolazine, tegafur, tellurapylammonium, telomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide (tetrachlorecaoxide), tetrazomib (tetrazomine), telipitin (thalistatin), thiocoraline, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotreonam, thyrotropin, puriftin, tirapazamine, titanium dichlorocyclopentadienide, topostin (topstein), toremifene, totipotential factor, translation inhibitor, tretinoin, triacetyluridine, tricitabine, trimetrefristin, triptorelin, trereocline, trereoline, tropisetron, tolteromide, tyrosine kinase inhibitors, tyrphostin (tyrphostin), UBC inhibitors, ubenimex, urogenital sinus derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, varelin (variolin) B, vector systems, erythrogene therapy, veraprodol, veratrmine, walnuts (verdins), verteporfin, vinorelbine, veclosartan (vinxatine), vitaxin (Vitaxin), vorozole, zanoteron, zeniplatin, bendamustine C and neat stastin.
The invention also provides a method for improving the positive immune response of an organism, which comprises the following steps: administering to a system or a subject in need thereof a therapeutically effective amount of the pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative and its isomer, prodrug, isotopic derivative, solvate, ester or salt or the composition of the present invention.
The present invention also provides a method of enhancing the effect of chemotherapy comprising administering to a system or subject in need thereof a therapeutically effective amount of a chemotherapeutic agent, concurrently or subsequently administering a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative and its isomer, prodrug, isotopic derivative, solvate, ester or salt or a composition according to the present invention.
The present invention also provides a method of enhancing immunotherapy comprising: administering to a system or a subject in need thereof a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative of formula (I) and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof, or a composition of the invention, simultaneously or subsequently infusing chimeric antigen receptor T cells (CAR-T) into the system or the subject.
In another aspect of the present invention, there is provided a method of treating or preventing a disease in a mammal, comprising: administering to a system or a subject in need thereof a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative and its isomer, prodrug, isotopic derivative, solvate, ester or salt or a composition according to the present invention.
Preferably, the mammal is a human or a pig.
Preferably, the disease is a disease or disorder associated with the activity of TLRs. More preferably, the disease or disorder associated with the activity of TLRs is a disease or disorder associated with the activity of TLRs 7 and 8, e.g., the disease or disorder associated with the activity of TLRs is selected from an infectious disease, an immune disease, a viral infection, or a tumor.
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, and the like.
Preferably, the viral infection includes, but is not limited to, infectious diseases caused by: ebola virus disease, anthrax disease, condyloma acuminatum, verruca simplex, plantar warts, respiratory Syncytial Virus (RSV), hepatitis b, hepatitis c, dengue virus, herpes simplex virus (e.g., HSV-I, HSV-II, CMV, or VZV), contagious molluscum, cowpox, smallpox, lentivirus, human Immunodeficiency Virus (HIV), human Papilloma Virus (HPV), cytomegalovirus (CMV), varicella Zoster Virus (VZV), rhinovirus, enterovirus, adenovirus, influenza, parainfluenza, mumps virus, measles virus, papova virus, flavivirus, retrovirus, coronavirus (e.g., novel coronavirus 2019-nCoV), arenavirus (e.g., LCM, junin virus, marjoram virus, melon narito virus, and lasalorex), and filovirus (e.g., ebola virus or marburg virus).
More preferably, the viral infection is an infectious disease caused by: human Immunodeficiency Virus (HIV), hepatitis B (HBV), african swine fever virus.
Preferably, the tumor includes but is not limited to: human sarcomas and carcinomas such as fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, wilms' tumor, cervical carcinoma, testicular tumor, lung carcinoma, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloblastic leukemia (myeloblasts, promyelocytes, myelomonocytic, monocytic and erythrocytic leukemias); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (hodgkin's disease and non-hodgkin's disease), multiple myeloma, waldenstrom's macroglobulinemia, and heavy chain disease.
More preferably, the neoplastic disease is preferably: colon cancer, bladder cancer, melanoma, meningioma, lung cancer, or pancreatic cancer.
The term "TLR" as used herein refers to a Toll-like receptor, i.e., a Toll-like receptor.
The term "pharmaceutically acceptable" as used herein refers to materials that are not biologically or otherwise undesirable, e.g., the materials may be incorporated into pharmaceutical compositions administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the compositions in which they are contained. When the term "pharmaceutically acceptable" is used in reference to a pharmaceutical carrier or excipient, it means that the carrier or excipient meets the required standards for toxicological and manufacturing testing or that it is included in the guidelines for inactive ingredients set forth by the U.S. food and drug administration.
The term "combination" or "pharmaceutical combination" as used herein refers to a product obtained by mixing or combining more than 1 active ingredient and comprising fixed and unfixed combinations of active ingredients.
The term "TLR disease" or "disease or disorder associated with TLR activity" as used herein refers to any disease state associated with toll-like receptors.
The room temperature of the invention is 20-30 ℃.
The term "alkyl" as used herein refers to a hydrocarbon chain radical that is straight or branched and free of unsaturation, and is attached 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, t-butyl, n-pentyl, n-hexyl, n-heptyl, and the like. If an alkyl group is substituted with a cycloalkyl group, it is correspondingly "cycloalkylalkyl", such as cyclopropylmethyl. If an alkyl group is substituted with an aryl group, it is correspondingly "aralkyl", such as benzyl, benzhydryl or phenethyl. If an alkyl group is substituted with a heterocyclyl group, it is correspondingly "heterocyclylalkyl".
The term "cycloalkyl" as used herein refers to alicyclic hydrocarbons. Typical cycloalkyl groups contain 1 to 4 single and/or fused rings, 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
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
The starting materials and reagents employed in the present invention are commercially available from commercial suppliers including, but not limited to: aldrich Chemical Company, lancaster Synthesis Ltd. Among them, dimethylformamide, tetrahydrofuran and dioxane were all super-dry solvents from the company welfare and stored in a glove box, dichloromethane and acetone were taken from the solvent purification system, respectively, and all glassware used for water sensitive reactions was first dried in an oven at 100 ℃. The starting materials and reagents used are used as such without further treatment, unless otherwise indicated.
1 H NMR and 13 the C NMR spectrum was determined using a Bruker DRX 400 nuclear magnetic resonance apparatus, and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00 ppm), CDCl was used 3 Or DMSO-d6 as a solvent (or other solvents). Method for 1H NMR: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublets, dt = doublet of triplets. If a coupling constant is provided, it is in Hz. Mass spectra were determined on a Finnigan Advantage type mass spectrometer in an ionization mode ESI. The compound was purified by column chromatography (silica gel GF254:200-400 mesh). The purity of the compound, as determined by HPLC unless otherwise indicated, is referred to as isolated yield.
EXAMPLE 1 Synthesis of Compound 2
Dissolving (bromodifluoromethyl) phosphonic acid diethyl ester (265mg, 1mmol) in 5mL tetrahydrofuran, placing the reaction liquid at-78 ℃, adding a tetrahydrofuran solution (containing 1mmol of LDA) of lithium diisopropylamide, reacting for 30min, dropwise adding a tetrahydrofuran (2 mL) solution of 1, 2-bis (2-iodoethoxy) ethane (400mg, 1.1mmol), storing the reaction liquid at-78 ℃, reacting for 12 h, and slowly adding ice water to quench the reaction after the reaction is finished. Adding hydrochloric acid to adjust the solution to become clear, extracting with ethyl acetate for three times, combining organic layers, spin-drying, and purifying by column chromatography to obtain compound 2 + ) Comprises the following steps: 431.2.
Figure BDA0003742604580000151
example 2 Synthesis of Compound 4
Compound 3 (350mg, 1.0 mmol) was dissolved in 5mL of dry acetonitrile, intermediate 2 (473mg, 1.1mmol) and potassium carbonate (350mg, 2.0 mmol) were added, and the reaction mixture was stirred at 75 ℃ for 12 hours, filtered, concentrated, and concentrated with DCM/H 2 O extraction, organic layer combination, anhydrous sulfuric acidDrying sodium, concentrating, and performing silica gel column chromatography to obtain a yellow solid compound 4, wherein the reaction formula is as follows:
Figure BDA0003742604580000152
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 C NMR(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.
EXAMPLE 3 Synthesis of Compound 5
Compound 24 (326mg, 0.5 mmol) was dissolved in 5mL of dry dichloromethane, and TMSBr (398 μ L,3 mmol) was added low in ice bath, warmed to room temperature and stirred for 1 hour, filtered, concentrated, and prepared on reverse phase by HPLC to give white solid 5 in 45% yield:
Figure BDA0003742604580000161
1 H 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。
example 4 Synthesis of Compound 6
Compound 6 (350mg, 1.0 mmol) was dissolved in 5mL of dry solutionTo the DMF of (5), intermediate 2 (473mg, 1.1mmol) and cesium carbonate (750mg, 2.0mmol) were added, and the reaction mixture was stirred at 75 ℃ for 12 hours, then, filtered, concentrated, and concentrated with DCM/H 2 O extraction, organic layers are combined, dried by anhydrous sodium sulfate, concentrated and chromatographed by a silica gel column to obtain a yellow solid compound 7, the yield is 80 percent, and the reaction formula is as follows:
Figure BDA0003742604580000162
1 H NMR(400MHz,Chloroform-d)δ7.81(d,J＝8.6Hz,1H),7.52(t,J＝5.8Hz,1H),7.42(dd,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),1.87–1.70(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。
example 4 Synthesis of Compound 8
Dissolving compound 7 (315mg, 0.5 mmol) in 5mL dry dichloromethane, adding TMSBr (398. Mu.L, 3 mmol) in low temperature in ice bath, heating to room temperature, stirring for 1 hour, filtering, concentrating, and preparing white solid 8 by HPLC reversed phase with 50% yield:
Figure BDA0003742604580000171
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),3.85–3.60(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。
example 5: EC of small molecule agonists 50 Measurement of
Reagent: HEK-Blue TM hTLR7、HEK-Blue TM hTLR8 and a contrast HEK-Blue Null2k cell culture and detection required reagent: DMEM (4.5 g/l glucose), bovine serum FBS, streptomycin (50. Mu.g/ml), penicillin (50U/ml) Blasticidin (10 mg/ml), zeocin TM (10 mg/ml), normocin (50 mg/ml), HEK-blue (TM) Detection. Basic culture medium: DMEM +10% FBS + streptomycin + penicillin + Normocin (100. Mu.g/ml); selective medium for resistance genes: DMEM +10% FBS + streptomycin + penicillin + Normocin (100. Mu.g/ml) + Blastidin (100. Mu.g/ml) and Zeocin TM (100μg/ml)。
Cell preparation: HEK-Blue TM hTLR7、HEK-Blue TM The hTLR8 cell line was selected from Invivogen. Taking out the cells frozen in the liquid nitrogen, quickly putting the cells into a water bath at 37 ℃, shaking the cells at intervals, and completely thawing the cells within 1 minute; transferring the cells into 15mL of a pre-preheated basal medium for resuspension; centrifuging for 5min at 1000r/min, and removing supernatant; resuspending 1ml of basic culture medium, transferring to a T25 culture flask, supplementing to 5ml of culture medium, and culturing in a 37 ℃ incubator; after two stable passages, selective antibiotic screening is added: HEK-Blue TM hTLR7 or HEK-Blue TM hTLR8: 100. Mu.g/ml Zeocin + Blastidin (30. Mu.g/ml), HEK-Blue Null2-k: zeocin at 100. Mu.g/ml; changing the solution 3 times a week; when the cell amount reaches 70% -80%, PBS is added to gently tap and blow to ensure that the cells are exfoliated.
And (3) activity detection: (1) Adding 20 mu L of sample to be detected in each hole of a 96-hole plate (three multiple holes are arranged); (2) adding 20 μ L of positive control (such as R848); add 20. Mu.L of negative control (e.g.: ddH) 2 O); (3) Removing the T75 culture bottle out of the incubator, removing the culture medium, adding pre-preheated 5-10mL PBS, and slightly blowing cells; adding 2-5mL PBS into the culture bottle, putting the culture bottle back to the incubator for incubation for 1-2min, and gently blowing and beating the culture bottle to scatter cells; and (4) mixing, counting cells and not centrifuging. With HEK-Blue TM The detection solution is used for resuspending and adjusting the cell number, and the incubation time is too long so as to avoid too deep background or false positive. The cell amount of HEK-Blue hTLR is about 2.2X 10 5 Perml, 180. Mu.L (about 40000 cells) per well, and about 2.8X 10 cells per HEK-Blue Null2-k 5 /ml, 180. Mu.L per well (about 50000 cells); (5) Culturing at 37 deg.C in incubator for 6-169, and detecting SEAP reading at 620-655 nm.
Effect% = (mean OD of administration group at each concentration-H) 2 Average value of OD in group O)/(OD in group of positive drugs max Average value-H 2 OD average O group) × 100. Using GraphPadPrism5 software fitting of Lg [ concentration ]]Effect curves, whereby EC is calculated 50 。
The experimental results are as follows: half maximum effect concentration (concentration for 50% of maximum effect 50 ) Refers to the concentration of small molecules that causes 50% of the maximal agonist effect. The experimental results show that: the half maximal effect concentrations of positive control R848 were hTLR7 (75 nM) and hTLR8 (480 nM), whereas the agonistic activity of TLR agonist nos. N1 of the invention was hTLR7= (1026 nM) and hTLR8= (640 nM), and the agonistic activity of N2 was hTLR7= (980 nM) and hTLR8= (750 nM). EC (EC) 50 The response stimulates the potency of TLR7 and TLR8 receptors, and the smaller value indicates the better effect. In the tables, the units of agonist activity are nanomolar unless otherwise indicated. The phosphate group-containing compounds N1 and N2 both have good biological activity, and the phosphate group can be coupled with an aluminum adjuvant so as to be used as a vaccine adjuvant combination with increased potency.
Compound number hTLR7(EC 50 ) hTLR8(EC 50 )
R848 (Positive control) 75nM 480nM
N1 1026nM 640nM
N2 980nM 750nM
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents and the like included within the spirit and scope of the present invention.
The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experiences, and preferences of one of ordinary skill in the art.
The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims (18)

1. Pyridine [3,2-d ] pyrimidine-2-amine based phosphoric acid derivatives of the general formula (I) and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof:
Figure FDA0003742604570000011
wherein R is 1 ,R 2 ,R 3 Independently selected from H, F, cl, br, I, substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl;
and, R 1 ,R 2 ,R 3 At least one of them is:
Figure FDA0003742604570000012
L 1 is selected from-OCH 2 CH 2 -、-CH 2 CH 2 CH 2 -and-OCH 2 CH 2 O-；
L 2 Is absent or selected from-NR 6 -、-(CH 2 ) m -and-NR 6 (CH 2 ) m -；
Y is- (CH) 2 ) t -；
R 4 And R 5 Independently selected from H, F, cl, br, I, substituted or unsubstituted alkyl or substituted or unsubstitutedA cycloalkyl group;
m is an integer selected from 1 to 10;
n is an integer from 1 to 10;
l is selected from- (CR) 7 R 8 ) p -、-NR 9 -、-O-、-S-；
X is selected from- (CR) 10 R 11 ) q -、-NR 12 -、-O-；
R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 Independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl;
R 14 selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl;
p is selected from an integer between 1 and 10;
q is an integer selected from 1 to 10;
t is selected from an integer between 1 and 10.
2. Pyridine [3,2-d ] -based according to claim 1]A phosphoric acid derivative of a pyrimidin-2-amine and its isomer, prodrug, isotopic derivative, solvate, ester or salt, wherein R is 1 ,R 2 ,R 3 Independently selected from H or C1-C3 alkyl, and at least one is:
Figure FDA0003742604570000021
more preferably, R 1 ,R 2 Independently selected from H or methyl, and R 3 Is composed of
Figure FDA0003742604570000022
3. Pyridine [3,2-d ] -based according to claim 1]A phosphoric acid derivative of pyrimidin-2-amine and its isomer, prodrug, isotopic derivative, solvate, ester or salt, wherein R 4 ，R 5 Independently selected from H, F or C1-C3 alkyl.
4. Pyridine [3, 2-d) -based according to claim 1]Phosphoric acid derivatives of pyrimidin-2-amine and isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof, wherein,
Figure FDA0003742604570000023
a group of the following structure:
Figure FDA0003742604570000024
5. pyridine [3, 2-d) -based according to claim 1]A phosphoric acid derivative of a pyrimidin-2-amine and its isomer, prodrug, isotopic derivative, solvate, ester or salt, wherein L is selected from the group consisting of- (CH) 2 )-、
Figure FDA0003742604570000026
-NH-or-O-.
6. Pyridine [3,2-d ] -based according to claim 1]A phosphoric acid derivative of a pyrimidin-2-amine and its isomer, prodrug, isotopic derivative, solvate, ester or salt, wherein X is selected from-CH 2 -, -NH-or-O-, R 14 Is C1-C3 alkyl.
7. The pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivative according to claim 1, wherein the pyridine [3,2-d ] pyrimidin-2-amine-based phosphoric acid derivative has the following structure:
Figure FDA0003742604570000025
Figure FDA0003742604570000031
8. use of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative according to any of claims 1-7 as a vaccine adjuvant, as well as isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof.
9. A pharmaceutical composition comprising a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative according to any one of claims 1 to 7 and its isomers, prodrugs, isotopic derivatives, solvates, esters, salts, and pharmaceutically acceptable excipients.
10. The pharmaceutical composition of claim 9, further comprising at least one additional therapeutic agent selected from the group consisting of chemotherapeutic agents, immunotherapies, 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 based on expression from recombinant viral vectors, and immunologically active fragments.
11. A vaccine comprising an antigenic or anti-tumor vaccine component against infections, viruses and parasitic diseases and a phosphoric acid derivative based on pyridine [3,2-d ] pyrimidin-2-amine according to any one of claims 1 to 7 as well as isomers, prodrugs, isotopic derivatives, solvates, esters or salts thereof.
12. The vaccine of claim 11, wherein the antigenic vaccine component against infections, viruses and parasitic diseases is selected from the group consisting of inactivated pathogens, pathogen surface antigens, mRNA or DNA encoding pathogen surface antigens; the anti-tumor vaccine component is selected from peptides, whole cells, genetically modified whole cells, recombinant proteins or tumor-associated antigens expressed by recombinant viral vectors.
13. The vaccine of claim 11, wherein the vaccine is selected from the group consisting of a human vaccine or a veterinary vaccine; preferably, the human vaccine is selected from the group consisting of a vaccine selected from the group consisting of a tumor vaccine, diphtheria vaccine, pertussis vaccine, tetanus vaccine, polio vaccine, hepatitis a vaccine, hepatitis B vaccine, rabies vaccine, measles vaccine, rubella vaccine, influenza vaccine, mumps vaccine, varicella vaccine, rotavirus vaccine, smallpox vaccine, yellow fever vaccine, mite encephalitis vaccine, haemophilus influenzae type B conjugate vaccine, typhoid vaccine, cholera vaccine, BCG vaccine, pneumococcal vaccine, coronavirus vaccine, norovirus vaccine, ebola virus vaccine, dengue virus vaccine, zika virus vaccine, coxsackie virus vaccine, and a vaccine against meningitis caused by neisseria meningitidis; more preferably, the tumor vaccine is useful for treating melanoma, basal cell carcinoma, large bowel cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, lymphoma, sarcoma, ovarian cancer, kaposi's sarcoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, malignant carcinoid cancer, bladder cancer, skin precancerous lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignant tumor, cervical cancer, endometrial cancer, glioblastoma, and adrenocortical cancer.
14. A method for enhancing a positive immune response in a body, comprising administering to a system or subject in need thereof a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative according to any one of claims 1 to 7, its isomer, prodrug, isotopic derivative, solvate, ester or salt, or a pharmaceutical composition according to claim 9 or 10.
15. A method of potentiating the effects of chemotherapy comprising administering to a system or subject in need thereof a therapeutically effective amount of a chemotherapeutic agent, simultaneously or subsequently administering a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative according to any one of claims 1-7 and its isomer, prodrug, isotopic derivative, solvate, ester or salt thereof, or a pharmaceutical composition according to claim 9 or 10.
16. A method of enhancing immunotherapy comprising administering to a system or a subject in need thereof a therapeutically effective amount of a pyridine [3,2-d ] pyrimidine-2-amine based phosphate derivative, isomer, prodrug, isotopic derivative, solvate, ester or salt thereof according to any one of claims 1-7, or a pharmaceutical composition according to claim 9 or 10, simultaneously or subsequently infusing chimeric antigen receptor T cells (CAR-T) into the system or the subject.
17. A method for treating or preventing a disease in a mammal, comprising administering to a system or a subject in need thereof a therapeutically effective amount of a pyridine [3,2-d ] pyrimidin-2-amine based phosphoric acid derivative according to any one of claims 1 to 7 and its isomer, prodrug, isotopic derivative, solvate, ester or salt, or a pharmaceutical composition according to claim 9 or 10; preferably, the disease is a disease or disorder associated with the activity of TLRs; more preferably, the disease or disorder associated with the activity of TLRs is a disease or disorder associated with the activity of TLRs 7 and 8.
18. A method of treating or preventing a disease in a mammal as claimed in claim 17, wherein the disease or condition associated with the activity of TLRs is selected from the group consisting of infectious diseases, immunological diseases, viral infections and tumours.
CN202210816438.1A 2021-07-12 2022-07-12 Water-soluble phosphoric acid derivative, preparation method thereof, pharmaceutical composition and application Pending CN115611945A (en)

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