CN108069963A - Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition and purposes - Google Patents

Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition and purposes Download PDF

Info

Publication number
CN108069963A
CN108069963A CN201711146690.1A CN201711146690A CN108069963A CN 108069963 A CN108069963 A CN 108069963A CN 201711146690 A CN201711146690 A CN 201711146690A CN 108069963 A CN108069963 A CN 108069963A
Authority
CN
China
Prior art keywords
compound
pharmaceutically acceptable
cell
formula
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711146690.1A
Other languages
Chinese (zh)
Other versions
CN108069963B (en
Inventor
廖学斌
贺磊
王志松
高燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Priority to CN201711146690.1A priority Critical patent/CN108069963B/en
Priority to PCT/CN2017/114611 priority patent/WO2019095455A1/en
Publication of CN108069963A publication Critical patent/CN108069963A/en
Application granted granted Critical
Publication of CN108069963B publication Critical patent/CN108069963B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition and purposes, more particularly to a kind of new TLR agonist compounds and the pharmaceutically acceptable salt or its composition of the compound, the preparation method that is directed to above-mentioned substance and the application in treatment infectious disease, respiratory disease and immunity disease, viral disease or tumour medicine is prepared, there is good clinical value.

Description

Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition and purposes
Technical field
The present invention relates to Toll-like receptor agonist (Toll-like receptors, TLRs) technical field, more particularly to It Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition as TLR7 and/or TLR8 agonists and is used to prepare Treat the application in communicable disease, respiratory disease, immune correlated disease, virosis or tumour medicine.
Background technology
Toll-like receptor is, including at least 13 members, to be found in evolving than a more conservative receptor family in people 10 kinds (TLR1-10), TLRI, TLR2, TLR4, TLR5, TLR6, TLR10 are expressed in cell surface, quickly identify bacterial metabolism Product, TLR3, TLR7, TLR8, TLR9 express in the cell, monitoring and identification mainly to viral nucleic acid, TLR3 identification Double-stranded RNA, and TLR7 and TLR8 identification single stranded RNAs, TLR9 identify unmethylated CG cozymases adjusts DNA of bacteria and some The reaction of virus.
TLRs energy special recognition pathogen associated molecular patterns (PAMP), all play in the innate immunity and acquired immunity Important role is the bridge for connecting the innate immunity and acquired immunity.Wherein, TLR7 combines the single stranded RNA of virus in identification Or after artificial synthesized small molecule purine compound, special adaptor protein can be recruited, a series of signal cascade reaction is activated, opens Dynamic high-caliber systemic adaptive immune response kills the cell of virus infection, so as to thoroughly remove virus.Clinically oneself passes through Start with TLR7 agonist treatment chronic viral infection diseases, such as hepatitis B, hepatitis C.In addition, TLR7 is exciting Agent can induce more rapid effective immunoprotection as influenza vaccines adjuvant.In anti-tumor aspect, TLR7 agonists not only may be used PDC directly to be stimulated to secrete IFN-α, it can also enhance the costimulation of pDC and antigen submission ability, the pDC of activation promotes CD4+T thin The multiplication of born of the same parents further activates CD8+T cells, killing tumor cell, so TLR7 agonists are known as immunologic adjuvant in body Also do not paid attention to the effect in killing neoplastic process gradually.
The content of the invention
The present invention provides Pyridopyrimidine derivatives or its salt, can be used as TLRs receptor stimulating agents, have selectivity The characteristics of good, active high, security is good, while provide Pyridopyrimidine derivatives or the preparation method and its medicine group of its salt Close object and purposes.
Pyridopyrimidine derivatives provided by the present invention or its salt, shown in structural formula such as formula (I)：
Wherein,
L represents linking group, to be not present or being C1-C6Alkylidene or C1-C6Alkenylene, wherein the group Optionally by C1-C4Alkyl substitutes；
R1It representsOr halogen, wherein R3Selected from H, halogen, aminomethylene, (substitution Amino) methylene or its quaternary ammonium salt, the methylene of heterocyclic substituted, nitrine methylene, phosphonic acids methylene, diethyl phosphonate base be sub- Methyl, methoxyl group acyl group, C1-C4Alkyl, C1-C4Alkoxy, CF3-、-COOH、-COOCH3、-CN、HO-CH2；
R2Expression-YR4, wherein Y represent N, S, O, R4Represent C1-C4Alkyl or C1-C4Alkoxy ,-YR4In Y and R4Also Heterocycle can be formed, heterocycle can be morpholine or piperidines；
Substituted-amino can be dimethylamino, Leu- amino, Leu Ala Ala Asn- amino, (trifluoromethyl) methylene Amino, methylamino；
Quaternary ammonium salt is quaternary ammonium salt；
Heterocycle is selected from
Halogen is fluorine, chlorine.
The L is C1-C6Alkylidene is preferably-CH2-。
The R1For
The R2For-NR4。
The compound of the formula (I) is selected from：
The pharmaceutically acceptable salt includes hydrochloride, sulfate, phosphate, carboxylate.
Another aspect of the present invention provides the preparation of the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of formula (I) Method, whereinStep includes：
(1) using -5 Bromopicolinic acid methyl esters of 3- amino and chlorine amitraz hydrochloride as starting material, dimethyl sulfone is added in, is made Compound 1, reaction equation are：
(2) compound 1 is taken to be dissolved in aceticanhydride, obtains compound 2, reaction equation is：
(3) at room temperature, compound 2 is taken to be dissolved in dioxane, DMF is added in, oxalyl chloride is then added dropwise, obtains compound 3, Reaction equation is：
(4) compound 3 obtained by step (3) is directly dissolved in dry tetrahydrofuran, add in organic compounds containing nitrogen or Organic compounds containing sulfur or oxygen-containing organic compound, then add in n,N-diisopropylethylamine, obtain compound 4, and reaction equation is：
(5) compound 4 is taken to be dissolved in methanol, adds in LiOHH2O, is made compound 5, and reaction equation is：
(6) under nitrogen protection, compound 5 is taken to be dissolved in dry tetrahydrofuran, sequentially add substitution benzyl bromine, zinc powder and Ni(PPh3)2Cl2, compound 6 is made, reaction equation is：
The organic compounds containing nitrogen is alkylamine, piperidines, alkoxyamine, preferably morpholine, n-butylamine；Sulfur-bearing organic compound Object is alkyl hydrosulfide, preferably n-butyl mercaptan；Oxygen-containing organic compound is alkylol, preferably n-butanol.
The substitution preferred 3- methyl benzyl bromine of benzyl bromine.
Another aspect of the present invention provides pharmaceutical composition, including：The Pyridopyrimidine derivatives of formula (I) or its pharmaceutically Acceptable salt and pharmaceutically acceptable auxiliary material.
The pharmaceutically acceptable auxiliary material include but not limited to sweetener, diluent, stabilizer, emulsifier, dispersant, Preservative, colorant, flavoring reinforcing agent, surfactant, wetting agent, disintegrant, suspending agent, isotonic agent, solvent.
The pharmaceutical composition of the present invention can prepare piece agent, pill, capsule, pulvis, granule, paste, emulsion, suspension Agent, solution, suppository, injection, inhalant, gelling agent.
Give the Pyridopyrimidine derivatives of the formula (I) of the present invention or its pharmaceutically acceptable salt or its pharmaceutical composition Approach include but not limited to oral, rectum, transmucosal, local, percutaneous, sucking, parenteral, sublingual, intravaginal, intranasal, flesh Interior, subcutaneous, intravenous administration.Dosage is 0.1-0.2mg/kg, preferably 0.1mg/kg, 1 time a day.
Further, the concentration of agonist is 0.1-0.2mg/kg in the composition, and the concentration of preferably agonist is 0.1mg/kg。
For the composition also containing at least one other therapeutic agents, the therapeutic agent is selected from chemotherapeutics, immunization therapy, anti-blood Pipe generating agent, cell factor, hormone, polynucleotides, antibody, immunologic competence segment.
Described preferred, antibody includes but not limited to：
(New Jersey Mai get Simon Rexs company (Medarex) is the anti-CTLA-4 antibody of humanization to MDX-010； SYNAGIS (Maryland State Medimmune Inc. (MedImmune)) is humanization anti respiratory syncytial virus (RSV) Dan Ke Grand antibody；HERCEPTIN (Herceptin) (California Genentech company), is Humanized anti-HER 2 monoclonal Antibody；The anti-CD18F of humanization (ab ') 2 (Genentech company (Genentech))；CDP860 is the anti-CD18F of humanization (ab ') 2 (Sai Er Imtech of Britain (Celltech, UK))；PRO542 is the AntiHIV1 RT activity gp120 antibody merged with CD4 (Pu Luojienike companies/Jian Zan transgenosis company (Progenics/GenzymeTransgenics))；Ostavir is people Anti-hepatitis B virus antibody (Protein Design Labs/Novartis Co., Ltd (ProteinDesignLab/Novartis))； PROTOVIRTM is the anti-CMVIgG1 antibody of humanization (Protein Design Labs/Novartis Co., Ltd)；MAK-195 (SEGARD), It is mouse anti-tnf-alpha F (ab ') 2 (Nore drugmaker of section/BASF AG (KnollPharma/BASF))；IC14 is Anti-CD 14 antibody (ICOS drugmakers (ICOSPharm))；Humanization anti-vegf IgG1 antibody (Genentech company)； OVAREXTM is mouse anti-CA 125 antibody (Ah tower's Thunder God department (Altarex))；PANOREXTM is the anti-17-IA cells table of mouse Face antigen I gG2a antibody (Glaxo Wellcome/mountain Tao Ke (GlaxoWellcome/Centocor))；BEC2 is the anti-uniqueness of mouse Type (GD3 epitopes) IgG antibody (immune clone system house (ImCloneSystem))；IMC-C225 is inosculating antibody EGFRIgG antibody (immune clone system house)；Wei Taxin (VITAXIN) TM is 3 alpha 2 integrin antibodies of humanized anti-alpha V β (applying molecular evolution/Medimmune Inc. (AppliedMolecularEvolution/MedImmune))；Alemtuzumab (Campath) 1H/LDP-03 is humanized anti-CD 52 IgG1 antibody (Liu Ke Saite company (Leukosite))； SmartM195 is Humanized CD 3-resisting 3IgG antibody (Protein Design Labs (ProteinDesignLab)/Kanebo company (Kanebo))；RITUXANTM (Rituximab TM), be inosculating antibody CD20IgG1 antibody (IDEC pharmacy (IDECPharm)/ Genentech, Roche/outstanding person inscribe gram (Zettyaku))；LYMPHOCIDETM is the anti-CD22IgG antibody of humanization (immune doctor Company (Immunomedics))；SmartID10 is humanization Anti-HLA antibodies (Protein Design Labs)； ONCOLYMTM (Lym-1) is the anti-HLADR antibody of radiolabeled mouse (Te Ni cloning companies (Techniclone))； ABX-IL8 is the anti-IL8 antibody of people (peace root Knicks (Abgenix))；Anti- CD11a is that (gene is safe for humanization IgG1 antibody Gram/Xoma Corporation (Xoma))；ICM3 is the anti-ICAM3 antibody of humanization (ICOS drugmakers)；IDEC-114 is primatized Anti-CD80 McAb (IDEC drugmakers/Mitsubishi (Mitsubishi))；ZEVALINTM, it is that radiolabeled mouse is anti- CD20 antibody (IDEC/ Schering Corp (ScheringAG))；IDEC-131 is that (IDEC/ defends material public affairs to humanization anti-CD40L antibodies It takes charge of (Eisai))；IDEC-151 is primatized anti-CD 4 antibodies (IDEC)；IDEC-152 is primatized anti-CD23 antibody (IDEC/SKK companies (Seikagaku))；The anti-CD3 of SMART are Humanized CD 3-resisting IgG (Protein Design Labs)； 5G1.1 is humanization anticomplement factor 5 (C5) antibody (brother Ya Li drugmaker (AlexionPharm))；D2E7 is humanization Anti-TNF-α antibody (CAT/ BASF AG)；CDP870 is humanization anti-tnf-alpha Fab segments (Sai Er Imtech)；IDEC- 151, it is primatized anti-CD4IgG1 antibody (IDEC drugmakers/SmithKline Beecham (SmithKlineBeecham))； MDX-CD4 is the anti-CD4IgG antibody of people (Mai get Simon Rexs company (Medarex)/Wei Cai companies/Ji Mai companies (Genmab))； CDP571 is humanization anti-tnf-alpha IgG4 antibody (Sai Er Imtech)；LDP-02 is 4 β of humanized anti-alpha, 7 antibody (Liu Ke Saite company/Genentech company)；OrthoCloneOKT4A is the anti-CD4IgG antibody (Aoduo Biotechnology Co., Ltd. of humanization (OrthoBiotech))；ANTOVATM is humanization anti-CD 40 L IgG antibody (Bayer genome company (Biogen))； ANTEGRENTM is the anti-VLA-4IgG antibody of humanization (Elan Co., Ltd (Elan))；MDX-33 is that people's anti-CD 64 (Fc γ R) is anti- Body (Mai get Simon Rexs company/Belling company (Centeon))；SCH55700 is anti-IL-5IgG4 antibody (the Sai Er Tykes of humanization Company/Schering Corp)；SB-240563 and SB-240683 is the anti-IL-5 of humanization and anti-IL-4 antibody (Smithkline Beecham's public affairs respectively It takes charge of (SmithKlineBeecham))；RhuMab-E25 is that (Genentech company/Novartis is public for the anti-IgEIgG1 antibody of humanization Take charge of (Norvartis)/Tan Nuo Biosys Corp. (TanoxBiosystems))；ABX-CBL is the anti-CD-147IgM antibody of mouse (An Gen Knicks company (Abgenix))；BTI-322 is that (Medimmune Inc./biological implantation is public for the anti-CD2IgG antibody of rat It takes charge of (BioTransplant))；Polyclonal/the OKT3 of Austria (Orthoclone/OKT3) is that mouse AntiCD3 McAb IgG2a antibody is (difficult to understand mostly biological Scientific ＆ technical corporation (orthoBiotech))；SIMULECTTM is inosculating antibody CD25IgG1 antibody (Novartis)；LDP-01 is The anti-β 2- integrins IgG antibody of humanization (Liu Ke Saite companies)；Anti- LFA-1 is anti-(the PM companies of CD18F (ab ') 2 of mouse (Pasteur-Merieux)/immunological technique company (Immunotech))；CAT-152 is that (Cambridge resists 2 antibody of people's anti-TGF-beta Body technique company (CambridgeAbTech))；CorsevinM is inosculating antibody factor Ⅴ II antibody (Shan Taoke companies)；MDX- 1106, it is PD-1 antibody (Bristol-Myers Squibb Co. (bristol-myerssquibb))；MDX-1105 is PDL1 antibody (sieve Family name company (Roche)).
Above-mentioned antibody is preferably PD-1 antibody, TIM-3 antibody, PD-1 antibody and TIM-3 antibody.
The therapeutic agent can be anticancer agent, and the anticancer agent includes but not limited to：Acivicin, Aclarubicin, hydrochloric acid Ah Examine up to azoles, acronine, Adozelesin, Aldesleukin, hemel, ambomycin, acetic acid Ametantrone, aminoglutethimide, Amsacrine, Anastrozole, Anthramycin, L-Asparaginasum, asperline, azacitidine, Azetepa, azotomycin, bar horse Take charge of he, Benzodepa, Bicalutamide, bisantrene hydrochloride, two methanesulfonic acid bisnafides, Bizelesin, Bleomycin Sulphate, cloth quinoline That sodium, Bropirimine, busulfan, act-C, Calusterone, Caracemide, Carbetimer, carboplatin, Carmustine, hydrochloric acid card It is soft than star, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, cis-platinum, Cladribine, methanesulfonic acid crisnatol, ring Phosphamide, cytarabine, Dacarbazine, actinomycin D, daunorubicin hydrochloride, Decitabine, Dexormaplatin, Dezaguanine, first Sulfonic acid Dezaguanine, diaziquone, docetaxel, Doxorubicin, doxorubicin hydrochloride, Droloxifene, droloxifene citrate, third Sour dromostanolone, duazomycin, Edatrexate, fenoperic acid hydrochloride, Elsamitrucin, Enloplatin, enpromate, according to a piperazine Pyridine, epirubicin hydrochloride, Erbulozole, esorubicin hydrochloride, Estramustine, estramustine phosphate sodium, etanidazole, support pool Glycosides, etoposide phosphate, etoprine, carbazole hydrochloride, fazarabine, Suwei A amine, azauridine, fludarabine phosphate, fluorine Uracil, flurocitabine, Fosquidone, Fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxycarbamide, idarubicin hydrochloride, It is ifosfamide, ilmofosine, interleukin I I (including recombinant interleukin II or rIL2), Interferon a2a, Interferon Alpha-2b, dry Disturb plain α-n1, Alferon N, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, acetic acid Lanreotide, come Bent azoles, Leuprolide Acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, Masoprocol, U.S.A step on Element, mustine hydrochlcride, megestrol acetate, acetic acid melengestrol, melphalan, menogaril, mercaptopurine, methotrexate, Methotrexate sodium, metoprine, Meturedepa, mitindomide, mitocarcin, mitocromin, Mitogillin, mitomalcin, Mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, Ormaplatin, former times difficult to understand relax Logical sequence, taxol, Pegaspargase, Peliomycin, neptamustine, peplomycin sulfate, Perfosfamide, pipobroman, piposulfan, Hydrochloric acid Piroxantrone, plicamycin, Plomestane, porfimer, porfiromycin, prednimustine, procarbazine hydrochloride, fast sieve Mycin, puromycin hydrochloride, pyrazofurin, riboprine, Rogletimide, Safingol, hydrochloric acid Safingol, Semustine, Simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, Spiroplatin, broneomycin, streptozotocin, sulphur Chlorobenzene urea, Talisomycin, tecogalan sodium, Tegafur, teloxandrone hydrochloride, Temoporfin, Teniposide, teroxirone, testis Interior junket, thiapurine, thioguanine, phosphinothioylidynetrisaziridine, Tiazofurine, Tirapazamine, citric acid toremifene, acetic acid Trestolone, phosphorus Sour triciribine, Trimetrexate, glucuronic acid Trimetrexate, Triptorelin, tubulozole hydrochloride, uracil mustard, Wu Rui are replaced Group, Vapreotide, Verteporfin, vinblastine sulfate, vincristine sulphate, eldisine, vindesine sulfate, sulfuric acid Changchun Fixed, sulfuric acid vinglycinate, sulfuric acid leurosine, vinorelbine tartrate, sulfuric acid vinrosidine, sulfuric acid vinzolidine, Vorozole, Zeniplatin, Zinostatin, zorubicin hydrochloride.Other anticancer agents that can be used include but not limited to：5-ethinyluracil, Ah Bit dragon, Aclarubicin, acyl group fulvene, gland cyclopentanol (adecypenol), Adozelesin, Aldesleukin, ALL-TK antagonisms Agent, hemel, Ambamustine, 2,4 dichlorphenoxyacetic acids (amidox), Amifostine, amino-laevulic acid, Amrubicin, peace Acridine, anagrelide, Anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, Antarelix, Anti- back sideization forms albumen 1, antiandrogen, prostate cancer, antiestrogenic, antineoplaston (antineoplaston), antisense widow's core Thuja acid, glycine aphidicolin, apoptogene conditioning agent, apoptosis regulator, apurinic nucleic acid, ara-CDP-DL-PTBA, Arginine deaminase, aniline bifurcation pyridine (asulacrine), atamestane, Atrimustine, A Xi statins (axinastatin) 1, Ah Western statin 2, A Xi statins 3, Azasetron, Azalomvcin, azatyrosine, baccatin III derivative, bar drawing alcohol (balanol), Batimastat, BCR/ABL antagonists, benzo chlorin (benzochlorin), benzoyl staurosporin, in β Amide derivatives, β-aricine (β-alethine), Asia aclacinomycin (betaclamycin) B, betulinic acid, bFGF inhibit Agent, Bicalutamide, bisantrene, double aziridine spermine, bisnafide, bit enlightening Buddhist nun (bistratene) A, Bizelesin, Bei Fu Special (breflate), Bropirimine, Budotitane, buthionine sulfoximine, Calcipotriol, Ka Futading (calphostin) C, happiness Set alkali derivant, canary pox IL2, capecitabine, carboxylic acid amides-amino-triazole, Carboxylamide triazole, CaRestM3, Inhibitor, Carzelesin, casein kinase 2 enzyme inhibitor (ICOS), Castanospermine, cecropin B, west derived from CARN700, cartilage Qu Ruike, chlorins (chlorlns), chloro quinoxaline sulfonamide, cicaprost, cis- porphyrin, Cladribine, grace chlorine rice Fragrant analog, clotrimazole, gram Citropten (collismycin) A, gram Citropten B, combretastatin A4, combretastatin analog, gram Receive peaceful (conagenin), section's Lay bass fourth (crambescidin) 816, crisnatol, cryptophycin (cryptophycin) 8, cryptophycin A derivatives, Curacao (curacin) A, penta anthraquinone of ring (cyclopentanthraquinone), Cycloplatin (cycloplatam), plug training mycin (cypemycin), cytarabine octadecane Base sodium phosphate, the cell cracking factor, hexestryl diphosphate (cytostatin), dacliximab, Decitabine, dehydrogenation didemnin B, Deslorelin, dexamethasone, right ifosfamide, dexrazoxane, Dexverapamil, diaziquone, didemnin B, 3,4- dihydroxy Benzo hydroxamic acid (didox), the positive spermine of diethyl, dihydro 5-azacitidine, dihydro taxol, 9-, two oxymycins (dioxamycin), diphenyl spiral shell not sting, docetaxel, tadenan, Dolasetron, doxifluridine, Droloxifene, Dronabinol, multi-kanamycin SA, Ebselen, Ecomustine, Edelfosine, edrecolomab, Eflornithine, elemene, Emitefur, epirubicin, Epristeride, Estramustine analog, estrogen agonist, estrogen antagonist, etanidazole, Etoposide phosphate, Exemestane, Fadrozole, fazarabine, Suwei A amine, Filgrastim, Finasteride, Flavopiridol (flavopiridol), Flezelastine, Fu Lusilong (fluasterone), fludarabine, hydrochloric acid fluoro daunorubicin (fluorodaunorunicin), forfenimex, formestane, Fostriecin, Fotemustine, moral porphyrin gadolinium (gadoliniumtexaphyrin), gallium nitrate, Galocitabine, Ganirelix, gelatinase inhibitor, gemcitabine, gluathione Inhibitor peptides, He Shu anti-(hepsulfam), heregulin, cyclohexyl diacetamide, hypericin, ibandronic acid, jaundice element, Idoxifene, Idramantone, ilmofosine, Ilomastat, imidazo acridone, imiquimod, immunostimulatory peptides, insulin 1 acceptor inhibitor of like growth factor, interferon agonist, interferon, interleukin, Iobenguane, iododoxorubicin, 4- ipomeanols, Iroplact, Irsogladine, different lattice azoles (isobengazole), different high halichondrins (isohomohalicondrin) B, Itasetron, knot Si Li get (jasplakinolide), card Harrar obtain (kahalalide) F, piece spiral shell element (lamellarin)-N Triacetic acid, Lanreotide, thunder receive mycin (leinamycin), Lenograstim, sulfuric acid lentinan, Lay support sting (leptolstatin), Letrozole, LIF ELISA, leucocyte alpha interferon, Leuprorelin+estrogen+progesterone, bright third Rayleigh, levamisol, straight polyamine analog, two glycopeptide of lipophilicity, lipophilicity platinum compounds, are agilely received Liarozole (lissoclinamide) 7, lobaplatin, lombricine, Lometrexol, Lonidamine, Losoxantrone, Lovastatin, Loxoribine, Lurtotecan, moral porphyrin lutetium (lutetiumtexaphyrin), the Lay rope film (lysofylline), cell cracking Peptide, Maitansine, slow promise statin (mannostatin) A, Marimastat, Masoprocol, maspin (maspin), the appropriate aniline of matrilysin inhibitor, Matrix Metalloproteinase Inhibitors, menogaril, sulphur bar, U.S. are for auspicious Woods, methioninase, Metoclopramide, MIF inhibitor, mifepristone, Miltefosine, Mirimostim, mispairing double-stranded RNA, Plain (mitotoxin) fibroblast life of mitoguazone, mitolactol, mitomycin analogs, mitonafide, eliminating toxic advanced in years The long factor-Saponaria officinalis toxalbumin, mitoxantrone, Mofarotene, Molgramostim, monoclonal antibody, human chorionic gonadotrophin, list Monophosphoryl lipid A+ Mycobacterial cell walls skeleton, Mopidamol, multidrug resistance gene inhibitor, based on more tumor inhibitors 1 Treatment, mustard class anticancer agent, Indian Ocean sponge (mycaperoxide) B, Mycobacterial cell wall extract, meter Ya Pulong (myriaporone), N- acetyl group dinaline, the benzamide of N- substitutions, nafarelin, nagrestipen (nagrestip), receive Lip river ketone+pentazocine, Na Paying (napavin), naphthalene terpinum (naphterpin), Nartograstim, Nedaplatin, Nemorubicin, Neridronic Acid, neutral endopeptidase, Nilutamide, Nysa mycin (nisamycin), nitrogen oxides conditioning agent, nitroxide are anti-oxidant Agent, Ni Duolin (nitrullyn), O6-BG, Octreotide, Ao Kesi ketone (okicenone), oligonucleotides, Ao Nasi Ketone, Ondansetron, Ondansetron, Aurion pungent (oracin), oral cytokine inducer, Ormaplatin, Osaterone, Ao Shali Platinum, oxa- Austria promise mycin (oxaunomycin), taxol, paclitaxel analogs, paclitaxel derivatives, Palau amine (palauamine), palmityl rhizomycin, Pamidronic Acid, panaxytiol, Panomifene, secondary bacterium iron plain (parabactin), Pazelliptine, Pegaspargase, peldesine, pentosan polysulfate sodium, Pentostatin, spray support azoles (pentrozole), Perflubron, training phosphinylidyne Amine, perillyl alcohol, benzene that mycin (phenazinomycin), phenylacetate (phenylacetate), inhibitors of phosphatases, skin Western Barney (picibanil), hydrochloric acid pilocarpine, pirarubicin, piritrexim, placental hormone (placetin) A, placental hormone B, fibre Plasminogen activator inhibitor, platinum complex, platinum compounds ,-three amine complex of platinum, porfimer, porfiromycin, chlorine sprinkle Buddhist nun The double acridones of pine, propyl, prostaglandin J2, proteasome inhibitor, the immunomodulator based on albumin A, protein kinase C inhibit Agent, inhibitors of protein kinase C, microalgae (microalgal), protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase Inhibitor, alizarinopurpurin, pyrazoloacridine, Pyridoxalated Hemoglobin Polyoxyethylene conjugate, raf antagonists, Raltitrexed, Ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitor, ras-GAP inhibitor, demethylation it is auspicious for general Spit of fland, Etidronic Acid rhenium Re186, rhizomycin, ribozyme, RII vitaminamides (retinamide), Rogletimide, rohitukine (rohitukine), Romurtide, roquinimex, rupee lattice ketone (rubiginone) B1, Lu Baixi (ruboxyl), Safingol, Umbrella support puts down (saintopin), SarCNU, Sa Kefei alcohol (sarcophytol) A, Sargramostim, Sdi1 analogies, Si Mosi Inhibitor 1 derived from spit of fland, aging has oligonucleotide, signal transduction inhibitor, signal transduction modulators, single chain antigen to combine Albumen, sizofiran, Sobuzoxane, Sodium Borocaptate, sodium, Sol alcohol (solverol), SM-binding protein, rope Receive bright, sparfosic acid, this Ka-7038Ⅶ (spicamycin) D, spiromustine, spleen pentapeptide (splenopentin), sponge statin (spongistatin) 1, squalamine, stem cell inhibitors, stem cell division inhibitor, this carry amide (stipiamide), base Matter lysin inhibitor, Si Feinuoxin (sulfinosine), potent vasoactive intestines peptide antagonists, plain Lardy tower (suradista), suramin, sphaerophysine, synthesis mucopolysaccharide, Tallimustine, tamoxifen methiodide, Tauromustine, Tazarotene, tecogalan sodium, Tegafur, tellurium pyrans foreign (tellurapyrylium), telomerase inhibitor, replace Temoporfin Muzolimine, Teniposide, ten oxidation tetrachloros (tetrachlorodecaoxide), four assistant amine (tetrazomine), Tai Lilating (thaliblastine), thiocoraline, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin Receptor stimulating agent, Thymotrinan, thyrotropic hormone, ethyl etiopurpurin tin, Tirapazamine, titanocene dichloride, topology Gloomy spit of fland (topsentin), Toremifene, the myeloid-lymphoid stem cell factor, translation inhibitor, vitamin A acid, triacetyl uridine, Qu Xili Shore, Trimetrexate, Triptorelin, Tropisetron, Turosteride, tyrosine kinase inhibitor, tyrphostin (tyrphostin), UBC inhibitor, ubenimex, growth inhibitory factor, urea kinases receptors antagonism derived from urogenital sinus Agent, Vapreotide, watt vertical Olympic (variolin) B, carrier system, red blood cell gene therapy, Velaresol, veramine, Wa Erding (verdins), Verteporfin, vinorelbine, Wei Kesating (vinxaltine), Wei Taxin (Vitaxin), Vorozole, Zha Nuo Te Long, Zeniplatin, Zilascorb and Zinostatin stimalamer.
A kind of method for improving body forward direction immune response, including：It gives and is treated effectively to system in need or individual Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of the formula (I) of amount, pharmaceutical composition or aforementioned pharmaceutical compositions, Or the compound prepared according to the above method, so as to adjust TLR7 and/or TLR8.
A kind of method for enhancing chemotherapy effect, the chemotherapeutic including giving therapeutically effective amount to system in need or individual Object subsequently or simultaneously gives the Pyridopyrimidine derivatives of formula (I) of therapeutically effective amount or its pharmaceutically acceptable salt, drug Composition or above-mentioned pharmaceutical composition or the compound prepared according to the above method.
A kind of method for improving immunization therapy, including：The formula (I) of therapeutically effective amount is given to system in need or individual Pyridopyrimidine derivatives or its pharmaceutically acceptable salt, pharmaceutical composition or above-mentioned pharmaceutical composition or according to upper Compound prepared by the method stated, subsequently or simultaneously by Chimeric antigen receptor T cell (CAR-T) input system or a internal.
Another aspect of the present invention provides a kind of method treated or prepay mammalian diseases, including：To in need System or individual give the Pyridopyrimidine derivatives of formula (I) of therapeutically effective amount or its pharmaceutically acceptable salt, medicine group Close object.
The method that another aspect of the present invention provides treatment cell proliferative disorders, including：To system in need or a Body gives the Pyridopyrimidine derivatives of formula (I) of therapeutically effective amount or its pharmaceutically acceptable salt, pharmaceutical composition, wherein Cell proliferative disorders are lymthoma, osteosarcoma, melanoma or mammary gland, kidney, prostate, colorectum, thyroid gland, ovary, pancreas Gland, neuron, lung, the tumour of uterus or gastrointestinal tract.
Another aspect of the present invention provides the Pyridopyrimidine derivatives of formula (I) or its pharmaceutically acceptable salt can be used as TLRs receptor stimulating agents, the application being used to prepare in treatment disease relevant with TLRs activity or disorder agent.
Preferred TLR is TLR7 and/or TLR8.
The Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or pharmaceutical composition of formula (I) are preparing pharmaceutical preparation In application, the pharmaceutical preparation can raise inflammation and cytokine mediated access, t cell activation, apoptotic signal access, B cell activation pathway etc. is conducive to the number gene of immunotherapy of tumors.
Further, pharmaceutical preparation improves the quantity of CD8+T cells.
Further, pharmaceutical preparation improves the ratio of CD8+T cells/Treg cells.
Further, pharmaceutical preparation is by improving PDL-1 expression.
Further, pharmaceutical preparation improves the level of interleukin-22 and/or the level for improving interferon gamma and/or reduces white be situated between Element 10 is horizontal.
The Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or pharmaceutical composition of formula (I) are preparing downward Wnt Application in signal path preparation.Preferably, β-catenin protein expression levels are lowered.
It is thin that the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or pharmaceutical composition of formula (I) prepare influence macrophage Application in born of the same parents in each levels of cytokine secretion pharmaceutical preparation, it is preferred that influence Il1b, Il6, Il12b, Tnf, Ifnb1, The expression of the isogenic mRNA level in-site of Cxcl1, Cxcl10 and Il10.
Another aspect of the present invention provides the Pyridopyrimidine derivatives of formula (I) or its pharmaceutically acceptable salt or above-mentioned Pharmaceutical composition be used to prepare application in treatment communicable disease, immunity disease, virosis or tumour medicine.
The preferred asthma of above-mentioned disease, cancer, HIV, HBV.
Now some researches show that TLR and communicable disease (Potent immune activation in chronic hepatitis C patients upon administration of an oral inducer of endogenous interferons that acts via Toll-like receptor.Antiviral Ther.,2012,17,4,P657- P667), immunity disease (A new era of targeting the ancient gatekeepers of the immune system: toll-like agonists in the treatment of allergic rhinitis and Asthma.Int.Arch. Allergy Immunol., 2014,164,1, P46-P63), virosis (.A nucleoside analog, 7-thia-8-oxoguanosine stimulates proliferation of thymocytes in Vitro.Immunol. Lett., 1999,69,3, P293-P300), tumour (Vaccine adjuvant activity of 3M-052: An imidazoquinoline designed for local activity without systemic cytokine induction.Vaccine,2011,29,33,P5434-P5442；Effective Innate and Adaptive Antimelanoma Immunity through Localized TLR7/8Activation, Immunol., 2014,193,9, P4722-P4731) closely related, TLR7 agonists TLR7 agonists provided by the invention pass through confirmatory experiment Show to can be used for treating above-mentioned disease.
Above-mentioned immunity disease is autoimmunity disease, and autoimmunity disease includes but not limited to：Systemic loupus erythematosus, class wind It is wet arthritis, inflammatory bowel disease, Si Yegelun syndrome, polymyositis, vasculitis, wegener granulomatosis, sarcoidosis, tatanic Rachitis, Reiter syndrome, arthritic psoriasis, Behcet syndrome etc..
Above-mentioned viral disease includes but not limited to：Ebola disease viral disease, anthrax-bacilus disease, condyloma acuminatum, simple wart, vola Wart, Respiratory Syncytial Virus(RSV) (RSV), hepatitis B, hepatitis C, dengue fever virus, herpes simplex virus (such as HSV-I, HSV-II, CMV or VZV), molluscum contagiosum, cowpox, smallpox, slow virus, human immunodeficiency virus (HIV), people's papillomatosis Malicious (HPV), cytomegalovirus (CMV), varicella virus (VZV), rhinovirus, enterovirus, adenovirus, influenza, secondary stream Sense, mumps virus, measles virus, papovavirus, flavivirus, retrovirus, arenavirus (such as LCM, peaceful disease recklessly Poison, malachi virus, guanarito virus and Lassa fever) and Filovirus (such as Ebola virus or Marburg virus).
Above-mentioned tumour includes but not limited to：People's sarcoma and cancer, as fibrosarcoma, muscle tumor, embryonal-cell lipoma, chondrosarcoma, It is osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, outstanding Because knurl, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, prostate cancer, squamous cell carcinoma, basal-cell carcinoma, gland cancer, Syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, cephaloma, bronchiolar carcinoma, hepatoma, cholangiocarcinoma, villus Film cancer, seminoma, embryonal carcinoma, the nephroblastoma, cervical carcinoma, testicular tumor, lung cancer, Small Cell Lung Cancer, epithelioma, colloid It is knurl, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, few Prominent glioma, meningioma, melanoma, neuroblastoma, retinoblastoma；Leukaemia, such as acute lymphoblastic Property leukaemia and acute myeloblastic leukemia (myeloblast, promyelocyte, Myelomonocyte, monocyte and red blood cell Leukaemia)；Chronic leukemia (chronic myelocytic (granulocyte) leukaemia and chronic lymphocytic leukemia)；It is red thin with true property Born of the same parents increase, lymthoma (Hodgkin's disease and non-Hodgkin lymphoma), Huppert's disease, macroglobulinemia Waldenstron and again Chain disease.
Preferably, tumour is colon cancer, carcinoma of urinary bladder, melanoma, meningioma, lung cancer or cancer of pancreas.
Now some researches show that TLR and intestinal cancer (Toll-like receptor (TLR) 7and TLR8expression on CD133+ cells in colorectal cancer points to a specific role for inflammation- induced TLRs in tumourigenesis and tumour progression[J].European Journal of Cancer,2010,46(15):2849-2857), carcinoma of urinary bladder (Intravesical Toll-like receptor 7agonist R‐837:Optimization of its formulation in an orthotopic mouse model of bladder cancer[J].International journal of urology, 2010,17(5):483-490), it is black Melanoma (Effective Innate and Adaptive Antimelanoma Immunity through Localized TLR7/8Activation.J.Immunol., 2014,193,9, P4722-P4731), meningioma (Vaccination for invasive canine meningioma induces in situ production of antibodies capable of antibody-dependent cell-mediated cytotoxicity[J].Cancer research,2013,73 (10):2987-2997.), lung cancer (Toll-like receptor agonists in cancer therapy.Immunotherapy.2009 November 1；1(6):949-964.) or cancer of pancreas (Toll-like receptor 7regulates pancreatic carcinogenesis in mice and humans.J Clin Invest.2012；122:4118–4129.[PubMed: 23023703]).
Further, tumour medicine extends tumor patient life cycle.
The Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or pharmaceutical composition of formula (I) are preparing pre- preventing tumor Recur the application in drug.Pharmaceutical composition can cause lasting systemic immunity to remember to prevent to recur.
Compared with prior art, the present invention has the advantages that：
(1) using medicine group made from the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of formula (I) of the present invention Object is closed for the recurrence of pre- anti-cancer, inhibition cancer metastasis, growth and/or diffusion, the extension for inhibiting cancer or cancer metastasis The prognosis time.
(2) it is the basic of immunization therapy that immune system generates powerful tumor-specific immunity for cancer cell.Toll-like Receptor (TLR) is the ultimate constituent of immune system.Due to the powerful stimulating innate immunity of TLR agonists and adaptive immunity Ability, can effectively tumors destroyed as single dose.
(3) in addition, with PD-1, Tim-3 are antibody combined can effectively enhance tumor killing effect each other.Drug combination group is from whole body It is horizontal that the immune response of body is improved in immune state, promotes killing of the immune system to tumour.
Related definition：
" pharmaceutically acceptable " refers to biology or other aspects do not have dysgenic material, such as the material can be whole It closes and gives in the pharmaceutical composition of patient, without causing any undesirable biological action or not with including its combination Any other ingredient is interacted with harmful way in object.When term it is " pharmaceutically acceptable " for refer to drug-carrier or During excipient, represent the carrier or excipient meets the required standard of toxicology and production test or it includes in the U.S. The Inactive Ingredient Guide that Food and Drug Admistraton formulates.
The term as used herein " combination " or " pharmaceutical composition " refer to obtain by mixing or combining more than a kind active ingredient It arrives and the product of the fixation including active ingredient and unfixed combination.
Term " TLR diseases " or " disease relevant with TLR activity or illness " refer to relevant any with toll sample receptors Morbid state.
Description of the drawings
Fig. 1 is the EC50 measurement charts of agonist D018 according to the present invention.
Fig. 2 is the growth curve chart of gross tumor volume according to the present invention.
Fig. 3 is the 34th day tumour each group mouse tumor volume diagram according to the present invention.
Fig. 4 is infiltrating lymphocytes detection figure in tumor tissues according to the present invention.
Fig. 5 is each experimental group CD 8+T lymphocyte quantity statistical charts according to the present invention.
Fig. 6 is each experimental group CD 8+T/Treg ratio statistical charts according to the present invention.
Fig. 7 is PDL-1 expression figures in each experimental group according to the present invention.
Fig. 8 is the secretion influence figure of each experimental mice Cytokine of Serum according to the present invention.
Fig. 9 is secretion influence figures of the agonist D018 according to the present invention on cell factor in macrophage.
Figure 10 is RT-PCR verifications candidate gene expression figure according to the present invention.
Figure 11 is IHC detections CCl according to the present invention4It expresses and schemes with β-catenin.
Figure 12 is different experiments group mice with tumor life cycle statistical chart according to the present invention.
Figure 13 is influence figure of the drug combination according to the present invention to immunological memory.
Figure 14 is the growth curve chart of agonist D018 according to the present invention and antibody PD-1 and TIM3 combination gross tumor volume.
Figure 15 is Ag104Ld mouse tumors inhibiting tumor assay result figure according to the present invention.
Figure 16 is that lymphocyte infiltration CD4T+ cells, CD8+T are thin in Ag104Ld mouse tumors tissue according to the present invention Born of the same parents and Treg cell flow cytometer detection figures.
Figure 17 is the DC cell flow cytometer detections of CD11c+CD103+ in Ag104Ld mouse tumors tissue according to the present invention Figure.
Figure 18 is memory T cells flow cytometer detection figure in center in Ag104Ld mouse tumors tissue according to the present invention.
Specific embodiment
Below in conjunction with the accompanying drawings, the specific embodiment of the present invention is described in detail, it is to be understood that the guarantor of the present invention Shield scope is not restricted by specific implementation.
Prepare embodiment：
The starting material and reagent that the present invention uses are all from commercially available, and supplier includes but not limited to：Aldrich Chemical Company、Lancaster Synthesis Ltd.Wherein, dimethylformamide, tetrahydrofuran and dioxane The super dry solvent in lark prestige company is available from, and is stored in glove box, it is pure that dichloromethane and acetone are taken respectively from solvent Change system, the glass apparatus for being useful for the reaction to water sensitive are all first dried in 100 DEG C of baking oven.In addition to especially indicating, Raw materials used after further treatment and reagent does not use directly.
1H NMR and 13C NMR spectras are obtained, chemical shift using 400 type nmr determinations of Bruker DRX It is represented with ppm.Use tetramethylsilane internal standard (0.00ppm), CDCl3Or DMSO-d6 makees solvent (or other solvents).1H The method for expressing of NMR：S=is unimodal, d=doublets, t=triplets, m=multiplets, what br=widened, pair of dd=doublets Weight peak, the doublet of dt=triplets.If coupling constant is provided, unit Hz.Mass spectrum Finnigan Advantage Type mass spectrograph measures, Ionization mode ESI.Compound purifies (silica GF254 by column chromatography:200-400 mesh).Chemical combination Object purity refers both to separation yield as without especially indicating, measured using HPLC.
Embodiment 1
The preparation method of compound 3, step include：
(1) -5 Bromopyrimidine methyl formate (6.2g, 27mmol) of 3- amino, chloromethane amidine hydrochloric acid are added in 250mL seal pipes Salt (3.4g, 30mmol) and dimethyl sulfone (50.8g, 20mmol), be heated to 140oC stirrings 6 it is small when postcooling to room temperature, toward anti- Addition 50mL water in liquid is answered, is filtered after ultrasonic 30min, filter residue is washed through water, acetone, is dried in vacuo to obtain sepia solid 1 (yield=65%), reaction equation is：
(2) compound 1 is taken to be dissolved in aceticanhydride (25mL), 140 DEG C of reflux 8h postcoolings is heated to room temperature, is concentrated under reduced pressure Crude product obtains white solid 2 (compound 2) through column chromatography, and reaction equation is：
(3) compound 2 (1193mg, 5.0mmol) is taken to be dissolved in the super dry dioxane of 25mL, adds in DMF (35mL), instead Liquid ice bath is answered to oxalyl chloride (514ul, 6.0mmol) is slowly added dropwise after 0 DEG C, reaction solution is warming up to be stirred at room temperature 2 it is small when, decompression Compound 3 is concentrated to give, reaction equation is：
Embodiment 2
The preparation method of compound N 21, step include：
(1) compound 3 of 1 gained of embodiment is directly dissolved in dry tetrahydrofuran (5mL), sequentially adds n-butylamine (731uL, 7.5mmol, 1.5equiv.) and n,N-diisopropylethylamine (1.6mL, 10.0mmol, 2.0equiv) reacts liquid chamber Temperature stirring 5 it is small when after be concentrated under reduced pressure, column chromatography purify compound 7, reaction equation are：
(2) compound 7 (882mg, 3mmol) is taken to be dissolved in methanol (80mL), adds in LiOHH2O (189mg, Be stirred at room temperature after 4.5mmol) 8 it is small when, be concentrated under reduced pressure, column chromatography obtains brown solid 8, and reaction equation is：
(3) under nitrogen protection, in 25mL seal pipes, compound 8 (125.9mg, 0.5mmol) is taken to be dissolved in 5mL dryings In tetrahydrofuran, 3- methyl benzyl bromines (141mg, 0.75mmol), zinc powder (98.1mg, 1.5mmol) and Ni (PPh3) are sequentially added 2Cl2 (138.8mg, 0.05mmol), be stirred at room temperature 24 it is small when, be concentrated under reduced pressure, silica gel column chromatography obtains pale yellow powder N21, reaction Formula is：
1H NMR (400MHz, CDCl3) δ 7.57 (d, J=8.6Hz, 1H), 7.28 (d, J=8.8Hz, 1H), 7.19 (t, J =7.4Hz, 1H), 7.10 (s, 1H), 7.04 (m, 3H), 5.20 (s, 2H), 4.14 (s, 2H), 3.57 (dd, J=13.1, 6.6Hz, 2H), 2.32 (s, 3H), 1.70 (dt, J=14.6,7.2Hz, 2H), 1.46 (dd, J=14.8,7.4Hz, 2H), 0.99 (t, J=7.3 Hz, 3H) .13C NMR (101MHz, CDCl3) δ 160.40 (s), 160.27 (s), 154.88 (s), 144.34(s),139.38(s), 138.30(s),132.85(s),129.92(s),128.57(s),128.06(s),127.97 (s),127.30(s),126.20(s),44.35 (s),40.34(s),31.58(s),21.50(s),20.31(s),13.97 (s).MS calculated for([M],C19H23N5)+: 321.2,found:322.2。
Embodiment 3
The preparation method of compound N 23 and compound N 25, step include：
To be raw material by compound 8 made from embodiment 2, under nitrogen protection, in 25mL seal pipes, compound 8 is taken (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially add 2- methoxyl groups bromobenzyl (150.8mg, 0.75mmol), zinc powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), it is small to be stirred at room temperature 24 When, it is concentrated under reduced pressure, silica gel column chromatography obtains pale yellow powder N23, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.56 (d, J=8.6Hz, 1H), 7.29 (d, J=8.6Hz, 1H), 7.23 (t, J =7.5Hz, 1H), 7.13 (m, 2H), 6.89 (m, 2H), 5.32 (s, 2H), 4.18 (s, 2H), 3.80 (s, 3H), 3.55 (dd, J =13.1,6.7Hz, 2H), 168 (m, 2H), 1.44 (dd, J=14.8,7.4Hz, 2H), 0.97 (t, J=7.3Hz, 3H) .13C NMR(101MHz,CDCl3)δ160.35(s),159.83(s),157.43(s),155.26(s),143.43(s),132.12 (s), 130.80(s),128.00(s),127.82(s),120.67(s),110.61(s),55.45(s),40.34(s), 38.46(s),31.51(s), 20.27(s),13.94(s).MS(EI)calculated for([M],C19H23N5O)+: 337.2,found:338.2。
To be raw material by compound 8 made from embodiment 2, under nitrogen protection, in 25mL seal pipes, compound 8 is taken (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially add 4- methoxyl groups bromobenzyl (150.8mg, 0.75mmol), zinc powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), it is small to be stirred at room temperature 24 When, it is concentrated under reduced pressure, silica gel column chromatography obtains compound N 25, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.65 (d, J=8.5Hz, 1H), 7.25 (d, J=8.6Hz, 2H), 7.18 (m, 4H), 5.89 (s, 2H), 4.19 (s, 2H), 3.57 (d, J=6.4Hz, 2H), 2.26 (s, 3H), 1.69 (m, 2H), 1.51- 1.36 (m, 2H), 0.98 (t, J=7.3Hz, 3H) .13C NMR (101MHz, CDCl3) δ 160.23 (s), 158.65 (s), 155.69(s), 140.47(s),137.20(s),136.80(s),130.71(s),130.59(s),130.07(s),128.19 (s),127.35(s), 127.08(s),126.32(s),42.02(s),40.58(s),31.35(s),20.22(s),19.89 (s),13.90(s).MS(EI) calculated for([M],C19H23N5O)+:337.2,found:338.2。
Embodiment 4
The preparation method of compound N 26, step include：
To be raw material by compound 8 made from embodiment 2, under nitrogen protection, in 25mL seal pipes, compound 8 is taken (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially adds 2-trifluoromethyl bromobenzyl (0.75mmol), zinc powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), be stirred at room temperature 24 it is small when, be concentrated under reduced pressure, silica gel Column chromatography obtains compound N 26, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.68 (d, J=7.6Hz, 1H), 7.62 (d, J=8.5Hz, 1H), 7.45 (t, J =7.4Hz, 1H), 7.34 (t, J=7.4Hz, 1H), 7.26 (m, 2H), 7.09 (s, 1H), 5.47 (s, 2H), 4.38 (s, 2H), 3.65-3.47 (m, 2H), 1.66 (dd, J=14.3,7.1Hz, 2H), 1.43 (dd, J=14.8,7.4Hz, 2H), 1.03-0.88 (m,3H).13C NMR(101MHz,CDCl3)δ160.27(s),159.84(s),153.83(s),143.17(s),137.63 (s), 132.26 (s), 132.04 (s), 131.87 (s), 128.89 (q, J=29.5Hz), 128.03 (s), 127.90 (s), 126.74 (s), 126.14 (q, J=5.8Hz), 124.60 (q, J=273.8Hz), 40.35 (d, J=5.4Hz), 31.39 (s),20.20(s),13.89 (s).MS(EI)calculated for([M],C19H20F3N5)+:375.2,found: 376.2。
Embodiment 5
The preparation method of compound N 32, step include：
To be raw material by compound 8 made from embodiment 2, under nitrogen protection, in 25mL seal pipes, compound 8 is taken (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially adds 4-bromobenzyl methyl benzoate (0.75mmol), zinc Powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), be stirred at room temperature 24 it is small when, be concentrated under reduced pressure, silicon Plastic column chromatography must obtain compound N 32, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.99 (d, J=8.3Hz, 2H), 7.59 (d, J=8.6Hz, 1H), 7.33 (d, J =8.2Hz, 2H), 7.28 (d, J=8.6Hz, 1H), 7.03 (s, 1H), 5.01 (s, 1H), 4.23 (s, 2H), 3.91 (s, 3H), 3.57 (dd, J=13.1,7.0Hz, 2H), 1.76-1.65 (m, 2H), 1.46 (dd, J=15.0,7.4Hz, 2H), 1.00 (t, J =7.3 Hz, 3H) .13C NMR (101MHz, Chloroform-d) δ 167.05 (s), 160.53 (s), 160.36 (s), 153.53(s), 144.95(s),144.86(s),133.36(s),129.98(s),129.20(s),128.53(s),128.36 (s),127.82(s),52.14(s), 44.30(s),40.32(s),31.57(s),20.30(s),13.95(s).MS(EI) calculated for([M],C20H23N5O2)+: 365.2,found:366.2.MS(EI)calculated for([M], C20H23N5O2)+:365.2,found:366.2.
Embodiment 6
The preparation method of compound N 33, step include：
To be raw material by compound 8 made from embodiment 2, under nitrogen protection, in 25mL seal pipes, compound 8 is taken (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially adds 3-bromobenzyl methyl benzoate (0.75mmol), zinc Powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), be stirred at room temperature 24 it is small when, be concentrated under reduced pressure, silicon Plastic column chromatography obtains compound N 33, and reaction equation is
1H NMR(400MHz,CDCl3) δ 7.96 (s, 1H), 7.90 (d, J=7.6Hz, 1H), 7.56 (d, J=8.6Hz, 1H), 7.43 (d, J=7.6Hz, 1H), 7.36 (t, J=7.6Hz, 1H), 7.26 ((d, J=8.6Hz, 1H, 7.02 (s, 1H), 5.02 (s, 2H), 4.21 (s, 2H), 3.89 (s, 3H), 3.55 (dd, J=13.3,6.8Hz, 2H), 1.73-1.57 (m, 2H), 1.44 (dd, J=15.0,7.4Hz, 2H), 0.97 (t, J=7.3Hz, 3H)13C NMR(101MHz,CDCl3)δ167.13 (s), 160.46(s),160.37(s),153.86(s),144.81(s),139.85(s),133.77(s),133.26(s), 130.55(s), 130.35(s),128.71(s),128.30(s),127.85(s),127.79(s),52.23(s),44.08 (s),40.32(s),31.56(s), 20.30(s),13.97(s).MS(EI)calculated for([M],C20H23N5O2)+: 365.2,found:366.2.
Embodiment 7
The preparation method of compound N 29, step include：
With by compound 8 made from embodiment 2 for raw material under nitrogen protection, in 25mL seal pipes, take compound 8 (125.9mg, 0.5mmol) is dissolved in 5mL dry tetrahydrofurans, sequentially adds 3-bromobenzyl benzoic acid (0.75mmol), zinc powder (98.1mg, 1.5mmol) and Ni (PPh3) 2Cl2 (138.8mg, 0.05mmol), be stirred at room temperature 24 it is small when, be concentrated under reduced pressure, silica gel Column chromatography obtains compound N 29, and reaction equation is：
1H NMR (400MHz, MeOD) δ 7.97 (s, 1H), 7.87 (d, J=7.7Hz, 1H), 7.76 (d, J=8.5Hz, 1H), 7.64 (d, J=8.6Hz, 1H), 7.56 (d, J=7.5Hz, 1H), 7.40 (t, J=7.7Hz, 1H), 4.93 (s, 2H), 4.31 (s, 2H), 3.68 (t, J=7.2Hz, 2H), 1.71 (dd, J=14.7,7.4Hz, 2H), 1.43 (dd, J=14.9, 7.4Hz, 2H), 0.99 (t, J=7.3Hz, 3H)13C NMR(101MHz,MeOD)δ169.54(s),161.18(s),159.16 (s), 156.05(s),140.41(s),134.50(s),134.38(s),132.27(s),131.05(s),130.53(s), 129.67(s), 128.87(s),127.46(s),126.73(s),44.22(s),41.79(s),31.78(s),20.94(s), 13.93(s).MS(EI) calculated for([M],C19H21N5O2)+:351.2,found:352.2。
Embodiment 8
The preparation method of compound N 10, step include：
(1) lithium aluminium hydride (75.9mg, 2.0mmol) is taken to be placed in arrow-necked bottle, dry tetrahydrochysene is added under condition of ice bath Furans (2mL), compound N 29 (182.7mg, 0.5mmol) is prepared into tetrahydrofuran solution made from embodiment 7, in ice bath item Under part, it is slowly added dropwise in the tetrahydrofuran solution of lithium aluminium hydride；After reaction, 76 microlitres of water, 76 micro- 10% hydrogen are added in Aqueous solution of sodium oxide, 3x76 microlitres of water quenching are gone out reaction, filtering, and DCM washing filter residues, anhydrous sodium sulfate drying concentrates the light of filtrate Yellow solid 9, reaction equation are：
(2) compound 9 is taken to be dissolved in dry DCM, thionyl chloride (182uL, 2.5mmol), heating are added dropwise under condition of ice bath To being stirred at room temperature 60 minutes, the yellow oil 10 of vacuum revolving, reaction equation is：
(3) compound 10 is dissolved in the tetrahydrofuran of 5mL dryings, adds in nafoxidine (207uL, 2.5mmol) and carbonic acid Potassium (207mg, 1.5mmol), reaction solution be stirred at room temperature 12 it is small when after, concentration, extracted with DCM/H2O, merge organic layer, anhydrous sulphur Sour sodium drying, concentration, silica gel column chromatography obtain faint yellow solid compound N 10, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.55 (d, J=8.5Hz, 1H), 7.26 (d, J=8.0Hz, 3H), 7.19 (d, J =7.4Hz, 2H), 7.07 (s, 1H), 5.09 (s, 2H), 4.15 (s, 2H), 3.56m, 4H), 2.49 (s, 4H), 1.77 (s, 4H), 1.73-1.59 (m, 3H), 1.46 (dd, J=14.5,7.2Hz, 2H), 0.98 (t, J=7.2Hz, 3H)13C NMR(101 MHz, CDCl3)δ160.42(s),160.38(s),154.70(s),144.76(s),138.00(s),137.71(s),133.09(s), 129.19(s),128.96(s),128.14(s),127.84(s),60.47(s),54.25(s),44.07(s),40.29(s), 31.58(s), 23.52(s),20.29(s),13.95(s).MS(EI)calculated for([M],C23H30N6)+:390.3, found:391.3。
Embodiment 9
The preparation method of compound N 01, step include：
To be raw material by compound N 10 made from embodiment 8, it is dissolved in the tetrahydrofuran of 5mL dryings, adds in dimethyl amine Hydrochloride (2.5mmol) and potassium carbonate (207mg, 1.5mmol), reaction solution be stirred at room temperature 12 it is small when after, concentration, use DCM/H2O Extraction merges organic layer, and anhydrous sodium sulfate drying concentrates, and silica gel column chromatography obtains compound N 01, and reaction equation is：
1H NMR (400MHz, Chloroform-d) δ 7.61 (d, J=8.6Hz, 1H), 7.30 (d, J=8.6Hz, 1H), 7.26 (d, J=8.5Hz, 2H), 7.20 (d, J=7.9Hz, 2H), 7.17 (s, 1H), 5.31 (s, 2H), 4.17 (s, 2H), 3.57 (q, J=6.8Hz, 2H), 3.44 (s, 2H), 2.26 (s, 6H), 1.70 (m, J=7.4Hz, 2H), 1.47 (m, J= 7.4Hz, 2H), 0.99 (t, J=7.4Hz, 3H)13C NMR(101MHz,Chloroform-d)δ160.37(s),159.71 (s),155.22(s), 143.13(s),138.29(s),136.62(s),132.24(s),129.61(s),129.11(s), 128.17(s),127.86(s),63.97(s), 45.27(s),44.08(s),40.46(s),31.53(s),20.31(s), 13.96(s).MS(EI)calculated for([M],C21H28N6) +:364.2,found:365.2。
Embodiment 10
The preparation method of compound N 02, step include：
The hydrochloric acid dioxane solution 1ml of 4M is configured, compound N 10 made from addition embodiment 8 (72.9mg, 0.2mmol), stir, be precipitated white solid, filtering, recrystallize white crystalline Compound N02, reaction equation are：
1H NMR (400MHz, Methanol-d4) δ 7.56 (d, J=8.6Hz, 1H), 7.54-7.48 (m, 4H), 7.45 (d, J=8.7Hz, 1H), 4.53 (s, 2H), 4.27 (s, 2H), 3.58 (t, J=7.2Hz, 2H), 3.11 (s, 9H), 1.70 (p, J=7.4 Hz, 2H), 1.47 (dt, J=14.9,7.4Hz, 2H), 1.00 (t, J=7.4Hz, 3H)13C NMR(101MHz, Methanol-d4)δ161.95(s),161.57(s),155.45(s),145.17(s),144.24(s),134.23(s), 132.93(s), 130.92(s),129.25(s),129.03(s),127.17(s),70.25(s),53.11(s),53.07 (s),53.03(s),44.61(s), 41.21(s),32.47(s),21.21(s),14.22(s).MS(EI)calculated for([M]+,C22H31N6)+:379.3,found: 379.3。
Embodiment 11
The preparation method of compound N 12, step include：
Under nitrogen protection, sodium azide (39.0mg, 0.6mmol) is taken to be dissolved in DMSO (1mL), adds in and makes embodiment 8 Compound 10 (177.9mg, 0.5mmol), reaction solution be stirred at room temperature 10 it is small when, be slowly added to 1mL water quenchings and go out reaction, treat instead Liquid is answered to be cooled to room temperature, reaction solution is poured into 1mL water, ethyl acetate extracts three times, merges organic layer, and anhydrous sodium sulfate is done Dry, filtering is concentrated to give compound N 12, reaction equation is：
1H NMR (400MHz, Chloroform-d) δ 7.52 (d, J=8.6Hz, 1H), 7.21 (d, J=10.6Hz, 5H), 7.01 (t, J=5.7Hz, 1H), 5.00 (s, 2H), 4.25 (s, 2H), 4.12 (s, 2H), 3.50 (td, J=7.2,5.8Hz, 2H), 1.70-1.59 (m, 2H), 1.47-1.33 (m, 2H), 0.93 (t, J=7.4Hz, 3H)13C NMR(101MHz, Chloroform-d)δ160.36(s),160.26(s),154.37(s),144.41(s),139.68(s),133.66(s), 133.02(s), 129.62(s),128.61(s),128.16(s),127.93(s),54.63(s),44.03(s),40.36 (s),31.56(s),20.31(s), 13.98(s).MS(EI)calculated for([M]+,C19H22N8)+:362.2, found:363.2。
Embodiment 12
The preparation method of compound N 14, step include：
In glove box, 25ml flasks is taken to add in ZnI2(191.5mg, 0.6mmol) and HF (5mL), obtains white liquid, Wherein there are some insoluble solids；Triethyl phosphine (129uL, 0.75mmol) and 8 compound 9 of embodiment are added in into flask (168.7mg, 0.5mmol)；After reaction mixture is heated to reflux 16h, cooling reaction solution is concentrated in vacuum to room temperature；Gained is residual After slag addition ethyl acetate (2x50mL) and 2NNaOH (2x10mL) are washed, merge organic phase and simultaneously use MgSO4It is dry, in vacuum Solvent evaporated obtains faint yellow solid after silica gel chromatography, i.e. compound N 14, reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.57 (d, J=8.5Hz, 1H), 7.24 (m, 5H), 7.09 (s, 1H), 5.05 (s, 2H), 4.15 (s, 2H), 4.09-3.94 (m, 4H), 3.57 (dd, J=13.0,6.5Hz, 2H), 1.79-1.60 (m, 2H), 1.47 (dd, J=14.6,7.2Hz, 2H), 1.24 (t, J=7.0Hz, 6H), 1.00 (t, J=7.2Hz, 3H)13C NMR (101MHz, CDCl3)δ160.27(s),160.14(s),154.56(s),144.33(s),138.02(s),138.00– 137.96(m),132.86 (s),130.00(s),129.94(s),129.21(s),129.18(s),128.01(s),127.79 (s), 62.06 (d, J=6.7Hz), 43.90 (s), 40.24 (s), 34.03 (s), 32.66 (s), 31.47 (s), 20.19 (s), 16.38(s),16.32(s),13.85(s).MS (EI)calculated for([M],C23H32N5O3P)+:457.2,found: 458.2。
Embodiment 13
The preparation method of compound N 35, step include：
(1) in the 100ml round-bottomed flasks of drying, compound 3 (128.5mg, 0.5mmol) is dissolved into anhydrous THF In (5ml), then 2- methoxyethyl amines (130uL, 1.5mmol) and DIEA (174uL, 1.0mmol) are added dropwise respectively into flask, 5h is stirred, is concentrated in vacuo, crude product purifies to obtain compound 11, reaction equation by silica gel column chromatography：
(2) compound 11 in methanol (0.1M) with LiOHH2O (31.5mg, 0.75mmol) stirs 8h, silicon at room temperature Compound 12 is obtained after rubber column gel column chromatographic purification, reaction equation is：
(3) 25ml tube sealings are taken, in glove box be packed into compound 12 (76.1mg, 0.3mmol), toluene bromide (54uL, 0.45mmol), zinc powder (58.9mg, 0.9mmol), Ni (PPh3)2Cl2(19.6mg, 0.03mmol) and THF (3mL, 0.1M), nothing Special sequence requirement；Reaction is stirred at room temperature for 24 hours, and silica gel chromatography obtains product light yellow solid N35, reaction equation For：
1H NMR (400MHz, CDCl3) δ 7.60 (d, J=8.6Hz, 1H), 7.54 (s, 1H), 7.34-7.26 (m, 3H), 7.26-7.18 (m, 3H), 5.68 (s, 2H), 4.17 (s, 2H), 3.78 (dd, J=10.5,5.2Hz, 2H), 3.64 (t, J= 5.1Hz, 2H),3.42(s,3H).13C NMR(101MHz,CDCl3)δ160.39(s),159.09(s),155.68(s), 142.23(s), 139.14(s),131.52(s),129.15(s),128.71(s),128.39(s),127.56(s),126.62 (s),70.86(s),59.02 (s),44.34(s),40.44(s).MS(EI)calculated for([M],C17H19N5O)+: 309.2,found:310.2。
Embodiment 14
The preparation method of compound N 37, step include：
(1) in the 25ml round-bottomed flasks of drying, compound 3 (128.5mg, 0.5mmol) is dissolved into anhydrous THF In (5ml), then piperidines (149uL, 1.5mmol) and DIEA (174uL, 1.0mmol) are added dropwise respectively into flask, stir 5h, very Sky concentration；Gained crude product purifies to obtain compound 13 by silica gel column chromatography, and reaction equation is：
(2) compound 13 stirs 8h at room temperature in methanol (0.1M) with LiOH.H2O (31.5mg, 0.75mmol)；Silica gel Compound 14 is obtained after column chromatography, reaction equation is：
(3) 25ml tube sealings are taken, in glove box be packed into compound 14 (79.1mg, 0.3mmol), toluene bromide (54uL, 0.45mmol), zinc powder (58.9mg, 0.9mmol), Ni (PPh3)2Cl2(19.6mg, 0.03mmol) and THF (3mL, 0.1M), nothing Special sequence requirement；Reaction is stirred at room temperature for 24 hours, and silica gel chromatography obtains product light yellow solid N37, reaction equation For：
1H NMR (400MHz, CDCl3) δ 7.59 (d, J=8.6Hz, 1H), 7.33-7.17 (m, 6H), 4.80 (s, 2H), 4.21 (s, 4H), 4.15 (s, 2H), 1.69 (dd, J=6.3,3.1Hz, 2H), 1.64 (d, J=4.8Hz, 4H) .13C NMR (101MHz,CDCl3)δ160.27(s),159.49(s),153.14(s),148.01(s),139.76(s),133.73(s), 130.59 (s),129.37(s),128.59(s),126.80(s),126.37(s),49.01(s),44.68(s),26.49 (s),25.09(s).MS(EI) calculated for([M],C19H21N5)+:319.2,found:320.2.
Embodiment 15
The preparation method of compound N 16, step include：
Take 25ml tube sealings, in glove box be packed into compound 5 (125.9mg, 0.5mmol), toluene bromide ((89uL, 0.75mmol), zinc powder (98.1mg, 1.5mmol), Ni (PPh3)2Cl2(32.7mg, 0.05mmol) and THF (5mL, 0.1M), nothing Special sequence requirement；Reaction is stirred at room temperature for 24 hours, and silica gel chromatography obtains product light yellow solid, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.56 (d, J=8.6Hz, 1H), 7.36-7.16 (m, 6H), 7.06 (s, 1H), 5.02 (s, 2H), 4.17 (s, 2H), 3.56 (dd, J=13.2,6.7Hz, 2H), 1.80-1.58 (m, 2H), 1.46 (dd, J= 14.9,7.4Hz, 2H), 0.98 (t, J=7.3Hz, 3H)13C NMR(101MHz,CDCl3)δ160.45(s),160.42(s), 154.63(s),144.82(s),139.53(s),133.19(s),129.18(s),128.68(s),128.21(s),127.89 (s), 126.54(s),44.41(s),40.32(s),31.60(s),20.31(s),13.98(s).MS(EI)calculated for([M], C18H21N5)+:307.2,found:308.2。
Embodiment 16
The preparation method of compound N 40, step include：25ml tube sealings are taken, compound 5 is packed into glove box (125.9mg, 0.5mmol), (1- bromoethyls) benzene (102mg, 0.75mmol), zinc powder (98.1mg, 1.5mmol), Ni (PPh3) 2Cl2(32.7mg, 0.05mmol) and THF (5mL, 0.1M), without special sequence requirement；Reaction is stirred at room temperature for 24 hours, silica gel Column chromatography purifies to obtain product light yellow solid N40, and reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.54 (d, J=8.6Hz, 1H), 7.28 (dd, J=17.3,8.1Hz, 5H), 7.20 (t, J=6.7Hz, 1H), 7.08 (s, 1H), 5.01 (s, 2H), 4.31 (d, J=7.1Hz, 1H), 3.57 (dd, J= 13.2,6.9Hz, 2H), 1.79-1.61 (m, 5H), 1.47 (dd, J=14.9,7.4Hz, 2H), 1.00 (t, J=7.3Hz, 3H).13C NMR(101MHz,CDCl3)δ160.50(s),160.45(s),158.50(s),145.13(s),144.78(s), 133.04(s), 128.59(s),127.82(s),127.33(s),126.50(s),47.04(s),40.32(s),31.64 (s),20.88(s),20.33(s), 14.00(s).MS(EI)calculated for([M],C19H23N5)+:321.2, found:322.2。
Embodiment 17
The preparation method of compound N 42, step include：
(1) in the suspension of the dioxane (2.44ml) to compound 4 (50mg, 0.171mmol) and water (0.86ml) Add in E- phenylvinylboronic acids (27.8mg, 0.188mmol, 1.1eq), Pd (PPh3)4(9.88mg, 0.0086mmol, 0.05equiv) and K2CO3(70.9mg, 0.513mmol, 3.0equiv)；Reaction mixture 120 DEG C of reactions under protection of argon gas 24h.Reaction mixture is diluted with EA, then is washed respectively with water and saturated salt and used Na2SO4It is concentrated after drying, in vacuum organic Phase；Residue after concentration does mobile phase with 4%methanol/DCM, and chromatography obtains compound 1550mg (92%), Reaction equation is：
(2) added in ethyl alcohol (0.28ml) suspension of compound 15 (45mg, 0.14mmol) Pd/ activated carbons (4.2mg, 5%), mixture is heated in hydrogen in 60 DEG C；It is concentrated by evaporation after filtering and obtains faint yellow solid N42, reaction equation is：
1H NMR (400MHz, CDCl3) δ 7.56 (d, J=8.5Hz, 1H), 7.31-7.22 (m, 3H), 7.20 (d, J= 6.7Hz, 3H), 7.05 (s, 1H), 5.08 (s, 2H), 3.56 (dd, J=13.3,6.9Hz, 2H), 3.23-2.99 (m, 4H), 1.77-1.63 (m, 2H), 1.46 (dd, J=15.0,7.4Hz, 2H), 0.99 (t, J=7.3Hz, 3H)13C NMR(101MHz, CDCl3)δ160.37(s),160.17(s),144.33(s),141.61(s),132.61(s),128.56(s),128.47(s), 128.19 (s),127.87(s),126.08(s),40.35(s),39.53(s),35.76(s),31.62(s),20.33(s), 13.99(s).MS(EI) calculated for([M],C19H23N5)+:321.2,found:322.2。
Embodiment 18
The preparation method of compound S01, step include：
(1) in the 25ml round-bottomed flasks of drying, compound 3 (128.5mg, 0.5mmol) is dissolved into anhydrous THF In (5ml), then n-butyl mercaptan (161uL, 1.5mmol) and DIEA (174uL, 1.0mmol) are added dropwise respectively into flask, stirring 5h is concentrated in vacuo；Gained crude product purifies to obtain compound 16 by silica gel column chromatography, and reaction equation is：
(2) compound 9 in methanol (0.1M) with LiOHH2O (31.5mg, 0.75mmol) stirs 8h at room temperature；Silica gel Compound 17 is obtained after column chromatography, reaction equation is：
(3) 25ml tube sealings are taken, the loading compound 17 (80.6mg, 0.3mmol) in glove box, toluene bromide (54uL, 0.45mmol), zinc powder (58.9mg, 0.9mmol), Ni (PPh3) 2Cl2 (19.6mg, 0.03mmol) and THF (3mL, 0.1M), Without special sequence requirement；Reaction is stirred at room temperature for 24 hours, and silica gel chromatography obtains product light yellow solid, LOLXB059, reaction equation are：
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=8.7Hz, 1H), 7.44-7.16 (m, 6H), 5.21 (d, J= 11.9Hz, 2H), 4.27 (s, 2H), 3.23 (t, J=7.3Hz, 2H), 1.77 (dd, J=14.8,7.5Hz, 2H), 1.54 (dd, J =14.7,7.4Hz, 2H), 0.99 (t, J=7.3Hz, 3H)13C NMR(101MHz,CDCl3)δ175.42(s),158.60 (s), 157.10(s),144.23(s),139.11(s),135.10(s),133.92(s),129.38(s),128.90(s), 128.78(s), 126.67(s),44.79(s),30.99(s),28.89(s),22.34(s),13.86(s).MS(EI) calculated for([M], C18H20N4S)+:324.2,found:325.2。
Embodiment 19
The preparation method of compound O01, step include：
(1) in the 100ml round-bottomed flasks of drying, compound 3 (128.5mg, 0.5mmol) is dissolved into anhydrous normal butyl alcohol In, NaH (24.1mg, 1.0mmol) is slowly added into flask；It is concentrated in a vacuum after gained reaction mixture stirring 5h； Crude product purifies to obtain compound 18 by silica gel column chromatography, and reaction equation is：
(2) compound 18 stirs 8h at room temperature in methanol (0.1M) with LiOH.H2O (31.5mg, 0.75mmol)；Silica gel Compound 19 is obtained after column chromatography, reaction equation is：
(3) a 25ml tube sealings are taken, in glove box be packed into compound 19 (75.6mg, 0.3mmol), toluene bromide (54uL, 0.45mmol), zinc powder (58.9mg, 0.9mmol), Ni (PPh3)2Cl2(19.6mg, 0.03mmol) and THF (3mL, 0.1M), nothing Special sequence requirement；Reaction is stirred at room temperature for 24 hours, and silica gel chromatography obtains product light yellow solid O01, reaction equation For：
1H NMR(400MHz,CDCl3) δ 7.65 (d, J=8.7Hz, 1H), 7.44-7.16 (m, 6H), 5.21 (d, J= 11.9Hz, 2H), 4.27 (s, 2H), 3.23 (t, J=7.3Hz, 2H), 1.77 (dd, J=14.8,7.5Hz, 2H), 1.54 (dd, J =14.7,7.4Hz, 2H), 0.99 (t, J=7.3Hz, 3H)13C NMR(101MHz,CDCl3)δ175.42(s),158.60 (s), 157.10(s),144.23(s),139.11(s),135.10(s),133.92(s),129.38(s),128.90(s), 128.78(s), 126.67(s),44.79(s),30.99(s),28.89(s),22.34(s),13.86(s).MS(EI) calculated for([M], C18H20N4S)+:307.2,found:308.2。
EXPERIMENTAL EXAMPLE：
First, the EC50 of agonist is measured
Reagent：HEK-BlueTM hTLR7、HEK-BlueTMHTLR8 and control HEK-Blue Null2-k cell culture and Reagent needed for detection：DMEM (4.5g/l glucose), cow's serum FBS, streptomysin (50 μ g/ml), penicillin (50U/ml) Blasticidin(10mg/ml)、ZeocinTM(10mg/ml)、Normocin(50mg/ml)、HEK-BlueTM Detection.Basal medium：DMEM+10%FBS+ streptomysins+penicillin+Normocin (100 μ g/ml)
Cell prepares：The cell frozen is put into rapidly 37 DEG C of water-baths, shakes frequently, it was made to melt completely in 1 minute； Cell is transferred to 15ml shifts to an earlier date in preheated basal medium and be resuspended；1000r/min centrifuges 5min, discards upper liquid； 1ml basal mediums are resuspended, and are transferred to T25 blake bottles, are supplemented to 5ml culture mediums, are placed in 37 DEG C of incubator cultures；Stablize passage two Selective antibiotic screening is added in after secondary：HEK-BlueTMHTLR7 or HEK-BlueTMhTLR8：100μg/ml Zeocin+ Blasticidin(30μg/ml)、HEK-Blue Null2-k：100μg/ml Zeocin；Change within one week liquid 2 times；When cell concentration reaches During to 70%-80%, PBS is added, which gently to pat, makes cell detachment.
Activity determination step：Add 20 μ l samples (setting multiple holes) per hole in 96 orifice plates；Add 20 μ l positive controls (such as： INF- α, 100ng/ml)；Add 20 μ l negative controls (such as：ddH2O)；It is light with the 5-10ml PBS (T75 blake bottles) preheated in advance Soft flushing cell；Add 2-5ml PBS (T75 blake bottles) in blake bottle and put back to incubator warm bath 1-2min, gently pat Make cell detachment；Cell count can not centrifuge；HEK-Blue hTLR cell concentrations are about 220000/ml, per 180 μ l of hole (about 40000 cells), HEK-Blue Null2-k cell concentrations are about 280000/ml, per 180 μ l of hole (about 50000 cells)；With Adjustment cell quantity is resuspended in HEK-Blue TM detection liquid, can not incubation time it is too long, in order to avoid background too depth or there is false positive； 37 DEG C of incubator culture 6-16h, 620-655nm detection SEAP readings.
Experimental result：Half-maximal effect concentration (concentration for 50%of maximal effect, EC50) Refer to the concentration that can cause 50% ceiling effect.Experimental result is shown：The half-maximal effect concentration of R848 is mTLR7 (75nM), The TLR agonist D018 (compound number N01) of hTLR (773nM) and the present invention：MTLR7 (33nM), hTLR (15nM) is (see figure 1), EC50 reacts the drug effect of agonist well, and value is smaller to illustrate that drug effect is higher.
2nd, the foundation of mouse tumor model and inhibiting tumor assay
Using MC38 colon-cancer cells in this experiment, MC38 is incubated in the DMEM culture mediums containing 10%FBS, in 5%CO2, It is cultivated in 37 DEG C of incubator.Routine passage pancreatin 0.25%Trypsin-EDTA (GIBCO:25200-056), 1- is digested 3min.Cell passage ratio is 1：3, cell density is less than 80% passage.Cell changes weekly liquid 2-3 times after passage.3 before kind of knurl Its cell passes on once, and one day culture solution more renewed before the injection.Take survival rate 80% 1 × 106A MC38 cells It is subcutaneous to be injected in 6-8 week old female C57BL/6 right side of mice.
Experiment packet：
The observation of mice tumors grew situation
It the 13rd after lotus knurl, starts with vernier caliper within 16,18,23,27,30,33 days, surveys the maximum diameter (a) of mouse tumor And most path (b), utilize formula V=1/2ab2(a is major diameter, and b is minor axis) calculates each group tumor average volume, draws each group and swells Tumor tissue growth curve (is specifically shown in Fig. 2)；Compared to first group IgG Isotype control group, the growth of other group of tumour are apparent It is suppressed (P<0.0001).It takes out tumor tissues and is observed within the 34th day after lotus knurl, as seen from Figure 3, PD-1+D018 groups are swollen Knurl size is far smaller than other groups.Data above synthesis shows that agonist D018 and R848, PD-1 antibodies on tumor have inhibition Effect, wherein, agonist D018 will be significantly better than activator group R848 and PD-1 antibody group to the therapeutic effect of tumour, especially It is that gross tumor volume maintains very low level always after PD-1 and D018 combinations, and is significantly less than other each groups, shows PD-1 + D018 tumor killing effects are optimal.Wherein 25 μ g groups self administration of medication of α PD-1+D018 processing beginning knurl volume is constantly in slowly growth very To being growth retardation state, therefore it is considered that the tumor killing effect of D018 there are apparent dose dependents between 10-25 μ g. D018 administration groups have better tumor killing effect and dose dependent compared with R848 administration groups, illustrate the drug of TLR7 agonists Curative effect will be significantly better than the R848 of commercialization.
3rd, infiltrating lymphocytes detect in tumor tissues
Tumor tissues infiltrating lymphocytes be made of panimmunity cell (CD4+T cells, CD8+T cells, NK cells, NKT cells etc.), key effect is played in tumour immunity.Antitumor immune function is played for further clear and definite agonist D018 Mechanism, we using Miltenyi mouse tumor dissociation kit and gentleMACSTMOcto Dissociator instrument Device prepares the single cell suspension of mouse tumor.Part is taken out to this single cell suspension and carries out FITC- anti-CD 4 antibodies, mark PE- The streaming dyeing of FOXP3 antibody, the anti-CD8 antibody of mark percp-, four color of mark APC- anti-CD 25 antibodies, with flow cytometer point The ingredient of tumor infiltrating lymphocyte therein is analysed, such as：CD4+T cells, CD8+T cells and Treg cells.Flow cytometer detection is shown (being specifically shown in Fig. 4).
CD8+T lymphocytes are also known as the main effects cell that Cytotoxic T lymphocytes (CTLs) is immune response, can quilt The compound that endogenous antigen peptide and MHCI quasi-molecules are formed activates, and specific killing target cell is simultaneously swollen in viral infection resisting with anti- It plays a significant role in knurl immune response.We set each mouse CD4+T cell numbers 10000 as door, each so as to uniform Sample, quantity PD-1+D018 (the 25 μ g) groups and PD-1+R848 (25 μ g) for analyzing CD8+T cells in each group loading organize tumor group Interior CD8+T quantity showed increased (Fig. 5) compared with other groups is knitted, while this two groups of mice tumors grew speed also significantly slow. CD8+T cells directly decide that mouse tumor is immunized as effector cell crucial in immunotherapy of tumors, the number of quantity Intensity and prognosis.It is limited to understand that α PD-1 and D018 independent role influence the quantity of CD8+T cells by result, and the two joins Sharing medicine but can significantly improve this state, thus it is speculated that may be that the agonist of TLR7 can enhance the quantity of T cells, increase The chance of its strong contact mouse tumor antigen, but these T cell fast deactivations for some reason, and when we add to mouse After entering α PD-1, the quantity of effector T cell can be obviously prolonged, therefore mouse tumor is greatly improved after the two drug combination Immune microenvironment, enhance immunological rejection of the mouse to tumour.
CD 8+ in PD-1+D018 (25 μ g) group, PD-1+R848 (10 μ g) groups and PD-1+R848 (25 μ g) group tumor tissues T/Treg ratios are higher (Fig. 6), while this 3 groups of mice tumors grew speed also significantly slow.The immune microenvironment of tumour except Outside the T cell of responsiveness, there is the regulatory T-cell of some inhibitions such as Treg cells, current research show tumour Immune microenvironment cannot only see the quantity of effector T cell, should also see both effect T cells and Treg cells in microenvironment Ratio, mean that immune microenvironment develops towards the direction for inhibiting tumour growth as ratio increases, meaned if ratio reduces The growth of tumour can be promoted by microenvironment.We compare the ratio of each group CD8+T cells/Treg cells therefore, can by figure Know 25 μ g groups of α PD-1+D018 its CD8+T cells/Treg cell compared with IgG Isotype control groups and α PD-1 groups Ratio be all significantly improved, p value is respectively p=0.0007 and p=0.0008, illustrates that drug combination group is micro- to tumour immunity There is the apparent positive inhibition for adjusting promotion mouse to tumour in environment.25 μ g groups of α PD-1+ R848 and IgG Isotype Control groups are also significantly improved compared to the ratio of its CD8+T cells/Treg cell, p value p=0.0172.And it individually gives Medicine group α PD-1,25 μ g groups of R848 25 μ g and D018 have no significantly immune enhancement of environment compared with the control group.
4th, the expression of cellular immunofluorescence detection PDL-1
The tumor tissues the being stripped out mouse tumor of Miltenyi is dissociated into kit and gentleMACSTMOcto After Dissociator instruments are prepared into the single cell suspension of tumour, the streaming that part carries out mouse is taken out to this single cell suspension PD-L1 antibody dyes, and washes the cell handled twice after dyeing half an hour, then filtering and upper machine testing is resuspended with 300ulPBS.
Its expression quantity is stepped up PD-1 General Expressions in the T cell of activation, and with the activation of T cell.As the T of activation Cell runs into the inactivation that its ligand PD-L1 then causes T cell, therefore the expression of PDL-1 also contributes to swell in tumor tissues Effector T cell quantity and function in microenvironment is immunized in knurl.We have detected the expression of tumor cell surface PD-L1 therefore (being specifically shown in Fig. 7).IgG Isotype control, α PD-1,25 μ g α PD-1+ D018 of D018,25 μ g are understood by result, respectively Group tumor cell surface has the expression of PD-L1, and α PD-1, D018 25 μ g, α compared with IgG Isotype control The gate of 25 μ g of PD-1+D018 expression PD-L1 is gradually deviated to the right, that is to say, that expression PD-L1 cells gradually increase.With this We analyze the mean values of each group simultaneously, and it is bright to understand that 25 μ g of α PD-1+ D018 mean compared with other groups have by result Aobvious raising, the reaction of mean values be the amount expressed on individual cells number, therefore that is 25 μ g groups of α PD-1+D018 Oncocyte on expression PD-L1 amounts be that compare p value with α PD-1 groups be respectively p=to its highest IgG Isotype control 0.0148 and p=0.0141.The PD-L1 expression of 25 μ g groups of α PD-1+D018 significantly improve with result not contradiction before, More and more researchs at present show patient that PDL-1 expression increases its receive α PD-1 treatments after there is better clinical treatment to imitate Fruit may have relation with more sensitive to α PD-1 treatments after the high expression of PD-L1.
5th, the detection of tumor-bearing mice Cytokine of Serum
Mouse put to death after, whole blood with No. 21 syringe needles of 1ml syringes in cardiac puncture collect blood, be placed at room temperature for 1 it is small when make Its condensation is blocking.3000 turns of collected after centrifugation serum.Storing frozen (- 80 DEG C) after serum harvest.ELISA detects murine interleukin Interleukin 2 (IL-2), the index of detection mouse IFN-γ, TNF-α and IL-6, each three secondary orifices of sample.By agents useful for same before experiment Place room temperature (18-25 DEG C) rewarming；Board-washing：The ELISA Plate being coated with is placed on board-washing machine and is rinsed 5 times with washing lotion；Then filtering Tapping on paper removes surplus liquid；Sample-adding：Add 100ul cell culture supernatant samples per hole, be placed in 37 DEG C of incubators, be incubated 1h； Board-washing：ELISA Plate is placed on board-washing machine and is rinsed 5 times with washing lotion, the then tapping on filter paper removes surplus liquid；Add Detection Ab：100ul (1 is added in per hole:200) diluted detection antibody；37 DEG C of incubators are placed in, are incubated 1h；Board-washing：By enzyme Target is placed on board-washing machine to be rinsed 5 times with washing lotion, and the then tapping on filter paper removes surplus liquid；Add Aiding-HRP：It adds in 100ul(1：1000) Aiding-HRP is diluted；37 DEG C of incubators are placed in, are incubated 30min；Board-washing：ELISA Plate is placed on board-washing machine It is rinsed 5 times with washing lotion, the then tapping on filter paper, removes surplus liquid；Substrate develops the color：100ul Substrate are added in per hole Solution F (are protected from light), 15min；It terminates：100ul terminate liquids are added in per hole and terminate reaction；Detection：450nm in microplate reader, OD values are measured in 30min, the cytokine concentrations in sample are calculated in the regression equation according to obtained by standard curve.
We have detected the various cell factors (the result is shown in Fig. 8) in mouse systemic blood, find drug combination group mouse The level of interleukin-22 is significantly raised in serum, interleukin-22 as stimulate and the cell factor of the differentiation and proliferation of maintenance T cell its Horizontal apparent increase functions most important for the effector cell in tumour immunity microenvironment.Drug combination group interleukin 6 level is significantly improved compared with control group, can stimulate the cell Proliferation for participating in immune response, differentiation and improve its work( Energy.The level of drug combination group interferon gamma is higher, and interferon has antiviral, immunological regulation and antitumor properties.Joint It is minimum in each group that medication group, which has the negative sense immunological regulation IL-10 level of inflammation and immunosuppressive factor,.Summarize with On, drug combination group is to be intended to improve the immune response level of body for the immune state of whole body, promotes immune system Killing to tumour.
6th, influences of the agonist D018 to levels of cytokine secretion in macrophage
The macrophage derived bone marrow cell in wild type C57BL/6, after taking-up in the conditioned medium containing M-CSF Culture obtains after 5 days.After culture period, bed board carries out cell experiment to adherent cell collecting again.Real-time quantitative PCR point The macrophage of wild type C57BL/6 bone marrow deriveds is analysed in different time and D018, R848 and Poly (I of various dose:C) It stimulates the isogenic mRNA of lower Il1b, Il6, Il12b, Tnf, Ifnb1, Cxcl1, Cxcl10 and Il10 horizontal, uses house keeper's base Because each sample is normalized in GAPDH.
BMDMs Il1b, Il6, Il12b in D018 (0.023 μ g/ml) or R848 (10 μ g/ml) are understood by result figure 9 Three kinds of cell factors are obviously improved when stimulating 1h, reach peak value in 6h.We use D018 (0.023 μ g/ ) or R848 (10 μ g/ml) or polyI ml:C (1 μ g/ml), respectively have detected stimulate BMDMs 0,1,3,6 small when after tnf Level, when stimulation the 1st is small, tnf has just directly reached peak, and factor level begins to decline since when the 3rd is small, and the 6th is small When just dropped it is very much.The D018 of 0.023 μ g/ml is stimulated from result it can be seen that compared with other two stimulant The level that BMDMs generates tnf is highest, and the dose active for illustrating D018 is that minimum activity in other words is highest.D018 (0.023 μ g/ml) or R848 (10 μ g/ml) stimulate BMDMs1 it is small when after Ifnb1, Cxcl1, Cxcl10 and Il10 arrived most High point is fallen after rise factor level since when the 6th is small.Tri- cell factor of Ifnb1, Cxcl1, Cxcl10 is substantially all D018 Activity be better than R848, and the activity of R848 is some higher when stimulating Il10.As a result confirm BMDMs in D018 (0.023 μ G/ml) or R848 (10 μ g/ml) stimulates the genes such as lower Il1b, Il6, Il12b, Tnf, Ifnb1, Cxcl1, Cxcl10 and Il10 MRNA level in-site be obviously improved, illustrate that D018 horizontal in vivo can generate mouse systemic immune system beneficial shadow It rings.
7th, influence of high-flux sequence detection of agonist D018 and PD-1 the antibody combination to tumor cell gene expression
Various different cell or tissues are harvested, is centrifuged and washed 3 times under conditions of 1000rpm, 5min with PBS.Extraction is total RNA, total rna concentration are measured through NanoDrop (ND-lOOO) UV detector, and RNA concentration is not less than 40ng/m, purity It is required that 0D 260/280 is more than 1.8.Each sample takes 5g, inverse with Reverse transcriptTM kit (Invitrogen) Transcribe synthesizing single-stranded cDNA library.Carry out high-flux sequence.
In order to further disclose the mechanism of drug combination, invention has carried out RNA-seq to the tumour of each group mouse, has as a result shown Show drug combination so that a series of variation has occurred in the gene expression of mouse tumor, raise inflammation and cytokine mediated Access, t cell activation, apoptotic signal access, B cell activation pathway etc. are conducive to the number gene of immunotherapy of tumors, another Aspect its infiltration of the immunocyte to tumor tissues is promoted to the downward of Wnt signal paths.We use glimmering under prompting herein Fluorescent Quantitative PCR method is verified.
It has been reported that the expression of β-catenin signal negative regulation Chemokines CCs cl4, Ccl4 can mediate DC cells to Tumor tissues infiltrate.Therefore activation β-catenin can cause the downward that Ccl4 is expressed, and be soaked so as to inhibit DC cells to tumor tissues Profit migration influences the t cell activation that DC cells are mediated.It can be seen that and IgG Isotype from our result at present Control is compared, and Ccl4 gene expressions are all raised in α PD-1 groups, 25 μ g and α PD-1+D018 of D018,25 μ g groups, but very It is apparent that the level of 25 μ g group Ccl4 gene expressions of drug combination α PD-1+D018 will be apparently higher than other groups.Therefore we The gene expression dose (see Figure 10) of β-catenin in each group is further had detected, from the results, it was seen that compared with the control group Apparent downward all has occurred in α PD-1, the gene expression dose of 25 μ g group β-catenin of D018 25 μ g and α PD-1+D018, Wherein α PD-1 and 25 μ g groups of α PD-1+D018 lower more obvious, 25 μ g groups of α PD-1+D018 so low-level expression The phenotypes that immune effector cells quantity increases and knurl volume is substantially suppressed such as β-catenin and its CD8+T cell are complete Consistent.
8th, IHC detects influence of the D018 and PD-1 antibody combination to ccl4 and β-catenin expression
It is as follows：1. it dewaxes：Organization chip, which is placed in dimethylbenzene, to be impregnated 20 minutes, is renewed fresh dimethylbenzene and is repeated one It is secondary；2. rehydration：Histotomy after dewaxing is impregnated 5 minutes 2 times in 100% ethyl alcohol, 95% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, 70% ethyl alcohol, distilled water respectively impregnate 5 minutes × 1 time；3. antigen retrieval：Antigen retrieval buffers are citric acid-citric acid Antigen retrieval buffers microwave high-temperature is heated to seething with excitement, section is put by sodium buffer solution, then is heated 5 minutes with the high fire of microwave, is added Distilled water is heated 5 minutes after recovering volume with microwave high-temperature again, cooled to room temperature；4. remove endogenous peroxydase： Fresh configuration 3%H2O2 drips piece, is protected from light incubation 10 minutes at room temperature；5. it closes：20 are closed with normal sheep serum working solution room temperature Minute；6. primary antibody reacts：Piece is dripped with 10 μ g/mL rabbit-anti people PHF23 primary antibodies (being diluted with PBS), when 37 DEG C of incubations 1 are small, control group Primary antibody is replaced with PBS；PBS is washed 5 minutes × 3 times；7. secondary antibody reacts：HRP- goat anti-rabbit antibodies 1：200 dilutions (being diluted with PBS), Room temperature reaction 30 minutes；8. PBS is washed 5 minutes × 3 times；9. benzidine ammonia (DAB) develops the color, piece, Microscopic observation reaction knot are dripped Fruit；10. haematoxylin redyeing nucleus, tap water oil blackeite；Ethyl alcohol uplink is dehydrated：50% ethyl alcohol 5 minutes, 75% ethyl alcohol 5 minutes, 95% ethyl alcohol 5 minutes, 100% ethyl alcohol 5 minutes × 2 times；Neutral gum mounting observes and records result under microscope.
It is partly the protein expression by reducing β-catenin to further confirm the effective mechanism of drug combination So as to reduce its inhibitory action to Ccl4 genes so that more Ccl4 play it and DC cells is promoted to infiltrate and swash to tumor tissue The effect of T cell living, we have detected IgG Isotype control, α PD-1,25 μ of D018 using the method for immunohistochemistry The protein expression level (see Figure 11) of Ccl4 and the β-catenin of 25 μ g each groups of g and α PD-1+D018, by result understand with it is right Rise successively in α PD-1,25 μ g groups of D018 25 μ g and α PD-1+D018 according to protein expression level of the group compared to Ccl4, and The protein expression level of β-catenin in control, α PD-1,25 μ g and α PD-1+D018 of D018,25 μ g each groups successively It reduces.As a result as it is anticipated that illustrating that the funtion part that drug combination plays is played a role by this access.Ccl4 becomes The property changed cytokine receptor promotes the infiltration of DC and T cell to tumor tissues so that the chance of DC cells contact tumour is more, more Good T cell tumour antigen deducted a percentage to lymphoid tissue so that the quantity of tumor specific lymphocytes increases in the circulatory system More, body enters the benign cycle of tumor-specific immunity, accelerates removing of the body to tumor tissues.
9th, each experimental group influences mice with tumor life cycle
We will be divided into mouse IgG Isotype control, α PD-1,25 μ g of R848, α PD-1+R84810 μ g, α 25 μ g of PD-1+R848, D018 25 μ g, 10 μ g of α PD-1+D018,8 groups of 25 μ g of α PD-1+D018, every group of 10 mouse, The tumour of tumor-bearing mice grows to PD-1 monoclonal antibodies after 100mm3 and corresponding drug therapy 200 μ g/mouse was carried out every 3 days, and TLR7 swashs Dynamic agent was divided into two dosage groups of 10 μ g and 25 μ g every 7 days immune courses for the treatment of.
It is other compared with IgG Isotype control (see Figure 12) according to the growth curve of each group mouse tumor Administration group mouse tumor has different degrees of diminution, and wherein 25 μ g groups self administration of medication of α PD-1+D018 processing starts knurl volume one Straight be in slowly grows even growth retardation state, compared with the control group p value<0.001.Either D018 or R848, Being administered alone group and therapeutic alliance group plays the role of significantly inhibiting tumour growth, and wherein administering drug combinations have more obviously suppression Knurl acts on, and there are apparent dose dependents between 10-25 μ g for its tumor killing effect.D018 is administered compared with R848 administration groups Group have better tumor killing effect and dose dependent, it is known that in MC38 groups at 33 days IgG Isotype control existence α PD-1 are 40% when rate is 0,41 day, D018 90%, α PD-1+D018 are 100%.This illustrates TLR7 agonists of the present invention Curative effect of medication will be significantly better than the R848 of commercialization.
Tenth, influence of the drug combination to immunological memory
Whether it is with anti-PD-1 and D018 connection in order to further determine the anamnestic response mediated for the T cell of tumour antigen The immune response that treatment triggers is closed, we have selected to receive to refuse the mouse (nothing of MC38 tumours after PD-1 and D018 therapeutic alliances Tumour ＞ 30 days).With 1 × 106MC38 cells are in the side-to-side inoculation of primary vaccination.
As a result the mouse tumor of visible (see Figure 13) those primary vaccinations is grown quickly, and those are refused after receiving therapeutic alliance The mouse of exhausted MC38 tumours is then fully against the pressure for having lived MC38 cells.These results clearly demonstrate, PD-1 and D018 Therapeutic alliance can cause lasting systemic immunity to remember to prevent to recur.
11, agonist D018, PD-1 antibody and TIM-3 antibody are combined the influence to mice tumors grew
In order to how probe into effect associated with D018 and other immune antiboidies, we select 6-8 week old females C57BL/6 small Mouse subcutaneously injects MC38 respectively in right side, builds bearing mouse model.Mouse is then divided into IgG Isotype control
, α Tim-3, D018, α PD-1+D018, α Tim-3+D018, α PD-1+D018+ α Tim-3 groups, it is every group 10 small Mouse is sorted at random after the tumour of tumor-bearing mice grows to 100mm3 as above 6 groups.PD-1 monoclonal antibodies carried out corresponding medicine every 3 days 200 μ g/ of object treatment only, TLR7 agonists every 7 days immune courses for the treatment of, every 3 days with vernier caliper measurement tumor size once.
It is understood according to the growth curve (Figure 14) of each group mouse tumor, it is other compared with IgG Isotype control to give Medicine group mouse tumor has different degrees of diminution, and wherein α PD-1+D018, the processing of α PD-1+D018+ α Tim-3 groups self administration of medication are opened Beginning knurl volume is constantly in slowly growth even growth retardation state, and there were significant differences compared with the control group.As seen from the figure Simple α Tim-3 administrations there are no apparent therapeutic effect, but it is after D018 therapeutic alliances with significantly improving to knurl volume The ability of inhibition.Therefore, D018 and α PD-1 and α Tim-3 antibody individually combine and triple combination treats and has apparent inhibition The effect of tumour growth, wherein two kinds of immune detection point administrations of joint have most apparent tumor-inhibiting action.
12, the foundation of Ag104Ld mouse tumor models and inhibiting tumor assay
Using Ag104Ld fibrocytes in this experiment, it is incubated in the DMEM culture mediums containing 10%FBS, in 5%CO2, It is cultivated in 37 DEG C of incubator.Routine passage pancreatin 0.25%Trypsin-EDTA (GIBCO:25200-056), 1- is digested 3min.Cell changes weekly liquid 2-3 times after passage.3 days cells pass on once before kind of knurl, and the training more renewed in one day before the injection Nutrient solution.Take survival rate 80% 1 × 105A Ag104Ld cell infusions are subcutaneous in 6-8 week old female CH1 right side of mice.
Experiment packet：
The observation of mice tumors grew situation (method is with content two).According to the literature, the PD-L1 of Ag104Ld cells Expression is similar to MC38, but it is weak in MC38 tumor models to the response ratio of PD-1 antibody, therefore IgG Isotype Control and PD-1 groups are not different.In our experiment as seen from Figure 15, PD-1 antibody is used alone and control group is true Real indifference, and D018 is used alone in treatment group just good tumor killing effect, with PD-1+D018 combination groups without significance difference It is different.This illustrates agonists of the D018 as TLR7, can activate corresponding access and generate a series of inflammatory factor, make The level of inflammation of the tumor microenvironment of Ag104Ld tumor model mouse improves, so as to more inhibit tumour using transferring immune response Growth.
13, infiltrating immune cells detect in Ag104Ld tumor models mouse tumor tissue
It can be in other tumor models, such as to PD-1 for further clear and definite agonist D018 and D018 and the combination of PD-1 antibody The weak tumor type of Antybody therapy response, can also play antitumor effect, our isolated Ag104Ld tumor models are small The single cell suspension of mouse tumor tissues.To this single cell suspension mark anti-CD45 antibody of APC cy7-, the anti-CD4 of flag F ITC- The streaming dyeing of antibody, mark PE-FOXP3 antibody, the anti-CD8 antibody of mark percp-, four color of mark APC- anti-CD 25 antibodies, is used The ingredient of flow cytometry analysis tumor infiltrating lymphocyte therein, such as：CD4+T cell, CD8+T cells and Treg cells, Flow cytometer detection shows and (is specifically shown in Figure 16).It is dyed with CD45 antibody, CD103 antibody, CD11c antibody streaming, analyzes wherein CD103+DC cells, flow cytometer detection shows and (is specifically shown in Figure 17).It is dyed with CD45, CD4, CD8, CD44, CD62L antibody streaming, point Analysis wherein memory t cell, center memory T cells (Tcm), effect memory T cells (Tem), flow cytometer detection show (specific See Figure 18).
CD8+T cell directly decides mouse as effector cell crucial in immunotherapy of tumors, the number of quantity The intensity of tumour immunity and prognosis situation.Analyze infiltration CD8 in Ag104Ld tumor model mouse each groups tumor tissues+T cell Quantity discovery, PD-1+D018 groups CD8+T quantity showed increased (figure compared with IgG Isotype control groups and α PD-1 groups 15), p value is respectively p=0.0302 and p=0.013.Its CD8+The ratio of T cell/Treg cells is also significantly improved, together When this two groups of mice tumors grew speed also significantly slow.
DC is the very capable full-time presenting cell of antigen submission, can start the reaction of T lymphocytes primary immune response.It grinds Study carefully and show that DC expresses a variety of phenotype molecules, CD11c is one of its specific marker object.CD103+DC is from intestinal mucosa by antigen Lamina propria is transported to the major modulatory DC subgroups of Peyer patches and lymphonodi mesenterici, can stimulate the T lymphs of activation Cell expresses 4 β of molecule alpha, 7 integrins of going back to the nest, so as to mediate the position that T lymphocyte homings are invaded to antigen.And CD103+ DC and CD8+T infiltrations are closely related to tumor microenvironment, therefore we have detected CD103 in each group+The content of DC.It can by result Know α PD-1+D018 and D018 independent roles group compared with the control group, there is a significant change, p value be respectively p=0.0404 and P=0.0102.
T cell is proliferated and is broken up, it is thin to form two functionally different classes by after the antigenic substance activation of specificity Born of the same parents, i.e. T immune effector cells and memory T cell.Wherein memory T cell then can be when antigen next time be invaded by supporting in memory Imperial mechanism is transferred out, destroys target cell again.Memory T cell includes central memory T cells (Tcm) and effect memory-type again T cell (Tem).The biomarker of Tcm is the double positive cells of CD62L and CD44, and this represent Tcm can pass through lymph screen It covers, returns lymph node, while in by the state of antigenic activation.Tcm cells have self-renewing and replication capacity, deposit in vivo Live time is long, can play long-term antitumor action.At present, Tcm cells can be carried by the monitoring of quantification for clinical procedureization treatment Index for reference.Tem is the Tcm cells being activated, under the stimulation again of antigen, can continue to generate largely carry it is of the same race The effect memory T cells of the cloning of antigen, the biomarker of Tem is CD62L-And CD44+Cell.Therefore we detect Tcm and Tem in tumor microenvironment, according to result (Figure 18) as it can be seen that compared with the control group, Tcm is in α PD-1+D018 and D018 Individually there is significant change, p value is respectively p=0.0454 and p=0.0359.This illustrate D018 drugs be used alone or Use in conjunction immunologic test point blocking antibody can make mouse generate apparent Tcm, carry out secondary immunity defence.
The description of the above-mentioned specific exemplary embodiment to the present invention is in order to illustrate and illustration purpose.These descriptions It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed And variation.The purpose of selecting and describing the exemplary embodiment is that explain that the certain principles of the present invention and its reality should With so that those skilled in the art can realize and utilize the present invention a variety of exemplary implementation schemes and Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.

Claims (20)

1. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of formula (I),
Wherein,
L represents linking group, to be not present or being C1-C6Alkylidene or C1-C6Alkenylene, wherein the group is optionally By C1-C4Alkyl substitutes；
R1It representsOr halogen, wherein R3Selected from H, halogen, aminomethylene, (substituted-amino) Methylene or its quaternary ammonium salt, the methylene of heterocyclic substituted, nitrine methylene, phosphonic acids methylene, diethyl phosphonate methylene, Methoxyl group acyl group, C1-C4Alkyl, C1-C4Alkoxy, CF3-、-COOH、-COOCH3、-CN、HO-CH2；
R2Expression-YR4, wherein Y represent N, S, O, R4Represent C1-C4Alkyl or C1-C4Alkoxy ,-YR4In Y and R4It can be with shape Into heterocycle, heterocycle can be morpholine or piperidines；
Substituted-amino can be dimethylamino, Leu- amino, Leu Ala Ala Asn- amino, (trifluoromethyl) methylene ammonia Base, methylamino；
Quaternary ammonium salt is quaternary ammonium salt；
Heterocycle is selected from
Halogen is fluorine, chlorine.
2. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt, feature of formula (I) according to claim 1 exist In：The L is C1-C6Alkylidene is preferably-CH2-。
3. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt, feature of formula (I) according to claim 1 exist In：The R1For
4. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt, feature of formula (I) according to claim 1 exist In：The R2For-NR4。
5. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of formula (I) according to claim 1, the compound choosing From：
6. the Pyridopyrimidine derivatives of formula (I) or the preparation method of its pharmaceutically acceptable salt, whereinStep Including：
(1) using -5 Bromopicolinic acid methyl esters of 3- amino and chlorine amitraz hydrochloride as starting material, dimethyl sulfone is added in, chemical combination is made Object 1, reaction equation are：
(2) compound 1 is taken to be dissolved in aceticanhydride, obtains compound 2, reaction equation is：
(3) at room temperature, compound 2 is taken to be dissolved in dioxane, DMF is added in, oxalyl chloride is then added dropwise, obtains compound 3, reaction equation For：
(4) compound 3 obtained by step (3) is directly dissolved in dry tetrahydrofuran, adds in organic compounds containing nitrogen or sulfur-bearing Organic compound or oxygen-containing organic compound, then add in n,N-diisopropylethylamine, obtain compound 4, and reaction equation is：
(5) compound 4 is taken to be dissolved in methanol, adds in LiOHH2O, is made compound 5, and reaction equation is：
(6) under nitrogen protection, compound 5 is taken to be dissolved in dry tetrahydrofuran, sequentially adds substitution benzyl bromine, zinc powder and Ni (PPh3)2Cl2, compound 6 is made, reaction equation is：
7. the Pyridopyrimidine derivatives of formula (I) according to claim 6 or the preparation side of its pharmaceutically acceptable salt Method, it is characterised in that：The organic compounds containing nitrogen is alkylamine, piperidines, alkoxyamine, preferably morpholine, n-butylamine；Sulfur-bearing has Machine compound is alkyl hydrosulfide, preferably n-butyl mercaptan；Oxygen-containing organic compound is alkylol, preferably n-butanol.
8. the Pyridopyrimidine derivatives of formula (I) according to claim 6 or the preparation side of its pharmaceutically acceptable salt Method, it is characterised in that：The substitution preferred 3- methyl benzyl bromine of benzyl bromine.
9. a kind of pharmaceutical composition, including：The Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of formula (I) and pharmaceutically Acceptable auxiliary material.
10. pharmaceutical composition according to claim 9, the composition is described also containing at least one other therapeutic agents Therapeutic agent is selected from chemotherapeutics, immunization therapy, anti-angiogenic agent, cell factor, hormone, polynucleotides, antibody, immunologic competence Segment.
11. pharmaceutical composition according to claim 10, which is characterized in that the antibody resists for PD-1 and/or TIM-3 Body, it is preferred that composition for the Pyridopyrimidine derivatives of PD-1 antibody and TIM-3 antibody and formula (I) or its can pharmaceutically connect The salt received.
12. a kind of method for improving body forward direction immune response, including：Therapeutically effective amount is given to system in need or individual The Pyridopyrimidine derivatives of claim 1-6 any one formula (I) or its pharmaceutically acceptable salt, pharmaceutical composition, Or compound prepared by the pharmaceutical composition described in claim 9-11 or the method according to claim 6-8, so as to adjust Save TLR7 and/or TLR8.
13. a kind of method for enhancing chemotherapy effect, the chemotherapeutic including giving therapeutically effective amount to system in need or individual Object subsequently or simultaneously gives the Pyridopyrimidine derivatives or its medicine of the claim 1-6 any one formula (I) of therapeutically effective amount Pharmaceutical composition on described in acceptable salt, pharmaceutical composition or claim 9-11 or according to claim 6-8 institutes Compound prepared by the method stated.
14. a kind of method for improving immunotherapy, including：The right that therapeutically effective amount is given to system in need or individual will Ask Pyridopyrimidine derivatives or its pharmaceutically acceptable salt of 1-6 any one formula (I), pharmaceutical composition or right will The compound that the pharmaceutical composition described in 9-11 or the method according to claim 6-8 is asked to prepare, subsequently or simultaneously will be embedding Close antigen receptor T cell input system or a internal.
15. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or right of claim 1-6 any one formula (I) will The pharmaceutical composition described in 9-11 any one is asked to be used to prepare treatment communicable disease, respiratory disease, immune related disease Application in disease, virosis or tumour medicine.
16. application according to claim 15, which is characterized in that the tumour includes but not limited to：Fibrosarcoma, muscle Knurl, embryonal-cell lipoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphatic endothelial Sarcoma, synovialoma, celiothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, prostate cancer, squamous are thin Born of the same parents' cancer, basal-cell carcinoma, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, cephaloma, bronchus Cancer, hepatoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, the nephroblastoma, cervical carcinoma, testicular tumor, Lung cancer, Small Cell Lung Cancer, epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal body Knurl, hemangioblastoma, acoustic neurinoma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retina are female Cytoma, leukaemia, polycythemia vera, lymthoma, Huppert's disease；Preferably, tumour is cold tumour, more preferably , tumour is colon cancer, carcinoma of urinary bladder, melanoma, meningioma, lung cancer, liver cancer or cancer of pancreas；The viral disease and correlation Virus includes but not limited to：Ebola disease viral disease, anthrax-bacilus disease, condyloma acuminatum, simple wart, plantar wart, Respiratory Syncytial Virus(RSV), Hepatitis B, hepatitis C, dengue fever virus, herpes simplex virus, molluscum contagiosum, cowpox, smallpox, slow virus, people are immunized Defective virus, human papilloma virus, cytomegalovirus, varicella virus, rhinovirus, enterovirus, adenovirus, stream Sense, parainfluenza, mumps virus, measles virus, papovavirus, flavivirus, retrovirus, arenavirus；Autoimmunity Disease includes but not limited to：Systemic loupus erythematosus, Si Yegelun syndrome, wegener granulomatosis, sarcoidosis, Lai Te synthesis Sign, Behcet syndrome；Respiratory disease includes but not limited to：Asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome are comprehensive Simulator sickness.
17. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or right of claim 1-6 any one formula (I) will The pharmaceutical composition described in 9-11 any one is asked to be used to prepare the application in vaccine adjuvant.
18. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or right of claim 1-6 any one formula (I) will Ask application of the pharmaceutical composition described in 9-11 any one in pharmaceutical preparation is prepared.Preferably, the pharmaceutical preparation up-regulation There are interests in each access such as inflammation and cytokine mediated access, TXi Baojihuo apoptotic signals access, B cell activation pathway The number gene of immunization therapy.It is furthermore preferred that the pharmaceutical preparation up-regulation inflammation and cytokine mediated access, T cell swash Be conducive to the number gene of immunotherapy of tumors in work, apoptotic signal access, B cell activation pathway.
19. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or right of claim 1-6 any one formula (I) will Seek each levels of cytokine secretion pharmaceutical preparation in influence macrophage is prepared of the pharmaceutical composition described in 9-11 any one In application, it is preferred that influence the isogenic mRNA water of Il1b, Il6, Il12b, Tnf, Ifnb1, Cxcl1, Cxcl10 and Il10 The pharmaceutical preparation of flat expression.
20. the Pyridopyrimidine derivatives or its pharmaceutically acceptable salt or right of claim 1-6 any one formula (I) will The pharmaceutical composition described in 9-11 any one is asked to be used to prepare and is preparing the application in preventing tumor recurrence drug.
CN201711146690.1A 2017-11-17 2017-11-17 Pyridopyrimidine derivative or salt thereof, and preparation method, pharmaceutical composition and application thereof Active CN108069963B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201711146690.1A CN108069963B (en) 2017-11-17 2017-11-17 Pyridopyrimidine derivative or salt thereof, and preparation method, pharmaceutical composition and application thereof
PCT/CN2017/114611 WO2019095455A1 (en) 2017-11-17 2017-12-05 Pyridopyrimidine derivatives or salts thereof, preparation method therefor, and pharmaceutical composition and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711146690.1A CN108069963B (en) 2017-11-17 2017-11-17 Pyridopyrimidine derivative or salt thereof, and preparation method, pharmaceutical composition and application thereof

Publications (2)

Publication Number Publication Date
CN108069963A true CN108069963A (en) 2018-05-25
CN108069963B CN108069963B (en) 2020-01-14

Family

ID=62157252

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711146690.1A Active CN108069963B (en) 2017-11-17 2017-11-17 Pyridopyrimidine derivative or salt thereof, and preparation method, pharmaceutical composition and application thereof

Country Status (2)

Country Link
CN (1) CN108069963B (en)
WO (1) WO2019095455A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114685491A (en) * 2020-12-31 2022-07-01 清华大学 Pyridine-2-amine derivative and pharmaceutical composition and application thereof
WO2022237884A1 (en) * 2021-05-13 2022-11-17 清华大学 Antibody drug conjugate, preparation method therefor and application thereof
CN115403510A (en) * 2022-08-11 2022-11-29 南方医科大学 PD-L1/CXCL12 double-target-point inhibitor, preparation method and application
CN115607550A (en) * 2021-07-14 2023-01-17 清华大学 Application of TLR7/8 agonist in inhibiting HIV
WO2023078241A1 (en) * 2021-11-05 2023-05-11 中国医药研究开发中心有限公司 Aromatic amine derivatives and preparation method and medical use thereof
WO2023160527A1 (en) * 2022-02-24 2023-08-31 广东东阳光药业股份有限公司 Pyrimido aromatic ring compound and use thereof in drug
CN117362286A (en) * 2023-12-08 2024-01-09 清华大学 Compounds with SIRT6 agonistic activity and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021185312A1 (en) * 2020-03-18 2021-09-23 正大天晴药业集团股份有限公司 Crystalline form of tlr8 agonist

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006135993A1 (en) * 2005-06-24 2006-12-28 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines and pharmaceutical compositions useful for treating hepatitis c.
WO2008077650A1 (en) * 2006-12-26 2008-07-03 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines useful for treating viral infections
WO2008077651A1 (en) * 2006-12-26 2008-07-03 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines useful for treating viral infections
WO2009003669A2 (en) * 2007-06-29 2009-01-08 4 Aza Ip Nv Pyrido(3,2-d)pyrimidines and pharmaceutical compositions useful for medical treatment
CN101365699A (en) * 2004-12-30 2009-02-11 4阿扎Ip股份有限公司 Pyrido(3,2-D)pyrimidines and pharmaceutical compositions useful for medical treatment
CN107108615A (en) * 2015-03-04 2017-08-29 吉利德科学公司 The diamino-pyridine of Toll-like receptor modulability 4,6 simultaneously [3,2 D] pyrimidine compound

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170071944A1 (en) * 2015-09-15 2017-03-16 Gilead Sciences, Inc. Modulators of toll-like receptors for the treatment of hiv

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101365699A (en) * 2004-12-30 2009-02-11 4阿扎Ip股份有限公司 Pyrido(3,2-D)pyrimidines and pharmaceutical compositions useful for medical treatment
WO2006135993A1 (en) * 2005-06-24 2006-12-28 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines and pharmaceutical compositions useful for treating hepatitis c.
WO2008077650A1 (en) * 2006-12-26 2008-07-03 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines useful for treating viral infections
WO2008077651A1 (en) * 2006-12-26 2008-07-03 Gilead Sciences, Inc. Pyrido(3,2-d)pyrimidines useful for treating viral infections
WO2009003669A2 (en) * 2007-06-29 2009-01-08 4 Aza Ip Nv Pyrido(3,2-d)pyrimidines and pharmaceutical compositions useful for medical treatment
CN107108615A (en) * 2015-03-04 2017-08-29 吉利德科学公司 The diamino-pyridine of Toll-like receptor modulability 4,6 simultaneously [3,2 D] pyrimidine compound

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MENG WANG: "Synthesis and antiproliferative activity of a series of novel 6-substituted pyrido[3,2-d]pyrimidines as potential nonclassical lipophilic antifolates targeting dihydrofolate reductase", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 *
邢雅玲: "人Toll样受体靶向药物研究进展", 《中国生物工程杂志》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114685491A (en) * 2020-12-31 2022-07-01 清华大学 Pyridine-2-amine derivative and pharmaceutical composition and application thereof
CN114685491B (en) * 2020-12-31 2024-01-12 清华大学 Pyridin-2-amine derivatives, pharmaceutical compositions and uses thereof
WO2022237884A1 (en) * 2021-05-13 2022-11-17 清华大学 Antibody drug conjugate, preparation method therefor and application thereof
CN115607550A (en) * 2021-07-14 2023-01-17 清华大学 Application of TLR7/8 agonist in inhibiting HIV
WO2023078241A1 (en) * 2021-11-05 2023-05-11 中国医药研究开发中心有限公司 Aromatic amine derivatives and preparation method and medical use thereof
WO2023160527A1 (en) * 2022-02-24 2023-08-31 广东东阳光药业股份有限公司 Pyrimido aromatic ring compound and use thereof in drug
CN115403510A (en) * 2022-08-11 2022-11-29 南方医科大学 PD-L1/CXCL12 double-target-point inhibitor, preparation method and application
CN115403510B (en) * 2022-08-11 2023-04-25 南方医科大学 PD-L1/CXCL12 double-target inhibitor, preparation method and application
CN117362286A (en) * 2023-12-08 2024-01-09 清华大学 Compounds with SIRT6 agonistic activity and uses thereof
CN117362286B (en) * 2023-12-08 2024-03-12 清华大学 Compounds with SIRT6 agonistic activity and uses thereof

Also Published As

Publication number Publication date
WO2019095455A1 (en) 2019-05-23
CN108069963B (en) 2020-01-14

Similar Documents

Publication Publication Date Title
CN108069963A (en) Pyridopyrimidine derivatives or its salt and its preparation method, pharmaceutical composition and purposes
Yang et al. The impact of VEGF on cancer metastasis and systemic disease
KR102053556B1 (en) Drug composition and its application
EP2621500B1 (en) Therapeutic use of a tlr agonist and combination therapy
BR112020007249A2 (en) B cell maturation antigen-binding proteins
CN108883187A (en) SMC conjoint therapy use for cancer treatment
EP1674479A1 (en) Modulation of Fc Gamma receptors for optimizing immunotherapy
KR20160093012A (en) Combinations of checkpoint inhibitors and therapeutics to treat cancer
MX2012010367A (en) Methods for the treatment of non-hodgkin&#39;s lymphomas using lenalidomide, and gene and protein biomarkers as a predictor.
TW201200149A (en) New uses of an immunomodulatory protein (GMI) from Ganoderma microsporum
KR20220072855A (en) Compositions and methods for increasing the efficacy of immunotherapeutic agents and vaccines
CN103721255A (en) Application of co-blocking of PD-1 and TIM-3 signal paths to anti-stomach-cancer treatment
CN109996544A (en) Treatment of cancer combination
WO2016176620A1 (en) Extracellular matirix compositions for the treatment of cancer or immunological diseases
US11602554B2 (en) P53-targeting vaccines and pd-1 pathway inhibitors and methods of use thereof
US20230192846A1 (en) Anti-ror-2 antibodies and methods of use
US20240117047A1 (en) Monoclonal antibodies specific for human ror1
US20200199681A1 (en) Mda-7 cancer therapies and methods of detecting biomolecules
CN110038009A (en) Canagliflozin application in preparation of anti-tumor drugs
US20230340089A1 (en) Smc1a antibodies and uses thereof
WO2023023600A1 (en) Anti-related-to-receptor tyrosine kinase (ryk) antibodies and uses thereof
WO2022197866A1 (en) Anti-hvem antibodies
WO2022183069A9 (en) Development of prmt-targeting therapy to enhance egfr-targeting drug efficacy in nsclc
CN117979997A (en) Anti-receptor-associated tyrosine kinase (RYK) antibodies and uses thereof
JP2017075137A (en) Novel immunostimulant

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant