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WO2018137614A1 - 杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及其在医药上的应用 - Google Patents

杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及其在医药上的应用 Download PDF

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Publication number
WO2018137614A1
WO2018137614A1 PCT/CN2018/073815 CN2018073815W WO2018137614A1 WO 2018137614 A1 WO2018137614 A1 WO 2018137614A1 CN 2018073815 W CN2018073815 W CN 2018073815W WO 2018137614 A1 WO2018137614 A1 WO 2018137614A1
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Prior art keywords
group
compound
formula
cycloalkyl
alkyl
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English (en)
French (fr)
Inventor
张国宝
陈一千
贺峰
陶维康
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Priority to CN201880004318.3A priority Critical patent/CN109937205B/zh
Publication of WO2018137614A1 publication Critical patent/WO2018137614A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention belongs to the field of medicine, and relates to a novel heteroarylthiathiadiazine-2,2-dioxide derivative represented by the general formula (I), a preparation method thereof and a pharmaceutical composition containing the same And its use as a therapeutic, especially as a TLR7 agonist.
  • TLRs Toll-like receptors
  • TLRs are important protein molecules involved in innate immunity.
  • TLRs are non-catalytic receptors for monomeric transmembranes, usually expressed in sentinel cells such as macrophages and dendritic cells, and can recognize structurally conserved molecules produced by microorganisms. Once these microorganisms break through physical barriers such as the skin or intestinal mucosa, they are recognized by TLRs, which in turn activate immune cell responses (Mahla, R S. et al., Front Immunol. 4:248 (2013)). The ability of the immune system to broadly recognize pathogenic microorganisms is due in part to the widespread presence of Toll-like immunoreceptors.
  • TLR7 is a member of a subset of TLRs (TLRs 3, 7, 8, and 9) and is restricted to the endosomal compartment of cells that specifically detect non-hexucleic acids. TLR7 plays a key role in recognizing ssRNA antiviral defense (Diebold S.S. et al, Science, 2004: 303, 1529-1531; and Lund J. M. et al, PNAS, 2004: 101, 5598-5603).
  • TLR7 has a limited expression profile in humans and is expressed primarily by B cells and plasmacytoid dendritic cells (pDC), but to a lesser extent by monocytes.
  • Plasmacytoid DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% of peripheral blood mononuclear cells (PBMCs)) that secrete high levels of interferon- ⁇ (IFN ⁇ ) and interferon in response to viral infections.
  • PBMCs peripheral blood mononuclear cells
  • IFN ⁇ interferon- ⁇
  • the first type I interferon-producing cell of ⁇ (IFN ⁇ ) Liu YJ, Annu. Rev. Immunol., 2005: 23, 275-306).
  • TLRs diseases and disorders are associated with abnormalities in TLRs such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD). ), ulcerative colitis, liver fibrosis, HBV, Flaviviridae virus, HCV, HPV, RSV, SARS, HIV or influenza virus infection. Therefore, the use of agonists for TLRs to treat related diseases is promising.
  • TLR7 and TLR8 are highly homologous, the TLR7 ligand, in most cases, is also a TLR8 ligand.
  • TLR8 stimulation primarily induces the production of cytokines such as tumor necrosis factor alpha (TNF-alpha) and chemokines.
  • TNF-alpha tumor necrosis factor alpha
  • Interferon alpha is one of the main drugs for the treatment of chronic hepatitis B or hepatitis C
  • TNF- ⁇ is a pro-inflammatory cytokine, and excessive secretion may cause serious side effects. Therefore, selectivity for TLR7 and TLR8 is critical for the development of TLR7 agonists for the treatment of viral infectious diseases.
  • TLR7 agonist patent applications such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, WO2012080730.
  • TLR7 agonists there are currently related TLR7 agonist patent applications, such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, WO2012080730.
  • TLR7 agonists such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, WO2012080730.
  • the present invention is directed to the above technical problems, and provides a drug compound having a lower incineration concentration, a better selectivity, and a more effective activation effect, and is a safer and more effective TLR7 agonist.
  • Ring A is selected from the group consisting of a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • G is selected from N and CR 6 ;
  • X 1 is selected from an alkylene group or S(O) m , wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, and nitrate Substituted by one or more substituents of a group, a cycloalkyl group and a heterocyclic group;
  • L 1 is selected from -NR 7 -, -O-, -S-, -C(O)-, -S(O) m -, -N(R 7 )C(O)-, -C(O)N (R 7 )-, -N(R 7 )S(O) 2 -, -S(O) 2 N(R 7 )- and a covalent bond;
  • R 1 is selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl group, the alkenyl group, the alkynyl group, and the cycloalkyl group are selected.
  • a heterocyclic group, an aryl group and a heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, hetero Substituting one or more substituents of a cyclic group, an aryl group, and a heteroaryl group;
  • R is the same or different and are each independently selected from hydrogen, halo, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl a base, a heteroaryl group, -C(O)R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O)NR 9 R 10 , wherein
  • the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitrate Substituted by one or more substituents of a group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • L 2 is selected from an alkylene group or a covalent bond, wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, Substituting one or more substituents of a cycloalkyl group and a heterocyclic group;
  • R 3 is selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, -C (O) R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O)NR 9 R 10 wherein the cycloalkyl group, heterocyclic group And the aryl and heteroaryl are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl group, a heteroaryl group, -
  • R 4 and R 5 are the same or different and are each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, and a heterocyclic ring.
  • Base aryl and heteroaryl;
  • R 4 and R 5 together form an oxo group
  • R 6 is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;
  • R 7 is selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • R 8 is selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an amino group, a hydroxyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • R 9 and R 10 are the same or different and are each independently selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein said alkyl, cycloalkyl,
  • the heterocyclic group, the aryl group and the heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aromatic Substituted with one or more substituents in the heteroaryl group;
  • R 9 and R 10 together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 heteroatoms which are the same or different from N, O and S, and
  • the heterocyclic group is optionally selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Substituted by a plurality of substituents;
  • n 0, 1, 2, 3 or 4;
  • n 0, 1, or 2.
  • the compound of the formula (I), wherein the ring A is selected from the group consisting of a phenyl group and a pyridyl group.
  • the compound of the formula (I), wherein the X 1 is an alkylene group is an alkylene group.
  • the compound of the formula (I) is a compound of the formula (II):
  • G, L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • the compound of the formula (I) is a compound of the formula (III):
  • R 9 and R 10 together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 hetero atoms which are the same or different and are selected from N, O and S, and the heterocyclic ring
  • the group is optionally substituted with one or more selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl Substituted, preferably 5-6 membered heterocyclic group, more preferably pyrrolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl or tetrahydropyranyl;
  • G, L 1 to L 2 and R 1 are as defined in the formula (I).
  • the compound of the formula (I), wherein the L 2 is an alkylene group, preferably a C 1-6 alkylene group, more preferably a methylene group. 1,2-ethylene, 1,1-ethylene or 1,3-propylene.
  • Typical compounds of the invention include, but are not limited to:
  • a tautomer a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof.
  • a compound of the formula (IB) is an intermediate for the preparation of a compound of the formula (I):
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • X is a halogen
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • the compound represented by the formula (IB) includes, but is not limited to:
  • a compound of the formula (IC) is used as an intermediate for the preparation of a compound of the formula (I):
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • Compounds of the formula (IC) include, but are not limited to:
  • Another aspect of the invention relates to a process for the preparation of a compound of the formula (IB) which comprises:
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • X is a halogen;
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • Another aspect of the invention relates to a process for the preparation of a compound of the formula (IC), the process comprising:
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • X is a halogen;
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • Another aspect of the invention relates to a process for the preparation of a compound of formula (I), which process comprises:
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesogen, a racemate thereof, Enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt or a pharmaceutical composition comprising the same in the manufacture of a medicament for a TLR7 agonist.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt or a pharmaceutical composition comprising the same for the preparation of a medicament for treating an infection caused by a virus selected from the group consisting of dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus , ⁇ ⁇ encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV , SARS and influenza viruses.
  • a virus selected from the group consisting of dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus , ⁇ ⁇ encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encepha
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a pharmaceutical composition comprising the same for use in the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD Use in drugs for ulcerative colitis and liver fibrosis.
  • the invention further relates to a method of agonizing TLR7 comprising the compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer
  • a method of agonizing TLR7 comprising the compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer
  • the invention further relates to a method of treating an infection caused by a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, ink Dementia encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses, the method comprising administering A therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, is required. Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.
  • a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, ink
  • the invention further relates to a treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis, autoimmune diseases a method of plaque psoriasis, systemic lupus erythematosus, actinic keratosis, and liver fibrosis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the formula (I) or a tautomer thereof, A meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
  • a compound of the formula (I) or a tautomer thereof A meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same, which is used as a medicament.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same, which is for use in a TLR7 agonist.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same for use in the treatment or prevention of an infection caused by a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, rumor Encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and flu virus.
  • a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, rumor Encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemo
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same for use in the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis and liver fibrosis.
  • the active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture.
  • Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives, To provide a pleasing and tasty pharmaceutical preparation. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for the preparation of a tablet for admixture.
  • the aqueous suspension contains the active substance and excipients suitable for the preparation of the aqueous suspension for mixing.
  • the aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents. Flavor.
  • Oil suspensions can be formulated by suspending the active ingredient in vegetable oil.
  • the oil suspension may contain a thickening agent.
  • the above sweeteners and flavoring agents may be added to provide a palatable preparation.
  • Dispersible powders and granules suitable for use in the preparation of aqueous suspensions may be employed in the preparation of the active ingredient and dispersion or dispersing or suspending agents or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or
  • compositions of the invention may also be in the form of an oil-in-water emulsion.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • acceptable vehicles or solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the patient by a local injection.
  • the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a parenterally acceptable non-toxic diluent or solvent.
  • sterile fixed oils may conveniently be employed as a solvent or suspension medium.
  • the compounds of the invention may be administered in the form of a suppository for rectal administration.
  • These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug.
  • suitable non-irritating excipient include a mixture of cocoa butter, glycerin gelatin, hydrogenated vegetable oil, polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.
  • the dosage of the drug to be administered depends on a variety of factors including, but not limited to, the following factors: the activity of the particular compound used, the age of the patient, the weight of the patient, the health of the patient, the behavior of the patient. , the patient's diet, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal treatment modality such as the mode of treatment, the daily dosage of the compound of formula (I) or the pharmaceutically acceptable salt
  • the type can be verified according to traditional treatment options.
  • alkyl refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group having from 1 to 12 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-methylpent
  • lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently selected from alkyl, alkenyl, alkynyl, Alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylthio, heterocycloalkylthio, oxo, -C(O)R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O Substituting one or more substituents in NR 9 R 10 .
  • alkylene refers to a saturated straight or branched aliphatic hydrocarbon radical having two residues derived from the removal of two hydrogen atoms from the same carbon atom of the parent alkane or two different carbon atoms.
  • alkylene examples include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 ) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • the alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently selected from alkyl, alkenyl, alkynyl groups.
  • alkenyl refers to a hydrocarbon group formed by one or more hydrogen atoms in the olefin molecule.
  • the alkenyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R 8, -C (O) OR 8, -S (O) m R Substituting one or more substituents of 8- , -NR 9 R 10 and -C(O)NR 9 R 10 .
  • alkynyl refers to a hydrocarbon containing a carbon-carbon triple bond in the molecule.
  • the alkynyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, Hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)R 8 , -C(O)OR 8 , -S(O) m R Substituting one or more substituents of 8- , -NR 9 R 10 and -C(O)NR 9 R 10 .
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, preferably from 3 to 10 The carbon atom, more preferably contains 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • a polycycloalkyl group includes a spiro ring, a fused ring, and a cycloalkyl group.
  • amino protecting group is intended to keep the amino group unchanged during the reaction of other parts of the molecule, and to protect the amino group with a group which is easily removed.
  • Non-limiting examples include t-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with from 1 to 3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably p-methoxybenzyl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O).
  • a hetero atom of m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1, 2.3.6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, High piperazinyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclic group, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy.
  • alkylthio alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio, oxo, -C(O)R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O)NR Substituting one or more substituents in 9 R 10 .
  • aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (ie, a ring that shares a pair of adjacent carbon atoms) having a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene. Base and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, One of a heterocycloalkylthio group, -C(O)R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O)NR 9 R 10 or Substituted by a plurality of substituents.
  • the substituent is preferably one or more of the following groups, which are independently optionally selected from the group consisting of alky
  • heteroaryl refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, Imidazolyl, pyrazolyl, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio
  • One of a heterocycloalkylthio group, -C(O)R 8 , -C(O)OR 8 , -S(O) m R 8 , -NR 9 R 10 and -C(O)NR 9 R 10 Or substituted with multiple substituents.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group.
  • a hydroxyl group substituted by one or more substituents of a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group.
  • haloalkyl refers to an alkyl group substituted by one or more halogens, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • hydroxyalkyl refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.
  • halogen means fluoro, chloro, bromo or iodo.
  • amino means -NH 2.
  • cyano refers to -CN.
  • nitro refers to -NO 2 .
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention which is safe and effective for use in a mammal and which possesses the desired biological activity.
  • a method for preparing a medicinal salt comprising the steps of:
  • a compound of the formula (I-1) and NH(W) s are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-2);
  • a compound of the formula (I-2) and a compound of the formula (I-3) are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-4);
  • the compound of the formula (I-4) is subjected to a reduction reaction in the presence of a reducing reagent to obtain a compound of the formula (IA);
  • the fourth step the compound of the formula (IA) and a nucleophilic substitution reaction under basic conditions to give a compound of the formula (IB);
  • the compound of the formula (IB) is subjected to ring closure in the presence of iodide under basic conditions to obtain a compound of the formula (IC);
  • the reagents providing basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl.
  • organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl.
  • said inorganic bases including but not limited to sodium hydride, Potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide;
  • Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane solution, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me 3 SiCl and TMSOTf;
  • the reducing reagents include, but are not limited to, iron powder, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH(Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na(AcO) 3 BH, B 2 H 5 . , Li(Et) 3 BH, Pd/C/H 2 and Raney Ni/H 2 ;
  • Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;
  • the above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;
  • a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • X is a halogen, preferably chlorine
  • S is 1 or 2;
  • Rings A, G, X 1 , L 1 to L 2 , R 1 to R 5 and n are as defined in the formula (I).
  • the fourth step the compound of the formula (II-A) and a nucleophilic substitution reaction under basic conditions to give a compound of the formula (II-B);
  • the reagents providing basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl.
  • organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl.
  • said inorganic bases including but not limited to sodium hydride, Potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide;
  • the reducing agents include, but are not limited to: iron, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot -Bu) 3, AlH 3, NaCNBH 3, Na (AcO) 3 BH, B 2 H 5 , Li(Et) 3 BH, Pd/C/H 2 and Raney Ni/H 2 ;
  • Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;
  • the above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;
  • a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • G, n, L 1 to L 2 and R 1 to R 5 are as defined in the formula (II).
  • a compound of the formula (I-1) and NH-(W) s are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-2);
  • a compound of the formula (I-2) and a compound of the formula (III-1) are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (III-2);
  • the fourth step the compound of the formula (III-A) and a nucleophilic substitution reaction under basic conditions to give a compound of the formula (III-B);
  • the compound of the formula (III-B) is subjected to ring closure in the presence of iodide under basic conditions to obtain a compound of the formula (III-C);
  • Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane solution, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me 3 SiCl and TMSOTf;
  • the reducing reagents include, but are not limited to, iron powder, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH(Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na(AcO) 3 BH, B 2 H 5 . , Li(Et) 3 BH, Pd/C/H 2 and Raney Ni/H 2 ;
  • Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;
  • W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;
  • S is 1 or 2;
  • G, L 1 to L 2 , R 1 and R 9 to R 10 are as defined in the formula (III).
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • NMR was measured using a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), internal standard was four.
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methyl silane
  • the measurement of the MS was carried out using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • ESI FINNIGAN LCQAd
  • the HPLC was measured using an Agilent 1200 DAD high pressure liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 x 4.6 mm column).
  • Chiral HPLC analysis assays were performed using LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.).
  • Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate.
  • the specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm.
  • the specification for thin layer chromatography separation and purification is 0.4mm. ⁇ 0.5mm.
  • the known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Companies such as Dare Chemicals.
  • the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L volume.
  • the pressurized hydrogenation reaction was carried out using a Parr Model 3916EKX hydrogenation apparatus and a clear blue QL-500 type hydrogen generator or a HC2-SS type hydrogenation apparatus.
  • the hydrogenation reaction is usually evacuated, charged with hydrogen, and operated three times.
  • reaction temperature is room temperature and is 20 ° C to 30 ° C.
  • the progress of the reaction in the examples was monitored by thin layer chromatography (TLC), the developing agent used for the reaction, the column chromatography eluent system used for the purification of the compound, and the thin layer chromatography developing solvent system including: A: Methylene chloride/methanol system, B: n-hexane/ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of an alkaline or acidic reagent such as triethylamine or acetic acid.
  • TLC thin layer chromatography
  • A Methylene chloride/methanol system
  • B n-hexane/ethyl acetate system
  • the volume ratio of the solvent is adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of an alkaline or acidic reagent such as triethylamine or acetic acid.
  • Determination of the compounds of the present invention adopts the following experimental method hTLR7 protein activation HEK-Blue TM hTLR7 expression in stably transfected cell lines of:
  • PBS Phosphate buffer pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).
  • HEK-Blue assay medium Take a bag of HEK-Blue dry powder, add 50ml to endotoxin water to dissolve, then put it into the 37 °C incubator, and then sterile filtration after 10 minutes.
  • the compound was first formulated into a 20 mM stock solution; it was diluted with pure DMSO to a maximum concentration of 6 x 106 nM and diluted by a 3-fold gradient for a total of 10 points.
  • the above-prepared compound was diluted 20-fold with a medium, and then 20 ⁇ l of the diluted compound was added to each well.
  • the cells were resuspended in HEK-Blue assay medium at a concentration of 2.2 ⁇ 10 5 cells/ml, and 180 ⁇ l of the cells were added to the above 96-well cell culture plate to which 20 ⁇ l of the drug had been added, and cultured at 37 ° C for 6-16 hours.
  • the microplate reader reads at a wavelength of 620 nm. Corresponding OD values obtained by Graphpad Prism calculated EC 50 value of the drug.
  • the activation of human TLR7 by the compounds of the present invention can be determined by the above test, and the measured EC 50 values are shown in Table 1.
  • Table 1 Compound of the present invention to the human EC TLR7 50.
  • the compounds of the present invention have a good activation effect on human TLR7.
  • Test Example 2 Determination of agonistic activity of human TLR8 by the compound of the present invention
  • Determination of the compounds of the present invention adopts the following experimental method hTLR8 protein activation HEK-Blue TM hTLR8 expression in stably transfected cell lines of:
  • PBS Phosphate buffer pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).
  • HEK-Blue assay medium Take a bag of HEK-Blue dry powder, add 50ml to endotoxin water to dissolve, then put it into the 37 °C incubator, and then sterile filtration after 10 minutes.
  • Compound is first formulated as a 20mM stock solution; neat DMSO and then diluted to a maximum concentration of 6 ⁇ 10 6 nM, and then diluted 3 fold gradient, a total of 10 points; first compound is diluted 20-fold with medium and then diluted with 20 ⁇ l added per well After the compound.
  • HEK-Blue TM hTLR8 cells Take HEK-Blue TM hTLR8 cells, first remove the supernatant, add 2-5 ml of pre-warmed PBS, put into the incubator for 1-2 minutes, gently pipe the cells, and trypan blue staining. The cells were resuspended in HEK-Blue assay medium at a concentration of 2.2 ⁇ 10 5 cells/ml, and 180 ⁇ l of the cells were added to the above 96-well cell culture plate to which 20 ⁇ l of the drug had been added, and cultured at 37 ° C for 6-16 hours.
  • the microplate reader reads at a wavelength of 620 nm.
  • the corresponding OD value can be obtained, and the EC 50 value of the drug is calculated by Graphpad Prism.
  • Table Compound 2 of the present invention to human TLR8, EC 50.
  • the compounds of the present invention have weak activation on human TLR8, indicating that the compounds of the present invention are selective for TLR7.
  • RMPI 1640 medium containing 10% FBS Resuspend and count with RMPI 1640 medium containing 10% FBS, adjust the amount of PBMC to 3.33 ⁇ 10 6 cells/ml, take 150 ⁇ l to the cell culture plate to which the compound has been added, and incubate at 37 ° C, 5.0% CO 2 The medium was cultured for 24 hours.
  • the cell culture plate was placed in a centrifuge, centrifuged at 1200 rpm for 10 minutes at room temperature. 150 ⁇ l of the supernatant was taken out per well.
  • the reagent in the human IFN- ⁇ kit is firstly equilibrated to room temperature, and anti-IFN- ⁇ -Eu 3+ - Cryptate conjugate and anti-IFN- ⁇ - are prepared according to the kit instructions in the dark.
  • the d2-conjugate both mixed in a 1:40 ratio with a conjugate buffer. Then, 16 ⁇ l of the supernatant obtained by centrifugation was added to each well.
  • the HTRF mode is read on the PHERAStar.
  • MEC Minimum Effective Concentration
  • Test Example 4 Inhibition of the Enzymatic Activity of the Compound of the Invention on the Metabolite of the Metabolite of CYP3A4 Midazolam in Human Liver Microsomes
  • PBS Phosphate buffer
  • CYP probe substrate (15 ⁇ M midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA K1003).
  • the compound of the present invention has no inhibitory effect on the midazolam metabolic site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolites based on CYP3A4 metabolism of midazolam metabolite sites do not occur. effect.
  • the activity of the compound of the present invention on human liver microsome CYP2D6 enzyme activity was determined by the following experimental method:
  • PBS Phosphate buffer
  • CYP probe substrate (20 ⁇ M dextromethorphan, SIGMA Q0750) and positive control inhibitor (Quinidine, SIGMA D9684).
  • Test Example 6 Inhibition of Enzyme Activity of Testosterone Metabolism Site of Human Liver Microsome CYP3A4 by Human Compound Microsomes
  • the enzymatic activity of the compound of the present invention on the testosterone metabolism site of human liver microsome CYP3A4 was determined by the following experimental method:
  • PBS Phosphate buffer
  • CYP probe substrate testosterone / 100 ⁇ M, SIGMA K1003
  • positive control inhibitor ketoconazole, Dr. Ehrenstorfer GmbH, C17322500
  • the compound of the present invention has no inhibitory effect on the testosterone metabolism site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolic drug interaction based on CYP3A4-based testosterone metabolism site does not occur.
  • the blocking effect of the compounds of the invention on hERG potassium current was tested using a fully automated patch clamp on a stable cell line transfected with hERG potassium channels.
  • the HEK293-hERG stable cell line was passaged at a density of 1:4 in MEM/EBSS medium (10% FBS, 400 ⁇ g/ml G418, 1% MEM non-essential amino acid solution (100 ⁇ ), 1% sodium pyruvate solution). Culture, culture within 48-72 hours for automated patch-clamp experiments.
  • the instrument When the whole cell mode is formed, the instrument will get the hERG current according to the set hERG current voltage program, and then the instrument will automatically perfuse the compound from low to high concentration.
  • the currents at each concentration of the compounds and the blank control current were analyzed by HEAK Patchmaster, HEAK EPC10 patch clamp amplifiers (Nanion) and Pathlinersoftware and data analysis software provided by Pathcontrol HTsoftware.
  • the compounds of the present invention have a weak inhibitory effect on hERG and can reduce side effects caused by the hERG pathway.
  • Test Example 8 Mouse pharmacokinetic test of the compound of the present invention
  • mice Using mice as test animals, the concentration of the drug in plasma at different times after administration of the compound of Example 1 by intragastric administration was determined by LC/MS/MS method. The pharmacokinetic behavior of the compounds of the invention in mice was investigated and their pharmacokinetic characteristics were evaluated.
  • a certain amount of the drug was weighed, and 5% by volume of DMSO, 5% by volume of tween 80, and 90% physiological saline were set to 1 mg/ml of a colorless clear liquid.
  • mice were intragastrically administered overnight after fasting, and the dose was 20.0 mg/kg, and the administration volume was 20 ml/kg.
  • Example 1 The compound of Example 1 was administered by gavage in mice, and 0.1 ml of blood was collected before administration and after 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after administration, and placed in a heparinized test tube, 3500 rpm. The plasma was separated by centrifugation for 10 minutes at /min and stored at -20 °C.
  • the content of the test compound in the plasma of the mice after the intragastric administration of different concentrations of the drug was determined: 25 ⁇ l of the mouse plasma at each time after administration, 50 ⁇ l (100 ng/mL) of the internal standard solution, and 200 ⁇ l of acetonitrile were added. The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (3600 rpm), and 10 ⁇ l of the supernatant was taken for LC/MS/MS analysis.
  • the pharmacokinetic parameters of the compounds of the invention are as follows:
  • the compounds of the present invention have better pharmacological absorption and have pharmacokinetic advantages.
  • Test sample Test Example 8 Pharmacokinetics The mice of Example 1 were administered intragastrically, and blood was collected before administration and at 0, 2.0, 4.0 and 8.0 hours after administration.
  • I.g. is administered by intragastric administration.
  • the compound of Example 1 was administered with 20 mpk in mice for 2 hours and had a strong ability to stimulate IFN- ⁇ expression.

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Abstract

本发明公开了杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及其在医药上的应用。具体而言，本发明公开了一种通式(I)所示的新的杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及含有该衍生物的药物组合物以及其作为治疗剂，特别是作为TLR7激动剂的用途，其中通式(I)的各取代基与说明书中的定义相同。

Description

杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及其在医药上的应用 技术领域
本发明属于医药领域，涉及一种通式(I)所示的新的杂芳基并噻二嗪-2,2-二氧化物类衍生物、其制备方法及含有该衍生物的药物组合物以及其作为治疗剂，特别是作为TLR7激动剂的用途。
背景技术
Toll样受体(toll-like receptors；TLRs)是参与先天免疫的一类重要蛋白质分子。TLRs是单体跨膜的非催化性受体，通常在岗哨细胞如巨噬细胞和树突状细胞中表达，可以识别由微生物产生的结构保守的分子。一旦这些微生物突破如皮肤或肠道粘膜的物理屏障，就会被TLRs识别，继而激活免疫细胞应答(Mahla,R S.等人,Front Immunol.4:248(2013))。免疫系统之所以具有广泛识别病原微生物的能力，某种程度上是由于Toll样免疫受体的广泛存在。
在哺乳动物中至少有10种不同的TLRs。一些此类受体的配体和相应的信号级联放大已经被鉴定出。TLR7是TLRs(TLRs 3、7、8和9)亚组的成员，局限于专门检测非己核酸的细胞的内涵体隔室。TLR7在通过识别ssRNA抗病毒防御方面起关键作用(Diebold S.S.等,Science,2004:303,1529-1531；和Lund J.M.等,PNAS,2004:101,5598-5603)。TLR7在人身上具有有限的表达分布，并主要通过B细胞和浆细胞样树突细胞(pDC)表达，而较低程度地通过单核细胞表达。浆细胞样DCs是淋巴衍生的树突细胞的唯一群体(0.2-0.8％的外周血单核细胞(PBMCs))，它是响应病毒感染而分泌高水平干扰素-α(IFNα)和干扰素-β(IFNβ)的最初的I型干扰素生成细胞(Liu Y-J,Annu.Rev.Immunol.,2005:23,275-306)。
很多疾病、障碍与TLRs的异常有关，比如黑色素瘤、非小细胞肺癌、肝细胞癌、基底细胞癌(basalcellcarcinoma)、肾细胞癌、骨髓瘤、变应性鼻炎、哮喘、慢性阻塞性肺炎(COPD)、溃疡性结肠炎、肝纤维化，HBV、黄病毒科(Flaviviridae)病毒、HCV、HPV、RSV、SARS、HIV或流行性感冒的病毒感染等。因此运用TLRs的激动剂治疗相关疾病是很有前景的。
由于TLR7和TLR8高度同源，因此TLR7配体，在大多数情况下也是TLR8配体。TLR8刺激主要诱导产生细胞因子如肿瘤坏死因子α(TNF-α)和趋化因子。干扰素α是治疗慢性乙型肝炎或丙型肝炎的主要药物之一，而TNF-α是一种促炎细胞因子，过多分泌可能导致严重的副作用。所以对TLR7和TLR8的选择性对于开发TLR7激动剂用于治疗病毒感染性疾病至关重要。
目前已有相关的TLR7激动剂专利申请，如WO2005025583、WO2007093901、WO2008011406、WO2009091032、WO2010077613、WO2010133882、 WO2011031965、WO2012080730。但是仍有必要继续研发安全的和治疗上更有效的TLR7激动剂。
本发明针对上述技术问题，提供一种起效浓度更低，选择性更好，激活效果更明显的药物化合物，是更安全和更有效的TLR7激动剂。
发明内容
本发明的目的在于提供一种通式(I)所示的化合物：
Figure PCTCN2018073815-appb-000001
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
环A选自环烷基、杂环基、芳基和杂芳基；
G选自N和CR 6；
X 1选自亚烷基或S(O) m，其中所述的亚烷基任选被选自卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基和杂环基中的一个或多个取代基所取代；
L 1选自-NR 7-、-O-、-S-、-C(O)-、-S(O) m-、-N(R 7)C(O)-、-C(O)N(R 7)-、-N(R 7)S(O) 2-、-S(O) 2N(R 7)-和共价键；
R 1选自氢原子、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，其中所述的烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
R 2相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10，其中所述的烷基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
L 2选自亚烷基或共价键，其中所述的亚烷基任选被选自卤素、烷基、烷氧基、 卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基和杂环基中的一个或多个取代基所取代；
R 3选自氢原子、烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10，其中所述的环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代；
R 4和R 5相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
或R 4和R 5一起形成氧代基；
R 6选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
R 7选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
R 8选自氢原子、烷基、卤代烷基、氨基、羟基、环烷基、杂环基、芳基和杂芳基；
R 9和R 10相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基、杂芳基，其中所述的烷基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
或者，所述R 9和R 10与相连接的氮原子一起形成杂环基，其中所述的杂环基内含有1～2个相同或不同选自N、O和S的杂原子，并且所述的杂环基任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
n为0、1、2、3或4；且
m为0、1或2。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的环A选自苯基和吡啶基。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的X 1为亚烷基。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其为通式(II)所示的化合物：
Figure PCTCN2018073815-appb-000002
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
G、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其为通式(III)所示的化合物：
Figure PCTCN2018073815-appb-000003
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
R 9和R 10与相连接的氮原子一起形成杂环基，其中所述的杂环基内含有1～2个相同或不同选自N、O和S的杂原子，并且所述的杂环基任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选为5-6元杂环基，更优选为吡咯烷基、四氢噻吩基、四氢呋喃基、哌啶基、哌嗪基、吗啉基或四氢吡喃基；
G、L 1～L 2和R 1如通式(I)中所定义。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的R 4和R 5均为氢或者R 4和R 5一起形成氧代基。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的L 1为-O-。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的R 1为烷基，优选为C 1-6的烷基，更优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基或正戊基。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的G为N。
在本发明一个优选的实施方案中，所述的通式(I)所示的化合物，其中所述的L 2为亚烷基，优选为C 1-6的亚烷基，更优选亚甲基、1,2-亚乙基、1,1-亚乙基或1,3-亚丙基。
本发明的典型化合物包括但不限于：
Figure PCTCN2018073815-appb-000004
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐。
在本发明一个优选的实施方案中，一种通式(IB)所示的化合物，其为制备通式(I)化合物的中间体：
Figure PCTCN2018073815-appb-000005
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
X为卤素；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
通式(IB)所示的化合物包括，但不限于：
Figure PCTCN2018073815-appb-000006
在本发明一个优选的实施方案中，一种通式(IC)所示的化合物，其作为制备通式(I)化合物的中间体：
Figure PCTCN2018073815-appb-000007
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
通式(IC)所示的化合物包括，但不限于：
Figure PCTCN2018073815-appb-000008
本发明的另一方面涉及一种制备通式(IB)所示的化合物的方法，该方法包括：
Figure PCTCN2018073815-appb-000009
通式(IA)的化合物和
Figure PCTCN2018073815-appb-000010
发生亲核取代反应得到通式(IB)的化合物；
其中：
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；X为卤素；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
本发明的另一方面涉及一种制备通式(IC)所示的化合物的方法，该方法包括：
Figure PCTCN2018073815-appb-000011
通式(IB)的化合物关环得到通式(IC)的化合物；
其中：
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；X为卤素；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
本发明的另一方面涉及一种制备通式(I)所示的化合物的方法，该方法包括：
Figure PCTCN2018073815-appb-000012
通式(IC)的化合物脱去保护基得到通式(I)的化合物；
其中：
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
本发明的另一方面涉及一种药物组合物，所述药物组合物含有治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明进一步涉及通式(I)所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或包含其的药物组合物在制备用于TLR7激动剂的药物中的用途。
本发明进一步涉及通式(I)所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或包含其的药物组合物在制备用于治疗由病毒引起的感染的药物中的用途，所述病毒选自：登革热病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、蜱传脑炎病毒、昆津病毒、墨累山谷脑炎病毒、圣路易脑炎病毒、鄂木斯克出血热病毒、牛病毒性腹泻病毒、寨卡病毒、HIV、HBV、HCV、HPV、RSV、SARS和流感病毒。
本发明进一步涉及通式(I)所示化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或包含其的药物组合物在制备用于治疗或预防黑色素瘤、非小细胞肺癌、肝细胞癌、基底细胞癌、肾细胞癌、骨髓瘤、变应性鼻炎、哮喘、COPD、溃疡性结肠炎和肝纤维化的药物中的用途。
本发明进一步涉及一种激动TLR7的方法，其包括将通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物与TLR7接触的步骤。
本发明进一步涉及一种治疗由病毒引起的感染的方法，所述的病毒选自：登革热病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、蜱传脑炎病毒、昆津病毒、 墨累山谷脑炎病毒、圣路易脑炎病毒、鄂木斯克出血热病毒、牛病毒性腹泻病毒、寨卡病毒、HIV、HBV、HCV、HPV、RSV、SARS和流感病毒，所述方法包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物。
本发明进一步涉及一种治疗或预防黑色素瘤、非小细胞肺癌、肝细胞癌、基底细胞癌、肾细胞癌、骨髓瘤、变应性鼻炎、哮喘、COPD、溃疡性结肠炎、自身免疫性疾病、斑块银屑病、系统性红斑狼疮、光化角质病和肝纤维化的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物，其用作药物。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物，其用于TLR7激动剂。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物，其用于治疗或预防由病毒引起的感染，所述病毒选自：登革热病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、蜱传脑炎病毒、昆津病毒、墨累山谷脑炎病毒、圣路易脑炎病毒、鄂木斯克出血热病毒、牛病毒性腹泻病毒、寨卡病毒、HIV、HBV、HCV、HPV、RSV、SARS和流感病毒。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物，其用于治疗或预防选黑色素瘤、非小细胞肺癌、肝细胞癌、基底细胞癌、肾细胞癌、骨髓瘤、变应性鼻炎、哮喘、COPD、溃疡性结肠炎和肝纤维化。
含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂。可按照本领域任何已知制备药用组合物的方法制备口服组合物，此类组合物可含有一种或多种选自以下的成分：甜味剂、矫味剂、着色剂和防腐剂，以提供悦目和可口的药用制剂。片剂含有活性成分和用于混合的适宜制备片剂的无毒的可药用的赋形剂。
水悬浮液含有活性物质和用于混合的适宜制备水悬浮液的赋形剂。水混悬液也可以含有一种或多种防腐剂例如尼泊金乙酯或尼泊金正丙酯、一种或多种着色 剂、一种或多种矫味剂和一种或多种甜味剂。
油混悬液可通过使活性成分悬浮于植物油中配制而成。油悬浮液可含有增稠剂。可加入上述的甜味剂和矫味剂，以提供可口的制剂。
通过加入水可使适用于制备水混悬液的可分散粉末和颗粒提供活性成分和用于混合的分散剂或湿润剂、悬浮剂或一种或多种防腐剂。适宜的分散剂或湿润剂和悬浮剂可说明上述的例子。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂例如抗坏血酸保存这些组合物。
本发明的药物组合物也可以是水包油乳剂的形式。
药物组合物可以是无菌注射水溶液形式。可以使用的可接受的溶媒或溶剂有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在肠胃外可接受的无毒稀释剂或溶剂中制备的无菌注射溶液或混悬液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。
可按用于直肠给药的栓剂形式给予本发明化合物。可通过将药物与在普通温度下为固体但在直肠中为液体，因而在直肠中会溶化而释放药物的适宜的无刺激性赋形剂混合来制备这些药物组合物。此类物质包括可可脂、甘油明胶、氢化植物油、各种分子量的聚乙二醇和聚乙二醇的脂肪酸酯的混合物。
如本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用具体化合物的活性、患者的年龄、患者的体重、患者的健康状况、患者的行为、患者的饮食、给药时间、给药方式、排泄的速率、药物的组合等；另外，最佳的治疗方式如治疗的模式、通式化合物(I)的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。
发明的详细说明
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二 甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“烯基”指烯烃分子中少一个或几个氢原子而成的烃基。烯基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自氢原子、烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“炔基”指分子中含有碳碳三键的碳氢化合物。炔基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自氢原子、烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、 杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，优选包含3至10个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。所述氨基保护基优选为对甲氧苄基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至10个环原子，其中1-4是杂原子；更优选包含5至6个环原子；其中1-3个是杂原子。单环杂环基的非限制性实例包括吡咯烷基、四氢吡喃基、1,2.3.6-四氢吡啶基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2018073815-appb-000013
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2018073815-appb-000014
Figure PCTCN2018073815-appb-000015
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、哒嗪基、咪唑基、吡唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2018073815-appb-000016
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代 时，取代基优选为一个或多个以下基团，其独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。术语“羟基”指-OH基团。
术语“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“氰基”指-CN。
术语“硝基”指-NO 2。
术语“氧代基”指＝O。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“可药用盐”是指本发明化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。
m、G和R 8～R 10如通式(I)化合物中所定义。
本发明化合物的合成方法
为了完成本发明的目的，本发明采用如下技术方案：
方案一
本发明通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2018073815-appb-000017
第一步，通式(I-1)的化合物和NH(W) s在碱性条件下，发生亲核取代反应得到通式(I-2)的化合物；
第二步，通式(I-2)的化合物和通式(I-3)的化合物在碱性条件下，发生亲核取代反应得到通式(I-4)的化合物；
第三步，通式(I-4)的化合物在还原试剂存在下，发生还原反应得到通式(IA)的化合物；
第四步，通式(IA)的化合物和
Figure PCTCN2018073815-appb-000018
在碱性条件下，发生亲核取代反应得到通式(IB)的化合物；
第五步，通式(IB)的化合物在碱性条件下，在碘化物存在下关环得到通式(IC)的化合物；
第六步，通式(IC)的化合物在酸性条件下，脱去保护基得到通式(I)的化合物；
其中：
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、吡啶、4-二甲氨基吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、双三甲基硅基胺基锂、醋酸钾、叔丁醇钠、叔丁醇钾和正丁醇钠，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、醋酸钾、碳酸铯、氢氧化钠和氢氧化锂；
提供酸性的条件的试剂包括但不限于氯化氢、氯化氢的1,4-二氧六环溶液、三氟乙酸、甲酸、乙酸、盐酸、硫酸、甲磺酸、硝酸、磷酸、对苯甲磺酸、Me 3SiCl和TMSOTf；
所述的还原试剂包括但不限于：铁粉、氢化铝锂、硼氢化钠、DIBAL-H、NaAlH(O-t-Bu) 3、AlH 3、NaCNBH 3、Na(AcO) 3BH、B 2H 5、Li(Et) 3BH、Pd/C/H 2和雷尼镍(Raney Ni)/H 2；
碘化物包括但不限于碘单质、碘化酮、碘化钾和碘化铯；
上述反应优选在溶剂中进行，所用溶剂包括但不限于：醋酸、甲醇、乙醇、正丁醇、甲苯、四氢呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亚砜、1,4-二氧六环、水、N,N-二甲基甲酰胺及其混合物；
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
X为卤素，优选为氯；
S为1或2；
环A、G、X 1、L 1～L 2、R 1～R 5和n如通式(I)中所定义。
方案二
本发明通式(II)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2018073815-appb-000019
第一步，通式(I-1)的化合物和NH(W) s在碱性条件下，发生亲核取代反应得到通式(I-2)的化合物；
第二步，通式(I-2)的化合物和通式(II-1)的化合物在碱性条件下，发生亲核取代反应得到通式(II-2)的化合物；
第三步，通式(II-2)的化合物在还原试剂存在下，发生还原反应得到通式(II-A)的化合物；
第四步，通式(II-A)的化合物和
Figure PCTCN2018073815-appb-000020
在碱性条件下，发生亲核取代反应得到通式(II-B)的化合物；
第五步，通式(II-B)的化合物在碱性条件下，在碘化物存在下关环得到通式(II-C)的化合物；
第六步，通式(II-C)的化合物在酸性条件下，脱去保护基得到通式(II)的化合物；
其中：
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、吡啶、4-二甲氨基吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、双三甲基硅基胺基锂、醋酸钾、叔丁醇钠、叔丁醇钾和正丁醇钠，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、醋酸钾、碳酸铯、氢氧化钠和氢氧化锂；
提供酸性的条件的试剂包括但不限于氯化氢、氯化氢的1,4-二氧六环溶液、三氟乙酸、甲酸、乙酸、盐酸、硫酸、甲磺酸、硝酸、磷酸、对苯甲磺酸、Me 3SiCl和TMSOTf；
所述的还原试剂包括但不限于：铁粉、氢化铝锂、硼氢化钠、DIBAL-H、NaAlH(O-t-Bu) 3、AlH 3、NaCNBH 3、Na(AcO) 3BH、B 2H 5、Li(Et) 3BH、Pd/C/H 2和雷尼镍(Raney Ni)/H 2；
碘化物包括但不限于碘单质、碘化酮、碘化钾和碘化铯；
上述反应优选在溶剂中进行，所用溶剂包括但不限于：醋酸、甲醇、乙醇、正丁醇、甲苯、四氢呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亚砜、1,4-二氧六环、水、N,N-二甲基甲酰胺及其混合物；
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
X为卤素，优选为氯；
S为1或2；
G、n、L 1～L 2和R 1～R 5如通式(II)中所定义。
方案三
本发明通式(III)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2018073815-appb-000021
第一步，通式(I-1)的化合物和NH-(W) s在碱性条件下，发生亲核取代反应得到通式(I-2)的化合物；
第二步，通式(I-2)的化合物和通式(III-1)的化合物在碱性条件下，发生亲核取代反应得到通式(III-2)的化合物；
第三步，通式(III-2)的化合物在还原试剂存在下，发生还原反应得到通式(III-A)的化合物；
第四步，通式(III-A)的化合物和
Figure PCTCN2018073815-appb-000022
在碱性条件下，发生亲核取代反应得到通式(III-B)的化合物；
第五步，通式(III-B)的化合物在碱性条件下，在碘化物存在下关环得到通式(III-C)的化合物；
第六步，通式(III-C)的化合物在酸性条件下，脱去保护基得到通式(III)的化合物；
其中：
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、吡啶、4-二甲氨基吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、双三甲基硅基胺基锂、醋酸钾、叔丁醇钠、叔丁醇钾和正丁醇钠，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、醋酸钾、碳酸铯、氢氧化钠和氢氧化锂；
提供酸性的条件的试剂包括但不限于氯化氢、氯化氢的1,4-二氧六环溶液、三氟乙酸、甲酸、乙酸、盐酸、硫酸、甲磺酸、硝酸、磷酸、对苯甲磺酸、Me 3SiCl和TMSOTf；
所述的还原试剂包括但不限于：铁粉、氢化铝锂、硼氢化钠、DIBAL-H、NaAlH(O-t-Bu) 3、AlH 3、NaCNBH 3、Na(AcO) 3BH、B 2H 5、Li(Et) 3BH、Pd/C/H 2和雷尼镍(Raney Ni)/H 2；
碘化物包括但不限于碘单质、碘化酮、碘化钾和碘化铯；
上述反应优选在溶剂中进行，所用溶剂包括但不限于：醋酸、甲醇、乙醇、正丁醇、甲苯、四氢呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亚砜、1,4-二氧六环、水、N,N-二甲基甲酰胺及其混合物；
W为氨基保护基，优选为叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基；
X为卤素，优选为氯；
S为1或2；
G、L 1～L 2、R 1和R 9～R 10如通式(III)中所定义。
附图说明
图1，本发明化合物的小鼠血清IFN-α值的时间曲线。
具体实施方式
实施例
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)，氘代氯仿(CDCl 3)，氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商:Thermo,型号：Finnigan LCQ advantage MAX)。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
手性HPLC分析测定使用LC-10A vp(Shimadzu)或者SFC-analytical(Berger Instruments Inc.)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
手性制备柱层析使用Prep Star SD-1(Varian Instruments Inc.)或SFC-multigram(Berger Instruments Inc.)。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
激酶平均抑制率及IC 50值的测定用NovoStar酶标仪(德国BMG公司)。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH & Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
实施例1
8-氨基-6-丁氧基-4-(4-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物
Figure PCTCN2018073815-appb-000023
第一步
2-丁氧基-6-氯-N,N-双(4-甲氧基苄基)-5-硝基嘧啶-4-胺1b
将2-丁氧基-4,6-二氯-5-硝基嘧啶1a(4.62g，17.43mmol，采用公知的方法“Journal of Medicinal Chemistry,2012,55(23),10387-10404”制备而得)溶解于50mL四氢呋喃溶液中，依次加入三乙胺(2.64g，26.14mmol)和N,N-双(4-甲氧基苄基)胺(4.49g，17.43mmol)，室温搅拌4小时。停止反应，减压蒸馏，残余物用CombiFlash快速制备仪以洗脱剂体系B纯化，得到标题产物1b(6.20g)，产率：73.8％。
MS m/z(ESI):487.2[M+1]
第二步
2-丁氧基-N 4,N 4-双(4-甲氧基苄基)-5-硝基-N 6-(4-(吡咯烷-1-基甲基)苄基)嘧啶-4,6-二
胺1d
将1b(1.20g，2.65mmol)溶于20mL四氢呋喃中，依次加入三乙胺(402mg，3.98mmol)和(4-(吡咯烷-1-基甲基)苯基)甲胺1c(469mg，2.65mmol，采用专利申请“WO2016044183”公开的方法制备而得)，室温搅拌2小时，停止反应。加入50mL水，用乙酸乙酯(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，再用薄层色谱法以展开剂体系A纯化，得到标题产物1d(1.00g)，产率：59.1％。
MS m/z(ESI):641.3[M+1]
第三步
2-丁氧基-N 4,N 4-双(4-甲氧基苄基)-N 6-(4-(吡咯烷-1-基甲基)苄基)嘧啶-4,5,6-三胺1e
依次将1d(1.00g，1.56mmol)、铁(350mg，6.25mmol)和氯化铵(675mg，12.50mmol)溶解于25mL乙醇和水(V/V＝4:1)的混合溶液中，加热至80℃，搅拌4小时。停止反应，冷却至室温，过滤，滤液加入30mL水，用二氯甲烷(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，得到标题产物1e(630mg)，产率：66.1％。
MS m/z(ESI):611.3[M+1]
第四步
N-(4-(双(4-甲氧基苄基)氨基)-2-丁氧基-6-((4-(吡咯烷-1-基甲基)苄基)氨基)嘧啶-5-基)-1-氯甲磺酰胺1f
将1e(305mg，0.50mmol)溶于10mL二氯甲烷中，依次加入吡啶(119mg，1.50mmol)、4-二甲氨基吡啶(61mg，0.50mmol)和氯甲基磺酰氯(149mg，1.00mmol)，室温反应2小时，停止反应。加入20mL水，用二氯甲烷(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，得到标题产物1f(260mg)，产率：72.0％。
MS m/z(ESI):723.3[M+1]
第五步
8-(双(4-甲氧基苄基)氨基)-6-丁氧基-4-(4-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物1g
将1f(260mg，0.36mmol)溶于5mLN,N-二甲基甲酰胺中，依次加入氢化钠(43mg，1.08mmol)和碘化钾(30mg，0.18mmol)，加热至50℃，搅拌反应16小时，停止反应。冷却至室温，加入20mL水，用乙酸乙酯(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(20mL×3)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用薄层色谱法以展开剂体系A纯化，得到标题产物1g(142mg)，产率： 57.4％。
MSm/z(ESI):687.3[M+1]
第六步
8-氨基-6-丁氧基-4-(4-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物1
将1g(300mg，0.55mmol)和10mL三氟乙酸加入反应瓶中，室温反应6小时，停止反应。滴加饱和碳酸氢钠溶液至pH为7～8，用二氯甲烷(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用薄层色谱法以展开剂体系A纯化，得到标题产物1(99mg)，产率：40.1％。
MS m/z(ESI):447.3[M+1]
1H NMR(400MHz,CD 3OD)δ7.49(s,4H),4.97(s,2H),4.61(s,2H),4.32(s,2H),4.15-4.12(t,3H),3.35-3.29(m,4H),2.89-2.87(m,4H),2.09-2.06(m,4H),1.65-1.61(m,2H),1.42-1.38(m,2H),0.93-0.89(t,3H).
实施例2
8-氨基-6-丁氧基-4-(3-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物
Figure PCTCN2018073815-appb-000024
Figure PCTCN2018073815-appb-000025
第一步
2-丁氧基-N 4,N 4-双(4-甲氧基苄基)-5-硝基-N 6-(3-(吡咯烷-1-基甲基)苄基)嘧啶-4,6-二
胺2b
将1b(4.5g，9.24mmol)溶于50mL四氢呋喃中，依次加入三乙胺(1.4g，13.86mmol)、4-二甲氨基吡啶(225mg，1.85mmol)和(3-(吡咯烷-1-基甲基)苯基)甲胺2a(1.76g，9.24mmol，采用专利申请“WO2010077613”公开的方法制备而得)，室温搅拌2小时，停止反应。反应液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，得到标题产物2b(5.0g)，产率：84.7％。
MS m/z(ESI):641.3[M+1]
第二步
2-丁氧基-N 4,N 4-双(4-甲氧基苄基)-N 6-(3-(吡咯烷-1-基甲基)苄基)嘧啶-4,5,6-三胺2c
依次将2b(3.3g，5.15mmol)、铁(1.15g，20.6mmol)和氯化铵(2.22g，41.20mmol)溶解于38mL乙醇和水(V/V＝15:4)的混合溶液中，加热至80℃，搅拌4小时。停止反应，冷却至室温，过滤，滤液加入50mL水，用二氯甲烷(30mL×4)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，得到标题产物2c(2.08g)，产率：66.2％。
MS m/z(ESI):611.3[M+1]
第三步
N-(4-(双(4-甲氧基苄基)氨基)-2-丁氧基-6-((3-(吡咯烷-1-基甲基)苄基)氨基)嘧啶-5- 基)-1-氯甲磺酰胺2d
将2c(600mg，0.98mmol)溶于20mL二氯甲烷中，依次加入4-二甲氨基吡啶(120mg，0.98mmol)、吡啶(232mg，2.94mmol)和氯甲基磺酰氯(293mg，1.97mmol)，室温反应2小时，停止反应。反应液减压浓缩，残余物用CombiFlash快速制备仪以洗脱剂体系A纯化，得到标题产物2d(480mg)，产率：67.8％。
MS m/z(ESI):723.3[M+1]
第四步
8-(双(4-甲氧基苄基)氨基)-6-丁氧基-4-(3-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物2e
将2d(72mg，0.10mmol)溶于5mL N,N-二甲基甲酰胺中，依次加入碘化钾(4mg，0.02mmol)和氢化钠(12mg，0.30mmol)，加热至50℃，搅拌反应16小时，停止反应。冷却至室温，加入20mL水，用乙酸乙酯(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL×2)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用薄层色谱法以展开剂体系A纯化，得到标题产物2e(38mg)，产率：55％。MS m/z(ESI):687.3[M+1]
第五步
8-氨基-6-丁氧基-4-(3-(吡咯烷-1-基甲基)苄基)-3,4-二氢-1H-嘧啶并[5,4-c][1,2,5]噻二嗪2,2-二氧化物2
将2e(68mg，0.10mmol)、5mL三氟乙酸和5mL二氯甲烷加入反应瓶中，室温反应2小时，停止反应。加入20mL水，用二氯甲烷(20mL×3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤，用无水硫酸镁干燥，过滤，滤液减压浓缩，残余物用薄层色谱法以展开剂体系A纯化后，再用高效液相色谱法以展开剂体系A纯化，得到标题产物2(12mg)，产率：27.2％。
MS m/z(ESI):447.5[M+1]
1H NMR(400MHz,CD 3OD)δ7.46(s,1H),7.41-7.32(m,3H),4.96(s,2H),4.50(m,2H),4.16-4.13(m,2H),3.96(s,2H),2.89-2.87(m,4H),1.94-1.91(m,4H),1.65-1.62(m,2H),1.43-1.31(m,2H),0.93-0.90(t,3H).
测试例：
生物学评价
测试例1、本发明化合物对人源TLR7激动活性的测定
本发明化合物对HEK-Blue TM hTLR7稳转株细胞表达的hTLR7蛋白激活作用采用如下实验方法测定：
一、实验材料及仪器
1.DMEM(Gibco,10564-029),
2.胎牛血清(GIBCO,10099),
3.台盼蓝溶液(Sigma,T8154-100ML),
4.Flexstation 3多功能酶标仪(Molecμlar Devices),
5.HEK-Blue TM hTLR7细胞系(InvivoGen,hkb-hTLR7),
6.HEK-Blue检测试剂(InvivoGen,hb-det3),
7.磷酸盐缓冲液(PBS)pH7.4(上海源培生物科技股份有限公司，B320)。
二、实验步骤
配置HEK-Blue检测培养基，取HEK-Blue检测干粉一袋，加入50ml去内毒素水溶解，再放入37℃培养箱，10分钟后无菌过滤。化合物先配制成20mM的原液；再用纯DMSO稀释至最高浓度为6x10 6nM，经3倍梯度稀释，共10个点。
用培养基先把上述配制好的化合物稀释20倍，然后每孔加入20μl稀释后的化合物。取HEK-Blue TM hTLR7细胞，先去掉上清，再加入2-5ml预热的PBS，放入培养箱1-2分钟，轻轻吹打细胞，台盼蓝染色计数。用HEK-Blue检测培养基重悬细胞调整浓度为2.2×10 5个细胞/ml，加180μl细胞至上述已加入20μl药物的96孔细胞培养板中，37℃，培养6-16h。
酶标仪读数，波长为620nm。可获得相应的OD值，经Graphpad Prism计算得到药物的EC 50值。
本发明化合物对人源TLR7激活作用可通过以上的试验进行测定，测得的EC 50值见表1。
表1 本发明化合物对人源TLR7的EC 50。
实施例编号 EC 50(nM)
1 443
结论：本发明化合物对人源TLR7具有较好的激活作用。
测试例2、本发明化合物对人源TLR8激动活性的测定
本发明化合物对HEK-Blue TM hTLR8稳转株细胞表达的hTLR8蛋白激活作用采用如下实验方法测定：
一、实验材料及仪器
1.DMEM(Gibco,10564-029),
2.胎牛血清(GIBCO,10099),
3.台盼蓝溶液(Sigma,T8154-100ML),
4.Flexstation 3多功能酶标仪(Molecμlar Devices),
5.HEK-Blue TM hTLR8细胞系(InvivoGen,hkb-hTLR8),
6.HEK-Blue检测试剂(InvivoGen,hb-det3),
7.磷酸盐缓冲液(PBS)pH7.4(上海源培生物科技股份有限公司，B320)。
二、实验步骤
配置HEK-Blue检测培养基，取HEK-Blue检测干粉一袋，加入50ml去内毒素水溶解，再放入37℃培养箱，10分钟后无菌过滤。化合物先配制成20mM的 原液；再用纯DMSO稀释至最高浓度为6×10 6nM，然后3倍梯度稀释，共10个点；用培养基先把化合物稀释20倍，然后每孔加入20μl稀释后的化合物。
取HEK-Blue TM hTLR8细胞，先去掉上清，加入预热的PBS 2-5ml，放入培养箱1-2分钟,轻轻吹打细胞，台盼蓝染色计数。用HEK-Blue检测培养基重悬细胞调整浓度为2.2×10 5个细胞/ml，加180μl细胞至上述已加入20μl药物的96孔细胞培养板中，37℃，培养6-16h。
酶标仪读数，波长为620nm。可获得相应的OD值，经Graphpad Prism计算得到药物的EC 50值。
本发明化合物对人源TLR8激活作用可通过以上的试验进行测定，测得的EC 50值见表2。
表2 本发明化合物对人源TLR8的EC 50。
实施例编号 EC 50(nM)
1 4877
2 >30000
结论：本发明化合物对人源TLR8激活作用较弱，说明本发明化合物对TLR7具有选择性。
测试例3、本发明中化合物刺激外周血单个核细胞(PBMC)分泌IFN-α能力的测定
本发明中化合物刺激PBMC分泌IFN-α能力采用如下实验方法测定：
一、实验材料及仪器
1.RPMI 1640(Invitrogen,11875),
2.FBS(Gibco,10099-141),
3.Ficoll-Paque PREMIUM(GE,17-5442-02),
4.台盼蓝溶液(Sigma,T8154-100ML),
5.SepMateTM-50(Stemcell,15460),
6.Bright-Line TM血细胞计数仪(Sigma,Z359629-1EA),
7. 96孔平底板(Corning,3599),
8. 96孔v底板(Corning,3894),
9.人源IFN-α试剂盒(cisbio,6FHIFPEB),
10.PHERAStar多功能酶标仪(BMG,PHERAStar)。
二、实验步骤
化合物用纯DMSO稀释，最高浓度为5mM，4倍梯度稀释，共9个点。然后取4μl化合物，加入到196μl含10％FBS的RMPI 1640培养基中，混匀。每孔取50μl至新的96孔细胞培养板。
所有试剂平衡到室温，取250ml培养瓶，将60ml血液和PBS+2％FBS加入其中，轻轻吹打混匀稀释。取50ml PBMC分离管SepMateTM-50，加入15ml淋巴 细胞分离液Ficoll-Paque PREMIUM，然后加入30ml稀释后血液。1200g离心10分钟，室温。取上清，然后300g，离心8分钟。用含10％FBS的RMPI 1640培养基重悬并计数，调整PBMC数量至3.33×10 6个细胞/ml，取150μl至已加入化合物的细胞培养板中，37℃，5.0％CO 2的培养箱中培养24h。
将细胞培养板放入离心机中，1200rpm，室温离心10分钟。每孔取出150μl上清。先平衡人源IFN-α试剂盒中的试剂至常温，在避光条件下根据试剂盒说明书配制抗-IFN-α-Eu 3+-穴状结合物(Cryptate conjugate)和抗-IFN-α-d2-结合物，两者均以1:40的比例与结合缓冲液(conjugate Buffer)混匀。然后每孔加入16μl的离心取得的上清液。再每孔加入2μl刚配好的抗-IFN-α-Eu 3+-穴状结合物和抗-IFN-α-d2-结合物，震荡混匀，室温避光孵育3h。
在PHERAStar上用HTRF模式读数。我们将刺激产生最低检测限至少3倍以上细胞因子水平的最低药物浓度，定义为该化合物在该细胞因子刺激实验上的MEC(最小有效浓度Minimal Effective Concentration)值。
本发明化合物刺激PBMC分泌IFN-α的能力通过以上的试验进行测定，测得的MEC值见表3。
表3 本发明化合物刺激PBMC分泌IFN-α的MEC
实施例编号 MEC(nM)
1 1.5
2 22
结论：从刺激PBMC分泌IFN-α的活性的数据上看，本发明化合物能够较好的引起IFN-α释放。
测试例4、本发明化合物对人肝微粒体CYP3A4咪达唑仑代谢位点的酶活性的抑制作用
本发明化合物对人肝微粒体CYP3A4咪达唑仑代谢位点的酶活性采用如下实验方法测定：
一、实验材料及仪器
1.磷酸缓冲液(PBS),
2.NADPH(Sigma N-1630),
3.人肝微粒体(Corning Gentest),
4.ABI QTrap 4000液质两用仪(AB Sciex),
5.Inertsil C8-3柱,4.6×50mm,5μm(美国迪马公司),
6.CYP探针底物(15μM的咪达唑仑，SIGMA UC429)和阳性对照抑制剂(酮康唑，SIGMA K1003)。
二、实验步骤
配置100mM的PBS缓冲液，用该缓冲液配制2.5mg/ml的微粒体溶液和5mM 的NADPH溶液，用PBS梯度稀释5X浓度的化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS梯度稀释5X浓度的酮康唑工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS稀释至15μM浓度的咪达唑仑工作液。
分别取2.5mg/ml的微粒体溶液、15μM的咪达唑仑工作液、MgCl 2溶液和化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM，每个浓度设置不同的反应体系)各20μl，混合均匀。阳性对照组用相同浓度的酮康唑代替化合物。同时将5mM的NADPH溶液一起在37℃预孵育5分钟。5分钟之后取20μl NADPH加入到个孔中，启动反应，孵育30分钟。所有孵育样品设双样本。30分钟后向所有样本中加入250μl含内标的乙腈，混匀，800rpm摇10分钟，然后3700 rpm离心10分钟。取80μl的上清液，转移至LC-MS/MS分析。
数值经Graphpad Prism计算得到药物对CYP3A4咪达唑仑代谢位点的IC 50值见表4。
表4 本发明化合物对CYP3A4咪达唑仑代谢位点的IC 50值
实施例编号 IC 50(μM)
1 31
2 17
结论：本发明化合物对人肝微粒体CYP3A4的咪达唑仑代谢位点没有抑制作用，表现出更好的安全性，提示不会发生基于CYP3A4代谢咪达唑仑代谢位点的代谢性药物相互作用。
测试例5、本发明化合物对人肝微粒体CYP2D6酶活性的抑制作用
本发明化合物对人肝微粒体CYP2D6酶活性采用如下实验方法测定：
一、实验材料及仪器
1.磷酸缓冲液(PBS),
2.NADPH(Sigma N-1630),
3.人肝微粒体(Corning Gentest),
4.ABI QTrap 4000液质两用仪(AB Sciex),
5.Inertsil C8-3柱,4.6×50mm,5μm(美国迪马公司),
6.CYP探针底物(20μM的右美沙芬，SIGMA Q0750)和阳性对照抑制剂(奎尼丁，SIGMA D9684)。
二、实验步骤
配置100mM的PBS缓冲液，用该缓冲液配制2.5mg/ml的微粒体溶液和5mM的NADPH溶液，用PBS梯度稀释5X浓度的化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS梯度稀释5X浓度的奎尼丁工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS稀释至20μM浓度的右美沙芬工作液。
分别取2.5mg/ml的微粒体溶液、20μM的右美沙芬工作液、MgCl 2溶液和化合 物工作液(150、50、15、5、1.5、0.15、0.015、0μM，每个浓度设置不同的反应体系)各20μl，混合均匀。阳性对照组用相同浓度的奎尼丁代替化合物。同时将5mM的NADPH溶液一起在37℃预孵育5分钟，5分钟之后取20μl NADPH加入到个孔中，启动反应，孵育30分钟。所有孵育样品设双样本。30分钟后向所有样本中加入250μl含内标的乙腈，混匀，800rpm摇10分钟。3700rpm离心10分钟。取80μl的上清液，转移至LC-MS/MS分析。
数值经Graphpad Prism计算得到药物对CYP2D6代谢位点的IC 50值见表5。
表5 本发明化合物对CYP2D6代谢位点的IC 50值
实施例编号 IC 50(μM)
1 36
2 22
结论：本发明化合物对人肝微粒体CYP2D6的酶活性没有抑制作用，表现出更好的安全性，提示不会发生基于CYP2D6发生代谢性药物相互作用。
测试例6、本发明化合物对人肝微粒体CYP3A4睾酮代谢位点的酶活性的抑制作用
本发明化合物对人肝微粒体CYP3A4睾酮代谢位点的酶活性采用如下实验方法测定：
一、实验材料及仪器
1.磷酸缓冲液(PBS),
2.NADPH(Sigma N-1630),
3.人肝微粒体(Corning Gentest),
4.ABI QTrap 4000液质两用仪(AB Sciex),
5.Inertsil C8-3柱,4.6×50mm,5μm(美国迪马公司),
6.CYP探针底物(睾酮/100μM，SIGMA K1003)和阳性对照抑制剂(酮康唑，Dr.Ehrenstorfer GmbH,C17322500)。
二、实验步骤
配置100mM的PBS缓冲液，用该缓冲液配制2.5mg/ml的微粒体溶液和5mM的NADPH溶液，用PBS梯度稀释5X浓度的化合物工作液(150,50,15,5,1.5,0.15,0.015,0μM)。用PBS梯度稀释5X浓度的酮康唑工作液(150,50,15,5,1.5,0.15,0.015,0μM)。用PBS稀释至50μM浓度的右美沙芬工作液。
分别取2.5mg/ml的微粒体溶液、50μM的睾酮工作液、MgCl 2溶液和化合物工作液(150,50,15,5,1.5,0.15,0.015,0μM，每个浓度设置不同的反应体系)各20μl，混合均匀。阳性对照组用相同浓度的酮康唑代替化合物。同时将5mM的NADPH溶液一起在37℃预孵育5分钟。5分钟之后取20μl NADPH加入到个孔中，启动反应，孵育30分钟。所有孵育样品设双样本。30分钟后向所有样本中加入250μl 含内标的乙腈，混匀，800rpm摇10分钟。3700rpm离心10分钟。取80μl的上清液，转移至LC-MS/MS分析。
数值经Graphpad Prism计算得到药物对CYP3A4睾酮代谢位点的IC 50值见表6。
表6 本发明化合物对CYP3A4睾酮代谢位点的IC 50值
实施例编号 IC 50(μM)
1 13
结论：本发明化合物对对人肝微粒体CYP3A4的睾酮代谢位点没有抑制作用，表现出更好的安全性，提示不会发生基于CYP3A4的睾酮代谢位点的代谢性药物相互作用。
测试例7
1、实验目的
应用全自动膜片钳在转染hERG钾通道的稳定细胞株上测试本发明化合物对hERG钾电流的阻断作用。
2、实验方法
2.1实验材料与仪器
2.1.1实验材料：
试剂名称 供货公司 货号
FBS GIBCO 10099
丙酮酸钠溶液 sigma S8636-100ML
MEM非必需氨基酸溶液(100×) sigma M7145-100ML
G418硫酸盐 Enzo ALX-380-013-G005
MEM Hyclone SH30024.01B
hERG cDNA Origene -
2.1.2实验仪器：
Figure PCTCN2018073815-appb-000026
2.2全自动膜片钳实验步骤
HEK293-hERG稳定细胞株按照1:4的密度在MEM/EBSS培养基(10％FBS，400μg/ml G418,1％ MEM非必需氨基酸溶液(100×),1％丙酮酸钠溶液)中进行传 代培养，培养48-72小时之内进行全自动膜片钳实验。实验当天将细胞用0.25％胰酶消化后，离心收集细胞，用细胞外液(140mM NaCl，4mM KCl，1mM MgCl 2，2mM CaCl 2，5mMD一水葡萄糖，10mM Hepes,pH7.4,298 mOsmol)重悬细胞制成细胞悬液。将细胞悬液放置在Patchliner仪器的细胞库上，Patchliner仪器利用负压控制器将细胞加到芯片(NPC-16)上，负压将单个细胞吸引在芯片的小孔上。当形成全细胞模式后，仪器将按照设定的hERG电流电压程序得到hERG电流，然后仪器自动的由低浓度到高浓度，进行化合物灌流。通过HEAK Patchmaster,HEAK EPC10膜片钳放大器(Nanion)和Pathlinersoftware以及Pathcontrol HTsoftware提供的数据分析软件，对化合物各浓度下的电流以及空白对照电流进行分析。
2.3测试结果
本发明化合物对hERG钾电流的阻断作用通过以上的试验进行测定，测得的IC 50值见表7。
表7 本发明化合物对hERG钾电流的阻断作用的IC 50
实施例编号 IC 50(μM)
1 ＞30
2 12
结论：本发明化合物对hERG的抑制作用弱，可降低由hERG通路引起的副作用。
药代动力学评价
测试例8、本发明化合物的小鼠药代动力学测试
1、摘要
以小鼠为受试动物，应用LC/MS/MS法测定了小鼠灌胃给予实施例1化合物后不同时刻血浆中的药物浓度。研究本发明化合物在小鼠体内的药代动力学行为，评价其药动学特征。
2、试验方案
2.1试验药品
实施例1化合物。
2.2试验动物
C57小鼠9只，雌性，购自上海杰思捷实验动物有限公司，动物生产许可证号：SCXK(沪)2013-0006。
2.3药物配制
称取一定量药物，加5％体积的DMSO、5％体积的tween80和90％生理盐水配置成1mg/ml无色澄清透明液体。
2.4给药
C57小鼠禁食过夜后灌胃给药，给药剂量均为20.0 mg/kg，给药体积均为20ml/kg。
3、操作
小鼠灌胃给药实施例1化合物，于给药前及给药后0.25，0.5，1.0，2.0，4.0，6.0，8.0，11.0，24.0小时采血0.1ml，置于肝素化试管中，3500转/分钟离心10分钟分离血浆，于-20℃保存。
测定不同浓度的药物灌胃给药后小鼠血浆中的待测化合物含量：取给药后各时刻的小鼠血浆25μl，加入内标溶液喜树碱50μl(100 ng/mL)，乙腈200μl，涡旋混合5分钟，离心10分钟(3600转/分钟)，取上清液10μl进行LC/MS/MS分析。
4、药代动力学参数结果
本发明化合物的药代动力学参数如下：
Figure PCTCN2018073815-appb-000027
结论：本发明化合物的药代吸收较好，具有药代动力学优势。
测试例9、本发明化合物的小鼠IFN-α值测定
一、实验材料及仪器
1、LumiKine TM Xpress mIFN-α(invivogen，luex-mifna)
2、酶标仪(PE，victor3)
3、试验样本：测试例8药代动力学中小鼠灌胃给药实施例1化合物，于给药前及给药后0，2.0，4.0和8.0小时采血。
二、实验步骤
1、包被：将100μl捕获抗体(用PBS稀释至终浓度为1μg/ml)加到96孔ELISA专用板中。贴膜后室温孵育过夜。
2、第二天倒掉板并拍干板内的液体。
3、每孔加200μl封闭缓冲液(PBS+2％BSA+0.05％Tween 20)，37℃孵育2小时.
4、倒掉板并拍干板内的液体。
5、加入用稀释缓冲液稀释到100μl的待测样本和标准曲线样本(最高浓度为500pg/ml，二倍梯度稀释七个点)。
6、用洗板机吸掉板内的液体，再每孔注入300μl洗涤缓冲液(PBS+0.05％Tween20)，震荡后吸掉液体，重复清洗3次，最后拍干板内的水滴。
7、每孔加入100μl连接有Lucia蛋白酶的抗体(终浓度为30 ng/ml),37℃孵育2小时。
8、重复步骤6。
9、每孔加入50μl QUANTI-Luc(化学发光试剂)，酶标仪读数。
三、检测结果：
本发明化合物的小鼠血清IFN-α值见如下表1和时间曲线见图1：
表1 小鼠血清中IFN-α值
Figure PCTCN2018073815-appb-000028
i.g.为灌胃给药。
四、结论：
实施例1化合物给药20mpk在小鼠体内2小时后，有较强的刺激IFN-α表达的能力。

Claims (22)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2018073815-appb-100001
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    环A选自环烷基、杂环基、芳基和杂芳基；
    G选自N和CR 6；
    X 1选自亚烷基和S(O) m，其中所述的亚烷基任选被选自卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基和杂环基中的一个或多个取代基所取代；
    L 1选自-NR 7-、-O-、-S-、-C(O)-、-S(O) m-、-N(R 7)C(O)-、-C(O)N(R 7)-、-N(R 7)S(O) 2-、-S(O) 2N(R 7)-和共价键；
    R 1选自氢原子、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基，其中所述的烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
    R 2相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10，其中所述的烷基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
    L 2选自亚烷基或共价键，其中所述的亚烷基任选被选自卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基和杂环基中的一个或多个取代基所取代；
    R 3选自氢原子、烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10，其中所述的环烷基、杂环基、芳基和杂芳基各自独立地任选被选 自烷基、烷氧基、卤素、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-C(O)R 8、-C(O)OR 8、-S(O) mR 8、-NR 9R 10和-C(O)NR 9R 10中的一个或多个取代基所取代；
    R 4和R 5相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
    或R 4和R 5一起形成氧代基；
    R 6选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
    R 7选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
    R 8选自氢原子、烷基、卤代烷基、氨基、羟基、环烷基、杂环基、芳基和杂芳基；
    R 9和R 10相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基和杂芳基，其中所述的烷基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
    或者，所述R 9和R 10与相连接的氮原子一起形成杂环基，其中所述的杂环基内含有1～2个相同或不同选自N、O和S的杂原子，并且所述的杂环基任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
    n为0、1、2、3或4；且
    m为0、1或2。
  2. 根据权利要求1所述的通式(I)所示的化合物，其中所述的环A选自苯基和吡啶基。
  3. 根据权利要求1或2所述的通式(I)所示的化合物，其中所述的X 1为亚烷基。
  4. 根据权利要求1～3中任一项所述的通式(I)所示的化合物，其为通式(II)所示的化合物：
    Figure PCTCN2018073815-appb-100002
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    G、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  5. 根据权利要求1～4中任一项所述的通式(I)所示的化合物，其为通式(III)所示的化合物：
    Figure PCTCN2018073815-appb-100003
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    R 9和R 10与相连接的氮原子一起形成杂环基，其中所述的杂环基内含有1～2个相同或不同选自N、O和S的杂原子，并且所述的杂环基任选被选自烷基、烷氧基、卤素、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
    G、L 1～L 2和R 1如权利要求1中所定义。
  6. 根据权利要求1～4中任一项所述的通式(I)所示的化合物，其中所述的R 4和R 5均为氢或者R 4和R 5一起形成氧代基。
  7. 根据权利要求1～6中任一项所述的通式(I)所示的化合物，其中所述的L 1为-O-。
  8. 根据权利要求1～7中任一项所述的通式(I)所示的化合物，其中所述的R 1为烷基。
  9. 根据权利要求1～8中任一项所述的通式(I)所示的化合物，其中所述的G为N。
  10. 根据权利要求1～9中任一项所述的通式(I)所示的化合物，其中所述的L 2为亚烷基。
  11. 根据权利要求1～10中任一项所述的通式(I)所示的化合物，其选自：
    Figure PCTCN2018073815-appb-100004
  12. 一种通式(IB)所示的化合物：
    Figure PCTCN2018073815-appb-100005
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    W为氨基保护基；
    X为卤素；
    S为1或2；
    环A、G、X 1、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  13. 根据权利要求12中所述的通式(IB)所示的化合物，其选自：
    Figure PCTCN2018073815-appb-100006
  14. 一种通式(IC)所示的化合物：
    Figure PCTCN2018073815-appb-100007
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    W为氨基保护基；
    S为1或2；
    环A、G、X 1、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  15. 根据权利要求14中所述的通式(IC)所示的化合物，其选自：
    Figure PCTCN2018073815-appb-100008
  16. 一种制备根据权利要求12所述的通式(IB)所示的化合物的方法，该方法包括：
    Figure PCTCN2018073815-appb-100009
    通式(IA)的化合物和
    Figure PCTCN2018073815-appb-100010
    发生亲核取代反应得到通式(IB)的化合物；
    其中：
    W为氨基保护基；
    X为卤素；
    S为1或2；
    环A、G、X 1、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  17. 一种制备根据权利要求14所述的通式(IC)所示的化合物的方法，该方法包括：
    Figure PCTCN2018073815-appb-100011
    通式(IB)的化合物关环得到通式(IC)的化合物；
    其中：
    W为氨基保护基；
    X为卤素；
    S为1或2；
    环A、G、X 1、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  18. 一种制备根据权利要求1所述的通式(I)所示的化合物的方法，该方法包括：
    Figure PCTCN2018073815-appb-100012
    通式(IC)的化合物脱去保护基得到通式(I)的化合物；
    其中：
    W为氨基保护基；
    S为1或2；
    环A、G、X 1、L 1～L 2、R 1～R 5和n如权利要求1中所定义。
  19. 一种药物组合物，所述药物组合物含有治疗有效量的根据权利要求1～11任一项所述的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  20. 根据权利要求1～11任一项所述的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药 用的盐或根据权利要求19所述的药物组合物在制备用于TLR7激动剂的药物中的用途。
  21. 根据权利要求1～11任一项所述的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求19所述的药物组合物在制备用于治疗由病毒引起的感染的药物中的用途，所述病毒选自：登革热病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、蜱传脑炎病毒、昆津病毒、墨累山谷脑炎病毒、圣路易脑炎病毒、鄂木斯克出血热病毒、牛病毒性腹泻病毒、寨卡病毒、HIV、HBV、HCV、HPV、RSV、SARS和流感病毒。
  22. 根据权利要求1～11任一项所述的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求19所述的药物组合物在制备用于治疗或预防黑色素瘤、非小细胞肺癌、肝细胞癌、基底细胞癌、肾细胞癌、骨髓瘤、变应性鼻炎、哮喘、慢性阻塞性肺炎、溃疡性结肠炎、自身免疫性疾病、斑块银屑病、系统性红斑狼疮、光化角质病和肝纤维化的药物中的用途。
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