CN109937205B

CN109937205B - Heteroaryl thiadiazine-2, 2-dioxide derivatives, preparation method and application thereof in medicines

Info

Publication number: CN109937205B
Application number: CN201880004318.3A
Authority: CN
Inventors: 张国宝; 陈一千; 贺峰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd; Chengdu Suncadia Pharmaceuticals Co Ltd
Priority date: 2017-01-24
Filing date: 2018-01-23
Publication date: 2021-12-21
Anticipated expiration: 2038-01-23
Also published as: TW201827442A; WO2018137614A1; CN109937205A

Abstract

The invention discloses a heteroaryl thiadiazine-2, 2-dioxide derivative, a preparation method and application thereof in medicine. The invention particularly discloses a novel heteroaryl thiadiazine-2, 2-dioxide derivative shown in a general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, particularly as a TLR7 agonist, wherein each substituent in the general formula (I) is defined as the same as that in the specification.

Description

Heteroaryl thiadiazine-2, 2-dioxide derivatives, preparation method and application thereof in medicines

Technical Field

The invention belongs to the field of medicines, and relates to a novel heteroaryl thiadiazine-2, 2-dioxide derivative shown in a general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative, and application of the derivative as a therapeutic agent, in particular as a TLR7 agonist.

Background

Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity. TLRs are non-catalytic receptors for single bodies to span membranes, are usually expressed in sentinel cells such as macrophages and dendritic cells, and recognize structurally conserved molecules produced by microorganisms. Once these microorganisms break through physical barriers such as skin or gut mucosa, they are recognized by TLRs, which in turn activate immune cell responses (Mahla, R s. et al, Front immunol. 4: 248 (2013)). The immune system has the ability to broadly recognize pathogenic microorganisms, in part due to the widespread existence of Toll-like immune receptors.

There are at least 10 different TLRs in mammals. Ligands for some of these receptors and the corresponding signal cascades have been identified. TLR7 is a member of the subset of TLRs (TLRs 3, 7, 8 and 9), restricted to the endosomal compartment of cells that specifically detect non-self nucleic acids. TLR7 plays a key role in antiviral defense through recognition of ssRNA (Diebold S.S. et al, Science, 2004: 303, 1529-. TLR7 has a limited expression profile in humans and is expressed predominantly by B cells and plasmacytoid dendritic cells (pdcs), and to a lesser extent by monocytes. Plasmacytoid DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% Peripheral Blood Mononuclear Cells (PBMCs)) that are the primary type I interferon-producing cells that secrete high levels of interferon-alpha (IFN α) and interferon-beta (IFN β) in response to viral infection (Liu Y-J, Annu. Rev. Immunol., 2005: 23, 275-.

Many diseases, disorders are associated with abnormalities of TLRs, such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma (basalcellcarcinosoma), renal cell carcinoma, myeloma, allergic rhinitis, asthma, Chronic Obstructive Pulmonary Disease (COPD), ulcerative colitis, liver fibrosis, HBV, Flaviviridae (Flaviviridae) virus, HCV, HPV, RSV, SARS, HIV or viral infection of the influenza, and the like. Therefore, agonists of TLRs are promising for the treatment of related diseases.

Because TLR7 and TLR8 are highly homologous, TLR7 ligand, and in most cases TLR8 ligand. TLR8 stimulation primarily induces production of cytokines such as tumor necrosis factor alpha (TNF-a) and chemokines. Interferon alpha is one of the main drugs for treating chronic hepatitis b or hepatitis c, and TNF-alpha is a proinflammatory cytokine, and excessive secretion may cause serious side effects. Selectivity to TLR7 and TLR8 is therefore critical for the development of TLR7 agonists for the treatment of viral infectious diseases.

Related TLR7 agonist patent applications are currently available, such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, WO 2012080730. There remains a need to continue to develop safe and therapeutically more effective TLR7 agonists.

Aiming at the technical problems, the invention provides a medicinal compound with lower acting concentration, better selectivity and more obvious activation effect, and the medicinal compound is a safer and more effective TLR7 agonist.

Disclosure of Invention

The invention aims to provide a compound shown in a general formula (I):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein:

ring a is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl;

g is selected from N and CR⁶；

X¹Selected from alkylene or S (O)_mWherein said alkylene groupOptionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, and heterocyclyl;

L¹is selected from-NR⁷-、-O-、-S-、-C(O)-、-S(O)_m-、-N(R⁷)C(O)-、-C(O)N(R⁷)-、-N(R⁷)S(O)₂-、 -S(O)₂N(R⁷) -and a covalent bond;

R¹selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, wherein said alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl groups, alkoxy groups, halogens, haloalkyl groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups;

R²are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R⁸、 -C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

L²selected from the group consisting of alkylene groups and covalent bonds, wherein said alkylene groups are optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, and heterocyclyl;

R³selected from the group consisting of hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Wherein said cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally selected from alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents of (1);

R⁴and R⁵The same or different, and each is independently selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

or R⁴And R⁵Together form an oxo group;

R⁶selected from the group consisting of hydrogen atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R⁷selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R⁸selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, amino groups, hydroxyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R⁹and R¹⁰The same or different, and each is independently selected from the group consisting of hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

or, said R⁹And R¹⁰Together with the nitrogen atom to which they are attached form a heterocyclic group containing 1 to 2 identical or different heteroatoms selected from N, O and S, and the heterocyclic group is optionally substitutedOne or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

n is 0, 1, 2, 3 or 4; and is

m is 0, 1 or 2.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein the ring a is selected from phenyl and pyridyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein X is¹Is an alkylene group.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (II):

wherein:

G、L¹～L²、R¹～R⁵and n is as defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (III):

wherein:

R⁹and R¹⁰Forming a heterocyclic group together with the nitrogen atom to which it is bonded, wherein the heterocyclic group contains 1 to 2 of the same or differentAnd a heteroatom selected from N, O and S, and said heterocyclyl is optionally substituted with one or more substituents selected from alkyl, alkoxy, halo, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably a 5-6 membered heterocyclyl, more preferably pyrrolidinyl, tetrahydrothienyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl or tetrahydropyranyl;

G、L¹～L²and R¹As defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is⁴And R⁵Are both hydrogen or R⁴And R⁵Together form an oxo group.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein L is¹is-O-.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is¹Is an alkyl group, preferably C_1-6More preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl or n-pentyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein G is N.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein L is²Is alkylene, preferably C_1-6More preferably a methylene group, a 1, 2-ethylene group, a 1, 1-ethylene group or a 1, 3-propylene group.

Typical compounds of the invention include, but are not limited to:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the present invention, a compound represented by the general formula (IB) is an intermediate for preparing the compound of the general formula (I):

wherein:

w is an amino protecting group, preferably tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Compounds of formula (IB) include, but are not limited to:

in a preferred embodiment of the present invention, a compound of formula (IC) is used as an intermediate for the preparation of a compound of formula (I):

wherein:

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Compounds of formula (IC) include, but are not limited to:

another aspect of the present invention relates to a method for preparing a compound represented by the general formula (IB), the method comprising:

compounds of the general formula (IA) and

nucleophilic substitution reaction to obtain the compound of general formula (IB);

wherein:

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Another aspect of the present invention relates to a method of preparing a compound represented by the general formula (IC), the method comprising:

ring closing the compound of formula (IB) to give a compound of formula (IC);

wherein:

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Another aspect of the present invention relates to a method of preparing a compound of formula (I), the method comprising:

removing the protecting group of the compound of the general formula (IC) to obtain a compound of the general formula (I);

wherein:

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The invention further relates to application of the compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same in preparation of a medicament for the TLR7 agonist.

The invention further relates to the use of a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for the preparation of a medicament for the treatment of 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, 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 virus.

The invention further relates to application of the compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound in preparation of medicines for treating or preventing melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis and hepatic fibrosis.

The invention further relates to a method for agonizing TLR7, comprising the step of contacting a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, with TLR 7.

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, murray valley encephalitis virus, saint louis encephalitis virus, ebosk hemorrhagic fever virus, bovine viral diarrhea virus, zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The present invention further relates to a method for treating or preventing melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis, autoimmune diseases, plaque psoriasis, systemic lupus erythematosus, actinic keratosis and liver fibrosis, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a medicament comprising the same, for use as a medicament.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt or a medicament containing the compound, which is used for the TLR7 agonist.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a medicament comprising 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, tick-borne 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 virus.

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof or a medicament containing the compound, which is used for treating or preventing melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis and hepatic fibrosis.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl paraben, one or more coloring agents, one or more flavoring agents and one or more sweetening agents.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickener. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent or one or more preservatives. Suitable dispersing or wetting agents and suspending agents are illustrative of the examples given above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Among the 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 oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding to a mixture of water and glycerol. The injection solution or microemulsion may be injected into the bloodstream of a patient by local bulk injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, non-toxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

The compounds of the present invention may be administered in the form of suppositories 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 therefore will melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as the mode of treatment, the daily amount of compound (I) of the formula or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylPropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylpropyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1-dimethylbutyl, 2-dimethylpentyl, 2-methylpentyl, 4-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2-methylpentyl, 4-methylpentyl, and so-methylpentyl, N-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof, and the like. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH)₂-), 1-ethylidene (-CH (CH)₃) -), 1, 2-ethylene (-CH)₂CH₂) -, 1-propylene (-CH (CH)₂CH₃) -), 1, 2-propylene (-CH)₂CH(CH₃) -), 1, 3-propylene (-CH)₂CH₂CH₂-) 1, 4-butylene (-CH₂CH₂CH₂CH₂-) and the like. Alkylene groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents preferably being independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) R⁸、-C(O)OR⁸、 -S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "alkenyl" refers to a hydrocarbon group having one or more fewer hydrogen atoms in the olefin molecule. The alkenyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from hydrogen atom, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "alkynyl" refers to the moleculeHydrocarbons containing a carbon-carbon triple bond. Alkynyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, preferably from 3 to 10 carbon atoms, more preferably from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "amino protecting group" is intended to protect an amino group with a group that can be easily removed in order to keep the amino group unchanged when the rest of the molecule is subjected to a reaction. Non-limiting examples include t-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, and the like. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably p-methoxybenzyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 10 ring atoms, of which 1-4 is a heteroatom; more preferably from 5 to 6 ring atoms; of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, tetrahydropyranyl, 1, 2.3.6-tetrahydropyridinyl, piperidinyl, and the like,Piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, 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 heterocyclyl, non-limiting examples of which include:

the heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、 -NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently optionally selected from alkyl, alkenylAlkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -C (O) R⁸、-C(O)OR⁸、-S(O)_mR⁸、-NR⁹R¹⁰and-C (O) NR⁹R¹⁰Is substituted with one or more substituents.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of 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 groups substituted with one or more substituents independently selected from hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above. The term "hydroxy" refers to an-OH group.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to the group-NH₂。

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

The term "oxo" refers to ═ O.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

m, G and R⁸～R¹⁰As defined for the compounds of general formula (I).

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

scheme one

The invention relates to a method for preparing a compound shown as a general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereoisomer or a mixture form thereof, or a pharmaceutically acceptable salt form thereof, which comprises the following steps:

in the first step, a compound of the formula (I-1) and NH- (W)_sCarrying out nucleophilic substitution reaction under alkaline condition to obtain a compound of a general formula (I-2);

secondly, carrying out nucleophilic substitution reaction on the compound of the general formula (I-2) and the compound of the general formula (I-3) under alkaline conditions to obtain a compound of the general formula (I-4);

thirdly, the compound of the general formula (I-4) is subjected to reduction reaction in the presence of a reducing agent to obtain a compound of a general formula (IA);

in a fourth step, compounds of the general formula (IA) and

carrying out nucleophilic substitution reaction under alkaline condition to obtain a compound of a general formula (IB);

fifthly, under the alkaline condition, the compound of the general formula (IB) is subjected to ring closing in the presence of iodide to obtain a compound of the general formula (IC);

sixthly, removing the protecting group of the compound of the general formula (IC) under an acidic condition to obtain a compound of a general formula (I);

wherein:

the reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide, and sodium N-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide;

reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, 1, 4-dioxane solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me₃SiCl and TMSOTf;

such reducing agents include, but are not limited to: iron powder, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (O-t-Bu)₃、AlH₃、NaCNBH₃、Na(AcO)₃BH、B₂H₅、Li(Et)₃BH、Pd/C/H₂And Raney nickel (Raney Ni)/H₂；

Iodides include, but are not limited to, elemental iodine, ketone iodide, 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-dimethylformamide and mixtures thereof;

x is halogen, preferably chlorine;

s is 1 or 2;

ring A, G, X¹、L¹～L²、R¹～R⁵And n is as defined in formula (I).

Scheme two

The invention relates to a method for preparing a compound shown as a general formula (II) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

secondly, carrying out nucleophilic substitution reaction on the compound of the general formula (I-2) and the compound of the general formula (II-1) under alkaline conditions to obtain a compound of the general formula (II-2);

thirdly, the compound of the general formula (II-2) is subjected to reduction reaction in the presence of a reducing agent to obtain a compound of the general formula (II-A);

in a fourth step, compounds of the formula (II-A) and

carrying out nucleophilic substitution reaction under alkaline condition to obtain a compound of a general formula (II-B);

fifthly, under the alkaline condition, the compound of the general formula (II-B) is subjected to ring closure in the presence of iodide to obtain a compound of the general formula (II-C);

sixthly, removing the protecting group of the compound of the general formula (II-C) under an acidic condition to obtain a compound of the general formula (II);

wherein:

x is halogen, preferably chlorine;

s is 1 or 2;

G、n、L¹～L²and R¹～R⁵As defined in formula (II).

Scheme three

The invention relates to a method for preparing a compound shown as a general formula (III) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

in the first step, a compound of the formula (I-1) and NH- (W)_sUnder alkaline condition, nucleophilic substitution reaction is carried out to obtainA compound of the general formula (I-2);

secondly, carrying out nucleophilic substitution reaction on the compound of the general formula (I-2) and the compound of the general formula (III-1) under alkaline conditions to obtain a compound of the general formula (III-2);

thirdly, carrying out reduction reaction on the compound of the general formula (III-2) in the presence of a reducing agent to obtain a compound of a general formula (III-A);

in a fourth step, compounds of the formula (III-A) and

carrying out nucleophilic substitution reaction under alkaline condition to obtain a compound of a general formula (III-B);

fifthly, under the alkaline condition, the compound of the general formula (III-B) is subjected to ring closing in the presence of iodide to obtain a compound of the general formula (III-C);

sixthly, removing the protecting group of the compound of the general formula (III-C) under an acidic condition to obtain a compound of the general formula (III);

wherein:

x is halogen, preferably chlorine;

s is 1 or 2;

G、L¹～L²、R¹and R⁹～R¹⁰As defined in formula (III).

Drawings

FIG. 1 is a time plot of mouse serum IFN- α values for compounds of the invention.

Detailed Description

Examples

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. delta.) of 10^-6The units in (ppm) are given. NMR was measured using a Brukeravence-400 nuclear magnetic spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS).

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

Chiral HPLC analytical determination using LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.).

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

Chiral preparative column chromatography used Prep Star SD-1(Varian Instruments Inc.) or SFC-multigram (Berger Instruments Inc.).

The CombiFlash rapid preparation instrument uses CombiFlash Rf200(TELEDYNE ISCO).

Average inhibition rate of kinase and IC₅₀The values were determined with a NovoStar microplate reader (BMG, Germany).

Known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co.KG, Acros Organics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, and the like.

In the examples, the reaction can be carried out in an argon atmosphere or a nitrogen atmosphere, unless otherwise specified.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction used a hydrogenation apparatus of Parr 3916EKX type and a hydrogen generator of Qinglan QL-500 type or a hydrogenation apparatus of HC2-SS type.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for purifying compounds and a developing solvent system for thin layer chromatography including: a: dichloromethane/methanol system, B: the volume ratio of the n-hexane/ethyl acetate system is adjusted according to the different polarities of the compounds, and a small amount of basic or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

Example 1

8-amino-6-butoxy-4- (4- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide

First step of

2-butoxy-6-chloro-N, N-bis (4-methoxybenzyl) -5-nitropyrimidin-4-amine 1b

2-butoxy-4, 6-dichloro-5-nitropyrimidine 1a (4.62g, 17.43mmol, prepared by the well-known method "Journal of Medicinal Chemistry, 2012, 55(23), 10387-. The reaction was stopped, distilled under reduced pressure and the residue was purified using CombiFlash flash prep with eluent system B to give the title product 1B (6.20g), yield: 73.8 percent.

MS m/z(ESI)：487.2[M+1]

Second step of

2-butoxy-N⁴，N⁴-bis (4-methoxybenzyl) -5-nitro-N⁶- (4- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4, 6-diamine 1d

1b (1.20g, 2.65mmol) was dissolved in 20mL tetrahydrofuran, triethylamine (402mg, 3.98mmol) and (4- (pyrrolidin-1-ylmethyl) phenyl) methylamine 1c (469mg, 2.65mmol, prepared as disclosed in the patent application "WO 2016044183") were added sequentially, and the reaction was stopped by stirring at room temperature for 2 hours. Addition of 50mL of water, extraction with ethyl acetate (20mL × 3), combination of the organic phases, washing with saturated sodium chloride solution (50mL), drying over anhydrous magnesium sulfate, filtration, concentration of the filtrate under reduced pressure, purification of the residue with CombiFlash flash prep apparatus with eluent system a and then with thin layer chromatography with developer system a gave the title product 1d (1.00g), yield: 59.1 percent.

MS m/z(ESI)：641.3[M+1]

The third step

2-butoxy-N⁴，N⁴-bis (4-methoxybenzyl) -N⁶- (4- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4, 5, 6-triamine 1e

1d (1.00g, 1.56mmol), iron (350mg, 6.25mmol) and ammonium chloride (675mg, 12.50 mmol) were dissolved in this order in a mixed solution of 25mL of ethanol and water (V/V4: 1), heated to 80 ℃ and stirred for 4 hours. The reaction was stopped, cooled to room temperature, filtered, 30mL of water was added to the filtrate, extracted with dichloromethane (20mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified with CombiFlash flash prep with eluent system a to give the title product 1e (630mg), yield: 66.1 percent.

MS m/z(ESI)：611.3[M+1]

The fourth step

N- (4- (bis (4-methoxybenzyl) amino) -2-butoxy-6- ((4- (pyrrolidin-1-ylmethyl) benzyl) amino) pyrimidin-5-yl) -1-chloromethanesulfonamide 1f

1e (305mg, 0.50mmol) was dissolved in 10mL of dichloromethane, and pyridine (119mg, 1.50mmol), 4-dimethylaminopyridine (61mg, 0.50mmol) and chloromethylsulfonyl chloride (149mg, 1.00 mmol) were added in this order to react at room temperature for 2 hours, and the reaction was stopped. 20mL of water was added, extracted with dichloromethane (20 mL. times.3), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified with a Combiflash flash Rapid prep using eluent System A to give the title product 1f (260mg), yield: 72.0 percent.

MS m/z(ESI)：723.3[M+1]

The fifth step

8- (bis (4-methoxybenzyl) amino) -6-butoxy-4- (4- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide 1g

1f (260mg, 0.36mmol) was dissolved in 5mL of N, N-dimethylformamide, and sodium hydride (43 mg, 1.08mmol) and potassium iodide (30mg, 0.18mmol) were added in this order, and the reaction was heated to 50 ℃ and stirred for 16 hours to stop the reaction. Cooled to room temperature, 20mL of water was added, extraction was performed with ethyl acetate (20mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (20mL × 3), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developer system a to give the title product 1g (142mg), yield: 57.4 percent.

MS m/z(ESI)：687.3[M+1]

The sixth step

8-amino-6-butoxy-4- (4- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide 1

1g (300mg, 0.55mmol) and 10mL of trifluoroacetic acid were added to a reaction flask, and reacted at room temperature for 6 hours, and the reaction was stopped. Saturated sodium bicarbonate solution was added dropwise to pH 7-8, extracted with dichloromethane (20mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developer system a to give the title product 1(99mg), yield: 40.1 percent.

MS m/z(ESI)：447.3[M+1]

¹H NMR(400MHz，CD₃OD)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).

Example 2

8-amino-6-butoxy-4- (3- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide

First step of

2-butoxy-N⁴，N⁴-bis (4-methoxybenzyl) -5-nitro-N⁶- (3- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4, 6-diamine 2b

1b (4.5g, 9.24mmol) was dissolved in 50mL tetrahydrofuran, triethylamine (1.4g, 13.86 mmol), 4-dimethylaminopyridine (225mg, 1.85mmol) and (3- (pyrrolidin-1-ylmethyl) phenyl) methylamine 2a (1.76g, 9.24mmol, prepared by the method disclosed in the patent application "WO 2010077613") were added sequentially, and the reaction was stirred at room temperature for 2 hours to stop the reaction. The reaction was concentrated under reduced pressure and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 2b (5.0g), yield: 84.7 percent.

MS m/z(ESI)：641.3[M+1]

Second step of

2-butoxy-N⁴，N⁴-bis (4-methoxybenzyl) -N⁶- (3- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4, 5, 6-triamine 2c

2b (3.3g, 5.15mmol), iron (1.15g, 20.6mmol) and ammonium chloride (2.22g, 41.20 mmol) were dissolved in this order in a mixed solution of 38mL ethanol and water (V/V15: 4), heated to 80 ℃ and stirred for 4 hours. The reaction was stopped, cooled to room temperature, filtered, 50mL of water was added to the filtrate, extracted with dichloromethane (30mL × 4), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified with CombiFlash flash prep with eluent system a to give the title product 2c (2.08g), yield: 66.2 percent.

MS m/z(ESI)：611.3[M+1]

The third step

N- (4- (bis (4-methoxybenzyl) amino) -2-butoxy-6- ((3- (pyrrolidin-1-ylmethyl) benzyl) amino) pyrimidin-5-yl) -1-chloromethanesulfonamide 2d

2c (600mg, 0.98mmol) was dissolved in 20mL of dichloromethane, and 4-dimethylaminopyridine (120mg, 0.98mmol), pyridine (232mg, 2.94mmol) and chloromethylsulfonyl chloride (293mg, 1.97 mmol) were added in this order to react at room temperature for 2 hours, and the reaction was stopped. The reaction was concentrated under reduced pressure and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 2d (480mg), yield: 67.8 percent.

MS m/z(ESI)：723.3[M+1]

The fourth step

8- (bis (4-methoxybenzyl) amino) -6-butoxy-4- (3- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide 2e

2d (72mg, 0.10mmol) was dissolved in 5mL of N, N-dimethylformamide, and potassium iodide (4 mg, 0.02mmol) and sodium hydride (12mg, 0.30mmol) were added in this order, and the reaction was heated to 50 ℃ and stirred for 16 hours to stop the reaction. Cooled to room temperature, 20mL of water was added, extraction was performed with ethyl acetate (20mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (50mL × 2), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developer system a to give the title product 2e (38mg), yield: and 55 percent.

MS m/z(ESI)：687.3[M+1]

The fifth step

8-amino-6-butoxy-4- (3- (pyrrolidin-1-ylmethyl) benzyl) -3, 4-dihydro-1H-pyrimido [5, 4-c ] [1, 2, 5] thiadiazine 2, 2-dioxide 2

2e (68mg, 0.10mmol), 5mL of trifluoroacetic acid and 5mL of dichloromethane were added to the reaction flask, and the reaction was stopped after reacting at room temperature for 2 hours. 20mL of water was added, extraction was performed with dichloromethane (20 mL. times.3), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by thin layer chromatography using developer system A and then by high performance liquid chromatography using developer system A to give the title product 2(12mg), yield: 27.2 percent.

MS m/z(ESI)：447.5[M+1]

¹H NMR(400MHz，CD₃OD)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).

Test example:

biological evaluation

Test example 1 determination of human TLR7 agonistic Activity of the Compound of the present invention

Compound of the invention p HEK-Blue^TMThe activation of hTLR7 protein expressed by hTLR7 stable transgenic cells was determined using the following experimental method:

first, experimental material and instrument

1.DMEM(Gibco，10564-029)，

2. Fetal bovine serum (GIBCO, 10099),

3. trypan blue solution (Sigma, T8154-100ML),

flexstation 3 Multi-functional microplate readers (molecular μ lar Devices),

5.HEK-Blue^TMthe hTLR7 cell line (InvivoGen, hkb-hTLR7),

HEK-Blue detection reagent (InvivoGen, hb-det3),

7. phosphate Buffered Saline (PBS) pH7.4 (Shanghai culture Biotech Co., Ltd., B320).

Second, the experimental procedure

Preparing a HEK-Blue detection culture medium, taking a bag of HEK-Blue detection dry powder, adding 50ml of endotoxin-removed water for dissolving, placing in an incubator at 37 ℃, and carrying out sterile filtration after 10 minutes. Preparing a 20mM stock solution by using the compound; further diluted with pure DMSO to a maximum concentration of 6X10⁶nM, diluted with a 3-fold gradient, for 10 dots.

The prepared compound was diluted 20-fold with the medium, and then 20. mu.l of the diluted compound was added to each well. Taking HEK-Blue^TMRemoving the supernatant of hTLR7 cells, adding 2-5ml of preheated PBS, placing in an incubator for 1-2 minutes, slightly blowing to beat the cells, and staining and counting by trypan blue. The cell suspension concentration was adjusted to 2.2X 10 by using HEK-Blue detection medium⁵At each cell/ml, 180. mu.l of the cells were added to the above 96-well cell culture plate to which 20. mu.l of the drug had been added, and cultured at 37 ℃ for 6 to 16 hours.

The microplate reader reads at a wavelength of 620 nm. Obtaining corresponding OD value, and calculating EC of the medicine by Graphpad Prism₅₀The value is obtained.

The activation of human TLR7 by the compounds of the invention can be determined by the above assay, the EC of which is measured₅₀The values are shown in Table 1.

Table 1 EC of the compounds of the invention against human TLR7₅₀。

Example numbering	EC₅₀(nM)
		1	443

And (4) conclusion: the compound has better activation effect on human TLR 7.

Test example 2 determination of human TLR8 agonistic Activity of the Compound of the present invention

Compound of the invention p HEK-Blue^TMThe activation of hTLR8 protein expressed by hTLR8 stable transgenic cells was determined using the following experimental method:

first, experimental material and instrument

1.DMEM(Gibco，10564-029)，

2. Fetal bovine serum (GIBCO, 10099),

3. trypan blue solution (Sigma, T8154-100ML),

flexstation 3 Multi-functional microplate readers (molecular μ lar Devices),

5.HEK-Blue^TMthe hTLR8 cell line (InvivoGen, hkb-hTLR8),

HEK-Blue detection reagent (InvivoGen, hb-det3),

Second, the experimental procedure

Preparing a HEK-Blue detection culture medium, taking a bag of HEK-Blue detection dry powder, adding 50ml of endotoxin-removed water for dissolving, placing in an incubator at 37 ℃, and carrying out sterile filtration after 10 minutes. Preparing a 20mM stock solution by using the compound; then diluted with pure DMSO to a maximum concentration of 6X10⁶nM, then 3-fold gradient dilution, 10 points total; the compounds were diluted 20-fold with medium and then 20. mu.l of the diluted compounds were added to each well.

Taking HEK-Blue^TMRemoving the supernatant of hTLR8 cells, adding 2-5ml of preheated PBS, placing the mixture into an incubator for 1-2 minutes, slightly blowing the cells, and staining and counting the cells by trypan blue. The cell suspension concentration was adjusted to 2.2X 10 by using HEK-Blue detection medium⁵At each cell/ml, 180. mu.l of the cells were added to the above 96-well cell culture plate to which 20. mu.l of the drug had been added, and cultured at 37 ℃ for 6 to 16 hours.

The activation of human TLR8 by the compounds of the invention can be determined by the above assay, the EC of which is measured₅₀The values are shown in Table 2.

Table 2 EC of the compounds of the invention against human TLR8₅₀。

Example numbering	EC₅₀(nM)
		1	4877
2	＞30000

And (4) conclusion: the compounds of the invention have weak activation effect on human TLR8, which indicates that the compounds of the invention have selectivity on TLR 7.

Test example 3 determination of the ability of the Compounds of the invention to stimulate IFN- α secretion from Peripheral Blood Mononuclear Cells (PBMCs)

The ability of the compounds of the invention to stimulate IFN- α secretion from PBMC is determined using the following assay:

first, experimental material and instrument

1.RPMI 1640(Invitrogen，11875)，

2.FBS(Gibco，10099-141)，

3.Ficoll-Paque PREMIUM(GE，17-5442-02)，

4. Trypan blue solution (Sigma, T8154-100ML),

5.SepMateTM-50(Stemcell，15460)，

6.Bright-Line^TMblood cell counter (Sigma, Z359629-1EA),

7.96 well flat bottom plate (Corning, 3599),

8.96 hole v-bottom plate (Corning, 3894),

9. human IFN-alpha kit (cisbio, 6FHIFPEB),

a PHERAStar multifunctional microplate reader (BMG, PHERAStar).

Second, the experimental procedure

Compounds were diluted in pure DMSO at a maximum concentration of 5mM, 4-fold gradient dilution, for a total of 9 points. Then, 4. mu.l of the compound was added to 196. mu.l of 10% FBS-containing RMPI 1640 medium, and mixed well. 50 μ l of each well was taken to a new 96 well cell culture plate.

All reagents were equilibrated to room temperature, and a 250ml flask was taken, to which 60ml of blood and PBS + 2% FBS were added, gently pipetted, and diluted well. 50ml of PBMC separation tube SepMateTM-50 is taken, 15ml of lymphocyte separation solution Ficoll-Paque PREMIUM is added, and then 30ml of diluted blood is added. Centrifuge at 1200g for 10 min at room temperature. The supernatant was removed, followed by centrifugation at 300g for 8 minutes. Resuspend and enumerate in RMPI 1640 medium containing 10% FBS, adjust PBMC to 3.33X 10⁶One cell/ml, 150. mu.l was added to the cell culture plate to which the compound had been added, at 37 ℃ and 5.0% CO₂The culture box is used for culturing for 24 hours.

The cell culture plate was placed in a centrifuge at 1200rpm and centrifuged for 10 minutes at room temperature. 150 μ l of supernatant was removed per well. Firstly, balancing the reagent in the human IFN-alpha kit to normal temperature, and preparing the anti-IFN-alpha-Eu according to the kit instruction under the condition of keeping out of the sun³⁺Cryptate conjugate (Cryptate conjugate) and anti-IFN-. alpha. -d 2-conjugate, both mixed in a 1: 40 ratio with binding Buffer (conjugate Buffer). Then 16. mu.l of the supernatant obtained by centrifugation was added to each well. Then 2 mul of freshly prepared anti-IFN-alpha-Eu are added into each hole³⁺The cryptate and the anti-IFN-alpha-d 2-conjugate were mixed by shaking and incubated for 3h at room temperature in the dark.

Readings were taken on a PHERAStar using HTRF mode. We define the lowest drug Concentration that stimulates a cytokine level at least 3-fold above the lowest detectable limit as the MEC (minimum Effective Concentration) value of the compound on the cytokine stimulation assay.

The ability of the compounds of the invention to stimulate IFN-. alpha.secretion from PBMCs was determined by the above assay and the MEC values determined are shown in Table 3.

TABLE 3 MECs that stimulate IFN- α secretion from PBMCs by compounds of the invention

Example numbering	MEC(nM)
		1	1.5
2	22

And (4) conclusion: from the data on the IFN-alpha secretion activity of PBMCs, the compound of the invention can better cause IFN-alpha release.

Test example 4 inhibition of enzymatic Activity at the site of metabolism of human liver microsomal CYP3A4 midazolam by Compounds of the invention

The enzyme activity of the compound on the metabolic site of human liver microsome CYP3A4 midazolam is measured by adopting the following experimental method:

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630)，

3. human liver microsomes (Corning Gentest),

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

inertsil C8-3 column, 4.6X 50mm, 5 μm (Dima, USA),

CYP probe substrate (15 μ M midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA k 1003).

Second, the experimental procedure

100mM PBS buffer was prepared, 2.5mg/ml microsome solution and 5mM NADPH solution were prepared using the buffer, and 5 Xconcentrated compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) was diluted in PBS gradient. Ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) at 5 Xconcentration was diluted with PBS gradient. Midazolam working solution diluted to 15 μ M concentration with PBS.

Respectively taking 2.5mg/ml microsome solution, 15 mu M midazolam working solution and MgCl₂The solution and the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M, each concentration setting different reaction system) each 20. mu.l, mixing evenly. The positive control group replaced the compound with ketoconazole at the same concentration. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes. After 5 min 20. mu.l NADPH was added to the wells, the reaction was started and incubated for 30 min. All incubated samples were set up in duplicate. After 30 minutes 250. mu.l of acetonitrile containing the internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and then centrifuged at 3700rpm for 10 minutes. 80 μ l of the supernatant was transferred to LC-MS/MS for analysis.

The numerical value is calculated by Graphpad Prism to obtain the medicineIC for CYP3A4 midazolam metabolic site₅₀The values are shown in Table 4.

TABLE 4 IC of the Compounds of the invention on the CYP3A4 midazolam metabolic site₅₀Value of

Example numbering	IC₅₀(μM)
		1	31
2	17

And (4) conclusion: the compound has no inhibition effect on the metabolic site of midazolam of human liver microsome CYP3A4, shows better safety, and prompts that the metabolic drug interaction based on the metabolic site of CYP3A4 metabolic midazolam does not occur.

Test example 5 inhibition of human liver microsomal CYP2D6 enzyme Activity by Compounds of the present invention

The compound of the invention adopts the following experimental method to measure the enzymatic activity of human liver microsome CYP2D 6:

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630)，

3. human liver microsomes (Corning Gentest),

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

inertsil C8-3 column, 4.6X 50mm, 5 μm (Dima, USA),

CYP probe substrate (20 μ M dextromethorphan, SIGMA Q0750) and positive control inhibitor (quinidine, SIGMA d 9684).

Second, the experimental procedure

100mM PBS buffer was prepared, 2.5mg/ml microsome solution and 5mM NADPH solution were prepared using the buffer, and 5 Xconcentrated compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) was diluted in PBS gradient. Quinidine working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μ M) at 5 Xconcentration was diluted with PBS gradient. Dextromethorphan working solution diluted to 20 μ M concentration with PBS.

Respectively taking 2.5mg/ml microsome solution, 20 mu M dextromethorphan working solution and MgCl₂The solution and the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M, each concentration setting different reaction system) each 20. mu.l, mixing evenly. The positive control group replaced the compound with quinidine at the same concentration. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes, 5 minutes later 20 u l NADPH added to the well, start the reaction, incubated for 30 minutes. All incubated samples were set up in duplicate. After 30 minutes, 250. mu.l of acetonitrile containing the internal standard was added to all samples, mixed well and shaken at 800rpm for 10 minutes. Centrifuge at 3700rpm for 10 minutes. 80 μ l of the supernatant was transferred to LC-MS/MS for analysis.

The value is calculated by Graphpad Prism to obtain the IC of the drug on the CYP2D6 metabolic site₅₀The values are shown in Table 5.

TABLE 5 IC of the Compounds of the invention on the site of CYP2D6 metabolism₅₀Value of

Example numbering	IC₅₀(μM)
		1	36
2	22

And (4) conclusion: the compound of the invention has no inhibition effect on the enzymatic activity of human liver microsome CYP2D6, shows better safety, and indicates that metabolic drug interaction based on CYP2D6 does not occur.

Test example 6 inhibition of enzymatic Activity at human liver microsomal CYP3A4 Testosterone metabolism site by Compounds of the present invention

The enzyme activity of the compound of the invention on the metabolic site of human liver microsomal CYP3A4 testosterone is determined by the following experimental method:

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630)，

3. human liver microsomes (Corning Gentest),

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

inertsil C8-3 column, 4.6X 50mm, 5 μm (Dima, USA),

CYP probe substrate (testosterone/100 μ M, SIGMA K1003) and positive control inhibitor (ketoconazole, dr. ehrenstorfer GmbH, C17322500).

Second, the experimental procedure

100mM PBS buffer was prepared, 2.5mg/ml microsome solution and 5mM NADPH solution were prepared using the buffer, and 5 Xconcentration compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) was diluted with PBS gradient. Ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μ M) at 5X concentration was diluted with PBS gradient. Dextromethorphan working solution diluted to 50 μ M concentration with PBS.

Respectively taking 2.5mg/ml microsome solution, 50 mu M testosterone working solution and MgCl₂The solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M, each concentration setting different reaction system) each 20. mu.l, mixing evenly. The positive control group replaced the compound with ketoconazole at the same concentration. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes. After 5 minutes 20. mu.l of NADPH were addedIn wells, the reaction was started and incubated for 30 min. All incubated samples were set up in duplicate. After 30 minutes, 250. mu.l of acetonitrile containing the internal standard was added to all samples, mixed well and shaken at 800rpm for 10 minutes. Centrifuge at 3700rpm for 10 minutes. 80 μ l of the supernatant was transferred to LC-MS/MS for analysis.

The value is calculated by Graphpad Prism to obtain the IC of the drug on the metabolic site of CYP3A4 testosterone₅₀The values are shown in Table 6.

TABLE 6 IC of Compounds of the invention on the site of metabolism of CYP3A4 testosterone₅₀Value of

Example numbering	IC₅₀(μM)
		1	13

And (4) conclusion: the compound of the invention has no inhibition effect on the testosterone metabolic site of human liver microsome CYP3A4, shows better safety, and suggests that metabolic drug interaction based on the testosterone metabolic site of CYP3A4 does not occur.

Test example 7

1. Purpose of experiment

The blocking effect of the compound on the hERG potassium current is tested on a stable cell strain transfected with the hERG potassium channel by using full-automatic patch clamp.

2. Experimental methods

2.1 Experimental materials and instruments

2.1.1 Experimental materials:

name of reagent	Supply company	Goods number
			FBS	GIBCO	10099
Sodium pyruvate solution	sigma	S8636-100ML
			MEM non-essential amino acid solution (100X)	sigma	M7145-100ML
G418 sulfate	Enzo	ALX-380-013-G005
			MEM	Hyclone	SH30024.01B
hERG cDNA	Origene	-

2.1.2 Experimental instruments:

2.2 full-automatic Patch Clamp test procedure

HEK293-hERG stable cell lines were subcultured at a density of 1: 4 in MEM/EBSS medium (10% FBS, 400. mu.g/ml G418, 1% MEM non-essential amino acid solution (100X), 1% sodium pyruvate solution) and cultured for a full-automatic patch clamp experiment within 48-72 hours. On the day of the experiment, cells were digested with 0.25% trypsin, harvested by centrifugation, and treated with extracellular fluid (140mM NaCl, 4mM KCl, 1mM MgCl)₂， 2mM CaCl₂5mMD dextrose monohydrate, 10mM Hepes, pH7.4, 298mOsmol) were resuspended into a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument, which applied the cells to the chip (NPC-16) using a negative pressure controller, which draws individual cells to the wells of the chip. After the whole cell mode is formed, the apparatus will obtain hERG current according to the set hERG current voltage program, and then the apparatus automatically carries out compound perfusion from low concentration to high concentration. The current at each concentration of compound and the blank control current were analyzed by data analysis software supplied by HEAK Patchmaster, HEAK EPC10 patch clamp amplifier (Nanion) and pathlersoft ware and Pathcontrol HTsoftware.

2.3 test results

The blocking effect of the compound of the present invention on hERG potassium current was measured by the above test, and the IC was determined₅₀The values are shown in Table 7.

TABLE 7 IC of the blocking effect of the compounds of the invention on hERG potassium current₅₀

Example numbering	IC₅₀(μM)
		1	＞30
2	12

And (4) conclusion: the compounds of the present invention have a weak inhibitory effect on hERG and can reduce side effects caused by the hERG pathway.

Pharmacokinetic evaluation

Test example 8 mouse pharmacokinetic testing of the Compound of the invention

1. Abstract

Using the mice as test animals, the drug concentrations in the plasma of the mice at various times after gavage administration of the compound of example 1 were determined by LC/MS/MS method. The pharmacokinetic behavior of the compounds of the invention in mice was studied and their pharmacokinetic profile was evaluated.

2. Test protocol

2.1 test drugs

The compound of example 1.

2.2 test animals

C57 mice 9, female, purchased from shanghai jessie laboratory animals ltd, animal production license number: SCXK (Shanghai) 2013 and 0006.

2.3 pharmaceutical formulation

An amount of drug was weighed and prepared as a 1mg/ml colorless clear transparent liquid by adding 5% by volume of DMSO, 5% by volume of tween80 and 90% physiological saline.

2.4 administration

C57 mice were fasted overnight and then gavaged at 20.0mg/kg for each dose and at 20ml/kg for each volume.

3. Operation of

Mice were gavaged with the compound of example 1, and 0.1ml of blood was collected before and after administration at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours, placed in heparinized tubes, centrifuged at 3500 rpm for 10 minutes to separate plasma, and stored at-20 ℃.

Determining the content of the compound to be tested in the plasma of the mouse after the drug with different concentrations is administered by gastric lavage: mu.l of mouse plasma at each time after administration was taken, 50. mu.l (100ng/mL) of camptothecin as an internal standard solution and 200. mu.l of acetonitrile were added, vortex-mixed for 5 minutes, centrifuged for 10 minutes (3600 rpm), and 10. mu.l of the supernatant was taken for LC/MS/MS analysis.

4. Pharmacokinetic parameter results

The pharmacokinetic parameters of the compounds of the invention are as follows:

and (4) conclusion: the compound of the invention has better drug absorption and pharmacokinetic advantage.

Test example 9 determination of mouse IFN-. alpha.value of the Compound of the present invention

First, experimental material and instrument

1、LumiKine^TM Xpress mIFN-α(invivogen，luex-mifna)

2. Enzyme mark instrument (PE, victor3)

3. Test samples: test example 8 pharmacokinetics mice were gavaged with the compound of example 1 and blood was collected before and at 0, 2.0, 4.0 and 8.0 hours after dosing.

Second, the experimental procedure

1. Coating: mu.l of capture antibody (diluted with PBS to a final concentration of 1. mu.g/ml) was added to a 96-well ELISA-dedicated plate. After film application, incubation was carried out overnight at room temperature.

2. The next day the plate was inverted and the liquid in the plate was patted dry.

3. Mu.l of blocking buffer (PBS + 2% BSA + 0.05% Tween 20) was added to each well and incubated at 37 ℃ for 2 hours.

4. The plate was inverted and the liquid in the plate was patted dry.

5. The test sample and the standard curve sample (maximum concentration of 500pg/ml, two-fold gradient dilution of seven points) diluted to 100. mu.l with dilution buffer were added.

6. The liquid in the plate was blotted with a plate washing machine, 300. mu.l of washing buffer (PBS + 0.05% Tween 20) was injected into each well, the liquid was blotted after shaking, washing was repeated 3 times, and finally water droplets in the plate were blotted.

7. Mu.l of antibody conjugated to Lucia protease (final concentration 30ng/ml) was added to each well and incubated at 37 ℃ for 2 hours.

8. And 6, repeating the step.

9. 50 μ l of QUANTI-Luc (chemiluminescent reagent) was added to each well and read by a microplate reader.

Thirdly, detecting results:

the mouse serum IFN- α values for the compounds of the invention are shown in Table 8 below and the time curves are shown in FIG. 1:

TABLE 8 IFN-. alpha.values in mouse sera

i.g. for intragastric administration.

Fourthly, conclusion:

example 1 administration of the compound at 20mpk showed a strong ability to stimulate IFN- α expression 2 hours in mice.

Claims

1. A compound of the general formula (I):

or a tautomer thereof or a pharmaceutically acceptable salt thereof,

wherein:

ring A is phenyl;

g is N;

X¹is methylene;

L¹is-O-;

R¹is C_1-6Alkyl or C_1-6A haloalkyl group;

R²selected from hydrogen atoms, halogens and C_1-6An alkyl group;

L²is methylene;

R³is pyrrolidinyl, wherein said pyrrolidinyl is optionally selected from C_1-6One or more of alkyl and halogenSubstituted by one substituent;

R⁴and R⁵Are each a hydrogen atom; and is

n is 0 or 1.

2. The compound of formula (I) according to claim 1, which is a compound of formula (III):

or a tautomer thereof or a pharmaceutically acceptable salt thereof,

wherein:

R⁹and R¹⁰Together with the nitrogen atom to which they are attached form a pyrrolidinyl group, wherein said pyrrolidinyl group is optionally selected from C_1-6Alkyl and halogen;

G、L¹、L²and R¹As defined in claim 1.

3. A compound of formula (I) according to claim 1 wherein R is¹Is C_1-6An alkyl group.

4.A compound of formula (I) according to claim 1, selected from:

5. a compound of the general formula (IB):

or a tautomer thereof or a pharmaceutically acceptable salt thereof,

wherein:

w is an amino protecting group;

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹、L²、R¹～R⁵And n is as defined in claim 1.

6. A compound of formula (IB) according to claim 5, selected from:

7. a compound of the general formula (IC):

or a tautomer thereof or a pharmaceutically acceptable salt thereof,

wherein:

w is an amino protecting group;

s is 1 or 2;

ring A, G, X¹、L¹、L²、R¹～R⁵And n is as defined in claim 1.

8. The compound of formula (IC) according to claim 7, selected from:

9. a process for the preparation of a compound of formula (IB) according to claim 5, which process comprises:

compounds of the general formula (IA) and

wherein:

w is an amino protecting group;

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹、L²、R¹～R⁵And n is as defined in claim 1.

10. A process for preparing a compound of formula (IC) according to claim 7, which process comprises:

ring closing the compound of formula (IB) to give a compound of formula (IC);

wherein:

w is an amino protecting group;

x is halogen;

s is 1 or 2;

ring A, G, X¹、L¹、L²、R¹～R⁵And n is as defined in claim 1.

11. A process for the preparation of a compound of formula (I) according to claim 1, which process comprises:

wherein:

w is an amino protecting group;

s is 1 or 2;

ring A, G, X¹、L¹、L²、R¹～R⁵And n is as defined in claim 1.

12. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 4, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

13. Use of a compound of general formula (I) according to any one of claims 1 to 4 or a tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 12, for the preparation of a medicament for a TLR7 agonist.

14. Use of a compound of general formula (I) according to any one of claims 1 to 4 or a tautomer thereof or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment of 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, 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 virus.

15. Use of a compound represented by the general formula (I) or a tautomer thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 12, for the preparation of a medicament for the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pulmonary disease, autoimmune disease, actinic keratosis, and liver fibrosis.

16. Use of a compound of general formula (I) according to any one of claims 1 to 4 or a tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 12, for the manufacture of a medicament for the treatment or prophylaxis of ulcerative colitis, plaque psoriasis and systemic lupus erythematosus.