TW201827442A - Heteroarylthiadiazine-2,2-dioxide derivative, a preparation method therefor, and a pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a heteroarylthiadiazine-2,2-dioxide derivative, a preparation method and a pharmaceutical use thereof. In particular, the present invention relates to a heteroarylthiadiazine-2,2-dioxide derivative as shown by general formula (I), the preparation method thereof and a pharmaceutical composition containing same, and the use thereof as a therapeutic agent. Particularly the use as an TLR7 receptor antagonist. The definition of each substituent of the general formula (I) is the same as that in the description.

Description

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

The invention belongs to the field of medicine and relates to a new heteroaryl thiadiazine-2,2-dioxide derivative represented by the general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative, and a pharmaceutical composition thereof. Use as a therapeutic agent, especially as a TLR7 agonist.

Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity. TLR is a monomeric transmembrane non-catalytic receptor, 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 activates the immune cell response (Mahla, RS, et al., Front Immunol. 4: 248 (2013)). The ability of the immune system to recognize pathogenic microorganisms is partly due to the widespread presence of Toll-like immune receptors.

There are at least 10 different TLRs in mammals. Ligands and corresponding signal cascades for some of these receptors have been identified. TLR7 is a member of the TLRs (TLRs 3, 7, 8, and 9) subgroup and is limited to the endosome compartment of cells that specifically detect non-nucleic acids. TLR7 plays a key role in antiviral defense by identifying ssRNAs (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 mainly expressed by B cells and plasma cell-like dendritic cells (pDC), and to a lesser extent by monocytes. Plasma cell-like DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% of peripheral blood mononuclear cells (PBMCs)). They secrete high levels of interferon-α (IFNα) and interferon- β (IFNβ) was the first type I interferon-producing cell (Liu YJ, Annu. Rev. Immunol., 2005: 23, 275-306).

Many diseases and disorders are related to abnormal TLRs, such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basalcellcarcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD ), Ulcerative colitis, liver fibrosis, HBV, flaviviridae (F1aviviridae) virus, HCV, HPV, RSV, SARS, HIV or influenza virus infection, etc. Therefore, the use of agonists of TLRs to treat related diseases is very promising.

Because TLR7 and TLR8 are highly homologous, TLR7 ligands are also TLR8 ligands in most cases. TLR8 stimulation mainly induces the production of cytokines such as tumor necrosis factor alpha (TNF-α) and chemokines. Interferon alpha is one of the main drugs for the treatment of chronic hepatitis B or C, and TNF-α is a pro-inflammatory cytokine. Excessive secretion may cause serious side effects. Therefore, selectivity for TLR7 and TLR8 is essential for the development of TLR7 agonists for the treatment of viral infectious diseases.

There are currently related patent applications for TLR7 agonists, such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, and WO2012080730. However, there is still a need to continue to develop safer and more therapeutically effective TLR7 agonists.

The invention aims at the above technical problems, and provides a drug compound with a lower effective concentration, better selectivity, and more obvious activation effect, and is a safer and more effective TLR7 agonist.

The object of the present invention is to provide a compound represented by the general formula (I): Or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein: ring A is selected from the ring Alkyl, heterocyclyl, aryl, and heteroaryl; G is selected from N and CR ⁶ ; X ^{1 is} selected from alkylene or S (O) _m , wherein the alkylene is optionally selected from halogen, alkyl , Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, and heterocyclyl; 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 covalent bond; R ^{1 is} selected from hydrogen atom, alkyl, haloalkyl, alkenyl, alkynyl, naphthenic Group, heterocyclic group, aryl group and heteroaryl group, wherein the alkyl group, alkenyl group, alkynyl group, cycloalkyl group, heterocyclic group, aryl group and heteroaryl group are each independently Substituted with one or more substituents of oxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ² the same or different, and Independently selected from hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl -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 group, cycloalkyl group , Heterocyclyl, aryl, and heteroaryl are each independently selected from alkyl, alkoxy, halogen, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amine, nitro, and cycloalkyl, as needed , Heterocyclyl, aryl, and heteroaryl are substituted with one or more substituents; L ^{2 is} selected from alkylene or covalent bond, wherein the alkylene is optionally selected from halogen, alkyl, and alkyl Substituted with one or more substituents of oxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, and heterocyclic; R ^{3 is} selected from hydrogen atom, alkyl, Alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -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, heterocyclyl, aryl and heteroaryl The radicals are each independently selected from alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl One or more substituents of -C (O) R ⁸ , -C (O) OR ⁸ , -S (O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ Substituted; R ⁴ and R ^{5 are the} same or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cyclic Alkyl, heterocyclyl, aryl, and heteroaryl; or R ⁴ and R ⁵ together form a pendant oxygen; R ^{6 is} selected from the group consisting of a hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, and hydroxyalkane Group, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ^{7 is} selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl And heteroaryl; R ^{8 is} selected from a hydrogen atom, an alkyl group, a haloalkyl group, an amine 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 each Independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, which The alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, Substituted with one or more substituents of cycloalkyl, heterocyclyl, aryl, and heteroaryl; or, R ⁹ and R ¹⁰ together with the nitrogen atom to which they are attached form a heterocyclyl, wherein the heterocyclic ring The group contains 1 to 2 heteroatoms which are the same or different and are selected from N, O and S, and the heterocyclic group is optionally selected from alkyl, alkoxy, halogen, amine, cyano, nitro, and hydroxyl groups. , Hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl with one or more substituents; n is 0, 1, 2, 3, or 4; and 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 ¹ is 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): Or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein: G, L ¹ ~ L ² , R ¹ to R ⁵ and n are as defined in the general 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): Or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein: R ⁹ and R ¹⁰ A heterocyclic group is formed together with the connected nitrogen atom, wherein the heterocyclic group contains 1 to 2 heteroatoms which are the same or different and selected from N, O and S, and the heterocyclic group is optionally selected from alkyl, Substituted with one or more substituents of alkoxy, halogen, amine, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, preferably 5 -6-membered heterocyclyl, more preferably pyrrolidinyl, tetrahydrothienyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl or tetrahydropyranyl; G, L ¹ to L ² and R ¹ As defined in general formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein R ⁴ and R ⁵ are both hydrogen or R ⁴ and R ⁵ together form a pendant oxygen group.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein L ¹ is -O-.

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein R ¹ is an alkyl group, preferably a C _1-6 alkyl group, more preferably a methyl group, an ethyl group, an n-group Propyl, isopropyl, n-butyl, isobutyl, third butyl, second 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 the L ² is an alkylene group, preferably a C _1-6 alkylene group, more preferably a methylene group, 1, 2-Ethyl, 1,1-Ethyl or 1,3-Propyl.

Typical compounds of the invention include, but are not limited to: Or a tautomer, a meso, a racemate, an enantiomer, a diastereomer, or a 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 a compound of the general formula (I): Or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein: W is an amino group protection Is preferably a third butoxycarbonyl group, an ethyl fluorenyl group, a benzyl group, an allyl group, and a p-methoxybenzyl group; X is a halogen; s is 1 or 2; rings A, G, X ¹ , and L ¹ to L ^2. R ¹ to R ⁵ and n are as defined in the general formula (I). The compound represented by the general formula (IB) includes, but is not limited to:

In a preferred embodiment of the present invention, a compound represented by the general formula (IC) is used as an intermediate for preparing the compound of the general formula (I): Or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof, wherein: W is an amino group protection Is preferably a third butoxycarbonyl group, an ethyl fluorenyl group, a benzyl group, an allyl group, and a p-methoxybenzyl group; s is 1 or 2; ring A, G, X ¹ , L ¹ to L ² , R ¹ ~ R ⁵ and n are as defined in general formula (I).

The compound represented by the general formula (IC) includes, but is not limited to:

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

Compounds of general formula (IA) and A nucleophilic substitution reaction occurs to obtain a compound of the general formula (IB); wherein: W is an amine protecting group, preferably a third butoxycarbonyl group, ethenyl group, benzyl group, allyl group and p-methoxybenzyl group; X Is 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 general formula (I).

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

The compound of the general formula (IB) is ring-closed to obtain the compound of the general formula (IC); wherein: W is an amine protecting group, preferably a third butoxycarbonyl group, ethenyl group, benzyl group, allyl group and p-methoxybenzyl X is 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 general formula (I).

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

The compound of the general formula (IC) is deprotected to obtain a compound of the general formula (I); wherein: W is an amine protecting group, preferably a third butoxycarbonyl group, an ethylfluorenyl group, a benzyl group, an allyl group, and 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 general formula (I).

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

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a medicine thereof. Use of the above-acceptable salt or a pharmaceutical composition comprising the same in the manufacture of a medicament for a TLR7 agonist.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a medicine thereof. Of an acceptable salt or a medicinal composition comprising the same in the manufacture 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, Saint Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV , SARS and influenza viruses.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a medicine thereof. Acceptable salts or medicinal compositions containing them are prepared for the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD , Medicine for ulcerative colitis and liver fibrosis.

The invention further relates to a method for stimulating TLR7, which comprises converting a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer , Or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, and a step of contacting TLR7.

The invention further relates to a method 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, tick-borne encephalitis virus, Kunjin virus, Murray valley brain Inflammatory virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses, said method comprising administering treatment to a patient in need An effective amount of a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a medicament An acceptable salt or pharmaceutical composition comprising the same.

The invention further relates to 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, autoimmune diseases, A method of 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 represented by the general formula (I) or a tautomer, meso Isomers, racemates, enantiomers, diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a medicament containing the same, which is used as a medicament.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a medicament containing the same, which is used for a TLR7 agonist.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a medicament containing the same for treating or preventing 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 brain Inflammatory virus, Kunjin virus, Murray valley encephalitis virus, Saint Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a drug containing the same, which is used for the treatment or prevention of selected 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, water or oil 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, and such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide pleasing and Delicious medicinal preparation. Tablets contain the active ingredients and non-toxic pharmaceutically acceptable excipients suitable for the preparation of tablets for mixing.

Aqueous suspensions contain the active substance and excipients suitable for the preparation of the aqueous suspension for mixing. The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl paraben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.

Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickener. The sweeteners and flavoring agents described above can be added to provide a palatable formulation.

Dispersible powders and granules suitable for use in the preparation of aqueous suspensions can be provided with active ingredients and dispersing or wetting agents, suspending agents or one or more preservatives for mixing by adding water. Suitable dispersing or wetting agents and suspending agents can illustrate the above examples. Other excipients such as sweeteners, flavors and colorants can also be added. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical composition of the present 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. 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 added to a mixture of water and glycerol to form a microemulsion. Injections or microemulsions can be injected into a patient's bloodstream by local, large injections. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of a compound of the invention. To maintain this constant concentration, continuous intravenous drug delivery devices 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 water or oily suspension for intramuscular and subcutaneous administration. This 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 prepared in a parenterally acceptable non-toxic diluent or solvent. In addition, a sterile fixed oil can be conveniently used as a solvent or suspension medium.

The compounds of the 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 thus will dissolve in the rectum to release the drug. Such materials include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As known to those skilled in the art, the dosage of a drug depends on a number of factors, including but not limited to the following: the activity of the specific compound used, the age of the patient, the weight of the patient, the patient's health, the patient's behavior, Patient's diet, time of administration, mode of administration, rate of excretion, combination of drugs, etc .; In addition, the optimal treatment method such as the mode of treatment, the daily dosage of the general compound (I) or the pharmaceutically acceptable salt The type can be verified according to the traditional treatment plan.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and the scope of patent applications 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, third butyl, second 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-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-di Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-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 2,2-diethyl-pentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl group, and various branched chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, second 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-di Methylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Amyl, 2,3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from alkyl, alkenyl, and alkyne Alkyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkane Oxygen, cycloalkylthio, heterocycloalkylthio, pendant oxygen, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O) _m R ⁸ , -NR ⁹ R ¹⁰ , and- C (O) NR ⁹ R ¹⁰ is 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 same carbon atom or two different carbon atoms of the parent alkane, which is derived from 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 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. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from alkyl, alkenyl, Alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocyclic Alkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxygen, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ is substituted with one or more substituents.

The term "alkenyl" refers to a hydrocarbon group formed by one or fewer hydrogen atoms in an olefin molecule. An alkenyl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a haloalkyl group, Hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O ) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ are substituted with one or more substituents.

The term "alkynyl" refers to a hydrocarbon containing a carbon-carbon triple bond in the molecule. The alkynyl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a haloalkyl group, Hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O ) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ are 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 contains 3 to 20 carbon atoms, preferably contains 3 to 12 carbon atoms, and preferably contains 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl.

The term "amino-protecting group" is to protect the amine group with an easily removable group in order to keep the amine group unchanged during the reaction of other parts of the molecule. Non-limiting examples include tertiary butoxycarbonyl, ethenyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro. The amine-protecting group is preferably p-methoxybenzyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that contains 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S (O) A heteroatom of _m (where m is an integer from 0 to 2), excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 10 ring atoms, of which 1-4 are heteroatoms; more preferably 5 to 6 ring atoms; of which 1- Three are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, tetrahydropyranyl, 1, 2.3.6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 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 connected to the parent structure is a heterocyclic group, and non-limiting examples thereof include:

The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected as necessary from alkyl, alkenyl, alkynyl, Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy Group, cycloalkylthio group, heterocycloalkylthio group, pendant oxygen 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 is substituted with one or more substituents.

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

The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ With one or more substituents.

The term "heteroaryl" refers to a heteroaromatic system containing 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 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 connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

Heteroaryl may be substituted or unsubstituted as required. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, and alkoxy , Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, heterocycloalkylthio, -C (O) R ⁸ , -C (O) OR ⁸ , -S (O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C (O) NR ⁹ R ¹⁰ With one or more substituents.

The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be substituted or unsubstituted as necessary. 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, Haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl are substituted with one or more substituents.

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

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

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

The term "amino" refers to -NH _2.

The term "cyano" refers to -CN.

The term "nitro" refers to -NO _2.

The term "lateral oxygen" refers to = O.

"As needed" or "as needed" means that the event or environment described later can, but does not have to occur, and the description includes situations where the event or environment occurs or does not occur. For example, "an optionally substituted heterocyclic group" means that the alkyl group may, but does not have to exist, and this 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 a group, preferably up to five, more preferably one to three hydrogen atoms independently of one another 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 can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or a physiological / 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 to the living body, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

"Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention. Such salts are safe and effective when used in mammals, and have due biological activity.

m, G, and R ⁸ to R ¹⁰ are as defined in the compound of the general formula (I).

Method for synthesizing compounds of the present invention

In order to achieve the object of the present invention, the present invention adopts the following technical solutions:

    Option One    

The compound represented by the general formula (I) of the present invention or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable compound thereof The method of preparing the accepted salt includes the following steps:

In the first step, a compound of the general formula (I-1) and NH- (W) _s undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (I-2); in the second step, the general formula (I -2) A compound of the general formula (I-3) and a compound of the general formula (I-3) undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (I-4); the third step is a compound of the general formula (I-4) In the presence of a reducing agent, a reduction reaction occurs to obtain a compound of general formula (IA); in a fourth step, the compound of general formula (IA) and Under basic conditions, a nucleophilic substitution reaction occurs to obtain a compound of the general formula (IB). In the fifth step, the compound of the general formula (IB) is ring-closed in the presence of an iodide to obtain a compound of the general formula (IC). Compound; in the sixth step, the compound of the general formula (IC) is deprotected under acidic conditions to obtain the compound of the general formula (I); wherein the reagent for providing basic conditions includes organic bases and inorganic bases, and the organic base Classes include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, n-butyllithium, lithium diisopropylamino, bistrimethylsilyl Lithium amino, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide. The inorganic bases include, but are 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, 1,4-dioxane solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, Phosphoric acid, p-benzenesulfonic acid, Me ₃ SiCl and TMSOTf; the reducing reagent includes, but is 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 simple substance, iodide ketone, potassium iodide, and cesium iodide; the above reaction is preferably performed in a solvent, and the solvent used includes, but is not limited to : Acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -di Methylformamide and mixtures thereof; W is an amine protecting group, preferably tertiary butoxycarbonyl, ethylfluorenyl, benzyl, allyl and p-methoxybenzyl; X is 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 general formula (I).

    Option II    

The compound represented by the general formula (II) of the present invention or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable compound thereof The method of preparing the accepted salt includes the following steps:

In the first step, a compound of the general formula (I-1) and NH- (W) _s undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (I-2); in the second step, the general formula (I -2) A compound of the general formula (II-1) and a compound of the general formula (II-1) undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (II-2); the third step is a compound of the general formula (II-2) In the presence of a reducing agent, a reduction reaction occurs to obtain a compound of the general formula (II-A); in a fourth step, the compound of the general formula (II-A) and Under basic conditions, a nucleophilic substitution reaction occurs to obtain a compound of the general formula (II-B); in the fifth step, the compound of the general formula (II-B) is ring-closed in the presence of iodide to obtain the general formula under basic conditions (II-C) compound; in the sixth step, the compound of general formula (II-C) is deprotected under acidic conditions to obtain a compound of general formula (II); wherein: the reagent for providing basic conditions includes an organic base And inorganic bases, the organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, n-butyllithium, diisopropylamine Lithium, lithium bistrimethylsilylamine, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide. The inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, carbonic acid. Potassium, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, toluenesulfonic acid, Me ₃ SiCl and TMSOTf; 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 simple substance, iodide ketone, potassium iodide, and cesium iodide; the above reaction is preferably performed in a solvent, and the solvent used includes, but Not limited to: acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -Dimethylformamide and mixtures thereof; W is an amine protecting group, preferably tertiary butoxycarbonyl, ethenyl, benzyl, allyl and p-methoxybenzyl; X is halogen, preferably Is chlorine; s is 1 or 2; G, n, L ¹ to L ² and R ¹ to R ⁵ are as defined in the general formula (II).

    third solution    

The compound represented by the general formula (III) of the present invention or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable compound thereof The method of preparing the accepted salt includes the following steps:

In the first step, a compound of the general formula (I-1) and NH- (W) _s undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (I-2); in the second step, the general formula (I -2) The compound of the general formula (III-1) and the compound of the general formula (III-1) undergo a nucleophilic substitution reaction under basic conditions to obtain a compound of the general formula (III-2); the third step is a compound of the general formula (III-2) In the presence of a reducing agent, a reduction reaction occurs to obtain a compound of the general formula (III-A); in a fourth step, the compound of the general formula (III-A) and Under basic conditions, a nucleophilic substitution reaction occurs to obtain a compound of the general formula (III-B); in the fifth step, the compound of the general formula (III-B) is ring-closed in the presence of an iodide to obtain the general formula (III-C) compound; in the sixth step, the compound of the general formula (III-C) is deprotected under acidic conditions to obtain the compound of the general formula (III); wherein: the reagent for providing basic conditions includes an organic base And inorganic bases, the organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, n-butyllithium, diisopropylamine Lithium, lithium bistrimethylsilylamine, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide. The inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, carbonic acid. Potassium, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, toluenesulfonic acid, Me ₃ SiCl and TMSOTf; the reducing agents include, but are not 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 ; Iodides include, but are not limited to, iodine simple substance, iodide ketone, potassium iodide, and cesium iodide; the above reaction is preferably performed in a solvent, and the solvent used includes , But not limited to: acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethylsulfine, 1,4-dioxane, water, N N -dimethylformamidine and mixtures thereof; W is an amine protecting group, preferably a third butoxycarbonyl group, acetamido group, benzyl group, allyl group and p-methoxybenzyl group; X is halogen, Preferred is chlorine; s is 1 or 2; G, L ¹ to L ² , R ¹ and R ⁹ to R ¹⁰ are as defined in the general formula (III).

Fig. 1 is a time curve of IFN-? Values in mouse serum of a compound of the present invention.

Examples

The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 ^-6 (ppm). The NMR measurement was performed using Bruker AVANCE-400 nuclear magnetometer. The measurement solvents were deuterated dimethylsulfinium (DMSO- d ₆ ), deuterated chloroform (CDCl ₃ ), and deuterated methanol (CD ₃ OD). Methylsilane (TMS).

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

The HPLC was measured using an Agilent 1200DAD high-pressure liquid chromatography (Sunfire C18 150 × 4.6mm column) and a Waters 2695-2996 high-pressure liquid chromatography (Gimini C18 150 × 4.6mm column).

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

The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silicone plate. The thin-layer chromatography (TLC) silicon plate uses a size of 0.15mm ~ 0.2mm. The thin-layer chromatography separation and purification product uses a size of 0.4mm. ~ 0.5mm.

Column chromatography generally uses Yantai Yellow Sea Silicone 200-300 mesh silica gel as the carrier.

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

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

The average inhibition rate of the kinase and the IC ₅₀ value were measured using a NovoStar microplate reader (BMG, Germany).

The known starting materials of the present invention can be synthesized by or in accordance with methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Chemicals and other companies.

There is no special description in the examples, and the reaction can be performed under an argon atmosphere or a nitrogen atmosphere.

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

The hydrogen atmosphere means that a reaction balloon is connected to a hydrogen gas balloon with a volume of about 1 L.

Pressurized hydrogenation reaction uses Parr 3916EKX type hydrogenation instrument and clear blue QL-500 type hydrogen generator or HC2-SS type hydrogenation instrument.

The hydrogenation reaction is usually evacuated and charged with hydrogen, and the operation is repeated 3 times.

For the microwave reaction, a CEM Discover-S 908860 microwave reactor was used.

There is no special description in the examples, and the solution means an aqueous solution.

There is no special description in the examples, and the reaction temperature is room temperature, which is 20 ° C to 30 ° C.

The monitoring of the reaction progress in the examples adopts thin layer chromatography (TLC), a developing agent used for the reaction, a column chromatography eluent system for purifying compounds, and a thin layer chromatography developing system including: A: Dichloromethane / methanol system, B: n-hexane / ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can be added to adjust.

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

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

2-butoxy-4,6-dichloro-5-nitropyrimidine 1a (4.62 g, 17.43 mmol, prepared by a known method " Journal of Medicinal Chemistry , 2012, 55 (23), 10387-10404" and )) Dissolved in 50 mL of tetrahydrofuran solution, triethylamine (2.64 g, 26.14 mmol) and N , N -bis (4-methoxybenzyl) amine (4.49 g, 17.43 mmol) were added in this order, and the mixture was stirred at room temperature for 4 hours. . The reaction was stopped, and the residue was distilled under reduced pressure. The residue was purified with a CombiFlash flash prep using eluent system B to give the title product 1b (6.20 g). Yield: 73.8%.

MS m / z (ESI): 487.2 [M + 1]

    Second step     2-butoxy- N ⁴ , N ⁴ -bis (4-methoxybenzyl) -5-nitro- N ^6- (4- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4 1,6-diamine 1 d

1b (1.20g, 2.65mmol) was dissolved in 20mL of tetrahydrofuran, and triethylamine (402mg, 3.98mmol) and (4- (pyrrolidin-1-ylmethyl) phenyl) methylamine 1c (469mg, 2.65) were added in this order. mmol, prepared by the method disclosed in patent application "WO2016044183"), and stirred at room temperature for 2 hours to stop the reaction. Add 50 mL of water, extract with ethyl acetate (20 mL x 3), combine the organic phases, wash with saturated sodium chloride solution (50 mL), dry over anhydrous magnesium sulfate, filter, concentrate the filtrate under reduced pressure, and use the CombiFlash rapid preparation instrument for the residue Purification with eluent system A followed by thin-layer chromatography with developing system A gave the title product 1d (1.00 g), yield: 59.1%.

MS m / z (ESI): 641.3 [M + 1]

    third step     2-butoxy- N ⁴ , N ⁴ -bis (4-methoxybenzyl) -N ^6- (4- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4,5,6- Triamine 1e

1d (1.00g, 1.56mmol), iron (350mg, 6.25mmol) and ammonium chloride (675mg, 12.50mmol) were dissolved in 25mL of ethanol and water (V / V = 4: 1) in this order, and heated to Stir at 80 ° C for 4 hours. Stop the reaction, cool to room temperature, filter, add 30 mL of water to the filtrate, extract with dichloromethane (20 mL x 3), combine the organic phases, wash with saturated sodium chloride solution (50 mL), dry over anhydrous magnesium sulfate, filter, and filter Concentrated under reduced pressure, and the residue was purified with a CombiFlash flash prep using eluent system A to give the title product 1e ( 630 mg), yield: 66.1%.

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) pyrimidine- 5-yl) -1-chloromethanesulfonamide 1f

1e (305 mg, 0.50 mmol) was dissolved in 10 mL of dichloromethane, and pyridine (119 mg, 1.50 mmol), 4-dimethylaminopyridine (61 mg, 0.50 mmol), and chloromethylsulfonyl chloride (149 mg, 1.00) were added in this order. mmol), the reaction was stopped at room temperature for 2 hours. Add 20 mL of water, extract with dichloromethane (20 mL x 3), combine the organic phases, wash with saturated sodium chloride solution (50 mL), dry over anhydrous magnesium sulfate, filter, concentrate the filtrate under reduced pressure, and use the CombiFlash rapid preparation instrument for the residue Purification with eluent system A gave the title product 1f ( 260 mg), yield: 72.0%.

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-1 H -pyrimido [5,4- c ] [1,2,5] thiadiazine 2,2-dioxide 1g

1f (260mg, 0.36mmol) was dissolved in 5mL of N , N -dimethylformamide, sodium hydride (43mg, 1.08mmol) and potassium iodide (30mg, 0.18mmol) were added in this order, and the mixture was heated to 50 ° C and stirred for 16 hours. Hours, stop the reaction. Cool to room temperature, add 20 mL of water, extract with ethyl acetate (20 mL × 3), combine the organic phases, wash with saturated sodium chloride solution (20 mL × 3), dry over anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue was purified by thin layer chromatography with developing system A to give 1 g ( 142 mg) of the title product, yield: 57.4%.

MS m / z (ESI): 687.3 [M + 1]

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

Add 1 g (300 mg, 0.55 mmol) and 10 mL of trifluoroacetic acid to the reaction flask, and react at room temperature for 6 hours to stop the reaction. Add saturated sodium bicarbonate solution dropwise to pH 7-8, extract with dichloromethane (20mL × 3), combine the organic phases, wash with saturated sodium chloride solution (50mL), dry over anhydrous magnesium sulfate, filter, and reduce the filtrate. The solution was concentrated under reduced pressure, and the residue was purified by thin layer chromatography using developing system A to obtain the title product 1 ( 99 mg). Yield: 40.1%.

MS m / z (ESI): 447.3 [M + 1]

¹ H NMR (400MHz, CD ₃ OD) δ 7.49 (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-1 H -pyrimido [5,4- c ] [ 1,2,5] thiadiazine 2,2-dioxide

    First step     2-butoxy- N ⁴ , N ⁴ -bis (4-methoxybenzyl) -5-nitro- N ^6- (3- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4 , 6-diamine 2b

1b (4.5 g, 9.24 mmol) was dissolved in 50 mL of tetrahydrofuran, and triethylamine (1.4 g, 13.86 mmol), 4-dimethylaminopyridine (225 mg, 1.85 mmol), and (3- (pyrrolidine-1) were added in this order. -Methylmethyl) phenyl) methylamine 2a (1.76 g, 9.24 mmol, prepared by the method disclosed in the patent application "WO2010077613"), stirred at room temperature for 2 hours to stop the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified with a CombiFlash rapid preparation device using eluent system A to obtain the title product 2b ( 5.0 g), yield: 84.7%.

MS m / z (ESI): 641.3 [M + 1]

    Second step     2-butoxy- N ⁴ , N ⁴ -bis (4-methoxybenzyl) -N ^6- (3- (pyrrolidin-1-ylmethyl) benzyl) pyrimidine-4,5,6- Triamine 2c

Dissolve 2b (3.3g, 5.15mmol), iron (1.15g, 20.6mmol), and ammonium chloride (2.22g, 41.20mmol) in 38mL of ethanol and water (V / V = 15: 4) in this order. Heat to 80 ° C and stir for 4 hours. Stop the reaction, cool to room temperature, filter, add 50 mL of water to the filtrate, extract with dichloromethane (30 mL x 4), combine the organic phases, wash with saturated sodium chloride solution (50 mL), dry over anhydrous magnesium sulfate, filter, and filter It was concentrated under reduced pressure, and the residue was purified with a CombiFlash flash prep using eluent system A to give the title product 2c ( 2.08 g), yield: 66.2%.

MS m / z (ESI): 611.3 [M + 1]

    third step     N- (4- (bis (4-methoxybenzyl) amino) -2-butoxy-6-((3- (pyrrolidin-1-ylmethyl) benzyl) amino) pyrimidine- 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) were added in this order. mmol), the reaction was stopped at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified with a CombiFlash rapid preparation device using eluent system A to obtain the title product 2d ( 480 mg), yield: 67.8%.

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-1 H -pyrimido [5,4- c ] [1,2,5] thiadiazine 2,2-dioxide 2e

2d (72mg, 0.10mmol) was dissolved in 5mL N , N -dimethylformamide, potassium iodide (4mg, 0.02mmol) and sodium hydride (12mg, 0.30mmol) were added in this order, heated to 50 ° C, and the reaction was stirred for 16 Hours, stop the reaction. Cool to room temperature, add 20 mL of water, extract with ethyl acetate (20 mL × 3), combine the organic phases, wash with saturated sodium chloride solution (50 mL × 2), dry over anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue was purified by thin layer chromatography with developing system A to give the title product 2e ( 38 mg), yield: 55%.

MS m / z (ESI): 687.3 [M + 1]

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

2e (68 mg, 0.10 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were added to the reaction flask, and the reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. 20 mL of water was added and extracted with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (50 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to thin layer chromatography After purification with the developer system A, the product was purified with the high performance liquid chromatography using the developer system A to obtain the title product 2 (12 mg). Yield: 27.2%.

MS m / z (ESI): 447.5 [M + 1]

¹ H NMR (400MHz, CD ₃ OD) δ 7.46 (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 agonistic activity of the compound of the present invention on human TLR7

The activation of hTLR7 protein expressed by the compounds of the present invention on HEK-Blue ^TM hTLR7 stable transfected cells is determined by the following experimental method:

I. Experimental materials and instruments

1. DMEM (Gibco, 10564-029), 2. Fetal bovine serum (GIBCO, 10099), 3. Trypan blue solution (Sigma, T8154-100ML), 4. Flexstation 3 multifunctional microplate reader (Molec μlar Devices), 5. HEK-Blue ^TM hTLR7 cell line (InvivoGen, hkb-hTLR7), 6. HEK-Blue detection reagent (InvivoGen, hb-det3), 7. Phosphate buffer (PBS) pH7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).

Experimental steps

Configure HEK-Blue detection medium, take a bag of HEK-Blue detection dry powder, add 50ml of endotoxin-free water to dissolve it, put it into a 37 ° C incubator, and filter it for 10 minutes. The compound was first prepared into a 20 mM stock solution; it was then diluted with pure DMSO to a maximum concentration of ⁶ × 10 ⁶ nM, and then diluted three-fold to a total of 10 points.

Dilute the compound prepared above 20-fold with culture medium, and then add 20 μl of the diluted compound to each well. Take HEK-Blue ^TM hTLR7 cells, remove the supernatant first, then add 2-5 ml of pre-warmed PBS, put in the incubator for 1-2 minutes, gently blow the cells, and trypan blue staining counts. The cells were resuspended in HEK-Blue detection medium to adjust the concentration to 2.2 × 10 ⁵ cells / ml, and 180 μl of cells were added to the above 96-well cell culture plate to which 20 μl of the drug had been added, and cultured at 37 ° C. for 6-16 h.

Microplate reader reads at 620nm. Corresponding OD values obtained by Graphpad Prism calculated EC ₅₀ value of the drug.

The activation of human-derived TLR7 by the compounds of the present invention can be determined through the above tests. The measured EC ₅₀ values are shown in Table 1.

Conclusion: The compound of the present invention has a better activation effect on human TLR7.

Test example 2. Determination of the agonistic activity of the compound of the present invention on human TLR8

The activation effect of the compound of the present invention on the hTLR8 protein expressed by HEK-Blue ^TM hTLR8 stable transfected cells is determined by the following experimental method:

I. Experimental materials and instruments

1. DMEM (Gibco, 10564-029), 2. Fetal bovine serum (GIBCO, 10099), 3. Trypan blue solution (Sigma, T8154-100ML), 4. Flexstation 3 multifunctional microplate reader (Molec μlar Devices), 5. HEK-Blue ^TM hTLR8 cell line (InvivoGen, hkb-hTLR8), 6. HEK-Blue detection reagent (InvivoGen, hb-det3), 7. Phosphate buffer (PBS) pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).

Experimental steps

Configure HEK-Blue detection medium, take a bag of HEK-Blue detection dry powder, add 50ml of endotoxin-free water to dissolve it, put it into a 37 ° C incubator, and filter it for 10 minutes. The compound was first prepared into a 20 mM stock solution; it was then diluted with pure DMSO to a maximum concentration of 6 × 10 ⁶ nM, followed by a 3-fold gradient dilution of 10 points; the compound was first diluted 20-fold with the medium, and then 20 μl of each well was added to the dilution After the compound.

Take HEK-Blue ^TM hTLR8 cells, remove the supernatant first, add 2-5ml of pre-warmed PBS, put in the incubator for 1-2 minutes, gently blow the cells, and trypan blue staining counts. The cells were resuspended in HEK-Blue detection medium to adjust the concentration to 2.2 × 10 ⁵ cells / ml, and 180 μl of cells were added to the above 96-well cell culture plate to which 20 μl of the drug had been added, and cultured at 37 ° C. for 6-16 h.

Microplate reader reads at 620nm. Corresponding OD values obtained by Graphpad Prism calculated EC ₅₀ value of the drug.

Compounds of the invention may be human TLR8 activation measured by the above test, the measured EC ₅₀ values are shown in Table 2.

Conclusion: The compounds of the present invention have relatively weak activation of human TLR8, which indicates that the compounds of the present invention are selective for TLR7.

Test Example 3. Determination of the ability of the compounds of the present invention to stimulate IFN-α secretion from peripheral blood mononuclear cells (PBMC)

The ability of the compounds in the present invention to stimulate PBMC to secrete IFN-α is determined by the following experimental methods:

I. Experimental materials and instruments

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 ^TM blood cell counter (Sigma, Z359629-1EA), 7. 96-well flat bottom plate (Corning, 3599), 8. 96-well v bottom plate (Corning, 3894), 9. Human-derived IFN-α kit (cisbio, 6FHIFPEB), 10. PHERAStar multifunctional microplate reader (BMG, PHERAStar).

Experimental steps

The compound was diluted with pure DMSO, with a maximum concentration of 5 mM and a 4-fold gradient dilution for a total of 9 points. Then 4 μl of the compound was added to 196 μl of RMPI 1640 medium containing 10% FBS, and mixed. Take 50 μl of each well to a new 96-well cell culture plate.

All reagents were equilibrated to room temperature. A 250 ml culture flask was taken. 60 ml of blood and PBS + 2% FBS were added to it, and gently mixed and diluted. Take 50ml PBMC separation tube SepMateTM-50, add 15ml Ficoll-Paque PREMIUM, and then add 30ml diluted blood. Centrifuge at 1200g for 10 minutes at room temperature. The supernatant was taken, then 300g, and centrifuged for 8 minutes. Resuspend and count in RMPI 1640 medium containing 10% FBS, adjust the number of PBMCs to 3.33 × 10 ⁶ cells / ml, take 150 μl into the cell culture plate with the compound added, in a 37 ° C, 5.0% CO ₂ incubator Cultivate for 24h.

The cell culture plate was placed in a centrifuge at 1200 rpm and centrifuged at room temperature for 10 minutes. Remove 150 μl of supernatant from each well. First balance the reagents in the human-derived IFN-α kit to room temperature, and prepare anti-IFN-α-Eu ³⁺ -Cryptate conjugate and anti-IFN-α- d2-conjugate, both are mixed with conjugate buffer at a ratio of 1:40. Then add 16 μl of the supernatant obtained by centrifugation to each well. Then add 2 μl of freshly prepared anti-IFN-α-Eu ³⁺ -hole conjugate and anti-IFN-α-d2-conjugate to each well, mix by shaking, and incubate at room temperature for 3 hours in the dark.

Read on PHERAStar in HTRF mode. We define the minimum drug concentration at which the stimulus produces a minimum detection limit of at least three times the cytokine level, as the compound's MEC (Minimal Effective Concentration) value on the cytokine stimulation experiment.

The ability of the compound of the present invention to stimulate PBMC to secrete IFN-α was determined by the above test. The measured MEC values are shown in Table 3.

Conclusion: From the data of stimulating the activity of PBMC to secrete IFN-α, the compound of the present invention can better induce the release of IFN-α.

Test Example 4. Inhibitory effect of the compound of the present invention on the enzyme activity of human liver microsome CYP3A4 midazolam metabolism site

The enzyme activity of the compound of the present invention on the metabolizing site of CYP3A4 midazolam in liver microsomes is determined by the following experimental method:

I. Experimental materials and instruments

1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inertsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (15 μM midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA K1003).

Experimental steps

Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsome solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). Ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted with PBS gradient. Midazolam working solution diluted to a concentration of 15 μM with PBS.

Take 2.5mg / ml microsome solution, 15μM midazolam working solution, MgCl ₂ solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0μM, each concentration setting is different). Reaction system) 20 μl each and mixed well. The positive control group replaced the compound with ketoconazole at the same concentration. At the same time, 5 mM NADPH solutions were pre-cultured together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and culture was performed for 30 minutes. All culture samples were set in double samples. After 30 minutes, 250 μl of acetonitrile containing internal standard was added to all samples, mixed, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.

The values are calculated by Graphpad Prism and the IC ₅₀ values of the drug on the CYP3A4 midazolam metabolism site are shown in Table 4.

Conclusion: The compound of the present invention has no inhibitory effect on the midazolam metabolism site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolic drugs based on the CYP3A4 metabolism midazolam metabolism site will not occur with each other effect.

Test Example 5. Inhibitory effect of the compound of the present invention on human liver microsomal CYP2D6 enzyme activity

The enzyme activity of the compound of the present invention on human liver microsomes CYP2D6 is determined by the following experimental method:

I. Experimental materials and instruments

1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inertsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (20 μM dextromethorphan, SIGMA Q0750) and positive control inhibitor (quinidine, SIGMA D9684).

Experimental steps

Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsomal solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). Quinine working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted 5X in PBS. Dextromethorphan working solution diluted to a concentration of 20 μM with PBS.

Take 2.5mg / ml microsome solution, 20μM dextromethorphan working solution, MgCl ₂ solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, respectively, and set different reactions for each concentration System) 20 μl each, mix well. The positive control group replaced the compound with quinidine at the same concentration. At the same time, 5 mM NADPH solution was pre-cultured together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and culture was performed for 30 minutes. All culture samples were set in double samples. After 30 minutes, add 250 μl of acetonitrile with internal standard to all samples, mix well, and shake at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.

The IC ₅₀ value of the drug on the CYP2D6 metabolic site was calculated by Graphpad Prism, as shown in Table 5.

Conclusion: The compounds of the present invention have no inhibitory effect on the enzyme activity of human liver microsomes CYP2D6, and show better safety, suggesting that metabolic drug interactions based on CYP2D6 will not occur.

Test Example 6. Inhibitory effect of the compound of the present invention on the enzyme activity of testosterone metabolism sites of human liver microsomes CYP3A4

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

I. Experimental materials and instruments

1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inerrtsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (testosterone / 100 μM, SIGMA K1003) and positive control inhibitor (ketoconazole, Dr. Ehrenstorfer GmbH, C17322500).

Experimental steps

Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsomal solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). A 5X concentration of ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted with PBS gradient. Dextromethorphan working solution diluted to a concentration of 50 μM with PBS.

Take 2.5mg / ml microsomal solution, 50μM testosterone working solution, MgCl ₂ solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, each reaction system is set for each concentration) 20 μl each, mix well. The positive control group replaced the compound with ketoconazole at the same concentration. At the same time, 5 mM NADPH solutions were pre-cultured together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and culture was performed for 30 minutes. All culture samples were set in double samples. After 30 minutes, add 250 μl of acetonitrile with internal standard to all samples, mix well, and shake at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.

The values are calculated by Graphpad Prism and the IC ₅₀ values of the drug on the CYP3A4 testosterone metabolism site are shown in Table 6.

Conclusion: The compound of the present invention has no inhibitory effect on testosterone metabolism sites of human liver microsomes CYP3A4, and shows better safety, suggesting that no metabolic drug interaction based on CYP3A4 testosterone metabolism sites will occur.

Test example 7

1.Experimental purpose

The blocking effect of the compound of the present invention on hERG potassium current was tested on a stable cell line transfected with hERG potassium channel using a fully automatic patch clamp.

2.Experimental methods

2.1 Experimental materials and instruments

2.1.1 Experimental materials:

2.1.2 Experimental equipment:

2.2 Fully automatic patch clamp experimental steps

HEK293-hERG stable cell line was in a density of 1: 4 in MEM / EB SS medium (10% FBS, 400 μg / ml G418, 1% MEM non-essential amino acid solution (100 ×), 1% sodium pyruvate solution). Subculture was performed, and fully automatic patch clamp experiments were performed within 48-72 hours. After the cells were digested with 0.25% trypsin on the day of the experiment, the cells were collected by centrifugation, and resuspended with extracellular fluid (140 mM NaCl, 4 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 5 mMD glucose monohydrate, 10 mM Hepes, pH 7.4, 298 mOsmol) The cells are made into a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument. The Patchliner instrument used a negative pressure controller to add cells to the chip (NPC-16). The negative pressure attracted individual cells to the small holes of the chip. When the whole-cell mode is formed, the instrument will obtain hERG current according to the set hERG current voltage program, and then the instrument will automatically perfuse the compound from low concentration to high concentration. Through the data analysis software provided by HEAK Patchmaster, HEAK EPC10 patch clamp amplifier (Nanion) and Pathlinersoftware and Patheontrol HTsoftware, the current at each concentration of the compound and the blank control current were analyzed.

2.3 Test results

The blocking effect of the compound of the present invention on the hERG potassium current was measured by the above test, and the IC ₅₀ values measured are shown in Table 7.

Conclusion: The compounds of the present invention have a weak inhibitory effect on hERG and can reduce the side effects caused by the hERG pathway.

Pharmacokinetic evaluation

Test Example 8. Mouse pharmacokinetic test of the compound of the present invention

1.Abstract

Using mice as test animals, LC / MS / MS method was used to determine the drug concentration in plasma of mice at different times after intragastric administration of the compound of Example 1. The pharmacokinetic behavior of the compound of the present invention in mice was studied, and its pharmacokinetic characteristics were evaluated.

2. Test plan

2.1 Test drugs

Example 1. Compound.

2.2 Test animals

Nine C57 mice, female, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0006.

2.3 Drug Formulation

Weigh a certain amount of drug, add 5% volume of DMSO, 5% volume of tween80 and 90% physiological saline to make up 1mg / ml colorless and clear liquid.

2.4 Administration

C57 mice were fasted orally after fasting overnight. The doses were 20.0 mg / kg and the volume was 20 ml / kg.

3.Operation

Mice were administered with the compound of Example 1 by gavage. 0.1 ml of blood was collected before and after 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, and 24.0 hours, and placed in a heparinized test tube at 3500 rpm. Centrifuge for 10 minutes per minute to separate plasma and store at -20 ° C.

To determine the content of test compounds in mouse plasma after drug administration at different concentrations: Take 25 μl of mouse plasma at each time after administration, add 50 μl of internal standard solution camptothecin (100 ng / mL), 200 μl of acetonitrile, Spin and mix for 5 minutes, centrifuge for 10 minutes (3600 rpm), and take 10 μl of the supernatant for LC / MS / MS analysis.

4. Results of pharmacokinetic parameters

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

Conclusion: The compound of the present invention has good drug metabolism and absorption, and has pharmacokinetic advantages.

Test Example 9. Determination of mouse IFN-α value of the compound of the present invention

    I. Experimental materials and instruments    

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

2. Microplate reader (PE, victor3)

3. Test sample: The mice of Example 8 in the pharmacokinetics of Test Example 8 were administered by gavage. The blood of the compound of Example 1 was collected before and at 0, 2.0, 4.0 and 8.0 hours after the administration.

    Experimental steps    

1. Coating: Add 100 μl capture antibody (diluted with PBS to a final concentration of 1 μg / ml) to a 96-well ELISA plate. After filming, the cells were incubated overnight at room temperature.

2. Discard the plate the next day and pat it dry.

3. Add 200 μl blocking buffer (PBS + 2% BSA + 0.05% Tween 20) to each well and incubate at 37 ° C for 2 hours.

4. Discard the plate and pat the liquid in the plate.

5. Add the sample to be tested and the standard curve sample diluted to 100 μl with dilution buffer (the highest concentration is 500 pg / ml, seven points of double gradient dilution).

6. Aspirate the liquid in the plate with a plate washer, and then inject 300 μl of washing buffer (PBS + 0.05% Tween 20) into each well. After shaking, aspirate the liquid, repeat the washing 3 times, and finally pat the water droplets in the plate.

7. Add 100 μl of Lucia protease-linked antibody (final concentration: 30ng / ml) to each well, and incubate at 37 ° C for 2 hours.

8. Repeat step 6.

9. Add 50μl QUANTI-Luc (chemiluminescent reagent) to each well, and read it on the microplate reader.

    Test results:    

The mouse serum IFN-α value of the compound of the present invention is shown in Table 1 below and the time curve is shown in Figure 1: ig is administered by gavage.

    Fourth, the conclusion:    

After the compound of Example 1 was administered with 20 mpk for 2 hours in mice, it had a strong ability to stimulate IFN-α expression.

Claims (22)

A compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable form thereof Salt of the compound: Wherein: ring A is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl; G is selected from N and CR ⁶ ; X ^{1 is} selected from alkylene and S (O) _m , wherein the alkylene group is as required Substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, and heterocyclic; 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 covalent bond; R ^{1 is} selected from hydrogen atom, alkyl, haloalkyl, olefin Alkynyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently as needed Via one or more selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl R ^{2 is the} same or different, and each is independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, and cyclic Alkyl, heterocyclyl, 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 alkoxy Group, cycloalkyl group, heterocyclic group, aryl group and heteroaryl group are each independently selected from alkyl, alkoxy, halogen, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amine, nitrate Substituted with one or more substituents of the group, cycloalkyl, heterocyclyl, aryl, and heteroaryl; L ^{2 is} selected from an alkylene group or a covalent bond, wherein the alkylene group is optionally selected from halogen , Alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, and heterocyclyl; R ^{3 is} selected from hydrogen Atom, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -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, heterocyclyl, aryl and hetero The aryl groups are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, and heterocyclic , The aryl, ^{heteroaryl, -C (O) R 8,} -C (O) OR 8, -S (O) m R 8, -NR 9 R 10 and -C (O) NR ⁹ R ¹⁰ Substituted by one or more substituents; R ⁴ and R ^{5 are the} same or different, and each is independently selected from a hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amine Group, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R ⁴ and R ⁵ together form a pendant oxygen group; R ^{6 is} selected from a hydrogen atom, halogen, alkyl, alkoxy, haloalkane Group, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ^{7 is} selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, Heterocyclyl, aryl, and heteroaryl; R ^{8 is} selected from hydrogen, alkyl, haloalkyl, amine, hydroxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ⁹ and R ¹⁰ The same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group And heteroaryl are each independently selected from alkyl, alkoxy, halogen, amine, cyano, nitro, hydroxy Or hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl with one or more substituents; or, R ⁹ and R ¹⁰ together with the nitrogen atom to form a heterocyclyl Wherein the heterocyclic group contains 1-2 heteroatoms which are the same or different and are selected from N, O and S, and the heterocyclic group is optionally selected from the group consisting of alkyl, alkoxy, halogen, amine, and cyano , Nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl with one or more substituents; n is 0, 1, 2, 3, or 4; and m is 0, 1, or 2.     The compound or the tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof or pharmaceutically acceptable as described in item 1 of the scope of patent application Accepted salts wherein the ring A is selected from phenyl and pyridyl.     A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above is an acceptable salt, wherein X ¹ is an alkylene group. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above acceptable salt, wherein the compound is represented by the general formula (II): , Where: G, L ¹ ~ L ² , R ¹ ~ R ⁵ and n are as defined in the first patent application scope. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 On the acceptable salt, the compound is represented by the general formula (III): , Wherein: R ⁹ and R ¹⁰ form a heterocyclic group together with the nitrogen atom connected thereto, wherein the heterocyclic group contains 1-2 heteroatoms which are the same or different and are selected from N, O and S, and the heterocyclic group Optionally substituted with one or more selected from the group consisting of alkyl, alkoxy, halogen, amine, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl Group; G, L ¹ to L ² and R ¹ are as defined in item 1 of the scope of patent application. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above acceptable salts, wherein R ⁴ and R ⁵ are both hydrogen or R ⁴ and R ⁵ together form a pendant oxygen group. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above is an acceptable salt, wherein the L ¹ is -O-. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 acceptable salt thereof, wherein R ¹ is the alkyl group.     A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above is an acceptable salt, wherein G is N.     A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 The above is an acceptable salt, wherein the L ² is an alkylene group. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in the scope of application for the patent item 1 or 2 Acceptable salt, wherein the compound is selected from: A compound represented by the general formula (IB) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable form thereof Salt of the compound: Wherein: W is an amine protecting group; X is 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 first scope of the patent application . The compound or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof or pharmaceutically acceptable as described in item 12 of the scope of patent application Accepted salts, wherein the compound is selected from: A compound represented by the general formula (IC) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable form thereof Salt of the compound: , Where: W is an amine protecting group; s is 1 or 2; ring A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the first item of the scope of patent application. A compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament as described in item 14 of the scope of patent application An acceptable salt of the compound is selected from: A method for preparing a compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicament thereof as described in item 12 of the scope of application for a patent Acceptable salt method, the method includes: Compounds of general formula (IA) and A nucleophilic substitution reaction occurs to obtain a compound of the general formula (IB); wherein: W is an amine protecting group; X is halogen; s is 1 or 2; ring A, G, X ¹ , L ¹ ~ L ² , R ¹ ~ R ⁵ and n are as defined in item 1 of the scope of patent application. A method for preparing a compound or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or a medicament thereof as described in item 14 of the scope of patent application Acceptable salt method, the method includes: The compound of the general formula (IB) is ring-closed to obtain the compound of the general formula (IC); wherein: W is an amine protecting group; X is 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 item 1 of the scope of patent application. A method for preparing a compound or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a medicine thereof as described in item 1 of the scope of patent application Acceptable salt method, the method includes: The compound of the general formula (IC) is deprotected to obtain the compound of the general formula (I); wherein: W is an amine protecting group; s is 1 or 2; ring A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in item 1 of the scope of patent application.     A medicinal composition comprising: a therapeutically effective amount of a compound as described in any one of claims 1 to 11 or a tautomer, meso, racemate, enantiomer thereof , Diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.         A compound or a tautomer, meso, racemate, enantiomer, diastereomer, or the The use in the form of a mixture, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in item 19 of the scope of patent application, for the preparation of a medicament for a TLR7 agonist.         A compound or a tautomer, meso, racemate, enantiomer, diastereomer, or the The use of a mixture or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in item 19 of the scope of patent application for preparing a medicament for treating an infection caused by a virus, the virus being 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, Omsk hemorrhagic fever virus, bovine viral diarrhea virus , Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses.         A compound or a tautomer, meso, racemate, enantiomer, diastereomer, or the Use of a mixture or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in item 19 of the scope of application for preparation for the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cells Cancer, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia, ulcerative colitis, autoimmune disease, plaque psoriasis, systemic lupus erythematosus, actinic keratosis, and liver fibrosis Drug.