WO2018137614A1 - Heteroarothiadiazide-2,2-dioxide derivative, and preparation method therefor and application thereof in medicament - Google Patents

Abstract

Disclosed are a heteroarothiadiazide-2,2-dioxide derivative, and a preparation method therefor and an application thereof in medicament. Specifically, disclosed are a novel heteroarothiadiazide-2,2-dioxide derivative represented by formula (I), a preparation method therefor, a pharmaceutical composition containing the derivative, and a use as a therapeutic agent, especially as a TLR7 agonist. Each substituent group in formula (I) is defined as that in the description.

Description

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

The present invention belongs to the field of medicine, and relates to a novel heteroarylthiathiadiazine-2,2-dioxide derivative represented by the general formula (I), a preparation method thereof and a pharmaceutical composition containing the same And its use as a therapeutic, especially as a TLR7 agonist.

Background technique

Toll-like receptors (TLRs) are important protein molecules involved in innate immunity. TLRs are non-catalytic receptors for monomeric transmembranes, usually expressed in sentinel cells such as macrophages and dendritic cells, and can recognize structurally conserved molecules produced by microorganisms. Once these microorganisms break through physical barriers such as the skin or intestinal mucosa, they are recognized by TLRs, which in turn activate immune cell responses (Mahla, R S. et al., Front Immunol. 4:248 (2013)). The ability of the immune system to broadly recognize pathogenic microorganisms is due in part to the widespread presence of Toll-like immunoreceptors.

There are at least 10 different TLRs in mammals. Some of these receptor ligands and corresponding signal cascade amplification have been identified. TLR7 is a member of a subset of TLRs (TLRs 3, 7, 8, and 9) and is restricted to the endosomal compartment of cells that specifically detect non-hexucleic acids. TLR7 plays a key role in recognizing ssRNA antiviral defense (Diebold S.S. et al, Science, 2004: 303, 1529-1531; and Lund J. M. et al, PNAS, 2004: 101, 5598-5603). TLR7 has a limited expression profile in humans and is expressed primarily by B cells and plasmacytoid dendritic cells (pDC), but to a lesser extent by monocytes. Plasmacytoid DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% of peripheral blood mononuclear cells (PBMCs)) that secrete high levels of interferon-α (IFNα) and interferon in response to viral infections. The first type I interferon-producing cell of β(IFNβ) (Liu YJ, Annu. Rev. Immunol., 2005: 23, 275-306).

Many diseases and disorders are associated with abnormalities in TLRs such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD). ), ulcerative colitis, liver fibrosis, HBV, Flaviviridae virus, HCV, HPV, RSV, SARS, HIV or influenza virus infection. Therefore, the use of agonists for TLRs to treat related diseases is promising.

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

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

The present invention is directed to the above technical problems, and provides a drug compound having a lower incineration concentration, a better selectivity, and a more effective activation effect, and is a safer and more effective TLR7 agonist.

Summary of the invention

It is an object of the present invention to provide a compound of the formula (I):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

among them:

Ring A is selected from the group consisting of a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

G is selected from N and CR ⁶ ;

X ^{1 is} selected from an alkylene group or S(O) _m , wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, and nitrate Substituted by one or more substituents of a group, a cycloalkyl group and a heterocyclic group;

L ^{1 is} selected from -NR ⁷ -, -O-, -S-, -C(O)-, -S(O) _m -, -N(R ⁷ )C(O)-, -C(O)N (R ⁷ )-, -N(R ⁷ )S(O) ₂ -, -S(O) ₂ N(R ⁷ )- and a covalent bond;

R ^{1 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl group, the alkenyl group, the alkynyl group, and the cycloalkyl group are selected. And a heterocyclic group, an aryl group and a heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, hetero Substituting one or more substituents of a cyclic group, an aryl group, and a heteroaryl group;

² R is the same or different and are each independently selected from hydrogen, halo, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl a base, a heteroaryl group, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ , wherein The alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitrate Substituted by one or more substituents of a group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

L ^{2 is} selected from an alkylene group or a covalent bond, wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, Substituting one or more substituents of a cycloalkyl group and a heterocyclic group;

R ^{3 is} selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, -C (O) R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ wherein the cycloalkyl group, heterocyclic group And the aryl and heteroaryl are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl group, a heteroaryl group, -C (O) R,, or a ^{8 -C (O) oR 8 -S} (O) m R 8, -NR 9 R 10 and -C (O) NR ⁹ R ¹⁰ in Substituted by a plurality of substituents;

R ⁴ and R ^{5 are the} same or different and are each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, and a heterocyclic ring. Base, aryl and heteroaryl;

Or R ⁴ and R ⁵ together form an oxo group;

R ^{6 is} selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R ^{7 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

R ^{8 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an amino group, a hydroxyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

R ⁹ and R ¹⁰ are the same or different and are each independently selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein said alkyl, cycloalkyl, The heterocyclic group, the aryl group and the heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aromatic Substituted with one or more substituents in the heteroaryl group;

Alternatively, R ⁹ and R ¹⁰ together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 heteroatoms which are the same or different from N, O and S, and The heterocyclic group is optionally selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Substituted by a plurality of substituents;

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

m is 0, 1, or 2.

In a preferred embodiment of the invention, the compound of the formula (I), wherein the ring A is selected from the group consisting of a phenyl group and a pyridyl group.

In a preferred embodiment of the invention, the compound of the formula (I), wherein the X ¹ is an alkylene group.

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

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

among them:

G, L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

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

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

among them:

R ⁹ and R ¹⁰ together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 hetero atoms which are the same or different and are selected from N, O and S, and the heterocyclic ring The group is optionally substituted with one or more selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl Substituted, preferably 5-6 membered heterocyclic group, more preferably pyrrolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl or tetrahydropyranyl;

G, L ¹ to L ² and R ¹ are as defined in the formula (I).

In a preferred embodiment of the invention, the compound of the formula (I) wherein R ⁴ and R ⁵ are all hydrogen or R ⁴ and R ⁵ together form an oxo group.

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

In a preferred embodiment of the invention, the compound of the formula (I), wherein the R ¹ is an alkyl group, preferably a C _1-6 alkyl group, more preferably a methyl group, a Base, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl or n-pentyl.

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

In a preferred embodiment of the invention, the compound of the formula (I), wherein the L ² is an alkylene group, preferably a C _1-6 alkylene group, more preferably a methylene group. 1,2-ethylene, 1,1-ethylene or 1,3-propylene.

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

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the invention, a compound of the formula (IB) is an intermediate for the preparation of a compound of the formula (I):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

among them:

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

X is a halogen;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

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

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

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

among them:

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

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

Another aspect of the invention relates to a process for the preparation of a compound of the formula (IB) which comprises:

发生亲核取代反应得到通式(IB)的化合物； a compound of the formula (IA) and

A nucleophilic substitution reaction occurs to give a compound of the formula (IB);

among them:

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl; X is a halogen;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

Another aspect of the invention relates to a process for the preparation of a compound of the formula (IC), the process comprising:

a compound of the formula (IB) is ring-closed to give a compound of the formula (IC);

among them:

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl; X is a halogen;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

Another aspect of the invention relates to a process for the preparation of a compound of formula (I), which process comprises:

Deprotection of a compound of formula (IC) to give a compound of formula (I);

among them:

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesogen, a racemate thereof, Enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

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

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt or a pharmaceutical composition comprising the same for the preparation of a medicament for treating an infection caused by a virus selected from the group consisting of dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus , 蜱 脑 encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV , SARS and influenza viruses.

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a pharmaceutical composition comprising the same for use in the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD Use in drugs for ulcerative colitis and liver fibrosis.

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

The invention further relates to a method of treating an infection caused by a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, ink Dementia encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses, the method comprising administering A therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, is required. Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

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

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same, which is used as a medicament.

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same, which is for use in a TLR7 agonist.

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same for use in the treatment or prevention of an infection caused by a virus selected from the group consisting of: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, rumor Encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and flu virus.

The invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a medicament comprising the same for use in the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis and liver fibrosis.

The active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture. Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives, To provide a pleasing and tasty pharmaceutical preparation. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for the preparation of a tablet for admixture.

The aqueous suspension contains the active substance and excipients suitable for the preparation of the aqueous suspension for mixing. The aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents. Flavor.

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

Dispersible powders and granules suitable for use in the preparation of aqueous suspensions may be employed in the preparation of the active ingredient and dispersion or dispersing or suspending agents or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or compositions or Suitable dispersing or wetting agents and suspending agents can be used to illustrate the above examples. Other excipients such as sweetening, flavoring, and coloring agents may also be added. These compositions are preserved by the addition of an anti-oxidant such as ascorbic acid.

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

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. 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 oily 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 glycerin to form a microemulsion. The injection or microemulsion can be injected into the bloodstream of the patient by a local injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention. To maintain this constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a parenterally acceptable non-toxic diluent or solvent. In addition, sterile fixed oils may conveniently be employed as a solvent or suspension medium.

The compounds of the invention may be administered in the form of a suppository for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug. Such materials include a mixture of cocoa butter, glycerin gelatin, hydrogenated vegetable oil, polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

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

Detailed description of the invention

Terms used in the specification and claims have the following meanings unless stated to the contrary.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group having from 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-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-dimethyl Pentyl, 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-B Hexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-decyl, 2-methyl-2-ethylhexyl, 2-methyl-3-B Hexyl group, 2,2- Diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently selected from alkyl, alkenyl, alkynyl, Alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylthio, heterocycloalkylthio, oxo, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O Substituting one or more substituents in NR ⁹ R ¹⁰ .

The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon radical having two residues derived from the removal of two hydrogen atoms from the same carbon atom of the parent alkane or two different carbon atoms. A linear or branched group having 1 to 20 carbon atoms, preferably having 1 to 12 carbon atoms, more preferably an alkylene group having 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, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently selected from alkyl, alkenyl, alkynyl groups. , alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , cycloalkylthio, heterocycloalkylthio, oxo, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C( O) Substituted by one or more substituents in NR ⁹ R ¹⁰ .

The term "alkenyl" refers to a hydrocarbon group formed by one or more hydrogen atoms in the olefin molecule. The alkenyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, ^{heteroaryl, -C (O) R 8,} -C (O) OR 8, -S (O) m R Substituting one or more substituents of ^8- , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ .

The term "alkynyl" refers to a hydrocarbon containing a carbon-carbon triple bond in the molecule. The alkynyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, Hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R Substituting one or more substituents of ^8- , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ .

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, preferably from 3 to 10 The carbon atom, more preferably contains 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene A polycycloalkyl group includes a spiro ring, a fused ring, and a cycloalkyl group.

The term "amino protecting group" is intended to keep the amino group unchanged during the reaction of other parts of the molecule, and to protect the amino group with a group which is easily removed. Non-limiting examples include t-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with from 1 to 3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably p-methoxybenzyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O). A hetero atom of _m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably comprising from 3 to 12 ring atoms, wherein from 1 to 4 are heteroatoms; more preferably from 3 to 10 ring atoms, wherein 1-4 are heteroatoms; more preferably from 5 to 6 ring atoms; They are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1, 2.3.6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, High piperazinyl and the like. Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.

The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclic group, non-limiting examples of which include:

The heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy. , alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio, oxo, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR Substituting one or more substituents in ⁹ R ¹⁰ .

The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (ie, a ring that shares a pair of adjacent carbon atoms) having a conjugated π-electron system, preferably 6 to 10 members, such as benzene. Base and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples of which include:

The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, One of a heterocycloalkylthio group, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ or Substituted by a plurality of substituents.

The term "heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, Imidazolyl, pyrazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:

The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio One of a heterocycloalkylthio group, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ Or substituted with multiple substituents.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group. Substituted by one or more substituents of a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group.

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

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

The term "halogen" means fluoro, chloro, bromo or iodo.

The term "amino" means -NH _2.

The term "cyano" refers to -CN.

The term "nitro" refers to -NO ₂ .

The term "oxo" refers to =0.

"Optional" or "optionally" means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur. For example, "heterocyclic group optionally substituted by an alkyl group" means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group. .

"Substituted" refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

"Pharmaceutically acceptable salt" refers to a salt of a compound of the invention which is safe and effective for use in a mammal and which possesses the desired biological activity.

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

Method for synthesizing the compound of the present invention

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

Option One

a compound of the formula (I) of the present invention or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A method for preparing a medicinal salt, comprising the steps of:

In a first step, a compound of the formula (I-1) and NH(W) _s are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-2);

In the second step, a compound of the formula (I-2) and a compound of the formula (I-3) are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-4);

In the third step, the compound of the formula (I-4) is subjected to a reduction reaction in the presence of a reducing reagent to obtain a compound of the formula (IA);

在碱性条件下，发生亲核取代反应得到通式(IB)的化合物； The fourth step, the compound of the formula (IA) and

a nucleophilic substitution reaction under basic conditions to give a compound of the formula (IB);

In the fifth step, the compound of the formula (IB) is subjected to ring closure in the presence of iodide under basic conditions to obtain a compound of the formula (IC);

In a sixth step, the compound of the formula (IC) is deprotected under acidic conditions to give a compound of the formula (I);

among them:

The reagents providing basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl. Lithium lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide and sodium n-butoxide, said inorganic bases including but not limited to sodium hydride, Potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide;

Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane solution, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me ₃ SiCl and TMSOTf;

The reducing reagents include, but are not limited to, iron powder, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH(Ot-Bu) ₃ , AlH ₃ , NaCNBH ₃ , Na(AcO) ₃ BH, B ₂ H _{5 .} , Li(Et) ₃ BH, Pd/C/H ₂ and Raney Ni/H ₂ ;

Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;

The above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

X is a halogen, preferably chlorine;

_S is 1 or 2;

Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in the formula (I).

Option II

a compound of the formula (II) of the present invention or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A method for preparing a medicinal salt, comprising the steps of:

In a first step, a compound of the formula (I-1) and NH(W) _s are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-2);

In the second step, the compound of the formula (I-2) and the compound of the formula (II-1) are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (II-2);

In the third step, the compound of the formula (II-2) is subjected to a reduction reaction in the presence of a reducing reagent to obtain a compound of the formula (II-A);

在碱性条件下，发生亲核取代反应得到通式(II-B)的化合物； The fourth step, the compound of the formula (II-A) and

a nucleophilic substitution reaction under basic conditions to give a compound of the formula (II-B);

In the fifth step, the compound of the formula (II-B) is subjected to ring closure in the presence of iodide under basic conditions to obtain a compound of the formula (II-C);

In a sixth step, the compound of the formula (II-C) is deprotected under acidic conditions to give a compound of the formula (II);

among them:

The reagents providing basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl. Lithium lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide and sodium n-butoxide, said inorganic bases including but not limited to sodium hydride, Potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide;

Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane solution, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me ₃ SiCl and TMSOTf;

The reducing agents include, but are not limited to: iron, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot -Bu) 3, AlH 3, NaCNBH 3, Na (AcO) 3 BH, B 2 H 5 , Li(Et) ₃ BH, Pd/C/H ₂ and Raney Ni/H ₂ ;

Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;

The above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

X is a halogen, preferably chlorine;

_S is 1 or 2;

G, n, L ¹ to L ² and R ¹ to R ⁵ are as defined in the formula (II).

third solution

a compound of the formula (III) of the present invention or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A method for preparing a medicinal salt, comprising the steps of:

In the first step, a compound of the formula (I-1) and NH-(W) _s are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (I-2);

In the second step, a compound of the formula (I-2) and a compound of the formula (III-1) are subjected to a nucleophilic substitution reaction under basic conditions to obtain a compound of the formula (III-2);

In the third step, the compound of the formula (III-2) is subjected to a reduction reaction in the presence of a reducing reagent to obtain a compound of the formula (III-A);

在碱性条件下，发生亲核取代反应得到通式(III-B)的化合物； The fourth step, the compound of the formula (III-A) and

a nucleophilic substitution reaction under basic conditions to give a compound of the formula (III-B);

In the fifth step, the compound of the formula (III-B) is subjected to ring closure in the presence of iodide under basic conditions to obtain a compound of the formula (III-C);

In a sixth step, the compound of the formula (III-C) is deprotected under acidic conditions to give a compound of the formula (III);

among them:

The reagents providing basic conditions include organic bases including, but not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and n-butyl. Lithium lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide and sodium n-butoxide, said inorganic bases including but not limited to sodium hydride, Potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide;

Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane solution, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me ₃ SiCl and TMSOTf;

The reducing reagents include, but are not limited to, iron powder, lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH(Ot-Bu) ₃ , AlH ₃ , NaCNBH ₃ , Na(AcO) ₃ BH, B ₂ H _{5 .} , Li(Et) ₃ BH, Pd/C/H ₂ and Raney Ni/H ₂ ;

Iodide includes, but is not limited to, iodine, iodinated ketone, potassium iodide, and cesium iodide;

The above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1 , 4-dioxane, water, N,N-dimethylformamide and mixtures thereof;

W is an amino protecting group, preferably t-butoxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl;

X is a halogen, preferably chlorine;

_S is 1 or 2;

G, L ¹ to L ² , R ¹ and R ⁹ to R ¹⁰ are as defined in the formula (III).

DRAWINGS

Figure 1. Time profile of mouse serum IFN-[alpha] values for compounds of the invention.

detailed description

Example

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). NMR was measured using a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), internal standard was four. Methyl silane (TMS).

The measurement of the MS was carried out using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

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

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

Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate. The specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm~0.2mm. The specification for thin layer chromatography separation and purification is 0.4mm. ~0.5mm.

Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as a carrier.

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

The CombiFlash Rapid Preparer uses the Combiflash Rf200 (TELEDYNE ISCO).

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

The known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Companies such as Dare Chemicals.

Unless otherwise specified in the examples, the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.

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

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

The pressurized hydrogenation reaction was carried out using a Parr Model 3916EKX hydrogenation apparatus and a clear blue QL-500 type hydrogen generator or a HC2-SS type hydrogenation apparatus.

The hydrogenation reaction is usually evacuated, charged with hydrogen, and operated three times.

The microwave reaction used a CEM Discover-S Model 908860 microwave reactor.

Unless otherwise stated in the examples, the solution means an aqueous solution.

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

The progress of the reaction in the examples was monitored by thin layer chromatography (TLC), the developing agent used for the reaction, the column chromatography eluent system used for the purification of the compound, and the thin layer chromatography developing solvent system including: A: Methylene chloride/methanol system, B: n-hexane/ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of an alkaline or acidic reagent such as triethylamine or acetic acid.

Example 1

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

first step

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 the well-known method "Journal of Medicinal Chemistry, 2012, 55(23), 10387-10404") It was dissolved in 50 mL of tetrahydrofuran solution, and triethylamine (2.64 g, 26.14 mmol) and N,N-bis(4-methoxybenzyl)amine (4.49 g, 17.43 mmol. The reaction was quenched, and the residue was purified with EtOAcjjjjjjj

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 , 6-two

Amine 1d

1b (1.20g, 2.65mmol) was dissolved in 20mL of tetrahydrofuran, followed by triethylamine (402mg, 3.98mmol) and (4-(pyrrolidin-1-ylmethyl)phenyl)methanamine 1c (469mg, 2.65) Ment, prepared by the method disclosed in the patent application "WO2016044183", stirred at room temperature for 2 hours, and the reaction was stopped. After adding 50 mL of water, the mixture was extracted with ethyl acetate (20 mL×3), EtOAc (EtOAc) Purification by eluent system A followed by purification by thin layer chromatography eluting with EtOAc EtOAc (EtOAc)

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 a mixed solution of 25mL of ethanol and water (V/V=4:1), and heated to Stir at 80 ° C for 4 hours. The reaction was quenched, cooled to room temperature, and filtered. EtOAc EtOAc (EtOAc m. The organic layer was concentrated under reduced pressure.

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- Base)-1-chloromethanesulfonamide 1f

1e (305 mg, 0.50 mmol) was dissolved in 10 mL of dichloromethane, then pyridine (119 mg, 1.50 mmol), 4-dimethylaminopyridine (61 mg, 0.50 mmol) and chloromethylsulfonyl chloride (149 mg, 1.00 mmol) The reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. After adding 20 mL of water and extracting with dichloromethane (20 mL × 3), the organic phase was combined, washed with saturated sodium chloride (50 mL), dried over anhydrous magnesium sulfate, filtered, Purification by eluent system A gave the title product 1f (260 mg).

MS m/z (ESI): 723.3 [M+1]

the fifth step

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

1f (260 mg, 0.36 mmol) was dissolved in 5 mL of N,N-dimethylformamide, sodium hydride (43 mg, 1.08 mmol) and potassium iodide (30 mg, 0.18 mmol) were sequentially added, and the mixture was heated to 50 ° C, and the reaction was stirred for 16 hours. Stop the reaction. After cooling to room temperature, 20 mL of water was added, and ethyl acetate (20 mL×3) was evaporated. The residue was purified by EtOAc EtOAc (EtOAc)

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

Step 6

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

1 g (300 mg, 0.55 mmol) and 10 mL of trifluoroacetic acid were placed in a reaction flask, and reacted at room temperature for 6 hours to stop the reaction. The mixture was added dropwise with aq. The organic layer was concentrated under reduced pressure.

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

_{^{1 H NMR (400MHz, CD 3}} OD) δ7.49 (s, 4H), 4.97 (s, 2H), 4.61 (s, 2H), 4.32 (s, 2H), 4.15-4.12 (t, 3H), 3.35 -3.29 (m, 4H), 2.89-2.87 (m, 4H), 2.09-2.06 (m, 4H), 1.65-1.61 (m, 2H), 1.42-1.38 (m, 2H), 0.93-0.89 (t, 3H).

Example 2

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

first step

2-butoxy-N ⁴ ,N ⁴ -bis(4-methoxybenzyl)-5-nitro-N ⁶ -(3-(pyrrolidin-1-ylmethyl)benzyl)pyrimidine-4 , 6-two

Amine 2b

1b (4.5 g, 9.24 mmol) was dissolved in 50 mL of tetrahydrofuran, followed by triethylamine (1.4 g, 13.86 mmol), 4-dimethylaminopyridine (225 mg, 1.85 mmol) and (3-(pyrrolidine-1-) Methyl)phenyl)methylamine 2a (1.76 g, 9.24 mmol, prepared by the method disclosed in the patent application "WO2010077613") was stirred at room temperature for 2 hours to stop the reaction. The reaction mixture was concentrated under reduced pressure. EtOAcjjjjjjj

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

2b (3.3g, 5.15mmol), iron (1.15g, 20.6mmol) and ammonium chloride (2.22g, 41.20mmol) were dissolved in a mixed solution of 38mL of ethanol and water (V/V=15:4). Heat to 80 ° C and stir for 4 hours. The reaction was quenched, cooled to room temperature, filtered, EtOAc EtOAc (EtOAc m. The organic layer was concentrated under reduced pressure. EtOAcjjjjjj

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- Base)-1-chloromethanesulfonamide 2d

2c (600 mg, 0.98 mmol) was dissolved in 20 mL of dichloromethane, then 4-dimethylaminopyridine (120 mg, 0.98 mmol), pyridine (232 mg, 2.94 mmol) and chloromethylsulfonyl chloride (293 mg, 1.97 mmol) The reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. The reaction mixture was concentrated under reduced pressure. EtOAcjjjjjjj

MS m/z (ESI): 723.3 [M+1]

the fourth step

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

2d (72mg, 0.10mmol) was dissolved in 5mL of N,N-dimethylformamide, potassium iodide (4mg, 0.02mmol) and sodium hydride (12mg, 0.30mmol) were added sequentially, heated to 50 ° C, stirring reaction for 16 hours , stop the reaction. After cooling to room temperature, 20 mL of water was added, and ethyl acetate (20 mL × 3) was evaporated. The residue was purified by EtOAc EtOAc (EtOAc) MS m/z (ESI): 687.3 [M+1]

the fifth step

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

2e (68 mg, 0.10 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were placed in a reaction flask, and reacted at room temperature for 2 hours to stop the reaction. After adding 20 mL of water and extracting with dichloromethane (20 mL × 3), the organic phase was combined, washed with saturated sodium chloride (50 mL), dried over anhydrous magnesium sulfate, filtered, After purification by the developer system A, the residue was purified by high-performance liquid chromatography to afford the title product 2 (12 mg), yield: 27.2%.

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

¹ H NMR (400 MHz, 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 case:

Biological evaluation

Test Example 1. Determination of agonistic activity of human TLR7 by the compound of the present invention

Determination of the compounds of the present invention adopts the following experimental method hTLR7 protein activation HEK-Blue ^TM hTLR7 expression in stably transfected cell lines of:

First, 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 multi-function microplate reader (Molecμlar Devices),

5.HEK-Blue ^TM hTLR7 cell lines (InvivoGen, hkb-hTLR7),

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

7. Phosphate buffer (PBS) pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).

Second, the experimental steps

Configure HEK-Blue assay medium, take a bag of HEK-Blue dry powder, add 50ml to endotoxin water to dissolve, then put it into the 37 °C incubator, and then sterile filtration after 10 minutes. The compound was first formulated into a 20 mM stock solution; it was diluted with pure DMSO to a maximum concentration of ⁶ x 106 nM and diluted by a 3-fold gradient for a total of 10 points.

The above-prepared compound was diluted 20-fold with a medium, and then 20 μl of the diluted compound was added to each well. Take HEK-Blue ^TM hTLR7 cells, first remove the supernatant, then add 2-5 ml of pre-warmed PBS, put into the incubator for 1-2 minutes, gently pipe the cells, and trypan blue staining. The cells were resuspended in HEK-Blue assay medium at a concentration of 2.2 × 10 ⁵ cells/ml, and 180 μl of the 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 hours.

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

The activation of human TLR7 by the compounds of the present invention can be determined by the above test, and the measured EC ₅₀ values are shown in Table 1.

Table 1 Compound of the present invention to the human EC TLR7 _50.

Example number EC ₅₀ (nM) 1 443

Conclusion: The compounds of the present invention have a good activation effect on human TLR7.

Test Example 2: Determination of agonistic activity of human TLR8 by the compound of the present invention

Determination of the compounds of the present invention adopts the following experimental method hTLR8 protein activation HEK-Blue ^TM hTLR8 expression in stably transfected cell lines of:

First, 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 multi-function microplate reader (Molecμlar 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).

Second, the experimental steps

Configure HEK-Blue assay medium, take a bag of HEK-Blue dry powder, add 50ml to endotoxin water to dissolve, then put it into the 37 °C incubator, and then sterile filtration after 10 minutes. Compound is first formulated as a 20mM stock solution; neat DMSO and then diluted to a maximum concentration of ⁶ × 10 6 nM, and then diluted 3 fold gradient, a total of 10 points; first compound is diluted 20-fold with medium and then diluted with 20μl added per well After the compound.

Take HEK-Blue ^TM hTLR8 cells, first remove the supernatant, add 2-5 ml of pre-warmed PBS, put into the incubator for 1-2 minutes, gently pipe the cells, and trypan blue staining. The cells were resuspended in HEK-Blue assay medium at a concentration of 2.2 × 10 ⁵ cells/ml, and 180 μl of the 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 hours.

The microplate reader reads at a wavelength of 620 nm. The corresponding OD value can be obtained, and the EC ₅₀ value of the drug is calculated by Graphpad Prism.

The activation of human TLR8 by the compounds of the present invention can be determined by the above test, and the measured EC ₅₀ values are shown in Table 2.

Table Compound 2 of the present invention to human TLR8, EC _50.

Example number EC ₅₀ (nM) 1 4877 2 >30000

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

Test Example 3: Determination of the ability of the compound of the present invention to stimulate peripheral blood mononuclear cells (PBMC) to secrete IFN-α

The ability of the compounds of the invention to stimulate the secretion of IFN-α by PBMC is determined by the following experimental method:

First, 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 hemocytometer (Sigma, Z359629-1EA),

7. 96-hole flat bottom plate (Corning, 3599),

8. 96-well v backplane (Corning, 3894),

9. Human IFN-α kit (cisbio, 6FHIFPEB),

10. PHERAStar multi-function microplate reader (BMG, PHERAStar).

Second, the experimental steps

Compounds were diluted in pure DMSO at a maximum concentration of 5 mM in 4 fold gradients for a total of 9 points. Then 4 μl of the compound was taken and added to 196 μl of RMPI 1640 medium containing 10% FBS and mixed. Take 50 μl per well into a new 96-well cell culture plate.

All reagents were equilibrated to room temperature, and a 250 ml culture flask was taken. 60 ml of blood and PBS + 2% FBS were added thereto, and the mixture was gently diluted by pipetting. Take 50 ml of PBMC separation tube SepMateTM-50, add 15 ml of lymphocyte separation solution Ficoll-Paque PREMIUM, and then add 30 ml of diluted blood. Centrifuge at 1200g for 10 minutes at room temperature. The supernatant was taken, then 300 g, and centrifuged for 8 minutes. Resuspend and count with RMPI 1640 medium containing 10% FBS, adjust the amount of PBMC to 3.33×10 ⁶ cells/ml, take 150 μl to the cell culture plate to which the compound has been added, and incubate at 37 ° C, 5.0% CO ₂ The medium was cultured for 24 hours.

The cell culture plate was placed in a centrifuge, centrifuged at 1200 rpm for 10 minutes at room temperature. 150 μl of the supernatant was taken out per well. The reagent in the human IFN-α kit is firstly equilibrated to room temperature, and anti-IFN-α-Eu ³⁺ - Cryptate conjugate and anti-IFN-α- are prepared according to the kit instructions in the dark. The d2-conjugate, both mixed in a 1:40 ratio with a conjugate buffer. Then, 16 μl of the supernatant obtained by centrifugation was added to each well. 2 μl of the well-prepared anti-IFN-α-Eu ³⁺ - cryptate and anti-IFN-α-d2-conjugate were added to each well, and the mixture was shaken and incubated at room temperature for 3 hours in the dark.

The HTRF mode is read on the PHERAStar. We will stimulate the lowest drug concentration that produces a minimum detection limit of at least 3 times the cytokine level, defined as the MEC (Minimal Effective Concentration) value of the compound in the cytokine stimulation test.

The ability of the compounds of the invention to stimulate PBMC secretion of IFN-[alpha] was determined by the above assay, and the measured MEC values are shown in Table 3.

Table 3 Compounds of the invention stimulate MECs secreting IFN-α by PBMC

Example number MEC(nM) 1 1.5 2 twenty two

Conclusion: From the data of stimulating the activity of IFN-α secreted by PBMC, the compound of the present invention can better cause IFN-α release.

Test Example 4 Inhibition of the Enzymatic Activity of the Compound of the Invention on the Metabolite of the Metabolite of CYP3A4 Midazolam in Human Liver Microsomes

The enzymatic activity of the compound of the present invention on the metabolite site of human liver microsomal CYP3A4 midazolam was determined by the following experimental method:

First, experimental materials and instruments

1. Phosphate buffer (PBS),

2. NADPH (Sigma N-1630),

3. Human liver microsomes (Corning Gentest),

4.ABI QTrap 4000 LC/MS (AB Sciex),

5.Inertsil C8-3 column, 4.6×50mm, 5μm (American Dima Company),

6. CYP probe substrate (15 μM midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA K1003).

Second, the experimental steps

Configure 100 mM PBS buffer, prepare 2.5 mg/ml microsome solution and 5 mM NADPH solution with this buffer, and dilute the 5X concentration of compound working solution with PBS (150, 50, 15, 5, 1.5, 0.15, 0.015). , 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. Dilute to a concentration of 15 μM midazolam working solution in 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 with different concentration settings). The reaction system was 20 μl each and mixed uniformly. The positive control group was replaced with the same concentration of ketoconazole instead of the compound. At the same time, 5 mM of NADPH solution was pre-incubated for 5 minutes at 37 °C. After 5 minutes, 20 μl of NADPH was added to the wells, and the reaction was started and incubated for 30 minutes. All samples were incubated for two samples. After 30 minutes, 250 μl of internal standard-containing acetonitrile was added to all samples, mixed, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant was taken and transferred to LC-MS/MS for analysis.

Value calculated by Graphpad Prism IC drugs on CYP3A4 Midazolam Metabolism sites ₅₀ are given in Table 4.

Table ₅₀ Compound 4 of the present invention the value of CYP3A4 Midazolam Metabolism site IC

Example number IC ₅₀ (μM) 1 31 2 17

Conclusion: The compound of the present invention has no inhibitory effect on the midazolam metabolic site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolites based on CYP3A4 metabolism of midazolam metabolite sites do not occur. effect.

Test Example 5: Inhibition of human liver microsomal CYP2D6 enzyme activity by the compound of the present invention

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

First, experimental materials and instruments

1. Phosphate buffer (PBS),

2. NADPH (Sigma N-1630),

3. Human liver microsomes (Corning Gentest),

4.ABI QTrap 4000 LC/MS (AB Sciex),

5.Inertsil C8-3 column, 4.6×50mm, 5μm (American Dima Company),

6. CYP probe substrate (20 μM dextromethorphan, SIGMA Q0750) and positive control inhibitor (Quinidine, SIGMA D9684).

Second, the experimental steps

Configure 100 mM PBS buffer, prepare 2.5 mg/ml microsome solution and 5 mM NADPH solution with this buffer, and dilute the 5X concentration compound working solution with PBS (150, 50, 15, 5, 1.5, 0.15, 0.015). , 0 μM). The 5× concentration of quinidine working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted with PBS. Dilute dextromethorphan working solution was diluted with PBS to a concentration of 20 μM.

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, 0μM, each reaction set differently System) 20 μl each, mixed evenly. The positive control group replaced the compound with the same concentration of quinidine. At the same time, 5 mM of NADPH solution was pre-incubated for 5 minutes at 37 ° C. After 5 minutes, 20 μl of NADPH was added to the wells, and the reaction was started and incubated for 30 minutes. All samples were incubated for two samples. After 30 minutes, 250 μl of internal standard acetonitrile was added to all samples, mixed, and shaken at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. 80 μl of the supernatant was taken and transferred to LC-MS/MS for analysis.

Graphpad Prism was obtained numerical calculation of the drug metabolized by CYP2D6 site IC ₅₀ values are shown in Table 5.

Table ₅₀ Compound 5 of the present invention value metabolized by CYP2D6 site IC

Example number IC ₅₀ (μM) 1 36 2 twenty two

Conclusion: The compound of the present invention has no inhibitory effect on the enzymatic activity of human liver microsome CYP2D6, and shows better safety, suggesting that no metabolic drug interaction based on CYP2D6 occurs.

Test Example 6, Inhibition of Enzyme Activity of Testosterone Metabolism Site of Human Liver Microsome CYP3A4 by Human Compound Microsomes

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

First, experimental materials and instruments

1. Phosphate buffer (PBS),

2. NADPH (Sigma N-1630),

3. Human liver microsomes (Corning Gentest),

4.ABI QTrap 4000 LC/MS (AB Sciex),

5.Inertsil C8-3 column, 4.6×50mm, 5μm (American Dima Company),

6. CYP probe substrate (testosterone / 100 μM, SIGMA K1003) and positive control inhibitor (ketoconazole, Dr. Ehrenstorfer GmbH, C17322500).

Second, the 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 with PBS (150, 50, 15, 5, 1.5, 0.15, 0.015). , 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. The dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.

Were taken 2.5mg / ml of microsomal solution, testosterone working fluid of 50μM, MgCl ₂ and the solution of compound working solution (150,50,15,5,1.5,0.15,0.015,0μM, each concentration of the reaction system set different) Each 20 μl was mixed well. The positive control group was replaced with the same concentration of ketoconazole instead of the compound. At the same time, 5 mM of NADPH solution was pre-incubated for 5 minutes at 37 °C. After 5 minutes, 20 μl of NADPH was added to the wells, and the reaction was started and incubated for 30 minutes. All samples were incubated for two samples. After 30 minutes, 250 μl of internal standard-containing acetonitrile was added to all samples, mixed, and shaken at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. 80 μl of the supernatant was taken and transferred to LC-MS/MS for analysis.

The IC ₅₀ values of the test for CYP3A4 testosterone metabolism sites calculated by Graphpad Prism are shown in Table 6.

Table ₅₀ Compound 6 of the present invention value CYP3A4 metabolism of testosterone site IC

Example number IC ₅₀ (μM) 1 13

Conclusion: The compound of the present invention has no inhibitory effect on the testosterone metabolism site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolic drug interaction based on CYP3A4-based testosterone metabolism site does not occur.

Test Example 7

1. Experimental purpose

The blocking effect of the compounds of the invention on hERG potassium current was tested using a fully automated patch clamp on a stable cell line transfected with hERG potassium channels.

2, experimental methods

2.1 Experimental materials and instruments

2.1.1 Experimental materials:

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

2.1.2 Experimental equipment:

2.2 automatic patch clamp experimental steps

The HEK293-hERG stable cell line was passaged at a density of 1:4 in MEM/EBSS medium (10% FBS, 400 μg/ml G418, 1% MEM non-essential amino acid solution (100×), 1% sodium pyruvate solution). Culture, culture within 48-72 hours for automated patch-clamp experiments. On the day of the experiment, the cells were digested with 0.25% trypsin, and 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 mM MD-water glucose, 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 apply the cells to the chip (NPC-16), and the negative pressure attracted individual cells to the wells of the chip. When the whole cell mode is formed, the instrument will get the hERG current according to the set hERG current voltage program, and then the instrument will automatically perfuse the compound from low to high concentration. The currents at each concentration of the compounds and the blank control current were analyzed by HEAK Patchmaster, HEAK EPC10 patch clamp amplifiers (Nanion) and Pathlinersoftware and data analysis software provided by Pathcontrol HTsoftware.

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 measured IC ₅₀ values are shown in Table 7.

Table 7 IC ₅₀ of the blocking effect of the compounds of the invention on hERG potassium current

Example number IC ₅₀ (μM) 1 >30 2 12

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

Pharmacokinetic evaluation

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

1. Summary

Using mice as test animals, the concentration of the drug in plasma at different times after administration of the compound of Example 1 by intragastric administration was determined by LC/MS/MS method. The pharmacokinetic behavior of the compounds of the invention in mice was investigated and their pharmacokinetic characteristics were evaluated.

2, the test plan

2.1 test drugs

The compound of Example 1.

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 preparation

A certain amount of the drug was weighed, and 5% by volume of DMSO, 5% by volume of tween 80, and 90% physiological saline were set to 1 mg/ml of a colorless clear liquid.

2.4 administration

C57 mice were intragastrically administered overnight after fasting, and the dose was 20.0 mg/kg, and the administration volume was 20 ml/kg.

3, operation

The compound of Example 1 was administered by gavage in mice, and 0.1 ml of blood was collected before administration and after 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after administration, and placed in a heparinized test tube, 3500 rpm. The plasma was separated by centrifugation for 10 minutes at /min and stored at -20 °C.

The content of the test compound in the plasma of the mice after the intragastric administration of different concentrations of the drug was determined: 25 μl of the mouse plasma at each time after administration, 50 μl (100 ng/mL) of the internal standard solution, and 200 μl of acetonitrile were added. The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (3600 rpm), and 10 μl of the supernatant was taken for LC/MS/MS analysis.

4, pharmacokinetic parameters results

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

Conclusion: The compounds of the present invention have better pharmacological absorption and have pharmacokinetic advantages.

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

First, experimental materials and instruments

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

2, microplate reader (PE, victor3)

3. Test sample: Test Example 8 Pharmacokinetics The mice of Example 1 were administered intragastrically, and blood was collected before administration and at 0, 2.0, 4.0 and 8.0 hours after administration.

Second, the experimental steps

1. Coating: 100 μl of capture antibody (diluted to a final concentration of 1 μg/ml with PBS) was added to a 96-well ELISA plate. The film was incubated overnight at room temperature.

2. Pour off the plate the next day and pat the liquid in the plate.

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

4. Pour off 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 the dilution buffer (the highest concentration is 500 pg/ml, and the double gradient is diluted by seven points).

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, absorb the liquid, repeat the washing 3 times, and finally take the water droplets in the dry plate.

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

8. Repeat step 6.

9. Add 50 μl of QUANTI-Luc (chemiluminescence reagent) to each well and read the microplate reader.

Third, the test results:

The serum IFN-α values of the compounds of the present invention are shown in Table 1 below and the time curve is shown in Figure 1:

Table 1 IFN-α value in mouse serum

I.g. is administered by intragastric administration.

Fourth, the conclusion:

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

Claims (22)

A compound of the formula (I):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, among them: Ring A is selected from the group consisting of a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; G is selected from N and CR ⁶ ; X ^{1 is} selected from the group consisting of alkylene and S(O) _m , wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, and nitrate Substituted by one or more substituents of a group, a cycloalkyl group and a heterocyclic group; L ^{1 is} selected from -NR ⁷ -, -O-, -S-, -C(O)-, -S(O) _m -, -N(R ⁷ )C(O)-, -C(O)N (R ⁷ )-, -N(R ⁷ )S(O) ₂ -, -S(O) ₂ N(R ⁷ )- and a covalent bond; R ^{1 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl group, the alkenyl group, the alkynyl group, and the cycloalkyl group are selected. And a heterocyclic group, an aryl group and a heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, hetero Substituting one or more substituents of a cyclic group, an aryl group, and a heteroaryl group; ² R is the same or different and are each independently selected from hydrogen, halo, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl a base, a heteroaryl group, -C(O)R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ , wherein The alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitrate Substituted by one or more substituents of a group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; L ^{2 is} selected from an alkylene group or a covalent bond, wherein the alkylene group is optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, Substituting one or more substituents of a cycloalkyl group and a heterocyclic group; R ^{3 is} selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, -C (O) R ⁸ , -C(O)OR ⁸ , -S(O) _m R ⁸ , -NR ⁹ R ¹⁰ and -C(O)NR ⁹ R ¹⁰ wherein the cycloalkyl group, heterocyclic group And the aryl and heteroaryl are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl group, a heteroaryl group, -C (O) R,, or a ^{8 -C (O) oR 8 -S} (O) m R 8, -NR 9 R 10 and -C (O) NR ⁹ R ¹⁰ in Substituted by a plurality of substituents; R ⁴ and R ^{5 are the} same or different and are each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, and a heterocyclic ring. Base, aryl and heteroaryl; Or R ⁴ and R ⁵ together form an oxo group; R ^{6 is} selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl; R ^{7 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; R ^{8 is} selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, an amino group, a hydroxyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; R ⁹ and R ^{10 are the} same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl 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 the heteroaryl group are each independently selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aromatic Substituted with one or more substituents in the heteroaryl group; Alternatively, R ⁹ and R ¹⁰ together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 heteroatoms which are the same or different from N, O and S, and The heterocyclic group is optionally selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Substituted by a plurality of substituents; n is 0, 1, 2, 3 or 4; m is 0, 1, or 2. The compound of the formula (I) according to claim 1, wherein the ring A is selected from the group consisting of a phenyl group and a pyridyl group. The compound of the formula (I) according to claim 1 or 2, wherein the X ¹ is an alkylene group. The compound of the formula (I) according to any one of claims 1 to 3, which is a compound represented by the formula (II):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, among them: G, L ¹ to L ² , R ¹ to R ⁵ and n are as defined in claim 1. The compound of the formula (I) according to any one of claims 1 to 4, which is a compound represented by the formula (III):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, among them: R ⁹ and R ¹⁰ together with a nitrogen atom to which they are bonded form a heterocyclic group, wherein the heterocyclic group contains 1 to 2 hetero atoms which are the same or different and are selected from N, O and S, and the heterocyclic ring The group is optionally substituted with one or more selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl Substituted by G, L ¹ to L ² and R ^{1 are} as defined in claim 1. The compound of the formula (I) according to any one of claims 1 to 4, wherein R ⁴ and R ⁵ are each hydrogen or R ⁴ and R ⁵ together form an oxo group. The compound of the formula (I) according to any one of claims 1 to 6, wherein the L ¹ is -O-. The compound of the formula (I) according to any one of claims 1 to 7, wherein the R ¹ is an alkyl group. The compound of the formula (I) according to any one of claims 1 to 8, wherein the G is N. The compound of the formula (I) according to any one of claims 1 to 9, wherein the L ² is an alkylene group. The compound of the formula (I) according to any one of claims 1 to 10, which is selected from the group consisting of:

A compound of the formula (IB):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, among them: W is an amino protecting group; X is a halogen; S is 1 or 2; Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in claim 1. A compound of the formula (IB) according to claim 12, which is selected from the group consisting of:

A compound of the formula (IC):

Or a tautomer, a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, among them: W is an amino protecting group; 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 claim 1. A compound of the formula (IC) according to claim 14 which is selected from the group consisting of:

A method of preparing a compound of the formula (IB) according to claim 12, the method comprising:

a compound of the formula (IA) and

A nucleophilic substitution reaction occurs to give a compound of the formula (IB); among them: W is an amino protecting group; X is a halogen; S is 1 or 2; Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in claim 1. A method of preparing a compound of the formula (IC) according to claim 14, the method comprising:

a compound of the formula (IB) is ring-closed to give a compound of the formula (IC); among them: W is an amino protecting group; X is a halogen; S is 1 or 2; Rings A, G, X ¹ , L ¹ to L ² , R ¹ to R ⁵ and n are as defined in claim 1. A method of preparing a compound of the formula (I) according to claim 1, the method comprising:

Deprotection of a compound of formula (IC) to give a compound of formula (I); among them: W is an amino protecting group; 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 claim 1. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I) according to any one of claims 1 to 11, or a tautomer thereof, a mesogen thereof, The racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. The compound of the formula (I) according to any one of claims 1 to 11, or a tautomer, a mesogen, a racemate, an enantiomer or a diastereomer thereof Use of the compound, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 19, in the manufacture of a medicament for a TLR7 agonist. The compound of the formula (I) according to any one of claims 1 to 11, or a tautomer, a mesogen, a racemate, an enantiomer or a diastereomer thereof Or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19 for use in the manufacture of a medicament for treating an infection caused by a virus selected from the group consisting of: a dengue virus, Yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses. The compound of the formula (I) according to any one of claims 1 to 11, or a tautomer, a mesogen, a racemate, an enantiomer or a diastereomer thereof , or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 19, prepared for the treatment or prevention of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell Cancer, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia, ulcerative colitis, autoimmune disease, plaque psoriasis, systemic lupus erythematosus, actinic keratosis, and liver fibrosis use.

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