CN110317202B

CN110317202B - Cyanopyrroloheteroaryl derivative, preparation method and medical application thereof

Info

Publication number: CN110317202B
Application number: CN201910247232.XA
Authority: CN
Inventors: 张国宝; 陈友喜; 陈一千; 黄智明; 贺峰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2018-03-30
Filing date: 2019-03-29
Publication date: 2021-12-24
Anticipated expiration: 2039-03-29
Also published as: CN110317202A

Abstract

The invention relates to cyano-pyrrolo-heteroaryl derivatives, a preparation method and application thereof in medicines. Specifically, the invention relates to a novel cyanopyrrolo-heteroaryl derivative shown in a general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, in particular as a TLR7 agonist, wherein each substituent group of the general formula (I) is as defined in the specification.

Description

Cyanopyrroloheteroaryl derivative, preparation method and medical application thereof

Technical Field

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

Background

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

There are at least 10 different TLRs in mammals. Ligands for some of these receptors and the corresponding signal cascades have been identified. TLR7 is a member of the subset of TLRs (TLRs 3, 7, 8 and 9), restricted to the endosomal compartment of cells that specifically detect non-self nucleic acids. TLR7 plays a key role in antiviral defense through recognition of ssRNA (Diebold S.S. et al, Science,2004:303, 1529-. TLR7 has a limited expression profile in humans and is expressed predominantly by B cells and plasmacytoid dendritic cells (pdcs), and to a lesser extent by monocytes. Plasmacytoid DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% Peripheral Blood Mononuclear Cells (PBMCs)) that are the primary type I interferon-producing cells that secrete high levels of interferon-alpha (IFN α) and interferon-beta (IFN β) in response to viral infection (Liu Y-J, Annu. Rev. Immunol.,2005:23, 275-one 306).

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

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

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

Aiming at the technical problems, the invention provides the TLR7 agonist with a novel structure, which has the advantages of good selectivity (selectivity to TLR7 and no activation effect to TLR8) and obvious activation effect. Meanwhile, the compound has weak inhibition effect on CYP and is a safer and more effective TLR7 agonist.

Disclosure of Invention

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

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

wherein:

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

g is N or CR⁴；

X¹Is alkylene, wherein said alkylene is optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, and heterocyclyl;

L¹selected from-O-, -S-, -C (O) -, -NR⁵-、-C(O)-OR⁵、-S(O)_m-、-N(R⁵)C(O)-、-C(O)N(R⁵)-、 -N(R⁵)S(O)_m-、-S(O)_mN(R⁵) -and a covalent bond;

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

R²the same or different, and each is independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁶R⁷and-CH₂NR⁶R⁷Is substituted with one or more substituents of (1);

L²is alkyleneWherein said alkylene is optionally selected from the group consisting of alkyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR⁵or-NR⁶R⁷Is substituted with one or more substituents of (1);

R³selected from the group consisting of haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl and-NR⁶R⁷Wherein said cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R⁴selected from the group consisting of hydrogen atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cyano groups, amino groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R⁵selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R⁶and R⁷Are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

or, said R⁶And R⁷Together with the attached nitrogen atom, form a heterocyclic group, wherein said heterocyclic group optionally contains 1 to 2 identical or different heteroatoms selected from N, O and S in addition to 1 nitrogen atom, and said heterocyclic group is optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

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

m is 0,1 or 2.

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

In a preferred embodiment of the invention, the compound of formula (I) wherein ring a is aryl or heteroaryl, preferably phenyl or pyridyl.

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

wherein:

G¹n, C or CH;

L¹～L²、X¹、R¹～R³and n is as defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is³Is a heterocyclic group, said heterocyclic group being optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl; r³Preferably selected from pyrrolidinyl, morpholine, piperidinyl, piperazinyl or methylpiperazinyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is³is-NR⁶R⁷，R⁶And R⁷Together with the nitrogen atom to which they are attached form a heterocyclic group, wherein said heterocyclic group optionally contains 1 to 2 identical or different heteroatoms selected from N, O and S in addition to 1 nitrogen atom, and said heterocyclic group is optionally selected from alkyl, alkoxy, halogen, amino, cyanoAryl, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

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

In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein L is²Is 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 (III):

wherein:

G²and G³Is N, C or CH, and G²And G³Not N at the same time;

R⁶and R⁷Together with the attached nitrogen atom, form a heterocyclic group, wherein said heterocyclic group optionally contains 1 to 2 identical or different heteroatoms selected from N, O and S in addition to 1 nitrogen atom, and said heterocyclic group is optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

L¹、R¹、R²and n is as defined in formula (I).

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

wherein:

G²and G³Is N, C or CH, and G²And G³Not N at the same time;

L¹and R¹As defined in formula (I).

In a preferred embodiment of the present invention, the compound of formula (I) wherein L¹is-O-or-C (O) -.

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

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is²Are the same or different and are each independently a hydrogen atom or a halogen.

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

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

Another aspect of the present invention relates to a compound represented by the general formula (IA):

wherein:

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

g is N or CR⁴；

L¹selected from-O-, -S-, -C (O) -, -NR⁵-、-C(O)-OR⁵、-S(O)_m-、-N(R⁵)C(O)-、-C(O)N(R⁵)-、 -N(R⁵)S(O)₂-、-S(O)₂N(R⁵) -and a covalent bond;

R²the same or different, and each is independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, hydroxyalkyl, hydroxy, cyano, hydroxyCycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁶R⁷and-CH₂NR⁶R⁷Is substituted with one or more substituents of (1);

L²is an alkylene group, wherein said alkylene group is optionally selected from the group consisting of alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR⁵or-NR⁶R⁷Is substituted with one or more substituents of (1);

or, said R⁶And R⁷Together with the nitrogen atom to which they are attached form a heterocyclic group, wherein said heterocyclic group optionally contains 1 to 2 identical or different heteroatoms selected from N, O and S in addition to 1 nitrogen atom, and said heterocyclic group is optionally selected fromAlkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

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

m is 0,1 or 2.

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

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

reacting a compound of formula (IA) to give a compound of formula (I);

wherein:

ring A, G, L¹～L²、X¹、R¹～R³And n is as defined in formula (I).

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

The invention further relates to application of the compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition containing the compound in preparation of a medicament for exciting TLR 7.

The invention further relates to the use of a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for the preparation of a medicament for the treatment of an infection caused by a virus selected from the group consisting of: dengue virus, yellow fever virus, west nile virus, japanese encephalitis virus, tick-borne encephalitis virus, kunjin virus, murray valley encephalitis virus, saint louis encephalitis virus, ebosk hemorrhagic fever virus, bovine viral diarrhea virus, checa virus, HIV, HBV, HCV, HPV, RSV, SARS, and influenza virus.

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

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

The invention further relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound, which is used for agonizing the TLR 7.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for use in 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, ebosk hemorrhagic fever virus, bovine viral diarrhea virus, checa 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, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, 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, ulcerative colitis, and liver fibrosis.

The invention further relates to a method of agonizing TLR7, the method comprising administering to a patient in need thereof a therapeutically effective dose of a compound of formula (I) of the present invention or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The present invention further relates to a method of treating an infection caused by a virus, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, wherein the virus is selected from the group consisting of: dengue virus, yellow fever virus, west nile virus, japanese encephalitis virus, tick-borne encephalitis virus, kunjin virus, murray valley encephalitis virus, saint louis encephalitis virus, ebosk hemorrhagic fever virus, bovine viral diarrhea virus, checa virus, HIV, HBV, HCV, HPV, RSV, SARS, and influenza virus.

The present invention further relates to a method for treating or preventing melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, COPD, ulcerative colitis, 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, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

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

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

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

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

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

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

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

The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

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

Detailed description of the invention

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

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a packageStraight or branched chain groups containing 1 to 20 carbon atoms, preferably alkyl groups containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-ethyl, 2-2, 2-2, 2-2, or, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, arylHeteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH)₂-), 1-ethylidene (-CH (CH)₃) -), 1, 2-ethylene (-CH)₂CH₂) -, 1-propylene (-CH (CH)₂CH₃) -), 1, 2-propylene (-CH)₂CH(CH₃) -), 1, 3-propylene (-CH)₂CH₂CH₂-) 1, 4-butylene (-CH₂CH₂CH₂CH₂-) and the like. Alkylene groups may be substituted OR unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、 -NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

The term "alkenyl" refers to a hydrocarbon group having one or more fewer hydrogen atoms in the olefin molecule. The alkenyl group may be substituted OR unsubstituted, and when substituted, the substituent is preferably one OR more groups independently selected from hydrogen atom, alkyl group, alkoxy group, halogen, haloalkyl group, hydroxyl group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents of (1);

the term "alkynyl" refers to a hydrocarbon containing a carbon-carbon triple bond in the molecule. Alkynyl groups may be substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

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

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

Such heterocyclyl rings include those wherein the above-described heterocyclyl groups (e.g., spiroheterocyclyl, fused heterocyclyl, and bridged heterocyclyl) are fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclyl group, non-limiting examples of which include:

the heterocyclyl group may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

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

aryl groups may be substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

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

heteroaryl may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR⁵、-C(O)R⁵、-S(O)_mR⁵、-NR⁶R⁷and-C (O) NR⁶R⁷Is substituted with one or more substituents.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more substituents independently selected from halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

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

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

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

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

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

The term "oxo" refers to ═ O.

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

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

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

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

m and R⁵～R⁷As defined for the compounds of general formula (I).

Synthesis of the Compounds of the invention

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

scheme one

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

in the first step, reacting a compound of the general formula (I-1) with a compound of the general formula (I-2) under alkaline conditions to obtain a compound of the general formula (I-3);

secondly, the compound of the general formula (I-3) is put into ammonia methanol solution for tube sealing reaction to obtain the compound of the general formula (IA);

thirdly, reacting the compound of the general formula (IA) with phosphorus oxychloride to obtain a compound of a general formula (I);

wherein:

x is halogen, preferably chlorine;

R^ais alkyl, preferably ethyl or methyl;

ring A, G, L¹～L²、X¹、R¹～R³And n is as defined in formula (I).

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

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

Scheme two

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

in the first step, a compound of a general formula (II-1) and a compound of a general formula (II-2) react under alkaline conditions to obtain a compound of a general formula (II-3);

secondly, the compound of the general formula (II-3) is put into ammonia methanol solution to be sealed and reacted to obtain the compound of the general formula (IIA);

thirdly, reacting the compound of the general formula (IIA) with phosphorus oxychloride to obtain a compound of a general formula (II);

wherein:

x is halogen, preferably chlorine;

R^ais alkyl, preferably ethyl or methyl;

G¹、L¹～L²、X¹、R¹～R³and n is as defined in formula (II).

Scheme three

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

in the first step, reacting a compound of a general formula (II-1) with a compound of a general formula (III-1) under alkaline conditions to obtain a compound of a general formula (III-2);

secondly, the compound of the general formula (III-2) is put into ammonia methanol solution to be sealed and reacted to obtain the compound of the general formula (IIIA);

thirdly, reacting the compound of the general formula (IIIA) with phosphorus oxychloride to obtain a compound of a general formula (III);

wherein:

x is halogen, preferably chlorine;

R^ais alkyl, preferably ethyl or methyl;

L¹、R¹、R²、R⁶、R⁷and n is as defined in formula (III).

Scheme four

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

in the first step, reacting a compound of a general formula (II-1) with a compound of a general formula (IV-1) under alkaline conditions to obtain a compound of a general formula (IV-2);

secondly, the compound of the general formula (IV-2) is in ammonia methanol solution, and the compound of the general formula (IVA) is obtained by tube sealing reaction;

thirdly, reacting the compound of the general formula (IVA) with phosphorus oxychloride to obtain a compound of a general formula (IV);

wherein:

x is halogen, preferably chlorine;

R^ais alkyl, preferably ethyl or methyl;

L¹、R¹、R²、R⁶、R⁷and n is as defined in formula (IV).

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

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

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

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

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

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

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

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

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

Average inhibition rate of kinase and IC₅₀The value was measured with a NovoStar microplate reader(BMG, Germany).

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

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

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

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

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

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

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

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

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

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

Example 1

4-amino-2-butoxy-7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile

First step of

2-butoxy-4, 6-dichloropyrimidine-5-carbaldehyde 1b

Phosphorus oxychloride (6.24g, 40.70mmol) was added to 75mL of dichloromethane, the temperature was cooled to 0 ℃ in an ice bath, N-dimethylformamide (2.98g, 40.70mmol) was slowly added, and after the addition, the reaction was stirred for 1 hour, 2-butoxypyrimidine-4, 6-diol 1a (6.24g, 40.70mmol, prepared by the method disclosed in the patent application "WO 201644183") was added, and the reaction was stirred at room temperature for 20 hours. The reaction solution is decompressed and concentrated, 50mL of phosphorus oxychloride is added into the residue, the temperature is reduced to 0 ℃ in an ice bath, N-diisopropylethylamine (6.99g, 54.20mmol) is added dropwise, and the temperature is raised to 60 ℃ after the addition, and the reaction is stirred for 2 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, the resulting residue was poured into 50mL of water, extracted with ethyl acetate (40 mL. times.3), the organic phases were combined, the organic phase was washed with a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 1b (1.8g, yield: 26.6%).

MS m/z(ESI):250.2[M+1]

Second step of

2- ((2-butoxy-6-chloro-5-formylpyrimidin-4-yl) - (4- (pyrrolidin-1-ylmethyl) benzyl) amino) acetic acid ethyl ester 1d

1b (300mg, 1.20mmol) was added to 5mL tetrahydrofuran, ethyl 2- ((4-pyrrolidin-1-ylmethyl) benzyl) amino) acetate 1c (366mg, 1.33mmol, prepared as disclosed in patent application "US 20160075707") was added, triethylamine (182mg, 1.81mmol) was added and the mixture was stirred at room temperature for 2 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), and the organic phases were combined, washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 1d (500mg, yield: 68.3%).

The third step

4-amino-2-butoxy-7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 1e

1d (300mg, 0.62mmol) was added to 5mL of 7M methanolic ammonia, and the reaction was stirred for 16 hours at 90 ℃ with the tube sealed. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of water was added to the resulting residue, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography developer system A to give the title product 1e (70mg, yield: 27.1%).

The fourth step

4-amino-2-butoxy-7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 1

1e (70mg, 0.166mmol) was added to 5mL of phosphorus oxychloride and the reaction mixture was warmed to 80 ℃ for 2 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and to the resulting residue was added 10mL of a saturated sodium bicarbonate solution, extracted with ethyl acetate (20 mL. times.3), the organic phases were combined, washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 1(30mg, yield: 44.8%).

MS m/z(ESI):405.5[M+1]

¹H NMR(400MHz,CD₃OD)δ7.32-7.34(m,2H),7.25-7.27(m,2H),7.24(s, 1H),5.36(s,2H),4.30-4.34(t,2H),3.79(s,2H),2.70-2.73(m,4H),1.81-1.84(m, 4H),1.67-1.75(m,2H),1.42-1.48(m,2H),0.92-0.95(m,3H).

Example 2

4-amino-2-butoxy-7- (4- (morpholinomethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 2

First step of

4- (Morpholinylmethyl) benzonitrile 2b

4- (bromomethyl) benzonitrile 2a (2.0g, 10.20mmol, prepared by the well-known method "Journal of Organic Chemistry,2014,79(1), 223-. The reaction solution was poured into 50mL of water, extracted with ethyl acetate (40 mL. times.3), the organic phases were combined, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 2B (1.5g, yield: 72.7%).

MS m/z(ESI):203.2[M+1]

Second step of

(4- (Morpholinylmethyl) phenyl) methylamine 2c

2b (1.5g,7.42mmol) was added to 30mL tetrahydrofuran, cooled in ice water, lithium aluminum hydride (563mg, 14.83mmol) was added in portions, and after the addition was completed, the mixture was warmed to room temperature and stirred for 2 hours. To the reaction solution was added 20mL of saturated brine, extracted with ethyl acetate (50 mL. times.3), the organic phases were combined, washed with water (50mL), saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to give the crude title product 2c (1.02g, yield: 68.3%) which was directly subjected to the next reaction without purification.

MS m/z(ESI):207.2[M+1]

The third step

2- ((4- (Morpholinylmethyl) benzyl) amino) acetic acid ethyl ester 2d

Crude 2c (1.0g, 4.85mmol) was added to 20mL tetrahydrofuran, ethyl bromoacetate (810 mg, 4.85mmol) was added, triethylamine (981mg, 9.7mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction solution was concentrated under reduced pressure, 30mL of water was added to the system, extraction was performed with methylene chloride (30 mL. times.3), the organic phases were combined, the organic phases were washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluent system B to obtain the title product 2d (1.42g, yield: 69.1%).

MS m/z(ESI):293.2[M+1]

The fourth step

Ethyl 2- ((2-butoxy-6-chloro-5-formylpyrimidin-4-yl) (4- (morpholinomethyl) benzyl) amino) acetate 2e

1B (249mg, 1.0mmol) was added to 5mL of tetrahydrofuran, 2d (292mg, 1.0mmol) was added, triethylamine (101mg, 1.0mmol) was added and stirred at room temperature for 2 hours, 20mL of water was added to the reaction solution, extraction was performed with dichloromethane (20 mL. times.3), the organic phases were combined, the organic phases were washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 2e (382mg, yield: 75.7%).

MS m/z(ESI):505.2[M+1]

The fifth step

4-amino-2-butoxy-7- (4- (morpholinomethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 2f

2e (350mg, 0.69mmol) was added to 10mL of 7M methanolic ammonia and the reaction stirred at 90 ℃ for 16 h. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and 10mL of water was added to the system, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), a saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin layer chromatography using developer system A to obtain the title product 2f (35mg, yield: 11.5%).

MS m/z(ESI):439.2[M+1]

The sixth step

2f (30mg, 0.048mmol) was added to 5mL of chloroform, phosphorus oxychloride (32mg, 0.21 mmol) was added, the temperature of the reaction solution was raised to 80 ℃ and the reaction was stirred for 3 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of a saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane (20 mL. times.3), the organic phases were combined, washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin layer chromatography using developer system A to give the title product 2(15mg yield: 52.1%).

MS m/z(ESI):421.5[M+1]

¹H NMR(400MHz,CDCl₃)δ7.35-7.39(m,2H),7.27-7.28(m,2H),7.24(s,1H), 5.39(s,2H),4.36-4.39(t,2H),3.77(s,2H),2.57-2.49(m,4H),1.76-1.80(m, 4H),1.49-1.52(m,2H),1.24-1.26(m,2H),0.95-0.99(m,3H).

Example 3

4-amino-2-butoxy-7- (4- ((4-methylpiperazin-1-yl) methyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 3

First step of

4- ((4-methylpiperazin-1-yl) methyl) benzonitrile 3a

2a (1.96g, 10.00mmol) was added to 20mL of chloroform, N-methylpiperazine (1.72g, 17.20 mmol) was added dropwise, and the reaction was stirred at room temperature for 4 hours after the addition. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 3a (1.7g, yield: 79.0%).

MS m/z(ESI):216.3[M+1]

Second step of

(4- ((4-methylpiperazin-1-yl) methyl) phenyl) methylamine 3b

3a (1.6g, 7.43mmol) was added to 30mL of tetrahydrofuran, cooled in ice water, lithium aluminum hydride (564mg, 14.86mmol) was added in portions, and the reaction was stirred at room temperature for 3 hours after the addition was completed. To the reaction mixture was added 20mL of saturated brine, extracted with ethyl acetate (50 mL. times.3), the organic phases were combined, washed with water (50mL), saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to give the title product 3b (1.1g, yield: 67.5%).

MS m/z(ESI):220.2[M+1]

The third step

Ethyl 2- ((4- ((4-methylpiperazin-1-yl) methyl) benzyl) amino) acetate 3c

3b (1.8g, 8.21mmol) was added to 20mL of tetrahydrofuran, ethyl bromoacetate (1.51g, 9.03mmol) and triethylamine (1.3g, 12.81mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the reaction mixture was concentrated under reduced pressure, 30mL of water was added to the system, extraction was performed with methylene chloride (30 mL. times.3), the organic phases were combined, the organic phases were washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 3c (920mg, yield: 36.7%).

MS m/z(ESI):306.2[M+1]

The fourth step

Ethyl 2- ((4- ((4-methylpiperazin-1-yl) methyl) benzyl) amino) acetate 3d

1b (500mg, 2.01mmol) was added to 20mL of tetrahydrofuran, 3e (613mg, 2.01mmol) was added, and triethylamine (305mg, 3.01mmol) was added and the mixture was stirred at room temperature for 2 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), and the organic phases were combined, washed with water (30mL), saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 3d (652mg, yield: 62.7%).

The fifth step

4-amino-2-butoxy-7- (4- ((4-methylpiperazin-1-yl) methyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 3e

3d (600mg, 1.16mmol) was added to 10mL of 7M methanolic ammonia and the reaction was stirred for 16 hours at 90 ℃ with a sealed tube. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and 10mL of water was added to the system, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 3e (50mg, yield: 9.6%).

MS m/z(ESI):439.2[M+1]

The sixth step

3e (50mg, 110.73. mu. mol) was added to 5mL of phosphorus oxychloride, the temperature of the reaction solution was raised to 80 ℃ and the reaction was stirred for 2 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of an ice saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane (20 mL. times.3), the organic phases were combined, washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 3(32mg yield: 66.7%).

MS m/z(ESI):434.6[M+1]

¹H NMR(400MHz,DMSO-d₆)δ7.56(s,1H),7.26-7.28(m,2H),7.16-7.18(m, 2H),5.30(s,2H),4.22-4.25(t,2H),3.45(s,2H),3.33(s,3H)2.74-2.85(m,4H), 2.43-2.47(m,4H),1.61-1.68(m,2H),1.35-1.41(m,2H),0.88-0.92(m,3H).

Example 4

4-amino-2-butoxy-7- ((5- (pyrrolidin-1-ylmethyl) pyridin-2-yl) methyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 4

First step of

5- (pyrrolidin-1-ylmethyl) pyridine-2-carbonitrile 4b

6- (bromomethyl) pyridine-2-carbonitrile 4a (1.6g, 8.12mmol, prepared by the well-known method "Organic Letters,2017,19(14), 3895-3898") was added to 20mL of THF, triethylamine (1.23g, 12.18mmol) was added, tetrahydropyrrole (1.19g, 16.70mmol) was added, and the reaction was carried out at room temperature for 4 hours. To the reaction solution was added 10mL of water, followed by extraction with dichloromethane (30 mL. times.3), and the organic phases were combined, washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 4B (1.0g, yield: 65.8%).

MS m/z(ESI):188.3[M+1]

Second step of

(5- (pyrrolidin-1-ylmethyl) pyridin-2-yl) methylamine 4c

4b (1.0g, 5.34mmol) was added to 20mL of methanol, palladium on carbon (300mg, 10%) was added, 6N hydrogen chloride solution (2mL) was added, the mixture was replaced with hydrogen three times, and the reaction was carried out at room temperature under hydrogen atmosphere for 72 hours. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to give the crude title product 4c (1.2g), which was directly used in the next reaction without purification.

MS m/z(ESI):192.4[M+1]

The third step

2- (((5- (pyrrolidin-1-ylmethyl) pyridin-2-yl) methyl) amino) acetic acid ethyl ester 4d

Crude 4c (1.2g, 6.26mmol) was added to 40mL tetrahydrofuran, triethylamine (953mg, 9.41mmol) was added, ethyl bromoacetate (1.1mg, 6.6mmol) was added dropwise, and after the addition was completed, the reaction was carried out at room temperature for 2 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), and the organic phases were combined, washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 4d (1.01 mg, yield: 71.9%).

MS m/z(ESI):278.4[M+1]

The fourth step

Ethyl 2- ((2-butoxy-6-chloro-5-formylpyrimidin-4-yl) (5- (pyrrolidin-1-ylmethyl) pyridin-2-yl) methyl) amino) acetate 4e

1b (500mg, 2.01mmol) was added to 20mL of tetrahydrofuran, 4d (557mg, 2.01mmol) was added, and triethylamine (305mg, 3.01mmol) was added and the mixture was stirred at room temperature for 2 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), and the organic phases were combined, washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 4e (389mg, yield: 39.6%).

The fifth step

4-amino-2-butoxy-7- ((5- (pyrrolidin-1-ylmethyl) pyridin-2-yl) methyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 4f

4e (370mg, 0.76mmol) was added to 10mL of 7M methanolic ammonia and the reaction stirred at 100 ℃ for 16 h. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and 10mL of water was added to the system, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), a saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin layer chromatography using developer system A to obtain the title product 4f (31mg, yield: 9.7%).

MS m/z(ESI):424.5[M+1]

The sixth step

4f (45mg, 0.11mmol) was added to 5mL of phosphorus oxychloride, the reaction mixture was warmed to 80 ℃ and stirred for 3 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, 10mL of a saturated sodium bicarbonate solution was added to the residue, extraction was performed with methylene chloride (10 mL. times.3), the organic phases were combined, washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin layer chromatography using developer system A to obtain the title product 4(15mg, yield: 34.8%).

MS m/z(ESI):406.5[M+1]

¹H NMR(400MHz,DMSO-d₆)δ7.74-7.79(m,2H),7.57(s,1H),7.14-7.20(m, 1H),,5.42(s,2H),4.16-4.19(t,2H),3.38(s,2H),2.28-2.38(m,4H),1.72-1.84(m, 4H),1.58-1.61(m,2H),1.32-1.37(m,2H),0.86-0.89(m,3H).

Example 5

4-amino-2-butoxy-7- ((6- (pyrrolidin-1-ylmethyl) pyridin-3-yl) methyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 5

First step of

6- (pyrrolidin-1-ylmethyl) pyridine-3-carbonitrile 5b

5a (4.0g, 20.30mmol, prepared by the well-known method "Journal of the American Chemical Society,2010,132(33), 11389-1139") was dissolved in tetrahydrofuran (30mL), triethylamine (3.08g, 30.45mmol) was added, tetrahydropyrrole (1.98g, 20.30mmol) was added, and the reaction was stirred at room temperature for 20 hours. To the system was added 50mL of water, extraction was performed with dichloromethane (30 mL. times.3), the organic phases were combined, the organic phases were washed with water (50mL), a saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 5B (1.5g, yield: 39.5%).

MS m/z(ESI):188.2[M+1]

Second step of

(6- (pyrrolidin-1-ylmethyl) pyridin-3-yl) methylamine 5c

5b (700mg, 3.74mmol) was added to 15mL of methanol, palladium on carbon (400mg, 10%) was added to 6N hydrogen chloride solution (1.5mL), and the mixture was replaced with hydrogen three times and reacted at room temperature under hydrogen atmosphere for 16 hours. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to give the title product 5c (730mg) which was carried on to the next reaction without purification.

MS m/z(ESI):192.4[M+1]

The third step

2- (((6- (pyrrolidin-1-ylmethyl) pyridin-3-yl) methyl) amino) acetic acid ethyl ester 5d

The crude product 5c (260mg, 1.36mmol) was added to THF (30mL), N-diisopropylethylamine (688mg, 6.80mmol) was added, ethyl bromoacetate (227mg, 1.36mmol) was added, and the reaction was carried out at room temperature for 24 hours after the addition. To the system was added 10mL of water, extraction was performed with dichloromethane (20 mL. times.3), the organic phases were combined, the organic phase was washed successively with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 5d (212mg, yield: 56.2%).

MS m/z(ESI):278.5[M+1]

The fourth step

Ethyl 2- ((2-butoxy-6-chloro-5-formylpyrimidin-4-yl) ((6- (pyrrolidin-1-ylmethyl) pyridin-3-yl) methyl) amino) acetate 5e

1b (150mg, 0.60mmol) and 5d (167mg, 0.60mmol) were added to THF (10mL), N-diisopropylethylamine (91mg, 0.90mmol) was added, and after completion of addition, the reaction was carried out at room temperature for 24 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), and the organic phases were combined, washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 5e (165 mg, yield: 55.9%).

The fifth step

4-amino-2-butoxy-7- ((6- (pyrrolidin-1-ylmethyl) pyridin-3-yl) methyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 5f

5e (160mg, 0.33mmol) was added to 10mL of 7M methanolic ammonia and the reaction stirred at 100 ℃ for 16 h. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and 10mL of water was added to the residue, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 5f (56mg, yield: 40.5%).

MS m/z(ESI):424.5[M+1]

The sixth step

5f (50mg, 0.12mmol) was added to 5mL of phosphorus oxychloride, the reaction solution was warmed to 80 ℃ and stirred for 3 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of a saturated sodium bicarbonate solution was added to the residue, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (30mL), a saturated sodium chloride solution (30mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 5(2mg yield: 2.2%).

MS m/z(ESI):406.6[M+1]

¹H NMR(400MHz,DMSO-d₆)δ7.62-7.66(m,2H),7.46(s,1H),7.18-7.22(m, 1H),5.43(s,2H),4.17-4.20(t,2H),3.33(s,2H),2.20-2.34(m,4H),1.63-1.78(m, 4H),1.58-1.61(m,2H),1.24-1.32(m,2H),0.87-0.91(m,3H).

Example 6

4-amino-2-pentanoyl-7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carbonitrile 6

First step of

2-Hydroxyiminocarboxylic acid ethyl ester hydrochloride 6b

2-Hydroxyhexanenitrile 6a (4.27g, 37.74mmol, prepared as disclosed in the patent application "CN 105924353") was dissolved in 60mL of diethyl ether, ethanol (1.91g, 41.51mmol) was added, a solution of hydrogen chloride in dioxane (4M, 94.34mL) was added dropwise at 0 ℃ and reacted for 5 hours at 0 ℃. The reaction was concentrated under reduced pressure to give the crude title product 6b (6.8g, yield: 92.1%) which was directly subjected to the next reaction without purification.

MS m/z(ESI):160.2[M+1]

Second step of

2-Hydroxyhexamethyleneimine amide 6c

Crude 6b (6.80g, 34.75mmol) was added to methanolic ammonia (7M, 18.72mL) and stirred at room temperature for 20 h. The reaction solution was concentrated under reduced pressure to give the crude title product 6c (5.5g, yield: 95.0%) which was directly subjected to the next reaction without purification.

MS m/z(ESI):131.2[M+1]

The third step

2- (1-hydroxypentyl) pyrimidine-4, 6-diol 6d

Crude 6c (4.8g, 28.80mmol) was dissolved in 100mL of ethanol, diethyl malonate (4.61g, 28.80mmol) was added, sodium ethoxide (5.88g, 86.41mmol) was added, and the reaction was heated under reflux for 20 hours. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and 50mL of water and concentrated hydrochloric acid were added to the residue to adjust the pH to 3, whereby a solid precipitated, filtered, the filter cake was washed with water (20mL × 3), and the filter cake was dried to obtain the title product 6d (3.9g, yield: 68.3%).

MS m/z(ESI):199.4[M+1]

The fourth step

1- (4, 6-dihydroxypyrimidin-2-yl) pentylacetate 6e

6d (3.0g, 15.13mmol) was added to 20mL of acetic acid, 20mL of acetic anhydride was added, the reaction solution was warmed to 105 ℃ and stirred for 2 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was taken up in toluene and concentrated under reduced pressure with the residual solvent to give the crude title product 6e (3.6g yield: 98.9%) which was subjected to the next reaction without purification.

MS m/z(ESI):241.4[M+1]

The fifth step

1- (5-formyl-4, 6-dihydroxypyrimidin-2-yl) pentylacetate 6f

The crude 6e (500mg, 2.08mmol) was added to 30mL tetrahydrofuran, the temperature of the reaction was cooled to 0 deg.C, N-dimethylformamide (228mg, 3.12mmol) was added, phosphorus oxychloride (479mg, 3.12mmol) was added dropwise, the temperature was raised to room temperature after completion of the addition, and the reaction was stirred for 20 hours. The reaction was concentrated under reduced pressure to give the crude title product 6f (700mg), which was directly used in the next reaction without purification.

The sixth step

1- (4, 6-dichloro-5-formylpyrimidin-2-yl) pentylacetate 6g

Crude 6f (500mg, 1.86mmol) was added to 5mL of phosphorus oxychloride, cooled in an ice bath, N-ethyldiisopropylamine (481mg, 3.73mmol) was added dropwise in the ice bath, the temperature of the reaction was raised to 80 ℃ and the reaction was stirred for 2 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of water was added to the reaction solution, extracted with ethyl acetate (20 mL. times.3), the organic phases were combined, washed with water (50mL), saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain 6g of the title product (212mg, yield: 37.3%).

Seventh step

Ethyl 2- ((2- (1-acetoxypentyl) -6-chloro-5-formylpyrimidin-4-yl) (4- (pyrrolidin-1-ylmethyl) benzyl) amino) acetate 6h

6g (200mg, 0.66mmol) was added to THF (10mL), N-diisopropylethylamine (99mg, 0.98mmol) and 1c (182mg, 0.66mmol) were added, and the mixture was stirred at room temperature for 20 hours. To the reaction solution was added 20mL of water, followed by extraction with dichloromethane (20 mL. times.3), the organic phases were combined, washed with water (50mL), saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered to remove the drying agent, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 6h (213mg, yield: 59.6%).

MS m/z(ESI):545.6[M+1]

Eighth step

4-amino-2- (1-hydroxypentyl) -7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 6i

6h (850mg, 1.56mmol) were added to 10mL of 7M methanolic ammonia and the reaction was stirred for 16h at 100 ℃ with a sealed tube. The reaction solution was cooled to room temperature, concentrated under reduced pressure, 10mL of water was added to the system, extracted with dichloromethane (10 mL. times.3), the organic phases were combined, washed with water (20mL), saturated sodium chloride solution (20mL) successively, dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by high performance liquid chromatography (Waters-2767, elution system: 10mmoL/L ammonium hydrogencarbonate, water, acetonitrile) to give the title product 6i (120mg, yield: 17.6%).

MS m/z(ESI):437.6[M+1]

The ninth step

4-amino-2-pentanoyl-7- (4- (pyrrolidin-1-ylmethyl) benzyl) -7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide 6j

6i (110mg, 0.25. mu. mol) was added to 10mL of methylene chloride, manganese dioxide (219mg, 2.52mmol) was added, and the reaction was carried out at room temperature for 20 hours. The reaction solution was filtered to remove excess manganese dioxide, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system A to give the title product 6j (20mg, yield: 18.3%).

MS m/z(ESI):435.6[M+1]

The tenth step

6j (20mg, 0.046. mu. mol) was added to 5mL of phosphorus oxychloride, the reaction solution was warmed to 100 ℃ and the reaction was stirred for 3 hours. The reaction solution was concentrated under reduced pressure, 10mL of a saturated sodium bicarbonate solution was added to the reaction solution, extraction was performed with ethyl acetate (20 mL. times.3), the organic phases were combined, washed with water (20mL), a saturated sodium chloride solution (20mL), dried over anhydrous magnesium sulfate, the drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin layer chromatography with developer system A to obtain the title product 6(8mg yield: 41.7%).

MS m/z(ESI):417.6[M+1]

¹H NMR(400MHz,CD₃OD)δ7.50-7.52(m,2H),7.40-7.44(m,3H),5.64(s, 2H),4.29(s,2H),2.02-2.05(m,4H),1.68-1.71(t,2H)，1.39-1.43(t,2H),1.28-1.32(m, 6H),0.94-0.97(m,3H).

Test example:

biological evaluation

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

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

first, experimental material and instrument

1.DMEM(Gibco，10564-029),

2. Fetal bovine serum (GIBCO,10099),

3. penicillin streptomycin (Gibco,15140-122),

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

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

6.HEK-Blue^TMHTLR7 cell line (InvivoGen, hkb-hTLR7),

HEK-Blue detection reagent (InvivoGen, hb-det 3).

Second, the experimental procedure

Preparing a HEK-Blue detection culture medium, taking a bag of HEK-Blue detection dry powder, adding 50ml of endotoxin-removed water for dissolving, placing in an incubator at 37 ℃, and carrying out sterile filtration after 10 minutes. Preparing a 20mM stock solution by using the compound; then diluted with pure DMSO to a maximum concentration of 6X 10⁶nM, diluted with a 3-fold gradient, for 10 dots.

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

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

The compound of the invention can activate human TLR7 through the aboveTest determination, EC determined₅₀The values are shown in Table 1.

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

Example numbering	EC₅₀(nM)	Emax(％)
			1	45	99
2	89	89
			3	131	83
4	143	84
			5	104	99

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

Test example 2 determination of the Activity of the Compounds of the present invention on human TLR8 agonist

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

first, experimental material and instrument

1.DMEM(Gibco，10564-029),

2. Fetal bovine serum (GIBCO,10099),

3. penicillin streptomycin (Gibco,15140-122),

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

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

6.HEK-Blue^TMHTLR8 cell line (InvivoGen, hkb-hTLR8),

HEK-Blue detection reagent (InvivoGen, hb-det 3).

Second, the experimental procedure

Preparing a HEK-Blue detection culture medium, taking a bag of HEK-Blue detection dry powder, adding 50ml of endotoxin-removed water for dissolving, placing in an incubator at 37 ℃, and carrying out sterile filtration after 10 minutes. Preparing a 20mM stock solution by using the compound; then diluted with pure DMSO to a maximum concentration of 6X 10⁶nM, then 3-fold gradient dilution, 10 points total; the compounds were diluted 20-fold with medium and then 20. mu.l of the diluted compounds were added to each well.

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

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

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

Example numbering	EC₅₀(μM)	Emax(％)
			1	>10	32
2	>30	0
			3	>30	21
4	>13	11

And (4) conclusion: the compound has weak activation effect on human TLR8, which indicates that the compound has high selectivity on TLR 7.

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

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

first, experimental material and instrument

1.RPMI 1640(Invitrogen,11875),

2.FBS(Gibco,10099-141),

3. Penicillin streptomycin (Gibco,15140-122),

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

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

6.SepMateTM-50(Stemcell,15460),

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

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

a PHERAStar multifunctional microplate reader (BMG, PHERAStar).

Second, the experimental procedure

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

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

The cell culture plate was placed in a centrifuge at 1200rpm and centrifuged for 10 minutes at room temperature. 150 μ l of supernatant was removed per well. Firstly, balancing the reagent in the human IFN-alpha kit to normal temperature, and preparing the anti-IFN-alpha-Eu according to the kit instruction under the condition of keeping out of the sun³⁺Cryptate (Anti-IFN-. alpha. -Eu)³⁺Cryptate conjugate) and Anti-IFN- α -d2-conjugate (Anti-IFN- α -d2-conjugate), both at a ratio of 1: 40 was mixed with binding Buffer (conjugate Buffer). Then 16. mu.l of the supernatant obtained by centrifugation was added to each well. Then 2 mul of freshly prepared anti-IFN-alpha-Eu are added into each hole³⁺The cryptate and the anti-IFN-alpha-d 2-conjugate were mixed by shaking and incubated for 3h at room temperature in the dark.

Readings were taken on a PHERAStar using HTRF mode. We define the minimum drug concentration that stimulates cytokine levels above the minimum detection limit by at least 3-fold, as the mec (minimum Effective concentration) value of the compound on the cytokine stimulation assay.

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

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

Example numbering	MEC(nM)
		1	3.7
2	0.4
		3	3
4	4.4

And (4) conclusion: from the data on the IFN-alpha secretion activity from PBMCs, the compounds of the present invention have the advantage of lower onset concentrations.

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

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

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630),

3. human liver microsomes (Corning Gentest),

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

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

CYP probe substrate (midazolam/10 μ M) and positive control inhibitor (ketoconazole).

Second, the experimental procedure

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

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

The value is calculated by Graphpad Prism to obtain the IC of the drug on CYP3A4 midazolam metabolic site₅₀The value is obtained.

IC of the compound of the invention on the midazolam metabolic site of human liver microsome CYP3A4₅₀The value is obtained.

Example numbering	IC₅₀(μM)
		4	15
6	17

And (4) conclusion: the compound has weak inhibition effect on the metabolic site of midazolam of human liver microsome CYP3A4, shows better safety, and prompts that the metabolic drug interaction based on the metabolic site of CYP3A4 metabolic midazolam does not occur.

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

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

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630),

3. human liver microsomes (Corning Gentest),

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

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

CYP probe substrate (dextromethorphan/10 μ M), and positive control inhibitor (quinidine).

Second, the experimental procedure

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

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

The value is calculated by Graphpad Prism to obtain the IC of the drug on the CYP2D6 metabolic site₅₀The value is obtained.

IC without inhibitory effect of the compound on human liver microsomal CYP2D6₅₀The value is obtained.

Example numbering	IC₅₀(μM)
		1	6
2	>30
		3	16
4	>30
		6	13

And (4) conclusion: the compound of the invention has weak inhibition effect on the enzyme activity of human liver microsome CYP2D6, shows better safety, and indicates that metabolic drug interaction based on CYP2D6 does not occur.

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

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

first, experimental material and instrument

1. A Phosphate Buffered Saline (PBS),

2.NADPH(Sigma N-1630),

3. human liver microsomes (Corning Gentest),

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

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

CYP probe substrate (testosterone/100 μ M) and positive control inhibitor (ketoconazole).

Second, the experimental procedure

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

The value is calculated by Graphpad Prism to obtain the IC of the drug on the metabolic site of CYP3A4 testosterone₅₀The value is obtained.

IC of the Compounds of the invention on the site of testosterone metabolism in human liver microsomal CYP3A4₅₀The value is obtained.

And (4) conclusion: the compound of the invention has weak inhibition on testosterone metabolic sites of human liver microsome CYP3A4, and shows better safety.

Test example 7

1. Purpose of experiment

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

2. Experimental methods

2.1 Experimental materials and instruments

2.1.1 Experimental materials:

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

2.1.2 Experimental instruments:

2.2 full-automatic Patch Clamp test procedure

HEK293-hERG stable cell lines were subcultured at a density of 1:4 in MEM/EBSS medium (10% FBS, 400. mu.g/ml G418, 1% MEM non-essential amino acid solution (100X), 1% sodium pyruvate solution) and cultured for a full-automatic patch clamp experiment within 48-72 hours. Cells were used 0 on the day of the experiment.After 25% pancreatin, the cells were harvested by centrifugation and washed with extracellular fluid (140mM NaCl, 4mM KCl, 1mM MgCl)₂， 2mM CaCl₂5mMD dextrose monohydrate, 10mM Hepes, pH7.4,298mOsmol) were resuspended in the cells to prepare a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument, which applied the cells to the chip (NPC-16) using a negative pressure controller, which draws individual cells to the wells of the chip. After the whole cell mode is formed, the apparatus will obtain hERG current according to the set hERG current voltage program, and then the apparatus automatically carries out compound perfusion from low concentration to high concentration. The current at each concentration of compound and the blank control current were analyzed by data analysis software supplied by HEAK Patchmaster, HEAK EPC10 patch clamp amplifier (Nanion) and pathlersoft ware and Pathcontrol HTsoftware.

2.3 test results

The blocking effect of the compound of the present invention on hERG potassium current was measured by the above test, and the IC was determined₅₀The values are shown in Table 4.

TABLE 4 IC of hERG potassium current blockade by the compounds of the invention₅₀

Example numbering	IC₅₀(μM)
		1	7
2	4
		3	17
4	6

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

Claims

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

or a pharmaceutically acceptable salt thereof,

wherein:

G¹n, C or CH;

X¹is C_1-6An alkylene group;

L¹selected from the group consisting of-O-, -S-, and-C (O) -;

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

R²are the same or different and are each independently a hydrogen atom or a halogen;

L²is C_1-6An alkylene group;

R³is a 5 or 6 membered heterocyclyl, said 5 or 6 membered heterocyclyl being optionally selected from C_1-6Alkyl radical, C_1-6Alkoxy, halogen, amino, cyano, nitro, hydroxy and C_1-6Substituted with one or more substituents in hydroxyalkyl; and is

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

2. A compound of formula (II) according to claim 1, wherein R³Selected from pyrrolidinyl, morpholine, piperidinyl, piperazinyl or methylpiperazinyl.

3. A compound of formula (II) according to claim 1 or 2, wherein L¹is-O-or-C (O) -.

4.A compound of formula (II) according to claim 1 or 2, wherein R¹Is C_1-6An alkyl group.

5. A compound of formula (II) according to claim 1 or 2, selected from:

6. a compound of formula (IIA):

or a pharmaceutically acceptable salt thereof,

wherein:

G¹n, C or CH;

X¹is C_1-6An alkylene group;

L¹selected from the group consisting of-O-, -S-, and-C (O) -;

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

L²is C_1-6An alkylene group;

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

7. A compound of formula (IIA) according to claim 6, selected from:

8. a process for preparing a compound of formula (II), the process comprising:

reacting the compound of the general formula (IIA) to obtain a compound of a general formula (II);

wherein:

G¹、L¹、L²、X¹、R¹to R³And n is as defined in claim 1.

9. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

10. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9, in the manufacture of a medicament for agonizing TLR 7.

11. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9, 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, ebosk hemorrhagic fever virus, bovine viral diarrhea virus, checa virus, HIV, HBV, HCV, HPV, RSV, SARS, and influenza virus.

12. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9, in the manufacture of a medicament for the treatment or prophylaxis 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.