CN111217816A

CN111217816A - FLT3 kinase inhibitor and preparation and application thereof

Info

Publication number: CN111217816A
Application number: CN201911157675.6A
Authority: CN
Inventors: 段文虎; 丁健; 陈运; 谢华; 张惠斌; 周金培
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2018-11-27
Filing date: 2019-11-22
Publication date: 2020-06-02
Anticipated expiration: 2039-11-22
Also published as: CN111217816B

Abstract

The invention relates to a FLT3 kinase inhibitor and preparation and application thereof. Specifically, the compound has a structure shown in a formula (I), wherein the definition of each group and substituent is described in the specification; the invention also discloses a preparation method of the compound and application of the compound in inhibiting FLT 3.

Description

FLT3 kinase inhibitor and preparation and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a compound with FLT3 and FLT3-ITD mutation inhibition activity, and preparation and application thereof.

Background

Protein Tyrosine Kinases (PTK) are kinases which catalyze the transfer of gamma-phosphate on ATP to protein tyrosine residues, can catalyze the phosphorylation of various substrate protein tyrosine residues, and play an important role in cell growth, proliferation and differentiation.

FMS-like tyrosine kinase 3(FLT3) belongs to the type III receptor tyrosine kinase family, and its protein structure comprises an extracellular region composed of 5 immunoglobulin (Ig) -like domains, 1 transmembrane region, 1 Juxtamembrane (JM) region and 2 Tyrosine Kinase (TK) regions separated by kinase insertion regions in cells. Multiple signaling pathways, including signal transducer and activator of transcription 5(STAT5), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways, are typically activated subsequently when FLT3 ligand binds to FLT 3. FLT3 has a very important role in the proliferation, differentiation and apoptosis of hematopoietic cells.

Activating mutations are present in acute myeloid leukemia cases up to 1/3. The most prominent activating mutation is the "internal tandem repeat" (ITD) in the juxtamembrane domain, resulting in sustained, ligand-independent kinase activation. Acute myeloid leukemia patients carrying the FLT3-ITD mutation had a significantly worse prognosis when receiving standard chemotherapy compared to wild-type FLT 3. In addition, activating loop point mutations in FLT3 kinase also occurred in other groups of acute myeloid leukemia patients, leading to associated poor prognosis manifested in overall survival. Therefore, the development of small molecule inhibitors with the activity of selectively inhibiting FLT3 kinase and FLT3-ITD mutant kinase has important significance for the treatment of acute myeloid leukemia.

Disclosure of Invention

The invention aims to provide a compound shown as a formula (I), a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvate thereof, a preparation method and application thereof, wherein the compound has FLT3 inhibitory activity.

In a first aspect of the present invention, there is provided a compound represented by formula (I), a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof, or a solvate thereof,

wherein,

n is an integer selected from 0, 1, 2, 3, 4;

y is C or N;

z is C or N;

R¹independently selected from the group consisting of: none, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, nitrile, carboxamide, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C6 cycloalkoxy, substituted or unsubstituted C3-C6 cycloalkylthio, said substitution being by one or more halogens;

R²selected from the group consisting of: substituted or unsubstituted Ring A, -NR_o-(CH₂)_m-NR_pR_q、-NR_o-(CH₂)_m-Ring A, -NR_pR_q、-(CH₂)_m-Ring A, -O- (CH)₂)_m-Ring A, -S- (CH)₂)_m-Ring A, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, nitrile, carboxamide, - (CH)₂)_m-R_b、-O-(CH₂)_m-R_b、-NH-(CH₂)_m-R_b、-S-(CH₂)_m-R_bSubstituted orUnsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 heteroaryl containing 1-3 heteroatoms selected from N, O and S; wherein said substitution means substitution with one or more substituents selected from the group consisting of: -C (═ O) - (C1-C6 alkyl), -S (═ O)₂- (C1-C6 alkyl), C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, amino, Ring A, halogen, hydroxy;

ring A is a 5-7 membered saturated heterocyclic group containing 1-3 heteroatoms selected from N, O and S;

R_o、R_pand R_qEach independently selected from the group consisting of: H. C1-C6 alkyl, halo C1-C6 alkyl;

m is independently an integer selected from 0, 1, 2, 3, 4, 5;

R_bselected from the group consisting of: H. amino, C1-C6 alkylamino, hydroxy, C1-C6 alkoxy, mercapto, C1-C6 alkylthio;

R³selected from the group consisting of: H. C1-C6 alkyl, halo C1-C6 alkyl;

R⁴selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-7 membered saturated heterocycle containing 1-3 members selected from N, O or S, - (CH)₂)_m-R_bA tricyclic bridged ring C6-C8 alkyl group sharing one N, said substitution being with one or more substituents selected from the group consisting of: halogen, -OH, -N (R)_c)₂C1-C3 alkyl;

or R³And R⁴Together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic ring containing 1-3N and 0-3 atoms selected from O, S, said heterocyclic ring optionally substituted with one or more R_aSubstitution;

R_aindependently selected from the group consisting of: C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl, hydroxyl, amino, nitrile group and halogen;

R_cindependently selected from the group consisting of: H. C1-C6 alkyl, halogenated C1-C6 alkyl.

In another preferred embodiment, the substituted C1-C6 alkyl is halogenated C1-C6 alkyl.

In another preferred embodiment, the halogenated C1-C6 alkyl group is selected from the group consisting of: trifluoromethyl, difluoromethyl.

In another preferred embodiment, the substituted C1-C6 alkoxy is a halogenated C1-C6 alkoxy.

In another preferred embodiment, the halogenated C1-C6 alkoxy group is selected from the group consisting of: trifluoromethoxy and difluoromethoxy.

In another preferred embodiment, R²Selected from the group consisting of: substituted or unsubstituted Ring A, -NR_o-(CH₂)_m-Ring A, - (CH)₂)_m-Ring A, -O- (CH)₂)_m-a ring a; the substituent means being substituted with one or more substituents selected from the group consisting of: -C (═ O) - (C1-C6 alkyl), -S (═ O)₂- (C1-C6 alkyl), C1-C6 alkyl, Ring A, halogen;

ring a is selected from the group consisting of: a 6-membered saturated heterocyclic group containing 1N and 1O, a 6-membered saturated heterocyclic group containing 2N, a 5-6-membered saturated heterocyclic group containing 1N;

m、R_oas defined above.

In another preferred embodiment, R_bSelected from the group consisting of: H. amino, methylamino, dimethylamino, diethylamino, diisopropylamino, hydroxyl, methoxy, ethoxy, mercapto, methylthio, ethylthio.

In another preferred embodiment, R³Is H;

R⁴is a substituted or unsubstituted 5-7 membered saturated heterocyclic ring containing 1-3N, said substitution being by one or more substituents selected from the group consisting of: halogen, -OH, -N (R)_c)₂；

Or R³And R⁴Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring, optionally substituted with one or more R_aSubstitution;

R_aindependently selected from the group consisting of: hydroxy, amino, halogen;

R_cindependently selected from the group consisting of: H. C1-C6 alkyl.

In another preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of:

an acid addition salt of a compound of formula (I) with an acid selected from the group consisting of: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, lactic acid, oxalic acid, adipic acid, glutaric acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid, phenylacetic acid, mandelic acid.

In another preferred embodiment, the compound is selected from the compounds listed in table 1.

In a second aspect of the present invention, there is provided a process for preparing a compound of the first aspect of the present invention, a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof or a solvate thereof, wherein the process is selected from the group consisting of:

the method comprises the following steps:

in a polar aprotic solvent, under the action of potassium fluoride, a compound (II) and a compound (III) undergo a substitution reaction to prepare a compound of a formula (I);

the second method comprises the following steps:

under the action of a condensing agent, carrying out condensation reaction on the compound (IV) and the compound (V) to prepare a compound shown in the formula (I);

wherein n, Y, Z, R are as defined above¹，R²，R³，R⁴According to the first aspect of the present invention.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the following components:

1) a therapeutically effective amount of one or more compounds of the first aspect of the present invention, stereoisomers, geometric isomers, tautomers thereof, pharmaceutically acceptable salts thereof, prodrugs thereof, hydrates thereof or solvates thereof; and

2) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the pharmaceutical composition is an injection, a capsule, a tablet, a pill, a powder or a granule.

In a fourth aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention, a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof or a solvate thereof, or a pharmaceutical composition according to the third aspect of the present invention, for a use selected from the group consisting of:

1) for the manufacture of a medicament for the prevention and/or treatment of FLT 3-mediated diseases;

2) preparing a medicament for inhibiting FLT 3.

In another preferred example, the FLT 3-mediated disease is responsive to FLT3 or mutant FLT3 kinase inhibition.

In another preferred embodiment, the FLT3 mutation is selected from the group consisting of: ITD mutation and TKD point mutation, preferably FLT3-ITD mutation.

In another preferred embodiment, the FLT 3-mediated disease is selected from the group consisting of: cancer, immune disease.

In another preferred embodiment, the cancer is selected from the group consisting of: leukemia, lymphoma, Hodgkin's disease, myeloma, acute lymphocytic leukemia, acute myelocytic leukemia, polar promyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic neutrophilic leukemia, polar undifferentiated cell leukemia, anaplastic large cell lymphoma, adult T cell ALL, AML with three lineage myelodysplasia, mixed lineage leukemia, myelodysplastic syndrome, myelodysplasia, multiple myeloma, myxosarcoma, chronic lymphocytic lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma.

In another preferred embodiment, the immune disease is selected from the group consisting of: arthritis, lupus, inflammatory bowel disease, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, still's disease, juvenile arthritis, diabetes, myasthenia gravis, hashimoto's thyroiditis, alder's thyroiditis, graves ' disease, rheumatoid arthritis syndrome, multiple sclerosis, infectious neuronitis, polar-transmissible encephalomyelitis, addison's disease, aplastic anemia, autoimmune hepatitis, optic neuritis, psoriasis, inhibitor anti-host disease, transfusion hypersensitivity, allergy, type I hypersensitivity, allergic rhinitis, atopic dermatitis.

In a fifth aspect of the invention, there is provided an inhibitor of FLT3, the inhibitor comprising one or more compounds of the first aspect of the invention, a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof or a solvate thereof.

In a sixth aspect of the invention, there is provided an in vitro method of non-therapeutically inhibiting FLT3 by contacting a compound of the first aspect of the invention, a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof or a solvate thereof, with FLT 3.

In a seventh aspect of the present invention, there is provided a method for inhibiting FLT3 by contacting a compound of the first aspect of the present invention, a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof or a solvate thereof with FLT 3.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of kinase selectivity of Compound 1-1 at a concentration of 0.1. mu.M.

FIG. 2 shows the results of kinase selectivity of Compound 1-1 at a concentration of 1. mu.M.

FIG. 3 is a graph showing the kinase selectivity results of compounds 1-19 at a concentration of 0.1. mu.M.

FIG. 4 is the kinase selectivity results for compounds 1-19 at a concentration of 1 μ M.

FIG. 5 is a compound

Kinase selectivity results at a concentration of 0.1. mu.M.

FIG. 6 is a compound

Kinase selectivity results at a concentration of 1 μ M.

Detailed Description

The present inventors have conducted extensive and intensive studies for a long time and have unexpectedly prepared a compound having a novel structure and excellent FLT3 inhibitory activity. Specifically, the inventors optimized R²The structure of the position is a hydrophilic structure, so that a compound with excellent FLT3 inhibitory activity and selectivity is obtained. On this basis, the inventors have completed the present invention.

Term(s) for

In the present invention, unless otherwise specified, the terms used have the ordinary meanings well known to those skilled in the art.

In the present invention, the term "halogen" means F, Cl, Br or I.

In the present invention, the term "C1-C6 alkyl" means a straight or branched chain alkyl group having 1 to 6 carbon atoms, preferably C1-C4 alkyl, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

In the present invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. C3-C7 cycloalkyl or C3-C6 cycloalkyl is preferred.

In the present invention, the term "C1-C6 alkoxy" means a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like. C1-C4 alkoxy is preferred. C1-C6 alkylthio have similar meanings.

In the present invention, the term "aromatic ring" or "aryl" has the same meaning, and is preferably "C6-C10 aryl", more preferably "C6-C8 aryl". The term "C6-C10 aryl" refers to an aromatic ring group having 6 to 10 carbon atoms, such as phenyl, naphthyl, and the like, that does not contain heteroatoms in the ring.

In the present invention, the term "heteroaromatic ring" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to more heteroatoms. For example, "C5-C10 heteroaryl" refers to an aromatic heterocycle containing 1-4 heteroatoms selected from oxygen, sulfur and nitrogen, and 5-10 carbon atoms. Non-limiting examples include: furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring joined to the parent structure is a heteroaryl ring. Heteroaryl groups may be optionally substituted or unsubstituted.

In the present invention, the term "halo" means substituted by halogen.

In the present invention, the term "tricyclic bridged C6-C8 alkyl group sharing one N" means, for example, the groups contained in the compounds I-34

In the present invention, the term "substituted" means that one or more hydrogen atoms on a specified group are replaced with a specified substituent. Particular substituents are those described correspondingly in the foregoing, or as appearing in the examples. Unless otherwise specified, a certain substituted group may have one substituent selected from a specific group at any substitutable site of the group, and the substituents may be the same or different at each position. It will be understood by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically achievable. Such substituents are for example (but not limited to): halogen, -C (═ O) - (C1-C6 alkyl), -C (═ O) -O- (C1-C6 alkyl), -S (═ O)₂- (C1-C6 alkyl), C1-C6 alkyl, halogenated C1-C6 alkylC1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl, amino, ring A, nitrile group, hydroxyl group and the like.

Compound (I)

The invention provides a compound shown as a formula (I), a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvate thereof,

wherein each group is as defined above.

In another preferred embodiment, in the compound, n, Y, Z and R¹、R²、R³、R⁴Each of which is a group corresponding to a specific compound described in table 1.

In another preferred embodiment, the compound is preferably the compound prepared in the examples.

TABLE 1

Salt form

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc.

Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

The term "prodrug" includes a class of compounds which are biologically active or inactive in nature and which undergo metabolic or chemical reactions in the body to become transformed, or a salt or solution of the compound, when administered by an appropriate method. The prodrugs include, but are not limited to, carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amide, carbamate, azo, phosphoramide, glucoside, ether, acetal forms of the compounds.

Preparation method

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

Typically, the process for the preparation of the compounds of the present invention is as follows, wherein the starting materials and reagents used are commercially available without specific reference.

Specifically, the invention relates to a preparation method of a compound shown in a formula (I), which is realized by the following steps:

the method 1 comprises the following steps:

hydrolyzing from the compound 1, condensing with corresponding aromatic amine to amide after chlorination, and then substituting with corresponding aliphatic amine to obtain the target product. Specifically, the preparation method comprises the following steps:

1. hydrolyzing the compound 1 under the action of lithium hydroxide to obtain a compound 2;

2. the compound 2 is chlorinated in phosphorus oxychloride and becomes acyl chloride to obtain a compound 3;

3. the compound 3 and a corresponding aromatic amine compound 4 form amide under the action of a condensing agent EDCI/HOBT/DIPEA or HATU/DIPEA to obtain a compound 5;

4. and carrying out substitution reaction on the compound 5 and corresponding fatty amine under the action of potassium fluoride to finally obtain the target compound (I).

The method 2 comprises the following steps:

after the compound 1 is chlorinated, the compound is substituted by corresponding aliphatic amine, and after hydrolysis, the compound and the compound 4 form amide to obtain the target compound.

Specifically, the preparation method comprises the following steps:

1. the compound 1 is chlorinated by phosphorus oxychloride under the catalysis of N, N-dimethylformamide to obtain a compound 6;

2. the compound 6 is substituted by corresponding fatty amine under the action of potassium fluoride to obtain a compound 7;

3. hydrolyzing the compound 7 under the action of lithium hydroxide to obtain a compound 8;

4. the compound 8 and corresponding aromatic amine compound 4 form amide under the action of condensing agents EDCI/HOBT/DIPEA or HATU/DIPEA to obtain the target compound (I).

Wherein

The preparation of (2) can be divided into method 3 and method 4.

The method 3 comprises the following steps:

x is a fluorine or chlorine atom

Specifically, the preparation method comprises the following steps:

1. SN generation of compound 9 under the action of alkali_ArReacting to generate a corresponding compound 10;

2. compound 10 is catalytically hydrogenated or reduced with iron powder/ammonium chloride to give compound 4.

The method 4 comprises the following steps:

x is a bromine atom

Specifically, the preparation method comprises the following steps:

1. pd (PPh) from Compound 11₃)₄Or Pd (dppf) Cl₂Carrying out Suzuki coupling reaction under the catalysis of the (1) to obtain a compound 12;

2. compound 12 is catalytically hydrogenated or reduced with iron powder/ammonium chloride to give compound 4.

Wherein n, Y, Z, R are as defined above¹，R²，R³，R⁴As defined above.

Pharmaceutical compositions and methods of administration

The invention also provides a pharmaceutical composition, which comprises the following components:

2) a pharmaceutically acceptable carrier or excipient.

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound as a main active ingredient can be used for treating, preventing and relieving diseases related to tumors because the compound of the present invention has excellent antitumor activity.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-1000mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically canThe accepted vector "refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compound can be independently administered or combined with other pharmaceutically acceptable compounds (such as antitumor drugs).

The treatment methods of the present invention can be administered alone or in combination with other therapeutic means or agents.

In the case of pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1-2000mg, preferably 50-1000 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Compared with the prior art, the invention has the following main advantages:

the compound has excellent FLT3 inhibitory activity and selectivity.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1

(R) -N- (2-methoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-1)

Step 1: preparation of 3-amino-1H-pyrazole-4-carboxylic acid ethyl ester

Ethyl 2-cyano-3-ethoxyacrylate (15.8g, 93.39mmol) was dissolved in ethanol, hydrazine hydrate (5.37ml, 85%) was added dropwise, reflux reacted overnight, concentrated, extracted with ethyl acetate and water, the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, and concentrated to dryness to give 11.5g of a yellow solid. Yield: 79 percent.

¹H NMR(300MHz,DMSO-d₆)δ11.88(s,1H),7.53(s,1H),5.91(s,2H),4.16(qd,J＝7.1,1.5Hz,2H),1.24(t,J＝7.1Hz,3H).

Step 2: preparation of ethyl 5-oxo-4, 5-dihydropyrazolo [1,5-a ] pyrimidine-3-carboxylate

Dissolving ethyl 3-amino-1H-pyrazole-4-carboxylate (10.76g, 69.35mmol), ethyl 3-ethoxyacrylate (15g, 104.02mmol) and cesium carbonate (20g, 104.02mmol) in N, N-dimethylformamide (120ml), reacting at 110 ℃ overnight, cooling to room temperature, adding acetic acid to adjust to subacidity, adding water to precipitate a solid, filtering, and drying to obtain 11g of a light yellow solid. Yield: 77 percent.

¹H NMR(300MHz,Chloroform-d)δ9.62(s,1H),8.12(dd,J＝8.1,0.8Hz,1H),8.04(s,1H),6.16(d,J＝8.0Hz,1H),4.37(q,J＝7.1Hz,2H),1.47–1.32(m,3H).

And step 3: preparation of ethyl 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylate

Dissolving 5-oxo-4, 5-dihydropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (3g, 14.48mmol) in N, N-dimethylformamide (20ml), adding phosphorus oxychloride (2ml), reacting at 90 ℃ for 2 hours, cooling to room temperature, adding water to precipitate a solid, filtering, and drying to obtain a white solid 2.5 g. Yield: 77 percent.

¹H NMR(300MHz,Chloroform-d)δ8.67–8.60(m,1H),8.56(s,1H),6.99(d,J＝7.2Hz,1H),4.52–4.33(m,2H),1.42(td,J＝7.1,0.9Hz,3H).

And 4, step 4: preparation of ethyl (R) -5- ((1- (tert-butoxycarbonyl) piperidin-3-yl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylate

Ethyl 5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.2g, 5.32mmol) and tert-butyl (R) -3-aminopiperidine-1-carboxylate (1.6g, 7.98mmol) were dissolved in dimethyl sulfoxide (20ml), potassium fluoride (3.09g, 53.2mmol) was added, the reaction was carried out at 100 ℃ for 2 hours, the mixture was cooled to room temperature, diluted with water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and subjected to silica gel column chromatography (dichloromethane/methanol) to obtain 1.5g of a white solid. The yield thereof was found to be 72%.

And 5: preparation of (R) -5- ((1- (tert-butoxycarbonyl) piperidin-3-yl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

Ethyl (R) -5- ((1- (tert-butoxycarbonyl) piperidin-3-yl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.5g, 3.85mmol) was dissolved in tetrahydrofuran/methanol/water (10ml/6ml/5ml), and lithium hydroxide hydrate (970mg, 23.11mmol) was added thereto to react at 60 ℃ for 8 hours, followed by concentration, acidification with 1mol/L dilute hydrochloric acid to precipitate a solid, suction filtration and drying to give 1.3g of a white solid. Yield: 93 percent.

¹H NMR(300MHz,DMSO-d₆)δ11.53(s,1H),8.55(d,J＝7.7Hz,1H),8.12(s,1H),7.80(d,J＝7.0Hz,1H),6.43(d,J＝7.5Hz,1H),3.99(s,1H),3.53(s,2H),1.99(s,1H),1.77(s,1H),1.34(m,11H).

Step 6: preparation of 4- (3-methoxy-4-nitrophenyl) morpholine

Dissolving 4-fluoro-2-methoxy-1-nitrobenzene (200mg, 1.17mmol) in N, N-dimethylformamide (4ml), adding potassium carbonate (323mg, 2.34mmol) and morpholine (203mg, 2.34mmol), reacting at 90 ℃ for 3 hours, diluting with water, extracting with ethyl acetate, drying over anhydrous sodium sulfate, concentrating under reduced pressure, and directly putting into the next reaction.

¹H NMR(300MHz,Chloroform-d)δ8.14–7.89(m,1H),6.43(d,J＝9.7Hz,1H),6.35(s,1H),4.05–3.78(m,7H),3.35(d,J＝5.0Hz,4H).

And 7: preparation of 2-methoxy-4-morpholinoaniline

Dissolving the product obtained in the previous step in methanol (25ml), adding palladium carbon (30mg), reducing for 4 hours under hydrogen atmosphere, filtering with diatomite, concentrating under reduced pressure to obtain the product, and directly putting the product into the next step for reaction.

And 8: preparation of (R) -N- (2-methoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-1)

(R) -5- ((1- (tert-butoxycarbonyl) piperidin-3-yl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (100mg, 276. mu. mol) and 2-methoxy-4-morpholinoaniline (63mg, 304. mu. mol) were dissolved in N, N-dimethylformamide (5ml), and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (63mg, 332. mu. mol), 1-hydroxybenzotriazole (44mg, 332. mu. mol) and DIPEA (89mg, 691. mu. mol) were added thereto, followed by reaction at 50 ℃ and monitoring by TLC, followed by extraction with ethyl acetate by dilution with water, drying over anhydrous sodium sulfate, filtration, and chromatography on silica gel (dichloromethane/methanol) to give 103mg of an off-white solid. Dissolving the obtained solid in dichloromethane (4ml), adding trifluoroacetic acid (1ml), reacting at room temperature for 2 hours, concentrating under reduced pressure, adding diethyl ether, pulping, filtering, and drying in vacuum to obtain the product.

¹H NMR(300MHz,DMSO-d₆)δ9.50(s,1H),8.67(d,J＝7.5Hz,2H),8.22(s,2H),8.00(d,J＝8.8Hz,1H),6.70(d,J＝2.5Hz,1H),6.60–6.43(m,2H),4.35(s,1H),3.86(s,3H),3.76(t,J＝4.8Hz,3H),3.34–2.97(m,7H),2.04(s,2H),1.74(s,2H).

Example 2

(R) -5- (3-Aminopiperidin-1-yl) -N- (2-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-2)

Step 1: preparation of ethyl (R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate

The tert-butyl (R) -3-aminopiperidine-1-carboxylate was replaced with tert-butyl (R) -piperidin-3-ylcarbamate, and the remaining required raw materials, reagents and preparation methods were the same as in step 4 of example 1, and the obtained white solid was ethyl (R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate.

Step 2: preparation of (R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

The ethyl (R) -5- ((1- (tert-butoxycarbonyl) piperidin-3-yl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylate was replaced with ethyl (R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylate, and the remaining necessary raw materials, reagents and preparation method were the same as in step 5 of example 1, and the obtained product was (R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid.

And step 3: preparation of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-2)

(R) -5- (3- ((tert-butoxycarbonyl) amino) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (85mg, 235. mu. mol), 2-methoxy-4-morpholinoaniline (58mg, 282. mu. mol) was dissolved in N, N-dimethylformamide (5ml), and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (54mg, 282. mu. mol), 1-hydroxybenzotriazole (38mg, 282. mu. mol) and DIPEA (76mg, 587. mu. mol) were added thereto, reacted at 50 ℃ for 4 hours, diluted with water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and column-chromatographed on silica gel (dichloromethane/methanol) to give 83mg of an oil. Dissolving the obtained oily substance in dichloromethane (4ml), adding trifluoroacetic acid (1ml), reacting at room temperature for 2 hours, concentrating under reduced pressure, adding diethyl ether, and pulping to obtain the product (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-formamide trifluoroacetate.

¹H NMR(300MHz,DMSO-d₆)δ9.59(s,1H),8.87(d,J＝7.9Hz,1H),8.42–8.20(m,2H),8.07(s,3H),6.84(d,J＝8.0Hz,1H),6.71(d,J＝2.5Hz,1H),6.53(dd,J＝9.0,2.5Hz,1H),4.19(s,2H),3.88(s,3H),3.82–3.57(m,5H),3.36(s,1H),3.11(t,J＝4.8Hz,3H),2.05(s,1H),1.87(s,1H),1.72(d,J＝8.7Hz,2H).

Example 3

(R) -N- (4- (4-acetylpiperazin-1-yl) -2-methoxyphenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-3)

The trifluoroacetate salt of (R) -N- (4- (4-acetylpiperazin-1-yl) -2-methoxyphenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 1.

¹H NMR(300MHz,DMSO-d₆)δ9.52(s,1H),8.64(dd,J＝26.2,11.3Hz,3H),8.35–8.13(m,2H),8.01(d,J＝8.8Hz,1H),6.74(s,1H),6.56(d,J＝8.9Hz,1H),6.49(d,J＝7.7Hz,1H),4.34(s,1H),3.86(s,3H),3.59(d,J＝5.7Hz,4H),3.32–2.95(m,7H),2.04(d,J＝4.7Hz,5H),1.74(s,2H).

Example 4

(R) -5- (3-Aminopiperidin-1-yl) -N- (2-methoxy-4- (4- (methylsulfonyl) piperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-4)

The trifluoroacetate salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (4- (methylsulfonyl) piperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that in example 1.

¹H NMR(300MHz,DMSO-d₆)δ9.59(s,1H),8.86(d,J＝7.9Hz,1H),8.28(t,J＝4.4Hz,2H),8.08(s,3H),6.83(d,J＝8.0Hz,1H),6.73(d,J＝2.5Hz,1H),6.56(dd,J＝9.0,2.4Hz,1H),4.21(d,J＝14.6Hz,2H),3.88(s,3H),3.66(d,J＝12.4Hz,2H),3.25(d,J＝3.0Hz,8H),2.93(s,3H),1.96(d,J＝55.5Hz,5H).

Example 5

(R) -5- (3-Aminopiperidin-1-yl) -N- (2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluoroacetate (I-5)

The bistrifluoroacetate salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (4-methylpiperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide can be prepared by a method similar to that in example 2.

¹H NMR(300MHz,DMSO-d₆)δ10.05(s,1H),9.60(s,1H),8.87(d,J＝7.9Hz,1H),8.29(d,J＝8.9Hz,2H),8.15(s,3H),6.84(d,J＝8.0Hz,1H),6.76(d,J＝2.5Hz,1H),6.57(dd,J＝8.9,2.5Hz,1H),4.20(s,2H),3.88(s,4H),3.65(d,J＝9.7Hz,2H),3.17(s,2H),2.87(s,4H),2.06(s,1H),1.87(s,1H),1.82–1.55(m,2H).

Example 6

(R) -5- (3-Aminopiperidin-1-yl) -N- (2-methoxy-4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluoroacetate (I-6)

The bistrifluoroacetate salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that in example 2.

¹H NMR(300MHz,DMSO-d₆)δ9.60(s,1H),8.87(d,J＝8.1Hz,3H),8.29(d,J＝8.7Hz,2H),8.13(s,3H),6.83(d,J＝8.0Hz,1H),6.75(d,J＝2.5Hz,1H),6.56(dd,J＝9.0,2.5Hz,1H),4.19(s,2H),3.88(s,3H),3.44–3.15(m,8H),2.06(s,1H),1.88(s,1H),1.74(d,J＝19.8Hz,2H).

Example 7

(R) -N- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluoroacetate (I-7)

The ditrifluoroacetate salt of (R) -N- (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 1.

¹H NMR(300MHz,DMSO-d₆)δ9.97(s,1H),9.45(s,1H),8.68(d,J＝7.3Hz,3H),8.21(s,2H),7.97(d,J＝8.9Hz,1H),6.44(d,J＝26.1Hz,3H),4.36(s,1H),3.88(s,3H),3.64(d,J＝7.9Hz,2H),3.27–3.02(m,4H),2.88(d,J＝20.6Hz,6H),2.03(s,2H),1.77(s,2H).

Example 8

Preparation of (R) -N- (2-methoxy-4- (4-morpholinopiperidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluoroacetate (I-8)

The bistrifluoroacetate salt of (R) -N- (2-methoxy-4- (4-morpholinopiperidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 1.

¹H NMR(300MHz,DMSO-d₆)δ9.49(s,1H),8.68(d,J＝7.6Hz,3H),8.23(d,J＝10.7Hz,2H),7.99(d,J＝8.7Hz,1H),6.71(s,1H),6.52(dd,J＝18.5,8.2Hz,2H),4.36(s,1H),3.86(s,8H),3.15(d,J＝33.9Hz,4H),2.68(t,J＝12.3Hz,2H),2.25–1.92(m,3H),1.72(d,J＝14.1Hz,3H).

Example 9

(R) -5- (3-Aminopiperidin-1-yl) -N- (3-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-9)

Step 1: preparation of 4- (2-methoxy-4-nitrophenyl) morpholine

Dissolving 1-fluoro-2-methoxy-4-nitrobenzene (200mg, 1.17mmol), morpholine (203mg, 2.34mmol) and potassium carbonate (323mg, 2.34mmol) in N, N-dimethylformamide (5ml), reacting at 80 ℃ for 6 hours, adding water to precipitate a solid, and filtering to obtain a yellow solid, namely 221 mg. Yield: 80 percent.

¹H NMR(300MHz,Chloroform-d)δ7.89(d,J＝8.9Hz,1H),7.02–6.81(m,1H),4.22–3.65(m,7H),3.25(q,J＝3.9Hz,4H).

Step 2: preparation of 3-methoxy-4-morpholinoaniline

4- (2-methoxy-4-nitrophenyl) morpholine (135mg, 556. mu. mol) was dissolved in methanol (15ml), palladium on carbon (25mg) was added, the reaction was carried out at room temperature for 2 hours under a hydrogen atmosphere, and the mixture was subjected to suction filtration with celite, and concentrated under reduced pressure and directly subjected to the next reaction.

And step 3: preparation of (R) -5- (3-aminopiperidin-1-yl) -N- (3-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-9)

The 2-methoxy-4-morpholinoaniline was replaced with 3-methoxy-4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 3 of example 2, and the obtained product was the trifluoroacetate salt of (R) -5- (3-aminopiperidin-1-yl) -N- (3-methoxy-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.72(s,1H),8.88(d,J＝7.8Hz,1H),8.30(s,1H),8.24–7.88(m,3H),7.37(s,1H),7.20(d,J＝8.4Hz,1H),6.91(dd,J＝14.8,8.2Hz,2H),4.24(d,J＝13.6Hz,1H),4.04(s,1H),3.93–3.48(m,8H),3.47–3.27(m,1H),2.98(s,4H),2.05(s,1H),1.89(s,1H),1.72(s,2H).

Example 10

(R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (morpholinomethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluorometalate (I-10)

Step 1: preparation of 4- (3-methoxy-4-nitrobenzyl) morpholine

4- (bromomethyl) -2-methoxy-1-nitrobenzene (200mg, 812. mu. mol), morpholine (141mg, 1.63mmol) and potassium carbonate (224mg, 1.63mmol) were dissolved in dimethyl sulfoxide (4ml) and reacted at 80 ℃ for 4 hours, diluted with water and extracted with ethyl acetate, dried over anhydrous sodium sulfate and chromatographed on silica gel (dichloromethane/petroleum ether) to give a brown oil.

¹H NMR(300MHz,Chloroform-d)δ7.84(s,1H),7.16(s,1H),7.01(d,J＝8.5Hz,1H),4.10–3.93(m,3H),3.75(s,4H),3.56(s,2H),2.71–2.23(m,4H).

Step 2: preparation of 2-methoxy-4- (morpholinomethyl) aniline

4- (3-methoxy-4-nitrobenzyl) morpholine (232mg, 919. mu. mol) was dissolved in methanol (25ml), palladium on carbon (42mg) was added, the reaction was carried out at room temperature for 2 hours under a hydrogen atmosphere, and the mixture was subjected to suction filtration with celite, and concentrated under reduced pressure and directly fed to the next reaction.

And step 3: preparation of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (morpholinomethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide bistrifluorometalate (I-10)

The 2-methoxy-4-morpholinoaniline was replaced with 2-methoxy-4- (morpholinomethyl) aniline, and the remaining required raw materials, reagents, and preparation methods were the same as in step 3 of example 2, and the obtained product was the bis-trifluoroformate of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (morpholinomethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ10.30(s,1H),9.82(s,1H),8.89(d,J＝7.9Hz,1H),8.51(d,J＝8.2Hz,1H),8.32(s,1H),8.12(s,3H),7.26(s,1H),7.07(d,J＝8.3Hz,1H),6.86(d,J＝8.0Hz,1H),4.44–4.09(m,4H),3.92(s,4H),3.68(s,4H),2.06(s,1H),1.88(s,1H),1.72(d,J＝9.3Hz,2H).

Example 11

(R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (2-morpholinoethoxy) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-11)

Step 1: preparation of 4- (2- (3-methoxy-4-nitrophenoxy) ethyl) morpholine

4-fluoro-2-methoxy-1-nitrobenzene (200mg, 1.17mmol), N-hydroxyethylmorpholine (306mg, 3.34mmol) and tetrabutylammonium chloride (53mg, 233. mu. mol) were dissolved in 25% potassium hydroxide (6ml) and toluene (6ml) and reacted at 60 ℃ for 24 hours, diluted with water and extracted with ethyl acetate, dried over anhydrous sodium sulfate and chromatographed on silica gel (dichloromethane/methanol) to give 140mg of a yellow oil. Yield: 42 percent.

Step 2: preparation of 2-methoxy-4- (2-morpholinoethoxy) aniline

4- (2- (3-methoxy-4-nitrophenoxy) ethyl) morpholine (140mg, 495. mu. mol) was dissolved in methanol (16ml), palladium on carbon (25mg) was added, and the reaction mixture was reacted at room temperature for 2 hours under a hydrogen atmosphere, filtered with suction using celite, and concentrated under reduced pressure to be directly put into the next reaction.

And step 3: preparation of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (2-morpholinoethoxy) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-11)

The 2-methoxy-4-morpholinoaniline was replaced with 2-methoxy-4- (2-morpholinoethoxy) aniline, and the other required raw materials, reagents and preparation methods were the same as in step 3 of example 2, to give a trifluoroacetate salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (2-morpholinoethoxy) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide. The resulting solid was dissolved in saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and subjected to preparative thin layer chromatography to give (R) -5- (3-aminopiperidin-1-yl) -N- (2-methoxy-4- (2-morpholinoethoxy) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.61(s,1H),8.88(d,J＝7.9Hz,1H),8.65–8.13(m,5H),6.89(d,J＝8.1Hz,1H),6.78–6.65(m,1H),6.56(d,J＝9.0Hz,1H),4.44–4.04(m,4H),3.88(s,3H),3.63(t,J＝4.6Hz,6H),2.78(d,J＝5.9Hz,2H),2.58(s,4H),2.05(d,J＝9.1Hz,1H),1.96–1.58(m,3H).

Example 12

(R) -N- (2-ethoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-12)

Step 1: preparation of 2-ethoxy-4-fluoro-nitrobenzene

Dissolving ethanol (83mg, 1.82mmol) in toluene (8ml), cooling to 0-5 deg.C in ice bath, slowly adding potassium tert-butoxide (205mg, 1.82mmol), keeping the temperature at 0-5 deg.C, stirring for 15 min, slowly adding 2, 4-difluoronitrobenzene (290mg, 1.82mmol), reacting in ice bath for 2hr, adding water to dilute ethyl acetate, extracting, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain light yellow solid.

¹H NMR(300MHz,Chloroform-d)δ7.93(dd,J＝9.0,6.0Hz,1H),6.83–6.63(m,2H),4.16(q,J＝7.0Hz,2H),1.64–1.42(m,4H).

Step 2: preparation of 4- (3-ethoxy-4-nitrophenyl) morpholine

The 4-fluoro-2-methoxy-1-nitrobenzene is replaced by 2-ethoxy-4-fluoro-1-nitrobenzene, and the rest of the required raw materials, reagents and preparation method are the same as the step 6 in the example 1, and the obtained product is 4- (3-ethoxy-4-nitrophenyl) morpholine.

¹H NMR(300MHz,Chloroform-d)δ7.98(dd,J＝9.3,0.7Hz,1H),6.49–6.38(m,1H),6.33(d,J＝2.6Hz,1H),4.15(q,J＝7.0Hz,2H),3.93–3.79(m,4H),3.40–3.26(m,4H),1.50(td,J＝7.0,0.6Hz,3H).

And step 3: preparation of 2-ethoxy-4-morpholinoaniline

The 4- (3-methoxy-4-nitrophenyl) morpholine was replaced with 4- (3-ethoxy-4-nitrophenyl) morpholine, and the remaining required raw materials, reagents and preparation methods were the same as in step 7 of example 1, and the obtained product was 2-ethoxy-4-morpholinoaniline.

And 4, step 4: preparation of (R) -N- (2-ethoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-12)

The 2-methoxy-4-morpholinobenzene was replaced with 2-ethoxy-4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 7 of example 1, and the obtained product was the trifluoroacetate salt of (R) -N- (2-ethoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.38(s,1H),8.86–8.49(m,3H),8.22(s,2H),7.85(d,J＝8.7Hz,1H),6.70(d,J＝2.5Hz,1H),6.64–6.38(m,2H),4.15(qq,J＝7.1,3.3Hz,2H),3.75(t,J＝4.6Hz,4H),3.34–2.90(m,7H),2.06(s,2H),1.69(d,J＝7.0Hz,2H),1.24(t,J＝6.9Hz,3H).

Example 13

(R) -N- (2-isopropoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-13)

The trifluoroacetate salt of (R) -N- (2-isopropoxy-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide can be obtained by a similar method to that in example 12.

¹H NMR(300MHz,DMSO-d₆)δ9.35(s,1H),8.80–8.50(m,3H),8.21(s,1H),8.15(s,1H),7.75(d,J＝8.7Hz,1H),6.69(d,J＝2.5Hz,1H),6.59(dd,J＝8.8,2.6Hz,1H),6.48(d,J＝7.6Hz,1H),4.54–4.41(m,1H),4.34(s,1H),3.74(t,J＝4.8Hz,4H),3.28–2.92(m,7H),2.00(s,2H),1.71(d,J＝9.8Hz,2H),1.25(d,J＝6.1Hz,3H),1.12(d,J＝6.0Hz,3H).

Example 14

(R) -N- (2- (methylthio) -4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-14)

Step 1: preparation of 2-methylthio-4-fluoro-1-nitrobenzene

Dissolving 2, 4-difluoronitrobenzene (870mg, 5.47mmol) in methanol (12ml), stirring for 10 minutes in ice bath, slowly dropwise adding 20% sodium methyl mercaptide solution (2ml) through a constant-pressure dropping funnel, reacting for 30 minutes in ice bath after dropwise adding, adding water to precipitate yellow solid, dissolving again, and performing silica gel column chromatography (ethyl acetate/petroleum ether) to obtain 707mg yellow solid. Yield: and 69 percent.

¹H NMR(300MHz,Chloroform-d)δ8.33(dd,J＝9.2,5.4Hz,1H),7.04(dd,J＝9.8,2.6Hz,1H),6.93(ddd,J＝9.3,7.0,2.6Hz,1H),2.49(s,3H).

Step 2: preparation of 4- (3- (methylthio) -4-nitrophenyl) morpholine

The 4-fluoro-2-methoxy-1 nitrobenzene is changed into 2-methylthio-4-fluoro-1-nitrobenzene, and the rest required raw materials, reagents and preparation method are the same as the step 6 in the example 1, and the obtained product is the 4- (3- (methylthio) -4-nitrophenyl) morpholine.

¹H NMR(300MHz,Chloroform-d)δ8.24(d,J＝9.4Hz,1H),6.64(dd,J＝9.4,2.5Hz,1H),6.54(d,J＝2.6Hz,1H),3.87(dd,J＝6.3,3.6Hz,4H),3.48–3.28(m,4H),2.46(s,3H).

And step 3: preparation of 2- (methylthio) -4-morpholinoaniline

The 4- (3-methoxy-4-nitrophenyl) morpholine was replaced by 4- (3- (methylthio) -4-nitrophenyl) morpholine, and the remaining required raw materials, reagents and preparation methods were the same as in step 7 of example 1, and the obtained product was 2- (methylthio) -4-morpholinoaniline.

¹H NMR(300MHz,Chloroform-d)δ6.99(d,J＝2.7Hz,1H),6.77(dd,J＝8.6,2.7Hz,1H),6.70(d,J＝8.6Hz,1H),4.02(s,2H),3.92–3.76(m,4H),3.15–2.95(m,4H),2.37(d,J＝0.6Hz,3H).

And 4, step 4: preparation of (R) -N- (2- (methylthio) -4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-14)

The 2-methoxy-4-morpholinobenzene was replaced with 2- (methylthio) -4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 8 of example 1, and the obtained product was the trifluoroacetate salt of (R) -N- (2- (methylthio) -4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.32(s,1H),8.67(d,J＝7.6Hz,3H),8.21(d,J＝11.1Hz,2H),7.64(d,J＝8.7Hz,1H),7.01–6.74(m,2H),6.45(d,J＝7.6Hz,1H),4.48(s,1H),3.75(t,J＝4.7Hz,4H),3.31(d,J＝11.1Hz,1H),3.23–2.85(m,6H),2.42(s,3H),2.08(d,J＝11.1Hz,1H),1.91(s,1H),1.66(m,2H).

Example 15

(R) -N- (2-bromo-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-15)

Step 1: preparation of 4- (3-bromo-4-nitrophenyl) morpholine

Dissolving 2-bromo-4-fluoro-1-nitrobenzene (145mg, 686 mu mol) in morpholine (4ml), reacting at room temperature for 30 minutes, adding water to precipitate a solid, filtering, and drying to obtain a yellow solid.

¹H NMR(300MHz,Chloroform-d)δ8.02(dd,J＝9.3,0.7Hz,1H),7.08(d,J＝2.7Hz,1H),6.78(dd,J＝9.3,2.8Hz,1H),3.97–3.77(m,4H),3.47–3.25(m,4H).

Step 2: preparation of 2-bromo-4-morpholinoaniline

Dissolving 4- (3-bromo-4-nitrophenyl) morpholine (197mg, 686. mu. mol) in ethanol/water (10ml/8ml), adding ammonium chloride (293mg, 5.49mmol) and reduced iron powder (306mg, 5.49mmol), refluxing for 2 hours, cooling to room temperature, suction-filtering with diatomaceous earth, concentrating under reduced pressure, dissolving with ethyl acetate, washing with water and saturated salt water, drying over anhydrous sodium sulfate, suction-filtering, and performing silica gel column chromatography (dichloromethane/methanol) to obtain 124mg of a light brown solid.

¹H NMR(300MHz,DMSO-d₆)δ6.92(d,J＝2.5Hz,1H),6.82–6.69(m,2H),4.79(s,2H),3.78–3.60(m,4H),2.95–2.80(m,4H).

And step 3: preparation of (R) -N- (2-bromo-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-15)

The 2-methoxy-4-morpholinobenzene was replaced with 2-bromo-4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 8 of example 1, and the obtained product was the trifluoroacetate salt of (R) -N- (2-bromo-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.29(s,1H),8.68(d,J＝7.6Hz,3H),8.25(s,2H),7.77(d,J＝9.0Hz,1H),7.21(d,J＝2.7Hz,1H),7.03(dd,J＝9.1,2.7Hz,1H),6.46(d,J＝7.6Hz,1H),3.74(t,J＝4.8Hz,4H),3.68–3.29(m,4H),3.12(t,J＝4.8Hz,4H),3.00(s,2H),2.07(s,1H),1.90(s,1H),1.76(s,1H),1.58(d,J＝10.4Hz,1H).

Example 16

(R) -N- (2-chloro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-16)

The trifluoroacetate salt of (R) -N- (2-chloro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide can be obtained by a similar method to that in example 15.

¹H NMR(300MHz,DMSO-d₆)δ9.41(s,1H),8.68(d,J＝7.5Hz,3H),8.26(s,2H),7.93(d,J＝9.0Hz,1H),7.06(d,J＝2.7Hz,1H),6.99(dd,J＝9.1,2.8Hz,1H),6.47(d,J＝7.6Hz,1H),4.41(s,1H),3.74(dd,J＝6.1,3.5Hz,4H),3.33(d,J＝12.2Hz,1H),3.12(t,J＝4.8Hz,6H),1.98(d,J＝37.9Hz,2H),1.67(dd,J＝40.2,10.1Hz,2H).

Example 17

(R) -N- (2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-17)

Step 1: preparation of 4- (3-methyl-4-nitrophenyl) morpholine

Dissolving 4-fluoro-2-methyl-1-nitrobenzene (312mg, 2.01mmol) in morpholine (4ml), reacting at 80 ℃ for 2 hours, monitoring the reaction by TLC, adding water to precipitate solid, filtering, and drying to obtain 449mg of yellow solid. Yield: 100 percent.

Step 2: preparation of 2-methyl-4-morpholinoaniline

Dissolving 4- (3-methyl-4-nitrophenyl) morpholine (446mg, 2.01mmol) in methanol (40ml), adding palladium carbon (77mg), reacting for 4 hours under a hydrogen atmosphere, filtering with diatomite, and concentrating under reduced pressure to dryness to obtain the product.¹H NMR(300MHz,DMSO-d₆)δ6.60(d,J＝2.3Hz,1H),6.58–6.46(m,2H),4.35(s,2H),3.79–3.61(m,4H),2.96–2.80(m,4H),2.02(s,3H).

And step 3: preparation of (R) -N- (2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-18)

The 2-methoxy-4-morpholinobenzene was replaced with 2-methyl-4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 8 of example 1, and the obtained product was the trifluoroacetate salt of (R) -N- (2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.02(s,1H),8.89–8.54(m,3H),8.23(s,2H),7.56(d,J＝8.7Hz,1H),6.87(d,J＝2.7Hz,1H),6.81(dd,J＝8.8,2.7Hz,1H),6.44(d,J＝7.6Hz,1H),3.75(t,J＝4.8Hz,4H),3.26–2.87(m,6H),2.28(s,3H),1.91(s,1H),1.76–1.48(m,2H).

Example 18

(R) -N- (4-Morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-18)

The trifluoroacetate salt of (R) -N- (4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide can be obtained by a similar method to that in example 17.

¹H NMR(300MHz,DMSO-d₆)δ9.61(s,1H),8.81(s,2H),8.67(d,J＝7.5Hz,1H),8.27(d,J＝17.7Hz,2H),7.54(d,J＝8.4Hz,2H),6.96(d,J＝8.9Hz,2H),6.46(d,J＝7.6Hz,1H),4.31(s,1H),3.75(t,J＝4.8Hz,4H),3.19(s,2H),3.07(t,J＝4.9Hz,4H),2.21(s,1H),1.95(s,1H),1.68(dd,J＝55.7,10.9Hz,2H).

Example 19

(R) -N- (2-cyano-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-19)

Step 1: preparation of 5-morpholino-2-nitrobenzonitrile

5-fluoro-2-nitrobenzonitrile (300mg, 1.81mmol) is dissolved in morpholine (5ml) and reacted for 30 minutes at 50 ℃, water is added to precipitate solid, and the solid is filtered and dried to obtain 383mg of yellow solid. Yield: 91 percent.

¹H NMR(300MHz,DMSO-d₆)δ7.36(d,J＝9.6Hz,1H),6.72(d,J＝2.9Hz,1H),6.42(dd,J＝9.6,2.9Hz,1H),2.88(dd,J＝5.9,4.0Hz,4H),2.67(dd,J＝5.8,4.1Hz,4H).

Step 2: preparation of 2-amino-5-morpholinobenzonitrile

5-Morpholino-2-nitrobenzonitrile (380mg, 1.63mmol) was dissolved in methanol (35ml), 10% palladium on carbon (45mg) was added, the reaction was carried out for 4 hours under a hydrogen atmosphere, suction filtration was carried out with celite, concentration was carried out under reduced pressure, and silica gel column chromatography (dichloromethane/methanol) was carried out to obtain 245mg of a pale yellow solid. Yield: 74 percent.

¹H NMR(300MHz,Chloroform-d)δ7.03(dd,J＝8.9,2.8Hz,1H),6.88(d,J＝2.8Hz,1H),6.71(d,J＝9.0Hz,1H),4.13(s,2H),3.97–3.72(m,4H),3.18–2.87(m,4H).

And step 3: preparation of 5-oxo-4, 5-dihydropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

Dissolving ethyl 5-oxo-4, 5-dihydropyrazolo [1,5-a ] pyrimidine-3-carboxylate (2g, 9.65mmol) in water (50ml), adding lithium hydroxide monohydrate (2.43g, 57.9mmol), reacting at 50 ℃ overnight, concentrating under reduced pressure, adjusting pH to weak acidity with 2mol/L dilute hydrochloric acid, precipitating a large amount of solid, filtering, and drying to obtain 1.52g of white solid. Yield: 88 percent.

¹H NMR(300MHz,DMSO-d₆)δ8.55(d,J＝8.0Hz,1H),8.08(s,1H),6.13(d,J＝7.9Hz,1H).

And 4, step 4: preparation of 5-chloropyrazolo [1,5-a ] pyrimidine-3-carbonyl chloride

Dissolving 5-oxo-4, 5-dihydropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (371mg, 2.07mmol) in phosphorus oxychloride (20ml), adding N, N-diisopropylethylamine (936mg, 7.5mmol), refluxing for 3 hours, concentrating under reduced pressure, dissolving dichloromethane, washing with saturated salt water, drying with anhydrous sodium sulfate, filtering with 200-300 mesh silica gel, concentrating under reduced pressure to dryness to obtain 420mg of yellow solid, and directly putting the yellow solid into the next reaction.

And 5:

preparation of 5-chloro-N- (2-cyano-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide

2-amino-5-morpholinobenzonitrile (144mg, 708. mu. mol) was dissolved in dichloromethane (8ml), N-diisopropylethylamine (274mg, 2.13mmol) was added, 5-chloropyrazolo [1,5-a ] pyrimidine-3-carbonyl chloride (214mg, 991. mu. mol) was added under ice bath, reacted overnight at room temperature, diluted with dichloromethane, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography (dichloromethane/methanol) to give 238mg of a yellow solid. Yield: 87 percent.

¹H NMR(300MHz,DMSO-d₆)δ9.74(s,1H),9.40(d,J＝7.2Hz,1H),8.76(s,1H),7.97(d,J＝9.1Hz,1H),7.44(d,J＝7.3Hz,1H),7.35(d,J＝8.1Hz,2H),3.74(t,J＝4.8Hz,4H),3.17(t,J＝4.8Hz,4H).

Step 6:

preparation of tert-butyl (R) -3- ((3- ((2-cyano-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate

5-chloro-N- (2-cyano-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (87mg, 227. mu. mol) and (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester (68mg, 340. mu. mol) were dissolved in DMSO (5ml), and anhydrous potassium fluoride (132mg, 2.27mmol) was added thereto, followed by reaction at 60 ℃ for 2 hours, dissolution in ethyl acetate, washing with water and saturated saline, drying over anhydrous sodium sulfate, filtration, concentration under reduced pressure, and chromatography on silica gel column (dichloromethane/methanol) gave 116mg of a yellow solid. Yield: 93 percent.

And 7: preparation of (R) -N- (2-cyano-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-19)

Dissolving tert-butyl (R) -3- ((3- ((2-cyano-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate (52mg, 95 mu mol) in dichloromethane (4ml), adding trifluoroacetic acid (1ml), reacting at room temperature for 2 hours, concentrating under reduced pressure, adding diethyl ether, pulping, filtering, and drying in vacuum to obtain the product.

¹H NMR(300MHz,DMSO-d₆)δ9.58(s,1H),9.00–8.47(m,3H),8.28(s,2H),7.83(d,J＝8.9Hz,1H),7.35(d,J＝8.8Hz,2H),6.46(d,J＝7.6Hz,1H),4.52(s,1H),3.90–3.62(m,4H),3.37(s,3H),3.16(t,J＝4.8Hz,5H),2.93(s,2H),1.96(d,J＝38.6Hz,2H),1.84–1.50(m,2H).

Example 20

(R) -N- (2-cyano-4-morpholinophenyl) -5- (pyrrolidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-21)

The procedure is as in example 19, except for the materials, reagents and preparation method used in step 6 and 7, tert-butyl (R) -3-aminopiperidine-1-carboxylate was changed to tert-butyl (R) -3-aminopyrrolidine-1-carboxylate, and the product obtained is the trifluoroacetate salt of (R) -N- (2-cyano-4-morpholinophenyl) -5- (pyrrolidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.71(s,1H),9.19(s,1H),8.98(s,1H),8.69(d,J＝7.6Hz,1H),8.53(d,J＝6.3Hz,1H),8.34(s,1H),7.81(d,J＝8.8Hz,1H),7.34(d,J＝8.6Hz,2H),6.45(d,J＝7.6Hz,1H),4.85(s,1H),3.89–3.65(m,4H),3.16(t,J＝4.8Hz,4H),2.29(dq,J＝14.5,7.4Hz,1H),2.02(dd,J＝13.0,6.3Hz,1H).

Example 21

(R) -N- (2-carbamoyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide hydrochloride (I-21)

Dissolving tert-butyl (R) -3- ((3- ((2-cyano-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate in 4.5mol/L hydrogen chloride dioxane solution, reacting for 2 hours at room temperature, concentrating under reduced pressure, pulping with diethyl ether, filtering, and drying in vacuum to obtain a yellow solid, namely the product.

¹H NMR(300MHz,DMSO-d₆)δ10.92(s,1H),9.40(s,1H),8.62(d,J＝7.6Hz,1H),8.43(d,J＝10.0Hz,3H),8.21(d,J＝7.4Hz,2H),7.91(s,1H),7.58(s,1H),7.45(d,J＝9.3Hz,1H),6.49(d,J＝7.6Hz,1H),4.75(s,1H),4.12–3.74(m,4H),3.56(s,1H),3.48(s,1H),3.35(s,3H),3.13(s,1H),2.94(s,2H),1.95(d,J＝17.4Hz,2H),1.67(dq,J＝18.9,10.6,8.8Hz,2H).

Example 22

(R) -N- (4-Morpholino-2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-22)

Step 1: preparation of 4- (4-nitro-3- (trifluoromethyl) phenyl) morpholine

Dissolving 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (2.01g, 9.61mmol) in morpholine (15ml), reacting at room temperature for 1 hour, adding water to precipitate a large amount of solid, filtering, and drying to obtain 2.61g of yellow solid. Yield: 98 percent.

¹H NMR(300MHz,Chloroform-d)δ8.03(d,J＝9.2Hz,1H),7.16(d,J＝2.8Hz,1H),6.95(dd,J＝9.2,2.8Hz,1H),3.96–3.79(m,4H),3.45–3.30(m,4H).

Step 2: preparation of 4-morpholino-2- (trifluoromethyl) aniline

4- (4-Nitro-3- (trifluoromethyl) phenyl) morpholine (880mg, 3.19mmol) was dissolved in methanol (45ml), 10% palladium on carbon (80mg) was added and the reaction was carried out under hydrogen atmosphere for 4 hours, filtered through celite and concentrated to dryness under reduced pressure to give a light brown oil, which was directly subjected to the next reaction.

And step 3: preparation of 5-chloro-N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide

4-Morpholino-2- (trifluoromethyl) aniline (795mg, 3.23mmol) was dissolved in dichloromethane (20ml), N-diisopropylethylamine (3.25g, 9.69mmol) was added, 5-chloropyrazolo [1,5-a ] pyrimidine-3-carbonyl chloride (976mg, 4.52mmol) was added under ice bath, reacted overnight at room temperature, diluted with dichloromethane, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and chromatographed on silica gel (dichloromethane/methanol) to give 1.27g of a yellow solid.

¹H NMR(300MHz,Chloroform-d)δ9.60(s,1H),8.73(s,1H),8.70(d,J＝7.3Hz,1H),8.18(d,J＝9.0Hz,1H),7.16(d,J＝2.9Hz,1H),7.11(dd,J＝8.9,2.9Hz,1H),7.01(d,J＝7.2Hz,1H),3.94–3.83(m,4H),3.25–3.13(m,4H).

And 4, step 4: preparation of tert-butyl (R) -3- ((3- ((4-morpholino-2- (trifluoromethyl) phenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate

5-chloro-N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (50mg, 117. mu. mol) and (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester (35mg, 176. mu. mol) were dissolved in DMSO (4ml), and anhydrous potassium fluoride (68mg, 1.17mmol) was added thereto, followed by reaction at 40 ℃ for 2 hours, dissolution in ethyl acetate, washing with water and saturated saline, drying over anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (dichloromethane/methanol) was performed to obtain 63mg of a pale yellow solid. Yield: 91 percent. ESI-MS: [ M + H ] ═ 590.1

And 5: preparation of (R) -N- (4-morpholino-2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-22)

Dissolving (R) -3- ((3- ((4-morpholino-2- (trifluoromethyl) phenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylic acid tert-butyl ester (63mg, 106 mu mol) in dichloromethane (4ml), adding trifluoroacetic acid (1ml), reacting at room temperature for 2 hours, concentrating under reduced pressure, adding diethyl ether, pulping, filtering, and drying in vacuum to obtain the product.

¹H NMR(300MHz,DMSO-d₆)δ9.18(s,1H),8.67(d,J＝7.7Hz,3H),8.22(d,J＝12.3Hz,2H),7.57(d,J＝8.9Hz,1H),7.27(d,J＝9.0Hz,1H),7.20(d,J＝2.8Hz,1H),6.44(d,J＝7.7Hz,1H),4.24(s,1H),3.76(t,J＝4.7Hz,4H),3.33(d,J＝46.4Hz,4H),3.23–3.08(m,4H),2.94(s,2H),1.94(d,J＝36.3Hz,2H),1.71–1.46(m,2H).

Example 23

5- (azetidin-3-ylamino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-23)

The procedure is as defined in example 22 except for the steps 4 and 5 of example 22, which are substituted for tert-butyl 3-aminopiperidine-1-carboxylate with tert-butyl 3-aminoazetidine-1-carboxylate, and the product is the trifluoroacetate salt of 5- (azetidin-3-ylamino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.29(s,1H),9.15–8.62(m,4H),8.31(s,1H),7.55(d,J＝8.7Hz,1H),7.39–7.11(m,2H),6.48(d,J＝7.6Hz,1H),4.78(d,J＝8.1Hz,1H),4.10(d,J＝46.7Hz,4H),3.76(s,4H),3.20(s,4H).

Example 24

(R) -N- (4-Morpholino-2- (trifluoromethyl) phenyl) -5- (pyrrolidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-24)

The procedure of example 22 was followed by steps 4 and 5 except for the raw materials, reagents and preparation method used in example 22, which were replaced with tert-butyl (R) -3-aminopiperidine-1-carboxylate, and the product obtained was trifluoroacetic acid salt of (R) -N- (4-morpholino-2- (trifluoromethyl) phenyl) -5- (pyrrolidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(400MHz,DMSO-d₆)δ9.29(s,1H),9.15(s,1H),8.93(s,1H),8.69(d,J＝7.6Hz,1H),8.48(d,J＝6.0Hz,1H),8.30(s,1H),7.56(d,J＝8.9Hz,1H),7.27(dd,J＝9.0,2.8Hz,1H),7.19(d,J＝2.8Hz,1H),6.43(d,J＝7.6Hz,1H),4.54(d,J＝8.1Hz,1H),3.76(t,J＝4.8Hz,4H),3.39–3.15(m,8H),2.23(dq,J＝14.9,7.6Hz,1H),2.00(dt,J＝12.2,6.3Hz,1H).

Example 25

(R) -5- (azepan-3-ylamino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-25)

The procedure is as in example 22, substituting (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester with (R) -3-aminoazepane-1-carboxylic acid tert-butyl ester, and the remaining required starting materials, reagents and preparation procedures are as in example 22, and the product obtained is the trifluoroacetate salt of (R) -5- (azepan-3-ylamino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.24(s,1H),8.89(d,J＝26.2Hz,2H),8.67(d,J＝7.6Hz,1H),8.25(d,J＝11.0Hz,2H),7.55(d,J＝8.8Hz,1H),7.33–7.22(m,1H),7.20(d,J＝2.7Hz,1H),6.46(d,J＝7.6Hz,1H),4.34(s,1H),3.76(t,J＝4.7Hz,4H),2.01(s,1H),1.92–1.62(m,4H),1.51(s,1H).

Example 26

N- (4-Morpholino-2- (trifluoromethyl) phenyl) -5- (piperidin-4-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-26)

The procedure of example 22 was repeated except for using 4-aminopiperidine-1-carboxylic acid tert-butyl ester instead of (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester and the other necessary raw materials, reagents and preparation methods in the same manner as in steps 4 and 5, to give a product, i.e., trifluoroacetic acid salt of N- (4-morpholino-2- (trifluoromethyl) phenyl) -5- (piperidin-4-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.20(s,1H),8.64(d,J＝7.7Hz,1H),8.51(s,2H),8.24(d,J＝16.2Hz,2H),7.61(d,J＝8.9Hz,1H),7.27(d,J＝9.3Hz,1H),7.19(d,J＝2.7Hz,1H),6.42(d,J＝7.6Hz,1H),4.15(s,1H),3.76(t,J＝4.8Hz,4H),3.31(d,J＝12.6Hz,2H),3.19(t,J＝4.9Hz,4H),2.05(d,J＝13.6Hz,2H),1.69(q,J＝11.6Hz,2H).

Example 27

5- (4-Aminopiperidin-1-yl) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-27)

The procedure of example 22 was repeated except for using tert-butyl (R) -3-aminopiperidine-1-carboxylate as the piperidine-4-ylcarbamate and the other necessary starting materials, reagents and preparation methods as in examples 4 and 5 to give a product, i.e., trifluoroacetic acid salt of 5- (4-aminopiperidin-1-yl) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.23(s,1H),8.85(d,J＝7.9Hz,1H),8.29(s,1H),7.94(s,3H),7.66(d,J＝9.0Hz,1H),7.34–7.23(m,1H),7.18(d,J＝2.8Hz,1H),6.95(d,J＝8.0Hz,1H),4.50(d,J＝13.4Hz,2H),3.76(t,J＝4.8Hz,4H),3.29–2.99(m,6H),1.99(d,J＝12.3Hz,2H),1.63–1.37(m,2H).

Example 28

5- ((2-aminoethyl) amino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-28)

The procedure of example 22 was repeated except for using tert-butyl (R) -3-aminopiperidine-1-carboxylate as tert-butyl (2-aminoethyl) carbamate and the other necessary raw materials, reagents and preparation methods as in steps 4 and 5, to give a product, i.e., trifluoroacetic acid salt of 5- ((2-aminoethyl) amino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.31(s,1H),8.67(d,J＝7.5Hz,1H),8.28(d,J＝9.3Hz,2H),7.92(s,3H),7.61(d,J＝8.9Hz,1H),7.33–7.23(m,1H),7.19(d,J＝2.8Hz,1H),6.45(d,J＝7.6Hz,1H),3.76(t,J＝4.7Hz,4H),3.59(q,J＝6.9,6.3Hz,2H),3.18(t,J＝4.8Hz,4H),3.04(t,J＝5.7Hz,2H).

Example 29

(R) -N- (4-morpholino-2- (trifluoromethyl) phenyl) -5- (quinuclidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-29)

The procedure of example 22 was followed except for using (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester as (R) -3-aminoquinine and the other necessary raw materials, reagents and preparation methods as in example 4 to obtain (R) -N- (4-morpholino-2- (trifluoromethyl) phenyl) -5- (quinuclidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,DMSO-d₆)δ9.22(s,1H),8.65(d,J＝7.8Hz,2H),8.22(s,1H),7.55(d,J＝8.9Hz,1H),7.26(d,J＝9.5Hz,1H),7.19(d,J＝2.8Hz,1H),6.57(d,J＝7.6Hz,1H),4.25(s,1H),3.75(t,J＝4.7Hz,4H),2.97(dd,J＝30.9,14.3Hz,5H),2.17(s,1H),1.99(s,1H),1.66(m,4H).

Example 305- ((2-Aminocyclohexyl) amino) -N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-30)

(R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester was replaced with (2-aminocyclohexyl) carbamic acid tert-butyl ester, and the remaining required raw materials, reagents and preparation methods were the same as in steps 4 and 5 of example 22, and the product was obtained.

¹H NMR(300MHz,DMSO-d₆)δ9.23(s,1H),8.68(d,J＝7.6Hz,1H),8.24(s,1H),7.99(d,J＝8.4Hz,1H),7.88(s,3H),7.61(d,J＝8.9Hz,1H),7.27(d,J＝8.8Hz,1H),7.19(d,J＝2.8Hz,1H),6.61(d,J＝7.7Hz,1H),4.52(s,1H),3.76(t,J＝4.8Hz,4H),3.43(s,1H),3.19(t,J＝4.8Hz,4H),1.69(m,6H),1.41(s,2H).

Example 31

(R) -N- (2-methoxy-4- (pyrrolidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-31)

Step 1: preparation of 1- (3-methoxy-4-nitrophenyl) pyrrolidine

The morpholine is replaced by the pyrrolidine, and the other needed raw materials, reagents and preparation methods are the same as the step 6 in the example 1, and the obtained product is the preparation of the 1- (3-methoxy-4-nitrophenyl) pyrrolidine.

¹H NMR(300MHz,Chloroform-d)δ8.04(d,J＝9.3Hz,1H),6.11(dd,J＝9.3,2.4Hz,1H),5.94(d,J＝2.4Hz,1H),3.52–3.30(m,4H),2.19–1.96(m,4H).

Step 2: preparation of 2-methoxy-4- (pyrrolidin-1-yl) aniline

The 4- (3-methoxy-4-nitrophenyl) morpholine was replaced by 1- (3-methoxy-4-nitrophenyl) pyrrolidine, the remaining required starting materials, reagents and preparation were the same as in example 1, step 7, and the product obtained was directly subjected to the next reaction.

And step 3: preparation of (R) -N- (2-methoxy-4- (pyrrolidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (I-31)

The trifluoroacetic acid salt of (R) -N- (2-methoxy-4- (pyrrolidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide can be obtained by replacing 2-methoxy-4-morpholinoaniline with 2-methoxy-4- (pyrrolidin-1-yl) aniline, and the remaining necessary starting materials, reagents and preparation method are the same as in step 8 of example 1. The resulting solid was dissolved in saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and subjected to preparative thin layer chromatography to give (R) -N- (2-methoxy-4- (pyrrolidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(300MHz,Chloroform-d)δ9.64(s,1H),8.39(s,1H),8.19(d,J＝8.5Hz,2H),6.18(dd,J＝18.6,9.3Hz,4H),4.38(s,1H),3.89(s,3H),3.28(s,4H),3.15–2.66(m,4H),1.94m,9H).

Example 32

(R) -N- (2-methoxy-4- (piperidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-32)

The trifluoroacetate salt of (R) -N- (2-methoxy-4- (piperidin-1-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 1.

¹H NMR(300MHz,DMSO-d₆)δ9.63(s,1H),8.68(d,J＝7.6Hz,3H),8.24(s,3H),7.03(d,J＝53.1Hz,2H),6.50(d,J＝7.6Hz,1H),4.37(s,1H),3.90(s,3H),3.53–2.95(m,8H),2.04(s,2H),1.71(m,7H).

Example 33

(R) -N- (2-methyl-6-morpholinopyridin-3-yl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-33)

The 2-methoxy-4-morpholinoaniline was replaced with 4-methyl-6-morpholinopyridin-3-amine, and the remaining required raw materials, reagents and preparation methods were the same as in step 8 of example 1, and the obtained product was (R) -N- (2-methyl-6-morpholinopyridin-3-yl) -5- (piperidin-3-ylamino) pyrazolo [1, 5-a)]Trifluoroacetic acid salt of pyrimidine-3-carboxamide.¹H NMR(300MHz,DMSO-d₆)δ9.04(s,1H),8.82(s,1H),8.67(d,J＝7.6Hz,2H),8.23(s,2H),7.82(d,J＝8.8Hz,1H),6.73(d,J＝8.9Hz,1H),6.45(d,J＝7.7Hz,1H),4.32(s,1H),3.71(t,J＝4.7Hz,4H),3.59–3.08(m,6H),2.97(s,2H),2.38(s,3H),2.13–1.84(m,2H),1.77–1.48(m,2H).

Example 34

(R) -N- (5-bromo-2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-34)

Step 1: preparation of 4- (2-bromo-5-methyl-4-nitrophenyl) morpholine

4- (2-bromo-5-methyl-4-nitrophenyl) morpholine was obtained by replacing 2-bromo-4-fluoro-1-nitrobenzene with 1-bromo-2-fluoro-4-methyl-5-nitrobenzene, and the remaining required raw materials, reagents and preparation methods were the same as in step 1 of example 15.

¹H NMR(400MHz,Chloroform-d)δ8.31(d,J＝1.3Hz,1H),6.83(s,1H),3.97–3.82(m,4H),3.24–3.09(m,4H),2.60(d,J＝1.3Hz,3H).

Step 2: preparation of 5-bromo-2-methyl-4-morpholinoaniline

5-bromo-2-methyl-4-morpholinoaniline can be obtained by replacing 4- (3-bromo-4-nitrophenyl) morpholine with 4- (2-bromo-5-methyl-4-nitrophenyl) morpholine and the remaining necessary raw materials, reagents and preparation method are the same as in step 2 of example 15.

¹H NMR(400MHz,Chloroform-d)δ6.91(s,1H),6.78(s,1H),3.92–3.77(m,4H),3.48(s,2H),2.99–2.86(m,4H),2.12(s,3H).

And step 3: preparation of N- (5-bromo-2-methyl-4-morpholinophenyl) -5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxamide

The 4-morpholino-2- (trifluoromethyl) aniline was replaced with 5-bromo-2-methyl-4-morpholino aniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 3 of example 22.

¹H NMR(400MHz,Chloroform-d)δ9.32(s,1H),8.80–8.67(m,2H),8.55(s,1H),7.03(d,J＝7.2Hz,1H),6.91(d,J＝0.8Hz,1H),3.94–3.78(m,4H),3.10–2.94(m,4H).

And 4, step 4: preparation of tert-butyl (R) -3- ((3- ((5-bromo-2-methyl-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate

Tert-butyl (R) -3- ((3- ((5-bromo-2-methyl-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-carboxylate can be obtained by substituting 5-chloro-N- (4-morpholino-2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide with N- (5-bromo-2-methyl-4-morpholinophenyl) -5-chloropyrazolo [1,5-a ] pyrimidine-3-carboxamide, and the remaining required starting materials, reagents and preparation method are the same as in step 4 of example 22.

¹H NMR(400MHz,DMSO-d₆)δ9.15(s,1H),8.63(d,J＝7.6Hz,1H),8.36(s,1H),8.23(s,1H),8.11(d,J＝7.3Hz,1H),7.06(s,1H),6.47(s,1H),4.02(s,1H),3.74(t,J＝4.5Hz,4H),2.90(t,J＝4.6Hz,4H),1.95(d,J＝31.9Hz,1H),1.79(s,1H),1.66–0.84(m,13H).

And 5: preparation of (R) -N- (5-bromo-2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-34)

(R) -tert-butyl 3- ((3- ((4-morpholino-2- (trifluoromethyl) phenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate was replaced by tert-butyl (R) -3- ((3- ((5-bromo-2-methyl-4-morpholinophenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate, and the remaining required starting materials, reagents and preparation were the same as in step 5 of example 24, to give (R) -N- (5-bromo-2-methyl-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1, trifluoroacetic acid salt of 5-a ] pyrimidine-3-carboxamide.

¹H NMR(400MHz,DMSO-d₆)δ9.18–8.98(m,1H),8.95–8.54(m,3H),8.25(s,2H),8.11(s,1H),7.11(s,1H),6.45(d,J＝7.7Hz,1H),4.37(s,1H),3.75(t,J＝4.5Hz,4H),3.35(d,J＝23.5Hz,6H),3.18(s,1H),2.94(t,J＝4.6Hz,6H),2.31(s,3H),2.04(s,1H),1.93(s,1H),1.74–1.51(m,2H).

Example 35

(R) -N- (2-bromo-3-fluoro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-35)

The trifluoroacetate salt of (R) -N- (2-bromo-3-fluoro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that in example 34.

¹H NMR(400MHz,DMSO-d₆)δ9.39(s,1H),8.69(q,J＝23.9,18.9Hz,3H),8.27(s,2H),7.73(d,J＝8.9Hz,1H),7.13(t,J＝9.2Hz,1H),6.47(d,J＝7.6Hz,1H),4.44(s,1H),3.75(t,J＝4.5Hz,4H),3.19(d,J＝12.1Hz,1H),3.01(t,J＝4.6Hz,6H),2.09(s,1H),1.91(s,1H),1.71(d,J＝11.4Hz,1H),1.59(t,J＝10.6Hz,1H).

Example 36

(R) -N- (2-fluoro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-36)

The 2-methoxy-4-morpholinobenzene was replaced with 2-fluoro-4-morpholinoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 8 of example 1, and the obtained product was the trifluoroacetate salt of (R) -N- (2-fluoro-4-morpholinophenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(400MHz,DMSO-d₆)δ9.62(s,1H),8.95–8.49(m,3H),8.26(d,J＝8.7Hz,2H),8.14(t,J＝9.2Hz,1H),6.96–6.71(m,2H),6.47(d,J＝7.6Hz,1H),4.33(s,1H),3.74(t,J＝4.7Hz,4H),3.26–2.90(m,7H),2.09(s,1H),1.96(s,1H),1.79(s,1H),1.64(d,J＝11.1Hz,1H).

Example 37

N- (4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) phenyl) -5- (((R) -piperidin-3-yl) amino) pyrazolo [1,5- ] pyrimidine-3-carboxamide trifluoroacetate (I-37)

Step 1: preparation of (2S, 6R) -2, 6-dimethyl-4- (4-nitro-3- (trifluoromethyl) phenyl) morpholine

4-fluoro-1-nitro-2- (trifluoromethyl) benzene (300mg, 1.43mmol) was dissolved in N, N-dimethylformamide (8ml), potassium carbonate (396mg, 2.87mmol) and cis-2, 6-dimethylmorpholine (247mg, 2.15mmol) were added and reacted at 40 ℃ for 2 hours, and a large amount of solid was precipitated by adding water, followed by suction filtration and drying to obtain 418mg of a yellow solid. Yield: 78 percent.

¹H NMR(400MHz,Chloroform-d)δ8.03(d,J＝9.2Hz,1H),7.14(d,J＝2.8Hz,1H),6.93(dd,J＝9.2,2.8Hz,1H),3.76(dtt,J＝12.5,6.3,3.1Hz,2H),3.69–3.60(m,2H),2.64(dd,J＝12.4,10.6Hz,2H),1.30(d,J＝6.2Hz,6H).

Step 2: 4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) aniline

Dissolving (2S, 6R) -2, 6-dimethyl-4- (4-nitro-3- (trifluoromethyl) phenyl) morpholine (150mg, 492 mu mmol) in ethanol/water (6ml/2ml), adding ammonium chloride (210mg, 3.94mmol) and reduced iron powder (220mg, 3.94mmol), reacting at 50 ℃ for 2 hours, cooling to room temperature, filtering with diatomite, concentrating under reduced pressure, dissolving with ethyl acetate, washing with water and saturated salt solution, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure to dryness, and directly putting into the next reaction.

And step 3: preparation of 5-chloro-N- (4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide

5-chloro-N- (4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be obtained by substituting 4-morpholino-2- (trifluoromethyl) aniline for 4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) aniline, and the remaining required starting materials, reagents, and preparation method are the same as in step 3 of example 22.

¹H NMR(400MHz,Chloroform-d)δ9.59(s,1H),8.73(s,1H),8.70(d,J＝7.2Hz,1H),8.16(d,J＝9.0Hz,1H),7.15(d,J＝2.9Hz,1H),7.10(dd,J＝8.9,2.9Hz,1H),7.01(d,J＝7.3Hz,1H),3.90–3.73(m,2H),3.54–3.38(m,2H),2.52–2.38(m,2H),1.28(d,J＝6.3Hz,6H).

And 4, step 4: preparation of (R) -3- ((3- ((4- ((2S, 6R) -2, 6-dimethylmorpholino) -2- (trifluoromethyl) phenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidin-tert-butyl-5-yl) amino) piperidine-1-carboxylic acid tert-butyl ester

5-chloro-N- (4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide (58mg, 128. mu. mol) and (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester (38mg, 191. mu. mol) were dissolved in DMSO (5ml), and anhydrous potassium fluoride (74mg, 1.28mmol) was added thereto, reacted at 40 ℃ for 2 hours, dissolved in ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography (dichloromethane/methanol) to give 70mg of an orange-yellow solid.

¹H NMR(400MHz,Chloroform-d)δ9.38(s,1H),8.43(s,1H),8.27(d,J＝7.6Hz,1H),7.93(s,1H),7.09(d,J＝8.0Hz,2H),6.15(s,1H),5.37(s,1H),3.81(ddd,J＝9.7,6.3,2.5Hz,2H),3.70–3.14(m,6H),2.44(dd,J＝11.9,10.4Hz,2H),1.33(m,9H).

And 5: preparation of N- (4- ((2S, 6R) -2, 6-dimethylmorpholine) -2- (trifluoromethyl) phenyl) -5- (((R) -piperidin-3-yl) amino) pyrazolo [1,5- ] pyrimidine-3-carboxamide trifluoroacetate (I-37)

Dissolving (R) -3- ((3- ((4- ((2S, 6R) -2, 6-dimethylmorpholino) -2- (trifluoromethyl) phenyl) carbamoyl) pyrazolo [1,5-a ] pyrimidine-tert-butyl-5-yl) amino) piperidine-1-carboxylic acid tert-butyl ester (65mg, 113 mu mol) in dichloromethane (4ml), adding trifluoroacetic acid (1ml), reacting at room temperature for 2 hours, concentrating under reduced pressure, adding diethyl ether, pulping, filtering, and drying under vacuum to obtain the product.

¹H NMR(400MHz,DMSO-d₆)δ9.17(s,1H),8.68(q,J＝18.4,13.5Hz,3H),8.22(d,J＝11.0Hz,2H),7.56(d,J＝8.8Hz,1H),7.26(d,J＝9.2Hz,1H),7.21(s,1H),6.44(d,J＝7.6Hz,1H),4.24(s,1H),3.69(d,J＝11.0Hz,4H),3.27(d,J＝11.9Hz,1H),3.13(s,1H),2.93(s,2H),2.30(t,J＝11.3Hz,2H),1.99(d,J＝11.6Hz,1H),1.89(s,1H),1.58(q,J＝13.7,11.1Hz,2H),1.17(d,J＝6.0Hz,6H).

Example 38

(R) -N- (4- (4-acetylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-38)

The trifluoroacetate salt of (R) -N- (4- (4-acetylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 37.

¹H NMR(400MHz,DMSO-d₆)δ9.18(s,1H),8.67(q,J＝17.5,16.4Hz,3H),8.32–8.17(m,2H),7.58(d,J＝8.9Hz,1H),7.28(d,J＝9.1Hz,1H),7.23(s,1H),6.44(d,J＝7.6Hz,1H),4.24(s,1H),3.60(s,4H),3.26(d,J＝4.6Hz,3H),3.16(d,J＝25.4Hz,3H),2.92(d,J＝12.9Hz,2H),2.08–2.03(m,3H),1.99(d,J＝12.2Hz,1H),1.89(s,1H),1.58(q,J＝13.1,11.1Hz,2H).

Example 39

(R) -N- (2-methyl-4- ((tetrahydro-2H-pyran-4-yl) amino) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-39)

The trifluoroacetate salt of (R) -N- (2-methyl-4- ((tetrahydro-2H-pyran-4-yl) amino) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method as in example 17.

¹H NMR(400MHz,DMSO-d₆)δ8.99(s,1H),8.69(t,J＝17.8Hz,3H),8.21(d,J＝15.3Hz,2H),7.51(s,1H),6.74(s,2H),6.44(d,J＝7.7Hz,1H),3.50(s,1H),3.38(t,J＝11.5Hz,3H),3.16(s,1H),2.98(s,2H),2.24(s,3H),2.07(s,1H),2.01–1.79(m,3H),1.60(dd,J＝24.4,13.6Hz,2H),1.44(d,J＝12.3Hz,2H).

Example 40

(R) -N- (4 '-fluoro-3-methyl- [1,1' -biphenyl ] -4-yl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt (I-40)

Step 1: 4- (4-fluorophenyl) -2-methylaniline

4-bromo-2-methylaniline (200mg, 1.07mmol) and 4-fluorobenzeneboronic acid (225mg, 1.61mmol) were dissolved in water/dioxane (5ml/10ml), potassium carbonate (297mg, 2.15mmol) and tetrakis (triphenylphosphine) palladium (62mg, 53. mu.M) were added, the reaction was carried out at 100 ℃ for 10 hours under argon substitution protection, suction filtration was carried out with celite, dilution with water was carried out, extraction was carried out with ethyl acetate, drying was carried out with anhydrous sodium sulfate, filtration was carried out, and silica gel column chromatography (petroleum ether/ethyl acetate) was carried out to obtain 128mg of a white solid. Yield: 84 percent.

¹H NMR(400MHz,DMSO-d₆)δ7.65–7.49(m,2H),7.33–7.10(m,4H),6.66(d,J＝8.1Hz,1H),4.97(s,2H),2.11(s,3H).

Step 2: preparation of (R) -N- (4 '-fluoro-3-methyl- [1,1' -biphenyl ] -4-yl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-40)

The 2-methoxy-4-morpholinobenzene was replaced with 4- (4-fluorophenyl) -2-methylaniline, and the remaining required raw materials, reagents and preparation method were the same as in step 8 of example 1, and the obtained product was trifluoroacetic acid salt of (R) -N- (4 '-fluoro-3-methyl- [1,1' -biphenyl ] -4-yl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide.

¹H NMR(400MHz,DMSO-d₆)δ9.20(s,1H),8.75(s,2H),8.69(d,J＝7.6Hz,1H),8.28(s,1H),8.24(d,J＝7.6Hz,1H),7.97(d,J＝8.4Hz,1H),7.80–7.66(m,2H),7.59(d,J＝2.2Hz,1H),7.51(dd,J＝8.3,2.2Hz,1H),7.38–7.20(m,2H),6.46(d,J＝7.6Hz,1H),4.40(s,1H),3.19(d,J＝12.7Hz,1H),3.00(dd,J＝18.9,10.1Hz,2H),2.42(s,3H),2.07(d,J＝11.7Hz,1H),1.94(s,1H),1.78–1.53(m,2H).

EXAMPLE 41

(R) -N- (2-methyl-4- (1-methyl-1H-pyrazol-4-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate (I-41)

The trifluoroacetate salt of (R) -N- (2-methyl-4- (1-methyl-1H-pyrazol-4-yl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a method similar to that in example 40.

¹H NMR(400MHz,DMSO-d₆)δ9.14(s,1H),8.80(s,1H),8.68(d,J＝7.7Hz,2H),8.25(d,J＝10.5Hz,2H),8.09(s,1H),7.94–7.72(m,2H),7.49(s,1H),7.40(d,J＝8.4Hz,1H),6.45(d,J＝7.7Hz,1H),4.38(s,1H),3.86(s,3H),3.18(s,1H),2.97(s,2H),2.36(s,3H),2.07(s,1H),1.93(s,1H),1.78–1.49(m,2H).

Example 42

(R) -N- (4- (dimethylamino) -2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt

(R) -N- (4- (dimethylamino) -2- (trifluoromethyl) phenyl) -5- (piperidin-3-ylamino) pyrazolo [1,5-a ] can be prepared by a method similar to that in example 37]Trifluoroacetic acid salt of pyrimidine-3-carboxamide.¹H NMR(400MHz,DMSO-d₆)δ9.16(s,1H),8.66(d,J＝7.6Hz,1H),8.52(d,J＝7.6Hz,1H),8.21(s,1H),7.49(d,J＝8.9Hz,1H),7.01(dd,J＝9.0,2.9Hz,1H),6.92(d,J＝3.0Hz,1H),6.49(d,J＝7.6Hz,1H),4.23(s,1H),3.25–3.19(m,1H),3.09–3.01(m,1H),2.97(s,6H),2.94–2.87(m,1H),1.95(d,J＝29.2Hz,2H),1.59(s,2H).

Example 43

(R) -5- (3-aminopiperidin-1-yl) -N- (2-chloro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt

Trifluoroacetic acid salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-chloro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that of example 19

¹H NMR(400MHz,DMSO-d₆)δ9.51(s,1H),8.89(d,J＝7.9Hz,1H),8.31(s,1H),8.24–7.90(m,4H),7.05(d,J＝2.7Hz,1H),6.99(dd,J＝9.1,2.8Hz,1H),6.86(d,J＝8.0Hz,1H),4.39–4.20(m,1H),4.19–4.05(m,1H),3.74(t,J＝4.7Hz,4H),3.67–3.49(m,2H),3.10(t,J＝4.8Hz,4H),2.12–2.00(m,1H),1.90–1.77(m,1H),1.75–1.52(m,2H).

Example 44

(R) -5- (3-aminopiperidin-1-yl) -N- (2-bromo-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt

Trifluoroacetic acid salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-bromo-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that of example 19

¹H NMR(400MHz,DMSO-d₆)δ9.39(s,1H),8.88(d,J＝7.9Hz,1H),8.31(s,1H),8.04(s,3H),7.92(d,J＝9.0Hz,1H),7.19(d,J＝2.7Hz,1H),7.03(dd,J＝9.1,2.8Hz,1H),6.87(d,J＝8.0Hz,1H),4.29(s,1H),4.09(d,J＝29.3Hz,1H),3.74(t,J＝4.8Hz,4H),3.64–3.48(m,2H),3.11(t,J＝4.8Hz,4H),2.02(s,1H),1.82(s,1H),1.75–1.53(m,2H).

Example 45

(R) -5- (3-aminopiperidin-1-yl) -N- (2-bromo-5-fluoro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate salt

Trifluoroacetic acid salt of (R) -5- (3-aminopiperidin-1-yl) -N- (2-bromo-5-fluoro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to that of example 19

¹H NMR(400MHz,DMSO-d₆)δ9.84(d,J＝2.6Hz,1H),8.88(d,J＝8.0Hz,1H),8.74(d,J＝8.3Hz,1H),8.32(s,1H),8.08(s,3H),7.18(d,J＝12.8Hz,1H),6.87(s,1H),4.21(d,J＝13.4Hz,1H),4.09(s,1H),3.75(t,J＝4.3Hz,4H),3.69–3.48(m,2H),2.94(t,J＝4.4Hz,4H),2.05(s,1H),1.85(s,1H),1.79–1.55(m,2H).

Example 46

5- ((3R, 5R) -3-amino-5-fluoropiperidin-1-yl) -N- (2-chloro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate

Trifluoroacetate salt of 5- ((3R, 5R) -3-amino-5-fluoropiperidin-1-yl) -N- (2-chloro-4-morpholinophenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to example 19

¹H NMR(400MHz,DMSO-d₆)δ9.48(s,1H),8.94(d,J＝7.9Hz,1H),8.34(s,1H),8.26–7.98(m,4H),7.05(d,J＝2.7Hz,1H),7.00(dd,J＝9.1,2.8Hz,1H),6.90(d,J＝7.7Hz,1H),5.13(d,J＝46.2Hz,1H),3.85–3.66(m,4H),3.55(d,J＝14.6Hz,1H),3.50–3.38(m,2H),3.30–3.20(m,1H),3.11(t,J＝4.8Hz,4H),2.37(s,1H),1.94(dt,J＝39.8,12.6Hz,1H).

Example 47

5- ((3R, 5R) -3-amino-5-fluoropiperidin-1-yl) -N- (2-chloro-4- ((2S, 6R) -2, 6-dimethylmorpholino) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide trifluoroacetate

Trifluoroacetate salt of 5- ((3R, 5R) -3-amino-5-fluoropiperidin-1-yl) -N- (2-chloro-4- ((2S, 6R) -2, 6-dimethylmorpholino) phenyl) pyrazolo [1,5-a ] pyrimidine-3-carboxamide may be prepared by a similar method to example 19

¹H NMR(400MHz,DMSO-d₆)δ9.46(s,1H),8.94(d,J＝7.9Hz,1H),8.34(s,1H),8.14(s,3H),8.07(d,J＝9.1Hz,1H),7.06(d,J＝2.7Hz,1H),6.99(dd,J＝9.1,2.7Hz,1H),6.91(s,1H),5.13(d,J＝46.2Hz,1H),3.75–3.63(m,2H),3.59(d,J＝11.7Hz,2H),3.48–3.35(m,4H),3.32–3.18(m,1H),2.37(s,1H),2.24(t,J＝11.1Hz,2H),1.95(dt,J＝39.9,12.5Hz,1H),1.16(d,J＝6.2Hz,6H).

As can be seen from fig. 1: compound I-1 is highly selective for protein tyrosine kinases at a concentration of 0.1. mu.M.

As can be seen from fig. 2: compound I-1 is highly selective for protein tyrosine kinases at a concentration of 1. mu.M.

As can be seen from fig. 3: the compound I-19 has better selectivity to protein tyrosine kinase under the concentration of 0.1 mu M.

As can be seen from fig. 4: the compound I-19 has better selectivity to protein tyrosine kinase under the concentration of 1 mu M.

FIG. 5 is a compound

Kinase selectivity results at a concentration of 0.1. mu.M.

As can be seen from fig. 5: the compound has poor selectivity to protein tyrosine kinase under the concentration of 0.1 mu M.

FIG. 6 is a compound

Kinase selectivity results at a concentration of 1 μ M.

As can be seen from fig. 6: the compound is poorly selective for protein tyrosine kinases at 1 μ M concentrations.

The above results show that: the selectivity of the compound on protein tyrosine kinase is obviously better than that of the IRAK4 inhibitor disclosed in WO 2016/144846.

Interleukin-1receptor-associated kinase 4 (IRAK 4) inhibitor compounds disclosed for WO 2016/144846.

Example 56

Effect of Compounds on cell proliferation of leukemia cell lines

1. Experimental Material

Leukemia cell lines: acute myelogenous leukemia cell strain MV4-11 (expressing FLT3-ITD mutant gene) MOLM-3 (expressing FLT3-ITD mutant gene and wild type FLT3 gene) acute monocytic leukemia cell strain THP-1

2. Test method

Leukemia cells in logarithmic growth phase were seeded at appropriate density into 96-well culture plates at 90. mu.L per well, after overnight incubation, compounds at different concentrations (1. mu.M for initial concentration, 5-fold dilution) were added for 72hr, and a solvent control group (negative control) was set. After the compound acts on cells for 72 hours, the influence of the compound on cell proliferation is detected by a CCK-8 cell counting kit (Shanghai Liji Biotech), 10 mu L of CCK-8 reagent is added into each hole, the hole is placed in an incubator at 37 ℃ for 2 to 4 hours, then a SpectraMax 190 reading is carried out by a full-wavelength micro-hole plate enzyme-labeling instrument, and the measurement wavelength is 450 nm. The inhibition (%) of the tumor cell growth by the compound was calculated using the following formula: the inhibition ratio (%) (OD negative control well-OD administration well)/OD negative control well × 100%. IC (integrated circuit)₅₀The values were determined by regression with a four parameter method using a microplate reader random plus software.

3. Results of the experiment

As can be seen from the results in Table 2, the compounds of the present invention significantly inhibited the cell proliferation activity of acute myeloid leukemia cell lines MV4-11 and MOLM-3. Wherein the cell proliferation inhibitory activity of the compounds I-14, I-16, I-17, I-20, I-24 and I-27 is slightly superior or approximately equivalent to that of the clinical stage III FLT3 inhibitor AC220 (Quizartinib). Meanwhile, the compound has no cell proliferation inhibition activity on the acute monocytic leukemia cell strain THP-1, which shows that the compound has higher selectivity, and inhibits the cell proliferation of the acute myeloid leukemia cell strain by inhibiting the mutation activity of FLT3 and FLT 3-ITD.

TABLE 2 Effect of Compounds on MV4-11, MOLM-3 and THP-1 cell proliferation

Example 57

Detection of kinase Activity of FLT3 kinase and FLT3-ITD mutant kinase Compounds

1. Test method

The inhibition of the kinase activity by the compound was calculated by measuring the ability of the kinase to phosphorylate substrates by Enzyme-Linked Immunosorbent Assay (ELISA). The kinase used Flt-3 and Flt-3^ITD(available from Eurofins). The ELISA main steps are as follows: enzyme reaction substrate Poly (Glu, Tyr)_4:1Diluting to 2.5 mu g/hole with PBS without potassium ions, reacting at 37 ℃ for 12-16h, and coating an enzyme label plate for later use. Reaction buffer (50mM HEPES pH7.4, 20mM MgCl) was added to each well₂，0.1mM MnCl₂，0.2mM Na₃VO₄1mM DTT) was added to a compound or solvent control, followed by addition of kinase to initiate the reaction, followed by shaking at 37 ℃ for 1 h. The plate was washed three times with T-PBS and shaken at 37 ℃ for 0.5h with the addition of the PY99 antibody. After washing the plate with T-PBS, horseradish peroxidase-labeled goat anti-mouse IgG was added and the mixture was subjected to shake reaction at 37 ℃ for 0.5 hour. After washing the plate again, 0.03% H was added₂O₂And 2mg/mL OPD developing solution, and reacting for 1-10min at 25 ℃ in a dark place. 2M H was added₂SO₄The reaction was stopped and read using a tunable wavelength microplate reader (SpectraMax Plus384, Molecular Devices) at a wavelength of 490 nm. IC (integrated circuit)₅₀Values were obtained from the inhibition curves.

2. Results of the experiment

As can be seen from the results in Table 3, the compound of the invention has obvious inhibition effect on FLT3 kinase and FLT3-ITD mutant kinase in vitro, and compared with the lead compound of WO2016144846, the inhibition activity of FLT3 kinase and FLT3-ITD mutant kinase is obviously improved. Wherein the in vitro inhibitory activity of the compounds I-14, I-16, I-17, I-20, I-24, I-27FLT3 kinase and FLT3-ITD mutant kinase is superior to or approximately equivalent to that of the clinical phase III FLT3 inhibitor AC220(Quizartinib), and corresponds to the cell proliferation inhibitory activity of the acute myeloid leukemia.

TABLE 3 detection of kinase Activity of the Compounds FLT3 kinase and FLT3-ITD mutant kinase

Wherein,

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. a compound shown in formula (I), its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate,

in,

n is an integer selected from 0, 1, 2, 3, and 4;

Y is C or N;

Z is C or N;

R ¹ is independently selected from the group consisting of none, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, nitrile, carboxamido, substituted or unsubstituted C1- C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C6 cycloalkoxy, substituted or unsubstituted C3-C6 cycloalkylthio, said substitution means being replaced by one or Multiple halogen substitutions;

R ² is selected from the group consisting of substituted or unsubstituted ring A, -NR _o -(CH ₂ ) _m -NR _p R _q , -NR _o -(CH ₂ ) _m -ring A, -NR _p R _q , - (CH ₂ ) _m -Ring A, -O-(CH ₂ ) _m -Ring A, -S-(CH ₂ ) _m -Ring A, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3 -C8cycloalkyl, nitrile, carboxamido, -(CH ₂ ) _m -R _b , -O-(CH ₂ ) _m -R _b , -NH-(CH ₂ ) _m -R _b , -S- (CH ₂ ) _m -R _b , substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 heteroaryl containing 1-3 heteroatoms selected from N, O and S; wherein, The substitution refers to substitution with one or more substituents selected from the group consisting of -C(=O)-(C1-C6 alkyl), -S(=O) ₂ -(C1-C6 alkyl), C1 -C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, amino, ring A, halogen, hydroxyl;

R _o , R _p and R _q are each independently selected from the group consisting of H, C1-C6 alkyl, halogenated C1-C6 alkyl;

m is independently an integer selected from 0, 1, 2, 3, 4, 5;

R _b is selected from the group consisting of H, amino, C1-C6 alkylamino, hydroxy, C1-C6 alkoxy, mercapto, C1-C6 alkylthio;

^R is selected from the group consisting of H, C1-C6 alkyl, halogenated C1-C6 alkyl;

R ⁴ is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-containing 1-3 selected from N, O or S 7-membered saturated heterocycle, -(CH ₂ ) _m -R _b , a tricyclic bridged C6-C8 alkyl group sharing one N, the substitution refers to being substituted by one or more substituents selected from the group consisting of halogen, -OH, -N(R _c ) ₂ , C1-C3 alkyl;

Or ^R3 and R4 together with the nitrogen atom to which they are attached form a ^4-8 membered heterocycle containing 1-3 N and 0-3 selected from O, S, optionally by one or more R _a substitutions;

R _is independently selected from the group consisting of C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl, hydroxy, amino, nitrile, halogen;

R _c is independently selected from the group consisting of H, C1-C6 alkyl, halogenated C1-C6 alkyl.

2. The compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate, wherein, R ² is selected from the group consisting of substituted or unsubstituted ring A, -NR _o -(CH ₂ ) _m -ring A, -(CH ₂ ) _m -ring A, -O-(CH ₂ ) _m -ring A; The substitution refers to substitution with one or more substituents selected from the group consisting of -C(=O)-(C1-C6 alkyl), -S(=O) ₂ -(C1-C6 alkyl), C1 -C6 alkyl, ring A, halogen;

Ring A is selected from the group consisting of: 6-membered saturated heterocyclyl containing 1 N and 1 O, 6-membered saturated heterocyclyl containing 1 O, 6-membered saturated heterocyclyl containing 2 N, 1-membered saturated heterocyclyl 5-6 membered saturated heterocyclic group of N;

m and R _o are as defined in claim 1 .

3. The compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate, wherein,

R ³ is H;

R ⁴ is a substituted or unsubstituted 5-7-membered saturated heterocycle containing 1-3 N, and the substitution refers to being substituted by one or more substituents selected from the group consisting of halogen, -OH, -N(R _c ) ₂ ;

or ^R3 and R4 together with the nitrogen atom to which they are attached form a 5-7 ^membered heterocycle optionally substituted with one or more _Ra ;

R _a is independently selected from the group consisting of hydroxy, amino, halogen;

R _c is independently selected from the group consisting of H, C1-C6 alkyl.

4. The compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate, wherein,

The pharmaceutically acceptable salt is selected from the group consisting of:

Acid addition salts of compounds of formula (I) with acids selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, lactic acid, oxalic acid, adipic acid, glutaric acid , Malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid, phenylacetic acid, mandelic acid.

5. The compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate, wherein, The compound is selected from the following compounds:

6. a preparation method of the compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate , is characterized in that, described method is selected from following method:

method one:

In a polar aprotic solvent, under the action of potassium fluoride, compound (II) and compound (III) undergo a substitution reaction to prepare the compound of formula (I);

Method Two:

Under the action of a condensing agent, compound (IV) and compound (V) undergo a condensation reaction to prepare the compound of formula (I);

Wherein, the definitions of the above n, Y, Z, R ¹ , R ² , R ³ , and R ⁴ are described in claim 1 .

7. A pharmaceutical composition, characterized in that, comprises the following components:

1) A therapeutically effective amount of one or more compounds of claim 1, their stereoisomers, geometric isomers, tautomers, their pharmaceutically acceptable salts, their prodrugs, and their hydrates or solvate; and

2) A pharmaceutically acceptable carrier or excipient.

8. a compound of claim 1, its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate or claim 7 purposes of the described pharmaceutical composition, it is characterised in that, for the purposes selected from the following group:

1) Preparation of a medicament for the prevention and/or treatment of FLT3-mediated diseases;

2) Preparation of a drug for inhibiting FLT3.

9. The use of claim 8, wherein the FLT3-mediated disease is selected from the group consisting of cancer, immune disease.

10. A FLT3 inhibitor, characterized in that the inhibitor comprises one or more compounds of claim 1, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable compounds thereof. Accepted salts, prodrugs thereof, hydrates or solvates thereof.