WO2020114465A1 - Preparation and application of heteroaromatic iminazole compound - Google Patents

Abstract

Provided are the preparation and application of an aromatic amide compound. Specifically, provided is a compound represented by the following formula I, the definition of each group being as described in the description. The compound has TRK kinase inhibitory activity and may be used as a pharmaceutical composition for treating TRK dysfunction-related diseases.

Description

Preparation and application of a class of imidazo aromatic ring compounds Technical field

The present invention relates to the field of small molecule drugs, in particular, the present invention relates to a TRK kinase inhibitor and its preparation and use.

Background technique

Tropomyosin receptor kinase (tropomyosin-receptorkinase, TRK) is a type of nerve growth factor receptor, which belongs to the receptor tyrosine kinase family, mainly including three highly homologous members TRKA, TRKB and TRKC, respectively It is encoded by the three genes NTRK1, NTRK2 and NTRK3. These receptor tyrosine kinases are mainly expressed in nerve tissues, and play an important role in the development and physiological functions of the nervous system through the activation of neurotrophins (neurotrophins). TRK acts as a tyrosine kinase receptor, and each TRK has its corresponding ligand to bind and activate its downstream signaling pathway. NGF (nerve growth factor) specifically binds and activates TRKA; TRKB ligands include BDGF (brain-derived growth factor) and NT-4/5 (neurotrophin-4/5); NT-3 specifically binds and activates TRKC . All three TRK receptors contain an extracellular domain for ligand binding, a transmembrane domain, and an intracellular domain with kinase activity.

When a specific ligand binds to the extracellular domain of the corresponding receptor, it will trigger oligomerization of the receptor and phosphorylation of specific tyrosine residues in the intracytoplasmic kinase domain, resulting in downstream signaling pathways such as Ras/ The activation of MAPK, PLCγ/PKC and PI3K/AKT signaling pathways, in turn, regulates a series of physiological processes such as neural cell proliferation, differentiation and survival (Bergman, et al. 1999). The TRK signaling pathway is usually precisely regulated, and its abnormal activation is closely related to tumorigenesis (Amatu, et al. 2016). The results show that there are many mechanisms that cause abnormal activation of the TRK pathway, including gene fusion, protein overexpression, and single nucleotide mutations. These abnormalities are closely related to the pathogenesis of tumors, especially NTRK gene fusion has been proven to cause multiple An important factor for tumorigenesis and does not depend on the tissue source and type of tumor. With the rapid development of current second-generation sequencing technology and precision medicine, more and more NTRK fusion genes have been discovered, such as ETV6-NTRK3, MPRIP–NTRK1, CD74–NTRK1, etc. The results of clinical trials in recent years have shown that these fusion genes are very effective anti-cancer targets, and tumors containing NTRK fusion genes have a very significant response rate to TRK inhibitors (Drilon, et al. 2018). Therefore, more and more TRK target inhibitors are reported, such as (WO2010048314, WO2011146336, WO2017004342). At the same time, in the clinical trial stage, it has been found that some patients receiving treatment have developed drug resistance and have been confirmed to be caused by some base mutations in the active region of the enzyme, such as NTRK1G595R or G667C mutation, NTRK3 G623R or G696A mutation, and The development of a new generation of TRK kinase inhibitors is expected to solve these problems.

In summary, there is an urgent need in the art to develop a new generation of TRK kinase inhibitors.

Summary of the invention

The object of the present invention is to provide a novel TRK kinase inhibitor.

In the first aspect of the present invention, a compound represented by the following formula I is provided:

among them,

R has the structure -L ₁ -R _A ;

L _{1 is} selected from the group consisting of substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms selected from N, S and O, or substituted or unsubstituted -(X) _y -, Wherein each of said X is independently selected from the group consisting of substituted or unsubstituted C ₁ -C ₈ alkylene, -O-, -C(=O)-, -CONH-, -NHCO-, -S -, -S(＝O)-, -S(＝O) ₂ -, -NH-;

y is selected from the group: 1, 2 or 3;

R _{A is} selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O;

Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , and Y ⁷ are each independently selected from the group consisting of CR ₁ or N; Z ¹ and Z ² are each independently selected from the group consisting of C or N;

And

Is an aromatic ring;

R _{1 is} independently selected from the group consisting of H, D, OH, Cl, F, and NH ₂ ;

R _{12 is} selected from the group consisting of H or C1-C4 alkyl;

Ring A is selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O (including Single ring, parallel ring);

L is selected from the group: none, -NR ₇ -, -O-;

R _{7 is} selected from: H, substituted C1-C6 alkyl; wherein the substitution means that one or more H atoms on the group are substituted by a substituent selected from the group consisting of halogen and hydroxyl;

Ring C is selected from the group consisting of substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted 3-12 membered cycloalkyl (including monocyclic, paracyclic, spiro or bridged ring), substituted or unsubstituted 4-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O (including monocyclic, paracyclic, spiro or bridged rings);

Unless otherwise specified, the "substituted" refers to being substituted by one or more (eg 2, 3, 4 etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halo C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methyl sulfone, -S(=O) ₂ NH ₂ , oxo(=O),- CN, hydroxyl, -NH ₂ , carboxyl, C1-C6 amide (-C(=O)-N(Rc) ₂ or -NH-C(=O)(Rc), Rc is H or C1-C5 alkane Group), C1-C6 alkyl-(C1-C6 amide group),

Or substituted or unsubstituted group selected from the group consisting of C1-C6 alkyl group, C3-C8 cycloalkyl group, C1-C6 amino group, C6-C10 aryl group, having 1-3 selected from N, S and 5-10 membered heteroaryl of hetero atom of O, 5-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH ₂ )-C6-C10 aryl,- (CH ₂ )-(5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S, and O), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 aryl, C1-C6 amine, C1-C6 amide, with 1-3 selected from N, S and O 5-10 membered heteroaryl of hetero atoms;

Is the attachment site of the group;

The additional condition is that the compound of formula I has a chemically stable structure.

In another preferred example, said L _{1 is} selected from the following group:

n is selected from the group: 0, 1, 2 or 3;

R ₂ , R _2a and R _2b are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted C ₁ -C ₈ alkyl;

X _{5 is} selected from the group consisting of NH, O, -CONH-, -NHCO-, S, -S(=O) ₂ -, -NHS(=O)-, -NHS(=O) ₂ -;

R _A is

Among them, the

Refers to the connection site of R _A and L ₁ ; X ⁴ is CH or N;

R _{3 is} selected from the group consisting of H, halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy.

In another preferred example, the ring C has the following structure:

Among them, k1, k2, k3 are independently selected from the following group: 0, 1, 2 or 3;

X ¹ and X ² are each independently C(R ₄ )R ₅ , NR ₆ or O;

Wherein R ₄ , R ₅ and R ₁₁ are independently selected from: H, OH, Me, halogen, oxo, CF ₃ , CN, NH ₂ , -OMe, COOH, CONH ₂ ; R _{6 is} selected from: none, H, Me, CONH ₂ .

In another preferred example, the ring C has the following structure:

Among them, m, p are independently selected from the following group: 0, 1, 2 or 3;

X ³ is C(R ₉ )R ₁₀ ;

Wherein, R ₈ , R ₉ and R ₁₀ are each independently selected from: H, OH, Me, halogen, CF ₃ , CN, NH ₂ , -OMe, COOH, CONH ₂ ;

In another preferred example, the ring C is connected to L through an N atom.

In another preferred example, the compound has the structure represented by the following formula II:

In another preferred example, the compound has the structure represented by the following formula III:

In another preferred example, the compound has a structure selected from the group consisting of:

According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising (1) the compound according to the first aspect of the present invention or its stereoisomer or tautomer, or a pharmaceutically acceptable thereof Salts, hydrates or solvates; (2) pharmaceutically acceptable carriers.

In another preferred example, the disease is selected from the group consisting of cancer, proliferative diseases, pain, skin diseases or disorders, metabolic diseases, muscle diseases, neurological diseases, autoimmune diseases, pruritus caused by dermatitis, inflammation-related Diseases, bone-related diseases.

In a third aspect of the present invention, there is provided a compound as described in the first aspect of the present invention or its stereoisomer or tautomer, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or The use of the pharmaceutical composition according to the second aspect of the present invention for the preparation and prevention of and/or treatment of TRK dysfunction (TRK gene amplification, or overexpression, or mutation, or dysfunctional activation caused by gene fusion) Pharmaceutical composition of related diseases.

In another preferred example, the disease is selected from the group consisting of cancer, proliferative diseases, pain, skin diseases or disorders, metabolic diseases, muscle diseases, neurological diseases, autoimmunity Itching caused by disease, dermatitis.

According to a fourth aspect of the present invention, there is provided a TRK inhibitor comprising the compound according to the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable thereof Salt, hydrate or solvate.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (eg, embodiments) can be combined with each other, thereby forming a new or preferred technical solution. Due to space limitations, I will not repeat them here.

detailed description

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

As used herein, when used in reference to specifically recited values, the term "about" means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including (including)" may be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

definition

As used herein, the term "alkyl" includes linear or branched alkyl groups. For example, C ₁ -C ₈ alkyl represents a linear or branched alkyl group having 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl Wait.

As used herein, the term "alkenyl" includes straight-chain or branched alkenyl. For example, C ₂ -C ₆ alkenyl refers to straight-chain or branched alkenyl with 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2 -Butenyl, or similar groups.

As used herein, the term "alkynyl" includes linear or branched alkynyl groups. For example, C ₂ -C ₆ alkynyl refers to a linear or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C ₃ -C ₈ cycloalkyl" means a cycloalkyl group having 3-8 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It may also be in the form of a double ring, such as a bridge ring or a spiral ring.

As used herein, the term "C ₁ -C ₈ alkoxy" refers to a linear or branched alkoxy group having 1-8 carbon atoms; for example, methoxy, ethoxy, propoxy, iso Propoxy, butoxy, isobutoxy, tert-butoxy, etc.

As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S, and O" means having 3-10 atoms and of which 1-3 atoms are A saturated or partially saturated cyclic group of heteroatoms selected from the group consisting of N, S, and O. It may be a single ring or a double ring, such as a bridge ring or a spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl and the like.

As used herein, the term "C ₆ -C ₁₀ aryl" refers to an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to having 5-10 atoms and wherein 1-3 atoms are selected from Cyclic aromatic groups of heteroatoms of N, S and O in the lower group. It can be monocyclic or fused ring. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2, 4)-Triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

Unless otherwise specified, the groups of the present invention can be substituted by substituents selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, C ₁ -C ₆ alkyl-amine, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkyl, halogenated C ₂ -C ₆ alkenyl, halogen Substituted C ₂ -C ₆ alkynyl, halogenated C ₁ -C ₆ alkoxy, allyl, benzyl, C ₆ -C ₁₂ aryl, C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkane Group, C ₁ -C ₆ alkoxy-carbonyl group, phenoxycarbonyl group, C ₂ -C ₆ alkynyl-carbonyl group, C ₂ -C ₆ alkenyl-carbonyl group, C ₃ -C ₆ cycloalkyl-carbonyl group, C ₁ -C ₆ alkyl-sulfonyl and the like.

As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halo" means substituted with an atom selected from F, Cl, Br, and I.

Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformers)): for example, containing asymmetry The R and S configurations of the center, the (Z) and (E) isomers of the double bond, etc. Therefore, individual stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformers) are within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers with different energies can exceed the low energy barrier and thus convert to each other. For example, proton tautomers (i.e., proton shift) include interconversion through proton transfer, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion through some bond-forming electron recombination.

As used herein, the term "solvate" refers to a compound of the present invention that coordinates with a solvent molecule to form a complex in a specific ratio.

Compound of formula I

The present invention provides a compound represented by the following formula I:

among them,

R has the structure -L ₁ -R _A ;

L _{1 is} selected from the group consisting of substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms selected from N, S and O, or substituted or unsubstituted -(X) _y -, Wherein each of said X is independently selected from the group consisting of substituted or unsubstituted C ₁ -C ₈ alkylene, -O-, -C(=O)-, -CONH-, -NHCO-, -S -, -S(＝O)-, -S(＝O) ₂ -, -NH-;

y is selected from the group: 1, 2 or 3;

R _{A is} selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O;

Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , and Y ⁷ are each independently selected from the group consisting of CR ₁ or N; Z ¹ and Z ² are each independently selected from the group consisting of C or N; And

Is an aromatic ring;

R _{1 is} independently selected from the group consisting of H, D, OH, Cl, F, and NH ₂ ;

Ring A is selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O (including (Single ring, parallel ring, spiral ring or bridge ring);

L is selected from the group: none, -NR ₇ -, -O-;

R _{7 is} selected from: H, substituted C1-C6 alkyl; wherein, the substitution means that one or more H atoms on the group are substituted with a substituent selected from the group consisting of halogen and hydroxyl;

Ring C is selected from the group consisting of substituted or unsubstituted 3-12 membered cycloalkyl (including monocyclic, paracyclic, spiro or bridged rings), substituted or unsubstituted having 1-3 selected from N, S and 4-12 membered heterocyclic group of hetero atom of O (including monocyclic, paracyclic, spiro or bridged ring);

Unless otherwise specified, the "substituted" refers to being substituted by one or more (eg 2, 3, 4 etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halo C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methyl sulfone, -S(=O) ₂ NH ₂ , oxo(=O),- CN, hydroxyl, -NH ₂ , carboxyl, C1-C6 amide (-C(=O)-N(Rc) ₂ or -NH-C(=O)(Rc), Rc is H or C1-C5 alkane Group), C1-C6 alkyl-(C1-C6 amide group),

Or substituted or unsubstituted group selected from the group consisting of C1-C6 alkyl group, C3-C8 cycloalkyl group, C1-C6 amino group, C6-C10 aryl group, having 1-3 selected from N, S and 5-10 membered heteroaryl of hetero atom of O, 5-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH ₂ )-C6-C10 aryl,- (CH ₂ )-(5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S, and O), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 aryl, C1-C6 amine, C1-C6 amide, with 1-3 selected from N, S and O 5-10 membered heteroaryl of hetero atoms;

Is the attachment site of the group;

The additional condition is that the compound of formula I has a chemically stable structure.

In another preferred example, the compound of formula I of the present invention is the compound prepared in the examples.

Preparation of compounds of formula I

The compound of formula I of the present invention can be prepared by the following method:

Pharmaceutical composition and method of administration

Since the compound of the present invention has excellent TRK kinase inhibitory activity, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and containing the compound of the present invention as the main active ingredient The pharmaceutical composition of can be used to prevent and/or treat diseases (eg, cancer) related to TRK kinase activity or expression level.

The pharmaceutical composition of the present invention contains the compound of the present invention within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound/dose of the present invention, and more preferably, 10-200 mg of the compound/dose of the present invention. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components of the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carrier parts are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween

), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

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

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) filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarding solvents, such as paraffin; (f) Absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain an opaque agent, and the release of the active compound or compound in this composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compound can also be formed into a microcapsule 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 compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oil, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and flavoring agents.

In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

The composition for parenteral injection may contain a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

When administered in combination, the pharmaceutical composition also includes one or more other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds can be administered simultaneously, separately, or sequentially with the compounds of the present invention.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is suitable for mammals in need of treatment (such as humans), wherein the dose when administered is the pharmaceutically effective dose, for 60kg body weight The dose to be administered is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples generally follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Synthesis of Intermediate A

(R,E)-N-(2,5-Difluorobenzylidene)-2-methylpropane-2-sulfinamide

Dissolve 2,5-difluorobenzaldehyde (5 g, 35.2 mmol) and (R)-2-methylpropane-2-sulfinamide (4.47 g, 36.9 mmol) in dichloromethane (50 mL) At room temperature, cesium carbonate (8.0 g, 24.6 mmol) was added, and then the temperature was raised to 50°C for 3 hours. The reaction was completed by TLC. The filter cake was washed with dichloromethane, the filtrate was washed with brine, and dried over anhydrous sodium sulfate. , Spin dry to give a yellow oily liquid (9g).

(R)-N-((R)-1-(2,5-difluorophenyl)-3-(1,3-dioxan-2-yl)propyl)-2-methylpropane-2 -Sulfinamide

Dissolve magnesium shavings (2 g, 83.3 mmol) in tetrahydrofuran (72 mL), protect with nitrogen, and add a solution of diisobutylaluminum hydride in tetrahydrofuran (0.1 mL, 1.5 M, 0.15 mmol) dropwise at 40°C. The system was reacted at 40°C for 0.5 hours, and then a solution of 2-(2-bromoethyl)-1,3-dioxane (14.3 g, 73.47 mmol) in tetrahydrofuran (40 mL) was slowly added dropwise to the system and the temperature was controlled at 40-50°C, keep at 40°C for 1 hour after dropping. The heating was removed, the reaction system was cooled to -30°C, and then (R,E)-N-(2,5-difluorobenzylidene)-2-methylpropane-2-sulfinamide (9 g , 36.73 mmol) of tetrahydrofuran (40 ml) solution was added dropwise to the system, the temperature was controlled at -30 ℃ -20 ℃, after the completion of the drop, stirred at -30 ℃ for 2 hours, TLC showed the reaction was completed, use 10% lemon The aqueous acid solution was quenched and the temperature was controlled at 10°C, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and vortexed to obtain a colorless oily liquid (15.8 g).

(R)-2-(2,5-Difluorophenyl)pyrrolidine

(R)-N-((R)-1-(2,5-Difluorophenyl)-3-(1,3-dioxan-2-yl)propyl)-2-methyl at room temperature Propane-2-sulfinamide (15.8 g, 43.76 mmol) was added to a mixed solution of trifluoroacetic acid (32 ml) and water (8 ml), stirred at room temperature for 1 hour, and then trifluoroacetic acid (60 ml) was added To the system, triethylsilane (15.2 g, 131.1 mmol) was added dropwise to the system. The reaction was overnight at room temperature. The reaction was completed by LCMS. Most of the trifluoroacetic acid was spun off, and the remainder was dissolved in hydrochloric acid (1N, 100 mL) and stirred for 0.5 hour. , Extracted with methyl tert-butyl ether, the organic phase was washed with hydrochloric acid (1N, 50 mL), the aqueous phases were combined, the aqueous phase was adjusted to pH=11 with 40% aqueous sodium hydroxide solution, then extracted with dichloromethane, and the organic phases were combined , Washed with saturated brine, dried over anhydrous sodium sulfate, and vortexed to give an oily liquid (6.7 g).

Synthesis of Intermediate B

(S)-N-((S)-1-(2,5-difluorophenyl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide

(R)-N-(2,5-Difluorobenzylidene)-2-methylpropane-2-sulfinamide (30 g, 122.45 mmol) was added to saturated sodium bromide (480 ML), add In (42 g, 367.35 mmol), then add allylmagnesium bromide (42 mL, 489.8 mmol), react at room temperature for 6 hours, complete the reaction by TLC, and use saturated sodium bicarbonate solution After quenching, filtering, the filtrate was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to give (S)-N-((S)-1-(2,5-difluorophenyl)butane- 3-en-1-yl)-2-methylpropane-2-sulfinamide, yellow solid (35 g).

(S)-N-((1S)-1-(2,5-difluorophenyl)-2-(oxiran-2-yl)ethyl)-2-methylpropane-2-sulfinic acid Amide

(S)-N-((S)-1-(2,5-difluorophenyl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide (35 g, 121.95 mmol) was dissolved in dichloromethane (800 mL), m-chloroperoxybenzoic acid (80 g, 365.85 mmol) was added in portions at room temperature, stirred overnight at room temperature, the reaction was completed by TLC, and the reaction solution was saturated with carbonic acid Wash with sodium hydrogen and saturated sodium thiosulfate solution, wash with saturated brine, dry with anhydrous sodium sulfate, spin-dry to get (S)-N-((S)-1-(2,5-difluorophenyl)butan-3 -En-1-yl)-2-methylpropane-2-sulfinamide, yellow solid (31 g, 79%).

(3R, 5R)-1-(tert-butylsulfonyl)-5-(2,5-difluorophenyl)pyrrolidine-3-ol

(S)-N-((S)-1-(2,5-difluorophenyl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide (31 g, 97.18 mmol) was dissolved in N,N-dimethylformamide (300 mL), potassium carbonate (40 g, 291.53 mmol), potassium iodide (16 g, 97.18 mmol) was added at room temperature, and the reaction was heated to 100°C After 1 hour, the reaction was monitored by TLC. After the reaction solution was cooled to room temperature, filtered, the filtrate was poured into water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by rotary drying (petroleum ether) /Ethyl acetate=10/1—5/1) to give compound (3R, 5R)-1-(tert-butylsulfonyl)-5-(2,5-difluorophenyl)pyrrolidine-3-ol ( 7.5g).

(2R, 4S)-1-(tert-butylsulfonyl)-2-(2,5-difluorophenyl)-4-fluoro

(3R, 5R)-1-(tert-butylsulfonyl)-5-(2,5-difluorophenyl)pyrrolidine-3-ol (2.0 g, 6.27 mmol) was dissolved in dichloromethane (50 ML), cooled to minus 60 ℃, DAST (2 mL) was added dropwise to the system, and then naturally raised to room temperature and stirred overnight, lcms monitor the completion of the reaction, the reaction solution was diluted with dichloromethane, slowly poured into ice water, Liquid separation, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by rotary drying (petroleum ether/ethyl acetate=10/1) to give (2R, 4S)-1-(tert-butylsulfonyl)- 2-(2,5-difluorophenyl)-4-fluoro, yellow solid (1.2 g, 60%).

(2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine

To a solution of (2R, 4S)-1-(tert-butylsulfonyl)-2-(2,5-difluorophenyl)-4-fluoro (500 mg, 1.55 mmol) in dichloromethane (20 mL) Add trifluoromethanesulfonic acid (0.7 mL), react for 2 hours, spin-dry the solvent, wash with 2 molar sodium hydroxide solution, extract with ethyl acetate, separate the liquid, wash the organic phase with saturated brine, anhydrous sodium sulfate Dry and spin through column purification (petroleum ether/ethyl acetate = 4/1) to give (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine, yellow solid (305 mg , 99%).

Synthesis of Intermediate C

(R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitroimidazo[1,2-b]pyridazine

At room temperature, dimethyl sulfoxide (30 ml) was added to a three-necked flask (100 ml), and the compound 6-chloro-3-nitroimidazo[1,2-b]pyridazine (1.0 g, 5.0 mmol) was added. (R)-2-(2,5-difluorophenyl)pyrrolidine (1.0 g, 5.5 mmol) and N,N-diisopropylethylamine (2.0 g, 15.1 mmol) at 110°C Stir overnight, pour into ice water, extract with ethyl acetate (100 mL*3), spin-dry the organic phase, and purify with a silica gel column (petroleum ether: ethyl acetate = 2:1) to give a yellow solid (650 mg, 37.6% ).

(R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine

Add ethanol (30 ml) to a three-necked flask (100 ml) at room temperature, and add compound (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitro Imidazo[1,2-b]pyridazine (650 mg, 1.9 mmol), iron powder (516.4 mg, 9.2 mmol) and ammonium chloride (985.5 mg, 18.5 mmol), stirred at 80°C for 3 hours , Cooled to room temperature, filtered, the filtrate was concentrated and extracted with ethyl acetate (50 ml * 3), the organic phase was spin-dried, purified with a silica gel column (dichloromethane: methanol = 50: 1), to give a green solid (200 mg, 33.7%).

¹ HNMR (400MHz, CDCl ₃ ): δ (ppm) 7.52-7.50 (m, 1H), 7.08-7.00 (m, 1H), 6.98 (s, 1H), 6.92-6.87 (m, 1H), 6.83-6.78 (m,1H), 6.27(s,1H), 5.30-5.27(m,1H), 3.92-3.87(m,1H), 3.70-3.63(m,1H), 2.49-2.44(m,1H), 2.10 -2.01(m,5H).

Synthesis of Intermediate D

6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-3-nitroimidazo[1,2-b]pyridazine

To (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine (150 mg, 0.75 mmol), 6-chloro-3-nitroimidazo[1,2-b ] Pyridazine (222 mg, 1.12 mmol) in dimethyl sulfoxide (2 mL) was added triethylamine (151 mg, 1.5 mmol). The reaction solution was stirred at 100°C for 16 hours. LCMS showed most of the starting material disappeared. The reaction solution was purified by preparative high-performance liquid chromatography to obtain a yellow solid (150 mg, 55%) as a yellow solid.

MS(ESI): m/z=363.9[M+H] ⁺ .

6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine

To 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-3-nitroimidazo[1,2-b]pyridazine ( 60 mg, 0.16 mmol) of methanol (20 mL) was added with palladium on carbon (15 mg). The reaction solution was replaced with a hydrogen balloon three times by a water pump, and stirred at room temperature for 8 hours. LCMS showed most of the starting material disappeared. The reaction solution was filtered through Celite and rinsed with dichloromethane (20 ml). The filtrate was concentrated and purified by preparative high-performance liquid chromatography to obtain a yellow solid (30 mg, 57%).

MS(ESI): m/z=333.9[M+H] ⁺ .

Synthesis of Intermediate E

6-(3-hydroxypyrrolidin-1-yl)nicotinic acid

Anhydrous n-butanol (10 ml) was added to the microwave tube (20 ml), compound 6-chloronicobutanoic acid (1.576 g, 10.0 mmol), compound pyrrolidine-3-ol (1.05 g, 12.0 mmol) was added And N,N-diisopropylethylamine (3.87 g, 30.0 mmol). The reaction system was stirred at 150°C for 16 hours. The reaction system was concentrated and purified by a silica gel column (dichloromethane: methanol = 4:1) to obtain a white solid (1.1 g, 52%).

¹ HNMR (400 MHz, CDCl ₃ ): δ (ppm) 12.29 (s, 1H), 8.61 (d, J=2.0 Hz, 1H), 7.90-7.87 (m, 1H), 6.46 (d, J=8.8 Hz, 1H), 5.0(s, 1H), 4.39(s, 1H), 3.53-2.43(m, 3H), 2.0-1.2(m, 2H), 1.19(s, 1H)

Example 1:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(3 -Hydroxypyrrolidine-1- Nic)amide

To (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine (25 mg, 0.08 mmol) , N,N-diisopropylethylamine (30 mg, 0.24 mmol), 6-(3-hydroxypyrrolidin-1-yl)nicotinic acid (50 mg, 0.24 mmol) in dichloromethane (6 ML) was added 2-(7-benzobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (130 mg, 0.24 mmol). The reaction solution was stirred at room temperature for 3 days. After concentration, the residue was purified by preparative high-performance liquid chromatography to obtain a white solid.

MS(ESI): m/z=506.2[M+H] ⁺ .

1HNMR (400MHz, CD ₃ OD) δ (ppm) 8.63 (s, 1H), 7.95 (dd, J = 9.0, 2.3 Hz, 1H), 7.65 (d, J = 9.9 Hz, 1H), 7.51 (s, 1H ), 6.97-6.82 (m, 3H), 6.79 (d, J = 10.0 Hz, 1H), 6.58 (d, J = 9.0 Hz, 1H), 5.32 (d, J = 5.6 Hz, 1H), 4.61-4.52 (m,1H), 3.98–3.87(m,1H), 3.72–3.49(m,5H), 2.51-2.42(m,1H), 2.26–2.14(m,1H),2.11-2.08(m,3H) , 1.99–1.87 (m, 1H).

Example 2:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( S)-3-Hydroxypyrrole Alkan-1-yl)nicoamide

(R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-flunik Amide

To (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine (150 mg, 0.476 mmol) , N,N-diisopropylethylamine (307 mg, 2.18 mmol), 6-fluoronicotinic acid (134 mg, 0.951 mmol) in methylene chloride solution (12 mL) was added 2-(7-oxidation Benzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (362 mg, 0.951 mmol). The reaction solution was stirred at room temperature for 3 days. Liquid quality monitoring showed product formation. Concentrate and purify through a column (ethyl acetate/dichloromethane=5/1) to obtain a brown oil (197 mg, 94.4%).

MS(ESI): m/z=439.1[M+H] ⁺ .

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( S)-3-Hydroxypyrrole Alkan-1-yl)nicoamide

To (R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-fluoro Nicotinamide (55 mg, 0.125 mmol), N,N-diisopropylethylamine (129 mg, 1.004 mmol) in N,N-dimethylformamide solution (2 mL) was added (S) -3-hydroxypyrrolidine hydrochloride (47 mg, 0.374 mmol). The temperature was raised to 90°C and stirred for 5 hours. Liquid quality monitoring showed product formation. After concentration, the residue was purified by preparative high-performance liquid chromatography to obtain a yellow solid. MS(ESI): m/z=506.2[M+H] ⁺ .

¹ HNMR (400 MHz, CD ₃ OD) δ (ppm) 8.61 (d, J = 1.7 Hz, 1H), 7.94 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 (d, J = 9.8 Hz, 1H) , 7.51 (s, 1H), 6.98–6.81 (m, 3H), 6.78 (d, J = 9.7 Hz, 1H), 6.57 (d, J = 9.0 Hz, 1H), 5.31 (dd, J = 8.2, 2.7 Hz, 1H), 4.56 (s, 3H), 3.97–3.87 (m, 1H), 3.72–3.50 (m, 4H), 2.54–2.38 (m, 1H), 2.23–2.13 (m, 1H), 2.11– 2.03(m, 2H), 1.98–1.86(m, 1H).

Example 3:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( R)-3-hydroxypyrrole Alkan-1-yl)nicoamide

The target compound was prepared from intermediate C and (R)-3-hydroxypyrrolidine hydrochloride under conditions similar to Example 2.

MS(ESI): m/z=506.5[M+H] ⁺ .

¹ HNMR (400MHz, CD ₃ OD) δ (ppm) 8.62 (s, 1H), 7.94 (d, J = 7.2Hz, 1H), 7.64 (d, J = 9.9Hz, 1H), 7.52 (s, 1H) , 6.95-6.85 (m, 3H), 6.77 (d, J = 9.7 Hz, 1H), 6.56 (d, J = 9.0 Hz, 1H), 5.32 (d, J = 5.9 Hz, 1H), 4.57 (s, 2H), 3.97–3.88 (m, 1H), 3.73–3.51 (m, 5H), 2.52-2.42 (m, 1H), 2.20-2.16 (m, 1H), 2.13-2.02 (m, 3H), 1.98- 1.90(m,1H).

Example 4:

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-6-((S)-3- Hydroxypyrrolidin-1-yl)nicoamide

The target compound was prepared from intermediate D and (S)-3-hydroxypyrrolidine hydrochloride under conditions similar to Example 2.

MS(ESI): m/z=523.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.62 (s, 1H), 8.66 (s, 1H), 8.00–7.92 (m, 1H), 7.78 (d, J=9.8 Hz, 1H), 7.46 (s,1H), 7.16–7.09 (m, 1H), 7.06–6.89 (m, 2H), 6.78–6.69 (m, 1H), 6.52 (d, J=9.0Hz, 1H), 5.53 (s, 0.5 H), 5.40 (s, 0.5H), 5.30 (m, 1H), 5.03–4.98 (m, 1H), 4.40 (s, 1H), 4.13–3.98 (m, 2H), 3.60–3.49 (m, 2H ), 3.05–2.86 (m, 1H), 2.79–2.67 (m, 1H), 2.24–1.86 (m, 4H).

Example 5:

(R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( 2-hydroxyethyl)amino)nikamide

The target compound was prepared from intermediate C and 2-aminoethane-1-ol under conditions similar to Example 2.

MS(ESI): m/z=480.0[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.53 (s, 1H), 8.51 (s, 1H), 7.76 (d, J=9.8 Hz, 2H), 7.43 (s, 1H), 7.26-7.19 (m,1H), 7.02(m,2H),6.92-6.84(m,1H),6.68(d,J=9.8Hz,1H),6.54(d,J=8.9Hz,1H),5.24(d, J = 6.0 Hz, 1H), 4.75 (m, 1H), 3.93–3.83 (m, 1H), 3.61–3.48 (m, 3H), 3.39 (dd, J=11.6, 5.8Hz, 2H), 2.43–2.30 (m,1H), 2.03-1.88(m,2H), 1.85-1.72(m,1H).

Example 6:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( 3-hydroxycyclopentyl)amino)nikamide

The target compound was prepared from intermediate C and 3-aminocyclopentane-1-ol hydrochloride under conditions similar to Example 2.

MS(ESI): m/z=519.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.51 (s, 1H), 8.50 (s, 1H), 7.77-7.74 (m, 2H), 7.43 (s, 1H), 7.21 (d, J= 7.4Hz, 1H), 7.08-6.96 (m, 2H), 6.91-6.85 (m, 1H), 6.68 (d, J = 9.6Hz, 1H), 6.50 (d, J = 8.9Hz, 1H), 5.25 ( d, J=5.9Hz, 1H), 4.64(d, J=3.9Hz, 1H), 4.26–4.07(m, 2H), 3.92-3.83(m, 1H), 3.55(dd, J=17.3, 8.1Hz , 1H), 2.42-2.19 (m, 2H), 2.05-1.88 (m, 3H), 1.86-1.76 (m, 1H), 1.75-1.52 (m, 3H), 1.45-1.33 (m, 1H).

Example 7:

(R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(di (Methylamino)nicotinamide

The target compound was prepared from intermediate C and 3-aminocyclopentane-1-ol hydrochloride under conditions similar to Example 2.

MS(ESI): m/z=464.0[M+H]+.

_{1HNMR (400MHz, DMSO-d 6} ) δ (ppm) 9.61 (s, 1H), 8.61 (s, 1H), 7.90 (d, J = 8.0Hz, 1H), 7.76 (d, J = 9.8Hz, 1H) , 7.44(s, 1H), 7.08-6.98(m, 2H), 6.92-6.86(m, 1H), 6.69(d, J=8.8Hz, 2H), 5.24(d, J=6.5Hz, 1H), 3.95-3.85(m, 1H), 3.55(dd, J=17.4, 7.8Hz, 1H), 3.27(s, 1H), 3.11(s, 5H), 2.42-2.29(m, 1H), 2.03-1.90( m, 2H), 1.85-1.76 (m, 1H).

Example 8:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-2-(( S)-3-hydroxypyrrolidin-1-yl)pyrimidine-5-carboxamide

The target compound was prepared from intermediate C and 2-chloropyrimidine-5-carboxylic acid under conditions similar to Example 2.

MS(ESI): m/z=506.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.81 (s, 1H), 8.75 (s, 1H), 7.77 (d, J = 10.0 Hz, 2H), 7.43 (s, 1H), 7.01-6.83 (m,3H),6.73–6.49(m,1H),5.21–5.19(m,1H),5.01(d,J=3.2Hz,1H),4.39(s,1H),3.88–3.86(m,2H ), 3.69-3.52 (m, 5H), 2.39-2.29 (m, 2H), 2.04-1.78 (m, 6H).

Example 9:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-2-(( S)-3-hydroxypyrrolidin-1-yl)thiazole-5-carboxamide

Methyl (S)-2-(3-hydroxypyrrolidin-1-yl)thiazole-5-carboxylate

To a solution of methyl 2-bromothiazole-5-carboxylate (500 mg, 2.27 mmol) in 1,4-dioxane (10 mL) was added (S)-3-hydroxypyrrolidine hydrochloride ( 234 mg, 2.72 mmol) and 1,8-diazabicycloundec-7-ene (690 mg, 4.54 mmol). The reaction system was stirred at 85°C for 16 hours. The reaction system was concentrated and purified on a silica gel column (ethyl acetate was used as the eluent) to obtain a white solid (465 mg, 90%).

MS(ESI): m/z=228.8[M+H] ⁺ .

(S)-2-(3-Hydroxypyrrolidin-1-yl)thiazole-5-carboxylic acid

Methyl(S)-2-(3-hydroxypyrrolidin-1-yl)thiazole-5-carboxylate (465 mg, 2.04 mmol), lithium hydroxide monohydrate (257 mg, 6.11 mmol) were added in this order Add a mixed solution of tetrahydrofuran/water (10 mL/4 mL). Stir at room temperature for 3 hours. LCMS showed the reaction was complete. The reaction solution was evaporated to dryness to remove tetrahydrofuran, 3 ml of water was added, and the pH of the solution was adjusted to 4 with 1N hydrochloric acid. The precipitated solid was filtered and washed with water four times. The solid was obtained as the target compound (400 mg, 91%).

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-2-(( S)-3-hydroxypyrrolidin-1-yl)thiazole-5-carboxamide

The target compound was prepared from intermediate C and (S)-2-(3-hydroxypyrrolidin-1-yl)thiazole-5-carboxylic acid under conditions similar to those in Example 1.

MS(ESI): m/z=511.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.61 (s, 1H), 7.92 (s, 1H), 7.74 (d, J = 9.6 Hz, 2H), 7.38 (s, 1H), 7.03-7.02 (m, 2H), 6.89–6.86 (m, 1H), 6.68–6.66 (m, 1H), 5.25–5.22 (m, 1H), 5.12 (d, J=3.6Hz, 1H), 4.40 (s, 1H ), 3.88–3.84(m, 2H), 3.55-3.47(m, 4H), 2.39–2.28(m, 2H), 2.12–1.79(m, 6H).

Example 10:

3-cyano-N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl) -4-((S)-3-hydroxypyrrolidin-1-yl)benzamide

The target compound was prepared from intermediate C and 3-cyano-4-fluorobenzoic acid under conditions similar to Example 2.

MS(ESI): m/z=529.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.76 (s, 1H), 8.02 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.77 (d, J=9.2 Hz, 1H ), 7.41 (s, 1H), 7.02-6.98 (m, 2H), 6.89-6.85 (m, 1H), 6.83 (d, J = 9.2Hz, 1H), 6.73-6.71 (m, 1H), 5.22- 5.21(m, 1H), 5.10(d, J=3.6Hz, 1H), 4.40(s, 1H), 3.89-3.85(m, 1H), 3.81-3.64(m, 3H), 3.56-3.48(m, 2H), 2.38–2.33 (m, 1H), 2.03–1.90 (m, 5H), 1.81–1.78 (m, 1H).

Example 11:

(R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(pyrrole Alkan-1-yl)nicoamide

The target compound was prepared from intermediate C and pyrrolidine under conditions similar to Example 2.

MS(ESI): m/z=489.9[M+H]+.

¹ HNMR (400 MHz, CD ₃ OD) δ (ppm) 8.61 (s, 1H), 7.92 (d, J = 7.0 Hz, 1H), 7.64 (d, J = 9.9 Hz, 1H), 7.52 (s, 1H) , 6.95-6.85 (m, 3H), 6.77 (d, J = 9.7 Hz, 1H), 6.55 (d, J = 9.0 Hz, 1H), 5.32 (d, J = 5.4 Hz, 1H), 3.96-3.88 ( m,1H), 3.65 (dd, J=17.5, 8.0Hz, 1H), 3.60-3.50 (m, 4H), 2.49-2.44 (m, 1H), 2.12–2.00 (m, 6H), 2.00–1.90 ( m,1H).

Example 12:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( S)-3-hydroxypyrrolidin-1-yl)pyridazine-3-carboxamide

Methyl (S)-6-(3-hydroxypyrrolidin-1-yl)pyridazine-3-carboxylate

To a solution of methyl 6-chloropyridazine-3-carboxylate (1.0 g, 5.8 mmol) in tetrahydrofuran (20 mL) was added (S)-3-hydroxypyrrolidine hydrochloride (610 mg, 7.0 mmol) ), potassium carbonate (1.2 g, 0.58 mmol) and tetrabutylamine iodide (210 mg, 0.58 mmol). The reaction system was stirred at 70°C for 16 hours. The reaction system was quenched by adding water, and extracted with dichloromethane and isopropanol (3:1). The organic phase was concentrated and purified by silica gel column (dichloromethane/methanol=95/5) to obtain a white solid (1 g, 76%) .

MS(ESI): m/z=224.3[M+H] ⁺ .

(S)-6-(3-Hydroxypyrrolidin-1-yl)pyridazine-3-carboxylic acid

Combine methyl (S)-6-(3-hydroxypyrrolidin-1-yl)pyridazine-3-carboxylate (300 mg, 1.3 mmol) and lithium hydroxide monohydrate (170 mg, 3.0 mmol) The mixture solution of tetrahydrofuran/water (13 ml/5 ml) was added sequentially. Stir at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction solution was evaporated to dryness to remove tetrahydrofuran, 3 ml of water was added, and the pH of the solution was adjusted to 4 with 1N hydrochloric acid. The precipitated solid was filtered and washed with water four times. The solid was obtained as the target compound (470 mg, 97%).

MS(ESI): m/z=209.9[M+H] ⁺ .

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( S)-3-hydroxypyrrolidin-1-yl)pyridazine-3-carboxamide

The target compound was prepared from intermediate C and (S)-6-(3-hydroxypyrrolidin-1-yl)pyridazine-3-carboxylic acid under conditions similar to Example 1.

MS(ESI): m/z=507.4[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 10.22 (s, 1H), 7.88 (d, J=9.4 Hz, 1H), 7.76 (d, J=9.9 Hz, 1H), 7.64 (s, 1H ), 7.31–7.22(m, 1H), 7.08–6.93(m, 3H), 6.67–6.57(m, 1H), 5.25(d, J=5.5Hz, 1H), 5.09(s, 1H), 4.46( s, 1H), 3.93 (s, 1H), 3.84–3.44 (m, 5H), 2.45–2.36 (m, 1H), 2.14–1.93 (m, 4H), 1.91–1.81 (m, 1H).

Example 13:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-5-(( S)-3-hydroxypyrrolidin-1-yl)pyrazine-2-carboxamide

The target compound was prepared from intermediate C and methyl 5-chloropyrazine-2-carboxylate under conditions similar to Example 12.

MS(ESI): m/z=507.3[M+H] ⁺ .

¹ HNMR (400MHz, DMSO-d6) δ (ppm) 9.84 (s, 1H), 8.68 (d, J = 1.1Hz, 1H), 7.95 (s, 1H), 7.77 (d, J = 9.8Hz, 1H) , 7.64 (s, 1H), 7.24 (td, J = 9.3, 4.5 Hz, 1H), 7.07-6.96 (m, 2H), 6.73-6.64 (m, 1H), 5.29 (d, J = 5.6 Hz, 1H) ), 5.09 (s, 1H), 4.44 (s, 1H), 3.99–3.89 (m, 1H), 3.72–3.43 (m, 5H), 2.45–2.35 (m, 1H), 2.16–1.81 (m, 5H) ).

Example 14:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-5-(( S)-3-hydroxypyrrolidin-1-yl)pyrimidine-2-carboxamide

The target compound was prepared from intermediate C and 5-bromopyrimidine-2-carboxylic acid under conditions similar to Example 12.

MS(ESI): m/z=501.8[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 10.17 (s, 1H), 8.19 (s, 2H), 7.77 (d, J=9.8 Hz, 1H), 7.67 (s, 1H), 7.28–7.19 (m,1H), 7.07–6.97 (m, 2H), 6.74–6.61 (m, 1H), 5.34–5.28 (m, 1H), 5.11 (d, J=3.8Hz, 1H), 4.46 (s, 1H ), 3.99–3.91(m, 1H), 3.65–3.43(m, 4H), 2.14–1.81(m, 5H).

Example 15:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-1-(3 -Hydroxycyclopentyl)- 1H-pyrazole-3-carboxamide (cis or tran)

Methyl 1-(3-carbonylcyclopentyl)-1H-pyrazole-3-carboxylate

To a mixture of methyl 1H-pyrazole-3-carboxylate (1 g, 7.9 mmol) and cyclopent-2-en-1-one (1.3 g, 16.0 mmol) was added p-toluenesulfonic acid monohydrate Substance (100 mg, 0.53 mmol). After the reaction solution was stirred at 60°C for 16 hours, it was purified by a flash separation column (dichloromethane to dichloromethane: methanol = 9:1). The resulting crude product was purified by high-performance liquid chromatography to obtain a light yellow oil (1.1 g, 67%).

MS(ESI): m/z=208.9[M+H] ⁺ .

Methyl 1-(3-hydroxycyclopentyl)-1H-pyrazole-3-carboxylate and Example 492C: Methyl 1-(3-hydroxycyclopentyl)-1H-pyrazole-3-carboxylic acid ester

Methyl 1- (3-cyclopentyl-carbonyl) lH-pyrazole-3-carboxylate (500 mg, 2.4 mmol) in methanol (20 ml) was cooled to 0 ℃. Sodium borohydride (500 mg, 13.0 mmol) was added to the reaction solution. The reaction solution was stirred at 0°C for 1.5 hours, poured into an ice-water mixture, and extracted three times with ethyl acetate. Dichloromethane: isopropanol = 3 :1 Extract three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by rapid separation and purification (dichloromethane/methanol=9/1) to obtain the target compounds as colorless oils 15A-2-1 (300 mg, 59%) and 15A-2-2 (100 mg, 22%) .

MS (ESI): m/z = 210.9 [M+H] ⁺ .

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-1-(3 -Hydroxycyclopentyl)-1H-pyrazole-3-carboxamide

To a solution of 15A-2-1 (84 mg, 0.4 mmol) in tetrahydrofuran (3 mL) and water (3 mL) was added lithium hydroxide (50 mg, 1.2 mmol). After the reaction solution was stirred at room temperature for 3 hours, use Acidified with concentrated hydrochloric acid, concentrated, and lyophilized. The resulting oil was dissolved in isopropanol and concentrated to give a white solid. The resulting white solid and (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine (50 mg, 0.16 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl uronic acid cation (91 mg, 0.24 mmol) , A mixture of N,N-diisopropylethylamine (62 mg, 0.48 mmol) and anhydrous N,N-dimethylformamide (1 mL) was stirred at 40 degrees for 16 hours. The reaction solution was purified by preparative high-performance liquid chromatography to obtain compound 15A (33 mg, 42%) as a white solid.

MS (ESI): m/z = 494.4 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.36 (s, 1H), 8.00 (d, J = 2.3 Hz, 1H), 7.77 (d, J = 9.7 Hz, 1H), 7.56 (s, 1H ), 7.21–7.09 (m, 1H), 7.09–7.01 (m, 1H), 7.01–6.91 (m, 1H), 6.75 (d, J=2.2 Hz, 1H), 6.66 (d, J=9.2 Hz, 1H), 5.30(d, J=6.7Hz, 1H), 4.93–4.75(m, 2H), 4.24(s, 1H), 3.97–3.89(m, 1H), 3.63–3.54(m, 1H), 2.46 –2.36(m,2H), 2.27–1.68(m,8H).

To a solution of 15A-2-2 (84 mg, 0.4 mmol) in tetrahydrofuran (3 mL) and water (3 mL) was added lithium hydroxide (50 mg, 1.2 mmol). After the reaction solution was stirred at room temperature for 3 hours, use Acidified with concentrated hydrochloric acid, concentrated, and lyophilized. The resulting oil was dissolved in isopropanol and concentrated to give a white solid. The resulting white solid and (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine (50 mg, 0.16 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl uronic acid cation (91 mg, 0.24 mmol) , A mixture of N,N-diisopropylethylamine (62 mg, 0.48 mmol) and anhydrous N,N-dimethylformamide (1 mL) was stirred at 40 degrees for 16 hours. The reaction solution was purified by preparative high-performance liquid chromatography to obtain 15B (34 mg, 43%) as a white solid.

MS (ESI): m/z = 494.4 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.56 (s, 1H), 8.00 (s, 1H), 7.94 (d, J = 10.0 Hz, 1H), 7.87 (s, 1H), 7.20-6.89 (m, 4H), 6.75 (d, J = 1.4 Hz, 1H), 5.33 (d, J = 7.8 Hz, 1H), 5.08–4.94 (m, 1H), 4.73 (brs, 1H), 4.39 (s, 1H), 4.01–3.91(m,1H), 3.66–3.55(m,1H), 2.46–2.37(m,1H), 2.36–2.25(m,1H), 2.22–1.79(m,7H), 1.69– 1.56 (m, 1H).

Example 16:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-1-(3 -Hydroxycyclopentyl)-1H-imidazole-4-carboxamide

The target compound was prepared from intermediate C and methyl 1H-imidazole-4-carboxylate under conditions similar to those of Example 15.

16A: MS (ESI): m/z = 494.3 [M+H] ⁺ .

¹ HNMR (400MHz, DMSO-d ₆ ) δ (ppm) 9.50 (s, 1H), 7.96 (d, J = 3.8Hz, 2H), 7.76 (d, J = 9.9Hz, 1H), 7.60 (s, 1H ), 7.30–7.20 (m, 1H), 7.08–7.01 (m, 1H), 7.00–6.92 (m, 1H), 6.71–6.61 (m, 1H), 5.31–5.24 (m, 1H), 4.87–4.77 (m,1H), 4.76–4.70(m,1H), 4.35–4.27(m,1H), 3.97–3.88(m,1H), 3.63–3.54(m,1H), 2.44–2.38(m,1H) , 2.34–2.23 (m, 1H), 2.14–1.93 (m, 5H), 1.91–1.82 (m, 1H), 1.82–1.69 (m, 1H), 1.57 (s, 1H).

16B: MS (ESI): m/z = 494.3 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.69 (s, 1H), 8.07–7.89 (m, 4H), 7.29–7.17 (m, 1H), 7.17–6.92 (m, 3H), 5.31 ( d, J=6.5Hz, 1H), 4.76–4.64(m, 1H), 4.27–4.17(m, 1H), 4.03–3.94(m, 1H), 3.68–3.59(m, 1H), 2.46–2.35( m, 2H), 2.26–2.16 (m, 1H), 2.08–1.83 (m, 4H), 1.78–1.69 (m, 3H).

Example 17:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-5-(( S)-3-hydroxypyrrolidin-1-yl)picoline

The target compound was prepared from intermediate C and 5-fluoroopicolinic acid under conditions similar to Example 2.

MS (ESI): m/z = 505.9 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 10.16 (s, 1H), 7.95 (d, J=2.7 Hz, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.77 (d, J = 9.8Hz, 1H), 7.65(s, 1H), 7.28–7.20(m, 1H), 7.08–6.97(m, 3H), 6.75–6.65(m, 1H), 5.35–5.29(m, 1H), 5.07 (d, J = 3.7 Hz, 1H), 4.45 (s, 1H), 4.00–3.91 (m, 1H), 3.65–3.44 (m, 4H), 3.28–3.22 (m, 1H), 2.17–1.83 ( m,6H).

Example 18:

(R)-N-(6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(ox Butyl-3-ylamino) nicotinic acid

The target compound was prepared from intermediate C and oxbutan-3-amine under conditions similar to Example 2.

MS (ESI): m/z = 492.4 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.59 (s, 1H), 8.32 (s, 1H), 8.24 (d, J=12.0 Hz, 1H), 7.76 (d, J=9.5 Hz, 1H ), 7.52 (t, J = 9.0 Hz, 1H), 7.38 (s, 1H), 7.13-7.00 (m, 2H), 6.93–6.82 (m, 1H), 6.75-6.67 (m, 1H), 6.46 ( dd, J = 9.5, 4.0 Hz, 1H), 5.22 (d, J = 8.1 Hz, 1H), 4.29 (t, J = 10.5 Hz, 1H), 4.25–4.15 (m, 1H), 4.13-4.05 (m , 1H), 3.92-3.83 (m, 1H), 3.57-3.48 (m, 2H), 3.45-3.40 (m, 1H), 2.45-2.32 (m, 1H), 2.00-1.90 (m, 2H), 1.85 -1.75(m,1H).

Example 19:

N-(6-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-6-(( S)-3-methoxypyrrolidin-1-yl)nikamide

The target compound was prepared from intermediate C and (S)-3-methoxypyrrolidine under conditions similar to Example 2.

MS (ESI): m/z = 520.0 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.61 (s, 1H), 8.61 (s, 1H), 7.90 (d, J=8.1 Hz, 1H), 7.76 (d, J=9.8 Hz, 1H ), 7.44 (s, 1H), 7.10-7.00 (m, 2H), 6.93-6.85 (m, 1H), 6.68 (d, J=9.0Hz, 1H), 6.52 (d, J=8.9Hz, 1H) , 5.24 (d, J = 6.2 Hz, 1H), 4.12-4.06 (m, 1H), 3.93-3.85 (m, 1H), 3.65–3.48 (m, 4H), 3.46-3.38 (m, 1H), 3.26 (s,3H), 2.43–2.30(m,1H), 2.15–2.02(m,2H), 2.00-1.90(m,2H), 1.85-1.77(m,1H).

Example 20:

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-hydroxypyrrolidin-1-yl)pyrazine-2-carboxamide

The target compound was prepared from intermediate D and methyl 5-chloropyrazine-2-carboxylate under conditions similar to Example 13.

MS (ESI): m/z = 524.9 [M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.81 (s, 1H), 8.69 (s, 1H), 8.04 (s, 1H), 7.81 (d, J = 9.8 Hz, 1H), 7.65 (s ,1H),7.29–7.23(m,1H),7.21–7.13(m,1H),7.02(mf,1H),6.77(d,J=9.6Hz,1H),5.52(d,J=52.3Hz, 1H), 5.38–5.32(m, 1H), 5.09(s, 1H), 4.45(s, 1H), 4.23–3.98(m, 2H), 3.73–3.46(m, 4H), 2.84–2.71(m, 1H), 2.31–1.91 (m, 3H).

Example 21:

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-hydroxypyrrolidin-1-yl)pyrimidine-2-carboxamide

The target compound was prepared from intermediate D and 5-bromopyrimidine-2-carboxylic acid under conditions similar to Example 14.

MS(ESI): m/z=524.9[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 10.13 (s, 1H), 8.31–8.18 (m, 2H), 7.82 (d, J=9.8 Hz, 1H), 7.67 (s, 1H), 7.36 –7.25(m,1H),7.21–7.11(m,1H),7.06–6.96(m,1H),6.77(d,J=9.8Hz,1H),5.52(d,J=52.5Hz,1H), 5.42–5.30(m, 1H), 5.12(s, 1H), 4.46(s, 1H), 4.23–3.98(m, 2H), 3.60–3.48(m, 3H), 3.31(d, J=10.3Hz, 1H), 2.87–2.73(m,1H), 2.31–2.12(m,1H), 2.11–2.01(m,1H), 2.00–1.92(m,1H).

Example 22:

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-methoxypyrrolidin-1-yl)pyrazine-2-carboxamide

Tert-Butyl(S)-3-methoxypyrrolidine-1-carboxylate

To a solution of (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine (1.0 g, 5.34 mmol) in N,N-dimethylformamide (10 mL) was added 60% in portions at 0°C After sodium hydride (320 mg, 6.01 mmol), the reaction was carried out at 0°C for half an hour. Methyl iodide (850 mg, 6.01 mmol) was added to the reaction solution, and the reaction was performed at room temperature for another 2 hours. Water (50 ml) was added, extracted with ethyl acetate (100 ml*1), and then washed with water (100 ml*1) and saturated brine (100 ml*1). The organic phase was dried, filtered and concentrated to give the title compound 22A Tert-Butyl(S)-3-methoxypyrrolidine-1-carboxylate (1.05 g, 97.7%) as a colorless oil.

MS(ESI): m/z=223.9[M+Na] ⁺ .

(S)-3-methoxypyrrolidine hydrochloride

A solution of tert-butyl(S)-3-methoxypyrrolidine-1-carboxylate (1.05 g, 5.22 mmol) in 3N hydrochloric acid in methanol (10 mL) was stirred at room temperature for 4 hours. The reaction solution was concentrated to obtain (S)-3-methoxypyrrolidine hydrochloride (710 mg, 98.8%) as a colorless oil.

MS(ESI): m/z=101.9[M+H] ⁺ .

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-methoxypyrrolidin-1-yl)pyrazine-2-carboxamide

To 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-amine (60 Mg, 0.18 mmol), 5-chloropyrazine-2-carboxylic acid (57 mg, 0.36 mmol) and N,N-diisopropylethylamine (116 mg, 0.90 mmol) in N,N-di To a solution of methylformamide (1 ml) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (110 Mg, 0.29 mmol). The reaction solution was stirred at room temperature overnight. To the reaction solution, N,N-diisopropylethylamine (234 mg, 1.8 mmol) and 22B(S)-3-methoxypyrrolidine hydrochloric acid (124 mg, 0.90 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated and prepared by reverse phase to obtain the title compound 22N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1, 2-b]pyridazin-3-yl)-5-((S)-3-methoxypyrrolidin-1-yl)pyrazine-2-carboxamide (55 mg, 56.7%) as a yellow solid. MS(ESI): m/z=539.0[M+H] ⁺ .

¹ HNMR(400MHz, DMSO-d ₆ ) δ9.81(s, 1H), 8.69(s, 1H), 8.05(s, 1H), 7.81(d, J=9.8Hz, 1H), 7.65(s, 1H ), 7.28-7.22 (m, 1H), 7.21-7.14 (m, 1H), 7.05-6.97 (m, 1H), 6.76 (d, J = 9.8Hz, 1H), 5.52 (d, J = 52.2Hz, 1H), 5.39–5.30(m, 1H), 4.24–3.97(m, 3H), 3.74–3.48(m, 4H), 3.27(s, 3H), 2.85–2.70(m, 1H), 2.31–2.19( m,1H), 2.19-2.04(m,2H).

Example 23:

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-methoxypyrrolidin-1-yl)-N-methylpyrazine-2-carboxamide

N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl )-5-((S)-3-methoxypyrrolidin-1-yl)pyrazine-2-carboxamide

According to the synthesis method in Example 22, the title compound 23A N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[ 1,2-b]pyridazin-3-yl)-5-((S)-3-methoxypyrrolidin-1-yl)pyrazine-2-carboxamide (140 mg, 85.8%), yellow solid.

MS(ESI): m/z=538.9[M+H] ⁺ .

Example 23: N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1,2-b]pyridazine -3-yl)-5-((S)-3-methoxypyrrolidin-1-yl)-N-methylpyrazine-2-carboxamide

To a solution of (40 mg, 0.074 mmol) in N,N-dimethylformamide (2 mL) was added sodium hydride (6 mg, 0.148 mmol) under ice bath. After reaction at room temperature for 1 hour, methyl iodide (21 Mg, 0.148 mmol), the reaction was continued for 4 hours at room temperature. Quench with water, add ethyl acetate, wash the organic phase with water and salt, dry over sodium sulfate, filter, and concentrate the filtrate. The residue was purified by reverse phase column to obtain the title compound 23N-(6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)imidazo[1, 2-b)pyridazin-3-yl)-5-((S)-3-methoxypyrrolidin-1-yl)-N-methylpyrazine-2-carboxamide (9 mg, 19.5%) , A white solid.

MS(ESI): m/z=553.0[M+H] ⁺ .

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 7.77 (d, J=9.8 Hz, 1H), 7.36 (s, 1H), 7.22–7.03 (m, 3H), 6.86 (t, J=23.6 Hz, 3H ), 5.49 (d, J = 52.9 Hz, 1H), 5.28-5.18 (m, 1H), 4.18-3.92 (m, 3H), 3.39 (s, 3H), 3.26 (d, J = 11.7 Hz, 1H) , 3.18 (s, 3H), 3.09-2.84 (m, 2H), 2.78-2.68 (m, 2H), 2.19-2.08 (m, 1H), 1.98-1.93 (m, 2H).

The following compounds were obtained by replacing the corresponding raw materials by a method similar to that in Example 2.

Biological Test Example 1 TRKA, TRKB, TRKC kinase in vitro activity test

Experimental Materials

Recombinant human-derived TRKA, TRKB, and TRKC proteins were purchased from Carna Biosciences. HTRFkinEASETK is purchased from CisbioBioassays. Use BioTek Microplate Reader Synergy Neo 2 to read the plate.

experimental method

The test compound was subjected to a three-fold concentration gradient dilution with a final concentration of 1 μM to 0.05 nM and 10 concentrations, each with two replicate wells; the content of DMSO in the detection reaction was 1%.

TRKA enzyme reaction:

0.2ng/μl TRKA protein kinase, 1μM TK Substrate-biotin peptide substrate, 14.68μM ATP, 1×enzymatic buffer, 5mM MgCl ₂ , 1mM DTT. The detection plate was White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 40 minutes, and the reaction system was 10 μl.

TRKB enzyme reaction:

0.037ng/μl TRKB protein kinase, 1μM TK Substrate-biotin peptide substrate, 4.77μM ATP, 1×enzymatic buffer, 5mM MgCl ₂ , 1mM MnCl ₂ , 1mM DTT. The detection plate was White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 50 minutes, and the reaction system was 10 μl.

TRKC enzyme reaction:

0.037ng/μl TRKC protein kinase, 1μM TK Substrate-biotin peptide substrate, 25.64μM ATP, 1×enzymatic buffer, 5mM MgCl ₂ , 1mM DTT. The detection plate was White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 40 minutes, and the reaction system was 10 μl.

Reaction detection:

Add 10μl of detection reagent to the reaction plate, containing the final concentration of 0.125μM SA-XL665 and 5μl 1×TK-Antibody, incubate at room temperature overnight, Synergy Neo2 read the plate.

data analysis

The value of 665/620Ratio is converted into the inhibition rate (%)=(1-Ratio _test /Ratio _max )×100% by the following formula. Ratio _max is a positive control without test compound, and Ratio _test is the test value of each concentration of different compounds. 4 IC50 (nM) data measured by parameter curve fitting, see Table 1 for details.

Table 1

Compound

TRKA(nm)

TRKB(nm)

TRKC(nm)

Example 1	<10	<10	<10
Example 2	<10	<10	<10
Example 3	<10	<10	<10
Example 4	<10	<10	<10
Example 5	<10	<10	<10
Example 6	<10	<10	<10
Example 7	<10	<10	<10
Example 8	<10	<10	<10
Example 9	<10	<10	<10
Example 10	<10	<10	<10
Example 11	<10	<10	<10
Example 12	<10	<10	<10
Example 13	<10	<10	<10
Example 14	<10	<10	<10
Example 15A	<10	<10	<10
Example 15B	<10	<10	<10
Example 16A	<10	<10	<10
Example 16B	<10	<10	<10
Example 17	<10	<10	<10
Example 18	<10	<10	<10
Example 19	<10	<10	<10
Example 20	<10	<10	<10
Example 21	<10	<10	<10
Example 22	<10	<10	<10
Example 23	<10	<10	<10
Example 24	<10	<10	<10
Example 25	<10	<10	<10
Example 26	<10	<10	<10
Example 27	<10	<10	<10
Example 28	<10	<10	<10
Example 29	<10	<10	<10
Example 30	<10	<10	<10
Example 31	<10	<10	<10
Example 32	<10	<10	<10
Example 33	<10	<10	<10
Example 34	<10	<10	<10
Example 35	<10	<10	<10
Example 36	<10	<10	<10

Example 37	<10	<10	<10
Example 38	<10	<10	<10
Example 39	<10	<10	<10
Example 40	<10	<10	<10
Example 41	<10	<10	<10
Example 42	<10	<10	<10
Example 43	<10	<10	<10
Example 44	<10	<10	<10
Example 45	<10	<10	<10

Biological test example 2: KM12-LUC cell proliferation experiment

Human colon cancer cell line KM12-LUC (LUC, stably expressing Luciferace) containing the TPM3-NTRK1 fusion gene was used as a model for evaluating the efficacy of the test compounds at the cytological level. The TRK fusion gene in KM12-LUC cells makes it independent of extracellular growth factor stimulation, and can continue to spontaneously activate and activate its downstream signaling pathways MAPK-ERK, PI3K-AKT and other signaling pathways that are closely related to cell proliferation. Therefore, inhibition of TRK activity in KM12-LUC cells can significantly inhibit cell proliferation. The method is as follows: on the first day, cells are seeded in a 384-well plate at 2000 cells/well; on the second day, different concentrations of the test compound are added; on the fifth day, CellTiter-Glo (Promega) is added to detect cell viability and the cells are counted for 72 hours Proliferation inhibition rate. Use prism5 to perform statistical analysis and obtain the IC ₅₀ value of the test compound. See Table 2 for details.

The results show that the compounds of the present invention can effectively inhibit the proliferation of KM12-LUC cells.

Table 2

Compound	KM12-LUC IC 50 (nm)
Example 4	<10
Example 10	<10
Example 13	<10
Example 27	<10
Example 41	<10

Biological test 3 Mutant TRKA (G595R), TRKA (G667C) and TRKC (G623R) kinase in vitro activity test

Experimental Materials

Recombinant human TRKA (G595R), TRKA (G667C), TRKC (G623R) proteins were purchased from SignalChem. HTRFkinEASETK is purchased from CisbioBioassays. Use BioTek Microplate Reader Synergy Neo 2 to read the plate.

experimental method

The test compound was subjected to 4 times concentration gradient dilution, with a final concentration of 1 μM to 0.004 nM and 10 concentrations, each with two replicate wells; the content of DMSO in the detection reaction was 1%.

TRKA (G595R) enzyme reaction:

0.12ng/μl TRKA (G595R) protein kinase, 1 μM TK Substrate-biotin peptide substrate, 4.5 μM ATP, 1×enzymatic buffer, 5 mM MgCl ₂ , 1 mM DTT. The detection plate was a White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 30 minutes, and the reaction system was 10 μl.

TRKA (G667C) enzyme reaction:

0.026ng/μl TRKA (G667C) protein kinase, 1 μM TK Substrate-biotin peptide substrate, 5.5 μM ATP, 1×enzymatic buffer, 5 mM MgCl ₂ , 1 mM DTT. The detection plate was a White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 30 minutes, and the reaction system was 10 μl.

TRKC (G623R) enzyme reaction:

1.0 ng/μl TRKC (G623R) protein kinase, 1 μM TK Substrate-biotin peptide substrate, 62.9 μM ATP, 1×enzymatic buffer, 5 mM MgCl ₂ , 1 mM DTT. The detection plate was White Proxiplate 384-Plus plate (PerkinElmer), which was reacted at room temperature for 50 minutes, and the reaction system was 10 μl.

Reaction detection:

Add 10μl of detection reagent to the reaction plate, containing the final concentration of 0.125μM SA-XL665 and 5μl 1×TK-Antibody, incubate at room temperature overnight, Synergy Neo2 read the plate.

data analysis

After subtracting the value of the negative control well without enzyme from the value of 665/620 Ratio, the reading is converted into the inhibition rate (%) = (1-Ratio _test / Ratio _max )×100% by the following formula. Ratio _max is a positive control without test compound, and Ratio _test is the test value of each concentration of different compounds. 4 IC50 (nM) data measured by parameter curve fitting, see Table 3 for details.

table 3

All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that, after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A compound represented by the following formula I:

   

   among them,

   R has the structure -L ₁ -R _A ;

   L _{1 is} selected from the group consisting of substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms selected from N, S and O, or substituted or unsubstituted -(X) _y -, Wherein each of said X is independently selected from the group consisting of substituted or unsubstituted C ₁ -C ₈ alkylene, -O-, -C(=O)-, -CONH-, -NHCO-, -S -, -S(＝O)-, -S(＝O) ₂ -, -NH-;

   y is selected from the group: 1, 2 or 3;

   R _{A is} selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O;

   Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , and Y ⁷ are each independently selected from the group consisting of CR ₁ or N; Z ¹ and Z ² are each independently selected from the group consisting of C or N;

   And

   

   Is an aromatic ring;

   R _{1 is} independently selected from the group consisting of H, D, OH, Cl, F, and NH ₂ ;

   R _{12 is} selected from the group consisting of H or C1-C4 alkyl;

   Ring A is selected from the group consisting of substituted or unsubstituted C ₆ -C ₁₀ aryl groups, substituted or unsubstituted 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O (including Single ring, parallel ring);

   L is selected from the group: none, -NR ₇ -, -O-;

   R _{7 is} selected from: H, substituted C1-C6 alkyl; wherein the substitution means that one or more H atoms on the group are substituted by a substituent selected from the group consisting of halogen and hydroxyl;

   Ring C is selected from the group consisting of substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted 3-12 membered cycloalkyl (including monocyclic, paracyclic, spiro or bridged ring), substituted or unsubstituted 4-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O (including monocyclic, paracyclic, spiro or bridged rings);

   Unless otherwise specified, the "substituted" refers to being substituted by one or more (eg 2, 3, 4 etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halo C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methyl sulfone, -S(=O) ₂ NH ₂ , oxo(=O),- CN, hydroxyl, -NH ₂ , carboxyl, C1-C6 amide (-C(=O)-N(Rc) ₂ or -NH-C(=O)(Rc), Rc is H or C1-C5 alkane Group), C1-C6 alkyl-(C1-C6 amide group),

   

   Or substituted or unsubstituted group selected from the group consisting of C1-C6 alkyl group, C3-C8 cycloalkyl group, C1-C6 amino group, C6-C10 aryl group, having 1-3 selected from N, S and 5-10 membered heteroaryl of hetero atom of O, 5-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O, -(CH ₂ )-C6-C10 aryl,- (CH ₂ )-(5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S, and O), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 aryl, C1-C6 amine, C1-C6 amide, with 1-3 selected from N, S and O 5-10 membered heteroaryl of hetero atoms;

   

   Is the attachment site of the group;

   The additional condition is that the compound of formula I has a chemically stable structure.
2. The compound of claim 1, wherein said L _{1 is} selected from the group consisting of:

   

   n is selected from the group: 0, 1, 2 or 3;

   R ₂ , R _2a and R _2b are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted C ₁ -C ₈ alkyl;

   X _{5 is} selected from the group consisting of NH, O, -CONH-, -NHCO-, S, -S(=O) ₂ -, -NHS(=O)-, -NHS(=O) ₂ -;

   R _A is

   

   Among them, the

   

   Refers to the connection site of R _A and L ₁ ; X ⁴ is CH or N;

   R _{3 is} selected from the group consisting of H, halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy.
3. The compound according to claim 1, wherein the compound has a structure represented by the following formula II:

   
4. The compound according to claim 1, wherein the compound has a structure represented by the following formula III:

   
5. The compound according to claim 1, wherein the compound has a structure selected from the group consisting of:

   

   

   

   

   

   
6. A pharmaceutical composition, characterized by comprising (1) the compound of claim 1 or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) A pharmaceutically acceptable carrier.
7. The use according to claim 6, wherein the diseases are selected from the group consisting of cancer, proliferative diseases, pain, skin diseases or disorders, metabolic diseases, muscle diseases, neurological diseases, autoimmune diseases, dermatitis Itching, inflammation-related diseases, bone-related diseases.
8. Use of the compound according to claim 1 or its stereoisomer or tautomer, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition according to claim 6, It is characterized by preparing a pharmaceutical composition for preventing and/or treating diseases related to TRK dysfunction (TRK gene amplification, or overexpression, or mutation, or dysfunctional activation caused by gene fusion).
9. The use according to claim 8, wherein the disease is selected from the group consisting of cancer, proliferative diseases, pain, skin diseases or disorders, metabolic diseases, muscle diseases, Neurological diseases, autoimmune diseases, itching caused by dermatitis.
10. A TRK inhibitor, characterized in that the inhibitor comprises the compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof .