CN112979646B

CN112979646B - Imidazopyridine derivative

Info

Publication number: CN112979646B
Application number: CN202110249800.7A
Authority: CN
Inventors: 卢法冠; 王磊; 杜镇建; 杜美静
Original assignee: Beijing Foreland Biopharma Co ltd
Current assignee: Beijing Foreland Biopharma Co ltd
Priority date: 2021-03-08
Filing date: 2021-03-08
Publication date: 2022-01-14
Anticipated expiration: 2041-03-08
Also published as: WO2022188203A1; CN112979646A

Abstract

The invention discloses an imidazopyridine derivative, relates to the field of bioactive small molecules, and particularly relates to a small molecule with Trk (Trk-like kinase) inhibition activity. The invention provides a novel substituted imidazopyridine compound as a Trk kinase inhibitor, which overcomes the problem of drug resistance of a generation of Trk inhibitor in clinical application.

Description

Imidazopyridine derivative

Technical Field

The invention relates to the field of bioactive small molecules, in particular to a small molecule with Trk (Trk-like kinase) inhibiting activity.

Background

Tropomyosin-related kinase (Trk) is a class of nerve growth factor receptors, and the family thereof consists of highly homologous tropomyosin-related kinase a (TrkA), tropomyosin-related kinase b (trkb), tropomyosin-related kinase c (trkc), encoded by NTRK1, NTRK2, and NTRK3, respectively. Among the neurotrophic factors are (i) Nerve Growth Factor (NGF) which activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activate TrkB, and (iii) NT3 which activates TrkC. Trk is widely expressed in neuronal tissue and is involved in the maintenance, signaling and survival of neuronal cells (Current Opinion in Neurobiology,2001,11: 272-280).

Trk kinases are thought to be involved in neuronal cell growth, differentiation, apoptosis, etc., and inhibitors of the Trk/neurotrophin pathway are effective in a variety of preclinical animal models of pain. For example, antagonistic TrkA/NGF pathway antibodies (e.g., RN-624) are effective in inflammatory and neuropathic pain animal models and in human clinical trials (Neuroscience,1994,62: 327-. In addition, the literature shows that BDNF content and TrkB signaling increase in dorsal root ganglia following inflammation (Brain Research,1997,749:358), and several studies show that antibodies that reduce signaling via the BDNF/TrkB pathway can inhibit neuronal hypersensitivity and associated Pain (Molecular Pain,2008,4: 27). Also, overexpression, activation, amplification and/or mutation of Trk have been described in the literature as being associated with many cancers, including melanoma (Journal of Investigative Dermatology,2008,128(8):2031-, 4(8) 1887-. In preclinical models of Cancer, non-selective small molecule inhibitors of Trk A, B and C and Trk/Fc chimeras are effective in inhibiting tumor growth and preventing tumor metastasis (Cancer Letters,2001,169: 107-114; Cancer Letters,2006,232: 90-98; Cancer Res,2008,68 (2) 346-351).

In a cancer clinical test, a LMNA-NTRK1 gene fusion type soft tissue sarcoma metastasis patient is treated by a small molecular Trk kinase inhibitor Larotretinib (LOXO-101), the lung sarcoma volume is obviously reduced within four weeks, the metastasis and diffusion of cancer cells are slowed down, the dyspnea of the patient is relieved, the lung tumor mass basically disappears after four months, and no obvious adverse reaction is found during the clinical treatment (cancer D dicov, 2015,5: 1049-. LOXO-101 has significant efficacy in adult/pediatric cancer patients who carry NTRK1/2/3 fusion positive, such as soft tissue malignancies, thyroid cancer, salivary gland cancer, gastrointestinal stromal tumors, childhood fibrosarcoma, non-small cell carcinomas, cholangiocarcinoma, renal cancer, salivary gland cancer, appendiceal cancer, pancreatic cancer, peripheral nerve sheath tumors, spindle cell tumors, etc., and the drug has low side effects (New England Journal of Medicine,2018,378(8): 731-. The Trk kinase inhibitor Entrectinib (NMS-P626) was studied in clinical trials in 18 patients with solid tumors with NTRK1/2/3, ROS1 or ALK gene rearrangements, and the results showed that 72% of patients responded to Entrectinib (J Target Ther Cancer,2015,12: 34-37).

Inhibition of the neurotrophin/Trk pathway may be effective in treating inflammatory diseases in a preclinical mode. For example, inhibition of the neurotrophin/Trk pathway is associated with preclinical patterns of: inflammatory lung diseases including asthma (Pharmacology & Therapeutics,2008,117(1):52-76), interstitial cystitis (The Journal of Urology,2005,173(3): 1016-.

The neurotrophin/Trk pathway, and particularly the BDNF/TrkB pathway, is also implicated in the etiology of neurodegenerative diseases, including multiple sclerosis, Parkinson's disease, and Alzheimer's disease (Frontiers in Neuroendocrinology,2006,27(4): 404-414). Modulation of the neurotrophin/Trk pathway may be useful in the treatment of these and related diseases.

Studies have shown that the TrkA receptor is crucial for the disease process in the infection of Trypanosoma cruzi (Typanosomacrui) (Chagasdisease) by parasitic infections in human hosts (Cell Host & Microbe,2007,1(4): 251-261). Thus, inhibition of TrkA may be useful in the treatment of chagas disease and related protozoal infections.

Also, Trk inhibitors may be useful in the treatment of diseases associated with imbalance in regulation of bone remodeling, such as osteoporosis, rheumatoid arthritis, and bone metastases. Bone metastases are frequent complications of cancer, occurring in patients with advanced breast or prostate cancer and in patients with lung, colon, stomach, bladder, uterus, rectum, thyroid or kidney cancer. Osteolytic metastases can cause severe pain, pathological fractures, life-threatening hypercalcemia, spinal cord compression, and other nerve compression syndromes. Therefore, drugs that induce apoptosis of proliferative osteoblasts would be extremely advantageous. Expression of the TrkA receptor and TrkC receptor has been observed in the osteogenic region in a fractured mouse model (Bone,2000,26(6): 625-633). In addition, NGF distribution was observed in almost all osteoblasts. The pan-Trk inhibitor can inhibit tyrosine signaling activated by neurotrophins bound to all 3 Trk receptors in human hFOB osteoblasts. These data support the theory of using Trk inhibitors for the treatment of bone remodeling diseases, such as bone metastasis in cancer patients.

The marketed first-generation TRK inhibitor drug Larotretinib (LOXO-101) and Entretinib (Entretinib) have broad-spectrum anticancer effects as TRK inhibitors, but related drug resistance reports appear along with the expansion of clinical application range (cancer research.2017, 7(9): 934.; part of synthesis research of aclonimine alkaloids and palladium-catalyzed aryne coupling reaction research [ D)]Lanzhou university 2018). These mutations include TRKA in the active site^G595R(and paralogues thereof TRKC)^G623R) TRKA adjacent to DFG sequence^G667C(and paralogues thereof TRKC)^G696A) And TRKA in a conserved area^F589L(NewEnglandJournalofMedicine.2018,378(8):731-9.；)。TRKA^G595RAnd TRKA^G667CRespectively similar to ALK^G1202RAnd ALK^G1269A(CancerDiscovery.2017,7(9):963.)。

With the increase of drug resistance reports, second-generation TRK inhibitors against drug resistance have been developed, such as LOXO-195 and TPX-0005, LOXO-195 and TPX-0005 limit the surface area of active site compounds through cyclization, and LOXO-195 and TPX-0005 have the same pyrazole [1,5-a ] pyrimidine structure as Rarotinib in terms of compound structure, but LOXO-195 and TPX-0005 limit the free rotation of substituents through cyclization. Although the second generation of inhibitors overcome the resistance of the first generation of inhibitors in use, their ring structures are relatively difficult to build. While LOXO-195, as a second generation selective TRK inhibitor, exhibits potent inhibition of TRK fusion proteins and is not affected by certain acquired resistance mutations (e.g., TRKAG595R, TRKAG667C, or TrkCG623R) that may be present in patients who have been challenged with lenotronil (LOXO-101) or multi-kinase inhibitors with anti-TRK activity. However, the IC50 of LOXO-195 for the mutant Trk enzyme was 5-10 times higher than the IC50 for the corresponding wild-type Trk enzyme.

In recent years, there is an urgent clinical need for Trk inhibitors with better therapeutic efficacy, fewer side effects, high metabolic stability and good bioavailability.

Disclosure of Invention

The invention provides a novel substituted imidazopyridine compound serving as a Trk kinase inhibitor, which aims to overcome the problems of insufficient stability, activity and the like in the prior art and meet the increasing clinical requirements.

The invention aims to provide an imidazopyridine derivative with activity on drug-resistant TRK kinase.

The purpose of the invention is realized by the following scheme

An imidazopyridine derivative with a structure of formula A

Or a tautomer, stereoisomer, enantiomer, diastereomer, salt, prodrug or N-oxide thereof, wherein

X, Y or Z is N, the others are CH.

R₃And R₄Each independently selected from C_1-3Alkyl or H; when R is₃And R₄When both are alkyl, R₃And R₄Can be further connected into a ring;

R₁selected from a 5-6 membered monocyclic aryl or a 5-6 membered monocyclic heteroaryl, unsubstituted or substituted by one or more substituents.

R₂Selected from alkyl or heterocycloalkyl, said R₂Unsubstituted or substituted with one or more substituents.

Further, R₃Is C_1-3Alkyl, preferably methyl, R₄Is H.

Further, R₃And R₄When both are alkyl, R₃And R₄Connecting to form a five-membered ring.

Further, formula A has the following formula A₁Structure of (1)

Further, R₁Wherein said substituents are each independently selected from halogen, C_1-4Alkoxy, -CF₃、-CHF₂、-OC_1-4Alkyl OC_1-3Alkyl or-OC_3-6A dihydroxyalkyl group.

Further, R₁Wherein each of said substituents is independently selected from halogen or C_1-4An alkoxy group.

Further, R₁Wherein each of said substituents is independently selected from F or methoxy.

Further, R₁Wherein said monocyclic aryl or monocyclic heteroaryl is selected from phenyl or pyridyl.

Further, said R₁Selected from phenyl, 3-fluorophenyl, 2, 5-difluorophenyl, 2-chloro-5-fluorophenyl, 2-methoxyphenyl, 2-methoxy-5-fluorophenyl, 2-trifluoromethyl-5-fluorophenyl, 2-difluoromethyl-5-fluorophenyl, 3-chloro-5-fluorophenyl, 3-fluoro-5-methoxyethoxyphenyl, 2-hydroxy-5-fluorophenyl or 5-fluoro-2-methoxyethoxyphenyl, 5-fluoro-pyridin-3-yl, 2, 5-difluoro-pyridin-3-yl, 2-methoxy-5-fluoro-pyridin-3-yl, 2-hydroxy-5-fluoro-pyridin-3-yl or 2-trifluoromethyl-5-fluoro-pyridin-3-yl.

Further, said R₁Selected from 2, 5-difluorophenyl, 2-methoxy-5-fluorophenyl, 2-hydroxy-5-fluorophenyl or 2-methoxy-5-fluoro-pyridin-3-yl.

Further, R₂Wherein said alkyl is selected from the group consisting of straight chain alkyl and monocycloalkyl.

Further, R₂Wherein said alkyl is selected from C_1-6Straight chain alkyl, C_4-8Cycloalkyl or 4-8 membered heterocycloalkyl.

Further, R₂Wherein said alkyl is selected from C_1-3Straight chain alkyl, C_5-6Cycloalkyl or 5-6 membered heterocycloalkyl.

Further, R₂Wherein said heterocycloalkyl is a 5-6 membered heterocycloalkyl containing one or more heteroatoms, each independently selected from N, O or S.

Further, R₂Wherein said substituent is selected from acyloxy, hydroxyalkyl, hydroxy, methoxy, halogen, alkyl halide, methyl, aryl unsubstituted or substituted with one or more substituents, heteroaryl unsubstituted or substituted with one or more substituents, cycloalkyl unsubstituted or substituted with one or more substituents, or heterocycloalkyl unsubstituted or substituted with one or more substituents.

Further, R₂Wherein the substituent is selected from formyloxy, hydroxy-C₂-alkyl, hydroxy, methoxy;

or aryl unsubstituted or substituted with one or more substituents, wherein in aryl substituted with one or more substituents, each of said substituents is independently selected from F or CF₃；

Or heteroaryl unsubstituted or substituted by one or more substituents selected from methyl;

or cycloalkyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of formyloxy, hydroxy;

or unsubstituted heterocycloalkyl.

or phenyl unsubstituted or substituted by one or more substituents, wherein in aryl substituted by one or more substituents, each of said substituents is independently selected from F or CF₃；

Or heteroaryl unsubstituted or substituted with one or more substituents selected from methyl, said heteroaryl being a 5-6 membered heteroaryl containing one or more heteroatoms each independently selected from N, O or S;

or C unsubstituted or substituted by one or more substituents_5-6Cycloalkyl, wherein in cycloalkyl substituted with one or more substituents selected from formyloxy, hydroxy;

or unsubstituted heterocycloalkyl, which is a 5-6 membered heterocyclic ring containing one or more heteroatoms, each independently selected from N, O or S.

Further, said R₂One selected from the following structures:

further, the formula AA has the formula A₁₁Absolute configuration shown

Further, the compound of formula a is selected from one of the following structures:

a preparation method of the imidazopyridine derivative comprises at least one step of the following steps,

step a) A_aAnd

reacting under alkaline condition to obtain A_bWherein L is selected from chlorine, bromine or iodine;

step b) A_bReacting with phenyl chloroformate or phenyl chloroformate derivative under alkaline condition to obtain A_cWherein R is^aSelected from H or-NO₂；

Step c) A_cAnd R₂-NH₂Reacting under alkaline condition to obtain A_d；

Step d) of reacting A_dReducing to obtain A_e；

Step e) A_eCyclization gives A.

Further, in step d), the reducing conditions are conventional reducing-NO known to those skilled in the art₂The conditions of (3) are not particularly limited, and examples thereof include hydrogenation reduction, iron powder reduction, zinc powder reduction, etc., and hydrogenation reduction is preferable.

Further, in step e), the cyclization is carried out by reacting A_eAnd triethyl orthoformate under the condition of acid catalysis to obtain A.

Further, in step e), the cyclization can be carried out by reacting A_eAnd ethyl formyl imine generates cyclization reaction under the condition of acid catalysis to obtain A.

Further, the preparation method may further include a functional group protection or deprotection step necessary to reduce side reactions, using a scheme well known to those skilled in the art. If phenolic hydroxyl is protected by benzyl, the benzyl is removed by hydrogenation after one or more steps of reaction to obtain the phenolic hydroxyl functional group. Such functional group transformation processes that do not affect the choice of functional groups in the final product should also be within the scope of the preparation process provided by the present invention.

The invention provides a pharmaceutical composition, which contains the compound or pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug thereof, and pharmaceutically acceptable auxiliary materials.

The invention provides the use of a compound or pharmaceutical composition of the invention as hereinbefore described in the manufacture of a medicament for use in inhibiting the activity of a Trk enzyme, particularly a mutant Trk enzyme.

Wherein the mutation site of TRK target point is located in at least one of G517R, A542V, V573M, F589L, F589C, G595R, G595S, D596V, F600L, F646V, C656Y, C656F, L657V, G667S, G667C, Y676S, G623R, G667C, G623E, L686M, G545M, A570M, Q596M, V601M, F617M, G623M, D M, R630M, C682M, L683M, G693M, G713, C685M, L686M, G696 366972 and G M. Preferably TRKA^G595R、TRKC^G623R、TRKB^G639R、TRKA^G667C、TRKC^G696AAnd TRKA^F589LAt least one of (1). More preferably TRKA^G595R、TRKC^G623RAnd TRKB^G639RAt least one of (1).

Further, the mode of action of said use is contacting the Trk enzyme with a compound described herein, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate thereof.

Further, the invention provides the use of the compound in a product for preventing and/or treating Trk enzyme mediated diseases.

Further, the disease includes, but is not limited to, at least one of pain and cancer.

The pain may be of any origin or etiology, including but not limited to one or more of inflammatory pain, visceral pain, cancer-induced pain, chemotherapy pain, trauma pain, surgical and post-surgical pain, labor pain, acute pain, chronic pain, intractable pain, somatic pain, nociceptive pain, neuropathic pain, blood-borne pain, immunoborne pain, endocrinologically-derived pain, metabolic-induced pain, cardiogenic pain, headache, phantom limb pain, and dental pain.

The compounds of formula I are useful for the treatment of acute pain. Acute pain, as defined by the international society for pain research, results from disease, inflammation, or tissue injury. Such pain typically occurs suddenly, for example, after trauma or surgery, and may be accompanied by anxiety or stress. The cause can often be diagnosed and treated, and the pain is limited to a given period and severity. In some instances, it can be converted to chronic.

The cancer includes, but is not limited to, one or more of lung cancer, colon cancer, prostate cancer, breast cancer, liver cancer, lymphatic cancer, thyroid cancer, multiple myeloma, soft tissue sarcoma, ovarian cancer, cervical cancer, fallopian tube cancer, renal cell carcinoma, gastric cancer, gastrointestinal stromal tumor, bone cancer, basal cell carcinoma, peritoneal cancer, skin fibroma, pancreatic cancer, esophageal cancer, glioblastoma, head and neck cancer, inflammatory myofibroblastoma, and anaplastic large cell lymphoma.

The compounds of the invention may be administered by any suitable route, for example, into the gastrointestinal tract (e.g. rectal or oral), nasal, pulmonary, muscle or blood vessels or transdermally or dermally. The compounds may be administered by any suitable administration form, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches and the like. Such compositions may contain components conventional in pharmaceutical formulation such as diluents, carriers, pH adjusting agents, sweeteners, fillers and additional active agents. If parenteral administration is desired, the composition will be sterile and in the form of a solution or suspension suitable for injection or infusion. Such compositions constitute a further aspect of the invention.

The term "pharmaceutically acceptable salt" as used herein is intended to mean a salt of the free acid or base of the compound represented by formula (a) which is non-toxic, biologically tolerable or otherwise biologically suitable for administration to a subject. Generally, see g.s.paulekuhn, et., "trends in pharmaceutical ingredient and salt selection base analysis of the orange book database", j.med.chem.,2007, 50: 6665-72, S.M.Berge, et., "pharmaceutical salts", JPharmSci., 1977, 66: 1-19, and handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahland Wermuth, eds., Wiley-VCHandVHCA, Zurich, 2002(G.S. Paulekuhn et al, "trends in the Selection of Salts of active Pharmaceutical ingredients based on analysis of the Hesperit database", "journal of Pharmaceutical chemistry,2007, Vol.50, p.6665 667 2; S.M.Berge et al," pharmaceutically acceptable Salts "," journal of Pharmaceutical sciences, 1977, Vol.66, p.1-19; and "pharmaceutically acceptable Salts: Properties, Selection and applications", edited by Stahl and Wermuth, Wiley-VCHandVHCA, Zurich, 2002). Examples of pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a patient without undue toxicity, irritation, or allergic response. The compounds of formula (I) may have sufficiently acidic groups, sufficiently basic groups, or both types of functional groups to react with various inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, alkylbenzoate, oxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, butyrate, citrate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, propionate, fumarate, butyrate, and so forth, Lactate, Y-hydroxybutyrate, glycolate, tartrate, mesylate, propanesulfonate, cai-1-sulfonate, tea-2-sulfonate, and mandelate.

The compound of formula (a) contains a basic nitrogen and the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example by treating the free base with an inorganic acid, for example hydrochloric acid, hydrobromic acid, sulphuric acid, sulphamic acid, nitric acid, boric acid, phosphoric acid and the like, or with an organic acid, for example acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosidyl acid (such as glucuronic or galacturonic acid), alpha-hydrocarbon acid (such as mandelic, citric or tartaric acid), amino acid (such as aspartic, glutaric or glutamic acid), aromatic acid (such as benzoic acid, 2-acetoxybenzoic acid), benzoic acid, fumaric acid, malonic acid, succinic acid, malic acid, succinic acid, fumaric acid, malonic acid, succinic acid, fumaric acid, succinic acid, tartaric acid, citric acid, tartaric acid, citric acid, Zetimoic or cinnamic acid), sulfonic acids (e.g., laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid), any compatible mixtures of acids such as those given herein by way of example, and any other acid and mixtures thereof that are considered equivalents or acceptable substitutes in accordance with the level of ordinary skill in the art.

Prodrugs described herein may include compounds having a polypeptide chain of amino acid residues or two or more (e.g., two, three, or four) amino acid residues covalently linked to a carboxylic acid group of formula (a) through an amide or ester bond. Examples of amino acid residues include the twenty naturally occurring amino acids commonly identified by three letter symbols, as well as 4-hydroxyproline, hydroxylysine, desmosine (demosine), isodesmosine (isodemosine), 3-methylhistidine, norvaline, β -alanine, γ -aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine.

Other types of prodrugs can be prepared by derivatizing the free carboxylic acid of formula (a) as an amide or alkyl ester. Examples of amides include those derived from ammonia, C_1-6Alkyl primary amines and di (C)_1-6Alkyl) secondary amines. Secondary amines include 5-or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of acid amines include those derived from ammonia C_1-3Alkyl primary amines and di (C)_l-2Alkyl) amines. Examples of the fats of the present invention include C_1-7Alkyl esters, C_5-7Cycloalkyl ester, phenyl ester and (C)_1-6Alkyl) phenyl ester. Preferably, the ester comprises a methyl ester. Also according to methods such as Fleisheretal, adv. drug Delivery Rev.1996,19,115-Methods described in reviews of drug delivery, 1996, volume 19, page 115-130) for the preparation of prodrugs by derivatizing the free hydroxyl group with groups including hemisuccinate, phosphate, dimethylaminoacetate, and phosphoryloxymethoxycarbonyl. Carbamate derivatives of hydroxy and amino groups may also produce prodrugs. Carbonate derivatives of hydroxyl groups, sulfonates and sulfates may also provide prodrugs. Derivatization of the radicals into (acyloxy) methyl and (acyloxy) ethyl esters, where the acyl group may be an alkyl ester optionally substituted with one or more ether, amine or dredged acid functional groups, or where the acyl group is an amino ester as described above, may also be used to produce prodrugs. Prodrugs of this type can be prepared as described in Robinsonetae 1, JMedChem.1996,39(1), 10-18(Robinson et al, J. Pharmacochemistry, 1996, vol. 39, No. 1, p. 10-18). The free amine may also be derivatized to an amide, sulfonamide, or phosphoramide. All of these prodrug moieties may incorporate groups including ether, amine, and carboxylic acid functional groups.

The invention has the advantages that

1) The structure of the first generation TRK inhibitor is used, so that the drug resistance problem of the first generation TRK inhibitor in the use process of the TRK inhibitor is solved, and the effect of the first generation TRK inhibitor is achieved or even surpassed that of the second generation TRK inhibitor.

2) A novel structural Trk inhibitor is provided.

3) The TRK inhibitors provided by the present invention exhibit an IC50 value for the mutant TRK inhibitor that is similar to or lower than the corresponding wild-type TRK inhibitor IC50 value.

4) The invention creatively selects the imidazopyridine derivatives to obtain the effect of inhibiting wild type and mutant Trk enzymes in a low concentration range, and although imidazopyrimidine or imidazopyrazine is a bioisostere of imidazopyridine, the derivatives of the imidazopyridine derivatives do not achieve the effect of the invention.

Detailed Description

In a specific embodiment of the invention, unless otherwise indicated, the reagents are purchased from commercial suppliers (e.g., Aldrich Chemical, Lancaster, TCI or Maybridge) and used without further purification.

Preparation A:

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2-aminopyridine

The compound 2-amino-3-nitro-6-chloropyridine (5g, 28.7mmol), the compound (R) -2- (2, 5-difluorophenyl) pyrrolidine hydrochloride (6.9g, 31.6mmol) and DIEA (11.1g, 86.1mmol) were dissolved in 100mL of ethanol and reacted with stirring at 80 ℃ for 12 hours. After TLC detection, the reaction is cooled to 0 ℃, filtered, and the solid is washed by ethanol and dried to obtain 8.6g of yellow solid product with the yield of 92.3%. LC-MS (ESI) M/z 321[ M + H ]]⁺。

Preparation B:

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (dibenzoyloxy) aminopyridine

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2-aminopyridine (8.6g, 26.5mmol) was dissolved in 100ml of pyridine, phenyl chloroformate (12.2g, 79.6mmol) was added dropwise, and the reaction was stirred at 70 ℃ for 5 hours. After TLC detection, the solvent is removed by rotary evaporation and concentration, 100ml water is added to quench the reaction, ethyl acetate (3X 100ml) is used for extraction, organic phases are combined, dried and dried by rotary drying to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/3, V/V), 12.0g of a yellow solid product was obtained in 80.7% yield. LC-MS (ESI) M/z 561[ M + H ]]⁺。

Example 1 Compound 1

Preparation M1

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3-benzylureido) pyridine

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (dibenzoyloxyformyl) aminopyridine (2g, 3.5mmol), benzylamine (0.56g, 5.3mmol) and sodium carbonate (0.74g, 7mmol) were suspended in 25mL acetonitrile and stirred at room temperature for 12 hours. After TLC detection, 20ml water is added to quench the reaction, extraction is carried out with ethyl acetate (3X 50ml), and the organic phases are combined, dried and spin-dried to obtain the crude product. The crude product was purified by silica gel column (ethyl acetate/petroleum ether ═ 1/3, V/V) to yield 1.4g of a yellow solid product in 85.7% yield. LC-MS (ESI) M/z 454[ M + H ]]⁺。

(R) -N-benzyl-5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Intermediate M1(200mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction is finished, filtering is carried out, and filtrate is reserved and is dried in a rotary mode to obtain a crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 40mg of a white solid product in 19.1% yield.

LC-MS(ESI)m/z:434.5[M+H]⁺

¹H NMR(600MHz,CDCl3)δ9.24(s,1H),8.44(s,1H),7.86(d,J＝8.7Hz,1H),7.36-7.33(m,2H),7.30-7.29(m,3H),6.87-6.84(m,1H),6.78-6.76(m,1H),6.69-6.67(m,1H),6.38-6.37(m,1H),5.27-5.25(m,1H),4.59-4.55(m,1H),4.49-4.47(m,1H),3.68(s,1H),3.45-3.44(m,1H),2.45-2.41(m,1H),2.04-1.97(m,3H).

Example 2 Compound 2

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-fluorobenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-fluorobenzyl) ureido) pyridine. Prepared by the method of preparation M1 using 4-fluorobenzylamine instead of benzylamine.

Step B preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-fluorobenzyl) -3H-imidazo [4,5-B ] pyridine-3-carboxamide. Intermediate (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-fluorobenzyl) ureido) pyridine (300mg, 0.63mmol) was dissolved in 5mL of THF, 10% palladium on carbon (30mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (15mg) was added to stir the reaction at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 95mg of a white solid product with a yield of 30.7%.

LC-MS(ESI)m/z:452.4[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.22(s,1H),8.43(s,1H),7.88–7.86(m,1H),7.26(s,2H),7.04–7.01(m,2H),6.88–6.85(m,1H),6.81–6.78(m,1H),6.71–6.68(m,1H),6.42–6.41(m,1H),5.28–5.27(m,1H),4.54–4.50(m,1H),4.43–4.40(m,1H),3.71(s,1H),3.48–3.46(m,1H),2.46–2.42(m,1H),2.06–1.98(m,3H).

Example 3 Compound 3

(R) -N- (3, 5-difluorobenzyl) -5- (2- (3, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3, 5-difluorobenzyl) ureido) pyridine. Prepared by the method of preparation M1, using 3, 5-difluorobenzylamine instead of benzylamine.

And B: (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3, 5-difluorobenzyl) ureido) pyridine (200mg, 0.41mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added thereto, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. And (5) detecting by TLC, filtering after the reaction is finished, and performing spin-drying to obtain a crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 135 ℃ for 4 hours. TLC assay, after completion of the reaction, the reaction was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 45mg of product as a white solid in 21.5% yield.

LC-MS(ESI)m/z:470.4[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.25(s,1H),8.42(s,1H),7.90(d,J＝8.9Hz,1H),6.84–6.70(m,6H),6.48–6.47(m,1H),5.32–5.31(m,1H),4.49–4.44(m,2H),3.81–3.78(m,1H),3.57–3.54(m,1H),2.47–2.44(m,1H),2.11–2.07(m,2H),2.01–1.98(m,1H).

Example 4 Compound 4

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-fluorophenethyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-fluorophenethyl) ureido) pyridine. Prepared by the method of preparation M1 using 4-fluorophenylethylamine instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-fluorophenethyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-fluorophenethyl) ureido) pyridine (200mg, 0.42mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 65mg of a white solid product in 31.1% yield.

LC-MS(ESI)m/z:466.5[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.87(s,1H),8.42(s,1H),7.86(d,J＝8.8Hz,1H),7.21–7.19(m,2H),7.05–7.00(m,3H),6.90–6.87(m,1H),6.69–6.66(m,1H),6.41–6.39(m,1H),5.22–5.20(m,1H),3.70–3.65(m,2H),3.44–3.41(m,2H),2.84–2.79(m,2H),2.46–2.44(m,1H),2.07–2.00(m,3H).

Example 5 Compound 5

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-trifluoromethylbenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-trifluoromethylbenzyl) ureido) pyridine. Prepared by the method of preparation M1 using 4-trifluoromethylbenzylamine instead of benzylamine.

And B: (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-trifluoromethylbenzyl) ureido) pyridine (300mg, 0.57mmol) was dissolved in 5mL of THF, 10% palladium on carbon (30mg) was added thereto, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (15mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 115mg of a white solid product in 37.2% yield.

LC-MS(ESI)m/z:502.5[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.28(s,1H),8.42(s,1H),7.91–7.89(m,1H),7.60–7.59(m,2H),7.37–7.36(m,2H),6.77–6.68(m,3H),6.46–6.45(m,1H),5.31–5.29(m,1H),4.57–4.54(m,2H),3.74(s,1H),3.50–3.48(m,1H),2.45–2.42(m,1H),2.07–1.97(m,3H).

Example 6 Compound 6

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-fluorobenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A is the preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-fluorobenzyl) ureido) pyridine. Prepared by the method of preparation M1 using 3-fluorobenzylamine instead of benzylamine.

Step B preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-fluorobenzyl) -3H-imidazo [4,5-B ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-fluorobenzyl) ureido) pyridine (300mg, 0.63mmol) was dissolved in 5mL of THF, 10% palladium on carbon (30mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (15mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 95mg of a white solid product with a yield of 30.6%.

LC-MS(ESI)m/z:452.4[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.24(s,1H),8.43(s,1H),7.89(d,J＝8.8Hz,1H),7.32–7.28(m,1H),7.06–7.05(m,1H),7.00–6.95(m,2H),6.84–6.82(m,1H),6.78–6.76(m,1H),6.71–6.68(m,1H),6.44–6.43(m,1H),5.30–5.29(m,1H),4.56–4.45(m,2H),3.76–3.73(m,1H),3.53–3.49(m,1H),2.47–2.43(m,1H),2.08–2.06(m,2H),2.00–1.97(m,1H).

Example 7 Compound 7

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (2-fluorobenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (2-fluorobenzyl) ureido) pyridine. Prepared by the method of preparation M1 using 2-fluorobenzylamine instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (2-fluorobenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (2-fluorobenzyl) ureido) pyridine (300mg, 0.63mmol) was dissolved in 5mL of THF, 10% palladium on carbon (30mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (15mg) was added to react at 135 ℃ for 4 hours. TLC assay, after completion of the reaction, the reaction was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 85mg of white solid product in 27.6% yield.

LC-MS(ESI)m/z:452.4[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.30(s,1H),8.43(s,1H),7.85(d,J＝8.9Hz,1H),7.30–7.27(m,2H),7.11–7.07(m,2H),6.90–6.87(m,1H),6.78–6.75(m,1H),6.72–6.69(m,1H),6.37–6.36(m,1H),5.27–5.25(m,1H),4.64–4.53(m,2H),3.79–3.76(m,1H),3.56–3.52(m,1H),2.48–2.43(m,1H),2.08–2.00(m,3H).

Example 8 Compound 8

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N-phenethyl-3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3-phenethylureido) pyridine.

Prepared by the method of preparation M1 using phenethylamine instead of benzylamine.

And B: (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3-phenethylureido) pyridine (200mg, 0.42mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 85mg of a white solid product with a yield of 40.1%.

LC-MS(ESI)m/z:448.5[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.89(s,1H),8.43(s,1H),7.85(d,J＝8.8Hz,1H),7.34–7.31(m,2H),7.26(s,1H),7.25(s,1H),7.06–7.02(m,1H),6.90–6.87(m,1H),6.69–6.66(m,1H),6.39–6.38(m,1H),5.23–5.22(m,1H),3.72–3.70(m,1H),3.62(s,1H),3.44–3.41(m,2H),2.88–2.82(m,2H),2.45–2.43(m,1H),2.06–2.01(m,3H).

Example 9 Compound 9

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-phenylpropyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3-phenylpropylureido) pyridine.

Prepared by the method of preparation M1, using amphetamine instead of benzylamine.

And step B, preparing (R) -5- (2- (2, 5-difluorophenyl) pyrrolidine-1-yl) -N- (3-phenylpropyl) -3H-imidazo [4,5-B ] pyridine-3-formamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3-phenylpropylureido) pyridine (250mg, 0.51mmol) was dissolved in 5mL of THF, 10% palladium on carbon (25mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (12.5mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 65mg of a white solid product in 31.1% yield.

LC-MS(ESI)m/z:462.5[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.72(s,1H),8.42(s,1H),7.88(d,J＝8.8Hz,1H),7.31–7.29(m,2H),7.21–7.20(m,3H),6.85–6.81(m,2H),6.73–6.71(m,1H),6.45–6.43(m,1H),5.34–5.32(m,1H),3.85–3.83(m,1H),3.61–3.57(m,1H),3.42–3.39(m,1H),3.24–3.23(m,1H),2.70–2.67(m,2H),2.48–2.45(m,1H),2.12–2.02(m,3H),1.88–1.80(m,2H).

Example 10 Compound 10

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-picolyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-picolyl) ureido) pyridine. Prepared by the method of preparation M1, using 3-aminomethylpyridine instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-picolyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-picolyl) ureido) pyridine (220mg, 0.46mmol) was dissolved in 5mL of THF, 10% palladium on carbon (22mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (11mg) was added to react at 135 ℃ for 4 hours. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 2/1, V/V) to obtain 62mg of a white solid product in 27.1% yield.

LC-MS(ESI)m/z:435[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.26(s,1H),8.58-8.55(m,2H),8.42(s,1H),7.89(d,J＝9.0Hz,1H),7.60(d,J＝7.2Hz,1H),7.28–7.26(m,1H),6.89–6.85(m,1H),6.79–6.76(m,1H),6.71–6.68(m,1H),6.45–6.44(m,1H),5.31–5.30(m,1H),4.58–4.55(m,1H),4.47–4.44(m,1H),3.77–3.74(m,1H),3.53–3.49(m,1H),2.47–2.43(m,1H),2.09–2.06(m,2H),2.00–1.97(m,1H).

Example 11 Compound 11

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-picolyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-picolyl) ureido) pyridine. Prepared by the method of preparation M1, using 4-aminomethylpyridine instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-picolyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-picolyl) ureido) pyridine (200mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (22mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (11mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 2/1, V/V) to obtain 78mg of a white solid product with a yield of 40.6%.

LC-MS(ESI)m/z:435[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.26(s,1H),8.56-8.55(m,2H),8.42(s,1H),7.93-7.91(m,1H),7.14–7.13(m,2H),6.70–6.69(m,3H),6.50–6.49(m,1H),5.32–5.31(m,1H),4.51–4.50(m,2H),3.81–3.78(m,1H),3.56–3.52(m,1H),2.48–2.42(m,1H),2.10–2.07(m,2H),1.98–1.96(m,1H).

Example 12 Compound 12

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-thienylmethyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-thienylmethyl) ureido) pyridine. Prepared by the method of preparation M1, using 3-aminomethylthiophene instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-thienylmethyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (3-thienylmethyl) ureido) pyridine (180mg, 0.41mmol) was dissolved in 5mL of THF, 10% palladium on carbon (18mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (9mg) was added to react at 135 ℃ for 4 hours. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 50mg of a white solid product in 26.4% yield.

LC-MS(ESI)m/z:440[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.21(s,1H),8.42(s,1H),7.85(d,J＝8.8Hz,1H),7.32–7.30(m,1H),7.17(s,1H),7.06–7.05(m,1H),6.94–6.90(m,1H),6.84–6.80(m,1H),6.71–6.68(m,1H),6.39–6.37(m,1H),5.28–5.26(m,1H),4.60–4.56(m,1H),4.45–4.43(m,1H),3.73–3.72(m,1H),3.50–3.46(m,1H),2.48–2.42(m,1H),2.07–1.99(m,3H).

Example 13 Compound 13

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((1-methyl-1-H-pyrazol-4-yl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- ((1-methyl-1-H-pyrazol-4-yl) methyl) ureido) pyridine. Prepared by the method of preparation M1 using 4- (aminomethyl) -1-methylpyrazole instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((1-methyl-1-H-pyrazol-4-yl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- ((1-methyl-1-H-pyrazol-4-yl) methyl) ureido) pyridine (220mg, 0.48mmol) was dissolved in 5mL of THF, 10% palladium on carbon (22mg) was added thereto, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (11mg) was added to react at 135 ℃ for 4 hours. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 62mg of a white solid product in 29.1% yield.

LC-MS(ESI)m/z:438[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.07(s,1H),8.41(s,1H),7.86(d,J＝8.8Hz,1H),7.45(s,1H),7.35(s,1H),7.03–6.99(m,1H),6.89–6.85(m,1H),6.73–6.70(m,1H),6.42–6.40(m,1H),5.30(d,J＝7.7Hz,1H),4.44–4.40(m,1H),4.24–4.21(m,1H),3.87(s,3H),3.78–3.75(m,1H),3.54–3.50(m,1H),2.49–2.43(m,1H),2.10–2.07(m,2H),2.03–2.00(m,1H).

Example 14 Compound 14

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((tetrahydro-2H-pyran-4-yl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (((tetrahydro-2H-pyran-4-yl) methyl) ureido) pyridine. Prepared by the method of preparation M1, using 4-aminomethyltetrahydropyran instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((tetrahydro-2H-pyran-4-yl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (((tetrahydro-2H-pyran-4-yl) methyl) ureido) pyridine (200mg, 0.43mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 100 ℃ for 1 hour. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 2/1, V/V) to obtain 56mg of a white solid product in 26.7% yield.

LC-MS(ESI)m/z:442[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.85(s,1H),8.42(s,1H),7.89(d,J＝8.9Hz,1H),7.07(td,J＝9.2,4.3Hz,1H),6.94–6.91(m,1H),6.75–6.72(m,1H),6.45–6.44(m,1H),5.35–5.33(m,1H),4.00–3.96(m,2H),3.87–3.84(m,1H),3.62–3.58(m,1H),3.39–3.35(m,2H),3.31–3.28(m,1H),3.03(s,1H),2.50–2.47(m,1H),2.14–2.05(m,3H),1.79–1.77(m,1H),1.67–1.65(m,1H),1.39–1.25(m,3H).

Example 15 Compound 15

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (tetrahydro-2H-pyran-4-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((tetrahydro-2H-pyran-4-yl) ureido) pyridine. Prepared by the method of preparation M1 using 4-aminotetrahydropyran instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (tetrahydro-2H-pyran-4-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((tetrahydro-2H-pyran-4-yl) ureido) pyridine (250mg, 0.56mmol) was dissolved in 5mL of THF, 10% palladium on carbon (25mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (13mg) was added to react at 100 ℃ for 1 hour. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 2/1, V/V) to obtain 89mg of a white solid product with a yield of 34.2%.

LC-MS(ESI)m/z:428[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.86(s,1H),8.44(s,1H),7.87(d,J＝8.9Hz,1H),7.12–7.08(m,1H),6.94–6.91(m,1H),6.73–6.70(m,1H),6.39–6.38(m,1H),5.31–5.30(m,1H),4.06–4.01(m,2H),3.96–3.95(m,1H),3.87–3.84(m,1H),3.64–3.60(m,1H),3.54–3.46(m,2H),2.51–2.44(m,1H),2.15–2.11(m,2H),2.05–2.01(m,3H),1.81(s,1H),1.64–1.57(m,1H),1.27–1.25(m,1H).

Example 16 Compound 16

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-methoxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((4-methoxycyclohexyl) ureido) pyridine. Prepared by the method of preparation M1, using 4-methoxycyclohexylamine hydrochloride instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-methoxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((4-methoxycyclohexyl) ureido) pyridine (210mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (21mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (11mg) was added to react at 100 ℃ for 1 hour. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 2/1, V/V) to obtain 49mg of a white solid product in 22.4% yield.

LC-MS(ESI)m/z:456[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.71(s,1H),8.43(s,1H),7.87(d,J＝8.8Hz,1H),7.08–7.04(m,1H),6.95–6.91(m,1H),6.73–6.70(m,1H),6.41(s,1H),5.33(s,1H),3.85–3.82(m,1H),3.79–3.77(m,1H),3.62–3.59(m,1H),3.40(s,3H),3.20–3.16(m,1H),2.49–2.43(m,1H),2.16–2.11(m,4H),2.06–2.01(m,2H),2.00–1.91(m,1H),1.38–1.26(m,3H),0.98–0.93(m,1H).

Example 17 Compound 17

(1S,4S) -4- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) cyclohexanecarboxylic acid

Step A: preparation of 6- ((R) - (2- (2, 5-difluorophenyl) pyrrolidin-1-yl)) -3-nitro-2- (((1S,4S) -4-hydroxycyclohexyl) ureido) pyridine. Prepared by the method of preparation M1 using cis-p-aminocyclohexanol hydrochloride in place of benzylamine.

And B: preparation of cyclohexyl (1S,4S) -4- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) carboxylate. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((1S,4S) - (4-hydroxycyclohexyl) ureido) pyridine (200mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 100 ℃ for 1 hour. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 31mg of a white solid product in 14.9% yield.

LC-MS(ESI)m/z:470[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.81(s,1H),8.46(s,1H),8.18(s,1H),7.88(s,1H),7.07–7.03(m,1H),6.93–6.90(m,1H),6.72–6.69(m,1H),6.39(s,1H),5.33–5.31(m,1H),5.16(s,1H),3.88–3.85(m,2H),3.66–3.63(m,1H),2.49–2.46(m,1H),2.14–2.12(m,2H),2.05–2.01(m,2H),1.99–1.96(m,2H),1.77–1.67(m,4H),1.43(s,1H).

Example 18 Compound 18

3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) methyl) acid cyclohexanecarboxylic acid

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (((3-hydroxycyclohexyl) methyl) ureido) pyridine. Prepared by the method of preparation M1 using 3- (aminomethyl) cyclohexanol instead of benzylamine.

And B: preparation of 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) methyl) acid cyclohexylcarboxylic acid. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (((3-hydroxycyclohexyl) methyl) ureido) pyridine (240mg, 0.50mmol) was dissolved in 5mL of THF, 10% palladium on carbon (24mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (12mg) was added to react at 100 ℃ for 1 hour. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 22mg of a white solid product in 8.9% yield.

LC-MS(ESI)m/z:484[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.85(s,1H),8.41(s,1H),8.03(s,1H),7.88(dd,J＝8.8,3.7Hz,1H),7.10–7.06(m,1H),6.94–6.92(m,1H),6.76–6.74(m,1H),6.45–6.42(m,1H),5.35–5.31(m,1H),4.85–4.84(m,1H),3.86(s,1H),3.61–3.59(m,1H),3.35–3.22(m,1H),3.13–2.95(m,1H),2.50–2.47(m,1H),2.14–2.05(m,5H),1.87–1.85(m,1H),1.76–1.74(m,1H),1.36–1.33(m,2H),1.27–1.23(m,1H),1.17-1.08(m,1H),0.95–0.93(m,1H).

Example 19 Compound 19

(1R,3S) -3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) methyl) acid cyclopentylcarboxylic acid

Step A: preparation of 6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((3- (1S,3R) -hydroxycyclohexyl) ureido) pyridine. Prepared by the method of preparation M1 using (1R,3S) -3-aminocyclopentanol hydrochloride instead of benzylamine.

And B: preparation of cyclopentylcarboxylic acid (1R,3S) -3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide) methyl) acid. 6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((3- (1S,3R) -hydroxycyclohexyl) ureido) pyridine (200mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (10mg) was added to react at 100 ℃ for 1 hour. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 26mg of a white solid product in 12.9% yield.

LC-MS(ESI)m/z:456[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.99(s,1H),8.44(s,1H),8.07(s,1H),7.85(d,J＝8.9Hz,1H),7.09–7.05(m,1H),6.94–6.91(m,1H),6.72–6.69(m,1H),6.37–6.35(m,1H),5.34–5.30(m,2H),4.38–4.34(m,1H),3.88–3.85(m,1H),3.67–3.62(m,1H),2.61–2.56(m,1H),2.50–2.43(m,1H),2.14–2.09(m,3H),2.02–1.95(m,3H),1.81–1.76(m,1H),1.48(s,1H).

Example 20 Compound 20

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4- (2-hydroxyethyl) cyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((4- (2-hydroxyethyl) cyclohexyl) ureido) pyridine. Prepared by the method of preparation M1, using 4-aminocyclohexaneethanol instead of benzylamine.

And B: preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4- (2-hydroxyethyl) cyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- ((4- (2-hydroxyethyl) cyclohexyl) ureido) pyridine (250mg, 0.51mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (25mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (110mg, 1.02mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to give 56mg of the product as a white solid in 21.9% yield.

LC-MS(ESI)m/z:470[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.91–8.80(m,1H),8.47–8.44(m,1H),7.87–7.80(m,1H),7.09–7.06(m,1H),6.93–6.91(m,1H),6.72–6.70(m,1H),6.37–6.24(m,1H),5.32–5.31(m,1H),3.84–3.59(m,5H),2.47–2.46(m,1H),2.14–2.00(m,4H),1.90–1.61(m,6H),1.55–1.47(m,3H),1.27–1.09(m,2H).

Example 21 Compound 21

5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((1R,4R) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of 6- ((R) - (2- (2, 5-difluorophenyl) pyrrolidin-1-yl)) -3-nitro-2- (((1R,4R) -4-hydroxycyclohexyl) ureido) pyridine. Prepared by the method of preparation M1 using trans-p-aminocyclohexanol hydrochloride instead of benzylamine.

And B: preparation of 5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((1R,4R) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. 6- ((R) - (2- (2, 5-difluorophenyl) pyrrolidin-1-yl)) -3-nitro-2- (((1R,4R) -4-hydroxycyclohexyl) ureido) pyridine (240mg, 0.52mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (24mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (110mg, 1.04mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to yield 46mg of the product as a white solid in 18.4% yield.

LC-MS(ESI)m/z:442[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.69(s,1H),8.43(s,1H),7.88(d,J＝8.8Hz,1H),7.08–7.04(m,1H),6.93–6.91(m,1H),6.72–6.69(m,1H),6.43–6.42(m,1H),5.34–5.32(m,1H),3.84–3.80(m,1H),3.76–3.74(m,1H),3.68–3.65(m,1H),3.61–3.56(m,1H),2.49–2.42(m,1H),2.14–2.07(m,4H),2.04–1.97(m,2H),1.88(s,1H),1.63(s,1H),1.48–1.25(m,3H),0.91(s,1H).

Example 22 Compound 22

5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((1S,4S) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

And B: 6- ((R) - (2- (2, 5-difluorophenyl) pyrrolidin-1-yl)) -3-nitro-2- (((1S,4S) -4-hydroxycyclohexyl) ureido) pyridine (250mg, 0.54mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (25mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (110mg, 1.08mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to give the product as a white solid, 48mg, 18.6% yield.

LC-MS(ESI)m/z:442[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.85(s,1H),8.45(s,1H),7.84(d,J＝8.9Hz,1H),7.11–7.07(m,1H),6.93–6.89(m,1H),6.72–6.69(m,1H),6.33–6.32(m,1H),5.24(t,J＝38.9Hz,1H),4.02–3.96(m,1H),3.90–3.87(m,2H),3.68–3.64(m,1H),2.51–2.44(m,1H),2.14–2.09(m,2H),2.05–1.97(m,1H),1.89–1.85(m,2H),1.82–1.72(m,6H),1.37–1.35(m,1H).

Example 23 Compound 23

5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((3-hydroxycyclohexyl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

And B: (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (((3-hydroxycyclohexyl) methyl) ureido) pyridine (210mg, 0.44mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (21mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (96mg, 0.88mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to yield 36mg of the product as a white solid in 16.6% yield.

LC-MS(ESI)m/z:456[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.86(s,1H),8.42(s,1H),7.88–7.87(m,1H),7.08–7.04(m,1H),6.93–6.89(m,1H),6.75–6.72(m,1H),6.43–6.42(m,1H),5.32(s,1H),3.87–3.85(m,1H),3.63–3.59(m,2H),3.33–3.30(m,1H),3.02(s,1H),2.50–2.47(m,1H),2.14–1.99(m,5H),1.84–1.79(m,1H),1.75–1.68(m,1H),1.60–1.55(m,1H),1.50–1.47(m,1H),1.31–1.26(m,1H),1.20–1.18(m,1H),1.01–0.92(m,2H).

Example 24 Compound 24

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-phenylcyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-phenylcyclohexyl) ureido) pyridine. Prepared by the method of preparation M1 using 4-phenylcyclohexylamine instead of benzylamine.

Step B preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-fluorobenzyl) -3H-imidazo [4,5-B ] pyridine-3-carboxamide. Intermediate (R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitro-2- (3- (4-phenylcyclohexyl) ureido) pyridine (200mg, 0.41mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (20mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (90mg, 0.82mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to yield 46mg of the product as a white solid in 18.9% yield.

LC-MS(ESI)m/z:502[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.27–8.93(m,1H),8.50(s,1H),7.84–7.74(m,1H),7.34–7.33(m,1H),7.32(s,1H),7.31(s,1H),7.28(s,1H),7.26(s,1H),7.13–7.05(m,1H),7.05–6.98(m,1H),6.97–6.82(m,2H),6.77–6.68(m,2H),6.28–6.06(m,1H),5.21–5.08(m,1H),4.38(s,1H),3.91–3.79(m,2H),3.70–3.49(m,2H),2.72(s,1H),2.60–2.50(m,1H),2.50–2.37(m,2H),2.21–2.10(m,3H),2.07–1.94(m,6H),1.94–1.86(m,4H),1.66–1.54(m,3H),1.47–1.35(m,1H),1.33–1.24(m,1H).

Preparation C:

(R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2-aminopyridine

The compound 2-amino-3-nitro-6-chloropyridine (2g, 11.5mmol), the compound (R) -5-fluoro-2-methoxy-3- (pyrrolidin-3-yl) pyridine hydrochloride (2.66g, 11.5mmol) and DIEA (4.4g, 34.5mmol) were dissolved in 50mL of ethanol and reacted with stirring at 80 ℃ for 12 hours. After TLC detection, the reaction is cooled to 0 ℃, filtered, washed by ethanol and dried by suction to obtain 4g of yellow solid product with 100 percent of yield. LC-MS (ESI) M/z 334[ M + H ]]⁺。

Preparation D:

(R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (dibenzoyloxy) aminopyridine

(R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2-aminopyridine (4g, 12mmol) was dissolved in 50ml of pyridine, phenyl chloroformate (5.6g, 36mmol) was added dropwise, and the reaction was stirred at 70 ℃ for 5 hours. After TLC detection, the solvent is removed by rotary evaporation and concentration, 50ml water is added to quench the reaction, ethyl acetate (3X 50ml) is used for extraction, organic phases are combined, dried and dried by rotary drying to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/3, V/V), 5.0g of the product was obtained as a yellow solid in 72.7% yield. LC-MS (ESI) M/z 574[ M + H ]]⁺。

Preparation M2

(R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (3- (3-fluorobenzyl) ureido) pyridine

(R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (diphenoxyformyl) aminopyridine (300mg, 0.52mmol), m-fluorobenzylamine (125mg, 1.04mmol) and sodium carbonate (159mg, 1.56mmol) were suspended in 10mL acetonitrile and stirred at room temperature for 12 hours. After TLC detection, 20ml water is added to quench the reaction, extraction is carried out with ethyl acetate (3X 50ml), and the organic phases are combined, dried and spin-dried to obtain the crude product. The crude product was purified by silica gel column (ethyl acetate/petroleum ether: 1/3, V/V) to obtain 230mg of a yellow solid product in 90.3% yield. LC-MS (ESI) M/z 485[ M + H ]]⁺。

Example 25 Compound 25

(R) -5- (2- (5-fluoro-2-methoxypyridin-3-yl) pyrrolidin-1-yl) -N- (3-fluorobenzyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Intermediate M2(230mg, 0.47mmol) was dissolved in 5mL of THF, 10% palladium on carbon (23mg) was added to replace hydrogen, and the reaction was stirred at room temperature for 12 hours. And (5) detecting by TLC, filtering after the reaction is finished, and performing spin-drying to obtain a crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (11mg) was added to react at 135 ℃ for 4 hours. TLC assay, after completion of the reaction, the reaction was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to yield 65mg of the product as a white solid in 29.7% yield.

LC-MS(ESI)m/z:465[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.91(s,1H),8.43(s,1H),7.90(s,1H),7.83(d,J＝8.7Hz,1H),7.33–7.30(m,2H),7.25–7.22(m,1H),6.98–6.96(m,1H),6.31(s,1H),5.11–5.10(m,1H),4.00(s,3H),3.59–3.49(m,2H),3.36(s,1H),2.82–2.74(m,2H),2.41–2.34(m,1H),2.00–1.97(m,2H),1.91–1.89(m,1H).

Example 26 Compound 26

(R) -5- (2- (5-fluoro-2-methoxypyridin-3-yl) pyrrolidin-1-yl) -N-phenethyl-3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (3-phenethylureido) pyridine. Prepared by the method of preparation M2, using phenethylamine instead of M-fluorobenzylamine.

And B: (R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (3-phenethylureido) pyridine (190mg,0.39mmol) was dissolved in 5mL of THF, 10% palladium on carbon (19mg) was added thereto to replace hydrogen, and the reaction was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in triethyl orthoformate (5mL), and p-toluenesulfonic acid (9mg) was added to react at 135 ℃ for 4 hours. TLC assay, after completion of the reaction, the reaction was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 43mg of a white solid product in 21.6% yield.

LC-MS(ESI)m/z:461[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.30(s,1H),8.42(s,1H),7.87(d,J＝8.7Hz,1H),7.79(d,J＝2.5Hz,1H),7.33–7.29(m,1H),7.26(s,1H),7.06–7.03(m,1H),7.00–6.97(m,3H),6.38(s,1H),5.17–5.16(m,1H),4.51–4.49(m,1H),4.38–4.35(m,1H),3.86(s,3H),3.72–3.70(m,1H),3.50–3.47(m,1H),2.43–2.38(m,1H),2.05–2.03(m,1H),1.98–1.92(m,2H).

Example 27 Compound 27

5- ((R) -2- (5-fluoro-2-methoxypyridin-3-yl) pyrrolidin-1-yl) -N- ((1S,4S) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (3- ((1S,4S) -4-hydroxycyclohexyl) ureido) pyridine. Prepared by the method of preparation M2, using (1S,4S) -4-hydroxycyclohexylamine instead of M-fluorobenzylamine.

And B: (R) -6- (2- (5-fluoro-2-methoxypyridine) pyrrolidin-1-yl) -3-nitro-2- (3- ((1S,4S) -4-hydroxycyclohexyl) ureido) pyridine (200mg,0.39mmol) was dissolved in 5mL of THF, 10% palladium on carbon (20mg) was added, hydrogen was replaced, and the reaction was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (90mg, 0.78mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by TLC preparation (ethyl acetate/petroleum ether: 3/1, V/V) to obtain 36mg of a white solid product in 13.9% yield.

LC-MS(ESI)m/z:455[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.45(s,1H),7.91–7.90(m,1H),7.83–7.82(m,1H),7.26(s,1H),7.04–7.02(m,1H),6.28(s,1H),5.12–5.11(m,1H),4.06(s,3H),4.00(s,1H),3.89–3.87(m,2H),3.65–3.64(m,1H),2.41–2.40(m,1H),2.10–2.07(m,2H),1.91–1.79(m,4H),1.74–1.69(m,5H),1.27–1.25(m,1H).

Preparation E:

(R) -2- (1- ((6-amino-5-nitropyridin-2-yl) amino) ethyl) -4-fluorophenol

The compound 2-amino-3-nitro-6-chloropyridine (1.24g, 7.1mmol), the compound (R) -2- (1-aminoethyl) -4-fluorophenol hydrochloride (1.37g, 7.1mmol) and DIEA (2.68g, 21.3mmol) were dissolved in 20mL of ethanol and the reaction was stirred at 80 ℃ for 24 hours. After the reaction was confirmed by TLC, it was dried by spinning and purified by silica gel column chromatography (ethyl acetate/petroleum ether: 1/3, V/V) to give 1.7g of a yellow solid product in 81.7% yield. LC-MS (ESI) M/z 293[ M + H ]]⁺。

Preparation F:

(R) -2- (1- ((6-amino-5-nitropyridin-2-yl) amino) ethyl) -1-benzyloxy-4-fluorobenzene

(R) -2- (1- ((6-amino-5-nitropyridin-2-yl) amino) ethyl) -4-fluorophenol (1.7g, 5.8mmol), bromobenzyl (2.0g, 11.6mmol), potassium carbonate (2.4g, 17.4mmol), sodium iodide (100mg) were added to a 50ml round-bottomed flask, DMF was added as a solvent to 20ml, and the reaction was stirred at room temperature for 18 hours. After completion of the reaction as confirmed by TLC detection, 100ml of water was added to quench the reaction, the product was extracted with ethyl acetate (30 ml. times.5 times), and the organic phase was washed with saturated brine, dried and spin-dried to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/2, V/V), 2.2g of the product was obtained as a yellow solid in 99.1% yield. LC-MS (ESI) M/z 383[ M + H ]]⁺。

Preparation G:

(R) -6- (1- (2-benzyloxy-5-fluorophenyl) ethyl) amino-3-nitro-2- (dibenzoyloxy) aminopyridine

(R) -2- (1- ((6-amino-5-nitropyridin-2-yl) amino) ethyl) -1-benzyloxy-4-fluorobenzene (2.2g, 5.7mmol) was dissolved in 20ml of pyridine, phenyl chloroformate (2.7g, 17.2mmol) was added dropwise, and the reaction was stirred in an oil bath at 50 ℃ for 18 hours. After TLC detection, removing solvent by rotary evaporation and concentration, adding 30ml water to quench reaction, extracting with ethyl acetate (30ml X3 times), combining organic phases, drying and rotary drying to obtain crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/2, V/V), 3.3g of a yellow solid product was obtained in 93.0% yield. LC-MS (ESI) M/z 623[ M + H ]]⁺。

Preparation M3

(R) -6- (1- (2-benzyloxy-5-fluorophenyl) ethyl) amino-3-nitro-2- (3- (4-methoxycyclohexyl) ureido) pyridine

(R) -6- (1- (2-benzyloxy-5-fluorophenyl) ethyl) amino-3-nitro-2- (dibenzoyloxy) aminopyridine (450mg, 0.72mmol), 4-methoxycyclohexylamine (192mg, 1.48mmol) and sodium carbonate (230mg, 2.17mmol) were suspended in 20mL acetonitrile and the reaction was stirred in a 50 ℃ oil bath for 18 hours. After the TLC detection reaction was complete, it was spun dry and purified by silica gel column (ethyl acetate/petroleum ether-1/2, V/V) to yield 260m g as a yellow solid in 66.7% yield. LC-MS (ESI) M/z 538[ M + H ]]⁺。

Preparation M4

(R) -5- ((1- (2-benzyloxy-5-fluorophenyl) ethyl) amino) -N- (4-methoxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Intermediate M3(260mg, 0.48mmol) was dissolved in 10mL of ethanol, 5% platinum carbon (30mg) was added to replace hydrogen, and the mixture was stirred at room temperature for 3 hours. Intermediate M3 was checked by TLC for complete reaction, filtered, and ethyl carboximide hydrochloride (106mg, 0.96mmol) was added to the filtrate and reacted for 18 hours with stirring in an oil bath at 50 ℃. The new product was formed by TLC, and the reaction mixture was concentrated and separated by silica gel column chromatography to obtain 110mg (ethyl acetate/petroleum ether: 5/1, V/V) of the product in 44.2% yield.

Example 28 Compound 28

(R) -5- ((1- (5-fluoro-2-hydroxyphenyl) ethyl) amino) -N- (4-methoxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Intermediate M4(R) -5- ((1- (2-benzyloxy-5-fluorophenyl) ethyl) amino) -N- (4-methoxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide (110mg, 0.21mmol) was dissolved in 10mL of methanol, 10% palladium on carbon (20mg) was added, hydrogen was replaced, and the reaction mixture was stirred in an oil bath at 60 ℃ for 18 hours. After the completion of the TLC detection reaction, filtration was performed, and the reaction solution was concentrated by rotary evaporation and purified by TLC plate separation (ethyl acetate/petroleum ether: 10/1, V/V) to obtain 49mg of a white solid product with a yield of 54.5%.

LC-MS(ESI)m/z:428[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.67-8.66(m,1H),8.50(s,1H),7.80(s,1H),6.99-6.96(m,2H),6.86-6.84(m,1H),6.80-6.78(m,1H),6.46-6.45(m,1H),5.09-5.07(m,1H),4.93-4.92(m,1H),3.86-3.85(m,1H),3.34(s,3H),2.17-2.15(m,1H),1.44-1.25(m,8H),1.65(s,3H)。

Example 29 Compound 29

(R) -5- ((1- (5-fluoro-2-hydroxyphenyl) ethyl) amino) -N- ((1s,4s) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide

Step A: preparation of (R) -6- (1- (2-benzyloxy-5-fluorophenyl) ethyl) amino-3-nitro-2- (3- ((1s,4s) -4-hydroxycyclohexyl) ureido) pyridine. Prepared by the method for preparing M3, using (1s,4s) -4-hydroxycyclohexylamine instead of 4-methoxycyclohexylamine, the yield is 54.8%.

And B: preparation of (R) -5- ((1- (2-benzyloxy-5-fluorophenyl) ethyl) amino) -N- ((1s,4s) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide. Prepared by the method for preparation M4 substituting M3 for (R) -6- (1- (2-benzyloxy-5-fluorophenyl) ethyl) amino-3-nitro-2- (3- ((1s,4s) -4-hydroxycyclohexyl) ureido) pyridine in 31.4% yield.

And C: (R) -5- ((1- (2-benzyloxy-5-fluorophenyl) ethyl) amino) -N- ((1s,4s) -4-hydroxycyclohexyl) -3H-imidazo [4,5-b ] pyridine-3-carboxamide (60mg, 0.12mmol) was dissolved in 10mL of methanol, 10% palladium on carbon (20mg) was added thereto to displace hydrogen, and then the reaction was stirred in an oil bath at 60 ℃ for 18 hours. After the TLC detection reaction was completed, the reaction solution was filtered, concentrated by rotary evaporation, and purified by TLC plate separation (ethyl acetate/petroleum ether: 10/1, V/V) to obtain 20mg of a white solid product with a yield of 40.3%.

LC-MS(ESI)m/z:414[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.72-8.70(m,1H),8.39(s,1H),8.28(s,1H),7.81(d,J＝12Hz,1H),6.95-6.93(m,1H),6.74-6.71(m,2H),6.55-6.53(m,1H),5.39-5.37(m,1H),5.00-4.99(m,1H),4.31-4.27(m,1H),4.01-3.99(m,1H),2.47(s,1H),2.15-1.65(m,8H),1.67(s,3H)。

Preparation M5

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4-aminopyridine

The compound 4-amino-5-nitro-2-chloropyridine (5g, 28.7mmol), the compound (R) -2- (2, 5-difluorophenyl) pyrrolidine hydrochloride (6.9g, 31.6mmol) and DIEA (11.1g, 86.1mmol) were dissolved in 100mL of ethanol and reacted with stirring at 80 ℃ for 12 hours. After TLC detection, the reaction is cooled to 0 ℃, filtered, and the solid is washed by ethanol and dried to obtain 8.9g of yellow solid product with 94.3 percent of yield. LC-MS (ESI) M/z 321[ M + H ]]⁺。

Preparation of M6:

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (dibenzoyloxy) aminopyridine

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4-aminopyridine (8.9g, 27.5mmol) was dissolved in 100ml of pyridine, phenyl chloroformate (12.2g, 79.6mmol) was added dropwise, and the reaction was stirred at 70 ℃ for 5 hours. After TLC detection, the solvent is removed by rotary evaporation and concentration, 100ml water is added to quench the reaction, ethyl acetate (3X 100ml) is used for extraction, organic phases are combined, dried and dried by rotary drying to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/3, V/V), the product was obtainedYellow solid product 10.0g, yield 70.7%. LC-MS (ESI) M/z 561[ M + H ]]⁺。

Preparation M7

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (3- (3-fluorobenzyl) ureido) pyridine

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (dibenzoyloxyformyl) aminopyridine (2g, 3.5mmol), 3-fluorobenzylamine (0.66g, 5.3mmol) and sodium carbonate (0.74g, 7mmol) were suspended in 25mL acetonitrile and stirred at room temperature for 12 h. After TLC detection, 20ml water is added to quench the reaction, extraction is carried out with ethyl acetate (3X 50ml), and the organic phases are combined, dried and spin-dried to obtain the crude product. The crude product was purified by silica gel column (ethyl acetate/petroleum ether ═ 1/3, V/V) to give 1.3g of a yellow solid product in 78.8% yield. LC-MS (ESI) M/z 472[ M + H ]]⁺。

Example 30 Compound 30

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-fluorobenzyl) -1H-imidazo [4,5-c ] pyridine-1-carboxamide

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (3- (3-fluorobenzyl) ureido) pyridine (300mg, 0.63mmol) was dissolved in 5mL of THF, 10% palladium on carbon (30mg) was added thereto to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (15mg) was added to react at 135 ℃ for 4 hours. After the TLC detection reaction was completed, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 75mg of a white solid product in 26.4% yield.

LC-MS(ESI)m/z:452[M+H]⁺。

¹H NMR(600MHz,CDCl₃)δ9.24(s,1H),8.43(s,1H),7.89(d,J＝8.8Hz,1H),7.32–7.29(m,1H),7.06–7.05(m,1H),6.99–6.95(m,2H),6.84–6.82(m,1H),6.78–6.75(m,1H),6.71–6.68(m,1H),6.44–6.43(m,1H),5.30–5.29(m,1H),4.56–4.52(m,1H),4.48–4.44(m,1H),3.76–3.73(m,1H),3.53–3.48(m,1H),2.47–2.43(m,1H),2.08–2.03(m,2H),2.00–1.97(m,1H).

Preparation M8

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -2-nitro-3-aminopyridine

The compound 3-amino-2-nitro-5-bromopyridine (1g, 4.5mmol), the compound (R) -2- (2, 5-difluorophenyl) pyrrolidine hydrochloride (1.1g, 5.1mmol) and DIEA (1.8g, 12.1mmol) were dissolved in 20mL of NMP and the reaction was stirred at 180 ℃ for 2 hours. And after TLC detection determines that the reaction is finished, adding water, extracting by using ethyl acetate, combining organic phases, drying, and performing rotary evaporation and concentration to obtain a crude product. The crude product was purified by silica gel column (ethyl acetate/petroleum ether ═ 1/3, V/V) to afford 1.5g of product as a yellow solid in 100% yield.

LC-MS(ESI)m/z:321[M+H]⁺。

Preparation of M9:

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -2-nitro-3- (dibenzoyloxy) aminopyridine

M8(1.5g, 4.6mmol) was dissolved in 10ml of pyridine, phenyl chloroformate (1.6g, 9.6mmol) was added dropwise, and the reaction was stirred at 70 ℃ for 5 hours. After TLC detection, the solvent is removed by rotary evaporation and concentration, 50ml water is added to quench the reaction, ethyl acetate (3X 50ml) is used for extraction, organic phases are combined, dried and dried by rotary drying to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/3, V/V), 2.3g of a yellow solid product was obtained in 89.3% yield.

LC-MS(ESI)m/z:561[M+H]⁺。

Preparation M10

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -2-nitro-3- (3- (4-methoxycyclohexyl) ureido) pyridine

M9(560mg, 1.0mmol), 4-methoxycyclohexylamine hydrochloride (260mg, 2.0mmol) and sodium carbonate (320mg, 3mmol) were suspended in 20mL of acetonitrile and stirred at room temperature for 12 hours. After the TLC detection reaction was completed, it was concentrated by rotary evaporation and purified by silica gel column (ethyl acetate/petroleum ether: 1/3, V/V) to obtain 350mg of a yellow solid product in 73.6% yield. LC-MS (ESI) M/z 476[ M + H ]]⁺。

Example 31 Compound 31

(R) -6- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (4-methoxycyclohexyl) -1H-imidazo [4,5-b ] pyridine-1-carboxamide

M10(230mg, 0.48mmol) was dissolved in 5mL of ethanol, 10% palladium on carbon (23mg) was added to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After completion of the TLC detection reaction, the mixture was filtered, and ethylformiminide hydrochloride (102mg, 0.96mmol) was added to the reaction mixture to conduct a reaction at 80 ℃ for 5 hours. After TLC detection reaction, concentrating the reaction solution, adding water, extracting with ethyl acetate, combining, drying and spin-drying to obtain a crude product. The crude product was purified by preparative TLC (ethyl acetate/petroleum ether-3/1, V/V) to give 44mg of the product as a white solid in 19.1% yield.

LC-MS(ESI)m/z:456[M+H]⁺。

¹H NMR(600MHz,CDCl₃)δ8.91–8.90(m,1H),8.64(s,1H),7.69–7.68(m,1H),7.19–7.18(m,1H),7.08–7.07(m,1H),6.92–6.91(m,1H),6.76–6.75(m,1H),5.00–4.98(m,1H),3.90(s,1H),3.81–3.79(m,1H),3.46–3.45(m,1H),3.36(s,3H),3.22(s,1H),2.51–2.50(m,1H),2.18–2.03(m,7H),1.45–1.44(m,4H).

Synthesis of comparative Compounds

Preparation H

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -4-amino-5-nitropyrimidine

The compound 2-chloro-4-amino-5-nitropyrimidine (1.0g, 5.7mmol), the compound (R) -2- (2, 5-difluorophenyl) pyrrolidine hydrochloride (1.2g, 5.7mmol) and DIEA (1.5g, 11.4mmol) were dissolved in 25mL of NMP and reacted with stirring at 180 ℃ for 2 hours. And after TLC detection determines that the reaction is finished, adding water, extracting by using ethyl acetate, combining organic phases, drying, and performing rotary evaporation and concentration to obtain a crude product. The crude product was purified by silica gel column (ethyl acetate/petroleum ether ═ 1/3, V/V) to afford 1.8g of product as a yellow solid in 98.4% yield.

LC-MS(ESI)m/z:322[M+H]⁺。

Preparation I:

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (dibenzoyloxy) aminopyrimidine

Intermediate H (1.8g, 6.1mmol) was dissolved in 20ml of pyridine, phenyl chloroformate (2.9g, 18.6mmol) was added dropwise, and the reaction was stirred at 70 ℃ for 5 hours. After TLC detection, the solvent is removed by rotary evaporation and concentration, 50ml water is added to quench the reaction, ethyl acetate (3X 50ml) is used for extraction, organic phases are combined, dried and dried by rotary drying to obtain a crude product. After purification on a silica gel column (ethyl acetate/petroleum ether: 1/3, V/V), 2.5g of the product was obtained as a yellow solid in 73.1% yield.

LC-MS(ESI)m/z:562[M+H]⁺。

Preparation M11

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (3- (3-fluorobenzyl) ureido) pyrimidine

Intermediate I (300mg, 0.53mmol), m-fluorobenzylamine (125mg, 1.06mmol) and sodium carbonate (158mg, 1.59mmol) were suspended in 10mL acetonitrile and stirred at room temperature for 12 hours. After the TLC detection reaction was completed, it was concentrated by rotary evaporation and purified by silica gel column (ethyl acetate/petroleum ether: 1/3, V/V) to obtain 210mg of a yellow solid product with a yield of 84.1%. LC-MS (ESI) M/z 473[ M + H ]]⁺。

Comparative example 1

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- (3-fluorobenzyl) -9H-purine-9-carboxamide

Intermediate M11(210mg, 0.44mmol) was dissolved in 5mL of THF, 10% palladium on carbon (21mg) was added to replace hydrogen, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (21mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 55mg of a white solid product with a yield of 23.6%.

LC-MS(ESI)m/z:453[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ9.20(s,1H),8.84(s,1H),8.64(s,1H),8.37(s,1H),7.30(s,1H),6.99–6.97(m,2H),6.87(s,1H),6.75–6.74(m,2H),5.32(s,1H),4.48–4.40(m,2H),4.03–3.90(m,2H),2.49(m,1H),2.08–1.98(m,3H)。

Comparative example 2

(R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((tetrahydro-2H-pyran-4-yl) methyl) -9H-purine-9-carboxamide

Step A: preparation of (R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (3- ((tetrahydro-2H-pyran-4-yl) methyl) ureido) pyrimidine. Prepared by the method of preparation M11, using 4-aminomethyltetrahydropyran instead of M-fluorobenzylamine.

And B: preparation of (R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -N- ((tetrahydro-2H-pyran-4-yl) methyl) -9H-purine-9-carboxamide. (R) -2- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -5-nitro-4- (3- ((tetrahydro-2H-pyran-4-yl) methyl) ureido) pyrimidine (250mg, 0.48mmol) was dissolved in 5mL THF, 10% palladium on charcoal (25mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After TLC detection reaction, filtering and spin-drying to obtain crude product. The crude product was dissolved in 5mL of triethyl orthoformate, and p-toluenesulfonic acid (25mg) was added to react at 135 ℃ for 4 hours. After the completion of the TLC detection reaction, the reaction solution was concentrated and purified by preparative TLC (ethyl acetate/petroleum ether: 1/1, V/V) to obtain 65mg of a white solid product in 27.6% yield.

LC-MS(ESI)m/z:443[M+H]⁺

¹H NMR(600MHz,CDCl₃)δ8.84(s,1H),8.37(s,1H),8.23(s,1H),7.05(s,1H),6.91(s,1H),6.76-6.74(m,1H),5.38(s,1H),3.98–3.87(m,4H),3.36–3.26(m,3H),2.93(s,1H),2.51(s,1H),2.10–2.05(m,3H),1.71(s,1H),1.55(m,1H),1.50(m,1H),1.33–1.23(m,2H).

Activity assay

CTG cell survival assay:

ATP is produced in living cell metabolic activity, and the content of ATP is linear with the number of living cells. The CTG chemiluminescence cell viability detection experiment is based on the principle and is a universal method for detecting the number of living cells in cultured cells. Addition of CellTiter-glo (CTG) reagent induces cell lysis and produces a chemiluminescent signal proportional to the amount of ATP in the well plate, which allows the viability of cell proliferation in the well plate to be measured by the chemiluminescent readout.

Experimental materials and instruments

Experimental materials:

test compound prepared in the above example, RPMI Medium 1640 (HyCl)one, Cat # SH30809.01), fetal bovine serum (FBS, GBICO, Cat #10099-

Luminecent Cell Viability Assay (Promega, Cat # G7572), 96-well flat-bottomed transparent blackboards (NUNC, Cat #165305), T25 flasks (NUNC, Cat #156367), T75 flasks (NUNC, Cat # 156439).

The cell lines used in the present invention are shown in Table 1.

TABLE 1 cell lines and culture media

Cell lines	Culture medium
		Ba/F3 LMNA-NTRK1	RPMI-1640+10％FBS
Ba/F3 LMNA-NTRK1-G595R	RPMI-1640+10％FBS
		Ba/F3 ETV6-NTRK2	RPMI-1640+10％FBS
Ba/F3 ETV6-NTRK2-G639R	RPMI-1640+10％FBS
		Ba/F3 ETV6-NTRK3	RPMI-1640+10％FBS
Ba/F3 ETV6-NTRK3-G623R	RPMI-1640+10％FBS

An experimental instrument:

carbon dioxide incubator, SANYO-MCO-20AIC, biosafety cabinet: BSC-1360-LIIA2, bench top high speed refrigerated centrifuge: sorvalst 16R, microplate fast oscillator: QB-9001, M3 trigger reader: SpectraMax M3, microscope: OLYMPUS-CKX41/CKX 31.

The experimental steps are as follows:

1. cell recovery:

taking out the cryopreservation tube from the liquid nitrogen container, directly immersing the tube in water bath at 37 ℃, quickly shaking to melt the tube as soon as possible, sucking out cell suspension, and adding the cell suspension into a centrifuge tube; centrifuging, removing supernatant, adding cell-containing growth medium to suspend cells, inoculating the whole cell suspension to a culture bottle, and performing static culture in a cell culture box.

2. Cell passage:

and (3) taking the cells out of the incubator to a biological safety cabinet when the cells grow to a logarithmic growth phase, transferring the cell suspension to a centrifugal tube for centrifugation, preparing the cells into a cell suspension, and then carrying out passage and experiment on the cells according to a certain proportion.

3. Cell plating

Cells in the logarithmic growth phase were harvested and counted using a platelet counter. Cell viability was checked by trypan blue exclusion, cell concentration was adjusted, and cell plating was performed. The cell plates were placed in a cell incubator for overnight incubation.

4. Drug dilution and dosing:

preparing a drug working solution, diluting the drug concentration from high to low by 3.16 times, adding the drug working solution into each hole of a 96-hole plate inoculated with cells, and repeating three holes for each drug concentration. Cells in the dosed 96-well plate were placed in a cell incubator for further culture, after which CTG analysis was performed.

5. Reading the plate at the end:

the CTG reagent was previously thawed. An equal volume of CTG solution was added to each well. The cells were lysed by shaking on a microplate fast shaker. The plate was placed at room temperature to stabilize the luminescence signal. And reading the cold light value.

6. Data processing

Data were analyzed using GraphPad Prism 5.0 software, fitted to the data using non-linear S-curve regression to derive a dose-effect curve, and IC50 values were calculated therefrom.

The results are shown in Table 2.

TABLE 2 inhibitory Effect (IC) of the Compounds of the present invention on mutant TRK enzymes₅₀)

It can be seen that the IC50 value of the first generation Trk inhibitor LOXO-101 on the mutant Trk enzyme reaches more than 1000, while the Trk inhibitor provided by the application has higher inhibitory activity on both wild type Trk kinase and mutant Trk kinase, and the IC50 value of part of examples on at least one mutant Trk enzyme is lower than the IC50 value on the wild type Trk enzyme, which indicates that the compound provided by the application has selectivity on the mutant Trk enzyme. However, although imidazopyrimidines or imidazopyrazines are bioisosteres of imidazopyridines, their derivatives do not achieve the effects of the compounds provided by the present invention.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An imidazopyridine derivative with a structure of formula A

Or a tautomer, pharmaceutically acceptable salt thereof, wherein,

x, Y or Z is N, the others are CH;

R₃is C_1-3Alkyl radical, R₄Is H; or R₃And R₄Are all C_1-3When alkyl, R₃And R₄Connecting to form a five-membered ring;

R₁selected from phenyl, 3-fluorophenyl, 2, 5-difluorophenyl, 2-chloro-5-fluorophenyl, 2-methoxyphenyl, 2-methoxy-5-fluorophenyl, 2-trifluoromethyl-5-fluorophenyl, 2-difluoromethyl-5-fluorophenyl, 3-chloro-5-fluorophenyl, 3-fluoro-5-methoxyethoxyphenyl or 5-fluoro-2-methoxyethoxyphenyl, 5-fluoro-pyridin-3-yl, 2, 5-difluoro-pyridin-3-yl, 2-methoxy-5-fluoro-pyridin-3-yl or 2-trifluoromethyl-5-fluoro-pyridin-3-yl;

R₂is selected from C_1-3Straight chain alkyl, C_5-6Monocyclic alkyl or 5-6 membered heteromonocycloalkyl, said R₂Unsubstituted or substituted by one or more substituents;

R₂wherein said heteromonocycloalkyl is a 5-6 membered heteromonocycloalkyl group containing one or more heteroatoms each independently selected from N, O or S;

R₂wherein the substituent is selected from formyloxy, hydroxy-C₂Alkyl, hydroxy, methoxy; or phenyl unsubstituted or substituted by one or more substituents, wherein in the phenyl substituted by one or more substituents, each of said substituents is independently selected from F or CF₃(ii) a Or heteroaryl unsubstituted or substituted with one or more substituents selected from methyl, said heteroaryl being a 5-6 membered heteroaryl containing one or more heteroatoms each independently selected from N, O or S; or C unsubstituted or substituted by one or more substituents_5-6Cycloalkyl, wherein in cycloalkyl substituted with one or more substituents selected from formyloxy, hydroxy; or unsubstituted heterocycloalkyl, which is a 5-6 membered heterocycloalkyl containing one or more heteroatoms, each independently selected from N, O or S.

2. The derivative of claim 1, or a tautomer, pharmaceutically acceptable salt thereof, wherein formula a has the following formula a₁Structure of (1)

3. The derivative of claim 1, or a tautomer, pharmaceutically acceptable salt thereof, wherein R is₁And 2, 5-difluorophenyl, 2-methoxy-5-fluorophenyl or 2-methoxy-5-fluoro-pyridin-3-yl is selected.

4. The derivative of any one of claim 1, or a tautomer, pharmaceutically acceptable salt thereof, wherein R is₂One selected from the following structures:

5. the derivative according to any one of claims 1 to 4, or a tautomer, a pharmaceutically acceptable salt thereof, wherein formula A is as shown in formula A₁₁Absolute configuration shown

6. An imidazopyridine derivative with a structure of formula A

Or a tautomer, pharmaceutically acceptable salt thereof, wherein the derivative is selected from the group consisting ofOne of the structures:

7. a process for the preparation of a derivative according to any one of claims 1 to 6, or a tautomer, a pharmaceutically acceptable salt thereof, comprising the steps of,

step a) A_aAnd

Step c) A_cAnd R₂-NH₂Reacting under alkaline condition to obtain A_d；

Step d) of reacting A_dReducing to obtain A_e；

Step e) A_eCyclization gives A.

8. A pharmaceutical composition comprising a derivative according to any one of claims 1 to 6, or a tautomer, a pharmaceutically acceptable salt thereof, or a derivative produced by the process according to claim 7, or a tautomer, a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

9. Use of a derivative according to any one of claims 1 to 6, or a tautomer, a pharmaceutically acceptable salt thereof, or a derivative prepared by the process according to claim 7, or a tautomer, a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment and/or prophylaxis of diseases and associated conditions mediated by the TRK enzyme.

10. The use according to claim 9, wherein the disease has developed at least partial resistance to at least one TRK enzyme therapeutically active agent.

11. The use according to claim 9, wherein the disease is selected from one of pain or cancer.