WO2022188203A1 - 一种咪唑并吡啶类衍生物 - Google Patents

一种咪唑并吡啶类衍生物 Download PDF

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WO2022188203A1
WO2022188203A1 PCT/CN2021/081904 CN2021081904W WO2022188203A1 WO 2022188203 A1 WO2022188203 A1 WO 2022188203A1 CN 2021081904 W CN2021081904 W CN 2021081904W WO 2022188203 A1 WO2022188203 A1 WO 2022188203A1
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substituents
pharmaceutically acceptable
reaction
tautomer
stereoisomer
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French (fr)
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卢法冠
王磊
杜镇建
杜美静
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北京富龙康泰生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of biologically active small molecules, in particular to a small molecule that inhibits Trk activity.
  • Tropomyosin-related kinase is a class of nerve growth factor receptors, and its family has highly homologous tropomyosin-related kinase A (TrkA), tropomyosin-related It consists of globulin-related kinase B (TrkB) and tropomyosin-related kinase C (TrkC), encoded by NTRK1, NTRK2 and NTRK3, respectively.
  • the neurotrophic factors are (i) nerve growth factor (NGF) that activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 that activate TrkB, and (iii) TrkC that activates NT3. 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 the growth, differentiation, and apoptosis of neuronal cells, and inhibitors of the Trk/neurotrophic factor pathway are effective in multiple preclinical animal models of pain.
  • antagonistic TrkA/NGF pathway antibodies eg, RN-624 are effective in animal models of inflammatory and neuropathic pain and in human clinical trials (Neuroscience, 1994, 62:327-331; J. Pain, 2004 , 5:157-163; Nat. Med. 1995, 1:774-780; Neuroreport, 1997, 8:807-810).
  • Trk is associated with many cancers, including melanoma (Journal of Investigative Dermatology, 2008, 128(8): 2031-2040), non-small cell lung cancer ( Molecular and Cellular Biochemistry, 2007, 295(1&2): 19-26), thyroid cancer (Neuroendocrinology Letters, 2007, 28(3): 221-229), acute myeloid leukemia (Cancer Lett, 2001, 169: 107-14) , malignant glioma (Journal of Neurochemistry, 2007, 103: 259-275), astrocytoma and medulloblastoma (Brain Pathology, 2006, 16: 304-310), colon cancer (Science, 2003, 300: 949), neuroblastoma (Nat.
  • 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).
  • Trk kinase inhibitor Larotrectinib (LOXO-101) was used to treat patients with LMNA-NTRK1 gene fusion soft tissue sarcoma metastases.
  • LOXO-101 Trk kinase inhibitor Larotrectinib
  • the size of the lung sarcoma was significantly reduced, and the metastasis of cancer cells was significantly reduced.
  • the spread was slowed down, and the patient's dyspnea was relieved.
  • the lung mass basically disappeared, and no obvious adverse reactions were found during clinical treatment (CancerDiscov, 2015, 5:1049-1057).
  • LOXO-101 has significant efficacy in NTRK1/2/3 fusion-positive adult/pediatric cancer patients, such as soft tissue malignancies, thyroid cancer, salivary gland cancer, gastrointestinal stromal tumor, childhood fibrosarcoma, non-small cell carcinoma, Cholangiocarcinoma, kidney cancer, salivary gland cancer, appendix cancer, pancreatic cancer, peripheral nerve sheath tumor, spindle cell tumor, etc., and the drug has few side effects (New England Journal of Medicine, 2018, 378(8):731-739.) .
  • Trk kinase inhibitor Entrectinib (NMS-P626) was studied in a clinical trial of 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 can be effective in the treatment of inflammatory diseases in a preclinical model.
  • inhibition of the neurotrophin/Trk pathway has been associated with preclinical patterns of the following diseases: inflammatory lung disease including asthma (Pharmacology & Therapeutics, 2008, 117(1):52-76), interstitial cystitis (The Journal of Urology, 2005, 173(3): 1016-1021), inflammatory bowel diseases including ulcerative colitis and Crohn's disease (Gut, 2000, 46(5): 670-678) and inflammatory skin diseases, such as atopic dermatitis (Archives of Dermatological Research, 2006, 298(1): 31-37), eczema and psoriasis (Journal of Investigative Dermatology, 2004, 122(3): 812-819) .
  • the neurotrophic factor/Trk pathway and specifically 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 can be used to treat these and related diseases.
  • TrkA receptor is essential for the disease process in the infection of the parasitic infection of Trypanosoma cruzi (Chagasdisease) in human hosts (Cell Host & Microbe, 2007, 1(4) :251-261).
  • inhibition of TrkA may be useful in the treatment of Chagas disease and related protozoal infections.
  • Trk inhibitors can also be used to treat diseases related to the imbalance of bone remodeling regulation, such as osteoporosis, rheumatoid arthritis and bone metastasis.
  • Bone metastases are a frequent complication of cancer, occurring in patients with advanced breast or prostate cancer and in patients with lung, colon, stomach, bladder, uterine, rectal, 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 can induce apoptosis of proliferative osteoblasts would be extremely beneficial.
  • TrkA receptors and TrkC receptors have been observed in osteogenic areas of the mouse model of fractures (Bone, 2000, 26(6):625-633). In addition, the distribution of NGF was observed in almost all osteoblasts. Pan-Trk inhibitors inhibit tyrosine signaling activated by neurotrophic factor binding to all three Trk receptors in human hFOB osteoblasts. These data support the theory of using Trk inhibitors for the treatment of bone remodeling disorders such as bone metastases in cancer patients.
  • TRKA G595R and its paralog TRKC G623R
  • TRKA G667C and its paralog TRKC G696A
  • TRKA F589L in the conserved region
  • second-generation TRK inhibitors for drug resistance have also begun to be developed, such as LOXO-195 and TPX-0005, LOXO-195 and TPX-0005 compounds that limit the active site by cyclization Surface area, from the compound structure, LOXO-195 and TPX-0005 have the same pyrazolo[1,5-a]pyrimidine structure as larotrectinib, but LOXO-195 and TPX-0005 have limited substituents by cyclization of free spins.
  • the second-generation inhibitors overcome the drug resistance in the use of the first-generation inhibitors, the construction of their ring structures is relatively difficult.
  • LOXO-195 exhibits strong inhibition of TRK fusion proteins and is not affected by certain acquired resistance mutations (such as TRKAG595R, TrkAG667C or TrkCG623R), which may appear in recipients in patients with larotrectinib (LOXO-101) or a multikinase inhibitor with anti-TRK activity.
  • TRKAG595R, TrkAG667C or TrkCG623R larotrectinib
  • LOXO-101 larotrectinib
  • the IC50 of LOXO-195 against the mutant Trk enzyme was 5-10 times higher than the IC50 of the corresponding wild-type Trk enzyme.
  • Trk inhibitors with better efficacy, fewer side effects, high metabolic stability and good bioavailability.
  • the present invention proposes a novel substituted imidazopyridine compound as a Trk kinase inhibitor, so as to overcome the problems of stability, activity and other deficiencies in the prior art and meet the growing clinical needs.
  • the object of the present invention is to provide an imidazopyridine derivative having activity against drug-resistant TRK kinase.
  • R 3 and R 4 are each independently selected from C 1-3 alkyl or H; when R 3 and R 4 are both alkyl, R 3 and R 4 may be further linked to form a ring;
  • R 1 is selected from 5-6 membered monocyclic aryl or 5-6 membered monocyclic heteroaryl unsubstituted or substituted with one or more substituents.
  • R2 is selected from alkyl or heterocycloalkyl, said R2 being unsubstituted or substituted with one or more substituents.
  • R 3 is C 1-3 alkyl, preferably methyl, and R 4 is H.
  • R 3 and R 4 are alkyl groups, R 3 and R 4 are connected to form a five-membered ring.
  • formula A has the structure of the following formula A 1
  • R 1 is independently selected from halogen, C 1-4 alkoxy, -CF 3 , -CHF 2 , -OC 1-4 alkyl, OC 1-3 alkyl or -OC 3 -6 Dihydroxyalkyl.
  • R 1 is independently selected from halogen or C 1-4 alkoxy.
  • substituents in R 1 are each independently selected from F or methoxy.
  • the monocyclic aryl or monocyclic heteroaryl in R 1 is selected from phenyl or pyridyl.
  • the R 1 is selected from phenyl, 3-fluorophenyl, 2,5-difluorophenyl, 2-chloro-5-fluorophenyl, 2-methoxyphenyl, 2-methoxyphenyl -5-fluorophenyl, 2-trifluoromethyl-5-fluorophenyl, 2-difluoromethyl-5-fluorophenyl, 3-chloro-5-fluorophenyl, 3-fluoro-5-methyl oxyethoxyphenyl, 2-hydroxy-5-fluorophenyl or 5-fluoro-2-methoxyethoxyphenyl, 5-fluoro-pyridin-3-yl, 2,5-difluoro- Pyridin-3-yl, 2-methoxy-pyridin-3-yl, 2-methoxy-5-fluoro-pyridin-3-yl, 2-hydroxy-5-fluoro-pyridin-3-yl or 2-tris Fluoromethyl-5-fluoro-pyridin-3-yl,
  • R 1 is selected from 2,5-difluorophenyl, 2-methoxy-5-fluorophenyl, 2-hydroxy-5-fluorophenyl or 2-methoxy-5-fluoro- Pyridin-3-yl.
  • alkyl group in R 2 is selected from straight chain alkyl group or monocyclic alkyl group.
  • alkyl group in R 2 is selected from C 1-6 straight chain alkyl group, C 4-8 cycloalkyl group or 4-8 membered heterocycloalkyl group.
  • alkyl group in R 2 is selected from C 1-3 straight chain alkyl group, C 5-6 cycloalkyl group or 5-6 membered heterocycloalkyl group.
  • heterocycloalkyl group in R 2 is a 5-6 membered heterocycloalkyl group containing one or more heteroatoms, each of which is independently selected from N, O or S.
  • substituents in R 2 are selected from acyloxy, hydroxyalkyl, hydroxy, methoxy, halogen, haloalkane, methyl, unsubstituted or aryl substituted by one or more substituents, unsubstituted or heteroaryl substituted with one or more substituents, unsubstituted or substituted with one or more substituents, cycloalkyl, or unsubstituted or substituted with one or more substituents, heterocycloalkyl.
  • R 2 is selected from formyloxy, hydroxy-C 2 -alkyl, hydroxy, and methoxy;
  • substituents are each independently selected from F or CF 3 ;
  • substituents are selected from methyl;
  • substituents are selected from formyloxy, hydroxyl;
  • R 2 is selected from formyloxy, hydroxy-C 2 -alkyl, hydroxy, and methoxy;
  • substituent groups are each independently selected from F or CF 3 ;
  • the heteroaryl is a 5-6 membered heteroaryl containing one or more heteroatoms, each of which is independently selected from N, O or S, in the heteroaryl group substituted by one or more substituents, the substituents are selected from methyl;
  • substituents C 5-6 cycloalkyl wherein in the cycloalkyl substituted by one or more substituents, the substituents are selected from formyloxy, hydroxyl;
  • heterocycloalkyl which is a 5-6 membered heterocycle containing one or more heteroatoms each independently selected from N, O or S.
  • R 2 is selected from one of the following structures:
  • formula A has an absolute configuration as shown in formula A 11
  • a preparation method of the imidazopyridine derivatives comprising at least one of the following steps, as shown in the following formula,
  • Step a) A and Reaction under alkaline conditions gives A b , wherein L is selected from chlorine, bromine or iodine;
  • Step b) reacting A b with phenyl chloroformate or phenyl chloroformate derivative under alkaline conditions to obtain A c , wherein R a is selected from H or -NO 2 ;
  • Step c) reacting A c and R 2 -NH 2 under alkaline conditions to obtain A d ;
  • Step d) obtains A e by reducing A d ;
  • Step e) cyclization of A e to obtain A.
  • the reduction conditions are the conventional reduction - NO conditions known to those skilled in the art, and are not particularly limited, such as hydrogenation reduction, iron powder reduction or zinc powder reduction, etc., preferably hydrogenation reduction. .
  • the cyclization is the cyclization reaction of A e and triethyl orthoformate under acid-catalyzed conditions to obtain A.
  • the cyclization can also be a cyclization reaction of A e and ethylformimide under acid-catalyzed conditions to obtain A.
  • the preparation method may further include a functional group protection or deprotection step necessary to reduce side reactions, and the functional group protection and deprotection adopts a scheme well known to those skilled in the art.
  • the phenolic hydroxyl group is protected by a benzyl group, and after one or more steps of reaction, the benzyl group is removed by hydrogenation to obtain a phenolic hydroxyl group.
  • Such functional group conversion processes that do not affect the selection of functional groups in the final product should also fall within the protection scope of the preparation method provided by the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug thereof, and a pharmaceutically acceptable excipient .
  • the present invention provides a use of the above-mentioned compound or pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting the activity of a Trk enzyme, especially the use of a medicament for inhibiting the activity of a mutant Trk enzyme.
  • the mutation sites of the TRK targets are located in G517R, A542V, V573M, F589L, F589C, G595R, G595S, D596V, F600L, F646V, C656Y, C656F, L657V, G667S, G667C, Y676S, G623R, G667C, G54R, L68 , at least one of A570V, Q596E, Q596P, V601G, F617L, F617C, G623S, D624V, R630K, C682Y, C682F, L683V, G693S, G713S, C685F, C685Y, L686V, G696A, and G639R.
  • TRKA G595R is at least one of TRKA G595R , TRKC G623R , TRKB G639R , TRKA G667C , TRKC G696A and TRKA F589L . More preferably, it is at least one of TRKA G595R , TRKC G623R and TRKB G639R .
  • Trk enzyme is contacted with a compound described herein or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate thereof.
  • the compounds provided by the present invention are used in products for preventing and/or treating Trk enzyme-mediated diseases.
  • 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 inflammatory pain, visceral pain, pain from cancer, chemotherapy pain, trauma pain, surgery and post-surgery pain, childbirth pain, acute pain, chronic pain, Intractable pain, somatic pain, nociceptive pain, neuropathic pain, hematogenous pain, immunogenic pain, endocrine pain, pain from metabolic lesions, cardiogenic pain, headache, phantom limb pain, and toothache one or more of.
  • Acute pain as defined by the International Association for the Study of Pain, results from disease, inflammation, or tissue damage. This pain usually occurs suddenly, for example, after trauma or surgery, and may be accompanied by anxiety or stress. The cause can usually be diagnosed and treated, and the pain is limited to a given period and severity. In some instances, it can become chronic.
  • the cancer includes, but is not limited to, lung cancer, colon cancer, prostate cancer, breast cancer, liver cancer, lymphoma, thyroid cancer, multiple myeloma, soft tissue sarcoma, ovarian cancer, cervical cancer, fallopian tube cancer, renal cell cancer, gastric cancer, gastric cancer intestinal stromal tumor, bone cancer, basal cell carcinoma, peritoneal carcinoma, dermatofibroma, pancreatic cancer, esophageal cancer, glioblastoma, head and neck cancer, inflammatory myofibroblastic tumor, and anaplastic large cell lymphoma one or more.
  • the compounds of the present invention may be administered by any suitable route, eg, into the gastrointestinal tract (eg, rectally or orally), nasally, pulmonary, intramuscular or vascular or transdermally or dermally.
  • the compounds can 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 formulations, such as diluents, carriers, pH adjusters, sweeteners, bulking agents, 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 another aspect of the present invention.
  • pharmaceutically acceptable salt in the present invention is intended to mean the free acid or base salt of the compound represented by formula (A), which is non-toxic, biologically tolerated or in other words biologically is suitable for administration to a subject.
  • formula (A) the compound represented by formula (A)
  • G.S.Paulekuhn, et al. "TrendsinActivePharmaceuticalIngredientSaltSelectionbasedAnalysis of the OrangeBookDatabase", J.Med.Chem., 2007, 50:6665-72, S.M.Berge, et al., “PharmaceuticalSalts", JPharmSci., 1977,66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahland Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002 (G.S.
  • Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride , Bromide, Iodide, Acetate, Propionate, Caprate, Caprylate, Acrylate, Formate, Isobutyrate, Caproate, Heptanoate, Propiolate, Oxalic Acid salt, malonate, phopoate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6- Diacid salts, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydrocarbyl benzoates, oxybenzoates, phthalates , sulfonate, xylene sulfonate, phenylacetate, phenylpropionate,
  • Compounds of formula (A) contain a basic nitrogen, and the desired pharmaceutically acceptable salts can be prepared by any suitable method available in the art, such as treating the free base with a mineral acid such as hydrochloric acid, hydrobromic acid, Sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc., or treating the free base with an organic acid such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxy horse Leric 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, pyranosic acid (eg glucuronic acid or galacturonic acid), alpha-hydrocarbon acids (eg mandelic acid, citric acid or tartaric acid), amino acids
  • Prodrugs described herein may include compounds having an amino acid residue covalently linked to a carboxylic acid group of formula (A) via an amide or ester bond or two or more (eg two, three or four) polypeptide chains of amino acid residues.
  • amino acid residues include the twenty naturally occurring amino acids commonly identified by a three-letter symbol, as well as 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methyl Histidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine.
  • prodrugs can be prepared by derivatizing the free carboxylic acid of formula (A) as an amide or alkyl ester.
  • amides include those derived from ammonia, C1-6 alkyl primary amines, and di(C 1-6 alkyl) secondary amines.
  • Secondary amines include 5- or 6-membered heterocycloalkyl or heteroaryl ring moieties.
  • acid amines include those amides derived from ammonia. Ci - 3 alkyl primary amines and di(C 1-2 alkyl) amines.
  • Examples of the lipids of the present invention include C 1-7 alkyl esters, C 5-7 cycloalkyl esters, phenyl esters and (C 1-6 alkyl)phenyl esters.
  • the lipids include methyl esters. It can also be carried out by using methods such as those described in Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130 (Fleisher et al., "Review of Advances in Drug Delivery", 1996, Vol. 19, pp. 115-130).
  • Prodrugs are prepared by derivatizing the free hydroxyl groups with groups including hemisuccinate, phosphate, dimethylaminoacetate, and phosphoryloxymethoxycarbonyl.
  • Carbamate derivatives of hydroxyl and amino groups can also yield prodrugs.
  • Carbonate derivatives of hydroxyl groups, sulfonates and sulfates can also provide prodrugs.
  • the acyl groups are derivatized to (acyloxy) methyl esters and (acyloxy) ethyl esters, where the acyl group can be an alkyl ester optionally substituted with one or more ether, amine, or acid functional groups, or where the acyl group is As described above for amino acid lipids, this can also be used to generate prodrugs. Prodrugs of this type can be found as in Robinson et al., JMedChem.
  • Free amines can also be derivatized as amides, sulfonamides or phosphoramides. All of these prodrug moieties can incorporate groups including ether, amine and carboxylic acid functionalities.
  • Trk inhibitor A novel structure of Trk inhibitor is provided.
  • the TRK inhibitor provided by the present invention shows that the IC50 value of the mutant Trk inhibitor is similar to or lower than the IC50 value of the corresponding wild-type Trk inhibitor.
  • the present invention creatively selects imidazopyridine derivatives to obtain the effect of inhibiting wild-type and mutant Trk enzymes at the same time in a low concentration range, while imidazopyrimidine or imidazopyrazine are biological substances of imidazopyridine. Isosteres, but their derivatives do not achieve the effect of the present invention.
  • reagents were purchased from commercial suppliers (eg, Aldrich Chemical, Lancaster, TCI or Maybridge) and used without further purification.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-fluorobenzyl)ureido) Preparation of pyridine. Prepared using the method for preparing M1 using 4-fluorobenzylamine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-fluorobenzyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • the intermediate (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-fluorobenzyl)ureido) Pyridine 300 mg, 0.63 mmol
  • 10% palladium on carbon (30 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (15 mg) was added, and the reaction was stirred at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3,5-difluorobenzyl) Preparation of ureido)pyridine. Prepared using the method for preparing M1 using 3,5-difluorobenzylamine instead of benzylamine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-fluorophenethyl)ureido ) Preparation of pyridine. Prepared using the method for preparing M1 using 4-fluorophenethylamine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-fluorophenethyl)-3H-imidazo[4,5- b] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-fluorophenethyl)ureido)pyridine 200 mg, 0.42 mmol
  • 10% palladium on carbon (20 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (10 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-trifluoromethylbenzyl) Preparation of ureido)pyridine. Prepared using the method for preparing M1 using 4-trifluoromethylbenzylamine instead of benzylamine.
  • TLC
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-fluorobenzyl)ureido) Preparation of pyridine. Prepared using the method for preparing M1 using 3-fluorobenzylamine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(3-fluorobenzyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-fluorobenzyl)ureido)pyridine 300 mg, 0.63 mmol
  • 10% palladium on carbon (30 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (15 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(2-fluorobenzyl)ureido) Preparation of pyridine. Prepared using the method for preparing M1 using 2-fluorobenzylamine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(2-fluorobenzyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(2-fluorobenzyl)ureido)pyridine 300 mg, 0.63 mmol
  • 10% palladium on carbon (30 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (15 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A Preparation of (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-phenethylureido)pyridine.
  • Step A Preparation of (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-phenylpropylureido)pyridine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(3-phenylpropyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-phenylpropylureido)pyridine 250 mg, 0.51 mmol
  • 10% palladium on carbon 25 mg
  • hydrogen was replaced
  • the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (12.5 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-pyridylmethyl)ureido) Preparation of pyridine. Prepared using the method for preparing M1 using 3-aminomethylpyridine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(3-pyridylmethyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-pyridylmethyl)ureido)pyridine 220 mg, 0.46 mmol
  • 10% palladium on carbon 22 mg
  • hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (11 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-pyridylmethyl)ureido) Preparation of pyridine. Prepared using the method for preparing M1 using 4-aminomethylpyridine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-pyridylmethyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-pyridylmethyl)ureido)pyridine 200 mg, 0.44 mmol
  • 10% palladium on carbon 22 mg
  • hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (11 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-thienylmethyl)ureido) Preparation of pyridine. Prepared using the method for the preparation of M1 using 3-aminomethylthiophene instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(3-thienylmethyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(3-thienylmethyl)ureido)pyridine 180 mg, 0.41 mmol
  • 10% palladium on carbon (18 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (9 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-((1-methyl-1-H - Preparation of pyrazol-4-yl)methyl)ureido)pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-((1-methyl-1-H - Preparation of pyrazol-4-yl)methyl)ureido)pyridine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-((1-methyl-1-H-pyrazol-4-yl) Preparation of methyl)-3H-imidazo[4,5-b]pyridine-3-carboxamide.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((tetrahydro-2H-pyran-4- Preparation of yl)methyl)ureido)pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((tetrahydro-2H-pyran-4- Preparation of yl)methyl)ureido)pyridine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)- Preparation of 3H-imidazo[4,5-b]pyridine-3-carboxamide.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((tetrahydro-2H-pyran-4-yl ) ureido) pyridine preparation. Prepared using the method for preparing M1 using 4-aminotetrahydropyran instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(tetrahydro-2H-pyran-4-yl)-3H-imidazo Preparation of [4,5-b]pyridine-3-carboxamide.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (13 mg) was added, and the reaction was carried out at 100° C. for 1 hour.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((4-methoxycyclohexyl)ureido) Preparation of pyridine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-methoxycyclohexyl)-3H-imidazo[4,5 -b] Preparation of pyridine-3-carboxamide.
  • (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((4-methoxycyclohexyl)ureido)pyridine 210 mg, 0.44 mmol
  • 10% palladium on carbon 21 mg
  • hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (11 mg) was added, and the reaction was carried out at 100° C. for 1 hour.
  • Step A 6-((R)-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1S,4S)-4-hydroxy Preparation of cyclohexyl)ureido)pyridine.
  • M1 6-((R)-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1S,4S)-4-hydroxy
  • Step B (1S,4s)-4-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3H-imidazo[4,5-b] Preparation of cyclohexyl pyridine-3-carboxamide)carboxylate.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (10 mg) was added, and the reaction was carried out at 100° C. for 1 hour.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step B 3-((5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3H-imidazo[4,5-b]pyridine-3-methyl Preparation of amide)methyl)acid cyclohexylcarboxylic acid.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (12 mg) was added, and the reaction was carried out at 100° C. for 1 hour.
  • Step A 6-((R)-2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((3-(1S,3R)-hydroxycyclohexyl ) ureido) pyridine preparation.
  • Step B (1R,3S)-3-((5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide)methyl)acid cyclopentylcarboxylic acid.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((4-(2-hydroxyethyl)cyclohexyl) ) ureido) pyridine preparation.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((4-(2-hydroxyethyl)cyclohexyl) ) ureido) pyridine preparation.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-((4-(2-hydroxyethyl)cyclohexyl) ) ureido) pyridine preparation.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-(2-hydroxyethyl)cyclohexyl)-3H-imidazo Preparation of [4,5-b]pyridine-3-carboxamide.
  • Step A 6-((R)-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1r,4R)-4-hydroxy Preparation of cyclohexyl)ureido)pyridine.
  • Step B 5-((R)-2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-((1r,4R)-4-hydroxycyclohexyl)-3H-imidazo
  • 6-((R)-(2-(2,5-difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1r,4R)-4-hydroxycyclohexyl )ureido)pyridine 240 mg, 0.52 mmol
  • 10% palladium on carbon 24 mg
  • hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours.
  • Step A 6-((R)-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1s,4S)-4-hydroxy Preparation of cyclohexyl)ureido)pyridine.
  • M1 6-((R)-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1s,4S)-4-hydroxy
  • Step B 6-((R)-(2-(2,5-difluorophenyl)pyrrolidin-1-yl))-3-nitro-2-(((1s,4S)-4- Hydroxycyclohexyl)ureido)pyridine (250 mg, 0.54 mmol) was dissolved in 5 mL of ethanol, 10% palladium on carbon (25 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After the reaction was detected by TLC, the mixture was filtered, ethylformimide hydrochloride (110 mg, 1.08 mmol) was added to the reaction solution, and the reaction was carried out at 80° C. for 5 hours.
  • reaction solution was concentrated, water was added, extracted with ethyl acetate, combined, dried, and spin-dried to obtain the crude product.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl)urea base) preparation of pyridine.
  • Step B (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(((3-hydroxycyclohexyl)methyl) ureido)pyridine (210 mg, 0.44 mmol) was dissolved in 5 mL of ethanol, 10% palladium on carbon (21 mg) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 12 hours. After the reaction was detected by TLC, the mixture was filtered, ethylformimide hydrochloride (96 mg, 0.88 mmol) was added to the reaction solution, and the reaction was carried out at 80° C. for 5 hours.
  • reaction solution was concentrated, water was added, extracted with ethyl acetate, combined, dried, and spin-dried to obtain the crude product.
  • Step A (R)-6-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-3-nitro-2-(3-(4-phenylcyclohexyl)ureido ) Preparation of pyridine. Prepared using the method for preparing M1 using 4-phenylcyclohexylamine instead of benzylamine.
  • Step B (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-fluorobenzyl)-3H-imidazo[4,5-b ] Preparation of pyridine-3-carboxamide.
  • Step A (R)-6-(2-(5-Fluoro-2-methoxypyridine)pyrrolidin-1-yl)-3-nitro-2-(3-phenethylureido)pyridine preparation. Prepared using the method for the preparation of M2, using phenethylamine instead of m-fluorobenzylamine.
  • Step A (R)-6-(2-(5-Fluoro-2-methoxypyridine)pyrrolidin-1-yl)-3-nitro-2-(3-((1s,4S)-4 - Preparation of hydroxycyclohexyl)ureido)pyridine.
  • M2 1,3-((1s,4S)-4-hydroxycyclohexylamine instead of m-fluorobenzylamine.
  • TLC ethyl acetate/
  • Step A (R)-6-(1-(2-Benzyloxy-5-fluorophenyl)ethyl)amino-3-nitro-2-(3-((1s,4s)-4-hydroxy Preparation of cyclohexyl)ureido)pyridine.
  • M3 was prepared using (1s,4s)-4-hydroxycyclohexylamine instead of 4-methoxycyclohexylamine in 54.8% yield.
  • Step B (R)-5-((1-(2-Benzyloxy-5-fluorophenyl)ethyl)amino)-N-((1s,4s)-4-hydroxycyclohexyl)-3H- Preparation of imidazo[4,5-b]pyridine-3-carboxamide.
  • (R)-6-(1-(2-benzyloxy-5-fluorophenyl)ethyl)amino-3-nitro-2-(3-((1s,4s) -4-Hydroxycyclohexyl)ureido)pyridine was prepared in 31.4% yield instead of M3.
  • Step C (R)-5-((1-(2-benzyloxy-5-fluorophenyl)ethyl)amino)-N-((1s,4s)-4-hydroxycyclohexyl)-3H -Imidazo[4,5-b]pyridine-3-carboxamide (60 mg, 0.12 mmol) was dissolved in 10 mL of methanol, 10% palladium on carbon (20 mg) was added, and after hydrogen was replaced, the reaction was heated and stirred in an oil bath at 60 °C for 18 hours .
  • Step A (R)-2-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-5-nitro-4-(3-((tetrahydro-2H-pyran- Preparation of 4-yl)methyl)ureido)pyrimidine.
  • M11 2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-5-nitro-4-(3-((tetrahydro-2H-pyran- Preparation of 4-yl)methyl)ureido)pyrimidine.
  • Step B (R)-2-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)- Preparation of 9H-purine-9-carboxamide.
  • the crude product was obtained by filtration and spin drying.
  • the crude product was dissolved in 5 mL of triethyl orthoformate, p-toluenesulfonic acid (25 mg) was added, and the reaction was carried out at 135° C. for 4 hours.
  • the CTG chemiluminescence cell viability assay is based on this principle and is a general method for detecting the number of viable cells in cultured cells.
  • the addition of CellTiter-Glo (CTG) reagent induces cell lysis and produces a chemiluminescent signal proportional to the amount of ATP in the plate, allowing the chemiluminescent readout to measure the viability of cell proliferation in the plate.
  • Test compounds prepared in the above examples RPMI Medium 1640 (HyClone, Cat#SH30809.01), fetal bovine serum (FBS, GBICO, Cat#10099-141), phosphate buffer (Solarbio, Cat#P1020-500) , Luminescent Cell Viability Assay (Promega, Cat#G7572), 96-well flat bottom transparent black plate (NUNC, Cat#165305), T25 flask (NUNC, Cat#156367), T75 flask (NUNC, Cat#156439).
  • the cell lines used in the present invention are shown in Table 1.
  • Cells in logarithmic growth phase were harvested and counted using a platelet counter. Cell viability was detected by trypan blue exclusion method, cell concentration was adjusted, and cells were plated. Place the cell plate in a cell incubator overnight.
  • the drug working solution was prepared, the drug concentration was from high to low, and the drug concentration was diluted 3.16 times.
  • the drug working solution was added to each well of the 96-well plate seeded with cells, and each drug concentration was three duplicate wells. Cells in the medicated 96-well plate were cultured in a cell incubator prior to CTG analysis.
  • CTG reagent Pre-thaw CTG reagent. An equal volume of CTG solution was added to each well. Cells were lysed by shaking on a microplate fast shaker. Place the cell plate at room temperature to stabilize the luminescent signal. Read the luminescence value.
  • the IC50 value of the first-generation Trk inhibitor LOXO-101 on the mutant Trk enzyme has reached more than 1000, while the Trk inhibitor provided in this application has high inhibitory activity against both wild-type Trk kinase and mutant Trk kinase.
  • the IC50 value for at least one mutant Trk enzyme in the examples is lower than the IC50 value for the wild-type Trk enzyme, indicating that the compounds provided in this application are selective for the mutant Trk enzyme.
  • imidazopyrimidine or imidazopyrazine are bioisosteres of imidazopyridine, their derivatives cannot achieve the effect of the compounds provided by the present invention.

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Abstract

本发明涉及生物活性小分子领域，具体涉及一种具有抑制Trk活性的小分子。提出一种作为Trk激酶抑制剂的新型的取代咪唑并吡啶化合物，克服了一代Trk抑制剂在临床应用中产生的耐药性问题，本发明提供的咪唑并吡啶化合物在给药剂量下，可以对突变型Trk激酶具有较强的抑制作用。

Description

一种咪唑并吡啶类衍生物 技术领域
本发明涉及生物活性小分子领域，具体涉及一种具有抑制Trk活性的小分子。
背景技术
原肌球蛋白相关激酶(tropomyosin-related kinase，Trk)是一类神经生长因子受体，其家族有高度同源性的原肌球蛋白相关激酶A(tropomyosin-related kinase A，TrkA)、原肌球蛋白相关激酶B(TrkB)、原肌球蛋白相关激酶C(TrkC)组成，分别由NTRK1、NTRK2和NTRK3编码。神经营养因子中有(i)可激活TrkA的神经生长因子(NGF)，(ii)可激活TrkB的脑源性神经营养因子(BDNF)及NT-4/5，及(iii)可激活TrkC的NT3。Trk广泛表现于神经元组织中且与神经元细胞的维持、信号传导及存活有关(Current Opinion in Neurobiology,2001,11:272-280)。
Trk激酶被认为与神经元细胞的生长、分化、凋亡等相关，Trk/神经营养因子途径的抑制剂在疼痛的多种临床前动物模式中有效。例如，在拮抗性TrkA/NGF途径抗体(例如，RN-624)在炎症性及神经性疼痛动物模式中及在人类临床试验中有效(Neuroscience,1994,62:327-331；J.Pain,2004,5:157-163；Nat.Med.1995,1:774-780；Neuroreport,1997,8:807-810)。另外，文献显示，发炎后，在背根神经节中BDNF含量及TrkB信号传导会增加(Brain Research,1997,749:358)，且若干研究显示使借助BDNF/TrkB途径的信号传导降低的抗体可抑制神经元过敏作用及有关疼痛(Molecular Pain,2008,4:27)。同时有文献亦已指出Trk的过表达、激活、扩增及/或突变与许多癌症有关，包括黑色素瘤(Journal of Investigative Dermatology,2008,128(8):2031-2040)，非小细胞肺癌(Molecular and Cellular Biochemistry,2007,295(1&2):19-26)，甲状腺癌(Neuroendocrinology Letters,2007,28(3):221-229)，急性骨髓白血病(Cancer Lett,2001,169:107-14)，恶性胶质瘤(Journal of Neurochemistry,2007,103:259-275)，星形细胞瘤与髓母细胞瘤(Brain Pathology,2006,16:304-310)，结肠癌(Science,2003,300:949)，神经母细胞瘤(Nat.Rev.Cancer,2003,3:203-216)、卵巢癌(Clin.Cancer Res.2003,9:2248-2259)、乳腺癌(Brain Pathology,2006,16:304-310)、前列腺癌(Clin.Cancer Res.1998,4(8):1887-1898)、胰腺癌(Journal ofGastroenterology and Hepatology,2006,21(5):850-858)、多发性骨髓瘤(Cancer Genetics and Cytogenetics,2007,178:1-10)、及大细胞神经内分泌瘤(Human Mutation,2008,29(5):609-616)。在癌症的临床前模式中，Trk A、B及C以及Trk/Fc嵌合体的非选择性小分子抑制剂在抑制肿瘤生长与阻止肿瘤转移方面有效(Cancer Letters,2001,169:107-114；Cancer Letters,2006,232:90-98；Cancer Res,2008,68:(2)346-351)。
在癌症临床试验中，小分子Trk激酶抑制剂Larotrectinib(LOXO-101)对LMNA-NTRK1基因融合型软组织肉瘤转移患者进行治疗，在四周时间内，肺部肉瘤体积明显变小，癌细胞的转移和扩散减缓，同时患者呼吸困难有所缓解，四个月后，肺部肿块基本消失，且在临床治疗期间并未发现明显不良反应(CancerDiscov,2015,5:1049-1057)。LOXO-101在携带NTRK1/2/3融合阳性的成人/儿童癌症患者上具有显著的疗效，如软组织恶性肿瘤、甲状腺癌、唾液腺癌、胃肠间质瘤、儿童纤维肉瘤、非小细胞癌、胆管癌、肾癌、唾液腺癌、阑尾癌、胰腺癌、外周神经鞘瘤、梭形细胞瘤等，且该药物副作用小(New England Journal of Medicine,2018,378(8):731-739.)。Trk激酶抑制剂Entrectinib(NMS-P626)在临床试验中对18名具有NTRK1/2/3、ROS1或ALK基因重排的实体肿瘤患者进行临床试验研究，结果表明72％的患者对Entrectinib都有反应(J Target Ther Cancer,2015,12:34-37)。
抑制神经营养因子/Trk途径可在临床前模式中有效治疗炎症性疾病。如，抑制神经营养因子/Trk途径与下述疾病的临床前模式有关：包括哮喘在内的炎性肺病(Pharmacology&Therapeutics,2008,117(1):52-76)、间质性膀胱炎(The Journal of Urology,2005,173(3):1016-1021)、包括溃疡性结肠炎及克罗恩病(Crohn's disease)在内的炎性肠病(Gut,2000,46(5):670-678)及炎性皮肤病，例如异位性皮炎(Archives of Dermatological Research,2006,298(1):31-37)、湿疹及干癣(Journal of Investigative Dermatology,2004,122(3):812-819)。
神经营养因子/Trk途径，具体而言BDNF/TrkB途径亦与神经变性疾病的病源学有关，这些疾病包括多发性硬化、帕金森氏病(Parkinson's disease)及阿兹海默氏病(Alzheimer's disease)(Frontiers in Neuroendocrinology,2006,27(4):404-414)。调节神经营养因子/Trk途径可用于治疗这些疾病及相关疾病。
研究表明TrkA受体对于克氏锥虫(Typanosomacruzi)(查加斯氏病(Chagasdisease))在人类宿主中的寄生虫感染的感染中的疾病过程至关重要(Cell Host&Microbe,2007,1(4):251-261)。因此，抑制TrkA可用于治疗查加斯氏病及相关的原虫感染。
同时，Trk抑制剂亦可用于治疗与骨重建调节失衡有关的疾病，例如骨质疏松症、类风湿性关节炎及骨转移。骨转移是癌症的频发性并发症，出现在晚期乳腺癌或前列腺癌患者中及肺癌、结肠癌、胃癌、膀胱癌、子宫癌、直肠癌、甲状腺癌或肾癌患者中。溶骨性转移可造成重度疼痛、病理性骨折、危及生命的高血钙症、脊髓压迫症及其它神经压迫症候群。因此，可诱发增殖性成骨细胞凋亡的药物将极为有利。已在骨折小鼠模式的成骨区域中观察到TrkA受体及TrkC受体的表现(Bone,2000,26(6):625-633)。另外，在几乎所有的成骨细胞中均观察到NGF的分布。pan-Trk抑制剂可在人类hFOB成骨细胞中，抑制由神经营养因子与 所有3个Trk受体结合后所激活的酪胺酸信号传导。这些数据支持使用Trk抑制剂治疗骨重建疾病(例如癌症患者中的骨转移)的理论。
已上市的一代TRK抑制剂药物拉罗替尼(Larotrectinib(LOXO-101))，恩曲替尼(Entrectinib)，作为TRK抑制剂，具有广谱抗癌的效果，但是随着临床使用范围的扩大，也出现了相关的耐药性报道(CancerDiscovery.2017,7(9):934.；部分阿枯米类生物碱的合成研究及钯催化的芳炔偶联反应研究[D].兰州大学.2018.)。这些突变包括在活性部位的TRKA G595R(及其旁系同源TRKC G623R)，毗邻DFG序列的TRKA G667C(及其旁系同源TRKC G696A)和在保守区的TRKA F589L(NewEnglandJournalofMedicine.2018,378(8):731-9.；)。TRKA G595R和TRKA G667C分别类似于ALK G1202R和ALK G1269A(CancerDiscovery.2017,7(9):963.)。
随着耐药性报道的增多，针对耐药性的二代TRK抑制剂也开始被研制出来，如LOXO-195和TPX-0005，LOXO-195和TPX-0005通过环合来限制活性部位的化合物表面积，从化合物结构上来看，LOXO-195和TPX-0005与拉罗替尼具有相同的吡唑[1,5-a]嘧啶结构，但是LOXO-195和TPX-0005通过环化限制了取代基的自由旋转。虽然二代抑制剂克服了一代抑制剂使用中的耐药性，但是其环状结构构建相对困难。同时LOXO-195作为二代选择性TRK抑制剂，表现出对TRK融合蛋白的强力抑制，并且不受某些获得性耐药突变(例如TRKAG595R、TrkAG667C或TrkCG623R)的影响，这些突变可能出现在接受拉罗替尼(LOXO-101)或具有抗TRK活性的多激酶抑制剂的患者中。但是LOXO-195对突变型Trk酶的IC50比相应野生型Trk酶的IC50高5-10倍。
Figure PCTCN2021081904-appb-000001
近年来，对于疗效更佳、副作用更小、代谢稳定性高、生物利用度好的Trk抑制剂存在迫切的临床需求。
发明内容
本发明提出一种作为Trk激酶抑制剂的新型的取代咪唑并吡啶化合物，以克服现有技术存在的稳定性、活性等不足的问题，满足日益增长的临床需求。
本发明的目的是提供一种针对耐药型TRK激酶具有活性的咪唑并吡啶类衍生物。
本发明的目的通过以下方案实现
一种咪唑并吡啶类衍生物，具有式A的结构
Figure PCTCN2021081904-appb-000002
或其互变异构体、立体异构体、对映异构体、非对映异构体、盐、前药或N-氧化物，其中
X、Y或Z其中一个为N时，其余的为CH。
R 3和R 4各自独立地选自C 1-3烷基或H；当R 3和R 4均为烷基时，R 3和R 4可进一步连接成环；
R 1选自未取代或被一个或多个取代基取代的5-6元单环芳基或5-6元单环杂芳基。
R 2选自烷基或杂环烷基，所述R 2未被取代或被一个或多个取代基取代。
进一步地，R 3为C 1-3烷基，优选为甲基，R 4为H。
进一步地，R 3和R 4均为烷基时，R 3和R 4连接成五元环。
进一步地，式A具有下式A 1的结构
Figure PCTCN2021081904-appb-000003
进一步地，R 1中所述取代基各自独立地选自卤素、C 1-4烷氧基、-CF 3、-CHF 2、-OC 1-4烷基OC 1-3烷基或-OC 3-6二羟烷基。
进一步地，R 1中所述取代基各自独立地选自卤素或C 1-4烷氧基。
进一步地，R 1中所述取代基各自独立地选自F或甲氧基。
进一步地，R 1中所述单环芳基或单环杂芳基选自苯基或吡啶基。
进一步地，所述R 1选自苯基、3-氟苯基、2，5-二氟苯基、2-氯-5-氟苯基、2-甲氧基苯基、2-甲氧基-5-氟苯基、2-三氟甲基-5-氟苯基、2-二氟甲基-5-氟苯基、3-氯-5-氟苯基、3-氟-5-甲氧基乙氧基苯基、2-羟基-5-氟苯基或5-氟-2-甲氧基乙氧基苯基、5-氟-吡啶-3-基、2,5-二氟-吡啶-3-基、2-甲氧基-吡啶-3-基、2-甲氧基-5-氟-吡啶-3-基、2-羟基-5氟-吡啶-3-基或2-三氟甲基-5-氟-吡啶-3-基。
进一步地，所述R 1选自2，5-二氟苯基、2-甲氧基-5-氟苯基、2-羟基-5-氟苯基或2-甲氧基-5-氟-吡啶-3-基。
进一步地，R 2中所述烷基选自直链烷基或单环烷基。
进一步地，R 2中所述烷基选自C 1-6直链烷基、C 4-8环烷基或4-8元杂环烷基。
进一步地，R 2中所述烷基选自C 1-3直链烷基、C 5-6环烷基或5-6元杂环烷基。
进一步地，R 2中所述杂环烷基为含有一个或多个杂原子的5-6元杂环烷基，所述杂原子各自独立地选自N、O或S。
进一步地，R 2中所述取代基选自酰氧基、羟基烷基、羟基、甲氧基、卤素、卤代烷、甲基、未取代或被一个或多个取代基取代的芳基、未取代或被一个或多个取代基取代的杂芳基未取代或被一个或多个取代基取代的环烷基、或未取代或被一个或多个取代基取代的杂环烷基。
进一步地，R 2中所述取代基选自甲酰氧基、羟基-C 2-烷基、羟基、甲氧基；
或未取代或被一个或多个取代基取代的芳基，其中被一个或多个取代基取代的芳基中，所述取代基各自独立地选自F或CF 3；
或未取代或被一个或多个取代基取代的杂芳基，其中被一个或多个取代基取代的杂芳基中，所述取代基选自甲基；
或未取代或被一个或多个取代基取代的环烷基，其中被一个或多个取代基取代的环烷基中，所述取代基选自甲酰氧基、羟基；
或未取代的杂环烷基。
进一步地，R 2中所述取代基选自甲酰氧基、羟基-C 2-烷基、羟基、甲氧基；
或未取代或被一个或多个取代基取代的苯基，其中被一个或多个取代基取代的芳基中，所述取代基各自独立地选自F或CF 3；
或未取代或被一个或多个取代基取代的杂芳基，所述杂芳基为含有一个或多个杂原子的5-6元杂芳基，所述杂原子各自独立地选自N、O或S，所述被一个或多个取代基取代的杂芳基中，所述取代基选自甲基；
或未取代或被一个或多个取代基取代的C 5-6环烷基，其中被一个或多个取代基取代的环烷基中，所述取代基选自甲酰氧基、羟基；
或未取代的杂环烷基，所述杂环烷基为含有一个或多个杂原子的5-6元杂环，所述杂原子各自独立地选自N、O或S。
进一步地，所述R 2选自以下结构中的一种：
Figure PCTCN2021081904-appb-000004
Figure PCTCN2021081904-appb-000005
进一步地，所述式A具有如式A 11所示的绝对构型
Figure PCTCN2021081904-appb-000006
进一步地，所述式A化合物选自以下结构中的一种：
Figure PCTCN2021081904-appb-000007
Figure PCTCN2021081904-appb-000008
一种所述咪唑并吡啶类衍生物的制备方法，包括以下步骤中的至少一步，如下式所示，
Figure PCTCN2021081904-appb-000009
步骤a)将A a和
Figure PCTCN2021081904-appb-000010
在碱性条件下反应得到A b，其中L选自氯、溴或碘；
步骤b)将A b和氯甲酸苯酯或氯甲酸苯酯衍生物在碱性条件下反应得到A c，其中R a选自H或-NO 2；
步骤c)将A c和R 2-NH 2在碱性条件下反应得到A d；
步骤d)将A d经过还原得到A e；
步骤e)将A e环化得到A。
进一步地，步骤d)中，所述还原条件为本领域技术人员所知的常规还原-NO 2的条件，不做特殊限定，如氢化还原、铁粉还原或锌粉还原等，优选为氢化还原。
进一步地，步骤e)中，所述环化是将A e和原甲酸三乙酯在酸催化的条件下发生环化反应得到A。
进一步地，步骤e)中，所述环化还可以是将A e和乙基甲酰亚胺在酸催化的条件下发生环化反应得到A。
进一步地，所述制备方法还可以包括为了减少副反应所必要的官能团保护或去保护步骤，所述官能团保护和去保护采用本领域技术人员所熟知的方案。如酚羟基使用苄基保护，在经过一步或多步反应后使用氢化脱除苄基保护得到酚羟基官能团。此类不影响最终产品官能团选择的官能团转化过程也应当在本发明提供的制备方法的保护范围内。
本发明提供一种药物组合物，所述组合物含有上述化合物或其药学上可接受的盐、立体异构体、互变异构体、溶剂合物或前药，及药学上可接受的辅料。
本发明提供一种本发明上文所述的化合物或药物组合物在制备用于抑制Trk酶的活性的药物中的用途，尤其是在抑制突变型Trk酶的活性的药物中的用途。
其中TRK靶点的突变位点位于G517R、A542V、V573M、F589L、F589C、G595R、G595S、D596V、F600L、F646V、C656Y、C656F、L657V、G667S、G667C、Y676S、G623R、G667C、G623E、L686M、G545R、A570V、Q596E、Q596P、V601G、F617L、F617C、G623S、D624V、R630K、C682Y、C682F、L683V、G693S、G713S、C685F、C685Y、L686V、G696A和G639R中的至少一种。优选为TRKA G595R、TRKC G623R、TRKB G639R、TRKA G667C、TRKC G696A和TRKA F589L中的至少一种。更优选为TRKA G595R、TRKC G623R和TRKB G639R中的至少一种。
进一步地，所述用途的作用方式为所述Trk酶与本文所述的化合物或其药学上可接受的盐、立体异构体、互变异构体、溶剂合物接触。
进一步地，本发明提供的化合物用于预防和/或治疗Trk酶介导的疾病的产品中的应用。
进一步地，所述疾病包括但不仅限于疼痛和癌症中的至少一种。
所述疼痛可以是任一来源或病因的疼痛，包括但不限于炎性疼痛、内脏疼痛、癌症引起的疼痛、化疗疼痛、创伤疼痛、手术及手术后疼痛、分娩疼痛、急性疼痛、慢性疼痛、顽固性疼痛、躯体疼痛、伤害性疼痛、神经性疼痛、血运源性疼痛、免疫源性疼痛、内分泌源性疼痛、代谢性病变引起的疼痛、心源性疼痛、头痛、幻肢痛和牙痛中的一种或多种。
式I的化合物用于治疗急性疼痛。急性疼痛，如国际疼痛研究协会所定义，起因于疾病、 炎症、或组织损伤。这种疼痛一般发生突然，例如，在外伤或手术后，并可能伴随着焦虑或压力。起因通常可被诊断与治疗，并且该疼痛局限在一段给定的期间与严重程度。在一些例子中，其可转变为慢性的。
所述癌症包括但不限于肺癌、结肠癌、前列腺癌、乳腺癌、肝癌、淋巴癌、甲状腺癌、多发性骨髓瘤、软组织肉瘤、卵巢癌、宫颈癌、输卵管癌、肾细胞癌、胃癌、胃肠道间质瘤、骨癌、基底细胞癌、腹膜癌、皮肤纤维瘤、胰腺癌、食管癌、胶质母细胞瘤、头颈癌、炎症性肌纤维母细胞瘤和间变性大细胞淋巴瘤中的一种或多种。
本发明的化合物可通过任何适合的途径施用，例如，进入胃肠道(例如直肠或口服)、鼻、肺、肌肉或血管或经皮肤或真皮。化合物可通过任何适合的施用形式施用，例如片剂、粉末、胶囊、溶液、分散液、混悬液、糖浆剂、喷雾剂、栓剂、凝胶、乳液、贴片等。此类组合物可包含药学制剂中传统的组分，例如稀释剂、载体、pH调节剂、甜味剂、填充剂和另外的活性剂。若需要肠胃外施用，该组合物将是无菌的并且呈适于注射或输注的溶液或悬浮液形式。此类组合物构成本发明另一方面。
本发明所称“药学上可接受的盐”，旨在表示式(A)表示的化合物的游离酸或碱的盐，其是无毒的、生物学耐受的或换句话说是在生物学上适于施用给受试者。一般来讲，参见G.S.Paulekuhn,etal.，“TrendsinActivePharmaceuticalIngredientSaltSelectionbasedAnalysis of the OrangeBookDatabase”，J.Med.Chem.,2007,50：6665～72,S.M.Berge,etal.，“PharmaceuticalSalts”，JPharmSci.，1977,66：1-19,andHandbookof Pharmaceutical Salts,Properties,Selection,and Use,Stahland Wermuth,Eds.,Wiley-VCHandVHCA,Zurich，2002(G.S.Paulekuhn等人，“根据橙皮书数据库的分析选择活性药物成分盐的趋势”，《药物化学杂志》，2007年，第50卷，第6665-6672页；S.M.Berge等人“，药用盐”,《药物科学杂志》，1977年，第66卷，第1-19页；以及《药用盐：特性、选择与应用》，Stahl和Wermuth编辑，Wiley-VCHandVHCA,Zurich，2002年)。可药用盐的例子是那些药理学有效且适于与患者组织接触而不会有不当毒性、刺激或变应性反应的盐。式(I)的化合物可以具有足够酸性的基团、足够碱性的基团或这两种类型的官能团，从而与多种无机碱或有机碱，以及无机酸和有机酸反应，形成可药用盐。
可药用盐的例子包括硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物、乙酸盐、丙酸盐、癸酸盐、辛酸盐、丙烯酸盐、甲酸盐、异丁酸盐、己酸盐、庚酸盐、丙炔酸盐、草酸盐、丙二酸盐、唬泊酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、丁炔-1,4-二酸盐、己炔-1,6-二酸盐、苯甲酸盐、氯代苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、烃基苯甲酸盐、氧基苯甲酸盐、邻苯二甲酸盐、磺酸盐、二甲苯磺酸盐、苯乙酸盐、苯丙酸盐、苯丁酸盐、 檬酸盐、乳酸盐、Y-轻丁酸盐、乙醇酸盐、酒石酸盐、甲磺酸盐、丙磺酸盐、蔡-1-磺酸盐、茶-2-磺酸盐和扁桃酸盐。
式(A)的化合物含有碱性氮，所需的可药用盐可通过本领域可用的任何合适方法制备，例如用无机酸处理该游离碱，所述无机酸例如是盐酸、氢溴酸、硫酸、氨基磺酸、硝酸、硼酸、磷酸等，或者用有机酸处理该游离碱，所述有机酸例如是乙酸、苯乙酸、丙酸、硬脂酸、乳酸、抗坏血酸、马来酸、羟基马来酸、羟乙磺酸、琥珀酸、戊酸、富马酸、丙二酸、丙酮酸、草酸、乙醇酸、水杨酸、油酸、棕桐酸、月桂酸、砒喃糖昔基酸(如葡糖醛酸或半乳糖醛酸)、α-烃酸(如扁桃酸、柠檬酸或酒石酸)、氨基酸(如天冬氨酸、戊二酸或谷氨酸)、芳族酸(如苯甲酸、2-乙酸氧基苯甲酸、蔡甲酸或肉桂酸)、磺酸(如月桂基磺酸、对甲苯磺酸、甲磺酸、乙磺酸)、诸如本文作为例子给出的那些酸的任何相容混合物、和根据本技术领域的普通技能水平被认为是等效物或可接受取代物的任何其他酸及其混合物。
本文所述的前药可以包括这样的化合物，其具有通过酰胺键或酯键共价连接到式(A)羧酸基团的氨基酸残基或者两个或更多个(例如两个、三个或四个)氨基酸残基的多肽链。氨基酸残基的例子包括通常用三个字母符号标识的二十种天然存在的氨基酸以及4-羟脯氨酸、羟基赖氨酸、锁链素(demosine)、异锁链素(isodemosine)、3-甲基组氨酸、正缬氨酸、β-丙氨酸、γ-氨基丁酸、瓜氨酸高半胱氨酸、高丝氨酸、鸟氨酸和甲硫氨酸。
其他类型的前药可以通过将式(A)结构的游离羧酸衍生为酰胺或烷基酯来制备。酰胺的例子包括衍生自氨、C 1-6烷基伯胺和二(C 1-6烷基)仲胺的那些。仲胺包括5-元或6-元杂环烷基或杂芳基环部分。酸胺的例子包括衍生自氨.C 1-3烷基伯胺和二(C l-2烷基)胺的那些酰胺。本发明的脂的例子包括C 1-7烷基酯,C 5-7环烷基酯、苯酯和(C 1-6烷基)苯酯。优选地，脂包括甲酯。还可以按照诸如Fleisheretal.,Adv.DrugDeliveryRev.1996,19,115-130(Fleisher等人，《药物递送进展综述》，1996年，第19卷，第115-130页)中所描述的那些方法，通过用包括半琥珀酸脂、磷酸脂、二甲基氨基乙酸脂、和磷酰氧基甲氧基羰基在内的基团使游离羟基衍生化来制备前药。羟基基和氨基的氨基甲酸酯衍生物也可产生前药。羟基的碳酸脂衍生物、磺酸脂和硫酸脂也可以提供前药。将经基衍生化为(酰氧基)甲脂和(酰氧基)乙脂，其中酰基可为任选被一个或多个醚、胺或浚酸官能团取代的烷基脂，或者其中酰基为如上所述的氨基酸脂，这也可用于产生前药。该类型的前药可如Robinsoneta1.，JMedChem.1996,39(1)，10-18(Robinson等人，《药物化学杂志》，1996年，第39卷第1期，第10-18页)中所述制备。游离胺也可衍生化为酰胺、磺酰胺或磷酰胺。所有这些前药部分都可掺入包括醚、胺和羧酸官能团在内的基团。
本发明的优势在于
1)使用一代TRK抑制剂的结构，解决了一代抑制剂在TRK抑制剂使用中的耐药性问题，达到甚至超过了二代抑制剂的效果。
2)提供了一种结构新颖的Trk抑制剂。
3)本发明提供的TRK抑制剂表现出对突变型Trk抑制剂IC50值比对相应的野生型Trk抑制剂IC50值相近或更低。
4)本发明创造性地选用咪唑并吡啶类衍生物，得到了在低浓度范围内同时对野生型和突变型Trk酶抑制的效果，而咪唑并嘧啶或咪唑并吡嗪虽然是咪唑并吡啶的生物电子等排体，但是他们的衍生物确达不到本发明的效果。
具体实施方式
本发明具体实施方式中，除非另有说明，则试剂是自商业供货商(例如Aldrich Chemical公司、Lancaster、TCI或Maybridge)购得且未经进一步纯化即使用。
制备A：
Figure PCTCN2021081904-appb-000011
(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-氨基吡啶
将化合物2-氨基-3硝基-6氯吡啶(5g，28.7mmol)、化合物(R)-2-(2,5-二氟苯基)吡咯烷盐酸盐(6.9g，31.6mmol)和DIEA(11.1g，86.1mmol)溶于100mL乙醇中，在80℃条件下搅拌反应12小时。TLC检测确定反应结束后，冷却至0℃，过滤，乙醇洗涤固体，抽干后获得黄色固体产品8.6g，产率92.3％。LC-MS(ESI)m/z:321[M+H] +。
制备B：
Figure PCTCN2021081904-appb-000012
(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(二苯氧甲酰基)氨基吡啶
将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-氨基吡啶(8.6g，26.5mmol)溶于100ml吡啶中，逐滴加入氯甲酸苯酯(12.2g，79.6mmol)，然后在70℃下搅拌反应5小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入100ml水淬灭反应，用乙酸乙酯(3×100ml)萃取，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品12.0g，产率80.7％。LC-MS(ESI)m/z:561[M+H] +。
实施例1-Compound 1
制备M1
Figure PCTCN2021081904-appb-000013
(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-苄基脲基)吡啶
将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(二苯氧甲酰基)氨基吡啶(2g，3.5mmol)，苄胺(0.56g，5.3mmol)和碳酸钠(0.74g，7mmol)悬浮于25mL乙腈中，室温搅拌12小时。TLC检测反应结束后，加入20ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有机相，干燥，旋干获得粗品。粗品经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品1.4g，产率85.7％。LC-MS(ESI)m/z:454[M+H] +。
Figure PCTCN2021081904-appb-000014
(R)-N-苄基-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
将中间体M1(200mg，0.44mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，保留滤液旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品40mg，产率19.1％。
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).
实施例2-Compound 2
Figure PCTCN2021081904-appb-000015
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-氟苄基)脲基)吡啶的制备。用制备M1的方法，使用4-氟苄胺代替苄胺进行制备。
步骤B:(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将中间体(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-氟苄基)脲基)吡啶(300mg，0.63mmol)溶于5mL THF中，加入10％钯碳(30mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(15mg)，在135℃条件下搅拌反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品95mg，产率30.7％。
LC-MS(ESI)m/z:452.4[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例3-Compound 3
Figure PCTCN2021081904-appb-000016
(R)-N-(3,5-二氟苄基)-5-(2-(3,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3,5-二氟苄基)脲基)吡啶的制备。用制备M1的方法，使用3,5-二氟苄胺代替苄胺进行制备。
步骤B：将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3,5-二氟苄基)脲基)吡啶(200mg，0.41mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测，反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在135℃下反应4小时。TLC检测，反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得产品45mg，白色固体，产率21.5％。
LC-MS(ESI)m/z:470.4[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例4-Compound 4
Figure PCTCN2021081904-appb-000017
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-氟苯乙基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-氟苯乙基)脲基)吡啶的制备。用制备M1的方法，使用4-氟苯乙胺代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-氟苯乙基)-3H-咪唑并[4,5-b]吡啶-3-甲 酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-氟苯乙基)脲基)吡啶(200mg，0.42mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品65mg，产率31.1％。
LC-MS(ESI)m/z:466.5[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例5-Compound 5
Figure PCTCN2021081904-appb-000018
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-三氟甲基苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-三氟甲基苄基)脲基)吡啶的制备。用制备M1的方法，使用4-三氟甲基苄胺代替苄胺进行制备。
步骤B：将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-三氟甲基苄基)脲基)吡啶(300mg，0.57mmol)溶于5mL THF中，加入10％钯碳(30mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(15mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品115mg，产率37.2％。
LC-MS(ESI)m/z:502.5[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例6-Compound 6
Figure PCTCN2021081904-appb-000019
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A:(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-氟苄基)脲基)吡啶的制备。用制备M1的方法，使用3-氟苄胺代替苄胺进行制备。
步骤B:(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-氟苄基)脲基)吡啶(300mg，0.63mmol)溶于5mL THF中，加入10％钯碳(30mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(15mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品95mg，产率30.6％。
LC-MS(ESI)m/z:452.4[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例7-Compound 7
Figure PCTCN2021081904-appb-000020
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(2-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(2-氟苄基)脲基)吡啶的制备。用制备M1的方法，使用2-氟苄胺代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(2-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰 胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(2-氟苄基)脲基)吡啶(300mg，0.63mmol)溶于5mL THF中，加入10％钯碳(30mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(15mg)，在135℃下反应4小时。TLC检测，反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品85mg，产率27.6％。
LC-MS(ESI)m/z:452.4[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例8-Compound 8
Figure PCTCN2021081904-appb-000021
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-苯乙基-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-苯乙基脲基)吡啶的制备。
用制备M1的方法，使用苯乙胺代替苄胺进行制备。
步骤B：将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-苯乙基脲基)吡啶(200mg，0.42mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品85mg，产率40.1％。
LC-MS(ESI)m/z:448.5[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例9-Compound 9
Figure PCTCN2021081904-appb-000022
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-苯丙基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-苯丙基脲基)吡啶的制备。
用制备M1的方法，使用苯丙胺代替苄胺进行制备。
步骤B:(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-苯丙基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-苯丙基脲基)吡啶(250mg，0.51mmol)溶于5mL THF中，加入10％钯碳(25mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(12.5mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品65mg，产率31.1％。
LC-MS(ESI)m/z:462.5[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例10-Compound 10
Figure PCTCN2021081904-appb-000023
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-吡啶甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-吡啶甲基)脲基)吡啶的制备。 用制备M1的方法，使用3-氨甲基吡啶代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-吡啶甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-吡啶甲基)脲基)吡啶(220mg，0.46mmol)溶于5mL THF中，加入10％钯碳(22mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(11mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝2/1，V/V)获得白色固体产品62mg，产率27.1％。
LC-MS(ESI)m/z:435[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例11-Compound 11
Figure PCTCN2021081904-appb-000024
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-吡啶甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-吡啶甲基)脲基)吡啶的制备。用制备M1的方法，使用4-氨甲基吡啶代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-吡啶甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-吡啶甲基)脲基)吡啶(200mg，0.44mmol)溶于5mL THF中，加入10％钯碳(22mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(11mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝2/1，V/V)获得白色固体产品78mg，产率40.6％。
LC-MS(ESI)m/z:435[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例12-Compound 12
Figure PCTCN2021081904-appb-000025
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-噻吩甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-噻吩甲基)脲基)吡啶的制备。用制备M1的方法，使用3-氨甲基噻吩代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-噻吩甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(3-噻吩甲基)脲基)吡啶(180mg，0.41mmol)溶于5mL THF中，加入10％钯碳(18mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(9mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品50mg，产率26.4％。
LC-MS(ESI)m/z:440[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例13-Compound 13
Figure PCTCN2021081904-appb-000026
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((1-甲基-1-H-吡唑-4-基)甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-((1-甲基-1-H-吡唑-4-基)甲基)脲基)吡啶的制备。用制备M1的方法，使用4-(氨基甲基)-1-甲基吡唑代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((1-甲基-1-H-吡唑-4-基)甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-((1-甲基-1-H-吡唑-4-基)甲基)脲基)吡啶(220mg，0.48mmol)溶于5mL THF中，加入10％钯碳(22mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(11mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品62mg，产率29.1％。
LC-MS(ESI)m/z:438[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例14-Compound 14
Figure PCTCN2021081904-appb-000027
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((四氢-2H-吡喃-4-基)甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((四氢-2H-吡喃-4-基)甲基)脲基)吡啶的制备。用制备M1的方法，使用4-氨甲基四氢吡喃代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((四氢-2H-吡喃-4-基)甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((四氢-2H-吡喃-4-基)甲基)脲基)吡啶(200mg，0.43mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL 原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝2/1，V/V)获得白色固体产品56mg，产率26.7％。
LC-MS(ESI)m/z:442[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例15-Compound 15
Figure PCTCN2021081904-appb-000028
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(四氢-2H-吡喃-4-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((四氢-2H-吡喃-4-基)脲基)吡啶的制备。用制备M1的方法，使用4-氨基四氢吡喃代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(四氢-2H-吡喃-4-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((四氢-2H-吡喃-4-基)脲基)吡啶(250mg，0.56mmol)溶于5mL THF中，加入10％钯碳(25mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(13mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝2/1，V/V)获得白色固体产品89mg，产率34.2％。
LC-MS(ESI)m/z:428[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例16-Compound 16
Figure PCTCN2021081904-appb-000029
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-甲氧基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((4-甲氧基环己基)脲基)吡啶的制备。用制备M1的方法，使用4-甲氧基环己胺盐酸盐代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-甲氧基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((4-甲氧基环己基)脲基)吡啶(210mg，0.44mmol)溶于5mL THF中，加入10％钯碳(21mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(11mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝2/1，V/V)获得白色固体产品49mg，产率22.4％。
LC-MS(ESI)m/z:456[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例17-Compound 17
Figure PCTCN2021081904-appb-000030
(1S,4s)-4-(5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)环己基 甲酸
步骤A：6-((R)-(2-(2,5-二氟苯基)吡咯烷-1-基))-3-硝基-2-(((1S,4S)-4-羟基环己基)脲基)吡啶的制备。用制备M1的方法，使用顺式对氨基环己醇盐酸盐代替苄胺进行制备。
步骤B：(1S,4s)-4-(5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)甲酸环己酯的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((1S,4S)-(4-羟基环己基)脲基)吡啶(200mg，0.44mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品31mg，产率14.9％。
LC-MS(ESI)m/z:470[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例18-Compound 18
Figure PCTCN2021081904-appb-000031
3-((5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)甲基)酸环己基甲酸
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((3-羟基环己基)甲基)脲基)吡啶的制备。用制备M1的方法，使用3-(氨甲基)环己醇代替苄胺进行制备。
步骤B：3-((5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)甲基)酸环己基甲酸的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((3-羟基环己基)甲基)脲基)吡啶(240mg，0.50mmol)溶于5mL THF中，加入10％钯碳(24mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(12mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品22mg，产率8.9％。
LC-MS(ESI)m/z:484[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例19-Compound 19
Figure PCTCN2021081904-appb-000032
(1R,3S)-3-((5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)甲基)酸环戊基甲酸
步骤A：6-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((3-(1S,3R)-羟基环己基)脲基)吡啶的制备。用制备M1的方法，使用(1R,3S)-3-氨基环戊醇盐酸盐代替苄胺进行制备。
步骤B：(1R,3S)-3-((5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺)甲基)酸环戊基甲酸的制备。将6-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((3-(1S,3R)-羟基环己基)脲基)吡啶(200mg，0.44mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(10mg)，在100℃下反应1小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品26mg，产率12.9％。
LC-MS(ESI)m/z:456[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例20-Compound 20
Figure PCTCN2021081904-appb-000033
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-(2-羟基乙基)环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((4-(2-羟基乙基)环己基)脲基)吡啶的制备。用制备M1的方法，使用4-氨基环己烷乙醇代替苄胺进行制备。
步骤B：(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-(2-羟基乙基)环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-((4-(2-羟基乙基)环己基)脲基)吡啶(250mg，0.51mmol)溶于5mL乙醇中，加入10％钯碳(25mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(110mg，1.02mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品56mg，产率21.9％。
LC-MS(ESI)m/z:470[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例21-Compound 21
Figure PCTCN2021081904-appb-000034
5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-N-((1r,4R)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3- 甲酰胺
步骤A：6-((R)-(2-(2,5-二氟苯基)吡咯烷-1-基))-3-硝基-2-(((1r,4R)-4-羟基环己基)脲基)吡啶的制备。用制备M1的方法，使用反式对氨基环己醇盐酸盐代替苄胺进行制备。
步骤B：5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-N-((1r,4R)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将6-((R)-(2-(2,5-二氟苯基)吡咯烷-1-基))-3-硝基-2-(((1r,4R)-4-羟基环己基)脲基)吡啶(240mg，0.52mmol)溶于5mL乙醇中，加入10％钯碳(24mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(110mg，1.04mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品46mg，产率18.4％。
LC-MS(ESI)m/z:442[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例22-Compound 22
Figure PCTCN2021081904-appb-000035
5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-N-((1s,4S)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：6-((R)-(2-(2,5-二氟苯基)吡咯烷-1-基))-3-硝基-2-(((1s,4S)-4-羟基环己基)脲基)吡啶的制备。用制备M1的方法，使用顺式对氨基环己醇盐酸盐代替苄胺进行制备。
步骤B：将6-((R)-(2-(2,5-二氟苯基)吡咯烷-1-基))-3-硝基-2-(((1s,4S)-4-羟基环己基)脲基)吡啶(250mg，0.54mmol)溶于5mL乙醇中，加入10％钯碳(25mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(110mg，1.08mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯 萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品48mg，产率18.6％。
LC-MS(ESI)m/z:442[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例23-Compound 23
Figure PCTCN2021081904-appb-000036
5-((R)-2-(2,5-二氟苯基)吡咯烷-1-基)-N-((3-羟基环己基)甲基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((3-羟基环己基)甲基)脲基)吡啶的制备。用制备M1的方法，使用3-(氨甲基)环己醇代替苄胺进行制备。
步骤B：将(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(((3-羟基环己基)甲基)脲基)吡啶(210mg，0.44mmol)溶于5mL乙醇中，加入10％钯碳(21mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(96mg，0.88mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品36mg，产率16.6％。
LC-MS(ESI)m/z:456[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例24-Compound 24
Figure PCTCN2021081904-appb-000037
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-苯基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-苯基环己基)脲基)吡啶的制备。用制备M1的方法，使用4-苯基环己胺代替苄胺进行制备。
步骤B:(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。将中间体(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-3-硝基-2-(3-(4-苯基环己基)脲基)吡啶(200mg，0.41mmol)溶于5mL乙醇中，加入10％钯碳(20mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(90mg，0.82mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品46mg，产率18.9％。
LC-MS(ESI)m/z:502[M+H] +
1H NMR(600MHz,CDCl 3)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).
制备C：
Figure PCTCN2021081904-appb-000038
(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-氨基吡啶
将化合物2-氨基-3-硝基-6-氯吡啶(2g，11.5mmol)、化合物(R)-5-氟-2-甲氧基-3-(吡咯烷-3-基)吡啶盐酸盐(2.66g，11.5mmol)和DIEA(4.4g，34.5mmol)溶于50mL乙醇中，在 80℃条件搅拌下反应12小时。TLC检测确定反应结束后，冷却至0℃下，过滤，乙醇洗涤，抽干，获得黄色固体产品4g，产率100％。LC-MS(ESI)m/z:334[M+H] +。
制备D：
Figure PCTCN2021081904-appb-000039
(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(二苯氧甲酰基)氨基吡啶
将(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-氨基吡啶(4g，12mmol)溶于50ml吡啶中，逐滴加入氯甲酸苯酯(5.6g，36mmol)，然后在70℃下搅拌反应5小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入50ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品5.0g，产率72.7％。LC-MS(ESI)m/z:574[M+H] +。
制备M2
Figure PCTCN2021081904-appb-000040
(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(3-(3-氟苄基)脲基)吡啶
将(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(二苯氧甲酰基)氨基吡啶(300mg，0.52mmol)，间氟苄胺(125mg，1.04mmol)和碳酸钠(159mg，1.56mmol)悬浮于10mL乙腈中，室温搅拌12小时。TLC检测反应结束后，加入20ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有机相，干燥，旋干获得粗品。粗品经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品230mg，产率90.3％。LC-MS(ESI)m/z:485[M+H] +。
实施例25-Compound 25
Figure PCTCN2021081904-appb-000041
(R)-5-(2-(5-氟-2-甲氧基吡啶-3-基)吡咯烷-1-基)-N-(3-氟苄基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
将中间体M2(230mg，0.47mmol)溶于5mL THF中，加入10％钯碳(23mg)，置换氢气后，室温搅拌反应12小时。TLC检测，反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(11mg)，在135℃下反应4小时。TLC检测，反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品65mg，产率29.7％。
LC-MS(ESI)m/z:465[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例26-Compound 26
Figure PCTCN2021081904-appb-000042
(R)-5-(2-(5-氟-2-甲氧基吡啶-3-基)吡咯烷-1-基)-N-苯乙基-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(3-苯乙基脲基)吡啶的制备。用制备M2的方法，使用苯乙胺代替间氟苄胺进行制备。
步骤B：将(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(3-苯乙基脲基)吡啶(190mg,0.39mmol)溶于5mL THF中，加入10％钯碳(19mg)，置换氢气后，室温搅拌反应12 小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于原甲酸三乙酯(5mL)中，加入对甲苯磺酸(9mg)，在135℃下反应4小时。TLC检测，反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品43mg，产率21.6％。
LC-MS(ESI)m/z:461[M+H] +
1H NMR(600MHz,CDCl 3)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).
实施例27-Compound 27
Figure PCTCN2021081904-appb-000043
5-((R)-2-(5-氟-2-甲氧基吡啶-3-基)吡咯烷-1-基)-N-((1s,4S)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(3-((1s,4S)-4-羟基环己基)脲基)吡啶的制备。用制备M2的方法，使用(1s,4S)-4-羟基环己胺代替间氟苄胺进行制备。
步骤B：将(R)-6-(2-(5-氟-2-甲氧基吡啶)吡咯烷-1-基)-3-硝基-2-(3-((1s,4S)-4-羟基环己基)脲基)吡啶(200mg,0.39mmol)溶于5mL THF中，加入10％钯碳(20mg)，置换氢气后，室温搅拌反应12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(90mg，0.78mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，通过TLC制备纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品36mg，产率13.9％。
LC-MS(ESI)m/z:455[M+H] +
1H NMR(600MHz,CDCl 3)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).
制备E：
Figure PCTCN2021081904-appb-000044
(R)-2-(1-((6-氨基-5-硝基吡啶-2-基)氨基)乙基)-4-氟苯酚
将化合物2-氨基-3-硝基-6-氯吡啶(1.24g，7.1mmol)、化合物(R)-2-(1-氨基乙基)-4-氟苯酚盐酸盐(1.37g，7.1mmol)和DIEA(2.68g，21.3mmol)溶于20mL乙醇中，在80℃条件下搅拌反应24小时。TLC检测确定反应结束后，旋干，经硅胶柱层析分离纯化(乙酸乙酯/石油醚＝1/3，V/V)，得到黄色固体产品1.7g，产率81.7％。LC-MS(ESI)m/z:293[M+H] +。
制备F：
Figure PCTCN2021081904-appb-000045
(R)-2-(1-((6-氨基-5-硝基吡啶-2-基)氨基)乙基)-1-苄氧基-4-氟苯
将(R)-2-(1-((6-氨基-5-硝基吡啶-2-基)氨基)乙基)-4-氟苯酚(1.7g，5.8mmol)，溴苄(2.0g，11.6mmol)，碳酸钾(2.4g，17.4mmol)，碘化钠(100mg)加入到50ml圆底烧瓶中，加入DMF作为溶剂20ml，室温搅拌反应18小时。TLC检测确定反应结束后，加入100ml水淬灭反应，用乙酸乙酯(30ml×5次)萃取产物，使用饱和食盐水洗涤有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/2，V/V)，获得黄色固体产品2.2g，产率99.1％。LC-MS(ESI)m/z:383[M+H] +。
制备G：
Figure PCTCN2021081904-appb-000046
(R)-6-(1-(2-苄氧基-5-氟苯基)乙基)氨基-3-硝基-2-(二苯氧甲酰基)氨基吡啶
将(R)-2-(1-((6-氨基-5-硝基吡啶-2-基)氨基)乙基)-1-苄氧基-4-氟苯(2.2g，5.7mmol)溶于20ml吡啶中，逐滴加入氯甲酸苯酯(2.7g，17.2mmol)，然后50℃油浴加热搅拌反应18小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入30ml水淬灭反应，用乙酸乙酯萃取(30ml×3次)，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/2，V/V)，获得黄色固体产品3.3g，产率93.0％。LC-MS(ESI)m/z:623[M+H] +。
制备M3
Figure PCTCN2021081904-appb-000047
(R)-6-(1-(2-苄氧基-5-氟苯基)乙基)氨基-3-硝基-2-(3-(4-甲氧基环己基)脲基)吡啶
将(R)-6-(1-(2-苄氧基-5-氟苯基)乙基)氨基-3-硝基-2-(二苯氧甲酰基)氨基吡啶(450mg，0.72mmol)，4-甲氧基环己胺(192mg，1.48mmol)和碳酸钠(230mg，2.17mmol)悬浮于20mL乙腈中，50℃油浴加热搅拌反应18小时。TLC检测反应结束后，旋干，经硅胶柱纯化(乙酸乙酯/石油醚＝1/2，V/V)，获得黄色固体产品260m g，产率66.7％。LC-MS(ESI)m/z:538[M+H] +。
制备M4
Figure PCTCN2021081904-appb-000048
(R)-5-((1-(2-苄氧基-5-氟苯基)乙基)氨基)-N-(4-甲氧基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
将中间体M3(260mg，0.48mmol)溶于10mL乙醇中，加入5％铂碳(30mg)，置换氢气后，室温搅拌3小时。TLC检测中间体M3全部反应，过滤，在滤液中加入乙基甲酰亚胺盐酸盐(106mg，0.96mmol)，在50℃油浴搅拌条件下反应18小时。TLC检测有新产物生成，浓缩反应液，通过硅胶柱层析分离得到产品110mg(乙酸乙酯/石油醚＝5/1，V/V)，产率44.2％。
实施例28-Compound 28
Figure PCTCN2021081904-appb-000049
(R)-5-((1-(5-氟-2-羟基苯基)乙基)氨基)-N-(4-甲氧基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
将中间体M4(R)-5-((1-(2-苄氧基-5-氟苯基)乙基)氨基)-N-(4-甲氧基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺(110mg，0.21mmol)溶于10mL甲醇中，加入10％钯碳(20mg)，置换氢气后，60℃油浴加热搅拌反应18小时。TLC检测反应结束后，过滤，旋蒸浓缩反应液，通过TLC制备板分离纯化(乙酸乙酯/石油醚＝10/1，V/V)获得白色固体产品49mg，产率为54.5％。
LC-MS(ESI)m/z:428[M+H] +
1H NMR(600MHz,CDCl 3)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)。
实施例29-Compound 29
Figure PCTCN2021081904-appb-000050
(R)-5-((1-(5-氟-2-羟基苯基)乙基)氨基)-N-((1s,4s)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺
步骤A：(R)-6-(1-(2-苄氧基-5-氟苯基)乙基)氨基-3-硝基-2-(3-((1s,4s)-4-羟基环己基)脲基)吡啶的制备。用制备M3的方法，使用(1s,4s)-4-羟基环己胺代替4-甲氧基环己胺进行制备，产率为54.8％。
步骤B：(R)-5-((1-(2-苄氧基-5-氟苯基)乙基)氨基)-N-((1s,4s)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺的制备。用制备M4的方法，将(R)-6-(1-(2-苄氧基-5-氟苯基)乙基)氨基-3-硝基-2-(3-((1s,4s)-4-羟基环己基)脲基)吡啶代替M3进行制备，产率为31.4％。
步骤C：将(R)-5-((1-(2-苄氧基-5-氟苯基)乙基)氨基)-N-((1s,4s)-4-羟基环己基)-3H-咪唑并[4,5-b]吡啶-3-甲酰胺(60mg，0.12mmol)溶于10mL甲醇中，加入10％钯碳(20mg)，置换氢气后，60℃油浴加热搅拌反应18小时。TLC检测反应结束后，过滤，旋蒸浓缩反应液，通过TLC制备板分离纯化(乙酸乙酯/石油醚＝10/1，V/V)获得白色固体产品20mg，产率为40.3％。
LC-MS(ESI)m/z:414[M+H] +
1H NMR(600MHz,CDCl 3)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)。
制备M5
Figure PCTCN2021081904-appb-000051
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-氨基吡啶
将化合物4-氨基-5硝基-2氯吡啶(5g，28.7mmol)、化合物(R)-2-(2,5-二氟苯基)吡咯烷盐酸盐(6.9g，31.6mmol)和DIEA(11.1g，86.1mmol)溶于100mL乙醇中，在80℃条件下搅拌反应12小时。TLC检测确定反应结束后，冷却至0℃，过滤，乙醇洗涤固体，抽干后获得黄色固体产品8.9g，产率94.3％。LC-MS(ESI)m/z:321[M+H] +。
制备M6：
Figure PCTCN2021081904-appb-000052
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(二苯氧甲酰基)氨基吡啶
将(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-氨基吡啶(8.9g，27.5mmol)溶于100ml吡啶中，逐滴加入氯甲酸苯酯(12.2g，79.6mmol)，然后在70℃下搅拌反应5小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入100ml水淬灭反应，用乙酸乙酯(3×100ml)萃取，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品10.0g，产率70.7％。LC-MS(ESI)m/z:561[M+H] +。
制备M7
Figure PCTCN2021081904-appb-000053
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(3-(3-氟苄基)脲基)吡啶
将(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(二苯氧甲酰基)氨基吡啶(2g，3.5mmol)，3-氟苄胺(0.66g，5.3mmol)和碳酸钠(0.74g，7mmol)悬浮于25mL乙腈中，室温搅拌12小时。TLC检测反应结束后，加入20ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有 机相，干燥，旋干获得粗品。粗品经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品1.3g，产率为78.8％。LC-MS(ESI)m/z:472[M+H] +。
实施例30-Compound 30
Figure PCTCN2021081904-appb-000054
(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-氟苄基)-1H-咪唑并[4,5-c]吡啶-1-甲酰胺
将(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(3-(3-氟苄基)脲基)吡啶(300mg，0.63mmol)溶于5mL THF中，加入10％钯碳(30mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(15mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品75mg，产率26.4％。
LC-MS(ESI)m/z:452[M+H] +。
1H NMR(600MHz,CDCl 3)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).
制备M8
Figure PCTCN2021081904-appb-000055
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-2-硝基-3-氨基吡啶
将化合物3-氨基-2硝基-5溴吡啶(1g，4.5mmol)、化合物(R)-2-(2,5-二氟苯基)吡咯烷盐酸盐(1.1g，5.1mmol)和DIEA(1.8g，12.1mmol)溶于20mL NMP中，在180℃条件下搅拌反应2小时。TLC检测确定反应结束后，加入水，用乙酸乙酯萃取，合并有机相，干燥， 旋蒸浓缩获得粗品。粗品经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)获得产品1.5g，黄色固体，产率100％。
LC-MS(ESI)m/z:321[M+H] +。
制备M9：
Figure PCTCN2021081904-appb-000056
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-2-硝基-3-(二苯氧甲酰基)氨基吡啶
将M8(1.5g，4.6mmol)溶于10ml吡啶中，逐滴加入氯甲酸苯酯(1.6g，9.6mmol)，然后在70℃下搅拌反应5小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入50ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品2.3g，产率89.3％。
LC-MS(ESI)m/z:561[M+H] +。
制备M10
Figure PCTCN2021081904-appb-000057
(R)-5-(2-(2,5-二氟苯基)吡咯烷-1-基)-2-硝基-3-(3-(4-甲氧基环己基)脲基)吡啶
将M9(560mg，1.0mmol)，4-甲氧基环己胺盐酸盐(260mg，2.0mmol)和碳酸钠(320mg，3mmol)悬浮于20mL乙腈中，室温搅拌12小时。TLC检测反应结束后，旋蒸浓缩，经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品350mg，产率为73.6％。LC-MS(ESI)m/z:476[M+H] +。
实施例31-Compound 31
Figure PCTCN2021081904-appb-000058
(R)-6-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(4-甲氧基环己基)-1H-咪唑并[4,5-b]吡啶-1-甲酰胺
将M10(230mg，0.48mmol)溶于5mL乙醇中，加入10％钯碳(23mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，在反应液中加入乙基甲酰亚胺盐酸盐(102mg，0.96mmol)，在80℃下反应5小时。TLC检测反应结束后，浓缩反应液，加入水，乙酸乙酯萃取，合并，干燥，旋干获得粗品。粗品经制备TLC纯化(乙酸乙酯/石油醚＝3/1，V/V)获得白色固体产品44mg，产率为19.1％。
LC-MS(ESI)m/z:456[M+H] +。
1H NMR(600MHz,CDCl 3)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).
对比化合物的合成
制备H
Figure PCTCN2021081904-appb-000059
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-4-氨基-5-硝基嘧啶
将化合物2-氯-4-氨基-5-硝基嘧啶(1.0g，5.7mmol)、化合物(R)-2-(2,5-二氟苯基)吡咯烷盐酸盐(1.2g，5.7mmol)和DIEA(1.5g，11.4mmol)溶于25mL NMP中，在180℃条件下搅拌反应2小时。TLC检测确定反应结束后，加入水，用乙酸乙酯萃取，合并有机相，干燥，旋蒸浓缩获得粗品。粗品经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)获得产品1.8g，黄色固体，产率98.4％。
LC-MS(ESI)m/z:322[M+H] +。
制备I：
Figure PCTCN2021081904-appb-000060
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(二苯氧甲酰基)氨基嘧啶
将中间体H(1.8g，6.1mmol)溶于20ml吡啶中，逐滴加入氯甲酸苯酯(2.9g，18.6mmol)，然后在70℃下搅拌反应5小时。TLC检测确定反应结束后，旋蒸浓缩除去溶剂，加入50ml水淬灭反应，用乙酸乙酯(3×50ml)萃取，合并有机相，干燥，旋干获得粗品。经硅胶柱纯化后(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品2.5g，产率73.1％。
LC-MS(ESI)m/z:562[M+H] +。
制备M11
Figure PCTCN2021081904-appb-000061
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(3-(3-氟苄基)脲基)嘧啶
将中间体I(300mg，0.53mmol)，间氟苄胺(125mg，1.06mmol)和碳酸钠(158mg，1.59mmol)悬浮于10mL乙腈中，室温搅拌12小时。TLC检测反应结束后，旋蒸浓缩，经硅胶柱纯化(乙酸乙酯/石油醚＝1/3，V/V)，获得黄色固体产品210mg，产率为84.1％。LC-MS(ESI)m/z:473[M+H] +。
对比例1
Figure PCTCN2021081904-appb-000062
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-(3-氟苄基)-9H-嘌呤-9-甲酰胺
将中间体M11(210mg，0.44mmol)溶于5mL THF中，加入10％钯碳(21mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(21mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品55mg，产率23.6％。
LC-MS(ESI)m/z:453[M+H] +
1H NMR(600MHz,CDCl 3)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)。
对比例2
Figure PCTCN2021081904-appb-000063
(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((四氢-2H-吡喃-4-基)甲基)-9H-嘌呤-9-甲酰胺
步骤A：(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(3-((四氢-2H-吡喃-4-基)甲基)脲基)嘧啶的制备。用制备M11的方法，使用4-氨甲基四氢吡喃代替间氟苄胺进行制备。
步骤B：(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-N-((四氢-2H-吡喃-4-基)甲基)-9H-嘌呤-9-甲酰胺的制备。将(R)-2-(2-(2,5-二氟苯基)吡咯烷-1-基)-5-硝基-4-(3-((四氢-2H-吡喃-4-基)甲基)脲基)嘧啶(250mg，0.48mmol)溶于5mL THF中，加入10％钯碳(25mg)，置换氢气后，室温搅拌12小时。TLC检测反应结束后，过滤，旋干获得粗品。将粗品溶于5mL原甲酸三乙酯中，加入对甲苯磺酸(25mg)，在135℃下反应4小时。TLC检测反应结束后，浓缩反应液，制备TLC纯化(乙酸乙酯/石油醚＝1/1，V/V)获得白色固体产品65mg，产率27.6％。
LC-MS(ESI)m/z:443[M+H] +
1H NMR(600MHz,CDCl 3)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).
活性实验
CTG细胞存活试验：
ATP是活细胞代谢活动中所必须产生的，其含量与活细胞数量存在线性关系。CTG化学发光细胞活力检测实验就是基于这个原理，是检测培养细胞中存活细胞数的通用方法。加入CellTiter-Glo(CTG)试剂后可诱导细胞裂解并产生与孔板中ATP数量成正比例关系的化学发光信号，从而可以通过化学发光的读数来衡量孔板中细胞增殖的活力。
实验材料与仪器
实验材料：
上述实施例制备的待测化合物，RPMI Medium 1640(HyClone，Cat#SH30809.01)，胎牛血清(FBS，GBICO，Cat#10099-141)，磷酸盐缓冲液(Solarbio，Cat#P1020-500)，
Figure PCTCN2021081904-appb-000064
Luminescent Cell Viability Assay(Promega,Cat#G7572)，96孔平底底透黑板(NUNC，Cat#165305)，T25培养瓶(NUNC，Cat#156367)，T75培养瓶(NUNC，Cat#156439)。
本发明所使用的细胞系见表1。
表1细胞系及培养基
细胞系 培养基
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
实验仪器：
二氧化碳培养箱，SANYO-MCO-20AIC，生物安全柜：BSC-1360-LIIA2，台式高速冷冻离心机：SorvallST 16R，微孔板快速振荡器：QB-9001，M3读板机：SpectraMax M3，显微镜：OLYMPUS-CKX41/CKX31。
实验步骤：
1.细胞复苏：
从液氮容器中取出冻存管，直接浸入37℃水浴中，并快速摇动令其尽快融化，吸出细胞悬液，加到离心管；离心，弃去上清液，加入含细胞生长培养基重悬细胞，将全部细胞悬液接种至培养瓶，细胞培养箱静置培养。
2.细胞传代：
待细胞生长至对数生长期，培养箱中取出细胞至生物安全柜中，将细胞悬液转移至离心管中离心，将细胞制成细胞悬液后按照一定比例将细胞传代和实验用。
3.细胞铺板
收获处于对数生长期的细胞并采用血小板计数器进行细胞计数。用台盼蓝排斥法检测细胞活力，调整细胞浓度，进行细胞铺板。将细胞板置于细胞培养箱中培养过夜。
4.药物稀释和加药：
配制药物工作溶液，药物浓度由高到低，3.16倍稀释，在接种有细胞的96孔板中每孔加入药物工作溶液，每个药物浓度三个复孔。将已加药的96孔板中的细胞置于细胞培养箱中继续培养，之后进行CTG分析。
5.终点读板：
预先融化CTG试剂。每孔加入等体积的CTG溶液。在微孔板快速振荡器上振动使细胞裂解。将细胞板放置于室温稳定冷光信号。读取冷光值。
6.数据处理
使用GraphPad Prism 5.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC50值。
实验结果见表2。
表2本发明化合物对突变型TRK酶的抑制效果(IC 50)
Figure PCTCN2021081904-appb-000065
Figure PCTCN2021081904-appb-000066
可以看出一代Trk抑制剂LOXO-101对突变型Trk酶的IC50值达到了1000以上，而本申请提供的Trk抑制剂对于野生型Trk激酶和突变型Trk激酶都具有较高的抑制活性，部分实施例至少对一种突变型Trk酶的IC50值低于对野生型Trk酶的IC50值，说明本申请提供的化合物对突变型Trk酶具有选择性。而咪唑并嘧啶或咪唑并吡嗪虽然是咪唑并吡啶的生物电子等排体，但是他们的衍生物确达不到本发明提供的化合物的效果。
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。

Claims (23)

  1. 一种咪唑并吡啶类衍生物，具有式A的结构
    Figure PCTCN2021081904-appb-100001
    或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其中，
    X、Y或Z其中一个为N，其余的为CH；
    R 3和R 4各自独立地选自C 1-3烷基或H；当R 3和R 4均为烷基时，R 3和R 4可进一步连接成环；
    R 1选自未取代或被一个或多个取代基取代的5-6元单环芳基或5-6元单环杂芳基；
    R 2选自烷基或杂环烷基，所述R 2未被取代或被一个或多个取代基取代。
  2. 根据权利要求1所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 3为C 1-3烷基，R 4为H。
  3. 根据权利要求1所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 3和R 4均为烷基时，R 3和R 4连接成五元环。
  4. 根据权利要求3所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，式A具有下式A 1的结构
    Figure PCTCN2021081904-appb-100002
  5. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 1中所述取代基各自独立地选自卤素、C 1-4烷氧基、-CF 3、-CHF 2、-OC 1-4烷基OC 1-3烷基或-OC 3-6二羟烷基。
  6. 根据权利要求5所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 1中所述取代基各自独立地选自卤素或C 1-4烷氧基。
  7. 根据权利要求6所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，所述R 1选自苯基、3-氟苯基、2，5-二氟苯基、2-氯-5-氟苯基、2-甲氧苯基、2-甲氧基-5-氟苯基、2-三氟甲基-5-氟苯基、2- 二氟甲基-5-氟苯基、3-氯-5-氟苯基、3-氟-5-甲氧基乙氧基苯基或5-氟-2-甲氧基乙氧基苯基、5-氟-吡啶-3-基、2,5-二氟-吡啶-3-基、2-甲氧基-吡啶-3-基、2-甲氧基-5-氟-吡啶-3-基或2-三氟甲基-5-氟-吡啶-3-基。
  8. 根据权利要求7所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，所述R 1选2，5-二氟苯基、2-甲氧基-5-氟苯基或2-甲氧基-5-氟-吡啶-3-基。
  9. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2中所述烷基选自直链烷基或单环烷基。
  10. 根据权利要求9所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2选自C 1-6直链烷基、C 4-8单环烷基或4-8元杂单环烷基，所述R 2未被取代或被一个或多个取代基取代。
  11. 根据权利要求10所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2选自C 1-3直链烷基、C 5-6单环烷基或5-6元杂单环烷基，所述R 2未被取代或被一个或多个取代基取代。
  12. 根据权利要求9所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2中所述杂单环烷基为含有一个或多个杂原子的5-6元杂单环烷基，所述杂原子各自独立地选自N、O或S。
  13. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2中所述取代基选自酰氧基、羟基烷基、羟基、甲氧基、卤素、卤代烷、甲基、未取代或被一个或多个取代基取代的芳基、未取代或被一个或多个取代基取代的杂芳基未取代或被一个或多个取代基取代的环烷基、或未取代或被一个或多个取代基取代的杂环烷基。
  14. 根据权利要求13所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2中所述取代基选自甲酰氧基、羟基-C 2-烷基、羟基、甲氧基；
    或未取代或被一个或多个取代基取代的芳基，其中被一个或多个取代基取代的芳基中，所述取代基各自独立地选自F或CF 3；
    或未取代或被一个或多个取代基取代的杂芳基，其中被一个或多个取代基取代的杂芳基中，所述取代基选自甲基；
    或未取代或被一个或多个取代基取代的环烷基，其中被一个或多个取代基取代的环烷基 中，所述取代基选自甲酰氧基、羟基；
    或未取代的杂环烷基。
  15. 根据权利要求14所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，R 2中所述取代基选自甲酰氧基、羟基-C 2烷基、羟基、甲氧基；
    或未取代或被一个或多个取代基取代的苯基，其中被一个或多个取代基取代的苯基中，所述取代基各自独立地选自F或CF 3；
    或未取代或被一个或多个取代基取代的杂芳基，所述杂芳基为含有一个或多个杂原子的5-6元杂芳基，所述杂原子各自独立地选自N、O或S，所述被一个或多个取代基取代的杂芳基中，所述取代基选自甲基；
    或未取代或被一个或多个取代基取代的C 5-6环烷基，其中被一个或多个取代基取代的环烷基中，所述取代基选自甲酰氧基、羟基；
    或未取代的杂环烷基，所述杂环烷基为含有一个或多个杂原子的5-6元杂环烷基，所述杂原子各自独立地选自N、O或S。
  16. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，所述R 2选自以下结构中的一种：
    Figure PCTCN2021081904-appb-100003
  17. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，所述式A具有如式A 11所示的绝对构型
    Figure PCTCN2021081904-appb-100004
  18. 根据权利要求1-4任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物，其特征在于，所述式A化合物选自以下结构中的一种：
    Figure PCTCN2021081904-appb-100005
    Figure PCTCN2021081904-appb-100006
  19. 一种如权利要求1-18任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物的制备方法，包括以下步骤中的至少一步，如下式所示，
    Figure PCTCN2021081904-appb-100007
    步骤a)将A a和
    Figure PCTCN2021081904-appb-100008
    在碱性条件下反应得到A b，其中L选自氯、溴或碘；
    步骤b)将A b和氯甲酸苯酯或氯甲酸苯酯衍生物在碱性条件下反应得到A c；
    步骤c)将A c和R 2-NH 2在碱性条件下反应得到A d；
    步骤d)将A d经过还原得到A e；
    步骤e)将A e环化得到A。
  20. 一种药物组合物，包含权利要求1-18任一项所述的衍生物，或其互变异构体、立体 异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物或权利要求19所述的制备方法制备得到的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物与药学上可接受的稀释剂或载体混合。
  21. 一种如权利要求1-18任一项所述的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物或权利要求19所述的制备方法制备得到的衍生物，或其互变异构体、立体异构体、对映异构体、非对映异构体、药学上可接受的盐、前药或N-氧化物的应用，所述应用是制备用于治疗和/或预防由TRK酶介导的疾病和相关病症的药物。
  22. 根据权利要求21所述的应用，其特征在于，所述疾病已经对至少一种TRK酶治疗活性剂产生至少部分耐药性。
  23. 根据权利要求21所述的应用，其特征在于，所述疾病选自疼痛或癌症中的一种。
PCT/CN2021/081904 2021-03-08 2021-03-19 一种咪唑并吡啶类衍生物 WO2022188203A1 (zh)

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