WO2022148317A1 - 2-氨基嘧啶类化合物及其药物组合物和应用 - Google Patents

2-氨基嘧啶类化合物及其药物组合物和应用 Download PDF

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WO2022148317A1
WO2022148317A1 PCT/CN2021/143765 CN2021143765W WO2022148317A1 WO 2022148317 A1 WO2022148317 A1 WO 2022148317A1 CN 2021143765 W CN2021143765 W CN 2021143765W WO 2022148317 A1 WO2022148317 A1 WO 2022148317A1
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alkyl
substituted
pharmaceutically acceptable
halogen
metabolites
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French (fr)
Inventor
丁克
李姗
张章
司鸿飞
涂正超
任小梅
雷冲
唐霞
高悦译
陈成斌
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Guangzhou Lixin Pharmaceutical Co Ltd
Jinan University
University of Jinan
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Guangzhou Lixin Pharmaceutical Co Ltd
Jinan University
University of Jinan
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Priority to AU2021416682A priority Critical patent/AU2021416682B2/en
Priority to EP21917356.4A priority patent/EP4279486A4/en
Priority to CN202180005255.5A priority patent/CN115087641B/zh
Priority to JP2023541526A priority patent/JP7621624B2/ja
Publication of WO2022148317A1 publication Critical patent/WO2022148317A1/zh
Priority to US18/343,732 priority patent/US20230348487A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems

Definitions

  • the invention relates to the technical field of chemical medicine, in particular to a class of 2-aminopyrimidine compounds and their pharmaceutical compositions and applications.
  • protein tyrosine kinases Since the late 1990s, protein tyrosine kinases have received increasing attention as emerging targets. Under normal circumstances, this type of protein with tyrosine kinase activity binds to ATP and phosphorylates on tyrosine residues at specific positions, which then causes the activation and transduction of important intracellular signaling pathways and participates in the regulation of cellular processes. Division, growth, proliferation, differentiation, aging, apoptosis and other life processes. The dysregulation of tyrosine kinases can lead to cell dysfunction, leading to a series of diseases in the body, including tumors and inflammatory diseases. Therefore, targeting protein tyrosine kinases has become an important aspect of precision medicine.
  • Protein tyrosine kinases include receptor tyrosine kinases and non-receptor tyrosine kinases.
  • the protein structure of receptor tyrosine kinases includes an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and an intracellular tyrosine kinase catalytic domain and regulatory sequences, rather than the subcellular localization of receptor tyrosine kinases Unlike receptor tyrosine kinases, it does not include extracellular and transmembrane structures and is a class of cytoplasmic tyrosine kinase proteins.
  • non-receptor tyrosine kinase After the non-receptor tyrosine kinase is activated in the cell, it binds to downstream signaling molecules and activates it, phosphorylates it, and exerts tyrosine kinase activity.
  • Janus kinase (JAK for short) is a non-receptor tyrosine kinase, which includes four family members: JAK1, JAK2, JAK3 and Tyk2.
  • this protein is named after the two-sided Roman god Janus (Janus). ) name.
  • extracellular specific ligands such as cytokines, drivers, growth factors, etc.
  • STATs signal transducers and activators of transcription
  • Phosphorylated STATs undergo homodimerization or heterodimerization and then translocate to the nucleus, where they bind to specific DNA binding sites and regulate gene transcription, resulting in changes in cellular functions.
  • JAK3 is only expressed in bone marrow, lymphoid and other tissue cells related to the hematopoietic system.
  • interleukins IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 specifically bind to receptors containing ⁇ c chain (common gamma chain) receptor subunits, it will cause Changes in receptor conformation, thereby promoting JAK3-related receptors and JAK1-related receptors to approach each other and undergo dimerization and autophosphorylation.
  • Activated JAK3 protein recruits and phosphorylates STAT1, STAT3, STAT5, or STAT6.
  • STATs are transferred to the nucleus, affecting the transcription and translation of downstream targeted DNA, and participating in lymphocytes, T cells, and B cells. and NK cell growth, proliferation, development and differentiation, as well as important life processes such as immune regulation.
  • Robust clinical studies have found that loss of function of JAK3 in humans causes severe combined immunodeficiency (SCID), while overactivation or mutation is associated with many autoimmune diseases and cancers (especially blood cancers such as leukemia) .
  • SCID severe combined immunodeficiency
  • overactivation or mutation is associated with many autoimmune diseases and cancers (especially blood cancers such as leukemia) .
  • the study found that the JAK3-STAT5 signaling cascade is an important signaling pathway in the process of hematopoiesis.
  • T-lymphocytic leukemia T-ALL
  • JAK3 is one of the most important signaling pathways in this pathway.
  • the most commonly mutated gene accounting for approximately 16.1% of T-ALL cases (Haematologica 2015, 100, 1301-1310).
  • JAK3(M511I) exhibited cytokine-independent cell proliferation and transformation ability in Ba/F3 cells;
  • the mice showed obvious clinical symptoms of T-ALL (dramatic increase in the number of white blood cells, enlargement of the spleen, thymus and lymph nodes, increased CD8+ T cells in peripheral blood and hematopoietic tissues, etc.), while the transplanted mice expressing wild-type JAK3 cells did not.
  • Onset (Blood 2014, 124, 3092-3100)).
  • JAK3 has become a potential new target for the treatment of hematological tumors, and the development of small-molecule inhibitors targeting JAK3 will provide an important strategy for alleviating or treating related diseases.
  • JAK inhibitors have been approved for marketing in the United States, the European Union or Japan, namely: ruxolitinib (ruxolitinib, a JAK1/JAK2 inhibitor, approved by the FDA in 2011), tofacitinib ( Pan-JAK inhibitor, approved by FDA in 2012), baricitinib (baricitinib, JAK1/JAK2 inhibitor, approved by EU and FDA in 2017 and 2018), peficitinib (pan-JAK inhibitor, released in Japan in 2019) Approved for listing in 2019), fizotinib (fedratinib, a JAK2 inhibitor, approved by the FDA in 2019), upadacitinib (upadacitinib, a JAK1 inhibitor, approved by the FDA in 2019), delgocitinib (delgocitinib, Pan-JAK inhibitor, approved for marketing in Japan in 2020), filgotinib (JAK1 inhibitor, approved for marketing in EU and Japan in 2020).
  • the five drugs approved by the FDA are all black-boxed due to serious side effects that may be clinically caused warn. Therefore, the development of highly selective JAK3 inhibitors can effectively reduce the toxic and side effects by reducing the interference to many cytokine pathways unrelated to the disease while maintaining the efficacy.
  • JAK3 has a higher ATP affinity relative to other family members, making the development of selective JAK3 inhibitors challenging, but not impossible accomplish.
  • JAK3 kinase contains a unique cysteine residue (Cys909) that has lipophilic function and can covalently bind to nucleophiles, while in the other three JAK subtypes The equivalent position of the type is serine.
  • JAK3 irreversible JAK3 inhibitor
  • PF-06651600 the IC50 value of the kinase level at the ATP concentration of 1mM: JAK3 is 33.1nM, other isoforms are all greater than 10 ⁇ M
  • the compound is currently in phase III clinical trials for the treatment of alopecia areata, and has been approved by the US FDA as a breakthrough therapy.
  • JAK3 selective inhibitor for clinical use, and it is of great clinical significance to develop high-activity, low-toxicity JAK3 small molecule inhibitors for the treatment of inflammatory diseases or hematological tumors.
  • the present invention provides a new class of 2-aminopyrimidine compounds, which can selectively inhibit the activity of JAK3 kinase with high activity, thereby inhibiting the growth of various tumor cells. Proliferation can be used to treat tumors or inflammatory diseases associated with JAK3 kinase. Specifically, the following technical solutions are included.
  • 2-aminopyrimidine compounds having the structure represented by formula (I) or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or Its prodrug molecules and metabolites:
  • R 1 is selected from: H, halogen, cyano, one or more R 11 substituted or unsubstituted C 1 -C 6 alkyl, one or more R 11 substituted or unsubstituted C 3 -C 6 cycloalkyl , one or more R 11 substituted or unsubstituted C 1 -C 6 alkoxy, one or more R 11 substituted or unsubstituted C 3 -C 6 cycloalkoxy, carboxamide;
  • n 0 or 1
  • L is O or S
  • each R 10 Each independently selected from: hydrogen, halogen, hydroxyl, one or more R 11 substituted or unsubstituted C 1 -C 3 alkyl, one or more R 11 substituted or unsubstituted C 1 -C 3 alkoxy , the heteroatom is O, S and/or N;
  • the configurations of the chiral carbon atoms marked with * are independently S configuration or R configuration.
  • the configurations of the chiral carbon atoms marked with * are independently S configuration or R configuration, and each R 10 is independently selected from: halogen, hydroxyl, C 1 -C 3 alkyl, C 1 -C 3 alkoxy.
  • each R 10 is independently selected from: F, hydroxyl, methyl, methoxy, ethoxy, and isopropoxy.
  • R 6 is selected from: hydrogen, halogen, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, -NH-CN,
  • W and X are both CH; Y and Z are independently selected from N or CR 6 , wherein R 6 is selected from: H, halogen, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, -NH-CN,
  • W and X are both CH; Y is CR 6 , wherein R 6 is selected from: H, halogen, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, Z is CH or N.
  • W, X, and Z are all CH; Y is CR 6 , wherein R 6 is:
  • R 2 is selected from: H, halogen, -(CH 2 ) m NR 3 R 4 , -(CH 2 ) m CR 3 R 4 R 5 ; wherein each m is independently 0, 1, 2 or 3;
  • Each R 3 and each R 4 is independently selected from: H, one or more R 12 substituted C 1 -C 3 alkyl groups, or R 3 , R 4 and the N or C to which they are attached together form one or more The 5-11-membered monocyclic, fused, bridged or spiro ring containing 0, 1, 2 or 3 heteroatoms in the ring atom substituted by R 12 ;
  • R 5 is selected from: H, cyano or C 1 -C 3 alkyl
  • Each R 12 is independently selected from: H, hydroxy, acetyl, R 13 substituted or unsubstituted 4-8 membered heterocyclyl, halogen, hydroxy, amino, C 1 -C 3 alkyl, C 1 -C 3 Alkoxy, -NHR 13 , -N(R 13 ) 2 ; R 13 is C 1 -C 3 alkyl.
  • R 2 is selected from the group consisting of: H, halogen, -(CH 2 ) m NR 3 R 4 , -(CH 2 ) m CR 3 R 4 R 5 ; wherein each m is independently 0 or 1;
  • Each R 3 and each R 4 are independently selected from: H, C 1 -C 3 alkyl, hydroxy substituted C 1 -C 3 alkyl, -NHR 13 substituted C 1 -C 3 alkyl, -N( R 13 ) 2 substituted C 1 -C 3 alkyl, or R 3 , R 4 and the N or C to which they are attached together form one or more R 12 substituted ring atoms containing 0, 1, 2 or 3 heterocyclic atoms A 5- to 11-membered monocyclic, bridged or spirocyclic ring;
  • R 5 is selected from: H, cyano or C 1 -C 3 alkyl
  • Each R 12 is independently selected from: H, hydroxy, acetyl, R 13 substituted piperazinyl, C 1 -C 3 alkyl, -NHR 13 , -N(R 13 ) 2 ; R 13 is C 1 - C3 alkyl.
  • R 2 is selected from the group consisting of: H, halogen,
  • R 1 is selected from the group consisting of: H, halogen, cyano, carboxamido, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 3 -C6cycloalkoxy, haloC1 - C6alkyl , haloC1 - C6alkoxy.
  • R 1 is selected from the group consisting of: H, halogen, C 1 -C 3 alkyl, C 1 -C 3 alkoxy.
  • R 1 is selected from the group consisting of: H, halogen, methyl, cyano, carboxamido, trifluoromethyl, difluoromethyl, methoxy, cyclopropyl, trifluoromethoxy.
  • the 2-aminopyrimidine compound has the structure shown in the following formula (II):
  • the present invention also provides the above-mentioned 2-aminopyrimidine compounds or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or their precursors Application of drug molecules and metabolites.
  • the tumor is a hematological tumor and a solid tumor
  • the hematological tumor is multiple myeloma, B lymphoma, myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid Leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, histiocytic lymphoma, acute megakaryocyte leukemia, prolymphocytic leukemia, T lymphoblastic leukemia, T lymphoblastic lymphoma; said solid tumor
  • non-small cell lung cancer small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, nasopharyngeal cancer, glioma
  • the inflammatory disease is rheumatoid arthritis, atopic fibrosis,
  • the present invention also provides a pharmaceutical composition for preventing and treating tumors and/or inflammatory diseases.
  • a pharmaceutical composition for preventing and treating tumors and/or inflammatory diseases prepared from active ingredients and pharmaceutically acceptable carriers or auxiliary materials, the active ingredients include the above-mentioned 2-aminopyrimidine compounds or their pharmaceutically acceptable
  • the present invention provides a class of 2-aminopyrimidine compounds with novel structures or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or Its prodrug molecules and metabolites can inhibit the kinase activity of Janus Kinase 3 (JAK3) with high efficiency and selectivity.
  • the cells have strong signal inhibitory effects and cell proliferation inhibition effects, and can be used for preparing anti-tumor and therapeutic drugs for inflammatory diseases related to JAK3 kinase.
  • Figure 1 shows the single crystal structure of Intermediate 3-1.
  • FIG. 2 shows the single crystal structure of Intermediate 9.
  • Figure 3 shows the results of the pharmacokinetic experiment of compound LS6-45.
  • Figure 4 shows that compound LS6-45 inhibits the activation of JAK3 pathway in U937 cells.
  • Figure 5 shows the results of the elution experiment of compound LS6-45.
  • Figure 6 shows the effect of compound LS6-45 on U937 cell cycle arrest and apoptosis.
  • Figure 7 shows the results of in vivo antitumor activity of the test compound LS6-45 in mice.
  • any variable eg, R3 , R4, etc.
  • the definition of each occurrence is independent of the definition of each other.
  • combinations of substituents and variables are permissible so long as such combinations stabilize the compound.
  • a line drawn into a ring system from a substituent indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atoms adjacent to the ring. It will be appreciated that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the present invention to provide compounds that are chemically stable and readily synthesized from readily available starting materials by the skill of the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stabilized.
  • alkyl as used herein is meant to include branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
  • the definition of “ C1 - C6 " in " C1 - C6 alkyl” includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain arrangement.
  • “ C1 - C6 alkyl” specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.
  • cycloalkyl refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms.
  • cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and the like.
  • alkoxy refers to groups having an -O-alkyl structure, such as -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 , -O-CH 2 CH(CH 3 ) 2 , - OCH 2 CH 2 CH 2 CH 3 , -O-CH(CH 3 ) 2 and the like.
  • heterocyclyl is a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent wherein one or more ring atoms are selected from N, O or S(O)m (where m is an integer from 0 to 2 ), the remaining ring atoms are carbon, such as: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydro oxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl , dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidine,
  • halo means chlorine, fluorine, bromine and iodine.
  • alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl substituents can be unsubstituted or substituted.
  • a C1 - C6 alkyl group can be substituted with one, two, or three substituents selected from OH, halogen, alkoxy, dialkylamino, or heterocyclyl groups such as morpholinyl, piperidinyl, and the like.
  • the present invention provides a 2-aminopyrimidine compound having the structure represented by formula (I):
  • R 1 is selected from: H, halogen, cyano, one or more R 11 substituted or unsubstituted C 1 -C 6 alkyl, one or more R 11 substituted or unsubstituted C 3 -C 6 cycloalkyl , one or more R 11 substituted or unsubstituted C 1 -C 6 alkoxy, one or more R 11 substituted or unsubstituted C 3 -C 6 cycloalkoxy, carboxamide;
  • n 0 or 1
  • L is O or S
  • each R 10 Each independently selected from: hydrogen, halogen, hydroxyl, one or more R 11 substituted or unsubstituted C 1 -C 3 alkyl, one or more R 11 substituted or unsubstituted C 1 -C 3 alkoxy , the heteroatom is O, S and/or N;
  • the present invention includes free forms of compounds of formula I or II, as well as pharmaceutically acceptable salts and stereoisomers thereof.
  • Some of the specific exemplary compounds herein are protonated salts of amine compounds.
  • the term "free form" refers to the amine compound in non-salt form.
  • Included pharmaceutically acceptable salts include not only exemplary salts of the particular compounds described herein, but also typical pharmaceutically acceptable salts of all compounds of formula I or II in free form.
  • the free forms of particular salts of the compounds can be isolated using techniques known in the art.
  • the free form can be regenerated by treating the salt with a suitable dilute aqueous base, such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate.
  • a suitable dilute aqueous base such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate.
  • the free forms differ somewhat from their respective salt forms in certain physical properties such as solubility in polar solvents, but for the purposes of the invention such acid and base salts are otherwise pharmaceutically equivalent to their respective free forms.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods.
  • salts of basic compounds are prepared by ion exchange chromatography or by reacting the free base and a stoichiometric amount or excess of an inorganic or organic acid in the desired salt form in a suitable solvent or combination of solvents.
  • salts of acidic compounds are formed by reaction with a suitable inorganic or organic base.
  • pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting a basic compound of the present invention with an inorganic or organic acid.
  • conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as those derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, hard Fatty acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetyl Salts prepared from oxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulf
  • salts derived from inorganic bases include aluminum salts, ammonium salts Salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Ammonium, calcium, magnesium, potassium and sodium salts are particularly preferred.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as Amino acid, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethyl Diamine, N-Ethylmorpholine, N-Ethylpiperidine, Glucosamine, Glucosamine, Histidine, Hydroxocobalamin, Isopropylamine, Lysine, Methylglucamine, Morpholine, Piperazine , piperidine, quack, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.
  • deprotonated acidic moieties such as carboxyl groups in compounds can be anionic under physiological conditions, such charged charges can then be replaced by internally cationic protonated or alkylated basic moieties such as tetravalent
  • the nitrogen atoms balance out, so it should be noted that the compounds of the present invention are potential inner salts or zwitterions.
  • the present application provides a method for treating tumors or inflammatory diseases in humans or other mammals with a compound of Formula I or Formula II and pharmaceutically acceptable salts thereof.
  • the compounds of the present application and pharmaceutically acceptable salts thereof can be used to treat or manage multiple myeloma, B lymphoma, myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloma Lineage leukemia, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, histiocytic lymphoma, acute megakaryocytic leukemia, prolymphocytic leukemia, T-lymphocytic leukemia, T-lymphoblastic lymphoma, non-small cell Lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell carcinoma, gastrointestinal stromal tumor, nasopharyngeal carcinoma, glioma, rheumatoid joint inflammation, atopic derma
  • the present invention also provides a pharmaceutical composition, which comprises an active ingredient in a safe and effective amount, and a pharmaceutically acceptable carrier or adjuvant.
  • the "active ingredient" in the present invention refers to the compound of formula I or formula II described in the present invention or a pharmaceutically acceptable salt, isotopic derivative, solvate thereof, or a stereoisomer, geometric isomer, Tautomers, or their prodrug molecules, metabolites.
  • the "active ingredients" and pharmaceutical compositions of the present invention can be used as JAK protein kinase inhibitors, and can be used to prepare medicaments for preventing and/or treating tumors and/or inflammatory diseases.
  • a “safe and effective amount” refers to an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of active ingredient/dose, more preferably 10-200 mg of active ingredient/dose.
  • the "one dose” is one tablet.
  • “Pharmaceutically acceptable carrier or excipient” means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity.
  • composition means that the components of the composition can be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.
  • Examples of pharmaceutically acceptable carriers or excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as hard Fatty acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate,
  • the compound of formula I or formula II of the present invention can form a complex with a macromolecular compound or macromolecule through non-bonding interaction.
  • the compound of formula I or formula II of the present invention as a small molecule, can also be linked with a macromolecular compound or a macromolecule through chemical bonds.
  • the macromolecular compounds can be biological macromolecules such as polysaccharides, proteins, nucleic acids, polypeptides, and the like.
  • the mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following:
  • fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid;
  • binders such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia;
  • humectants for example, glycerin
  • disintegrating agents for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate;
  • absorption accelerators for example, quaternary amine compounds
  • humectants such as cetyl alcohol and glyceryl monostearate
  • adsorbents for example, kaolin
  • Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof.
  • the dosage form may also contain buffering agents.
  • the solid dosage forms can also be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances and the like.
  • the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the compounds of the present invention may be administered alone or in combination with other therapeutic agents such as hypoglycemic agents.
  • a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is
  • the administration dose is usually 1 to 2000 mg, preferably 20 to 500 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the compounds of Formula I or Formula II may be used in combination with other drugs known to treat or ameliorate similar conditions.
  • the administration mode and dosage of the original drug remain unchanged, while the compound of formula I or formula II is administered simultaneously or subsequently.
  • a pharmaceutical composition containing one or more known drugs and a compound of formula I or formula II it is preferred to use a pharmaceutical composition containing one or more known drugs and a compound of formula I or formula II.
  • Drug combinations also include administration of a compound of formula I or formula II with one or more other known drugs at overlapping time periods.
  • the dose of the compound of formula I or formula II or known drugs may be lower than when they are administered alone.
  • Drugs or active ingredients that can be used in combination with the compounds of formula I or II include, but are not limited to:
  • the drugs or active ingredients that can be used in combination with the compounds of formula I or II include, but are not limited to: aldesleukin, alendronate, interferon, atranoin, allopurinol, Sodium allopurinol, Palonosetron hydrochloride, Hexamethylmelamine, Aminoglutide, Amifostine, Amrubicin, Amridine, Anastrozole, Dorasetron, aranesp, arglabin , Arsenic trioxide, Arnosine, 5-azacytidine, azathioprine, BCG or tic BCG, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromine Urethane, bortezomib, busulfan, calcitonin, alezolizumab injection, capecitabine, carboplatin, casodex, cefesone, simole
  • a novel 2-aminopyrimidine compound is provided.
  • the compounds have a strong selective inhibitory effect on JAK3 kinase, can effectively inhibit the growth of various tumor cells, and can be used to prepare antitumor drugs.
  • the compounds can effectively prevent the transmission of the JAK3-STAT5 signaling pathway, and can be used for the preparation of drugs for anti-inflammatory diseases.
  • Reduced iron powder (1.42g, 25.3mmol) and ammonium chloride (4.6g, 84.3mmol) were added to the mixed solvent of ethanol/water (volume ratio 2:1) of intermediate 7 (2.85g, 8.43mmol), refluxed React for 2 hours. After the reaction was completed, it was cooled to room temperature, filtered through celite, and most of the solvent was spin-dried, extracted with dichloromethane three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, spin-dried, and subjected to column chromatography. Solid 4 (2.2 g, 87% yield) was isolated.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol is used instead of isosorbide (2) to participate in the reaction.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 4-fluoro-2-methoxy-1-nitrobenzene is used instead of 2-chloro-1-fluoro-4-nitrobenzene (5) to participate in the reaction.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1-fluoro-2-methoxy-4-nitrobenzene is used instead of 2-chloro-1-fluoro-4-nitrobenzene (5), and N- Methylpiperazine was used in the reaction instead of 1-methyl-4-(piperidinyl-4-yl)piperazine hydrochloride (6).
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 1-(4-aminophenyl)cyclopentane-1-methyl Nitrile was involved in the reaction in place of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4).
  • the synthetic method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 4-(4-methylpiperazin-1-yl)aniline 3-Chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) was involved in the reaction.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 3-chloro-4-(4-methylpiperazine-1- yl)aniline instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) was involved in the reaction.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 4-(4-(4-methylpiperazin-1-yl) ) piperidin-1-yl)aniline instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) participated in the reaction.
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 3-methoxy-4-(4-(4-methyl) Piperazin-1-yl)piperidin-1-yl)aniline instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) participated in the reaction .
  • the synthetic method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 2-chloro-4-(4-(4-methylpiperazine) -1-yl)piperidin-1-yl)aniline was involved in the reaction instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4).
  • the synthesis method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 3,5-dichloro-4-(4-(4-methyl) Piperazin-1-yl)piperidin-1-yl)aniline instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) reaction.
  • the synthetic method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol replaces isosorbide (2), 5-methyl-6-(4-(4-methylpiperidine) Azin-1-yl)piperidin-1-yl)pyridin-3-amino instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4 )Reacted.
  • the synthetic method is the same as that of Example 1LS 3-96, except that 1,4:3,6-dianhydromannitol is used instead of isosorbide (2), 5-((4-ethylpiperazin-1-yl)methane yl)pyridin-2-amino instead of 3-chloro-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (4) was involved in the reaction.
  • the synthesis method is the same as the synthesis of compound 3-1 in Example 1, except that 1,4:3,6-dianhydromannitol (8) is used instead of isosorbide (2) to participate in the reaction.
  • the synthesis method is the same as the synthesis of compound 11, except that the raw material 14 is used instead of m-nitroaniline (10) to participate in the reaction.
  • Reduced iron powder (9 mg, 0.16 mmol) and ammonium chloride (3 mg, 0.055 mmol) were added to the mixed solvent of ethanol/water (volume ratio 2:1) of intermediate 17 (32 mg, 0.055 mmol), and the reaction was refluxed for 2 hours . After the reaction was completed, it was cooled to room temperature, filtered through celite, and most of the solvent was spin-dried, extracted with dichloromethane three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, spin-dried, and subjected to column chromatography. 18 was isolated as a light grey solid.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 4-fluoro-2-methoxy-5-nitroaniline is used instead of 4-fluoro-3-nitroaniline (14) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that morpholine is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 2-methylaminoethanol is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 2-butenoic acid is used instead of acrylic acid (13) to participate in the reaction.
  • the synthesis method is the same as the synthesis of Example 16LS 5-12, except that methacrylic acid is used instead of acrylic acid (13) to participate in the reaction.
  • the synthesis method is the same as the synthesis of Example 16LS 5-12, except that 4-bromocrotonic acid is used instead of acrylic acid (13) to participate in the reaction, and the obtained intermediate 19 and 2M dimethylamine tetrahydrofuran solution are substituted to obtain.
  • the synthetic method is the same as that of Example 16LS 5-12, except that N,N,N'-trimethylethylenediamine replaces 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6 )Reacted.
  • the synthetic method is the same as that of Example 16LS 5-12, except that 3-methyl-3,9-diazaspiro[5,5]undecane is used instead of 1-methyl-4-(piperidin-4-yl ) piperazine hydrochloride (6) participates in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 4-(dimethylamino)piperidine is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 2,5-dichloropyrimidine is used instead of 2,4,5-trichloropyrimidine to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 5-methyl-2,4-dichloropyrimidine is used instead of 2,4,5-trichloropyrimidine to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that N-ethylpiperazine is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 3-(dimethylamino)pyrrole is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that (1S,4S)-2-oxo-5-azabicyclo[2.2.1]heptane is used instead of 1-methyl-4-(piperidine-4- yl)piperazine hydrochloride (6) participates in the reaction.
  • the synthesis method is the same as that of Example 16LS 5-12, except that 2-methyl-2,5-diazabicyclo[2.2.1]heptane is used instead of 1-methyl-4-(piperidin-4-yl ) piperazine hydrochloride (6) participates in the reaction.
  • the method of subsequent synthesis steps is the same as the synthesis of Example 16LS 5-12, except that the reaction raw materials are replaced to participate in the reaction.
  • the synthesis method is the same as that of Example 32LS 6-16, except that N,N,N'-trimethylethylenediamine replaces 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6 )Reacted.
  • the synthesis method is the same as that of Example 32LS 6-16, except that morpholine is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • the method of subsequent synthesis steps is the same as the synthesis of Example 32LS 6-16, except that the reaction raw materials are replaced to participate in the reaction.
  • the synthesis method is the same as that of Example 36LS 6-59, except that N,N,N'-trimethylethylenediamine replaces 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6 )Reacted.
  • the synthesis method is the same as that of Example 36LS 6-59, except that N-ethylpiperazine is used instead of 1-methyl-4-(piperidin-4-yl)piperazine hydrochloride (6) to participate in the reaction.
  • Example 36LS 6-59 The synthetic method is the same as that of Example 36LS 6-59, except that compound 9 is replaced by intermediate 3-2, and 1-methyl-4-(piperidine) is replaced by N,N,N'-trimethylethylenediamine (34).
  • Perid-4-yl)piperazine hydrochloride (6) participates in the reaction.
  • the synthetic method is the same as that of Example 39 LS 6-88, except that N-ethylpiperazine is used instead of N,N,N'-trimethylethylenediamine (34) to participate in the reaction.
  • Step 4 The synthesis of intermediates 45, 46, 47 and the target product LS6-121 is the same as that of LS5-12, except that the reaction raw materials are correspondingly replaced to participate in the reaction.
  • the synthetic method is the same as that of Example 32LS 6-16, except that iodoethane is used to replace methyl iodide, and N,N,N'-trimethylethylenediamine is used to replace 1-methyl-4-(piperidin-4-yl ) piperazine hydrochloride (6) participates in the reaction.
  • the synthetic method is the same as the synthesis of Example 32LS 6-16, except that iodoisopropane is used to replace methyl iodide, and N,N,N'-trimethylethylenediamine is used to replace 1-methyl-4-(piperidine-4- yl)piperazine hydrochloride (6) participates in the reaction.
  • the kinase inhibitory activity of the compounds of the present invention against four members of the JAK family was tested with reference to the Z'-Lyte assay method, wherein the ATP concentration of each kinase was Km ; Family members have higher ATP affinity, so this example also tested the kinase inhibitory activity of the compounds against JAK3 when the ATP concentration was 1 mM.
  • Test method First, the test compound was prepared into a 10 mM stock solution with DMSO, and serially diluted to 10 concentrations with a 3-fold gradient, for use. After diluting 5X reaction buffer into 1X reaction buffer (50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl 2 , 1 mM EGTA) with deionized water, a mixture of kinase and peptide substrate was prepared with 1X reaction buffer , and phosphorylated peptide substrate solution, the kinase concentration was determined according to enzyme titration, and the final concentration of peptide substrate and phosphorylated peptide substrate was 2 ⁇ M.
  • 1X reaction buffer 50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl 2 , 1 mM EGTA
  • ATP ATP to meet the test requirements (Note: According to the recommended concentration of the instructions, the ATP concentration of JAK1 kinase test is 75 ⁇ M, the ATP concentration of JAK2 kinase test is 25 ⁇ M, the ATP concentration of JAK3 kinase test is 10 ⁇ M, and the ATP concentration of TYK2 kinase test is 25 ⁇ M).
  • the emission wavelengths at 460nm and 535nm were detected respectively.
  • the phosphorylation rate of the substrate was obtained, and the activity of the kinase and the inhibitory effect on the kinase were further calculated. Impact.
  • Table 3 Listed in Table 3 are compound numbers (corresponding to compound numbers in Examples 1-44) and corresponding kinase activity results.
  • a ATP concentration is Km ; b ATP concentration is 1 mM
  • kinase activity assay (Table 3) that the compounds of the present invention exhibit relatively high selective inhibitory activity against JAK3 subtype kinases.
  • Some compounds (such as LS 5-12, LS 5-77, LS 5-62, LS 5-66, LS 5-74, LS 5-88, LS 5-91, LS 5-102, LS 5-143, LS 5-150, LS 5-152, LS 5-154, LS 6-45, LS 6-49, LS 6-77, LS 6-88, LS 6-105, LS 6-121, LS 7-13, LS 7-18, etc.) showed potent and selective JAK3 kinase inhibitory activity, and also maintained potent activity when the ATP concentration was 1 mM.
  • Cell lines human chronic myeloid leukemia cell K562, human acute myeloid leukemia cell U937, human T lymphoblastic leukemia cell HuT78, human T lymphoblastic lymphoma cell line Jurkat. Cells were purchased from the Chinese Academy of Sciences Stem Cell Bank or ATCC.
  • CCK-8 cell counting kit-8
  • tumor cells in logarithmic growth phase were seeded in 96-well plates at a density of 1*10 4 cells/well, adherent cells were cultured overnight, and cells were suspended Direct drug stimulation.
  • Test compounds of different concentrations maximum working concentration 10 ⁇ M, 10 gradients diluted at a ratio of 1:3) were added, and two duplicate wells were set for each concentration, with a final volume of 200 ⁇ L.
  • 10 ⁇ L of CCK-8 reagent was added to each well, and the incubation was continued for 1-3 hours.
  • the drug-induced inhibition of cell growth was calculated according to the following formula:
  • Inhibition rate (%) (OD control-OD drug addition)/OD control ⁇ 100%
  • the median inhibitory concentration ( IC50 ) was calculated.
  • Vehicle intravenous 5% DMSO+10% polyethylene glycol-15 hydroxystearate+85% normal saline, oral 0.5% hydroxypropyl methylcellulose.
  • Animals in the oral administration group were fasted overnight (10-14 hours) before administration, and fed 4 hours after administration. Weigh before administration, and calculate the dosage according to body weight.
  • the drugs were administered by intravenous injection (iv, 5 mg/kg) or oral gavage (po, 15 mg/kg), respectively.
  • Blood was collected from the jugular vein at time points 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, and 8h after administration. About 0.20 mL of each sample was collected, anticoagulated with heparin sodium, placed on ice after collection, and kept at 1 Plasma was centrifuged within hours (centrifugation conditions: 6800 g, 6 minutes, 2-8°C).
  • Plasma samples were stored in a -80°C freezer prior to analysis.
  • the biological sample analysis method and the analysis of all samples are completed by the analytical laboratory of Medicipua Pharmaceutical Technology (Shanghai) Co., Ltd., and the intra-day accuracy evaluation of the quality control samples is performed at the same time as the samples are analyzed, and more than 66.7% of the quality control samples are required. The accuracy is between 80-120%.
  • the pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0, and the pharmacokinetic parameters and their mean and standard deviation were provided.
  • Example 48 Stability test of rat liver microsomes for representative molecule LS6-45
  • Prepare spiking solutions of test and reference compounds 500 ⁇ M spiking solution: add 5 ⁇ L of 10 mM stock to 95 ⁇ L acetonitrile; 1.5 ⁇ M microsome spiking solution (0.75 mg/mL): add 479.75 ⁇ L K/ 1.5 ⁇ L of 500 ⁇ M spiked solution and 18.75 ⁇ L of 20 mg/mL liver microsomes were added to Mg buffer).
  • NADPH was then dissolved in K buffer to make a 6 mM, 5 mg/mL NADPH stock.
  • liver microsome stability (Table 6) showed that the stable half-life T 1/2 of liver microsomes of the reference molecule Ketanserin was 18.71 minutes, which was consistent with historical data, indicating the reliability of the experiment.
  • the half-life T 1/2 of the example molecule LS6-45 is greater than 120 minutes, indicating that this molecule has high liver microsomal stability.
  • Example 49 Representative molecule LS6-45 effectively downregulates JAK3-STAT signaling in U937 cells
  • FIG 4 The experimental results are shown in Figure 4, in which, Western Blot analysis of JAK3, STAT3, STAT5 proteins and their time/dose-dependent phosphorylation by compound LS6-45, with GAPDH as an internal reference.
  • A is the time-dependent treatment of U937 cell line with compound LS6-45 (100 nM)
  • B is the dose-dependent treatment of U937 cell line with compound LS6-45 for 10 h (800 nM 1:4 diluted five concentrations).
  • Cell cycle detection Take well-growing cells and adjust the density to 8 ⁇ 10 5 cells/mL, and evenly divide them into 6-well plates, with 2 mL per well. The compound was then diluted in a gradient manner, added to the cell suspension, and incubated in an incubator for 24 hours. In this experiment, BD CycletestTM Plus DNA Reagent Kit was used for staining. After 24 h, the cells were collected into a 15 mL centrifuge tube, the 6-well plate was washed twice with PBS, the washes were combined into a 15 mL centrifuge tube, centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. Add 5 mL of buffer solution to each tube and gently resuspend the cells.
  • PI solution C
  • PI-stained samples need to be filtered with gauze before being loaded into the machine; it is recommended that the stained samples be placed on ice; the cells should not be over-digested, and the overall operation time should be as little as possible; A thin layer of cells is sufficient.
  • Apoptosis detection Take well-growing cells and adjust the density to 8 ⁇ 10 5 cells/mL, and evenly divide them into 6-well plates, with 2 mL per well. Then the compound was serially diluted, added to the cell suspension, and placed in the incubator for 48h. In this experiment, PE-coupled Annexin-V apoptosis detection kit was used for staining. After 48 h, the cells were collected into a 15 mL centrifuge tube, the 6-well plate was washed twice with PBS, the washings were combined into a 15 mL centrifuge tube, centrifuged (1200 rpm ⁇ 5 min), and the supernatant was discarded.
  • FIG. 6 shows the changes of cell cycle (A) and cell cycle-related proteins (B) after treating U937 cells with compound LS6-45 for 24 h, and the changes of apoptosis in U937 cells treated with compound LS6-45 for 48 h (C ).
  • the results (Fig. 6) showed that U937 had obvious G0/G1 phase arrest after treatment with different concentrations of compound LS6-45, and showed a dose-dependent manner (A in Fig. 6); meanwhile, cell cycle-related proteins (CDK2, CDK4, CDK6, Cyclin B1, Cyclin D3 and Cyclin E1) were also significantly down-regulated after treatment (B in Figure 6). No apoptosis was observed in U937 cells treated with compound LS6-45 (C in Figure 6).
  • mice 5 to 7 days after inoculation (U937 tumor grows very fast, observe once every 3 days), when the tumor grows to 100-200mm 3 in size, it is administered in groups, and animals that are too large or too small are excluded. Mice were randomly divided into administration group and solvent group, with 6 mice in each group. 50mg/kg, 25mg/kg and 12.5mg/kg doses of the drug were administered twice a day (bid), and 10mg/kg of the drug was intraperitoneally injected once (qd), and the control group was given an equal volume of solvent.
  • the dosing cycle was 10 days, daily administration, and the animal body weight was weighed every 2 days to measure the tumor volume (the initial tumor volume and body weight were recorded on the day of grouping).
  • the next day after the administration the animals were weighed and the tumor volume was measured, and the animals were sacrificed to dissect the tumor and weighed. Tumors were fixed with neutral formalin for pathological observation. Animal blood samples could be collected for routine blood analysis and major organs of animals were collected for pathological analysis according to experimental needs. At the end of the study, all animals were euthanized and tumors, livers, kidneys and lungs from nude mice were removed for further analysis.

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Abstract

一种具有式(Ⅰ)所示结构的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，及其药用组合物和应用。涉及的化合物可以高效、高选择性地抑制Janus Kinase 3(JAK3)的激酶活性，对多种血液肿瘤细胞(尤其是人急性髓系白血病细胞U937细胞)和实体瘤细胞均具有较强的信号抑制作用和细胞增殖抑制作用，可用于制备抗肿瘤药物，并且可以用于制备防治炎症性疾病的药物。

Description

2-氨基嘧啶类化合物及其药物组合物和应用 技术领域
本发明涉及化学医药技术领域，具体涉及一类2-氨基嘧啶类化合物及其药物组合物和应用。
背景技术
自20世纪90年代末开始，蛋白酪氨酸激酶作为新兴靶点开始日益受到关注。正常情况下，这类具有酪氨酸激酶活性的蛋白与ATP结合并在特定位置的酪氨酸残基上发生磷酸化，之后会引起细胞内重要信号通路的激活和转导，参与调节细胞的分裂、生长、增殖、分化、衰老、凋亡等生命过程。而酪氨酸激酶的失调则会引起细胞功能紊乱，导致机体发生一系列疾病，包括肿瘤和炎症性疾病等。因此，靶向蛋白酪氨酸激酶已经成为精准医疗的重要方面。
蛋白酪氨酸激酶包括受体酪氨酸激酶和非受体酪氨酸激酶。受体酪氨酸激酶的蛋白结构包括胞外配体结合区、疏水性的跨膜区和胞内的酪氨酸激酶催化结构域及调控序列，而非受体酪氨酸激酶的亚细胞定位与受体酪氨酸激酶不同，不包括胞外和跨膜结构，是一类胞质酪氨酸激酶蛋白。非受体酪氨酸激酶在细胞内被激活后，通过与下游信号分子结合并将其激活，使其磷酸化，发挥酪氨酸激酶活性。Janus激酶(简称JAK)是一种非受体酪氨酸激酶，该家族包括JAK1、JAK2、JAK3和Tyk2四个家族成员。由于该类激酶的蛋白结构包含两个激酶域，与ATP结合并发挥激酶催化活性的“真”激酶域以及没有催化活性的假激酶域，因此，这种蛋白质以两面罗马神贾纳斯(Janus)的名字命名。胞外特定的配体(如细胞因子、驱动因子、生长因子等)与受体的结合会导致JAKs的活化以及JAKs相关受体的磷酸化。受体磷酸化后，通过对含有特定序列的SH2结构域进行识别，启动对相应STATs(信号转导子和转录激活子)的募集，并随后对STATs蛋白进行磷酸化。磷酸化的STATs发生同源二聚化或异源二聚化后转移至细胞核，与特定的DNA结合位点结合，调节基因转录，从而导致细胞功能发生变化。
与其他家族成员在组织、细胞中有广泛表达不同，JAK3仅在骨髓、淋巴等与造血系统相关的组织细胞中表达。当白介素IL-2、IL-4、IL-7、IL-9、IL-15和IL-21特异性地与含γ c链(common gamma chain)受体亚基的受体结合后，会引起受体构象的变化，从而促进JAK3相关受体和JAK1相关受体互相靠近并发生二聚化和自磷酸化。活化了的JAK3蛋白征募STAT1、STAT3、STAT5或STAT6并将其磷酸化，在发生二聚化后，STATs转移至细胞核，影响下游靶向DNA的转录和翻译，参与淋巴细胞、T细胞、B细胞及NK细胞的生长、增殖、 发育与分化，以及免疫调节等重要生命过程。强有力的临床研究发现，人体JAK3功能性缺失(loss of function)会造成严重综合免疫缺陷(SCID)，而过度激活或突变则与诸多自身免疫性疾病及癌症(特别是白血病等血液癌症)相关。研究发现，JAK3-STAT5信号级联是造血过程中重要的信号通路，在T-淋巴细胞白血病(T-ALL)病例中，该信号通路发生异常的概率高达27.7％；其中，JAK3是该通路中最常见的突变基因，约占T-ALL病例的16.1％(Haematologica 2015,100,1301-1310)。作为JAK3最常见的激活突变，JAK3(M511I)在Ba/F3细胞中表现出细胞因子非依赖性的细胞增殖转化能力；在骨髓移植小鼠模型中，移植含JAK3(M511I)突变造血细胞的小鼠明显表现出T-ALL的临床症状(白细胞数量急剧升高，脾、胸腺和淋巴结肿大，外周血和造血组织的CD8+T细胞增加等)，而移植表达野生型JAK3细胞的小鼠未发病(Blood 2014,124,3092-3100))。此外，研究表明，约1/3含JAK3突变的T-ALL病例含有两个JAK3突变(纯合子突变或两个不同的突变)；双突变的发生在293T细胞、Ba/F3细胞模型中均表现出更强的通路激活作用和细胞转化能力，在致血液肿瘤活性上存在更大威胁(Blood 2018,131,421-425)。因此，JAK3已成为血液肿瘤治疗的潜在新靶标，靶向JAK3开发小分子抑制剂将为缓解或治疗相关疾病提供重要策略。
目前已有8款JAK抑制剂在美国、欧盟或日本被获批上市，分别是：鲁索替尼(ruxolitinib，JAK1/JAK2抑制剂，FDA于2011年批准上市)、托法替尼(tofacitinib，泛JAK抑制剂，FDA于2012年批准上市)、巴瑞替尼(baricitinib，JAK1/JAK2抑制剂，欧盟、FDA分别于2017年、2018年批准上市)、peficitinib(泛JAK抑制剂，日本于2019年批准上市)、菲卓替尼(fedratinib，JAK2抑制剂，FDA于2019年批准上市)、乌帕替尼(upadacitinib，JAK1抑制剂，FDA于2019年批准上市)、迪高替尼(delgocitinib，泛JAK抑制剂，日本于2020年批准上市)、filgotinib(JAK1抑制剂，欧盟、日本于2020年批准上市)。其中，经FDA批准上市的五款药物(鲁索替尼、托法替尼、巴瑞替尼、乌帕替尼和菲卓替尼)均因临床上可能引起的严重副反应被加以黑框警告。因此，研发高选择性的JAK3抑制剂可以在保持疗效的同时，通过减少对众多与疾病不相关细胞因子通路的干扰，有效降低毒副作用。
尽管高选择性JAK抑制剂的研发将会是未来药物化学家进行分子优化的方向和趋势，但是，由于JAKs家族的蛋白结构有高度的序列同源性，尤以ATP催化区域内观察到的同源性最高，使得在JAK家族中难以发现高选择性的ATP竞争性抑制剂；此外，JAK3相对于其他家族成员具有更高的ATP亲和力，导致选择性JAK3抑制剂的开发充满挑战，但并非不能实现。通过对比ATP结合口袋的特定氨基酸序列发现，JAK3激酶含有一个独特的、具有亲脂功能且可以与亲核试剂发生共价结合的半胱氨酸残基(Cys909)，而在其他三种JAK亚型的等效位置为丝氨酸。基于此，辉瑞公司(Pfizer)通过基于结构的药物设计策略开发出选择性 极高的JAK3不可逆抑制剂PF-06651600(ATP浓度为1mM时激酶水平IC 50值：JAK3为33.1nM，其他亚型均大于10μM)，目前该化合物处于临床III期试验用于治疗斑秃，并获得了美国FDA突破性疗法的认定。除此之外，尚无JAK3选择性抑制剂进入临床使用，开发高活性、低毒性的JAK3小分子抑制剂用于炎症性疾病或血液肿瘤的治疗具有重要的临床意义。
综上所述，本领域迫切需要研发新类型的，尤其是骨架新颖的小分子化合物来解决目前临床上JAK抑制剂选择性低、毒副作用大等问题，并解决临床需求。
发明内容
针对上述问题，本发明提供了一类新的2-氨基嘧啶类化合物，这类新的2-氨基嘧啶类化合物，能够高活性的选择性抑制JAK3激酶的活性，从而能够抑制多种肿瘤细胞的增殖，可用于治疗与JAK3激酶相关的肿瘤或者炎症性疾病。具体包括如下技术方案。
具有式(Ⅰ)所示结构的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物：
Figure PCTCN2021143765-appb-000001
其中，
R 1选自：H、卤素、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基、一个或多个R 11取代或未取代的C 3-C 6环烷基、一个或多个R 11取代或未取代的C 1-C 6烷氧基、一个或多个R 11取代或未取代的C 3-C 6环烷氧基、甲酰胺基；
R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5或-(CH 2) mOCR 3R 4R 5；其中，各m分别独立地为0、1、2或3；各R 3和各R 4分别独立地选自：H、一个或者多个R 12取代的C 1-C 6烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的3-12元单环、稠环、桥环或螺环；R 5选自：H、氰基或C 1-C 3烷基；各R 12分别独立地选自：H、卤素、羟基、氨基、C 1-C 3烷基、-C(＝O)NHR 13取代的C 1-C 3烷基、羟基取代的C 1-C 3烷基、C 3-C 6环烷基取代的C 1-C 3烷基、C 3-C 8杂环基取代的C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13、R 13取代或未取代的含有0、1、2或3个杂原子的4-8元单环、稠环、桥环或螺环；R 13为C 1-C 3烷基；所述杂原子为O、S和/或N；
W、X、Y、Z各自独立地为N或-CR 6；其中，R 6选自：氢、卤素、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基、-NH-CN、 -NHC(O)-CR 7＝CR 8R 9、-NHS(O) 2-CR 7＝CR 8R 9；R 7、R 8和R 9分别独立地选自：H、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基；
n为0或1；
L为O或S；
Figure PCTCN2021143765-appb-000002
为一个或多个R 10取代的4-12元饱和或部分饱和的环原子中含有0、1、2或3个杂原子的单环、桥环、螺环或者稠环；其中，各R 10分别独立地选自：氢、卤素、羟基、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基，所述杂原子为O、S和/或N；
R 11选自：卤素、羟基、氨基、C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13。
在其中一些实施例中，
Figure PCTCN2021143765-appb-000003
为一个或多个R 10取代的8-10元饱和或部分饱和的含有1、2或3个氧原子的稠合双环。
在其中一些实施例中，
Figure PCTCN2021143765-appb-000004
选自以下基团：
Figure PCTCN2021143765-appb-000005
Figure PCTCN2021143765-appb-000006
其中，用*标记的手性碳原子的构型分别独立地为S构型或者R构型。
在其中一些实施例中，
Figure PCTCN2021143765-appb-000007
选自以下基团：
Figure PCTCN2021143765-appb-000008
Figure PCTCN2021143765-appb-000009
其中，用*标记的手性碳原子的构型分别独立地为S构型或者R构型，各R 10分别独立地选自：卤素、羟基、C 1-C 3烷基、C 1-C 3烷氧基。
在其中一些实施例中，
Figure PCTCN2021143765-appb-000010
选自以下基团：
Figure PCTCN2021143765-appb-000011
在其中一些实施例中，
Figure PCTCN2021143765-appb-000012
选自以下基团：
Figure PCTCN2021143765-appb-000013
其中，各R 10分别独立地选自：F、羟基、甲基、甲氧基、乙氧基、异丙氧基。
在其中一些实施例中，
Figure PCTCN2021143765-appb-000014
选自以下基团：
Figure PCTCN2021143765-appb-000015
在其中一些实施例中，R 6选自：氢、卤素、C 1-C 3烷基、C 1-C 3烷氧基、-NH-CN、
Figure PCTCN2021143765-appb-000016
Figure PCTCN2021143765-appb-000017
在其中一些实施例中，W、X均为CH；Y和Z分别独立地选自N或CR 6，其中，R 6选 自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基、-NH-CN、
Figure PCTCN2021143765-appb-000018
Figure PCTCN2021143765-appb-000019
在其中一些实施例中，W、X均为CH；Y为CR 6，其中，R 6选自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基、
Figure PCTCN2021143765-appb-000020
Z为CH或者N。
在其中一些实施例中，W、X、Z均为CH；Y为CR 6，其中，R 6为：
Figure PCTCN2021143765-appb-000021
在其中一些实施例中，R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5；其中，各m分别独立地为0、1、2或3；
各R 3和各R 4分别独立地选自：H、一个或者多个R 12取代的C 1-C 3烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的5-11元单环、稠环、桥环或螺环；
R 5选自：H、氰基或C 1-C 3烷基；
各R 12分别独立地选自：H、羟基、乙酰基、R 13取代或未取代的4-8元杂环基、卤素、羟基、氨基、C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2；R 13为C 1-C 3烷基。
在其中一些实施例中，R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5；其中，各m分别独立地为0或1；
各R 3和各R 4分别独立地选自：H、C 1-C 3烷基、羟基取代的C 1-C 3烷基、-NHR 13取代的C 1-C 3烷基、-N(R 13) 2取代的C 1-C 3烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的5-11元单环、桥环或螺环；
R 5选自：H、氰基或C 1-C 3烷基；
各R 12分别独立地选自：H、羟基、乙酰基、R 13取代的哌嗪基、C 1-C 3烷基、-NHR 13、-N(R 13) 2；R 13为C 1-C 3烷基。
在其中一些实施例中，R 2选自：H、卤素、
Figure PCTCN2021143765-appb-000022
Figure PCTCN2021143765-appb-000023
在其中一些实施例中，R 1选自：H、卤素、氰基、甲酰胺基、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、C 3-C 6环烷氧基、卤代C 1-C 6烷基、卤代C 1-C 6烷氧基。
在其中一些实施例中，R 1选自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基。
在其中一些实施例中，R 1选自：H、卤素、甲基、氰基、甲酰胺基、三氟甲基、二氟甲基、甲氧基、环丙基、三氟甲氧基。
在其中一些实施例中，所述2-氨基嘧啶类化合物具有如下式(II)所示结构：
Figure PCTCN2021143765-appb-000024
本发明还提供了上述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物的应用。
具体技术方案如下：
上述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物在制备JAK3抑制剂 中的应用。
上述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物在制备预防和/或治疗肿瘤和/或炎症性疾病的药物中的应用。
在其中一些实施例中，所述肿瘤为血液瘤和实体瘤，所述血液瘤为多发性骨髓瘤、B淋巴瘤、骨髓纤维化、真性红细胞增多症、原发性血小板增多症、慢性髓系白血病、急性髓性白血病、急性淋巴细胞性白血病、慢性粒细胞白血病、组织细胞淋巴瘤、急性巨核细胞白血病、幼淋巴细胞白血病、T淋巴细胞白血病、T淋巴母细胞性淋巴瘤；所述实体瘤为非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、胰腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、鼻咽癌、胶质瘤；所述炎症性疾病为类风湿性关节炎、特应性皮炎、接触性皮炎、牛皮癣、银屑病、溃疡性结肠炎、克罗恩病、湿疹、盘状红斑狼疮、全身性红斑狼疮、斑秃、移植物抗宿主病、强直性脊柱炎、弥漫性皮肤系统性硬化、皮肌炎。
本发明还提供了一种防治肿瘤和/或炎症性疾病的药用组合物。
具体技术方案如下：
一种防治肿瘤和/或炎症性疾病的药用组合物，由活性成分和药学上可接受的载体或者辅料制备得到，所述活性成分包括上述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物。
本发明提供了一类结构新颖的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，该类化合物可以高效、高选择性地抑制Janus Kinase 3(JAK3)的激酶活性，对多种血液肿瘤细胞(尤其是人急性髓系白血病细胞U937细胞)和实体瘤细胞均具有较强的信号抑制作用和细胞增殖抑制作用，可用于制备抗肿瘤以及与JAK3激酶相关的炎症性疾病的治疗药物。
附图说明
图1为中间体3-1的单晶结构。
图2为中间体9的单晶结构。
图3为化合物LS6-45的药代动力学实验结果。
图4为化合物LS6-45抑制U937细胞内JAK3通路的活化。
图5为化合物LS6-45的洗脱实验结果。
图6为化合物LS6-45对U937细胞周期阻滞及凋亡的影响。
图7为受试化合物LS6-45的小鼠体内抗肿瘤活性结果。
具体实施方式
本发明所述化合物中，当任何变量(例如R 3、R 4等)在任何组分中出现超过一次，则其每次出现的定义独立于其它每次出现的定义。同样，允许取代基及变量的组合，只要这种组合使化合物稳定。自取代基划入环系统的线表示所指的键可连接到任何能取代的环原子上。如果环系统为多环，其意味着这种键仅连接到邻近环的任何适当的碳原子上。要理解本领域普通技术人员可选择本发明化合物的取代基及取代型式而提供化学上稳定的并可通过本领域技术和下列提出的方法自可容易获得的原料容易的合成的化合物。如果取代基自身被超过一个基团取代，应理解这些基团可在相同碳原子上或不同碳原子上，只要使结构稳定。
本文所用术语“烷基”意指包括具有特定碳原子数目的支链的和直链的饱和脂肪烃基。例如，“C 1-C 6烷基”中“C 1-C 6”的定义包括以直链或支链排列的具有1、2、3、4、5或6个碳原子的基团。例如，“C 1-C 6烷基”具体包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、戊基、己基。术语“环烷基”指具有特定碳原子数目的单环饱和脂肪烃基。例如“环烷基”包括环丙基、环丁基、环戊基或环己基等。术语“烷氧基”指具有-O-烷基结构的基团，如-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3、-O-CH 2CH(CH 3) 2、-OCH 2CH 2CH 2CH 3、-O-CH(CH 3) 2等。术语“杂环基”为饱和或部分不饱和的单环或多环环状取代基，其中一个或多个环原子选自N、O或S(O)m(其中m是0-2的整数)的杂原子，其余环原子为碳，例如：吗啉基、哌啶基、四氢吡咯基、吡咯烷基、二氢咪唑基、二氢异噁唑基、二氢异噻唑基、二氢噁二唑基、二氢噁唑基、二氢吡嗪基、二氢吡唑基、二氢吡啶基、二氢嘧啶基、二氢吡咯基、二氢四唑基、二氢噻二唑基、二氢噻唑基、二氢噻吩基、二氢三唑基、二氢氮杂环丁烷基、四氢呋喃基、四氢噻吩基等，及其N-氧化物，杂环取代基的连接可通过碳原子或通过杂原子实现。
正如本领域技术人员所理解的，本文中所用“卤素”(“halo”)或“卤”意指氯、氟、溴和碘。
除非另有定义，烷基、环烷基、芳基、杂芳基和杂环烷基取代基可为未被取代的或取代的。例如，C 1-C 6烷基可被一个、两个或三个选自OH、卤素、烷氧基、二烷基氨基或杂环基例如吗啉基、哌啶基等的取代基取代。
本发明提供了一种具有式(Ⅰ)所示结构的2-氨基嘧啶类化合物：
Figure PCTCN2021143765-appb-000025
其中，
R 1选自：H、卤素、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基、一个或多个R 11取代或未取代的C 3-C 6环烷基、一个或多个R 11取代或未取代的C 1-C 6烷氧基、一个或多个R 11取代或未取代的C 3-C 6环烷氧基、甲酰胺基；
R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5或-(CH 2) mOCR 3R 4R 5；其中，各m分别独立地为0、1、2或3；各R 3和各R 4分别独立地选自：H、一个或者多个R 12取代的C 1-C 6烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的3-12元单环、稠环、桥环或螺环；R 5选自：H、氰基或C 1-C 3烷基；各R 12分别独立地选自：H、卤素、羟基、氨基、C 1-C 3烷基、-C(＝O)NHR 13取代的C 1-C 3烷基、羟基取代的C 1-C 3烷基、C 3-C 6环烷基取代的C 1-C 3烷基、C 3-C 8杂环基取代的C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13、R 13取代或未取代的含有0、1、2或3个杂原子的4-8元单环、稠环、桥环或螺环；R 13为C 1-C 3烷基；所述杂原子为O、S和/或N；
W、X、Y、Z各自独立地为N或-CR 6；其中，R 6选自：氢、卤素、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基、-NH-CN、-NHC(O)-CR 7＝CR 8R 9、-NHS(O) 2-CR 7＝CR 8R 9；R 7、R 8和R 9分别独立地选自：H、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基；
n为0或1；
L为O或S；
Figure PCTCN2021143765-appb-000026
为一个或多个R 10取代的4-12元饱和或部分饱和的环原子中含有0、1、2或3个杂原子的单环、桥环、螺环或者稠环；其中，各R 10分别独立地选自：氢、卤素、羟基、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基，所述杂原子为O、S和/或N；
R 11选自：卤素、羟基、氨基、C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13。
本发明包括式Ⅰ或者式II化合物的游离形式，也包括其药学上可接受的盐及立体异构体。本文中一些特定的示例性化合物为胺类化合物的质子化了的盐。术语“游离形式”指以非盐形式的胺类化合物。包括在内的药学上可接受盐不仅包括本文所述特定化合物的示例性盐，也包括所有式Ⅰ或者式II化合物游离形式的典型的药学上可接受的盐。可使用本领域已知技术分 离所述化合物特定盐的游离形式。例如，可通过用适当的碱稀水溶液例如NaOH稀水溶液、碳酸钾稀水溶液、稀氨水及碳酸氢钠稀水溶液处理该盐使游离形式再生。游离形式在某些物理性质例如在极性溶剂中溶解度上与其各自盐形式多少有些区别，但是为发明的目的这种酸盐及碱盐在其它药学方面与其各自游离形式相当。
可通过常规化学方法自含有碱性部分或酸性部分的本发明化合物合成本发明的药学上可接受的盐。通常，通过离子交换色谱或通过游离碱和化学计算量或过量的所需盐形式的无机或有机酸在适当溶剂或多种溶剂的组合中反应制备碱性化合物的盐。类似的，通过和适当的无机或有机碱反应形成酸性化合物的盐。
因此，本发明化合物的药学上可接受的盐包括通过碱性本发明化合物和无机或有机酸反应形成的本发明化合物的常规无毒盐。例如，常规的无毒盐包括得自无机酸例如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等的盐，也包括自有机酸例如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、对氨基苯磺酸、2-乙酰氧基苯甲酸、富马酸、甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、羟乙基磺酸、三氟乙酸等制备的盐。
如果本发明化合物为酸性的，则适当的“药学上可接受的盐”指通过药学上可接受的无毒碱包括无机碱及有机碱制备的盐.得自无机碱的盐包括铝盐、铵盐、钙盐、铜盐、铁盐、亚铁盐、锂盐、镁盐、锰盐、亚锰盐、钾盐、钠盐、锌盐等。特别优选铵盐、钙盐、镁盐、钾盐和钠盐。得自药学上可接受的有机无毒碱的盐，所述碱包括伯胺、仲胺和叔胺的盐，取代的胺包括天然存在的取代胺、环状胺及碱性离子交换树脂例如精氨酸、甜菜碱、咖啡因、胆碱、N,N'-二苄基乙二胺、二乙胺、2-二乙基氨基乙醇、2-二甲基氨基乙醇、氨基乙醇、乙醇胺、乙二胺、N-乙基吗啉、N-乙基哌啶、葡萄糖胺、氨基葡萄糖、组氨酸、羟钴胺、异丙基胺、赖氨酸、甲基葡萄糖胺、吗啉、哌嗪、哌啶、呱咤、多胺树脂、普鲁卡因、嘌呤、可可碱、三乙胺、三甲胺、三丙胺、氨基丁三醇等。
Berg等，“Pharmaceutical Salts，”J.Pharm.Sci.’1977：66：1-19更详细描述了上文所述药学上可接受的盐及其它典型的药学上可接受的盐的制备。
由于在生理条件下化合物中脱质子化的酸性部分例如羧基可为阴离子的，而这种带有的电荷然后可被内部带有阳离子的质子化了的或烷基化的碱性部分例如四价氮原子平衡抵消，所以应注意本发明化合物是潜在的内盐或两性离子。
在一个实施方案中，本申请提供了一种利用具有式Ⅰ或者式II的化合物及其药学可接受的盐治疗人或其它哺乳动物肿瘤或炎症性疾病。
在一个实施方案中，本申请的化合物及其药学可接受的盐可以用于治疗或控制多发性骨 髓瘤、B淋巴瘤、骨髓纤维化、真性红细胞增多症、原发性血小板增多症、慢性髓系白血病、急性髓性白血病、急性淋巴细胞性白血病、慢性粒细胞白血病、组织细胞淋巴瘤、急性巨核细胞白血病、幼淋巴细胞白血病、T淋巴细胞白血病、T淋巴母细胞性淋巴瘤、非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、胰腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、鼻咽癌、胶质瘤、类风湿性关节炎、特应性皮炎、接触性皮炎、牛皮癣、银屑病、溃疡性结肠炎、克罗恩病、湿疹、盘状红斑狼疮、全身性红斑狼疮、斑秃、移植物抗宿主病、强直性脊柱炎、弥漫性皮肤系统性硬化、皮肌炎。
药物代谢物及前药
本发明所涉及的化合物及其药学可接受的盐的代谢产物，以及可以在体内转变为本申请所涉及的化合物及其药学可接受的盐的结构的前药，也包含在本发明的权利要求中。
药物组合物
本发明还提供了一种药物组合物，它包含安全有效量范围内的活性成分，以及药学上可接受的载体或者辅料。
本发明所述的“活性成分”是指本发明所述的式I或者式II化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物。
本发明所述的“活性成分”和药物组合物可用作JAK蛋白激酶抑制剂，可用于制备预防和/或治疗肿瘤和/或炎症性疾病的药物。
“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg活性成分/剂，更佳地，含有10-200mg活性成分/剂。较佳地，所述的“一剂”为一个药片。
“药学上可接受的载体或者辅料”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。
“相容性”在此指的是组合物中各组分能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
药学上可以接受的载体或者辅料部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2021143765-appb-000027
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
在另一优选例中，本发明式I或者式II化合物可与大分子化合物或高分子通过非键合作 用形成复合物。在另一优选例中，本发明式I或者式II化合物作为小分子还可通过化学键与大分子化合物或高分子相连接。所述大分子化合物可以是生物大分子如高聚糖、蛋白、核酸、多肽等。
本发明的活性成分或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)等。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。
在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：
(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；
(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；
(c)保湿剂，例如，甘油；
(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐和碳酸钠；
(e)缓溶剂，例如，石蜡；
(f)吸收加速剂，例如，季胺化合物；
(g)润湿剂，例如，鲸蜡醇和单硬脂酸甘油酯；
(h)吸附剂，例如，高岭土；和
(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
所述的固体剂型还可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例如，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物可以单独给药，或者与其他治疗药物(如降糖药)联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
联合用药
式Ⅰ或者式II化合物可以与已知的治疗或改进相似病状的其它药物联用。联合给药时，原来药物的给药方式和剂量保持不变，而同时或随后服用式Ⅰ或者式II化合物。当式Ⅰ或者式II化合物与其它一种或几种药物同时服用时，优选使用同时含有一种或几种已知药物和式I或者式II化合物的药用组合物。药物联用也包括在重叠的时间段服用式Ⅰ或者式II化合物与其它一种或几种已知药物。当式Ⅰ或者式II化合物与其它一种或几种药物进行药物联用时，式Ⅰ或者式II化合物或已知药物的剂量可能比它们单独用药时的剂量较低。
可以与式Ⅰ或者式II化合物进行药物联用的药物或活性成分包括但不局限为：
雌激素受体调节剂、雄激素受体调节剂、视网膜样受体调节剂、细胞毒素/细胞抑制剂、抗增殖剂、蛋白转移酶抑制剂、HMG-CoA还原酶抑制剂、HIV蛋白激酶抑制剂、逆转录酶抑制剂、血管生成抑制剂、细胞增殖及生存信号抑制剂、干扰细胞周期关卡的药物和细胞凋亡诱导剂，细胞毒类药物、酪氨酸蛋白抑制剂、EGFR抑制剂、VEGFR抑制剂、丝氨酸/苏氨酸蛋白抑制剂、Bcr-Abl抑制剂、c-Kit抑制剂、Met抑制剂、Raf抑制剂、MEK抑制剂、MMP抑制剂、拓扑异构酶抑制剂、组氨酸去乙酰化酶抑制剂、蛋白酶体抑制剂、CDK抑制剂、Bcl-2家族蛋白抑制剂、MDM2家族蛋白抑制剂、IAP家族蛋白抑制剂、STAT家族蛋白抑制剂、PI3K抑制剂、AKT抑制剂、整联蛋白阻滞剂、干扰素-α、白介素-12、COX-2抑制剂、p53、p53激活剂、VEGF抗体、EGF抗体、JAK抑制剂等。
在一个实施方案中，可以与式Ⅰ或者式II化合物进行药物联用的药物或活性成分包括但不局限为：阿地白介素、阿仑膦酸、干扰素、阿曲诺英、别嘌醇、别嘌醇钠、帕洛诺司琼盐酸盐、六甲蜜胺、氨基格鲁米特、氨磷汀、氨柔比星、安丫啶、阿纳托唑、多拉司琼、aranesp、arglabin、三氧化二砷、阿诺新、5-氮胞苷、硫唑嘌呤、卡介苗或tice卡介苗、贝他定、醋酸倍他米松、倍他米松磷酸钠制剂、贝沙罗汀、硫酸博来霉素、溴尿甘、bortezomib、白消安、降钙素、阿来佐单抗注射剂、卡培他滨、卡铂、康士得、cefesone、西莫白介素、柔红霉素、 苯丁酸氮芥、顺铂、克拉屈滨、克拉屈滨、氯屈磷酸、环磷酰胺、阿糖胞昔、达卡巴嗪、放线菌素D、柔红霉素脂质体、地塞米松、磷酸地塞米松、戊酸雌二醇、地尼白介素2、狄波美、地洛瑞林、地拉佐生、己烯雌酚、大扶康、多西他奇、去氧氟尿苷、阿霉素、屈大麻酚、钦-166-壳聚糖复合物、eligard、拉布立酶、盐酸表柔比星、阿瑞吡坦、表阿霉素、阿法依伯汀、红细胞生成素、依铂、左旋咪唑片、雌二醇制剂、17-β-雌二醇、雌莫司汀磷酸钠、炔雌醇、氨磷汀、羟磷酸、凡毕复、依托泊甙、法倔唑、他莫昔芬制剂、非格司亭、非那司提、非雷司替、氟尿苷、氟康唑、氟达拉滨、5-氟脱氧尿嘧啶核苷一磷酸盐、5-氟尿嘧啶、氟甲睾酮、氟他胺、福麦斯坦、1-β-D-阿糖呋喃糖胞噻啶-5’-硬脂酰磷酸酯、福莫司汀、氟维司群、丙种球蛋白、吉西他滨、吉妥单抗、甲磺酸伊马替尼、卡氮芥糯米纸胶囊剂、戈舍瑞林、盐酸格拉尼西隆、组氨瑞林、和美新、氢化可的松、赤型-羟基壬基腺嘌呤、羟基脲、替坦异贝莫单抗、伊达比星、异环磷酰胺、干扰素α、干扰素-α2、干扰素α-2A、干扰素α-2B、干扰素α-nl、干扰素α-n3、干扰素β、干扰素γ-la、白细胞介素-2、内含子A、易瑞沙、依立替康、凯特瑞、硫酸香菇多糖、来曲唑、甲酰四氢叶酸、亮丙瑞林、亮丙瑞林醋酸盐、左旋四咪唑、左旋亚叶酸钙盐、左甲状腺素钠、左甲状腺素钠制剂、洛莫司汀、氯尼达明、屈大麻酚、氮芥、甲钴胺、甲羟孕酮醋酸酯、醋酸甲地孕酮、美法仑、酯化雌激素、6-琉基嘌呤、美司钠、氨甲蝶呤、氨基乙酰丙酸甲酯、米替福新、美满霉素、丝裂霉素C、米托坦、米托葱醌、曲洛司坦、柠檬酸阿霉素脂质体、奈达铂、聚乙二醇化非格司亭、奥普瑞白介素、neupogen、尼鲁米特、三苯氧胺、NSC-631570、重组人白细胞介素1-β、奥曲肽、盐酸奥丹西隆、去氢氢化可的松口服溶液剂、奥沙利铂、紫杉醇、泼尼松磷酸钠制剂、培门冬酶、派罗欣、喷司他丁、溶链菌制剂、盐酸匹鲁卡品、毗柔比星、普卡霉素、卟吩姆钠、泼尼莫司汀、司替泼尼松龙、泼尼松、倍美力、丙卡巴脐、重组人类红细胞生成素、雷替曲塞、利比、依替膦酸铼-186、美罗华、力度伸-A、罗莫肽、盐酸毛果芸香碱片剂、奥曲肽、沙莫司亭、司莫司汀、西佐喃、索布佐生、唬钠甲强龙、帕福斯酸、干细胞治疗、链佐星、氯化锶-89、左旋甲状腺素钠、他莫昔芬、坦舒洛辛、他索那明、tastolactone、泰索帝、替西硫津、替莫唑胺、替尼泊苷、丙酸睾酮、甲睾酮、硫鸟嘌呤、噻替哌、促甲状腺激素、替鲁膦酸、拓扑替康、托瑞米芬、托西莫单抗、曲妥珠单抗、曲奥舒凡、维A酸、甲氨喋呤片剂、三甲基密胺、三甲曲沙、乙酸曲普瑞林、双羟萘酸曲普瑞林、优福定、尿苷、戊柔比星、维司力农、长春碱、长春新碱、长春酰胺、长春瑞滨、维鲁利秦、右旋丙亚胺、净司他丁斯酯、枢复宁、紫杉醇蛋白质稳定制剂、acolbifene、干扰素r-lb、affinitak、氨基喋呤、阿佐昔芬、asoprisnil、阿他美坦、阿曲生坦、BAY43-9006、阿瓦斯丁、CCI-779、CDC-501、西乐葆、西妥昔单抗、克立那托、环丙孕酮醋酸酯、地西他滨、DN-101、阿霉素-MTC、dSLIM、度他雄胺、edotecarin、 依氟鸟氨酸、依喜替康、芬维A胺、组胺二盐酸盐、组氨瑞林水凝胶植入物、钬-166DOTMP、伊班膦酸、干扰素γ、内含子-PEG、ixabepilone、匙孔形血蓝蛋白、L-651582、兰乐肽、拉索昔芬、libra、lonafamib、米泼昔芬、米诺屈酸酯、MS-209、脂质体MTP-PE、MX-6、那法瑞林、奈莫柔比星、新伐司他、诺拉曲特、奥利默森、onco-TCS、osidem、紫杉醇聚谷氨酸酯、帛米酸钠、PN-401、QS-21、夸西洋、R-1549、雷洛昔芬、豹蛙酶、13-顺维A酸、沙铂、西奥骨化醇、T-138067、tarceva、二十二碳六烯酸紫杉醇、胸腺素αl、嘎唑呋林、tipifarnib、替拉扎明、TLK-286、托瑞米芬、反式MID-lo7R、伐司朴达、伐普肽、vatalanib、维替泊芬、长春氟宁、Z-100和唑来麟酸或它们的组合。
本发明的有益之处在于：
(1)提供一种结构新颖的2-氨基嘧啶类化合物。
(2)该类化合物对JAK3激酶产生较强的选择性抑制作用，可以有效抑制多种肿瘤细胞的生长，可用于制备抗肿瘤药物。
(3)该类化合物有效阻止JAK3-STAT5信号通路的传递，可用于抗炎症性疾病的药物的制备。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域技术人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
下列实施例中所用试剂均可购买得到。
实施例1
(3R,3aR,6S,6aR)-6-((5-氯-2-((3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 3-96)和(3S,3aR,6R,6aR)-6-((5-氯-2-((3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-97)的合成
Figure PCTCN2021143765-appb-000028
步骤1. 6-((2,5-二氯-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(3-1和3-2)的合成
在冰浴下，向异山梨醇(2,0.48g,3.27mmol)的四氢呋喃(THF,20mL)溶液中分批加入60％氢化钠(0.13g,3.27mmol)，继续搅拌10分钟后，向体系中滴加2,4,5-三氯嘧啶(1,0.5g,2.72mmol)，并于室温搅拌过夜。待反应完全，向反应体系中缓慢滴加冰水淬灭。旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得白色固体3-1(227mg，收率：28.5％)和3-2(123mg，收率15.5％)。
1H NMR for 3-1(400MHz,DMSO-d 6)δ8.69(s,1H),5.44-5.40(m,1H),5.00(d,J＝6.0Hz,1H),4.60(d,J＝4.4Hz,1H),4.49(t,J＝4.8Hz,1H),4.20-4.11(m,1H),4.08-4.00(m,2H),3.78(dd,J＝8.8,6.4Hz,1H),3.45-3.38(m,1H).MS(ESI)m/z 293.0[M+H] +.
中间体3-1的晶体结构及其解析如图1(CCDC号为2119312)和表1所示，确证其化学结构(立体构型)正确。
表1中间体3-1的单晶参数
Figure PCTCN2021143765-appb-000029
Figure PCTCN2021143765-appb-000030
1H NMR for 3-2(400MHz,CDCl 3)δ8.34(s,1H),5.59(td,J＝5.6,3.6Hz,1H),5.05(t,J＝5.2Hz,1H),4.44(d,J＝4.8Hz,1H),4.36(s,1H),4.08(dd,J＝10.8,3.6Hz,1H),4.00-3.88(m,2H),3.84(d,J＝10.0Hz,1H),2.31(d,J＝4.4Hz,1H).MS(ESI)m/z 293.0[M+H] +.
步骤2. 3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)的合成
向2-氯-1-氟-4-硝基苯(5,0.3g,1.71mmol)和碳酸钾(0.48g,3.42mmol)的乙腈(10mL)溶液中加入1-甲基-4-(哌啶基-4-基)哌嗪盐酸盐(6,0.45g,2.0mmol)，体系升温至80摄氏度反应过夜。待反应完全，冷却至室温，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得黄色固体7(0.53g,收率92％)。 1H NMR(400MHz,DMSO-d 6)δ8.21(d,J＝2.8Hz,1H),8.13(dd,J＝9.2,2.8Hz,1H),7.27(d,J＝9.2Hz,1H),3.57(d,J＝12.0Hz,2H),2.82(t,J＝11.6Hz,2H),2.51-2.40(m,4H),2.44-2.19(m,5H),2.14(s,3H),1.88(d,J＝11.6Hz,2H),1.64-1.49(m,2H).
向中间体7(2.85g,8.43mmol)的乙醇/水(体积比2:1)的混合溶剂中加入还原铁粉(1.42g,25.3mmol)和氯化铵(4.6g,84.3mmol)，回流反应2小时。待反应完全，冷却至室温，经硅藻土抽滤后，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得固体4(2.2g,收率87％)。 1HNMR(400MHz,CDCl 3)δ6.87(d,J＝8.4Hz,1H),6.74(d,J＝2.8Hz,1H),6.53(dd,J＝8.4,2.8Hz,1H),3.51(s,2H),3.29(d,J＝11.6Hz,2H),2.89-2.22(m,14H),1.89(d,J＝11.6Hz,2H),1.80-1.73(m,2H).MS(ESI)m/z 309.2[M+H] +.
步骤3.(3R,3aR,6S,6aR)-6-((5-氯-2-((3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 3-96)的合成
Figure PCTCN2021143765-appb-000031
向中间体3-1(63mg,0.214mmol)的乙二醇单甲醚溶剂中加入中间体4(60mg,0.195mmol)和2.5M的氯化氢乙醇溶液(0.2mL)，反应体系升温至120摄氏度反应过夜。待反应完全，冷却至室温，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得白色固体LS 3-96(33mg,收率30％)。 1HNMR(400MHz,CDCl 3)δ8.15(s,1H),7.65(d,J＝2.4Hz,1H),7.29(dd,J＝8.8,2.4Hz,1H),7.08(s,1H),6.99(d,J＝8.8Hz,1H),5.51(d,J＝3.6Hz,1H),4.76(t,J＝4.8Hz,1H),4.69(d,J＝4.4Hz,1H),4.35(q,J＝6.0Hz,1H),4.25(d,J＝10.8Hz,1H),4.18(dd,J＝10.8,4.0Hz,1H),3.94(dd,J＝9.2,6.0Hz,1H),3.66(dd,J＝9.2,5.6Hz,1H),3.46-3.34(m,2H),2.82-2.33(m,11H),2.30(s,3H),1.93(d,J＝11.9Hz,2H),1.83-1.68(m,2H). 13C NMR(151MHz,CDCl 3)δ163.32(s),157.32(s),156.96(s),145.19(s),134.40(s),129.12(s),121.75(s),120.53(s),118.60(s),107.03(s),85.70(s),82.13(s),81.22(s),73.60(s),73.46(s),72.32(s),61.72(s),55.45(s),51.70(d,J＝3.6Hz),49.03(s),46.06(s),28.58(s).HRMS(ESI)calcd for C 26H 35Cl 2N 6O 4[M+H] +565.2091；found 565.2080.
(3S,3aR,6R,6aR)-6-((5-氯-2-((3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-97)的合成
Figure PCTCN2021143765-appb-000032
合成方法同LS 3-96的合成，只是以中间体3-2代替3-1参与反应，收率37％。 1H NMR(400MHz,CDCl 3)δ8.14(s,1H),7.78(d,J＝2.4Hz,1H),7.16(dd,J＝8.8,2.4Hz,1H),7.02-6.96(m,1H),6.88(s,1H),5.51-5.44(m,1H),5.06(t,J＝5.2Hz,1H),4.47(d,J＝4.8Hz,1H),4.39(d,J＝2.8Hz,1H),4.08(dd,J＝10.4,4.0Hz,1H),4.03-3.94(m,2H),3.88(d,J＝10.4Hz,1H),3.40(d,J＝12.0Hz,2H),3.01-2.18(m,14H),2.05-1.97(m,1H),1.93(d,J＝12.4Hz,2H),1.84-1.71(m,2H). 13C NMR(151MHz,CDCl 3)δ163.93(s),157.35(s),156.53(s),144.91(s),134.60(s), 128.81(s),121.82(s),120.33(s),118.58(s),106.42(s),88.72(s),80.62(s),76.64(s),75.87(s),75.67(s),71.10(s),61.69(s),55.37(s),51.67(d,J＝10.6Hz),48.94(s),46.01(s),31.63(s).HRMS(ESI)calcd for C 26H 35Cl 2N 6O 4[M+H] +565.2091；found 565.2084.
实施例2
(3R,3aR,6R,6aR)-6-((5-氯-2-((3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-104)的合成
Figure PCTCN2021143765-appb-000033
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)参与反应。
1H NMR(400MHz,CDCl 3)δ8.14(s,1H),7.79(d,J＝2.4Hz,1H),7.13(dd,J＝8.8,2.4Hz,1H),7.06-6.89(m,2H),5.49(q,J＝5.2Hz,1H),4.94(t,J＝5.2Hz,1H),4.54(t,J＝5.2Hz,1H),4.30(dd,J＝13.2,6.4Hz,1H),4.24-4.09(m,2H),3.98(dd,J＝9.2,6.4Hz,1H),3.63(dd,J＝8.8,8.0Hz,1H),3.39(d,J＝11.6Hz,2H),2.96-2.26(m,14H),1.93(d,J＝12.0Hz,2H),1.85-1.74(m,2H). 13C NMR(151MHz,CDCl 3)δ163.78(s),157.46(s),156.82(s),145.17(s),134.53(s),128.94(s),122.08(s),120.45(s),118.82(s),106.65(s),81.63(s),80.73(s),76.68(s),73.45(s),72.22(s),71.53(s),61.69(s),55.44(s),51.72(d,J＝1.0Hz),49.04(s),46.06(s),28.61(s).HRMS(ESI)calcd for C 26H 35Cl 2N 6O 4[M+H] +565.2091；found 565.2087.
实施例3
(3R,3aR,6S,6aR)-6-((5-氯-2-((2-甲氧基-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-100)的合成
Figure PCTCN2021143765-appb-000034
合成方法同实施例1LS 3-96的合成，只是以4-氟-2-甲氧基-1-硝基苯代替2-氯-1-氟-4-硝基苯(5)参与反应。
1H NMR(400MHz,CDCl 3)δ8.13(s,1H),8.07(d,J＝9.6Hz,1H),7.40(s,1H),6.59-6.49 (m,2H),5.57(d,J＝3.2Hz,1H),4.76(t,J＝4.8Hz,1H),4.70(d,J＝4.4Hz,1H),4.36(q,J＝1.6Hz,1H),4.27(d,J＝10.8Hz,1H),4.15(dd,J＝10.7,3.7Hz,1H),3.94(dd,J＝9.2,5.6Hz,1H),3.88(s,3H),3.74-3.61(m,3H),2.79-2.45(m,14H),1.95(d,J＝12.8Hz,2H),1.77-1.68(m,2H).MS(ESI)m/z 561.3[M+H] +.
实施例4
(3R,3aR,6S,6aR)-6-((5-氯-2-((3-甲氧基-4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-106)的合成
Figure PCTCN2021143765-appb-000035
合成方法同实施例1LS 3-96的合成，只是以1-氟-2-甲氧基-4-硝基苯代替2-氯-1-氟-4-硝基苯(5)、以N-甲基哌嗪代替1-甲基-4-(哌啶基-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.14(s,1H),7.11(d,J＝2.4Hz,1H),7.06(dd,J＝8.8,2.4Hz,1H),6.98(s,1H),6.91(d,J＝8.4Hz,1H),5.56(d,J＝3.6Hz,1H),4.75(t,J＝4.8Hz,1H),4.67(d,J＝4.4Hz,1H),4.40-4.31(m,1H),4.23(d,J＝10.8Hz,1H),4.12(dd,J＝10.8,4.0Hz,1H),3.93(dd,J＝9.6,6.0Hz,1H),3.88(s,3H),3.64(dd,J＝9.6,6.0Hz,1H),3.08(s,4H),2.63(s,5H),2.36(s,3H).MS(ESI)m/z 478.2[M+H] +.
实施例5
1-(4-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)苯基)环戊烷-1-甲腈(LS 5-58)的合成
Figure PCTCN2021143765-appb-000036
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、1-(4-氨基苯基)环戊烷-1-甲腈代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.16(s,1H),7.52(d,J＝8.4Hz,2H),7.44(s,1H),7.40(d,J＝8.4Hz,2H),5.52(q,J＝5.6Hz,1H),4.89(t,J＝5.2Hz,1H),4.53(t,J＝5.2Hz,1H),4.34-4.25(m,1H),4.17(d,J＝5.2Hz,2H),3.98(dd,J＝8.8,6.4Hz,1H),3.67-3.58(m,1H),2.52-2.44(m, 2H),2.09-1.90(m,6H).HRMS(ESI)calcd for C 22H 24ClN 4O 4[M+H] +443.1481；found 443.1465.
实施例6
(3R,3aR,6R,6aR)-6-((5-氯-2-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-144)的合成
Figure PCTCN2021143765-appb-000037
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、4-(4-甲基哌嗪-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.12(s,1H),7.40-7.33(m,2H),6.95-6.85(m,3H),5.46(q,J＝5.6Hz,1H),4.85(t,J＝5.2Hz,1H),4.51(t,J＝5.2Hz,1H),4.33-4.25(m,1H),4.17(dd,J＝10.0,6.0Hz,1H),4.10(dd,J＝10.0,5.6Hz,1H),3.98(dd,J＝9.2,6.4Hz,1H),3.63(dd,J＝9.2,8.0Hz,1H),3.29-3.20(m,4H),2.78-2.68(m,4H),2.46(s,3H).HRMS(ESI)calcd for C 21H 27ClN 5O 4[M+H] +448.1746；found 448.1734.
实施例7
(3R,3aR,6R,6aR)-6-((5-氯-2-((3-氯-4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-142)的合成
Figure PCTCN2021143765-appb-000038
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、3-氯-4-(4-甲基哌嗪-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.15(s,1H),7.79(d,J＝2.8Hz,1H),7.17(dd,J＝8.8,2.8Hz,1H),7.06(s,1H),7.02(d,J＝8.8Hz,1H),5.49(q,J＝5.6Hz,1H),4.93(t,J＝5.6Hz,1H),4.54(t,J＝5.6Hz,1H),4.30(dd,J＝12.8,6.4Hz,1H),4.22-4.12(m,2H),3.98(dd,J＝9.2,6.4Hz,1H),3.63(dd,J＝8.8,8.0Hz,1H),3.11(s,4H),2.71(s,4H),2.43(s,3H).HRMS(ESI)calcd for C 21H 26Cl 2N 5O 4[M+H] +482.1356；found 482.1330.
实施例8
(3R,3aR,6R,6aR)-6-((5-氯-2-((4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-145)的合成
Figure PCTCN2021143765-appb-000039
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.14(s,1H),7.40-7.32(m,2H),6.97-6.90(m,2H),6.87(s,1H),5.48(q,J＝5.6Hz,1H),4.87(t,J＝5.6Hz,1H),4.53(t,J＝5.6Hz,1H),4.31(m,1H),4.19(dd,J＝9.6,5.6Hz,1H),4.12(dd,J＝10.0,5.6Hz,1H),4.00(dd,J＝9.2,6.8Hz,1H),3.76-3.61(m,3H),2.91-2.56(m,10H),2.50-2.44(m,1H),2.42(s,3H),1.99(d,J＝12.8Hz,2H),1.77-1.67(m,2H).HRMS(ESI)calcd for C 26H 36ClN 6O 4[M+H] +531.2481；found 531.2457.
实施例9
(3R,3aR,6R,6aR)-6-((5-氯-2-((3-甲氧基-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-143)的合成
Figure PCTCN2021143765-appb-000040
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、3-甲氧基-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.13(s,1H),7.06-6.94(m,3H),6.88(d,J＝8.4Hz,1H),5.47(q,J＝5.6Hz,1H),4.83(t,J＝5.2Hz,1H),4.49(t,J＝5.6Hz,1H),4.32-4.23(m,1H),4.16(dd,J＝10.0,6.0Hz,1H),4.08(dd,J＝10.0,5.6Hz,1H),3.97(dd,J＝9.2,6.4Hz,1H),3.85(s,3H),3.63(dd,J＝9.2,8.0Hz,1H),3.49(d,J＝11.6Hz,2H),2.75-2.45(m,11H),2.31(s,3H),1.90(d,J＝11.2Hz,2H),1.85-1.75(m,2H).HRMS(ESI)calcd for C 27H 38ClN 6O 5[M+H] +561.2587；found 561.2556.
实施例10
(3R,3aR,6R,6aR)-6-((5-氯-2-((2-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-148)的合成
Figure PCTCN2021143765-appb-000041
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、2-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.14(s,1H),7.93(d,J＝9.2Hz,1H),7.08(s,1H),6.94(d,J＝2.8Hz,1H),6.84(dd,J＝9.2,2.8Hz,1H),5.47(q,J＝5.6Hz,1H),4.84(t,J＝5.2Hz,1H),4.50(t,J＝5.2Hz,1H),4.33-4.24(m,1H),4.16(dd,J＝10.0,6.0Hz,1H),4.09(dd,J＝9.6,5.2Hz,1H),3.97(dd,J＝9.2,6.8Hz,1H),3.72-3.59(m,3H),2.76-2.45(m,10H),2.34-2.28(m,4H),1.94(d,J＝12.0Hz,2H),1.70-1.60(m,2H).HRMS(ESI)calcd for C 26H 35Cl 2N 6O 4[M+H] +565.2091；found 565.2068.
实施例11
(3R,3aR,6R,6aR)-6-((5-氯-2-((3,5-二氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 5-16)的合成
Figure PCTCN2021143765-appb-000042
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、3,5-二氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.17(s,1H),7.55(d,J＝2.4Hz,1H),7.43(d,J＝2.4Hz,1H),7.05(s,1H),5.49(q,J＝5.2Hz,1H),4.96(t,J＝5.2Hz,1H),4.55(t,J＝5.6Hz,1H),4.36-4.26(m,1H),4.18(d,J＝5.6Hz,2H),3.98(dd,J＝9.2,6.8Hz,1H),3.63(dd,J＝8.8,8.0Hz,1H),3.33(t,J＝11.6Hz,2H),3.03(d,J＝11.6Hz,2H),2.85-2.25(m,13H),1.87(d,J＝10.8Hz,5H), 1.74-1.67(m,2H).HRMS(ESI)calcd for C 26H 34Cl 3N 6O 4[M+H] +599.1702；found 599.1694.
实施例12
(3R,3aR,6R,6aR)-6-((5-氯-2-((5-甲基-6-(4-(4-甲基哌嗪-1-基)哌啶-1-基)吡啶-3-基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 4-150)的合成
Figure PCTCN2021143765-appb-000043
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、5-甲基-6-(4-(4-甲基哌嗪-1-基)哌啶-1-基)吡啶-3-氨基代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.23(d,J＝2.4Hz,1H),8.11(s,1H),7.62(d,J＝2.0Hz,1H),7.06(s,1H),5.46(q,J＝5.6Hz,1H),4.84(t,J＝5.2Hz,1H),4.49(t,J＝5.6Hz,1H),4.27(dt,J＝8.0,6.4Hz,1H),4.17-4.06(m,2H),3.96(dd,J＝9.2,6.4Hz,1H),3.61(dd,J＝8.8,8.4Hz,1H),3.41(d,J＝12.8Hz,2H),2.87-2.57(m,7H),2.43-2.13(m,10H),1.95(d,J＝11.6Hz,2H),1.74-1.58(m,2H).MS(ESI)m/z 546.3[M+H] +.
实施例13
(3R,3aR,6R,6aR)-6-((5-氯-2-((5-((4-乙基哌嗪-1-基)甲基)吡啶-2-基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(LS 5-54)的合成
Figure PCTCN2021143765-appb-000044
合成方法同实施例1LS 3-96的合成，只是以1,4:3,6-双脱水甘露醇代替异山梨醇(2)、5-((4-乙基哌嗪-1-基)甲基)吡啶-2-氨基代替3-氯-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯胺(4)参与反应。
1H NMR(400MHz,CDCl 3)δ8.29-8.22(m,2H),8.22(d,J＝1.6Hz,1H),8.19(d,J＝8.4Hz,1H),7.66(dd,J＝8.8,2.4Hz,1H),5.53(q,J＝5.6Hz,1H),4.90(t,J＝5.2Hz,1H),4.54(t,J＝5.6Hz,1H),4.35-4.26(m,1H),4.21(dd,J＝10.0,5.6Hz,1H),4.15(dd,J＝10.0,5.2Hz,1H),3.97(dd,J＝8.8,6.4Hz,1H),3.64(dd,J＝8.8,8.0Hz,1H),3.49(s,2H),2.74-2.45(m,10H),1.14(t,J ＝7.2Hz,3H).HRMS(ESI)calcd for C 22H 30ClN 6O 4[M+H] +477.2012；found 477.2020.
实施例14
N-(3-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)苯基)丙烯酰胺(LS 5-77)的合成
Figure PCTCN2021143765-appb-000045
步骤1.(3R,3aR,6R,6aR)-6-((2,5-二氯嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(9)的合成
合成方法同实施例1化合物3-1的合成，只是以1,4:3,6-双脱水甘露醇(8)代替异山梨醇(2)参与反应。
1H NMR(400MHz,DMSO-d 6)δ8.68(s,1H),5.51(td,J＝5.2,4.0Hz,1H),4.80(t,J＝5.2Hz,1H),4.28(t,J＝4.8Hz,1H),4.14-3.90(m,3H),3.70(t,J＝7.6Hz,1H),3.34(dd,J＝9.6,8.0Hz,1H).MS(ESI)m/z 293.0[M+H] +.
中间体9的晶体结构及其解析如图2(CCDC号为2119315)和表2所示，确证其化学结构(立体构型)正确。
表2中间体9的单晶参数
Figure PCTCN2021143765-appb-000046
Figure PCTCN2021143765-appb-000047
步骤2.(3R,3aR,6R,6aR)-6-((5-氯-2-((3-硝基苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(11)的合成
向中间体9(0.3g,1.0mmol)的异丙醇溶液中加入2-硝基苯胺(10,0.13g,0.98mmol)和浓盐酸(1.13mmol)，升温至80摄氏度反应过夜。待反应完全，冷却至室温，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得固体11(0.18g,46％)。
1H NMR(400MHz,DMSO-d 6)δ10.26(s,1H),8.85(t,J＝2.4Hz,1H),8.43(s,1H),7.95(d,J＝8.4Hz,1H),7.83(dd,J＝8.0,2.4Hz,1H),7.59(t,J＝8.2Hz,1H),5.53(dd,J＝10.8,5.2Hz,1H),4.99(d,J＝6.8Hz,1H),4.88(t,J＝5.2Hz,1H),4.34(t,J＝4.8Hz,1H),4.14-4.04(m,2H),3.99(dd,J＝10.0,4.8Hz,1H),3.79-3.69(m,1H),3.40(t,J＝8.4Hz,1H).MS(ESI)m/z 395.1[M+H] +.
步骤3.(3R,3aR,6R,6aR)-6-((2-((3-氨基苯基)氨基)-5-氯嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(12)的合成
向中间体11(0.15g,0.38mmol)的乙醇/水(体积比2:1)的混合溶剂中加入还原铁粉(64mg,1.14mmol)和氯化铵(0.2g,3.8mmol)，回流反应2小时。待反应完全，冷却至室温，经硅藻土抽滤后，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得固体12(0.11g,79％)。
1H NMR(400MHz,DMSO-d 6)δ9.39(s,1H),8.25(s,1H),6.96(t,J＝2.0Hz,1H),6.91(t,J＝8.0Hz,1H),6.77(d,J＝8.0Hz,1H),6.20(dd,J＝8.0,1.2Hz,1H),5.44(dd,J＝11.2,5.6Hz,1H),5.00(s,2H),4.95(d,J＝6.8Hz,1H),4.82(t,J＝5.2Hz,1H),4.31(t,J＝4.8Hz,1H),4.13-4.00(m,2H),3.93(dd,J＝9.6,5.2Hz,1H),3.76-3.68(m,1H),3.42-3.35(m,1H).MS(ESI)m/z 365.1[M+H] +.
步骤4.N-(3-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)苯基)丙烯酰胺(LS 5-77)的合成
向中间体12(90mg,0.25mmol)的DMF溶液中加入丙烯酸(13,20mg,0.27mmol)、O-(7-偶氮苯并三氮唑-1-氧)-N,N”,N”-四甲基脲六氟磷酸酯(HATU,0.12g,0.3mmol)和N,N-二异丙基乙胺(DIPEA,64mg,0.5mmol)，反应于室温条件下搅拌过夜。待反应完全，减压旋干大部分溶剂，粗品经二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，经柱层析分离得白色固体。
1H NMR(400MHz,DMSO-d 6)δ10.10(s,1H),9.75(s,1H),8.31(s,2H),7.28(d,J＝8.0Hz,1H),7.22(t,J＝8.0Hz,1H),7.13(d,J＝7.6Hz,1H),6.46(dd,J＝16.8,10.0Hz,1H),6.25(dd,J＝16.8,2.0Hz,1H),5.75(dd,J＝10.0,2.0Hz,1H),5.65(q,J＝5.2Hz,1H),4.94(d,J＝6.8Hz,1H),4.82(t,J＝5.2Hz,1H),4.26(t,J＝4.8Hz,1H),4.13-4.00(m,2H),3.94(dd,J＝9.6,5.2Hz,1H),3.72(t,J＝7.4Hz,1H),3.38(t,J＝8.4Hz,1H). 13C NMR(151MHz,DMSO-d 6)δ163.76(s),163.52(s),158.17(s),157.11(s),140.78(s),139.63(s),132.36(s),129.15(s),127.27(s),115.16(s),113.73(s),110.72(s),105.22(s),81.98(s),80.72(s),77.40(s),72.33(s),71.47(s),70.95(s).HRMS(ESI)calcd for C 19H 20ClN 4O 5[M+H] +419.1117；found 419.1126.
实施例15
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-氟苯基)丙烯酰胺(LS 5-62)的合成
Figure PCTCN2021143765-appb-000048
步骤1.(3R,3aR,6R,6aR)-6-((5-氯-2-((4-氟-3-硝基苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(15)的合成
合成方法同化合物11的合成，只是以原料14代替间硝基苯胺(10)参与反应。
1H NMR(400MHz,CDCl 3)δ8.84(dd,J＝6.4,2.8Hz,1H),8.20(s,1H),7.81(s,1H),7.45(dt,J＝8.8,3.2Hz,1H),7.26-7.19(m,1H),5.68-5.59(m,1H),5.00(t,J＝5.6Hz,1H),4.58(t,J＝5.6Hz,1H),4.33-4.27(m,1H),4.24(dd,J＝10.4,4.4Hz,1H),4.18(dd,J＝10.0,5.2Hz,1H),3.96(dd,J＝8.8,6.4Hz,1H),3.62(dd,J＝8.8,8.0Hz,1H).MS(ESI)m/z 413.1[M+H] +.
步骤2.(3R,3aR,6R,6aR)-6-((2-((3-氨基-4-氟苯基)氨基)-5-氯嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(16)的合成
合成方法同化合物12的合成。
1H NMR(400MHz,DMSO-d 6)δ9.44(s,1H),8.25(s,1H),7.12(dd,J＝8.4,2.4Hz,1H),6.88(dd,J＝11.2,8.8Hz,1H),6.79-6.70(m,1H),5.44(dd,J＝10.8,5.2Hz,1H),5.09(s,2H), 4.95(d,J＝7.2Hz,1H),4.80(t,J＝5.2Hz,1H),4.31(t,J＝5.2Hz,1H),4.12-4.01(m,2H),3.93(dd,J＝9.6,4.8Hz,1H),3.75-3.68(m,1H),3.42-3.35(m,1H).MS(ESI)m/z 383.1[M+H] +.
步骤3.N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-氟苯基)丙烯酰胺(LS 5-62)的合成
向中间体16(62mg,0.16mmol)的DMF溶液中加入丙烯酸(13,13mg,0.18mmol)、O-(7-偶氮苯并三氮唑-1-氧)-N,N”,N”-四甲基脲六氟磷酸酯(HATU,74mg,0.19mmol)和N,N-二异丙基乙胺(DIPEA,42mg,0.32mmol)，反应于室温条件下搅拌过夜。待反应完全，减压旋干大部分溶剂，粗品经二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，经柱层析分离得白色固体。
1H NMR(400MHz,CDCl 3)δ8.76(s,1H),8.07(s,1H),7.06-6.92(m,2H),6.42-6.26(m,2H),5.83(q,J＝5.2Hz,1H),5.75(dd,J＝8.8,2.8Hz,1H),4.87(t,J＝5.2Hz,1H),4.39(t,J＝5.2Hz,1H),4.25-4.04(m,3H),3.87(dd,J＝8.8,6.4Hz,1H),3.55(t,J＝8.4Hz,1H),3.36-3.30(m,1H). 13C NMR(151MHz,DMSO-d 6)δ163.91(s),163.79(s),158.08(s),157.21(s),149.32(d,J＝240.7Hz),136.60(s),131.79(s),127.92(s),126.02(d,J＝12.4Hz),116.45(d,J＝6.9Hz),115.66(d,J＝20.6Hz),114.98(s),105.18(s),81.99(s),80.73(s),77.38(s),72.29(s),71.44(s),71.00(s).HRMS(ESI)calcd for C 19H 19ClFN 4O 5[M+H] +437.1023；found437.1033.
实施例16
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 5-12)的合成
Figure PCTCN2021143765-appb-000049
步骤1.(3R,3aR,6R,6aR)-6-((5-氯-2-((4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)-3-硝基苯基)氨基)嘧啶-4-基)氧)六氢呋喃并[3,2-b]呋喃-3-醇(17)的合成
向中间体15(0.4g,0.97mmol)的乙腈溶液中加入1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6,0.32g,1.45mmol)、N,N-二异丙基乙胺(DIPEA,0.38g,2.9mmol)，升温至90摄氏度反应过夜。待反应完全，冷却至室温，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得固体17。
1H NMR(400MHz,CDCl 3)δ8.51(d,J＝2.4Hz,1H),8.17(s,1H),7.34(dd,J＝8.8,2.4Hz,1H),7.22(s,1H),7.12(d,J＝8.8Hz,1H),5.59(q,J＝5.2Hz,1H),4.97(t,J＝5.4Hz,1H),4.57(t,J＝5.4Hz,1H),4.34-4.24(m,1H),4.18(d,J＝5.2Hz,2H),3.96(dd,J＝8.8,6.4Hz,1H),3.65-3.60(m,1H),3.30-3.25(m,2H),3.16-2.98(m,14H),2.80(t,J＝11.6Hz,2H),2.65(d,J＝9.2Hz,2H).MS(ESI)m/z 576.2[M+H] +.
步骤2.N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 5-12)的合成
向中间体17(32mg,0.055mmol)的乙醇/水(体积比2:1)的混合溶剂中加入还原铁粉(9mg,0.16mmol)和氯化铵(3mg,0.055mmol)，回流反应2小时。待反应完全，冷却至室温，经硅藻土抽滤后，旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得浅灰色固体18。向中间体18(30mg,0.055mmol)的2毫升DMF溶液中加入丙烯酸(13,4.4mg,0.06mmol)、O-(7-偶氮苯并三氮唑-1-氧)-N,N”,N”-四甲基脲六氟磷酸酯(HATU,26mg,0.066mmol)和N,N-二异丙基乙胺(DIPEA,15mg,0.11mmol)，反应于室温条件下搅拌过夜。待反应完全，减压旋干大部分溶剂，粗品经二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，经柱层析分离得白色固体。
1H NMR(400MHz,CDCl 3)δ8.99(s,1H),8.76(s,1H),8.14(s,1H),7.15-7.05(m,2H),6.97(d,J＝7.2Hz,1H),6.37(dd,J＝16.8,1.6Hz,1H),6.25(dd,J＝16.8,10.0Hz,1H),5.99(q,J＝5.6Hz,1H),5.77(dd,J＝10.0,1.2Hz,1H),4.98(t,J＝5.2Hz,1H),4.49(t,J＝5.6Hz,1H),4.31-4.20(m,2H),4.13(dd,J＝9.6,5.2Hz,1H),3.95(dd,J＝8.8,6.4Hz,1H),3.62(dd,J＝9.2,8.0Hz,1H),3.01(d,J＝12.0Hz,2H),2.88-2.49(m,10H),2.42-2.32(m,4H),2.06(d,J＝12.4Hz,2H),1.70-1.60(m,2H). 13C NMR(151MHz,CDCl 3)δ164.01(s),163.02(s),157.48(s),156.73(s),136.59(s),136.46(s),133.77(s),131.82(s),127.21(s),120.80(s),114.14(s),109.73(s),106.59(s),81.66(s),81.01(s),76.85(s),73.41(s),72.37(s),71.70(s),61.29(s),55.38(s),52.63(d,J＝13.3Hz),49.52(s),46.01(s),30.03(s).HRMS(ESI)calcd for C 29H 39ClN 7O 5[M+H] +600.2696；found 600.2693.
实施例17
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-4-甲氧基-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 5-3)的合成
Figure PCTCN2021143765-appb-000050
合成方法同实施例16LS 5-12的合成，只是以4-氟-2-甲氧基-5-硝基苯胺代替4-氟-3-硝基苯胺(14)参与反应。
1H NMR(400MHz,CDCl 3)δ9.44(s,1H),8.46(s,1H),8.15(s,1H),7.71(s,1H),6.71(s,1H),6.45-6.16(m,3H),5.76(dd,J＝9.2,2.4Hz,1H),5.04(t,J＝5.6Hz,1H),4.48(t,J＝5.2Hz,1H),4.30(dd,J＝9.6,6.0Hz,1H),4.22(s,1H),4.16(dd,J＝9.6,4.8Hz,1H),3.93(dd,J＝8.8,6.4Hz,1H),3.86(s,3H),3.75-3.64(m,2H),3.64-3.56(m,1H),3.13-2.86(m,8H),2.77-2.58(m,6H),2.10-2.02(m,2H),1.90-1.85(m,2H). 13C NMR(151MHz,CDCl 3)δ164.18(s),162.60(s),157.24(s),156.61(s),144.22(s),135.76(s),131.77(s),126.78(s),126.40(s),125.78(s),109.53(s),106.21(s),103.14(s),81.73(s),81.23(s),72.94(s),72.26(s),71.84(s),61.32(s),56.05(s),55.04(s),52.48(d,J＝29.1Hz),49.12(s),45.74(s),29.67(s).HRMS(ESI)calcd for C 30H 41ClN 7O 6[M+H] +630.2801；found 630.2820.
实施例18
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-吗啉苯基)丙烯酰胺(LS 5-66)的合成
Figure PCTCN2021143765-appb-000051
合成方法同实施例16LS 5-12的合成，只是以吗啉代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ9.05(s,1H),8.87(s,1H),8.12(s,1H),7.89(s,1H),7.16(d,J＝8.8Hz,1H),7.08-6.97(m,1H),6.43-6.25(m,2H),6.07(q,J＝5.2Hz,1H),5.80(dd,J＝9.6,1.6Hz,1H),5.01(t,J＝5.6Hz,1H),4.48(t,J＝5.2Hz,1H),4.31-4.13(m,3H),3.95(dd,J＝8.8,6.4Hz,1H),3.92-3.77(m,4H),3.72(q,J＝6.8Hz,1H),3.64-3.55(m,1H),2.96-2.76(m,4H). 13C NMR(151MHz,CDCl 3)δ164.03(s),162.98(s),157.42(s),156.74(s),137.08(s),135.50(s),133.91(s),131.76(s),127.33(s),121.31(s),114.28(s),109.72(s),106.74(s),81.66(s),81.02(s),76.85(s),73.40(s),72.36(s),71.71(s),67.73(s),52.88(s).HRMS(ESI)calcd for C 23H 27ClN 5O 6[M+H] +504.1644；found 504.1637.
实施例19
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-羟乙基)(甲基)氨基)苯基)丙烯酰胺(LS 5-74)的合成
Figure PCTCN2021143765-appb-000052
合成方法同实施例16LS 5-12的合成，只是以2-甲基氨基乙醇代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.84(s,1H),8.09(s,1H),7.21-6.99(m,2H),6.52-6.27(m,2H),5.88(d,J＝4.8Hz,1H),5.75-5.64(m,1H),4.92(t,J＝5.2Hz,1H),4.42(t,J＝5.2Hz,1H),4.28-4.13(m,2H),4.09(dd,J＝9.6,5.2Hz,1H),3.90(dd,J＝8.8,6.8Hz,1H),3.68-3.56(m,3H),3.35(dt,J＝3.2,1.6Hz,1H),2.93(s,2H),2.80-2.70(m,3H).HRMS(ESI)calcd for C 22H 27ClN 5O 6[M+H] +492.1644；found492.1649.
实施例20
(E)-N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)-2-丁烯酰胺(LS 5-72)的合成
Figure PCTCN2021143765-appb-000053
合成方法同实施例16LS 5-12的合成，只是以2-丁烯酸代替丙烯酸(13)参与反应。
1H NMR(400MHz,CDCl 3)δ8.96(s,1H),8.52(s,1H),8.13(s,1H),7.13-7.02(m,2H),7.02-6.87(m,2H),6.06-5.89(m,2H),4.98(t,J＝5.6Hz,1H),4.49(t,J＝5.2Hz,1H),4.32-4.19(m,2H),4.13(dd,J＝10.0,5.2Hz,1H),3.95(dd,J＝8.8,6.4Hz,1H),3.65-3.57(m,1H),3.10-2.46(m,16H),2.08(d,J＝10.0Hz,2H),1.96(dd,J＝6.8,1.6Hz,3H),1.65-1.55(m,2H). 13C NMR(151MHz,DMSO-d 6)δ159.23(s),158.68(s),152.76(s),151.94(s),136.42(s),131.79(s),131.58(s),129.28(s),121.20(s),115.90(s),109.08(s),104.99(s),101.72(s),76.93(s),76.25(s),68.60(s),67.63(s),66.95(s),56.57(s),50.57(s),47.78(d,J＝14.0Hz),44.80(s),41.19(s),25.38(s),13.16(s).HRMS(ESI)calcd for C 30H 41ClN 7O 5[M+H] +614.2852；found 614.2855.
实施例21
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)甲基丙烯酰胺(LS 5-73)的合成
Figure PCTCN2021143765-appb-000054
合成方法同实施例16LS 5-12的合成，只是以甲基丙烯酸代替丙烯酸(13)参与反应。
1H NMR(400MHz,CDCl 3)δ9.19(s,1H),9.03(s,1H),8.14(s,1H),7.19-7.00(m,2H),6.94(d,J＝8.0Hz,1H),6.02(q,J＝5.2Hz,1H),5.87(s,1H),5.48(s,1H),4.99(t,J＝5.6Hz,1H),4.48(t,J＝5.2Hz,1H),4.32-4.19(m,2H),4.14(dd,J＝10.0,5.2Hz,1H),3.95(dd,J＝9.2,6.4Hz,1H),3.66-3.57(m,1H),3.02(d,J＝12.0Hz,2H),2.91-2.44(m,10H),2.43-2.27(m,4H),2.13-2.00(m,5H),1.70-1.59(m,2H). 13C NMR(151MHz,CDCl 3)δ165.52(s),164.02(s),157.48(s),156.71(s),140.48(s),136.71(s),136.44(s),134.02(s),120.88(s),120.58(s),113.86(s),109.35(s),106.52(s),81.65(s),81.04(s),76.85(s),73.33(s),72.37(s),71.75(s),61.52(s),55.32(s),52.71(d,J＝15.8Hz),49.41(s),45.99(s),29.90(s),18.78(s).HRMS(ESI)calcd for C 30H 41ClN 7O 5[M+H] +614.2852；found 614.2856.
实施例22
(E)-N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)-4-(二甲氨基)-2-丁烯酰胺(LS 5-81)的合成
Figure PCTCN2021143765-appb-000055
合成方法同实施例16LS 5-12的合成，只是以4-溴巴豆酸代替丙烯酸(13)参与反应后，得到的中间体19与2M的二甲胺四氢呋喃溶液发生取代可得。
Figure PCTCN2021143765-appb-000056
化合物LS 5-81： 1H NMR(400MHz,CDCl 3)δ8.99(s,1H),8.69(s,1H),8.13(s,1H),7.15(s,1H),7.08(d,J＝8.4Hz,1H),7.06-6.84(m,2H),6.15(d,J＝15.2Hz,1H),5.98(q,J＝5.6Hz,1H),4.97(t,J＝5.2Hz,1H),4.48(t,J＝5.2Hz,1H),4.33-4.19(m,2H),4.11(dd,J＝10.0,5.4Hz,1H),3.95(dd,J＝8.8,6.4Hz,1H),3.68-3.57(m,1H),3.20(d,J＝6.0Hz,2H),3.01(d,J＝11.6Hz,2H),2.94-2.63(m,9H),2.47-2.39(m,5H),2.34(s,6H),2.08(d,J＝12.0Hz,2H),1.80-1.68(m,2H).HRMS(ESI)calcd for C 32H 46ClN 8O 5[M+H] +657.3274；found 657.3296.
实施例23
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 5-88)的合成
Figure PCTCN2021143765-appb-000057
合成方法同实施例16LS 5-12的合成，只是以N,N,N'-三甲基乙二胺代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ10.11(s,1H),9.03(s,1H),8.14(s,1H),7.14(d,J＝8.0Hz,2H),7.01(d,J＝8.4Hz,1H),6.56-6.27(m,2H),6.02(d,J＝5.4Hz,1H),5.72(d,J＝11.7Hz,1H),4.99(t,J＝5.2Hz,1H),4.50(t,J＝5.2Hz,1H),4.35-4.19(m,2H),4.14(dd,J＝9.6,5.1Hz,1H),4.00-3.88(m,1H),3.69-3.57(m,1H),2.92(s,2H),2.69(s,3H),2.55-2.15(m,7H),2.05-1.94(m,1H). 13C NMR(151MHz,CDCl 3)δ164.02(s),163.66(s),157.52(s),156.71(s),136.70(d,J＝14.1Hz),136.10(s),132.00(s),129.89(s),126.72(s),122.51(s),114.27(s),110.31(s),106.53(s),81.65(s),81.03(s),76.85(s),73.43(s),72.37(s),71.71(s),57.24(s),45.41(s),43.32(s),29.33(s).HRMS(ESI)calcd for C 24H 32ClN 6O 5[M+H] +519.2117；found 519.2106.
实施例24
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨 基)-2-(9-甲基-3,9-二氮杂螺[5.5]十一烷-3-基)苯基)丙烯酰胺(LS 5-91)的合成
Figure PCTCN2021143765-appb-000058
合成方法同实施例16LS 5-12的合成，只是以3-甲基-3,9-二氮杂螺[5,5]十一烷代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.98(s,1H),8.83(s,1H),8.14(s,1H),7.17-7.06(m,2H),7.02-6.91(m,1H),6.37(dd,J＝16.8,1.6Hz,1H),6.27(dd,J＝16.8,10.0Hz,1H),5.99(d,J＝5.6Hz,1H),5.77(dd,J＝10.0,1.6Hz,1H),4.99(t,J＝5.2Hz,1H),4.49(t,J＝5.2Hz,1H),4.30-4.20(m,2H),4.13(dd,J＝10.0,5.2Hz,1H),3.95(dd,J＝9.2,6.4Hz,1H),3.62(t,J＝8.4Hz,1H),2.88-2.67(m,4H),2.44(s,4H),2.32(s,3H),1.95-1.65(m,8H). 13C NMR(151MHz,CDCl 3)δ164.00(s),162.98(s),157.51(s),156.73(s),136.94(s),136.46(s),133.71(s),131.92(s),127.06(s),120.84(s),114.19(s),109.75(s),106.52(s),81.67(s),81.01(s),76.85(s),73.38(s),72.38(s),71.69(s),53.44(s),51.19(s),48.64(s),46.41(s),28.58(s).HRMS(ESI)calcd for C 29H 38ClN 6O 5[M+H] +585.2587；found 585.2582.
实施例25
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)苯基)丙烯酰胺(LS 5-102)的合成
Figure PCTCN2021143765-appb-000059
合成方法同实施例16LS 5-12的合成，只是以4-(二甲氨基)哌啶代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.87(s,1H),8.09(s,1H),7.10-6.95(m,2H),6.39-6.26(m,2H),5.92(d,J＝4.8Hz,1H),5.76(dd,J＝7.3,4.1Hz,1H),4.94(t,J＝5.2Hz,1H),4.44(t,J＝5.2Hz,1H),4.26-4.15(m,2H),4.10(dd,J＝10.0,5.2Hz,1H),3.91(dd,J＝8.8,6.8Hz,1H),3.59(t,J＝8.4Hz,1H),3.01(d,J＝12.0Hz,2H),2.76-2.63(m,2H),2.56-2.43(m,7H),2.07(d,J＝12.4Hz,2H),1.80-1.66(m,2H). 13C NMR(151MHz,CDCl 3)δ164.02(s),163.05(s),157.46(s),156.72 (s),136.65(s),136.34(s),133.80(s),131.86(s),127.17(s),120.84(s),114.13(s),109.74(s),106.62(s),81.66(s),81.01(s),76.85(s),73.42(s),72.36(s),71.70(s),61.92(s),52.40(d,J＝12.6Hz),41.96(s),30.00(s).HRMS(ESI)calcd for C 26H 34ClN 6O 5[M+H] +545.2274；found 545.2257.
实施例26
N-(5-((5-氯嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 5-126)的合成
Figure PCTCN2021143765-appb-000060
合成方法同实施例16LS 5-12的合成，只是以2,5-二氯嘧啶代替2,4,5-三氯嘧啶参与反应。
1H NMR(400MHz,CDCl 3)δ8.72(s,1H),8.54(s,1H),8.35(s,2H),7.48(dd,J＝8.8,2.5Hz,1H),7.20-7.10(m,2H),6.40(d,J＝16.4Hz,1H),6.26(dd,J＝16.8,10.0Hz,1H),5.78(d,J＝10.4Hz,1H),3.02(d,J＝12.0Hz,2H),2.92-2.30(m,14H),2.07(d,J＝12.4Hz,2H),1.67(d,J＝10.8Hz,2H).HRMS(ESI)calcd for C 23H 31ClN 7O[M+H] +456.2273；found 456.2274.
实施例27
N-(5-((4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)-5-甲基嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 5-133)的合成
Figure PCTCN2021143765-appb-000061
合成方法同实施例16LS 5-12的合成，只是以5-甲基-2,4-二氯嘧啶代替2,4,5-三氯嘧啶参与反应。
1H NMR(400MHz,CDCl 3)δ8.97(s,1H),8.78(s,1H),7.96(s,1H),7.18(s,1H),7.07(d,J＝8.6Hz,1H),7.04-6.95(m,1H),6.36(d,J＝16.8Hz,1H),6.24(dd,J＝16.8,10.0Hz,1H),5.91(q,J＝6.4Hz,1H),5.75(d,J＝11.2Hz,1H),4.95(t,J＝5.1Hz,1H),4.51(t,J＝5.1Hz,1H),4.33-4.22(m,2H),4.04-3.88(m,2H),3.62-3.54(m,1H),2.98(d,J＝11.6Hz,2H),2.75-2.29(m,14H),2.05-2.00(m,5H),1.63(d,J＝8.8Hz,2H).HRMS(ESI)calcd for C 30H 42N 7O 5[M+H] +580.3242；found 580.3250.
实施例28
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-乙基哌嗪-1-基)苯基)丙烯酰胺(LS 5-143)的合成
Figure PCTCN2021143765-appb-000062
合成方法同实施例16LS 5-12的合成，只是以N-乙基哌嗪代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.98(s,1H),8.76(s,1H),8.15(s,1H),7.19(d,J＝8.4Hz,1H),7.10-6.94(m,2H),6.43-6.33(m,1H),6.27(dd,J＝16.9,10.0Hz,1H),5.98(d,J＝6.0Hz,1H),5.79(dd,J＝10.0,1.2Hz,1H),4.99(t,J＝5.3Hz,1H),4.50(t,J＝5.3Hz,1H),4.32-4.18(m,2H),4.14(dd,J＝9.8,5.2Hz,1H),3.96(dd,J＝9.0,6.5Hz,1H),3.65-3.58(m,1H),3.19-2.45(m,10H),1.55-1.44(m,3H). 13C NMR(151MHz,CDCl 3)δ164.01(s),162.96(s),157.47(s),156.70(s),136.82(s),135.87(s),133.87(s),131.89(s),127.11(s),121.25(s),114.27(s),109.64(s),106.58(s),81.68(s),81.01(s),76.85(s),73.38(s),72.38(s),71.69(s),53.76(s),52.49(s),52.42(s),12.06(s).HRMS(ESI)calcd for C 25H 32ClN 6O 5[M+H] +531.2117；found 531.2126.
实施例29
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(3-(二甲氨基)吡咯-1-基)苯基)丙烯酰胺(LS 5-150)的合成
Figure PCTCN2021143765-appb-000063
合成方法同实施例16LS 5-12的合成，只是以3-(二甲氨基)吡咯代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.92(s,1H),8.73(s,1H),8.14(s,1H),7.11(d,J＝8.2Hz,1H),7.02-6.94(m,2H),6.58-6.27(m,2H),5.97(d,J＝6.0Hz,1H),5.76(d,J＝11.6Hz,1H),5.01-4.93(m,1H),4.52-4.45(m,1H),4.26(dd,J＝9.8,6.0Hz,2H),4.14(dd,J＝9.8,5.2Hz,1H),3.95(dd,J＝9.2,6.4Hz,1H),3.65-3.58(m,1H),3.24-2.95(m,5H),2.53-2.24(m,6H),2.20(s,1H),2.07(s, 1H). 13C NMR(151MHz,CDCl 3)δ164.00(s),163.15(s),157.53(s),156.70(s),136.18(s),134.15(s),134.04(d,J＝7.7Hz),131.76(s),127.26(s),120.52(d,J＝4.9Hz),114.67(s),110.56(s),106.44(s),81.68(d,J＝3.0Hz),81.02(s),73.33(d,J＝2.6Hz),72.38(s),71.71(d,J＝4.2Hz),65.77(s),57.33(s),52.48(d,J＝4.4Hz),44.02(s),30.14(s).HRMS(ESI)calcd for C 25H 32ClN 6O 5[M+H] +531.2117；found 531.2111.
实施例30
N-(2-((1S,4S)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)苯基)丙烯酰胺(LS 5-152)的合成
Figure PCTCN2021143765-appb-000064
合成方法同实施例16LS 5-12的合成，只是以(1S,4S)-2-氧-5-氮杂双环[2.2.1]庚烷代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.82(s,2H),8.14(s,1H),7.51(s,1H),7.23-6.96(m,2H),6.54-6.29(m,2H),5.94(d,J＝4.0Hz,1H),5.80(d,J＝11.2Hz,1H),5.00(t,J＝5.3Hz,1H),4.71-4.59(m,1H),4.51(t,J＝5.2Hz,1H),4.37-4.11(m,3H),4.11-3.99(m,1H),3.98-3.82(m,2H),3.81-3.69(m,1H),3.67-3.45(m,2H),3.28(s,1H),2.63(d,J＝7.2Hz,1H),2.27-2.13(m,1H),2.10-1.99(m,1H). 13C NMR(151MHz,CDCl 3)δ164.01(s),162.97(s),157.50(s),156.75(s),135.85(s),133.97(s),133.68(s),131.71(s),127.42(s),121.42(s),114.47(s),111.01(s),106.56(s),81.66(s),81.03(s),73.41(s),72.36(s),72.28(s),71.71(s),62.09(s),59.74(s),36.42(s).HRMS(ESI)calcd for C 24H 27ClN 5O 6[M+H] +516.1644；found 516.1640.
实施例31
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-羟基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((1R,4R)-5-甲基-2,5-二氮杂二环[2.2.1]庚烷-2-基)苯基)丙烯酰胺(LS 5-154)的合成
Figure PCTCN2021143765-appb-000065
合成方法同实施例16LS 5-12的合成，只是以2-甲基-2,5-二氮杂二环[2.2.1]庚烷代替1- 甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.93(s,1H),8.52(s,1H),8.13(s,1H),7.17-7.04(m,2H),6.99(d,J＝6.4Hz,1H),6.69-6.54(m,1H),6.40(dd,J＝16.8,1.4Hz,1H),5.97(d,J＝5.2Hz,1H),5.78(dd,J＝10.2,1.4Hz,1H),4.96(t,J＝5.3Hz,1H),4.48(t,J＝5.3Hz,1H),4.25(dd,J＝9.8,5.9Hz,2H),4.14(dd,J＝9.9,5.1Hz,1H),4.07-3.91(m,2H),3.79(d,J＝17.2Hz,2H),3.67-3.57(m,1H),2.96(d,J＝10.3Hz,1H),2.82-2.56(m,5H),2.15-2.06(s,2H). 13C NMR(151MHz,CDCl 3)δ163.99(s),162.97(s),157.54(s),156.75(s),135.47(s),134.96(s),133.84(s),131.78(s),127.29(s),121.19(s),114.46(s),110.90(s),106.43(s),81.64(s),81.02(s),73.37(s),72.36(s),71.73(s),63.91(s),63.34(s),60.85(s),56.21(s),42.15(s),33.96(s).HRMS(ESI)calcd for C 25H 30ClN 6O 5[M+H] +529.1961；found 529.1965.
实施例32
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-甲氧基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 6-16)的合成
Figure PCTCN2021143765-appb-000066
步骤1.(3R,3aR,6R,6aR)-6-甲氧基六氢呋喃[3,2-b]呋喃-3-醇(20)的合成
将1,4:3,6-双脱水甘露醇(8,1.0g,6.84mmol)溶于30毫升二氯甲烷中，向体系中加入氧化银(2.37g,10.2mmol)，避光搅拌10分钟后，滴加碘甲烷(0.43mL,6.84mmol)后，体系避光反应过夜。次日，待反应完全，经硅藻土抽滤后，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物20(0.8g,77％)。 1H NMR(400MHz,CDCl 3)δ4.57(t,J＝4.8Hz,1H),4.52(t,J＝5.2Hz,1H),4.28(q,J＝6.0Hz,1H),4.08(dd,J＝8.6,6.4Hz,1H),4.02-3.91(m,2H),3.76-3.62(m,2H),3.47(d,J＝7.7Hz,3H).MS(ESI)m/z  162.1[M+H] +.
步骤2. 2,5-二氯-4-(((3R,3aR,6R,6aR)-6-甲氧基六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶(21)的合成
将中间体20(0.2g,1.25mmol)溶于5毫升1,4-二氧六环溶剂中，向体系加入叔丁醇钠(0.14g,1.38mmol)，室温搅拌10分钟后，向体系滴加原料1(143μL,1.25mmol)，反应于室温搅拌过夜。待反应完全，减压旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物21(0.29g,80％)。 1H NMR(400MHz,CDCl 3)δ8.34(s,1H),5.56(dd,J＝10.2,5.7Hz,1H),4.96(t,J＝5.4Hz,1H),4.57(t,J＝4.9Hz,1H),4.21(dd,J＝10.4,4.4Hz,1H),4.09(dd,J＝10.4,5.7Hz,1H),4.01-3.95(m,1H),3.95-3.87(m,1H),3.67(t,J＝8.4Hz,1H),3.50(s,3H).MS(ESI)m/z 307.0[M+H] +.
后续合成步骤的方法同实施例16LS 5-12的合成，只是将反应原料进行替代参与反应。
化合物22： 1H NMR(400MHz,CDCl 3)δ8.81(dd,J＝6.4,2.7Hz,1H),8.19(s,1H),7.82(s,1H),7.50-7.41(m,1H),7.30-7.20(m,1H),5.59(dd,J＝9.5,5.6Hz,1H),5.02(t,J＝5.4Hz,1H),4.61(t,J＝4.9Hz,1H),4.29(dd,J＝10.5,3.7Hz,1H),4.09(dd,J＝10.6,5.4Hz,1H),4.02-3.95(m,1H),3.95-3.87(m,1H),3.75-3.66(m,1H),3.50(s,3H).MS(ESI)m/z 427.1[M+H] +.
化合物23： 1H NMR(400MHz,CDCl 3)δ8.47(d,J＝2.4Hz,1H),8.14(s,1H),7.29(dd,J＝8.8,2.5Hz,1H),7.23(s,1H),7.11(d,J＝8.8Hz,1H),5.54(dd,J＝10.1,5.6Hz,1H),4.99(t,J＝5.4Hz,1H),4.61(t,J＝4.9Hz,1H),4.22(dd,J＝10.3,4.3Hz,1H),4.09(dd,J＝10.3,5.6Hz,1H),4.02-3.95(m,1H),3.95-3.86(m,1H),3.74-3.67(m,1H),3.49(s,3H),3.33-3.22(m,2H),2.72-2.25(m,14H),1.91(d,J＝11.6Hz,2H),1.78-1.69(m,2H).MS(ESI)m/z 591.2[M+H] +.
化合物24： 1H NMR(400MHz,CDCl 3)δ8.11(s,1H),6.96(d,J＝2.4Hz,1H),6.91(d,J＝8.4Hz,1H),6.85-6.75(m,2H),5.44(q,J＝5.6Hz,1H),4.90(t,J＝5.3Hz,1H),4.58(t,J＝4.9Hz,1H),4.13(dd,J＝5.6,1.5Hz,2H),4.06-3.95(m,3H),3.95-3.90(m,1H),3.72(t,J＝8.7Hz,1H),3.50(s,3H),3.17(d,J＝12.0Hz,2H),2.85-2.43(m,14H),2.01(d,J＝12.0Hz,2H),1.72(d,J＝10.8Hz,2H).MS(ESI)m/z 561.3[M+H] +.
化合物LS 6-16： 1H NMR(400MHz,CDCl 3)δ8.92(s,1H),8.75(s,1H),8.11(s,1H),7.22(s,1H),7.08(d,J＝8.8Hz,1H),7.01(d,J＝8.4Hz,1H),6.36(dd,J＝16.9,1.2Hz,1H),6.25(dd,J＝16.9,10.0Hz,1H),5.90(q,J＝5.2Hz,1H),5.76(dd,J＝10.0,1.2Hz,1H),5.00(t,J＝5.3Hz,1H),4.53(t,J＝4.8Hz,1H),4.22-4.09(m,2H),4.04-3.93(m,1H),3.92-3.82(m,1H),3.75-3.67(m,1H),3.47(s,3H),2.99(d,J＝12.0Hz,2H),2.80-2.26(m,14H),2.06(d,J＝11.6Hz,2H),1.69-1.61(m,2H).MS(ESI)m/z 614.3[M+H] +.
实施例33
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-甲氧基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)以乙基)(甲基)氨基)苯基)丙烯酰胺(LS 6-45)的合成
Figure PCTCN2021143765-appb-000067
合成方法同实施例32LS 6-16的合成，只是以N,N,N'-三甲基乙二胺代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ10.19(s,1H),8.97(s,1H),8.13(s,1H),7.22-6.96(m,3H),6.50-6.25(m,2H),5.93(d,J＝5.6Hz,1H),5.78-5.62(m,1H),5.02(t,J＝5.3Hz,1H),4.55(t,J＝4.8Hz,1H),4.28-4.13(m,2H),3.99(t,J＝7.2Hz,1H),3.94-3.85(m,1H),3.72(t,J＝8.4Hz,1H),3.48(s,3H),2.87(t,J＝4.8Hz,2H),2.68(s,3H),2.52-2.06(m,8H). 13C NMR(151MHz,CDCl 3)δ164.21(s),163.57(s),157.53(s),156.59(s),136.80(s),136.64(s),136.16(s),132.04(s),126.58(s),122.57(s),114.25(s),110.29(s),106.64(s),81.46(s),80.04(s),76.94(s),71.93(s),69.89(s),58.37(s),57.38(s),56.62(s),45.55(s),43.30(s).HRMS(ESI)calcd for C 25H 34ClN 6O 5[M+H] +533.2274；found 533.2251.
实施例34
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-甲氧基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-吗啡啉苯基)丙烯酰胺(LS 6-48)的合成
Figure PCTCN2021143765-appb-000068
合成方法同实施例32LS 6-16的合成，只是以吗啡啉代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.96(s,1H),8.83(s,1H),8.13(s,1H),7.38(s,1H),7.14(d,J＝8.4Hz,1H),7.07(d,J＝8.0Hz,1H),6.38(dd,J＝16.8,1.6Hz,1H),6.28(dd,J＝16.9,10.0Hz,1H),5.93(q,J＝5.0Hz,1H),5.79(dd,J＝10.0,1.4Hz,1H),5.01(t,J＝5.3Hz,1H),4.54(t,J＝4.8Hz,1H),4.25-4.11(m,2H),4.03-3.95(m,1H),3.93-3.78(m,5H),3.76-3.67(m,1H),3.48(s,3H),2.93-2.75(m,4H).HRMS(ESI)calcd for C 24H 29ClN 5O 6[M+H] +518.1801；found 518.1791.
实施例35
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-甲氧基六氢呋喃并[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((S)-3,4-二甲基哌嗪-1-基)苯基)丙烯酰胺(LS 6-49)的合成
Figure PCTCN2021143765-appb-000069
合成方法同实施例32LS 6-16的合成，只是以(S)-1,2-二甲基哌嗪代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ8.92(s,1H),8.79(s,1H),8.13(s,1H),7.19-6.98(m,3H),6.39(d,J＝16.8Hz,1H),6.26(dd,J＝16.9,10.1Hz,1H),5.89(q,J＝5.2Hz,1H),5.78(dd,J＝10.0,1.2Hz,1H),5.01(t,J＝5.3Hz,1H),4.55(t,J＝4.8Hz,1H),4.22-4.08(m,2H),4.02-3.95(m,1H),3.93-3.86(m,1H),3.76-3.68(m,1H),3.49(s,3H),3.13-2.97(m,2H),2.94-2.77(m,2H),2.77-2.68(m,1H),2.56-2.36(m,5H),1.19(d,J＝6.0Hz,3H).HRMS(ESI)calcd for C 26H 34ClN 6O 5[M+H] +545.2274；found 545.2265.
实施例36
N-(5-((5-氯-4-(((3R,3aR,6S,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)丙烯酰胺(LS 6-59)的合成
Figure PCTCN2021143765-appb-000070
步骤1. 2,5-二氯-4-(((3R,3aR,6S,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶(26)的合成
将中间体9(0.58g,2.0mmol)溶于10毫升无水二氯甲烷中，于-78摄氏度下缓慢滴加二乙胺基三氟化硫(DAST,1.6g,10.0mmol)。加毕，继续反应30分钟后，将反应体系升至室温搅拌过夜。待反应完全，冰浴环境下向体系中滴加水淬灭反应，以二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物26(0.22g,37％)。 1H NMR(400MHz,CDCl 3)δ8.36(s,1H),5.63(td,J＝5.4,3.8Hz,1H),5.22-5.00(m,2H),4.66(dd,J＝11.2,5.2Hz,1H),4.16-3.87(m,4H). 19F NMR(376MHz,CDCl 3)δ-188.91–-189.36(m,1F).MS(ESI)m/z 295.0[M+H] +.
后续合成步骤的方法同实施例32LS 6-16的合成，只是将反应原料进行替代参与反应。
化合物27： 1H NMR(400MHz,CDCl 3)δ8.83(dd,J＝6.4,2.6Hz,1H),8.21(s,1H),7.85(s,1H),7.53-7.45(m,1H),7.30-7.20(m,1H),5.70-5.62(m,1H),5.22-5.05(m,2H),4.70(dd,J＝11.6,5.1Hz,1H),4.18-3.92(m,4H).MS(ESI)m/z415.1[M+H] +.
化合物28： 1H NMR(400MHz,CDCl 3)δ8.50(d,J＝2.8Hz,1H),8.14(s,1H),7.45(s,1H),7.39(dd,J＝8.8,2.4Hz,1H),7.10(d,J＝8.9Hz,1H),5.62(td,J＝5.3,3.5Hz,1H),5.19-5.01(m,2H),4.68(dd,J＝11.7,5.1Hz,1H),4.12-3.90(m,4H),3.32-3.24(m,2H),2.87-2.39(m,14H),1.93(d,J＝10.9Hz,2H),1.80-1.66(m,2H).MS(ESI)m/z 578.2[M+H] +.
化合物29： 1H NMR(600MHz,CDCl 3)δ8.07(s,1H),7.04(s,1H),6.96(d,J＝2.4Hz,1H),6.85(d,J＝8.4Hz,1H),6.77(dd,J＝8.4,2.4Hz,1H),5.47(dd,J＝9.5,5.4Hz,1H),5.16-5.02(m,2H),4.62(dd,J＝11.7,5.0Hz,1H),4.18-3.82(m,6H),3.15-3.12(m,2H),3.05-2.43(m,14H),2.06-1.96(m,2H),1.81-1.67(m,2H).MS(ESI)m/z 548.3[M+H] +.
化合物LS 6-59： 1H NMR(400MHz,CDCl 3)δ9.02(s,1H),8.76(s,1H),8.14(s,1H),7.21-7.05(m,2H),6.95(d,J＝7.6Hz,1H),6.36(dd,J＝16.9,1.4Hz,1H),6.25(dd,J＝16.9,10.0Hz,1H),6.04(d,J＝4.7Hz,1H),5.77(dd,J＝9.9,1.5Hz,1H),5.20-4.95(m,2H),4.62(dd,J＝11.5,4.9Hz,1H),4.19-3.90(m,4H),3.01(d,J＝10.6Hz,2H),2.96-2.22(m,14H),2.07(d,J＝12.0Hz,2H),1.66(d,J＝11.6Hz,2H).HRMS(ESI)calcd for C 29H 38ClFN 7O 4[M+H] +602.2652；found 602.2641.
实施例37
N-(5-((5-氯-4-(((3R,3aR,6S,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 6-77)的合成
Figure PCTCN2021143765-appb-000071
合成方法同实施例36LS 6-59的合成，只是以N,N,N'-三甲基乙二胺代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(600MHz,CDCl 3)δ10.23(s,1H),9.07(s,1H),8.14(s,1H),7.25-7.08(m,2H),6.98(s,1H),6.50-6.26(m,2H),6.08(s,1H),5.77-5.66(m,1H),5.22-5.02(m,2H),4.63(dd,J＝11.5,5.0Hz,1H),4.19-3.92(m,4H),2.87(s,2H),2.69(s,3H),2.65-1.89(m,8H). 13C NMR(126MHz,CDCl 3)δ164.03(s),163.57(s),157.58(s),156.68(s),136.79(s),136.62(s),136.13(s),132.07(s),126.52(s),122.51(s),114.27(s),110.33(s),106.30(s),96.37(s),94.94(s),85.84(d,J＝30.9Hz),81.31(s),76.51(s),73.35(d,J＝21.9Hz),71.35(s),57.28(s),45.43(s),43.31(s). 19F NMR(376MHz,CDCl 3)δ-188.71(s).HRMS(ESI)calcd for C 24H 31ClFN 6O 4[M+H] +521.2074；found 521.2056.
实施例38
N-(5-((5-氯-4-(((3R,3aR,6S,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-乙基哌嗪-1-基)苯基)丙烯酰胺(LS 6-78)的合成
Figure PCTCN2021143765-appb-000072
合成方法同实施例36LS 6-59的合成，只是以N-乙基哌嗪代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ9.03(s,1H),8.74(s,1H),8.15(s,1H),7.20(d,J＝8.8Hz,1H),7.09(s,1H),7.01(d,J＝7.2Hz,1H),6.38(dd,J＝16.9,1.4Hz,1H),6.27(dd,J＝16.8,10.1Hz,1H),6.04(d,J＝4.3Hz,1H),5.79(dd,J＝10.0,1.2Hz,1H),5.18-5.03(m,2H),4.63(dd,J＝11.6,5.0Hz,1H),4.14-4.04(m,3H),4.00-3.94(m,1H),3.18-2.57(m,10H),1.30-1.25(m,3H).HRMS(ESI)calcd for C 25H 31ClFN 6O 4[M+H] +533.2074；found 533.2059.
实施例39
N-(5-((5-氯-4-(((3R,3aR,6R,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 6-88)的合成
Figure PCTCN2021143765-appb-000073
合成方法同实施例36LS 6-59的合成，只是以中间体3-2代替化合物9、以N,N,N'-三甲基乙二胺(34)代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
化合物30： 1H NMR(400MHz,CDCl 3)δ8.36(s,1H),5.64(d,J＝4.4Hz,1H),5.28-5.02(m,2H),4.67(dd,J＝11.4,4.9Hz,1H),4.15-3.89(m,4H). 19F NMR(376MHz,CDCl 3)δ-188.95–-189.30(m,1F).MS(ESI)m/z 295.0[M+H] +.
化合物31： 1H NMR(400MHz,CDCl 3)δ8.83(d,J＝6.0Hz,1H),8.20(s,1H),7.52(s,1H),7.46(d,J＝7.2Hz,1H),7.23(d,J＝9.6Hz,1H),5.66(s,1H),5.23-5.07(m,2H),4.70(dd,J＝11.2,4.7Hz,1H),4.16-4.00(m,4H).MS(ESI)m/z 415.1[M+H] +.
化合物32： 1H NMR(400MHz,CDCl 3)δ8.44(s,1H),8.17(s,1H),7.32(d,J＝9.0Hz,1H),7.19(d,J＝8.8Hz,1H),7.09(s,1H),5.62(d,J＝4.8Hz,1H),5.23-5.04(m,2H),4.71(dd,J＝12.0,4.7Hz,1H),4.19-3.91(m,4H),3.34(t,J＝6.4Hz,2H),2.84(s,3H),2.70(s,2H),2.41(s,6H). 19F NMR(376MHz,CDCl 3)δ-188.99–-189.05(m,1F).MS(ESI)m/z 497.1[M+H] +.
化合物LS 6-88： 1H NMR(600MHz,CDCl 3)δ10.22(s,1H),9.07(s,1H),8.14(s,1H),7.21-7.07(m,2H),6.98(d,J＝6.0Hz,1H),6.46-6.21(m,2H),6.07(s,1H),5.77-5.66(m,1H),5.16(t,J＝5.4Hz,1H),5.10(dd,J＝50.4,2.4Hz,1H),4.63(dd,J＝11.4,5.0Hz,1H),4.23-3.85(m,4H),2.98-2.75(m,2H),2.69(s,3H),2.61-1.91(m,8H). 13C NMR(126MHz,CDCl 3)δ164.04(s),163.62(s),157.54(s),156.68(s),136.77(s),136.59(s),136.13(s),132.07(s),126.54(s), 122.48(s),114.23(s),110.35(s),106.36(s),96.37(s),94.94(s),85.85(d,J＝30.8Hz),81.31(s),76.51(s),73.36(d,J＝21.9Hz),71.36(s),57.22(s),45.38(s),43.34(s). 19F NMR(376MHz,CDCl 3)δ-188.74(s).HRMS(ESI)calcd for C 24H 31ClFN 6O 4[M+H] +521.2074；found 521.2059.
实施例40
N-(5-((5-氯-4-(((3R,3aR,6R,6aS)-6-氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-(4-乙基哌嗪-1-基)苯基)丙烯酰胺(LS 6-89)的合成
Figure PCTCN2021143765-appb-000074
合成方法同实施例39 LS 6-88的合成，只是以N-乙基哌嗪代替N,N,N'-三甲基乙二胺(34)参与反应。
1H NMR(600MHz,CDCl 3)δ9.04(s,1H),8.82(s,1H),8.14(s,1H),7.17(d,J＝8.4Hz,1H),7.13(s,1H),6.98(d,J＝6.0Hz,1H),6.38(d,J＝17.4Hz,1H),6.27(dd,J＝16.9,10.1Hz,1H),6.05(s,1H),5.78(dd,J＝10.1,1.0Hz,1H),5.20-5.02(m,2H),4.63(dd,J＝11.5,4.9Hz,1H),4.18-3.89(m,4H),3.23-2.35(m,10H),1.25-1.07(m,3H).HRMS(ESI)calcd for C 25H 31ClFN 6O 4[M+H] +533.2074；found 533.2058.
实施例41
N-(5-((5-氯-4-(((3R,3aR,6aS)-6,6-二氟六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 6-105)的合成
Figure PCTCN2021143765-appb-000075
步骤1.中间体35的合成
向原料8(10g,68.4mmol)的80毫升DMF溶液中加入氢氧化钾(4.8g,85.5mmol)，室温搅拌30分钟后，向体系中缓慢滴加溴化苄(6.77mL,57mmol)，室温搅拌过夜。待反应完成，减压旋干大部分溶剂，乙酸乙酯萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物35(8.5g,53％)。 1H NMR(600MHz,DMSO-d 6)δ7.54-7.25(m,5H),4.81(d,J＝6.9Hz,1H),4.62(d,J＝11.6Hz,1H),4.53(t,J＝4.6Hz,1H),4.47(d,J＝11.6Hz,1H),4.29(t,J＝4.7Hz,1H),4.14-4.04(m,2H),3.88(dd,J＝8.3,7.0Hz,1H),3.84-3.79(m,1H),3.50(t,J＝8.4Hz,1H),3.40-3.35(m,1H).MS(ESI)m/z 237.1[M+H] +.
步骤2.中间体36的合成
向中间体35(0.62g,2.62mmol)的无水二氯甲烷(30毫升)溶液中分批加入戴斯-马丁氧化剂(DMP,5.55g,13.10mmol)，室温搅拌30分钟后，将反应体系升温至50摄氏度反应过夜。待反应完成，向体系中加入硫代硫酸钠水溶液淬灭反应，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物36(0.38g,61％)。 1H NMR(600MHz,CDCl 3)δ7.42-7.28(m,5H),4.93(t,J＝5.4Hz,1H),4.75(d,J＝11.9Hz,1H),4.63(d,J＝11.9Hz,1H),4.32-4.23(m,2H),4.16(dd,J＝10.9,6.0Hz,1H),4.07(d,J＝17.4Hz,1H),3.99(dd,J＝9.3,5.8Hz,1H),3.84(dd,J＝9.3,6.5Hz,1H).MS(ESI)m/z 235.1[M+H] +.
步骤2.中间体37的合成
将中间体36(0.4g,1.7mmol)溶于10毫升无水二氯甲烷中，于-78摄氏度下缓慢滴加二乙胺基三氟化硫(DAST,1.38g,8.5mmol)。加毕，继续反应30分钟后，将反应体系升至室温搅拌过夜。待反应完全，冰浴环境下向体系中滴加水淬灭反应，以二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物37(0.15g,35％)。 1H NMR(600MHz,CDCl 3)δ7.44-7.28(m,5H),4.75(dd,J＝10.5,4.4Hz,2H),4.57(d,J＝11.8Hz,1H),4.41(dd,J＝10.0,4.8Hz,1H),4.13-3.96(m,4H),3.74(t,J＝8.6Hz,1H). 19F NMR(376MHz,CDCl 3)δ-106.76(d,J＝246.3Hz,1F),-126.84(d,J＝246.6Hz,1F).MS(ESI)m/z 257.1[M+H] +.
步骤3.中间体38的合成
将中间体37(0.21g,0.82mmol)溶于无水乙醇(8mL)中，以氢气球保护反应，加入催化量10％钯/碳。将反应体系升温至60摄氏度反应约3小时。待反应完全，经硅藻土抽滤后，旋干大部分溶剂，以二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得化合物38(0.12g,85％)。 1H NMR(400MHz,CDCl 3)δ4.68(t,J＝4.9Hz,1H),4.43(dd,J＝9.6,4.8Hz,1H),4.40-4.35(m,1H),4.14-4.01(m,2H),3.94(ddd,J＝25.2,10.5,6.0Hz,1H),3.60(dd,J＝9.2,7.5Hz,1H),2.51(s,1H). 19F NMR(376MHz,CDCl 3)δ-107.40(d,J＝247.4Hz,1F),-126.72(d,J＝247.4Hz,1F).MS(ESI)m/z 167.1[M+H] +.
步骤4.中间体39的合成
在冰浴下，向中间体38(0.12g,0.72mmol)的四氢呋喃(THF,4mL)溶液中分批加入60％氢化钠(58mg,1.44mmol)，继续搅拌10分钟后，向体系中滴加2,4,5-三氯嘧啶(1,0.14g,0.72mmol)，并于室温搅拌过夜。待反应完全，向反应体系中缓慢滴加冰水淬灭。旋干大部分溶剂，二氯甲烷萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得白色固体39(144mg，64％)。 1HNMR(400MHz,CDCl 3)δ8.37(s,1H),5.60(q,J＝5.5Hz,1H),5.15(t,J＝5.5Hz,1H),4.49(dd,J＝10.6,5.3Hz,1H),4.24-4.15(m,2H),4.04-3.93(m,2H). 19F NMR(376MHz,CDCl 3)δ-107.24–-108.04(m,1F),-127.41–-128.09(m,1F).MS(ESI)m/z 313.0[M+H] +.
中间体40、41、42及LS 6-105的合成方法同实施例39，只是将反应原料进行替代参与反应。
化合物40： 1H NMR(400MHz,CDCl 3)δ8.81(dd,J＝6.6,2.7Hz,1H),8.22(s,1H),7.52(s,1H),7.46(dt,J＝8.9,3.1Hz,1H),7.29-7.21(m,1H),5.65(q,J＝5.3Hz,1H),5.20(t,J＝5.5Hz,1H),4.53(dd,J＝10.8,5.4Hz,1H),4.30-4.15(m,2H),4.12-3.96(m,2H). 19F NMR(376MHz,CDCl 3)δ-107.31–-108.10(m,1F),-124.39(s,1F),-127.80–-128.52(m,1F).MS(ESI)m/z433.1[M+H] +.
化合物41： 1H NMR(400MHz,CDCl 3)δ8.41(d,J＝2.6Hz,1H),8.18(s,1H),7.30-7.26(m,1H),7.17(d,J＝9.0Hz,1H),7.11(s,1H),5.60(q,J＝5.5Hz,1H),5.18(t,J＝5.5Hz,1H),4.53(dd,J＝10.6,5.3Hz,1H),4.20(d,J＝5.4Hz,2H),4.0-3.95(m,2H),3.28(t,J＝7.2Hz,2H),2.84(s,3H),2.61(t,J＝7.2Hz,2H),2.33(s,6H).MS(ESI)m/z 515.2[M+H] +.
化合物LS 6-105： 1H NMR(400MHz,CDCl 3)δ10.24(s,1H),9.14(s,1H),8.15(s,1H),7.24-7.11(m,2H),6.88(d,J＝7.2Hz,1H),6.496.25(m,2H),6.12(d,J＝5.7Hz,1H),5.77-5.68(m,1H),5.24(t,J＝5.4Hz,1H),4.44(dd,J＝10.5,5.1Hz,1H),4.29(dd,J＝9.5,6.5Hz,1H),4.16(dd,J＝9.9,5.5Hz,1H),4.10-3.92(m,2H),2.87(s,2H),2.69(s,3H),2.42-2.10(m,8H). 13C NMR(151MHz,CDCl 3)δ163.89(s),163.61(s),157.55(s),156.89(s),136.74(s),136.69(s),136.21(s),131.97(s),126.74(s),122.65(s),114.23(s),110.05(s),106.32(s),81.71(dd,J＝37.6,17.8Hz),81.47(d,J＝3.8Hz),76.09(s),71.64(s),70.88(dd,J＝31.4,28.8Hz),57.35(s),56.59(s),45.52(s),43.29(s). 19F NMR(376MHz,CDCl 3)δ-107.03–-108.06(m,1F),-127.65–-128.43(m,1F).HRMS(ESI)calcd for C 24H 30ClF 2N 6O 4[M+H] +539.1980；found 539.1962.
实施例42
N-(5-((5-氯-4-(((3R,3aS,6aR)-六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 6-121)的合成
Figure PCTCN2021143765-appb-000076
步骤1.中间体42的合成
冰浴下，向中间体35(0.2g,0.85mmol)的5毫升吡啶溶液中缓慢加入三氟甲磺酸酐(0.17mL,1.0mmol)。反应升至室温后，继续搅拌2小时。以4N盐酸溶液淬灭反应，乙酸乙酯萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得42(0.24g,77％yield)； 1H NMR(400MHz,CDCl 3)δ7.46-7.27(m,5H),5.20(dd,J＝10.0,5.2Hz,1H),4.73(dd,J＝11.2,4.8Hz,2H),4.58(d,J＝11.8Hz,1H),4.49(t,J＝5.0Hz,1H),4.17(dd,J＝10.9,4.2Hz,1H),4.11-4.05(m,1H),4.04-3.97(m,2H),3.73(t,J＝8.7Hz,1H).MS(ESI)m/z369.1[M+H] +.
步骤2.中间体43的合成
室温下，向中间体42(0.2g,0.54mmol)的乙腈溶液中加入硼氢化钠(62mg,1.63mmol)，反应升温至50摄氏度搅拌过夜。次日，反应液以水淬灭后，乙酸乙酯萃取三次，合并有机相，以饱和食盐水洗涤、无水硫酸钠干燥，旋干，柱层析分离得43(86mg,72％yield)； 1HNMR(400MHz,CDCl 3)δ7.41-7.26(m,5H),4.78(d,J＝12.0Hz,1H),4.65(td,J＝4.8,1.7Hz,1H),4.58(d,J＝12.0Hz,1H),4.49(t,J＝4.7Hz,1H),4.08-3.90(m,3H),3.84(dd,J＝8.8,6.5Hz,1H),3.69(dd,J＝8.7,7.7Hz,1H),2.11-1.97(m,2H).MS(ESI)m/z 221.1[M+H] +.
步骤3.中间体44的合成
室温下，将50毫克10％钯/碳加入到中间体43(0.3g,1.36mmol)的乙醇溶液(5mL)中，反应体系在氢气条件下，于60摄氏度反应4小时。反应结束后，硅藻土抽滤并浓缩可得无色油状物44， 1H NMR(400MHz,CDCl 3)δ4.59(t,J＝4.8Hz,1H),4.46(t,J＝5.1Hz,1H),4.31-4.19(m,1H),4.05(td,J＝8.3,2.4Hz,1H),3.89-3.76(m,2H),3.66(dd,J＝9.6,4.9Hz,1H),2.79(d,J＝6.2Hz,1H),2.14(dd,J＝13.0,5.3Hz,1H),2.09-1.96(m,1H).MS(ESI)m/z 131.1[M +H] +.
步骤4.中间体45、46、47及目标产物LS6-121的合成同LS5-12，只是将反应原料进行相应替代参与反应。
化合物45： 1H NMR(400MHz,CDCl 3)δ8.34(s,1H),5.58(td,J＝5.4,3.2Hz,1H),4.86(t,J＝5.4Hz,1H),4.64(t,J＝5.2Hz,1H),4.15(dd,J＝10.7,3.2Hz,1H),4.01-3.88(m,2H),3.87-3.78(m,1H),2.13(dd,J＝13.4,5.2Hz,1H),2.05-1.89(m,1H).MS(ESI)m/z 277.0[M+H] +.
化合物46： 1H NMR(400MHz,CDCl 3)δ8.83(dd,J＝6.6,2.8Hz,1H),8.19(s,1H),7.44(dt,J＝8.9,3.2Hz,1H),7.31(s,1H),7.25-7.17(m,1H),5.59(td,J＝5.3,2.8Hz,1H),4.93(t,J＝5.4Hz,1H),4.68(t,J＝5.2Hz,1H),4.20(dd,J＝10.7,2.8Hz,1H),3.98-3.84(m,3H),2.15(dd,J＝13.2,4.6Hz,1H),2.01-1.87(m,1H).MS(ESI)m/z 397.1[M+H] +.
化合物47： 1H NMR(400MHz,CDCl 3)δ8.43(d,J＝2.6Hz,1H),8.15(s,1H),7.32-7.26(m,1H),7.13(d,J＝8.9Hz,1H),7.03(s,1H),5.56(td,J＝5.2,3.2Hz,1H),4.90(t,J＝5.4Hz,1H),4.69(t,J＝5.1Hz,1H),4.17(dd,J＝10.6,3.1Hz,1H),4.00-3.83(m,3H),3.22(t,J＝7.2Hz,2H),2.83(s,3H),2.57-2.48(m,2H),2.26(s,6H),2.14(dd,J＝13.1,4.0Hz,1H),1.98-1.90(m,1H).MS(ESI)m/z 479.2[M+H] +.
LS 6-121： 1H NMR(400MHz,CDCl 3)δ10.22(s,1H),8.93(s,1H),8.14(s,1H),7.24-6.97(m,3H),6.41(dd,J＝17.0,1.9Hz,1H),6.36-6.26(m,1H),5.90(d,J＝3.7Hz,1H),5.71(dd,J＝9.9,1.9Hz,1H),4.91(t,J＝5.3Hz,1H),4.63(t,J＝5.0Hz,1H),4.14(dd,J＝10.3,3.6Hz,1H),4.02(dd,J＝10.3,5.3Hz,1H),3.96-3.83(m,2H),2.92-2.77(m,2H),2.69(s,3H),2.50-2.13(m,8H),2.15-2.07(m,1H),1.95-1.90(m,1H). 13C NMR(151MHz,CDCl 3)δ164.30(s),163.60(s),157.61(s),156.52(s),136.87(s),136.63(s),136.15(s),132.06(s),126.57(s),122.54(s),114.30(s),110.44(s),106.59(s),83.69(s),81.89(s),71.49(s),69.01(s),57.38(s),56.58(s),45.54(s),43.33(s),34.21(s).HRMS(ESI)calcd for C 24H 32ClN 6O 4[M+H] +503.2168；found 503.2160.
实施例43
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-乙氧基六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 7-13)的合成
Figure PCTCN2021143765-appb-000077
合成方法同实施例32LS 6-16的合成，只是以碘乙烷代替碘甲烷、以N,N,N'-三甲基乙二胺代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ10.22(s,1H),8.95(s,1H),8.13(s,1H),7.16(d,J＝8.6Hz,1H),7.08(d,J＝8.8Hz,1H),7.04(s,1H),6.45-6.26(m,2H),5.95-5.87(m,1H),5.70(dd,J＝9.9,1.7Hz,1H),5.00(t,J＝5.3Hz,1H),4.51(t,J＝4.5Hz,1H),4.22(dd,J＝10.0,4.5Hz,1H),4.15(dd,J＝10.1,5.8Hz,1H),4.03-3.90(m,2H),3.79-3.67(m,2H),3.60-3.51(m,1H),2.85(t,J＝4.8Hz,2H),2.68(s,3H),2.49-2.00(m,8H),1.30-1.25(m,3H). 13C NMR(151MHz,CDCl 3)δ164.25(s),163.55(s),157.54(s),156.56(s),136.83(s),136.62(s),136.16(s),132.04(s),126.57(s),122.57(s),114.25(s),110.28(s),106.63(s),81.37(s),80.41(s),79.94(s),71.97(s),69.89(s),66.32(s),57.40(s),56.63(s),45.56(s),43.31(s),15.39(s).HRMS(ESI)calcd for C 26H 36ClN 6O 5[M+H] +547.2430；found 547.2420.
实施例44
N-(5-((5-氯-4-(((3R,3aR,6R,6aR)-6-异丙氧基六氢呋喃[3,2-b]呋喃-3-基)氧)嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)(甲基)氨基)苯基)丙烯酰胺(LS 7-18)的合成
Figure PCTCN2021143765-appb-000078
合成方法同实施例32LS 6-16的合成，只是以碘代异丙烷代替碘甲烷、以N,N,N'-三甲基乙二胺代替1-甲基-4-(哌啶-4-基)哌嗪盐酸盐(6)参与反应。
1H NMR(400MHz,CDCl 3)δ10.22(s,1H),8.94(s,1H),8.13(s,1H),7.16(d,J＝8.6Hz,1H),7.09(d,J＝7.8Hz,1H),7.05(s,1H),6.41(dd,J＝16.9,1.9Hz,1H),6.30(dd,J＝17.1,9.8Hz,1H),5.90(dd,J＝10.0,5.1Hz,1H),5.70(dd,J＝9.8,1.9Hz,1H),4.98(t,J＝5.2Hz,1H),4.45(t,J＝4.7Hz,1H),4.22(dd,J＝10.1,4.4Hz,1H),4.13(dd,J＝10.1,5.8Hz,1H),4.07-3.97 (m,1H),3.97-3.88(m,1H),3.81-3.65(m,2H),2.85(t,J＝5.6Hz,2H),2.69(s,3H),2.50-1.94(m,8H),1.25(d,J＝6.1Hz,3H),1.20(d,J＝6.1Hz,3H). 13C NMR(151MHz,CDCl 3)δ164.28(s),163.53(s),157.56(s),156.53(s),136.85(s),136.62(s),136.15(s),132.04(s),126.56(s),122.56(s),114.27(s),110.30(s),106.61(s),81.21(s),80.95(s),77.94(s),77.14(s),72.18(s),71.94(s),69.99(s),57.41(s),56.66(s),45.58(s),43.30(s),22.54(d,J＝69.9Hz).HRMS(ESI)calcd for C 27H 38ClN 6O 5[M+H] +561.2587；found 561.2571.
实施例45 2-氨基嘧啶类化合物激酶抑制活性测试
参照Z'-Lyte分析测试方法测试了本发明化合物针对JAK家族中四个成员(JAK1，JAK2，JAK3以及TYK2)的激酶抑制活性，其中每种激酶的ATP浓度为K m；由于JAK3相对于其他家族成员具有更高的ATP亲和力，因此本实施例还测试了当ATP浓度为1mM时，化合物对JAK3的激酶抑制活性。
测试方法：首先用DMSO将测试化合物配置成10mM母液，以3倍梯度连续稀释10个浓度，备用。用去离子水将5X反应缓冲液稀释成1X反应缓冲液(50mM HEPES pH 7.5,0.01％Brij-35,10mM MgCl 2,1mM EGTA)后，用1X反应缓冲液配置激酶与肽底物的混合液，以及磷酸化肽底物溶液，激酶浓度根据enzyme titration确定，肽底物和磷酸化肽底物的终浓度为2μM。于384孔板上每孔加入5μL激酶与肽底物的混合液，再用Echo520加样器加5nL测试化合物(终浓度为10μM起始)，在室温震荡混匀15min后，向孔内加入适量ATP以达到测试需要(备注：按照说明书推荐浓度，JAK1激酶测试时ATP浓度为75μM，JAK2激酶测试时ATP浓度为25μM，JAK3激酶测试时ATP浓度为10μM，TYK2激酶测试时ATP浓度为25μM)。每次试验需再设100％磷酸化孔(加5μL磷酸化肽底物溶液)，0％磷酸化孔(加5μL激酶与肽底物的混合液，不加测试化合物和ATP)，0％抑制孔(加5μL激酶与肽底物的混合液以及合适浓度的ATP)，均做复孔。将上述反应体系置30℃烘箱里反应1.5小时。用试剂盒Development buffer A缓冲液1：128稀释Development reagent B，每孔加入2.5μL稀释后的混合液，30℃反应1小时后。放置于Envision仪器上进行检测，在400nm的激发波长下，分别检测在460nm和535nm处的发射波长，通过计算比值，得出底物的磷酸化率，进一步计算出激酶的活性及抑制剂对激酶的影响。
表3中所列为化合物编号(对应于实施例1-44中的化合物编号)以及对应激酶活性结果。
表3化合物激酶抑制活性
Figure PCTCN2021143765-appb-000079
Figure PCTCN2021143765-appb-000080
Figure PCTCN2021143765-appb-000081
aATP浓度为K m； bATP浓度为1mM
由激酶活性检测结果可知(表3)，本发明化合物对JAK3亚型激酶表现出较高的选择性抑制活性。部分化合物(如LS 5-12，LS 5-77，LS 5-62，LS 5-66，LS 5-74，LS 5-88，LS 5-91，LS 5-102，LS 5-143，LS 5-150，LS 5-152，LS 5-154，LS 6-45，LS 6-49，LS 6-77，LS 6-88，LS 6-105、LS 6-121、LS 7-13、LS 7-18等)显示强效、选择性JAK3激酶抑制活性，并且在ATP浓度为1mM时，也能保持强效活性。
实施例46 2-氨基嘧啶类化合物对肿瘤细胞的体外增殖抑制活性
细胞株：人慢性髓系白血病细胞K562，人急性髓系白血病细胞U937，人T淋巴细胞白血病细胞HuT78，人T淋巴母细胞性淋巴瘤细胞系Jurkat。细胞购买于中国科学院干细胞库或ATCC。
方法：CCK-8(cell counting kit-8)法，具体如下：处于对数生长期的肿瘤细胞按1*10 4个细胞/孔的密度接种于96孔板，贴壁细胞过夜培养，悬浮细胞直接加药刺激。加入不同浓度的受试化合物(最大工作浓度10μM，按1：3比例稀释10个梯度)，每个浓度设置两个复孔，终体积200μL。药物处理72小时后，每孔加入10μL CCK-8试剂，继续孵育1-3小时。用超级酶标仪测量在450nm和650nm处的吸光值，导出增量Z(A450-A650)。使用GraphPad Prism 8.0.0按以下公式计算药物对细胞生长的抑制率：
抑制率(％)＝(OD对照-OD加药)/OD对照×100％
计算出其半数抑制浓度(IC 50)。
表4化合物对细胞增殖的抑制活性IC 50(μΜ)
Figure PCTCN2021143765-appb-000082
细胞实验结果发现(见表4)，本发明的2-氨基嘧啶类化合物可显著抑制多种血液癌细胞的增殖，部分化合物对肿瘤细胞的抑制活性优于阳性对照化合物Tofacitinib和PF-06651600。
实施例47代表性分子LS6-45的大鼠体内药代动力学性质
溶媒：静脉5％DMSO+10％聚乙二醇-15羟基硬脂酸酯+85％生理盐水，口服0.5％羟丙基甲基纤维素。
种属：SD大鼠，SPF级。动物转移自实验机构动物储备库(999M-017)，上海西普尔-必凯实验动物有限公司。
大鼠数量：每组3只，雄性。
口服给药组动物给药前禁食过夜(10-14小时)，给药4小时后给食。给药前称重，根据体重，计算给药量。通过静脉注射(iv,5mg/kg)或灌胃口服(po,15mg/kg)分别给药。给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h时间点于颈静脉采血，每个样品采集约0.20mL，肝素钠抗凝，采集后放置冰上，并于1小时之内离心分离血浆(离心条件：6800g，6分钟，2-8℃)。血浆样本在分析前存放时则放于-80℃冰箱内。生物样品分析方法及所有样品的分析由美迪西普亚医药科技(上海)有限公司分析实验室完成，分析样品的同时进行质控样品的日内准确度评价，并要求超过66.7％的质控样品的准确度在80-120％之间。通过不同时间点的血药浓度数据，运用Phoenix WinNonlin7.0计算药代动力学参数，提供药代动力学参数及其平均值和标准差。
实验中与动物处理、护理和治疗相关的所有程序均遵循实验动物护理伦理委员会协议和中国上海实验动物护理和使用指南。
实验结果如表5和图3所示。
表5化合物LS 6-45的药代动力学参数
LS6-45 iv(5mg/kg) po(15mg/kg)
动物数量 3 3
C max(ng/mL) 911.33±139.73 238.28±13.68
AUC (0-∞)(h*ng/mL) 536.99±9.89 333.50±45.52
CL(mL/min/kg) 155.22±2.83  
F(％)   20.66±2.83
大鼠体内药代动力学实验数据(表5和图3)显示，化合物LS6-45具有较高的C max、曲线下面积(AUC)，以及口服利用度(F＝20.66％)，表明该化合物具有良好的药代动力学性质，具有可口服的潜力。
实施例48代表性分子LS6-45的大鼠肝微粒体稳定性测试
首先预热0.1M磷酸盐钾缓冲液与5mM MgCl 2(即K/Mg缓冲液)，pH 7.4±0.1。准备待测化合物和参比化合物的加标溶液(500μM加标溶液：将5μL的10mM储备液加到95μL 的乙腈中；1.5μM微粒体加标溶液(0.75mg/mL)：向479.75μL K/Mg缓冲液中加入1.5μL的500μM加标溶液和18.75μL的20mg/mL肝微粒体)。然后将NADPH溶于K缓冲液中，制得6mM，5mg/mL的NADPH储备液。不同时间点(0-、5-、15-、30-、45-min)，将30μL含0.75mg/mL微粒体溶液的1.5μM的加标液分配到指定的检测板上。0min时，在0min板孔中加入150μL的含内标(IS)的乙腈，再加入15μL的NADPH储备液(6mM)。然后，将所有板在37摄氏度预孵育5分钟，加入15μL的NADPH原液(6mM)，启动反应并计时。在5min、15min、30min、45min时，分别在相应板孔中加入150μL含IS的乙腈溶液，停止反应。淬灭后，摇板10分钟(600rpm/min)，然后6000rpm/min离心15分钟。将每孔上清液80μL转入含有140μL超纯水的96孔样品板中进行LC/MS分析。生物样品分析方法及所有样品的分析由美迪西普亚医药科技(上海)有限公司分析实验室完成。
表6大鼠肝微粒体稳定性实验结果
Figure PCTCN2021143765-appb-000083
肝微粒体稳定性实验数据(表6)表明，参照分子Ketanserin的肝微粒体稳定半衰期T 1/2为18.71分钟，与历史数据一致，表明实验的可靠性。实施例分子LS6-45的半衰期T 1/2大于120分钟，表明该分子具有较高的肝微粒体稳定性。
实施例49代表性分子LS6-45可有效下调U937细胞中JAK3-STAT信号传导
细胞株：人急性髓系白血病细胞U937
时间依赖性实验：将受试分子LS6-45在固定浓度(100nM)与U937细胞共孵育，并于特定的作用时间进行细胞总蛋白的提取；剂量依赖性实验：将不同浓度下的受试分子LS6-45与U937细胞株共孵育10小时后，进行细胞总蛋白的提取。悬浮细胞U937离心去上清，用PBS洗2次后，加入适量裂解液混合物(RIPA裂解液(强)：磷酸酶抑制剂混合物(50×):PMSF＝100:1:1)，裂解30min，离心(4℃ 12000rpm×15min)。取上清，弃沉淀。取适量上清按BCA法测定蛋白浓度后按5：1的比例加入loading buffer，100℃煮沸7min，用于Western Blot实验或-80℃冰箱储存备用。
实验结果如图4所示，其中，Western Blot分析化合物LS6-45对JAK3、STAT3、STAT5蛋白及其时间/剂量依赖性磷酸化情况，以GAPDH作为内参。图4中，A为化合物LS6-45 (100nM)时间依赖处理U937细胞系，B为化合物LS6-45剂量依赖处理U937细胞系10h(800nM 1:4稀释五个浓度)。
Western blot实验结果(图4)显示，化合物LS6-45(100nM)与U937细胞共孵育4小时后可有效降低JAK3蛋白磷酸化水平，并在8小时左右完全抑制JAK3的磷酸化，同时下游蛋白STAT3和STAT5的磷酸化水平也呈时间依赖性减弱(图4中的A)。不同浓度的化合物LS6-45处理U937细胞系10h后，发现JAK3的磷酸化水平在50nM时就已经观察到明显的减弱，在200nM时JAK3的磷酸化水平几乎完全消失，同时下游蛋白STAT3和STAT5的磷酸化水平也呈剂量依赖性减弱(图4中的B)。说明受试分子LS6-45可时间/剂量依赖性地下调U937细胞中的JAK3信号通路。
实施例50 Wash-out(洗脱)实验
细胞株：人急性髓系白血病细胞U937
200nM受试化合物LS6-45与U937细胞共孵育10小时后，离心弃上清，收集细胞并以PBS清洗细胞以去除残留化合物，加入新鲜培养基后，继续于37摄氏度培养箱内进行细胞培养，并于0、0.5、1、2、4、8、12、16h收取细胞，提取蛋白进行western blot分析。
洗脱实验(图5)结果显示，在培养体系中去除化合物LS6-45后，U937细胞内的JAK3及下游蛋白STAT5的磷酸化水平在8小时内被持续抑制，并于8-12小时之间缓慢恢复。表明受试化合物LS6-45可以共价结合JAK3蛋白，并在8小时内持续发挥激酶抑制活性，是一类不可逆的JAK3小分子抑制剂。
实施例51化合物LS6-45抑制U937细胞增殖的机制研究
细胞周期检测：取生长状态良好的细胞调整密度至8×10 5个/mL，均匀地分入6孔板中，每孔2mL。然后将化合物梯度稀释后加入细胞悬液中，放培养箱培养24h。本实验采用BD CycletestTM Plus DNA Reagent Kit试剂盒进行染色。24h后收集细胞到15mL离心管，用PBS洗6孔板2次，合并洗液到15mL离心管，离心12000rpm×5min，弃去上清液。各管中加入5mL缓冲溶液，温和地重悬细胞。离心：12000rpm×5min，小心吸去上清液，留下约50μL液体不用弃去。各管中加入1.5mL缓冲溶液，温和地重悬细胞，小心转移到1.5mL EP管中。调整细胞浓度到1×10 6个/mL，本实验有5×10 5个细胞即可完成实验。离心：1200rpm×5min，小心吸去上清液。加入125μL的solutionA，温和地混匀，室温反应10min。加入100μL的solution B，温和地混匀，室温反应10min。加入200μL的solution C(PI)，温和地混匀，避光反应10min后上流式细胞仪检测。注意：PI染色的样品上机前样品需要纱网过滤；建议染色后的样品置冰上；细胞不要消化过度，操作总体用时尽量少；细胞不要过多，离心后1.5mL EP管底部看到薄薄的一层细胞即可。
细胞凋亡检测：取生长状态良好的细胞调整密度至8×10 5个/mL，均匀地分入6孔板中，每孔2mL。然后将化合物梯度稀释后加入细胞悬液中，放培养箱48h。本实验采用PE偶联Annexin-V凋亡检测试剂盒进行染色。48h后收集细胞到15mL离心管，用PBS洗6孔板2次，合并洗液到15mL离心管，离心(1200rpm×5min)，弃去上清液。1.5mL冷的PBS洗细胞重悬细胞并转移到1.5mL EP管中。用冷的PBS重复洗一次，1200rpm×5min离心后，弃去上清液。加入100μL 1×Binding Buffer(PBS稀释)，重悬细胞。加入2.5μL AnnexinV-PE和2.5μL 7-AAD。温和混匀，避光室温反应15min。加入400μL 1×Binding Buffer。1h内上流式细胞仪检测。注意：需要额外用有凋亡的细胞设置单标对照管，所有条件同样品管。染色后的样品置冰上。
图6为用化合物LS6-45处理U937细胞24h后，细胞周期(A)及细胞周期相关蛋白(B)的变化情况，以及用化合物LS6-45处理U937细胞48h后细胞凋亡的变化情况(C)。结果(图6)显示，不同浓度的化合物LS6-45处理后U937发生明显的G0/G1期阻滞，并呈现剂量依赖性(图6中的A)；同时细胞周期相关蛋白(CDK2、CDK4、CDK6、Cyclin B1、Cyclin D3和Cyclin E1)也在处理后显著下调(图6中的B)。化合物LS6-45处理后的U937细胞未见凋亡发生(图6中的C)。
实施例52化合物LS6-45小鼠体内抗肿瘤活性
本研究均按照暨南大学《实验动物饲养与使用规范》进行动物饲养和实验操作，并得到暨南大学动物伦理委员会的许可。
动物模型：4-6周龄的雄性CB-17 SCID小鼠。
U937细胞体外培养(1640+10％FBS+1％双抗)放大到50皿(10cm)，收集细胞到50mL离心管中，800rpm×5min，弃去上清，细胞富集到1个50mL无菌离心管中，PBS洗1次，800rpm×5min，用适量无菌PBS混悬细胞，计数并将细胞稀释到2×10 7个/mL；0.2mL/只，接种于动物右前腋皮下(尽量在动物20g之前接种)。
接种后5～7天(U937肿瘤生长速度很快，3天左右观察一次)，待瘤体增长至100～200mm 3大小时分组给药，注意剔除瘤体过大或过小的动物。将小鼠随机分为给药组和溶剂组，每组设置6只小鼠。每日灌胃50mg/kg，25mg/kg和12.5mg/kg剂量的药物两次(bid)，腹腔注射10mg/kg的药物一次(qd)，对照组给予等体积的溶剂。
给药周期10天，每天给药，每2天称取一次动物体重，量取瘤体积(分组当天记录初始瘤体积和体重)。瘤体积计算公式为：V＝π/6*a*b*b(a和b分别为肿瘤的长和宽)。给药结束后次日称重、量取瘤体积后处死动物解剖瘤体称重。瘤体用中性福尔马林固定备病理观察，可根据试验需要采集动物血液样本作血常规分析，采集动物主要脏器作病理分析。研究 结束时，所有动物都实施安乐死，取裸鼠的肿瘤、肝脏、肾脏和肺脏进行进一步分析。
实验结果如图7所示，其中A为各组小鼠体重变化曲线；B为各剂量组小鼠瘤体积生长曲线；C为实验终点时各处理组肿瘤大小；D为各处理组肿瘤组织中JAK3通路活化水平及周期相关蛋白的变化情况。**p<0.01。
体内活性结果(图7)显示，给药期间肿瘤的生长速度显著低于对照组，其中50mg/kg剂量组的肿瘤生长完全停滞甚至消退(图7中的B/C)。给药期间未见毒副作用，体重也没有下降的迹象(图7中的A)。对实验小鼠肿瘤组织进行western blot分析，与对照相比，化合物LS6-45对JAK3、STAT3和STAT5磷酸化以及细胞周期相关蛋白的抑制作用显著(图7中的D)。表明受试化合物具有良好的体内抗肿瘤活性。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对以下实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (23)

  1. 具有式(Ⅰ)所示结构的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物：
    Figure PCTCN2021143765-appb-100001
    其中，
    R 1选自：H、卤素、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基、一个或多个R 11取代或未取代的C 3-C 6环烷基、一个或多个R 11取代或未取代的C 1-C 6烷氧基、一个或多个R 11取代或未取代的C 3-C 6环烷氧基、甲酰胺基；
    R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5或-(CH 2) mOCR 3R 4R 5；其中，各m分别独立地为0、1、2或3；各R 3和各R 4分别独立地选自：H、一个或者多个R 12取代的C 1-C 6烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的3-12元单环、稠环、桥环或螺环；R 5选自：H、氰基或C 1-C 3烷基；各R 12分别独立地选自：H、卤素、羟基、氨基、C 1-C 3烷基、-C(＝O)NHR 13取代的C 1-C 3烷基、羟基取代的C 1-C 3烷基、C 3-C 6环烷基取代的C 1-C 3烷基、C 3-C 8杂环基取代的C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13、R 13取代或未取代的含有0、1、2或3个杂原子的4-8元单环、稠环、桥环或螺环；R 13为C 1-C 3烷基；所述杂原子为O、S和/或N；
    W、X、Y、Z各自独立地为N或-CR 6；其中，R 6选自：氢、卤素、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基、-NH-CN、-NHC(O)-CR 7＝CR 8R 9、-NHS(O) 2-CR 7＝CR 8R 9；R 7、R 8和R 9分别独立地选自：H、氰基、一个或多个R 11取代或未取代的C 1-C 6烷基；
    n为0或1；
    L为O或S；
    Figure PCTCN2021143765-appb-100002
    为一个或多个R 10取代的4-12元饱和或部分饱和的环原子中含有0、1、2或3个杂原子的单环、桥环、螺环或者稠环；其中，各R 10分别独立地选自：氢、卤素、羟基、一个或多个R 11取代或未取代的C 1-C 3烷基、一个或多个R 11取代或未取代的C 1-C 3烷氧基，所述杂原子为O、S和/或N；
    R 11选自：卤素、羟基、氨基、C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2、-C(＝O)R 13。
  2. 根据权利要求1所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其 特征在于，
    Figure PCTCN2021143765-appb-100003
    为一个或多个R 10取代的8-10元饱和或部分饱和的含有1、2或3个氧原子的稠合双环。
  3. 根据权利要求2所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，
    Figure PCTCN2021143765-appb-100004
    选自以下基团：
    Figure PCTCN2021143765-appb-100005
    Figure PCTCN2021143765-appb-100006
    其中，用*标记的手性碳原子的构型分别独立地为S构型或者R构型。
  4. 根据权利要求3所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，
    Figure PCTCN2021143765-appb-100007
    选自以下基团：
    Figure PCTCN2021143765-appb-100008
    其中，用*标记的手性碳原子的构型分别独立地为S构型或者R构型，各R 10分别独立地选自：卤素、羟基、C 1-C 3烷基、C 1-C 3烷氧基。
  5. 根据权利要求4所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，
    Figure PCTCN2021143765-appb-100009
    选自以下基团：
    Figure PCTCN2021143765-appb-100010
    Figure PCTCN2021143765-appb-100011
  6. 根据权利要求5所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，
    Figure PCTCN2021143765-appb-100012
    选自以下基团：
    Figure PCTCN2021143765-appb-100013
    其中，各R 10分别独立地选自：F、羟基、甲基、甲氧基、乙氧基、异丙氧基。
  7. 根据权利要求6所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，
    Figure PCTCN2021143765-appb-100014
    选自以下基团：
    Figure PCTCN2021143765-appb-100015
  8. 根据权利要求1所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 6选自：氢、卤素、C 1-C 3烷基、C 1-C 3烷氧基、-NH-CN、
    Figure PCTCN2021143765-appb-100016
    Figure PCTCN2021143765-appb-100017
  9. 根据权利要求1所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，W、X均为CH；Y和Z分别独立地选自N或CR 6，其中，R 6选自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基、-NH-CN、
    Figure PCTCN2021143765-appb-100018
    Figure PCTCN2021143765-appb-100019
  10. 根据权利要求9所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，W、X均为CH；Y为CR 6，其中，R 6选自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基、
    Figure PCTCN2021143765-appb-100020
    Z为CH或者N。
  11. 根据权利要求10所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，W、X、Z均为CH；Y为CR 6，其中，R 6为：
    Figure PCTCN2021143765-appb-100021
  12. 根据权利要求1-11任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5；其中，各m分别独立地为0、1、2或3；
    各R 3和各R 4分别独立地选自：H、一个或者多个R 12取代的C 1-C 3烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的5-11元单环、稠环、桥环或螺环；
    R 5选自：H、氰基或C 1-C 3烷基；
    各R 12分别独立地选自：H、羟基、乙酰基、R 13取代或未取代的4-8元杂环基、卤素、羟基、氨基、C 1-C 3烷基、C 1-C 3烷氧基、-NHR 13、-N(R 13) 2；R 13为C 1-C 3烷基。
  13. 根据权利要求12所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 2选自：H、卤素、-(CH 2) mNR 3R 4、-(CH 2) mCR 3R 4R 5；其中，各m分别独立地为0或1；
    各R 3和各R 4分别独立地选自：H、C 1-C 3烷基、羟基取代的C 1-C 3烷基、-NHR 13取代的C 1-C 3烷基、-N(R 13) 2取代的C 1-C 3烷基，或者R 3、R 4和与其相连的N或C一同形成一个或者多个R 12取代的环原子中含有0、1、2或3个杂原子的5-11元单环、桥环或螺环；
    R 5选自：H、氰基或C 1-C 3烷基；
    各R 12分别独立地选自：H、羟基、乙酰基、R 13取代的哌嗪基、C 1-C 3烷基、-NHR 13、-N(R 13) 2； R 13为C 1-C 3烷基。
  14. 根据权利要求1-11任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 2选自：H、卤素、
    Figure PCTCN2021143765-appb-100022
    Figure PCTCN2021143765-appb-100023
  15. 根据权利要求1-11任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 1选自：H、卤素、氰基、甲酰胺基、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、C 3-C 6环烷氧基、卤代C 1-C 6烷基、卤代C 1-C 6烷氧基。
  16. 根据权利要求15所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 1选自：H、卤素、C 1-C 3烷基、C 1-C 3烷氧基。
  17. 根据权利要求1-11任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，R 1选自：H、卤素、甲基、氰基、甲酰胺基、三氟甲基、二氟甲基、甲氧基、环丙基、三氟甲氧基。
  18. 根据权利要求1-7任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、 代谢产物，其特征在于，具有如下式(II)所示结构：
    Figure PCTCN2021143765-appb-100024
  19. 根据权利要求1所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物，其特征在于，所述2-氨基嘧啶类化合物选自：
    Figure PCTCN2021143765-appb-100025
    Figure PCTCN2021143765-appb-100026
    Figure PCTCN2021143765-appb-100027
    Figure PCTCN2021143765-appb-100028
  20. 权利要求1-19任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物在制备JAK3抑制剂中的应用。
  21. 权利要求1-19任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物在制备预防和/或治疗肿瘤和/或炎症性疾病的药物中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述肿瘤为血液瘤和实体瘤，所述血液瘤为多发性骨髓瘤、B淋巴瘤、骨髓纤维化、真性红细胞增多症、原发性血小板增多症、慢性髓系白血病、急性髓性白血病、急性淋巴细胞性白血病、慢性粒细胞白血病、组织细胞淋巴瘤、急性巨核细胞白血病、幼淋巴细胞白血病、T淋巴细胞白血病、T淋巴母细胞性淋巴瘤；所述实体瘤为非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、胰腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、鼻咽癌、胶质瘤；所述炎症性疾病为类风湿性关节炎、特应性皮炎、接触性皮炎、牛皮癣、银屑病、溃疡性结肠炎、克罗恩病、湿疹、盘状红斑狼疮、全身性红斑狼疮、斑秃、移植物抗宿主病、强直性脊柱炎、弥漫性皮肤系统性硬化、皮肌炎。
  23. 一种防治肿瘤和/或炎症性疾病的药用组合物，其特征在于，由活性成分和药学上可接受的载体或者辅料制备得到，所述活性成分包括权利要求1-19任一项所述的2-氨基嘧啶类化合物或者其药学上可接受的盐、同位素衍生物、溶剂化物，或者其立体异构体、几何异构体、互变异构体，或者其前药分子、代谢产物。
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