WO2020233716A1 - 咪唑并哒嗪类mnk1/mnk2激酶抑制剂及其制备方法和应用 - Google Patents

咪唑并哒嗪类mnk1/mnk2激酶抑制剂及其制备方法和应用 Download PDF

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WO2020233716A1
WO2020233716A1 PCT/CN2020/091893 CN2020091893W WO2020233716A1 WO 2020233716 A1 WO2020233716 A1 WO 2020233716A1 CN 2020091893 W CN2020091893 W CN 2020091893W WO 2020233716 A1 WO2020233716 A1 WO 2020233716A1
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substituted
compound
unsubstituted
alkyl
pharmaceutically acceptable
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PCT/CN2020/091893
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English (en)
French (fr)
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陈春麟
张惠斌
袁新睿
周金培
洪菊
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上海道熵生物科技有限公司
上海美迪西生物医药股份有限公司
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Publication of WO2020233716A1 publication Critical patent/WO2020233716A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention belongs to the technical field of medicine. Specifically, the present invention provides a new class of imidazopyridazine compounds or their stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof , Hydrates, prodrugs or solvates, and their use as inhibitors of MNK1 and MNK2 kinases.
  • MAPKs Mitogen-activated protein kinases
  • MAPKAPK MAPK-activated protein kinases family
  • MNKs MAPK-activated protein kinases
  • MNK kinase is a serine/threonine protein kinase, which can specifically phosphorylate Ser209 of the eukaryotic initiation factor eIF4E (eukaryotic initiation factor 4E), thereby indirectly regulating mRNA translation.
  • eIF4E eukaryotic initiation factor 4E
  • the eukaryotic initiation factor eIF4E can strengthen the coding and regulation of the transcription of cyclin and oncoprotein mRNA, thereby causing the up-regulation of tumor-related protein expression.
  • eIF4E, the backbone protein eIF4G and RNA helicase eIF4A constitute the eukaryotic initiation factor complex eIF4F. Since eIF4E is responsible for the binding of this complex to the 5'end cap structure of mRNA, it plays an irreplaceable regulatory role in the process of RNA translation.
  • eIF4E In normal cells, the activity of eIF4E is restricted, and the transcription of these tumor-related mRNAs is inhibited; while in tumor cells or tissues, the high expression or over-activation of eIF4E can cause the up-regulation of these mRNA transcription levels.
  • Up-regulation of eIF4E expression levels has been detected in a variety of tumors and tumor cell lines, including colon cancer, breast cancer, bladder cancer, lung cancer, prostate cancer, gastric cancer, Hodg's lymphoma and neuroblastoma.
  • the up-regulation or over-activation of MNK kinase and eIF4E expression is often accompanied by drug resistance and poor prognosis. More importantly, although MNKs are an indispensable condition for eIF4E-regulated tumor formation, inhibiting MNKs activity will not affect the survival and growth of normal cells and animals. Therefore, inhibiting MNKs by small molecule drugs is a promising tumor treatment method.
  • MNK/eIF4E signaling pathway can affect the synthesis of a variety of chemokines, cytokines, and immune checkpoint proteins, thereby regulating the immune response.
  • MNK kinase inhibitors can significantly reduce the content of PD-L1 on the surface of tumor cells, but have no effect on the expression of PD-L1 mRNA.
  • MNK inhibitors can reduce the immune checkpoint proteins PD-1, TIM-3 and the surface of activated T cells. Expression of LAG3.
  • MNK kinase inhibitors can reduce 50% of the PD-L1 content in mice, and MNK inhibitors are more sensitive to tumor models with high PD-L1 expression.
  • MNK kinase inhibitors can significantly enhance the function of cytotoxic CD8 + T cells, inhibit the differentiation of activated regulatory T cells, and promote the formation of central memory T cells.
  • MNK inhibitors almost completely inhibited tumor proliferation. The same tumor was re-inoculated 29 days after the drug was stopped, and the tumor proliferation of the mice was still completely controlled without the administration. This also reflects from the side that MNK inhibitors can activate the durable anti-tumor immune response of model mice.
  • MNK inhibitors have been reported, among which BAY1143269, Tomivosertib and ETC-206 have entered the clinic.
  • BAY1143269, Tomivosertib and ETC-206 can significantly inhibit the growth of tumor mass and prolong the survival time of mice.
  • both Tomivosertib and ETC-206 can significantly reduce the phosphorylation level of eIF4E in patients and show initial symptom relief.
  • MNK inhibitors can exert anti-tumor effects by regulating the tumor microenvironment.
  • the understanding of the MNK/eIF4E pathway continues to deepen, the development of inhibitors based on this pathway will play a more important role in targeted therapy and tumor immunotherapy.
  • the purpose of the present invention is to develop a new MNK kinase inhibitor.
  • the first aspect of the present invention provides a compound represented by general formula (I), or its stereoisomer, geometric isomer, tautomer, its pharmaceutically acceptable salt, its prodrug and its Hydrate or solvate:
  • X is S or O
  • R 1 and R 2 are each independently selected from the following group: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C10 aryl , Substituted or unsubstituted 5-10 membered heteroaryl with 1-3 heteroatoms selected from N, S and O, substituted or unsubstituted with 1-3 heteroatoms selected from N, S and O
  • Each R is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted having 1- 3 5-10 membered heteroaryl groups selected from N, S and O heteroatoms, substituted or unsubstituted 5-10 membered heterocyclic groups having 1 to 3 heteroatoms selected from N, S and O;
  • R 3 is H, or has a structure represented by -NR 4 R 5 , wherein R 4 and R 5 are each independently selected from the following group: hydrogen, substituted or unsubstituted C1-C6 alkyl; or R 1 and R 2 It forms a substituted or unsubstituted 5-6 membered heterocyclic group with the N atom to which it is connected;
  • substituted refers to being substituted by one or more (for example, 2, 3, 4, etc.) substituents selected from the following group: halogen, C1-C6 alkyl, halogenated C1- C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 alkylamino, -C(O)C1-C6 alkyl, C3-C8 cycloalkyl, halogenated C3 -C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, unsubstituted or substituted by one or more substituents selected from the following group: C6-C10 aryl, C3-C8 cycloalkyl , 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclic groups having 1-3 heteroatoms selected from N, S and O; so
  • substituents selected from the following group: C
  • the X is O.
  • R 1 and R 2 are each independently a substituted or unsubstituted C1-C6 alkyl; or R 1 and R 2 and the N atom to which they are connected together form a substituted or unsubstituted 5- 6-membered heterocyclic group.
  • the -NR 1 R 2 has the structure shown below:
  • Y is selected from the following group: none (ie chemical bond), O, S, NR 6 , or CHR 6 ;
  • Said R 6 is selected from the following group: C1-C6 alkyl, C1-C6 alkylamino, -C(O)C1-C6 alkyl, amino, with 1-3 heterocycles selected from N, S and O Atomic 5-10 membered heterocyclic group.
  • the compound has the structure shown in the following formula II:
  • R 3 is a five-membered or six-membered N-containing heterocyclic ring.
  • the compound is selected from I-1 to I-17.
  • the second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (i) a therapeutically effective amount of the compound of formula (I) described in the first aspect of the present invention, or a stereoisomer thereof, One or more of geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates or solvates thereof, and (ii) pharmaceutically acceptable carriers or excipients .
  • the pharmaceutical composition has a preparation form selected from the group consisting of tablets, pills, granules, films, dripping pills, capsules, injections, soft capsules, emulsions, liposomes, and freezers. Dry powder, polymer microspheres, or polyethylene glycol derivatives.
  • the pharmaceutical composition is used to treat diseases or disorders related to the activity or expression of MNK.
  • the MNK is MNK1 or MNK2.
  • the third aspect of the present invention provides a compound of formula (I) as described in the first aspect of the present invention, or its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable
  • the use of the salt, its prodrug, hydrate or solvate is characterized in that the compound is used to prepare a pharmaceutical composition for treating or preventing diseases or disorders related to the activity or expression of MNK.
  • the disease is cancer.
  • the cancer is selected from the group consisting of leukemia, lymphoma, Hodgkin's disease, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, polar promyelocytic leukemia, chronic lymphocytes Leukemia, chronic myelogenous leukemia, chronic neutrophil leukemia, polar undifferentiated cell leukemia, degenerative large cell lymphoma, adult T-cell ALL, AML with three-lineage myelodysplasia, mixed-lineage leukemia, Myelodysplastic syndrome, myelodysplasia, multiple myeloma and myelosarcoma, chronic lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia, mantle cell lymphoma, mediastinum (Thymus) Large B-cell lymphoma, intravascular large B-cell lymphoma, primary exu
  • the fourth aspect of the present invention provides a method for preparing the compound of formula (I) as described in the first aspect of the present invention, the method comprising the steps:
  • MNK1 and MNK2 MNK1 and MNK2
  • the compound can be used as an active ingredient to prepare a pharmaceutical composition for treating diseases related to the activity or expression of MNK kinase.
  • the inventor completed the present invention.
  • alkyl includes linear or branched alkyl groups.
  • C 1 -C 8 alkyl means a straight or branched alkyl group having 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl Wait.
  • C 3 -C 8 cycloalkyl means a cycloalkyl group having 3-8 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It may also be in the form of a double ring, such as a bridged ring or a spiro ring.
  • C 1 -C 6 alkoxy refers to a straight or branched alkoxy group having 1 to 6 carbon atoms; for example, methoxy, ethoxy, propoxy, iso Propoxy, butoxy, isobutoxy, tert-butoxy, etc.
  • 10-20 membered aryl group refers to an aryl group having 10-20 carbon atoms, for example, naphthyl, anthryl, phenanthryl, or the like.
  • the term "5-12 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to those having 5-10 atoms and wherein 1-3 atoms are selected from The following groups are cyclic aromatic groups with heteroatoms of N, S and O. It may be a monocyclic ring or a condensed ring form.
  • pyridyl pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2, 4)-Triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, etc.
  • the groups of the present invention can be substituted by substituents selected from the following group: halogen, nitrile, nitro, hydroxyl, amino , C 1 -C 6 alkyl-amino, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 alkoxy, halogenated C 1- C 6 alkyl, halo C 2 -C 6 alkenyl, halo C 2 -C 6 alkynyl, halo C 1 -C 6 alkoxy, allyl, benzyl, C 6 -C 12 aryl , C 1 -C 6 alkoxy-C 1 -C 6 alkyl, C 1 -C 6 alkoxy-carbonyl, phenoxycarbonyl, C 2 -C 6 alkynyl-carbonyl, C 2 -C 6 alken
  • halogen or halogen atom refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halogenated” refers to substitution with an atom selected from F, Cl, Br, and I.
  • the structural formula described in the present invention is intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, containing asymmetry The R and S configuration of the center, the (Z) and (E) isomers of the double bond, etc. Therefore, a single stereochemical isomer of the compound of the present invention or a mixture of its enantiomers, diastereomers or geometric isomers (or conformational isomers) belongs to the scope of the present invention.
  • tautomers means that structural isomers with different energies can exceed the low energy barrier to convert into each other.
  • proton tautomers ie, proton transfer
  • Valence tautomers include interconversion through some bond-forming electron recombination.
  • solvate refers to a complex in which the compound of the present invention coordinates with solvent molecules to form a specific ratio.
  • hydrate refers to a complex formed by coordination of the compound of the present invention with water.
  • the term "pharmaceutically acceptable salt” includes acid addition salts of compounds of formula (I) with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, lactic acid , Oxalic acid, adipic acid, glutaric acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid, Phenylacetic acid, mandelic acid, in addition also include acid salts of inorganic bases.
  • the present invention synthesizes a series of compounds of general structural formula (I) or their stereoisomers, geometric isomers, tautomers and their pharmaceutically acceptable salts, hydrates, prodrugs or solvates. Things:
  • X is S or O
  • R 1 and R 2 are independently selected from hydrogen, halogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, Optionally substituted heterocycloalkyl, -CN, -OR, -SR, -N(R) 2 , -NO 2 , -COR, -CO 2 R, -CON(R) 2 , -CONROR,- SOR, -SO 2 R, -SO 2 N(R) 2 , -OCOR, -NRCOR, -NRSO 2 R or -NRCON(R) 2 ;
  • Each R is independently selected from hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted Substituted heterocycloalkyl;
  • R 1 and R 2 together may form an optionally substituted 5-6 membered cycloalkane, aryl, heterocycloalkyl, or heteroaryl.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or its stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof, One or more of its prodrug and its hydrate or solvate, and optionally a pharmaceutically acceptable carrier or excipient.
  • the "pharmaceutically acceptable salt” of the present invention includes acid addition salts formed by compounds of formula (I) with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid , Lactic acid, oxalic acid, adipic acid, glutaric acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid Acid, phenylacetic acid, mandelic acid, and also acid salts of inorganic bases.
  • the prodrugs of the derivatives of the present invention are derivatives of the general formula (I), and they may have weak or even no activity by themselves, but after administration, under physiological conditions (for example, by metabolism, solvolysis or other means) ) Is converted into the corresponding biologically active form.
  • the compounds of the present invention or their pharmaceutically acceptable salts, hydrates and prodrugs can be used alone as the only anti-tumor drugs, or can be used in combination with existing anti-tumor drugs for the treatment and/prevention of tumors, etc. .
  • the present invention relates to a preparation method of the compound of formula (I), which is achieved by the following steps:
  • the target compound is obtained through ring closure, coupling with methoxycarbonyl phenylboronic acid, bromination, hydrolysis, condensation with the corresponding amine and coupling.
  • the preparation method includes the following steps:
  • Compound 3 is coupled with methoxycarbonylphenylboronic acid under the catalysis of Pd(PPh 3 ) 4 or Pd(dppf)Cl 2 to obtain compound 4;
  • Compound 7 is coupled with benzofuran-2-boronic acid or benzothiophene-2-boronic acid under the catalysis of Pd(PPh 3 ) 4 or Pd(dppf)Cl 2 to obtain the target compound (I);
  • the compound represented by formula (I) of the present invention is selected from the compounds in Table 1 below:
  • the compound of formula (I) of the present invention can be prepared by the following steps:
  • the target compound is obtained through ring closure, coupling with methoxycarbonyl phenylboronic acid, bromination, hydrolysis, condensation with the corresponding amine and coupling.
  • the preparation method includes the following steps:
  • Compound 7 is coupled with benzofuran-2-boronic acid or benzothiophene-2-boronic acid under the catalysis of Pd(PPh 3 ) 4 or Pd(dppf)Cl 2 to obtain the target compound (I);
  • the pharmaceutical composition can be used to prevent and/or treat (stabilize, reduce or cure) MNK kinase activity or expression related diseases (for example, colorectal cancer, gastric cancer, thyroid cancer, lung cancer, leukemia, B cell lymphoma, T cell lymphoma Tumor, hair cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, pancreatic cancer, melanoma, multiple myeloma, brain cancer, CNS cancer, kidney cancer, prostate cancer, Ovarian cancer, breast cancer, uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response, inappropriate cellular inflammatory response, leukemia and myelodysplastic syndrome, mal
  • MNK kinase activity or expression related diseases for example, colorectal cancer, gastric cancer, thyroid cancer, lung cancer, leukemia, B cell lymphoma, T cell lymphoma Tumor, hair cell lymphoma, Hodgkin
  • the pharmaceutical composition of the present invention includes a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention/agent, more preferably, 10-200 mg of the compound of the present invention/agent.
  • the "one dose" is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that each component of the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as Tween
  • wetting agents such as sodium lauryl sulfate
  • coloring agents such as sodium lauryl sulfate
  • flavoring agents such as pepperminophen, sorbitol, etc.
  • antioxidants
  • the method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and gly
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous 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 can be administered alone or in combination with other pharmaceutically acceptable compounds (such as other anti-tumor or anti-inflammatory drugs).
  • the pharmaceutical composition When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (such as other anti-tumor or anti-inflammatory drugs) .
  • One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds can be used to prevent and/or treat MNK simultaneously, separately or sequentially with the compound of the present invention Kinase activity or expression related diseases.
  • a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) in need of treatment, wherein the dosage when administered is the effective dosage considered pharmaceutically, and in order to achieve the desired result, per kilogram
  • the total amount of the compound of formula (I) administered for 24 hours is about 0.01-800 mg, preferably the total amount is 0.1-80 mg/kg. If necessary, it is administered in several single doses.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • 3-Amino-6-chloropyridazine (2.80g, 21.61mmol) was dissolved in a mixed solution of 30mL ethanol and 18mL water, and bromoacetaldehyde diethyl acetal (5.11g, 25.94mmol) and 48% hydrobromic acid were added Aqueous solution (3mL), heated to 100°C and reacted for 8h. After the completion of the reaction was monitored by the TLC plate, the reaction solution was cooled to room temperature and concentrated under reduced pressure. 100 mL of water was added, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product was separated by column chromatography to obtain 2.05 g of white solid powder, which was the intermediate 6-chloroimidazo[1,2-b]pyridazine, with a yield of 61.8%.
  • Step 2 Preparation of methyl 4-(imidazo[1,2-b]pyridazinyl-6-yl)benzoate
  • Step 3 Preparation of methyl 4-(3-bromoimidazo[1,2-b]pyridazinyl-6-yl)benzoate
  • Methyl 4-(3-bromoimidazo[1,2-b]pyridazinyl-6-yl)benzoate (1.6g, 4.82mmol) was dissolved in a mixed solution of 20mL tetrahydrofuran and 10mL methanol, and slowly added dropwise In 10mL LiOH (0.92g, 38.54mmol) aqueous solution, stir at room temperature for 4h. After the reaction was monitored by TLC plate, it was concentrated under reduced pressure, diluted with 20 mL of water, and 1M hydrochloric acid was slowly added dropwise to adjust the pH to 2 to 3. A large amount of white solid precipitated.
  • Step 5 (4-(3-Bromoimidazo[1,2-b]pyridazinyl-6-yl)phenyl)(morpholinyl)methanone
  • Tetratriphenylphosphine palladium (30 mg, 26 ⁇ mol) was added, the air in the reaction device was fully replaced with nitrogen, and stirring was continued at room temperature for 15 minutes, and then the temperature was raised to 95° C. for reaction for 8 hours. After the completion of the reaction was monitored by TLC panel, the reaction solution was cooled to room temperature, the insoluble matter was filtered off with Celite, the filtrate was concentrated under reduced pressure, and the residue was separated by column chromatography to obtain 67 mg of white solid powder, (4-(3-(benzothiophene) -2-yl)imidazo[1,2-b]pyridazinyl-6-yl)phenyl)(morpholinyl)methanone, the yield was 55.6%.
  • ADP-Glo Kinase Test Kit (Promega, catalog No. V6930) was used to screen compound activity. All reactions were kinase reaction buffer HEPES (15mM HEPES pH7.4,20mM NaCl 2, 1mM EGTA, 10mM MgCl 2, 0.1mg / mL BGG and 0.02% Tween-20) in progress.
  • the final MNK1 reaction solution contains 10nM MNK1 (Carna, catalog No.02-145), 100 ⁇ M peptide substrate (TATKSGSTTKNR, Genscript), 300 ⁇ M ATP and different concentrations of compounds; the final MNK2 reaction solution contains 3nM MNK1 (Carna, catalog No.02- 146), 50 ⁇ M peptide substrate (TATKSGSTTKNR, Genscript), 10 ⁇ M ATP and different concentrations of compounds.
  • the final DMSO concentration in each reaction was 1%.
  • the specific implementation method is:
  • substrate mixture polypeptide substrate and ATP
  • the compound of the present invention has excellent inhibitory activity on MNK1 and MNK2.
  • Tolbutamide was selected as the reference compound.
  • the specific method is as follows:
  • spiking solution for test compound and reference compound add 5 ⁇ L of compound stock solution (10nM) to 95 ⁇ L of acetonitrile;
  • 1.5 ⁇ M spiking solution in microsomes (0.75mg/mL) configuration add 1.5 ⁇ L spiking solution and 18.75 ⁇ L liver particle solution (20mg/mL) into 479.75 ⁇ L K 3 PO 4 buffer;
  • the compound of the present invention has good metabolic stability in the liver microsomal model.
  • TMD-8 and DOHH-2 cells were cultured with RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibody. All cells were completed in a 37°C constant temperature incubator with 95% humidity and 5% CO 2 .
  • the cells adhere to the wall, discard the culture medium, add 150 ⁇ L of culture medium containing different concentrations of the test compound to each well, and continue culturing for 48 hours (set the solvent group as a control, set the blank group to zero);
  • Compound inhibition rate (%) (OD control -OD administration) / (OD control -OD blank) ⁇ 100%, inhibition rate of the compound and then plotted curve Graphpad Prism 5, calculate IC 50.
  • the compound was tested for its anti-tumor cell proliferation activity (see Table 4 for the results).
  • the compound of the present invention has good anti-tumor cell proliferation activity.

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Abstract

本发明提供了一类咪唑并哒嗪类MNK1/MNK2激酶抑制剂及其制备方法和应用。具体地，本发明提供了一种式(I)所示的化合物：或其立体异构体、互变异构体或药学上可接受的盐。本发明的化合物可以用于制备治疗MNK(优选地包括MNK1和MNK2)活性或表达量相关的疾病或病症的药物组合物。

Description

咪唑并哒嗪类MNK1/MNK2激酶抑制剂及其制备方法和应用 技术领域
本发明属于医药技术领域，具体而言，本发明提供了一类新的咪唑并哒嗪类化合物或其立体异构体、几何异构体、互变异构体及其药学上可接受的盐、水合物、前药或溶剂合物，及其作为MNK1和MNK2激酶抑制剂的用途。
背景技术
蛋白激酶(protein kinases)在多种细胞功能中发挥着重要的作用，其异常活化与多种疾病相关。丝裂原活化蛋白激酶(mitogen-activated protein kinases，MAPKs)能够接收多种细胞信号(包括生长因子、环境刺激和细胞因子)并被激活，从而调控细胞的增殖、分化、存活、细胞周期控制以及程序性细胞死亡。在接收这些刺激信号后，MAPKs能够激活下游的靶蛋白，即MAPK激活蛋白激酶家族(MAPK-activated protein kinases family，MAPKAPK)，该激酶家族其中包括丝裂原活化蛋白激酶作用激酶(MAPK-interacting kinases，MNKs)。MNK激酶属于丝氨酸/苏氨酸蛋白激酶，能够特异性的将真核起始因子eIF4E(eukaryotic initiation factor 4E)的Ser209磷酸化，从而间接的调控mRNA翻译。
真核起始因子eIF4E作为一种重要的转录因子，能够加强编码调控细胞周期蛋白与致癌蛋白mRNA的转录从而引起肿瘤相关蛋白表达的上调。eIF4E与骨架蛋白eIF4G和RNA解旋酶eIF4A组成了真核起始因子复合体eIF4F。由于eIF4E负责该复合体与mRNA的5’末端帽子结构结合，所以其在RNA翻译过程中发挥着不可替代的调控作用。在正常细胞中，eIF4E的活性被限制，这些肿瘤相关mRNA的转录得到抑制；而在肿瘤细胞或组织中，eIF4E的高表达或过度活化会引起这些mRNA转录水平的上调。在多种肿瘤和肿瘤细胞株中均检测到了eIF4E表达水平的上调，其中包括结肠癌、乳腺癌、膀胱癌、肺癌、前列腺癌、胃癌、霍奇经淋巴瘤和成神经细胞瘤。而在临床上，MNK激酶与eIF4E表达上调或过度激活往往伴随着耐药以及不良预后。更为重要的是，虽然MNKs是eIF4E调控的肿瘤形成的必不可少的条件，但抑制MNKs活性并不会影响正常细胞和动物的存活与生长。所以，通过小分子药物抑制MNKs是一种极具前景的肿瘤治疗手段。
eIF4E作为一个重要的翻译限速因子，MNK/eIF4E信号通路能够影响多种趋化因子、细胞因子以及免疫检查点蛋白的合成，进而调控免疫应答。MNK激酶抑制剂能够显著降低肿瘤细胞表面PD-L1的含量，而对PD-L1 mRNA的表达无影响，同时MNK抑制剂能够降低激活性T细胞表面免疫检查点蛋白PD-1、TIM-3和LAG3的表达。肿瘤异种移植模型结果也证实，MNK激酶抑制剂能够降低小鼠体内50％的PD-L1含量，并且MNK抑制剂对PD-L1高表达的肿瘤模型更为敏感。同时，MNK激酶抑制剂能够显著增强细胞毒CD8 +T细胞的功能，抑制激活的调节性T细胞的分化，并促进中央记忆T细胞的形成。在小鼠结肠癌同种移植模型中，MNK抑制剂几乎完全抑制肿瘤的增殖。而在停药29天后再次接种相同的肿瘤，在不给药的情况下，小鼠的肿瘤增殖依旧得到了完全的控制。这也从侧面反映了MNK抑制剂能够激活模型鼠持久的抗肿瘤免疫应答。
随着近几年对MNKs结构及功能了解的深入，陆续有多个MNK抑制剂得到了报道，其中BAY1143269、Tomivosertib和ETC-206陆续进入了临床。在小鼠异种移植 模型中，BAY1143269、Tomivosertib以及ETC-206均能够显著地抑制瘤块的生长，并延长小鼠的生存期。从已经公布的I期临床结果来看，Tomivosertib和ETC-206均能够显著降低病人体内eIF4E的磷酸化水平，并表现出初步的症状缓解。而目前临床结果也进一步证实，在体内条件下，MNK抑制剂能够通过调节肿瘤微环境发挥抗肿瘤效果。随着对MNK/eIF4E通路了解的不断深入，基于该通路的抑制剂开发将在靶向治疗和肿瘤免疫疗法中发挥更为重要作用。
综上所述，本领域迫切需要开发新的MNK激酶抑制剂。
发明内容
本发明的目的是开发一种新的MNK激酶抑制剂。
本发明的第一方面，提供了通式(I)所示的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物：
Figure PCTCN2020091893-appb-000001
其中，
X为S或O；
R 1和R 2各自独立地选自下组：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂环基、-CN、-OR、-SR、-N(R) 2、-NO 2、-COR、-CO 2R、-CON(R) 2、-CONROR、-SOR、-SO 2R、-SO 2N(R) 2、-OCOR、-NRCOR、-NRSO 2R或-NRCON(R) 2；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基；
各个R各自独立选自氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂环基；
R 3为H，或具有-NR 4R 5所示的结构，其中，R 4和R 5各自独立地选自下组：氢、取代或未取代的C1-C6烷基；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基；
其中，所述的“取代”是指被选自下组的一个或多个(例如2个、3个、4个等)取代基所取代：卤素、C1-C6烷基、卤代的C1-C6烷基、C1-C6烷氧基、卤代的C1-C6烷氧基、C1-C6烷胺基、-C(O)C1-C6烷基、C3-C8环烷基、卤代的C3-C8环烷基、氧代、-CN、羟基、氨基、羧基、未取代或被一个或多个取代基取代的选自下组的基团：C6-C10芳基、C3-C8环烷基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、具有1-3个选自N、S和O的杂原子的5-10元杂环基；所述的取代基选自下组：卤素、C1-C6烷氧基。
在另一优选例中，所述的X为O。
在另一优选例中，所述的R 1和R 2各自独立地为取代或未取代的C1-C6烷基；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基。
在另一优选例中，所述的-NR 1R 2具有如下所示的结构：
Figure PCTCN2020091893-appb-000002
其中，Y选自下组：无(即化学键)、O、S、NR 6、或CHR 6；
所述的R 6选自下组：C1-C6烷基、C1-C6烷胺基、-C(O)C1-C6烷基、氨基、具有1-3个选自N、S和O的杂原子的5-10元杂环基。
在另一优选例中，所述的化合物具有如下式II所示的结构：
Figure PCTCN2020091893-appb-000003
在另一优选例中，R 3为含N的五元或六元杂环。
在另一优选例中，所述的化合物选自I-1至I-17。
本发明的第二方面，提供了一种药物组合物，所述药物组合物包括：(i)治疗有效量的本发明第一方面所述的式(I)化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药、水合物或溶剂合物的一种或多种，以及(ii)药学上可接受的载体或赋形剂。
在另一优选例中，所述药物组合物具有选自下组的制剂形式：片剂、丸剂、颗粒剂、膜剂、滴丸、胶囊剂、注射剂、软胶囊、乳剂、脂质体、冻干粉、高分子微球，或者聚乙二醇衍生物。
在另一优选例中，所述的药物组合物用于治疗与MNK的活性或表达量相关的疾病或病症。
在另一优选例中，所述的MNK为MNK1或MNK2。
本发明的第三方面，提供了一种如本发明第一方面所述的式(I)化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药、水合物或溶剂合物的用途，其特征在于，所述的化合物用于制备治疗或预防与MNK的活性或表达量相关的疾病或病症的药物组合物。
在另一优选例中，所述的疾病为癌症。
在另一优选例中，所述的癌症选自下组：白血病，淋巴瘤，霍奇金病，骨髓瘤、急性淋巴细胞白血病、急性粒细胞白血病、极性早幼粒细胞白血病、慢性淋巴细胞白血病、慢性粒细胞白血病、慢性嗜中性粒细胞白血病、极性未分化细胞白血病、退行发育性大细胞性淋巴瘤、成人T细胞ALL、伴有三谱系脊髓发育不良的AML、混合型谱系白血病、脊髓发育不良综合征、骨髓增生异常、多发性骨髓瘤和脊髓肉瘤、慢性淋巴细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤或慢性淋巴细胞白血病、套细胞淋巴瘤、纵膈(胸腺)大B细胞淋巴瘤、血管内大B细胞淋巴瘤、原发性渗出性淋巴瘤、伯基特淋巴瘤。
本发明的第四方面，提供了如本发明第一方面所述的式(I)化合物的制备方法，所述方法包括步骤：
Figure PCTCN2020091893-appb-000004
在金属钯催化剂(优选为Pd(PPh 3) 4或Pd(dppf)Cl 2)催化下，用式7化合物与
Figure PCTCN2020091893-appb-000005
进行偶联反应，得到目标化合物(I)。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过长期而深入的研究，发现了一类对MNK激酶(优选为MNK1和MNK2)具有优异的抑制效果的新型化合物。该化合物可以作为活性成分，用于制备治疗与MNK激酶活性或表达量相关的疾病的药物组合物。在此基础上，发明人完成了本发明。
定义
如本文所用，术语“烷基”包括直链或支链的烷基。例如C 1-C 8烷基表示具有1-8个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基等。
如本文所用，术语“C 3-C 8环烷基”指具有3-8个碳原子的环烷基。其可以是单环，例如环丙基、环丁基、环戊基、环己基、或类似基团。也可以是双环形式，例如桥环或螺环形式。
如本文所用，术语“C 1-C 6烷氧基”是指具有1-6个碳原子的直链或支链的烷氧基；例如，甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基等。
如本文所用，术语“10-20元芳基”是指具有10-20个碳原子的芳基，例如，萘基、蒽基、菲基或类似基团。
如本文所用，术语“具有1-3个选自下组N、S和O的杂原子的5-12元杂芳基”指具有5-10个原子的且其中1-3个原子为选自下组N、S和O的杂原子的环状芳香基团。其可以是单环，也可以是稠环形式。具体的实例可以为吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、吡咯基、吡唑基、咪唑基、(1,2,3)-三唑基以及(1,2,4)-三唑基、四唑基、呋喃基、噻吩基、异恶唑基、噻唑基、恶唑基等。
本发明所述的基团除非特别说明是“取代的或未取代的”，否则本发明的基团均可被选自下组的取代基所取代：卤素、腈基、硝基、羟基、氨基、C 1-C 6烷基-胺基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 1-C 6烷氧基、卤代C 1-C 6烷基、卤代C 2-C 6烯基、卤代C 2-C 6炔基、卤代C 1-C 6烷氧基、烯丙基、苄基、C 6-C 12芳基、C 1-C 6烷氧基-C 1-C 6烷基、C 1-C 6烷氧基-羰基、苯氧羰基、C 2-C 6炔基-羰基、C 2-C 6烯基-羰基、C 3-C 6环烷基-羰基、C 1-C 6烷基-磺酰基等。
如本文所用，“卤素”或“卤原子”指F、Cl、Br、和I。更佳地，卤素或卤原子选自F、Cl和Br。“卤代的”是指被选自F、Cl、Br、和I的原子所取代。
除非特别说明，本发明所描述的结构式意在包括所有的同分异构形式(如对映异构，非对映异构和几何异构体(或构象异构体))：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
如本文所用，术语“互变异构体”表示具有不同能量的结构同分异构体可以超过低能垒，从而互相转化。比如，质子互变异构体(即质子移变)包括通过质子迁移进行互变，如1H-吲唑与2H-吲唑。化合价互变异构体包括通过一些成键电子重组而进行互变。
如本文所用，术语“溶剂合物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
如本文所用，术语“水合物”是指本发明化合物与水进行配位形成的配合物。
如本文所用，术语“药学上可接受的盐”包括式(I)化合物与下列酸形成的酸加成盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、甲酸、乙酸、三氟乙酸、乳酸、草酸、己二酸、戊二酸、丙二酸、马来酸、琥珀酸、富马酸、酒石酸、柠檬酸、棕榈酸、苯甲酸、甲磺酸、对甲苯磺酸、水杨酸、苯基乙酸、杏仁酸，此外还包括无机碱的酸式盐。
活性成分
本发明合成了一系列结构通式(I)所述的化合物或其立体异构体、几何异构体、互变异构体及其药学上可接受的盐、水合物、前药或溶剂合物：
Figure PCTCN2020091893-appb-000006
其中，
X为S或O；
R 1和R 2独立选自氢、卤素、任选地被取代的脂肪基、任选地被取代的芳香基、任选地被取代的杂芳香基、任选地被取代的环烷基、任选地被取代的杂环烷基、-CN、-OR、-SR、-N(R) 2、-NO 2、-COR、-CO 2R、-CON(R) 2、-CONROR、-SOR、-SO 2R、-SO 2N(R) 2、-OCOR、-NRCOR、-NRSO 2R或-NRCON(R) 2；
每个R独立选自氢、任选地被取代的脂肪基、任选地被取代的芳香基、任选地被取代的杂芳香基、任选地被取代的环烷基、任选地被取代的杂环烷基；
R 1和R 2可以一起形成任选地被取代的5-6元环烷烃、芳香基、杂环烷基或杂芳香基。
在另一方面，本发明提供一种药物组合物，其包含治疗有效量式(I)化合物或其立体异构体、几何异构体、互变异构体及其药学上可接受的盐、其前药及其水合物或溶剂合物的一种或多种，以及任选的药学上可接受的载体或赋形剂。
在又一方面，本发明“药学上可接受的盐”包括式(I)化合物与下列酸形成的酸加成盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、甲酸、乙酸、三氟乙酸、乳酸、草酸、己二酸、戊二酸、丙二酸、马来酸、琥珀酸、富马酸、酒石酸、柠檬酸、棕榈酸、苯甲 酸、甲磺酸、对甲苯磺酸、水杨酸、苯基乙酸、杏仁酸，此外还包括无机碱的酸式盐。
本发明衍生物的前药是通式(I)的衍生物，它们自身可能具有较弱的活性甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
本发明涉及的化合物或其药学上可接受的盐、水合物、前药可作为唯一的抗肿瘤药物单独使用，或者可以与现已上市的抗肿瘤药物联合使用，用于治疗和/预防肿瘤等。
在又一方面，本发明涉及式(I)化合物的制备方法，通过以下步骤实现：
Figure PCTCN2020091893-appb-000007
从化合物1和2出发，经过关环、与甲氧羰基苯硼酸偶联、溴代、水解、与相应的胺缩合再偶联得到目标化合物。具体的说，所述制备方法包括如下步骤：
以3-氨基6-氯吡嗪1和溴代乙醛缩二乙醇2为原料，在氢溴酸中关环得到化合物3；
化合物3在Pd(PPh 3) 4或Pd(dppf)Cl 2催化作用下与甲氧基羰基苯硼酸偶联得到化合物4；
化合物4在N-溴代丁二酰亚胺作用下溴代得到化合物5；
化合物5在氢氧化锂作用下水解得到化合物6；
化合物6在缩合剂EDCI/HOBT或HATU作用下与相应的胺缩合得到化合物7。
化合物7在Pd(PPh 3) 4或Pd(dppf)Cl 2催化作用下与苯并呋喃-2-硼酸或苯并噻吩-2-硼酸偶联得到目标化合物(I)；
其中，以上X、R 1和R 2的定义如前所述。
优选的，本发明式(I)所示的化合物选自下表1中的化合物：
表1.化合物结构
Figure PCTCN2020091893-appb-000008
Figure PCTCN2020091893-appb-000009
Figure PCTCN2020091893-appb-000010
式(I)化合物的制备
本发明所述的式(I)化合物可以通过以下步骤进行制备：
Figure PCTCN2020091893-appb-000011
从化合物1和2出发，经过关环、与甲氧羰基苯硼酸偶联、溴代、水解、与相应的胺缩合再偶联得到目标化合物。具体的说，所述制备方法包括如下步骤：
1.以2-氨基5-溴吡啶1和溴代乙醛缩二乙醇2为原料，在氢溴酸中关环得到化合物3；
2.化合物3在Pd(PPh 3) 4或Pd(dppf)Cl 2催化作用下与甲氧基羰基苯硼酸偶联得到化合物4；
3.化合物4在N-溴代丁二酰亚胺作用下溴代得到化合物5；
4.化合物5在氢氧化锂作用下水解得到化合物6；
5.化合物6在缩合剂EDCI/HOBT或HATU作用下与相应的胺缩合得到化合物7。
6.化合物7在Pd(PPh 3) 4或Pd(dppf)Cl 2催化作用下与苯并呋喃-2-硼酸或苯并噻吩-2-硼酸偶联得到目标化合物(I)；
其中，以上X、R 1和R 2的定义如前所述。
药物组合物和施用方法
由于本发明化合物具有优异的MNK激酶的抑制活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于预防和/或治疗(稳定、减轻或治愈)MNK激酶活性或表达量相关疾病(例如，结直肠癌，胃癌，甲状腺癌，肺癌，白血病，B细胞淋巴瘤，T细胞淋巴瘤，毛细胞淋巴瘤，霍奇金淋巴瘤，非霍奇金淋巴瘤，伯基特淋巴瘤，胰腺癌，黑素瘤，多发性骨髓瘤，脑癌，CNS癌，肾癌，前列腺癌，卵巢癌、乳腺癌、不受控制的细胞生长、增殖和/或存活、不适宜的细胞免疫应答、不适宜的细胞炎症应答、白血病和骨髓增生异常综合征、恶性淋巴瘤、头颈部肿瘤、肺肿瘤和肺转移瘤、胸部肿瘤、非小细胞肿瘤和小细胞肺肿瘤、胃肠肿瘤、内分泌肿瘤、乳房和其他妇科肿瘤、泌尿系肿瘤、肾、膀胱和前列腺肿瘤、皮肤肿瘤、肉瘤、肿瘤转移、神经变性病症)。
本发明的药物组合物包含安全有效量范围内的本发明化合物及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物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本发明化合物/剂，更佳地，含有10-200mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”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份能和本发明化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体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 PCTCN2020091893-appb-000012
)、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。
本发明化合物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、肠胃外(静脉内、肌肉内或皮下)。
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，活性化合物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。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂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，这种组合物中活性化合物或化合物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。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
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。除了活性化合物外，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，例知，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。
除了活性化合物外，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，或者与其他药学上可接受的化合物(例如其他抗肿瘤或抗炎症药物)联合给药。
联合给药时，所述药物组合物还包括与一种或多种(2种，3种，4种，或更多种)其他药学上可接受的化合物(例如其他抗肿瘤或抗炎症药物)。该其他药学上可接受的化合物中的一种或多种(2种，3种，4种，或更多种)可与本发明的化合物同时、分开或顺序地用于预防和/或治疗MNK激酶活性或表达量相关疾病。
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，为达到所需结果，每千克每24小时给药的式(I)化合物的总量为约0.01-800mg，优选的总量为0.1-80mg/kg。如果必要，以几次单剂量的形式给药。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。
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，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。
实施例1
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮(I-1)
Figure PCTCN2020091893-appb-000013
步骤1：6-氯咪唑并[1,2-b]哒嗪的制备
Figure PCTCN2020091893-appb-000014
将3-氨基-6-氯哒嗪(2.80g，21.61mmol)溶于30mL乙醇和18mL水的混合溶液中，加入溴代乙醛缩二乙醇(5.11g，25.94mmol)和48％氢溴酸水溶液(3mL)，升温至100℃反应8h。TLC板监测反应完全后，冷却反应液至室温后减压浓缩，加入100mL水，乙酸乙酯萃取，合并有机相后用饱和食盐水洗，有机相用无水硫酸钠干燥，过滤减压浓缩，残留物经柱层析分离，得到白色固体粉末2.05g，为中间体6-氯咪唑并[1,2-b]哒嗪，收率61.8％。
1H NMR(300MHz,DMSO-d 6)δ(ppm):8.35(s,1H),8.23(d,J＝9.5Hz,1H),7.85(d,J＝1.1Hz,1H),7.36(d,J＝9.5Hz,1H).
步骤2：4-(咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯的制备
Figure PCTCN2020091893-appb-000015
将6-氯咪唑并[1,2-b]哒嗪(2.0g，13.01mmol)溶于45mL 1,4-二氧六环和4.5mL水的混合溶液中，加入4-甲氧羰基苯硼酸(2.81g，15.63mmol)和三水磷酸钾(6.94g，26.05mmol)，以氮气充分置换反应装置内的空气，室温搅拌15min，加入四三苯基膦钯(0.72g，0.65mmol)，以氮气充分置换反应装置内的空气，室温继续搅拌15min后升温至95℃反应8h。TLC板监测反应完全后，冷却反应液至室温，硅藻土滤去不溶物，滤液减压浓缩，残留物经柱层析分离，得到白色固体1.9g，为中间体4-(咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯，收率为57.6％。
1H NMR(300MHz,CDCl 3)δ(ppm):8.42(dd,J＝1.6,0.9Hz,1H),8.19–8.17(m,1H),8.16–8.14(m,1H),7.78(d,J＝9.4Hz,1H),7.72(d,J＝1.1Hz,1H),7.70(s,1H),7.69–7.67(m,1H),7.67–7.64(m,1H),3.98(s,3H).
步骤3：4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯的制备
Figure PCTCN2020091893-appb-000016
将4-(咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯(1.5g，5.92mmol)溶于24mL乙腈和8mL二氯甲烷的混合溶液中，加入N-溴代丁二酰亚胺(1.26g，7.11mmol)，室温下搅拌反应8h。TLC板监测反应完全后，减压浓缩，加入100mL水，二氯甲烷萃取，合并有机相后用饱和食盐水洗，有机相用无水硫酸钠干燥，过滤减压浓缩，残留物经柱层析分离，得到黄色固体粉末1.62g，为中间体4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯，收率为82.3％。
步骤4：4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸的制备
Figure PCTCN2020091893-appb-000017
将4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸甲酯(1.6g，4.82mmol)溶于20mL四氢呋喃和10mL甲醇的混合溶液中，缓慢滴加10mL LiOH(0.92g，38.54mmol)水溶液中，室温下搅拌4h。TLC板监测反应完全后，减压浓缩，加入20mL水稀释，缓慢滴加1M盐酸，将pH调至2～3，有大量白色固体析出，过滤收集滤饼，水洗，烘干得白色固体1.52g，为中间体4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸，收率为100％。
步骤5：(4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮
Figure PCTCN2020091893-appb-000018
将4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯甲酸(360mg，1.14mmol)溶于3mL N,N-二甲基甲酰胺，依次加入吗啉(119mg，1.36mmol)、EDCI(261mg，1.36mmol)、HOBT(184mg，1.36mmol)、三乙胺(138mg，1.36mmol)和DMAP(14mg，0.11μmol)，升温至40℃反应8h。TLC板监测反应完全后，加入200mL二氯甲烷稀释，饱和食盐水洗(20mL×3)，有机相用无水硫酸钠干燥，过滤减压浓缩，残留物经柱层析(二氯甲烷/甲醇＝100/1～100/3)分离，得到黄色固体粉末356mg，为中间体(4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮，收率为65.9％。
步骤6：(4-(3-(苯并噻吩-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮(I-1)的制备
Figure PCTCN2020091893-appb-000019
将(4-(3-溴咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮(110mg，0.28mmol)溶于2mL1,4-二氧六环和0.2mL水的混合溶液中，加入苯并呋喃-2-硼酸(55mg，0.34mmol)和三水磷酸钾(138mg，0.52mmol)，以氮气充分置换反应装置内的空气，室温搅拌15min，加入四三苯基膦钯(30mg，26μmol)，以氮气充分置换反应装置内的空气，室温继续搅拌15min后升温至95℃反应8h。TLC板监测反应完全后，冷却反应液至室温，硅藻土滤去不溶物，滤液减压浓缩，残留物经柱层析分离，得到白色固体粉末67mg，(4-(3-(苯并噻吩-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(吗啉基)甲酮，收率为55.6％。
1H NMR(300MHz,CDCl 3)δ(ppm):8.60(s,1H),7.86–7.76(m,2H),7.64(d,J＝7.9 Hz,2H),7.52(dd,J＝12.7,6.7Hz,4H),7.35(d,J＝3.5Hz,1H),7.31–7.20(m,2H),3.67(d,J＝50.8Hz,8H)；MS:found m/z[M+H] +425.04,calcd.m/z[M]424.15.
实施例2
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-甲基哌嗪-1-基)甲酮(I-2)
Figure PCTCN2020091893-appb-000020
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-甲基哌嗪-1-基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.16(dd,J＝8.7,4.1Hz,3H),7.80–7.53(m,6H),7.35(dd,J＝17.6,7.8Hz,2H),3.74(d,J＝89.8Hz,4H),2.51(s,4H),2.39(s,3H)；MS:found m/z[M+H] +438.17,calcd.m/z[M]437.19.
实施例3
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌啶基)甲酮(I-3)
Figure PCTCN2020091893-appb-000021
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌啶基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.39(s,1H),8.14(d,J＝5.3Hz,3H),7.76(s,1H),7.72(d,J＝7.2Hz,1H),7.69–7.53(m,4H),7.34(dd,J＝17.8,8.4Hz,2H),3.62(s,4H),1.71(d,J＝19.0Hz,4H),1.59(s,2H)；MS:found m/z[M-H] -421.02,calcd.m/z[M]422.17.
实施例4
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(N,N-二甲基胺基)甲酮(I-4)
Figure PCTCN2020091893-appb-000022
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(N,N-二甲基胺基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.41(s,1H),8.23(d,J＝9.4Hz,1H),8.16(d,J＝8.4Hz,2H),7.79(d,J＝0.8Hz,1H),7.76–7.63(m,4H),7.60(d,J＝8.2Hz,1H),7.41–7.30(m,2H),3.21(s,3H),3.11(s,3H)；MS:found m/z[M+H] +383.08,calcd.m/z[M]382.14.
实施例5
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(吡咯烷基)甲酮(I-5)
Figure PCTCN2020091893-appb-000023
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(吡咯烷基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.38(s,1H),8.15(t,J＝9.0Hz,3H),7.89–7.64(m,4H),7.64–7.46(m,2H),7.34(dd,J＝17.9,7.3Hz,2H),3.74(t,J＝6.5Hz,2H),3.55(t,J＝6.2Hz,2H),2.08–1.92(m,4H)；MS:found m/z[M+H] +408.54,calcd.m/z[M]408.16.
实施例6
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-乙酰基哌嗪-1-基)甲酮(I-6)
Figure PCTCN2020091893-appb-000024
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-乙酰基哌嗪-1-基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.18(dd,J＝8.8,3.5Hz,3H),7.71(dd,J＝15.7,8.3Hz,4H),7.60(t,J＝8.5Hz,2H),7.42–7.28(m,2H),3.65(d,J＝32.4Hz,8H),2.18(s,3H)；MS:found m/z[M+H] +465.97,calcd.m/z[M]465.18.
实施例7
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-二甲氨基哌啶基)甲酮(I-7)
Figure PCTCN2020091893-appb-000025
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-二甲氨基哌啶基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.41(s,1H),8.17(dd,J＝8.8,7.1Hz,3H),7.67(ddd,J＝20.4,17.7,8.9Hz,6H),7.35(dd,J＝18.0,7.2Hz,2H),4.81(s,1H),3.93(s,1H),3.02(d,J＝63.5Hz,2H),2.52(dd,J＝15.5,9.5Hz,1H),2.38(s,6H),2.01(d,J＝45.9Hz,4H)；MS:found m/z[M+H] +466.30,calcd.m/z[M]465.22.
实施例8
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-甲基哌啶基)甲酮(I-8)
Figure PCTCN2020091893-appb-000026
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-甲基哌啶基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.39(s,1H),8.16(dd,J＝13.3,5.7Hz,3H),7.80–7.53(m,6H),7.35(dd,J＝17.7,8.2Hz,2H),4.73(s,1H),3.82(s,1H),2.96(d,J＝68.5Hz,2H),1.79(d,J＝47.1Hz,5H),1.03(s,3H)；MS:found m/z[M+H] +437.13,calcd.m/z[M]436.19.
实施例9
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-异丙基哌嗪-1-基)甲酮(I-9)
Figure PCTCN2020091893-appb-000027
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-异丙基哌嗪-1-基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.23–8.10(m,3H),7.67(ddd,J＝30.9,15.6,7.3Hz,6H),7.35(dd,J＝17.5,8.2Hz,2H),3.88(s,2H),3.57(s,2H),2.82(dd,J＝13.3,6.4Hz,1H),2.62(d,J＝33.5Hz,4H),1.11(d,J＝6.3Hz,6H)；MS:found m/z[M+H] +466.30,calcd.m/z[M]465.22.
实施例10
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-哌啶基哌啶基)甲酮(I-10)
Figure PCTCN2020091893-appb-000028
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-哌啶基哌啶基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.15(t,J＝8.0Hz,3H),7.66(ddd,J＝19.0,16.6,8.0Hz,6H),7.35(dd,J＝17.7,7.9Hz,2H),4.85(s,1H),3.93(s,1H),3.09(s,1H),2.84(s,1H),2.62(s,5H),1.69(s,4H),1.27(s,6H)；MS:found m/z[M+H] +506.26,calcd.m/z[M]505.25.
实施例11
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-吗啉基哌啶基)甲酮(I-11)
Figure PCTCN2020091893-appb-000029
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-吗啉基哌啶基)甲酮。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.16(dd,J＝8.8,7.0Hz,3H),7.66(ddd,J＝19.4,16.2,7.9Hz,6H),7.42–7.29(m,2H),4.79(s,1H),3.92(s,1H),3.77(s,4H),3.04(d,J＝55.0Hz,2H),2.59(d,J＝15.4Hz,4H),2.51(d,J＝6.2Hz,1H),1.90(s,2H),1.58(s,2H)；MS:found m/z[M+H] +508.34,calcd.m/z[M]507.23.
实施例12
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐(I-12)
Figure PCTCN2020091893-appb-000030
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(300MHz,CDCl 3)δ(ppm):8.40(s,1H),8.23–8.10(m,3H),7.67(ddd,J＝30.9,15.6,7.3Hz,6H),7.35(dd,J＝17.5,8.2Hz,2H),5.98(s,1H),3.49(d,J＝9.8Hz,8H)；MS:found m/z[M+H] +424.14,calcd.m/z[M]423.17.
实施例13
(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-氨基哌啶基)甲酮三氟乙酸盐(I-13)
Figure PCTCN2020091893-appb-000031
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(300MHz,CDCl 3)δ(ppm):8.48–8.38(m,2H),8.33(d,J＝8.3Hz,2H), 8.04(d,J＝9.6Hz,1H),7.87–7.79(m,2H),7.70(d,J＝7.9Hz,1H),7.64(d,J＝8.3Hz,2H),7.36(dt,J＝15.6,7.2Hz,2H),4.40(s,1H),3.65(s,1H),3.16–2.92(m,3H),1.84(s,2H),1.35(s,2H)；MS:found m/z[M+H] +438.04,calcd.m/z[M]437.19.
实施例14
(R)-(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-氨基哌啶基)甲酮三氟乙酸盐(I-14)
Figure PCTCN2020091893-appb-000032
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(300MHz,CDCl 3)δ(ppm):8.44(dd,J＝12.3,6.3Hz,2H),8.34(d,J＝7.1Hz,2H),8.05(d,J＝9.6Hz,1H),7.88–7.80(m,2H),7.70(t,J＝7.4Hz,3H),7.37(dt,J＝14.9,6.7Hz,2H),4.23(d,J＝98.7Hz,2H),3.08–2.91(m,3H),1.99(s,1H),1.74(s,1H),1.51(s,2H)；MS:found m/z[M+H] +438.04,calcd.m/z[M]437.19.
实施例15
(S)-(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(4-氨基哌啶基)甲酮三氟乙酸盐(I-15)
Figure PCTCN2020091893-appb-000033
可通过与实施例1中类似的方法制备(4-(3-(苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(300MHz,CDCl 3)δ(ppm):8.41(dd,J＝35.2,9.5Hz,4H),8.07(d,J＝9.2Hz,1H),7.75(dd,J＝31.3,22.7Hz,5H),7.52–7.25(m,2H),4.31(d,J＝42.1Hz,2H),2.99(d,J＝62.7Hz,3H),1.87(d,J＝58.0Hz,2H),1.45(d,J＝40.8Hz,2H)；MS:found m/z[M+H] +438.17,calcd.m/z[M]437.19.
实施例16
(4-(3-(7-(吡咯烷-1-基)苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐(I-16)
Figure PCTCN2020091893-appb-000034
可通过与实施例1中类似的方法制备(4-(3-(7-(吡咯烷-1-基)苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(400MHz,CDCl 3)δ(ppm):9.07(s,1H),8.14(s,1H),7.99(d,J＝11.0Hz,1H),7.77(d,J＝7.4Hz,2H),7.61(d,J＝8.1Hz,2H),7.30(s,2H),7.10(s,1H),6.90(d,J＝7.0Hz,1H),4.01(s,4H),3.35(d,J＝31.2Hz,8H),1.66(s,4H)；MS:found m/z[M+H] +492.94,calcd.m/z[M]492.23.
实施例17
(4-(3-(7-(哌啶-1-基)苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐(I-17)
Figure PCTCN2020091893-appb-000035
可通过与实施例1中类似的方法制备(4-(3-(7-(哌啶-1-基)苯并呋喃-2-基)咪唑并[1,2-b]哒嗪基-6-基)苯基)(哌嗪基)甲酮三氟乙酸盐。
1H NMR(400MHz,CDCl 3)δ(ppm):9.05(s,1H),8.14(s,1H),7.98(d,J＝11.1Hz,1H),7.75(d,J＝7.3Hz,2H),7.59(d,J＝8.1Hz,2H),7.29(s,2H),7.07(s,1H),6.89(d,J＝7.0Hz,1H),3.99(s,4H),3.30(d,J＝31.2Hz,8H),1.79(s,4H),1.64(s,2H)；MS:found m/z[M+H] +507.04,calcd.m/z[M]506.24.
生物活性测试例1激酶水平测试
利用ADP-Glo激酶测试试剂盒(Promega,catalog No.V6930)，筛选化合物活性。所有激酶反应均在HEPES反应缓冲液(15mM HEPES pH7.4，20mM NaCl 2，1mM EGTA，10mM MgCl 2，0.1mg/mL BGG和0.02％Tween-20)中进行。最终MNK1反应液包含10nM MNK1(Carna，catalog No.02-145)、100μM多肽底物(TATKSGSTTKNR，Genscript)、300μM ATP和不同浓度化合物；最终MNK2反应液包含3nM MNK1(Carna，catalog No.02-146)、50μM多肽底物(TATKSGSTTKNR，Genscript)、10μM ATP和不同浓度化合物。各反应中最终DMSO浓度为1％。具体实施方法为：
384孔板每孔加入4μL蛋白溶液，再加入2μL化合物溶液，1000rpm离心5min，摇床上室温孵育10min；
每孔加入4μL底物混合物(多肽底物和ATP)，1000rpm离心5min，摇床上室温孵育60min；
每孔加入10μL ADP-Glo试剂，1000rpm离心5min，摇床上室温孵育40min(避光)；
每孔加入20μL Detection试剂，1000rpm离心5min，摇床上室温孵育40min(避光)；
使用多功能酶标仪冷发光模块读取RLU(Relative luminescence unit)值。信号强度用以表征MNK酶的活性强度，并利用化合物稀释的8个浓度来计算实现50％酶活抑制所需的化合物浓度(IC 50)。(实验结果见表2)
表2.化合物对MNK1和MNK2酶活的抑制活性
Figure PCTCN2020091893-appb-000036
从上述结果中可见，本发明的化合物对于MNK1和MNK2均有优异的抑制活性。
生物活性测试例2肝微粒稳定性测试
选用Tolbutamide为参比化合物。具体方法如下：
配置0.1M K 3PO 4(pH 7.4)缓冲液及3×NADPH储存液(6mM，5mg/mL)，并在37℃水浴锅中预热；
受试化合物及参比化合物spiking solution的配置：将5μL化合物储备液(10nM)加入到95μL乙腈中；
1.5μM spiking solution in microsomes(0.75mg/mL)的配置：将1.5μL spiking solution和18.75μL肝微粒溶液(20mg/mL)加入到479.75μL K 3PO 4缓冲液中；
取30μL spiking solution in microsomes加入到多孔板中，并在37℃条件下孵育5min；
每孔加入15μL NADPH储存液开始反应，并计时；
分别在0min、5min、15min、30min和45min加入150μL含IS的乙腈溶液，终止反应；
振荡10min后6000rpm离心15min；每孔取80μL上清LC/MS检测，计算T 1/2。(实验结果见表3)
表3.化合物肝微粒体稳定性
Figure PCTCN2020091893-appb-000037
从上述结果中可见，本发明的化合物在肝微粒体模型中具有良好的代谢稳定性。
生物活性测试例3化合物的体外抗肿瘤细胞增殖活性测试
用MTT法测试了优选化合物的体外抗肿瘤细胞增殖活性。TMD-8和DOHH-2细胞用含10％胎牛血清和1％双抗的RPMI 1640培养基培养。所有细胞均在95％湿度、5％CO 2的37℃恒温培养箱中完成。实验步骤：
贴壁细胞
1.取对数生长期的肿瘤细胞，细胞计数后以1×10 5个细胞/mL密度接种于96孔板中，每孔100μL，设置3个复孔；
2. 12h后细胞贴壁，弃去培养基，每孔加入含有不同浓度受试化合物的培养基150μL，继续培养48h(设置溶剂组作为对照，设置空白组调零)；
3. 48h后，每孔加入5mg/mL的MTT 20μL，37℃培养箱中继续孵育4h；
4.弃去上清，每孔加入DMSO 150μL溶解甲瓒，室温振荡15min充分溶解后，用多功能酶标仪570nm波长处测定吸光度。
悬浮细胞
加入20μL的MTT孵育4h后，每孔加入三联液100μL，并于37℃培养箱中放置过夜后，用多功能酶标仪570nm波长处测定吸光度。
实验数据处理方法：
化合物抑制率(％)＝(OD对照-OD给药)/(OD对照-OD空白)×100％，再通过Graphpad Prism 5绘制化合物的抑制率曲线，计算IC 50。测试了化合物抗肿瘤细胞增殖活性(结果见表4)。
表4.化合物抗肿瘤细胞增殖活性(IC 50，μM)
Figure PCTCN2020091893-appb-000038
从上述结果中可见，本发明的化合物具有好的抗肿瘤细胞增殖活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 通式(I)所示的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物：
    Figure PCTCN2020091893-appb-100001
    其中，
    X为S或O；
    R 1和R 2各自独立地选自下组：氢、卤素、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂环基、-CN、-OR、-SR、-N(R) 2、-NO 2、-COR、-CO 2R、-CON(R) 2、-CONROR、-SOR、-SO 2R、-SO 2N(R) 2、-OCOR、-NRCOR、-NRSO 2R或-NRCON(R) 2；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基；
    各个R各自独立选自氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、取代或未取代的具有1-3个选自N、S和O的杂原子的5-10元杂环基；
    R 3为H，或具有-NR 4R 5所示的结构，其中，R 4和R 5各自独立地选自下组：氢、取代或未取代的C1-C6烷基；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基；
    其中，所述的“取代”是指被选自下组的一个或多个(例如2个、3个、4个等)取代基所取代：卤素、C1-C6烷基、卤代的C1-C6烷基、C1-C6烷氧基、卤代的C1-C6烷氧基、C1-C6烷胺基、-C(O)C1-C6烷基、C3-C8环烷基、卤代的C3-C8环烷基、氧代、-CN、羟基、氨基、羧基、未取代或被一个或多个取代基取代的选自下组的基团：C6-C10芳基、C3-C8环烷基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、具有1-3个选自N、S和O的杂原子的5-10元杂环基；所述的取代基选自下组：卤素、C1-C6烷氧基。
  2. 如权利要求1所述的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物，其特征在于，所述的X为O。
  3. 如权利要求1所述的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物，其特征在于，所述的R 1和R 2各自独立地为取代或未取代的C1-C6烷基；或R 1和R 2与其相连的N原子同形成取代或未取代的5-6元杂环基。
  4. 如权利要求1所述的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物，其特征在于，所述的-NR 1R 2具有如下所示的结构：
    Figure PCTCN2020091893-appb-100002
    其中，Y选自下组：无(即化学键)、O、S、NR 6、或CHR 6；
    所述的R 6选自下组：C1-C6烷基、C1-C6烷胺基、-C(O)C1-C6烷基、氨基、具有1-3 个选自N、S和O的杂原子的5-10元杂环基。
  5. 如权利要求1所述的化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药及其水合物或溶剂合物，其特征在于，所述的化合物选自下组：
    Figure PCTCN2020091893-appb-100003
    Figure PCTCN2020091893-appb-100004
    Figure PCTCN2020091893-appb-100005
  6. 一种药物组合物，其特征在于，所述药物组合物包括：(i)治疗有效量的权利要求1-5中任一所述的式(I)化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药、水合物或溶剂合物的一种或多种，以及(ii)药学上可接受的载体或赋形剂。
  7. 如权利要求6所述的药物组合物，其特征在于，所述的药物组合物用于治疗与MNK的活性或表达量相关的疾病或病症。
  8. 如权利要求1-5任一所述的式(I)化合物，或其立体异构体、几何异构体、互变异构体、其药学上可接受的盐、其前药、水合物或溶剂合物的用途，其特征在于，所述的化合物用于制备治疗或预防与MNK的活性或表达量相关的疾病或病症的药物组合物。
  9. 如权利要求8所述的用途，其特征在于，所述的疾病为癌症。
  10. 如权利要求1-5任一所述的式(I)化合物的制备方法，其特征在于，所述方法包括步骤：
    Figure PCTCN2020091893-appb-100006
    在金属钯催化剂(优选为Pd(PPh 3) 4或Pd(dppf)Cl 2)催化下，用式7化合物与
    Figure PCTCN2020091893-appb-100007
    进行偶联反应，得到目标化合物(I)。
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