WO2023143206A1 - 香豆素类化合物及其应用 - Google Patents

香豆素类化合物及其应用 Download PDF

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WO2023143206A1
WO2023143206A1 PCT/CN2023/072434 CN2023072434W WO2023143206A1 WO 2023143206 A1 WO2023143206 A1 WO 2023143206A1 CN 2023072434 W CN2023072434 W CN 2023072434W WO 2023143206 A1 WO2023143206 A1 WO 2023143206A1
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compound
acid
compounds
pharmaceutically acceptable
group
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PCT/CN2023/072434
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English (en)
French (fr)
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张冬凯
谭海忠
伍文韬
徐洋洋
孙继奎
高娜
张杨
黎健
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202380018548.6A priority Critical patent/CN118574826A/zh
Publication of WO2023143206A1 publication Critical patent/WO2023143206A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the invention discloses a series of coumarin compounds and applications thereof, and specifically discloses compounds represented by formula (I) and pharmaceutically acceptable salts thereof.
  • the mitogen-activated protein kinase (MAPK) signaling pathway is one of the important pathways in the eukaryotic signal transmission network.
  • MAPK The key signaling pathway of the response, MAPK is a group of evolutionarily conserved serine-threonine kinases, the RAS/RAF/MEK/ERK signaling pathway is one of the MAPK pathways, and its protein overexpression or mutation is found in many malignant tumors.
  • MAPK mitogen-activated protein kinase
  • RAF small molecule inhibitors and MEK small molecule inhibitors have been approved for indications focused on melanoma with BRAF V600E mutation.
  • BRAF has kinase activity only after being phosphorylated by RAS and undergoes dimerization. Mutant BRAF is always in an activated state.
  • BRAF V600E monomer can phosphorylate MEK.
  • BRAF and CRAF form Heterodimers play a role.
  • Existing RAF and MEK drugs are only effective against BRAF V600E mutations, but are ineffective against tumors with RAS mutations upstream of MAPK. New second-generation RAF small molecule inhibitors are needed.
  • the MEK inhibitors on the market at the same time are all allosteric kinase inhibitors. Although allosteric inhibitors are highly specific to MEK, they are susceptible to the influence of upstream kinase signals, and the feedback regulation of MEK phosphorylation will lead to the reactivation of RAS pathway signals. , MEK inhibition alone is not effective in treating RAS-mutated solid tumors. Therefore, novel Dual RAF/MEK inhibitors may become the choice for the treatment of MAPK pathway activation diseases.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof
  • T1 is selected from CH and N;
  • R 2 is selected from halogen
  • R 3 is selected from halogen, C 1-3 alkyl, -CH 2 -C 1-4 alkylamino and -CH 2 -4-6 membered N-containing heterocycloalkyl, the C 1-3 alkyl, -CH 2 -C 1-4 alkylamino and -CH 2 -4-6 membered N-containing heterocycloalkyl are independently optionally substituted by 1, 2 or 3 halogens;
  • R 4 is selected from halogen and C 1-3 alkyl, said C 1-3 alkyl is optionally substituted by 1, 2 or 3 halogens;
  • R 5 is selected from C 1-3 alkyl optionally substituted by 1 , 2 or 3 halogens.
  • said R 1 is selected from Other variables are as defined herein.
  • the R 2 is selected from F, and other variables are as defined in the present invention.
  • the R 3 is selected from F, Cl, Br, CH 3 , CH 2 CH 3 , CH 2 NHCH 3 and CH 2 N(CH 3 ) 2 , and the CH 3 , CH 2 CH 3.
  • CH 2 NHCH 3 and CH 2 N(CH 3 ) 2 are optionally substituted by 1, 2 or 3 halogens, other variables are as defined herein.
  • the R 3 is selected from CH 3 and CH 2 N(CH 3 ) 2 , and other variables are as defined in the present invention.
  • the R 4 is selected from F, and other variables are as defined in the present invention.
  • the R 5 is selected from CH 3 , and other variables are as defined in the present invention.
  • the present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
  • the compound of the present invention can inhibit MEK target and RAF target, have good proliferation inhibition on HCT116 cells, have high unbound plasma exposure and good oral bioavailability in mice, and have significant antitumor effect .
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and similar acids; also salts of amino acids (such as arginine, etc.) , and salts of organic acids such as glucuronic acid.
  • Certain specific compounds of the present invention contain basic and acidic functional groups and can
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include deuterium and hydrogen A variant, as long as the valences of the specified atoms are normal and the compound is stable after substitution.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically realizable basis.
  • any variable eg, R
  • its definition is independent at each occurrence.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • linking group listed does not indicate its linking direction
  • its linking direction is arbitrary, for example,
  • the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
  • any one or more sites of the group can be linked to other groups through chemical bonds.
  • connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
  • the chemical bonds that the site is connected with other groups can use straight solid line bonds Straight dotted key or tilde express.
  • the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted line bond indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups;
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
  • halogen or halogen by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • C 1-3 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n - propyl and isopropyl), and the like.
  • C 1-4 alkylamino denotes those alkyl groups containing 1 to 4 carbon atoms attached to the rest of the molecule through an amino group.
  • the C 1-4 alkylamino group includes C 1-3 , C 1-2 , C 2-4 , C 4 , C 3 and C 2 alkylamino groups and the like.
  • C 1-4 alkylamino examples include, but are not limited to, -NHCH 3 , -N(CH 3 ) 2 , -NHCH 2 CH 3 , -N(CH 3 )CH 2 CH 3 , -N(CH 2 CH 3 )( CH 2 CH 3 ), -NHCH 2 CH 2 CH 3 , -NHCH 2 (CH 3 ) 2 , -NHCH 2 CH 2 CH 2 CH 3 and the like.
  • 4-6 membered N-containing heterocycloalkyl by itself or in combination with other terms represents a saturated cyclic group consisting of 4 to 6 ring atoms, of which 1, 2, 3 or 4 ring atoms are independently selected Heteroatoms from O, S, and N, at least one of which is an N atom and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S (O) p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, fused and bridged rings.
  • a heteroatom may occupy the linking position of the heterocycloalkyl group with the rest of the molecule.
  • the 4-6-membered heterocycloalkyl group includes 5-6-membered, 4-membered, 5-membered and 6-membered heterocycloalkyl groups and the like.
  • 4-6 membered heterocycloalkyl groups include, but are not limited to, azetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), hexahydropyridazine Base etc.
  • C n-n+m or C n -C n+m includes any specific instance of n to n+m carbons, for example C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also including any range from n to n+m, for example, C 1-12 includes C 1- 3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 etc.; similarly, n to n +m means that the number of atoms on the ring is from n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-member
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD), the Bruker D8venture diffractometer was used to collect the diffraction intensity data of the grown single crystal. The light source was CuK ⁇ radiation, and the scanning method was ⁇ / ⁇ scanning. After collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed. .
  • SXRD single crystal X-ray diffraction
  • Bruker D8venture diffractometer was used to collect the diffraction intensity data of the grown single crystal.
  • the light source was CuK ⁇ radiation
  • the scanning method was ⁇ / ⁇ scanning.
  • the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed. .
  • the solvent used in the present invention is commercially available.
  • Fig. 1 is the change curve of the tumor volume of mice under different dosages of compound 3 in 28 days;
  • Fig. 2 is a graph showing the change of body weight of mice under different dosages of compound 3 in 28 days.
  • HPLC preparative chromatography HPLC preparation method: Phenomenex preparative chromatograph; Chromatographic column: C18 75*30mm*3 ⁇ m; Mobile phase A: 10mM ammonium bicarbonate aqueous solution (containing 0.05% ammoniacal liquor), mobile phase B: acetonitrile; running gradient: B%: 25%-60%, running for 8 min), to obtain compound 1.
  • Dissolve compound 2-2 (100 mg) in N,N-dimethylformamide (3 mL), add pyridine (203.67 mg) and methylsulfonamido chloride (250.21 mg) in acetonitrile (1.5 mL), and the reaction system Stir at 20°C for 2 hours. After the reaction was completed, saturated brine (15 mL) was added to the system to quench, extracted with ethyl acetate (15 mL*5), separated, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • HPLC preparative chromatography HPLC preparation method: Phenomenex preparative chromatograph; Chromatographic column: C18 75*30mm*3 ⁇ m; Mobile phase A: 10mM ammonium bicarbonate aqueous solution (containing 0.05% ammoniacal liquor), mobile phase B: acetonitrile; running gradient: B%: 25%-60%, running 8min), to obtain compound 2.
  • Test Example 1 Evaluation of HCT116 Cytological Activity in Vitro
  • McCoy's 5A medium penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • HCT116 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision Multilabel Analyzer (PerkinElmer).
  • HCT116 cells were seeded in ultra-low adsorption 96-well U-plates, 80 ⁇ L of cell suspension per well, which contained 1000 HCT116 cells. Cell plates were cultured overnight in a carbon dioxide incubator.
  • the compound to be tested was diluted 5 times to the 9th concentration with a row gun, that is, diluted from 2mM to 5.12nM, and a double-well experiment was set up.
  • Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.0256 nM.
  • Cell plates were cultured in a carbon dioxide incubator for 5 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • the compound of the present invention exhibits better inhibitory activity on cell proliferation in HCT116 cell line.
  • test compound was mixed with 10% dimethyl sulfoxide/30% PEG-400/60% aqueous solution, vortexed and sonicated to prepare a 0.20 mg/mL clear solution, which was filtered through a microporous membrane for use.
  • Male CD-1 mice aged 7 to 10 weeks were selected and given the candidate compound solution and the reference compound VS-6766 solution by intravenous injection.
  • the reference compound and the test compound were respectively mixed with 20% sulfobutyl- ⁇ -cyclodextrin/80% aqueous solution, vortexed and ultrasonically prepared to obtain a nearly transparent solution containing fine particles at 0.20 mg/mL, and the candidate compound solution was orally administered.
  • mice BALB/c Nude mice, female, 6-8 weeks old, weighing 18-22 grams
  • Tumor inoculation 5 ⁇ 10 6 NCI-H358 cells were inoculated on the right side of the neck of each mouse, the inoculation volume was 0.1 mL, and the cell suspension was a mixture of PBS and Matrigel (3:1). When the average volume of the tumor reaches 150-200mm 3 , the drugs are divided into random groups for administration. The dosing regimen and the number of experimental animals in each group are shown in Table 3 below: animal experiment grouping and dosing regimen
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured twice a week with vernier calipers. tumor volume
  • V 0.5a ⁇ b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively.
  • TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume of a certain treatment group at a certain measurement-The average tumor volume of this treatment group at the same time)/(Average tumor volume of the vehicle control group at the same measurement-Solution The average tumor volume when the control group was grouped)] ⁇ 100%.
  • Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C% T RTV /C RTV ⁇ 100% (T RTV : RTV of the treatment group; C RTV : RTV of the negative control group).
  • TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [(1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group))/(Average tumor volume at the end of treatment of the solvent control group-Start of treatment of the solvent control group Time-average tumor volume)] ⁇ 100%.
  • the compound of the present invention has a significant tumor-inhibiting effect, and the current dosage and intermittent administration may have reached the maximum tumor-inhibiting effect of this target.
  • in vivo drug efficacy studies will consider reducing the dosage, or taking intermittent administration.
  • Sexual dosing exhibited a dose-response and improved tolerance in animals.

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Abstract

本发明公开了一系列香豆素类化合物及其应用，具体公开了式(I)所示化合物及其药学上可接受的盐。

Description

香豆素类化合物及其应用
本申请主张以下优先权申请
申请号：CN202210093816.8，申请日：2022年1月26日；
申请号：CN202210405730.4，申请日：2022年4月18日。
技术领域
本发明公开了一系列香豆素类化合物及其应用，具体公开了式(I)所示化合物及其药学上可接受的盐。
背景技术
促分裂素原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路是真核生物信号传递网络中的重要途径之一，是细胞增殖、分化、细胞凋亡以及正常条件和病理条件下应激反应的关键信号通路，MAPK是一组进化保守的丝氨酸-苏氨酸激酶，RAS/RAF/MEK/ERK信号通路是MAPK通路之一，在很多恶性肿瘤中发现其蛋白过度表达或突变，是肿瘤领域的经典信号通路之一。
统计结果显示，非小细胞肺癌有31％病人由KRAS突变驱动的，90％胰腺癌病人由KRAS突变驱动，此外21％的子宫内膜癌、45％的结直肠癌、5％的卵巢癌等都是由KRAS突变驱动的；28％的黑色素瘤和20％的多发性骨髓瘤由NRAS突变驱动；60％的黑色素瘤、35-60％的卵巢癌、30-80％的乳状头甲腺癌等都是由BRAF突变驱动的，病人群体巨大。
靶向RAF和MEK已有多个药物上市，但目前上市的RAF小分子抑制剂和MEK小分子抑制剂获批适应症集中在BRAFV600E突变的黑色素瘤。正常情况下，BRAF只有被RAS磷酸化后发生二聚化才有激酶活性，突变的BRAF则一直都处于激活状态，BRAFV600E单体即可磷酸化MEK，在RAS突变情况下，BRAF和CRAF形成异二聚体发挥作用，现有的RAF和MEK药物只针对BRAFV600E突变有效，对于MAPK上游的RAS突变的肿瘤无效，需要新的二代RAF小分子抑制剂。同时上市的MEK抑制剂都是变构激酶抑制剂，虽然变构抑制剂对MEK具有高度的特异性，但易受上游激酶信号的影响，MEK磷酸化的反馈调节，导致RAS通路信号会重新激活，单独抑制MEK无法有效治疗RAS突变的实体瘤。因此新型的Dual RAF/MEK的抑制剂有可能成为治疗MAPK通路激活疾病的选择。
发明内容
本发明提供了式(I)所示化合物或其药学上可接受的盐
其中，
T1选自CH和N；
R1选自嘧啶基和-C(＝O)-C1-4烷氨基；
R2选自卤素；
R3选自卤素、C1-3烷基、-CH2-C1-4烷氨基和-CH2-4-6元含N杂环烷基，所述C1-3烷基、-CH2-C1-4烷氨基和-CH2-4-6元含N杂环烷基分别独立地任选被1、2或3个卤素取代；
R4选自卤素和C1-3烷基，所述C1-3烷基任选被1、2或3个卤素取代；
R5选自C1-3烷基，所述C1-3烷基任选被1、2或3个卤素取代。
在本发明的一些方案中，所述R1选自其他变量如本发明所定义。
在本发明的一些方案中，所述R2选自F，其他变量如本发明所定义。
在本发明的一些方案中，所述R3选自F、Cl、Br、CH3、CH2CH3、CH2NHCH3和CH2N(CH3)2，所述CH3、CH2CH3、CH2NHCH3和CH2N(CH3)2任选被1、2或3个卤素取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R3选自CH3和CH2N(CH3)2，其他变量如本发明所定义。
在本发明的一些方案中，所述R4选自F，其他变量如本发明所定义。
在本发明的一些方案中，所述R5选自CH3，其他变量如本发明所定义。
本发明还有一些方案是由上述各变量任意组合而来。
本发明还提供了下式所示化合物或其药学上可接受的盐，
技术效果
本发明化合物可抑制MEK靶点和RAF靶点，对HCT116细胞具有较好的增殖抑制，在小鼠中具有较高的非结合血浆暴露量和良好的口服生物利用度以及具有显著的抑瘤作用。
相关定义
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的 变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR)0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键直形虚线键或波浪线表示。例如-OCH3中的直形实线键表示通过该基团中的氧原子与其他基团相连；中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括 这4种连接方式，即使-N-上画出了H原子，但是仍包括 这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
除非另有说明，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键或直形虚线键
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。
除非另有规定，术语“C1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C1-3烷基包括C1-2和C2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C1- 3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C1-4烷氨基”表示通过氨基连接到分子的其余部分的那些包含1至4个碳原子的烷基基团。所述C1-4烷氨基包括C1-3、C1-2、C2-4、C4、C3和C2烷氨基等。C1-4烷氨基的实例包括但不限于-NHCH3、-N(CH3)2、-NHCH2CH3、-N(CH3)CH2CH3、-N(CH2CH3)(CH2CH3)、-NHCH2CH2CH3、-NHCH2(CH3)2、-NHCH2CH2CH2CH3等。
术语“4-6元含N杂环烷基”本身或者与其他术语联合分别表示由4至6个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，至少有一个杂原子为N原子，其余为碳原子，其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“4-6元含N杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述4-6元杂环烷基包括5-6元、4元、5元和6元杂环烷基等。4-6元杂环烷基的实例包括但不限于氮杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、六氢哒嗪基等。
除非另有规定，Cn-n+m或Cn-Cn+m包括n至n+m个碳的任何一种具体情况，例如C1-12包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、和C12，也包括n至n+m中的任何一个范围，例如C1-12包括C1- 3、C1-6、C1-9、C3-6、C3-9、C3-12、C6-9、C6-12、和C9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培 养出的单晶用Bruker D8venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：φ/ω扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本发明所使用的溶剂可经市售获得。
化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。
附图说明
图1为化合物3不同给药剂量下小鼠的肿瘤体积在28天的变化曲线图；
图2为化合物3不同给药剂量下小鼠的体重在28天的变化曲线图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
合成路线：
步骤1：化合物1-2的合成
将化合物1-1(50g)加入到冰乙酸(500mL)中，依次加入N-溴代丁二酰亚胺(68.84g)，过氧化苯甲酰(2.76g)，反应体系在85℃搅拌12小时。反应结束后将反应液浓缩至干，加入水(300mL)，用乙酸乙酯(200mL*3)萃取，分液，合并有机相并用饱和食盐水(150mL*3)洗涤，有机相用无水硫酸钠干燥，过滤，滤液浓缩得到粗品经柱层析分离纯化(洗脱剂：石油醚/乙酸乙酯＝100:0-70:30)得到化合物1-2。化合物1-2的表征如下：1H NMR(400MHz,CDCl3)δ:8.07-7.99(m,1H),7.76-7.69(m,1H),7.34-7.28(m,1H),4.58-4.53(m,2H)。
步骤2：化合物1-3的合成
0℃氮气保护下将60％纯度氢化钠(512.72mg)加入到无水四氢呋喃(15mL)中，缓慢滴加乙酰乙酸乙酯(1.67g)，反应体系在0℃搅拌0.5小时，控温0℃下将上述反应液加入至化合物1-2(3g)的无水四氢呋喃(15mL)中，加完后反应体系升至20℃搅拌12小时。反应结束后，向反应体系中加入饱和氯化铵水溶液(30mL)淬灭，用乙酸乙酯(30mL*3)萃取分液，合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩得到产物粗品经柱层析(洗脱剂：石油醚/乙酸乙酯＝100:0-80:20)纯化得到化合物1-3。
化合物1-3的表征：LCMS:m/z(ESI)＝284.0[M+H]+。1H NMR(400MHz,CDCl3)δ:7.95-7.88(m,1H),7.58-7.51(m,1H),7.23-7.16(m,1H),4.22-4.11(m,2H),3.86-3.66(m,1H),3.33-3.18(m,2H),2.27(s,3H),1.22(t,J＝7.2Hz,3H)。
步骤3：化合物1-4的合成
将化合物1-3(600mg)溶于70％纯度的硫酸水溶液中(6mL)，加入5-氟间苯二酚(271.35mg)，反应体系在25℃搅拌16小时。反应结束后，将反应体系直接过滤，滤饼用水(10mL)淋洗，固体减压浓缩至干。经柱层析(洗脱剂：二氯甲烷/甲醇＝100:0-98:2)纯化，得到化合物1-4。
化合物1-4的表征：LCMS:m/z(ESI)＝348.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:10.97-10.77(m,1H),7.99(t,J＝7.2Hz,1H),7.57(t,J＝7.2Hz,1H),7.32(t,J＝8.0Hz,1H),6.69-6.56(m,2H),4.03(s,2H),2.47(s,3H)。
步骤4：化合物1-5的合成
将化合物1-4(100mg)溶于N,N-二甲基甲酰胺(2.5mL)，加入碳酸铯(112.59mg)和2-溴嘧啶(183.12mg)，反应体系在80℃搅拌反应1小时。反应结束后向体系中加入饱和食盐水(5mL)，用乙酸乙酯(5mL*3)萃取，分液，合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩得到产物粗品经柱层析分离(洗脱剂：二氯甲烷/甲醇＝100:0-98:2)得到化合物1-5。
化合物1-5的表征：LCMS:m/z(ESI)＝426.1[M+H]+。1H NMR(400MHz,DMSO-d6)δ:8.71(d,J＝4.4Hz,2H),8.00(t,J＝7.2Hz,1H),7.63(t,J＝7.2Hz,1H),7.41-7.25(m,4H),4.10(s,2H),2.56(d,J＝6.0Hz,3H)。
步骤5：化合物1-6的合成
将化合物1-5(35mg)溶于乙酸乙酯(2mL)，加入二氯化锡二水合物(74.27mg)，反应体系在80℃搅拌反应4小时。反应结束后向体系中加入饱和碳酸氢钠水溶液(5mL)淬灭，乙酸乙酯(5mL*3)萃取，分液，合并有机相，过滤，滤液减压浓缩得到化合物1-6。
化合物1-6的表征：LCMS:m/z(ESI)＝396.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:8.71(d,J＝4.8Hz,2H),7.37(t,J＝4.8Hz,1H),7.31-7.24(m,2H),6.78-6.68(m,1H),6.66-6.57(m,1H),6.28-6.26(m,1H),5.08(s,2H),3.93(s,2H),2.51-2.49(m,3H)。
步骤6：化合物1的合成
将化合物1-6(40mg)溶于N,N-二甲基甲酰胺(2mL)，加入吡啶(80.03mg)和甲基磺酰胺基氯(98.32mg)的乙腈(1mL)溶液，反应体系在20℃搅拌2小时。反应结束后向体系中加入饱和食盐水(5mL)淬灭，用乙酸乙酯(5mL*5)萃取，分液，合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩得到产物粗品。粗品通过高效液相制备色谱分离、纯化(高效液相制备方法:Phenomenex制备色谱仪；色谱柱:C18 75*30mm*3μm；流动相A:10mM碳酸氢铵水溶液(含有0.05％氨水)，流动相B:乙腈；运行梯度:B％:25％-60％，运行8min)，得到化合物1。
化合物1的表征：LCMS:m/z(ESI)＝489.1[M+H]+。1H NMR(400MHz,DMSO-d6)δ:9.36(br s,1H),8.71(d,J＝4.8Hz,2H),7.37(t,J＝4.8Hz,1H),7.32-7.25(m,3H),7.22-7.20(m,1H),7.04-7.02(m,1H),6.95-6.92(m,1H),3.99(s,2H),2.57-2.51(m,6H)。
实施例2
合成路线：
步骤1：化合物2-1的合成
将化合物1-4(100mg)溶于N,N-二甲基甲酰胺(2.5mL)，加入碳酸钾(119.39mg)，0℃搅拌0.5小时，再加入二甲氨基甲酰氯(37.16mg)，20℃搅拌反应2小时。反应结束后向体系中加入饱和食盐水(5mL)，用乙酸乙酯(5mL*3)萃取，分液，合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩得到产物粗品经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝100:0-60:40)得到化合物2-1。
化合物2-1的表征：LCMS:m/z(ESI)＝419.1[M+H]+。1H NMR(400MHz,DMSO-d6)δ:8.07-8.00(m,1H),7.68-7.62(m,1H),7.34-7.27(m,1H),7.21-7.14(m,2H),4.08(s,2H),3.05(s,3H),2.93(s,3H),2.54(d,J＝6.0Hz,3H)。
步骤2：化合物2-2的合成
将化合物2-1(100mg)溶于乙酸乙酯(5mL)，加入二氯化锡二水合物(215.75mg)，反应体系在80℃搅拌反应4小时。反应结束后向体系中加入饱和碳酸氢钠水溶液(5mL)淬灭，乙酸乙酯(5mL*3)萃取，分液，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物2-2。
化合物2-2的表征：LCMS:m/z(ESI)＝389.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:7.19-7.11(m,2H),6.75-6.68(m,1H),6.65-6.56(m,1H),6.33-6.24(m,1H),5.08(s,2H),3.91(s,2H),3.05(s,3H),2.93(s,3H),2.56-2.47(m,3H)。
步骤3：化合物2的合成
将化合物2-2(100mg)溶于N,N-二甲基甲酰胺(3mL)，加入吡啶(203.67mg)和甲基磺酰胺基氯(250.21mg)的乙腈(1.5mL)溶液，反应体系在20℃搅拌2小时。反应结束后向体系中加入饱和食盐水(15mL)淬灭，用乙酸乙酯(15mL*5)萃取，分液，合并有机相并用无水硫酸钠干燥，过滤，滤液减压浓缩得到产物粗品。粗品通过高效液相制备色谱分离、纯化(高效液相制备方法:Phenomenex制备色谱仪；色谱柱:C18 75*30mm*3μm；流动相A:10mM碳酸氢铵水溶液(含有0.05％氨水)，流动相B:乙腈；运行梯度:B％:25％-60％，运行8min)，得到化合物2。
化合物2的表征：LCMS:m/z(ESI)＝482.1[M+H]+。1H NMR(400MHz,CDCl3)δ:7.41(t,J＝8.0Hz,1H),7.03(t,J＝8.0Hz,1H),7.00-6.87(m,3H),6.60(s,1H),4.44-4.35(m,1H),4.08(s,2H),3.12(s,3H),3.04(s,3H),2.77(d,J＝5.2Hz,3H),2.56(d,J＝6.0Hz,3H)。
实施例3
合成路线：
步骤1：化合物3-2的合成
将化合物3-1(41g)溶于无水甲醇(410mL)，0℃下分批加入硼氢化钠(10.77g)，氮气保护下在20℃搅拌2小时，在氮气流下向反应液中各缓慢滴入丙酮(50mL)，搅拌1小时后，减压浓缩溶剂至干，向粗品中加入乙酸乙酯(500mL)和饱和食盐水(200mL)，水(200mL)，分液，合并有机相，有机相用无水硫酸钠干燥后过滤，减压浓缩溶剂至干得到化合物3-2。
化合物3-2的表征如下：LCMS:m/z(ESI)＝161.9[M+H]+。1H NMR(400MHz,CDCl3)δ:8.18(d,J＝4.8Hz,1H),7.47(t,J＝4.8Hz,1H),4.86(s,2H),2.77(s,1H)。
步骤2：化合物3-3的合成
将化合物3-2(10g)溶于无水四氢呋喃(100mL)，然后加入三乙胺(12.53g)，分批加入甲基磺酸酐(12.94g)，20℃搅拌1小时。减压浓缩溶剂至干，粗品经硅胶柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-70:30)得到化合物3-3。
化合物3-3的表征如下：LCMS:m/z(ESI)＝239.9[M+H]+。1H NMR(400MHz,CDCl3),δ:8.28(d,J＝4.8Hz,1H),7.40(t,J＝4.8Hz,1H),5.34(s,2H),3.13(s,3H)。
步骤3：化合物3-4的合成
将碘化钠(625.45mg)，乙酰乙酸乙酯(1.09g)溶于无水四氢呋喃(10mL)，滴加2.2M叔丁醇锂的四氢呋喃溶液(2.09mL)滴加完毕搅拌1小时后，降温至0℃后，在氮气保护下将化合物3-3(1g)的无水四氢呋喃(5mL)溶液加入，滴加完毕后20℃搅拌1小时。加入饱和食盐水(20mL)，水(20mL)，乙酸乙酯(20mL)，分液，有机相用无水硫酸钠干燥后过滤，减压浓缩溶剂至干。粗品经氧化铝柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-90:10)得到化合物3-4。
化合物3-4的表征如下：LCMS:m/z(ESI)＝273.9[M+H]+。
步骤4：化合物3-5的合成
将化合物3-4(0.11g)溶于98％浓硫酸(788.39mg)，降温至0℃，加入5-氟间苯二酚(51.49mg)，20℃搅拌16小时。反应结束后，向反应液中加入乙酸乙酯(5mL)、饱和食盐水(3mL)、水(3mL)，分液，有机相用无水硫酸钠干燥后过滤，减压浓缩溶剂至干。粗品经硅胶柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-0:100)得到化合物3-5。
化合物3-5的表征如下：LCMS:m/z(ESI)＝337.9[M+H]+。1H NMR(400MHz,CDCl3)δ:11.00(s,1H),8.13-8.11(d,J＝5.2Hz,1H),7.30-7.20(t,J＝5.2Hz,1H),6.69-6.51(m,2H),4.02(s,2H),2.50-2.45(d,J＝6.0Hz,3H)。
步骤5：化合物3-6的合成
将化合物3-5(310mg)溶于N,N-二甲基甲酰胺(3mL)，再加入碳酸铯(358.91mg)和2-溴嘧啶(175.13mg)，80℃搅拌8小时。向反应液中加入乙酸乙酯(20mL)和水(20mL)，分液，有机相用无水硫酸钠干燥后过滤，减压浓缩溶剂至干。粗品经硅胶柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-70:30)得到化合物3-6。
化合物3-6的表征如下：LCMS:m/z(ESI)＝416.0[M+H]+。1H NMR(400MHz,CDCl3)δ:8.63(d,J＝4.8Hz,2H),8.10(d,J＝4.8Hz,1H),7.18-7.14(m,2H),7.12-7.07(m,1H),6.96(m,1H),4.13(s,2H),2.61(d,J＝6.0Hz,3H)。
步骤6：化合物3-7的合成
将化合物3-6(140mg)，二苯甲酮亚胺(82.47mg)，三(二苄基丙酮)二钯(30.83mg)，4,5-双(二苯基磷)-9,9-二甲基氧杂蒽(38.97mg)，碳酸铯(329.13mg)溶于无水甲苯(5mL)，氮气保护下110℃搅拌3小时。减压浓缩溶剂至干，粗品经硅胶柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-50:50)得到化合物3-7。化合物3-7的表征如下：LCMS:m/z(ESI)＝561.2[M+H]+。1H NMR(400MHz,CDCl3)δ:8.63(d,J＝4.8Hz,2H),8.02(d,J＝5.2Hz,1H),7.47-7.28(m,9H),7.17(t,J＝4.8Hz,2H),7.07(s,1H),6.98-6.92(m,1H),6.81(s,1H),3.94(s,2H),2.30(m,3H)。
步骤7：化合物3-8的合成
将化合物3-7(130mg)溶于无水四氢呋喃(5.2mL)，然后加入1M的稀盐酸(260.00μL)，20℃搅拌2小时。向反应液中加入乙酸乙酯(10mL)，饱和食盐水(5mL)和水(5mL)，分液，收集水相用饱和碳酸钠溶液调pH至9后加入乙酸乙酯(10mL)，分液，有机相用无水硫酸钠干燥后过滤，减压浓缩溶剂至干得到化合物3-8。
化合物3-8的表征如下：LCMS:m/z(ESI)＝397.0[M+H]+。1H NMR(400MHz,CDCl3)δ:ppm 8.63(d,J＝4.8Hz,2H),7.70(d,J＝5.4Hz,1H),7.17(t,J＝4.8Hz,1H),7.11-7.02(m,1H),7.00-6.90(m,1H),6.53(t,J＝5.4Hz,1H),5.19-5.01(m,2H),4.07(s,2H),2.58(d,J＝6.0Hz,3H)。
步骤8：化合物3的合成
将化合物3-8(103mg)溶于N,N-二甲基甲酰胺(1.2mL)，加入吡啶(113.06mg)、甲基磺酰胺基氯(101.01mg)的乙腈(0.6mL)溶液，20℃搅拌2小时。反应液经高效液相制备色谱分离、纯化(高效液 相制备方法:Waters Xbridge BEH制备色谱仪；色谱柱:C18 100*30mm*10μm；流动相A:0.1％碳酸氢铵水溶液，流动相B:乙腈；运行梯度:B％:25％-55％，运行8min)得到化合物3。
化合物3的表征如下：LCMS:m/z(ESI)＝490.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:ppm 8.71(d,J＝4.8Hz,2H),7.87(d,J＝5.0Hz,1H),7.44-7.23(m,3H),6.80-6.60(m,1H),4.00(s,2H),2.53(m,3H),2.48(s,3H)。
实施例4
合成路线：
步骤1：化合物4-1的合成
氮气保护下将化合物3-5(300mg)溶于N,N-二甲基甲酰胺(3mL)，加入碳酸钾(368.34mg)，0℃下加入二甲氨基甲酰氯(124.19mg)，20℃搅拌3小时。向反应液中加入水(6mL)，搅拌30分钟后过滤，滤饼用水(2mL)淋洗，收集滤饼。真空旋干得到化合物4-1。
化合物4-1的表征如下：LCMS:m/z(ESI)＝409.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:8.13(d,J＝5.0Hz,1H),7.32(s,1H),7.25-7.10(m,2H),4.08(s,2H),3.05(s,3H),2.93(s,3H),2.53(d,J＝6.0Hz,3H)。
步骤2：化合物4-2的合成
将化合物4-1(360mg)，二苯甲酮亚胺(215.47mg)，三(二苄基丙酮)二钯(80.64mg)，4,5-双(二苯基磷)-9,9-二甲基氧杂蒽(101.91mg)，碳酸铯(860.81mg)溶于甲苯(5mL)，氮气保护下110℃搅拌3小时。减压浓缩溶剂至干，粗品经硅胶柱层析纯化(梯度洗脱：石油醚/乙酸乙酯＝100:0-50:50)得到化合物4-2。化合物4-2的表征如下：LCMS:m/z(ESI)＝554.2[M+H]+。1H NMR(400MHz,CDCl3)δ:8.01(d,J＝5.0Hz,1H),7.91-7.72(m,2H),7.53-7.32(m,4H),7.23-7.06(m,4H),7.00-6.97(m,1H),6.95-6.90(m,1H),6.80(t,J＝5Hz,1H),3.92(s,2H),3.13(s,3H),3.05(s,3H),2.27(d,J＝6.0Hz,3H)。
步骤3：化合物4-3的合成
将化合物4-2(190mg)溶于无水四氢呋喃(7.6mL)，然后加入1M的稀盐酸水溶液(384.80μL)，20℃搅拌2小时。反应液直接过滤，滤饼用无水四氢呋喃(0.5mL)淋洗。滤饼真空旋干得到化合物4-3的盐酸盐。
化合物4-3的表征如下：LCMS:m/z(ESI)＝390.0[M+H]+。1H NMR(400MHz,DMSO-d6)δ:7.65(d,J＝6.0Hz,1H),7.26-7.12(m,2H),6.60(s,1H),4.03(s,2H),3.05(s,3H),2.93(s,3H),2.53(s,3H)。
步骤4：化合物4的合成
将化合物4-3的盐酸盐(98mg)溶于N,N-二甲基甲酰胺(1mL)，加入吡啶(100.13mg)和甲基磺酰胺基氯(89.46mg)的乙腈(0.5mL)溶液，20℃搅拌2小时。反应液经高效液相制备色谱分离、纯化(高效液相制备方法:Phenomenex制备色谱仪；色谱柱:C18 75*30mm*3μm；流动相A:0.1％碳酸氢铵水溶液，流动相B:乙腈；运行梯度:B％:25％-45％，运行8min)得到化合物4。
化合物4的表征如下：LCMS:m/z(ESI)＝483.2[M+H]+。1H NMR(400MHz,DMSO-d6)δ:7.80(d,J＝4.6Hz,1H),7.23-7.06(m,2H),6.62(s,1H),3.97(s,2H),3.06(s,3H),2.94(s,3H),2.55-2.51(m,3H),2.45(s,3H)。
评价体系
试验例1：体外HCT116细胞学活性评价
实验材料：
McCoy's 5A培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。3D CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。HCT116细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将HCT116细胞种于超低吸附96孔U型板中，80μL细胞悬液每孔，其中包含1000个HCT116细胞。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进5倍稀释至第9个浓度，即从2mM稀释至5.12nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.0256nM。细胞板置于二氧化碳培养箱中培养5天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。
向细胞板中加入每100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。本发明的化合物对HCT116细胞增殖的抑制活性试验结果见表1。
表1：本发明化合物体外筛选试验结果
结论：本发明化合物在HCT116细胞系中，对细胞的增殖展现出较好的抑制活性。
试验例2：化合物3的体内药代动力学研究
CD-1雄性小鼠口服及静脉注射受试化合物的药代动力学研究
受试化合物与10％二甲基亚砜/30％PEG-400/60％水溶液混合，涡旋并超声，制备得到0.20mg/mL澄清溶液，微孔滤膜过滤后备用。选取7至10周龄的雄性CD-1小鼠，静脉注射给予候选化合物溶液和参考化合物VS-6766溶液。参考化合物和受试化合物分别与20％磺丁基-β-环糊精/80％水溶液混合，涡旋并超声制备得到0.20mg/mL含细小颗粒的近似透明的溶液，口服给予候选化合物溶液。收集一定时间的全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件(美国Pharsight公司)计算药代参数。实验结果如表2所示：
表2.受试化合物的药代动力学结果
实验结论：PK研究显示，本发明化合物在小鼠中具有较高的非结合血浆暴露量和良好的口服生物利用度。
实验例3：化合物3的体内药效学研究
受试药物对雌性BALB/c Nude小鼠皮下异种移植人非小细胞肺癌NCI-H358肿瘤模型的体内药效学研究
1.细胞培养：人肺癌细胞NCI-H358(ECACC-95111733)复苏后体外单层培养，培养条件为RPMI 1640培养基中加10％胎牛血清和1％双抗，37℃5％CO2细胞培养箱中培养，一周两次传代。当NCI-H358细胞饱和度为80％-90％时，收取细胞，计数，接种
2.动物：BALB/c Nude小鼠，雌性，6-8周龄，体重18-22克
3.肿瘤接种：在每只小鼠的右侧颈背部接种5×106个NCI-H358细胞，接种体积为0.1mL，细胞悬液为PBS和Matrigel(3:1)混合液。肿瘤平均体积达到150-200mm3时随机分组给药。给药方案与每组实验动物数见下表3：动物实验分组和给药方案
表3.实验动物分组及给药方案
注：QD代表每日一次。
4.肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的
计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组某一次测量时平均瘤体积－该处理组分组时平均瘤体积)/(溶媒对照组同一次测量时平均瘤体积－溶媒对照组分组时平均瘤体积)]×100％。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组时(即PG-D0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。
TGI(％)，反映肿瘤生长抑制率。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
5.实验结果
实验结果如图1和图2所示。
给药28天时结果如表4所示
表4.给药第28天下的T/C及TGI

实验结论：本发明化合物具有显著的抑瘤作用，目前的给药剂量和间歇性给药可能已经达到本靶点的最大抑瘤效果，未来体内药效研究将考虑降低给药剂量，或采取间歇性给药展现出剂量相关性和提高动物的耐受性。

Claims (8)

  1. 式(I)所示化合物或其药学上可接受的盐
    其中，
    T1选自CH和N；
    R1选自嘧啶基和-C(＝O)-C1-4烷氨基；
    R2选自卤素；
    R3选自卤素、C1-3烷基、-CH2-C1-4烷氨基和-CH2-4-6元含N杂环烷基，所述C1-3烷基、-CH2-C1-4烷氨基和-CH2-4-6元含N杂环烷基分别独立地任选被1、2或3个卤素取代；
    R4选自卤素和C1-3烷基，所述C1-3烷基任选被1、2或3个卤素取代；
    R5选自C1-3烷基，所述C1-3烷基任选被1、2或3个卤素取代。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R1选自
  3. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R2选自F。
  4. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R3选自F、Cl、Br、CH3、CH2CH3、CH2NHCH3和CH2N(CH3)2，所述CH3、CH2CH3、CH2NHCH3和CH2N(CH3)2任选被1、2或3个卤素取代。
  5. 根据权利要求1或4所述化合物或其药学上可接受的盐，其中，R3选自CH3和CH2N(CH3)2。
  6. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R4选自F。
  7. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R5选自CH3。
  8. 下式所示化合物或其药学上可接受的盐，

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CN101384577A (zh) * 2006-02-09 2009-03-11 中外制药株式会社 具有抗肿瘤活性的新型香豆素衍生物
US20110009398A1 (en) * 2007-07-20 2011-01-13 Toshiyuki Sakai p27 Protein Inducer
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CN102827124A (zh) * 2011-06-17 2012-12-19 北京韩美药品有限公司 香豆素类衍生物及其药物组合物及用途
CN103172606A (zh) * 2011-12-23 2013-06-26 北京韩美药品有限公司 具有抗肿瘤活性的香豆素类化合物
CN109824640A (zh) * 2019-01-28 2019-05-31 浙江省医学科学院 一种香豆素类化合物及其药物组合物、制备方法和应用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008217A2 (en) * 2000-07-21 2002-01-31 Chugai Seiyaku Kabushiki Kaisha COUMARIN DERIVATIVES USEFUL AS TNFα INHIBITORS
CN101384577A (zh) * 2006-02-09 2009-03-11 中外制药株式会社 具有抗肿瘤活性的新型香豆素衍生物
US20110009398A1 (en) * 2007-07-20 2011-01-13 Toshiyuki Sakai p27 Protein Inducer
JP2012092038A (ja) * 2010-10-26 2012-05-17 F Hoffmann La Roche Ag 糖尿病、糖尿病関連疾患又は糖尿病性合併症の予防又は治療剤
CN102827124A (zh) * 2011-06-17 2012-12-19 北京韩美药品有限公司 香豆素类衍生物及其药物组合物及用途
CN103172606A (zh) * 2011-12-23 2013-06-26 北京韩美药品有限公司 具有抗肿瘤活性的香豆素类化合物
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