CN107200716B - Benzoxazine compound and preparation method and application thereof - Google Patents

Benzoxazine compound and preparation method and application thereof Download PDF

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CN107200716B
CN107200716B CN201610154444.XA CN201610154444A CN107200716B CN 107200716 B CN107200716 B CN 107200716B CN 201610154444 A CN201610154444 A CN 201610154444A CN 107200716 B CN107200716 B CN 107200716B
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CN107200716A (en
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徐萍
彭宜红
孙静
牛彦
张�浩
许凤荣
王超
梁磊
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    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
    • C07D265/12—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems
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Abstract

The invention discloses a benzoxazine compound, the structure of which is shown as formula (I) or pharmaceutically acceptable salt thereof, and the definition of each substituent group in the formula (I) is shown in the specification. In addition, the invention also discloses a preparation method and application of the compound or the pharmaceutically acceptable salt thereof. The benzoxazine compound has an MEK inhibition function, has an inhibition effect on non-phosphorylated MEK, has low toxicity, and can be used as an anti-tumor and antiviral drug.
Figure DDA0000943832800000011

Description

Benzoxazine compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a benzoxazine compound and a preparation method and application thereof. The compounds have MEK inhibiting function and certain antitumor and antiviral effects, so that the compounds can be used for treating tumor diseases and viral diseases.
Background
The mitogen-activated protein kinase (MAPK) signal pathway family is a central building block of intracellular signal cascade, mammals have at least four different pathways, and the Raf/MEK/ERK pathway is the first discovered mitogen-activated protein kinase pathway and is one of deeper studied signal transduction pathways. It is mainly composed of three cascade core protein kinases Raf, MEK (Mitogen-activated and Extracellular signal-regulated Kinase) and ERK, which are sequentially activated in turn to transmit signals to various intracellular targets, thereby regulating various cellular processes, such as transcription, translation, proliferation, differentiation and apoptosis. [ Shaul YD, Seger R.Biochimica et Biophysica Acta,2007,1773:1213-
The Raf/MEK/ERK pathway is a key intracellular signaling pathway and one of the frequently abnormally activated signaling pathways in a variety of human tumors, among which the common cancers are melanoma, thyroid cancer, colon cancer, bone marrow cancer, metastatic biliary tract cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, and the like. Therefore, inhibition of this pathway has become one of the research hotspots for antitumor drugs, for example, the FDA approved Raf inhibitor sorafenib is marketed in 2005 for treating solid tumors such as advanced kidney cancer, the FDA approved MEK inhibitor trametinib is marketed in 2013 for treating unresectable or metastatic melanoma, and besides the marketed drugs, some kinase inhibitors are currently in different stages of antitumor clinical research. [ Wortzel I, Segar R.genes & Cancer,2011,2(3):195- & 209.], [ Isshiki Y, Kohci Y, Iikura H, et al.Bioorganic & Medicinal Chemistry Letters,2011,21:1795-
In addition, more and more studies have demonstrated that activation of the Raf/MEK/ERK pathway is essential for efficient replication of viruses, such as hepatitis virus, vaccinia virus, herpes simplex II virus, simian virus 40, HIV-1 virus, herpes simian virus, coxsackie virus, Kaposi sarcoma virus, influenza virus, etc., all rely on this pathway for replication. Thus, selective blockade of this pathway can prevent the replication and propagation of multiple viruses. For example, the MEK1 inhibitor U0126 can strongly inhibit all influenza a, b viruses currently monitored. [ Akinleye A, Furqan M, Mukhi N, et al. journal of Hematology&Oncology,2013,6(27):1-11.],[Giambartolomei S,Covone F,Levreo M,etal.Oncogene,2001,20:2606-2610.],[
Figure BDA0000943832780000021
L,Berting A,Malkowsk B,et al.Oncogene,2003,22:2604-2610.],[
Figure BDA0000943832780000022
JC,Andrade AA,Silva PNG,et al.the Journal ofBiological Chemistry,2001,276(42):38353-38360.],[Smith CC,Nelson J,AurelianL,et al.Journal of Virology,2000,74(22):10417-10429.],[Walia NS,Krishnan HH,Naranatt PP,et al.Journal of Virology,2005,79(16):10308-10329.],[Pleschka S,Wolff T,Ehrhardt C,et al.Nature Cell Biology,2001,3:301-305.]
MEK kinase has unique structural and functional characteristics, and in view of the position of the MEK kinase in a pathway, MEK is the only known kinase which can activate ERK at present, and the ERK is also the only known substrate of the MEK, so the MEK has high substrate specificity and is also positioned at the node position of the pathway, and thus the MEK inhibitor which selects the MEK as a molecular target can improve the selectivity of the pathway. From the structure of MEK, it contains an allosteric pocket different from the ATP binding pocket, which is highly conserved in MEK1 and MEK2, and very low sequence homology in the similar region of other kinases, which results in an increased selectivity of the inhibitor for the kinase, and when the inhibitor binds to this pocket, induces the conformation of the enzyme in an inactive state, reducing the ability of the enzyme to be activated or activate downstream substrates, thereby inhibiting the activity of the enzyme. At present, most MEK inhibitors act on the pocket and belong to non-ATP competitive inhibitors. From the drug resistance perspective, proteins in the ERK pathway are encoded by human genes and have little possibility of mutation, so that inhibition of the pathway can avoid the problem of drug resistance caused by antiviral drugs against the virus itself and can also avoid the defects of vaccines. Therefore, it is desired in the art to develop a new MEK inhibitor as a novel antitumor drug and antiviral drug, which has the characteristics of low toxicity, difficulty in drug resistance generation, high kinase selectivity, good pathway selectivity, and the like. [ Smith CK, Carr D, Mayhood TW, et al protein Express and Purification,2007,52:446- & 456 ] ], [ Ohren JF, Che HF, Pavlovsky A, et al. Nature Structural & Molecular Biology,2004,11(12):1192- & 1197 ] ], [ Ludwig S, Planz O, esPlchka S, et al. trends in Molecular Medicine,2003,9(2) & 46-52 ]
Disclosure of Invention
The invention develops a benzoxazine compound or pharmaceutically acceptable salt thereof, wherein the compound has an MEK (methyl ethyl ketone) inhibition function, and not only has antitumor and antiviral effects, but also is low in toxicity and high in safety.
The invention aims to provide a novel benzoxazine compound or pharmaceutically acceptable salt thereof.
A second object of the present invention is to provide a process for the preparation of such compounds or pharmaceutically acceptable salts thereof.
The third purpose of the invention is to provide a pharmaceutical composition containing the compound or the pharmaceutically acceptable salt thereof.
A fourth object of the invention is to provide the use of such compounds or pharmaceutically acceptable salts thereof.
Specifically, the invention provides a benzoxazine compound, the structure of which is shown as formula (I), or pharmaceutically acceptable salt thereof:
Figure BDA0000943832780000031
wherein R is1Is unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
R2、R3、R4And R5Each independently hydrogen or halogen;
R6is unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl or aryl;
R7and R8Each independently hydrogen or C1-C6 alkyl;
a is hydrogen or of the formula:
Figure BDA0000943832780000032
here, Y is-NH-;
l is-S (O)2-or-CO-;
z is-NH-or-CH2-；
R9Is hydrogen, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl, C3-C6 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, arylalkyl, or heteroaryl; here, the substituted C1-C6 alkyl group means substituted at an optional position of the alkyl carbon chain by optionally one or more cyano, halogen, hydroxy or phenyl groups.
In an embodiment of the present invention, the present invention provides a benzoxazine-like compound, wherein the unsubstituted C1-C6 alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, 2-methylpropyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, or the like.
In an embodiment of the present invention, the present invention provides a benzoxazine-based compound, wherein the halogen is selected from fluorine, chlorine, bromine or iodine, preferably fluorine.
In an embodiment of the present invention, the benzoxazine-based compound provided by the present invention is one in which the aromatic hydrocarbon group is phenyl or naphthyl, preferably phenyl.
In an embodiment of the present invention, the present invention provides a benzoxazine-based compound, wherein the heteroaryl group is selected from furyl, imidazolyl, indolyl, oxadiazolyl, oxazinyl, oxazolyl, isoxazolyl, pyranyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazinyl, thiadiazolyl, thiatriazolyl, thiazolyl, isothiazolyl, thienyl, triazinyl, or triazolyl.
In an embodiment of the present invention, the present invention provides a benzoxazine-based compound, wherein the pharmaceutically acceptable salt refers to an organic salt and an inorganic salt of the compound of formula (I); can be made in situ during the final isolation and purification of the compound, or can be made by: separately reacting a compound of formula (I) with a suitable organic or inorganic acid and then isolating the salt formed; the organic or inorganic acid may be selected from the group consisting of hydrobromide, hydrochloride, hydroiodide, sulphate, bisulphate, nitrate, acetate, trifluoroacetate, oxalate, benzenesulphonate, malonate, stearate, laurate, malate, borate, benzoate, lactate, phosphate, hexafluorophosphate, tosylate, formate, citrate, maleate, fumarate, succinate, tartrate, naphthenate, methanesulphonate, lactobionate and laurylsulphonate and the like.
In one embodiment of the invention, the invention provides a benzoxazine compound or a pharmaceutically acceptable salt thereof, wherein R1Is unsubstituted C1-C6 alkyl, S (O)2R6,C(O)R6,C(O)NR7R8Or S (O)2NR7R8；
R2And R4Are all hydrogen; r6Is unsubstituted C1-C6 alkyl or aromatic hydrocarbon radical.
In one embodiment of the invention, the invention provides a benzoxazine compound or a pharmaceutically acceptable salt thereof, wherein R1Is unsubstituted C1-C6 alkyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
R2、R4And R5Are all hydrogen; r3Is halogen.
In one embodiment of the invention, the invention provides a benzoxazine compound or a pharmaceutically acceptable salt thereof, wherein R1Is methyl, ethyl, n-propyl, n-butyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
Here, R6Is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;
R7and R8Each independently hydrogen, methyl or ethyl.
In one embodiment of the invention, the benzoxazine compound or pharmaceutically acceptable salt thereof is provided, wherein A is hydrogen and R is2And R4Are all hydrogen; r3And R5One is hydrogen and the other is halogen (preferably fluorine).
In a preferred embodiment of the present invention, the benzoxazine compound provided by the present invention or a pharmaceutically acceptable salt thereof is represented by formula (II):
Figure BDA0000943832780000041
wherein R is1、R3、R4、R5And A is as defined for the compound of formula (I) above.
In a preferred embodiment of the present invention, the benzoxazine compound provided by the present invention or a pharmaceutically acceptable salt thereof is represented by formula (III):
Figure BDA0000943832780000051
wherein R is1、R3、R4、R5Y, L, Z and R9As described for the compounds of formula (I) above.
In a preferred embodiment of the present invention, the benzoxazine compound provided by the present invention or a pharmaceutically acceptable salt thereof is represented by formula (IV):
Figure BDA0000943832780000052
wherein R is1、R3Y, L, Z and R9As described for the compounds of formula (I) above.
In a particularly preferred embodiment of the present invention, the present invention provides the following benzoxazine-based compounds or pharmaceutically acceptable salts thereof:
Figure BDA0000943832780000053
or
Figure BDA0000943832780000054
Wherein R is4And R5Are all hydrogen, Y is-NH-, and R1、R3L, Z and R9Is defined as follows:
Figure BDA0000943832780000055
Figure BDA0000943832780000061
Figure BDA0000943832780000071
in another aspect, the present invention provides a method for preparing the above benzoxazine compound or pharmaceutically acceptable salt thereof, wherein when a in the compound of formula (I) is hydrogen, the method comprises the following steps:
(1) a compound of formula (V) and a halide R1X reacts under alkaline conditions to obtain a compound shown as a formula (VI);
Figure BDA0000943832780000072
(2) reacting a compound shown as a formula (VI) with a compound shown as a formula (VII) to obtain a compound shown as a formula (I);
Figure BDA0000943832780000073
here, the halides R1R in X, formula (V), formula (VI) and formula (VII)1、R2、R3、R4And R5As described for the compounds of formula (I) above; x is halogen, preferably chlorine, bromine or iodine.
The invention provides a preparation method of the benzoxazine compound or pharmaceutically acceptable salt thereof, wherein A in the compound of formula (I) is-Y-L-Z-R9When the temperature of the water is higher than the set temperature,
which comprises the following steps:
(1) a compound of formula (V) and a halide R1X reacts under alkaline conditions to obtain a compound shown as a formula (VI);
Figure BDA0000943832780000081
(2) reacting a compound shown in a formula (VI) with a compound shown in a formula (VIII) to obtain a compound shown in a formula (IX);
Figure BDA0000943832780000082
(3) carrying out reduction reaction on the compound shown in the formula (IX) to obtain a compound shown in the formula (X);
Figure BDA0000943832780000083
(4) reacting a compound shown as a formula (X) with a compound shown as a formula (XI) to obtain a compound shown as a formula (I);
Figure BDA0000943832780000084
here, the halides R1R related to X, formula (V), formula (VI), formula (VIII), formula (IX), formula (X) and formula (XI)1、R2、R3、R4、R5、R9The definitions of Y, L and Z are as described for the compound of formula (I) above; x is halogen, preferably chlorine, bromine or iodine; q is a leaving group, preferably chloro, bromo, iodo or:
Figure BDA0000943832780000085
in an embodiment of the present invention, the present invention provides a method for preparing the above benzoxazine-based compound or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (V) in step (1) can be obtained by:
nucleophilic substitution reaction is carried out on 2, 4-dihydroxy benzamide and ethyl chloroformate under the action of pyridine to obtain the compound shown in the formula V.
It should be noted that the synthetic routes described above are intended to illustrate the preparation of the compounds of the present invention, and the preparation is by no means limited thereto, and other synthetic methods are equally possible under the teaching of the present invention.
In a third aspect, the present invention provides a pharmaceutical composition, where the pharmaceutical composition includes a pharmacologically effective amount of the above benzoxazine compound or its pharmaceutically acceptable salt, and may further include a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally in the form of tablets, capsules, pills, powders, granules, powders, or syrups, or parenterally in the form of injections. The unit dose of the pharmaceutical composition is 0.1mg to 1 g. The pharmaceutical composition can be prepared by conventional pharmaceutical methods.
In a fourth aspect, the invention provides the application of the benzoxazine compound or pharmaceutically acceptable salt thereof in preparing MEK inhibitor drugs, wherein the application includes but is not limited to the application as antiviral drugs (including but not limited to anti-EV 71 virus) or antitumor drugs. The dosage is 0.1mg-1g per time, and the times are from once per week to three or four times per day. The administration route is oral, topical or parenteral.
Experiments prove that the compound has the effect of inhibiting the MEK activity, has good MEK inhibition activity, can be used as a MEK inhibitor drug with a new structural type, is not only used for treating tumors and viral diseases, but also has low toxicity.
Drawings
FIG. 1 is a graph showing the inhibition of survival of RD cells by compounds 31 and 33 of the present invention.
FIG. 2 is a graph showing the results of the compound 31 and 33 of the present invention and the control drug on the cytopathic effect of EV71 treatment and the control group.
FIG. 3 is a graph showing the results of Western blotting experiments on compounds 31 and 33 of the present invention, control drugs, and control-treated groups.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative only and are not to be construed as limiting the scope of the invention.
Abbreviations: ph represents phenyl, Ar represents aryl hydrogen, δ represents chemical shift, unit ppm, J represents coupling constant, unit Hz, mp represents melting point, s represents singlet, d represents doublet, t represents triplet, q represents quartet, m represents multiplet, brs represents broad singlet, dd represents doublet;
conditions of NMR: bruker angle III 400MHz NMR spectrometer. TMS is an internal standard, and deuterated DMSO and deuterated chloroform are solvents;
mass spectrometry: waters ACQ-SQD LC-MS ESI mass spectrometer, Bruker Apex IV FTMS high resolution ESI mass spectrometer;
the starting materials in this application are all known products available, or purchased from reagent companies, or synthetic methods have been reported in the literature;
except the intermediates reported in the prior literature, the preparation method and the characterization data of the other intermediates are given;
scheme 1 for the synthesis of the compounds of the invention:
Figure BDA0000943832780000101
reaction conditions are as follows:
(a) ethyl chloroformate, pyridine, acetonitrile;
(b)R1-Cl，NaH，DMF；
(c) bromobenzyl or 3-nitrobenzyl bromide, K2CO3，DMF；
(d)5％Pd/C，H2；
(e) Reaction reagents: CH (CH)3COCl,CH3SO2Cl,CH3CH2SO2Cl or N-substituted-2-oxo-oxazoline 3-sulfonamides (i.e., compound 5 in scheme 1 above); basic catalyst: pyridine or triethylamine; solvent: CH (CH)2Cl2Or acetonitrile.
Scheme 2 for the synthesis of compounds of the invention:
Figure BDA0000943832780000111
reaction conditions are as follows:
(a) ethyl chloroformate, pyridine, acetonitrile;
(b)R1-Cl,NaH,DMF；
(c) NBS, Peroxybenzoic acid, CDCl3Refluxing;
(d)K2CO3,DMF；
(e)5％Pd/C,H2；
(f) reaction reagents: CH (CH)3SO2Cl,CH3SO2Cl or N-substituted-2-oxo-oxazoline 3-sulfonamide; basic catalyst: pyridine or triethylamine; solvent: CH (CH)2Cl2Or acetonitrile;
(g) strong ammonia water;
(h)MeI,NaH,DMF。
examples 1 to 19
Examples 1-19 Compounds the above scheme 1 is employed
EXAMPLE 13 Synthesis of- (3-Methanesulfonamidobenzyl) -7- (N, N-Dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 6)
Synthetic route 1 step (a)
To a suspension of 2, 4-dihydroxybenzamide (6.53mmol, 1g) and pyridine (40.49mmol, 3.26mL) in 9mL of acetonitrile was added dropwise a solution of ethyl chloroformate (7.183mmol, 0.78g) in 3mL of acetonitrile at 0 ℃ in portions so that the temperature of the system did not exceed 5 ℃. After the dropwise addition, heating and stirring in an oil bath, distilling at normal pressure, stopping distillation when no solvent is distilled, changing into reflux, reducing the temperature to 90 ℃, and reacting for 2 hours. The reaction solution was cooled to room temperature, and then 20mL of water and concentrated hydrochloric acid were added to acidify the reaction solution, followed by filtration to obtain 7-hydroxy-1, 3-benzoxazine-2, 4(3H) -dione (Compound 1) as a pale gray solid (0.94 g, yield 80.55%).1H NMR(400MHz,DMSO-d6)δ11.77(s,1H,OH),10.95(s,1H,NH),7.76(d,J＝8.5Hz,1H,Ar),6.81(d,J＝8.6Hz,1H,Ar),6.65(s,1H,Ar)；MS(ESI)m/z：178.13(M–H+)。
Synthetic route 1 step (b)
To a solution of 7-hydroxy-1, 3-benzoxazine-2, 4(3H) -dione (2.23mmol, 0.4g) in 10mL of DMF at 0 deg.C was added NaH (80%, 4.47mmol,0.1341g), N-dimethylcarbamoyl chloride (4.47mmol, 0.41mL) was added dropwise, and the mixture was stirred at room temperature for 3H. Adding water to quench the reaction, extracting with ethyl acetate for 3 times, washing the organic phase with saturated salt water, and drying with anhydrous sodium sulfate. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-100/1) (v/v) gave 7- (N, N-dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 2a) as a white solid in an amount of 0.396g, 70.97% yield.1H NMR(400MHz,DMSO-d6)δ12.03(s,1H,NH),7.93(d,J＝8.4Hz,1H,Ar),7.25(s,1H,Ar),7.19(d,J＝8.4Hz,1H,Ar),3.06(s,3H,NCH3),2.94(s,3H,NCH3)；MS(ESI)m/z:249.31(M–H+)。
Synthetic route 1 step (c)
7- (N, N-Dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (1.49mmol, 0.373g) and K were added at room temperature2CO3To a suspension of 3-nitrobenzyl bromide (1.68mmol, 0.363g) in 8mL of DMF (2.98mmol, 0.411g) was added and reacted for 9 h. Adding water to quench the reaction, extracting with ethyl acetate for 3 times, and separating the organic phaseWashed with saturated brine and dried over anhydrous sodium sulfate. Column chromatography (gradient elution: petroleum ether, petroleum ether/ethyl acetate 3/1, dichloromethane/methanol 100/1, dichloromethane/methanol 40/1) (v/v) gave 3- (3-nitrobenzyl) -7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 3a) as a white solid 0.5387g, crude yield 57.41%.1H NMR(400MHz,DMSO-d6)δ8.28(s,1H,NH),8.14(d,J＝8.4Hz,1H,Ar),8.01(d,J＝8.6Hz,1H,Ar),7.87(d,J＝7.6Hz,1H,Ar),7.63(t,J＝7.9Hz,1H,Ar),7.32(s,1H,Ar),7.24(d,J＝8.6Hz,1H,Ar),5.18(s,2H,CH2),3.06(s,3H,NCH3),2.94(s,3H,NCH3)；MS(ESI)m/z:386.32(M+H+),408.33(M+Na+)。
Synthetic route 1 step (d)
To a solution of 3- (3-nitrobenzyl) -7- (N, N-dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (1.2mmol, 0.462g) in 70mL of methanol at room temperature was added 5% Pd/C (0.1eq, 0.05g) and the mixture was stirred in a hydrogenation reactor and allowed to react for 9H. The catalyst is removed by filtration, and the mixture is concentrated under reduced pressure to obtain a crude product of the 3- (3-aminobenzyl) -7- (N, N-dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -diketone (compound 4a) and a yellow sticky solid. The reaction mixture is directly put into the next step without separation and purification. MS (ESI) M/z 356.28(M + H)+),378.43(M+Na+)。
Synthetic route 1 step (e)
Pyridine (1.42mmol, 0.11mL) was added to a solution of 3- (3-aminobenzyl) -7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (1.29mmol, 0.46g) in 10mL of dichloromethane at room temperature, methanesulfonyl chloride (1.42mmol, 0.11mL) was added dropwise in a solution of 2mL of dichloromethane, and the reaction was stirred at room temperature for 6H. Adding water to quench the reaction, extracting with ethyl acetate for 3 times, combining organic phases, washing with water, washing with saturated salt solution, and drying with anhydrous sodium sulfate. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-200/1, dichloromethane/methanol-100/1) (v/v) gave 3- (3-methanesulfonamidobenzyl) -7- (N, N-dimethylaminobenzoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 6) as a white solid, 0.2233g, 61.4% yield over two steps. 1H NMR (400MHz, DMSO-d)6)δ9.70(s,1H,NH),8.01(d,J＝8.6Hz,1H,Ar),7.03–7.38(m,6H,Ar),5.03(s,2H,CH2),3.06(s,3H,SO2CH3),2.97(s,3H,NCH3),2.94(s,3H,NCH3)；HRMS(ESI)m/z:434.10192(M+H+),456.08390(M+Na+)。
EXAMPLE 37 Synthesis of- (N, N-Dimethylcarbamoyloxy) -3- (3-sulfamoylaminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 8)
Formic acid (15mmol, 0.6905g) was slowly added dropwise to chlorosulfonyl isocyanate (15mmol, 2.123g) at 0 ℃, followed by stirring at room temperature for 1H, 10mL of dichloromethane was added, and the mixture was added dropwise to a flask containing 3- (3-aminobenzyl) -7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 4a) (3mmol, 1.066g), pyridine (30mmol, 2.4mL) and 11mL of dichloromethane at 0 ℃ and stirred at room temperature for 17H. The reaction was quenched with water and extracted 3 times with dichloromethane. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-100/1, dichloromethane/methanol-40/1) (v/v) afforded 0.279g of a white solid in 26.83% yield.1H NMR(400MHz,DMSO-d6)δ9.42(s,1H,NH),8.01(d,J＝8.6Hz,1H,Ar),7.34(d,J＝2.0Hz,1H,Ar),7.25(dd,J＝8.6,2.0Hz,1H,Ar),7.21(d,J＝7.8Hz,1H,Ar),7.11(d,J＝8.2Hz,1H,Ar),7.08(s,1H,Ar),7.06(s,2H,NH2),6.97(d,J＝7.7Hz,1H,Ar),5.02(s,2H,CH2),3.06(s,3H,CH3),2.94(s,3H,CH3)；HRMS(ESI)m/z:435.09640(M+H+),457.07803(M+Na+)。
EXAMPLE 43 Synthesis of (3- (N-methyl) sulfamoylaminobenzyl) -7- (N, N-dimethylcarbamoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 9)
A solution of 2-bromoethanol (10mmol, 0.71mL) in 3mL of anhydrous dichloromethane was slowly added dropwise to a solution of chlorosulfonyl isocyanate (10mmol, 1.415g) in 13mL of anhydrous dichloromethane at 0 ℃ and stirred for 2 h. The mixture was added dropwise to a two-necked flask containing methylamine in THF (2M, 11mmol, 5.5mL), triethylamine (16mmol, 2.22mL) and 5mL of dichloromethane at 0 deg.C, and the mixture was stirred at room temperature for 18 h. Adding hydrochloric acid aqueous solution (1mol/L) to quench the reaction, extracting with dichloromethane, washing with water, drying with anhydrous sodium sulfate, and reducing pressureConcentrating to obtain N-methyl-2-oxo-oxazolidine-3-sulfonamide (compound 5a), colorless and transparent crystals, and recrystallizing with ethanol to obtain 0.8551g of white solid with yield of 47.76%.1H NMR(400MHz,DMSO-d6)δ8.18(d,J＝4.4Hz,1H,NH),4.39(t,J＝7.8Hz,2H,OCH2),3.94(t,J＝7.8Hz,2H,NCH2),2.62(d,J＝4.8Hz,3H,CH3)；MS(ESI)m/z:179.15(M-H+)。
Synthetic route 1 step (e)
To a solution of 3- (3-aminobenzyl) -7- (N, N-dimethylaminobenzoyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 4a) (1.33mmol, 0.4726g) in 17mL acetonitrile was added dropwise triethylamine (3.99mmol, 0.56mL) and N-methyl-2-oxooxazolidine-3-sulfonamide (compound 5a) (2.66mmol, 0.48g) and heated at 80 ℃ under reflux for 18H. Diluting with ethyl acetate, washing with hydrochloric acid aqueous solution (1mol/L), washing with saturated sodium bicarbonate solution, washing with saturated salt solution, and drying with anhydrous sodium sulfate. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-200/1, dichloromethane/methanol-100/1) (v/v) afforded 7- (N, N-dimethylcarbamoyloxy) -3- (3- (N-methyl) sulfamoylaminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 9) as an off-white solid, 0.2665g, yield 44.68%.1H NMR(400MHz,DMSO-d6)δ9.60(s,1H,NHSO2NHCH3),8.01(d,J＝8.6Hz,1H,Ar),7.34(d,J＝2.0Hz,1H,Ar),7.30-7.19(m,3H,Ar),7.10(d,2H,Ar,NHSO2NHCH3),7.00(d,J＝7.5Hz,1H,Ar),5.02(s,2H,CH2),3.06(s,3H,NHCH3),2.94(s,3H,CH3),2.43(d,J＝5.0Hz,3H,CH3)；HRMS(ESI)m/z:449.11206(M+H+)。
Compounds 7, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 of examples 2, 5-19 were synthesized with compounds 6, 8 or 9 and the results are shown in table 1:
TABLE 1 inventive examples 2, 5 to 19
Figure BDA0000943832780000151
Figure BDA0000943832780000161
Examples 20-58 Using scheme 2
EXAMPLE 207 Synthesis of- (N, N-Dimethylcarbamoyloxy) -3- (2-fluoro-3-methanesulfonamidobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 29)
Synthetic route 2 step (c)
To 100mL of CCl of 2-fluoro-3-nitrotoluene (64.5mmol, 10g) and N-bromosuccinimide (77.4mmol, 13.78g)4Benzoyl peroxide (6.45mmol, 1.56g) was added to the suspension and heated at 80 ℃ under reflux for 12 h. Cooled to room temperature, filtered to remove insoluble matter, and concentrated under reduced pressure. And (3) performing column chromatography separation (gradient elution: petroleum ether, petroleum ether/ethyl acetate: 30/1) (v/v), and collecting a pure product to obtain 2-fluoro-3-nitrobenzyl bromide, 6g of yellow oily liquid, and the yield is 39.76%.1H NMR(400MHz,DMSO-d6)δ8.14(t,J＝7.7Hz,1H,Ar),7.97(t,J＝7.1Hz,1H,Ar),7.46(t,J＝8.0Hz,1H,Ar),4.81(s,2H,CH2)。
Synthetic route 2 step (d)
To a solution of 7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 2a) (9.83mmol, 2.46g) in 20mL DMF at room temperature was added K2CO3(19.66mmol, 2.72g) and 2-fluoro-3-nitrobenzyl bromide (11.11mmol, 2.6g) were stirred at room temperature for 5 h. Adding water to quench the reaction, filtering under reduced pressure to obtain yellow solid, and infrared drying. Column chromatography (gradient elution: petroleum ether/ethyl acetate-5/1, dichloromethane/methanol-200/1) (v/v) gave 7- (N, N-dimethylcarbamoyloxy) -3- (2-fluoro-3-nitrobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 27a) as a white solid in 2.87g, 72.4% yield.1H NMR(400MHz,DMSO-d6)δ8.07(t,J＝7.0Hz,1H,Ar),8.00(d,J＝8.6Hz,1H,Ar),7.88(t,J＝6.5Hz,1H,Ar),7.44-7.30(m,2H,Ar),7.24(dd,J1＝8.6Hz,J1＝2.1Hz,1H,Ar),5.17(s,2H,CH2),3.07(s,3H,CH3),2.94(s,3H,CH3)；MS(ESI)m/z:404.35(M+H+),426.35(M+Na+)。
Synthetic route 2 step (e)
7- (N, N-Dimethylcarbamoyloxy) -3- (2-fluoro-3-nitrobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 27a) (3.03mmol, 1.223g), 5% Pd/C (0.17g) and 70mL of methanol were added to a hydrogenation reaction tube at room temperature and stirred in the hydrogenation reactor for 8H. Taking out from the hydrogenation reactor, adding ethyl acetate, heating in water bath until the system becomes clear, filtering to remove insoluble substances, and concentrating the liquid under reduced pressure. Column chromatography (gradient elution, dichloromethane/methanol-200/1, dichloromethane/methanol-100/1) (v/v) gave crude 7- (N, N-dimethylcarbamoyloxy) -3- (2-fluoro-3-aminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 28a) as a yellow solid of 0.9648g with a crude yield of 85.23%. MS (ESI) M/z 374.37(M + H)+),396.38(M+Na+)。
Synthetic route 2 step (f)
Pyridine (1.034mmol, 0.08mL) and methanesulfonyl chloride (1.034mmol, 0.08mL) were added dropwise to a solution of 7- (N, N-dimethylaminocarbonyloxy) -3- (2-fluoro-3-aminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 28a) (0.94mmol, 0.35g) in 10mL of dichloromethane at 0 deg.C, turned to room temperature and stirred for 35H. Adding saturated sodium bicarbonate solution into the reaction solution to quench the reaction, extracting with ethyl acetate for 3 times, washing with hydrochloric acid aqueous solution (1mmol/L), washing with saturated sodium bicarbonate solution, washing with saturated salt solution, and drying with anhydrous sodium sulfate. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-200/1, dichloromethane/methanol-100/1, dichloromethane/methanol-40/1) (v/v) gave 7- (N, N-dimethylcarbamoyloxy) -3- (2-fluoro-3-methanesulfonamidobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 29) as a white solid, 0.379g, 93.03% yield.1H NMR(400MHz,DMSO-d6)δ9.62(s,1H,NH),8.00(d,J＝8.6Hz,1H,Ar),7.30-7.34(m,2H,Ar),7.28-7.18(m,2H,Ar),7.10(t,J＝7.9Hz,1H,Ar),5.11(s,2H,CH2),3.05(d,6H,CH3NCH3),2.94(s,3H,CH3SO2)；HRMS(ESI)m/z:452.09223(M+H+),474.07417(M+Na+),490.04811(M+K+)。
EXAMPLE synthesis of 217- (N, N-Dimethylcarbamoyloxy) -3- (2-fluoro-3-ethanesulfonylaminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 30)
Formic acid (4.7mmol, 0.22g) was slowly added dropwise to chlorosulfonyl isocyanate (4.7mmol, 0.6652g) at 0 ℃, after stirring at room temperature for 1H, 5mL of dichloromethane was added, and the mixture was added dropwise to a bottle containing 3- (2-fluoro-3-aminobenzyl) -7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 28a) (0.94mmol, 0.35g), pyridine (9.4mmol, 0.75mL) and 10mL of dichloromethane at 0 ℃ and stirred at room temperature for 2.5H. The reaction was quenched with water, extracted 1 time with dichloromethane and the organic phase concentrated under reduced pressure. The mixture was concentrated for column chromatography (gradient elution: dichloromethane, dichloromethane/methanol 200/1, dichloromethane/methanol 100/1, dichloromethane/methanol 80/1, dichloromethane/methanol 40/1) (v/v), slurried with an organic solvent (dichloromethane/methanol 40/1) (v/v) to wash unseparated pyridine, and filtered to give 0.1157g of a white solid in 28.33% yield.1H NMR(400MHz,DMSO-d6)δ9.18(s,1H,NH),8.00(d,J＝8.6Hz,1H,H-5),7.39(t,J＝6.9Hz,1H,Ar),7.34(d,J＝1.9Hz,1H,H-8),7.24(dd,J1＝8.6Hz,J2＝1.7Hz,1H,H-6),7.15-7.01(m,4H,NH2,Ar),5.09(s,2H,CH2),3.07(s,3H,CH3),2.94(s,3H,CH3)；HRMS(ESI)m/z:453.08747(M+H+),475.06942(M+Na+),491.04336(M+K+)。
Example 247 Synthesis of- (N, N-Dimethylcarbamoyloxy) -3- (2-fluoro-3- (N- (2-cyanoethyl)) sulfamoylaminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (Compound 33)
A solution of 2-bromoethanol (30mmol, 2.13mL) in 5mL of anhydrous dichloromethane was slowly added dropwise to a solution of chlorosulfonyl isocyanate (30mmol, 4.25g) in 26mL of anhydrous dichloromethane at 0 ℃ and stirred for 2 h. The mixture was added dropwise to a two-necked flask containing 3-aminopropionitrile (33mmol, 2.3133g), triethylamine (48mmol, 6.66mL) and 15mL of dichloromethane at 0 ℃ and stirred at room temperature for 19 hours. Adding hydrochloric acid aqueous solution (1mmol/L) to quench reaction, extracting with dichloromethane for 2 times, washing with water, drying with anhydrous sodium sulfate to obtain colorless transparent crystals, and recrystallizing with ethanol to obtain N- (2-cyanoethyl) -2-oxooxazolidine-3-sulfonamide (compound 5c) as white solid 2.719g with yield of 36.52%.1H NMR(400MHz,DMSO-d6)δ8.74(t,J＝5.5Hz,1H,NH),4.38(t,J＝7.8Hz,2H,OCH2),3.96(t,J＝7.8Hz,2H,NCH2),3.31(q,J＝6.2Hz,2H,CH2CN),2.69(t,J＝6.4Hz,2H,NHCH2)；MS(ESI)m/z:218.25(M-H+)。
Synthetic route 2 step (f)
To a solution of 3- (2-fluoro-3-aminobenzyl) -7- (N, N-dimethylaminocarbonyloxy) -1, 3-benzoxazine-2, 4(3H) -dione (compound 28a) (1.21mmol, 0.45g) in 15mL acetonitrile was added dropwise triethylamine (6.05mmol, 0.83mL) and N-methyl-2-oxooxazolidine-3-sulfonamide (compound 5c) (3.87mmol, 0.848g), and heated at 80 ℃ under reflux for 24H. Diluting with ethyl acetate, washing with hydrochloric acid aqueous solution (1mmol/L), washing with saturated sodium bicarbonate solution, washing with saturated salt solution, and drying with anhydrous sodium sulfate. Column chromatography (gradient elution: dichloromethane, dichloromethane/methanol-200/1, dichloromethane/methanol-100/1) (v/v), dichloromethane trituration, filtration afforded 7- (N, N-dimethylcarbamoyloxy) -3- (2-fluoro-3- (N- (2-cyanoethyl)) sulfamoylaminobenzyl) -1, 3-benzoxazine-2, 4(3H) -dione (compound 30) as a white solid, 0.292g, 49.5% yield.1H NMR(400MHz,DMSO-d6)δ9.56(s,1H,PhNH),8.01(d,J＝8.6Hz,1H,Ar),7.81(t,J＝5.8Hz,1H,NHCH2),7.34-7.38(m,2H,Ar),7.25(dd,J＝8.6,1.3Hz,1H,Ar),7.18(t,J＝7.0Hz,1H,Ar),7.08(t,J＝7.9Hz,1H,Ar),5.10(s,2H,CH2Ph),3.17(q,J＝6.3Hz,2H,NHCH2),3.07(s,3H,CH3),2.95(s,3H,CH3),2.66(t,J＝6.5Hz,2H,CH2CN)；HRMS(ESI)m/z:506.11402(M+H+),528.09597(M+Na+),544.06991(M+K+)。
Compounds 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 of examples 22-23,25-58, the synthetic method with compounds 29, 30, or 33, the results of which are shown in table 2:
TABLE 2 inventive examples 22-23,25-58
Figure BDA0000943832780000191
Figure BDA0000943832780000201
Figure BDA0000943832780000211
Figure BDA0000943832780000221
Example 59 inhibition of non-phosphorylated MEK1 by Compounds of the invention
The molecular level experiment adopts a homogeneous time-resolved fluorescence (HTRF) method, the experiment is divided into two steps, namely a kinase reaction step and a detection reaction step, the kinase reaction step is a step of BRAF phosphorylating MEK1, and a compound diluted by kinase reaction buffer 50mM pH 7.0HEPES buffer, 10mM MgCl2, 1mM DTT, 0.5mM Orthofanadate, 0.01% BSA, 0.27 ng/. mu.l active BRAF (09-122) (Carna), 30nM inactive MEK1GST tagged- (07-141-10) (Carna), 100. mu.M ATP, and room temperature reaction for 2 hours; the second step is a detection reaction step, antibodies MAb Anti-phosphorus MEK1/2-K (61P17KAE) and MAb Anti GST-XL665(61 GSTMALA) from Cisbio are added, signals are detected by a microplate reader FlexStation 3(Molecular Devices) after 3 hours of reaction, and data are processed by using graph Padprism 5 as mapping software. The experiment was repeated 2 times. The known MEK inhibitor PD0325901 was used as a positive control. Some of the compound test results are shown in table 3 below.
TABLE 3 inhibitory Activity of the Compounds of the invention on non-phosphorylated MEK1
Figure BDA0000943832780000231
EXAMPLE 60 evaluation of toxicity of the Compound of the present invention on rhabdomyosarcoma cells (RD cells)
The experimental process is divided into four steps: (1) preparing cell suspension, mixing, adding 100 μ L/well into 96-well plate, and culturing the inoculated cell culture plate in incubator;(2) when the cell density in each well reached 60-70%, different concentration gradients of drug (pH 6.010% FBS medium) were added, 3 multiple wells per concentration of drug, 100. mu.L per well, 5% CO2Incubation at 37 ℃ for 24 hours; (3) sucking off the culture medium in each well, adding 100 μ L of serum-free DMEM culture medium, adding 20 μ L of LMTT solution (5mg/ml) in each well, and culturing for 2 h; (4) the culture medium was aspirated off, 150. mu.L of DMSO was added to each well, the mixture was shaken for 10 minutes, and the absorbance (OD value) at 490nm was measured with a microplate reader.
Calculating CC by improved kouzhui method50：lgCC50＝Xm-I(P-(3-Pm-Pn)/4)
In the formula:
xm: lg (maximum dose); i: lg (maximum dose/adjacent dose); p: sum of positive reaction rates; pm: the maximum positive reaction rate; pn: minimal positive reaction rate.
Cytotoxicity results for representative compounds 31 and 33 are shown in table 4 below, CC50The graphs are shown in FIG. 1 of the specification, and illustrate the CC of two compounds due to the fact that a maximum concentration (409.6 μ M) has just reached about 50% cell death50The value is more than or equal to 400 mu M.
TABLE 4 toxicity results of representative compounds of the invention on RD cells
Compound numbering CC50/μM
31 ≥400
33 >400
And (4) conclusion: CC of two Compounds50The values are all above 400 mu MThus, the compound has higher safety.
EXAMPLE 61 evaluation of antiviral (EV71) Activity of the Compound of the present invention
After determining the toxicity of the compounds of the present invention to RD cells, the inventors selected to be less than CC50The antiviral effect of the compound of the present invention was examined by the drug concentration gradient of the values. Normal growing cells without added virus were used as well plate control (Mock); cells with added virus and no drug were used as negative controls; recording the cytopathic result of each hole, and calculating the EC of the drug-inhibited virus according to the Reed-Muench two-law method50The value is obtained.
The operation process comprises two steps: (1) preparing cell suspension, mixing, adding 100 μ L/well into 96-well plate, and culturing the inoculated cell culture plate in incubator; (2) when the cell density in each well reached 80-90%, the stock culture was aspirated, and a virus (3MOI) containing drug (pH 6.02% FBS medium) was added in different concentration gradients (9 gradients (0.195-50. mu.M) in 8 duplicate wells at 100. mu.L/well with 5% CO2After incubation at 37 ℃ until the negative control wells reached +++ and above the lesion level, the lesion effect was recorded, see table 5.
lgEC50Distance scale x difference between log of dilutions + log of dilutions above 50% disease rate
In the formula
Distance scale ═ (percentage above 50% variability-50%)/(percentage above 50% variability-percentage below 50% variability)
TABLE 5 EC for inhibition of viral infection by Compounds 31 and 3350Results
Compound numbering EC50/μM EC50/μM EC50Mean value/. mu.M SD/μM
31 0.96 1.08 1.02 0.085
33 3.39 4.44 3.92 0.742
The above results indicate that both drugs inhibit 50% of virus replication at micromolar concentrations in 100 μ L solution, with a greater safe therapeutic range (CC)50/EC50Both greater than 100).
Example 62 evaluation of Effect of Compounds of the present invention on inhibition of intracellular ERK pathway
The above experiments have demonstrated that compounds 31 and 33 are effective in inhibiting viral replication. In this experiment, after cells were pretreated with compounds 31 and 33 for 1h, 3MOI of Enterovirus 71 (EV71) was added to the cells and the effect of compounds 31 and 33 on the ERK pathway and viral replication was examined. The method comprises the following specific steps: preparing rhabdomyosarcoma cells (RD) into cell suspension at 2-3 × 105The density of each cell/well is inoculated on a 6-well plate, when the monolayer coverage reaches a density of 80-90%, the plate is replaced by 2% FBS culture medium (pH 6.0) pretreatment for 1h containing different concentrations (6.25-25 μ M) of compounds 31 and 33, EV71 virus solution with 3MOI is added, the cytopathic condition is observed in an inverted microscope (OLYMPUS) after the virus infection for 24h (see figure 2), the cells are collected and lysed to obtain total protein, and BCA egg is used for obtaining total proteinThe white concentration determination kit detects the content of the protein, and the activation condition of an ERK channel and the expression level of the virus protein (VP1) are detected by using a protein immunoblotting method (Western blot).
FIG. 2 shows the cytomorphograms of the negative control group (MOCK), EV71 control group, compound 31 (25. mu.M), compound 33 (25. mu.M), control PD0325901 (10. mu.M; PD0325901 used at a concentration less than compounds 31 and 33 because PD0325901 inhibits the activity at the enzyme level 10 times better than 31 and 33, and therefore the concentration of PD0325901 is selected to be less than 31 and 33 at the time of cell level detection), and the control U0126 (30. mu.M) and EV71 co-acted experimental groups, respectively. The growth state of cells in a negative control group (MOCK) is good; the EV71 control group has obvious cell death phenomenon, the cell gap is enlarged, and the cells become round, which indicates that the EV71 infected cells cause serious pathological effect on the cells; the remaining four compounds clearly organized EV 71-induced cytopathic effects and reduced cell death, and as a result, the cells in PD0325901 and U0126-treated groups were somewhat diseased, while the cytopathic effects in compounds 31 and 33-treated groups were minimal. Compounds 31 and 33 of the invention have better activity than the control drug in preventing EV 71-induced cytopathic effects.
The results of the Western blot experiment are shown in FIG. 3, wherein EV71VP1 is structural protein of EV71, the inhibition effect of the compound on virus replication is indirectly measured by detecting the expression level of the protein, p-MEK1/2 is phosphorylated MEK1/2 protein, t-MEK1/2 is total MEK1/2 protein, the degree of the compound inhibiting the expression of p-MEK1/2 is measured by comparing the expression levels of the two, p-ERK1/2 is phosphorylated ERK1/2 protein, t-ERK1/2 is total ERK1/2 protein, the degree of the compound inhibiting the expression of p-ERK1/2 and the inhibition effect on the pathway are measured by comparing the expression levels of the two, β -actin protein is used as an internal standard of the whole experiment, the expression amount of 12 wells is basically consistent, and the expression amount of the protein in different groups of the experiment is used for showing that the same concentration is used, so that the expression effect and the effect of the drug action of the several proteins are truly reflected.
As can be seen from the experimental results, compounds 31 and 33 at 6.25 μ M can strongly inhibit the expression of p-MEK1/2, while t-MEK1/2 is unchanged compared with the control group, which indicates that compounds 31 and 33 prevent the conversion of non-phosphorylated MEK into t-MEK1/2, while the p-MEK1/2 levels of PD0325901 and U0126 are higher than those of MOCK group, which is consistent with the report that some inhibitors can induce ERK-dependent feedback inhibition phenomenon in the literature; all four compounds strongly inhibited the expression of p-ERK1/2, indicating that all four compounds can block the ERK1/2 pathway.
Specifically, PD0325901 and U0126 have certain inhibitory activity on p-MEK1/2 in addition to the inhibitory effect on non-phosphorylated MEK 1/2. Although PD0325901 and U0126 inhibit non-phosphorylated MEK1/2, the negative feedback effect existing in the cell still causes the accumulation of p-MEK1/2, just as the expression level of p-MEK1/2 protein corresponding to PD0325901 and U0126 in FIG. 3 is much higher than that of MOCK group, but unlike compound 31 and compound 33, PD0325901 and U0126 can still bind with p-MEK1/2 protein (but will not reduce the expression level of p-MEK 1/2) to inhibit the phosphorylation of p-MEK1/2 to its downstream target ERK1/2, so as shown in FIG. 3, the expression level of p-ERK1/2 protein corresponding to PD0325901 and U0126 can still be inhibited, thus still blocking the pathway.
Compared with a negative control group (MOCK), the EV 71-treated group has higher p-ERK1/2 level, which is also consistent with the fact that EV71 infection reported in the literature can cause abnormal activation of ERK1/2 in RD cells; for virus structural protein VP1, it can be seen that compounds 31 and 33 strongly inhibit the expression of VP1 at a concentration of 25 μ M, and both compounds show dose-dependent inhibition effect, and PD0325901 also shows slight dose-dependent inhibition effect, although the activity of PD0325901 is better than that of compounds 31 and 33, the inhibition effect on virus replication is not as good as that of compounds 31 and 33, and the expression level of VP1 of U0126 is equivalent to that of EV71 treated group, and no inhibition effect on virus replication is shown.
And (4) conclusion: the 1, 3-benzoxazine-2, 4-dione compounds represented by the compounds 31 and 33 can obviously inhibit activation of ERK (see figure 3) caused by viruses, and different from positive drugs PD0325901 and U0126, the compounds 31 and 33 can not only inhibit expression of p-MEK1/2, but also effectively relieve the increase of p-MEK1/2 expression caused by ERK-dependent feedback inhibition. In conclusion, compounds 31 and 33 have better antiviral effects and have the potential to be safer antiviral drugs than the positive drugs U0126 and PD 0325901.
Example 63 evaluation of inhibitory Activity of Compounds of the present invention on tumor cells
By using
Figure BDA0000943832780000261
The cell activity is detected by a luminescence method, and the experimental process comprises the following steps: (1) preparing a 1 mu M ATP solution by using a cell culture medium, carrying out a series of gradient dilution (the dilution multiple is 10), adding 100 mu L/hole into a 96-hole plate, adding a CellTiter Glo reagent (100 mu L/hole), placing the micro-hole plate on an oscillator, carrying out gentle oscillation and uniform mixing for 2 minutes, incubating for 10 minutes at room temperature, detecting and recording the result, and drawing an ATP standard curve; (2) preparing cell suspension, uniformly mixing and counting, adding 100 mu L/hole into a 96 black-hole plate, wherein the cell density is 24/hole, adding a culture medium without cells into a blank hole, adding a solution (the concentration is 20 mu M) of a compound to be detected into an experimental hole, incubating for 30 minutes at room temperature, and incubating for 72 hours at 37 ℃ in an incubator; (3) adding CellTiter Glo reagent (100 mu L/hole) to induce cell lysis, placing the microplate on an oscillator, gently oscillating and uniformly mixing for 2 minutes, incubating for 10 minutes at room temperature, detecting and recording results, and calculating the cell survival rate. TABLE 6 inhibitory Activity results (20. mu.M) for partial Compounds on 8 different cell lines
Figure BDA0000943832780000262
Note: PC3M1E8 is a human prostate cancer cell high-transfer subline, Hela is a human cervical cancer cell line, HEK293 is a human embryonic kidney cell line, and A375 is a human melanoma cell line.
And (4) conclusion: 5 compounds (9, 30, 31, 32 and 33) have certain inhibition effect on the A375 cell line; 2 compounds (10 and 58) have certain inhibitory effect on a PC3M1E8 cell line; 1 compound (40) has certain inhibition effect on Hela cell line; these compounds did not inhibit HEK293 cells by more than 50% demonstrating less toxicity to normal cells. The pH of the experimental system was not adjusted for this experiment.
The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.

Claims (11)

1. A benzoxazine compound, the structure of which is shown in formula (I), or pharmaceutically acceptable salt thereof:
Figure FDA0002325490140000011
wherein R is1Is unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
R2、R3、R4And R5Each independently hydrogen or halogen;
R6is unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl or aryl; here, the aromatic hydrocarbon group is a phenyl group or a naphthyl group;
R7and R8Each independently hydrogen or C1-C6 alkyl;
a is hydrogen or of the formula:
Figure FDA0002325490140000012
here, Y is-NH-;
l is-S (O)2-or-CO-;
z is-NH-or-CH2-；
R9Is hydrogen, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, aryl or heteroaryl; here, the substituted C1-C6 alkyl refers to an alkyl carbon chain optionally substituted at an optional position with one or more cyano, halogen, hydroxy or phenyl groups; the aromatic hydrocarbon group is phenyl or naphthyl; said heteroaryl groupSelected from furyl, imidazolyl, indolyl, oxadiazolyl, oxazinyl, oxazolyl, isoxazolyl, pyranyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazinyl, thiadiazolyl, thiatriazolyl, thiazolyl, isothiazolyl, thienyl, triazinyl, or triazolyl.
2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1Is unsubstituted C1-C6 alkyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
R2And R4Are all hydrogen;
R6is unsubstituted C1-C6 alkyl or aromatic hydrocarbon radical; here, the aromatic hydrocarbon group is a phenyl group or a naphthyl group.
3. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1Is unsubstituted C1-C6 alkyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
R2、R4And R5Are all hydrogen; r3Is halogen.
4. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1Is methyl, ethyl, n-propyl, n-butyl, S (O)2R6、C(O)R6、C(O)NR7R8Or S (O)2NR7R8；
Here, R6Is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;
R7and R8Each independently hydrogen, methyl or ethyl.
5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (III):
Figure FDA0002325490140000021
wherein R in the formula (III)1、R3、R4、R5Y, L, Z and R9As described for the compounds of formula (I) as defined in claim 1.
6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (IV):
Figure FDA0002325490140000022
wherein R in the formula (IV)1、R3Y, L, Z and R9As described for the compounds of formula (I) as defined in claim 1.
7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from one of the following compounds:
Figure FDA0002325490140000031
and compounds of the structure of formula (III):
Figure FDA0002325490140000032
R4and R5Are all hydrogen, Y is-NH-, and R1、R3L, Z and R9Is defined as follows:
Figure FDA0002325490140000033
Figure FDA0002325490140000041
8. a process for the preparation of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, which process comprises the following steps when a is hydrogen in the compound of formula (I):
(1) a compound of formula (V) and a halide R1X reacts under alkaline conditions to obtain a compound shown as a formula (VI);
Figure FDA0002325490140000051
(2) reacting a compound shown as a formula (VI) with a compound shown as a formula (VII) to obtain a compound shown as a formula (I);
Figure FDA0002325490140000052
here, the halides R1X, R in the formulae (V), (VI) and (VII)1、R2、R3、R4And R5As described for compounds of formula (I) in claim 1; x is chlorine, bromine or iodine;
when A in the compound of formula (I) is-Y-L-Z-R9The preparation method comprises the following steps:
(1) a compound of formula (V) and a halide R1X reacts under alkaline conditions to obtain a compound shown as a formula (VI);
Figure FDA0002325490140000053
(2) reacting a compound shown in a formula (VI) with a compound shown in a formula (VIII) to obtain a compound shown in a formula (IX);
Figure FDA0002325490140000054
(3) carrying out reduction reaction on the compound shown in the formula (IX) to obtain a compound shown in the formula (X);
Figure FDA0002325490140000055
(4) reacting a compound shown as a formula (X) with a compound shown as a formula (XI) to obtain a compound shown as a formula (I);
Figure FDA0002325490140000061
here, the halides R1R related to X, formula (V), formula (VI), formula (VIII), formula (IX), formula (X) and formula (XI)1、R2、R3、R4、R5、R9The definitions of Y, L and Z are as described for compounds of formula (I) in claim 1; x is chlorine, bromine or iodine; q is chlorine, bromine, iodine or
Figure FDA0002325490140000062
9. A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof.
10. Use of a compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for a MEK inhibitor.
11. Use of a compound as claimed in any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of an anti-tumour or anti-viral disease.
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