CN107200716A

CN107200716A - Benzo oxazinyl compound and preparation method and application

Info

Publication number: CN107200716A
Application number: CN201610154444.XA
Authority: CN
Inventors: 徐萍; 彭宜红; 孙静; 牛彦; 张�浩; 许凤荣; 王超; 梁磊
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2016-03-17
Filing date: 2016-03-17
Publication date: 2017-09-26
Anticipated expiration: 2036-03-17
Also published as: CN107200716B

Abstract

The invention discloses a kind of benzo oxazinyl compound, structure such as formula (I) is shown, or its pharmaceutically acceptable salt, and the definition of each substituent refers to specification in formula (I).In addition, the invention also discloses the compound or the preparation method and application of its pharmaceutically acceptable salt.There is the benzo oxazinyl compound of the present invention MEK to suppress function, and inhibited to non-phosphorylating MEK, meanwhile, toxicity is low, can be used as antitumor and antiviral drugs.

Description

Benzo oxazinyl compound and preparation method and application

Technical field

The invention belongs to field of pharmaceutical chemistry technology, a kind of benzo oxazinyl compound and its preparation side are related in particular to Method and application.There is such compound MEK to suppress function, there is certain antitumor action and antivirus action, thus can by with In the treatment of tumor disease and viral disease.

Background technology

MAPK (MAPK) signal path family is a center construction block of intracellular signal cascades, At least four kinds different paths of mammal, Raf/MEK/ERK paths are that the MAPK found first leads to Road, is also one of deeper signal transduction pathway of research.It is main by three cascade core protein kinases Raf, MEK (Mitogen-activated and Extracellular signal-regulated Kinase) and ERK are constituted, and three are swashed Sequencing is activated enzyme successively, and signal is passed into a variety of intracellular targets, so as to regulate and control various cell processes, for example, turned Record, translation, propagation, differentiation and apoptosis.[Shaul YD,Seger R.Biochimica et Biophysica Acta,2007, 1773:1213-1226.],[Li Y,Zhang LQ,He FC.International Journal of Virology,2006, 13(1):19-22.],[Wortzel I,Segar R.Genes&Cancer,2011,2(3):195-209.],[Frémin C, Meloche S.Journal of Hematology&Oncology,2010,3(8):1-11.]

Raf/MEK/ERK paths are intracellular crucial signal transduction pathways, are also often to be sent out in a variety of human tumors One of signal transduction pathway of raw abnormal activation, wherein common cancer has melanoma, thyroid cancer, colon cancer, bone marrow cancer, turned Shifting property cancer of bile ducts, oophoroma, non-small cell lung cancer and prostate cancer etc..Therefore suppressing this path has become antineoplastic One of study hotspot, FDA approval Raf inhibitor Sorafenib listings in such as 2005, for treating the entities such as advanced renal cell cancer Knurl, FDA approval mek inhibitor Sibutramine Hydrochlorides list for Buddhist nun within 2013, for treating unresectable or transfer melanoma, except upper Outside city's medicine, there are some kinase inhibitors to be currently in the different phase of antitumor clinical research.[Wortzel I, Segar R.Genes&Cancer,2011,2(3):195-209.],[Isshiki Y,Kohchi Y,Iikura H,et al.Bioorganic&Medicinal Chemistry Letters,2011,21:1795-1801.],[Dong Q,Dougan DR,Cong XC,et al.Bioorganic&Medicinal Chemistry Letters,2011,21:1315-1319.], [Pinto R,Herioid S,Cakarova L,et al.Antiviral Research,2011,92:45-56.], [Bekaii-Saab T,Phelps MA,Li XB,et al.Journal of Clinical Oncology,2011,29 (17):2357-2363.],[Bromberg-White JL,Anderson NJ,Duesbery NS.Briefings in Functional Genomics,2012,11(4):300-310.],[Droebner K,Pleschka S,Ludwig S,et al.Antiviral Research,2011,92:195-203.]

In addition, increasing research is confirmed, the activation of Raf/MEK/ERK paths is necessary to viral effective duplication , such as hepatitis viruse, vaccinia virus, II herpes simplex virus types, simian virus 40, HIV-1 viruses, herpesvirus saimiri, Ke Sa Qi viruses, Kaposi's sarcoma virus, influenza virus etc. all rely on this path and replicated.Therefore, selective exclusion this Bar path can prevent a variety of viral duplications and breeding.For example MEK1 inhibitor U0126 can at present be monitored with strong inhibition All first, influenza B virus.[Akinleye A,Furqan M,Mukhi N,et al.Journal of Hematology& Oncology,2013,6(27):1-11.],[Giambartolomei S,Covone F,Levreo M,et al.Oncogene,2001,20:2606-2610.],[L,Berting A,Malkowsk B,et al.Oncogene, 2003,22:2604-2610.],[JC,Andrade AA,Silva PNG,et al.the Journal of Biological Chemistry,2001,276(42):38353-38360.],[Smith CC,Nelson J,Aurelian L,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 kinases has unique 26S Proteasome Structure and Function feature, and from the point of view of position of the MEK kinases in path, MEK is current It is known it is unique can activate ERK kinases, ERK is also MEK the only known substrate, therefore MEK has the substrate of height special One property, while be also at the node location of this path, therefore selection MEK can be improved pair as the mek inhibitor of molecular target The selectivity of path.From the point of view of MEK structure, it contains an allosteric pocket for being different from ATP binding pockets, and this pocket exists It is highly conserved in MEK1 and MEK2, then very low in the sequence homology of other kinases similar areas, this causes inhibitor pair The selectivity of kinases is improved, when inhibitor is attached to this pocket, and the conformation of induced enzyme is in a kind of inactive state, makes Enzyme is activated or activated the ability reduction of stream substrates, so that the activity of inhibitory enzyme.Overwhelming majority mek inhibitor is acted at present In the pocket, belong to non ATP competitive inhibitor.In terms of drug resistance angle, the albumen in ERK paths is encoded by human gene , the possibility very little undergone mutation, therefore suppressing the path can avoid the antiviral drugs of itself for virus from being brought Drug resistance problems, the defect of vaccine can also be avoided.Therefore, this area wishes to develop new mek inhibitor, as new anti- Tumour medicine and antiviral drugs, and with toxicity is low, be not likely to produce drug resistance, Kinase Selectivity is high and selectivity of path The features such as good.[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,Pleschka S,et al.Trends in Molecular Medicine,2003,9(2):46-52.]

The content of the invention

The present inventor have developed a kind of benzo oxazinyl compound, or its pharmaceutically acceptable salt, and the compound has MEK suppresses function, and not only with antitumor, antiviral effect, and toxicity is low, safe.

It is an object of the present invention to provide a kind of new benzo oxazinyl compound, or its pharmaceutically acceptable salt.

The second object of the present invention is to provide this kind of compound or the preparation method of its pharmaceutically acceptable salt.

Third object of the present invention is to provide the pharmaceutical composition containing such compound or its pharmaceutically acceptable salt.

Fourth object of the present invention is to provide this kind of compound or the application of its pharmaceutically acceptable salt.

Specifically, the invention provides a kind of benzo oxazinyl compound, shown in structure such as formula (I), or it is pharmaceutically Acceptable salt：

Wherein, R₁For unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl groups, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₂、R₃、R₄And R₅It is each independently hydrogen or halogen；

R₆For unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl groups or aryl；

R₇And R₈It is each independently hydrogen or C1-C6 alkyl；

A is hydrogen or following formula：

Here, Y is-NH-；

L is-S (O)₂- or-CO-；

Z is-NH- or-CH₂-；

R₉For hydrogen, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl, C3-C6 cycloalkyl, C2-C4 alkenyls, C2-C4 Alkynyl, C3-C6 cycloalkyl groups, aryl or heteroaryl；Here, the substituted C1-C6 alkyl refers in the alkyl carbon chain Replaced in optional position by optional one or more cyano group, halogen, hydroxyl or phenyl.

In embodiments of the invention, the benzo oxazinyl compound that the present invention is provided, wherein, it is described unsubstituted C1-C6 alkyl be selected from methyl, ethyl, propyl group, isopropyl, normal-butyl, 2- methyl-propyls, the tert-butyl group, n-pentyl, neopentyl or N-hexyl etc..

In embodiments of the invention, the benzo oxazinyl compound that the present invention is provided, wherein, the halogen is selected from Fluorine, chlorine, bromine or iodine, it is preferable that be fluorine.

In embodiments of the invention, the benzo oxazinyl compound that the present invention is provided, wherein, the aryl is benzene Base or naphthyl, it is therefore preferable to phenyl.

In embodiments of the invention, the benzo oxazinyl compound that the present invention is provided, wherein, the heteroaryl is selected from Furyl, imidazole radicals, indyl, oxadiazolyl, oxazinyl, oxazolyl, isoxazolyls, pyranose, pyrazinyl, pyrazolyl, pyrrole Piperidinyl, pyrimidine radicals, pyrrole radicals, thiadiazine base, thiadiazolyl group, thiatriazole base, thiazolyl, isothiazolyl, thienyl, triazine radical, Or triazolyl.

In embodiments of the invention, the benzo oxazinyl compound that the present invention is provided, wherein, it is described pharmaceutically to connect The salt received refers to the organic salt and inorganic salts of formula (I) compound；It can be made original position during final separation and purifying compound, Or can could be made by：Individually formula (I) compound and appropriate organic or inorganic acid are reacted, then separated The salt formed；Organic or inorganic acid may be selected from hydrobromate, hydrogen chlorate, hydriodate, sulfate, disulfate, nitric acid Salt, acetate, trifluoroacetate, oxalates, benzene sulfonate, malonate, stearate, laruate, malate, boric acid Salt, benzoate, lactate, phosphate, hexafluorophosphate, toluene fulfonate, formates, citrate, maleate, rich horse Hydrochlorate, succinate, tartrate, naphthoate, mesylate, Lactobionate and lauryl sulfonate etc..

In one embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its is pharmaceutically acceptable Salt, wherein, R₁For unsubstituted C1-C6 alkyl, S (O)₂R₆,C(O)R₆,C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₂And R₄All it is hydrogen；R₆For unsubstituted C1-C6 alkyl or aryl.

In one embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its is pharmaceutically acceptable Salt, wherein, R₁For unsubstituted C1-C6 alkyl, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₂、R₄And R₅All it is hydrogen；R₃For halogen.

In one embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its is pharmaceutically acceptable Salt, wherein, R₁For methyl, ethyl, n-propyl, normal-butyl, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

Here, R₆For methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group or the tert-butyl group；

R₇And R₈It is each independently hydrogen, methyl or ethyl.

In one embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its is pharmaceutically acceptable Salt, wherein, A is hydrogen, R₂And R₄All it is hydrogen；R₃And R₅In one be hydrogen, and another is halogen (be preferably fluorine).

In a kind of preferred embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its pharmaceutically may be used The salt of receiving, as shown in formula (II)：

Wherein, R₁、R₃、R₄、R₅And A is as defined above described by formula (I) compound in face.

In a kind of preferred embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its pharmaceutically may be used The salt of receiving, as shown in formula (III)：

Wherein, R₁、R₃、R₄、R₅, Y, L, Z and R₉It is as defined above described by formula (I) compound in face.

In a kind of preferred embodiment of the present invention, the benzo oxazinyl compound that provides of the present invention or its pharmaceutically may be used The salt of receiving, as shown in formula (IV)：

Wherein, R₁、R₃, Y, L, Z and R₉It is as defined above described by formula (I) compound in face.

The present invention a kind of particularly preferred embodiment in, the invention provides following benzo oxazinyl compound or its Pharmaceutically acceptable salt：

Or

Wherein, R₄And R₅All it is hydrogen, Y is-NH-, and R₁、R₃, L, Z and R₉It is defined as follows：

On the other hand, the invention provides the preparation side of above-mentioned benzo oxazinyl compound or its pharmaceutically acceptable salt Method, when A is hydrogen in formula (I) compound, it comprises the following steps：

(1) compound shown in formula (V) and halides R₁X reacts in the basic conditions, obtains compound shown in formula (VI)；

(2) compound shown in formula (VI) is reacted with compound shown in formula (VII), obtains compound shown in formula (I)；

Here, halides R₁The R being related in X, formula (V), formula (VI) and formula (VII)₁、R₂、R₃、R₄And R₅It is as defined above face Formula (I) compound described by；X is halogen, preferably chlorine, bromine or iodine.

The invention provides the preparation method of above-mentioned benzo oxazinyl compound or its pharmaceutically acceptable salt, work as formula (I) A is-Y-L-Z-R in compound₉When,

It comprises the following steps：

(2) compound shown in formula (VI) is reacted with compound shown in formula (VIII), obtains compound shown in formula (IX)；

(3) compound shown in formula (IX) carries out reduction reaction, obtains compound shown in formula (X)；

(4) compound shown in formula (X) is reacted with formula (XI) compound, obtains compound shown in formula (I)；

Here, halides R₁The R being related in X, formula (V), formula (VI), formula (VIII), formula (IX), formula (X) and formula (XI)₁、 R₂、R₃、R₄、R₅、R₉, Y, L and Z be as defined above described by formula (I) compound in face；X is halogen, preferably chlorine, bromine or iodine；Q For leaving group, it is therefore preferable to chlorine, bromine, iodine or：

In embodiments of the invention, the above-mentioned benzo oxazinyl compound that provides of the present invention or its is pharmaceutically acceptable Salt preparation method, wherein, compound shown in step (1) formula (V) can be obtained in the following way：

With ethyl chloroformate nucleophilic substitution occurs in the presence of pyridine for 2,4- dihydroxy benzoyl amines, obtains Formula V Compound.

It should be noted that synthesis path described above is to illustrate the preparation of the compounds of this invention, and prepare This is not restricted to, under the teachings of the present invention, other synthetic methods are equally possible.

The third aspect, the invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes pharmacologically effective The above-mentioned benzo oxazinyl compound or its pharmaceutically acceptable salt of amount, can also include and pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally in the form of tablet, capsule, pill, powder, granule, powder or syrup, or with The form parenteral introduction of injection.The unit dose of described pharmaceutical composition is 0.1mg to 1g.Aforementioned pharmaceutical compositions can lead to Cross conventional pharmaceutical method preparation.

Fourth aspect, the invention provides above-mentioned benzo oxazinyl compound, or prepared by its pharmaceutically acceptable salt Application in mek inhibitor medicine, the application include but is not limited to as antiviral (include but is not limited to anti-EV71 virus) or Antineoplastic.Dosage is each 0.1mg-1g, and number of times is to be weekly to daily three or four times.Method of administration is mouth Clothes, external application or parenteral.

Experiments prove that, the compounds of this invention has the effect for suppressing MEK activity, with good MEK inhibitory activity, Can as new construction type mek inhibitor medicine, be applied not only to the treatment of tumour and viral disease, meanwhile, toxicity is low.

Brief description of the drawings

Fig. 1 is the survival rate suppression curve figure of the compounds of this invention 31 and 33 pairs of RD cells.

Fig. 2 is the compounds of this invention 31,33, control drug to cytopathic effect result figure and right caused by EV71 processing According to a group result figure.

Fig. 3 is the compounds of this invention 31,33, the protein immunoblot experiment result figure of control drug and control treatment group.

Embodiment

Below by experiment and the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments The purpose of illustration is only used for, is not limited the scope of the invention.

Abbreviation：Ph represents phenyl, and Ar represents aryl hydrogen, and δ represents chemical shift, and unit ppm, J represent coupling constant, single Hz, mp represent fusing point for position, and s represents unimodal, and d represents doublet, and t represents triplet, and q represents quartet, and m represents multiplet, br S represents width unimodal, and dd represents double doublet；

NMR condition：Bruker avance III 400MHz type NMRs.TMS is internal standard, deuterated DMSO and deuterium It is solvent for chloroform；

Mass spectrograph：Waters ACQ-SQD LC-MS type ESI mass spectrographs, Bruker Apex IV FTMS type high-resolution ESI Mass spectrograph；

In the application initiation material be it is known obtain product, or be commercially available from Reagent Company, or existing document report Road synthetic method；

Except the centre of existing document report is external, remaining intermediate provides preparation method and characterize data；

The synthetic route 1 of the compounds of this invention：

Reaction condition：

(a) ethyl chloroformate, pyridine, acetonitrile；

(b)R₁- Cl, NaH, DMF；

(c) bromobenzyl or 3- nitro bromobenzyls, K₂CO₃, DMF；

(d) 5%Pd/C, H₂；

(e) reaction reagent：CH₃COCl,CH₃SO₂Cl,CH₃CH₂SO₂Cl or N- substitution -2- Yang oxazolines 3- sulfonamide (that is, compound 5 in said synthesis route 1)；Base catalyst：Pyridine or triethylamine；Solvent：CH₂Cl₂Or acetonitrile.

The synthetic route 2 of the compounds of this invention：

Reaction condition：

(a) ethyl chloroformate, pyridine, acetonitrile；

(b)R₁-Cl,NaH,DMF；

(c) NBS, benzoyl hydroperoxide, CDCl₃, backflow；

(d)K₂CO₃,DMF；

(e) 5%Pd/C, H₂；

(f) reaction reagent：CH₃SO₂Cl,CH₃SO₂Cl or N- substitution -2- Yang oxazolines 3- sulfonamide；Base catalyst： Pyridine or triethylamine；Solvent：CH₂Cl₂Or acetonitrile；

(g) concentrated ammonia liquor；

(h)MeI,NaH,DMF。

Embodiment 1-19

Embodiment 1-19 compounds use synthetic route 1 above

The 3- of embodiment 1 (3- methylsulfonyl amidos benzyl) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazine -2, The synthesis of 4 (3H)-diketone (compound 6)

The step (a) of synthetic route 1

0 DEG C, to the 9mL second of 2,4- dihydroxy benzoyl amines (6.53mmol, 1g) and pyridine (40.49mmol, 3.26mL) The 3mL acetonitrile solutions of ethyl chloroformate (7.183mmol, 0.78g) are added dropwise in nitrile suspension, are added dropwise by several times, it is ensured that system temperature No more than 5 DEG C.After completion of dropping, oil bath heating stirring, air-distillation when being distilled out there is no solvent, stops distillation, is changed to Backflow, temperature is reduced to 90 DEG C, reacts 2h.Reaction solution is cooled to room temperature, plus 20mL water, concentrated hydrochloric acid acidifying, filtering, obtain 7- hydroxyls- 1,3- benzoxazine -2,4 (3H)-diketone (compound 1), light gray solid 0.94g, yield 80.55%.¹H NMR(400MHz, DMSO-d₆) δ 11.77 (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⁺)。

The step (b) of synthetic route 1

0 DEG C, into the 10mL DMF solutions of 7- hydroxyls -1,3- benzoxazine -2,4 (3H)-diketone (2.23mmol, 0.4g) NaH (80%, 4.47mmol, 0.1341g) is added, N is added dropwise, N- dimethylaminoethyl chlorides (4.47mmol, 0.41mL) go to room Temperature stirring, reacts 3h.Add water and reaction is quenched, ethyl acetate is extracted 3 times, the washing of organic phase saturated common salt, anhydrous sodium sulfate drying. Column chromatography for separation (gradient elution：Dichloromethane, methylene chloride/methanol=100/1) (v/v), obtain 7- (N, N- dimethylamino formyls Epoxide) -1,3- benzoxazines -2,4 (3H)-diketone (compound 2a), white solid 0.396g, yield 70.97%.¹H NMR (400MHz,DMSO-d₆) δ 12.03 (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⁺)。

The step (c) of synthetic route 1

Room temperature, to 7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H)-diketone (1.49mmol, 0.373g) and K₂CO₃Added in the 8mL DMF suspensions of (2.98mmol, 0.411g) 3- nitros bromobenzyls (1.68mmol, 0.363g), 9h is reacted.Add water and reaction is quenched, ethyl acetate is extracted 3 times, organic phase saturated common salt washing, anhydrous sodium sulfate is done It is dry.Column chromatography for separation (gradient elution：Petroleum ether, petrol ether/ethyl acetate=3/1, dichloromethane, methylene chloride/methanol= 100/1, methylene chloride/methanol=40/1) (v/v), 3- (3- nitrobenzyls) -7- (N, N- formyl oxygen dimethylamino) -1 is obtained, 3- benzoxazines -2,4 (3H)-diketone (compound 3a), white solid 0.5387g, thick yield 57.41%.¹H NMR (400MHz,DMSO-d₆) δ 8.28 (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⁺)。

The step (d) of synthetic route 1

Room temperature, to 3- (3- nitrobenzyls) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H) - 5%Pd/C (0.1eq, 0.05g) is added in the 70mL methanol solutions of diketone (1.2mmol, 0.462g), is stirred in hydrogenation instrument Mix, react 9h.Filtration of catalyst, is concentrated under reduced pressure, obtain 3- (3- aminobenzyls) -7- (N, N- formyl oxygen dimethylamino) - 1,3- benzoxazine -2,4 (3H)-diketone (compound 4a) crude product, yellow, viscous solid.Do not isolated and purified, directly thrown Next step is reacted.MS(ESI)m/z:356.28(M+H⁺),378.43(M+Na⁺)。

The step (e) of synthetic route 1

Room temperature, to 3- (3- aminobenzyls) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H) - Pyridine (1.42mmol, 0.11mL) is added in the 10mL dichloromethane solutions of diketone (1.29mmol, 0.46g), methylsulfonyl is added dropwise The 2mL dichloromethane solutions of chlorine (1.42mmol, 0.11mL), are stirred at room temperature, and react 6h.Add water and reaction is quenched, ethyl acetate extraction Take 3 times, organic phase merges, washing, saturated common salt washing, anhydrous sodium sulfate drying.Column chromatography for separation (gradient elution：Dichloromethane Alkane, methylene chloride/methanol=200/1, methylene chloride/methanol=100/1) (v/v), obtain 3- (3- methylsulfonyl amidos benzyl) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H)-diketone (compound 6), white solid 0.2233g, two Walk yield 61.4%.1H NMR(400MHz,DMSO-d₆) δ 9.70 (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⁺)。

The 7- of embodiment 3 (N, N- formyl oxygen dimethylamino) -3- (3- sulfonamides amidos benzyl) -1,3- benzoxazine -2, The synthesis of 4 (3H)-diketone (compound 8)

0 DEG C, formic acid (15mmol, 0.6905g), room are slowly added dropwise into chlorosulphonyl isocyanate (15mmol, 2.123g) After temperature stirring 1h, 10mL dichloromethane is added, 0 DEG C, this mixed liquor is added dropwise to containing 3- (3- aminobenzyls) -7- (N, N- bis- Methylamino formyloxy) -1,3- benzoxazines -2,4 (3H)-diketone (compound 4a) (3mmol, 1.066g), pyridine In the bottle of (30mmol, 2.4mL) and 11mL dichloromethane, 17h is stirred at room temperature.Add water and reaction is quenched, dichloromethane is extracted 3 times. Column chromatography for separation (gradient elution：Dichloromethane, methylene chloride/methanol=100/1, methylene chloride/methanol=40/1) (v/v), Obtain white solid 0.279g, yield 26.83%.¹H NMR(400MHz,DMSO-d₆) δ 9.42 (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⁺)。

The 3- of embodiment 4 (3- (N- methyl) sulfonamides amidos benzyl) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzene The synthesis of Bing oxazines -2,4 (3H)-diketone (compound 9)

0 DEG C, 2- is slowly added dropwise into the 13mL anhydrous methylene chloride solution of chlorosulphonyl isocyanate (10mmol, 1.415g) The 3mL anhydrous methylene chloride solution of bromoethanol (10mmol, 0.71mL), stirs 2h.0 DEG C, this mixed liquor is added dropwise to containing first In the two-mouth bottle of the THF solution (2M, 11mmol, 5.5mL) of amine, triethylamine (16mmol, 2.22mL) and 5mL dichloromethane, turn To being stirred at room temperature, 18h is reacted.Add aqueous hydrochloric acid solution (1mol/L) and reaction, dichloromethane extraction, washing, anhydrous slufuric acid is quenched Sodium is dried, and is concentrated under reduced pressure, is obtained N- methyl -2- oxooxazolidine -3- sulfonamide (compound 5a), colourless transparent crystal, ethanol weight Crystallization, obtains white solid 0.8551g, yield 47.76%.¹H NMR(400MHz,DMSO-d₆) δ 8.18 (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⁺)。

The step (e) of synthetic route 1

To 3- (3- aminobenzyls) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H)-diketone Triethylamine (3.99mmol, 0.56mL) and N- first are added dropwise in the 17mL acetonitrile solutions of (compound 4a) (1.33mmol, 0.4726g) Base -2- oxooxazolidine -3- sulfonamide (compound 5a) (2.66mmol, 0.48g), 80 DEG C are heated to reflux 18h.Add acetic acid second Ester is diluted, and aqueous hydrochloric acid solution (1mol/L) is washed, and saturated sodium bicarbonate solution is washed, saturated common salt washing, anhydrous sodium sulfate drying.Post Chromatography (gradient elution：Dichloromethane, methylene chloride/methanol=200/1, methylene chloride/methanol=100/1) (v/v), obtain 7- (N, N- formyl oxygen dimethylamino) -3- (3- (N- methyl) sulfonamides amidos benzyl) -1,3- benzoxazines -2,4 (3H)-two Ketone (compound 9), khaki 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⁺)。

Embodiment 2,5-19 compound 7,10,11,14,15,16,17,18,19,20,21,22,23,24,25,26 are closed Into method with compound 6,8 or 9,1 the results are shown in Table：

The embodiment of the present invention 2,5-19 of table 1

Embodiment 20-58 uses synthetic route 2

The 7- of embodiment 20 (N, N- formyl oxygen dimethylamino) -3- (the fluoro- 3- methylsulfonyls amido benzyls of 2-) -1,3- benzos The synthesis of oxazine -2,4 (3H)-diketone (compound 29)

The step (c) of synthetic route 2

To 2- fluorin-3-nitrotoluenes (64.5mmol, 10g) and N-bromosuccinimide (77.4mmol, 13.78g) 100mL CCl₄Benzoyl peroxide (6.45mmol, 1.56g) is added in suspension, 80 DEG C are heated to reflux 12h.It is cooled to room Temperature, is filtered to remove insoluble matter, is concentrated under reduced pressure.Column chromatography for separation (gradient elution：Petroleum ether, petrol ether/ethyl acetate=30/1) (v/v) sterling, is collected, the fluoro- 3- nitrobenzyls bromides of 2-, yellow oily liquid 6g, yield 39.76% is obtained.¹H NMR(400MHz, DMSO-d₆) δ 8.14 (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)。

The step (d) of synthetic route 2

Room temperature, to 7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H)-diketone (compound 2a) K is added in the 20mL DMF solutions of (9.83mmol, 2.46g)₂CO₃The fluoro- 3- nitrobenzyls bromide of (19.66mmol, 2.72g) and 2- (11.11mmol, 2.6g), is stirred at room temperature 5h.Add water and reaction is quenched, be filtered under diminished pressure to obtain yellow solid, infra-red drying.Column chromatography point From (gradient elution：Petrol ether/ethyl acetate=5/1, dichloromethane, methylene chloride/methanol=200/1) (v/v), obtain 7- (N, N- formyl oxygen dimethylaminos) -3- (the fluoro- 3- nitrobenzyls of 2-) -1,3- benzoxazines -2,4 (3H)-diketone (compound 27a), White solid 2.87g, yield 72.4%.¹H NMR(400MHz,DMSO-d₆) δ 8.07 (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⁺)。

The step (e) of synthetic route 2

Room temperature, by 7- (N, N- formyl oxygen dimethylamino) -3- (the fluoro- 3- nitrobenzyls of 2-) -1,3- benzoxazines -2,4 (3H)-diketone (compound 27a) (3.03mmol, 1.223g), 5%Pd/C (0.17g) and 70mL methanol are added to hydrogenation Guan Zhong, 8h is stirred in hydrogenation instrument.Taken out from hydrogenation instrument, add ethyl acetate and heating water bath to system becomes clear Clearly, insoluble matter, liquid pressure-reducing concentration are filtered to remove.Column chromatography for separation (gradient elution, dichloromethane, methylene chloride/methanol= 200/1, methylene chloride/methanol=100/1) (v/v), obtain 7- (N, N- formyl oxygen dimethylamino) -3- (fluoro- 3- amino benzyls of 2- Base) -1,3- benzoxazines -2,4 (3H)-diketone (compound 28a) crude product, yellow solid 0.9648g, thick yield 85.23%.MS (ESI)m/z:374.37(M+H⁺),396.38(M+Na⁺)。

The step (f) of synthetic route 2

0 DEG C, to 7- (N, N- formyl oxygen dimethylamino) -3- (the fluoro- 3- aminobenzyls of 2-) -1,3- benzoxazines -2,4 In the 10mL dichloromethane solutions of (3H)-diketone (compound 28a) (0.94mmol, 0.35g) be added dropwise pyridine (1.034mmol, 0.08mL) with mesyl chloride (1.034mmol, 0.08mL), go to and 35h is stirred at room temperature.Unsaturated carbonate hydrogen is added into reaction solution Reaction is quenched in sodium solution, and ethyl acetate is extracted 3 times, and aqueous hydrochloric acid solution (1mmol/L) is washed, and saturated sodium bicarbonate solution is washed, saturation Salt is washed, anhydrous sodium sulfate drying.Column chromatography for separation (gradient elution：Dichloromethane, methylene chloride/methanol=200/1, two Chloromethanes/methanol=100/1, methylene chloride/methanol=40/1) (v/v), obtain 7- (N, N- formyl oxygen dimethylamino) -3- (2- Fluoro- 3- methylsulfonyls amido benzyl) -1,3- benzoxazines -2,4 (3H)-diketone (compound 29), white solid 0.379g, yield 93.03%.¹H NMR(400MHz,DMSO-d₆) δ 9.62 (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⁺)。

The 7- of embodiment 21 (N, N- formyl oxygen dimethylamino) -3- (the fluoro- 3- ethanesulfonamide groups benzyls of 2-) -1,3- benzos The synthesis of oxazine -2,4 (3H)-diketone (compound 30)

0 DEG C, formic acid (4.7mmol, 0.22g), room are slowly added dropwise into chlorosulphonyl isocyanate (4.7mmol, 0.6652g) Temperature stirring 1h after, add 5mL dichloromethane, 0 DEG C, by this mixed liquor be added dropwise to containing 3- (the fluoro- 3- aminobenzyls of 2-) -7- (N, N- formyl oxygen dimethylaminos) -1,3- benzoxazines -2,4 (3H)-diketone (compound 28a) (0.94mmol, 0.35g), pyridine In the bottle of (9.4mmol, 0.75mL) and 10mL dichloromethane, 2.5h is stirred at room temperature.Add water and reaction is quenched, dichloromethane extraction 1 Secondary, organic phase is concentrated under reduced pressure.Concentrate mixture column chromatography for separation (gradient elution：Dichloromethane, methylene chloride/methanol=200/ 1, methylene chloride/methanol=100/1, methylene chloride/methanol=80/1, methylene chloride/methanol=40/1) (v/v), using organic Solvent (methylene chloride/methanol=40/1) (v/v) mashing does not separate clean pyridine to wash away, and filters to obtain white solid 0.1157g, yield 28.33%.¹H NMR(400MHz,DMSO-d₆) δ 9.18 (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⁺)。

The 7- of embodiment 24 (N, N- formyl oxygen dimethylamino) -3- (the fluoro- 3- of 2- (N- (2- cyano ethyls)) sulfonamides amine Base benzyl) -1,3- benzoxazines -2,4 (3H)-diketone (compound 33) synthesis

0 DEG C, 2- is slowly added dropwise into the 26mL anhydrous methylene chloride solution of chlorosulphonyl isocyanate (30mmol, 4.25g) The 5mL anhydrous methylene chloride solution of bromoethanol (30mmol, 2.13mL), stirs 2h.0 DEG C, this mixed liquor is added dropwise to containing 3- In the two-mouth bottle of aminopropionitrile (33mmol, 2.3133g), triethylamine (48mmol, 6.66mL) and 15mL dichloromethane, room is gone to Temperature stirring, reacts 19h.Add aqueous hydrochloric acid solution (1mmol/L) and reaction is quenched, dichloromethane is extracted 2 times, washing, anhydrous slufuric acid Sodium is dried, and obtains colourless transparent crystal, ethyl alcohol recrystallization obtains N- (2- cyano ethyls) -2- oxooxazolidine -3- sulfonamide (chemical combination Thing 5c), white solid 2.719g, yield 36.52%.¹H NMR(400MHz,DMSO-d₆) δ 8.74 (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, CH₂), CN 2.69 (t, J=6.4Hz, 2H, NHCH₂)；MS(ESI)m/z:218.25(M-H⁺)。

The step (f) of synthetic route 2

To 3- (the fluoro- 3- aminobenzyls of 2-) -7- (N, N- formyl oxygen dimethylamino) -1,3- benzoxazines -2,4 (3H) - In the 15mL acetonitrile solutions of diketone (compound 28a) (1.21mmol, 0.45g) be added dropwise triethylamine (6.05mmol, 0.83mL) and N- methyl -2- oxooxazolidine -3- sulfonamide (compound 5c) (3.87mmol, 0.848g), 80 DEG C are heated to reflux 24h.Add Ethyl acetate is diluted, and aqueous hydrochloric acid solution (1mmol/L) is washed, and saturated sodium bicarbonate solution is washed, saturated common salt washing, anhydrous sodium sulfate Dry.Column chromatography for separation (gradient elution：Dichloromethane, methylene chloride/methanol=200/1, methylene chloride/methanol=100/1) (v/v), dichloromethane is beaten, and filters to obtain 7- (N, N- formyl oxygen dimethylamino) -3- (fluoro- 3- of 2- (N- (2- cyano ethyls)) Sulfonamides amido benzyl) -1,3- benzoxazines -2,4 (3H)-diketone (compound 30), white solid 0.292g, yield 49.5%.¹H NMR(400MHz,DMSO-d₆) δ 9.56 (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⁺)。

The compound 31 of embodiment 22-23,25-58,32,34,35,36,37,38,39,40,41,42,43,44,45, 46th, 47,48,49,50,51,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70, synthetic method is same Compound 29,30 or 33, the results are shown in Table 2：

The 22-23,25-58 of the embodiment of the present invention of table 2

The inhibitory action of embodiment 59, the compounds of this invention to non-phosphorylating MEK1

Molecular level experiment uses homogeneous phase time discrimination fluorescence (HTRF) method, and experiment is divided into two steps, is kinases respectively Reactions steps and detection reactions steps, the step of kinase reaction step is BRAF phosphorylation MEK1, addition are buffered by kinase reaction Liquid 50mM pH=7.0HEPES buffer, 10mM MgCl2,1mM DTT, 0.5mM Orthovanadate, 0.01%BSA are dilute The compound released, 0.27ng/ μ l active BRAF (09-122) (Carna), 30nM inactive MEK1GST tagged- (07-141-10) (Carna), 100 μM of ATP are reacted at room temperature 2 hours；Second step is detection reactions steps, adds Cisbio public Take charge of antibody MAb Anti-phospho MEK1/2-K (61P17KAE) and MAb Anti GST-XL665 (61GSTXLA), reaction 3 Signal is detected using ELIASA FlexStation 3 (Molecular Devices) after hour, mapping software GraphPad is used Prism 5 is handled data.Experiment is repeated 2 times.Positive control is used as using known mek inhibitor PD0325901.Partization Compound test result is as shown in table 3 below.

Inhibitory activity of the compounds of this invention of table 3 to non-phosphorylating MEK1

The toxicity assessment of embodiment 60, the compounds of this invention to human rhabdomyosarcoma cells (RD cells)

Experimentation is divided into four steps：(1) prepare cell suspension and mix, 100 μ L/ holes are added in 96 orifice plates, will be inoculated with Good Tissue Culture Plate is put into incubator and cultivated；(2) when the cell density length in every hole is to 60-70%, add different dense Spend the medicine (the 10%FBS culture mediums of pH 6.0) of gradient, 3 multiple holes of medicine of each concentration, per hole 100 μ L, 5%CO₂、37 DEG C be incubated 24 hours；(3) culture medium in every hole is siphoned away, the DMEM culture mediums that 100 μ L are free of serum is added, 20 μ L is added per hole MTT solution (5mg/ml), continues to cultivate 2h；(4) nutrient solution is all siphoned away, 150 μ L DMSO is added per hole, vibrated 10 minutes, Absorbance (OD values) under ELIASA detection 490nm.

CC is calculated using improvement bandit's formula method₅₀：lgCC₅₀=Xm-I (P- (3-Pm-Pn)/4)

In formula：

Xm：Lg (maximum dose)；I：Lg (maximum dose/adjacent dosage)；P：Positive reaction rate sum；Pm：It is maximum positive Reactivity；Pn：Minimum positive reaction rate.

The cytotoxicity result of representative compound 31 and 33 is as shown in table 4 below, CC₅₀Curve map is shown in Figure of description 1, because Cmax (409.6 μM) has just reached about 50% cell death, illustrates the CC of two compound things₅₀>=400 μM of value.

Toxicity data of the representation compound of the present invention of table 4 to RD cells

Compound number	CC₅₀/μM
		31	≥400
33	>400

Conclusion：The CC of two compounds₅₀Value illustrates that compound security is higher more than 400 μM.

Antiviral (EV71) activity rating of embodiment 61, the compounds of this invention

After toxicity of the compounds of this invention to RD cells is determined, the present inventor's selection is less than CC₅₀The drug concentration of value Gradient, detects the antiviral effect of the compounds of this invention.Compareed using the cell for being not added with viral, normal growth as orifice plate (Mock)；Using addition virus, the cell of drug containing is not used as negative control；Cytopathic effect of the record per hole, according to Reed-Muench Liang Shi methods calculate the EC of Drug inhibition virus₅₀Value.

Operating process is in two steps：(1) prepare cell suspension and mix, 100 μ L/ holes are added in 96 orifice plates, will be inoculated with Tissue Culture Plate be put into incubator cultivate；(2) when the cell density length in every hole is to 80-90%, original fluid is siphoned away, The medicine (the 2%FBS culture mediums of pH 6.0) of the various concentrations gradient containing viral (3MOI) is added, totally 9 gradient (0.195-50 μ M), 8 multiple holes, per hole 100 μ L, 5%CO₂, 37 DEG C be incubated to negative control hole and reach +++ and after above lesion degree, record Lesion effect, is shown in Table 5.

lgEC₅₀Difference between=distance proportion × dilution factor logarithm+higher than 50% lesion rate dilution factor logarithm

In formula

Distance proportion=(percentage -50% for being higher than 50% lesion rate)/(percentage for being higher than 50% lesion rate-it is less than The percentage of 50% lesion rate)

The compound 31 and 33 of table 5 suppresses the EC of virus infection₅₀As a result

Compound number	EC₅₀/μM	EC₅₀/μM	EC₅₀Average value/μM	SD/μM
					31	0.96	1.08	1.02	0.085
33	3.39	4.44	3.92	0.742

The above results show, in 100 μ L solution, and two medicines can suppress answering for 50% virus under micro-molar concentration System, with larger safe treatment scope (CC₅₀/EC₅₀It is all higher than 100).

Embodiment 62, the compounds of this invention are evaluated intracellular ERK paths inhibition

Above-mentioned experiment has verified that compound 31 and 33 can effectively suppress the duplication of virus.This experiment uses compound After 31 and 33 pretreatment cell 1h, 3MOI enterovirns type 71 (EV71), detection compound 31 and 33 pairs are added into cell The influence of ERK paths and virus replication.Comprise the following steps that：Human rhabdomyosarcoma cells (RD) are configured to cell suspending liquid, pressed 2-3×10⁵The density of individual cells/well is inoculated on 6 orifice plates, when cell monolayer coverage rate reaches 80-90% density, is changed 1h is pre-processed for the 2%FBS culture mediums (pH 6.0) containing various concentrations (6.25-25 μM) compound 31 and 33,3MOI is added EV71 viral solutions, treat virus infection 24h after inverted microscope (OLYMPUS) observation cytopathy situation (referring to Fig. 2), Collect cell and crack acquisition total protein, detect protein content using BCA determination of protein concentration kit, printed using protein immunization The Activation of mark method (Western blot) detection ERK paths and the expression of virus protein (VP1).

Fig. 2 lists negative control group (MOCK), EV71 control groups, compound 31 (25 μM), (25 μ of compound 33 respectively M), (10 μM of comparison medicine PD0325901；The concentration ratio compound 31 and 33 that PD0325901 is used is small, is because PD0325901 exists The inhibitory activity of enzyme level is compared with 31 and 33 10 times, therefore in cell-based assay, and PD0325901 concentration selects 31 Hes of ratio 33 is small) and comparison medicine U0126 (30 μM) and the cellular morphology figure of EV71 collective effect experimental groups.Negative control group (MOCK) cell Growth conditions are good；EV71 control groups occur in that obvious Cell death, and space between cells becomes big, cell rounding, explanation EV71 infection cells cause cell to occur serious lesion effect；Remaining four compound can substantially organize the thin of EV71 inductions Born of the same parents' lesion effect, reduces cell death, from the results of view, the cell of PD0325901 and U0126 treatment groups there occurs to a certain degree Lesion, and the treatment group cytopathic effect very little of compound 31 and 33.In terms of the cytopathic effect for preventing EV71 from inducing, The compounds of this invention 31 and 33 has preferably active compared with control drug.

The result of protein immunoblot experiment is shown in Fig. 3, wherein, EV71VP1 is EV71 structural proteins, by detecting the egg The inhibition that white expression indirect measure compounds on viral is replicated；P-MEK1/2 is the MEK1/2 albumen of phosphorylation, T-MEK1/2 is total MEK1/2 albumen, by comparing both expressions, and p-MEK1/2 expression is suppressed to weigh compound Degree；P-ERK1/2 is the albumen of phosphorylated CREB 1/2, and t-ERK1/2 is total ERK1/2 albumen, by comparing both expression water It is flat, to weigh compound to the p-ERK1/2 degree expressed and the inhibition to the path；β-actin albumen is used as whole reality The internal standard tested, the expression quantity in 12 holes is basically identical, to illustrate that different groups of the experiment uses the albumen of same concentrations, so that very The expression effect and drug effectiveness of above-mentioned several albumen are reacted on the spot.

From experimental result as can be seen that can consumingly suppress p-MEK1/2 table in 6.25 μM of compound 31 and 33 Reach, and t-MEK1/2 is then constant compared with control group, illustrates that compound 31 and compound 33 prevent non-phosphorylating MEK to be converted into T-MEK1/2, and PD0325901 and U0126 p-MEK1/2 levels are then higher compared with MOCK groups, this some suppression with document report The feedback inhibition phenomenon that preparation can induce ERK to rely on is consistent；The p-ERK1/2 expression of four compound equal strong inhibitions, shows Four compounds can block ERK1/2 paths.

Specifically, PD0325901 and U0126 to the MEK1/2 of non-phosphorylating in addition to having inhibitory action, to p-MEK1/2 Also there is certain inhibitory activity.Although PD0325901 and U0126 inhibit non-phosphorylating MEK1/2, intracellular presence it is negative Feedback effect has still resulted in p-MEK1/2 accumulation, as PD0325901 and U0126 corresponding positions p-MEK1/2 albumen in Fig. 3 Expression quantity is much higher compared with MOCK groups, but from unlike compound 31 and compound 33, PD0325901 and U0126 still can be with With p-MEK1/2 protein bindings (but not reduce p-MEK1/2 expression quantity) and suppress p-MEK1/2 to target ERK1/ downstream The expression of corresponding p-ERK1/2 albumen still can be with 2 phosphorylation, therefore as shown in Figure 3, PD0325901 and U0126 It is suppressed, therefore can still blocks the path.

Compared with negative control group (MOCK), the p-ERK1/2 levels of EV71 treatment groups are higher, this also with document report EV71 infection can cause the intracellular ERK1/2 abnormal activations of RD to be consistent；For virus structural protein VP1, it can be seen that compound 31 and 33 under 25 μM of concentration, strong inhibition VP1 expression, and the two compounds show dose-dependent suppression Effect, PD0325901 also shows slight dose-dependant inhibition, although PD0325901 activity is compared with compound 31 It is good with 33, but the effect of its suppressing virus replication is not so good as compound 31 and 33, U0126 VP1 expression quantity and EV71 treatment group phases When not showing the effect of suppressing virus replication.

Conclusion：Can substantially it be suppressed for the 1,3- benzoxazine -2,4- cyclohexadione compounds of representative with compound 31 and 33 The activation (referring to Fig. 3) of ERK paths caused by viral, different from positive drug PD0325901 and U0126, compound 31 and 33 is not P-MEK1/2 expression can only be suppressed, while also can effectively alleviate p-MEK1/2 tables caused by the feedback inhibition that ERK is relied on Increase up to amount.In a word, compared to positive drug U0126 and PD0325901, compound 31 and 33 has more preferable antiviral effect, had It is used as the potentiality of safer antiviral drugs.

The inhibitory activity evaluation of embodiment 63, the compounds of this invention to tumour cell

UsingLuminescence method detects cell viability, and experimentation is divided into the following steps：(1) trained using cell 1 μM of ATP solution of the basigamy system of supporting, carries out a series of gradient dilutions (extension rate is 10), 100 μ L/ holes is added in 96 orifice plates, CellTiter Glo reagents (100 μ L/ holes) are added, microwell plate is put into gentle vibration on the oscillator is mixed 2 minutes, and room temperature is incubated Educate 10 minutes, detect and record result, draw ATP standard curves；(2) prepare cell suspension and mix counting, in 96 black holes plates 100 μ L/ holes of middle addition, cell density is 24/hole, and not celliferous culture medium is added in blank well, and experimental port adds to be measured Compound solution (concentration is 20 μM), is incubated at room temperature 30 minutes, 37 DEG C of incubator culture 72h；(3) CellTiter Glo examinations are added Agent (100 μ L/ holes) inducing cell lysis, puts gentle vibration on the oscillator by microwell plate and mixes 2 minutes, be incubated at room temperature 10 points Clock, detects and records result, calculates cells survival rate.Inhibitory activity result of the part of compounds of table 6 to 8 kinds of different cell lines (20μM)

Note：PC3M1E8 is Human Prostate Cancer Cells height transfer subbreed, and Hela is Human cervical cancer cell lines, and HEK293 is people Embryonic kidney cells system, A375 is human melanoma cell line.

Conclusion：5 compounds (9,30,31,32 and 33) have certain inhibitory action to A375 cell lines；2 chemical combination Thing (10 and 58) has certain inhibitory action to PC3M1E8 cell lines；1 compound (40) has certain to Hela cell lines Inhibitory action；These compounds are not above 50% suppression to HEK293 cells, it was demonstrated that smaller to normal cytotoxicity.This The pH of the non-regulation experiment system of experiment.

The preferred embodiments of the present invention are the foregoing is only, are merely illustrative for the purpose of the present invention, and it is nonrestrictive； Those of ordinary skill in the art understand that can carry out many to it in the spirit and scope that the claims in the present invention are limited changes Become, modification, or even equivalent change, but fall within protection scope of the present invention.

Claims

1. a kind of benzo oxazinyl compound, shown in structure such as formula (I), or its pharmaceutically acceptable salt：

R₂、R₃、R₄And R₅It is each independently hydrogen or halogen；

R₆For unsubstituted C1-C6 alkyl, C3-C6 cycloalkyl groups or aryl；

R₇And R₈It is each independently hydrogen or C1-C6 alkyl；

A is hydrogen or following formula：

Here, Y is-NH-；

L is-S (O)₂- or-CO-；

Z is-NH- or-CH₂-；

R₉For hydrogen, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl, C3-C6 cycloalkyl, C2-C4 alkenyls, C2-C4 alkynyls, C3-C6 cycloalkyl groups, aryl or heteroaryl；Here, the substituted C1-C6 alkyl refers in the optional position of the alkyl carbon chain Put and replaced by optional one or more cyano group, halogen, hydroxyl or phenyl.

2. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, R₁For unsubstituted C1-C6 alkyl, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₂And R₄All it is hydrogen；

R₆For unsubstituted C1-C6 alkyl or aryl.

3. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, R₁For unsubstituted C1-C6 alkyl, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₂、R₄And R₅All it is hydrogen；R₃For halogen.

4. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, R₁For methyl, ethyl, n-propyl, just Butyl, S (O)₂R₆、C(O)R₆、C(O)NR₇R₈Or S (O)₂NR₇R₈；

R₇And R₈It is each independently hydrogen, methyl or ethyl.

5. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, formula (I) compound is changed for formula (III) Compound：

Wherein, R in formula (III)₁、R₃、R₄、R₅, Y, L, Z and R₉Definition formula (I) compound is retouched as described in the appended claim 1 State.

6. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, formula (I) compound is changed for formula (IV) Compound：

Wherein, R in formula (IV)₁、R₃, Y, L, Z and R₉Definition as described in the appended claim 1 described by formula (I) compound.

7. the preparation method of the compound or its pharmaceutically acceptable salt any one of claim 1 to 6, it is included such as Lower step：

Here, halides R₁The R being related in compound shown in compound shown in X, the formula (V) and the formula (VI)₁And R₂Determine Justice is as described by formula (I) compound in claim 1；X is halogen, preferably chlorine, bromine or iodine.

8. the pharmaceutical composition of a kind of compound comprising any one of claim 1-6 or its salt pharmaceutically received.

9. the compound or its salt pharmaceutically received any one of claim 1-6 are in mek inhibitor medicine is prepared Application.

10. compound or its salt pharmaceutically received any one of claim 1-6 prepare it is antitumor or antiviral Application in medicine.