CN110526907B - Benzoxazinone derivatives and uses thereof - Google Patents

Benzoxazinone derivatives and uses thereof Download PDF

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CN110526907B
CN110526907B CN201810736569.2A CN201810736569A CN110526907B CN 110526907 B CN110526907 B CN 110526907B CN 201810736569 A CN201810736569 A CN 201810736569A CN 110526907 B CN110526907 B CN 110526907B
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李锐
魏于全
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Sichuan University
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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
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
    • C07D265/36—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings condensed with one six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Abstract

The invention relates to a benzoxazinone derivative and application thereof, belonging to the technical field of antitumor drugs. The invention aims to provide a novel benzoxazinone derivative, and the structural formula of the compound is shown as a formula I. The invention designs and synthesizes a series of new compounds taking benzoxazinone as a parent nucleus, and the compounds can inhibit PI3K/Akt/mTOR signal pathways, thereby achieving better effect of inhibiting tumor cell proliferation.
Figure DDA0001722173430000011

Description

Benzoxazinone derivatives and uses thereof
Technical Field
The invention relates to a benzoxazinone derivative and application thereof, belonging to the technical field of antitumor drugs.
Background
The PI3K/Akt/mTOR signaling pathway mainly comprises three members of phosphatidylinositol 3-kinase (phosphoside-3-kinase, PI3K), protein kinase B (PKB/Akt), and target rapamycin (mTOR). PI3K is an esterase, which is mainly present in the cytoplasm at rest; PI3K, when fully activated, catalyzes the conversion of the substrate PIP2 to PIP3, with PIP3 acting as a second messenger activating Akt downstream. Akt is a key protein downstream of PI3K, is a serine/threonine protein kinase highly conserved in evolution, and is located in cytoplasm in a resting state; activated Akt phosphorylates and inhibits TSC1/2 formation, which in turn releases Rheb, ultimately activating downstream mTORC. mTOR belongs to the serine/threonine protein kinase family, possesses two subtypes, mTORC1 and mTORC2, which are important regulators of cell growth and proliferation; when the cells are stimulated by external trophic factors or growth factor signals, intracellular mTOR can activate some related target proteins at the downstream, and further positively or negatively regulate the metabolism and growth of the whole cells. The PI3K/Akt/mTOR signaling pathway is involved in regulating various cellular life processes, such as: growth and proliferation of cells, synthesis of proteins, transcription and metabolic processes. The existing research shows that the PI3K/Akt/mTOR signaling pathway plays a crucial role in the occurrence and development of tumors, and the inhibition of the PI3K/Akt/mTOR signaling pathway is considered to be a promising anticancer therapy.
At present, the antitumor drugs designed aiming at the PI3K/Akt/mTOR signaling pathway are not exhaustive, such as ATP competitive PI3K and mTOR inhibitor BEZ235, multi-target PI3K inhibitor PI-103, mTOR single inhibitor rapamycin and the like. However, in actual studies, it was found that the existing compounds have some drawbacks. For example, a single mTOR inhibitor is easy to cause drug resistance, and a rapamycin derivative can generate feedback activation of Akt when inhibiting mTOR, so that the PI3K signal pathway is reactivated, and therefore, rapamycin has limited tumor cell inhibition capacity in vitro tumor cell inhibition experiments.
Patent US2010/0311736a1 discloses compounds with N-3-pyridyl-4-fluorobenzenesulfonamide as a parent nucleus, two of which contain a benzoxazinone structure, and the N-6 position is linked to a benzene ring through a methylene group. However, through research, the compound has limited inhibition rate to PI3K alpha, even partial inhibition activity is very low, and through research on the crystal structure of PI3K alpha, the molecular activity obtained by directly coupling the N-6 position with a benzene ring is far superior to that obtained by coupling methylene with the benzene ring.
Disclosure of Invention
The invention aims to research and develop a novel compound which inhibits a PI3K/Akt/mTOR signaling pathway and is used for treating and preventing cancers related to PI3K/Akt/mTOR signaling abnormality.
The invention aims to provide a novel benzoxazinone derivative which can successfully inhibit a PI3K/Akt/mTOR signaling pathway.
The structural formula of the benzoxazinone derivative is shown as the formula I:
Figure BDA0001722173410000021
formula I
Wherein R is1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000022
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro;
R2is composed of
Figure BDA0001722173410000023
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000025
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
The invention also provides isomers, pharmaceutically acceptable salts and hydrates of the benzoxazinone derivatives.
The invention also provides application of the benzoxazinone derivative in producing a medicament for inhibiting a PI3K/Akt/mTOR signal pathway in a mammal.
The invention also provides application of the benzoxazinone derivatives in preparation of antitumor drugs.
The invention also provides a pharmaceutical composition, which consists of an effective component and pharmaceutically acceptable auxiliary materials, wherein the effective component contains therapeutically effective amount of the benzoxazinone derivative or the isomer thereof or pharmaceutically acceptable salt or hydrate thereof.
The invention also provides application of the benzoxazinone derivative in preparation of a DNA-PK inhibitor.
Preferably, the benzoxazinone derivative is compound C5-1.
The invention designs and synthesizes a series of new compounds taking benzoxazinone as a parent nucleus, and the compounds can inhibit PI3K/Akt/mTOR signal pathways, thereby achieving better effect of inhibiting tumor cell proliferation. In addition, the compound can be used as a DNA-PK inhibitor and has good inhibitory activity on DNA-PK.
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FIG. 1 is a photograph of plate clones of Hela cells at various concentrations of Compound C5-1.
FIG. 2 shows the plate clone colony count statistics of Hela cells with different concentrations of compound C5-1.
FIG. 3 shows the time of administration and the change in tumor volume in the Hela transplanted tumor model.
Fig. 4 shows the variation of the administration time and tumor volume of a549 transplantable tumor model.
FIG. 5 shows the change in the administration time and the body weight of mice in the Hela transplantation tumor model.
Fig. 6 shows the variation of the administration time and the body weight of mice in the a549 transplantable tumor model.
Detailed Description
The structural formula of the benzoxazinone derivative is shown as the formula I:
Figure BDA0001722173410000031
formula I
Wherein R is1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000032
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000033
R3Is hydrogen, C1-C4 alkoxy, halogenOr hydroxy, R4Is hydrogen or
Figure BDA0001722173410000034
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000035
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000036
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000037
R5Is hydrogen or halogen, X1Is C or N. Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000038
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000039
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000310
R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA00017221734100000311
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA00017221734100000312
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000041
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000042
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000043
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000044
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R1Is C3-C6 cycloalkyl, heterocyclyl or
Figure BDA0001722173410000045
R6Is a mono-or polysubstituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000046
X2Is C or N. Further preferred is X2Is N.
Further preferably, R1Is C3-C6 cycloalkyl or
Figure BDA0001722173410000047
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000048
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000049
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA00017221734100000410
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA00017221734100000411
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000412
R5Is hydrogen or halogen, X1Is C or N. Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA00017221734100000413
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000051
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000052
R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA0001722173410000053
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000054
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA0001722173410000055
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000056
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA0001722173410000057
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000058
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R1Is C3-C6 cycloalkyl or
Figure BDA0001722173410000059
R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA00017221734100000510
X2Is C or N. Further preferred is X2Is N.
Further preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA00017221734100000511
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000512
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000061
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000062
R5Is hydrogen or halogen, X1Is C or N. Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, orPhenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000063
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000064
R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000065
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000066
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000067
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R1Is cyclopropyl, cyclohexyl, a benzene ring, or phenyl monosubstituted by halogen, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000068
X2Is C or N. Further preferred is X2Is N.
As one of the preferable modes, the structural formula is shown as a formula II:
Figure BDA0001722173410000069
formula II
Wherein R is6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA00017221734100000610
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000611
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000071
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000072
R5Is hydrogen or halogen, X1Is C or N. Preferably, R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000073
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000074
R5Is hydrogen or halogen. Preferably, R6Is a single substituent group, and is a mono-substituent,R6selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000075
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000076
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000077
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R6Is a mono substituent, R6Selected from hydrogen, halogen, C1-C6 alkyl, C1-C4 alkoxy, hydroxyl, amino, nitro or C1-C4 alkyl substituted by hydroxyl, amino and nitro; r2Is composed of
Figure BDA0001722173410000078
X2Is C or N. Further preferred is X2Is N.
Further preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000079
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000710
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000081
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000082
R5Is hydrogen or halogen, X1Is C or N. Preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000083
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000084
R5Is hydrogen or halogen. Preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000085
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000086
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R6Is chlorine, bromine, methyl or methoxy, R2Is composed of
Figure BDA0001722173410000087
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R6Is chlorine, bromine or methylOr methoxy radical, R2Is composed of
Figure BDA0001722173410000088
X2Is C or N. Further preferred is X2Is N.
More preferred structural formulas are shown in formula III:
Figure BDA0001722173410000089
formula III
The radicals in formula III are selected as in formula II, except that R in formula II6Defined as para substituents.
As another preferred mode, R in the formula I1Is C3-C6 cycloalkyl; r2Is composed of
Figure BDA00017221734100000810
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000091
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000092
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000093
R5Is hydrogen or halogen, X1Is C or N. Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000094
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA0001722173410000095
R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000096
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000097
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000098
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R1Is C3-C6 cycloalkyl, R2Is composed of
Figure BDA0001722173410000099
X2Is C or N. Further preferred is X2Is N.
Further preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA00017221734100000910
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000911
R5Is hydrogen or halogen, X1Is C or N, X2Is C or N.
Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA00017221734100000912
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxyRadical, R4Is hydrogen or
Figure BDA00017221734100000913
R5Is hydrogen or halogen, X1Is C or N. Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA00017221734100000914
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R4Is hydrogen or
Figure BDA00017221734100000915
R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA0001722173410000101
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA0001722173410000102
R3Is hydrogen, C1-C4 alkoxy, halogen or hydroxy, R5Is hydrogen or halogen. Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA0001722173410000103
R3Is chlorine or methoxy, R5Is hydrogen or halogen.
Preferably, R1Is cyclopropyl or cyclohexyl, R2Is composed of
Figure BDA0001722173410000104
X2Is C or N. Further preferred is X2Is N.
The following are preferred structural formulae of the present invention.
Figure BDA0001722173410000105
Figure BDA0001722173410000111
Figure BDA0001722173410000121
Figure BDA0001722173410000131
The preparation method of the benzoxazinone derivative can adopt a conventional method, and the specific preparation method is described in example 1.
The invention also relates to an isomer, a pharmaceutically acceptable salt and a hydrate of the compound shown as the general formula I. Among the pharmaceutically acceptable salts, the compounds of formula I include, but are not limited to, salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid, hydrobromic acid and nitric acid, and salts with various organic acids such as malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, acetic acid, lactic acid, p-toluenesulfonic acid, methanesulfonic acid, palmitic acid, and the like. Some of the compounds of the present invention may be crystallized or recrystallized using water or various organic solvents, in which case various solvates may be formed. The present invention includes those stoichiometric solvates, including hydrates, as well as compounds containing variable amounts of water that are formed when prepared by a low pressure sublimation drying process.
The benzoxazinone derivative can be used for producing medicaments for inhibiting PI3K/Akt/mTOR signal pathways in mammals.
The benzoxazinone derivatives can also be used for preparing antitumor drugs.
The compound of the present invention or a pharmaceutically acceptable salt thereof may be used alone or in the form of a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient, and when used in the form of a pharmaceutical composition, a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt or hydrate thereof and one or more pharmaceutically acceptable carriers or diluents are usually combined to make an appropriate administration form or dosage form. Accordingly, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a benzoxazinone derivative according to the invention, all possible isomers thereof or a pharmaceutically acceptable salt or hydrate thereof and at least one pharmaceutically acceptable carrier.
Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrapin, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.
The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or warm suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, warm suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, implant, patch, liniment, etc.
The pharmaceutical compositions of the present invention may also contain conventional carriers, including but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium oxide, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1 to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.
The invention also provides application of the benzoxazinone derivative in preparation of a DNA-PK inhibitor.
Preferably, the benzoxazinone derivative is compound C5-1.
In the present invention, the "C1-C6 alkyl group" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and the like. "C1-C4 alkyl" refers to a straight or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and the like.
"C1-C4 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, and the like.
"monosubstituted" means that only one hydrogen atom is substituted.
"polysubstituted" means that at least two hydrogen atoms are substituted, and the substituents may be the same or different.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Synthesis of the Compound of example 1
1. Synthesis of intermediates
Preparation of intermediate 2-chloro-N-cyclohexylacetamide (a1)
Figure BDA0001722173410000141
The starting material cyclohexylamine (3g, 30mmol) was dissolved in 25mL acetonitrile and potassium carbonate (6.2g, 45mmol) was added. Stirring for 20min under ice bath to mix thoroughly. Chloroacetyl chloride (3mL, 36mmol) was slowly added dropwise to generate a large amount of white smoke, and stirring was continued for 30min until the white smoke disappeared and the solution was pale yellow. The reaction was continued at room temperature for 3h and monitored by TLC until the reaction was complete. The treatment method comprises the following steps: most of the acetonitrile was removed by distillation under reduced pressure, and 100mL of water was addedExtraction was performed with EA (4X 25mL), the organic layer was collected, washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated by distillation under reduced pressure to give a white crude product, which was recrystallized from a PE/EA system to give a white powder of needle-like crystals, about 3.5g, in 67% yield. ESI-MS 198.1[ M + Na ]]+.
Synthesis of intermediate (5-bromo-2-chlorophenoxy) -N-cyclohexylacetamide (b1)
Figure BDA0001722173410000151
Dissolving 5-bromo-2-chlorophenol (2.9g, 14.3mmol) serving as a raw material in 50mL of acetonitrile, adding potassium carbonate (2.5g, 17.1mmol), stirring for 30min, adding the intermediate a1 into the reaction solution, moving the reaction solution into an oil bath kettle at 80 ℃, heating and refluxing for 4 hours, changing the mixed solution from clear to turbid (separating out white solid), and monitoring the progress of the reaction by TCL. The treatment method comprises the following steps: the reaction was cooled to room temperature, part of the acetonitrile was removed by swirling, water was added to remove potassium carbonate, extraction was performed 4 times with EA, the organic layer was collected, washed three times with water and saturated brine respectively, and distillation was performed under reduced pressure to give a pink solid, which was recrystallized from PE/EA system to give 4.7g of a white solid with a yield of about 80%.1H NMR(400MHz,DMSO)δ7.88(s,1H),7.40(d,J＝8.4Hz,1H),7.26–7.11(m,2H),4.64(s,2H),3.67–3.54(m,1H),1.81–1.61(m,4H),1.55(d,J＝12.5Hz,1H),1.40–1.07(m,5H).ESI-MS:346.1,348.1[M+H]+.
Synthesis of intermediate 2-chloro-N-cyclopropylacetamide (a2)
Figure BDA0001722173410000152
The preparation method of the intermediate is the same as the preparation operation method of the intermediate a1, and only the raw material is changed into cyclopropylamine. The organic layer was distilled under reduced pressure to give a pale yellow oil, which was recrystallized from PE/EA to give white crystals in 53.9% yield. ESI-MS 134.1[ M + H ]]+.
Synthesis of intermediate (5-bromo-2-chlorophenoxy) -N-cyclopropylacetamide (b2)
Figure BDA0001722173410000153
The reaction was run as intermediate b1 using a2 as starting material and PE/EA system recrystallized to a white solid in powder form with 88.2% yield.1H NMR(400MHz,DMSO)δ8.12(s,1H),7.40(d,J＝8.3Hz,1H),7.26–7.13(m,2H),4.61(s,2H),2.67(s,1H),0.79–0.55(m,2H),0.55–0.35(m,2H).ESI-MS:342.3,344.2[M+K]+.
Synthesis of intermediate N- (5-bromo-2-chloropyridine-3) -4-fluorobenzenesulfonamide (h)
Figure BDA0001722173410000154
Dissolving 5-bromo-2-chloro-3-aminopyridine (f, 530mg, 2.57mmol) as a raw material in 8mL of pyridine, slowly adding dropwise a pyridine solution of 4-fluorobenzenesulfonyl chloride (4g, 19.4mmol) in an amount of about 5mL under an ice bath condition, supplementing a small spoon of DMAP (dimethyl formamide) as a catalyst into the solution, and continuing the reaction at room temperature overnight after dropwise addition, wherein the solution is yellow brown. TCL monitors the progress of the reaction. The treatment method comprises the following steps: adding 200mL of water into the reaction solution to generate a large amount of precipitate, filtering, washing the filter cake layer with water until no pyridine smell exists, washing with ethanol once, drying to obtain milky solid powder g, 940mg, dissolving the intermediate g in 10mL of methanol, and adding about 3mL of potassium carbonate aqueous solution. The reaction was stirred at room temperature for 5-6h and TCL was monitored to completion. The treatment method comprises the following steps: adding water into the reaction solution, extracting with EA for 4 times, combining organic layers, washing with saturated salt solution for 2 times, drying with anhydrous sodium sulfate, concentrating under reduced pressure, performing column chromatography with EA/PE elution system to obtain light yellow solid, and recrystallizing with PE/EA system to obtain white powder. The yield was about 75%. ESI-MS 363.1[ M-H ]]-.
Synthesis of intermediate N- (5-bromo-2-methoxypyridin-3-yl) -4-fluorobenzenesulfonamide (j)
Figure BDA0001722173410000161
Firstly, raw material 5-bromo-2-chloro-3-aminopyridine (f, 500mg, 2.42mmol) is dissolved in 20mL dioxane, sodium methoxide (300mg, 5.55mmol) is added, the temperature is raised to 100 ℃ for reflux, the reaction lasts for about 10h, and the reaction progress is detected by TCL. The treatment method comprises the following steps: adding water to quench and react, extracting by EA, collecting an organic layer, concentrating, and passing an EA/PE elution system through a column to obtain the 5-bromo-2-methoxy-3-aminopyridine (i). Intermediate i (500mg, 2.45mmol) was dissolved in 10mL pyridine, 8mL pyridine solution of 4-fluorobenzenesulfonyl chloride (4g, 19.4mmol) was added slowly under ice bath conditions, and a small amount of DMAP was added. The reaction is continued for 5-6h at room temperature. The post-treatment method is the same as the intermediate h after the reaction is completed. After drying, 720mg of a white solid was obtained in about 81% yield. ESI-MS: 259.2[ M-H]-.
Intermediate 3- (4-fluorophenyl sulfonylamino) phenylboronic acid (k)
Figure BDA0001722173410000162
Dissolving 3-aminobenzeneboronic acid (1g, 7.3mmol) and 4-fluorobenzenesulfonyl chloride (2g, 9.7mmol) in 10mL of dichloromethane, adding 1.5mL of triethylamine, reacting at room temperature overnight, changing the solution from suspension to a yellow clear solution, and treating the reaction solution: distilling the reaction solution under reduced pressure to remove DCM, adding water, adding alkene HCl dropwise to adjust the pH to acidity, extracting with EA for 3 times, collecting an organic layer, drying, and concentrating. This gave a brown oil which was recrystallized from the PE/EA system to give about 1.88g of a yellow solid powder in about 92% yield. ESI-MS 294.1[ M-H ]]-.
Synthesis of intermediate N- (5-boronate-2-methoxypyridin-3-yl) -4-fluorobenzenesulfonamide (l)
Figure BDA0001722173410000163
Taking N- (5-bromo-2-methoxy-3-pyridyl) -4-fluorobenzenesulfonamide (j, 400mg, 1.12mmol), pinacol diboron (340mg, 1.32mmol), PdCl2(dppf) (60mg, 0.084mmol) and potassium acetate (328mg, 3.46mmol), mixing in 10mL1, 4-dioxane, vacuumizing,introducing nitrogen for protection, and placing in an oil bath kettle at 100 ℃ for reaction for 5 h. TCL monitors the progress of the reaction. And (3) treatment reaction: the reaction was cooled to room temperature, 30mL of water was added, extraction was performed with EA (4X 20mL), the organic layers were combined, dried over anhydrous sodium sulfate, distilled under reduced pressure, and column chromatography was performed using EA/PE (1: 5) elution system to give a crude product which was then recrystallized to give 360mg of white solid powder with a yield of 79.6%. ESI-MS 409.0[ M + H ]]+,431[M+Na]+.
2. Synthesis of benzoxazinones
Preparation of 6-bromo-2H-1, 4-benzoxazin-3 (4H) -one (C0)
Figure BDA0001722173410000171
Chloroacetyl chloride (4.7mL, 60mmol) was dissolved in 25mL of THF solution while on ice, and the solution was added dropwise to a solution of 2-amino-4-bromophenol (9g, 48mmol) in THF. Then NaHCO is added3(6g, 72mmol), the reaction was stirred for 1h on ice. When the TCL detection reaction is finished, potassium carbonate (10g, 72mmol) is added, the reaction solution is heated to 80 ℃ for reaction for 3h, and the reaction is continued overnight at room temperature. And (3) treatment reaction: diluting the reaction solution with water, extracting with EA for 4 times, collecting organic layer, and respectively adding diluted hydrochloric acid and saturated NaHCO3The resulting extract was washed once with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and recrystallized to give 6.9g of a brown solid with a yield of about 63%.1H NMR(400MHz,DMSO)δ10.82(s,1H),7.07(dd,J＝8.5,2.4Hz,1H),7.03(d,J＝2.3Hz,1H),6.91(d,J＝8.5Hz,1H),4.59(s,2H)ppm.HRMS(DART-TOF)calculated for C8H6BrNO2Na[M+Na]+m/z 249.9480,251.9459,found 249.9448,251.9419.
Preparation of 6-bromo-4- (3-methoxyphenyl) -2H-benzoxazin-3 (4H) -one (C1)
Figure BDA0001722173410000172
The C1 is prepared from 6-bromo-2H-1, 4-benzoxazine-3 (4H) -one (C0) and 3-methoxybenzeneBoric acid, other materials and procedures were the same as C2, with a yield of about 19.2%.1H NMR(400MHz,DMSO)δ7.51(t,J＝8.1Hz,1H),7.18(dd,J＝8.5,2.3Hz,1H),7.11(dd,J＝8.4,3.4Hz,1H),7.05(d,J＝8.5Hz,1H),6.99–6.96(m,1H),6.92(d,J＝8.7Hz,1H),6.36(d,J＝2.3Hz,1H),4.83(s,2H),3.79(s,3H)ppm.HRMS(DART-TOF)calculated for C15H12BrNO3Na[M+Na]+m/z 355.9898,357.9878,found 355.9861,357.9856.
Preparation of 6-bromo-4-phenyl-2H-1, 4-benzoxazin-3 (4H) -one (C2)
Figure BDA0001722173410000181
6-bromo-2H-1, 4-benzoxazin-3 (4H) -one (C0, 350mg, 1.54mmol) was used as a reaction raw material, raw material C0 was dissolved in 20mL of THF, phenylboronic acid (400mg, 3.28mmol) and copper acetate (420mg, 2.31mmol) were added under stirring at room temperature, triethylamine (0.5mL, 4.62mmol) was added dropwise, and a little of 4-mesh molecular sieve was added to the reaction solution. The reaction was transferred to a 60 ℃ oil bath for about 6h and the reaction was monitored for completion by TCL. Treating the reaction solution: distilling the reaction solution under reduced pressure to remove part of THF, adding water, filtering, extracting the filtrate with EA 4 times, collecting organic layer, drying, concentrating, dry-loading, performing column chromatography with PE/EA (6: 1) elution system, collecting the target compound, and recrystallizing to obtain white crystal 102mg with a yield of about 22%.1H NMR(400MHz,DMSO)δ7.63–7.58(m,1H),7.53(t,J＝7.4Hz,1H),7.37(d,J＝7.1Hz,1H),7.18(dd,J＝8.5,2.3Hz,1H),7.06(d,J＝8.5Hz,1H),6.31(d,J＝2.3Hz,1H),4.85(s,1H)ppm.HRMS(DART-TOF)calculated for C14H10BrNO2Na[M+Na]+m/z 325.9793,327.9772found 325.9771,327.9762.
Preparation of 6-bromo-4- (4-chlorophenyl) -2H-benzoxazin-3 (4H) -one (C3)
Figure BDA0001722173410000182
The raw materials for preparing C3 are 6-bromo-2H-1, 4-benzoxazine-3 (4H) -one (C0) and4-Chlorobenzeneboronic acid, the other starting materials and procedures were identical to C2, giving a yield of about 19.8%.1H NMR(400MHz,CDCl3)δ7.53(d,J＝8.7Hz,2H),7.22(d,J＝8.6Hz,2H),7.11(dd,J＝8.6,2.2Hz,1H),6.93(d,J＝8.6Hz,1H),6.55(s,1H),4.75(s,2H)ppm.HRMS(DART-TOF)calculated for C14H9BrClNO2Na[M+Na]+m/z 336.9505,338.9485found 336.9501,338.9481.
Preparation of 6-bromo-4- (4-methylphenyl) -2H-benzoxazin-3 (4H) -one (C4)
Figure BDA0001722173410000183
The C4 was prepared from 6-bromo-2H-1, 4-benzoxazin-3 (4H) -one (C0) and 4-methylphenylboronic acid in the same manner as C2, at a yield of about 21.6%.1H NMR(400MHz,CDCl3)δ7.35(d,J＝8.1Hz,2H),7.14(d,J＝8.2Hz,2H),7.08(dd,J＝8.5,2.2Hz,1H),6.91(d,J＝8.5Hz,1H),6.56(d,J＝2.2Hz,1H),4.75(s,2H),2.44(s,3H)ppm.HRMS(DART-TOF)calculated for C15H12BrNO2Na[M+Na]+m/z 339.9949,341.9929,found 339.9931,341.9906.
Preparation of 6-bromo-4- (4-methoxyphenyl) -2H-benzoxazin-3 (4H) -one (C5)
Figure BDA0001722173410000191
The C5 was prepared from 6-bromo-2H-1, 4-benzoxazin-3 (4H) -one (C0) and 4-methoxyphenylboronic acid in the same manner as C2, at a yield of about 21.6%. The yield was 25.2%.1H NMR(400MHz,DMSO)δ7.28(d,J＝8.9Hz,2H),7.16(dd,J＝8.5,2.3Hz,1H),7.12(d,J＝8.9Hz,2H),7.03(d,J＝2.3Hz,1H),6.35(d,J＝2.3Hz,1H),4.82(s,2H),3.84(s,3H)ppm.HRMS(DART-TOF)calculated for C15H12BrNO3Na[M+Na]+m/z 355.9898,357.9878,found 355.9876,357.9855.
Preparation of 6-bromo-4- (4-fluorophenyl) -2H-benzoxazin-3 (4H) -one (C6)
Figure BDA0001722173410000192
The C6 was prepared from 6-bromo-2H-1, 4-benzoxazin-3 (4H) -one (C0) and 4-tolylboronic acid in the same manner as C2, giving a yield of about 18.7%.1H NMR(400MHz,DMSO)δ7.47–7.40(m,4H),7.19(dd,J＝8.5,2.3Hz,1H),7.06(d,J＝8.5Hz,1H),6.34(d,J＝2.2Hz,1H),4.84(s,2H)ppm.HRMS(DART-TOF)calculated for C14H9BrFNO2Na[M+Na]+m/z 343.9698,345.9678,found 343.9612,345.9655.
Preparation of 6-bromo-4-cyclohexyl-2H-benzoxazin-3 (4H) -one (C7)
Figure BDA0001722173410000193
The intermediate (5-bromo-2-chlorophenoxy) -N-cyclohexylacetamide (b1) was used as a starting material, and b1(220mg, 0.64mmol) and cesium carbonate (500mg, 1.53mmol) were placed in a microwave reaction tube, and dissolved in 3mL of DMF. And (3) placing the reaction test tube in a microwave instrument for reaction, wherein the microwave condition is set to be 150 ℃, the power is 50W, and the time is 60 min. The reaction was monitored by TCL for completion after the reaction was complete. And (2) treating the reaction, adding water into the reaction, extracting with EA, washing with water for three times, drying with anhydrous sodium sulfate, concentrating under reduced pressure, loading by a dry method, performing column chromatography by using a PE/EA (10: 1) elution system, collecting a target compound, concentrating to obtain a yellow oily substance, and recrystallizing PE to obtain 80mg of light yellow crystals with the yield of about 50.6%.1H NMR(400MHz,CDCl3)δ7.25(d,J＝1.9Hz,1H),7.08(d,J＝10.5Hz,1H),6.86(d,J＝8.5Hz,1H),4.45(s,2H),4.12–4.01(m,1H),2.40–2.25(m,2H),1.98–1.67(m,5H),1.47–1.20(m,3H)ppm.HRMS(DART-TOF)calculated for C14H17BrNO2[M+H]+m/z 310.0443,312.0422,found 310.0436,312.0415.
Preparation of 6-bromo-4-cyclopropyl-2H-benzoxazin-3 (4H) -one (C8)
Figure BDA0001722173410000201
Intermediate 2- (5-bromo-2-chlorophenoxy) -N-cyclopropylacetamide (b2)
The other raw materials and operations were the same as those of C7. The yield was about 43.6%.1H NMR(400MHz,CDCl3)δ7.44(d,J＝2.2Hz,1H),7.10(dd,J＝8.5,2.2Hz,1H),6.84(d,J＝8.5Hz,1H),4.54(s,2H),2.76–2.65(m,1H),1.22–1.16(m,2H),0.82–0.75(m,2H)ppm.HRMS(DART-TOF)calculated for C11H11BrNO2[M+H]+m/z 267.9973,269.9953,found 267.9952,269.9932.
3. Synthesis of target Compound
Synthesis of target Compound C1-1
Figure BDA0001722173410000202
The starting material 6-bromo-4- (3-methoxyphenyl) -2H-benzoxazin-3 (4H) -one (C1, 60mg, 0.18mmol), intermediate l (80mg, 0.21mg), catalyst PdCl2(dppf) (10mg, 0.013mmol) and potassium acetate base (60mg, 0.6mmol) were added to the reaction tube and dissolved in 4mL of dioxane. Vacuumizing, filling nitrogen, moving into an oil bath kettle at 80 ℃ for reaction for 5 hours, and monitoring the reaction progress by TCL. Treating the reaction solution: cooling to room temperature, adding water, extracting with EA, mixing organic layers, washing with saturated NaCl solution twice, drying with anhydrous sodium sulfate, distilling under reduced pressure, dry-loading, separating and purifying by thin layer chromatography with PE/EA (4: 1) developer, collecting the target product, and recrystallizing to obtain white powder 40 mg. The yield was 41.7%.1H NMR(400MHz,DMSO)δ10.06(s,1H),δ8.29(d,J＝2.3Hz,1H),7.89(dd,J＝9.0,5.0Hz,2H),7.61(d,J＝2.3Hz,1H),7.55–7.48(m,3H),7.28(dd,J＝8.3,2.1Hz,1H),7.21(d,J＝8.3Hz,1H),7.11(dd,J＝8.1,2.7Hz,1H),7.01(t,J＝2.1Hz,1H),6.97(d,J＝7.8Hz,1H),6.53(d,J＝2.0Hz,1H),4.87(s,2H),3.79(s,3H),3.47(s,3H)ppm.HRMS(DART-TOF)calculated for C27H22FN3O6S[M+H]+m/z 535.1213,found 535.1211.
Synthesis of target Compound C1-2
Figure BDA0001722173410000211
The compound C1-2 is prepared from the compound C1, the intermediate k and other raw materials and operations as C1-1. The PE/EA system was recrystallized to give 56mg of a pale yellow solid. The yield was 61.5%.1H NMR(400MHz,DMSO)δ10.41(s,1H),7.72(dd,J＝8.9,5.2Hz,2H),7.53(t,J＝8.1Hz,1H),7.33(t,J＝8.8Hz,2H),7.21(t,J＝7.9Hz,1H),7.17–7.11(m,2H),7.06(s,1H),7.04–6.99(m,2H),6.96(d,J＝8.7Hz,2H),6.42(s,1H),4.84(s,2H),3.80(s,3H)ppm.HRMS(DART-TOF)calculated for C27H21FN2O5S[M+H]+m/z 504.1155,found 504.1149.
Synthesis of target Compound C1-3
Figure BDA0001722173410000212
The compound C1-3 is prepared from the compound C1, the intermediate h and the diboron pinacol ester, and other raw materials and operations are the same as those of the compound C7-3. Separating and purifying by thin layer chromatography, and collecting the target compound. Recrystallization afforded a milky white solid with a yield of 16.5%.1H NMR(400MHz,DMSO)δ10.56(s,1H),8.60(d,J＝2.3Hz,1H),7.96(dd,J＝9.0,5.0Hz,2H),7.69(d,J＝2.3Hz,1H),7.61–7.47(m,3H),7.35(dd,J＝8.3,2.1Hz,1H),7.26(d,J＝8.3Hz,1H),7.10(dd,J＝8.7,2.9Hz,1H),7.01(t,J＝2.1Hz,1H),6.96(d,J＝10.3Hz,1H),6.62(d,J＝2.1Hz,1H),4.90(s,2H),3.78(s,3H)ppm.HRMS(DART-TOF)calculated for C25H17ClF2N3O4S[M+H]+m/z 528.0596,found 528.0579.
Synthesis of target Compound C1-4
Figure BDA0001722173410000221
The compound C1-4 is prepared from compound C1 and 3-quinolineboronAnd (4) acid. The other raw materials and operations were the same as those of C1-1. A white solid was obtained. The yield was about 39.6%.1H NMR(400MHz,DMSO)δ8.91(d,J＝2.3Hz,1H),8.33(d,J＝2.3Hz,1H),7.99(d,J＝9.4Hz,2H),7.74(t,J＝8.3Hz,1H),7.61(t,J＝8.0Hz,1H),7.56–7.47(m,2H),7.28(d,J＝8.3Hz,1H),7.10(dd,J＝8.1,2.8Hz,1H),7.05–6.97(m,2H),6.70(d,J＝2.1Hz,1H),4.90(s,2H),3.79(s,3H)ppm.HRMS(DART-TOF)calculated for C24H18N2O3[M+H]+m/z 382.1317,found 382.1309.
Synthesis of target Compound C1-5
Figure BDA0001722173410000222
The compound C1-5 is prepared from compound C1 and 4-hydroxyphenylboronic acid. The other raw materials and operations were the same as those of C1-1. A white solid was obtained. The yield was about 15.8%.1H NMR(400MHz,DMSO)δ9.69(s,1H),7.49(t,J＝8.1Hz,1H),7.20–7.07(m,5H),7.00–6.92(m,2H),6.75(d,J＝8.6Hz,2H),6.43(d,J＝2.0Hz,1H),4.82(s,2H),3.78(s,3H)ppm.HRMS(DART-TOF)calculated for C20H15FNO3[M+H]+m/z 336.1036,found 336.1029.
Synthesis of target Compound C2-1
Figure BDA0001722173410000223
Weighing 6-bromo-4-phenyl-2H-1, 4-benzoxazine-3 (4H) -one (C2, 50mg, 0.17mmol), intermediate l (80mg,0.20mmol) and catalyst PdCl2(dppf) (9mg,0.012mmol), potassium acetate base (50mg,0.49mmol) were added to 3mL of 1, 4-dioxane, heated to 80 ℃ for reaction for 5h, and the reaction was detected to be complete by TCL. The treatment method comprises the following steps: cooling the reaction solution, adding water, extracting with EA for 4 times, collecting organic layer, washing with saturated NaCl solution twice, drying with anhydrous sodium sulfate, concentrating under reduced pressure, separating and purifying by thin layer chromatography, using PE/EA (4: 1) system as developing agent, collecting target product band, eluting with EA, distilling under reduced pressure, recrystallizing PE/EA to obtain yellow powder 15mg, and collecting the productThe ratio was 18.1%.1H NMR(400MHz,DMSO)δ10.02(s,1H),7.93(s,1H),7.70(dd,J＝7.9,4.1Hz,2H),7.63(t,J＝7.5Hz,2H),7.58–7.52(m,1H),7.45(d,J＝2.3Hz,1H),7.42(d,J＝7.1Hz,2H),7.37(t,J＝8.9Hz,2H),7.23(dd,J＝8.3,2.0Hz,1H),7.18(d,J＝8.3Hz,1H),6.36(d,J＝2.0Hz,1H),4.87(s,2H),3.64(s,3H)ppm.HRMS(DART-TOF)calculated for C26H20FN3O5S[M+H]+m/z 505.1108,found 505.1101.
Synthesis of target Compound C2-2
Figure BDA0001722173410000231
The compound C2-2 is prepared from C2, intermediate k and other raw materials and is operated similarly to the compound C2-1. The yield was 61.5%.1H NMR(400MHz,DMSO)δ10.40(s,1H),7.71(dd,J＝8.9,5.1Hz,2H),7.63(t,J＝7.4Hz,2H),7.56(t,J＝6.7Hz,1H),7.41(d,J＝7.0Hz,2H),7.35(t,J＝8.8Hz,2H),7.22(t,J＝7.9Hz,1H),7.16(s,2H),7.07–7.00(m,2H),6.98(d,J＝8.1Hz,1H),6.37(s,1H),4.86(s,2H)ppm.HRMS(DART-TOF)calculated for C26H19FN2O4S[M+H]+m/z 474.1050,found 474.1025.
Synthesis of target Compound C2-3
Figure BDA0001722173410000232
The starting materials used for the synthesis of compound C2-3 were C2(60mg, 0.2mmol), intermediate h (87mg, 0.24mmol) and pinacol diboron (76, 0.3mmol) in "one pot", other starting materials and procedures were the same as C1-3, giving a yield of 11.8%.1H NMR(400MHz,DMSO)δ10.41(s,1H),8.19(s,1H),8.03(dd,J＝8.9,4.9Hz,1H),7.96(d,J＝9.2Hz,1H),7.87(d,J＝2.2Hz,1H),7.70(dd,J＝8.8,5.2Hz,2H),7.65–7.60(m,2H),7.55(s,2H),7.44–7.38(m,2H),7.22(d,J＝8.3Hz,1H),6.43(s,1H),4.86(s,2H).HRMS(DART-TOF)calculated for C25H17ClFN3O4S[M+H]+m/z 509.0612,found 509.0611.
Synthesis of target Compound C2-4
Figure BDA0001722173410000241
Compound C2-4 was prepared starting from C2(45mg, 0.15mmol) and 3-quinolineboronic acid (31mg, 0.18mmol), with the other starting materials and procedures as for compound C2-1 in 36.6% yield.1H NMR(400MHz,DMSO)δ8.89(d,J＝2.4Hz,1H),8.32(d,J＝2.2Hz,1H),7.98(d,J＝8.3Hz,2H),7.77–7.70(m,1H),7.64–7.57(m,3H),7.56–7.47(m,2H),7.44(d,J＝7.0Hz,2H),7.29(d,J＝8.3Hz,1H),6.65(d,J＝2.1Hz,1H),4.92(s,2H)ppm.HRMS(DART-TOF)calculated for C23H16N2O2[M+H]+m/z 352.1212,found 352.1210.
Synthesis of target Compound C2-5
Figure BDA0001722173410000242
Compound C2-5 was prepared starting from C2(45mg, 0.15mmol) and 4-hydroxyphenylboronic acid (30mg, 0.22mmol), with the other starting materials and procedures as for compound C2-1 in 21.5% yield.1H NMR(400MHz,DMSO)δ9.56(s,1H),7.59(t,J＝7.9Hz,2H),7.52(t,J＝7.4Hz,1H),7.39(d,J＝7.0Hz,2H),7.18(dd,J＝8.3,2.1Hz,1H),7.12(d,J＝8.5Hz,3H),6.74(d,J＝8.6Hz,2H),6.38(d,J＝2.0Hz,1H),4.83(s,2H)ppm.HRMS(DART-TOF)calculated for C20H15NO3[M+H]+m/z 317.1052,found 317.1041.
Synthesis of target Compound C3-1
Figure BDA0001722173410000243
6-bromo-4- (4-chlorophenyl) -2H-benzoxazine-3 (4H) -one (C3) and intermediate l were used to synthesize C3-1, and the other starting materials were recrystallized from C2-1 to give a pale yellow powder with a yield of 24.2%.1H NMR(400MHz,DMSO)δ10.04(s,1H),7.96(d,J＝2.3Hz,1H),7.75–7.66(m,4H),7.51(d,J＝2.3Hz,1H),7.47(d,J＝8.7Hz,2H),7.38(t,J＝8.9Hz,2H),7.24(dd,J＝8.3,2.0Hz,1H),7.19(d,J＝8.3Hz,1H),6.40(d,J＝2.0Hz,1H),4.86(s,2H),3.65(s,3H)ppm.HRMS(DART-TOF)calculated for C26H19ClFN3O5S[M+H]+m/z 539.0718,found 539.0713.
Synthesis of target Compound C3-2
Figure BDA0001722173410000251
The compound C3-2 is prepared from C3 and an intermediate k, and other raw materials and operations are the same as those of C2-1. A white powder was obtained with a yield of about 34.7%.1H NMR(400MHz,DMSO)δ10.42(s,1H),7.76–7.65(m,4H),7.47(d,J＝8.7Hz,2H),7.36(t,J＝8.8Hz,2H),7.24(t,J＝7.9Hz,1H),7.17(d,J＝1.1Hz,2H),7.10–7.03(m,2H),7.00(d,J＝10.0Hz,1H),6.38(s,1H),4.86(s,2H)ppm.HRMS(DART-TOF)calculated for C26H18ClFN2O4S[M+H]+m/z 508.0660,found 508.0645.
Synthesis of target Compound C3-3
Figure BDA0001722173410000252
The compound C3-3 is prepared from C3 as raw material, an intermediate h and pinacol diboron by a one-pot method, and other raw materials and operation methods are the same as those of C1-3, and the yield is 15.6%.1H NMR(400MHz,DMSO)δ10.49(s,1H),8.30(d,J＝2.3Hz,1H),7.72(dd,J＝8.9,5.1Hz,1H),7.68(d,J＝8.7Hz,2H),7.64(d,J＝2.3Hz,1H),7.58(t,J＝8.8Hz,1H),7.47(d,J＝8.7Hz,2H),7.41(dd,J＝10.9,6.8Hz,2H),7.33(dd,J＝8.3,2.1Hz,1H),7.24(d,J＝8.3Hz,1H),6.50(d,J＝2.1Hz,1H),4.89(s,2H)ppm.HRMS(DART-TOF)calculated for C25H16Cl2FN3O4S[M+H]+m/z 543.0223,found 543.0221.
Synthesis of target Compound C3-4
Figure BDA0001722173410000253
Compound C3-4 was prepared starting from C3(45mg, 0.13mmol) and 3-quinolineboronic acid (30mg, 0.16mmol), with the other starting materials and procedures analogous to those for compound C2-1 to give 20mg of a white solid in 39.2% yield.1H NMR(400MHz,DMSO)δ8.95(d,J＝2.4Hz,1H),8.36(d,J＝2.2Hz,1H),8.00(d,J＝8.6Hz,2H),7.74(t,J＝7.6Hz,1H),7.66(d,J＝8.7Hz,2H),7.61(t,J＝7.0Hz,1H),7.55–7.44(m,3H),7.29(d,J＝8.3Hz,1H),6.73(d,J＝2.1Hz,1H),4.91(s,2H)ppm.HRMS(DART-TOF)calculated for C23H15ClN2O2[M+H]+m/z 386.0822,found 386.0816.
Synthesis of target Compound C3-5
Figure BDA0001722173410000261
Compound C3-5 was prepared starting from C3(45mg, 0.13mmol) and 4-hydroxyphenylboronic acid (30mg, 0.22mmol), with the other starting materials and procedures as for compound C2-1 in 19.4% yield.1H NMR(400MHz,DMSO)δ9.65(s,1H),7.65(d,J＝8.7Hz,2H),7.45(d,J＝8.7Hz,2H),7.17(ddd,J＝22.2,11.0,5.2Hz,4H),6.76(d,J＝8.6Hz,2H),6.41(d,J＝2.0Hz,1H),4.83(s,2H)ppm.HRMS(DART-TOF)calculated for C20H14ClNO3[M+H]+m/z 351.0662,found 351.0639.
Synthesis of target Compound C4-1
Figure BDA0001722173410000262
6-bromo-4- (4-methylphenyl) -2H-benzoxazine-3 (4H) -one (C4) and intermediate l were used to synthesize C4-1, and the other starting materials were recrystallized from C2-1, PE/EA system to give a white powder with a yield of 39.2%.1H NMR(400MHz,DMSO)δ10.03(s,1H),7.91(d,J＝2.3Hz,1H),7.71(dd,J＝8.9,5.2Hz,2H),7.47(d,J＝2.3Hz,1H),7.42(d,J＝8.1Hz,2H),7.37(t,J＝8.8Hz,2H),7.28(d,J＝8.2Hz,2H),7.21(dd,J＝8.3,2.0Hz,1H),7.17(d,J＝8.3Hz,1H),6.38(d,J＝1.9Hz,1H),4.85(s,2H),3.65(s,3H),2.40(s,3H)ppm.HRMS(DART-TOF)calculated for C27H22FN3O5S[M+H]+m/z 519.1264,found 519.1243.
Synthesis of target Compound C4-2
Figure BDA0001722173410000271
The compound C4-2 is prepared from C4 and an intermediate k, and other raw materials and operations are the same as those of C2-1. A milky white powder was obtained with a yield of about 30.3%.1H NMR(400MHz,DMSO)δ10.41(s,1H),7.72(dd,J＝8.9,5.1Hz,2H),7.42(d,J＝8.0Hz,2H),7.34(t,J＝8.8Hz,2H),7.27(d,J＝8.2Hz,2H),7.22(t,J＝7.9Hz,1H),7.15(d,J＝2.7Hz,2H),7.06(s,1H),7.01(d,J＝7.8Hz,1H),6.97(d,J＝8.1Hz,1H),6.40(s,1H),4.84(s,2H),2.40(s,3H)ppm.HRMS(DART-TOF)calculated for C27H21FN2O4S[M+H]+m/z 488.1206,found 488.1206.
Synthesis of target Compound C4-3
Figure BDA0001722173410000272
The compound C4-3 is prepared from C4 as raw material, an intermediate h and pinacol diboron by a one-pot method, and other raw materials and operation methods are the same as those of C1-3, and the yield is 12.2%.1H NMR(400MHz,DMSO)δ10.49(s,1H),8.16(s,1H),8.03(dd,J＝10.5,3.4Hz,1H),7.70(dd,J＝8.9,5.2Hz,2H),7.61–7.52(m,2H),7.41(t,J＝7.6Hz,3H),7.36(d,J＝8.8Hz,2H),7.28(d,J＝8.3Hz,3H),7.21(d,J＝8.3Hz,1H),6.44(d,J＝2.0Hz,1H),4.87(s,2H),2.40(s,3H)ppm.HRMS(DART-TOF)calculated for C26H19ClFN3O4S[M+H]+m/z 523.0769,found 523.0761.
Synthesis of target Compound C4-4
Figure BDA0001722173410000273
Compound C4-4 was prepared starting from C4(50mg, 0.16mmol) and 3-quinolineboronic acid (41mg, 0.24mmol), with the other starting materials and procedures analogous to those for compound C2-1 to give 16mg of a white solid in 28.1% yield.1H NMR(400MHz,DMSO)δ8.89(d,J＝2.4Hz,1H),8.33(d,J＝2.2Hz,1H),7.99(d,J＝8.5Hz,2H),7.77–7.71(m,1H),7.66–7.53(m,1H),7.49(dd,J＝8.3,2.1Hz,1H),7.40(d,J＝8.1Hz,2H),7.29(dd,J＝12.6,8.3Hz,3H),6.67(d,J＝2.1Hz,1H),4.90(s,2H),2.40(s,3H)ppm.HRMS(DART-TOF)calculated for C24H18N2O2[M+H]+m/z 351.0662,found 351.0649.
Synthesis of target Compound C4-5
Figure BDA0001722173410000281
Compound C4-5 was prepared starting from C4(50mg, 0.16mmol) and 4-hydroxyphenylboronic acid (35mg, 0.25mmol), with the other starting materials and procedures as for compound C2-1 in 27% yield.1H NMR(400MHz,DMSO)δ9.72(s,1H),7.37(t,J＝8.9Hz,2H),7.25(d,J＝8.2Hz,2H),7.19–7.08(m,3H),6.77(dd,J＝16.6,8.6Hz,3H),6.41(d,J＝2.0Hz,1H),4.81(s,2H),2.40(s,3H)ppm.HRMS(DART-TOF)calculated for C21H17NO3[M+H]+m/z 331.1208,found 331.1202.
Synthesis of target Compound C5-1
Figure BDA0001722173410000282
6-bromo-4- (4-methoxyphenyl) -2H-benzoxazine-3 (4H) -one (C5) and intermediate l were used to synthesize C5-1, and the other raw materials were recrystallized from C2-1, PE/EA system to give a pale yellow powder with a yield of 39.5%.1H NMR(400MHz,DMSO)δ10.04(s,1H),7.92(d,J＝2.3Hz,1H),7.71(dd,J＝8.9,5.2Hz,2H),7.48(d,J＝2.3Hz,1H),7.38(d,J＝8.8Hz,2H),7.35–7.30(m,2H),7.21(dd,J＝8.3,2.0Hz,1H),7.16(dd,J＝8.6,1.9Hz,3H),6.40(s,1H),4.85(s,2H),3.83(s,3H),3.65(s,3H)ppm.HRMS(DART-TOF)calculated for C27H22FN3O6S[M+H]+m/z 535.1213,found 535.1201.
Synthesis of target Compound C5-2
Figure BDA0001722173410000283
The compound C5-2 is prepared from C5 and an intermediate k, and other raw materials and operations are the same as those of C2-1. A white powder was obtained with a yield of about 33.8%.1H NMR(400MHz,DMSO)δ10.42(s,1H),7.72(dd,J＝8.9,5.1Hz,2H),7.39–7.28(m,4H),7.23(t,J＝7.9Hz,1H),7.15(d,J＝6.9Hz,4H),7.08(s,1H),7.03(d,J＝7.8Hz,1H),6.98(d,J＝8.1Hz,1H),6.41(s,1H),4.84(s,2H),3.83(s,3H)ppm.HRMS(DART-TOF)calculated for C27H21FN2O5S[M+H]+m/z 504.1155,found 504.1142.
Synthesis of target Compound C5-3
Figure BDA0001722173410000291
The compound C5-3 is prepared from C5 as raw material, an intermediate h and pinacol diboron by a one-pot method, and other raw materials and operation methods are the same as those of C1-3, and the yield is 16.7%.1H NMR(400MHz,DMSO)δ10.53(s,1H),8.44(s,1H),8.19(s,1H),7.88(d,J＝2.2Hz,1H),7.83(dd,J＝8.9,5.2Hz,2H),7.71(dd,J＝8.9,5.2Hz,1H),7.59(d,J＝2.3Hz,1H),7.40(t,J＝8.4Hz,2H),7.32(d,J＝8.9Hz,1H),7.20(d,J＝8.3Hz,1H),7.15(d,J＝8.9Hz,1H),6.47(s,1H),4.87(s,2H),3.83(s,3H)ppm.HRMS(DART-TOF)calculated for C26H19ClFN3O5S[M+H]+m/z 539.0718,found 539.0703.
Synthesis of target Compound C5-4
Figure BDA0001722173410000292
Compound C5-4 was prepared starting from C5 and 3-quinolineboronic acid, with the other starting materials and procedures analogous to those for compound C2-1, to give a white solid in 27.5% yield. 1H NMR (400MHz, DMSO) δ 8.91(d, J ═ 2.4Hz,1H),8.33(d, J ═ 2.2Hz,1H),8.00(d, J ═ 8.4Hz,2H), 7.77-7.70 (m,1H), 7.64-7.58 (m,1H),7.49(dd, J ═ 8.3,2.1Hz,1H),7.35(d, J ═ 8.9Hz,2H),7.27(d, J ═ 8.3Hz,1H),7.13(d, J ═ 9.0Hz,2H),6.70(d, J ═ 2.1Hz,1H),4.89(s,2H),3.83(s,3H) ppm24H18N2O3[M+H]+m/z 382.1317,found 382.1309.
Synthesis of target Compound C5-5
Figure BDA0001722173410000301
Compound C5-5 was prepared starting from C5 and 4-hydroxyphenylboronic acid, and the other starting materials and procedures were the same as for compound C2-1, giving a white solid with a yield of 18.4%.1H NMR(400MHz,DMSO)δ9.59(s,1H),7.30(d,J＝8.9Hz,2H),7.20–7.14(m,2H),7.11(dd,J＝9.0,4.8Hz,4H),6.75(d,J＝8.6Hz,2H),6.43(d,J＝2.0Hz,1H),4.81(s,2H),3.83(s,3H)ppm.HRMS(DART-TOF)calculated for C21H17NO4[M+H]+m/z 347.1158,found 347.1143.
Synthesis of target Compound C6-1
Figure BDA0001722173410000302
6-bromo-4- (4-fluorophenyl) -2H-benzoxazin-3 (4H) -one (C6, 50mg, 0.16mmol) and intermediate l (70mg, 0.18mmol) were used for the synthesis of C6-1, and the other starting materials were recrystallized from C2-1, PE/EA system to give 40mg of a white solid in 48.7% yield.1H NMR(400MHz,DMSO)δ10.03(s,1H),7.93(d,J＝2.3Hz,1H),7.75–7.65(m,4H),7.48(dd,J＝8.7,5.5Hz,3H),7.37(t,J＝8.8Hz,2H),7.23(dd,J＝8.3,2.0Hz,1H),7.18(d,J＝8.3Hz,1H),6.40(s,1H),4.86(s,2H),3.65(s,3H)ppm.HRMS(DART-TOF)calculated for C26H19F2N3O5S[M+H]+m/z 523.1013,found 523.1011.
Synthesis of target Compound C6-2
Figure BDA0001722173410000303
The compound C6-2 was prepared starting from C6(50mg, 0.16mmol) and intermediate k (68mg, 0.24mmol), using the same procedures as C2-1. 60mg of a milky white powder was obtained in a yield of about 65.2%. 1H NMR (400MHz, DMSO) δ 10.43(s,1H),7.73(dd, J ═ 8.9,5.2Hz,2H),7.69(d, J ═ 8.7Hz,2H),7.47(d, J ═ 8.7Hz,2H),7.35(t, J ═ 8.8Hz,2H),7.23(t, J ═ 7.9Hz,1H),7.17(d, J ═ 1.0Hz,2H), 7.10-7.03 (m,2H),6.99(d, J ═ 8.1Hz,1H),6.37(s,1H),4.85(s,2H), ppm26H18F2N2O4S[M+H]+m/z 492.0955,found 492.0949.
Synthesis of target Compound C6-3
Figure BDA0001722173410000311
Compound C6-3 was prepared starting from C6(50mg, 0.16mmol), intermediate h (70mg, 0.19mmol) and pinacol diboron (60mg,0.24mmol) using the "one pot" procedure, the other starting materials and procedures were the same as C1-3 to give 10mg of a white solid in 12.1% yield.1H NMR(400MHz,DMSO)δ10.49(s,1H),8.16(s,1H),8.03(dd,J＝9.0,5.0Hz,1H),7.85–7.79(m,1H),7.73–7.64(m,2H),7.61–7.54(m,2H),7.46(dd,J＝8.5,3.0Hz,2H),7.36(t,J＝8.8Hz,2H),7.31–7.26(m,1H),7.22(dd,J＝8.3,2.4Hz,1H),6.46(d,J＝12.1Hz,1H),4.88(s,2H)ppm.HRMS(DART-TOF)calculated for C25H16ClF2N3O4S[M+H]+m/z 527.0518,found 527.0516.
Synthesis of target Compound C6-4
Figure BDA0001722173410000312
Compound C6-4 was prepared starting from C6(50mg, 0.16mmol) and 3-quinolineboronic acid (41mg, 0.24mmol), and the other starting materials and procedures were the same as for compound C2-1, and was recrystallized to give 36mg of white crystals in 62% yield.1H NMR(400MHz,DMSO)δ8.95(d,J＝2.4Hz,1H),8.36(d,J＝2.2Hz,1H),8.00(d,J＝8.3Hz,2H),7.78–7.71(m,1H),7.66(d,J＝8.7Hz,2H),7.61(t,J＝7.5Hz,1H),7.55–7.47(m,3H),7.29(d,J＝8.3Hz,1H),6.73(d,J＝2.1Hz,1H),4.87(s,2H)ppm.HRMS(DART-TOF)calculated for C23H15FN2O2[M+H]+m/z 370.1118,found 370.1116.
Synthesis of target Compound C6-5
Figure BDA0001722173410000321
Compound C6-5 was prepared starting from C6(50mg, 0.16mmol) and 4-hydroxyphenylboronic acid (33mg, 0.24mmol), and the other starting materials and procedures were the same as for compound C2-1 to give 13mg of a white solid in 25% yield.1H NMR(400MHz,DMSO)δ9.74(s,1H),7.65(d,J＝6.6Hz,2H),7.45(d,J＝8.7Hz,2H),7.24–7.08(m,4H),6.76(d,J＝8.6Hz,2H),6.41(d,J＝2.0Hz,1H),4.83(s,2H)ppm.HRMS(DART-TOF)calculated for C20H14FNO3[M+H]+m/z 335.0958,found 335.0939.
Synthesis of target Compound C7-1
Figure BDA0001722173410000322
Weighing the raw materials of 6-bromo-4-cyclohexyl-2H-benzoxazine-3 (4H) -ketone (C7, 60mg, 0.19mmol), intermediate l (95mg, 0.23mmol) and catalyst PdCl2(dppf) (10.6mg, 0.015mmol) and potassium acetate base (60mg, 0.6mmol) were added to the reaction tube and dissolved in 4mL of dioxane. Vacuumizing, filling nitrogen gas, and moving inThe reaction was carried out in an oil bath at 80 ℃ for 5h, and the progress of the reaction was monitored by TCL. Treating the reaction solution: cooling to room temperature, adding water, extracting with EA, combining organic layers, washing twice with saturated NaCl solution, drying with anhydrous sodium sulfate, distilling under reduced pressure, loading by dry method, and performing column chromatography with PE/EA (10: 1) eluent. The objective compound was collected and recrystallized to obtain 42mg of white crystals, which was 42.4% in yield.1H NMR(400MHz,DMSO)δ10.07(s,1H),8.28(d,J＝2.3Hz,1H),7.86–7.77(m,3H),7.45–7.37(m,3H),7.21(dd,J＝8.3,1.9Hz,1H),7.12(d,J＝8.3Hz,1H),4.54(s,2H),4.26–4.15(m,1H),3.67(s,3H),2.39–2.27(m,2H),1.85–1.63(m,5H),1.50–1.20(m,3H)ppm.HRMS(DART-TOF)calculated for C26H26FN3O5S[M+H]+m/z 511.1577,found 511.1559.
Synthesis of target Compound C7-2
Figure BDA0001722173410000323
Compound C7-2 was prepared starting from C7(60mg, 0.19mmol) and intermediate k (68mg, 0.23 mmol). Other raw materials and operation methods are the same as those of the compound C7-1. Recrystallization afforded 50mg of a white solid. The yield was 54.6%.1H NMR(400MHz,DMSO)δ10.47(s,1H),7.85(dd,J＝8.9,5.1Hz,2H),7.41(t,J＝8.8Hz,2H),7.36–7.30(m,4H),7.15(dd,J＝8.3,1.8Hz,1H),7.12–7.08(m,2H),4.55(s,2H),4.25–4.15(m,1H),2.38–2.24(m,2H),1.89–1.61(m,5H),1.50–1.20(m,3H)ppm.HRMS(DART-TOF)calculated for C26H25FN2O4S[M+H]+m/z 480.1519,found 480.1503.
Synthesis of target Compound C7-3
Figure BDA0001722173410000331
C7(60mg, 0.19mmol), intermediate h (83mg, 0.23mmol), pinacol diboride (74mg, 0.29mmol) and PdCl catalyst2(dppf) (10.6mg, 0.015mmol) and potassium acetate base (60mg, 0.6mmol) were added to a reaction tube, anddissolve in 4mL dioxane. The method comprises the steps of vacuumizing by a one-pot method, filling nitrogen, moving into an oil bath kettle at 80 ℃ for reaction for 6 hours, and monitoring the progress of the reaction by TCL. Treating the reaction solution: cooling to room temperature, adding water, extracting with EA, combining organic layers, washing twice with saturated NaCl solution, drying with anhydrous sodium sulfate, distilling under reduced pressure, loading by dry method, and performing column chromatography with PE/EA (8: 1) eluent. The title compound was collected and recrystallized to give 12mg of a pale yellow solid. The yield was about 12.1%.1H NMR(400MHz,CDCl3)δ10.49(s,1H)，8.32(d,J＝2.3Hz,1H),8.15(d,J＝2.2Hz,1H),7.82(dd,J＝8.9,4.9Hz,1H),7.31(d,J＝1.8Hz,1H),7.20–7.10(m,4H),7.04(d,J＝6.4Hz,1H),4.55(s,2H),4.35–4.21(m,1H),2.47–2.31(m,2H),2.01–1.68(m,5H),1.47–1.34(m,3H)ppm.HRMS(DART-TOF)calculated for C25H23ClFN3O4S[M+H]+m/z 515.1082,found 515.1065.
Synthesis of target Compound C7-4
Figure BDA0001722173410000332
The compound C7-4 was prepared starting from compound C7 and 3-quinolineboronic acid. The other raw materials and operations were the same as those of C7-1. Recrystallization afforded a white solid. The yield was about 68.5%.1H NMR(400MHz,DMSO)δ9.25(d,J＝2.4Hz,1H),8.61(d,J＝2.2Hz,1H),8.07(t,J＝8.6Hz,2H),7.82–7.75(m,1H),7.69–7.63(m,2H),7.50(dd,J＝8.3,2.0Hz,1H),7.20(d,J＝8.3Hz,1H),4.58(s,2H),4.31–4.19(m,1H),2.47–2.35(m,2H),1.86–1.58(m,5H),1.53–1.18(m,3H)ppm.HRMS(DART-TOF)calculated for C23H22N2O2[M+H]+m/z 358.1681,found 358.1663.
Synthesis of target Compound C7-5
Figure BDA0001722173410000341
The compound C7-5 is prepared from compound C7 and 4-hydroxyphenylboronic acid. The other raw materials and operations were the same as those of C7-1. To obtain whiteA colored solid. The yield was about 32%.1H NMR(400MHz,DMSO)δ9.52(s,1H),7.45(d,J＝8.6Hz,2H),7.33(d,J＝1.8Hz,1H),7.18(dd,J＝8.3,1.9Hz,1H),7.05(d,J＝8.3Hz,1H),6.85(d,J＝8.6Hz,2H),4.51(s,2H),4.24–4.12(m,1H),2.41–2.28(m,2H),1.85–1.61(m,5H),1.48–1.16(m,3H)ppm.HRMS(DART-TOF)calculated for C20H21NO3[M+H]+m/z 323.1521,found 323.1509.
Synthesis of target Compound C8-1
Figure BDA0001722173410000342
The compound C8-1 is prepared from 6-bromo-4-cyclopropyl-2H-benzoxazine-3 (4H) -one (C8) and intermediate l, and the other raw materials and operations are the same as those of C7-1. A white solid was obtained. The yield was 26.1%.1H NMR(400MHz,DMSO)δ10.06(s,1H),8.25(d,J＝2.3Hz,1H),7.83(dd,J＝8.9,5.2Hz,2H),7.78(d,J＝2.3Hz,1H),7.50(d,J＝2.1Hz,1H),7.42(t,J＝8.9Hz,2H),7.22(dd,J＝8.3,2.1Hz,1H),7.07(d,J＝8.3Hz,1H),4.62(s,2H),3.68(s,3H),2.91–2.80(m,1H),1.15–1.09(m,2H),0.70–0.62(m,2H)ppm.HRMS(DART-TOF)calculated for C23H20FN3O5S[M+H]+m/z 469.1108,found 469.1105.
Synthesis of target Compound C8-2
Figure BDA0001722173410000343
The compound C8-2 is prepared from the compound C8 and an intermediate k, and other raw materials and operations are the same as those of the compound C7-1. A yellow oil was obtained. The yield was about 43.1%.1H NMR(400MHz,DMSO)δ10.46(s,1H),7.86(dd,J＝8.9,5.1Hz,2H),7.47–7.38(m,3H),7.35–7.30(m,3H),7.14(dd,J＝8.3,2.1Hz,1H),7.10–7.04(m,2H),4.62(s,2H),2.86–2.78(m,1H),1.15–1.09(m,2H),0.70–0.63(m,2H)ppm.HRMS(DART-TOF)calculated for C23H19FN2O4S[M+H]+m/z 438.1050,found 438.1039.
Synthesis of target Compound C8-3
Figure BDA0001722173410000351
The compound C8-3 is prepared from the compound C8, the intermediate h and the diboron pinacol ester, and other raw materials and operations are the same as those of the compound C7-3. Recrystallization afforded a milky white solid with a yield of 17.3%.1H NMR(400MHz,DMSO)δ10.51(s,1H),8.52(s,1H),7.88(d,J＝2.2Hz,1H),7.82(dd,J＝8.8,5.2Hz,2H),7.56(d,J＝1.8Hz,1H),7.43(t,J＝8.8Hz,2H),7.30(dd,J＝8.3,1.9Hz,1H),7.11(d,J＝8.3Hz,1H),4.65(s,2H),2.90–2.78(m,1H),1.15–1.07(m,2H),0.71–0.64(m,2H)ppm.HRMS(DART-TOF)calculated for C22H17ClFN3O4S[M+H]+m/z 473.0612,found 473.0601.
Synthesis of target Compound C8-4
Figure BDA0001722173410000352
The compound C8-4 was prepared starting from compound C8 and 3-quinolineboronic acid. Other raw materials and operations were the same as those of Compound C7-1. White crystals were obtained. The yield was 33.5%.1H NMR(400MHz,DMSO)δ9.25(d,J＝2.3Hz,1H),8.62(d,J＝2.2Hz,1H),8.07(dd,J＝7.6,5.6Hz,2H),7.84(d,J＝2.0Hz,1H),7.78(t,J＝8.2Hz,1H),7.66(t,J＝7.6Hz,1H),7.53(dd,J＝8.3,2.0Hz,1H),7.16(d,J＝8.3Hz,1H),4.67(s,2H),2.93–2.86(m,1H),1.26–1.18(m,2H),0.74–0.67(m,2H)ppm.HRMS(DART-TOF)calculated for C20H16N2O2[M+H]+m/z 316.1212,found 316.1206.
Synthesis of target Compound C8-5
Figure BDA0001722173410000353
The compound C8-5 is prepared from compound C8 and 4-hydroxyphenylboronic acid. The other raw materials and operations were the same as those of C7-1. To obtainTo a white solid. The yield was about 22.4%.1H NMR(400MHz,DMSO)δ9.52(s,1H),7.51(d,J＝2.1Hz,1H),7.45(d,J＝8.6Hz,2H),7.18(dd,J＝8.3,2.1Hz,1H),7.01(d,J＝8.3Hz,1H),6.85(d,J＝8.6Hz,2H),4.59(s,2H),2.86–2.80(m,1H),1.17–1.10(m,2H),0.69–0.62(m,2H)ppm.HRMS(DART-TOF)calculated for C17H15NO3[M+H]+m/z 281.1052,found 281.1041.
EXAMPLE 2 inhibitory Activity of the object Compound on tumor cells
The target compounds used in the in vitro experiment are all prepared by DMSO to be stored at the concentration of 20mg/mL, the compounds are all stored in a refrigerator at 4 ℃ in a dark place, and the target compounds are diluted by complete culture media according to the required concentration during the experiment. The tumor cell strain comprises: human colon cancer cell line (HCT-116), human lung adenocarcinoma cell line (A549), human breast cancer cell line (MCF-7), human liver tumor cell line (HepG2), human malignant melanoma cell line (A375), human cervical cancer cell line (Hela) and the like, which are purchased from American ATCC company. The specific test method comprises the following steps:
plate paving: tumor cells in logarithmic growth phase were first collected and the concentration of the cell suspension was adjusted with the culture medium. Then 2 to 4 x 104Cell density per well was seeded in 96-well plates, 100. mu.L of cell suspension per well was placed at 37 ℃ with 5% CO2The incubator is incubated for 24 h.
Adding medicine: using a two-fold dilution method, 20mg/mL of the target compound was diluted to different concentrations with complete medium, where the concentration of the primary screen was set at 40,20, 10. mu.M and the concentration of the fine screen was set at 20, 10, 5, 2.5, 1.25, 0.625. mu.M. Solutions of the target compounds at different concentrations were added to 96-well plates in an amount of 100. mu.L/well. In addition, a 2mg/mL BEZ235 positive drug was set as a control group and a culture medium only blank control group. To reduce errors, each concentration was triplicated. The culture was continued for 48 h.
And (3) treatment: and (3) taking out the 96-well plate, adding a 5mg/mL solution prepared from MTT into the 96-well plate in an amount of 20 muL/well, continuing to incubate for 2-4 h, discarding the supernatant, adding 150 muL DMSO into each well, and fully shaking for 10-15 min.
And (3) detection: after each well is vibrated and stained uniformly, the absorbance value of each well is measured by an enzyme-labeling instrument under the wavelength of 570nm, and the average value is taken. According to the formula: the relative cell proliferation inhibition rate (%) was defined as (blank control group-experimental group)/blank control group × 100%, and the relative tumor cell proliferation inhibition rate for each target compound was calculated and IC was used50Calculation software to determine the median Inhibitory Concentration (IC)50μ mol/L). The test results are shown in Table 1.
TABLE 1 (Unit. mu. mol/L)
Figure BDA0001722173410000361
Figure BDA0001722173410000371
From the above IC50The data show that most target compounds have certain proliferation inhibition activity on different tumor cell lines. Among them, C2-1, C3-1, C5-1 and C8-1 have better activities.
EXAMPLE 3 study of the kinase Activity of the target Compounds on PI3K alpha
The above molecules were subjected to purity measurement, and then weighed and sent to Shanghai Ruizi chemical company for measurement of the kinase activity of PI3K alpha. The IC50 of these molecules to PI3K α was determined. The results are shown in Table 2.
TABLE 2
Figure BDA0001722173410000372
Figure BDA0001722173410000381
According to the enzyme activity test result, a batch of molecules capable of effectively targeting PI3K alpha is obtained, wherein the IC50 of the compound C5-1 to PI3K alpha kinase is obviously better than the IC50(9.57nM) of PI-103 serving as a positive control drug in the experiment and is also better than the control molecule BEZ235(80.5 nM).
IC50 calculations for C5-1 and PI-103 versus PI3K α are shown in Table 3. The inhibitory activity of C5-1, PI-103 and BEZ235 on PI3K/mTOR targets is shown in Table 4. The values in Table 4 are all IC50 in nM.
TABLE 3
Figure BDA0001722173410000382
Figure BDA0001722173410000391
The IC50 calculated for C5-1 was 0.627nM for PI3K α and the IC50 calculated for PI3K α was 9.57 nM.
Table 4(IC50: nM)
Target spot PI3Kα PI3Kβ PI3Kγ PI3Kδ mTOR
C5-1 0.63 94.54 22 9.2 13.85
PI-103 9.6 11.98 64.03 11 5.3
BEZ235 80.5 703.9 104.2 85.3 1.43
It can be seen that C5-1 not only targets PI3K α, but also has better inhibitory activity against PI3K β, PI3K γ, PI3K δ, and mTOR.
EXAMPLE 4 comparison of the kinase Activity of the target Compound with the control Compound on PI3K alpha
A part of the compounds was taken and a reported comparative compound (US2010/0311736a1) was synthesized at the same time, and the inhibition ratio of PI3K α was measured at a concentration of 100nM, and the results thereof are shown in table 5. The nuclear magnetic characterization of the comparative compounds is as follows:
C3-6-1：1H NMR(400MHz,CDCl3)δ＝8.13(d,J＝2.3,1H),8.05(d,J＝2.3,1H),7.72(dd,J＝8.4,1.1,2H),7.63–7.56(m,1H),7.44(dd,J＝10.8,4.9,2H),7.37–7.27(m,4H),7.16(dd,J＝8.3,2.0,1H),7.10(d,J＝8.3,1H),7.04(d,J＝2.0,1H),5.21(s,2H),4.79(s,2H).
C3-6：1H NMR(400MHz,CDCl3)δ＝8.05(d,J＝2.3,1H),7.96(d,J＝2.2,1H),7.75–7.68(m,2H),7.61–7.54(m,3H),7.48–7.41(m,2H),7.34–7.28(m,2H),7.17(d,J＝1.1,2H),6.53(t,J＝1.1,1H),4.83(s,2H).
C2-1-1：1H NMR(400MHz,CDCl3)δ＝8.20(d,J＝2.2,1H),7.99–7.91(m,4H),7.40(d,J＝2.2,1H),7.35–7.29(m,4H),7.25–7.17(m,5H),7.08–6.99(m,3H),5.21(s,2H),4.78(s,2H),3.61(s,3H).
C2-6-1：1H NMR(400MHz,CDCl3)δ＝8.05(dd,J＝17.3,2.3,1H),7.97(dd,J＝8.5,1.1,1H),7.75–7.67(m,2H),7.60–7.53(m,2H),7.43(t,J＝7.8,1H),7.35(t,J＝4.7,2H),7.29(d,J＝5.0,1H),7.12–7.05(m,2H),5.22(d,J＝14.0,2H),4.80(s,2H).
C2-6：1H NMR(400MHz,CDCl3)δ＝8.04(s,1H),7.96–7.90(m,1H),7.68(dd,J＝11.2,3.8,2H),7.60(t,J＝7.6,2H),7.53(dd,J＝14.8,7.2,2H),7.41(t,J＝7.6,2H),7.35(dd,J＝10.5,3.3,2H),7.16(s,2H),6.56(d,J＝5.8,1H),4.85(d,J＝5.6,2H).
TABLE 5
Figure BDA0001722173410000401
Figure BDA0001722173410000411
The structural comparison fully shows that the structural introduction of the benzylidene group is completely less advantageous than the direct coupling with the benzene ring in activity.
EXAMPLE 5 inhibitory Activity of Compound C5-1 on DNA-PK
DNA-dependent protein kinase (DNA-PK) is a key protein kinase in the process of repairing genome DNA damage, participates in and determines the whole process of connecting non-homologous ends with a DNA damage repairing pathway.
The inhibition rate of the C5-1 molecule pair on the DNA-PK is measured at the concentration of 100nM, and the result shows that the inhibition rate of the C5-1 molecule pair reaches 75 percent at 100nM, and the inhibitor is a powerful DNA-PK inhibitor.
EXAMPLE 6 plate cloning of Compound C5-1
Plate cloning is also called plate cloning formation experiment, and the method is suitable for the experiment of adherent cells. The proliferation capacity of the tumor cells is mainly examined and reflected by the number of clones formed by the survival of cells attached to the wall after the tumor cells are inoculated, namely the cell clone formation rate (cell inoculation survival rate). A clonal cell is a set of cells that is formed from a single cell, after culture for more than 6 generations of continuous growth, and from its progeny. The set contains more than 50 cells with the size of 0.3-1.0. After tumor cells are treated with drugs, their proliferative capacity is affected by the drugs, resulting in changes in the number and morphology of the clones. After the nucleic acid in the cloned cell is stained into blue-violet by a staining agent (usually crystal violet), the condition of the tumor cell clone formation can be visually judged from the color, and the effect of the drug for inhibiting the cell proliferation is reflected.
Plate paving: human cervical cancer cells Hela in the logarithmic growth phase were taken, digested with pancreatin, and suspended in DMEM complete medium containing 10% fetal bovine serum for use. The cells were seeded at a density of 100 cells/well in 24-well plates, and the plates were incubated at 37 ℃ in an incubator with 5% CO2 for 12 h.
Adding medicine: the well plate was removed, and after observation of cell adherence, the supernatant medium was removed and the experimental and control groups were set. The medicine is diluted with complete culture medium to six concentrations of 20, 10, 5, 2.5, 1.25, 0.625 and 0.375 mu M of the target compound C5-1, and 1.5mL of the medicine liquid is added into each hole. The control group was added with an equal volume of medium without drug solution. The well plate was placed in an incubator at 37 ℃ with 5% CO2 for 2-3 weeks.
And (3) treatment: and stopping cell culture when macroscopic cell colonies are formed in the observation hole plate, removing supernate, repeatedly washing twice by using PBS (phosphate buffer solution), fixing for 15min by using 4% paraformaldehyde fixing solution, removing the fixing solution, finally dyeing for 20-30 min by using 0.5% crystal violet staining solution, removing the staining solution, and washing away redundant staining by using the PBS buffer solution. Drying at room temperature, taking a picture with a camera and performing statistical analysis on the number of colonies formed, as detailed in FIGS. 1 and 2.
As seen from the figure, the experimental group of Hela cells showed a significantly decreased colony formation rate and a significantly concentration-dependent behavior, as compared with the control group. Its colony formation is inversely proportional to the drug concentration, i.e., the higher the drug concentration, the lower its colony formation rate. In the drug concentration well plates above 5 μ M, tumor cells formed few colonies and no significant cell colony formation, whereas at 1.25 μ M concentration, more than half of the colonies formed had been inhibited compared to the cells of the negative control group.
EXAMPLE 7 pharmacokinetic Studies of Compound C5-1 molecule
Experimental animals: healthy adult male SD rats, 6, with a body weight of 180-250 g. The administration route is as follows: tail vein (i.v.); gavage (p.o.) dosing: 1mg/kg (i.v.), 10mg/kg (p.o.), the administration volume was 5 ml/kg. The administration dosage form is intravenous administration: DMA/Solutol HS 15/physiological saline (5:10:85, v/v/v); oral administration: 0.5% CMC-Na.
The preparation of the medicine comprises the following steps: i.v. administration group: precisely weighing appropriate amount of medicine (equivalent to 1.0mg of original medicine), adding DMA 0.25ml and Solutol HS150.5 ml, ultrasonically dissolving, slowly adding normal saline to final volume of 5ml, ultrasonically and vortexing, and mixing.
p.o. dosing group: accurately weighing appropriate amount of medicine (equivalent to 10.0mg of original medicine) and 0.5% CMC-Na 5ml, and mixing by ultrasonic and vortex.
The experimental scheme is as follows: healthy adult SD rats 6 in two groups (3 each per group i.v. and p.o.); after fasting overnight (free drinking), the tail vein and the gavage were administered with a volume of 5 ml/kg; the intravenous injection administration group is used for collecting 0.2ml blood from retroorbital venous plexus 5min,15min,0.5,1,2,4,8,12 and 24h before and after administration, centrifuging at 4 deg.C for 5min to separate blood plasma, and storing at-20 deg.C for testing. The group for intragastric administration is treated by collecting 0.2ml of blood from retroorbital venous plexus 0.5,1,2,4,6,8,12 and 24 hours before and after administration by the same method as the group for intravenous injection.
And (3) sample analysis: the drug concentration in plasma was measured by LC/MS/MS method, and the results are shown in Table 6.
TABLE 6
Figure BDA0001722173410000421
EXAMPLE 8 in vivo anti-tumor Activity study of Compound C5-1 molecule
In order to research the anti-tumor activity of C5-1 in an animal body, a xenograft tumor model is constructed by selecting cell strains Hela and A549, and an experimental animal is a Balb/C nude mouse.
Laboratory animal
The mice used in the subject were purchased from Beijing Huafukang Biotechnology GmbH and bred in the important laboratory animal House (SPF grade) of the biotherapy country of Sichuan university. Mice were of SPF grade and were bred according to the national institutes of Health Guide for the Care and Use of Laboratory Animals standards. The use of the experimental animals was approved by the ethical Committee of Sichuan University (Institutional Ethics Committee of Sichuan University). Experimental animal feeding conditions: the temperature is 16-26 ℃, the relative humidity is 40-70%, an artificial lighting system is adopted, the light and shade are alternated for 12h, and the feed (Beijing Ke Australian cooperative feed Co., Ltd.) is sterilized by Co60 and is freely drunk.
The specific method comprises the following steps:
1) after the Hela and A549 tumor cells in logarithmic growth phase are digested by pancreatin, the cells are collected by centrifugation; cells were washed 3 times with double-null (no antibiotics and serum) medium; adding a proper amount of double-culture-free basis suspension tumor cells, and carrying out density measurement on the tumor cells; the cell density is adjusted to 1 × 10 according to the experimental requirements7one/mL.
2) The resuspended cells were 1X 10 per mouse6Individual cells (0.1mL) the cells were inoculated subcutaneously into the right axilla of mice (6-8 weeks old, 18-22g in weight).
3) About 10 days after inoculation, the tumor at the inoculated part of the mouse forms tumor, and the volume is 150-200 mm3At time, all mice with similar tumor volumes were randomly grouped. The Hela model experiment sets 5 groups (control group, masculine medicine group BEZ 23520 mg/kg, C5-110 mg/kg, C5-120 mg/kg, C5-140 mg/kg and C5-150 mg/kg), and the A549 model experiment sets 4 groups (control group, masculine medicine group BEZ 23520 mg/kg, C5-120 mg/kg and C5-140 mg/kg) with 6 pieces per group. The drug solvent adopts a mixed solvent of PEG300 and NMP 9, and the drug is configured to the corresponding concentration
And (3) recording experimental data: tumor volume and mouse body weight were measured every 3 days, including tumor major axis and minor axis perpendicular to major axis, in units: millimeters (mm); mouse body weight in grams (g). During the experiment, the health conditions of the mice, such as the animal activity, water intake and food intake, the glossiness and color of the hair of the mice, diarrhea, inflammation of tumor parts and the like, need to be observed.
Tumor volume (mm)3) Long diameter x short diameter/2
The tumor inhibition rate (%) is (mean tumor volume in control group-mean tumor volume in experimental group)/mean tumor volume in control group x 100%.
The experiment is carried out for 30 days, and the test results are shown in figures 3-6.
According to the experimental results, the tumor inhibition rate of C5-1 to the Hela transplanted tumor model is 87.7%, 79% and 66.6% at the administration concentration of 50,40 and 20mg/kg, and the tumor inhibition rate of the positive drug is 66.5% at the administration concentration of 20 mg/kg. The tumor inhibition rates of the C5-1 to an A549 transplantation tumor model are 70.16% and 66.6% under the administration concentration of 40 and 20mg/kg, and the tumor inhibition rate of the positive drug is 56.08% under the administration concentration of 20 mg/kg. At equivalent concentrations, the in vivo antitumor activity of C5-1 was close to or slightly higher than that of BEZ235, a positive drug. However, during the administration, we observed that the body weight of mice in the C5-1 administration group was stable, indicating low toxicity of the molecule. The weight of the mice of the positive drug group is always lower than that of the C5-1 administration group, which shows that the molecule has the advantages of high efficiency and low toxicity compared with the positive drug.

Claims (6)

1. The benzoxazinone derivative is characterized in that the structural formula is as follows:
Figure FDA0002972268650000011
2. a pharmaceutically acceptable salt of the benzoxazinone derivative according to claim 1.
3. The use of a benzoxazinone derivative according to claim 1 in the manufacture of a medicament for inhibiting the PI3K/Akt/mTOR signaling pathway in a mammal.
4. Use of the benzoxazinone derivatives according to claim 1 in the preparation of an antitumor medicament.
5. A pharmaceutical composition characterized by: consists of an effective component and pharmaceutically acceptable auxiliary materials, wherein the effective component comprises a therapeutically effective amount of the benzoxazinone derivative or the pharmaceutically acceptable salt thereof according to claim 1.
6. The use of a benzoxazinone derivative according to claim 1 which is compound C5-1 in the preparation of a DNA-PK inhibitor.
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