CN113493471B

CN113493471B - Heteroaromatic kinase inhibitors

Info

Publication number: CN113493471B
Application number: CN202110358716.9A
Authority: CN
Inventors: 刘斌
Original assignee: Xuanzhu Pharma Co Ltd
Current assignee: Shandong Xuanzhu Pharma Co Ltd
Priority date: 2020-04-03
Filing date: 2021-04-02
Publication date: 2024-06-11
Anticipated expiration: 2041-04-02
Also published as: CN113493471A

Abstract

The invention belongs to the technical field of medicines, and in particular relates to a heteroaromatic ring DNA-PK kinase inhibitor compound shown in a general formula (I), pharmaceutically acceptable salt or isomer thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salt or isomer thereof, a method for preparing the compound, the pharmaceutically acceptable salt or isomer thereof, and application of the compound, the pharmaceutically acceptable salt or isomer thereof.

Description

Heteroaromatic kinase inhibitors

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a heteroaromatic ring DNA-PK inhibitor compound, pharmaceutically acceptable salt or isomer thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salt or isomer thereof, a method for preparing the compound, the pharmaceutically acceptable salt or isomer thereof, and application of the compound, the pharmaceutically acceptable salt or isomer thereof.

Background

Cancer is a malignant disease which is difficult to treat worldwide, has high treatment difficulty and high death rate, brings heavy burden to patients and families, and is a main disease affecting the health of residents in China. In recent years, the incidence rate of cancer in China is obviously increased, the death rate of the cancer is gradually increased, and the prevention and treatment of the cancer face a severe situation.

Currently, radiation therapy and chemotherapy are the most effective means of treating cancer in addition to surgical resection, while radiation therapy is the most effective non-surgical treatment for malignant tumors. Both radiation and considerable anticancer drugs can act directly or indirectly on DNA or DNA metabolic processes, resulting in DNA damage, with DNA double strand breaks (DNA double strand break, DSBs) being the most fatal to cancer cells. After DNA damage, a series of cellular reactions such as damaged DNA repair can be initiated, and the repair result is to improve the survival of cancer cells, which is one of the mechanisms of tumor cells for resisting chemoradiotherapy. If the DNA double strand breaks are not timely and completely repaired, cancer cells can cause cell death due to apoptosis or/and mitotic disorders. Therefore, the sensitivity of cancer cells to radiotherapy and chemotherapy can be improved and the proliferation of cells can be inhibited as long as the repair of the DNA damage is inhibited.

In human higher eukaryotic cells, repair of DSBs is mainly performed by DNA non-homologous end joining (nonhomologous end joining, NHEJ) dominated by DNA-dependent protein kinase (DNA-DEPENDENT PROTEIN KINASE, DNA-PK), thereby repairing damaged DNA, maintaining cellular activity and genomic stability. NHEJ repair is mainly involved in G1/S phase DNA damage repair and does not require DNA end ligation templates. NHEJ repair requires the co-ordination of many proteins and signaling pathways. The heterodimer of the Ku70/80 subunit and the catalytic subunit DNA-dependent protein kinase (DNA-PKcs) together constitute an active DNA-PK enzyme complex.

DNA-PKcs, a member of the phosphatidylinositol 3 kinase (PI 3K) superfamily, is a serine/threonine protein kinase; the PI3K superfamily also includes ATM, ATR, mTOR and 4 PI3K subtypes. When DNA-PK binds to fragmented DNA, its kinase activity is activated. The important function of Ku is to bind to the ends of DNA, recruit DNA-PKcs, both of which constitute DNA-PK holoenzymes and activate DNA-PKcs; the activated DNA-PKcs guide Artemis protein (an endonuclease) to bind to the damaged site, and DNA end-cutting treatment is carried out by virtue of the ribozyme activity so as to facilitate connection repair, then the XRCC 4/DNA-ligase IV complex is recruited by the activated DNA-PKcs, and finally the broken DNA double-chain end is positioned and connected by the DNA-ligase IV to complete repair. XRCC4 is a protein that forms a complex with DNA-ligase IV and can increase the activity of DNA-ligase IV. DNA-PKcs have 40 autophosphorylated amino acid residues, most typically autophosphorylation sites occur at Ser2056 (POR cluster) and Thr2609 (ABCDE cluster). NHEJ is believed to develop through three key steps: recognizing DSB-Ku 70/80 binds to incomplete DNA ends, recruiting two molecules of DNA-PKcs to adjacent sides of the DSB; performing DNA processing to remove the end-point-out non-ligatable ends or other forms of damage; finally, DNA ends are ligated.

Tumor cells are more sensitive to DNA-PK due to their higher basal levels of endogenous replication pressure (oncogene-induced replication pressure) and DNA damage, and the lower efficiency of DNA repair mechanisms in tumor cells.

At present, the development of the DNA-PK inhibitor with high efficiency and good selectivity has important clinical significance, can synergistically enhance the radiotherapy and chemotherapy effects, effectively inhibit the growth of tumors, and simultaneously can effectively reduce the damage to normal cells and reduce side effects.

Disclosure of Invention

The invention aims to provide a heteroaromatic ring compound with a novel structure and good inhibition effect on DNA-PK. Further, such compounds may be useful for increasing the sensitivity of a subject to radiation therapy and/or one or more anticancer agents. Further, the compounds may be used in combination with radiation therapy and/or one or more anticancer agents for the prevention and/or treatment of benign tumors or cancers.

The technical scheme of the invention is as follows:

in one aspect, the present invention provides a compound of the following formula (I), a pharmaceutically acceptable salt thereof, or an isomer thereof,

Wherein,

X ₁、X₂、X₃ are each independently selected from CH or N;

X ₄ is selected from CR ²R³、NR⁴, O, or S;

X ₅ is selected from CR ⁵ or N;

Y ₂、Y₃、Y₄、Y₅ is independently selected from CH or N;

Y is selected from S, NH or CH ₂;

Ring a is selected from 3-8 membered cycloalkyl optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, halo C _1-6 alkoxy, halo C _1-6 alkylthio, hydroxy C _1-6 alkoxy, hydroxy C _1-6 alkylthio, amino C _1-6 alkoxy, amino C _1-6 alkylthio;

R ¹、R²、R³、R⁴、R⁵ is each independently selected from H, halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halo C _1-6 alkoxy, halo C _1-6 alkylthio, hydroxy C _1-6 alkoxy, hydroxy C _1-6 alkylthio, amino C _1-6 alkoxy, amino C _1-6 alkylthio.

In certain embodiments, ring A is selected from 3-6 membered cycloalkyl optionally substituted with 1-2 substituents or 3-6 membered heterocyclyl; the substituents are selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, C _1-6 alkylcarbonyl, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halo C _1-6 alkoxy or halo C _1-6 alkylthio.

In certain embodiments, ring A is selected from 3-6 membered cycloalkyl or 3-6 membered heterocyclyl.

In certain embodiments, ring A is selected from 3-6 membered saturated cycloalkyl optionally substituted with 1-2 substituents or 3-6 membered saturated heterocyclyl; the substituents are selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, C _1-6 alkylcarbonyl, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halo C _1-6 alkoxy or halo C _1-6 alkylthio.

In certain embodiments, ring A is selected from 3-6 membered saturated cycloalkyl or 3-6 membered saturated heterocyclyl.

In certain embodiments, ring a is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyridinyl, tetrahydropyrazolidinyl, tetrahydroimidazolidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, morpholinyl optionally substituted with 1-2 substituents; the substituents are selected from halogen, hydroxy, amino, nitro, cyano, methyl, ethyl, propyl, isopropyl, methylamino, dimethylamino, methylcarbonyl, monofluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, aminomethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

In certain embodiments, ring a is selected from cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyridinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl optionally substituted with 1-2 substituents; the substituent is selected from halogen, hydroxy, amino, methyl, ethyl, propyl, isopropyl, methyl carbonyl, monofluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, aminomethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

In certain embodiments, ring a is selected from cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydropyridinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl.

In certain embodiments, ring A is selected from the group consisting of optionally substituted with 1-2 substituents The substituent is selected from halogen, hydroxy, amino, methyl, ethyl, propyl, isopropyl, methyl carbonyl, monofluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, aminomethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

In certain embodiments, R ¹、R²、R³、R⁴ is each independently selected from H, halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, halogenated C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halogenated C _1-6 alkoxy, or halogenated C _1-6 alkylthio;

R ⁵ is selected from H, halogen, hydroxy, amino, C _1-6 alkyl or halogenated C _1-6 alkyl.

In certain embodiments, R ¹、R²、R³ is each independently selected from fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, propyl, isopropyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy.

In certain embodiments, R ⁴ is selected from H, methyl, ethyl, propyl, isopropyl, or trifluoromethyl.

In certain embodiments, R ⁵ is selected from H, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, or methoxy.

In certain embodiments, Y ₂、Y₃、Y₅ is N, and Y ₄ is selected from CH or N, respectively.

In certain embodiments, Y ₂、Y₃、Y₅ is N and Y ₄ is CH, respectively.

In certain embodiments, Y is S.

In certain embodiments, each X ₁、X₂、X₃ is independently selected from CH or N.

In certain embodiments, each X ₁、X₂ is independently selected from CH or N; x ₃ is CH.

In certain embodiments, X ₁ is selected from CH or N; x ₂ is N; x ₃ is CH.

In certain embodiments, each X ₁、X₂、X₃ is independently selected from CH or N;

X ₄ is selected from CR ²R³、NR⁴, O, or S;

X ₅ is CR ⁵ or N;

y ₂、Y₃、Y₅ is N and Y ₄ is CH;

y is selected from S or NH;

ring a is selected from cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyridinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, optionally substituted with 1-2 substituents; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl, propyl, isopropyl, methylcarbonyl, trifluoromethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio or trifluoromethoxy;

r ¹、R²、R³ is independently selected from fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl, propyl, isopropyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy;

R ⁴ is selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl;

R ⁵ is selected from H, fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl or methoxy.

In certain embodiments, each X ₁、X₂ is independently selected from CH or N; x ₃ is CH;

X ₄ is selected from CR ²R³、NR⁴, O, or S;

X ₅ is CR ⁵ or N;

y ₂、Y₃、Y₅ is N and Y ₄ is CH;

y is selected from S or NH;

Ring a is selected from cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl optionally substituted with 1-2 substituents; the substituent is selected from fluorine, chlorine, bromine, iodine, hydroxyl, methyl, ethyl, isopropyl, methylcarbonyl, trifluoromethyl, methoxy, ethoxy or trifluoromethoxy;

R ¹、R²、R³ is independently selected from fluorine, chlorine, bromine, iodine, methyl, trifluoromethyl, methoxy or trifluoromethoxy;

R ⁴ is selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl;

In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt or isomer thereof, further has a structure represented by formula (II),

Wherein ,R¹、X₁、X₂、X₄、R²、R³、R⁴、Y、Y₃、Y₄、Y₅ and ring a are as described in any of the previous schemes.

In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt or isomer thereof, further has a structure represented by formula (III-1) or formula (III-2) below,

Wherein R ¹、X₁、X₂、X₄、R²、R³、R⁴, Y and ring A are as described in any of the previous schemes.

The technical schemes in the invention can be mutually combined to form new technical schemes, and the formed new technical schemes are also included in the scope of the invention.

In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt or isomer thereof, is selected from the group consisting of:

In another aspect, the present invention also provides a pharmaceutical formulation comprising a compound of formula (I), formula (II), formula (III-1) or formula (III-2), a pharmaceutically acceptable salt or isomer thereof, and one or more pharmaceutically acceptable excipients, which may be in any pharmaceutically acceptable dosage form. Pharmaceutically acceptable excipients are non-toxic, compatible with the active ingredient and otherwise biologically compatible substances for use in the organism. The choice of a particular excipient will depend on the mode of administration or type and state of disease used to treat a particular patient.

In certain embodiments, the above pharmaceutical formulations may be administered orally, parenterally, rectally, or pulmonary, etc., to a patient or subject in need of such treatment. For oral administration, the pharmaceutical composition may be formulated into oral preparations, for example, into conventional oral solid preparations such as tablets, capsules, pills, granules, etc.; can also be made into oral liquid preparation such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. may be added. For parenteral administration, the pharmaceutical preparations may also be formulated as injections, including injectable solutions, injectable sterile powders, and injectable concentrated solutions. When the injection is prepared, the conventional method in the existing pharmaceutical field can be adopted for production, and when the injection is prepared, no additive can be added, and the proper additive can be added according to the property of the medicine. For rectal administration, the pharmaceutical composition may be formulated as suppositories and the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalation, aerosol, powder spray or spray.

In a further aspect, the present invention also relates to the use of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof for the manufacture of a medicament for the prevention and/or treatment of benign tumors or cancers, including carcinoma in situ and metastatic cancers.

Furthermore, the invention also relates to the use of a pharmaceutical preparation containing the compound of the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), pharmaceutically acceptable salts or isomers thereof for preparing medicines for preventing and/or treating benign tumors, cancers and other diseases, wherein the cancers comprise in-situ cancers and metastatic cancers.

In a further aspect, the present invention also relates to the use of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, for the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancers, etc. in combination with radiation therapy and/or one or more anticancer agents, including cancer in situ and metastatic cancers.

Furthermore, the invention also relates to the use of a pharmaceutical preparation containing the compound of the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), pharmaceutically acceptable salts or isomers thereof for preparing a medicament for preventing and/or treating benign tumors or cancers and other diseases, wherein the medicament can be combined with radiotherapy and/or one or more anticancer agents, and the cancers comprise in-situ cancers and metastatic cancers.

In a further aspect, the invention also relates to the use of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof for the manufacture of a medicament for sensitizing cancer cells to an anticancer agent and/or ionizing radiation.

Furthermore, the present invention also relates to the use of a pharmaceutical formulation comprising a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof for the preparation of a medicament for sensitizing cancer cells to an anticancer agent and/or ionizing radiation.

The ionizing radiation refers to radiation of rays of various energies received by a patient during radiotherapy.

In another aspect, the invention also provides a pharmaceutical composition comprising a compound of the foregoing formula (I), formula (II), formula (III-1) or formula (III-2), a pharmaceutically acceptable salt or isomer thereof, and one or more second therapeutically active agents selected from the group consisting of anti-cancer agents, including mitotic inhibitors, alkylating agents, antimetabolites, DNA intercalators, antitumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal agents, angiogenesis inhibitors, cytostatic agents, targeting antibodies, HMG-CoA reductase inhibitors, and prenyl protein transferase inhibitors.

In certain embodiments, the second therapeutically active agent may be a drug that reduces or reduces one or more side effects of the compounds of the invention when used to treat a disease in a subject, or may be a drug that enhances the efficacy of the compounds of the invention.

In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, as described above.

In a further aspect, the present invention also relates to the use of a pharmaceutical composition comprising a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, for the manufacture of a medicament for the prevention and/or treatment of benign tumors or cancers, including carcinoma in situ and metastatic cancers.

In a further aspect, the present invention also relates to the use of a pharmaceutical composition comprising a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, for the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancer and the like, which medicament may be used in combination with radiation therapy and/or one or more anticancer agents, including cancer in situ and metastatic cancers.

Further, the present invention also relates to the use of a pharmaceutical composition comprising the aforementioned compound of formula (I), formula (II), formula (III-1) or formula (III-2), a pharmaceutically acceptable salt thereof or an isomer thereof for the preparation of a medicament for sensitizing cancer cells to an anticancer agent and/or ionizing radiation.

In another aspect, the present invention also provides a method of treating a disease associated with excessive activation of DNAPK, the method comprising administering to a patient in need thereof an effective amount of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical preparation or pharmaceutical composition; the disease associated with excessive activation of DNAPK is selected from benign tumors or cancers, including carcinoma in situ and metastatic cancers.

Further, the present invention provides a method for treating a disease associated with excessive activation of DNAPK, comprising administering to a patient before/after receiving radiation therapy an effective amount of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical preparation or pharmaceutical composition; the disease associated with excessive activation of DNAPK is selected from benign tumors or cancers, including carcinoma in situ and metastatic cancers.

Further, the present invention provides a method for treating a disease associated with excessive activation of DNAPK, comprising administering to a patient before/after receiving chemotherapy an effective amount of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical preparation or pharmaceutical composition; the disease associated with excessive activation of DNAPK is selected from benign tumors or cancers, including carcinoma in situ and metastatic cancers.

In another aspect, the present invention also provides a method of enhancing the sensitivity of a patient to an anticancer agent or radiation therapy, the method comprising administering to a patient in need thereof an effective amount of a compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical preparation or pharmaceutical composition; the anticancer agent is as described below.

Further, the present invention provides a method for enhancing sensitivity of a patient to an anticancer agent or radiation therapy, which comprises administering to a patient before/after receiving radiation therapy an effective amount of the compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition; the anticancer agent is as described below.

Further, the present invention provides a method for enhancing the sensitivity of a patient to an anticancer agent or radiation therapy, which comprises administering to a patient before/after receiving chemotherapy an effective amount of the compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt or isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition; the anticancer agent is as described below.

In another aspect, the invention also provides a kit comprising:

(a) An effective amount of one or more of the compounds of the aforementioned formula (I), formula (II), formula (III-1) or formula (III-2), pharmaceutically acceptable salts or isomers thereof,

And (b) an effective amount of one or more anticancer agents.

The "anticancer agent" as used herein refers to an agent having a therapeutic effect on tumors, including but not limited to mitotic inhibitors, alkylating agents, antimetabolites, DNA intercalators, antitumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal agents, angiogenesis inhibitors, cell growth inhibitors, targeting antibodies, HMG-CoA reductase inhibitors, and prenyl protein transferase inhibitors, etc.; the tumors include benign tumors and cancers. By "effective amount" is meant an amount of a drug that is capable of preventing, alleviating, delaying, inhibiting or curing a condition in a subject. The size of the dose administered is related to the mode of administration of the drug, the pharmacokinetics of the agent, the severity of the disease, the individual sign (sex, weight, height, age) of the subject, etc.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, however, for a better understanding of the present invention, the following definitions of some terms are provided. When the definition and interpretation of terms provided by the present invention are not identical to the meanings commonly understood by those skilled in the art, the definition and interpretation of terms provided by the present invention is in control.

"Halogen" as used herein refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

"C _1-6 alkyl" as used herein means straight or branched chain alkyl groups containing 1 to 6 carbon atoms, including for example "C _1-4 alkyl", "C _1-3 alkyl", "C _1-2 alkyl", "C _2-6 alkyl", "C _2-5 alkyl", "C _2-4 alkyl", "C _2-3 alkyl", "C _3-6 alkyl", "C _3-5 alkyl", "C _3-4 alkyl" and the like, specific examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 1, 2-dimethylpropyl, and the like. As used herein, "C _1-4 alkyl" refers to a specific example of C _1-6 alkyl containing 1 to 4 carbon atoms.

As used herein, "C _1-6 alkoxy" refers to "C _1-6 alkyl-O-", and "C _1-6 alkyl" is as defined above. As used herein, "C _1-4 alkoxy" refers to "C _1-4 alkyl-O-", and "C _1-4 alkyl" is as defined above.

The term "C _1-6 alkylthio" as used herein refers to "C _1-6 alkyl-S-", and "C _1-6 alkyl" is as defined above. The term "C _1-4 alkylthio" as used herein refers to "C _1-4 alkyl-S-", and "C _1-4 alkyl" is as defined above.

As used herein, "hydroxy C _1-6 alkyl, amino C _1-6 alkyl, halo C _1-6 alkyl" means that one or more hydrogens of the C _1-6 alkyl are replaced with one or more hydroxy, amino or halogen groups, respectively. C _1-6 alkyl is as defined above

The term "hydroxy C _1-6 alkoxy, amino C _1-6 alkoxy, halo C _1-6 alkoxy" as used herein means that one or more hydrogens of the "C _1-6 alkoxy" are replaced with one or more hydroxy, amino or halogen groups.

The term "hydroxy C _1-6 alkylthio, amino C _1-6 alkylthio, halogenated C _1-6 alkylthio" as used herein means that one or more hydrogens in the "C _1-6 alkylthio" are replaced with one or more hydroxy, amino or halogen groups.

The term "C _1-6 alkylamino group, di (C _1-6 alkyl) amino group" as used herein refers to C _1-6 alkyl-NH-

As used herein, "C _1-6 alkylcarbonyl" refers to C _1-6 alkyl-C (O) -.

"3-8 Membered cycloalkyl" as used herein refers to a saturated or partially saturated and non-aromatic monocyclic ring group containing 3-8 ring atoms, and "3-8 membered cycloalkyl" as used herein includes "3-8 membered saturated cycloalkyl" and "3-8 membered partially saturated cycloalkyl", for example, "3-6 membered cycloalkyl", "3-6 membered saturated cycloalkyl", "5-6 membered saturated cycloalkyl", and the like. Examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and the like.

"3-8 Membered heterocyclic group" as used herein refers to a saturated or partially saturated and non-aromatic monocyclic ring group containing at least one (e.g., 1,2,3, 4 or 5) heteroatom(s) and having 3-8 ring atoms, the heteroatom being a nitrogen atom, an oxygen atom and/or a sulfur atom, optionally a ring atom in the ring structure (e.g., a carbon atom, a nitrogen atom or a sulfur atom) may be oxo. The "3-8 membered heterocyclic group" as used herein includes "3-8 membered saturated heterocyclic group" and "3-8 membered partially saturated heterocyclic group". The "3-8 membered heterocyclic group" is, for example, "3-6 membered heterocyclic group", "3-6 membered saturated heterocyclic group", "3-5 membered saturated heterocyclic group", "5-6 membered saturated heterocyclic group", or the like. Specific examples thereof include, but are not limited to: aziridinyl, 2H-aziridinyl, diazinoalkyl, 3H-diazinopropenyl, azetidinyl, oxetanyl, 1, 4-dioxanyl, 1, 3-dioxolanyl, 1, 4-dioxadienyl, tetrahydrofuranyl, dihydropyrrolyl, tetrahydropyrrolyl, tetrahydropyrazolidinyl, tetrahydroimidazolyl, 4, 5-dihydroimidazolyl, pyrazolidinyl, 4, 5-dihydropyrazolyl, 2, 5-dihydrothienyl, tetrahydrothienyl, 4, 5-dihydrothiazolyl, thiazolidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, tetrahydropyridinyl, piperidonyl, tetrahydropyridonyl, dihydropyridonyl, piperazinyl, hexahydropyrimidinyl, morpholinyl, and the like.

"Optionally substituted" as used herein refers to both cases where one or more hydrogen atoms on the substituted group are "substituted" or "unsubstituted" with one or more substituents.

The invention relates to chemotherapy, which is short for chemical drug treatment, and the aim of treatment is achieved by killing cancer cells by using chemical therapeutic drugs.

The invention relates to a method for treating tumor, namely tumor radiotherapy, which mainly utilizes radioactive rays to treat tumor locally, wherein the radioactive rays comprise alpha rays, beta rays, gamma rays generated by radioactive isotopes, x rays, electron rays, proton beams, other particle beams generated by various x-ray therapeutic machines or accelerators, and the like.

The term "pharmaceutically acceptable salt" as used herein refers to salts of acidic functional groups (e.g., -COOH, -OH, -SO ₃ H, etc.) present in the compound with suitable inorganic or organic cations (bases), including salts with alkali metals or alkaline earth metals, ammonium salts, salts with nitrogen-containing organic bases; and salts of basic functional groups (e.g., -NH2, etc.) present in the compounds with suitable inorganic or organic anions (acids), including salts with inorganic or organic acids (e.g., carboxylic acids, etc.).

"Isomers" as used herein means that the compounds of the present invention contain one or more asymmetric centers and are therefore useful as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The compounds of the present invention may have asymmetric centers that each independently produce two optical isomers. The scope of the present invention includes all possible optical isomers and mixtures thereof. The compounds of the present invention, if they contain olefinic double bonds, include cis-isomers and trans-isomers unless specified otherwise. The compounds described herein may exist in tautomeric (one of the functional group isomers) forms having different points of attachment of hydrogen through displacement of one or more double bonds, for example, the keto and enol forms thereof are keto-enol tautomers. The compounds of the invention contain spiro structures, which are affected by the steric structure of the ring, and substituents on the ring may be present on both sides of the ring to form the opposite cis (cis) and trans (trans) isomers. Each tautomer and mixtures thereof are included within the scope of the present invention. Enantiomers, diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof and the like of all compounds are included within the scope of the present invention.

The compounds of the invention may be prepared by enantiospecific synthesis or resolution from mixtures of enantiomers to give the individual enantiomers. Conventional resolution techniques include resolution of mixtures of enantiomers of the starting material or final product using various well-known chromatographic methods.

When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 99 wt%, or 99.9 wt% pure relative to the other stereoisomers. When a single isomer is named or depicted by structure, the depicted or named enantiomer is at least 60% by weight, 70% by weight, 80% by weight, 90% by weight, 99% by weight, or 99.9% by weight pure. Optical purity wt% is the ratio of the weight of an enantiomer to the weight of the enantiomer plus the weight of its optical isomer.

Advantageous effects of the invention

1. The compound, the pharmaceutically acceptable salt or the isomer thereof has excellent DNA-PK inhibition effect, good pharmacokinetic property in organisms, lasting effect and high bioavailability, and can enhance the sensitivity of cancer cells to radiotherapy and/or one or more anticancer agents.

2. The compound, the pharmaceutically acceptable salt or the isomer thereof has high enzymatic selectivity, good treatment effect on benign tumors and cancers, and high stability of liver microsomes.

3. The compound has the advantages of simple preparation process, high purity of the medicine, stable quality and easy mass industrial production.

Detailed description of the preferred embodiments

The technical scheme of the present invention will be described in detail below with reference to specific embodiments, but the scope of the subject matter of the present invention should not be construed as being limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.

Abbreviations:

DMFDMA: n, N-dimethylformamide dimethyl acetal; DPPA: diphenyl azide phosphate; lawson reagent: 2, 4-bis (p-methoxyphenyl) -1, 3-dithio-2, 4-diphosphatetidine-2, 4-sulfide; DIEA: n, N-diisopropylethylamine; DCM: dichloromethane; meOH: methanol; brettPhos Pd G3: methanesulfonic acid (2-dicyclohexylphosphine) -3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II).

Preparation example one: preparation of 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine

1. Preparation of (E) -N, N-dimethyl-N' - (4-methyl-5-nitropyridin-2-yl) formamidine

4-Methyl-5-nitropyridin-2-amine (20 g,130.6 mmol), toluene (400 mL), N-dimethylformamide dimethyl acetal (47 g,394.3 mmol) were added, heated to 110℃and reacted for 3 hours, dried by spinning, washed with N-heptane (300 mL), filtered and the solid dried to give the desired product (26 g, yield: 95.6%).

2. Preparation of (E) -N-hydroxy-N' - (4-methyl-5-nitropyridin-2-yl) carboxamidine

(E) -N, N-dimethyl-N' - (4-methyl-5-nitropyridin-2-yl) formamidine (16.0 g,76.8 mmol), methanol (200 mL), hydroxylamine hydrochloride (10.7 g,153.9 mmol) was added, heated to 65 ℃, cooled for 3 hours, water (300 mL), ethyl acetate (300 mL), separated, the organic phase dried over anhydrous sodium sulfate, filtered, spin-dried, the solid slurry washed with methyl tert-butyl ether (100 mL) and the solid dried to give the title product (9.2 g, yield: 61.3%).

3. Preparation of 7-methyl-6-nitro- [1,2,4] triazolo [1,5-a ] pyridine

(E) -N-hydroxy-N' - (4-methyl-5-nitropyridin-2-yl) formamidine (10.8 g,55.0 mmol), tetrahydrofuran (110 mL), trifluoroacetic anhydride (13.9 g,66.2 mmol) was added, the reaction was monitored by LC-MS at 10 ℃ for 12 hours, the solvent was spun dry, mixed solvent (100 mL, ethyl acetate: petroleum ether=1:4) was added to the solid, filtration and the solid dried in vacuo to give the desired product (5.6 g, yield: 57.1%).

4. Preparation of 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine

7-Methyl-6-nitro- [1,2,4] triazolo [1,5-a ] pyridine (4.4 g,24.7 mmol) was dissolved in ethanol (50 mL), palladium on carbon (528 mg), ammonium formate (7.8 g,124.0 mmol) was added, the reaction was heated to 70℃for 4 hours, suction filtration and the filtrate was spun dry to give the product (3.6 g, yield: 98.3%).

Preparation example II: preparation of 2-chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one

1. Preparation of ethyl 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylate

Ethyl 2, 4-dichloropyrimidine-5-carboxylate (40.0 g,181 mmol) was dissolved in acetonitrile (1000 mL), tetrahydro-2H-pyran-4-amine hydrochloride (24.9 g,181 mmol), potassium carbonate (62.5 g,452 mmol), 20℃and the reaction was carried out for 16 hours, LC-MS detection reaction was complete, suction filtration and filtrate concentration gave the target product (45 g, yield: 87.0%).

2. Preparation of 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylic acid

Ethyl 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylate (15.0 g,52.4 mmol) was dissolved in a system of tetrahydrofuran (150.0 mL) and water (150.0 mL), lithium hydroxide (4.8 g,114.3 mmol) was added, the reaction was carried out at 25℃for 1 hour, concentration was carried out, pH=3 was adjusted to precipitate a white solid from the aqueous phase, filtration was carried out, and the solid was dried to give the objective product (11.5 g, yield: 85.2%).

3. Preparation of 2-chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one

2-Chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylic acid (10.0 g,38.8 mmol) was dissolved in N, N-dimethylacetamide (60.0 mL), triethylamine (4.0 g,39.6 mmol) and diphenyl azide phosphate (11.0 g,40.0 mmol) were added, the reaction was continued for 1 hour at 25℃and then the temperature was raised to 110℃for 16 hours, after lowering to 25℃the reaction was poured into ice water, a pale yellow solid was precipitated, and the mixture was dried by filtration to give a crude product (7.1 g, yield: 71.7%).

Preparation example III: preparation of 2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one

2-Chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (7.4 g,29.0 mmol) was dissolved in N, N-dimethylformamide (100.0 mL), 60% NaH (1.5 g,37.5 mmol) was added after the system had cooled to 0deg.C, CH ₃ I (5.0 g,35.2 mmol) was added after 30 minutes of reaction, the reaction was continued at 0deg.C for 2 hours, water (150.0 mL) and ethyl acetate (220.0 mL) were added, and the organic phase was concentrated and dried to give the crude product (6.9 g, yield: 88.5%).

Embodiment one: preparation of 7-methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purine-8-thione (Compound 1)

1. Preparation of 7-methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one

2-Chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (3.3 g,12.3 mmol) was dissolved in dioxane (50.0 mL), 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine (1.8 g,12.2 mmol), 2- (dicyclohexylphosphine) 3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl (760.0 mg,0.784 mmol) and cesium carbonate (7.8 g,23.9 mmol) were added and the system was reacted at 100℃for 2.0 hours under nitrogen and concentrated, washed with methanol (50.0 mL). Column chromatography of the solid (DCM: meoh=20:1) afforded the product (3.4 g, yield: 73.9%).

The molecular formula: molecular weight of C ₁₈H₂₀N₈O₂: 380.4 LC-MS (M/e): 381.2 (M+H ⁺)

¹HNMR(400MHz,DMSO):δ9.10(s,1H),8.66(s,1H),8.35(s,1H),8.06(s,1H),7.69(s,1H),4.41-4.37(m,1H),3.96-3.92(m,2H),3.42-3.39(m,2H),3.28(s,3H),2.53-2.50(m,1H),2.48-2.35(m,1H),2.37(s,3H),1.67-1.64(m,2H)

2. Preparation of 7-methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purine-8-thione

7-Methyl-2- ((7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) amino) -9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-one (250.0 mg, 0.650 mmol), lawsen reagent (1400.0 mg, 3.458 mmol) was dissolved in dioxane (10.0 mL), the system was microwaved at 155 ℃ for 4.0 hours and the residue after concentration was subjected to column chromatography (dichloromethane: methanol=20:1) to give the product (35.0 mg, 13.5% yield).

Molecular formula C ₁₈H₂₀N₈ OS molecular weight 396.5 LC-MS (M/e): 397.2 (M+H ⁺)

¹H-NMR(400MHz,DMSO)δ:9.06(s,1H),8.47(s,1H),8.40(s,1H),7.75(s,1H),5.25(s,1H),4.0-13.91(m,2H),3.7(s,3H),3.52-3.42(m,2H),2.72-2.63(m,2H),2.48(s3H),1.77-1.68(m,2H).

Embodiment two: preparation of 8-imino-7-methyl-N- (7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) -9- (tetrahydro-2H-pyran-4-yl) -8, 9-dihydro-7H-purin-2-amine (Compound 2)

1. Preparation of 2, 8-dichloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purine

2-Chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (6.7 g,26.3 mmol) was dissolved in POCl ₃ (60.0 mL), DIEA (5.9 g,45.8 mmol) was added to the system, then reacted at 110℃for 11.0H, concentrated to dryness followed by pH adjustment to 7 with saturated aqueous NaHCO ₃, and the crude product was purified by column chromatography (DCM: meOH=10:1) (3.82 g, yield: 53.2%).

2. Preparation of 2-chloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purin-8-amine

2, 8-Dichloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purine (3.2 g,11.7 mmol) was dissolved in methanolic amine (7M, 50.0 mL) and the system was concentrated after 3.0 hours at 100deg.C and purified by column chromatography (MeOH: DCM=1:10) to give the product (1.5 g, yield: 50.5%).

3. Preparation of 2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-imine

2-Chloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purin-8-amine (530 mg,2.1 mmol) was dissolved in ethanol (15.0 mL) and then concentrated after microwave reaction at 130℃for 2.0 hours with the addition of CH ₃ I (2.0 g,14.1 mmol) to give the crude product (120.0 mg, yield: 21.3%) which was purified by column chromatography (MeOH: DCM=1:10).

4. Preparation of 8-imino-7-methyl-N- (7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-yl) -9- (tetrahydro-2H-pyran-4-yl) -8, 9-dihydro-7H-purin-2-amine

2-Chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-imine (89.9 mg,0.34 mmol), 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine (50.0 mg,0.34 mmol), cesium carbonate (220.0 mg,0.67 mmol) and BrettPhos Pd G3 (25.0 mg,0.028 mmol) were dissolved in dioxane (5.0 mL), and the system was concentrated after 3.0 hours reaction at 100℃under N ₂ and purified by column chromatography (MeOH: DCM=1:10) to give the product (7.5 mg, yield: 5.8%).

The molecular formula: molecular weight of C ₁₈H₂₁N₉ O: 379.4 LC-MS (M/e): 380.0 (M+H ⁺)

¹HNMR(400MHz,CDCl₃):δ:9.84(s,1H),8.25(s,1H),7.68(s,1H),7.56(s,1H),7.26(s,1H),6.59(s,1H),4.67-4.45(m,1H),4.17-4.15(m,2H),3.58-3.49(m,2H),3.31(s,3H),2.91-2.75(m,2H),2.53(s,3H),1.87-1.76(m,2H).

Using the same or similar methods as the examples above, compounds shown in the following tables were prepared:

Experimental protocol

Exemplary protocols for some of the compounds of the present invention are provided below to demonstrate the advantageous activity and beneficial technical effects of the compounds of the present invention. It should be understood that the following experimental schemes are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure.

Experimental example 1 in vitro cytological Activity of the Compounds of the invention

Abbreviations (abbreviations)

EDTA: ethylenediamine tetraacetic acid

DMSO: dimethyl sulfoxide

Tris: trimethylolaminomethane

Brij-35: laurinol polyoxyethylene ether

DTT: dithiothreitol

Test article: the structural formula and the preparation method of the compound are shown in the examples.

Experimental reagent:

Name of the name	Branding
		ADP-Glo Kinase Assay	Promege
DNA-PK	Promege

The experimental method comprises the following steps:

1. 1-fold preparation of kinase buffer

1) 1-Fold kinase buffer

40mM Tris,pH 7.5

0.0055％Brij-35

20mM MgCl₂

0.05mM DTT

2. Compound formulation

1) The initial concentration of the compound was 1. Mu.M, which was formulated at a 100-fold concentration, i.e., 100. Mu.M. Mu.l of 10mM compound was taken and 198. Mu.l of 100% DMSO was added to prepare a 100. Mu.M compound solution. Mu.l of 100-fold compound was added to the second well on a 96-well plate, and 60. Mu.l of 100% DMSO was added to the other wells. Mu.l of the compound was taken from the second well and added to the third well, followed by 3-fold dilution down in sequence for a total of 10 concentrations.

2) Transfer 100 μl of 100% dmso and the highest concentration of positive control worth (400 nM) into two empty wells as Max well and Min well, respectively.

3) 50Nl of compound was transferred into 384 well plates using Echo.

3. Preparation of 2x kinase solution

1) A 2-fold DNA-PK kinase solution was prepared using a 1-fold kinase buffer.

2) Transfer 2.5. Mu.l of 2-fold enzyme solution into 384-well plate reaction wells.

3) Oscillating, mixing, and standing at room temperature.

4. Preparation of 2x substrate solution

1) A 2-fold substrate solution was prepared using a 1-fold kinase buffer.

2) Transfer 2.5 μl of 2-fold substrate solution into 384-well plate reaction wells to initiate the reaction.

3) Oscillating and mixing evenly.

5. Kinase reaction and termination

1) 384 Well plates were capped and incubated for 3 hours at 28 ℃.

2) Transfer 5. Mu.l ADP-Glo reagent and incubate at 28℃for 2 hours.

6. Detection of reaction results

1) Transfer 10 μl kinase assay reagent to 384 well plate reaction wells to terminate the reaction.

2) Resting for 30 minutes at room temperature.

7. Data reading

Sample values were read at Envision.

8. Inhibition rate calculation

After converting the data into inhibition rate data, curve fitting is performed.

Percent inhibition = (max-conversion)/(max-min) ×100, where max refers to the conversion of DMSO control, min refers to the conversion of no enzyme activity control, and conversion refers to the conversion of test compound at each concentration.

Experimental results:

TABLE 1 in vitro enzymatic Activity data for Compounds of the invention

Conclusion of experiment:

the results show that the compound has better inhibition effect on the activity of DNA-PK kinase.

Experimental example 2 experiments on the metabolic stability of the Compound of the invention in liver microsomes in different species

Test article: the compound 1 is self-made, and the chemical name and the preparation method are shown in the preparation examples of the compound.

Experimental materials:

Cynomolgus monkey mixed liver microsomes were purchased from the rad liver disease research center (Shanghai limited), lot number: ZXBZ the concentration of hepatic microsomal protein was 20 mg.mL ^-1.

Mixed liver microsomes of SD rats and CD-1 mice were purchased from Xeno Tech, lot numbers: 1610290 (SD rat), 1710069 (CD-1 mouse). The concentration of the hepatic microsomal protein is 20 mg.mL ^-1.

Human mixed liver microsomes were purchased from corning company under the trade designation 452117, lot number 38294, and liver microsome protein concentration was 20 mg/mL ^-1.

The experimental initiation factor beta-NADPH is purchased from Solarbio company; phosphate Buffer (PBS) at pH 7.4 was self-made by the present laboratory.

Sample solution preparation:

accurately weighing a proper amount of sample powder, adding a proper amount of dimethyl sulfoxide (DMSO) to dissolve to 1mM, and diluting with methanol to 20 times to 50 μm working solution.

The experimental method comprises the following steps:

TABLE 2 composition of liver microsome metabolic stability experiment incubation System

The experimental operation steps are as follows:

(1) According to the ratio of "experimental incubation system composition" in Table 2 above, a mixed solution 1 (without microsomes, test sample and. Beta. -NADPH) of incubation system was prepared by taking 100mM PBS 5.85mL,20mM MgCl ₂ mL of solution and 3.57mL of H ₂ O for each compound. The experiment is carried out simultaneously with the positive drug verapamil of the experiment incubation system to prove that the activity of the liver microsome enzyme is normal.

(2) Liver microsomes (20 mg protein/mL) were removed from the-80℃refrigerator and pre-incubated for 3min on a 37℃water bath thermostated shaker.

(3) 1.9ML of the incubation system mixed solution 1 was taken for each species of the compound, and 56. Mu.L of microsomes of different species were added to prepare an incubation system mixed solution 2 (containing no test sample and. Beta. -NADPH).

(4) Sample group (containing microsomes and β -NADPH): mu.L of the mixed solution 2 of the incubation system was taken, 14. Mu.L of the working solution of the test sample having a concentration of 50. Mu.M was added, and 70. Mu.L of the working solution of 10mM of beta-NADPH was added. Mixing, and repeating. Sampling time points are 0min,5min,10min,20min,30min and 60min. This sample set was used to evaluate the metabolic stability of compounds via β -NADPH.

(5) Control group (microsome-containing, beta-NADPH-free, water instead of beta-NADPH): 264. Mu.L of the mixed solution 2 of the incubation system was added with 6. Mu.L of the working solution of the test sample having a concentration of 50. Mu.M, and 30. Mu.L of water was added. Mixing, and repeating. Sampling time points were 0min and 60min. The negative control group was used to evaluate whether the compound was present in the liver microsome incubation system for non- β -NADPH mediated metabolism.

(6) At each predetermined time point 50 μl was sampled from the incubation sample tube and added to the stop sample tube (300 μl of cold stop containing internal standard tolbutamide 50ng/mL acetonitrile solution), vortexed, and the reaction stopped.

(7) After vortexing for 10min, centrifugation was carried out for 5min (12000 rpm).

(8) 100 Μl of the supernatant was taken, 100 μl of water was added, mixed well by vortexing, and analyzed by LC-MS/MS.

Data analysis:

the ratio of the test sample to the internal standard peak area is converted into the residual percentage by the following formula.

Experimental results:

TABLE 3 results of hepatic microsomal stability of the compounds of the invention

Conclusion of experiment:

The compound has good stability in liver microsomes of human, monkey, rat and mouse.

Experimental example 3 in vivo pharmacokinetic experiments in CD1 mice of Compounds of the invention

Abbreviations (abbreviations)

HPC: hydroxypropyl cellulose

DMA: dimethylacetamide test article: the compound 1 is self-made, and the chemical name and the preparation method are shown in the preparation examples of the compound. Test animals: CD1 mice, females, purchased from Beijing Vitrehua laboratory animal technologies Co., ltd, had a weight of 24-29g and a total of 12.

Sample solution preparation:

The preparation method of the blank solvent (1) comprises the following steps: 28g of HP-beta-CD is weighed, a proper amount of water for injection is added for dissolution, the volume of the water for injection is fixed to 100mL, and the water for injection is uniformly mixed by vortex, thus obtaining 28 percent of HP-beta-CD.

The preparation method of the blank solvent (2) comprises the following steps: weighing 20g of HPC, slowly adding into 500mL of stirred purified water, adding 1mL of Tween 80, stirring to be clear and transparent, fixing the volume to 1000mL, and uniformly stirring to obtain 2% HPC+0.1% Tween 80.

IV (intravenous bolus) administration:

Taking compound 1 (2.57 mg), adding DMA (491 mu L), shaking for dissolving, then adding PEG400 (982 mu L), vortex mixing, finally adding blank solvent (1) (3.44 mL), vortex mixing, and preserving heat at 50 ℃ for 20min to obtain a clear solution of 0.5mg/mL, and taking the clear solution as an IV administration solution of compound 1.

PO (gastric lavage) administration:

compound 1 (3.83 mg) was weighed, placed in a tissue grinder, and 3.66mL of a blank solvent (2) was added, and the mixture was uniformly ground at 1200 rpm to obtain a suspension drug solution having a concentration of 1mg/mL, as a PO drug solution for compound 1.

Experimental method

IV is administered at a dose of 2.5mg/kg, at a concentration of 0.5mg/mL, and at a volume of 5mL/kg.

PO administration dosage is 10mg/kg, administration concentration is 1mg/mL, and administration volume is 10mL/kg.

Blood collection time point: blood was collected at 0.083, 0.25, 0.5, 1, 2,4, 6, 8, and 24 hours after administration, specifically in the manner shown in the following table.

At each time point, about 50. Mu.L of whole blood was collected through the inner canthus of the eye, placed in an anticoagulant tube containing EDTA-K ₂ anticoagulant, and centrifuged at 8000 rpm for 6 minutes at 4℃to obtain a plasma sample, which was stored in a freezer at-80℃for analysis.

Plasma sample analysis

The protein precipitation method is adopted: plasma samples were taken at 20. Mu.L, 200. Mu.L of an internal standard (containing 50ng/mL of tolbutamide in acetonitrile) was added, vortexed for 10min, centrifuged at 4000 rpm for 20min, 100. Mu.L of supernatant was taken, 100. Mu.L of water was added, vortexed and mixed for 3min, and LC-MS/MS analysis was performed.

Experimental results

TABLE 4 evaluation results of CD1 mouse PK

AUC _0-t represents the area under the drug time curve 0- & gt; CL represents clearance; v _ss represents the steady state apparent distribution volume; t _1/2 represents terminal elimination half-life; expression peak time of T _max generation; expression peak concentration of generation C _max; f% represents absolute bioavailability.

Conclusion of the experiment

From the data in table 4, the compounds of the present invention have good pharmacokinetic properties and have higher exposure and bioavailability.

Claims

1. A compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof,

Wherein,

X ₁ is selected from CH or N; x ₂ is N; x ₃ is CH;

X ₄ is selected from CR ²R³ or NR ⁴;

X ₅ is N;

Y ₂、Y₃、Y₅ is N, Y ₄ is selected from CH or N;

y is selected from S or NH;

R ¹、R²、R³、R⁴ is each independently selected from H, halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halo C _1-6 alkoxy, halo C _1-6 alkylthio, hydroxy C _1-6 alkoxy, hydroxy C _1-6 alkylthio, amino C _1-6 alkoxy, amino C _1-6 alkylthio.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein,

Ring a is selected from 3-6 membered cycloalkyl optionally substituted with 1-2 substituents or 3-6 membered heterocyclyl; the substituents are selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, C _1-6 alkylamino, di (C _1-6 alkyl) amino, C _1-6 alkylcarbonyl, halo C _1-6 alkyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halo C _1-6 alkoxy or halo C _1-6 alkylthio.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein,

Ring a is selected from 3-6 membered saturated cycloalkyl optionally substituted with 1-2 substituents or 3-6 membered saturated heterocyclyl.

4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein,

Ring a is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyridinyl, tetrahydropyrazolidinyl, tetrahydroimidazolidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, morpholinyl optionally substituted with 1-2 substituents; the substituents are selected from halogen, hydroxy, amino, nitro, cyano, methyl, ethyl, propyl, isopropyl, methylamino, dimethylamino, methylcarbonyl, monofluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, aminomethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

5. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein,

R ¹、R²、R³、R⁴ is independently selected from H, halogen, hydroxy, amino, nitro, cyano, C _1-6 alkyl, halogenated C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, halogenated C _1-6 alkoxy or halogenated C _1-6 alkylthio.

6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein,

X ₁ is selected from CH or N; x ₂ is N; x ₃ is CH;

X ₄ is selected from CR ²R³ or NR ⁴;

X ₅ is N;

y ₂、Y₃、Y₅ is N and Y ₄ is CH;

y is selected from S or NH;

r ⁴ is selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl.

7. The compound according to any one of claim 1 to 6, or a pharmaceutically acceptable salt thereof, which further has a structure represented by the following general formula (III-1) or general formula (III-2),

Wherein R ¹、X₁、X₂、X₄, Y and ring A are as defined in any one of claims 1 to 6.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

9. a pharmaceutical formulation comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in any one of pharmaceutically acceptable dosage forms, comprising one or more pharmaceutically acceptable excipients.

10. A pharmaceutical composition comprising a compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, comprising one or more second therapeutically active agents selected from the group consisting of anti-cancer agents comprising mitotic inhibitors, alkylating agents, antimetabolites, DNA intercalators, antitumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal agents, angiogenesis inhibitors, cytostatic agents, targeting antibodies, HMG-CoA reductase inhibitors, and prenyl protein transferase inhibitors.

11. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation according to claim 9, or a pharmaceutical composition according to claim 10, in the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancers, including carcinoma in situ and metastatic cancers.

12. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation according to claim 9, or a pharmaceutical composition according to claim 10, in the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancers, in combination with radiation therapy and/or one or more anticancer agents, including cancer in situ and metastatic.

13. Use of a compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation according to claim 9, or a pharmaceutical composition according to claim 10, in the manufacture of a medicament for sensitizing cancer cells to anticancer agents and/or radiation therapy.

14. A kit comprising:

(a) An effective amount of one or more compounds according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof,

And (b) an effective amount of one or more anticancer agents.