CN113493471A - Heteroaromatic kinase inhibitors - Google Patents

Heteroaromatic kinase inhibitors Download PDF

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Publication number
CN113493471A
CN113493471A CN202110358716.9A CN202110358716A CN113493471A CN 113493471 A CN113493471 A CN 113493471A CN 202110358716 A CN202110358716 A CN 202110358716A CN 113493471 A CN113493471 A CN 113493471A
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amino
pharmaceutically acceptable
alkyl
compound
isomer
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刘斌
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Shandong Xuanzhu Pharma Co Ltd
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Xuanzhu Pharma Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Abstract

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

Description

Heteroaromatic kinase inhibitors
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a heteroaromatic DNA-PK inhibitor compound, pharmaceutically acceptable salts or isomers thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salts or isomers thereof, a method for preparing the compound, the pharmaceutically acceptable salts or isomers thereof, and application of the compound, the pharmaceutically acceptable salts or isomers thereof.
Background
Cancer is a malignant disease which is difficult to treat all over the world, has high treatment difficulty and high mortality 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 of cancer in our country has increased significantly, the mortality rate has also gradually increased, and cancer prevention and treatment face a severe situation.
Currently, radiotherapy and chemotherapy are the most effective means of treating cancer in addition to surgical resection, while radiotherapy is the most effective non-surgical treatment for malignancies. Radiation and a considerable number of anticancer drugs can act directly or indirectly on DNA or DNA metabolic processes, resulting in DNA damage, of which DNA Double Strand Break (DSB) is the most lethal for cancer cells. After DNA damage, a series of cellular responses such as damaged DNA repair can be initiated, and the repair results in the improvement of cancer cell survival, which is one of the mechanisms of tumor cells resisting to radiotherapy and chemotherapy. If a DNA double strand break is not repaired in time and integrity, cancer cells die as a result of apoptosis or/and mitotic disturbances. Therefore, by inhibiting the repair of such DNA damage, the sensitivity of cancer cells to radiotherapy and chemotherapy can be improved, and the proliferation of cells can be inhibited.
In human and other higher eukaryotes, DSB repair is mainly performed by DNA-Dependent Protein Kinase (DNA-PK) dominated DNA non-homologous end joining (NHEJ), thereby repairing damaged DNA and maintaining cellular activity and genomic stability. NHEJ repair is primarily involved in G1/S phase DNA damage repair and does not require DNA end-joining templates. NHEJ repair requires an economic coordination 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 belongs to the phosphatidylinositol 3 kinase (PI3K) superfamily member, is a serine/threonine protein kinase; the PI3K superfamily also includes ATM, ATR, mTOR and 4 PI3K subtypes. When DNA-PK binds to the fragmented DNA, its kinase activity is activated. The important function of Ku is to combine with the end of DNA, recruit DNA-PKcs, and the two compose DNA-PK holoenzyme and activate DNA-PKcs; activated DNA-PKcs directs the Artemis protein (an endonuclease) to bind to the damaged site, DNA end-breaking is performed by virtue of its ribozyme activity to facilitate ligation repair, then the XRCC 4/DNA-ligase IV complex is recruited by the activated DNA-PKcs, and finally the broken DNA double-stranded end is targeted and ligated by DNA-ligase IV to complete repair. XRCC4 is a protein that forms a complex with DNA-ligase IV and increases the activity of DNA-ligase IV. DNA-PKcs present 40 amino acid residues that can be autophosphorylated, with the most typical autophosphorylation sites occurring at Ser2056(POR cluster) and Thr2609(ABCDE cluster). NHEJ is thought to develop through three key steps: recognition of DSB-binding of Ku70/80 to incomplete DNA ends recruits two molecules of DNA-PKcs to the adjacent side of the DSB; performing DNA processing to remove the end-pointed non-ligatable ends or other forms of damage; finally, the DNA ends are ligated.
Tumor cells are more sensitive to DNA-PK because they have a higher basal level of endogenous replication stress (oncogene-induced replication stress) and DNA damage, and the DNA repair mechanisms are less efficient in tumor cells.
At present, the development of a high-efficiency and good-selectivity DNA-PK inhibitor has important clinical significance, can synergistically enhance the effects of radiotherapy and chemotherapy, 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 solve the technical problem of providing a heteroaromatic ring compound which has a novel structure and a good inhibition effect on DNA-PK. Further, such compounds may be used to increase the sensitivity of a subject to radiation therapy and/or one or more anti-cancer agents. Furthermore, the compounds can be used for preventing and/or treating benign tumors or cancers by combining with radiotherapy and/or one or more anticancer agents.
The technical scheme of the invention is as follows:
in one aspect, the present invention provides a compound represented by the following general formula (I), a pharmaceutically acceptable salt thereof, or an isomer thereof,
Figure BDA0003004645000000021
wherein the content of the first and second substances,
X1、X2、X3each independently selected from CH or N;
X4selected from the group consisting of CR2R3、NR4O or S;
X5selected from the group consisting of CR5Or N;
Y2、Y3、Y4、Y5each independently selected from CH or N;
y is selected from S, NH or CH2；
Ring A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocyclyl optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, nitro, cyano, C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio radical, C1-6Alkylcarbonyl, halo C1-6Alkoxy, halo C1-6Alkylthio, hydroxy C1-6Alkoxy, hydroxy C1-6Alkylthio, amino C1-6Alkoxy, amino C1-6An alkylthio group;
R1、R2、R3、R4、R5each independently selected from H, halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy, halo C1-6Alkylthio, hydroxy C1-6Alkoxy, hydroxy C1-6Alkylthio, amino C1-6Alkoxy, amino C1-6An alkylthio group.
In certain embodiments, ring a is selected from a 3-6 membered cycloalkyl or 3-6 membered heterocyclyl optionally substituted with 1-2 substituents; the substituent is selected from halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, C1-6Alkylcarbonyl, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy or halo C1-6An alkylthio group.
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 or 3-6 membered saturated heterocyclyl optionally substituted with 1-2 substituents; the substituent is selected from halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, C1-6Alkylcarbonyl, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy or halo C1-6An alkylthio group.
In certain embodiments, ring a is selected from a 3-6 membered saturated cycloalkyl or a 3-6 membered saturated heterocyclyl.
In certain embodiments, ring a is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyrrolyl, tetrahydropyrazolidinyl, tetrahydroimidazolidyl, 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, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyrrolyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, optionally substituted with 1-2 substituents; the substituents are selected from halogen, hydroxy, amino, methyl, ethyl, propyl, isopropyl, 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, tetrahydrofuryl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl.
In certain embodiments, ring A is selected from optionally substituted with 1-2 substituents
Figure BDA0003004645000000041
Figure BDA0003004645000000042
The substituents are selected from halogen, hydroxy, amino, methyl, ethyl, propyl, isopropyl, methylcarbonyl, monofluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, aminomethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.
In certain embodiments, R1、R2、R3、R4Each independently selected from H, halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl, halo C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy or halo C1-6An alkylthio group;
R5selected from H, halogen, hydroxy, amino, C1-6Alkyl or halo C1-6An alkyl group.
In certain embodiments, R1、R2、R3Each independently selected from fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, propyl, isopropyl, trifluoromethyl, methoxy, ethoxy or trifluoromethoxy.
At a certain pointIn some embodiments, R4Selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl.
In certain embodiments, R5Selected from H, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl or methoxy.
In certain embodiments, Y2、Y3、Y5Are respectively N, Y4Selected from CH or N.
In certain embodiments, Y2、Y3、Y5Are respectively N, Y4Is CH.
In certain embodiments, Y is S.
In certain embodiments, X1、X2、X3Each independently selected from CH or N.
In certain embodiments, X1、X2Each independently selected from CH or N; x3Is CH.
In certain embodiments, X1Selected from CH or N; x2Is N; x3Is CH.
In certain embodiments, X1、X2、X3Each independently selected from CH or N;
X4selected from the group consisting of CR2R3、NR4O or S;
X5is CR5Or N;
Y2、Y3、Y5are respectively N, Y4Is CH;
y is selected from S or NH;
ring A is selected from cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyrrolyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, optionally substituted with 1-2 substituents; the substituents are selected from fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl, propyl, isopropyl, methylcarbonyl, trifluoromethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio or trifluoromethoxy;
R1、R2、R3each independently selected from fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, propyl, isopropyl, trifluoromethyl, methoxy, ethoxy or trifluoromethoxy;
R4selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl;
R5selected from H, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl or methoxy.
In certain embodiments, X1、X2Each independently selected from CH or N; x3Is CH;
X4selected from the group consisting of CR2R3、NR4O or S;
X5is CR5Or N;
Y2、Y3、Y5are respectively N, Y4Is 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;
R1、R2、R3each independently selected from fluoro, chloro, bromo, iodo, methyl, trifluoromethyl, methoxy or trifluoromethoxy;
R4selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl;
R5selected from H, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl or methoxy.
In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or an isomer thereof, further has a structure as shown in formula (II),
Figure BDA0003004645000000061
wherein R is1、X1、X2、X4、R2、R3、R4、Y、Y3、Y4、Y5And ring a is as described in any of the previous schemes.
In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or an isomer thereof, further has a structure represented by formula (III-1) or formula (III-2),
Figure BDA0003004645000000062
wherein R is1、X1、X2、X4、R2、R3、R4Y and ring a are as described in any of the previous schemes.
The technical solutions of the present invention can be combined with each other to form a new technical solution, and the formed new technical solution is also included in the scope of the present invention.
In certain embodiments, the compound of formula (I), a pharmaceutically acceptable salt thereof, or an isomer thereof, is selected from the group consisting of:
Figure BDA0003004645000000063
Figure BDA0003004645000000071
in another aspect, the invention further provides a pharmaceutical preparation, which contains the compound described in the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), a pharmaceutically acceptable salt thereof or an isomer thereof, and one or more pharmaceutically acceptable excipients, and the pharmaceutical preparation can be any pharmaceutically acceptable dosage form. Pharmaceutically acceptable excipients are substances which are non-toxic, compatible with the active ingredient and otherwise biologically suitable for use in the organism. The choice of a particular excipient will depend on the mode of administration or disease type and state used to treat a particular patient.
In certain embodiments, the pharmaceutical formulations described above may be administered to a patient or subject in need of such treatment by oral, parenteral, rectal, or pulmonary administration, among others. For oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, conventional oral solid preparations such as tablets, capsules, pills, granules and the like; it can also be made into oral liquid, such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, appropriate filler, binder, disintegrating agent, lubricant, etc. can be added. For parenteral administration, the above pharmaceutical preparations may also be prepared into injections, including injections, sterile powders for injection and concentrated solutions for injection. The injection can be prepared by conventional method in the existing pharmaceutical field, and can be prepared without adding additives or adding suitable additives according to the properties of the medicine. For rectal administration, the pharmaceutical composition may be formulated as a suppository or the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalation formulation, aerosol, powder spray, or the like.
In another aspect, the invention also relates to the use of the compound of the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), the pharmaceutically acceptable salt thereof or the isomer thereof in the preparation of medicines for preventing and/or treating diseases such as benign tumors or cancers, wherein the cancers comprise carcinoma in situ and metastatic cancers.
Furthermore, the invention also relates to application of a pharmaceutical preparation containing the compound shown in the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), pharmaceutically acceptable salt thereof or isomer thereof in preparing a medicament for preventing and/or treating diseases such as benign tumors or cancers, wherein the cancers comprise carcinoma in situ and metastatic cancers.
In another aspect, the present invention also relates to the use of the compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), its pharmaceutically acceptable salt or its isomer in the preparation of a medicament for preventing and/or treating diseases such as benign tumor or cancer in combination with radiotherapy and/or one or more anticancer agents, wherein the cancer includes carcinoma in situ and metastatic cancer.
Furthermore, the invention also relates to application of a pharmaceutical preparation containing the compound shown in the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), pharmaceutically acceptable salt thereof or isomer thereof in preparing a medicament for preventing and/or treating diseases such as benign tumor or cancer, wherein the medicament can be combined with radiotherapy and/or one or more anticancer agents, and the cancer comprises in-situ cancer and metastatic cancer.
In another aspect, the present invention also relates to the use of a compound described by the aforementioned general formula (I), general formula (II), general formula (III-1) or general 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.
Further, the invention also relates to application of a pharmaceutical preparation containing the compound shown in the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), and pharmaceutically acceptable salts or isomers thereof in preparing a medicament for sensitizing cancer cells to anticancer agents and/or ionizing radiation.
The ionizing radiation refers to the radiation of various energy rays received by a patient during the radiotherapy process.
In another aspect, the present invention also provides 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 thereof, or an isomer thereof, and one or more second therapeutically active agents selected from the group consisting of anti-cancer agents including mitotic inhibitors, alkylating agents, anti-metabolites, DNA chimerics, anti-tumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal drugs, angiogenesis inhibitors, cell growth inhibitors, targeting antibodies, HMG-CoA reductase inhibitors, and prenyl protein transferase inhibitors.
In certain embodiments, the second therapeutically active agent can be a drug that reduces or reduces one or more side effects of a compound of the invention when used to treat a disease in a subject, or can enhance the efficacy of a compound of the invention.
In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, as described above.
In another aspect, the present invention also relates to the use of a pharmaceutical composition containing the compound of the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt thereof, or an isomer thereof, in the preparation of a medicament for preventing and/or treating diseases such as benign tumor or cancer, including carcinoma in situ and metastatic carcinoma.
In another aspect, the present invention also relates to the use of a pharmaceutical composition containing a compound described in the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt thereof, or an isomer thereof, for the preparation of a medicament for the prevention and/or treatment of diseases such as benign tumors or cancer, which can be used in combination with radiotherapy and/or one or more anticancer agents, including carcinoma in situ and metastatic cancer.
Further, the invention also relates to application of a pharmaceutical composition containing the compound shown in the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), and pharmaceutically acceptable salts or isomers thereof in preparing a medicament for sensitizing cancer cells to anticancer agents and/or ionizing radiation.
In another aspect, the present invention also provides a method for treating a disease associated with DNAPK overactivation, the method comprising administering to a patient in need thereof an effective amount of a compound of formula (I), formula (II), formula (III-1) or formula (III-2), a pharmaceutically acceptable salt thereof or an isomer thereof, a pharmaceutical preparation or a pharmaceutical composition as described above; the disease associated with DNAPK over-activation is selected from benign tumors or cancers, including carcinoma in situ and metastatic carcinoma.
Further, the present invention provides a method for treating a disease associated with DNAPK overactivation, which comprises administering 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 thereof or an isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition to a patient before/after receiving radiotherapy; the disease associated with DNAPK over-activation is selected from benign tumors or cancers, including carcinoma in situ and metastatic carcinoma.
Further, the present invention provides a method for treating a disease associated with DNAPK overactivation, which comprises administering 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 thereof or an isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition to a patient before/after receiving chemotherapy; the disease associated with DNAPK over-activation is selected from benign tumors or cancers, including carcinoma in situ and metastatic carcinoma.
In another aspect, the present invention also provides a method for enhancing the sensitivity of a patient to an anticancer agent or radiation therapy, which comprises administering to a patient in need thereof an effective amount of a compound represented by the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt thereof or an 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 an effective amount of a compound represented by the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt thereof or an isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition to the patient before/after receiving radiation therapy; 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 an effective amount of a compound represented by the aforementioned general formula (I), general formula (II), general formula (III-1) or general formula (III-2), a pharmaceutically acceptable salt thereof or an isomer thereof, the aforementioned pharmaceutical preparation or pharmaceutical composition to the patient before/after receiving chemotherapy; the anticancer agent is as described below.
In another aspect, the present invention also provides a kit comprising:
(a) an effective amount of one or more compounds described by the general formula (I), the general formula (II), the general formula (III-1) or the general formula (III-2), pharmaceutically acceptable salts thereof or isomers thereof,
and (b) an effective amount of one or more anti-cancer agents.
The "anticancer agent" of the present invention refers to an agent having a certain therapeutic effect on tumors, including, but not limited to, mitotic inhibitors, alkylating agents, antimetabolites, DNA chimerics, antitumor antibiotics, growth factor inhibitors, signal transduction inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal drugs, angiogenesis inhibitors, cell growth inhibitors, targeting antibodies, HMG-CoA reductase inhibitors, prenyl protein transferase inhibitors, and the like; the tumor includes benign tumor and cancer. By "effective amount" is meant a dosage of a drug that prevents, alleviates, retards, inhibits or cures a condition in a subject. The size of the administered dose is related to the administration mode of the drug, the pharmacokinetics of the medicament, the severity of the disease, the individual signs (sex, weight, height, age) of the subject, and the like.
In the present invention, unless otherwise defined, scientific and technical terms used herein have meanings commonly understood by those skilled in the art, however, in order to better understand the present invention, definitions of some terms are provided below. To the extent that the definitions and explanations of terms provided herein do not conform to the meanings commonly understood by those skilled in the art, the definitions and explanations of terms provided herein shall control.
The "halogen" as referred to herein means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
"C" according to the invention1-6Alkyl "denotes straight or branched alkyl having 1 to 6 carbon atoms, including for example" C1-4Alkyl group "," C1-3Alkyl group "," C1-2Alkyl group "," C2-6Alkyl group "," C2-5Alkyl group "," C2-4Alkyl group "," C2-3Alkyl group "," C3-6Alkyl group "," C3-5Alkyl group "," C3-4Alkyl "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. "C" according to the invention1-4Alkyl "means C1-6Specific examples of the alkyl group having 1 to 4 carbon atoms.
"C" according to the invention1-6Alkoxy "means" C1-6alkyl-O- ", said" C1-6Alkyl "is as defined above. "C" according to the invention1-4Alkoxy "means" C1-4alkyl-O- ", said" C1-4Alkyl "is as defined above.
"C" according to the invention1-6Alkylthio "means" C1-6alkyl-S- ", said" C1-6Alkyl "is as defined above. "C" according to the invention1-4Alkylthio "means" C1-4alkyl-S- ", said" C1-4Alkyl "is as defined above.
The "hydroxy group C" of the present invention1-6Alkyl, amino C1-6Alkyl, halo C1-6Alkyl "means C1-6One or more hydrogens of the alkyl group are each replaced by one or more hydroxyl groups, amino groups or halogens. C1-6Alkyl is as previously defined
The "hydroxy group C" of the present invention1-6Alkoxy, amino C1-6Alkoxy, halo C1-6Alkoxy "means" C1-6One or more hydrogens of "alkoxy" are replaced with one or more hydroxy, amino, or halogen.
The "hydroxy group C" of the present invention1-6Alkylthio, amino C1-6Alkylthio, halo C1-6Alkylthio "means" C1-6Alkylthio "is one in which one or more hydrogens are replaced with one or more hydroxy, amino, or halogen.
"C" according to the invention1-6Alkylamino radical, di (C)1-6Alkyl) amino "means independently C1-6alkyl-NH-),
Figure BDA0003004645000000111
"C" according to the invention1-6Alkylcarbonyl "means C1-6alkyl-C (O) -.
The "3-to 8-membered cycloalkyl" as referred to herein means a saturated or partially saturated monocyclic cyclic group having 3 to 8 ring atoms and having no aromaticity, and the "3-to 8-membered cycloalkyl" as referred to herein includes "3-to 8-membered saturated cycloalkyl" and "3-to 8-membered partially saturated cycloalkyl", and is exemplified by "3-to 6-membered cycloalkyl", "3-to 6-membered saturated cycloalkyl", "5-to 6-membered saturated cycloalkyl", and the like. Examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cyclohexenyl and the like.
The "3-to 8-membered heterocyclic group" as used herein means a saturated or partially saturated and non-aromatic monocyclic cyclic group containing at least one (e.g., 1,2, 3, 4 or 5) hetero atom which is a nitrogen atom, an oxygen atom and/or a sulfur atom and has 3 to 8 ring atoms, and optionally, a ring atom (e.g., a carbon atom, a nitrogen atom or a sulfur atom) in the cyclic structure may be oxo. The "3-to 8-membered heterocyclic group" described in the present invention includes "3-to 8-membered saturated heterocyclic group" and "3-to 8-membered partially saturated heterocyclic group". The "3-to 8-membered heterocyclic group" is exemplified by "3-to 6-membered heterocyclic group", "3-to 6-membered saturated heterocyclic group", "3-to 5-membered saturated heterocyclic group", "5-to 6-membered saturated heterocyclic group", etc. Specific examples thereof include, but are not limited to: aziridinyl, 2H-aziridinyl, diazacyclopropenyl, 3H-diazacyclopropenyl, 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, dihydropiperidonyl, piperazinyl, azetidinyl, piperazinyl, Hexahydropyrimidyl, morpholinyl, and the like.
The term "optionally substituted with a substituent" as used herein refers to both the case where one or more hydrogen atoms on a substituted group are "substituted" or "unsubstituted" with one or more substituents.
The chemotherapy is the abbreviation of chemical drug therapy, and achieves the purpose of treatment mainly by using chemical therapeutic drugs to kill cancer cells.
The "radiotherapy" in the invention refers to a tumor treatment method, i.e. tumor radiotherapy, which mainly uses radioactive rays to perform local tumor treatment, wherein the "radioactive rays" include alpha, beta and gamma rays generated by radioactive isotopes, and x rays, electron beams, proton beams and other particle beams generated by various x-ray treatment machines or accelerators.
"pharmaceutically acceptable salt" as used herein refers to an acidic functional group (e.g., -COOH, -OH, -SO) present in a compound3H, etc.) with a suitable inorganic or organic cation (base), including salts with alkali or alkaline earth metals, ammonium salts, salts with nitrogen-containing organic bases; and salts of basic functional groups present in the compounds (e.g., -NH2, etc.) 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 thus are available 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 an olefinic double bond, include cis-isomers and trans-isomers, unless otherwise specified. The compounds of the invention may exist in tautomeric (one of the functional group isomers) forms having different points of attachment of hydrogen through one or more double bond shifts, e.g., a ketone and its enol form are keto-enol tautomers. The compounds of the present invention contain a spiro ring structure, and substituents on the ring may be present on both sides of the ring to form the opposite cis (cis) and trans (trans) isomers, depending on the steric structure of the ring. Each tautomer and mixtures thereof are included within the scope of the present invention. All enantiomers, diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof and the like of the compounds are included within the scope of the present invention.
The compounds of the invention may be prepared by enantiospecific synthesis or by resolution from a mixture of enantiomers in such a way as to give the individual enantiomers. Conventional resolution techniques include resolving mixtures of enantiomers of the starting material or the final product using various well-known chromatographic methods.
When the stereochemistry of the disclosed compounds is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight relative to the other stereoisomers. When a single isomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. The 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 inhibitory effect, has good pharmacokinetic property in organisms, has 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 and the pharmaceutically acceptable salt or the isomer thereof have high enzymological selectivity, have better treatment effect on benign tumors and cancers, and have high stability of liver microsomes.
3. The compound of the invention has simple preparation process, high medicine purity, stable quality and easy large-scale industrial production.
Detailed description of the preferred embodiments
The technical solutions of the present invention will be described below in conjunction with the specific embodiments, and the above-mentioned contents of the present invention will be further described in detail, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Abbreviations:
DMFDMA: n, N-dimethylformamide dimethyl acetal; DPPA: diphenyl phosphorazidate; lawson's reagent: 2, 4-bis (p-methoxyphenyl) -1, 3-dithia-2, 4-diphosphetane-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) carboxamidine
Figure BDA0003004645000000141
4-methyl-5-nitropyridin-2-amine (20g,130.6mmol), toluene (400mL), N-dimethylformamide dimethyl acetal (47g,394.3mmol) was added, heated to 110 ℃ for 3 hours, spun dry, washed with N-heptane (300mL), filtered, and the solid was dried to give the desired product (26g, yield: 95.6%).
2. Preparation of (E) -N-hydroxy-N' - (4-methyl-5-nitropyridin-2-yl) carboxamidine
Figure BDA0003004645000000142
(E) -N, N-dimethyl-N' - (4-methyl-5-nitropyridin-2-yl) formamidine (16.0g,76.8mmol), methanol (200mL), hydroxylamine hydrochloride (10.7g,153.9mmol) added thereto, heating to 65 deg.C, 3 hours, cooling, addition of water (300mL), ethyl acetate (300mL), separation, drying of the organic phase over anhydrous sodium sulfate, filtration, spin-drying, washing of the solid with methyl tert-butyl ether (100mL), and drying of the solid to give the desired product (9.2g, yield: 61.3%).
3. Preparation of 7-methyl-6-nitro- [1,2,4] triazolo [1,5-a ] pyridine
Figure BDA0003004645000000143
(E) -N-hydroxy-N' - (4-methyl-5-nitropyridin-2-yl) formamidine (10.8g,55.0mmol), tetrahydrofuran (110mL), trifluoroacetic anhydride (13.9g,66.2mmol) added, reaction at 10 ℃ for 12 hours, LC-MS check reaction complete, solvent spin dried, mixed solvent (100mL, ethyl acetate: petroleum ether ═ 1:4) added to the solid, filtered, and solid dried in vacuo to give the desired product (5.6g, yield: 57.1%).
4. Preparation of 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine
Figure BDA0003004645000000144
7-methyl-6-nitro- [1,2,4] triazolo [1,5-a ] pyridine (4.4g,24.7mmol) was dissolved in ethanol (50mL), palladium on carbon (528mg), ammonium formate (7.8g,124.0mmol) were added, the mixture was heated to 70 ℃ for 4 hours to react, and the reaction solution was filtered with suction, and the filtrate was dried by spin-drying to obtain the product (3.6g, yield: 98.3%).
Preparation example two: 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
Figure BDA0003004645000000151
Ethyl 2, 4-dichloropyrimidine-5-carboxylate (40.0g,181mmol) was dissolved in acetonitrile (1000mL), and tetrahydro-2H-pyran-4-amine hydrochloride (24.9g,181mmol), potassium carbonate (62.5g,452mmol) were added, reacted at 20 ℃ for 16 hours, LC-MS detected that the reaction was complete, suction filtered, and the filtrate was concentrated to give the objective product (45g, yield: 87.0%).
2. Preparation of 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylic acid
Figure BDA0003004645000000152
Ethyl 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylate (15.0g,52.4mmol) was dissolved in a system of tetrahydrofuran (150.0mL) and water (150.0mL), lithium hydroxide (4.8g,114.3mmol) was added, reaction was carried out at 25 ℃ for 1 hour, concentration was carried out, a white solid was precipitated by adjusting pH of the aqueous phase to 3, filtration was carried out, and the solid was dried to obtain the objective product (11.5g, yield: 85.2%).
3. Preparation of 2-chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one
Figure BDA0003004645000000153
Dissolving 2-chloro-4- ((tetrahydro-2H-pyran-4-yl) amino) pyrimidine-5-carboxylic acid (10.0g,38.8mmol) in N, N-dimethylacetamide (60.0mL), adding triethylamine (4.0g,39.6mmol) and diphenylphosphorylazide (11.0g,40.0mmol), reacting the system at 25 ℃ for 1 hour, raising the temperature to 110 ℃ for further reaction for 16 hours, cooling to 25 ℃, pouring into ice water, precipitating a light yellow solid, filtering and spin-drying to obtain a crude product (7.1g, yield: 71.7%).
Preparation example three: preparation of 2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one
Figure BDA0003004645000000161
2-chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (7.4g,29.0mmol) was dissolved in N, N-dimethylformamide (100.0mL), the temperature of the system was reduced to 0 deg.C, 60% NaH (1.5g,37.5mmol) was added to react for 30 minutes, and CH was added3I (5.0g,35.2mmol), after 2 hours at 0 ℃ and addition of water (150.0mL) and ethyl acetate (220.0mL), the organic phase is concentrated and spin dried to give the crude product (6.9g, yield: 88.5%).
The first embodiment is as follows: 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
Figure BDA0003004645000000162
2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (3.3g,12.3mmol) was dissolved in dioxane (50.0mL), and a system of 7-methyl- [1,2,4] triazolo [1,5-a ] pyridin-6-amine (1.8g,12.2mmol), 2- (dicyclohexylphosphine) 3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl (760.0mg,0.784mmol) and cesium carbonate (7.8g,23.9mmol) was added, reacted at 100 ℃ under a nitrogen atmosphere for 2.0 hours, concentrated, and washed with methanol (50.0 mL). Column chromatography of the solid (DCM: MeOH: 20:1) afforded the product (3.4g, yield: 73.9%).
The molecular formula is as follows: c18H20N8O2Molecular weight: 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
Figure BDA0003004645000000171
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 (250.0mg, 0.656mmol), lawson's reagent (1400.0mg, 3.456mmol) was dissolved in dioxane (10.0mL), the system was microwave-reacted at 155 ℃ for 4.0 hours, and after concentration, column chromatography was performed on the residue (dichloromethane: methanol ═ 20:1) to obtain a product (35.0mg, yield 13.5%).
Molecular formula C18H20N8OS 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).
Example 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
Figure BDA0003004645000000172
2-chloro-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-one (6.7g,26.3mmol) was dissolved in POCl3(60.0mL), DIEA (5.9g,45.8mmol) was added to the system, the temperature was raised to 110 ℃ to react for 11.0 hours, the mixture was concentrated and dried, and saturated NaHCO was used3The aqueous solution was adjusted to pH 7, and after concentration the crude product was purified by column chromatography (DCM: MeOH ═ 10:1) to give the product (3.82g, yield: 53.2%).
2. Preparation of 2-chloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purin-8-amine
Figure BDA0003004645000000173
2, 8-dichloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purine (3.2g,11.7mmol) was dissolved in an amine methanol solution (7M, 50.0mL), reacted at 100 ℃ for 3.0 hours, concentrated, and purified by column chromatography (MeOH: DCM ═ 1:10) to give the product (1.5g, yield: 50.5%).
3. Preparation of 2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-imine
Figure BDA0003004645000000181
2-chloro-9- (tetrahydro-2H-pyran-4-yl) -9H-purin-8-amine (530mg,2.1mmol) was dissolved in ethanol (15.0mL) and CH was added3The system I (2.0g,14.1mmol) was microwave reacted at 130 ℃ for 2.0 h, concentrated and purified by column chromatography (MeOH: DCM ═ 1:10) to give the crude product (120.0mg, yield: 21.3%).
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
Figure BDA0003004645000000182
2-chloro-7-methyl-9- (tetrahydro-2H-pyran-4-yl) -7, 9-dihydro-8H-purin-8-imine (89.9mg,0.34mmol), 7-methyl- [1,2,4]Triazolo [1,5-a]Pyridin-6-amine (50.0mg,0.34mmol), cesium carbonate (220.0mg,0.67mmol) and Brettphos Pd G3(25.0mg,0.028mmol) were dissolved in dioxane (5.0mL) and the system was then heated in N2After 3.0 h at 100 ℃ under ambient conditions, it is concentrated and purified by column chromatography (MeOH: DCM ═ 1:10) to give the product (7.5mg, yield: 5.8%).
The molecular formula is as follows: c18H21N9O molecular weight: 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).
The compounds shown in the following table were prepared using the same or similar methods as the examples above:
Figure BDA0003004645000000191
experimental protocol
An exemplary experimental scheme of a portion of the compounds of the invention is provided below to show the advantageous activity and advantageous technical effects of the compounds of the invention. It should be understood, however, that the following experimental protocols are only 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 Compounds of the invention
Abbreviations
EDTA: ethylenediaminetetraacetic acid
DMSO, DMSO: dimethyl sulfoxide
Tris (Tris): tris (hydroxymethyl) aminomethane
Brij-35: polyoxyethylene lauryl ether
DTT: dithiothreitol
And (3) testing the sample: the structural formula and the preparation method of the compound are shown in the examples.
Experimental reagent:
name (R) Brand
ADP-Glo Kinase Assay Promege
DNA-PK Promege
The experimental method comprises the following steps:
1. 1-fold kinase buffer solution is prepared
1) 1-fold kinase buffer
40mM Tris,pH 7.5
0.0055％Brij-35
20mM MgCl2
0.05mM DTT
2. Compound preparation
1) The initial concentration of the compound to be tested was 1. mu.M, and the compound was prepared at 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 solution of the compound. 100 μ l of 100-fold compound was added to the second well of the 96-well plate, and 60 μ l of 100% DMSO was added to the other wells. Mu.l of compound from the second well was added to the third well and diluted sequentially 3-fold further down for a total of 10 concentrations.
2) Transfer the highest concentration (400nM) of 100. mu.l of 100% DMSO and the positive control Wortmannin to two empty wells as Max and Min wells, respectively.
3) Echo was used to transfer 50nl of compound to 384-well plates.
3. Preparation of 2 Xkinase solution
1) A2-fold DNA-PK kinase solution was prepared using a 1-fold kinase buffer.
2) Transfer 2.5. mu.l of 2-fold enzyme solution to 384-well reaction wells.
3) Shaking, mixing, and standing at room temperature.
4. Preparation of 2 Xsubstrate solution
1) A2-fold substrate solution was prepared using 1-fold kinase buffer.
2) Transfer 2.5. mu.l of 2-fold substrate solution to 384-well reaction wells to initiate the reaction.
3) Oscillating and mixing.
5. Kinase reaction and termination
1) The 384 well plates were capped and incubated at 28 ℃ for 3 hours.
2) Transfer 5. mu.l ADP-Glo reagent and incubate at 28 ℃ for 2 hours.
6. Detection of reaction results
1) The reaction was stopped by transferring 10. mu.l of the kinase detection reagent to reaction wells of a 384-well plate.
2) Rest for 30 minutes at room temperature.
7. Data reading
Sample values were read at Envision.
8. Inhibition rate calculation
And converting the data into inhibition rate data and then performing curve fitting.
Percent inhibition is (max-conversion)/(max-min) 100. where max refers to the conversion rate of the DMSO control, min refers to the conversion rate of the no enzyme control, and conversion refers to the conversion rate at each concentration of test compound.
The experimental results are as follows:
TABLE 1 in vitro enzymatic Activity data for Compounds of the invention
Figure BDA0003004645000000211
And (4) experimental conclusion:
the result shows that the compound has better inhibition effect on the activity of DNA-PK kinase.
Experimental example 2 Metabolic stability of liver microsomes in various species of the Compound of the present invention
And (3) testing the sample: the chemical name and the preparation method of the compound 1 are shown in the preparation examples of the compound.
Experimental materials:
cynomolgus monkey mixed liver microsomes were purchased from the research center for liver disease of reed (shanghai ltd) under the batch number: ZXBZ, liver microsomal protein concentration 20 mg/mL-1。
Mixed liver microsomes from SD rats and CD-1 mice were purchased from Xeno Tech, under respective batch numbers: 1610290(SD rat), 1710069(CD-1 mouse). The concentration of the liver microsome protein is 20 mg/mL-1。
Human mixed liver microsomes were purchased from corning corporation under the cat # 452117, lot # 38294, and a liver microsomes protein concentration of 20 mg/mL-1。
The experimental initiation factor beta-NADPH is purchased from Solarbio company; phosphate Buffered Saline (PBS) pH 7.4 was self-prepared by the laboratory.
Preparing a test solution:
a proper amount of test powder is precisely weighed, a proper amount of dimethyl sulfoxide (DMSO) is added to dissolve the test powder to 1mM, and the test powder is diluted by 20 times to 50 mu M of working solution by using methanol.
The experimental method comprises the following steps:
TABLE 2 liver microsome metabolic stability experiment incubation system composition
Figure BDA0003004645000000221
The experimental operation steps are as follows:
(1) according to the above ratio of "constitution of Experimental incubation System" in Table 2, 5.85mL of 100mM PBS and 20mM MgCl were used for each compound2Solution 0.585mL and H2O3.57 mL, and a mixed solution 1 (not containing microsomes, a sample and. beta. -NADPH) for incubation was prepared. The positive pair drug verapamil of the experimental incubation system was also performed to demonstrate normal liver microsomal enzyme activity.
(2) Liver microsomes (20mg protein/mL) were removed from the-80 ℃ freezer and placed on a 37 ℃ water bath constant temperature shaker for pre-incubation for 3 min.
(3) For each compound, 1.9mL of mixed solution 1 of incubation system was taken for each species, and 56. mu.L of microsomes of different species was added to prepare mixed solution 2 of incubation system (containing no test substance and. beta. -NADPH).
(4) Sample set (microsome and β -NADPH containing): and adding 14 mu L of the test sample working solution with the concentration of 50 mu M into 616 mu L of the mixed solution 2 of the incubation system, and adding 70 mu L of 10mM beta-NADPH working solution. Mixing, and repeating the steps. The sampling time points are 0min, 5min, 10min, 20min, 30min and 60 min. This sample set was used to evaluate the metabolic stability of compounds mediated via β -NADPH.
(5) Control group (microsome-containing, no β -NADPH, water instead of β -NADPH): 264 mu L of the mixed solution 2 of the incubation system is taken, 6 mu L of the working solution of the test article with the concentration of 50 mu M is added, and 30 mu L of water is added. Mixing, and repeating the steps. Sampling time points were 0min and 60 min. This negative control group was used to evaluate whether compounds present non- β -NADPH mediated metabolism in the liver microsome incubation system.
(6) At each predetermined time point, 50 μ L of sample was taken from the incubation sample tube, added to a stop sample tube (containing 300 μ L of cold stop reagent, containing 50ng/mL acetonitrile as internal standard of tolbutamide), vortexed, and the reaction was stopped.
(7) After vortexing for 10min, centrifuge for 5min (12000 rpm).
(8) Taking 100 mu L of supernatant, adding 100 mu L of water, mixing uniformly by vortex, and carrying out LC-MS/MS sample injection analysis.
And (3) data analysis:
the percent residual was converted by the ratio of the peak area of the test article to the internal standard in the following equation.
Figure BDA0003004645000000231
The experimental results are as follows:
TABLE 3 hepatic microsome stability results for the compounds of the invention
Figure BDA0003004645000000232
And (4) experimental conclusion:
the compound of the invention has good stability in liver microsomes of human, monkey, rat and mouse.
Experimental example 3 CD1 mouse pharmacokinetics experiment of the Compound of the present invention
Abbreviations
HPC: hydroxypropyl cellulose
DMA: dimethylacetamide test sample: the chemical name and the preparation method of the compound 1 are shown in the preparation examples of the compound. The test animals were: CD1 mice, female, purchased from Beijing Wittiulihua laboratory animals technologies, Inc., weighing 24-29g, for a total of 12 mice.
Preparing a test solution:
the preparation method of the blank solvent (1) comprises the following steps: weighing 28g of HP-beta-CD, adding a proper amount of water for injection to dissolve, then fixing the volume to 100mL by using the water for injection, and uniformly mixing by vortex to obtain 28% HP-beta-CD.
The preparation method of the blank solvent (2) comprises the following steps: weighing 20g of HPC, slowly adding 500mL of stirred purified water, then adding 1mL of Tween 80, stirring until the mixture is clear and transparent, diluting to 1000mL, and uniformly stirring to obtain 2% of HPC + 0.1% of Tween 80.
IV (bolus IV) administration:
taking the compound 1(2.57mg), adding DMA (491 muL), shaking for dissolving, then adding PEG400(982 muL), vortex and mixing uniformly, finally adding a blank solvent (1) (3.44mL), vortex and mixing uniformly, and keeping the temperature at 50 ℃ for 20min to obtain a clear solution of 0.5mg/mL, which is used as an IV administration solution of the compound 1.
PO (intragastric) administration:
weighing the compound 1(3.83mg), placing the compound in a tissue grinder, adding 3.66mL of blank solvent (2), and grinding uniformly at 1200 r/min to obtain suspension liquid medicine with the concentration of 1mg/mL, wherein the suspension liquid medicine is used as PO administration liquid medicine of the compound 1.
Experimental methods
The IV dose was 2.5mg/kg, the concentration was 0.5mg/mL, and the volume was 5 mL/kg.
PO was administered at a dose of 10mg/kg, at a concentration of 1mg/mL, and at a volume of 10 mL/kg.
Blood sampling time points: blood was collected at 0.083, 0.25, 0.5, 1,2,4, 6, 8, 24h after administration, in particular in the manner shown in the following table.
Figure BDA0003004645000000241
Approximately 50. mu.L of whole blood was collected at each time point by the canthus and placed in the eye containing EDTA-K2In the anticoagulation tube of anticoagulantPlasma samples were obtained by centrifugation at 8000 rpm for 6 minutes at 4 ℃ and were frozen at-80 ℃ in a freezer for analysis.
Plasma sample analysis
Adopting a protein precipitation method: and taking 20 mu L of a plasma sample, adding 200 mu L of an internal standard (acetonitrile solution containing 50ng/mL of tolbutamide), vortexing for 10min, then centrifuging for 20min at 4000 rpm, taking 100 mu L of supernatant, adding 100 mu L of water, vortexing and uniformly mixing for 3min, and then carrying out LC-MS/MS analysis.
Results of the experiment
TABLE 4 evaluation results of CD1 mouse PK
Figure BDA0003004645000000251
AUC0-tArea under curve 0 → t when drug is represented; CL represents clearance; vssRepresenting the steady state apparent distribution volume; t is1/2Represents a terminal elimination half-life; t ismaxRepresents the time to peak; cmax(ii) surrogate expression peak concentration; f% represents the absolute bioavailability.
Conclusion of the experiment
As can be seen from the data in table 4, the compound of the present invention has good pharmacokinetic properties and high exposure and bioavailability.

Claims (15)

1. A compound represented by the general formula (I), a pharmaceutically acceptable salt thereof or an isomer thereof,
Figure FDA0003004644990000011
wherein the content of the first and second substances,
X1、X2、X3each independently selected from CH or N;
X4selected from the group consisting of CR2R3、NR4O or S;
X5selected from the group consisting of CR5Or N;
Y2、Y3、Y4、Y5each independently selected from CH or N;
y is selected from S, NH or CH2；
Ring A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocyclyl optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, nitro, cyano, C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio radical, C1-6Alkylcarbonyl, halo C1-6Alkoxy, halo C1-6Alkylthio, hydroxy C1-6Alkoxy, hydroxy C1-6Alkylthio, amino C1-6Alkoxy, amino C1-6An alkylthio group;
R1、R2、R3、R4、R5each independently selected from H, halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy, halo C1-6Alkylthio, hydroxy C1-6Alkoxy, hydroxy C1-6Alkylthio, amino C1-6Alkoxy, amino C1-6An alkylthio group.
2. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to claim 1,
ring A is selected from 3-6 membered cycloalkyl or 3-6 membered heterocyclyl optionally substituted with 1-2 substituents; the substituent is selected from halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl radical, C1-6Alkylamino radical, di (C)1-6Alkyl) amino, C1-6Alkylcarbonyl, halo C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy or halo C1-6An alkylthio group;
preferably, ring A is selected from 3-6 membered saturated cycloalkyl or 3-6 membered saturated heterocyclyl optionally substituted with 1-2 substituents.
3. The compound, a pharmaceutically acceptable salt thereof, or an isomer thereof according to any one of claims 1 to 2,
ring A is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyrrolyl, tetrahydropyrazolidinyl, tetrahydroimidazolidyl, 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.
4. The compound, a pharmaceutically acceptable salt thereof, or an isomer thereof according to any one of claims 1 to 3,
R1、R2、R3、R4each independently selected from H, halogen, hydroxyl, amino, nitro, cyano and C1-6Alkyl, halo C1-6Alkyl radical, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy or halo C1-6An alkylthio group;
R5selected from H, halogen, hydroxy, amino, C1-6Alkyl or halo C1-6An alkyl group.
5. The compound, a pharmaceutically acceptable salt thereof, or an isomer thereof according to any one of claims 1 to 4,
Y2、Y3、Y5are respectively N, Y4Selected from CH or N。
6. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to any one of claims 1-5,
X1、X2、X3each independently selected from CH or N;
X4selected from the group consisting of CR2R3、NR4O or S;
X5is CR5Or N;
Y2、Y3、Y5are respectively N, Y4Is CH;
y is selected from S or NH;
ring A is selected from cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyrrolyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, optionally substituted with 1-2 substituents; the substituents are selected from fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl, propyl, isopropyl, methylcarbonyl, trifluoromethyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio or trifluoromethoxy;
R1、R2、R3each independently selected from fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, propyl, isopropyl, trifluoromethyl, methoxy, ethoxy or trifluoromethoxy;
R4selected from H, methyl, ethyl, propyl, isopropyl or trifluoromethyl;
R5selected from H, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl or methoxy.
7. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to any of claims 1-6, further having a structure represented by the following general formula (II),
Figure FDA0003004644990000021
wherein R is1、X1、X2、X4、R2、R3、R4、Y、Y3、Y4、Y5And ring A is as defined in any one of claims 1 to 6.
8. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to any one of claims 1 to 7, which further has a structure represented by the following general formula (III-1) or general formula (III-2),
Figure FDA0003004644990000031
wherein R is1、X1、X2、X4、R2、R3、R4Y and Ring A are as defined in any one of claims 1 to 7.
9. The compound of claim 1, a pharmaceutically acceptable salt thereof, or an isomer thereof, selected from the group consisting of:
Figure FDA0003004644990000032
10. a pharmaceutical formulation comprising a compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt thereof or an isomer thereof, wherein the pharmaceutical formulation comprises one or more pharmaceutically acceptable excipients, and wherein the pharmaceutical formulation is in any pharmaceutically acceptable dosage form.
11. A pharmaceutical composition comprising a compound of any one of claims 1-9, a pharmaceutically acceptable salt thereof, or an isomer thereof, comprising one or more second therapeutically active agents selected from the group consisting of anti-cancer agents, including mitotic inhibitors, alkylating agents, anti-metabolites, DNA chimerics, anti-tumor 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.
12. Use of a compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical formulation according to claim 10, or a pharmaceutical composition according to claim 11 for the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancers, including carcinoma in situ and metastatic cancers.
13. Use of a compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical formulation according to claim 10, or a pharmaceutical composition according to claim 11, in the manufacture of a medicament for the prevention and/or treatment of benign tumours or cancers, including in situ and metastatic cancers, in combination with radiotherapy and/or one or more anti-cancer agents.
14. Use of a compound of any one of claims 1-9, a pharmaceutically acceptable salt or isomer thereof, a pharmaceutical formulation of claim 10, or a pharmaceutical composition of claim 11, in the manufacture of a medicament for sensitizing cancer cells to an anti-cancer agent and/or radiation therapy.
15. A kit, comprising:
(a) an effective amount of one or more compounds of any one of claims 1-9, a pharmaceutically acceptable salt thereof, or an isomer thereof,
and (b) an effective amount of one or more anti-cancer agents.
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