7,9-dihydropurine derivatives and pharmaceutical uses thereof

Abstract

The application discloses a 7,9-dihydropurine derivative and a pharmaceutical application thereof. Specifically provided are a compound shown in formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. Experiments show that the compound provided by the application has good inhibitory activity on DNA-PK, and especially, the inhibitory activity of compounds CLJ1, 4, 8, 15, 22-25, 56 and 59 is even superior to that of a known DNA-PK inhibitor AZD-7648. The compound provided by the application has a wide application prospect in preparation of a DNA-PK inhibitor. The application provides a new selection for an anti-tumor drug sensitizer, a radiotherapy sensitizer and a tumor treatment drug, and also provides a new selection for a tumor treatment method.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to a 7,9-dihydropurine derivative and its pharmaceutical uses. Background Technology

[0002] Cancer is one of the deadliest diseases affecting human health and lifespan, and researching effective ways to combat it is a constant goal for scientists. Currently, there are numerous methods and drugs for treating cancer, with relatively effective methods including surgery, radiotherapy, and drug therapy. Treatment methods for cancer include directly killing cancer cells and regulating the body's immune system. The specific mechanisms by which cancer cells are directly killed include directly damaging DNA, inhibiting DNA synthesis, and inhibiting protein synthesis. Radiotherapy and many anticancer drugs achieve their therapeutic goals by directly damaging DNA. This DNA damage triggers the body to repair the damaged DNA, which in turn increases the survival rate of cancer cells, making them more resistant. Therefore, if DNA damage repair can be inhibited, the sensitivity of cancer cells can be increased.

[0003] Among DNA damage types, DNA double-strand breaks (DSBs) are the most severe, being a major cause of gene mutations and chromosome breakage, and significantly impacting tumorigenesis and development. DSB repair primarily occurs through DNA-dependent protein kinase (DNA-PK)-dominated nonhomologous end joining (NHEJ). Therefore, inhibiting the function and activity of DNA-PK is an effective way to suppress DSB repair and enhance the sensitivity of tumor cells to radiation and antitumor drugs.

[0004] DNA-PK is a complex composed of the catalytic subunit DNA-PKcs and the Ku70 / 80 heterodimer. Ku70 and Ku80 (also known as Ku86) are encoded by the human XRCC6 and XRCC5 genes, respectively, and have a strong affinity for the free ends of DNA. This heterodimer can recognize DSBs and recruit the kinase subunit DNA-PKcs. Currently, the role of DNA-PK inhibitors in radiosensitization and chemotherapy sensitization has been confirmed. For example, Boeckman et al. found that the mechanism by which cisplatin promotes cellular radiosensitization is mainly through preventing DNA-PKcs from phosphorylating the target protein, thus inhibiting the NHEJ repair of DSBs. Therefore, DNA-PK inhibitors, as radiosensitizers and antitumor drug sensitizers, have important clinical value in improving the efficacy of tumor treatment.

[0005] Furthermore, research has found that DNA-PK inhibitors can directly inhibit the proliferation of tumor cells and also have anti-tumor effects when used alone. Studies have already demonstrated that inhibiting Ku70 or DNA-PKcs expression can delay the growth of cervical cancer cells.

[0006] Currently reported DNA-PK inhibitors include AZD-7648, KU-57788, NU-7441, NU-7026, etc. Among them, AZD-7648 (CAS:2230820-11-6) is a potent and highly selective DNA-PK inhibitor developed by AstraZeneca. Studies have shown that AZD-7648 can enhance DNA damage induced by radiotherapy and doxorubicin chemotherapy. Furthermore, the team also demonstrated that combining AZD-7648 with the PARP inhibitor olaparib increases genomic instability in ATM-deficient cells, thereby inhibiting cell growth and promoting apoptosis. The study also found that AZD-7648 can enhance the efficacy of olaparib in xenograft PDX tumor models, sustainably inhibiting tumor growth.

[0007]

[0008] However, there is still room for improvement in the inhibitory activity of AZD-7648 against DNA-PK. It also exhibits significant inhibitory activity against PI3K family subtypes. Furthermore, its oral exposure is relatively low, and its half-life and clearance rate require further improvement. Therefore, developing compounds with superior inhibitory activity against DNA-PK and more balanced in vitro and in vivo properties has significant clinical application value and social benefits. Summary of the Invention

[0009] The purpose of this invention is to provide a 7,9-dihydropurine derivative and its use in the preparation of DNA-PK inhibitors.

[0010] This invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0011]

[0012] Where R1 is LR5, and L is C 1～4 Alkylene, NH, or none; R5 is selected from substituted or unsubstituted 3- to 6-membered saturated heterocyclic groups, substituted or unsubstituted 3- to 6-membered cycloalkyl groups, substituted or unsubstituted bridged cycloalkyl groups, substituted or unsubstituted C-membered groups. 1～6 Alkyl group, wherein the substituent is selected from C10. 1～6 Alkyl, SO2R6, COR6, halogen, hydroxyl; R6 is selected from halogenated or unsubstituted C. 1～6 Alkyl, halogenated or unsubstituted 3- to 6-membered saturated cycloalkyl groups;

[0013] R2 is selected from C 1～6 Alkyl groups, 3-6 membered saturated heterocyclic groups, 3-6 membered saturated cycloalkyl groups;

[0014] X is NH or none;

[0015] Ring A is

[0016] Among them, M1, M2, M3, M4, and M5 are each independently selected from CH or N;

[0017] m is an integer from 1 to 5, and R3 is independently selected from substituted or unsubstituted C. 1～5 Alkyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted 5-6 membered aryl; each substituent is independently selected from halogenated or unsubstituted C. 1～5 Alkyl, C 1～5 alkoxy, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, SO2R7, COR7, or two substituents linked to form a ring; R7 is selected from hydrogen or C. 1～5 alkyl;

[0018] n is an integer from 1 to 4;

[0019] R4 is independently selected from hydrogen, halogenated or unsubstituted C4. 1～6 Alkyl, halogenated or unsubstituted C 1～6 Alkyl, cyano, nitro, halogen, COOR8, COR8, SO2R8, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted 5-6 membered aryl; each substituent is independently selected from halogenated or unsubstituted C 1～5 Alkyl group, R8 is C 1～6 alkyl;

[0020] Y is selected from N or CH;

[0021] h is an integer from 0 to 2; R y Selected from C 1～6 alkyl.

[0022] Furthermore,

[0023] R1 is LR5, L is C 1～4 Alkylene, NH, or none; R5 is selected from substituted or unsubstituted 3- to 6-membered saturated heterocyclic groups, substituted or unsubstituted 3- to 6-membered cycloalkyl groups, and substituted or unsubstituted bridged cycloalkyl groups, wherein the substituent is selected from C... 1～6 Alkyl, SO2R6, COR6; R6 is selected from halogenated or unsubstituted C 1～6 Alkyl, halogenated or unsubstituted 3- to 6-membered saturated cycloalkyl groups;

[0024] R2 is selected from C1～6 alkyl;

[0025] X is NH or none;

[0026] Ring A is

[0027] Among them, M1, M2, M3, M4, and M5 are each independently selected from CH or N;

[0028] m is an integer from 1 to 5, and R3 is independently selected from substituted or unsubstituted C. 1～5 Alkyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted 5-6 membered aryl; each substituent is independently selected from halogenated or unsubstituted C. 1～5 Alkyl, C 1～5 alkoxy, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, SO2R7, aldehyde group, or two substituents linked to form a ring; R7 is selected from C 1～5 alkyl;

[0029] n is an integer from 1 to 4;

[0030] R4 is independently selected from hydrogen, halogenated or unsubstituted C4. 1～6 Alkyl, halogenated or unsubstituted C 1～6 Alkyl, cyano, nitro, halogen, COOR8, COR8, SO2R8, substituted or unsubstituted 5- to 6-membered heteroaryl groups, wherein each substituent is independently selected from C10. 1～5 Alkyl group, R8 is C 1～6 alkyl.

[0031] Furthermore,

[0032] The structure of the compound is shown in Formula II:

[0033]

[0034] Where R1 is LR5, and L is C 1～3 Alkylene, NH, or none; R5 is selected from substituted or unsubstituted 3- to 6-membered saturated heterocyclic groups, substituted or unsubstituted 3- to 6-membered cycloalkyl groups, and substituted or unsubstituted bridged cycloalkyl groups, wherein the substituent is selected from C... 1～5 Alkyl, SO2R6, COR6; R6 is selected from halogenated or unsubstituted C 1～5 Alkyl, halogenated or unsubstituted 3- to 6-membered saturated cycloalkyl groups;

[0035] R2 is selected from C 1～3 alkyl;

[0036] M1, M2, M3, M4, and M5 are each independently selected from CH or N;

[0037] m is 2, and one of R3 is C. 1～3 The alkyl group, and the other R3, are selected from substituted or unsubstituted 5- to 6-membered heteroaryl groups, substituted or unsubstituted 5- to 6-membered aryl groups; each of the substituents is independently selected from halogenated or unsubstituted C4 groups. 1～5 Alkyl, C 1～5 alkoxy, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, SO2R7, aldehyde group, or two substituents linked to form a ring; R7 is selected from C 1～5 alkyl.

[0038] Furthermore,

[0039] The structure of the compound is shown in Formula III-a:

[0040]

[0041] Alternatively, the structure of the compound may be as shown in formula III-b1 or formula III-b2:

[0042]

[0043] Alternatively, the structure of the compound may be as shown in formula III-c1, III-c2, or III-c3:

[0044]

[0045] Alternatively, the structure of the compound is as shown in Formula III-d:

[0046]

[0047] Alternatively, the structure of the compound may be as shown in formula III-e1 or formula III-e2:

[0048]

[0049] Alternatively, the structure of the compound is as shown in Formula III-f:

[0050]

[0051] Wherein, R1 is selected from the following groups, whether substituted or unsubstituted: Where L is methylene, NH, or none; the substituent is selected from C. 1～3 Alkyl, SO2R6, COR6; R6 is selected from halogenated or unsubstituted C 1～3 Alkyl, halogenated, or unsubstituted cyclopropyl;

[0052] R a1 R b1 R b2 R c1R c2 R d1 R e1 R e2 R f1 Each of the following groups, individually selected from substituted or unsubstituted groups: Each of the substituents is independently selected from halogenated or unsubstituted C. 1～3 Alkyl, C 1～3 alkoxy, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, SO2R7, aldehyde group, or two substituents linked to form a ring; R7 is selected from C 1～3 alkyl.

[0053] Furthermore,

[0054] The structure of the compound is shown in Formula IV:

[0055]

[0056] Where R1 is LR5, and L is C 1～3 Alkylene, NH, or none; R5 is selected from substituted or unsubstituted 3- to 6-membered saturated heterocyclic groups, substituted or unsubstituted 3- to 6-membered cycloalkyl groups, and substituted or unsubstituted bridged cycloalkyl groups, wherein the substituent is selected from C... 1～5 Alkyl, SO2R6, COR6; R6 is selected from halogenated or unsubstituted C 1～5 Alkyl, halogenated or unsubstituted 3- to 6-membered saturated cycloalkyl groups;

[0057] R2 is selected from C 1～3 alkyl;

[0058] n is an integer from 1 to 4;

[0059] R4 is independently selected from hydrogen, halogenated or unsubstituted C4. 1～5 Alkyl, halogenated or unsubstituted C 1～5 Alkyl, cyano, nitro, halogen, COOR8, COR8, SO2R8, substituted or unsubstituted 5- to 6-membered heteroaryl groups, wherein each substituent is independently selected from C10. 1～5 Alkyl group, R8 is C 1～5 alkyl.

[0060] Furthermore,

[0061] The structure of the compound is shown in Formula V:

[0062]

[0063] Where n is an integer from 1 to 2;

[0064] R4 is independently selected from hydrogen, halogenated or unsubstituted C4. 1～3Alkyl, halogenated or unsubstituted C 1～3 Alkyl, cyano, nitro, halogen, COOR8, COR8, SO2R8, substituted or unsubstituted Each of the substituents is independently selected from C. 1～3 Alkyl group, R8 is C 1～3 alkyl.

[0065] Furthermore,

[0066] The compound is one of the following compounds:

[0067]

[0068]

[0069]

[0070]

[0071] Furthermore,

[0072] The compound is one of the following compounds:

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] The present invention also provides the use of the above-described compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of DNA-PK inhibitors.

[0081] Furthermore, the DNA-PK inhibitor is a drug that inhibits the repair of damaged DNA in tumor cells.

[0082] Furthermore, the DNA-PK inhibitor is an antitumor drug sensitizer, a radiotherapy sensitizer, or a drug for treating tumors.

[0083] Furthermore, the antitumor drug sensitizer is a chemotherapy drug sensitizer.

[0084] Furthermore, the chemotherapy drugs include cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

[0085] Furthermore, the tumors include malignant tumors of the hematologic system, myelodysplastic syndrome, breast cancer, lung cancer, endometrial cancer, central nervous system tumors, gastric cancer, esophageal cancer, liver cancer, cholangiocarcinoma, colon cancer, rectal cancer, small bowel cancer, pancreatic cancer, melanoma, thyroid cancer, head and neck cancer, salivary gland cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, uterine cancer, vulvar cancer, bladder cancer, kidney cancer, squamous cell carcinoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, soft tissue sarcoma, Ewing's sarcoma, gastrointestinal stromal tumor, Kaposi's sarcoma, rhabdomyosarcoma, and neuroblastoma.

[0086] The malignant tumors of the hematopoietic system are preferably leukemia, multiple myeloma, or lymphoma; the lung cancer is preferably non-small cell lung cancer, small cell lung cancer, or squamous cell carcinoma; the central nervous system tumors are preferably glioma, dysplastic neuroepithelial tumor, glioblastoma multiforme, mixed glioma, medulloblastoma, retinoblastoma, neuroblastoma, germ cell tumor, or teratoma; the renal cancer is preferably renal cell carcinoma, clear cell renal cancer, or renal eosinophilic tumor.

[0087] The present invention also provides a combination antitumor drug comprising the above-mentioned compounds and antitumor drugs in the same or different strength units for simultaneous or separate administration, and a pharmaceutically acceptable carrier.

[0088] Furthermore, the antitumor drug is a chemotherapy drug.

[0089] Further, the chemotherapy drugs are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

[0090] The present invention also provides an antitumor composition comprising the above-described compounds and an antitumor drug.

[0091] Furthermore, the antitumor drug is a chemotherapy drug.

[0092] Further, the chemotherapy drugs are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

[0093] The present invention also provides an antitumor drug, characterized in that it is a formulation prepared by using the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof as the active ingredient, plus a pharmaceutically acceptable carrier.

[0094] The present invention also provides a method for treating tumors, wherein the method comprises combining the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, with an antitumor drug or radiotherapy.

[0095] Furthermore, the antitumor drug is a chemotherapy drug.

[0096] Further, the chemotherapy drugs are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736 (dvorumab), AZD1775, AZD6738, AZD1390, or AZD0156.

[0097] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0098] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0099] Halogens are fluorine, chlorine, bromine or iodine.

[0100] "Pharmaceutical acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0101] A "salt" is an acidic and / or basic salt formed by reacting a compound or its stereoisomer with an inorganic and / or organic acid and / or base. It also includes zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. These salts can be obtained directly during the final separation and purification of the compound. Alternatively, they can be obtained by mixing the compound, or its stereoisomer, with an appropriate (e.g., equimolar) amount of acid or base. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or prepared by freeze-drying after reaction in an aqueous medium.

[0102] In this invention, pharmaceutically acceptable salts can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate of the compound.

[0103] Bridged cycloalkyl refers to a polycyclic cycloalkyl group in which two rings share two non-adjacent carbon atoms.

[0104] "Heterocyclic group" refers to a substituent in a saturated or unsaturated cyclic hydrocarbon; the cyclic hydrocarbon can be monocyclic or polycyclic and carries at least one cyclic heteroatom (including but not limited to O, S, or N). "Saturated heterocyclic group" refers to a saturated heterocyclic group. For example, "3- to 6-membered saturated heterocyclic group" refers to a saturated heterocyclic group with 3 to 6 ring atoms.

[0105] "Cycloalkyl" refers to a substituent in a saturated or unsaturated cyclic hydrocarbon; the cyclic hydrocarbon can be monocyclic or polycyclic. "Saturated cycloalkyl" refers to a saturated cycloalkyl group. For example, "3- to 6-membered saturated cycloalkyl" refers to a saturated cycloalkyl group with 3 to 6 carbon atoms in its ring.

[0106] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a conjugated π-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent group must be on a carbon atom of a ring with a conjugated π-electron system. The aryl group can be substituted or unsubstituted.

[0107] "Heteroaryl" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0108] DNA-PK inhibitors refer to agents that inhibit DNA-PK-dominated DNA damage repair, as well as agents that inhibit DNA-PK expression.

[0109] Anti-tumor drugs include chemotherapy drugs, molecularly targeted drugs, and tumor immunotherapy drugs.

[0110] Chemotherapy is a method of treating tumors using chemically synthesized drugs, and it is one of the main methods of cancer treatment. Chemotherapy drugs are the medications used in chemotherapy; they can act on different stages of tumor cell growth and reproduction, inhibiting or killing tumor cells.

[0111] Experiments show that the compounds of the present invention have good inhibitory activity against DNA-PK, especially compounds CLJ1, 4, 8, 15, 22-25, 56, and 59, whose inhibitory activity is even better than that of the known DNA-PK inhibitor AZD-7648.

[0112] The compounds provided by this invention have broad application prospects in the preparation of DNA-PK inhibitors. This invention also provides a new option for antitumor drug sensitizers, radiotherapy sensitizers, and drugs for treating tumors, as well as a new method for treating tumors.

[0113] As is well known to those skilled in the art, the combined use of DNA-PK inhibitors with antitumor drugs such as cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156 can enhance the therapeutic effect of antitumor drugs. Therefore, the compounds provided by this invention, when used in combination with the aforementioned antitumor drugs, offer a new option for combination antitumor therapy.

[0114] This specification also relates to the use of such compounds and their salts in the treatment or prevention of DNA-PK-mediated diseases (including cancer); in addition, the compounds of the present invention have good inhibitory activity against the proliferation of various tumor cells, and some compounds show better antiproliferative activity than AZD-7648.

[0115] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0116] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0117] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0118] Example 1: Preparation of CLJ1

[0119]

[0120] Step a: Preparation of intermediate M1

[0121] At room temperature, potassium carbonate (15.63 g, 2.5 eq) was added in portions to an acetonitrile solution (250 mL) of ethyl 2,4-dichloro-5-pyrimidinecarboxylate (10.00 g, 1.0 eq) and 4-aminotetrahydropyran hydrochloride (6.23 g, 1.0 eq). The reaction was allowed to proceed overnight at room temperature. After the reaction was completed as indicated by TLC, the potassium carbonate was removed by filtration. The filtrate was concentrated under vacuum and subjected to rapid column chromatography using a PE / EA system (3-10%) to obtain a white powder M1 with a yield of approximately 70%. 1 H NMR(400MHz,Chloroform-d)δ8.66(d,J=1.4Hz,1H),8.38(d,J=7.8Hz,1H),4.43–4.26(m,3H),3.98(dt,J=12.0,3.7Hz, 2H),3.56(td,J=11.5,2.2Hz,2H),2.07–1.95(m,2H),1.60(dtd,J=12.4,10.8,4.3Hz,2H),1.38(td,J=7.2,1.2Hz,3H).

[0122] Step b: Preparation of intermediate M2

[0123] At room temperature, a 90 mL aqueous solution of lithium hydroxide (2.64 g, 2 eq) was slowly added to a 90 mL tetrahydrofuran solution of M1 (9 g, 1 eq). After the reaction was completed by TLC, the tetrahydrofuran and some water were removed by concentration. The remaining mixture was then slowly added dropwise with 3 M hydrochloric acid solution while stirring at room temperature. The addition was stopped when the pH paper showed that the pH was about 5. The large amount of solid precipitated was collected by vacuum filtration and dried overnight to obtain a white powder M2, which can be used as a raw material for the next step without further purification. 1 H NMR (400MHz, DMSO-d6) δ8.59(d,J=1.2Hz,1H),8.54(d,J=7.7Hz,1H),4.17(dtt,J=10.8,6.9,4.1Hz,1 H),3.84(dt,J=11.9,3.6Hz,2H),3.47(dd,J=11.4,2.3Hz,2H),1.93–1.82(m,2H),1.60–1.49(m,2H).

[0124] Step c: Preparation of intermediate M3

[0125] At room temperature, diphenyl azidophosphate (7.2 mL, 1 eq) and triethylamine (4.3 mL, 1 eq) were added to a solution of N,N-dimethylformamide (8 g, 1 eq) in M2 (125 mL). Nitrogen gas was evacuated three times, and the temperature was raised to 80 °C. After the reaction was completed as shown by TLC, most of the N,N-dimethylformamide was removed by vacuum concentration. An appropriate amount of ethyl acetate and a saturated ammonium chloride solution with a volume three times that of ethyl acetate were added for extraction. The organic layer was separated, concentrated under vacuum, and intermediate M3 was obtained by rapid column chromatography using a PE / EA (10-100%) system with a yield of 49% as a white powder. 1 H NMR (400MHz, DMSO-d6) δ8.13(s,1H),4.41(tt,J=12.2,4.2Hz,1H),3.97(dd,J=11.6,4. 5Hz, 2H), 3.45 (dd, J=12.1, 1.9Hz, 2H), 2.43 (tt, J=12.4, 6.5Hz, 2H), 1.72–1.63 (m, 2H).

[0126] Step d: Preparation method of intermediate M4

[0127] At 0℃, NaH (1.97 g, 2 eq) was slowly added in portions to M3 (6.3 g, 1 eq) of N,N-dimethylformamide solution (15 mL). The mixture was stirred at 0℃ for 20-30 min. Then, iodomethane (4.62 mL, 3 eq) of N,N-dimethylformamide solution (5 mL) was slowly added dropwise. The temperature was slowly restored to room temperature. After the reaction was completed as shown by TLC, a small amount of saturated saline solution was slowly added under ice bath. After the mixture stopped producing bubbles, a large amount of saturated saline solution was added, and a large amount of solid precipitated. The solid was filtered and dried under vacuum overnight to obtain M4, a white powder with a yield of 90%. 1 H NMR (400MHz, DMSO-d6) δ8.36 (s, 1H), 4.45 (tt, J=12.2, 4.2Hz, 1H), 3.97 (dd, J=11.6, 4.5Hz, 2H), 3.50–3.41(m,2H),3.36(s,3H),2.43(tt,J=12.5,6.3Hz,2H),1.68(ddd,J=12.3,4.3,1.8Hz,2H).

[0128] Step e: Preparation of intermediate M5

[0129] A mixture of 2-bromo-4-methyl-5-aminopyridine (0.4 g, 1 eq), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxapentylborane-2-yl)-1H-pyrazole (0.53 g, 1.2 eq), potassium carbonate (0.98 g, 3 eq), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (10%, 0.16 g) was added to a solution of dioxane (14 mL), ethanol (6 mL), and water (12 mL). Nitrogen gas was evacuated three times, and the mixture was heated to 70 °C. The reaction was allowed to proceed for about 2 hours. TLC showed that the reaction was complete. Insoluble solids were removed by diatomaceous earth filtration. The filtrate was concentrated and subjected to rapid column chromatography in a PE / EA system (40-80%) to give intermediate M5, a light yellow solid, with a yield of 69%. 1 H NMR (400MHz, Chloroform-d) δ7.95(s,1H),7.84(s,1H),7.77(s,1H),7.15(s,1H),3.89(s,3H),2.16(s,3H).

[0130] Step f: Preparation of the final product CLJ1

[0131] 10 mL of dioxane was added to a mixture of M4 (0.1 g, 1 eq), M5 (0.084 g, 1 eq), cesium carbonate (0.243 g, 2 eq), tris(dibenzylene-BASE-acetone)dipalladium (0.051 g, 15%), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (0.065 g, 30%). Nitrogen gas was evacuated three times, and the mixture was heated to 100 °C and reacted overnight. After TLC showed that M4 had reacted completely, 5 mL of ethyl acetate was added to the reaction solution. Insoluble solids were removed by diatomaceous earth filtration. The filtrate was concentrated, and the final product CLJ1, a light brown solid, was obtained by rapid column chromatography in a PE / EA system (50-100%), with a yield of 52%. 1 H NMR(400MHz,Chloroform-d)δ9.06(s,1H),7.88(d,J=8.2Hz,2H),7.81(s,1H),7.29(s,1H),6.76(s,1H),4.49(tt,J=12.2,4.2Hz,1H),4.09(dd, J=11.6,4.4Hz,2H),3.90(s,3H),3.49(td,J=12.2,1.9Hz,2H),3.35(s,3 H),2.71(qd,J=12.5,4.6Hz,2H),2.32(s,3H),1.74–1.65(m,2H).ESI-MS m / z:421.2[M+H] + .

[0132] Examples 2-25: Preparation of CLJ2-CLJ25

[0133] The preparation method of CLJ2-CLJ 25 is the same as in Example 1, except that the methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxapentoboran-2-yl)-1H-pyrazole used to prepare M5 is replaced with the corresponding borate ester. The structure and characterization of CLJ2-CLJ 25 are as follows:

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Examples 26-32: Preparation of CLJ26-CLJ32

[0150] The preparation method of CLJ26-CLJ32 is the same as in Example 1, except that the 2-bromo-4-methyl-5-aminopyridine used to prepare M5 is replaced with the corresponding amino compound, and the 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxapentoboran-2-yl)-1H-pyrazole is replaced with the corresponding borate ester. Characterization is as follows:

[0151]

[0152] CLJ26, 1 H NMR (400MHz, DMSO-d6) δ12.96(s,1H),8.57(s,1H),8.09(s,1H),8.04(s,1H),7.87(d,J=8.3Hz,1H),7.47(d,J=8.3Hz,1H),4.39(ddt,J=12.1,8. 3,4.1Hz,1H),3.96(dd,J=11.6,4.4Hz,2H),3.47–3.40(m,2H),3.29(s,3 H),2.58–2.50(m,2H),2.45(s,3H),1.65(dd,J=13.0,3.9Hz,2H).ESI-Ms m / z:407.1[M+H] +

[0153]

[0154] CLJ27, 1H NMR(400MHz,Chloroform-d)δ8.73(d,J=2.6Hz,1H),8.57(d,J=8.6Hz,1H),8.24(dd,J=8.6,2.5Hz,1H),7.88(s,1H),7.55(d,J=8.5Hz,1H),6.87–6.75(m,2H),4.55(tt,J=12.2,4.2Hz,1H),4.18–4.07(m,2H),3.99(s,3H),3.59–3.50(m,2H),3.41(s,3H),2.77(qd,J=12.6,4.7Hz,2H),2.67(s,3H),1.79–1.71(m,2H).ESI-Ms m / z:448.2[M+H] +

[0155]

[0156] CLJ28, 1 H NMR(400MHz,Chloroform-d)δ8.30(d,J=8.4Hz,1H),7.92–7.86(m,1H),7.53–7.46(m,1H),7.30(dd,J=8.4,2.2Hz,1H),6.81(s,1H),6.28(t,J=1.4Hz,1H),4.55(ddt,J=12.2,8.0,4.3Hz,1H),4.13(dd,J=11.8,4.5Hz,2H),3.91(d,J=1.0Hz,3H),3.54(t,J=12.0Hz,2H),3.40(d,J=1.0Hz,3H),2.78(qd,J=12.5,4.6Hz,2H),2.41(s,3H),1.74(dd,J=13.1,4.0Hz,2H).ESI-MS m / z:420.2[M+H] + .

[0157]

[0158] CLJ29, 1H NMR(400MHz,Chloroform-d)δ8.27(d,J=1.7Hz,1H),7.89(s,1H),7.79–7.72(m,2H),7. 19(d,J=7.8Hz,1H),7.10(dd,J=7.8,1.8Hz,1H),6.80(s,1H),4.56(tt,J=12.2,4.1Hz,1 H), 4.10(dd,J=11.7,4.5Hz,2H), 3.94(s,3H), 3.53(td,J=12.2,1.9Hz,2H), 3.40(s,3H), 2.78(qd,J=12.5,4.6Hz,2H), 2.35(s,3H), 1.75(ddd,J=12.3,4.2,1.8Hz,2H).ESI-MS m / z:420.4[M+H] + .

[0159]

[0160] CL30 1 H NMR(400MHz,Chloroform-d)δ8.64(d,J=2.0Hz, 1H), 8.31(d,J=1.9Hz,1H), 7.92(s,1H), 7.86(s,1H), 7.81(s,1H), 6.84(s,1H), 4.57(ddt,J=12.3,8.4,4.1Hz,1H), 4.12(dd,J=11.8,4.5Hz,2H), 3.95(s,3H), 3.55(td,J=12.2,1.9Hz,2H), 3.42(s,3H), 2.76(qd,J=12.5,4.6Hz,2H), 2.61(s,3H), 1.76(dd,J=13.3,3.9Hz,2H).ESI-MSm / z:420.2[M+H] + .

[0161]

[0162] CLJ31 1H NMR(400MHz,Chloroform-d)δ8.58(s,1H),8.23(s,1H),8.08(s,1H),8.03(s,1H),7.98(s,1H),7.06(s,1H),4.61(tt,J=12.3,4.1Hz,1H),4.16(dd,J=1 1.7,4.8Hz,2H),3.94(s,3H),3.58(td,J=12.1,1.8Hz,2H),3.43(s,3H),2.8 0(qd,J=12.5,4.6Hz,2H),2.32(s,3H),1.79(dd,J=13.2,4.2Hz,2H).ESI-MS m / z:420.2[M+H] + .

[0163]

[0164] CLJ32, 1 H NMR(400MHz,Chloroform-d)δ9.32(s,1H),8.14(s,1H),8.06(s,1H),7.85(s,1H),6.63(s,1H),4.53(tt,J=12.3,4.2Hz,1H),4.13(dd, J=11.8,4.5Hz,2H),3.95(s,3H),3.53(td,J=12.2,1.9Hz,2H),3.40(s,3H),2.82–2.69(m,2H),2.57(s,3H),1.77–1.66(m,2H).ESI-MS m / z:421.1[M+H] + .

[0165] Example 33: Preparation of CLJ33

[0166] The preparation method of CLJ33 is the same as in Example 1, except that the 4-aminotetrahydropyran hydrochloride used to prepare M2 is replaced with memantine hydrochloride, and the subsequent intermediates are replaced accordingly.

[0167]

[0168] EM1, 1H NMR(400MHz,Chloroform-d)δ8.58(d,J=2.0Hz,1H),8.35(s,1H),4.29(qd,J=7.1,1.7Hz,2H),2.19–2.13(m,1H),2.02–1.97(m,2H),1.83(d,J=11.6Hz,2H),1.66(d,J=11.6Hz,2H),1.43–1.14(m,13H),0.83–0.78(m,2H).

[0169] EM3, 1 H NMR(400MHz,Chloroform-d)δ10.25(s,1H),8.14(s,1H),2.53–2.47(m,2H),2.35–2.26(m,3H),2.25–2.17(m,2H),1.51(dt,J=12.4,2.8Hz,2H),1.37(dt,J=12.3,2.5Hz,2H),1.31(dt,J=12.4,2.4Hz,1H),1.22(dt,J=12.5,2.1Hz,1H),0.94(s,6H).

[0170] EM4, 1 H NMR(400MHz,Chloroform-d)δ7.93(d,J=1.1Hz,1H),3.35(d,J=1.0Hz,3H),2.50–2.42(m,2H),2.32–2.22(m,3H),2.18(dt,J=11.9,1.7Hz,2H),1.48(dt,J=12.7,2.7Hz,2H),1.39–1.26(m,3H),1.19(dq,J=12.5,1.9Hz,1H),0.91(d,J=1.7Hz,6H).

[0171]

[0172] CLJ33, 1H NMR(400MHz,Chloroform-d)δ8.83(s,1H),7.86(d,J=15.6Hz,2H),7.76(s,1H),7 .30(s,1H),6.73(s,1H),3.93(s,3H),3.29(s,3H),2.35(d,J=3.1Hz,2H),2.31(s, 3H),2.24(d,J=11.9Hz,2H),2.20–2.15(m,1H),2.11(d,J=12.0Hz,2H),1.41(dd,J =11.9,3.3Hz,2H),1.27(d,J=12.8Hz,2H),1.22–1.08(m,2H),0.84(s,6H).ESI-MS m / z: 499.2 [M+H] +

[0173] Example 34: Preparation of CLJ34

[0174] The preparation method of CLJ34 is the same as in Example 1, except that the 4-aminotetrahydropyran hydrochloride used to prepare M2 is replaced with memantine hydrochloride, and the intermediate M6 is replaced with FM2. The preparation method of FM2 is as follows:

[0175]

[0176] Preparation of FM1

[0177] At -5℃, a solution of 1.5 mL concentrated nitric acid in glacial acetic acid (5 mL) was slowly added dropwise to a solution of 1 g of 3,4-(methylenedioxy)toluene in glacial acetic acid (15 mL). The mixture was stirred at this temperature for 30 min. The reaction solution was then slowly brought to room temperature. After the TLC reaction was completed, ice water was slowly added dropwise, and a large amount of solid precipitated. The solid was filtered and dried under vacuum overnight. The resulting light yellow solid was used in the next step without further processing. 1 H NMR (400MHz, DMSO-d6) δ7.59(s,1H),7.05(s,1H),6.19(s,2H),2.47(s,3H).

[0178] Preparation of FM2

[0179] At room temperature, hydrazine hydrate (1 mL) was slowly added dropwise to a methanol solution of FM1 (1.2 g, 1 eq) and palladium on carbon (10%). The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the product was filtered through diatomaceous earth and concentrated under vacuum to obtain the target product, a light yellow solid, which was used in the next step without further processing. 1H NMR (400MHz, Chloroform-d) δ6.57(s,1H),6.28(s,1H),5.82(s,2H),3.49–3.22(m,2H),2.08(s,3H).

[0180]

[0181] CLJ34, 1 H NMR(400MHz,Chloroform-d)δ7.76(s,1H),7.30(s,1H),6.68(s,1H),6.46(s,1H),5.90(s,2H),3.29(s,3H),2.41(d,J=3.3Hz,2H),2.24(d,J=12.0Hz,2 H),2.21(s,3H),2.18(d,J=12.1Hz,2H),1.47–1.41(m,2H),1.30(d,J=12.5H z,2H),1.27–1.20(m,2H),1.14(dt,J=12.4,2.0Hz,1H),0.87(s,6H).ESI-MS m / z:462.2[M+H] +

[0182] Examples 35-45: Preparation of CLJ35-CLJ45

[0183] The preparation method of CLJ35-CLJ45 is the same as in Example 1, except that the 4-aminotetrahydropyran hydrochloride used to prepare M2 is replaced with memantine hydrochloride, and the subsequent intermediates are replaced accordingly. The structure and characterization of CLJ35-CLJ45 are shown below:

[0184]

[0185] CLJ35, 1H NMR(400MHz,Chloroform-d)δ9.04(d,J=17.2Hz,1H),7.90(d,J=7.7Hz,2H),7.81(d,J=6.9Hz,1H),7.32(s,1H),6.71(d,J=13.5Hz,1H),3.95(s,4H),3.38(d,J=5.6Hz,3H),2.48(dq,J=12.0,6.0Hz,1H),2.34(d,J=3.9Hz,4H),1.91–1.81(m,2H),1.73(tt,J=11.1,5.1Hz,3H),1.18–1.11(m,2H),0.97(dd,J=7.0,4.6Hz,1H),0.76(d,J=6.5Hz,3H).ESI-MS m / z:433.2[M+H] +

[0186]

[0187] CLJ36, 1 H NMR(400MHz,Chloroform-d)δ8.94(s,1H),7.88(d,J=3.8Hz,2H),7.82(s,1H),7.31(s,1H),6.79(s,1H),5.06(dtd,J=9.9,7.4,5.7Hz,1H),4.19–3.97(m,4H),3.92(s,3H),3.36(s,3H),2.52(ddt,J=13.1,7.8,5.4Hz,1H),2.31(s,3H),2.28–2.21(m,1H).ESI-MS m / z:407.1[M+H] +

[0188]

[0189] CLJ37, 1 H NMR(400MHz,Chloroform-d)δ9.10(s,1H),7.91(s,2H),7.84(s,1H),7.33(s,1H),6.59(s,1H),4.00–3.93(m,5H),3.78(d,J=7.2Hz,2H),3.40(s,3H),3.35(td,J=11.7,2.2Hz,2H),2.35(s,3H),2.18(ddt,J=11.3,7.8,3.9Hz,1H),1.62–1.54(m,2H),1.51–1.38(m,2H).ESI-MS m / z:435.2[M+H] +

[0190]

[0191] CLJ38, 1 H NMR(400MHz,Chloroform-d)δ9.10(s,1H),7.91(s,2H),7.84(s,1H),7.33(s,1H),6.59(s,1H),4.00–3.93(m,5H),3.78(d,J=7.2Hz,2H),3.40(s,3H),3.35(td,J=11.7,2.2Hz,2H),2.35(s,3H),2.18(ddt,J=11.3,7.8,3.9Hz,1H),1.62–1.54(m,2H),1.51–1.38(m,2H).ESI-MS m / z:377.1[M+H] +

[0192]

[0193] CLJ39, 1 H NMR(400MHz,Chloroform-d)δ8.95(s,1H),7.91(s,2H),7.83(s,1H),7.32(s,1H),6.61(s,1H),4.82(d,J=13.6Hz,1H),4.49(ddt,J=12.2,8.1,4.2Hz,1H),3.94(s,4H),3.38(s,3H),3.16(td,J=13.2,2.4Hz,1H),2.59(dtd,J=21.3,12.6,3.4Hz,2H),2.45(td,J=12.6,4.3Hz,1H),2.34(s,3H),2.09(s,3H),1.81(t,J=13.5Hz,2H).ESI-MS m / z:498.1[M+H] +

[0194]

[0195] CLJ40, 1H NMR(400MHz,Chloroform-d)δ9.02(s,1H),7.89(d, J=3.5Hz,2H),7.83(s,1H),7.32(s,1H),6.74(s,1H),4.40(tt,J=12.2,4.0Hz,1H),4.00–3.94(m,2H),3.93(s,3H),3.37(s,3H),2.97(td,J=12.5,2.3Hz,2H),2.73(qd,J=12.6,4.2Hz,2H),2.35(s,4H),1.88–1.79(m,2H),1.13(dt,J=6.9,3.4Hz,2H),0.96–0.88(m,2H).ESI-MS m / z:524.2[M+H] +

[0196]

[0197] CLJ41, 1 H NMR(400MHz,Chloroform-d)δ9.31(s,1H),8.17(s,1H),7.96(s,1H),7.90(s,1H),7.52(s,1H),6.86(s,1H),4.85–4.78(m,1H),4.55(ddt,J=12.2,8.0,4.2Hz,1H),3.96(s,4H),3.40(s,3H),3.24(dd,J=13.4,2.6Hz,1H),2.70–2.63(m,1H),2.54(qd,J=12.1,11.7,4.4Hz,2H),2.47(s,3H),2.12(s,3H),1.84(dd,J=28.4,12.8Hz,2H).ESI-MSm / z:552.1[M+H] +

[0198]

[0199] CLJ42, 1H NMR(400MHz,DMSO-d6)δ8.60(d,J=46.2Hz,1H),8.51(d,J=5.6Hz,1H),8.16(s,1H),8.01(d,J=5.0Hz,1H),7.91(s,1H),7.48(d,J=2.9Hz,1H),4.55–4.40(m,2H),4.17(t,J=17.1Hz,1H),3.87(s,3H),3.17(s,3H),2.74(t,J=12.9Hz,1H),2.59(ddd,J=13.9,10.5,8.3Hz,1H),2.29(dd,J=12.3,4.7Hz,2H),2.22(d,J=2.5Hz,3H),1.94–1.84(m,3H),1.78(d,J=13.0Hz,2H),1.73–1.62(m,1H).ESI-MS m / z:524.2[M+H] +

[0200]

[0201] CLJ43, 1 H NMR(400MHz,Chloroform-d)δ9.00(d,J=11.8Hz,1H),7.91(d,J=6.8Hz,2H),7.83(s,1H),7.34(s,1H),6.89–6.79(m,1H),4.83–4.62(m,2H),4.53(tt,J=12.2,4.1Hz,1H),4.31(d,J=13.9Hz,1H),3.95(s,3H),3.38(s,3H),3.31–3.16(m,3H),2.75–2.41(m,3H),2.36(s,3H),2.29–2.18(m,1H),1.86(dd,J=38.3,12.8Hz,2H).ESI-MSm / z:506.2[M+H] +

[0202]

[0203] CLJ44, 1H NMR(400MHz,Chloroform-d)δ8.99(s,1H),7.90(d,J=5.6Hz,2H),7.83(s,1H),7.3 4(s,1H),6.81(s,1H),4.77(d,J=13.2Hz,1H),4.51(ddt,J=12.1,8.2,4.2Hz,1H),4 .37(d,J=13.6Hz,1H),3.93(s,3H),3.37(s,3H),3.17(q,J=7.3Hz,2H),2.60–2.45 (m,2H),2.35(s,3H),1.91–1.72(m,3H),0.94(d,J=7.9Hz,2H),0.71(s,2H).ESI-MS m / z: 488.2 [M+H] +

[0204]

[0205] CLJ45, 1 H NMR(400MHz,Chloroform-d)δ8.93(s,1H),7.90(d,J=2.1Hz,2H),7.82(s,1H),7.3 1(s,1H),6.70(s,1H),4.85–4.76(m,1H),4.47(tt,J=12.1,4.1Hz,1H),3.92(s,4H) ,3.36(s,3H),3.14(td,J=13.3,2.5Hz,1H),2.57(dtd,J=21.4,12.7,3.5Hz,2H),2. 46(d,J=4.4Hz,1H),2.32(s,3H),2.06(s,3H),1.78(td,J=13.7,7.6Hz,2H).ESI-MS m / z:462.2[M+H] +

[0206] Examples 46-53, 59: Preparation of CLJ46-CLJ54

[0207] The preparation method of CLJ46-CLJ54 is the same as in Example 1, except that M5 is replaced with the corresponding commercially available secondary amine raw material. The structure and characterization of CLJ46-CLJ54 are as follows:

[0208]

[0209]

[0210]

[0211]

[0212]

[0213] Example 55: Preparation of CLJ55

[0214] The preparation of CLJ55 is the same as in Example 1, except that intermediate SM6 is replaced with GM3. The preparation method of GM3 is as follows:

[0215]

[0216] Preparation of GM1

[0217] Add 20 mL of N,N-dimethylformamide dimethyl acetal to 1 g of 3-methyl-4-nitrobenzamide. After the mixture is dissolved, evacuate the nitrogen gas three times, raise the temperature to 85 °C, and maintain this temperature while stirring for 3 h. After the reaction is complete as shown by TLC, concentrate the reaction solution under vacuum to obtain a yellow oily substance. Add 30 mL of glacial acetic acid at room temperature, and slowly add 2.5 mL of hydrazine hydrate. A large amount of white solid precipitates. Raise the temperature to 90 °C and stir for 1 h. After the reaction is dissolved, concentrate the solution under vacuum as shown by TLC. Add an appropriate amount of diethyl ether, and a large amount of white solid precipitates. Filter the solid to obtain the solid, which can be used for the next step without further purification. 1 H NMR (400MHz, DMSO-d6) δ8.63(s,1H),8.15–8.00(m,3H),2.59(s,3H).

[0218] Preparation of GM2

[0219] 3,4-dihydro-2H-pyran (1 mL) was added to a tetrahydrofuran solution of GM1 (1 g) in one go. A small amount of p-toluenesulfonic acid (0.1 g) was added while stirring at room temperature. The mixture was stirred at room temperature for 30 min. The reaction was shown to be complete by TLC. The reaction solution was concentrated and subjected to rapid column chromatography in a PE / EA system to obtain a white solid. 1 H NMR(400MHz,Chloroform-d)δ8.34(s,1H),8.13–8.01(m,3H),5.51(dd,J=8.5,3.7Hz,1H) ,4.15–4.03(m,1H),3.81–3.69(m,1H),2.66(s,3H),2.21–2.02(m,3H),1.77–1.65(m,3H).

[0220] Preparation of GM3

[0221] 1.5 mL of hydrazine hydrate solution was slowly added dropwise to a methanol solution of GM2 (1.3 g) and an appropriate amount of palladium on carbon. The reaction was carried out overnight in a sealed container at room temperature. After the reaction was completed, the palladium on carbon was removed by diatomaceous earth filtration and the target product was obtained by vacuum concentration. The product was a white solid.

[0222]

[0223] CLJ55, 1 H NMR(400MHz,Chloroform-d)δ8.32–8.25(m,2H),8.04–7.94(m,2H),7.89(s,1H),6.9 1(s,1H),5.48(dd,J=7.5,4.7Hz,1H),4.54(tt,J=12.2,4.1Hz,1H),4.13(dd,J=11.6, 4.9Hz,3H),3.80–3.70(m,1H),3.57–3.51(m,2H),3.39(s,3H),2.77(qd,J=12.5,4.6H z,2H),2.41(d,J=2.1Hz,3H),2.25–1.96(m,4H),1.73(dt,J=11.9,3.0Hz,4H).ESI-MS m / z:491.2[M+H] +

[0224] Example 56: Preparation of CLJ56

[0225] Add 0.5 mL of concentrated hydrochloric acid to a methanol solution of CLJ44 (0.2 g), react overnight at room temperature, concentrate under vacuum, add 10 mL of water, slowly add sodium hydroxide solid particles, adjust the pH to about 8, add 30 mL of ethyl acetate for extraction, separate the organic layer, dry with anhydrous sodium sulfate, concentrate, and obtain a light yellow solid, namely CLJ55.

[0226]

[0227] CLJ56, 1 H NMR (400MHz, DMSO-d6) δ8.33(s,1H),8.04(s,1H),7.82(s,2H),7.75(dd,J=8.3,2.0Hz,1H),7.58(d,J=8.3Hz,1H),4.40(ddt,J=12.2,8.0,4. 1Hz,1H),3.97(dd,J=11.3,4.4Hz,2H),3.44–3.37(m,2H),3.29(s,3H),2.54(dd,J=15.9,11.3Hz,2H),2.26(s,3H),1.70–1.63(m,2H).ESI-MS m / z:407.1[M+H] +

[0228] Example 57: Preparation of CLJ57

[0229] The preparation of CLJ57 is the same as in Example 1, except that intermediate SM6 is replaced with HM2. The preparation method of HM2 is as follows:

[0230]

[0231] Preparation of HM1

[0232] Under ice bath conditions, excess NaH (2 eq) was added to a 5 mL solution of GM1 (1 eq) in N,N-dimethylformamide. The mixture was stirred in an ice bath for 30 min, and then a 5 mL solution of iodomethane in N,N-dimethylformamide was slowly added dropwise. The reaction was allowed to proceed overnight. After the reaction was complete as indicated by TLC, saturated brine was slowly added under ice bath conditions, resulting in the precipitation of a large amount of solid. The solid was filtered and dried under vacuum overnight. It was ready for use in the next step without further purification.

[0233] Preparation of HM2

[0234] 1.2 mL of hydrazine hydrate solution was slowly added dropwise to a methanol solution of HM1 (1 g) and an appropriate amount of palladium on carbon. The reaction was carried out overnight in a sealed container at room temperature. After the reaction was completed, the palladium on carbon was removed by diatomaceous earth filtration and the target product was obtained by vacuum concentration. The product was a white solid. 1 H NMR (400MHz,

[0235] Chloroform-d)δ7.98(s,1H),7.80(d,J=1.9Hz,1H),7.76(dd,J=8.1,2.0Hz,1H),6.71(d,J=8.2Hz,1H),3.92(s,3H),3.59(s,2H),2.21(s,3H).

[0236]

[0237] CLJ57, 1 H NMR(400MHz,Chloroform-d)δ8.31(d,J=8.5Hz,1H),8.04(s,1H),8.01–7.93(m,2H),7.90(d,J=1.0Hz,1H),6.87(s,1H),4.54(tt,J=12.3,4.3Hz,1H),4 .17–4.10(m,2H),3.96(s,3H),3.53(t,J=12.0Hz,2H),3.40(d,J=1.0Hz,3H ),2.78(qd,J=12.5,4.6Hz,2H),2.41(s,3H),1.73(d,J=10.7Hz,2H).ESI-MS m / z:421.2[M+H] +

[0238] Example 58: Preparation of CLJ58

[0239] The preparation of CLJ58 is the same as in Example 1, except that intermediate SM6 is replaced with FM2.

[0240]

[0241] CLJ58, 1 H NMR(400MHz,Chloroform-d)δ7.81(s,1H),7.40(s, 1H),6.69(s,1H),6.46(s,1H),5.92(s,2H),4.50(tt,J=12.2,4.2Hz,1H),4.11(dd,J=11.7,4.6Hz,2H),3.57–3 .47(m,2H),3.37(s,3H),2.75(qd,J=12.5,4.6Hz,2H),2.23(s,3H),1.71(ddd,J=12.2,4.3,1.9Hz,2H).ESI-MS m / z:384.1[M+H] +

[0242] Example 59: Preparation of CLJ59

[0243] The preparation of CLJ59 is the same as in Example 1, except that the 4-aminotetrahydropyran hydrochloride used to prepare M2 is replaced with 1-tert-butoxycarbonyl-4-aminopiperidine, and the subsequent intermediates are replaced accordingly.

[0244]

[0245] IM1, 1 H NMR(400MHz,Chloroform-d)δ8.65(s,1H),8.37(d,J=7.8Hz,1H),4.33(q,J=7.1Hz,2H),4.30–4.19( m,1H),3.99(s,2H),3.00(t,J=12.3Hz,2H),2.03–1.91(m,2H),1.45(s,11H),1.36(t,J=7.1Hz,3H).

[0246] IM2, 11H NMR (400 MHz, Chloroform-d) δ 8.76 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 4.30 (dp, J = 15.0, 6.1, 5.1 Hz, 1H), 4.07 (d, J = 13.5 Hz, 2H), 3.00 (t, J = 12.7 Hz, 2H), 2.00 (d, J = 12.7 Hz, 2H), 1.47 (s, 11H).

[0247] IM3 1 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 8.04 (s, 1H), 4.32 (tt, 1H), 4.07 (d, 2H), 2.79 (s, 2Hs), 2.14 - 2.30 (m, 2H), 1.61–1.73 (m, 2H), 1.41 (s, 9H).

[0248] IM4 1 1H NMR (400 MHz, Chloroform-d) δ 8.00 (s, 1H), 4.48 (tt, J = 12.3, 4.1 Hz, 1H), 4.28 (s, 2H), 3.43 (s, 3H), 2.83 (d, J = 11.4 Hz, 2H), 2.52 (qd, J = 12.6, 4.5 Hz, 2H), 1.75 (s, 2H), 1.48 (s, 9H).

[0249] IM5 1 1H NMR (400 MHz, Chloroform-d6) δ 9.23 (s, 1H), 8.78 (s, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.24 (s, 1H), 7.06 (d, J = 1.6 Hz, 1H), 4.73 (s, 1H), 4.41 (dt, J = 12.5, 7.1 Hz, 2H), 3.96 (s, 3H), 3.26 (s, 3H), 3.05 (dt, J = 12.5, 7.1 Hz, 2H), 2.93 (s, 1H), 2.87 (q, J = 7.0 Hz, 1H), 2.50 (dt, J = 13.4, 7.0 Hz, 2H), 2.44 (s, 3H), 1.65 (dt, J = 13.0, 7.1 Hz, 2H), 1.47 (s, 9H).

[0250] Preparation of CLJ59:

[0251] At room temperature, 1.5 mL of concentrated hydrochloric acid was slowly added dropwise to a methanol solution of 1 g of IM5. The mixture was stirred overnight at room temperature. After the reaction was completed as shown by TLC, the reaction solution was concentrated under vacuum. Water was added, and sodium hydroxide solid particles were slowly added in batches to adjust the pH to about 8. Ethyl acetate with a volume three times that of water was added. The organic layer was extracted and separated, dried over anhydrous sodium sulfate, and concentrated to obtain the target compound, which was light yellow-brown. 1 H NMR(400MHz,Chloroform-d)δ9.20(s,1H),7.96(s,1H),7.88–7.84(m,2H),7.31(s,1H),4.59–4.46(m,1H),3.95(s,3H), 3.54(d,J=12.4Hz,2H),3.40(s,3H),3.00–2.80(m,4H),2.37(s,3H),2.00–1.90(m,2H).

[0252] The following biological experimental examples demonstrate the beneficial effects of the present invention.

[0253] Experimental Example 1: Effect of Compounds on DNA-PK Enzyme Activity

[0254] 1. Experimental Methods

[0255] Enzyme activity detection and IC 50 Calculation method:

[0256] DNA-PK inhibitory activity assay: DNA-PK enzyme was added to an assay buffer containing 50 nM GST-cMyc-p53 and a given concentration of Mg / ATP and incubated. The reaction was initiated by adding the Mg / ATP mixture. After incubation at room temperature for 30 minutes, the reaction was terminated by adding a stop solution containing EDTA. Finally, an assay buffer containing d2-labeled anti-GST monoclonal antibody and Europium-labeled anti-Ser15 phosphate antibody against phosphorylated p53 was added. The plate was then read using time-resolved fluorescence analysis. The homogeneous time-resolved fluorescence (HTRF) signal was determined according to the formula HTRF = 10000 × (Em665nm / Em620nm).

[0257] The enzyme activity data of each compound prepared in the embodiments of the present invention at different concentrations against DNA-PK enzyme were measured according to the above method, and the results are shown in Table 1; and the IC50 of some compounds inhibiting DNA-PK activity was calculated. 50 The values ​​are shown in Table 2. The known DNA-PK inhibitor AZD-7648 was used as a positive control.

[0258] 2. Experimental Results

[0259] Table 1. Enzymatic activities of each compound against DNA-PK at different concentrations.

[0260]

[0261]

[0262] Table 2 shows the IC50 values ​​of each compound for inhibiting DNA-PK enzyme. 50 value

[0263] Cmpd <![CDATA[DNA-PKIC 50 (nM)]]> Cmpd <![CDATA[DNA-PKIC 50 (nM) <!-- 49 -->]]> AZD-7648 1 CLJ29 10 CLJ1 0.1 CLJ31 2 CLJ2 8 CLJ33 1 CLJ3 2 CLJ34 3 CLJ4 0.4 CLJ35 24 CLJ5 6 CLJ36 35 CLJ8 0.8 CLJ39 17 CLJ13 2 CLJ40 3 CLJ15 0.7 CLJ41 143 CLJ17 6 CLJ44 21 CLJ18 4 CLJ46 6 CLJ20 7 CLJ47 13 CLJ22 0.1 CLJ50 3 CLJ23 0.9 CLJ51 1 CLJ24 0.4 CLJ54 4 CLJ25 0.3 CLJ56 0.4 CLJ26 25 CLJ58 1 CLJ27 161 CLJ59 0.9

[0264] The experimental results in Tables 1 and 2 demonstrate that the compounds provided by this invention exhibit good inhibitory activity against DNA-PK. In particular, compounds CLJ1, 4, 8, 15, 22-25, 56, and 59 of this invention show high IC50 values ​​for inhibiting DNA-PK. 50 It is lower than that of the known DNA-PK inhibitor AZD-7648.

[0265] Experimental Example 2: Determination of the inhibitory rate of the compound on radiosensitization of tumor cells in a clonal experiment.

[0266] 1. Experimental Methods

[0267] Cloning experiments:

[0268] (1) Cell seeding: Hct116 cells were collected from culture dishes in a clean bench using centrifuge tubes, centrifuged at 1000 rpm for 3 min, resuspended in DMED complete medium, and counted. The cells were seeded into 24-well plates at a density of 400 cells / well, with a volume of 500 μL per well. The 24-well plates were placed in a 5% CO2 incubator and incubated at 37°C for three days.

[0269] (2) Drug treatment: Hct116 cells were treated with the test compound at a pre-set concentration for 1 hour and then irradiated. The known DNA-PK inhibitor AZD-7648 was used as a positive control.

[0270] (3) Irradiation: The cells treated with the drug were irradiated at 2 Gy, and then the cells were placed in an incubator for one week.

[0271] (4) Crystal violet staining: Remove the cell supernatant from the 24-well plate, gently wash the cells twice with PBS, and slowly add methanol to fix at room temperature for 20 min. Discard the methanol and place the 24-well plate on the lab bench to allow the methanol to evaporate. Add crystal violet and stain for 20 min, recover the crystal violet, and wash the 24-well plate three times with ultrapure water.

[0272] (5) Taking pictures: Use a chemiluminescence imaging system to take pictures.

[0273] The results of the cloning experiment were analyzed using the software Image J for quantitative analysis. Then, after calculating the inhibition rate of each concentration of the compound on cell clone formation, the compound treatment concentration was converted into a logarithmic form. With the logarithmized concentration as x and the inhibition rate as y, the dose-response curve was fitted using GraphPad Prism 8.0 software, and the IC 50 value was fitted.

[0274] 2. Experimental results

[0275] Table 3 Determination of the inhibition rate of each compound on the radiosensitization of tumor cells induced by radiation in the cloning experiment

[0276]

[0277] Table 4 Determination of the IC 50 value of each compound on the radiosensitization of tumor cells induced by radiation in the cloning experiment

[0278]

[0279] From the results of Table 3 and Table 4, it can be seen that the compounds provided by the present invention can effectively inhibit the radiosensitization of tumor cells induced by radiation; in particular, the compounds CLJ1, 22, 23, 31, and 54 of the present invention have an IC 50 lower than that of the known DNA-PK inhibitor AZD-7648. The compounds provided by the present invention can be used to prepare a radiosensitizer for radiotherapy.

[0280] Experimental Example 3: In vivo pharmacokinetic properties of the compound in rats

[0281] 1. Experimental method

[0282] Weigh an appropriate amount of the test compound, first dissolve it in a small amount of 1% DMSO, and then add a physiological saline solution to prepare a compound solution of 1 mg·mL -1 . Wait for drug administration. Six SD rats (Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030), all male rats, each weighing 200-250 g, were intravenously administered at 5 mg·kg -1 and 5 mg·kg -1Blood samples were collected from each animal via cardiac puncture at 0 min before administration and at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after administration, and stored in a refrigerator (-20°C). Plasma was separated from the blood by centrifugation (4°C, 4000 g, 15 min) and stored in a refrigerator at -80°C. Samples were analyzed using an ultra-high-speed liquid chromatography (UFLC) system (SIL-30AC autosampler, LC-30AD chromatograph, CBM-20A communications bus module, CTO-20AC prominence column oven, Shimadzu Corporation, Japan), and plasma concentration data were analyzed using a non-compartmental analysis method.

[0283] 2. Experimental Results

[0284] Table 5. In vivo pharmacokinetic properties of each compound.

[0285]

[0286]

[0287] As can be seen from the results in Table 5, the compounds provided by this invention are superior to the known DNA-PK inhibitor AZD-7648 in terms of pharmacokinetic properties, including exposure, half-life, and clearance rate. In particular, compounds CLJ1, CLJ13, and CLJ31 also have better bioavailability than the known DNA-PK inhibitor AZD-7648, in addition to the above parameters.

[0288] In summary, this invention provides the 7,9-dihydropurine derivatives shown in Formula I and their pharmaceutical uses. Experiments show that the compounds provided by this invention exhibit good inhibitory activity against DNA-PK, particularly compounds CLJ1, 4, 8, 15, 22-25, 56, and 59, whose inhibitory activity is even superior to the known DNA-PK inhibitor AZD-7648. The compounds provided by this invention have broad application prospects in the preparation of DNA-PK inhibitors. This invention provides a new option for antitumor drug sensitizers, radiotherapy sensitizers, and drugs for treating tumors, and also provides a new option for methods of treating tumors.

Claims

1. The compound represented by formula V, or a pharmaceutically acceptable salt thereof: Formula V in, n is an integer from 1 to 2; R4 is independently selected from hydrogen, halogenated or unsubstituted C4. 1~3 Alkyl, halogenated or unsubstituted C 1~3 Alkyl, cyano, halogen, COOR8, substituted or unsubstituted The substituted groups are each independently selected from C 1~3 Alkyl group, R8 is C 1~3 alkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: The compound is one of the following compounds: CLJ54.

3. Use of the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, in the preparation of a DNA-PK inhibitor.

4. The use according to claim 3, characterized in that: The DNA-PK inhibitor is a drug that inhibits the repair of damaged DNA in tumor cells.

5. The use according to claim 4, characterized in that: The DNA-PK inhibitor is a radiosensitizer or a drug for treating tumors.

6. The use according to claim 5, characterized in that: The tumors mentioned are malignant tumors of the hematologic system, myelodysplastic syndrome, breast cancer, lung cancer, endometrial cancer, central nervous system tumors, gastric cancer, esophageal cancer, liver cancer, cholangiocarcinoma, colon cancer, rectal cancer, small bowel cancer, pancreatic cancer, melanoma, thyroid cancer, head and neck cancer, salivary gland cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, uterine cancer, vulvar cancer, bladder cancer, kidney cancer, squamous cell carcinoma, osteosarcoma, chondrosarcoma, soft tissue sarcoma, gastrointestinal stromal tumor, Kaposi's sarcoma, rhabdomyosarcoma, or neuroblastoma.

7. The use according to claim 6, characterized in that: The soft tissue sarcoma is either leiomyosarcoma or Ewing's sarcoma.

8. The use according to claim 6, characterized in that: The malignant tumors of the hematopoietic system are leukemia, multiple myeloma, and lymphoma; the lung cancer is non-small cell lung cancer or small cell lung cancer; the tumors of the central nervous system are glioma, dysplastic neuroepithelial tumor, medulloblastoma, retinoblastoma, germ cell tumor, or teratoma; and the kidney cancer is renal cell carcinoma.

9. The use according to claim 8, characterized in that: The non-small cell lung cancer is squamous cell carcinoma; the glioma is glioblastoma multiforme or mixed glioma; the renal cell carcinoma is clear cell carcinoma.

10. A combination antitumor drug, characterized in that: It contains the compounds and antitumor drugs described in any one of claims 1 to 2, in the same or different unit formulations for simultaneous or separate administration, and a pharmaceutically acceptable carrier.

11. The combination drug according to claim 10, characterized in that: The anti-tumor drug is a chemotherapy drug.

12. The combination drug according to claim 11, characterized in that: The chemotherapy drugs mentioned are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

13. An antitumor composition, characterized in that: It consists of the compound described in any one of claims 1 to 2 and an antitumor drug.

14. The composition according to claim 13, characterized in that: The anti-tumor drug is a chemotherapy drug.

15. The composition according to claim 14, characterized in that: The chemotherapy drugs mentioned are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

16. An antitumor drug, characterized in that: It is a formulation made with the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, as the active ingredient, plus a pharmaceutically acceptable carrier.

17. Use of the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, in combination with an antitumor drug or radiotherapy in the preparation of an antitumor drug.

18. The use according to claim 17, characterized in that: The anti-tumor drug is a chemotherapy drug.

19. The use according to claim 18, characterized in that: The chemotherapy drugs mentioned are cisplatin, doxorubicin, olaparib, bleomycin, doxorubicin, etoposide, oxaliplatin, carboplatin, pentorubicin, idarubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, MEDI4736, AZD1775, AZD6738, AZD1390, or AZD0156.

Images