Preparation and Application of Wee1 Kinase Inhibitor

Abstract

The present invention discloses the preparation and application of Wee1 kinase inhibitors. Specifically, it relates to a compound represented by formula (I) or formula (II) and its pharmaceutically acceptable salts, a pharmaceutical composition containing the compound or its pharmaceutically acceptable salts, the use of the compound or its pharmaceutically acceptable salts in the preparation of a medicament for treating or preventing Wee1 kinase-related diseases, especially tumors. This is a class of heterocyclic compounds. At the same time, a preparation method of the pharmaceutical composition of the compound or its pharmaceutically acceptable salts is disclosed. Each substituent in formula (I) or formula (II) is the same as the definition in the specification.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and more specifically, to novel compounds having Wee1 kinase inhibitory activity, their preparation methods, and the use of such compounds in the preparation of anti-tumor drugs. Background Art

[0002] Wee-1 protein kinase is an important negative regulatory protein in the cell cycle checkpoint. The cell cycle checkpoints include the G1 checkpoint for the transition from G1 (cell resting phase) to S phase (DNA synthesis phase), the G2 checkpoint for the transition from G2 (cell division preparation phase) to M (cell division phase), and the spindle checkpoint for the transition from metaphase to anaphase in M phase. Wee-1 protein kinase plays an important role in the G2 checkpoint. The entry of cells into M phase depends on the activity of CDK1 kinase. Wee-1 inhibits the activity of CDK1 by phosphorylating Tyr15 of CDK1 protein, preventing cells from entering M phase (cell division phase). While Polo kinase phosphorylates Wee-1, activating the degradation of Wee-1 protein and promoting cells to enter M phase. Thus, it can be seen that the activity of Wee-1 kinase determines the activity of the G2 checkpoint, and further regulates the transition of cells from G2 to M phase [Cell Cycle, 2013, 12(19): 3159-64.].

[0003] When the DNA of cells is damaged, the G1, S, and G2 checkpoints delay the entry of cells into the division phase, gaining time for the repair of damaged DNA before cells enter division, thus ensuring the integrity of the genome. The key regulatory factor P53 of the G1 checkpoint is in a mutant form in many malignant tumor cells (PNAS, 2007, 104(10): 3753-3758). Tumor cells with defective P53 function, when their DNA is damaged, cannot arrest the cell cycle in G1 phase, and thus are more dependent on the G2 checkpoint. In response to DNA damage, the G2 checkpoint inhibits the phosphorylation of CDK1 through two parallel and interconnected pathways, thereby delaying the entry of cells into the division phase. According to the type of DNA damage, the ataxia-telangiectasia mutated (ATM) protein kinase or the ataxia-telangiectasia related (ATR) protein kinase is activated (Oncotarget, 2016, 7(31): 49902-49916).

[0004] Therefore, inhibiting the G2 checkpoint can selectively kill tumor cells. The important role of Wee-1 kinase activity in the G2 checkpoint suggests that Wee-1 kinase determines the repair or death of tumor cells after DNA damage. Inhibiting Wee-1 activity can promote tumor cells with unrepaired DNA damage after DNA damage to enter the M phase and induce apoptosis [Curr Clin Pharmacol, 2010.5(3):186-91].

[0005] WEE1 is overexpressed in many malignant tumors, such as liver cancer, breast cancer, malignant glioma, melanoma, adult and pediatric brain tumors. Some of these tumor cells have abnormal G1 checkpoints. If the activity of WEE1 is inhibited, it will lead to G2 checkpoint failure. At this time, cells with damaged DNA that have not been repaired will continue to divide and ultimately die by division. (Molecular Cancer Therapeutics, 2013, 12(12):2675-2684.) Whether by pyrimidine derivatives (PD0166285) or small interfering RNA knockdown, inhibiting the activity of WEE1 will make ovarian cancer, colon cancer, cervical cancer, osteosarcoma, malignant glioma and lung cancer cells more sensitive to DNA damage caused by radiation and topoisomerase inhibition. Therefore, both single-agent and combination use of WEE1 inhibitors have broad development prospects (Cancer Biology & Therapy, 2010, 9(7):523-525).

[0006] AstraZeneca's Wee1 inhibitor AZD1775 has entered clinical phase II. More than 30 clinical trials are under development and have shown good therapeutic effects. Currently, the relevant Wee1 inhibitor patents mainly include: WO2007126122, WO2008133866, WO2011034743, WO2013039854, WO2018090939, WO2018133829, WO2019011228, WO2019037678, WO2019085933, WO2019173082, WO2020083404, WO2020192581, WO2020221358, WO2021043152. However, there is currently no marketed product for this target. Therefore, it is of great significance to develop more efficient inhibitors that inhibit this target. Summary of the Invention

[0007] A compound represented by the general formula (I), its stereoisomers, pharmaceutically acceptable salts, polymorphs or isomers, wherein the structure of the compound represented by the general formula (I) is as follows:

[0008]

[0009] Wherein,

[0010] each L1 is independently selected from a bond, O, NH, CH2, CO or S each time it appears;

[0011] each L2 is independently selected from a bond, O, NH, CH2, CO or S each time it appears;

[0012] each X1 and X2 is independently selected from N or CR9 each time it appears;

[0013] when a is a single bond, X3 is selected from NR1 or NH;

[0014] when a is a double bond, X3 is selected from N;

[0015] each ring B is independently selected from 3- to 8-membered heterocycles each time it appears, and each 3- to 8-membered heterocycle independently contains 1, 2, 3 or 4 heteroatoms selected from N, O, or S each time it appears; each B is independently optionally substituted with 1, 2, 3, 4, 5 or 6 R 11 substituted or unsubstituted;

[0016] each Ar1 is independently selected from phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl or 10-membered heteroaryl each time it appears, and each heteroaryl independently contains 1, 2, 3 or 4 heteroatoms selected from N, O, or S each time it appears; each Ar1 is independently optionally substituted with 1, 2, 3, 4, 5 or 6 R 12 substituted or unsubstituted;

[0017] each Ar2 is independently selected from phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl or 10-membered heteroaryl each time it appears, and each heteroaryl independently contains 1, 2, 3 or 4 heteroatoms selected from N, O, or S each time it appears; each Ar2 is independently optionally substituted with 1, 2, 3, 4, 5 or 6 R 13 substituted or unsubstituted;

[0018] each R 10 is independently selected from deuterium, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 、C 1-6 heteroalkyl, -C 1-6 alkylene-(OR6) 1-3 、-C 1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7 or -C 3-6Carbocyclic group; each R9 is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5 or 6 substituents selected from deuterium, halogen, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7 or -S(O)2NR6R7;

[0019] each R9, R 11 , R 12 , R 13 is independently selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -OR6, -C 1-6 alkylene-(OR6) 1-3 , -O-C 1-6 alkylene-(halogen) 1-3 , -SR6, -S-C 1-6 alkylene-(halogen) 1-3 , -NR6R7, -C 1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, -S(O)2NR6R7 or -C 3-6 carbocyclic group; each R9, R 11 , R 12 , R 13 is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5 or 6 substituents selected from deuterium, halogen, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7 or -S(O)2NR6R7;

[0020] each R1 is independently selected from or -C 1-6 alkyl, -C 2-6 alkenyl, -C 2- alkynyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -OR6, -C 1-6Alkylene-(OR6) 1-3 、-O-C 1-6 Alkylene-(halogen) 1-3 、-SR6、-S-C 1-6 Alkylene-(halogen) 1-3 、-NR6R7、-C 1-6 Alkylene-NR6R7、-C(=O)R6、-C(=O)OR6、-OC(=O)R6、-C(=O)NR6R7、-NR6C(=O)R7、-S(O)2NR6R7、-C 3-6 Carbocyclic group, 3- to 8-membered heterocycle; each 3- to 8-membered heterocycle independently contains 1, 2, 3, or 4 heteroatoms selected from N, O, or S at each occurrence; each R1 is independently optionally substituted or unsubstituted with 1, 2, 3, 4, 5, or 6 substituents selected from deuterium, halogen, -C 1-6 Alkyl, -C 1-6 Alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, or -S(O)2NR6R7;

[0021] Wherein:

[0022] Each L3 is independently selected from a bond, O, NH, CO, or S at each occurrence;

[0023] Each ring A is a C 3-10 Carbocyclic ring, and the Can be attached to the same carbon atom or different atoms of the ring A;

[0024] Each R2 is -OR6, -NR6R7, -SR6, -S(=O)R6, -S(=O)2R6, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclic group, each heterocyclic group and heteroaryl independently contains 1, 2, 3, or 4 heteroatoms selected from N, O, S, S=O, or S(=O)2 at each occurrence, and each R3 is independently optionally substituted or unsubstituted with 1, 2, 3, 4, 5, or 6 R 19 Substituents;

[0025] Each R3 and R4 are independently selected from deuterium, hydrogen, halogen, -C 1-6 Alkyl, ,, -C 2-6 Alkenyl, -C 2-6 Alkynyl, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, or -S(O)2NR6R7, or -C 3-10The carbocyclic group, each heterocyclic group and heteroaryl group each independently contain 1, 2, 3 or 4 heteroatoms selected from N, O, S, S=O or S(=O)2 upon each occurrence; each R3 and R4 are each independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5 or 6 substituents selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7 or -S(O)2NR6R7;

[0026] Each R5 is independently selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -OR6, -C 1-6 alkylene-(OR6) 1-3 , -O-C 1-6 alkylene-(halogen) 1-3 , -SR6, -S-C 1-6 alkylene-(halogen) 1-3 , -NR6R7, -C 1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, -S(O)2NR6R7 or -C 3-6 carbocyclic group, each heterocyclic group and heteroaryl group each independently contain 1, 2, 3 or 4 heteroatoms selected from N, O, S, S=O or S(=O)2 upon each occurrence; each R3 and R4 are each independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5 or 6 substituents selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7 or -S(O)2NR6R7;

[0027] Each Ar3 is independently selected from phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl or 10-membered heteroaryl, each heteroaryl group each independently contain 1, 2, 3 or 4 heteroatoms selected from N, O, or S upon each occurrence; each Ar3 is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5 or 6 R 13 substituents;

[0028] Each R 13 is independently selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -OR6, -C 1-6 alkylene-(OR6) 1-3 , -O-C 1-6 alkylene-(halogen) 1-3 , -SR6, -S-C 1-6 alkylene-(halogen) 1-3 , -NR6R7, -C1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, -S(O)2NR6R7 or -C 3-6 carbocyclic group; each R 12 is independently optionally substituted or unsubstituted with 1, 2, 3, 4, 5 or 6 substituents selected from deuterium, halogen, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7 or -S(O)2NR6R7;

[0029] Each R6 and R7 is independently selected from hydrogen or -C1-6 alkyl each time it appears, and each R6 and R7 is independently optionally substituted or unsubstituted with 1, 2, 3, 4, 5 or 6 R8; or R6 and R7 together with the N atom to which they are commonly attached form a 3-10 membered heterocycle, the 3-10 membered heterocycle may further contain 1, 2, 3 or 4 heteroatoms selected from N, O, S, S(=O) or S(=O)2, and the 3-10 membered heterocycle is independently optionally substituted or unsubstituted with 1, 2, 3, 4, 5 or 6 R8;

[0030] Each R8 is independently selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -O-C 1-6 alkylene-(halogen) 1-3 , -SC 1-6 alkyl, -S-C 1-6 alkylene-(halogen) 1-3 , -NC 1- 6C 1-6, -C 1-6 Alkylene - NC 1-6 Alkyl C 1-6 Alkyl, -C(=O)C 1-6 Alkyl, -C(=O)OC 1-6 Alkyl, -OC(=O)C 1-6 Alkyl, -C(=O)NC 1-6 Alkyl C 1-6 Alkyl, -NC 1-6 Alkyl C(=O)C 1-6 Alkyl, -S(O)2NC 1-6 Alkyl C 1-6 Alkyl or -C 3-6 Carbocyclic group;

[0031] n is selected from 0, 1, 2, 3, 4, 5 or 6;

[0032] s is selected from 0, 1, 2, 3, 4, 5 or 6;

[0033] p is selected from 0, 1, 2, 3, 4, 5 or 6;

[0034] q is selected from 0, 1, 2, 3, 4, 5 or 6.

[0035] In some embodiments, each R1 is selected from the following structures:

[0036]

[0037]

[0038] Pharmaceutically acceptable salts are selected from the following compounds, their isomers, solvates or their precursors, or their pharmaceutically acceptable salts:

[0039]

[0040]

[0041]

[0042]

[0043] A compound having the formula (II), its stereoisomers, pharmaceutically acceptable salts, polymorphs or isomers, wherein the structure of the compound having the formula (II) is as follows:

[0044]

[0045] Wherein,

[0046] Each L1 is independently selected from a bond, O, NH, CH2, CO or S each time it appears;

[0047] Each L2 is independently selected from a bond, O, NH, CH2, CO, or S each time it appears;

[0048] Each X1, X2 is independently selected from N or C each time it appears;

[0049] Each R1 is independently selected from deuterium, -C 1-6 alkyl, -C 1-6 alkenyl, -C 1-6 alkynyl, phenyl, 6-membered heteroaryl, -C 1-6 alkylene-(halogen) 1-3 、-C 1-6 alkenylene-(halogen) 1-3 、-C 1-6 alkynylene-(halogen) 1-3 、C 1-6 heteroalkyl, -C 1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, -S(O)2NR6R7, or -C 3-6 carbocyclic group; each R1 is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5, or 6 substituents selected from deuterium, halogen, -C 1-6 alkyl, -C 1-6 alkoxy, oxo, -OR6, -NR6R7, -CN, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, or -S(O)2NR6R7;

[0050] Each R2 is independently selected from phenyl, naphthyl, 6-membered heteroaryl, 9-membered heteroaryl, or 10-membered heteroaryl each heteroaryl independently contains 1, 2, 3, or 4 heteroatoms selected from N, O, or S each time it appears; each Ar2 is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5, or 6 R8 each time it appears;

[0051] Each ring B is independently selected from 3-8 membered heterocycles, the 3-8 membered heterocycles independently contain 1, 2, 3, or 4 heteroatoms selected from N, O, or S each time it appears; each B is independently optionally substituted or unsubstituted by 1, 2, 3, 4, 5, or 6 R9 each time it appears;

[0052] Each Ar2, each occurrence independently, is selected from phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl or 10-membered heteroaryl, and each heteroaryl, each occurrence independently, contains 1, 2, 3 or 4 heteroatoms selected from N, O, or S; each Ar2, each occurrence independently, is optionally substituted with 1, 2, 3, 4, 5 or 6 R 10 substituted or unsubstituted;

[0053] Each R8, R9, R 10 , each occurrence independently, is selected from deuterium, halogen, oxo, -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN, -OR6, -C 1-6 alkylene-(OR6) 1-3 , -O-C 1-6 alkylene-(halogen) 1-3 , -SR6, -S-C 1-6 alkylene-(halogen) 1-3 , -NR6R7, -C 1-6 alkylene-NR6R7, -C(=O)R6, -C(=O)OR6, -OC(=O)R6, -C(=O)NR6R7, -NR6C(=O)R7, -S(O)2NR6R7 or -C 3-6 carbocyclic group;

[0054] Each R6 and R7, each occurrence independently, is selected from hydrogen or -C 1-6 alkyl, -C 1-6 alkylene-(halogen) 1-3 , C 1-6 heteroalkyl, -CN;

[0055] In some embodiments, the compound of formula (I) or its isomer, solvate or precursor, or a pharmaceutically acceptable salt thereof is selected from the following compounds, their isomers, solvates or precursors, or their pharmaceutically acceptable salts:

[0056]

[0057] Another object of the present invention is a pharmaceutical composition, which contains a pharmacologically acceptable excipient or carrier, and a compound represented by formula (1A) or formula (1B) of the present invention, its isomer or a pharmaceutically acceptable salt thereof as an active ingredient.

[0058] Another object of the present invention provides the use of the above-mentioned compound of the present invention, its isomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases mediated by Wee1.

[0059] Certain chemical terms

[0060] Unless otherwise stated, the following terms used in the specification and claims

[0061] The representation “C x-y ” used herein to denote a range of carbon atom numbers, where x and y are both integers. For example, C 3-8 Cycloalkyl means a cycloalkyl group having 3 to 8 carbon atoms, i.e., a cycloalkyl group having 3, 4, 5, 6, 7, or 8 carbon atoms. It should also be understood that “C 3-8 ” also includes any sub-range therein. For example, C 3-7 、C 3-6 、C 4-7 、C 4-6 、C 5-6 etc.

[0062] “Alkyl” refers to a straight-chain or branched-chain hydrocarbon group containing 1 to 20 carbon atoms, such as 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, and 2-ethylbutyl. The alkyl may be substituted or unsubstituted.

[0063] “Alkenyl” refers to a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon double bond and usually 2 to 20 carbon atoms, such as 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Non-limiting examples of alkenyl include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1,4-pentadienyl, and 1,4-butadienyl. The alkenyl may be substituted or unsubstituted.

[0064] “Alkynyl” refers to a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon triple bond and usually 2 to 20 carbon atoms, such as 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl. The alkynyl may be substituted or unsubstituted.

[0065] "Cycloalkyl" refers to a saturated cyclic hydrocarbon radical substituent containing 3 to 14 carbon ring atoms. The cycloalkyl can be a monocyclic carbon ring, usually containing 3 to 7 carbon ring atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Optionally, the cycloalkyl can be a bicyclic or tricyclic fused together, such as decahydronaphthyl, and the cycloalkyl can be substituted or unsubstituted.

[0066] "Heterocyclic group", "heterocycloalkyl", "heterocycle" refer to a stable 3- to 18-membered monovalent non-aromatic ring, including 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, the heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, which may contain fused rings, spiro rings, or bridged ring systems. The nitrogen, carbon, or sulfur on the heterocyclic group can be optionally oxidized, the nitrogen atom can be optionally quaternized, and the heterocyclic group can be partially or fully saturated. The heterocyclic group can be connected to the rest of the molecule through a single bond via a carbon atom or a heteroatom on the ring. A heterocyclic group containing fused rings can contain one or more aromatic rings or heteroaromatic rings, as long as the atom on the non-aromatic ring is connected to the rest of the molecule. For the purposes of this application, the heterocyclic group is preferably a stable 4- to 11-membered monovalent non-aromatic monocyclic or bicyclic ring, which contains 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur, and more preferably a stable 4- to 8-membered monovalent non-aromatic monocyclic ring, which contains 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Non-limiting examples of heterocyclic groups include azepanyl, azetidinyl, decahydroisoquinolinyl, dihydrofuranyl, dihydroindolyl, dioxolanyl, 1,1-dioxo-thiomorpholinyl, imidazolidinyl, imidazolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazinyl, piperazinyl, piperidinyl, 4-piperidinone, pyranyl, pyrazolidinyl, pyrrolidinyl, quinuclidinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, etc.

[0067] "Spiroheterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members, where a single atom (called the spiro atom) is shared between monocyclic rings, and one or more of the ring atoms are selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. These can contain one or more double bonds, but none of the rings have a completely conjugated electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of spiro atoms shared between rings, the spirocycloalkyls are divided into monospiroheterocyclic groups, bispiroheterocyclic groups, or multispiroheterocyclic groups, preferably monospirocycloalkyls and bispirocycloalkyls. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiro group. Non-limiting examples of spiroheterocyclic groups include:

[0068]

[0069] "Fused heterocyclic group" refers to a polycyclic heterocyclic group having 5 to 20 ring atoms, in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π - electron system, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it has 6 to 14 ring atoms, more preferably 7 to 10 ring atoms. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic alkyl groups, preferably bicyclic or tricyclic, more preferably 5 - membered / 5 - membered or 5 - membered / 6 - membered bicyclic fused heterocyclic groups. Non - limiting examples of the fused heterocyclic group include:

[0070]

[0071] "Aryl" or "aromatic group" refers to an aromatic monocyclic or fused polycyclic group containing 6 to 14 carbon atoms, preferably having 6 to 10 ring atoms, such as phenyl and naphthyl, and more preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, and the ring connected to the parent structure is the aryl ring.

[0072] "Heteroaryl" or "heteroaromatic group" refers to a 5 - 16 - membered ring system containing 1 - 15 carbon atoms, preferably 1 - 10 carbon atoms, 1 - 4 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. Unless otherwise specified, the heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic system, which may contain fused or bridged ring systems, provided that the point of attachment to the rest of the molecule is an aromatic ring atom. The nitrogen, carbon and sulfur atoms on the heteroaryl ring may be selectively oxidized, and the nitrogen atom may be selectively quaternized. For the purposes of this invention, the heteroaryl is preferably a stable 4 - 11 - membered monocyclic aromatic ring containing 1 - 3 heteroatoms selected from nitrogen, oxygen and sulfur, and more preferably a stable 5 - 8 - membered monocyclic aromatic ring containing 1 - 3 heteroatoms selected from nitrogen, oxygen and sulfur. Non - limiting examples of the heteroaryl include acridinyl, azepinyl, benzimidazolyl, benzindolyl, benzodioxolyl, benzodioxolyl, benzofuranone, benzofuranyl, benzonaphthofuranyl, benzopyrone, benzopyranyl, benzopyrazolyl, benzothiadiazolyl, benzothiazolyl, benzotriazolyl, furyl, imidazolyl, indazolyl, indolyl, oxazolyl, purinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinuclidinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazinyl, triazolyl, etc. In this application, the heteroaryl is preferably a 5 - 8 - membered heteroaryl containing 1 - 3 heteroatoms selected from nitrogen, oxygen and sulfur, and more preferably pyridyl, pyrimidinyl, thiazolyl. The heteroaryl may be substituted or unsubstituted.

[0073] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0074] "Hydroxyl" means -OH, "amino" means -NH2, "amide group" means -NHCO-, "cyano" means -CN, "nitro" means -CN, "isocyano" means -NC, and "trifluoromethyl" means -CF3.

[0075] As used herein, the term "heteroatom" or "hetero" when used alone or as part of other components refers to atoms other than carbon and hydrogen, and the heteroatoms are independently selected from oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and tin, but are not limited to these atoms. In embodiments where two or more heteroatoms are present, the two or more heteroatoms may be the same as each other, or some or all of the two or more heteroatoms may be different from each other.

[0076] As used herein, the term "fused" or "fused ring" when used alone or in combination refers to a cyclic structure in which two or more rings share one or more bonds.

[0077] As used herein, the term "spiro" or "spiro ring" when used alone or in combination refers to a cyclic structure in which two or more rings share one or more atoms.

[0078] "Optional" or "optionally" means that the subsequent described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and this description includes the cases where the heterocyclic group is substituted with an alkyl group and the cases where the heterocyclic group is not substituted with an alkyl group.

[0079] "Substituted" means that one or more atoms in a group, preferably 5, more preferably 1 to 3 atoms, are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are at their possible chemical positions, and those skilled in the art can determine (by experiment or theory) the possible or impossible substitutions without excessive effort. For example, a free amino group or hydroxyl group may be unstable when combined with a carbon atom having an unsaturated (such as an olefin) bond. The substituents include but are not limited to hydroxyl, amino, halogen, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, etc.

[0080] "Pharmaceutical composition" refers to a composition containing one or more compounds described herein or their pharmaceutically acceptable salts or prodrugs, as well as other components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to an organism, facilitate the absorption of the active ingredient, and thereby exert biological activity.

[0081] "Isomers" refer to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the spatial arrangement of their atoms. Isomers with different spatial arrangements of their atoms are called "stereoisomers". Stereoisomers include optical isomers, geometric isomers, and conformational isomers. The compounds of the present invention may exist in the form of optical isomers. These optical isomers are of the "R" or "S" configuration according to the configuration of the substituents around the chiral carbon atom. Optical isomers include enantiomers and diastereomers. Methods for preparing and separating optical isomers are known in the art.

[0082] The compounds of the present invention may also exist as geometric isomers. The present invention contemplates various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl groups, or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen bonds are designated as the Z or E configuration, and substituents around cycloalkyl or heterocyclic groups are designated as the cis or trans configuration.

[0083] The compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism.

[0084] It should be understood that the present invention includes any tautomeric or stereoisomeric forms and mixtures thereof, and is not limited to any one tautomeric or stereoisomeric form used in the naming or chemical structural formula of the compound.

[0085] "Isotopes" are all isotopes of atoms that occur in the compounds of the present invention. Isotopes include those atoms that have the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example but not limited to 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. Isotope-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples using appropriate isotope-labeled reagents in place of non-isotope-labeled reagents. Such compounds have various potential uses, such as as standards and reagents in the determination of biological activity. In the case of stable isotopes, such compounds have the potential to favorably alter biological, pharmacological, or pharmacokinetic properties.

[0086] "Prodrug" means that the compounds of the present invention can be administered in the form of a prodrug. A prodrug refers to a derivative that is converted into the bioactive compound of the present invention under physiological conditions in vivo, such as by oxidation, reduction, hydrolysis, etc. (each of which utilizes enzymes or proceeds without the participation of enzymes). Examples of prodrugs are the following compounds: wherein the amino group in the compound of the present invention is acylated, alkylated or phosphorylated, such as eicosanoylamino group, propanamide group, pivaloyloxymethylamino group, or wherein the hydroxyl group is acylated, alkylated, phosphorylated or converted into a borate, such as acetoxy group, palmitoyloxy group, pivaloyloxy group, succinyloxy group, fumaroyloxy group, propanoyloxy group, or wherein the carboxyl group is esterified or amidated, or wherein the mercapto group forms a disulfide bridge with a carrier molecule that selectively delivers the drug to the target and / or to the cytosol of the cell, such as a peptide. These compounds can be prepared from the compounds of the present invention according to known methods.

[0087] "Pharmaceutically acceptable salt" or "pharmaceutically acceptable" means made from a pharmaceutically acceptable base or acid, including inorganic bases or acids and organic bases or acids. In the case where the compounds of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Thus, the compounds of the present invention containing acidic groups can exist in the form of salts and can be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with amines or organic amines, such as primary amines, secondary amines, tertiary amines, cyclic amines, etc., such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, ethanolamine, dicyclohexylamine, ethylenediamine, purine, piperazine, piperidine, choline and caffeine, etc. Particularly preferred organic bases are salts of isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. The compounds of the present invention containing basic groups can exist in the form of salts and can be used according to the present invention in the form of their addition with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, phosphorous acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to those skilled in the art. If the compounds of the present invention contain both acidic and basic groups in the molecule, the present invention includes inner salts or inner ammonium salts in addition to the salt forms mentioned above. Each salt is obtained by conventional methods known to those skilled in the art, such as by contacting these with organic or inorganic acids or bases in a solvent or dispersant or by anion exchange or cation exchange with other salts.

[0088] Accordingly, as used in the present application, when referring to "compound", "compounds of the present invention" or "compounds of the invention", it includes all forms of said compounds, such as their prodrugs, stable isotope derivatives, pharmaceutically acceptable salts, isomers, meso forms, racemates, enantiomers, diastereoisomers and mixtures thereof.

[0089] As used herein, the term "tumor" includes benign tumors and malignant tumors (such as cancer).

[0090] As used herein, the term "cancer" includes various malignant tumors in which Bruton's tyrosine kinase is involved in its occurrence, including but not limited to non-small cell lung cancer, esophageal cancer, melanoma, rhabdomyosarcoma, carcinoma, multiple myeloma, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer and liver cancer (such as hepatocellular carcinoma), more specifically liver cancer, gastric cancer and bladder cancer.

[0091] As used herein, the term "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of at least one agent or compound that, when administered, is sufficient to alleviate to some extent one or more symptoms of the disease or disorder being treated. The result can be the reduction and / or alleviation of signs, symptoms or causative factors and / or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant alleviation of the disorder. Techniques such as dose escalation trials can be used to determine the effective amount suitable for any individual case.

[0092] As used in the present invention, the term "polymorph" or "polymorphism" means that the compounds of the present invention have multiple crystal lattice forms, and some compounds of the present invention may have more than one crystal form. The present invention encompasses all polymorphic forms or mixtures thereof.

[0093] Intermediate compounds of the compounds of the present invention and their polymorphs are also within the scope of the present invention.

[0094] Crystallization often produces solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an entity formed by the combination of one or more molecules of a compound of the present invention and one or more solvent molecules.

[0095] The solvent can be water, in which case the solvate is a hydrate. Additionally, it can also be an organic solvent. Therefore, the compounds of the present invention can exist as hydrates, including monohydrates, dihydrates, hemihydrates, trihydrates, tetrahydrates, etc., as well as the corresponding solvated forms. The compounds of the present invention can be true solvates, but in some other cases, the compounds of the present invention may only accidentally retain water or a mixture of water and some other solvents. The compounds of the present invention can react in a solvent or precipitate or crystallize in a solvent. The solvates of the compounds of the present invention are also included within the scope of the present invention.

[0096] As used herein, the term "acceptable" in relation to a formulation, composition or ingredient means that it has no continuing harmful effect on the overall health of the treated subject.

[0097] As used herein, the term "pharmaceutically acceptable" means a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition.

[0098] "Pharmaceutically acceptable carriers" include, but are not limited to, adjuvants, carriers, excipients, auxiliaries, deodorants, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants and wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the relevant government administrative departments for use in humans and domestic animals.

[0099] As used herein, the terms "subject", "patient", "object" or "individual" refer to an individual suffering from a disease, disorder or condition, etc., including mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (such as chimpanzees and other apes and monkeys); domestic animals, such as cows, horses, sheep, goats, pigs; household animals, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. Examples of non-human mammals include, but are not limited to, birds and fish, etc. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0100] As used herein, the term "treatment" refers to the treatment of a relevant disease or condition in a mammal, particularly a human, including

[0101] (i) preventing a mammal, particularly a mammal that has been previously exposed to a disease or condition but has not been diagnosed with the disease or condition, from developing the corresponding disease or condition;

[0102] (ii) inhibiting a disease or condition, i.e., controlling its development;

[0103] (iii) alleviating a disease or disorder, i.e., causing the regression of the disease or disorder to slow down;

[0104] (iv) alleviating the symptoms caused by a disease or disorder.

[0105] As used herein, the terms "disease" and "disorder" may be used interchangeably or may have different meanings, because for certain specific diseases or disorders, there are no known causative agents (so the cause of the disease is still unclear), and thus they cannot be recognized as diseases but can only be regarded as unwanted conditions or syndromes, and more or less specific symptoms of such syndromes have been confirmed by clinical researchers.

[0106] As used herein, the terms "administering", "applying", "dosing", etc. refer to methods that can deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral route, duodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), topical administration, and rectal administration. In a preferred embodiment, the compounds and compositions discussed herein are administered orally.

[0107] Synthesis of Compounds

[0108] The preparation methods of the compounds of formula (I) or formula (II) of the present invention are specifically described below, but these specific methods do not constitute any limitation to the present invention.

[0109] The compounds of formula (I) or formula (II) described above can be synthesized using standard synthetic techniques or well-known techniques in combination with the methods described herein. In addition, the solvents, temperatures, and other reaction conditions mentioned herein can be changed. The starting materials for the synthesis of the compounds of formula (I) or formula (II) can be obtained by synthesis or from commercial sources. The compounds described herein and other related compounds with different substituents can be synthesized using well-known techniques and starting materials, including those found in March, ADVANCED ORGANIC CHEMISTRY 4th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4th Ed., Vols. A and B (Plenum 2000, 2001), Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3 <rd>The method in Ed., (Wiley 1999). The general method for preparing the compound can be changed by using appropriate reagents and conditions for introducing different groups in the molecular formula provided herein.

[0110] On the one hand, the compounds described herein are according to methods known in the art. However, the conditions of the method, such as reactants, solvents, bases, the amounts of the compounds used, reaction temperature, reaction time required, etc. are not limited to the following explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention pertains. On the one hand, the present invention also provides a method for preparing the compound of formula (II) or formula (II), which is prepared by the following method:

[0111] Method A

[0112]

[0113] Method B

[0114]

[0115] Method C

[0116]

[0117] Method D

[0118] Specific implementation methods

[0119] The present invention also provides a method for preparing the compound. The preparation of the compound of general formula (I) of the present invention can be completed by the following exemplary methods and examples, but these methods and examples should not be considered as limiting the scope of the present invention in any way. The compounds of the present invention can also be synthesized by synthetic techniques known to those skilled in the art, or by comprehensively using methods known in the art and the methods described in the present invention. The product obtained in each step is obtained by separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation, etc. The starting materials and chemical reagents required for the synthesis can be synthesized conventionally according to the literature (reaxys) or purchased.

[0120] Unless otherwise specified, the temperature is in degrees Celsius. The reagents are purchased from commercial suppliers such as Chem blocks Inc, Astatech Inc or Macklin, and these reagents can be used directly without further purification unless otherwise specified.

[0121] Unless otherwise specified, the following reactions are carried out at room temperature, in an anhydrous solvent, under a positive pressure of nitrogen or argon or using a drying tube; the glassware is dried by baking and / or heating.

[0122] Unless otherwise specified, silica gel of 200 - 300 mesh from Qingdao Marine Chemical Factory was used for column chromatography purification; precast thin-layer chromatography silica gel plates (HSGF254) produced by Yantai Chemical Industry Research Institute were used for preparative thin-layer chromatography; a Thermo LCD Fleet type (ESI) liquid chromatography - mass spectrometry combined instrument was used for the determination of MS.

[0123] Nuclear magnetic resonance data (1H NMR) were obtained using a Bruker Avance - 400 MHz or Varian Oxford - 400 Hz nuclear magnetic resonance spectrometer. Solvents used for nuclear magnetic resonance data were CDCl3, CD3OD, D2O, DMS - d6, etc. Tetramethylsilane (0.000 ppm) or residual solvent was used as the reference (CDCl3: 7.26 ppm; CD3OD: 3.31 ppm; D2O: 4.79 ppm; d6 - DMSO: 2.50 ppm). When indicating the diversity of peak shapes, the following abbreviations were used to represent different peak shapes: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak), dd (doublet of doublets), dt (doublet of triplets). If coupling constants were given, they were in Hertz (Hz).

[0124] Example 1

[0125] Preparation of 3-(2,6 - dichlorophenyl)-7-((4-(4 - methylpiperazin - 1 - yl)phenyl)amino)-2-((hexahydro - 1H - pyrrolizin - 7a - yl)methoxy)pyrimido[4,5 - d]pyrimidin - 4(3H)-one (Compound 1)

[0126]

[0127] First step: Preparation of 3-(2,6 - dichlorophenyl)-7 - (methylthio)pyrimido[4,5 - d]pyrimidine - 2,4(1H,3H)-dione

[0128] 10 g of ethyl 4 - amino - 2 - (methylthio)pyrimidine - 5 - carboxylate was dissolved in 150 mL of DMF, then 3.15 g of NaH was added and stirred at room temperature for 5 minutes. 9.70 g of 2,6 - dichlorophenyl isocyanate was added to the reaction solution and stirred at room temperature for 1 hour. Ethyl acetate and 1N hydrochloric acid aqueous solution were added to the reaction solution, and the organic layer was separated. It was washed with saturated brine, dried with anhydrous sodium sulfate, and then the solvent was evaporated. The precipitated solid was solidified with methanol, and the solid was taken out by filtration to obtain 13.7 g of 3-(2,6 - dichlorophenyl)-7 - (methylthio)pyrimido[4,5 - d]pyrimidine - 2,4(1H,3H)-dione as a white solid.

[0129] LC / MS(ESI): m / z = 355 [M+H] + .

[0130] Step 2: Preparation of 2-chloro-3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0131] Dissolve 3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (5.31 g, 15 mmol) in POCl3 (80 mL), add a small amount of N,N-dimethylaniline, and heat under reflux with stirring for 4 h. Then pour it into ice water to quench the reaction, filter to obtain a solid product, wash it with water, and dry it to obtain the crude product, yellow solid 2-chloro-3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one (4.86 g, 78%), which can be used for the next reaction without further purification.

[0132] LC / MS(ESI): m / z = 374 [M+H] + .

[0133] Step 3: Preparation of 2-((tetrahydro-1H-pyrrolo[3,4-c]pyrrol-7a(5H)-yl)methoxy)-3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0134] Mix 2-chloro-3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one (373 mg, 1 mmol), hexahydropyrrolo[3,4-c]pyrrole-7a-methanol (53 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol), a catalytic amount of potassium iodide, and DMF (10 mL), heat to 120 °C, and stir the reaction for 4 h. Cool to room temperature, evaporate the solvent under reduced pressure, and perform column chromatography to obtain a white solid, 2-((tetrahydro-1H-pyrrolo[3,4-c]pyrrol-7a(5H)-yl)methoxy)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one (416 mg, 87%). LC / MS(ESI): m / z = 479 [M+H] + .

[0135] Step 4: Preparation of 2-((tetrahydro-1H-pyrrolo[3,4-c]pyrrol-7a(5H)-yl)methoxy)-3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0136] 2-(Tetrahydro-1H-pyrano[3,4-d]pyrimidin-7a(5H)-yl)methoxy)-7-(methylthio)pyrimido[4,5-d]pyrimidin-4(3H)-one (382 mg, 0.8 mmol) was dissolved in 10 mL of CH2Cl2. m-Chloroperbenzoic acid (522 mg, 3 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with CH2Cl2 and washed with saturated NaHCO3 and Na2S2O3. The organic layer was dried over MgSO4, evaporated under reduced pressure, and purified by column chromatography to obtain the yellow solid 2-(Tetrahydro-1H-pyrano[3,4-d]pyrimidin-7a(5H)-yl)methoxy)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidin-4(3H)-one (392 mg, 96%). LC / MS (ESI): m / z = 511 [M+H] + .

[0137] Step 5: Preparation of 2-(Tetrahydro-1H-pyrano[3,4-d]pyrimidin-7a(5H)-yl)methoxy)-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0138] 2-(Tetrahydro-1H-pyrano[3,4-d]pyrimidin-7a(5H)-yl)methoxy)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidin-4(3H)-one (51 mg, 0.1 mmol) was dissolved in 10 ml of DMF. DIPEA (65 mg, 0.5 mmol) and 4-(4-methylpiperazin-1-yl)aniline (23 mg, 0.12 mmol) were added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was poured into ice water, and the crude product was filtered and then purified by flash column chromatography to obtain the yellow solid 2-(Tetrahydro-1H-pyrano[3,4-d]pyrimidin-7a(5H)-yl)methoxy)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (50 mg, 83%). 1 H NMR (300 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.12 (s, 1H), 7.42 - 7.99 (m, 4H), 6.88 - 7.29 (m, 3H), 4.41 (s, 2H), 3.46 - 3.61 (m, 4H), 3.01 - 3.15 (m, 4H), 2.48–2.58 (m, 4H), 2.01 - 2.27 (m, 5H), 1.87 - 1.96 (m, 4H), 1.78 - 1.80 (m, 2H), 1.69 - 1.76 (m, 2H); LC / MS (ESI): m / z = 622.2 [M+H] + .

[0139] Example 2

[0140] Preparation of 3-(2,6-difluorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 2)

[0141]

[0142] Compound 2 (51 mg, yield 86%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 589.3 [M+H] + 。

[0143] Example 3

[0144] Preparation of 3-(2,6-dimethylphenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 3)

[0145]

[0146] Compound 3 (45 mg, yield 78%) was obtained by a method similar to that of Example 1. 1 H NMR (300 MHz, CDCl3) δ 9.22 (s, 1H), 7.06 - 7.78 (m, 7H), 4.19 (s, 2H), 3.31 (m, 4H), 2.60 - 2.68 (m, 6H), 2.40 (s, 3H), 2.15 (s, 6H), 2.07 - 2.19 (m, 2H), 1.87 - 1.98 (m, 4H), 1.66 - 1.80 (m, 2H); LC / MS (ESI): m / z = 581.3 [M+H] + 。

[0147] Example 4

[0148] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((adamantan-1-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 4)

[0149]

[0150] Compound 4 (53 mg, yield 82%) was obtained by a method similar to that of Example 1. 1 1H NMR (300 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.12 (s, 1H), 7.42 - 7.80 (m, 4H), 6.88 - 7.28 (m, 3H), 4.04 (s, 2H), 3.46 - 3.61 (m, 4H), 3.01 - 3.15 (m, 4H), 2.01 (m, 3H), 1.69 - 1.72 (m, 12H); LC / MS (ESI): m / z = 647.2 [M+H] + 。

[0151] Example 5

[0152] Preparation of 3-(2,6-difluorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((adamantan-1-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 5)

[0153]

[0154] Compound 5 (56 mg, yield 91%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 614 [M+H] + 。

[0155] Example 6

[0156] Preparation of 3-(2,6-dimethylphenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((adamantan-1-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 6)

[0157]

[0158] Compound 6 (50 mg, yield 83%) was obtained by a method similar to that of Example 1. 1 1H NMR (300 MHz, CDCl3) δ 9.22 (s, 1H), 7.06 - 7.78 (m, 7H), 3.93 (s, 2H), 2.01 (m, 3H), 1.70 - 1.76 (m, 12H); LC / MS (ESI): m / z = 606.3 [M+H] + 。

[0159] Example 7

[0160] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dichlorophenyl)-7-((3-methyl-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazin-8-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 7)

[0161]

[0162] Compound 7 (53 mg, yield 78%) was obtained by a method similar to that of Example 6. LC / MS (ESI): m / z = 675.3 [M+H] + 。

[0163] Example 8

[0164] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dichlorophenyl)-7-((4-(1-methylpiperidin-4-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 8)

[0165]

[0166] Compound 8 (49 mg, yield 76%) was obtained by a method similar to that of Example 6. LC / MS (ESI): m / z = 646 [M+H] + 。

[0167] Example 9

[0168] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dichlorophenyl)-7-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 9)

[0169]

[0170] Compound 9 (56 mg, yield 87%) was obtained by a method similar to that of Example 6. LC / MS (ESI): m / z = 646.3 [M+H] + 。

[0171] Example 10

[0172] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dichlorophenyl)-7-((2'-methyl-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 10)

[0173]

[0174] Compound 10 (46 mg, yield 72%) was obtained by a method similar to that of Example 6. LC / MS (ESI): m / z = 644.2 [M+H] + 。

[0175] Example 11

[0176] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dimethylphenyl)-7-((2'-methyl-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 11)

[0177]

[0178] Compound 11 (41 mg, yield 69%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 603.3 [M+H] + 。

[0179] Example 12

[0180] Preparation of 2-(adamantan-1-ylmethoxy)-3-(2,6-dimethylphenyl)-7-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 12)

[0181]

[0182] Compound 12 (51 mg, yield 84%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 605.3 [M+H] + 。

[0183] Example 13

[0184] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-methylpyrrolidin-2-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 13)

[0185]

[0186] First step: Preparation of 3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

[0187] 3-(2,6-Dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (3.82 g, 8 mmol) was dissolved in 50 mL of CH2Cl2. m-Chloroperbenzoic acid (5.22 g, 30 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with CH2Cl2 and washed with saturated NaHCO3 and Na2S2O3. The organic layer was dried over MgSO4 and concentrated under reduced pressure. Column chromatography gave 3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione as a yellow solid (2.84 g, 92%). LC / MS (ESI): m / z = 388 [M+H] + .

[0188] Step 2: Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

[0189] 3-(2,6-Dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (1.16 g, 3 mmol) was dissolved in 25 mL of DMF. DIPEA (1.94 g, 15 mmol) and 4-(4-methylpiperazin-1-yl)aniline (0.48 g, 0.12 mmol) were added, and the mixture was stirred at room temperature for 24 h. The mixture was poured into ice water, and the crude product was filtered. Then, flash column chromatography gave 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione as a yellow solid (1.33 g, 89%).

[0190] LC / MS (ESI): m / z = 499 [M+H] + 。

[0191] Step 3: Preparation of 2-chloro-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0192] 3-(2,6-Dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (1.25 g, 2.5 mmol) was dissolved in POCl3 (20 mL), a small amount of N,N-dimethylaniline was added, and the mixture was heated under reflux with stirring for 4 h. Then it was poured into ice water for quenching, and the solid product was obtained by filtration, washed with water, and dried to obtain the crude product, yellow solid 2-chloro-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (0.93 g, 72%), which was used for the next reaction without further purification. LC / MS (ESI): m / z = 517 [M+H] + .

[0193] Step 4: Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-methylpyrrolidin-2-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one

[0194] 2-Chloro-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (52 mg, 0.1 mmol), N-methylpyrrolidin-2-methanol (23 mg, 0.2 mmol), potassium carbonate (62 mg, 0.45 mmol), catalytic amount of potassium iodide and DMF (10 mL) were mixed, heated to 120 °C, and stirred for 4 h. After cooling to room temperature, it was poured into water, and the crude product was obtained by filtration and purified by column chromatography to obtain a white solid, 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-methylpyrrolidin-2-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (53 mg, 92%). LC / MS (ESI): m / z = 576 [M+H] + .

[0195] Example 14

[0196] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(((1-(dimethylaminomethyl)cyclopropane)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 14)

[0197]

[0198] Compound 14 (51 mg, yield 84%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 610.2 [M+H] + 。

[0199] Example 15

[0200] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-(pyrrolidin-1-ylmethyl)cyclopropane)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 15)

[0201]

[0202] Compound 15 (58 mg, yield 91%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 636.2 [M+H] + .

[0203] Example 16

[0204] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-methylpiperidin-3-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 16)

[0205] Compound 16 (47 mg, yield 88%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 610 [M+H] + .

[0206] Example 17

[0207] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1,4-dimethylpiperazin-2-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 17)

[0208] Compound 17 (49 mg, yield 79%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 625 [M+H] + .

[0209] Example 18

[0210] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(3-(dimethylamino)propoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 18)

[0211] Compound 18 (49 mg, yield 84%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 584 [M+H] + .

[0212] Example 19

[0213] Preparation of 2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dichlorophenyl)-6-((3-methyl-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazin-8-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 19)

[0214] Compound 19 (60 mg, yield 92%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 650.2 [M+H] + 。

[0215] Example 20

[0216] Preparation of 2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dichlorophenyl)-7-((4-(1-methylpiperidin-4-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 20)

[0217] Compound 20 (50 mg, yield 81%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 621.2 [M+H] + 。

[0218] Example 21

[0219] Preparation of 2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dichlorophenyl)-7-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 21)

[0220] Compound 21 (53 mg, yield 86%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 621.2 [M+H] + 。

[0221] Example 22

[0222] Preparation of 2-((hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dichlorophenyl)-7-((2'-methyl-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 22)

[0223] Compound 22 (46 mg, yield 74%) was obtained by a method similar to that of Example 1. LC / MS (ESI): m / z = 619.2 [M+H] + 。

[0224] Example 23

[0225] Preparation of 2-((Hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dimethylphenyl)-7-((2'-methyl-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 23)

[0226] Compound 23 (50 mg, yield 86%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 578.3 [M+H] + 。

[0227] Example 24

[0228] Preparation of 2-((Hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dimethylphenyl)-7-((3-methyl-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazin-8-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 24)

[0229] Compound 24 (55 mg, yield 91%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 609.3 [M+H] + 。

[0230] Example 25

[0231] Preparation of 2-((Hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dimethylphenyl)-7-((4-(1-methylpiperidin-4-yl)phenyl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 25)

[0232] Compound 25 (50 mg, yield 89%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 580 [M+H] + 。

[0233] Example 26

[0234] Preparation of 2-((Hexahydro-1H-pyrrolizin-7a-yl)methoxy)-3-(2,6-dimethylphenyl)-7-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 26)

[0235] Compound 26 (45 mg, yield 77%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 580 [M+H] + 。

[0236] Example 27

[0237] Preparation of 3-(2,6-Dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-benzyloxypyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 27)

[0238] Compound 27 (52 mg, yield 88%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 589 [M+H] + 。

[0239] Example 28

[0240] Preparation of 3-(2,6-Dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((pyridin-3-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (Compound 28)

[0241] Compound 28 (49 mg, yield 83%) was obtained by a method similar to that of Example 13. LC / MS (ESI): m / z = 590 [M+H] + 。

[0242] Example 29

[0243] Preparation of N-(3-(2,6-Dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-2-yl)nicotinamide (Compound 29)

[0244] Compound 29 (48 mg, yield 79%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 603 [M+H] + 。

[0245] Example 30

[0246] Preparation of N-(3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-2-yl)furan-2-carboxamide (Compound 30)

[0247] Compound 30 (50 mg, yield 84%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 592 [M+H] + 。

[0248] Example 31

[0249] Preparation of N-(3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-2-yl)oxazole-2-carboxamide (Compound 31)

[0250] Compound 31 (53 mg, yield 89%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 593 [M+H] + 。

[0251] Example 32

[0252] Preparation of N-(3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (Compound 32)

[0253] Compound 32 (51 mg, yield 85%) was obtained by a method similar to that of Example 3. LC / MS (ESI): m / z = 606 [M+H] + 。

[0254] Example 33

[0255] Preparation of 1-benzyl-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 33)

[0256]

[0257] First step: Preparation of 1-benzyl-3-(2,6-dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

[0258] 3-(2,6-Dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (354 mg, 1 mmol), benzyl chloride (151 mg, 1.2 mmol), potassium carbonate (620 mg, 4.5 mmol), a catalytic amount of potassium iodide, and DMF (25 mL) were mixed, heated to 120 °C, and stirred for 4 h. After cooling to room temperature, the reaction mixture was poured into water and filtered to obtain a pale white solid, 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-((1-methylpyrrolidin-2-yl)methoxy)pyrimido[4,5-d]pyrimidin-4(3H)-one (347 mg, 78%). LC / MS (ESI): m / z = 446 [M+H] + .

[0259] Step 2: Preparation of 1-benzyl-3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

[0260] 3-(2,6-Dichlorophenyl)-7-(methylthio)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (311 mg, 0.7 mmol) was dissolved in 10 mL of CH2Cl2, and m-chloroperbenzoic acid (359 mg, 2.1 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with CH2Cl2 and washed with saturated NaHCO3 and Na2S2O3. The organic layer was dried over MgSO4, concentrated under reduced pressure, and purified by column chromatography to obtain a yellow solid, 1-benzyl-3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (290 mg, 87%). LC / MS (ESI): m / z = 478 [M+H] + .

[0261] Step 3: Preparation of 1-benzyl-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

[0262] Dissolve 1-benzyl-3-(2,6-dichlorophenyl)-7-(methylsulfonyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (95 mg, 0.2 mmol) in 10 ml of DMF, add DIPEA (78 mg, 0.6 mmol) and 4-(4-methylpiperazin-1-yl)aniline (46 mg, 0.24 mmol), and stir at room temperature for 24 hours. Pour into ice water, filter to obtain the crude product, and then obtain the off-white solid 1-benzyl-3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (104 mg, 89%) by flash column chromatography.

[0263] LC / MS (ESI): m / z = 589 [M+H] + 。

[0264] Example 34

[0265] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(pyridin-4-ylmethyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 34)

[0266]

[0267] Compound 96 (88 mg, yield 74%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 590 [M+H] + 。

[0268] Example 35

[0269] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-((hexahydro-1H-pyrrolizin-7a-yl)methyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 36)

[0270]

[0271] Compound 36 (97 mg, yield 78%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 622 [M+H] + 。

[0272] Example 36

[0273] Preparation of 1-benzyl-3-(2,6-dimethylphenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 36)

[0274]

[0275] Compound 36 (95 mg, yield 87%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 548 [M+H] + 。

[0276] Example 37

[0277] Preparation of 3-(2,6-dimethylphenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(pyridin-4-ylmethyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 37)

[0278]

[0279] Compound 37 (83 mg, yield 76%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 549.3 [M+H] + 。

[0280] Example 38

[0281] Preparation of 3-(2,6-dimethylphenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-((hexahydro-1H-pyrrolizin-7a-yl)methyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 38)

[0282]

[0283] Compound 38 (72 mg, yield 62%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 581 [M+H] + 。

[0284] Example 39

[0285] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(((1-((dimethylamino)methyl)cyclopropyl)methyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 39)

[0286] Compound 39 (84 mg, yield 69%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 610 [M+H] + 。

[0287] Example 40

[0288] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 40)

[0289] Compound 40 (91 mg, yield 72%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 636 [M+H] + 。

[0290] Example 41

[0291] Preparation of 3-(2,6-dichlorophenyl)-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(adamantan-1-ylmethyl)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 41)

[0292] Compound 41 (87 mg, yield 67%) was obtained by a method similar to that of Example 33. LC / MS (ESI): m / z = 647 [M+H] + 。

[0293] Similar to the synthesis of 1-41, the following compounds can be obtained:

[0294]

[0295]

[0296] Example 42

[0297] Preparation of 2-propenyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 42)

[0298]

[0299] First step: Preparation of 2-propenyl-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0300] Methyl 4,6-dichloropyridazine-3-carboxylate (9.89 g, 47.8 mmol) was dissolved in 150 mL of THF, and DIPEA (20.8 ml, 120 mmol) and tert-butyl 1-allylhydrazinecarboxylate (8.22 g, 47.8 mmol) were added. The reaction mixture was refluxed for 72 hours and then concentrated in vacuo. Et2O (50 ml) was added to the residue, and the mixture was filtered. The filtrate was concentrated by evaporation, and the residue was cooled in an ice bath. Then, TFA (40 ml) was added. The resulting solution was stirred at room temperature for 1 h and then at 70 °C for 1 h. The solution was concentrated by evaporation, and the residue was dissolved in THF (50 ml) and cooled in an ice bath. Then, NaH (75 ml) was added. The resulting solution was stirred at RT for 15 minutes and then adjusted to pH = 3 by adding acetic acid. The solution was concentrated by evaporation, and then 100 ml of chloroform and 100 ml of water were added. The organic phase was washed with 50 ml of saturated brine, dried over anhydrous Mg2SO4, concentrated in vacuo, and slurried with n-hexane. The solid precipitate was washed with ethanol and Et2O and then dried in vacuo to obtain the yellow solid compound 2-propenyl-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (4.41 g, 44%). LC / MS (ESI): m / z = 211 [M+H] + .

[0301] Step 2: Preparation of 2-propenyl-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0302] 2-Propenyl-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (2.1 g, 10 mmol), 5-(4-methylpiperazin-1-yl)-2-aminopyridine (1.92 g, 10 mmol), potassium carbonate (6.21 g, 45 mmol), a catalytic amount of potassium iodide, and DMF (80 mL) were mixed and heated to 120 °C. The reaction mixture was stirred for 4 hours. After cooling to room temperature, it was poured into water, and the crude product was obtained by filtration. The crude product was purified by column chromatography to obtain the off-white solid 2-propenyl-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (2.89 g, 79%). LC / MS (ESI): m / z = 367 [M+H] + .

[0303] Step 3: Preparation of 2-propenyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0304] To a stirred solution of 2-propenyl-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (73 mg, 0.2 mmol) and 2-(6-bromo-2-pyridyl)propan-2-ol (52 mg, 0.24 mmol) in 10 mL of dioxane were added copper(I) iodide (38 mg, 0.20 mmol), potassium carbonate (42 mg, 0.3 mmol) and N,N'-dimethylethylenediamine (20 mg, 0.22 mmol), and the mixture was stirred at 80 °C overnight. After completion of the reaction, the solvent was removed under reduced pressure; the residue was diluted with water and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product, which was purified by flash chromatography to give 2-propenyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (48 mg, yield 48%).

[0305] LC / MS(ESI): m / z = 502 [M+H] + .

[0306] Example 43

[0307] Preparation of 2-propenyl-1-(6-(2-hydroxypropyl-2-yl)phenyl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 43)

[0308]

[0309] Compound 43 (62 mg, yield 62%) was obtained by a method similar to that of Example 42. LC / MS(ESI): m / z = 501 [M+H] + 。

[0310] Example 44

[0311] (R)-2-propenyl-1-(7-ethyl-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 44) Preparation

[0312]

[0313] Compound 44 (54 mg, yield 51%) was obtained by a method similar to that of Example 42. LC / MS (ESI): m / z = 528 [M+H] + 。

[0314] Example 45

[0315] Preparation of (R)-2-propenyl-1-(7-ethyl-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-2-yl)-6-((4-(4-methylpiperazin-1-yl)phenyl)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 45)

[0316]

[0317] Compound 45 (60 mg, yield 57%) was obtained by a method similar to that of Example 44. LC / MS (ESI): m / z = 527 [M+H] + 。

[0318] Example 46

[0319] Preparation of 2-propenyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((3-methyl-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazin-8-yl)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 46)

[0320]

[0321] Compound 46 (48 mg, yield 45%) was obtained by a method similar to that of Example 45. LC / MS (ESI): m / z = 529 [M+H] + 。

[0322] Example 47

[0323] Preparation of (R)-2-propenyl-1-(7-ethyl-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-2-yl)-6-((3-methyl-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazin-8-yl)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 47)

[0324]

[0325] Compound 47 (53 mg, yield 48%) was obtained by a method similar to that of Example 45. LC / MS (ESI): m / z = 555 [M+H] + 。

[0326] Example 48

[0327] (R)-2-Propenyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 48) Preparation

[0328]

[0329] Compound 48 (57 mg, yield 58%) was obtained by a method similar to that of Example 43. LC / MS (ESI): m / z = 500 [M+H] + .

[0330] Example 49

[0331] (R)-2-Propenyl-1-(7-ethyl-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-2-yl)-6-(2'-methyl-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 49) Preparation

[0332]

[0333] Compound 49 (56 mg, yield 54%) was obtained by a method similar to that of Example 45. LC / MS (ESI): m / z = 524 [M+H] + .

[0334] Example 50

[0335] (R)-2-Propenyl-1-(7-ethyl-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-2-yl)-6-((2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 51) Preparation

[0336]

[0337] Compound 50 (50 mg, yield 48%) was obtained by a method similar to that of Example 45. LC / MS (ESI): m / z = 526 [M+H] + .

[0338] Example 51

[0339] Preparation of 2-(2,6-dichlorophenyl)-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (Compound 94)

[0340]

[0341] First step: Preparation of 2-(2,6-dichlorophenyl)-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0342] To a solution of methyl 4,6-dichloropyridazine-3-carboxylate (9.89 g, 47.8 mmol) in 150 mL of THF was added DIPEA (20.8 ml, 120 mmol) and 2,6-dichlorophenylhydrazine hydrochloride (10.18 g, 47.8 mmol). The reaction mixture was refluxed for 72 h and then concentrated in vacuo. Et2O (50 ml) was added to the residue, and the mixture was filtered. The filtrate was concentrated by rotary evaporation and dissolved. The residue was cooled in an ice bath, and then TFA (40 ml) was added. The resulting solution was stirred at room temperature for 1 h and then at 70 °C for 1 h. The solution was concentrated by rotary evaporation, and the residue was dissolved in THF (50 ml) and cooled in an ice bath. Then NaH (75 ml) was added. The resulting solution was stirred at RT for 15 min and then adjusted to pH = 3 by adding acetic acid. The solution was concentrated by rotary evaporation, and then 100 ml of chloroform and 100 ml of water were added. The organic phase was washed with 50 ml of saturated brine, dried over anhydrous Mg2SO4, concentrated in vacuo, and slurried with n-hexane. The solid precipitate was washed with ethanol and Et2O and then dried in vacuo to give the yellow solid compound 2-(2,6-dichlorophenyl)-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (8.13 g, 54%).

[0343] LC / MS (ESI): m / z = 316 [M+H] + .

[0344] Second step: Preparation of 2-(2,6-dichlorophenyl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0345] 2-(2,6-dichlorophenyl)-6-chloro-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (1.58 g, 5 mmol), 5-(4-methylpiperazin-1-yl)-2-aminopyridine (0.96 g, 5 mmol), potassium carbonate (3.45 g, 25 mmol), a catalytic amount of potassium iodide, and DMF (50 mL) were mixed, heated to 120 °C, and stirred for 4 h. After cooling to room temperature, it was poured into water, and the crude product was obtained by filtration and purified by column chromatography to obtain a white solid, 2-(2,6-dichlorophenyl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (1.86 g, 79%).

[0346] LC / MS (ESI): m / z = 471 [M+H] + 。

[0347] Step 3: Preparation of 2-(2,6-dichlorophenyl)-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one

[0348] CuI (38 mg, 0.2 mmol), K2CO3 (42 mg, 0.3 mmol), and N,N'-dimethylethylenediamine (20 mg, 0.22 mmol) were added to a stirred solution of 2-(2,6-dichlorophenyl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (94 mg, 0.2 mmol) and 2-(6-bromo-2-pyridyl)propan-2-ol (52 mg, 0.24 mmol) in 10 mL of dioxane, and the mixture was stirred at 80 °C overnight. After completion of the reaction, the solvent was removed under reduced pressure; the residue was diluted with water and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with a saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was purified by flash chromatography to obtain 2-(2,6-dichlorophenyl)-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-1,2-dihydro-3H-pyrazolo[4,3-c]pyridazin-3-one (52 mg, yield 43%).

[0349] LC / MS (ESI): m / z = 607 [M+H] + 。

[0350] Example 52

[0351] Preparation of 6 - propenyl - 5-(6-(2 - hydroxypropyl - 2 - yl)pyridin - 2 - yl)-3-((5-(4 - methylpiperazin - 1 - yl)pyridin - 2 - yl)amino)-5,6 - dihydro - 3H - pyrazolo[3,4 - e][1,2,4]triazin - 7 - one (Compound 101)

[0352]

[0353] First step: Preparation of 6 - propenyl - 3-(methylthio)-5,6 - dihydro - 3H - pyrazolo[3,4 - e][1,2,4]triazin - 7 - one

[0354] To a solution of ethyl 3 - (methylthio)-5 - chloro - [1,2,4]triazine - 6 - carboxylate (11.1 g, 47.8 mmol) in 150 mL of THF was added DIPEA (20.8 ml, 120 mmol) and tert - butyl 1 - allylhydrazinecarboxylate (8.23 g, 47.8 mmol). The reaction mixture was refluxed for 72 hours and then concentrated in vacuo. Et2O (50 ml) was added to the residue, and the mixture was filtered. The filtrate was concentrated by evaporation and the residue was cooled in an ice bath. Then TFA (40 ml) was added. The resulting solution was stirred at room temperature for 1 h and then at 70 °C for 1 h. The solution was concentrated by evaporation, and the residue was dissolved in THF (50 ml) and cooled in an ice bath. Then NaOH (75 ml) was added. The resulting solution was stirred at RT for 15 minutes and then adjusted to pH = 3 by adding acetic acid. The solution was concentrated by evaporation, then 100 ml of chloroform and 100 ml of water were added, and the organic phase was washed with 50 ml of saturated brine, dried over anhydrous Mg2SO4, concentrated in vacuo, and triturated with n - hexane. The solid precipitate was washed with ethanol and Et2O and then dried in vacuo to obtain the yellow solid compound 6 - propenyl - 3-(methylthio)-5,6 - dihydro - 3H - pyrazolo[3,4 - e][1,2,4]triazin - 7 - one (5.0 g, 47%).

[0355] LC / MS(ESI): m / z = 224 [M + H] + 。

[0356] Second step: Preparation of 6 - propenyl - 5-(6-(2 - hydroxypropyl - 2 - yl)pyridin - 2 - yl)-3-(methylthio)-5,6 - dihydro - 3H - pyrazolo[3,4 - e][1,2,4]triazin - 7 - one

[0357] To a solution of 6-propenyl-3-(methylthio)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (224 mg, 1 mmol) and 2-(6-bromo-2-pyridyl)propan-2-ol (260 mg, 1.2 mmol) in 20 mL of dioxane was added CuI (190 mg, 1 mmol), K2CO3 (210 mg, 1.5 mmol) and N,N'-dimethylethylenediamine (100 mg, 1.1 mmol) with stirring, and the mixture was stirred overnight at 80 °C. After completion of the reaction, the solvent was removed under reduced pressure; the residue was diluted with water and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by flash chromatography to obtain 6-propenyl-5-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-(methylthio)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (258 mg, yield 72%).

[0358] LC / MS(ESI): m / z = 359 [M + H] + 。

[0359] Step 3: Preparation of 6-propenyl-3-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one

[0360] 6-Propenyl-5-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-(methylthio)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (180 mg, 0.5 mmol) was dissolved in 10 mL of CH2Cl2, m-chloroperbenzoic acid (522 mg, 6 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with CH2Cl2 and washed with saturated NaHCO3 and Na2S2O3. The organic layer was dried over MgSO4, evaporated under reduced pressure, and purified by column chromatography to obtain a yellow solid, 6-propenyl-3-(methylsulfonyl)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (183 mg, 94%). LC / MS(ESI): m / z = 391 [M + H] + 。

[0361] Step 4: Preparation of 6-propenyl-5-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one

[0362] 6-Allyl-3-(methylsulfonyl)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (117 mg, 0.3 mmol) was dissolved in 25 ml of DMF. DIPEA (194 mg, 1.5 mmol) and 4-(4-methylpiperazin-1-yl)aniline (70 mg, 0.36 mmol) were added, and the mixture was stirred at room temperature for 24 h. It was poured into ice water, and the crude product was filtered. Then, yellow solid 6-allyl-3-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-5,6-dihydro-3H-pyrazolo[3,4-e][1,2,4]triazin-7-one (133 mg, 89%) was obtained by flash column chromatography. 1H NMR (400 MHz, DMSO-d6) 10.13 (m, 1H), 7.56 - 8.10 (m, 6H), 5.67 (dd, 1H), 5.31 (br, 1H), 5.01 (dd, 1H), 4.69 - 4.84 (m, 3H), 3.51 - 3.88 (m, 4H), 3.11 - 3.23 (m, 4H), 2.43 (s, 3H), 1.47 (s, 6H); LC / MS (ESI): m / z = 502 [M+H] + .

[0363] Similar to the synthetic route of 43 - 52, the following compounds can be obtained:

[0364]

[0365]

[0366] Example 53: Biological activity test

[0367] The present invention will be further described and explained below in combination with test examples, but these examples do not mean to limit the scope of the present invention.

[0368] Test example 1: Determination of the inhibitory activity of the compound on Wee-1 enzyme

[0369] Using the Lanthra Screen Wee-1 kinase kit (invitrogen), the inhibitory effect of the compound on Wee-1 kinase activity was determined. Four-fold serial dilutions were made from a working concentration of 0.2 mM in DMSO, with 10 concentrations diluted. After thoroughly mixing 5 μL of Wee-1 kinase (final concentration 5 nM), 5 μL of Eu-Anti-GST Antibody (final concentration 2 nM) mixture and 5 μL of kinase Tracer 178 (final concentration 50 nM), the mixture was incubated at room temperature for one hour and then the plate was read. The minimum signal value was obtained from a separate culture medium (cell count was zero). Inhibition rate % = (maximum signal value - compound signal value) / (maximum signal value - minimum signal value) × 100%. Data was processed using Graphpad prism 5 software. The IC 50 value was calculated by fitting an S-shaped dose-response curve. Where "A" represents IC 50 ≤ 10 nM; "B" represents 10 nM < IC 50 ≤ 500 nM; "C" represents 500 nM < IC 50 ≤ 2000 nM; "D" represents 2000 nM < IC 50 .

[0370] Table 1. IC 50

[0371] Compound <![CDATA[IC 50 > Compound <![CDATA[IC 50 > Compound <![CDATA[IC 50 > 1 A 8 A 15 A 2 A 9 A 16 A 3 A 10 A 17 A 4 A 12 A 18 A 5 A 13 A 101 A 6 A 14 A 103 A

[0372] Test Example 2. Determination of the cell proliferation activity of the compound

[0373] CellTiter-Glo was used <tm>Live cell detection kit for determining the inhibitory effect of a test compound on the proliferation of human non-small cell lung cancer cell line NCI-H23. Among them, the culture medium is McCoy's 5A medium supplemented with fetal bovine serum at a final concentration of 10%.

[0374] Experimental procedure: Digest NCI-H23 cells that have reached 80% confluence with trypsin, centrifuge, resuspend, and count. Prepare NCI-H23 cell suspensions at 6000 cells / mL with the culture medium, add them to a 96-well cell culture plate (90 μL / well), and incubate in a cell culture incubator containing 5% CO2 at 37°C. After 24 hours of cell culture, dissolve the reference compound table and the test compound in DMSO to prepare a stock solution with a concentration of 30 mM. Further dilute the diluted compound stock solution with the culture medium of NCI-H23, and transfer the diluted mixture to the corresponding cell plates respectively. The final concentration of the test compound is 1 μM (as the starting concentration for IC 50 testing), and perform nine-fold serial dilutions with a four-fold dilution factor. The nine concentrations are: 10 μM, 2.5 μM, 0.625 μM, 0.156 μM, 0.039 μM, 0.0098 μM, 0.0024 μM, 0.0006 μM, and 0.000015 μM. Mix well, centrifuge, and incubate in a cell culture incubator containing 5% CO2 at 37°C for 3 days. Take out the 96-well cell culture plate, add CellTiterGlo (CTG, a chemiluminescent cell viability detection kit) reagent (100 μL / well), mix well, centrifuge, and incubate at room temperature for 10 minutes. After gently shaking, measure the absorbance at a wavelength of 450 nm on a SpectraMax M5 Reader, using the absorbance at 650 nm as a reference (i.e., 450 nm absorbance - 650 nm absorbance), and calculate the inhibition rate. Use the software Graphpad Prism 6 and adopt the calculation formula XY-analysis / Nonlinear regression (curve fit) / Dose response-Inhibition / log(inhibitor) vs. response-Variable slope (four parameters) to perform IC 50 curve fitting and calculate the IC 50 value. Among them, "A" indicates IC 50 ≤10 nM; "B" indicates 10 nM < IC 50 ≤500 nM; "C" indicates 500 nM < IC 50 ≤2000 nM; "D" indicates 2000 nM < IC 50 .

[0375] Table 2. IC of the compounds of the present invention for inhibiting the growth of NCI-H23 cells 50

[0376] Compound <![CDATA[IC 50 > Compound <![CDATA[IC 50 > Compound <![CDATA[IC 50 > 1 B 4 B 34 C 3 B 33 C 101 B

[0377] Although the present invention has been described in detail above, those skilled in the art will understand that the present invention can be variously modified and changed without departing from the spirit and scope of the present invention. The scope of the rights of the present invention is not limited to the detailed description above, but shall be attributed to the claims.< / tm> < / rd>

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1.

3. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 1, and the pharmaceutical composition according to claim 2 in the manufacture of a medicament for treating a disease associated with WEE1 activity.

4. Use according to claim 3, wherein the disease associated with WEE1 activity is liver cancer, breast cancer, malignant glioma, melanoma, adult brain tumor, pediatric brain tumor, ovarian cancer, colon cancer, cervical cancer, osteosarcoma, lung cancer, gastric cancer, head and neck cancer or leukemia.

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