WO2023093840A1 - Use of wee1 kinase inhibitors in the treatment of cancer - Google Patents

Use of wee1 kinase inhibitors in the treatment of cancer Download PDF

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WO2023093840A1
WO2023093840A1 PCT/CN2022/134300 CN2022134300W WO2023093840A1 WO 2023093840 A1 WO2023093840 A1 WO 2023093840A1 CN 2022134300 W CN2022134300 W CN 2022134300W WO 2023093840 A1 WO2023093840 A1 WO 2023093840A1
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amino
pyrimido
phenyl
pyrimidin
dihydroimidazo
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PCT/CN2022/134300
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French (fr)
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Chih Yi Hsieh
Ye Edward Tian
Sui Xiong Cai
Cong Cong ZHANG
Ming Chuan GUO
Yu Xiao ZHAO
Huan XIA
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Impact Therapeutics (Shanghai) , Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings

Definitions

  • This disclosure is in the field of medicinal chemistry and relates to the field of cancer treatment.
  • the disclosure relates to the use of Wee1 kinase inhibitors in the treatment of cancer with FBXW7 or CHD4 mutation.
  • the process of proliferation and division of eukaryotic cell is referred to as cell cycle.
  • a cell grows from a newly divided cell to an adult cell to give birth of a daughter cell through cell cycle.
  • Cell cycle can be roughly divided to 4 distinct phases, the G1 phase, the S phase, the G2 phase and the M phase.
  • the G1 phase is also referred to as the phase of growth, which is characterized by active cell metabolism, large scale of synthesis of protein, carbohydrate, lipid and RNA and rapid growth from newly divided young cells to mature cells.
  • the S stage of cell cycle is characterized by DNA synthesis and replication.
  • the cell chromosome changes from diploid to tetraploid. Then, cells enter the G2 phase.
  • the characteristic of G2 phase is that cells in this phase continue to grow, and the synthesized DNA is checked for damages and mutations. The damaged DNA is repaired and cells prepare for mitosis.
  • the last phase of the cell cycle is the M phase. In this phase, the cells divide from one mother cell to two identical daughter cells through mitosis, completing cell reproduction cycle.
  • there are multiple cell cycle checkpoints including G1/S or G1 checkpoint and G2/M or G2 checkpoint. The main functions of these checkpoints are to check and ensure the accurate replication of genetic information (DNA) during the cell cycle and to determine whether the cells enter the next phase of cell cycle.
  • Each cell cycle checkpoint is a system composed of multiple factors that work through complex mechanisms.
  • the G2/M checkpoint uses a complex process to check for DNA damage.
  • Cdk1 that forms a complex with Cyclin-B1 (Nurse, P., Nature, 1990, 344: 503-508) .
  • the activation and inactivation of Cdk1 play a vital role in the cell’s entering mitosis (M) from G2 phase and the subsequent completion of mitosis.
  • M mitosis
  • the activity of Cdk1 is regulated by multiple mechanisms, including the binding of Cyclin A or Cyclin B, phosphorylation and dephosphorylation.
  • Wee1 kinase phosphorylates Cdk1 and inhibits its activity, as a result it delays the cell from entering mitosis.
  • Wee1 is a tyrosine kinase that regulates the activity of Cdk1 by phosphorylating tyrosine 15 (Y15) on Cdk1 molecule (McGowan, C. H. and Russell, P., EMBO J, 1993, 12: 75- 85; Parker, L. L. and Piwnica-Worms, H., Science, 1992, 257: 1955-1957) . Accordingly, Wee1 is a key inhibitory regulator of Cdk1 activity and plays an important role in G2-M phase checkpoints (O’ Connell, M. J. et al, EMBO J, 1997, 16: 545-554) .
  • Wee1 kinase inhibitors may be used as targeted therapy for the treatment of cancers and other disorders that have cell cycle checkpoint defects.
  • Adavosertib (AZD1775) is the first Wee1 kinase inhibitor to enter the clinic and showed good clinical efficacy data in several tumors including ovarian, colon, and uterine carcinoma.
  • Wee1 kinase inhibitors that are currently in clinical phase include ZN-c3 and Debio-0123, which are in clinical phase I.
  • Wee1 kinase inhibitors currently in the preclinical research stage such as DN-1609, NUV-569 and the like.
  • WO2018090939 discloses the following compounds of Formula I or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
  • A is N or CR 15 ;
  • R 1 is hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
  • R 2 is optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 3 -R 7 and R 15 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1- C 10 alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
  • WO2019011228 discloses the following compounds of Formula II or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
  • A is N or CR 6 ;
  • R 1 is hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
  • R 2 is optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 3 -R 6 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1- C 10 alkyl (such as haloalkyl, hydroxyalkyl, aminoalkyl and carboxyalkyl) , alkenyl, alkynyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
  • WO2021073491 discloses the following compounds of Formula III or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
  • R 1 and R 2 are independently halo;
  • R 3 is halo, C 1-4 alkyl or C 1-4 alkoxy;
  • R 4 and R 6 each are independently H or C 1-4 alkyl;
  • R 5 is H or C 1-4 alkyl;
  • R 7 is H, halo, C 1-4 alkyl or C 1-4 alkoxy; and
  • X is CH or N.
  • FBXW7 belongs to the F-box protein family which is the target protein recognition component of the SCF (SKP1 cullin-F box) type ubiquitin ligase and contains several conservative domains for protein interaction. In most cases, FBXW7 recognizes the phosphorylated substrate and promotes it ubiquitination and degradation. Many substrates of FBXW7 are oncogenes, as one of the important factors of ubiquitin proteasome degradation pathway. For example, Cyclin E (the protein encoded by CCNE1 gene) , c-myc, c-jun and Notch are degraded through the ubiquitin proteasome pathway mediated by FBXW7.
  • SCF SCF
  • Mutations and deletions of FBXW7 can cause the accumulation of these genes related to cancer cell proliferation, and have been found in ovarian cancer, breast cancer and colorectal cancer. Nearly 9%of colorectal cancers have mutations or deletions of FBXW7 gene. It has been confirmed that FBXW7 recognizes and participates in the ubiquitination and degradation of a variety of oncoproteins in colorectal cancer, gastric cancer, ovarian cancer and liver cancer, and plays a role in inhibiting the biological functions of tumor cells. Therefore, mutations of FBXW7 gene often lead to the occurrence and progression of tumors.
  • cyclin E cannot be degraded through the ubiquitin proteasome pathway due to the mutation of FBXW7, thus forming the accumulation of cyclin E (B. T. Hughes et al., 2013, Proc Natl Acad Sci U S A 110; 8954 –8959) .
  • Both CCNE1 amplification and cyclin E accumulation will increase the speed of cells passing G1/Sphase due to their overexpression, thus depending on the checkpoint of G2/M phase to ensure normal cell proliferation.
  • the checkpoint of G2/M phase of the cell that causes abnormality thus making the cell sensitive to DNA damage, thus causing cell death.
  • Wee1 kinase inhibitor (AZD1775) has good anti-tumor activity in CCNE1 amplified refractory solid tumors, especially in high-grade serous ovarian cancer.
  • the study showed that in 27 cases of refractory solid tumors, confirmed partial remission was observed in 7 patients, and the objective response rate (ORR) was 25.9% (95% CI 15.1-47.5%) .
  • ORR objective response rate
  • 13 patients with high-grade serous ovarian cancer 5 patients had objective remission (38.5%) , and 8 patients were stable for more than 6 months or achieved objective remission (61.5%) . Therefore, FBXW7 mutation, as an important reason for cyclin E accumulation, can be used as a predictive biomarker to screen potential patients of Wee1 kinase inhibitors.
  • CHD4 Chromododomain Helicase DNA binding protein 4
  • Nucleosome remodeling and deacetylase (NuRD) complex contains a helicase/atpase domain, which can promote histone deacetylation, thus controlling chromatin reorganization and transcriptional regulation.
  • Nucleosome remodeling and deacetylase (NuRD) complex is one of the major chromatin remodeling complexes found in cells. It plays an important role in regulating gene transcription, genome integrity and cell cycle progression. Through the influence on these basic cellular processes, an increasing body of evidence shows that the activity change of NuRD complex can lead to developmental defects, tumorigenesis and accelerated aging.
  • CHD4 plays an important role in DNA damage response and regulation of cell cycle checkpoint activation. CHD4 knockdown will increase the sensitivity of this cell line to chemotherapy (Kouki Neo, et al, Journal of Hepatology, 2015; 63: 1164-72) , and the loss of CHD4 function will make it sensitive to PARP inhibitors (Mei ren Pan et al, J Bio Chem, 2012; 287 (67) : 64-72) .
  • Wee1 kinase inhibitor has good efficacy in a variety of cell lines carrying specific FBXW7 mutations or CHD4 mutations, as well as in colon cancer mouse models with human FBXW7 or CHD4 mutations. It provides a potential therapeutic method for a variety of tumor patients carrying FBXW7 or CHD4 mutations
  • the present disclosure finds that the growth of various tumor cells with FBXW7 and/or CHD4 mutations can be effectively inhibited by inhibiting Wee1 kinase activity, which is of great significance in the treatment of tumors with FBXW7 and/or CHD4 mutation.
  • this disclosure provides use of a Wee1 kinase inhibitor in the preparation of medicament for the treatment of a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , a Wee1 kinase inhibitor for use in a method for the treatment of a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , and a method for treatment of a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , comprising administration of an effective amount of a Wee1 kinase inhibitor or its pharmaceutical composition to a subject in need.
  • the Wee1 kinase inhibitors described herein are small-molecule Wee1 kinase inhibitors. These inhibitors include, but are not limited to, AZD1775, Zc-03, Debio-0123, IMP7068, DN-1609 and NUV-569; and compounds disclosed in WO2007126122, WO2019028008, WO2019173082, WO202021032, WO2015092431, WO2018162932, WO2019037678, WO2020210377, WO2020210383, WO2020210375, WO2020210380, WO2020210381, WO2018133829, WO2019085933, WO2018090939, WO2019011228 and WO2021073491.
  • the Wee1 kinase inhibitor is a compound of Formula (I) , Formula (II) or Formula (III) as described herein.
  • the said tumor is a solid tumor or a hematologic lymphoma.
  • the said FBXW7 mutation is R465C mutation and/or R505C mutation.
  • the tumor is colorectal cancer, preferably colorectal cancer with R465C mutation and/or R505C mutation in FBXW7 protein.
  • the method of treatment described herein for tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) further comprises detecting the presence of a mutation in the FBXW7 and/or the CHD4, particularly the mutation (s) as described herein.
  • Fig. 1 FBXW7 was overexpressed in LoVo.
  • Fig. 2 Overexpression of FBXW7 in LoVo cells reduced the sensitivity of the cells to Wee1 kinase inhibitors (IC 50 : 67 nM in the control group and 121 nM in the FBXW7 -overexpressed group) .
  • Fig. 3 CHD4 expression was decreased in OVCAR3 CHD4 knockdown group (shRNA#1) .
  • Fig. 4 Knockdown of CHD4 in OVCAR3 cells increased the sensitivity of cells to Wee1 kinase inhibitors (IC 50 : 1881 nM in the control group and 334 nM in the CHD4 knockdown group) .
  • Fig. 5 A bar graph illustrating cancer types and their occurrence of alteration of FBXW7.
  • Fig. 6 A bar graph illustrating cancer types and their occurrence of alteration of CHD4.
  • Tumors described herein refer to new organisms formed by local histiocytosis of the body under the action of various tumorigenic factors. According to the cellular characteristics of new organisms and the degree of harm to the body, tumors can be divided into benign tumors and malignant tumors.
  • Malignant tumor can be divided into cancer and sarcoma, cancer is defined as a malignant tumor that comes from epithelial tissue.
  • Sarcoma is defined as a malignant tumor that mesenchymal tissue produces.
  • the malignant tumor that forms by epithelium of large intestine mucosa calls carcinoma of large intestine mucosa epithelium, simply called large intestine carcinoma.
  • Tumor cells or cancer cells are generally characterized by abnormal proliferation relative to normal cells and formation of clusters or tumors in individuals with tumors. Tumors include solid tumors and hematologic tumors.
  • FBXW7 and CHD4 described herein have well-known meanings in the art.
  • the wild-type sequence of human FBXW7 is showed in Genbank access number AAI43945.1
  • the wild-type sequence of human CHD4 is showed in Ganbank access number AAH38596.1. Mutation described herein refer to mutation relative to wild-type sequence.
  • the cancer carrying one or more FBXW7 mutations and/or one or more CHD4 mutations described herein can include any type of solid tumor or malignant lymphoma.
  • Cancer can be familial or sporadic.
  • Mutations can be one or more of substitutions, deletions, and insertions.
  • Frameshift mutations at the gene level can result in parts of the encoded protein completely different from the wild-type protein, and such mutations can be regarded as deletion mutations compared with the wild-type protein.
  • the mutation occurs in the WD40 domain of FBXW7 (residues 378-659 of FBXW7) .
  • the FBXW7 mutation described herein includes, in particular, one or more mutations occurring at positions 27, 117, 200, 226, 336, 441, 460, 465, 479, 505, 514, and 616. In the preferred embodiments, the mutation occurs at one or more of positions 465, 479, and 505.
  • the mutation is one or more of H27Q, deletion of amino acid at position 117, V200I, T226I, I336M, R441W, H460Y, R465C, R465H, R479Q, R505C, R505H, V514D, and T616I.
  • the mutation includes or is R465C, R465H, R479Q, R505C, or R505H.
  • the mutation is a truncated (deletion) mutation, including, for example, truncation of amino acid residues at positions 1-40 of the N-terminus of FBXW7, truncation of amino acid residues at positions 1-103 of the N-terminus of FBXW7, truncation starting from position 118 (including position 118) of FBXW7, truncation starting from position 279 (including position 279) of FBXW7, and frameshift mutation starting from position 404 (including position 404) of FBXW7.
  • truncated (deletion) mutation including, for example, truncation of amino acid residues at positions 1-40 of the N-terminus of FBXW7, truncation of amino acid residues at positions 1-103 of the N-terminus of FBXW7, truncation starting from position 118 (including position 118) of FBXW7, truncation starting from position 279 (including position 279) of
  • the CHD4 mutation includes one or more mutations occurring at positions 19, 57, 279, 538, 732, 841, 852, 869, 895, 966, 1038, 1042, 1068, 1105, 1272, 1384, 1541, 1886 and 1894, and truncation mutation.
  • the mutations include D19G, P57S, R279H, R538W, G723S, A841V, Y852H, V869M, Q895K, M966I, G1038D, G1042D, R1068C, R1105W, G1272D, P1384H, P1541H, Q1886H, S1894T and frameshift mutation starting from position 1488 (including position 1488) of CHD4.
  • the CHD4 mutation includes a mutation at position 279 or position 1042, preferably R279H or G1042D.
  • tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) can be tumors occurring in the urinary tract, cervix, esophagus, lung, colorectal, ovary, endometrium, pancreas, prostate, or lymph.
  • tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) are solid cancer and hematoma, including but not limited to, liver cancer, melanoma, Hodgkin's disease, non-hodgkin's lymphoma, acute lymphatic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, Wilms’ tumor, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary gigantic globulin hematic disease, bladder cancer, chronic granulocyte leukemia, primary Brain cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, colon cancer, malignant pancreatic islet cell tumor, malignant carcinoid sex cancer, malignant melanoma, choriocarcinoma, mycosis granuloma, head and neck cancer
  • the tumor having FBXW7 mutation (s) includes skin cutaneous melanoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, esophageal adenocarcinoma, bladder urothelial carcinoma, stomach adenocarcinoma, cervical squamous cell carcinoma, colorectal adenocarcinoma, uterine corpus endometrial carcinoma, and uterine carcinosarcoma; and the tumor having CHD4 mutation (s) include lung squamous cell carcinoma, cervical squamous cell carcinoma, brain lower grade glioma, testicular germ cell tumor, colorectal adenocarcinoma, bladder urothelial carcinoma, ova
  • the tumors described herein are those shown in the following Table A and having the FBXW7 mutation (s) and/or the CHD4 mutations listed in the corresponding rows of the table A below:
  • the tumors carrying the FBXW7 mutation (s) are those carrying R465C or R505C, including, in particular, colorectal cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, lymphoma, urethral cancer, bladder cancer, and T-lymphocytic leukemia.
  • the tumor is rectal cancer with a mutated FBXW7, wherein the mutated FBXW7 contains one or more mutations selected from the group consisting of T616I, R465C, truncation starting from position 118 and truncation starting from position 279.
  • the rectal cancer further comprises one or more mutations occurred in APC, TP53, KRAS and ARID1A.
  • the mutations in APC are selected from the group consisting of frameshift mutation starting from position 541 (including position 541) or from 660 (including position 660) , truncation starting from position 902 or 1292.
  • the mutations in TP53 are selected from the group consisting of R273H, R175H, R248Q and R248W/Loss.
  • the mutations in KRAS are selected from the group consisting of G12C and G12S.
  • the mutation in ARID1A is a truncation starting from position 1086.
  • the terms “individual, “subject” and “subject” are used interchangeably to refer to patients with tumors with FBXW7 or CHD4 mutations.
  • An individual may have heterozygotes or polymorphisms with FBXW7 mutations or CHD4 mutations.
  • Wee1 kinase inhibitor used herein can be any compound or entity that inhibits, reduces, or eliminates Wee1 kinase, such as small organic molecules, peptides, or nucleic acids.
  • Examples of compounds that are known as Wee1 kinase inhibitors and can be used according to the present disclosure include, but are not limited to: AZD1775, Zc-03, Debio-0123, DN-1609, NUV-569, and compounds disclosed in WO2007126122, WO2019028008, WO2019173082, WO202021032, WO2015092431, WO2018162932, WO2019037678, WO2020210377, WO2020210383, WO2020210375, WO2020210380, WO2020210381, WO2018133829, WO2019085933, WO2018090939, WO2019011228 and WO2021073491.
  • the Wee1 kinase inhibitor is the inhibitor disclosed in WO2018090939, which is incorporated herein by reference in its entirety. More specifically, the Wee1 kinase inhibitor is a compound represented by Formula I:
  • A is N or CR 15 ;
  • R 1 is hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
  • R 2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
  • R 3 -R 7 and R 15 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1 -C 10 alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
  • A is N.
  • R 1 and R 2 are optionally substituted aryl.
  • R 3 -R 7 are each independently H, halo, or C 1 -C 6 alkyl.
  • R 15 is H or C 1 -C 6 alkyl.
  • the substituents on R 1 are selected from any one, two or three of the following groups: halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and halo C 1 -C 6 alkyl.
  • R 1 is selected from: H, C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 3 -C 8 cycloalkyl, heteroaryl, and aryl which is optionally substituted by 1-4 groups selected from halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and halo C 1 -C 6 alkyl.
  • R 1 is selected from phenyl which is optionally substituted by 1-4 groups selected from halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and halo C 1 -C 6 alkyl; in some embodiments, the number of substituents is 2; in some embodiments, at least one substituent is in the ortho position; in some embodiments, at least one substituent is halo; in some embodiments, the number of substituents on the phenyl is 2, both of which are located adjacent to each other, and wherein at least one is halo.
  • R 1 is selected from optionally substituted pyridyl, pyrimidyl, thiophenyl, furanyl, pyrrolyl and imidazolyl.
  • R 1 is selected from H, optionally substituted C 1- C 8 alkyl, C 3- C 8 cycloalkyl, and C 2- C 8 alkenyl.
  • the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 acyl, optionally substituted heterocyclic group, halo, optionally substituted oxy group, nitro, and optionally substituted C 1 -C 6 alkylamino; preferably, the substituents on these substituted group may be 1-4 groups selected from the following group: C 1 -C 6 alkyl, C 1 -C 6 acyl, a heterocyclic group optionally substituted by 1-4 of C 1 -C 6 alkyl, halo, -NR a R b and hydroxy, wherein R a and R b are each independently H and C 1 -C 6 alkyl; preferably, the heterocyclic group is selected from piperazinyl, piperidinyl, morpholinyl, and 1, 4-diazacycloheptyl.
  • the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted piperazinyl, optionally substituted piperazinyl-C 1 -C 4 alkyl, optionally substituted piperidinyl, imidazolyl, optionally substituted 1, 4-diazacycloheptyl, C 1 -C 6 alkyl, C 1 -C 6 acyl, optionally substituted morpholinyl, morpholinyl-C 1 -C 4 alkyl, halo, halo C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted hydroxy C 1 -C 6 alkyl, optionally substituted amino C 1 -C 6 alkyl, optionally substituted piperidinylamino, optionally substituted C 1 -C 6 alkyl amino, optionally substituted heterocyclic alkyl-O-and nitro; preferably, the substituent
  • the optionally substituted piperazinyl is the piperazinlyl which can be substituted by 1, 2 or 3 groups selected from: C 1 -C 6 alkyl, hydroxy C 1 -C 6 alkyl, and C 1 -C 6 acyl.
  • the piperazine group has at least one substituent at the para-position, and optionally, one or two substituents at the meta-position.
  • the optionally substituted piperidinyl is the piperidinyl which can be substituted by 1 group selected from C 1 -C 6 alkyl and C 1 -C 6 alkyl amino.
  • the optionally substituted morpholinyl is the morpholinyl which can be substituted by 1 or 2 groups selected from C 1 -C 6 alkyl.
  • R 2 is selected from optionally substituted phenyl, pyridyl, piperazinyl, tetrahydroisoquinolinyl, 2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl and 4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl.
  • R 2 is selected from phenyl substituted by optionally substituted piperazinyl, phenyl substituted by optionally substituted pyridinyl, and tetrahydroisoquinolinyl optionally substituted by one to three C 1 -C 6 alkyls or halos.
  • the piperazinyl is optionally substituted by one to three groups selected from C 1 -C 6 alkyl, hydroxy C 1 -C 6 alkyl and C 1 -C 6 acyl.
  • the piperidinyl is optionally substituted by 1 group selected from C 1 -C 6 alkyl and C 1 -C 6 alkyl amino.
  • R 4 and R 5 are each independently H, C 1 -C 6 alkyl and halo, preferably, both H.
  • R 6 and R 7 are each independently H, C 1 -C 6 alkyl and halo, preferably, both H.
  • preferred compounds of Formula I include, without limitation:
  • the Wee1 kinase inhibitor is the imidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one compound disclosed in WO2019011228. More specifically, the Wee1 kinase inhibitor suitable for the present disclosure is a compound represented by the following Formula II:
  • A is N or CR 6 ;
  • R 1 is hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
  • R 2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
  • R 3 -R 6 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1 -C 10 alkyl (such as haloalkyl, hydroxyalkyl, aminoalkyl and carboxyalkyl) , alkenyl, alkynyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
  • A is N.
  • R 1 and R 2 are each optionally substituted aryl.
  • R 3 is H.
  • R 4 and R 5 are each H and optionally substituted C 1- C 6 alkyl.
  • R 4 is H or unsubstituted C 1- C 6 alkyl.
  • R 5 is H or C 1 -C 6 alkyl optionally substituted by hydroxy, such as hydroxy C 1 -C 6 alkyl.
  • R 6 is H.
  • the substituents on R 1 are selected from any one, two, three or four of the following groups: halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and halo C 1 -C 6 alkyl.
  • R 1 is selected from: C 2- C 8 alkenyl, and phenyl which is optionally substituted by one to four substituents selected from halo and C 1- C 6 alkyl.
  • R 1 is selected from phenyl which is optionally substituted by one to four substituents selected from halo and C 1 -C 6 alkyl; in some embodiments, the number of substituents is 2; in some embodiments, at least one substituent is in the ortho position; in some embodiments, at least one substituent is halo; in some embodiments, the number of substituents on the phenyl is 2, both of which are located adjacent to each other, and wherein at least one is halo.
  • R 1 is selected from optionally substituted C 2 -C 8 alkenyl.
  • the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted C 1 -C 6 alkyl, optionally substituted oxy group, halo, and optionally substituted heterocyclic group; preferably, the substituents on these optionally substituted group may be one to four groups selected from the following groups: C 1 -C 6 alkyl and -NR a R b , wherein R a and R b are each independently H and C 1 -C 6 alkyl; preferably, the heterocyclic group is selected from piperazinyl and piperidinyl.
  • the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted piperazinyl, optionally substituted piperidinyl, C 1 -C 6 alkyl, halo, and C 1 -C 6 alkoxy; preferably, the substituents on the optionally substituted group may be one to four groups selected from the following groups: C 1 -C 6 alkyl and -NR a R b , wherein R a and R b are each independently H and C 1 -C 6 alkyl.
  • the optionally substituted piperazinyl is piperazinyl which can be substituted by 1, 2 or 3 groups selected from: C 1 -C 6 alkyl.
  • the piperazine group has at least one substituent at the para-position, and optionally, one or two substituents at the meta-position.
  • the optionally substituted piperidinyl is piperidinyl which can be substituted by 1 group selected from C 1 -C 6 alkyl and -NR a R b , wherein R a and R b are each independently H and C 1 -C 6 alkyl.
  • R 2 is selected from optionally substituted phenyl and optionally substituted tetrahydroisoquinolinyl.
  • R 2 is selected from phenyl substituted by optionally substituted piperazinyl, phenyl substituted by optionally substituted pyridinyl, and tetrahydroisoquinolinyl optionally substituted by one to three C 1 -C 6 alkyls.
  • the piperazinyl is optionally substituted by one to three groups selected from C 1 -C 6 alkyl.
  • the piperidinyl is optionally substituted by one group selected from C 1 -C 6 alkyl and -NR a R b , wherein R a and R b are each independently H and C 1 -C 6 alkyl.
  • R 4 and R 5 are independently selected from H and optionally substituted C 1- C 6 alkyl.
  • R 4 and R 5 are H and optionally substituted C 1 -C 6 alkyl.
  • preferred compounds of Formula II include, without limitation:
  • the Wee1 kinase inhibitor suitable for the present disclosure is the dihydroimidazopyrimidopyrimidinone compound disclosed in WO2021073491. More specifically, the Wee1 kinase inhibitor suitable for the present disclosure is a compound represented by the following Formula III:
  • R 1 and R 2 are independently halo;
  • R 3 is halo, C 1-4 alkyl or C 1-4 alkoxy;
  • R 4 and R 6 are each independently H or C 1-4 alkyl;
  • R 5 is H or C 1-4 alkyl;
  • R 7 is H, halo, C 1-4 alkyl or C 1-4 alkoxy; and
  • X is CH or N.
  • R and R 2 are both chloro.
  • R 3 is halo, methyl or ethyl.
  • R 7 is H, halo, methyl or methoxy.
  • R 4 and R 6 are each independently H or methyl.
  • R 5 is H, methyl or methyl-d3.
  • R 4 , R 5 and R 6 are not H at the same time; preferably, R 4 and R 6 are C 1-4 alkyl, and R 5 is H or C 1-4 alkyl; more preferably, R 4 and R 6 are methyl, and R 5 is H, methyl or methyl-d3.
  • preferred compounds of Formula III include, without limitation:
  • stereoisomers including optical isomers.
  • the disclosure includes all stereoisomers and the racemic mixtures of such stereoisomers as well as the individual enantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
  • Examples of pharmaceutically acceptable salts include inorganic and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic base salts formed with bases, such as sodium hydroxy, tris (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methyl-glucamine.
  • inorganic and organic acid salts such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate
  • inorganic and organic base salts formed with bases such as sodium hydroxy, tris (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methyl-glucamine.
  • prodrugs of the compounds of the disclosure include the simple esters of carboxylic acid-containing compounds (e.g., those obtained by condensation with a C 1- 4 alcohol according to methods known in the art) ; esters of hydroxy-containing compounds (e.g., those obtained by condensation with a C 1-4 carboxylic acid, C 3-6 diacid or anhydride thereof such as succinic anhydride and fumaric anhydride, according to methods known in the art) ; imines of amino-containing compounds (e.g., those obtained by condensation with a C 1-4 aldehyde or ketone according to methods known in the art) ; carbamate of amino-containing compounds, such as those described by Leu, et al. (J. Med. Chem.
  • Wee1 kinase inhibitors of the present disclosure can be prepared using method compounds known to those skilled in the art or methods in the references (including patents, patent applications and patent publications) cited in the present disclosure, including the synthesis methods disclosed in WO2018090939, WO2019011228 and WO2021073491.
  • the Wee1 kinase inhibitor of the present disclosure can be administered in a pharmaceutical composition containing a pharmaceutically acceptable carrier, wherein the pharmaceutical composition includes all pharmaceutical preparations containing the compound of the present disclosure in an amount that can effectively achieve its intended goal. While individual needs vary, determination of the optimal amount of each part in the pharmaceutical preparation is within the skill of the art.
  • the compounds or the pharmaceutically acceptable salts thereof may be administered to mammals orally at a dose of about 0.0025 to 50 mg per kg body weight per day. Preferably, from approximately 0.01 mg/kg to approximately 10 mg/kg body weight is orally administered.
  • a known anticancer agent is also administered, it is administered in an amount that is effective to achieve its intended purpose. The optimal amounts of such known anticancer agents are well known to those skilled in the art.
  • the unit oral dose may comprise from approximately 0.01 to approximately 50 mg, preferably approximately 0.1 to approximately 10 mg of the compound of the disclosure.
  • the unit dose may be administered one or more times, with one or more tablets daily, each containing from approximately 0.1 to 50 mg, conveniently approximately 0.25 to 10 mg of the compound of the disclosure or solvates thereof.
  • the compound (s) of the disclosure may be present at a concentration of approximately 0.01 to 100 mg per gram of carrier.
  • the compound (s) of the disclosure may be administered as a raw chemical.
  • the compounds of the disclosure may also be administered as part of a suitable pharmaceutical preparation containing pharmaceutically acceptable carriers (comprising excipients and auxiliaries) .
  • pharmaceutically acceptable carriers facilitate the manufacture of pharmaceutically acceptable preparations from the compound (s) .
  • the pharmaceutical preparations particularly oral preparations and those used for the preferred administration routes, such as tablets, dragees, and capsules, as well as solutions suitable for injection or oral administration, contain from approximately 0.01%to 99%, preferably from approximately 0.25%to 75%of active compound (s) , together with excipient (s) .
  • Acid addition salts are formed by mixing a solution of the compound (s) of the present disclosure with a solution of a pharmaceutically acceptable non-toxic acid, such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, and the like.
  • a pharmaceutically acceptable non-toxic acid such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, and the like.
  • Base addition salts are formed by mixing a solution of the compounds of the present disclosure with a solution of a pharmaceutically acceptable non-toxic base, such as sodium hydroxide, potassium hydroxide, hydrocholine, sodium carbonate, tris (hydroxymethyl) aminomethane, N-methyl-glucosamine and the like.
  • a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, hydrocholine, sodium carbonate, tris (hydroxymethyl) aminomethane, N-methyl-glucosamine and the like.
  • the pharmaceutical preperations of the disclosure may be administered to any mammal, so long as they may experience the therapeutic effects of the compound (s) of the disclosure.
  • mammals Foremost among such mammals are humans and veterinary animals, although the disclosure is not intended to be so limited.
  • the pharmaceutical preperations of the present disclosure may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, frequency of treatment, and the nature of the effect desired.
  • the pharmaceutical preparations of the present disclosure can be manufactured in a known manner, e.g., by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • Pharmaceutical preparations for oral use may be obtained by combining the active compounds with solid excipient (s) , optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers, such as saccharides, e.g. lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate; as well as binders, such as starch paste, including maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, e.g. lactose or sucrose, mannitol or sorbitol
  • cellulose preparations and/or calcium phosphates e.g. tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch paste, including maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose
  • disintegrating agents may be added, such as the above-mentioned starches and carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, e.g., silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose solutions such as cellulose acetate phthalate or hydroxypropylmethyl-cellulose phthalate, are used.
  • Dyes or pigments may be added to the tablets or dragee core coatings, e.g., for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which may be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active compounds in the form of granules, which may be mixed with fillers, such as lactose; binders, such as starches; and/or lubricants, such as talc or magnesium stearate, and stabilizers.
  • the active compound (s) are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin, in which stabilizers may be added.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds, e.g., aqueous solutions and alkaline solutions of water-soluble salts.
  • appropriate oily injection suspensions of the active compounds may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides or polyethylene glycol-400, or cremophor, or cyclodextrins.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, e.g., sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the topical formulations of this disclosure can be formulated as oils, creams, lotions, ointments and the like by choice of appropriate carriers.
  • Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin) , branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C 12 ) .
  • Preferred carriers are those in which the active ingredient (s) are soluble.
  • Emulsifiers, stabilizers, humectants and antioxidants may also be included, as well as agents imparting color or fragrance, if desired.
  • transdermal penetration enhancers may be employed in these topical formulations. Examples of such enhancers are found in U.S. Patent Nos. 3,989,816 and 4,444,762.
  • Creams are preferably formulated from a mixture of mineral oil, self-emulsifying beeswax and water in which mixture of the active ingredient, dissolved in a small amount of an oil, such as almond oil, is admixed.
  • An oil such as almond oil
  • a typical example of such a cream is one which includes approximately 40 parts of water, approximately 20 parts of beeswax, approximately 40 parts of mineral oil and approximately one part of almond oil.
  • Ointments may be formulated by mixing a solution of the active ingredient (s) in a vegetable oil, such as almond oil, with warm soft paraffin and allowing the mixture to cool.
  • a vegetable oil such as almond oil
  • a typical example of such an ointment is one which includes approximately 30%by weight of almond oil and approximately 70%by weight of white soft paraffin.
  • an "effective amount” refers to an amount of the active compound (s) or pharmaceutical agent (s) that cause the biological or medical response of the tissue, system, animal, or individual human being sought by researchers, veterinarians, physicians, or other clinicians, and the biological or medical response includes one or more of the following: (1) preventing diseases: for example, preventing diseases, conditions, or disorders in an individual who may be susceptible to the disease, condition, or disorder but still not experiencing or presenting the pathology or symptoms of the disease, (2) inhibiting diseases: for example, inhibiting diseases, conditions or disorders in an individual experiencing or presenting the pathology or symptoms of the disease, condition, or disorder (i.e., preventing the further development of the pathology or symptoms of the pathology and/or disorder) , and (3) improving diseases: for example, improving diseases, conditions, or disorders in an individual experiencing or presenting the pathology or symptoms of the disease, condition, or disorder (i.e., reverse the pathology and/or symptom) . Therefore, a non-limiting example of an "effective dose" of the composition of
  • the composition containing the Wee1 kinase inhibitor can be used in combination with at least one known anti-cancer drug or a pharmaceutically acceptable salt of an anti-cancer drug.
  • the composition can be used in combination with other anti-cancer drugs related to the mechanism of DNA damage and repair, including PARP inhibitors olaparib, niraprib, rucaparib, talazoparib, pamiparib, fluzoparib and senaparib; HDAC inhibitors Volinota, Romididesin, Papiseta and Bailesta; and so on.
  • composition can be used in combination with other anti-cancer drugs related to cell division checkpoints, including Chk1/2 inhibitors, CDK4/6 inhibitors such as Palbociclib, ATM inhibitors, ATR inhibitors, DNA-PK inhibitors and so on.
  • Other known targeted anti-cancer drugs which may be used for anti-cancer combination therapy include, but are not limited to, PRMT5 inhibitors, Pol ⁇ inhibitors, RAD51 inhibitors and so on.
  • anti-cancer drugs which may be used for anticancer combination therapy include tamoxifen, letrozole, fulvestrant, mitoguazone, octreotide, retinoic acid, arsenic trioxide, zoledronic acid, bortezomib, carfilzomib, Ixazomib, vismodegib, sonidegib, denosumab, thalidomide, lenalidomide, Venetoclax, Aldesleukin (recombinant human interleukin-2) and Sipueucel-T (prostate cancer treatment vaccine) .
  • the present disclosure also provides use of the reagent (s) described herein to detect the presence of mutation (s) in FBXW7 and/or CHD4 of a subject, in particular in the preparation of a detection kit to determine the subject's sensitivity or efficacy to a treatment with a Wee1 kinase inhibitor, in particular with a Wee1 kinase inhibitor described herein.
  • the reagent (s) may include those routinely used to detect FBXW7 or CHD4 gene and/or protein. For example, in order to detect whether there is a change in a gene leading to the protein mutation (s) described in the present disclosure, PCR and sequencing can be used to determine whether there is a mutation in the gene.
  • the reagents include but are not limited to the reagents required for PCR and sequencing, such as primers, probes, polymerases, dNTP, etc.
  • the technique of designing genes and probes based on known gene sequences is well known in the field.
  • Tissue samples containing one or more types of cells such as cancerous tissue mentioned above, or non-cancerous tissue biopsy samples, such as a control, may be obtained from the subject.
  • the samples were then tested for the presence of FBXW7 and/or CHD4 mutation (s) , especially those described herein, using a conventional method.
  • the presence of the mutation (s) described herein indicates that the subject is sensitive to a treatment with the Wee1 kinase inhibitor described herein or that the subject's tumor could be effectively treated with the Wee1 kinase inhibitor described herein.
  • the present disclosure provides a method for treating a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) in a subject, comprising administering to the subject an effective amount of a Wee1 kinase inhibitor or its pharmaceutical composition.
  • the method further comprises detecting whether the subject has the FBXW7 mutation (s) and/or a CHD4 mutation (s) , especially those described herein, or whether the subject has a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein.
  • the Wee1 kinase inhibitors are those described herein, especially the compounds having the structure represented by Formula I, II or III.
  • the subject is suffered from any of the cancers with the indicated mutations listed in Table A.
  • the subject is suffered from a rectal cancer with a mutated FBXW7, wherein the mutated FBXW7 contains one or more mutations selected from the group consisting of T616I, R465C, truncation starting from position 118 and truncation starting from position 279.
  • the rectal cancer further comprises one or more mutations occurred in APC, TP53, KRAS and ARID1A.
  • the mutations in APC are selected from the group consisting of frameshift mutation starting from position 541 (including position 541) or from 660 (including position 660) , truncation starting from position 902 or 1292.
  • the mutations in TP53 are selected from the group consisting of R273H, R175H, R248Q and R248W/Loss.
  • the mutations in KRAS are selected from the group consisting of G12C and G12S.
  • the mutation in ARID1A is a truncation starting from position 1086.
  • the present disclosure provides a method for evaluating an individual with a cancer to determine his treatment regime, comprising identifying whether cancer cells obtained from the individual have a FBXW7 mutation and/or a CHD4 mutation, and/or whether the cancer cells have a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein, and administering to an individual with the FBXW7 mutation and/or the CHD4 mutation and/or with a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein a Wee1 kinase inhibitor, especially those inhibitors described herein.
  • the cancer cells may have an FBXW7 and/or CHD4 mutant phenotype.
  • Identification of cancer cells with FBXW7 and/or CHD4 mutation (s) may also include exposing a cancer cell sample obtained from an individual with cancer to a Wee1 kinase inhibitor and determining a number of dead cells in the sample relative to dead cells in a control sample.
  • a higher number of dead cancer cells than that of dead control cells demonstrates increased sensitivity of cancer cell to the Wee1 kinase inhibitor, indicating that cancer cells may have a FBXW7 mutation or a CHD4 mutation, such as reduced expression or activity of FBXW7 or CHD4 or loss of function.
  • the present disclosure provides a method for treating cancer in an individual with a Wee1 kinase inhibitor, wherein the individual is a heterozygote or polymorphism with a FBXW7 mutation, or a CHD4 mutation.
  • the treatment method further comprises determining whether the cancer tissue or cancer cell of the individual has a FBXW7 mutation and/or a CHD4 mutation by a genetic testing method, and then administering to the individual with the Wee1 kinase inhibitor if his cancer is identified as a cancer with a FBXW7 mutation and/or a CHD4 mutation.
  • the present disclosure provides a method for determining whether or not a cancer patient is responsive or sensitive to or benefits from a treatment with a Wee1 kinase inhibitor, especially the Wee1 kinase inhibitor described herein, comprising identifying whether the patient’s cancer cells have a FBXW7 mutation and/or a CHD4 mutation, and/or a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein, wherein presence of a FBXW7 mutation and/or a CHD4 mutation, especially any of the mutations as described herein, and/or presence of reduced expression or reduced activity of the FBXW7 protein and/or the CHD4 protein, indicates that the patient is sensitive to the treatment with a Weel kinase inhibitor.
  • the identification is performed with a sample, such as a surgical sample or a biopsy sample, from the cancer patient.
  • the expression level and activity of the FBXW7 protein and the CHD4 protein could readily be determined by, such as comparing the expression level or activity of the protein in the cancer cell to that in the normal cell of the same tissue with a conventional technique.
  • Mini patient-derived xenograft (MiniPDX TM ) model is a new method to test drug efficacy on human tumors in animals.
  • MiniPDX TM minimal patient-derived xenograft
  • a proprietary preparation method was used to isolate tumor cells from fresh human tumor specimens.
  • the cell preparation was placed in a dedicated MiniPDX TM device and transplanted subcutaneously in mice.
  • the specially designed pore size of the MiniPDX TM device allows small molecules below 500 KD to enter and exit freely while tumor cells remain in the device.
  • mice were administered with testing drugs systemically for 7 days. After treatment, the MiniPDX TM devices were taken out, and the tumor cell viability was determined by an in vitro ATP method. The effectiveness of dosage regimens was calculated based on the overall viability of tumor cells.
  • mice of Balb/c-nude strain of 6-8 weeks of age and weighing 18-22g were purchased from GemPharmatech Co., Ltd. Animals were housed for at least 3 days for adaption before experiment.
  • mice used in the experiments were provided with food and water ad libitum.
  • the mouse pellet feed was cobalt-60 radio-sterilized and the drinking water was sterilized by high pressure.
  • Sample cases Sample type Patient information Pathological results 1 Surgical sample male, 63 years old rectal cancer 2 Biopsy sample male, 63 years old rectal cancer 3 Surgical sample male, 44 years old rectal cancer 4 Biopsy sample male rectal cancer 5 Biopsy sample male, 65 years old rectal cancer 6 Surgical sample female, 49 years old rectal cancer 7 Chest fluid sample male, 65 years old rectal cancer 8 Biopsy sample male, 70 years old rectal cancer 9 Surgical sample male, 47 years old rectal cancer 10 surgical sample female, 63 years old rectal cancer 11 biopsy sample male, 57 years old rectal cancer 12 surgical samples male, 80 years old rectal cancer 13 Surgical sample female, 57 years old rectal cancer 14 biopsy sample male, 51 years old rectal cancer 15 biopsy sample male, 50 years old rectal cancer 16 Surgical sample female rectal cancer
  • the patient's surgical sample was immersed in HBSS sample buffer.
  • cell suspension was prepared with MiniPDX TM sample preparation buffer and the cells were counted.
  • MiniPDX TM device Cells were re-suspended and filled into MiniPDX TM device.
  • the MiniPDX TM device containing human tumor cells was inoculated into mice subcutaneously.
  • the treatment group was administered with compound A in a vehicle of 0.5%MC containing 0.2%Tween 80 suspension in water at 60 mg/kg, P. O., QD for 7 days and the control group was administered with an equal volume of vehicle.
  • the treatment started on the inoculation day that was designated as day 0.
  • the pharmacological efficacy of Compound A was evaluated based on the relative increase rate (T/C %) of tumor cells.
  • T/C % T /C ⁇ 100%, where T and C are the cell numbers of the treated group and the control group, respectively.
  • FBXW E117* indicates that FBXW7 is truncated at 117-E, and the gene encodes a truncated protein.
  • FBXW7 mutation may be used as a biomarker to screen patients for Wee1 inhibitors in clinical treatment of cancer.
  • the 50%inhibition concentration (IC 50 ) values on cell proliferation were determined using CTG cell viability assay. Human cancer cell lines were treated with multiple concentrations of the testing compound and viable cells were detected under each drug concentration after 72 hours of treatment. The IC 50 values were determined by plotting viable cells against compound concentrations and calculated using commercial curve fitting programs.
  • Fetal bovine serum was purchased from ExCell Bio. (Cat #FND500) .
  • CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega (Cat#G7573) .
  • the cells were resuscitated and cultured in recommended culture mediums.
  • the cells in 96 well plates were returned to a CO 2 incubator and cultured overnight at 37 °C, 5%CO 2 and 95%humidity.
  • testing compounds were dissolved with corresponding solvents to make stock solutions. A serial dilution was made to the concentrations that were 10 times the final testing concentrations.
  • the cells in the 96 well plates with the testing compound added were return to CO 2 incubator and cultured for additional 72 hours at 37 °C, 5%CO 2 and 95%humidity.
  • the CellTiter Glo reagent and cell culture plates containing cells treated with the testing compound were placed at room temperature for 30 minutes.
  • the plate was placed on an orbit shaking table for 2 minutes to fully lyse the cells.
  • the plate with lysed cells was placed at room temperature for 10 minutes.
  • the plate was loaded to a EnVision plate reader to read chemiluminescence values.
  • Cell survival rate (%) (Lum drug to be tested -Lum culture medium control ) / (Lum cell control -Lum culture medium control ) ⁇ 100%.
  • Lum cell control -Lum culture medium control was set as 100%, and Lum Medium control value was set as 0%.
  • Amplification multiple (Lum None treated –Lum Medium control of Day 5) / (Lum None treated –Lum Medium control of Day 2)
  • E40Ter means the protein was truncated and has only N-terminal 1-40 amino acids (AA)
  • E103Ter means the protein truncated and has only N-terminal 1-103 AAs
  • E117del refers to the absence of an E in the 117th position
  • L403fs means that there is a reading frame shift mutation at the 404th AA (including the 404th AA)
  • F1488fs refers to a reading frame shift after position 1488
  • K50K refers to a synonymous mutation.
  • the Wee1 inhibitor Compound A was found to have inhibitory effects on cell growth of cell lines that carry mutations in FBXW7 gene or in CHD4 gene or in both genes. Therefore, mutations in FBXW7 gene or CHD4 gene or both may be used as a biomarker to screen cancer patients for Wee1 inhibitor treatment.
  • Fetal bovine serum was purchased from ExCell Bio. (Cat #FND500) .
  • CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega (Cat#G7573) .
  • FBXW7 defective LoVo cells were introduced with wild type sequence of FBXW7 gene to exam the sensitivity to wee1 inhibitors.
  • G plasmid (addgene #12259) and 7.5 ⁇ g psPAX2 plasmid were mixed with either 10 ⁇ g FBXW7 expression plasmid (genecopiea, EX-T3614-Lv128) or the empty plasmid (EX-NEG-LV128) in control group in 0.5 ml DMEM medium and incubated at room temperature for 5 minutes.
  • 70 ⁇ l of 1 mg/ml PEI (sigma #764965) was mixed with 0.5 ml DMEM and incubated at room temperature for 5 minutes. Then the plasmid mixtures were mixed with PEI solutions, and incubated at room temperature for an additional 20 minutes.
  • the plasmid/PEI mixture was added dropwise to a 10 cm cell culture plate containing 80%confluent HEK293T cells in 5 ml DMEM (10%FBS) . Eight hours later, the culture medium was replaced with 10 ml fresh DMEM medium (10%FBS) and cells were return to CO 2 incubator. After 48 hours, the culture medium was removed and centrifuged at 300g for 5 minutes. The supernatant was retained and centrifuged again at 2000g for 20 minutes. About 1/3 volume of 40%PEG8000 was added into the supernatant and the mixture was incubated on an ice shaker for 4 hours. Then the mixture was centrifugated again at 4 °C at 1500g for 1 hour.
  • the precipitate was resuspended in 100 ⁇ l PBS, and added into a six-well plate containing LOVO cells at 80%confluence (each well contained 1ml DMEM with 10%FBS and 8 ⁇ g/ml polybrene) .
  • 1 ml DMEM (10%FBS+8 ⁇ g/ml polybrene) fresh medium was added to each well.
  • cell culture medium was replaced with 2 ml fresh culture medium (10%FBS and 4 ⁇ g/ml puromycin) .
  • FBXW7 Verification of FBXW7 overexpression: the cell lysate was detected by Western blot.
  • FBXW7 R&D NB100-88138
  • primary antibody was incubated at 1: 1000 dilution for 4 °Covernight, and then secondary antibody was incubated at room temperature for 1 hour, then incubated with ECL substrate, and then the protein expression was detected with an imager (Fig. 1) .
  • Results show that there are overexpressed FBXW7 in the lysates of LoVo cells with FBXW7 knock-in.
  • FBXW7 defective LoVo cells expressed more FBXW7 proteins.
  • the IC 50 values of Compound A tested with FBXW7 overexpressed LoVo cells or FBXW7 defective LoVo cells (control) were 121 nM and 67 nM, respectively (Fig. 2) . Therefore, introduction of wild type FBXW7 gene sequence into FBXW7 defective LoVo cells reduces the sensitivity of these cells to Wee1 inhibitors Compound A.
  • FBXW7 defective LoVo cells are more sensitive toWee1 inhibitor (Compound A) treatment.
  • OVCAR3 cells expressing wild-type CHD4 gene were introduced with CHD4 knockdown virus using CRISPR technique. These OVCAR3 cells either expressing or not expressing CHD4 gene were tested for sensitivity to the Wee1 inhibitor Compound A treatment.
  • G (addgene #12259) plasmid and 7.5 ⁇ g psPAX2 plasmid were mixed with either 10 ⁇ g CHD4 knockdown plasmid (genecopiea, HSH091375-LVRU6GP) or a control plasmid (CSHCTR001-LVRU6GP) in 0.5 ml DMEM and left at room temperature for 5 minutes.
  • 70 ⁇ l 1 mg/ml PEI was mixed with 0.5 ml DMEM and left at room temperature for 5 minutes. Then, the plasmid mixtures were mixed with the PEI solution and incubated at room temperature for 20 minutes.
  • the plasmids/PEI mixtures were added dropwise to 10 cm cell culture plates containing HEK293T cells at 80%confluence in 5 ml DMEM (10%FBS) . After 8 hours incubation, the culture medium was replaced with 10 ml fresh DMEM medium (10%FBS) and incubated for another 48 hours. Afterwards, the culture medium was collected and centrifuged at 300g for 5 minutes, the supernatant was collected and centrifuged at 2000g for 20 minutes. About 1/3 volume of 40%PEG8000 was added to the supernatant and the mixture was incubated on an ice shaker for 4 hours. After incubation, the mixture was centrifugated at 4 °C at 1500g for 1 hour.
  • the precipitate was resuspended with 100 ⁇ l PBS and added dropwise to a six-well plate containing OVCAR3 cells at 80%confluence (each well contained 1 ml DMEM with 10%FBS and 8 ⁇ g/ml polybrene) .
  • the cells were incubated at 37°C in a 5%CO 2 incubator.
  • 1 ml DMEM (10% FBS + 8 ⁇ g/ml polybrene) was added to each well.
  • culture medium was replaced with 2 ml fresh medium (10%FBS+4 ⁇ g/ml puromycin) .
  • the knockdown level of CHD4 was validated using Western blot.
  • the primary antibody against CHD4 was from Sinobiological (#102312-T34) .
  • Knockdown of CHD4 genes in OVCAR3 cells decreased the CHD4 protein expression (Fig. 3) .
  • the IC 50 values of Compound A tested in the CHD4 knockdown OVCAR3 cells and the OVCAR3 cells expressing wild-type CHD4 gene were 334 nM and 1881 nM, respectively. Therefore, down-regulation of the CHD4 expression increased the sensitivity of OVCAR3 cells to Wee1 inhibitor Compound A.

Abstract

This disclosure relates to the use of Wee1 kinase inhibitors in the treatment of cancer. In particular, the disclosure provides the use of Wee1 kinase inhibitors in the preparation of drugs for the treatment of cancers with FBXW7 or CHD4 mutation.

Description

USE OF WEE1 KINASE INHIBITORS IN THE TREATMENT OF CANCER
Field of the Disclosure
This disclosure is in the field of medicinal chemistry and relates to the field of cancer treatment. In particular, the disclosure relates to the use of Wee1 kinase inhibitors in the treatment of cancer with FBXW7 or CHD4 mutation.
Background of the Invention
The process of proliferation and division of eukaryotic cell is referred to as cell cycle. A cell grows from a newly divided cell to an adult cell to give birth of a daughter cell through cell cycle. Cell cycle can be roughly divided to 4 distinct phases, the G1 phase, the S phase, the G2 phase and the M phase. The G1 phase is also referred to as the phase of growth, which is characterized by active cell metabolism, large scale of synthesis of protein, carbohydrate, lipid and RNA and rapid growth from newly divided young cells to mature cells. There are also some cells that do not go on the cell cycle after completing the G1 phase and stay in the phase, and those cells are referred to as staying in G0 phase. The S stage of cell cycle is characterized by DNA synthesis and replication. In this phase, the cell chromosome changes from diploid to tetraploid. Then, cells enter the G2 phase. The characteristic of G2 phase is that cells in this phase continue to grow, and the synthesized DNA is checked for damages and mutations. The damaged DNA is repaired and cells prepare for mitosis. The last phase of the cell cycle is the M phase. In this phase, the cells divide from one mother cell to two identical daughter cells through mitosis, completing cell reproduction cycle. During the whole process of the cell cycle, there are multiple cell cycle checkpoints, including G1/S or G1 checkpoint and G2/M or G2 checkpoint. The main functions of these checkpoints are to check and ensure the accurate replication of genetic information (DNA) during the cell cycle and to determine whether the cells enter the next phase of cell cycle.
Each cell cycle checkpoint is a system composed of multiple factors that work through complex mechanisms. For instance, the G2/M checkpoint uses a complex process to check for DNA damage. In this process, there is an important kinase Cdk1 that forms a complex with Cyclin-B1 (Nurse, P., Nature, 1990, 344: 503-508) . The activation and inactivation of Cdk1 play a vital role in the cell’s entering mitosis (M) from G2 phase and the subsequent completion of mitosis. The activity of Cdk1 is regulated by multiple mechanisms, including the binding of Cyclin A or Cyclin B, phosphorylation and dephosphorylation. Wee1 kinase phosphorylates Cdk1 and inhibits its activity, as a result it delays the cell from entering mitosis.
Wee1 is a tyrosine kinase that regulates the activity of Cdk1 by phosphorylating tyrosine 15 (Y15) on Cdk1 molecule (McGowan, C. H. and Russell, P., EMBO J, 1993, 12: 75- 85; Parker, L. L. and Piwnica-Worms, H., Science, 1992, 257: 1955-1957) . Accordingly, Wee1 is a key inhibitory regulator of Cdk1 activity and plays an important role in G2-M phase checkpoints (O’ Connell, M. J. et al, EMBO J, 1997, 16: 545-554) . Loss or inactivation of Wee1 may result in premature entry into mitosis, leading to mitotic failure and cell death (Stumpff, J. et al, Curr Biol, 2004, 14: 2143-2148) . Some tumor cells have functional deficiency or loss of G1/S cell cycle checkpoint function and heavily rely on G2/M phase checkpoints to ensure the progress of cell growth and division (Sancar, A. et al, Annu Rev Biochem, 2004, 73: 39-85) . In cancer cells the function of G1/S checkpoint is often lost due to reasons such as mutations in p53. These cells are heavily relying on G2/M checkpoint function when DNA damage happens and sensitive to Wee1 function loss (Wang, Y., et al, Cancer Biol&Ther, 2004, 3: 305-313) .
Inhibition of Wee1 activity may selectively promote the death of cancer cells with defective cell cycle checkpoints, but have less effect on normal cells with normal cell cycle checkpoint function. Therefore, Wee1 kinase inhibitors may be used as targeted therapy for the treatment of cancers and other disorders that have cell cycle checkpoint defects.
In recent years, several Wee1 kinase inhibitors have been developed and reported including compounds disclosed in WO2007126122 (substituted 1, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidin-3-one compounds) , WO2019028008, WO2019173082 and WO202021032 (substituted 1, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidin-3-one compounds) , WO2015092431 and WO2018162932 (substituted 2, 3-dihydropyrimido [4, 5-d] pyrimidin-4(1H) -one compounds) , WO2019037678 (substituted 1, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidin-3-one compounds) , WO2020210377 and WO2020210383 (substituted heterocyclic compounds) , WO2020210375, WO2020210380, WO2020210381 (substituted 1, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidin-3-one compounds) , WO2018133829 (l, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidin-3-one compounds) and WO2019085933 (l, 2-dihydro-3H-pyrazolo [3, 4-d] pyrimidine-3-one macrocyclic compounds) . Among them, Adavosertib (AZD1775) is the first Wee1 kinase inhibitor to enter the clinic and showed good clinical efficacy data in several tumors including ovarian, colon, and uterine carcinoma. Wee1 kinase inhibitors that are currently in clinical phase include ZN-c3 and Debio-0123, which are in clinical phase I. There are also a variety of Wee1 kinase inhibitors currently in the preclinical research stage, such as DN-1609, NUV-569 and the like.
WO2018090939 discloses the following compounds of Formula I or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
Figure PCTCN2022134300-appb-000001
wherein, A is N or CR 15;
R 1 is hydrogen, optionally substituted C 1-C 8 alkyl, optionally substituted C 2-C 8 alkenyl, optionally substituted C 3-C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
R 2 is optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted aryl, or optionally substituted heteroaryl;
R 3-R 7 and R 15 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-C 10 alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
In addition, WO2019011228 discloses the following compounds of Formula II or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
Figure PCTCN2022134300-appb-000002
wherein, A is N or CR 6;
R 1 is hydrogen, optionally substituted C 1-C 8 alkyl, optionally substituted C 2-C 8 alkenyl, optionally substituted C 3-C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
R 2 is optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted aryl, or optionally substituted heteroaryl;
R 3-R 6 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-C 10 alkyl (such as haloalkyl, hydroxyalkyl, aminoalkyl and carboxyalkyl) , alkenyl, alkynyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
WO2021073491 discloses the following compounds of Formula III or pharmaceutically acceptable salts or prodrugs thereof as Wee1 kinase inhibitors.
Figure PCTCN2022134300-appb-000003
wherein, R 1 and R 2 are independently halo; R 3 is halo, C 1-4 alkyl or C 1-4 alkoxy; R 4 and R 6 each are independently H or C 1-4 alkyl; R 5 is H or C 1-4 alkyl; R 7 is H, halo, C 1-4 alkyl or C 1-4 alkoxy; and X is CH or N.
FBXW7 belongs to the F-box protein family which is the target protein recognition component of the SCF (SKP1 cullin-F box) type ubiquitin ligase and contains several conservative domains for protein interaction. In most cases, FBXW7 recognizes the phosphorylated substrate and promotes it ubiquitination and degradation. Many substrates of FBXW7 are oncogenes, as one of the important factors of ubiquitin proteasome degradation pathway. For example, Cyclin E (the protein encoded by CCNE1 gene) , c-myc, c-jun and Notch are degraded through the ubiquitin proteasome pathway mediated by FBXW7. Mutations and deletions of FBXW7 can cause the accumulation of these genes related to cancer cell proliferation, and have been found in ovarian cancer, breast cancer and colorectal cancer. Nearly 9%of colorectal cancers have mutations or deletions of FBXW7 gene. It has been confirmed that FBXW7 recognizes and participates in the ubiquitination and degradation of a variety of oncoproteins in colorectal cancer, gastric cancer, ovarian cancer and liver cancer, and plays a role in inhibiting the biological functions of tumor cells. Therefore, mutations of FBXW7 gene often lead to the occurrence and progression of tumors.
As the main substrate of FBXW7, cyclin E cannot be degraded through the ubiquitin proteasome pathway due to the mutation of FBXW7, thus forming the accumulation of cyclin E (B. T. Hughes et al., 2013, Proc Natl Acad Sci U S A 110; 8954 –8959) . Both CCNE1 amplification and cyclin E accumulation will increase the speed of cells passing G1/Sphase due to their overexpression, thus depending on the checkpoint of G2/M phase to ensure normal cell proliferation. By targeting the inhibition of Wee1 kinase, the checkpoint of G2/M phase of the cell that causes abnormality, thus making the cell sensitive to DNA damage, thus causing cell death. At present, it is found in a phase II clinical trial (Shiqing Fu et al., AACR Annual Meeting 2021; 974-974) that Wee1 kinase inhibitor (AZD1775) has good anti-tumor activity in CCNE1 amplified refractory solid tumors, especially in high-grade serous ovarian cancer. The study showed that in 27 cases of refractory solid tumors, confirmed partial remission was observed in 7 patients, and the objective response rate (ORR) was 25.9% (95% CI 15.1-47.5%) . Among 13 patients with high-grade serous ovarian cancer, 5 patients had objective remission (38.5%) , and 8 patients were stable for more than 6 months or achieved objective remission (61.5%) . Therefore, FBXW7 mutation, as an important reason for cyclin E accumulation, can be used as a predictive biomarker to screen potential patients of Wee1 kinase inhibitors.
CHD4 (Chromododomain Helicase DNA binding protein 4) is a major subunit that inhibits nucleosome remodeling and deacetylase (NuRD) complex. It contains a helicase/atpase domain, which can promote histone deacetylation, thus controlling chromatin reorganization and transcriptional regulation. Nucleosome remodeling and deacetylase (NuRD) complex is one of the major chromatin remodeling complexes found in cells. It plays an important role in regulating gene transcription, genome integrity and cell cycle progression. Through the influence on these basic cellular processes, an increasing body of evidence shows that the activity change of NuRD complex can lead to developmental defects, tumorigenesis and accelerated aging.
CHD4 plays an important role in DNA damage response and regulation of cell cycle checkpoint activation. CHD4 knockdown will increase the sensitivity of this cell line to chemotherapy (Kouki Neo, et al, Journal of Hepatology, 2015; 63: 1164-72) , and the loss of CHD4 function will make it sensitive to PARP inhibitors (Mei ren Pan et al, J Bio Chem, 2012; 287 (67) : 64-72) .
In a phase II clinical trial of Wee1 kinase inhibitor AZD1775, the data indicates the proof of concept on uterine serous cancer. However, in exploratory biomarker analysis of this trial, no relationship between clinical activity and specific molecular biomarker was observed. Therefore, the discovery of biomarkers is of great significance for understanding the mechanism of Wee1 kinase inhibitors and expanding the indications.
The present disclosure finds that Wee1 kinase inhibitor has good efficacy in a variety of cell lines carrying specific FBXW7 mutations or CHD4 mutations, as well as in colon cancer mouse models with human FBXW7 or CHD4 mutations. It provides a potential therapeutic method for a variety of tumor patients carrying FBXW7 or CHD4 mutations
Summary of the Disclosure
The present disclosure finds that the growth of various tumor cells with FBXW7 and/or CHD4 mutations can be effectively inhibited by inhibiting Wee1 kinase activity, which is of great significance in the treatment of tumors with FBXW7 and/or CHD4 mutation.
Therefore, this disclosure provides use of a Wee1 kinase inhibitor in the preparation of medicament for the treatment of a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , a Wee1 kinase inhibitor for use in a method for the treatment of a tumor  with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , and a method for treatment of a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) , comprising administration of an effective amount of a Wee1 kinase inhibitor or its pharmaceutical composition to a subject in need.
In one or more embodiments, preferably, the Wee1 kinase inhibitors described herein are small-molecule Wee1 kinase inhibitors. These inhibitors include, but are not limited to, AZD1775, Zc-03, Debio-0123, IMP7068, DN-1609 and NUV-569; and compounds disclosed in WO2007126122, WO2019028008, WO2019173082, WO202021032, WO2015092431, WO2018162932, WO2019037678, WO2020210377, WO2020210383, WO2020210375, WO2020210380, WO2020210381, WO2018133829, WO2019085933, WO2018090939, WO2019011228 and WO2021073491.
In one or more embodiments, the Wee1 kinase inhibitor is a compound of Formula (I) , Formula (II) or Formula (III) as described herein.
In one or more embodiments, the said tumor is a solid tumor or a hematologic lymphoma.
In one or more embodiments, the said FBXW7 mutation is R465C mutation and/or R505C mutation.
In one or more embodiments, the tumor is colorectal cancer, preferably colorectal cancer with R465C mutation and/or R505C mutation in FBXW7 protein.
In one or more embodiments, the method of treatment described herein for tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) further comprises detecting the presence of a mutation in the FBXW7 and/or the CHD4, particularly the mutation (s) as described herein.
Also provided is use of the reagents described herein to detect the presence of a mutation (s) in a subject's FBXW7 and/or CHD4, in particular in the preparation of a detection kit to determine the subject's sensitivity or efficacy to treatment with a Wee1 kinase inhibitor, particularly the Wee1 kinase inhibitor described herein.
Description of drawing
Fig. 1: FBXW7 was overexpressed in LoVo.
Fig. 2: Overexpression of FBXW7 in LoVo cells reduced the sensitivity of the cells to Wee1 kinase inhibitors (IC 50: 67 nM in the control group and 121 nM in the FBXW7 -overexpressed group) .
Fig. 3: CHD4 expression was decreased in OVCAR3 CHD4 knockdown group (shRNA#1) .
Fig. 4: Knockdown of CHD4 in OVCAR3 cells increased the sensitivity of cells to Wee1 kinase inhibitors (IC 50: 1881 nM in the control group and 334 nM in the CHD4 knockdown group) .
Fig. 5: A bar graph illustrating cancer types and their occurrence of alteration of FBXW7.
Fig. 6: A bar graph illustrating cancer types and their occurrence of alteration of CHD4.
Detailed description of the disclosure
Tumors described herein refer to new organisms formed by local histiocytosis of the body under the action of various tumorigenic factors. According to the cellular characteristics of new organisms and the degree of harm to the body, tumors can be divided into benign tumors and malignant tumors. Malignant tumor can be divided into cancer and sarcoma, cancer is defined as a malignant tumor that comes from epithelial tissue. Sarcoma is defined as a malignant tumor that mesenchymal tissue produces. For example, the malignant tumor that forms by epithelium of large intestine mucosa calls carcinoma of large intestine mucosa epithelium, simply called large intestine carcinoma. Tumor cells or cancer cells are generally characterized by abnormal proliferation relative to normal cells and formation of clusters or tumors in individuals with tumors. Tumors include solid tumors and hematologic tumors.
FBXW7 and CHD4 described herein have well-known meanings in the art. The wild-type sequence of human FBXW7 is showed in Genbank access number AAI43945.1, and the wild-type sequence of human CHD4 is showed in Ganbank access number AAH38596.1. Mutation described herein refer to mutation relative to wild-type sequence.
The cancer carrying one or more FBXW7 mutations and/or one or more CHD4 mutations described herein can include any type of solid tumor or malignant lymphoma. Cancer can be familial or sporadic. Mutations can be one or more of substitutions, deletions, and insertions. Frameshift mutations at the gene level can result in parts of the encoded protein completely different from the wild-type protein, and such mutations can be regarded as deletion mutations compared with the wild-type protein.
In some embodiments, the mutation occurs in the WD40 domain of FBXW7 (residues 378-659 of FBXW7) . In some embodiments, the FBXW7 mutation described herein includes, in particular, one or more mutations occurring at positions 27, 117, 200, 226, 336, 441, 460, 465, 479, 505, 514, and 616. In the preferred embodiments, the mutation occurs at one or more of positions 465, 479, and 505. Preferably, the mutation is one or more of H27Q, deletion of amino acid at position 117, V200I, T226I, I336M, R441W, H460Y, R465C, R465H, R479Q, R505C, R505H, V514D, and T616I. Preferably, the mutation includes or is R465C, R465H,  R479Q, R505C, or R505H. In some embodiments, the mutation is a truncated (deletion) mutation, including, for example, truncation of amino acid residues at positions 1-40 of the N-terminus of FBXW7, truncation of amino acid residues at positions 1-103 of the N-terminus of FBXW7, truncation starting from position 118 (including position 118) of FBXW7, truncation starting from position 279 (including position 279) of FBXW7, and frameshift mutation starting from position 404 (including position 404) of FBXW7.
In some embodiments, The CHD4 mutation includes one or more mutations occurring at positions 19, 57, 279, 538, 732, 841, 852, 869, 895, 966, 1038, 1042, 1068, 1105, 1272, 1384, 1541, 1886 and 1894, and truncation mutation. Preferably, the mutations include D19G, P57S, R279H, R538W, G723S, A841V, Y852H, V869M, Q895K, M966I, G1038D, G1042D, R1068C, R1105W, G1272D, P1384H, P1541H, Q1886H, S1894T and frameshift mutation starting from position 1488 (including position 1488) of CHD4. In some embodiments, the CHD4 mutation includes a mutation at position 279 or position 1042, preferably R279H or G1042D.
In some embodiments, tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) can be tumors occurring in the urinary tract, cervix, esophagus, lung, colorectal, ovary, endometrium, pancreas, prostate, or lymph.
In some embodiments, tumors with a FBXW7 mutation (s) and/or a CHD4 mutation (s) are solid cancer and hematoma, including but not limited to, liver cancer, melanoma, Hodgkin's disease, non-hodgkin's lymphoma, acute lymphatic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, Wilms’ tumor, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary gigantic globulin hematic disease, bladder cancer, chronic granulocyte leukemia, primary Brain cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, gastric cancer, colon cancer, malignant pancreatic islet cell tumor, malignant carcinoid sex cancer, malignant melanoma, choriocarcinoma, mycosis granuloma, head and neck cancer, osteogenic sarcoma, pancreatic cancer, acute myelocytic leukemia, hairy cell leukemia, rhabdomyosarcoma, kaposi's sarcoma, tumors of genitourinary tract, thyroid cancer, esophageal cancer, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial carcinoma, polycythemia vera, idiopathic thrombocythemia, adrenocortical carcinoma, skin cancer and prostate cancer.
Figs. 5 and 6 show some cancers having FBXW7 mutation (s) and their occurrence of alternation of FBXW7, and some cancers having CHD4 mutation (s) and their occurrence of alternation of CHD4, respectively. Therefore, in some embodiments, the tumor having FBXW7 mutation (s) includes skin cutaneous melanoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, esophageal adenocarcinoma, bladder urothelial  carcinoma, stomach adenocarcinoma, cervical squamous cell carcinoma, colorectal adenocarcinoma, uterine corpus endometrial carcinoma, and uterine carcinosarcoma; and the tumor having CHD4 mutation (s) include lung squamous cell carcinoma, cervical squamous cell carcinoma, brain lower grade glioma, testicular germ cell tumor, colorectal adenocarcinoma, bladder urothelial carcinoma, ovarian serous cystadenocarcinoma, stomach adenocarcinoma, skin cutaneous melanoma, uterine carcinosarcoma, and uterine corpus endometrial carcinoma.
In some embodiments, the tumors described herein are those shown in the following Table A and having the FBXW7 mutation (s) and/or the CHD4 mutations listed in the corresponding rows of the table A below:
Table A
Figure PCTCN2022134300-appb-000004
Figure PCTCN2022134300-appb-000005
In some embodiments, the tumors carrying the FBXW7 mutation (s) are those carrying R465C or R505C, including, in particular, colorectal cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, lymphoma, urethral cancer, bladder cancer, and T-lymphocytic leukemia.
In some embodiments, the tumor is rectal cancer with a mutated FBXW7, wherein the mutated FBXW7 contains one or more mutations selected from the group consisting of T616I, R465C, truncation starting from position 118 and truncation starting from position 279. In some embodiments, the rectal cancer further comprises one or more mutations occurred in APC, TP53, KRAS and ARID1A. Preferably, the mutations in APC are selected from the group consisting of frameshift mutation starting from position 541 (including position 541) or from 660 (including position 660) , truncation starting from position 902 or 1292. Preferably, the mutations in TP53 are selected from the group consisting of R273H, R175H, R248Q and R248W/Loss. Preferably, the mutations in KRAS are selected from the group consisting of G12C and G12S. Preferably, the mutation in ARID1A is a truncation starting from position 1086.
As used herein, the terms “individual, “subject” and “subject” are used interchangeably to refer to patients with tumors with FBXW7 or CHD4 mutations. An individual may have heterozygotes or polymorphisms with FBXW7 mutations or CHD4 mutations.
Wee1 kinase inhibitor used herein can be any compound or entity that inhibits, reduces, or eliminates Wee1 kinase, such as small organic molecules, peptides, or nucleic acids.
Examples of compounds that are known as Wee1 kinase inhibitors and can be used according to the present disclosure include, but are not limited to: AZD1775, Zc-03, Debio-0123, DN-1609, NUV-569, and compounds disclosed in WO2007126122, WO2019028008, WO2019173082, WO202021032, WO2015092431, WO2018162932, WO2019037678, WO2020210377, WO2020210383, WO2020210375, WO2020210380, WO2020210381, WO2018133829, WO2019085933, WO2018090939, WO2019011228 and WO2021073491.
In preferred embodiments, the Wee1 kinase inhibitor is the inhibitor disclosed in WO2018090939, which is incorporated herein by reference in its entirety. More specifically, the Wee1 kinase inhibitor is a compound represented by Formula I:
Figure PCTCN2022134300-appb-000006
or a pharmaceutically acceptable salt or a prodrug thereof, wherein A is N or CR 15;
R 1 is hydrogen, optionally substituted C 1-C 8 alkyl, optionally substituted C 2-C 8 alkenyl, optionally substituted C 3-C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
R 2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
R 3-R 7 and R 15 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-C 10 alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
In one or more embodiments, A is N.
In one or more of the foregoing embodiments, R 1 and R 2 are optionally substituted aryl.
In one or more of the foregoing embodiments, R 3-R 7 are each independently H, halo, or C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 15 is H or C 1-C 6 alkyl.
In one or more of the foregoing embodiments, the substituents on R 1 are selected from any one, two or three of the following groups: halo, C 1-C 6 alkyl, C 1-C 6 alkoxy, and halo C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 1 is selected from: H, C 1-C 8 alkyl, C 2-C 8 alkenyl, C 3-C 8 cycloalkyl, heteroaryl, and aryl which is optionally substituted by 1-4 groups selected from halo, C 1-C 6 alkyl, C 1-C 6 alkoxy, and halo C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 1 is selected from phenyl which is optionally substituted by 1-4 groups selected from halo, C 1-C 6 alkyl, C 1-C 6 alkoxy, and halo C 1-C 6 alkyl; in some embodiments, the number of substituents is 2; in some embodiments, at least one substituent is in the ortho position; in some embodiments, at least one substituent is halo; in some embodiments, the number of substituents on the phenyl is 2, both of which are located adjacent to each other, and wherein at least one is halo.
In one or more of the foregoing embodiments, R 1 is selected from optionally substituted pyridyl, pyrimidyl, thiophenyl, furanyl, pyrrolyl and imidazolyl.
In one or more of the foregoing embodiments, R 1 is selected from H, optionally substituted C 1-C 8 alkyl, C 3-C 8 cycloalkyl, and C 2-C 8 alkenyl.
In one or more of the foregoing embodiments, the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted C 1-C 6 alkyl, optionally substituted C 1-C 6 acyl, optionally substituted heterocyclic group, halo, optionally substituted oxy group, nitro, and optionally substituted C 1-C 6 alkylamino; preferably, the substituents on these substituted group may be 1-4 groups selected from the following group: C 1-C 6 alkyl, C 1-C 6 acyl, a heterocyclic group optionally substituted by 1-4 of C 1-C 6 alkyl, halo, -NR aR b and hydroxy, wherein R a and R b are each independently H and C 1-C 6 alkyl; preferably, the heterocyclic group is selected from piperazinyl, piperidinyl, morpholinyl, and 1, 4-diazacycloheptyl.
In one or more of the foregoing embodiments, the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted piperazinyl, optionally substituted piperazinyl-C 1-C 4 alkyl, optionally substituted piperidinyl, imidazolyl, optionally substituted 1, 4-diazacycloheptyl, C 1-C 6 alkyl, C 1-C 6 acyl, optionally substituted morpholinyl, morpholinyl-C 1-C 4 alkyl, halo, halo C 1-C 6 alkyl, optionally substituted C 1-C 6 alkoxy, optionally substituted hydroxy C 1-C 6 alkyl, optionally substituted amino C 1-C 6 alkyl, optionally substituted piperidinylamino, optionally substituted C 1-C 6 alkyl amino, optionally substituted heterocyclic alkyl-O-and nitro; preferably, the substituents on the optionally substituted group may be 1-4 groups selected from the following groups: C 1-C 6 alkyl, C 1-C 6 acyl, halo, -NR aR b and C 1-C 6 alkyl substituted by hydroxy, wherein R a and R b are each independently H and C 1-C 6 alkyl.
In one or more of the foregoing embodiments, the optionally substituted piperazinyl is the piperazinlyl which can be substituted by 1, 2 or 3 groups selected from: C 1-C 6 alkyl, hydroxy C 1-C 6 alkyl, and C 1-C 6 acyl.
In one or more of the foregoing embodiments, the piperazine group has at least one substituent at the para-position, and optionally, one or two substituents at the meta-position.
In one or more of the foregoing embodiments, the optionally substituted piperidinyl is the piperidinyl which can be substituted by 1 group selected from C 1-C 6 alkyl and C 1-C 6 alkyl amino.
In one or more of the foregoing embodiments, the optionally substituted morpholinyl is the morpholinyl which can be substituted by 1 or 2 groups selected from C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 2 is selected from optionally substituted phenyl, pyridyl, piperazinyl, tetrahydroisoquinolinyl, 2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl and 4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl.
In one or more of the foregoing embodiments, R 2 is selected from phenyl substituted by optionally substituted piperazinyl, phenyl substituted by optionally substituted pyridinyl, and tetrahydroisoquinolinyl optionally substituted by one to three C 1-C 6 alkyls or halos.
In one or more of the foregoing embodiments, the piperazinyl is optionally substituted by one to three groups selected from C 1-C 6 alkyl, hydroxy C 1-C 6 alkyl and C 1-C 6 acyl.
In one or more of the foregoing embodiments, the piperidinyl is optionally substituted by 1 group selected from C 1-C 6 alkyl and C 1-C 6 alkyl amino.
In one or more of the foregoing embodiments, R 4 and R 5 are each independently H, C 1-C 6 alkyl and halo, preferably, both H.
In one or more of the foregoing embodiments, R 6 and R 7 are each independently H, C 1-C 6 alkyl and halo, preferably, both H.
In one or more embodiments, preferred compounds of Formula I include, without limitation:
6- (2-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
4- (2-chlorophenyl) -8- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -1, 2-dihydroimidazo [1, 2-a] pyrido [3, 4-e] pyrimidin-5 (4H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-acetylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2'-acetyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dimethylphenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dimethylphenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dimethylphenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-isopropyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (tert-butyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-cyclopropyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-cyclohexyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-allyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (thiophen-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (furan-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (1H-pyrrol-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (1H-Imidazol-5-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 8-dimethyl-8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -9, 9-dimethyl-8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2S, 6R) -2, 6-dimethylmorpholino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (morpholinomethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-fluoro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-chloro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-trifluoromethyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-trifluoromethyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3-nitrophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (6- (4-methylpiperazin-1-yl) pyridin-3-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2'-isopropyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (5-methyl-4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-fluoro-6- (trifluoromethyl) phenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2-trifluoromethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-trifluoromethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- (3-methyl- (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (5-methyl-4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-trifluoromethylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methoxyphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dimethylphenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dimethylphenyl) -2- ( (4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (4-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (3-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 4-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-4-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-3-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 5-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 3-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (pyrimidin-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-cyclobutyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-cyclopentyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6-phenyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (pyridin-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (pyridin-3-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (pyridin-4-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (1H-imidazol-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methyl-1, 4-diazacyclohept-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (4-methylpiperazin-1-yl) methyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (2- (dimethylamino) ethoxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (3- (dimethylamino) propoxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (1-methylpiperidin-4-yl) oxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2- (dimethylamino) ethyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2- (dimethylamino) ethyl) (methyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3- (dimethylamino) propyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3- (dimethylamino) propyl) (methyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (1-methylpiperidin-4-yl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (methyl (1-methylpiperidin-4-yl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-bromo-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (5- (4-methylpiperazin-1-yl) pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-bromo-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-5-trifluoromethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methoxy-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (5-chloro-2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2, 5-dimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (5-chloro-2, 4, 4-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4, 5-tetramethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (5'-chloro-2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2', 5'-dimethyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-trifluoromethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-bromo-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-bromo-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3- (hydroxymethyl) -4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3- (hydroxymethyl) -4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3- (hydroxymethyl) -4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- (4- (2-hydroxyethyl) piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4-morpholinophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3- ( (methylamino) methyl) -4-morpholinophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one dihydrochloride;
or a pharmaceutically acceptable salt or a prodrug thereof.
In other preferred embodiments, the Wee1 kinase inhibitor is the imidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one compound disclosed in WO2019011228. More specifically, the Wee1 kinase inhibitor suitable for the present disclosure is a compound represented by the following Formula II:
Figure PCTCN2022134300-appb-000007
or a pharmaceutically acceptable salt or a prodrug thereof, wherein A is N or CR 6;
R 1 is hydrogen, optionally substituted C 1-C 8 alkyl, optionally substituted C 2-C 8 alkenyl, optionally substituted C 3-C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
R 2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
R 3-R 6 are independently hydrogen, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-C 10 alkyl (such as haloalkyl, hydroxyalkyl, aminoalkyl and carboxyalkyl) , alkenyl, alkynyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, hydroxyamino or optionally substituted alkylthio.
In one or more embodiments, A is N.
In one or more of the foregoing embodiments, R 1 and R 2 are each optionally substituted aryl.
In one or more of the foregoing embodiments, R 3 is H.
In one or more of the foregoing embodiments, R 4 and R 5 are each H and optionally substituted C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 4 is H or unsubstituted C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 5 is H or C 1-C 6 alkyl optionally substituted by hydroxy, such as hydroxy C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 6 is H.
In one or more of the foregoing embodiments, the substituents on R 1 are selected from any one, two, three or four of the following groups: halo, C 1-C 6 alkyl, C 1-C 6 alkoxy, and halo C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 1 is selected from: C 2-C 8 alkenyl, and phenyl which is optionally substituted by one to four substituents selected from halo and C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 1 is selected from phenyl which is optionally substituted by one to four substituents selected from halo and C 1-C 6 alkyl; in some embodiments, the number of substituents is 2; in some embodiments, at least one substituent is in the ortho position; in some embodiments, at least one substituent is halo; in some embodiments, the number of substituents on the phenyl is 2, both of which are located adjacent to each other, and wherein at least one is halo.
In one or more of the foregoing embodiments, R 1 is selected from optionally substituted C 2-C 8 alkenyl.
In one or more of the foregoing embodiments, the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted C 1-C 6 alkyl, optionally substituted oxy group, halo, and optionally substituted heterocyclic group; preferably, the substituents on these optionally substituted group may be one to four groups selected from the following groups: C 1-C 6 alkyl and -NR aR b, wherein R a and R b are each independently H and C 1-C 6 alkyl; preferably, the heterocyclic group is selected from piperazinyl and piperidinyl.
In one or more of the foregoing embodiments, the substituents on R 2 are selected from any one, two, three or four of the following groups: optionally substituted piperazinyl, optionally substituted piperidinyl, C 1-C 6 alkyl, halo, and C 1-C 6 alkoxy; preferably, the substituents on the optionally substituted group may be one to four groups selected from the following groups: C 1-C 6 alkyl and -NR aR b, wherein R a and R b are each independently H and C 1-C 6 alkyl.
In one or more of the foregoing embodiments, the optionally substituted piperazinyl is piperazinyl which can be substituted by 1, 2 or 3 groups selected from: C 1-C 6 alkyl.
In one or more of the foregoing embodiments, the piperazine group has at least one substituent at the para-position, and optionally, one or two substituents at the meta-position.
In one or more of the foregoing embodiments, the optionally substituted piperidinyl is piperidinyl which can be substituted by 1 group selected from C 1-C 6 alkyl and -NR aR b, wherein R a and R b are each independently H and C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 2 is selected from optionally substituted phenyl and optionally substituted tetrahydroisoquinolinyl.
In one or more of the foregoing embodiments, R 2 is selected from phenyl substituted by optionally substituted piperazinyl, phenyl substituted by optionally substituted pyridinyl, and tetrahydroisoquinolinyl optionally substituted by one to three C 1-C 6 alkyls.
In one or more of the foregoing embodiments, the piperazinyl is optionally substituted by one to three groups selected from C 1-C 6 alkyl.
In one or more of the foregoing embodiments, the piperidinyl is optionally substituted by one group selected from C 1-C 6 alkyl and -NR aR b, wherein R a and R b are each independently H and C 1-C 6 alkyl.
In one or more of the foregoing embodiments, R 4 and R 5 are independently selected from H and optionally substituted C 1-C 6 alkyl. Preferably, R 4 and R 5 are H and optionally substituted C 1-C 6 alkyl.
In one or more embodiments, preferred compounds of Formula II include, without limitation:
6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-difluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4, 5-tetramethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (2, 5-dimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-fluorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -9-ethylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-ethylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-isopropylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -9- (hydroxymethyl) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
6-allyl-2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
or a pharmaceutically acceptable salt or prodrug thereof.
In other preferred embodiments, the Wee1 kinase inhibitor suitable for the present disclosure is the dihydroimidazopyrimidopyrimidinone compound disclosed in WO2021073491. More specifically, the Wee1 kinase inhibitor suitable for the present disclosure is a compound represented by the following Formula III:
Figure PCTCN2022134300-appb-000008
or a stereoisomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, wherein R 1 and R 2 are independently halo; R 3 is halo, C 1-4 alkyl or C 1-4 alkoxy; R 4 and R 6 are each independently H or C 1-4 alkyl; R 5 is H or C 1-4 alkyl; R 7 is H, halo, C 1-4 alkyl or C 1-4 alkoxy; and X is CH or N.
In preferred embodiments of Formula III, R and R 2 are both chloro.
In preferred embodiments of Formula III, R 3 is halo, methyl or ethyl.
In preferred embodiments of Formula III, R 7 is H, halo, methyl or methoxy.
In preferred embodiments of Formula III, R 4 and R 6 are each independently H or methyl.
In preferred embodiments of Formula III, R 5 is H, methyl or methyl-d3.
In preferred embodiments of Formula III, when X is N, R 4, R 5 and R 6 are not H at the same time; preferably, R 4 and R 6 are C 1-4 alkyl, and R 5 is H or C 1-4 alkyl; more preferably, R 4 and R 6 are methyl, and R 5 is H, methyl or methyl-d3.
In one or more embodiments, preferred compounds of Formula III include, without limitation:
6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 5-dimethylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 5-dimethyl-4- (methyl-d3) piperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
2- ( (3-bromo-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
2- ( (3-bromo-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
2- ( (3-chloro-4- (1-methylpiperidin-4-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
or a pharmaceutically acceptable salt or prodrug thereof.
Some of the compounds of the present disclosure may exist as stereoisomers including optical isomers. The disclosure includes all stereoisomers and the racemic mixtures of such stereoisomers as well as the individual enantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
Examples of pharmaceutically acceptable salts include inorganic and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic base salts formed with bases, such as sodium hydroxy, tris (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methyl-glucamine.
Examples of prodrugs of the compounds of the disclosure include the simple esters of carboxylic acid-containing compounds (e.g., those obtained by condensation with a C 1- 4 alcohol according to methods known in the art) ; esters of hydroxy-containing compounds (e.g., those obtained by condensation with a C 1-4 carboxylic acid, C 3-6 diacid or anhydride thereof such as succinic anhydride and fumaric anhydride, according to methods known in the art) ; imines of amino-containing compounds (e.g., those obtained by condensation with a C 1-4 aldehyde or ketone according to methods known in the art) ; carbamate of amino-containing compounds, such as those described by Leu, et al. (J. Med. Chem. 42: 3623-3628 (1999) ) and Greenwald, et al. (J. Med. Chem. 42: 3657-3667 (1999) ) ; and acetals and ketals of alcohol-containing compounds (e.g., those obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art) .
The Wee1 kinase inhibitors of the present disclosure can be prepared using method compounds known to those skilled in the art or methods in the references (including patents, patent applications and patent publications) cited in the present disclosure, including the synthesis methods disclosed in WO2018090939, WO2019011228 and WO2021073491.
The Wee1 kinase inhibitor of the present disclosure can be administered in a pharmaceutical composition containing a pharmaceutically acceptable carrier, wherein the pharmaceutical composition includes all pharmaceutical preparations containing the compound of the present disclosure in an amount that can effectively achieve its intended goal. While individual needs vary, determination of the optimal amount of each part in the pharmaceutical preparation is within the skill of the art. Typically, the compounds or the pharmaceutically acceptable salts thereof may be administered to mammals orally at a dose of about 0.0025 to 50 mg per kg body weight per day. Preferably, from approximately 0.01 mg/kg to approximately 10 mg/kg body weight is orally administered. If a known anticancer agent is also administered, it is administered in an amount that is effective to achieve its intended purpose. The optimal amounts of such known anticancer agents are well known to those skilled in the art.
The unit oral dose may comprise from approximately 0.01 to approximately 50 mg, preferably approximately 0.1 to approximately 10 mg of the compound of the disclosure. The unit dose may be administered one or more times, with one or more tablets daily, each containing from approximately 0.1 to 50 mg, conveniently approximately 0.25 to 10 mg of the compound of the disclosure or solvates thereof.
In topical preparations, the compound (s) of the disclosure may be present at a concentration of approximately 0.01 to 100 mg per gram of carrier.
The compound (s) of the disclosure may be administered as a raw chemical. The compounds of the disclosure may also be administered as part of a suitable pharmaceutical preparation containing pharmaceutically acceptable carriers (comprising excipients and auxiliaries) . Such pharmaceutically acceptable carriers facilitate the manufacture of pharmaceutically acceptable preparations from the compound (s) . Preferably, the pharmaceutical preparations, particularly oral preparations and those used for the preferred administration routes, such as tablets, dragees, and capsules, as well as solutions suitable for injection or oral administration, contain from approximately 0.01%to 99%, preferably from approximately 0.25%to 75%of active compound (s) , together with excipient (s) .
Also included within the scope of the present disclosure are the pharmaceutically acceptable salts of the Wee1 kinase inhibitor (s) of the present disclosure. Acid addition salts are formed by mixing a solution of the compound (s) of the present disclosure with a solution of a pharmaceutically acceptable non-toxic acid, such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, and the like. Base addition salts are formed by mixing a solution of the compounds of the present disclosure with a solution of a pharmaceutically acceptable non-toxic base, such as sodium hydroxide, potassium hydroxide, hydrocholine, sodium carbonate, tris (hydroxymethyl) aminomethane, N-methyl-glucosamine and the like.
The pharmaceutical preperations of the disclosure may be administered to any mammal, so long as they may experience the therapeutic effects of the compound (s) of the disclosure. Foremost among such mammals are humans and veterinary animals, although the disclosure is not intended to be so limited.
The pharmaceutical preperations of the present disclosure may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, frequency of treatment, and the nature of the effect desired.
The pharmaceutical preparations of the present disclosure can be manufactured in a known manner, e.g., by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Pharmaceutical preparations for oral use may be obtained by combining the active compounds with solid excipient (s) , optionally grinding the resulting  mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers, such as saccharides, e.g. lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate; as well as binders, such as starch paste, including maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches and carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, e.g., silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose solutions, such as cellulose acetate phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dyes or pigments may be added to the tablets or dragee core coatings, e.g., for identification or in order to characterize combinations of active compound doses.
Other pharmaceutical preparations, which may be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active compounds in the form of granules, which may be mixed with fillers, such as lactose; binders, such as starches; and/or lubricants, such as talc or magnesium stearate, and stabilizers. In soft capsules, the active compound (s) are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin, in which stabilizers may be added.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds, e.g., aqueous solutions and alkaline solutions of water-soluble salts. In addition, appropriate oily injection suspensions of the active compounds may be administered. Suitable lipophilic solvents or vehicles include fatty oils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides or polyethylene glycol-400, or cremophor, or cyclodextrins. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, e.g., sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
The topical formulations of this disclosure can be formulated as oils, creams, lotions, ointments and the like by choice of appropriate carriers. Suitable carriers include  vegetable or mineral oils, white petrolatum (white soft paraffin) , branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C 12) . Preferred carriers are those in which the active ingredient (s) are soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included, as well as agents imparting color or fragrance, if desired. Additionally, transdermal penetration enhancers may be employed in these topical formulations. Examples of such enhancers are found in U.S. Patent Nos. 3,989,816 and 4,444,762.
Creams are preferably formulated from a mixture of mineral oil, self-emulsifying beeswax and water in which mixture of the active ingredient, dissolved in a small amount of an oil, such as almond oil, is admixed. A typical example of such a cream is one which includes approximately 40 parts of water, approximately 20 parts of beeswax, approximately 40 parts of mineral oil and approximately one part of almond oil.
Ointments may be formulated by mixing a solution of the active ingredient (s) in a vegetable oil, such as almond oil, with warm soft paraffin and allowing the mixture to cool. A typical example of such an ointment is one which includes approximately 30%by weight of almond oil and approximately 70%by weight of white soft paraffin.
In the present disclosure, "effective amount" refers to an amount of the active compound (s) or pharmaceutical agent (s) that cause the biological or medical response of the tissue, system, animal, or individual human being sought by researchers, veterinarians, physicians, or other clinicians, and the biological or medical response includes one or more of the following: (1) preventing diseases: for example, preventing diseases, conditions, or disorders in an individual who may be susceptible to the disease, condition, or disorder but still not experiencing or presenting the pathology or symptoms of the disease, (2) inhibiting diseases: for example, inhibiting diseases, conditions or disorders in an individual experiencing or presenting the pathology or symptoms of the disease, condition, or disorder (i.e., preventing the further development of the pathology or symptoms of the pathology and/or disorder) , and (3) improving diseases: for example, improving diseases, conditions, or disorders in an individual experiencing or presenting the pathology or symptoms of the disease, condition, or disorder (i.e., reverse the pathology and/or symptom) . Therefore, a non-limiting example of an "effective dose" of the composition of the present disclosure can be used to inhibit, block or reverse the activation, migration or proliferation of cells, or to effectively treat cancer or improve symptoms of cancer.
The composition containing the Wee1 kinase inhibitor can be used in combination with at least one known anti-cancer drug or a pharmaceutically acceptable salt of an anti-cancer drug. In particular, the composition can be used in combination with other anti-cancer drugs related to the mechanism of DNA damage and repair, including PARP inhibitors olaparib, niraprib, rucaparib, talazoparib, pamiparib, fluzoparib and senaparib; HDAC  inhibitors Volinota, Romididesin, Papiseta and Bailesta; and so on. And the composition can be used in combination with other anti-cancer drugs related to cell division checkpoints, including Chk1/2 inhibitors, CDK4/6 inhibitors such as Palbociclib, ATM inhibitors, ATR inhibitors, DNA-PK inhibitors and so on. Other known targeted anti-cancer drugs which may be used for anti-cancer combination therapy include, but are not limited to, PRMT5 inhibitors, Polθinhibitors, RAD51 inhibitors and so on. Other known anti-cancer drugs which may be used for anti-cancer combination therapy include, but are not limited to, alkylating agents, such as busulfan, melphalan, chlorambucil, cyclophosphamide, ifosfamide, temozolomide, bendamustine, cis-platin, mitomycin C, bleomycin and carboplatin; topoisomerase I inhibitors, such as camptothecin, irinotecan and topotecan; topoisomerase II inhibitors, such as doxorubicin, epirubicin, aclacinomycin, mitoxantrone, elliptinium and etoposide; RNA/DNA antimetabolites, such as 5-azacytidine, gemcitabine, 5-fluorouracil and methotrexate; DNA antimetabolites, such as 5-fluoro-2'-deoxy-uridine, fludarabine, nelarabine, ara-C, thioguanine, pralatrexate, pemetrexed, hydroxyurea and thioguanine; antimitotic agent, such as colchicine, vinblastine, vincristine, vinorelbine, paclitaxel, ixabepilone, cabazitaxel and docetaxel; antibodies, such as mAb, panitumumab, necitumumab, nivolumab, pembrolizumab, ramucirumab, bevacizumab, pertuzumab, trastuzumab, cetuximab, obinutuzumab, ofatumumab, rituximab, alemtuzumab, ibritumomab, tositumomab, brentuximab, daratumumab, elotuzumab, Ofatumumab, Dinutuximab, Blinatumomab, ipilimumab, avastin, herceptin and mabthera; Antibody–Drug Conjugates (ADC) such as T-DM1, Trastuzumab Deruxtecan, Trastuzumab Emtansine, Datopotamab Deruxtecan, Gemtuzumab Ozogamicin, Brentuximab Vedotin, Inotuzumab Ozogamicin, Sacituzumab govitecan, Enfortumab Vedotin, Belantamab Mafodotin; kinase inhibitors, such as imatinib, gefitinib, erlotinib, osimertinib, afatinib, ceritinib, alectinib, crizotinib, erlotinib, lapatinib, sorafenib, regorafenib, vemurafenib, dabrafenib, aflibercept, sunitinib, nilotinib, dasatinib, bosutinib, ponatinib, ibrutinib, cabozantinib, lenvatinib, vandetanib, trametinib, cobimetinib, axitinib, temsirolimus, Idelalisib, pazopanib, Torisel and everolimus. Other known anti-cancer drugs which may be used for anticancer combination therapy include tamoxifen, letrozole, fulvestrant, mitoguazone, octreotide, retinoic acid, arsenic trioxide, zoledronic acid, bortezomib, carfilzomib, Ixazomib, vismodegib, sonidegib, denosumab, thalidomide, lenalidomide, Venetoclax, Aldesleukin (recombinant human interleukin-2) and Sipueucel-T (prostate cancer treatment vaccine) .
The present disclosure also provides use of the reagent (s) described herein to detect the presence of mutation (s) in FBXW7 and/or CHD4 of a subject, in particular in the preparation of a detection kit to determine the subject's sensitivity or efficacy to a treatment with a Wee1 kinase inhibitor, in particular with a Wee1 kinase inhibitor described herein. The reagent (s) may include those routinely used to detect FBXW7 or CHD4 gene and/or protein.  For example, in order to detect whether there is a change in a gene leading to the protein mutation (s) described in the present disclosure, PCR and sequencing can be used to determine whether there is a mutation in the gene. Therefore, the reagents include but are not limited to the reagents required for PCR and sequencing, such as primers, probes, polymerases, dNTP, etc. The technique of designing genes and probes based on known gene sequences is well known in the field.
Tissue samples containing one or more types of cells, such as cancerous tissue mentioned above, or non-cancerous tissue biopsy samples, such as a control, may be obtained from the subject. The samples were then tested for the presence of FBXW7 and/or CHD4 mutation (s) , especially those described herein, using a conventional method. The presence of the mutation (s) described herein indicates that the subject is sensitive to a treatment with the Wee1 kinase inhibitor described herein or that the subject's tumor could be effectively treated with the Wee1 kinase inhibitor described herein.
In some embodiments, the present disclosure provides a method for treating a tumor with a FBXW7 mutation (s) and/or a CHD4 mutation (s) in a subject, comprising administering to the subject an effective amount of a Wee1 kinase inhibitor or its pharmaceutical composition. In some embodiments, the method further comprises detecting whether the subject has the FBXW7 mutation (s) and/or a CHD4 mutation (s) , especially those described herein, or whether the subject has a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein. Preferably, the Wee1 kinase inhibitors are those described herein, especially the compounds having the structure represented by Formula I, II or III. In some embodiments, the subject is suffered from any of the cancers with the indicated mutations listed in Table A. In some embodiments, the subject is suffered from a rectal cancer with a mutated FBXW7, wherein the mutated FBXW7 contains one or more mutations selected from the group consisting of T616I, R465C, truncation starting from position 118 and truncation starting from position 279. In some embodiments, the rectal cancer further comprises one or more mutations occurred in APC, TP53, KRAS and ARID1A. Preferably, the mutations in APC are selected from the group consisting of frameshift mutation starting from position 541 (including position 541) or from 660 (including position 660) , truncation starting from position 902 or 1292. Preferably, the mutations in TP53 are selected from the group consisting of R273H, R175H, R248Q and R248W/Loss. Preferably, the mutations in KRAS are selected from the group consisting of G12C and G12S. Preferably, the mutation in ARID1A is a truncation starting from position 1086.
In some embodiments, the present disclosure provides a method for evaluating an individual with a cancer to determine his treatment regime, comprising identifying whether cancer cells obtained from the individual have a FBXW7 mutation and/or a CHD4 mutation,  and/or whether the cancer cells have a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein, and administering to an individual with the FBXW7 mutation and/or the CHD4 mutation and/or with a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein a Wee1 kinase inhibitor, especially those inhibitors described herein.
In the embodiment, the cancer cells may have an FBXW7 and/or CHD4 mutant phenotype.
Identification of cancer cells with FBXW7 and/or CHD4 mutation (s) may also include exposing a cancer cell sample obtained from an individual with cancer to a Wee1 kinase inhibitor and determining a number of dead cells in the sample relative to dead cells in a control sample. A higher number of dead cancer cells than that of dead control cells demonstrates increased sensitivity of cancer cell to the Wee1 kinase inhibitor, indicating that cancer cells may have a FBXW7 mutation or a CHD4 mutation, such as reduced expression or activity of FBXW7 or CHD4 or loss of function.
In some embodiments, the present disclosure provides a method for treating cancer in an individual with a Wee1 kinase inhibitor, wherein the individual is a heterozygote or polymorphism with a FBXW7 mutation, or a CHD4 mutation.
In some embodiments, the treatment method further comprises determining whether the cancer tissue or cancer cell of the individual has a FBXW7 mutation and/or a CHD4 mutation by a genetic testing method, and then administering to the individual with the Wee1 kinase inhibitor if his cancer is identified as a cancer with a FBXW7 mutation and/or a CHD4 mutation.
In some embodiments, the present disclosure provides a method for determining whether or not a cancer patient is responsive or sensitive to or benefits from a treatment with a Wee1 kinase inhibitor, especially the Wee1 kinase inhibitor described herein, comprising identifying whether the patient’s cancer cells have a FBXW7 mutation and/or a CHD4 mutation, and/or a reduced expression or activity of the FBXW7 protein and/or the CHD4 protein, wherein presence of a FBXW7 mutation and/or a CHD4 mutation, especially any of the mutations as described herein, and/or presence of reduced expression or reduced activity of the FBXW7 protein and/or the CHD4 protein, indicates that the patient is sensitive to the treatment with a Weel kinase inhibitor. In some embodiments, the identification is performed with a sample, such as a surgical sample or a biopsy sample, from the cancer patient.
In the present disclosure, the expression level and activity of the FBXW7 protein and the CHD4 protein could readily be determined by, such as comparing the expression level or activity of the protein in the cancer cell to that in the normal cell of the same tissue with a conventional technique.
The following examples are illustrative, but not limiting, of the methods and preparations of the present disclosure. Other suitable modifications and adaptations of various conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the disclosure.
Example 1
The effects of Wee1 kinase inhibitor (Compound A) on human colorectal cancer samples in a MiniPDX TM efficacy model
Compound A: 6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimidino [5, 4-e] pyrimidin-5 (6H) -one (Example 77 of WO2018090939) .
Mini patient-derived xenograft (MiniPDX TM) model is a new method to test drug efficacy on human tumors in animals. In the model cells prepared from human tumor samples were tested for sensitivity to testing drugs, either alone or in combination, in animals.
A proprietary preparation method was used to isolate tumor cells from fresh human tumor specimens. The cell preparation was placed in a dedicated MiniPDX TM device and transplanted subcutaneously in mice. The specially designed pore size of the MiniPDX TM device allows small molecules below 500 KD to enter and exit freely while tumor cells remain in the device. In the experiments described below, mice were administered with testing drugs systemically for 7 days. After treatment, the MiniPDX TM devices were taken out, and the tumor cell viability was determined by an in vitro ATP method. The effectiveness of dosage regimens was calculated based on the overall viability of tumor cells.
The effect of Wee1 kinase inhibitor Compound A on colorectal cancer cells was tested using the MiniPDX TM animal model in the present disclosure.
Experimental animals:
Female mice of Balb/c-nude strain of 6-8 weeks of age and weighing 18-22g were purchased from GemPharmatech Co., Ltd. Animals were housed for at least 3 days for adaption before experiment.
Animal housing and care:
All the animal studies were carried out in experimental animal center which has passed AAALAC international certification and following AAALAC guidance. All experimental animals were housed at 24-27 ℃, 40-60%humidity, 10-20 times/hour ventilation rate and a 12h/12h day/night cycle. All mice used in the experiments were provided with food and water  ad libitum. The mouse pellet feed was cobalt-60 radio-sterilized and the drinking water was sterilized by high pressure.
After inoculation with MiniPDX TM devices, animals were observed and recorded daily for body weight, and abnormal food and water intake, hair color and behavior changes.
Sample information used in MiniPDX TM studies:
Sample cases Sample type Patient information Pathological results
1 Surgical sample male, 63 years old rectal cancer
2 Biopsy sample male, 63 years old rectal cancer
3 Surgical sample male, 44 years old rectal cancer
4 Biopsy sample male rectal cancer
5 Biopsy sample male, 65 years old rectal cancer
6 Surgical sample female, 49 years old rectal cancer
7 Chest fluid sample male, 65 years old rectal cancer
8 Biopsy sample male, 70 years old rectal cancer
9 Surgical sample male, 47 years old rectal cancer
10 surgical sample female, 63 years old rectal cancer
11 biopsy sample male, 57 years old rectal cancer
12 surgical samples male, 80 years old rectal cancer
13 Surgical sample female, 57 years old rectal cancer
14 biopsy sample male, 51 years old rectal cancer
15 biopsy sample male, 50 years old rectal cancer
16 Surgical sample female rectal cancer
Experimental methods:
1. The patient's surgical sample was immersed in HBSS sample buffer. 
2. After washing and removing tissue debris, cell suspension was prepared with MiniPDX TM sample preparation buffer and the cells were counted. 
3. Cells were re-suspended and filled into MiniPDX TM device. The MiniPDX TM device containing human tumor cells was inoculated into mice subcutaneously. The treatment group was administered with compound A in a vehicle of 0.5%MC containing 0.2%Tween 80 suspension in water at 60 mg/kg, P. O., QD for 7 days and the control group was administered with an equal volume of vehicle. The treatment started on the inoculation day that was designated as day 0.
4. In the end of experiment, the MiniPDX TM devices were taken out and cell viability was evaluated using a CellTiter-Glo test method.
The pharmacological efficacy of Compound A was evaluated based on the relative increase rate (T/C %) of tumor cells.
The definition of relative increasing rate is: T/C %= T /C× 100%, where T and C are the cell numbers of the treated group and the control group, respectively.
5. Whole Exome sequencing (WES) was also performed on the samples in the MiniPDX TM studies.
Results:
The WES sequencing results are summarized in Table 1. Mutations of FBXW7 gene were found in 5 of 16 rectal cancer samples. Compound A had good inhibitory effect on cell growth in 4 of the 5 samples with T/C %values less than 50%. No good inhibitory activity of Compound A was observed for the 11 rectal cancer samples having no FBXW7 gene mutation, and the T/C%values were greater than 50%. As a result, there was a good correlation between the inhibition of compound A on colorectal cancer cell growth and the mutations in FBXW7 gene.
Table 1
Sample No.  T/C% FBXW7 APC TP53 KRAS ARID1A TMB AURKA ATRX
1 29% T616I Q541fs R273H   S1085*   Amp  
2 37% E117* N660fs R248W/Loss G12C        
3 39% R465C Q1291* R175H     High Amp  
4 45% R278* Q901* R248Q G12S        
5 54%   Q1367fs Splice site Q61H   High Amp  
6 55%     R175H G12D       D1264del
7 60%                
8 61%   L1482fs R248Q G12D   High    
9 70%   R805* R282W   Q799H      
10 71% R479Q R216* G266*       Amp  
11 78%           High   E1464del
12 94%   N1797fs   G12C Splice site High/MSI   Loss
13 96%   T1556fs   G12D        
14 102%   Q789*   G13D        
15 111%   T1079* H214R Q61R G330dup      
16 157%   R213* Q38*          
Note: *indicates that the protein encoded by the gene is terminated prematurely at the corresponding amino acid number. For example, FBXW E117*indicates that FBXW7 is truncated at 117-E, and the gene encodes a truncated protein.
In summary, we found that the Wee1 inhibitor Compound A had an inhibitory effect on human rectal cancer cell growth in a MiniPDX TM efficacy animal model that correlates with mutations in FBXW7 gene. Therefore, FBXW7 mutation may be used as a biomarker to screen patients for Wee1 inhibitors in clinical treatment of cancer.
Example 2
The effect of Wee1 kinase inhibitor Compound A on cell proliferation of human tumor cell lines
The 50%inhibition concentration (IC 50) values on cell proliferation were determined using CTG cell viability assay. Human cancer cell lines were treated with multiple concentrations of the testing compound and viable cells were detected under each drug concentration after 72 hours of treatment. The IC 50 values were determined by plotting viable cells against compound concentrations and calculated using commercial curve fitting programs.
Reagents and consumables
1. Conventional culture mediums and consumables were used in cell culture.
2. Fetal bovine serum (FBS) was purchased from ExCell Bio. (Cat #FND500) .
3. CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega (Cat#G7573) .
4. 96 well black wall transparent flat bottom cell culture plates were purchased from Corning (Cat #3340) .
Instruments
EnVision multi label microplate tester, PerkinElmer, 2104-0010A;
Cell counter, Inno Alliance Biotech, Countstar;
CO 2 incubator, Thermo Scientific, Model 3100 Series;
Biosafety cabinet, Thermo Scientific, Model 1300 Series A2;
Inverted microscope, Olympius, CKX41SF;
Refrigerator, SIEMENS, KK25E76TI.
Experimental methods
Cell culture
The cells were resuscitated and cultured in recommended culture mediums.
Cell planking (Day 1) :
1. Cells were collected in logarithmic growth period and counted with cell counter. Trypan blue exclusion method was used to determine viable cells.
2. Cells were diluted with culture medium to adjust cell numbers. Ninety μl of cell suspension containing appropriate cells was aliquoted into each well of a 96 well cell culture plate.
3. The cells in 96 well plates were returned to a CO 2 incubator and cultured overnight at 37 ℃, 5%CO 2 and 95%humidity.
T 0 cell vitality reading (Day 2) :
4. Ten μl of cell culture medium was added to each well of a 96-well cell culture plate.
5. CellTiter Glo reagent and cell culture plates were left at room temperature for 30 minutes.
6. Fifty μl of CellTiter Glo reagent was added to each well.
7. Plates were placed on an orbit shaking table to shake for 2 minutes to fully lyse cells.
8. Plates were placed at room temperature for 10 minutes.
9. Read the plate for chemiluminescence with EnVision.
Drug treatment (Day 2) :
10. The testing compounds were dissolved with corresponding solvents to make stock solutions. A serial dilution was made to the concentrations that were 10 times the final testing concentrations.
11. Ten μl of dilutions containing the testing compound were added to each well of a 96-well plate with cells inoculated before. Each concentration of compounds was tested in triplicates. The highest concentration of compound tested was 100 μM. Nine concentrations of serially diluted compounds were tested with a dilution factor of 3.16.
12. The cells in the 96 well plates with the testing compound added were return to CO 2 incubator and cultured for additional 72 hours at 37 ℃, 5%CO 2 and 95%humidity.
Cell vitality reading (Day 5)
13. The CellTiter Glo reagent and cell culture plates containing cells treated with the testing compound were placed at room temperature for 30 minutes.
14. Fifty μl of CellTiter Glo reagent were added to each well of the cell culture plate.
15. The plate was placed on an orbit shaking table for 2 minutes to fully lyse the cells.
16. The plate with lysed cells was placed at room temperature for 10 minutes.
17. The plate was loaded to a EnVision plate reader to read chemiluminescence values.
Data processing
GraphPad Prism 5.0 commercial software was used to analyze data. A non-linear S curve regression algorism was used to fit the data to calculate IC 50 values. The following equations were used for the calculations.
Cell survival rate (%) = (Lum  drug to be tested -Lum  culture medium control) / (Lum  cell control -Lum  culture  medium control) × 100%.
Lum  cell control -Lum  culture medium control was set as 100%, and Lum  Medium control value was set as 0%.
Amplification multiple = (Lum None treated –Lum  Medium control of Day 5) / (Lum None treated –Lum  Medium control of Day 2)
Result:
The WES sequences of 29 cell lines were obtained, and some of the key results are shown in Table 2. Mutations of FBXW7 gene were found in 15 of 29 cell lines, and of the 15 cell lines 11 cell lines were sensitive to Compound A to certain degree (IC 50 < 2 μM) . Mutations of CHD4 gene were also found in 17 of the 29 cell lines, and of the 17 cell lines 12 cell lines were sensitive to compound A (IC 50 < 2 μM) . Of these cell lines that had good sensitivity to Compound A, 7 cell lines have CGD4 mutations, 9 cell lines have FBXW7 mutations, and 4 cell lines have both CHD4 and FBXW7 mutations. Based on these observations, it may be concluded that Compound A has better inhibitory effect on the growth of rectal cancer cells that carry mutations in FBXW7 gene or CHD4 gene, or both.
Table 2
Figure PCTCN2022134300-appb-000009
Figure PCTCN2022134300-appb-000010
Note: Y indicates that the gene has mutations; E40Ter means the protein was truncated and has only N-terminal 1-40 amino acids (AA) ; E103Ter means the protein truncated and has only N-terminal 1-103 AAs ; E117del refers to the absence of an E in the 117th position; L403fs means that there is a reading frame shift mutation at the 404th AA (including the 404th AA) ; F1488fs refers to a reading frame shift after position 1488; K50K refers to a synonymous mutation.
In summary, in this study of multiple cell lines the Wee1 inhibitor Compound A was found to have inhibitory effects on cell growth of cell lines that carry mutations in FBXW7 gene or in CHD4 gene or in both genes. Therefore, mutations in FBXW7 gene or CHD4 gene or both may be used as a biomarker to screen cancer patients for Wee1 inhibitor treatment.
Example 3
The effect of Wee1 inhibitor Compound A on the cell proliferation of cell lines having different FBXW7 or CHD4 expression
Reagents and consumables
1. Conventional culture mediums and consumables were used in cell culture.
2. Fetal bovine serum (FBS) was purchased from ExCell Bio. (Cat #FND500) .
3. CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega (Cat#G7573) .
4. 96 well black wall transparent flat bottom cell culture plates were purchased from Corning (Cat #3340) .
Test Compound
Compound A: 6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimidino [5, 4-e] pyrimidin-5 (6H) -one (Example 77 of WO2018090939) .
Instruments
EnVision multi label microplate tester, PerkinElmer, 2104-0010A;
Cell counter, Inno Alliance Biotech, Countstar;
CO 2 incubator, Thermo Scientific, Model 3100 Series;
Biosafety cabinet, Thermo Scientific, Model 1300 Series A2;
Inverted microscope, Olympius, CKX41SF;
Refrigerator, SIEMENS, KK25E76TI.
Experimental methods
1. Correlation between FBXW7 expression and sensitivity to Wee1 inhibitor.
FBXW7 defective LoVo cells were introduced with wild type sequence of FBXW7 gene to exam the sensitivity to wee1 inhibitors.
5.0 μg pMD2. G plasmid (addgene #12259) and 7.5 μg psPAX2 plasmid were mixed with either 10 μg FBXW7 expression plasmid (genecopiea, EX-T3614-Lv128) or the empty plasmid (EX-NEG-LV128) in control group in 0.5 ml DMEM medium and incubated at room temperature for 5 minutes. At the same time, 70 μl of 1 mg/ml PEI (sigma #764965) was mixed with 0.5 ml DMEM and incubated at room temperature for 5 minutes. Then the plasmid mixtures were mixed with PEI solutions, and incubated at room temperature for an additional 20 minutes. The plasmid/PEI mixture was added dropwise to a 10 cm cell culture plate containing 80%confluent HEK293T cells in 5 ml DMEM (10%FBS) . Eight hours later, the culture medium was replaced with 10 ml fresh DMEM medium (10%FBS) and cells were return to CO 2 incubator. After 48 hours, the culture medium was removed and centrifuged at 300g for 5 minutes. The supernatant was retained and centrifuged again at 2000g for 20 minutes. About 1/3 volume of 40%PEG8000 was added into the supernatant and the mixture was incubated on an ice shaker for 4 hours. Then the mixture was centrifugated again at 4 ℃ at 1500g for 1 hour. The precipitate was resuspended in 100 μl PBS, and added into a six-well plate containing LOVO cells at 80%confluence (each well contained 1ml DMEM with 10%FBS and 8 μg/ml polybrene) . In the next day 1 ml DMEM (10%FBS+8 μg/ml polybrene) fresh medium was added to each well. After two days of incubation, cell culture medium was replaced with 2 ml fresh culture medium (10%FBS and 4 μg/ml puromycin) . Cells were incubated for another two days and were spread on 96 well plates (2000 cells/well; 90 μl medium/well) , and incubated at 37 ℃, 5%CO 2 incubator overnight. In the next day, the Wee1 inhibitor Compound A stock solution (10 μM) was diluted with DMSO to 8 concentrations in a serial patter of 1: 3.16. Ten μl of each concentration were mixed with 90 μl of medium (10 times dilution) , and the control had DMSO only. Serially diluted Compound A was added to cells and cells were incubated at 37 ℃, 5%CO 2 incubator for 4 days. After 4 days incubation, 50 μl CellTiter Glo reagent were added to each well, the cells were fully lysed by vibrating on a fixed  orbit shaking table for 2 minutes, and incubated at room temperature for 10 minutes. The chemiluminescence values were obtained using an EnVision plate reader.
Verification of FBXW7 overexpression: the cell lysate was detected by Western blot. FBXW7 (R&D NB100-88138) primary antibody was incubated at 1: 1000 dilution for 4 ℃overnight, and then secondary antibody was incubated at room temperature for 1 hour, then incubated with ECL substrate, and then the protein expression was detected with an imager (Fig. 1) . Results show that there are overexpressed FBXW7 in the lysates of LoVo cells with FBXW7 knock-in.
Results:
The introduction of wild type FBXW7 into FBXW7 defective LoVo cells expressed more FBXW7 proteins. The IC 50 values of Compound A tested with FBXW7 overexpressed LoVo cells or FBXW7 defective LoVo cells (control) were 121 nM and 67 nM, respectively (Fig. 2) . Therefore, introduction of wild type FBXW7 gene sequence into FBXW7 defective LoVo cells reduces the sensitivity of these cells to Wee1 inhibitors Compound A. FBXW7 defective LoVo cells are more sensitive toWee1 inhibitor (Compound A) treatment.
2. Correlation between CHD4 expression and sensitivity to Wee1 inhibitor.
OVCAR3 cells expressing wild-type CHD4 gene were introduced with CHD4 knockdown virus using CRISPR technique. These OVCAR3 cells either expressing or not expressing CHD4 gene were tested for sensitivity to the Wee1 inhibitor Compound A treatment.
5.0 μg pMD2. G (addgene #12259) plasmid and 7.5 μg psPAX2 plasmid were mixed with either 10 μg CHD4 knockdown plasmid (genecopiea, HSH091375-LVRU6GP) or a control plasmid (CSHCTR001-LVRU6GP) in 0.5 ml DMEM and left at room temperature for 5 minutes. At the same time, 70 μl 1 mg/ml PEI (sigma #764965) was mixed with 0.5 ml DMEM and left at room temperature for 5 minutes. Then, the plasmid mixtures were mixed with the PEI solution and incubated at room temperature for 20 minutes. The plasmids/PEI mixtures were added dropwise to 10 cm cell culture plates containing HEK293T cells at 80%confluence in 5 ml DMEM (10%FBS) . After 8 hours incubation, the culture medium was replaced with 10 ml fresh DMEM medium (10%FBS) and incubated for another 48 hours. Afterwards, the culture medium was collected and centrifuged at 300g for 5 minutes, the supernatant was collected and centrifuged at 2000g for 20 minutes. About 1/3 volume of 40%PEG8000 was added to the supernatant and the mixture was incubated on an ice shaker for 4 hours. After incubation, the mixture was centrifugated at 4 ℃ at 1500g for 1 hour. The precipitate was resuspended with 100 μl PBS and added dropwise to a six-well plate containing OVCAR3 cells at 80%confluence (each well contained 1 ml DMEM with 10%FBS and 8 μg/ml polybrene) . The cells were incubated at 37℃ in a 5%CO 2 incubator. In the next day, 1 ml DMEM (10% FBS + 8μg/ml polybrene) was added to each well. After two days of incubation, culture medium was replaced with 2 ml fresh medium (10%FBS+4 μg/ml puromycin) . After another two days of incubation, the cells under puromycin were spread in a 96-well plates (2000 cells/well; 90 μl medium/well) , and incubated at 37 ℃, 5%CO 2 overnight. In the next day, Wee1 inhibitor Compound A was serially diluted to 8 concentrations using DMSO at a ratio of 1: 3.16. Ten μl of the DMSO dilution was added to 90 μl medium (10 times dilution) . In the DMSO control, 10 μl DMSO was mixed with 90 μl medium. Compound dilutions were added to the 96-well plate containing cells. Then, the cells were incubated at 37 ℃, 5%CO 2 for 4 days. Four days later, 50 μl CellTiter Glo reagent was added to each well and cells were fully lysed by vibrating on a fixed orbit shaking table for 2 minutes. The lysed cells were incubated at room temperature for 10 minutes. The chemiluminescence value was read using an EnVision plate reader.
The knockdown level of CHD4 was validated using Western blot. The primary antibody against CHD4 was from Sinobiological (#102312-T34) .
Results:
Knockdown of CHD4 genes in OVCAR3 cells decreased the CHD4 protein expression (Fig. 3) . The IC 50 values of Compound A tested in the CHD4 knockdown OVCAR3 cells and the OVCAR3 cells expressing wild-type CHD4 gene were 334 nM and 1881 nM, respectively. Therefore, down-regulation of the CHD4 expression increased the sensitivity of OVCAR3 cells to Wee1 inhibitor Compound A.

Claims (15)

  1. Use of a Wee1 kinase inhibitor in the manufacture of a medicament for the treatment of a cancer with a FBXW7 mutation and/or a CHD4 mutation.
  2. The use of claim 1, wherein the Wee1 kinase inhibitor is selected from compounds of Formula I:
    Figure PCTCN2022134300-appb-100001
    or pharmaceutically acceptable salts or prodrugs thereof, wherein:
    A is N or CR 15;
    R 1 is H, optionally substituted C 1-8 alkyl, optionally substituted C 2-8 alkenyl, optionally substituted C 3-8 cycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
    R 2 is an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
    R 3-R 7 and R 15 are independently H, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-10 alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, carbonylamido or optionally substituted alkylthio.
  3. The use of claim 2, wherein the compound of Formula I is selected from the group consisting of:
    6- (2-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    4- (2-chlorophenyl) -8- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -1, 2-dihydroimidazo [1, 2-a] pyrido [3, 4-e] pyrimidin-5 (4H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-acetylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2'-acetyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dimethylphenyl) -2- ( (2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dimethylphenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dimethylphenyl) -2- ( (2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-isopropyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (tert-butyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-cyclopropyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-cyclohexyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-allyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (thiophen-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (furan-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (1H-pyrrol-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (1H-Imidazol-5-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 8-dimethyl-8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -9, 9-dimethyl-8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2S, 6R) -2, 6-dimethylmorpholino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (morpholinomethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-fluoro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-chloro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-trifluoromethyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2-methyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-trifluoromethyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3-nitrophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (6- (4-methylpiperazin-1-yl) pyridin-3-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2'-isopropyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (5-methyl-4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-fluoro-6- (trifluoromethyl) phenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- ( (3R, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2-trifluoromethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) -3- (trifluoromethyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-trifluoromethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- (3-methyl- (4- (4-isopropylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (5-methyl-4, 5, 6, 7-tetrahydropyrazolo [1, 5-a] pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-trifluoromethylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methoxyphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dimethylphenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dimethylphenyl) -2- ( (4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (4-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (3-chlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 4-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-4-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-3-fluorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 5-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 3-dichlorophenyl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (pyrimidin-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-cyclobutyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-cyclopentyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6-phenyl-2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (pyridin-2-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (pyridin-3-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (pyridin-4-yl) -2- ( (4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (1H-imidazol-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4-methyl-1, 4-diazacyclohept-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (4-methylpiperazin-1-yl) methyl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (2- (dimethylamino) ethoxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (3- (dimethylamino) propoxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (1-methylpiperidin-4-yl) oxy) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2- (dimethylamino) ethyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (2- (dimethylamino) ethyl) (methyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3- (dimethylamino) propyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (3- (dimethylamino) propyl) (methyl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- ( (1-methylpiperidin-4-yl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (methyl (1-methylpiperidin-4-yl) amino) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-bromo-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (5- (4-methylpiperazin-1-yl) pyrazin-2-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-bromo-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-5-trifluoromethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methoxy-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (5-chloro-2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2, 5-dimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (5-chloro-2, 4, 4-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4, 5-tetramethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (5'-chloro-2'-methyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2', 5'-dimethyl-2', 3'-dihydro-1'H-spiro [cyclopropane-1, 4'-isoquinolin] -7'-yl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-trifluoromethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-bromo-5-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-bromo-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-bromo-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dichloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3- (hydroxymethyl) -4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3- (hydroxymethyl) -4- (piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3- (hydroxymethyl) -4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- (4- (2-hydroxyethyl) piperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4-morpholinophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3- ( (methylamino) methyl) -4-morpholinophenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one dihydrochloride;
    or a pharmaceutically acceptable salt or prodrug thereof.
  4. The use of claim 1, wherein the Wee1 kinase inhibitor is selected from compounds of Formula II:
    Figure PCTCN2022134300-appb-100002
    or pharmaceutically acceptable salts or prodrugs thereof, wherein:
    A is N or CR 6;
    R 1 is H, optionally substituted C 1-C 8 alkyl, optionally substituted C 2-C 8 alkenyl, optionally substituted C 3-C 8 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic group or optionally substituted heteroaryl;
    R 2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl, or an optionally substituted heteroaryl;
    R 3-R 6 are independently H, halo, optionally substituted amino, optionally substituted alkoxy, optionally substituted C 1-C 10 alkyl (such as haloalkyl, hydroxyalkyl, aminoalkyl and carboxyalkyl) , alkenyl, alkynyl, nitro, cyano, acylamido, hydroxy, thiol, acyloxy, azido, carboxy, ethylenedioxo, carbonylamido or optionally substituted alkylthio.
  5. The use of claim 4, wherein the compound of Formula II is selected from the group consisting of:
    6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (2, 4, 4-trimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3, 5-dimethyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-difluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-fluorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -4-isopropyl-3, 5-dimethylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-5-methoxy-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2, 4, 4, 5-tetramethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (2, 5-dimethyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-fluorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-chloro-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-bromo-6-chlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-fluoro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-fluoro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2-chloro-6-methylphenyl) -2- ( (3-chloro-5-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -8-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-chloro-4- (4-methylpiperazin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-methylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -9-ethylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-ethylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -9-isopropylimidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (4-methylpiperazin-1-yl) phenyl) amino) -9- (hydroxymethyl) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    6-allyl-2- ( (3-methyl-4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1, 2-b] pyrimido [4, 5-d] pyridazin-5 (6H) -one;
    or a pharmaceutically acceptable salt or prodrug thereof.
  6. The use of claim 1, wherein the Wee1 kinase inhibitor is a compound represented by Formula III:
    Figure PCTCN2022134300-appb-100003
    or a stereoisomer thereof, or a pharmaceutically acceptable salt or prodrug thereof,
    wherein R 1 and R 2 are independently halo; R 3 is halo, C 1-4 alkyl or C 1-4 alkoxy; R 4 and R 6 are each independently H or C 1-4 alkyl; R 5 is H or C 1-4 alkyl; R 7 is H, halo, C 1-4 alkyl or C 1-4 alkoxy; and X is CH or N.
  7. The use of claim 6, wherein the compound of Formula III is selected from the group consisting of:
    6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 5-dimethylpiperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (4- ( (3S, 5R) -3, 5-dimethyl-4- (methyl-d3) piperazin-1-yl) -3-methylphenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    2- ( (3-bromo-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    2- ( (3-bromo-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3, 5-dimethyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methoxy-5-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5S) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (piperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    2- ( (3-chloro-4- (1-methylpiperidin-4-yl) phenyl) amino) -6- (2, 6-dichlorophenyl) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    6- (2, 6-dichlorophenyl) -2- ( (3-fluoro-5-methyl-4- (1-methylpiperidin-4-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one;
    or a pharmaceutically acceptable salt or prodrug thereof.
  8. The use of any of claims 1-7, wherein:
    the FBXW7 mutation includes one or more mutations occurring at positions 27, 117, 200, 226, 336, 441, 460, 465, 479, 505, 514 and 616, as well as truncation mutation and frameshift mutation;
    the CHD4 mutation includes one or more mutations occurring at positions 19, 57, 279, 538, 732, 841, 852, 869, 895, 966, 1038, 1042, 1068, 1105, 1272, 1384, 1541, 1886 and 1894, and frameshift mutation.
  9. The use of claim 8, wherein:
    the FBXW7 mutation includes one or more mutations selected from: H27Q, deletion of amino acid at position 117, V200I, T226I, I336M, R441W, H460Y, R465C, R465H, R479Q, R505C, R505H, V514D, T616I, truncation of amino acid residues at positions 1-40 of the N-terminus, truncation of amino acid residues at positions 1-103 of the N-terminus, truncation from position 118 (including position 118) , truncation starting from position 279 (including position 279) , and frameshift mutation starting from position 404 (including position 404) ; preferably, the FBXW7 mutation includes or is R465C, R465H, R479Q, R505C, or R505H;
    the CHD4 mutation includes one or more mutations selected from: D19G, P57S, R279H, R538W, G723S, A841V, Y852H, V869M, Q895K, M966I, G1038D, G1042D, R1068C, R1105W, G1272D, P1384H, P1541H, Q1886H, S1894T, and frameshift mutations starting from position 1488.
  10. The use of claim 1, wherein the Wee1 kinase inhibitor is 6- (2, 6-dichlorophenyl) -2- ( (3-methyl-4- ( (3S, 5R) -3, 4, 5-trimethylpiperazin-1-yl) phenyl) amino) -8, 9-dihydroimidazo [1, 2-a] pyrimido [5, 4-e] pyrimidin-5 (6H) -one, or a pharmaceutically acceptable salt or prodrug thereof.
  11. The use of any one of claims 1-10, wherein the cancer is selected from the group consisting of: diffuse midline glioma, childhood diffuse pontine glioma, midline glioma, liver cancer, melanoma, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, Wilm’s tumor, cervical cancer, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder cancer, chronic myeloid leukemia, primary brain cancer, malignant melanoma, small cell lung cancer, gastric cancer, colon cancer, malignant pancreatic islet tumor, malignant carcinoid cancer, malignant melanoma, choriocarcinoma, mycosis fungoides, head and neck cancer, osteogenic sarcoma, pancreatic cancer, acute myeloid leukemia, hairy cell leukemia, rhabdomyosarcoma, Kaposi's sarcoma, urogenital tumors, thyroid cancer, esophageal cancer, malignant hypercalcemia, cervical hyperplasia, renal cell carcinoma, endometrial cancer, polycythemia vera, idiopathic thrombocythemia, adrenocortical carcinoma, skin cancer, and prostate cancer;
    preferably, the cancer is selected from gynecological oncology, colorectal cancer, and gastric carcinoma.
  12. The use of any one of claims 1-10, wherein:
    the cancer having FBXW7 mutation (s) is selected from a group consisting of skin cutaneous melanoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, esophageal adenocarcinoma, bladder urothelial carcinoma, stomach adenocarcinoma, cervical squamous cell carcinoma, colorectal adenocarcinoma, uterine corpus endometrial carcinoma, and uterine carcinosarcoma;
    the cancer having CHD4 mutation (s) is selected from a group consisting of lung squamous cell carcinoma, cervical squamous cell carcinoma, brain lower grade glioma, testicular germ cell tumor, colorectal adenocarcinoma, bladder urothelial carcinoma, ovarian serous cystadenocarcinoma, stomach adenocarcinoma, skin cutaneous melanoma, uterine carcinosarcoma, and uterine corpus endometrial carcinoma.
  13. Use of a reagent for detecting the presence of a FBXW7 mutation and/or a CHD4 mutation in cancer cells or a cancer tissue of an individual in the preparation of a detection kit  for determining whether the individual is responsive to or benefits from treatment with a Wee1 kinase inhibitor.
  14. Use of claim 13, wherein the Wee1 kinase inhibitor is the compound as defined in any of claims 2-7.
  15. Use of claim 13, wherein the FBXW7 mutation and the CHD4 mutation are the mutation (s) as defined in any one of claims 8 and 9.
PCT/CN2022/134300 2021-11-26 2022-11-25 Use of wee1 kinase inhibitors in the treatment of cancer WO2023093840A1 (en)

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