Use of KIF18a inhibitor for treating ovarian cancer

Abstract

Provided is use of a KIF18A inhibitor in preparing a medicament for treating ovarian cancer disease. Pharmacological studies have demonstrated that compounds 1-3 can inhibit the growth of ovarian cancer heterogeneous tumors, and a high dose of the compounds can cause tumor regression in mice. Compared with AMG650, a lower drug exposure of compounds 1-3 reduces the potential drug accumulation toxicity and possesses a significant therapeutic effect on ovarian cancer with good safety.

Description

Uses of KIF18A inhibitors in the treatment of ovarian cancer

[0001] This application requires the applicant to have:

[0002] Priority rights to the earlier application filed with the China National Intellectual Property Administration on July 2, 2024, with patent application number 202410881197.8 and title "Use of KIF18A inhibitor in the treatment of ovarian cancer";

[0003] Priority rights to the earlier application filed with the China National Intellectual Property Administration on September 29, 2024, with patent application number 202411375549.9 and title "Use of KIF18A inhibitor in the treatment of ovarian cancer";

[0004] The entire contents of the aforementioned prior applications are incorporated herein by reference. Technical Field

[0005] This invention belongs to the pharmaceutical field, specifically relating to the use of a KIF18A inhibitor in the preparation of a drug for treating ovarian cancer. Background Technology

[0006] Cancer is one of the most serious diseases affecting human health, with mortality and morbidity rates often ranking among the highest of all diseases. Although the quality of life for some patients has been greatly improved with the continuous development and progress of medical technology and drug research, there are still many unmet clinical needs in the search for effective treatments or cures for different cancers, and new targets will provide new possibilities for future cancer drug development.

[0007] Cancer cells exhibit unregulated cell proliferation due to damage or loss of one or more genes that regulate the cell cycle. Various kinases and kinesins have been identified as playing key roles in the regulation and progression of cell cycle and mitosis in both normally dividing cells and cancer cells.

[0008] Kinesin molecules are kinetic proteins that use intracellular microtubules as their orbital pathways; also known as molecular motors, they convert ATP energy into mechanical energy and are closely related to eukaryotic cell division, mitosis, meiosis, tissue and organ growth and development, neuronal development, and signal transduction. Kinesin members share a relatively conserved motor domain. Based on the location of the motor domain in the molecule, the kinesin family is broadly divided into three categories: N-type kinesins, where the amino (-NH2) terminal region of the polypeptide chain contains a motor domain; M-type kinesins, where the middle region contains a motor domain; and C-type kinesins, where the carboxyl (-COOH) terminal region contains a motor domain.

[0009] KIF18A is a member of N-type Kinesin-8 motor protein family. KIF18A protein is highly expressed in various tumors. Therefore, the development of KIF18A protein inhibitors can be a new breakthrough in cancer treatment drugs.

[0010] Ovarian cancer is a malignant tumor growing on the ovary, which is a serious threat to women's health. At present, it is still necessary to develop drugs for effectively treating ovarian cancer. SUMMARY

[0011] In order to improve the deficiencies of the prior art, the purpose of the present application is to provide a drug with definite therapeutic effect on ovarian cancer. Specifically, the present application provides a use of a KIF18A inhibitor in the preparation of a drug for treating ovarian cancer.

[0012] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0013] The present application provides a use of a compound 1, a compound 2 and / or a compound 3 as shown below, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for treating ovarian cancer,

[0014] The present application also provides a use of a compound 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating ovarian cancer,

[0015] According to an embodiment of the present application, the pharmaceutically acceptable salt is selected from one or more of a sodium salt, a potassium salt, a hydrochloride salt, a p-toluenesulfonate salt, a maleate salt, a methanesulfonate salt and an ethanesulfonate salt of the compound, and is preferably a p-toluenesulfonate salt.

[0016] According to an embodiment of the present application, the ovarian cancer is high-grade serous ovarian cancer.

[0017] According to an embodiment of the present application, the ovarian cancer is platinum-resistant ovarian cancer.

[0018] According to an embodiment of the present application, the ovarian cancer is platinum-resistant high-grade serous ovarian cancer.

[0019] According to an embodiment of the present application, the ovarian cancer is ovarian cancer with disease progression after platinum drug chemotherapy.

[0020] According to an embodiment of the present application, the ovarian cancer is platinum-resistant high-grade serous ovarian cancer.

[0021] According to an embodiment of the present application, the ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer.

[0022] According to an embodiment of the present application, the ovarian cancer is ovarian cancer with disease progression after chemotherapy with a PARP inhibitor.

[0023] According to an embodiment of the present application, the ovarian cancer is high-grade serous ovarian cancer with disease progression after chemotherapy with a PARP inhibitor.

[0024] According to an embodiment of the present application, the ovarian cancer is olaparib-resistant ovarian cancer.

[0025] According to an embodiment of the present application, the ovarian cancer is ovarian cancer with disease progression after chemotherapy with olaparib.

[0026] According to an embodiment of the present application, the ovarian cancer is high-grade serous ovarian cancer with disease progression after chemotherapy with olaparib.

[0027] According to an embodiment of the present application, the ovarian cancer is recurrent or metastatic advanced ovarian cancer.

[0028] According to an embodiment of the present application, the ovarian cancer is TP53-mutated ovarian cancer.

[0029] According to an embodiment of the present application, the ovarian cancer is TP53-mutated recurrent or metastatic advanced ovarian cancer.

[0030] According to an embodiment of the present application, the medicament comprises a pharmaceutically acceptable carrier or excipient, and is suitable for preparation into a form of intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, in-situ administration to a tumor, etc.

[0031] According to an embodiment of the present application, the medicament is prepared into an oral preparation, such as a tablet, a capsule, a pill, a granule, a solution, a suspension, a syrup, an injection (including an injection solution, a sterile powder for injection, or a concentrated solution for injection), a suppository, an inhalant, or a spray.

[0032] According to an embodiment of the present application, the medicament is administered orally.

[0033] According to an embodiment of the present application, the medicament is administered once a day.

[0034] The present application also provides a method for preventing and / or treating ovarian cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of Compound 1, Compound 2, and / or Compound 3, or a pharmaceutically acceptable salt thereof, or a composition containing Compound 1, Compound 2, and / or Compound 3, or a pharmaceutically acceptable salt thereof.

[0035] According to an embodiment of the present application, the pharmaceutically acceptable salt is selected from one or more of a sodium salt, a potassium salt, a hydrochloride salt, a p-toluenesulfonic acid salt, a maleic acid salt, a methanesulfonic acid salt, and an ethanesulfonic acid salt of the compound, preferably a p-toluenesulfonic acid salt.

[0036] According to an embodiment of the present application, the ovarian cancer is a high-grade serous ovarian cancer.

[0037] According to an embodiment of the present application, the ovarian cancer is a platinum-resistant ovarian cancer.

[0038] According to an embodiment of the present application, the ovarian cancer is a platinum-resistant high-grade serous ovarian cancer.

[0039] According to an embodiment of the present application, the ovarian cancer is an ovarian cancer with disease progression after platinum chemotherapy.

[0040] According to an embodiment of the present application, the ovarian cancer is a high-grade serous ovarian cancer with disease progression after platinum chemotherapy.

[0041] According to an embodiment of the present application, the ovarian cancer is a PARP inhibitor (PARPi)-resistant ovarian cancer.

[0042] According to an embodiment of the present application, the ovarian cancer is an ovarian cancer with disease progression after PARP inhibitor chemotherapy.

[0043] According to an embodiment of the present application, the ovarian cancer is a high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy.

[0044] According to an embodiment of the present application, the ovarian cancer is an Olaparib-resistant ovarian cancer.

[0045] According to an embodiment of the present application, the ovarian cancer is an ovarian cancer with disease progression after Olaparib chemotherapy.

[0046] According to an embodiment of the present application, the ovarian cancer is a high-grade serous ovarian cancer with disease progression after Olaparib chemotherapy.

[0047] According to an embodiment of the present application, the ovarian cancer is a recurrent or metastatic advanced ovarian cancer.

[0048] According to an embodiment of the present application, the ovarian cancer is a TP53-mutated ovarian cancer.

[0049] According to an embodiment of the present application, the ovarian cancer is a TP53-mutated recurrent or metastatic advanced ovarian cancer.

[0050] According to an embodiment of the present application, the compound or the pharmaceutically acceptable salt thereof or the composition is administered once a day.

[0051] According to the embodiments of the present application, the compound or its pharmaceutically acceptable salt or the composition is administered orally. According to the embodiments of the present application, the daily administration dose of the compound 1 or compound 2 or / and compound 3 is 0.1-1000 mg, which can be 0.1 mg, 0.3 mg, 0.5 mg, 0.7 mg, 0.9 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg.

[0052] According to the embodiments of the present application, the compound 1 or compound 2 or compound 3 or its pharmaceutically acceptable salt or the composition is administered alone or in combination with other anti-tumor drugs.

[0053] According to the embodiments of the present application, the combination administration can be simultaneous or sequential administration.

[0054] According to the embodiments of the present application, the subject is a mammal, preferably a human.

[0055] The present application also provides a medicine for preventing and / or treating ovarian cancer disease in a subject, which is compound 1, compound 2 and / or compound 3, or a pharmaceutically acceptable salt thereof, or a composition containing a therapeutically effective amount of compound 1, compound 2 and / or compound 3 or a pharmaceutically acceptable salt thereof.

[0056] Advantages of the present application:

[0057] The present application proves that compounds 1-3 can inhibit the growth of ovarian cancer heterogenous tumors, and high-dose compounds can make the tumors of mice regress. In particular, compound 1 has good therapeutic effect on platinum-resistant ovarian cancer. Compared with AMG650, the lower drug exposure of compounds 1-3 can reduce potential drug accumulation toxicity. It is shown that compounds 1-3 have obvious therapeutic effect on ovarian cancer and good safety. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is a line graph showing the influence of tumor volume in OVCAR-3 xenograft tumor model mice.

[0059] Figure 2 is a schematic diagram showing the tumor weight of OVCAR-3 xenograft tumor model mice.

[0060] Figure 3 is a line graph showing the influence of body weight in OVCAR-3 xenograft tumor model mice.

[0061] Figure 4 is a line graph showing the influence of tumor volume in OVCAR-3 xenograft tumor model mice.

[0062] Figure 5 is a schematic diagram showing the tumor weight of OVCAR-3 xenograft tumor model mice.

[0063] Figure 6 is a line graph showing the influence of body weight in OVCAR-3 xenograft tumor model mice.

[0064] Figure 7 is a schematic diagram showing the positive rate of phosphorylated histone PH3 in tumor tissue of mice.

[0065] Figure 8 is a schematic diagram showing the influence of tumor volume in human-derived ovarian cancer in vivo xenograft tumor model mice.

[0066] Figure 9 is a schematic diagram showing the tumor weight of human-derived ovarian cancer in vivo xenograft tumor model mice.

[0067] Figure 10 is a schematic diagram showing the influence of body weight in human-derived ovarian cancer in vivo xenograft tumor model mice.

[0068] Explanation or definition of terms

[0069] The term "subject" herein refers to an animal, such as a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cows, and humans.

[0070] The term "therapeutically effective amount" or "effective amount" herein refers to the amount of a compound disclosed and / or described herein that, when administered to a patient in need of such treatment, is sufficient to effect treatment as defined herein. The therapeutically effective amount of a compound can be an amount sufficient to treat a disease responsive to modulation (e.g., inhibition) of KIF18A or an amount sufficient to treat ovarian cancer. The therapeutically effective amount will vary depending, for example, on the subject and disease condition being treated, the subject's body weight and age, the severity of the disease condition, the particular compound, the dosing regimen to be followed, the timing and mode of administration, factors which can readily be determined by one of ordinary skill in the art.

[0071] The term "treatment" herein includes one or more of: inhibiting the disease or condition; slowing or arresting the development of clinical symptoms of the disease or condition; and / or relieving the disease or condition (i.e., causing regression or remission of clinical symptoms), and complete or partial reduction of clinical symptoms of the disease or condition.

[0072] The term "therapeutic effect" herein refers to the effect caused by the treatment, which is manifested at the cellular level as the inhibition rate of cell growth or the mortality rate of cells, at the animal level as its change, usually alleviating or improving the symptoms of the disease or disease condition, or curing the disease or disease condition.

[0073] The term "pharmaceutically acceptable salt" herein refers to the fact that the compound can exist in the form of various pharmaceutically acceptable salts. If the compound has a basic center, it can form an acid addition salt; if the compound has an acidic center, it can form a base addition salt; if the compound contains both an acidic center and a basic center, it can also form an internal salt. DETAILED DESCRIPTION

[0074] The application will be further described in conjunction with specific examples. It should be understood that the following examples are merely illustrative and explanatory of the application and should not be interpreted as limiting the scope of the application. Any technology realized based on the above description of the application is encompassed within the scope intended to be protected by the application.

[0075] The preparation methods of compounds 1-3 are referred to the patent documents of WO2024051755A1, WO2024067675A1, CN117510463A respectively.

[0076] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0077] Example 1

[0078] Experimental name Pharmacodynamic effect test of compound 1 and compound 2 on human ovarian cancer OVCAR-3 cell xenograft tumor mice

[0079] 1.1 Purpose of the experiment

[0080] To observe the therapeutic effect of Compound 1 and Compound 2 on human ovarian cancer OVCAR-3 cell xenograft tumor mice and compare with AMG650.

[0081] 1.2 Experimental reagents

[0082] Experimental instruments

[0083] 1.3 Main test drugs

[0084] AMG650, batch number: EB2302070-048P1; storage condition: 2-8℃; production and sample sending unit: Changchun Jin Sai Pharmaceutical Co., Ltd.

[0085] Compound 1, batch number: EB2214099-062P1; storage condition: 2-8℃; production and sample sending unit: Changchun Jin Sai Pharmaceutical Co., Ltd.

[0086] Compound 2, batch number: EB2214099-078P1; storage condition: 2-8℃; production and sample sending unit: Changchun Jin Sai Pharmaceutical Co., Ltd.

[0087] Solvent components: 20% PEG400 + 10% VE-TPGS + 70% aqueous solution containing 10% hydroxypropyl-β-cyclodextrin.

[0088] The preparation method is as follows: accurately weigh an appropriate amount of test product into a suitable container, add the above solvent components step by step, and fully ultrasonic vortex, each step should be clear solution before adding the next step, the final solution after preparation is clear and transparent solution for administration.

[0089] Storage condition of the administration solution: the preparation frequency is once a week, each time after preparation, store at-20℃. Before administration, put it at room temperature.

[0090] 1.4 Cell strains

[0091] Human ovarian cancer cell line OVCAR-3 is derived from ATCC.

[0092] 1.5 Experimental animals

[0093] Species and strain: Balb / c Nude mice.

[0094] Gender and age: female, 6-7 weeks old.

[0095] Animal source: Jiangsu Jicui Yakang Biotechnology Co., Ltd. Production License Number: SCXK (Jiangsu) 2018-0008, Animal Qualification Certificate Number: 202302595 / 202303296.

[0096] Feeding conditions: Animals are housed in polypropylene mouse cages. The cage size is length × width × height = 33 cm × 21 cm × 17 cm, and at most 5 animals are housed in each cage. Temperature: The set temperature range is 20 - 26 °C, humidity: The set humidity range is 40% - 70%, air change rate: not less than 15 times of fresh air per hour, lighting: 12 hours light and 12 hours dark in alternation.

[0097] Establishment of animal model: OVCAR-3 cells are cultured in PRMI1640 medium containing 10% FBS and maintained in a 37 °C saturated humidity incubator containing 5% CO2. Logarithmic growth phase OVCAR-3 cells are collected and resuspended in PRMI1640 basal medium containing 50% Matrigel, and the cell concentration is adjusted to 6.66×10 7 / mL. Under sterile conditions, 0.1 mL of cell suspension is inoculated subcutaneously into the right dorsal side of the mouse, and the inoculation concentration is 1×10 7 / 0.15 mL / mouse.

[0098] Grouping of experimental animals: 27 days after animal inoculation, tumors that are too large, too small, or irregular are removed. Animals with regular tumor volumes of 111 - 264 mm 3 are selected for grouping. There are 10 animals in each group, a total of 8 groups. After grouping, the average tumor volume of each group of animals is about 149 mm 3 At this time, the animals are randomly grouped according to tumor volume, so that the difference in tumor volume between groups is less than 10% of the mean value. The day of grouping is recorded as Day 0, and drug administration starts according to the animal body weight.

[0099] The experimental grouping is as follows in the table:

[0100] According to the relevant regulations on animal welfare, during the experiment, if an individual experimental animal meets any of the following conditions, the animal will be removed from the experimental group and euthanized. (1) The animal's body weight has decreased by more than 20% compared with Day 0 (BWL ≥ 20%); (2) The animal shows serious adverse reactions, such as blindness, paralysis, etc.; (3) The tumor volume is greater than 2000 mm 3 ; (4) An open ulcer forms on the tumor surface.

[0101] After the last administration, blood was collected at different time points and the collected whole blood was put into a centrifuge tube containing K2-EDTA anticoagulant and centrifuged (centrifugation conditions: 3200 g for 10 minutes at 2-8°C) within 1 hour after collection. After centrifugation, the supernatant plasma sample was taken for PK analysis. At the end of the last weighing, the remaining animals were euthanized with CO2, samples were collected, tumors were weighed and photographed.

[0102] 1.6 Experimental observation index and detection method

[0103] During the experiment, the body weight and tumor volume of the animals were measured twice a week, and the clinical symptoms of the animals were observed and recorded daily.

[0104] The formula for calculating the tumor volume (TV) is: 1 / 2×a×b 2 , where a and b are the length and width of the tumor measurement, respectively.

[0105] The tumor inhibition rate (%TGI TV ) is calculated by the formula: 1-(TV Tn -TV T0 ) / (TV Cn -TV C0 )*100%, TV C is the average tumor volume of the negative control group, TV T is the average tumor volume of the treatment group, TV T0 is the average tumor volume of the negative control group before administration, and TV C0 is the average tumor volume of the treatment group before administration.

[0106] The formula for calculating the body weight change (%BWC) is: (BW t -BW0) / BW0×100%, where BW t is the body weight of the animal at each measurement, and BW0 is the body weight of the animal at the time of grouping.

[0107] According to the Chinese NMPA "Technical Guidelines for Nonclinical Studies of Cytotoxic Antitumor Drugs" (November 2006), %T / C≤40% and P<0.05 by statistical analysis are effective. If the number of drug-related animal deaths exceeds 20%, the drug dose is considered to have serious toxicity.

[0108] 1.7 Statistical analysis

[0109] In this study, the experimental data are expressed as Mean±SEM.

[0110] The tumor volume (mm 3) Tumor growth curves were plotted for Y axis; animal body weight change curves were plotted for X axis with time point as X axis and animal body weight (g) as Y axis. Two-tailed t-test was used for comparison between groups, P < 0.05 was significant difference, P < 0.01 was extremely significant difference (Microsoft Excel 2013, Redmond, WA, USA).

[0111] 1.8 Experimental results

[0112] As shown in Figure 1, the TGI of AMG650 at 15 mg / kg and 30 mg / kg was 72.58% and 115.11%, respectively, the TGI of Compound 1 at 10 mg / kg and 30 mg / kg was 74.92% and 110.45%, respectively, and the TGI of Compound 2 at 10 mg / kg, 20 mg / kg and 30 mg / kg was 47.35%, 106.60% and 115.58%, respectively.

[0113] As shown in Figure 2, the tumor weight of the model group mice was 607 ± 270 mg, the tumor weight of AMG650 at 15 mg / kg and 30 mg / kg was 274 ± 146 mg and 32 ± 16 mg, respectively, the tumor weight of Compound 1 at 10 mg / kg and 30 mg / kg was 205 ± 140 mg and 37 ± 7 mg, respectively, and the tumor weight of Compound 2 at 10 mg / kg, 20 mg / kg and 30 mg / kg was 385 ± 139 mg, 66 ± 33 mg and 27 ± 10 mg, respectively.

[0114] As shown in Figure 3, AMG650, Compound 1 and Compound 2 all dose groups had no significant effect on animal body weight change relative to the solvent group, and no obvious toxicity was observed.

[0115] In summary, in the OVCAR-3 ovarian cancer heterograft mouse model, Compound 1 and Compound 2 significantly inhibited tumor growth, and high concentrations of the compounds could cause the tumor to regress in mice, showing a significant therapeutic effect on ovarian cancer.

[0116] As shown in Table 1, compared with AMG650, the lower absolute exposure of Compound 1 and Compound 2 can reduce potential drug accumulation toxicity.

[0117] Table 1 Comparison of mouse plasma exposure

[0118] Example 2

[0119] Experimental name Pharmacodynamic effect of Compound 3 on human ovarian cancer OVCAR-3 cell xenograft tumor mice

[0120] 2.1 Purpose of the experiment

[0121] To observe the therapeutic effect of compound 3 on human ovarian cancer OVCAR-3 xenograft tumor mice, evaluate its effect, and preliminarily explore its mechanism of action, and compare it with AMG650, to provide experimental basis for clinical trials.

[0122] 2.2 Experimental reagents and experimental instruments

[0123] The same as Example 1.

[0124] 2.3 Main test drugs

[0125] AMG650, batch number: EB2206963-025P1; storage condition: 2-8℃; production and sample sending unit: Changchun Jin Sai Pharmaceutical Co., Ltd.

[0126] Compound 3, batch number: EB2211229-031P1; storage condition: 2-8℃; production and sample sending unit: Changchun Jin Sai Pharmaceutical Co., Ltd.

[0127] Solvent components: 20% PEG400 + 10% VE-TPGS + 70% aqueous solution containing 10% hydroxypropyl-β-cyclodextrin.

[0128] Preparation method, administration solution storage condition The same as Example 1.

[0129] 2.4 Cell strains

[0130] Human ovarian cancer cell line OVCAR-3 was derived from ATCC.

[0131] 2.5 Experimental animals

[0132] Species and strain: Balb / c Nude mice.

[0133] Gender and age at arrival: female, 6-7 weeks old.

[0134] Animal source: Jiangsu Jicui Yekang Biotechnology Co., Ltd. Production license number: SCXK(Su)2018-0008, animal qualification certificate: 202302595 / 202303296.

[0135] Raising conditions, animal model establishment method The same as Example 1.

[0136] Grouping of experimental animals: 27 days after inoculation of animals, remove tumors that are too large, too small, and irregular, and select regular tumors with a volume of 119-227mm 3 for grouping, 10 animals per group, and the average tumor volume of each group of animals after grouping was 158mm 3When the animals were randomly grouped according to the tumor volume, the tumor volume difference of each group was less than 10% of the average value, and the grouping date was recorded as Day 0, and the drug administration was started according to the animal weight.

[0137] The experimental grouping is as follows:

[0138] After the last weighing at the end of the experiment, the remaining animals were euthanized and sacrificed with CO2, samples were collected, and the tumors were weighed and photographed.

[0139] 2.6 Experimental observation index and detection method

[0140] The same as Example 1.

[0141] 2.7 Statistical analysis

[0142] The same as Example 1.

[0143] 2.8 Experimental results

[0144] As shown in Figure 4, the TGI of AMG650 10mg / kg and 30mg / kg was 7.93% and 107.96%, respectively, and the TGI of compound 3 10mg / kg and 30mg / kg was 94.91% and 111.24%, respectively.

[0145] As shown in Figure 5, the tumor weight of the model group mice was 852±67mg, the tumor weight of AMG650 10mg / kg and 30mg / kg was 768±88mg and 50±10mg, respectively, and the tumor weight of compound 3 10mg / kg and 30mg / kg was 143±24mg and 20±3mg, respectively.

[0146] As shown in Figure 6, AMG650 and compound 3 had no significant effect on the body weight change of the animals in all dose groups compared with the solvent group, and no obvious toxicity was observed.

[0147] In summary, in the OVCAR-3 ovarian cancer heterograft mouse model, compound 3 significantly inhibited tumor growth, and high concentrations of the compound could cause the tumor to regress in mice, showing a significant therapeutic effect on ovarian cancer.

[0148] Table 2

[0149] As shown in Table 2, the plasma exposure of compound 3 was much lower than that of AMG650, indicating that the drug had lower exposure under the same efficacy, which could reduce the potential drug accumulation toxicity.

[0150] Example 3

[0151] 3.1 Experimental animals

[0152] 3.1.1 Animal information

[0153] Species and strain: Balb / c Nude mice.

[0154] Gender and age at arrival: Female, 6 - 7 weeks old.

[0155] Source of animals: Jiangsu Jicui Yakang Biotechnology Co., Ltd. Production License Number: SCXK (Su) 2018 - 0008, Animal Qualification Certificate Number: 202302595 / 202303296.

[0156] 3.1.2 Animal feeding

[0157] Living conditions: SPF environment, IVC mouse cages, 5 mice per cage.

[0158] Temperature: 20 - 26 °C.

[0159] Humidity: 40 - 70%.

[0160] Lighting: 12 - hour day - night cycle.

[0161] Feed: Irradiated rat and mouse feed, purchased from Beijing Keao Xieli Feed Co., Ltd., free access to food.

[0162] Drinking water: City tap water, filtered and autoclaved before drinking.

[0163] Litter: Corn cob, purchased from Beijing Keao Xieli Feed Co., Ltd., used after autoclaving, changed once a week.

[0164] Adaptive feeding: Mice were given an adaptive feeding period of no less than 7 days before the experiment.

[0165] Animal identification: Each mouse cage was hung with an experimental information label card, including mouse information, cell inoculation information, animal experiment information, and experimenter information, etc. Mice were marked by ear tagging method.

[0166] 3.2 Experimental materials

[0167] 3.2.1 Test drugs

[0168] AMG650, batch number: EB2302070 - 048P1; Storage condition: 2 - 8 °C; Manufacturer and sample - sending unit: Changchun Jinsai Pharmaceutical Co., Ltd.

[0169] Compound 1, batch number: EB2214099 - 144P1; Storage condition: 2 - 8 °C; Manufacturer and sample - sending unit: Changchun Jinsai Pharmaceutical Co., Ltd.

[0170] Solvent components: 20% PEG400 + 10% VE - TPGS + 70% aqueous solution containing 10% hydroxypropyl - β - cyclodextrin.

[0171] The preparation method is as follows: an appropriate amount of test sample is accurately weighed and placed in a suitable container, and the above-mentioned solvent components are added step by step and ultrasonically stirred, and a clear solution is obtained after each step, and then the next step is added. The final solution after preparation is a clear and transparent solution for administration.

[0172] The storage condition of the administration solution is as follows: after each preparation, it is divided and stored at -20°C. Before administration, it is placed at room temperature.

[0173] 3.3 Cell lines

[0174] The human ovarian cancer cell line OVCAR-3 is derived from ATCC.

[0175] 3.4 Experimental reagents and experimental instruments

[0176] The same as in Example 1.

[0177] 3.5 Experimental design

[0178] The experimental design is shown in the following table:

[0179] 3.6 Experimental method

[0180] 3.6.1 Model establishment

[0181] The same as in Example 1.

[0182] 3.6.2 Grouping, administration observation, and sample collection

[0183] The average tumor volume of the experimental animals was 630mm 3 The animals were grouped and administered according to the above table. The mice were euthanized 8 hours after administration, and tumor tissue samples were collected. They were stored in formalin, made into paraffin sections, and subjected to immunohistochemical staining to detect cell phosphorylated histone.

[0184] 3.7 Experimental results

[0185] As shown in Figure 7, the expression of phosphorylated histone PH3 in all dose groups of AMG650 and Compound 1 increased relative to the solvent group, and the phosphorylated histone in all groups of Compound 1 was significantly higher than that in the solvent group (P<0.001), indicating that Compound 1 caused abnormal increase of mitosis in the process of tumor cell replication, and Compound 1 had better effect on inhibiting tumor mitosis and cell proliferation compared with the positive control drug AMG650.

[0186] Example 4

[0187] The purpose of this experiment is to evaluate the pharmacodynamic effect of the test compound on the human ovarian cancer xenograft model.

[0188] 4.1 Experimental animals

[0189] NCG mice, female, 6 - 8 weeks old, weighing about 18 - 22 g. Purchased from Chengdu Yakang Biotech Co., Ltd. (SCXK(Chuan)2020 - 034). Experimental animal use license number: SYXK(Shaan)2023 - 008.

[0190] 4.2 Animal feeding

[0191] All mice will be housed in the SPF - level animal room with an IVC constant temperature and pressure system, where the temperature is 20 - 26 °C, the humidity is 40 - 70%, and the light cycle is 12 hours light and 12 hours dark. No more than 5 mice will be housed in each cage, and the bedding in the cage is corncob, which is changed twice a week. Throughout the experiment, all experimental mice can eat and drink freely, and the feed and water are both sterilized by high - pressure steam and changed twice a week. The animals are numbered using a mouse ear puncher. All experimental animals are acclimated and observed for at least 3 days before the experiment.

[0192] 4.3 Establishment and grouping design of human - derived ovarian cancer xenograft tumors

[0193] Ovarian cancer tumor tissues from platinum - resistant ovarian cancer patients are inoculated subcutaneously in immunodeficient mice. Continuously observe until tumors form and the tumor volume reaches approximately 1000 mm 3 . Then, dissect the tumor tissue and cut it into tissue pieces of approximately 3 mm×3 mm×3 mm evenly. After that, inoculate the tumor tissue pieces subcutaneously in mice and observe the growth of the tumors.

[0194] When the average tumor volume reaches approximately 100 - 300 mm 3 , group the tumor - bearing mice and observe the administration. Before grouping, measure the tumor volume and weigh the body weight of all tumor - bearing mice. According to the measured tumor volume size, randomly group the tumor - bearing mice. Adopt the principle of randomized block design. First, divide the mice into blocks according to the tumor volume, and then randomly assign the mice within each block to each treatment group. The grouping and administration information are as follows in the table:

[0195] 4.4 Experimental observation

[0196] After inoculating the tumor tissue, observe the experimental animals daily and record their morbidity, death, etc. During the regular experiment, monitor the behavior, food intake, water intake, body weight change, hair luster, and other abnormal conditions of all experimental animals. After the experiment, dissect the tumors, weigh the tumor blocks, and take pictures.

[0197] Record the long diameter D Length and short diameter D Width of the subcutaneous tumors in mice twice a week using a vernier caliper, the tumor volume (TV) of mice was calculated according to the following formula: TV = 0.5 * D Length *D Width 2

[0198] According to the tumor growth, the tumor growth inhibition rate (TGI%) and the relative tumor proliferation rate (T / C%) were calculated in real time, and the formula was as follows:

[0199] TGI% = 100% - (TV treatment-Dn -TV treatment-D0 ) / (TV Control-Dn -TV Control-D0 )*100%

[0200] Where TV Treatment-Dn and TV treatment-D0 are the average tumor volumes of the administration group on the nth day and the 0th day of administration, and TV Control-Dn -TV Control-D0 are the average tumor volumes of the control group on the nth day and the 0th day of administration.

[0201] 4.5 Experimental data analysis

[0202] All data were expressed as Mean ± SEM, SEM = SD / SQRT(n), n = number of animals in the experimental group. One-Way ANOVA test was used to compare the differences in tumor volume between the treatment group and the control group. All data were analyzed using Graphpad. *P < 0.05 was considered to be significantly different.

[0203] 4.6 Experimental results

[0204] As shown in Figure 8, the TGI of Olaparib 100mg / kg was -5.78%, and the TGI of Compound 1 100mg / kg was 98.79%. That is, Compound 1 also has obvious therapeutic effect on platinum-resistant ovarian cancer.

[0205] As shown in Figure 9, the tumor weight of the model group mice was 470±232mg, and the tumor weight of the Olaparib 100mg / kg group and the Compound 1 100mg / kg group mice was 472±154mg and 123±54mg, respectively.

[0206] As shown in Figure 10, Olaparib and Compound 1 dose groups had no significant effect on the body weight change of the animals relative to the vehicle group, and no obvious toxicity was observed.

[0207] 4.7 Experimental conclusion

[0208] Based on the above experimental results, it can be known that the compound 1 can be used for treating platinum drug-resistant ovarian cancer, i.e., the compound 1 can be used for treating ovarian cancer patients with disease progression after platinum drug chemotherapy, especially high-grade serous ovarian cancer patients with disease progression after platinum drug chemotherapy; at the same time, the compound 1 can also be used for treating PARP inhibitor (PARPi) drug-resistant ovarian cancer, i.e., the compound 1 can be used for treating ovarian cancer patients with disease progression after PARP inhibitor drug chemotherapy, especially high-grade serous ovarian cancer patients with disease progression after PARP inhibitor drug chemotherapy. For example, the compound 1 can be used for treating olaparib-resistant ovarian cancer, i.e., the compound 1 can be used for treating ovarian cancer patients with disease progression after olaparib chemotherapy, especially high-grade serous ovarian cancer patients with disease progression after olaparib chemotherapy.

[0209] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of Compound 1, Compound 2, and / or Compound 3, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of ovarian cancer, Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salt, potassium salt, hydrochloride, p-toluenesulfonate, maleate, methanesulfonate and ethanesulfonate of the compound, preferably p-toluenesulfonate.

2. Use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of ovarian cancer, Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salt, potassium salt, hydrochloride, p-toluenesulfonate, maleate, methanesulfonate and ethanesulfonate of the compound, preferably p-toluenesulfonate.

3. Use according to claim 1 or 2, wherein, The ovarian cancer is high-grade serous ovarian cancer.

4. Use according to any one of claims 1 to 3, wherein, The ovarian cancer is platinum-resistant ovarian cancer, preferably platinum-resistant high-grade serous ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after platinum chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after platinum chemotherapy. The ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after PARP inhibitor chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy. The ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after PARP inhibitor chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy. The ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after PARP inhibitor chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy.

5. Use according to any one of claims 1 to 4, wherein, The ovarian cancer is recurrent or metastatic advanced ovarian cancer, preferably TP53-mutated ovarian cancer; preferably, the ovarian cancer is TP53-mutated recurrent or metastatic advanced ovarian cancer.

6. Use according to any one of claims 1 to 5, wherein, The drug comprises a pharmaceutically acceptable carrier or excipient, and is suitable for preparation into a formulation form of intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, tumor in situ administration, etc.; preferably, the drug is administered once a day; preferably, the drug is administered orally.

7. A method of preventing and / or treating ovarian cancer disease in a subject, wherein, The methods include administering to the subject a therapeutically effective amount of Compound 1, Compound 2, and / or Compound 3, or a pharmaceutically acceptable salt thereof, or a composition containing Compound 1, Compound 2, and / or Compound 3, or a pharmaceutically acceptable salt thereof, The drug is prepared into an oral preparation, such as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers or sprays.

8. The method of claim 7, wherein, Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salt, potassium salt, hydrochloride, p-toluenesulfonate, maleate, methanesulfonate and ethanesulfonate of the compound, preferably p-toluenesulfonate. The ovarian cancer is high-grade serous ovarian cancer; and / or, the ovarian cancer is platinum-resistant ovarian cancer, preferably platinum-resistant high-grade serous ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after platinum chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after platinum chemotherapy. The ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after PARP inhibitor chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy. The ovarian cancer is PARP inhibitor (PARPi) drug-resistant ovarian cancer; preferably, the ovarian cancer is ovarian cancer with disease progression after PARP inhibitor chemotherapy; preferably, the ovarian cancer is high-grade serous ovarian cancer with disease progression after PARP inhibitor chemotherapy. and / or the ovarian cancer is an Olaparib-resistant ovarian cancer; preferably, the ovarian cancer is an ovarian cancer with disease progression after Olaparib chemotherapy; preferably, the ovarian cancer is a high-grade serous ovarian cancer with disease progression after Olaparib chemotherapy; and / or the ovarian cancer is a recurrent or metastatic, platinum-resistant, ovarian cancer; preferably, the ovarian cancer is a TP53-mutated ovarian cancer; preferably, the ovarian cancer is a TP53-mutated, recurrent or metastatic, platinum-resistant, ovarian cancer.

9. The method of claim 7 or 8, wherein, the compound or the pharmaceutically acceptable salt thereof or the composition is administered once daily; the compound or the pharmaceutically acceptable salt thereof or the composition is administered orally.

10. The method according to any one of claims 7-9, wherein, the subject is a mammal, preferably a human.

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