WO2024255830A1 - Use of akt inhibitor in preparation of drug for preventing or treating ovarian cancer - Google Patents

Abstract

Use of an AKT inhibitor in the preparation of a drug for preventing or treating ovarian cancer, and use of an AKT inhibitor in combination with other drugs for treating ovarian cancer comprising one or more of a PARP inhibitor, a plant-based chemotherapy drug, an antibiotic chemotherapy drug, a platinum drug and an alkylating agent in the preparation of a drug for preventing or treating ovarian cancer. The combined administration can effectively inhibit the growth of ovarian tumor cells and cell colony formation, and is significantly superior to the administration of a single drug. In addition, the combined administration has an additive or synergistic effect, has few toxic side effects on normal cells and has a high safety, provides a more effective method for treating ovarian cancer, and has an important clinical value.

Description

Use of AKT inhibitors in the preparation of drugs for preventing or treating ovarian cancer

This application claims the priority of Chinese patent application 202310707215.6 with an application date of June 14, 2023 and Chinese patent application 202311223529.5 with an application date of September 21, 2023, and this application cites the full text of the above-mentioned Chinese patent applications.

Technical Field

The present invention belongs to the field of biomedicine. Specifically, the present application relates to the use of an AKT inhibitor in the preparation of a drug for preventing or treating ovarian cancer.

Background Art

Cancer is a disease with a high mortality rate second only to cardiovascular disease. The number of cancer patients and deaths is increasing worldwide. Ovarian cancer is a common malignant tumor in women, and its mortality rate ranks first among all tumor-related deaths in women. Since ovarian cancer lacks obvious external symptoms in the early stages and the disease is hidden, it is very easy to be misdiagnosed. Most ovarian cancer patients have metastases when diagnosed, and simple surgical treatment is difficult to completely eliminate the tumor, so postoperative recurrence is very likely to occur. More than 70% of patients are already in the advanced stage when diagnosed, and about 70% of patients relapse within two years after surgery.

Drug therapy is an adjunct to surgical treatment of ovarian cancer, and commonly used therapeutic drugs are platinum compounds. Platinum compounds (such as cisplatin and carboplatin) can activate the DNA damage response, causing DNA damage, thereby inducing cell apoptosis and exerting significant anti-cancer activity. Although tumor cells are initially sensitive to platinum treatment, ovarian cancer patients are prone to drug resistance, and resistance to platinum may be attributed to upregulation of the ABCB1 gene, reversal of BRCA1 and BRCA2 germline alleles, or impaired apoptosis pathways. Therefore, there is an urgent need to develop a reliable drug to treat drug-resistant ovarian cancer.

Currently, the anti-ovarian cancer drugs under development are mainly targeted drugs. Targeted therapy specifically targets the altered genes or proteins in tumor cells, and has the advantages of strong specificity, significant efficacy, and low toxicity and side effects. It has become a new treatment method besides the three traditional treatment methods of surgery, radiotherapy, and chemotherapy.

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors are new molecular targeted anti-tumor drugs developed and marketed in recent years. They are clinically applicable to patients with advanced ovarian cancer with or without mutations in the tumor suppressor gene BRCA. PARP-1 inhibitors work through a "synergistic lethal effect", that is, in the case of DNA homologous recombination defects caused by BRCA mutations, the simultaneous action of PARP-1 inhibitors and BRCA gene mutations can further aggravate DNA damage, causing ovarian cancer cells with BRCA mutations to die because their DNA cannot be repaired by recombination. Currently, a number of PARP-1 inhibitors, including olaparib, rucaparib, niraparib, talazoparib and fluzoparib, have been approved for marketing or are in clinical research.

AKT is a type of serine/threonine kinase that affects cell survival, growth, metabolism, proliferation, migration and differentiation through numerous downstream effectors. More than 50% of human tumors have overactivated AKT. Overactivation of AKT can lead to tumorigenesis, metastasis and drug resistance. AKT has three subtypes: AKT1, AKT2 and AKT3. As a typical protein kinase, each subtype consists of an amino-terminal PH domain (Pleckstrin homology domain), a middle ATP-binding kinase domain and a carboxyl-terminal regulatory domain. About 80% of the amino acid sequences of the three subtypes are homologous, with only large changes in the PH domain and kinase domain connection region. Therefore, molecular targeted therapy targeting AKT is also an important direction for the current development of tumor targeted drugs.

WO2020156437A1 discloses a compound represented by formula (A), the chemical name of which is (R)-4-((1S, 6R)-5-((S)-2-(4-chlorophenyl)-3-(isopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one, and the chemical molecular formula is shown in the following formula (A):

The compound represented by formula (A) is an AKT protein kinase inhibitor, which can effectively inhibit the activity of AKT kinase and is used for the treatment of locally advanced or metastatic solid tumors. However, there is no report on the use of the compound represented by formula (A) in combination with other ovarian cancer therapeutic drugs in the preparation of drugs for the prevention or treatment of ovarian cancer.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art, and to provide a use of an AKT inhibitor in the preparation of a drug for preventing or treating ovarian cancer, and in particular to provide a use of an AKT inhibitor in combination with other ovarian cancer therapeutic drugs in the preparation of a drug for preventing or treating ovarian cancer.

To achieve the above object, the present invention provides the following technical solutions:

A use of an AKT inhibitor in the preparation of a drug for preventing or treating ovarian cancer, wherein the AKT inhibitor is a compound represented by formula (I) (hereinafter collectively referred to as the compound of formula (I)) or a pharmaceutically acceptable salt or hydrate thereof,

In some embodiments, the compound of formula (I) is a compound of formula (A) or a compound of formula (B):

The present invention also provides a use of an AKT inhibitor in the preparation of a drug for preventing or treating ovarian cancer, wherein the AKT inhibitor is of formula (A): The compound shown (hereinafter collectively referred to as the compound of formula (A)) or a pharmaceutically acceptable salt or hydrate thereof.

In one embodiment of the present invention, the ovarian cancer is platinum-resistant ovarian cancer.

In one embodiment of the present invention, the ovarian cancer is platinum-resistant high-grade serous carcinoma, platinum-resistant ovarian adenocarcinoma, platinum-resistant epithelial serous carcinoma, platinum-resistant clear cell carcinoma, platinum-resistant endometrioid ovarian cancer, platinum-resistant fallopian tube cancer or platinum-resistant primary peritoneal cancer.

In one embodiment of the present invention, the ovarian cancer is platinum-resistant high-grade serous carcinoma or platinum-resistant ovarian adenocarcinoma.

In one embodiment of the present invention, the ovarian cancer is locally advanced or metastatic ovarian cancer.

In one embodiment of the present invention, the ovarian cancer is locally advanced or metastatic ovarian cancer that has failed standard treatment, or has no standard treatment options, or is not currently suitable for standard treatment.

In one embodiment of the present invention, the ovarian cancer is platinum-resistant locally advanced or metastatic ovarian cancer.

In one embodiment of the present invention, the ovarian cancer is locally advanced or metastatic ovarian cancer that is platinum-resistant and has failed standard treatment, or has no standard treatment options, or is not currently suitable for standard treatment.

In one embodiment of the present invention, the compound of formula (A) is used in combination with other drugs for treating ovarian cancer.

In one embodiment of the present invention, the administration of the combined use is selected from simultaneous, concurrent, independent or sequential application.

In one embodiment of the present invention, the combined administration route is selected from oral, parenteral, rectal, pulmonary or local administration, and the parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection.

In one embodiment of the present invention, the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs.

In one embodiment of the present invention, the compound of formula (A) is used in combination with a PARP inhibitor.

In one embodiment of the present invention, the PARP inhibitor includes one or more of Niraparib, Olaparib, Rucaparib, Talazoparib or Fluzoparib; preferably Niraparib or Olaparib.

In a preferred embodiment of the present invention, the PARP inhibitor is niraparib.

In a preferred embodiment of the present invention, the PARP inhibitor is olaparib.

In one embodiment of the present invention, the dosage of the compound of formula (A) is 0.1 to 1000 mg, specifically 1 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg , 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 700mg, 750mg, 800mg, 900mg or 1000mg and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated); preferably 20-500mg; more preferably 200-400mg; more preferably 100, 200, 300, 400 or 500mg; further preferably 300, 400 or 500mg.

In one embodiment of the present invention, the administration frequency of the compound of formula (A) can be once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks, once a month, 21+7 (28 days as a cycle, once a day or twice a day for the first 21 days, rest for 7 days), 4+3 (7 days as a cycle, once a day or twice a day for the first 4 days, rest for 3 days) or 5+2 (7 days as a cycle, once a day or twice a day for the first 5 days, rest for 2 days); preferably once a day, twice a day, 21+7 (28 days as a cycle, once a day or twice a day for the first 21 days, rest for 7 days), 4+3 (7 days as a cycle, once a day or twice a day for the first 4 days, rest for 3 days) or 5+2 (7 days as a cycle, once a day or twice a day for the first 5 days, rest for 2 days). Days constitute a cycle, once a day or twice a day for the first 5 days, and rest for 2 days).

In one embodiment of the present invention, the administration frequency of the compound of formula (A) is once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks or once a month; preferably once a day or twice a day.

In one embodiment of the present invention, the compound of formula (A) is administered once a day, twice a day or three times a day for 21 consecutive days, followed by an interval of 1 to 2 weeks without administration of the compound; or is administered for 5 or 4 consecutive days, followed by an interval of 1 to 3 days without administration of the compound.

In one embodiment of the present invention, the administration frequency of the compound of formula (A) is once a day or twice a day, for 21 consecutive days, followed by a 1-week interval without administration of the compound; or for 5 consecutive days, followed by a 2-day interval without administration of the compound; or for 4 consecutive days, followed by a 3-day interval without administration of the compound.

In one embodiment of the present invention, the dosage of the PARP inhibitor is 100-1000 mg, specifically 100 mg, 105 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, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg 0mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg or 1000mg and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated); preferably 100-600mg; more preferably 200mg, 400mg, 300mg or 600mg.

In one embodiment of the present invention, the administration frequency of the PARP inhibitor may be once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks, or once a month; preferably once a day, twice a day, or three times a day.

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21+7, and the dosage is 200-400 mg/time; the administration frequency of the PARP inhibitor is once a day, and the dosage is 300 mg/time.

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of the PARP inhibitor is once a day, and the dosage is 300 mg/time.

In one embodiment of the present invention, 28 days is one cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of niraparib is once a day, and the dosage is 300 mg/time.

In one embodiment of the present invention, 28 days is one cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of Olaparib is once a day, and the dosage is 300 mg/time.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to the PARP inhibitor is selected from 1:0.1-300, preferably 1:0.1, 1:0.2, 1:0.5, 1:1, 1:2.5, 1:1.5, 1:2, 1:3, 1:5, 1:6.5, 1:8, 1:10, 1:15, 1:20, 1:30, 1:45, 1:50, 1:60, 1:75, 1:85, 1:90, 1:100, 1:120, 1:150, 1:200, 1:250 or 1:300, and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated).

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to niraparib is selected from 1:0.01-300, preferably 1:0.01, 1:0.1, 1:0.2, 1:0.5, 1:0.75, 1:1, 1:2.5, 1:1.5, 1:2, 1:3, 1:5, 1:6.5, 1:8, 1:10, 1:15, 1:20, 1:30, 1:45, 1:50, 1:60, 1:75, 1:85, 1:90, 1:100, 1:120, 1:150, 1:200, 1:25 0 or 1:300 and values between any two of the above values (although not listed one by one, but deemed to be clearly stated); preferably 1:0.75, 1:1, 1:1.3, 1:1.5, 1:2.7, 1:3.1, 1:4.6, 1:6.3, 1:6.6, 1:7, 1:8, 1:10, 1:13, 1:20, 1:21, 1:25, 1:30, 1:33, 1:40, 1:50 or 1:60 and values between any two of the above values (although not listed one by one, but deemed to be clearly stated); more preferably 1:0.75, 1:1 or 1:0.5.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to olaparib is selected from 1:0.01-300, preferably 1:0.01, 1:0.1, 1:0.2, 1:0.5, 1:0.75, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7:1:8, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:75, 1:85, 1:90, 1:100, 1:120, 1:130, 1:140, 1:150, 1:160, 1:175, 1:185, 1:190, 1:100, 1:1 ...5, 1:120, 1:130, 1:140, 1:150, 1:160, 1:175, 1:185, 1:190, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:1 :150, 1:200, 1:250 or 1:300 and values between any two of the above values (although not listed one by one, it is deemed to be clearly stated); preferably 1:0.75, 1:1, 1:1.6, 1:2.5, 1:3.1, 1:4, 1:5, 1:6, 1:6.3, 1:6.6, 1:7, 1:8, 1:25, 1:39, 1:40, 1:50 or 1:100 and values between any two of the above values (although not listed one by one, it is deemed to be clearly stated); more preferably 1:0.75, 1:1 or 1:1.5.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to olaparib is selected from 1:5 or 1:6.3 by mass ratio.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to olaparib is selected from 1:1.25 by mass ratio.

In one embodiment of the present invention, the compound of formula (A) is used in combination with a platinum drug.

In one embodiment of the present invention, the platinum drug includes one or more of cisplatin, carboplatin, lobaplatin or nedaplatin; preferably carboplatin.

In one embodiment of the present invention, the dosage of carboplatin is 50 to 1000 mg, specifically 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 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 The present invention relates to an oral dosage form of at least 200 mg to 300 mg of an oral dosage form. The oral dosage form of at least 200 mg to 300 mg of an oral dosage form is preferably 400 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg or 1000 mg, and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated); preferably 400-800 mg; more preferably 400 mg, 500 mg, 600 mg, 700 mg or 800 mg.

In one embodiment of the present invention, the administration frequency of carboplatin can be once a day, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three weeks, once every four weeks, once every three weeks (administered once over 5 days), once every four weeks (administered once over 5 days).

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound; the administration frequency of carboplatin may be once a week for 3 consecutive weeks.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to carboplatin, measured by mass ratio, is selected from 1:0.1-300; preferably 1:0.1, 1:0.2, 1:0.5, 1:1, 1:2.5, 1:1.5, 1:2, 1:3, 1:5, 1:6.5, 1:8, 1:10, 1:15, 1:20, 1:30, 1:45, 1:50, 1:60, 1:75, 1:85, 1:90, 1:100, 1:120, 1:150, 1:200, 1:250 or 1:300 and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated); preferably 1:0.2, 1:0.5, 1:1, 1:2.5, 1:1.5, 1:2 or 1:3; more preferably 1:1, 1:1.5 or 1:2.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to carboplatin is selected from 1:1 by mass ratio.

In one embodiment of the present invention, the compound of formula (A) is used in combination with a phytochemical chemotherapeutic agent.

In one embodiment of the present invention, the plant-based chemotherapy drug includes one or more of vincristine, vinblastine, etoposide or paclitaxel; preferably paclitaxel; more preferably paclitaxel injection.

In one embodiment of the present invention, the dosage of paclitaxel is 1-300 mg/m ² , which can be selected from 10 mg/m ² , 30 mg/m ² , 50 mg/m ² , 55 mg/m ² , 60 mg/m ^{2 , 65 mg/m 2 ,} 70 mg/m ² , 75 mg/m ² , 80 mg/m ^{2 , 85 mg/m 2 ,} 90 mg/m ² , 95 mg/m ² , 100 mg/m ² , 105 mg/m ² , 110 mg/m ² , 115 mg/m ² , 120 mg/m ² , 125 mg/m ² , 130 mg/m ² , 135 mg/m ² , 140 mg/m ² , 145 mg/m ² , 150 mg/m ² , 155mg/m ² , 160mg/m ² , 165mg/m ² , 170mg/m ² , 175mg/m ² , 180mg/m ² , 185mg/m ² , 190mg/m ² , 195mg/m ² , 200mg/m ² , 205mg/m ² , 210mg/m ² , 215mg/m ² , 220mg/m ² , 225mg/m ² , 230mg/m ² , 235mg/m ² , 240mg/m ² , 245mg/m ² , 250mg/m ² , 255mg/m ² , 260mg/m ² , 265mg/m ² , 270mg/m ² , 275mg/m ² , 280mg/m ² , 285mg/m ² , 290mg/m ² , 295mg/m ² or 300mg/m ² and values between any two of the above values (although not listed one by one, but deemed to be clearly indicated); preferably 10-150mg/m ² ; more preferably 40-100mg/m ² ; further preferably 60mg/m ² , 70mg/m ² , 80mg/m ² , 90mg/m ² or 100mg/m ² ; further preferably 80mg/m ² .

In one embodiment of the present invention, the administration frequency of paclitaxel can be once a day, once a week, once every two weeks, once every three weeks, once a month, once every two months, once a week (use for 2 weeks and stop for 1 week), once a week (use for 3 weeks and stop for 1 week), once a week (use for 6 weeks and stop for 2 weeks), once every 3 weeks or once every 4 weeks; preferably once a week (use for 3 weeks and stop for 1 week).

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21+7, and the dosage is 200-400 mg/time; the administration frequency of paclitaxel is once a week (3 weeks on and 1 week off, administered on the 1st, 8th and 15th days of each cycle), and the dosage is 80 mg/m2 ^/ time.

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of paclitaxel is 1, 8, and 15 days of each cycle, and the dosage is 80 mg/m2 ^/ time.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to paclitaxel is selected from 0.01-300:1, preferably 0.01:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 30:1, 40:1, 50:1, 100:1, 130:1, 150:1, 180:1. 1, 200:1, 220:1, 250:1 or 300:1 and values between any two of the above values (although not listed one by one, they are deemed to be clearly stated); preferably 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1 or 20:1 and values between any two of the above values (although not listed one by one, they are deemed to be clearly stated); more preferably 1:1, 1.5:1, 2:1, 3:1 or 4:1.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to paclitaxel is selected from 4:1 in terms of mass ratio.

In a preferred embodiment of the present invention, the compound of formula (A) is used in combination with an antibiotic chemotherapeutic drug.

In one embodiment of the present invention, the antibiotic chemotherapy drug includes one or more of actinomycin D, mitomycin, doxorubicin, doxorubicin, epirubicin or daunorubicin; preferably doxorubicin; more preferably doxorubicin liposomes.

In one embodiment of the present invention, the dosage of doxorubicin is 0.1-300 mg/m ² , specifically 1 mg/m ² , 2.5 mg/m ² , 5 mg/m ² , 7.5 mg/m ² , 10 mg/m ² , 12.5 mg/m ² , 15 mg/m ² , 17.5 mg/m ² , 20 mg/m ² , 22.5 mg/m ² , 25 mg/m ² , 30 mg/m ² , 32.5 mg/m ² , 35 mg/m 2 , 37.5 mg/m ² , 40 mg/m ² , 50 mg/m ² , 52.5 mg/m ² , 55 mg/m ² , 60 mg/m ² , 65 mg/m ² , 70 mg ^/ m ² , 75 mg/m ² , 80mg/m ² , 85mg/m ² , 90mg/m ² , 95mg/m ² , 100mg/m ² , 105mg/m ² , 110mg/m ² , 120mg/m ² , 130mg/m ² , 140mg/m ² , 150mg/m ² , 160mg/m ² , 170mg/m ² , 180mg/m ² , 190mg/m ² , 200mg/m ² , 210mg/m ² , 220mg/m ² , 230mg/m ² , 240mg/m ² , 250mg/m ² , 260mg/m ² , 270mg/m ² , 280mg/m ^2. 290mg/m ² or 300 mg/m ² and values between any two of the above values (although not listed one by one, they are deemed to be clearly indicated); preferably 1-100 mg/m ² ; more preferably 10-60 mg/m ² ; further preferably 10 mg/m ² , 20 mg/m ² , 30 mg/m ² , 40 mg/m ² or 50 mg/m ² ; further preferably 40 mg/m ² .

In one embodiment of the present invention, the administration frequency of doxorubicin can be once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every month, or once every two months; preferably once every two weeks, once every three weeks, or once every four weeks; more preferably once every four weeks; and further preferably once every four weeks (administered on the first day of each cycle).

In one embodiment of the present invention, one cycle is 28 days; the administration frequency of the compound of formula (A) is 21+7, and the dosage is 200-400 mg/time; the administration frequency of doxorubicin is once every four weeks (administered on the first day of each cycle), and the dosage is 40 mg/m2 ^/ time.

In one embodiment of the present invention, 28 days is one cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of doxorubicin is on the first day of each cycle, The dosage is 80 mg/m2 ^/ time.

In one embodiment of the present invention, the dosage ratio of the compound of formula (A) to doxorubicin is selected from 0.01-300:1, preferably 0.01:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 3:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 30:1, 40:1, 50:1, 10 0:1, 130:1, 150:1, 180:1, 200:1, 220:1, 250:1 or 300:1 and values between any two of the above values (although not listed one by one, it is deemed to be clearly stated); preferably 3:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1 or 20:1 and values between any two of the above values (although not listed one by one, it is deemed to be clearly stated).

In one embodiment of the present invention, the compound of formula (A) is used in combination with one or more of a PARP inhibitor, a plant-based chemotherapeutic drug or an antibiotic-based chemotherapeutic drug.

In one embodiment of the present invention, the compound of formula (A) is used in combination with one or more of niraparib, paclitaxel or doxorubicin.

In one embodiment of the present invention, the compound of formula (A) is used in combination with niraparib, paclitaxel or doxorubicin.

In one embodiment of the present invention, the above-mentioned combined use method can be further used in combination with other treatment methods besides PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs, for example, further combined with other chemotherapy, hormones, antibody agents, targeted drugs, and surgery and/or radiotherapy.

In one embodiment of the present invention, the compound of formula (A) can be administered to the patient in the form of a free base, or in the form of a pharmaceutically acceptable salt, hydrate or prodrug (which will be converted into a free base form in vivo), such as a fumarate salt, preferably a monofumarate salt.

In one embodiment of the present invention, the pharmaceutically acceptable salt of the compound of formula (A) is a fumarate dihydrate, the chemical name of which is (R)-4-((1S, 6R)-5-((S)-2-(4-chlorophenyl)-3-(isopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one fumarate dihydrate, and the chemical formula is shown in the following formula (I-1):

In one embodiment of the present invention, other ovarian cancer therapeutic drugs can be administered to patients in the form of free bases, or in the form of pharmaceutically acceptable salts, hydrates or prodrugs (which will be converted to free base forms in vivo), such as salts of PARP inhibitors.

The present invention also provides a drug combination or pharmaceutical composition, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and other ovarian cancer therapeutic drugs, and one or more pharmaceutically acceptable carriers; the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs;

In some embodiments, the compound of formula (I) is a compound of formula (A) or a compound of formula (B):

The present invention also provides a drug combination or pharmaceutical composition, comprising a compound of formula (A) and other ovarian cancer therapeutic drugs, and one or more pharmaceutically acceptable carriers; the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs.

In the drug combination or pharmaceutical composition of the present invention, the PARP inhibitor includes one or more of niraparib, olaparib, rucaparib, talazoparib or fluzoparib, preferably niraparib or olaparib; the plant-based chemotherapy drugs include one or more of vincristine, vinblastine, etoposide or paclitaxel; preferably paclitaxel; the platinum drugs include one or more of cisplatin, carboplatin, lobaplatin or nedaplatin; preferably carboplatin; the antibiotic chemotherapy drugs include one or more of actinomycin D, mitomycin, doxorubicin, doxorubicin, epirubicin or daunorubicin; preferably doxorubicin.

In the pharmaceutical combination or pharmaceutical composition of the present invention, the mass ratio of the compound of formula (A) to other ovarian cancer therapeutic drugs is as described above.

In the pharmaceutical combination or pharmaceutical composition of the present invention, the administration method, dosage and frequency of the compound of formula (A) and other ovarian cancer therapeutic drugs are as described above.

The pharmaceutical composition of the present invention can be prepared into any pharmaceutically acceptable dosage form, for example, tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection and concentrated solutions for injection), liposomes, suppositories, inhalants or sprays.

The pharmaceutical composition of the present invention can also be administered to patients or subjects in need of such treatment in any suitable manner, such as oral, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc.; or, oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When prepared into oral preparations, the pharmaceutical preparations may also contain suitable fillers, binders, disintegrants, lubricants, etc.

The pharmaceutical composition of the present invention can be administered alone, or can be further administered in combination with PARP inhibitors, plant-based chemotherapeutic drugs, antibiotics, The drug is used in combination with other treatments other than chemotherapy drugs or platinum drugs, such as other chemotherapy, hormones, antibodies, targeted drugs and other therapeutic agents, as well as surgery and/or radiation therapy.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include one or more of niraparib, olaparib, paclitaxel, doxorubicin or carboplatin.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include niraparib, olaparib, paclitaxel, doxorubicin or carboplatin.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include one or more of niraparib, olaparib, paclitaxel, doxorubicin or carboplatin.

The present invention also provides a method for preventing and/or treating ovarian cancer, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition of a compound of formula (A) and other ovarian cancer therapeutic drugs, wherein the other ovarian cancer therapeutic drugs include niraparib, olaparib, paclitaxel, doxorubicin or carboplatin.

Beneficial effects:

The present invention provides a use of an AKT inhibitor alone or in combination with other ovarian cancer therapeutic drugs in the preparation of a drug for preventing or treating ovarian cancer, which can effectively inhibit the growth of ovarian tumor cells and cell clone formation, and the combined effect is significantly better than that of a single drug, with an additive or synergistic effect, while having low toxic and side effects on normal cells and high safety, providing a more effective method for treating ovarian cancer and having important clinical value.

Related definitions

Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

The "combination" described herein is a mode of administration, which includes various situations in which two drugs are administered sequentially or simultaneously, and refers to administering at least one dose of the compound of formula (A) and at least one dose of other ovarian cancer therapeutic drugs (such as PARP inhibitors) in any manner within a certain time period. The time period can be within a dosing cycle, and the compound of formula (A) inhibitor and other ovarian cancer therapeutic drugs can be administered simultaneously or sequentially, and any of the methods described includes administering the compound of formula (A) and other ovarian cancer therapeutic drugs through the same administration route or different administration routes. For example, in one embodiment, one or more doses of the compound of formula (A) are administered simultaneously or separately with one or more doses of other ovarian cancer therapeutic drugs; in one embodiment, multiple doses of the compound of formula (A) are administered simultaneously or separately with multiple doses of other ovarian cancer therapeutic drugs; in one embodiment, multiple doses of the compound of formula (A) are administered simultaneously or separately with one dose of other ovarian cancer therapeutic drugs; in one embodiment, one dose of the compound of formula (A) is administered simultaneously or separately with multiple doses of other ovarian cancer therapeutic drugs; in one embodiment, one dose of the compound of formula (A) is administered simultaneously or separately with one dose of other ovarian cancer therapeutic drugs; in all embodiments, the compound of formula (A) may be administered first or the other ovarian cancer therapeutic drugs may be administered first. The term "simultaneous administration" refers to The term "concurrent administration" refers to the administration of two or more drugs to a patient at the same time or very close in time. The term "concurrent administration" refers to the administration of two or more drugs in the same period of time, such as on the same day, but not necessarily at the same time; the term "independent administration" refers to the independent administration of a drug over a period of time, such as the independent administration of a drug over the course of several days or a week, and then the independent administration of other drugs over a subsequent period of time; the term "sequential administration" refers to the administration of drugs to a patient in a specific order, with each drug administered after the previous drug, forming an ordered sequence.

The "oral administration" mentioned herein refers to taking the drug into the body by oral administration, the drug is absorbed into the blood circulation through the gastrointestinal tract, and then distributed throughout the body or acts on specific organs.

The "parenteral administration" mentioned herein refers to an administration method that does not pass through the gastrointestinal tract, including subcutaneous injection, intramuscular injection, intravenous injection, etc.

The term "rectal administration" as used herein refers to a method in which the drug is delivered into the rectum through the anus, and the drug is absorbed into the blood circulation through the rectal mucosa.

The term "pulmonary administration" as used herein refers to delivering the drug directly into the lungs by inhalation, and the drug is absorbed into the blood circulation through the alveoli.

As used herein, "topical administration" refers to administration by coating or applying the drug directly to epidermal tissue, including the skin or mucous membranes of the mouth or vagina.

"Use in combination" or "use in combination with..." as described herein is not intended to imply that the therapies or therapeutic agents must be physically mixed or administered simultaneously and/or formulated for delivery together, although these delivery methods are within the scope described herein. The therapeutic agents in these combinations can be administered simultaneously with, before, or after one or more other additional therapies or therapeutic agents. The therapeutic agents can be administered in any order. In general, each therapeutic agent will be administered at a dose and/or time schedule determined for that therapeutic agent. It should also be understood that the additional therapeutic agents used in the combination can be administered together in a single composition or separately in different compositions. In general, it is expected that the additional therapeutic agents used in the combination are used at levels not exceeding those when they are used alone. In one embodiment, the levels used in the combination will be lower than the levels used in the single agent therapy.

The "combined use" and "combination use" described herein can be used together with a compound of formula (A) or a pharmaceutically acceptable salt thereof and other ovarian cancer therapeutic drugs. Depending on the expected dosage and frequency of administration, the two compounds can be administered together or separately. The dosage and frequency of administration can vary depending on the compound used and the specific condition to be treated. Generally, it is preferred to use the minimum dose sufficient to provide effective treatment, and it can be determined by the following criteria, such as the patient's age, weight and gender; the extent and severity of the cancer to be treated; and the judgment of the treating physician. Usually, an assay suitable for the condition being treated can be used to monitor the therapeutic effect of the patient, or conventional tests and procedures well known in the art can be used to determine the optimal dose. For example, as indicated by the urgency of the treatment situation, a single bolus can be given, several divided doses can be given over time, or the dose can be proportionally reduced or increased. These assays are well known to those of ordinary skill in the art. In addition, it should also be understood that the therapeutic agent of the combination of the present invention can be administered by any suitable route, and the route of administration can vary according to, for example, the health status of the recipient of the combination and the cancer to be treated.

The "pharmaceutical composition" described herein refers to a mixture containing at least one therapeutic agent and at least one pharmaceutically acceptable carrier, and the "pharmaceutically acceptable carrier" includes but is not limited to diluents, binders, disintegrants, and lubricants.

The "dosage" described herein refers to the amount of the therapeutic agent. For example, when a 2 mg dose of the compound of formula (A) is administered and the compound of formula (A) is administered in a tablet containing a fumarate of the compound of formula (A), the tablet will contain a fumarate of the compound of formula (A) equivalent to 2 mg of the compound of formula (A).

The "salt" described herein may exist alone or in a mixture with the free compound of the present invention, and is preferably a pharmaceutically acceptable salt. The "pharmaceutically acceptable salt" refers to an organic acid salt or an inorganic acid salt commonly used in pharmacy, wherein the organic acid salt includes but is not limited to fumarate, methanesulfonate, isethionate, α-naphthalenesulfonate, p-toluenesulfonate, 1,2-ethanedisulfonate, oxalate, maleate, citrate, succinate, L-(+)-tartrate, hippurate, L-ascorbate, L-malate, benzoate or gentisate, and the inorganic acid salt includes but is not limited to hydrochloride, sulfate or phosphate. In one embodiment, the "pharmaceutically acceptable salt" is a fumarate; preferably a fumarate; more preferably a fumarate hydrate; and further preferably a fumarate dihydrate.

The "standard treatment" mentioned in this article refers to the evaluation of the patient's condition before treatment, and the selection of an appropriate treatment plan based on the patient's condition. During the treatment process, the doctor will adjust the treatment plan based on changes in the patient's condition. The treatment model for ovarian cancer is surgery/neoadjuvant chemotherapy + surgery-postoperative adjuvant chemotherapy-maintenance therapy and multiple lines of treatment for recurrence after initial treatment.

The term “failure of standard treatment” as used herein refers to tumor progression or the appearance of new tumors after standard treatment.

In this article, according to the Chinese Anti-Cancer Association, Guidelines for the Diagnosis and Treatment of Ovarian Malignancies (Fifth Edition), 6.1 Classification of Recurrent Ovarian Cancer: Refer to the standards of the American Gynecologic Oncology Group (GOG): "Platinum resistance" refers to a response to initial chemotherapy, but progression or recurrence within 6 months after completion of chemotherapy.

Herein, the term "locally advanced" means that the tumor is confined to the primary organ or adjacent tissues and organs, but the onset time may be longer and the tumor is larger, but metastasis to distant organs has not yet occurred.

As used herein, a "therapeutically effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a disorder. In the case of cancer, a therapeutically effective dose of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down to a certain extent and preferably prevent) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to a certain extent and preferably prevent) tumor metastasis; inhibit tumor growth to a certain extent; and/or alleviate one or more symptoms associated with the disorder to a certain extent. Depending on the extent to which a drug can prevent the growth of existing cancer cells and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer treatment, in vivo efficacy can be measured by assessing duration of survival, duration of progression-free survival (PFS), response rate (RR), duration of response, and/or quality of life.

The “DLT” mentioned in this article is defined as the following toxic reactions related to the study drug (including definitely related, probably related, or possibly related) that occur in subjects in each dose group from the start of drug administration to the end of the first course of treatment (a total of 28 days): (1) Hematological toxicity: Grade 4 neutropenia lasting for more than 3 days; Grade 3 febrile neutropenia; Grade 4 thrombocytopenia; Grade 3 thrombocytopenia with bleeding; Grade 4 anemia; (2) Non-hematological toxicity: Grade 4 non-hematological toxicity; Grade 3 non-hematological toxicity that persists for 3 days after treatment Grade 4 hyperglycemia: blood glucose >27.8mmol/L or 500mg/dL; Grade 3 hyperglycemia: blood glucose >13.9mmol/L or 250mg/dL, which persists for ≥7 days after treatment; (3) Other toxic reactions that require permanent discontinuation of the study drug or result in the total dose of the study drug received during the DLT observation period being less than 75% of the planned dose as determined by the investigator.

The “objective response rate (ORR)” mentioned in this article is defined as the proportion of subjects with complete remission (CR) and partial remission (PR) of their tumors after treatment.

The “disease control rate (DCR)” mentioned in this article is defined as the proportion of subjects whose tumors achieved complete remission (CR), partial remission (PR) and stable disease (SD) after treatment.

The duration of response (DOR) mentioned in this article is defined as the time from the start of objective response to the first occurrence of tumor progression or death due to any cause.

The "progression-free survival (PFS)" mentioned in this article is defined as the time from the start of treatment to tumor progression or death due to any cause.

The "CA-125" mentioned in this article refers to tumor antigen 125, which is currently recognized as the most valuable observation indicator for preoperative diagnosis of ovarian cancer, observation of postoperative recurrence and prognosis evaluation.

As used herein, "GCIG" means Gynecologic Cancer Institute.

The “RP2D” mentioned herein means the recommended dose for Phase II clinical trials.

The "synergistic effect" described herein refers to the phenomenon that the effects of two drugs used in combination are more effective than their individual effects, relative to antagonism. The compound of formula (I) or formula (A) disclosed herein is an effective AKT inhibitor, and its preparation method is published in the international application WO2020156437A1. The "compound" described herein includes all stereoisomers and tautomers.

The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present invention.

Herein, unless otherwise specified, when referring to the dosage of the compound of formula (A) or fulvestrant, it refers to the dosage of the active compound. It is understood that when the application form is a pharmaceutically acceptable salt or hydrate, the dosage or the ratio of the two can still be calculated based on the active compound.

PG: Propylene glycol.

Solutol HS-15: Polyethylene glycol-15-hydroxystearic acid.

HPMC: Hydroxypropyl methylcellulose.

DMSO: dimethyl sulfoxide.

HP-β-CD: Hydroxypropyl-β-cyclodextrin.

PBS: Phosphate buffered saline.

FBS: fetal bovine serum.

MW: molecular weight.

COLO-704: a high-grade serous carcinoma cell line.

SK-OV-3: epithelial serous carcinoma cell line.

OVCAR-3: a high-grade serous carcinoma cell line, platinum-resistant.

TOV-21G: clear cell carcinoma cell line.

A2780: Ovarian adenocarcinoma cell line, platinum-resistant.

ES-2: clear cell carcinoma cell line.

DETAILED DESCRIPTION

The following typical examples are used to illustrate the present invention. Simple replacements or improvements made to the present invention by those skilled in the art all belong to the technical solutions protected by the present invention.

Example 1 Experiment on the effect of the compound of formula (A) alone or in combination on the proliferation of ovarian cancer cells (COLO-704 and SK-OV-3) 1. Experimental purpose: To investigate the effect of the compound of formula (A), combined with niraparib/olaparib, on the proliferation of COLO-704 and SK-OV-3 cell lines.

2. Experimental Methods

2.1 Cell culture

a) COLO-704 cells were cultured in RPMI-1640 medium, supplemented with 1% double antibody and 10% FBS, and cultured at 37°C _and 5% CO2;

b) SK-OV-3 cells were cultured in McCoy's 5A medium, supplemented with 1% double antibody and 10% FBS, and cultured at 37°C and 5% CO ₂ .

2.2 Cell plating

a) The cells were routinely cultured until the cell confluence reached 80%-90%. When the number reached the required level, the cells were collected; b) The cells were resuspended in the corresponding culture medium, counted, and prepared into a cell suspension of appropriate density; c) The COLO-704 and SK-OV-3 cell suspensions were added to a 384-well plate and cultured in an incubator overnight.

2.3 Preparation of compounds

a) The compound of formula (A) (using the monofumarate dihydrate of the compound of formula (A), the concentration is calculated based on the compound of formula (A); refer to the preparation method disclosed in WO2022017448A1 to prepare the compound of formula (A) or its salt) was diluted 3-fold for 10 concentrations starting from the stock solution of 100mM. b) The compound of formula (A) was diluted with DMSO from 100mM to 12mM, and then 3-fold diluted for 10 concentrations. c) Niraparib was diluted with DMSO from 100mM to 32mM, and then 2-fold diluted for 10 concentrations. d) Olaparib was diluted with DMSO from 100mM to 80mM, 75mM, and then 2-fold diluted for 10 concentrations starting from 80mM. e) Niraparib was diluted with DMSO from 32mM to 24mM. f) The final concentration of DMSO was 0.5%. g) Cells plus DMSO were used as high reading control wells. h) Cell-free culture medium was used as low reading control wells.

2.4 Compound treatment of cells

a) Single medication:

24 hours after COLO-704 and SK-OV-3 cells were plated, the compound was used alone. Compound (A), Niraparib, Olaparib: 75 nL of the compound prepared in step 2.3 b)-d) + 75 nL of DMSO were added.

b) Combination medication:

24 hours after COLO-704 and SK-OV-3 cells were plated, the compound of formula (A) was combined with niraparib: 75 nL of the compound prepared in step 2.3b) + 75 nL of the compound prepared in step 2.3c) and e). The compound of formula (A) was combined with olaparib: 75 nL of the compound prepared in step 2.3b) + 75 nL of the compound prepared in step 2.3d).

c) Final concentration of test compound:

COLO-704, SK-OV-3 cells:

Compound of formula (A): 30000, 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52 nM;

Niraparib: 80000, 40000, 20000, 10000, 5000, 2500, 1250, 625, 312.5, 156.25 nM;

Niraparib: 20000, 40000, 60000 nM, respectively, in combination with gradient concentrations of the compound of formula (A);

Olaparib: 200000, 150000, 100000, 50000, 25000, 12500, 6250, 3125, 1562.5, 781.25 nM;

Olaparib: 25000, 50000, 100000 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

e) The cell culture plate was centrifuged at 1000 rpm for 1 min and placed in a 37°C, 5% CO ₂ incubator for 72 hours.

2.5 CTG method detection

a) Add 30 μL CTG reagent (CelltiterGlo kit) to each well, place on a fast shaker for 2 minutes, centrifuge at 100 rpm for 1 min, and place at room temperature away from light for 30 minutes.

b) Read the chemiluminescent signal using Envision instrument.

2.6 Data Analysis

a) Calculation of compound _IC50 :

_IC50 was calculated using GraphPadPrism 8 software, and the following nonlinear fitting formula was used to obtain the _IC50 (half-maximal inhibitory concentration) of the compound: Y = minimum value + (maximum value - minimum value) / (1 + 10^(( _LogIC50 -X)*slope))

X: log value of compound concentration, Y: inhibition rate (%inhibition)

Inhibition rate (%inhibition) = 100-(compound well reading-low reading control reading)/(high reading control reading-low reading control reading)*100

b) Compound combination evaluation index (Synergy.score)

The calculated cell viability inhibition rate was imported into the SynergyFinder website for analysis of the synergy index. Website: https://tangsoftwarelab.shinyapps.io/synergyfinder/#!/dashboard.

Evaluation index: Synergy.score<-10, the interaction between the two compounds is antagonistic; -10<Synergy.score<10, the interaction between the two compounds is additive; Synergy.score>10, the interaction between the two compounds is synergistic.

3. Experimental results

3.1 Cell proliferation activity IC ₅₀

The experiment was carried out according to the above steps, and the inhibition rate was calculated based on the readings.

Table 1. Study on the inhibitory effect of compound of formula (A) combined with PARP inhibitor on the proliferation of ovarian cancer cells

4. Experimental Conclusion

As shown in Table 1, the compound of formula (A) alone or in combination with niraparib/olaparib showed good in vitro anti-cell proliferation effect on the above tumor cells.

Example 2 Experimental study on the effect of the compound of formula (A) alone or in combination on the proliferation of ovarian cancer cells (OVCAR-3, TOV-21G, A2780)

1. Experimental purpose: To investigate the effects of the compound of formula (A) alone or in combination with niraparib/olaparib on the proliferation of three cell lines (OVCAR-3, TOV-21G, A2780).

2. Experimental Methods

2.1 Cell culture

a) OVCAR-3 cells were cultured in RPMI 1640 medium containing 20% FBS at 37°C and 5% CO _2. b) TOV-21G cells were cultured in RPMI 1640 medium containing 15% FBS at 37°C and 5% CO _2. c) A2780 cells were cultured in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO ₂ .

2.2 Cell plating

a) The cells were routinely cultured until the cell confluence reached 80%-90%. When the number reached the required level, the cells were collected; b) The cells were resuspended in the corresponding culture medium, counted, and prepared into a cell suspension of appropriate density; c) The OVCAR-3, TOV-21G, and A2780 cell suspensions were inoculated into a 96-well plate at 150 μL/well and cultured in an incubator overnight.

2.3 Preparation of compounds

Table 2. Compound information

a) Drugs were added 24 hours after cell plating.

b) Compound test concentration:

1) OVCAR-3 cells:

Compound of formula (A): 30, 15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, 0.12, 0.06 μM;

Olaparib: 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μM;

Olaparib: 100, 50, 25 μM, respectively, combined with gradient concentrations of the compound of formula (A);

Niraparib: 80, 70, 60, 50, 40, 30, 20, 10, 5, 2.5 μM;

Niraparib: 40, 30, 20 μM, respectively, used in combination with gradient concentrations of the compound of formula (A).

2) TOV-21G cells:

Compound of formula (A): 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, 0.01 μM;

Olaparib: 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μM;

Olaparib: 6.25, 3.13, 1.56 μM, respectively, combined with gradient concentrations of the compound of formula (A);

Niraparib: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10 μM;

Niraparib: 3.13, 1.56, 0.78 μM, respectively, used in combination with gradient concentrations of the compound of formula (A).

3) A2780 cells:

Compound of formula (A): 30, 15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, 0.12, 0.06 μM;

Olaparib: 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μM.

c) Cells plus DMSO were used as high reading control wells, and cell culture medium-free wells were used as low reading control wells. The final concentration of DMSO in each well was 0.5%.

d) After drug addition, the cell culture plate was placed in a 37°C, 5% CO ₂ incubator for 72 h.

2.4 Detection

a) After the drug incubation is completed, remove the cell culture plate and equilibrate at room temperature for 30 minutes. Add 50 μL of CellCounting-Lite solution that has been pre-melted and equilibrated to room temperature to each well, shake it at 450 rpm on a microplate shaker for 5 minutes, and leave it at room temperature for 10 minutes. b) Read the chemiluminescent signal value using a multifunctional microplate reader.

2.5 Data Analysis

a) _IC50 was calculated using GraphPadPrism 8 software, and the _IC50 (half maximal inhibitory concentration) of the compound was obtained using the following nonlinear fitting formula: Y = minimum value + (maximum value - minimum value) / (1 + 10^(( _LogIC50 -X)*slope))

X: log value of compound concentration, Y: inhibition rate (%inhibition)

Inhibition rate (%inhibition) = 100-(compound well reading-low reading control reading)/(high reading control reading-low reading control reading)*100

b) The Combination Index (CI value) was calculated using CompuSyn software.

Evaluation criteria: CI < 1, the interaction between the two compounds is synergistic; CI = 1, the interaction between the two compounds is additive CI>1, the interaction between the two compounds is antagonistic.

3. Experimental results

Table 3. Study on the inhibitory effect of the compound of formula (A) combined with PARP inhibitor on the proliferation of ovarian cancer cells (OVCAR-3, TOV-21G)

Table 4. Study on the inhibitory effect of the compound of formula (A) combined with PARP inhibitor on the proliferation of ovarian cancer cells (A2780)

Table 5. Evaluation index of the combination of the compound of formula (A) and PARP inhibitor in ovarian cancer cells (OVCAR-3)

Table 6. Evaluation index of the combination of the compound of formula (A) and PARP inhibitor in ovarian cancer cells (TOV-21G)

Table 7. Evaluation index (A2780) of the combination of the compound of formula (A) and PARP inhibitor in ovarian cancer cells

4. Experimental conclusion:

As shown in the table above, the compound of formula (A) was used in combination with the PARP inhibitors olaparib and niraparib on the above ovarian cancer cells, and both had synergistic efficacy; the above experiments were carried out twice in parallel, and the results were consistent.

Example 3 Pharmacodynamic study of the compound of formula (A) alone or in combination with olaparib in the treatment of SK-OV-3 human ovarian cancer xenograft model

1. Experimental purpose: The purpose of this study is to evaluate the efficacy of compound (A) combined with olaparib in the SK-OV-3 human ovarian cancer xenograft model in female NOD SCID mice.

2. Experimental animals: NOD SCID female mice, 6-8 weeks old, weighing 18-20 g, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd., in an SPF environment with a temperature of (23±3)℃ and a humidity of 40-70%.

3. Test products

3.1 Solvent selection:

Solvent of compound of formula (A): PG: 50% HS-15: 0.5% HPMC=2:1:7

Olaparib solvent: 10% DMSO + 90% (10% HP-β-CD in PBS pH 7.4)

3.2 Preparation method: The preparation and storage conditions of the drug are shown in Table 8 below:

Table 8 Drug preparation information

4. Experimental methods and steps

4.1 Cell culture

SK-OV-3 tumor cells were cultured in McCoy's 5a medium containing inactivated 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO ₂ incubator. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

4.2 Tumor cell inoculation and grouping

SK-OV-3 tumor cells resuspended in serum-free McCoy's 5a culture medium were inoculated at 1×10 ⁷ (0.1 mL + Matrigel) on the right rib of the experimental animals, and a total of 48 animals were inoculated. When the tumor grew to 239 mm ³ , 32 animals with relatively uniform tumor volumes were selected and divided into 4 groups, with 8 animals in each group. The specific dosing regimen is shown in the table below:

Table 9 Compound Dosage Scheme

Note: po means oral administration; QD x 28 days means administration once a day for 28 consecutive days; the group of compound of formula (A) and olaparib combined administration was first given compound of formula (A), and then olaparib was administered after an interval of 1 hour; the dose of olaparib from the 1st day to the 17th day after grouping was 100 mg/kg; after the 17th day: the dose of olaparib was adjusted to 125 mg/kg.

4.3 Detection indicators

4.3.1 Tumor volume: The tumor volume was measured twice a week using a vernier caliper to measure the long and short diameters of the tumor. The volume was calculated using the formula: volume = 0.5 × long diameter × short diameter ² .

4.3.2 Animal response after administration: While measuring the tumor volume, weigh the mice. Record the relationship between the change in the weight of the mice and the administration time. At the same time, observe the survival and health status of the mice, such as the general status of animal activity and eating during the administration period.

4.4 Drug evaluation indicators

4.4.1 Relative tumor volume increase ratio T/C (%)

T/C (%) = mean relative tumor volume of the treatment group/mean relative tumor volume of the control group × 100

4.4.2 Tumor growth inhibition rate (TGI, %)

Tumor growth inhibition rate (TGI,%) = (1-T/C) × 100

4.5 Statistical analysis

4.5.1 Data collection: Measure and observe as required by the experimental plan, and record manually or directly in a computer database.

4.5.2 Statistical analysis: SPSS 16.0 statistical software and One-Way ANOVA test were used to perform statistical analysis of tumor volume among groups, and p < 0.05 was considered to be a significant difference.

5 Experimental results

Table 10. Pharmacodynamic data of the compound of formula (A) alone or in combination with olaparib in the treatment of SK-OV-3 human ovarian cancer

Note: a Mean ± standard error; b Compared with the control group; c One-way ANOVA combined with LSD test, P < 0.05 has significant difference, P < 0.001 has extremely significant difference 6. Experimental conclusion

During the experiment, the animals in the experimental group were in good general condition in terms of activity and feeding, and there were no abnormalities in the animals. The animals were divided into groups and given medication on the 19th day after the inoculation of SK-OV-3 tumor cells, and the average tumor volume of each group was 239 mm ³ . On the 28th day after grouping, the mean tumor volume of the solvent control group was 1,962 mm ³ ; the tumor volume of the 20 mg/kg compound of formula (A) monotherapy group was 933 mm ³ (T/C=46.0%, TGI=54.0%, compared with the solvent control group P<0.001); the tumor volume of the 100 mg/kg/125 mg/kg olaparib monotherapy group was 1,597 mm ³ (T/C=81.5%, TGI=18.5%, compared with the solvent control group P=0.029); the tumor volume of the 20 mg/kg compound of formula (A) and 100 mg/kg/125 mg/kg olaparib combination therapy group was 759 mm ³ (T/C=36.9%, TGI=63.1%, compared with the solvent control group P<0.001).

Compared with the solvent control group and each single treatment group, the anti-tumor effect of the combination treatment group of the compound of formula (A) and olaparib was stronger.

Example 4 Pharmacodynamic study of the compound alone or in combination with paclitaxel in the treatment of TOV-21G human ovarian cancer xenograft model

1. Study purpose: To test the inhibitory effect of compounds on tumor growth in the TOV-21G human ovarian cancer xenograft model.

2. Experimental animals: NOD SCID female mice, 6-8 weeks old, weighing 18-20 g, were purchased from Beijing Ankai Yibo Biotechnology Co., Ltd. The breeding environment was SPF grade, with a temperature of (23±3)°C and a humidity of 40-70%.

3. Preparation of test samples

3.1 Solvent selection:

Solvent of compound of formula (A): PG: 50% HS-15: 0.5% HPMC=2:1:7

Paclitaxel solvent: normal saline

3.2 Preparation method: The preparation and storage conditions of the drug are as follows:

Table 11. Drug preparation information

Note: Compounds are corrected and calculated according to conversion factors and purity.

4 Experimental plan

4.1 Cell culture

TOV-21G tumor cells were cultured in a mixed medium (1:1) of MCDB 105 culture medium (1.5 g/L sodium bicarbonate) and 199 culture medium (2.2 g/L sodium bicarbonate) containing inactivated 15% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO ₂ incubator. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

4.2 Tumor cell inoculation and grouping

TOV-21G tumor cells resuspended in a mixed culture medium (1:1) of serum-free MCDB 105 culture medium (1.5g/L sodium bicarbonate) and 199 culture medium (2.2g/L sodium bicarbonate) were inoculated subcutaneously on the right rib of the experimental animals at 1×10 ⁷ /0.1mL+Matrigel (1:1), and a total of 60 animals were inoculated. When the tumor grew to about 150mm ^3, 40 animals with relatively uniform tumor volumes were selected for group administration, a total of 4 groups, and the specific dosing regimen is shown in the table below:

Table 12. Compound Dosing Scheme

Note: p.o. means oral administration; i.v. means intravenous injection; QD x 21 days means administration once a day for 21 consecutive days; QWx3 weeks means administration once a week for 3 consecutive weeks.

On the 21st day of treatment, the animal plasma was continuously collected and stored at the designated time points, totaling 100 plasma samples and 16 tumor samples. The detailed sampling plan is shown in the table below.

Table 13. Sample collection plan

4.3 Detection indicators

4.3.1 Tumor volume: The tumor volume was measured twice a week using a vernier caliper to measure the long and short diameters of the tumor. The volume was calculated using the formula: volume = 0.5 × long diameter × short diameter ² .

4.3.2 Animal response after administration: While measuring the tumor volume, weigh the mice. Record the relationship between the change in the weight of the mice and the administration time. At the same time, observe the survival and health status of the mice, such as the general status of animal activity and eating during the administration period.

4.4 Drug evaluation indicators

4.4.1 Relative tumor volume increase ratio T/C (%)

T/C (%) = mean relative tumor volume of the treatment group/mean relative tumor volume of the control group × 100

4.4.2 Tumor growth inhibition rate (TGI, %)

Tumor growth inhibition rate (TGI,%) = (1-T/C) × 100

4.4.3 Collection of animal plasma and PK analysis

4.5 Statistical analysis

4.5.1 Data collection: Measure and observe as required by the experimental plan, and record manually or directly in a computer database.

4.5.2 Statistical analysis: SPSS 16.0 statistical software and One-Way ANOVA test were used to perform statistical analysis of tumor volume among groups, and p < 0.05 was considered to be a significant difference.

5 Experimental results

Table 14. Tumor inhibition data of the compound of formula (A) combined with paclitaxel in the TOV-21G human ovarian cancer subcutaneous xenograft model

Note: a Mean ± standard error; b Compared with the control group; c One-way ANOVA combined with Dunnett T3 test, P < 0.05 indicates significant difference

Table 15. Pharmacodynamic data of the compound of formula (A) combined with paclitaxel in the TOV-21G human ovarian cancer subcutaneous xenograft model

Compared with the group treated with the compound of formula (A) alone, the combination treatment group showed a significant inhibitory effect on tumor growth (P=0.016); compared with the group treated with paclitaxel alone, the combination treatment group showed a significant inhibitory effect on tumor growth (P=0.001). The results of the accompanying PK study showed that after the compound of formula (A) was combined with paclitaxel in the TOV-21G human ovarian cancer subcutaneous xenograft model, the main PK parameters of paclitaxel were not affected by each other. During the experiment, the general state of animals in all experimental groups, such as activity and eating, was good, and the animals had no abnormalities. Example 5 Combination experiment of compound alone or in combination with paclitaxel or doxorubicin in ovarian cancer cells

1. Study objective: To evaluate the effect of the compound of formula (A) alone or in combination with paclitaxel or doxorubicin on the proliferation of ovarian cancer cell lines (TOV-21G, SK-OV-3, OVCAR-3)

2. Experimental Methods

2.1 Cell culture

a) TOV-21G cells were cultured in RPMI1640 medium containing 15% FBS at 37°C and 5% CO ₂ ; b) SK-OV-3 cells were cultured in McCoy's 5a medium containing 10% FBS at 37°C and 5% CO ₂ ;

c) OVCAR-3 cells were cultured in RPMI1640 medium containing 20% FBS at 37°C and 5% CO ₂ .

2.2 Cell plating

a) The cells were routinely cultured until the cell confluence reached 80%-90%. When the number reached the required level, the cells were collected; b) The cells were resuspended with the corresponding culture medium, counted, and prepared into a cell suspension of appropriate density; c) TOV-21G cells were inoculated in a 96-well plate at a cell density of 2×10 ⁴ cells/well and 150 μL/well using complete culture medium; SK-OV-3 cells were inoculated in a 96-well plate at a cell density of 2×10 ³ cells/well and 150 μL/well using complete culture medium; OVCAR-3 cells were inoculated in a 96-well plate at a cell density of 5000 cells/well and 150 μL/well using complete culture medium.

2.3.1 Preparation of compounds

Table 16. Compound information

Note: The monofumarate dihydrate of the compound of formula (A) is used, and the preparation method disclosed in WO2022017448A1 is referred to to prepare the compound of formula (A) or its salt

2.3.2 Compound treatment of cells

1) Single medication:

a) 24 hours after plating, the compound was treated alone and the sample was added using a compound titrator.

b) Compound test concentration:

TOV-21G cells:

Compound of formula (A): 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52 nM;

SK-OV-3 cells, OVCAR-3 cells:

Compound of formula (A): 100000, 33333.33, 11111.11, 3703.70, 1234.57, 411.52, 137.17, 45.72, 15.24, 5.08 nM; Paclitaxel (consistent in three cell lines):

1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05nM;

Doxorubicin (consistent in all three cell lines):

30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52nM.

c) Set up Medium+DMSO wells and cell+DMSO wells at the same time. Set 2 replicate wells for each concentration, set the DMSO concentration in each well (0.3% for TOV-21G cells and OVCAR-3 cell lines, and 0.5% for SK-OV-3 cell lines); set up 3 parallel plates for each compound, and detect cell viability after 48h, 72h, and 96h of drug action.

2) Combination medication (Matrix):

a) 24 hours after plating, the cells were treated with the compound of formula (A) and paclitaxel or doxorubicin at the same time, and the samples were added using a compound titrator.

b) Compound test concentration:

TOV-21G cells:

Compound of formula (A): 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52 nM.

Paclitaxel: 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05 nM.

Doxorubicin: 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52 nM.

SK-OV-3 cells:

Compound of formula (A): 100000, 33333.33, 11111.11, 3703.70, 1234.57, 411.52, 137.17, 45.72, 15.24, 5.08 nM.

Paclitaxel: 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05 nM.

Doxorubicin: 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52 nM.

OVCAR-3 cells:

Compound of formula (A): 100000, 33333.33, 11111.11, 3703.70, 1234.57, 411.52, 137.17, 45.72, 15.24, 5.08 nM.

Paclitaxel: 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37 nM.

Doxorubicin: 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15 nM.

c) Set up Medium+DMSO wells and Cell+DMSO wells at the same time. Set up 2 replicate wells for each concentration, set the DMSO concentration in each well to 0.5%, and detect cell viability 96 hours after drug administration. Repeat the experiment 3 times.

3) Combination therapy (paclitaxel or doxorubicin fixed concentration):

a) 24 hours after cell plating, the cells were treated with the compound of formula (A) and paclitaxel or doxorubicin at the same time. The concentration of paclitaxel or doxorubicin was fixed and the samples were added using a compound titrator.

b)TOV-21G cells:

Compound of formula (A): 30000, 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52 nM.

Paclitaxel: 3nM, 4nM, 5nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

Doxorubicin: 20 nM, 40 nM, 80 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

SK-OV-3 cells:

Paclitaxel: 4 nM, 8 nM, 12 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

Compound of formula (A): 100000, 33333.33, 11111.11, 3703.70, 1234.57, 411.52, 137.17, 45.72, 15.24, 5.08 nM;

Doxorubicin: 40 nM, 80 nM, 120 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

OVCAR-3 cells:

The final concentrations of the compound of formula (A) are: 100000, 33333.33, 11111.11, 3703.70, 1234.57, 411.52, 137.17, 45.72, and 15.24 nM.

Paclitaxel: 1 nM, 2 nM, 3 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

Doxorubicin: 50 nM, 100 nM, 200 nM, respectively, used in combination with gradient concentrations of the compound of formula (A).

c) Set up Medium+DMSO wells and Cell+DMSO wells at the same time. Set up 2 replicate wells for each concentration, set the DMSO concentration in each well to 0.5%, and detect cell viability 96 hours after drug administration. Repeat the experiment 3 times.

2.4 Cell viability assay

a) About 2 hours before the test, take out an appropriate amount of Cell Counting-Lite 2.0 Luminescent Cell Viability Test Reagent (hereinafter referred to as the Test Reagent) in advance and restore it to room temperature;

b) After the incubation of drugs and cells, take out the cell culture plate and equilibrate at room temperature for 30 minutes;

c) Add 50 μL of detection reagent to each well of the cell culture plate in a light-proof condition, place on a fast shaker for 3 min, and leave at room temperature for 10 min;

d) Read the chemiluminescent signal using Envision instrument.

2.5 Data Analysis

a) Calculation of compound _IC50 :

_IC50 was calculated using GraphPadPrism 8 software, and the following nonlinear fitting formula was used to obtain the _IC50 (half maximal inhibitory concentration) of the compound:

Y=Bottom+(Top-Bottom)/(1+10^((LogIC ₅₀ -X)*HillSlope)).

X: log value of compound concentration, Y: inhibition rate (%inhibition).

Inhibition rate (%inhibition) = 100-(compound well reading-low reading control reading)/(high reading control reading-low reading control reading)*100.

b) Compound combination evaluation index (Synergy.score)

The calculated cell viability inhibition rate was imported into the SynergyFinder website for analysis of the combination index. Website: https://tangsoftwarelab.shinyapps.io/synergyfinder/#！ / dashboard . If Synergy.score<-10, the interaction between the two compounds is antagonistic; if -10<Synergy.score<10, the interaction between the two compounds is additive; if Synergy.score>10, the interaction between the two compounds is synergistic.

3. Experimental results

Combination therapy (paclitaxel or doxorubicin fixed concentration)

Table 17. Inhibitory activity of the compound of formula (A) combined with paclitaxel or doxorubicin on ovarian cancer cells

Experimental conclusion: The compound of formula (A) combined with paclitaxel or doxorubicin exhibited good cell inhibitory activity in the above ovarian cancer cells.

Example 6 Experimental study on the effect of the compound of formula (A) combined with carboplatin on the proliferation of ovarian cancer cells (ES-2)

1. Experimental purpose: To study the effect of the compound of formula (A) combined with carboplatin on the proliferation of ovarian cancer cell line (ES-2).

2. Experimental methods:

2.1 Cell culture: ES-2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences): human ovarian clear cell carcinoma cell line, cultured in McCoy's 5A medium with 10% FBS.

2.2 Cell plating: On day 0 of the experiment, the density of live cells in the logarithmic growth phase was adjusted to 10,000 cells/mL and inoculated into a 96-well plate at 100 μL/well. The inoculated cell plate was placed in an incubator at 37°C and 5% _CO2 for overnight culture.

2.3 Drug incubation

24 hours after cell plating, remove the cell plate cultured overnight and add 50 μL of McCoy's 5A medium containing 10% FBS to each well. Set the cell control group as the high reading control well and the cell culture medium without cell culture medium as the low reading control well. After the addition of drugs, place the cell culture plate in a 37°C, 5% CO ₂ incubator for 72 hours. The final drug concentration is as follows:

Solution 1:

Compound of formula (A): 40.0, 29.3, 25.0, 20.0, 15.0, 7.5, 3.8, 1.9, 0.9 μM;

Carboplatin: 25.0, 20.0, 17.5, 15.0, 10.0, 7.5, 5.0, 2.5, 1.3 μM.

Option 2:

Compound of formula (A): 50.0, 29.3, 20.0, 15.0, 7.5, 3.8, 1.9, 0.9 μM;

Carboplatin: 10, 15, 20 μM, respectively, used in combination with graded concentrations of the compound of formula (A).

After the drug incubation, remove the cell culture plate and equilibrate at room temperature for 30 minutes. Add 50 μL of CellCounting-Lite solution that has been melted and equilibrated to room temperature to each well, shake it at 450 rpm for 5 minutes on a microplate shaker, and leave it at room temperature for 10 minutes. Read the chemiluminescent signal value with a multifunctional microplate reader.

2.4 Data Analysis

GraphPadPrism 5 software was used to fit the dose-effect curve, and the IC ₅₀ (half-maximal inhibitory concentration) of the compound on cell proliferation inhibition was obtained using the following nonlinear fitting formula:

Y = minimum value + (maximum value - minimum value) / (1 + 10^((LogIC ₅₀ -X) * slope))

X: log value of compound concentration, Y: inhibition rate (%inhibition)

Inhibition rate (%inhibition) = 100-(compound well reading-low reading control reading)/(high reading control reading-low reading control reading)*100

The Q value is the combination index, and the Q value is calculated using the following formula:

Q value = EA+B/(EA+EB-EA×EB), where EA+B is the inhibition rate of combined medication, and EA and EB are the inhibition rates of drug A and drug B, respectively.

Evaluation criteria for the combination index: Q value>1.15, the interaction between the two compounds is synergistic; 0.85≤Q value≤1.15, the interaction between the two compounds is additive; Q value<0.85, the interaction between the two compounds is antagonistic.

3 Experimental results

3.1 _IC50 value

Table 18. Study on the inhibitory effect of the compound of formula (A) combined with carboplatin on the proliferation of ovarian cancer (ES-2) cells

3.2 Compound Coupling Index

Table 19. Evaluation index of the combination of the compound of formula (A) and carboplatin in ES-2 ovarian cancer cells

Example 7 Pharmacodynamic study of the compound of formula (A) in combination with olaparib and carboplatin in the treatment of OVCAR-3 human ovarian cancer xenograft model

1. Study purpose: To test the inhibitory effect of the compound of formula (A) in combination with olaparib and carboplatin on the growth of tumors in the OVCAR-3 human ovarian cancer xenograft model.

2. Experimental animals: NOD SCID female mice, 6-8 weeks old, weighing 18-20 g, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd., in an SPF environment with a temperature of (23±3)℃ and a humidity of 40-70%.

3. Preparation of test samples

3.1 Solvent selection:

Solvent of compound of formula (A): PG: 50% HS-15: 0.5% HPMC=2:1:7

Olaparib solvent: 10% DMSO + 90% (10% HP-β-CD in PBS pH 7.4)

Carboplatin solvent: normal saline

3.2 Preparation method: The preparation and storage conditions of the drug are as follows:

Table 20. Drug preparation information

4. Experimental methods and procedures

4.1 Cell culture

OVCAR-3 tumor cells were cultured in RPMI1640 medium containing inactivated 20% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO ₂ incubator. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

4.2 Tumor cell inoculation and grouping

OVCAR-3 tumor cells resuspended in serum-free RPMI1640 culture medium were inoculated subcutaneously at the right flank of experimental animals at 2×10 ⁷ (0.2 mL + Matrigel), and a total of 60 animals were inoculated. When the tumor grew to about 222 mm ³ , 36 animals with relatively uniform tumor volume were selected and divided into groups for drug administration, for a total of 6 groups.

Table 21. Compound Dosage Scheme

Note: p.o. means oral administration; i.p. means intraperitoneal injection; QD x21 days means administration once a day for 21 consecutive days; QW x3 weeks means administration once a week for 3 consecutive weeks. In the group treated with the compound of formula (A) combined with olaparib and carboplatin, the compound of formula (A) was administered first, and then olaparib and carboplatin were administered 1 hour later.

4.3 Detection indicators

4.3.1 Tumor volume: The tumor volume was measured twice a week using a vernier caliper to measure the long and short diameters of the tumor. The volume was calculated using the formula: volume = 0.5 × long diameter × short diameter ² .

4.3.2 Animal response after administration: While measuring the tumor volume, weigh the mice. Record the changes in the weight of the mice and the time of administration. At the same time, the survival and health status of the mice, such as animal activity and eating during the administration period, were observed.

4.4 Drug evaluation indicators

4.4.1 Relative tumor volume increase ratio T/C (%)

T/C (%) = mean relative tumor volume of the treatment group/mean relative tumor volume of the control group × 100

4.4.2 Tumor growth inhibition rate (TGI, %)

Tumor growth inhibition rate (TGI,%) = (1-T/C) × 100

4.5 Statistical analysis

4.5.1 Data collection: Measure and observe as required by the experimental plan, and record manually or directly in a computer database.

4.5.2 Statistical analysis: SPSS 16.0 statistical software and One-Way ANOVA test were used to perform statistical analysis of tumor volume among groups, and p < 0.05 was considered to be a significant difference.

5. Results

Table 22. Tumor inhibition data of compound of formula (A) in combination with olaparib and carboplatin on OVCAR-3 ovarian cancer transplanted mice

Note: a Mean ± standard error; b Compared with the control group; c One-way ANOVA combined with Dunnett T3 test, P < 0.05 indicates significant difference

Table 23. Pharmacodynamic data of the compound of formula (A) in combination with olaparib and carboplatin on OVCAR-3 ovarian cancer transplanted mice

6. Experimental Conclusion

OVCAR-3 tumor cells were inoculated and divided into groups for drug administration 21 days after inoculation. The mean tumor volume of each group was 222 mm ³ . After 21 days of administration, the mean tumor volume of the solvent control group was 2,370 mm ³ ; the mean tumor volume of the group treated with the compound of formula (A) at a dose of 40 mg/kg alone was 1,370 mm ³ (T/C=56.1%, TGI was 43.9%, compared with the solvent control group p=0.124); the mean tumor volume of the group treated with olaparib at a dose of 50 mg/kg alone was 1,982 mm ³ (T/C=77.5%, TGI was 22.5%, compared with the solvent control group p=0.978); the mean tumor volume of the group treated with the compound of formula (A) at a dose of 40 mg/kg and olaparib at a dose of 50 mg/kg was 1,156 mm ³ (T/C=46.1%, TGI was 53.9%, compared with the solvent control group p=0. Compared with the solvent control group, p=0.057); the mean tumor volume of the carboplatin 40 mg/kg monotherapy group was 896 mm ³ (T/C=36.0%, TGI was 64.0%, compared with the solvent control group, p=0.028); the mean tumor volume of the formula (A) compound 40 mg/kg and carboplatin 40 mg/kg combined administration group was 512 mm ³ (T/C=20.3%, TGI was 79.7%, compared with the solvent control group, p=0.013).

The 40 mg/kg carboplatin monotherapy group and the 40 mg/kg carboplatin combined therapy group with the compound of formula (A) showed significant tumor growth inhibition effects. The combined group had a stronger tumor inhibition effect than each monotherapy group.

Example 8 Safety, tolerability, pharmacokinetic properties and preliminary efficacy evaluation of the compound of formula (A) tablets combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin hydrochloride liposome injection in patients with locally advanced or metastatic ovarian cancer who have failed standard treatment Study population:

Cohort A: Patients with locally advanced/metastatic ovarian cancer confirmed by cytology or histology who have failed standard treatment, or have no standard treatment options, or are not currently suitable for standard treatment.

Cohort B and C: patients with locally advanced/metastatic ovarian cancer confirmed by cytology or histology and platinum-resistant, who have failed standard treatment, have no standard treatment options, or are not currently suitable for standard treatment.

Experimental design: This study was an open, single-arm, non-randomized trial.

Table 24. Dosage regimen

Final indicator:

ORR, DCR, DOR, PFS, CA-125 response rate (GCIG standard), and pharmacokinetic parameters.

Example 9 An open, randomized, positive-controlled, phase II clinical study on the efficacy and safety of the compound of formula (A) tablets combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin hydrochloride liposome injection in the treatment of platinum-resistant ovarian cancer

Study purpose: To evaluate the clinical efficacy of the compound of formula (A) combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin liposome injection in the treatment of platinum-resistant ovarian cancer subjects; to further evaluate the clinical efficacy of the compound of formula (A) combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin liposome injection in the treatment of platinum-resistant ovarian cancer subjects; to investigate the pharmacokinetic (PK) characteristics of the compound of formula (A) combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin liposome injection in the treatment of platinum-resistant ovarian cancer subjects at steady state; to explore the pharmacokinetic (PK) characteristics of the compound of formula (A) combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin liposome injection in the treatment of platinum-resistant ovarian cancer subjects; To investigate the potential effect of combined use of niraparib tosylate capsules/paclitaxel injection/doxorubicin hydrochloride liposome injection on the PK of niraparib/paclitaxel/doxorubicin at steady state, with each group of single-drug treatment as a control; to evaluate the safety and tolerability of the compound of formula (A) combined with niraparib tosylate capsules/paclitaxel injection/doxorubicin hydrochloride liposome injection in the treatment of subjects with platinum-resistant ovarian cancer.

Experimental design: This study is an open, parallel, randomized, phase II multicenter clinical trial of safety and efficacy.

Trial Groups:

This study included 6 cohorts:

Cohort 1: Tablets of the compound of formula (A) combined with niraparib tosylate capsules;

Cohort 2: Tablets of the compound of formula (A) combined with paclitaxel injection;

Cohort 3: Tablets of the compound of formula (A) combined with doxorubicin hydrochloride liposome injection;

Cohort 4: niraparib tosylate capsules monotherapy;

Cohort 5: paclitaxel injection monotherapy;

Cohort 6: monotherapy with doxorubicin hydrochloride liposome injection.

Dosage regimen for specific drugs:

Tablets of the compound of formula (A): Specification: 100 mg, oral, once a day, dosage: 200 mg/time to 400 mg/time; continuous administration for 21 days, rest for 7 days, 28 days as a dosing cycle, treatment until intolerance or disease progression or subject withdrawal.

Niraparib Tosylate Capsules: Specification: 100 mg, Dosage: 300 mg/time, oral, once a day, with a dosing cycle of 28 days, treatment until intolerance or disease progression or subject withdrawal.

Paclitaxel injection: Specification: 30 mg (5 mL)/bottle, dosage: 80 mg/m ² /time, intravenous infusion, administered on the 1st, 8th and 15th days of each cycle, 28 days as a dosing cycle, treatment until intolerance or disease progression or subject withdrawal.

Doxorubicin hydrochloride liposome injection: Specifications: 10mL: 20mg, 40mg/ ^m2 /time, intravenous infusion, administered on the first day of each cycle, 28 days as a dosing cycle, treatment until intolerance or disease progression or subject withdrawal.

Final indicator:

ORR, DCR, DOR, PFS, CA-125 response rate (GCIG criteria), pharmacokinetic parameters, adverse events, vital signs, electrocardiogram (ECG), physical examination, and laboratory tests.

Claims (11)

Use of the compound represented by formula (I) or its pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for preventing or treating ovarian cancer, wherein the ovarian cancer is platinum-resistant ovarian cancer,
Preferably, the ovarian cancer is platinum-resistant high-grade serous carcinoma, platinum-resistant ovarian adenocarcinoma, platinum-resistant epithelial serous carcinoma, platinum-resistant clear cell carcinoma, platinum-resistant endometrioid ovarian cancer, platinum-resistant fallopian tube cancer or platinum-resistant primary peritoneal cancer; More preferably, the ovarian cancer is platinum-resistant high-grade serous carcinoma or platinum-resistant ovarian adenocarcinoma. Use of a compound represented by formula (I) or a pharmaceutically acceptable salt or a hydrate thereof in the preparation of a drug for preventing or treating ovarian cancer, wherein the ovarian cancer is locally advanced or metastatic ovarian cancer,
Preferably, the ovarian cancer is locally advanced or metastatic ovarian cancer that has failed standard treatment, or has no standard treatment options, or is not currently suitable for standard treatment; Preferably, the ovarian cancer is locally advanced or metastatic ovarian cancer that is platinum-resistant; More preferably, the ovarian cancer is locally advanced or metastatic ovarian cancer that is platinum-resistant and has failed standard treatment, or has no standard treatment options, or is not currently suitable for standard treatment. The use according to claim 1 or 2, wherein the compound of formula (I) is a compound of formula (A) or a compound of formula (B): The use according to any one of claims 1 to 3, wherein the compound of formula (A) is used in combination with other ovarian cancer therapeutic drugs; the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs. The use according to claim 4, wherein the combined administration method is selected from simultaneous, parallel, independent or sequential application; the combined administration route is selected from oral, parenteral, rectal, pulmonary or local administration. The use according to any one of claims 3 to 5, wherein the pharmaceutically acceptable salt of the compound of formula (A) is an organic acid salt or an inorganic acid salt; wherein the organic acid salt includes fumarate, methanesulfonate, hydroxyethanesulfonate, α-naphthalenesulfonate, p-toluenesulfonate, 1,2-ethanedisulfonate, oxalate, maleate, citrate, succinate, L-(+)-tartrate, hippurate, L-ascorbate, L-malate, benzoate or gentisate; the inorganic acid salt includes hydrochloride, sulfate or phosphate; preferably fumarate; more preferably monofumarate; further preferably monofumarate dihydrate; Preferably, the dosage of the compound of formula (A) is 0.1 to 1000 mg; preferably 20 to 500 mg; more preferably 200 to 400 mg; more preferably 100, 200, 300, 400 or 500 mg; further preferably 300, 400 or 500 mg; Preferably, the frequency of administration of the compound of formula (A) is once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks or once a month; preferably once a day or twice a day; Preferably, the compound of formula (A) is administered once a day, twice a day or three times a day for 21 consecutive days, followed by an interval of 1 to 2 weeks without administration of the compound; or administered for 5 or 4 consecutive days, followed by an interval of 1 to 3 days without administration of the compound; Further preferably, the administration frequency of the compound of formula (A) is once a day or twice a day, for 21 consecutive days, followed by a 1-week interval without administration of the compound; or for 5 consecutive days, followed by a 2-day interval without administration of the compound; or for 4 consecutive days, followed by a 3-day interval without administration of the compound. The use according to claim 4, characterized in that the compound of formula (A) is used in combination with a PARP inhibitor; The PARP inhibitor includes one or more of niraparib, olaparib, rucaparib, talazoparib or fluzoparib; preferably, the PARP inhibitor is niraparib or olaparib; more preferably, the PARP inhibitor is niraparib; more preferably, the PARP inhibitor is olaparib; Preferably, the PARP inhibitor is administered at a dosage of 100 to 1000 mg; preferably 100 to 600 mg; more preferably 200 mg, 400 mg, 300 mg or 600 mg; Preferably, the PARP inhibitor is administered once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks, or once a month; preferably once a day, twice a day, or three times a day; Preferably, the dosage ratio of the compound of formula (A) to the PARP inhibitor is selected from 1:0.1-300 by mass ratio; more preferably, the dosage ratio of the compound of formula (A) to niraparib is selected from 1:0.01-300 by mass ratio; more preferably 1:0.75, 1:1 or 1:0.5; more preferably, the dosage ratio of the compound of formula (A) to olaparib is selected from 1:0.01-300 by mass ratio; more preferably 1:0.75, 1:1 or 1:1.5; Preferably, the administration of the combination therapy is 28 days as a cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of the PARP inhibitor is once a day, and the dosage is 300 mg/time; More preferably, the combination is administered for a period of 28 days; the compound of formula (A) is administered for 21 consecutive days, followed by a one-week interval without administration of the compound, and the dosage is 200-400 mg/time; the niraparib is administered once a day, and the dosage is 300 mg/time; More preferably, the combination is administered in a cycle of 28 days; the compound of formula (A) is administered continuously for 21 days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the frequency of olaparib is once a day, and the dosage is 300 mg/time. The use according to claim 4, characterized in that the compound of formula (A) is used in combination with a platinum drug; the platinum drug comprises one or more of cisplatin, carboplatin, lobaplatin or nedaplatin; preferably carboplatin; Preferably, the dosage of carboplatin is 50 to 1000 mg; preferably 400 to 800 mg; more preferably 400 mg, 500 mg, 600 mg, 700 mg or 800 mg; Preferably, the administration frequency of carboplatin is once a day, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three weeks, once every four weeks, once every three weeks, divided into 5 days, or once every four weeks, divided into 5 days; More preferably, the administration of the combination therapy is 28 days as a cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound; the administration frequency of carboplatin may be once a week for 3 consecutive weeks; Preferably, in terms of mass ratio, the dosage ratio of the compound of formula (A) to carboplatin is selected from 1:0.1-300; more preferably 1:1, 1:1.5 or 1:2; further preferably 1:1. The use according to claim 4, characterized in that the compound of formula (A) is used in combination with a plant-based chemotherapeutic drug; the plant-based chemotherapeutic drug comprises one or more of vincristine, vinblastine, etoposide or paclitaxel; preferably paclitaxel; more preferably paclitaxel injection; Preferably, the dosage of paclitaxel is 1-300 mg/m ² ; preferably 10-150 mg/m ² ; more preferably 40-100 mg/m ² ; further preferably 60 mg/m ² , 70 mg/m ² , 80 mg/m ² , 90 mg/m ² or 100 mg/m ² ; further preferably 80 mg/m ² ; Preferably, the administration frequency of paclitaxel is once a day, once a week, once every two weeks, once every three weeks, once a month, once every two months, once a week, use for 2 weeks and rest for 1 week, once a week, use for 3 weeks and rest for 1 week, once a week, use for 6 weeks and rest for 2 weeks, once every 3 weeks or once every 4 weeks; preferably once a week, use for 3 weeks and rest for 1 week; Preferably, the administration of the combination therapy is 28 days as a cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of paclitaxel is 1, 8, and 15 days of each cycle, and the dosage is 80 mg/ ^m2 /time; Preferably, in terms of mass ratio, the dosage ratio of the compound of formula (A) to paclitaxel is selected from 0.01-300:1; more preferably 1:1, 1.5:1, 2:1, 3:1 or 4:1; further preferably 4:1. The use according to claim 4, characterized in that the compound of formula (A) is used in combination with an antibiotic chemotherapy drug; the antibiotic chemotherapy drug comprises one or more of actinomycin D, mitomycin, adriamycin, doxorubicin, epirubicin or daunorubicin; preferably doxorubicin; more preferably doxorubicin liposomes; Preferably, the dosage of doxorubicin is 0.1-300 mg/m ² ; preferably 1-100 mg/m ² ; more preferably 10-60 mg/m ² ; further preferably 10 mg/m ² , 20 mg/m ² , 30 mg/m ² , 40 mg/m ² or 50 mg/m ² ; further preferably 40 mg/m ² ; Preferably, the administration frequency of doxorubicin can be once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every month, or once every two months; preferably once every two weeks, once every three weeks, or once every four weeks; more preferably once every four weeks; further preferably once every four weeks, on the first day of each cycle; Preferably, the administration of the combination is 28 days as a cycle; the administration frequency of the compound of formula (A) is 21 consecutive days, followed by a 1-week interval without administration of the compound, and the dosage is 200-400 mg/time; the administration frequency of doxorubicin is on the first day of each cycle, and the dosage is 80 mg/ ^m2 /time; Preferably, the dosage ratio of the compound of formula (A) to doxorubicin is selected from 0.01-300:1 by mass ratio; preferably 3:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1 or 20:1. A pharmaceutical composition or drug combination, characterized in that it contains a compound of formula (I) or a pharmaceutically acceptable salt thereof and other ovarian cancer therapeutic drugs, and one or more pharmaceutically acceptable carriers; the other ovarian cancer therapeutic drugs include one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs, platinum drugs or alkylating agents; preferably one or more of PARP inhibitors, plant-based chemotherapy drugs, antibiotic chemotherapy drugs or platinum drugs;
Preferably, the compound of formula (I) is a compound of formula (A) or a compound of formula (B):
Preferably, the pharmaceutically acceptable salt of the compound of formula (A) is an organic acid salt or an inorganic acid salt; wherein the organic acid salt includes one or more of fumarate, methanesulfonate, hydroxyethanesulfonate, α-naphthalenesulfonate, p-toluenesulfonate, 1,2-ethanedisulfonate, oxalate, maleate, citrate, succinate, L-(+)-tartrate, hippurate, L-ascorbate, L-malate, benzoate or gentisate; the inorganic acid salt includes one or more of hydrochloride, sulfate or phosphate; preferably fumarate; more preferably monofumarate; further preferably monofumarate dihydrate.