WO2023131305A1 - Prmt5抑制剂和抗癌治疗剂的组合 - Google Patents

Prmt5抑制剂和抗癌治疗剂的组合 Download PDF

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WO2023131305A1
WO2023131305A1 PCT/CN2023/071046 CN2023071046W WO2023131305A1 WO 2023131305 A1 WO2023131305 A1 WO 2023131305A1 CN 2023071046 W CN2023071046 W CN 2023071046W WO 2023131305 A1 WO2023131305 A1 WO 2023131305A1
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cancer
compound
pharmaceutical composition
inhibitors
drug combination
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French (fr)
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周峰
杨桂梅
唐锋
薛黎婷
杨文清
陈平
唐任宏
任晋生
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江苏先声药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Patent application No. 202210270226.8 submitted to the State Intellectual Property Office of China on March 18, 2022.
  • the present disclosure relates to a pharmaceutical composition or drug combination of a PRMT5 inhibitor compound and other anticancer therapeutic agents, and a method of using the pharmaceutical composition or drug combination for preventing or treating related pathological conditions.
  • Epigenetic changes are key mediators that drive and maintain the malignant phenotype of tumors. Changes in DNA methylation, histone acetylation and methylation, non-coding RNA, and post-translational modifications are all epigenetic drivers of cancer development independent of changes in DNA sequence. Arginine methylation is an important class of post-translational modifications that affect cell growth and proliferation, apoptosis, angiogenesis and metastasis by regulating transcription and post-transcriptional RNA processing.
  • methylarginine There are three types of methylarginine, namely ⁇ -NG-monomethylarginine (MMA), ⁇ -NG, N'G-asymmetric dimethylarginine (ADMA) and ⁇ -NG, N'G-symmetric dimethylarginine (SDMA).
  • MMA ⁇ -NG-monomethylarginine
  • ADMA N'G-asymmetric dimethylarginine
  • SDMA N'G-symmetric dimethylarginine
  • PRMT protein arginine methyltransferase
  • AdoMet S-adenosylmethionine
  • PRMT5 is primarily a type II enzyme that catalyzes the symmetric dimethylation of arginine. PRMT5 was first discovered in a two-hybrid assay to detect proteins interacting with Janus tyrosine kinase (JAK2).
  • PRMT5 is a general transcriptional repressor that forms a complex with other transcription factors, including BRG1 and hBRM, Blimp1, and Snail. PRMT5 participates in a variety of different cellular biological processes through the methylation of a variety of substrates in the cytoplasm and nucleus, including histone H4 residue Arg3 (H4R3) and H3 residue Arg8 (H3R8). H4R3 methylation is associated with transcriptional repression, while H3R8 methylation is thought to be associated with both transcriptional activation and transcriptional repression.
  • H4R3 methylation is associated with transcriptional repression
  • H3R8 methylation is thought to be associated with both transcriptional activation and transcriptional repression.
  • PRMT5 In addition to the direct induction of repressive histone marks by PRMT5, the enzyme's role in gene silencing is also mediated through the formation of multiple arrestin complexes, including NuRD components, HDACs, MDB proteins, and DNA methyltransferases. PRMT5 affects its substrate specificity by interacting with some binding proteins. A central component in this protein complex is MEP50. MEP50 is required for the enzymatic activity of PRMT5. Studies have found that PRMT5 can methylate proteins involved in RNA splicing, such as SmD3, which can be used to track the biological activity of PRMT5 in cells.
  • SmD3 RNA splicing
  • PRMT5 plays an important role in tumorigenesis. Studies have found that the expression of PRMT5 is upregulated in a variety of tumors, including lymphoma, lung cancer, breast cancer and colorectal cancer. In addition, PRMT5 expression was increased in mantle cell lymphoma (MCL) patient samples, and PRMT5 knockout could inhibit the proliferation of MCL cells, indicating that PRMT5 plays an important role in MCL. PRMT5 overexpression promotes cell proliferation, and in melanoma, breast cancer, and lung cancer cell lines, PRMT5 knockdown can inhibit the proliferation of these cells. Therefore, PRMT5 is a potential target for cancer therapy.
  • MCL mantle cell lymphoma
  • MTAP methylthioadenosine phosphorylase
  • PCT/CN2021/103597 (application date June 30, 2021) describes a compound 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4 -Tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro -5H-Benzo[e][1,4]diazepine -5-keto (compound I) hydrochloride, the study found that the compound I hydrochloride has good PRMT5 enzyme inhibition, cell proliferation inhibition and cell SDMA inhibitory activity, and good pharmacokinetic properties and liver function. Cell metabolism stability, and showed significant tumor growth inhibition in mouse subcutaneous xenograft Z-138 model, and showed a good dose-response relationship.
  • PRMT5 inhibitors have promising results in cancer treatment as monotherapy, the field still needs to study the combination therapy of PRMT5 inhibitors and other anticancer therapeutic agents in order to obtain better and more effective clinical treatment drugs and programs.
  • the present disclosure provides a combined drug combination comprising a PRMT5 inhibitor and at least one anticancer therapeutic agent for treating cancer, wherein the PRMT5 inhibitor is selected from compound I and pharmaceutically acceptable salts thereof.
  • the present disclosure also provides a pharmaceutical composition for treating cancer, which includes a PRMT5 inhibitor and at least one anticancer therapeutic agent, and a pharmaceutically acceptable adjuvant, the PRMT5 inhibitor being selected from Compound I and its pharmaceutically acceptable salt.
  • the present disclosure also provides a method of treating cancer comprising administering to an individual in need of such treatment a therapeutically effective amount of a combined pharmaceutical combination or pharmaceutical composition comprising a PRMT5 inhibitor and at least one anticancer therapeutic agent , wherein the PRMT5 inhibitor is selected from compound I and pharmaceutically acceptable salts thereof.
  • the present disclosure also provides the use of a combined pharmaceutical combination or pharmaceutical composition comprising a PRMT5 inhibitor and at least one anticancer therapeutic agent in the preparation of a medicament for treating tumor diseases, wherein the PRMT5 inhibitor is selected from Compound I and its pharmaceutically acceptable salts.
  • the disclosure also provides the use of a combined pharmaceutical combination or pharmaceutical composition comprising a PRMT5 inhibitor and at least one anticancer therapeutic agent in the treatment of tumor diseases, wherein the PRMT5 inhibitor is selected from Compound I and its pharmaceutically acceptable Accepted salt.
  • the disclosure also provides a combined pharmaceutical combination or pharmaceutical composition
  • a PRMT5 inhibitor and at least one anticancer therapeutic agent for the treatment of tumor diseases, wherein the PRMT5 inhibitor is selected from Compound I and pharmaceutically acceptable salts thereof .
  • the pharmaceutically acceptable salt of Compound I of the present disclosure is selected from hydrochloride, hydrobromide, 1,5-naphthalene disulfonate, oxalate, citrate, sulfuric acid Salt, Phosphate, L-Tartrate, L-Malate, Succinate, Adipate, Fumarate, Oxalate, Propionate, Benzoate, Acetate, Formate Or L-arginine salt, preferably hydrochloride, hydrobromide or 1,5-naphthalene disulfonate, more preferably hydrochloride, even more preferably monohydrochloride.
  • cancers of the present disclosure include, but are not limited to, lymphoma, pancreatic cancer, colon cancer, malignant melanoma, ovarian cancer, lung cancer, cervical cancer, or head and neck cancer.
  • cancers of the present disclosure include, but are not limited to, lymphoma, pancreatic cancer, colon cancer, or malignant melanoma.
  • the lymphoma of the present disclosure is selected from mantle cell lymphoma.
  • the lung cancer of the present disclosure is selected from non-small cell lung cancer.
  • the head and neck cancer of the present disclosure is selected from submandibular squamous cell carcinoma.
  • the at least one anticancer therapeutic agent is selected from the group consisting of platinum derivatives, alkylating agents, topoisomerase inhibitors, paclitaxels, antitumor antibiotics, plant alkaloids, nucleoside analogs, B One or more of a cellular lymphoma 2 (BCL-2) inhibitor, a type I PRMT inhibitor, and a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, preferably selected from B-cell lymphoma 2 One or more of (BCL-2) inhibitors, type I PRMT inhibitors, and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors.
  • the at least one anti-cancer therapeutic agent is selected from B-cell lymphoma 2 (BCL-2) inhibitors, type I PRMT inhibitors, cyclin-dependent kinase 4/6 (CDK4/6 ) inhibitors, paclitaxel and platinum derivatives.
  • BCL-2 B-cell lymphoma 2
  • type I PRMT inhibitors type I PRMT inhibitors
  • CDK4/6 cyclin-dependent kinase 4/6
  • paclitaxel platinum derivatives.
  • the at least one anti-cancer therapeutic agent is selected from a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor.
  • the platinum derivative is selected from cisplatin, carboplatin, oxaliplatin and satraplatin.
  • the platinum derivative is selected from cisplatin and carboplatin.
  • the alkylating agent is selected from streptozotocin, dacarbazine, procarbazine, semustine, lomustine, formustine, nimustine, bendamol Uramustine, uramustine, cyclophosphamide, ifosfamide, melphalan, and hexamethylmelamine.
  • the topoisomerase inhibitor is selected from the group consisting of etoposide, teniposide, camptothecin, 10-hydroxycamptothecin, irinotecan and topotecan.
  • the paclitaxel is selected from docetaxel, cabazitaxel and paclitaxel.
  • the paclitaxel is selected from docetaxel and paclitaxel.
  • the antitumor antibiotic is selected from the group consisting of doxorubicin, daunorubicin, actinomycin, bleomycin, amatoxonin, elsamitrucin and mitoxantrone.
  • the plant alkaloid is selected from vinblastine, vincristine and vindesine.
  • the nucleoside analog is selected from the group consisting of cytarabine, fludarabine, iodooxyuridine, bromodeoxyuridine, fludeoxyuridine, and gemcitabine.
  • the BCL-2 inhibitor is selected from the group consisting of venetoclax (ABT-199), ABT-737, ABT-263, APG-1252, S-055746, BDA-366, HA14-1, BH3I-1, apogossypol, TW-37, TM12-06 and obatoclax, preferably ABT-199.
  • the type I PRMT inhibitor is selected from a PRMT1 inhibitor, a PRMT3 inhibitor, a PRMT4 inhibitor, a PRMT6 inhibitor and a PRMT8 inhibitor, preferably a PRMT1 inhibitor, more preferably GSK3368715.
  • the CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Ribociclib, Abemaciclib, ebvaciclib and trilaciclib, preferably Palbociclib.
  • the PRMT5 inhibitor and at least one anti-cancer therapeutic agent in the combined drug combination may be packaged separately or packaged together.
  • the PRMT5 inhibitor and at least one anti-cancer therapeutic agent in the use or treatment method are each in the form of a pharmaceutical composition, and can be administered simultaneously, sequentially or at intervals.
  • the PRMT5 inhibitor and the at least one anti-cancer therapeutic agent in the use or method of treatment are each administered at intervals.
  • the PRMT5 inhibitor and at least one anticancer therapeutic agent in the use or treatment method are administered in the same or different dosage regimens, respectively.
  • the PRMT5 inhibitor and at least one anticancer therapeutic agent in the use or treatment method are administered in different dosage regimens.
  • the PRMT5 inhibitor in the use or method of treatment may be administered at a frequency of 3 times a day (t.i.d), 2 times a day (b.i.d) or 1 time a day (q.d); 0.01 to 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight.
  • At least one anti-cancer therapeutic agent in the use or method of treatment can be administered 3 times a day (t.i.d), 2 times a day (b.i.d), 1 time a day (q.d), 1 time a week Once (q1w), once every 2 weeks (q2w), once every 3 weeks (q3w), or once every 4 weeks (q4w).
  • the PRMT5 inhibitor and at least one anti-cancer therapeutic agent have the same or different treatment cycles, for example, every 1 week, every 2 weeks, every 3 weeks or every 4 weeks is a treatment cycle.
  • a combined pharmaceutical combination or composition of the present disclosure comprising a PRMT5 inhibitor and at least one anti-cancer therapeutic agent facilitates:
  • the treated patients have a longer survival period (such as median survival period, progression-free survival period or overall survival period).
  • drug combination in combination refers to two or more active ingredients administered simultaneously or sequentially (administered in the form of their respective active ingredients themselves or their pharmaceutical compositions, or in the form of their respective pharmaceutically acceptable salts or esters and other derivatives, prodrugs or pharmaceutical compositions thereof).
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts of alkali ions with free acids or salts of acid ions with free bases for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids can be mentioned salts formed with organic acids, salts formed with basic or acidic amino acids, and the like.
  • composition refers to a mixture of the active ingredients described in the present disclosure and pharmaceutically acceptable excipients, which can be prepared by combining the active ingredients described in the present disclosure with pharmaceutically acceptable excipients.
  • compositions of the present disclosure can be administered in various suitable routes, or the components in the combined pharmaceutical combination can be administered independently in various suitable routes, typical routes include but are not limited to: oral, rectal, topical, inhalation, Parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, subcutaneous, subcapsular, subarachnoid, intravenous, intraarterial, intrathecal, intralymphatic, intralesional, intracystic , intraorbital, intracardiac, intradermal, intraarticular, intraspinal, transtracheal, epidural, and intrasternal.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious stimulating effect on the organism and will not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • the term "individual" is a mammal. In some embodiments, the individual is a mouse. In some embodiments, the individual is a human.
  • the components in the combined drug combination in the present disclosure can each independently exist in the form of a pharmaceutical composition.
  • the components in the combined drug combination in the present disclosure may be each independently, or some or all of them together are suitable dosage forms, including but not limited to tablets, pills, lozenges, dragees, capsules, liquids, Oral forms such as gels, slurries, and suspensions, or parenteral forms such as sterile solutions, suspensions, or lyophilized products.
  • treatment generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect partially or completely stabilizes or cures the disease and/or side effects due to the disease and may be therapeutic.
  • Treatment in this disclosure encompasses any treatment of a disease in a patient, including: (a) inhibiting the symptoms of the disease, ie arresting its development; or (b) relieving the symptoms of the disease, ie causing regression of the disease or symptoms.
  • the term "effective amount” means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying the The amount of a compound of the application for the onset of one or more symptoms of a particular disease, condition or disorder described in .
  • the amount of a compound of the present disclosure that constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art according to its own knowledge and this disclosure.
  • administering means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art.
  • the compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include, but are not limited to, the examples in this disclosure.
  • Figure 1 is a ball-and-stick diagram of compound 1-8 single crystal.
  • Fig. 2 is the tumor growth curve of the mice receiving the test compound in the Z-138 subcutaneous tumor model, and the compound 002 in the figure refers to the compound of Example 2.
  • Fig. 3 is the body weight change curve of the mice receiving the test compound in the Z-138 subcutaneous tumor model, and compound 002 in the figure refers to the compound of Example 2.
  • Fig. 4A is a matrix diagram of the synergistic effect of the compound of Example 4 combined with the combined drug GSK3368715 on HCT-116 cell proliferation inhibition.
  • Fig. 4B is a matrix diagram of the synergistic effect of the combination of the compound of Example 4 and the combined drug GSK3368715 on MIA PaCa-2 cell proliferation inhibition.
  • Fig. 5 is a matrix diagram of the synergistic effect of the combination of the compound of Example 4 and the combined drug ABT199 on Z138 cell proliferation inhibition.
  • Fig. 6 is a matrix diagram of the synergistic effect of the combination of the compound of Example 4 and the combined drug Palbociclib on the inhibition of A375 cell proliferation.
  • Fig. 7 is a matrix diagram of the synergistic effect of the combination of the compound of Example 4 and the combined drug Palbociclib on the inhibition of A2780 cell proliferation.
  • Fig. 8A is a matrix diagram of the synergistic effect of the compound of Example 4 combined with the combined drug docetaxel on the inhibition of A2780 cell proliferation.
  • Fig. 8B is a matrix diagram of the synergistic effect of the compound of Example 4 combined with the combined drug docetaxel on the inhibition of OVCAR3 cell proliferation.
  • Fig. 9 is a matrix diagram of the synergistic effect of the combination of the compound of Example 4 and the combined drug docetaxel on the inhibition of A549 cell proliferation.
  • Figure 10A is a matrix diagram of the synergistic effect of the compound of Example 4 combined with paclitaxel on the inhibition of A2780 cell proliferation.
  • Figure 10B is a matrix diagram of the synergistic effect of the compound of Example 4 combined with paclitaxel on the inhibition of OVCAR3 cell proliferation.
  • Figure 11A is a matrix diagram of the synergistic effect of the compound of Example 4 combined with paclitaxel on Hela cell proliferation inhibition.
  • Figure 11B is a matrix diagram of the synergistic effect of the compound of Example 4 combined with paclitaxel on the inhibition of A253 cell proliferation.
  • Example 12 is a matrix diagram of the synergistic effect of the compound of Example 4 combined with carboplatin on A253 cell proliferation inhibition.
  • Fig. 13 is a matrix diagram of the synergistic effect of the compound of Example 4 combined with the combined drug cisplatin on Hela cell proliferation inhibition.
  • Fig. 14 is a graph showing the relative tumor volume and body weight change rate (%) of each group in the A375 xenograft tumor model.
  • NMR nuclear magnetic resonance
  • MS mass spectroscopy
  • the eluent in the embodiment can form a mixed eluent from two or more solvents, and the ratio is the volume ratio of each solvent.
  • “0-10% methanol/dichloromethane” means that during the gradient elution process, the volume ratio of methanol and dichloromethane in the mixed eluent is 0:100-10:100.
  • Reagents used in this disclosure are commercially available. All solvents used in this disclosure were commercially available and used without further purification.
  • ratios indicated for mixed solvents are volume mixing ratios.
  • % refers to mass percent wt%.
  • reaction solution was reacted at room temperature for 4 hours. After the reaction was complete, the excess N,N-diisopropylethylamine and N,N-dimethylformamide were removed by rotary evaporation, then cooled in an ice bath, diluted with saturated brine (1L), extracted with ethyl acetate (200mL X 2), the combined organic phases were washed with 5% aqueous sodium carbonate solution (500mL X 2), and then washed with saturated brine (500mL).
  • the reaction system was cooled to room temperature, and Boc anhydride (122 mg, 0.56 mmol) was added to react for 1.5 hours.
  • the reaction system was cooled to 0°C, and the pH of the reaction solution was adjusted to 5.0 with 1mol/L hydrochloric acid aqueous solution, and then extracted with ethyl acetate (30mL X 3), the organic phase was dried with anhydrous sodium sulfate, filtered, The filtrate was concentrated under reduced pressure.
  • Single crystal preparation method Weigh compound 1-8 (10.0 mg) into a 3 mL screw-top glass bottle, add 2 mL of methanol, stir for 5 minutes, and then the solid dissolves. Add 0.5 mL of water to the glass bottle and continue stirring for 5 minutes. The solution was filtered through a 0.22 ⁇ m microporous membrane into a 3 mL screw-top glass bottle, and the mouth of the glass bottle was covered with plastic wrap. Prick 8 small holes at the mouth of the bottle with a needle and leave it at room temperature for 7 days to obtain a single crystal of the above compound.
  • the obtained single crystal sample was subjected to X-ray analysis, and the test results are shown in Table 1 and Fig. 1 .
  • Chromatographic information Chromatographic column - Shim-pack IC-A3; guard column - Shim-pack IC-GA3; eluent - 8mmol/L p-hydroxybenzoic acid, 3.2mmol/L Bis-Tris, 50mmol/L boric acid Solution; flow rate - 1.5mL/min; column temperature - 40°C; injection volume - 50 ⁇ L; conductivity detector mode - positive ion mode;
  • Reference solution (containing 6 ⁇ g/mL of chloride ion): Accurately measure 50 mg of sodium chloride into a 50 mL measuring bottle, add water to dissolve and dilute to the mark, shake well, precisely pipette 1.0 mL into a 100 mL measuring bottle, add water to dilute to the mark , shake well.
  • Test solution (0.1mg/mL): Accurately weigh 10mg of the sample to be tested into a 100mL measuring bottle, dissolve and dilute to the mark with water, and shake well.
  • Determination method Accurately measure the reference substance solution and the test solution respectively, inject them into a high-performance liquid chromatograph, use the eluent as the eluent to elute, and record the chromatogram.
  • the experimental instruments, reagents and methods for determining the content of hydrochloric acid by potentiometric titration are as follows.
  • Blank titration Measure 50 mL of solvent (60% methanol) into a 100 mL beaker, and titrate to the end point with sodium hydroxide titration solution (0.1 mol/L).
  • Titration of the sample to be tested Accurately weigh 300 mg of the sample to be tested, place it in a 100 mL beaker, add 50 mL of solvent (60% methanol), and titrate to the end point with sodium hydroxide titration solution (0.1 mol/L).
  • the hydrochloric acid content is calculated by the following formula:
  • V 0 blank consumes the volume of sodium hydroxide titration solution, unit (mL);
  • V the volume of sodium hydroxide titration solution consumed by the sample to be tested, unit (mL);
  • m the weighing amount of the sample to be tested, unit (mg);
  • PRMT5/MEP50 protein was purchased from BPS bioscience (USA); histone H4 peptide (Histone H4 Peptide) substrate was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; Anti-Histone H4 (symmetric dimethyl R3) antibody- ChIP Grade was purchased from Abcam (US); S-(5'-adenosyl)-L-methionine chloride dihydrochloride was purchased from Sigma (US); 384-well plate, AlphaScreen streptavidin AlphaScreen Streptavidin Donor beads, AlphaScreen Protein A Acceptor beads and Envision 2104 multi-label Reader were purchased from PerkinElmer Instruments Co., Ltd. ( U.S.); Echo 550 pipette (Echo 550 Liquid Handler) was purchased from Labcyte (U.S.).
  • Detection of enzymatic activity the compound was injected into a 384-well plate by Echo, so that the final concentration was 0-1000 nM (initial concentration 1000 nM, 3-fold dilution, 10 points), and the DMSO content was 0.5%.
  • Prepare 6X detection reagent containing AlphaScreen Protein A Acceptor beads and Anti-Histone H4 (symmetric dimethyl R3) antibody add 5 ⁇ L to each well, and incubate at room temperature for 60 minutes.
  • Prepare 6X detection reagent containing AlphaScreen Streptavidin Donor beads add 5 ⁇ L to each well, and incubate at room temperature for 60 minutes.
  • Envision detection signal value The test results are shown in Table 2.
  • Test Test Example 2 Experiment of Inhibitory Activity of Compounds on Tumor Cell Proliferation
  • Z-138 cells were purchased from ATCC (US); IMDM medium and penicillin-streptomycin were purchased from Sigma (US); horse serum was purchased from Hyclone (US); 96-well plates were purchased from Corning ( U.S.); Cell-Titer Glo reagent was purchased from Promega (U.S.).
  • Cell culture Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO 2 . Cells in the logarithmic growth phase can be used for experiments.
  • Cell-Titer Glo reagent was used to detect the proliferation inhibitory activity of the compound on Z-138 cells. Adjust the cell concentration, inoculate 96-well plates with 180 ⁇ L per well (500/well), and place them at 37° C. and 5% CO 2 to equilibrate for 10-15 minutes. 20 ⁇ L of compound-containing culture solution was added to each well to make the final concentration 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content was 0.1%. Cell plates were incubated at 37°C, 5% CO 2 for 8 days.
  • the medium was changed on the fourth day: 100 ⁇ L of the supernatant was slowly aspirated, and 100 ⁇ L of fresh culture medium containing the compound was added to keep the concentration of the compound unchanged. Cell viability was detected by Cell-Titer Glo reagent. The test results are shown in Table 2.
  • Test Test Example 3 Compound's inhibitory activity experiment on SDMA
  • Z-138 cells were purchased from ATCC (US); IMDM medium and penicillin-streptomycin were purchased from Sigma (US); horse serum was purchased from Hyclone (US); Hoechst antibody was purchased from Invitrogen (US) ); Alexa Fluor 488 goat anti-rabbit IgG antibody was purchased from Invitrogen (U.S.); Anti-dimethyl-Arginine symmetric (SYM11) antibody was purchased from Merck (U.S.); DPBS was purchased from Gibco (U.S.); From Cell signaling technology company (USA); paraformaldehyde was purchased from Beijing Suo Laibao Technology Co., Ltd.; 384-well plate and Echo 550 Liquid Handler were purchased from Labcyte company (USA); ImageXpress Nano was purchased from Molecular Devices company (USA).
  • Cell culture Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO 2 . Cells in the logarithmic growth phase can be used for experiments.
  • Immunofluorescence detection Immunofluorescence was used to detect the effect of compounds on SDMA in Z-138 cells. The cell concentration was adjusted to 1*10 5 /mL, 40 ⁇ L per well was inoculated into a 384-well plate (4000/well), and placed at 37° C., 5% CO 2 to equilibrate for 10-15 minutes. The compound was injected into a 384-well plate by Echo, so that the final concentration was 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content was 0.1%. Cell plates were incubated at 37°C, 5% CO 2 for 2 days.
  • CB17-SCID mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; DMSO, HP- ⁇ -CD (hydroxypropyl- ⁇ -cyclodextrin), MC (methylcellulose), and acetonitrile were purchased from From Merck (USA), K 2 EDTA anticoagulant tube was purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.
  • mice 6 female CB17-SCID mice (20-30g, 4-6 weeks) were randomly divided into 2 groups, 3 mice in each group.
  • Group 1 was administered the compound by tail vein injection, the dose was 2mg/kg, and the vehicle was 5% DMSO+95% 10% HP- ⁇ -CD aqueous solution, and the second group was orally administered the compound, the dose was 10 mg/kg, and the vehicle was 0.5% MC aqueous solution.
  • Animals were fed and watered normally before the experiment.
  • Venous blood was collected from mice in each group before administration and at 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The collected whole blood samples were placed in K 2 EDTA anticoagulant tubes, centrifuged for 5 minutes (4000 rpm, 4° C.) and the plasma was collected for testing.
  • mice plasma sample Take 10 ⁇ L of mouse plasma sample, add 150 ⁇ L of acetonitrile solvent (which contains internal standard compound) to precipitate protein, vortex for 0.5 min, centrifuge (4700 rpm, 4 ° C) for 15 min, and supernatant with 0.05% (v/v) formic acid Dilute 2 times with water, inject 3 ⁇ L into LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection.
  • LC-MS/MS system ABS Sciex Triple Quad 6500+
  • human hepatocytes were purchased from Biopredic Company; mouse hepatocytes were purchased from BioIVT Company; acetonitrile and methanol were purchased from Merck Company; AOPI stain was purchased from Nexcelom Company; dexamethasone was purchased from NIFDC Company; Bao Technology Co., Ltd.; DPBS (10x), GlutaMAX TM -1 (100x) and human recombinant insulin were purchased from Gibco by Life Technologies; fetal bovine serum was purchased from Corning; formic acid was purchased from DIKMAPURE; Isotonic Percoll was purchased from GE Healthcare; Alprazolam was purchased from Supelco; caffeine was purchased from ChromaDex.inc; HEPES, tolbutamide and Williams' Medium E were purchased from Sigma.
  • the specific preparation information of the liver cell resuscitation solution is shown in Table 4 below.
  • the hepatocytes were quickly placed in a 37°C water bath and shaken until all ice crystals were dispersed, sprayed with 70% ethanol and transferred to a biological safety cabinet.
  • the contents of the hepatocyte tubules were poured into a centrifuge tube containing 50 mL of resuscitation medium, which was centrifuged at 100 g for 10 minutes. After centrifugation, aspirate the recovery medium and add enough incubation medium to obtain a cell suspension with a cell density of about 1.5 ⁇ 10 6 cells/mL.
  • Use Cellometer Vision to count liver cells and determine the density of live cells. The survival rate of liver cells must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5 ⁇ 106 viable cells/mL.
  • Z138 cells were purchased from ATCC; IMDM medium, penicillin and streptomycin and 0.25% trypsin-EDTA were purchased from Gibco; horse serum and PBS were purchased from Hyclone; Matrigel was purchased from Corning.
  • mice Female, 6-7 weeks old, weighing about 14-20 grams, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were raised in an SPF-grade environment, and each cage was individually All animals had free access to a standard certified commercial laboratory diet and water ad libitum.
  • PBS phosphate-buffered saline without calcium and magnesium ions
  • Matrigel Matrigel
  • the dosage of the compound of Example 2 is 1.5mg/kg, 5mg/kg or 15mg/kg, PO, administered twice a day (BID) x 3 weeks. 6 mice per group.
  • Tumor diameters were measured twice a week with vernier calipers.
  • Mouse body weights were measured twice a week.
  • the antitumor efficacy of compounds was evaluated by tumor growth inhibition rate TGI (%).
  • TGI (%) [(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment of the solvent control group-at the beginning of treatment of the solvent control group Mean tumor volume)] x 100%.
  • the compound of Example 2 of the present disclosure had a significant inhibitory effect on tumor growth when administered twice a day at 1.5 mg/kg, 5 mg/kg and 15 mg/kg, and showed a better Dose-response relationship, administration of 15mg/kg twice a day has the effect of shrinking tumors.
  • the compound of Example 2 did not significantly affect the body weight of the mice at the dose tried in this pharmacodynamic experiment, nor did it cause any death of the mice, and the mice could tolerate it.
  • Test Test Example 7 Effects of Compounds of the Disclosure Combined with Type I PRMT Inhibitor GSK3368715 on Cell Proliferation Inhibition
  • the plasticware and consumables required for this experiment include: cell culture medium DMEM (Gibco #21068028); McCoy's 5A medium (Gibco #16600108); fetal bovine serum (FBS) (Gibco #10099-141C); PBS phosphate Buffer (Hyclone #SH30256.01); 100% DMSO (Sigma #D2650); 96-well clear bottom sterile culture plate (Corning #3599), 1.5 mL opaque brown Eppendorf tubes; Type I PRMT inhibitor GSK3368715 (Selleckchem #S8858) 0.25% Trypsin (Gibco #25200-072); Horse Serum (Gibco #16050-130); Luminescent Cell Viability Assay Kit (CTG) (Promega, #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips Head (Axy
  • the equipment required for this experiment includes: Eppendorf pipette, Eppendorf pipette gun, constant temperature carbon dioxide incubator (ThermoFisher), FlexStation 3 multifunctional microplate reader (Molecular Devices).
  • the cells needed for this experiment include: MIA PaCa-2 (ATCC#CRM-CRL-142), the medium is McCoy's 5A medium containing 10% FBS; HCT116 (ATCC#CCL-247), the medium is containing 10% FBS, DMEM medium with 2.5% horse serum.
  • the compound of Example 4 was diluted to 40 ⁇ M with DMSO, and GSK3368715 was diluted to 100 ⁇ M; the diluted compound was diluted 40 times with the medium corresponding to HCT 116 or MIA PaCa-2, and the concentrations were 1 ⁇ M and 2.5 ⁇ M respectively; This is the initial concentration.
  • the prepared 2.5% DMSO culture solution was used for 2-fold serial dilution.
  • the compound of Example 4 was serially diluted to 7 concentration gradients, and GSK3368715 was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 was transferred in an orthogonal manner to the culture plates of HCT116 and MIA PaCa-2 cells plated the day before.
  • the positive control group is the medium wells containing 0.25% DMSO without cells; the negative control group is the wells treated with 0.25% DMSO with cells.
  • the cell plate was placed in a 37°C, CO 2 cell incubator, and the cells were treated with drugs and cultured for 7 days. Take out the drug-treated cell plate, add 100 ⁇ L of CTG reagent to each well, and shake at room temperature for 0.5 hours in the dark.
  • the model assumes probabilistic independence of drugs.
  • the theoretical curve for the expected effect of the combination is calculated from the following equation:
  • Effect A and Effect B are the effects of individual drugs A and B at a specific concentration x.
  • Test Test Example 8 Effects of Compounds of the Disclosure Combined with BCL-2 Inhibitor ABT199 on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium IMDM (Gibco#12440-053); PBS phosphate buffered saline (Hyclone#SH30256.01); horse serum (Hyclone#SH30074.03); 100% DMSO (Sigma #D2650); ABT-199 (MCE #HY-15531); 96-well bottom-through sterile culture plate (Costar #3610), 1.5 mL opaque brown Eppendorf tube; Luminescent Cell Viability Assay Kit (CTG) (Promega, #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips Head (Axygen).
  • CCG Luminescent Cell Viability Assay Kit
  • the equipment required for this experiment includes: Eppendorf pipette, Eppendorf pipette gun, constant temperature carbon dioxide incubator (ThermoFisher), FlexStation 3 multifunctional microplate reader (Molecular Devices), cell counter (CountStar).
  • the cells required in this experiment include: Z138 (ATCC#CRL-3001), and the medium is IMDM containing 10% horse serum.
  • the positive control group is the medium wells containing 0.5% DMSO without cells; the negative control group is the wells treated with 0.5% DMSO with cells.
  • Test Test Example 9 Effects of Compounds of the Disclosure Combined with CDK4/6 Inhibitor Palbociclib on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium DMEM (Gibco #21068028); fetal bovine serum (FBS) (Gibco #10099-141C); PBS phosphate buffer (Hyclone #SH30256.01); 100 % DMSO (Sigma #D2650); 96 well bottomed sterile culture plates (Corning #3599), 1.5 mL opaque brown Eppendorf tubes; Palbociclib (Selleckchem #S1116); 0.25% trypsin (Gibco #25200-072); Luminescent Cell Viability Assay Kit (CTG) (Promega #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips (Axygen).
  • CCG Luminescent Cell Viability Assay Kit
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • the medium is DMEM medium containing 10% FBS.
  • Digest A375 cells in a cell culture flask (with 0.25% trypsin), resuspend with the corresponding fresh medium, adjust the corresponding cell density to 300 cells/80 ⁇ L/well after counting, plate into a 96-well plate, put Incubate overnight at 37°C in a CO 2 cell incubator.
  • the compound of Example 4 was diluted to 200 ⁇ M and Palbociclib was diluted to 1000 ⁇ M with DMSO; the diluted compound was diluted 40 times with DMEM medium containing 10% FBS, and the concentrations were 5 ⁇ M and 25 ⁇ M; Concentration, the prepared 2.5% DMSO culture solution was used for 2-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and Palbociclib was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and Palbociclib were respectively transferred to the culture plate of A375 cells plated the day before in an orthogonal manner.
  • the positive control group is the medium wells containing 0.25% DMSO without cells; the negative control group is the wells treated with 0.25% DMSO with cells.
  • Test Test Example 10 Effects of Compounds of the Disclosure Combined with CDK4/6 Inhibitor Palbociclib on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium RPMI-1640 (Gibco#A10491-01); fetal bovine serum (FBS) (Gibco#10099-141C); PBS phosphate buffered saline (Hyclone#SH30256.
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • Cells needed in this experiment are RPMI-1640 medium containing 10% FBS.
  • A2780 cells were digested in cell culture flasks (with 0.25% trypsin), resuspended with the corresponding fresh medium, adjusted the cell density to 600 cells/80 ⁇ L/well after counting, plated into 96-well plates, and placed in 37 °C, CO 2 cell incubator, cultivate overnight.
  • the compound of Example 4 was diluted to 100 ⁇ M with DMSO, and Palbociclib was diluted to 2 mM; the diluted compound was diluted 100 times with the RPMI-1640 medium containing 10% FBS, and its concentration was 1 ⁇ M and 20 ⁇ M respectively; For the initial concentration, the prepared 1% DMSO culture solution was used for 2-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and Palbociclib was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and Palbociclib were transferred in an orthogonal manner to the corresponding cell culture plates plated the day before.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • Test Test Example 11 Effects of Compounds of the Disclosure Combined with Docetaxel on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium RPMI-1640 (Gibco#A10491-01); fetal bovine serum (FBS) (Gibco#10099-141C); PBS phosphate buffered saline (Hyclone#SH30256.
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • OVCAR3 ATCC#HTB-161
  • culture medium is RPMI-1640 medium containing 20% FBS, 0.01mg/ml bovine insulin (Yeasen#40107ES60); A2780 (Cobioer#CBP60283), culture The base is RPMI-1640 medium containing 10% FBS.
  • Digest OVCAR3 and A2780 cells in cell culture flasks (with 0.25% trypsin), resuspend with the corresponding fresh medium, adjust the corresponding cell density to 1500 and 600 cells/80 ⁇ L/well after counting, and spread to 96-well plates Inside, put into 37°C, CO 2 cell incubator, culture overnight.
  • the compound of Example 4 was diluted to 40 ⁇ M with DMSO, and the docetaxel was diluted to 4 ⁇ M; the diluted compound was diluted 40 times with RPMI-1640 medium containing 10% FBS, and the concentrations were 100 nM and 10 nM respectively; Using this as the initial concentration, the prepared 2.5% DMSO culture solution was used for 2-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and docetaxel was serially diluted to 4 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and docetaxel were transferred in an orthogonal manner to the corresponding cell culture plates plated the day before.
  • the positive control group is the medium wells containing 0.5% DMSO without cells; the negative control group is the wells treated with 0.5% DMSO with cells.
  • Test Test Example 12 Effects of Compounds of the Disclosure Combined with Docetaxel on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium DMEM (Gibco #21068028); fetal bovine serum (FBS) (Gibco #10099-141C); PBS phosphate buffer (Hyclone #SH30256.01); 100 % DMSO (Sigma #D2650); 96 well bottomed sterile culture plates (Corning #3610), 1.5 mL opaque brown Eppendorf tubes; Docetaxel (MCE #HY-B0011); 0.25% trypsin (Gibco #25200-072); Luminescent Cell Viability Assay Kit (CTG) (Promega #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips (Axygen).
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • PBS phosphate buffer Hyclone #SH
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • A549 (ATCC#CCL-185), the medium is DMEM medium containing 10% FBS.
  • A549 cells were digested in a cell culture flask (with 0.25% trypsin), resuspended with the corresponding fresh medium, adjusted the cell density to 200 cells/80 ⁇ L/well after counting, plated into a 96-well plate, and placed in 37 °C, CO 2 cell incubator, cultivate overnight.
  • the compound of Example 4 was diluted to 50 ⁇ M with DMSO, and Docetaxel was diluted to 20 ⁇ M; the diluted compound was diluted 100 times with DMEM medium containing 10% FBS, and its concentration was 0.5 ⁇ M and 0.2 ⁇ M respectively; Initial concentration, carry out 2-fold serial dilution (compound of embodiment 4) and 3-fold serial dilution (Docetaxel) with prepared 1% DMSO culture fluid, embodiment 4 serial dilution 7 concentration gradients, Docetaxel serial dilution 6 concentrations gradient. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and Docetaxel were transferred in an orthogonal manner to the corresponding cell culture plate plated the day before.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • Test Test Example 13 Effect of Compounds of the Disclosure Combined with Paclitaxel on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium RPMI-1640 (Gibco#A10491-01); fetal bovine serum (FBS) (Gibco#10099-141C); PBS phosphate buffered saline (Hyclone#SH30256.
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • OVCAR3 ATCC#HTB-161
  • culture medium is RPMI-1640 medium containing 20% FBS, 0.01mg/ml bovine insulin (Yeasen#40107ES60); A2780 (Cobioer#CBP60283), culture The base is RPMI-1640 medium containing 10% FBS;
  • Digest OVCAR3 and A2780 cells in cell culture flasks (with 0.25% trypsin), resuspend with the corresponding fresh medium, adjust the corresponding cell density to 1500 and 600 cells/80 ⁇ L/well after counting, and plate to 96 wells Plates were placed in a 37°C, CO 2 cell incubator and incubated overnight.
  • the compound of Example 4 was diluted to 100 ⁇ M with DMSO, and paclitaxel was diluted to 50 ⁇ M; the diluted compound was diluted 100 times with RPMI-1640 medium containing 10% FBS, and the concentrations were 1 ⁇ M and 0.5 ⁇ M respectively; As the initial concentration, carry out 2 times (embodiment 4 compound) gradient dilution and 3 times (paclitaxel) gradient dilution with the prepared 1% DMSO culture fluid, embodiment 4 compound serial dilution 7 concentration gradients, paclitaxel serial dilution 6 Concentration gradient.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • the cell plate was placed in a 37°C, CO 2 cell incubator, and the cells were treated with drugs and cultured for 7 days. Take out the drug-treated cell plate, add 100 ⁇ L of CTG reagent to each well, and shake at room temperature for 0.5 hours in the dark. The luminescence value was read with an Envision multifunctional microplate reader.
  • Test Test Example 14 Effect of Compounds of the Disclosure Combined with Paclitaxel on Cell Proliferation Inhibition
  • the plasticware and consumables required for this experiment include: cell culture medium McCoy's 5a (ATCC#30-2007); cell culture medium Eagle's Minimum Essential Medium (ATCC#30-2003); fetal bovine serum (FBS) (Gibco#10099 -141C); PBS phosphate buffered saline (Hyclone#SH30256.01); 100% DMSO (Sigma#D2650); 96 well bottomed sterile culture plates (Corning#3610), 1.5mL opaque brown Eppendorf tubes; paclitaxel (Selleck# S1150); 0.25% trypsin (Gibco #25200-072); Luminescent Cell Viability Assay Kit (CTG) (Promega #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips (Axygen).
  • CCG Luminescent Cell Viability Assay Kit
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • HeLa ATCC#HTB-41
  • the medium is Eagle's Minimum Essential Medium medium containing 10% FBS
  • A253 ATCC#HTB-41
  • the medium is McCoy's 5a containing 10% FBS Medium.
  • the compound of Example 4 was diluted to 25mM with DMSO, paclitaxel was diluted to 20 ⁇ M; the diluted compound was diluted 100 times with the medium containing 10% FBS, and its concentration was 250 ⁇ M and 200nM respectively; this was used as the starting concentration respectively , with the prepared 1% DMSO culture solution for 2-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and paclitaxel was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and paclitaxel were respectively transferred to the corresponding HeLa cell culture plate plated the day before in an orthogonal manner.
  • the compound of Example 4 was diluted to 5mM with DMSO, paclitaxel was diluted to 200 ⁇ M; the diluted compound was diluted 100 times with the medium containing 10% FBS, and its concentration was 50 ⁇ M and 2000nM respectively; this was used as the starting concentration respectively , with the prepared 1% DMSO culture solution for 3-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and paclitaxel was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and paclitaxel were transferred in an orthogonal manner to the corresponding A253 cell culture plates plated the day before.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • Test Test Example 15 Effects of Compounds of the Disclosure Combined with Carboplatin on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium McCoy's 5a (ATCC#30-2007); fetal bovine serum (FBS) (Gibco#10099-141C); PBS phosphate buffered saline (Hyclone#SH30256.01 ); 100% DMSO (Sigma #D2650); 96-well clear-bottom sterile culture plates (Corning #3610), 1.5 mL opaque brown Eppendorf tubes; carboplatin (MCE #HY-17393); 0.25% trypsin (Gibco #25200- 072); Luminescent Cell Viability Assay Kit (CTG) (Promega #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips (Axygen).
  • CCG Luminescent Cell Viability Assay Kit
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • Digest A253 cells in a cell culture flask (with 0.25% trypsin), resuspend with the corresponding fresh medium, adjust the corresponding cell density to 800 cells/80 ⁇ L/well after counting, plate into a 96-well plate, put Incubate overnight at 37°C in a CO 2 cell incubator.
  • the compound of Example 4 was diluted to 5 mM with DMSO, and Carboplatin was diluted to 1 mM with the cell culture medium containing 1% DMSO; the diluted compound of Example 4 was diluted 100 times with the McCoy's 5a medium containing 10% FBS, and its The concentration was 50 ⁇ M; using this as the initial concentration, the prepared 1% DMSO culture solution was used for 3-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and carboplatin was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and carboplatin were transferred in an orthogonal manner to the corresponding cell culture plates plated the day before.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • Test Test Example 16 Effect of Compounds of the Disclosure Combined with Cisplatin on Cell Proliferation Inhibition
  • the plastic utensils and consumables required for this experiment include: cell culture medium Eagle's Minimum Essential Medium (ATCC#30-2003); fetal bovine serum (FBS) (Gibco#10099-141C); PBS phosphate buffered saline (Hyclone#SH30256 .01); 100% DMSO (Sigma #D2650); 96-well bottomed sterile culture plates (Corning #3610), 1.5 mL opaque brown Eppendorf tubes; cisplatin (MCE #HY-17394); 0.25% trypsin (Gibco # 25200-072); Luminescent Cell Viability Assay Kit (CTG) (Promega #G7573), 25mL Serological Pipette Tips (JET), 5mL Serological Pipette Tips (JET), P1000 Pipette Tips and P200 Pipette Tips (Axygen).
  • CCG Luminescent Cell Viability Assay Kit
  • CCG Luminescent Cell Viability Assay Kit
  • the equipment required for this experiment includes: Eppendorf pipette; Eppendorf pipette gun; constant temperature carbon dioxide incubator (ThermoFisher); Envision multi-functional microplate reader (Perkin Elmer).
  • HeLa ATCC#HTB-41
  • the medium is Eagle's Minimum Essential Medium medium containing 10% FBS.
  • the compound of Example 4 was diluted to 25 mM with DMSO, and the cisplatin was diluted to 200 ⁇ M with the cell culture medium containing 1% DMSO; the diluted compound of Example 4 was diluted 100 times with the medium containing 10% FBS, and its concentration was 250 ⁇ M; using this as the initial concentration, the prepared 1% DMSO culture solution was used for 2-fold serial dilution, the compound of Example 4 was serially diluted to 7 concentration gradients, and cisplatin was serially diluted to 6 concentration gradients. Finally, 10 ⁇ l of the serially diluted compound of Example 4 and cisplatin were transferred in an orthogonal manner to the corresponding cell culture plates plated the day before.
  • the positive control group is the medium wells containing 0.2% DMSO without cells; the negative control group is the wells treated with 0.2% DMSO with cells.
  • Test Example 17 The efficacy of the disclosed compound combined with CDK4/6 inhibitor Palbociclib in A375 xenograft tumor model
  • Tumor diameters were measured twice a week with vernier calipers.
  • V tumor volume
  • RTV relative tumor volume
  • mice were weighed twice a week.
  • Example 4 of the present disclosure can significantly inhibit tumor growth, and the tumor volume after combined with Palbociclib is significantly smaller than that of the single drug group.
  • the mice were in good condition throughout the experiment, and there was no significant change in body weight.
  • This experiment shows that Example 4 combined with Palbociclib has a synergistic anti-tumor effect on human melanoma A375 cells, and has good safety.

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Abstract

本发明提供了选自化合物I及其药学上可接受的盐的PRMT5抑制剂化合物与至少一种其他抗癌治疗剂的药物组合物或联用药物组合，以及所述PRMT5抑制剂化合物与至少一种其他抗癌治疗剂在制备用于治疗肿瘤疾病的药物中的用途。

Description

PRMT5抑制剂和抗癌治疗剂的组合
相关申请的交叉引用
本申请要求以下2件中国发明专利申请的权益和优先权，在此将其全部内容以援引的方式整体并入本文中：
2022年1月6日向中国国家知识产权局提交的第202210013989.4号专利申请，和
2022年3月18日向中国国家知识产权局提交的第202210270226.8号专利申请。
技术领域
本公开内容涉及PRMT5抑制剂化合物与其他抗癌治疗剂的药物组合物或联用药物组合，以及使用所述药物组合物或联用药物组合用于预防或治疗相关病理学病症的方法。
背景技术
表观遗传学改变是驱动和维持肿瘤恶性化表型的关键介质。DNA甲基化、组蛋白乙酰化和甲基化、非编码RNA、翻译后修饰的变化都是癌症发生的表观遗传驱动力，而与DNA序列的变化无关。精氨酸甲基化是一类重要的翻译后修饰，通过调节转录和转录后RNA处理影响细胞生长和增殖，凋亡，血管生成和转移。存在三种类型的甲基精氨酸，即ω-NG-单甲基精氨酸(MMA)、ω-NG，N’G-不对称二甲基精氨酸(ADMA)和ω-NG，N’G-对称二甲基精氨酸(SDMA)。这种修饰是由蛋白质精氨酸甲基转移酶(PRMT)家族催化的，从S-腺苷甲硫氨酸(AdoMet)把甲基转移到组蛋白和非组蛋白的精氨酸侧链。在人类基因组中注释了九个PRMT基因，基于产生的甲基精氨酸类型分为I型(PRMT1,2,3,4,6和8)、II型(PRMT5和PRMT9)和III型酶(PRMT7)。PRMT5主要是II型酶，可催化精氨酸的对称二甲基化。PRMT5是在检测与Janus酪氨酸激酶(JAK2)相互作用蛋白的双杂交实验中被首次发现的。
PRMT5是一种通用的转录抑制因子，与其他转录因子形成复合物，包括BRG1和hBRM，Blimp1以及Snail。PRMT5通过对多种细胞质和细胞核中的底物的甲基化，包括组蛋白H4残基Arg3(H4R3)和H3残基Arg8(H3R8)而参与多种不同的细胞生物学过程。H4R3甲基化与转录抑制相关，而H3R8甲基化被认为既与转录激活有关，又和转录抑制有关。PRMT5除了直接诱导抑制性组蛋白标记外，该酶在基因沉默中的作用还通过形成多抑制蛋白复合物来介导，包括NuRD组分、HDACs、MDB蛋白和DNA甲基转移酶。PRMT5通过与一些结合蛋白的相互作用进而影响其底物特异性。这种蛋白质复合物中的核心成分是MEP50。MEP50对于PRMT5的酶学活性是必须的。研究发现，PRMT5可以甲基化参与RNA剪接的蛋白，比如SmD3，可用于跟踪细胞内PRMT5的生物学活性。
PRMT5在肿瘤发生中起重要作用。研究发现PRMT5在多种肿瘤中的表达上调，包括淋巴瘤、肺癌、乳腺癌和结直肠癌。此外，PRMT5表达在套细胞淋巴瘤(MCL)病人样本中增高，而PRMT5敲除则可以抑制MCL细胞增殖，表明PRMT5在MCL中起重要作用。PRMT5过表达促进细胞增生，在黑色素瘤、乳腺癌和肺癌细胞系中，PRMT5敲除则可以抑制这些细胞的增殖。因此，PRMT5是癌症治疗的潜在靶点。
甲基硫腺苷磷酸化酶(MTAP)的丧失赋予了细胞对PRMT5及其结合蛋白WDR77的选择性依赖。MTAP由于与通常缺失的肿瘤抑制基因CDKN2A靠近而经常丢失。携带MTAP缺失的细胞的胞内甲硫基腺苷(MTA，被MTAP裂解的代谢物)浓度增加。此外，MTA特异性抑制PRMT5的酶活性。与MTAP表达的细胞相比，MTA或PRMT5小分子抑制剂显著抑制MTAP缺失的癌细胞系的细胞活力。
因此，本领域需要开发能够抑制PRMT5的活性并治疗各种PRMT5相关疾病的小分子活性化合物。
PCT/CN2021/103597(申请日2021年6月30日)描述了一种化合物1-乙基-4-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000001
-5-酮(化合物I)盐酸盐，研究发现，所述化合物I盐酸盐具有较好的PRMT5酶学抑制、细胞增殖抑制和细胞SDMA抑制活性，以及良好的药代动力学性质和肝细胞代谢稳定性，并在小鼠皮下移植瘤Z-138模型中显示出显著的肿瘤生长抑制作用，并 呈现较好的剂量反应关系。
Figure PCTCN2023071046-appb-000002
尽管PRMT5抑制剂作为单一疗法在癌症治疗方面具有有希望的结果，本领域仍需要研究PRMT5抑制剂和其他抗癌治疗剂的组合疗法，以期获得更优、更有效的临床治疗药物和方案。
发明内容
本公开内容提供了一种用于治疗癌症的包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合，其中所述PRMT5抑制剂选自化合物I及其药学上可接受的盐。
本公开内容还提供了用于治疗癌症的药物组合物，其包括PRMT5抑制剂和至少一种抗癌治疗剂，以及药学上可接受的辅料，所述PRMT5抑制剂选自化合物I和其药学上可接受的盐。
本公开内容还提供了一种治疗癌症的方法，所述方法包含向需要所述治疗的个体给予治疗有效量的包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合或药物组合物，其中所述PRMT5抑制剂选自化合物I和其药学上可接受的盐。
本公开内容还提供了包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合或药物组合物在制备用于治疗肿瘤疾病的药物中的用途，其中所述PRMT5抑制剂选自化合物I和其药学上可接受的盐。
本公开内容还提供了包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合或药物组合物在治疗肿瘤疾病方面的用途，其中所述PRMT5抑制剂选自化合物I和其药学上可接受的盐。
本公开内容还提供了治疗肿瘤疾病的包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合或药物组合物，其中所述PRMT5抑制剂选自化合物I和其药学上可接受的盐。
在可选实施方案中，本公开所述化合物I的药学上可接受的盐选自盐酸盐、氢溴酸盐、1,5-萘二磺酸盐、草酸盐、柠檬酸盐、硫酸盐、磷酸盐、L-酒石酸盐、L-苹果酸盐、琥珀酸盐、己二酸盐、富马酸盐、草酸盐、丙酸盐、苯甲酸盐、乙酸盐、甲酸盐或L-精氨酸盐，优选盐酸盐、氢溴酸盐或1,5-萘二磺酸盐，更优选盐酸盐，更加优选一盐酸盐。
在可选实施方案中，本公开所述癌症包括但不限于淋巴瘤、胰腺癌、结肠癌、恶性黑色素瘤、卵巢癌、肺癌、宫颈癌或头颈癌。
在可选实施方案中，本公开所述癌症包括但不限于淋巴瘤、胰腺癌、结肠癌或恶性黑色素瘤。
在可选实施方案中，本公开所述淋巴瘤选自套细胞淋巴瘤。
在可选实施方案中，本公开所述肺癌选自非小细胞肺癌。
在可选实施方案中，本公开所述头颈癌选自颌下腺鳞癌。
在可选实施方案中，所述至少一种抗癌治疗剂选自铂衍生物、烷化剂、拓扑异构酶抑制剂、紫杉醇类、抗肿瘤抗生素、植物生物碱、核苷类似物、B细胞淋巴瘤2(BCL-2)抑制剂、I型PRMT抑制剂和细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂中的一种或多种，优选选自B细胞淋巴瘤2(BCL-2)抑制剂、I型PRMT抑制剂和细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂中的一种或多种。
在可选实施方案中，所述至少一种抗癌治疗剂选自B细胞淋巴瘤2(BCL-2)抑制剂、I 型PRMT抑制剂、细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂、紫杉醇类和铂衍生物中的一种或多种。
在可选实施方案中，所述至少一种抗癌治疗剂选自细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂。
在可选实施方案中，所述铂衍生物选自顺铂、卡铂、奥沙利铂和沙铂。
在可选实施方案中，所述铂衍生物选自顺铂和卡铂。
在可选实施方案中，所述烷化剂选自链脲霉素、达卡巴嗪、丙卡巴嗪、司莫司汀、洛莫司汀、福莫司汀、尼莫司汀、苯达莫司汀、乌拉莫司汀、环磷酰胺、异环磷酰胺、美法仑和六甲基三聚氰胺。
在可选实施方案中，所述拓扑异构酶抑制剂选自依托泊苷、替尼泊苷、喜树碱、10-羟基喜树碱、伊立替康和托泊替康。
在可选实施方案中，所述紫杉醇类选自多西他赛、卡巴他赛和紫杉醇。
在可选实施方案中，所述紫杉醇类选自多西他赛和紫杉醇。
在可选实施方案中，所述抗肿瘤抗生素选自阿霉素、柔红霉素、放线菌素、博来霉素、麦曲霉素、依沙芦星和米托蒽醌。
在可选实施方案中，所述植物生物碱选自长春碱、长春新碱和长春地辛。
在可选实施方案中，所述核苷类似物选自阿糖胞苷、氟达拉滨、碘脱氧尿苷、溴脱氧尿苷、氟脱氧尿苷和吉西他滨。
在可选实施方案中，所述BCL-2抑制剂选自维奈托克(ABT-199)、ABT-737、ABT-263、APG-1252、S-055746、BDA-366、HA14-1、BH3I-1、阿朴棉子酚、TW-37、TM12-06和obatoclax，优选ABT-199。
在可选实施方案中，所述I型PRMT抑制剂选自PRMT1抑制剂、PRMT3抑制剂、PRMT4抑制剂、PRMT6抑制剂和PRMT8抑制剂，优选PRMT1抑制剂，更优选为GSK3368715。
在可选实施方案中，所述CDK4/6抑制剂选自帕博西尼(Palbociclib)、Ribociclib、Abemaciclib、ebvaciclib和trilaciclib，优选Palbociclib。
在可选实施方案中，所述联用药物组合中的PRMT5抑制剂和至少一种抗癌治疗剂可分开地包装或包装在一起。
在可选实施方案中，所述用途或治疗方法中PRMT5抑制剂和至少一种抗癌治疗剂各自呈药物组合物形式，可同时、顺序或间隔给药。
在可选实施方案中，所述用途或治疗方法中PRMT5抑制剂和至少一种抗癌治疗剂各自以间隔给药的形式给药。
在可选实施方案中，所述用途或治疗方法中PRMT5抑制剂和至少一种抗癌治疗剂分别以相同或不同的给药方案进行给药。
在可选实施方案中，所述用途或治疗方法中PRMT5抑制剂和至少一种抗癌治疗剂分别以不同的给药方案进行给药。
在可选实施方案中，所述用途或治疗方法中PRMT5抑制剂可以每日3次(t.i.d)、每日2次(b.i.d)或每日1次(q.d)的频率施用；每天给药的剂量为0.01到100mg/kg体重，优选为0.05到50mg/kg体重，更优选0.1到30mg/kg体重。
在可选实施方案中，所述用途或治疗方法中至少一种抗癌治疗剂可以以每日3次(t.i.d)、每日2次(b.i.d)、每日1次(q.d)、每周1次(q1w)、每2周1次(q2w)、每3周1次(q3w)或每4周一次(q4w)的频率施用。
在可选实施方案中，所述PRMT5抑制剂和至少一种抗癌治疗剂分别具有相同或不同的治疗周期，例如每1周、每2周、每3周或者每4周为一个治疗周期。
施用本公开所述的包括PRMT5抑制剂和至少一种抗癌治疗剂的联用药物组合或药物组合物有助于：
(1)与单独给予PRMT5抑制剂或所述至少一种抗癌治疗剂相比，在减少肿瘤的生长或 甚至消除肿瘤方面产生更好的疗效，或提供更少量的给药；
(2)提供在患者中具有良好耐受的治疗，与单独给予PRMT5抑制剂或所述至少一种抗癌治疗剂相比，其不良反应和/或并发症更少；
(3)提供相比于标准的化疗而言，所治疗患者具有更长的生存期(如中位生存期、无进展生存期或总生存期)。
定义
术语“联用药物组合”是指同时或先后施用的两种或两种以上的活性成分(以其各自的活性成分本身或其药物组合物的形式施用，或者以其各自的药学上可接受的盐或酯等衍生物、前药或其药物组合物的形式施用)的组合。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”包括碱根离子与自由酸形成的盐或酸根离子与自由碱形成的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
本公开内容中涉及的化合物I或其药学上可接受的盐的剂量，除非另有说明，均基于游离形式的化合物I的分子量计算。
术语“药物组合物”是指本公开所述的活性成分与药学上可接受的辅料组成的混合物，其可通过将本公开所述的活性成分与药学上可接受的辅料组合而制备。
本公开的药物组合物可以以适合的各种途径，或者联用药物组合中的组分可以各自独立地以适合地各种途径施用，典型途径包括但不限于：口服、直肠、局部、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、表皮下、囊下、蛛网膜下、静脉内、动脉内、鞘内、淋巴管内、病灶内、囊内、眶内、心内、真皮内、关节内、脊柱内、经气管、硬膜外和胸骨内给药。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
术语“联用”或“联合使用”是指两种或以上的活性物质可以各自作为单一制剂同时地、或各自作为单一制剂以任何顺序依次地施用于有需要的个体。
术语“个体”是哺乳动物。在部分实施方案中，所述个体是小鼠。在部分实施方案中，所述个体是人。
本公开内容中的联用药物组合中的组分可以各自独立地以药物组合物形式存在。
本公开内容中的联用药物组合中的组分可以各自独立地，或者其中的部分或全部共同是适合的剂型，包括但不限于片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等口服形式，或者无菌溶液剂、混悬剂或冻干产品等肠胃外给药形式。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
术语“治疗”一般是指获得需要的药理和/或生理效应。该效应部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本公开内容中的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)抑制疾病的症状，即阻止其发展；或(b)缓解疾病的症状，即导致疾病或症状退化。
术语“有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本公开化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“施用”表示使用本领域技术人员已知的多种方法和递送系统中的任一种，向主体物理引入包含治疗剂的组合物。
本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开中的实施例。
本公开具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本公开的化学变化及其所需的试剂和物料。为了获得本公开的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
附图说明
图1为化合物1-8单晶的球棍图。
图2为Z-138皮下瘤模型中接受受试化合物的小鼠的肿瘤生长曲线，图中化合物002即指实施例2化合物。
图3为Z-138皮下瘤模型中接受受试化合物的小鼠的体重变化曲线，图中化合物002即指实施例2化合物。
图4A为实施例4化合物与联用药物GSK3368715联用对HCT-116细胞增殖抑制的协同作用的矩阵图。
图4B为实施例4化合物与联用药物GSK3368715联用对MIA PaCa-2细胞增殖的抑制协同作用的矩阵图。
图5为实施例4化合物与联用药物ABT199联用对Z138细胞增殖抑制的协同作用的矩阵图。
图6为实施例4化合物与联用药物Palbociclib联用对A375细胞增殖抑制的协同作用的矩阵图。
图7为实施例4化合物与联用药物Palbociclib联用对A2780细胞增殖抑制的协同作用的矩阵图。
图8A为实施例4化合物与联用药物多西他赛联用对A2780细胞增殖抑制的协同作用的矩阵图。
图8B为实施例4化合物与联用药物多西他赛联用对OVCAR3细胞增殖抑制的协同作用的矩阵图。
图9为实施例4化合物与联用药物多西他赛联用对A549细胞增殖抑制的协同作用的矩阵图。
图10A为实施例4化合物与联用药物紫杉醇联用对A2780细胞增殖抑制的协同作用的矩阵图。
图10B为实施例4化合物与联用药物紫杉醇联用对OVCAR3细胞增殖抑制的协同作用的矩阵图。
图11A为实施例4化合物与联用药物紫杉醇联用对Hela细胞增殖抑制的协同作用的矩阵图。
图11B为实施例4化合物与联用药物紫杉醇联用对A253细胞增殖抑制的协同作用的矩阵图。
图12为实施例4化合物与联用药物卡铂联用对A253细胞增殖抑制的协同作用的矩阵图。
图13为实施例4化合物与联用药物顺铂联用对Hela细胞增殖抑制的协同作用的矩阵图。
图14为A375异种移植瘤模型中各组的相对肿瘤体积和体重变化率(％)曲线图。
具体实施方式
下面结合具体实施例对本公开进行进一步的描述，然而，本公开内容中的这些实施例仅用于阐明而不限制本公开的范围。同样，本公开不限于本文描述的任何具体优选的实施方案。本领域技术人员应该理解，对本申请技术特征所作的等同替换或相应的改进仍属于本公开的保护范围之内。
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用BRUKER AV-400型核磁共振仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)等，内标为四甲基硅烷(TMS)；“IC 50”指半数抑制浓度，指达到最大抑制效果一半时的浓度。
实施例中的洗脱剂可由两种或多种溶剂形成混合洗脱剂，其比值为各溶剂的体积比。例如，“0～10％甲醇/二氯甲烷”表示梯度洗脱过程中，混合洗脱剂中的甲醇与二氯甲烷的体积用量比为0:100～10:100。
本公开中所用的试剂可通过商业途径获得。本公开中使用的所有溶剂均是市售的，无需进一步纯化即可使用。
除非另作说明，混合溶剂表示的比例是体积混合比例。
除非另作说明，否则％是指质量百分比wt％。
化合物经手工命名，市售化合物采用供应商目录名称。
实施例1：(S)-3-((S)-环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(中间体1)的制备
Figure PCTCN2023071046-appb-000003
步骤1：化合物b的制备
Figure PCTCN2023071046-appb-000004
室温下将(S)-2-(叔丁氧羰基)-1,2,3,4-四氢异喹啉-3-羧酸(a)(55g，200mmol)，二甲羟胺盐酸盐(29.4g，300mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(91g，240mmol)加入到1L单口瓶中，再加入无水N,N-二甲基甲酰胺(500mL)，氮气保护后冰浴冷却，然后滴加N,N-二异丙基乙胺(104mL，600mmol)。反应液在室温反应4小时。反应完全后旋蒸除去过量的N,N-二异丙基乙胺和N,N-二甲基甲酰胺，然后冰浴冷却，用饱和食盐水(1L)稀释，用乙酸乙酯萃取(200mL X 2)，有机相合并后用5％碳酸钠水溶液洗涤(500mL X 2)，然后用饱和食盐水洗涤(500mL)。无水硫酸钠干燥，过滤，滤液减压浓缩后硅胶柱层析(洗脱剂梯度：石油醚/乙酸乙酯＝2/1)纯化，得到目标中间体(S)-3-(甲氧基(甲基)氨基甲酰)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(b)(62g，收率：97％)。
LCMS:Rt:1.76min；MS m/z(ESI):321.3[M+H] +。
手性HPLC:Rt:3.159min。
步骤2：化合物c的制备
Figure PCTCN2023071046-appb-000005
室温下将(S)-3-(甲氧基(甲基)氨基甲酰)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(b)(20g，62.5mmol)称量到500mL三口瓶中，加入无水四氢呋喃(200mL)，冷却至-70℃，缓慢滴加二 异丁基氢化铝(DIBAL-H)甲苯溶液(1.5mol/L，83mL，125mmol)，反应液在-70℃搅拌1小时。反应完全后在-70℃缓慢加入饱和氯化铵溶液(100mL)淬灭，然后加入0.5mol/L盐酸水溶液稀释(200mL)。分层后有机相用饱和氯化钠水溶液洗涤(200mL X 2)，然后用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物用硅胶柱层析(洗脱剂梯度：石油醚/乙酸乙酯＝8/1)纯化，得中间体(S)-3-甲酰基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(c)(15g，收率:92％)。
LCMS:Rt:1.93min；MS m/z(ESI):206.1[M-56+H] +。
手性HPLC:Rt:2.018min。
步骤3：化合物d的制备
Figure PCTCN2023071046-appb-000006
将甲基三苯基溴化膦(238g，0.67mol)分散到无水四氢呋喃(1.5L)中，氮气保护下冷却至-70℃，缓慢滴加二(三甲基硅基)氨基钠(334mL，0.67mol)，控制温度低于-50℃，滴加完毕后缓慢升至室温，搅拌2小时。重新冷却至-70℃，缓慢滴加(S)-3-甲酰基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(c)(87g，0.33moL)的四氢呋喃溶液(200mL)，控制温度低于-50℃，滴加完毕后缓慢升至室温过夜。TLC检测反应完毕后，冷却至0℃，加入饱和氯化铵溶液淬灭，缓慢加入1mol/L盐酸水溶液调节pH到3-4，加入乙酸乙酯(500mL)萃取。有机相用饱和食盐水洗涤(200mL X 2)，无水硫酸钠干燥后过滤，滤液减压浓缩。残余物加入混合溶剂(乙酸乙酯/石油醚＝1/4)重结晶，过滤除去析出的三苯基氧化膦，滤液浓缩后硅胶柱层析(乙酸乙酯/石油醚＝1/8)得目标产物(S)-3-乙烯基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(d)(85g，收率:98％)。
手性HPLC:Rt:1.883min。
步骤4：化合物e的制备
Figure PCTCN2023071046-appb-000007
将(S)-3-乙烯基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(d)(25.9g，0.1mol)溶于乙酸乙酯/乙腈(500mL/500mL)溶液中，冷却至0℃，在10分钟内加入高碘酸钠(32.1g，0.15mol)与三氯化钌水合物(1.6g，7.7mmol)的水溶液，反应液在0℃搅拌10分钟。TLC检测反应完毕，加入硫代硫酸钠饱和溶液(150mL)淬灭，搅拌30分钟后分层，有机相用饱和食盐水洗涤(200mL X 2)，无水硫酸钠干燥，过滤后滤液减压浓缩，残余物经硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/2-1/1)，得产物(S)-3-((S)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(e)(极性较小的产物，14g，收率48％)。加大洗脱剂极性(乙酸乙酯/石油醚＝2/1-1/0)，得产物(S)-3-((R)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(e-1)(极性较大的产物，8g，收率28％)。
步骤5：化合物f的制备
Figure PCTCN2023071046-appb-000008
将(S)-3-((S)-1,2-二羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(e)(14g，0.047mol)溶于二氯甲烷(150mL)中，加入三乙胺(9.90mL，0.072mol)后在搅拌状态下分批次加入对甲苯磺酰氯(10.0g，0.052mol)，反应液在40℃搅拌过夜。将反应液冷却至室温，用饱和食盐水洗涤(100mL X 2)，有机相用无水硫酸钠干燥。过滤后滤液减压浓缩，残余物经硅胶柱层析(乙酸乙酯/石油醚＝1/4)得目标产物(S)-3-((S)-1-羟基-2-(甲苯磺酰氧基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(f)(12.6g，收率59％)。
步骤6：化合物中间体1的制备
Figure PCTCN2023071046-appb-000009
将(S)-3-((S)-1-羟基-2-(甲苯磺酰氧基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(f)(12.6g，28.2mmol)溶于N,N-二甲基甲酰胺(150mL)中，氮气保护下分批次加入氢化钠(1.70g，42.3mmol)，反应液在40℃搅拌1小时，TLC检测反应完全。冷却至0℃，滴加饱和食盐水淬灭，反应液直接反相硅胶色谱柱(水/乙腈＝50/10)纯化，柱层析所得溶液用乙酸乙酯萃取(200mL X 3)，有机相经无水硫酸钠干燥后过滤，滤液减压浓缩得到中间体(S)-3-((S)-环氧乙烷-2-基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(中间体1)(6.5g，收率72％)。
实施例2：1-乙基-4-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000010
-5-酮(化合物I)盐酸盐的制备
Figure PCTCN2023071046-appb-000011
步骤1：化合物1-2的制备
Figure PCTCN2023071046-appb-000012
室温下将4-溴-2-氟苯甲酸(1-1)(7.5g，34.2mmol)和N-叔丁氧羰基-1,2-乙二胺(16.4g，102.7mmol)加入到N-甲基吡咯烷酮(30mL)中。加入完毕后，升温至120℃反应16小时。反应完全后，将反应液缓慢加入到水(150mL)中，用2mol/L稀盐酸调节溶液pH至5～6，然后用乙酸乙酯萃取(150mL X 2)。合并有机相，用饱和食盐水(150mL X 2)洗涤，无水硫酸钠干燥，过滤。将滤液减压浓缩得到目标中间体4-溴-2-((2-((叔丁氧羰基)氨基)乙基) 氨基)苯甲酸(1-2)(粗品，11.0g,收率：89％)。
LCMS:Rt:1.836min；MS m/z(ESI):359.0[M+H] +。
步骤2：化合物1-3的制备
Figure PCTCN2023071046-appb-000013
室温下将4-溴-2-((2-((叔丁氧羰基)氨基)乙基)氨基)苯甲酸(1-2)(11.0g，30.6mmol)，加入到4mol/L盐酸/二氧六环溶液(30mL)中，然后在室温反应1小时。反应完全后将反应液减压浓缩，得到目标中间体2-((2-氨基乙基)氨基)-4-溴苯甲酸(1-3)(粗品，8g,收率：100％)。
LCMS:Rt:0.688min；MS m/z(ESI):259.0[M+H] +。
步骤3：化合物1-4的制备
Figure PCTCN2023071046-appb-000014
室温下将2-((2-氨基乙基)氨基)-4-溴苯甲酸(1-3)(3g，11.6mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(8.8g,23.2mmol)和三乙胺(4.7g,46.4mmol)加入到超干N,N-二甲基甲酰胺(20mL)中，室温反应1.5小时。反应完全后加入饱和食盐水(100mL)，用乙酸乙酯萃取(100mL X 2)，有机相用饱和食盐水洗涤(50mL X 2)，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物用正相硅胶色谱柱(洗脱剂梯度：二氯甲烷/甲醇＝20/1)纯化，得到目标中间体8-溴-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000015
-5-酮(1-4)(1.6g,收率：57％)。
LCMS:Rt:1.173min；MS m/z(ESI):241.0[M+H] +。
步骤4：化合物1-5的制备
Figure PCTCN2023071046-appb-000016
室温下将8-溴-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000017
-5-酮(1-4)(1.6g,6.6mmol)，氰基硼氢化钠(834mg，13.3mmol)和乙醛(584mg，13.3mmol)加入到醋酸(2mL)和甲醇(20mL)混合溶液中，室温反应1小时。反应完全后加入2mol/L稀盐酸(1mL)和水(50.0mL)，用乙酸乙酯(50.0mL X 2)萃取。有机相合并，用饱和食盐水(50mL X 2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物经正相硅胶色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝1/2)纯化，得到目标中间体8-溴-1-乙基-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000018
-5-酮(1-5)(1.1g,收率：62％)。
LCMS:Rt:1.499min；MS m/z(ESI):269.0[M+H] +。
步骤5：化合物1-6的制备
Figure PCTCN2023071046-appb-000019
室温下将8-溴-1-乙基-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000020
-5-酮(1-5)(400mg，1.52mmol)加入到超干N,N-二甲基甲酰胺(20mL)中，然后加入氢化钠(90.9mg，2.27mmol)。添加完毕，加热至40℃搅拌1h后，再加入实施例1制得的中间体1(500mg，1.82mmol)，反应16小时。反应完全后，加入水(100mL)，用乙酸乙酯(100mL X 2)萃取。有机相合并，用饱和食盐水洗涤(50mL X 2)，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物经正相硅胶色谱柱(洗脱剂梯度：二氯甲烷/甲醇＝20/1)纯化，得到目标中间体(1R,10aS)-1-((8-溴-1-乙基-5-氧代-1,2,3,5-四氢-4H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000021
-4-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(1-6)(150mg,收率：21％)。
LCMS:Rt:1.872min；MS m/z(ESI):470.1[M+H] +。
步骤6：化合物1-7的制备
Figure PCTCN2023071046-appb-000022
室温下将中间体(1R,10aS)-1-((8-溴-1-乙基-5-氧代-1,2,3,5-四氢-4H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000023
-4-基)甲基)-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-3-酮(1-6)(150mg,0.32mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯(11.7mg，0.02mmol)和醋酸钾(94mg，0.96mmol)加入到无水乙醇(10mL)中，CO置换3次，加热至70℃反应3.0小时。反应完全后冷却至室温，将反应液减压浓缩，加入饱和食盐水(50mL)，然后用乙酸乙酯(50mL X 2)萃取，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。残余物经正相硅胶色谱柱(洗脱剂梯度：石油醚/乙酸乙酯＝1/2)纯化，得到目标中间体1-乙基-5-氧代-4-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-2,3,4,5-四氢-1H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000024
-8-羧酸乙酯(1-7)(130mg,收率：87％)。
LCMS:Rt:1.784min；MS m/z(ESI):464.1[M+H] +。
步骤7：化合物1-8的制备
Figure PCTCN2023071046-appb-000025
室温下将1-乙基-5-氧代-4-(((1R,10aS)-3-羰基-1,5,10,10a-四氢-3H-噁唑并[3,4-b]异喹啉-1-基)甲基)-2,3,4,5-四氢-1H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000026
-8-羧酸乙酯(1-7)(130mg，0.28mmol)加入 到甲醇(4mL)和水(4mL)的混合溶液中，然后加入氢氧化钠(90mg，2.24mmol)，加热至70℃，反应16.0小时。反应完全后，将反应体系降温至室温，加入Boc酸酐(122mg，0.56mmol)反应1.5小时。反应完全后，将反应体系降温至0℃，用1mol/L的盐酸水溶液调节反应液的pH至5.0，后用乙酸乙酯(30mL X 3)萃取，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。残余物经反相硅胶色谱柱(洗脱剂梯度：乙腈/水＝43％)得到目标中间体4-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-1-乙基-5-氧代-2,3,4,5-四氢-1H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000027
-8-羧酸(1-8)(粗品，60mg,收率：42％)。
LCMS:Rt:1.722min；MS m/z(ESI):510.3[M+H] +。
化合物1-8的单晶X-射线结构测定和单晶X-射线分析：
单晶制备方法：称取化合物1-8(10.0mg)放入3mL螺口玻璃瓶中，加入甲醇2mL，搅拌5分钟后固体溶清。向玻璃瓶中加入0.5mL水，继续搅拌5分钟。溶液经0.22μm微孔滤膜滤至3mL螺口玻璃瓶中，玻璃瓶口用保鲜膜覆盖。用针头在瓶口处扎8个小孔，室温放置7天，制得上述化合物单晶。
所得单晶样品进行X-射线分析，测试结果见表1和图1。
表1化合物1-8的单晶样品和晶体数据
Figure PCTCN2023071046-appb-000028
通过上述X-射线晶体衍射实验，可以确定化合物1-8的化学结构和绝对构型如下：
Figure PCTCN2023071046-appb-000029
步骤8：化合物2-2的制备
Figure PCTCN2023071046-appb-000030
室温下将4-((R)-2-((S)-2-(叔丁氧基羰基)-1,2,3,4-四氢异喹啉-3-基)-2-羟基乙基)-1-乙基-5-氧代-2,3,4,5-四氢-1H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000031
-8-羧酸(1-8)(100mg,0.196mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(231mg,0.608mmol)和N,N-二异丙基乙胺(157mg,1.216mmol)加入到超干N,N-二甲基甲酰胺(2.5mL)中，然后加入2-甲氧基-7-氮杂螺[3.5]壬烷盐酸盐(53mg,0.275mmol)，室温搅拌反应1.0小时。反应完全后加入水(30mL)，然后用乙酸乙酯(30mL X 3)萃取，合并有机相并用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物用正相硅胶色谱柱(洗脱剂：二氯甲烷/甲醇＝20/1)纯化，得到目标中间体(S)-3-((R)-2-(1-乙基-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-5-氧代-1,2,3,5-四氢-4H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000032
-4-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(2-2)(110mg,收率：86.7％)。
LCMS:Rt:2.062min；MS m/z(ESI):647.3[M+H] +.
步骤9：化合物I盐酸盐的制备
Figure PCTCN2023071046-appb-000033
室温下将(S)-3-((R)-2-(1-乙基-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-5-氧代-1,2,3,5-四氢-4H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000034
-4-基)-1-羟基乙基)-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯(2-2)(110mg,0.170mmol)加入到4mol/L盐酸/二氧六环(2.5mL)溶液中。室温搅拌反应1.0小时。反应完全后将反应液减压浓缩，残余物用高效液相制备色谱法(洗脱剂梯度：
Figure PCTCN2023071046-appb-000035
)纯化，得到目标化合物1-乙基-4-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000036
-5-酮(化合物I)盐酸盐(39.81mg，收率：42.8％)。
1H NMR(400MHz,CD 3OD)：δ7.64(d,J＝7.6Hz,1H),7.35-7.22(m,4H),7.12(d,J＝8Hz,2H),4.51-4.36(m,2H),4.33-4.30(m,1H),4.04-3.89(m,2H),3.73-3.61(m,7H),3.51-3.50(m,1H),3.38-3.34(m,4H),3.31-3.30(m,2H),3.22(s,3H),2.29-2.23(m,2H),1.71-1.68(m,4H),1.55(s,2H),1.17(t,J＝7.0Hz,3H).
LCMS:Rt:1.355min；MS m/z(ESI):547.4[M+H] +.
通过电导法测定所得化合物I盐酸盐中的盐酸含量，实验仪器、试剂、方法和结果如下。
实验仪器与试剂：高效液相色谱仪(岛津LC-20AD)、电导检测器(CDD-10Avp)、电子天平(Sartorius，MAS125P-1CE-DU)、氯化钠、对羟基苯甲酸、Bis-Tris(HPLC级，阿拉丁A1910012)、硼酸、超纯水；
色谱信息：色谱柱——Shim-pack IC-A3；保护柱——Shim-pack IC-GA3；淋洗液——8mmol/L对羟基苯甲酸、3.2mmol/L Bis-Tris、50mmol/L硼酸溶液；流速——1.5mL/min；柱温——40℃；进样量——50μL；电导检测器模式——阳离子模式；
溶液配制：
对照品溶液(含氯离子6μg/mL)：精密量取氯化钠50mg至50mL量瓶中，加水溶解并稀释至刻度，摇匀，精密移取1.0mL至100mL量瓶中，加水稀释至刻度，摇匀。
供试品溶液(0.1mg/mL)：精密称取待测样品10mg至100mL量瓶中，用水溶解并稀释至刻度，摇匀。
测定方法：分别精密量取对照品溶液与供试品溶液，注入高效液相色谱仪，以淋洗液作为洗脱剂进行洗脱，记录色谱图。
实验结果：结果测得所述化合物I盐酸盐中的HCl含量为10.57％(化合物I一盐酸盐的HCl含量理论值为6.25％)。
实施例3 1-乙基-4-((R)-2-羟基-2-((S)-1,2,3,4-四氢异喹啉-3-基)乙基)-8-(2-甲氧基-7-氮杂螺[3.5]壬烷-7-羰基)-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂
Figure PCTCN2023071046-appb-000037
-5-酮(化合物I)的制备
室温下将按实施例2方法制得的3.0g化合物I盐酸盐加入100mL单口瓶中，加入12mL二氯甲烷，溶清后加入12mL纯化水，搅拌中加入20％氢氧化钠溶液调pH至水相pH为10左右，分液，用3ml二氯甲烷萃取三次水相，将有机相合并，用10mL纯化水反洗一次有机相，将有机相用无水硫酸钠干燥，减压浓缩至干，得2.5g泡沫状固体，即化合物I。
1H NMR(400MHz,DMSO-d 6):δ7.49-7.41(m,1H),7.17-7.04(m,3H),7.01(d,J＝5.5Hz,1H),6.90-6.79(m,2H),5.01(d,J＝5.5Hz,1H),4.03(q,J＝7.1Hz,1H),3.98-3.79(m,4H),3.75(s,1H),3.63-3.35(m,6H),3.32-3.19(m,3H),3.18-3.12(m,2H),3.10(s,3H),2.84-2.65(m,3H),2.15(s,2H),1.70-1.39(m,6H),1.07(t,J＝7.0Hz,3H).
实施例4化合物I一盐酸盐的制备
室温下将实施例3制得的化合物I(4.31g)加入500mL单口瓶中，加入异丙醇(43mL)，搅拌溶清后置于冰水浴中，缓慢滴加3.9mL 2mol/L氯化氢-乙酸乙酯溶液，搅拌至析出大量固体后抽滤，将滤饼用5mL异丙醇淋洗、25℃真空干燥，得到固体(3.74g)。通过电位滴定法对所得固体中的盐酸含量进行测定，确定所述盐中化合物I与HCl的摩尔比约为1：1。
电位滴定法测定盐酸含量的实验仪器、试剂和方法如下。
实验仪器与试剂：电位滴定仪(Metrohm，型号888)、电子天平(Mettler Toledo，型号XS205DU)；甲醇(色谱级，稀释至60％甲醇作为溶剂和空白)、0.1mol/L氢氧化钠滴定液(南京化学试剂股份有限公司，批号210926428W)；
滴定参数：
Figure PCTCN2023071046-appb-000038
滴定过程：
空白滴定：量取溶剂(60％甲醇)50mL置于100mL烧杯中，用氢氧化钠滴定液(0.1mol/L)滴定至终点。
待测样品滴定：精密称取待测样品300mg，置于100mL烧杯中，加入溶剂(60％甲醇)50mL，用氢氧化钠滴定液(0.1mol/L)滴定至终点。
盐酸含量通过以下公式计算：
Figure PCTCN2023071046-appb-000039
备注：
V 0:空白消耗氢氧化钠滴定液的体积，单位(mL)；
V:待测样品消耗氢氧化钠滴定液的体积，单位(mL)；
C：氢氧化钠滴定液的浓度，单位(mol/L)；
m：待测样品的称样量，单位(mg)；
3.646：氢氧化钠滴定液滴定度，mg/mL。
生物学活性及相关性质测试试验例
测试试验例1：PRMT5酶学活性抑制实验
材料：PRMT5/MEP50蛋白购于BPS bioscience公司(美国)；组蛋白H4多肽(Histone H4 Peptide)底物购于生工生物工程(上海)股份有限公司；Anti-Histone H4(symmetric dimethyl R3)抗体-ChIP Grade购于艾博抗公司(美国)；S-(5’-腺苷)-L-甲硫氨酸氯化物二盐酸盐购于西格玛公司(美国)；384孔板、AlphaScreen链霉亲和素供体微珠(AlphaScreen Streptavidin Donor beads)、AlphaScreen蛋白A受体微珠(AlphaScreen Protein A Acceptor beads)和多功能酶标仪Envision 2104 multi-label Reader购于珀金埃尔默仪器有限公司(美国)；Echo 550移液器(Echo 550 Liquid Handler)购于Labcyte公司(美国)。
酶学活性检测：利用Echo将化合物打入384孔板中，使终浓度为0-1000nM(起始浓度1000nM，3倍稀释，10个点)，DMSO含量为0.5％。每孔加入10μL 2X PRMT5/MEP50溶液，常温孵育30分钟。每孔加入10μL 2X PRMT5/MEP50底物溶液启动反应，常温孵育60 分钟。准备含AlphaScreen Protein A Acceptor beads和Anti-Histone H4(symmetric dimethyl R3)抗体的6X检测试剂，每孔加入5μL，常温孵育60分钟。准备含AlphaScreen Streptavidin Donor beads的6X检测试剂，每孔加入5μL，常温孵育60分钟。Envision检测信号值。测试结果见表2。
测试试验例2：化合物对肿瘤细胞增殖的抑制活性实验
材料与细胞：Z-138细胞购于ATCC(美国)；IMDM培养基和青霉素-链霉素购于西格玛公司(美国)；马血清购于Hyclone公司(美国)；96孔板购于康宁公司(美国)；Cell-Titer Glo试剂购于普洛麦格公司(美国)。
细胞培养：Z-138细胞用含10％马血清+1％青霉素-链霉素的IMDM培养液于37℃、5％CO 2条件下培养。处于对数生长期细胞方可用于实验。
细胞增殖活性检测：利用Cell-Titer Glo试剂检测化合物对Z-138细胞的增殖抑制活性。调整细胞浓度，每孔180μL接种96孔板(500/孔)，置于37℃、5％CO 2条件下平衡10-15分钟。每孔加入20μL含化合物的培养液，使终浓度达到0-300nM(起始浓度300nM，3倍稀释，10个点)，DMSO含量为0.1％。细胞板置于37℃、5％CO 2条件下孵育8天。其中第四天换液：缓慢吸去100μL上清，并补充100μL含有化合物的新鲜培养液，保持化合物浓度不变。通过Cell-Titer Glo试剂检测细胞活性。测试结果见表2。
测试试验例3：化合物对SDMA的抑制活性实验
材料与细胞：Z-138细胞购于ATCC(美国)；IMDM培养基和青霉素-链霉素购于西格玛公司(美国)；马血清购于Hyclone公司(美国)；Hoechst抗体购于invitrogen公司(美国)；Alexa Fluor 488 goat anti-rabbit IgG抗体购于Invitrogen公司(美国)；Anti-dimethyl-Arginine symmetric(SYM11)抗体购于Merck公司(美国)；DPBS购于Gibco公司(美国)；Nonfat Dry milk购于Cell signaling technology公司(美国)；多聚甲醛购于北京索莱宝科技有限公司；384孔板和Echo 550 Liquid Handler购于Labcyte公司(美国)；ImageXpress Nano购于Molecular Devices公司(美国)。
细胞培养：Z-138细胞用含10％马血清+1％青霉素-链霉素的IMDM培养液于37℃、5％CO 2条件下培养。处于对数生长期的细胞方可用于实验。
免疫荧光检测：利用免疫荧光检测化合物对Z-138细胞中SDMA的影响。调整细胞浓度为1*10 5/mL，每孔40μL接种384孔板(4000/孔)，置于37℃、5％CO 2条件下平衡10-15分钟。利用Echo将化合物打入384孔板中，使终浓度为0-300nM(起始浓度300nM，3倍稀释，10个点)，DMSO含量为0.1％。细胞板置于37℃、5％CO 2条件下孵育2天。每孔加入40μL 8％多聚甲醛，常温孵育30分钟。弃上清，DPBS洗板，每孔加入40μL 0.5％PBST(磷酸盐吐温缓冲液)，常温孵育60分钟。弃上清，0.05％PBST洗板，每孔加入40μL封闭液(1％Nonfat Dry milk in 0.05％PBST)，常温孵育60分钟。弃上清，每孔加入20μL一抗(SYM11，1:500封闭液稀释)，4℃过夜。弃上清，0.05％PBST洗板，每孔加入20μL二抗(Alexa Fluor 488 goat anti-rabbit 1:1000和Hoechst 1:5000封闭液稀释)，常温孵育60分钟。弃上清，0.05％PBST洗板，ImageXpress Nano检测荧光强度。测试结果见表2。
表2
Figure PCTCN2023071046-appb-000040
测试试验例4：小鼠药代动力学实验
实验材料：CB17-SCID小鼠购自北京维通利华实验动物技术有限公司；DMSO、HP-β-CD(羟丙基-β-环糊精)、MC(甲基纤维素)、乙腈购自Merck公司(美国)、K 2EDTA抗凝管购自江苏新康医疗器械有限公司。
实验方法：雌性CB17-SCID小鼠6只(20-30g，4-6周)，随机分成2组，每组3只。第1组尾静脉注射给予化合物，剂量为2mg/kg，溶媒为5％DMSO+95％10％HP-β-CD的水溶 液，第2组口服给予化合物，剂量10mg/kg，溶媒为0.5％MC水溶液。动物实验前正常喂食喂水。每组小鼠于给药前及给药后0.083(仅静脉注射组)、0.25、0.5、1、2、4、6、8和24h进行静脉采血。收集的全血样品置于K 2EDTA抗凝管中，离心5min后(4000rpm，4℃)取血浆待测。
取小鼠血浆样品10μL，加入150μL乙腈溶剂(其中含内标化合物)沉淀蛋白，涡旋0.5min后，离心(4700rpm，4℃)15min，上清液用含0.05％(v/v)甲酸的水稀释2倍，进样3μL于LC-MS/MS系统(AB Sciex Triple Quad 6500+)进行定量检测。在测定样品浓度时随行CB17-SCID小鼠血浆标准曲线(线性范围：0.5-1000ng/mL)和质控样品。对于10倍稀释样品的制备，取2μL小鼠血浆样品加入18μL的空白血浆，涡旋0.5min后，加入300μL乙腈溶剂(其中含内标化合物)沉淀蛋白，其余处理步骤同不稀释样品。
药代动力学测试结果如表3所示。
表3小鼠药代动力学测试结果
Figure PCTCN2023071046-appb-000041
测试试验例5：肝细胞代谢稳定性测试
实验材料：人肝细胞购自Biopredic公司；小鼠肝细胞购自BioIVT公司；乙腈和甲醇购自Merck公司；AOPI染色剂购自Nexcelom公司；地塞米松购自NIFDC公司；DMSO购自北京索莱宝科技有限公司；DPBS(10x)、GlutaMAX TM-1(100x)和人重组胰岛素购自Gibco by Life Technologies；胎牛血清购自Corning公司；甲酸购自DIKMAPURE公司；Isotonic Percoll购自GE Healthcare公司；阿普唑仑购自Supelco公司；咖啡因购自ChromaDex.inc；HEPES、甲苯磺丁脲和Williams’Medium E购自Sigma公司。
实验准备：
将受试物粉末用DMSO配制高浓度储备液，使用前用乙腈稀释到100μM的工作液，受试物终浓度为1μM。
肝细胞复苏液的具体制备信息见下表4。混合49.5mL Williams’E Medium和0.5mL GlutaMAX作为孵育液。将肝细胞复苏液和孵育液于使用前置于37℃水浴中至少预热15分钟。取一管超低温保存的肝细胞，确保肝细胞在复苏之前仍处于低温冰冻状态。将肝细胞迅速置于37℃水浴中并轻摇直至所有冰晶全部分散，喷洒70％乙醇后转移至生物安全柜中。将肝细胞小管的内容物倾入盛有50mL复苏培养基的离心管中，将其于100g离心10分钟。离心后，吸出复苏培养基并加入足量孵育培养基得到细胞密度约1.5×10 6个细胞/mL的细胞混悬液。用Cellometer Vision对肝细胞进行计数及确定活细胞密度，肝细胞成活率必须大于75％。利用孵育培养基稀释肝细胞混悬液至活细胞密度为0.5×10 6个活细胞/mL。
表4肝细胞复苏液制备
Figure PCTCN2023071046-appb-000042
Figure PCTCN2023071046-appb-000043
实验方法：
转移247.5μL活细胞(人肝细胞或鼠肝细胞)的混悬液或培养基到96孔深孔板，将深孔板置于涡旋上孵箱中预热10分钟。所有样品均采用双平行孵育。每孔加入2.5μL 100μM受试物进行反应起始，将深孔板放回孵箱涡旋器上。孵育样品，分别于0、15、30、60、90和120分钟，取25μL混悬液，加入125μL含内标的乙腈(100nM阿普唑仑，200nM咖啡因，100nM甲苯磺丁脲)终止反应。涡旋10分钟，于3220g、4℃条件离心30分钟，离心结束后转移100μL上清液到进样板，加入150μL纯水混匀，用于LC-MS/MS分析。
所有的数据计算均通过Microsoft Excel软件进行。通过提取离子图谱检测峰面积。通过对母药消除百分比的自然对数与时间进行线性拟合，检测母药的体外半衰期(t 1/2)。
体外半衰期(t 1/2)通过斜率计算：
in vitro t 1/2＝0.693/k
实验结果如表5所示。
表5肝细胞代谢稳定性测试结果
化合物 人t 1/2(min) 鼠t 1/2(min)
实施例2 252.37 94.62
测试试验例6：小鼠体内药效实验
实验材料：Z138细胞购自ATCC；IMDM培养液、青链霉素和0.25％胰酶-EDTA购自Gibco公司；马血清、PBS购自Hyclone公司；Matrigel购自Corning公司。
动物信息：CB17-SCID小鼠，雌性，6-7周，体重约14-20克，动物购自上海灵畅生物科技有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
实验方法：
细胞培养：人套细胞淋巴瘤Z-138细胞株体外培养，培养条件为IMDM(细胞培养液)中加入10％马血清，1％青链霉素溶液，37℃、5％CO 2孵箱。一周两次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为85％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1mL/(含1×10 7)Z-138细胞悬液(PBS:Matrigel＝1：1)皮下接种于每只小鼠的右后背。在接种后第18天，测量肿瘤平均体积达到约125mm 3时，依据肿瘤体积和动物体重采用随机分层分组方法开始分组给药。PBS为无钙镁离子的磷酸缓冲盐溶液，Matrigel是基质胶。
给药：实施例2化合物的给药剂量为1.5mg/kg、5mg/kg或15mg/kg，PO，每天两次给药(BID)x 3周。每组6只小鼠。
肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b 2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。
化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。
TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。
实验结果：
见表6、图2和图3。实验过程中无小鼠发病或死亡。
表6 Z-138皮下瘤模型肿瘤体积
Figure PCTCN2023071046-appb-000044
实验结论：
在小鼠皮下移植瘤Z-138模型中，本公开实施例2化合物在1.5mg/kg、5mg/kg和15mg/kg一天两次给药对肿瘤生长都具有显著抑制作用，并呈现较好的剂量反应关系，15mg/kg一天两次给药，具有缩小肿瘤的效果。实施例2化合物在本次药效实验所尝试剂量下未显著影响小鼠体重，也未引起任何小鼠死亡，小鼠可以耐受。
测试试验例7：本公开化合物联合I型PRMT抑制剂GSK3368715对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基DMEM(Gibco#21068028)；McCoy's5A培养基(Gibco#16600108)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3599)，1.5mL不透明棕色Eppendorf管；I型PRMT抑制剂GSK3368715(Selleckchem#S8858)；0.25％胰酶(Gibco#25200-072)；马血清(Horse Serum)(Gibco#16050-130)；
Figure PCTCN2023071046-appb-000045
Luminescent细胞活力检测试剂盒(CTG)(Promega，#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器，Eppendorf移液枪，恒温二氧化碳培养箱(ThermoFisher)，FlexStation 3多功能酶标仪(Molecular Devices)。
本实验所需要的细胞包括：MIA PaCa-2(ATCC#CRM-CRL-142)，培养基为含10％FBS的McCoy’s 5A培养基；HCT116(ATCC#CCL-247)，培养基为含10％FBS、2.5％马血清的DMEM培养基。
实验方法：
将HCT 116和MIA PaCa-2细胞从细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度为300个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。用DMSO分别将实施例4化合物稀释至40μM，GSK3368715稀释至100μM；将稀释后的化合物用HCT 116或MIA PaCa-2对应的培养基进行40倍稀释，其浓度分别为1μM和2.5μM；分别以此为起始浓度用配制好的2.5％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，GSK3368715连续稀释6个浓度梯度。最后以正交的方式分别转移10μl梯度稀释后的化合物实施例4化合物至前一天铺板的HCT116和MIA PaCa-2细胞的培养板中。阳性对照组为未种植细胞的含0.25％DMSO的培养基孔；阴性对照组为种有细胞的以0.25％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用FlexStation3多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)(Bliss,C.I.,Bacteriol.Rev.,1956,20,243-258)。该模型假设药物具有机率独立性。由下列方程式计算组合的预期作用的理论曲线：
Bliss作用＝作用A+作用B-作用A×作用B
其中：作用A和作用B为单独的药物A和B在特定浓度x下的作用。
当精确添加两种药物的组合时，Bliss作用为预期的作用。如果观察的作用(即实验检测的组合的作用)小于Bliss作用时，Bliss评分(δ＝观察的作用-Bliss作用)为负值，代表药物之间存在拮抗作用；如果观察的作用大于Bliss作用，Bliss评分为正值，代表药物之间存在协同作用；如果观察的作用等于Bliss作用，Bliss评分等于零，代表药物之间相互独立。
实验结果：如图4A和图4B所示，实施例4化合物联合I型PRMT抑制剂GSK3368715对人结肠癌细胞HCT 116和人胰腺癌细胞MIA PaCa-2细胞的增殖有强协同抑制作用。
测试试验例8：本公开化合物联合BCL-2抑制剂ABT199对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基IMDM(Gibco#12440-053)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；马血清(Hyclone#SH30074.03)；100％DMSO(Sigma#D2650)；ABT-199(MCE#HY-15531)；96孔透底无菌培养板(Costar#3610)，1.5mL不透明棕色Eppendorf管；
Figure PCTCN2023071046-appb-000046
Luminescent细胞活力检测试剂盒(CTG)(Promega，#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器，Eppendorf移液枪，恒温二氧化碳培养箱(ThermoFisher)，FlexStation 3多功能酶标仪(Molecular Devices)，细胞计数仪(CountStar)。
本实验所需要的细胞包括：Z138(ATCC#CRL-3001)，培养基为含10％马血清的IMDM。
实验方法：
将Z138细胞1000rpm，离心3min，用新鲜培养基重悬，计数后调整细胞密度为1200个细胞/80μL/孔，铺板至96孔板内，用含10％马血清的IMDM分别将实施例4化合物稀释至30nM，ABT-199稀释至300nM；分别以此为起始浓度用配制好的2.5％DMSO培养液进行3倍梯度稀释，实施例4化合物连续稀释5个浓度梯度，ABT-199连续稀释8个浓度梯度。最后分别转移10μl梯度稀释后的化合物实施例4化合物及ABT-199至正交的Z138细胞的培养板中。阳性对照组为未种植细胞的含0.5％DMSO的培养基孔；阴性对照组为种有细胞的以0.5％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入50μL CTG试剂，室温避光震荡0.5小时。用FlexStation3多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图5所示，本公开实施例4化合物联合BCL-2抑制剂ABT199对人套细胞淋巴癌Z138细胞的增殖有强协同抑制作用。
测试试验例9：本公开化合物联合CDK4/6抑制剂Palbociclib对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基DMEM(Gibco#21068028)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3599)，1.5mL不透明棕色Eppendorf管；Palbociclib(Selleckchem#S1116)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000047
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：A375(ATCC#CRL-1619)，培养基为含10％FBS的DMEM培养基。
实验方法：
将A375细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度为300个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。用DMSO分别将实施例4化合物稀释至200μM，Palbociclib稀释至1000μM；将稀释后的化合物用含10％FBS的DMEM培养基进行40倍稀释，其浓度分别为5μM和25μM；分别以此为起始浓度，用配制好的2.5％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，Palbociclib连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及Palbociclib至前一天铺板的A375细胞的培养板中。阳性对照组为未种植细胞的含0.25％DMSO的培养基孔；阴性对照组为种有细胞的以0.25％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图6所示，本公开实施例4化合物联合Palbociclib对A375细胞的增殖有弱协同抑制作用。
测试试验例10：本公开化合物联合CDK4/6抑制剂Palbociclib对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基RPMI-1640(Gibco#A10491-01)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；Palbociclib(Selleckchem#S1116)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000048
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：A2780(Cobioer#CBP60283)，培养基为含10％FBS的RPMI-1640培养基。
实验方法：
将A2780细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整细胞密度为600个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。
用DMSO分别将实施例4化合物稀释至100μM，Palbociclib稀释至2mM；将稀释后的化合物用含10％FBS的RPMI-1640培养基进行100倍稀释，其浓度分别为1μM和20μM；分别以此为起始浓度，用配制好的1％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，Palbociclib连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及Palbociclib至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图7所示，本公开实施例4化合物联合Palbociclib对A2780细胞的增殖有协同抑制作用。
测试试验例11：本公开化合物联合多西他赛对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基RPMI-1640(Gibco#A10491-01)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；多西他赛(MCE#HY-B0011)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000049
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：OVCAR3(ATCC#HTB-161)，培养基为含20％FBS，0.01mg/ml bovine insulin(Yeasen#40107ES60)的RPMI-1640培养基；A2780(Cobioer#CBP60283)，培养基为含10％FBS的RPMI-1640培养基。
实验方法：
将OVCAR3及A2780细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度为1500及600个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。用DMSO分别将实施例4化合物稀释至40μM，多西他赛稀释至4μM；将稀释后的化合物用含10％FBS的RPMI-1640培养基进行40倍稀释，其浓度分别为100nM和10nM；分别以此为起始浓度，用配制好的2.5％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，多西他赛连续稀释4个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及多西他赛至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.5％DMSO的培养基孔；阴性对照组为种有细胞的以0.5％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图8A和图8B所示，本公开实施例4化合物联合多西他赛对OVCAR3及A2780细胞的增殖有协同抑制作用。
测试试验例12：本公开化合物联合多西他赛对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基DMEM(Gibco#21068028)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；Docetaxel(MCE#HY-B0011)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000050
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：A549(ATCC#CCL-185)，培养基为含10％FBS的DMEM培养基。
实验方法：
将A549细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整细胞密度为200个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。
用DMSO分别将实施例4化合物稀释至50μM，Docetaxel稀释至20μM；将稀释后的化 合物用含10％FBS的DMEM培养基进行100倍稀释，其浓度分别为0.5μM和0.2μM；分别以此为起始浓度，用配制好的1％DMSO培养液进行2倍梯度稀释(实施例4化合物)和3倍梯度稀释(Docetaxel)，实施例4化合物连续稀释7个浓度梯度，Docetaxel连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及Docetaxel至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图9所示，本公开实施例4化合物联合多西他赛对A549细胞的增殖有协同抑制作用。
测试试验例13：本公开化合物联合紫杉醇对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基RPMI-1640(Gibco#A10491-01)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；紫杉醇(Selleck#S1150)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000051
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：OVCAR3(ATCC#HTB-161)，培养基为含20％FBS，0.01mg/ml bovine insulin(Yeasen#40107ES60)的RPMI-1640培养基；A2780(Cobioer#CBP60283)，培养基为含10％FBS的RPMI-1640培养基；
实验方法：
将OVCAR3及A2780细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度分别为1500及600个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。
用DMSO分别将实施例4化合物稀释至100μM，紫杉醇稀释至50μM；将稀释后的化合物用含10％FBS的RPMI-1640培养基进行100倍稀释，其浓度分别为1μM和0.5μM；分别以此为起始浓度，用配制好的1％DMSO培养液进行2倍(实施例4化合物)梯度稀释和3倍(紫杉醇)梯度稀释，实施例4化合物连续稀释7个浓度梯度，紫杉醇连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及紫杉醇至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图10A和10B所示，本公开实施例4化合物联合紫杉醇对A2780及OVCAR3细胞的增殖有协同抑制作用。
测试试验例14：本公开化合物联合紫杉醇对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基McCoy's 5a(ATCC#30-2007)；细胞培养基Eagle's Minimum Essential Medium(ATCC#30-2003)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；紫杉醇(Selleck#S1150)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000052
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：HeLa(ATCC#HTB-41)，培养基为含10％FBS的Eagle's Minimum Essential Medium培养基；A253(ATCC#HTB-41)，培养基为含10％FBS的McCoy's 5a培养基。
实验方法：
将HeLa及A253细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后均调整相应细胞密度为800个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。
用DMSO将实施例4化合物稀释至25mM，将紫杉醇稀释至20μM；将稀释后的化合物用含10％FBS的培养基进行100倍稀释，其浓度为别250μM和200nM；分别以此为起始浓度，用配制好的1％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，紫杉醇连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及紫杉醇至前一天铺板的相应HeLa细胞的培养板中。
用DMSO将实施例4化合物稀释至5mM，将紫杉醇稀释至200μM；将稀释后的化合物用含10％FBS的培养基进行100倍稀释，其浓度为别50μM和2000nM；分别以此为起始浓度，用配制好的1％DMSO培养液进行3倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，紫杉醇连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及紫杉醇至前一天铺板的相应A253细胞的培养板中。
阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图11A和11B所示，本公开实施例4化合物联合紫杉醇对HeLa和A253细胞的增殖有协同抑制作用。
测试试验例15：本公开化合物联合卡铂对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基McCoy's 5a(ATCC#30-2007)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；卡铂(MCE#HY-17393)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000053
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：A253(ATCC#HTB-41)，培养基为含10％FBS的McCoy's 5a培养 基。
实验方法：
将A253细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度为800个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。用DMSO将实施例4化合物稀释至5mM，用含1％DMSO的细胞培养基将Carboplatin稀释至1mM；将稀释后的实施例4化合物用含10％FBS的McCoy's 5a培养基进行100倍稀释，其浓度为50μM；分别以此为起始浓度，用配制好的1％DMSO培养液进行3倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，卡铂连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及卡铂至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃、CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图12所示，本公开实施例4化合物联合卡铂对A253细胞的增殖有协同抑制作用。
测试试验例16：本公开化合物联合顺铂对细胞增殖抑制的影响
实验材料和仪器：
本实验所需的塑料器皿及消耗品包括：细胞培养基Eagle's Minimum Essential Medium(ATCC#30-2003)；胎牛血清(FBS)(Gibco#10099-141C)；PBS磷酸盐缓冲液(Hyclone#SH30256.01)；100％DMSO(Sigma#D2650)；96孔透底无菌培养板(Corning#3610)，1.5mL不透明棕色Eppendorf管；顺铂(MCE#HY-17394)；0.25％胰酶(Gibco#25200-072)；
Figure PCTCN2023071046-appb-000054
Luminescent细胞活力检测试剂盒(CTG)(Promega#G7573)，25mL血清移液管吸头(JET)，5mL血清移液管吸头(JET)，P1000移液管吸头和P200移液管吸头(Axygen)。
本实验所需的仪器设备包括：Eppendorf移液器；Eppendorf移液枪；恒温二氧化碳培养箱(ThermoFisher)；Envision多功能酶标仪(Perkin Elmer)。
本实验所需要的细胞：HeLa(ATCC#HTB-41)，培养基为含10％FBS的Eagle's Minimum Essential Medium培养基。
实验方法：
将HeLa细胞在细胞培养瓶内消化(用0.25％胰酶)，用对应的新鲜培养基重悬，计数后调整相应细胞密度为800个细胞/80μL/孔，铺板至96孔板内，放入37℃，CO 2细胞培养箱，培养过夜。用DMSO将实施例4化合物稀释至25mM，用含1％DMSO的细胞培养基将顺铂稀释至200μM；将稀释后的实施例4化合物用含10％FBS的培养基进行100倍稀释，其浓度为250μM；分别以此为起始浓度，用配制好的1％DMSO培养液进行2倍梯度稀释，实施例4化合物连续稀释7个浓度梯度，顺铂连续稀释6个浓度梯度。最后分别以正交的方式转移10μl梯度稀释后的实施例4化合物及顺铂至前一天铺板的相应细胞的培养板中。阳性对照组为未种植细胞的含0.2％DMSO的培养基孔；阴性对照组为种有细胞的以0.2％DMSO处理的孔。细胞板放入37℃，CO 2细胞培养箱，细胞药物处理培养7天。取出药物处理后的细胞板，每孔加入100μL CTG试剂，室温避光震荡0.5小时。用Envision多功能酶标仪读取发光值。计算细胞存活率(％)＝100-100×(阴性对照孔发光值的平均值-化合物孔发光值)/(阴性对照发光值的平均值-阳性对照发光值的平均值)。使用Combenefit软件来分析矩阵的存活率，并通过测试试验例7中相同的布利斯独立模型(Bliss Independence model)的统计分析来确定药物之间的作用(协同、独立或者拮抗)。
实验结果：如图13所示，本公开实施例4化合物联合顺铂对HeLa细胞的增殖有协同抑 制作用。
测试试验例17：本公开化合物联合CDK4/6抑制剂Palbociclib在A375异种移植瘤模型中的药效
实验材料和方法：
利用A375皮下肿瘤模型，对实施例4化合物与CDK4/6抑制剂Palbociclib(南京先声东元制药有限公司)联用的抗肿瘤活性进行研究。对32只雌性NPG小鼠(6-7周龄，购自北京维通达生物技术有限公司)皮下接种5.0×10 6个人黑色素瘤A375细胞(购自北纳生物，BNCC100266)，待肿瘤生长至平均体积99.8mm 3时，随机分为4组，每组8只；第一组给予10ml/kg溶媒对照(bid)，第二组灌胃给予10mg/kg实施例4化合物溶液(bid)，第三组灌胃给予20mg/kg化合物Palbociclib混悬液(qd)，第四组灌胃给予10mg/kg实施例4化合物溶液(bid)同时灌胃给予20mg/kg化合物Palbociclib混悬液(qd)；以上溶媒均为含0.5％甲基纤维素(MC)的水溶液(Sigma-Aldrich,M0512-250g)。整个实验过程中小鼠可自由进食、饮水。
肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。
肿瘤体积(V)的计算公式：V＝0.5a×b 2，其中a和b分别表示肿瘤的长径和短径。
相对肿瘤体积(RTV)的计算公式：RTV＝V t/V 0，其中V 0是第0天分组给药时测得的肿瘤体积，V t为某一次测量时的肿瘤体积。
每周两次称量小鼠体重。
用Graphpad Prism 9.0软件进行分析。用Two-way ANOVA对相对肿瘤体积进行统计学分析；每个时间点的数据中，组间两两比较使用Tukey进行分析。
实验结果：如图14所示，其中*P<0.05，***P<0.001，可见本公开实施例4化合物能显著抑制肿瘤生长，且联合Palbociclib后肿瘤体积显著小于单药组。整个实验过程中小鼠状态良好，没有明显体重变化。该实验表明，实施例4联合Palbociclib对人黑色素瘤A375细胞有协同抗肿瘤效果，且安全性良好。

Claims (16)

  1. 一种用于治疗癌症的包括PRMT5抑制剂和至少一种抗癌治疗剂的药物组合物或联用药物组合，其中所述PRMT5抑制剂选自化合物I及其药学上可接受的盐
    Figure PCTCN2023071046-appb-100001
  2. 根据权利要求1所述的药物组合物或联用药物组合，其中所述至少一种抗癌治疗剂选自铂衍生物、烷化剂、拓扑异构酶抑制剂、紫杉醇类、抗肿瘤抗生素、植物生物碱、核苷类似物、B细胞淋巴瘤2(BCL-2)抑制剂、I型PRMT抑制剂和细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂中的一种或多种，优选选自B细胞淋巴瘤2(BCL-2)抑制剂、I型PRMT抑制剂、细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂、紫杉醇类和铂衍生物中的一种或多种，更加优选选自B细胞淋巴瘤2(BCL-2)抑制剂、I型PRMT抑制剂和细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂中的一种或多种。
  3. 根据权利要求2所述的药物组合物或联用药物组合，其中所述铂衍生物选自顺铂、卡铂、奥沙利铂和沙铂，优选顺铂和卡铂。
  4. 根据权利要求2所述的药物组合物或联用药物组合，其中所述烷化剂选自链脲霉素、达卡巴嗪、丙卡巴嗪、司莫司汀、洛莫司汀、福莫司汀、尼莫司汀、苯达莫司汀、乌拉莫司汀、环磷酰胺、异环磷酰胺、美法仑和六甲基三聚氰胺。
  5. 根据权利要求2所述的药物组合物或联用药物组合，其中所述拓扑异构体抑制剂选自依托泊苷、替尼泊苷、喜树碱、10-羟基喜树碱、伊立替康和托泊替康。
  6. 根据权利要求2所述的药物组合物或联用药物组合，其中所述紫杉醇类选自多西他赛、卡巴他赛和紫杉醇，优选多西他赛和紫杉醇。
  7. 根据权利要求2所述的药物组合物或联用药物组合，其中所述抗肿瘤抗生素选自阿霉素、柔红霉素、放线菌素、博来霉素、麦曲霉素、依沙芦星和米托蒽醌。
  8. 根据权利要求2所述的药物组合物或联用药物组合，其中所述植物生物碱选自长春碱、长春新碱和长春地辛。
  9. 根据权利要求2所述的药物组合物或联用药物组合，其中所述核苷类似物选自阿糖胞苷、氟达拉滨、碘脱氧尿苷、溴脱氧尿苷、氟脱氧尿苷和吉西他滨。
  10. 根据权利要求2所述的药物组合物或联用药物组合，其中所述BCL-2抑制剂选自维奈托克(ABT-199)、ABT-737、ABT-263、APG-1252、S-055746、BDA-366、HA14-1、BH3I-1、阿朴棉子酚、TW-37、TM12-06和obatoclax，优选ABT-199。
  11. 根据权利要求2所述的药物组合物或联用药物组合，其中所述I型PRMT抑制剂选自PRMT1抑制剂、PRMT3抑制剂、PRMT4抑制剂、PRMT6抑制剂和PRMT8抑制剂，优选PRMT1抑制剂，更优选为GSK3368715。
  12. 根据权利要求2所述的药物组合物或联用药物组合，其中所述细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂选自帕博西尼(Palbociclib)、Ribociclib、Abemaciclib、ebvaciclib和trilaciclib，优选Palbociclib。
  13. 根据权利要求1-12任一项所述的联用药物组合，其中PRMT5抑制剂和至少一种抗癌治疗剂可分开地包装或包装在一起。
  14. 一种治疗癌症的方法，所述方法包含向需要所述治疗的个体给予治疗有效量的如权利要求1-12任一项所述的药物组合物或权利要求1-13任一项所述的联用药物组合，所述癌症优选为淋巴瘤、胰腺癌、结肠癌、恶性黑色素瘤、卵巢癌、肺癌、宫颈癌和头颈癌，更加优选 选自淋巴瘤、胰腺癌、结肠癌或恶性黑色素瘤。
  15. 根据权利要求14所述的方法，其中PRMT5抑制剂和至少一种抗癌治疗剂各自呈药物组合物形式，可同时、顺序或间隔给药。
  16. 权利要求1-12任一项所述的药物组合物或权利要求1-13任一项所述的联用药物组合在制备用于治疗癌症的药物中的用途，所述癌症优选选自淋巴瘤、胰腺癌、结肠癌、恶性黑色素瘤、卵巢癌、肺癌、宫颈癌和头颈癌，更加优选选自淋巴瘤、胰腺癌、结肠癌和恶性黑色素瘤。
PCT/CN2023/071046 2022-01-06 2023-01-06 Prmt5抑制剂和抗癌治疗剂的组合 WO2023131305A1 (zh)

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WO2019173804A1 (en) * 2018-03-09 2019-09-12 Pharmablock Sciences (Nanjing), Inc. Inhibitors of protein arginine methyltransferase 5 (prmt5), pharmaceutical products thereof, and methods thereof
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