WO2022021784A1 - 一种parp抑制剂tsl-1502中间体tsl-1502m的用途 - Google Patents

一种parp抑制剂tsl-1502中间体tsl-1502m的用途 Download PDF

Info

Publication number
WO2022021784A1
WO2022021784A1 PCT/CN2020/140553 CN2020140553W WO2022021784A1 WO 2022021784 A1 WO2022021784 A1 WO 2022021784A1 CN 2020140553 W CN2020140553 W CN 2020140553W WO 2022021784 A1 WO2022021784 A1 WO 2022021784A1
Authority
WO
WIPO (PCT)
Prior art keywords
tsl
cells
cancer
tumor
azd2281
Prior art date
Application number
PCT/CN2020/140553
Other languages
English (en)
French (fr)
Inventor
杨海龙
李德馨
马晓慧
唐海
蔡金勇
周水平
郭建飞
范立君
沈伟生
王萍
Original Assignee
江苏天士力帝益药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏天士力帝益药业有限公司 filed Critical 江苏天士力帝益药业有限公司
Publication of WO2022021784A1 publication Critical patent/WO2022021784A1/zh

Links

Images

Classifications

    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the invention belongs to the technical field of pharmaceutical preparation, in particular to the use of TSL-1502M, an intermediate of PARP inhibitor TSL-1502.
  • PARP inhibitor TSL-1502 chemical name is (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(3-methyl-1-((S)-1-propyl) Pyrrolidin-3-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinoline-5-oxy)tetrahydro-2H-pyran-2-
  • the compound of formic acid set its number as TSL-1502
  • this compound first appeared in 201180002886.8 (publication number CN102510863A, authorized announcement number CN102510863B, hereinafter referred to as the 2011 patent), the compound of paragraph 0177, its structural formula is shown in formula I.
  • the above patent discloses specific tumor types in claim 16, such as head cancer, thyroid cancer, neck cancer, eye cancer, skin cancer, oral cancer, throat cancer, esophagus cancer, breast cancer, bone cancer, blood cancer, bone marrow cancer, lung cancer , colon cancer, sigmoid colon cancer, rectal cancer, stomach cancer, prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, brain cancer, colon cancer, heart cancer, adrenal cancer, subcutaneous tissue cancer, lymph node cancer, pigment cancer , malignant glioma, etc.
  • the therapeutic effect of melanoma and human breast cancer cell lines was confirmed only in Example 19, and other tumors were not involved.
  • TSL-1502M is an intermediate for the preparation of TSL-1502, its application number is 201910465780.X, and the name of the invention is an intermediate TSL-1502M of a PARP inhibitor and its preparation method, (application date: May 31, 2019 ,, the publication number is CN112010850A), the structural formula is shown in formula II,
  • TSL-1502M The compound of formula II is designated as TSL-1502M.
  • TSL-1502M As an intermediate, it can be used for the preparation of TSL-1502 and the detection of TSL-1502. It is useful as a reference substance. However, there is no information on the pharmaceutical use of TSL-1502M disclosed in this document.
  • the present invention provides the application of TSL-1502M in the preparation of medicaments for preventing and treating anti-tumor.
  • TSL-1502M The structure of the TSL-1502M is shown in formula II:
  • the compound has an anti-tumor effect, mainly by inhibiting the proliferation of tumor cells and inducing cell apoptosis to play an anti-tumor effect.
  • the tumor cells include: tumor cells with defective BRCA function and tumor cells with BRCA gene mutation , or tumor cells with normal BRCA function.
  • the tumor includes the following tumors:
  • the tumor is specifically breast cancer, pancreatic cancer, ovarian cancer, and colon cancer.
  • the tumor is preferably breast cancer, ovarian cancer, pancreatic cancer.
  • the present invention also provides a pharmaceutical preparation composition containing TSL-1502M.
  • the pharmaceutical preparation composition can be in any ingestible pharmaceutical form: such as: tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet preparations, capsules, hard capsules, soft capsules, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, ointments, Plasters, creams, sprays, drops, patches.
  • the pharmaceutical preparation of the present invention is preferably in the form of a unit dose pharmaceutical preparation.
  • the unit dose of the medicament may contain 0.1-1000 mg of the TSL-1502M of the present invention, and the rest are pharmaceutically acceptable auxiliary materials.
  • the pharmaceutically acceptable adjuvants can be 0.01-99.99% by weight of the total weight of the formulation.
  • the dosage of the pharmaceutical preparation of the present invention is determined according to the patient's condition, such as 1-3 times a day, 1-20 tablets at a time, and the like.
  • the pharmaceutical preparation of the present invention is an oral preparation or an injection.
  • the oral preparation is selected from capsules, tablets, dropping pills, granules, concentrated pills, and oral liquids.
  • the injection is selected from liquid, semi-solid, solid, powder form, preferably injection, powder injection.
  • the pharmaceutical preparation of the present invention may contain adjuvants, such as binders, fillers, diluents, tableting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, if necessary Tablets are coated.
  • adjuvants such as binders, fillers, diluents, tableting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, if necessary Tablets are coated.
  • Suitable fillers include cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable humectants include sodium lauryl sulfate.
  • the pharmaceutical preparation of the present invention can be prepared as a solid oral composition by mixing, filling, tableting and other common methods. Repeated mixing allows the active to be distributed throughout those compositions where large amounts of fillers are used.
  • Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, Emulsifiers such as lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives agents such as methylparaben or propylparaben or sorbic acid and, if desired, conventional flavoring or coloring agents.
  • suspending agents for example sorbitol, syrup, methylcellulose, gelatin, hydroxy
  • liquid unit dosage forms are prepared containing the active substance of the present invention and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solutions are usually prepared by dissolving the active substance in a carrier, filter sterilizing before filling into a suitable vial or ampoule, and sealing. Adjuvants such as a local anesthetic, preservatives and buffering agents can also be dissolved in the carrier. To improve its stability, the composition can be frozen after filling into a vial and the water removed under vacuum.
  • the dosage of TSL-1502M can be 0.01 mg-2000 mg per day.
  • TSL-1502M is mainly Better than TSL-1502.
  • TSL-1502M has a synergistic effect on cytotoxic drugs.
  • TSL-1502M and olaparib can inhibit DNA damage repair (preferably breast cancer), and the activity of TSL-1502M is stronger than that of olaparib.
  • TSL-1502 and TSL-1502M induce breast cancer cell cycle arrest in G 2 /M phase (preferably breast cancer), and the activity of TSL-1502M is significantly stronger than that of TSL-1502.
  • TSL-1502 and TSL-1502M can induce breast cancer cell apoptosis, and the activity of TSL-1502M is stronger than that of olaparib.
  • FIG. 1 Effects of TSL-1502 and TSL-1502M on cell proliferation in vitro
  • FIG. 1 The inhibitory effect of TSL-1502, TSL-1502M and cytotoxic drugs on the proliferation of MDA-MB-436 and SW620 cells;
  • FIG. 1 Inhibitory effect of TSL-1502 and ABT-888 on the colony formation of MDA-MB-436 cells
  • FIG. 7 Effects of TSL-1502, TSL-1502M and AZD2281 on induction of apoptosis in MDA-MB-436 cells.
  • the reference application number is 201910465780.X, the invention name is a kind of PARP inhibitor intermediate TSL-1502M and Example 1 of its preparation method:
  • Quenching after the reaction is completed, slowly add hydrochloric acid (73 g, 2 mol) with a mass percentage concentration of 36 to 38% to the reaction mixture, adjust the pH to between 4 and 5, control the rate of addition, and add 20% sodium hydroxide to the reaction mixture. Absorb a large amount of gas, and control the temperature to 20 °C ⁇ 30 °C in order to prevent the material from being punched.
  • hydrochloric acid 73 g, 2 mol
  • Post-processing S1, the quenched reaction solution is concentrated under reduced pressure to remove ethanol, and the temperature does not exceed 70 °C;
  • the combined organic phase obtained in S7 and S6 is washed twice with 3% aqueous sodium bicarbonate solution and then dried with anhydrous sodium sulfate, filtered to remove the sodium sulfate solid, and the filtrate is concentrated under reduced pressure and then vacuum-dried to obtain the intermediate TSL of the PARP inhibitor. -1502M 226g.
  • TSL-1502 white powder, batch number 120301, purity 99.56%, water content 16.65%, sealed, stored at 2-8°C, provided by the applicant;
  • TSL-1502M white powder, batch number 20150801, purity 98.6%, sealed, stored at 2-8°C, provided by the applicant;
  • ABT-888 (Veliparib, Veliparib): white powder, batch number HM-069-8-20101112, purity 98.12%, sealed, stored at 2-8°C, purchased from Shanghai Haoyuan Company, ABT-888 is a new type of potent PARP-1 and PARP-2 inhibitors, mainly for breast cancer;
  • AZD2281 (Olaparib, Olaparib): white powder, batch number 20131105, purity 99.15%, sealed, stored at 2-8°C, purchased from Shanghai Demo Company, AZD2281 is a PARP inhibitor, approved indications include ovarian cancer , breast cancer, pancreatic cancer, prostate cancer;
  • Temozolomide (abbreviation: TMZ): white powder, batch number 20150418, sealed, stored at 2-8°C, temozolomide is an imidazole tetrazine derivative, mainly used for glioblastoma multiforme;
  • SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan.
  • the cas number is 86639-52-3): pale yellow powder, purity 99.1%, batch number E060206, sealed, 2-8°C Preserved, purchased from Shanghai Junjie Biotechnology Company, irinotecan is a semi-synthetic derivative of camptothecin, mainly used for colon (rectal) cancer.
  • TSL-1502, TSL-1502M, AZD2281 and ABT-888 were made into 10 mM stock solution with DMSO, and stored in aliquots at -70°C; TMZ was made into 200 mM stock solution with DMSO, and aliquoted and stored at -70°C.
  • RPMI1640, L-15, F-10, DMEM and IMDM were purchased from Gibco BRL; FBS was purchased from Gibco BRL; SRB was purchased from Sigma; anti-Rad51 primary antibody was purchased from Santa Cruz; anti-P- ⁇ -H2AX and PARP Primary antibody was purchased from Cell Signaling Technology; primary anti- ⁇ -Tubulin antibody was purchased from Sigma; horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Calbiochem; Alexa 488 goat anti-rabbit fluorescent secondary antibody was purchased from Molecular Probes Company; Immobilon Western HRP Substrate luminal reagent was purchased from Millipore Company; other common chemical reagents were domestic analytical pure (AR) reagents;
  • Multifunctional microplate reader Synergy H4 was purchased from BioTek Company; carbon dioxide incubator (Model 3111) was purchased from Thermo Company; inverted microscope XDS-1B was purchased from Chongqing Optoelectronics Instrument Co., Ltd.; flow cytometer (FACS Calibur flow cytometer) was purchased from Beckman Dickson company; laser confocal microscope from Olympus; Western blot imager from GlinxScience instruments.
  • SRB method Sulfonylrhodamine B protein staining method
  • a certain number of cells in logarithmic growth phase were seeded in 96-well culture plates. After 24 hours of adherent growth, different concentrations (1, 3, 10, 30, 100, 300, 1000, 3000, 10000 nM) of drugs were added. After 10 days of drug action, cells were fixed with trichloroacetic acid. Then the SRB solution was stained; finally, Tris solution was added to dissolve the SRB, and the OD value was measured at a wavelength of 510 nm by a microplate reader, and the cell growth inhibition rate was calculated by the following formula:
  • Inhibition rate (OD value control well - OD value administration well )/OD value control well ⁇ 100%
  • the median inhibitory concentration IC 50 was calculated.
  • Cells were seeded in 6-well plates at a density of 200 per well. After 24 hours of adherent growth, different concentrations (0.3, 1, 3, 10, 30, 100 nM) of drugs were added, and the culture medium and drugs were changed every 5 days. After 20 days, they were fixed with methanol: glacial acetic acid (3:1). Stained with crystal violet, counted, and photographed.
  • the cells attached to the small coverslip were treated with drugs, fixed with 4% paraformaldehyde, penetrated the membrane in phosphate buffer (pH 7.4) containing 0.3% Triton X-100, and added anti-Rad51 after blocking.
  • the primary antibody was incubated at 4°C, followed by Alexa 488goat anti-rabbit fluorescent secondary antibody was incubated, and finally stained with DAPI. After mounting, the specimens were observed and photographed with a laser confocal microscope.
  • MDA-MB-436 was inoculated in a six-well plate (2 ⁇ 10 5 /ml), and different concentrations of TSL-1502M or AZD2281 (100, 1000, 10000nM) were added for 1.5 hours, and then 1mM TMZ was added for 0.5 hours.
  • Cells were lysed with ⁇ SDS gel loading buffer (50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue). The cell lysate was denatured by heating in a boiling water bath, and subjected to SDS-PAGE electrophoresis.
  • the protein was transferred to a PVDF membrane using a wet transfer system, and the PVDF membrane was placed in blocking solution (5% nonfat dry milk diluted in TBS/T). Block at room temperature, and then react with I and II antibodies; after washing the membrane, use Immobilon Western HRP Substrate luminal reagent to develop color, and take pictures with a Western blot imager.
  • blocking solution 5% nonfat dry milk diluted in TBS/T.
  • MDA-MB-436 was inoculated in a six-well plate (1 ⁇ 10 5 /ml), and after adding different concentrations of TSL-1502, TSL-1502M (0.01, 0.1, 1 ⁇ M) or AZD2281 (0.1, 1, 10 ⁇ M) for 120 hours, Cells were lysed by adding 1 ⁇ SDS gel loading buffer (50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue).
  • 1 ⁇ SDS gel loading buffer 50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue.
  • Cell lysates were denatured by heating in a boiling water bath, Carry out SDS-PAGE electrophoresis, after electrophoresis, transfer the protein to PVDF membrane with wet transfer system, put the PVDF membrane in blocking solution (5% nonfat milk powder diluted in TBS/T) to block at room temperature, and then react with I and II antibodies; After washing the membrane, use Immobilon Western HRP Substrate luminal reagent to develop color and take pictures with Western blot imager.
  • blocking solution 5% nonfat milk powder diluted in TBS/T
  • MDA-MB-436 cells were seeded in 6-well plates (2 ⁇ 10 5 /ml), and 48 h after drug treatment, the cells were collected. Fix with ethanol overnight, then add RNase and propidium iodide, mix well, and stain at 37°C in the dark for 30 minutes. Finally, the DNA content in the cells is detected by FACSCalibur flow cytometer. Each group of samples is 1 ⁇ 10 4 cell. The experimental results were analyzed with ModFit Lt Mac V3.0 software.
  • TSL-1502 and TSL-1502M inhibit the proliferation of various in vitro cultured tumor cells
  • TSL-1502M has a very significant inhibitory effect on the proliferation of BRCA gene-deficient or mutant cells (V-C8, MDA-MB-436, Capan-1 and UWB1.289), with IC 50 of 0.2 ⁇ 0.0nM and 0.9 ⁇ 0.2nM, respectively , 100.6 ⁇ 73.8nM, 1.9 ⁇ 0.4nM), the inhibitory effect was significantly stronger than its prodrug TSL-1502 (IC 50 were 132.1 ⁇ 4.7nM, 79.6 ⁇ 12.5nM, ⁇ 10000nM, 935.7 ⁇ 526.6nM, respectively), and the reference Than drugs AZD2281 (IC 50 were 18.6 ⁇ 1.3nM, 4.0 ⁇ 0.7nM, 586.1 ⁇ 89.4nM, ⁇ 1nM (inhibition rate was slightly weaker than TSL-1502M)) and ABT-888 (IC 50 were 314.2 ⁇ 24.4nM, 114.7 ⁇ 60.8nM, NA, 2102.5 ⁇ 1470.1nM);
  • TSL-1502M TSL-1502
  • AZD2281 and ABT-888 all had weaker proliferation inhibition.
  • BRCA gene normal cells V-C8#13-5 and UWB1.289BRCA1
  • TSL-1502M TSL-1502
  • AZD2281 ABT-888
  • 468 cells, TSL-1502M and AZD2281 had certain inhibitory effects on proliferation (IC 50 were 73.6 ⁇ 19.5nM and 541.1 ⁇ 93.8nM, respectively).
  • TSL-1502M is more sensitive to BRCA1/2-deficient cells, and has a selective inhibitory effect on cell proliferation. , AZD2281 and ABT-888 strong.
  • TSL-1502 and TSL-1502M synergistic cytotoxic drugs
  • BRCA1 mutant MDA-MB-436 cells 30, 100, 300 nM of TSL-1502, 3, 10, 30 nM of TSL-1502M, 10, 30, 100 nM of AZD2281 or 30, 100, 300 nM of ABT-888 with 10 , 30 ⁇ M TMZ for 120 hours.
  • TSL-1502, TSL-1502M and reference compounds AZD2281 and ABT-888 could significantly enhance the inhibitory effect of TMZ on the proliferation of MDA-MB-436 cells.
  • TSL-1502M In SW620 cells with normal BRCA gene, 3, 10 ⁇ M of TSL-1502M or 3, 10 ⁇ M of AZD2281 combined with 0.1, 1 nM of irinotecan active metabolite SN38 for 144 hours, TSL-1502M and the reference compound AZD2281 can significantly Enhancement of SN38's proliferation-inhibitory effect on SW620 cells.
  • TSL-1502M has a synergistic effect on cytotoxic drugs in BRCA gene mutation or normal tumor cells.
  • Table 3-1 Proliferation inhibitory effect on MDA-MB-436 cells. Inhibition rate (%, Mean ⁇ SD)
  • TSL-1502 inhibits the clone formation of BRCA function-deficient tumor cells
  • TSL-1502 prodrug takes a long time to produce anti-tumor effect. Therefore, when we tested the inhibitory effect of TSL-1502 on clonogenicity of MDA-MB-436 cells (BRCA1 gene mutation), the effect time was extended to 20 days. The results are shown in FIG. 3 , both TSL-1502 and the reference compound were able to inhibit the colony formation of MDA-MB-436 cells in a dose-dependent manner. TSL-1502 inhibited clonogenicity with IC50 of 9.5 ⁇ 1.4 nM, and AZD2281 and ABT-888 inhibited clonogenicity with IC50 of 1.7 ⁇ 0.1 and 9.5 ⁇ 1.4 nM, respectively.
  • TSL-1502 and the reference drugs AZD2281 and ABT-888 could inhibit the clonogenicity of BRCA function-deficient tumor cells, and the inhibitory effect of TSL-1502 was stronger than that of ABT-888 and weaker than that of AZD2281.
  • TSL-1502M can selectively induce the formation of Rad51 foci in cells with normal BRCA1/2 function, and its effect is similar to that of AZD2281.
  • TSL-1502M and TMZ significantly enhanced the phosphorylation of ⁇ -H2AX in MDA-MB-436 cells
  • Phosphorylated histone H2AX ( ⁇ -H2AX) is a marker of DNA damage, and we examined the effect of TSL-1502M combined with TMZ on DNA damage repair in BRCA1-mutated MDA-MB-436 cells. After TSL-1502M and the reference compound AZD2281 were combined with TMZ, the effect of ⁇ -H2AX on ⁇ -H2AX was shown in Figure 5. The results showed:
  • TSL-1502 induces cell arrest in G2/M phase
  • MDA-MB-436 cells were treated with TSL-1502, TSL-1502M and the reference compound AZD2281. After 48 hours, the effects on cell cycle were detected by flow cytometry. The result is shown in Figure 6:
  • TSL-1502, TSL-1502M and the reference compound AZD2281 were all able to dose - dependently induce an increase in cells in the G2/M phase, ie, induce cell cycle arrest in the G2/M phase.
  • TSL-1502M induces G 2 /M cycle arrest similar to that of AZD2281: 10 ⁇ M dose can significantly induce G 2 /M arrest; TSL-1502 induces G 2 /M cycle arrest is weaker than TSL-1502M And the reference compound AZD2281: 10 ⁇ M dose induces weak G 2 /M arrest.
  • TSL-1502 and TSL-1502M induced MDA-MB-436 cell cycle arrest in G 2 /M phase, and the activity of TSL-1502M was significantly stronger than that of TSL-1502, which was similar to the reference compound AZD2281.
  • TSL-1502 and TSL-1502M induce apoptosis
  • the MDA-MB-436 cells were treated with TSL-1502, TSL-1502M and the reference drug AZD2281. After 120 hours, the cleavage changes of the apoptosis marker protein PARP were detected by Western Blot. The result is shown in Figure 7:
  • TSL-1502 has a weaker induction of PARP cleavage at a dose of 1 ⁇ M, and it is more pronounced at 10 ⁇ M;
  • TSL-1502M can significantly induce PARP cleavage at a dose of 0.01 ⁇ M
  • the reference drug AZD2281 had a weaker induction of PARP cleavage at a dose of 0.1 ⁇ M, and was more pronounced at 1 ⁇ M and 10 ⁇ M.
  • TSL-1502, TSL-1502M and the reference drug AZD2281 could induce apoptosis of MDA-MB-436 cells.
  • the activity of TSL-1502 was weaker than that of AZD2281, and the activity of TSL-1502M was stronger than that of AZD2281.
  • the present invention studies the in vitro antitumor activity and action mechanism of TSL-1502 and its metabolite TSL-1502M. mainly includes:
  • TSL-1502M can significantly inhibit the proliferation of BRCA gene-deficient or mutant cells, such as V-C8, MDA-MB-436 and UWB1.289 cells, with IC50 of 0.2 ⁇ 0.0nM, 0.9 ⁇ 0.2nM and 1.9, respectively ⁇ 0.4nM, the inhibitory effect was significantly stronger than its prodrug TSL-1502 ( IC50 of 132.1 ⁇ 4.7nM, 79.6 ⁇ 12.5nM and 935.7 ⁇ 526.6nM, respectively), and the reference drug AZD2281 ( IC50 of 18.6 ⁇ 1.3, respectively) nM, 4.0 ⁇ 0.7 nM and ⁇ 1 nM (slightly weaker inhibition than TSL-1502M) and ABT-888 (IC 50 of 314.2 ⁇ 24.4 nM, 114.7 ⁇ 60.8 nM and 2102.5 ⁇ 1470.1 nM, respectively).
  • BRCA gene normal cells such as V-C8#13-5 and UWB1.289BRCA1 cells, TSL-1502M, TSL-1502, AZD2281 and ABT-888 were weak.
  • TSL-1502 can inhibit the colony formation of MDA-MB-436 cells.
  • TSL-1502M significantly potentiates the proliferation-inhibitory effect of temozolomide on MDA-MB-436 cells (BRCA1 mutant) and SW620 cells (BRCA1/2 normal) by the active metabolite SN38 of irinotecan; selectively induces BRCA1/2 normal cells.
  • the formation of Rad51 focal point in MDA-MB-436 cells; the phosphorylation level of ⁇ -H2AX in MDA-MB-436 cells is significantly increased after combined use with temozolomide; it can induce G 2 /M phase arrest and apoptosis in MDA-MB-436 cells;
  • the action characteristics of TSL-1502M were similar to those of the reference compound AZD2281, and the activity of TSL-1502M was stronger than that of AZD2281.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种PARP抑制剂TSL-1502的中间体TSL-1502M的抗肿瘤用途。TSL-1502M的结构式见式II。

Description

一种PARP抑制剂TSL-1502中间体TSL-1502M的用途 技术领域
本发明属于药物制备技术领域，具体涉及一种PARP抑制剂TSL-1502的中间体TSL-1502M的用途。
背景技术
PARP抑制剂TSL-1502，化学名为(2S,3S,4S,5R,6S)-3,4,5-三羟基-6-(3-甲基-1-((S)-1-丙基吡咯烷-3-基)-6,7,8,9-四氢-3H-吡唑并[3,4-c]异喹啉-5-氧基)四氢-2H-吡喃-2-甲酸的化合物(设定其编号为TSL-1502)，该化合物最早出现在201180002886.8(公开号为CN102510863A，授权公告号为CN102510863B，以下简称为2011年专利)的0177段化合物，其结构式见式Ⅰ。
Figure PCTCN2020140553-appb-000001
上述专利在权利要求16公开了具体肿瘤类型，如头部癌、甲状腺癌、颈癌、眼癌、皮肤癌、口腔癌、咽喉癌、食道癌、胸癌、骨癌、血癌、骨髓癌、肺癌、结肠癌、乙状结肠癌、直肠癌、胃癌、前列腺癌、乳腺癌、卵巢癌、肾癌、肝癌、胰腺癌、脑癌、肠癌、心脏癌、肾上腺癌、皮下组织癌、淋巴结癌、色素癌、恶性神经胶质瘤等。然而实际上，也仅在实施例19证实了黑色素瘤、人乳腺癌细胞株的治疗效果，对其他肿瘤没有涉及。
TSL-1502M，是制备TSL-1502的中间体，其申请号为201910465780.X，发明名称为一种PARP抑制剂的中间体TSL-1502M及其制备方法，(申请日为2019年05月31日，，公开号为CN112010850A)，结构式见式Ⅱ，
Figure PCTCN2020140553-appb-000002
式Ⅱ化合物设定其编号为：TSL-1502M，其作为中间体即可以用于制备TSL-1502，又可以用于TSL-1502的检测，作为对照品是有用的。但是该文献中并没有公开TSL-1502M的药物用途信息。
发明内容
本发明是在研究TSL-1502M时，意外发现其药物用途，特别是抗肿瘤的用途。
具体的，本发明提供了TSL-1502M在制备预防和治疗抗肿瘤的药物中的应用。
所述TSL-1502M的结构见式Ⅱ：
Figure PCTCN2020140553-appb-000003
本发明经过研究，意外发现该化合物具有抗肿瘤的作用，主要通过抑制肿瘤细胞增殖并诱导细胞凋亡发挥抗肿瘤作用，所述肿瘤细胞包括：BRCA功能缺陷的肿瘤细胞、BRCA基因突变的肿瘤细胞，或BRCA功能正常的肿瘤细胞。
为此，本发明所述的抗肿瘤作用，其中的肿瘤包括以下肿瘤：
所述肿瘤具体为乳腺癌、胰腺癌、卵巢癌、结肠癌。
所述肿瘤优选为乳腺癌、卵巢癌、胰腺癌。
本发明还提供了一种含有TSL-1502M药物制剂组合物，所述药物制剂组合物，可以是任何可服用的药物形式：如：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂、贴剂。
本发明的药物制剂，优选的是单位剂量的药物制剂形式，如在制成药剂时，单位剂量的药剂可含有本发明的TSL-1502M 0.1-1000mg，其余为药学上可接受的辅料。药学上可接受的辅料以重量计可以是制剂总重量的0.01-99.99％。
本发明的药物制剂在使用时根据病人的情况确定用法用量，如一日1-3次，一次1-20片等。
优选的，本发明的药物制剂为口服制剂或注射剂。
其中，所述口服制剂选自胶囊剂、片剂、滴丸、颗粒剂、浓缩丸、口服液中的一种。
其中，所述注射剂选自液体、半固体，固体，粉剂形式，优选注射液，粉针中的一种。
本发明的药物制剂，其口服给药的制剂可含有辅料，诸如粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂，必要时可对片剂进行包衣。
适用的填充剂包括纤维素、甘露糖醇、乳糖和其它类似的填充剂。适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠。适宜的润滑剂包括，例如硬脂酸镁。适宜的药物可接受的湿润剂包括十二烷基硫酸钠。
本发明的药物制剂可通过混合，填充，压片等常用的方法制备固体口服组合物。进行反复混合可使活性物质分布在整个使用大量填充剂的那些组合物中。
口服液体制剂的形式例如可以是水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或者可以是一种在使用前可用水或其它适宜的载体复配的干燥产品。这种液体制剂可含有常规的添加剂，诸如悬浮剂，例如山梨醇、糖浆、甲基纤维素、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶或氢化食用脂肪，乳化剂，例如卵磷脂、脱水山梨醇一油酸酯或阿拉伯胶；非水性载体(它们可以包括食用油)，例如杏仁油、分馏椰子油、诸如甘油的酯的油性酯、丙二醇或乙醇；防腐剂，例如对羟基苯甲酯或对羟基苯甲酸丙酯或山梨酸，并且如果需要，可含有常规的香味剂或着色剂。
对于注射剂，制备的液体单位剂型含有本发明的活性物质和无菌载体。根据载体和浓度，可以将此化合物悬浮或者溶解。溶液的制备通常是通过将活性物质溶解在一种载体中，在将其装入一种适宜的小瓶或安瓿前过滤消毒，然后密封。辅料例如一种局部麻醉剂、防腐剂和缓冲剂也可以溶解在这种载体中。为了提高其稳定性，可在装入小瓶以后将这种组合物冰冻，并在真空下将水除去。
所述应用时，TSL-1502M的使用剂量可以是一天0.01mg-2000mg。
本发明提供的抗肿瘤作用，具有以下特点：
1、发明人试验发现，对BRCA1/2功能缺陷的细胞(尤其是乳腺癌细胞、胰腺癌细胞、卵巢癌细胞)更为敏感，对细胞的增殖抑制作用具有选择性，TSL-1502M的效果要优于TSL-1502。
2、在BRCA基因突变或正常的肿瘤细胞(尤其是乳腺癌细胞)中，TSL-1502M对细胞毒药物均有增效作用。
3、TSL-1502M及奥拉帕利能抑制DNA损伤修复(优选乳腺癌)，TSL-1502M活性强于奥拉帕利。
4、TSL-1502和TSL-1502M诱导乳腺癌细胞周期阻滞在G 2/M期(优选乳腺癌)，TSL-1502M活性明显强于TSL-1502。
5、TSL-1502，TSL-1502M和阳性对照药物奥拉帕利一样，都能诱导乳腺癌细胞凋亡，其中TSL-1502M的活性比奥拉帕利强。
附图说明
图1：TSL-1502及TSL-1502M对体外培养细胞增殖的影响；
图2：TSL-1502、TSL-1502M与细胞毒药物合用对MDA-MB-436、SW620细胞的增殖抑制作用；
图3：TSL-1502及ABT-888对MDA-MB-436细胞克隆形成的抑制作用；
图4：TSL-1502M及AZD2281对Rad51聚集点形成的影响；
图5：TSL-1502M对MDA-MB-436细胞γ-H2AX磷酸化的影响；
图6：TSL-1502，TSL-1502M及AZD2281,ABT888对MDA-MB-436细胞周期的影响；
图7：TSL-1502，TSL-1502M及AZD2281诱导MDA-MB-436细胞凋亡的作用。
具体实施方式
实施例1：TSL-1502M的制备
参考申请号为201910465780.X，发明名称为一种PARP抑制剂的中间体TSL-1502M及其制备方法的实施例1：
反应：反应釜中，加入主原料(S)-3-甲基-1-(吡咯烷-3-基)-3,4,6,7,8,9-六氢-5H-吡唑并[3,4-c]异喹啉-5-酮(2S,3S)-2,3-二(苯甲酰氧基)丁二酸盐(630.0g，1mol)和乙醇(3800mL)，搅拌，降温至10℃，加入氰基硼氢化钠(125.7g，2mol)，再滴加丙醛(116.2g，2mol)，滴加过程中温度不超过20℃，滴加完毕后，继续在10℃～25℃搅拌反应1h～2h。
淬灭：反应结束后，向反应混合物中缓慢滴加质量百分浓度为36～38％的盐酸(73g，2mol)，调节pH至4-5之间，控制滴加速度，以20％氢氧化钠吸收大量冒出的气体，为防止冲料，同时控制温度为20℃～30℃。
后处理：S1、淬灭后的反应液减压浓缩蒸除乙醇，温度不超过70℃；
S2、向S1的残留物中加入水与二氯甲烷(水与二氯甲烷的重量比为1：4)，使用量为式Ⅰ化合物重量的7倍，在5℃～15℃温度下用40％的氢氧化钠水溶液调节混合液的pH至9～10，搅拌，静置，分层，有机相保留；
S3、S2所得水相用二氯甲烷继续萃取1次，所得有机相与S2所得有机相合并，水相保留；
S4、S3所得合并后的有机相用40％的氢氧化钠水溶液萃取，所得水相与S3所得水相合并，有机相保留；
S5、S4所得合并后的水相用36～38％的盐酸调节pH至4～5，再加入二氯甲烷，二氯甲烷 使用量为式Ⅰ化合物重量的5倍，搅拌后静置分层，有机相保留；
S6、S5所得水相用二氯甲烷继续萃取2次，所得有机相与S4、S5所得有机相合并；
S7、S6所得合并后的有机相用3％碳酸氢钠水溶液洗涤2次后再用无水硫酸钠干燥，过滤除去硫酸钠固体，滤液减压浓缩后真空干燥，得到PARP抑制剂的中间体TSL-1502M 226g。
实验例1
一、实验材料
1、受试药物
TSL-1502：白色粉末，批号120301，纯度99.56％，含水量16.65％，密封，2-8℃保存，申请人自备；
TSL-1502M:白色粉末，批号20150801，纯度98.6％，密封，2-8℃保存，申请人自备；
ABT-888(维利帕尼，Veliparib)：白色粉末，批号HM-069-8-20101112，纯度98.12％，密封，2-8℃保存，购自上海皓元公司，ABT-888是一种新型的强效PARP-1和PARP-2抑制剂，主要用于乳腺癌；
AZD2281(奥拉帕利，Olaparib)：白色粉末，批号20131105，纯度99.15％，密封，2-8℃保存，购自上海德默公司，AZD2281是一种PARP抑制剂，已批准适应症包括卵巢癌、乳腺癌、胰腺癌、前列腺癌；
替莫唑胺(简称：TMZ)：白色粉末，批号20150418,密封，2-8℃保存，替莫唑胺是咪唑四嗪衍生物，主要用于多形性胶质母细胞瘤；
SN-38(伊立替康的活性代谢物7-乙基-10-羟基喜树碱。cas号为86639-52-3)：淡黄色粉末，纯度99.1％，批号E060206，密封，2-8℃保存，购自上海骏杰生物技术公司，伊立替康是喜树碱的半合成衍生物，主要用于结肠(直肠)癌。
2、药品配制方法：
TSL-1502、TSL-1502M、AZD2281和ABT-888用DMSO配成10mM的原液，分装保存于-70℃；TMZ用DMSO配成200mM的原液，分装保存于-70℃。
3、细胞株选择依据及细胞株来源
参考FDA批准上市的PARP抑制剂Olaparib(LYNPARZA)、Rucaparib(RUBRACA)和Niraparib(ZEJULA)的药效学研究情况，选择BRCA1/2突变型及野生型细胞来评价TSL-1502及其代谢产物的体外抗肿瘤活性。细胞来源及培养条件见表1
表1：细胞来源及类型
Figure PCTCN2020140553-appb-000004
Figure PCTCN2020140553-appb-000005
4、试剂及仪器
RPMI1640，L-15，F-10，DMEM及IMDM购自Gibco BRL公司；FBS购自Gibco BRL公司；SRB购自Sigma公司；抗Rad51一抗购自Santa Cruz公司；抗P-γ-H2AX和PARP一抗购自Cell Signaling Technology公司；抗β-Tubulin一抗购自Sigma公司；辣根过氧化酶标记的羊抗兔和羊抗鼠二抗购自Calbiochem公司；Alexa
Figure PCTCN2020140553-appb-000006
488 goat anti-rabbit荧光二抗购自Molecular Probes公司；Immobilon Western HRP Substrate luminal reagent购自Millipore公司；其它普通的化学试剂系国产分析纯(AR)试剂；
多功能酶标仪Synergy H4购自BioTek公司；二氧化碳培养箱(Model 3111)购自Thermo公司；倒置显微镜XDS-1B购自重庆光电仪器有限公司；流式细胞仪(FACS Calibur flow cytometer)购自Beckman Dickson公司；激光共聚焦显微镜购自奥林巴斯公司；Western blot成像仪购自GlinxScience instruments。
二、实验方法
1、磺酰罗丹明B蛋白染色法(SRB法)
接种一定数量的对数生长期细胞于96孔培养板。贴壁生长24小时后，加入不同浓度(1、3、10、30、100、300、1000、3000、10000nM)的药物。药物作用10天后，用三氯乙酸固定细胞。然后SRB溶液染色；最后加入Tris溶液溶解SRB，酶标仪510nm波长下测定OD值，以下列公式计算细胞生长抑制率：
抑制率＝(OD值 对照孔－OD值 给药孔)/OD值 对照孔×100％
根据各浓度抑制率，计算半数抑制浓度IC 50。
2、克隆形成实验
细胞按每孔200个的密度接种于6孔板中。贴壁生长24小时后，加入不同浓度(0.3、1、3、10、30、100nM)的药物，每5天更换培养液和药物，20天后，以甲醇：冰醋酸(3：1)固定，用结晶紫染色，计数，拍照。
3、免疫荧光法
贴在小盖玻片上的细胞经药物处理后，用4％多聚甲醛固定，在含0.3％Triton X-100的 磷酸盐缓冲液(pH7.4)中穿透破膜，封闭后加入抗Rad51的一抗4℃孵育，再用Alexa
Figure PCTCN2020140553-appb-000007
488goat anti-rabbit荧光二抗孵育，最后用DAPI染色。封片后用激光共聚焦显微镜观察拍照。
4、Western blot法
4.1γ-H2AX的磷酸化的检测：
MDA-MB-436接种于六孔板(2×10 5/ml)，加入不同浓度的TSL-1502M或AZD2281(100、1000、10000nM)作用1.5小时后，再加入1mM TMZ作用0.5小时，加入1×SDS凝胶上样缓冲液(50mM Tris-HCl(pH 6.8)，100mM DTT，2％SDS，10％甘油，0.1％溴酚蓝)裂解细胞。细胞裂解物在沸水浴中加热变性，进行SDS-PAGE电泳，电泳结束后，用湿转系统将蛋白转移至PVDF膜，将PVDF膜置于封闭液(5％脱脂奶粉稀释于TBS/T)中室温封闭，然后I，II抗反应；洗膜后，用Immobilon Western HRP Substrate luminal reagent试剂发色，Western blot成像仪拍照。
4.2细胞凋亡的检测：
MDA-MB-436接种于六孔板(1×10 5/ml)，加入不同浓度的TSL-1502、TSL-1502M(0.01、0.1、1μM)或AZD2281(0.1、1、10μM作用120小时后，加入1×SDS凝胶上样缓冲液(50mM Tris-HCl(pH 6.8)，100mM DTT，2％SDS，10％甘油，0.1％溴酚蓝)裂解细胞。细胞裂解物在沸水浴中加热变性，进行SDS-PAGE电泳，电泳结束后，用湿转系统将蛋白转移至PVDF膜，将PVDF膜置于封闭液(5％脱脂奶粉稀释于TBS/T)中室温封闭，然后I，II抗反应；洗膜后，用Immobilon Western HRP Substrate luminal reagent试剂发色，Western blot成像仪拍照。
5、流式细胞术
MDA-MB-436细胞接种于6孔板中(2×10 5/ml)，经药物处理后48h，收集细胞。用乙醇固定过夜，然后加入RNase及碘丙啶，混匀，37℃避光染色30分钟,最后用流式细胞仪(FACSCalibur flow cytometer)检测细胞中DNA含量，每组样品为1×10 4个细胞。实验结果用ModFit Lt Mac V3.0软件分析。
三、实验结果
1、TSL-1502、TSL-1502M抑制多种体外培养肿瘤细胞的增殖
TSL-1502、AZD2281、ABT-888作用于细胞240小时(120小时更换新配药物一次)后，检测其对细胞的增殖抑制作用。结果如表2和图1所示：
对BRCA基因缺陷或突变细胞(V-C8、MDA-MB-436、Capan-1和UWB1.289)，TSL-1502M的增殖抑制作用十分显著，IC 50分别为0.2±0.0nM、0.9±0.2nM、100.6±73.8nM、1.9±0.4nM)，该抑制作用显著强于其前药TSL-1502(IC 50分别为132.1±4.7nM、79.6±12.5nM、≈10000nM、 935.7±526.6nM)，以及参比药物AZD2281(IC 50分别为18.6±1.3nM、4.0±0.7nM、586.1±89.4nM、≈1nM(抑制率稍弱于TSL-1502M))和ABT-888(IC 50分别为314.2±24.4nM、114.7±60.8nM、NA、2102.5±1470.1nM)；
但对BRCA1突变的HCC1937细胞，TSL-1502M、TSL-1502、AZD2281和ABT-888的增殖抑制均较弱。
对BRCA基因正常细胞(V-C8#13-5和UWB1.289BRCA1)，TSL-1502M、TSL-1502、AZD2281和ABT-888的增殖抑制作用均较弱；但对BRCA功能正常的MDA-MB-468细胞，TSL-1502M和AZD2281有一定增殖抑制作用(IC 50分别为73.6±19.5nM和541.1±93.8nM)。
总体而言，TSL-1502M对BRCA1/2功能缺陷的细胞更为敏感，对细胞的增殖抑制作用具有选择性，其选择性的作用特点与AZD2281和ABT-888相似，其抑制作用比TSL-1502、AZD2281和ABT-888强。
表2：对体外培养细胞增殖的影响(n＝2)
Figure PCTCN2020140553-appb-000008
2、TSL-1502、TSL-1502M增效细胞毒药物抗肿瘤活性
检测了TSL-1502和TSL-1502M与细胞毒药物的联合抗肿瘤作用。结果如表3-1、3-2和图2所示。
在BRCA1突变的MDA-MB-436细胞中，30、100、300nM的TSL-1502，3、10、30nM的TSL-1502M，10、30、100nM的AZD2281或30、100、300nM ABT-888与10、30μM的TMZ合用120小时。
TSL-1502、TSL-1502M和参比化合物AZD2281、ABT-888均能够明显增效TMZ对MDA-MB-436细胞的增殖抑制作用。
在BRCA基因正常的SW620细胞中，3、10μM的TSL-1502M或3、10μM的AZD2281与0.1、1nM的伊立替康活性代谢产物SN38联合作用144小时，TSL-1502M和参比化合物AZD2281均能够明显增效SN38对SW620细胞的增殖抑制作用。
上述结果表明，在BRCA基因突变或正常的肿瘤细胞中，TSL-1502M对细胞毒药物均有增效作用。
2.1 TSL-1502和TSL-1502M对MDA-MB-436细胞的增殖抑制作用：见表3-1
表3-1：对MDA-MB-436细胞的增殖抑制作用。抑制率(％，Mean±SD)
Figure PCTCN2020140553-appb-000009
2.2：TSL-1502和TSL-1502M对SW620细胞的增殖抑制作用。见表3-2
表3-2：对SW620细胞的增殖抑制作用。抑制率(％，Mean±SD)
Figure PCTCN2020140553-appb-000010
3、TSL-1502抑制BRCA功能缺陷肿瘤细胞的克隆形成
TSL-1502前药需要较长时间产生抗肿瘤作用，因此，我们检测TSL-1502对MDA-MB-436细胞(BRCA1基因突变)克隆形成抑制作用时，将作用时间延长到20天。结果如图3所示， TSL-1502和参比化合物均能够剂量依赖地抑制MDA-MB-436细胞的克隆形成。TSL-1502抑制克隆形成的IC 50为9.5±1.4nM，AZD2281和ABT-888抑制克隆形成的IC 50分别为1.7±0.1和9.5±1.4nM。总体而言，TSL-1502和参比药物AZD2281和ABT-888均能抑制BRCA功能缺陷肿瘤细胞的克隆形成，TSL-1502的抑制作用比ABT-888强，比AZD2281弱。
4、TSL-1502M选择性地诱导Rad51聚焦点的形成
PARP抑制能够诱导DNA损伤，进而增加同源重组功能正常细胞中自发的Rad51聚集点的形成。而在BRCA1/2缺陷的细胞中，同源重组修复功能障碍，Rad51聚集点不会被诱导形成。我们用免疫荧光法检测了TSL-1502M及AZD2281对Rad51聚集点形成的影响。如图4所示：
10μM的的TSL-1502M或30μM的AZD2281作用24小时后，在BRCA1/2功能正常的细胞(V-C8#13-5)中，Rad51聚集点被明显诱导，而在BRCA1/2功能缺陷的细胞(V-C8)中，Rad51聚集点不能被诱导。
可见，TSL-1502M能够选择性地诱导BRCA1/2功能正常的细胞中Rad51聚焦点的形成，其作用特点与AZD2281相似。
5、TSL-1502M与TMZ合用明显增强MDA-MB-436细胞γ-H2AX的磷酸化
磷酸化的组蛋白H2AX(γ-H2AX)是DNA损伤的标志物，我们在BRCA1突变的MDA-MB-436细胞中检测了TSL-1502M与TMZ合用后对DNA损伤修复的影响。TSL-1502M以及参比对照化合物AZD2281合用TMZ后，对γ-H2AX的影响结果见图5，结果显示：
TSL-1502M或参比化合物AZD2281与TMZ合用后γ-H2AX的磷酸化明显增强。
上述结果表明，TSL-1502M及AZD2281能抑制DNA损伤修复，TSL-1502M活性强于AZD2281。
6、TSL-1502诱导细胞阻滞在G2/M期
以TSL-1502、TSL-1502M和参比化合物AZD2281作用于MDA-MB-436细胞，48小时后，以流式细胞术检测对细胞周期的影响。结果如图6所示：
TSL-1502，TSL-1502M和参比化合物AZD2281都能够剂量依赖地诱导G 2/M期细胞增加，即诱导细胞周期G 2/M期阻滞。
TSL-1502M诱导G 2/M期周期阻滞作用和AZD2281近似：10μM剂量下都能明显诱导G 2/M期阻滞；TSL-1502诱导G 2/M期周期阻滞作用弱于TSL-1502M和参比化合物AZD2281：10μM剂量下诱导G 2/M期阻滞作用较弱。
上述结果表明，TSL-1502和TSL-1502M诱导MDA-MB-436细胞周期阻滞在G 2/M期，TSL-1502M活性明显强于TSL-1502，和参比化合物AZD2281近似。
7、TSL-1502、TSL-1502M诱导细胞凋亡
以TSL-1502，TSL-1502M和参比药物AZD2281作用于MDA-MB-436细胞，120小时后，以Western Blot法检测凋亡标志物蛋白PARP的切割变化。结果如图7所示：
TSL-1502在1μM剂量下，有较弱的诱导PARP切割作用，10μM更明显；
TSL-1502M在0.01μM剂量下，即能明显诱导PARP切割；
参比药物AZD2281在0.1μM剂量下，有较弱的诱导PARP切割作用，1μM和10μM更明显。
结果表明，TSL-1502，TSL-1502M和参比药物AZD2281一样，都能诱导MDA-MB-436细胞凋亡，其中TSL-1502的活性比AZD2281弱，TSL-1502M的活性比AZD2281强。
试验结论：
本发明研究了TSL-1502及其代谢产物TSL-1502M的体外抗肿瘤活性及作用机制。主要包括：
1)TSL-1502M对BRCA基因缺陷或突变细胞，如V-C8、MDA-MB-436和UWB1.289细胞有显著的增殖抑制作用，IC 50分别为0.2±0.0nM、0.9±0.2nM和1.9±0.4nM，该抑制作用显著强于其前药TSL-1502(IC 50分别为132.1±4.7nM、79.6±12.5nM和935.7±526.6nM)，以及参比药物AZD2281(IC 50分别为18.6±1.3nM、4.0±0.7nM和≈1nM(抑制率稍弱于TSL-1502M))和ABT-888(IC 50分别为314.2±24.4nM、114.7±60.8nM和2102.5±1470.1nM)。
对BRCA基因正常细胞，如V-C8#13-5和UWB1.289BRCA1细胞，TSL-1502M、TSL-1502、AZD2281和ABT-888的增殖抑制均较弱。
2)TSL-1502能够抑制MDA-MB-436细胞的克隆形成。TSL-1502M明显增效替莫唑胺对MDA-MB-436细胞(BRCA1突变)和伊立替康活性代谢产物SN38对SW620细胞(BRCA1/2正常)的增殖抑制作用；选择性地诱导BRCA1/2功能正常细胞中Rad51聚焦点的形成；与替莫唑胺合用后诱导MDA-MB-436细胞的γ-H2AX磷酸化水平显著增加；能够诱导MDA-MB-436细胞G 2/M期阻滞，能够诱导细胞凋亡；TSL-1502M的作用特点与参比化合物AZD2281相似，TSL-1502M的活性比AZD2281强。

Claims (10)

  1. 式Ⅱ化合物在制备预防和治疗抗肿瘤的药物中的应用：
    Figure PCTCN2020140553-appb-100001
  2. 根据权利要求1所述的应用，其特征在于，通过抑制肿瘤细胞增殖并诱导细胞凋亡发挥抗肿瘤作用，所述肿瘤细胞包括：BRCA功能缺陷的肿瘤细胞、BRCA基因突变的肿瘤细胞，或BRCA功能正常的肿瘤细胞。
  3. 根据权利要求1所述的应用，其特征在于，所述肿瘤选自以下肿瘤：乳腺癌、胰腺癌、卵巢癌、结肠癌。
  4. 根据权利要求3所述的应用，其特征在于，所述肿瘤选自以下肿瘤：乳腺癌、卵巢癌、胰腺癌。
  5. 根据权利要求1所述的应用，其特征在于，所述药物是任何可服用的药物形式。
  6. 根据权利要求5所述的应用，其特征在于，所述药物选自片剂、胶囊剂、口服液、口含剂、颗粒剂、丸剂、散剂、膏剂、丹剂、粉剂、注射剂、栓剂、膏剂、喷雾剂、滴剂、贴剂、滴丸剂。
  7. 根据权利要求6所述的应用，其特征在于，所述注射剂选自液体、半固体，固体，粉剂形式。
  8. 根据权利要求7所述的应用，其特征在于，所述注射剂为注射液，粉针中的一种。
  9. 根据权利要求5所述的应用，其特征在于，所述药物是单位剂量的药物制剂形式，单位剂量的药剂含有式Ⅱ化合物0.1-1000mg，其余为药学上可接受的辅料。
  10. 根据权利要求9所述的应用，其特征在于，所述药学上可接受的辅料以重量计是制剂总重量的0.01-99.99％。
PCT/CN2020/140553 2020-07-30 2020-12-29 一种parp抑制剂tsl-1502中间体tsl-1502m的用途 WO2022021784A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010748321.5A CN114053276B (zh) 2020-07-30 2020-07-30 一种parp抑制剂tsl-1502中间体tsl-1502m的用途
CN202010748321.5 2020-07-30

Publications (1)

Publication Number Publication Date
WO2022021784A1 true WO2022021784A1 (zh) 2022-02-03

Family

ID=80037078

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/140553 WO2022021784A1 (zh) 2020-07-30 2020-12-29 一种parp抑制剂tsl-1502中间体tsl-1502m的用途

Country Status (2)

Country Link
CN (1) CN114053276B (zh)
WO (1) WO2022021784A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115073544A (zh) * 2022-06-30 2022-09-20 上海应用技术大学 一种parp抑制剂吡唑并奎啉衍生物及其合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007144669A1 (en) * 2006-06-15 2007-12-21 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Pyrazolo[1,5-a]quinazolin-5(4h)-ones as inhibitors of poly(adp-ribose)polymerase (parp)
CN101421268A (zh) * 2006-02-15 2009-04-29 艾博特公司 作为有效parp抑制剂的吡唑并喹诺酮
CN102510863B (zh) * 2010-05-24 2014-09-03 苏州汉德森医药科技有限公司 吡唑衍生物

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2612979A1 (en) * 2005-06-10 2006-12-21 Bipar Sciences, Inc. Parp modulators and treatment of cancer
KR101596526B1 (ko) * 2007-10-03 2016-02-22 에이자이 아이엔씨. Ｐａｒｐ 억제제 화합물, 조성물 및 사용 방법
JP2011500684A (ja) * 2007-10-19 2011-01-06 バイパー サイエンシズ，インコーポレイティド ベンゾピロン系ｐａｒｐ阻害剤を用いる癌の処置方法および組成物
TWI482621B (zh) * 2009-12-23 2015-05-01 Sigma Tau Ind Farmaceuti 青蒿素基藥物與其他化學治療劑的抗癌組合物
EP2918292B1 (en) * 2012-11-08 2019-12-11 Nippon Kayaku Kabushiki Kaisha Polymeric compound having camptothecin compound and anti-cancer effect enhancer bound thereto, and use of same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101421268A (zh) * 2006-02-15 2009-04-29 艾博特公司 作为有效parp抑制剂的吡唑并喹诺酮
WO2007144669A1 (en) * 2006-06-15 2007-12-21 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Pyrazolo[1,5-a]quinazolin-5(4h)-ones as inhibitors of poly(adp-ribose)polymerase (parp)
CN102510863B (zh) * 2010-05-24 2014-09-03 苏州汉德森医药科技有限公司 吡唑衍生物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOPKINS TODD A., AINSWORTH WILLIAM B., ELLIS PAUL A., DONAWHO CHERRIE K., DIGIAMMARINO ENRICO L., PANCHAL SANJAY C., ABRAHAM VIVEK: "PARP1 Trapping by PARP Inhibitors Drives Cytotoxicity in Both Cancer Cells and Healthy Bone Marrow", MOLECULAR CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 17, no. 2, 1 February 2019 (2019-02-01), US , pages 409 - 419, XP055891336, ISSN: 1541-7786, DOI: 10.1158/1541-7786.MCR-18-0138 *
ZHANG TENG, WANG CUI-LING, ZHANG NING, LIU JIAN-LI: "Progress in the study of PAＲP-1 inhibitors", CHINESE JOURNAL OF NEW DRUGS, GAI-KAN BIANJIBU, BEIJING, CN, vol. 23, no. 7, 12 May 2014 (2014-05-12), CN , pages 793 - 801, XP055891348, ISSN: 1003-3734 *

Also Published As

Publication number Publication date
CN114053276B (zh) 2024-05-07
CN114053276A (zh) 2022-02-18

Similar Documents

Publication Publication Date Title
EP3440067B1 (en) Tetrahydroisoquinoline estrogen receptor modulators and uses thereof
EP3233852B1 (en) Tetrahydro-pyrido[3,4-b]indoles as estrogen receptor modulators and uses thereof
EP3472162B1 (en) Heteroaryl estrogen receptor modulators and uses thereof
JP6401773B2 (ja) Ｂｅｔブロモドメイン阻害剤およびこれを用いる治療方法
TWI290046B (en) Phenanthroindolizidine alkaloids
CN108137539B (zh) 取代的苯并咪唑、它们的制备和它们作为药物的用途
US10399939B2 (en) Tetrahydronaphthalene estrogen receptor modulators and uses thereof
KR20240014585A (ko) 악성 종양 치료용 제제 및 조성물
CN111655261A (zh) 非环状cxcr4抑制剂和其用途
US10654867B2 (en) Heteroaryl estrogen receptor modulators and uses thereof
WO2023025116A1 (zh) 杂环类衍生物、其制备方法及其医药上的用途
CN113387962A (zh) 吡唑并[3,4-d]嘧啶-3-酮衍生物、其药物组合物及应用
WO2014059314A1 (en) Treating brain cancer using agelastatin a (aa) and analogues thereof
AU2019311121B2 (en) Fused ring derivative used as FGFR4 inhibitor
WO2022021784A1 (zh) 一种parp抑制剂tsl-1502中间体tsl-1502m的用途
WO2022021785A1 (zh) 一种含有tsl-1502m的药物组合物及其应用
WO2022002243A1 (zh) 咪唑并嘧啶类衍生物、其制备方法及其在医药上的应用
EP3733206A1 (en) Anticancer agent
WO2023222011A1 (zh) 一种治疗三阴乳腺癌的联合用药物
TW202140474A (zh) 稠合雜芳基類衍生物、其製備方法及其在醫藥上的應用
WO2022072645A2 (en) Methods for treating cancer
WO2022206724A1 (zh) 杂环类衍生物及其制备方法和用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20946974

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20946974

Country of ref document: EP

Kind code of ref document: A1