WO2021238007A1 - 可诱导prc2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用 - Google Patents

可诱导prc2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用 Download PDF

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WO2021238007A1
WO2021238007A1 PCT/CN2020/119438 CN2020119438W WO2021238007A1 WO 2021238007 A1 WO2021238007 A1 WO 2021238007A1 CN 2020119438 W CN2020119438 W CN 2020119438W WO 2021238007 A1 WO2021238007 A1 WO 2021238007A1
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formula
pharmaceutical composition
prc2
ezh2
bifunctional compound
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French (fr)
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余洛汀
刘志昊
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四川大学华西医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

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  • the present disclosure relates to the technical field of chemical medicine, in particular, to a bifunctional compound that can induce degradation of the core subunit of the PRC2 protein complex, a pharmaceutical composition and an application thereof.
  • the polycomb gene family (PcG) protein was originally discovered to be a type of protein that regulates the establishment of the Drosophila organism by participating in the regulation of the expression of Hox genes during the development and cell differentiation of Drosophila. Function critical epigenetic regulator.
  • PRC1 and PRC2 are the two main polyphobic inhibitory complexes in PcG proteins, which regulate gene transcription through histone post-translational modification. Different combinations of multiple subunits can form PRC1 with different functions.
  • the classic PRC1 is composed of CBX (chrombox), PCGF (polycomb group factor), HPH (human polyhomeotic homolog), and RING1A/B (really interesting new gene).
  • Subunit composition mainly through the catalytic subunit RING1A/B to mediate H2AK119ub1, which plays an important role in embryonic development, maintenance of stem cell characteristics, and tumorigenesis;
  • PRC2 includes EZH2, SUZ12, EED, and RbAp46/48 (retinoblastoma-associated proteins 46).
  • /48 also known as RBBP4/7)
  • Several core subunits mainly through the catalytic subunit EZH2 mediate H3K27me2 and H3K27me3, and regulate the transcription of genes related to cell cycle, senescence, differentiation and tumorigenesis.
  • EZH2 as the catalytic subunit of PRC2, can catalyze H3K27me3 with the participation of the other two core subunits of the complex, SUZ12 and EED, and together with PRC1-mediated H2AK119ub1 to form a complex that hinders transcription extension on the nucleosome In order to maintain the transcriptional silencing of downstream genes.
  • EZH2 itself cannot exert its catalytic activity alone. It must at least depend on the SUZ12 and EED subunits of PRC2 to exert HMTase activity and inhibit the transcription of many downstream genes.
  • SUZ12 is a core subunit that is essential to maintain the integrity of PRC2, the stability of EZH2, and the activity of HMTase.
  • the C-terminal VEFS (Vrn2-Emf2-Fis2-SUZ12) domain can bind to EZH2 stably and maintain the PRC2 complex. Assembly, on the other hand, can assist the HMTase activity of PRC2 through allosteric action.
  • the interaction and binding between the repeated WD domain of EED subunits and EZH2 are necessary conditions to ensure that EZH2 has HMTase activity, and the specific binding of the C-terminal WD domain of EED to H3K36me3 is a necessary condition for HMTase allosteric activation, so EED It is also a core subunit of PRC2 to exert HMTase activity.
  • Retinoblastoma-associated protein 46 and 48 (RbAp46/48) subunits are highly homologous histone chaperone proteins, and they play an important role in the maintenance of chromatin structure.
  • EZH2 does not require the direct participation of RbAp46 and RbAp48 to exert HMTase activity
  • the process of introducing PRC2 into nucleosomes requires the combination of RbAp48 and histone H3-H4 heterodimers to achieve, so RbAp48 also ensures that PRC2 is normal
  • the core subunit that exerts HMTase activity is highly homologous histone chaperone proteins
  • the core subunits of the PRC2 complex not only participate in the formation of the PRC2 complex to catalyze H3K27me3 and up-regulate the transcriptional silencing tumor suppressor gene, but also perform their own non-PRC2 catalytic functions.
  • EZH2 can not only mediate gene silencing by catalyzing H3K27me3, but also mediate transcriptional activation of genes in certain tumors in a manner that does not depend on HMTase activity.
  • a number of studies have shown that EZH2 can also act as a transcription activator to methylate non-histone proteins or directly interact with other proteins to form transcription complexes to activate gene transcription.
  • pEZH2 can act as a co-activator of androgen receptor (AR) and its related complexes to promote castration-resistant prostate cancer (castration).
  • -resistant prostate cancer (CRPC) cell growth that is, AKT mediates the Ser21 phosphorylation of EZH2, promotes EZH2 to bind to AR and catalyzes the methylation of AR or AR-related proteins, thereby activating the transcription of a series of target genes downstream of AR. It provides a new combination therapy idea for the treatment of metastatic and hormone refractory prostate cancer.
  • phosphorylated EZH2 can also directly bind and methylate Lys180 of STAT3, mediating the phosphorylation of tyrosine (Tyr) at position 705 of STAT3 to enhance the activity of STAT3, thereby contributing to glioblastoma and glial The tumorigenicity of blastoma stem cells.
  • Tyr tyrosine
  • EZH2 has also been found in colorectal cancer cells to activate transcription in a manner independent of methyltransferase activity.
  • the human proliferating cell nuclear antigen-related factor PAF recruits EZH2, promotes the binding and interaction of EHZ2 with TCF/ ⁇ -catenin, and forms the transcription initiation together with the transcription factor TCF/LEF at the promoters of genes such as c-myc, cyclinD1, and Axin2
  • the complex induces the transcriptional activation of tumor formation-related target genes in the Wnt pathway, and promotes the progression of colorectal cancer.
  • EED does not depend on the function of PRC2 to directly interact with androgen receptors, regulates the expression level of AR and AR downstream targets, and promotes the development of prostate cancer. EED also does not depend on the function of PRC2 and the direct action of histone deacetyltransferase HDAC, which affects the catalytic activity of HDAC.
  • the current EZH2 inhibitors or EED inhibitors are only used to inhibit the activity of PRC2, but they cannot effectively inhibit EZH2, EED and other carcinogenic activities that do not depend on the catalytic function of PRC2, and cannot simultaneously perform on multiple core subunits of PRC2. Degradation to completely block the carcinogenic activity of the subunits of the PRC2 complex.
  • the purpose of the present disclosure includes, for example, providing a bifunctional compound that can induce degradation of the core subunit of the PRC2 protein complex and a pharmaceutical composition and application, so as to improve the above-mentioned problems.
  • the embodiments of the present disclosure provide a bifunctional compound that can induce degradation of the core subunit of the PRC2 protein complex, which includes a compound represented by any one of formula I-III, a pharmaceutically acceptable salt thereof, or Prodrugs, their solvates, their hydrates, their polymorphs, their tautomers, stereoisomers or isotopically substituted compounds;
  • n in formula I-III is an integer of 1-10, X is all O, N or S, and Y is all O, H 2 or S.
  • n in formula I-III is an integer of 2-6.
  • n in formula I-III is an integer from 2 to 10, and X and Y are both O; in one or more embodiments, n in formula I-III is both Is an integer of 2-6, X and Y are both O; in one or more embodiments, the chemical formula of the bifunctional compound is
  • the embodiments of the present disclosure also provide a method for preparing a bifunctional compound.
  • X is N, S, and Y in formula I-III
  • the synthetic route of the compound represented by formula I-III is:
  • the embodiments of the present disclosure also provide a pharmaceutical composition, which includes a pharmaceutically acceptable auxiliary component and the bifunctional compound of the foregoing embodiment.
  • the pharmaceutical composition is an aqueous solution, powder, granule, tablet or lyophilized powder. In one or more embodiments, when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition The composition also contains water for injection, saline solution, aqueous glucose solution, saline for injection or infusion, glucose for injection or infusion, Green's solution, or Green's solution containing lactate.
  • the embodiments of the present disclosure also provide the application of the bifunctional compound or pharmaceutical composition of the foregoing embodiment in the preparation of kinase inhibitors.
  • the embodiments of the present disclosure also provide the application of the bifunctional compound or the pharmaceutical composition of the foregoing embodiment in the preparation of a drug for treating tumors; optionally, the tumors include breast cancer, colorectal cancer, prostate cancer, Ovarian cancer, pancreatic cancer or stomach cancer.
  • the embodiments of the present disclosure also provide the application of the bifunctional compound or the pharmaceutical composition of the foregoing embodiment in the preparation of a degrading agent that degrades the core subunit of the PRC2 protein complex, optionally, degrades the core subunit of the PRC2 protein complex
  • a degrading agent that degrades the core subunit of the PRC2 protein complex
  • the base is the EZH1, EZH2, EED, SUZ12 and RbAp46/48 subunits that simultaneously degrade the PRC2 protein complex.
  • the embodiments of the present disclosure also provide the application of the bifunctional compound or pharmaceutical composition of the foregoing embodiment in the preparation of oral or intravenous injection preparations.
  • the oral or intravenous injection at least includes the bifunctional compound or pharmaceutical composition, which can be Optionally, excipients and/or adjuvants are also included.
  • the bifunctional compound can effectively induce the degradation of the core subunits of the PRC2 protein complex, thereby achieving treatment by the PRC2 complex and its subunits including EZH2, EED, SUZ12, Cancers mediated by RbAp46 and RbAp48 completely block the carcinogenic activity of PRC2 complex subunits.
  • the bifunctional compound can effectively induce the degradation of the core subunits of the PRC2 protein complex, thereby achieving treatment by the PRC2 complex and its subunits including EZH2, EED, SUZ12, Cancers mediated by RbAp46 and RbAp48 completely block the carcinogenic activity of PRC2 complex subunits.
  • EZH2 inhibitors and EED inhibitors they have better anticancer activity.
  • the bifunctional compound or pharmaceutical composition is used as a kinase inhibitor to treat a variety of human tumors, and it has good anti-tumor activity and low toxicity.
  • Figure 1 and Figure 2 show the Western blot method to detect the protein levels of EZH2, SUZ12, EED, RbAp48 and histone H3K27me3 in WSU-DLCL-2 cells;
  • Figure 3 is a synthetic molecule that degrades the core subunits of PRC2 and reduces the level of H3K27me3;
  • FIG. 4 shows that E7 effectively degrades PRC2 and inhibits H3K27me3.
  • FIG. 5 shows that E7 binds to the EZH2 subunit of PRC2 and plays a role.
  • CETSA detects the thermal stability of EZH2, SUZ12, EED and RbAp48 proteins in WSU-DLCL-2 cells treated with E7 and the corresponding gray-scale statistical results of western blotting. The statistical results are expressed in the form of three average ⁇ standard deviation (SD).
  • SD three average ⁇ standard deviation
  • B Western blotting to detect the competitive effects of EZH2 inhibitor and EED inhibitor with E7 in WSU-DLCL-2 cells.
  • C Western blot detection of E7 for multiple methylation modification products of histone H3 in WSU-DLCL-2 cells;
  • FIG. 6 shows that E7 degrades PRC2 through the ubiquitin proteasome pathway.
  • the immunoprecipitation experiment detects the effect of 1 ⁇ M E7 on the ubiquitination modification of EZH2(A), SUZ12(B) and EED(C) for 48h.
  • D After WSU-DLCL-2 cells were pretreated with 1 ⁇ M lenalidomide or 5 ⁇ M MLN4924/MG-132 for 4 hours, the cells were treated with 1 ⁇ M E7 for 48 hours, and the cell lysate was collected for western blot analysis;
  • FIG. 7 shows that E7 activates WSU-DLCL-2 (A), and the catalytic function of EZH2 in Pfeiffer (B) and A549 (C) cells mediates the transcription of silenced genes. After the cells were treated with the designated compound for 48 hours, the mRNA level was detected by RT-qPCR. The statistical results are expressed in the form of three average ⁇ SD, *P ⁇ 0.05, **p ⁇ 0.01;
  • Figure 8 shows that E7 inhibits the transcription of genes activated by the non-catalytic function of EZH2 in A549 (A), NCI-H1299 (B) and MDA-MB-468 (C) cells. After the cells were treated with the designated compound for 48 hours, the mRNA level was detected by RT-qPCR. The statistical results are expressed in the form of three average ⁇ SD, *P ⁇ 0.05, **p ⁇ 0.01;
  • FIG. 9 shows that E7 effectively inhibits the proliferation of WSU-DLCL-2 (A), A549 (B) and NCI-H1299 (C) cells.
  • Figure 10 is the MTT experiment to detect the inhibitory effect of E7 on WSU-DLCL-2 (A), Pfeiffer (B), A549 (C) and NCI-H1299 (D) cell viability for 3d, 5d and 7d.
  • the survival curve and IC50 value are fitted by GraphPad Prism 5.0 software.
  • the statistical results are expressed in the form of two average values ⁇ SD.
  • the bifunctional compound and pharmaceutical composition that can induce degradation of the core subunit of the PRC2 protein complex provided by the present disclosure will be specifically described below.
  • Some embodiments of the present disclosure provide a bifunctional compound that can induce degradation of the core subunit of the PRC2 protein complex, which includes a compound represented by any one of formula I-III, a pharmaceutically acceptable salt or prodrug thereof , Its solvates, its hydrates, its polymorphs, its tautomers, stereoisomers or isotopically substituted compounds;
  • n in formula I-III is an integer of 1-10, X is all O, N or S, and Y is all O, H 2 or S.
  • hydrate refers to a compound that further binds stoichiometric or non-stoichiometric water through non-covalent intermolecular forces.
  • polymorph refers to a solid crystalline form of a compound or a complex thereof, which can be characterized by physical methods, such as X. ray powder diffraction patterns or infrared spectroscopy.
  • EED inhibitors In the existing EZH2 inhibitors, EED inhibitors only inhibit the activity of the PRC2 complex, but they cannot effectively inhibit EZH2. EED and other carcinogenic activities that do not depend on the catalytic function of PRC2, let alone the other core sub-substances of the PRC2 complex. In the case of simultaneous inhibition of bases such as EZH1, SUZ12, and RbAp46/48, a lot of research and practice have been carried out, and the bifunctional compounds described in the above three structural formulas have been creatively discovered, which can effectively induce the core subunits of the PRC2 protein complex.
  • bifunctional compound that can induce degradation of the core subunits of the PRC2 protein complex can also be tautomers, stereoisomers, and mixtures of all ratios of the compounds shown in any one of I-III. .
  • n in the above formula I-III is an integer of 2-6.
  • n in formula I-III is an integer of 2-10, and X and Y are both O; in one or more embodiments, n in formula I-III is both 2.
  • the chemical formula of the bifunctional compound may be
  • some embodiments of the present disclosure also provide a pharmaceutical composition, which includes a pharmaceutically acceptable auxiliary ingredient and the bifunctional compound of the foregoing embodiment.
  • auxiliary components are general as long as the conventional components are added in the process.
  • the above-mentioned pharmaceutical composition may be in a liquid form or a solid form. That is, the pharmaceutical composition includes, but is not limited to, aqueous solutions, powders, granules, tablets or freeze-dried powders.
  • the pharmaceutical composition when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition also contains injections. Water, saline solution, aqueous glucose solution, saline for injection or infusion, glucose for injection or infusion, Green's solution or Green's solution containing lactate.
  • some embodiments of the present disclosure also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of kinase inhibitors. Especially for EZH2 enzyme, it has better inhibitory performance.
  • some embodiments of the present disclosure also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of drugs for treating tumors; optionally, the tumors include breast cancer, colorectal cancer, Solid tumors such as prostate cancer, ovarian cancer, pancreatic cancer or gastric cancer.
  • some embodiments of the present disclosure also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of a degrading agent that degrades the core subunit of the PRC2 protein complex.
  • the core subunits of the degraded PRC2 protein complex are EZH1, EZH2, EED, SUZ12 and RbAp46/48 subunits that simultaneously degrade the PRC2 protein complex.
  • some embodiments of the present disclosure also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of oral or intravenous injection preparations.
  • Oral or intravenous injections include at least the bifunctional compound or the pharmaceutical composition, and optionally, excipients and/or adjuvants.
  • the first step preparation 1b, ammonolysis reaction.
  • the second step 1c-1k general synthesis step.
  • G4 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(4- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ butyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G5 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(5- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ pentyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G6 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(6- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ hexyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G7 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(7- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ heptyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G8 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(8- ⁇ [2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ octyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G9 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(9- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ nonyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G10 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(10- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ decyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G11 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(11- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ undecyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • G12 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(12- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy ⁇ dodecyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • TLC thin layer chromatography
  • the second step 2b (4g, 17.4mmol, 1.0eq) and tetrahydropyrone (4.4g, 52.2mmol, 3.0eq) dissolved in chloroform (50mL), add acetic acid (2.1g, 34.8mmol, 2.0eq) at room temperature After stirring for 3 h, sodium triacetyl borohydride (2.7 g, 43.6 mmol, 2.5 eq) was added, and stirring was continued overnight. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to obtain product 2c (3.3 g, 57.8%).
  • the third step Under the protection of nitrogen, take 2c (3g, 9.1mmol, 1.0eq) into a 50mL round bottom flask and add 30mL of 1,2-dichloroethane, and carefully inject anhydrous acetaldehyde (2.3g, 27.3mmol, 3.0eq) was placed below the surface of the solution, and acetic acid (1.1g, 18.2mmol, 2.0eq) was added within 30min, and the reaction solution was orange. After the reaction mixture was naturally warmed to room temperature, it was stirred for 1 h. Then the mixture was cooled to 0°C, and sodium triacetoxyborohydride (1.48g, 23mmol) was slowly added in batches.
  • the feeding rate was controlled to keep the temperature of the reaction system below 5°C. After 2h, it was reversed to room temperature and stirred overnight. After the TLC reaction is completed, cool the reaction system to 0°C, add 100 mL of ice water, and slowly add excess sodium bicarbonate saturated aqueous solution while stirring. After the addition is complete, stir for 30 minutes and then stand for liquid separation, and extract the aqueous phase with dichloromethane. The organic layers were combined, washed twice with water, and then separated. The organic phase was separated and concentrated under reduced pressure to constant weight to obtain a yellow to light red oily liquid.
  • Step 4 Under the protection of nitrogen, add 2d (2g, 5.6mmol) to 25mL of methanol at one time. After the temperature is raised to 60°C, slowly add 20mL of sodium hydroxide aqueous solution (2M) dropwise while keeping warm. The color of the reaction liquid changes from light green The clear liquid gradually becomes an emulsion, and finally becomes a light green clear liquid. After the reaction was incubated for 1 hour, TLC monitored the completion of the reaction. The reaction solution was transferred to a rotary evaporator, most of the methanol was removed under reduced pressure, 100 mL of water was added to the residue, and the mixture was stirred for 10 min. The solids were completely dissolved.
  • Step 5 3-(Aminomethyl)-4,6-lutidine-2(1H)-one (0.61g, 4mmol) and 2e (1.5g, 4.4mmol) dissolved in DMSO (10mL), add HOAT (0.55g, 1.5mmol) and EDCI (0.84g, 2.2mmol), the reaction solution was stirred at 45°C for 20h. After the reaction was monitored by TLC, the reaction solution was poured into ice water (100mL), stirred for 30 minutes, and then precipitated out, filtered, washed with water, dried and dissolved in a mixture of methanol and chloroform (10:1), mixed with samples, and passed through a silica gel column.
  • Step 6 Take 2f(1.2g,2.5mmol,1.0eq), tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine -1-carboxylate (1.21g, 3mmol, 1.2eq) is dissolved in a mixed solution of 1,4-dioxane and water (4:1, 30mL), K 2 CO 3 (3.75mmol, 0.52g) is added, Pd(dppf)Cl 2 (0.2mmol, 146mg), protected by nitrogen, was transferred to 100°C and reacted for 8h, and then cooled to room temperature.
  • the reaction solution was distilled under reduced pressure to remove the solvent, and then dissolved in ethyl acetate, and filtered with Celite.
  • the filtrate was extracted, dried with anhydrous sodium sulfate and concentrated, mixed with samples, and purified by silica gel column chromatography to obtain the product.
  • add directly to 25mL of trifluoroacetic acid/dichloromethane solution (10%) and react for 1h at room temperature, then distill under reduced pressure to remove the solvent, and the residue is neutralized to pH 8 with saturated sodium carbonate solution (2M) ,
  • the precipitate is separated out, filtered with suction, washed with water, and dried to obtain 2 g (1.04 g, 74%) of the product.
  • Step 7 Synthesis of E4-E12, nucleophilic substitution reaction.
  • E4 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(4- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)butyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E5 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(5- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)pentyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E6 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(6- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)hexyl)piperazin-1-yl ]Methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E7 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(7- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)heptyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E8 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(8- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)octyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E9 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(9- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)nonyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E10 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(10- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)decyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E11 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(11- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)undecyl)piperazine- 1-yl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • E12 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(12- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)oxy)dodecyl)piperazine- 1-yl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • This example provides the synthesis and related chemical data of 9 bifunctional compounds S4-S12.
  • the synthetic route of S4-S12 is as follows:
  • the first step S1 (4mmol) and S2 (4.4mmol) were dissolved in DMSO (10mL), HOAT (0.55g, 1.5mmol) and EDCI (0.84g, 2.2mmol) were added, and the reaction solution was stirred at 45°C for 20h. After the reaction was monitored by TLC, the reaction solution was poured into ice water (100mL), stirred for 30 minutes, and then precipitated out, filtered, washed with water, dried and dissolved in a mixture of methanol and chloroform (10:1), mixed with samples, and passed through a silica gel column. Analyze and purify to obtain yellow solid S3 (1.57g).
  • Step 2 Take S3 (2.5mmol, 1.0eq), borate (3mmol, 1.2eq) and dissolve it in a mixed solution of 1,4-dioxane and water (4:1, 30mL), add K 2 CO 3 (3.75mmol, 0.52g), Pd(dppf)Cl 2 (0.2mmol, 146mg), protected by nitrogen, were transferred to 100°C and reacted for 8h, and then cooled to room temperature. The reaction solution was distilled under reduced pressure to remove the solvent, and then dissolved in ethyl acetate, and filtered with Celite. The filtrate was extracted, dried with anhydrous sodium sulfate and concentrated, mixed with samples, and purified by silica gel column chromatography to obtain the product.
  • the third step Synthesis of S6-S11.
  • K7 N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'- ⁇ [4-(7- ⁇ [2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl)thio)heptyl)piperazine-1- Alkyl]methyl ⁇ -5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.
  • M5 1-[(2S)-Butane-2-yl]-N-[(4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6- ⁇ 6-[4-(5- ⁇ [2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]thioether group ⁇ pentyl)piperazin-1-yl]pyridin-3-yl ⁇ -3-methyl-1H-indole-4-carboxamide.
  • N4 1-((S)-sec-butyl)-N-((4,6-Dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl)-6-( 6-(4-(4-((2-(2,6-dioxapiperidin-3-yl)-1,3-dioxaisoindolin-4-yl)amino)butyryl)piper Azin-1-yl)pyridin-3-yl)-3-methyl-1H-indole-4-carboxamide.
  • the instruments used in the biological experiments in the embodiments of the present disclosure are as follows: Ultra-clean workbench BHC-1000IIA/B3: Sujing Antai Biotechnology Company; Constant temperature water bath PolyScience 9505: PolyScience Company; Sterilizer MLS-3780: SANYO Company; Oven : Binder company; ultrapure water meter Milli-Q Integral 10: Millipore company; microplate reader Multiscan MK3, cell incubator, low-speed centrifuge Sorvall ST1: Thermofisher company; flow cytometer: BD company; pH meter ORION STAR A211: ThermoScientific; Sonic Materials Inc: Danbury; 37°C constant temperature shaker Thermolyne, small vertical electrophoresis tank Mini-PROTEAN 3, transfer film tank Mini Trans-blot: Bio-Rad company; X-ray photography cassette AX-II : Guangdong Yuehua Medical Equipment Factory Co., Ltd.; automatic X-ray developing machine HQ
  • the cell strain used in the embodiment of the present disclosure was purchased from American Type Culture Collection (ATCC). 6, 24, 96-well plates for cell culture, 15 mL, 50 mL centrifuge tubes, 25 cm 2 culture square flasks and 75 cm 2 culture flasks were all purchased from Chengdu Abidin Company. The 10 mL petri dish was purchased from WHB Company. Matrigel was purchased from BD Company. Dimethyl maplein (DMSO), MTT, SDS, polysorbate Tween-20, sodium dodecyl sulfonate SDS, glycine, Tris, PEG-400 were purchased from Sigma.
  • DMSO dimethyl maplein
  • MTT MTT
  • SDS polysorbate Tween-20
  • sodium dodecyl sulfonate SDS glycine
  • Tris Tris, PEG-400 were purchased from Sigma.
  • the skimmed milk powder was purchased from Yili Dairy Company; the self-developing film was purchased from Kodak Company.
  • Common reagents such as 10% APS, 1mol/L Tris-HCl (pH: 6.8), 1.5mol/L Tris-HCl (pH: 8.8), electrophoresis buffer transfer buffer, TBS buffer, TBST buffer, etc. are prepared in this experiment .
  • All antibodies were purchased from Cell signaling Technology (Beverly, MA).
  • the blocking goat serum, rabbit serum and DAB color reagent kit were purchased from Beijing Zhongshan Jinqiao Company.
  • the TUNEL detection kit was purchased from Promega (Roche Applied Science).
  • the degradation of the core subunits is significantly weaker than that of E4.
  • E7 7 carbon atoms
  • E8-E12 shows a strong degradation effect on the subunits of PRC2
  • the degradation ability of E8-E12 on PRC2 core subunits gradually weakened; while the same dose of EZH2 enzyme inhibitors GSK126 and EPZ6438 under the same conditions hardly changed the protein levels of PRC2 core subunits in the cell.
  • the inhibition of EZH2 enzyme activity in WSU-DLCL-2 cells was evaluated by detecting the levels of H3K27me3 in the cells.
  • 1 ⁇ M test molecule can almost inhibit H3K27me3 to different degrees after acting on WSU-DLCL-2 cells for 48h, and the trend of the decrease of H3K27me3 protein level is roughly the same as the trend of PRC2 subunit degradation, among which G8-G12,
  • the inhibitory rate of E4 and E7-E11 on H3K27me3 is above 60%, and they have relatively strong inhibitory activity.
  • the two types of PRC2 degradation molecules that target EZH2 can almost degrade the core subunits of PRC2 and inhibit H3K27me3, but their ability to degrade PRC2 and inhibit H3K27me3 varies depending on the length of the Linker alkyl chain. difference.
  • PROTAC E7 not only showed the best in vitro EZH2 enzyme inhibitory activity, but also showed the best degradation of PRC2 core subunits (degradation rate: EZH2 72%, SUZ12 75%, EED 81%, RbAp48 74%) and inhibit H3K27me3 (Inhibition rate 86%), therefore, the compound E7 was selected to continue the subsequent chemical biology research.
  • E7 mainly degrades free EZH2 that is not involved in the formation of PRC2 complex in the initial stage of action (0-12h), and it takes longer to induce degradation of EZH2 and other subunits of PRC2 complex to form a complete ternary complex .
  • E7 acts for 24 hours, the protein levels of PRC2 core subunits EZH2, SUZ12, EED, and RbAp48 and its catalytic product H3K27me3 begin to decrease significantly, and after that, with the extension of the action time, E7 degrades and affects the PRC2 subunits.
  • the inhibition of H3K27me3 is gradually enhanced, until the effect of 96h is almost complete degradation of PRC2.
  • the ligases formed binary complexes respectively, but reduced the effective concentration involved in the formation of the ternary complex of EZH2-E7-E3 ubiquitin ligase, which was manifested in the degradation of PRC2 core subunits and the inhibitory effect on H3K27me3 Weaken. From the above results, it can be concluded that 1 ⁇ M E7 can significantly and stably degrade EZH2, SUZ12, EED and RbAp48 subunits of PRC2 when acting on WSU-DLCL-2 cells for 48 hours and effectively inhibit H3K27me3.
  • RT-qPCR fluorescence real-time quantitative PCR
  • E7 ability of E7 to degrade PRC2 in several other tumor cell lines driven by EZH2 dysfunction was further investigated to rule out the specific effect of E7 on DLBCL cell line WSU-DLCL-2.
  • the results are shown in D in Figure 4, 1 ⁇ M E7 can significantly degrade the core of PRC2 in DLBCL (WSU-DLCL-2, Pfeiffer), PCa (LNCaP, DU 145) and ovarian cancer (A2780, SKOV3) cells for 48 hours.
  • the Cell Thermal Drift Test can determine the binding of the drug to the protein by detecting the change in the thermal stability of the protein caused by the drug in the cell.
  • the principle is: the combination of the drug in the cell with the corresponding protein will enhance the structural stability of the protein , So that the protein can withstand higher temperatures without being degraded. Therefore, the combination of E7 and PRC2 core subunits was investigated by CETSA. Incubate E7 and WSU-DLCL-2 cells pretreated with MG-132 for a certain period of time.
  • E7 After E7 binds to the corresponding protein in the cell, extract the cell lysate to detect EZH2 and SUZ12 in the control group and E7-treated cells , EED and RbAp48 proteins incubate for 6min at different temperatures (45, 48, 51, 54, 57, 60°C).
  • E7 and EZH2 inhibitor and EED inhibitor further verified the binding of E7 and EZH2 subunits. It can be seen from B in Figure 5 that in WSU-DLCL-2 cells, E7 can significantly reduce the protein levels of EZH2, SUZ12, EED, RbAp48 and H3K27me3, when the EZH2 inhibitor EPZ6438 or GSK126 and E7 are used to treat the cells simultaneously
  • the degradation effect of E7 on the core subunits of PRC2 is weakened, but H3K27me3 is still strongly inhibited; while treating the cells with EED inhibitor EED226 and E7 at the same time does not affect the degradation effect of E7 on PRC2 subunits, nor does it affect E7.
  • EPZ6438 and GSK126 interfere with the degradation of each subunit of PRC2 by E7 are competing with E7 to bind to the SAM binding pocket of EZH2, hinder the binding of E7 and EZH2, resulting in a decrease in the actual concentration of E7; while EPZ6438 and GSK126 do not Influencing the inhibitory effect on H3K27me3 is because as EZH2 methyltransferase inhibitors, they have the activity of inhibiting H3K27me3, so they occupy the binding site of E7 and also play the role of inhibiting H3K27me3, and can still maintain a strong inhibition of H3K27me3.
  • EED226 The binding site of EED226 is the H3K27me3 binding pocket of EED. Its binding to EED does not occupy the binding site of E7, so it does not affect the degradation of PRC2 subunits and the inhibition of H3K27me3 by E7. This indirectly proves that E7 binds to the EZH2 subunit of PRC2.
  • E7 induces degradation of EZH2 and other subunits of PRC2, and it is the premise that ubiquitin molecules are first recruited to the target protein, and the recognition and degradation of polyubiquitinated proteins by UPS is required to achieve this. Therefore, the ubiquitination modification of EZH2, SUZ12 and EED subunits in WSU-DLCL-2 cells treated with E7 was detected by immunoprecipitation experiment (IP). The results are shown in Figure 6.
  • lenalidomide, MLN4924 and MG-132 were used to verify whether blocking the ubiquitination modification of the target protein or inhibiting the activity of the proteasome can destroy the degradation of E7, so as to further determine the way E7 degrades PRC2.
  • Lenalidomide is a CRBN ligand with a structure very similar to thalidomide.
  • MG-132 is a proteasome inhibitor.
  • the D in Figure 6 shows that pre-inhibiting the activity of the proteasome in WSU-DLCL-2 cells with MG-132 can also prevent the subunits of PRC2 from being degraded by E7 and stably exist.
  • EZH2 plays a role in two aspects in the process of driving tumorigenesis and development: one is as a transcription repressor, which mediates transcriptional silencing of downstream genes in a manner that depends on its methyltransferase catalytic activity; As a transcriptional co-activator, it mediates transcriptional activation of downstream target genes in a manner independent of methyltransferase activity.
  • E7 activates the transcriptional silencing mediated by the catalytic function of EZH2
  • EZH2 mutations cause abnormally high levels of histone H3K27 methylation modification, which makes the chromatin structure highly constricted, leading to the transcriptional silencing of downstream tumor suppressor genes and triggering tumor formation. Therefore, we first tested the regulation of E7 on several H3K27me3 enriched genes ADRB2, CDKN2A, TXINP and TNFRSF21 in two EZH2 mutant DLBCL cells WSU-DLCL-2 (EZH2 Y641F ) and Pfeiffer (EZH2 A677G). .
  • RT-qPCR results of A and B in Figure 7 show that after WSU-DLCL-2 and Pfeiffer cells were treated with E7 for 48 hours, the mRNA levels of several detected genes were almost all significantly up-regulated, and E7 had a significant effect on ADRB2 and TNFRSF21.
  • the up-regulation was stronger than that of EZH2 enzyme inhibitors EPZ6438 and GSK126, but the up-regulation of CDKN2A and TXINP was slightly weaker than that of EPZ6438 and GSK126.
  • E7 can effectively activate the catalytic function-mediated transcriptional silencing of EZH2 and inhibit the catalytic activity of EZH2.
  • EZH2 can significantly promote the proliferation of tumor cells, and this proliferation promotion is partly dependent on the catalytic function of EZH2 in PRC2, so we are also in NSCLC with SWI/SNF mutations.
  • the regulation of E7 on the transcription of the above genes was tested in the cell line A549. The results are shown in C in Figure 7.
  • E7 Unlike the results observed in DLBCL, E7 only increased the mRNA levels of CDKN2A and TXINP2 genes, but failed to up-regulate the expression of ADRB2 and TNFRSF21.
  • the degree of upregulation of these genes by E7 in A549 is not as obvious as in WSU-DLCL-2 and Pfeiffer.
  • the reason is that the catalytic activity of EZH2 does not play a leading role in the proliferation of tumor cells with SWI/SNF mutations such as A549. Activity is the main cause of the proliferation of such tumor cells. Therefore, in A549 cells, the transcription of genes such as ADRB2 and CDKN2A is not inhibited by the catalytic activity of EZH2.
  • PROTAC E7 degrades EZH2 and other core subunits of PRC2, either inhibits gene silencing mediated by EZH2 as a transcription repressor, or inhibits EZH2 as transcription
  • the abnormal activation of transcription mediated by co-activators can ultimately effectively inhibit the driving role of EZH2 in tumorigenesis and development.
  • E7 can not only destroy the catalytic function of EZH2 that depends on PRC2, but also destroy its non-catalytic function that does not depend on PRC2, so it can completely inhibit the carcinogenic activity of EZH2.
  • E7 inhibits the proliferation of tumor cells with abnormal EZH2
  • E7 can almost completely inhibit the growth of WSU-DLCL-2 cells, showing good proliferation inhibitory activity; although EPZ6438 and GSK126 also show certain proliferation inhibitory activity on WSU-DLCL-2 cells , But its degree of inhibition is significantly inferior to that of E7. It can be seen that E7 can effectively inhibit the growth of tumor cells driven by the catalytic function of EZH2.
  • the proliferation activity of SWI/SNF mutant A549 and NCI-H1299 cells mainly depends on the non-catalytic function of EZH2 in PRC2.
  • E7 has very significant proliferation inhibitory activity on both A549 and NCI-H1299 cell lines; although GSK126 can also be obvious It inhibits the proliferation of the two cell lines, but the inhibitory activity is not as good as E7; EPZ6438 only shows very limited proliferation inhibitory activity on these two cell lines.
  • the MTT test results in Figure 10 also show that the tumor cells WSU-DLCL-2 driven by the catalytic function of EZH2, Pfeiffer, and the cell viability of the tumor cells A549 and NCI-H1299 driven by the non-catalytic function of EZH2 all showed a good time.
  • Dependent inhibition, IC 50 values are all at low micromolar levels, especially for the DLBCL cell line Pfeiffer, the IC 50 of E7 for 7 days is only 0.17 ⁇ M.
  • the above results further prove that E7 has a good inhibitory effect on the viability of tumor cells driven by the catalytic and non-catalytic functions of EZH2.
  • the above biological experimental data shows that the compounds of the embodiments of the present disclosure have a degrading effect on each subunit of the PRC2 complex, and exhibit a more extensive and powerful anti-tumor effect than inhibitors.
  • the bifunctional compound can effectively induce the degradation of the core subunits of the PRC2 protein complex, thereby achieving the treatment of cancer mediated by the PRC2 complex and its subunits including EZH2, EED, SUZ12, RbAp46, and RbAp48, completely blocking the PRC2 complex
  • the carcinogenic activity of the substance subunits such as EZH2 inhibitors and EED inhibitors
  • EZH2 inhibitors and EED inhibitors has better anti-cancer activity. It has the ability to treat breast cancer, colorectal cancer, prostate cancer, and pancreas.
  • Use of various solid tumors such as cancer and ovarian cancer, as well as various tumor diseases such as hematoma.
  • the bifunctional compound or pharmaceutical composition is used as a kinase inhibitor to treat a variety of human tumors, and it has good anti-tumor activity and low toxicity.

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Abstract

一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用，该双功能化合物包括如式（I）-（III）任一项所示的化合物、其药学上可接受的盐或前药、其溶剂化物、其水合物、其多晶型物、其互变异构体、立体异构体或同位素取代的化合物式（I），式（II），式（I）：其中式（I）-（III）中的n均为1～10的整数，X均为O，N或S，Y均为O，H 2或S。该双功能化合物能够诱导PRC2蛋白复合物核心亚基降解，进而达到治疗由PRC2复合物和其亚基EZH2，EED，SUZ12，RbAp46，RbAp48介导的癌症，阻断PRC2复合物亚基的致癌活性。

Description

可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用
相关申请的交叉引用
本申请要求于2020年05月25日提交中国专利局的申请号为2020104518316、名称为“可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及化学医药技术领域，具体而言，涉及一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用。
背景技术
随着对肿瘤发病机制研究的深入，人类已经深刻意识到肿瘤的发生和发展不仅与细胞内基因突变或缺失等所导致的DNA序列改变相关，而且与其他可遗传物质发生改变引起的基因失调有关，也就是所谓的表观遗传现象。表观遗传修饰可以通过DNA甲基化、组蛋白修饰、染色质重塑或非编码RNA干扰的方式调控机体内原癌基因激活、抑癌基因失活、DNA损伤修复及肿瘤干细胞分化等过程，从而调控机体的生长、发育和病理改变。
多疏基因家族(polycomb group，PcG)蛋白最初被发现是一类在果蝇发育和细胞分化过程中通过参与调控Hox基因的表达而调节果蝇机体建立的蛋白，后来被鉴定为对多种细胞功能至关重要的表观遗传调控因子。在哺乳动物中，PRC1和PRC2是PcG蛋白中两种主要的多疏抑制复合物，它们通过组蛋白翻译后修饰调控基因的转录。多个亚基不同组合可以组成功能各异的PRC1，其中经典的PRC1由CBX(chrombox)、PCGF(polycomb group factor)、HPH(human polyhomeotic homolog)和RING1A/B(really interesting new gene)几个核心亚基组成，主要通过催化亚基RING1A/B介导H2AK119ub1，在胚胎发育、维持干细胞特性以及肿瘤发生等过程中发挥重要作用；PRC2包括EZH2、SUZ12、EED和RbAp46/48(retinoblastoma-associated proteins 46/48，也称RBBP4/7)几个核心亚基，主要通过催化亚基EZH2介导H3K27me2和H3K27me3，调控与细胞周期、衰老、分化和肿瘤发生相关的基因的转录。
其中，EZH2作为PRC2的催化亚基，可在复合物的另外两个核心亚基SUZ12和EED的共同参与下催化H3K27me3，并与PRC1介导的H2AK119ub1共同在核小体上形成阻碍转录延伸的复合体，进而维持下游基因的转录沉默。EZH2本身不能单独发挥催化活性，它必须至少依赖于PRC2的SUZ12和EED两个亚基才能发挥HMTase活性而抑制下游多种基因的转录。SUZ12是维持PRC2完整性、EZH2稳定性和HMTase活性至关重要的一个核心亚基，其C端的VEFS(Vrn2-Emf2-Fis2-SUZ12)结构域一方面可与EZH2稳定结合，维系PRC2复合物的组装，另一方面可通过变构作用辅助发挥PRC2的HMTase活性。EED亚基的重复WD结构域与EZH2的相互作用和结合是确保EZH2具有HMTase活性的必需条件，并且EED的C端WD结构域与H3K36me3的特异性结合是HMTase变构激活的必需条件，因而EED也是PRC2发挥HMTase活性的一个核心亚基。成视网膜细胞瘤相关蛋白46和48(RbAp46/48)亚基是高度同源的组蛋白伴侣蛋白，它们对于染色质结构的维持具有重要作用。在PRC2中，虽然EZH2发挥HMTase活性不需要RbAp46和RbAp48的直接参与，但将PRC2引入核小体的过程需要RbAp48与组蛋白H3-H4异源二聚体结合才能实现，所以RbAp48也是保证PRC2正常发挥HMTase活性的核心亚基。
但是，PRC2复合物各核心亚基除参与形成PRC2复合物催化H3K27me3并上调转录沉默的抑癌基因之外，还可发挥自身的非PRC2催化功能。例如，EZH2作为一种多功能蛋白不仅可以通过催化H3K27me3介导基因沉默，还能以不依赖于HMTase活性的方式在某些肿瘤中介导基因的转录激活。多项研究表明EZH2还可以作为转录激活因子甲基化非组蛋白或直接与其他蛋白相互作用形成转录复合物，激活基因的转录。EZH2的21位丝氨酸(Ser)被蛋白激酶B(AKT)磷酸化后，pEZH2可作为雄激素受体(androgen receptor，AR)及其相关复合物的共激活因子促进去势抵抗性前列腺癌(castration-resistant prostate cancer，CRPC)细胞的生长，即AKT介导EZH2的Ser21磷酸化，促进EZH2结合AR并催化AR或AR相关蛋白甲基化，从而激活AR下游一系列靶基因的转录，这一发现为治疗转移性、激素难治性前列腺癌提供了新的联合治疗思路。还有研究发现磷酸化的EZH2也可以直接结合并甲基化STAT3的Lys180，介导STAT3的705位酪氨酸(Tyr)磷酸化而增强STAT3的活性，从而促成胶质母细胞瘤和胶质母细胞瘤干细胞的致瘤性。这些结果与EZH2催化非组蛋白GATA4和RORα而抑制基因转录的结果全然相反。
此外，EZH2在结直肠癌细胞中也被发现可以以不依赖于甲基转移酶活性的方式激活转录。人增殖细胞核抗原相关因子PAF招募EZH2，促进EHZ2与TCF/β-连环蛋白结合并发生相互作用，在c-myc、cyclinD1、Axin2等基因的启动子处与转录因子TCF/LEF共同形成转录起始复合物，诱导Wnt通路中肿瘤形成相关靶基因的转录激活，促进结直肠癌进展。另外，在AR阳性前列腺癌中，EED不依赖于PRC2功能直接与雄激素受体发生相互作用，调控AR的表达水平和AR下游靶标，促进前列腺癌的发展。EED还可不依赖于PRC2功能与组蛋白去乙酰基转移酶HDAC直接作用，影响HDAC的催化活性。
因此，目前的EZH2抑制剂或EED抑制剂仅用于抑制PRC2的活性，但是并不能有效抑制EZH2，EED等不依赖于PRC2催化功能的致癌活性，更不能同时对PRC2的多种核心亚基进行降解，来完全阻断PRC2复合物亚基的致癌活性。
鉴于此，特提出本公开。
发明内容
本公开的目的包括，例如提供一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用，以改善上述问题。
本公开是这样实现的：
第一方面，本公开实施例提供了一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物，其包括如式I-III任一项所示的化合物、其药学上可接受的盐或前药、其溶剂化物、其水合物、其多晶型物、其互变异构体、立体异构体或同位素取代的化合物；
Figure PCTCN2020119438-appb-000001
其中，式I-III中的n均为1～10的整数，X均为O，N或S，Y均为O，H 2或S。
在一种或多种实施方式中，式I-III中的n均为2～6的整数。
在一种或多种实施方式中，式I-III中的n均为2～10的整数，X和Y均为O；在一种或多种实施方式中，式I-III中的n均为2～6的整数，X和Y均为O；在一种或多种实施方式中，所述双功能化合物的化学式为
Figure PCTCN2020119438-appb-000002
第二方面，本公开实施例还提供了一种双功能化合物的制备方法，式I-III中X为N，S，Y为O时，式I-III所示的化合物的合成路线为：
Figure PCTCN2020119438-appb-000003
式I-III中X和Y均为O时，式I化合物的合成路线为：
Figure PCTCN2020119438-appb-000004
式II化合物的合成路线为：
Figure PCTCN2020119438-appb-000005
式III化合物的合成路线为：
Figure PCTCN2020119438-appb-000006
第三方面，本公开实施例还提供了一种药物组合物，其包括药学上可接受的辅助性成分和前述实施例的双功能化合物。
在一种或多种实施方式中，所述药物组合物为水溶液、粉末、颗粒、片剂或冻干粉，在一种或多种实施方式中，所药物组合物为水溶液时，所述药物组合物还含有注射用水、盐水溶液、葡萄糖水溶液、注射或输注用盐水、注射或输注用葡萄糖、格林氏溶液或含有乳酸盐的格林氏溶液。
第四方面，本公开实施例还提供了前述实施方式的双功能化合物或药物组合物在制备激酶抑制剂中的应用。
第五方面，本公开实施例还提供了前述实施方式的双功能化合物或药物组合物在制备治疗肿瘤的药物中的应用；可选地，所述肿瘤包括乳腺癌、结直肠癌、前列腺癌、卵巢癌、胰腺癌或胃癌。
第六方面，本公开实施例还提供了前述实施方式的双功能化合物或药物组合物在制备降解PRC2蛋白复合物核心亚基的降解剂中的应用，可选地，降解PRC2蛋白复合物核心亚基为同时降解PRC2蛋白复合物的EZH1，EZH2，EED，SUZ12以及RbAp46/48亚基。
第七方面，本公开实施例还提供了前述实施方式的双功能化合物或药物组合物在制备口服或静脉注射制剂中的应用，口服或静脉注射剂至少包括所述双功能化合物或药物组合物，可选地，还包括赋形剂和/或佐剂。
本公开上述实施例的方案之一至少具有以下有益效果：该双功能化合物能够有效地诱导PRC2蛋白复合物核心亚基降解，进而达到治疗由PRC2复合物和其亚基包括EZH2，EED，SUZ12，RbAp46，RbAp48在内介导的癌症，完全阻断PRC2复合物亚基的致癌活性，相比于单纯地抑制PRC2复合物活性，例如EZH2抑制剂，EED抑制剂，具有更好地抗癌活性，其具有能够治疗乳腺癌、结直肠癌、前列腺癌、胰腺癌、卵巢癌等多种实体瘤以及血液瘤等多种肿瘤疾病的用途。该双功能化合物或药物组合物作为激酶抑制剂用于治疗人的多种肿瘤，并且其具有较好的抗肿瘤活性和较低的毒性。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1和图2为蛋白免疫印迹法检测WSU-DLCL-2细胞内EZH2、SUZ12、EED、RbAp48以及组蛋白H3K27me3的蛋白水平；
图3为合成分子降解PRC2各核心亚基并降低H3K27me3水平；
图4为E7有效降解PRC2并抑制H3K27me3。A.蛋白免疫印迹检测E7在不同时间点对WSU-DLCL-2细胞内PRC2各亚基和H3K27me3的抑制情况。B.蛋白免疫印迹检测不同浓度E7作用于WSU-DLCL-2细胞48h对PRC2各亚基和H3K27me3的抑制情况。C.RT-qPCR检测WSU-DLCL-2细胞内PRC2各核心亚基的mRNA水平。统计结果以三次平均值±SD的形式表示。D.蛋白免疫印迹检测E7在DLBCL、PCa和卵巢癌细胞株中对PRC2各亚基的降解作用；
图5为E7与PRC2的EZH2亚基结合发挥作用。A.CETSA检测E7处理的WSU-DLCL-2细胞内EZH2、SUZ12、EED和RbAp48蛋白的热稳定性及相应的免疫印迹灰度统计结果。统计结果均以三次平均值±标准差(SD)的形式表示。B.蛋白免疫印迹检测WSU-DLCL-2细胞中EZH2抑制剂和EED抑制剂与E7的竞争性作用。C.蛋白免疫印迹检测E7对于WSU-DLCL-2细胞中组蛋白H3的多种甲基化修饰产物；
图6为E7通过泛素蛋白酶体途径降解PRC2。免疫沉淀实验检测1μM E7作用48h对EZH2(A)、SUZ12(B)和EED(C)泛素化修饰的影响。D.WSU-DLCL-2细胞经1μM来那度胺或5μM MLN4924/MG-132预处理4h后，用1μM E7处理细胞48h，收集细胞裂解液进行免疫印迹分析；
图7为E7激活WSU-DLCL-2(A)，Pfeiffer(B)和A549(C)细胞中EZH2的催化功能介导沉默的基因的转录。细胞经指定化合物处理48h后利用RT-qPCR检测mRNA水平。统计结果以三次平均值±SD的形式表示，*P＜0.05，**p＜0.01；
图8为E7抑制A549(A)，NCI-H1299(B)和MDA-MB-468(C)细胞中EZH2的非催化功能介导激活的基因的转录。细胞经指定化合物处理48h后利用RT-qPCR检测mRNA水平。统计结果以三次平均值±SD的形式表示，*P＜0.05，**p＜0.01；
图9为E7有效抑制WSU-DLCL-2(A)、A549(B)和NCI-H1299(C)细胞的增殖。经10μM E7,EPZ6438 或GSK126处理的细胞的增殖曲线(左)和在不同时间点的光学显微镜图像(右)，统计结果以三次平均值±SD的形式表示；
图10为MTT实验检测E7作用3d、5d、7d对WSU-DLCL-2(A)、Pfeiffer(B)、A549(C)和NCI-H1299(D)细胞活力的抑制作用。存活曲线及IC50值由GraphPad Prism 5.0软件拟合。统计结果以两次平均值±SD的形式表示。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开提供的一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物和药物组合物及应用进行具体说明。
本公开的一些实施方式提供了一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物，其包括如式I-III任一项所示的化合物、其药学上可接受的盐或前药、其溶剂化物、其水合物、其多晶型物、其互变异构体、立体异构体或同位素取代的化合物；
Figure PCTCN2020119438-appb-000007
其中，式I-III中的n均为1～10的整数，X均为O，N或S，Y均为O，H 2或S。
上述术语“药学上可接受的”是指在在合理的医学判断范围，能适于用来与人类和其他哺乳动物的组织接触，而没有不当毒性、刺激、过敏反应等，其在对受者给药时能直接或间接地提供本公开上述实施方式的双功能化合物。术语“水合物”表示进一步通过非共价分子间作用力结合化学计量或非化学计量的水的化合物。术语“多晶型物”表示化合物或其复合物的固体结晶形式，其可以通过物理方法，例如X.射线粉末衍射图或红外光谱进行表征。
发明人在现有的EZH2抑制剂，EED抑制剂仅抑制PRC2复合物的活性，但是并不能有效抑制EZH2，EED等不依赖于PRC2催化功能的致癌活性，更不能对PRC2复合物的其他核心亚基例如EZH1、SUZ12以及RbAp46/48等同时进行抑制的情况下，进行了大量的研究和实践，创造性地发现了以上三种结构式所述的双功能化合物，能够有效地诱导PRC2蛋白复合物核心亚基降解，进而达到治疗由PRC2复合物和其亚基包括EZH2，EED，SUZ12，RbAp46，RbAp48在内介导的癌症，完全阻断PRC2复合物亚基的致癌活性，相比于单纯地抑制PRC2复合物活性，例如EZH2抑制剂，EED抑制剂，具有更好地抗癌活性，其具有能够治疗乳腺癌、结直肠癌、前列腺癌、胰腺癌、卵巢癌等多种实体瘤以及血液瘤等多种肿瘤疾病的用途。
需要说明的是，该可诱导PRC2蛋白复合物核心亚基降解的双功能化合物还可以是I-III任一项所示的化合物的互变异构体、立体异构体及其所有比例的混合物。
为了进一步优化双功能化合物的降解能力以及综合性能，一些实施方式中，上述式I-III中的n均为2～6的整数。
一些较佳的实施方式中，式I-III中的n均为2～10的整数，X和Y均为O；在一种或多种实施方式中，式I-III 中的n均为2～6的整数，X和Y均为O。例如，一些实施方式中，该双功能化合物的化学式可为
Figure PCTCN2020119438-appb-000008
当式I-III中X为N，S，Y为O时，式I-III所示的化合物的合成路线为：
Figure PCTCN2020119438-appb-000009
当式I-III中X和Y均为O时，其三类化合物可以通过一些合成路线进行合成，具体地，式I化合物的合成路线为：
Figure PCTCN2020119438-appb-000010
式II化合物的合成路线为：
Figure PCTCN2020119438-appb-000011
式III化合物的合成路线为：
Figure PCTCN2020119438-appb-000012
在一种或多种实施方式中，本公开的一些实施方式还提供了一种药物组合物，其包括药学上可接受的辅助性成分和前述实施方式的双功能化合物。其中辅助性成分为一般性只要过程中添加的常规成分。
在一种或多种实施方式中，上述药物组合物可以为液体形式，也可以为固体形式。即药物组合物包括但不限于水溶液、粉末、颗粒、片剂或冻干粉，为了使得药物组合物能够充分作用于机体，一些实施方式中，药物组合物为水溶液时，药物组合物还含有注射用水、盐水溶液、葡萄糖水溶液、注射或输注用盐水、注射或输注用葡萄糖、格林氏溶液或含有乳酸盐的格林氏溶液。
在一种或多种实施方式中，本公开的一些实施方式还提供了前述双功能化合物或药物组合物在制备激酶抑制剂中的应用。特别是对于EZH2酶具有较佳的抑制性能。
在一种或多种实施方式中，本公开的一些实施方式还提供了前述双功能化合物或药物组合物在制备治疗肿瘤的药物中的应用；可选地，肿瘤包括乳腺癌、结直肠癌、前列腺癌、卵巢癌、胰腺癌或胃癌等实体瘤。
在一种或多种实施方式中，本公开的一些实施方式还提供了前述双功能化合物或药物组合物在制备降解PRC2蛋白复合物核心亚基的降解剂中的应用。可选地，降解PRC2蛋白复合物核心亚基为同时降解PRC2蛋白复合物的EZH1，EZH2，EED，SUZ12以及RbAp46/48亚基。
在一种或多种实施方式中，本公开的一些实施方式还提供了前述双功能化合物或药物组合物在制备口服或静脉注射制剂中的应用。口服或静脉注射剂至少包括该双功能化合物或该药物组合物，可选地，还包括赋形剂和/或佐剂。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供了9种双功能化合物G4-G12的合成及其相关化学数据。G4-G12的合成路线如下：
Figure PCTCN2020119438-appb-000013
具体制备过程为：
第一步：制备1b，氨解反应。
1a(3.28g,20mmol,1.0eq)，3-氨基哌啶-2,6-二酮(3.1g,24mmol,1.2eq)溶解于50mL乙酸酐中，140℃加热回流6h，反应完成后减压蒸馏，除去反应溶剂，将剩余物倒入水中，浅灰色沉淀析出，抽滤，将滤饼水洗、干燥获得产品1b(3.84g,71％)。HRMS m/z calculated for C13H10N2O5[M+H]+:275.0662,found:275.0686。
第二步：1c-1k通用合成步骤。
1b(1mmol,1.0eq),DIPEA(3mmol,3.0eq)以及二溴烷烃(1.2mmol,1.2eq)溶解于DMF中(10mL)，于85-100℃反应3-6h。反应完成后，加入乙酸乙酯(50mL)及水萃取，Na 2SO 4干燥后浓缩获得残余物，经柱层析纯化得到相应产品。
化合物1c：白色固体(195.8mg,48％)。1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.82(dd,J＝8.5,7.2Hz,1H),7.52(d,J＝8.5Hz,1H),7.45(d,J＝7.2Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.26(t,J＝6.1Hz,2H),3.66(t,J＝6.7Hz,2H),2.88(m,J＝17.1,13.9,5.4Hz,1H),2.57(dd,J＝16.4,12.1Hz,2H),2.03(ddd,J＝12.5,8.5,5.8Hz,3H),1.90(dt,J＝8.8,6.1Hz,2H).HRMS m/z calculated for C 17H 17BrN 2O 5[M+Na]+:431.0213,found:431.0219。
化合物1d：白色固体(236.3mg,56％)。1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.81(dd,J＝8.5,7.2Hz,1H),7.52(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.22(t,J＝6.3Hz,2H),3.56(t,J＝6.7Hz,2H),2.94–2.82(m,1H),2.67–2.53(m,2H),2.03(m,J＝13.5,6.0,3.4,2.9Hz,1H),1.90(m,J＝6.8Hz,2H),1.80(m,J＝6.7Hz,2H),1.59(m,J＝9.6,6.2Hz,2H).HRMS m/z calculated for C 18H 19BrN 2O 5[M+Na] +:445.0370,found:445.0367。
化合物1e：白色固体(161.3mg,37％)。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.81(dd,J＝8.5,7.3Hz,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.21(t,J＝6.3Hz,2H),3.54(t,J＝6.7Hz,2H),2.96–2.82(m,1H),2.67–2.53(m,2H),2.03(m,J＝13.5,6.5,6.0,3.4Hz,1H),1.84(q,J＝6.8Hz,2H),1.76(q,J＝6.7Hz,2H),1.47(dq,J＝7.8,4.5,4.1Hz,4H).HRMS m/z calculated for C 19H 21BrN 2O 5[M+Na] +:459.0526,found:459.0523。
化合物1f：白色固体(284.0mg,63％)。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.81(t,J＝7.9Hz,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.21(t,J＝6.4Hz,2H),3.53(t,J＝6.7Hz,2H),2.89(m,J＝19.1,14.4,5.4Hz,1H),2.59(d,J＝17.3Hz,2H),2.11–1.96(m,1H),1.79(dp,J＝20.7,6.6Hz,4H),1.54–1.30(m,6H).HRMS m/z calculated for C 20H 23BrN 2O 5[M+H] +:451.0863,found:451.0852。
化合物1g：白色固体(232.4mg,50％)。1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.80(dd,J＝8.5,7.2Hz,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.20(t,J＝6.4Hz,2H),3.52(t,J＝6.7Hz,2H),2.94–2.81(m,1H),2.69–2.52(m,2H),2.03(m,J＝14.1,6.7,3.3,2.9Hz,1H),1.77(tq,J＝12.4,6.7Hz,4H),1.52–1.28(m,8H).HRMS m/z calculated for C 21H 25BrN 2O 5[M+H] +:465.1020,found:465.0994。
化合物1h：白色固体(292.0mg,61％)。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.81(dd,J＝8.5,7.3Hz,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.20(t,J＝6.4Hz,2H),3.52(m,J＝6.7,2.0Hz,2H),2.89(m J＝16.8,13.9,5.3Hz,1H),2.58(dd,J＝16.9,12.4Hz,2H),2.09–1.99(m,1H),1.84–1.72(m,4H),1.46(t,J＝7.6Hz,2H),1.37(d,J＝7.2Hz,4H),1.33–1.25(m,4H).HRMS m/z calculated for C 22H 27BrN 2O 5[M+H] +:479.1176,found:479.1164。
化合物1i：白色固体(201.7mg,41％)。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.84–7.75(m,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.20(t,J＝6.4Hz,2H),3.52(td,J＝6.7,2.2Hz,2H),2.88(m,J＝17.0,13.9,5.4Hz,1H),2.58(dd,J＝16.3,12.2Hz,2H),2.03(tt,J＝8.0,4.4Hz,1H),1.76(m,J＝14.1,11.4,6.7Hz,4H),1.45(q,J＝7.4Hz,2H),1.36(q,J＝6.3Hz,4H),1.30(d,J＝18.4Hz,6H).HRMS m/z calculated for C 23H 29BrN 2O 5[M+Na] +:515.1152,found:515.1147。
化合物1j：白色固体(293.5mg,58％)。1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.80(t,J＝7.9Hz,1H),7.51(d,J＝8.6Hz,1H),7.44(d,J＝7.3Hz,1H),5.07(dd,J＝13.0,5.4Hz,1H),4.20(t,J＝6.4Hz,2H),3.51(t,J＝6.7Hz, 2H),2.88(s,1H),2.61(s,2H),2.03(d,J＝13.5Hz,1H),1.76(t,J＝9.2Hz,4H),1.45(t,J＝7.8Hz,2H),1.41–1.33(m,4H),1.27(s,8H).HRMS m/z calculated for C 24H 31BrN 2O 5[M+Na] +:529.1308,found:529.1298。
化合物1k：白色固体(176.8mg,34％)。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.81(t,J＝7.8Hz,1H),7.47(dd,J＝27.8,7.8Hz,2H),5.08(dd,J＝13.0,5.5Hz,1H),4.20(t,J＝6.5Hz,2H),3.51(t,J＝6.7Hz,2H),2.98–2.80(m,1H),2.60(d,J＝17.2Hz,2H),2.15–1.96(m,1H),1.77(q,J＝8.6,8.2Hz,4H),1.53–1.41(m,2H),1.35(s,4H),1.26(s,10H).HRMS m/z calculated for C 25H 33BrN 2O 5[M+Na] +:543.1465,found:543.1472。
第三步：制备G4-G12
GSK126(0.25mmol,1eq)，NaHCO 3(0.5mmol,2.0eq)以及1c-1k(0.3mmol,1.2eq)溶于5mL DMF中，在85℃下反应5h。反应完成后,经乙酸乙酯萃取，Na 2SO 4干燥，减压蒸馏除去溶剂后经硅胶柱层析分离获得相应产品G4-G12。
G4：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(4-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}丁基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000014
白色固体(72.1mg,34％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.50(d,J＝2.6Hz,1H),8.12(t,J＝5.2Hz,1H),7.91(dd,J＝8.8,2.7Hz,1H),7.81(t,J＝7.9Hz,1H),7.72(s,1H),7.53(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.17(s,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.09(dd,J＝12.9,5.4Hz,1H),4.59(p,J＝7.0Hz,1H),4.35(d,J＝5.1Hz,2H),4.26(t,J＝6.3Hz,2H),3.51(d,J＝4.9Hz,4H),2.90(td,J＝17.3,15.5,5.3Hz,1H),2.70–2.53(m,2H),2.47(s,2H),2.41(t,J＝6.8Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.08–1.96(m,1H),1.82(q,J＝6.9Hz,4H),1.69(q,J＝7.4Hz,2H),1.40(d,J＝6.6Hz,3H),1.23(s,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.23,170.41,169.18,167.33,165.82,163.57,160.55,158.47,158.47,156.49,149.77,146.00,143.12,138.19,137.49,136.37,133.72,131.17,130.22,126.48,124.86,123.42,122.24,120.28,116.73,116.50,115.60,110.18,108.15,107.82,107.36,69.20,57.79,52.98,52.09,49.24,45.40,35.54,31.44,29.97,26.85,22.93,22.50,21.31,19.44,18.66,12.15,11.20.HRMS m/z calculated for C 48H 54N 8O 7[M+H] +:855.4188,found:855.4185。
G5：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(5-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}戊基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000015
白色固体(45.6mg,21％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.50(d,J＝2.4Hz,1H),8.12(t,J＝5.1Hz,1H),7.91(dd,J＝8.8,2.6Hz,1H),7.81(t,J＝7.9Hz,1H),7.72(s,1H),7.52(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.18(d,J＝1.4Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.59(p,J＝6.9Hz,1H),4.36(d,J＝5.1Hz,2H),4.23(t,J＝6.3Hz,2H),3.51(t,J＝4.7Hz,4H),2.89(m,J＝17.3,14.0,5.4Hz,1H),2.66–2.53(m,2H),2.48(s,2H),2.34(d,J＝7.4Hz,2H),2.24(s,3H),2.17(s,3H),2.11(s,3H),2.07–1.99(m,1H),1.80(m,J＝8.4,4.8Hz,4H),1.53(m,J＝19.9,7.1Hz,4H),1.41(d,J＝6.6Hz,3H),1.24(d,J＝5.1Hz,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.22,170.40,169.18,167.32,165.78,163.57,158.48,156.50,149.77,145.99,143.12,138.20,137.49,136.37,133.72,131.17,130.22,126.48,124.87,123.42,122.24,120.29,116.71,116.50,115.60,110.18,108.15,107.83,107.37,69.28,58.29,53.04,52.09,49.23,45.36,35.54,31.44,29.97,28.81,26.37,23.80,22.50,21.31,19.44,18.65,12.15,11.20.HRMS m/z calculated for C 49H 56N 8O 7[M+H] +:869.4345,found:869.4343。
G6：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(6-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}己基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000016
浅黄色固体(63.3mg,29％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.49(d,J＝2.5Hz,1H),8.12(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.81(t,J＝7.9Hz,1H),7.72(d,J＝1.5Hz,1H),7.52(d,J＝8.6Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.17(d,J＝1.4Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.60(m,J＝6.9Hz,1H),4.35(d,J＝5.1Hz,2H),4.21(t,J＝6.4Hz,2H),3.50(t,J＝4.8Hz,4H),2.89(m,J＝17.5,14.1,5.4Hz,1H),2.66–2.54(m,2H),2.46(s,4H),2.32(d,J＝7.2Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.07–2.00(m,1H),1.86–1.72(m,4H),1.50(m,J＝7.5Hz,4H),1.40(t,J＝7.3Hz,3H),1.23(s,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.23,170.40,169.17,167.31,165.78,163.57,158.47,156.51,149.77,145.99,143.12,138.20,137.49,136.36,133.73,131.17,130.22,126.48,124.86,123.42,122.24,120.29,116.72,116.49,115.60,110.18,108.14,107.82,107.36,69.26,58.33,53.07,52.08,49.22,45.36,35.54,31.44,29.97,28.86,27.04,26.66,25.68,22.50,21.31,19.44,18.66,12.15,11.20.HRMS m/z calculated for C 50H 58N 8O 7[M+H] +:883.4501,found:882.4502。
G7：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(7-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}庚基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000017
灰色固体(73.6mg,33％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.09(s,1H),8.49(d,J＝2.5Hz,1H),8.12(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.81(dd,J＝8.5,7.3Hz,1H),7.72(d,J＝1.5Hz,1H),7.52(d,J＝8.6Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.17(d,J＝1.5Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.60(q,J＝6.9Hz,1H),4.35(d,J＝5.1Hz,2H),4.21(t,J＝6.4Hz,2H),3.50(s,4H),2.88(m,J＝17.3,14.0,5.4Hz,1H),2.69–2.54(m,2H),2.49–2.40(m,4H),2.31(d,J＝7.6Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.03(m,J＝11.9,6.1,3.5Hz,1H),1.78(m,J＝19.7,6.9Hz,4H),1.47(q,J＝7.6Hz,4H),1.40(d,J＝6.6Hz,3H),1.34(dt,J＝9.1,4.3Hz,2H),1.24(d,J＝4.8Hz,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.22,170.39,169.17,167.31,165.77,163.56,158.47,156.51,149.77,145.99,143.11,138.19,137.48,136.37,133.73,131.17,127.92,126.49,123.41,122.24,120.27,116.71,116.50,115.59,110.18,108.14,107.81,107.37,69.28,58.40,53.07,52.08,49.23,45.36,35.53,31.44,29.97,29.04,28.86,27.36,25.74,22.49,21.31,19.44,18.66,12.14,11.20.HRMS m/z calculated for C 51H 60N 8O 7[M+H] +:897.4658,found:897.4656。
G8：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(8-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}辛基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000018
白色固体(41.6mg,18％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.49(d,J＝2.6Hz,1H), 8.13(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.80(dd,J＝8.5,7.2Hz,1H),7.72(d,J＝1.5Hz,1H),7.51(d,J＝8.6Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.17(d,J＝1.4Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.60(q,J＝6.9Hz,1H),4.35(d,J＝5.0Hz,2H),4.21(t,J＝6.3Hz,2H),3.50(t,J＝5.7Hz,4H),2.88(m,J＝17.3,14.1,5.4Hz,1H),2.58(dd,J＝20.8,6.8Hz,2H),2.45(s,4H),2.31(d,J＝7.4Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.07–1.98(m,1H),1.78(m,J＝13.8,6.7Hz,4H),1.48(d,J＝7.1Hz,4H),1.40(d,J＝6.7Hz,3H),1.31(d,J＝10.9Hz,4H),1.23(s,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.22,170.39,169.17,167.31,165.77,163.57,158.47,156.50,149.77,145.99,143.12,138.20,137.48,136.36,133.72,131.17,130.22,126.47,124.86,123.41,122.24,120.27,116.70,116.50,115.58,108.14,107.82,107.35,69.30,58.45,53.07,52.08,49.22,45.36,35.53,31.44,29.97,29.36,29.10,28.87,27.36,26.71,25.72,22.49,21.30,19.44,18.66,12.14,11.20.HRMS m/z calculated for C 52H 62N 8O 7[M+H] +:911.4814,found:911.4819。
G9：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(9-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}壬基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000019
白色固体(85.6mg,37％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.11(s,1H),8.49(d,J＝2.5Hz,1H),8.13(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.80(t,J＝7.9Hz,1H),7.72(s,1H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),7.22–7.13(m,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.60(q,J＝6.9Hz,1H),4.35(d,J＝5.1Hz,2H),4.20(t,J＝6.3Hz,2H),3.50(s,4H),2.88(m,J＝17.8,14.4,5.3Hz,1H),2.68–2.54(m,2H),2.45(s,4H),2.30(s,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.03(dd,J＝11.6,5.8Hz,1H),1.86–1.69(m,4H),1.45(d,J＝7.2Hz,4H),1.40(d,J＝6.6Hz,3H),1.38–1.27(m,6H),1.23(s,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.22,170.39,169.18,167.31,165.76,163.58,158.47,156.50,149.78,145.99,143.12,138.20,137.47,136.35,133.72,131.17,130.23,126.47,124.86,123.42,122.24,120.25,116.70,116.50,115.58,110.19,108.14,107.84,107.35,69.28,58.47,56.50,53.07,52.09,49.23,45.35,35.54,31.44,29.97,29.43,29.37,29.10,28.89,27.44,26.75,25.73,22.50,21.30,19.44,18.65,12.15,11.19.HRMS m/z calculated for C 53H 64N 8O 7[M+H] +:925.4971,found:925.4975。
G10：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(10-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}葵基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000020
白色固体(54.1mg,23％)。 1H NMR(400MHz,DMSO-d 6)δ11.46(s,1H),11.12(s,1H),8.49(d,J＝2.5Hz,1H),8.13(t,J＝5.2Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.84–7.76(m,1H),7.72(d,J＝1.5Hz,1H),7.50(d,J＝8.6Hz,1H),7.43(d,J＝7.2Hz,1H),7.25(s,1H),7.18(d,J＝1.4Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.59(m,J＝6.8Hz,1H),4.36(d,J＝5.1Hz,2H),4.20(t,J＝6.4Hz,2H),3.50(t,J＝4.9Hz,4H),2.89(m,J＝17.3,14.0,5.3Hz,1H),2.65–2.53(m,2H),2.45(t,J＝5.0Hz,4H),2.29(t,J＝7.4Hz,2H),2.24(s,3H),2.17(s,3H),2.11(s,3H),2.03(m,J＝13.3,6.3,5.8,3.2Hz,1H),1.86–1.71(m,4H),1.45(d,J＝7.7Hz,4H),1.40(d,J＝6.7Hz,3H),1.29(s,8H),1.23(s,2H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.32,170.41,169.32,167.35,165.79,163.67,158.45,156.49,150.14,145.91,143.24,138.17,137.54,136.41,133.63,131.03,130.20,126.45,124.95,123.37,122.12,120.22,116.60,116.43,115.60,110.12,108.15,107.45,69.28,58.43,52.98,52.14,49.20,45.26,35.54,31.38,29.94,29.36,29.06,28.83,27.41,26.61,25.71,22.47,21.27,19.44,18.62,12.08,11.16.HRMS m/z calculated for  C 54H 66N 8O 7[M+H] +:939.5127,found:939.5124。
G11：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(11-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}十一烷基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000021
浅黄色固体(97.6mg,41％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.49(d,J＝2.5Hz,1H),8.12(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.80(t,J＝7.9Hz,1H),7.72(s,1H),7.50(d,J＝8.5Hz,1H),7.43(d,J＝7.2Hz,1H),7.25(s,1H),7.20–7.11(m,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.60(p,J＝6.7Hz,1H),4.35(d,J＝5.1Hz,2H),4.20(t,J＝6.4Hz,2H),3.51(s,4H),2.96–2.83(m,1H),2.58(dd,J＝15.9,11.8Hz,2H),2.47(s,4H),2.31(d,J＝7.8Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.03(dd,J＝9.3,4.6Hz,1H),1.86–1.70(m,4H),1.46(t,J＝7.5Hz,4H),1.40(d,J＝6.7Hz,3H),1.28(s,12H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.21,170.38,169.17,167.31,165.76,163.57,158.46,156.50,149.76,145.99,143.12,138.19,137.47,136.36,133.72,131.17,130.21,126.49,124.87,123.42,122.24,120.26,116.70,116.50,115.58,110.18,108.14,107.82,107.37,69.28,58.43,53.04,52.08,49.22,45.33,35.54,31.44,29.97,29.42,29.12,28.89,27.44,26.71,25.74,22.49,21.30,19.44,18.66,12.14,11.19.HRMS m/z calculated for C 55H 68N 8O 7[M+H] +:953.5283,found:953.5284。
G12：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(12-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]氧基}十二烷基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000022
黄色固体(33.8mg,14％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.49(d,J＝2.5Hz,1H),8.12(t,J＝5.1Hz,1H),7.90(dd,J＝8.8,2.6Hz,1H),7.80(dd,J＝8.5,7.3Hz,1H),7.72(d,J＝1.5Hz,1H),7.50(d,J＝8.6Hz,1H),7.43(d,J＝7.3Hz,1H),7.25(s,1H),7.17(d,J＝1.4Hz,1H),6.89(d,J＝8.9Hz,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.59(q,J＝6.9Hz,1H),4.34(t,J＝5.7Hz,2H),4.19(t,J＝6.4Hz,2H),3.50(t,J＝5.0Hz,4H),2.88(m,J＝17.4,14.1,5.4Hz,1H),2.69–2.52(m,2H),2.46(d,J＝4.8Hz,4H),2.30(t,J＝7.5Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.03(m,J＝15.1,7.9,4.2Hz,1H),1.85–1.70(m,4H),1.51–1.43(m,4H),1.40(d,J＝6.7Hz,3H),1.27(s,14H),0.73(t,J＝7.3Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.21,170.38,169.17,167.31,165.76,163.57,158.48,156.50,149.76,145.99,138.19,137.47,136.36,133.72,131.17,130.21,126.48,123.42,122.24,120.26,116.71,116.50,115.58,110.18,108.14,107.81,107.36,69.28,58.45,53.07,52.08,49.22,45.36,35.54,31.44,29.97,29.50,29.45,29.12,28.88,27.44,26.75,25.73,22.49,21.30,19.44,18.66,12.14,11.19.HRMS m/z calculated for C 56H 70N 8O 7[M+H] +:967.5440,found:967.5438。
实施例2
本实施例提供了9种双功能化合物E4-E12的合成及其相关化学数据。E4-E12的合成路线如下：
Figure PCTCN2020119438-appb-000023
具体制备过程为：
第一步：2-甲基-3-溴-5-硝基苯甲酸甲酯2a(5.5g,20mmol,1.0eq)，氯化铵(5.6g,100mmol,5.0eq)溶于乙醇水溶液中(60mL,H 2O:EtOH＝1:3)，升温至80℃后分三次加入铁粉(11.2g,200mmol,10.0eq)。反应1h后薄层色谱(TLC)检测反应完成，硅藻土助滤，滤液减压蒸馏除去溶剂,剩余物经DCM萃取，Na 2SO 4干燥后浓缩获得产品2b(4.41g,92％)，无需进一步纯化。 1H NMR(400MHz,CDCl 3)δ7.31(d,J＝2.6Hz,1H),6.92(d,J＝2.6Hz,1H),3.87(s,3H),3.82(s,2H),2.26(s,3H).HRMS m/z calculated for C 9H 10BrNO 2[M+H] +:243.9967,found:243.9958。
第二步：2b(4g,17.4mmol,1.0eq)以及四氢吡喃酮(4.4g,52.2mmol,3.0eq)溶于氯仿(50mL),加入乙酸(2.1g,34.8mmol,2.0eq)室温下搅拌3h后加入三乙酰基硼氢化钠(2.7g,43.6mmol,2.5eq)，继续搅拌过夜。反应完成后，减压蒸馏除去溶剂，残余物经硅胶柱层析纯化获得产品2c(3.3g,57.8％)。 1H NMR(400MHz,CDCl 3)δ7.23(d,J＝2.0Hz,1H),6.84(d,J＝2.0Hz,1H),4.01(dt,J＝11.9,3.6Hz,2H),3.87(s,3H),3.66(d,J＝7.4Hz,1H),3.54(td,J＝11.6,2.3Hz,2H),3.48(m,1H),2.23(s,3H),2.10–1.99(m,2H),1.51(m,J＝13.3,10.6,4.3Hz,2H).HRMS m/z calculated for C 14H 18BrNO 3[M+H] +:328.0542,found:328.0547。
第三步：氮气保护下，取2c(3g,9.1mmol,1.0eq)在50mL圆底烧瓶中加入30mL的1,2-二氯乙烷，缓慢搅拌下小心注入无水乙醛(2.3g,27.3mmol,3.0eq)于溶液液面下，再于30min内加入乙酸(1.1g,18.2mmol,2.0eq)，反应溶液呈橘黄色。待反应混合物自然升至室温，搅拌1h。之后将混合物降温至0℃，分批慢慢加入三乙酰氧基硼氢化钠(1.48g,23mmol)，控制加料速度保持反应体系温度低于5℃，2h后反转至室温并搅拌过夜。TLC反应完成后将反应体系冷至0℃，加入100mL冰水，搅拌下慢慢加入过量的碳酸氢钠饱和水溶液，加毕，搅拌30min后静置，分液，并用二氯甲烷萃取水相，合并有机层并用水洗涤两次后分液。有机相分出并减压浓缩至恒重，得黄色至浅红色油状液体。 1H NMR(400MHz,CDCl 3)δ7.71(d,J＝2.0Hz,1H),7.37(d,J＝2.0Hz,1H),3.96(d,J＝12.1Hz,2H),3.89(s,3H),3.32(td,J＝11.3,2.9Hz,2H),3.05(q,J＝7.1Hz,2H),2.99–2.85(m,1H),2.45(s,3H),1.80–1.54(m,4H),0.87(t,J＝7.0Hz,3H).HRMS m/z calculated for C 16H 23BrNO 3[M+H] +:356.0861,found:356.0854。
第四步：氮气保护下，在25mL甲醇中一次性加入2d(2g,5.6mmol)，升温至60℃后，保温下慢慢滴加20mL氢氧化钠的水溶液(2M)，反应液颜色由浅绿色清液逐渐变为乳浊液，最后变为浅绿色清液。保温反应1h后，TLC监测反应完成。将反应液转移至旋转蒸发仪中，减压下除去大部分甲醇，往剩余物中加入100mL水，搅拌10min，固体物完全溶解。升温至65℃，加入盐酸(2M)调节pH＝2～3，沉淀析出，停止热浴，降温至室温后搅拌0.5h。抽滤，滤饼用冰水充分洗涤，滤饼抽干后于真空干燥箱中以五氧化二磷为干燥剂，60℃干燥10h，得2e(1.75g,91.2％)白色目标化合物。 1H NMR(400MHz,DMSO-d 6)δ13.15(s,1H),7.62(d,J＝2.1Hz,1H),7.49(d,J＝2.1Hz,1H),3.83(dt,J＝9.5,2.3Hz,2H),3.26(td,J＝11.6,2.1Hz,2H),3.04(q,J＝7.1Hz,2H),3.01–2.92(m,1H),2.40(s,3H),1.66–1.58(m,2H),1.50(m,J＝11.7,4.3Hz,2H),0.80(t,J＝7.0Hz,3H).HRMS m/z calculated for C 15H 20BrNO 3[M+H] +:342.0699,found:342.0706。
第五步：3-(氨甲基)-4,6-二甲基吡啶-2(1H)-酮(0.61g,4mmol)以及2e(1.5g,4.4mmol)溶于DMSO(10mL), 加入HOAT(0.55g,1.5mmol)和EDCI(0.84g,2.2mmol)，反应液在45℃下搅拌20h。TLC监测反应完成后,将反应液倒入冰水中(100mL),搅拌30min后析出沉淀，过滤，水洗，干燥后经甲醇氯仿的混合液(10：1)溶解后，拌样，经硅胶柱层析纯化获得黄色固体2f(1.42g,68％)。 1H NMR(400MHz,DMSO-d 6)δ11.47(s,1H),8.23(t,J＝5.0Hz,1H),7.31(d,J＝2.0Hz,1H),7.09(d,J＝2.0Hz,1H),5.86(s,1H),4.25(d,J＝4.9Hz,2H),3.83(dd,J＝10.8,3.5Hz,2H),3.28–3.18(m,2H),3.01(q,J＝7.0Hz,2H),2.97–2.89(m,1H),2.54(s,1H),2.19(s,3H),2.15(s,3H),2.11(s,3H),1.60(d,J＝12.4Hz,2H),1.49(m,J＝11.7,4.2Hz,2H).HRMS m/z calculated for C 23H 30BrN 3O 3[M+H] +:476.1543,found:476.1552。
第六步：取2f(1.2g,2.5mmol,1.0eq),tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine-1-carboxylate(1.21g,3mmol,1.2eq)溶于1,4-二氧六环与水的混合溶液(4:1,30mL)中，加入K 2CO 3(3.75mmol,0.52g)，Pd(dppf)Cl 2(0.2mmol,146mg)，氮气保护后转至100℃下反应8h，然后冷却至室温。反应液减压蒸馏除去溶剂后用乙酸乙酯溶解，硅藻土助滤。萃取滤液，用无水硫酸钠干燥后浓缩，拌样，经硅胶柱层析纯化获得产物。无需进一步纯化，直接加入至25mL的三氟乙酸/二氯甲烷溶液(10％)中室温下反应1h，然后减压蒸馏除去溶剂，剩余物经饱和碳酸钠溶液(2M)中和至pH＝8,析出沉淀,抽滤，水洗，干燥，获得产品2g(1.04g,74％)。 1H NMR(400MHz,DMSO-d 6)δ11.47(s,1H),8.63(s,1H),8.20(t,J＝5.0Hz,1H),7.60(d,J＝7.9Hz,2H),7.38(d,J＝8.0Hz,3H),7.28–7.16(m,1H),5.86(s,1H),4.30(d,J＝4.9Hz,2H),3.89–3.77(m,2H),3.57(s,2H),3.25(t,J＝11.4Hz,2H),3.09(d,J＝6.2Hz,6H),3.04–2.98(m,1H),2.57(t,J＝4.8Hz,4H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.66(d,J＝12.3Hz,2H),1.53(m,J＝12.4,12.0,4.1Hz,2H),0.84(t,J＝6.9Hz,3H).HRMS m/z calculated for C 34H 45N 5O 3[M+H] +:572.3595,found:572.3601。
第七步：合成E4-E12，亲核取代反应。
2g(0.25mmol,1eq),NaHCO 3(0.5mmol,2.0eq)以及1c-1k(0.3mmol,1.2eq)溶于DMF(5mL)，在85℃下反应3-8h.反应完成后,乙酸乙酯萃取后，Na 2SO 4干燥，减压蒸馏除去溶剂后经硅胶柱层析分离获得相应产品E4-E12。
E4：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(4-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}丁基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000024
黄色固体(36.0mg,15％)。 1H NMR(400MHz,CDCl 3)δ12.00(s,1H),9.66(s,1H),8.35(d,J＝2.5Hz,1H),7.71–7.57(m,2H),7.44(d,J＝7.2Hz,1H),7.27–7.15(m,4H),5.91(s,1H),4.93(dd,J＝11.9,5.4Hz,1H),4.54(d,J＝5.9Hz,2H),4.19(q,J＝6.2Hz,2H),3.94(dt,J＝11.6,3.3Hz,2H),3.65–3.52(m,4H),3.41(s,1H),3.31(td,J＝11.3,2.9Hz,2H),3.08(q,J＝7.0Hz,2H),3.04–2.95(m,1H),2.88–2.68(m,3H),2.58(t,J＝5.0Hz,4H),2.45(t,J＝7.1Hz,2H),2.39(s,3H),2.33(s,3H),2.14(s,3H),2.13–2.05(m,1H),1.92(t,J＝6.8Hz,2H),1.81–1.50(m,9H),0.88(t,J＝7.0Hz,3H). 13C NMR(101MHz,CDCl 3)δ171.65,168.71,167.05,165.09,158.69,156.61,150.67,149.55,145.91,142.60,139.33,136.45,135.87,135.58,133.86,132.83,125.55,123.17,122.11,120.12,118.81,115.69,109.86,106.90,69.25,67.31,58.40,58.31,52.83,49.11,45.25,41.64,36.13,31.43,30.52,28.76,26.33,23.96,22.71,19.69,18.65,14.68,12.77.HRMS m/z calculated for C 51H 62N 7O 8[M+H] +:900.4654,found:900.4653。
E5：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(5-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}戊基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000025
黄色固体(61.7mg,27％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.08(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.57(d,J＝7.9Hz,2H),7.51(d,J＝8.5Hz,1H),7.43(d,J＝7.2Hz,1H),7.42–7.38(m,1H),7.35(d,J＝7.8Hz,2H),7.22(d,J＝1.7Hz,1H),5.86(s,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.30(d,J＝5.0Hz,2H),4.23(t,J＝6.3Hz,2H),3.88–3.78(m,2H),3.46(s,2H),3.25(t,J＝11.4Hz,2H),3.09(q,J＝7.2Hz,2H),3.01(d,J＝10.6Hz,1H),2.94–2.82(m,1H),2.57(dd,J＝15.3,11.4Hz,2H),2.45–2.29(m,8H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.07–1.97(m,1H),1.83–1.71(m,2H),1.71–1.59(m,4H),1.59–1.48(m,2H),1.25(d,J＝13.0Hz,4H),0.84(t,J＝6.9Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.21,170.38,169.51,167.31,165.79,163.46,156.47,149.94,149.34,143.19,140.08,138.94,137.89,137.49,137.47,133.71,133.07,129.86,126.83,123.35,122.09,121.28,120.27,116.71,115.57,107.80,69.16,66.79,62.22,58.34,57.66,53.18,49.21,41.67,35.36,31.42,30.79,29.45,26.79,22.92,22.48,19.42,18.66,15.02,13.21.HRMS m/z calculated for C 52H 64N 7O 8[M+H] +:914.4811,found:914.4812。
E6：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(6-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}己基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000026
黄色固体(46.4mg,20％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.09(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.57(d,J＝7.8Hz,2H),7.51(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),7.40(s,1H),7.35(d,J＝7.9Hz,2H),7.22(s,1H),5.86(s,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.29(d,J＝5.0Hz,2H),4.20(t,J＝6.4Hz,2H),3.83(d,J＝11.2Hz,2H),3.47(s,2H),3.24(d,J＝11.3Hz,2H),3.06(dd,J＝18.9,11.1Hz,3H),2.88(q,J＝12.5Hz,1H),2.58(d,J＝17.5Hz,2H),2.38(s,6H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.03–1.99(m,1H),1.75(m,J＝7.0Hz,2H),1.67(d,J＝12.0Hz,2H),1.49(dt,J＝24.8,6.0Hz,4H),1.38–1.31(m,2H),1.24(d,J＝6.6Hz,6H),0.84(q,J＝6.9Hz,3H). 13C NMR(101MHz,CDCl 3)δ173.31,170.41,169.65,167.34,165.81,163.54,156.49,150.27,149.38,143.31,139.97,137.57,133.65,133.08,130.00,126.84,123.41,121.97,121.20,120.95,120.27,116.62,115.64,108.07,69.21,66.78,58.28,52.92,49.20,41.69,35.37,31.37,30.75,30.73,29.42,29.10,28.74,25.56,22.47,19.41,18.63,15.01,13.14.HRMS m/z calculated for C 53H 66N 7O 8[M+H] +:928.4967,found:928.4982。
E7：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(7-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}庚基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000027
黄色固体(101.3mg,43％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.56(d,J＝7.8Hz,2H),7.50(d,J＝8.6Hz,1H),7.43(d,J＝7.3Hz,1H),7.40(s,1H),7.35(d,J＝7.8Hz,2H),7.22(s,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.30(d,J＝4.9Hz,2H),4.20(t,J＝6.4Hz,2H),3.83(d,J＝11.1Hz,2H),3.47(s,2H),3.28–3.20(m,2H),3.09(q,J＝7.6,7.1Hz,2H),3.01(d,J＝10.7Hz,1H),2.89(m,J＝13.6,12.5,6.9Hz,1H),2.59(d,J＝17.0Hz,2H),2.37(s,6H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.03(d,J＝12.5Hz,1H),1.75(t,J＝7.2Hz,2H),1.67(d,J＝12.0Hz,2H),1.53(dt,J＝12.5,7.6Hz,2H),1.43(p,J＝7.4Hz,4H),1.33(d,J＝6.3Hz,2H),1.28(d,J＝7.8Hz,2H),1.23(s,4H),0.83(t,J＝7.0Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.31,170.41,169.65,167.34,165.80,163.55,156.49,150.28,149.38,143.31,139.95,138.96,137.62,137.55,137.50,133.65,133.08,129.98,126.82,123.41,121.97,121.20,120.24,116.61,115.61,108.09,69.26,66.78,62.02,58.28,58.07,52.99,52.64,49.20,41.69,35.37,31.39,30.75,29.42,28.92,28.78,27.20,26.23,25.67,22.47,19.41,18.63,15.01,13.14.HRMS  m/z calculated for C 54H 68N 7O 8[M+H] +:942.5124,found:942.5135。
E8：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(8-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}辛基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000028
黄色固体(69.3mg,29％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.09(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.56(d,J＝7.8Hz,2H),7.50(d,J＝8.6Hz,1H),7.43(d,J＝7.2Hz,1H),7.40(d,J＝2.1Hz,1H),7.35(d,J＝7.9Hz,2H),7.22(d,J＝1.9Hz,1H),5.86(s,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.30(d,J＝5.0Hz,2H),4.19(t,J＝6.4Hz,2H),3.88–3.77(m,2H),3.47(s,2H),3.25(t,J＝11.8Hz,2H),3.09(q,J＝7.1Hz,2H),3.02(q,J＝6.9,5.4Hz,1H),2.88(m,J＝17.5,14.2,5.3Hz,1H),2.63–2.54(m,2H),2.46–2.30(m,6H),2.25(s,5H),2.21(s,3H),2.11(s,3H),2.06–2.00(m,1H),1.75(t,J＝7.3Hz,2H),1.66(d,J＝12.0Hz,2H),1.54(td,J＝11.8,4.0Hz,2H),1.43(dt,J＝11.6,6.5Hz,4H),1.36–1.20(m,8H),0.83(t,J＝6.9Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.20,170.38,169.51,167.31,165.75,163.47,156.49,149.95,149.33,143.19,140.07,138.96,137.49,133.71,133.07,129.85,126.83,123.36,122.09,121.28,120.26,116.70,115.58,107.82,69.28,66.79,62.17,58.34,53.22,49.21,41.68,35.37,31.43,30.79,29.33,29.08,28.86,27.30,26.64,25.70,22.49,19.41,18.66,15.02,13.20.HRMS m/z calculated for C 55H 70N 7O 8[M+H] +:956.5280,found:956.5281。
E9：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(9-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}壬基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000029
黄色固体(97.1mg,40％)。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),11.10(s,1H),8.18(q,J＝5.6,5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.56(d,J＝7.8Hz,2H),7.51(d,J＝8.5Hz,1H),7.43(d,J＝7.3Hz,1H),7.40(d,J＝1.9Hz,1H),7.35(d,J＝7.9Hz,2H),7.22(d,J＝1.8Hz,1H),5.86(s,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.29(d,J＝4.9Hz,2H),4.19(t,J＝6.4Hz,2H),3.83(d,J＝11.1Hz,2H),3.47(s,2H),3.24(d,J＝11.3Hz,2H),3.09(q,J＝7.1Hz,2H),3.01(q,J＝7.2,5.6Hz,1H),2.89(td,J＝13.4,12.1,6.9Hz,1H),2.57(dd,J＝15.9,12.0Hz,2H),2.37(s,6H),2.25(s,5H),2.21(s,3H),2.11(s,3H),2.05–1.99(m,1H),1.75(p,J＝6.6Hz,2H),1.66(d,J＝12.1Hz,2H),1.54(td,J＝11.9,4.1Hz,2H),1.45(t,J＝7.7Hz,2H),1.40(d,J＝8.9Hz,2H),1.33(d,J＝5.8Hz,2H),1.25(d,J＝11.4Hz,8H),0.84(q,J＝7.0Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.29,170.40,169.63,167.33,165.78,163.53,156.49,150.22,149.37,143.29,139.97,138.97,137.54,133.66,133.08,129.97,126.83,123.40,121.98,121.21,120.25,116.62,115.61,108.04,69.27,66.78,62.05,58.29,52.99,52.64,49.20,41.68,35.38,31.40,30.75,29.33,29.24,29.02,28.83,27.26,25.68,22.47,19.41,18.64,15.01,13.15.HRMS m/z calculated for C 56H 72N 7O 8[M+H] +:970.5437,found:970.5474。
E10：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(10-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}葵基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000030
黄色固体(81.2mg,33％)。 1H NMR(400MHz,DMSO-d 6)δ11.44(s,1H),11.10(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.56(d,J＝7.9Hz,2H),7.50(d,J＝8.5Hz,1H),7.43(d,J＝7.3Hz,1H),7.39(d,J＝1.8Hz,1H),7.35(d,J＝7.9Hz,2H),7.22(d,J＝1.8Hz,1H),5.85(s,1H),5.07(dd,J＝12.9,5.3Hz,1H),4.29(d,J＝5.0Hz,2H),4.19(d,J＝6.5Hz,2H),3.86–3.78(m,2H),3.47(s,2H),3.24(d,J＝11.3Hz,2H),3.09(q,J＝7.2Hz,2H),3.01(d,J＝11.0Hz,1H),2.88(m,J＝18.0,14.1,5.3Hz,1H),2.68–2.54(m,2H),2.35(d,J＝16.6Hz,6H),2.25(s,5H),2.21(s,3H),2.10(s,3H),2.06–1.98(m,1H),1.75(m,J＝6.6Hz,2H),1.66(d,J＝11.4Hz,2H),1.53(dt,J＝12.2,5.9Hz,2H),1.45(t,J＝7.8Hz,2H),1.42–1.31(m,4H),1.25(d,J＝8.1Hz,10H),0.83(t,J＝6.9Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.21,170.38,167.31,166.11,165.75,163.99,161.20,156.49,149.94,149.33,137.49,133.07,129.85,126.83,121.28,120.27,116.70,115.59,106.37,76.61,69.30,67.68,64.13,58.35,53.14,49.20,35.36,34.70,31.43,29.39,29.09,28.88,26.26,25.51,22.48,22.23,19.00,18.43,17.95,16.80,15.02,14.56,11.44.HRMS m/z calculated for C 57H 74N 7O 8[M+H] +:984.5593,found:984.5593。
E11：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(11-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}十一烷基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000031
黄色固体(119.8mg,47％)。 1H NMR(400MHz,DMSO-d 6)δ11.44(s,1H),11.09(s,1H),8.17(t,J＝5.0Hz,1H),7.80(t,J＝7.9Hz,1H),7.56(d,J＝7.8Hz,2H),7.51(d,J＝8.6Hz,1H),7.43(d,J＝7.3Hz,1H),7.39(d,J＝1.8Hz,1H),7.35(d,J＝8.0Hz,2H),7.22(d,J＝1.8Hz,1H),5.86(s,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.29(d,J＝4.9Hz,2H),4.19(t,J＝6.4Hz,2H),3.83(d,J＝11.2Hz,2H),3.47(s,2H),3.24(d,J＝11.3Hz,2H),3.09(q,J＝7.1Hz,2H),3.01(d,J＝10.7Hz,1H),2.88(m,J＝18.6,14.5,5.3Hz,1H),2.58(d,J＝17.4Hz,2H),2.37(s,6H),2.24(s,4H),2.21(s,3H),2.10(s,3H),2.06–1.96(m,2H),1.75(t,J＝7.3Hz,2H),1.66(d,J＝12.4Hz,2H),1.52(dd,J＝12.1,3.9Hz,2H),1.45(s,2H),1.34(s,4H),1.24(d,J＝4.5Hz,12H),0.83(t,J＝7.5Hz,3H). 13C NMR(101MHz,CDCl 3)δ171.64,170.14,168.71,167.12,165.66,165.16,158.71,156.73,150.71,149.55,145.96,142.63,139.36,136.40,136.14,135.81,135.64,133.85,132.78,125.51,123.20,122.08,120.05,118.89,117.16,115.62,109.90,106.77,77.26,69.47,67.31,58.71,58.41,52.89,49.14,45.18,41.63,36.15,31.51,30.52,29.47,29.41,29.31,29.15,28.89,27.49,26.65,25.73,22.67,19.70,18.65,14.68,12.76.HRMS m/z calculated for C 58H 76N 7O 8[M+H] +:998.5750,found:998.5775。
E12：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(12-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]氧}十二烷基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000032
黄色固体(48.1mg,19％)。 1H NMR(400MHz,DMSO-d 6)δ11.44(s,1H),11.09(s,1H),8.17(t,J＝4.9Hz,1H),7.82–7.75(m,1H),7.60–7.53(m,2H),7.50(dd,J＝8.6,1.9Hz,1H),7.43(dd,J＝7.3,1.6Hz,1H),7.39(d,J＝2.0Hz, 1H),7.38–7.31(m,2H),7.22(d,J＝1.8Hz,1H),5.85(s,1H),5.07(dd,J＝12.9,5.3Hz,1H),4.29(d,J＝4.9Hz,2H),4.22–4.14(m,2H),3.83(d,J＝11.1Hz,2H),3.46(s,2H),3.23(d,J＝11.3Hz,2H),3.08(q,J＝7.1Hz,2H),3.00(d,J＝11.1Hz,1H),2.88(m,J＝18.9,14.2,5.4Hz,1H),2.57(dd,J＝15.5,11.9Hz,2H),2.37(s,6H),2.24(s,5H),2.21(s,3H),2.10(s,3H),2.06–1.97(m,1H),1.74(t,J＝7.2Hz,2H),1.66(d,J＝12.3Hz,2H),1.53(m,J＝11.9,4.0Hz,2H),1.49–1.41(m,2H),1.35(dd,J＝16.7,5.5Hz,4H),1.25(d,J＝7.7Hz,14H),0.83(t,J＝6.9Hz,3H). 13C NMR(101MHz,DMSO-d 6)δ173.20,170.37,169.51,167.31,165.76,163.46,156.50,149.94,149.33,143.19,140.08,138.96,137.50,133.72,129.85,126.83,122.09,121.28,120.26,116.70,115.58,107.81,69.28,66.80,62.20,58.34,53.26,53.11,49.21,41.68,35.36,31.44,30.79,29.48,29.42,29.40,29.11,28.88,27.39,26.72,25.72,22.49,19.41,18.65,15.02,13.20.HRMS m/z calculated for C 59H 78N 7O 8[M+H] +:1012.5906,found:1012.5910。
实施例3
本实施例提供了9种双功能化合物S4-S12的合成及其相关化学数据。S4-S12的合成路线如下：
Figure PCTCN2020119438-appb-000033
具体制备过程为：
第一步：S1(4mmol)以及S2(4.4mmol)溶于DMSO(10mL),加入HOAT(0.55g,1.5mmol)和EDCI(0.84g,2.2mmol)，反应液在45℃下搅拌20h。TLC监测反应完成后,将反应液倒入冰水中(100mL),搅拌30min后析出沉淀，过滤，水洗，干燥后经甲醇氯仿的混合液(10：1)溶解后，拌样，经硅胶柱层析纯化获得黄色固体S3(1.57g)。 1H NMR(400MHz,Chloroform-d)δ12.78(s,1H),7.41(t,J＝5.7Hz,1H),7.22(d,J＝2.0Hz,1H),7.18(d,2.0Hz,1H),4.54(d,J＝5.7Hz,2H),3.94(dt,J＝11.6,3.3Hz,2H),3.36–3.25(m,2H),3.02(q,J＝7.0Hz,2H),2.95(m,1H),2.93(dd,J＝7.4,4.8Hz,2H),2.44(t,J＝6.0Hz,2H),2.27(s,3H),2.19(s,3H),1.76(dd,J＝7.5,4.3Hz,4H),1.68–1.58(m,4H),0.85(t,J＝7.0Hz,3H). 13C NMR(101MHz,CDCl 3)δ168.51,163.56,151.17,150.82,140.70,140.36,133.52,128.40,125.01,120.94,118.74,114.96,67.28,58.57,41.39,35.99,30.35,27.42,24.97,22.31,22.22,16.72,14.78,12.79。
第二步：取S3(2.5mmol,1.0eq),硼酸酯(3mmol,1.2eq)溶于1,4-二氧六环与水的混合溶液(4:1,30mL)中，加入K 2CO 3(3.75mmol,0.52g)，Pd(dppf)Cl 2(0.2mmol,146mg)，氮气保护后转至100℃下反应8h，然后冷却至室温。反应液减压蒸馏除去溶剂后用乙酸乙酯溶解，硅藻土助滤。萃取滤液，用无水硫酸钠干燥后浓缩，拌样，经硅胶柱层析纯化获得产物。无需进一步纯化，直接加入至25mL的三氟乙酸/二氯甲烷溶液(10％)中室温下反应1h，然后减压蒸馏除去溶剂，剩余物经饱和碳酸钠溶液(2M)中和至pH＝8,析出沉淀,抽滤，水洗，干燥，获得产品2g S4(1.04g,74％)。HRMS m/z calculated for C 35H 46N 6O 3[M+H] +:599.3704,found:599.3711。
第三步：合成S6-S11。
S4(0.25mmol,1eq),NaHCO 3(0.5mmol,2.0eq)以及1c-1k(0.3mmol,1.2eq)溶于DMF(5mL)，在85℃下反应5h。反应完成后,乙酸乙酯萃取后，Na 2SO 4干燥，减压蒸馏除去溶剂后经硅胶柱层析分离获得相应产品S6-S11。
S6：5-(6-(4-(6-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢-2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000034
黄色固体(48.1mg,13％)。12.48(s,1H),8.33(d,J＝2.4Hz,1H),7.60(dd,J＝8.8,2.6Hz,1H),7.41(t,J＝5.8Hz,1H),7.25(d,J＝1.9Hz,1H),7.20(d,J＝1.8Hz,1H),6.62(d,J＝8.8Hz,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.57(d,J＝5.7Hz,2H)，4.30(d,J＝5.0Hz,2H),4.23(t,J＝6.3Hz,2H),3.94(dt,J＝11.5,3.2Hz,2H)，3.88–3.78(m,2H),3.57(t,J＝5.0Hz,4H)，3.46(s,2H),3.31(td,J＝11.2,3.1Hz,2H),3.25(t,J＝11.4Hz,2H),3.09(q,J＝7.2Hz,2H),3.01(d,J＝10.6Hz,1H),2.94–2.82(m,1H),2.57(dd,J＝11.4Hz,2H),2.45–2.29(m,8H),2.21(s,3H),2.11(s,3H),2.07–1.97(m,1H),1.83–1.71(m,2H),1.59–1.48(m,2H),1.25(d,J＝13.0Hz,3H),0.84(t,J＝6.9Hz,3H).HRMS m/z calculated for C 54H 66N 8O 8[M+H] +:955.5076,found:955.5071。
S7：5-(6-(4-(7-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢-2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000035
黄色固体(48.1mg,13％)。HRMS m/z calculated for C 55H 68N 8O 8[M+H] +:969.5233,found:969.5243。
S8：5-(6-(4-(8-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢-2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000036
黄色固体(83mg,24％)。HRMS m/z calculated for C 56H 70N 8O 8[M+H] +:1012.5906,found:1012.5910。
S9：5-(6-(4-(7-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢-2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000037
黄色固体(48.1mg,13％)。HRMS m/z calculated for C 57H 72N 8O 8[M+H] +:997.5946,found:997.5938。
S10：5-(6-(4-(10-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢 -2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000038
黄色固体(58.1mg,17％)。HRMS m/z calculated for C 58H 74N 8O 8[M+H] +:1011.5702,found:1011.5710。
S11：5-(6-(4-(11-((2-(2,6-吡啶二酮-3-基)-1,3-二氧杂异吲哚-4-基)氧杂)环己基)哌嗪-1-基)吡啶-3-yl)-3-(乙基(四氢-2H-吡喃-4-基)氨基)-2-甲基-N-((1-甲基-3-氧杂-2,3,5,6,7,8-六氢异喹啉-4-基)甲基)苯甲酰胺。
Figure PCTCN2020119438-appb-000039
黄色固体(91mg,27％)。HRMS m/z calculated for C 59H 76N 8O 8[M+H] +:1025.5859,found:1025.5863。
实施例4
K7：N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-4'-{[4-(7-{[2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂-2,3-二氢-1H-异吲哚-4-基]硫}庚基)哌嗪-1-基]甲基}-5-[乙基(氧杂-4-基)氨基]-4-甲基-[1,1'-二苯基]-3-甲酰胺。
Figure PCTCN2020119438-appb-000040
棕褐色固体(42.3mg,13％)。 1H NMR(400MHz,DMSO-d 6)δ11.43(s,1H),11.12(s,1H),8.16(t,1H),7.81(t,J＝7.9Hz,1H),7.52(d,J＝7.9Hz,2H),7.51(d,1H),7.44(d,J＝7.3Hz,1H),7.41(s,1H),7.36(d,2H),7.23(s,1H),5.86(s,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.30(d,J＝4.9Hz,2H),4.20(t,J＝6.4Hz,2H),3.83(d,J＝11.1Hz,2H),3.47(s,2H),3.28–3.20(m,2H),3.09(q,J＝7.6,7.1Hz,2H),3.01(d,J＝10.7Hz,1H),2.89(m,J＝13.6,12.5,6.9Hz,1H),2.59(d,J＝17.0Hz,2H),2.37(s,6H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.03(d,J＝12.5Hz,1H),1.75(t,J＝7.2Hz,2H),1.67(d,J＝12.0Hz,2H),1.53(dt,2H),1.43(p,6H),1.27(d,2H),1.24(s,4H),0.79(t,3H).HRMS m/z calculated for C 54H 68N 7O 8[M+H] +:958.4895,found:958.4893。
实施例5
M5：1-[(2S)-丁烷-2-基]-N-[(4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基]-6-{6-[4-(5-{[2-(2,6-二碳基哌啶-3-基)-1,3-二氧杂-2,3-氢-1H-异吲哚-4-基]硫醚基}戊基)哌嗪-1-基]吡啶-3-基}-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000041
浅褐色固体。HRMS m/z calculated for C 49H 56N 8O 7[M+H] +:871.3960,found:871.3962。
实施例6
N4：1-((S)-仲丁基)-N-((4,6-二甲基-2-氧杂-1,2-二氢吡啶-3-基)甲基)-6-(6-(4-(4-((2-(2,6-二氧杂哌啶-3-基)-1,3-二氧杂异吲哚啉-4-基)氨基)丁酰基)哌嗪-1-基)吡啶-3-基)-3-甲基-1H-吲哚-4-甲酰胺。
Figure PCTCN2020119438-appb-000042
浅黄色固体。 1H NMR(400MHz,DMSO-d 6)δ11.46(s,1H),11.08(s,1H),8.52(d,J＝2.6Hz,1H),8.12(t,J＝5.2Hz,1H),7.91(m,1H),7.80(t,1H),7.74(s,1H),7.51(d,J＝8.5Hz,1H),7.41(d1H),7.28(s,1H),7.13(s,1H),6.92(d,1H),5.86(s,1H),5.12(m,1H),4.52(m,1H),4.33(d,2H),4.26(t,J＝6.3Hz,2H),3.48(d,4H),2.92(m,,1H),2.66(m,2H),2.47(s,2H),2.41(t,J＝6.8Hz,2H),2.27(s,3H),2.18(s,3H),2.09(s,3H),2.03(m,1H),1.82(m,4H),1.67(q,J＝7.4Hz,2H),1.39(d,3H),0.76(t,J＝7.3Hz,3H).HRMS m/z calculated for C 49H 56N 8O 7[M+H] +:871.3960,found:871.3962。
生物学试验
一、实验仪器及材料
本公开实施方式中生物实验所用到的仪器如下，超净工作台BHC-1000IIA/B3：苏净安泰生物技术公司；恒温水浴箱PolyScience 9505：PolyScience公司；灭菌锅MLS-3780：SANYO公司；烘箱：Binder公司；超纯水仪Milli-Q Integral 10：Millipore公司；酶标仪Multiscan MK3、细胞培养箱、低速离心机Sorvall ST1：Thermofisher公司；流式细胞仪：BD公司；pH计ORION STAR A211：ThermoScientific公司；超声破碎仪Sonic Materials Inc：Danbury公司；37℃恒温摇床Thermolyne、小型垂直电泳槽Mini-PROTEAN 3、转膜槽Mini Trans-blot：Bio-Rad公司；X射线摄影暗匣AX-Ⅱ：广东粤华医疗医械厂有限公司；全自动X光洗片机HQ-320XT：虎丘影像有限公司；切片烘片机Leica RM 2125：Leica公司；普通光学显微镜及倒置显微镜：Olympus公司；荧光倒置相差显微镜：Carl Zeiss公司；荧光正置相差显微镜：Leica DM2500(莱卡)公司和Carl Zeiss(卡尔蔡司)公司；游标卡尺(0-150mm)：上海申韩量具有限公司。
本公开实施方式所采用的细胞株购自美国ATCC(American Type Culture Collection)公司。细胞培养用6、24、96孔板，15mL、50mL离心管、25cm 2培养方瓶和75cm 2培养瓶均购自成都阿比丁公司。10mL培养皿购自WHB公司。基质胶(Matrigel)购自BD公司。二甲基亚枫(DMSO)、MTT、SDS、聚山梨酸酯Tween-20、十二烷基磺酸钠SDS，甘氨酸，三羟甲基氨基甲烷Tris、PEG-400购自Sigma公司。过硫酸铵APS，氢氧化钠、过硫酸铵、浓盐酸、异丙醇、甲醇等常见分析纯化学试剂为科龙化学公司。生理盐水购自科伦制药有限公司。Annexin V-FITC/PI试剂盒、PI试剂、结晶紫染料、RIPA细胞裂解缓冲液(强)和丙烯酰胺缓冲液、苏木素染液购自碧云天生物技术公司。N,N,N',N'-四甲基乙二胺TEMED，G250蛋白定量液购自Bio-Rad公司。PVDF膜和显影发光底物购自Millipore公司。脱脂奶粉购自伊利乳业公司；自显影胶片购自Kodak公司。10％APS、1mol/L Tris-HCl(pH:6.8)、1.5mol/L Tris-HCl(pH:8.8)及电泳缓冲液转膜缓冲液、TBS缓冲液、TBST缓冲液等常见试剂由本实验配制。所有抗体均购自Cell signaling Technology公司(Beverly,MA)。封闭用羊血清、兔血清和DAB显色试剂盒购自北京中杉金桥公司。TUNEL检测试剂盒购自promega公司(Roche Applied Science)。
首先，采用AlphaScreen技术测试实施例1～5中的产品化合物以及其阳性对照GSK126和EPZ6438对EZH2酶的抑制活性。检测结果如表1所示。
表1
Figure PCTCN2020119438-appb-000043
Figure PCTCN2020119438-appb-000044
注：每个化合物测试2次，表格中数值表示平均值。
由表1结果可知，G4-G12、E4-E12对EZH2酶的抑制活性都在纳摩尔水平，其中，n为2～6的G4-G8、E4-E8对EZH2的抑制活性均与各自的阳性对照GSK126和EPZ6438相当，当n为7～10时，G9-G12、E9-E12对EZH2的抑制活性相比于其阳性对照分别减弱了大约6-60和4-14倍。总体来看，绝大部分分子都能保持EZH2抑制剂本身的EZH2酶抑制活性，并且E系列比G系列保持了更好的活性；在所有合成的分子中，E7对EZH2酶具有最好的抑制活性(IC 50＝2.7nM)，E7对于EZH2酶抑制活性优于阳性对照。
二、PRC2蛋白复合物亚基降解能力
首先通过蛋白免疫印迹法检测WSU-DLCL-2细胞内EZH2、SUZ12、EED、RbAp48以及组蛋白H3K27me3的蛋白水平。WSU-DLCL-2细胞分别与1μM待测化合物孵育48h，以等量的EPZ6438、GSK126、DMSO作为对照，结果如图1所示。
通过免疫印迹实验检测合成的两类PRC2降解剂在DLBCL细胞株WSU-DLCL-2中对EZH2及PRC2其他核心亚基SUZ12、EED、RbAp48的降解效果。结果如图1所示，1μM待测化合物处理48h后，WSU-DLCL-2细胞内EZH2、SUZ12、EED以及RbAp48蛋白水平的量化。C.1μM降解剂处理48h后，WSU-DLCL-2细胞内H3K27me3蛋白水平的量化。应用Image J软件对蛋白表达量进行定量分析，统计结果均以三次平均值±标准差(SD)的形式表示，*P＜0.05，**P＜0.01，***P＜0.001。在G系列中，G4对EZH2、SUZ12、EED和RbAp48亚基都只表现出较弱的降解作用，G5-G7仅对SUZ12和EED亚基有微弱的降解，而G8-G12对EZH2、SUZ12、EED和RbAp48亚基表现出明显的降解作用；在E系列中，烷基链最短的E4对于PRC2的全部核心亚基EZH2、SUZ12、EED和RbAp48具有非常明显的降解作用，E5和E6对PRC2各核心亚基的降解则明显弱于E4，虽然中间烷基链延长至7个碳原子(E7)时又表现了对PRC2各亚基的强烈降解作用，但之后随着中间烷基链的继续延长，E8-E12对PRC2各核心亚基的降解能力反而逐渐减弱；而相同剂量的EZH2酶抑制剂GSK126和EPZ6438在相同条件下几乎完全不改变细胞内PRC2各核心亚基的蛋白水平。
此外，对于M5,K7为代表的两类经氮原子以及硫原子连接烷基链的降解分子而言，均展现良好的PRC2亚基的降解效果，但是对于另外以N4为代表的经碳烷基链的降解分子，并未展现出对于PRC2亚基的降解效果。对于N4为代表的碳烷基链类型连接子或者两端的配体不同的例子，其效果相比于实施例4和实施例5的化合物效果更差。
通过检测细胞内H3K27me3水平评价了合成的两类PRC2降解剂在WSU-DLCL-2细胞内对于EZH2酶活性的抑制作用。如图3所示，1μM测试分子作用于WSU-DLCL-2细胞48h后几乎都能不同程度地抑制H3K27me3，并且H3K27me3蛋白水平降低的趋势与PRC2亚基降解的趋势大体一致，其中G8-G12、E4、E7-E11对H3K27me3的抑制率在60％以上，具有相对强效的抑制活性。总体而言，两类靶向EZH2的PRC2降解分子几乎都能降解PRC2各核心亚基并抑制H3K27me3，但它们对PRC2的降解能力和对H3K27me3的抑制能力因Linker烷基链长度的不同而有所差异。其中的PROTAC E7不仅表现了最好的体外EZH2酶抑制活性，而且表现了最好的降解PRC2核心亚基(降解率：EZH2 72％，SUZ12 75％，EED 81％，RbAp48 74％)和抑制H3K27me3(抑制率86％)的活性，因此，选择化合物E7继续进行后续的化学生物学研究。
三、E7有效降解PCR2
在WSU-DLCL-2细胞中评价了E7诱导PRC2降解的时效和量效关系。时效方面，考察1μM E7作用不同时间对PRC2各核心亚基的降解和对H3K27me3的抑制情况，结果显示图4中的A所示，在0-1h内，E7对EZH2的降解随着作用时间的延长逐渐增强，但对PRC2其他亚基和H3K27me3几乎没有影响；在作用的2-12h，E7对PRC2各亚基的降解作用消失，并且对H3K27me3的抑制作用较弱且不稳定。据此推测，E7在作用初期(0-12h)主要降解未参与形成PRC2复合物的游离EZH2，而它诱导PRC2复合物的EZH2及其他亚基降解需要更长的时间形成完整的三元复合物。E7作用24h后，PRC2核心亚基EZH2、SUZ12、EED和RbAp48以及其催化产物H3K27me3的蛋白水平开始显著下降，并且在此之后，随着作用时间的延长，E7对PRC2各亚基的降解和对H3K27me3的抑制都逐渐增强，直到作用到96h几乎实现了对PRC2的完全降解。量效方面，考察不同浓度的E7作用48h对PRC2各核心亚基的降解和对H3K27me3的抑制情况。结果如图4中的B所示，低浓度E7(0-0.5μM)对PRC2各亚基仅有微弱的降解作用；当浓度达到1μM时，E7能显著降解PRC2各亚基并抑制H3K27me3，而且这种显著效果在 作用浓度增大到5μM时都能持续保持；但当E7的浓度过高，达到10μM时，产生了“钩子效应”(hook effect)，即E7过量导致其与EZH2和E3泛素连接酶分别形成了二元复合物，反而降低其参与形成EZH2-E7-E3泛素连接酶三元复合物的有效作用浓度，表现为对PRC2各核心亚基的降解和对H3K27me3的抑制作用减弱。从上述结果可以得出：1μM的E7作用于WSU-DLCL-2细胞48h能非常明显且稳定地降解PRC2的EZH2、SUZ12、EED以及RbAp48亚基并有效抑制H3K27me3。
此外，利用荧光实时定量PCR(RT-qPCR)实验检测了经1μM E7处理48h的WSU-DLCL-2细胞内EZH2、SUZ12、EED和RbAp48的mRNA水平，以确定其表达的减少是E7在蛋白层面而非基因层面发挥作用导致的结果。如图4中的C显示，E7跟单纯的EZH2甲基转移酶抑制剂EPZ6438和GSK126一样，几乎不改变PRC2的EZH2、SUZ12、EED和RbAp48亚基的mRNA水平，说明E7确实不影响表达这些蛋白的基因的转录，而是在这些蛋白的翻译或翻译后阶段发挥作用。
进一步考察E7在其他几种EZH2功能异常所驱动的肿瘤细胞株中降解PRC2的能力，以排除E7对DLBCL细胞株WSU-DLCL-2的特异性作用。结果如图4中的D所示，1μM E7在DLBCL(WSU-DLCL-2，Pfeiffer)、PCa(LNCaP，DU 145)和卵巢癌(A2780，SKOV3)细胞中作用48h均能显著降解PRC2的核心亚基EZH2、SUZ12、EED和RbAp48，并有效降低催化产物H3K27me3的水平，表明E7降解PRC2各亚基和抑制H3K27me3的作用能在多种EZH2功能异常所驱动的肿瘤类型中得以发挥，而不仅仅局限于DLBCL中。目前的EZH2酶抑制剂在临床研究中主要对淋巴瘤和肉瘤等少数类型的肿瘤展现了良好的抑制活性，因而这也提示，相比于绝大多数EZH2酶抑制剂，E7可能能够在更多类型的肿瘤细胞中发挥作用。
四、E7通过结合EZH2降解PRC2
细胞热漂移实验(CETSA)可以通过检测药物在细胞内引起的蛋白质热稳定性变化判断药物与蛋白的结合情况，其原理为：药物在细胞内与相应蛋白的结合会提升该蛋白的结构稳定性，使得该蛋白能耐受更高的温度而不被降解。因而通过CETSA考察了E7与PRC2各核心亚基的结合情况。将E7与经MG-132预处理过的WSU-DLCL-2细胞共同孵育一定时间，待E7与细胞内相应蛋白结合后，提取细胞裂解液，检测对照组细胞和E7处理的细胞内EZH2、SUZ12、EED和RbAp48蛋白在不同温度(45、48、51、54、57、60℃)条件下孵育6min的降解情况。结果如图5中的A所示，对照组EZH2蛋白在51℃条件下孵育6min时已有明显降解，而E7处理的EZH2蛋白加热至57℃时才发生相当水平的降解，可见E7明显提高了EZH2蛋白的热稳定性，表明E7在细胞内与EZH2发生了结合；而其他几种蛋白SUZ12、EED和RbAp48，无论E7处理与否，几乎都是在相同的温度条件下发生相同程度的降解，即E7不改变这些蛋白的热稳定性，说明E7与这些蛋白没有结合。由此证明E7选择性结合PRC2的EZH2亚基导致其降解。
通过E7与EZH2抑制剂和EED抑制剂的竞争性结合实验进一步验证了E7与EZH2亚基的结合。从图5中的B可以看出，在WSU-DLCL-2细胞中，E7能显著降低EZH2、SUZ12、EED、RbAp48和H3K27me3的蛋白水平，当用EZH2抑制剂EPZ6438或GSK126与E7同时处理细胞时，E7对PRC2核心亚基的降解作用被削弱，但H3K27me3仍然受到强烈的抑制；而用EED抑制剂EED226与E7同时处理细胞则丝毫不影响E7对PRC2各亚基的降解效果，也不影响E7对H3K27me3的抑制。EPZ6438和GSK126之所以会干扰E7对PRC2各亚基的降解，是因为它们都会与E7竞争结合EZH2的SAM结合口袋，阻碍E7与EZH2的结合，导致E7的实际作用浓度降低；而EPZ6438和GSK126不影响对H3K27me3的抑制作用是因为作为EZH2甲基转移酶抑制剂，它们本身具有抑制H3K27me3的活性，因而它们占据E7的结合位点同样发挥抑制H3K27me3的作用，仍然能够维持对H3K27me3的强烈抑制。EED226的结合位点是EED的H3K27me3结合口袋，它与EED的结合并不占据E7的结合位点，所以并不影响E7对PRC2各亚基的降解和对H3K27me3的抑制。由此间接证明E7结合PRC2的EZH2亚基。
目前PRC2复合物的三维结构已被解析，因而能最直接且明确地证明E7与PRC2各亚基之间的结合情况的方法是通过E7与PRC2的共晶结构进行表征。因此后续我们还将通过E7与PRC2形成共晶的方式来进一步确认E7与PRC2的EZH2亚基的结合。
在WSU-DLCL-2细胞内检测组蛋白H3其他Lys位点的甲基化修饰水平，以考察E7对于EZH2HMTase活性的选择性抑制作用，图5中的C显示E7只选择性抑制EZH2所催化的H3K27me3和H3K27me2，而对其他几种HMTase催化产物H3K27me1、H3K9me3和H3K4me3几乎没有影响，表明E7选择性抑制EZH2HMTase活性；而且检测E7对与EZH2高度同源的EZH1的体外酶抑制活性显示，E7对EZH2的抑制活性(IC 50＝2.7nM)比对EZH1的抑制活性(IC 50＝180nM)强66倍，可见E7对EZH2HMTase活性具有高度的选择性抑制作用。这一点也为说明E7通过靶向EZH2发挥作用提供了辅助证明。
五、E7通过泛素蛋白酶体途径降解PRC2
根据PROTACs的作用原理，E7诱导EZH2及PRC2其他亚基降解首先将泛素分子招募到靶蛋白上为前提，并 且需要借助UPS对多聚泛素化蛋白的识别和降解作用才能实现。因而通过免疫沉淀实验(IP)检测了经E7处理的WSU-DLCL-2细胞内的EZH2、SUZ12和EED亚基的泛素化修饰情况。结果如图6所示，E7处理的细胞内，EZH2(图6中的A)、SUZ12(图6中的B)和EED(图6中的C)亚基的泛素化水平都明显高于对照组各蛋白亚基的泛素化水平，说明E7的作用导致细胞内EZH2、SUZ12和EED亚基发生了无差别的泛素化修饰。
此外，通过干扰UPS成员的功能，反向验证E7通过泛素蛋白酶体途径降解PRC2各亚基。本实验使用来那度胺、MLN4924和MG-132逐一验证阻碍靶蛋白的泛素化修饰或抑制蛋白酶体的活性是否能够破坏E7的降解作用，以进一步确定E7降解PRC2的途径。来那度胺是一种与沙利度胺结构十分相似的CRBN配体，能与E7竞争结合E3泛素连接酶而阻碍EZH2-E7-E3泛素连接酶三元复合物的形成，如图6的D所示，在用E7处理细胞之前，先用来那度胺对WSU-DLCL-2细胞进行预处理，结果会削弱E7对PRC2的EZH2、SUZ12、EED以及RbAp48亚基的降解；MLN4924能通过特异性靶向NEDD活化酶(NAE)抑制靶蛋白的泛素化修饰，因而用MLN4924对WSU-DLCL-2细胞进行预处理也会比较明显地抑制E7对于PRC2各亚基的降解。MG-132是一种蛋白酶体抑制剂，图6中的D显示，用MG-132预先抑制WSU-DLCL-2细胞内蛋白酶体的活性同样可以使PRC2各亚基不被E7降解而稳定存在。这些结果表明抑制UPS中的泛素化修饰过程或蛋白酶体的活性都能有效干扰E7对PRC2各蛋白亚基的降解，说明E7确实介导PRC2各亚基发生无差别泛素化，实现对PRC2各亚基的降解。
六、E7对EZH2下游基因转录的调控
确定了E7能通过结合EZH2，经由泛素蛋白酶体途径有效降解EZH2及PRC2其他核心亚基之后，进一步考察E7对于EZH2致癌功能的影响。目前的研究已经发现EZH2在驱动肿瘤发生发展的过程中主要表现为两个方面的作用：一是作为转录抑制因子，以依赖于其甲基转移酶催化活性的方式介导下游基因转录沉默；二是作为转录共激活因子，以不依赖于甲基转移酶活性的方式介导下游靶基因转录激活。鉴于EZH2在不同肿瘤类型中以全然不同的角色调控不同基因的转录而驱动肿瘤的进展，本公开实施方式中在不同类型的肿瘤细胞株中分别考察了E7对EZH2调控多种基因转录的影响。
七、E7激活EZH2的催化功能介导的转录沉默
在一些DLBCL中，EZH2突变引起组蛋白H3K27发生异常高水平的甲基化修饰，使得染色质结构高度固缩，导致下游抑癌基因转录沉默而触发肿瘤形成。由此，我们首先在两株EZH2突变的DLBCL细胞WSU-DLCL-2(EZH2 Y641F)和Pfeiffer(EZH2 A677G)中检测了E7对于几种H3K27me3上富集的基因ADRB2、CDKN2A、TXINP和TNFRSF21的调控。图7中的A和B的RT-qPCR结果显示，WSU-DLCL-2和Pfeiffer细胞经E7处理48h后，几种检测的基因的mRNA水平几乎都有显著的上调，而且E7对于ADRB2和TNFRSF21的上调分别强于EZH2酶抑制剂EPZ6438和GSK126，但对于CDKN2A和TXINP的上调略弱于EPZ6438和GSK126。总之，在EZH2突变的DLBCL细胞株WSU-DLCL-2和Pfeiffer中，E7能够有效激活EZH2的催化功能介导的转录沉默，抑制EZH2的催化活性。在一些SWI/SNF突变的肿瘤中，EZH2对于肿瘤细胞的增殖有明显的促进作用，并且这种增殖促进作用部分依赖于EZH2在PRC2中的催化功能，所以我们也在具有SWI/SNF突变的NSCLC细胞株A549细胞中检测了E7对于以上几种基因转录的调控。结果如图7中的C所示，与在DLBCL中观察到的结果不同，E7只提高了CDKN2A和TXINP2基因的mRNA水平，而没能上调ADRB2和TNFRSF21的表达。E7在A549中上调这些基因的程度不如在WSU-DLCL-2和Pfeiffer中明显，其原因在于，EZH2的催化活性对A549这类SWI/SNF突变的肿瘤细胞的增殖不起主导作用，其非催化活性才是导致这类肿瘤细胞增殖的主要原因，因而在A549细胞中，诸如ADRB2、CDKN2A等基因的转录本身并没有受到EZH2催化活性的抑制，所以即使E7抑制EZH2的催化活性也不会影响这些基因的表达。上述结果可以证明E7通过降解EZH2及PRC2其他核心亚基，有效抑制EZH2的催化活性及其介导的转录沉默。
八、E7抑制EZH2的非催化功能介导的转录激活
在由EZH2的非催化功能驱动的肿瘤细胞A549、NCI-H1299和MDA-MB-468中检测E7对于EZH2的非催化活性所激活的几种下游基因ARL6IP、BRIC5、CENPK、CEP76、CHEK1和TACC3的转录的影响。如图8显示，在这几种细胞中，E7处理48h使得上述EZH2激活的基因都有较为明显的下调，而EZH2甲基转移酶抑制剂EPZ6438和GSK126对这些基因的表达几乎没有影响。上述结果证明，E7通过降解EZH2，有效抑制EZH2的非催化活性及其对下游基因转录的异常激活。
上述结果表明，无论EZH2通过怎样的方式驱动肿瘤发生发展，PROTAC E7通过降解EZH2以及PRC2的其他核心亚基，或是能抑制EZH2作为转录抑制因子介导的基因沉默，或是能抑制EZH2作为转录共激活因子介导的转录异常激活，最终都能有效地抑制EZH2在肿瘤发生发展中的驱动作用。也就是说，E7不仅能破坏EZH2依赖于PRC2的催化功能，而且可以破坏其不依赖于PRC2的非催化功能，所以能够全面彻底地抑制EZH2的致癌活性。因而对于EZH2驱动的肿瘤，尤其是其非催化功能驱动的肿瘤，这种诱导EZH2降解的策略应该比单纯地抑制EZH2 甲基转移酶活性有更好的抑制效果，所以接下来我们初步评价并比较了E7与EZH2酶抑制剂EPZ6438和GSK126在抑制EZH2驱动的肿瘤细胞增殖方面的活性差异。
九、E7抑制EZH2异常的肿瘤细胞的增殖
通过细胞计数和观察的方法监测E7和EPZ6438、GSK126对几种EZH2驱动的肿瘤细胞的生长抑制情况。如图9中的A显示，E7几乎能完全抑制WSU-DLCL-2细胞的生长，展现了良好的增殖抑制活性；EPZ6438和GSK126虽然也都对WSU-DLCL-2细胞展现出一定的增殖抑制活性，但其抑制程度相比于E7明显逊色许多，可见E7可以有效抑制EZH2的催化功能所驱动的肿瘤细胞生长。SWI/SNF突变的A549和NCI-H1299细胞的增殖活性主要依赖于EZH2在PRC2中的非催化功能，因而也考察E7和EPZ6438、GSK126对这两种细胞株的增殖抑制情况。结果如图9中的B和C，与在WSU-DLCL-2细胞中观察到的结果相似，E7对于A549和NCI-H1299两种细胞株都有非常显著的增殖抑制活性；GSK126虽然也能明显地抑制两种细胞株的增殖，但抑制活性不如E7；EPZ6438对这两种细胞株则只表现出非常有限的增殖抑制活性。这些结果充分证明E7对EZH2的催化和非催化功能所驱动的肿瘤细胞的生长具有良好的抑制作用。
图10中的MTT检测结果也表明E7对EZH2的催化功能驱动的肿瘤细胞WSU-DLCL-2、Pfeiffer和EZH2的非催化功能驱动的肿瘤细胞A549、NCI-H1299的细胞活力均展现了良好的时间依赖性抑制作用，IC 50值均在低微摩尔水平，尤其对于DLBCL细胞株Pfeiffer，E7作用7天的IC 50仅为0.17μM。上述结果进一步证明E7对EZH2的催化和非催化功能所驱动的肿瘤细胞的活力具有良好的抑制作用。
综上所述，通过以上的生物实验数据表明，本公开实施方式的化合物对于PRC2复合物各亚基均具有降解效果，并且展现了相比于抑制剂更为广泛和强大的抗肿瘤效果。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
该双功能化合物能够有效地诱导PRC2蛋白复合物核心亚基降解，进而达到治疗由PRC2复合物和其亚基包括EZH2，EED，SUZ12，RbAp46，RbAp48在内介导的癌症，完全阻断PRC2复合物亚基的致癌活性，相比于单纯地抑制PRC2复合物活性，例如EZH2抑制剂，EED抑制剂，具有更好地抗癌活性，其具有能够治疗乳腺癌、结直肠癌、前列腺癌、胰腺癌、卵巢癌等多种实体瘤以及血液瘤等多种肿瘤疾病的用途。该双功能化合物或药物组合物作为激酶抑制剂用于治疗人的多种肿瘤，并且其具有较好的抗肿瘤活性和较低的毒性。

Claims (21)

  1. 一种可诱导PRC2蛋白复合物核心亚基降解的双功能化合物，其特征在于，其包括如式I-III任一项所示的化合物、其药学上可接受的盐或前药、其溶剂化物、其水合物、其多晶型物、其互变异构体、立体异构体或同位素取代的化合物；
    Figure PCTCN2020119438-appb-100001
    其中，式I-III中的n均为1～10的整数，X均为O，N或S，Y均为O，H 2或S。
  2. 根据权利要求1所述的双功能化合物，其特征在于，式I-III中的n均为2～6的整数。
  3. 根据权利要求1所述的双功能化合物，其特征在于，式I-III中的n均为2～10的整数，X和Y均为O。
  4. 根据权利要求3所述的双功能化合物，其特征在于，式I-III中的n均为2～6的整数，X和Y均为O。
  5. 根据权利要求4所述的双功能化合物，其特征在于，所述双功能化合物的化学式为
    Figure PCTCN2020119438-appb-100002
  6. 根据权利要求1所述的双功能化合物，其特征在于，式I-III中的n均为2～10的整数，X和Y均为O；优选地，式I-III中的n均为2～6的整数，X和Y均为O；更优选地，所述双功能化合物的化学式为
    Figure PCTCN2020119438-appb-100003
  7. 如权利要求1所述的双功能化合物的制备方法，其特征在于，式I-III中X为N，S，Y为O时，式I-III所示的化合物的合成路线为：
    Figure PCTCN2020119438-appb-100004
  8. 根据权利要求7所述的双功能化合物的制备方法，其特征在于，式I-III中X和Y均为O时，式I化合物的合成路线为：
    Figure PCTCN2020119438-appb-100005
    式II化合物的合成路线为：
    Figure PCTCN2020119438-appb-100006
    式III化合物的合成路线为：
    Figure PCTCN2020119438-appb-100007
  9. 如权利要求1所述的双功能化合物的制备方法，其特征在于，式I-III中X为N，S，Y为O时，式I-III所示的化合物的合成路线为：
    Figure PCTCN2020119438-appb-100008
    优选地，式I-III中X和Y均为O时，式I化合物的合成路线为：
    Figure PCTCN2020119438-appb-100009
    式II化合物的合成路线为：
    Figure PCTCN2020119438-appb-100010
    式III化合物的合成路线为：
    Figure PCTCN2020119438-appb-100011
  10. 一种药物组合物，其特征在于，其包括药学上可接受的辅助性成分和如权利要求1～3任一项所述的双功能化合物。
  11. 根据权利要求10所述的药物组合物，其特征在于，所述药物组合物为水溶液、粉末、颗粒、片剂或冻干粉。
  12. 根据权利要求11所述的药物组合物，其特征在于，所药物组合物为水溶液时，所述药物组合物还含有注射用水、盐水溶液、葡萄糖水溶液、注射或输注用盐水、注射或输注用葡萄糖、格林氏溶液或含有乳酸盐的格林氏溶液。
  13. 根据权利要求10所述的药物组合物，其特征在于，所述药物组合物为水溶液、粉末、颗粒、片剂或冻干粉，优选地，所药物组合物为水溶液时，所述药物组合物还含有注射用水、盐水溶液、葡萄糖水溶液、注射或输注用盐水、注射或输注用葡萄糖、格林氏溶液或含有乳酸盐的格林氏溶液。
  14. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备激酶抑制剂中的应用。
  15. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备治疗肿瘤的药物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述肿瘤包括乳腺癌、结直肠癌、前列腺癌、卵巢癌、胰腺癌或胃癌。
  17. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备治疗肿瘤的药物中的应用；优选地，所述肿瘤包括乳腺癌、结直肠癌、前列腺癌、卵巢癌、胰腺癌或胃癌。
  18. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备降解PRC2蛋白复合物核心亚基的降解剂中的应用。
  19. 根据权利要求18所述的应用，其特征在于，降解PRC2蛋白复合物核心亚基为同时降解PRC2蛋白复合物的EZH1，EZH2，EED，SUZ12以及RbAp46/48亚基。
  20. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备降解PRC2蛋白复合物核心亚基的降解剂中的应用，优选地，降解PRC2蛋白复合物核心亚基为同时降解PRC2蛋白复合物的EZH1， EZH2，EED，SUZ12以及RbAp46/48亚基。
  21. 如权利要求1～6任一项所述的双功能化合物或权利要求5～6任一项所述的药物组合物在制备口服或静脉注射制剂中的应用，所述口服或静脉注射剂至少包括所述双功能化合物或药物组合物，优选地，还包括赋形剂和/或佐剂。
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