CN113845484A - Novel quinazoline small molecule inhibitor and application thereof in antitumor drugs - Google Patents

Novel quinazoline small molecule inhibitor and application thereof in antitumor drugs Download PDF

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CN113845484A
CN113845484A CN202111046514.7A CN202111046514A CN113845484A CN 113845484 A CN113845484 A CN 113845484A CN 202111046514 A CN202111046514 A CN 202111046514A CN 113845484 A CN113845484 A CN 113845484A
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欧阳亮
张吉发
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Abstract

The invention relates to a double-targeting BRD4-CK2 small molecule inhibitor and application thereof in a triple negative breast cancer resistant drug, belonging to the technical field of antitumor science. The invention solves the technical problem of providing a compound serving as a double-targeting BRD4-CK2 small-molecule inhibitor. The compound or the pharmaceutically acceptable salt thereof can be used as a double-targeting BRD4-CK2 inhibitor, has certain triple negative breast cancer resistance activity, and can effectively inhibit the growth of triple negative breast cancer cells.

Description

Novel quinazoline small molecule inhibitor and application thereof in antitumor drugs
Technical Field
The invention discloses a double-targeting BRD4-CK2 small molecule inhibitor and application thereof in tumor treatment, belonging to the technical field of antitumor medicine.
Background
Recent statistics by the international cancer research center indicate that about 170 million women are diagnosed with breast cancer every year worldwide and result in the death of 50 million patients. In China, breast cancer has a high rate of growth of about 4% in recent years, and has a trend of further rejuvenation, seriously threatening the health of women. Triple Negative Breast Cancer (TNBC) refers to breast cancer which is negative for Estrogen Receptor (ER), progestational hormone receptor (PR) and human epidermal growth factor receptor 2(Her-2), accounts for 15-20% of all breast cancer pathological types, and has extremely high lethality rate. Due to the special biological behavior and clinical pathological characteristics of TNBC, the prognosis of TNBC is worse than that of other types of breast cancer, and the TNBC has the characteristics of high malignancy, younger onset age, high invasiveness, easy relapse and transfer and the like. Because ER, PR are not expressed and there is a lack of HER2 amplification, TNBC generally has no recognized molecular therapeutic targets and lacks clinically effective therapeutic drugs and therapeutic strategies.
The TNBC treatment means commonly used in clinic at present are still surgery and conventional systemic cytotoxic chemical treatment, the treatment effect is not ideal, and the prognosis is still poor. Bromodomain accompanying protein 4(BRD4) has an attractive epigenetic target in cancer and other chronic diseases. More and more BRD4 inhibitors are being discovered and used in the treatment of TNBC, but the emergence of drug resistance is the most major problem limiting the clinical use of BRD4 inhibitors. The protein kinase casein kinase 2(CK2) is a molecular switch that mediates BRD4 phosphorylation and dephosphorylation and thus functions. In addition, CK2 inhibition also results in increased uptake of known drugs by multidrug resistant cells. Therefore, designing synthetic dual-targeting inhibitors of BRD4-CK2 may provide new strategies for TNBC therapy.
Disclosure of Invention
The technical problem solved by the invention is to provide a novel compound serving as a dual-targeting BRD4-CK2 inhibitor.
A compound of formula I or formula II:
Figure BDA0003251074340000011
wherein R is1Is 3,5-diCH3H or 3, 5-diBr; r2Is 4-F, 4-OCH34-Cl, 5-Cl or 5-CH3；R3Is composed of
Figure BDA0003251074340000012
Figure BDA0003251074340000021
The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparing antitumor drugs.
Further, the anti-tumor therapeutic drug is preferably a triple negative breast cancer therapeutic drug.
Further, the triple negative breast cancer treatment drug is a double-targeting BRD4-CK2 small molecule inhibitor, and the application of the triple negative breast cancer treatment drug is used for triple negative breast cancer related treatment.
The present invention also provides a pharmaceutical composition which is a preparation comprising an effective dose of the above-mentioned compound or a pharmaceutically acceptable salt thereof.
The compound or the pharmaceutically acceptable salt thereof prepared by the invention can be used as a double-target BRD4-CK2 small molecule inhibitor, and has a relatively obvious triple negative breast cancer treatment effect.
Drawings
FIG. 1A is a picture of the autophagy flux changes observed under an inverted microscope after transfection of MDA-MB-231 cells with autophagy double-standard adenovirus GFP-mRFP-LC3, and treatment with 5. mu.M Compound 32(44e) for 24 hours.
FIG. 1B 44e is a picture of the inhibition of colony formation in MDA-MB-231 cells at 0. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M concentrations.
FIG. 1C 44e is a graph of MDA-MB-231 cell death promoted at 0. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M concentrations.
FIG. 1D 44e is a picture showing that MDA-MB-231 cells promoted apoptosis at 0. mu.M and 5. mu.M concentrations.
FIG. 2A is a photograph showing the change in tumor volume of nude mice by oral administration of 44e to a nude mouse model of MDA-MB-231 xenograft.
FIG. 2B photographs of the change in tumor volume of nude mice xenografted with MDA-MB-468 by oral administration of 44 e.
Detailed Description
The present invention provides compounds of formula I or formula II:
Figure BDA0003251074340000022
wherein R is1Is 3,5-diCH3H or 3, 5-diBr; r2Is 4-F, 4-OCH34-Cl, 5-Cl or 5-CH3；R3Is composed of
Figure BDA0003251074340000023
Figure BDA0003251074340000031
As a preferred embodiment, R is preferred1Is H; r3Is composed of
Figure BDA0003251074340000032
Figure BDA0003251074340000033
More preferably R1Is H; r3Is composed of
Figure BDA0003251074340000034
The following are some preferred structures of the compounds of the present invention.
Figure BDA0003251074340000041
The invention also provides pharmaceutically acceptable salts of the compounds of the invention. The salt may be nitrate, hydrochloride, sulfate or phosphate, etc.
The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating triple negative breast cancer. Further, the triple negative breast cancer treatment drug is preferably a double-targeting BRD4-CK2 small molecule inhibitor.
The present invention also provides a pharmaceutical composition which is a preparation comprising an effective dose of the above-mentioned compound or a pharmaceutically acceptable salt thereof. The compounds of the invention can be prepared in the following forms by methods known in the art: tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols or sprays for inhalation, sterile aqueous or oily solutions or suspensions or sterile emulsions for parenteral (including intravenous, intramuscular or infusion). The liquid formulations can be prepared using sterile water or water-propylene glycol solutions as solvents, or the active ingredient can be formulated in aqueous polyethylene glycol solutions. Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be prepared by dispersing the finely divided active component in water together with viscous materials such as natural synthetic gums, resins, methyl cellulose, carboxymethyl cellulose and other suspending agents known in the pharmaceutical arts.
The pharmaceutical composition may be in unit dosage form. In these forms, the composition is divided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged preparation comprising discrete quantities of the preparation, such as tablets, capsules, and powders in vials or ampoules . The unit dosage form can also be a capsule, cachet, or tablet or it can be the appropriate number of any of these packaged forms.
The active ingredient of the pharmaceutical composition of the present invention may be only the compound of the present invention, or may be combined with other anti-triple negative breast cancer compounds as an active ingredient.
In the treatment of triple negative breast cancer, such combination therapy may be achieved by the simultaneous, sequential or separate administration of the various therapeutic ingredients. Such combinations employ the compounds of the present invention in an effective dosage range and the other pharmaceutically active agents in a permitted dosage range.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1 Synthesis of Compounds 1-41.
Compounds 1-41 were synthesized using the following reaction scheme:
Figure BDA0003251074340000051
Reagents and conditions：(a)K2CO3，DMF，80℃，3h；(b)DCM，TFA，r.t，2h.；(c)DMAC，PTSA，NaHSO3，120℃，4-8h；(d)DMF，Et3N，HOBt，EDCI，r.t.；24h。
general procedure for the Synthesis of intermediates 1 a-c.
Tert-butyl bromoacetate (3.0mL, 1.2equiv) and p-hydroxybenzaldehyde analog (2.5g, 1equiv) were dissolved in DMF (10mL) and 5.2g K was added2CO3The reaction was carried out at 80 ℃ for 1.5 h. After cooling the reaction, 80mL of water was added, filtered and dried to give the crude product, which was purified by silica gel column Chromatography (CH)2Cl2/CH3OH 80: 1-5: 1) to give intermediates 1a-c (55-60% yield) without further purification.
Intermediate (1a)1H-NMR(400MHz，CD3OD)，δ(ppm)：9.84(1H，s)，7.86(2H，d，J＝8.8Hz)，7.06(2H，d，J＝8.8Hz)，4.71(2H，s)，1.48(9H，s)。HRMS(ESI)+Calculated for C13H17O3，[M+H]+：m/z 221.1178，found 221.1170。
General procedure for the Synthesis of intermediates 2 a-c.
Intermediates 1a-c (2g, 9mmol) were dissolved in CH2Cl2(5mL), TFA (1.5mL) was added dropwise, and the reaction was carried out at room temperature for 1 h. The reaction was concentrated under reduced pressure to afford intermediates 2a-c (72-77% yield) as white solids without further purification.
Intermediate (2a)1H-NMR(400MHz，CD3OD)，δ(ppm)：12.28(1H，s)，9.82(1H，s)，7.84(2H，d，J＝8.8Hz)，7.04(2H，d，J＝8.8Hz)，3.68(2H，s)。HRMS(ESI)+Calculated for C9H9O4，[M+H]+：m/z 181.0501，found 181.0507。
Intermediates 3 a-h.
Intermediate 2a (0.9mmol, 1equiv) and benzaldehyde derivative (0.9mmol, 1equiv) were dissolved in DMAc (15 mL)) Adding NaHSO4(1.05mmol, 1.2equiv) and PTSA (0.22mmol, 0.24 equiv). The reaction mixture was stirred at 120 ℃ under reflux for 4-6 h. After completion of the reaction, water (100mL) was added to the reaction, and the precipitate was obtained by filtration. Purification of the solid by column chromatography on silica gel (CH)2Cl2/CH3OH 80: 1-10: 1) to yield intermediates 3a-h, respectively (41-65% yield).
Intermediate (3a)1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.97(1H，s)，8.13(2H，d，J＝8.8Hz)，7.04(2H，d，J＝8.8Hz)，6.72(1H，d，J＝2.0Hz)，6.51(1H，d，J＝2.0Hz)，4.79(2H，s)，3.88(3H，s)，3.84(3H，s)。HRMS(ESI)+Calculated for C18H16NaN2O6，[M+Na]+：m/z 379.0906，found 379.0910。
Compounds 1 to 41.
Dissolving the intermediate 3a-h (0.42mmol, 1.2eq) in DMF (3mL), adding HOBT (0.54mmol, 1.3equiv) at 0 ℃, stirring for 10min, adding EDCI (0.54mmol, 1.3equiv), adding the reactant containing amino substitution, continuing the reaction, adding TEA (1.5mmol, 3.5equiv) for reaction at 0 ℃ for 1h, finally reacting at normal temperature for 24h, adding 80mL of water into the reaction solution, filtering and drying to obtain a crude product. Purification of the solid by column chromatography on silica gel (CH)2Cl2/CH3OH 30: 1-1: 1) to give 1-41 (43-66% yield), respectively, as the final product.
Compound 1, mp 262-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.29(1H，s)，8.90(1H，s)，8.09-8.15(2H，m)，7.08(1H，d，J＝8.8Hz)，7.02(1H，d，J＝8.8Hz)，6.71-6.73(1H，m)，6.53(1H，d，J＝2.4Hz)，5.00(1H，s)，4.56(2H，s)，3.89(3H，s)，3.85(3H，s)，3.62(4H，t，J＝4.4Hz)，2.79(4H，t，J＝4.4Hz)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：170.0，167.2，166.5，164.1，163.1，157.8，143.1，133.0，132.8，118.9，116.4，116.3，103.7，99.8，95.8，67.2，67.1，65.6，57.2，57.0，56.8，56.0。HRMS(ESI)+Calculated for C22H24NaN4O6，[M+Na]+：m/z 463.1594，found463.1601。
Compound 2, mp 265-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.39(1H，s)，8.81(1H，s)，7.92(2H，s)，6.69(1H，s)，6.45(1H，s)，4.60(1H，s)，4.25(2H，s)，3.87(3H，s)，3.63(3H，s)，3.64(4H，t，J＝4.4Hz)，2.82(4H，t，J＝4.4Hz)，2.30(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：169.3，164.8，163.6，160.7，158.1，157.4，153.7，130.4(2)，128.3(2)，128.2，105.0，100.7，97.0，70.5，65.9(2)，55.8，55.5，54.5(2)，16.1(2)。HRMS(ESI)+Calculated for C24H29N4O6，[M+H]+：m/z 469.2087，found469.2103。
Compound 3, mp 253-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.93(1H，s)，8.16(3H，d，J＝8.8Hz)，7.07(2H，d，J＝8.8Hz)，6.71(1H，d，J＝2.4Hz)，6.52(1H，d，J＝2.4Hz)，4.59(2H，s)，3.89(3H，s)，3.84(3H，s)，3.79-3.81(2H，m)，3.23(2H，t，J＝8.8Hz)，3.03(2H，t，J＝8.8Hz)，1.49-1.52(2H，m)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：167.9，166.8，164.0，162.8，157.8，157.6，142.9，132.6(2)，118.4，116.1(2)，103.4，99.5，95.6，67.8，67.6(2)，57.1，55.6，44.9，35.8，31.2(2)。HRMS(ESI)+Calculated for C24H28N3O6，[M+H]+：m/z454.1978，found454.1978。
Compound 4, mp 224-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.78(1H，s)，8.24(1H，t，J＝6.0Hz)，7.90(2H，s)，6.73(1H，d，J＝2.4Hz)，6.51(1H，d，J＝2.4Hz)，4.28(2H，s)，3.88(3H，s)，3.84(3H，s)，3.26(2H，t，J＝11.6Hz)，3.09(2H，t，J＝7.6Hz)，2.31(6H，s)，1.55(2H，d，J＝8.8Hz)，1.16-1.19(2H，m)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：168.1，166.5，162.5，160.8，157.4，157.3，143.0，132.5(2)，131.0(2)，125.0，103.4，99.4，95.7，71.3，67.4(2)，57.0，56.5，44.6，35.5，31.1(2)，16.5(2)。HRMS(ESI)+Calculated for C26H31NaN3O6，[M+Na]+：m/z 504.2111，found 504.2106。
Compound 5, mp 267-269 ℃, yield 45.5%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.50(1H，s)，8.50(2H，s)，6.80(1H，s)，6.57(1H，s)，4.48(2H，s)，3.90(3H，s)，3.86(3H，s)，3.45-3.47(2H，m)，3.07-3.08(2H，m)，2.75-2.77(2H，m)，2.97-2.99(2H，m)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.0，164.4，160.9，159.5，154.1，152.4，149.7，132.0(2)，131.2，117.6(2)，104.8，101.5，98.2，71.0，56.0，55.7，52.7(2)，43.5，42.6，28.8(2)。HRMS(ESI)+Calculated for C24H27Br2N2O5，[M+H]+：m/z 611.0328，found 611.0320。
Compound 6, mp 270-.1H-NMR(400MHz，CDCl3)，δ(ppm)：7.78(1H，t，J＝6.0Hz)，6.78(1H，s)，6.41(1H，s)，4.27(2H，s)，3.91(6H，s)，2.80(2H，d，J＝4.8Hz)，2.29(6H，s)，2.14(2H，t，J＝8.8Hz)，2.01-2.03(2H，m)，1.57-1.60(2H，m)；13C-NMR(100MHz，CDCl3)，δ(ppm)：167.0(2)，165.0，164.8，161.0，157.2，154.1，131.1，128.5(2)，125.1，123.0，105.0，101.2，98.2，70.6，56.2，55.7，54.5(2)，46.3，45.8，32.3(2)，16.5(2)。HRMS(ESI)+Calculated for C26H33N4O5，[M+H]+：m/z 481.2451，found481.2443。
Compound 7, mp 256-258 ℃, yield 62.5%.1H-NMR(400MHz，CDCl3+CD3OD)，δ(ppm)：8.25(2H，d，J＝2.4Hz)，7.35(2H，d，J＝2.4Hz)，7.01(1H，s)，6.72(1H，s)，4.82(2H，s)，4.16(3H，s)，4.15(3H，s)，3.09-3.12(4H，m)，2.85-2.87(4H，m)，2.57(3H，s)；13C-NMR(100MHz，CDCl3+CD3OD)，δ(ppm)：166.0，165.9，165.2，161.1，159.9，153.5，152.7，129.2(2)，126.0，114.8(2)，104.5，100.8，97.9，66.7，55.8，55.5，53.9(2)，49.2(2)，45.2。HRMS(ESI)+Calculated for C23H27NaN5O5，[M+Na]+：m/z 476.1910，found476.1904。
Compound 8, mp 269-271 ℃, yield 63.7%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.07(1H，s)，8.15(2H，d，d，J＝8.8Hz)，7.13(2H，d，J＝8.8Hz)，7.06(2H，s)，6.74(1H，d，J＝2.4Hz)，6.53(1H，d，J＝2.4Hz)，4.80(2H，s)，3.89(3H，s)，3.85(3H，s)，3.74(6H，s)，3.62(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.0，164.4，161.0，160.7，159.5，153.0，152.7(2)，151.7，134.5，133.7，129.7(2)，124.2，114.7(2)，104.3，100.2，97.6，97.4(2)，67.1，60.1(2)，56.0，55.7(2)。HRMS(ESI)+Calculated for C27H27NaN3O8，[M+Na]+：m/z 544.1696，found 544.1694。
Compound 9, mp 240-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.98(1H，s)，7.92(2H，s)，7.18(2H，s)，6.76(1H，d，J＝2.4Hz)，6.54(1H，d，J＝2.4Hz)，4.48(2H，s)，3.89(3H，s)，3.85(3H，s)，3.75(6H，s)，3.63(3H，s)，2.36(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.3，164.4，161.0，159.5，157.8，152.8，152.7(2)，152.0，134.5，133.8，130.9(2)，128.6(2)，127.3，104.5，100.6，97.7，97.6(2)，71.0，60.1，56.0，55.8(2)，55.7，16.2(2)。HRMS(ESI)+Calculated for C29H31NaN3O8，[M+Na]+：m/z 572.2009，found 572.2007。
Compound 10, mp 257 ℃ and 260 ℃, yield 57.2%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：7.93(2H，s)，7.45-7.47(2H，m)，7.10-7.11(1H，m)，6.74(1H，d，J＝2.4Hz)，6.50(1H，d，J＝2.4Hz)，4.67(2H，s)，3.89(3H，s)，3.84(3H，s)，2.37(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：168.3，164.2，160.9，160.1，159.7，159.1，158.0，153.1，152.7，146.6，130.6(2)，130.4，128.7，128.4(2)，128.0，121.2(2)，104.7，101.1，97.5，70.9，55.9，55.6，16.3(2)。HRMS(ESI)+Calculated for C27H26N5O5，[M+Na]+：m/z 522.1753，found 522.1752。
Compound 11, mp 244-246 ℃, yield 64.4%.1H-NMR(400MHz，CDCl3)，δ(ppm)：8.26(2H，d，J＝8.4Hz)，7.06(2H，d，J＝8.4Hz)，6.46(1H，d，J＝2.4Hz)，6.42(1H，d，J＝8.4Hz)，4.56(2H，s)，3.98(3H，s)，3.93(3H，s)，4.03-4.06(4H，m)，2.87(2H，t，J＝12.4Hz)，1.94(2H，t，J＝12.4Hz)，1.45(9H，s)；13C-NMR(100MHz，CDCl3)，δ(ppm)：166.9，165.4，161.9，161.5，159.9，154.7，152.2，129.8(2)，115.0(2)，104.9，101.1，98.5，79.9，67.5，56.6，55.9，46.7，42.8，42.5，32.1(2)，28.6(5)。HRMS(ESI)+Calculated for C28H35N4O7，[M+H]+：m/z 539.2506，found539.2514。
Compound 12, mp 236-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：8.13(1H，d，J＝8.4Hz)，7.90(2H，s)，6.72(1H，d，J＝2.4Hz)，6.48(1H，d，J＝2.4Hz)，4.26(2H，s)，3.91-3.93(2H，m)，3.89(3H，s)，3.83(3H，s)，3.31-3.33(2H，m)，2.80-2.82(2H，m)，2.29(6H，s)，1.70-1.72(2H，m)，1.39(9H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.8，164.0，161.9，160.9，160.5，157.6，153.9，153.2，152.9，130.6(2)，128.3(2)，104.8，100.9，97.4，78.6，70.8，55.9，55.5，45.7，42.7，42.2，31.2(2)，28.1(3)，16.1(2)。HRMS(ESI)+Calculated for C30H39N4O4，[M+Na]+：m/z 589.2638，found 589.2635。
Compound 13, mp 240-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.86(1H，s)，9.86(1H，s)，7.92(2H，s)，7.55(2H，d，J＝8.8Hz)，6.90(2H，d，J＝8.8Hz)，6.74(1H，d，J＝2.0Hz)，6.51(1H，d，J＝2.0Hz)，4.47(2H，s)，3.88(3H，s)，3.84(3H，s)，3.08(4H，t，J＝4.8Hz)，2.44(4H，t，J＝4.8Hz)，2.35(6H，s)，2.21(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：165.9，164.3，161.0，159.8，157.8，153.1，152.4，147.6，130.8(2)，130.2，128.4(2)，127.9，121.1(2)，115.6(2)，104.7，101.2，97.6，71.2，56.0，55.7，54.6(2)，48.5(2)，45.8，16.8(2)。HRMS(ESI)+Calculated for C31H36N5O5，[M+H]+：m/z 558.2716，found 558.2719.
Compound 14, mp 237-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.88(1H，s)，10.01(1H，s)，7.93(2H，s)，7.42(1H，s)，7.28(1H，t，J＝8.0Hz)，7.23(1H，t，J＝8.0Hz)，6.77(1H，d，J＝2.4Hz)，6.73(1H，s)，6.51(1H，d，J＝2.4Hz)，4.49(2H，s)，3.88(3H，s)，3.84(3H，s)，3.35-3.37(4H，m)，3.17(4H，s)，2.86(3H，s)，2.36(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.4，165.6，164.2，160.9，159.7，157.6，153.0，152.4，149.9，139.2，130.7(2)，129.3，128.3(2)，127.9，111.7，107.5，104.6，101.1，97.6，71.0，57.8，55.9，55.6，52.2(2)，45.7(2)，16.2(2)。HRMS(ESI)+Calculated for C31H35NaN5O5，[M+Na]+：m/z 580.2536，found580.2534。
Compound 15, mp 270-.1H-NMR(400MHz，AceticAcid-d4)，δ(ppm)：8.58(2H，s)，7.58(2H，d，J＝8.8Hz)，7.22(2H，d，J＝8.8Hz)，7.05(1H，d，J＝2.0Hz)，6.71(1H，d，J＝2.0Hz)，5.01(2H，s)，4.10(3H，s)，4.11(3H，s)，3.92(4H，t，J＝4.8Hz)，3.41(4H，t，J＝4.8Hz)，3.12(3H，s)；13C-NMR(100MHz，AceticAcid-d4)，δ(ppm)：168.7，167.8，164.9.0，163.1，156.1，154.8，151.0，148.8，133.9(2)，132.9，132.4，123.9(2)，119.7(2)，119.2(2)，105.7，103.0，100.2，72.6，57.2，57.1，54.7(2)，48.7(2)，44.3。HRMS(ESI)+Calculated for C29H30Br2N5O5，[M+H]+：m/z 688.0593，found 688.0596。
Compound 16, mp 267-269 ℃, yield 58.2%.1H-NMR(400MHz，AceticAcid-d4)，δ(ppm)：8.31(2H，d，J＝8.0Hz)，7.82(2H，d，J＝8.0Hz)，7.39(2H，d，J＝8.0Hz)，7.23(2H，d，J＝8.0Hz)，7.12(1H，s)，6.72(1H，s)，5.00(2H，s)，4.12(3H，s)，4.09(3H，s)，3.94(4H，brs)，2.44(4H，brs)，3.16(3H，s)；13C-NMR(100MHz，AceticAcid-d4)，δ(ppm)：169.6，167.9，165.0，163.2，162.2，154.9，154.4，148.8，132.5，131.6(2)，126.8，124.2(2)，119.2(2)，116.8(2)，105.4，102.3，100.0，68.8，57.2，57.1，54.6(2)，48.7(2)，44.4。HRMS(ESI)+Calculated for C29H32N5O5，[M+H]+：m/z530.2403，found 530.2401。
Compound 17, mp 256-258 ℃, yield 66.0%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.95(1H，s)，10.12(1H，s)，8.17(2H，d，J＝8.4Hz)，7.38(1H，s)，7.21(1H，t，J＝8.0Hz)，7.11-7.15(2H，m)，6.77(1H，dd，J＝8.0，2.4Hz)，6.72(1H，d，J＝2.4Hz)，6.52(1H，d，J＝2.0Hz)，4.82(2H，s)，3.85(3H，s)，3.82(3H，s)，3.74-3.76(2H，m)，3.51-3.53(2H，m)，3.16(2H，s)，2.96-2.98(2H，m)，2.86(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.8，166.6，164.7，161.5，160.9，153.5，152.9，150.5，139.8，129.9(2)，125.5，115.1(2)，112.3，112.0，107.8，105.0，101.5，97.9，89.7，67.5，56.0，55.7，52.7(2)，46.2(2)，42.5。HRMS(ESI)+Calculated for C29H31NaN5O5，[M+Na]+：m/z 552.2223，found 552.2222。
Compound 18, mp 252-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.96(1H，s)，8.23(2H，d，J＝2.4Hz)，8.15(1H，d，J＝8.4Hz)，7.14-7.28(4H，m)，6.71(1H，d，J＝2.0Hz)，6.52(1H，d，J＝2.0Hz)，4.90(2H，s)，3.89(3H，s)，3.85(3H，s)，3.51-3.53(4H，m)，2.98-3.03(4H，m)，2.87(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.0，164.3，161.1，160.0，159.9，158.7，152.8，152.5，141.0，132.0，129.8(2)，125.5，125.3，124.8，120.8，114.8(2)，104.6，100.9，97.5，67.4，56.0，55.6，53.3(2)，48.5(2)，42.4。HRMS(ESI)+Calculated for C29H31NaN5O5，[M+Na]+：m/z 552.2223，found 552.2215。
Compound 19, mp 273-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.87(1H，s)，9.89(1H，s)，7.92(2H，s)，7.57(2H，d，J＝8.4Hz)，6.92(2H，d，J＝8.4Hz)，6.74(1H，d，J＝2.0Hz)，6.52(1H，d，J＝2.0Hz)，4.45(2H，s)，3.89(3H，s)，3.84(3H，s)，3.73(4H，t，J＝4.8Hz)，3.05(4H，t，J＝4.8Hz)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.4，165.3，161.6，159.5，157.8，155.4，145.9，142.0，131.5(2)，129.9(2)，123.4(2)，121.1，121.0(2)，119.5，103.5，98.5，97.1，71.2，65.0(2)，56.4，56.1，52.0(2)，16.3(2)。HRMS(ESI)+Calculated for C30H33N4O4，[M+H]+：m/z 545.2400，found 545.2400。
Compound 20, mp 264-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.87(1H，s)，9.91(1H，s)，7.92(2H，s)，7.34(1H，s)，7.15-7.23(2H，m)，6.74(1H，d，J＝2.4Hz)，6.69(1H，dd，J＝8.4，2.4Hz)，6.51(1H，d，J＝2.8Hz)，4.47(2H，s)，3.88(3H，s)，3.84(3H，s)，3.73(4H，t，J＝4.8Hz)，3.08(4H，t，J＝4.8Hz)，2.35(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：167.7，166.4，164.3，162.5，161.0，157.7，152.4，151.5，139.1，130.8(2)，129.1，128.4(2)，127.9，110.9(2)，106.，104.6，101.2，97.6，71.0，66.1(2)，56.0，55.7，48.5(2)，16.2(2)。HRMS(ESI)+Calculated for C30H32NaN4O6，[M+Na]+：m/z 567.2220，found 567.2211。
Compound 21, mp 242-244 ℃ and yield 49.7%.1H-NMR(400MHz，DMSO-d6+CF3COOD)，δ(ppm)：10.34(1H，s)，8.39(2H，s)，7.87(2H，d，J＝8.4Hz)，7.62(2H，d，J＝8.4Hz)，6.80(1H，d，J＝2.0Hz)，6.62(1H，d，J＝2.0Hz)，4.74(2H，s)，3.95(4H，t，J＝4.8Hz)，3.86(6H，s)，3.58(4H，t，J＝4.8Hz)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.6，166.4，162.6，156.9，156.8，153.8，139.9，138.6，134.6(2)，122.5(2)，122.2(2)，118.6(2)，116.8，115.0，104.5，99.8，98.4，72.4，65.0(2)，57.0，56.6，55.4(2)。HRMS(ESI)+Calculated for C28H27Br2N4O6，[M+H]+：m/z 675.0277，found 675.0275。
Compound 22, mp 276-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.92(1H，s)，9.93(1H，s)，7.50(2H，d，J＝8.8Hz)，7.12(2H，d，J＝8.8Hz)，6.91(2H，d，J＝8.8Hz)，6.71(1H，d，J＝2.4Hz)，6.50(1H，d，J＝2.4Hz)，4.76(2H，s)，3.88(3H，s)，3.84(3H，s)，3.72(4H，t，J＝4.8Hz)，3.05(4H，t，J＝4.8Hz)；13C-NMR(100MHz，CDCl3+CF3COOD)，δ(ppm)：169.0，163.2，162.0，160.0，156.4，137.5，131.3，131.0，123.4，123.3，121.8，121.5，119.0，117.0，116.9，116.1，116.0，113.3，101.0，98.5，94.8，67.0，64.5，64.2，56.9，56.3，56.1(2)。HRMS(ESI)+Calculated for C28H29N4O4，[M+H]+：m/z 517.2087，found 517.2086。
The compound 23, mp 272-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.02(1H，s)，8.16(2H，d，J＝8.4Hz)，7.28(1H，t，J＝2.4Hz)，7.09-7.16(4H，m)，6.72(1H，d，J＝2.4Hz)，6.68(1H，dd，J＝8.4，2.4Hz)，6.51(1H，d，J＝2.4Hz)，4.79(2H，s)，3.88(3H，s)，3.84(3H，s)，3.72(4H，t，J＝4.8Hz)，3.06(4H，t，J＝4.8Hz)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.1(2)，164.3，161.0，160.5，152.9，152.5，151.5，139.2，129.4(2)，129.2，125.0，114.6(2)，110.9，110.7，106.4，104.5，100.9，97.5，67.1，66.1(2)，56.0，55.6，48.5(2)，。HRMS(ESI)+Calculated for C28H29N4O6，[M+H]+：m/z 517.2087，found 517.2053。
Compound 24, mp 265-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.87(1H，s)，9.58(1H，s)，7.92(2H，s)，7.53(2H，d，J＝8.8Hz)，6.89(2H，d，J＝8.8Hz)，6.74(1H，s)，6.52(1H，s)，4.45(2H，s)，3.89(3H，s)，3.84(3H，s)，3.06-3.08(4H，m)，2.35(6H，s)，1.58-1.63(4H，m)，1.51-1.52(2H，m)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：165.8，164.2，160.9，157.8，153.1，152.4，148.4，130.8(2)，129.9，128.7(2)，127.9，121.1(2)，116.1(2)，104.7，101.2，97.6，71.1，56.0，55.6，50.0(2)，35.8，25.3(2)，23.9，16.2(2)。HRMS(ESI)+Calculated for C31H35N4O5，[M+H]+：m/z 543.2607，found 535.2607。
Compound 25, mp 271-273 ℃, yield 62.0%.1H-NMR(400MHz，DMSO-d6+CF3COOD)，δ(ppm)：10.48(1H，s)，8.52(2H，s)，7.94(2H，d，J＝8.8Hz)，7.73(2H，d，J＝8.8Hz)，6.85(1H，d，J＝2.4Hz)，6.65(1H，d，J＝2.4Hz)，4.78(2H，s)，3.93(3H，s)，3.91(3H，s)，3.58-3.61(4H，m)，1.94-1.97(4H，m)，1.70(2H，s)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.0，165.4，165.3，161.7，155.2，151.8，142.1，139.8，138.2，133.2(2)，122.4(2)，121.5，121.4(2)，118.0(2)，104.6，99.8，98.9，71.7，56.8(2)，56.5，56.1，23.8(2)，21.0。HRMS(ESI)+Calculated for C29H29Br2N4O5，[M+H]+：m/z 673.0484，found 673.0490。
Compound 26, mp 253-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.93(1H，s)，9.89(1H，s)，8.17(2H，d，J＝8.8Hz)，7.45(1H，d，J＝8.8Hz)，7.12(2H，d，J＝8.8Hz)，6.88(1H，d，J＝8.8Hz)，6.71(1H，d，J＝2.4Hz)，6.51(1H，d，J＝2.4Hz)，4.75(2H，s)，3.88(3H，s)，3.84(3H，s)，3.04-3.07(4H，m)，1.57-1.62(4H，m)，1.50-1.51(2H，m)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.6，165.8，162.8，161.9，157.6，156.4，144.2，139.7，137.9，131.7(2)，122.3(2)，120.9，120.8(2)，119.2，115.3(2)，103.1，98.7，96.1，67.2，56.5(2)，56.2，23.6(2)，20.8。HRMS(ESI)+Calculated for C29H31N4O5，[M+H]+：m/z 515.2294，found 515.2300。
Compound 27, mp 243-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：9.80(1H，s)，8.16(2H，d，J＝9.2Hz)，7.42(2H，d，J＝9.2Hz)，7.14(2H，d，J＝8.8Hz)，6.72(1H，d，J＝2.4Hz)，6.50-6.53(2H，m)，4.74(2H，s)，3.89(3H，s)，3.85(3H，s)，3.17-3.19(4H，m)，1.92-1.95(4H，m)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.2，163.2，162.1(2)，157.3，156.9，143.3，141.4，132.0(2)，121.1(2)，119.9，119.8(2)，118.6，115.5(2)，103.0，98.9，95.6，67.4，56.8，56.3，55.3(2)，23.9(2)。HRMS(ESI)+Calculated for C28H29N4O5，[M+H]+：m/z 501.2138，found 501.2133。
Compound 28, mp 258-260 ℃, yield 55.9%.1H-NMR(400MHz，DMSO-d6+CF3COOD)，δ(ppm)：7.80(1H，dd，J＝14.0，2.4Hz)，7.68(2H，s)，7.47(1H，dd，J＝9.2，2.4Hz)，7.36(1H，t，J＝9.2Hz)，6.83(1H，d，J＝2.0Hz)，6.55(1H，d，J＝2.0Hz)，4.46(2H，s)，3.82-3.85(4H，m)，3.81(3H，s)，3.78(3H，s)，3.34-3.36(4H，m)，2.30(6H，s)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：169.2，168.8，164.3，162.6，159.3，158.9，155.8(250.2Hz)，143.9，134.7(2)，130.6(2)，132.6(2)，126.4，126.2，124.6，122.9，104.8，101.2，97.0，72.7，65.9，65.5，58.0，57.7，56.2(2)，17.7(2)。HRMS(ESI)+Calculated for C30H32FN4O6，[M+Na]+：m/z 585.2125，found 585.2122。
Compound 29, mp 236-238 ℃, yield 58.0%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.1(1H，s)，8.15(2H，d，J＝8.8Hz)，7.56(1H，dd，J＝14.8，2.4Hz)，7.32(1H，dd，J＝14.8，2.4Hz)，7.14(2H，d，J＝14.8Hz)，7.02(1H，t，J＝9.6Hz)，6.73(1H，d，J＝2.4Hz)，6.53(1H，d，J＝2.4Hz)，4.80(2H，s)，3.89(3H，s)，3.85(3H，s)，3.71-3.74(4H，m)，2.94-2.96(4H，m)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.1，164.5，161.1，160.7，159.6，155.6，153.2，153.0，151.8，135.7(40.6Hz)，133.5(36.0Hz)，129.7(2)，124.3，119.2，115.8，114.7，108.2(100.8Hz)，104.4，100.3，97.6，67.1，66.2(2)，56.1，55.7，50.8(2)。HRMS(ESI)+Calculated for C28H28FN4O6，[M+H]+：m/z 535.1993，found 535.1992。
Compound 30, mp 245-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：11.94(1H，s)，10.16(1H，s)，8.17(2H，d，J＝8.8Hz)，7.55(1H，dd，J＝14.8，2.4Hz)，7.30(2H，dd，J＝8.8，2.4Hz)，7.13(1H，d，J＝2.4Hz)，7.00(1H，t，J＝8.8Hz)，6.71(1H，d，J＝2.4Hz)，6.51(1H，d，J＝2.4Hz)，4.79(2H，s)，3.88(3H，s)，3.84(3H，s)，2.96(4H，t，J＝4.8Hz)，2.46(4H，brs)，2.22(3H，s)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.2，166.1，163.3，162.8，162.2，157.4，157.0，156.2，153.7，143.2，134.7(42.8Hz)，134.5(36.8Hz)，120.2，118.6，116.0，115.5(2)，108.5(100.8Hz)，103.1，98.9，95.5，67.4，56.8，56.7，56.4，56.3，53.1(2)，47.9。HRMS(ESI)+Calculated for C29H31FN5O5，[M+H]+：m/z 548.2309，found 548.2311。
Compound 31, mp 253-.1H-NMR(400MHz，CDCl3)，δ(ppm)：8.60(1H，s)，7.85(2H，s)，7.58(1H，dd，J＝14.0，2.4Hz)，7.26-7.27(1H，m)，6.94(1H，d，J＝8.8Hz)，6.81(1H，s)，6.44(1H，s)，4.40(2H，s)，3.92(6H，s)，3.10(4H，t，J＝4.4Hz)，2.61(4H，t，J＝4.4Hz)，2.36(9H，s)；13C-NMR(100MHz，CDCl3)，δ(ppm)：166.1，165.2，161.4，157.0，156.7，154.2，153.9，152.3，137.3(36.4Hz)，131.9(36.8Hz)，131.3(2)，129.1，128.5(2)，119.1，115.9，109.2(56.4Hz)，105.0，101.4，98.4，70.5，56.3，55.8，55.2(2)，50.7(2)，46.2，16.5(2)。HRMS(ESI)+Calculated for C31H35FN5O5，[M+H]+：m/z 576.2622，found 576.2624。
Compound 32, mp 273-276 ℃ and yield 62.9%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.29(1H，s)，8.17(2H，d，J＝8.4Hz)，7.85(1H，s)，7.55(1H，d，J＝8.8Hz)，7.21(1H，d，J＝8.8Hz)，7.12(2H，d，J＝8.8Hz)，6.71(1H，s)，6.50(1H，s)，4.81(2H，s)，3.88(3H，s)，3.84(3H，s)，3.30-3.37(8H，m)，2.86(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.3，164.2，160.9，160.3，159.8，153.1，152.3，143.1，135.1，129.3(2)，127.5，125.1，121.3(2)，119.3，114.6(2)，104.5，101.1，97.4，67.0，55.9，55.6，52.9(2)，48.1(2)，42.2。HRMS(ESI)+Calculated for C29H31ClN5O5，[M+H]+：m/z 564.2014，found 564.2011。
Compound 33, mp 260-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.22(1H，s)，7.93(2H，s)，7.91(1H，d，J＝2.4Hz)，7.61(1H，dd，J＝8.8，2.4Hz)，7.14(1H，d，J＝8.8Hz)，6.73(1H，d，J＝2.4Hz)，6.50(1H，d，J＝2.4Hz)，4.49(2H，s)，3.88(3H，s)，3.84(3H，s)，3.17(4H，brs)，2.93(4H，brs)，2.35(6H，s)，2.23(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.6，164.1，160.9，157.6，153.3，153.0，145.0，134.3，130.7(2)，128.4(2)，127.4，121.7，120.8，119.6，104.8，101.0，97.4，79.2，71.0，56.0，55.6，54.9(2)，51.1，48.6，45.8，16.3(2)。HRMS(ESI)+Calculated for C31H35ClN5O5，[M+H]+：m/z 614.2148，found 614.2174。
Compound 34, mp 268-271 ℃, yield 47.6%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：12.17(1H，s)，10.21(1H，s)，8.52(2H，s)，7.88(1H，d，J＝2.4Hz)，7.56(1H，dd，J＝8.8，2.4Hz)，7.16(1H，d，J＝8.8Hz)，6.81(1H，s)，6.56(1H，s)，4.67(2H，s)，3.89(3H，s)，3.85(3H，s)，3.74(4H，t，J＝4.4Hz)，2.94(4H，t，J＝4.0Hz)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：165.1，164.4，160.9，154.0，152.4，149.8，144.8，134.3，132.0(2)，131.3，127.4，122.8，121.7，120.9，119.7，117.7(2)，104.8，101.5，98.2，71.3，66.4(2)，56.1，55.7，51.5(2)。HRMS(ESI)+Calculated for C28H25Br2ClNaN4O6，[M+Na]+：m/z 730.9707，found 730.9714。
Compound 35, mp 247-249 ℃ yield 56.0%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.20(1H，s)，8.15(2H，d，J＝8.8Hz)，7.52(1H，dd，J＝8.8，2.4Hz)，7.13-7.16(3H，m)，6.73(1H，d，J＝2.4Hz)，6.54(1H，d，J＝2.4Hz)，4.81(2H，s)，3.89(3H，s)，3.85(3H，s)，3.73(4H，t，J＝4.4Hz)，2.92(4H，t，J＝4.0Hz)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.1，164.4，161.1，160.6，159.5，153.0，151.8，144.6，134.4，129.7(2)，127.5，124.3，121.4，120.9，119.3，114.7(2)，104.3，100.3，97.6，67.0，66.4(2)，56.0，55.7，51.5(2)。HRMS(ESI)+Calculated for C28H27ClNaN4O6，[M+Na]+：m/z 573.1517，found 573.1533。
Compound 36, mp 258-261 ℃ and yield 51.5%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.17(1H，s)，7.90(3H，s)，7.62(2H，dd，J＝8.8，2.4Hz)，7.15(1H，d，J＝8.8Hz)，6.75(1H，d，J＝2.4Hz)，6.54(1H，d，J＝2.4Hz)，4.49(2H，s)，3.88(3H，s)，3.85(3H，s)，3.73(4H，t，J＝4.4Hz)，2.93(4H，t，J＝4.0Hz)，2.35(6H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：165.5，164.4，161.0，159.5，157.9，152.9，151.9，144.7，134.4，130.9(2)，128.6(2)，127.4，127.2，121.7，120.8，119.6，104.5，100.5，97.7，71.0，66.4(2)，56.0，55.7，51.5(2)，16.2(2)。HRMS(ESI)+Calculated for C30H32ClN4O6，[M+H]+：m/z 579.2010，found 579.2018。
Compound 37, mp 255-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.28(1H，s)，8.18(2H，d，J＝8.8Hz)，7.87(1H，d，J＝2.4Hz)，7.76-7.80(2H，m)，7.70(1H，td，J＝8.8，2.4Hz)，7.56(1H，dd，J＝8.8，2.4Hz)，7.22(1H，d，J＝8.8Hz)，7.15(1H，d，J＝8.8Hz)，3.52(2H，d，J＝11.2Hz)，3.38(2H，d，J＝11.2Hz)，3.19-3.22(2H，m)，3.29(2H，t，J＝13.2Hz)，2.89(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.3，161.8，161.0，158.6，151.4，145.7，143.1，135.1，130.0(32Hz)，129.4(2)，127.5，125.3，123.1(97.2Hz)，121.9(33.2Hz)，121.3(2)，119.3，114.7(2)，110.6(93.2Hz)，67.0，52.8(2)，48.1(2)，42.5。HRMS(ESI)+Calculated for C27H25ClFNaN5O3，[M+Na]+：m/z 544.1528，found 544.1526。
Compound 38, mp 243-245 ℃ and yield 47.7%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：10.28(1H，s)，8.03(2H，d，J＝8.8Hz)，7.85(1H，d，J＝2.4Hz)，7.80(1H，d，J＝8.8Hz)，7.60(1H，d，J＝2.4Hz)，7.51-7.56(2H，m)，7.28(2H，d，J＝8.8Hz)，7.17(1H，d，J＝8.8Hz)，4.88(2H，s)，3.90(3H，s)，3.52(2H，d，J＝12.0Hz)，3.36(2H，d，J＝12.0Hz)，3.21(2H，td，J＝12.0，2.8Hz)，2.97(2H，td，J＝12.0，2.8Hz)，2.87(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.4，162.1，160.0(2)，157.5，149.7，143.1，135.1，129.1(2)，127.5，125.7，124.2，124.1，121.5.121.4，121.3，119.3，114.7(2)，105.9，67.0，55.7，52.9(2)，48.1(2)，42.2。HRMS(ESI)+Calculated for C28H29ClN5O4，[M+H]+：m/z 534.1908，found 534.1899。
Compound 39, mp 281-283 ℃ and yield 54.5 percent.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：12.60(1H，s)，10.30(1H，s)，10.05(1H，s)，8.20(2H，d，J＝8.8Hz)，8.06(1H，d，J＝2.4Hz)，7.86(1H，d，J＝2.4Hz)，7.83(1H，dd，J＝8.8，2.4Hz)，7.72(1H，d，J＝8.8Hz)，7.55(1H，dd，J＝8.8，2.4Hz)，7.22(1H，d，J＝8.8Hz)，7.15(2H，d，J＝8.8Hz)，4.83(2H，s)，3.52-3.55(2H，m)，3.38(2H，d，J＝12.0Hz)，3.18(2H，td，J＝12.0，2.8Hz)，2.99(2H，td，J＝12.0，2.8Hz)，2.89(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.3，161.4，160.5，152.3，147.6，143.1，135.1，134.6，130.3，129.6(2)，129.5，127.5，125.2，124.9，121.9，121.3(2)，119.3，114.7(2)，67.0，52.9(2)，48.1(2)，42.2。HRMS(ESI)+Calculated for C27H26Cl2N5O3，[M+H]+：m/z 538.1413，found 538.1408。
Compound 40, mp 280-.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：12.51(1H，s)，10.18(1H，s)，8.19(2H，d，J＝8.8Hz)，8.12(1H，d，J＝8.8Hz)，7.80(1H，d，J＝2.4Hz)，7.45(1H，d，J＝2.4Hz)，7.51(2H，dd，J＝8.8，2.4Hz)，7.13-7.17(3H，m)，4.81(2H，s)，3.33(4H，brs)，2.91-2.93(4H，m)，2.25(3H，s)；13C-NMR(100MHz，DMSO-d6+CF3COOD)，δ(ppm)：166.3，161.6，160.9，153.6，149.4，143.2，139.4，135.2，129.9(2)，128.1，127.7，126.7，125.9，124.7，121.5，121.4，119.6，119.5，114.9(2)，67.1，53.0(2)，48.2(2)，42.2。HRMS(ESI)+Calculated for C27H26Cl2N5O3，[M+H]+：m/z 538.1413，found 538.1415。
Compound 41, mp 279-282 ℃ and yield 55.5%.1H-NMR(400MHz，DMSO-d6)，δ(ppm)：12.33(1H，s)，10.27(1H，s)，9.91(1H，s)，8.17(1H，d，J＝8.8Hz)，8.01(1H，d，J＝8.4Hz)，7.93(2H，s)，7.85(1H，d，J＝2.4Hz)，7.56(1H，dd，J＝8.8，2.4Hz)，7.52(1H，s)，7.31(1H，dd，J＝8.4，2.4Hz)，7.22(1H，d，J＝8.8Hz)，7.14(1H，d，J＝8.8Hz)，4.82(2H，s)，3.52(2H，d，J＝11.6Hz)，3.38(2H，d，J＝11.6Hz)，3.20(2H，t，J＝12.0Hz)，2.97(2H，t，J＝12.0Hz)，2.88(3H，s)，2.46(3H，s)；13C-NMR(100MHz，DMSO-d6)，δ(ppm)：166.3，162.2，160.3，151.9，148.8，145.0，143.1，135.1，129.4(2)，127.7，127.5，126.8，125.7，125.5，121.3(2)，119.3，118.3，114.7(2)，67.0，52.9(2)，48.1(2)，42.7，21.4。HRMS(ESI)+Calculated for C28H29ClN5O3，[M+H]+：m/z 518.1959，found 518.1953。
Test example 1 Compounds 1-41 were tested for inhibitory activity against BRD4 and CK2 and antiproliferative activity against MDA-MB-231 and MDA-MB-468 cells at a concentration of 1. mu.M.
Figure BDA0003251074340000151
aCompounds were tested in triplicate. Data are presented as mean ± SD.
bIC50Values were determined by cell viability for 24 hours.
Experimental results show that the compounds of the invention have inhibitory activity on BRD4 and CK2 and inhibitory activity on triple negative breast cancer cell proliferation, wherein the compounds 31, 32, 33, 34 and 35 have good effects, and the optimal compound 32(44e) has stronger inhibitory activity on BRD4 and CK2 and triple negative breast cancer cell inhibitory activity.
Test example 2 Compound 32(44e) induces MDA-MB-231 autophagy in vitro, promoting cell death and apoptosis
The MDA-MB-231 cells were transfected with GFP-mRFP-LC3 adenovirus, and the effect of 44e on the cells was observed by inverted microscopy. It was clearly observed that compound 44e induced the autophagy flux of MDA-MB-231 cells, suggesting that it was able to induce autophagy in MDA-MB-231 cells (FIG. 1A).
The results of the clonogenic experiments show that 44e significantly inhibited the growth and clonogenic activity of MDA-MB-231 cells in a concentration-dependent manner at concentrations of 0. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M (FIG. 1B).
FIG. 1C MDA-MB-231 cells treated with different concentrations of compound 44e, and after 24h of compound and cell action, both cells were stained with Hoechst 33258 fluorescent dye, and a significant increase in the number of cell deaths was observed in a dose-dependent manner (FIG. 1C).
Compound 44e was able to induce apoptosis of MDA-MB-231 cells in a concentration-dependent manner at 0 μ M and 5 μ M concentrations using flow cytometry to determine the apoptosis rate of the cells (fig. 1D).
Test example 3 evaluation of Effect of Compound 32(44e) on in vivo treatment of triple negative Breast cancer
MDA-MB-468 and MDA-MB-231 transplanted tumor models were established and randomly divided into 4 groups of 6 tumors, and an Intragastric administration (Intragastric administration) mode was performed for 19 consecutive days. Both doses of 44e and the drug combination group showed dose-dependent Tumor Growth Inhibition (TGI) effects at the tested doses (fig. 2A, 2B). Particularly, in an MDA-MB-231 transplantation tumor model, when the application dose is 50mg/kg 44e, the inhibition activity is strongest, and the growth inhibition rate is 63.8%.

Claims (7)

1. A dual-targeting BRD4-CK2 small molecule inhibitor, which is characterized in that: a compound of formula I or formula II:
Figure FDA0003251074330000011
wherein R is1Is 3,5-diCH3H or 3, 5-diBr; r2Is 4-F, 4-OCH34-Cl, 5-Cl or 5-CH3；R3Is composed of
Figure FDA0003251074330000012
Figure FDA0003251074330000013
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the structural formula is shown as formula I.
3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein:
R1is 3,5-diCH3Or H; r3Is composed of
Figure FDA0003251074330000014
Figure FDA0003251074330000015
Preferably R1Is H; r3Is composed of
Figure FDA0003251074330000016
Figure FDA0003251074330000017
More preferably R1Is H; r3Is composed of
Figure FDA0003251074330000018
4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the compound of formula I is as follows:
Figure FDA0003251074330000021
5. use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of triple negative breast cancer.
6. Use according to claim 5, characterized in that: the triple negative breast cancer treatment drug is an anti-tumor drug; preferably, the anti-tumor drug is a dual-targeting BRD4-CK2 inhibitor drug.
7. A pharmaceutical composition characterized by: a formulation comprising an effective amount of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.
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