Coumarin derivatives with anti-breast cancer activity and use thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a coumarin derivative with anti-breast cancer activity and application thereof. The chemical structural formula of the coumarin derivative is shown in formula (I) or formula (II), wherein R1 is an electron-withdrawing or electron-donating group, and R2 is a saturated nitrogen heterocycle or an aromatic nitrogen heterocycle. The coumarin analog prepared by the application has a certain inhibitory effect on breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a preparation method of a coumarin analogue and an application thereof in the preparation of an anti-breast cancer drug. Background Art

[0002] Breast cancer (BC) is the most common and deadly type of cancer in women worldwide. Breast cancer can be divided into four subtypes based on three immunophenotypes: progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor-2 (HER2): luminal A (PR + , ER + , HER2 - ), luminal B type (PR - , ER + , HER2 + ), HER-2 type (PR - , ER - , HER2 + ) and triple negative (PR - , ER - , HER2 - ). Among them, triple-negative breast cancer (TNBC) has the characteristics of high heterogeneity, easy recurrence and easy metastasis, making it the most malignant subtype. According to the latest clinical studies, the proportion of TNBC patients accounts for about 15-20% of the total number of breast cancer patients. As of now, chemotherapy is still the most important treatment for TNBC. Anthracyclines and taxanes occupy an important position in the treatment of TNBC, but the efficacy of these two types of drugs in TNBC is not better than that of "non-triple-negative" breast cancer. In addition, studies have found that some BC patients (especially TNBC patients) are prone to drug resistance to certain conventional chemotherapy drugs, resulting in poor clinical treatment effects and poor prognosis. Therefore, the exploration of novel and effective potential anti-BC drug molecules remains an important scientific problem that needs to be solved urgently.

[0003] Coumarins are natural compounds containing benzo-α-pyrone structural units and possess a variety of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, and antiviral activities. Given their advantages, such as low toxicity, high bioavailability, and amenable structural modification, numerous recent reports have demonstrated the inhibitory effects of coumarin analogs on gastric, colon, ovarian, non-small cell lung, breast, and malignant melanoma cancers. These findings suggest that the coumarin scaffold can serve as an effective template for the search and discovery of novel anticancer lead drugs. While most coumarin compounds have been modified at positions C-3, C-4, and C-7, only a few studies have examined structural derivatization at the C-6 position, and none have investigated their inhibitory effects on breast cancer cells.

[0004] The present invention takes the coumarin skeleton as the research object, designs and synthesizes a series of novel coumarin analogs, and discovers a lead molecule with anti-breast cancer (BC) activity. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for preparing novel coumarin analogs to further enrich the structural types of coumarin compounds; another purpose is to provide uses of the coumarin analogs, including the use of the analogs in breast cancer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The general chemical structures of coumarin derivatives are shown in Formula (I) and Formula (II):

[0008]

[0009] Wherein, R1 is fluorine, chlorine, bromine, methyl, methoxy, tert-butyl, nitro, trifluoromethyl, trifluoromethoxy, 1,3-dimethoxy-5-vinylbenzene;

[0010] R2 is one of the following structural formulas:

[0011]

[0012] The synthesis route is shown below:

[0013]

[0014] The preparation method of coumarin derivatives comprises the following steps:

[0015] (1) Synthesis of 6-methylcoumarin: 5-methylsalicylaldehyde and sodium acetate were placed in a round-bottom flask, degassed with Ar for three times, and N,N-dimethylformamide solution was added. The mixture was stirred at room temperature for 30 min, and acetic anhydride was added dropwise in small amounts. The temperature was raised to 170°C and the stirring was continued for 6 h. The mixture was detected by TLC. After the raw materials were completely removed, the reaction system was cooled to room temperature and washed with dichloromethane (3×10 mL). The organic layer was collected and washed with distilled water and saturated brine, respectively. The organic layer was dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to obtain the target compound 2. The molar ratio of 5-methylsalicylaldehyde, sodium acetate, and acetic anhydride was 1:3:2.5.

[0016] (2) Synthesis of 6-bromomethylcoumarin:

[0017] 6-Methylcoumarin (target compound 2) was weighed and placed in a round-bottom flask. The mixture was degassed three times with Ar, and a carbon tetrachloride solution was added. The temperature was raised to 85°C. Simultaneously, an acetonitrile solution containing N-bromosuccinimide and azobisisobutyronitrile was slowly added dropwise and allowed to react overnight. After TLC was performed, the reaction system was cooled to room temperature after the reaction of the raw materials was complete. The mixture was extracted with dichloromethane (3×10 mL), and the DCM layer was collected and dried over anhydrous Na2SO4. The crude product was recrystallized from n-hexane and ethyl acetate (1:1), filtered, and the filter cake was vacuum dried to obtain target compound 3. The molar ratio of 6-methylcoumarin, N-bromosuccinimide, and azobisisobutyronitrile was 1:1:0.2.

[0018] (3) Synthesis of compounds 4a-4k:

[0019] The phenol derivative and K2CO3 were dissolved in acetonitrile and stirred at room temperature for 1 hour. 6-Bromomethylcoumarin (target compound 3) was slowly added dropwise while the reaction system was heated to 85°C. After overnight reaction, TLC confirmed the reaction was complete. The mixture was filtered, the combined filtrates were spin-dried to dryness, and then extracted with DCM (3 × 10 mL), washed with distilled water and saturated brine, and the organic layer was dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to yield target compounds 4a-4k. The molar ratio of 6-bromomethylcoumarin, phenol derivative, and potassium carbonate was 1:1.5:2.

[0020] (4) Synthesis of compounds 5a-5u:

[0021] Synthesis of Compounds 5a-5l: An appropriate amount of a nitrogen heterocyclic compound and potassium carbonate were dissolved in acetonitrile and stirred at room temperature for 1 hour. 6-Bromomethylcoumarin (target compound 3) was then slowly added dropwise while the reaction system was heated to 85°C. The reaction was continued for 12 hours. Complete reaction was confirmed by TLC. The filtrate was filtered and dried, extracted with dichloromethane (3 × 10 mL), washed with distilled water and saturated sodium chloride, and dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) to afford target compounds 5a-5l. The molar ratio of 6-Bromomethylcoumarin, indole derivative, and sodium hydride was 0.9:1:1.1.

[0022] Synthesis of Compounds 5m-5u: Weigh sodium hydride into a Shrek tube and add N,N-dimethylformamide solution. Slowly add the indole compound dissolved in DMF dropwise at 0°C. Stir at room temperature for 30 minutes, then add 6-bromomethylcoumarin (target compound 3) dissolved in DMF dropwise. Continue stirring overnight until the reaction is complete. Extract with ethyl acetate (3 x 10 mL), wash with distilled water and saturated brine, combine the EtOAc layers, and dry over anhydrous Na₂SO₄. The crude product is purified by flash chromatography (petroleum ether and ethyl acetate as eluants) and concentrated under reduced pressure to yield target compounds 5m-5u. The molar ratio of 6-bromomethylcoumarin, nitrogen heterocyclic compound, and potassium carbonate is 1:1.5:2.

[0023] Application of coumarin derivatives in the preparation of drugs for preventing or treating breast cancer.

[0024] Further application of coumarin derivatives in the preparation of drugs for preventing or treating triple-negative breast cancer.

[0025] As a preferred embodiment: the structural formula of coumarin derivatives is

[0026] This study, based on coumarin as the basic skeleton, investigated derivatization at the C-6 position. Compounds 5e and 5f are the first reported derivatives with novel structures and high selectivity for triple-negative breast cancer cells (MDA-MB-231), providing important insights for the subsequent development of lead molecules against TNBC. Furthermore, this study tested for the first time the inhibitory effect of the known compound 6-bromomethylcoumarin on breast cancer cells (MDA-MB-231 and MCF-7). 6-Bromomethylcoumarin exhibited moderate inhibitory effects against both breast cancer cell lines, but its selectivity for MDA-MB-231 cells was inferior to that of 5e and 5f, providing valuable insights into its potential use in anti-breast cancer drugs.

[0027] The coumarin derivatives synthesized by the invention have good solubility in most organic solvents such as ethyl acetate, dichloromethane, tetrahydrofuran, chloroform, methanol and dimethyl sulfoxide.

[0028] The present invention achieves the following beneficial effects: first, it provides novel coumarin analogs with significant inhibitory effects on breast cancer cells (MDA-MB-231 and MCF-7), with high sensitivity to MDA-MB-231 cells, demonstrating the potential of coumarin compounds as lead anti-TNBC drugs. Second, it provides the chemical structures and corresponding synthetic routes of these analogs, providing useful guidance for the subsequent construction of coumarin backbone analogs. DETAILED DESCRIPTION

[0029] The present invention is described in detail below by means of specific examples. It should be understood that the following examples are for explanation and illustration only and do not limit the scope of the present invention in any form. In the following embodiments, biochemical reagents not otherwise specified are conventional reagents in the art and can be prepared according to conventional methods in the art or commercially available, and the specifications are laboratory pure grade.

[0030] Preparation of compounds 4a-4k

[0031]

[0032] Preparation of compound 5a-Su

[0033]

[0034] The preparation steps of 6-bromomethylcoumarin are as follows:

[0035] (1) 1.00 g (7.34 mM) of 5-methylsalicylaldehyde and 1.51 g (18.36 mM) of sodium acetate were weighed and placed in a round-bottom flask. The mixture was degassed three times with Ar. 15 mL of DMF solution was added and stirred at room temperature for 30 min. The system was heated to 170°C and 1.39 mL of acetic anhydride solution (22.02 mM) was slowly added dropwise. Stirring was continued for 6 h. After TLC was performed, the reaction system was cooled to room temperature and extracted with dichloromethane (3 × 10 mL). The mixture was then washed with distilled water and saturated NaCl solution and dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to obtain the target compound 2.

[0036] (2) Weigh 1.00 g (6.24 mM) of 6-methylcoumarin into a 50 mL round-bottom flask, degas with Ar three times, add 15 mL of CCl4 solution, raise the reaction temperature to 85°C, slowly add 1.11 g (6.24 mM) of N-bromosuccinimide and 0.21 g (1.25 mM) of azobisisobutyronitrile dissolved in acetonitrile (15 mL), and continue stirring for 6 h. Extract with dichloromethane (3 × 10 mL), wash with distilled water and saturated brine, combine the organic layers, and dry over anhydrous Na2SO4. The crude product is recrystallized with n-hexane and EtOAc (1:1), filtered, and the filter cake is vacuum-dried to obtain the target compound 3, i.e., 6-bromomethylcoumarin.

[0037] Example 1

[0038] 4a:

[0039] Pterostilbene (CAS No. 537-42-8) (0.44 mM) and potassium carbonate (0.58 mM) were weighed into a 20 mL Shrek tube. 1 mL of acetonitrile solution was added and stirred at room temperature for 1 hour. 6-Bromomethylcoumarin (0.29 mM) dissolved in acetonitrile was slowly added dropwise. The mixture was refluxed at 85°C for 12 hours, and the reaction system was cooled to room temperature. Subsequently, the mixture was extracted with dichloromethane (3 × 10 mL), washed with distilled water and saturated brine, and the DCM layer was dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to obtain the desired product 4a in a 96.2% yield.

[0040] 4a: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.71 (d, J = 9.6Hz, 1H), 7.61-7.55 (m, 2H), 7.45 (d, J = 8.8Hz, 2H), 7.36 (d, J = 8.4Hz, 1H), 7.03 (d, J = 16Hz, 1H), 6.96(d,J=8.8Hz,2H), 6.91(d,J=16Hz,1H), 6.65(d,J=2.4Hz,2H), 6.45(d,J=9.6Hz,1H), 6.38(t,J=2.2Hz,1H), 5.11(s,2H), 3.83(s,6H). 13 C NMR (75MHz, CDCl3): δ (ppm) 161.0 (2C), 160.7, 158.1, 153.7, 143.3, 139.5, 133.4, 131.0, 130.6, 12 8.5,127.9(2C),127.0,126.7,118.9,117.2,117.1,115.0(2C),104.4(2C),99.7,69.0,55.4(2C).

[0041] Example 2

[0042] 4b:

[0043] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-nitrophenol to obtain the target product 4b with a yield of 85%.

[0044] 4b: 1H NMR (400MHz, DMSO-d6): δ (ppm) 8.22 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 9.6 Hz, 1H), 7.84 (brs, 1H), 7.72 (d,J=8Hz,1H),7.44(d,J=8.4Hz,1H),7.24(d,J=9.2Hz,2H),6.52(d,J=9.6Hz,1H),5.32(s,2H). 13 C NMR (101MHz, DMSO-d6): δ (ppm) 163.5, 159.9, 153.3, 144.1, 141.1, 132.3, 131.7, 127.9, 125.9 (2C), 124.2, 118.8, 116.6, 115.4 (2C), 69.3.

[0045] Example 3

[0046] 4c:

[0047] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-tert-butylphenol to obtain the target product 4c with a yield of 90.7%.

[0048] 4c: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.72 (d, J = 9.6Hz, 1H), 7.60 (d, J = 2.4Hz, 1H), 7.57 (brs, 1H), 7.35 (d, J =8.0Hz,1H),7.34-7.31(m,2H),6.93-6.89(m,2H),6.44(d,J=9.6Hz,1H),5.09(s,2H),1.30(s,9H). 13 C NMR (75MHz, CDCl3) δ (ppm) 160.8, 156.2, 153.6, 144.1, 143.4, 133.8, 131.0, 126.7, 126.4 (2C), 118.9, 117.2, 117.0, 114.2 (2C), 69.0, 34.2, 31.5.

[0049] Example 4

[0050] 4d:

[0051] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with 3,4-difluorophenol to obtain the target product 4d with a yield of 96.9%.

[0052] 4d: 1H NMR (400MHz, DMSO-d6): δ (ppm) 8.35 (d, J = 2.8Hz, 1H), 8.26 (m, 1H), 8.11 (d, J = 9.6Hz, 1H), 7.84 (d, J=2.0Hz,1H),7.74(m,1H),7.50(d,J=9.2Hz,1H),7.47(s,1H),6.53(d,J=9.2Hz,1H),5.44(s,2H). 13 C NMR (101MHz, DMSO-d6): δ (ppm) 159.9, 158.8, 153.4, 144.1, 140.9, 131.9, 131.6, 127.8, 125.5, 124.6, 122.1, 118.8, 116.8, 116.7, 113.9, 70.3. 19 F NMR (376MHz, DMSO-d6): δ (ppm) -170.8 (brs), -187.4 (brs).

[0053] Example 5

[0054] 4e:

[0055] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-methoxyphenol to obtain the target product 4e with a yield of 92.1%.

[0056] 4e: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.70 (d, J = 9.6Hz, 1H), 7.58 (d, J = 2Hz, 2H), 7.56 (s, 1H), 7.34 (d, J = 8 .4Hz,1H),6.92-6.88(m,2H),6.86-6.82(m,2H),6.43(d,J=9.2Hz,1H),5.04(s,2H),3.76(s,3H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.8, 154.3, 153.7, 152.6, 143.4, 133.9, 131.1, 126.7, 118.9, 117.2, 117.1, 115.9 (2C), 114.8 (2C), 69.7, 55.8.

[0057] Example 6

[0058] 4f:

[0059] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-fluorophenol to obtain the target product 4f with a yield of 89.7%.

[0060] 4f: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.71 (d, J = 9.2Hz, 1H), 7.58 (d, J = 2.4Hz, 1H), 7.56 (s, 1H), 7.34 (d,J=8.4Hz,1H),7.01-6.95(m,2H),6.93-6.87(m,2H),6.43(d,J=9.6Hz,1H),5.05(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.7, 157.5 (d, J = 239.9Hz), 154.6 (d, J = 1.9Hz), 153.8, 143.4, 13 3.4,131.1,126.8,119.0,117.2(d,J=10.7Hz,2C),116.1(d,J=30.6Hz,2C),116.0,116.0,69.7. 19 F NMR (282MHz, CDCl3): δ (ppm)-123.1 (brs).

[0061] Example 7

[0062] 4g:

[0063] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-chlorophenol, to obtain 4 g of the target product with a yield of 95.8%.

[0064] 4g: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.72 (d, J = 9.6 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.56 (s, 1H), 7.36 (d,J=8.0Hz,1H),7.28-7.24(m,2H),6.92-6.88(m,2H),6.46(d,J=9.6Hz,1H),5.08(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.7, 157.0, 153.8, 143.3, 133.2, 131.0, 129.6 (2C), 126.8, 126.4, 119.0, 117.4, 117.3, 116.2 (2C), 69.3.

[0065] Example 8

[0066] 4h:

[0067] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-trifluoromethoxyphenol to obtain the target product 4h with a yield of 89.1%.

[0068] 4h: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.71 (d, J = 9.6Hz, 1H), 7.59 (d, J = 2.0Hz, 1H), 7.56 (s, 1H), 7.34 (d,J=8.0Hz,1H),7.16-7.14(m,2H),6.97-6.93(m,2H),6.45(d,J=9.6Hz,1H),5.08(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.7, 157.0, 153.8, 143.3, 133.1, 131.0, 126. 8,122.7(2C),120.6(q,J=257.3Hz),119.0,117.3,117.2,115.7(2C),69.5. 19 F NMR: (282MHz, CDCl3): δ (ppm)-58.4 (brs).

[0069] Example 9

[0070] 4i:

[0071] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-bromophenol to obtain the target product 4i with a yield of 90.1%.

[0072] 4i: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.70 (d, J = 9.6 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.54 (s, 1H), 7.39 -7.35(m,2H),7.33(d,J=8.4Hz,1H),6.86-6.82(m,2H),6.43(d,J=9.6Hz,1H),5.06(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.6, 157.5, 153.7, 143.3, 133.0, 132.4 (2C), 130.9, 126.7, 118.9, 117.2, 117.1, 116.7 (2C), 113.5, 69.2.

[0073] Example 10

[0074] 4j:

[0075] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-methylphenol to obtain the target product 4j with a yield of 93.1%.

[0076] 4j: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.70 (d, J = 9.6Hz, 1H), 7.59 (d, J = 2.0Hz, 1H), 7.57 (s, 1H), 7.34 (d, J = 8.4Hz,1H),7.12-7.08(m,2H),6.89-6.85(m,2H),6.44(d,J=9.6Hz,1H),5.07(s,2H),2.29(s,3H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.8, 156.3, 153.7, 143.4, 133.8, 131.0, 130.7, 130.1 (2C), 126.7, 118.9, 117.2, 117.0, 114.7 (2C), 69.1, 20.6.

[0077] Example 11

[0078] 4k:

[0079] The synthesis method was the same as that in Example 1, except that pterostilbene was replaced with p-trifluoromethylphenol to obtain the target product 4k with a yield of 87.4%.

[0080] 4k: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.72 (d, J = 9.6Hz, 1H), 7.60 (d, J = 2.2Hz, 1H), 7.57 (s, 1H), 7.55 (d, J=8.9Hz,2H),7.35(d,J=8.3Hz,1H),7.03(d,J=8.8Hz,2H),6.44(d,J=9.5Hz,1H),5.14(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.8, 160.7, 153.9, 143.3, 132.8, 131.0, 127.1 (q, J = 3.8Hz, 2C ),126.8,124.4(q,J=272.1Hz),123.5(q,J=33.3Hz),119.0,117.4,117.2,114.9(2C),69.2. 19 F NMR (282MHz, CDCl3): δ (ppm)-61.5 (brs).

[0081] Example 12

[0082] 5a:

[0083] N-Methylpiperazine (0.44 mM) and K2CO3 (0.58 mM) were dissolved in acetonitrile and stirred at room temperature for 1 hour. Compound 3 (0.29 mM) dissolved in acetonitrile (0.5 mL) was slowly added dropwise. After stirring at 85°C for 12 hours, the reaction was complete as determined by TLC. The system was then cooled to room temperature. The product was then extracted with dichloromethane (3 × 10 mL), washed with distilled water and saturated brine, and dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to obtain the target compound 5a in a 92.7% yield.

[0084] 5a: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.70 (d, J = 9.6Hz, 1H), 7.50 (dd, J = 8.8, 2.4Hz, 1H), 7.44 (brs, 1H ),7.28(d,J=1.2Hz,1H),6.41(d,J=9.6Hz,1H),3.53(s,2H),2.72-2.40(m,8H),2.29(s,3H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.9, 153.2, 143.5, 134.8, 132.7, 128.0, 118.6, 116.7, 116.6, 62.0, 55.0 (2C), 53.0 (2C), 46.0.

[0085] Example 13

[0086] 5b:

[0087] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by N-acetylpiperazine to obtain the target product 5b with a yield of 90.5%.

[0088] 5b: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.69 (d, J = 9.2Hz, 1H), 7.50 (dd, J = 8.4, 2.0Hz, 1H), 7.44 (brs, 1H), 7.3 0(d,J=8.4Hz,1H),6.43(d,J=9.2Hz,1H),3.55(s,2H),3.45-3.63(m,4H),2.43(m,4H),2.08(s,3H). 13C NMR (75MHz, CDCl3): δ (ppm) 168.0, 159.8, 152.3, 142.3, 131.6, 127.0, 122.0, 117.7, 115.9, 115.8, 60.8, 52.0, 51.6, 45.1, 40.2, 20.3.

[0089] Example 14

[0090] 5c:

[0091] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by N-phenylpiperazine to obtain the target product 5c with a yield of 85.4%.

[0092] 5c: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.71 (d, J = 9.6Hz, 1H), 7.54 (dd, J = 8.4, 2.1Hz, 1H), 7.49 (brs, 1H), 7.31 (d, J = 8.4Hz, 2H), 7.27 (t, J = 7. 8Hz,2H),6.93(d,J=7.6Hz,2H),6.86(t,J=14.4,1H),6.43(d,J=9.6Hz,1H),3.60(s,2H),3.20(t,J=10Hz,4H),2.62(t,J=12Hz,4H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.9, 153.3, 151.3, 143.5, 134.6, 132.7, 129.2 ( 2C),128.1,119.8,118.7,116.8,116.8,116.1(2C),62.1,53.2(2C),49.2(2C).

[0093] Example 15

[0094] 5d:

[0095] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by 1-(4-fluorobenzyl)piperazine to obtain the target product 5d with a yield of 81.0%.

[0096] 5d: 1H NMR (400MHz, CDCl3): δ (ppm) 7.68 (d, J = 9.6Hz, 1H), 7.49 (dd, J = 8.4, 2.0Hz, 1H), 7.43 (brs, 1H), 7.27 (d, J = 6.4Hz ,2H),7.25(d,J=5.2Hz,1H),6.98(t,J=8.6Hz,2H),6.41(d,J=9.6Hz,1H),3.54(s,2H),3.47(s,2H),2.47(m,8H). 13 C NMR (101MHz, CDCl3): δ (ppm) 162.1 (d, J = 245.7Hz), 161.0, 153.3, 143.6, 134.9, 133.8 (d, J = 3.0Hz), 132.8, 1 30.7(d,J=7.8Hz,2C),128.1,118.7,116.8,116.8,115.1(d,J=21.2Hz,2C),62.3,62.1,53.2(2C),53.0(2C). 19 F NMR (282MHz, CDCl3): δ (ppm)-115.8 (brs).

[0097] Example 16

[0098] 5e:

[0099] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by 1-[4-(trifluoromethyl)benzyl]piperazine to obtain the target product 5e with a yield of 80.3%.

[0100] 5e: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.67 (d, J = 9.6Hz, 1H), 7.53 (d, J = 8.0Hz, 2H), 7.47 (dd, J = 8.7, 2.0Hz, 1H), 7.42 (s, 1H),7.41(d,J=7.2Hz,2H),7.24(d,J=8.4Hz,1H),6.38(d,J=9.6Hz,1H),3.54(s,2H),3.52(s,2H),2.46(m,8H). 13C NMR (101MHz, CDCl3): δ (ppm) 160.9, 153.2, 143.5, 142.5, 134.8, 132.7, 129.3 (q, J = 32.3Hz, 2C), 129 .2,128.0,125.1(q,J=3.7Hz,2C),124.3(q,J=272.9Hz)118.6,116.7,116.6,62.4,62.0,53.1(4C). 19 F NMR (282MHz, CDCl3): δ (ppm)-62.3 (brs).

[0101] Example 17

[0102] 5f:

[0103] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by 1-[4-(trifluoromethoxy)benzyl]piperazine to obtain the target product 5f with a yield of 83.0%.

[0104] 5f: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.69 (d, J = 9.5Hz, 1H), 7.49 (dd, J = 8.5, 2.2Hz, 1H), 7.44 (s, 1H), 7.33 (d, J = 8.6Hz, 2H),7.28(d,J=8.9Hz,1H),7.15(d,J=7.5Hz,2H),6.41(d,J=9.5Hz,1H),3.55(s,2H),3.51(s,2H),2.48(s,8H). 13 C NMR (75MHz, CDCl3): δ (ppm) 161.0, 153.3, 148.4, 143.6, 135.9 (q, J = 167.7Hz), 132.9, 130.5 (2 C),128.2,126.1,122.3,120.9,118.9,118.7,116.9,116.8,62.2,62.1,53.1(2C),53.0(2C). 19 F NMR (282MHz, CDCl3): δ (ppm)-57.9 (brs).

[0105] Example 18

[0106] 5g:

[0107] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced with 4-piperidinylpiperidine to obtain 5 g of the target product with a yield of 83.3%.

[0108] 5g: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.65 (d, J = 9.6Hz, 1H), 7.42 (dd, J = 8.8, 2.0Hz, 1H), 7.39 (s, 1H), 7.21 (d, J = 8.4Hz, 1H), 6.35 (d, J = 9.6Hz ,1H),3.45(s,2H),2.49(t,J=5.2Hz,4H),2.28(m,4H),1.93(tt,J=135.6,11.6Hz,4H),1.56-1.50(m,4H),1.38(quint,J=5.9Hz,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.9, 153.1, 143.6, 135.2, 132.6, 127.9, 118. 6,116.6,116.6,62.8,62.0,53.4(2C),50.1(2C),27.6(2C),26.1(2C),24.6.

[0109] Example 19

[0110] 5h:

[0111] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced with 3,5-dimethylpiperidine to obtain the target product 5h with a yield of 79.3%.

[0112] 5h: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.70 (d, J = 9.6Hz, 1H), 7.48 (dd, J = 8.4, 2.0Hz, 1H), 7.44 (brs, 1H), 7.27 (d, J = 6.0 Hz,1H),6.41(d,J=9.6Hz,1H),3.49(s,2H),1.46(t,J=10.8Hz,2H),0.82(d,J=6.4Hz,6H),1.67-0.47(m,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 161.0, 153.1, 143.6, 135.3, 132.8, 128.0, 118.6, 116.6, 116.6, 62.4, 61.5 (2C), 42.1, 31.1 (2C), 19.6 (2C).

[0113] Example 20

[0114] 5i:

[0115] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by morpholine to obtain the target product 5i with a yield of 82.1%.

[0116] 5i: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.69 (d, J = 9.6Hz, 1H), 7.50 (dd, J = 8.4, 2.1Hz, 1H), 7.44 (d, J = 1.8Hz, 1H), 7.27(d,J=8.1Hz,1H),6.41(d,J=9.6Hz,1H),3.70(t,J=4.7Hz,4H),3.52(s,2H),2.44(t,J=4.7Hz,4H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.9, 153.3, 143.4, 134.4, 132.7, 128.0, 118.7, 116.8, 116.7, 66.9 (2C), 62.4, 53.6 (2C).

[0117] Example 21

[0118] 5j:

[0119] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by pyrazole to obtain the target product 5j with a yield of 82.0%.

[0120] 5j: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.65 (d, J = 9.6Hz, 1H), 7.57 (d, J = 1.8Hz, 1H), 7.46 (d, J = 2.4Hz, 1H), 7.39 (dd, J = 8.4, 2 .1Hz,1H),7.31(d,J=2.7Hz,1H),7.28(d,J=1.8Hz,1H),6.42(d,J=9.6Hz,1H),6.32(t,J=2.1Hz,1H),5.37(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.6, 153.7, 143.2, 140.1, 133.4, 131.1, 129.5, 126.9, 119.0, 117.4, 117.2, 106.4, 55.0.

[0121] Example 22

[0122] 5k:

[0123] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by imidazole to obtain the target product 5k with a yield of 84.9%.

[0124] 5k: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.61 (d, J = 9.6Hz, 1H), 7.54 (s, 1H), 7.32 (dd, J = 8.8, 2.0Hz, 1H), 7.25 (d, J = 4 .4Hz,1H),7.18(d,J=2.4Hz,1H),7.06(s,1H),6.88(d,J=2.4Hz,1H),6.38(d,J=9.6Hz,1H),5.15(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.3, 153.7, 143.0, 137.4, 132.8, 130.6, 130.2, 126.4, 119.2, 119.1, 117.6, 117.4, 49.8.

[0125] Example 23

[0126] 5l:

[0127] The synthesis method was the same as that of Example 12, except that N-methylpiperazine was replaced by 4-iodopyrazole to obtain the target product 51 with a yield of 85.3%.

[0128] 5l: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.65 (d, J = 9.6 Hz, 1H), 7.54 (s, 1H), 7.47 (s, 1H), 7.38 (dd, J = 8.4 ,2.0Hz,1H),7.32(d,J=2.0Hz,1H),7.29(d,J=8.8Hz,1H),6.41(d,J=9.6Hz,1H),5.33(s,2H). 13 C NMR (101MHz, CDCl3): δ (ppm) 160.4, 153.8, 145.1, 143.1, 133.8, 132.4, 131.2, 127.1, 119.0, 117.5, 117.3, 57.0, 55.4.

[0129] Example 24

[0130] 5m:

[0131] To a DMF solution containing sodium hydride (0.46 mmol) at 0°C, indole (0.42 mM) dissolved in DMF (0.5 mL) was added dropwise. The mixture was brought to room temperature and stirred for 30 min. Then, a DMF solution of compound 3 (0.38 mM) was slowly added dropwise, and stirring was continued for 12 h. The mixture was then extracted with EtOAc (3 × 10 mL), washed with distilled water and saturated brine, and the organic layer was dried over anhydrous Na2SO4. The crude product was purified by column chromatography (petroleum ether and ethyl acetate as eluents) and concentrated under reduced pressure to obtain the target compound 5m in a yield of 76.4%.

[0132] 5m: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.66 (m, 1H), 7.54 (d, J = 9.6Hz, 1H), 7.29 (dd, J = 8.7, 2.1Hz ,1H),7.25(d,J=5.1Hz,1H),7.24(d,J=3.3Hz,1H),7.22-7.21(m,J=3.3Hz,1H),7.18(dd ,J=6.6,1.5Hz,1H),7.15(d,J=1.8Hz,1H),7.14(d,J=3.3Hz,1H),7.12(d,J=1.5Hz,1H) ,7.10(d,J=2.1Hz,1H),6.58(dd,J=3.2,0.8Hz,1H),6.36(d,J=9.6Hz,1H),5.36(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.6, 153.4, 143.2, 136.1, 134.1, 130.2, 128.8, 128.1,125.7,122.0,121.2,119.9,119.0,117.4,117.1,109.5,102.3,49.3.

[0133] Example 25

[0134] 5n:

[0135] The synthesis method was the same as that of Example 24, except that indole was replaced by 6-chloroindole to obtain the target product 5n with a yield of 75.6%.

[0136] 5n: 1H NMR (300MHz, CDCl3): δ (ppm) 7.57 (d, J = 9.6Hz, 1H), 7.56 (dd, J = 8.4, 0.54Hz, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 7.23-7.22 (m, 1H), 7. 13(d,J=3.2Hz,1H),7.10(d,J=1.7Hz,1H),7.08(d,J=1.8Hz,1H),6.56(dd,J=3.2,0.9Hz,1H),6.39(d,J=9.6Hz,1H),5.32(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.5, 153.5, 143.1, 136.5, 133.5, 130.1, 128.8, 128.1,127.3,125.6,122.1,120.6,119.1,117.5,117.2,109.5,102.5,49.3.

[0137] Example 26

[0138] 5o:

[0139] The synthesis method was the same as that of Example 24, except that indole was replaced by 4-bromoindole, and the target product 5o was obtained with a yield of 77.0%.

[0140] 5o: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.54 (d, J = 9.6Hz, 1H), 7.30-7.27 (m, 1H), 7.26 (d, J = 2.1Hz, 1H), 7.25 (d, J = 5.1Hz, 1H), 7.19 (t, J = 14.6Hz, 1 H),7.17(d,J=8.1Hz,1H),7.08(d,J=1.2Hz,1H),7.02(t,J=8.0Hz,1H),6.62(dd,J=3.3,0.9Hz,1H),6.36(d,J=9.6Hz,1H),5.34(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.5, 153.5, 143.1, 136.4, 133.5, 130.1, 129.5, 128.8,125.7,123.0,122.8,119.0,117.4,117.1,115.1,108.8,102.6,49.6.

[0141] Example 27

[0142] 5p:

[0143] The synthesis method was the same as that of Example 24, except that indole was replaced by 6-bromoindole to obtain the target product 5p with a yield of 77.2%.

[0144] 5p: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.55 (d, J = 9.6Hz, 1H), 7.51 (d, J = 8.4Hz, 1H), 7.38 (s, 1H), 7.26 (d, J = 1.2Hz, 1H), 7.26 (s, 1H), 7. 21(dd,J=8.4,1.8Hz,1H),7.11(d,J=3.3Hz,1H),7.09(m,1H),6.55(dd,J=3.3,0.9Hz,1H),6.38(d,J=9.6Hz,1H),5.30(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.5, 153.5, 143.1, 137.0, 133.5, 130.1, 128.8, 127.6,125.6,123.2,122.4,119.1,117.5,117.2,115.7,112.5,102.6,49.3.

[0145] Example 28

[0146] 5q:

[0147] The synthesis method was the same as that of Example 24, except that indole was replaced by 5-methylindole, and the target product 5q was obtained with a yield of 75.3%.

[0148] 5q: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.57 (d, J = 9.6Hz, 1H), 7.47 (brs, 1H), 7.31 (dd, J = 8.4, 1.8Hz, 1H), 7.28 (d, J = 4.5Hz, 1H), 7.14 (d, J = 4.8Hz, 1H), 7.13(s,1H),7.12(d,J=1.5Hz,1H),7.02(dd,J=8.4,1.5Hz,1H),6.52(dd,J=3.0,0.9Hz,1H),6.39(d,J=9.6Hz,1H),5.36(s,2H),2.46(s,3H). 13C NMR (75MHz, CDCl3): δ (ppm) 160.6, 153.4, 143.2, 134.5, 134.3, 130.1, 129.1, 12 9.1,128.2,125.6,123.6,120.9,119.0,117.4,117.1,109.2,101.7,49.3,21.4.

[0149] Example 29

[0150] 5r:

[0151] The synthesis method was the same as that of Example 24, except that indole was replaced by 5-methoxyindole to obtain the target product 5r with a yield of 74.6%.

[0152] 5r: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.58 (d, J = 9.6Hz, 1H), 7.30 (d, J = 1.8Hz, 1H), 7.29 (s, 1H), 7.28 (d, J = 4.2Hz, 1H), 7.14 (d, J = 2.1Hz, 1H), 7.13 (s,1H),7.11(d,J=6.3Hz,1wH),6.85(dd,J=9.0,2.7Hz,1H),6.52(dd,J=3.3,0.9Hz,1H),6.40(d,J=9.6Hz,1H),5.35(s,2H),3.86(s,3H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.6, 154.3, 153.4, 143.2, 134.2, 131.4, 130.1, 12 9.3,128.7,125.6,119.0,117.4,117.1,112.3,110.3,102.7,101.8,55.8,49.5.

[0153] Example 30

[0154] 5s:

[0155] The synthesis method was the same as that of Example 24, except that indole was replaced by 5-bromoindole to obtain the target product 5s with a yield of 74.7%.

[0156] 5s: 1H NMR (300MHz, CDCl3): δ (ppm) 7.74 (d, J = 1.8Hz, 1H), 7.52 (d, J = 9.6Hz, 1H), 7.23 (s, 1H), 7.22 (d, J = 2.1Hz, 1H), 7.19 (d, J = 2.1Hz, 1H), 7.11(d,J=3.0Hz,1H),7.06(d,J=4.8Hz,1H),7.04(d,J=2.7Hz,1H),6.49(dd,J=3.0,0.6Hz,1H),6.34(d,J=9.6Hz,1H),5.31(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.5, 153.5, 143.1, 134.8, 133.6, 130.5, 130.1, 129.3,125.6,124.9,123.7,119.0,117.5,117.2,113.2,111.0,101.9,49.5.

[0157] Example 31

[0158] 5t:

[0159] The synthesis method was the same as that of Example 24, except that indole was replaced by 5-fluoroindole to obtain the target product 5t with a yield of 72.4%.

[0160] 5t: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.59 (d, J = 9.6Hz, 1H), 7.31 (dd, J = 9.6, 2.4Hz, 1H), 7.29 (s, 1H), 7.28 (d, J = 2.1Hz, 1H), 7.21 (d, J = 3.0Hz, 1H), 7.16 ( d,J=4.2Hz,1H),7.13(s,1H),7.12(d,J=2.4Hz,1H),6.93(td,J=9.1,2.5 Hz, 1H), 6.56 (dd, J=3.0, 0.9Hz, 1H), 6.40 (d, J=9.6Hz, 1H), 5.37 (s, 2H). 13C NMR (75MHz, CDCl3): δ (ppm) 160.6, 158.1 (d, J = 233.3Hz), 153.5, 143.2, 133.9, 132.8, 130.2, 129.8, 129.2 (d, J = 10.2Hz) ,125.7,119.1,117.4(d,J=20.4Hz),110.4(d,J=37.4Hz),110.3,110.3,106.0(d,J=23.2Hz),102.3(d,J=4.7Hz),49.6. 19 F NMR (282MHz, CDCl3): δ (ppm)-124.8 (brs).

[0161] Example 32

[0162] 5u:

[0163] The synthesis method was the same as that of Example 24, except that indole was replaced by 5-trifluoromethylindole to obtain the target product 5u with a yield of 76.7%.

[0164] 5u: 1 H NMR (300MHz, CDCl3): δ (ppm) 7.98 (brs, 1H), 7.59 (d, J = 9.6Hz, 1H), 7.43 (dd, J = 8.7, 1.8Hz, 1H), 7.35-7.31 (m, 1H), 7.32 (d, J = 4.8H) z,1H),7.30(d,J=1.8Hz,1H),7.29(s,1H),7.16(d,J=1.8Hz,1H),6.71(dd,J=3.2,0.9Hz,1H),6.40(d,J=9.6Hz,1H),5.43(s,2H). 13 C NMR (75MHz, CDCl3): δ (ppm) 160.5, 153.6, 143.1, 137.4, 133.5, 130.2, 130.0, 128.2, 125.7, 125.4 (q, J = 269. 8Hz), 122.3 (q, J = 31.6Hz), 119.1, 119.0 (q, J = 4.2Hz), 118.8 (q, J = 3.4Hz), 117.5, 117.2, 109.9, 103.4, 49.5. 19 F NMR (282MHz, CDCl3): δ (ppm)-60.2 (brs).

[0165] Anti-breast cancer activity experimental methods and results:

[0166] Screening method: Breast cancer cells MDA-MB-231 and MCF-7 were cultured at 3-5×10 3 The cells were seeded in a 96-well plate at a density of 100 cells / well and placed in a 37°C, 5% CO2 cell culture incubator. After attachment, different concentrations of the test compound were added in sequence to make the final concentration of the drug 1, 3, 10, 30 and 100 μM, with 3 replicates for each concentration. At the same time, DMSO was used as a blank control and doxorubicin (Dox) was used as a positive control. After incubation for 48 hours, 10 μL of CCK-8 solution was added to each well. After 2 hours of treatment, the absorbance (OD value) at 450 nm was detected using an automatic microplate reader. The IC value of the compound was calculated using SPSS13.0 (SPSS, Inc., Chicago, IL) software. 50 Cell proliferation inhibition rate (%) = (1-OD 药物组 / OD 空白组 )×100%

[0167] Experimental results: As shown in Table 1, compounds with anti-breast cancer activity are listed. At the same time, according to their inhibitory effects on MDA-MB-231 and MCF-7 cells (IC 50 value), and the corresponding selectivity index (SI) was calculated.

[0168] Table 1 Coumarin analogs with anti-breast cancer activity

[0169]

[0170] The above results show that at a maximum concentration of 100 μM, derivatives 4a-4k had no inhibitory effect on MDA-MB-231 and MCF-7 cells. Among derivatives 5a-5u, most derivatives (5a-5d and 5g-5u) had little or weak inhibitory effect on the two breast cancer cell lines, and only derivatives 5e and 5f had moderate inhibitory activity against TNBC cells MDA-MB-231 (IC 50 =32-36 μM). It is noteworthy that the selectivity index of derivatives 5e and 5f for MDA-MB-231 cells was significantly improved compared with compounds 3, 5g, 5h, 5j, 5n and Dox, with SI values ​​of 0.28 and 0.36, respectively. Combined with the above analysis, preliminary structure-activity relationship showed that: (1) the introduction of nitrogen heterocyclic compounds at the C-6 position of the coumarin parent nucleus can maintain the anti-mammary activity of such compounds (4a-4k vs. 5a-5u). (2) the introduction of aromatic rings containing -CF3 and -OCF3 at the C-6 position of piperazine as a linker can significantly enhance the sensitivity of TNBC cells to such compounds (5e, 5f vs. 5a-5u; 5e, 5f vs. 3, Dox).

Claims

1. Use of a coumarin derivative in the preparation of a medicament for preventing or treating breast cancer, characterized in that: The coumarin derivatives are selected from the following compounds: ； 。 2. The use of the coumarin derivative according to claim 1, characterized in that: The breast cancer is triple-negative breast cancer.

3. The use of the coumarin derivatives according to claim 1, characterized in that: The dosage form of the medicine is powder, granule, tablet, capsule, pill, solution, suspension or injection.

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