Inhibitors targeting c-myc and uses thereof

Abstract

The present application relates to the inhibitor of targeting c-Myc and its application, belong to the technical field of targeted anti-tumor drugs.The technical problem solved by the present application is to provide an inhibitor of targeting c-Myc with better treatment effect on colorectal cancer.The structural formula of the inhibitor of targeting c-Myc of the present application is shown in formula I or formula II.A series of new inhibitors of targeting c-Myc are synthesized by optimizing design, these compounds have superior antitumor activity and biological safety, and are an ideal c-Myc small molecule inhibitor, which has good prospects in the treatment of colorectal cancer.

Description

Technical Field

[0001] This invention relates to inhibitors targeting c-Myc and their applications, belonging to the field of targeted anti-tumor drug technology. Background Technology

[0002] Colorectal cancer has become the second leading cause of cancer death and the third most common cancer worldwide. In China, colorectal cancer is one of the most prevalent cancers. In 2020, it was estimated that there were approximately 560,000 new cases and 280,000 new deaths from colorectal cancer in China. Clinically, there are various treatments for cancer, primarily chemotherapy, radiotherapy, and surgery. Currently, traditional chemotherapy drugs for colorectal cancer mainly include 5-fluorouracil, irinotecan, and platinum-based compounds; however, these drugs generally suffer from high toxicity and drug resistance. With ongoing research, targeted therapies have emerged, including monoclonal antibodies and small molecule targeted drugs. However, targeted drugs still have drawbacks such as high cost, numerous side effects, and low selectivity. Therefore, there is a need to develop highly selective targeted drugs to treat colorectal cancer. Previous studies have confirmed that c-Myc is almost always highly expressed in colorectal cancer patients, leading to research into c-Myc-targeted therapy for colorectal cancer. Simultaneously, the development and design of small molecule inhibitors targeting the c-Myc protein for colorectal cancer treatment has become a research hotspot.

[0003] Currently, reported small molecule inhibitors of c-Myc protein mainly include stabilizers of G-quadruplex such as T-BFQ and IZCZ-3; small molecule inhibitors of c-Myc / Max dimer such as 10058-F4, 10074-G5, and JY-3-094; and small molecule inhibitors targeting the c-Myc / Max / DNA complex such as Mycro3 and KJ-Pyr-9. Although some progress has been made in the research of c-Myc small molecule inhibitors, most of them are at the protein and cellular levels, and their in vitro activity is poor, their toxicity is high, and their drug-likeness is too low to enter the clinical stage.

[0004] Chinese invention patent application number 202010066925.1 discloses a phenoxy-N-phenylaniline derivative and its application. Compound 42 designed by the patent exhibits superior antitumor activity in in vitro and in vivo experiments. Mechanism studies (gel electrophoresis migration assay) have also confirmed that it can interfere with the binding of the c-Myc / Max complex to its E-box DNA sequence. Its in vitro activity IC50 against HT29 tumor cells is 2.48 μM, which requires further optimization. Summary of the Invention

[0005] To address the above deficiencies, the technical problem solved by this invention is to provide a c-Myc-targeting inhibitor that has a better therapeutic effect on colorectal cancer.

[0006] The structural formula of the c-Myc-targeting inhibitor of this invention is shown in Formula I or Formula II:

[0007]

[0008]

[0009] Wherein, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0010] R2 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, or... Or R1 and R2 can be connected to form a benzene ring;

[0011] R3 is

[0012] In one embodiment of the present invention, the structural formula is shown in Formula I.

[0013] In one specific implementation, R3 is

[0014] In one specific embodiment, R2 is H, fluorine, methyl, methoxy, or...

[0015] In some specific embodiments, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0016] In another embodiment, the structural formula is shown in Formula II.

[0017] In one specific implementation, R3 is

[0018] In one specific implementation, R2 is H.

[0019] This invention also provides the use of the c-Myc-targeting inhibitor described herein in the preparation of drugs for treating colorectal cancer.

[0020] The c-Myc inhibitor described in this invention is a small molecule c-Myc inhibitor that has excellent activity in inhibiting colorectal cancer cells and can be used to treat colorectal cancer.

[0021] The present invention also provides a pharmaceutical composition.

[0022] The pharmaceutical composition of the present invention comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a therapeutically effective amount of the c-Myc-targeting inhibitor of the present invention or a pharmaceutically acceptable salt thereof.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention synthesizes a series of novel c-Myc inhibitors through optimized design. These compounds exhibit superior antitumor activity and biosafety, making them ideal small-molecule c-Myc inhibitors with promising potential for the treatment of colorectal cancer. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the optimization of c-Myc inhibitor compounds.

[0026] Figure 2 This is a graph showing the toxicity of compounds B13 and 5-fluorouracil to normal human hepatocytes.

[0027] Figure 3 This is a cell cycle distribution diagram of compound B13 acting on HT29 cells.

[0028] Figure 4 This is a cell cycle distribution diagram of HCT116 cells treated with compound B13.

[0029] Figure 5 The image shows the results of apoptosis detection in HT29 cells by compound B13.

[0030] Figure 6 The inhibition efficiency (A, B) of compound c-Myc / Max dimer binding to DNA was detected by EMSA, where A is compound 42 synthesized previously and B is compound B13. Detailed Implementation

[0031] The structural formula of the c-Myc-targeting inhibitor of this invention is shown in Formula I or Formula II:

[0032]

[0033] Wherein, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0034] R2 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, or... Or R1 and R2 can be connected to form a benzene ring;

[0035] R3 is

[0036] In order to find small molecule compounds with better in vitro activity against colorectal cancer cells, this invention optimized existing compounds and designed and synthesized a total of 42 novel small molecule c-Myc inhibitor compounds. A schematic diagram of the optimization is shown below. Figure 1 Through optimization, the antitumor activity of the compound was improved, especially the B13 molecular structure, and its IC29 activity against colorectal cancer tumor cells was enhanced. 50 The concentration was 0.38 μM, which showed a 7-fold increase in activity compared to compound 42 (i.e., compound A3 of the present invention) in 202010066925.1, and a higher activity than the positive control drug 5-fluorouracil (IC50). 50 =9.19μM) is 24 times higher than that of cisplatin (IC50) 50 =11.63μM) is 31 times higher.

[0037] In one embodiment of the present invention, the structural formula is shown in Formula I, that is, the structural formula of the inhibitor targeting c-Myc is:

[0038]

[0039] In one specific embodiment, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0040] R2 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, or... Or R1 and R2 can connect to form a benzene ring; R3 is...

[0041] In one specific embodiment, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0042] R2 is H, fluorine, methyl, methoxy, or R3 is

[0043] In some specific embodiments, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0044] R2 is H, fluorine, methoxy, or... R3 is

[0045] In another embodiment, the structural formula is shown in Formula II, that is, the structural formula of the inhibitor targeting c-Myc is:

[0046]

[0047] In one specific embodiment, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0048] R2 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, or... Or R1 and R2 can connect to form a benzene ring; R3 is...

[0049] In one specific embodiment, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, benzene ring,

[0050] R2 is H; R3 is

[0051] Specifically, the structural formula of the c-Myc-targeting inhibitor of the present invention is any one of the following structural formulas:

[0052]

[0053]

[0054]

[0055] This invention also provides the use of the c-Myc-targeting inhibitor described herein in the preparation of drugs for treating colorectal cancer.

[0056] The c-Myc inhibitor described in this invention is a small molecule c-Myc inhibitor that has excellent activity in inhibiting colorectal cancer cells and can be used to treat colorectal cancer.

[0057] The present invention also provides a pharmaceutical composition.

[0058] The pharmaceutical composition of the present invention comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a therapeutically effective amount of the c-Myc-targeting inhibitor of the present invention or a pharmaceutically acceptable salt thereof.

[0059] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0060] Example 1: Synthesis of the compound

[0061] 1. Synthesis of compound A1-11

[0062] Synthesis route:

[0063]

[0064] Reagents and conditions: (i) 4-aminophenol, bromobenzene, t-BuONa, Brettphos Pd G3, ultra-dry reagent 1,4-dioxane, 90℃, 3h. 75.1%-81.3%; (ii) 4-bromo-2,6-dimethoxybenzaldehyde, 4-aminophenol, Pd2(dba )3 , t-BuXPhos, Cs2CO3, toluene, nitrogen protection, 100℃, 12h, 60.0%-65.7%; (iii) aromatic ethylamine, methanol, 65℃, reflux reflux 2h, NaBH4, 1h, 55%-65%.

[0065] Synthesis method M1 for intermediate compound AM-1:

[0066]

[0067] p-Aminophenol (2 g, 18.3 mmol), bromobenzene (4.3 g, 27.45 mmol), sodium tert-butoxide (3.5 g, 36.6 mmol), and BrettPhos Pd G3 (82 mg, 0.0915 mmol, 0.5 mmol%) were added to a 50 mL round-bottom flask containing 15 mL of anhydrous 1,4-dioxane. The mixture was reacted under nitrogen protection in a 90 °C oil bath with a magnetic stirrer for 4 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction solution was filtered through diatomaceous earth, and the filter cake was washed several times with a small amount of methanol. An appropriate amount of silica gel powder was added to the collected filtrate, and the mixture was dried and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain 2.67 g of the target product as a reddish-brown solid. The yield was 79.2%. 1 H NMR(400MHz,Chloroform-d)δ7.24-7.18(m,2H),7.05-7.00(m,2H),6.93-6.88(m,2H), 6.83(tt,J=7.3,1.1Hz,1H),6.80-6.76(m,2H),5.46(s,1H),4.71(s,1H).ESI-MS:mass calcd for[M+H] + (186.0919); found m / z,186.0916.

[0068] Synthesis method M2 for intermediate compound AM-2:

[0069]

[0070] Intermediate AM-1 (4-(phenylamino)phenol) (185 mg, 1 mmol), 4-bromo-2,6-dimethoxybenzaldehyde (294 mg, 1.2 mmol), Pd2(dba)3 (45.75 mg, 0.05 mmol), t-Bu XPhos (24.8 mg, 0.05 mmol), and Cs2CO3 (650 mg, 2 mmol) were added to a 25 mL round-bottom flask containing 5 mL of toluene solution. The reaction was carried out under nitrogen protection in an oil bath with a magnetic stirrer at 100 °C for 12 h. The reaction progress was monitored using a thin-layer chromatography plate. After the reaction was completed, the reaction mixture was cooled for a period of time, and then the toluene solution was removed by rotary evaporation under vacuum. 25 mL of saturated brine was added, and the mixture was extracted three times with ethyl acetate (8 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Column chromatography was used to separate and purify the organic phase to give 205 mg of a reddish-brown solid. The yield was 58.70%. 1 H NMR(400MHz,Chloroform-d)δ10.37(s,1H),7.31–7.27(m,2H),7.13–7.06(m,4H),7.04– 6.99(m,2H),6.96(t,J=7.4Hz,1H),6.12(s,2H),5.72(s,1H),3.81(s,6H).ESI-MS:mass calcd for[M+Na] + (372.1212); found m / z,372.1208.

[0071] General method M3 for the preparation of final products A1-A11:

[0072] Intermediate AM-2 (80 mg, 0.22 mmol) and various phenylethylamines (0.26 mmol) were added to 4 mL of methanol solution and refluxed in an oil bath with a magnetic stirrer at 65 °C for 2 h. The mixture was then removed and cooled to room temperature for a period of time, followed by the addition of NaBH4 (16 mg, 0.44 mmol) and a reaction time of 30 min. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove methanol, and 15 mL of saturated brine was added. The mixture was then extracted three times with ethyl acetate (5 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain the desired target compound.

[0073] Synthesis of target compound A1:

[0074]

[0075] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-hydroxyphenylethylamine. The product was a brown solid with a yield of 56.76%. 1H NMR(400MHz,Chloroform-d)δ7.24(dd,J=8.5,7.2Hz,2H),7.05(ddd,J=16.8,7.6,1.7Hz,4H),6.95–6.88(m,3H),6.88–6 .82(m,2H),6.71(d,J=8.4Hz,2H),6.10(s,2H),5.92(s,1H),4.03(s,2H),3.58(s,6H),2.88(dp,J=11.7,6.0,5.2Hz,4H). 13 CNMR(101MHz,Chloroform-d)δ159.89,159.43,155.93,150.21,143.64,139.33,129.68,129.39,128.62, 120.67,120.57,119.82,117.12,115.88,105.94,94.04,55.73,55.69,48.42,40.42,33.20.ESI-MS:mass calcd for C 29 H 30 N₂O₄[M+H] + (471.2284); found m / z471.2230.

[0076] Synthesis of target compound A2:

[0077]

[0078] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-aminophenylethylamine. The product was a brown solid with a yield of 58.62%. 1 H NMR(400MHz,Chloroform-d)δ7.30–7.24(m,2H),7.13–7.02(m,4H),6.98–6.89(m,5H) ,6.62–6.57(m,2H),6.13(s,2H),5.77(s,1H),4.09(s,2H),3.65(s,6H),2.93(s,4H). 13C NMR(101MHz,Chloroform-d)δ159.66,159.47,150.42,144.85,143.66,139.22,129.66,129.40,128.42,120 .72,120.52,119.90,117.10,115.31,106.55,94.10,55.75,55.71,48.92,40.59,33.58,22.67.ESI-MS:mass calcd for C 29 H 31 N3O3[M+H] + (470.2444); found m / z,470.2445.

[0079] Synthesis of target compound A3:

[0080]

[0081] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-methoxyphenylethylamine. The product was a reddish-brown powder crystal with a yield of 60.34%. 1 H NMR(400MHz,Chloroform-d)δ7.28–7.23(m,2H),7.11–7.06(m,4H),7.05–7.00(m,2H),6.97–6.88(m, 3H),6.83–6.79(m,2H),6.16(s,2H),5.72(s,1H),3.95(s,2H),3.77(s,3H),3.66(s,6H),2.88(s,4H). 13 C NMR(101MHz,Chloroform-d)δ178.88,159.46,158.14,150.53,143.68,139.15,131.09,129.75,129.40,12 0.70,120.47,119.93,117.07,113.92,94.15,55.72,55.69,55.30,49.15,40.67,33.89.ESI-MS: masscalcd for C 30 H 32 N₂O₄[M+H] + (485.2240); found m / z,485.2245.

[0082] Synthesis of target compound A4:

[0083]

[0084] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-methylphenethylamine. The product was a brown powdery crystal with a yield of 55.23%. 1 H NMR(400MHz,Chloroform-d)δ7.29–7.23(m,2H),7.11–7.03(m,8H),6.97–6.88(m,3H),6.13(s, 2H),5.77(s,1H),4.10(s,2H),3.67(s,6H),3.08–3.01(m,2H),3.00–2.93(m,2H),2.30(s,3H). 13 C NMR(101MHz,Chloroform-d)δ160.55,159.64,149.93,143.54,139.50,136.18,134.78,129.41,129.33,128.73,120.8 0,119.74,117.22,103.70,93.79,78.22,59.00,55.79,55.75,47.88,40.05,32.89,21.04,21.01,18.67.ESI-MS:mass calcd for C 30 H 32 N₂O₃[M+H] + (469.2491); found m / z,469.2450.

[0085] Synthesis of target compound A5:

[0086]

[0087] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-fluorophenylethylamine. The product was a brown powdery crystal with a yield of 57.44%. 1 H NMR(400MHz,Chloroform-d)δ7.26(dd,J=8.6,7.2Hz,2H),7.16–7.10(m,2H),7.10–7.05(m,2H),7.05–7.00(m,2 H),6.99–6.93(m,3H),6.93–6.88(m,2H),6.15(s,2H),5.75(s,1H),3.99(s,2H),3.68(s,6H),3.00–2.87(m,4H). 13C NMR(101MHz,Chloroform-d)δ162.78,160.35,159.86,159.53,150.26,143.62,139.31,134.56,134.53,130.26,130.18,129 .40,120.75,120.60,119.84,117.14,115.38,115.17,106.23,94.01,78.23,55.76,55.72,48.67,40.39,33.53.ESI-MS:mass calcd for C 29 H 29 FN2O3[M+H] + (473.2240); found m / z,473.2246

[0088] Synthesis of target compound A6:

[0089]

[0090] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-chlorophenylethylamine. The product was a brownish-black powder, with a yield of 60.64%. 1 H NMR(400MHz,Chloroform-d)δ7.28–7.22(m,4H),7.13–7.06(m,4H),7.05–7.01(m,2H),6.97– 6.88(m,3H),6.16(s,2H),5.82–5.71(m,1H),3.93(s,2H),3.67(s,6H),2.89(q,J=3.7Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ159.54,159.46,150.40,143.67,139.23,137.74,132.05,130.15,129.40,128 .55,120.70,120.52,119.89,117.10,107.34,94.08,55.73,55.69,50.61,48.77,40.57,34.15.ESI-MS:mass calcd forC 29 H 29 ClN2O3[M+H] + (489.1945); found m / z,489.1943.

[0091] Synthesis of target compound A7:

[0092]

[0093] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-bromophenylethylamine. The product was a brownish-red powder, with a yield of 56.48%. 1 H NMR(400MHz,Chloroform-d)δ7.42–7.34(m,2H),7.29–7.22(m,2H),7.12–7.00(m,6H),6.98–6.86(m,3H),6.15(s,2H) ,5.75(s,1H),4.01(s,2H),3.69(s,6H),3.39(s,1H),2.99(dd,J=11.3,4.6Hz,2H),2.91(ddd,J=8.9,5.5,1.9Hz,2H). 13 C NMR(101MHz,Chloroform-d)δ160.13,159.58,150.11,143.58,139.38,137.67,131.61,130.57,129.41,120.78 ,120.69,120.30,119.81,117.18,93.91,78.23,58.99,55.80,55.76,48.19,40.28,33.44,18.67.ESI-MS:mass calcd for C 29 H 29 BrN2O3[M+H] + (533.1440); found m / z,533.1466.

[0094] Synthesis of target compound A8:

[0095]

[0096] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and 3,4-dimethoxyphenylethylamine. The product was a brownish-red powder, with a yield of 55.28%. 1 H NMR(400MHz,Chloroform-d)δ7.30–7.23(m,2H),7.13–7.03(m,4H),6.97–6.89(m,3H),6.78–6.66(m,3H),6.13(s ,2H),5.83(s,1H),4.18(s,2H),3.82(d,J=9.5Hz,6H),3.68(s,6H),3.11(t,J=7.5Hz,2H),3.01(t,J=7.2Hz,2H). 13CNMR(101MHz,Chloroform-d)δ161.22,159.75,149.46,149.12,147.92,143.40,139.80,129.65,129.40,120.99,120.91,119.55,117.35 ,111.88,111.38,101.64,93.53,78.21,66.31,58.98,55.95,55.92,55.89,55.85,55.81,47.28,39.77,32.17,18.66.ESI-MS: masscalcd for C 31 H 34 N₂O₅[M+H] + (515.2546); found m / z,515.2539.

[0097] Synthesis of target compound A9:

[0098]

[0099] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and 2-pyridineethylamine. The product was a brownish-red powder, with a yield of 52.96%. 1 H NMR(400MHz,Chloroform-d)δ7.59(td,J=7.7,1.9Hz,1H),7.26(ddd,J=9.6,6.9,2.1Hz,3H),7.13(dd,J=7.7,4.5Hz,2H),7.11–7 .07(m,2H),7.04(d,J=7.9Hz,2H),6.97–6.91(m,3H),6.17(s,2H),5.77(s,1H),4.14(s,2H),3.76(s,6H),3.22(h,J=5.0Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ160.50,159.69,159.35,149.95,148.42,143.52,139.50,136.83,129.40,123.51 ,121.73,120.82,120.77,119.73,117.23,94.23,93.86,78.22,55.88,55.84,45.92,40.14,33.50.ESI-MS:mass calcd forC 28 H 29 N3O3[M+H] + (456.2287); found m / z,456.2257.

[0100] Synthesis of target compound A10:

[0101]

[0102] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and 2-thiopheneethylamine. The product was a brownish-red powder, with a yield of 56.23%. 1 H NMR(400MHz,Chloroform-d)δ7.21–7.15(m,2H),7.05(dd,J=5.1,1.2Hz,1H),7.03–6.92(m,4H),6.91–6.86(m,2H),6.86–6.80(m, 2H),6.74(dd,J=3.4,1.1Hz,1H),6.10(s,2H),5.64(s,1H),3.84(s,2H),3.61(s,6H),3.04(t,J=7.1Hz,2H),2.84(t,J=7.1Hz,2H). 13 C NMR(101MHz,Chloroform-d)δ159.43,159.14,150.73,143.74,142.14,139.02,129.40,126.81,125.24, 123.61,120.65,120.38,120.01,117.01,108.56,94.28,55.72,55.68,49.44,40.70,29.54.ESI-MS:mass calcd for C 27 H 28 N₂O₃S[M+H] + (461.1899); found m / z,461.1858.

[0103] Synthesis of target compound A11:

[0104]

[0105] The product was prepared according to general synthetic method M3, from intermediate compound AM-2 and p-trifluoromethylphenylethylamine. The product was a brownish-red crystalline solid with a yield of 60.23%. 1H NMR(400MHz,Chloroform-d)δ7.52(d,J=8.0Hz,2H),7.27(dd,J=17.7,7.8Hz,4H),7.11–7.05(m,2H),7.05–6.98(m,2H) ,6.99–6.86(m,3H),6.16(s,2H),5.82–5.52(m,1H),3.94(s,2H),3.67(s,6H),3.00(t,J=7.1Hz,2H),2.96–2.85(m,2H). 13 CNMR(101MHz,Chloroform-d)δ159.53,159.46,150.44,143.64,143.60,139.20,129.40,129.13,128.80,128.47,125.62,125.36 ,125.33,125.29,122.92,120.73,120.52,119.90,117.10,107.44,94.09,55.71,55.67,48.66,40.60,34.72.ESI-MS: masscalcd for C 30 H 29 F3N2O3[M+H] + (523.2209); found m / z,523.2212.

[0106] 2. Synthesis of compounds B1-B25

[0107] Synthesis route:

[0108]

[0109] Reagents and conditions: (i) 4-Aminophenol, t-BuONa, Brettphos Pd G3, ultra-dry reagent 1,4-dioxane, 90℃, 3h, 75.1%-81.3%. (ii) 4-Bromo-2,6-dimethoxybenzaldehyde, Pd2(dba)3, t-BuXPhos, Cs2CO3, toluene, nitrogen protection, 100℃, 12h, 60.0%-65.7%. (iii) 4-Trifluoromethoxyphenylethylamine, methanol, 65℃, reflux 2h, NaBH4, 1h, 55%-65%.

[0110] Synthesis of intermediate B1-M1:

[0111]

[0112] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-fluorobromobenzene. The product was a brownish-red solid with a yield of 75.22%. 1 H NMR(400MHz,Chloroform-d)δ6.96–6.89(m,4H),6.89–6.83(m,2H),6.81–6.74(m,2H),5.35(s,1H).

[0113] Synthesis of intermediate B1-M2:

[0114]

[0115] The product was prepared from intermediates B1-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brownish-red solid with a yield of 48.97%. 1 H NMR(400MHz,Chloroform-d)δ10.36(s,1H),7.06–7.03(m,2H),7.01(d,J=2.3Hz, 2H),6.98(dt,J=8.9,2.7Hz,4H),6.11(s,2H),5.87(d,J=6.3Hz,1H),3.79(s,6H).

[0116] Synthesis of target compound B1:

[0117]

[0118] The product was prepared according to general synthetic method M3, from intermediates B1-M2 and trifluoromethylphenylethylamine. The product was a brownish-red solid with a yield of 65.32%. 1 H NMR(400MHz,Chloroform-d)δ7.45(s,2H),7.19(s,2H),6.93–6.90(m,2H),6.90–6.87(m,4H),6.87–6.84(m, 2H),6.08(s,2H),5.66–5.46(m,1H),3.78(s,2H),3.58(s,6H),2.84(d,J=5.2Hz,2H),2.81(d,J=5.3Hz,2H). 13C NMR(101MHz,Chloroform-d)δ159.37,159.08,158.98,156.60,150.31,144 .16,139.93,139.60,139.58,129.10,128.61,128.29,125.67,125.27,125 .23,125.20,122.97,120.51,119.65,119.58,118.87,116.07,115.84,109 .11,94.14,55.63,55.60,50.56,50.54,49.16,40.86,35.39.ESI-MS:mass calcd for C 30 H 28 F4N2O3[M+H] + (541.2114); found m / z,541.2116.

[0119] Synthesis of intermediate B2-M1:

[0120]

[0121] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-chlorobromobenzene. The product was a brownish-red solid with a yield of 72.54%. 1 H NMR (400MHz, Chloroform-d) δ7.14 (d, J = 6.6 Hz, 2H), 7.00–6.94 (m, 2H), 6.83–6.75 (m, 4H), 5.43 (s, 1H), 5.05 (s, 1H).

[0122] Synthesis of intermediate B2-M2:

[0123]

[0124] The product was prepared from intermediates B2-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 47.32%. 1 H NMR(400MHz,Chloroform-d)δ10.36(d,J=6.0Hz,1H),7.25–7.18(m,2H),7.12 –7.06(m,2H),7.05–6.95(m,4H),6.11(s,2H),5.95–5.84(m,1H),3.79(s,6H).

[0125] Synthesis of target compound B2:

[0126]

[0127] The product was prepared according to general synthetic method M3, from intermediate B2-M2 and trifluoromethylphenylethylamine. The product was a brownish-red solid with a yield of 62.36%. 1 H NMR(400MHz,Chloroform-d)δ7.45(d,J=8.0Hz,2H),7.22(d,J=8.0Hz,2H),7.15–7.10(m,2H),7.01–6.95(m ,2H),6.90–6.84(m,4H),6.09(s,2H),5.61(s,1H),3.79(s,2H),3.59(s,6H),2.84(dq,J=11.2,6.2Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ159.39,158.81,151.22,144.14,142.51,138.45,129.30,129.11,128.64,125. 29,125.25,125.10,120.39,120.32,117.98,109.27,94.39,55.66,55.63,49.18,40.86,35.38.ESI-MS:mass calcd for C 30 H 28 ClF3N2O3[M+H] + (557.1819); found m / z,557.1821.

[0128] Synthesis of intermediate B3-M1:

[0129]

[0130] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with 1,4-dibromobenzene. The product was a brownish-red solid with a yield of 62.22%. 1 H NMR (400MHz, Chloroform-d) δ7.26 (dd, J = 8.5, 3.4Hz, 2H), 7.01–6.95 (m, 2H), 6.82–6.76 (m, 2H), 6.76–6.70 (m, 2H), 5.50 (s, 1H).

[0131] Synthesis of intermediate B3-M2:

[0132]

[0133] The product was prepared from intermediates B3-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 46.39%.1 H NMR(400MHz,Chloroform-d)δ10.36(d,J=6.0Hz,1H),7.25–7.18(m,2H),7.12 –7.06(m,2H),7.05–6.95(m,4H),6.11(s,2H),5.95–5.84(m,1H),3.79(s,6H).

[0134] Synthesis of target compound B3:

[0135]

[0136] The product was prepared according to general synthetic method M3, from intermediate B3-M2 and trifluoromethylphenylethylamine. The product was a brownish-red solid with a yield of 65.36%. 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=8.0Hz,2H),7.35–7.27(m,3H),7.13–7.01(m,3H),6.98–6.92(m,2H),6.92 –6.80(m,2H),6.16(d,J=9.7Hz,2H),5.68(s,1H),3.87(d,J=1.8Hz,2H),3.66(d,J=4.3Hz,6H),2.94–2.89(m,4H). 13 C NMR(101MHz,Chloroform-d)δ159.38,158.76,151.37,150.68,143.70,143.05,139.05,138.22,132.20,129.40,129.11,125.66,125.29,125.25 ,121.48,120.69,120.62,120.39,120.29,119.99,118.23,117.04,112. 19,94.42,94.23,93.88,55.67,55.62,49.09,40.85,35.38.ESI-MS:mass calcd for C 30 H 28 BrF3N2O3[M+H] + (601.1314); found m / z,601.1317.

[0137] Synthesis of intermediate B4-M1:

[0138]

[0139] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-methoxybromobenzene. The product was a brownish-red solid with a yield of 58.87%. 1 H NMR (400MHz, Chloroform-d) δ6.95–6.86(m,4H),6.85–6.79(m,2H),6.77–6.71(m,2H),3.78(s,3H).

[0140] Synthesis of intermediate B4-M2:

[0141]

[0142] The product was prepared from intermediate B4-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 53.35%. 1 H NMR(400MHz,Chloroform-d)δ10.29(s,1H),7.07–6.97(m,2H),6.92–6.82(m ,4H),6.83–6.76(m,2H),6.03(s,2H),5.52(s,1H),3.73(s,3H),3.72(s,6H).

[0143] Synthesis of target compound B4:

[0144]

[0145] The product was prepared according to general synthetic method M3, from intermediate B4-M2 and trifluoromethylphenylethylamine. The product was a brownish-red solid with a yield of 55.37%. 1 H NMR(400MHz,Chloroform-d)δ7.44(d,J=8.0Hz,2H),7.21(d,J=7.9Hz,2H),6.99–6.94(m,2H),6.84(s,4H ),6.81–6.74(m,2H),6.08(s,2H),5.40(s,1H),3.74(s,2H),3.72(s,3H),3.57(s,6H),2.83–2.77(m,4H). 13C NMR(101MHz,Chloroform-d)δ159.29,159.09,155.08,149.49,144.54,141.27,136.33,129.09,128.50,128.18,125.70,125 .22,125.18,123.01,121.40,120.59,117.44,114.75,109.88,93.99,55.63,55.61,55.58,49.52,41.05,35.85.ESI-MS:mass calcd for C 31 H 31 F3N2O4[M+H] + (552.2236);foundm / z,552.2238.

[0146] Synthesis of intermediate B5-M1:

[0147]

[0148] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with 3,4-dimethoxybromobenzene. The product was a brown solid with a yield of 62.13%. 1 H NMR (400MHz, Chloroform-d) δ6.94–6.87(m,2H),6.76(dd,J=8.7,2.6Hz,3H),6.56(d,J=2.6Hz,1H),6.49(dd,J=8.5,2.6Hz,1H),3.83(s,3H),3.79(s,3H).

[0149] Synthesis of intermediate B5-M2:

[0150]

[0151] The product was prepared from intermediates B5-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 55.87%. 1 H NMR(400MHz,Chloroform-d)δ10.36(s,1H),6.98(s,4H),6.87–6.80(m,1H),6.7 5–6.68(m,2H),6.11(s,2H),5.67(s,1H),3.87(s,3H),3.85(s,3H),3.79(s,6H).

[0152] Synthesis of target compound B5:

[0153]

[0154] The product was prepared according to general synthetic method M3, from intermediate B5-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 52.98%. 1 H NMR(400MHz,Chloroform-d)δ7.52(d,J=8.0Hz,2H),7.30(s,2H),6.98–6.88(m,4H),6.81(d,J=8.4Hz,1H),6.72–6.61(m,2H),6 .14(s,2H),5.58(s,1H),3.99(s,2H),3.86(s,3H),3.84(s,3H),3.68(s,6H),3.06(t,J=7.6Hz,2H),2.94(dd,J=8.3,6.2Hz,2H). 13 C NMR(101MHz,Chloroform-d)δ160.19,159.52,149.74,149.13,144.57,143.17,141.26,136.78,129.13,128.89,128.57,125.58,125.41,125.37,1 22.87,120.83,117.74,112.31,111.43,105.88,104.88,93.74,90.28,56 .36,56.30,55.92,55.87,55.73,55.68,48.26,40.37,34.15.ESI-MS:mass calcd for C 32 H 33 F3N2O5[M+H] + (583.2420);foundm / z,583.2421.

[0155] Synthesis of intermediate B6-M1:

[0156]

[0157] The product was prepared by replacing bromobenzene with p-methylbromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a brown solid with a yield of 70.13%. 1 H NMR (400MHz, Chloroform-d) δ7.03(d,J=8.1Hz,2H),7.00–6.94(m,2H),6.89–6.81(m,2H),6.80–6.74(m,2H),5.37(s,1H),4.54(s,1H),2.27(s,3H).

[0158] Synthesis of intermediate B6-M2:

[0159]

[0160] The product was prepared from intermediate B6-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 62.37%. 1 H NMR(400MHz,Chloroform-d)δ10.37(s,1H),7.11(d,J=8.2Hz,2H),7.07–7.03( m,2H),7.03–6.95(m,4H),6.11(s,2H),5.63(s,1H),3.80(s,6H),2.32(s,3H).

[0161] Synthesis of target compound B6:

[0162]

[0163] The product was prepared according to general synthetic method M3, from intermediate B6-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 54.98%. 1 H NMR(400MHz,Chloroform-d)δ7.51(d,J=8.0Hz,2H),7.28(s,2H),7.06(d,J=8.1Hz,2H),7.03–6.97(m,2H),6.97–6.9 0(m,4H),6.16(s,2H),5.74(s,1H),3.88(s,2H),3.64(s,6H),3.51(s,1H),2.91(dq,J=11.5,6.1Hz,4H),2.28(s,3H). 13 C NMR(101MHz,Chloroform-d)δ159.41,149.86,144.07,140.90,140.16,130.5 5,129.91,129.13,128.63,128.31,127.99,125.70,125.33,125.29,125.26,1 25.22,123.00,120.57,119.58,118.77,118.16,108.58,94.03,92.85,55.69 ,55.65,55.60,50.27,49.03,40.79,35.24,20.68,20.64,20.60.ESI-MS:mass calcd for C 31 H 31 F3N2O3[M+H] + (536.2287); found m / z,536.2288.

[0164] Synthesis of intermediate B7-M1:

[0165]

[0166] The product was prepared by replacing bromobenzene with 3-methyl-4-methoxybromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a brown solid with a yield of 67.73%. 1 H NMR (400MHz, Chloroform-d) δ6.77 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 7.3 Hz, 2H), 6.63 (d, J = 7.4 Hz, 3H), 5.59 (s, 1H), 3.69 (s, 3H), 2.09 (s, 3H).

[0167] Synthesis of intermediate B7-M2:

[0168]

[0169] The product was prepared from intermediates B7-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 56.37%. 1 H NMR(400MHz,Chloroform-d)δ10.29(s,1H),6.90(d,J=2.7Hz,1H),6.87(d,J=1.9Hz,3H),6.85(d, J=9.2Hz,2H),6.75–6.71(m,1H),6.03(s,2H),5.41(s,1H),3.76(s,3H),3.73(s,6H),2.15(s,3H).

[0170] Synthesis of target compound B7:

[0171]

[0172] The product was prepared according to general synthetic method M3, from intermediate B7-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 54.98%. 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=7.9Hz,2H),7.29(d,J=7.9Hz,3H),6.96–6.85(m,6H),6.82–6.75(m,1H),6.15(s ,2H),5.38(d,J=60.6Hz,1H),3.93(s,2H),3.82(s,3H),3.66(s,6H),2.99–2.95(m,2H),2.95–2.91(m,2H),2.21(s,3H). 13C NMR(101MHz,Chloroform-d)δ160.09,159.45,153.43,148.77,143.49,141.82 ,135.71,129.14,128.79,128.47,128.14,127.73,125.63,125.40,125.36,12 5.32,125.28,123.36,122.93,121.78,120.84,118.38,117.17,110.93,106.8 1,93.69,77.30,55.71,55.64,50.44,48.52,40.52,34.55,16.33.ESI-MS:mass calcd for C 32 H 33 F3N2O4[M+H] + (567.2471); found m / z,567.2482.

[0173] Synthesis of intermediate B8-M1:

[0174]

[0175] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-ethylbromobenzene. The product was a brown solid with a yield of 74.23%. 1 H NMR(400MHz,Chloroform-d)δ7.01–6.96(m,2H),6.92–6.87(m,2H),6.82–6.76(m, 2H), 6.74–6.59 (m, 2H), 5.31 (s, 1H), 2.50 (q, J = 7.6Hz, 2H), 1.13 (t, J = 7.6Hz, 3H).

[0176] Synthesis of intermediate B8-M2:

[0177]

[0178] The product was prepared from intermediate B8-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 53.10%. 1 H NMR (400MHz, Chloroform-d) δ10.30 (s, 1H), 7.06 (d, J = 8.4Hz, 2H), 7.00–6.94 (m, 4H), 6.93–6. 89(m,2H),6.04(s,2H),5.59(s,1H),3.73(s,6H),2.54(q,J=7.6Hz,2H),1.16(t,J=7.6Hz,3H).

[0179] Synthesis of target compound B8:

[0180]

[0181] The product was prepared according to general synthetic method M3, from intermediate B8-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 56.23%. 1 H NMR(400MHz,Chloroform-d)δ7.43(d,J=8.0Hz,2H),7.20(s,2H),7.01(d,J=8.3Hz,2H),6.96–6.82(m,6H),6.08(d,J=1.0H z,2H),5.58(s,1H),3.74(d,J=1.3Hz,2H),3.56(s,6H),2.78(d,J=3.4Hz,4H),2.50(q,J=7.6Hz,2H),1.13(t,J=7.6Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ159.32,158.95,150.18,144.53,141.19,139.91,137.05,129.10,128.83,128.72,128.51,128.19,125.73 ,125.23,125.19,125.16,123.03,120.43,118.98,118.01,110.01,94.17,55.61,55.57,49.50,41.04,35.83,28.15,15.82.ESI-MS:mass calcd for C 32 H 33 F3N2O3[M+H] + (551.2522); found m / z,551.2529.

[0182] Synthesis of intermediate B9-M1:

[0183]

[0184] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-propylbromobenzene. The product was a brown solid with a yield of 71.84%. 1H NMR(400MHz,Chloroform-d)δ6.98–6.93(m,2H),6.91–6.87(m,2H),6.80–6.74(m,2H),6.71–6.65 (m,2H),5.30(s,1H),4.81(s,1H),2.47–2.39(m,2H),1.53(q,J=7.5Hz,2H),0.86(t,J=7.3Hz,3H).

[0185] Synthesis of intermediate B9-M2:

[0186]

[0187] The product was prepared from intermediates B9-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 56.50%. 1 H NMR(400MHz,Chloroform-d)δ10.30(s,1H),7.08–7.02(m,2H),7.01–6.97(m,2H),6.97–6.93(m,2H),6.93–6.89(m ,2H),6.04(s,2H),5.58(s,1H),3.73(s,6H),2.48(dd,J=8.5,6.7Hz,2H),1.59–1.55(m,2H),0.88(t,J=7.3Hz,3H).

[0188] Synthesis of target compound B9:

[0189]

[0190] The product was prepared from intermediate B9-M2 and trifluoromethylphenylethylamine according to general synthetic method M3. The product was a brown solid with a yield of 52.19%. 1 H NMR(400MHz,Chloroform-d)δ7.42(d,J=7.9Hz,2H),7.18(d,J=8.1Hz,2H),6.98( d,J=8.2Hz,2H),6.94–6.91(m,2H),6.88(d,J=8.3Hz,2H),6.82(d,J=8.8Hz,2H),6 .06(s,2H),5.66(s,1H),3.91(s,2H),3.59(s,6H),2.98(t,J=7.6Hz,2H),2.84(t, J=7.5Hz,2H),2.43(t,J=7.6Hz,2H),1.52(q,J=7.5Hz,2H),0.85(d,J=7.3Hz,3H). 13CNMR(101MHz,Chloroform-d)δ160.20,159.96,159.59,149.50,143.24,141 .04,140.29,135.59,129.32,129.15,129.05,128.87,128.55,125.63,125.3 9,122.93,120.75,120.45,119.94,118.95,118.78,118.04,117.83,105.89, 93.81,55.72,48.28,40.35,37.34,34.13,24.74,13.89,13.84.ESI-MS:mass calcd for C 33 H 35 F3N2O3[M+H] + (564.2600);foundm / z,564.2622.

[0191] Synthesis of intermediate B10-M1:

[0192]

[0193] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-butylbromobenzene. The product was a brown solid with a yield of 68.24%. 1 H NMR(400MHz,Chloroform-d)δ6.97–6.94(m,2H),6.92–6.88(m,2H),6.81–6.75(m,2H),6.71–6.66(m,2H),5. 30(s,1H),4.58(s,1H),2.46(dd,J=9.1,6.4Hz,2H),1.52–1.47(m,2H),1.29–1.25(m,2H),0.86–0.83(m,3H).

[0194] Synthesis of intermediate B10-M2:

[0195]

[0196] The product was prepared from intermediates B10-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 53.52%. 1H NMR(400MHz,Chloroform-d)δ10.30(s,1H),7.08–7.02(m,2H),7.00–6.89(m,6H),6.04(s,2H),5.58 (s,1H),3.73(s,6H),2.54–2.47(m,2H),1.52–1.47(m,2H),1.32–1.26(m,2H),0.86(t,J=7.3Hz,3H).

[0197] Synthesis of target compound B10:

[0198]

[0199] The product was prepared according to general synthetic method M3, from intermediate B10-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 57.28%. 1 H NMR(400MHz,Chloroform-d)δ7.43(d,J=7.9Hz,2H),7.20(d,J=8.0Hz,2H),6.99(d,J=8 .1Hz,2H),6.95–6.92(m,2H),6.89(d,J=8.3Hz,2H),6.83(d,J=8.6Hz,2H),6.06(s,2H), 5.60(s,1H),3.90(s,2H),3.59(s,6H),2.97(t,J=7.5Hz,2H),2.85(t,J=7.5Hz,2H),2. 47(t,J=7.7Hz,2H), 1.49(t,J=7.9Hz,2H), 1.27(q,J=7.4Hz,2H), 0.85(d,J=7.2Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ159.32,158.92,150.18,144.56,141.21,139.89,135.47,129.32,129.10,128.82,128.50,128.18,125.73,125.2 2,125.18,125.15,123.03,120.42,119.00,117.87,110.12,94.16,55.5 9,55.56,50.38,49.48,41.04,37.34,35.87,24.75,13.84.ESI-MS:mass calcd for C 34 H 37 F3N2O3[M+H] + (579.2835); found m / z,579.2837.

[0200] Synthesis of intermediate B11-M1:

[0201]

[0202] The intermediate compound AM-1 was prepared by replacing bromobenzene with p-bromobenzyl alcohol, following the synthetic method M1. The product was a brown solid with a yield of 65.89%. 1 H NMR (400MHz, DMSO-d6) δ7.59 (s, 1H), 7.08 (d, J = 8.5Hz, 2H), 6.95–6.88 (m, 2H), 6. 85–6.78(m,2H),6.73–6.66(m,2H),4.90(t,J=5.6Hz,1H),4.35(d,J=5.0Hz,2H).

[0203] Synthesis of intermediate B11-M2:

[0204]

[0205] The product was prepared from intermediates B11-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 57.92%. 1 H NMR (400MHz, Chloroform-d) δ10.31(s,1H),7.08–7.01(m,4H),6.98–6.94(m,4H),6.15(s,1H),6.08(s,2H),3.75(s,6H),3.42(s,2H).

[0206] Synthesis of target compound B11:

[0207]

[0208] The product was prepared according to general synthetic method M3, from intermediate B11-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 49.22%. 1 H NMR(400MHz,Chloroform-d)δ7.45(dd,J=8.0,5.7Hz,4H),7.12–7.06(m,2H),6.98–6.95(m,2H),6.91 –6.85(m,4H),6.09(s,2H),5.59(s,1H),3.76(s,2H),3.66(s,2H),3.57(s,6H),2.81(d,J=4.4Hz,4H). 13C NMR(101MHz,Chloroform-d)δ159.32,158.78,150.77,144.22,142.73,139.06,132.17,129.29,129.10,129.04,125.38,125 .34,125.24,125.20,120.32,119.80,117.15,109.95,94.31,55.62,55.58,53.36,50.05,49.42,36.18,35.72.ESI-MS:mass calcd for C 31 H 31 F3N2O4[M+H] + (553.2314); found m / z,553.2318.

[0209] Synthesis of intermediate B12-M1:

[0210]

[0211] The intermediate compound AM-1 was prepared by replacing bromobenzene with p-bromophenylethanol, following the synthetic method M1. The product was a brown solid with a yield of 75.29%. 1 H NMR (400MHz, Methanol-d4) δ7.03–6.98(m,2H),6.97–6.90(m,2H),6.87–6.80(m,2H),6.74–6.66(m,2H),3.68(t,J=7.2Hz,2H),2.70(t,J=7.2Hz,2H).

[0212] Synthesis of intermediate B12-M2:

[0213]

[0214] The product was prepared from intermediates B12-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 52.85%. 1 H NMR(400MHz,Chloroform-d)δ10.30(s,1H),7.12–7.07(m,2H),7.03–6.99(m,2H),6.99–6.95(m,2H),6 .95–6.91(m,2H),6.05(s,2H),5.63(s,1H),3.79(d,J=3.9Hz,2H),3.73(s,6H),2.76(t,J=6.5Hz,2H).

[0215] Synthesis of target compound B12:

[0216]

[0217] The product was prepared according to general synthetic method M3, from intermediate B12-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 59.82%. 1 H NMR(400MHz,Chloroform-d)δ7.44(d,J=8.0Hz,2H),7.02–6.98(m,2H),6.96–6.91(m,2H),6.89–6.84(m,4H),6.08(s,2H ),5.54(s,1H),3.74(s,2H),3.57(s,6H),2.79(d,J=2.9Hz,4H),2.47(t,J=7.7Hz,2H),1.49(ddt,J=9.1,7.7,3.5Hz,2H). 13 C NMR(101MHz,Chloroform-d)δ159.31,158.91,150.22,144.57,141.14,139.86,135.72,129.26,129.10,128.51,128.19,125.72,125.22,12 5.19,125.15,123.02,120.41,119.01,117.91,110.09,94.18,55.61,55.58,49.55,41.06,35.87,34.92,33.86,22.36,14.00.ESI-MS:mass calcd for C 32 H 33 F3N2O4[M+H] + (567.2471); found m / z,567.2475.

[0218] Synthesis of intermediate B13-M1:

[0219]

[0220] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-bromophenylpropanol. The product was a brown solid with a yield of 79.09%. 1 H NMR(400MHz,Methanol-d4)δ7.03–6.96(m,2H),6.96–6.88(m,2H),6.86–6.78(m,2H),6 .73–6.64(m,2H),3.55(t,J=6.6Hz,2H),2.56(dd,J=8.6,6.8Hz,2H),1.83–1.73(m,2H).

[0221] Synthesis of intermediate B13-M2:

[0222]

[0223] The product was prepared from intermediates B13-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 48.80%. 1 H NMR (400MHz, Methanol-d4) δ10.21(s,1H),7.10(t,J=2.3Hz,2H),7.08(d,J=2.4Hz,2H),7.05–7.01(m,2H),6.98–6.96 (m,2H),6.18(s,2H),5.48(s,1H),3.77(s,6H),3.57(t,J=6.5Hz,2H),2.61(dd,J=8.7,6.7Hz,2H),1.85–1.77(m,2H).

[0224] Synthesis of target compound B13:

[0225]

[0226] The product was prepared according to general synthetic method M3, from intermediate B13-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 48.24%. 1 H NMR(400MHz,Chloroform-d)δ7.52–7.44(m,2H),7.22(s,2H),7.08–7.03(m,2H),7 .01–6.96(m,2H),6.91(ddt,J=15.3,6.6,1.6Hz,4H),6.10(d,J=1.5Hz,2H),3.85( d,J=1.5Hz,2H),3.63(td,J=6.4,1.5Hz,2H),3.59(d,J=1.6Hz,6H),2.93–2.82(m, 4H), 2.61 (td, J=7.6, 1.4Hz, 2H), 1.97 (s, 1H), 1.83 (dtd, J=11.3, 6.4, 3.2Hz, 2H). 13C NMR(101MHz,Chloroform-d)δ159.56,159.41,149.98,143.57,141.35,139.85,134.56,129.32,129.15,125.36,125.3 2,120.59,119.11,117.93,107.55,93.98,77.24,62.23,55.63,48.18,40.34,34.70,34.41,31.35,22.96.ESI-MS:mass calcd for C 33 H 35 F3N2O4[M+H] + (580.2549); found m / z,580.2551.

[0227] Synthesis of intermediate B14-M1:

[0228]

[0229] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-ethoxybromobenzene. The product was a brown solid with a yield of 75.09%. 1 H NMR(400MHz,Chloroform-d)δ6.87–6.78(m,4H),6.77–6.71(m,2H),6.70–6.64 (m,2H),5.18(s,1H),4.55(s,1H),3.92(q,J=7.0Hz,2H),1.32(t,J=7.0Hz,3H).

[0230] Synthesis of intermediate B14-M2:

[0231]

[0232] The product was prepared from intermediate B14-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 53.20%. 1 H NMR(400MHz,Chloroform-d)δ10.29(s,1H),7.02–6.98(m,2H),6.90–6.85(m,4H),6.82–6.79 (m,2H),6.03(s,2H),5.45(s,1H),3.95(q,J=7.0Hz,2H),3.73(s,6H),1.35(t,J=7.0Hz,3H).

[0233] Synthesis of target compound B14:

[0234]

[0235] The product was prepared according to general synthetic method M3, from intermediate B14-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 52.24%. 1 H NMR(400MHz,Chloroform-d)δ7.43(d,J=8.0Hz,2H),6.97–6.90(m,2H),6.82(s,4H),6.78–6.72(m,2H),6.07( s,2H),5.53–5.33(m,1H),3.91(q,J=7.0Hz,2H),3.73(s,2H),3.55(s,6H),2.78(s,4H),1.31(t,J=7.0Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ159.29,159.09,154.39,149.42,144.61,141.35,136.25,129.10,128.47,128.15,125.73,125.21,12 5.17,123.03,121.38,120.60,117.39,115.43,110.04,93.98,63.85,55.60,55.56,49.57,41.06,35.91,14.95,14.93.ESI-MS:mass calcd forC 32 H 33 F3N2O4[M+H] + (566.2392); found m / z,566.2393.

[0236] Synthesis of intermediate B15-M1:

[0237]

[0238] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-bromobenzoate. The product was a brown solid with a yield of 67.09%. 1 H NMR (400MHz, Methanol-d4) δ7.79–7.75(m,2H),7.05–7.00(m,2H),6.84–6.75(m,4H),3.82(s,3H).

[0239] Synthesis of intermediate B15-M2:

[0240]

[0241] The product was prepared from intermediates B15-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 54.90%. 1 H NMR(400MHz,Chloroform-d)δ10.31(s,1H),7.90–7.81(m,2H),7.18–7.12(m,2H),7.0 5–6.98(m,2H),6.95–6.89(m,2H),6.07(s,2H),5.95(s,1H),3.81(s,3H),3.75(s,6H).

[0242] Synthesis of target compound B15:

[0243]

[0244] The product was prepared according to general synthetic method M3, from intermediate B15-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 58.45%. 1 H NMR(400MHz,Chloroform-d)δ7.93–7.88(m,2H),7.53(d,J=7.8Hz,2H),7.29(d,J=8.1Hz,2H),7.16(dt,J=8.3,2.3Hz,2H),6.99(dd,J=8.6,1.6Hz,2H), 6.94–6.90(m,2H),6.19(d,J=1.4Hz,2H),6.08–6.07(m,1H),3.87(d,J=1.3 Hz,3H),3.71(d,J=1.5Hz,2H),3.67(d,J=1.4Hz,6H),2.91(d,J=3.4Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ166.98,159.40,158.31,152.74,148.68,144.16,136.38,131.53,129.13,129.10,125 .28,125.24,122.84,120.80,119.98,113.97,94.78,90.33,77.24,55.65,51.72,49.26,40.91,35.46.ESI-MS:mass calcd for C 32 H 31 F3N2O5[M+H] + (581.2263); found m / z,581.2279.

[0245] Synthesis of intermediate B16-M1:

[0246]

[0247] The product was prepared by replacing bromobenzene with p-cyanobromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a brown solid with a yield of 72.19%. 1 H NMR (400MHz, Methanol-d4) δ7.43–7.37(m,2H),7.07–6.99(m,2H),6.86–6.81(m,2H),6.81–6.74(m,2H).

[0248] Synthesis of intermediate B16-M2:

[0249]

[0250] The product was prepared from intermediate B16-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 56.89%. 1 H NMR (400MHz, Chloroform-d) δ10.31(s,1H),7.06–6.99(m,4H),6.93–6.87(m,2H),6.74–6.68(m,2H),6.07(s,2H),5.79(s,1H),3.75(s,6H).

[0251] Synthesis of target compound B16:

[0252]

[0253] The product was prepared according to general synthetic method M3, from intermediate B16-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 62.87%. 1 H NMR(400MHz,Chloroform-d)δ7.44(d,J=8.0Hz,2H),7.39–7.35(m,2H),7.20(d,J=8.0Hz,2H),7.09–7.05(m,2H),6.94– 6.90(m,2H),6.84–6.80(m,2H),6.34(s,1H),6.11(s,2H),3.80(s,2H),3.59(s,6H),3.09(s,1H),2.84(q,J=3.6Hz,4H). 13C NMR(101MHz,Chloroform-d)δ159.44,158.16,153.36,148.70,144.05,135.53,133.76,129.10,128.65,128.33,125.29, 125.25,123.57,122.95,120.01,119.90,114.35,109.72,100.94,94.93,55.68,55.65,49.26,40.87,35.34.ESI-MS:mass calcd for C 31 H 28 F3N3O3[M+H] + (547.2083); found m / z,547.2086.

[0254] Synthesis of intermediate B17-M1:

[0255]

[0256] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with p-tert-butylbromobenzene. The product was a yellowish-brown solid with a yield of 68.19%. 1 H NMR (400MHz, Chloroform-d) δ7.26 (d, J = 4.3Hz, 2H), 7.01 (d, J = 8.2Hz, 2H), 6.89 (d, J = 8.2Hz, 2H), 6.82–6.75 (m, 2H), 1.31 (s, 9H).

[0257] Synthesis of intermediate B17-M2:

[0258]

[0259] The product was prepared from intermediates B17-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown oily substance with a yield of 52.91%. 1 H NMR(400MHz,Chloroform-d)δ10.37(s,1H),7.33(d,J=8.7Hz,2H),7.11–7.07(m, 2H),7.06–7.04(m,2H),7.01–6.98(m,2H),6.12(s,2H),3.81(s,6H),1.33(s,9H).

[0260] Synthesis of target compound B17:

[0261]

[0262] The product was prepared according to general synthetic method M3, from intermediate B17-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 57.28%. 1 H NMR(400MHz,Chloroform-d)δ7.56–7.52(m,2H),7.32–7.29(m,4H),7.07–7.04(m,2H),7.02–6.99(m,2H) ,6.97–6.94(m,2H),6.18(s,2H),5.63(s,1H),3.84(s,2H),3.67(s,6H),2.91–2.88(m,4H),1.32(s,9H). 13 C NMR(101MHz,Chloroform-d)δ159.33,159.04,150.21,144.36,143.91,140.95,139.74,129.11,128.57,128.40,128.25,126.18,125.70,125 .29,125.25,125.22,125.18,122.99,120.43,119.19,117.38,109.58,94.14,77.26,55.61,49.32,40.95,35.64,34.16,31.48.ESI-MS:mass calcd for C 34 H 37 F3N2O3[M+H] + (579.2882); found m / z579.2834.

[0263] Synthesis of intermediate B18-M1:

[0264]

[0265] The product was prepared by replacing bromobenzene with 3-fluoro-4-trifluoromethylbromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a brownish-black solid with a yield of 65.12%. 1 H NMR (400MHz, Chloroform-d) δ7.29(dd,J=11.6,2.1Hz,1H),7.20(ddt,J=8.5,1.9,0.9Hz,1H),7.14–7.08(m,2H),7.03–6.95(m,1H),6.90–6.85(m,2H).

[0266] Synthesis of intermediate B18-M2:

[0267]

[0268] The product was prepared from intermediate B18-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown oily substance with a yield of 54.19%. 1 H NMR(400MHz,Chloroform-d)δ10.39(s,1H),7.35(dd,J=11.4,1.9Hz,1H),7.28( d,J=1.6Hz,1H),7.26–7.23(m,3H),7.13–7.10(m,2H),6.15(s,2H),3.82(s,6H).

[0269] Synthesis of target compound B18:

[0270]

[0271] The product was prepared according to general synthetic method M3, from intermediate B18-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 52.19%. 1 H NMR(400MHz,Chloroform-d)δ7.55–7.48(m,2H),7.30–7.26(m,3H),7.25–7.21(m,1H),7.19 –7.14(m,3H),7.04–7.01(m,2H),6.21(s,2H),3.82(s,2H),3.66(s,6H),2.88–2.84(m,4H). 13 C NMR(101MHz,Chloroform-d)δ159.39,157.81,153.48,144.64,136.61,136.51,135.33,129.08,128.80,,128.16,125.73,125.26,125.15,125 .12,123.03,122.59,122.57,121.89,121.82,120.62,,112.54,112.50 ,111.20,94.98,77.30,55.57,50.19,49.64,41.07,35.95.ESI-MS:mass calcd for C 31 H 27 F7N2O3[M+H]+(609.1920); found m / z 609.1929.

[0272] Synthesis of intermediate B19-M1:

[0273]

[0274] The product was prepared by replacing bromobenzene with 4-trifluoromethylbromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a red solid with a yield of 62.12%. 1 H NMR(400MHz,Chloroform-d)δ7.31(s,2H),7.02–6.96(m,2H),6.82–6.72(m,4H),5.63(s,1H),4.96(s,1H).

[0275] Synthesis of intermediate B19-M2:

[0276]

[0277] The product was prepared from intermediates B19-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 56.83%. 1 H NMR (400MHz, Chloroform-d) δ10.31(s,1H),7.42(d,J=8.4Hz,2H),7.13(d,J=8.8Hz,2H),7.02–6.95(m,4H),6.07(s,2H),5.94(s,1H),3.74(s,6H).

[0278] Synthesis of target compound B19:

[0279]

[0280] The product was prepared according to general synthetic method M3, from intermediate B19-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 58.48%. 1 H NMR(400MHz,Chloroform-d)δ7.42(d,J=8.0Hz,2H),7.34(d,J=8.5Hz,2H),7.17(s,2H),7.05–7.01(m,2 H),6.91–6.86(m,4H),6.16(s,1H),6.10(s,2H),3.80(d,J=1.9Hz,2H),3.57(s,6H),2.87–2.78(m,4H). 13C NMR(101MHz,Chloroform-d)δ159.45,158.64,152.29,147.49,143.95,136.95,129.10,128.67,128.35,126.69,126.66,126.03,125. 67,125.30,125.26,123.34,122.96,122.32,120.94,120.14,114.63,109.14,94.62,55.65,55.61,49.08,40.79,35.19.ESI-MS:mass calcd for C 31 H 28 F6N2O3[M+H]+(591.2334); found m / z 591.2397.

[0281] Synthesis of intermediate B20-M1:

[0282]

[0283] The product was prepared by replacing bromobenzene with 3,4-ditrifluoromethylbromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a red solid with a yield of 63.81%. ¹H NMR (400 MHz, Chloroform-d) δ 7.30–7.22 (m, 2H), 6.93–6.86 (m, 2H), 6.83 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 2.8 Hz, 1H), 6.69 (dd, J = 8.6, 2.8 Hz, 1H).

[0284] Synthesis of intermediate B20-M2:

[0285]

[0286] The product was prepared from intermediate B20-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 46.47%. ¹H NMR (400 MHz, Chloroform-d) δ 10.39 (s, ¹H), 7.81–7.80 (m, ¹H), 7.57 (dd, J = 8.8, 2.1 Hz, ¹H), 7.27–7.24 (m, 2H), 7.20 (d, J = 8.8 Hz, 1H), 7.16–7.12 (m, 2H), 6.34 (s, ¹H), 6.17 (s, 2H), 3.83 (s, 6H).

[0287] Synthesis of target compound B20:

[0288]

[0289] The product was prepared according to general synthetic method M3, from intermediate B20-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 53.29%. 1 H NMR(400MHz,Chloroform-d)δ7.79–7.76(m,1H),7.52(dd,J=8.3,2.8Hz,3H),7.30(d,J=8.0Hz,2H),7.18(d,J=8.9 Hz,2H),7.10(d,J=8.8Hz,1H),7.07–7.02(m,2H),6.30(s,1H),6.23(s,2H),3.83(s,2H),3.69(s,6H),2.89(s,4H). 13 C NMR(101MHz,Chloroform-d)δ159.43,157.41,154.80,146.39,144.61,134.49,129.8 5,129.82,129.08,128.83,128.51,128.19,125.71,125.61,125.31,125.25,125.21, 125.17,125.13,123.01,122.87,122.62,120.26,119.93,119.74,114.83,114.73,11 4.43,114.13,111.43,95.28,95.01,77.25,55.63,49.71,41.09,35.93.ESI-MS:mass calcd for C 32 H 27 F9N2O3[M+H]+(659.1934); found m / z 659.1937.

[0290] Synthesis of intermediate B21-M2:

[0291]

[0292] The intermediate compound M2 was prepared from 6-hydroxyquinoline according to the synthetic method for AM-2. The product was a brown solid with a yield of 46.47%. 1 H NMR (400MHz, Chloroform-d) δ10.38(s,1H),7.79–7.76(m,2H),7.25(d,J=8.9Hz,2H),7.12–7.09(m,2H),6.15(s,2H),6.02(s,1H),3.82(s,6H).

[0293] Synthesis of target compound B21:

[0294]

[0295] The product was prepared according to general synthetic method M3, from intermediate B21-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 62.97%. 1 H NMR(400MHz,Chloroform-d)δ8.84(dd,J=4.2,1.7Hz,1H),8.10(d,J=9.2Hz,1H),8.03(ddd,J=8.5,1.9,0.8Hz,1H),7.57–7.53(m,2H),7.4 9(dd,J=9.2,2.7Hz,1H),7.38(dd,J=8.3,4.2Hz,1H),7.35–7.30(m,2H),7.24(s,1H),6.29(s,2H),3.86(s,2H),3.68(s,6H),2.92(s,4H). 13 C NMR(101MHz,Chloroform-d)δ159.53,157.22,155.46,149.15,145.15,144.25,135.21,131.36,129.10,128. 33,125.29,125.25,123.03,121.60,112.80,110.95,95.82,77.24,55.71,49.56,41.02,35.60.ESI-MS:mass calcd forC 27 H 25 F3N2O3[M+H]+(483.1896); found m / z: 483.1876.

[0296] Synthesis of intermediate B22-M1:

[0297]

[0298] The product was prepared by replacing bromobenzene with p-trifluoromethoxybromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a red solid with a yield of 58.97%. 1 H NMR (400MHz, Chloroform-d) δ6.99–6.94(m,2H),6.94–6.90(m,2H),6.77–6.70(m,4H),5.63(d,J=15.6Hz,1H),5.43(s,1H).

[0299] Synthesis of intermediate B22-M2:

[0300]

[0301] The product was prepared from intermediate B22-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 55.24%. 1 H NMR (400MHz, Chloroform-d) δ10.37(s,1H),7.14–7.10(m,4H),7.04(dd,J=8.9,2.2Hz,4H),6.12(s,2H),5.77(s,1H),3.81(s,6H).

[0302] Synthesis of target compound B22:

[0303]

[0304] The product was prepared according to general synthetic method M3, from intermediate B22-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 57.23%. 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=7.9Hz,2H),7.30(d,J=7.8Hz,2H),7.12–7.06(m,4H),6.98(td,J=6.5,6.1,1.6Hz ,4H),6.18(d,J=1.4Hz,2H),5.83(s,1H),3.90(s,2H),3.67(d,J=1.4Hz,6H),2.95(d,J=5.5Hz,2H),2.92(d,J=5.7Hz,2H). 13 CNMR(101MHz,Chloroform-d)δ161.27,159.45,159.15,151.14,143.75,142 .81,142.47,142.45,138.50,129.11,128.74,128.42,125.63,125.33,125.2 9,125.25,122.93,122.40,121.89,120.56,120.42,119.35,117.29,108.17, 94.28,90.31,77.25,55.65,55.59,55.29,48.86,40.68,34.91.ESI-MS:mass calcd for C 31 H 28 F6N2O4[M+H]+(607.2032); found m / z: 607.2045.

[0305] Synthesis of intermediate B23-M1:

[0306]

[0307] The product was prepared by replacing bromobenzene with p-thiomethylbromobenzene, following the synthetic method M1 for intermediate compound AM-1. The product was a brown solid with a yield of 55.37%. 1 H NMR (400MHz, Chloroform-d) δ7.16–7.10(m,2H),6.92–6.88(m,2H),6.80–6.74(m,2H),6.72–6.68(m,2H),5.39(s,1H),2.35(s,3H).

[0308] Synthesis of intermediate B23-M2:

[0309]

[0310] The product was prepared from intermediate B23-M2 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 52.56%. 1 H NMR(400MHz,Chloroform-d)δ10.30(s,1H),7.18(d,J=2.2Hz,2H),7.03–7.00(m,2H),6.95(d, J=4.1Hz,2H),6.94(dd,J=4.3,2.1Hz,2H),6.05(s,2H),5.63(s,1H),3.74(s,6H),2.40(s,3H).

[0311] Synthesis of target compound B23:

[0312]

[0313] The product was prepared according to general synthetic method M3, from intermediate B23-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 57.23%. 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=8.0Hz,2H),7.30(d,J=8.0Hz,2H),7.26(d,J=5.2Hz,2H),7.07–7.03(m,2H),6.98(d,J =4.7Hz,2H),6.96(dd,J=4.8,2.0Hz,2H),6.18(s,2H),5.73(s,1H),3.85(s,2H),3.67(s,6H),2.92–2.88(m,4H),2.46(s,3H). 13CNMR(101MHz,Chloroform-d)δ159.35,158.83,150.84,144.34,142.10,138. 88,130.08,129.10,128.55,128.54,128.23,127.91,125.70,125.29,125.25 ,125.21,125.17,123.00,122.46,120.34,119.94,117.87,117.74,115.51,1 09.68,94.80,94.32,77.30,55.62,49.36,40.96,35.61,17.99.ESI-MS:mass calcd for C 31 H 31 F3N2O3S[M+H]+(569.2086); found m / z: 569.2058.

[0314] Synthesis of intermediate B24-M1:

[0315]

[0316] The intermediate compound AM-1 was synthesized using method M1, by replacing bromobenzene with 2-bromonaphthalene. The product was a brown solid with a yield of 62.31%. 1 H NMR(400MHz,Chloroform-d)δ7.71(dd,J=8.5,3.0Hz,2H),7.59(d,J=8.2Hz,1H),7.40–7.35(m,1H),7 .27–7.23(m,1H),7.20(d,J=2.3Hz,1H),7.11(dd,J=9.0,2.8Hz,3H),6.87–6.82(m,2H),5.65(s,1H).

[0317] Synthesis of intermediate B24-M2:

[0318]

[0319] The product was prepared from intermediate B24-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 46.12%. 1H NMR(400MHz,Chloroform-d)δ10.39(s,1H),7.77(t,J=7.7Hz,2H),7.67(d,J=8.2Hz,1H),7.49(s,1H),7.44( dd,J=7.0,1.4Hz,1H),7.37–7.32(m,1H),7.24(d,J=5.7Hz,3H),7.09–7.04(m,2H),6.15(s,2H),3.83(s,6H).

[0320] Synthesis of target compound B24:

[0321]

[0322] The product was prepared according to general synthetic method M3, from intermediate B24-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 46.98%. 1 H NMR (400MHz, Chloroform-d) δ7.75(d,J=8.6Hz,2H),7.64(dd,J=8.3,1.0Hz,1H),7.56–7.52(m,2H),7.41(ddd,J=8.2,6.8,1.3Hz,1H),7.37(d,J=2. 3Hz,1H),7.32–7.29(m,3H),7.18(dd,J=9.3,2.7Hz,3H),7.03–7.00(m,2H ),6.22(s,2H),5.91(s,1H),3.87(s,2H),3.68(s,6H),2.93–2.89(m,4H). 13 C NMR(101MHz,Chloroform-d)δ159.38,158.79,151.15,144.35,141.53,138.78 ,134.69,129.26,129.12,129.00,128.57,128.25,127.69,126.53,126.38,125 .72,125.31,125.27,125.23,125.19,123.38,123.02,120.44,120.34,119.56 ,110.54,109.82,94.43,77.29,55.64,49.39,40.99,35.65.ESI-MS:masscalcd for C 34 H 31 F3N2O3[M+H]+(573.2365); found m / z: 573.2387.

[0323] Synthesis of intermediate B25-M1:

[0324]

[0325] The product was prepared by replacing bromobenzene with 4-bromobiphenyl, following the synthetic method M1 for intermediate compound AM-1. The product was a brown solid with a yield of 53.68%. 1 H NMR(400MHz,Chloroform-d)δ7.56(d,J=7.7Hz,2H),7.47(d,J=8.3Hz,2H),7.41(t,J=7.6Hz,2 H),7.28(s,1H),7.11–7.05(m,2H),6.97(d,J=8.4Hz,2H),6.82(d,J=8.4Hz,2H),5.57(s,1H).

[0326] Synthesis of intermediate B25-M2:

[0327]

[0328] The product was prepared from intermediate B25-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a brown solid with a yield of 56.29%. 1 H NMR(400MHz,Chloroform-d)δ10.38(s,1H),7.60–7.53(m,4H),7.43(t,J=7.7Hz,2H),7.35 –7.29(m,1H),7.21–7.11(m,4H),7.08–7.02(m,2H),6.14(s,2H),5.82(s,1H),3.82(s,6H).

[0329] Synthesis of target compound B25:

[0330]

[0331] The product was prepared according to general synthetic method M3, from intermediate B25-M2 and trifluoromethylphenylethylamine. The product was a brown solid with a yield of 54.82%. 1H NMR(400MHz,Chloroform-d)δ7.60–7.55(m,3H),7.52(dd,J=8.6,2.1Hz,3H),7.43(t,J=7.7Hz,2H),7.31(dd,J=7.7,1.6Hz,3H),7.15–7.12 (m,2H),7.12–7.08(m,2H),7.01–6.97(m,2H),6.19(s,2H),5.74(s,1H ),3.92(s,2H),3.68(s,6H),2.97(d,J=5.9Hz,2H),2.95–2.89(m,2H). 13 C NMR(101MHz,Chloroform-d)δ159.36,158.74,151.06,144.42,143.21,140.82,138.67,133.40,129.11,128.76,128.56,128.24,128.03,126. 59,126.49,125.71,125.25,125.22,125.18,123.01,120.32,117.15,1 17.02,109.97,94.39,77.26,55.64,49.44,41.01,35.73.ESI-MS:mass calcd for C 36 H 34 F3N2O3[M+H]+(599.2522); found m / z: 599.2545.

[0332] 3. Synthesis of compounds C1-C7

[0333] Synthesis route:

[0334]

[0335] Reagents and conditions: (i) 4-bromophenol, Pd(dppf)Cl2, AcOK, 1,4-dioxane, 100℃, 4h, 67.6%-78%; (ii) 4-bromo-2,6-dimethoxybenzaldehyde, Pd2(dba)3, t-BuXPhos, Cs2CO3, toluene, nitrogen protection, 100℃, 12h, 60.0%-65.7%; (iii) 4-trifluoromethoxyphenylethylamine, methanol, 65℃, reflux 2h, NaBH4, 1h, 55%-65%. General method for the synthesis of intermediate C1-7-M1:

[0336] 1.5 mmol of phenylboronic acid with different substituents, 1 mmol of p-bromophenol, 3 mmol of potassium acetate, and 0.2 mmol of Pd(dppf)Cl2 were added to a 25 mL round-bottom flask containing a suitable amount of 1,4-dioxane solvent. After removing air by nitrogen purging, the reaction was carried out at 100 °C for 4–5 h. The reaction was monitored using thin-layer chromatography (TLC), and the magnetic stirrer was turned off after the reaction was completed. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed several times with a small amount of ethyl acetate. The filtrate was collected, and the organic phase was evaporated under vacuum to obtain the crude product. A suitable amount of saturated brine was added to the crude product, followed by three consecutive extractions with equal volumes of ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. A large amount of organic solvent was removed by vacuum rotary evaporation, and the remaining 2–3 mL was used for high-performance thin-layer chromatography (HPLC) to separate and purify the intermediate compound.

[0337] Synthesis of intermediate compound C1-M1:

[0338]

[0339] The product was prepared by general synthesis method M4 from p-methoxyphenylboronic acid and p-bromophenol. The product was a yellowish-white solid with a yield of 74.13%. 1 H NMR (400MHz, Chloroform-d) δ7.50–7.46(m,2H),7.45–7.41(m,2H),6.99–6.94(m,2H),6.92–6.87(m,2H),4.79(s,1H),3.85(s,3H).

[0340] Synthesis of intermediate compound C1-M2:

[0341]

[0342] The product was prepared from intermediates C1-M1 according to the synthetic method M2 for intermediate compound AM-2. The product was a yellowish-white solid with a yield of 52.31%. 1 H NMR(400MHz,Chloroform-d)δ10.40(s,1H),7.61–7.58(m,2H),7.54(d,J=8.8Hz,2H ),7.16–7.14(m,2H),7.02(d,J=4.2Hz,2H),6.19(s,2H),3.97(s,3H),3.82(s,6H).

[0343] Synthesis of target compound C1:

[0344]

[0345] The product was prepared according to general synthetic method M3, from intermediate C1-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 54.82%. 1 H NMR (400MHz, Methanol-d4) δ7.53(dd,J=16.1,8.2Hz,4H),7.34(d,J=7.6Hz,3H),7.04(d,J=7.7Hz,2H),6.97(d,J=7.8Hz, 2H),6.77(s,1H),6.28(s,2H),3.72(s,3H),3.65(s,6H),3.34–3.29(m,2H),2.87(d,J=6.2Hz,2H),2.82(d,J=6.9Hz,2H). 13 CNMR(101MHz,Chloroform-d)δ174.22,159.40,159.02,158.79,157.93,156 .01,141.87,136.09,133.95,132.98,129.12,129.10,128.15,127.95,127. 88,125.28,125.24,118.93,114.26,114.19,110.12,102.44,95.19,77.24, 55.68,55.65,55.39,55.36,49.35,49.22,41.04,40.96,35.56.ESI-MS:mass calcd for C 31 H 30 F3NO4[M+H]+(538.2205); found m / z: 538.2242.

[0346] Synthesis of intermediate compound C2-M2:

[0347]

[0348] The intermediate compound M2 was prepared from 4-hydroxy-4-bromobiphenyl using the same method as AM-2. The product was a yellowish-white solid with a yield of 58.26%. 1 H NMR (400MHz, Chloroform-d) δ10.40(s,1H),7.63–7.57(m,4H),7.49–7.45(m,2H),7.19–7.14(m,2H),6.19(s,2H),3.83(s,6H).

[0349] Synthesis of target compound C2:

[0350]

[0351] The product was prepared according to general synthetic method M3, from intermediate C2-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 47.28%. 1 H NMR(400MHz,Chloroform-d)δ7.60–7.55(m,4H),7.52(d,J=8.8Hz,2H),7.46–7.42(m,2H),7.32(d,J=7.9 Hz,2H),7.10–7.06(m,2H),6.25(d,J=2.3Hz,2H),3.88(d,J=2.1Hz,2H),3.69(s,6H),2.94–2.90(m,4H). 13 C NMR(101MHz,Chloroform-d)δ159.46,157.65,156.93,139.32,135.05,131.91,129.12,128.82,128.44,128.42,128 .24,126.87,125.31,125.27,121.32,118.86,114.26,95.40,95.29,77.24,55.71,49.30,40.92,35.42.ESI-MS:mass calcd for C 30 H 27 BrF3NO3[M+H]+(586.1205); found m / z: 586.1234.

[0352] Synthesis of intermediate compound C3-M2:

[0353]

[0354] The intermediate compound M2 was prepared from 4-hydroxybiphenyl using the same method as AM-2. The product was a yellowish-white solid with a yield of 53.73%. 1 H NMR(400MHz,Chloroform-d)δ10.40(s,1H),7.67–7.64(m,2H),7.61(d,J=7.3H z,2H),7.47(t,J=7.6Hz,3H),7.18(d,J=8.6Hz,2H),6.20(s,2H),3.83(s,6H).

[0355] Synthesis of target compound C3:

[0356]

[0357] The product was prepared according to general synthetic method M3, from intermediate C3-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 54.34%. 1H NMR(400MHz,Chloroform-d)δ7.61–7.54(m,6H),7.45(t,J=7.8Hz,2H),7.38–7.31(m,3H),7.13 –7.08(m,2H),6.28(d,J=2.6Hz,2H),3.88(d,J=3.3Hz,2H),3.70(s,6H),2.92(d,J=2.7Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ159.41,157.55,156.73,144.56,140.42,136.28,129.11,128.83,128.54,128.40,128.22,127.90,127.11,126 .87,125.72,125.28,125.25,125.21,125.17,123.02,119.07,118.78, 111.03,95.41,77.28,55.83,55.67,49.62,41.08,35.88.ESI-MS:mass calcd for C 30 H 28 F3NO3[M+H]+(508.2100); found m / z: 508.2124.

[0358] Synthesis of intermediate compound C4-M1:

[0359]

[0360] The product was prepared by reacting p-methylphenylboronic acid and p-bromophenol according to general synthetic method M4. The product was a yellowish-white solid with a yield of 74.13%. 1 H NMR (400MHz, Chloroform-d) δ7.49–7.42(m,4H),7.24(s,2H),6.93–6.88(m,2H),2.39(s,3H).

[0361] Synthesis of intermediate compound C4-M2:

[0362]

[0363] The product was prepared from 4-hydroxybiphenyl according to the synthetic method for intermediate compound AM-2. The product was a yellowish-white solid with a yield of 49.20%. 1H NMR (400MHz, Chloroform-d) δ10.40(s,1H),7.64–7.61(m,2H),7.51(d,J=8.0Hz,2H),7.18–7.14(m,2H),6.19(s,2H),3.82(s,6H),2.42(s,3H).

[0364] Synthesis of target compound C4:

[0365]

[0366] The product was prepared according to general synthetic method M3, from intermediate C4-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 55.27%. 1 H NMR(400MHz,Chloroform-d)δ7.58–7.56(m,2H),7.55–7.54(m,2H),7.51–7.46(m,2H),7.34–7.31(m,2H),7.26(d,J=3.1Hz,2H),7. 08(dq,J=7.6,3.1Hz,2H),6.26(t,J=2.7Hz,2H),3.86–3.84(m,2H),3.72–3.68(m,6H),2.90(t,J=2.7Hz,4H),2.41(t,J=2.7Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ159.39,157.60,156.44,144.56,137.55,136.87 ,136.26,130.23,129.74,129.54,129.10,128.53,128.18,127.89,126.87,12 6.70,125.71,125.24,125.20,125.16,123.01,119.07,118.77,118.62,111.0 0,95.34,95.27,94.76,77.25,55.66,49.62,41.08,35.90,21.09.ESI-MS:mass calcd forC 31 H 30 F3NO3[M+H]+(522.2256); found m / z: 522.2234.

[0367] Synthesis of intermediate C5-M1:

[0368]

[0369] The product was prepared by general synthesis method M4 from p-trifluoromethylphenylboronic acid and p-bromophenol. The product was a white solid with a yield of 64.97%. 1 H NMR (400MHz, Chloroform-d) δ7.70(d,J=8.4Hz,2H),7.64(d,J=8.5Hz,2H),7.52–7.48(m,2H),6.97–6.93(m,2H).

[0370] Synthesis of intermediate C5-M2:

[0371]

[0372] The product was prepared from C5-M2 according to the synthesis method of intermediate compound AM-2. The product was a yellowish-white solid with a yield of 49.20%. 1 H NMR (400MHz, Chloroform-d) δ10.40(s,1H),7.63–7.57(m,4H),7.49–7.45(m,2H),7.19–7.14(m,2H),6.19(s,2H),3.83(s,6H).

[0373] Synthesis of target compound C5:

[0374]

[0375] The product was prepared according to general synthetic method M3, from intermediate C5-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 52.43%. 1 H NMR(400MHz,Chloroform-d)δ7.69(d,J=1.3Hz,4H),7.59–7.56(m,2H),7.54(dd,J=8.6,3.5 Hz,3H),7.31(s,1H),7.12–7.09(m,2H),6.27(s,2H),3.86(s,2H),3.70(s,6H),2.91(s,4H). 13C NMR(101MHz,Chloroform-d)δ159.45,159.40,157.62,157.19,152.22,144.43,143.91, 134.58,132.68,129.74,129.10,128.96,128.64,128.60,128.25,127.07,125.81,125.7 7,125.74,125.70,125.25,125.21,122.99,120.73,120.20,118.72,118.66,118.50,111 .16,95.63,95.31,95.23,94.84,77.25,55.68,55.64,49.58,41.05,35.78.ESI-MS:mass calcd for C 31 H 27 F6NO3[M+H]+(576.1973); found m / z: 576.1929.

[0376] Synthesis of intermediate C6-M1:

[0377]

[0378] The product was prepared by general synthesis method M4 from p-fluorophenylboronic acid and p-bromophenol. The product was a white solid with a yield of 58.23%. 1 H NMR (400MHz, Chloroform-d) δ7.51–7.46(m,2H),7.45–7.40(m,2H),7.14–7.07(m,2H),6.93–6.89(m,2H).

[0379] Synthesis of intermediate C6-M2:

[0380]

[0381] The product was prepared by reacting intermediate C6-M1 with trifluoromethylphenylethylamine according to general synthetic method M2. The product was a brownish-yellow solid with a yield of 47.83%. 1 H NMR(400MHz,Chloroform-d)δ10.40(s,1H),7.61–7.53(m,4H),7.19–7.12(m,4H),6.19(s,2H),3.83(s,6H).

[0382] Synthesis of target compound C6:

[0383]

[0384] The product was prepared according to general synthetic method M3, from intermediate C6-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 56.43%. 1 H NMR(400MHz,Chloroform-d)δ7.56–7.52(m,4H),7.52–7.49(m,2H),7.34–7.30(m,2H),7.16 –7.10(m,2H),7.10–7.06(m,2H),6.25(s,2H),3.88(s,2H),3.69(s,6H),2.95–2.91(m,4H). 13 C NMR(101MHz,Chloroform-d)δ163.56,161.12,159.47,157.86,156.54,144.0 9,136.55,136.52,135.42,129.77,129.12,128.68,128.44,128.36,128.29, 125.66,125.34,125.30,125.27,125.23,122.96,118.91,118.76,115.77,11 5.56,109.78,95.29,95.13,77.25,55.70,49.23,40.87,35.33.ESI-MS:mass calcd for C 30 H 27 F4NO3[M+H]+(526.2005); found m / z: 526.2033.

[0385] Synthesis of intermediate C7-M1:

[0386]

[0387] The product was prepared by general synthesis method M4 from p-chlorophenylboronic acid and p-bromophenol. The product was a white solid with a yield of 53.49%. 1 H NMR (400MHz, Chloroform-d) δ7.51–7.42(m,4H),7.41–7.36(m,2H),6.94–6.89(m,2H).

[0388] Synthesis of intermediate C7-M2:

[0389]

[0390] The product was prepared by reacting intermediate C7-M1 with trifluoromethylphenylethylamine according to general synthetic method M2. The product was a brownish-yellow solid with a yield of 47.83%. 1H NMR (400MHz, Chloroform-d) δ10.40(s,1H),7.62–7.59(m,2H),7.55–7.52(m,2H),7.45–7.42(m,2H),7.19–7.16(m,2H),6.19(s,2H),3.83(s,6H).

[0391] Synthesis of target compound C7:

[0392]

[0393] The product was prepared according to general synthetic method M3, from intermediate C6-M2 and trifluoromethylphenylethylamine. The product was a brownish-yellow oil with a yield of 53.29%. 1 H NMR(400MHz,Chloroform-d)δ7.57–7.53(m,3H),7.51(dd,J=9.0,2.2Hz,3H),7.45–7.39(m,2H),7.32(d, J=8.0Hz,2H),7.08(dd,J=9.2,2.5Hz,2H),6.25(s,2H),3.86(s,2H),3.69(s,6H),2.91(d,J=2.9Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ159.44,157.52,156.96,144.33,138.86,135.00,133.18,129.11,128.98,128.95,128.82,128.60,128. 27,128.09,125.68,125.31,125.27,125.23,125.20,122.98,118.82,110.60,95.43,77.25,55.69,49.45,40.98,35.63.ESI-MS:mass calcd for C 30 H 27 ClF3NO3[M+H]+(542.1710); found m / z: 542.1745.

[0394] Example 2: Inhibitory activity of compounds against tumor cells

[0395] The inhibitory activity of the compounds against tumor cells was determined using the MTT assay. The cell lines used included HT29 (human colon cancer cells), HCT116 (human colon cancer cells), HCT115 (human colon cancer cells), and HCT-8 (human ileocecal cancer cells). The specific steps were as follows:

[0396] Cell seeding: Prepare a single-cell suspension using RPMI 1640 complete medium containing 10% fetal bovine serum, and seed 3000-5000 cells per well into 80 μL of 96-well plates. Cell culture: Incubate the 96-well plates in a CO2 incubator at 37°C and 5% CO2 for 24 hours. Drug administration: After tumor cells adhere, administer the drug by serially diluting DMSO solution with culture medium to set concentrations of 40, 13.3, 4.44, 1.48, 0.493, 0.164, 0.0548, and 0.0182 μM, with three replicates for each concentration. Continue culturing for 72 hours after drug administration. Color development: After 3 days of culture, add 20 μL of MTT solution (5 mg / mL prepared with PBS) to each well. Continue incubation for 4 hours, then terminate the culture and carefully aspirate the culture supernatant from the wells. For suspension cells, centrifuge before aspirating the culture supernatant. Add 150 μL of DMSO to each well and shake for 10 minutes to fully dissolve the crystals. Colorimetric analysis: Select 570 nm as the measurement wavelength and measure the absorbance of each well using an ELISA reader. Calculate the in vitro proliferation inhibition rate of each target compound using the formula: Cell inhibition rate % = (OD value of control group - OD value of treated group) / (OD value of control group - OD value of blank group) × 100%. Then, calculate the IC50 value of the corresponding compound using GraphPad Prism8 software. 50 The values ​​(in μM) are shown in Table 1.

[0397] Table 1

[0398]

[0399]

[0400] a IC 50 The average value of three independent replicate experiments was used to determine the cell inhibitory activity after 72 hours of drug administration.

[0401] Experimental results showed that when R3 was a halogenated substituent such as fluorine, chlorine, or bromine (A5-A7), the antitumor activity was not significantly improved and even decreased. Changing the substituent to amino or hydroxyl groups (A1-A2) was detrimental to antitumor activity. Changing the D ring to aromatic heterocyclic pyridine or thiophene (A9, A10) was also detrimental to antitumor activity. Enhancing the hydrophobicity of the substituents on the D ring, such as A8 (dimethoxy) or A11 (trifluoromethyl), significantly improved the activity. The antiproliferative activity of compound A11 against HT29 tumor cells was found to be IC50. 50=1.35 μM. Then, based on the para-substitution of trifluoromethyl on ring D, the substituents on ring A were further modified to study its antitumor activity. Experimental results revealed that increasing the halogen substituents on ring A (B1-B3) did not significantly enhance antitumor activity. Changing the substituents on ring A to alkyl chains or methoxy groups (B4-B10) had little effect on its activity; only B5 showed a slight increase (IC5). 50 =0.92 μM). When the A ring is changed to an alcohol alkyl chain, ethoxy group, or cyano group (B11-B13, B14, B16), the activity is significantly improved. Among them, compound B13 with propyl alcohol substitution has the best activity (IC50 of HT29). 50 =0.38 μM). Subsequent optimization of ring A (including adding substituents such as tert-butyl, trifluoromethyl, methylthio, and trifluoromethoxy) did not yield compounds with significantly improved activity. Later, changes were made to the connection method between rings A and B, and studies showed that only the connection via an aromatic amine was most beneficial to activity.

[0402] Ultimately, we screened 42 compounds and selected B13 as the optimal target molecule, whose activity against colorectal cancer tumor cells (HT29) was determined by an IC50 value. 50 The activity was 0.38 μM, which was 7 times higher than that of compound 42 (A3) and higher than that of the positive control drug 5-fluorouracil (5-FU) (IC50). 50 =9.19μM) is 24 times higher than that of cisplatin (IC50) 50 =11.63μM) is 31 times higher.

[0403] Example 3 Toxicity test of compound B13

[0404] To verify the toxicity of the target compound B13 to normal cells, normal human hepatocytes (LO2) were selected as a reference. 5-FU, a current first-line chemotherapy drug for colorectal cancer, was used as a control. Dosage gradients were set at 40, 13, 4.4, 1.4, 0.49, and 0.16 μM. Cell viability of LO2 cells under these drug administration conditions was measured. The test results are as follows: Figure 2 As shown.

[0405] As shown in the figure, when the positive control drug 5-FU was administered at concentrations of 40, 13, and 4.4 μM, almost all normal human hepatocytes died, while the cell survival rates at the corresponding concentrations of compound B13 were 15.19%, 32.28%, and 49.41%, respectively. When the concentration was further decreased to 1.4, 0.49, and 0.16 μM, the survival rate of normal human hepatocytes corresponding to 5-FU gradually increased, reaching a maximum of 55.40%. In contrast, when compound B13 was administered at a concentration of 0.16 μM, all cells survived (survival rate 99.96%). The above analysis indicates that the toxicity of compound B13 is significantly lower than that of the positive control drug 5-FU, suggesting that compound B13, as a targeted drug, has high selectivity and biocompatibility.

[0406] Example 4: Cell cycle experiment with compound B13

[0407] Human colorectal cancer cells HT29 and HCT116 in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged, and then resuspended in 1640 complete culture medium containing 10% fetal bovine serum to prepare a cell suspension. The cell concentration was determined by dilution at 1×10⁻⁶. 6 Cells were seeded at a density of / well in 6-well plates. After seeding, the 6-well plates were placed in a CO2 incubator and cultured at 37°C and 5% CO2 for 24 hours. Once the cells had adhered and reached a certain density, the target compound B13 solution (prepared with DMSO) was serially diluted with culture medium to achieve concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 μM. After drug administration, the cells were incubated in a CO2 incubator for 48 hours. Cells were then digested with trypsin, and the cell suspension was collected in 10 mL centrifuge tubes and centrifuged for 5 minutes. The supernatant was aspirated, and 1 mL of pre-chilled PBS was added to resuspend the cells. The cells were centrifuged again for 5 minutes, and this washing and centrifugation process was repeated twice. After removing the PBS, 300 μL of pre-chilled PBS was added to resuspend the cells. The centrifuge tubes were placed on a vortex mixer, and pre-chilled anhydrous ethanol was added while vortexing. After mixing, the cells were fixed for 12 hours. The ethanol was removed, and the cells were centrifuged for 5 minutes. The cells were washed three times with PBS. Resuspend cells in 200 μL of pre-chilled PBS, stain with PI dye, and analyze. Results are shown below. Figure 3 and Figure 4 .

[0408] Analysis of the two cell cycle distribution maps revealed that in the untreated control group, HT29 cells had a cell cycle distribution of 65.37% in G1 phase, 12.01% in S phase, and 16.33% in G2 phase. However, after administration of compound B13, a significant change in the cell cycle distribution was observed. The proportion of cells in G1 phase gradually increased with increasing concentration, exhibiting a drug concentration-dependent effect. When the concentration reached 5 μM, the proportion of HT29 cells in G1 phase was 76.86%, an increase of 11.49% compared to the control group. This indicates that compound B13 arrests the cell cycle in HT29 cells at the G1 phase. In the untreated control group, HCT116 cells had a cell cycle distribution of 51.72% in G1 phase, 33.67% in S phase, and 12.97% in G2 phase. The cell cycle distribution also changed after administration, with a gradual increase in G1 phase cells and a corresponding decrease in S1 phase cells. When the drug concentration reached 5 μM, the proportion of HCT116 cells in the G1 phase was 66.42%, which was 14.7 percentage points higher than that in the control group. This demonstrates that compound B13 can arrest HCT116 cells in the G1 phase. These effects suggest that compound B13 may downregulate the c-Myc signaling pathway in cells, thereby inducing cell cycle arrest.

[0409] Example 5: Apoptosis experiment of compound B13

[0410] HT29 cells in logarithmic growth phase were harvested, the culture medium was discarded, and the cells were digested with trypsin and diluted with 1640 medium containing 10% fetal bovine serum at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of / well in 6-well plates. Incubation was performed at 37°C and 5% CO2 for 24 hours. The prepared target compound was serially diluted to specific concentrations, resulting in final drug concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 μM. After drug administration, the cells were incubated in a CO2 incubator for 24 hours. After trypsin digestion, the cell suspension was transferred to centrifuge tubes and centrifuged for 5 minutes. The supernatant was aspirated, and the cells were washed with pre-chilled PBS buffer and centrifuged. Washing was repeated twice. Samples for flow cytometry analysis were prepared according to the instructions of the apoptosis detection kit. Results are shown in [Figure number missing]. Figure 5 All experimental results were obtained from three independent replicates. *P<0.05 vs DMSO; **P<0.01 vs DMSO; ***P<0.001 vs DMSO; ****P<0.0001.

[0411] Apoptosis assays showed that without drug administration, the survival rate of HT29 cells was 96.94%, with no significant apoptosis. When compound B13 was administered, the cell survival rate gradually decreased from 89.51% to 55.56% with increasing concentrations. Furthermore, HT29 tumor cells gradually underwent apoptosis with increasing drug concentrations, exhibiting both early and late apoptosis, with the total apoptosis rate gradually increasing from 9.04% to 42.58%. This indicates that compound B13 can induce apoptosis to a certain extent, and this effect is concentration-dependent. Therefore, compound B13 demonstrates excellent antitumor activity in vitro.

[0412] Example 6: Gel electrophoretic migration experiment of compound B13

[0413] Electrophoresis was performed using a 4% polyacrylamide gel (Acr:Bis = 29:1) with 0.5×Tris-borate EDTA (TBE) as the electrophoresis buffer. The binding reaction buffer contained 1×PBS (pH 7.3), 1 mmol / L EDTA, 0.1% NP40, 5% glycerol, and 1 mmol / L DTT. Compounds were dissolved in DMSO for testing, with concentration gradients of 20, 10, and 5 μM. The biotinylated double-stranded DNA oligonucleotide 5'-CACCCGGTCACGTGGCCTACAC-3' probe, containing a 22 bp E-Box sequence, was synthesized by General Biotechnology. In the binding reaction buffer, the oligonucleotide probe concentration was 10 nmol / L, and the purified c-Myc bHLH-LZ and Max(S) proteins were both at 60 nmol / L. The protein complexes and compounds were incubated at room temperature for 90 min, followed by incubation with the oligonucleotide probe for 25 min. Samples were then loaded and run on the gel, and images were developed using a Bio-Rad FX. The results are shown in [Figure number missing]. Figure 6 .

[0414] Gel electrophoresis migration assays showed that the DNA sequence of the E-Box could bind to the c-Myc / Max dimer complex, exhibiting a significant migration band. In the absence of the transcription factor protein, only a free DNA band was observed. The addition of either compound 42 or B13 inhibited DNA migration, with the effect becoming more pronounced with increasing drug concentration. Even at a concentration of 20 μM, compound 42 still showed a small migration band. However, at a concentration of 20 μM, compound B13 almost completely eliminated the migration band, indicating that B13 at 20 μM completely inhibited the binding of the c-Myc / Max dimer complex to DNA. EMSA experiments validated that the designed compound B13 effectively targets the c-Myc / Max dimer complex, demonstrating superior efficacy compared to the previously studied compound 42.

Claims

1. An inhibitor targeting c-Myc, characterized in that: Its structural formula is shown in Formula II: ， Wherein, R1 is H, halogen, C1-C4 alkyl, C1-C3 alkoxy, ; R2 is H, halogen, or C1-C4 alkyl; R3 is .

2. The c-Myc-targeting inhibitor according to claim 1, characterized in that: R2 is H.

3. The c-Myc-targeting inhibitor according to claim 1, characterized in that: Its structural formula is any one of the following compound structural formulas:

4. The use of the c-Myc-targeting inhibitor according to any one of claims 1 to 3 in the preparation of a drug for treating colorectal cancer.

5. A pharmaceutical composition, characterized in that: It consists of an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a therapeutically effective amount of an inhibitor targeting c-Myc as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

Images