CN116410120A - A kind of 2-phenylindole amides antitumor compound and application thereof - Google Patents

Abstract

The invention relates to a 2-phenyl indole amide compound, which has a formula

Is characterized by comprising the following structure:

wherein the substituents R ₁ ，R ₂ Hydrogen, hydroxy, halogen, C1-6 alkoxy such as methoxy or ethoxy, C1-6 alkanoyloxy such as acetyl; ar is aryl, substituted aryl, such as phenyl, naphthyl, pyridyl, thienyl, quinolylA group selected from the group consisting of a pinyl group, a pyrimidinyl group, a benzimidazolyl group, a thiadiazolyl group, an isoxazolyl group, a benzothiazolyl group, a pyridyl vinyl group, a styryl group, a m-dinitrophenyl group, a m-dichlorobenzyl group, o-chloronitrophenyl, o-fluorotrifluoromethylphenyl, pentafluorophenyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, p-fluorophenyl, naphthylmethyl and the like, wherein n=0 to 2; r is R ₁ ，R ₂ When substituted simultaneously, each substituent may be the same or different. Experiments prove that the compounds have good inhibition effect on breast cancer cells, particularly canine breast cancer cells, and have potential for canine breast cancer treatment.

Description

A kind of 2-phenylindole amides antitumor compound and application thereof

technical field

The present invention relates to a small molecule compound, especially a small molecule compound with anti-tumor activity.

Background technique

Breast cancer is one of the globally recognized public health problems, which seriously threatens the physical and mental health of women all over the world. Similar to human breast cancer, dogs, as human companion animals, live in the same environment as humans and are exposed to the same carcinogenic factors. Canine breast cancer is also the most common malignant tumor in female dogs, and it is the second most frequently diagnosed tumor in dogs. High cancer, the average incidence rate can be as high as 25% to 42%, and the incidence rate can even be as high as 50% to 70% in unsterilized female dogs, which is three times the malignant rate of human mammary gland tumors, and this ratio increases with age. increased by growth.

Drug therapy occupies an important position both in advanced breast cancer and before and after surgery. In my country's veterinary clinics, antineoplastic drugs such as doxorubicin and paclitaxel are often used directly in human clinical clinics, which are not only expensive, but also violate the "Veterinary Drug Administration Regulations" 41 "prohibiting the use of human drugs for animals" "Provisions. At the same time, foreign countries have approved masitinib, togranib phosphate and other chemical small molecule drugs as anti-tumor drugs for pet clinical use, while my country has not yet approved the marketing of pet-specific anti-cancer drugs. The status of anti-breast tumor drug use.

On the other hand, the same carcinogenic factors also make the clinical and pathological features of canine breast cancer and human breast cancer very similar. The tumor spontaneous, tumor incidence, age of onset, hormones, course of disease, tumor size, clinical stage, lymph node invasion, and even the pathology of breast intraepithelial lesions are similar between the two. The canine breast cancer model is increasingly recognized as an excellent model for translational cancer research and has been recognized as an excellent predictive model for human drug development.

Therefore, accelerating the development of pet-specific anti-tumor drugs will meet the clinical needs of pets for anti-cancer drugs, save human loyal companions, and undoubtedly provide new research ideas for human tumor medicine.

Aiming at the problem of lack of special anti-tumor chemical drugs for pets in the prior art, the invention synthesizes and prepares a series of 2-phenylindoleamide compounds, and screens the compounds with low toxicity and high efficiency against breast cancer.

Contents of the invention

The purpose of the present invention is to provide an anti-tumor compound and its application in the preparation of anti-breast cancer drugs in view of the lack of pet-specific anti-tumor chemical drugs in the prior art.

In order to achieve the above object, the technical scheme adopted in the present invention is:

所示 （图1）：A kind of antitumor compound, its structure is as formula

Shown (Figure 1):

It is characterized in that: R ₁ and R ₂ are hydrogen, hydroxyl, halogen, C1-6 alkoxy such as methoxy or ethoxy, C1-6 alkanoyloxy such as acetyl; Ar is aryl, substituted aryl , such as phenyl, naphthyl, pyridyl, thienyl, quinolinyl, pyrimidinyl, benzimidazolyl, thiadiazolyl, isoxazolyl, benzothiazolyl, pyridinevinyl, styryl, m- Dinitrophenyl, m-dichlorophenyl, o-chloronitrophenyl, o-fluorotrifluoromethylphenyl, pentafluorophenyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, p-fluorophenyl, naphthylmethyl, etc., where n=0~2.

所示结构（图2）。As a preferred embodiment of the present invention, the compound has the formula

structure shown (Fig. 2).

R ₁ , R ₂ are preferably hydrogen, methoxy and hydroxyl; Ar is preferably p-chlorophenyl, p-bromophenyl, p-fluorophenyl, m-dinitrophenyl, m-dichlorophenyl, ortho-chloronitrophenyl , o-fluorotrifluoromethylphenyl, pentafluorophenyl, p-methoxyphenyl, 1-naphthyl, 1-benzothiazolyl, 4-pyridyl, 2-quinolinyl, 2-thiazolyl, 5-methylisoxazol-3-yl; n is preferably 0.

When R ₁ and R ₂ are substituted at the same time, each substituent may be the same or different.

The compound core and branched chain structure of the compound of the present invention have been optimized and designed, and have a good selective anti-tumor effect on breast cancer cell lines, especially canine breast cancer cell lines, with a half inhibitory concentration as low as 2.44 μM. A good inhibition rate was obtained, and the cell cycle was arrested in the G0G1 phase. Potential for the treatment of canine breast cancer.

化合物选自下述化合物中的一种 （见图3）：The preferred formula of the present invention

The compound is selected from one of the following compounds (see Figure 3):

Another object of the present invention is to provide the active ingredient used in the preparation of medicines and its application in the preparation of medicines for treating tumors.

Specifically, the application of the above compound in the preparation of drugs for treating breast cancer.

Preferably, the drug for treating tumor is an anti-tumor drug for treating canine breast cancer.

In summary, owing to adopting above-mentioned technical scheme, the beneficial effect of the present invention is:

1. The small molecular compound synthesized by the present invention has good anti-tumor activity, has the same anti-tumor activity as the existing chemical drug component Bazedoxifene, and has better tumor cell selectivity than Bazedoxifene, and can achieve anti-tumor at a lower concentration effect.

2. The compounds synthesized by the present invention can block the cycle of tumor cells in the G0G1 phase, and the block rate is high.

Description of drawings

的结构式。Figure 1 Compound formula

the structural formula.

化合物的结构式。Fig. 2 preferred formula of the present invention

The structural formula of the compound.

Fig. 3 Structural formula of preferred compounds of the present invention.

Fig. 4 The route diagram for the preparation of synthetic derivatives.

Figure 5 Synthesis of A series derivative structures.

Figure 6 Synthesis of B series derivative structures.

Figure 7 Synthesis of C series derivative structures.

Figure 8 Preliminary screening of anti-proliferation activity of 2-phenylindole derivatives on CMT-7346 cells (20 μM).

Figure 9 The half inhibitory concentration of 2-phenylindole compounds on CMT-7346 and NIH3T3 cells.

Figure 10 Quantification results of the effect of compound C5 on the cell cycle.

Figure 11 Quantification results of the effect of compound C5 on apoptosis.

Figure 12 Effect of compound C5 on the ultrastructure of canine breast cancer cells.

Specific implementation plan:

In order to make the object, technical solution and advantages of the invention clearer, the following examples further describe the present invention in detail. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1: The synthetic route of the compound is shown in Figure 4. Add benzonitrile derivatives (100 mmoL), 2-fluorotoluene derivatives (300 mmoL), lithium bistrimethylsilylamide (200 mmoL) and cesium fluoride (100 mmoL) into a 500 mL round bottom flask, add 200mL of cyclopentyl methyl ether, stirred and dissolved at room temperature, placed the round bottom flask in an oil bath stirrer, refluxed at 110 °C for 12 h, cooled to room temperature, added 30 mL of water to quench the reaction, and then used ethyl acetate Extraction is carried out, the obtained organic layer is dried by anhydrous sodium sulfate, and the solvent is recovered under reduced pressure to obtain a residue, and the crude product is separated by silica gel column chromatography to obtain the 2-phenylindole skeleton product 2-phenyl-1H-indole (A), 5-methoxy-2-(4-methoxyphenyl)-1H-indole (B). Subsequently, the 2-phenylindole compound activation solution was added dropwise to the carboxyl compound activation solution with a rubber dropper, then the flask was sealed, and the reaction was stirred at room temperature for 12 h. After the reaction was completed, it was dissolved with ethyl acetate, and saturated bicarbonate Wash three times with sodium solution and saturated sodium chloride solution, dry the organic layer with anhydrous sodium sulfate, recover the solvent under reduced pressure to obtain a residue, and separate by silica gel column chromatography to obtain the products A1-A22 (Figure 5), B1-B20 (Figure 6). Add the obtained B-type N-substituted 5-methoxy-2-(4-methoxyphenyl)-1 H -indole derivative (0.125 mmoL) into a 5 mL microwave reaction tube, seal the container, and fill it with Nitrogen, repeated three times, added 1 mL of dichloromethane, then cooled over acetone/liquid nitrogen, then added _BBr3 (0.625 mmoL) with stirring. The solution was warmed to 0 °C, the reaction mixture was suspended in saturated NaHCO ₃ , extracted with ethyl acetate, the organic extract was washed 3 times with saturated sodium chloride solution, the organic layer was dried with anhydrous sodium sulfate and the solvent was recovered under reduced pressure to obtain The residue was separated by silica gel column chromatography to obtain the product C1-C15 (Fig. 7). The structure of the derivative was identified by thin layer chromatography (TLC), ¹ H-NMR, ¹³ C-NMR and HR-MS.

A series derivatives

Compound A1 (yellow solid, yield: 35%), HRMS calculated for C ₂₁ H ₁₃ Cl ₂ NONa388.0272, found 388.0264 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.06 (d , J =5.6 Hz), 7.97 (d, J =5.6 Hz),7.68 – 7.62 (m, 1H), 7.36 (s, 1H), 7.35 (s, 1H),7.35 – 7.31 (m, 1H), 7.25 – 7.19 (m, 5H), 7.17 – 7.13 (m, 1H), 6.78 (d, J =0.8 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.39, 140.60, 138.19, 137.92,134.96 , 132.95, 131.99, 129.35, 128.62 (×3, two quaternary carbons, one tertiary carbon), 128.47 (×4),128.13,125.15, 124.09, 121.00, 114.60, 110.66.

Compound A2 (yellow solid, yield: 43%), HR-MS m/z: calculated for C ₂₁ H ₁₃ N ₃ O ₅ 387.0855, found 387.0866 [M] ^- ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.84 (t, J = 2.1 Hz, 1H), 8.56 (d, J = 2.1 Hz, 2H), 8.29 (dd, J = 8.3, 0.9 Hz, 1H), 7.74 – 7.69 (m, 1H), 7.44 (dtd , J = 29.9, 7.4,1.2 Hz, 2H), 7.23 – 7.17 (m,2H), 7.12 – 7.01 (m, 3H), 6.86 (d, J = 0.8 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.99,147.57 (×2), 139.23, 138.45, 138.06, 132.53, 129.47(×2), 129.27, 129.01 (×2), 128.66 (×2), 128.55, 125.86, 1 24.86, 121.16, 120.88 , 115.01, 112.10.

Compound A3 (yellow solid, yield: 51%), HR-MS m/z: calculated for C ₂₂ H ₁₃ F ₄ NO383.0933, found 384.1005 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.04 (dq, J =8.2, 0.9 Hz, 1H), 7.80(ddd, J = 8.6, 4.7, 2.3 Hz, 1H), 7.71 – 7.63 (m, 2H),7.41 – 7.32 (m, 2H) , 7.23 – 7.19 (m, 2H), 7.19 – 7.10(m, 3H), 7.02 (t, J =9.2 Hz, 1H), 6.79 (d, J = 0.8 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.38,161.62 (C4', d, J= 267.3 Hz), 140.30, 138.06, 135.78 (d, J=10.5 Hz), 132.67,131.53 (d, J=3 Hz), 129.70 (m) , 129.14, 128.55 (×2), 128.34 (×2), 127.91,124.94, 123.83, 121.53 (C7', d, J=278.6 Hz), 120.83, 118.01 (dd, J,=3.3Hz, J ₂ =1.2 Hz), 116.90 (d, J=21 Hz), 114.34, 110.31.

Compound A4 (yellow solid, yield: 55%), HR-MS m/z: calculated for C ₂₁ H ₁₃ ClN ₂ O ₃ 376.0615, found 377.0666 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ( m, 5H), 6.80 (d, J = 0.8 Hz, 1H). ¹³ C NMR (151MHz, Chloroform-d) δ 166.48, 146.69, 139.91, 137.93, 134.83, 133.62, 132.46, 131.69, 130.63, 129.14, 128.61( ×2), 128.44 (×2), 128.07, 127.08, 125.17,124.11, 120.90, 114.40, 110.81.

Compound A5 (white solid, yield: 37%), HR-MS m/z: calculated for C ₂₁ H ₁₀ F ₅ NO387.0683, found 388.0754 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.49 (dd, J =8.3, 1.0 Hz, 1H), 7.61 (dt, J= 7.6, 0.9 Hz, 1H), 7.48 – 7.37 (m, 2H), 7.29(dq, J = 5.1, 2.8 Hz , 2H), 7.26 – 7.23 (m, 3H), 6.67 (d, J = 0.7 Hz, 1H). ¹³ C NMR(151 MHz, Chloroform-d) δ 157.46, 143.23 (×2, dm, J = 247.3 Hz ), 142.22 (dm, J =253.05 Hz), 136.82 (×2, dm, J = 254.9 Hz), 138.97, 137.47, 132.09, 129.37,128.74, 128.65, 128.01, 125.83, 125.07 , 120.77, 116.16 (×2) , 113.04 (×2),112.62 (m).

Compound A6 (yellow solid, yield: 64%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O298.1106, found 299.1179 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.52 – 8.43 (m,2H), 8.09 – 7.99 (m, 1H), 7.69 – 7.62 (m, 1H), 7.39 – 7.32 (m, 2H), 7.32 –7.29 (m, 2H), 7.24– 7.20 (m, 2H), 7.18 – 7.09 (m, 3H), 6.78 (s, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 168.26, 149.85 (×2), 142.34, 140.26, 137.90, 132.54 ,129.18, 128.52 (×2),128.26 (×2), 128.01, 124.95, 123.92, 122.83 (×2),120.77, 114.44, 110.59.

Compound A7 (white solid, yield: 53%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O298.1106, found 299.1179 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.70 (dd, J =2.3, 0.9 Hz, 1H), 8.49 (dd, J = 4.9, 1.7 Hz, 1H), 8.05 – 7.99 (m, 1H), 7.82 –7.77 (m, 1H), 7.69 – 7.63 (m, 1H), 7.39 – 7.30 (m, 2H), 7.26 – 7.23(m, 2H),7.20 – 7.13 (m, 2H), 7.13 – 7.07 (m, 2H), 6.79 (d, J = 0.8 Hz, 1H). ¹³ C NMR(151 MHz, Chloroform-d) δ 169.65, 151.74, 148.70, 138.69, 137.57, 136.23, 134.97, 132.04, 131.34, 129.09(×2), 128. 74, 128.60 (×2), 124.53, 124.16, 123.59, 122.61, 116.78, 109.85.

Compound A8 (yellow solid, yield: 59%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O298.1106, found 321.0998 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.29 (ddd, J =4.8, 1.7, 0.9 Hz, 1H), 8.10 (dd, J = 8.1, 1.0 Hz, 1H), 7.75 (dt, J = 7.8, 1.1Hz, 1H), 7.66 – 7.58 (m, 2H), 7.33 (dtd, J = 25.2, 7.3,1.2 Hz, 2H), 7.25 (t, J = 1.3 Hz, 2H), 7.14 – 7.07 (m, 3H), 7.07 – 7.01 (m, 1H ), 6.75 (s, 1H). ¹³ CNMR (151 MHz, Chloroform-d) δ 168.76, 152.56, 148.90, 141.19, 138.26, 136.37,133.76, 129.54, 128.36 (×2),127.96 (×2 ), 127.19, 125.73, 125.34, 124.75, 123.71, 120.74, 114.66, 110.22.

Compound A9 (yellow solid, yield: 33%), HR-MS m/z: calculated for C ₂₃ H ₁₇ NO,323.1310, found 346.1202 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.45 – 8.38(m, 1H), 7.78 – 7.69 (m, 1H), 7.64 – 7.60 (m, 1H), 7.54 (ddd, J = 8.2, 2.3,1.2 Hz,2H), 7.47 – 7.43 (m, 2H), 7.41 – 7.36 (m, 2H), 7.34 – 7.29 (m, 2H),7.26 – 7.23 (m, 2H), 7.03 (ddd, J = 8.4,3.0, 1.4 Hz, 2H), 6.76 – 6.72 ( ( × ² ), 128.75 (×2), 128.64, 128.34 (×2), 128.02, 124.85, 123.53, 122.23, 120.46, 115.52,110.55.

Compound A10 (yellow solid, yield: 36%), HR-MS m/z: calculated for C ₂₄ H ₁₆ N ₂ O348.1263, found 371.1155 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.29 – 8.24(m, 1H), 8.05 (dd, J = 8.4, 0.8 Hz, 1H), 7.84 (dd, J = 12.3, 8.5 Hz, 2H), 7.71 –7.60 (m,3H), 7.52 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.36 (dtd, J = 24.3, 7.3, 1.2Hz, 2H), 7.22 – 7.17 (m, 2H), 6.84 (t, J = 7.8 Hz, 2H ), 6.75 (s, 1H), 6.72 (td, J = 7.3, 1.3 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 169.36, 152.41, 146.66,142.06, 138.68, 136.97, 134.36, 130.30 , 130.20, 130.03, 128.61, 128.49 (×2),128.38, 127.88 (×2),127.67, 126.97, 125.09, 124.14, 121.45, 120.95, 115.43,110.54.

Compound A11 (white solid, yield: 42%), HR-MS m/z: calculated for C ₃₄ H ₄₇ FNO ₉ 303.0718, found 326.0610 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.80(ddd, J = 7.2, 2.1, 0.8 Hz, 1H), 7.67 – 7.63 (m, 1H), 7.53 (dd, J = 4.9, 1.2 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.31 – 7.26 (m, 4H), 7.25 – 7.23 (m, 1H), 7.20 –7.15 (m, 1H), 6.84 (dd, J = 4.9, 3.8 Hz, 1H), 6.81 (d, J = 0.8 Hz, 1H) ^.13 C NMR(151 MHz, Chloroform-d) δ 163.29, 140.61, 138.59, 138.20, 135.51, 134.29, 133.00, 129.01, 128.37 (×2), 127.85 (×2), 127.51, 12 7.48, 124.13, 122.97, 120.69 , 113.52, 109.01.

Compound A12 (yellow solid, yield: 35%), HR-MS m/z: calculated for C ₁₉ H ₁₄ N ₂ O ₂ 302.1055, found 325.0947 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.15(dq, J = 8.2, 0.9 Hz, 1H), 7.61 (ddd, J = 7.5, 1.5, 0.7 Hz, 1H), 7.39 – 7.28 (m,4H), 7.26 – 7.16 (m, 3H), 6.74 (d, J = 0.8 Hz, 1H), 6.09 (q, J = 0.9 Hz, 1H),2.26 (d, J = 0.9 Hz, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 170.58, 161.42, 159.56, 140.71, 138.04, 133.34, 129.65, 128.71 (×2), 128.29 (×2), 127.73, 125.19, 124.39, 120.97, 114.96, 111.54, 102. 32, 12.10.

Compound A13 (yellow solid, yield: 41%), HR-MS m/z: calculated for C ₁₉ H ₁₃ N ₃ O299.1059, found 322.0951 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.88 (d, J =1.4 Hz, 1H), 8.65 (dd, J = 5.1, 1.3 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 7.64(dd, J = 7.8, 1.3 Hz, 1H), 7.55 (dq, J = 5.1, 1.1 Hz, 1H), 7.38 (dtd, J =31.0, 7.4, 1.2 Hz, 2H), 7.21 (dd, J = 7.9, 1.7 Hz, 2H), 7.14 –7.06 (m, 3H),6.75 (s, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.93, 159.44, 158.01, 157.78, 140.31, 137.94, 133.15, 129.47, 128.61 (×2), 128.10 (×2), 127.85, 125.21, 124.32, 120.77, 120.72, 114.91, 111.06.

Compound A14 (red solid, yield: 32%), HR-MS m/z: calculated for C ₂₅ H ₁₇ NO347.1310, found 370.1202 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.07 – 7.98(m, 2H), 7.78 – 7.69 (m, 2H), 7.69 – 7.62 (m, 1H), 7.56 – 7.43 (m, 3H), 7.37– 7.30 (m, 2H), 7.22 (dd, ¹³ C NMR (151 MHz, Chloroform-d ) , 127.51, 126.70, 125.25, 125.01, 124.50, 123.95, 121.02, 115.26, 110.67.

Compound A15 (pale yellow solid, yield: 28%), HR-MS m/z: calculated for C ₂₆ H ₁₉ NO361.1467, found 362.1538 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.30 – 8.21 (m,1H), 7.82 – 7.78 (m, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.60 – 7.57 (m, 1H), 7.56– 7.52 (m, 2H), 7.45– 7.28 (m, 9H), 7.12 – 7.08 (m, 1H), 6.69 (d, J = 0.8 Hz,1H), 4.05 (s, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 172.64 , 139.49, 138.10, 134.29, 133.83, 132.02, 130.85, 129.19, 129.05 (×4), 128.90, 128.83, 128.20, 127.68, 126.39, 125.81, 125.44 , 125.36, 123.87, 123.49, 120.56, 116.05, 112.09, 43.97.

Compound A16 (white solid, yield: 22%), HR-MS m/z: calculated for C ₂₅ H ₁₈ N ₂ O362.1419, found 363.1492 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.90 – 8.81 (m,1H), 8.31 (ddt, J = 8.3, 1.7, 0.9 Hz, 1H), 7.98 (dd, J = 30.7, 8.5 Hz, 2H), 7.59 – 7.55 (m, 1H) , 7.54 – 7.44 (m, 5H), 7.35 (dddd, J = 12.3, 6.7, 2.8, 1.5Hz, 3H), 7.32– 7.27 (m, 2H), 6.68 (d, J = 1.5 Hz, 1H), 3.85 (d, J = 1.6Hz,2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 172.42, 150.44, 147.45, 139.36, 138.16, 136.00, 134.14, 132.36, 131.11, 129.57, 129.2 0 (×2), 129.14 , 129.01 (×2), 128.93, 128.16, 127.96, 125.42, 123.97, 121.40, 120.65, 115.79, 112.14, 45.90.

Compound A17 (yellow solid, yield: 36%), HR-MS m/z: calculated for C ₁₈ H ₁₃ N ₃ OS319.0779, found 320.0851 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.17 – 8.12 (m,1H), 7.67 – 7.62 (m, 1H), 7.45 – 7.35 (m, 2H), 7.25 – 7.15 (m, 5H),6.78 (s,1H), 2.57 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 160.39, 159.60, 144.64,139.21, 137.63, 131.78, 129.13, 128.59, 128.37 (×2), 128.10 (×2), 125.59, 124.62, 121.05, 114.56 ,112.19, 13.22.

Compound A18 (pale yellow solid, yield: 24%), HR-MS m/z: calculated for C ₂₂ H ₁₆ N ₂ O3 24.1263, found 325.1335 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform -d) δ 8.51 (dd, J =4.9, 1.6 Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.31 – 8.23 (m, 1H), 7.66 (d, J =15.6 Hz, 1H) , 7.61 (d, J = 7.7 Hz, 1H), 7.55 – 7.50 (m, 2H), 7.48 – 7.37 (m,4H), 7.32 (td, J = 7.5, 1.0 Hz, 1H), 7.24 (dt, J = 7.9, 2.0 Hz, 1H), 7.18 (dd, J = 8.0, 4.7 Hz, 1H), 6.75 (s, 1H), 6.39 (d, J = 15.6 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform -d) δ 165.63, 150.78, 149.31, 139.75, 139.18, 137.75, 134.31, 133.72, 130.28, 129.14, 128.99(×2), 128.88 (×2), 128.59, 125.0 8, 124.20, 123.82, 123.51, 120.54, 115.64, 110.95.

Compound A19 (yellow solid, yield: 21%), HR-MS m/z: calculated for C ₂₄ H ₁₉ N ₃ O365.1528, found 388.1420 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.31 (dq, J =8.4, 0.9 Hz, 1H), 7.83 (s, 1H), 7.76 (dt, J = 8.1, 1.0 Hz, 1H), 7.53 (ddd, J =7.7, 1.4, 0.7 Hz , 1H), 7.38 – 7.28 (m, 7H), 7.26 – 7.19 (m, 2H), 7.05 (dt, J =7.9, 0.9 Hz, 1H), 6.60 (d, J = 0.7 Hz, 1H), 4.46 ( T, J = 6.4 Hz, 2H), 2.82 (T, J = 6.4 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-D) Δ 171.20, 143.43, 143.26, 138.72,137.53, 133.64, 133.16, 128.95 ( ×2), 128.93,128.78, 128.53 (×2), 125.37,124.03, 122.90, 122.17, 120.52, 120.28, 115.71, 112.33, 109.20, 40.09, 39.03.

Compound A20 (white solid, yield: 31%), HR-MS m/z: calculated for C ₁₉ H ₁₃ NOS303.0718, found 326.0610 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.85 – 7.80(m, 1H), 7.72 (dd, J = 3.0, 1.3 Hz, 1H), 7.68 – 7.62 (m, 1H), 7.35 – 7.32 (m,2H), 7.32 – 7.26 (m,3H) , 7.25 – 7.21 (m, 2H), 7.20 – 7.15 (m, 1H), 7.12 (dd, J = 5.1, 3.0 Hz, 1H), 6.79 (d, J = 0.8 Hz, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.43, 140.92, 138.32, 137.78, 134.53, 133.19, 129.26, 128.51, 128.44 (×2),128.25 (×2), 127.67, 126.17, 124.41 , 123.24, 120.87, 114.06, 109.48.

Compound A21 (white solid, yield: 33%), HR-MS m/z: calculated for C ₂₃ H ₁₅ NOS353.0874, found 376.0767 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.16 (dt, J =8.2, 1.0 Hz, 1H), 7.91 – 7.84 (m, 1H), 7.79 – 7.73 (m, 2H), 7.69 – 7.63 (m,1H), 7.42 (dddd, J = 40.1, 8.3, 7.1, 1.2 Hz, 2H), 7.32 – 7.29 (m, 2H), 7.27 (d, J = 1.7 Hz, 1H), 7.25 (d, J = 1.3 Hz, 1H), 7.11 – 7.02 (m, 3H ), 6.79 (d, J = 0.7Hz,1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.17, 140.91, 139.55, 138.05, 137.49, 136.58, 132.86, 131.77, 129.13, 128.0 3 (×2), 127.78 (×2), 127.37, 125.57, 125.37, 124.30, 123.87, 123.16, 122.30, 120.73, 113.90, 109.29.

Compound A22 (pale pink solid, yield: 48%), HR-MS m/z: calculated for C ₂₂ H ₁₇ NO ₂ 327.1259, found 350.1151 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.69– 7.61 (m, 3H), 7.55 (dq, J = 7.4, 0.9 Hz, 1H), 7.36 – 7.31 (m, 2H), 7.25 –7.19 (m, 4H), 7.19 – 7.14 (m, 1H) , 6.78 (dd, J = 8.6, 1.6 Hz, 3H), 3.81 (s,3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 169.23, 163.45, 141.29, 138.19, 132.94,132.72 (×2), 129.11, 128.17 (×2), 128.09 (×2), 127.46, 127.10, 123.80, 122.63, 120.62, 113.66, 113.63 (×2), 108.63, 55.40.

Series B Derivatives

Compound B1 (white solid, yield: 30%), HR-MS m/z: calculated for C ₂₃ H ₁₇ C _l2 NO ₃ 425.0585, found 448.0469 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 7.97 (d, J = 2.0 Hz, 1H), 7.93(d, J = 9.0 Hz, 1H), 7.32 (d, J = 1.9 Hz, 2H),7.24 (t, J = 1.9 Hz, 1H), 7.15 – 7.10 (m, 2H), 7.08 (d, J = 2.6 Hz, 1H), 6.95 (dd, J = 9.0, 2.6 Hz, 1H), 6.74 – 6.71 (m, 2H), 6.62 (d, J = 0.7 Hz, 1H), 3.89(s, 3H), 3.75 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.05, 159.17, 156.68, 140.84, 137.84, 134.64 (×2), 132.56, 131.53, 130.13, 129.78, 128.18, 125.27, 115.35, 113.72, 113.21, 109.77, 103.10, 55.70, 55.29.

Compound B2 (orange solid, yield: 34%), HR-MS m/z: calculated for C ₂₃ H ₁₇ N ₃ O ₇ 447.1067, found 448.1141 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.85(t, J = 2.1 Hz, 1H), 8.53 (d, J = 2.1Hz, 2H), 8.20 (d, J = 9.0 Hz, 1H), 7.12(d, J = 2.5 Hz, 1H), 7.10 – 7.07 (m, 2H), 7.03 (dd, J = 9.0, 2.6 Hz, 1H), 6.69 (d, J = 0.7 Hz, 1H), 6.61 – 6.57 (m, 2H), 3.92 (s, 3H) , 3.67 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.71, 159.51, 157.29, 147.44, 139.64, 138.43, 132.44, 131.76, 130.40 (×2), 130.28, 129 .43 (×2), 124.74, 120.60, 115.97, 113.94 (×2), 113.85, 111.39, 103.46, 55.73, 55.28.

Compound B3 (yellow-white solid, yield: 38%), HR-MS m/z: calculated for C ₂₄ H ₁₇ F ₄ NO ₃ 443.1145, found 444.1217 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 7.97(d, J = 9.0 Hz, 1H), 7.77 (ddd, J = 8.5, 4.6, 2.3 Hz, 1H), 7.65 (dd, J = 6.8,2.3 Hz, 1H), 7.11 – 7.07 (m ,3H), 7.02 (t, J = 9.1 Hz, 1H), 6.97 (dd, J = 9.0,2.6 Hz, 1H),6.68 – 6.65 (m, 2H), 6.63 (d, J = 0.6 Hz, 1H) , 3.90 (s, 3H), 3.72(s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.25, 161.31 (d, J=262.5 Hz), 159.22, 156.66, 140.74, 135.69 (d, J = 10.5 Hz), 132.66, 131.60 (d, J= 3 Hz), 130.11, 129.92 (×2), 129.62 (m), 125.12, 121.69 (d, J= 271.5 Hz), 117.98(m), 116.79 (d , J=21 Hz), 115.33, 113.77 (×2), 113.27,109.61, 103.07, 55.70,55.21.

Compound B4 (yellow solid, yield: 42%), HR-MS m/z: calculated for C ₂₃ H ₁₇ ClN ₂ O ₅ 436.0826, found 437.0900 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.01(d, J = 9.0 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.65 (dd, J = 8.3, 2.1 Hz,1H), 7.36 (d, J = 8.3 Hz, 1H ), 7.12 – 7.07 (m, 3H), 6.98 (dd, J = 9.0, 2.6Hz, 1H),6.70 – 6.66 (m, 2H), 6.64 (s, 1H), 3.90 (s, 3H), 3.74 ( s, 3H). ¹³ C NMR(151 MHz, Chloroform-d) δ 166.35, 159.41, 156.86, 146.66, 140.38, 133.59, 132.50, 131.59, 130.18, 130.06(×2), 129.82, 127.10, 125.92, 124.91, 115.45 ,113.90 (×2), 113.43, 110.15, 103.21, 55.70, 55.33.

Compound B5 (yellow-white solid, yield: 38%), HR-MS m/z: calculated for C ₂₃ H ₁₄ F ₅ NO ₃ 447.0894, found 470.0789 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.39(d, J = 9.0 Hz, 1H), 7.20 – 7.17 (m, 2H), 7.05 (d, J = 2.5 Hz, 1H), 7.01 (dd, J = 9.0, 2.6 Hz, 1H), 6.77 – 6.72 (m, 2H), 6.54 (s, 1H), 3.90 (s, 3H), 3.77 (s,3H) ^.13 C NMR (151 MHz, Chloroform-d) δ 159.94, 157.44, 157.05,143.02 ( ×2, dm, J =250.5 Hz), 142.01 (dm, J = 256.5 Hz), 136.84 (×2, dm, J = 253.5 Hz), 139.51,131.87, 130.51, 130.20 (×2), 124.19, 117.16, 113.43, 113.37 (×2), 112.73(m), 112.54, 103.64, 55.65,55.40.

Compound B6 (white solid, yield: 60%), HR-MS m/z: calculated for C ₂₂ H ₁₈ N ₂ O ₃ 358.1317, found 381.1210 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.46(ddd, J = 5.9, 3.6, 1.8 Hz, 2H), 7.96 (dt, J = 9.7, 4.9 Hz, 1H), 7.31 – 7.24 (m,2H), 7.08 (ddt, J = 9.5, 6.3 , 2.7 Hz, 3H), 6.95 (dq, J = 10.1, 4.2, 3.6 Hz, 1H),6.68 – 6.58 (m, 3H), 3.89 (t, J = 4.9 Hz, 3H), 3.72 – 3.67 (m, 3H). ¹³ C NMR (151MHz, Chloroform-d) δ 167.86, 159.43, 156.88, 149.22, 149.20, 140.72, 132.54, 130.33, 130.03, 125.01, 123.24, 115.57, 113.81, 113.29, 110.16, 103.26, 55.76, 55.25.

Compound B7 (yellow-black solid, yield: 57%), HR-MS m/z: calculated for C ₂₂ H ₁₈ N ₂ O ₃ 358.1317, found 381.1210 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.67(dd, J = 2.2, 1.0 Hz, 1H), 8.49 (dd, J = 4.9, 1.7Hz, 1H), 7.94 (d, J = 9.0Hz, 1H), 7.76 (dt, J = 7.9 , 1.9 Hz,1H), 7.16 – 7.12 (m, 2H), 7.12 – 7.08 (m,2H), 6.97 – 6.93 (m, 1H), 6.70 – 6.66 (m, 2H), 6.63 (s, 1H), 3.89 (d, J= 0.9Hz, 3H), 3.71 (d, J = 0.8 Hz, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.75,159.09, 156.64, 151.95, 150.35, 140.80, 137.14, 132.63, 131.50, 130.16, 129.97 (×2), 125.08, 122.74, 115.31, 113.81(×2), 113.18, 109.71, 103.08, 55.69, 55.19.

Compound B8 (yellow solid, yield: 53%), HR-MS m/z: calculated for C ₂₂ H ₁₈ N ₂ O ₃ 358.1317, found 381.1210 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.31(ddd, J = 4.8, 1.8, 1.0Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.68 (dt, J = 7.8,1.1 Hz,1H), 7.59 (td, J = 7.7, 1.7 Hz, 1H), 7.16 – 7.11 (m, 3H), 7.06 (d, J=2.5 Hz, 1H), 6.93 (dd, J = 9.0, 2.5 Hz, 1H), 6.64 – 6.60 (m, 2H ), 6.59 (d, J= 0.7 Hz, 1H), 3.88 (s, 3H), 3.70 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 168.47, 158.61, 156.52, 152.68, 148.80, 141.51, 136.21, 132.78, 130.44, 129.61 (×2), 126.24, 125.45, 125.12, 115.48, 113.34 (×2), 112.82, 109.52, 103.14, 55.68, 55.1 7.

Compound B9 (white solid, yield: 32%), HR-MS m/z: calculated for C ₂₅ H ₂₁ NO ₃ 383.1521, found 406.1414 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.31(d, J = 9.0 Hz, 1H), 7.68(d, J = 15.5 Hz, 1H), 7.45 – 7.42 (m, 2H), 7.33 – 7.29 (m, 1H), 7.28 – 7.24 (m, 2H) , 7.06 (dt, J = 10.2, 1.9 Hz, 3H), 6.99 –6.94(m, 3H), 6.59 (d, J = 0.7 Hz, 1H), 6.35 (d, J = 15.5 Hz, 1H),3.88 ( s,3H), 3.81 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.18, 159.82, 156.42, 143.33, 139.91, 134.59, 132.40, 130.33 (×2), 130.16, 130. 07, 128.61 ( ×2),127.99 (×2), 126.19, 122.18,116.55, 114.21 (×2), 113.02, 109.83, 102.88,55.64, 55.38.

Compound B10 (yellow-white solid, yield: 46%), HR-MS m/z: calculated for C ₂₆ H ₂₀ N ₂ O ₃ 408.1474, found 430.9151 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ8.16 (dd, J = 9.0, 1.5 Hz, 1H), 8.03 (dt, J = 8.4, 1.1 Hz, 1H), 7.89 (d, J = 8.4Hz, 1H), 7.76 – 7.68 (m, 2H), 7.67 – 7.62 (m, 1H), 7.53 (ddt, J = 8.1, 6.8, 1.4Hz, 1H), 7.10 (dd, J = 2.6, 1.5 Hz, 1H), 7.08 – 7.05 (m, 2H) , 6.96 (ddd, J = 8.9,2.6, 1.5 Hz, 1H), 6.59 (dd, J = 1.5, 0.7 Hz, 1H), 6.38 – 6.33 (m, 2H), 3.90 (d, J = 1.3 Hz, 3H ), 3.45 (d, J = 1.4 Hz, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 168.69,158.29, 156.65, 152.34, 146.39, 141.93, 136.45, 132.84, 130.59, 129 .88,129.75, 129.54 ( ×2), 128.03, 127.94, 127.26, 126.44, 120.97, 116.03, 113.05(×2), 112.79, 109.71, 103.18, 55.70, 55.04.

Compound B11 (white solid, yield: 33%), HR-MS m/z: calculated for C ₂₃ H ₁₄ F ₅ NO ₃ 363.0929, found 386.0824 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ7.68 (d, J = 9.0 Hz, 1H), 7.52 (dd, J = 4.9, 1.2 Hz, 1H), 7.30 – 7.27 (m, 2H),7.26 – 7.24 (m, 1H), 7.07 (d , J = 2.5 Hz, 1H), 6.88 (dd, J = 9.0, 2.5 Hz, 1H),6.84 (dd, J = 4.9, 3.8 Hz, 1H), 6.80 – 6.75 (m, 2H), 6.67 – 6.63 ( m, 1H), 3.88(s, 3H), 3.76(s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 163.25, 158.98, 156.10, 141.10, 138.55, 135.36, 134.01, 132.94, 129 .89, 129.09 (×2), 127.44, 125.65, 114.35, 113.83 (×2), 112.74, 108.18, 102.76, 55.70, 55.19.

Compound B12 (tan solid, yield: 36%), HR-MS m/z: calculated for C ₂₁ H ₁₈ N ₂ O ₄ 362.1267, found 385.1159 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform- d) δ 8.07(d, J = 9.0 Hz, 1H), 7.22 – 7.16 (m, 2H), 7.04 (d, J = 2.6 Hz, 1H), 6.94 (dd, J = 9.0, 2.6 Hz, 1H), 6.79 – 6.74 (m, 2H), 6.58 (d, J = 0.7 Hz, 1H), 6.02 (d, J = 1.0 Hz, 1H), 3.88 (s, 3H), 3.78(s, 3H), 2.27 (d , J = 0.9 Hz, 3H). ¹³ C NMR(151 MHz, Chloroform-d) δ 170.09, 160.92, 159.35, 159.15, 156.89, 140.91,132.35, 130.48, 129.93 (×2), 125.53, 115.77, 113.46 (× 2), 113.00, 110.80, 103.31, 102.07, 55.66, 55.26, 11.88.

Compound B13 (yellow solid, yield: 55%), HR-MS m/z: calculated for C ₂₁ H ₁₇ N ₃ O ₃ 359.1270, found 382.1162 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ8.91 (q, J = 1.6 Hz, 1H), 8.65 (dt, J = 4.6, 2.2 Hz, 1H), 8.18 (dt, J = 9.0, 2.3Hz, 1H), 7.48 (td, J = 3.2 , 1.5 Hz, 1H), 7.10 (dt, J = 8.9, 2.3 Hz, 2H), 7.07 (t, J = 2.4 Hz, 1H), 6.98 (dd, J = 9.0, 2.3 Hz, 1H), 6.63 (dt , J = 9.0, 2.3 Hz, 2H), 6.60 (d, J = 1.7 Hz, 1H), 3.89 (t, J = 2.2 Hz, 3H), 3.71 (t, J = 2.3 Hz, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.66, 159.81, 159.22, 158.03, 157.67, 157.03, 140.76, 132.46, 130.60, 130.07(×2), 125.53, 120.66, 115.92 , 113.54 (×2), 113.18, 110.55, 103.44 , 55.69, 55.20.

Compound B14 (yellow-white solid, yield: 28%), HR-MS m/z: calculated for C ₂₇ H ₂₁ NO ₃ 407.1521, found 430.1414 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.96– 7.91 (m, 2H), 7.73 (td, J = 7.3, 6.7, 1.2 Hz, 2H), 7.51 – 7.45 (m, 2H), 7.43(dd, J = 7.1, 1.2 Hz, 1H), 7.22 (dd, J = 8.2, 7.1 Hz, 1H), 7.08 (d, J = 2.6 Hz, 1H), 6.94 – 6.90 (m, 3H), 6.53 (s, 1H), 6.38 – 6.34 (m, 2H), 3.89 (s,3H),3.59 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 169.64, 158.57, 156.47, 141.72,133.26, 133.22, 132.57, 131.83, 130.74, 130. 30, 129.41 (×2 ), 128.76, 128.16, 127.44, 126.20, 125.35, 124.85, 124.17, 115.87, 112.83, 112.80 (×2), 109.72, 103.03, 55.66, 55.09.

Compound B15 (yellow solid, yield: 36%), HR-MS m/z: calculated for C ₂₈ H ₂₃ NO ₃ 421.1678, found 422.1748 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 8.22(dt, J = 9.1, 0.6 Hz, 1H), 7.81 (dd, J= 8.6, 1.2 Hz, 1H), 7.73 (d, J = 8.5Hz, 1H), 7.44 – 7.31 (m, 6H), 7.12 (dd, J = 7.0, 1.1 Hz, 1H), 7.03 (d, J =2.6 Hz, 1H), 6.93 – 6.87 (m, 3H), 6.54 (d, J = 0.7 Hz, 1H), 4.05 (s, 2H),3.87(s, 3H), 3.79 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 172.03, 159.91, 156.44, 139.82, 133.57, 132.52, 131.82, 130.92, 130.20 (×2) , 129.97, 128.58, 127.85, 127.27, 126.24, 126.08, 125.56, 125.24, 123.25, 117.04, 114.13 (×2), 113.11, 111.36, 102.89, 55.61, 55.23, 43.33.

Compound B16 (white solid, yield: 18%), HR-MS m/z: calculated for C ₂₇ H ₂₂ N ₂ O ₃ 422.1630, found 423.1703 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d ) δ 8.86(dd, J = 4.3, 1.7 Hz, 1H), 8.23 (d, J = 9.1 Hz, 1H), 8.02 (dd, J = 8.1, 1.5 Hz, 1H), 7.97 (d, J = 8.6 Hz , 1H), 7.42 – 7.34 (m, 4H), 7.32 (dd, J = 8.7, 2.0 Hz,1H), 7.02 – 6.96 (m, 3H), 6.93 (dd, J = 9.1, 2.6 Hz, 1H), 6.53 (s, 1H), 3.86(d, J = 1.1 Hz, 6H), 3.84 (s, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 172.06,160.24, 156.74, 150.42, 147.47, 139.91, 135.99, 132.81, 132.65, 131.16, 130.42 (×2), 130.14, 129.56, 128.18, 127.89, 126.43, 121.39, 116.93, 114.55 (×2), 113.44, 111 .59, 103.17, 55.84, 55.56, 45.61.

Compound B17 (white solid, yield: 29%) HR-MS m/z: calculated for C ₂₀ H ₁₇ N ₃ O ₃ S379.0991, found 380.1063 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform -d) δ 8.06 (d, J =9.0 Hz, 1H), 7.15– 7.10 (m, 2H), 7.07 (d, J = 2.5 Hz, 1H), 6.99 (dd, J = 9.0,2.5 Hz, 1H) , 6.73 – 6.68 (m, 2H), 6.61 (s, 1H), 3.89 (s, 3H), 3.74 (s, 3H),2.56 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 160.15, 159.72, 159.29, 157.19, 144.78, 139.79, 132.16, 130.25, 129.47, 126.33, 115.60, 113.82, 113.58, 111.49, 103.50, 55. 69, 55.23, 13.21.

Compound B18 (white solid, yield: 82%), HR-MS m/z: calculated for C ₂₃ H ₁₈ FNO ₃ 375.1271, found 398.1163 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.65(d, J = 9.0 Hz, 1H), 7.61– 7.57 (m, 2H), 7.20 – 7.16 (m, 2H), 7.07 (d, J =2.5 Hz, 1H), 6.94 – 6.86 (m, 3H) , 6.73 – 6.69 (m, 2H), 6.63 (d, J = 0.7 Hz,1H), 3.88 (s,3H), 3.74 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 168.70, 165.07(d, J=252 Hz), 159.02, 156.21, 141.40, 132.79, 132.71, 132.65, 131.29 (d, J=3Hz), 130.04, 129.52 (×2), 125.39, 115.38, 115 .23, 114.79, 113.66 (× 2), 112.79, 108.66, 102.86, 55.68, 55.19.

Compound B19 (white solid, yield: 76%), HR-MS m/z: calculated for C ₂₃ H ₁₈ ClNO ₃ 391.0975, found 392.1047 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.65(d, J = 9.0 Hz, 1H), 7.52 – 7.49 (m, 2H), 7.22 – 7.19 (m, 2H), 7.18 – 7.15 (m, 2H), 7.07 (d, J = 2.5 Hz, 1H) , 6.88 (dd, J = 9.0, 2.6 Hz, 1H), 6.73 – 6.69 (m,2H), 6.63 (d, J = 0.7 Hz, 1H), 3.88 (s, 3H), 3.75 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 169.02, 159.32, 156.49, 141.56, 139.00, 133.73, 132.93, 131.64(×2), 130.31, 129.79 (×2), 128.60 (×2), 125.54, 115.08, 113.94(×2),113.04, 109.06, 103.13, 55.90, 55.46.

Compound B20 (yellow solid, yield: 87%), HR-MS m/z: calculated for C ₂₃ H ₁₈ BrNO435.0470, found 436.0545 [M+H] ⁺ ; ¹ H NMR (600 MHz, Chloroform-d) δ 7.65 (dt, J =9.0, 0.6 Hz, 1H), 7.44 – 7.35 (m, 4H), 7.17 – 7.14 (m, 2H), 7.07 (d, J= 2.5Hz, 1H), 6.88 (dd, J = 9.0, 2.6 Hz, 1H), 6.73 – 6.70 (m, 2H), 6.63 (d, J =0.7 Hz, 1H), 3.88 (s, 3H), 3.75 (s, 3H). ¹³ C NMR (151 MHz , Chloroform-d) δ 168.94, 159.12, 156.28, 141.34, 133.97, 132.69, 131.51 (×2), 131.36 (×2), 130.10, 129.60 (×2), 127.40, 125.30, 114 .90, 113.73 (×2), 112.83, 108.90, 102.92, 55.68, 55.27.

C series derivatives

Compound C1 (yellow solid, yield: 88%), HR-MS m/z: calculated for C ₂₁ H ₁₃ Cl ₂ NO ₃ 397.0273, found 420.0165 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.94(dt, J = 8.9, 0.7 Hz, 1H), 7.36 (t, J = 1.9 Hz, 1H), 7.29 (d, J = 1.9 Hz, 2H), 7.05– 7.01 (m, 2H), 6.99 – 6.97 (m, 1H), 6.81 (dd, J = 8.9, 2.5 Hz, 1H), 6.60 –6.57 (m, 2H), 6.57 (d, J = 0.7 Hz, 1H). ¹³ C NMR (151 MHz , Methanol-d ₄ ) δ 168.53,158.51, 155.40, 142.58, 139.92, 135.79 (×2), 133.50, 132.13, 132.07, 131.28(×2), 129.31 (×2), 125.57, 116.35,116.11 (×2) , 114.26, 110.49, 106.38.

Compound C2 (red solid, yield: 90%), HR-MS m/z: calculated for C ₂₁ H ₁₃ N ₃ O ₇ 419.0754, found 420.0826 [M+H] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.57 (s,1H), 9.41 (s, 1H), 8.72 (t, J = 2.1 Hz, 1H), 8.54 (d, J = 2.1 Hz, 2H), 8.04 (d, J =8.8 Hz, 1H), 7.06 – 6.97 (m, 3H), 6.83 (dd, J = 8.9, 2.5 Hz, 1H), 6.68 (s, 1H),6.45 – 6.37 (m, 2H). ¹³ C NMR (151 MHz, DMSO -d ₆ ) δ 164.81, 157.27, 154.51, 147.00(×2), 140.40, 138.02, 131.20, 130.91 (×2),130.54, 129.70 (×2), 122.73,120.11, 116.17, 114.82 (×2), 113.41 , 110.38, 105.37.

Compound C3 (yellow solid, yield: 89%), HR-MS m/z: calculated for C ₂₂ H ₁₃ F ₄ NO ₃ 415.0832, found 416.0903 [M+H] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.93(d, J = 8.8 Hz, 1H), 7.73 (ddd, J = 8.6, 4.7, 2.3 Hz, 1H), 7.61 (dd, J = 6.8, 2.3Hz, 1H), 7.14 (dd, J = 10.2, 8.7 Hz, 1H), 7.00 – 6.96 (m, 3H), 6.82 (dd, J = 8.8,2.5 Hz, 1H), 6.56 (s, 1H), 6.55 – 6.52 (m, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 168.85, 162.46 (d, J = 261 Hz), 158.49, 155.23, 142.51, 137.43 (d, J = 10.5 Hz), 133.67 (d, J = 3 Hz), 133.57 , 131.99, 131.38 (×2), 130.37 (d, J = 6 Hz), 126.06,123.35 (d, J = 270 Hz), 118.67 (dd, J ₁ = 33 Hz, J ₂ = 13.5 Hz), 117.96 ( d, J = 21 Hz), 116.22, 116.16 (×2), 114.25, 110.22, 106.34.

Compound C4 (yellow solid, yield: 93%), HR-MS m/z: calculated for C ₂₁ H ₁₃ ClN ₂ O ₅ 408.0513, found 431.0405 [M+H] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 8.00– 7.95 (m, 1H), 7.85 (d, J = 2.1 Hz, 1H), 7.60 (dd, J = 8.3, 2.1 Hz, 1H), 7.44(d, J = 8.3 Hz, 1H), 7.02 – 6.99 (m, 2H), 6.98 (d, J = 2.4 Hz, 1H), 6.82 (dd, J =8.9, 2.5 Hz, 1H), 6.58 – 6.52 (m, 3H). ¹³ C NMR (151 MHz , Methanol-d ₄ ) δ 168.09,158.68, 155.44, 148.34, 142.32, 137.01, 134.95, 133.45, 132.66, 132.10,131.61 (×2), 130.40, 127.91, 1 25.28, 116.43, 116.19 (×2), 114.33, 110.66 ,106.41.

Compound C5 (white solid, yield: 94%), HR-MS m/z: calculated for C ₂₁ H ₁₀ F ₅ NO ₃ 419.0581, found 420.0654 [M+H] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 8.21(d, J = 8.9 Hz, 1H), 7.10 – 7.04 (m, 2H), 6.94 (d, J = 2.5 Hz, 1H), 6.84 (dd, J =8.9, 2.5 Hz, 1H), 6.65 – 6.61 (m, 2H), 6.49 (s, 1H). ¹³ C NMR (151 MHz, Chloroform-d) δ 159.55, 158.27, 156.29, 144.40 (×2, dm, J = 250.5 Hz), 143.47(dm , J = 256.5 Hz), 143.90, 138.36 (×2, dm, J = 250.50 Hz), 135.09, 134.94,134.07 (×2), 126.83, 120.34, 118.35 (×2), 117.17,116.80 (m), 115.78 , 109.37

Compound C6 (white solid, yield: 95%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O ₃ 330.1004, found 353.0897 [M+Na] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.53 (s,1H), 9.33 (s, 1H), 8.49 – 8.41 (m, 2H), 7.78 (d, J = 8.8 Hz, 1H), 7.35 – 7.27(m, 2H), 7.07 – 7.01 (m, 2H), 6.97 (d, J = 2.5 Hz, 1H), 6.77 (dd, J = 8.9, 2.5Hz, 1H), 6.65 (s, 1H), 6.55 – 6.46 (m, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 167.91, 157.07, 154.19, 149.66 (×2), 142.34, 140.92, 131.12, 130.43, 130.13 (×2), 123.16, 122.81 (×2), 115 .04,114.92 (× 2), 113.17, 109.26, 105.24.

Compound C7 (white solid, yield: 92%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O ₃ 330.10044, found 331.1077 [M+H] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.51 (s,1H), 9.30 (s, 1H), 8.56 (dd, J = 2.3, 0.8 Hz, 1H), 8.51 (dd, J = 4.9, 1.7 Hz,1H), 7.77 (dt, J = 7.9, 2.0 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.24 (ddd, J = 8.0,4.9, 0.9 Hz, 1H), 7.07 – 7.02 (m, 2H), 6.97 (d, ¹³ C NMR (151 MHz, DMSO - d ₆ ) δ 167.71, 156.91, 154.01, 152.21, 149.87, 141.01, 136.92, 131.29, 131.20, 130.31, 130.00 (×2), 123.11, 123.02, 115.05 (×2), 1 14.81, 113.08, 108.80, 105.15.

Compound C8 (white solid, yield: 87%), HR-MS m/z: calculated for C ₂₀ H ₁₄ N ₂ O ₃ 330.1004, found 353.0897 [M+Na] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.43 (s,1H), 9.26 (s, 1H), 8.26 – 8.22 (m, 1H), 7.78 (td, J = 7.6, 1.7 Hz, 1H), 7.75 –7.71 (m, 2H), 7.30 (ddd, J = 7.5, 4.6, 1.3 Hz, 1H), 7.02 – 6.97 (m, 2H), 6.94(d, J = 2.5 Hz, 1H), 6.75 (dd, J = 8.8, 2.5 Hz, 1H), 6.56 (s, 1H), 6.51 – 6.45(m, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 168.53, 156.54, 153.89, 152.40, 148.39, 141.91, 136.87, 131.25, 13 0.44, 129.39(× 2), 125.91, 125.02, 123.92, 114.70(×2), 114.64, 112.80, 108.15, 105.06.

Compound C9 (white solid, yield: 89%), HR-MS m/z: calculated for C ₂₃ H ₁₇ NO ₃ 355.1208, found 378.1101 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 8.14(d, J = 8.9 Hz, 1H), 7.57 (d, J = 15.5 Hz, 1H), 7.33 – 7.23 (m, 5H), 7.12 – 7.04(m, 2H), 6.93 (d, J = 2.5 Hz, 1H), 6.89 – 6.84 (m, 2H), 6.79 (dd, J = 8.9, 2.5Hz, 1H), 6.51 (s, 1H), 6.38 (d, J = 15.5 Hz, 1H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 167.87, 159.32, 155.03, 144.20, 141.76, 136.03, 133.16, 132.07, 131.66 (×2),131.37, 129.80 (×2), 129.07 ( ×2), 126.35, 123.47, 117.34, 116.68 (×2), 114.06, 110.67, 106.20.

Compound C10 (white solid, yield: 63%), HR-MS m/z: calculated for C ₂₄ H ₁₆ N ₂ O ₃ 380.1161, found 403.1053 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 8.19(dd, J = 8.5, 0.8 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.86 (dq, J = 8.5, 1.0 Hz, 1H), 7.82 (dd, J = 8.3 , 1.4 Hz, 1H), 7.71 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.68(d, J = 8.4 Hz, 1H), 7.59 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H) , 7.00 (d, J = 2.4 Hz,1H), 6.97 – 6.93 (m, 2H), 6.83 (dt, J = 8.7, 2.5 Hz, 1H), 6.52 (d, J = 0.7 Hz,1H), 6.26 – 6.21 (m, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 170.07, 157.79, 155.43, 154.31, 147.48, 143.51, 138.43, 133.56, 132.53, 131.43, 130. 99 (×2), 129.96, 129.65, 129.41, 128.82, 126.18, 122.29, 116.70, 115.53 (×2), 114.00, 110.63, 106.41.

Compound C11 (white solid, yield: 97%), HR-MS m/z: calculated for C ₁₉ H ₁₃ NO ₃ S335.0616, found 358.0508 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Methanol- d ₄ ) δ 7.70 (dd, J =5.0, 1.2 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.22 (dd, J = 3.8, 1.2 Hz, 1H), 7.18– 7.14 (m, 2H), 6.97 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 5.0, 3.8 Hz, 1H), 6.74(dd, J = 8.8, 2.4 Hz, 1H), 6.66 – 6.62 (m, 2H ), 6.60 (s, 1H). ¹³ C NMR (151 MHz,Methanol-d ₄ ) δ 165.23, 158.31, 154.65, 142.86, 139.79, 136.91, 135.56,133.81, 131.82, 130.30(×2 ), 128.76, 125.96, 116.30 (×2), 114.83, 113.77, 108.67, 106.12.

Compound C12 (dark brown solid, yield: 96%), HR-MS m/z: calculated for C ₁₉ H ₁₄ N ₂ O ₄ 334.0954, found 357.0846 [M+Na] ⁺ ; ¹ H NMR (600 MHz, Methanol- d ₄ ) δ 8.01(d, J = 8.8 Hz, 1H), 7.10 – 7.03 (m, 2H), 6.95 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 8.8, 2.5 Hz, 1H) , 6.68 – 6.61 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.09 (q, J = 0.9Hz, 1H), 2.25 (d, J = 0.9 Hz, 3H). ¹³ C NMR ( 151 MHz, Methanol-d ₄ ) δ 171.87, 162.51,161.07, 158.64, 155.73, 142.74, 133.03, 132.37, 131.52 (×2), 125.45, 116.68,115.89 (×2), 114.18, 111.44, 106.54, 103.42, 11.62 .

Compound C13 (white solid, yield: 96%), HR-MS m/z: calculated for C ₂₁ H ₁₄ FNO ₃ 347.0958, found 348.1031 [M+H] ⁺ ; ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.66(d, J = 8.9 Hz, 1H), 7.51 – 7.46 (m, 2H), 7.04 – 7.00 (m, 2H), 6.96 (d, J = 2.4Hz, 1H), 6.95 – 6.90 (m, 2H ), 6.75 (dd, J = 8.8, 2.5 Hz, 1H), 6.58 – 6.52 (m,3H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 170.62, 166.38 (d, J= 250.5 Hz), 158.25, 154.84, 143.09, 133.87 (×2, d, J = 9 Hz), 133.72, 133.20 (d, J = 3 Hz), 131.94, 130.99 (×2), 125.73, 116.25, 116.10, 116.09 (× 2) , 115.59, 113.89, 109.28, 106.20.

Compound C14 (white solid, yield: 94%), HR-MS m/z: calculated for C ₂₁ H ₁₄ ClNO ₃ 363.0662, found 364.0733 [M+H] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.54 (s,1H), 9.25 (s, 1H), 7.52 – 7.47 (m, 3H), 7.38 – 7.34 (m, 2H), 7.08 – 7.04 (m,2H), 6.95 (d, J = 2.4 Hz , 1H), 6.72 (dd, J = 8.9, 2.5 Hz, 1H), 6.66 (d, J = 0.7 Hz,1H), 6.60 – 6.55 (m, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) , 114.25, 112.90, 108.05, 105.03.

Compound C15 (white solid, yield: 95%), HR-MS m/z: calculated for C ₂₁ H ₁₄ BrNO ₃ 407.0157, found 408.0230 [M+H] ⁺ ; ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 9.54 (s,1H), 9.25 (s, 1H), 7.53 – 7.46 (m, 3H), 7.43 – 7.39 (m, 2H), 7.09 – 7.03 (m,2H),6.95 (d, J = 2.4 Hz , 1H), 6.72 (dd, J = 8.8, 2.4 Hz, 1H), 6.66 (d, J = 0.7 Hz,1H), 6.60 – 6.56 (m, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) , 114.23, 112.90, 108.05, 105.03.

Example 2:

Cytotoxicity test of synthetic compounds on tumor cells (canine breast cancer cell CMT-7346) and normal cells (mouse embryonic fibroblast NIH3T3) in vitro:

1. Experimental samples and experimental methods

The compounds prepared in Example 1 were used for activity experiments.

The cytotoxicity of compounds A1-A22, B1-B20, and C1-C15 to tumor cells (canine breast cancer cell CMT-7346) and normal cells (mouse embryonic fibroblast NIH3T3) was determined by MTS method with Bazedoxifene as a positive control .

The principle of MTS method for detecting cell activity: MTS is a brand new MTT analogue, its full name is 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxy-methoxyphenyl)-2-(4-sulfopheny)- 2H-tetrazolium, is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan (Formazan) compounds. The content of formazan can be measured at 490nm with a microplate reader. Under normal circumstances, the amount of formazan produced is directly proportional to the number of living cells, so the number of living cells can be inferred from the optical density OD value.

Inoculate cells

When the CMT-7364 cells grow well and are in the logarithmic growth phase, according to the passage operation, the cells are resuspended in 1 mL of complete culture medium, diluted with an appropriate amount of complete culture medium, and the count is 5×10 ⁴ cells/mL. The prepared cell suspension was inoculated into a 96-well cell culture plate, 100 μL was added to each well, sealed and transferred to a 37 °C, 5% _CO2 constant temperature incubator for 24 h.

Preparing and administering medicines

The compound was dissolved in DMSO, and the corresponding solvent was used as a negative control group. The control group was added with 100 μL of complete medium, and the experimental group was supplemented with complete medium to 200 μL, so that the final concentration of the drug solution in each well was 20 μM, and 5 groups were paralleled. Derivatives showing good inhibitory activity at a concentration of 20 μM were re-screened for active drugs using CMT-7346 and NIH3T3 cells according to a certain concentration gradient.

Kit to detect cell viability

After the cells were treated with drugs for 48 h, the MTS solution and the complete culture solution were mixed at a ratio of 1:10, the medium containing the drug solution in the 96-well plate was discarded, 200 μL of the mixed solution was added to each well, and 5 wells without The cell mixture blank group was placed in an incubator and reacted for 2–4 h. The absorbance of each well at the absorption wavelength of 490 nm was detected with a microplate reader, the data was exported and the inhibition rate was calculated, and the IC ₅₀ was calculated by the Bliss method through GraphPad Prism 8 software.

2. Experimental results (see Figure 8, Figure 9)

3. Conclusion

When the concentration of the drug solution was 20 μM, among the 59 synthetic compounds tested, 21 compounds (compounds A1, A4, A10, A14, A21, A22, B4, B5, B11, C1, C2, C3, C4, C5, C6, C7, C10, C11, C13, C14, C15) showed good inhibitory activity of canine breast cancer cell proliferation (inhibition rate>50%) (see Figure 8).

The above 21 compounds were screened by IC ₅₀ , and the IC 50 of 15 compounds ( _A1 , A4, A10, A14, B4, B11, C1, C3, C4, C6, C7, C11, C13, C14, C15) was less than 20 μM, The IC ₅₀ of 6 compounds (A21, A22, B5, C2, C5, C10) was less than 10 μM. Among them, the IC ₅₀ of B5, C2, and C5 were comparable to the positive drug Bazedoxifene, and there was no significant difference (see Fig. 9).

The degree of selectivity of a compound can be expressed by its SI (Selectivity Index) value. When the SI value (>10) indicates high selectivity to cancer cells, when the SI value (>2) indicates selectivity to cancer cells, and A compound is considered moderately toxic when the SI value (<2) means it can also cause cytotoxicity in normal cells. Therefore, calculate each SI value using the formula given below (see Figure 9).

SI = IC ₅₀ for normal cells/IC ₅₀ for cancer cells

Among the 21 compounds tested and screened, except for A22, B11, and C13, the other 18 compounds were all selective (SI>2), and 11 of them (A1, A14, B4, B5, C1, C3, The SI values of C5, C7, C10, C14, and C15) are greater than 10, showing high selectivity for CMT-7346 cells (SI>10). It is particularly worth mentioning that among these compounds, 14 compounds have higher selectivity to CMT-7346 cells than the positive control (SI = 3.57), including compound B5 ( SI = 29.18), C2 (SI = 7.50) and C5 (SI = 16.41).

Example 3

Effect of Compound C5 on Canine Breast Cancer Cells.

1. The effect of compound C5 on CMT-7346 cell cycle detected by flow cytometry

(1) Cell inoculation and administration

When the CMT-7346 cells grow well and are in the logarithmic growth phase, according to the passage operation, the cells are resuspended in 1 mL of complete culture medium, diluted with an appropriate amount of complete culture medium, and the count is 5×10 ⁴ cells/mL. The prepared cell suspension was inoculated into a 6-well cell culture plate, 1 mL per well, sealed and transferred to a 37 °C, 5% _CO2 constant temperature incubator for 24 h. The medium was discarded and the prepared medicinal solution was added to make the final concentration of the medicinal solution in each well its IC ₅₀ , and corresponding solvents were added to the blank control group. The culture was continued and samples were taken at 48 h.

(2) Sampling

After being cultured for 48 hours, the CMT-7346 cells were taken, and according to the passage operation, the cells were resuspended in 1 mL of complete culture medium, diluted with an appropriate amount of complete culture medium, counted to 2× ¹⁰⁶ /mL, and set aside.

(3) Fixed and sieved

After centrifuging again, discard the PBS, add pre-cooled 70% ethanol to fix, and react overnight in a refrigerator at 4 °C. Discard the fixative after centrifugation, resuspend with 3 mL PBS for 5 min, and discard the PBS after centrifugation. Filter once with a 400-mesh sterile sieve for cells, wash with 3 mL of PBS, and discard the PBS after centrifugation.

(4) Dyeing

Stain evenly with 1 mL of PI staining solution, and react at 4 °C in the dark for 30 min. After the reaction, detect with a flow cytometer and record the red fluorescence at an excitation wavelength of 488 nm.

(5) Experimental results

Flow cytometry was used to determine the effect of compound C5 on the cell cycle after treatment of CMT-7346 cells for 24 h and 48 h. As shown in Figure 10, after C5 treated cells for 24 h and 48 h, the proportion of G0G1 phase increased from 52.81±0.04% in the control group to 61.33±0.05% and 81.14±0.12%, respectively, and the proportion of G0G1 phase increased significantly. Compound C5 has the effect of arresting CMT-7346 cells in G0G1 phase in a time-dependent manner.

2. The effect of compound C5 on the apoptosis of CMT-7346 cells detected by flow cytometry

(1) Cell inoculation and administration

When the CMT-7346 cells grow well and are in the logarithmic growth phase, according to the passage operation, the cells are resuspended in 1 mL of complete culture medium, diluted with an appropriate amount of complete culture medium, and the count is 5×10 ⁴ cells/mL. The prepared cell suspension was inoculated into a 6-well cell culture plate, 1 mL per well, sealed and transferred to a 37 °C, 5% _CO2 constant temperature incubator for 24 h. The medium was discarded and the prepared medicinal solution was added to make the final concentration of the medicinal solution in each well its IC ₅₀ , and corresponding solvents were added to the blank control group. Continue to cultivate and take samples at 24 h and 48 h.

(2) Sampling

After being cultured for 48 h, the CMT-7346 cells treated with different dosage concentrations were taken respectively, and the cells were resuspended in 1 mL of complete medium according to the subculture operation, and an appropriate amount of complete medium was added to dilute, and the count was 2×10 ⁶ pc/mL, spare.

(3) Dyeing

After centrifuging again, discard the PBS, resuspend the cells with 500 μL of Binding Buffer working solution, and add 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide to each group of cell suspensions in turn. After vortex mixing, stain for 15‒20min, and the whole reaction process should be protected from light. Flow cytometer for fluorescence detection (Annexin V-FITC maximum excitation light is 488 nm, emission light is 520 nm; PI maximum excitation light is 535 nm, emission light is 617 nm).

(4) Experimental results

Apoptosis is an important regulatory program for cancer cells to maintain cell homeostasis. In this study, the effect of compounds on CMT-7364 cell apoptosis was detected by Annexin V-FITC/PI double staining method. Compared with the blank control group, after being treated with compound C5 for 24 h and 48 h, the apoptosis rates of the cells reached 17.42±2.76% and 51.97%±1.56%, respectively, which were significantly higher than the blank control group, showing that compound C5 significantly induced Apoptosis of CMT-7364 cells (Fig. 11).

3. Transmission electron microscope observation of the effect of compound C5 on the ultrastructure of CMT-7346 cells

(1) Cell inoculation and administration

When the CMT-7346 cells grow well and are in the logarithmic growth phase, according to the passage operation, the cells are resuspended in 1 mL of complete culture medium, diluted with an appropriate amount of complete culture medium, and the count is 5×10 ⁴ cells/mL. The prepared cell suspension was inoculated into a 6-well cell culture plate, 1 mL per well, sealed and transferred to a 37 °C, 5% _CO2 constant temperature incubator for 24 h, the medium was discarded and the prepared drug was added. solution, so that the final concentration of the drug solution in each well was its IC ₅₀ , and the corresponding solvent was added to the blank control group. Continue to cultivate and take samples at 24 h and 48 h.

(2) Fixed

Pour off the medium, collect the cells with a cell scraper, add PBS and transfer to a centrifuge tube, discard the supernatant after centrifugation, wash with PBS twice, and then perform the fixation operation in the following order: 4% glutaric acid fixation for 2‒4 h — Wash 3 times with PBS, 15 min each time—fix with 2% osmic acid for 1 h—wash 3 times with PBS, 15 min each time.

(3) Dehydration

The cells were dehydrated step by step with different concentrations of ethanol: 50% ethanol—80% ethanol—90% ethanol—absolute ethanol—xylene, 15 min each time, repeated 3 times.

(4) Embedding

Infiltrate the epoxy resin overnight in a fume hood. Cell aggregates were cut to a suitable size, placed in a mold containing embedding agent, and polymerized at 60 °C for 72 h.

(5) slice

Cut the aggregated cells into ultra-thin sections with a thickness of about 10–20 μm using an ultramicrotome.

dyeing

The cell sections were stained in the following order: uranyl acetate staining in the dark for 20‒30 min—washing with distilled water, blotted dry on filter paper—staining with lead citrate for 15‒30 min. After the sections were dry, they were detected with a transmission electron microscope and photographed.

(6) Experimental results

The effect of compound C5 on the ultrastructure of canine breast cancer CMT-7364 cells was observed by transmission electron microscopy, and the results are shown in FIG. 12 . Under the transmission electron microscope, it can be seen that the cells in the blank group have intact cell membranes, complete nuclear membrane structure, clear nucleoli, and organelles in the cytoplasm, such as mitochondria, Golgi apparatus, endoplasmic reticulum, etc., have complete structures and normal shapes, and can clearly Double membranes and cristae of mitochondria were observed (Fig. 12A). After compound C5 was treated for 24 h, although the nuclear membranes of most cells remained intact, some changes occurred in the nuclei, such as slightly uneven distribution of chromatin and marginalization, and the color of chromatin around the nuclear membrane changed. The cell membrane has a little budding phenomenon, and the number of vesicles has also increased; the shape of mitochondria is elongated and swollen compared with the blank group. body (Fig. 12B). After 48 hours of administration, the nuclear membrane structure of most cells was relatively complete, the nuclear shape changed seriously, the nuclear chromatin edge set deepened, the shape of mitochondria changed, lipid droplets began to increase, and more autophagosomes and autophagy lysozymes appeared. body (Figure 12).

Claims (6)

1. A compound, characterized in that the compound has the formula

Structure:

Wherein the substituents R ₁ and R ₂ are hydrogen, hydroxyl, halogen, C1-6 alkoxy such as methoxy or ethoxy, C1-6 alkanoyloxy such as acetyl; Ar is aryl, substituted aryl, Such as phenyl, naphthyl, pyridyl, thienyl, quinolinyl, pyrimidinyl, benzimidazolyl, thiadiazolyl, isoxazolyl, benzothiazolyl, pyridinyl, styryl, m-di Nitrophenyl, m-dichlorophenyl, o-chloronitrophenyl, o-fluorotrifluoromethylphenyl, pentafluorophenyl, p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, p- Fluorophenyl, naphthylmethyl, etc., where n=0~2; when R ₁ and R ₂ are substituted at the same time, each substituent can be the same or different. 2. The compound according to claim 1, characterized in that the preferred structure of the compound is as follows:

,

R ₁ , R ₂ are preferably hydrogen, methoxy and hydroxyl; Ar is preferably p-chlorophenyl, p-bromophenyl, p-fluorophenyl, m-dinitrophenyl, m-dichlorophenyl, ortho-chloronitrophenyl , o-fluorotrifluoromethylphenyl, pentafluorophenyl, p-methoxyphenyl, 1-naphthyl, 1-benzothiazolyl, 4-pyridyl, 2-quinolinyl, 2-thiazolyl, 5-methylisoxazol-3-yl; n is preferably 0. 3. The compound according to claim 1, characterized in that it is selected from the following compounds:

. 4. Use of the compound according to any one of claims 1-3 in the preparation of a drug for treating tumors. 5. The application according to claim 4, characterized in that the drug for treating tumor is an anti-tumor drug for treating breast cancer. 6. The application according to claim 4, characterized in that, the anti-breast cancer drug is a canine breast cancer drug.

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