Spiroquinoline derivatives having anti-breast cancer activity, methods of making and use thereof

Abstract

The application belongs to the technical field of synthetic methods of pharmaceutical chemistry, and discloses a spiro quinoline derivative with anti-breast cancer activity, a structural general formula of which is shown as chemical formula 1. The application further discloses a preparation method and application of the spiro quinoline derivative. The application provides a novel spiro quinoline derivative containing a pyrrolidine side chain, and the spiro quinoline derivative has anti-breast cancer activity and can be used as a candidate active molecule for preparing anti-breast cancer drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical chemical synthesis methods, specifically relating to spirocyclic quinoline derivatives with anti-breast cancer activity, as well as the preparation method and application of the above-mentioned spirocyclic quinoline derivatives. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and drug treatment is the primary approach. With in-depth research, anti-breast cancer drugs have evolved from traditional chemotherapy drugs to targeted therapy, endocrine therapy, and novel molecular drugs. Currently, a large number of marketed anti-breast cancer drugs are used clinically; however, drug resistance and toxic side effects severely limit drug screening in clinical practice. Therefore, designing and developing more structurally novel anti-breast cancer drugs is of great significance.

[0003] Spirocyclic compounds possess rigid and stable structures, exhibiting unique properties not found in typical organic compounds, such as end-group isomerism, spiroconjugation, and spirohyperconjugation. Compared to monocyclic or planar aromatic structures, spirocyclic structures possess larger three-dimensional spatial structures. Heterocyclic spirocyclic structures are also considered bioisosteres of certain groups, capable of altering the water solubility, lipophilicity, preferred conformation, and ADMET properties of drug molecules to a certain extent, making optimized lead molecules easier to drug. Therefore, spirocyclic compounds occupy a very important position in drug development. Spirocyclic compounds of different sizes and ring systems possess rich three-dimensional structures, providing possibilities for improving drug efficacy and enhancing drug patent innovation; they can both break existing drug patents and be used to design novel small molecule compounds with new structures or skeletons. Therefore, designing and synthesizing quinoline derivatives containing spirocyclic structures and exploring their antitumor activity is of great significance. Currently, there are no literature reports on the synthesis and antitumor activity of quinoline derivatives containing spirocyclic structures. The class of compounds reported in this study belongs to a new structure, and its synthetic method has not been reported before. Summary of the Invention

[0004] The purpose of this invention is to provide a spirocyclic quinoline derivative with anti-breast cancer activity, thereby providing an anti-breast cancer compound with a novel structure.

[0005] The second objective of this invention is a method for preparing the above-mentioned spirocyclic quinoline derivative.

[0006] A third objective of this invention is the application of the aforementioned spirocyclic quinoline derivative in the preparation of drugs for treating breast cancer.

[0007] The first technical solution adopted in this invention is: a spirocyclic quinoline derivative with anti-breast cancer activity, the general structural formula of which is shown in chemical formula 1:

[0008] Chemical Formula 1 Wherein, R is selected from hydrogen and an alkylamine group having 1 to 5 carbon atoms; n is selected from 0 or 4 methylene groups; m is selected from 0 or 4 methylene groups; When n=0, m=4; when n=4, m=0.

[0009] The first technical solution adopted in this invention is also characterized by: The above-mentioned spirocyclic quinoline derivatives are selected from one of the following structures:

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016] .

[0017] The second technical solution adopted in this invention is: the preparation method of the above-mentioned spirocyclic quinoline derivative, firstly, using compound (1): 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid and compound (2): spiro[3,4]oct-6-one and spiro[3,4]oct-6-one as starting materials, through a phosphine oxychloride-mediated Friedländer condensation cyclization reaction, to obtain compound (3): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[ b Quinoline and compound (4): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadien[ b The target compound was prepared by mixing two spirocyclic quinoline isomers, namely [quinoline], and then by Buchwald-Hartwig coupling reaction or dehalogenation reaction.

[0018] The second technical solution adopted in this invention is further characterized by: The synthetic reaction route is as follows: .

[0019] The above preparation method includes the following steps: Step 1, Compound (3): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline and compound (4): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadien[ b Synthesis of mixtures of quinoline: Under anhydrous conditions, compound (1): 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid and compound (2): spiro[3.4]oct-6-one were added sequentially to phosphorus oxychloride. After stirring evenly, the mixture was refluxed. When compound (1) was completely consumed, the mixture was concentrated under reduced pressure and diluted with anhydrous dichloromethane. After the diluted mixture cooled, it was adjusted to alkaline and extracted. The organic phase was dried, concentrated under reduced pressure, and purified by column chromatography to obtain a mixture of compounds (3) and (4). The molar ratio of compound (2) to compound (1) ranges from 1.0 to 2.0:1.0; The volume / molar ratio of phosphorus oxychloride to compound (1) is 2 mL to 5 mL: 1 mmol / L; The volume / molar ratio of dichloromethane to compound (1) is 2 mL to 6 mL: 1 mmol / L; Step 2, Compound (6a): N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentano[ b Quinoline-9'-amine and compound (7a): N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine, or, compound (6b): N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadienyl[] b Quinoline-9'-amine and compound (7b): N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine, or, compound (6c): N-pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentan[ b Quinoline-9'-amine and compound (7c): N -pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentan[ b Synthesis of quinoline-9'-amine: Under nitrogen protection, a mixture of compounds (3) and (4), a tetrahydrofuran solution of primary amine compound (5), sodium tert-butoxide, 3rd BrettPhos precatalyst, and dioxane were added sequentially. After mixing evenly, the mixture of compounds (3) and (4) was reacted until it was completely consumed. The mixture was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain two isomers (6a) and (7a) or isomers (6b) and (7b) or isomers (6c) and (7c). The molar ratio of compound (5) to the mixture of compounds (3) and (4) ranges from 8.0 to 20.0:1.0; The molar ratio of sodium tert-butoxide to the mixture of compounds (3) and (4) ranges from 2.0 to 5.0:1.0; The molar ratio of the 3rd BrettPhos precatalyst to the mixture of compounds (3) and (4) ranged from 0.05 to 0.2:1.0; The volume / molar ratio of the mixture of dioxane with compounds (3) and (4) is: 4 mL to 10 mL: 1 mmol / L; Step 3, Compound (8): 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline and compound (9): 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopenta[ b Synthesis of mixtures of quinoline: Under nitrogen protection, the mixture of compounds (3) and (4) was added to methanol, and then 10% palladium / carbon was added. After mixing evenly, the mixture was replaced with hydrogen three times. After the reaction was completed under hydrogen atmosphere, the mixture was filtered under reduced pressure, concentrated, and purified by silica gel column chromatography to obtain a mixture of two isomer compounds (8) and (9). The mass ratio of palladium / carbon to compounds (3) and (4) is in the range of 0.1 to 0.3:1.0; The volume / molar ratio of the methanol mixture with compounds (3) and (4) is 10 mL to 20 mL: 1 mmol / L.

[0020] The primary amine compound (5) mentioned above is ethylamine, cyclopropylmethylamine, or n-pentylamine; When the primary amine compound (5) is ethylamine, two isomers (6a) and (7a) are generated. When the primary amine compound (5) is cyclopropylmethylamine, two isomers (6b) and (7b) are generated. When the primary amine compound (5) is n-pentylamine, two isomers (6c) and (7c) are generated.

[0021] The third technical solution adopted in this invention is the application of the above-mentioned spirocyclic quinoline derivative in the preparation of anti-breast cancer drugs.

[0022] The third technical solution adopted in this invention is further characterized in that: The above-mentioned spirocyclic quinoline derivatives are used as active ingredients in the preparation of anti-breast cancer drugs.

[0023] The dosage forms of the above-mentioned drugs are oral preparations, mucosal delivery preparations, or injections.

[0024] The aforementioned spirocyclic quinoline derivatives can inhibit breast cancer cell activity.

[0025] The beneficial effects of this invention are: This invention synthesizes a novel class of spirocyclic quinoline derivatives containing pyrrolidine side chains. These compounds exhibit inhibitory activity against four types of tumor cells: HepG2 liver cancer, MCF-7 breast cancer, A549 lung cancer, and HeLa cervical cancer. Compound 6a, in particular, showed the strongest inhibitory effect against MCF-7 breast cancer (IC50). 50 = 1.4 ± 0.2 μmol / L), its activity is 3 times that of the positive control drug cisplatin. It can be used as a candidate drug for anti-breast cancer. In addition, compounds 6-9 showed no toxicity to normal MCF-10A cells, while cisplatin showed some toxicity. Attached Figure Description

[0026] Figure 1 This is the synthetic route for quinoline derivatives (compounds 6-9) containing spirocyclic structures; Figure 2 This is a high-performance liquid chromatography (HPLC) image of compound (6a) (detection wavelength 254 nm). Figure 3 This is a high-performance liquid chromatography (HPLC) image of compound (6a) (detection wavelength 220 nm). Figure 4 This is the mass spectrum of compound (6a); Figure 5 This is the 1H NMR spectrum of compound (6a); Figure 6 This is a high-performance liquid chromatography (HPLC) image of compound (6b) (detection wavelength 254 nm). Figure 7 This is a high-performance liquid chromatography (HPLC) image of compound (6b) (detection wavelength 220 nm). Figure 8 This is the mass spectrum of compound (6b); Figure 9 This is the 1H NMR spectrum of compound (6b); Figure 10 This is a high-performance liquid chromatography (HPLC) image of compound (7a) (detection wavelength 254 nm). Figure 11 This is the evaporation purity diagram of compound (7a); Figure 12 This is the mass spectrum of compound (7a); Figure 13 This is the 1H NMR spectrum of compound (7a); Figure 14 This is a high-performance liquid chromatography (HPLC) image of compound (7b) (detection wavelength 254 nm). Figure 15 This is a high-performance liquid chromatography (HPLC) image of compound (7b) (detection wavelength 220 nm). Figure 16 This is the mass spectrum of compound (7b); Figure 17 This is the 1H NMR spectrum of compound (7b); Figure 18 This is a high-performance liquid chromatography (HPLC) image of compound (8) (detection wavelength 254 nm). Figure 19 This is a high-performance liquid chromatography (HPLC) image of compound (8) (detection wavelength 220 nm). Figure 20 This is the mass spectrum of compound (8); Figure 21 This is the 1H NMR spectrum of compound (8); Figure 22 This is a high-performance liquid chromatography (HPLC) image of compound (9) (detection wavelength 254 nm). Figure 23 This is the high performance liquid chromatography (HPLC) image of compound (9) (detection wavelength 220 nm). Figure 24 This is the mass spectrum of compound (9); Figure 25 This is the 1H NMR spectrum of compound (9). Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0028] like Figure 1As shown, this invention designed and synthesized a series of quinoline derivatives containing spirocyclic structures (compounds 6-9), and evaluated their in vitro antitumor activity. First, using 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid (compound 1) and spiro[3,4]oct-6-one (compound 2) as starting materials, a mixture of spirocyclic quinoline isomers (compounds 3 and 4) was obtained via a phosphine oxychloride-mediated Friedländer condensation cyclization reaction. Then, six target compounds (compounds 6-9) were successfully prepared via Buchwald-Hartwig coupling or dehalogenation reactions. Next, the MTT assay was used to test the cytotoxicity of the target compounds against four types of tumor cells (HepG2, MCF-7, A549, and HeLa) and normal breast epithelial cells (MCF-10A).

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: The preparation method of spirocyclic quinoline derivatives involves first using 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid (compound 1) and spiro[3,4]oct-6-one (compound 2) as starting materials, and then conducting a phosphine oxychloride-mediated Friedländer condensation cyclization reaction to obtain 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 3) and 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadiene[ b The target compound was prepared by mixing quinoline (compound 4) and then by Buchwald-Hartwig coupling reaction or dehalogenation reaction.

[0031] Example 2 The preparation method of spirocyclic quinoline derivatives specifically includes the following steps: Step 1: 9'-Chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 3) and 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadiene[b The synthetic route for the mixture of quinoline (compound 4) is as follows:

[0032] Under anhydrous conditions, 2.94 g (10 mmol) of 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid (compound 1) and 1.36 g (11 mmol) of spiro[3.4]oct-6-one (compound 2) were added sequentially to phosphorus oxychloride (20 mL). After stirring, the mixture was refluxed (106 °C) for 8 h. After complete consumption of compound 1 by TLC, the mixture was concentrated under reduced pressure. The crude product was diluted with anhydrous dichloromethane (30 mL). The diluted product was slowly poured into an ice-water mixture (30 mL) with stirring. After the ice was completely melted, 2 mol / L sodium hydroxide solution was added dropwise to adjust the mixture to alkaline (pH = 8). The mixture was then extracted with dichloromethane (3 × 30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio 1:1). V / V = 20 / 1), yielding 2.12 g of a mixture of two isomers (compounds 3 and 4) as a brown semi-oily solid, with a yield of 53.0% (the two isomers have small polarity differences, making separation difficult, so they were used directly in the next reaction). ESI-MS [M+1] + m / z 401.2.

[0033] Step 2 N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentano[ b Quinoline-9'-amine (compound 6a) and N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b The specific synthetic route for quinoline-9'-amine (compound 7a) is as follows:

[0034] Under nitrogen protection, 800 mg (2 mmol) of compound 3 (a mixture of 3 and 4), 10 mL (20 mmol) of tetrahydrofuran solution of ethylamine (5a), 577 mg (6 mmol) of sodium tert-butoxide, and 91 mg (0.1 mmol) of 3 were added sequentially to the sealed tube. rdIn a mixture of BrettPhos precatalyst and dioxane (10 mL), the mixture was stirred until homogeneous. The reaction system was then sealed in a tube and reacted at 100°C for 2 h. After complete consumption of compounds (a mixture of compounds 3 and 4) by TLC, the mixture was concentrated under reduced pressure. The crude product was then purified by silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio 1:1). V / V =15 / 1), resulting in two isomers, 6a and 7a.

[0035] N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentano[ b Quinoline-9'-amine (compound 6a): brown solid, yield 29.2%. 1 H NMR (300 MHz, CD3OD): δ 7.68 (s, 1H), 7.18 (s, 1H), 4.35 (t, J =5.7 Hz, 2H), 4.11-4.06 (m, 2H), 4.03(s, 3H), 3.88-3.80 (m, 2H), 3.53-3.48 (m, 2H), 3.22-3.08 (m, 4H), 2.94-2.84(m, 2H), 2.58-2.53 (m, 2H), 2.43-2.32 (m, 2H), 2.29-2.18 (m, 6H), 2.15-2.05(m, 2H), 1.55-1.51 (m, 3H). ESI-MS [M+1] + m / z 410.4.

[0036] N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine (compound 7a): brown solid, yield 33.6%. 1 H NMR (300 MHz, CD3OD): δ 7.67 (s, 1H), 7.14 (s, 1H), 4.33 (t, J=5.7 Hz, 2H), 4.04 (s, 3H), 3.90-3.83(m, 4H), 3.52-3.50 (m, 2H), 3.47-3.45 (m, 2H), 3.21-3.09 (m, 4H), 2.42-2.33(m, 2H), 2.23-2.02 (m, 10H), 1.41-1.37 (m, 3H). ESI-MS [M+1] + m / z 410.4.

[0037] The high-performance liquid chromatography (HPLC) chromatogram of compound (6a) (detection wavelength 254 nm) is shown below. Figure 2 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (6a) (detection wavelength 220 nm) is shown below. Figure 3 As shown; The mass spectrum of compound (6a) is as follows: Figure 4 As shown; The proton NMR spectrum of compound (6a) is shown below. Figure 5 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (6b) (detection wavelength 254 nm) is shown below. Figure 6 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (6b) (detection wavelength 220 nm) is shown below. Figure 7 As shown; The mass spectrum of compound (6b) is as follows: Figure 8 As shown; The proton NMR spectrum of compound (6b) is shown below. Figure 9 As shown.

[0038] Step 3 N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadienyl[] b Quinoline-9'-amine (compound 6b) and N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b The synthetic route for the mixture of quinoline-9'-amine (compound 7b) is as follows:

[0039] Under nitrogen protection, 800 mg (2 mmol) of compound 3 and 4, 1.42 g (20 mmol) of cyclopropylmethylamine (compound 5b), 577 mg (6 mmol) of sodium tert-butoxide, 91 mg (0.1 mmol) of 3rd BrettPhos precatalyst, and dioxane (20 mL) were added sequentially to a sealed tube. After stirring thoroughly, the reaction system was incubated in the sealed tube at 100 °C for 2 h. After complete consumption of compound 3 and 4 by TLC, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio 1:1). V / V = 15 / 1), resulting in two isomers, 6b and 7b.

[0040] N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadienyl[] b Quinoline-9'-amine (compound 6b): yellow solid, yield 23.5%. 1 H NMR (300MHz, CD3OD): δ 7.66 (s, 1H), 7.18 (s, 1H), 4.35 (t, J=5.4 Hz, 2H), 4.03 (s,3H), 3.96-3.94 (m, 2H), 3.84-3.82 (m, 2H), 3.52-3.48 (m, 2H), 3.19-3.10 (m,4H), 2.96-2.94 (m, 2H), 2.59-2.54 (m, 2H), 2.40-2.07 (m, 10H), 1.45-1.35 (m,1H), 0.77-0.73 (m, 2H), 0.52-0.50 (m, 2H). ESI-MS [M+1] + m / z 436.5.

[0041] N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine (compound 7b): yellow solid, yield 28.9%. ¹H NMR (300 MHz, DMSO-d6): δ8.29 (br, 1H), 7.75 (s, 1H), 7.21 (s, 1H), 4.20 (t, J=5.7 Hz, 2H), 3.95 (s, 3H), 3.63-3.59 (m, 4H), 3.42-3.37 (m, 2H), 3.29-3.21 (m, 2H), 3.15-3.08 (m, 2H), 3.05-2.98 (m, 2H), 2.22-2.20 (m, 2H), 2.08-2.04 (m, 5H), 1.98-1.94 (m, 5H), 1.18-1.10 (m, 1H), 1.55-1.50 (m, 2H), 1.49-1.34 (m, 2H). ESI-MS[M+1] + m / z 436.5.

[0042] The high-performance liquid chromatography (HPLC) chromatogram of compound (7a) (detection wavelength 254 nm) is shown below. Figure 10 As shown; The evaporative purity diagram of compound (7a) is shown below. Figure 11 As shown; The mass spectrum of compound (7a) is shown below. Figure 12 As shown; The proton NMR spectrum of compound (7a) is shown below. Figure 13 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (7b) (detection wavelength 254 nm) is shown below. Figure 14 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (7b) (detection wavelength 220 nm) is shown below. Figure 15 As shown; The mass spectrum of compound (7b) is as follows: Figure 16 As shown; The proton NMR spectrum of compound (7b) is shown below. Figure 17 As shown.

[0043] Step 4, 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 8) and 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopenta[ b The specific synthetic route for the mixture of quinoline (compound 9) is as follows:

[0044] Under nitrogen protection, 400 mg (1 mmol) of compounds (a mixture of compounds 3 and 4) was added to methanol (15 mL), followed by 10% palladium / carbon (40 mg). After stirring thoroughly, the mixture was purged three times with hydrogen, and then stirred under a hydrogen atmosphere for 6 h. After the reaction was complete, the mixture was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio V / V = 20 / 1) to obtain a mixture of two isomers (compounds 8 and 9) of 307 mg as a brown solid, with a yield of 83.8% (the two isomers have small polarity differences, making column chromatography separation difficult). The mixture of compounds 8 and 9 was separated by chiral chromatography (DAICEL chiral AD-H column, mobile phase: n-hexane: ethanol = 30: 70, detection wavelength 190–500 nm, 5.2 min).

[0045] 7'-Methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 8): brown solid, yield 30.1%. 1 H NMR (300 MHz, CD3OD): δ 8.13 (s,1H), 7.30 (s, 1H), 7.28 (s, 1H), 4.27 (t, J=6.3 Hz, 2H), 3.96 (s, 3H), 3.02(t, J=7.2 Hz, 2H), 2.79-2.74 (m, 2H), 2.65-2.63 (m, 4H), 2.45-2.13 (m, 10H), 1.86-1.82 (m, 4H). ESI-MS [M+1] + m / z 367.2.

[0046] 7'-Methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopenta[ b Quinoline (compound 9): pale yellow solid, yield 37.3%. 1 H NMR (300 MHz, CD3OD): δ7.91 (s,1H), 7.28 (s, 1H), 7.22 (s, 1H), 4.22 (t, J=6.3 Hz, 2H), 3.96 (s, 3H), 3.15-3.13 (m, 4H), 2.82-2.77 (m, 2H), 2.70-2.66 (m, 4H), 2.20-1.98 (m, 8H), 1.88-1.84 (m, 4H). ESI-MS [M+1] + m / z 367.2.

[0047] The high-performance liquid chromatography (HPLC) chromatogram of compound (8) (detection wavelength 254 nm) is shown below. Figure 18 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (8) (detection wavelength 220 nm) is shown below. Figure 19 As shown; The mass spectrum of compound (8) is shown below. Figure 20 As shown; The proton NMR spectrum of compound (8) is shown below. Figure 21 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (9) (detection wavelength 254 nm) is shown below. Figure 22 As shown; The high-performance liquid chromatography (HPLC) chromatogram of compound (9) (detection wavelength 220 nm) is shown below. Figure 23 As shown; The mass spectrum of compound (9) is shown below. Figure 24 As shown; The proton NMR spectrum of compound (9) is shown below. Figure 25 As shown.

[0048] Example 3: The preparation method of spirocyclic quinoline derivatives specifically includes the following steps: Step 1: 9'-Chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 3) and 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadiene[ b The synthetic route for the mixture of quinoline (compound 4) is as follows:

[0049] Under anhydrous conditions, 2.94 g (10 mmol) of 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid (compound 1) and 2.48 g (20 mmol) of spiro[3.4]oct-6-one (compound 2) were added sequentially to phosphorus oxychloride (50 mL). After stirring, the mixture was refluxed (106 °C) for 8 h. After complete consumption of compound 1 by TLC, the mixture was concentrated under reduced pressure. The crude product was diluted with anhydrous dichloromethane (60 mL). While stirring, the diluted product was slowly poured into an ice-water mixture (50 mL). After the ice was completely melted, 2 mol / L sodium hydroxide solution was added dropwise to adjust the mixture to alkaline (pH = 8). Dichloromethane (3 × 50 mL) was added for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio 1:1). V / V = 20 / 1), yielding 2.01 g of a mixture of two isomers (compounds 3 and 4) as a brown semi-oily solid, with a yield of 50.2% (the two isomers have small polarity differences, making separation difficult, so they were used directly in the next reaction). ESI-MS [M+1] + m / z 401.2.

[0050] Step 2 N -pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentan[ b Quinoline-9'-amine (compound 6c) and N -pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentan[ b The specific synthetic route for quinoline-9'-amine (compound 7c) is as follows:

[0051] Under nitrogen protection, 800 mg (2 mmol) of compound 3 (a mixture of 3 and 4), 20 mL (40 mmol) of tetrahydrofuran solution of n-pentylamine (5c), 961 mg (10 mmol) of sodium tert-butoxide, and 362 mg (0.4 mmol) of 3 were added sequentially to the sealed tube. rd In a mixture of BrettPhos precatalyst and dioxane (20 mL), the mixture was stirred thoroughly and then reacted in a sealed tube at 100 °C for 2 h. After complete consumption of compounds (a mixture of compounds 3 and 4) by TLC, the mixture was concentrated under reduced pressure. The crude product was then purified by silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio 1:1). V / V =15 / 1), resulting in two isomers, 6c and 7c.

[0052] Example 4: Step 4, 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline (compound 8) and 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopenta[ b The specific synthetic route for the mixture of quinoline (compound 9) is as follows:

[0053] Under nitrogen protection, 400 mg (1 mmol) of compounds (a mixture of compounds 3 and 4) was added to methanol (20 mL), followed by 120 mg of 10% palladium / carbon. After thorough mixing, the mixture was purged three times with hydrogen and stirred under a hydrogen atmosphere for 6 h. After the reaction was complete, the mixture was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, volume ratio V / V = 20 / 1) to obtain 332 mg of a mixture of two isomers (compounds 8 and 9) as a brown solid, with a yield of 90.7% (the two isomers have small polarity differences, making column chromatography separation difficult). The mixture of compounds 8 and 9 was separated by chiral chromatography (DAICEL chiral AD-H column, mobile phase: n-hexane: ethanol = 30: 70, detection wavelength 190–500 nm, 5.2 min).

[0054] Example 5: The above-mentioned spirocyclic quinoline derivatives are used in the preparation of anti-breast cancer drugs.

[0055] Example 6: The above-mentioned spirocyclic quinoline derivatives are used as active ingredients in the preparation of anti-breast cancer drugs.

[0056] The following is an in vitro antitumor activity test of the product obtained in the above embodiments: Five cell lines were selected to test the in vitro antitumor activity of the target compound: HepG2 liver cancer cells, human breast cancer MCF-7 cells, human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and normal breast cancer cells MCF-10A. The MTT assay was used to evaluate the in vitro antitumor activity of the target compound. Procedure: The sample was dissolved in DMSO and then diluted with DMEM medium to different concentrations of 50, 20, 10, 5, 1, 0.5, 0.1, and 0.01 μmol / L. The test cell lines in the logarithmic growth phase were suspended in DMEM medium without phenol red containing 10% fetal bovine serum and seeded into 96-well cell culture plates. 100 μL (4-10)*10⁻¹⁰ μL was added to each well.4 mL -1 Cell suspension. After the cells were fully adhered, the original culture medium was discarded, and 100 µL of culture medium containing the test drug was added. After culturing for 3 days, 30 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for another 4 h. Then, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the cells. The absorbance (OD) value of each well was measured at 490 nm using a microplate reader. The inhibition rate (%) of the test drug against tumor cells was calculated according to the formula, and the half-maximal inhibitory concentration (IC50) was calculated using OriginPro software. 50 )value.

[0057] Cell inhibition rate (%) = (normal OD value - drug-treated OD value) / normal OD value × 100%.

[0058] Five cell lines were selected as test cells: HepG2 liver cancer cells, human breast cancer MCF-7 cells, human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and normal breast epithelial cells MCF-10A. The in vitro antitumor activity of compounds (6–9) was tested (Table 2). The results showed that compounds 6–9 exhibited inhibitory activity against all four tumor cell lines: HepG2, MCF-7, A549, and HeLa. Compound 6a, in particular, showed the strongest inhibitory effect against MCF-7 (IC50). 50 = 1.4 ± 0.2 mol / L), its activity is 3 times that of the positive control drug cisplatin. Furthermore, compounds 6–9 are not toxic to normal MCF-10A cells, while cisplatin exhibits some toxicity.

[0059] Table 1. In vitro antitumor activity of the products (n = 3)

[0060] The results showed that all target compounds possessed broad-spectrum antitumor activity, with compound 6a exhibiting the strongest inhibitory activity against MCF-7 breast cancer cells (IC50). 50 The concentration of the target compound was 1.4 ± 0.2 μmol / L, which is three times that of the positive control drug cisplatin. Simultaneously, the target compound showed no significant toxicity to normal MCF-10A cells, demonstrating good tumor cell selectivity. The spirocyclic quinoline derivative synthesized in this study provides a new compound basis for antitumor drug development, and compound 6a can be further studied as a candidate drug for highly selective breast cancer treatment.

Claims

1. A spirocyclic quinoline derivative with anti-breast cancer activity, characterized in that, The general structural formula is shown in chemical formula 1: Chemical Formula 1 Wherein, R is selected from hydrogen and an alkylamine group having 1 to 5 carbon atoms; n is selected from 0 or 4 methylene groups; m is selected from 0 or 4 methylene groups; When n=0, m=4; when n=4, m=0.

2. The spirocyclic quinoline derivative according to claim 1, characterized in that, The spirocyclic quinoline derivative is selected from one of the following structures: 。 3. The method for preparing the spirocyclic quinoline derivative according to claim 1 or 2, characterized in that, First, starting with compound (1): 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid and compound (2): spiro[3,4]oct-6-one and spiro[3,4]oct-6-one, a phosphine oxychloride-mediated Friedländer condensation cyclization reaction was carried out to obtain compound (3): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline and compound (4): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadien[ b The target compound was prepared by mixing two spirocyclic quinoline isomers, namely [quinoline], and then by Buchwald-Hartwig coupling reaction or dehalogenation reaction.

4. The method for preparing the spirocyclic quinoline derivative according to claim 3, characterized in that, The synthetic reaction route is as follows: 。 5. The method for preparing the spirocyclic quinoline derivative according to claim 3, characterized in that, The preparation method includes the following steps: Step 1, Compound (3): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline and compound (4): 9'-chloro-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentadien[ b Synthesis of mixtures of quinoline: Under anhydrous conditions, compound (1): 2-amino-5-methoxy-4-[3-(pyrrolidone-1-yl)propoxy]benzoic acid and compound (2): spiro[3.4]oct-6-one were added sequentially to phosphorus oxychloride. After stirring evenly, the mixture was refluxed. When compound (1) was completely consumed, the mixture was concentrated under reduced pressure and diluted with anhydrous dichloromethane. After the diluted mixture cooled, it was adjusted to alkaline and extracted. The organic phase was dried, concentrated under reduced pressure, and purified by column chromatography to obtain a mixture of compounds (3) and (4). The molar ratio of compound (2) to compound (1) ranges from 1.0 to 2.0:1.0; The volume / molar ratio of phosphorus oxychloride to compound (1) is 2 mL to 5 mL: 1 mmol / L; The volume / molar ratio of dichloromethane to compound (1) is 2 mL to 6 mL: 1 mmol / L; Step 2, Compound (6a): N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentano[ b Quinoline-9'-amine and compound (7a): N -Ethyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine, or, compound (6b): N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadienyl[] b Quinoline-9'-amine and compound (7b): N -(cyclopropylmethyl)-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentano[ b Quinoline-9'-amine, or, compound (6c): N -pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentan[ b Quinoline-9'-amine and compound (7c): N -pentyl-7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopentan[ b Synthesis of quinoline-9'-amine: Under nitrogen protection, a mixture of compounds (3) and (4), a tetrahydrofuran solution of primary amine compound (5), sodium tert-butoxide, 3rd BrettPhos precatalyst, and dioxane were added sequentially. After mixing evenly, the mixture of compounds (3) and (4) was reacted until it was completely consumed. The mixture was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain two isomers (6a) and (7a) or isomers (6b) and (7b) or isomers (6c) and (7c). The molar ratio of compound (5) to the mixture of compounds (3) and (4) ranges from 8.0 to 20.0:1.0; The molar ratio of sodium tert-butoxide to the mixture of compounds (3) and (4) ranges from 2.0 to 5.0:1.0; The molar ratio of the 3rd BrettPhos precatalyst to the mixture of compounds (3) and (4) ranged from 0.05 to 0.2:1.0; The volume / molar ratio of the mixture of dioxane with compounds (3) and (4) is: 4 mL to 10 mL: 1 mmol / L; Step 3, Compound (8): 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-2',3'-dihydrospiro[cyclobutane-1,1'-cyclopentadien[] b Quinoline and compound (9): 7'-methoxy-6'-[3-(pyrrolidone-1-yl)propoxy]-1',3'-dihydrospiro[cyclobutane-1,2'-cyclopenta[ b Synthesis of mixtures of quinoline: Under nitrogen protection, the mixture of compounds (3) and (4) was added to methanol, and then 10% palladium / carbon was added. After mixing evenly, the mixture was replaced with hydrogen three times. After the reaction was completed under hydrogen atmosphere, the mixture was filtered under reduced pressure, concentrated, and purified by silica gel column chromatography to obtain a mixture of two isomer compounds (8) and (9). The mass ratio of palladium / carbon to compounds (3) and (4) is in the range of 0.1 to 0.3:1.0; The volume / molar ratio of the methanol mixture with compounds (3) and (4) is 10 mL to 20 mL: 1 mmol / L.

6. The method for preparing the spirocyclic quinoline derivative according to claim 5, characterized in that, The primary amine compound (5) is ethylamine, cyclopropylmethylamine, or n-pentylamine; When the primary amine compound (5) is ethylamine, two isomers (6a) and (7a) are generated. When the primary amine compound (5) is cyclopropylmethylamine, two isomers (6b) and (7b) are generated. When the primary amine compound (5) is n-pentylamine, two isomers (6c) and (7c) are generated.

7. The use of the spirocyclic quinoline derivative according to claim 1 or 2 in the preparation of anti-breast cancer drugs.

8. The use of the spirocyclic quinoline derivative according to claim 7 in the preparation of anti-breast cancer drugs, characterized in that, Application of spirocyclic quinoline derivatives as active ingredients in the preparation of anti-breast cancer drugs.

9. The use of the spirocyclic quinoline derivative according to claim 7 in the preparation of anti-breast cancer drugs, characterized in that, The dosage form of the drug is oral preparation, mucosal delivery preparation or injection.

10. The use of the spirocyclic quinoline derivative according to claim 7 in the preparation of anti-breast cancer drugs, characterized in that, The spirocyclic quinoline derivative can inhibit breast cancer cell activity.

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