Synthesis and application of a fused ring compound with anti-triple negative breast cancer

Abstract

The application discloses a fused ring compound with anti-triple negative breast cancer, a synthesis method and application thereof, and relates to the field of organic synthesis. 50 The application efficiently constructs a 4H-benzoindene skeleton through a Lewis acid catalyzed cyclization reaction of propargyl alcohol and 1-methylindole-3-carboxylic acid derivatives, and the method has the advantages of easy raw material, mild condition and high yield. In vitro anti-tumor activity test shows that the compound has an IC 50 Value of 15.44±2.25 μM on triple negative breast cancer cells MDA-MB-231, and can concentration-dependently up-regulate the Bax / Bcl-2 protein ratio and induce tumor cell apoptosis. The compound provided by the application has the advantages of novel structure, green and efficient synthesis method, significant anti-triple negative breast cancer activity and clear mechanism, and has good potential to be developed into an anti-triple negative breast cancer drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and organic synthesis technology, specifically relating to a fused-ring compound with a 4H-benzoindene skeleton, its synthesis method, and its application in the preparation of anti-triple-negative breast cancer drugs. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. It is characterized by rapid proliferation, high invasiveness, high recurrence rate, and poor prognosis, and is more common in young women. Due to the lack of specific targets, traditional endocrine therapy and targeted therapy are largely ineffective against TNBC, and the mainstream clinical treatment relies solely on systemic chemotherapy. However, existing chemotherapy drugs have limitations such as limited efficacy, significant side effects, and a tendency to develop drug resistance, making them unsuitable for long-term treatment.

[0003] Currently, the development of small molecule targeted drugs for TNBC still faces significant technical shortcomings: existing anti-tumor small molecule drugs have limited scaffold types, and no 4H-benzo[a]indene fused-ring scaffold compounds have been applied to anti-triple-negative breast cancer research. Furthermore, there is a lack of novel chemical entities that can specifically induce TNBC cell apoptosis by regulating the mitochondrial apoptosis pathway and upregulating the Bax / Bcl-2 protein ratio. Polycyclic aromatic fused-ring compounds, with their rigid planar structure and extended π-conjugated system, possess excellent drug-likeness and tunable bioactivity, making them a core research scaffold for anti-tumor lead compounds. Among them, the 4H-benzo[a]indene scaffold is widely found in various natural active products, possessing advantages such as low toxicity and high biocompatibility. However, current research on its anti-tumor properties is limited, and there are no reports on its use in targeted therapy for triple-negative breast cancer or regulation of the Bax / Bcl-2 apoptosis pathway.

[0004] To address the shortcomings of existing technologies, such as single drug structure, poor targeting, unclear mechanism of action, and limited drug options, this invention designs and synthesizes a novel 4H-benzoindene skeleton fused ring compound. It verifies that this compound exhibits highly selective inhibitory activity against triple-negative breast cancer cells and exerts anti-tumor effects by regulating the Bax / Bcl-2 apoptosis pathway. This provides novel structural candidate compounds and new therapeutic strategies for the development of targeted drugs for TNBC. Summary of the Invention

[0005] This invention designs and synthesizes a fused-ring compound (compound A) containing a 4H-benzo[a]indene backbone. In vitro antitumor activity tests show that this compound has significant inhibitory activity against triple-negative breast cancer cells MDA-MB-231 and can induce apoptosis by upregulating the Bax / Bcl-2 ratio. It can be considered a potential drug for the treatment of triple-negative breast cancer. The structure of the compound is as follows: , The method for synthesizing compound A includes the following steps: Step (1) Synthesis of compound 1a: Compound 1-1 and compound 1-2 undergo a nucleophilic addition reaction to obtain compound 1a; Step (2) Synthesis of compound 2a: Compound 2-1 and iodomethane undergo methylation to give compound 2-2, and compound 2-2 undergoes ester hydrolysis to give compound 2a; Step (3) Synthesis of compound A: Compound 1a and compound 2a react to obtain compound A.

[0006] The reaction conditions in step (1) are -78℃, and carried out under anhydrous and oxygen-free conditions.

[0007] In step (3), the catalyst is copper trifluoromethanesulfonate Cu(OTf)2, and its amount is 10-30 mol% of the molar amount of compound 1a; the organic solvent is dichloromethane (DCM); the reaction temperature is 30-60 ℃; and the reaction time is 3-6 hours.

[0008] Furthermore, the 4H-benzoindene skeleton compound obtained in this invention can be used to prepare anti-triple-negative breast cancer drugs.

[0009] The beneficial effects of this invention are: (1) This invention designs and synthesizes a novel fused-ring compound with a 4H-benzoindene skeleton, the structure of which simultaneously contains an indene ring, an indole ring, and multiple aryl substituents. This skeleton has significant structural originality in the field of anti-triple-negative breast cancer, providing a novel chemical entity for TNBC treatment.

[0010] (2) This invention utilizes a Cu(OTf)₂-catalyzed cyclization reaction of propargyl alcohol with a 1-methylindole-3-carboxylic acid derivative to construct the 4H-benzoindene skeleton in one step. Compared with existing technologies, this method has the following advantages: readily available raw materials, no need for expensive metals or complex ligands, mild reaction conditions (40°C, air atmosphere), short reaction time (4 hours), high yield, and single regioselectivity. This method overcomes the disadvantages of traditional fused ring synthesis involving multiple steps, high temperatures, and strong acid or strong base conditions, making it suitable for large-scale production and demonstrating significant inventiveness.

[0011] (3) In vitro cell experiments showed that compound A of the present invention has an IC50 effect on triple-negative breast cancer cells MDA-MB-231. 50 The concentration was as low as 15.44 ± 2.25 μM, and the inhibitory effect on lung cancer, colon cancer, and prostate cancer cell lines was relatively weak, demonstrating good selectivity for TNBC. Further mechanistic studies revealed that this compound can upregulate the Bax / Bcl-2 protein ratio in a concentration-dependent manner, inducing apoptosis in MDA-MB-231 cells through the mitochondrial apoptosis pathway. This clear mechanism of action provides solid experimental evidence for its development as a drug targeting TNBC, and has significant clinical application value. Attached Figure Description

[0012] Figure 1 The 1H NMR spectrum of the 4H-benzo[a]indene skeleton compound (A) obtained in Example 1 of this invention; Figure 2 The carbon NMR spectrum of the 4H-benzo[a]indene skeleton compound (A) obtained in Example 1 of this invention; Figure 3 The image shows the electrophoresis results of the 4H-benzoindene skeleton compound (A) obtained in Example 1 on MDA-MB-231 cells labeled with Bax and Bcl-2. Detailed Implementation

[0013] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. The components of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0014] Example 1: Preparation of compound A Preparation of compound 1a Under nitrogen protection, 1.32 g of compounds 1-2 were dissolved in 5 mL of anhydrous tetrahydrofuran. 4.8 mL of n-butyllithium was slowly added at -78 °C. After reacting for 12 hours, 1.82 g of benzophenone was added. The temperature was then raised to 0 °C and reacted for 6 hours. The reaction was quenched by slowly adding saturated ammonium chloride solution. The solvent was evaporated, and the sample was loaded onto a dry plate and subjected to column chromatography to obtain compound 1a with a yield of 67%.

[0015] Preparation of compound 2a Methyl 3-indolecarboxylate 2-1 (0.7 g) was dissolved in tetrahydrofuran solution (THF, 20 mL). Sodium hydride (NaH, 0.24 g) dissolved in tetrahydrofuran (10 mL) was added at 0 °C. The mixture was stirred for 0.5 h under these conditions, and then allowed to return to room temperature to continue the reaction. After gradually returning to room temperature, iodomethane was added, and the mixture was stirred overnight. The reaction was monitored by TLC. After the reaction was complete, 10 mL of saturated NH4Cl aqueous solution was added to quench the reaction. The mixture was extracted with ethyl acetate (3 × 15 mL), and the organic layers were combined and washed with brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was subjected to column chromatography (petroleum ether:ethyl acetate = 5:1) to separate and obtain 1-substituted methyl 3-indolecarboxylate 2-2, with a yield of 92%.

[0016] The above product 2-2 was dissolved in a mixed solution of tetrahydrofuran:methanol:water = 1:1:1 (15 mL), and then 6 mL of 2 N sodium hydroxide solution (NaOH) was added. The mixture was refluxed at 90 °C for 9 h. The reaction was monitored by TLC. After the reaction was complete, water (20 mL) and ethyl acetate (3 × 15 mL) were added for extraction. The mixture was acidified with 1 N HCl solution to pH = 2.0. The organic layers were combined and washed with brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was subjected to column chromatography (petroleum ether:ethyl acetate = 3:1) to separate and obtain 1-substituted 3-indolecarboxylic acid 2a in 95% yield.

[0017] Preparation of compound A At 40°C, 1a (0.3 mmol) and 2a (0.1 mmol), Cu(OTf)₂ (20 mol%) were added to a sealed tube and dissolved in DCM. The reaction was carried out in air for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was cooled to room temperature, and DCM was removed under reduced pressure. The crude product was purified by silica gel column chromatography with a ratio of petroleum ether to ethyl acetate of 3:1 to obtain the corresponding product A in 89% yield. 1 H NMR (600 MHz, CDCl3) δ: 8.46 (s, 1H), 7.73-7.66 (m, 7H), 7.63-7.51 (m, 13H), 7.47-7.43 (m, 2H), 7.38 (d, J=12.0 Hz, 1H), 3.35 (s, 3H),1.07-1.01 (m, 1H), 0.90-0.84 (m, 1H), 0.16-0.00 (m, 8H). 13C NMR (150 MHz, CDCl3) δ: 163.13, 148.29, 145.57, 142.75, 141.31, 141.21, 140.75, 139.56,138.23, 135.57, 135.46, 134.18, 132.05, 132.01, 130.42, 130.31, 130.27,130.11, 128.70, 128.04, 127.96, 127.91, 127.72, 127.55, 126.49, 126.34,126.14, 126.07, 125.90, 57.98, 32.10, 11.30, 10.75, 8.57, 7.94, 7.88, 6.90. (Appendix) Figure 1 Appendix Figure 2 .

[0018] Example 2: In vitro antitumor activity test MDA-MB-231 cells were purchased from Beyotime, the culture medium was complete MDA-MB-231 cell culture medium purchased from Beyotime, and the CCK-8 kit was purchased from Beyotime.

[0019] The inhibitory effect of compound A on the proliferation of various tumor cell lines was detected using the CCK-8 assay. Cell lines included human lung cancer A549, human colon cancer Caco-2, human prostate cancer PC-3, and human triple-negative breast cancer MDA-MB-231. Cells were seeded at 3000-5000 cells / well in 96-well plates. After adhesion, different concentrations of the compound (0-100 μM) were added for 48 h. CCK-8 reagent was then added, and absorbance was measured at 450 nm to calculate the IC50. 50 Value. The results are as follows: Example 3: Bax / Bcl-2 protein expression regulation experiment (Western Blot) To investigate the molecular mechanism of apoptosis-induced by compound A, the expression of Bax and Bcl-2 proteins was detected by Western blotting. MDA-MB-231 cells were treated with different concentrations of compound A for 24 hours, using 0.1% DMSO as a control. Results (see attached). Figure 3 The results showed that with increasing compound concentration, the expression of the anti-apoptotic protein Bcl-2 decreased, while the expression of the pro-apoptotic protein Bax increased, and the Bax / Bcl-2 ratio increased in a concentration-dependent manner. These results indicate that compound A induces apoptosis in TNBC cells through the mitochondrial apoptosis pathway (upregulating the Bax / Bcl-2 ratio), and the mechanism is clear.

[0020] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. A fused-ring compound, characterized in that, The general structural formula of the compound is shown in Formula A:

2. A method for preparing the fused-ring compound of claim 1, characterized in that, The method for synthesizing compound A includes the following steps: Step (1) Synthesis of compound 1a: Compound 1-1 and compound 1-2 undergo a nucleophilic addition reaction to obtain compound 1a; Step (2) Synthesis of compound 2a: Compound 2-1 and iodomethane undergo methylation to give compound 2-2, and compound 2-2 undergoes ester hydrolysis to give compound 2a; Step (3) Synthesis of compound A: Compound 1a and compound 2a react to obtain compound A with a 4H-benzoindene skeleton.

3. The preparation method according to claim 2, characterized in that, The reaction conditions in step (1) are -78℃, and carried out under anhydrous and oxygen-free conditions.

4. The preparation method according to claim 2, characterized in that, In step (3), the catalyst is copper trifluoromethanesulfonate Cu(OTf)2, and its amount is 10-30 mol% of the molar amount of compound 1a; the organic solvent is dichloromethane; the reaction temperature is 30-60 ℃; and the reaction time is 3-6 hours.

5. The use of the fused-ring compound of claim 1 in the preparation of an antitumor drug for treating triple-negative breast cancer.

6. The application according to claim 5, characterized in that, The fused-ring compound can induce apoptosis in triple-negative breast cancer cells by upregulating the Bax / Bcl-2 protein expression ratio.

Images