Curcumin analogues, methods of preparation and uses thereof

Abstract

The application discloses a curcumin analogue, a preparation method and application thereof, and has a structural formula of (E)-3-((1H-indol-3-yl)methylene)-5-((E)-4-mercaptobenzylidene)-1-methylpiperidine-4-thione. The compound is synthesized through a plurality of reaction steps, including aldehyde acylation, cyclization, thio reaction and condensation reaction. In-vitro experiments show that the IC50 value of the compound to triple-negative breast cancer cells MDA-MB-231 is 3.69+ / -0.89 muM, which is significantly better than existing chemotherapeutic drugs. Cell experiments show that the compound can effectively inhibit tumor growth and induce cell apoptosis, and has the potential to be developed into an anti-triple-negative breast cancer drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a curcumin analogue, a preparation method and application thereof. BACKGROUND

[0002] Triple-negative breast cancer (TNBC) is a special type of breast cancer characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This type of breast cancer is more commonly seen in young women and is associated with a poorer prognosis. Triple-negative breast cancer is biologically distinct from other types of breast cancer, often exhibiting higher proliferation rates and greater invasiveness. This makes TNBC patients often at a later stage at diagnosis, with relatively limited treatment options. Since there is a lack of specific hormone receptors, traditional endocrine therapy (such as tamoxifen or aromatase inhibitors) is ineffective for TNBC. Chemotherapy is currently the main treatment, but may not be effective for some patients, and has significant side effects. Therefore, it is necessary to develop new treatment methods for triple-negative breast cancer.

[0003] Prostate cancer is a major disease that seriously threatens men's health worldwide and has become a very serious public health problem. In the statistics of male malignant tumor incidence, prostate cancer has long occupied the top position. Currently, there are many treatment methods for prostate cancer in clinical practice, such as surgical resection, radiotherapy, and chemotherapy. However, these conventional treatment methods have certain limitations. Surgical treatment has poor effect on patients in the advanced stage or with metastasis, and surgery itself has high risk, which can cause serious complications such as urinary incontinence and sexual dysfunction; radiotherapy can kill cancer cells while causing significant damage to surrounding normal tissues, resulting in adverse reactions such as radiation cystitis and proctitis; chemotherapy drugs lack specific targeting effects on cancer cells, and have significant toxic effects on normal cells while inhibiting cancer cells, resulting in serious side effects such as nausea, vomiting, hair loss, and bone marrow suppression in patients during treatment, which greatly reduces the patient's quality of life and treatment compliance.

[0004] Studies have shown that natural product curcumin has various pharmacological effects in vitro, including anti-inflammatory, antioxidant, anticoagulant, anti-aging, hypolipidemic, anti-atherosclerotic, and anti-tumor effects, which have received extensive attention and research at home and abroad. However, there is currently no report that curcumin analogues can be effectively used for the treatment of triple-negative breast cancer and prostate cancer.

[0005] Existing patent CN 113956227A discloses a flavonoid compound for the treatment of triple-negative breast cancer; however, this compound has a large molecular weight, a complex preparation process, and high drug costs. Patent CN113582978A discloses a compound containing a thiobenzofuran structure, but does not involve the treatment of triple-negative breast cancer cells (TNBC). This invention, through structural modification, designs and synthesizes novel monocarbonyl curcumin analogs to achieve inhibitory effects on triple-negative breast cancer cells and human prostate cancer cells, and can serve as potential drugs for the treatment of triple-negative breast cancer. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention modifies the structure of turmeric and, through repeated experiments, discovers a turmeric analog that specifically inhibits triple-negative breast cancer cells. The structure of the analog is as follows:

[0007]

[0008] More preferably, the turmeric analogue can be prepared into a variety of pharmaceutically acceptable salts, including mesylate, citrate, phosphate, or acetate.

[0009] Furthermore, the present invention optimizes the synthesis method of turmeric analogues, specifically employing the following steps:

[0010]

[0011] Step 1: React 3-indolecarboxaldehyde with benzoyl chloride to generate intermediate compound 2;

[0012] Step 2: Compound 2 is condensed with piperidine and N-methyl-4-piperidinone in tetrahydrofuran to generate intermediate compound 3;

[0013] Step 3: Under nitrogen protection, compound 3 reacts with Lawson's reagent to generate thiointermediate compound 4;

[0014] Step 4: Compound 4 is condensed with p-mercaptobenzaldehyde under the catalysis of trifluoroacetic acid to obtain the target turmeric analog.

[0015] More preferably, in step one, the reaction temperature is 0-5℃ and the reaction time is 1-3 hours; in step two, the reaction temperature is 40-60℃ and the reaction time is 2-5 hours; in step three, the molar ratio of Lawson's reagent to compound 3 is 1-1.5:1; and in step four, the molar ratio of trifluoroacetic acid to compound 4 is 3-5:1.

[0016] A further preferred application is the use of the turmeric analogue obtained in the present invention in the preparation of anti-triple-negative breast cancer drugs.

[0017] Furthermore, the drug used in this invention is a tablet, capsule, injection, or lyophilized powder preparation.

[0018] The beneficial effects of this invention are:

[0019] (1) The turmeric analog provided by the present invention has a significant inhibitory effect on triple-negative breast cancer cells (IC50 of 3.69±0.89μM for MDA-MB-231 cells) and prostate cancer cells (IC50 of 7.36±1.05 for PC-3 cells), and can achieve a high-efficiency inhibitory effect on cancer cells at a low dose.

[0020] (2) The method for preparing turmeric analogs provided by the present invention is simple, has a high reaction yield, and produces high-purity products, making it suitable for large-scale industrial production. Attached Figure Description

[0021] Figure 1 The first image shows the hydrogen NMR spectrum of the turmeric analog obtained in Example 1 of this invention.

[0022] Figure 2 The carbon NMR spectrum of the turmeric analog obtained in Example 2 of this invention;

[0023] Figure 3 This is the mass spectrum of the turmeric analog obtained in Example 3 of the present invention;

[0024] Figure 4 The IC50 values ​​of the turmeric analogue obtained in Example 1 against four types of human breast cancer cells, human liver cancer cells, and human prostate cancer cells;

[0025] Figure 5 The figure shows the results of the liveness and death experiment of the turmeric analog obtained in Example 1 on MDA-MB-231 cells;

[0026] Figure 6 The image shows the electrophoresis results of the turmeric analog obtained in Example 1 on MDA-MB-231 cells labeled with Bax and Bcl-2. Detailed Implementation

[0027] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. 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.

[0028] Example 1: Preparation of Curcuma analogues

[0029] The raw materials 3-indolecarboxaldehyde, benzoyl chloride, and Lawson reagent were purchased from Anhui Zesheng Technology Co., Ltd., and 4-mercaptobenzaldehyde was purchased from Beijing Bailingwei Technology Co., Ltd.

[0030] Step 1:

[0031]

[0032] The starting material 3-indolecarboxaldehyde (10 mmol) was dissolved in a solution of dichloromethane (10 mL), and triethylamine (12 mmol) was added. Benzoyl chloride (10 mmol) was slowly added to the mixture at 0 °C. After reacting for 1 h, the solvent was directly evaporated to obtain compound 2 with a yield of 95%.

[0033] Step 2:

[0034]

[0035] Compound 2 (5 mmol) was dissolved in a solution of tetrahydrofuran (10 mL), and piperidine (7.5 mmol) and N-methyl-4-piperidinone (7.5 mmol) were added. The mixture was reacted at 50 °C for 4 hours. TLC was monitored until the reaction of the starting material was complete. The reaction was quenched by adding 10 mL of ammonium chloride aqueous solution, and extracted with 50 mL × 3 ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and after vacuum distillation, silica gel column chromatography (EA:MeOH = 8:1) gave product compound 3 in 76% yield.

[0036] Step 3:

[0037]

[0038] Under nitrogen protection, compound 3 (3 mmol) was dissolved in 5 mL of tetrahydrofuran, and Lawson's reagent (4 mmol) was added at 30 °C. The reaction was carried out at this temperature for 12 hours. TLC monitoring was performed until the reaction of the starting material was complete. The reaction was quenched by adding 10 mL of ammonium chloride aqueous solution, extracted with 50 mL × 3 ethyl acetate, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (EA:MeOH = 6:1) under reduced pressure to give compound 4 in 89.2% yield.

[0039] Step 4:

[0040]

[0041] Compound 4 (1 mmol) was dissolved in 3 ml of acetic acid, and trifluoroacetic acid (5 mmol) and p-mercaptobenzaldehyde (2.5 mmol) were added. The mixture was reacted at 80 °C for 5 hours. The solvent was then directly evaporated, and silica gel column chromatography (EA:MeOH = 7:1) was performed to give the curcumin analog (compound 5) with a yield of 85% and a purity of 99.2%. 1 H NMR(400MHz,MeOD)δ8.25(s,1H),7.83(d,J=7.6Hz,1H),7.79(s,1H),7.63(s,1H),7.46(d,J=7.7Hz,1H ),7.35(d,J=8.5Hz,2H),7.28–7.17(m,2H),6.88(d,J=8.5Hz,2H),3.86(d,J=10.3Hz,4H),2.59(s,3H). 13 C10 NMR (150MHz, DMSO) δ 174.91, 147.46, 142.47, 139.47, 136.21, 131.26, 131.17, 131.07, 129.43, 129.21, 128.68, 127.06, 124.86, 122.72, 122.64, 119.79, 117.91, 112.44, 58.85, 33.96. HRMS (m / z): 377.1624. The 1H NMR spectrum, 1C NMR spectrum, and mass spectrum are shown below. Figure 1 , Figure 2 , Figure 3 .

[0042] Example 2: Cell Experiment

[0043] MDA-MB-231 cells, MCF-7 cells, SK-BR-3 cells, BT-474 cells, Hepg-2 cells, and PC-3 cells, along with culture media and CCK-8 kits, were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0044] 1. CCK-8 Viability Experiment

[0045] Cells at a density of 3000-5000 cells / well were seeded in 96-well plates. After cell attachment, cells were treated with different dilutions or the control solvent DMSO for 48 hours. Then, CCK-8 reagent was added, and the cells were cultured at 37°C and 5% CO2 for 1 hour. Absorbance was measured at 450 nm using a microplate spectrophotometer (Thermo). The control group had 100% cell viability.

[0046] For IC50, cells were treated with different concentrations of the curcumin analogue obtained in Example 1 (0, 0.78125 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) for 48 h, cell viability was measured, and inhibition rate was calculated. CCK-8 assays were performed three times, with each assay repeated at least three times. The IC50 of the compounds was studied using Prism statistical software.

[0047] Cell viability assays were performed on the turmeric analogue obtained in Example 1 to test its toxicity to various tumor cell types, including four types of human breast cancer cells (MDA-MB-231, MCF-7, SK-BR-3, BT-474), human liver cancer cells (Hepg-2), and human prostate cancer cells (PC-3). The IC50 results are shown in [Figure 1]. Figure 4 .

[0048] Table 1. IC50 values ​​of turmeric analogues against human breast cancer cells, hepatocytes, and prostate cancer cells.

[0049]

[0050] The turmeric analog prepared in this invention has an IC50 (half-maximal inhibitory concentration) of 3.69 ± 0.89 μM for MDA-MB-231, which is significant.

[0051] Epirubicin, at the same dose as the aforementioned turmeric analogue, was tested using a CCK-8 assay. The IC50 values ​​against the four different types of human breast cancer cells were 15% higher than those of the turmeric analogue prepared in this invention. Doubling the dose of epirubicin could achieve the same IC50 values ​​as the aforementioned turmeric analogue.

[0052] 2. Live-Death Experiments

[0053] The liveness and death experiment of the turmeric analogue obtained in Example 1 on MDA-MB-231 cells.

[0054] Procedure: Cells were exposed to a turmeric analogue for 48 hours and stained with a LIVE / DEAD kit. NC was used as a negative control, and DMSO was used as the solvent. See below for detailed experimental results. Figure 5 .

[0055] 3. Mechanism study of the turmeric analogue obtained in Example 1 on the apoptosis of MDA-MB-231 cells.

[0056] Electrophoresis experiments were performed on Bax and Bcl-2 markers. Using 0.1% dimethyl sulfoxide (DMSO) as a control, other test samples were treated with different concentrations (5 μM, 10 μM, 15 μM) of compound 4 for 24 h. With increasing drug concentration, the expression of Bacl-2 and Bax proteins decreased. The Bax / Bcl-2 ratio increased with increasing compound 4 concentration. Previous studies have shown that inhibiting Bax protein function through Akt protein phosphorylation can induce apoptosis. Therefore, the curcumin analogue obtained in Example 1 can inhibit Akt protein phosphorylation, increase the Bax / Bcl-2 apoptosis protein ratio, and promote apoptosis in MDA-MB-231 cells. Specific results are shown in [link to example]. Figure 6 .

[0057] 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 turmeric analogue, characterized in that, The structural formula is as follows:

2. A pharmaceutically acceptable salt of the turmeric analogue of claim 1, wherein the pharmaceutically acceptable salt includes citrate, phosphate, mesylate, or acetate.

3. A method for preparing the turmeric analogue of claim 1, characterized in that, The preparation route is as follows: Step 1: React 3-indolecarboxaldehyde with benzoyl chloride to generate intermediate compound 2; Step 2: Compound 2 is condensed with piperidine and N-methyl-4-piperidinone in tetrahydrofuran to generate intermediate compound 3; Step 3: Under nitrogen protection, compound 3 reacts with Lawson's reagent to generate thiointermediate compound 4; Step 4: Compound 4 is condensed with p-mercaptobenzaldehyde under the catalysis of trifluoroacetic acid to obtain the product turmeric analog.

4. The preparation method according to claim 3, characterized in that, In step one, the reaction temperature is 0-5℃ and the reaction time is 1-3 hours; in step two, the reaction temperature is 40-60℃ and the reaction time is 2-5 hours; in step three, the molar ratio of Lawson's reagent to compound 3 is 1-1.5:1; in step four, the molar ratio of trifluoroacetic acid to compound 4 is 3-5:

1.

5. The use of the turmeric analogue of claim 1 in the preparation of drugs for treating triple-negative breast cancer and prostate cancer.

6. The application according to claim 5, characterized in that, The drug is a tablet, capsule, injection, or lyophilized powder preparation.

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