A mitochondria-targeted beta-anhydroicariside derivative, and a preparation method and pharmaceutical use thereof

Abstract

The application belongs to the technical field of natural medicinal chemistry, and discloses a mitochondria-targeting beta-dehydrated icariin derivative as well as a preparation method and medicinal use thereof. The application discloses a beta-dehydrated icariin derivative with a mitochondria-targeting function. The derivative is a monosubstituted compound, is a yellow solid powder at normal temperature, and is easily soluble in organic solvents such as methanol and dichloromethane. The beta-dehydrated icariin derivative can target into tumor cells and mitochondria thereof, induce cell apoptosis by affecting a mitochondrial membrane potential, and improve an anti-tumor effect. The beta-dehydrated icariin derivative has the advantages of high efficiency in targeting mitochondria, good anti-tumor effect, simple preparation method, easy operation, and convenience in subsequent development and industrialization.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and in particular to a mitochondrial-targeted β-dehydrated epimedium derivative, its preparation method, and its medicinal uses. Background Technology

[0002] Statistics show a clear upward trend in cancer mortality rates. The high mortality rate is not only due to the severe damage it causes to physiological tissues, but also because of its high metastatic potential and difficulty in treatment. Despite significant advancements in medical technology and treatment methods, cancer treatment remains one of the greatest challenges facing global public health systems.

[0003] Currently, most cancer treatments rely on traditional surgical resection and radiotherapy / chemotherapy. However, existing problems include the high risk of recurrence after surgery, as many cancer patients have metastases; chemotherapy drugs have issues such as poor tumor targeting, high toxicity, decreased quality of life for patients, and drug resistance with long-term use; radiotherapy, through high-energy photon radiation, can directly and indirectly destroy cancer cells or tumor tissue, and more than half of cancer patients receive radiotherapy alone or in combination with surgery for various types of cancer. However, due to the strong self-renewal and proliferation capabilities of tumor cells, and the varying sensitivities of different tumor cells to radiation, it is difficult to completely eliminate all tumor cells during radiotherapy. With advancements in medical technology, various new therapies have been applied clinically in recent years, such as immunotherapy, targeted therapy, and gene therapy, which are continuously improving patient survival rates and enhancing the treatment prospects for an increasing number of patients.

[0004] Mitochondria, as vital organelles within cells, participate in various pathophysiological metabolic processes, including energy production, apoptosis, tumorigenesis, and aging. In-depth research on tumor cells has revealed that impaired programmed apoptosis is the primary reason for their ability to replicate indefinitely and evade natural apoptosis. Mitochondria play a crucial role in apoptosis, making mitochondrial-induced tumor cell apoptosis a current research hotspot. Mitochondria are the cell's main energy factories, constantly providing the cell with the necessary energy, thus requiring continuous ATP synthesis. However, ATP synthesis in mitochondria necessitates the pumping of H+ into the cytoplasm. + Na + K +The mitochondrial cells generate ATP by driving a positive force across the mitochondrial membrane. This process creates a chemical gradient (acidic on the outside, alkaline on the inside) and a potential gradient (positively charged on the outside, negatively charged on the inside), resulting in a potential difference (ΔΨm) between the inner and outer membranes of the mitochondria. Because tumor cells have a stronger proliferative capacity than normal cells, they require more energy to reproduce. Therefore, the potential gradient (mitochondrial membrane potential, ΔΨm) of tumor cells is much higher than that of normal cells. Since DLC molecules can easily cross the hydrophobic barrier between the cell membrane and the mitochondrial membrane, they accumulate in the mitochondrial matrix under the influence of membrane potential. Theoretically, according to the Nernst equation, for every 61.5 mV increase in ΔΨm, the concentration of charged ions entering the inner membrane increases tenfold. Since the driving force provided by tumor cells for DLC is more than ten times that of normal cells, this means that the concentration of DLC in the mitochondria of tumor cells is 100-1000 times higher than its concentration in the cytoplasm.

[0005] The intermitochondrial space carries a large number of positive charges, while the matrix carries a large number of negative charges, thus forming the mitochondrial transmembrane potential (MTP). MTP is negative inside and positive outside, with a value of -130mV to 150mV in normal cells. When lipophilic cations accumulate in the mitochondria to the point where the potential across the mitochondrial membrane is balanced, they will accumulate within the mitochondria. The mitochondrial membrane potential of tumor cells is often higher than that of normal cells. Therefore, directly linking lipophilic cations to small molecules via covalent bonds can rely solely on electrostatic adsorption, bypassing complex mechanisms to penetrate the mitochondrial membrane. Subsequently, the released bioactive molecules exert their effects directly within the mitochondria, inducing tumor cell death or apoptosis. Lipophilic cation carriers mainly include triphenylphosphine (TPP), berberine (BBR), rhodamine 123, and MKT-077. TPP is the most typical and widely used of these. The active group at the end of TPP can connect with drugs and maintain drug activity. The overall structure is composed of hydrophilic charged groups and hydrophobic groups. The delocalization of the π electron clouds of the three benzene rings makes the whole molecule positively charged. Thus, driven by the mitochondrial membrane potential, it can carry small molecule drugs to target the inside of mitochondria.

[0006] In the history of anti-tumor treatment, traditional Chinese medicine (TCM) has always been very active. Many TCM active ingredients possess strong anti-tumor effects. Due to their significant therapeutic effects, low toxicity, and multi-target advantages, they are widely used in clinical treatment, bringing hope to many patients and becoming a hot research topic in the field of oncology. Epimedium is a traditional Chinese medicine, and its main active ingredients are flavonoids. Among them, icariin and epimedium glycoside are flavonoid compounds that have been extensively studied and proven to have rich medicinal value. β-dehydrated epimedium glycoside can be prepared by hydrolyzing epimedium glycoside. Currently, the anti-tumor effects of epimedium glycoside have been reported in many publications, but the pharmacological effects of β-dehydrated epimedium glycoside as a hydrolysate of epimedium glycoside are rarely reported. Furthermore, like many other biologically active flavonoid compounds, β-dehydrated epimedium glycoside suffers from low absolute bioavailability and lack of selectivity for tumor cell mitochondria, which limits its clinical application. Therefore, there is an urgent need in this field to develop a β-dehydrated epimedium structure modifier that has mitochondrial targeting for tumor cells and high bioactivity. Summary of the Invention

[0007] The purpose of this invention is to provide a mitochondrial-targeted β-dehydrated epimedium derivative, its preparation method, and its pharmaceutical uses, in order to solve the problems of low absolute bioavailability and lack of selectivity for tumor cell mitochondria in existing β-dehydrated epimedium, which limits its clinical application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a mitochondrial-targeted β-dehydrated epimedium derivative, the structure of which is shown in Formula I:

[0010]

[0011] Among them, R1 and R4 are independently -OH or -OA, and A is -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are independently -H, -CH3, -C2H5, -C3H7 or -C4H9; X is one of the halogen atoms; n is any integer from 1 to 11.

[0012] This invention also provides a method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative, comprising the following steps:

[0013] (1) The compound of formula II, the compound of formula III, the condensing agent, the catalyst and the solvent are subjected to an esterification reaction to obtain the compound of formula V;

[0014] (2) Nucleophilic substitution was performed on compound V, triphenylphosphine and solvent to obtain β-dehydrated epimedium derivative;

[0015] The structural formula of compound II is: The structural formula of compound III is: The structural formula of compound V is: Among them, R1 and R4 are independently -OH or -OA, and A is -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are independently -H, -CH3, -C2H5, -C3H7 or -C4H9; X is one of the halogen atoms; n is any integer from 1 to 11.

[0016] Preferably, the solvents used in steps (1) and (2) are one or more of dichloromethane, chloroform, acetone, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

[0017] Preferably, the condensing agent is one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the catalyst is 4-dimethylaminopyridine.

[0018] Preferably, in step (1), the molar volume ratio of compound II, compound III, condensing agent, catalyst and solvent is 1.2-3 mmol: 1 mmol: 1.2-3 mmol: 0.2-1 mmol: 4-10 mL.

[0019] Preferably, in step (2), the molar volume ratio of compound V, triphenylphosphine, and solvent is 1 mmol: 3-15 mmol: 5-15 mL.

[0020] Preferably, the esterification reaction and nucleophilic substitution are carried out at temperatures of 0–90°C; the esterification reaction takes 1–5 h; and the nucleophilic substitution takes 48–72 h.

[0021] The present invention also provides the use of the mitochondrial-targeted β-dehydrated epimedium derivative in the preparation of an antitumor drug comprising the β-dehydrated epimedium derivative and a pharmaceutically acceptable salt thereof.

[0022] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) This invention discloses a β-dehydrated epimedium derivative with mitochondrial targeting function. This derivative is a monosubstituted compound, a yellow solid powder at room temperature, and readily soluble in organic solvents such as methanol and dichloromethane. Using β-dehydrated epimedium as a lead compound and industrially sourced bromoalkylcarboxylic acid as an excipient, this invention first completed and improved the synthesis method of an important intermediate for the mitochondrial targeting compound of β-dehydrated epimedium. Furthermore, triphenylphosphine with mitochondrial targeting function was linked to this intermediate, optimizing the experimental design and synthesizing a series of mitochondrial-targeting antitumor drugs.

[0024] (2) Compared with β-dehydrated icariin, the β-dehydrated icariin derivative obtained in this invention exhibits improved solubility and increased efficiency in entering the mitochondria of tumor cells, providing a research basis for the industrial production and clinical application of β-dehydrated icariin. The β-dehydrated icariin derivative can target and enter tumor cells and their mitochondria, inducing apoptosis by affecting mitochondrial membrane potential and improving antitumor efficacy. The β-dehydrated icariin derivative has advantages such as high mitochondrial targeting efficiency, good antitumor efficacy, simple preparation method, ease of operation, and suitability for subsequent development and industrialization. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 The β-dehydrated epimedium derivative obtained in Example 1 1 H-NMR spectrum;

[0027] Figure 2 The β-dehydrated epimedium derivative obtained in Example 1 13 C-NMR spectrum;

[0028] Figure 3 The β-dehydrated epimedium derivative obtained in Example 2 1 H-NMR spectrum;

[0029] Figure 4 The β-dehydrated epimedium derivative obtained in Example 2 13 C-NMR spectrum.

[0030] Figure 5 The β-dehydrated epimedium derivative obtained in Example 3 1 H-NMR spectrum;

[0031] Figure 6 The β-dehydrated epimedium derivative obtained in Example 3 13 C-NMR spectrum;

[0032] Figure 7 The β-dehydrated epimedium derivative obtained in Example 4 1 H-NMR spectrum;

[0033] Figure 8 The β-dehydrated epimedium derivative obtained in Example 4 13 C-NMR spectrum;

[0034] Figure 9 The β-dehydrated epimedium derivative obtained in Example 5 1 H-NMR spectrum;

[0035] Figure 10 The β-dehydrated epimedium derivative obtained in Example 5 13 C-NMR spectrum;

[0036] Figure 11 The β-dehydrated epimedium derivative obtained in Example 6 1 H-NMR spectrum;

[0037] Figure 12 The β-dehydrated epimedium derivative obtained in Example 6 13 C-NMR spectrum;

[0038] Figure 13 The β-dehydrated epimedium derivative obtained in Example 7 1 H-NMR spectrum;

[0039] Figure 14 The β-dehydrated epimedium derivative obtained in Example 7 13 C-NMR spectrum. Detailed Implementation

[0040] This invention provides a mitochondrial-targeted β-dehydrated epimedium derivative, the structure of which is shown in Formula I:

[0041]

[0042]

[0043] In the β-dehydrated epimedium derivative, R1 and R4 are preferably -OH or -OA, further, R1 is -OH and R4 is -OA; A is preferably -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are preferably -H, -CH3, -C2H5, -C3H7 or -C4H9; X is preferably one of the halogen atoms; n is preferably any integer from 1 to 11.

[0044] This invention also provides a method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative, comprising the following steps:

[0045] (1) The compound of formula II, the compound of formula III, the condensing agent, the catalyst and the solvent are subjected to an esterification reaction to obtain the compound of formula V;

[0046] (2) Nucleophilic substitution was performed on compound V, triphenylphosphine and solvent to obtain β-dehydrated epimedium derivative;

[0047] The structural formula of compound II is: The structural formula of compound III is: The structural formula of compound V is: Wherein, R1 and R4 are independently preferably -OH or -OA, further, R1 is -OH and R4 is -OA; A is preferably -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are independently preferably -H, -CH3, -C2H5, -C3H7 or -C4H9; X is preferably one of the halogen atoms; n is preferably any integer from 1 to 11.

[0048] In this invention, the solvents used in steps (1) and (2) are preferably one or more of dichloromethane, chloroform, acetone, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

[0049] In this invention, the condensing agent is preferably one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the catalyst is preferably 4-dimethylaminopyridine.

[0050] In this invention, in step (1), the molar volume ratio of the compound of formula II, the compound of formula III, the condensing agent, the catalyst and the solvent is preferably 1.2-3 mmol: 1 mmol: 1.2-3 mmol: 0.2-1 mmol: 4-10 mL; more preferably 1.5-2 mmol: 1 mmol: 1.5-2 mmol: 0.5-0.8 mmol: 5-8 mL.

[0051] In this invention, in step (2), the molar volume ratio of compound V, triphenylphosphine and solvent is preferably 1 mmol: 3-15 mmol: 5-15 mL, and more preferably 1 mmol: 5-10 mmol: 10-14 mL.

[0052] In this invention, the temperatures for the esterification reaction and nucleophilic substitution are independently 0–90°C. More preferably, the temperature for the esterification reaction is 0–20°C, and the temperature for the nucleophilic substitution is 70–90°C. The time for the esterification reaction is preferably 0.5–5 h, more preferably 1–3 h. The time for the nucleophilic substitution is preferably 48–72 h, more preferably 52–60 h.

[0053] In this invention, after the esterification reaction and nucleophilic substitution are completed, the products obtained are sequentially subjected to solvent drying and purification. Solvent drying is performed on a rotary evaporator. The solvent drying temperature after esterification is preferably 20-60°C, more preferably 25-30°C. The solvent drying temperature after nucleophilic substitution is preferably 30-60°C, more preferably 40-50°C. The solvent drying time after esterification and nucleophilic substitution is preferably 2-10 min, more preferably 2-5 min. Purification involves passing the solvent-dried product through a silica gel column. In the purification after esterification, the reagent used is a mixture of petroleum ether and ethyl acetate, and the concentration ratio of petroleum ether to ethyl acetate is preferably 5-30:1, more preferably 10-20:1. In the purification after nucleophilic substitution, the reagent used is a mixture of chloroform and methanol, and the concentration ratio of chloroform to methanol is preferably 10-50:1, more preferably 20-40:1.

[0054] The reaction formula for the preparation method of the mitochondrial-targeted β-dehydrated icariin derivative is as follows:

[0055]

[0056]

[0057] The present invention also provides the use of the mitochondrial-targeted β-dehydrated epimedium derivative in the preparation of an antitumor drug comprising the β-dehydrated epimedium derivative and a pharmaceutically acceptable salt thereof.

[0058] In this invention, the tumors in the antitumor drugs include, but are not limited to, breast cancer cell lines, lung cancer cell lines, and pancreatic cancer cell lines.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] (1) Preparation of intermediate compound V (denoted as β4B): 0.1 mmol of β-dehydrated epimedium and 0.15 mmol of 4-bromobutyric acid were mixed and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 2 h. At this time, β-dehydrated epimedium completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β4B. The crude β4B product was passed through a silica gel column (petroleum ether: ethyl acetate = 10:1) to obtain β4B.

[0062] The structural formula of β4B is:

[0063] β4B is a pale yellow solid powder with a yield of 68.83%.

[0064] (2) Preparation of β-dehydrated epimedium derivative (denoted as β4T): 0.1 mmol β4B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β4T. The crude β4T was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β4T.

[0065] The structural formula of β4T is:

[0066] β4T is a pale yellow, viscous solid with a yield of 51.19%.

[0067] 1 H NMR (300MHz, CDCl3) δ11.85 (s, 1H), 7.92-7.76 (m, 11H), 7.71-7.66 (m, 6H), 7.14-7.09 (m, 2H), 6.24 (s, 1H), 4.11 (t, J=14.8H z, 2H), 3.90 (s, 3H), 3.23 (t, J=6.1Hz, 2H), 2.86 (t, J=6.6Hz, 2H), 2.15 (s, 2H), 1.88 (d, J=6.5Hz, 2H), 1.38 (d, J=2.4Hz, 6H). 13CNMR (75MHz, CDCl3) δ175.40, 170.28, 162.08, 160.42, 158.99, 155.77, 153.87, 134.85, 133.61, 133.48, 130.36, 1 30.19, 129.79, 121.40, 118.44, 117.30, 114.57, 100.26, 99.88, 76.05, 55.47, 31.41, 29.48, 26.40, 18.65, 16.06.

[0068] Example 2

[0069] (1) Preparation of intermediate compound V (denoted as β5B): 0.1 mmol of β-dehydrated epimedium and 0.15 mmol of 5-bromopentanoic acid were mixed and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 2 h. At this time, β-dehydrated epimedium completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β5B. The crude β5B product was passed through a silica gel column (petroleum ether: ethyl acetate = 10:1) to obtain β5B.

[0070] The structural formula of β5B is:

[0071] β5B is a pale yellow solid powder with a yield of 65.38%.

[0072] 1 H NMR (300MHz, CDCl3) δ11.94 (s, 1H), 7.85-7.82 (m, 2H), 7.04-7.01 (m, 2H), 6.27, (s, 1H), 3.89 (s, 3H), 3.43 (d, J=6.3Hz, 2H ), 2.84(d, J=6.8Hz, 2H), 2.68(t, J=6.8Hz, 2H), 2.01-1.96(m, 2H), 1.95–1.92(m, 2H), 1.86(d, J=6.8Hz, 2H), 1.38(s, 6H). 13 C NMR (75MHz, CDCl3) δ175.71, 170.21, 161.96, 160.49, 159.38, 154.07, 129.79, 122.14, 114. 23, 105.17, 100.49, 99.80, 76.05, 55.46, 32.99, 32.84, 31.63, 29.65, 26.56, 23.32, 16.21.

[0073] (2) Preparation of β-dehydrated epimedium derivative (denoted as β5T): 0.1 mmol β5B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β5T. The crude β5T product was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β5T.

[0074] The structural formula of β5T is:

[0075] β5T is a pale yellow solid powder with a yield of 42.95%.

[0076] 1 H NMR (300MHz, CDCl3) δ11.80 (s, 1H), 7.88-7.75 (m, 2H), 7.85-7.74 (m, 9H), 7.70-7.75 (m, 6H), 7.07-7.03 (m, 2H) ), 6.24 (s, 1H), 3.89 (s, 3H), 3.85 (s, 2H), 2.85 (t, J=6.8Hz, 2H), 2.78 (s, 2H), 2.04-1.93 (m, 4H), 1.38 (s, 6H). 13 C NMR (75MHz, CDCl3) δ175.42, 170.20, 161.97, 160.35, 158.99, 155.63, 153.90, 134.80, 133.58, 130.32, 129.66, 121.60, 118.65, 114.35, 104.81, 100.17, 99.83, 76.03, 55.46, 31.43, 32.81, 29.47, 26.41, 22.37, 21.16, 16.04.

[0077] Example 3

[0078] (1) Preparation of intermediate compound V (denoted as β6B): 0.1 mmol of β-dehydrated epimedium and 0.15 mmol of 6-bromohexanoic acid were mixed and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 2 h. At this time, β-dehydrated epimedium completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β6B. The crude β6B product was passed through a silica gel column (petroleum ether: ethyl acetate = 10:1) to obtain β6B.

[0079] The structural formula of β6B is:

[0080] β6B is a pale yellow solid powder with a yield of 86.94%.

[0081] 1 H NMR (300MHz, CDCl3) δ11.95 (s, 1H), 7.86-7.82 (m, 2H), 7.04-7.01 (m, 2H), 6.27 (s, 1H), 3.90 (s, 3H), 3.40 (t, J=6.7Hz, 2H), 2.85 (t, J=6.8Hz, 2H), 2.66 (t, J=7.4Hz, 2H), 1.55-1.47 (m, 2H), 1.93-1.89 (m, 2H), 1.87 (d, J=4.2Hz, 2H) 1.78 (d, J=7.7Hz, 2H), 1.38 (s, 6H). 13 C NMR (75MHz, CDCl3) δ175.80, 170.97, 161.96, 160.50, 159.39, 154.10, 129.85, 122.52, 114.22, 105.25, 100.49, 99.83, 76.08, 55.48, 33.40, 32.65, 32.35, 31.64, 27.53, 26.59, 23.91, 16.24.

[0082] (2) Preparation of β-dehydrated epimedium derivative (denoted as β6T): 0.1 mmol β6B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β6T. The crude β6T was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β6T.

[0083] The structural formula of β6T is:

[0084] β6T is a pale yellow solid powder with a yield of 35.69%.

[0085] 1 H NMR (300MHz, CDCl3) δ11.92 (s, 1H), 7.88-7.84 (m, 2H), 7.84-7.74 (m, 9H), 7.71-7.66 (m, 6H), 7.07-7.03 (m, 2H), 6.23 (s, 1H) , 3.89 (s, 3H), 3.81 (s, 2H), 2.84 (t, J = 6.7Hz, 2H), 2.62 (t, J = 6.6Hz, 2H), 1.86 (s, 2H), 1.84 (s, 2H), 1.80 (s, 2H), 1.36 (s, 6H). 13 C NMR (75MHz, CDCl3) δ175.59, 170.39, 161.29, 160.30, 159.06, 155.6, 153.86, 134.85, 133.49, 130.40, 129.62, 121. 71, 117.45, 114.29, 104.90, 100.18, 99.76, 75.97, 55.50, 33.18, 31.43, 29.44, 26.39, 23.92, 22.75, 22.08, 16.03.

[0086] Example 4

[0087] (1) Preparation of intermediate compound V (denoted as β7B): 0.1 mmol of β-dehydrated epimedium and 0.15 mmol of 7-bromoheptanoic acid were mixed and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 2 h. At this time, β-dehydrated epimedium completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β7B. The crude β7B product was passed through a silica gel column (petroleum ether: ethyl acetate = 12:1) to obtain β7B.

[0088] The structural formula of β7B is:

[0089] β7B is a pale yellow solid powder with a yield of 74.34%.

[0090] 1H NMR (300MHz, CDCl3) δ11.96 (s, 1H), 7.88-7.78 (m, 2H), 7.06-6.97 (m, 2H, m), 6.26 (s, 1H), 3.89 (s, 3H), 3.40 (t, J=6.8Hz, 2H), 2.85 (t, J=6.7Hz, 2H), 2.64 (t, J=7.4Hz, 2H), 1.86 (s, 2H), 1.80-1.73 (m, 2H), 1.61 (s, 2H), 1.37 (s, 6H), 1.25 (s, 4H). 13 C NMR (75MHz, CDCl3) δ175.77, 170.59, 161.89, 160.43, 159.36, 155.64, 154.04, 136.69, 129.78, 122.19, 114 .15, 105.15, 100.42, 99.75, 76.00, 55.43, 33.67, 32.41, 31.60, 29.61, 27.99, 27.66, 26.53, 24.47, 16.19.

[0091] (2) Preparation of β-dehydrated epimedium derivative (denoted as β7T): 0.1 mmol β7B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β7T. The crude β7T was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β7T.

[0092] The structural formula of β7T is:

[0093] β7T is a pale yellow solid powder with a yield of 47.44%.

[0094] 1 H NMR (300MHz, CDCl3) δ11.93 (s, 1H), 7.86-7.84 (m, 2H), 7.84-7.75 (m, 9H), 7.71-7.65 (m, 6H), 7.06-7.01 (m, 2H), 6.23 (s, 1H), 3.89 (s, 3H), 3.79 ( d, J=14.6Hz, 2H), 2.84 (t, J=6.7Hz, 2H), 2.60 (t, J=7.2Hz, 2H), 1.87 (d, J=6.6Hz, 2H), 1.84 (s, 2H), 1.73-1.63 (m, 6H), 1.61 (s, 2H), 1.37 (s, 6H).13 C NMR (75MHz, CDCl3) δ175.66(C-4), 170.55(16-C), 161.91(C-4'), 160.29(C-7), 159.07, 155.64, 153.87, 134.86, 133.47, 130.41, 12 9.65, 121.76, 117.46, 114.23, 104.92, 100.17, 99.76, 75.99, 55.51, 33.41, 31.43, 29.49, 29.28, 27.74, 26.40, 23.89, 21.98, 16.03.

[0095] Example 5

[0096] (1) Preparation of intermediate compound V (denoted as β8B): 0.1 mmol of β-dehydroepiandrocin was mixed with 0.15 mmol of 8-bromooctanoic acid, and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 1.5 h, at which point β-dehydroepiandrocin completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β8B. The crude β8B product was passed through a silica gel column (petroleum ether: ethyl acetate = 12:1) to obtain β8B.

[0097] The structural formula of β8B is:

[0098] β8B is a pale yellow solid powder with a yield of 63.38%.

[0099] 1 H NMR (300MHz, CDCl3) δ11.97 (s, 1H), 7.87-7.81 (m, 2H), 7.04-6.98 (m, 2H), 6.26 (s, 1H), 3.89 (s, 3H), 3.40 (t, J=6.8Hz, 2H), 2.85 (t, J=6.7Hz), 2 .63 (t, J=7.5Hz, 2H), 1.88 (d, J=6.8Hz, 2H), 1.84 (d, J=6.4Hz, 2H), 1.74 (q, J=7.3Hz, 2H), 1.60 (s, 2H), 1.38 (6s, 1H), 1.34 (s, 2H), 1.25 (s, 2H). 13C NMR (75MHz, CDCl3) δ175.80, 170.68, 161.88, 160.42, 159.36, 155.63, 154.04, 131.07, 129.79, 122.21, 114.13 , 105.16, 100.42, 99.74, 75.99, 55.42, 33.80, 33.75, 32.57, 31.60, 28.70, 28.26, 27.83, 26.53, 24.54, 16.18.

[0100] (2) Preparation of β-dehydrated epimedium derivative (denoted as β8T): 0.1 mmol β8B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β8T. The crude β8T was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β8T.

[0101] The structural formula of β8T is:

[0102] β8T is a pale yellow solid powder with a yield of 44.27%.

[0103] 1 H NMR (300MHz, CDCl3) δ11.95 (s, 1H), 7.86-7.84 (m, 2H), 7.83-7.74 (m, 9H) ,7.71-7.65(m,6H),7.05-7.00(m,2H),6.23(s,1H),3.88(s,3H),3.74(2H , s, H-23), 2.84 (t, J = 6.7Hz, 2H), 2.57 (t, J = 7.3Hz, 2H), 1.94-1.91 (m, 2H ), 1.89 (d, J=6.5Hz, 2H), 1.85 (d, J=6.7Hz, 4H), 1.37 (s, 6H), 1.24 (s, 4H). 13C NMR (75MHz, CDCl3) δ175.67, 170.58, 161.88, 160.28, 159.11, 155.61, 153.88, 134.84, 134.48, 130.40, 129.63, 121.83, 11 7.52, 114.17, 104.94, 100.18, 99.73, 75.96, 55.47, 33.50, 31.44, 29.46, 29.70, 28.27, 28.12, 26.40, 24.23, 22.17, 16.03.

[0104] Example 6

[0105] (1) Preparation of intermediate compound V (denoted as β9B): 0.1 mmol of β-dehydroepiandrocin was mixed with 0.15 mmol of 9-bromononanoic acid, and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 1.5 h, at which point β-dehydroepiandrocin completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β9B. The crude β9B product was passed through a silica gel column (petroleum ether: ethyl acetate = 12:1) to obtain β9B.

[0106] The structural formula of β9B is:

[0107] β9B is a pale yellow solid powder with a yield of 87.57%.

[0108] 1 H NMR (300MHz, CDCl3) δ11.96 (s, 1H), 7.88-7.78 (m, 2H), 7.06-6.97 (m, 2H), 6.26 (s, 1H), 3.89 (s, 3H), 3.40 (t, J=6.8Hz, 2 H), 2.85 (t, J=6.7Hz, 2H), 2.64 (t, J=7.4Hz, 2H), 1.86 (s, 2H), 1.80-1.73 (m, 2H), 1.61 (s, 2H), 1.37 (s, 6H), 1.25 (s, 4H). 13C NMR (75MHz, CDCl3) δ175.81, 170.73, 161.87, 160.41, 159.36, 155.62, 154.04, 131.07, 129.78, 122.22, 114.12, 10 5.12, 100.41, 99.74, 75.99, 55.36, 33.81, 33.79, 32.68, 31.59, 28.93, 28.81, 28.45, 27.98, 26.52, 24.61, 16.17.

[0109] (2) Preparation of β-dehydrated epimedium derivative (denoted as β9T): 0.1 mmol β9B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β9T. The crude β9T product was then passed through a silica gel column (chloroform:methanol = 30:1) to obtain β9T.

[0110] The structural formula of β9T is:

[0111] β9T is a pale yellow solid powder with a yield of 38.34%.

[0112] 1 H NMR (300MHz, CDCl3) δ11.99 (s, 1H), 7.87-7.85 (m, 2H), 7.84-7.76 (m, 9H, m), 7.72-7.66 (m, 6H), 7.04-7.00 (m, 2H), 6.23 (s, 1H), 3.88 (s, 3H), 3 .81 (s, 2H), 2.84 (t, J=6.8Hz, 2H), 2.57 (t, J=7.4Hz, 2H), 1.88 (d, J=6.8Hz, 2H), 1.84 (s, 2H), 1.69 (t, J=7.0Hz, 4H), 1.37 (s, 6H), 1.25 (m, 6H). 13C NMR (75MHz, CDCl3) δ175.69, 170.65, 161.87, 160.28, 159.12, 155.71, 153.75, 137.19, 133.48, 130.41, 129.65, 121.83, 118.6 8, 114.14, 104.85, 100.19, 99.74, 75.96, 55.44, 33.60, 31.44, 29.84, 29.47, 28.48, 28.41, 28.35, 26.41, 24.36, 22.34, 16.04.

[0113] Example 7

[0114] (1) Preparation of intermediate compound V (denoted as β10B): 0.1 mmol of β-dehydrated epimedium and 0.15 mmol of 10-bromodecanoic acid were mixed and 5 mL of dichloromethane was added. After stirring, 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 mmol of 4-dimethylaminopyridine were added. After the addition was complete, the reaction was carried out at 0 °C for 1.5 h. At this time, β-dehydrated epimedium completely disappeared. After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 30 °C with a water pump under reduced pressure to obtain crude β10B. The crude β10B product was passed through a silica gel column (petroleum ether: ethyl acetate = 15:1) to obtain β10B.

[0115] The structural formula of β10B is:

[0116] β10B is a pale yellow solid powder with a yield of 84.73%.

[0117] (2) Preparation of β-dehydrated epimedium derivative (denoted as β10T): 0.1 mmol β10B and 0.5 mmol triphenylphosphine were mixed and 10 mL acetone was added to dissolve them completely. The mixture was then heated under reflux at 70 °C for 48 h. During the reflux, the reaction was monitored by TLC (chloroform:methanol = 10:1). After the reaction was completed, the product was placed in a rotary evaporator and the solvent was removed by rotary evaporation at 40 °C with a water pump under reduced pressure to obtain crude β10T. The crude β10T product was then passed through a silica gel column (chloroform:methanol = 50:1) to obtain β10T.

[0118] The structural formula of β10T is:

[0119] β10T is a pale yellow, viscous solid with a yield of 37.75%.

[0120] 1H NMR (300MHz, CDCl3) δ11.98 (s, 1H), 7.86-7.74 (m, 12H), 7.72-7.67 (m, 5H), 7.03-6.98 (m, 2H), 6.23 (s, 1H), 3.88 (s, 3H), 3.73 (s, 2H), 2.88-2.81 (m, 2H), 2.58 (t, J=7.1Hz, 2H), 1.85 (m, J=9.4Hz, 8H), 1.37 (m, 6H), 1.24 (m, 8H). 13 C NMR (75MHz, CDCl3) δ175.70, 170.67, 161.86, 160.27, 159.13, 134.84, 133.48, 133.35, 130.40, 130.23, 129.65, 121.90, 117.5 6, 114.11, 100.13, 99.73, 75.96, 55.39, 33.65, 31.45, 30.18, 29.97, 29.47, 28.71, 28.63, 28.55, 26.40, 24.42, 22.29, 16.03.

[0121] Application Example 1

[0122] The antitumor activity of the β-dehydrated epimedium derivatives obtained in Examples 1-7 was tested:

[0123] (1) Selection of test cells and drugs:

[0124] β4B, β4T, β5B, β5T, β6B, β6T, β7B, β7T, β8B, β8T, β9B, β9T, β10B, β10T, and commercially available β-dehydrated epimedium (CAS: 38226-86-7) were selected as drugs; A549 (human non-small cell lung cancer cells), MCF-7 (human breast cancer cells), and PANC-1 (human pancreatic cancer cells) were selected as test cells.

[0125] (2) The testing method is as follows:

[0126] MTT assay: A549 (human non-small cell lung cancer cells), MCF-7 (human breast cancer cells), and PANC-1 (human pancreatic cancer cells) cells in logarithmic growth phase were collected and seeded into 96-well plates at a density of 5.0 × 10⁶ cells per well. 3 / 100μL, incubated at 37℃ in a 5% CO2 incubator. The next day, the culture medium was removed, and 100μL of different concentrations of β4B, β4T, β5B, β5T, β6B, β6T, β7B, β7T, β8B, β8T, β9B, β9T, β10B, β10T and commercially available β-dehydrated epimedium (CAS: 38226-86-7) were added respectively (the drug concentrations were double-diluted, and the concentrations of each drug were set at 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM and 1.5625μM. Each test was repeated 3 times with 3 parallel wells). The above-mentioned drugs were used as positive control groups. A negative control group without any drug was set up. After 48 hours, 10 μL of thiazolyl blue (MTT) was added to each well, and the cells were cultured for another 4 hours. Then, 150 μL of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction. The cells were then shaken at 37°C for 10 minutes. Immediately afterward, the absorbance (OD) value of each well at 490 nm was measured using a microplate reader, and the cell growth inhibition rate of the drug was calculated. The cell growth inhibition rate (%) was calculated as follows: (Mean OD value of the positive control group - Mean OD value of the blank control group) / (Mean OD value of the negative control group - Mean OD value of the blank control group). Then, the IC50 of the drug was calculated based on the cell growth inhibition rate at different drug concentrations. 50 The results are shown in Table 1.

[0127] Table 1. In vitro antitumor activity of Examples 1-7 and commercially available β-dehydrated icariin (CAS: 38226-86-7).

[0128]

[0129] As shown in Table 1, the IC50 of commercially available β-dehydrated epimedium (CAS: 38226-86-7) is significantly lower than that of commercially available β-dehydrated epimedium. 50 At a concentration of 50.39 μM, the seven β-dehydrated epimedium derivatives (numbered 2, 4, 6, 8, 10, 12, and 14) obtained in this invention significantly enhanced the inhibitory activity against A549 (human non-small cell lung cancer cells) cell proliferation, particularly reducing β7T (numbered 8) by nearly 43-fold, IC50 value. 50 The value was 1.15 μM, indicating that β7T was more sensitive to A549 (human non-small cell lung cancer cells).

[0130] Similarly, commercially available β-dehydrated epimedium (CAS: 38226-86-7) showed an IC50 of 1,500 against MCF-7 (human breast cancer cells). 50The concentration was 27.63 μM. The seven β-dehydrated icariin derivatives obtained in this invention (numbered 2, 4, 6, 8, 10, 12, and 14) all showed superior inhibitory effects against MCF-7 (human breast cancer cells) compared to β-dehydrated icariin. In particular, β7T (numbered 8) showed a nearly 23-fold (IC50) reduction in concentration compared to β-dehydrated icariin. 50，β-脱水淫羊藿素 / IC 50，β7T =23.62), which showed strong anti-tumor activity.

[0131] Structure-activity relationship analysis showed that the length of the carbon chain can affect the cytotoxicity of all these derivatives. The antitumor activity of the 4-carbon chain derivatives was relatively weaker compared to derivatives with other carbon chain lengths, possibly due to steric hindrance caused by the shorter carbon chain, such as the reduced IC50 of β4T in human breast cancer cells MCF-7. 50 The value was 10.02 μM, while the IC50 of other derivatives was... 50 The values ​​were all less than 4 μM; chains with 5 or more carbons significantly enhanced antitumor activity. The effect of chain length on activity generally showed a gradual increase from 5 carbon chains to 7 or 8 carbon chains, followed by a gradual decrease in activity as the carbon chain length increased. For example, when the chain length increased from 5 (β5T) to 7 (β7T), the corresponding IC50 of the compound on human non-small cell lung cancer cells A549 decreased. 50 The value decreased from 17.20 μM to 1.15 μM; when the chain length increased from 7 (β7T) to 10 (β10T), the corresponding IC50 value of the compound on human non-small cell lung cancer cells A549 decreased. 50 The value increased from 1.15 μM to 17.98 μM. However, this trend was not obvious in human pancreatic cancer cells PANC-1. The antitumor activity of the derivative was slightly increased compared with β-dehydrated epimedium, and the chain length had no obvious effect on the activity.

[0132] During the testing process, it was found that when the concentration of intermediates (numbered 1, 3, 5, 7, 9, 11, and 13) was 100 μM, the tumor cells observed under the microscope still showed good growth status, and no obvious shrinkage or vacuolation phenomenon was observed in the cells. The MTT results also showed that the intermediates did not show good effects, indicating that the introduction of different groups at the same site of dehydrated epimedium significantly affected the binding of the active site, proving the necessity of introducing the triphenylphosphine group to improve the biological activity of dehydrated epimedium.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mitochondrial-targeted β-dehydrated icariin derivative, characterized in that, The structure of the β-dehydrated epimedium derivative is shown in Formula I: Among them, R1 and R4 are independently -OH or -OA, and A is -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are independently -H, -CH3, -C2H5, -C3H7 or -C4H9; X is one of the halogen atoms; n is any integer from 1 to 11.

2. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to claim 1, characterized in that, The steps include the following: (1) The compound of formula II, the compound of formula III, the condensing agent, the catalyst and the solvent are subjected to an esterification reaction to obtain the compound of formula V; (2) Nucleophilic substitution was performed on compound V, triphenylphosphine and solvent to obtain β-dehydrated epimedium derivative; The structural formula of compound II is: The structural formula of compound III is: The structural formula of compound V is: Among them, R1 and R4 are independently -OH or -OA, and A is -CH3, -C2H5, -C3H7 or -C4H9; R2, R3, R5 and R6 are independently -H, -CH3, -C2H5, -C3H7 or -C4H9; X is one of the halogen atoms; n is any integer from 1 to 11.

3. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to claim 2, characterized in that, In steps (1) and (2), the solvents are independently one or more of dichloromethane, chloroform, acetone, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

4. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to claim 3, characterized in that, The condensing agent is one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the catalyst is 4-dimethylaminopyridine.

5. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to any one of claims 2 to 4, characterized in that, In step (1), the molar volume ratio of compound II, compound III, condensing agent, catalyst and solvent is 1.2-3 mmol: 1 mmol: 1.2-3 mmol: 0.2-1 mmol: 4-10 mL.

6. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to claim 5, characterized in that, In step (2), the molar volume ratio of compound V, triphenylphosphine and solvent is 1 mmol: 3-15 mmol: 5-15 mL.

7. The method for preparing the mitochondrial-targeted β-dehydrated epimedium derivative according to claim 6, characterized in that, The esterification reaction and nucleophilic substitution are carried out at temperatures ranging from 0 to 90°C; the esterification reaction takes 1 to 5 hours; and the nucleophilic substitution takes 48 to 72 hours.

8. The use of the mitochondrial-targeted β-dehydrated epimedium derivative of claim 1 in the preparation of antitumor drugs, characterized in that, The antitumor drug comprises β-dehydrated epimedium derivatives and their pharmaceutically acceptable salts.

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