Process for the preparation of a steroidal compound having the ability to induce DNA damage in hepatoma cells and to alleviate neuroinflammation

Abstract

The application discloses a preparation method of a steroid compound with the functions of inducing DNA damage of liver cancer cells and relieving neuroinflammation, and belongs to the technical field of drug synthesis. The technical solution of the application is characterized by the following: the molecular structure of the steroid compound is: or, wherein R is an aryl compound, an alkyl compound, a heterocyclic compound, or wherein X is an aryl compound. The application designs and synthesizes a novel steroid compound. The compound not only has the ability of inducing DNA damage and apoptosis of HepG2 cells, but also can effectively relieve the release of NO of microglial cells induced by LPS.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to a method for preparing a steroidal compound that induces DNA damage in liver cancer cells and alleviates neuroinflammation. Background Technology

[0002] Steroids, as a class of drug molecules with polycyclic skeletons, play a crucial role in maintaining normal physiological functions and treating diseases. The development of steroid-based new drugs has long been a hot area in drug development, and significant progress has been made in recent years, with over 300 steroid compounds applied clinically for disease treatment. These include the anti-prostate cancer drug abiraterone, the contraceptive levonorgestrel, the hypogonadism treatment methanogenone, and the anti-inflammatory drug dexamethasone, demonstrating their broad pharmaceutical potential. Constructing structurally novel and diverse steroid compounds and evaluating their biological activities is a hot topic in steroid drug development. Among these, the construction of heterocyclic steroid compounds is a focus of steroid chemical modification. Introducing heterocyclic skeletons into steroid skeleton structures can enrich the variety of steroid compounds, increase their structural novelty, complexity, and diversity, and simultaneously endow them with new or improved biological activities, laying the foundation for steroid drug development.

[0003] In recent years, research on the structural modification of steroidal compounds has continued to deepen, especially the introduction of heterocyclic structures at their D-ring sites, which has become an important strategy for expanding their pharmacodynamic spectrum and enhancing their biological activity. For example, the POPAM-OH-substituted steroidal alkylating agents developed by Sarli's group have shown excellent antitumor activity in various tumor xenograft models; while the compounds constructed by Wang's group by introducing a 1,3,4-thiadiazole ring into diosgenin showed significant toxicity in HepG2 and A549 cells, with the mechanism of action involving the activation of the mitochondrial apoptosis pathway, showing high development potential. Among the many isomeric ring modification groups, 1,2,3-triazole has attracted much attention due to its excellent physicochemical properties and biological activity. This type of five-membered nitrogen-containing heterocyclic structure has a rigid planar skeleton and good electronic properties, and can form stable non-covalent interactions with various enzymes, proteins, and nucleic acids, such as hydrogen bonds and hydrophobic interactions, showing broad application prospects in antibacterial, anti-inflammatory, antiviral, and antitumor research. Most importantly, 1,2,3-triazole can insert into the DNA double helix or disrupt its structural stability, inducing DNA damage in tumor cells, thereby triggering cell cycle arrest and apoptosis. This mechanism has become a research hotspot in the current development of anti-tumor drugs.

[0004] The principle of structural splicing involves combining two or more molecules with the same or similar pharmacological activities through coupling or chemical bonding to form a hybrid molecule. This new molecule combines the properties of both molecules, enhances their efficacy, improves drug absorption, reduces toxicity, increases solubility, and prolongs drug half-life, thus achieving better therapeutic effects. This method has become an important tool in new drug development. Given the important role of steroidal skeletons and 1,2,3-triazoles in antitumor drug development, this study, based on the principle of structural splicing, used testosterone and methyltestosterone as the parent skeleton. By introducing an azide group at the 17-hydroxyl position and coupling it with various terminal alkyne units through a "click chemistry" reaction, a series of novel steroid-triazole hybrid molecules were constructed. Preliminary in vitro cell activity screening showed that some compounds exhibited significant DNA damage-inducing activity in HepG2 liver cancer cells and simultaneously demonstrated the potential to alleviate neuroinflammation. This provides an important chemical basis and candidate structures for developing novel steroidal lead compounds with both anti-liver cancer and neuroprotective functions. Summary of the Invention

[0005] The present invention discloses a method for preparing a steroidal compound that induces DNA damage in liver cancer cells and alleviates neuroinflammation, characterized in that the molecular structure of the steroidal compound is as follows: or Where R is an aryl compound, an alkyl compound, a heterocyclic compound, or , where X is an aryl compound.

[0006] The present invention discloses a method for preparing a steroidal compound that induces DNA damage in liver cancer cells and alleviates neuroinflammation, characterized in that:

[0007] 1. Add testosterone or methylhydrotestosterone to dichloromethane, then add potassium carbonate, and slowly add a dichloromethane solution containing chloroacetyl chloride. After the reaction, separate the product by silica gel column chromatography. or .

[0008] 2. Put or Add acetonitrile, then add sodium azide, heat to reflux, react for a period of time, and then concentrate to obtain... or .

[0009] 3. Take a certain amount or Azide compounds, sodium L-ascorbate, and anhydrous CuSO4 were added to a round-bottom flask. A mixed solvent of water, tert-butanol, and tetrahydrofuran was then added. The mixture was stirred at room temperature and then heated to reflux. After stirring for a period of time, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted multiple times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and after thorough stirring and standing, the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain the final product. or .

[0010] The steroidal compounds described in this invention have the following technical advantages: (1) This invention modifies the molecular structures of the reported marketed drugs testosterone and methylhydrotestosterone to obtain novel compounds; (2) These compounds have the ability to induce DNA damage and apoptosis in HepG2 cells; (3) These compounds can effectively alleviate the release of NO from microglia induced by LPS. Attached Figure Description

[0011] Figure 1 This is the 1H NMR spectrum of compound 5b.

[0012] Figure 2 It involves the docking of compound 5b with human topoisomerase II. Detailed Implementation

[0013] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0014] Example 1

[0015]

[0016] Testosterone (1, 0.01 mol) was added to 200 mL of dichloromethane (DCM) and stirred until completely dissolved. After adding potassium carbonate (0.01 mol), a solution of dichloroacetyl chloride (0.011 mol) in DCM was slowly added dropwise. The mixture was stirred at room temperature for 1 hour after the addition was complete, and the reaction was monitored by TLC. Water was added to the reaction mixture, and the organic phase was separated and extracted three times with the aqueous DCM phase. The combined organic layers were concentrated and separated by silica gel chromatography to obtain compound 2 (2.55 g).

[0017] Example 2

[0018]

[0019] Compound 2 (0.01 mol) was dissolved in 200 mL of acetonitrile, and sodium azide (0.02 mol) was added. The mixture was heated under nitrogen protection and refluxed for 5 hours. After the reaction was completed, the mixture was concentrated, DCM was added, and the mixture was washed with water. The organic phase was concentrated again to obtain compound 3 (3.07 g).

[0020] Example 3

[0021]

[0022] Add compound 3 (1 mmol) to the reaction flask The reaction mixture consisted of 1.1 mmol of tert-butanol (50 mL), 50 mL of water, 50 mL of tetrahydrofuran (50 mL), 0.2 mmol of anhydrous copper sulfate, and 0.4 mmol of sodium ascorbate. The mixture was stirred and refluxed for 6 hours, monitored by TLC. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 2). The combined organic layers were washed again with brine (30 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, and separated by silica gel column chromatography to obtain compound 4a (0.261 g). 1 H NMR (400MHz, CDCl3): 7.57 (s, 1H), 7.18 (t, J=8.0Hz, 2H), 6.74 (t, J=8.0Hz, 1H), 6.67 (d, J=8.0Hz, 2H), 5.73 (s, 1H), 5.13(d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.50 (s, 2H), 2.43-2.16 (m, 5H), 2.04-1.99 (m, 1H), 1.87-1.81 (m, 1H), 1.74-1.49 (m, 7H), 1.40-1.31 (m, 2H),1.19 (s, 3H), 1.15-0.89 (m, 4H), 0.73 (s, 3H).

[0023] Example 4

[0024] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4b (0.209 g). 1H NMR (400MHz, CDCl3): 7.57 (s, 1H), 7.08 (t, J=8.0Hz, 1H), 6.32-6.27 (m, 2H), 6.22 (t, J=4.0Hz, 1H), 5.73 (s, 1H), 5.13 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.48 (s,2H), 2.45-2.15 (m, 5H), 2.04-1.99 (m, 1H), 1.85-1.81 (m, 1H), 1.75-1.47 (m,7H), 1.41-1.31 (m, 2H), 1.18 (s, 3H), 1.15-0.89 (m, 4H), 0.73 (s, 3H).

[0025] Example 5

[0026] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4c (0.331 g). 1 H NMR (400MHz, CDCl3): 7.59(s, 1H), 7.00-6.95 (m, 2H), 6.76 (t, J=8.0Hz, 1H), 6.68-6.63 (m, 1H), 5.73(s, 1H), 5.14 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.54 (s, 2H), 2.45-2.16 (m, 5H), 2.04-1.99 (m, 1H), 1.85-1.81 (m, 1H), 1.73-1.32 (m, 9H), 1.18(s, 3H), 1.14-0.89 (m, 4H), 0.71 (s, 3H).

[0027] Example 6

[0028] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), and finally compound 4d (0.192 g) was obtained. 1H NMR (400MHz, CDCl3): 7.62(s, 1H), 7.17 (d, J=8.0Hz, 1H), 6.70 (d, J=4.0Hz, 1H), 6.65-6.62 (m, 1H), 5.73 (s, 1H), 5.15 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.54 (s, 2H),2.42-2.17 (m, 5H), 2.03-1.99 (m, 1H), 1.86-1.80 (m, 1H), 1.73-1.49 (m, 7H),1.38-1.32 (m, 2H), 1.18 (s, 3H), 1.14-0.89 (m, 4H), 0.70 (s, 3H).

[0029] Example 7

[0030] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4e (0.289 g). 1 H NMR (400MHz, CDCl3): 7.58(s, 1H), 7.17 (d, J=8.0Hz, 1H), 6.70 (d, J=4.0Hz, 1H), 6.65-6.62 (m, 1H), 5.73 (s, 1H), 5.15 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.54 (s, 2H),2.42-2.17 (m, 5H), 2.03-1.99 (m, 1H), 1.86-1.80 (m, 1H), 1.73-1.49 (m, 7H),1.38-1.32 (m, 2H), 1.18 (s, 3H), 1.14-0.89 (m, 4H), 0.70 (s, 3H).

[0031] Example 8

[0032] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4f (0.331 g). 1H NMR (400MHz, CDCl3): 7.58 (s, 1H), 7.21 (d, J=8.0Hz, 2H), 6.62 (d, J=8.0Hz, 2H), 5.73 (s,1H), 5.13 (d, J=4.0Hz, 2H), 4.67 (t, J=8.0Hz, 1H), 4.48 (s, 2H), 2.43-2.16(m, 5H), 2.05-1.99 (m, 1H), 1.87-1.81 (m, 1H), 1.73-1.53 ​​(m, 6H), 1.43-1.33(m, 3H), 1.29-1.25 (m, 9H), 1.19 (s, 3H), 1.11-0.91 (m, 4H), 0.74 (s, 3H).

[0033] Example 9

[0034] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding 4 g (0.154 g) of the compound. 1 H NMR (400MHz, CDCl3): 7.59 (s, 1H), 7.41 (d, J=12.0Hz, 2H), 6.67 (d, J=8.0Hz, 2H), 5.73 (s, 1H), 5.15 (d, J=4.0Hz, 2H), 4.67 (t, J=8.0Hz, 1H), 4.53 (s, 2H), 2.43-2.15 (m,5H), 2.04-1.99 (m, 1H), 1.87-1.81 (m, 1H), 1.73-1.49 (m, 7H), 1.38-1.28 (m,2H), 1.18 (s, 3H), 1.16-0.90 (m, 4H), 0.71 (s, 3H).

[0035] Example 10

[0036] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), and the final compound was obtained 4h (0.218 g). 1H NMR (400MHz, CDCl3): 7.59 (s, 1H), 6.89 (t, J=8.0Hz, 2H), 6.59 (s, 2H), 5.73 (s, 1H), 5.14 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.48 0.72 (s, 3H).

[0037] Example 11

[0038] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4i (0.306 g). 1 H NMR (400MHz, CDCl3): 7.57(s, 1H), 7.13-7.08 (m, 2H), 6.76-6.67 (m, 2H), 5.73 (s, 1H), 5.13 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.54 (s, 2H), 2.52-2.18 (m, 7H), 2.04-1.99(m, 1H), 1.87-1.81 (m, 1H), 1.73-1.25 (m, 12H), 1.18 (s, 3H), 1.14-0.92 (m,4H), 0.72 (s, 3H).

[0039] Example 12

[0040] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4j (0.232 g). 1H NMR (400MHz, CDCl3): 7.45 (s, 1H), 6.75-6.40 (m, 2H), 5.73 (s, 1H), 5.11 (d, J=4.0Hz, 2H), 4.66-4.62 (m, 3H), 3.81 (d, J=8.0Hz, 3H), 2.45-2.12 (m, 5H), 2.04-1.99 (m, 1H), 1.86-1.82 (m, 1H), 1.73-1.25 (m, 12H), 1.19 (s, 3H), 1.14-0.88 (m, 4H), 0.72(s, 3H).

[0041] Example 13

[0042] The operation steps in this embodiment are completely the same as in embodiment 3, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 4k (0.175 g). 1 H NMR (400MHz, CDCl3): 7.67(d, J=8.0Hz, 1H), 7.58 (s, 1H), 7.19 (t, J=8.0Hz, 1H), 6.65 (d, J=4.0Hz, 1H), 6.48 (t, J=8.0Hz, 1H), 5.73 (s, 1H), 5.14 (d, J=4.0Hz, 2H), 4.67-4.57 (m,3H), 2.43-2.17 (m, 6H), 2.03-1.99 (m, 1H), 1.85-1.81 (m, 1H), 1.71-1.57 (m,6H), 1.41-1.32 (m, 3H), 1.18 (s, 3H), 1.17-0.94 (m, 3H), 0.69 (s, 3H).

[0043] Example 14

[0044]

[0045] Add compound 3 (1 mmol) to the reaction flask The reaction mixture consisted of 1.1 mmol of tert-butanol (50 mL), 50 mL of water, 50 mL of tetrahydrofuran (50 mL), 0.2 mmol of anhydrous copper sulfate, and 0.4 mmol of sodium ascorbate. The mixture was stirred under reflux and monitored by TLC. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 2). The combined organic layers were washed again with brine (30 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, and separated by silica gel column chromatography to obtain compound 5a (0.206 g). 1 H NMR (400MHz, CDCl3): 7.88 (s, 1H), 7.76 (d, J=8.0Hz, 2H), 7.28 (d, J=4.0Hz, 2H), 5.72 (s, 1H), 5.20 (d, J=4.0Hz, 2H), 4.69(d, J=12.0Hz, 1H), 2.69 (dd, J1=8.0Hz, J2=8.0Hz, 2H), 2.42-2.19 (m, 5H), 2.04-1.98 (m, 1H), 1.85-1.54 (m, 8H), 1.42-1.33 (m, 2H), 1.27 (t, J=4.0Hz,3H), 1.17 (s, 3H), 1.09-0.90 (m, 3H), 0.77 (s, 3H).

[0046] Example 15

[0047] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5b (0.235 g). 1 H NMR (400MHz, CDCl3): 8.01(s, 1H), 7.97 (d, J=8.0Hz, 2H), 7.69 (d, J=8.0Hz, 2H), 5.72 (s, 1H), 5.25 (d,J=4.0Hz, 2H), 4.71 (d, J=8.0Hz, 1H), 2.45-2.21 (m, 5H), 2.04-1.99 (m, 1H),1.87-1.53 ​​(m, 9H), 1.43-1.34 (m, 2H), 1.22-1.15 (m, 4H), 1.11-0.90 (m, 3H),0.79 (s, 3H).

[0048] Example 16

[0049] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5c (0.144 g). 1 H NMR (400MHz, CDCl3): 9.02 (s,1H), 8.60 (d, J=4.0Hz, 1H), 8.25-8.22 (m, 1H), 8.02 (s, 1H), 7.40 (dd, J1=4.0Hz, J2=4.0Hz, 1H), 5.73 (s, 1H), 5.26 (d, J=4.0Hz, 2H), 4.71 (t, J=12.0Hz,1H), 2.43-2.21 (m, 5H), 2.04-1.99 (m, 1H), 1.87-1.54 (m, 8H), 1.43-1.34 (m,2H), 1.18 (s, 3H), 1.11-0.92 (m, 3H), 0.79 (s, 3H).

[0050] Example 17

[0051] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), and finally compound 5d (0.163 g) was obtained. 1 H NMR (400MHz, CDCl3): 7.82 (s,1H), 7.71 (s, 1H), 7.47 (d, J=4.0Hz, 1H), 7.40 (dd, J1=4.0Hz, J2=4.0Hz, 1H), 5.73 (s, 1H), 5.21 (d, J=4.0Hz, 2H), 4.70 (t, J=12.0Hz, 1H), 2.42-2.21 (m,5H), 2.04-1.99 (m, 1H), 1.83-1.56 (m, 8H), 1.43-1.32 (m, 2H), 1.18 (s, 3H), 1.10-0.90 (m, 3H), 0.78 (s, 3H).

[0052] Example 18

[0053] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5e (0.358 g). 1H NMR (400MHz, CDCl3): 7.93 (s,1H), 7.39 (d, J=4.0Hz, 1H), 7.16 (s, 1H), 6.80-6.72 (m, 2H), 5.73 (s, 1H), 5.23 (s, 2H), 4.71 (t, J=8.0Hz, 1H), 2.43-2.20 (m, 5H), 2.04-1.99 (m, 1H),1.86-1.55 (m, 8H), 1.43-1.33 (m, 2H), 1.18 (s, 3H), 1.09-0.90 (m, 3H), 0.77(s, 3H).

[0054] Example 19

[0055] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5f (0.336 g). 1 H NMR (400MHz, CDCl3):7.95-7.91 (m, 3H), 7.69-7.63 (m, 4H), 7.46 (t, J=8.0Hz, 2H), 7.36 (t, J=8.0Hz, 1H), 5.72 (s, 1H), 5.23 (s, 2H), 4.71 (t, J=8.0Hz, 1H), 2.45-2.18 (m,5H), 2.04-1.98 (m, 1H), 1.85-1.56 (m, 8H), 1.43-1.33 (m, 2H), 1.17 (s, 3H),1.10-0.90 (m, 3H), 0.78 (s, 3H).

[0056] Example 20

[0057] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding 5 g (0.290 g) of the compound. 1H NMR (400MHz, CDCl3): 7.86(s, 1H), 7.28 (s, 1H), 7.23-7.13 (m, 2H), 6.68 (d, J=8.0Hz, 1H), 5.72 (s,1H), 5.20 (s, 2H), 4.70 (t, 0.76 (s, 3H).

[0058] Example 21

[0059] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), and finally the compound was prepared for 5 h (0.369 g). 1 H NMR (400MHz, CDCl3): 7.76(s, 1H), 7.63 (d, J=8.0Hz, 2H), 6.74 (d, J=8.0Hz, 2H), 5.72 (s, 1H), 5.19 (d,J=4.0Hz, 2H), 4.69 (t, J=8.0Hz, 1H), 3.85 (s, 1H), 2.46-2.18 (m, 5H), 2.04-1.99 (m, 1H), 1.86-1.54 (m, 8H), 1.42-1.31 (m, 2H), 1.17 (s, 3H), 1.07-0.89(m, 3H), 0.77 (s, 3H).

[0060] Example 22

[0061] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5i (0.197 g). 1H NMR (400MHz, CDCl3):7.94-7.89 (m, 3H), 7.67 (d, J=12.0Hz, 2H), 7.56 (d, J=8.0Hz, 2H), 7.29 (d, J=8.0Hz, 2H), 5.72 (s, 1H), 5.23 (d, J=4.0Hz, 2H), 4.71 (t, J=8.0Hz, 1H), 2.72(dd, J1=4.0Hz, J2=4.0Hz, 2H), 2.42-2.18 (m, 5H), 2.04-1.98 (m, 1H), 1.85-1.53(m, 8H), 1.40-1.26 (m, 5H), 1.17 (s, 3H), 1.10-0.89 (m, 3H), 0.78 (s, 3H).

[0062] Example 23

[0063] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5j (0.299 g). 1 H NMR (400MHz, CDCl3): 8.30(d, J=12.0Hz, 2H), 8.09 (s, 1H), 8.03 (d, J=8.0Hz, 2H), 5.73 (s, 1H), 5.27(d, J=4.0Hz, 2H), 4.72 (t, J=8.0Hz, 1H), 2.45-2.19 (m, 5H), 2.05-1.99 (m,1H), 1.87-1.55 (m, 8H), 1.44-1.34 (m, 2H), 1.19 (s, 3H), 1.11-0.92 (m, 3H), 0.81 (s, 3H).

[0064] Example 24

[0065] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5k (0.302 g). 1H NMR (400MHz, CDCl3): 8.64(s, 1H), 8.25 (dd, J1=8.0Hz, J2=8.0Hz, 2H), 8.07 (s, 1H), 7.63 (t, J=8.0Hz,1H), 5.73 (s, 1H), 5.26 (d, J=4.0Hz, 2H), 4.73 (t, J=8.0Hz, 1H), 2.43-2.22(m, 5H), 2.05-1.99 (m, 1H), 1.86-1.55 (m, 8H), 1.44-1.34 (m, 2H), 1.19 (s,3H), 1.12-0.91 (m, 3H), 0.81 (s, 3H).

[0066] Example 25

[0067] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding 5 l (0.227 g) of compound. 1 H NMR (400MHz, CDCl3): 7.81(s, 1H), 7.75 (d, J=8.0Hz, 2H), 6.96 (d, J=8.0Hz, 2H), 5.73 (s, 1H), 5.20 (d,J=4.0Hz, 2H), 4.70 (t, J=8.0Hz, 1H), 4.08 (dd, J1=8.0Hz, J2=8.0Hz, 2H), 2.43-2.21 (m, 5H), 2.04-2.00 (m, 1H), 1.84-1.55 (m, 8H), 1.44-1.33 (m, 5H), 1.18(s, 3H), 1.08-0.90 (m, 3H), 0.77 (s, 3H).

[0068] Example 26

[0069] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5m (0.177 g). 1H NMR (400MHz, CDCl3): 7.91(s, 1H), 7.46 (s, 1H), 7.39-7.31 (m, 2H), 6.90 (d, J=8.0Hz, 1H), 5.72 (s,1H), 5.22 (d, J=4.0Hz, 2H), 4.70 (t, J=8.0Hz, 1H), 3.87 (s, 3H), 2.42-2.18(m, 5H), 2.04-1.98 (m, 1H), 1.85-1.53 ​​(m, 7H), 1.42-1.26 (m, 3H), 1.17 (s, 3H), 1.09-0.90 (m, 3H), 0.77 (s, 3H).

[0070] Example 27

[0071] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5n (0.058 g). 1 H NMR (400MHz, CDCl3): 7.93(s, 1H), 7.85 (s, 1H), 7.73 (d, J=8.0Hz, 1H), 7.39-7.31 (m, 2H), 5.73 (s,1H), 5.23 (d, J=4.0Hz, 2H), 4.71 (t, J=8.0Hz, 1H), 2.41-2.20 (m, 4H), 2.04-1.99 (m, 1H), 1.84-1.54 (m, 7H), 1.43-1.33 (m, 2H), 1.28-1.23 (m, 2H), 1.18(s, 3H), 1.08-0.91 (m, 3H), 0.78 (s, 3H).

[0072] Example 28

[0073] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5o (0.096 g). 1H NMR (400MHz, CDCl3): 7.83 (s,1H), 7.41-7.40 (m, 1H), 7.33-7.31 (m, 1H), 7.10-7.08 (m, 1H), 5.73 (s, 1H), 5.20 (d, J=4.0Hz, 2H), 4.71 (t, J=8.0Hz, 1H), 2.43-2.20 (m, 5H), 2.05-1.99(m, 1H), 1.83-1.54 (m, 7H), 1.40-1.26 (m, 3H), 1.18 (s, 3H), 1.09-0.91 (m, 3H), 0.78 (s, 3H).

[0074] Example 29

[0075] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5p (0.104 g). 1 H NMR (400MHz, CDCl3): 8.60 (d,J=4.0Hz, 1H), 8.28 (s, 1H), 8.19 (d, J=8.0Hz, 1H), 7.80 (t, J=8.0Hz, 1H), 7.25 (d, J=8.0Hz, 1H), 5.72 (s, 1H), 5.23 (s, 2H), 4.71 (t, J=8.0Hz, 1H),2.42-2.21 (m, 4H), 2.04-1.99 (m, 1H), 1.87-1.53 ​​(m, 8H), 1.42-1.31 (m, 2H),1.17 (s, 3H), 1.08-0.90 (m, 3H), 0.77 (s, 3H).

[0076] Example 30

[0077] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5q (0.307 g). 1H NMR (400MHz, CDCl3): 7.92 (s,1H), 7.85 (d, J=8.0Hz, 2H), 7.44 (t, J=8.0Hz, 2H), 7.35 (t, J=8.0Hz, 1H), 5.73 (s, 1H), 5.13 (d, J=4.0Hz, 2H), 4.66 (t, J=8.0Hz, 1H), 4.50 (s, 2H),2.43-2.16 (m, 5H), 2.04-1.99 (m, 1H), 1.87-1.81 (m, 1H), 1.74-1.49 (m, 7H),1.40-1.31 (m, 2H), 1.19 (s, 3H), 1.15-0.89 (m, 4H), 0.73 (s, 3H).

[0078] Example 31

[0079] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5r (0.264 g). 1 H NMR (400MHz, CDCl3): 7.60 (s, 1H), 5.73 (s, 1H), 5.15 (s, 2H), 4.67 (t, J=8.0Hz, 1H), 2.47-2.16 (m, 6H), 2.05-1.98 (m, 3H), 1.90-1.54 (m, 15H), 1.43-1.33 (m, 3H), 1.19 (s, 3H), 1.09-0.90 (m, 3H), 0.75 (s, 3H).

[0080] Example 32

[0081] The operation steps in this embodiment are completely the same as those in Embodiment 14, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 5s (0.214 g). 1H NMR (400MHz, CDCl3): 7.93 (s,1H), 7.62-7.55 (m, 1H), 7.42-7.37 (m, 1H), 7.04 (t, J=8.0Hz, 1H), 5.72 (s,1H), 5.23 (d, J=4.0Hz, 2H), 4.71 (t, J=8.0Hz, 1H), 2.41-2.22 (m, 4H), 2.03-1.99 (m, 1H), 1.84-1.55 (m, 7H), 1.41-1.33 (m, 2H), 1.26 (s, 2H), 1.18 (s,3H), 1.08-0.90 (m, 3H), 0.78 (s, 3H).

[0082] Example 33

[0083]

[0084] Methylhydrotestosterone (6, 0.01 mol) was added to 200 mL of dichloromethane (DCM) and stirred until completely dissolved. After adding potassium carbonate (0.01 mol), a solution of dichloroacetyl chloride (0.011 mol) was slowly added dropwise. The mixture was stirred at room temperature for 1 hour after the addition was complete, and the reaction was monitored by TLC to indicate completion. Water was added to the reaction mixture, and the organic phase was separated and extracted three times with the aqueous DCM phase. The combined organic layers were concentrated to give compound 7 (2.73 g).

[0085] Example 34

[0086]

[0087] Compound 7 (0.01 mol) was dissolved in 200 mL of acetonitrile, and sodium azide (0.02 mol) was added. The mixture was heated under reflux for 5 hours under nitrogen protection. After the reaction was completed, the mixture was concentrated, DCM was added, and the mixture was washed with brine to separate the organic phase. The organic phase was then concentrated again to give compound 8 (2.69 g).

[0088] Example 35

[0089]

[0090] Add compound 8 (1 mmol) to the reaction flask. The reaction mixture consisted of 1.1 mmol of tert-butanol (50 mL), 50 mL of water, 50 mL of tetrahydrofuran (50 mL), 0.2 mmol of anhydrous copper sulfate, and 0.4 mmol of sodium ascorbate. The mixture was stirred under reflux and monitored by TLC. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 2). The combined organic layers were washed again with brine (30 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, and separated by silica gel column chromatography to obtain compound 9a (0.325 g). 1 H NMR (400Hz, CDCl3): 7.91 (s, 1H), 7.85 (d, J=4.0Hz, 2H), 7.44 (t, J1=8.0Hz, J2=8.0Hz, 2H), 7.35 (t, J1=8.0Hz, J2=8.0Hz, 1H), 5.22 (d, J=4.0Hz, 2H), 4.71 (t, J1=8.0Hz, J2=8.0Hz, 1H), 2.70 (dd, J1=8.0Hz, J2=4.0Hz, 1H), 2.26-2.04 (m, 5H), 1.82-1.64 (m, 4H), 1.60 (s, 1H),1.55-1.46 (m, 3H), 1.38-1.28 (m, 4H), 1.17 (t, J1=12.0Hz, J2=16.0Hz, 1H), 1.11(s, 3H), 0.98 (t, J1=12.0Hz, J2=12.0Hz, 1H), 0.91-0.83 (m, 4H), 0.75 (s, 3H).

[0091] Example 36

[0092] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9b (0.152 g). 1H NMR (400Hz, CDCl3): 7.58 (s, 1H), 7.41 (d, J=12.0Hz, 2H), 6.67 (d, J=8.0Hz, 2H), 5.14 (s, 2H), 4.67 (t, J1=8.0Hz, J2=8.0Hz, 2H), 4.52 (s, 2H), 2.71 (dd, J1=4.0Hz, J2=8.0Hz,1H), 2.23-2.04 (m, 5H), 1.82-1.73 (m, 1H), 1.70-1.60 (m, 4H), 1.52-1.44 (m,3H), 1.38-1.24 (m, 4H), 1.16 (d, J=12.0Hz, 1H), 1.11 (s, 3H), 1.07-0.93 (m, 2H), 0.85 (d, J=8.0Hz, 3H), 0.69 (s, 3H).

[0093] Example 37

[0094] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9c (0.227 g). 1 H NMR (400Hz, CDCl3): 7.57(s, 1H), 7.11 (t, J1=8.0Hz, J2=12.0Hz, 2H), 6.74 (t, J1=8.0Hz, J2=8.0Hz, 1H), 6.68 (d, J=8.0Hz, 1H), 5.13 (d, J=4.0Hz, 2H), 4.66 (t, J1=8.0Hz, J2=8.0Hz,1H), 4.54 (s, 2H), 4.20 (s, 1H), 2.71 (dd, J1=4.0Hz, J2=8.0Hz, 1H), 2.50 (dd,J1=8.0Hz, J2=8.0Hz, 2H), 2.22-2.04 (m, 5H), 1.82-1.73 (m, 1H), 1.69-1.60 (m,4H), 1.52-1.44 (m, 3H), 1.38-1.22 (m, 7H), 1.11 (s, 3H), 1.07-0.93 (m, 2H), 0.86 (d, J=4.0Hz, 4H), 0.69 (s, 3H).

[0095] Example 38

[0096] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), and finally compound 9d (0.194 g) was obtained. 1 H NMR (400Hz, CDCl3): 7.91 (t, J1=8.0Hz, J2=8.0Hz, 3H), 7.67 (d, J=8.0Hz, 2H), 7.56 (d, J=8.0Hz, 2H), 7.29 (d, J=8.0Hz, 2H), 5.23 (d, J=4.0Hz, 2H), 4.72 (t, J1=8.0Hz, J2=8.0Hz, 1H), 2.74-2.67 (m, 3H), 2.26-2.04 (m, 5H), 1.78-1.65 (m, 3H), 1.57-1.47 (m, 5H), 1.38-1.27 (m, 7H), 1.18 (d, J=12.0Hz, 1H), 1.11 (s, 3H), 0.98 (t, J1=12.0Hz, J2=12.0Hz, 1H), 0.86 (d, J=8.0Hz, 4H), 0.76 (s, 3H).

[0097] Example 39

[0098] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9e (0.088 g), MS: 549 [M+H] + .

[0099] Example 40

[0100] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9f (0.185 g), MS: 537 [M+H] + .

[0101] Example 41

[0102] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding 9 g (0.277 g) of compound, MS: 537 [M+H] + .

[0103] Example 42

[0104] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), the final compound was obtained 9 h (0.113 g), MS: 597 [M+H] + .

[0105] Example 43

[0106] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9i (0.206 g), MS: 558 [M+H] + .

[0107] Example 44

[0108] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9j (0.191 g), MS: 491 [M+H] + .

[0109] Example 45

[0110] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9k (0.104 g), MS: 496 [M+H] + .

[0111] Example 46

[0112] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9 l (0.317 g), MS: 505 [M+H] + .

[0113] Example 47

[0114] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9m (0.162 g), MS: 518 [M+H] + .

[0115] Example 48

[0116] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9n (0.247 g), MS: 535 [M+H] + .

[0117] Example 49

[0118] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9o (0.113 g), MS: 505 [M+H] + .

[0119] Example 50

[0120] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9p (0.153 g).

[0121] Example 51

[0122] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... The compound was replaced with trimethylsilylacetylene (1.1 mmol) to finally obtain compound 9q (0.247 g). 1 H NMR (400Hz, CDCl3): 4.71 (t, J=8.0Hz, 1H), 3.86 (s, 2H), 2.70 (dd, J1=8.0Hz, J2=4.0Hz, 1H), 2.27-2.04 (m,4H), 1.83-1.48 (m, 8H), 1.40-1.19 (m, 5H), 1.12 (s, 3H), 1.09-0.84 (m, 9H).

[0123] Example 52

[0124] The operation steps in this embodiment are completely the same as those in embodiment 35, except that the raw materials are... Replace with (1.1 mmol), finally yielding compound 9r (0.172 g). 1H NMR (400Hz, CDCl3): 7.59 (s,1H), 5.15 (d, J=4.0Hz, 2H), 4.59 (t, J=8.0Hz, 1H), 2.70 (dd, J1=8.0Hz, J2=4.0Hz, 1H), 2.30-1.97 (m, 8H), 1.87-1.46 (m, 15H), 1.39-1.27 (m, 5H), 1.11 (s, 3H), 1.08-0.85 (m, 6H), 0.72 (s, 3H).

[0125] Example 53

[0126] To examine the antitumor activity of steroidal compounds, the survival rate of HepG2 liver cancer cells was detected using the MTT assay. We first screened the activity of compounds 4a-4k, 5a-5s, and 9a-9t on HepG2 cells at a concentration of 40 μM. We found that compounds 4a-4k and 5a-5s generally showed better inhibitory effects on HepG2 cells than 9a-9t, and testosterone itself did not show an inhibitory effect. Therefore, we determined the half-maximal inhibitory concentrations (IC50) of compounds 4a-4k and 5a-5s. The IC50 values ​​of the six compounds were also measured. 50 Values ​​below 20 μM: 4f (19.61 μM), 4g (19.16 μM), 5b (4.84 μM), 5c (7.31 μM), 5f (19.81 μM), and 5p (17.92 μM). Given the outstanding performance of 5b, its effects on the esophageal cancer cell line KYS-150 and the normal hepatocyte cell line L02 were further tested. The results showed that 5b exhibited inhibitory activity against both KYS-150 and L02 cells (IC50). 50 The value was 8.01 μM, but it was not toxic to L02 cells (IC50). 50 > 40 μM), indicating good selectivity. Replacing the testosterone structure in compound 5b with a androstenone structure, we found that the resulting compound also lacked activity (IC50 > 40 μM). 50 > 40 μM), structural analysis revealed that the six-membered rings in androstatin and methylhydrotestosterone lack double bonds with testosterone, making them relatively flexible and unable to form a sufficiently rigid planar structure to effectively enter the target site, thus resulting in relatively weak activity.

[0127]

[0128] Example 54

[0129] The colony formation assay is used to evaluate the ability of adherent cells to proliferate and form cell colonies on agar plates, and is one of the important methods for detecting cell proliferation capacity. This assay was used to further investigate the inhibitory effect of compound 5b on hepatocellular carcinoma cell proliferation. Hepatocellular carcinoma cells were treated with low, medium, and high concentrations (2, 4, and 8 μmol / L) of the compound for 9–12 days, and the number of cell colonies was counted to analyze the proliferation inhibition rate. The results showed that the number of cell colonies in the compound 5b treatment group was significantly reduced in a concentration-dependent manner. At a medium concentration of 4 μM, it began to significantly inhibit HepG2 cell colony formation, achieving an inhibition rate of over 60% compared to the control group. At a concentration of 8 μM, colony formation was almost completely inhibited, indicating that compound 5b has a strong anti-proliferative effect on HepG2 cells.

[0130] Example 55

[0131] HepG2 cells in logarithmic growth phase were digested with 0.25% trypsin-EDTA digestion solution, followed by termination of digestion with DMEM complete medium containing 10% FBS. The cell density was adjusted to 2.5 × 10⁻⁶ cells / year. 6 Cells / mL. Cells were seeded at 500 μL / well in 48-well cell culture plates and pre-cultured at 37°C in a 5% CO2 incubator for 24 h until complete cell attachment. Hepatocellular carcinoma cells were treated with low, medium, and high concentrations (2, 4, and 8 μmol / L) of compound 5b for 48 h. Flow cytometry analysis revealed that at 2 μmol / L, the apoptosis rate of 5b reached approximately 30%, and at 8 μmol / L, the apoptosis rate reached approximately 80%.

[0132] Example 56

[0133] To verify whether compound 5b induces apoptosis in HepG2 cells by promoting DNA damage, Western blot analysis showed that, compared with the control group, the expression of p-H2AX protein was significantly increased in the compound 5b treatment group in HepG2 cells. At 8 μmol / L, 5b expression exceeded 3, while at 0 μmol / L it was approximately 1. These results indicate that compound 5b can increase the expression of the DNA damage marker p-H2AX protein in HepG2 cells.

[0134] Example 57

[0135] To verify the effect of compound 5b on the HepG2 cell cycle, HepG2 cells were treated with compound 5b (2-8 μM). With increasing concentration, the proportion of cells in the S phase gradually decreased from 32.06% to 23.43%, while the proportion of cells in the G2 / M phase significantly increased from 23.22% to 33.00%. This indicates that compound 5b can induce HepG2 cells to arrest in the G2 / M phase, thereby inhibiting the proliferation of HepG2 cells.

[0136] Example 58

[0137] To investigate whether compound-induced DNA damage is related to the inhibition of human topoisomerase II, we conducted molecular docking studies. The DNA binding site in human topoisomerase IIβ exhibits shape complementarity around the docking ligand conformation. Two-dimensional interaction diagrams analyzed the non-covalent interactions between the ligand and the binding site residues: it was clearly observed that the 1,2,3-triazole ring and benzene ring of compound 5b form π-π stacks with DT D:9 and DG F:13, respectively. These binding characteristics suggest that this type of compound may act on human topoisomerase II through non-covalent interactions with the active site DNA, providing a potential mechanism for explaining its ability to induce DNA damage in tumor cells.

[0138] Example 59

[0139] NO generation rate detection: BV2 cells (mouse microglia) were seeded into 96-well culture plates using DMEM medium and cultured in a 37°C incubator containing 5% CO2. Cells were divided into a drug-treated group, a positive control group, and an LPS model group. After 24 hours of culture, the drug-treated group was incubated with the corresponding concentrations (1 μM or 10 μM) of all compounds prepared in the examples for 2 hours. The positive control group was incubated with the corresponding concentration (20 μM) of resveratrol. Subsequently, LPS was added to the drug-treated group, the positive control group, and the LPS model group at a final concentration of 100 ng / mL to induce cellular inflammatory response. After 24 hours of further culture, the supernatant from each group was reacted with an equal volume of Griess buffer, and the OD value of each group was measured at 540 nm using a BioTek microplate reader. The NO generation rate of the LPS model group was set as 100% (n=3), and the NO generation rates of the other groups were calculated accordingly.

[0140] Cell viability assay: BV2 cells were seeded in 96-well plates using DMEM medium and cultured in a 37°C incubator containing 5% CO2. Cells were divided into a drug-treated group and an LPS model group. After 24 hours of culture, the drug-treated group was incubated for 2 hours with the corresponding concentrations (1 μM or 10 μM) of all compounds prepared in the examples. Subsequently, LPS was added to both the drug-treated group and the LPS model group at a final concentration of 100 ng / mL to induce cellular inflammatory responses. After another 24 hours of culture, MTT at a final concentration of 0.5 mg / mL was added to each well for viable cell staining. After incubation in the incubator, the culture medium was discarded, and 100 µL of DMSO was added to each well. The cells were shaken thoroughly to dissolve the DMSO, and the OD value of each group was measured at 490 nm using a BioTek microplate reader. The cell viability of the LPS model group was set as 100% (n=3), and the cell viability of the other groups was calculated accordingly.

[0141] Detection results: The NO generation rate detection results are shown in the table below. It can be seen that 5b has a very significant effect on inhibiting NO generation. When we replaced the testosterone structure in compound 5b with the androstenone structure, we found that the ability to alleviate NO release was weakened. Further molecular docking experiments revealed that this is because compound 5b contains an acidic group that chelates with the ferrous ions of heme in the IDO1 target site. It may inhibit IDO1 activity, thereby reducing inflammation and inhibiting NO release. The positive control drug 20μM resveratrol reduced the NO in the supernatant to about 50% of that in the LPS group.

[0142]

[0143]

[0144] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for preparing a steroidal compound that induces DNA damage in liver cancer cells and alleviates neuroinflammation, characterized in that... The structure of this steroid compound is as follows: or Where R is an aryl compound, an alkyl compound, a heterocyclic compound, or , where X is an aryl compound.

2. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: A certain amount of testosterone is added to dichloromethane, followed by the addition of potassium carbonate. A dichloromethane solution containing chloroacetyl chloride is then slowly added dropwise. After the reaction, the mixture is separated by silica gel column chromatography to obtain the final product. .

3. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: a certain amount of methylhydrotestosterone is added to dichloromethane, followed by the addition of potassium carbonate. A dichloromethane solution containing chloroacetyl chloride is then slowly added dropwise. After the reaction, the mixture is separated by silica gel column chromatography to obtain... .

4. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: Take a certain amount of Add acetonitrile, then add sodium azide, heat to reflux, react for a period of time, and then concentrate to obtain... .

5. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: Take a certain amount of Add acetonitrile, then add sodium azide, heat to reflux, react for a period of time, and then concentrate to obtain... .

6. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: Take a certain amount Azide compounds, sodium L-ascorbate, and anhydrous CuSO4 were added to a round-bottom flask. A mixed solvent of water, tert-butanol, and tetrahydrofuran was then added. The mixture was stirred at room temperature and then heated to reflux. After stirring for a period of time, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted multiple times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and after thorough stirring and standing, the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain the final product. .

7. The method for preparing steroidal compounds according to claim 1, characterized in that... The specific process is as follows: Take a certain amount Azide compounds, sodium L-ascorbate, and anhydrous CuSO4 were added to a round-bottom flask. A mixed solvent of water, tert-butanol, and tetrahydrofuran was then added. The mixture was stirred at room temperature and then heated to reflux. After stirring for a period of time, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted multiple times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and after thorough stirring and standing, the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain the final product. .

8. The use of the steroidal compounds as described in claim 1 in antitumor activity.

9. The use of the steroidal compound as described in claim 1 in relieving neuroinflammation.

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