A yeast-derived ergosterol nitrogen heterocycle derivative, its preparation method and use in preparing anti-tumor drugs

Abstract

This invention discloses a yeast-derived ergosterol nitrogen-containing heterocyclic derivative, its preparation method, and its application in the preparation of antitumor drugs. The synthesis uses yeast-derived ergosterol as a starting material. First, the three carbon-carbon double bonds within the molecule are reduced to saturation. Then, the hydroxyl group of ring A is selectively oxidized. The resulting intermediate undergoes a condensation reaction with malononitrile. Subsequently, under mild conditions, it undergoes a ring-expansion reaction with various aldehydes to efficiently prepare the target derivative. Antitumor studies show that all target compounds exhibit superior in vitro antitumor activity compared to ergosterol, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a method for preparing a yeast-derived ergosterol nitrogen heterocyclic derivative and its application. Background Technology

[0002] Ergosterol is the main sterol substance in fungal cell walls. Its structure is based on a steroidal core and contains C, H, and O elements; it contains multiple double bonds at C-5 (6), C-7 (8), and C-22 (23) positions, and a hydroxyl group at C-3 position. This unique structure not only makes it a precursor to vitamin D2 biosynthesis, but also endows it with various potential biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects.

[0003] Currently, ergosterol research mainly focuses on its extraction and separation, pharmacological activity, biosynthetic pathways, and the preparation of derivatives through simple modifications. However, in-depth research is still lacking in the deep skeletal modification of its rigid steroidal core, especially in systematically exploring the structure-activity relationship between the design of novel nitrogen heterocyclic structures and pharmacological activity. Therefore, this study focuses on biomimetic ring-expansion reactions of the ergosterol core. This strategy aims to overcome the limitations of its original four-ring skeleton by constructing new ring systems to significantly regulate the electron distribution, spatial conformation, and reactive sites of the molecule, thus achieving both clear structural novelty and synthetic innovation. Systematically evaluating the antioxidant activity of such ring-expansion derivatives will not only provide key structure-activity relationships for a deeper understanding of the antitumor mechanism of ergosterol compounds, but also holds promise for discovering novel antitumor lead compounds with significantly superior activity to the parent compound based on this inexpensive and readily available natural steroidal skeleton, laying a chemical and biological foundation for its further applications in functional materials, drug development, and other fields. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a method for preparing and applying yeast-derived ergosterol nitrogen heterocyclic derivatives. These compounds exhibit good antioxidant activity and have the potential to be developed into novel drugs.

[0005] This invention provides an ergosterol nitrogen heterocyclic derivative having the structure shown in general formula (I):

[0006]

[0007] General Formula (I) Among them, the double bonds of the ergosterol skeleton can be reduced to single bonds; In some preferred embodiments, in the structure shown in general formula (I), R is selected from a benzene ring or a benzene ring that is mono- or poly-substituted by methyl, ethyl, methoxy, ethoxy, halogen, nitro, cyano, or phenyl. In some preferred embodiments, the ergosterol derivative is selected from any one of the following compounds:

[0008] The present invention also provides a method for preparing the above-mentioned yeast-derived ergosterol nitrogen heterocyclic derivative, comprising the following steps: (1) Ergosterol was reduced with hydrogen to obtain ergosterol reduction product 1; (2) Ergosterol reduction product 1 was oxidized under PCC catalysis to obtain oxidation intermediate 2; (3) The oxidation product 2 was subjected to a nucleophilic reaction with malononitrile to obtain malononitrile intermediate 3; (4) The malononitrile substrate 3 is subjected to a cycloaddition reaction with an aldehyde compound (such as an aromatic aldehyde or an aliphatic aldehyde) to obtain an ergosterol ring-expanded derivative.

[0009] The preparation process of yeast-derived ergosterol of the present invention is carried out according to the following synthetic route, and the specific synthetic route and steps of the derivative can be referred to the examples.

[0010] .

[0011] In step (1), the catalytic hydrogenation reaction is carried out at a hydrogen pressure of 3-10 bar, a catalyst of 5-10% Pd / C, a reaction temperature of 15-30 °C, and a reaction time of 8-15 h; the molar ratio of intermediate 1 to Pd / C is 1-5:0.1-0.6; and the solvent used in the reaction is one or more of methanol, ethanol, and dichloromethane.

[0012] In step (2), the oxidant used in the oxidation reaction is PCC, and CaCO3 is added as a buffer; the reaction temperature is 15-30 ℃, and the reaction time is 2-5 h; the molar ratio of intermediate 1, PCC and CaCO3 is 1-2:4-6:2-5; the solvent used in the reaction process is one or more of methanol, ethanol and dichloromethane.

[0013] In step (3), the nucleophilic addition reaction is carried out at a temperature of 50-80 °C for 2-5 h; the molar ratio of intermediate 2 to malononitrile is 1-2:1-3; and the solvent used in the reaction is one or more of methanol, ethanol, and dichloromethane.

[0014] In step (4), the aldehyde compound is an aromatic aldehyde or an aliphatic aldehyde; the cycloaddition reaction is carried out in an alcohol solvent under reflux in the presence of ammonium acetate.

[0015] The purpose of this invention is to provide a series of ergosterol nitrogen heterocyclic skeleton derivatives to improve the bioactivity of ergosterol, increase its bioavailability, and increase the possibility of ergosterol's clinical application.

[0016] Another object of the present invention is to provide a method for its preparation and its application in the development of antitumor drugs.

[0017] The present invention also provides an antitumor pharmaceutical composition comprising the aforementioned yeast-derived ergosterol nitrogen heterocyclic derivative.

[0018] To achieve the objectives of this invention, ergosterol derived from yeast cell walls was subjected to a cycloaddition reaction with aldehyde derivatives to obtain a series of ergosterol nitrogen-containing heterocyclic derivatives. These compounds exhibit good cytotoxic activity and can be used to prepare antitumor drugs for clinical treatment of various diseases, demonstrating promising development prospects. Attached Figure Description

[0019] Figure 1 This is the 1H NMR spectrum of compound a8.

[0020] Figure 2 This is the carbon NMR spectrum of compound a8. Detailed Implementation

[0021] The invention is further illustrated by the following specific examples, but it should be noted that the scope of the invention is not limited by these embodiments.

[0022] Example 1 Synthesis Route:

[0023] Synthesis of Intermediate 1 Ergosterol (1.0 mmol) was dissolved in 4 mL of methanol, and 10% Pd / C (0.1 mmol) was added. The apparatus was set up and checked, hydrogen gas (7 bar) was introduced, and the mixture was stirred at room temperature for 12 h. The mixture was stained with phosphomolybdic acid, and the reaction was monitored by TLC until it was complete. The palladium on carbon was removed by filtration, the methanol solvent was removed by vacuum distillation, the mixture was washed with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain a white solid, which is intermediate 1, with a yield of 95%.

[0024] Synthesis of intermediate 2 Add 5 mL of dichloromethane to a 50 mL reaction flask, then add intermediate 1 (1.0 mmol), PCC (4.0 mmol), and CaCO3 (2.0 mmol) in sequence. Stir at room temperature for 3 h. Stain the reaction plate with phosphomolybdic acid, monitor the reaction for completeness by TLC, filter to remove precipitate, wash with water 3 times, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, and precipitate by column chromatography to obtain a white solid, which is intermediate 2, with a yield of 90%.

[0025] Synthesis of intermediate 3 5 mL of ethanol was added to a 100 mL round-bottom flask, followed by intermediate 2 (1.0 mmol) and then malondicyandiamide (1.0 mmol). The mixture was heated to 60 °C and refluxed for 2 h. The mixture was then concentrated by vacuum distillation and column chromatography was used to obtain target intermediate 3 in 60% yield.

[0026] Synthesis of target derivative a1 5 mL of anhydrous ethanol was added to a 50 mL round-bottom flask, followed by 0.5 g of ammonium acetate. After partial dissolution, 1 mmol of intermediate 3 and 1.5 mol of benzaldehyde were added. The mixture was heated to reflux at 80 °C for 12 h. Thin-layer chromatography was used to monitor the complete reaction. The ethanol was evaporated under reduced pressure after cooling. The mixture was dissolved in ethyl acetate, extracted three times with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting compound a1 was obtained by thin-layer chromatography with a yield of 30%. Derivatives a2-a11 can be synthesized using a similar method. NMR characterization is as follows: compound a1 1 H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 4.0 Hz, 5H), 5.04 (s, 2H), 2.98(dd, J = 19.0, 5.7 Hz, 1H), 2.60 – 2.44 (m, 2H), 2.38 – 2.25 (m, 2H), 2.05 (d, J = 15.2 Hz, 1H), 1.96 – 1.88 (m, 2H), 1.61 (s, 10H), 1.34 – 1.23 (m, 7H), 0.94 – 0.85 (m, 8H), 0.83 – 0.78 (m, 10H), 0.54 (s, 3H). 13C NMR (101 MHz, CDCl3) δ162.09, 157.01, 152.69, 151.10, 143.41, 138.77, 128.60, 128.49, 128.24,125.39, 120.40, 77.32, 77.01, 76.69, 56.75, 48.64, 42.72, 40.30, 40.07,39.12, 37.10, 36.93, 34.75, 33.55, 33.07, 31.54, 30.39, 29.69, 29.27, 28.91,27.00, 25.87, 20.47, 19.59, 19.25, 18.34, 17.64, 15.45, 11.58. Example 2 Replacing benzaldehyde with 25 mg of 3,5-dimethoxybenzaldehyde, and following the same procedures as in Example 1, yielded 12 mg of pale yellow solid product a2, with a yield of 79%. The reaction procedure was as described in Example 1.

[0027] compound a2 1 H NMR (400 MHz, CDCl3) δ 6.62 – 6.39 (m, 3H), 5.05 (s, 2H), 3.84 (d, J = 4.5 Hz, 6H), 2.95 (td, J = 17.9, 5.7 Hz, 1H), 2.71 – 2.41 (m, 2H), 2.34 –2.21 (m, 2H), 2.07 (d, J = 2.9 Hz, 1H), 1.71 (d, J = 9.7 Hz, 3H), 1.51 – 1.39 (m,5H), 1.32 – 1.23 (m, 8H), 0.95 – 0.85 (m, 10H), 0.84 – 0.76 (m, 10H), 0.54(s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.65, 156.92, 151.47, 143.42, 125.39,120.36, 106.63, 106.48, 100.75, 89.73, 77.33, 77.01, 76.69, 56.75, 55.48,48.60, 42.71, 40.27, 39.91, 39.12, 36.89, 34.76, 33.55, 33.07, 31.53, 30.38,29.70, 29.27, 28.93, 27.01, 25.88, 20.48, 19.65, 19.26, 18.35, 17.64, 15.45, 11.59. Example 3 Replacing benzaldehyde with 25 mg of 3,4-dimethoxybenzaldehyde, and following the same procedures as in Example 1, yielded 15 mg of pale yellow solid product a3, with a yield of 82%. The reaction procedure was as described in Example 1.

[0028] Compound a3 1 H NMR (400 MHz, CDCl3) δ 7.10 – 6.87 (m, 3H), 5.04 (s, 2H), 4.01 –3.87 (m, 6H), 2.96 (td, J = 18.0, 5.8 Hz, 1H), 2.69 (dd, J = 27.8, 16.0 Hz, 2H),2.36 – 2.27 (m, 2H), 2.07 (s, 1H), 1.84 (s, 3H), 1.63 (s, 6H), 1.27 (t, J = 3.4Hz, 8H), 0.95 – 0.85 (m, 10H), 0.84 – 0.74 (m, 10H), 0.53 (s, 3H). 13C NMR (101MHz, CDCl3) δ 148.78, 125.38, 121.57, 120.37, 112.21, 110.65, 56.78, 55.99,48.71, 40.40, 40.29, 39.12, 37.13, 36.99, 34.74, 33.56, 33.12, 31.54, 30.40,29.70, 29.37, 29.28, 28.93, 27.00, 25.88, 21.86, 20.47, 19.75, 19.28, 18.34,17.65, 16.71, 15.45, 11.55. Example 4 Replacing benzaldehyde with 25 mg of 4-fluorobenzaldehyde, and following the same procedures as in Example 1, yielded 16 mg of pale yellow solid product a4, with a yield of 72%. The reaction procedure was as described in Example 1.

[0029] Compound a4 1 H NMR (400 MHz, CDCl3) δ 7.53 – 7.41 (m, 2H), 7.16 (dd, J = 9.8, 7.7Hz, 2H), 5.03 (s, 2H), 2.98 (dd, J = 19.1, 5.8 Hz, 1H), 2.60 – 2.49 (m, 2H), 2.34 (d, J = 16.0 Hz, 2H), 2.07 (s, 1H), 1.95 (d, J = 12.5 Hz, 1H), 1.78 – 1.67(m, 3H), 1.62 – 1.57 (m, 5H), 1.44 – 1.38 (m, 3H), 1.28 (d, J = 2.1 Hz, 5H), 0.90 (ddd, J = 22.4, 6.6, 2.2 Hz, 10H), 0.85 – 0.75 (m, 10H), 0.53 (s, 3H). 13CNMR (101 MHz, CDCl3) δ 156.97, 151.60, 143.52, 130.59, 130.50, 125.28,115.37, 115.15, 89.75, 56.73, 48.63, 42.72, 40.26, 40.16, 39.11, 37.07,36.95, 34.75, 33.55, 33.06, 31.53, 30.37, 29.70, 29.25, 28.89, 27.01, 25.88,20.48, 19.63, 19.25, 18.34, 17.64, 15.45, 11.59. Example 5 Replacing benzaldehyde with 25 mg of 4-bromobenzaldehyde, and following the same procedures as in Example 1, yielded 11 mg of pale yellow solid product a5, with a yield of 65%. The reaction procedure was as described in Example 1.

[0030] Compound a5 1 H NMR (400 MHz, CDCl3) δ 7.64 – 7.57 (m, 2H), 7.38 – 7.32 (m, 2H), 5.04 (s, 2H), 2.98 (dd, J = 19.1, 5.7 Hz, 1H), 2.54 (d, J = 15.6 Hz, 2H), 2.34(d, J = 16.4 Hz, 2H), 2.07 (d, J = 2.5 Hz, 1H), 1.93 (d, J = 10.6 Hz, 1H), 1.82 (d, J = 10.0 Hz, 3H), 1.72 (d, J = 12.5 Hz, 4H), 1.62 – 1.57 (m, 3H), 1.28 (s, 6H), 0.96 – 0.86 (m, 10H), 0.80 (dd, J = 6.9, 1.3 Hz, 10H), 0.53 (s, 3H). 13C NMR (101MHz, CDCl3) δ 160.32, 151.70, 143.56, 138.42, 131.42, 130.32, 125.25, 123.04,120.30, 56.74, 48.63, 42.73, 40.26, 40.08, 39.12, 37.06, 36.94, 34.76, 33.56,33.05, 31.93, 31.54, 30.38, 29.70, 29.24, 28.89, 27.02, 25.88, 20.48, 19.67,19.24, 18.35, 17.65, 15.45, 11.59. Example 6 Replacing benzaldehyde with 25 mg of 4-bromobenzaldehyde, and following the same procedures as in Example 1, yielded 11 mg of pale yellow solid product a6, with a yield of 65%. The reaction procedure was as described in Example 1.

[0031] Compound a6 1 H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 2.0 Hz, 1H), 7.01 – 6.92 (m, 2H), 5.02 (s, 2H), 3.97 (s, 3H), 2.96 (dd, J = 12.2, 6.8 Hz, 1H), 2.62 (d, J = 16.1Hz, 2H), 2.33 (s, 2H), 2.07 (s, 2H), 1.91 (s, 1H), 1.40 (s, 3H), 1.28 (s,12H), 0.89 – 0.86 (m, 10H), 0.83 – 0.79 (m, 10H), 0.53 (s, 3H). 13C NMR (101MHz, CDCl3) δ 145.31, 143.35, 125.45, 120.84, 120.35, 115.15, 110.30, 77.31,76.99, 76.68, 56.78, 56.04, 48.68, 42.73, 40.29, 39.14, 37.15, 36.96, 34.76,33.57, 33.10, 31.92, 31.56, 30.42, 29.97, 29.69, 29.34, 29.27, 28.92, 27.08,26.41, 25.87, 22.67, 20.45, 19.67, 19.26, 18.35. Example 7 Replacing benzaldehyde with 25 mg of 3-pyridinecarboxaldehyde, and following the same procedures as in Example 1, yielded 14 mg of pale yellow solid product a7, with a yield of 49%. The reaction procedure was as described in Example 1.

[0032] Compound a7 1 H NMR (400 MHz, CDCl3) δ 8.74 (td, J = 4.6, 1.7 Hz, 1H), 8.59 – 8.47(m, 1H), 7.62 (ddt, J = 20.4, 7.8, 2.0 Hz, 1H), 7.48 (m, 1H), 5.11 (s, 2H), 3.10 – 2.95 (m, 1H), 2.64 – 2.44 (m, 2H), 2.28 – 2.22 (m, 2H), 2.09 – 1.94(m, 2H), 1.84(dt, J = 14.0, 6.3 Hz, 3H), 1.71 (d, J = 8.7 Hz, 6H), 1.40 (s, 3H), 1.30 – 1.25 (m, 6H), 0.91 – 0.85 (m, 8H), 0.80 (td, J = 7.0, 1.9 Hz, 10H), 0.55(d, J = 12.9 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 150.11, 135.91, 133.05, 125.64,125.03, 124.96, 123.34, 123.26, 115.24, 96.80, 56.71 (d, J = 8.3 Hz), 48.40,42.69, 40.80, 40.56, 40.42, 36.92, 36.55, 34.71, 33.82, 31.54, 29.97, 29.69,29.17, 28.78, 26.97, 25.87, 20.46, 20.19, 19.47, 19.34, 19.23, 18.36, 18.33, 17.64, 15.44, 11.78. Example 8 Replacing benzaldehyde with 25 mg of 4-pyridinecarboxaldehyde, and performing the same procedures as in Example 1, yielded 17 mg of pale yellow solid product a8, with a yield of 58%. The reaction procedure was as described in Example 1.

[0033] Compound a8 1 H NMR (400 MHz, CDCl3) δ 8.89 – 8.74 (m, 2H), 7.19 (dd, J = 14.2, 4.9Hz, 2H), 5.09 (d, J = 2.6 Hz, 2H), 3.11 – 2.95 (m, 1H), 2.51 (dd, J = 40.9, 13.2Hz, 2H), 2.26 (d, J = 12.0 Hz, 2H), 2.11 – 2.04 (m, 2H), 1.89 (d, J = 12.7 Hz, 3H), 1.64 (s, 6H), 1.28 (s, 9H), 0.91 – 0.87 (m, 8H), 0.82 – 0.75 (m, 10H), 0.57 – 0.51 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 150.61, 150.25, 149.66, 147.07,146.65, 145.15, 143.79, 124.93, 124.76, 77.28, 76.97, 76.65, 48.47, 42.72,40.49, 39.15, 36.96, 36.52, 34.74, 33.81, 33.56, 31.59, 30.47, 29.66, 29.16,28.79, 26.92, 25.85, 20.40, 19.41, 19.24, 18.34, 17.67, 15.45, 11.75. Example 9 Replacing benzaldehyde with 25 mg of 4-hydroxybenzaldehyde, and following the same procedures as in Example 1, yielded 10 mg of pale yellow solid product a9, with a yield of 47%. The reaction procedure was as described in Example 1.

[0034] Compound a9 1 H NMR (400 MHz, CDCl3) δ 7.38 – 7.29 (m, 2H), 6.83 – 6.74 (m, 2H), 5.10 (s, 2H), 3.04 – 2.91 (m, 1H), 2.64 – 2.50 (m, 2H), 2.34 (d, J = 15.7 Hz,2H), 1.84 (s, 2H), 1.72 (dd, J = 10.3, 5.8 Hz, 3H), 1.62 – 1.56 (m, 2H), 1.42(d, J = 14.1 Hz, 4H), 1.28 (s, 6H), 1.17 – 1.10 (m, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 – 0.85 (m, 6H), 0.83 – 0.79 (m, 10H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 161.16, 156.98, 156.52, 151.72, 143.45, 130.27, 127.35, 125.52,125.36, 120.41, 116.27, 115.45, 115.30, 89.41, 56.75, 48.67, 43.38, 42.73,42.57, 40.27, 40.21, 39.12, 37.11, 36.96, 34.75, 33.55, 33.14, 32.39, 31.54,30.39, 29.70, 29.26, 28.90, 27.00, 25.88, 20.48, 20.21, 19.67. Example 10 Replacing benzaldehyde with 25 mg of 3,5-fluorobenzaldehyde, and following the same procedures as in Example 1, yielded 12 mg of pale yellow solid product a10, with a yield of 56%. The reaction procedure was as described in Example 1.

[0035] compound a10 1 H NMR (400 MHz, CDCl3) δ 7.03 – 6.86 (m, 2H), 6.78 (ddd, J = 14.1, 8.6,2.2 Hz, 1H), 5.07 (d, J = 3.7 Hz, 2H), 3.00 (ddd, J = 18.6, 12.7, 5.7 Hz, 1H), 2.64 – 2.48 (m, 2H), 2.27 (d, J = 5.8 Hz, 2H), 2.06 (d, J = 2.7 Hz, 1H), 1.94 (s,1H), 1.89 – 1.79 (m, 3H), 1.71 (dt, J = 10.5, 4.7 Hz, 3H), 1.61 – 1.54 (m, 2H), 1.42 (d, J = 15.9 Hz, 4H), 1.27 (q, J = 8.7 Hz, 6H), 0.94 – 0.92 (m, 2H), 0.90 –0.85 (m, 6H), 0.84 – 0.78 (m, 10H), 0.55 (d, J= 5.0 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 156.94, 152.14, 149.57, 146.59, 140.09, 125.15, 125.02, 120.26,115.89, 111.70, 111.48, 104.31, 103.83, 56.82, 56.73, 56.71, 48.55, 48.39,42.71, 40.36, 40.33, 40.19, 39.86, 39.11, 37.05, 36.96, 36.91, 36.49, 29.18,28.79, 19.42, 19.25, 19.22, 19.03, 18.36, 18.22, 17.64, 15.45, 15.38, 14.20, 14.12, 11.77, 11.61. Example 11 Replacing benzaldehyde with 25 mg of 3,5-chlorobenzaldehyde, and following the same procedures as in Example 1, yielded 15 mg of a pale yellow solid product a11, with a yield of 62%. The reaction procedure was as described in Example 1.

[0036] Compound a11 1 H NMR (400 MHz, CDCl3) δ 7.43 (t, J = 1.9 Hz, 1H), 7.35 (d, J = 1.9 Hz,2H), 5.06 (s, 2H), 3.01 – 2.92 (m, 1H), 2.47 (s, 2H), 2.34 (d, J = 15.9 Hz,2H), 2.07 (s, 1H), 1.95 (s, 1H), 1.84 (s, 2H), 1.73 (d, J = 4.2 Hz, 2H), 1.60(s, 4H), 1.40 (s, 3H), 1.29 – 1.25 (m, 6H), 0.94 (d, J = 6.5 Hz, 3H), 0.87 (dd, J = 6.9, 1.5 Hz, 6H), 0.80 (dt, J = 6.3, 1.2 Hz, 10H), 0.54 (s, 3H). 13C NMR (101MHz, CDCl3) δ 158.50, 156.96, 152.16, 143.66, 142.30, 134.93, 128.69, 127.18,125.15, 120.34, 115.86, 90.59, 56.75, 48.52, 42.71, 40.18, 39.86, 37.08,36.97, 34.76, 33.55, 33.04, 31.54, 30.39, 29.97, 29.70, 29.23, 28.90, 27.01,25.90, 20.49, 19.64, 19.26, 18.34, 17.64, 15.45, 15.38, 11.61. The following is an evaluation of the in vitro antitumor activity of the ergosterol nitrogen heterocyclic derivatives of this invention, mainly including triple-negative breast cancer MDA-MB-231 and liver cancer HepG2.

[0037] Experimental methods (1) Cell preparation: Take logarithmic growth phase cells, collect them and resuspend them in complete culture medium to prepare a single cell suspension.

[0038] (2) Cell seeding: Seed the cell suspension into 96-well plates, adjusting the density to 4000-5000 cells per well. Fill the edge wells with PBS to reduce evaporation. Incubate at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0039] (3) Compound preparation: Prepare a 10 mM stock solution of the compound using sterile DMSO and perform serial dilutions (maximum concentration 10 μM). Add the serially diluted compound solutions (8 concentrations in total, 3 replicates per concentration) to adherent cells. Continue culturing for 72 hours.

[0040] (4) Cell viability test: Add MTT reagent to each well and incubate in the dark for 4 hours.

[0041] (5) Absorbance measurement: The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0042] (6) Data analysis: The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting the data with SPSS software. The results are shown in Table 1.

[0043] Table 1. Antitumor activity of ergosterol derivatives n = 3, ± s

[0044] As shown in Table 1, ergosterol exhibited in vitro antiproliferative activity greater than 32 μM against both types of tumor cells. Most derivatives showed superior antitumor activity compared to the parent compound ergosterol. The structure-activity relationship indicated that changes in the benzene ring substituents of ergosterol A ring expansion had little effect on its activity. Pyridine ring substitution resulted in better in vitro activity than substituted benzene rings, with 3-pyridine substitution showing the best activity, indicating promising development prospects.

[0045] The above embodiments are merely illustrative examples of specific implementations of the present invention and are not intended to limit the scope of protection of the present invention in any way. Although the present invention has been described in detail through preferred embodiments, those skilled in the art should understand that appropriate adjustments, modifications, or equivalent substitutions can be made to the specific embodiments based on the above technical teachings without departing from the essential scope of the technical solution of the present invention. Any simple modifications, equivalent changes, and reasonable alterations made to the above embodiments based on the technical essence of the present invention should be considered within the scope of protection defined by the claims of the present invention.

Claims

1. A yeast-derived ergosterol azacyclic derivative, characterized in that, It has the structure shown in general formula (I): General Formula (I) In this embodiment, the double bonds in the ergosterol skeleton are optionally reduced to single bonds; R is selected from any one of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl.

2. The yeast-derived ergosterol azacyclic derivative according to claim 1, characterized in that, The aryl group is phenyl or a substituted aryl group; the heteroaryl group is pyridyl or pyranyl.

3. The yeast-derived ergosterol azacyclic derivative of claim 1, wherein, The substituent is selected from one or more of methyl, ethyl, methoxy, ethoxy, halogen, nitro, cyano, and phenyl, and the substituent is monosubstituted or polysubstituted.

4. The yeast-derived ergosterol azacyclic derivative of claim 1, wherein, The ergosterol ring-expanded derivative is selected from any one of the following structural formulas: 。 5. The process for the preparation of aza derivatives of ergosterol of yeast origin according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Ergosterol was subjected to catalytic hydrogenation to obtain ergosterol reduction product 1; (2) Ergosterol reduction product 1 was oxidized under PCC catalysis to obtain oxidation product 2; (3) The oxidation product 2 was subjected to a nucleophilic reaction with malononitrile to obtain malononitrile intermediate 3; (4) The malononitrile intermediate 3 was subjected to a cycloaddition reaction with an aldehyde compound to obtain the ergosterol nitrogen heterocyclic derivative shown in general formula (I).

6. The production method according to claim 5, characterized by In step (1), the catalytic hydrogenation reaction is carried out at a hydrogen pressure of 3-10 bar, a catalyst of 5-10% Pd / C, a reaction temperature of 15-30 °C, and a reaction time of 8-15 h; the molar ratio of intermediate 1 to Pd / C is 1-5:0.1-0.6; and the solvent used in the reaction is one or more of methanol, ethanol, and dichloromethane.

7. The production method according to claim 5, characterized by, In step (2), the oxidant used in the oxidation reaction is PCC, and CaCO3 is added as a buffer; the reaction temperature is 15-30 ℃, and the reaction time is 2-5 h; the molar ratio of intermediate 1, PCC and CaCO3 is 1-2:4-6:2-5; the solvent used in the reaction process is one or more of methanol, ethanol and dichloromethane.

8. The production method according to claim 5, characterized by, In step (3), the reaction temperature of the nucleophilic addition reaction is 50-80 °C and the reaction time is 2-5 h; the molar ratio of intermediate 2 to malononitrile is 1-2:1-3, and the solvent used in the reaction is one or more of methanol, ethanol, and dichloromethane.

9. The preparation method according to claim 3, characterized in that, In step (4), the aldehyde compound is an aromatic aldehyde or an aliphatic aldehyde; the cycloaddition reaction is carried out in an alcohol solvent under reflux in the presence of ammonium acetate.

10. An antitumor drug or composition, characterized in that, Including claim 1 4. Yeast-derived ergosterol nitrogen heterocyclic derivatives as described in any one of the above.

Images