A c17 sesquiterpene alkaloid, methods of preparation and uses

Abstract

The application relates to the technical field of plant chemistry and active ingredient heterocyclic compounds of traditional Chinese medicine, and discloses a C17 sesquiterpene alkaloid, a preparation method and application, wherein the C17 sesquiterpene alkaloid and a pharmaceutically acceptable salt thereof are shown as formula I; the C17 sesquiterpene alkaloid of formula I is separated from an ethanol extract n-butanol extraction phase of Dendrobium huoshanense stems through three-step operations of silica gel column chromatography, acid solution and base precipitation and recrystallization; the structure of the compound is determined through nuclear magnetic resonance, high-resolution mass spectrometry and single crystal diffraction analysis; experiments show that the C17 sesquiterpene alkaloid can effectively inhibit the survival and proliferation of A549 lung cancer cells, has good anti-lung cancer activity, and can be used for preparing anti-lung cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of phytochemistry and heterocyclic compounds of active ingredients in traditional Chinese medicine, specifically to a C17 sesquiterpene alkaloid, its preparation method, and its application. Background Technology

[0002] Sesquiterpene alkaloids are a class of natural products with diverse structures and significant biological activities. However, there are few reports on such compounds isolated from Dendrobium huoshanense, especially C17 sesquiterpene alkaloids with novel carbon skeleton structures. Therefore, the discovery of sesquiterpene alkaloids from Dendrobium huoshanense and the exploration of their anti-lung cancer activity are of great significance for the development of novel anti-tumor drugs. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a C17 sesquiterpene alkaloid, wherein the C17 sesquiterpene alkaloid has the structure shown in Formula I and its pharmaceutically acceptable salt:

[0004] Formula I.

[0005] Preferably, the molecular formula of the C17 sesquiterpene alkaloid is C 17 H 27 NO4.

[0006] Preferably, the pharmaceutically acceptable salt is obtained by reacting a C17 sesquiterpene alkaloid with an acid, wherein the acid includes an inorganic acid or an organic acid.

[0007] Preferably, the pharmaceutically acceptable salt includes any one of hydrobromide, hydrochloride, phosphate, nitrate, formate, propionate, butyrate, isobutyrate, valerate, hexanoate, oxalate, malonate, succinate, fumarate, maleate, tartrate, malate, citrate, mandelate, benzoate, salicylate, cinnamate, ascorbate, and glutamate.

[0008] A method for preparing the C17 sesquiterpene alkaloid includes the following steps: Step 1: After crushing and sieving the dried stems of Dendrobium huoshanense, reflux extraction is performed, the extracts are combined, and concentrated under reduced pressure to obtain an ethanol extract. Step 2: Dissolve the ethanol extract in water and extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase; Step 3: The n-butanol phase was separated by silica gel column chromatography, and gradient elution was performed with a chloroform / methanol mixed solvent. Fractions of the same type were combined to obtain 12 fractions, which were numbered Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, and Fr.12. Step 4: Fraction Fr.11 is treated with acid dissolution and alkali precipitation to obtain subfraction Fr.11.1; subfraction Fr.11.1 is recrystallized to obtain C17 sesquiterpene alkaloids.

[0009] Preferably, in step one, the reflux extraction method involves heating and refluxing an 85%-95% (v / v) ethanol aqueous solution for 3-5 times.

[0010] Preferably, in step two, the mass ratio of the ethanol extract to water is (1.0-2.0):(10.0-20.0).

[0011] Preferably, in step three, the volume ratio of chloroform to methanol in the chloroform / methanol mixed solvent changes from 80:1 to 0:100.

[0012] Preferably, in step four, the method for preparing the C17 sesquiterpene alkaloid is as follows: Fraction Fr.11 was dissolved in a 0.2wt% hydrochloric acid aqueous solution and extracted with dichloromethane to obtain an aqueous layer; the aqueous layer was adjusted to pH 9.5-10.5 with a 25wt%-28wt% ammonia aqueous solution, and then extracted with chloroform 3-5 times. The chloroform extracts were combined and concentrated to obtain subfraction Fr.11.1; wherein the mass ratio of fraction Fr.11 to 0.2wt% hydrochloric acid aqueous solution was (1.0-2.0):(10-20); The sub-fraction Fr.11.1 is dissolved in chloroform, and an inert solvent, petroleum ether, is added externally for liquid-phase diffusion crystallization; wherein the mass ratio of the sub-fraction Fr.11.1, chloroform, and petroleum ether is (1.0-2.0):(20.0-30.0):(30.0-40.0).

[0013] The C17 sesquiterpene alkaloid can be used to prepare anti-lung cancer drugs.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolates a C17 sesquiterpene alkaloid (DHS-DA-2) from Dendrobium huoshanense. This compound effectively inhibits the survival and proliferation of A549 lung cancer cells and promotes their apoptosis in a concentration-dependent manner, with an IC50 value of 159.17 μmol / L. Molecular docking and molecular dynamics simulations show that the C17 sesquiterpene alkaloid of this invention can stably bind to the active pockets of Akt1 and mTOR kinases. By intervening in the key PI3K / Akt / mTOR signaling pathway in lung cancer cells, it induces cell cycle arrest and apoptosis, thereby exerting an anti-proliferative effect and exhibiting good anti-lung cancer activity. It can be used to prepare anti-lung cancer drugs. As a potential resource of anti-lung cancer sesquiterpene alkaloids, DHS-DA-2 provides theoretical insights into the derivation of functional components from natural products for the prevention and treatment of lung cancer, and provides a new lead compound for the development of anti-lung cancer drugs. In addition, this invention is of great significance for further research on the medicinal properties of the rare and endangered traditional Chinese medicine Dendrobium huoshanense. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 2 This is the mass spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 3 The Fourier transform infrared spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention is shown below. Figure 4 This is a thin-layer chromatography image of potassium bismuth iodide for the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. Figure 5 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 1 1H NMR spectrum (600MHz, DMSO-) d6 ); Figure 6 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 13 C10 NMR spectrum (151 MHz, DMSO-) d6 ); Figure 7 This is the DEPT 135° spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 8 HSQC spectrum of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 9 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 1 H- 1 H COSY spectrum; Figure 10 This is the HMBC spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 11 The Key HMBC of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and 1 H- 1 HCOSY related diagrams; Figure 12 This is the XRD single-crystal diffraction pattern of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 13 The results show the anti-lung cancer activity of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention with cisplatin. Figure 14 This is a diagram showing the molecular docking binding sites between the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention and the Akt1 protein (PDB:3O96); Figure 15 A diagram showing the molecular docking binding sites of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention with the mTOR protein (PDB:4JSV); Figure 16 RMSD diagram of MD simulation of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention and 4JSV protein; Figure 17 RMSF diagram of MD simulation of C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of the present invention; Figure 18 Rg plot of MD simulation of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention and 4JSV protein; Figure 19 MD-simulated hydrogen bond diagram of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention and 4JSV protein; Figure 20 SASA diagram of MD simulation of C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of the present invention; Figure 21Energy landscape diagram of C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of the present invention, as simulated by MD. Figure 22 MD-simulated MM / PBSA binding free energy diagram of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention with 4JSV protein; Figure 23 The energy breakdown diagram of MM / PBSA residues of C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of the present invention. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0017] Example 1 This embodiment discloses a method for preparing C17 sesquiterpene alkaloids, including the following steps: Step 1: After crushing and sieving the dried stems (80.0 kg) of Dendrobium huoshanense, extract them three times by heating and reflux with 90% ethanol aqueous solution. Combine the extracts and concentrate under reduced pressure to obtain ethanol extract (10.5 kg). Step 2: Dissolve the ethanol extract (10.5 kg) in water (105.0 kg), and then fractionally extract with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether phase (0.65 kg), ethyl acetate phase (0.95 kg), n-butanol phase (1.2 kg) and aqueous phase (7.7 kg). Step 3: Separate the n-butanol phase by silica gel column chromatography using a chloroform / methanol mixed solvent as a gradient elution system. The volume ratio of chloroform to methanol in the chloroform / methanol mixed solvent is set at the following nine ratios: 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:100. Between adjacent gradients, the proportion of chloroform gradually decreases while the proportion of methanol gradually increases. In the early stage (80:1 to 20:1), the chloroform decreases by 20 parts each time, with a slow change; in the middle stage (20:1 to 2:1), the decrease gradually becomes smaller; and in the later stage (2:1 to 0:100), only 1-2 parts are decreased each time, ensuring a smooth transition in elution polarity and facilitating the separation of different components. Then, the phase is monitored by thin-layer chromatography (TLC) and the R-values ​​are combined. fFractions with consistent values ​​were divided into 12 major fractions, which were numbered Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, and Fr.12. Step 4: Dissolve the fraction Fr.11 (20.0 g) obtained in Step 3 in 0.2wt% hydrochloric acid aqueous solution (200.0 g), and extract with dichloromethane 3 times to obtain the aqueous layer; The aqueous layer was adjusted to pH 10.0 with a 28 wt% ammonia solution, and then extracted three times with chloroform. The chloroform extracts were combined and concentrated to obtain sub-fraction Fr.11.1 (420.0 mg). The sub-fraction Fr.11.1 (420.0 mg) was dissolved in chloroform (8.4 g) in portions, and petroleum ether (12.6 g) was added externally as an inert solvent. Liquid-phase diffusion crystallization was carried out to obtain C17 sesquiterpene alkaloid (26.2 mg), which was named DHS-DA-2.

[0018] Experimental Example I. The C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 was characterized, and the characterization results are shown in Table 1. Figures 2-12 As shown: Table 1

[0019] Example 1 of the present invention isolated a novel C17 sesquiterpene alkaloid from Dendrobium huoshanense, named DHS-DA-2 (structural schematic diagram shown). Figure 1 As shown), it is a colorless crystal; its molecular formula, determined by high-resolution electrospray ionization mass spectrometry (HRESIMS), is C. 17 H 27 NO4.

[0020] Figure 2 This is the mass spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention, wherein the measured [M+H]... + The peak value was m / z 310.2048 (calculated value 310.1833), verifying that the molecular formula of DHS-DA-2 is C. 17 H 27 NO4.

[0021] Figure 3 This is the Fourier transform infrared spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. Figure 3 This indicates that DHS-DA-2 has three characteristic functional groups: two carbonyl groups (1706 cm⁻¹). -1 1792 cm -1) and a hydroxyl group (3464 cm) -1 ).

[0022] Figure 4 This is a thin-layer chromatography (TLC) image of bismuth potassium iodide for C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. Figure 4 It can be seen that DHS-DA-2 reacts positively with potassium bismuth iodide reagent.

[0023] Table 1 shows the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 1 H NMR (600 MHz) and 13 C NMR (151 MHz) data; Figure 5 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 1 H NMR spectrum (600 MHz, DMSO- d6 ); Figure 6 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 13 C10 NMR spectrum (151 MHz, DMSO-) d6 ); Figure 7 This is the DEPT 135° spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 8 HSQC spectrum of C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 9 This refers to the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention. 1 H- 1 H COSY spectrum; Figure 10 This is the HMBC spectrum of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention; Figure 11 The Key HMBC of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and 1 H- 1 H COSY related graphs; Figure 12 This is the XRD single-crystal diffraction pattern of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention.

[0024] From Table 1 and Figure 5 It can be seen that DHS-DA-2 1 1H NMR spectrum (600 MHz, DMSO-) d6 ) showed a hydroxyl signal [δ H 5.38 (1H, s)], five methyl signals [δH 2.03 (6H, s, H-11, 12), δ H 1.16 (3H, s, H-13),δ H 0.98 (3H, d, J = 10.4 Hz, H-15), δ H 0.75 (3H, d, J = 6.7 Hz, H-16), three methylene signals [(δ H 2.22–2.17 (1H, m, H⁻⁷), δ H 1.92 (1H, dd, J = 11.7, 4.9 Hz, H-10), δ H 1.84(1H, t, J = 8.8 Hz, H-10), δ H 1.81 (1H, t, J = 7.1 Hz, H-7), δ H 1.78 (1H, m, H-8), δ H 1.07–1.15 (1H, m, H-8)], five methine signals [δ H 4.49 (1H, d, J = 5.5 Hz, H-2), δ H 2.79 (1H, d, J = 3.9 Hz, H-4), δ H 2.48 (1H, dd, J = 5.5, 4.0 Hz, H-3), δ H 2.02(1H, s, H-9), δ H 1.76 (1H, s, H-14)).

[0025] From Table 1 and Figure 6 , Figure 7 It can be seen that ¹³C NMR (151 MHz, DMSO- d6 The DEPT-135° spectrum and other spectra showed a total of 17 carbon signals, including 4 quaternary carbons (δ¹⁸Cs). C 208.4, C-1; δ C 175.6, C-17; δ C 82.3, C-5; δ C 58.8, C-6;), 5 primary carbons (δ C 45.3, C-11, 12; δ C 23.7, C-13; δC 21.8, C-15; δ C 18.9, C-16), 3 secondary carbons (δ C 61.2, C-10; δ C 40.1, C-7; δ C 27.9, C-8) and 5 tertiary carbons (δ C 83.4, C-2; δ C 53.3, C-3; δ C 51.6, C-4;δ C 51.1, C-9, δ C 24.8, C-14); the above spectral data suggest that DHS-DA-2 may be a sesquiterpene alkaloid with 17 skeletal carbons.

[0026] From Table 1 and Figure 8 , Figure 9 , Figure 11 It can be seen that through HSQC and 1 H- 1 H COSY spectral data analysis precisely assigned the hydrogen and hydrogen-containing carbon resonances of DHS-DA-2. Based on the COSY spectral analysis, cross-peaks were observed between H3 and H2, H4, and H14, and between H14 and H15, H16. 1 H- 1 The H COSY correlation profile describes fragment A, which contains H2-H3-H4-H14-H15-H16; furthermore, the cross-peaks between H8 and H7, H9, and between H9 and H10 indicate the presence of a fragment, named fragment B. Figure 11 ).

[0027] From Table 1 and Figure 10 , Figure 11 It can be seen that in the HMBC spectrum of DHS-DA-2, according to the HMBC correlation ( Figure 11 From C-17 to H-2 and C-17 to H-4, a five-membered oxygen heterocycle is constructed with fragment A. According to the HMBC cross peaks C-6, C-1 and H-2; C-1, C-6, C-5, C-9 and H-13; C-5, C-6 and H-4, a six-membered ring is formed. Similarly, according to the HMBC correlation from C-7 to H-4, a five-membered ring is formed with fragment B. In summary, DHS-DA-2 prepared in Example 1 of this invention is a novel sesquiterpene alkaloid.

[0028] Depend on Figure 12Analysis of single-crystal diffraction data revealed the absolute configuration of DHS-DA-2, showing that the molecule has six chiral centers with absolute configurations of 2R, 3S, 4S, 5S, 6S, and 9S. A search of the Sci-Finder database confirmed that it is a new compound.

[0029] II. Evaluation of the in vitro anti-lung cancer cell activity of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1. Evaluation methods: Human lung cancer A549 cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ℃ and 5% CO2. Cell viability was determined using the CCK-8 assay. A549 cells were cultured at a density of 4 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates. After 24 hours of adhesion, cells were treated with serially diluted test compounds and cultured for another 24 hours. Dimethyl sulfoxide (DMSO, 0.1% v / v) was used as a solvent control, and cisplatin (DPP) was used as a reference control. After treatment, 10 μL of CCK-8 solution was added to each well and incubated (37 °C, 2 h). Finally, the absorbance was measured at 450 nm using a microplate reader. The results are shown in Table 2. Figure 13 As shown: Table 2

[0030] Figure 13 The results show the anti-lung cancer activity of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention with cisplatin; Figure 13 As shown in Tables A and 2, within the concentration range of 25-400 μmol / L, DHS-DA-2 inhibited lung cancer cell proliferation in a concentration-dependent manner. When the concentration reached 400 μmol / L, cell viability decreased to 38.9%, and the inhibition rate reached 61.1%, with an IC50 value of 159.17 μmol / L (P<0.01). All concentration groups showed highly significant differences compared to the control group (P<0.01). The positive control drug cisplatin (…) Figure 13 -B) showed stronger cytotoxicity in the concentration range of 1.25-20 μmol / L, with an IC50 value of 3.67 μmol / L (P<0.01). In summary, the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention can effectively inhibit the survival and proliferation of A549 lung cancer cells. This is because DHS-DA-2 exerts its anti-proliferative effect by intervening in the key PI3K / Akt / mTOR signaling pathway in lung cancer cells, inducing cell cycle arrest and apoptosis.

[0031] III. Molecular docking simulation of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1. To further understand the key binding interactions, molecular docking simulations were performed on DHS-DA-2, focusing on key lung cancer-related proteins: protein kinase Akt1 (PDB: 3O96) and the mTOR kinase domain (PDB: 4JSV). This evaluation aimed to determine whether the docking analysis results supported the results of anti-lung cancer experiments. Flexible molecular docking analysis was performed using AutoDock Vina software to demonstrate the binding mode of DHS-DA-2 within the hydrophobic pockets of the full-length protein kinase Akt1 (PDB: 3O96) and the mTOR kinase domain (PDB: 4JSV). The results are as follows: Figures 14-15 As shown: Figure 14 This is a diagram showing the molecular docking binding sites between the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention and the Akt1 protein (PDB:3O96); Figure 14 Docking results of the small molecule compound DHS-DA-2 with Akt1 (PDB: 3O96) showed that the docking free binding energy was -7.8 kcal / mol, and DHS-DA-2 bound to the allosteric pocket between the domains. Figure 14 -A), interaction analysis shows ( Figure 14 -B), DHS-DA-2 forms hydrogen bonds with Thr-82 and Gln-79, respectively, and exhibits hydrophobic interactions with Leu-264 and Val-270. It also has van der Waals contact with Ile-84. Figure 14 -C); Figure 15 This is a diagram showing the molecular docking binding sites of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of the present invention with the mTOR protein (PDB:4JSV); Figure 15 Docking results of the small molecule compound DHS-DA-2 with the mTOR kinase domain (PDB:4JSV) showed a docking free binding energy of -8.1 kcal / mol, indicating that the compound is located in the ATP-competitive binding pocket of mTOR. Figure 15 -A), interaction analysis shows ( Figure 15 -B), DHS-DA-2 forms hydrogen bonds with Thr-2279 and Asn-46, exhibits hydrophobic interactions with Met-2281, Trp-274, and Leu-224, and also has van der Waals contact with Val-316 and Gln-225 ( Figure 15 -C); Therefore, combining Figures 14-15It is known that DHS-DA-2 binds to the allosteric pocket between the PH domain and the kinase domain of Akt1, forming hydrogen bonds with Thr-82 and Gln-79, interacting hydrophobically with Leu-264 and Val-270, and forming van der Waals forces, π-cations, and C-H bonds with residues such as Ile-84, Lys-268, and Asp-292, stabilizing the kinase in an inactive conformation. In mTOR, the compound is located in the ATP competitive binding pocket, forming hydrogen bonds with Thr-2279 and Asn-46, interacting hydrophobically with Met-2281, Trp-274, and Leu-224, and is stably bound through π-alkyl and alkyl interactions, suggesting that it inhibits mTOR activity by competitively blocking ATP.

[0032] IV. Molecular dynamics simulations were performed to assess the stability of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 in binding to the target protein. Molecular dynamics simulations of the DHS-DA-2 / mTOR (PDB: 4JSV) complex were performed using the GROMACS 2025 software package for 100 ns. The results are as follows: Figures 16-23 As shown: Figure 16 The RMSD diagram of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and the 4JSV protein was obtained from MD simulation. Figure 16 It can be seen that the root mean square deviation (RMSD) of the system tends to stabilize after about 10 ns, and the overall fluctuation is in the range of 3-6 Å. Figure 17 The RMSF plot of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and the 4JSV protein was obtained by MD simulation. Figure 17 As can be seen, the root mean square fluctuation (RMSF) analysis shows that the RMSF values ​​of most residues fall between 1.0 and 4.0 Å; Figure 18 The Rg plot of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and the MD simulation of 4JSV protein is shown in the figure. Figure 18 It can be seen that the radius of gyration (Rg) remained between 37.5 and 39.5 Å during the simulation, with an average value of approximately 38.5 Å; Figure 19 The MD-simulated hydrogen bond diagram of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention with 4JSV protein is shown in the figure. Figure 19 As can be seen, hydrogen bond analysis shows that during the 100 ns simulation period, the ligand and protein maintained a stable hydrogen bond interaction, with the number of hydrogen bonds mostly concentrated between 2 and 5. Figure 20The SASA diagram of MD simulation of C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of this invention is shown in the figure. Figure 20 It can be seen that the solvent accessible surface area (SASA) increases rapidly in the initial stage of the simulation and then remains stable. Figure 21 The energy landscape diagram of the C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of this invention was obtained from MD simulation. Figure 21 It can be seen that there is a "valley" in the energy potential surface, which shows that the free energy of the system is low at certain RMSD values, meaning that the system is more stable in this conformation; Figure 22 The MD-simulated MM / PBSA binding free energy diagram of the C17 sesquiterpene alkaloid (DHS-DA-2) prepared in Example 1 of this invention and 4JSV protein is obtained from... Figure 22 The analysis of the MM / PBSA fusion free energy showed that the total binding free energy (ΔGTotal) reached -15.37 kcal / mol, indicating that DHS-DA-2 has a high binding affinity for mTOR. Figure 23 The energy breakdown diagram of MM / PBSA residues of the C17 sesquiterpene alkaloid (DHS-DA-2) and 4JSV protein prepared in Example 1 of this invention is obtained from the MD-simulated diagram. Figure 23 The results of residue energy decomposition show that Val316 and Cys317 make the strongest binding contributions. Therefore, combining Figures 16-23 It can be seen that by performing 100 ns molecular dynamics simulations and MM / PBSA binding free energy analysis on the DHS-DA-2 complex with the mTOR kinase domain, the binding stability and mechanism of action of DHS-DA-2 were systematically evaluated. DHS-DA-2 forms a high-affinity binding with the ATP-competitive pocket of the mTOR kinase domain through a stable hydrogen bond network and strong van der Waals / hydrophobic interactions, providing a structural basis and clear direction for its development as a novel selective mTOR inhibitor.

[0033] In summary, this invention presents a C17 sesquiterpene alkaloid, DHS-DA-2, which was isolated from the n-butanol fraction of *Dendrobium huoshanense* stems using silica gel column chromatography, acid extraction and alkali precipitation, and recrystallization techniques. Its structure was elucidated by NMR, high-resolution mass spectrometry, and single-crystal diffraction analysis. Anti-lung cancer studies showed that DHS-DA-2 significantly inhibited the survival and proliferation of A549 lung cancer cells and promoted their apoptosis in a concentration-dependent manner. Molecular docking analysis revealed significant binding interactions between DHS-DA-2 and proteins such as Akt1 and mTOR. Molecular dynamics simulations further confirmed the stability and feasibility of DHS-DA-2 binding to target proteins. DHS-DA-2 serves as a potential resource for discovering anti-lung cancer sesquiterpene alkaloids, providing theoretical insights into deriving functional components from natural products for the prevention and treatment of lung cancer. Therefore, this study is of great significance for further research into the medicinal properties of this rare and endangered traditional Chinese medicine, *Dendrobium huoshanense*.

[0034] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A C17 sesquiterpene alkaloid, characterized in that, The C17 sesquiterpene alkaloid has the structure shown in Formula I and its pharmaceutically acceptable salt: Formula I.

2. The C17 sesquiterpene alkaloid according to claim 1, characterized in that, The molecular formula of the C17 sesquiterpene alkaloid is C 17 H 27 NO4.

3. The C17 sesquiterpene alkaloid according to claim 1, characterized in that, The pharmaceutically acceptable salt is obtained by reacting a C17 sesquiterpene alkaloid with an acid, wherein the acid is an inorganic or organic acid.

4. The C17 sesquiterpene alkaloid according to claim 1, characterized in that, The pharmaceutically acceptable salt is any one of hydrobromide, hydrochloride, phosphate, nitrate, formate, propionate, butyrate, isobutyrate, valerate, hexanoate, oxalate, malonate, succinate, fumarate, maleate, tartrate, malate, citrate, mandelate, benzoate, salicylate, cinnamate, ascorbate, and glutamate.

5. A method for preparing the C17 sesquiterpene alkaloid as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: After crushing and sieving the dried stems of Dendrobium huoshanense, reflux extraction is performed, the extracts are combined, and concentrated under reduced pressure to obtain an ethanol extract. Step 2: Dissolve the ethanol extract in water and extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase; Step 3: The n-butanol phase was separated by silica gel column chromatography, and gradient elution was performed with a chloroform / methanol mixed solvent. Fractions of the same type were combined to obtain 12 fractions, which were numbered Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, and Fr.

12. Step 4: Fraction Fr.11 is treated with acid dissolution and alkali precipitation to obtain subfraction Fr.11.1; subfraction Fr.11.1 is then subjected to liquid-phase diffusion crystallization to obtain C17 sesquiterpene alkaloids.

6. The method according to claim 5, characterized in that, In step one, the reflux extraction method involves heating and refluxing an 85%-95% (v / v) ethanol aqueous solution 3-5 times.

7. The method according to claim 5, characterized in that, In step two, the mass ratio of the ethanol extract to water is (1.0-2.0):(10.0-20.0).

8. The method according to claim 5, characterized in that, In step three, the volume ratio of chloroform to methanol in the chloroform / methanol mixed solvent changes from 80:1 to 0:

100.

9. The method according to claim 5, characterized in that, In step four, the method for preparing the C17 sesquiterpene alkaloid is as follows: Fraction Fr.11 was dissolved in a 0.2wt% hydrochloric acid aqueous solution and extracted with dichloromethane to obtain an aqueous layer. The aqueous layer was adjusted to pH 9.5-10.5 with a 25wt%-28wt% ammonia aqueous solution and then extracted with chloroform 3-5 times. The chloroform extracts were combined and concentrated to obtain subfraction Fr.11.

1. The mass ratio of fraction Fr.11 to the 0.2wt% hydrochloric acid aqueous solution was (1.0-2.0):(10.0-20.0). The sub-fraction Fr.11.1 is dissolved in chloroform, and petroleum ether is added externally for liquid-phase diffusion crystallization; wherein the mass ratio of the sub-fraction Fr.11.1, chloroform and petroleum ether is (1.0-2.0):(20.0-30.0):(30.0-40.0).

10. The application of the C17 sesquiterpene alkaloid according to claim 1 in the preparation of anti-lung cancer drugs.

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