P-gp and microtubule protein inhibitors and preparation methods and applications thereof

Abstract

The present invention provides a P-gp and microtubule protein inhibitor, wherein the P-gp and microtubule protein inhibitor comprises a terpenoid compound having an indole ring; or the P-gp and microtubule protein inhibitor comprises a pharmaceutically acceptable enantiomer or salt of a terpenoid compound having an indole ring. The present invention also provides an extraction method and application of the terpenoid compound having an indole ring.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and more specifically, to a P-gp and tubulin inhibitor and a preparation method and use thereof. Background Art

[0002] Xuefeng Cordyceps is a dried complex of the fruiting body and larval corpse of the fungus Xuefeng Cordyceps (TCWen, RC Zhu, JC Kang & K. D. Hyde) of the Nematode family parasitizing on the larvae of the bat moth family insects. Fungal taxonomy and molecular biology studies have shown that Xuefeng Cordyceps is the species with the closest genetic relationship with Cordyceps sinensis in the same genus. Xuefeng Cordyceps has the effects of nourishing the lungs and kidneys, relieving cough and reducing phlegm. It is mainly used to treat kidney deficiency, impotence, spermatorrhea, back pain, chronic cough and asthma. It is used by the people to enhance immunity, strengthen the body, prevent and treat cancer, and has significant effects. Modern pharmacological studies have shown that Xuefeng Cordyceps has anti-tumor and antioxidant activities, and it is clear that its characteristic anti-tumor components are indole alkaloid compounds. Whether it is the extraction part rich in indole alkaloid compounds or the monomer compound, it has strong anti-tumor activity. For example, the IC50 of its ethyl acetate part on NB4 cells and U937 cells is 3.76 and 1.91 μg / mL, and its in vitro anti-tumor activity is better than the positive control cisplatin. The xuefengins AD and xuefenglasins AC separated from it have strong in vitro anti-tumor activity, especially the activity on MCF-7 cells is better than the positive control 5-fluorouracil. Animal experiments have also proved that this type of component has strong in vivo anti-tumor activity. Studies have shown that the indole alkaloids of Cordyceps sinensis have a certain inhibitory effect on the subcutaneous transplanted tumors of human non-small cell lung cancer PC9 nude mice.

[0003] At present, the research of indole alkaloids has become one of the hot spots in the development of anti-tumor drugs, especially in the field of new drug lead compound discovery. Therefore, the targeted separation, purification and structural identification of anti-tumor alkaloid compounds of Cordyceps sinensis provide lead compounds for the development of new resources and innovative drugs, which has important application prospects. Summary of the invention

[0004] The first object of the present invention is to provide an inhibitor that can simultaneously inhibit P-gp and tubulin.

[0005] The second object of the present invention is to provide an application of P-gp and tubulin inhibitors.

[0006] The third object of the present invention is to provide a pharmaceutical composition comprising P-gp and a tubulin inhibitor.

[0007] The fourth object of the present invention is to provide a method for extracting P-gp and tubulin inhibitors.

[0008] A P-gp and microtubule protein inhibitor, wherein the P-gp and microtubule protein inhibitor comprises compound I; or

[0009] The P-gp and tubulin inhibitors include pharmaceutically acceptable enantiomers or salts of compound I;

[0010] The structure of the compound I is as follows:

[0011] .

[0012] The P-gp and microtubule protein inhibitor is used for preparing drugs for treating cancer.

[0013] The cancer includes lymphoma, leukemia, adenocarcinoma or liver cancer.

[0014] A pharmaceutical composition comprises the P-gp and microtubule protein inhibitor and at least one pharmaceutically acceptable excipient, diluent or carrier.

[0015] A method for extracting P-gp and tubulin inhibitors comprises the following steps:

[0016] 1) Extracting the Xuefeng Cordyceps powder with alcohol under reflux, filtering, and obtaining a filtrate and a filter residue;

[0017] 2) adding the filter residue into alcohol again for extraction, filtering, and obtaining a filtrate;

[0018] 3) combining the filtrates in steps 1) and 2), concentrating under reduced pressure, and freeze-drying to obtain an extract;

[0019] 4) dissolving the extract in water, and extracting with ethyl acetate and n-butanol in sequence to obtain ethyl acetate extract, n-butanol extract, and raffinate;

[0020] 5) Concentrating the ethyl acetate extract, the n-butanol extract, and the raffinate under reduced pressure and freeze-drying them to obtain an ethyl acetate fraction, an n-butanol fraction, and a raffinate fraction;

[0021] 6) dissolving the ethyl acetate fraction in methanol, pouring the sample into a chromatography column, eluting with 50% to 100% methanol to obtain fractions; subdividing the fractions again using a chromatography column; purifying using analytical preparative liquid chromatography to obtain compound I.

[0022] The alcohol includes ethanol;

[0023] The concentration of the ethanol is not less than 95%.

[0024] The extraction time is 2h.

[0025] The chromatography column comprises an ODS chromatography column.

[0026] The mobile phase of the analytical preparative liquid chromatography is ethanol-water.

[0027] The volume ratio of methanol to water is 75:25.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The P-gp and tubulin inhibitor provided by the present invention can inhibit P-gp and tubulin at the same time, thereby resisting the cell transport of cancer cells to them, thereby producing a stronger anti-cancer effect.

[0030] The P-gp and microtubule protein inhibitors provided by the present invention have a broad anti-cancer spectrum and can produce a therapeutic effect on lymphoma, leukemia, adenocarcinoma or liver cancer.

[0031] The pharmaceutical composition provided by the present invention can be used to prepare P-gp and tubulin inhibitors into pharmaceutical preparations.

[0032] The P-gp and microtubule protein inhibitor provided by the invention are extracted from Cordyceps sinensis, have a wide source, and the extraction method is relatively simple and low in cost, and has commercial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The 1H-1H COSY and HMBC nuclear magnetic resonance correlation spectra of compound I are shown.

[0034] Figure 2 shows the cell morphology of A549 cells observed under a 40x phase contrast microscope after the incubation with compound I. Figure 2a is a morphology of cells incubated with 0 μM compound I; Figure 2b is a morphology of cells incubated with 40 μM compound I; Figure 2c is a morphology of cells incubated with 60 μM compound I; and Figure 2d is a morphology of cells incubated with 80 μM compound I.

[0035] Figure 3 shows the cell morphology of A549 cells observed under a 40x phase contrast microscope after the incubation with compound II. Figure 3a is a morphology of cells incubated with 0 μM compound II; Figure 3b is a morphology of cells incubated with 40 μM compound II; Figure 3c is a morphology of cells incubated with 60 μM compound II; and Figure 3d is a morphology of cells incubated with 80 μM compound II.

[0036] FIG4 shows the Hoechst staining observation photos of compound I and compound II.

[0037] FIG5 shows the effect of compound I on A549 apoptosis.

[0038] FIG6 shows the regulatory effect of compound I on p38 and caspase-3.

[0039] FIG7 shows the blotting diagram of the effects of the control substance, compound I and compound II on the expression of P-gp protein. DETAILED DESCRIPTION

[0040] In view of the strong anti-tumor activity exhibited by Cordyceps sinensis, and the fact that its characteristic anti-tumor components are indole alkaloid compounds, separation, purification, identification and study of the anti-cancer mechanism of indole alkaloid compounds in Cordyceps sinensis will help to find new anti-cancer compounds and prepare them into corresponding preparations.

[0041] Specifically, the preparation method of the terpene compound having an indole ring comprises the following steps:

[0042] 1) The Xuefeng Cordyceps powder was extracted with 8 times the amount of 95% ethanol under reflux for 2 hours, and filtered to obtain a filtrate and a filter residue;

[0043] 2) adding the filter residue into 95% ethanol again for extraction, filtering, and obtaining a filtrate;

[0044] 3) combining the filtrates in steps 1) and 2), concentrating under reduced pressure, and freeze-drying to obtain an extract;

[0045] 4) dissolving the extract in water, and extracting with ethyl acetate and n-butanol in sequence to obtain ethyl acetate extract, n-butanol extract, and raffinate;

[0046] 5) Concentrating the ethyl acetate extract, the n-butanol extract, and the raffinate under reduced pressure and freeze-drying them to obtain an ethyl acetate fraction, an n-butanol fraction, and a raffinate fraction;

[0047] 6) The ethyl acetate fraction was dissolved in methanol, and the sample was poured into an ODS chromatography column, and eluted with 50%-100% methanol to obtain 60 fractions (Fr.A1-10-Fr.F1-10).

[0048] Fr.D4~5 was further subdivided using an ODS column to obtain 20 fractions (Fr.D4~5-1~20). HPLC-Q-TOF-MS technology was used to track these 20 fractions and Fr.D4~5-6~8 was identified as the target polarity segment.

[0049] The product was purified by analytical preparative liquid chromatography with the mobile phase being methanol:water in a volume ratio of 75:25 to obtain compound I.

[0050] The specific process of structural identification of compound Ⅰ is as follows:

[0051] Compound Ⅰ, white powder, λmax: 228nm, 280nm. High resolution mass spectrometry ESI-Q-TOF-MS / MS m / z: 512.2677 [M+H]+ (Calcd for C 29 H 37 NO7, 512.2643), indicating that the molecular formula of the compound is C 29 H 37 NO7, the degree of unsaturation is Ω=12.

[0052] The 1H NMR spectrum (600 MHz, CDCl3-d) of compound Ⅰ showed a group of typical indole ring signals in the low field [δH7.70, 1H, s, H-1; 7.47, 1H, t, H-20; 7.37, 1H, dd, J1=6.62 Hz, J2=1.31 Hz, H-23; 7.14, 1H, m, overlapping, H-21; 7.15, 1H, m, overlapping, H-22], and the corresponding 13C NMR was [δC153.1 (C-2), 141.2 (C-24), 125.7 (C-19), 120.5 (C-21), 119.5 (C-22), 118.6 (C-20), 116.8 (C-18), 112.4 (C-23)]. In addition, the 1H NMR spectrum also showed five groups of methyl signals in the high field [δH 2.14, 3H, s, Me-31; 1.45, 3H, s, Me-29; 1.32, 3H, s, Me-25; 1.25, 3H, s, Me-26; 1.24, 3H, s, Me-28].

[0053] The 13C spectrum (125MHz, CDCl3-d) of compound I shows a total of 29 carbon signals, including 6 quaternary carbon signals [δC172.2, 77.4, 74.0, 66.1, 52.0, 44.5], 4 methylene carbon signals [δC31.9, 29.6, 27.8, 26.6], 6 methine carbon signals [δC79.9, 72.8, 71.1, 69.8, 63.9, 50.7], and 5 methyl carbon signals [δC27.0, 23.6, 21.4, 20.9, 16.6], in addition to the above 8 typical indole ring signals. Its specific 1H and 13C data are shown in Table 1.

[0054] Table 1

[0055]

[0056] refer to Figure 1By analyzing the 1H-1H COSY spectrum of compound I, it can be determined that the main structure of compound I is the same as the indole terpene compound provided in the invention patent with publication number CN110642919A, except that C-6 and C-7 are connected. A careful analysis of the HMBC spectrum revealed that H-14 is related to C-15, C-16, C-12, and C-30, H-15 is related to C-3, C-16, and C-14, and H-31 is related to C-30. It can be inferred that the C-14 position of compound I is substituted by an acetyl group, resulting in the appearance of a methine carbon signal [δC71.1 (C-14); δH5.19, 1H, dd, J1=11.41 Hz, J2=4.69 Hz], and Me-31 is connected to C-30. In addition, Me-25 is related to C-2, C-3, C-4 and C-16, indicating that Me-25 is connected to C-3; Me-26 is related to C-3, C-4, C-5 and C-13, and it can be inferred that Me-26 is connected to C-4; Me-28 is related to C-27, C-9, C-29, and it can be inferred that Me-28 is connected to C-27; Me-29 is related to C-27, C-9, C-28, and it can be inferred that Me-29 is connected to C-27; H-6 is related to C-7, C-12, C-6, C-4, and it can be inferred that there is an unsaturated double bond between C-6 and C-7. In addition, H-11 is related to C-10 and C-9; H-10 is related to C-11, C-9 and C-27; H-9 is related to C-10, C-11, C-27, C-28 and C-29; H-5 is related to C-26, C-4, C-6, C-7 and C-13. In the 1H-1H COSY spectrum, the existence of spin coupling systems H-5 / H-6, H-6 / H-7, H-9 / H-10 / H-11, H-14 / H-15, H-16 / H-17, H-20 / H-21 / H-22 / H-23 can be obtained. Therefore, the planar structure of compound Ⅰ is determined. Its relative stereostructure can be inferred from NOESY spectrum, Me-26 is related to H-16, H-14, H-10, H-11 and H-9, H-14 is related to H-16, H-11, H-10 is related to H-9, H-14, OH-13 is related to Me-25, Ha-5. Combined with the 13C NMR [δC 77.4 (C-13)] of compound I and its biosynthetic pathway at C-13, it can be inferred that Me-26, H-16, H-14, H-10, H-11 and H-9 are β-configuration, and Me-25, OH-13 are α-configuration. Compound I is a new compound that has not been reported, named Xuefengin Z. The compound is very similar to the structure of the indole terpenoid compound provided in the invention patent with publication number CN110642919A (referred to as compound II in the present invention).However, in the process of comparing the anti-cancer effects of the two, the difference between the two became apparent.

[0057] Specifically, the present application uses the following method to compare the anticancer activities of compound I and compound II.

[0058] Monomer activity detection:

[0059] 1. Cell lines: U937, MCF-7, Hep G2, A549 cell lines were selected.

[0060] 2. Experimental Methods

[0061] 1) Add cells in the growth phase to a 96-well plate at a concentration of 1.25×105 cells / mL, 90 μL per well. The negative control is saline, and the positive controls are cisplatin and 5-fluorouracil (5-Fu), and the sample addition volume is 10 μL per well.

[0062] 2) Incubate in a 37°C, 5% carbon dioxide incubator for 48 h, add 10 μL / well of MTS (5 mg / mL), and continue incubation for 4 h; then add 100 μL / well of a triple solution (10% SDS-5% isopropanol-0.012 mol / L HCl). After standing at room temperature for 12 h, measure the OD value of each well at 595 nm in a microplate reader.

[0063] Inhibition rate (%) = (OD value of control group - OD value of test group) / OD value of control group. Samples with inhibition rate greater than 50% were screened again and IC50 values ​​were calculated. The results are shown in Table 2.

[0064] Table 2

[0065]

[0066] Furthermore, since the occurrence of cancer is mostly caused by the blockage of cell apoptosis, inducing cancer cells to undergo apoptosis is helpful for the treatment of cancer. Therefore, the present invention compares the effects of compound I and compound II on apoptosis of lung cancer cells.

[0067] Specifically, in order to further explore the anti-tumor mechanism of compound I and compound II, the inventors observed the changes in cell morphology caused by compound I and compound II through phase contrast microscopy, and used Hoechst 33258 staining to further detect the effects of compound I and compound II on the cell nucleus to confirm whether compound I and compound II can induce apoptosis in lung cancer cells. The specific steps are as follows:

[0068] (1) Inoculation of cells

[0069] Place a clean cell slide in a six-well plate. Take cells with good growth status and inoculate them in the six-well plate at 4.0×105 (A549) to make the cells evenly distributed. Place the six-well plate at 37°C, 5% CO2 and saturated humidity to culture the cells for 24 hours.

[0070] (2) Dosing treatment

[0071] After the cells adhered to the wall, they were treated with compound Ⅰ and compound Ⅱ (0 μM, 40 μM, 60 μM, 80 μM) for 24 h.

[0072] (3) Observation of cell morphology using phase contrast microscopy

[0073] After the drug incubation, the cells were placed under a 40x phase contrast microscope to observe cell morphology and take photos.

[0074] The results are shown in Figure 2. Under high drug concentration treatment, the morphology of cells treated with compound I became rounded and gradually showed a phenomenon of detaching from the culture dish wall. However, as shown in Figure 3, cells treated with compound II did not undergo obvious changes.

[0075] (3) Hoechst staining observation

[0076] After the drug incubation, discard the old culture medium, add 1 ml of cell fixative to each well, and fix the cells for 10 minutes. After the fixation, discard the fixative, wash three times with pre-cooled PBS, and add 500ul Hoechst 33258 for light-proof staining for 5 minutes. After the staining, discard the staining solution, wash three times with PBS, and observe and photograph under an inverted fluorescence microscope (20×).

[0077] The results are as follows Figure 4 As shown in the figure, in the samples treated with compound I, the number of cell nuclei decreased, and they showed different degrees of fragmentation, and the staining results became brighter at high concentrations, indicating that the cell nuclei were shrunk. This process is a typical nuclear change characteristic during cell apoptosis. The above experimental results show that compound I can effectively induce apoptosis of lung cancer cells. In the samples treated with compound II, the number of cell nuclei did not change significantly.

[0078] On this basis, the invention further carried out cell cycle detection and related apoptosis protein detection.

[0079] The specific steps and results are as follows:

[0080] (1) Cell cycle detection

[0081] Collect the cells treated in the same way and make a single cell suspension of 1×106 / ml. Set up a negative control and a blank control. Wash the cells once with PBS, discard the supernatant, leave 100ul PBS to blow the cells apart, and then add pre-cooled 75% ethanol to the cell suspension while vortexing to fix the cells overnight. Centrifuge the fixed cells, wash them with PBS, centrifuge again, discard the supernatant, and add the prepared IP staining working solution to stain for 10 minutes. Place them on a flow cytometer for detection.

[0082] The results are as follows Figure 5 It was found that compound Ⅰ could effectively arrest the A549 cell cycle at the G2 / M phase at concentrations of 80μM and 120μM.

[0083] (2) Detection of related apoptosis proteins

[0084] The supernatant of the treated cells was discarded, the cells were washed with PBS containing PMSF, and about 100ul of SDS cell lysis solution was added. After the cells were fully lysed, they were collected and then heated and denatured in a 100-degree metal bath for 10 minutes. The collected cell proteins were separated by SDS-PAGE of different concentrations (8%-15%) according to the molecular weight of the target protein, and then the proteins in the gel were transferred to NC membrane or PVDF membrane. The membrane was blocked with 5% milk solution for 1 hour and then the target protein was incubated with the primary antibody overnight. After the incubation, TBST (TBS containing 1‰ Tween 20) was used to wash three times, each for 5 minutes. After the washing, the secondary antibody was incubated immediately at room temperature for 1 hour. After the incubation, TBST was used to wash three times, each for 5 minutes. Finally, after being treated with an enhanced chemiluminescence detection kit, the target protein blot was obtained using the Bio-Rad gel imager Gel Docxrs imaging system.

[0085] The results are as follows Figure 6 As shown in the figure, compound I can activate classic apoptosis-related proteins such as Caspase 3 and p38 protein after acting on A549 cells, which is manifested by increased expression of pro-apoptotic protein bax, decreased expression of anti-apoptotic protein bcl-2 protein, increased expression of cleaved PARP, cleaved caspase 3, and cleaved caspase 9 proteins, and it is dose-dependent for compound I. Combined with the previous changes in cell morphology and cell nucleus, it proves that compound I can induce apoptosis of lung cancer cells in a dose-dependent manner.

[0086] Furthermore, in order to explore the difference between the two, the present invention compares the effects of the two on the transport activity of P-gp protein.

[0087] Specifically, in order to study the transport activity of compound I and compound II on P-gp protein, the present invention provides experimental data for fluorescence detection of P-gp substrate detection. Rhodamine 6g is a classic substrate of drug-resistant protein P-gp, and its fluorescence intensity can be detected by flow cytometry to measure the transport activity of P-gp protein. The present invention treats A549 / DTX cells by setting up a rhodamine 6g control group and compound I and compound II respectively. Through the analysis of the experimental results, it can be seen that the fluorescence intensity value of the rhodamine 6g control group is 64±5. After adding compound I, the intracellular accumulation of fluorescence intensity value increased significantly, and the fluorescence intensity value increased to 365±10. After adding compound II, the fluorescence intensity value did not increase significantly, and the fluorescence intensity value was maintained at 65±6. It can be seen from this that

[0088] Therefore, the P-gp transport activity of A549 / DTX cells was significantly inhibited after the addition of compound Ⅰ.

[0089] To study the effects of compound I and compound II on the expression level of P-gp protein, the expression of P-gp by the P-gp inhibitor was further tested by Western blot. The experiment was divided into A549 / DTX cell blank control group, compound I group (20 μmol·L -1 ), compound II group (20 μg·mL -1 ). After grouping, membrane proteins were extracted and analyzed. The results are as follows Figure 7 The P-gp protein expression blot is shown. Compared with the control group, there was no significant difference in the P-gp protein expression level in the compound II group, and the P-gp protein expression in A549 / DTX cells in the compound I group was reduced. Therefore, P-gp inhibitors can reduce P-gp protein expression.

[0090] For this reason, after compound I enters cancer cells, it is more difficult to be transported by P-gp and can be enriched in cancer cells, thereby producing a longer-lasting and stronger inhibitory effect on cancer cells.

[0091] In order to study the effects of compound I and compound II on the microtubule distribution of A549 / DTX cells and the mechanism of promoting microtubule polymerization and inhibiting microtubule depolymerization to inhibit mitosis, the inventors fluorescently labeled β-tubulin and set up a compound I group (10 μmol·L -1 ), compound Ⅱ (10 μmol·L -1 ) group, laser confocal analysis of the distribution of β-tubulin in microtubules of A549 / DTX cells. From the laser confocal analysis, it can be seen that β-tubulin is mainly located in the cell nucleus and diffusely distributed in the cytoplasm. Compared with the compound II group, the β-tubulin in the compound I group increased in the cell nucleus and decreased in the cytoplasm, which shows that compound I can inhibit microtubule depolymerization.

[0092] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A method for extracting P-gp and tubulin inhibitors, characterized in that: The steps include: 1) The Xuefeng Cordyceps powder was extracted with 8 times the amount of 95% ethanol under reflux for 2 hours, and filtered to obtain a filtrate and a filter residue; 2) adding the filter residue into 95% ethanol again for extraction, filtering, and obtaining a filtrate; 3) combining the filtrates in steps 1) and 2), concentrating under reduced pressure, and freeze-drying to obtain an extract; 4) dissolving the extract in water, and extracting with ethyl acetate and n-butanol in sequence to obtain ethyl acetate extract, n-butanol extract, and raffinate; 5) Concentrating the ethyl acetate extract, the n-butanol extract, and the raffinate under reduced pressure and freeze-drying them to obtain an ethyl acetate fraction, an n-butanol fraction, and a raffinate fraction; 6) dissolving the ethyl acetate fraction in methanol, pouring the sample into an ODS chromatography column, and eluting with 50%-100% methanol to obtain 60 fractions, which are numbered as Fr.A1-10-Fr.F1-10; The fraction numbered Fr.D4~5 was further subdivided using an ODS chromatography column to obtain 20 fractions, which were numbered Fr.D4~5-1~20; The 20 fractions were tracked using HPLC-Q-TOF-MS technology, and the fractions numbered Fr.D4~5-6~8 were determined to be the target polarity segment; Purification was performed by analytical preparative liquid chromatography with a mobile phase of methanol:water in a volume ratio of 75:25 to obtain P-gp and tubulin inhibitors; The structures of the P-gp and tubulin inhibitors are as follows: 。

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