Diterpenoid compound as well as preparation method and application thereof

Abstract

The invention provides a diterpenoid compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: fermenting rice by using bipolaris sp., so as to obtain a rice fermentation product; extracting the rice leavening with ethanol to obtain an ethanol extract; extracting the ethanol extract with ethyl acetate to obtain an ethyl acetate extract; and separating the ethyl acetate extract to obtain the diterpenoid compounds (compounds 1-7). The compounds 1-6 provided by the invention are enantiomeric-kaurane diterpenes of a 5 / 6 / 6 / 5 ring system formed after C-2 site degradation, and the compound 7 is enantiomeric-kaurane diterpenes of which epoxy is formed at C-2 and C-3 sites. The compound provided by the invention can inhibit the transport function of P-gp and reduce efflux of paclitaxel, so that the treatment effect of paclitaxel is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a diterpenoid compound, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human health. Current main treatment methods include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. While surgical resection has a clear therapeutic effect, it is only suitable for 10%-20% of patients and is prone to recurrence, with limited effectiveness in patients with metastatic disease at an advanced stage. Radiotherapy can locally control sensitive tumors but is difficult to eradicate and may damage bodily functions. Targeted and immunotherapies have limitations such as narrow applicability, long treatment duration, and high cost. Therefore, chemotherapy remains one of the important clinical treatments for malignant tumors. However, many chemotherapy drugs, including vinblastine and arubicin, are prone to drug resistance. Even the first-line drug paclitaxel has shown resistance in various tumor types. Tumor drug resistance has become a major cause of treatment failure and patient death.

[0003] Tumor drug resistance is divided into primary drug resistance and multidrug resistance. Primary drug resistance refers only to resistance to the inducing drug, while multidrug resistance (MDR) occurs when tumor cells, after prolonged exposure to a particular chemotherapeutic drug, not only develop resistance to that drug but also to multiple other chemotherapeutic drugs with different structures and functions. MDR is the most important defense mechanism for tumor cells against chemotherapeutic drug attacks and one of the main causes of chemotherapy failure. Therefore, research on drugs that reverse tumor drug resistance has become a key focus of anti-tumor research.

[0004] Overexpression of P-glycoprotein (P-gp) is a key mechanism of multidrug resistance in tumors, primarily by actively effluxing chemotherapeutic drugs, thereby reducing intracellular drug concentrations and leading to treatment failure. To overcome this resistance, three generations of P-gp inhibitors have been developed: first-generation inhibitors such as verapamil and cyclosporine A; second-generation inhibitors such as dextrovertamil; and third-generation inhibitors such as zosuquidar and tariquidar. However, these inhibitors generally suffer from low selectivity, unsatisfactory pharmacokinetics, or significant toxic side effects, and most have failed to be successfully applied clinically. Therefore, developing novel, highly effective, and low-toxicity P-gp inhibitors remains an urgent need to overcome multidrug resistance. Summary of the Invention

[0005] In view of this, the present invention aims to provide a diterpenoid compound, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is a diterpenoid compound having the structures shown in compounds 1 to 7: .

[0007] The second technical solution of the present invention is a method for preparing the above-mentioned diterpenoid compound, comprising the following steps: Using fungi of the genus Helicobacter. Bipolar sp. Ferment rice to obtain rice fermentation product; The rice fermentation product was extracted with ethanol to obtain an ethanol extract; The ethanol extract was extracted with ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract was separated to obtain the diterpenoid compound.

[0008] The third technical solution of the present invention is a drug that reverses the resistance of colon cancer cells to paclitaxel, comprising the above-mentioned diterpenoid compounds.

[0009] The fourth technical solution of the present invention is the application of the above-mentioned diterpenoid compound in the preparation of a drug that reverses the resistance of colon cancer cells to paclitaxel.

[0010] The fifth technical solution of the present invention is a P-gp inhibitor, comprising the above-mentioned diterpenoid compounds.

[0011] The sixth technical solution of the present invention is the application of the above-mentioned diterpenoid compound in the preparation of P-gp inhibitor drugs.

[0012] The present invention discloses the following technical effects: The compounds 1–6 provided by this invention are enantio-kaurane diterpenes that form a 5 / 6 / 6 / 5 ring system after degradation at the C-2 position, while compound 7 is an enantio-kaurane diterpene with epoxides formed at the C-2 and C-3 positions. The compounds provided by this invention can inhibit P-gp transport function, reduce paclitaxel efflux, and thus significantly improve the therapeutic effect of paclitaxel, providing an important approach for finding new lead compounds to overcome paclitaxel resistance. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is the crystal structure of compound 1 in this invention.

[0015] Figure 2 This is the crystal structure of compound 2 in this invention.

[0016] Figure 3This is the crystal structure of compound 3 in this invention.

[0017] Figure 4 This is the crystal structure of compound 4 in this invention.

[0018] Figure 5 This is the crystal structure of compound 5 in this invention.

[0019] Figure 6 This is the crystal structure of compound 6 in this invention.

[0020] Figure 7 This refers to the derivatization synthesis of compounds 2 and 3 in this invention.

[0021] Figure 8 This invention relates to the effects of compound 1 on the proliferation, cell cycle, and rhodamine 123 accumulation of SW620 / AD300 cells, as well as the thermostability of P-gp. Specifically, A and B show images and quantitative analysis of SW620 / AD300 cell colonies after the use of paclitaxel and compound 1 alone and in combination; C and D show cell cycle analysis of SW620 / AD300 cells after the use of paclitaxel and compound 1 alone and in combination; E and F show flow cytometry analysis of rhodamine 123 accumulation in SW620 / AD300 cells; G shows the expression levels of P-gp in SW620 / AD300, SW620 / AD300 (treated with compound 1), and SW620; Na... + / K + ATPase was used as a loading control. H: Melting curve of P-gp treated with compound 1 in a cell thermal displacement assay. Data are expressed as mean ± standard deviation, n=3. <0.005, compared with the negative control group.

[0022] Figure 9 The results show the molecular docking between compound 1 and P-gp in this invention.

[0023] Figure 10 The effect of compound 1 in this invention on the in vivo reversal activity of paclitaxel resistance is shown in the figure. A: Daily tumor volume in mice during treatment; B: Tumor weight in mice after treatment; C: Image of tumor in mice after treatment; D: Daily body weight of mice; E: HE staining of tumor, liver, and kidney tissues, scale bar 50µm; F: TUNEL assay of tumor tissue, scale bar 5µm. <0.005, compared with the negative control group. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The first aspect of this invention provides a diterpenoid compound having the structures shown in compounds 1 to 7: .

[0030] A second aspect of the present invention provides a method for preparing the above-mentioned diterpenoid compound, comprising the following steps: Using fungi of the genus Helicobacter. Bipolar sp. Ferment rice to obtain rice fermentation product; The rice fermentation product was extracted with ethanol to obtain an ethanol extract; The ethanol extract was extracted with ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract was separated to obtain the diterpenoid compound.

[0031] In this invention, the fermentation specifically involves: mixing rice and water, and then inoculating with fungi of the genus *Helicobacter*. Bipolar sp., then incubated and fermented at 28°C for 30 days; The mass-to-volume ratio of the rice to the water is 1g:1mL; The fungi of the genus *Hydroceta* Bipolar sp. was purchased from the China Agricultural Microbial Culture Collection Center, accession number ACCC38958. The ITS sequence data of this strain has been submitted to GenBank, accession number PQ771450.

[0032] In this invention, the separation includes the following steps: (1) The ethyl acetate extract was subjected to normal phase silica gel column chromatography, and gradient elution was performed using a mixed solvent of petroleum ether, ethyl acetate and methanol with a volume ratio of 10:1:0–5:5:2. Similar fractions were detected by thin-layer chromatography to obtain five fractions, Fr.1-Fr.5. Specifically, the elution gradients are: petroleum ether: ethyl acetate: methanol = 10:1:0, 8:1:0, 5:1:0, 3:1:0, 2:1:0, 1:1:0, 40:40:1, 25:25:1, 15:15:1, 5:5:1, 5:5:2; (2) The Fr.3 is passed through an inverting C 18 Column chromatography was performed using a gradient elution with a mixed solvent of methanol and water, wherein methanol accounted for 30%-100% of the volume of the mixed solvent, yielding 11 fractions from Fr.3.1 to Fr.3.11. Specifically, the elution gradient is as follows: the volume ratio of methanol to the mixed solvent is 30%, 50%, 70%, 90%, and 100%. Fr.3.8 was subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 15:1 to 0:1 to obtain 13 fractions from Fr.3.8.1 to Fr.3.8.13. Specifically, the elution gradients are: petroleum ether: ethyl acetate = 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1; Fr.3.8.11 was further subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1-0:1, yielding a total of 8 fractions from Fr.3.8.11.1 to Fr.3.8.11.8. Among them, Fr.3.8.11.2 was purified by reversed-phase high-performance liquid chromatography to obtain compound 1. Specifically, the elution gradients are: petroleum ether: ethyl acetate = 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1; (3) The Fr.3.8.11.3 was purified by reversed-phase high-performance liquid chromatography to obtain compound 2; (4) The Fr.3.7 was subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 15:1-0:1 to obtain 13 fractions from Fr.3.7.1 to Fr.3.7.13; then, compound 4 and compound 6 were separated from Fr.3.7.1 by reversed-phase high-performance liquid chromatography. Specifically, the elution gradients are: petroleum ether: ethyl acetate = 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1; (5) The Fr.3.8.5 was purified by reversed-phase high-performance liquid chromatography to obtain compound 7; (6) The Fr.4 is first subjected to reversed-phase column chromatography, and gradient elution is performed using a mixed solvent of methanol and water, wherein the volume ratio of methanol to the mixed solvent is 30%-100%, and 19 components from Fr.4.1 to Fr.4.19 are obtained. Specifically, the elution gradient is as follows: the volume ratio of methanol to the mixed solvent is 30%, 50%, 70%, 90%, and 100%. Fr.4.17 was then subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of dichloromethane and methanol in a volume ratio of 100:1-0:1, yielding a total of 8 fractions from Fr.4.17.1 to Fr.4.17.8. Specifically, the elution gradients are: dichloromethane:methanol = 100:1, 80:1, 60:1, 40:1, 20:1, 0:1; Fr.4.17.7 was subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1-0:1 to obtain five fractions: Fr.4.17.7.1-Fr.4.17.7.5. Fr.4.17.7.3 was then separated by reversed-phase high-performance liquid chromatography to obtain compounds 3 and 5. Specifically, the elution gradients are: petroleum ether: ethyl acetate = 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1.

[0033] A third aspect of the present invention provides a drug for reversing the resistance of colon cancer cells to paclitaxel, comprising the above-mentioned diterpenoid compounds.

[0034] The fourth aspect of this invention provides the use of the above-mentioned diterpenoid compound in the preparation of a drug that reverses the resistance of colon cancer cells to paclitaxel.

[0035] A fifth aspect of the present invention provides a P-gp inhibitor comprising the above-mentioned diterpenoid compounds.

[0036] The sixth aspect of this invention provides the use of the above-mentioned diterpenoid compounds in the preparation of P-gp inhibitor drugs.

[0037] The drugs or preparations provided by this invention also include pharmaceutically acceptable excipients.

[0038] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0039] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0040] Example 1 Preparation and structural identification of compounds 1-7 (a) Preparation of compounds 1–7 1. Strain Information Fungi of the genus *Hylocereus* Bipolar sp. was purchased from the China Agricultural Microbial Culture Collection Center, accession number ACCC38958. The ITS sequence data of this strain has been submitted to GenBank, accession number PQ771450.

[0041] 2. Extraction and separation The strain was cultured on PDA medium for 5 days, then cut into small pieces and inoculated into 1L Erlenmeyer flasks, each containing 230g of rice and 230mL of distilled water (a total of 60kg of rice was cultured). The strain was fermented in the rice at 28°C for one month. The fermentation product was extracted with ethanol ten times to obtain a total ethanol extract. The total extract was dispersed in water and extracted with ethyl acetate to obtain 1kg of ethyl acetate extract.

[0042] The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography (100–200 mesh) using gradient elution with petroleum ether:ethyl acetate:methanol ratios of 10:1:0, 8:1:0, 5:1:0, 3:1:0, 2:1:0, 1:1:0, 40:40:1, 25:25:1, 15:15:1, 5:5:1, and 5:5:2. Similar fractions were analyzed by thin-layer chromatography, yielding five fractions (Fr.1–Fr.5). Fr.1 showed a dark spot under UV 254 nm and a yellow color with concentrated sulfuric acid-ethanol reagent, exhibiting tailing and poor spot formation, suggesting it is an oily compound. Fr.2 showed significant absorption under UV 254 nm and a green and yellow color with concentrated sulfuric acid-ethanol reagent, also exhibiting poor spot formation, suggesting it is a mixture of steroids and oils. Fr.3 shows significant absorption at UV254nm and exhibits brown, purplish-red, pink, and blue-violet colors after being developed with concentrated sulfuric acid-ethanol, demonstrating good spot formation; it is presumed to be a terpene compound. Fr.4 also shows significant absorption at UV254nm and exhibits brown and purplish-red colors after being developed with concentrated sulfuric acid-ethanol, demonstrating good spot formation; it is presumed to be a terpene compound. Fr.5 shows little absorption at UV254nm and appears black after being developed with concentrated sulfuric acid-ethanol, exhibiting poor spot formation; it is a highly polar component.

[0043] Fr.3 first passes through the inverting C 18 Column chromatography was performed using a gradient elution with a mixed solution of methanol and water, wherein the methanol content was 30%, 50%, 70%, 90%, and 100%, yielding 11 fractions: Fr.3.1-Fr.3.11.

[0044] Fr.3.8 was subjected to normal-phase silica gel column chromatography again, with gradient elution using petroleum ether:ethyl acetate ratios of 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and 0:1, yielding 13 fractions (Fr.3.8.1-Fr.3.8.13).

[0045] Fr.3.8.11 was further subjected to normal-phase silica gel column chromatography, with gradient elution using petroleum ether:ethyl acetate ratios of 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and 0:1, yielding 8 fractions (Fr.3.8.11.1-Fr.3.8.11.8).

[0046] Fr.3.8.11.2 Compound 1 was obtained by reversed-phase high-performance liquid chromatography (RP-HPLC) with 70% acetonitrile, and the retention time was 44 min. Fr.3.8.11.3 Compound 2 was also obtained by reversed-phase HPLC with 62% acetonitrile, and the retention time was 32 min.

[0047] Component Fr.3.8.5 was purified by reversed-phase high-performance liquid chromatography with 82% methanol to obtain compound 7, with a retention time of 12 min.

[0048] Fr.3.7 was subjected to normal-phase silica gel column chromatography with gradient elution of petroleum ether:ethyl acetate at ratios of 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and 0:1, yielding 13 fractions (Fr.3.7.1–Fr.3.7.13). Finally, compounds 4 and 6 were separated from Fr.3.7.1 by reversed-phase high-performance liquid chromatography (RP-HPLC) with 71% methanol, with retention times of 34 min and 32 min, respectively.

[0049] Fr.4 first passes through the inverting C 18 Column chromatography was performed using a gradient elution with a mixed solution of methanol and water, wherein the methanol content was 30%, 50%, 70%, 90%, and 100%, resulting in the separation of 19 fractions (Fr.4.1-Fr.4.19).

[0050] Fr.4.17 was then subjected to normal-phase silica gel column chromatography with gradient elution using dichloromethane:methanol = 100:1, 80:1, 60:1, 40:1, 20:1, and 0:1 to obtain 8 fractions (Fr.4.17.1-Fr.4.17.8).

[0051] Fr.4.17.7 was further separated by normal-phase silica gel column chromatography with gradient elution using petroleum ether:ethyl acetate ratios of 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and 0:1. Fr.4.17.7.3 was separated by reversed-phase high-performance liquid chromatography, eluting with 73% methanol to obtain compounds 3 and 5, with retention times of 55 min and 40 min, respectively.

[0052] (II) Structural identification of compounds 1-7 The structures of compounds 1-7 were determined by comprehensive analysis of high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, optical rotation, nuclear magnetic resonance, circular dichroism spectroscopy, and X-ray single crystal diffraction data.

[0053] Compound 1: Colorless crystals; melting point 137.5-139.0 °C; [ α ]25 D -96 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 201 (3.76) nm; ECD (CH3CN) l max (Δ e ) = 203 (+9.23), 226 (-0.84) nm; IR (KBr) n max3361, 3191, 2925, 2853, 1819, 1744, 1731, 1660, and 1633cm -1 HRESIMS m / z 439.2088 [M + Na] + (calcd for C 24 H 32 O6Na + , 439.2097). The nuclear magnetic resonance (NMR) data of compound 1 are shown in Table (1).

[0054] Compound 2: Colorless crystals; melting point 154.9-156.6 °C; [ α ]25 D -114 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 202 (3.75) nm; ECD (CH3CN) l max (Δ e ) = 201 (+10.04), 225 (-0.83) nm; IR (KBr) n max 3364, 3207, 2927, 2855, 1820, 1733, 1715, and 1659 cm -1 HRESIMS m / z 425.1939 [M + Na] + (calcd for C 23 H 30 O6Na + , 425.1940). The nuclear magnetic resonance (NMR) data of compound 2 are shown in Table (1).

[0055] Compound 3: Colorless crystals; melting point 91.1-92.5 °C; [ α ]25 D +16 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 203 (3.78) nm; ECD (CH3CN) l max (Δ e ) = 204 (+11.73) nm; IR (KBr) n max3435, 2928, 2863, 1734, 1442, and 1162 cm -1 HRESIMS m / z 457.2201 [M + Na] + (calcd for C 24 H 34 O7Na + , 457.2202). The nuclear magnetic resonance (NMR) data of compound 3 are shown in Table (1).

[0056] Compound 4: colorless crystals; melting point 177.3-178.8 °C; [ α ]25 D -86 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 205 (3.53) nm; ECD (CH3CN) l max (Δ e ) = 203 (+7.28) nm; IR (KBr) n max 3516, 2920, 2852, 1808, and 1656 cm -1 HRESIMS m / z 325.1778 [M + Na] + (calcdfor C 19 H 26 O3Na + , 325.1780). The nuclear magnetic resonance (NMR) data of compound 4 are shown in Table (2).

[0057] Compound 5: Colorless crystals; melting point 164.4-165.6 °C; [ α ]25 D +12 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 205 (3.69) nm; ECD (CH3CN) l max (Δ e ) = 204 (+10.96) nm; IR(KBr) n max 3433, 2925, 2858, 1713, 1668, 1445, 1254, and 1058 cm-1 HRESIMS m / z 357.2053 [M + Na] + (calcd for C 20 H 30 O4Na, 357.2042). The nuclear magnetic resonance (NMR) data of compound 5 are shown in Table (2).

[0058] Compound 6: Colorless crystals; melting point 171.3-172.5 °C; [ α ]25 D-163 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 198 (3.64) nm; ECD (CH3CN) l max (Δ e ) = 218 (-0.90) nm; IR (KBr) n max 3529, 2931, 2859, 1806, and 1656 cm -1 HRESIMS m / z 325.1780 [M + Na] + (calcdfor C 19 H 26 O3Na, 325.1780). The nuclear magnetic resonance (NMR) data of compound 6 are shown in Table (2).

[0059] Compound 7: Amorphous powder; [ α ]25 D-538 ( c 0.1, CH3OH); UV (CH3CN) l max (log e ) = 200(4.00) nm; ECD (CH3CN) l max (Δ e ) = 207 (+20.40) nm; IR (KBr) n max 2925, 2854, 1706,1472, 1446, and 1063 cm -1 HRESIMS m / z 371.2202 [M + Na] + (calcd for C 21H 32 O4Na + ,371.2198). The nuclear magnetic resonance (NMR) data of compound 7 are shown in Table (2).

[0060] Table 1. Proton and carbon spectral data of compounds 1-3

[0061] Table 2. Proton and carbon spectral data of compounds 4-7

[0062] Example 2 Derivatization studies of compounds 2 and 3 (a) Synthesis of compound 1 and compound 8-compound 11 from compound 2 Compound 2 was divided into five fractions and added to five 4 mL round-bottom flasks containing a solution of dry dichloromethane (DCM, 1.00 mL) pre-contained with EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.10 equivalent) and DMAP (4-dimethylaminopyridine) (0.10 equivalent). Then, under argon protection, dry methanol, ethanol, 1-propanol, 1-butanol, and 2-methyl-1-propanol (each 2.00 equivalent) were added to each flask. The reaction mixture was stirred at room temperature for 3 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). The target compounds were purified by reversed-phase high-performance liquid chromatography (94% acetonitrile / water, 3.5 mL / min) to obtain compounds 1, 8, 9, 10, and 11.

[0063] (ii) Synthesis of compound 12 from compound 3 Compound 3, potassium carbonate (2.50 equivalents), and iodomethane (2 equivalents) were dissolved in 1.00 mL of anhydrous N,N-dimethylformamide (DMF) in a 4 mL flask. Then, anhydrous methanol (2.00 equivalents) was added under argon protection. The reaction was stirred at room temperature for 3 hours (monitored by TLC), followed by purification by reversed-phase high-performance liquid chromatography (94% acetonitrile / water, 3.5 mL / min). Compound 12 was obtained in 92% yield.

[0064] Example 3 Effects of compounds 1-12 in combination with paclitaxel on SW620 / AD300 cells and tumors at cellular and animal levels (a) In vitro activity evaluation 1. SW620 / AD300 cell culture The cells were provided by Professor Liu Hongmin (Department of Oncology, Cancer Hospital Affiliated to Zhengzhou University, China) and cultured in Dulbeccos Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (Wuhan, China), with 100 U / mL penicillin and 100 μg / mL streptomycin at 37 °C and 5% CO2.

[0065] 2. CCK-8 test Cells were spaced at 5 × 10⁶ cells per well. 3 The test compound was seeded at a density in 96-well plates and pre-incubated for 24 hours. Then, different concentrations of the test compound were added to the designated wells and incubated for 72 hours. 10 μL of CCK-8 was added to each well and incubated at 37°C for 1 hour. The absorbance of each well was measured at 450 nm using a Varioskan LUX microplate spectrophotometer. IC50 was calculated using GraphPad. 50 Value. The reversal fold (RF) is the half-maximal inhibitory concentration (IC50) of paclitaxel. 50 IC50 value of paclitaxel (PTX) and other compounds used in combination 50 The ratio of values.

[0066] 3. Settlement formation experiment SW620 / AD300 cells were seeded in 6-well plates (2000 cells / well) and treated for 7 days with compound 1 in combination with different concentrations of paclitaxel. After removing the culture medium and washing with PBS, the cells were fixed with 4% paraformaldehyde, stained with 0.1% (w / v) crystal violet, washed with PBS, photographed, and counted.

[0067] 4. Flow cytometry SW620 / AD300 cells were co-cultured with the corresponding test compounds in 6-well plates for 24 hours. Cells were then collected and fixed overnight at 4°C with 70% ethanol. Subsequently, samples were stained with propidium iodide solution containing RNase A for 30 minutes and analyzed using a BD flow cytometer.

[0068] 5. Rhodamine 123 Accumulation Experiment SW620 / AD300 cells were seeded in 6-well plates (3 × 10⁶ cells / well). 5 Cells were treated with different concentrations of compound 1 or verapamil (VPM) at 37°C for 24 hours, followed by co-incubation with rhodamine 123 at 37°C for 30 minutes. Cells were analyzed using BDFACS flow cytometry.

[0069] 6. Cell thermal displacement experiment Membrane proteins were extracted from SW620 / AD300 cells and divided into two equal parts. One part was treated with DMSO, and the other part was treated with compound 1. After incubation at 37°C for 30 minutes, the membrane proteins were further aliquoted into smaller equal parts and then heated at specified temperatures ranging from 40°C to 65°C for 3 minutes each. The expression level of P-gp in the membrane proteins after heating was analyzed by Western blotting.

[0070] (ii) Molecular docking Molecular docking analysis was performed using Schrödinger Maestro software. The protein model 7A6E was retrieved from the Protein Database (PDB) and preprocessed using the Protein Preparation Workflow tool. Compound structures were preprocessed using the LigPrep tool. The receptor grid was centered between the substrate binding site and the channel entrance, ensuring complete coverage of both regions. Ligand docking was performed using default settings. The interaction and structural superposition results were visualized and analyzed using PyMOL software.

[0071] (III) Evaluation of in vivo activity 1. Animal modeling and processing Male BALB / c nude mice (weighing 18-22 grams, 6-7 weeks old) were housed in ventilated cages and provided with food and sterilized water. Each mouse was subcutaneously inoculated with approximately 1×10-1 ... 7 SW620 / AD300 cells. Mice were randomly divided into four groups (n=5 per group) and treated according to one of the following protocols: (i) Blank control (containing 90% corn oil and 10% DMSO, 10 mL / kg / every 3 days, by gavage).

[0072] (ii) Paclitaxel (5 mg / kg every 3 days, dissolved in 10% DMSO and suspended in 90% corn oil, administered by gavage).

[0073] (iii) Paclitaxel combined with verapamil (5 mg / kg every 3 days, dissolved in 10% DMSO and suspended in 90% corn oil, administered by gavage).

[0074] (iv) Paclitaxel combined with compound 1 (50 mg / kg every 3 days, dissolved in 10% DMSO and suspended in 90% corn oil, administered by gavage).

[0075] Animal weight and tumor volume were measured daily using an analytical balance and vernier calipers. After 14 days of treatment, animals were euthanized, and tumor, liver, and kidney tissues were fixed in 4% paraformaldehyde for further analysis.

[0076] 2. TUNEL staining to detect in vivo cell apoptosis Tumor tissue sections were treated with proteinase K for 20 minutes at room temperature and then rinsed with PBS. Subsequently, the sections were incubated with TdT enzyme and FITC-labeled dUTP in a humidified environment at 37°C for 1 hour. Afterward, the sections were mounted with anti-fluorescence quenching mounting medium, and the fluorescence of positive staining was observed using a fluorescence microscope.

[0077] 3. HE staining was performed for histopathological analysis.

[0078] The tissue sections were dewaxed, stained with hematoxylin for 5 minutes, rinsed, and differentiated with 1% hydrochloric acid alcohol. Next, they were stained with eosin solution for 2 minutes. After rinsing and dehydration to clear, they were mounted with neutral resin. Finally, the tissue on the sections was observed using a fluorescence microscope.

[0079] (iv) Results Analysis 1. Initial Activity Screening: The cytotoxicity of compounds 1-7 in combination with paclitaxel against SW620 / AD300 cells was first tested using a CCK-8 assay. As shown in Table 3, after 72 hours of treatment with a combination of paclitaxel (1 μM) and compound 1 (20 μM), the survival rate of SW620 / AD300 cells was the lowest, indicating that compound 1 has a good potential to reverse multidrug resistance in tumors. Notably, the reversal effect of compound 1 was significantly better than that of the positive control verapamil (VPM). Furthermore, compound 1, when used alone, did not exhibit cytotoxicity. These activity screening results indicate that the presence of the four-membered lactone ring and succinic acid group is crucial for conferring multidrug resistance activity. However, a structural comparison of compounds 1 and 2 shows that esterification of the C-4′ carboxyl group can significantly enhance the activity of the compounds.

[0080] Table 3 Activity screening results of compounds 1-12

[0081] 2. Activity Screening of Derivatized Products: The same method was used to evaluate the anti-tumor multidrug resistance and cytotoxic activity of derivatized products 8-12. However, none of these analogues showed significant activity compared to compound 1 (Table 3). These findings suggest that increasing the size and hydrophobicity of the ester group at C-4′ is detrimental. The activity results of compound 12 further demonstrate the importance of the presence of a four-membered lactone ring in enhancing compound activity.

[0082] 3. Colony formation assay to evaluate the effect of compound 1 on cell proliferation and survival: such as Figure 8 As shown in Figures A and B, compound 1, when used in combination with paclitaxel, significantly inhibited the proliferation of SW620 / AD300 cells, even at a concentration of 0.25 μM.

[0083] 4. Flow cytometry to assess the effects of compounds on cell cycle progression: such as Figure 8 As shown in C and D, combined treatment with compound 1 (20 μM) and paclitaxel reduced the percentage of cells in the G1 phase of SW620 / AD300 cells from 35% to 20%, the percentage in the S phase from 45% to 27%, and the percentage in the G2 / M phase from 15% to 47%.

[0084] 5. Rhodamine 123 accumulation assay to detect the effect of compounds on P-gp efflux function: such as Figure 8 As shown in Figures E and F, compound 1 can increase the intracellular accumulation of the P-gp efflux pump substrate rhodamine 123 in a dose-dependent manner, indicating that compound 1 can enhance the sensitivity of SW620 / AD300 cells to paclitaxel by inhibiting the transport of P-gp protein.

[0085] 6. Cellular thermal displacement assay to evaluate the effect of compound 1 on the thermal stability of P-gp: such as Figure 8 As shown in Figure H, compound 1 significantly enhances the thermal stability of P-gp at high temperatures. These results suggest that compound 1 may be a potential P-gp inhibitor.

[0086] 7. Molecular docking: Using the complex structure of P-gp and the inhibitor tariquidar as a reference, one molecule of compound 1 was docked to the vestibule between the P-gp binding bag and the pathway tunnel. This indicates that compound 1 forms a stable complex with P-gp, with a binding affinity of -6.799 kcal / mol. Figure 9 Compound 1 shown forms three hydrogen bonds with Trp232 (TM4), Gln347 (TM6) and Gln990 (TM12), and an aromatic hydrogen bond with Trp223.

[0087] 8. Analysis of animal experimental results: The results showed that the combination of compound 1 and paclitaxel significantly reduced tumor weight and volume in nude mice, even exceeding that of the positive control drug verapamil. Figure 10 (A–C). However, during treatment with the test dose, there was no significant change in body weight in each group of mice, indicating that compound 1 is safe for mice at the therapeutic dose. Figure 10 (Middle D). HE and TUNEL results of tumor tissue ( Figure 10 Studies E and F in the study showed that the combined use of compound 1 and paclitaxel led to increased tumor cell death (nuclear condensation, vacuolation in HE staining) and apoptosis (bright green in TUNEL staining). However, HE staining analysis of liver and kidney tissues indicated that the combined administration of compound 1 and paclitaxel did not increase hepatotoxicity or nephrotoxicity. Figure 10 (E).

[0088] In summary, this invention provides seven enantiocarbazine-type diterpenoid compounds. Compounds 1-6 are the first reported C-2 position-degraded enantiocarbazine diterpenoids. In bioactivity studies, compound 1 significantly reversed paclitaxel resistance in colon cancer cells, with a reversal fold of 204. Further structure-activity relationship analysis revealed that the four-membered lactone ring and the monomethyl succinate group are key pharmacophores. The presence of these two groups is essential for the compound to exhibit potent multidrug resistance reversal activity, while their absence leads to a significant reduction or complete loss of activity. These studies not only expand the diversity of enantiocarbazine diterpenoid structures but also provide important targets for finding new lead compounds to overcome paclitaxel resistance.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A diterpenoid compound, characterized in that, It has the structures shown in compounds 1 through 7: 。 2. A method for preparing the diterpenoid compound according to claim 1, characterized in that, Includes the following steps: Using fungi of the genus Helicobacter. Bipolaris sp. Ferment rice to obtain rice fermentation product; The rice fermentation product was extracted with ethanol to obtain an ethanol extract; The ethanol extract was extracted with ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract was separated to obtain the diterpenoid compound.

3. The preparation method according to claim 2, characterized in that, The fermentation process specifically involves mixing rice and water, and then inoculating the mixture with fungi of the genus *Helicobacter*. Bipolaris sp., then incubated and fermented at 28°C for 30 days.

4. The preparation method according to claim 2, characterized in that, The separation includes the following steps: (1) The ethyl acetate extract was subjected to normal phase silica gel column chromatography, and gradient elution was performed using a mixed solvent of petroleum ether, ethyl acetate and methanol with a volume ratio of 10:1:0–5:5:

2. Similar fractions were detected by thin-layer chromatography to obtain five fractions, Fr.1-Fr.

5. (2) The Fr.3 is passed through an inverting C 18 Column chromatography was performed using a gradient elution with a mixed solvent of methanol and water, wherein methanol comprised 30%-100% of the mixed solvent, yielding 11 fractions Fr.3.1-Fr.3.

11. Fr.3.8 was then subjected to normal-phase silica gel column chromatography using a gradient elution with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 15:1-0:1, yielding 13 fractions Fr.3.8.1-Fr.3.8.

13. Fr.3.8.11 was further subjected to normal-phase silica gel column chromatography using a gradient elution with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1-0:1, yielding 8 fractions Fr.3.8.11.1-Fr.3.8.11.

8. Fr.3.8.11.2 was purified by reversed-phase high-performance liquid chromatography to obtain compound 1. (3) The Fr.3.8.11.3 was purified by reversed-phase high-performance liquid chromatography to obtain compound 2; (4) The Fr.3.7 was subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 15:1-0:1 to obtain 13 fractions from Fr.3.7.1 to Fr.3.7.13; then, compound 4 and compound 6 were separated from Fr.3.7.1 by reversed-phase high-performance liquid chromatography. (5) The Fr.3.8.5 was purified by reversed-phase high-performance liquid chromatography to obtain compound 7; (6) Fr.4 was first subjected to reversed-phase column chromatography with gradient elution using a mixed solvent of methanol and water, wherein methanol accounted for 30%-100% of the volume of the mixed solvent, and 19 components, Fr.4.1-Fr.4.19, were obtained. Fr.4.17 was then subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of dichloromethane and methanol with a volume ratio of 100:1-0:1, and 8 components, Fr.4.17.1-Fr.4.17.8, were obtained. Fr.4.17.7 was subjected to normal-phase silica gel column chromatography with gradient elution using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 10:1-0:1, and 5 components, Fr.4.17.7.1-Fr.4.17.7.5, were obtained. Then Fr.4.17.7.3 was separated by reversed-phase high-performance liquid chromatography to obtain compound 3 and compound 5.

5. A drug for reversing paclitaxel resistance in colon cancer cells, characterized in that, Includes the diterpenoid compounds as described in claim 1.

6. The use of the diterpenoid compound of claim 1 in the preparation of a medicament for reversing paclitaxel resistance in colon cancer cells.

7. A P-gp inhibitor, characterized in that, Includes the diterpenoid compounds of claim 1.

8. The use of the diterpenoid compound of claim 1 in the preparation of a P-gp inhibitor drug.

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