Steroid-indole alkaloid hybrid dimer compounds, preparation method thereof and application thereof in preparing antitumor chemotherapy drug sensitizer

Abstract

The application discloses a steroid-indole alkaloid hybrid dimer compound, a preparation method thereof and application of the compound in preparation of an antitumor chemotherapy drug sensitizer, and first prepares the steroid-indole diketopiperazine alkaloid hybrid compound from a fungus Aspergillus sp. EGF15-0-3 rice culture medium. The compound is a new skeleton compound first discovered in nature. It is first discovered that the hybrid compound has Tdp1 inhibitory activity and can be applied as a tyrosine-DNA phosphodiesterase 1 (Tdp1) inhibitor. It is first discovered that the hybrid compound has chemotherapy sensitization activity on a tumor chemotherapy drug topotecan TPT, which provides a drug source molecule and data support for development of a new natural antitumor combination inhibitor, and has important significance for development and utilization of marine Aspergillus sp. fungus resources.

Description

Technical fields:

[0001] This invention relates to steroid-indole alkaloid hybrid dimers, their preparation methods, and their application in the preparation of sensitizers for antitumor chemotherapy drugs. Background technology:

[0002] Malignant tumors have always been a major threat to human health. Despite continuous progress and in-depth research into tumor treatment, current clinical treatment methods for malignant tumors primarily rely on chemotherapy drugs to induce DNA damage in tumor cells, thereby killing them. Topoisomerase 1 (Top1) inhibitors are widely used clinically as chemotherapy drugs for treating tumors such as lung cancer, colon cancer, and bladder cancer, including topotecan (TPT), irinotecan (IRT), and hydroxycamptothecin (HCPT). These drugs usually achieve significant efficacy in the early stages of treatment, but as treatment progresses, tumor cells easily develop resistance to these drugs, significantly reducing their therapeutic effect. Studies have shown that TPT treatment leads to an increase in the expression of the DNA repair enzyme tyrosine-DNA phosphodiesterase 1 (Tdp1), resulting in tolerance to the same dose of the drug after a period of treatment. Marine fungi, due to their high species diversity, have gradually become an important source of drug molecules. The search for highly effective, low-toxicity Tdp1 inhibitors from marine fungi that can synergize with chemotherapy drugs for tumors holds promise as an important strategy to overcome tumor drug resistance and provide more possibilities for cancer treatment. Summary of the Invention:

[0003] The purpose of this invention is to provide a steroid-indole alkaloid hybrid dimer compound, its preparation method, and its application in the preparation of sensitizers for antitumor chemotherapy drugs.

[0004] This invention is achieved through the following technical solutions:

[0005] The structural formula of the steroid-indole alkaloid heterodimer is as follows:

[0006]

[0007] The preparation method of the steroid-indole alkaloid heterodimer compound is as follows: it is prepared from rice culture medium of Aspergillus sp. EGF 15-0-3, a marine soft coral fungus with accession number GDMCC60476. The specific steps are as follows:

[0008] 1) Strain activation: Inoculate the strain onto a plate containing PDA medium and incubate at 28°C for 2-3 days. After single colonies grow on the plate, use an inoculation loop to pick a single colony and transfer it to an Erlenmeyer flask containing PDB medium. Incubate at 165 rpm for 2-3 days in a shaker at 28°C to obtain activated Aspergillus sp. EGF15-0-3 seed culture.

[0009] 2) Fermentation of the strain: The activated seed liquid was added to an Erlenmeyer flask containing rice culture medium and incubated at 28°C for 35 days. The strain was compared with the blank culture medium to observe whether there was obvious growth. Fermentation was stopped after growth was completed. The strain was inactivated with EtOAc, extracted by shaking on a shaker, and then concentrated under reduced pressure to obtain EtOAc phase extract.

[0010] 3) The EtOAc phase extract obtained in step 2) was subjected to silica gel column chromatography with V... PE :V EtOAc After gradient elution with a petroleum ether-ethyl acetate system of 50:1 to 1:1, the column was flushed with ethyl acetate and pure methanol. TLC thin-layer chromatography was performed, and combined with NMR and LC-MS analysis, yielding eight fractions Fr.1-Fr.8. Fr.6, based on NMR and LC-MS analysis, was identified as an enriched site for a steroidal indole-dione-piperazine hybrid. Further MCI column chromatography was then performed using V... 甲醇 :V 水 A crude separation was performed using a 40%-100% methanol-water system as the eluent to obtain eight sub-fractions: Fr.6-1-Fr.6-8. Fr.6-8 was then subjected to gel column chromatography with V... 甲醇 :V 二氯甲烷 A methanol-dichloromethane system with a ratio of 7:3 was used as the eluent, followed by silica gel column chromatography, with V... PE :V EtOAc Using a petroleum ether-ethyl acetate system of 50:1 to 10:1 as the eluent, Fr.6-8-1-Fr.6-8-10 was obtained. Among them, Fr.6-8-9 was repeatedly prepared by HPLC to obtain compounds 1-4.

[0011] PDA medium: 2.0% glucose, 200g / L potato juice, 10% agar. The potato juice was prepared using aged seawater as a solvent, with a salinity of 3.5% and a natural pH.

[0012] PDB is the culture medium for PDA without agar.

[0013] Rice culture medium: 100g rice, 0.3% peptone, 100mL saline seawater.

[0014] The inventors have, for the first time, prepared a heterozygous steroidal indole-diketopiramate alkaloid compound from rice culture medium containing the fungus Aspergillus sp. EGF15-0-3. Compounds 1-4 are novel skeletal compounds discovered in nature for the first time. They have also discovered for the first time that this type of hybrid compound possesses Tdp1 inhibitory activity. Therefore, this invention also protects the application of the said compounds as inhibitors of tyrosine-DNA phosphodiesterase 1 (Tdp1).

[0015] This invention is the first to discover the chemosensitizing activity of this type of hybrid compound against tumor chemotherapy drugs.

[0016] Therefore, this invention also protects the use of the compound in the preparation of sensitizers for antitumor chemotherapy drugs.

[0017] The tumors include human lung cancer, human breast cancer, and human cervical cancer.

[0018] Antitumor chemotherapy drugs include topotecan (TPT), irinotecan (IRT), and hydroxycamptothecin (HCPT).

[0019] The present invention also protects a composition comprising the steroid-indole alkaloid hybrid dimer compound and the tumor chemotherapy drug TPT.

[0020] The beneficial effects of this invention are as follows:

[0021] 1) A steroidal indole-dione-piperazine alkaloid hybrid compound was prepared for the first time from rice culture medium of the fungus Aspergillus sp. EGF15-0-3. This compound is a new skeletal compound discovered in nature for the first time.

[0022] 2) This type of hybrid was first discovered to have tyrosine-DNA phosphodiesterase 1 (Tdp1) inhibitory activity, and can be used as a Tdp1 inhibitor.

[0023] 3) This study first discovered the chemosensitizing activity of this type of hybrid compound against the tumor chemotherapy drug topotecan (TPT), providing drug source molecules and data support for the development of novel natural anti-tumor combination inhibitors, and is of great significance for the development and utilization of marine Aspergillus fungal resources.

[0024] The marine soft coral Aspergillus sp. EGF15-0-3, with accession number GDMCC 60476, has been disclosed in CN 111139185 B, Aspergillus fungi and their applications. Attached image description:

[0025] Figure 1 It is compound 1 1 H- 1 Schematic diagram of H COSY, key HMBC and NOE;

[0026] Figure 2 These are the single-crystal X-ray diffraction structures of compounds 1 and 2;

[0027] Figure 3 It is compound 2. 1 H- 1 Schematic diagram of H COSY, key HMBC and NOE;

[0028] Figure 4 It is compound 3. 1 H- 1 Schematic diagram of H COSY, key HMBC and NOE;

[0029] Figure 5 These are the single-crystal X-ray diffraction structures of compounds 3 and 4;

[0030] Figure 6 These are the measured and calculated ECD spectra of compounds 3 and 4;

[0031] Figure 7 It is compound 4. 1 H- 1 Schematic diagram of H COSY, key HMBC and NOE;

[0032] Figure 8 The Tdp1 enzyme inhibitory activities of compounds 1-4 are shown in (A), and the reproductive inhibition curves of compound 1 on different tumor cells are shown in (B).

[0033] Figure 9 This describes the antitumor effects of different concentrations of compound 1 combined with gradient concentrations of TPT. Among them, (A) cell survival curves of MCF-7, HeLa, and H460; (B) H460 cell effect synergy index; and (C) H460 cell colony formation. Detailed implementation method:

[0034] The following is a further description of the invention, but not a limitation thereof.

[0035] Example 1: Preparation and structural identification of compounds 1-4

[0036] Microbial origin: The strain EGF15-0-3 in the patent was isolated from soft corals collected in the waters of Sanya, South China Sea. It was identified as a fungus of the genus Aspergillus and deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 60476.

[0037] Culture medium formulation:

[0038] PDA medium: 2.0% glucose, 200g / L potato juice, 10% agar. The potato juice is prepared using aged seawater as a solvent, with a salinity of 3.5% and a natural pH. PDB is a medium for PDA without agar.

[0039] Rice culture medium: 100g rice, 0.3% peptone, 100mL saline seawater.

[0040] Strain activation: After thawing the inoculum tubes from the refrigerator, use an inoculation loop to take an appropriate amount of bacterial solution and inoculate it onto a plate containing PDA medium. A total of 5 plates are placed in a constant temperature incubator at 28℃ for 2-3 days. After single colonies grow on the plates, use an inoculation loop to pick a single colony and transfer it to a 1000mL Erlenmeyer flask containing 200mL of PDA medium. A total of 1000mL is activated. Incubate in a shaker at 28℃ for 165r / min for 2-3 days to obtain activated EGF15-0-3 seed culture.

[0041] Fermentation of the strain: 5.0 mL of the activated seed culture was added to a 1000 mL Erlenmeyer flask containing 100 g of rice culture medium, and a total fermentation volume of 900 L was achieved. The culture was incubated statically at 28 °C for 35 days, and the strain was observed to show obvious growth compared to a blank culture medium. Fermentation was stopped once growth was complete.

[0042] Post-treatment of secondary metabolites: After the strain had grown to maturity, it was directly inactivated with EtOAc (250 mL / 100 g rice medium) and then extracted three times by shaking on a shaker (165 r / min), each time for 8 hours. The EtOAc solutions were combined and concentrated under reduced pressure to obtain EtOAc extract (900.0 g).

[0043] Separation of steroidal indole alkaloid hybrid compounds: The crude extract of EtOAc fraction was subjected to silica gel column chromatography with V... PE :V EtOAc After gradient elution with a petroleum ether-ethyl acetate system of 50:1 to 1:1, the column was flushed with ethyl acetate and pure methanol. TLC thin-layer chromatography was performed, and combined with NMR and LC-MS analysis, yielding eight fractions Fr.1-Fr.8. Fr.6, based on NMR and LC-MS analysis, was identified as an enriched site for a steroidal indole-dione-piperazine hybrid. Further MCI column chromatography was then performed using V... 甲醇 :V 水 A crude separation was performed using a 40%-100% methanol-water system as the eluent to obtain eight sub-fractions (Fr. 6-1-Fr. 6-8). Fr. 6-8 was subjected to gel column chromatography followed by silica gel column chromatography to obtain Fr. 6-8-1-Fr. 6-8-10. The gel column chromatography was performed with V... 甲醇 :V 二氯甲烷 A methanol-dichloromethane system with a ratio of 7:3 was used as the eluent, and silica gel column chromatography was performed with V PE :VEtOAc A petroleum ether-ethyl acetate system with a ratio of 50:1 to 10:1 was used as the eluent. Fr.6-8-9 was repeatedly prepared by HPLC to obtain compounds 1-4, with the following structural formulas:

[0044]

[0045] Structural assessment:

[0046] Compound 1, a white-brown powder, has a quasi-molecular ion peak at m / z of 712.4463 [M+H] according to its high-resolution mass spectrometry (HRESIMS). + (The calculated value is C) 47 H 58 N3O3712.4478), indicating its molecular formula is C 47 H 57 N3O3 has 21 degrees of unsaturation in its molecule. Ultraviolet spectroscopy shows its maximum absorption wavelength λ. max =224, 293, and 336 nm. Infrared spectroscopy shows the presence of amino groups (3358, 3192, and 3050 cm⁻¹) in the structure. -1 ), carbonyl (1681 and 1635cm) -1 ) and benzene rings (1583, 1456 and 1373 cm) -1 ) group. Comprehensive analysis of the 1D NMR and HSQC (Table 1) of compound 1 revealed the presence of three amino proton signals [δ] in its structure. H [8.52(1H,s), 7.51(1H,s) and 6.10(1H,s)], an isolated ketone carbonyl signal (δ C =199.7), two amide carbonyl signals (δ C =168.2 and 159.4), a quaternary substitution double bond signal (δ C =154.0 and 125.4), 3 ternary substitution double bond signals [δ H 7.18(1H,m,overlapped), 5.76(1H,s) and 5.51(1H,s); δ C =164.3,135.1,124.7,123.4,122.5 and 122.4], a cis-binary substitution double bond signal [δ H 6.61(1H,d,J=9.6Hz) and 6.07(1H,m,overlapped); δ C =133.7 and 125.0], a terminal single or double bond signal [δ H 6.05(1H,m,overlapped) and 5.20(2H,m); δ C=144.4 and 113.4], a 2,3-disubstituted indole fragment [δ H 7.36(1H,d,J=8.0Hz), 7.23(1H,d,J=8.0Hz), 7.18(1H,t,J=8.0Hz) and 7.13(1H,t,J=8.0Hz); δ C =143.8,134.5,126.3,121.0,118.8,111.5,111.5 and 103.0], four quaternary carbons, five methines, eight methylenes and seven methyl signals. Based on the above information, it was found that compound 1 also contains the characteristic signal of indole-dikepiperazine (i.e. Figure 1 (1a). Furthermore, based on the key correlations observed in HMBC: H-1′ with C-3′ / C-5′, H-2′ with C-4′, H-4′ with C-6′, H-6′ with C-10′, H-7′ with C-9′, H-9′ with C-12′ / C-14′, H-11′ with C-8′, H-12′ with C-14′, H-15′ with C-8′ / C-17′, H-17′ with C-21′ / C-22′, H-18′ with C-12′ / C-17′, and H-19′ with C-1′ / C-9′, the remaining fragments (i.e., Figure 1 1b) is a known steroid compound (22E)-ergosta-4,6,8(14),22-tetraen-3-one [see J.Nat.Prod.2011,74,1281-1283.]. Based on the key correlations between H-20 and C-21′ / C23′ and H-23′ and C-12 / C13 / C20 / C20′ in HMBC, it is deduced that the indole-dione-piperazine fragment 1a and the steroid fragment 1b are linked by C-20-C-12′, C-12-C-9′ and N-11-C-7′ bonds. Figure 1 Thus, the planar structure of compound 1 is the first heterodimer compound of indole-dikepiperazine alkaloid and steroid skeleton.

[0047] The relative configurations of compound 1 were inferred from its NOESY spectra. For the steroid nucleus fragment (I-1), the NOESY spectra clearly show correlations between H3-19′ / H3-18′ and H-11′a, H3-18′ and H-16′a, H-17′ and H-16′b, and H-9′ and H-11′b, suggesting that H3-19′, H3-18′, H-11′a, and H-16′a are in the same plane of the molecule, while H-9′, H-16′b, and H-17′ are in another plane. This leads to the conclusion that the relative configurations of the steroid nucleus fragment are 9′R*, 10′R*, 13′R*, and 17′R*. For the indole-diketopiperazine fragment (II-1), another key set of correlations between H-23′ and H-20 suggests that H-23′ and NH-11 are in opposite planes of the molecule. Figure 1 The NOE correlation between 14-NH and H-4 indicates that Δ 8,9 The double bond configuration is also Z-configuration. Further single-crystal data confirmed the absolute configuration of compound 1 to be 12S,9′S,10′R,13′R,17′S,23′R,24′R. Figure 2 ).

[0048] Table 1. Compound 1 1 H (400MHz) and 13 C (100MHz) NMR data (δin ppm, J in Hz, solvent CDCl3) a

[0049]

[0050]

[0051] Compound 2, a pale yellowish-brown powder, has a quasi-molecular ion peak at m / z of 712.4406 [M+H] according to its high-resolution mass spectrometry (HRESIMS). + (The calculated value is C) 47 H 58 N3O3712.4478), indicating its molecular formula is C 47 H 57 N3O3 contains 21 degrees of unsaturation in its molecule. Based on a comprehensive analysis of the 1D and 2D NMR spectra of compound 2 (Table 2) and its molecular weight information, compound 2 and compound 1 have the same planar structure. The main difference between their spectra lies in C-12[δ] C =60.6 (compound 2), 62.1 (compound 1)], C-20 [δ C =29.5 (compound 2), 34.4 (compound 1)] and C-23′[δ C=49.9 (compound 2), 41.1 (compound 1)] The difference in signal intensity was observed, and the correlation between H-23′ and NH-11 / H-20 was clearly observed in the NOESY spectrum of compound 2. Figure 3 This suggests that compound 2 may be a diastereomer of the C-12 spirocyclic ring of compound 1. Similarly, we also determined the absolute configuration of compound 2 to be 12R,9′S,10′R,13′R,17′S,23′R,24′R using single-crystal data. Figure 2 ), is the epimer of the C-12 spirocyclic compound 1.

[0052] Table 2. Compound 2 1 H (400MHz) and 13 C (100MHz) NMR data (δin ppm, J in Hz, solvent CDCl3)

[0053]

[0054]

[0055] Compound 3, a white-brown powder, has a quasi-molecular ion peak at m / z of 712.4453 [M+H] according to its high-resolution mass spectrometry (HRESIMS). + (The calculated value is C) 47 H 58 N3O3712.4478), indicating its molecular formula is C 47 H 57 N3O3 contains 21 degrees of unsaturation in its molecule. Based on a comprehensive analysis of the 1D and 2D NMR spectra of compound 3 (Table 3) and its molecular weight information, compound 3 and compound 2 also have the same planar structure. The main difference between their spectra lies in C-20[δ] C =34.3 (compound 3), 29.5 (compound 2)], C-23′[δ C =43.5 (compound 3), 49.9 (compound 2)], C-24′[δ C =40.7 (compound 3), 39.4 (compound 2)] and C-25′[δ C =28.2 (compound 3), 33.3 (compound 2)] The difference in signals, and only the correlation between NH-11 and H-20 was observed in the NOESY spectrum of compound 3. Figure 4 This suggests that compound 3 may be the C-23′ epimer of compound 2. The relative configuration of compound 3, determined based on its single-crystal data, is 12R*,9′S*,10′R*,13′R*,17′S*,23′S*,24′R*. Figure 5Its absolute configuration was determined using quantum chemical ECD calculations as 12R,9′S,10′R,13′R,17′S,23′S,24′R. Figure 6 ), which is the C-23′ epimer of compound 2.

[0056] Table 3. Compound 3 1 H (400MHz) and 13 C (100MHz) NMR data (δin ppm, J in Hz, solvent CDCl3)

[0057]

[0058] Compound 4, a pale yellow powder, has a quasi-molecular ion peak at m / z of 712.4429 [M+H] according to its high-resolution mass spectrometry (HRESIMS). + (The calculated value is C) 47 H 58 N3O3712.4478), indicating its molecular formula is C 47 H 57 N3O3 contains 21 degrees of unsaturation in its molecule. Based on a comprehensive analysis of the 1D and 2D NMR spectra of compound 4 (Table 4) and its molecular weight information, compound 4 and compound 3 also have the same planar structure. The main difference between their spectra lies in C-20[δ] C =27.3 (compound 4), 34.3 (compound 3)] and C-23′[δ C =51.3 (compound 4), 43.5 (compound 3)] The difference in signals, and the correlation between NH-11 and H-23′ can be clearly observed in the NOESY spectrum of compound 4. Figure 7 This suggests that compound 4 may be a diastereomer of the C-12 spirocyclic ring of compound 3. Similarly, the relative configuration of compound 4 was determined based on its single-crystal data as 12S*, 9′S*, 10′R*, 13′R*, 17′S*, 23′S*, 24′R*. Figure 5 Its absolute configuration was determined using the same quantum chemical ECD calculation method as 12S,9′S,10′R,13′R,17′S,23′S,24′R. Figure 6 ), is the epimer of the C-12 spirocyclic compound 3.

[0059] Table 4. Compound 4 1 H (400MHz) and 13 C (100MHz) NMR data (δin ppm, J in Hz, solvent CDCl3)

[0060]

[0061] Physical constants and spectral data:

[0062] Compound 1: pale white powder, (c0.10,MeOH); UV(MeOH)λ max (logε)223(0.60),282(0.47),347(0.54)nm; IR(KBr)ν max 3358,3192,2958,2926,1670,1635,1583,1419,1219,1191,966,742cm -1 HRESIMS(ESI-TOF)m / z[M+H] + 712.4453(Calcd for C 47 H 58 N3O3712.4478); CD(MeOH):λ max (Δε)210(-22.61),256(8.49),318(-16.29); 1 H NMR and 13 The C NMR values ​​are shown in Table 1.

[0063] Compound 2: pale yellow powder, (c0.10,MeOH); UV(MeOH)λ max (logε)224(1.36),283(0.52),348(1.26)nm; IR(KBr)ν max 3325,3273,2960,2918,1681,1583,1456,1354,1261,1193,1099,1020,800,742cm -1 HRESIMS(ESI-TOF)m / z[M+H] + 712.4406 (Calcd for C) 47 H 58 N3O3712.4478); CD(MeOH):λ max (Δε)215(28.69),262(-12.26),364(38.60); 1 HNMR and 13 The C NMR values ​​are shown in Table 2.

[0064] Compound 3: pale white powder, (c0.10,MeOH); UV(MeOH)λ max (logε)222(1.05),282(0.38),348(0.66)nm; IR(KBr)νmax 3327,3215,2960,2914,1670,1668,1583,1417,1219,871,738cm -1 HRESIMS(ESI-TOF)m / z[M+H] + 712.4432 (Calcd for C) 47 H 58 N3O3); CD(MeOH):λ max (Δε)242(18.33),317(-9.83); 1 H NMR and 13 The C NMR values ​​are shown in Table 3.

[0065] Compound 4: pale yellow powder, (c0.10,MeOH); UV(MeOH)λ max (logε)224(1.33),284(0.52),346(1.20)nm; IR(KBr)ν max 3334,3199,2960,2924,1670,1662,1583,1417,1219,964,742cm -1 HRESIMS(ESI-TOF)m / z[M+H] + 712.4429(Calcd for C 47 H 58 N3O3712.4478); CD(MeOH):λ max (Δε)223(4.83),284(-3.02),364(12.58); 1 H NMR and 13 CNMR is shown in Table 4.

[0066] Example 2: Inhibitory activity of compounds 1-4 against tyrosine-DNA phosphodiesterase 1 (Tdp1)

[0067] The Tdp1 enzyme inhibitory activity of steroidal indole-diketopiramate hybrid compounds (compounds 1-4) was evaluated using a fluorescence quantitative detection method. The results showed (see...) Figure 8 Steroidal indole-diketopiramate hybrid compounds exhibited varying degrees of inhibitory activity against Tdp1 enzyme, with compound 1 showing the best activity (IC50). 50 =5.04±0.48μM).

[0068] Example 3: Application of compounds 1-4 in the preparation of sensitizers for the antitumor chemotherapy drug topotecan (TPT)

[0069] Dosage alone

[0070] The inhibitory effect of compound 1 on the proliferation of human lung cancer cells H460, human breast cancer cells MCF-7, and human cervical cancer cells HeLa was evaluated using the CCK-8 assay. The study found that compound 1 had weak cytotoxicity against these three cell lines, and no significant cytotoxicity was observed at a concentration of 100 μM. Figure 9 For studying the synergistic lethal antitumor effects of two drugs, combining them with compounds with weak cytotoxic activity can achieve chemotherapy sensitization at low doses without increasing drug toxicity, thus having certain advantages in the development of antitumor sensitizers.

[0071] Combined administration

[0072] To further evaluate whether compound 1 can serve as a sensitizer for clinical antitumor drugs, we selected the Top1 inhibitor topotecan (TPT) as a combination drug to detect its synergistic antitumor effect in the three cell types mentioned above. The results showed (see...). Figure 9 In combination with compound 1 (A and B), TPT showed weak killing ability against the lung cancer cell line H460; however, when TPT was used in combination with compound 1, it selectively enhanced the sensitivity of H460 cells to TPT, indicating a significant synergistic effect. Studies have shown that with prolonged administration of Top1 inhibitors, the expression level of Tdp1 gradually increases, thereby promoting drug resistance in tumor cells, which is a self-protective mechanism of tumor cells. To further investigate the long-term (10-day) anti-proliferative ability of the active compound against tumor cells, we used a colony formation assay to evaluate the synergistic effect of active compound 1 on TPT. Figure 9 As shown in Figure C, at non-toxic concentrations, the combined administration of compound 1 and TPT significantly reduced the formation of H460 cell clones in a concentration-dependent manner.

Claims

1. A hybrid dimeric compound of a steroid-indole alkaloid characterized in that, The structural formula is as follows:

2. The method for preparing the steroid-indole alkaloid hybrid dimeric compound of claim 1, characterized in that, The rice medium was prepared from the marine soft coral Aspergillus sp. EGF 15-0-3 of the preservation number GDMCC 60476, and the specific steps are as follows: 1) The strain was inoculated on a plate containing PDA medium, and placed in a constant temperature incubator at 28℃ for 2-3 days. After single colonies grew on the plate, a single colony was picked up with an inoculation ring and transferred to a triangular flask containing PDB medium, and cultured in a constant temperature shaking flask cabinet at 28℃ at 165r / min for 2-3 days to obtain the activated seed culture solution of Aspergillus sp. EGF 15-0-3; PDA medium: glucose 2.0%, potato juice 200g / L, agar 10%, potato juice prepared with old seawater as solvent, salinity 3.5%, pH natural; PDB is a medium without agar in PDA; 2) The activated seed solution was taken and added to a triangular flask containing rice medium, and cultured at 28℃ for 35 days. After inactivation with EtOAc, the EtOAc phase extract was obtained by shaking extraction and reduced pressure concentration; 3) The EtOAc phase extract obtained from step 2) was subjected to silica gel column chromatography, eluting with petroleum ether-ethyl acetate system in gradient of V PE :V EtOAc = 50:1 to 1:1, followed by elution with ethyl acetate and pure methanol system, TLC tracking, combined with NMR and LC-MS analysis to obtain 8 fractions Fr.1-Fr.

8. Fr.6 was enriched with steroidal indole dione piperazine hybrid compounds according to NMR and LC-MS analysis, and was selected for further MCI column chromatography, eluting with V 甲醇 :V 水 = 40% to 100% methanol-water system as eluent for crude separation to obtain 8 sub-fractions: Fr.6-1-Fr.6-8. Fr.6-8 was subjected to gel column chromatography, eluting with V 甲醇 :V 二氯甲烷 = 7:3 methanol-dichloromethane system as eluent, followed by silica gel column chromatography, eluting with V PE :V EtOAc = 50:1 to 10:1 petroleum ether-ethyl acetate system as eluent to obtain Fr.6-8-1-Fr.6-8-10. Fr.6-8-9 was subjected to repeated HPLC liquid phase preparation to obtain compounds 1-4.

3. The method of claim 2, wherein, Rice medium: rice 100g, peptone 0.3%, salt seawater 100mL.

4. The use of the steroid-indole alkaloid hybrid dimer compound of claim 1 in the preparation of a tyrosine-DNA phosphodiesterase 1 inhibitor.

5. The use of the steroid-indole alkaloid hybrid dimer compound of claim 1 in the preparation of an antitumor chemotherapy drug sensitizer.

6. Use according to claim 5, characterized in that, The tumor includes human lung cancer, human breast cancer and human cervical cancer.

7. Use according to claim 5, characterized in that, The antitumor chemotherapy drug is topotecan, irinotecan and hydroxycamptothecine.

8. A composition comprising the steroid-indole alkaloid hybrid dimer compound of claim 1 and the tumor chemotherapy drug topotecan.

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