6,6'-diisoprenylated indigorubin and its preparation method and application

Abstract

The present invention discloses 6,6′-diisopentenyl indirubin and a preparation method and application thereof. The structure of 6,6′-diisopentenyl indirubin is shown in Formula I: The 6,6′-diisopentenyl indirubin of the present invention is obtained by microbial fermentation, and has mild preparation conditions and simple operation. Experiments have shown that 6,6′-diisopentenyl indirubin has excellent anti-tumor activity, and has better anti-tumor effect than indirubin.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to 6,6'-diisopentenyl indirubin and a preparation method thereof and application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Indirubin is a bisindole alkaloid and an isomer of indigo, which can be isolated from the traditional Chinese medicine Indigo. Indigo is mainly used in the treatment of chronic myeloid leukemia in clinical practice. It has the characteristics of reliable clinical efficacy, small side effects, and no obvious inhibitory effect on the bone marrow. However, the main component of Indigo prepared by traditional methods is indigo, and the content of indirubin in Indigo is low. In addition, indigo has low water solubility and poor pharmacokinetic properties for cancer treatment, which greatly limits its clinical application. Therefore, many researchers continue to explore ways to obtain indirubin from other sources or develop new indirubin derivatives with better pharmacokinetic properties and improved pharmacological characteristics. It is necessary to quickly obtain indirubin and obtain indirubin derivatives with better pharmacological effects.

[0003] Actinomycetes are an important treasure trove for natural product discovery. About 70% of the antibiotics reported so far are produced by actinomycetes. Saccharopolyspora belongs to the order Actinomycetales and the genus Pseudonocardia, and has the potential to produce antibiotics and drug lead compounds. For example, the broad-spectrum antibiotic erythromycin was isolated from Saccharopolyspora rubrum and the biological pesticide spinosad was isolated from Saccharopolyspora spinosa, which reflects the ability of Saccharopolyspora to produce effective bioactive compounds and also provides a reference for the exploration of secondary metabolites in other Saccharopolyspora.

[0004] Through the prediction and analysis of secondary metabolites, we found that Shandong Saccharopolyspora has a gene cluster related to indole metabolism, which is a fragment of indigo carmine production, and may promote the production of indigo carmine or its derivatives. Based on this, there is great potential for mining secondary metabolites such as indigo carmine or indigo carmine derivatives in Shandong Saccharopolyspora. Summary of the invention

[0005] The object of the present invention is to provide 6,6'-diisopentenyl indirubin.

[0006] The second object of the present invention is to provide a method for preparing 6,6'-diisopentenyl indirubin.

[0007] The third object of the present invention is to provide the use of 6,6'-diisopentenyl indirubin in the preparation of anti-tumor drugs.

[0008] The technical solution of the present invention is summarized as follows:

[0009] 6,6′-diisopentenyl indigorubin, as shown in formula I:

[0010]

[0011] The preparation method of the above 6,6′-diisopentenyl indigorubin comprises the following steps:

[0012] (1) inoculating Saccharopolyspora shandongensis CGMCC4.3530 cultured on a solid culture medium into a seed culture medium, performing shake flask culture to obtain a seed culture, inoculating the seed culture into a fermentation medium for culture, and collecting the fermentation liquid;

[0013] (2) The fermentation liquid obtained in step (1) is centrifuged to obtain a precipitate, and the precipitate is extracted with solvent A to obtain an extract, the extract is evaporated to dryness, extracted with water and solvent B, the solvent B layer is evaporated to dryness to obtain a crude extract, loaded with 200-300 mesh silica gel column, the crude extract and 100-200 mesh silica gel are mixed in a mass ratio of 1:1.5-3, dry loading, and the crude extract is subjected to silica gel column chromatography separation using solvent C as the mobile phase, the purple fraction is collected, concentrated and dried, and the fraction is re-dissolved with solvent D, added to a gel column, separated using solvent D as the mobile phase, the purple fraction is collected, and dried to obtain pure 6,6′-diisopentenyl indigorubin.

[0014] Step (1) is preferably: inoculating Saccharopolyspora shandongensis CGMCC4.3530 cultured on ISP4 solid culture medium into a seed culture medium, culturing in a shake flask at 28-30° C. and 180-240 rpm for 36-70 h to obtain a seed culture, inoculating the seed culture into a fermentation culture medium, culturing in a shake flask at 28-30° C. and 180-240 rpm for 7-10 days, and collecting the fermentation broth.

[0015] The fermentation medium is preferably: soluble starch 18-22 g / L, glucose 8-12 g / L, peptone 3-7 g / L, yeast extract 3-7 g / L, NaCl 3-7 g / L, K2HPO4 0.3-0.7 g / L, MgSO4·7H2O 0.3-0.7 g / L, CaCO3 1-3 g / L, HP20 macroporous adsorption resin 10-40 g / L, add sterile water to 1 L, and adjust the pH to 7.0-7.4.

[0016] Solvent A is preferably at least one of methanol, ethyl acetate, dichloromethane and petroleum ether.

[0017] Solvent B is preferably ethyl acetate or petroleum ether.

[0018] Solvent C is preferably a mixture of petroleum ether and ethyl acetate, or a mixture of petroleum ether and dichloromethane.

[0019] Solvent D is preferably methanol or a mixture of methanol and dichloromethane.

[0020] The application of the above 6,6'-diisopentenyl indirubin I in the preparation of anti-tumor drugs.

[0021] Advantages of the present invention:

[0022] The 6,6′-diisopentenyl indirubin of the present invention is obtained by microbial fermentation, and the preparation conditions are mild and the operation is simple. Experiments have shown that 6,6′-diisopentenyl indirubin has excellent anti-tumor activity, and has a good in vitro inhibitory effect on the proliferation of leukemia cell line K562, breast cancer cell MDA-MB-231, non-small cell lung cancer A549, and oral epidermoid cancer cell KB. 50 The values ​​reached 6.01±0.15μΜ, 8.53±0.37μΜ, 11.73±0.42μΜ, and 8.64±0.32μΜ, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the structure of indigo red II.

[0024] Figure 2 This is the high-resolution mass spectrum of 6,6′-diisopentenyl indigorubin.

[0025] Figure 3 This is the hydrogen spectrum of 6,6′-diisopentenyl indigorubin.

[0026] Figure 4 This is the carbon spectrum of 6,6′-diisopentenyl indigorubin.

[0027] Figure 5 This is the DEPT spectrum of 6,6′-diisopentenyl indigorubin.

[0028] Figure 6 This is the HMBC spectrum of 6,6′-diisopentenyl indigorubin.

[0029] Figure 7 This is the HSQC spectrum of 6,6′-diisopentenyl indigorubin.

[0030] Figure 8 This is the COSY spectrum of 6,6′-diisopentenyl indigorubin.

[0031] Fig. 9 The figure is a graph showing the anti-tumor effect of 6,6′-diisoprenylated indigorubin on leukemia cell line K562, wherein DYH is the compound indigorubin and Compound 1 is the compound 6,6′-diisoprenylated indigorubin.

[0032] Fig.10The figure shows the anti-tumor effect of 6,6′-diisopentenyl indirubin on breast cancer cells MDA-MB-231. Among them, DYH is the compound indirubin, and Compound 1 is the compound 6,6′-diisopentenyl indirubin.

[0033] Fig.11 The figure is a graph showing the anti-tumor effect of 6,6′-diisopentenyl indirubin on non-small cell lung cancer A549. Among them, DYH is the compound indirubin, and Compound 1 is the compound 6,6′-diisopentenyl indirubin.

[0034] Fig.12 The figure is a graph showing the anti-tumor effect of 6,6′-diisopentenyl indigorubin on oral epidermoid carcinoma cell KB. Among them, DYH is the compound indigorubin, and Compound 1 is the compound 6,6′-diisopentenyl indigorubin. DETAILED DESCRIPTION

[0035] The Shandong Saccharopolyspora CGMCC4.3530 used in the present invention was purchased from

[0036] China General Microbiology Center (CGMCC), website: https: / / cgmcc.net

[0037] The present invention is described in detail below by specific examples. The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and belong to descriptive or explanatory words, rather than restrictive words, and the present invention is not limited by the following examples. Equivalent replacements or corresponding improvements made to the content of the present invention still belong to the protection scope of the present invention.

[0038] Example 1

[0039] ISP4 solid culture medium: soluble starch medium 17g / L, agar 20g / L, add sterile water to 1L.

[0040] Example 2

[0041] Seed culture medium: glucose 20 g / L, yeast extract 15 g / L, malt extract 10 g / L, MgSO4·7H2O 0.244 g / L, FeSO4 0.3 g / L, add sterile water to 1 L, and adjust the pH to 7.2.

[0042] Example 3

[0043] Fermentation medium: soluble starch 20 g / L, glucose 10 g / L, peptone 5 g / L, yeast extract 5 g / L, NaCl 5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 2 g / L, HP20 macroporous adsorption resin 30 g / L, add sterile water to 1 L, and adjust the pH to 7.2.

[0044] Example 4

[0045] Fermentation medium: soluble starch 18 g / L, glucose 12 g / L, peptone 3 g / L, yeast extract 7 g / L, NaCl 3 g / L, K2HPO4 0.7 g / L, MgSO4·7H2O 0.3 g / L, CaCO3 3 g / L, HP20 macroporous adsorption resin 10 g / L, add sterile water to 1 L, and adjust the pH to 7.0.

[0046] Example 5

[0047] Fermentation medium: soluble starch 22 g / L, glucose 8 g / L, peptone 7 g / L, yeast extract 3 g / L, NaCl 7 g / L, K2HPO4 0.3 g / L, MgSO4·7H2O 0.7 g / L, CaCO3 1 g / L, HP20 macroporous adsorption resin 40 g / L, add sterile water to 1 L, and adjust the pH to 7.4.

[0048] Example 6

[0049] The preparation method of 6,6′-diisopentenyl indigorubin comprises the following steps:

[0050] (1) Saccharopolyspora shandongensis CGMCC4.3530 cultured on the ISP4 solid medium prepared in Example 1 was inoculated into the seed medium prepared in Example 2, and cultured in a shaking flask at 29° C. and 220 rpm for 54 h to obtain a seed culture, and the seed culture was inoculated into the fermentation medium prepared in Example 3 and cultured in a shaking flask at 29° C. and 220 rpm for 9 days, and the fermentation broth was collected;

[0051] (2) 10 L of the fermentation broth obtained in step (1) was centrifuged to obtain a precipitate, and extracted with 500 mL of methanol to obtain an extract. The extract was extracted multiple times until the extract had no obvious color. The extracts were combined and dried by rotary evaporation. 500 mL of water and 500 mL of ethyl acetate were extracted multiple times until the extract had no obvious color. The ethyl acetate layer was taken and dried by rotary evaporation to obtain a crude extract. 200-300 mesh silica gel was used to load the column. The crude extract was mixed with 100-200 mesh silica gel in a mass ratio of 1:1.5, and the sample was loaded by dry method. The crude extract was subjected to silica gel column chromatography analysis using a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the mobile phase, and the purple fraction was collected. The concentrated and dried fraction was reconstituted with methanol, and added to a gel column (LH-20). Methanol was used as the mobile phase for separation. The purple fraction was collected and dried by rotary evaporation to obtain pure 6,6′-diisopentenyl indigo red.

[0052] Example 7

[0053] The preparation method of 6,6′-diisopentenyl indigorubin comprises the following steps:

[0054] (1) Saccharopolyspora shandongensis CGMCC4.3530 cultured on the ISP4 solid medium prepared in Example 1 was inoculated into the seed medium prepared in Example 2, and cultured in a shaking flask at 28° C. and 180 rpm for 70 h to obtain a seed culture. The seed culture was inoculated into the fermentation medium prepared in Example 4 and cultured in a shaking flask at 28° C. and 180 rpm for 10 days, and the fermentation broth was collected.

[0055] (2) 10 L of the fermentation broth obtained in step (1) was centrifuged to obtain a precipitate, and extracted with 500 mL of a mixed solvent of methanol and ethyl acetate to obtain an extract. The extracts were extracted multiple times until the extract had no obvious color. The extracts were combined and dried by rotary evaporation. 500 mL of water and 500 mL of petroleum ether were extracted multiple times until the extract had no obvious color. The petroleum ether layer was taken and dried by rotary evaporation to obtain a crude extract, which was loaded on a 200-300 mesh silica gel column. The crude extract was mixed with 100-200 mesh silica gel in a mass ratio of 1:2, and the sample was loaded by dry method. The crude extract was subjected to silica gel column chromatography analysis using a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the mobile phase, and the purple fraction was collected. The fraction was concentrated and dried, and the fraction was reconstituted with a mixed solvent of methanol and dichloromethane in a volume ratio of 1:1, and a gel column (LH-20) was added. The fraction was separated using a mixed solvent of methanol and dichloromethane in a volume ratio of 1:1 as the mobile phase. The purple fraction was collected and dried by rotary evaporation to obtain pure 6,6′-diisopentenyl indigo red.

[0056] Example 8

[0057] The preparation method of 6,6′-diisopentenyl indigorubin comprises the following steps:

[0058] (1) Saccharopolyspora shandongensis CGMCC4.3530 cultured on the ISP4 solid medium prepared in Example 1 was inoculated into the seed medium prepared in Example 2, and cultured in a shaking flask at 30° C. and 240 rpm for 36 h to obtain a seed culture. The seed culture was inoculated into the fermentation medium prepared in Example 5 and cultured in a shaking flask at 30° C. and 240 rpm for 7 days, and the fermentation broth was collected.

[0059] (2) 10 L of the fermentation broth obtained in step (1) was centrifuged to obtain a precipitate, and extracted with 500 mL of a mixed solvent of petroleum ether and dichloromethane in a volume ratio of 1:1 to obtain an extract, and extracted multiple times until the extract had no obvious color, and the combined extracts were rotary evaporated and dried, and extracted with 500 mL of water and 500 mL of ethyl acetate, and extracted multiple times until the extract had no obvious color, and the ethyl acetate layer was rotary evaporated and dried to obtain a crude extract, and the crude extract was mixed with 100-200 mesh silica gel in a mass ratio of 1:3 using a 200-300 mesh silica gel column, and the sample was dry loaded, and the crude extract was subjected to silica gel column chromatography analysis using a mixed solution of petroleum ether and dichloromethane in a volume ratio of 1:3 as the mobile phase, and the purple fraction was collected, concentrated and dried, and reconstituted with methanol, and added to a gel column (LH-20), and separated with methanol as the mobile phase, and the purple fraction was collected, and rotary evaporated and dried to obtain pure 6,6′-diisopentenyl indigorubin.

[0060] Example 9

[0061] Structural Identification of 6,6′-Diisoprenylindigorubin Prepared in Example 6

[0062] The structures of indirubin derivatives were obtained by mass spectrometry and nuclear magnetic resonance (NMR). Figure 1-Figure 9 , the physical and chemical data are as follows:

[0063] Reddish purple solid, [α] D 25 0 (c = 0.0005, MeOH), the product was further analyzed using LC-MS-IT-TOF, and the ion peak of the compound [M+H] was detected in MS + m / z = 399.2025 (calculated value C 26 H 26 N2O2, 398.1994), and its molecular formula was determined to be C 26 H 26 N2O2.

[0064] 1 H NMR (500 MH Z,CDCl3),δ10.30(s,1H),8.76(d,J=8.0Hz,1H),7.97(s,1H),7.62(d,J=7.8Hz,1H),6.92(d,J=8.0Hz,1H),6.8 2(d,J=7.8Hz,1H),6.80(s,1H),6.73(s,1H),5.31(m,2H),3.36(m,4H),1.78(s,3H),1.76(s,3H),1.73(s,6H)。

[0065] 13 C NMR(125MH Z ,CDCl3),δ C 139.74(C-2), d C 187.77(C-3), d C 118.35(C-3a), d C 125.28(C-4), d C 122.27(C-5), d C 152.77(C-6), d C 111.66(C-7), d C 152.36(C-7a), d C 35.31(C-8), d C 122.74(C-9), d C 134.35(C-10), d C 25.91(C-11), d C 18.07(C-12), d C 172.65(C-2′), δ C 106.76(C-3′), δ C 119.95(C-3a′), δ C 125.93(C-4′), δ C 122.91(C-5′), δ C 144.08(C-6′), δ C 109.62(C-7′), δ C 139.57(C-7a′), δ C 34.93(C-8′), δ C 121.57(C-9′), δ C 133.27(C-10′), δ C 25.90(C-11′), δ C 18.03(C-12′).

[0066] According to the displacement of hydrogen (δ Ph-H =6~9) and COSY spectrum signals, it can be inferred that 4-H, 5-H and 7-H are hydrogens on the same benzene ring, and similarly 4′-H, 5′-H and 7′-H are hydrogens on the same benzene ring; since 1-NH in HMBC is related to 2-C, 3-C, 3a-C, 7a-C, 4-H is related to 3-C and 6-C, 5-H is related to 3a-C and 7-C, and 7-H is related to 5-C, it can be preliminarily inferred that the parent nucleus of the compound is an indigo red structure; according to the split peaks of 4-H, 5-H and 7-H, it can be inferred that the substituent is substituted at the 6-position. Finally, the compound was determined to be indigo red substituted by isopentenyl at the 6-position and 6′-position.

[0067] Example 10

[0068] Anticancer Effects of 6,6′-Diisopentenyl Indigorubin

[0069] The CCK8 method was used to study the compounds 6,6′-diisoprenylindigorubin and indirubin ( Figure 1 )'s antitumor activity:

[0070] Inoculating cells: Use cell culture medium containing 10wt% fetal bovine serum (the cell culture medium is the leukemia cell line K562 cultured in RMPI 1640; and the cell culture medium of breast cancer cells MDA-MB-231, non-small cell lung cancer A549, and oral epidermoid carcinoma cell KB cultured in DMEM) to make a cell suspension with a density of 30,000 cells / mL. Then, the cells are inoculated into a 96-well plate with a volume of 100 μL per well. The cells are then cultured for 24 hours.

[0071] Add the test compounds 6,6′-diisoprenylindigrubin and indirubin respectively: dissolve the compound 6,6′-diisoprenylindigrubin or indirubin with DMSO to prepare the compound into a mother solution concentration of 10 mM. Then, use cell culture medium (DMEM or RMPI 1640) containing 10wt% fetal bovine serum to dilute the mother solution at different times to prepare a culture medium with a final concentration of 10 times.

[0072] The prepared culture medium containing different concentrations of 6,6′-diisopentenyl indigo carmine or indigo carmine was added to the 96-well plate containing cells, with 10 μL added to each well, and 6 replicate wells were set for each treatment. The culture was then continued for 48 hours.

[0073] Color development: After culturing at 37°C for 48 hours, pour out the culture medium in the wells of adherent cells, and then add 10 μL of CCK8 solution and 100 μL of culture medium to each well; add 10 μL of CCK8 solution to each well of suspended cells; set up 4 blank replicate wells and incubate for 4 hours.

[0074] Colorimetry: After sufficient color development, select 450nm wavelength, read the light absorption value of each well on a multifunctional microplate reader, record the results, and after calculation, obtain the survival rate of cells in 6,6′-diisopentenyl indigorubin or indigorubin at different concentrations.

[0075] The cell survival rates of leukemia cell line K562, breast cancer cell line MDA-MB-231, non-small cell lung cancer cell line A549, and oral epidermoid carcinoma cell line KB in different concentrations of 6,6′-diisopentenyl indirubin and indirubin are shown in the figure. Fig. 9 , Fig.10 , Fig.11 , Fig.12 As shown, IC 50 The value results are shown in Table 1:

[0076] Table 1 IC values ​​of 6,6′-diisopentenyl indirubin against different tumor cell lines 50 value

[0077]

Claims

1. 6,6′-diisoprenylated indigorubin, characterized by As shown in formula (I):

2. The method for preparing 6,6′-diisopentenyl indigorubin according to claim 1, characterized in that The following steps are involved: (1) inoculating Saccharopolyspora shandongensis CGMCC4.3530 cultured on a solid culture medium into a seed culture medium, performing shake flask culture to obtain a seed culture, inoculating the seed culture into a fermentation medium for culture, and collecting the fermentation liquid; (2) The fermentation liquid obtained in step (1) is centrifuged to obtain a precipitate, and the precipitate is extracted with solvent A to obtain an extract, the extract is evaporated to dryness, extracted with water and solvent B, the solvent B layer is evaporated to dryness to obtain a crude extract, loaded with 200-300 mesh silica gel column, the crude extract and 100-200 mesh silica gel are mixed in a mass ratio of 1:1.5-3, dry loading, and the crude extract is subjected to silica gel column chromatography separation using solvent C as the mobile phase, the purple fraction is collected, concentrated and dried, and the fraction is re-dissolved with solvent D, added to a gel column, separated using solvent D as the mobile phase, the purple fraction is collected, and dried to obtain pure 6,6′-diisopentenyl indigorubin.

3. The method according to claim 2, characterized in that The step (1) comprises: inoculating Saccharopolyspora shandongensis CGMCC4.3530 cultured on ISP4 solid culture medium into a seed culture medium, culturing in a shaking flask at 28-30° C. and 180-240 rpm for 36-70 hours to obtain a seed culture, inoculating the seed culture into a fermentation culture medium, culturing in a shaking flask at 28-30° C. and 180-240 rpm for 7-10 days, and collecting the fermentation liquid.

4. The method according to claim 2 or 3, characterized in that The fermentation medium comprises: 18-22 g / L soluble starch, 8-12 g / L glucose, 3-7 g / L peptone, 3-7 g / L yeast extract, 3-7 g / L NaCl, 0.3-0.7 g / L K2HPO4, 0.3-0.7 g / L MgSO4·7H2O, 1-3 g / L CaCO3, 10-40 g / L HP20 macroporous adsorption resin, sterile water is added to 1 L, and the pH value is adjusted to 7.0-7.

4.

5. The method according to claim 2, characterized in that The solvent A is at least one of methanol, ethyl acetate, dichloromethane and petroleum ether.

6. The method according to claim 2, characterized in that The solvent B is ethyl acetate or petroleum ether.

7. The method according to claim 2, characterized in that The solvent C is a mixture of petroleum ether and ethyl acetate, or a mixture of petroleum ether and dichloromethane.

8. The method according to claim 2, characterized in that The solvent D is methanol or a mixture of methanol and dichloromethane.

9. Use of the 6,6'-diisopentenyl indirubin according to claim 1 in the preparation of anti-tumor drugs.

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