Process for the preparation of indolinedione piperazine alkaloids and uses thereof

Abstract

This invention discloses a method for preparing indole-diketopiramate alkaloids and their uses. Specifically, this invention discloses the use of indole-diketopiramate alkaloids 12R,13S-Dihydroxyfumitremorgin C and / or tryprostatin A (as shown in Formula (I)) in the preparation of LXRα agonists, and the use of indole-diketopiramate alkaloids 12R,13S-Dihydroxyfumitremorgin C and / or tryprostatin A (as shown in Formula (I)) in the preparation of drugs for the prevention and / or treatment of diseases regulated by LXRα agonists. This invention provides new candidate compounds for the development of drugs for the prevention and treatment of hypercholesterolemia and is of great significance for the development of marine-derived drugs in China.

Description

Technical Field

[0001] This invention belongs to the field of natural products, specifically relating to the preparation method and uses of indole-diketopiramate alkaloids. Background Technology

[0002] Hypercholesterolemia is a metabolic disorder characterized by abnormally elevated levels of cholesterol, particularly low-density lipoprotein cholesterol (LDL-C), in the blood. Elevated LDL-C is an independent risk factor for atherosclerosis, leading to coronary heart disease and heart failure, seriously endangering human health. Since atherosclerosis is the pathological basis of cardiovascular disease, lipid-lowering therapy is of great significance in preventing and treating coronary heart disease and other cardiovascular diseases. Studies have confirmed that lowering plasma total cholesterol (TC) can effectively prevent coronary heart disease. Furthermore, lipid-lowering therapy is also an important clinical measure for preventing and treating cerebral infarction.

[0003] LXRs are members of the nuclear receptor superfamily of ligand-activated transcription factors, and exist in two subtypes: LXRα and LXRβ. As cholesterol sensors in the human body, their activation mobilizes cholesterol from the periphery, promotes its excretion by the liver and restricts its absorption, reduces its uptake by cells, and enhances its conversion to bile acids (in mouse liver), thereby achieving net cholesterol removal from the body and improving plasma lipoprotein profiles.

[0004] Since its first cloning in the 1990s, the liver X receptor (LXR) has been studied for approximately 30 years and has shown great potential for treating metabolic diseases such as atherosclerosis, hypercholesterolemia, and NAFLD. Furthermore, numerous clinical trials are underway for conditions such as diarrhea, cancer, atherosclerosis, and atopic dermatitis. LXR activation can maintain lipid homeostasis, improve atherosclerosis, modulate immunity, and possess anti-inflammatory activity.

[0005] Several natural compounds have been found to alleviate hypercholesterolemia by targeting LXRα or through the LXRα pathway, showing promising therapeutic potential. Compared to compounds, natural products have more complex and diverse structures and biological activities; therefore, many natural products have fewer side effects when exerting therapeutic effects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing indole-diketopiperazine alkaloids and their uses. This invention obtains two indole-diketopiperazine alkaloids, 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A, isolated from rice fermentation cultures of the sponge-associated epiphytic fungus Aspergillus sp. SCSIO 41420, by screening LXRα ligands derived from marine microbial natural products.

[0007] The first aspect of this invention provides the use of the indole-dione-piperazine alkaloids 12R,13S-Dihydroxyfumitremorgin C and / or tryprostatin A, as shown in formula (I), in the preparation of LXRα agonists.

[0008]

[0009] Furthermore, the indole-dikepiperazine alkaloid shown in formula (I) enhances the expression levels of the ABCA1 and CYP7A1 genes.

[0010] A second aspect of the present invention provides the use of the indole-dikepiperazine alkaloids 12R,13S-Dihydroxyfumitremorgin C and / or tryprostatin A of formula (I) in the preparation of medicaments for the prevention and / or treatment of diseases regulated by LXRα agonists.

[0011] Furthermore, the diseases regulated by LXRα agonists include hypercholesterolemia and its induced diseases, hypertriglyceridemia and its induced diseases, and mixed hyperlipidemia and its induced diseases.

[0012] Furthermore, the induced diseases include non-alcoholic fatty liver disease, atherosclerosis, and cardiovascular disease.

[0013] Furthermore, the cardiovascular diseases mentioned include the prevention and treatment of coronary heart disease, stroke, myocardial infarction, and ischemic kidney disease.

[0014] The third aspect of this invention provides a method for preparing the indole-dionepiperazine alkaloids 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A as shown in formula (I), comprising the following steps:

[0015] (1) Prepare rice fermentation culture of sponge symbiotic fungus Aspergillus sp.SCSIO 41420. Extract the rice fermentation culture with ethyl acetate, concentrate the ethyl acetate extract, and obtain supernatant fermentation broth extract.

[0016] (2) The extract was chromatographically analyzed using a normal-phase silica gel column with a gradient elution system of petroleum ether / ethyl acetate / methanol. Petroleum ether-ethyl acetate was eluted at volume ratios of 100:0, 90:10, 80:20, 60:40, 20:80, 10:90, and 0:100. Ethyl acetate-methanol was eluted at volume ratios of 99:1, 98:2, 95:5, 90:10, 85:15, 80:20, and 50:50. The petroleum ether / ethyl acetate mixture was collected at a volume ratio of... Fraction Fr4, eluted at 80:20 and 60:40, was purified to obtain 12R,13S-Dihydroxyfumitremorgin C. Fraction Fr6, eluted from petroleum ether / ethyl acetate at volume ratios of 10:90 and 0:100, was collected. Fraction Fr6 was eluted using a methanol-water gradient. Fractions Fr6-9, eluted from methanol-water at volume ratios of 60:40 and 80:20, were collected and purified to obtain tryprostatin A.

[0017] Furthermore, the rice fermentation culture of the sponge-associated epiphytic fungus Aspergillus sp. SCSIO 41420 was prepared by the following method:

[0018] A. Strawberry strain Aspergillus sp.SCSIO 41420 was streaked onto MB solid medium plates and cultured until sporulation occurred. The spores were then inoculated into MA liquid seed medium and cultured on a shaker at 26°C for 4 days to obtain seed culture solution.

[0019] B. Under aseptic conditions, the cultured seed culture was inoculated into a rice fermentation medium and cultured at 26°C on a shaker for 30 days to obtain the fermentation culture of strain Aspergillus sp.SCSIO 41420.

[0020] Furthermore, the formulation of the MB solid culture medium is as follows: 15 g / L malt extract powder, 24 g / L crude sea salt, 15 g / L agar, pH 7.0;

[0021] The formulation of the MA liquid seed culture medium is as follows: 15 g / L malt extract powder, 24 g / L crude sea salt, pH 7.0;

[0022] The formula for the fermented rice culture medium is: 800g / L rice and 24g / L coarse sea salt.

[0023] Further, in step A, the spores are inoculated into 100 mL of MA liquid seed culture medium;

[0024] In step B, 10 mL of the cultured seed culture solution is inoculated into 250 mL of rice culture medium for large-scale fermentation.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] 1. This invention utilizes the HepG2 cell in vitro model and employs qPCR real-time fluorescence quantitative technology and WB protein immunoblotting technology to confirm that compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A can effectively activate LXRα and induce increased expression of its downstream target genes ABCA1 and CYP7A1.

[0027] 2. In this invention, a high cholesterol cell model was constructed in vitro. Through Oil Red staining and determination of intracellular TG and T-CHO content, it was confirmed that compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A can reduce lipid content in the high cholesterol cell model.

[0028] 3. This invention is the first to demonstrate the LXRα agonist effect of compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A, providing new candidate compounds for the preparation of drugs to prevent and treat hypercholesterolemia.

[0029] 4. The raw material of this invention is derived from symbiotic fungi of marine sponges. It is a brand-new discovery of marine-derived compounds in the field of prevention and treatment of hypercholesterolemia, which broadens the horizons of marine-derived drugs for disease treatment and has broad prospects for development and application. Attached Figure Description

[0030] Figure 1 It is compound 1 1 1H NMR (500MHz, DMSO-d6) spectrum;

[0031] Figure 2 It is compound 1 13 C NMR (125MHz, DMSO-d6) spectrum;

[0032] Figure 3 It is compound 2. 1 1H NMR (500MHz, DMSO-d6) spectrum;

[0033] Figure 4 It is compound 2. 13 C10 NMR (125MHz, DMSO-d6) spectrum.

[0034] Figure 5 These are the qPCR results for compounds 1 and 2, showing their effects on the levels of LXRα, ABCA1, and CYP7A1 mRNA in HepG2 cells.

[0035] Figure 6The results are Western blot (WB) results for compounds 1 and 2, showing their effects on the levels of LXRα, ABCA1, and CYP7A1 proteins in HepG2 cells.

[0036] Figure 7 The values ​​represent the levels of TG and T-CHO in HepG2 high-cholesterol model cells, specifically compounds 1 and 2.

[0037] Figure 8 These are the results of Oil Red staining of compounds 1 and 2 in HepG2 high-cholesterol model cells. Detailed implementation method:

[0038] The following examples are further illustrations of the present invention, but not limitations thereof. In the examples, all original reagents and materials are commercially available, and experimental methods not specifically described are conventional methods and conditions well known in the art, or as recommended by the instrument manufacturer.

[0039] Example 1:

[0040] This embodiment provides the preparation and structural identification of compounds 1 and 2 as shown in formula (Ⅰ).

[0041]

[0042] The preparation methods of compounds 1 and 2 include the following steps:

[0043] 1. Seed culture

[0044] (1) Seed culture medium formula (MA medium): Dissolve 15g of malt extract powder, 24g of crude sea salt, 1L of distilled water, and pH 7.0. Mix thoroughly and dispense 100mL into 250mL Erlenmeyer flasks. Sterilize at 121℃ for 30 minutes to obtain the seed culture medium.

[0045] (2) Seed culture: Aspergillus sp.SCSIO 41420 was streaked onto MB solid medium (15g malt extract powder, 24g crude sea salt, 15g agar, 1L distilled water, pH 7.0) plates and cultured until sporulation. Then, the spores were inoculated into MA liquid seed medium and cultured on a shaker at 180 rpm and 26°C for 4 days to obtain seed culture.

[0046] 2. Scale-up fermentation culture

[0047] (1) Formula for large-scale fermentation medium: 200g rice, 6g coarse sea salt, 250mL distilled water.

[0048] (2) Scale-up fermentation culture: Under aseptic conditions, 10 mL of the cultured seed culture was inoculated into the scale-up fermentation rice culture medium and cultured at 26°C on a shaker for 30 days to obtain the fermentation culture of strain Aspergillus sp.SCSIO 41420.

[0049] 3. Extraction and separation

[0050] Extraction of fermentation culture: After the fermentation was completed, an equal volume of ethyl acetate was added to each fermentation flask and soaked for 1 hour. The rice was crushed, sonicated for 15 minutes, and then extracted with ethyl acetate 3 times. The supernatant of ethyl acetate was collected and concentrated into an extract under a fume hood, about 39g.

[0051] Compound separation: The extract was dissolved in a small amount of ethyl acetate and mixed with 100-200 mesh silica gel. Normal silica gel column chromatography was performed using a petroleum ether / ethyl acetate / methanol gradient elution system. Petroleum ether-ethyl acetate eluted at volume ratios of 100:0, 90:10, 80:20, 60:40, 20:80, 10:90, and 0:100. Ethyl acetate-methanol eluted at volume ratios of 99:1, 98:2, 95:5, 90:10, 85:15, 80:20, and 50:50, yielding 13 fractions Fr.1-Fr.13. Fraction Fr4, eluted from petroleum ether / ethyl acetate at volume ratios of 80:20 and 60:40, was collected. Fr.4 was purified by semi-preparative high-performance liquid chromatography to obtain compound 3 (4.48 mg / t). R 22 min, 70% acetonitrile / water, 2.5 mL / min, 210 nm). Fractions Fr6 and Fr.6 eluted from petroleum ether / ethyl acetate at a volume ratio of 10:90, 0:100 were collected and subjected to medium-pressure reversed silica gel column chromatography with a methanol-water gradient elution system, yielding 13 fractions Fr.6-1-Fr.6-13. Fractions Fr6-9 eluted from methanol-water at a volume ratio of 60:40, 80:20 were collected. Fr.6-9 fractions were purified by semi-preparative HPLC to give compound 2 (4.90 mg, t). R 14 min, 35% acetonitrile / water, 2.5 mL / min, 210 nm).

[0052] 4. Physicochemical data of compound 1 and compound 2

[0053] Figure 1 It is compound 1 1 1H NMR (500MHz, DMSO-d6) spectrum;

[0054] Figure 2 It is compound 1 13 C NMR (125MHz, DMSO-d6) spectrum;

[0055] Figure 3It is compound 2. 1 1H NMR (500MHz, DMSO-d6) spectrum;

[0056] Figure 4 It is compound 2. 13 C10 NMR (125MHz, DMSO-d6) spectrum.

[0057] The physicochemical data of compounds 1 and 2 are as follows:

[0058] Compound 1 is a white powder. The compound's... 1 The H NMR spectrum shows three methyl groups [δ] H 1.92(s,3H,H-23), 1.58(s,3H,H-24), 3.75(s,3H,H-25)], 3 methylene groups [δ H 2.29(m,1H,H2-14),1.87(m,2H,H-15),1.87(m,1H,H2-14),3.51(m,1H,H2-16),3.46(m,1H,H2-16)], 4 olefinic protons [δ H 4.76(m, 1H, H-21), 7.61(d, J = 8.7 Hz, 1H, H-5), 6.62(dd, J = 8.7, 2.3 Hz, 1H, H-6), 6.82(d, J = 2.3 Hz, 1H, H-8)], 3 methines [δ H 5.75(dd,J=9.5,1.1Hz,1H,H-10), 4.39(dd,J=9.5,6.9Hz,1H,H-13), 5.51(dd,J=3.5,1.1Hz,1H,H-20)]. 13 The C10 NMR spectrum showed 22 carbon signals, including 3 methyl groups, 3 methylene groups, 4 enmethyl groups, 3 methine groups, 7 quaternary carbons, and 2 carbonyl groups. (Compared with literature...) [1] Compound 3 was identified as 12R,13S-Dihydroxyfumitremorgin C.

[0059] Compound 2 is a brown oil. This compound's... 1 The HNMR spectrum showed three methyl groups [δ] H 1.69(s,3H,H-23), 1.71(s,3H,H-24), 3.72(s,3H,H-25)], 5 methylene groups [δ H3.48 (dd, J = 14.6, 7.7 Hz, 1H, H2-10), 2.96 (dd, J = 14.6, 6.1 Hz, 1H, H2-10), 1.40 (m, 1H, H2-15), 1.90 (m, 1H, H2-15), 1.60 (m, 1H, H2-16), 1.11 (m, 1H, H2-16), 3.17 (m, 1H, H2-17), 3.14 (m, 1H, H2-17), 3.40 (m, 1H, H2-20), 3.40 (m, 1H, H2-20)], 4 olefinic protons [δ H 7.32(d, J = 8.0 Hz, 1H, H-5), 6.57(dd, J = 8.0, 2.3 Hz, 1H, H-6), 6.77(d, J = 2.3 Hz, 1H, H-8), 5.3(m, 1H, H-21)], 2 methines [δ H 4.00 (dd, J=8.0, 2.3Hz, 1H, H-11), 4.22 (t, J=5.7Hz, 1H, H-14)]. 13 The C10 NMR spectrum showed 22 carbon signals, including 3 methyl groups, 5 methylene groups, 4 enmethyl groups, 2 methine groups, 6 quaternary carbons, and 2 carbonyl groups. (Compared with literature...) [2] Compound 2 was identified as tryprostatin A.

[0060] [1]Zhang DH, Noviendri D, Nursid M, Yang XD, Son BW*.12,13-dihydroxyfumitremorgin C, fumitremorgin C, and brevianamide F, antibacterial diketopiperazine alkaloids from the marine-derived fungus Pseudallescheriasp. Natural Product Sciences.2007,13(3):251-254.

[0061] [2]Liu R, Li H, Yang J, Zhou F. Indole Diketopiperazines from EndophyticFungus ofAstragalus membranaceus and Biological Evaluation. ChemistryofNatural Compounds. 2018, 54(6): 1196-1198.

[0062] Analysis of the NMR data of the compounds confirmed that compounds 1 and 2 are the known compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A, with structures as shown in formula (I):

[0063]

[0064] Example 2:

[0065] This embodiment investigates the effects of compounds 1-2 on the in vitro stimulation of LXRα and its downstream target genes ABCA1 and CYP7A1 in HepG2 cells, as well as their influence on lipid content in a high-cholesterol cell model.

[0066] Experimental cells: HepG2 human hepatoblastoma cells were used in this invention and cultured in DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin, and then cultured in a CO2 incubator (37°C, 5% CO2).

[0067] Abbreviations: OA (Oleic acid); PA (Palmitic acid); T-Cho (serum cholesterol); TG (triglycerides).

[0068] 1. Effects of Compound 1 and Compound 2 on LXRα and its downstream target genes ABCA1 and CYP7A1 1.1 Cellular Intervention

[0069] HepG2 cells in the logarithmic growth phase, cultured in vitro, were used at a concentration of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded at a density of cells / well in six-well plates and cultured for 24 hours until they adhered. Then, the cells were divided into three groups and different compounds were added for co-culture for 24 hours.

[0070] Control group: Co-culture with an equal amount of DMSO.

[0071] Group 1: Add 25 μM of compound 1 for co-culture.

[0072] Compound 2 group: 25 μM of compound 2 was added for co-culture.

[0073] 1.2 Result Indicator Detection

[0074] To investigate the agonistic effects of LXRα and its downstream target genes ABCA1 and CYP7A1 at the transcriptional level, total RNA was extracted from HepG2 cells after 24 hours of co-culture, reverse transcribed into cDNA, and then the transcriptional expression changes were detected by qPCR.

[0075] To investigate the agonistic effects of LXRα and its downstream target genes ABCA1 and CYP7A1 on protein translation, total protein was extracted from HepG2 cells after 24 hours of co-culture, and changes in protein translation expression were detected by Western blotting.

[0076] 1.3 Experimental Results

[0077] The transcriptional expression levels of LXRα and its downstream target genes ABCA1 and CYP7A1 were detected using qPCR. Figure 5 As shown, the mRNA expression levels of LXRα increased by 1.16-fold and 1.37-fold, respectively, while the mRNA levels of CYP7A1 and ABCA1 increased significantly as expected, by 5-fold and 5.02-fold, and 2.05-fold and 2.13-fold, respectively. The data were statistically significant (P<0.001).

[0078] Western blot analysis was used to detect changes in the translational expression levels of LXRα and its downstream target genes ABCA1 and CYP7A1. Figure 6 As shown, the protein expression levels of LXRα increased by 1.15-fold and 1.41-fold, respectively, while the mRNA levels of CYP7A1 and ABCA1 increased significantly as expected, by 2.32-fold and 1.69-fold, and 1.91-fold and 3.04-fold, respectively. The data were statistically significant (P<0.001).

[0079] The results showed that although compounds 1 and 2 did not directly and significantly increase the expression levels of LXRα at the transcriptional and translational levels, they significantly increased the expression levels of its downstream target genes ABCA1 and CYP7A1 at both the transcriptional and translational levels. This demonstrates that compounds 1 and 2 stimulate the expression of their downstream target genes by increasing LXRα's activity through binding.

[0080] 2. Effects of Compound 1 and Compound 2 on lipid content in a high-cholesterol cell model

[0081] 2.1 High cholesterol cell model

[0082] HepG2 cells in the logarithmic growth phase, cultured in vitro, were used at a concentration of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of cells / well in cell spreaders or six-well plates and cultured for 24 hours until the cells adhered. Then, different compounds were added to the four groups for co-culture for 48 hours.

[0083] Control group: Co-culture was carried out with an equal amount of solvent (dimethyl sulfoxide).

[0084] Model group: Co-cultured with 0.5mM OA and 0.25mM PA.

[0085] Group 1: Co-culture with 0.5 mM OA, 0.25 mM PA and 25 μM compound 1.

[0086] Compound 2 group: Co-culture with 0.5 mM OA, 0.25 mM PA and 25 μM compound 2.

[0087] 2.2 Measurement of intracellular TG and T-CHO content

[0088] According to the Nanjing Jiancheng reagent kit, after adding the standard, sample, and control dd H2O, add the corresponding reagents, incubate at 37 degrees for 10 min, and then measure the absorbance at 510 nm.

[0089] 2.3 Oil Red staining of cells

[0090] Frozen sections were rewarmed and dried, fixed in fixative for 15 minutes, washed with tap water, and air-dried.

[0091] Oil Red staining: Immerse the sections in oil red staining solution for 8-10 minutes (cover and protect from light), then wash with distilled water.

[0092] Background differentiation: slight differentiation with 75% alcohol, followed by washing with distilled water.

[0093] Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with blueing solution, and rinse with running water.

[0094] Mounting: Mount the slide with glycerin gelatin mounting medium.

[0095] Microscopic examination, image acquisition and analysis.

[0096] 2.4 Experimental Results

[0097] The results of intracellular TG and T-CHO content measurements are as follows: Figure 7 In a high cholesterol model, co-culturing with compounds 1 and 2 resulted in a significant decrease in intracellular triglyceride and total cholesterol levels (data were statistically significant).

[0098] Cell oil red staining results as follows Figure 8 The number of red oil droplets in high-cholesterol cells co-cultured with compounds 1 and 2 was significantly reduced.

[0099] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of this invention should also fall within the protection scope of this invention.

Claims

1. The indole-dione-piperazine alkaloid 12 shown in formula (I) R , 13 S The use of -Dihydroxyfumitremorgin C and / or tryprostatin A in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease. Equation (I).

Images