A new chlorinated xanthones compound and its preparation method and use

Abstract

The present invention discloses a novel chlorinated xanthones (named Austocystin S) and its application in inhibiting PTP1B and anti-tumor activity. The xanthones are isolated from fungi. Aspergillus puniceus Austocystin S was isolated from fermentation products. It was prepared using normal-phase silica gel column chromatography and semi-preparative liquid chromatography, a simple preparation method. Its chemical structure was identified by spectroscopic techniques such as NMR, MS, and CD, and it was identified as a new compound. PTP1B inhibitory activity testing showed that the compound, Austocystin S, exhibited strong inhibitory activity against PTP1B and exhibited selective inhibitory activity against HepG2 liver cancer cells, suggesting that it may be an anti-tumor PTP1B immunomodulator with promising application prospects.

Description

Technical Field

[0001] The present invention relates to a fungus extract, in particular to a novel separation method of chlorinated xanthones and its application in inhibiting activity on PTP1B and anti-tumor activity. Background Art

[0002] Literature reports indicate that the secondary metabolites of the fungus Aspergillus puniceus contain compounds with two typical structures: diketopiperazine alkaloids and xanthones (Cai Songyan. Study on the secondary metabolites of deep-sea fungus Aspergillus puniceus A2 and their anti-inflammatory activity. Shanghai Ocean University, 2022; Bioorganic Chemistry, 2021, 107:104571-104582; Journal of Natural Products, 2019, 82(6):1558-1564.). Xanthones are widely found in plants, but there are relatively few reports of their presence in microbial metabolites. Plant-derived xanthones have been reported to have multiple pharmacological activities, including hepatoprotection, anti-tumor, and treatment of cardiovascular diseases (Cardiovascular Drug Reviews, 2004, 22(2):91-102.).

[0003] Studies have found that protein tyrosine phosphatase 1B (PTP1B) can inhibit insulin signal transduction by inhibiting insulin receptor substrates and the phosphoinositide-3-kinase / protein kinase B (PI3K / Akt) signaling pathway (Nutrition & Metabolism, 2021, 18(1):48; Frontiers in Pharmacology, 2021, 12:632-169). PTP1B is a potential and effective target for the treatment of type 2 diabetes and obesity. Recently, many foreign researchers have found that inhibiting PTP1B in T cells can mobilize the body's immune response to cancer, thereby inhibiting tumor growth (Cancer Discovery, 2022; 12(3):752-773). Therefore, PTP1B is receiving widespread attention and research as a potential therapeutic target for solid tumors. Summary of the Invention

[0004] The first purpose of the present invention is to provide a new chlorine-containing xanthones compound, the second purpose is to provide a method for preparing the compound, and the third purpose is to provide its application in PTP1B inhibition and anti-tumor.

[0005] A chlorinated xanthonoid compound named Austocystin S has the following structure:

[0006]

[0007] The invention relates to an application of the chlorinated xanthones in the preparation of drugs for inhibiting the activity of PTP1B.

[0008] The chlorinated xanthones are used in the preparation of drugs for inhibiting tumors, wherein the tumor diseases include HGC-27 gastric cancer cells and HepG2 liver cancer cells.

[0009] The preparation method of the chlorine-containing xanthones comprises the following steps:

[0010] Step A: Inoculating Aspergillus puniceus strains into PDA solid medium (potato dextrose agar solid medium) for activation, inoculating the activated Aspergillus puniceus strains into PDB medium (potato dextrose liquid medium) to obtain a seed solution, and inoculating the seed solution into corn / water solid medium for static culture to obtain a fermentation product;

[0011] Step B: soaking the fermentation product in step A overnight and extracting to obtain a crude fermentation extract;

[0012] Step C: The crude fermentation extract obtained in step B is passed through a normal phase silica gel column and gradient eluted with a dichloromethane-methanol mixture. The obtained fractions are separated using a semi-preparative high performance liquid chromatography to obtain the compound Austocystin S.

[0013] In step B, the fermentation product is dried at 40-50° C. and then soaked in a dichloromethane-methanol mixture overnight, wherein the volume ratio of dichloromethane to methanol is 1:1-3.

[0014] In step C, during the gradient elution process, dichloromethane-methanol volume ratios of 100:0, 50:1, 40:1, 30:1, 10:1, 5:1, 1:1, and 0:1 were used for elution.

[0015] In step C, a semi-preparative high performance liquid chromatograph was used for separation, a C18 reverse phase column was used, the mobile phase was a mixed solvent of methanol / water with a volume ratio of 85:15, and the peak was collected at a retention time of 11.5 minutes to obtain the product.

[0016] Beneficial effects:

[0017] The present invention discovers a new compound, Austocystin S, for the first time, provides a method for preparing the compound, and finds that the compound has a good inhibitory effect on PTP1B and also has a certain selective inhibitory activity against HepG2 liver cancer cells, suggesting that the compound can be used as an anti-tumor PTP1B immunomodulator. Therefore, the compound can be used to prepare drugs against related tumors or as a lead compound for such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the structural formula of the compound Austocystin S.

[0019] Figure 2 The HMBC and 1 H- 1 H COSY correlation diagram.

[0020] Figure 3 This is the CD pattern of compound Austocystin S.

[0021] Figure 4 Austocystin S 1 H-NMR spectrum (solvent: DMSO-d6).

[0022] Figure 5 Austocystin S 13 C-NMR spectrum (solvent: DMSO-d6).

[0023] Figure 6 This is the DEPT 135 image of the compound Austocystin S (the solvent is DMSO-d6).

[0024] Figure 7 Austocystin S 1 H- 1 H COSY pattern (solvent: DMSO-d6).

[0025] Figure 8 This is the HSQC chart of the compound Austocystin S (the solvent is DMSO-d6).

[0026] Figure 9 This is the HMBC chart of the compound Austocystin S (the solvent is DMSO-d6).

[0027] Figure 10 This is the NOESY pattern of the compound Austocystin S (the solvent is DMSO-d6).

[0028] Figure 11 This is the HR-ESI-MS spectrum of the compound Austocystin S.

[0029] The following examples are used to further illustrate the present invention, but are not intended to limit the scope of protection of the present invention. DETAILED DESCRIPTION

[0030] Example 1: Preparation and structural analysis of the compound Austocystin S

[0031] The preparation method comprises the following steps:

[0032] The frozen Aspergillus puniceus strain was inoculated onto a sterilized PDA medium (potato dextrose agar solid medium) plate and placed in a 28°C incubator for activation. After 3 days of incubation, a seed plug was obtained. A 0.5 cm x 0.5 cm seed plug was selected and inoculated into PDB medium (potato dextrose liquid medium) and cultured at 150 rpm. After 2-3 days, a small number of mycelial pellets formed to obtain a seed liquid. The seed liquid was inoculated into a corn / water solid medium (40 g corn + 100 mL water) and incubated at 28°C for 30 days to obtain a fermentation product.

[0033] The fermentation product was dried in a drying oven at 45°C, soaked overnight in dichloromethane:methanol (volume ratio 1:1), extracted three times, and the extracts were combined and concentrated to obtain 83.2 g of crude fermentation extract.

[0034] The crude fermentation extract was mixed with normal-phase silica gel (200-300 mesh) in a 1:1 ratio and then powdered. The sample was passed through a normal-phase silica gel column (10 cm × 70 cm) using a dry-loading method and a wet-flow method. Elution was performed using a gradient of dichloromethane and methanol (volume ratios: 100:0, 50:1, 40:1, 30:1, 10:1, 5:1, 1:1, and 0:1), yielding 16 fractions, Fr.1 to 13. Fraction Fr.1 was dissolved in acetone and separated by semi-preparative HPLC using a C18 reverse-phase column and a mobile phase consisting of a methanol / water mixture (volume ratio: 85:15). The peak at a retention time of 11.5 minutes was collected to yield austocystin S (5 mg).

[0035] Structural identification of the compound Austocystin S

[0036] Compound Austocystin S: Crystalline (dichloromethane). Optical rotation data [α] 25 D -25.9 (c 0.10, methanol), UV (methanol) λmax =203, 248, 270, 350 nm; CD (0.50 mM, methanol) λ max (Δε): 228 (+0.89), 242 (–2.83), 268 (–1.43), 300 (+1.01) nm; by high-resolution mass spectrometry HR-ESI-MS: m / z 381.0138 [M+H] + (Calculated value: 381.0142), its molecular formula is determined to be C 18 H 11 ClO6, the degree of unsaturation is 13. The carbon spectrum of this compound 13 C-NMR and H spectroscopy 1 The H-NMR data are very similar to those of the reference compound Austocystin A (Bioorganic Chemistry, 2021, 107: 104571-104582), but one methoxyl signal is obviously missing. A careful comparison of the carbon spectrum data of the two compounds revealed that δ C 157.1(C-1), 110.1(C-2), 180.3(C-9), 104.7(C-9a) and the reference compound δ C 156.1 (C-1), 117.5 (C-2), 172.7 (C-9), 110.6 (C-9a) are very different (see Table 1), and it is speculated that there is one less methoxy group at C-1. H 6.66(H-4) / 4.72(H-2') and δ C 157.1 (C-1) respectively, which proves that this compound has one less methoxy group at C-1 compared with Austocystin A. The HMBC and 1H-1H COSY spectra (see Figure 2 ), verified the planar structure of the compound, and found that the NMR data of the 1', 2', 3' and 4' positions of the compound were consistent with those of the known compounds, and the coupling constant J H-1' / H-2' =7.0 Hz is also consistent with the known compounds, and it is speculated that the configurations of C-1' and C-2' are R and S, respectively. In order to further verify its stereostructure, the CD spectrum of the compound was tested (see Figure 3 ), with negative cotton effects at 242 and 268 nm and positive cotton effects at 228 and 300 nm. These data were consistent with those of Austocystin R and Austocystin A, confirming the configurations of C-1' and C-2' to be R and S, respectively. A search revealed that the compound was new and named Austocystin S.

[0037] Table 1. Compound Austocystin S and Austocystin A in the literature 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data (solvent: DMSO-d6)

[0038]

[0039]

[0040] Example 2: PTP1B inhibitory activity test of compound Austocystin S

[0041] 1. Experimental Materials

[0042] 1.1 Experimental instruments: electronic balance (AL204-IC, Shanghai Mettler-Toledo Instrument Co., Ltd.); Infinite M200 Pro microplate reader (TECAN Group, Switzerland).

[0043] 1.2 Experimental drugs: recombinant human PTP1B protein, sodium orthovanadate (Na3VO4), dithiothreitol (DTT), p-nitrophenyl phosphate (p-NPP), ethylenediaminetetraacetic acid (EDTA), sodium hydroxide, citric acid, and sodium chloride.

[0044] 2. Experimental Methods

[0045] 2.1 Follow the literature method (Journal of Functional Foods, 2018, 41: 232-239.).

[0046] 2.2 The compound Austocystin S (10 μL) was added to 170 μL of reaction buffer solution, which consists of 50 mM citric acid (pH 7.4), 50 mM NaCl, 2 mM dithiothreitol (DTT), and 1.1 mM EDTA. 20 μL of recombinant PTP1B solution (1 mg / mL, 1 μL) was added and mixed in each well. The reaction mixture was preheated for 15 min using a 37°C block heater. 10 μL of the reaction substrate p-nitrophenyl phosphate (pNPP) (33 mM) was added and reacted at 37°C for 15 min. NaOH solution (10 μL, 0.1 M) was added to stop the reaction. The absorbance was recorded at 405 nm.

[0047] 2.3 Set up the enzyme activity group (enzyme + reaction buffer + substrate), enzyme blank group (reaction buffer + reaction substrate), sample group (sample + reaction buffer + enzyme + reaction substrate), and sample blank group (sample + reaction buffer + reaction substrate), and use sodium orthovanadate aqueous solution as the positive control.

[0048] 3. Experimental Results

[0049] The results of the inhibitory activity of the fermentation crude extract on PTP1B are shown in Table 2, and the results of the inhibitory activity of the compound Austocystin S on PTP1B are shown in Table 3.

[0050] Table 2. Inhibitory activity of fermentation crude extracts against PTP1B

[0051]

[0052] Table 3. Inhibitory activity of compound Austocystin S on PTP1B

[0053]

[0054]

[0055] Example 3: Antitumor activity test of compound Austocystin S

[0056] 1. Experimental Materials

[0057] Preparation of compound mother solution: The isolated compound Austocystin S was prepared into a mother solution with a concentration of 50 mM using DMSO.

[0058] To prepare MTT solution: Dissolve 0.5 g of MTT in 50 mL of heated PBS. Mix thoroughly, aliquot, and store in a -20°C refrigerator protected from light. All operations should be performed in the dark.

[0059] 2. Experimental Methods

[0060] The MTT colorimetric method was used to detect the inhibitory effect of Austocystin S on the proliferation of 9 cell types. Cells in the logarithmic growth phase (about 1×10 4 100 μL of the compound (cells / well) was plated in a 96-well plate. After cells adhered overnight, medium containing the compound was added to achieve final concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μM. After 72 hours of culture, 5 mg / mL MTT reagent was added and incubated for 4 hours. The medium was discarded, 150 μL of DMSO was added, and the cells were shaken for 10 minutes. The absorbance (OD) was measured at 490 nm on a microplate reader. A blank group (medium), a control group (cells + medium), and an experimental group (cells + medium + compound) were set up. Inhibition rate / % = (OD value of control group - OD value of experimental group) / (OD value of control group - OD value of blank group) × 100%.

[0061] The experimental results are shown in Table 4.

[0062] 3. Experimental Results

[0063] Table 4. Cytotoxic activity of compound Austocystin S against 9 tumor cell lines IC 50 (μM)

[0064]

[0065] The experimental results show that the compound Austocystin S has a certain inhibitory activity only against HGC-27 gastric cancer cells and HepG2 liver cancer cells, among which the inhibitory activity against HepG2 liver cancer cells is the best, IC 50 The value was 20.68 μM, which proved that the compound had strong selectivity in inhibiting 9 tumor cell lines.

[0066] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The embodiments and features therein may be combined arbitrarily unless they conflict. The scope of protection of the present invention shall be the technical solutions set forth in the claims, including equivalent alternatives to the technical features of the technical solutions set forth in the claims. Equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A method for preparing a chlorine-containing xanthones compound, characterized in that: The steps include: Step A: Aspergillus puniceus The bacteria were inoculated into potato dextrose agar solid medium for activation. Aspergillus puniceus The bacterial strain is then inoculated into a potato glucose liquid culture medium to obtain a seed solution, and the seed solution is inoculated into a corn / water solid culture medium and statically cultured to obtain a fermentation product; Step B: soaking the fermentation product in step A overnight and extracting to obtain a crude fermentation extract; Step C: The crude fermentation extract obtained in step B was passed through a normal phase silica gel column and gradient eluted with a dichloromethane-methanol mixture. The obtained fractions were separated using a semi-preparative high performance liquid chromatography to obtain the compound Austocystin S. The structural formula of the compound Austocystin S is as follows: 。 2. The method for preparing chlorine-containing xanthones according to claim 1, wherein: In step B, the fermentation product is dried at 40-50° C. and then soaked in a dichloromethane-methanol mixture overnight, wherein the volume ratio of dichloromethane to methanol is 1:1-3.

3. The method for preparing chlorine-containing xanthones according to claim 1, wherein: In step C, during the gradient elution process, dichloromethane-methanol volume ratios of 100: 0, 50: 1, 40: 1, 30: 1, 10: 1, 5: 1, 1: 1, and 0: 1 were used for elution.

4. The method for preparing chlorine-containing xanthones according to claim 1, wherein: In step C, a semi-preparative high performance liquid chromatograph was used for separation, a C18 reverse phase column was used, the mobile phase was a mixed solvent of methanol / water with a volume ratio of 85:15, and the peak was collected at a retention time of 11.5 minutes to obtain the product.

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