Ploroglucinol compound, separation and application

Abstract

The invention relates to a phloroglucinol compound, separation and application of the phloroglucinol compound in preparation of drugs for resisting acute myeloid leukemia, and belongs to the technical field of medicines. The novel compound long-column polycycline A separated from the plant hypericum has good anti-tumor activity, especially has good anti-tumor activity on AML cell lines, and can be used as a lead compound for development of anti-AML drugs. The invention provides an alternative compound for research and development of new drugs for resisting acute myeloid leukemia cells, and has very important significance for development and utilization of natural product resources.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to phloroglucinol compounds, separation and preparation of anti-acute myeloid leukemia drugs, and in particular to the separation and purification process, structure confirmation and anti-acute myeloid leukemia activity of compound 1. Background Art

[0002] Acute myeloid leukemia (AML) is a common clinical hematological malignancy. It is a group of diseases that are transformed from hematopoietic progenitor cells at different stages during the differentiation and development of myeloid cells (J. Clin. Invest., 2020, 130, 1552–1564). Its origins are from the granulocyte, monocyte, erythroid or megakaryocyte lines. The disease is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood and inhibition of normal hematopoiesis, and can widely infiltrate various organs such as the liver, spleen, and lymph nodes. Clinical manifestations include anemia, bleeding, infection, and fever. Most cases are in critical condition with a poor prognosis (Blood Rev., 2021, 47, 100773). The exact cause of AML is not yet fully understood, but it is related to regional environmental factors, ionizing radiation, chemical exposure, alcoholism and smoking, and mainly to the body's special response to certain viral infections (Future Oncol., 2020, 16, 961-972). In addition, in recent years, studies on gene mutation frequency and genetic susceptibility biomarkers have found that it may be the result of the combined effects of genetic and environmental factors, and the high incidence of acute myeloid leukemia is related to close contact with the above factors (Br J Haematol., 2022, 196, 316-328). Therefore, exploring and developing effective drugs for the treatment of AML has long been an important topic in this field.

[0003] Natural products are the most common source of drugs. According to statistics, from the 1980s to the present, 30-40% of the drugs marketed each year are directly or indirectly derived from natural products. In 2010, the proportion of drugs from natural product-related sources reached 50%. There are a wide variety of plant secondary metabolites. With the increasing research on them in recent years, more and more compounds with novel chemical structures and strong activity have been discovered, laying a certain foundation for the development of new drugs. Natural products derived from plant secondary metabolites are also an important source of anti-tumor drugs. The classical anti-tumor drugs commonly used in clinical practice, such as paclitaxel, camptothecin, vinblastine, and vincristine, are all derived from plant secondary metabolites. Therefore, it is of great significance to isolate new natural products with anti-AML effects from plants. Summary of the invention

[0004] The task of the present invention is to provide a separation and purification method of a novel compound in Hypericum plants having anti-acute myeloid leukemia (AML) activity and its application. The compound in the present invention has good anti-tumor activity, especially good anti-tumor activity against AML cell lines, and can be used as a lead compound for the development of anti-AML drugs.

[0005] According to a first aspect of the present invention, a phloroglucinol compound is provided, and the structural formula of the phloroglucinol compound is shown in Formula 1:

[0006]

[0007] According to another aspect of the present invention, a method for preparing the phloroglucinol compound is provided, comprising the following steps:

[0008] (1) extracting the Hypericum plant with alcohol, concentrating under reduced pressure to recover the alcohol, and then extracting with dichloromethane to obtain a dichloromethane extract;

[0009] (2) The dichloromethane extract obtained in step (1) is subjected to column chromatography, and then gradient eluted with petroleum ether and ethyl acetate to obtain 7 components arranged in order from small to large polarity, and the fourth component is subjected to gel chromatography, positive and reverse phase silica gel column chromatography and high performance liquid chromatography in sequence to obtain the phloroglucinol compound.

[0010] Preferably, the Hypericum plant is Hypericum longifolium.

[0011] Preferably, during the elution process, the petroleum ether and ethyl acetate are eluted in a volume ratio of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1, in sequence.

[0012] According to another aspect of the present invention, there is provided the use of the phloroglucinol compound for preparing anti-tumor drugs.

[0013] Preferably, the tumor is an acute myeloid leukemia tumor.

[0014] Preferably, the phloroglucinol compound is used to inhibit the activity of mitochondrial complex V in acute myeloid leukemia tumor cells, causing inflammation and oxidative stress disorders in the acute myeloid leukemia tumor cells, thereby causing apoptosis of the acute myeloid leukemia tumor cells.

[0015] Preferably, the tumor cells are NB4 cells and / or THP-1 cells.

[0016] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0017] (1) The present invention discovered a novel phloroglucinol compound with a completely new carbon skeleton from Hypericum plants. The structural type of the compound is discovered for the first time and is innovative in chemical structure. Further biological activity evaluation results show that the novel compound has a good anti-AML effect, and the main mechanism is to significantly increase the mitochondrial membrane potential of THP-1 and NB4 cell lines and significantly inhibit the activity of mitochondrial complex V of tumor cells.

[0018] (2) The mesotriol compound of the present invention can significantly inhibit the phosphorylation of S6 in THP-1 and NB4 cell lines and significantly induce apoptosis of tumor cells.

[0019] (3) The mesotriol compound of the present invention can significantly upregulate the mRNA levels of multiple cytokines or chemokines, promote endogenous inflammation of THP-1 and NB4 cells, and cause tumor cell growth inhibition or apoptosis.

[0020] (4) The mesotriol compound of the present invention can increase the oxidative stress of THP-1 and NB4 cells to exert its apoptosis-inducing effect.

[0021] (5) The mesotriol compound of the present invention can significantly increase the mitochondrial membrane potential of THP-1 and NB4 cell lines at 12 hours and inhibit the activity of mitochondrial complex V of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a single crystal diffraction pattern of the mesotriol compound of the present invention.

[0023] Figure 2 The pharmacological effects of phloroglucinol compounds against AML were verified by using Western-blot and flow cytometry tests; A is the dose-effect curve of phloroglucinol compounds against two hematological malignant tumor cells, NB4 and THP-1; B is the effect of phloroglucinol compounds on total ribosomal protein S6 and phosphorylated ribosomalprotein S6 in NB4 and THP-1 cells; C is the flow cytometry detection of cell apoptosis rate after double staining with Annexin-V and PI.

[0024] Figure 3 The method is to test the induction effect of phloroglucinol compounds on endogenous inflammation in AML cell lines; wherein: AH is NB4 or THP-1 cells treated with DMSO or compound 1 for 12 hours, and qPCR is used to analyze the mRNA expression of cytokines or chemokines.

[0025] Figure 4The method is to test the induction effect of pyrogallol compounds on oxidative stress in AML cell lines; wherein: A is the analysis of mRNA expression by qPCR; B is the analysis of the expression of AMPK and phospho-AMPK by Western blot; C is the analysis of ROS content in AML cell lines by flow cytometry.

[0026] Figure 5 It is to test the inhibitory effect of phloroglucinol compounds on mitochondrial complex V; wherein: AD is flow cytometry analysis of mitochondrial membrane potential of AML cell line; E is mitochondrial complex V activity analysis; F is a schematic diagram of the pharmacological mechanism of the compound shown in formula 1. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The new compound separated and purified from Hypericum plants in the present invention belongs to phloroglucinol compounds, and its chemical name is (2R, 6S)-5-hydroxy-6-(((4aR, 5S, 8S, 8aR)-5-hydroxy-8-isopropyl-5-methyl-3, 4, 4a, 5, 6, 7, 8, 8a-octahydronaphthalen-2-yl) methyl)-2-methyl-2-(3-methylbut-2-en-1-yl)-4-(3-methylbutanol)-6-(((1R, 4S, 7S)-1, 3, 3-trimethyl-2-oxabicyclo [2.2.1] heptane-7-yl) cyclohexyl-4-ene-1, 3-dione, and is named as long-column polycycline A. Its structural formula is as shown in Formula 1;

[0029]

[0030] The preparation method of the phloroglucinol compound of the present invention comprises the following steps: using industrial alcohol (95% ethanol) to extract Hypericum plant (preferably Hypericum longifolium), decompressing and concentrating to recover the industrial alcohol, and extracting with dichloromethane to obtain a dichloromethane extract; subjecting the dichloromethane extract to column chromatography: specifically, mixing the sample with silica gel and loading the column by dry method; then eluting with petroleum ether-ethyl acetate gradient, combining the same components, and obtaining 7 components with small to large polarity; wherein component 4 is separated by repeated gel chromatography, positive and reverse phase silica gel column chromatography and high performance liquid chromatography to obtain a phloroglucinol compound (named as long-column polycycline A).

[0031] The invention discloses an application of the mesopyrogallol compound in preventing acute myeloid leukemia. The compound can inhibit the activity of mitochondrial complex V in acute myeloid leukemia cells, induce inflammation and oxidative stress disorder in tumor cells, and thus cause apoptosis of tumor cells.

[0032] Preferably, the tumor cells are NB4 cells and / or THP-1 cells.

[0033] In summary, the present invention preferably separates and purifies the industrial alcohol (95% ethanol) extract of Hypericum longifolium produced in Enshi, Hubei, to obtain a new compound, which is a secondary metabolite of Hypericum longifolium. Its structure is determined by using a variety of spectral analysis methods and other methods, and the specific structure is shown in Formula 1. By evaluating the anti-AML activity of the compound of Formula 1, it was found that the compound of Formula 1 has extremely strong cytotoxic activity against two AML cell lines (NB4 cells and THP-1 cells), and can be used as a lead compound for the development of drugs for the treatment of AML.

[0034] The following are specific embodiments

[0035] Example 1

[0036] 1. Separation and preparation of phloroglucinol compound as shown in Formula 1

[0037] 15 kg of long-column Hypericum was extracted 7 times with industrial alcohol, concentrated under reduced pressure at below 50°C to recover industrial alcohol, and extracted with dichloromethane to obtain 650 g of dichloromethane extract. The dichloromethane extract was subjected to column chromatography: specifically, the sample was mixed with 200-300 mesh silica gel, and the column was loaded by dry method, and the gradient was eluted with petroleum ether-ethyl acetate (volume ratio 30:1-0:1, such as eluting in accordance with the volume ratio of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1, and 5 columns were added for each volume ratio), TLC detection, and the same components were combined to obtain a total of 7 components with small to large polarity. Component 4 was then separated by repeated gel chromatography, positive and reverse phase silica gel column chromatography and high performance liquid chromatography to obtain a phloroglucinol compound (named as long-column polycycline A) (1.3 mg).

[0038] 2. Structural identification of the compound long-column polycycline A as shown in formula 1

[0039] The compound long-column polycycline A was subjected to nuclear magnetic resonance, mass spectrum, optical rotation, infrared spectrum, ultraviolet spectrum, circular dichroism and other data tests, and the long-column polycycline A was subjected to X-ray single crystal diffraction data test to determine the structure of the compound.

[0040] Long-column polycycline A: Colorless needle crystals; mp 119.2–123.0℃; [α] 2D 5 –14.8(c0.3,MeOH); UV(MeOH)λ max (logε):283(4.04)nm; IR(ν max ):3454,2960,2934,2870,1711,1669,1382,1165and 1093cm –1 ;ECD(MeOH)λ max (Δε) 203(-3.94), 211(-0.49), 224(-3.55), 261(+1.57), 275(-0.06), 287(+0.75) and 307(-2.94)nm; the absolute configuration of long-column polycycline A was determined by X-ray single crystal diffraction. Figure 1 is the single crystal diffraction pattern of the long column polycycline A of the present invention, and the NMR data are as described in Table 1.

[0041] Table 1. H and C spectra of long-column polycycline A (δ in ppm and J in Hz)

[0042]

[0043]

[0044] Example 2

[0045] Anti-AML effect of the compound polycycline A in the present invention and its inhibitory activity on mitochondrial complex V

[0046] ① The CCK8 method was used to test the cytotoxic activity of the compound polycycline A against various malignant tumor cells

[0047] The cells were placed in a 96-well culture plate (4 × 10 3 / well) for 48 h, and then incubated with CCK8 reagent at 37°C for 4 h. The absorbance at OD 450nm was measured using a plate reader (Synergy HT, Biotek). Cell viability and 50% inhibitory concentration (IC 50 As shown in Table 2, polycycline A has cytotoxic effects on a variety of hematological tumors and solid tumor cells, among which it has better cytotoxic effects on two hematological malignant tumor cells, NB4 and THP-1.

[0048] Table 2 Cytotoxic activity results of long-column polycycline A against various malignant tumor cells (48h, μM)

[0049]

[0050] ②The antitumor activity of the compound long-column polycycline A against NB4 and THP-1 was tested using Western-blot, flow cytometry and other methods.

[0051] The results are as follows:

[0052] like Figure 2 As shown in A, the IC of long-column polycycline A for NB4 cells 50 The value is 3.588 μM, and the IC 50 The value is 5.760 μM. Ribosomal protein S6 is an important regulator of ribosome biogenesis and mRNA translation enhancement, and its phosphorylation is closely related to tumor cell growth. Figure 2 As shown in Figure B, polycycline A treatment significantly inhibited the phosphorylation of S6, which provided evidence for the tumor cell growth inhibitory effect of polycycline A at the molecular level. Figure 2 As shown in C, the compound long-column polycycline A can significantly induce tumor cell apoptosis, and the average apoptosis ratio of the high-dose group exceeds 60%.

[0053] AML is a cancer caused by the canceration of immune cells, and is deeply affected by inflammation during its development. Therefore, we tested the changes of multiple cytokines or chemokines after treatment with the compound polycycline A. Figure 3 As shown, in THP-1 and NB4 cell lines, the compound polycycline A significantly upregulated the mRNA levels of multiple cytokines or chemokines, promoted endogenous inflammation of AML cells, and could cause tumor cell growth inhibition or apoptosis.

[0054] Given that endogenous cellular inflammation is often accompanied by oxidative stress and that interference with cellular oxidative stress is considered an effective target for anticancer drugs, we analyzed whether the compounds exert their pharmacological effects by inducing oxidative stress. Figure 4 As shown in Figure A, after 48 hours of treatment with the compound polycycline A, the transcription of oxidative stress-related genes such as Trib3, Gdf15, Sesn2, ATF4, EIF4EBP1 and TXNIP in AML cells increased significantly. AMPK plays an important role in cell metabolism. As an oxidative stress-sensitive protein kinase, it opens or closes pathways under energy stress to maintain system energy balance. Figure 4As shown in B, western blot analysis results showed that after 48 hours of compound treatment, phosphorylated AMPK increased significantly, while total AMPK was almost unaffected. The increase in phosphorylated AMPK represents the activation of AMPK, which is a response to oxidative stress. This data indicates that the oxidative stress of AML cells increased after treatment with the compound polycycline A. These data indicate that the compound polycycline A exerts its apoptosis-inducing effect by increasing the oxidative stress of AML cells. One of the main causes of oxidative stress is the excessive formation of reactive oxygen species (ROS), and changes in ROS are accompanied by a series of metabolic changes in cells, which play a key role in cell apoptosis. Therefore, we detected changes in ROS content in AML cells. As Figure 4 As shown in Figure C, after 12 hours of compound treatment, the level of ROS in AML cells increased, which may be the cause of oxidative stress and the initiation of cell apoptosis. After that, when AML cells were treated with the compound for 48 hours, the level of ROS in the cells was lower than that in the DMSO group.

[0055] Mitochondria are the central organelles of cellular respiration, and the maintenance of mitochondrial membrane potential is crucial for the normal function of the mitochondrial respiratory chain. Figure 5 As shown in Figures AD, the compounds significantly increased the mitochondrial membrane potential in THP-1 and NB4 cell lines at 12 hours. This effect is unique because most anti-cancer drugs reduce mitochondrial membrane potential by directly damaging mitochondria. Mitochondrial membrane potential is maintained by the mitochondrial respiratory chain, which consists of five mitochondrial complexes that maintain mitochondrial charge homeostasis through electron or proton transfer. Notably, mitochondrial complex V is the only proton pump in the respiratory chain, which reduces the mitochondrial potential caused by the actions of other complexes by transporting protons into the mitochondrial matrix. Therefore, we tested whether the compounds directly reduce the activity of mitochondrial complex V. Figure 5 As shown in Figure E, we found that the compound significantly inhibited the activity of mitochondrial complex V in mitochondria isolated from mouse liver. Figure 5 F in FIG. 1 is a schematic diagram of the anti-tumor mechanism of the compound.

[0056] Here’s how:

[0057] Acute leukemia cells were treated with a concentration gradient of 2.5-10 μM of the compound long-column polycycline A for 48 hours, the cells were collected, the total protein was extracted, and the protein concentration of the lysate was determined using the BCA protein assay reagent. The normalized amount of lysate samples was loaded on 10% SDS-PAGE and transferred to the NC membrane. Different antibodies were used for immunoblotting, and α-tubulin was used as a loading control. The bands were detected using the LI-COR near-infrared imaging system.

[0058] The fluorescent probe DCFH-DA kit was used to analyze the generation of ROS in tumor cells. 5 The cells were incubated with compound 1 for 48 h in a 12-well plate with 10 μM DCFH-da at 37°C for 20 min, washed twice with PBS, and the fluorescence signal of DCFH was detected by flow cytometry.

[0059] The mitochondrial membrane potential was analyzed using the fluorescent probe TMRE kit. The cells were seeded at 1×10 5 The cells were incubated with compound 1 for 12 h in a 12-well plate with 4% paraformaldehyde and 1% paraformaldehyde per well. The cells were then harvested. The cells were incubated with TMRE at 37°C for 5 min. The positive control CCCP was incubated with TMRE at a concentration of 5 uM. The fluorescence signal of TMRE was detected by flow cytometry.

[0060] The activity of mitochondrial complex V / ATP synthase activity was detected using the mitochondrial respiratory chain complex V / ATP synthase activity detection kit. The test compound and positive drug were incubated with mitochondrial lysate for 30 minutes, and then the activity of mitochondrial complex V was detected.

[0061] Annexin V-FITC apoptosis detection kit was used to test the effect of the test compound on tumor cell apoptosis. After the compound was treated for 48 hours, the cells were collected and stained with light at 25°C for 10 minutes, and the cell apoptosis rate was detected by flow cytometry.

[0062] Total RNA from cells was isolated using TRIzol TM Reagent separation, cDNA II Q RT SuperMix synthesis. SYBR Green qPCR Mix purchased from biosharp. The primers used are as follows:

[0063]

[0064] The analysis is as follows:

[0065] The new compound long-stalked polycycline A isolated from the plant Hypericum has good anti-tumor activity, especially against AML cell lines, and can be used as a lead compound for the development of anti-AML drugs.

[0066] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A phloroglucinol compound, characterized in that: The structural formula of the phloroglucinol compound is shown in Formula 1:

2. The method for preparing the phloroglucinol compound according to claim 1, wherein The following steps are involved: (1) extracting the Hypericum plant with alcohol, concentrating under reduced pressure to recover the alcohol, and then extracting with dichloromethane to obtain a dichloromethane extract; (2) The dichloromethane extract obtained in step (1) is subjected to column chromatography, and then gradient eluted with petroleum ether and ethyl acetate to obtain 7 components arranged in order from small to large polarity, and the fourth component is subjected to gel chromatography, positive and reverse phase silica gel column chromatography and high performance liquid chromatography in sequence to obtain the phloroglucinol compound.

3. The preparation method according to claim 2, characterized in that: The Hypericum plant is Hypericum longifolium.

4. The preparation method according to claim 2, characterized in that: During the elution process, the petroleum ether and ethyl acetate are eluted in sequence according to volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:

1.

5. Use of the phloroglucinol compound as claimed in claim 1 for preparing anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor is an acute myeloid leukemia tumor.

7. The use according to claim 6, characterized in that The phloroglucinol compound is used to inhibit the activity of mitochondrial complex V in acute myeloid leukemia tumor cells, causing inflammation and oxidative stress disorders in the acute myeloid leukemia tumor cells, thereby causing apoptosis of the acute myeloid leukemia tumor cells.

8. The use according to claim 6, characterized in that: The tumor cells are NB4 cells and / or THP-1 cells.

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