Phloroglucinol compound, separation and application

Abstract

The present application relates to phloroglucinol compounds, separation and preparation of anti acute myelocytic leukemia drug application, belong to the medical technical field. The new compound long column polycyclic compound A separated from the plant hypericum genus has good antitumor activity, especially good antitumor activity to AML cell line, and can be used as a lead compound for developing anti-AML drug. The present application provides an alternative compound for developing new anti-acute myelocytic leukemia cell drug, and has very important significance for the development and utilization of natural product resources.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to the application of phloroglucinol compounds, their isolation and preparation as drugs against acute myeloid leukemia, and particularly to the process of isolation and purification of compound 1, structural confirmation and its activity against acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a common hematologic malignancy, a group of diseases arising from the malignant transformation of hematopoietic progenitor cells at different stages of myeloid cell differentiation and development (J. Clin. Invest., 2020, 130, 1552–1564). Its origins include granulocytic, monocytic, erythroid, or megakaryotic cell lines. The disease is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood, suppressing normal hematopoiesis, and can extensively infiltrate various organs such as the liver, spleen, and lymph nodes. Clinical manifestations include anemia, bleeding, infection, and fever; most cases are critically ill with a poor prognosis (Blood Rev., 2021, 47, 100773). The exact cause of AML is not fully understood, but it is associated with geographical and environmental factors, ionizing radiation, chemical exposure, alcohol abuse, and smoking, primarily related to specific responses of the body to certain viral infections (Future Oncol., 2020, 16, 961-972). Furthermore, recent studies using gene mutation frequencies and genetic susceptibility biomarkers have suggested that it may be the result of a combination of genetic and environmental factors, with a high incidence of acute myeloid leukemia linked to close exposure to these factors (Br J Haematol., 2022, 196, 316-328). Therefore, exploring and developing effective drugs for the treatment of AML has long been an important task in this field.

[0003] Natural products are the most common source of drugs. Statistics show that since the 1980s, 30-40% of marketed drugs each year are directly or indirectly derived from natural products, with the proportion reaching as high as 50% in 2010. Plant secondary metabolites are extremely diverse, and with increasing research in recent years, more and more compounds with novel chemical structures and strong activity have been discovered, laying a foundation for new drug development. Natural products derived from plant secondary metabolites are also an important source of antitumor drugs. Commonly used classic antitumor drugs such as paclitaxel, camptothecin, vincristine, and vinblastine are all derived from plant secondary metabolites. Therefore, isolating new natural products with anti-AML activity from plants is of paramount importance. Summary of the Invention

[0004] The objective of this invention is to provide a method for isolating and purifying novel compounds from the *Hypericum* genus that exhibit activity against acute myeloid leukemia (AML) and their applications. The compounds of this invention possess good antitumor activity, particularly against AML cell lines, and can serve as lead compounds for the development of anti-AML drugs.

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

[0006]

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

[0008] (1) The plants of the genus Hypericum were extracted with alcohol, and the alcohol was recovered by vacuum concentration. Then, the extract was obtained by dichloromethane extraction.

[0009] (2) The dichloromethane fraction extract obtained in step (1) was subjected to column chromatography and then eluted with petroleum ether and ethyl acetate gradient to obtain 7 components with polarity from smallest to largest. The fourth component was then separated by gel chromatography, reversed-phase silica gel column chromatography and high performance liquid chromatography 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 sequentially at volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1.

[0012] According to another aspect of the present invention, the use of the aforementioned phloroglucinol compound in the preparation of antitumor drugs is provided.

[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, thereby causing intracellular inflammation and oxidative stress disorders in acute myeloid leukemia tumor cells, resulting in apoptosis of acute myeloid leukemia tumor cells.

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

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0017] (1) This invention discovers a novel phloroglucinol compound with a completely new carbon skeleton from Hypericum plants. The structure of this compound is the first discovery and is innovative in chemical structure. Further bioactivity 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 in tumor cells.

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

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

[0020] (4) The phloroglucinol compound of the present invention can increase the oxidative stress of THP-1 and NB4 cells to induce apoptosis.

[0021] (5) The phloroglucinol 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 in tumor cells. Attached Figure Description

[0022] Figure 1 This is a single-crystal diffraction pattern of the phloroglucinol compound of this invention.

[0023] Figure 2 The pharmacological effects of phloroglucinol on AML were verified using Western blot and flow cytometry. Specifically: A shows the dose-response curves of phloroglucinol against NB4 and THP-1 hematologic malignancies; B shows the effects of phloroglucinol on total ribosomal protein S6 and phosphorylated ribosomal protein S6 in NB4 and THP-1 cells; and C shows the apoptosis rate detected by flow cytometry after Annexin-V and PI double staining.

[0024] Figure 3 The study tested the induction of endogenous inflammation in AML cell lines by phloroglucinol compounds; where AH refers to NB4 or THP-1 cells treated with DMSO or compound 1 for 12 h, and the mRNA expression of cytokines or chemokines was analyzed by qPCR.

[0025] Figure 4This study tested the induction of oxidative stress in AML cell lines by phloroglucinol compounds; where: A is the analysis of mRNA expression using qPCR; B is the analysis of AMPK and phospho-AMPK expression levels using Western blot; and C is the analysis of ROS content in AML cell lines using flow cytometry.

[0026] Figure 5 This test examines the inhibitory effect of phloroglucinol on mitochondrial complex V; where: AD represents the mitochondrial membrane potential of AML cell lines analyzed by flow cytometry; E represents the activity analysis of mitochondrial complex V; and F is a schematic diagram of the pharmacological mechanism of the compound shown in Formula 1. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The novel compound isolated and purified from Hypericum plants in this invention belongs to the phloroglucinol compound family. 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-octahydronaphth-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-en-1,3-dione. It is named long-column polycycline A, and its structural formula is shown in Formula 1.

[0029]

[0030] The present invention provides a method for preparing the phloroglucinol compound, comprising the following steps: extracting Hypericum plants (preferably Hypericum longifolium) with industrial alcohol (95% ethanol), concentrating under reduced pressure to recover the industrial alcohol, and extracting with dichloromethane to obtain a dichloromethane fraction extract; subjecting the dichloromethane fraction extract to column chromatography: specifically, mixing with silica gel and dry packing the column; then eluting with a gradient of petroleum ether-ethyl acetate, combining the same components to obtain a total of 7 components with increasing polarity; wherein component 4 is further separated by repeated gel chromatography, reversed-phase silica gel column chromatography, and high-performance liquid chromatography to obtain the phloroglucinol compound (named longifolium polycycline A).

[0031] The present invention relates to the application of the phloroglucinol compound in the treatment of acute myeloid leukemia. The compound can inhibit the activity of mitochondrial complex V in acute myeloid leukemia cells, causing inflammation and oxidative stress disorders in tumor cells, thereby inducing tumor cell apoptosis.

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

[0033] In summary, this invention preferably obtains a new compound, a secondary metabolite of *Hypericum longichum*, by separating and purifying an industrial alcohol (95% ethanol) extract from *Hypericum longichum* produced in Enshi, Hubei Province. Its structure was determined using various spectroscopic analysis methods and other techniques, and the specific structure is shown in Formula 1. Evaluation of the anti-AML activity of the compound of Formula 1 revealed that it exhibits extremely strong cytotoxic activity against two AML cell lines (NB4 cells and THP-1 cells), and can serve as a lead compound for the development of drugs to treat AML.

[0034] The following are specific embodiments.

[0035] Example 1

[0036] 1. Isolation and preparation of the resorcinol compound as shown in Formula 1

[0037] 15 kg of Hypericum perforatum was extracted seven times with industrial alcohol. The industrial alcohol was then concentrated under reduced pressure below 50°C to recover the extract. Dichloromethane was then used for extraction to obtain 650 g of the dichloromethane fraction extract. The dichloromethane fraction extract was subjected to column chromatography: specifically, the sample was mixed with 200-300 mesh silica gel and dry-packed into a column. A gradient elution with petroleum ether-ethyl acetate was used (volume ratio 30:1–0:1, eluents were added sequentially at volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1, with five columns added for each volume ratio). TLC analysis was performed, and identical fractions were combined, yielding seven fractions with increasing polarity. Fraction 4 was further separated by repeated gel permeation chromatography, reversed-phase silica gel column chromatography, and high-performance liquid chromatography to obtain a phloroglucinol compound (named long-column polycycline A) (1.3 mg).

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

[0039] The structure of the compound long-column polycycline A was determined by nuclear magnetic resonance, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, circular dichroism spectroscopy, and X-ray single-crystal diffraction.

[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 the long-pillared polycycline A was determined by X-ray single-crystal diffraction. Figure 1 This is the single-crystal diffraction pattern of the long-column polycycline A of the present invention, and the NMR data are shown in Table 1.

[0041] Table 1. HCl and C-ray spectral data (δin ppm and J in Hz) of compound long-column polycycline A

[0042]

[0043]

[0044] Example 2

[0045] The anti-AML activity and inhibitory activity against mitochondrial complex V of the compound long-column polycycline A in this invention.

[0046] ① The cytotoxic activity of compound long-column polycycline A against various malignant tumor cells was tested using the CCK8 assay.

[0047] Cells were placed in 96-well culture plates (4 × 10⁶ cells per well). 3 Cells were treated in wells for 48 hours, then incubated with CCK8 reagent at 37°C for 4 hours. Absorbance at OD 450 nm was measured using a plate reader (Synergy HT, Biotek). Cell viability and 50% inhibitory concentration (IC50) were calculated. 50 As shown in Table 2, long-column polycycline A has cytotoxic effects on various hematologic malignancies and solid tumor cells, among which it has better cytotoxic effects on NB4 and THP-1 hematologic malignancies.

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

[0049]

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

[0051] The results are as follows:

[0052] like Figure 2 As shown in Figure A, the effect of long-column polycycline A on the IC50 of NB4 cells... 50 The value is 3.588μM, IC of THP-1 50 The value was 5.760 μM. Ribosomal protein S6 is an important regulator of ribosome biogenesis and enhanced mRNA translation; its phosphorylation is closely related to tumor cell growth. For example... Figure 2 As shown in B, treatment with long-column polycycline A significantly inhibited S6 phosphorylation, providing molecular evidence for the tumor cell growth inhibitory effect of long-column polycycline A. Figure 2 As shown in C, the compound long-column polycycline A can significantly induce tumor cell apoptosis, and the average apoptosis rate in the high-dose group exceeds 60%.

[0053] AML, a cancer caused by the malignant transformation of immune cells, is significantly influenced by inflammation during its development. Therefore, we examined changes in various cytokines and chemokines after treatment with the compound long-column polycycline A. For example... Figure 3 As shown, in THP-1 and NB4 cell lines, the compound long-column polycycline A significantly upregulated the mRNA levels of various cytokines or chemokines, promoted endogenous inflammation in AML cells, and could induce tumor cell growth inhibition or apoptosis.

[0054] Given that endogenous cellular inflammation is often accompanied by oxidative stress, and that interfering with cellular oxidative stress is considered an effective target for anticancer drugs, we analyzed whether compounds exert their pharmacological effects by inducing oxidative stress. Figure 4 As shown in Figure A, treatment with compound long-column polycycline A for 48 h significantly increased the transcription of oxidative stress-related genes such as Trib3, Gdf15, Sesn2, ATF4, EIF4EBP1, and TXNIP in AML cells. AMPK plays an important role in cellular metabolism; as an oxidative stress-sensitive protein kinase, it switches pathways on or off under energy stress to maintain systemic energy balance. Figure 4As shown in Figure B, Western blot analysis revealed a significant increase in phosphorylated AMPK after 48 hours of compound treatment, while total AMPK remained largely unaffected. The increase in phosphorylated AMPK represents AMPK activation, a response to oxidative stress. This data indicates that treatment with the compound long-column polycycline A increased oxidative stress in AML cells. These data suggest that long-column polycycline A exerts its apoptosis-inducing effect by increasing oxidative stress in 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 intracellular metabolic changes that play a crucial role in apoptosis. Therefore, we examined changes in ROS levels in AML cells. Figure 4 As shown in Figure C, after 12 hours of compound treatment, ROS levels in AML cells increased, which may be the reason for oxidative stress and the initiation of apoptosis. Subsequently, after 48 hours of compound treatment, ROS levels in AML cells were lower than in the DMSO group.

[0055] Mitochondria are the central organelles for cellular respiration, and maintaining the mitochondrial membrane potential is crucial for the normal function of the mitochondrial respiratory chain. For example... Figure 5 As shown in the AD diagram, the compound significantly increased the mitochondrial membrane potential of THP-1 and NB4 cell lines at 12 hours. This effect is unique because most antitumor drugs reduce mitochondrial membrane potential by directly disrupting 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, reducing the mitochondrial potential induced by the effects of other complexes by delivering protons into the mitochondrial matrix. Therefore, we tested whether the compound directly reduced the activity of mitochondrial complex V. Figure 5 As shown in E, in mitochondria isolated from mouse liver, we found that the compound significantly inhibited the activity of mitochondrial complex V. Figure 5 F in the diagram represents the mechanism of action of the compound against tumors.

[0056] The method is as follows:

[0057] Acute leukemia cells were treated with a compound called long-column polycycline A at concentration gradients of 2.5–10 μM for 48 h. Cells were collected, total protein was extracted, and the protein concentration of the lysate was determined using the BCA protein assay reagent. Normalized amounts of the lysate sample were loaded onto a 10% SDS-PAGE plate and transferred to an NC membrane. Immunoblots were performed using different antibodies, with α-tubulin as a loading control. The wavelength range was detected using a LI-COR near-infrared imaging system.

[0058] The generation of ROS in tumor cells was analyzed using a fluorescent probe DCFH-DA kit. Cells were seeded at 1×10⁶ cells / day. 5 Cells were incubated with compound 1 in 12-well plates for 48 h, and then harvested. The cells were then incubated 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] Mitochondrial membrane potential was analyzed using a fluorescent probe TMRE kit. Cells were seeded at 1×10⁶ cells / day. 5 Cells were incubated with compound 1 in 12-well plates for 12 h, and then harvested. Cells were incubated with TMRE at 37 °C for 5 min. The positive control CCCP was incubated with TMRE at a concentration of 5 μM. The fluorescence signal of TMRE was detected by flow cytometry.

[0060] The activity of mitochondrial complex V was detected using a mitochondrial respiratory chain complex V / ATP synthase activity assay kit. The test compound and positive control were incubated with mitochondrial lysis buffer for 30 minutes, and then the activity of mitochondrial complex V was measured.

[0061] The effect of the test compound on tumor cell apoptosis was tested using the Annexin V-FITC apoptosis detection kit. After treating the compound for 48 h, cells were collected, stained with light at 25 °C for 10 min, and the apoptosis rate was detected by flow cytometry.

[0062] Total RNA from cells was analyzed using TRIzol. TM Reagent isolation, cDNA used Synthesis of II Q RT SuperMix. SYBR Green qPCR Mix was purchased from Biosharp. The primers used are as follows:

[0063]

[0064] The analysis is as follows:

[0065] The novel compound long-column polycycline A, isolated from the Hypericum genus, exhibits good antitumor activity, particularly against AML cell lines, and can serve as a lead compound for the development of antiAML drugs.

[0066] Those skilled in the art will readily understand that the above description is merely 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 within the scope of protection of the present invention.

Claims

1. The application of phloroglucinol compounds in the preparation of drugs for treating acute myeloid leukemia tumors, wherein the structural formula of the phloroglucinol compound is shown in Formula 1: Formula 1.

2. The application as described in claim 1, characterized in that, The phloroglucinol compound is used to inhibit the activity of mitochondrial complex V in acute myeloid leukemia tumor cells, thereby causing intracellular inflammation and oxidative stress disorders in acute myeloid leukemia tumor cells, resulting in apoptosis of acute myeloid leukemia tumor cells.

3. The application according to claim 2, characterized in that, The tumor cells are NB4 cells and / or THP-1 cells.

4. The application according to claim 1, characterized in that, The resorcinol compound was prepared by the following steps: (1) The plants of the genus Hypericum were extracted with alcohol, and the alcohol was recovered by vacuum concentration. Then, the extract was obtained by dichloromethane extraction. (2) The dichloromethane fraction extract obtained in step (1) was subjected to column chromatography and then eluted with petroleum ether and ethyl acetate gradient to obtain 7 components in order of increasing polarity. The fourth component was then separated by gel chromatography, reversed-phase silica gel column chromatography and high performance liquid chromatography to obtain the phloroglucinol compound.

5. The application according to claim 4, characterized in that, The St. John's wort plant in question is St. John's wort.

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

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