A phloroglucinol compound, its separation and application in preparing anti-psoriasis drugs

Abstract

The present invention relates to phloroglucinol compounds, separation and application thereof in the preparation of anti-psoriatic drugs, and belongs to the field of medical technology. The new compound hypericum polyketone A isolated from Hypericum sibiricum of the present invention is a novel TNF-α small molecule inhibitor that can target and inhibit TNF-α to effectively block multiple inflammatory factors and improve the symptoms of psoriasis epidermal hyperplasia. It has good anti-psoriatic activity and can be used as a lead compound for the development of drugs for the treatment of psoriasis. The present invention provides alternative compounds for the development of new anti-psoriatic drugs and is of great significance to the 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 the separation of a phloroglucinol compound and its application in the preparation of anti-psoriatic drugs, especially to the separation and purification process, structural confirmation and anti-psoriatic activity of compound 1. Background Art

[0002] Psoriasis is a chronic, immune-mediated inflammatory skin disease triggered by a combination of genetic and environmental factors. Its global prevalence is approximately 2% to 3%, and has been increasing annually in recent years. It has become a major health hazard to patients, both physically and mentally. Treatments include topical, physical, and systemic therapies, but these remain incurable (The Lancet, 2021, 397, 1301). Therefore, it is urgent to gain a deeper understanding of the pathogenesis of psoriasis, explore new therapeutic targets, and identify highly effective and low-toxic anti-psoriatic drugs.

[0003] Research has shown that the pathogenesis of psoriasis is linked to multiple immune cells, inflammatory cells, and inflammatory pathways. Tumor necrosis factor α (TNF-α) plays a key role, participating in multiple stages of psoriasis development. It is a key proinflammatory cytokine and can interact with other cytokines to amplify the inflammatory response. Therefore, inhibiting TNF-α can reduce excessive keratinocyte proliferation and inflammation, making it an important target for the treatment of psoriasis (Drug Discov. Today, 2023, 28, 103575). At present, macromolecular drugs targeting TNF-α, such as infliximab, adalimumab, etanercept, and benzaluzumab, have been widely used in the clinical treatment of psoriasis. However, biological macromolecular drugs have inherent disadvantages, such as high price, single administration method, and long-term use will increase the risk of tumors and infections (Int.J.Mol.Sci., 2019, 20, 1475; Ann.Rheum.Dis., 2020, 79, 285). In contrast, TNF-α small molecule inhibitors have the advantages of low price and convenient oral administration, making TNF-α small molecule inhibitors a new strategy for the development of highly effective and low-toxic anti-psoriasis drugs.

[0004] Natural products are an important source of drugs. Among the small molecule drugs launched between 1981 and 2019, up to 68% of the drug molecules were directly or indirectly derived from natural products and their derivatives. Natural products themselves have many natural advantages, such as complex and diverse structures, significant activity, low toxicity and side effects, and short R&D cycles. These advantages provide more options for their transformation into drugs, making them a hot topic in drug research and development (J. Nat. Prod., 2020, 83, 770). It is worth noting that many drugs used clinically to treat psoriasis are derived from medicinal plants, such as tripterygium wilfordii glycosides, glycyrrhizic acid, and total glucosides of white paeony (Chin. J. Dermatol., 2023, 56, 573). Therefore, it is of great significance to find lead compounds with novel structures for the treatment of psoriasis from medicinal plants. Summary of the Invention

[0005] The present invention aims to provide novel compounds from Hypericum plants with anti-psoriatic activity, as well as methods for their isolation, purification, and application. The compounds described in this invention are potential small-molecule TNF-α inhibitors with excellent anti-psoriatic activity and could serve as lead compounds for the development of anti-psoriatic drugs.

[0006] 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:

[0007]

[0008] According to another aspect of the present invention, a method for separating the phloroglucinol compound is provided, comprising the following steps:

[0009] (1) crushing a Hypericum plant and then performing alcohol extraction, wherein the Hypericum plant is Hypericum davidianum, concentrating under reduced pressure to recover alcohol, and then extracting with dichloromethane to obtain a dichloromethane extract;

[0010] (2) The dichloromethane extract obtained in step (1) is subjected to column chromatography, and then gradient elution with petroleum ether-ethyl acetate is performed to obtain 7 components with increasing polarity. The fourth component is sequentially subjected to MCI column chromatography, gel chromatography, positive and reverse phase silica gel column chromatography, and high performance liquid chromatography to obtain the phloroglucinol compound.

[0011] Preferably, during the elution process, the petroleum ether and ethyl acetate are eluted in the volume ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 3:1 and 0:1 in sequence.

[0012] According to another aspect of the present invention, there is provided a use of the phloroglucinol compound for preparing a TNF-α inhibitory drug.

[0013] According to another aspect of the present invention, there is provided the use of the phloroglucinol compound for preparing an anti-psoriasis drug.

[0014] Preferably, the phloroglucinol compound is used to inhibit the effects of inflammatory factors in HaCaT cells after TNF-α stimulation.

[0015] Preferably, the phloroglucinol compound is used to improve the symptoms of psoriasis epidermal hyperplasia.

[0016] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0017] (1) A novel phloroglucinol compound was discovered from the medicinal plant Hypericum scabra. The structural type of this compound is discovered for the first time and has certain innovation in its chemical structure.

[0018] (2) Further biological activity evaluation results showed that the novel compound is a small molecule inhibitor of TNF-α, has strong binding affinity with TNF-α, can significantly inhibit the death of L929 cells induced by TNF-α and actinomycin D, and has a strong improvement effect on the severity of skin erythema and scaling, skin thickness and CSS scores of psoriatic mice, so it has good anti-psoriatic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the structural formula of the compound hypericum ketone A.

[0020] Figure 2 This is the single crystal diffraction pattern of the compound hypericum ketone A.

[0021] Figure 3 The CCK-8 method, DAPI staining analysis, surface plasmon resonance and other methods were used to verify that the compound hypericum ketone A can target and inhibit the activity of TNF-α; among them: A is the effect of compound hypericum ketone A on the viability of L929 cells; B–C are the viability, morphology and DAPI staining analysis of L929 cells after treatment with compound hypericum ketone A, TNF-α and actinomycin D; D–E are the effects of compound hypericum ketone A on the thermal stability of TNF-α protein analyzed by CETSA and TSA; F is the SPR experiment to determine the binding affinity of compound hypericum ketone A to TNF-α; G is the analysis of the binding mode and binding energy of compound hypericum ketone A to TNF-α by computer molecular docking technology.

[0022] Figure 4Figure 1 shows the effects of the test compound hypericum ketone A on inflammatory factors in TNF-α-induced HaCaT cells. Figure A shows the effect of hypericum ketone A on HaCaT cell viability; Figures B–D show the mRNA expression levels of IL-23, CXCL1, and S100A9 analyzed by RT-qPCR; and Figure E shows the effect of hypericum ketone A on TNF-α-induced NF-κB p65 phosphorylation and nuclear translocation.

[0023] Figure 5 This study analyzed the pharmacodynamic effects of hypermetholone A on mice with imiquimod (IMQ)-induced psoriasis. Figure A shows the appearance of skin lesions in the control, hypermetholone A, and tacrolimus-treated mice after 7 consecutive days of treatment. Figure B shows the quantification of CSS in each group. Figure C shows the appearance and histological characteristics of the skin lesions in each group. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is 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 for the purpose of explaining 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 may be combined with each other as long as they do not conflict with each other.

[0025] The new compound isolated and purified from Hypericum plants in the present invention belongs to the phloroglucinol class of compounds, has a chemical name of (1R,3a'R,5S,6a'R)-3-benzoyl-1',1',4-trimethyl-3a',5-bis(3-methylbut-2-en-1-yl)-6',6a'-dihydro-1'H,3'H-spiro[cyclohexane-1,5'-cyclopenta[c]furan]-3-ene-2,3',4'(3a'H)-trione, and is named hypericum pyrogallol A. The structural formula is shown in Formula 1.

[0026]

[0027] The preparation method of the mesoglucosidol compound of the present invention comprises the following steps: using industrial alcohol (95% ethanol) to extract Hypericum plant (preferably Hypericum sibiricum) and recovering the industrial alcohol by decompression concentration, and extracting with dichloromethane to obtain a dichloromethane extract; subjecting the dichloromethane extract to column chromatography: specifically, mixing the sample with silica gel and packing the column by dry method; then eluting with petroleum ether-ethyl acetate gradient, combining similar fractions, and obtaining a total of 7 components with increasing polarity; wherein component 4 is separated and purified repeatedly by MCI chromatography column, gel chromatography column, positive and reverse phase silica gel column chromatography column, and high performance liquid chromatography to obtain compound hypericum polyol A.

[0028] The present invention discloses an application of the mesopyrogallol compound in treating psoriasis. The compound can inhibit the activity of TNF-α through targeted inhibition, thereby inhibiting the inflammatory response, and can inhibit the excessive proliferation of epidermal cells to alleviate the epidermal hyperplasia symptoms of psoriasis.

[0029] In summary, the present invention preferably isolates and purifies an industrial alcohol (95% ethanol) extract of Hypericum sibiricum, a plant native to Enshi, Hubei Province, to obtain a new compound, a secondary metabolite of Hypericum sibiricum. Its structure was determined using various spectral analysis methods and other methods, and its specific structure is shown in Formula 1. Evaluation of the anti-psoriatic activity of the compound of Formula 1 revealed that it is a small molecule inhibitor of TNF-α and is able to significantly alleviate the symptoms of psoriasis epidermal hyperplasia, making it a potential lead compound for the development of psoriasis treatment drugs.

[0030] The following are specific embodiments

[0031] Example 1

[0032] 1. Separation and preparation of the compound hypericum polyketone A as shown in formula 1

[0033] 35 kg of Hypericum leaf parts were extracted 10 times with industrial alcohol, concentrated under reduced pressure below 50°C to recover the industrial alcohol, and then extracted with dichloromethane to obtain 2.5 kg of dichloromethane extract. The dichloromethane extract was subjected to column chromatography: specifically, the sample was mixed with 80–120 mesh silica gel and dry-packed on a 1.5 m high and 20 cm diameter column. Elution was performed with a petroleum ether-ethyl acetate gradient using the following solvent ratios: petroleum ether:ethyl acetate = 50:1, 40:1, 30:1, 20:1, 10:1, 3:1, and 0:1, with 20 L of elution at each ratio. TLC analysis was performed, and similar fractions were combined to yield seven fractions (fractions 1 to 7) in increasing polarity. Component 4 was subjected to gradient elution on a reverse phase medium pressure chromatography column with a column height of 0.6 m and a column diameter of 10 cm, with a methanol-water gradient elution, the solvent ratios being methanol:water = 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, and 1:0, with 5 L of elution at each ratio, and TLC detection was performed. Similar components were combined to obtain a total of 6 subcomponents (components 4.1 to 4.6) with increasing polarity. Component 4.3 was separated by high performance liquid chromatography using a Yuexu AQ-C18 column (column length 20 cm, column diameter 2 cm) with separation conditions of acetonitrile:water = 23:77, a flow rate of 2 ml / min, a detection wavelength of 254 nm, and a retention time of 97 min to obtain hypericum polyketone A.

[0034] 2. Structural identification of the compound hypericum polyketone A shown in formula 1

[0035] The compound hypericum ketone A was subjected to data tests such as nuclear magnetic resonance, mass spectrum, optical rotation, infrared spectrum, ultraviolet spectrum, circular dichroism, etc., and hypericum ketone A was subjected to data test of X-ray single crystal diffraction to determine the structure of the compound.

[0036] Hypericum peroxidase A: Colorless block crystals; mp 139.4–143.8℃; UV(CH3OH)λ max (logε)=201(4.52)nm; IR(KBr)v max 2980,2919,2856,1776,1729,1679,1656,1619,1597,1449,1378,1324,1264,1168,1131,954and 691cm –1 ;ECD(CH3OH)λ max(Δε) 204 (+8.12), 241 (-7.14), 249 (-0.41), 269 (-4.45), 337 (+1.05) nm. The absolute configuration of hypericum ketone A was determined by X-ray single crystal diffraction. The crystal structure is as follows Figure 1 The NMR data are shown in Table 1. The single crystal diffraction pattern is shown in Figure 2 shown.

[0037] Table 1. Compound Hypericum quinone A 1 H and 13 C NMR data (δ in ppm and J in Hz)

[0038]

[0039] a 1 H NMR (600 MHz) and 13 C NMR(150MHz)recorded in CD3OD

[0040] Example 2

[0041] The inhibitory activity of the compound hypericum polyketone A on TNF-α and its anti-psoriasis activity

[0042] ① CCK-8 assay, DAPI staining analysis, surface plasmon resonance and other methods were used to verify that the compound hypericum polyketone A can target and inhibit the activity of TNF-α

[0043] The inhibitory activity of the compound hypericum ketone A against TNF-α was tested in L929 cells. First, the compound hypericum ketone A had no significant cytotoxicity to L929 cells at a concentration of 40 μM, and had a significant protective effect against L929 cell death induced by TNF-α and actinomycin D in a concentration-dependent manner ( Figure 3 A in Figure 3 and B in Figure 3). DAPI staining results also showed that compound hypericum polyketone A can effectively protect L929 cells from cell death and inhibit TNF-α, even better than the positive control SPD304 ( Figure 3 In addition, the cell thermal shift assay (CETSA), thermal shift assay (TSA) and surface plasmon resonance (SPR) assay were used to evaluate the binding ability of the compound hypericum polyketone A to TNF-α. The results showed that compared with the DMSO group ( Figure 3 Compared with D in Figure 3 and E in Figure 3), the compound hypericum ketone A increased the cell thermal stability of TNF-α from the tolerable temperature of 82°C to ℃, and the tolerable temperature was significantly improved. SPR experiments also showed that the compound hypericum ketone A can directly bind to TNF-α with a KD value of 5.48μM, with a strong affinity ( Figure 3 F in). At the same time, the binding mode and binding energy of the compound hypericum multiolone A with TNF-α were determined by computer molecular docking, such as Figure 3 As shown in Figure 1 (G), the binding pocket of the TNF-α trimer is relatively sharp and spindle-shaped. Hypericum glutinosin A completely occupies the binding site of the crystallographic ligand SPD304, preventing the assembly of a biologically active trimeric complex. Hypericum glutinosin A forms multiple hydrophobic interactions with Ile133, Leu233, Tyr135, and Tyr227 of the TNF-α monomer. Notably, the benzyl group in hypericum glutinosin A exhibits a strong π-π interaction with the Tyr195C residue, which plays a crucial role in stabilizing ligand binding.

[0044] ②RT-qPCR and immunofluorescence were used to test the effect of compound hypericum polyketone A on inflammatory-related factors in HaCaT cells induced by TNF-α

[0045] The effects of the compound hypericum polyketone A on key psoriasis-related signaling pathways in HaCaT cells were examined. First, the compound hypericum polyketone A showed significant inhibitory activity against TNF-α-induced inflammatory factors IL-23, CXCL1, and S100A9, with low toxicity ( Figure 4 In addition, immunofluorescence results showed that the compound hypericum ketone A could inhibit the nuclear translocation of NF-κB p65 induced by TNF-α ( Figure 4 The above results indicate that the compound hypericum polyketone A can effectively block multiple inflammatory factors by targeting and inhibiting TNF-α.

[0046] ③ Detect the pharmacodynamic effect of compound hypericum ketone A on imiquimod (IMQ)-induced psoriasis mice

[0047] The anti-psoriatic activity of the compound hypermelone A was evaluated in an IMQ-induced psoriasis mouse model. Figure 5 A and Figure 5 As shown in Figure B, the skin of mice in the IMQ group was significantly thickened and erythematous, and the CSS score continued to increase. However, the severity of skin erythema and scaling, skin thickness, and CSS score of mice in the hypericum A treatment group were reduced. HE staining results showed that mice in the IMQ group had obvious epidermal hyperplasia, scaling, and severe inflammatory reactions, while hypericum A and the positive drug tapinarof could improve these symptoms ( Figure 5 C in the figure). Further immunohistochemical results showed that ( Figure 5C), Ki67 expression was upregulated and IL-17 was continuously expressed in the upper layer of the skin of mice in the IMQ group, while Ki67 expression was downregulated and IL-17 expression was discontinuous in the group treated with compound hypericum peroxidase A, indicating that compound hypericum peroxidase A can alleviate the epidermal hyperplasia symptoms of psoriasis by inhibiting the excessive proliferation of epidermal cells.

[0048] Results and Analysis:

[0049] Hypericum perforatum A, a new compound isolated from Hypericum plants, can effectively block multiple inflammatory factors by targeting and inhibiting TNF-α, and can improve the symptoms of psoriasis epidermal hyperplasia by inhibiting the excessive proliferation of epidermal cells. It has excellent anti-psoriatic activity and can be used as a lead compound for the development of drugs for the treatment of psoriasis.

[0050] It will be easily understood by those skilled in the art 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 in the scope of protection of the present invention.

Claims

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

2. The phloroglucinol compound as claimed in claim 1 is used to prepare TNF- α Inhibit the use of drugs.

3. Use of the phloroglucinol compound according to claim 1 for preparing an anti-psoriasis drug.

4. The use according to claim 3, characterized in that The phloroglucinol compound is used for improving the symptoms of psoriasis epidermal hyperplasia.

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