Phloroglucinol compounds and use thereof in the preparation of antiviral drugs

Abstract

This invention belongs to the field of natural and chemical medicine technology, and discloses two types of *Symplocos edulis* (a type of plant) from the genus *Symplocos*. Bag Philippians The resorcinol compounds were isolated from [the organism]. The structures of the resorcinol compounds are shown in formula (I) or formula (II). The resorcinol compounds of this invention exhibit significant inhibitory effects on respiratory syncytial virus (RSV) and herpes simplex virus (HSV) even at low concentrations, and show low cytotoxicity and high selectivity, demonstrating promising applications in antiviral drugs and medications for treating diseases caused by RSV and HSV infections.

Description

Technical Field

[0001] This invention belongs to the field of natural and chemical drugs, specifically relating to two phloroglucinol compounds with significant antiviral activity, their preparation methods, and their application in the preparation of antiviral drugs. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading pathogens causing severe lower respiratory tract infections in infants, young children, and the elderly worldwide, characterized by high infectivity and seasonal prevalence. This virus primarily attacks respiratory epithelial cells, causing cell fusion to form syncytia, which in turn leads to serious diseases such as bronchitis and pneumonia. Current treatment options for RSV are extremely limited. While monoclonal antibody drugs (such as nisecumab) and vaccines are available for passive immunization and prevention, the development of specific antiviral drugs for already infected patients is slow, and a mature clinical treatment system has not yet been established, relying heavily on symptomatic treatment and immune modulation.

[0003] Herpes simplex virus (HSV) is a DNA virus that commonly causes skin and mucous membrane herpes and severe eye infections. HSV infection is characterized by significant latency; the virus can remain dormant in nerve ganglia for many years, and recurrence is highly likely once the body's immunity is weakened or stimulated. Currently, clinical treatment mainly relies on nucleoside analogues for antiviral therapy. While effective in the acute phase, long-term use may lead to drug resistance, and the eradication effect on the virus is limited. Furthermore, some patients are insensitive to existing antiviral drugs or face the risk of nephrotoxicity, making new, safe, and effective treatments urgently needed. Therefore, the development of novel drugs with broad-spectrum antiviral activity and fewer side effects has significant clinical application value.

[0004] Coarse chaff firewood ( Mallotus philippensis ) belongs to the genus *Pterocarya* of the family Euphorbiaceae. Mallotus This plant, widely distributed in tropical and subtropical Asia, has a long history of use in traditional medicine, primarily for treating skin parasitic infections and gastrointestinal diseases. Modern pharmacological studies have shown that extracts of *Branchium coronarium* possess various biological activities, including antibacterial, anti-inflammatory, antitumor, and antiviral activity. However, its antiviral active components remain unclear and require further research and exploration. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides two phloroglucinol compounds isolated from coarse firewood, their preparation methods, and their applications.

[0006] The technical solution adopted in this invention is: A compound of formula (I) or formula (II) or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate, or a prodrug:

[0007] This invention provides a method for preparing the aforementioned phloroglucinol compounds, wherein the fruit of *Cinnamomum camphora* is extracted with an organic solvent to obtain a crude extract; the crude extract is separated by column chromatography and purified by high performance liquid chromatography to obtain the aforementioned phloroglucinol compounds.

[0008] Specifically, the following steps are included: S1. The fruit of *Cinnamomum camphora* was extracted by reflux with methanol, and the crude extract was obtained by concentration under reduced pressure. S2. The crude extract described in S1 was subjected to silica gel column chromatography, using a petroleum ether-ethyl acetate mixed solution (20:1→0:100) as the mobile phase for gradient elution to obtain fractions Fr.A~Fr.J; S3. Select the active fraction Fr.I for silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate mixed solution (50:1→0:100) to obtain 14 sub-fractions (Fr.I1~Fr.I14). S4. The active fractions Fr.I8 and Fr.I6 obtained in step S3 were subjected to reverse-phase ODS column chromatography. Fr.I8 was eluted with a gradient using a methanol-water mixture (50:50→100:0) as the mobile phase, and Fr.I6 was eluted with a methanol-water mixture (40:60→100:0) as the mobile phase, to obtain sub-fractions Fr.I8a~Fr.I8r and Fr.I6a~Fr.I6h, respectively. S5. The active fraction obtained in step S4 is separated and purified by preparative high performance liquid chromatography, with acetonitrile-water as the mobile phase.

[0009] In step S1, methanol reflux extraction is performed 2-3 times.

[0010] In step S5, Fr.I8g was purified by semi-preparative HPLC (acetonitrile-water 60:40-70:30) to obtain compound (I); Fr.I6f was eluted with methanol as the mobile phase by gel HW-40F column chromatography to obtain fractions Fr.I6f1~Fr.I6f7, of which fraction Fr.I6f3 was purified by semi-preparative HPLC (acetonitrile-water 70:30-80:20) to obtain compound (II).

[0011] The present invention provides a pharmaceutical composition comprising one or two of the above-mentioned compounds, their stereoisomers, pharmaceutically acceptable salts or solvates, or prodrugs as active ingredients.

[0012] The pharmaceutical composition comprises the active ingredient as described above and a pharmaceutically acceptable carrier or excipient.

[0013] This invention provides the use of the above-mentioned compounds, their stereoisomers, pharmaceutically acceptable salts, solvates, or prodrugs in the preparation of medicaments for treating and / or preventing viral infections.

[0014] The present invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for treating and / or preventing viral infections.

[0015] The viral infection mentioned is either respiratory syncytial virus (RSV) infection or herpes simplex virus (HSV) infection.

[0016] This invention provides the use of the above-mentioned compounds, their stereoisomers, pharmaceutically acceptable salts, solvates, prodrugs, or pharmaceutical compositions thereof in the preparation of medicaments for treating diseases such as bronchitis, pneumonia, oral herpes, and genital herpes caused by RSV or HSV.

[0017] This invention isolates and identifies two novel phloroglucinol compounds from *Branchum paniculatum* extract. These compounds exhibit significant inhibitory effects against respiratory syncytial virus (RSV) and herpes simplex virus (HSV) at low concentrations, with low cytotoxicity to host cells and high selectivity. Therefore, the compounds of this invention can be used to prepare antiviral drugs, particularly anti-RSV or HSV drugs, and can also be used to prepare drugs for treating diseases caused by RSV or HSV infection. Attached Figure Description

[0018] Figure 1 For the compound Mallophilone A 1 1H NMR (400MHz, CDCl3) spectrum.

[0019] Figure 2 For the compound Mallophilone A 13 C10 NMR (100MHz, CDCl3) spectrum.

[0020] Figure 3 The image shows the HR-ESI-MS spectrum of compound Mallophilone A.

[0021] Figure 4 For the compound Mallophilone J 1 1H NMR (400MHz, CDCl3) spectrum.

[0022] Figure 5 For the compound Mallophilone J 13 C10 NMR (100MHz, CDCl3) spectrum.

[0023] Figure 6The image shows the HR-ESI-MS spectrum of the compound Mallophilone J. Detailed Implementation

[0024] To better understand the essence of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0025] Example 1: Extraction, separation, and structural identification of compounds Mallophilone A and Mallophilone J: (1) Coarse chaff firewood ( Mallotus philippensis 24 kg of dried fruit was crushed and extracted three times by reflux with methanol. After concentration under reduced pressure, 655 g of crude extract of total extract was obtained.

[0026] (2) The crude extract was subjected to silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate mixture (20:1→0:100) as the mobile phase. The fractions were analyzed by thin-layer chromatography (TLC) and combined to obtain 10 fractions Fr.A~Fr.J. Among them, fraction Fr.I (1:1 fraction) 119.7 g was subjected to silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate mixture (50:1→0:100) to obtain 14 sub-fractions Fr.I1~Fr.I14. The fractions Fr.I8 (3:1 fraction) and Fr.I6 (5:1 fraction) were subjected to reversed-phase ODS column chromatography, with gradient elution using methanol-water mixed solutions (50:50→100:0) and (40:60→100:0) as mobile phases, to obtain sub-fractions Fr.I8a~Fr.I8r and Fr.I6a~Fr.I6h.

[0027] The fraction Fr.I8g (75:25 fraction) was separated and purified by reversed-phase semi-preparative HPLC. The mobile phase was acetonitrile-water with a volume ratio of 65:35 and the flow rate was 4 mL / min. The eluent with a retention time of 33 min was obtained, concentrated and dried to obtain the compound Mallophilone A.

[0028] The fraction Fr.I6f (80:20 fraction) was eluted by HW-40F gel column chromatography with methanol as the mobile phase to obtain fractions Fr.I6f1~Fr.I6f7. Among them, fraction Fr.I6f3 (85:15-90:10 fraction) was purified by reversed-phase semi-preparative HPLC with acetonitrile-water at a volume ratio of 79:21 as the mobile phase to obtain an eluent with a retention time of 26 min. The eluent was concentrated and dried to obtain the compound Mallophilone J.

[0029] Structural identification of Mallophilone A: Yellow powder; HR-ESI-MS showed quasi-molecular ion peaks. m / z 685.2985 [M+H] + (calcd for C 40 H 45 O 10 + (685.3007), see the atlas. Figure 3 ; 1 H NMR and 13 The C NMR data are shown in Table 1, and the spectra are shown in Table 2. Figures 1-2 .

[0030] Structural identification of Mallophilone J: Yellow powder; HR-ESI-MS showed quasi-molecular ion peaks. m / z 477.2634 [M+H] + (calcd for C 30 H 37 O5 + (477.2636), see the atlas. Figure 6 ; 1 H NMR and 13 The C NMR data are shown in Table 1, and the spectra are shown in Table 2. Figures 4-5 .

[0031] Based on the above physicochemical and spectral data, the structures of Mallophilone A and Mallophilone J were identified as shown in formulas (I) and (II).

[0032]

[0033] Table 1. Mallophilone A and Mallophilone J 1 H NMR (400 MHz) and 13 C NMR (100MHz) data

[0034]

[0035] Example 2 Inhibitory effects of Mallophilone A and Mallophilone J on respiratory syncytial virus (RSV) and herpes simplex virus (HSV) The inhibitory effects of the compounds on respiratory syncytial virus and herpes simplex virus were tested using the cytopathic effect method, with ribavirin and acyclovir as positive control drugs. The specific procedures are as follows: Assay for drug cytotoxicity: Drug cytotoxicity was determined using the Cell Counting Kit-8 (CCK-8) assay. HEp-2 cells were grown in Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HEp-2 cells were seeded in 96-well plates (1.8 × 10⁶ cells / well). 4 Cells were incubated overnight at 37°C with 5% CO2 until a monolayer formed. Cells were then treated with different concentrations (0.75, 1.5, 3.125, 6.25, 12.5, 25, 50, 100 μM) of either a compound or ribavirin. After 24 hours, 10 μL of CCK-8 solution was added to each well, and the culture plate was placed in the dark for a period of time. During this period, color changes were observed, and absorbance (OD value) was measured at 450 nm using an enzyme immunoassay analyzer. The median lethal concentration (LD50) of the compound was calculated by regression analysis of the dose-response curve derived from the OD values. 50 ).

[0036] Antiviral activity screening of drugs: The anti-RSV or HSV activity of compounds was determined by cytopathic effects. HEp-2 cells were seeded in 96-well plates (1.8 × 10⁻⁶). 4 Cells were incubated in wells at 37°C and 5% CO2 overnight until they formed a monolayer. Different RSV / RSV-RFP virus strains were diluted with cell maintenance medium and used to infect the cells. Different concentrations of compounds or ribavirin were then used for treatment. After culturing for 24 hours at 37°C and 5% CO2, cell viability in each well was observed and recorded under a microscope. After 30–48 hours, when the virus control group showed complete cytopathic effects, the RSV or HSV-infected groups were observed under a microscope for cell cytopathic effects in each well, and the half-maximal effective concentration (IC50) of the compound was recorded. 50 The results are shown in Tables 2 and 3.

[0037]

[0038]

[0039] The above experimental results show that Mallophilone A and Mallophilone J obtained in Example 1 of this invention have significant inhibitory effects on respiratory syncytial virus (RSV) and herpes simplex virus (HSV), and both have an inhibitory effect on the IC50 of RSV. 50 The values ​​were 2.98 ± 0.34 μM and 3.10 ± 0.29 μM, respectively, and no significant toxicity was observed to host cells (CC). 50 The values ​​were high, and the selectivity index (SI) was greater than 32; Mallophilone J had an IC50 value for herpes simplex virus. 50The effective concentration was 3.19 ± 0.43 μM, with a high selectivity index (SI > 31.3); Mallophilone A exhibited moderate anti-HSV activity. This indicates that Mallophilone A and Mallophilone J possess good safety and excellent antiviral efficacy.

[0040] In summary, the phloroglucinol compounds Mallophilone A and Mallophilone J isolated and purified in this invention exhibit significant inhibitory effects against both respiratory syncytial virus (RSV) and herpes simplex virus (HSV). Mallophilone J shows excellent inhibitory effects against both RSV and HSV, with high selectivity and good safety. Mallophilone A demonstrates particularly strong inhibitory activity and high selectivity against RSV (SI=33.6). Therefore, the phloroglucinol compounds of this invention, or compositions containing such compounds, can be used in the preparation of antiviral drugs. These antiviral drugs can be used to treat and / or prevent RSV and HSV infections, and can also be used to treat bronchitis and pneumonia caused by RSV, as well as oral herpes, genital herpes, and other diseases caused by HSV.

[0041] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A phloroglucinol compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition, characterized in that: It comprises one or two of the phloroglucinol compounds of claim 1 or their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier or excipient.

3. A pharmaceutical preparation, characterized in that: It comprises the phloroglucinol compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2.

4. The method for preparing the phloroglucinol compound according to claim 1, characterized in that, Includes the following steps: S1. Extract the fruit of *Cynanchum paniculatum* using an organic solvent, and concentrate under reduced pressure to obtain a crude extract; S2. The crude extract described in S1 was subjected to silica gel column chromatography, using a petroleum ether-ethyl acetate mixed solution with a volume ratio of 20:1→0:100 as the mobile phase for gradient elution, and the fractions Fr.A~Fr.J were separated. S3. Select the active fraction Fr.I for silica gel column chromatography, using a gradient elution of a petroleum ether-ethyl acetate mixed solution with a volume ratio of 50:1 to 0:100 to obtain sub-fractions Fr.I1~Fr.I14; S4. The active fractions Fr.I8 and Fr.I6 obtained in step S3 were subjected to reverse-phase ODS column chromatography. Fr.I8 was eluted with a gradient of a methanol-water mixture with a volume ratio of 50:50 to 100:0, and Fr.I6 was eluted with a methanol-water mixture with a volume ratio of 40:60 to 100:0, to obtain sub-fractions Fr.I8a~Fr.I8r and Fr.I6a~Fr.I6h, respectively. S5. The active fractions Fr.I8g and Fr.I6f obtained in step S4 were separated and purified by preparative high performance liquid chromatography with acetonitrile-water as the mobile phase, to obtain compounds of formula (I) and formula (II).

5. The preparation method according to claim 4, characterized in that: In step S1, the organic solvent is 90-100% methanol, the extraction method is reflux extraction, and the extraction is performed 2-3 times.

6. The preparation method according to claim 4, characterized in that: In step S5, Fr.I8g is purified by semi-preparative HPLC to obtain compound (I), with the mobile phase being acetonitrile-water at a volume ratio of 60:40-70:

30.

7. The preparation method according to claim 4, characterized in that, In step S5, Fr.I6f is eluted by gel column chromatography with methanol as the mobile phase to obtain fractions Fr.I6f1~Fr.I6f7, wherein fraction Fr.I6f3 is purified by semi-preparative HPLC to obtain compound (II), and the mobile phase is acetonitrile-water with a volume ratio of 70:30-80:

20.

8. The use of the phloroglucinol compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, or the pharmaceutical preparation of claim 3, in the preparation of a medicament for the treatment and / or prevention of viral infections.

9. The application according to claim 8, characterized in that, The virus in question is either respiratory syncytial virus or herpes simplex virus.

10. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, or the pharmaceutical preparation of claim 3 in the preparation of a medicament for treating bronchitis, pneumonia, oral herpes, or genital herpes caused by respiratory syncytial virus or herpes simplex virus.

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