TWI898254B - Perylenequinone derivatives, and preparation method and use thereof - Google Patents

Abstract

A compound represented by formula (I) and its stereoisomer and/or its salt, and preparation method thereof, and use thereof in the preparation of antiviral or anti-inflammatory medicines

Description

Perylenequinone derivatives and preparation methods and uses thereof

The present invention relates to a perylenquinone derivative, particularly a pharmacologically active perylenquinone derivative.

Each year, nearly 90% of the world's population is infected with the human herpes simplex virus (Epstein-Barr virus (EBV)). This virus not only causes infectious leukocytosis but is also thought to be associated with lymphoproliferative disorders, head and neck cancer, breast cancer, systemic lupus erythematosus, vitamin D deficiency, chronic fatigue syndrome, thyroid disease, rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases. Currently, other antiviral drugs (aciclovir, ganciclovir, valganciclovir, etc.) or other natural products (resveratrol, luteolin, epigallocatechin gallate, curcumin, etc.) are mostly used clinically to combat EBV. Although EBV is not currently a critical issue for treatment, the symptoms it causes and its association with cancer warrant the development of drugs targeting this virus.

Perylenequinone, on the other hand, is primarily a pentacyclic natural product derived from the 4,9-dihydroxyperylene-3,10-quinone skeleton. 85% of the currently known derivatives are derived from fungal secondary metabolites, while the remaining 15% originate from the animal and plant kingdoms. Based on their structure, they can be divided into three main categories: type A (without carbon substituents), type B (with carbon substituents), and type C (with additional aliphatic rings) (Weiss, U; Merlini, L.; Nasini, G.; Naturally occurring perylenequinones. In Forschritte der chemie organischer naturstoffe / Progress in the chamistry of organic natural products , Springer Vienna: Vienna, 1987; Vol. 70, pp 1-71, ISBN: 9783709189061). In the fungal kingdom, 82% of perylenequinones come from the Dothideomycetes, with Alternaria producing the most and most unique structures within Class A.

Chain Sporophytes can be found in plants, for example, as endophytes of marine plants. Compared to terrestrial fungi, marine fungi were explored and researched relatively recently. However, since 1990, at least 100 compounds have been isolated annually from their secondary metabolites. To date, over 1,000 marine fungal secondary metabolites have been identified as candidate compounds for drug development. Furthermore, natural products possess greater chemical diversity and biocompatibility than synthetic drugs, making them an important source for the development of drugs for cancer, hypertension, infection, and immunosuppression.

The present invention provides a compound represented by formula (I), its stereoisomers and/or salts thereof as active ingredients for the development of antiviral and anti-inflammatory drugs.

The present invention provides a compound represented by formula (I), its stereoisomers and/or its salts Formula (I), wherein: R1 and R2 are independently selected from -OR8, =0, R3 and R4 are independently selected from -OR9, halogen; and Ring A or Ring B is a benzene ring; wherein, when Ring B is a benzene ring, C-1 has a substituent of R5; when Ring A is a benzene ring and Ring C is not a benzene ring, C-6 has a substituent of R6, and C-6a has a substituent of R7; wherein R6 is -SR10, and R5, R6, R7, R8, R9 and R10 are each independently selected from hydrogen, halogen, hydroxyl, cyano, amino, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amide, ester, acyl, carboxyl, sulfonyl, sulfonamide, substituted or unsubstituted alkenyl, and substituted or unsubstituted alkynyl.

In one embodiment of the present invention, R8 is hydrogen, and R9 is selected from hydrogen and substituted or unsubstituted lower alkyl.

In one embodiment of the present invention, R3 and R4 are independently selected from chloro, hydroxyl and -OCH ₃ , and R3 and R4 are different.

In one embodiment of the present invention, at least one of R3 and R4 is a hydroxyl group.

In one embodiment of the present invention, the aforementioned R10 is a substituted or unsubstituted C1-C6 carboxyl group, and the substituent is selected from a halogen, a hydroxyl, an amino, a C1-C6 alkyl, a 2-7 membered heteroalkyl, an ester, an acyl, and an amide group.

In one embodiment of the present invention, when the aforementioned ring A is a benzene ring, R1 is -OR8, and R2 is =O; when the ring B is a benzene ring, R1 is =O, and R2 is -OR8.

In one embodiment of the present invention, the compound represented by the above formula (I), its stereoisomers and/or its salts further have the structure represented by formula (I-1), formula (I-2) or formula (I-3): Formula (I-1); Formula (I-2); Formula (I-3).

In one embodiment of the present invention, the compound represented by the above formula (I), its stereoisomers and/or its salts further have the structure represented by formula (i-1), formula (i-2), formula (i-3) or formula (i-4): Formula (i-1); Formula (i-2); Formula (i-3); Formula (i-4).

In one embodiment of the present invention, the compound represented by the above formula (I), its stereoisomers and/or its salts further have the structure represented by formula (i-3-1) or formula (i-3-2) Formula (i-3-1); Formula (i-3-2).

In one embodiment of the present invention, the compound represented by the above formula (I), its stereoisomers and/or its salts are obtained by isolating the secondary metabolites of the fungus of the genus Chain Sporangium.

In one embodiment of the present invention, the aforementioned Chain Sporophora fungus undergoes aeration and fermentation in an environment containing seawater to produce secondary metabolites.

In one embodiment of the present invention, the compound of formula (I), its stereoisomers, and/or salts thereof are obtained by extraction of secondary metabolites, column chromatography, normal phase thin layer chromatography, and high performance liquid chromatography separation; wherein the Alternaria fungus is Alternaria alstroemeriae .

The present invention also provides a method for preparing the compound represented by formula (I), its stereoisomers and/or its salts.

The present invention also provides a method for preparing an antiviral or anti-inflammatory drug using the compound represented by formula (I), its stereoisomers and/or its salts.

In order to make the above and other purposes, features and advantages of the present invention more clearly understood, the following embodiments are specifically cited and described in detail with reference to the accompanying drawings.

The present invention can be more readily understood through the various embodiments and examples described in detail below, as well as through the chemical diagrams and tables described above and below. Although the present invention's compounds, compositions, and/or methods have been disclosed and described herein, it should be understood that unless otherwise specified in the claims, the present invention is not limited to a specific method of preparation, as those skilled in the art will readily appreciate that such methods are inherently variable. It should also be understood that the terminology used herein is intended only to describe specific embodiments and is not intended to be limiting.

As used herein, unless otherwise specified, the terms "isolated" or "isolated" mean that the substance has been removed from its native environment (e.g., the natural environment, if the substance occurs naturally). The term "isolated" does not necessarily extend to mean that the substance has been purified.

1. Compounds of the Present Invention

The present invention provides a compound represented by formula (I), its stereoisomers and/or its salts Formula (I), wherein: (1) R1 and R2 are independently selected from -OR8, =0, R3 and R4 are independently selected from -OR9, halogen, (2) Ring A or Ring B is a benzene ring. When Ring B is a benzene ring, C-1 has a substituent of R5. When Ring A is a benzene ring and Ring C is not a benzene ring, C-6 has a substituent of R6, and C-6a has a substituent of R7. R6 is -SR10, and R5, R6, R7, R8, R9, and R10 are each independently selected from hydrogen, a halogen, a hydroxyl group, a cyano group, an amino group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted amide group, an ester group, an acyl group, a carboxyl group, a sulfonyl group, a sulfonamide group, a substituted or unsubstituted alkenyl group, and a substituted or unsubstituted alkynyl group.

In certain aspects of the compound represented by formula (I), R8 is hydrogen, and R9 is selected from hydrogen and substituted or unsubstituted lower alkyl.

In certain embodiments of the compound of formula (I), R3 and R4 are independently selected from chloro, hydroxyl, and -OCH ₃ , and R3 and R4 are different. In a preferred embodiment, at least one of R3 and R4 is hydroxyl.

In certain aspects of the compound represented by formula (I), R10 is a substituted or unsubstituted C1-C6 carboxyl group, and the substituent is selected from a halogen, a hydroxyl, an amino, a C1-C6 alkyl, a 2-7 membered heteroalkyl, an ester, an acyl, and an amide group.

In certain aspects of the compound represented by formula (I), when ring A is a benzene ring, R1 is -OR8, and R2 is =0. When ring B is a benzene ring, R1 is =0, and R2 is -OR8.

In certain aspects, the compound represented by formula (I) further has a structure represented by formula (I-1), formula (I-2) or formula (I-3) Formula (I-1); Formula (I-2); Formula (I-3).

In certain embodiments, the compound represented by formula (I-1) further has the structure represented by formula (i-1) or formula (i-2), the compound represented by formula (I-2) further has the structure represented by formula (i-3), and the compound represented by formula (I-3) further has the structure represented by formula (i-4).

2. Preparation Methods of the Compounds of the Present Invention

In one embodiment of the present invention, the compound represented by formula (I) is derived from a fungus, such as Alternaria . Alternaria encompasses saprophytic, endophytic, and pathogenic fungi in nature and can be found in seeds, plants, agricultural products, animals, soil, and the atmosphere. In a preferred embodiment of the present invention, the Alternaria fungus from which the compound of formula (I) is derived is an endophytic fungus of a coastal plant, such as Hibiscus maritima.

There are two main species of sea hibiscus, one of which is Atriplex maximowicziana Makino, a member of the Chenopodiaceae family. Atriplex maximowicziana Makino is primarily found on the coastal beaches of Kinmen and is a medicinal plant. In one embodiment of the present invention, the compound represented by formula (I) is derived from an endophytic fungus of the genus Alternaria from Atriplex maximowicziana Makino. Endophytic fungi can be found in plant tissues such as stems, leaves, roots, and buds. In a preferred embodiment of the present invention, the compound represented by formula (I) is derived from the endophytic fungus strain Km2286 of Atriplex maximowicziana Makino.

In one embodiment of the present invention, a method for preparing a compound represented by formula (I) comprises the following steps: (a) cultivating Alternaria alstroemeriae and performing aerated fermentation to produce a fermentation broth; (b) separating the fermentation broth and performing extraction to obtain an extract; and (c) separating and purifying the extract to obtain the compound represented by formula (I).

Examples of Preparation Methods of the Compounds of the Present Invention

Example 1: Fungal culture and aerated fermentation

1.1 Fungal Acquisition, Strain Isolation, and Screening: Fresh samples of T. truncatula were collected from Lotus Lake in Kinmen. After harvest, the plants were washed with plenty of tap water. The stems and leaves were then cut into small pieces (0.5 ^cm² ) and placed flat on PDA culture medium supplemented with penicillin (50 ppm) and streptomycin (50 ppm) to inhibit the growth of contaminants. Once hyphae emerged, they were immediately inoculated onto fresh PDA culture medium. Repeated isolation was performed until the hyphae maintained color and maintained a consistent appearance, confirming the isolate as pure.

1.2 Fungal Culture and Aerated Fermentation 1.2.1 Culture Medium Preparation 1.2.1.1 Solid Culture Medium: PDA Culture Medium. Prepare 2g of potato dextrose agar per 100mL of deionized water. Sterilize at 121°C for 20 minutes. Pour the mixture into separate culture dishes and allow to cool before using as the solid culture medium. 1.2.1.2 Liquid Culture Medium: PDY Culture Medium. Prepare 2g of peptone, 10g of glucose, and 1g of yeast extract per 1L of deionized water. Pour 3.5L of the culture medium into a 5L serum bottle and sterilize at 121°C for 20 minutes. Allow to cool before using as the fermentation tank for fungal culture. 1.2.2 Inoculate six 5.5 cm diameter plates of PDA medium with the strain Alternaria alstroemeriae Km2286 isolated in 1.1. After incubation at 37°C for 7 days, cut each plate of PDA medium, which is full of hyphae, into 16 equal pieces. Inoculate 8 of these pieces into 5 L serum bottles containing PDY medium supplemented with 3.5 L of seawater. Ferment with extensive aeration at room temperature for 14 days, bringing the total culture volume to 42 L in 12 bottles.

Example 1 may further include antiviral activity screening and strain identification. Strain identification may include using microscopy and 28S rRNA sequence analysis to identify the isolated strain as Alternaria alstroemeriae .

Example 2: Fermentation Broth Extraction Remove the aeration system from the 12 serum bottles after 14 days of incubation in 1.2.2. Mycelium and fermentation broth were separated by vacuum filtration. The fermentation broth was then divided and extracted twice with 1.3 times the volume of the extraction solvent. The broth was concentrated by decompression to obtain approximately 4.8 g of ethyl acetate extract. The extraction solvent can be, but is not limited to, ethyl acetate, n-butanol, chloroform, or dichloromethane. In this example, ethyl acetate is preferred.

Example 3: Separation and purification of the extract

3.1 Column Chromatography Separation of Extracts The extract from Example 2, for example, 4.8 g of the ethyl acetate extract, was redissolved in an extraction solvent. The extraction solvent may be, but is not limited to, methanol or ethanol. In this example, the ethyl acetate extract was preferably redissolved in 10 mL of methanol and subjected to column chromatography on a Sephadex LH-20 (2.8 cm i.d. × 68.3 cm) column using methanol as the eluent at a flow rate of 2.5 mL/min. One tube was collected every 25 mL (approximately 10 minutes), for a total of 60 tubes. The fractions were then pooled into 11 fractions (fractions 1-11) based on the results of normal phase thin layer chromatography (TLC) analysis, for example, based on Rf values. Among the 11 fractions, the TLC analysis results of fractions 7 and 10 were relatively pure, facilitating separation and purification.

3.2 Separation and Purification of Fractions 1–11 by High-Performance Liquid Chromatography (HPLC) 3.2.1 HPLC System: RI Detector: Bischoff RI-8120 (Bischoff, Leonberg, Germany) Pump: Hitachi L-7100 (Hitachi, Tokyo, Japan) 3.2.2 Separation and Purification of Compound 1: Fraction 7 was purified by reverse-phase HPLC using a Phenomenex Luna 5μ PFP semi-preparative column (10 × 250 mm) with an RI detector. The mobile phase consisted of 40% ACN + 0.1% FA at a flow rate of 2 mL/min. Compound 1 (5.0 mg) was obtained at a retention time of 16.0 min. The Rf value of compound 1 on normal phase TLC was 0.40 (DCM:MeOH = 10:1). 3.2.3 Isolation and Purification of Compound 2: Fraction 10 was purified by reverse phase HPLC using a SunFire 5μ C18 semi-preparative column (10×250 mm) with an RI detector for detection. The mobile phase consisted of 40% ACN + 0.1% FA at a flow rate of 2 mL/min. Compound 2 (3.4 mg) was obtained at a retention time of 32.5 min. The Rf value of compound 2 on normal phase TLC was 0.53 (DCM:MeOH = 10:1). 3.2.4 Isolation and Purification of Compound 3: Fraction 7 was purified by reverse-phase HPLC using a Phenomenex Luna 5μ PFP semi-preparative column (10×250 mm) with an RI detector. The mobile phase consisted of 40% ACN + 0.1% FA at a flow rate of 2 mL/min. Compound 3 (6.4 mg) was obtained at a retention time of 9.4 min. The Rf value of compound 3 on normal-phase TLC was 0.53 (DCM:MeOH = 10:1). 3.2.5 Isolation and Purification of Compound 4: Fraction 7 was purified by reverse-phase HPLC using a Phenomenex Luna 5μ PFP semi-preparative column (10×250 mm) with an RI detector. The mobile phase consisted of 40% ACN + 0.1% FA at a flow rate of 2 mL/min. The compound-containing fraction was collected between 5 and 14 minutes and then further purified using a Phenomenex Luna 5μ PFP semi-preparative column (10×250 mm) with a RI detector at a flow rate of 2 mL/min. Compound 4 (8.2 mg) was obtained at a retention time of 14.1 minutes. The Rf value of compound 4 on normal-phase TLC was 0.23 (DCM:MeOH = 10:1).

3. Physical Properties and Structure of the Compounds of the Present Invention

Examples of Physical Property Measurement and Structural Analysis of the Compounds of the Present Invention

Example 4: Physical property determination and structural analysis of compounds 1 to 4

4.1 Instruments for measuring physical properties HRESIMS: Q Extractive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fischer Scientific, Bremen, Germany) IR: JASCO FT/IR 4100 spectrometer (Tokyo, Japan) NMR: Bruker AVIII HD 400 and AVIII-500 spectrometer (Bruker, Ettlingen, Germany) Optional rotation: JASCO P-2000 Digital Polarimeter (JASCO, Tokyo, Japan) UV: Thermo UV-Visible Heλios α Spectrophotometer (Thermo Scientific, Waltham, MA, USA) CD: JASCO J-815 Spectropolarimeter (Tokyo, Japan)

4.2 Appearance and physical data of compound 1: dark red powder [α] ²⁵ _D =+11.1 ( c 0.1, MeOH) IR (ATR) _{V max} ：3382, 2927, 2861, 1622, 1591, 1460, 1231, 1061 cm ^-1 UV (MeOH): λ _max (log ε )=256 (4.51) nm, 396 (4.16) nm ECD ( c 0.14 μm, MeOH) λ _max (Δ ε )：224 (-0.08), 277 (0.09), 371 (-0.02), 429 (0.06), 464 (0.06) ¹ H, ¹³ C NMR data: as shown in Table 1 HRESIMS m/z 365.1015 [H+M] ⁺ (calcd for C ₂₁ H ₁₇ O ₆ , 365.1025), 363.0862 [H–M] ^– (calcd for C ₂₁ H ₁₅ O ₆ , 363.0869) Chemical structure: Formula (i-1).

4.3 Structural elucidation of compound 1

Compound 1 is a reddish-brown powder with specific rotation [α] ²⁵ _D = +11.1 ( c 0.1, MeOH). UV spectra (MeOH) are λ _max (log ε ) = 256 (4.51) nm, 396 (4.16) nm. High-resolution electron-scattering ionization mass spectrometry (HRESIMS) revealed a [H+M] ⁺ ion peak at m/z 365.1015 (calcd for C ₂₁ H ₁₇ O ₆ 365.1025); a [H–M] ^– ion peak at m/z 363.0862 (calcd for C ₂₁ H ₁₅ O ₆ 363.0869). The molecular formula is inferred to be C ₂₁ H ₁₆ O ₆ . Infrared spectroscopy (IR) revealed the presence of functional groups such as hydroxyl groups (3382 cm ^-1 ) and conjugated carbonyl groups (1622 cm ^-1 ).

Based on the UV absorption peaks at 256 and 396 nm, compound 1 is inferred to have a polycyclic aromatic structure. For further information, see Table 1 and Figures 1-3 below. The hydrogen spectrum of compound 1 (pyridine -d ₅ , 600 MHz) showed a methoxy proton signal at δ _H 3.67 (3H, s, H ₃ -13); a methine proton signal at δ _H 5.99 (1H, d, J =3.6 Hz, H-7); two oxymethine proton signals at δ _H 5.40 (1H, t, J =3.6 Hz, H-8) and δ _H 6.20 (1H, d, J =3.6 Hz, H-9); and two oxymethine proton signals at δ _H 7.15 (1H, d, J =10.2 Hz, H-5) and δ _H 8.83 (1H, d, J =10.2 Hz, H-6) is the signal of the methine proton on the double bond; at δ _H 7.55 (1H, d, J =9.0 Hz, H-2), δ _H 7.82 (1H, d, J =9.0 Hz, H-11), δ _H 8.75 (1H, d, J =9.0 Hz, H-12), and δ _H 9.05 (1H, d, J =9.0 Hz, H-1) are the signals of two sets of AB coupled protons on the benzene ring. Table 1: ^1H , ^13C , and 2D NMR data of compound 1 (δ in ppm, J in Hz) No. ¹ H m ( J Hz) ^{ac 13} C ^b COSY HMBC NOSEY 1 9.05 d (9.0) 133.8 H-2 3, 12a, 3b H-2, H-12 2 7.55 d (9.0) 119.7 H-1 3, 3a, 12b H-1 3 167.6 3a 112.6 3b 125.7 4 189.3 5 7.15 d (10.2) 127.7 H-6 3a, 6a H-6 6 8.83 d (10.2) 140.2 H-5 4, 3b, 6b H-5, H-7, H-13 6a 125.6 6b 138.8 7 5.99 d (3.6) 77.8 H-8 6a, 6b, 8, 9b, 13 H-6, H-8, H-9, H-13 8 5.40 t (3.6) 68.6 H-7, H-9 6b, 9a H-7, H-9, H-13 9 6.20 d (3.6) 68.6 H-8 7, 8, 9a, 9b, 10 H-7, H-8 9a 120.7 9b 127.7 10 157.1 11 7.82 d (9.0) 122.5 H-12 9a, 10, 12a H-12 12 8.75 d (9.0) 124.3 H-11 9b, 10, 12b H-1, H-11 12a 126.7 12b 122.9 13 3.67 s 57.3 7 H-6, H-7, H-8 ^a Measured in pyridine- d ₅ (600 MHz), ^b Measured in pyridine- d ₅ (150 MHz), ^c Multiplicities were obtained from HSQC.

Carbon spectrum (pyridine -d ₅ , 150 MHz) showed that compound 1 has 21 carbon atoms. HSQC analysis (Figure 1) revealed a methoxy signal at δ _C 57.3 (C-13); nine methine signals at δ _C 68.6 (C-8), 68.6 (C-9), 77.8 (C-7), 119.7 (C-2), 122.5 (C-11), 124.3 (C-12), 127.7 (C-5), 133.8 (C-1), and 140.2 (C-6). Of these, δ _C 119.7 (C-2), 122.5 (C-11), 124.3 (C-12), and 133.8 (C-1) are carbon atoms on the benzene ring; and 11 quaternary carbon signals are located at δ _C δ C 157.1 (C-10) and 167.6 (C-3) represent the carbon atoms with oxygen attached to the benzene ring, while δ _C 189.3 ( _C -4) represents the carbonyl group.

According to the preliminary interpretation of the hydrogen and carbon spectra, the relevant signals of COSY include: H-1/H-2; H-5/H-6; H-7/H-8; H-8/H-9; H-11/H-12 and the relevant signals of HMBC include: H-1/C-3, C-12a and C-3b; H-2/C-3a and C-12b; H-5/C-3a and C-6a; H-6/C-4, C-3b and C-6b; H-7/C-6a, C-9b and C-13; H-8/C-6b, C-9a and C-13; H-9/C-7, C-9b and C-10; H-11/C-9a and C-12a; H-12/C-9b, C-10 and C-12b; H ₃ -13/C-7 (Table 1). Through the relevant signals of COSY and HMBC, it can be known that C-1, C-2, C-3, C-3a, C-3b and C-12b are carbon atoms on ring A, and C-9a, C-9b, C-10, C-11, C-12 and C-12a are carbon atoms on ring D; from the signals of H-1/C-12a and H-12/C-12b, it can be known that C-12a on ring D is connected to C-12b on ring A; from the signals of H-5/C-3a, C-6a and H-6/C-3b, COSY The results of COSY show that C-4, C-5, C-6, and C-6a are connected to C-3a and C-3b on ring A to form ring B. The signals of H-8/C-6b, C-9a, and H-9/C-9b combined with the results of COSY show that C-6b, C-7, C-8, and C-9 are connected to C-9a and C-9b on ring D to form ring E. The signals of H-6/C-6b and H-7/C-6a show that C-6b of ring E is connected to C-6a on ring B to form ring C between rings A-B and D-E. The _3-13 /C-7 signal indicates that the methoxy group is attached to the C-7 position of ring E. COSY signals H-1/H-2 and H-11/H-12, as well as coupling constants J _H-1/H-2 (9.0 Hz) and J _H-11/H-12 (9.0 Hz), indicate that the two hydrogen atoms are ortho relative to each other on the benzene ring. C-3 and C-10 are both carbon atoms on the benzene ring that bear hydroxyl groups, but C-3 (δ _C 167.6) is further downfield than C-10 (δ _C 157.1), suggesting that this is related to the hydrogen bond between the C-3 hydroxyl group and the C-4 carbonyl group. The structure obtained from the above structural analysis was compared using Reaxys and Scifinder ⁿ . Its planar structure was identical to altertoxin IX in previous literature, confirming that it is a perylenequinone derivative.

The NOSEY pattern revealed signals associated with H-1/H-2 and H-12; H-5/H-6; H-6/H-7 and H-13; H-7/H-8, H-9, and H-13; H-8/H-9; and H-11/H-12 (Table 1). The prominent signal in H-1/H-12 confirmed the ipsilateral location of loops A and D, while the prominent signal in H-6/H-7 confirmed the ipsilateral location of loops B and E. Based on the coupling constants J _H-7/H-8 (3.6 Hz) and J _H-8/H-9 (3.6 Hz) and the construction of a molecular model (ChemBio3D Ultra 14.0), the dihedral angles of H(7)-C(7)-C(8)-H(8) and H(8)-C(8)-C(9)-H(9) of compound 1 were inferred to be between 50° and 70°. Based on the H-7/H-9 key signal in the NOSEY spectrum, it was determined that H-7, H-8, and H-9 are located in the β-pseudo-axial, β-pseudo-equatorial, and β-pseudo-axial positions, respectively. Therefore, the C-7-C-9 relative stereoisomers of compound 1 are 7 S *, 8 R *, and 9 R *.

For the absolute isomers, Gaussian 16 DFT calculations were used to obtain UV and ECD spectra for the 7 R , 8 S , 9 S and 7 S , 8 R , 9 R configurations (Figure 3). After generating the UV spectra using Gaussview 6, the eight excited states (S1-S8) were assigned to the experimental UV and CD spectra. The configuration of the compound was determined by comparing the S1-S8 trends in the CD spectra (Figure 3). To facilitate observation of the CD trends, the adjusted ECD calculation results were subsequently compared with the experimental CD spectra (Figure 2). As shown in Figure 2, compound 1 exhibits positive CE at approximately 430 and 460 nm, negative CE at approximately 371 nm, and positive CE at approximately 277 nm. These positions correspond to S1, S2-S3, and S5-S7, respectively, as shown in Figure 3. Their trends are consistent with 7S , 8R , and 9R . Therefore, the absolute stereoisomers of compound 1's C-7-C-9 configurations are confirmed to be 7S , 8R , and 9R . Based on the above structural elucidation and comparison with literature in the Reaxys and Scifinder ⁿ databases, compound 1 is confirmed to be a previously unreported novel compound.

4.4 Appearance and physical data of compound 2: reddish-brown powder [α] ²⁵ _D =+32.6 ( c 0.1, MeOH) IR (ATR) _{V max} ：3367, 2925, 1618, 1587, 1464, 1230, 1037 cm ^-1 UV (MeOH): λ _max (log ε )=256 (4.37) nm, 398 (4.18) nm ECD ( c 0.14 μm, MeOH) λ _max (Δ ε )：216 (-4.82), 320 (0.17), 391 (-0.95) ¹ H, ¹³ C NMR data: as shown in Table 2 HRESIMS m/z 369.0522 [H+M] ⁺ (calcd for C ₂₀ H ₁₄ ClO ₅ , 369.0530), 367.0367 [H–M] ^– (calcd for C ₂₀ H ₁₂ ClO ₅ , 367.0373) Chemical structure: Formula (i-2).

4.5 Structural Elucidation of Compound 2 Based on the aforementioned method, Table 2, and Figures 4-6, Compound 2 was determined to be a novel, previously unreported compound, a perylenequinone derivative. Table 2: ¹ H, ¹³ C and 2D NMR data of compound 2 (δ in ppm, J in Hz) No. ¹ H m ( J Hz) ^{ac 13} C ^b COSY HMBC NOSEY 1 9.06 d (9.6) 133.9 H-2 3, 3b, 12a H-2, H-12 2 7.56 d (9.6) 120.0 H-1 3, 3a, 12b H-1 3 167.9 3a 112.5 3b 125.8 4 189.1 5 7.12 d (9.6) 127.7 H-6 3a, 6a H-6 6 9.02 d (9.6) 140.4 H-5 3b, 4 H-5, H-7 6a 125.0 6b 139.2 7 6.52 d (4.8) 66.4 H-8 6a, 6b, 8, 9, 9b H-6, H-8, H-9 8 5.43 t (4.8) 69.5 H-7, H-9 H-7, H-9 9 6.76 d (4.8) 68.6 H-8 H-7, H-8 9a 116.9 9b 127.2 10 156.8 11 7.73 d (9.6) 123.1 H-12 9a, 10, 12a H-12 12 8.79 d (9.6) 124.7 H-11 9b, 10, 12b H-1, H-11 12a 127.1 12b 122.8 ^a Measured in pyridine- d ₅ (600 MHz), ^b Measured in pyridine- d ₅ (150 MHz), ^c Multiplicities were obtained from HSQC.

4.6 Appearance and physical data of compound 3: Reddish-brown powder [α] ²⁴ _D =+162.8 ( c 0.1, MeOH) IR (ATR) _{V max} ：3256, 2929, 2868, 1638, 1460, 1234, 1019 cm ^-1 UV (MeOH): λ _max (log ε )=258 (4.16) nm ECD ( c 0.13 μm, MeOH) λ _max (Δ ε )：236 (-2.99), 253 (0.44), 284 (1.38), 314 (0.88) ¹ H, ¹³ C NMR data: as shown in Table 3 HRESIMS m/z 389.078 [H+M] ⁺ (calcd for C ₂₀ H ₁₈ ClO ₆ , 389.0792), 387.0629 [H–M] ^– (calcd for C ₂₀ H ₁₆ ClO ₆ , 387.0635) Chemical structure: Formula (i-3).

4.7 Structural elucidation of compound 3

According to the above method and Table 3, Figures 7-9, compound 3 was confirmed to be a perylenequinone derivative. The dihedral angles of compound 3 were determined by coupling constants and molecular model (ChemBio3D Ultra 14.0) (Figure 10) (Table 4). The dihedral angles of H(1)-C(1)-C(12b)-H(12b) and H(6b)-C(6b)-C(7)-H(7) were 173.3° and 167.1°, respectively. Combined with the coupling constants J _H-1/H-12b (8.4 Hz) and J _H-6b/H-7 (9.0 Hz) and H-1-H-12b and H-6b-H-7 are all in axial-axial relationship. H-1, H-6b, H-7 and H-12b are located at α-pseudo-axial, β-pseudo-axial, α-pseudo-axial and β-pseudo-axial respectively. The dihedral angles of H(7)-C(7)-C(8)-H(8) and H(8)-C(8)-C(9)-H(9) are 60.9° and 70.9° respectively. The coupling constants J _H-7H-8 (3.0 Hz), J _H-8/H-9 (3.0 Hz) and the absence of H-7/H-9 signals in the NOSEY spectrum suggest that H-7, H-8, and H-9 are located in the axial (α-pseudo-axial), equatorial (α-pseudo-equatorial), and equatorial (β-pseudo-equatorial) positions, respectively. Table 3: ¹ H, ¹³ C and 2D NMR data of compound 3 (δ in ppm, J in Hz) No. ¹ H m ( J Hz) ^{ac 13} C ^b COSY HMBC NOSEY 1 4.56 ddd (12.0, 8.4, 4.2) 66.7 H ₂ -2, H-12b H ₂ -2, H-12 2 2.90 dd (15.6, 12.0) 3.10 dd (15.6, 12.0) 46.9 H-1 1, 3, 3a, 12b H-1 3 203.7 3a 114.8 3b 143.6 4 158.8 5 6.84 d (9.0) 114.0 H-6 3a, 4, 6a H-6 6 7.88 d (9.0) 133.9 H-5 3b, 4, 6b H-5, H-7 6a 130.4 6b 3.43 dd (9.0, 3.0) 40.4 H-7 6a, 7, 9b 7 4.87 dd (9.0, 3.0) 65.0 H-6b, H-8, 7-OH 8 H-6, H-8 8 4.53 t (3.0) 66.4 H-7, H-9 6b, 7, 9, 9a H-7, H-9 9 5.06 d (3.0) 66.6 H-8, 9-OH H-8 9a 119.5 9b 136.8 10 154.6 11 6.71 d (8.4) 112.4 H-12 9a, 10, 12a H-12 12 7.58 d (8.4) 126.0 H-11 9b, 10, 12b H-1, H-11 12a 128.4 12b 3.61 dd (8.4, 3.0) 45.1 H-1 1, 12a, 3b H-1 4-OH 11.90 7-OH 5.62 H-7 9-OH 5.67 H-9 10-OH 9.42 ^a Measured in DMSO- d ₆ (600 MHz), ^b Measured in DMSO- d ₆ (150 MHz), ^c Multiplicities were obtained from HSQC. Table 4: Dihedral angles and coupling constants of compound 3 Atoms Dihedral angles (゜) ^a Coupling constants ( J in Hz) ^b H(1)-C(1)-C(2)-H _a (2) 55.6 4.2 H(1)-C(1)-C(2)-H _b (2) 173.3 12.0 H(1)-C(1)-C(12b)-H(12b) 167.2 8.4 H(6b)-C(6b)-C(7)-H(7) 167.1 9.0 H(7)-C(7)-C(8)-H(8) 60.9 3.0 H(8)-C(8)-C(9)-H(9) 70.9 3.0 ^a The dihedral angles were predicted by ChemBio3D Ultra 14.0. ^b The J values were obtained from ¹ H NMR.

H-6b and H-12b have three relative isotropic orientations. Molecular modeling (ChemBio3D Ultra 14.0) was used to predict the distances between hydrogen atoms and compared them with the distances calculated from the NOSEY signal intensity (Table 5). When H-6b and H-12b are both in β form (Equation (i-3) in Figure 11A), the distances between H(1)-H(12) and H(6)-H(7) in space are 2.2 Å. Although the distance between H-7/H-8 is larger, it is only 0.4 Å different from the value calculated by NOSEY. When H-6b and H-12b are in β form and α form respectively (Equation (i-3-1) in Figure 11B), the distance between H(1)-H(12) in space increases to 3.9 Å, which is 1.7 Å different from the value calculated by NOSEY. When the H(1)-H(12) and H(6)-H(7) distances in space increase to 3.6-3.7 Å, which differs from the NOSEY calculated values by 1.4-1.5 Å. Therefore, based on the H-1/H-12 and H-6/H-7 key signals of NOSEY, it is inferred that H-6b and H-12b should be located on the same side (Figure 11A). Compound 3 was subsequently subjected to homonuclear decoupling experiments. The results, shown in Figure 12, indicate that decoupling of H-6b affects the mutually coupled H-7 and H-12b, with the splitting pattern changing from doublet of doublets (dd) to doublets (d). Decoupling of H-12b also affects the mutually coupled H-1 and H-6b, with the splitting pattern changing from doublet of doublets of doublets (ddd) and doublet of doublets (dd) to doublet of doublets (dd) and doublets (d). This confirms that H-6b and H-12b are two pairs of long-range coupled methine groups ( ⁵ J _H-6b/H-12b = 3.0 Hz). The generation of long-range coupling in compound 3 is speculated to be related to the shape of ring C in the structure. Ring C is connected to the two benzene rings (ring B and ring D) on both sides and folded at a specific angle, forming not only a boat-like structure, but also believed to contain a dual homoallylic path. In addition, H-6b and H-12b are located in pseudo-axial positions (Figure 10). This type of ⁵ J _Cis coupling constant is usually large (≥3.0 Hz). Based on the above information, the relative stereochemistry of C-1, C-6b, C-7, C-8, C-9, and C-12b of compound 3 is S *, S *, R *, R *, R *, and S *, respectively. Table 5: NOESY signals and interatomic distances of compound 3 NOSEY Atomic distances (Å) 6bβ, 12bβ ^b 6bβ, 12bα ^b 6bα, 12bβ ^{b Experimental} H-1/H-12 2.2 3.9 3.6 2.2 H-5/H-6 2.4 2.4 2.4 2.6 H-6/H-7 2.2 2.5 3.7 2.2 H-7/H-8 2.5 2.5 3.0 2.9 H-8/H-9 2.6 2.6 2.4 2.6 H-11/H-12 2.4 2.4 2.4 2.4 ^a The intensity values were obtained by integrating the cross peaks in the NOSEY spectrum by MestReNova. According to the distance between the two hydrogens orthocoupled on the benzene ring in Chembio3D is 2.4 Å, we determined that the H-11/H-12 distance is 2.5Å, and the other atomic distances are calculated by the formula I is the intensity of cross peak; R is the atomic distance. ^b Predicted by ChemBio3D Ultra 14.0.

For the absolute stereoisomer, Gaussian 16 DFT calculations were used to obtain UV and ECD spectra for the two configurations: 1 S , 6b S , 7 R , 8 R , 9 R , 12b S and 1 R , 6b R , 7 S , 8 S , 9 S , 12b R (Figure 9). After generating the UV spectrum using Gaussview 6, the eight excited states (S1-S8) were assigned to the experimental UV and CD spectra. The configuration of the compound was determined by comparing the S1-S8 states in the CD spectra (Figure 9). To facilitate observation of CD trends, the adjusted ECD calculation results were subsequently compared with the experimental CD spectra (Figure 8). Figure 8 shows compound 3 exhibiting positive CE at approximately 314, 284, and 254 nm, and negative CE at 236 nm. These positions correspond to S2-S4 and S5-S6 in Figure 9, respectively, and their trends are consistent with 1S , 6bS , 7R , 8R , 9R , 12bS . Therefore, the absolute stereoisomers of compound 3, C-1, C-6b, C-7, C-8, C-9, and C-12b, are confirmed to be 1S , 6bS , 7R , 8R , 9R , 12bS . Based on the above structural elucidation and comparison with literature in the Reaxys and Scifinder ⁿ databases, compound 3 is confirmed to be a previously unreported novel compound.

4.8 Appearance and physical data of compound 4 Reddish brown powder [α] ²⁵ _D =+108.4 ( c 0.1, MeOH) IR (ATR) _{V max} ：3356, 2923, 1635, 1594, 1458, 1236, 1021 cm ^-1 UV (MeOH)： λ _max (log ε )=258 (4.89), 289 (4.86), 386 (4.15) nm ECD ( c 0.10 μm, MeOH) λ _max (Δ ε )：216 (-2.63), 317 (0.13), 396 (-0.17) ¹ H, ¹³ C NMR data: as shown in Table 6 HRESIMS m/z 507.0508 [H–M] ^– (calcd for C ₂₃ H ₂₀ ClO ₉ S, 507.0517) Chemical structure: Formula (i-4).

4.9 Structural Elucidation of Compound 4

Based on the aforementioned methods, Table 6, and Figures 13–15, compound 4 was confirmed to be a perylenequinone derivative. The dihedral angles of compound 4 were determined (Table 7) using coupling constants and molecular modeling (ChemBio3D Ultra 14.0) (Figure 16). From the coupling constants J _Ha-5/H-6 (3.6 Hz) and J _Hb-5/H-6 (3.6 Hz) combined with the dihedral angles of _Ha (5)-C(5)-C(6)-H(6) and H _b (5)-C(5)-C(6)-H(6) (52.3～64.3°), it is inferred that H-6 and 6a-OH are located in the equatorial position (β-pseudo-equatorial) and the axial position (β-pseudo-axial), respectively. From the coupling constants J _H-6b/H-7 (3.6 Hz) and the obvious signal of NOSEY spectrum H-7/H-13, it is inferred that H-6b and H-7 are located in the axial position (α-pseudo-axial) and the equatorial position (α-pseudo-equatorial), respectively. From the coupling constants J _H-7/H-8 (3.6 Hz) and J _H-8/H-9 (3.6 Hz) inferred that the dihedral angles of H(7)-C(7)-C(8)-H(8) and H(8)-C(8)-C(9)-H(9) were smaller (44.7-58.0°), and no signals of H-6b/H-8 and H-7/H-9 were observed in the NOSEY spectrum. Therefore, H-8 and H-9 are located in the equatorial position (β-pseudo-equatorial) and axial position (β-pseudo-axial), respectively. Combining this information with molecular modeling (ChemBio3D Ultra 14.0) (Figure 16), the stereochemistry of C-6, C-6a, C-6b, C-7, C-8, and C-9 of compound 4 was confirmed to be S *, S *, S *, R *, R *, and R *. However, the stereochemistry of C-14 could not be determined using this method. Table 6: ¹ H, ¹³ C and 2D NMR data of compound 4 (δ in ppm, J in Hz) No. ¹ H m ( J , Hz) ^{ac 13} C ^b COSY HMBC NOSEY 1 7.96 d (9.0) 134.3 H-2 3, 3b, 12a H-2, H-12 2 7.00 d (9.0) 119.8 H-1 3, 3a, 12b H-1 3 162.2 3a 114.9 3b 137.2 4 204.7 5 3.10 dd (16.8, 3.6) 3.72 dd (16.8, 3.6) 40.4 H-6 3a, 4, 6, 6a H-6 6 4.22 t (3.6) 48.4 H-5 3b, 4, 6a, 13 H-5, H-7 6a 73.7 6b 4.14 d (3.6) 40.5 H-7 3b, 6a, 7, 9a, 9b, 12a H-7 7 5.31 t (3.6) 55.4 H-6b, H-8 8, 9, 9b H-6, H-6b, H-8, H-13 8 4.43 t (3.6) 73.9 H-7, H-9 6b, 7, 9, 9a H-7, H-9 9 4.86 d (3.6) 68.7 H-8 7, 8, 9a, 9b, 10 H-8 9a 122.9 9b 129.8 10 158.5 11 6.89 d (8.4) 115.2 H-12 9a, 10, 12a H-12 12 7.65 d (8.4) 125.5 H-11 9b, 10, 12b H-1, H-11 12a 125.4 12b 126.8 13 3.08‒3.10 ^d 36.6 H-14 6, 14, 15 H-7, H-14 14 4.43 dd (5.4, 4.2) 73.0 H-13 13, 15 H-13 15 176.4 ^a Measured in CD ₃ OD - d ₄ (600 MHz), ^b Measured in CD ₃ OD - d ₄ (150 MHz), ^c Multiplicities were obtained from HSQC, ^d Signal without multiplicities were overlapped. Table 7: Dihedral angles and coupling constants of compound 4 Atoms Dihedral angles (゜) ^a Coupling constants ( J in Hz) ^b _Ha (5)-C(5)-C(6)-H(6) 64.3 3.6 H _b (5)-C(5)-C(6)-H(6) 52.3 3.6 H(6b)-C(6b)-C(7)-H(7) 54.9 3.6 H(7)-C(7)-C(8)-H(8) 58.0 3.6 H(8)-C(8)-C(9)-H(9) 44.7 3.6 ^a The dihedral angles were predicted by ChemBio3D Ultra 14.0. ^b The J values were obtained from ¹ H NMR.

In the absolute stereo section, the DFT calculation method of Gaussian 16 was used to obtain the UV and CD spectra of four configurations: 6 S , 6a S , 6b S , 7R , 8 R , 9 R , 14 S , 6 S , 6a S , 6b S , 7 R , 8 R , 9 R , 14 R , 6 R , 6a R , 6b R , 7 S , 8 S , 9 S , 14 S and 6 R , 6a R , 6b R , 7 S , 8 S , 9 S , 14 R (Figure 15). After generating the UV spectrum, the eight excited states (S1-S8) were assigned to the experimental UV and CD spectra. The configuration of the compound was determined by comparing the S1-S8 trends in the CD spectrum (Figure 15). To facilitate observation of the CD trend, the adjusted ECD calculation results were subsequently compared with the experimental CD spectrum (Figure 14). Figure 14 shows that compound 4 exhibits negative CE around 396 nm, positive CE around 317 nm, and a distinct negative CE at 216 nm. These positions correspond to S2, S3, and S4 in Figure 15 , respectively. Their trends are consistent with both 6 S , 6a S , 6b S , 7R , 8 R , 9 R , 14 S and 6 S , 6a S , 6b S , 7 R , 8 R , 9 R , 14 R. Therefore, only the absolute isomers of C-6, C-6a, C-6b, C-7, C-8, and C-9 of compound 4 can be confirmed as 6 S , 6a S , 6b S, 7R , 8 R , and 9 R ; the absolute isomer of C-14 remains unidentified. The obtained structure was compared with the literature in the Reaxys and Scifinder ⁿ databases, confirming that compound 4 was a new compound that had not been reported before.

In addition to the plants and fungi of Example 1, the preparation methods of the compounds of the present invention can also use other plants and fungi to cultivate, isolate, and purify to obtain the compounds of the present invention. The cultivation, isolation, and purification methods are not limited to those exemplified in Example 1. Fermentation broth extraction and the separation and purification of the extract are also not limited to those exemplified in Examples 2 and 3. In addition, compounds 1-4 can also be prepared by methods other than those exemplified herein, including chemical synthesis. As long as the obtained compounds have the structure and physical properties as described above, they are within the scope of the present invention. The determination method and analytical steps of Example 4 are merely illustrative and the present invention is not limited thereto.

IV. Activity Evaluation of the Compounds of the Present Invention

Activity Evaluation Examples of the Compounds of the Present Invention

Example 5: Activity Evaluation of Compounds

5.1 Antiviral activity of compounds The inhibitory effects of compounds from fungal secondary metabolites on EBV in cell lines were evaluated by analyzing the expression of EBV lytic proteins Rta, Zta, and EA-D. 5.1.1 Experimental materials: P3HR1 cell line containing EBV. 5.1.2 Experimental methods 5.1.2.1 Cell treatment (1): P3HR1 cells were cultured at a concentration of 6×10 ⁵ cell/mL at 37°C and 5% CO _2. After 24 hours of culture, compounds 1 to 4 were added to a final concentration of 10 μM. Then, SB (sodium butyrate) was added 1 hour later to induce the cells to enter the lytic cycle. After 24 hours, cells were collected and lysed with lysis buffer. Protein supernatant was obtained by centrifugation for Western blotting. 5.1.2.2 Cell treatment (2): P3HR1 cells were cultured at 37°C and 5% _CO2 . After 24 hours, different concentrations of compounds 1-4 were added and allowed to stand for 24 hours. MTT assay was then performed. 5.1.2.2 Western Blot: After obtaining protein supernatants, the expression of EBV lytic proteins Rta, Zta, and EA-D was analyzed by Western blot (Figure 17). Image analysis software (ImageJ) was used to scan the image intensity for data analysis. The relative expression levels of β-tublin, Rta, Zta, and EA-D were converted, and the half-effective concentrations ( _EC50 ) of compounds 1-4 were estimated based on the expression level of Rta, as shown in Table 8. 5.1.2.3 MTT Assay: The effects of compounds 1-4 at different concentrations on P3HR1 cell viability were detected using MTT assay, and the 50% cytotoxic concentration ( _CC50 ) was calculated, as shown in Table 8. 5.1.3 Experimental Results Table 8: Half-effective concentration, 50% cytotoxicity concentration and selectivity index of compounds 1-4 Compound 24-hour processing _EC50 (μM) ^a CC ₅₀ (μM) ^b SI ^c 1 1.21±0.10 60.15±5.11 49.71 2 1.51±0.29 39.35±7.38 26.06 3 2.42±0.56 61.17±18.09 25.28 4 0.17±0.07 2.30±0.11 13.53 ^a EC ₅₀ =concentration that reduces EBV replication by 50%. ^b CC ₅₀ =concentration that reduces cell viability by 50%. ^c SI= CC ₅₀ / EC ₅₀ ; the higher the SI ratio, the theoretically more effective and safer a drug would be during in vivo treatment for a given vital infection. SI value ≧ 10 was assumed to belong of a selected potential sample that can be further investigated. As shown in Figure 17, compounds 1, 2, 3, and 4 exhibited significant inhibitory effects against EBV at a concentration of 10 μM, with EC ₅₀ values listed in Table 8. The expression of tubulin in Figure 17 reflects the cytotoxicity of each treatment group. Compounds 1, 2, and 3 all showed tubulin expression, indicating no cytotoxicity. Compound 4 showed no β-tubulin expression, making it impossible to determine whether its antiviral effect is due to cytotoxicity. The cytotoxicity fraction and CC ₅₀ values are listed in Table 8. Furthermore, the selectivity index (SI) was calculated based on the EC ₅₀ and CC ₅₀ values. As shown in Table 8, all SI values were greater than 10, indicating that compounds 1-4 have the potential for further study.

5.2 Anti-inflammatory Activity of Compounds The inhibitory effect of compounds derived from fungal secondary metabolites on NO production in cell lines was evaluated by analyzing nitric oxide concentrations. 5.2.1 Experimental Materials: Murine microglial cell line (BV-2) 5.2.2 Experimental Methods 5.2.2.1 Cell Treatment: BV-2 cells were cultured in DMEM supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, HEPES, and _NaHCO₃ at 37°C, 5% _CO₂ , and 95% humidified air. 5.2.2.2 Griess reagent assay: Quantitatively plate BV-2 cells (5 × 10 ⁵ cells/well) in a 24-well culture plate. After incubation in a constant temperature incubator for 24 hours, remove the culture medium and add fresh DMEM medium without phenol red. Then, add samples dissolved in DMSO, including the following: a blank control (0.1% dimethyl sulfoxide) and compounds 1–4 (10 μM). 30 minutes later, add lipopolysaccharide (LPS) at 150 ng/mL and incubate for 24 hours. The supernatant of the cell culture medium was removed and an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine) was added. The reaction was allowed to proceed at room temperature for 10 minutes. The absorbance (OD) was measured at a fluorescent wavelength of 550 nm using an ELISA reader, and the inhibitory effect of the compound on NO production in BV-2 cells was calculated. The results are shown in Figure 18. 5.2.2.3 MTT Assay: Compounds 1-4 were added to a 96-well plate containing BV-2 cells and incubated for 24 hours. The supernatant was then removed and DMEM containing MTT was added. The cells were incubated at 37°C, 5% _CO₂ , and 95% humidified air for 1 hour. The absorbance was measured at 550 nm using an ELISA reader to determine the cytotoxicity of Compounds 1-4 against BV-2 cells. The results are shown in Figure 19. 5.2.2.4 Half-inhibitory concentrations ( _IC₅₀ ) of Compounds 1-4: Based on the results in Figure 18, with curcumin ("R" in Figure 18) as a control, the half-inhibitory concentrations ( _IC₅₀ ) of Compounds 1-4 were calculated. The results are shown in Table 9. 5.1.3 Experimental results Table 9: Half-inhibitory concentrations of compounds 1-4 and curcumin Compound IC ₅₀ (μM) ^ab 1 2.73±1.06 2 0.73±0.18 ^** 3 4.08±0.53 ^* 4 0.33±0.01 ^*** Curcumin 2.69±0.34 ^a IC ₅₀ =concentration that reduces NO production by 50%. ^b Asterisks denote significance compared to curcumin (positive control group) according to two-tailed t-test. * p ＜ 0.05, ** p ＜ 0.01 and *** p ＜ 0.001. As shown in Figure 18, compounds 1, 2, 3, and 4 exhibited significant inhibitory effects at a concentration of 10 μM, with percentage inhibitions of 104.7%, 94.9%, 92.1%, and 101.6%, respectively. The _IC₅₀ values are shown in Table 9. Notably, compounds 2 and 4 required only 0.73±0.18 and 0.33±0.01 μM, respectively, to achieve a 50% inhibition. The cytotoxicity fraction and _CC₅₀ values, shown in Figure 19, demonstrate that compounds 1-4 exhibited no significant cytotoxic activity at a concentration of 10 μM, with cell viability exceeding 90%.

In summary, the present invention provides a perylenequinone compound represented by formula (I) and a method for preparing the compound represented by formula (I). In applications, the compound represented by formula (I) of the present invention exhibits antiviral and anti-inflammatory activity. Regarding the former, the compound represented by formula (I) of the present invention exhibits particular antiviral activity against the human herpes virus (Epstein-Barr virus, EBV). Human herpes virus has a high infection rate in the human population and can also cause infectious leukocytosis. It is also believed to be associated with lymphoproliferative diseases, head and neck cancer, breast cancer, systemic lupus erythematosus, vitamin D deficiency, chronic fatigue syndrome, thyroid disease, rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases. Therefore, the development of the compound represented by formula (I) holds great promise.

Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Those skilled in the art may make modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the attached patent application.

without

Figure 1 shows the HSQC spectrum of compound 1. Figure 2 shows the CD spectrum and calculated ECD spectrum of compound 1. Figure 3 shows the excited state positions of the UV and CD spectra of compound 1. Figure 4 shows the HSQC spectrum of compound 2. Figure 5 shows the CD spectrum and calculated ECD spectrum of compound 2. Figure 6 shows the excited state positions of the UV and CD spectra of compound 2. Figure 7 shows the HSQC spectrum of compound 3. Figure 8 shows the CD spectrum and calculated ECD spectrum of compound 3. Figure 9 shows the excited state positions of the UV and CD spectra of compound 3. Figure 10 shows the molecular model of compound 3 (ChemBio3D Ultra 14.0). Figure 11 shows different stereoconfigurations of compound 3. Figure 12 shows the homonuclear decoupling spectrum of compound 3 (pyridine- d ₅ , 600 MHz). Figure 13 shows the HSQC spectrum of compound 4. Figure 14 shows the CD spectrum and calculated ECD spectrum of compound 4. Figure 15 shows the excited state positions of UV and CD spectra of compound 4. Figure 16 shows the molecular model of compound 4 (ChemBio3D Ultra 14.0). Figure 17 shows the effects of compounds 1-4 on Rta and tublin expression. Figure 18 shows the effects of compounds 1-4 on LPS-stimulated NO production. Figure 19 shows the effects of compounds 1-4 on BV-2 cell viability.

Claims (10)

A compound represented by formula (I), its stereoisomers and/or its salts Formula (I), wherein: R1 and R2 are independently selected from -OH and =O, R3 and R4 are independently selected from -OH, -OR9 and Cl, wherein R9 is a substituted or unsubstituted alkyl group; and when R1 is -OH, R2 is =O, R3 is -OR9 and R4 is -OH, the compound shown in formula (I) further has the structure shown in formula (i-1); when R1 is -OH, R2 is =O, R3 is -OH and R4 is Cl, the compound shown in formula (I) further has the structure shown in formula (i-2); when R1 is =O, R2 is -OH, R3 is -OH and R4 is Cl, the compound shown in formula (I) further has the structure selected from formula (i-3), formula (i-3-1) and formula (i-3-2); when R1 is -OH, R2 is =O, R3 is Cl and R4 is -OH, the compound shown in formula (I) further has the structure shown in formula (i-4) Formula (i-1); Formula (i-2); Formula (i-3); Formula (i-3-1); Formula (i-3-2) Formula (i-4). The compound represented by formula (I) as described in claim 1, its stereoisomers and/or its salts are obtained by isolating a secondary metabolite of a fungus of the genus Chain Sporangium. The compound represented by formula (I), its stereoisomers and/or salts thereof as described in claim 2, wherein the Chain Sporangium fungus undergoes aeration fermentation in an environment to produce the secondary metabolite, and the environment contains seawater. The compound represented by formula (I) as described in claim 2, its stereoisomers and/or salts thereof are obtained by extracting, column chromatography, normal phase thin layer chromatography analysis and high performance liquid chromatography separation and purification of the secondary metabolite; wherein the Alternaria fungus is Alternaria alstroemeriae . A method for preparing a compound represented by formula (I), its stereoisomers and/or its salts Formula (I), wherein: R1 and R2 are independently selected from -OH and =O, R3 and R4 are independently selected from -OH, -OR9 and Cl, wherein R9 is a substituted or unsubstituted alkyl group; when R1 is -OH, R2 is =O, R3 is -OR9 and R4 is -OH, the compound shown in formula (I) further has the structure shown in formula (i-1); when R1 is -OH, R2 is =O, R3 is -OH and R4 is Cl, the compound shown in formula (I) further has the structure shown in formula (i-2); when R1 is =O, R2 is -OH, R3 is -OH and R4 is Cl, the compound shown in formula (I) further has the structure shown in formula (i-3); when R1 is -OH, R2 is =O, R3 is Cl and R4 is -OH, the compound shown in formula (I) further has the structure shown in formula (i-4) Formula (i-1); Formula (i-2); Formula (i-3); Formula (i-4); The preparation method comprises: culturing a Chain Sporangium fungus, performing aeration fermentation of the Chain Sporangium fungus to produce a fermentation broth; separating the fermentation broth from hyphae of the Chain Sporangium fungus, extracting the fermentation broth to obtain an extract; and separating and purifying the extract, comprising: re-dissolving the extract in an organic solvent; performing column chromatography using the organic solvent as an eluent, and obtaining a plurality of collected solutions from the column chromatography; performing normal phase thin layer chromatography on the plurality of collected solutions, and distributing the plurality of collected solutions into a plurality of fractions according to the normal phase thin layer chromatography; wherein the plurality of fractions include a first fraction and a second fraction; and performing reverse phase high performance liquid chromatography on the first fraction and the second fraction, and purifying the first fraction and the second fraction to obtain a compound represented by formula (i-1), a compound represented by formula (i-2), and a compound represented by formula (i-3); wherein the first fraction is purified by reverse phase high performance liquid chromatography at a retention time of 16.0 min to obtain the compound of formula (i-1), and at a retention time of 9.4 min to obtain the compound of formula (i-3); and the second fraction is purified by reverse phase high performance liquid chromatography at a retention time of 32.5 min to obtain the compound of formula (i-2); The step of performing reversed-phase high-performance liquid chromatography on the first fraction further includes performing reversed-phase high-performance liquid chromatography on the first fraction using a Phenomenex Luna 5μ PFP semi-preparative column with a retention time of 5-14 minutes, and then obtaining the compound represented by formula (i-4) using a Phenomenex Luna 5μ PFP semi-preparative column at a retention time of 14.1 minutes. The method for preparing the compound represented by formula (I), its stereoisomers, and/or salts thereof as described in claim 5, wherein the step of culturing the Alternaria fungus further comprises: obtaining a plant having an endophytic fungus of the genus Alternaria; treating the stem or leaf of the plant into at least one block, and culturing the stem or leaf block with mycelium; after the mycelium grows, separating mycelium of different colors and inoculating the different mycelium into multiple culture media; repeating the separation until the mycelium color no longer changes and the mycelium morphology is uniform, thereby obtaining the strain of the Alternaria fungus; and culturing the Alternaria strain in a liquid culture medium. The method for preparing the compound represented by formula (I), its stereoisomers, and/or salts thereof as described in claim 5, wherein the step of performing aerobic fermentation of the Alternaria fungus further comprises: preparing a liquid culture medium with seawater; and inoculating the Alternaria fungus into the liquid culture medium, culturing the Alternaria fungus at room temperature, and performing aerobic fermentation. The method for preparing the compound represented by formula (I), its stereoisomers and/or salts thereof as described in claim 5, wherein the step of extracting the fermentation broth to obtain the extract further comprises extracting with ethyl acetate to obtain an ethyl acetate extract. The method for preparing the compound represented by formula (I), its stereoisomers, and/or salts thereof as described in claim 5, wherein the steps of performing high-performance liquid chromatography on the first and second fractions further comprise: Purifying the first fraction by reversed-phase high-performance liquid chromatography using a Phenomenex Luna 5μ PFP semi-preparative column; wherein the mobile phase is 30-40% ACN + 0.1% FA at a flow rate of 2 mL/min; and Purifying the second fraction by reversed-phase high-performance liquid chromatography using a SunFire 5μ C18 semi-preparative column; wherein the mobile phase is 30-40% ACN + 0.1% FA at a flow rate of 2 mL/min. A use of a compound represented by formula (I) according to any one of claims 1 to 4, its stereoisomers and/or its salts for preparing an antiviral or anti-inflammatory drug.