A method for preparing a compound of formula (I) comprising the steps of: (a) reacting a compound of formula (II) with a compound

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a derivative of ergosterol, a preparation method and application thereof. The application provides a group of ergosterol structural derivatives isolated from a fermentation product of a fungus Penicillium oxalicum, and the ergosterol structural derivatives show type I interferon inhibitory activity and can be used in drug application for treating type I interferon regulation related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to ergosterol derivatives and their preparation methods. Background Technology

[0002] Type I interferon (IFN-I) is a class of cytokines widely expressed in human nucleated cells. Various factors, such as viral infections, innate immune diseases, or interferon therapy, can cause IFN-I dysregulation, leading to tissue damage and inflammation. Therefore, IFN-I plays a crucial role in anti-inflammation, antiviral activity, antitumor activity, and immune regulation. Recent studies have shown that IFN-I diseases are associated with inappropriate or excessive activation of the interferon gene-stimulating factor (STING) pathway. STING pathway modulators have become a hot topic in the treatment of type I IFN-I diseases, and their role in the pathogenesis of autoinflammatory and innate immune diseases is receiving increasing attention. (YJCrow, DBStetson. The type I interferonopathies: 10 years on [J]. Nature Reviews Immunology, 2022, 22(8): 471-483; Zhang Tianyu, Song Hongmei. Research progress on interferon gene stimulating factor and type I interferon diseases [J]. Chinese Journal of Rheumatology, 2022, 26(6): 396-400; J.Hadjadj, N.Yatim, L.Barnabei, et al. Impaired type I interferonopathies activityandinflammatoryresponsesinsevereCOVID-19patients[J].Science,2020,369(6504):718-724; K.Nganou-Makamdop,DCDoue k.Manipulating theinterferonsignalingpathway:implicationsforHIVInfection[J].VirologicaSinica,2019,34(2):192-196.)

[0003] Ergosterol is a steroid with a 28-carbon skeleton, accounting for a significant proportion of steroid compounds. Its basic building blocks are a tetracyclic steroid nucleus and a 9-carbon (C-20-C-28) steroid side chain, and it is commonly found in fungi and higher plants. Ergosterol derivatives exhibit rich chemical diversity due to high oxidation and skeleton rearrangement, displaying various biological activities such as cytotoxicity, anti-inflammation, antibacterial (fungal), and shrimp toxicity. However, currently, there are no publicly reported methods for isolating and extracting ergosterol derivatives from the fermentation broth of the fungus *Penicillium oxalicum*, nor are there any reports of applying ergosterol derivatives to diseases caused by excessively high levels of type I interferon. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide an ergosterol derivative and a method for preparing the same, wherein the prepared ergosterol derivative has type I interferon inhibitory activity and can be used to prepare drugs for treating diseases related to type I interferon regulation.

[0005] Specifically, the present invention provides an ergosterol derivative, characterized in that it has the structure shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0006]

[0007] in,

[0008] R1 is OH or =O;

[0009] When C-4 and C-5 form a carbon-carbon single bond and C-5 and C-6 form a carbon-carbon single bond, R2 is OH. When C-4 and C-5 form a carbon-carbon double bond or C-5 and C-6 form a carbon-carbon double bond, R2 does not exist.

[0010] When C-5 and C-6 form a carbon-carbon single bond and C-6 and C-7 form a carbon-carbon single bond, R3 is H, OH, OAc or =O; when C-5 and C-6 form a carbon-carbon double bond or C-6 and C-7 form a carbon-carbon double bond, R3 is H.

[0011] When C-6 and C-7 form a carbon-carbon single bond and C-7 and C-8 form a carbon-carbon single bond, R4 is H and =O. When C-6 and C-7 form a carbon-carbon double bond or C-7 and C-8 form a carbon-carbon double bond, R4 is H.

[0012] R5 is OH when C-7 and C-8 form a carbon-carbon single bond, C-8 and C-9 form a carbon-carbon single bond, and C-8 and C-14 form a carbon-carbon single bond. R5 does not exist when C-7 and C-8 form a carbon-carbon double bond, or C-8 and C-9 form a carbon-carbon double bond, or C-8 and C-14 form a carbon-carbon double bond.

[0013] When C-8 and C-9 form a carbon-carbon single bond, R6 is H or OH; when C-8 and C-9 form a carbon-carbon double bond, R6 does not exist.

[0014] R7 is H, OH, or palmitoyl group;

[0015] C-22 and C-23 can form carbon-carbon single bonds or carbon-carbon double bonds;

[0016] C-24 is available in either R or S configuration;

[0017] The B ring can be oxidized and rearranged to form a C-5-OC-6 ether ring structure;

[0018] The C / D rings can be rearranged to form a 5 / 6 ring compound structure;

[0019] The A / B rings can degrade into a five-membered lactone ring structure;

[0020] R2 and R3 can form a C-5-OC-6 structure;

[0021] R2 and R5 can form a C-5-OOC-8 structure;

[0022] R2 and R6 can form a C-5-OOC-9 structure.

[0023] In some specific embodiments of the present invention, the structure of formula (I) does not include the following structure:

[0024]

[0025] In some specific embodiments of the present invention, R7 is palmitoyl.

[0026] In some specific embodiments of the present invention, the structure of formula (I) is as follows:

[0027]

[0028] In some specific embodiments of the present invention, the structure of formula (I) is as follows:

[0029]

[0030] in,

[0031] R1 is OH or =O;

[0032] When C-4 and C-5 form a carbon-carbon single bond and C-5 and C-6 form a carbon-carbon single bond, R2 is OH. When C-4 and C-5 form a carbon-carbon double bond or C-5 and C-6 form a carbon-carbon double bond, R2 does not exist.

[0033] When C-5 and C-6 form a carbon-carbon single bond and C-6 and C-7 form a carbon-carbon single bond, R3 is H, OH, OAc or =O; when C-5 and C-6 form a carbon-carbon double bond or C-6 and C-7 form a carbon-carbon double bond, R3 is H.

[0034] When C-6 and C-7 form a carbon-carbon single bond and C-7 and C-8 form a carbon-carbon single bond, R4 is H and =O. When C-6 and C-7 form a carbon-carbon double bond or C-7 and C-8 form a carbon-carbon double bond, R4 is H.

[0035] R5 is OH when C-7 and C-8 form a carbon-carbon single bond, C-8 and C-9 form a carbon-carbon single bond, and C-8 and C-14 form a carbon-carbon single bond. R5 does not exist when C-7 and C-8 form a carbon-carbon double bond, or C-8 and C-9 form a carbon-carbon double bond, or C-8 and C-14 form a carbon-carbon double bond.

[0036] When C-8 and C-9 form a carbon-carbon single bond, R6 is H or OH; when C-8 and C-9 form a carbon-carbon double bond, R6 does not exist.

[0037] R7 is H or OH;

[0038] C-22 and C-23 can form carbon-carbon single bonds or carbon-carbon double bonds;

[0039] C-24 is available in either R or S configuration;

[0040] The B ring can be oxidized and rearranged to form a C-5-OC-6 ether ring structure;

[0041] The C / D rings can be rearranged to form a 5 / 6 ring compound structure;

[0042] The A / B rings can degrade into a five-membered lactone ring structure;

[0043] R2 and R3 can form a C-5-OC-6 structure;

[0044] R2 and R5 can form a C-5-OOC-8 structure;

[0045] R2 and R6 can form a C-5-OOC-9 structure.

[0046] In some specific embodiments of the present invention, R6 is H and R7 is OH.

[0047] This invention also provides a method for preparing the above-mentioned ergosterol derivative, characterized in that,

[0048] The total crude extract was obtained from the fermentation broth of the fungus Penicillium oxalicum, and then separated and purified.

[0049] In some specific embodiments of the present invention, the preparation method of ergosterol derivatives includes the following steps:

[0050] (1) Preparation of strain fermentation broth: Inoculate the fungus Penicillium oxalicum into the culture medium and ferment at 25-30℃ to obtain the strain fermentation broth; the culture medium may contain malt extract, glucose, sucrose, peptone, potato, rice or agar, etc.

[0051] (2) Extraction of fermentation broth: The fermentation broth of the strain was extracted with an organic solvent, and the extract was concentrated under reduced pressure to obtain the total crude extract;

[0052] (3) Separation and purification: The total crude extract was separated and purified to obtain the ergosterol derivative.

[0053] In some specific embodiments of the present invention, the organic solvent is ethyl acetate, diethyl ether, methanol, dichloromethane, or chloroform, and the separation and purification method is chromatographic separation, which includes normal phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and high performance liquid chromatography.

[0054] In some specific embodiments of the present invention, the fungus *Penicillium oxalicum* is inoculated into a culture medium and cultured at a constant temperature of 25–30°C for a certain period of time. The culture medium is then pulverized, soaked in an organic solvent, and then extracted at least once by ultrasonic extraction with an organic solvent. The extracts are combined and concentrated under reduced pressure to obtain a crude extract. The crude extract is then separated by silica gel thin-layer chromatography. Normal-phase silica gel is selected, and a gradient elution is performed using a dichloromethane / methanol system. The fractions are collected, and similar components are separated and combined by silica gel thin-layer chromatography to obtain 10 components, namely Fr.1-10.

[0055] Fraction Fr.1 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol; followed by normal-phase silica gel column chromatography, eluted with petroleum ether / acetone, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 1 [(22E,24S)-ergoster-9α,15α-dihydroxy-4,6,8(14),22-tetraen-3-one-15-palmitate], 6 [herbarulide], and 11 [(22E,24R)-ergoster]. -6,8(14),22-trien-3β,5α,9α-triol(11)】, 12

(22E,24R)-7α-methoxy-5α,6α-epoxyergoster-8(14),22-dien-3β-ol(12)

(22E,24R)-5α,8α-peroxyergoster-6,9(11),22-trien-3β-ol

(22E,24R)-5α,8α-peroxyergoster-6,22-dien-3β-ol

[0056] Fraction Fr.2 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 4 [(22E,24R)-ergoster-4,6,8(14),22-tetraen-3-one] and 5 [isocyathisterol];

[0057] Fraction Fr.4 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal-phase silica gel column chromatography, eluted with petroleum ether / acetone, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 3 [ganodermaside], 7 [dankasterones A] and 16 [demethylincisterol A3];

[0058] Fraction Fr.5 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compound 10 [(22E,24R)-ergoster-3β-hydroxy-5,8,22-trien-7-one];

[0059] Fraction Fr.7 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal-phase silica gel column chromatography, eluted with petroleum ether / acetone, to give compound 13 [(22E,24R)-6-acetoxy-ergoster-7,22-diene-3β,5α,6β-triol];

[0060] Fraction Fr.9 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, to obtain compound 2 [(22E,24R)-ergoster-9α,15α-dihydroxy-4,6,8(14),22-tetraen-3-one];

[0061] Fraction Fr.10 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal-phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, to give compounds 8 [(22E,24R)-ergoster-3β,5α-dihydroxy-7,22-dien-6-one] and 9 [(22E,24R)-ergoster-3β,5α,9α-trihydroxy-7,22-dien-6-one];

[0062] The culture medium includes one or more of the following: PDA medium, Czapek's medium (CZA), peptone yeast extract glucose medium (GPY), potato dextrose agar medium (PDA), Bengal red medium (RBM), and rice.

[0063] Compounds 6, 7 and 16 are derivatives of ergosterol produced through extensive oxidation and structural rearrangement.

[0064] Compared with existing technologies, this invention provides an ergosterol derivative with type I interferon inhibitory activity isolated from the fermentation product of the fungus *Penicillium oxalicum*. The preparation method provided by this invention is simple, the raw materials are widely available, and mass production and industrialization are possible. Furthermore, in vitro activity evaluation experiments showed that the prepared ergosterol derivative significantly inhibited the expression of cytokines Tnfα and Ifnb1 induced by lipopolysaccharide (LPS) or 5,6-dimethylxanthone-4-acetic acid (DXMAA) in Raw264.7 cells, indicating that the above derivative has type I interferon inhibitory activity and can therefore be used in drug applications for the treatment of diseases related to type I interferon regulation. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 The cytotoxic activity test data for compounds 1-16 are compared with the control group. * P<0.05, ** P<0.01, n=4.

[0067] Figure 2 Data on the inhibition of LPS-induced expression of inflammatory cytokines Tnfα and Ifnb1 in Raw264.7 cells by compound 1-16; * indicates comparison with the control group. * P<0.05, ** P<0.01; # indicates comparison with the blank group. ## P<0.01, n=3.

[0068] Figure 3 Data on the inhibition of DXMAA-induced expression of inflammatory factors Tnfα and Ifnb1 in Raw264.7 cells by compound 1-16, * indicates comparison with the control group. **P<0.01; # indicates comparison with the blank group. ## P<0.01, n=3. Detailed Implementation

[0069] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Example 1: Preparation of a group of ergosterol derivatives 1-16

[0071] The symbiotic fungus *Penicillium oxalicum*, collected from the South China Sea coral *Sinulariagaweli*, can grow on Czapek's Agar (CZA), Peptone Yeast Extract Glucose Agar (GPY), Potato Gluconate Agar (PDA), Bengal Red Agar (RBM), and rice. After optimization, PDA medium was selected. After 28 days of incubation at 28°C, the medium was pulverized, soaked in ethyl acetate for 24 hours, and then extracted five times (30 minutes each time) with an equal volume of ethyl acetate using ultrasonication. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. Thin-layer chromatography (TLC) was performed on the crude extract, using normal-phase silica gel (200-300 mesh) and a dichloromethane / methanol system for preliminary separation. Gradient elution was performed using a dichloromethane / methanol system (v / v 100:0, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1). The fractions were collected, and similar components were combined by TLC analysis to obtain 10 fractions (Fr.1-10).

[0072] Fraction Fr.1 was subjected to Sephadex LH-20 gel column chromatography with dichloromethane / methanol (2:1) as the eluent; followed by normal phase silica gel column chromatography (300-400 mesh) with petroleum ether / acetone as the eluent; and then semi-preparative high performance liquid chromatography was used to separate compounds 1, 6, 11, 12, 14 and 15.

[0073] Fraction Fr.2 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 4 and 5.

[0074] Fraction Fr.4 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 3, 7 and 16.

[0075] Fraction Fr.5 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compound 10.

[0076] Fraction Fr.7 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, to obtain compound 13.

[0077] Fraction Fr.9 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, to obtain compound 2.

[0078] Fraction Fr.10 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography (300-400 mesh), eluted with petroleum ether / acetone system, to obtain compounds 8 and 9.

[0079] The properties and structural characterization of compounds 1-16 are as follows:

[0080] Compound 1:

[0081]

[0082] A pale yellow oily substance, named (22E,24S)-ergoster-9α,15α-dihydroxy-4,6,8(14),22-tetraen-3-one-15-palmitate,[α] 2 D 5+43.40(c0.10,CH3OH); UV(CH3OH)λ max (logε)330(2.49),265(2.02)nm; ECD(CH3OH,c1.5×10 -4 )λ max (Δε)241(+1.80),320(-1.43),361(+2.76)nm; IR(micro)ν max3443,2929,2854,1733,1664,1595,1460,1260,1094,1033,970,801cm -1 The precise molecular weight [M+H] is provided by the quasi-molecular ion peak in high-resolution mass spectrometry (HRESIMS). + Based on m / z 663.53448 and the analysis of proton and carbon spectra, the molecular formula of this compound was determined to be C63. 44 H 70 O7, through 1 H-NMR, 13 The structure of the compound was determined by C-NMR, DEPT and a full set of two-dimensional nuclear magnetic resonance; 1 H and 13 The C-NMR data are shown in Table 1.

[0083] Table 1. Compound 1 1 H and 13 C NMR data (CDCl3, 500MHz / 125MHz)

[0084]

[0085]

[0086] Note: J: coupling constant; s: singlet; d: doublet; t: triplet; m: multiplet; dd: double doublet.

[0087] Compound 2:

[0088]

[0089] A pale yellow powder; its structure was determined by comparing it with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Shi Q., ​​Huang Y., Su H., et al. C(28)steroids from the fruiting bodies of Ganoderma resinaceum with potential anti-inflammatory activity[J]. Phytochemistry, 2019, 168: 112-109.)

[0090] Compound 3:

[0091]

[0092] A pale yellow powder; its structure was determined by comparing it with data reported in previous literature. 1 H-NMR and 13C-NMR data can be found in literature reports (WengY.F., LuJ., XiangL., etal.GanodermasidesCandD, twonewanti-agingergosterolsfromsporesofthemedicinalmushroomGanodermalucidum[J].BioscienceBiotechnologyandBiochemistry,2011,75(4):800-803.)

[0093] Compound 4:

[0094]

[0095] A pale yellow powder; its structure was determined by comparing it with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Kwon H.C., Zee S.D., Cho S.Y., et al. Cytotoxicergosterols from Paecilomycess p. J300[J]. Archives of Pharmacal Research, 2002, 25(6): 851-855.)

[0096] Compound 5:

[0097]

[0098] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Liu X.H., Miao F.P., Liang X.R., et al. Ergosteroid derivatives from analgicolous strain of Aspergillusus[J]. Natural Product Research, 2014, 28(15):1182-1186.)

[0099] Compound 6:

[0100]

[0101] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13C-NMR data can be found in the literature report (Duecker F.L., Heinze R.C., Mueller M., et al. Synthesis of the alleged structures of her barulide and her barulide and structural revision of her barulide[J]. Organic Letters, 2020, 22(4):1585-1588.)

[0102] Compound 7:

[0103]

[0104] The pale yellow oily substance was identified by comparing its structure with previously reported data. 1 H-NMR and 13 C-NMR data can be found in the literature report (Amagata T., Tanaka M., Yamada T., et al. Variation in cytostatic constituents of asponge-derived Gymnascella ank aliensis by manipulating the carbon source[J]. Journal of Natural Products, 2007, 70(11):1731-1740.)

[0105] Compound 8:

[0106]

[0107] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in literature reports (Fangkrathok N., Sripanidkulchai B., Umehara K., etal. BioactiveergostanoidsandanewpolyhydroxyoctanefromLentinuspolychro usmyceliaandtheirinhibitoryeffectsonE2-enhancedcellproliferationofT47Dcells[J].NaturalProductResearch,2013,27(18):1611-1619.)

[0108] Compound 9:

[0109]

[0110] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Hoque N., Afroz F., Khatun F., et al. Physicochemical, pharmacokinetic and cytotoxicity of the compounds isolated from an endophyte Fusarium oxysporum: In vitro and insilico approaches [J]. Toxins, 2022, 14(3): 159.)

[0111] Compound 10:

[0112]

[0113] The pale yellow oily substance was identified by comparing its structure with previously reported data. 1 H-NMR and 13 C-NMR data can be found in the literature report (Wang F.Z., Fang Y.C., Zhang M., et al. Six newer gosterols from the marine-derived fungus Rhizopus sp. [J]. Steroids, 2008, 73(1): 19-26.)

[0114] Compound 11:

[0115]

[0116] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Tan Z., Zhao J.L., Liu J.M., et al. Lanostanetriol penoidsandergostane-type steroids from the cultured mycelia of Ganoderm acapense[J]. Journal of Asian Natural Products Research, 2018, 20(9): 844-851.)

[0117] Compound 12:

[0118]

[0119] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Gao H., Hong K., Chen G.D., et al. New oxidized sterols from Aspergillus ammonia and the endo-boat conformation adopted by the cyclohexene oxide system[J]. Magnetic Resonance in Chemistry, 2010, 48(1):38-43.)

[0120] Compound 13:

[0121]

[0122] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Zhang W., Draeger S., Schulz B., et al. Ring Baromatic steroids from an endophytic fungus, Colletotrichum sp. [J]. Natural Product Communications, 2009, 4(11): 1449-1454.)

[0123] Compound 14:

[0124]

[0125] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Kim K.H., Choi S.U., Park K.M., et al. Cytotoxic constituents of Amanita subjunquillea [J]. Archives of Pharmacal Research, 2008, 31(5): 579-586.)

[0126] Compound 15:

[0127]

[0128] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Du Z.Z., Shen Y.M. Arare new cleistanthan editorerpene from the pericarp of Trewiana diflora[J]. Helvetica Chimica Acta, 2006, 89(11):2841-2845.)

[0129] Compound 16:

[0130]

[0131] The structure of the white amorphous powder was determined by comparison with data reported in previous literature. 1 H-NMR and 13 C-NMR data can be found in the literature report (Yajima A., Kagohara Y., Shikai K., et al. Synthesis of twoosteoclast-forming suppressors, demethylincisterol A(3) and chaxine A[J]. Tetrahedron, 2012, 68(6): 1729-1735.)

[0132] Example 2: In vitro detection of the cytotoxic activity of compounds 1-16 prepared in Example 1 against macrophage Raw264.7 cells.

[0133] I. Experimental Methods

[0134] The cytotoxic activity of compound 1-16 against Raw264.7 macrophages was tested using a CCK-8 assay. 3 × 10⁻⁶ compounds were added to each well of a 96-well plate. 4 Raw264.7 cells were cultured overnight in a CO2 incubator. Compound 1-16 was added at concentrations of 1.25, 2.5, 5, 10, 20, and 40 μM, respectively. After 24 hours of incubation, the cells were centrifuged and the supernatant was discarded. CCK-8 was diluted with DMEM medium and added to 96-well plates. After 1 hour, the fluorescence value was read at 450 nm using a microplate reader, and cell viability was calculated.

[0135] II. Experimental Results

[0136] The potential toxicity of compounds 1–16 was evaluated in Raw264.7 cells. Except for compounds 2, 7, and 16 at a concentration of 40 μM, most compounds did not reduce cell viability in Raw264.7 cells. Detection data are attached. Figure 1As shown.

[0137] Example 3: In vitro detection of the effects of compounds 1-16 prepared in Example 1 on the expression of pro-inflammatory factors Tnfα and Ifnb1 in LPS-induced macrophage Raw264.7 cells.

[0138] I. Experimental Methods

[0139] Raw264.7 cells were pretreated with 20 μM compound 1-16 for 2 h, followed by stimulation with 100 ng / mLLPS for 6 h. Total RNA was extracted from Raw264.7 cells using triazole reagent. cDNA was synthesized using SuperScri ptIII reverse transcriptase. qRT-PCR was performed using the PrimeScript RT kit. The relative expression of the target gene and Gapdh was quantified using the ΔΔct method. Primer sequences were: Ifnb1, forward 5'-GCACTGGGTGGAATGAGACT-3' and reverse 5'-AGTGGAGAGCAGTTGAGGACA-3'; Tnfα, forward 5'-GTCCCCAAAGGGATGAGAAGTT-3' and reverse 5'-GTTTGCTA CGACGTGGGCTACA-3'.

[0140] GraphPadPrism 8.0 was used for statistical analysis. One-way ANOVA and Tukey's test were used to analyze the data, comparing them with the blank group and the control group. A p-value <0.05 was considered statistically significant.

[0141] II. Experimental Results

[0142] Compounds 1-16 inhibited the expression of the inflammatory cytokines Tnfα and Ifnb1 in LPS-induced Raw264.7 cells. Detection data are attached. Figure 2 As shown.

[0143] Example 4: In vitro detection of the effects of compounds 1-16 prepared in Example 1 on the expression of pro-inflammatory factors Tnfα and Ifnb1 in DXMAA-induced macrophage Raw264.7 cells.

[0144] I. Experimental Methods

[0145] Raw264.7 cells were pretreated with 20 μM compound 1-16 for 2 h, followed by stimulation with 25 μg / mL cGAS-STING pathway-specific agonist DMXAA for 6 h. Total RNA was extracted from Raw264.7 cells using triazole reagent. cDNA was synthesized using SuperScript III reverse transcriptase. qRT-PCR was performed using the PrimeScriptRT kit. The relative expression of the target gene and Gapdh was quantified using the ΔΔct method. Primer sequences were: Ifnb1, forward 5'-GCACTGGGTGGAATGAGACT-3' and reverse 5'-AGTGGAGAGCAGTTGAGGACA-3'; Tnfα, forward 5'-GTCCCCAAAGGGATGAGAGTT-3' and reverse 5'-GTTTGCTACGACGTGGGCTACA-3'.

[0146] GraphPadPrism 8.0 was used for statistical analysis. One-way ANOVA and Tukey's test were used to analyze the data, comparing them with the blank group and the control group. A p-value <0.05 was considered statistically significant.

[0147] II. Experimental Results

[0148] Compounds 1-16 inhibited the expression of inflammatory cytokines Tnfα and Ifnb1 in Raw264.7 cells induced by DXMAA. Detection data are attached. Figure 3 As shown.

[0149] From the appendix Figure 2 and attached Figure 3 As can be seen, compounds 1-16 can significantly inhibit the expression of pro-inflammatory factors Tnfα and Ifnb1 in macrophages Raw264.7 induced by LPS or DXMAA. This result indicates that compounds 1-16 have good inhibitory activity against type I interferon, which provides a basis for the preparation of type I interferon inhibitors.

[0150] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An ergosterol derivative, characterized in that, Having the structure shown in compound 1, or a pharmaceutically acceptable salt thereof: 。 2. A method for simultaneously preparing the ergosterol derivative as described in claim 1 and compounds 2-16, characterized in that, The total crude extract was obtained from the fermentation broth of the fungus *Penicillium oxalicum*, and then purified to obtain compounds 1-16, the structures of which are as follows: ； The preparation of the fermentation broth includes the following steps: inoculating the fungus *Penicillium oxalate* into a culture medium and fermenting it at 25-30°C to obtain the strain fermentation broth; the culture medium contains malt extract, glucose, sucrose, peptone, potato, rice, or agar; The fermentation broth extraction includes the following steps: extracting the fermentation broth of the strain using an organic solvent, and concentrating the extract under reduced pressure to obtain a total crude extract; The separation and purification process includes the following steps: the total crude extract is separated and purified to obtain compounds 1-16; The organic solvent is ethyl acetate, diethyl ether, methanol, dichloromethane, or chloroform. The separation and purification method is chromatographic separation, specifically normal-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and high-performance liquid chromatography.

3. The preparation method according to claim 2, characterized in that, The fungus *Penicillium oxalicum* was inoculated into a culture medium and cultured at a constant temperature of 25–30°C for a certain period of time. The culture medium was then pulverized, soaked in an organic solvent, and then extracted with an organic solvent by ultrasonication at least once. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. The crude extract was then separated by silica gel thin-layer chromatography. Normal-phase silica gel was selected, and a gradient elution was performed using a dichloromethane / methanol system. The fractions were collected, and similar components were separated and combined by silica gel thin-layer chromatography to obtain 10 fractions, namely Fr.1–10. Fraction Fr.1 was subjected to Sephadex LH-20 gel column chromatography with dichloromethane / methanol as the eluent; followed by normal-phase silica gel column chromatography with petroleum ether / acetone as the eluent, and then semi-preparative high performance liquid chromatography to obtain compounds 1, 6, 11, 12, 14 and 15. Fraction Fr.2 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 4 and 5. Fraction Fr.4 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compounds 3, 7 and 16. Fraction Fr.5 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, and then separated by semi-preparative high performance liquid chromatography to obtain compound 10. Fraction Fr.7 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, to obtain compound 13; Fraction Fr.9 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol, followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, to obtain compound 2; Fraction Fr.10 was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane / methanol (2:1), followed by normal phase silica gel column chromatography, eluted with petroleum ether / acetone system, to obtain compounds 8 and 9; The culture medium includes one or more of PDA medium, Czapek's agar (CZA), peptone yeast extract glucose medium (GPY), Bengal red medium (RBM), and rice.

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