Preparation method and application of a class of sponge-derived pyrrole imidazole alkaloid dimers

Abstract

The application discloses a preparation method and application of a pyrrole imidazole alkaloid dimer from a sponge, and belongs to the technical field of biological medicines. The technical problem that PIAs in marine sponges are difficult to separate and purify efficiently is successfully overcome by a specific method, and a series of pyrrole imidazole alkaloid dimers with novel structures are separated from the sponges for the first time. The pyrrole imidazole alkaloid dimers are divided into four different skeletons according to structures, including two new skeletons and ten new compounds. Compounds 3, 4, 7, 8, 9 and 11 have anti-inflammatory activity of targeting TNF-alpha, and can significantly reduce the gene transcription level of TNF-alpha in a lipopolysaccharide-induced microglial cell inflammation model in a concentration-dependent manner. The ten compounds also have biological activities such as anti-tumor, immunosuppression and anti-neovascularization. The application provides new compounds for development of marine medicinal molecules and provides a new scientific basis for development and utilization of marine medicinal resources.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying a type of sponge-derived pyrrolimidazole alkaloid dimer. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The ocean nurtures abundant biological resources, among which sponges and other organisms have evolved unique chemical defense systems due to their special living environment, producing a large number of bioactive secondary metabolites, providing an important source for new drug development. Pyrrolimidazole alkaloids (PIAs) are secondary metabolites unique to marine sponges, isolated only from sponges, and possess diverse structural frameworks (more than 30 types) and significant anti-inflammatory and immunosuppressive biological activities. These compounds are all formed from the precursor monomer oroidin through biosynthetic pathways to form monomers, dimers, or tetramers, with dimers being the most abundant and diverse in structure. Representative complex dimers (such as Palau'amine and its analogues) have been shown to have specific target bioactivity, demonstrating their significant application potential.

[0004] However, the precise separation of novel PIA dimers from marine sponges faces significant challenges. On one hand, the guanidine groups in these substances result in high polarity, making conventional separation methods (such as column chromatography and thin-layer chromatography) ineffective. On the other hand, the complex structures lead to significant differences in the UV absorption spectra of PIAs with different skeletons, resulting in densely overlapping peaks in HPLC chromatograms and extremely low separation and analysis efficiency. Existing technologies struggle to achieve the precise separation and identification of these complex active ingredients, hindering the efficient discovery of novel PIA dimers. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing and applying a class of sponge-derived pyrrolimidazole alkaloid dimers. This invention utilizes ultraviolet (UV) tracing to create a "UV tracing map" of various PIA backbone types. This map allows for the elimination of duplicates of known backbone types, thereby focusing on backbone types indicated by "new UV spectra" and efficiently identifying new PIA backbones. Through research, this invention has identified concentrated sites of PIA dimers. Further purification yielded 13 PIA dimer compounds (compounds 1-13), of which compounds 1-4 are new backbone compounds, and compounds 1-10 are new compounds. Furthermore, activity studies on the isolated PIA dimer compounds showed that compounds 3, 4, 7, 8, 9, and 11 could reduce the transcriptional level of the TNF-α gene in an LPS-induced BV2 cell inflammation model in a concentration-dependent manner.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a class of sponge-derived pyrrolimidazole alkaloid dimers having any one or more of the following structures:

[0008]

[0009]

[0010] A second aspect of the present invention provides a method for preparing the sponge-derived pyrrolimidazole alkaloid dimer described in the first aspect, comprising:

[0011] The sponge was extracted with ethyl acetate to obtain a crude extract;

[0012] The crude extract was subjected to gradient elution through a forward silica column to obtain a primary fraction containing the target pyrrolimidazole alkaloid dimer.

[0013] The primary fraction was eluted by medium-pressure liquid chromatography to obtain a secondary fraction containing the target pyrrolimidazole alkaloid dimer.

[0014] The secondary fraction was subjected to gradient elution through a forward silica column to obtain a tertiary fraction containing the target pyrrolimidazole alkaloid dimer;

[0015] The tertiary fraction was eluted by gel column chromatography to obtain the quaternary fraction containing the target pyrrolimidazole alkaloid dimer;

[0016] The four fractions were separated by high performance liquid chromatography to obtain the target pyrrolimidazole alkaloid dimer.

[0017] In some embodiments of the present invention, the gradient elution via a forward silica column is performed using a petroleum ether / ethyl acetate mixture and a dichloromethane / methanol mixture as the eluents; first, the petroleum ether / ethyl acetate mixture is used for elution, with the ratio of petroleum ether to ethyl acetate changing from 5:1 to 0:1, and then the dichloromethane / methanol mixture is used for elution, with the ratio of dichloromethane to methanol changing from 10:1 to 0:1.

[0018] In some embodiments of the present invention, the elution is performed by medium-pressure liquid chromatography for rapid preparative chromatography, and the eluent is a methanol / water mixture; during elution, the volume percentage of methanol increases from 5% to 100%.

[0019] In some embodiments of the present invention, the elution by gel column chromatography uses methanol as the eluent.

[0020] In some embodiments of the present invention, the high-performance liquid chromatography separation uses a mobile phase of a methanol / water mixture or an acetonitrile / water mixture containing 0.09-0.11% trifluoroacetic acid, an Agilent XDB-C18 column or a COSMOSIL πNAP column, a flow rate of 1.9-2.1 mL / min, an injection volume of 9-11 μL, and a column temperature of 24.5-25.5 °C.

[0021] A third aspect of the present invention provides a pharmaceutical preparation in which the active ingredient is the sponge-derived pyrrolimidazole alkaloid dimer or its pharmaceutical salt as described in the first aspect.

[0022] In some embodiments of the present invention, the pharmaceutical preparation further includes pharmaceutically acceptable excipients.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers and / or excipients.

[0024] In some embodiments of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, at least one of alumina, aluminum stearate, polyvinylpyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, and lanolin.

[0025] In some embodiments of the present invention, the excipients include, but are not limited to, at least one of adhesives, fillers, lubricants, disintegrants, and wetting agents.

[0026] In some embodiments of the present invention, the dosage form of the pharmaceutical preparation is an oral preparation, an injectable preparation, or a topical preparation.

[0027] In some embodiments of the present invention, the drug preparation is administered orally, by spray inhalation, by intravenous injection, by rectal administration, or by intraperitoneal instillation.

[0028] A fourth aspect of the present invention provides the use of the sponge-derived pyrrolimidazole alkaloid dimer described in the first aspect or the above-described pharmaceutical preparation or compound konbu'acidinA in the preparation of a TNF-α-related inflammation inhibitor.

[0029] A fifth aspect of the present invention provides a method for screening sponge-derived pyrrolimidazole alkaloid dimers, comprising:

[0030] The sponge was extracted with ethyl acetate to obtain a crude extract;

[0031] The crude extract was separated to obtain several fractional components;

[0032] The ultraviolet tracing method was used to screen several components to identify the components containing the target pyrrolimidazole alkaloid dimer, which was then separated by high performance liquid chromatography to obtain the target pyrrolimidazole alkaloid dimer.

[0033] In some embodiments of the present invention, the ultraviolet tracking method includes: performing full-wavelength high-performance liquid chromatography analysis on each component to obtain UV spectra and retention times, and determining the skeleton type of the target pyrrolimidazole alkaloid dimer and the component containing the target pyrrolimidazole alkaloid dimer by using the UV spectra and retention times.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention successfully overcomes the technical challenge of efficiently separating and purifying pyrrolimidazole alkaloid dimers (PIAs) from marine sponges due to their high polarity, significant differences in structural framework and UV absorption. Using this technique, a series of novel pyrrolimidazole alkaloid dimer compounds were precisely isolated from the sponge *Stylissa massa* for the first time. These pyrrolimidazole alkaloid dimers were classified into four different frameworks, including two new frameworks and ten new compounds. The obtained compounds (compounds 3, 4, 7, 8, 9, and 11) were verified to significantly reduce the gene transcription level of the key pro-inflammatory factor TNF-α in a lipopolysaccharide (LPS)-induced microglia (BV2) inflammation model in a concentration-dependent manner, exhibiting potential anti-inflammatory activity. However, the newly discovered macrocyclic framework compounds 1 and 2 showed weaker inhibitory activity. Based on existing technology, the pyrrolimidazole alkaloids 1-13 isolated in this invention may also possess antitumor, immunosuppressive, and anti-angiogenic biological activities, warranting further investigation. This invention provides new compounds for the development of marine drug molecules and offers new scientific evidence for the development and utilization of marine medicinal resources. It also provides efficient technical support for the in-depth exploration of structurally diverse marine natural products (especially complex PIAs dimers) and yields several novel compound skeletons with clear anti-inflammatory activities, providing an important material basis for the development of new drug lead compounds for treating inflammation-related diseases.

[0036] This invention employs an ultraviolet tracking method. Based on the ultraviolet spectral maps of known PIAs, it first discovers new skeletal PIA dimers by identifying new ultraviolet absorption spectra. This method enables target tracking and precise acquisition of target pyrrolimidazole alkaloid dimers, solving the problem of repeated separation of known compounds. The discovery efficiency and accuracy of PIA dimers are significantly improved. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1 This is the ultraviolet tracking map in Embodiment 1 of the present invention (where skeletons 1 and 2 are new skeletons).

[0039] Figure 2 The image shows the ultraviolet spectrum of the PIAs dimer structure in Example 1 of this invention.

[0040] Figure 3 The HPLC chromatograms of six common PIA skeletons in the embodiments of the present invention show different retention times (5-100% methanol / water, 0.1% trifluoroacetic acid added, 1h) under the same conditions.

[0041] Figure 4 The HPLC chromatogram of the concentrated PIAs dimer in Example 1 of the present invention (5-100% methanol / water, with the addition of 0.1% trifluoroacetic acid, 1h).

[0042] Figure 5 This is a high-resolution mass spectrometry of compound 1 extracted in Example 1 of the present invention.

[0043] Figure 6 This is an X-ray single-crystal diffraction pattern of compound 1 extracted in Example 1 of the present invention.

[0044] Figure 7 This is a high-resolution mass spectrometry of compound 2 extracted in Example 1 of the present invention.

[0045] Figure 8 This is an X-ray single-crystal diffraction pattern of compound 2 extracted in Example 1 of the present invention.

[0046] Figure 9 This is a high-resolution mass spectrometry of compound 3 extracted in Example 1 of the present invention.

[0047] Figure 10 This is a high-resolution mass spectrometry of compound 4 extracted in Example 1 of the present invention.

[0048] Figure 11 This is a high-resolution mass spectrometry of compound 5 extracted in Example 1 of the present invention.

[0049] Figure 12 This is a high-resolution mass spectrometry of compound 6 extracted in Example 1 of the present invention.

[0050] Figure 13 This is a high-resolution mass spectrometry of compound 7 extracted in Example 1 of the present invention.

[0051] Figure 14 This is a high-resolution mass spectrometry of compound 8 extracted in Example 1 of the present invention.

[0052] Figure 15 This is a high-resolution mass spectrometry of compound 9 extracted in Example 1 of the present invention.

[0053] Figure 16 This is a high-resolution mass spectrometry of compound 10 extracted in Example 1 of the present invention.

[0054] Figure 17 This is a high-resolution mass spectrometry of compound 11 extracted in Example 1 of the present invention.

[0055] Figure 18 This is a high-resolution mass spectrum of compound 12 extracted in Example 1 of the present invention.

[0056] Figure 19 This is a high-resolution mass spectrometry of compound 13 extracted in Example 1 of the present invention.

[0057] Figure 20 The experimental and calculated ECD curves of compound 3 extracted in Example 1 of this invention are shown.

[0058] Figure 21 The experimental and calculated ECD curves of compound 4 extracted in Example 1 of this invention are shown.

[0059] Figure 22 Experimental and calculated ECD curves of compounds 5 and 6 extracted in Example 1 of this invention.

[0060] Figure 23 The experimental ECD curves are for compounds 7, 8, 9, and 11 extracted in Example 1 of this invention.

[0061] Figure 24 The results of the test on the transcriptional activity of compounds 1-13 on the TNF-α gene in Example 2 of this invention are shown. Detailed Implementation

[0062] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0063] This invention provides a class of sponge-derived pyrrolimidazole alkaloid dimers, the structure of which is any one or more of the following:

[0064]

[0065]

[0066] This invention utilizes ultraviolet (UV) tracing to create a "UV tracing map" for each PIA skeleton type. This map allows for the elimination of duplicate known skeleton types, thereby focusing on the skeleton type indicated by "new UV spectra" and efficiently identifying new PIA skeletons. In this regard, this invention discovered concentrated sites of PIA dimers in sponges, and through further purification, yielded 13 PIA dimer compounds. To distinguish between the compounds, they are named compounds 1-13, and their structures are shown below:

[0067]

[0068] Compounds 1-4 are novel skeletal compounds, and compounds 1-10 are also novel compounds. Compounds 11-13 are known compounds konbu'acidin A, benzosceptrin C, and benzosceptrin B, respectively. Activity studies on the isolated PIAs dimers showed that the novel skeletal compounds 3 and 4, and konbu'acidin skeletal derivatives 7, 8, 9, and 11, possess anti-inflammatory activity targeting TNF-α, and can reduce the transcriptional level of the TNF-α gene in an LPS-induced BV2 cell inflammation model in a concentration-dependent manner. The newly discovered macrocyclic skeletal compounds 1 and 2 showed weaker inhibitory activity, providing an important material basis for developing novel drug lead compounds for treating inflammation-related diseases. Based on previous research, the PIAs compounds 1-13 isolated in this invention may also possess anti-tumor, immunosuppressive, and anti-angiogenic biological activities, warranting further investigation. This invention provides new compounds for the development of marine drug molecules and offers new scientific evidence for the development and utilization of marine medicinal resources.

[0069] The present invention also provides a method for preparing the above-mentioned sponge-derived pyrrolimidazole alkaloid dimer, comprising:

[0070] The sponge was extracted with ethyl acetate to obtain a crude extract;

[0071] The crude extract was subjected to gradient elution through a forward silica column to obtain a primary fraction containing the target pyrrolimidazole alkaloid dimer.

[0072] The primary fraction was eluted by medium-pressure liquid chromatography to obtain a secondary fraction containing the target pyrrolimidazole alkaloid dimer.

[0073] The secondary fraction was subjected to gradient elution through a forward silica column to obtain a tertiary fraction containing the target pyrrolimidazole alkaloid dimer;

[0074] The tertiary fraction was eluted by gel column chromatography to obtain the quaternary fraction containing the target pyrrolimidazole alkaloid dimer;

[0075] The four fractions were separated by high performance liquid chromatography to obtain the target pyrrolimidazole alkaloid dimer.

[0076] This invention extracts pyrrolimidazole alkaloid dimers from marine sponges. After identifying the target pyrrolimidazole alkaloid dimer, a step-by-step elution process is performed to enrich the target pyrrolimidazole alkaloid dimer, thereby accurately obtaining the target pyrrolimidazole alkaloid dimer. The sponge-derived pyrrolimidazole alkaloid dimer preparation method of this application has the advantages of high efficiency and high accuracy in the discovery of target pyrrolimidazole alkaloid dimers.

[0077] In this invention, the sponge is Stylissa massa.

[0078] In this invention, there are no special restrictions on the source of any raw materials; they can be commercially available.

[0079] In this invention, gradient elution is performed using a forward silica gel column, with the eluent being a mixture of petroleum ether / ethyl acetate and a mixture of dichloromethane / methanol. First, the petroleum ether / ethyl acetate mixture is used for elution, with the ratio of petroleum ether to ethyl acetate decreasing from 5:1 to 0:1. Then, the dichloromethane / methanol mixture is used for elution, with the ratio of dichloromethane to methanol decreasing from 10:1 to 0:1.

[0080] Specifically, the elution was first performed using a petroleum ether / ethyl acetate mixed solution with a ratio of 5:1 to 1:1 to 0:1; then, the elution was performed using a dichloromethane / methanol mixed solution with a ratio of 10:1 to 5:1 to 3:1 to 0:1.

[0081] In this invention, arrows indicate that the proportion of eluent changes from one proportion to another during the elution process.

[0082] In this invention, the elution is performed by rapid preparative chromatography at medium pressure liquid chromatography, and the eluent is a methanol / water mixture; during elution, the volume percentage of methanol increases from 5% to 100%.

[0083] Specifically, during elution, the volume percentage of methanol in the methanol / water mixture changes from 5% to 10% to 30% to 50% to 70% to 100%. The arrows indicate the change in the volume percentage of methanol during elution.

[0084] In this invention, the elution is performed by gel column chromatography, and the eluent is methanol.

[0085] Specifically, Sephadex G-10 gel column can be used for chromatographic elution.

[0086] In this invention, the high-performance liquid chromatography separation uses a mobile phase of a methanol / water mixture or an acetonitrile / water mixture containing 0.09-0.11% (v / v) trifluoroacetic acid, an Agilent XDB-C18 column or a COSMOSIL πNAP column, a flow rate of 1.9-2.1 mL / min, an injection volume of 9-11 μL, and a column temperature of 24.5-25.5 °C.

[0087] Specifically, when performing high-performance liquid chromatography (HPLC) separation on the aforementioned fourth-order fractions, the mobile phase was a methanol / water mixture containing 0.09-0.11% trifluoroacetic acid, with a methanol-to-water volume ratio of 9:91-11:89. The chromatographic column was an Agilent XDB-C18 column, the flow rate was 1.9-2.1 mL / min, the injection volume was 9-11 μL, and the column temperature was 24.5-25.5 °C, yielding compound 1 and compound 2. The retention times for compound 1 and compound 2 were 49.4 ± 0.5 min and 55.6 ± 0.5 min, respectively.

[0088] Specifically, when performing high-performance liquid chromatography (HPLC) separation on the four fractions, the mobile phase was a methanol / water mixture containing 0.09-0.11% trifluoroacetic acid, with a methanol-to-water volume ratio of 56:44-58:42. The chromatographic column was an Agilent XDB-C18 column, the flow rate was 1.9-2.1 mL / min, the injection volume was 9-11 μL, and the column temperature was 24.5-25.5℃, yielding compounds 3, 4, 10, and 12. The retention times for compounds 3, 4, 10, and 12 were 11.6±0.5 min, 18.1±0.5 min, 14.2±0.5 min, and 23.1±0.5 min, respectively.

[0089] Specifically, when performing high-performance liquid chromatography (HPLC) separation on the aforementioned fourth-order fractions, the mobile phase was a methanol / water mixture containing 0.09-0.11% trifluoroacetic acid, with a methanol-to-water volume ratio of 31:69-33:67. The chromatographic column was an Agilent XDB-C18 column, the flow rate was 1.9-2.1 mL / min, the injection volume was 9-11 μL, and the column temperature was 24.5-25.5 °C, yielding compounds 5 and 6. The retention times for compounds 5 and 6 were 57.9 ± 0.5 min and 62.6 ± 0.5 min, respectively.

[0090] Specifically, when performing high-performance liquid chromatography (HPLC) separation on the four fractions, the mobile phase was a methanol / water mixture containing 0.09-0.11% trifluoroacetic acid, with a methanol-to-water volume ratio of 49:51-51:49. The chromatographic column was an Agilent XDB-C18 column, the flow rate was 1.9-2.1 mL / min, the injection volume was 9-11 μL, and the column temperature was 24.5-25.5 °C, yielding compounds 7, 9, and 13. The retention times for compounds 7, 9, and 13 were 14.3 ± 0.5 min, 27.3 ± 0.5 min, and 21.3 ± 0.5 min, respectively.

[0091] Specifically, when performing high-performance liquid chromatography (HPLC) separation on the aforementioned fourth-order fractions, the mobile phase was an acetonitrile / water mixture containing 0.09-0.11% trifluoroacetic acid, with a volume ratio of acetonitrile to water of 29:71-31:69. The chromatographic column was a COSMOSIL πNAP column, the flow rate was 1.9-2.1 mL / min, the injection volume was 9-11 μL, and the column temperature was 24.5-25.5℃, yielding compounds 8 and 11. The retention times for compounds 8 and 11 were 21.8 ± 0.5 min and 23.7 ± 0.5 min, respectively.

[0092] The present invention also provides a pharmaceutical preparation in which the active ingredient is the above-mentioned sponge-derived pyrrolimidazole alkaloid dimer or its pharmaceutical salt.

[0093] In this invention, the medicinal salt can be a salt formed by the sponge-derived pyrrolimidazole alkaloid dimer and an inorganic acid (hydrochloric acid, sulfuric acid, nitric acid, etc.), or a salt formed by the sponge-derived pyrrolimidazole alkaloid dimer and an organic acid (acetic acid, oxalic acid, citric acid, fumaric acid, etc.).

[0094] In this invention, the pharmaceutical preparation further includes pharmaceutically acceptable excipients that can be determined by those skilled in the art to meet clinical standards.

[0095] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers and / or excipients.

[0096] The pharmaceutically acceptable carriers include, but are not limited to, at least one of serum proteins, chitosan, polylactic acid, stearic acid, alumina, aluminum stearate, polyvinylpyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, and lanolin. Typically, the properties of the carrier depend on the specific route of administration. For example, parenteral preparations often contain an injectable fluid as a vehicle, which includes pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerin, etc. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate. No specific limitations are imposed here.

[0097] The excipients include, but are not limited to, at least one of binders, fillers, lubricants, disintegrants, emulsifiers, stabilizers, antioxidants, pH adjusters, preservatives, fragrances, and humectants. The binders include, but are not limited to, at least one of syrups, gum arabic, and sorbitol. The fillers include, but are not limited to, at least one of lactose, corn starch, and glycine. The lubricants include, but are not limited to, at least one of magnesium stearate, talc, and silica. The humectants include, but are not limited to, sodium lauryl sulfate.

[0098] In this invention, the dosage form of the pharmaceutical preparation is an oral preparation, an injectable preparation, or a topical preparation.

[0099] In this invention, the oral preparations are tablets, capsules, granules, drops, concentrated pills, or liquids; the injectable preparations are liquid injections, powders for injection, or tablets for injection; and the external preparations are external solvents, ointments, patches, powders for external use, or inhalers.

[0100] In this invention, the drug preparation is administered orally, by spray inhalation, by intravenous injection, by rectal administration, or by intraperitoneal instillation.

[0101] In this invention, the pharmaceutical preparation can be administered into the body in a known manner, such as via intravenous systemic delivery. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in this invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0102] In this invention, the drug preparation can be administered to humans and non-human mammals, including mice, rats, guinea pigs, cattle, sheep, cats, dogs, horses, monkeys, and orangutans, with humans being the preferred species.

[0103] The present invention also provides a method for preparing the above-mentioned pharmaceutical preparation, comprising: mixing the above-mentioned sponge-derived pyrrolimidazole alkaloid dimer or its pharmaceutical salt and pharmaceutically acceptable excipients in a certain proportion, and preparing the pharmaceutical preparation according to the requirements of different dosage forms.

[0104] The present invention also provides the use of the above-mentioned sponge-derived pyrrolimidazole alkaloid dimer or the above-mentioned pharmaceutical preparation or compound konbu'acidinA in the preparation of TNF-α-related inflammation inhibitors.

[0105] TNF-α, a core pro-inflammatory cytokine, is secreted by macrophages, monocytes, and other cells. In inflammatory responses, it synergistically regulates the production of other cytokines, influencing cell survival and death. In many inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, TNF-α expression is significantly increased and closely related to disease progression. Elevated levels can also serve as indicators for assessing disease activity and treatment efficacy, thus making it a key target for inflammation suppression. Anti-TNF-α therapy has achieved some success in the treatment of related diseases.

[0106] This invention also provides a method for screening sponge-derived pyrrolimidazole alkaloid dimers, comprising:

[0107] The sponge was extracted with ethyl acetate to obtain a crude extract;

[0108] The crude extract was separated to obtain several fractional components;

[0109] The ultraviolet tracing method was used to screen several components to identify the components containing the target pyrrolimidazole alkaloid dimer. The sponge-derived pyrrolimidazole alkaloid dimer was then separated by high performance liquid chromatography.

[0110] In this invention, the ultraviolet tracking method includes: performing full-wavelength high-performance liquid chromatography analysis on each component to obtain UV spectra and retention times, comparing with a UV map of a known skeleton, and determining the skeleton type of the target pyrrolimidazole alkaloid dimer and the component containing the target pyrrolimidazole alkaloid dimer by using UV spectra and retention times.

[0111] In this invention, the sponge is Stylissa massa.

[0112] This invention utilizes high-performance liquid chromatography (HPLC) to detect different pyrrolimidazole alkaloids in fractional components. Based on the detection results, an ultraviolet (UV) tracking map is obtained relating the skeleton type of pyrrolimidazole alkaloids to UV spectra. This UV tracking map is then used for UV tracing. Specifically, the structural information provided by the HPLC separation conditions, mobile phase ratios, retention times, and UV absorption spectra of the separated PIAs compounds is summarized and organized to obtain characteristic chromatographic and spectral information of compounds with different skeleton types containing characteristic functional groups. Using the established HPLC-DAD online molecular characteristic spectrum detection method, an UV tracking map relating the skeleton type of pyrrolimidazole alkaloids to UV spectra is constructed (e.g., ...). Figure 1 As shown in the figure, UV-1 and UV-2 are two novel ultraviolet absorbers discovered in this invention, representing two previously unreported new PIAs frameworks, and therefore ultraviolet tracking can be performed using the ultraviolet tracking map.

[0113] In this invention, the crude extract is pre-separated using a forward silica gel column to obtain several multi-stage fractions.

[0114] Specifically, the pre-separation is performed using a forward silica gel column, with the eluent being a petroleum ether / ethyl acetate mixture and a dichloromethane / methanol mixture. First, the petroleum ether / ethyl acetate mixture is used for elution, with the ratio of petroleum ether to ethyl acetate decreasing from 5:1 to 1:1 and then to 0:1. Then, the dichloromethane / methanol mixture is used for elution, with the ratio of dichloromethane to methanol decreasing sequentially from 10:1 to 5:1, 3:1, and 0:1.

[0115] Specifically, elution is first performed using a petroleum ether / ethyl acetate mixture with a ratio of 5:1 → 1:1 → 0:1; then, elution is performed using a dichloromethane / methanol mixture with a ratio of 10:1 → 5:1 → 3:1 → 0:1. The arrows indicate changes in the eluent ratio during the elution process.

[0116] Different skeletal structures exhibit characteristic ultraviolet (UV) absorption spectra, which can be used to infer the structural type of a substance. PIAs (polycyclic aromatic hydrocarbons) have a high N / C ratio and, after polymerization using oroidin as a biosynthetic precursor, form complex polycyclic compounds. Compounds with the same skeleton but different substituents exhibit similar UV absorption spectra, while polymers with different skeletons correspond to distinct UV absorption spectra. Based on this characteristic, this invention uses the UV spectra of HPLC absorption peaks and their retention times to determine the compound type of the chromatographic peaks, enabling UV tracking of target PIAs. This significantly improves separation efficiency and accuracy, effectively avoiding redundant separations.

[0117] This invention identified the concentrated sites of PIAs dimers using the aforementioned screening method. Further purification yielded 13 PIAs dimer compounds (compounds 1-13), resulting in four different pyrrolimidazole alkaloid dimers with varying skeletons, including two novel pyrrolimidazole alkaloid dimer skeletons and ten novel pyrrolimidazole alkaloid dimers. Compounds 1-4 are novel skeleton compounds, and compounds 1-10 are novel compounds. Activity studies on the isolated PIAs dimer compounds showed that novel skeleton compounds 3 and 4, and konbu'acidin skeleton-derived compounds 7, 8, 9, and 11, exhibited anti-inflammatory activity targeting TNF-α, and could reduce the transcriptional level of the TNF-α gene in an LPS-induced BV2 cell inflammation model in a concentration-dependent manner, providing an important material basis for developing novel drug lead compounds for the treatment of inflammation-related diseases. However, the newly discovered macrocyclic skeleton compounds 1 and 2 showed weak inhibitory activity. Based on previous research, the PIAs compounds 1-13 isolated in this invention may also possess antitumor, immunosuppressive, and anti-angiogenic biological activities, warranting further in-depth investigation. This invention provides new compounds for the development of marine drug molecules and offers new scientific basis for the development and utilization of marine medicinal resources.

[0118] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0119] Example 1

[0120] S1: The sponge sample *Stylissa massa* was soaked in ethyl acetate for 10 days, during which time ultrasonic-assisted extraction was performed to obtain the first extract. The sponge sample was then cut into pieces of approximately 1 cm. 3 Small pieces were then soaked in ethyl acetate for 10 days, during which ultrasonic-assisted extraction was performed to obtain a second extract. The extracts were combined and concentrated to obtain a crude extract. The ultrasonic-assisted extraction was carried out at room temperature, with an ultrasonic power of 150W and an ultrasonic time of 15 minutes each time. The extraction was performed twice in total, and the extract was named SM-Y.

[0121] S2: The crude extract SM-Y obtained in step S1 was passed through a normal silica gel column (silica gel mesh size 200-300) for gradient elution. The eluents were a mixture of petroleum ether / ethyl acetate and a mixture of dichloromethane / methanol, with the following ratios: petroleum ether:ethyl acetate = 5:1 → 1:1 → 0:1 → dichloromethane:methanol = 10:1 → 5:1 → 3:1 → 0:1. The total elution time was approximately 200 minutes. After elution, the collected filtrate was concentrated, yielding 14 primary fractions, which were named SM-Y-1, SM-Y-2, SM-Y-3…SM-Y-14 in the order of elution. Since different skeletons of pyrrolimidazole alkaloids (PIAs) show different retention times in HPLC chromatograms under the same conditions, the skeleton type of the compound can be determined by HPLC analysis of its UV absorption and retention time. High-performance liquid chromatography (HPLC) fingerprints were plotted for each of the 14 primary fractions. Analysis of the fingerprints revealed several unique chromatographic peaks in the SM-Y-2 to SM-Y-7 fractions with retention times of 38-50 min. Upon reviewing the UV absorption spectra and comparing them with previously plotted UV absorption maps, these peaks were found to be previously unseen, suggesting potential novel PIAs with a new skeletal framework. The focus then shifted to the isolation and preparation of these compounds. These peaks were then combined and segmented using medium-pressure liquid chromatography (HPLC) to remove extreme polarity impurities.

[0122] S3: The six primary fractions containing the target component obtained in step S2, namely SM-Y-2, SM-Y-3, SM-Y-4, SM-Y-5, SM-Y-6, and SM-Y-7, are combined and renamed SM-Y-2. SM-Y-2 is then eluted by reversed-phase medium-pressure liquid chromatography (RPLC) with a methanol / water mixture as the eluent. During gradient elution, the volume ratio of methanol in the methanol / water mixture is 5% → 10% → 30% → 50% → 70% → 100%. The total elution time is approximately 200 minutes. After elution, the collected filtrate was concentrated to obtain eight secondary fractions, which were named SM-Y-2-1, SM-Y-2-2, SM-Y-2-3...SM-Y-2-8 in the order of elution. HPLC fingerprints were plotted for each of these secondary fractions to determine that the target component was located in fractions SM-Y-2-2 to SM-Y-2-7. These fractions containing the target component were combined, renamed SM-Y-2-2, and then subjected to further separation and purification.

[0123] S4: Separate SM-Y-2-2 obtained in step S3 using a normal silica gel column (silica gel mesh size 200-300). The eluents are a mixture of petroleum ether / ethyl acetate and a mixture of dichloromethane / methanol, with the following eluent ratios: petroleum ether:ethyl acetate = 5:1 → 1:1 → 0:1 → dichloromethane:methanol = 10:1 → 5:1 → 3:1 → 0:1. The total elution time is approximately 200 minutes. After elution, the collected filtrate is concentrated to obtain 15 tertiary fractions, which are named SM-Y-2-2-1, SM-Y-2-2-2, SM-Y-2-2-3…SM-Y-2-2-15 according to the elution order. High-performance liquid chromatography (HPLC) fingerprints were plotted for each of the 15 tertiary fractions to identify the target component in fractions SM-Y-2-2-7 to SM-Y-2-2-15. These fractions containing the target component were then combined, renamed SM-Y-2-2-7, and subjected to further separation and purification.

[0124] S5: Pass the SM-Y-2-2-7 obtained in step S4 through a Sephadex G-10 gel column with methanol as the eluent. After elution, the collected filtrate is concentrated to obtain 126 quaternary fractions, namely SM-Y-2-2-7-1, SM-Y-2-2-7-2, SM-Y-2-2-7-3...SM-Y-2-2-7-126. HPLC fingerprint chromatograms are plotted for each fraction, and the target chromatographic peaks are determined to be in the ranges of SM-Y-2-2-7-6 to SM-Y-2-2-7-35 and SM-Y-2-2-7-49 to SM-Y-2-2-7-122.

[0125] S6: SM-Y-2-2-7-6 to SM-Y-2-2-7-35 and SM-Y-2-2-7-49 to SM-Y-2-2-7-122 were precisely purified by HPLC to obtain compounds 1-13. Compound 1 was prepared using a 10% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 49.4 min; Compound 2 was prepared using a 10% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 55.6 min; Compound 3 was prepared using a 57% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 11.6 min; Compound 4 was prepared using a 57% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 18.1 min; Compound 5 was prepared using a 32% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 57.9 min; Compound 6 was prepared using a 32% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 57.9 min; The retention time for compound 7 was 62.6 min using an XDB-C18 column; the preparation conditions for compound 7 were 50% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 14.3 min; the preparation conditions for compound 8 were 30% acetonitrile / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 62.6 min.

[0126] The retention time for compound 9 was 21.8 min using a COSMOSIL column; the preparation conditions for compound 10 were 50% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 27.3 min; the preparation conditions for compound 10 were 57% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 14.2 min; the preparation conditions for compound 11 were 30% acetonitrile / water solution with 0.1% trifluoroacetic acid, using a COSMOSIL πNAP column, with a retention time of 23.7 min; the preparation conditions for compound 12 were 57% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 23.1 min; the preparation conditions for compound 13 were 50% methanol / water solution with 0.1% trifluoroacetic acid, using an Agilent XDB-C18 column, with a retention time of 21.3 min.

[0127] S7: The obtained 13 compounds were subjected to NMR, HRESIMS, ECD and SXRD to determine their planar and stereo structures. Compounds 1-10 were identified as new PIAs dimers, of which compounds 1-4 were new skeletal compounds and compounds 11-13 were known PIAs dimers.

[0128] The structural characterization of compounds 1-13 is shown in the table below:

[0129] Table 1. NMR structural characterization data of compound 1 and compound 2

[0130]

[0131]

[0132] a Signals were recorded at 500MHz; b Signals were recorded at 125MHz;

[0133] Table 2. NMR structural characterization data of compounds 3 and 4

[0134]

[0135]

[0136] a Signals were recorded at 600MHz; b Signals were recorded at 150MHz;

[0137] Table 3. NMR structural characterization data of compounds 5, 6, and 7.

[0138]

[0139]

[0140] a Signals were recorded at 600MHz; b Signals were recorded at 150MHz;

[0141] Table 4. NMR structural characterization data of compounds 8, 9, and 11.

[0142]

[0143]

[0144] a Signals recorded at 600MHz in DMSO-d6; b Signals recorded at 150MHz in DMSO-d6;

[0145] Table 5. NMR structural characterization data of compounds 10, 12, and 13.

[0146]

[0147]

[0148] a Signals were recorded at 600MHz; b Signals were recorded at 150MHz;

[0149] In this embodiment, the crude extract of Stylissa massa was obtained by ultrasonic extraction with ethyl acetate. The crude extract was then separated stepwise to obtain several fractions. During the stepwise separation process, the target pyrrolimidazole alkaloid dimer was screened using ultraviolet tracing.

[0150] The ultraviolet tracing process is as follows: First, the components of each part of the sponge sample Stylissa massa obtained through primary separation are analyzed.

[0151] HPLC-DAD analysis was performed by retrieving UV absorption spectra and plotting a PIAs UV tracking map. Figure 1 In comparison, several new types of PIAs (indicating UV absorption spectroscopy) were discovered. Figure 2 This suggests that the target PIAs may be new scaffold components. Therefore, in subsequent stepwise separation processes, HPLC-DAD analysis was performed on each fraction, and UV absorption spectra were retrieved to locate and track fractions containing the target PIAs. Figure 4 ).

[0152] Different substances have characteristic ultraviolet absorption spectra, and their structures can be inferred from these spectra. This invention utilizes the comparison between UV absorption spectra and existing ultraviolet tracking maps to discover new skeletal PIAs by identifying new UV absorption spectra. Furthermore, different skeletal PIAs show different retention times in their HPLC analysis chromatograms under the same conditions. Figure 3 Therefore, the retention time in the HPLC chromatogram can also be used to determine the PIAs skeleton type. Target tracking of the target PIAs greatly improves the efficiency and accuracy of separation.

[0153] The separation and purification process of the sponge sample Stylissa massa in this embodiment can be roughly divided into the following main parts:

[0154] The first step is to extract crude extract from the sponge sample.

[0155] The second step is to plot HPLC fingerprints of the obtained primary fractions, observe their retention times, compare them with the retention times of common PIA skeletons under the same HPLC analysis conditions, and compare the UV absorption spectrum of each chromatographic peak with the plotted UV tracking map to determine if it is a previously undiscovered UV absorption, suggesting that it may be a new PIA skeleton. Therefore, such compounds are identified as target compounds.

[0156] The third step involved separating the target component using various natural product separation methods, including forward silica column chromatography, medium-pressure rapid preparation, and gel column chromatography. During the separation process, HPLC fingerprints were generated in real time to pinpoint the location of the target component and maximize its collection to ensure the smooth progress of subsequent experiments.

[0157] Finally, the target components were precisely purified and prepared by high performance liquid chromatography to obtain compounds 1-13. Their planar structures were confirmed by NMR and HRESIMS detection, and their stereoconfigurations were confirmed by ECD and SXRD methods. Finally, compounds 1-13 were determined to be PIAs dimers, among which compounds 1-4 were new skeletal PIAs dimer compounds, compounds 1-10 were new PIAs dimer compounds, and compounds 11-13 were known PIAs dimers konbu'acidin A, benzosceptrin C, and benzosceptrin B, respectively.

[0158] Example 2

[0159] Test of PIAs dimer's transcriptional activity against TNF-α gene:

[0160] Experimental materials:

[0161] Mouse glial cell line (BV2 cells), lipopolysaccharide (LPS), TRIzol cell lysate, isopropanol (analytical grade), anhydrous ethanol (analytical grade), chloroform (analytical grade), DEPC-treated water (DNA / RNase-free, molecular biology grade), HiScript 111 RT SuperMix for qPCR (+gDNAwiper), ChamQ Universal SYBR qPCRMaster Mix.

[0162] Experimental principle:

[0163] TNF-α (tumor necrosis factor α) is a ligand of the TNF superfamily and a pleiotropic cellular molecule that plays a central role in inflammation, apoptosis, and immune system development. It is secreted by various cell types, including macrophages, monocytes, and transformed cells. As a pro-inflammatory cytokine, TNF-α participates in normal inflammatory and immune responses, maintaining tissue homeostasis by synergistically regulating the production of other cytokines and cell survival and death. It exists in two biologically active forms: transmembrane TNF-α (tmTNF-α) and secreted TNF-α (sTNF-α). Its expression increases in various pathological states such as sepsis and malignant tumors, and due to its association with the development of diseases such as arthritis, it has become an important drug target. Anti-TNF-α therapy has shown some effectiveness in treating various inflammatory diseases.

[0164] qPCR (real-time quantitative polymerase chain reaction) is an important technique in molecular biology, its core significance being the precise quantitative analysis of specific nucleic acids (DNA or RNA, after reverse transcription into cDNA). In the drug screening stage, qPCR can detect the effect of drugs on target gene expression, assessing the drug's specificity and efficacy.

[0165] Experimental steps:

[0166] Drug administration and sample collection: Discard the culture medium in the BV2 cells after overnight culture. Add 2 mL of fresh culture medium (DMEM + 10% FBS + 1% P / S) to each well of a six-well plate, and then add a certain volume of the compound stock solution (10 mM) to the desired final concentration. Incubate at 37°C. After 1 h, add 2 μL of LPS (1 mg / mL, filtered through a 0.22 μm filter membrane) and incubate at 37°C. After 6 h, discard the liquid in the culture dish, add 1 mL of TRIzol lysis buffer to each well, collect the cell lysis buffer, transfer it to a centrifuge tube, and place it in a -80°C freezer overnight.

[0167] Table 6. Composition of each hole in a six-hole plate

[0168] Hole number of a six-hole plate 1 2 3 4 5 6 processing method Ctrl LPS LPS+5μM LPS+10μM LPS+20μM LPS+40μM Compound (μL) / / 1 2 4 8 DMSO (μL) 2 2 / / / / LPS (μL) / 2 2 2 2 2

[0169] RNA extraction: Add 200 μL of chloroform to the sample, vortex to mix (2500 rpm), and let stand at room temperature for 3 min; centrifuge at 14000 g for 15 min at 4 °C, take 500 μL of the upper aqueous phase (containing RNA), add 700 μL of pre-chilled isopropanol, mix well, and incubate at room temperature for 10 min; centrifuge at 14000 g for 10 min to obtain RNA precipitate, wash with 500 μL of pre-chilled 70% ethanol, and centrifuge under the same conditions for 10 min; discard the ethanol, dry for 5 min, and dissolve in RNase-free water.

[0170] Reverse transcription: 20 μL system: 4 μL 4×gDNAwiper Mix + RNA + RNase-free water (to a final volume of 16 μL), treated at 42℃ for 2 min; add 4 μL 5×qRT Super Mix, incubate at 37℃ for 15 min and at 85℃ for 5 sec to obtain 20 μL cDNA; dilute with 180 μL DEPC water to a final volume of 200 μL.

[0171] qPCR reaction: Prepare the mixture according to the ratio (10 μL qPCR Super Mix / replica + 0.5 μL each of primers 1 and 2 / replica), with 3 replicates per sample; add 33 μL of the mixture + 27 μL of cDNA diluent to each well (60 μL in total, divided into 3 wells, 20 μL per well), and transfer to an eight-tube strip for detection.

[0172] Experimental results show that ( Figure 24 The novel scaffold compounds 3 and 4, and konbu'acidin-derived compounds 7, 8, 9, and 11 provided in this embodiment exhibit anti-inflammatory activity targeting TNF-α, and can reduce the transcriptional level of the TNF-α gene in an LPS-induced BV2 cell inflammation model in a concentration-dependent manner. However, novel scaffold compounds 1 and 2 show weaker transcriptional repression. Although the inhibitory activity of other PIAs is weak, based on previous research, the PIAs 1-13 isolated in this invention may also possess antitumor, immunosuppressive, and anti-angiogenic biological activities, warranting further investigation. This invention provides new compounds for the development of marine drug molecules and offers new scientific basis for the development and utilization of marine medicinal resources.

[0173] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of sponge-derived pyrrolimidazole alkaloid dimer, characterized in that, Its structure can be any one or more of the following: 、 、 、 。 2. A method for preparing the sponge-derived pyrrolimidazole alkaloid dimer according to claim 1, characterized in that, include: The sponge was extracted with ethyl acetate to obtain a crude extract; The crude extract was subjected to gradient elution via a forward silica gel column to obtain the primary fraction containing the target pyrrolimidazole alkaloid dimer. The gradient elution via the forward silica gel column used a petroleum ether / ethyl acetate mixture and a dichloromethane / methanol mixture as eluents. Elution was first performed with the petroleum ether / ethyl acetate mixture, with the ratio of petroleum ether to ethyl acetate decreasing from 5:1 to 0:1, followed by elution with the dichloromethane / methanol mixture, with the ratio of dichloromethane to methanol decreasing from 10:1 to 0:

1. The primary fraction was eluted by medium-pressure liquid chromatography (LC-PC) to obtain a secondary fraction containing the target pyrrolimidazole alkaloid dimer; the elution was performed using a methanol / water mixture as the eluent; during elution, the volume percentage of methanol increased from 5% to 100%. The secondary fraction was subjected to gradient elution using a forward silica gel column to obtain a tertiary fraction containing the target pyrrolimidazole alkaloid dimer. The gradient elution using the forward silica gel column was performed with a petroleum ether / ethyl acetate mixture and a dichloromethane / methanol mixture. Elution was first performed with the petroleum ether / ethyl acetate mixture, with the ratio of petroleum ether to ethyl acetate decreasing from 5:1 to 0:1, followed by elution with the dichloromethane / methanol mixture, with the ratio of dichloromethane to methanol decreasing from 10:1 to 0:

1. The tertiary fraction was eluted by gel column chromatography to obtain a quaternary fraction containing the target pyrrolimidazole alkaloid dimer; the eluent for the gel column chromatography was methanol. The fourth fraction was separated by high performance liquid chromatography (HPLC) to obtain the target pyrrolimidazole alkaloid dimer. The HPLC separation was performed using a mobile phase of a methanol / water mixture or an acetonitrile / water mixture containing 0.09-0.11% trifluoroacetic acid, an Agilent XDB-C18 column or a COSMOSIL πNAP column, a flow rate of 1.9-2.1 mL / min, an injection volume of 9-11 μL, and a column temperature of 24.5-25.5℃.

3. A pharmaceutical preparation, characterized in that, Its active ingredient is the sponge-derived pyrrolimidazole alkaloid dimer or its pharmaceutical salt as described in claim 1.

4. The pharmaceutical formulation according to claim 3, characterized in that, The pharmaceutical preparation also includes pharmaceutically acceptable excipients.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutically acceptable excipients are pharmaceutically acceptable carriers and / or excipients.

6. The pharmaceutical formulation as described in claim 5, characterized in that, The pharmaceutically acceptable carriers include at least one of alumina, aluminum stearate, polyvinylpyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, and lanolin.

7. The pharmaceutical formulation as described in claim 5, characterized in that, The excipient is at least one of the following: binder, filler, lubricant, disintegrant, and wetting agent.

8. The pharmaceutical preparation according to claim 3, characterized in that, The dosage form of the pharmaceutical preparation is an oral preparation, an injectable preparation, or a topical preparation.

9. The pharmaceutical preparation according to claim 3, characterized in that, The drug formulation can be administered orally, by spray inhalation, by intravenous injection, by rectal administration, or by intraperitoneal instillation.

10. The use of a sponge-derived pyrrolimidazole alkaloid dimer according to claim 1 or a pharmaceutical preparation according to any one of claims 3-9 in the preparation of a TNF-α-related inflammation inhibitor.

11. A method for screening sponge-derived pyrrolimidazole alkaloid dimers according to claim 1, characterized in that, include: The sponge was extracted with ethyl acetate to obtain a crude extract; The crude extract was separated to obtain several fractional components; The ultraviolet tracing method was used to screen several components to identify the components containing the target pyrrolimidazole alkaloid dimer, which were then separated by high performance liquid chromatography to obtain the target pyrrolimidazole alkaloid dimer. The ultraviolet tracking method includes: performing full-wavelength high-performance liquid chromatography analysis on each component to obtain UV spectra and retention times, and determining the skeleton type of the target pyrrolimidazole alkaloid dimer and the component containing the target pyrrolimidazole alkaloid dimer by using UV spectra and retention times.

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