A cyclic tetrapeptide compound, and a preparation method and application thereof

Abstract

The application belongs to the technical field of microbial medicine, and specifically discloses a cyclic tetrapeptide compound, a preparation method and application thereof. Five cyclic tetrapeptide compounds are isolated from a fermentation product of a fungus-bacterium symbiotic body of Beibuwan conch for the first time, including four new compounds Nectriatidels A-D and compound 5 (Nectriatidel). The application also provides a preparation method of the compounds. Activity tests show that the compounds 1-4 have weak antibacterial activity, but can significantly enhance the antibacterial activity of amphotericin B on Candida albicans, and in particular, the compounds 4 and 5 can reduce the MIC of amphotericin B by 8 times, and meanwhile, the series of compounds have no toxicity to various human and mouse tumor cells. Therefore, the compounds can be used as safe and efficient antifungal synergists, and have good application prospects in the preparation of medicines for treating Candida albicans infection.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, and specifically relates to a method for preparing a cyclic tetrapeptide compound derived from a marine fungal-bacterial symbiont and its application in the preparation of synergistic anti-Candida albicans drugs. Background Technology

[0002] With the widespread use of organ transplantation, immunosuppressive therapy, radiotherapy, and chemotherapy, as well as the increase in immune-related diseases, the incidence of fungal infections has risen significantly. Immunocompromised patients are particularly susceptible to invasive Candida infections. These infections often originate from the host's endogenous flora and are induced when the host's defense mechanisms are compromised. Pathogens include Candida albicans and non-Candida albicans species. Candida albicans is a common opportunistic pathogen, frequently colonizing the oral cavity, respiratory tract, digestive tract, and vagina. When the host's defense mechanisms are compromised due to decreased immune function, dysbiosis, or chronic inflammation, this bacterium can cause infections ranging from local superficial infections to invasive systemic infections, the latter often accompanied by high morbidity and mortality. Currently, the main antifungal drugs used clinically include azoles (such as fluconazole), polyenes (such as amphotericin B), and echinocandins. However, long-term use leads to increasingly serious drug resistance, and the toxic reactions and side effects of these drugs themselves severely limit their efficacy. Therefore, developing novel treatment strategies for drug-resistant Candida albicans infections has become an urgent need in the field of antifungal medicine.

[0003] Combination therapy is a common clinical approach for managing complex or drug-resistant Candida albicans infections. By synergistically combining drugs with different mechanisms of action, it can enhance efficacy while potentially reducing the dosage of individual drugs and the associated toxicity risks. Currently, combination therapy mainly includes two categories: combinations of different classes of antifungal drugs and combinations of antifungal drugs with potentiators. The former may face problems such as unstable synergistic effects, additive toxicity, and the emergence of new drug resistance, while the latter, by adding potentiators that do not have significant antifungal activity themselves, can synergistically enhance the antifungal effect of existing drugs and may improve their safety profile. Therefore, finding structurally novel and uniquely acting potentiators is one of the key breakthrough directions for optimizing combination therapy.

[0004] Marine microorganisms, due to their unique living environment, can produce a large number of novel bioactive metabolites, making them an important resource for active drug molecules. However, current research on marine-derived fungal-bacterial symbionts (especially those from the Beibu Gulf in my country) is still insufficient, and reports on the extraction of active cyclic tetrapeptides from these symbionts are even rarer.

[0005] Based on this, in order to more effectively develop and utilize my country's marine microbial resources, especially the unique marine biological resources of the Beibu Gulf, this invention aims to find and identify novel cyclic tetrapeptide compounds with synergistic amphotericin B activity against Candida albicans from a specific fungal-bacterial symbiont from the Beibu Gulf, in order to provide new candidate substances for addressing the above-mentioned drug development needs. Summary of the Invention

[0006] The purpose of this invention is to provide a cyclic tetrapeptide compound, its preparation method, and its application in the preparation of synergistic anti-Candida albicans drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a cyclic tetrapeptide compound isolated from rice fermentation products of the fungal-bacterial symbionts GXIMD 04541-GXIMD04532. This symbiont was isolated from the Arabian cowrie (Mauritia arbica) in the Beibu Gulf of my country and was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42405.

[0009] One of the following compounds is a cyclic tetrapeptide compound or a pharmaceutically acceptable salt thereof:

[0010]

[0011] The present invention also provides a method for preparing the above-described cyclic peptide compounds, comprising the following steps:

[0012] Step 1: Inoculate the fungal-bacterial symbionts GXIMD 04541-GXIMD 04532 into PDB liquid medium for fermentation culture to obtain rice culture medium fermentation products;

[0013] Step 2: The rice culture medium fermentation product is extracted multiple times with an organic solvent and concentrated to obtain a crude extract;

[0014] Step 3: The crude extract is sequentially separated and purified by normal-phase silica gel column chromatography, reverse-phase column chromatography and high-performance liquid chromatography to obtain the cyclic tetrapeptide compound described in claim 1.

[0015] To further clarify, the fungal-bacterial symbionts GXIMD 04541-GXIMD 04532 are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42405.

[0016] To further explain, in step 1, the fermentation culture is carried out by static culture in rice culture medium at 26-28°C for 25-35 days.

[0017] To further clarify, in step 2, the organic solvent is ethyl acetate.

[0018] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the said cyclic tetrapeptide compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0019] The present invention also provides the use of the aforementioned cyclic tetrapeptide compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for enhancing the antifungal activity of amphotericin B.

[0020] To further clarify, the fungus in question is Candida albicans.

[0021] To further clarify, the drug is used to treat or prevent infections caused by Candida albicans.

[0022] The present invention also provides a pharmaceutical combination for enhancing antifungal efficacy, comprising amphotericin B and the cyclic tetrapeptide compound of claim 1 or a pharmaceutically acceptable salt thereof.

[0023] The present invention has the following beneficial effects:

[0024] 1. This invention is the first to isolate four novel cyclic tetrapeptide compounds (Nectriatidels AD) from a specific fungal-bacterial symbiont from the Beibu Gulf, which significantly enriches the structural diversity library of cyclic tetrapeptide compounds.

[0025] 2. This invention reveals for the first time the application potential of the cyclic tetrapeptide compounds in enhancing the antifungal activity of AmB against Candida albicans. Activity tests showed that compounds 1-3 all exhibited clear synergistic antifungal activity, with compound 4 (32 μg / mL) reducing the MIC value of AmB from 1.0 μg / mL to 0.125 μg / mL, demonstrating a clear inhibitory effect of synergistic activity, comparable to similar compounds reported in the prior art. Meanwhile, compounds 1-5 showed no cytotoxic activity against colorectal adenocarcinoma cell lines (DLD-1, HT29), human thyroid cancer cells (KTC cells), mouse melanoma cells (B16F10 cells), and human breast cancer cells (MCF7 cells). These findings indicate that the compounds of this invention are potential lead compounds for novel, non-toxic, synergistic antifungal drugs.

[0026] 3. The preparation method provided by this invention is based on large-scale culturable microbial fermentation, with readily available raw materials and a clear and reproducible process route. The entire process, from symbiotic fermentation to compound separation and purification, is detailed and feasible, laying a solid technical foundation for further research and potential industrialization development of this type of compound.

[0027] Biomaterials Information

[0028] Fungal-bacterial symbionts GXIMD 04541-GXIMD 04532 were deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 17, 2025, with accession number CGMCC No. 42405, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0029] Figure 1 The key HMBCs of compounds 1-4 1 H- 1 Schematic diagram of H COSY signal.

[0030] Figure 2 Schematic diagram of the key NOESY signals of compounds 1-4.

[0031] Figure 3 Compound 1 1 1H NMR spectrum (solvent: methanol-d4).

[0032] Figure 4 Compound 1 13 C10 NMR spectrum (solvent: methanol-d4).

[0033] Figure 5 Compound 2 1 1H NMR spectrum (solvent: methanol-d4).

[0034] Figure 6 Compound 2 13 C10 NMR spectrum (solvent: methanol-d4).

[0035] Figure 7 Compound 3 1 1H NMR spectrum (solvent: methanol-d4).

[0036] Figure 8 Compound 3 13 C10 NMR spectrum (solvent: methanol-d4).

[0037] Figure 9 Compound 4 11H NMR spectrum (solvent: methanol-d4).

[0038] Figure 10 Compound 4 13 C10 NMR spectrum (solvent: methanol-d4). Detailed Implementation

[0039] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0040] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0041] Example 1

[0042] This example describes the preparation of cyclic tetrapeptide compounds.

[0043] A method for preparing a cyclic tetrapeptide compound includes the following steps:

[0044] (1) Seed culture preparation and fermentation: fungal-bacterial symbiosis Remove the freshly cut, bacterial-containing agar from the 4°C freezer and inoculate it into sterilized PDB liquid medium (200 g potato, 20 g glucose, 30 g sea salt, and 1 L water). Incubate at 28°C with shaking at 180 rpm for 3 days to prepare the seed culture. Inoculate the bacterial seed culture at a rate of 15 mL per bottle into rice medium (80.0 g rice, 0.4 g yeast extract, 0.4 g glucose, 1.6 g sea salt, and 120 mL pure water). Incubate at room temperature for 30 days to obtain the rice fermentation product.

[0045] (2) The rice culture medium fermentation product was crushed with a wooden stick and extracted with ethyl acetate multiple times until the extract was nearly colorless. The extract was collected and concentrated under reduced pressure to obtain crude extract.

[0046] (3) The crude extract was separated by normal-phase silica gel column chromatography, with gradient elution using chloroform-methanol (volume ratio 100:0, 98:2, 95:5, 85:15, 80:20, 70:30, 0:100). The eluent was collected and concentrated under reduced pressure. After analysis by TLC and HPLC, all fractions were combined to obtain 5 fractions Fr.1-Fr.5. Fr.3 (30.8 g) was separated by medium-pressure reversed-phase column chromatography, with gradient elution using methanol-water (volume ratio 10:90-100:0) to obtain 6 fractions Fr 3.1-Fr 3.6. Fr 3.3 (11.7 g) was separated by medium-pressure reversed-phase column chromatography, with gradient elution using methanol-water (volume ratio 10:90-100:0) as the mobile phase to obtain 7 subfractions (Fr 3.3.1-Fr 3.3.7).

[0047] (4) Fr 3.3.3 was prepared and purified by semi-preparative high-performance liquid chromatography (HPLC). The preparation conditions were as follows: YMC semi-preparative column (10 mm × 250 mm, 5 μm), mobile phase CH3CN / H2O (v / v 30:70), flow rate 2 mL / min, and isocratic elution to obtain compound 1 (3.2 mg, t R = 24.72 min), compound 2 (3.8 mg, t R =25.17 min) and compound 3 (2.9 mg, t R = 25.99 min); Fr 3.3.4 Compound 4 (2.5 mg, t) was prepared by HPLC using CH3OH / H2O (v / v 70:30) as the mobile phase and a flow rate of 2 mL / min. R = 28.16 min) and compound 5 (12.7 mg, t) R = 26.30 min).

[0048] The structural formulas of compounds 1-5 are shown below:

[0049]

[0050] Example 2

[0051] This example illustrates the identification of the structures of compounds 1-5.

[0052] This embodiment involves spectroscopic analysis of the compound obtained in Example 1.

[0053] The chemical structure of the cyclic tetrapeptide compound of this invention was determined by nuclear magnetic resonance (NMR) detection and mass spectrometry analysis. Its physicochemical properties and spectral data are as follows:

[0054] Compound 1: White powder, soluble in methanol. UV (MeOH)λ max 220, 253, 284 nm; HR-ESI-MS shows molecular ion peaks. 505.2072 [M+Na] + It is speculated that its molecular formula is C 25 H 30 N4O6, with an unsaturation degree of 13. 1 H NMR spectrum (as attached) Figure 3 As shown, seven aromatic hydrogens (δ) are given in the low-field region. H 8.34, 7.56, 7.48, 7.16, 6.70, 6.69, 6.54), 4 methines (δ H 4.49, 4.13, 4.11, 2.03), 1 methylene (δ H 3.28, 3.08), 4 methyl groups (δ) H 2.95, 1.46, 0.94, 0.89). 13 C NMR (with appendix) Figure 4 Four amide carbons (δ) were observed. C 172.1, 170.2, 173.0, 176.1), 12 aromatic hydrocarbon carbons (δ C (125.4, 127.7, 124.4, 132.6, 122.1, 138.1, 131.0, 121.7, 116.4, 145.2, 146.6, 117.5), one methylene carbon signal δ C 34.0, 4 methylene carbon signals (δ) C 70.3, 56.2, 31.2, 54.9), and 4 methyl carbon signals (δ C (40.7, 20.0, 18.7, 16.1). The observed NMR data are very similar to those of nectriatidel, suggesting that compound 1 is a cyclic peptide.

[0055] Detailed analysis of COSY and HMBC data revealed the presence of one anthranilic acid (ABA), one N-methyl-3-hydroxytyrosine (N-Me-3-OH-Tyr), one valine (Val), and one alanine (Ala) in compound 1. Furthermore, HMBC spectroscopy showed a correlation between −NCH3 and C-18, and between H-19 and C-23, indicating the amino acid sequence Aad-N-Me-3-OH-Tyr-Val-Ala, thus confirming the planar structure of compound 1. The absolute configuration of compound 1 was then determined using the Marfey reaction and NOESY spectroscopy, and it was named Nectriatidel A.

[0056] Compound 2: White powder, soluble in methanol. UV (MeOH)λ max 220, 254, 284 nm; HR-ESI-MS at m / z 491.1907 [M+Na] + Based on the molecular ion peak shown and combined with the proton and carbon spectra, its molecular formula is deduced to be C13. 24 H 28 N4O6 has an unsaturation degree of 13. The NMR data of compound 2 are very similar to those of nectriatidel, the difference being that 2 lacks one methine and one methyl group, and has one additional oxygen-containing methine (δ-methyl group). H 3.94, m; δc 68.4). In the ¹H–¹H COSY spectrum, H-20 (δ H 3.94) and H-19 (δ) H 4.16) / H-21 (δ H The presence of threonine was confirmed by the correlation between H-20 and C-18 (δc 172.7) and HMBC correlation between 1.16). Furthermore, NCH3 (δ H The presence of HMBC correlations between C-18, H-19, and C-22 (δc 176.5) at 2.95 indicates that the amino acid sequence is Aad-N-Me-Tyr-Thr-Ala, thus determining the planar structure of compound 2. Finally, the absolute configuration of compound 2 was determined by the Marfey reaction and NOESY spectroscopy.

[0057] Compound 3: White powder, soluble in methanol. UV (MeOH)λ max 220, 255, 284 nm; HR-ESI-MS shows molecular ion peaks. 461.1801 [M+Na] + It is speculated that its molecular formula is C 23 H 26N4O5, with an unsaturation degree of 13. The NMR data of compound 3 are very similar to those of compound 2, the difference being that compound 3 lacks an oxygen-containing methine (δ-methyl group). H 3.94, m; δc 68.4). 1 H− 1 H-19 (δ) was observed in the HCOSY spectrum. H 5.02) and H-20 (δ) H 1.23) related, combined with H-20 and C-18 (δ C 173.6) / C-19 (δ C The HMBC correlation of 45.2) indicates that the amino acid fragment of 3 is alanine rather than threonine. Detailed analysis of its 2D NMR data shows that the linkage sequence of the amino acid fragment of 3 is Aad-N-Me-Tyr-Ala-Ala. Subsequently, the absolute configuration of compound 3 was determined by Marfey reaction and NOESY spectroscopy.

[0058] Compound 4: White powder, soluble in methanol. UV (MeOH)λ max 220, 253, 284 nm; HR-ESI-MS shows molecular ion peaks. 503.2272 [M+Na] + The molecular formula is presumed to be C. 26 H 32 N4O5, with an unsaturation degree of 13. Comparison of C1H NMR data revealed that compound 4 is very similar to compound 2. The difference lies in the fact that the third amino acid fragment of compound 4 is leucine, not threonine. Comprehensive analysis of its two-dimensional NMR data confirmed the planar structure of compound 4. The absolute configuration of compound 4 was determined using the Marfey reaction and NOESY spectroscopy.

[0059] Compound 5: White powder, soluble in methanol. UV (MeOH)λ max 220, 254, 285 nm; 1 H NMR and 13 CNMR data are consistent with reported nectriatidel values. HR-ESI-MS yields quasi-molecular ion peaks. 489.2108 [M+Na] + It is speculated that the molecular formula of compound 5 is C. 25 H 30 N4O5. 1 ¹H NMR (500 MHz, MeOH) δ H8.33 (1H, dd, J = 8.3, 1.0 Hz, H-6), 7.56 (1H, dd, J = 7.7, 1.6 Hz, H-3), 7.48 (1H, ddd, J =8.8, 7.5, 1.6 Hz, H-5), 7.16 (1H, dd, J = 7.6, 1.1 Hz, H-4), 7.05 (2H, d, J =8.4 Hz, H-12 / 16), 6.73 (2H, d, J = 8.5 Hz, H-13 / 15), 4.47 (1H, d, J = 10.3Hz, H-19), 4.14 (1H, d, J = 3.4 Hz, H-9), 4.12 (1H, d, J = 4.5 Hz, H-24), 3.33 (1H, m, H-10a), 3.15 (1H, dd, J = 14.2, 11.1 Hz, H-10b), 2.91 (3H, s, H-17), 2.03 (1H, m, H-20), 1.45 (3H, d, J = 7.3 Hz, H-25), 0.92 (3H, d, J = 6.6Hz, H-21), 0.89 (3H, d, J = 6.8 Hz, H-22). 13 C NMR (125 MHz, MeOH) δ C 172.0 (C,C-1), 125.3 (C, C-2), 127.8 (CH, C-3), 124.3 (CH, C-4), 132.6 (CH, C-5), 122.1 (CH, C-6), 138.0 (C, C-7), 170.1 (C, C-8), 70.3 (CH, C-9), 33.6 (CH2,C-10), 130.2 (C, C-11), 131.4 (CH, C-12 / 16), 116.4 (CH, C-13 / 15), 157.3 (C,C-14), 40.6 (CH3, C-17), 173.0 (C, C-18), 56.2 (CH, C-19), 31.1 (CH, C-20), 20.0 (CH3, C-21), 18.7 (CH3, C-22), 172.4 (C, C-23), 54.9 (CH, C-24), 15.5 (CH3, C-25). NMR data were obtained from nectriatidel. [2019, 82, 2673-2681] Comparison was performed, and the structure of compound 5 was identified as compound nectriatidel.

[0060] Table 1. Compounds 1-2 1 H NMR (500 MHz) and 13 C10 NMR (125MHz) data (Methanol-d4, δ ppm)

[0061]

[0062] Table 2. Compounds 3-4 1 H NMR (500 MHz) and 13 C10 NMR (125MHz) data (Methanol-d4, δ ppm)

[0063]

[0064] Example 3

[0065] This example demonstrates the activity test of compound 1-5, which enhances the activity of amphotericin B against Candida albicans.

[0066] The activity of amphotericin B (AmB) combined with compounds 1-5 against Candida albicans was tested using the microdilution method. The specific experimental method is as follows:

[0067] Following the guidelines in the CLSI document, the broth microdilution method was performed using a 96-well plate (Corning). First, *Candida albicans* was inoculated into Sabouraud dextrose agar and cultured on a shaker. The bacterial suspension was diluted with sterile 0.85% physiological saline and adjusted to 0.5 McFarland standard by spectrophotometry. OD0.05 530 The value was approximately 0.12–0.13. The seed culture was diluted 1000-fold with RPMI 1640 medium (ThermoFisher Scientific, Waltham, MA, USA). In the first-instance single-compound / drug activity assay against Candida albicans, the diluted Candida albicans seed culture (100 μL), RPMI 1640 medium (99 μL), and the test compound (dissolved in 1 μL DMSO, final concentration 0.125–50 μg / mL) were added to each well of a 96-well plate and incubated at 37°C for 24 h. OD was measured using a microplate reader. 550The minimum inhibitory concentration (MIC) was used to evaluate antifungal activity. The MIC is the lowest concentration required to inhibit fungal growth by 90% relative to the control group (no drug). In the second antifungal synergistic activity assay, diluted seed culture (100 μL), RPMI 1640 medium (98 μL), AmB (dissolved in 1 μL DMSO, final concentration 0.0313 to 1 μg / mL), and test compounds (dissolved in 1 μL DMSO, final concentrations of 0, 2, 4, 8, 16, or 32 μg / mL) were used. The 96-well plates were incubated at 30°C for 24 h, with three replicates for each different concentration. Turbidity was observed in the 96-well plates. The combined minimum inhibitory concentration (MIC) of AmB and the compound at each specified concentration (0–32 μg / mL) was recorded.

[0068] Experimental results showed that in the antifungal activity against Candida albicans by testing amphotericin B and the compounds alone, amphotericin B exhibited antifungal activity with a minimum inhibitory concentration (MIC) of 1.0 μg / mL, while compounds 1-5 showed no activity against Candida albicans at a concentration of 50 μg / mL. Compounds 4 and 5 (32 μg / mL) reduced the MIC of AmB from 1.0 μg / mL to 0.125 μg / mL, showing an 8-fold synergistic effect, which was essentially comparable to the synergistic antifungal activity of compound nectriatidel reported in the literature [The Journal of Antibiotics 2024, 77: 214–220]. Compounds 1, 2, and 3 (32 μg / mL) reduced the MIC of AmB from 1.0 μg / mL to 0.25, 0.50, and 0.25 μg / mL, respectively, demonstrating a certain synergistic effect. Replacing N-methyl-3-hydroxytyrosine (compound 1) with N-methyltyrosine (compound 5) induces a 4-fold increase in AmB activity, indicating that the presence of an additional hydroxyl group in tyrosine reduces its synergistic activity. When valine (Val) in compound 5 is replaced with threonine (Thr) in compound 2, alanine (Ala) in compound 3, and leucine (Leu) in compound 4, the synergistic activity of AmB against Candida albicans varies, with compound 4 exhibiting the strongest synergistic activity. In summary, although the synergistic activities of compounds 1-3 are weaker than those of compounds 4 and 5, they still retain some degree of synergistic activity.

[0069] Table 3. MIC values ​​of AmB combination compounds against Candida albicans

[0070]

[0071] Example 4

[0072] This embodiment is a test of the cytotoxic activity of compounds 1-5.

[0073] Assay Methods: Human colorectal adenocarcinoma cells HT29 and DLD1, human thyroid cancer cells KTC, mouse melanoma cells B16F10, and human breast cancer cells MCF7 were selected as test subjects. The cytotoxic activity of the compounds was determined by the MTT assay. The test compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium to prepare a series of concentrations. Cells were collected using trypsin and seeded in 96-well microplates (5 × 10³ cells per well) and cultured at 37°C in a 5% CO2 incubator until cell adhesion. After pre-incubation, cells were treated with a series of diluted compounds for 48 h, with cisplatin and 0.01% DMSO serving as positive and negative controls, respectively. After treatment, MTT solution was added to each well for further incubation. During this period, succinate dehydrogenase in the mitochondria of live cells reduced the yellow MTT to purple formazan crystals. Subsequently, the supernatant was discarded and DMSO was added to dissolve the formazan crystals. Finally, the OD value of each well at 490 nm was measured using an ELISA reader. Cell viability was calculated by comparing the OD values ​​of the experimental group and the control group, thereby assessing the cytotoxicity of the compound.

[0074] Experimental results showed that compounds 1-5 did not exhibit cytotoxic activity against any cell line at a concentration of 40 μM, indicating that these cyclic tetrapeptide compounds can serve as a safe and non-toxic synergist against Candida albicans.

[0075] This invention isolated four novel cyclic tetrapeptide compounds and one known compound from rice fermentation products of the fungal-bacterial symbiont GXIMD 04541-GXIMD 04532 (accession number CGMCC No. 42405) derived from the snail *Cypripedium spp.* in the Beibu Gulf. These compounds exhibit novel structures, and experiments have confirmed their ability to effectively enhance the antifungal activity of amphotericin B against *Candida albicans*, while demonstrating no significant cytotoxic activity of their own. Therefore, the cyclic tetrapeptide compounds provided by this invention can serve as safe and highly effective antifungal synergists, showing significant potential for application in the preparation of pharmaceutical compositions for treating *Candida albicans* infections (especially drug-resistant infections).

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A cyclic tetrapeptide compound or a pharmaceutically acceptable salt thereof, characterized in that, It is one of the following compounds:

2. A method for preparing the cyclic peptide compound of claim 1, characterized in that, Includes the following steps: Step 1: Inoculate the fungal-bacterial symbionts GXIMD 04541-GXIMD 04532 into PDB liquid medium for fermentation culture to obtain rice culture medium fermentation products; Step 2: The rice culture medium fermentation product is extracted multiple times with an organic solvent and concentrated to obtain a crude extract; Step 3: The crude extract is sequentially separated and purified by normal-phase silica gel column chromatography, reverse-phase column chromatography and high-performance liquid chromatography to obtain the cyclic tetrapeptide compound described in claim 1.

3. The method according to claim 2, characterized in that, The fungal-bacterial symbionts GXIMD 04541-GXIMD04532 are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42405.

4. The method according to claim 2, characterized in that, In step 1, the fermentation culture is carried out by static culture in rice culture medium at 26-28°C for 25-35 days.

5. The preparation method according to claim 2, characterized in that, In step 2, the organic solvent is ethyl acetate.

6. A pharmaceutical composition, characterized in that, The active ingredient comprises a therapeutically effective amount of the cyclic tetrapeptide compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use of the cyclic tetrapeptide compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the antifungal activity of amphotericin B.

8. The application according to claim 7, characterized in that, The fungus in question is Candida albicans.

9. The application according to claim 7 or 8, characterized in that, The drug is used to treat or prevent infections caused by Candida albicans.

10. A pharmaceutical combination for synergistic antifungal effects, characterized in that, It comprises amphotericin B and the cyclic tetrapeptide compound of claim 1 or a pharmaceutically acceptable salt thereof.

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