Use of a class of pyrrolidinol acids in the preparation of drugs for inhibiting pathogenicity of candida albicans

Abstract

The application discloses application of a kind of pyrrolidinol acid compounds in preparation of drug for inhibiting pathogenicity of Candida albicans. The pyrrolidinol acid compound is shown as formula (I). The pyrrolidinol acid compounds-compounds 1-7 of the application are new compounds, have significant inhibiting effect on pathogenicity of tested pathogenic Candida albicans, can be used for preparing drug for inhibiting pathogenicity of Candida albicans or as precursor of drug for inhibiting pathogenicity of Candida albicans, for treating infection caused by Candida albicans, thus the application provides new alternative compounds for developing drug for inhibiting pathogenicity of Candida albicans, and has important significance for developing marine drug resources in China.

Description

Technical fields:

[0001] This invention belongs to the field of natural products, specifically relating to seven pyrrolidone acid compounds and their application in the preparation of drugs that inhibit the pathogenicity of Candida albicans. Background technology:

[0002] Candida albicans is a common fungal pathogen that exhibits two distinct growth states in different environments: yeast and hyphal forms. The pathogenicity of this organism within the host is closely related to its ability to transform into the hyphal form and form biofilms within host tissues. The hyphal form plays a crucial role in the invasion and deep infection caused by Candida albicans. Compared to yeast, hyphae are more invasive, making the control of infection within the host more challenging. Furthermore, the pathogenicity of Candida albicans is also manifested in its ability to form biofilms. By constructing complex biofilm structures through polymers in and around its cells, Candida albicans can form a robust three-dimensional network that both protects itself and increases its resistance to antimicrobial agents. Therefore, biofilm formation is considered a key factor promoting the pathogenicity of Candida albicans. Treatment with low concentrations of antibiotics is insufficient to kill bacteria deep within the biofilm and fundamentally inhibit its pathogenicity, while the use of high concentrations of antibiotics, in addition to their side effects and toxicity, may lead to antibiotic resistance in the long term. Therefore, the discovery of novel bioactive compounds that can inhibit the formation of Candida albicans hyphae and biofilms without producing bactericidal effects is crucial for addressing the threat posed by the pathogenicity of Candida albicans to humans.

[0003] Due to its unique ecological environment, the ocean is generally considered to possess special biosynthetic gene clusters, making it an important source of lead compounds with novel structures and functions. Targeting the pathogenicity of Candida albicans, the discovery of lead drugs from marine microorganisms that inhibit hyphal and biofilm formation without killing the fungus represents an effective treatment strategy for various diseases caused by Candida albicans. Summary of the Invention:

[0004] The first objective of this invention is to provide seven pyrrolidone acid compounds with inhibitory activity against Candida albicans and their pharmaceutical salts.

[0005] The pyrrolidone acid compound or its pharmaceutical salt of the present invention has the structure shown in formula (I):

[0006]

[0007] The inventors conducted co-culture fermentation of marine actinomycetes from different sources in a specific culture medium. Subsequently, through scaled-up fermentation, extraction, and purification, seven compounds 1-7 containing pyrrole units were obtained. Using (+)-HR-ESIMS, 1D, and 2DNMR techniques, the seven monomers were identified as compounds containing different types of alkyl, hydroxyethyl, and carboxyl groups. Their specific structures are shown in formula (I).

[0008] Evaluation of the inhibitory activity of compounds 1-7 against Candida albicans revealed that they exhibited good inhibitory activity against the formation of Candida albicans biofilm and the growth of hyphae, as represented by C. albicans SC5314. Thus, they can inhibit the pathogenicity of Candida albicans without killing its precursors, demonstrating their potential as lead compounds for developing anti-Candida albicans pathogenic drugs.

[0009] Therefore, a second objective of the present invention is to provide the use of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, or compound 7 in the preparation of antibacterial drugs.

[0010] Preferably, the antibacterial drug is an anti-Candida albicans pathogenic drug.

[0011] A third object of the present invention is to provide an anti-Candida albicans pathogenic drug, characterized in that it comprises an effective amount of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, or compound 7 as an active ingredient, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a method for preparing a pyrrolidone acid compound, which is prepared and isolated from a mixed fermentation broth of Streptomyceskoyangensis SCSIO 5802 and Nocardiopsis sp. SCSIO SX5N9, wherein Nocardiopsis sp. SCSIO SX5N9 has accession number GDMCC NO: 64065.

[0013] The compounds 1-7 of this invention, containing different types of alkyl, hydroxyethyl, and carboxyl groups, are novel compounds that exhibit significant inhibitory effects on tested Candida albicans. They can be used to prepare drugs that inhibit the pathogenicity of Candida albicans for the treatment of various diseases caused by Candida albicans infection. Therefore, this invention provides candidate compounds for the development of new drugs that inhibit the pathogenicity of Candida albicans and is of great significance for the development of marine drug resources in China.

[0014] Streptomyces koyangensis SCSIO 5802 (hereinafter referred to as strain SCSIO 5802), a deep-sea sediment actinomycete (published in Tetrahedron. 2017, 73, 5366-5372, which the applicant also holds and guarantees to make available to the public within 20 years from the application date) and Nocardiopsis sp. SCSIO SX5N9 (hereinafter referred to as strain SCSIO SX5N9), a coral symbiotic actinomycete, are deposited at the South China Sea Institute of Oceanology, Chinese Academy of Sciences (SCSIO), with accession numbers SXA2012001 and SXA20180N9, respectively. Address: South China Sea Institute of Oceanology, No. 164, Xingang West Road, Haizhu District, Guangzhou, Guangdong Province. Meanwhile, Nocardiopsissp.SCSIO SX5N9 was deposited on November 24, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China, with accession number GDMCC NO: 64065. Attached image description:

[0015] Figure 1 It is compound 1 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0016] Figure 2 It is compound 1 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol;

[0017] Figure 3 It is compound 2. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0018] Figure 4 It is compound 2. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0019] Figure 5 It is compound 3. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0020] Figure 6 It is compound 3. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0021] Figure 7 It is compound 4. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0022] Figure 8 It is compound 4. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0023] Figure 9 It is compound 5. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0024] Figure 10 It is compound 5. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0025] Figure 11 It is compound 6. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0026] Figure 12 It is compound 6. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0027] Figure 13 It is compound 7. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol;

[0028] Figure 14 It is compound 7. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol;

[0029] Figure 15 These are HMBC-related and HH COSY-related graphs;

[0030] Figure 16 This is a graph showing the hyphal inhibition rate of the compound;

[0031] Figure 17 This is a graph showing the mycelial formation rate of compounds 1 and 3 at different concentrations;

[0032] Figure 18 This is a diagram showing the effects of compounds 1 and 3 on Candida albicans hyphal formation;

[0033] Figure 19 This is a graph showing the biofilm formation rate of the compounds;

[0034] Figure 20 This is a graph showing the effect of pyrrolidone glycol compounds on the growth of Candida albicans strain SC5314;

[0035] Figure 21 This is a graph showing the effect of pyrrolidine glycol compounds on the virulence of Candida albicans strain SC5314. Detailed implementation method:

[0036] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0037] Example 1:

[0038] Preparation and structural identification of compounds 1-7 as shown in formula (I)

[0039] I. Preparation of compounds 1-7 as shown in formula (I)

[0040] 1. Seed culture:

[0041] (1) Take out the bacterial strains SCSIO 5802 and SCSIO SX5N9 from the -80℃ freezer, thaw them and inoculate them into MISP2 plates (glucose 4g / L, yeast extract 4g / L, malt extract 10g / L, sea salt 30g / L, pH 7.2-7.4, agar powder 15g / L), and place them in a 28℃ incubator for 5-7 days to activate the bacterial strains; transfer the activated bacterial strains into a 250mL shake flask containing 50mL LAM2ab medium (soluble starch 0.5%, soybean flour 0.5%, glucose 2%, yeast extract 0.2%, bacteriological peptone 0.2%, KH2PO4 0.05%, MgSO4·7H2O 0.05%, crude sea salt 3%, CaCO3 0.2%, NaCl 0.4%, pH 7.2-7.4), and incubate at 28℃ and 200rpm for 48h to obtain the seed culture. The seed culture was inoculated at 5% of the fermentation volume, with a total volume of 2.5 ml (5802 / SX5N9 at a ratio of 2 / 0.5) into MAM2ab medium. Strains SCSIO 5802 and SCSIO SX5N9 were cultured separately as controls. After inoculation, the culture was incubated at 28°C and 200 rpm for 7 days before harvesting and analysis.

[0042] 2. Scale-up fermentation of the strain, product extraction and separation:

[0043] (1) Scale-up fermentation of strains:

[0044] Following the above seed culture protocol, prepare MAM2ab medium and scale up the fermentation to 14L.

[0045] (2) Extraction of fermentation products:

[0046] The fermentation product (co-cultured 5802 / SX5N9) was centrifuged at 3400 rpm for 10 min to separate the bacterial culture and cells. The bacterial culture was extracted four times with an equal volume of butanone, and the bacterial cells were extracted three times with twice the volume of acetone. The extracts were then concentrated to obtain crude extracts of bacterial cells (M) and bacterial culture (L). The target product was mainly distributed in the bacterial culture (L).

[0047] (3) Separation of fermentation products:

[0048] The crude extract of bacterial culture (L) was sequentially processed in a CHCl3 / MeOH system (100 / 0, 98 / 2, 96 / 4, 94 / 6, 92 / 8, 9 / 1, 8 / 2, 1 / 1, V / V, 150 ml) to obtain 8 fractions, Frs.LA1-Frs.LA8. Frs.LA3-Frs.LA5 were combined and passed through a reverse-phase Zhongsu B column (acetonitrile / water 30 / 700~100 / 0, 20 mL / min, 60 min), collecting one fraction every 6 min, to obtain 10 fractions, Frs.LB1-Frs.LB10. Frs.LB9 water / acetonitrile was subjected to SP-HPLC (semi-preparative high performance liquid chromatography) (85 / 15~30 / 70, 0~19 min; 0 / 100, 19.1-23.0 min; 85 / 15 23.1-26.0 min; 2.5 mL·min -1 (270nm), approximately 4.1 mg (t) of target compound 1 was obtained. R =22.8min) and 2 about 5.0mg (t) R =22.4 min). Frs.LB8 was prepared by SP-HPLC with water / acetonitrile (85 / 15-30 / 70, 0-15 min; 0 / 100, 15.1-23.0 min; 85 / 15, 23.1-26.0 min; 2.5 mL·min). -1 (270nm), approximately 8.8 mg (t) of the target compound 3 was obtained. R =19.0min), 4 about 1.0mg (t) R =18.4min) and 7 about 2.0mg (t) R =17.2 min). Frs.LB7 was sequentially passed through a normal C-column in a CHCl3 / MeOH system (100 / 0, 98 / 2, 97 / 3, 96 / 4, 95 / 5, 93 / 7, 8 / 2, 1 / 1, V / V, 100 mL) to obtain eight fractions, Frs.LC1-Frs.LC8. Among them, Frs.LC5 was prepared by SP-HPLC semi-precipitation with water / acetonitrile (95 / 5~55 / 45, 0~19 min; 0 / 100, 19.1-23.0 min; 95 / 5 23.1-26.0 min; 2.5 mL·min). -1 (270nm), approximately 1.0 mg (t) of the target compound 5 was obtained. R =19.8min) and 6 about 1.0mg (t) R =19.2min).

[0049] II. Physicochemical data of compounds 1-7

[0050] II. Structural Identification of Compounds 1-7

[0051] Figure 1 It is compound 1 1 ¹H NMR (700MHz) spectrum, solvent: deuterated methylform; Figure 2 It is compound 1 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol; Figure 3 It is compound 2. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 4 It is compound 2. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol. Figure 5 It is compound 3. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 6 It is compound 3. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol. Figure 7 It is compound 4. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 8 It is compound 4. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol. Figure 9 It is compound 5. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 10 It is compound 5. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol. Figure 11 It is compound 6. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 12 It is compound 6. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol. Figure 13 It is compound 7. 1 1H NMR (700MHz) spectrum, solvent: deuterated methanol; Figure 14 It is compound 7. 13 C10 NMR (175MHz) spectrum, solvent: deuterated methanol.

[0052] Structural analysis was performed on compounds 1-7. Based on high-resolution mass spectrometry and one-dimensional and two-dimensional NMR data, their structures were identified as follows:

[0053] Structural identification diagram as shown Figure 15As shown: a single-direction arrow represents HMBC correlation, and a double-direction arrow represents HH COSY correlation.

[0054] Based on the above identification and analysis, the structures of compounds 1-7 are as shown in formula (Ⅰ).

[0055]

[0056] Example 2:

[0057] Experiments on the inhibition of Candida albicans pathogenicity of compounds 1-7 containing different types of alkyl, hydroxyethyl and carboxyl groups in Example 1.

[0058] Activity detection solution

[0059] (1) Activation of Candida albicans strain SC5314:

[0060] The standard strain of Candida albicans SC5314 was streaked onto LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar) for activation and incubated overnight at 30°C.

[0061] (2) Effects of pyrrolidone glycol compounds on the hyphae of Candida albicans strain SC5314:

[0062] Single colonies of strain SC5314 were picked from LB agar plates and inoculated into GMM medium (6.7 g / LYNB, 0.2% glucose). The culture was incubated overnight at 30°C with shaking at 200 rpm. The OD value of the bacterial culture was then measured. 600 Dilute the bacterial culture to OD using GMM medium. 600 =0.1. Take 500 μL of bacterial culture into a 1.5 mL EP tube, add the compound to a final concentration of 100 μM, vortex to mix, and incubate in a 37°C water bath (DMSO is set at 30°C and 37°C as positive and negative controls, respectively). After 6 h, centrifuge at 5000 rpm for 10 min, discard the supernatant, add 70 μL of GMM culture medium to resuspend the bacterial cells, observe the formation of hyphae under a microscope, and take pictures of different fields of view.

[0063] (3) Effects of pyrrolidone compounds on the biofilm of Candida albicans strain SC5314

[0064] Single colonies of strain SC5314 were picked from LB agar plates and inoculated into GMM medium (6.7 g / LYNB, 0.2% glucose). The culture was incubated overnight at 30°C with shaking at 200 rpm. The OD value of the bacterial culture was then measured. 600 Dilute the bacterial culture to OD using GMM medium. 600=0.1. Take 200 μL of bacterial culture into a 96-well plate, add a compound to a final concentration of 100 μM, and incubate at 30℃ for 16 h. Set DMSO as a negative control. Discard the supernatant and wash the culture plate with sterile PBS buffer. Add 200 μL of 0.5% crystal violet staining solution to each well and stain for 45 min. Discard the staining solution, wash with water, and after the culture plate is completely dry, add 200 μL of 95% ethanol solution to each well to completely dissolve the crystal violet bound to the biofilm. Measure the absorbance (A) at 600 nm using a microplate reader. With the control group containing DMSO as 100%, calculate the relative values ​​of the added compounds. Repeat the experiment three times and record the results.

[0065] (4) Effects of pyrrolidone glycol compounds on the growth of Candida albicans strain SC5314:

[0066] A single colony of Candida albicans strain SC5314 was picked and incubated overnight at 30°C in GMM liquid medium. Its OD value was then measured. 600 Dilute to 0.05 and add the compound to achieve a final concentration of 100 μM. Add 200 μL / well to a 96-well plate and incubate at 30°C with moderate shaking in a full-band microplate reader. The instrument automatically measures the OD of each well every hour. 600 The value was continuously monitored for 12 hours.

[0067] (5) Effects of pyrrolidone compounds on Candida albicans SC5314 invasion of A549 cells:

[0068] After resuscitation and adherence, A549 cells were digested with trypsin and then cultured in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum at a concentration of 1×10⁻⁶. 4 Cells were cultured overnight in 96-well plates at a concentration of 100 cells / well. When the cells reached 80% confluence, the culture medium was discarded, and the cells were washed three times with PBS (0.01M, pH 7.4). Single colonies of *Candida albicans* strain SC5314 were picked and cultured overnight in GMM medium containing 0.2% glucose at 30°C using a shaker. The cells were collected by centrifugation, washed three times with PBS, and then... 8 CFU / mL of the compound was dispersed in DMEM cell maintenance medium containing 1% (v / v) FBS, with a final concentration of 100 μM added to each well. 200 μL of each well was added to a 96-well cell culture plate, with three replicates, and incubated for 8 hours. DMSO and wells without the compound were included as controls. The content of lactate dehydrogenase (LDH) released from human lung cancer A549 cells was used for detection.

[0069] Experimental results

[0070] (1) Pyrrolidone compounds inhibit Candida albicans hyphae formation.

[0071] The morphological transformation from yeast to hyphae in *Candida albicans* is crucial for its pathogenicity. We investigated the effects of these compounds on this morphological transformation in vitro. *Candida albicans* was inoculated into GMM liquid medium and cultured at 30°C with shaking to maintain its yeast morphology. The culture was then diluted and cultured at 37°C to promote hyphal formation. With or without the addition of compounds, after 6 hours of incubation, the vast majority of cells in the negative control group (with DMSO) formed hyphae, while hyphal formation was reduced in all compounds-added groups, with a hyphal inhibition rate exceeding 40% at a final concentration of 100 μM. Figure 16 Compounds 1 and 3 showed significant inhibitory effects on Candida albicans hyphae, and their inhibition of hyphal formation was concentration-dependent. Figure 17 Microscopic observation of hyphal formation revealed that compounds 1 and 3 at a concentration of 100 μM significantly affected Candida albicans hyphal formation, resulting in almost no hyphal production. Figure 18 ).

[0072] (2) Pyrrolidone compounds inhibit the formation of Candida albicans biofilm.

[0073] Candida albicans biofilm is also one of the key factors promoting its pathogenicity. By examining the effects of compounds 1-7 on Candida albicans biofilm formation, it was found that all compounds inhibited biofilm formation to varying degrees, such as... Figure 19 Compound 1 showed an inhibition rate of over 70% against Candida albicans biofilm formation, while compound 3 showed an inhibition rate of approximately 60%.

[0074] (3) Pyrrolidone compounds had almost no effect on the growth of Candida albicans strain SC5314.

[0075] The results are as follows Figure 20 As shown, with DMSO as a control, 100 μM of compounds 1 and 3 had no effect on the growth of Candida albicans strain SC5314. This result indicates that compounds 1 and 3 do not kill the bacteria in Candida albicans strain SC5314, and therefore are unlikely to induce drug resistance.

[0076] (4) Pyrrolidone compounds have a certain inhibitory effect on the virulence of Candida albicans strain SC5314.

[0077] Cytotoxicity assays showed that, with DMSO as a control, compounds 1-7 were not toxic to cells under sterile conditions. Figure 21 As attached Figure 21As shown in Figure a, DMSO was used as a control when Candida albicans SC5314 was added; Figure b shows that compounds 1 and compounds 3-6 all had a certain protective effect against the infection of cells by strain SC5314, and compound 3 reduced the virulence of Candida albicans to below 12%.

[0078] In summary, this invention provides a new lead compound for the development of novel drugs to inhibit the pathogenicity of Candida albicans strains. Furthermore, it is of great significance for the further development of China's marine drug resources using co-culture methods.

Claims

1. Pyrrolidine alcohol compounds with structures as shown in formula (Ⅰ) and their pharmaceutical salts: Equation (Ⅰ).

2. The use of the compound of claim 1 in the preparation of an antibacterial drug, wherein the drug is an inhibitor of Candida albicans.

3. An antibacterial drug, characterized in that, It includes an effective amount of the compound of claim 1 as an active ingredient, or a pharmaceutical salt thereof, and a pharmaceutically acceptable carrier.

4. The drug according to claim 3, characterized in that, The drug mentioned is an inhibitor of Candida albicans.

5. A method for preparing the pyrrolidone acid compound according to claim 1, characterized in that, From Streptomyces koyangensis SCSIO 5802 and Nocardiopsis The isolate was prepared and isolated from the mixed fermentation broth of sp. SCSIO SX5N9. Nocardiopsis sp. SCSIO SX5N9, accession number GDMCC NO: 64065.

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