Application of pyrrolidine alcohol acid compounds in preparation of drugs for inhibiting pathogenicity of candida albicans

Abstract

The invention discloses an application of pyrrolidine alcohol acid compounds in preparation of drugs for inhibiting pathogenicity of candida albicans. The structure of the pyrrolidine alcohol acid compound is shown as a formula (I). The pyrrolidine alcohol acid compounds 1-7 are novel compounds, have a remarkable inhibiting effect on the pathogenicity of tested pathogenic candida albicans, can be used for preparing drugs for inhibiting the pathogenicity of the candida albicans or used as precursors of the drugs for inhibiting the pathogenicity of the candida albicans, and are used for treating infection caused by the candida albicans. Therefore, the invention provides a new alternative compound for developing drugs for inhibiting pathogenicity of Candida albicans, and has important significance for developing marine drug resources in China. # imgabs0 #

Description

Technical Field:

[0001] The present invention belongs to the field of natural products, and specifically relates to seven pyrrolidine carboxylic acid compounds and their applications in the preparation of drugs for inhibiting the pathogenicity of Candida albicans. Background Art:

[0002] Candida albicans is a common fungal pathogen that exhibits two different growth states in different environments: yeast and hyphal forms. The pathogenicity of this organism in 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 key role in invasive and deep infections caused by Candida albicans. Compared with yeast cells, hyphae are more invasive, making it more challenging to control infections within the host. In addition, the pathogenicity of Candida albicans also lies in its ability to form biofilms. By constructing a complex biofilm structure with polymers around its cells, Candida albicans can form a strong three-dimensional network that can both protect itself and increase its resistance to antibacterial agents. Therefore, biofilm formation is considered a key factor promoting the pathogenicity of Candida albicans. If treatment with low concentrations of antibiotics is insufficient to kill the bacteria deep within the biofilm and cannot fundamentally inhibit its pathogenicity, while using high concentrations of antibiotics may lead to antibiotic resistance in the long term in addition to their side effects and toxicity. Therefore, discovering novel bioactive compounds that can inhibit the formation of Candida albicans hyphae and biofilms without having a bactericidal effect 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 have special biosynthetic gene clusters and is an important source of lead compounds with new structures and new functions. Aiming to solve the pathogenicity of Candida albicans, exploring lead drugs from marine microorganisms that can inhibit the formation of its hyphae and biofilms without killing Candida albicans is an effective solution for treating various diseases caused by Candida albicans. Summary of the Invention:

[0004] The first object of the present invention is to provide seven pyrrolidine carboxylic acid compounds with Candida albicans inhibitory activity and their pharmaceutically acceptable salts.

[0005] The pyrrolidine carboxylic acid compound or its pharmaceutically acceptable salt of the present invention has a structure as shown in formula (I):

[0006]

[0007] The inventor of the present invention carried out co-culture fermentation of marine actinomycetes from different sources in a specific culture medium. Furthermore, through large-scale fermentation, extraction, separation and purification, seven compounds 1-7 containing pyrrole units were obtained. Through techniques such as (+)-HR-ESIMS, 1D and 2D NMR, it was determined that the seven monomers were compounds containing different types of alkyl groups, hydroxyethyl groups and carboxyl groups. The specific structures are shown in formula (I).

[0008] By evaluating the inhibitory activity of compounds 1-7 against Candida albicans, it was found that they had good inhibitory activity against the formation of Candida albicans biofilm and the growth of hyphae represented by C. albicans SC5314. Therefore, they could inhibit its pathogenicity without killing the Candida albicans pathogenic bacteria precursor, and had the potential to develop lead compounds for anti-Candida albicans pathogenic drugs.

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

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

[0011] The third object of the present invention is to provide an anti-Candida albicans pathogenic drug, which is characterized by comprising an effective amount of compound 1 or compound 2 or compound 3 or compound 4 or compound 5 or compound 6 or compound 7 shown in formula (I) as an active ingredient, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a preparation method of a pyrrolidone carboxylic acid compound, which is prepared and separated from the mixed fermentation broth of Streptomyces koyangensis SCSIO 5802 and Nocardiopsis sp. SCSIO SX5N9, and the Nocardiopsis sp. SCSIO SX5N9 has a preservation number of GDMCC NO: 64065.

[0013] The compounds 1-7 containing different types of alkyl groups, hydroxyethyl groups and carboxyl groups of the present invention are new compounds, have a significant inhibitory effect on the tested Candida albicans, can be used to prepare drugs for inhibiting the pathogenicity of Candida albicans, and are used to treat various diseases caused by Candida albicans infection. Therefore, the present invention provides alternative compounds for developing new drugs for inhibiting the pathogenicity of Candida albicans, and has important significance for developing Chinese marine drug resources.

[0014] Streptomyces koyangensis SCSIO 5802 (hereinafter referred to as strain SCSIO 5802), an actinomycete isolated from deep-sea sediment (published in Tetrahedron. 2017, 73, 5366 - 5372, also held by the applicant, and guaranteed to be available to the public within 20 years from the filing date), and Nocardiopsis sp. SCSIO SX5N9 (hereinafter referred to as strain SCSIO SX5N9), an actinomycete isolated from coral, were deposited at the South China Sea Institute of Oceanology, Chinese Academy of Sciences (SCSIO), with the deposit numbers: SXA2012001 and SXA20180N9 respectively. Address: South China Sea Institute of Oceanology, No. 164 Xingang West Road, Haizhu District, Guangzhou, Guangdong Province. Meanwhile, Nocardiopsis sp. SCSIO SX5N9 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on November 24, 2023. Address: 5th Floor, Building 59, No. 100 Dashanyuan, Xianlie Middle Road, Guangzhou, Postcode: 510070, Deposit Number GDMCC NO: 64065. Brief Description of the Drawings:

[0015] Figure 1 It is the 1 HNMR (700 MHz) spectrum of Compound 1, with the solvent being: deuterated methanol;

[0016] Figure 2 It is the 13 C NMR (175 MHz) spectrum of Compound 1, with the solvent being: deuterated methanol;

[0017] Figure 3 It is the 1 H NMR (700 MHz) spectrum of Compound 2, with the solvent being: deuterated methanol;

[0018] Figure 4 It is the 13 C NMR (175 MHz) spectrum of Compound 2, with the solvent being: deuterated methanol.

[0019] Figure 5 It is the 1 H NMR (700 MHz) spectrum of Compound 3, with the solvent being: deuterated methanol;

[0020] Figure 6 It is the 13 C NMR (175 MHz) spectrum of Compound 3, with the solvent being: deuterated methanol.

[0021] Figure 7 It is the 1 H NMR (700 MHz) spectrum of Compound 4, with the solvent being: deuterated methanol;

[0022] Figure 8 is the 13 13C NMR (175 MHz) spectrum of Compound 4, solvent: deuterated methanol.

[0023] Figure 9 is the 1 1H NMR (700 MHz) spectrum of Compound 5, solvent: deuterated methanol;

[0024] Figure 10 is the 13 13C NMR (175 MHz) spectrum of Compound 5, solvent: deuterated methanol.

[0025] Figure 11 is the 1 1H NMR (700 MHz) spectrum of Compound 6, solvent: deuterated methanol;

[0026] Figure 12 is the 13 13C NMR (175 MHz) spectrum of Compound 6, solvent: deuterated methanol.

[0027] Figure 13 is the 1 1H NMR (700 MHz) spectrum of Compound 7, solvent: deuterated methanol;

[0028] Figure 14 is the 13 13C NMR (175 MHz) spectrum of Compound 7, solvent: deuterated methanol; Figure 15 is the HMBC correlation and H-H COSY correlation diagram; Figure 16 is the mycelial inhibition rate diagram of the compound; Figure 17 is the mycelial formation rate diagram of Compounds 1 and 3 at different concentrations; Figure 18 is the diagram of the effect of Compounds 1 and 3 on the mycelial formation of Candida albicans; Figure 19 is the biofilm formation rate diagram of the compound; Figure 20 is the diagram of the effect of pyrrolidone carboxylic acid compound on the growth of Candida albicans strain SC5314; Figure 21 is the diagram of the effect of pyrrolidone carboxylic acid compound on the virulence of Candida albicans strain SC5314. Specific implementation method:

[0029] The following examples are further illustrations of the present invention, rather than limitations thereof.

[0030] Example 1:

[0031] Preparation and Structural Identification of Compounds 1-7 Shown in Formula (Ⅰ)

[0032] I. Preparation of Compounds 1-7 Shown in Formula (Ⅰ)

[0033] 1. Seed Culture:

[0034] (1) Take out the strains SCSIO 5802 and SCSIO SX5N9 from the -80°C refrigerator, thaw and inoculate them on the MISP2 plate (glucose 4 g / L, yeast extract powder 4 g / L, malt extract powder 10 g / L, sea salt 30 g / L, pH 7.2 - 7.4, agar powder 15 g / L), place them in the 28°C incubator for 5 - 7 d to activate the strains; transfer the activated strains to a 250 mL shake flask containing 50 mL of MAM2ab (soluble starch 0.5%, soybean powder 0.5%, glucose 2%, yeast extract powder 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) medium, and culture at 28°C and 200 rpm for 48 h to obtain the seed liquid. The seed liquid is inoculated at 5% of the fermentation volume, with a total of 2.5 ml in the ratio of 2 / 0.5 (mL / mL) of 5802 / SX5N9, and inoculated into the MAM2ab medium respectively. The strains SCSIO 5802 and SCSIO SX5N9 are cultured separately as controls. After inoculation, culture at 28°C and 200 rpm for 7 d to harvest the bacteria, and perform extraction and detection.

[0035] 2. Strain Scale-up Fermentation, Product Extraction and Separation:

[0036] (1) Strain Scale-up Fermentation:

[0037] According to the above seed culture protocol, prepare the MAM2ab medium and perform scale-up fermentation of 14 L.

[0038] (2) Fermentation Product Extraction:

[0039] After centrifuging the fermentation product (co-culture of 5802 / SX5N9) at 3400 rpm for 10 min, separate the bacterial liquid and the bacterial cells. Extract the bacterial liquid with an equal volume of butanone 4 times, and extract the bacterial cells with 2 times the volume of acetone 3 times. Concentrate them respectively to obtain the crude extract extracts of the bacterial cells (M) and the bacterial liquid (L). The target product is mainly distributed in the bacterial liquid (L).

[0040] (3) Fermentation Product Separation:

[0041] The crude extract of the bacterial liquid (L) is treated with CHCl 3The 8 fractions Frs. LA1 - Frs. LA8 were obtained in sequence from the / MeOH system (100 / 0, 98 / 2, 96 / 4, 94 / 6, 92 / 8, 9 / 1, 8 / 2, 1 / 1, V / V, 150 ml); Frs. LA3 - Frs. LA5 were combined and passed through a reverse-phase medium B column (acetonitrile / water 30 / 700 - 100 / 0, 20 mL / min, 60 min), and one fraction was collected every 6 min, yielding 10 fractions Frs. LB1 - Frs. LB10 from the column. Frs. LB9 was subjected to semi-preparative SP-HPLC (semi-preparative high performance liquid chromatography) with water / acetonitrile (85 / 15 - 30 / 70, 0 - 19 min; 0 / 100, 19.1 - 23.0 min; 85 / 15 23.1 - 26.0 23.1 - 26.0 min; 2.5 mL·min -1 , 270 nm), obtaining about 4.1 mg of target compound 1 (t R = 22.8 min) and about 5.0 mg of 2 (t R = 22.4 min). Frs. LB8 was subjected to semi-preparative 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 23.1 - 26.0 min; 2.5 mL·min -1 , 270 nm), obtaining about 8.8 mg of target compound 3 (t R = 19.0 min), about 1.0 mg of 4 (t R = 18.4 min) and about 2.0 mg of 7 (t R = 17.2 min). Frs. LB7 was passed through a normal-phase C column in sequence from the CHCl 3 / MeOH system (100 / 0, 98 / 2, 97 / 3, 96 / 4, 95 / 5, 93 / 7, 8 / 2, 1 / 1, V / V, 100 ml) to obtain 8 fractions Frs. LC1 - Frs. LC8 in total. Among them, Frs. LC5 was subjected to semi-preparative SP-HPLC 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 , 270 nm), obtaining about 1.0 mg of target compound 5 (t R = 19.8 min) and about 1.0 mg of 6 (t R = 19.2 min).

[0042] II. Physicochemical data of compounds 1 - 7

[0043] II. Structure Identification of Compounds 1-7

[0044] Figure 1 This is the 1 1H NMR (700 MHz) spectrum of Compound 1. The solvent is deuterochloroform; Figure 2 This is the 13 13C NMR (175 MHz) spectrum of Compound 1. The solvent is deuterated methanol; Figure 3 This is the 1 1H NMR (700 MHz) spectrum of Compound 2. The solvent is deuterated methanol; Figure 4 This is the 13 13C NMR (175 MHz) spectrum of Compound 2. The solvent is deuterated methanol. Figure 5 This is the 1 1H NMR (700 MHz) spectrum of Compound 3. The solvent is deuterated methanol; Figure 6 This is the 13 13C NMR (175 MHz) spectrum of Compound 3. The solvent is deuterated methanol. Figure 7 This is the 1 1H NMR (700 MHz) spectrum of Compound 4. The solvent is deuterated methanol; Figure 8 This is the 13 13C NMR (175 MHz) spectrum of Compound 4. The solvent is deuterated methanol. Figure 9 This is the 1 1H NMR (700 MHz) spectrum of Compound 5. The solvent is deuterated methanol; Figure 10 This is the 13 13C NMR (175 MHz) spectrum of Compound 5. The solvent is deuterated methanol. Figure 11 This is the 1 1H NMR (700 MHz) spectrum of Compound 6. The solvent is deuterated methanol; Figure 12 This is the 13 13C NMR (175 MHz) spectrum of Compound 6. The solvent is deuterated methanol. Figure 13 This is the 1 1H NMR (700 MHz) spectrum of Compound 7. The solvent is deuterated methanol; Figure 14 This is the 13 13C NMR (175 MHz) spectrum of Compound 7. The solvent is deuterated methanol.

[0045] Structural analysis tests were carried out on Compounds 1-7. Based on high-resolution mass spectrometry, one-dimensional and two-dimensional NMR data, their structural identifications are as follows:

[0046] The schematic diagram of structural identification is as shown in Figure 15 the figure: The unidirectional arrow represents HMBC correlation, and the bidirectional arrow represents H-H COSY correlation

[0047] According to the above identification and analysis, the structures of Compounds 1-7 are as shown in Formula (I).

[0048]

[0049] Example 2:

[0050] Experiment on inhibiting the pathogenicity of Candida albicans by compounds containing different types of alkyl, hydroxyethyl and carboxyl groups - Compounds 1-7 in Example 1.

[0051] Activity detection scheme

[0052] (1) Activation of Candida albicans strain SC5314:

[0053] Streak the standard strain of Candida albicans SC5314 on LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) for activation, and place it in an incubator at 30 °C for overnight culture.

[0054] (2) Effect of pyrrolidone carboxylic acid compounds on the hyphae of Candida albicans strain SC5314:

[0055] Pick a single colony of SC5314 strain on the LB solid plate, inoculate it into GMM medium (6.7 g / L YNB, 0.2% glucose), and culture it overnight at 30 °C with shaking at 200 rpm. Measure the OD of the bacterial solution 600 , and dilute the bacterial solution with GMM medium to OD 600 = 0.1. Take 500 μL of the bacterial solution into a 1.5 mL EP tube, add compounds with a final concentration of 100 μM respectively, shake well, and incubate in a water bath at 37 °C (at the same time, set DMSO as the positive and negative controls at 30 °C and 37 °C respectively). After 6 h, centrifuge at 5000 rpm for 10 min, discard the supernatant, add 70 μL of GMM culture medium to resuspend the cells, observe the formation of hyphae under a microscope, and take photos in different fields of view.

[0056] (3) Effect of pyrrolidone carboxylic acid compounds on the biofilm of Candida albicans strain SC5314

[0057] Pick a single colony of SC5314 strain on the LB solid plate, inoculate it into GMM medium (6.7 g / L YNB, 0.2% glucose), and culture it overnight at 30 °C with shaking at 200 rpm. Measure the OD of the bacterial solution 600 , and dilute the bacterial solution with GMM medium to OD 600= 0.1. Take 200 μL of the bacterial solution and place it in a 96-well plate. Add the compound with a final concentration of 100 μM, and then incubate it statically in an incubator at 30 °C for 16 h. Set DMSO as the negative control. Discard the supernatant, 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 it 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) value at a wavelength of 600 nm using a microplate reader. Take the control group with DMSO added as 100%, and calculate the relative values of the added compounds respectively. Repeat the experiment 3 times and record the results.

[0058] (4) Effect of pyrrolidone carboxylic acid compounds on the growth of Candida albicans strain SC5314:

[0059] Pick a single colony of Candida albicans SC5314 strain and inoculate it into GMM liquid medium, and culture it overnight at 30 °C. Dilute its OD 600 value to 0.05, and add the compound to make its final concentration 100 μM. Add it to a 96-well plate at 200 μL / well, place the culture plate in a full-wavelength microplate reader, and culture it with moderate shaking at 30 °C. The instrument automatically measures the OD 600 value of each well every 1 hour and continuously monitor for 12 h.

[0060] (5) Effect of pyrrolidone carboxylic acid compounds on the invasion of A549 cells by Candida albicans SC5314:

[0061] After the A549 cells are recovered and adherent, digest them with trypsin, and culture them overnight in high-glucose medium DMEM containing 10% (v / v) fetal bovine serum at a concentration of 1 × 10 4 cells / well in a 96-well plate. When the cells grow to 80% confluence, discard the culture medium and wash the cells three times with PBS (0.01 M, pH 7.4). Pick a single colony of Candida albicans SC5314 strain and inoculate it into GMM culture medium containing 0.2% glucose, and culture it overnight on a shaker at 30 °C. Centrifuge to collect the bacteria, wash them three times with PBS, and disperse them at a concentration of 10 8 CFU / mL in DMEM cell maintenance medium containing 1% (v / v) FBS, and add the compound with a final concentration of 100 μM respectively. Take 200 μL and add it to a 96-well cell plate respectively, set three parallels, and incubate in a cell culture incubator for 8 h. At the same time, set DMSO and the wells without adding the compound as control wells. Detect by measuring the content of lactate dehydrogenase LDH released by human lung cancer A549 cells.

[0062] Experimental results

[0063] (1) Pyrrolidone carboxylic acid compounds inhibit the hyphal formation of Candida albicans

[0064] The morphological transition of Candida albicans from yeast to hyphal form is very important for its pathogenicity. We detected the effects of these compounds on the morphological transition of Candida albicans in vitro. Candida albicans was inoculated into GMM liquid medium and cultured in a shaker at 30 °C to maintain its yeast form. The culture solution was diluted at a certain ratio and then cultured at 37 °C to promote hyphal formation. With or without the compound added to the culture solution, after 6 h of incubation, the vast majority of cells in the negative control group with DMSO added formed hyphae, while the hyphal formation in all groups with the compound added decreased, and the hyphal inhibition rate exceeded at least 40% at a final concentration of 100 μM( Figure 16 ). Compounds 1 and 3 had a significant inhibitory effect on the hyphae of Candida albicans and showed a concentration-dependent inhibition of hyphal formation( Figure 17 ). When observing the hyphal formation under a microscope, it could be seen that the effects of compounds 1 and 3 on the hyphal formation of Candida albicans at a concentration of 100 μM were significant, and almost no hyphae were produced( Figure 18 ).

[0065] (2) Pyrrolidone carboxylic acid compounds inhibit the biofilm formation of Candida albicans

[0066] The biofilm of Candida albicans is also one of the key factors promoting its pathogenicity. By detecting the effects of compounds 1 - 7 on the biofilm formation of Candida albicans, it was found that all compounds could inhibit its biofilm formation to varying degrees, such as Figure 19 . Among them, the inhibition rate of compound 1 on the biofilm formation of Candida albicans reached more than 70%, and the inhibition rate of compound 3 reached about 60%.

[0067] (3) Pyrrolidone carboxylic acid compounds have little effect on the growth of Candida albicans strain SC5314

[0068] The results are as Figure 20 shown. Taking DMSO as the control, 100 μM of compound 1 and compound 3 had no effect on the growth of Candida albicans strain SC5314. This result indicates that the effects of compound 1 and compound 3 on Candida albicans strain SC5314 are not to kill bacteria, so drug resistance is not easily generated.

[0069] (4) Pyrrolidone carboxylic acid compounds have a certain inhibitory effect on the virulence of Candida albicans strain SC5314

[0070] The results of the cytotoxicity experiment showed that taking DMSO as the control, in the absence of bacteria, compounds 1 - 7 had no toxicity to cells, such as Figure 21 . As shown in the appendix Figure 21As shown in Figure a, DMSO was used as a control under the condition of adding Candida albicans SC5314; Figure b shows that Compounds 1, 3-6 have a certain protective effect on inhibiting the invasion of strain SC5314 into cells, and Compound 3 reduces the virulence of Candida albicans by less than 12%.

[0071] In summary, the present invention provides a new lead compound for the development of novel drugs for inhibiting the pathogenicity of Candida albicans strains. On this basis, it is of great significance to further develop Chinese marine drug resources using the co-culture method.

Claims

1. Pyrrolidine carboxylic acid compounds with the structure shown in formula (I) and their pharmaceutically acceptable salts:

2. Use of the compound according to claim 1 in the preparation of antibacterial drugs.

3. According to the use described in claim 2, characterized in that, the drug is for Candida albicans.

4. An antibacterial drug, characterized in that, it comprises an effective amount of the compound according to claim 1 as an active ingredient, or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

5. According to the drug described in claim 4, characterized in that, the drug is for inhibiting Candida albicans.

6. A preparation method of the pyrrolidine carboxylic acid compound according to claim 1, characterized in that, it is prepared and isolated from the mixed fermentation broth of Streptomyces koyangensis SCSIO 5802 and Nocardiopsis sp. SCSIO SX5N9, and the Nocardiopsis sp. SCSIO SX5N9 has a preservation number of GDMCC NO: 64065.

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