Application of compound 345-F-7 in preparation of drugs for inhibiting candida tropicalis

Abstract

The invention relates to application of a compound 345-F-7 in preparation of drugs for inhibiting candida tropicalis, and belongs to the technical field of medicines.The diversity library of drug-like compounds is screened to obtain that the compound 345-F-7 has an obvious inhibiting effect on a candida tropicalis drug-resistant strain, the inhibition concentration of the compound to Ctr0029 is 4 micrograms / ml, and the inhibition concentration of the compound to Ctr0029 is 4 micrograms / ml, and the inhibition concentration of the compound to Ctr0029 is 2 micrograms / ml. And the inhibition of the 345-F-7 on the Ctr0029 can reach IC50. A further test on the inhibition effect of the 345-F-7 on the biological membranes of the fluconazole-resistant candida tropicalis Ctr0029 and the candida tropicalis standard strain ATCC1369 shows that the inhibition effect of the 345-F-7 on the candida tropicalis biological membranes is very good, and the inhibition effect exceeds the effect of fluconazole with the same concentration.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically, it relates to the use of compound 345-F-7 in the preparation of a drug for inhibiting Candida tropicalis. Background Technology

[0002] Candidemia, a type of bloodstream infection caused by Candida species, is one of the most common fungal sepsis cases in clinical practice, with a particularly high mortality rate among hospitalized patients with hematological disorders. In recent years, the infection rate of Candida tropicalis has increased significantly in the Asia-Pacific and Latin American regions, with associated morbidity and mortality also showing a continuous upward trend. Compared to other Candida species, Candida tropicalis exhibits stronger biofilm formation ability, higher tissue invasiveness, and multidrug resistance, resulting in a significant increase in its incidence in bloodstream infections, urinary tract infections, and deep tissue infections. Therefore, the development of new anti-Candida tropicalis drugs is imperative. Summary of the Invention

[0003] To address the problems existing in the background technology, this invention, through screening a diverse compound library of 50K drugs from MedChemExpress, found that compound 345-F-7 has a significant inhibitory effect on drug-resistant strains of Candida tropicalis. Further analysis revealed…

[0004] The inhibition tests on biofilm formation of ATCC1369 and Ctr0029 and the inhibition of hyphal formation of fluconazole-resistant Candida tropicalis Ctr0029 verified their antibacterial effect on Candida tropicalis, providing a new direction for the treatment of drug-resistant Candida tropicalis.

[0005] To achieve the above objectives, the first objective of the present invention is to provide compound 345-F-7 for use in the preparation of drugs for inhibiting Candida tropicalis.

[0006] The structural formula of compound 345-F-7 is as follows:

[0007]

[0008] A second object of the present invention is to provide a medicament for inhibiting Candida tropicalis, the medicament comprising compound 345-F-7.

[0009] The beneficial effects of this invention are:

[0010] This invention screens compounds from a diverse library of drug-like compounds to identify those resistant to fluconazole-resistant Candida tropicalis.

[0011] Compound 345-F-7 exhibits significant inhibitory activity against both Ctr0029 and the standard strain of Candida tropicalis ATCC1369. The inhibitory concentration of this compound against Ctr0029 is 4 μg / ml, and at a concentration of 2 μg / ml, 345-F-7 shows an inhibitory effect of up to IC50 on Ctr0029. 50 .

[0012] Further experiments on the biofilm inhibition of different concentrations of 345-F-7 on fluconazole-resistant Candida tropicalis Ctr0029 and the standard strain of Candida tropicalis ATCC1369 revealed that 345-F-7 had a very good biofilm inhibition effect on Candida tropicalis, even exceeding the effect of fluconazole at the same concentration.

[0013] The inhibition test of different concentrations of 345-F-7 on the mycelium of fluconazole-resistant Candida tropicalis Ctr0029 revealed that 345-F-7 can significantly inhibit the formation of mycelium of fluconazole-resistant Candida tropicalis Ctr0029 even at low concentrations.

[0014] This invention provides a new direction for drug research in the treatment of tropical Candida. Attached Figure Description

[0015] Figure 1 The inhibitory effect of compound 345-F-7 of the present invention on Ctr0029 and ATCC1369;

[0016] Figure 2 This invention describes the inhibition of biofilm formation in ATCC1369 and Ctr0029 by different concentrations of 345-F-7.

[0017] Figure 3 This invention describes the inhibition of hyphal formation of fluconazole-resistant Candida tropicalis Ctr0029 by different concentrations of 345-F-7.

[0018] Figure 4 This invention describes the effects of different concentrations of 345-F-7 on the degree of hemolysis of red blood cells.

[0019] Figure 5 This is a comparison of the antibacterial effects of 345-F-7 against different bacterial strains. Detailed Implementation

[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0021] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0022] Example 1

[0023] We screened drug-like compounds with inhibitory effects on tropical Candida strains from MedChemExpress's 50K drug-like diverse compound library.

[0024] Initial screening:

[0025] The drug screening method was performed in accordance with the People's Republic of China Health Industry Standard "Standard for Drug Susceptibility Testing against Yeast-like Fungi - Broth Dilution Method" WS / T 421—2024, as follows:

[0026] (1) Preparation of compound working solution:

[0027] Take a sterile 96-well cell culture plate and add 200 μL of RPMI 1640 (containing MOPS) medium as a protective solution to column 1. Add compounds from MedChemExpress's 50K class drug library to columns 2-11 respectively. The compound solution is diluted as follows: take 6 μL of the original compound solution and add 94 μL of RPMI 1640 medium containing MOPS to make the compound concentration 20 μM, i.e., a 2-fold working solution. Add 128 μg / ml fluconazole to the first well of column 12. Dilute the solutions 2-fold to obtain 2-fold working solutions of 64, 32, 16, 8, 4, and 2 μg / ml respectively as drug controls. The eighth well of column 12 is the growth control.

[0028] (2) Preparation of bacterial culture:

[0029] Fluconazole-resistant Candida tropicalis Ctr0029 and the standard Candida tropicalis strain ATCC1369 were inoculated into PDA and cultured at 35°C for 24 h. A bacterial suspension was prepared using sterile PBS, and the concentration was adjusted to 0.5 McFarland units (i.e., 1 × 10⁻⁶). 6 ~5×10 6 (cfu / ml), then diluted 1000-fold with RPMI 1640 to a final concentration of 2 times the working solution (i.e., 1×10⁻⁶). 3 ~5×10 3 (cfu / ml). (3) Add the prepared 2x bacterial suspension to 2x compound solution to obtain a bacterial concentration of 0.5×10⁻⁶. 3 ~2.5×10 3The final working solution was prepared at a concentration of 10 μM (CFU / ml) and incubated at 35°C for 24 hours. Results were interpreted using the following initial screening criteria: microscopic observation of bacterial inhibition; no bacterial growth at 80% was considered effective; and a clear culture medium indicated complete inhibition, indicating significant inhibitory effect. Initial screening revealed that compound 345-F-7 significantly inhibited both fluconazole-resistant Candida tropicalis Ctr0029 and the standard Candida tropicalis strain ATCC1369, with clear culture medium. Compound 345-F-7 at a concentration of 10 μM was active against Ctr0029 (clear culture medium with no bacterial growth).

[0030] Secondary screening:

[0031] (1) Preparation of compound working solution:

[0032] Compound 345-F-7 obtained from the initial screening was prepared as a working solution with an initial concentration of 215 ug / ml (2-fold). One row of 12 wells in a 96-well plate was taken and diluted 2-fold serially to prepare 2-fold working solutions, namely: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0 (concentration unit ug / ml). The drug control fluconazole was diluted in the same way.

[0033] (2) Preparation of bacterial culture

[0034] The method is the same as the initial screening.

[0035] (3) Add the prepared 2x bacterial suspension to the 2x compound solution, and add 20ul of Resorufin indicator with a concentration of 100ug / ml to each well. Incubate at 35℃ for 24h and interpret the results. If the culture medium does not turn red, the concentration of the well is the MIC interpretation concentration. Repeat the operation 3 times. If the MIC interpretation does not exceed the range of one well, the value is considered stable and reliable.

[0036] Results (attached) Figure 1 The study showed that compound 345-F-7 exhibited a minimum inhibitory concentration (MIC) of 4 μg / mL against the drug-resistant clinical strain of Candida tropicalis Ctr0029. At a concentration of 2 μg / mL, it achieved an inhibition rate of 50% against this strain, i.e., an IC50 value. 50 The value is 2 μg / mL.

[0037] The minimum inhibitory concentration (MIC) of compound 345-F-7 against ATCC1369 is 2 μg / ml.

[0038] Example 2

[0039] Inhibitory effect of 345-F-7 on biofilm formation of Candida tropicalis

[0040] (1) Preparation of bacterial culture:

[0041] Fluconazole-resistant Candida tropicalis Ctr0029 and the standard Candida tropicalis strain ATCC1369 were inoculated into PDA and cultured at 35°C for 24 h. A bacterial suspension was prepared using sterile PBS and adjusted to a concentration of 1×10⁻⁶. 4 -1×10 5 cfu / ml.

[0042] (2) Inoculate 100 μL of bacterial suspension into a sterile 96-well plate, incubate at 37°C for 90 min to promote initial adhesion, wash three times with PBS to remove unadhered cells, add RPMI1640 medium containing different concentrations of 345-F-7 and fluconazole, and continue incubation for 24 hours.

[0043] (3) After the culture is completed, discard the culture medium, wash 3 times with PBS, add 150 μL of XTT (0.5 mg / mL) / PMS (1 μmol / L) mixed solution, incubate in the dark for 2 hours, take 70 μL of supernatant and transfer it to a new 96-well plate, and detect the absorbance at 495 nm wavelength to evaluate metabolic activity.

[0044] Biofilm formation rate is calculated using the following formula: Biofilm formation rate (%) = 100 × (Drug group A) 495 - Blank A 495 ) / (Group A without medication) 495 - Blank A 495 ).

[0045] Results (attached) Figure 2 The results showed that 345-F-7 concentrations greater than 4 ug / ml had a good inhibitory effect on the formation of ATCC1369 biofilm, and 8-64 ug / ml had a good inhibitory effect on the formation of fluconazole-resistant Candida tropicalis Ctr0029 biofilm.

[0046] Example 3

[0047] 345-F-7 inhibits hyphal formation in Candida tropicalis

[0048] (1) Select the fluconazole-resistant tropical Candida Ctr0029 clone and culture it in SDY medium at 35°C with shaking for 16 h.

[0049] (2) Centrifuge to remove supernatant, wash 3 times with PBS. Mix with RPMI 1640 + 10% fetal bovine serum culture medium, and adjust the bacterial culture to 1×10⁻⁶. 6 cfu / ml.

[0050] (3) Place the sterilized coverslip in a 6-well plate, take 1 mL of bacterial solution in the 6-well plate, add different concentrations of 345-F-7 and mix well, and incubate at 35°C.

[0051] (4) After 4 hours, remove the coverslip, use filter paper to absorb the culture from the edge, and then add a drop of lactic acid cotton blue. Observe the effect of 345-F-7 on the mycelial morphology of Ctr0029 under a 40x microscope.

[0052] Results (attached) Figure 3 The results showed that 345-F-7 could significantly inhibit the mycelial formation of fluconazole-resistant Candida tropicalis Ctr0029 at low concentrations.

[0053] Example 4

[0054] Toxicity testing of 345-F-7

[0055] To evaluate the hemolytic activity of compound 345-F-7, different concentrations (1–256 μg / mL) of 345-F-7 were mixed with 2% human erythrocytes and incubated at 37°C for 1 hour. The hemolytic activity was then assessed by measuring the OD (oxidative stress). 540 The absorbance value at nm reflects the degree of hemolysis. 1% Triton X-100 was used as a positive control, and 10mM PBS as a negative control. Figure 4 As shown, within the concentration range of ≤64 μg / mL, the hemolysis rate induced by 345-F-7 was similar to that of the PBS group, with almost no hemolysis. Slight hemolysis occurred at 128 μg / mL, while moderate hemolysis was observed at 256 μg / mL, but still significantly lower than that of the Triton X-100 group. These results indicate that 345-F-7 exhibits good biocompatibility with human erythrocytes in the low to medium concentration range, suggesting its high safety profile.

[0056] Example 5

[0057] Comparison of the antibacterial effects of 345-F-7 against filamentous fungi, Candida, and Candida tropicalis (antibacterial test method is the same as in Example 1).

[0058] Further testing revealed that compound 345-F-7 exhibits antibacterial activity against a variety of clinically common yeast-type and filamentous fungi. Figure 5See Table 1. The minimum inhibitory concentrations (MICs) against the fluconazole-resistant clinical isolate Ctr0029 and the standard strain ATCC1369 of Candida tropicalis were 4 μg / mL and 2 μg / mL, respectively. The MICs against Cryptococcus neoformans ATCC32045 and Trichophyton rubrum ATCC MYA-3638 were also 4 μg / mL, demonstrating strong antifungal activity. The MICs against Candida albicans, Candida glabrata, Candida parapsilosis, and T. marneffei were 8–32 μg / mL, indicating that this compound has certain antifungal activity against various yeasts and filamentous fungi, especially showing superior performance against some drug-resistant strains compared to fluconazole, demonstrating good application potential.

[0059] Table 1. Minimum inhibitory concentrations of compound 345-F-7 against different bacterial strains.

[0060]

[0061] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of compound 345-F-7 in the preparation of drugs that inhibit Candida tropicalis.

2. The application as described in claim 1, characterized in that, The structural formula of the compound 345-F-7 is as follows:

3. A drug for inhibiting Candida tropicalis, characterized in that, It contains compound 345-F-7.

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