WO2019019049A1 - Anti-candida albicans piperazine derivative, preparation method therefor and application thereof - Google Patents

Abstract

The present invention provides an anti-Candida albicans piperazine derivative, a preparation method therefor and application thereof. The structural formula of the piperazine derivative is as represented by formula I, and the derivative can be used for preparing an anti-Candida albicans infection drug.

Description

Piperazine derivative against Candida albicans and preparation method and application thereof Technical field

The invention relates to the field of compound synthesis, in particular to a piperazine derivative against Candida albicans and a preparation method and application thereof.

Background technique

Candida albicans is a fungal disease widely spread in humans and is one of the most important pathogens for hospital-acquired infections. The fungus can infect and colonize a wide range of microbial environments in the human body, including blood vessels, mucosal surfaces and major internal organs. As a conditional pathogen, Candida albicans is not only the cause of thrush and vaginitis, but also causes serious systemic infections in immunodeficient patients and leads to higher mortality. At present, the most effective treatment and prevention of Candida albicans infection is the topical and systematic use of azole antifungal drugs, which can directly kill the bacteria, but with the widespread use of azole drugs, resistance is also increasing. serious. For this reason, it is very valuable to develop a drug with a novel antibacterial strategy to treat Candida albicans infection.

Candida albicans has a very special characteristic, yeast-hyphal dimorphism. The morphological transformation of Candida albicans from the yeast state to the mycelial state is an important factor in the infection of cells. The free yeast state of Candida albicans adheres first and promotes tissue invasion through morphological transformation. Patients infected with Candida albicans usually penetrate the infected tissue from mycelial pathogens. Defective strains with morphological mutations during infection are non-toxic, and morphological changes are important for the pathogenicity of Candida albicans. Therefore, we use the importance of Candida albicans adhesion and morphological transformation in its pathogenic role to screen for new drugs that inhibit this strain.

Summary of the invention

The primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a piperazine derivative against Candida albicans.

Another object of the present invention is to provide a process for producing the above piperazine derivative against Candida albicans.

A further object of the present invention is to provide the use of the above piperazine derivatives against Candida albicans.

The object of the present invention is achieved by the following technical scheme: a piperazine derivative against Candida albicans, the structural formula is as shown in Formula I:

Wherein R is 2-X, 4-X or 4-CX ₃ ; X is a halogen element including F, Cl, Br and I; 2 and 4 represent the position of the substituent group.

The R is preferably 4-CF ₃ , 2-Cl, 2-Br or 4-Br.

The preparation method of the piperazine derivative against Candida albicans comprises the following steps:

(1) Mix 4-hydroxycarbazole and epichlorohydrin, add NaOH solution at -10 °C to 10 °C, heat to 60 °C to continue the reaction, TLC to detect the progress of the reaction, add appropriate amount of water after the reaction, use Extracting with ethyl acetate, combining the organic phases, washing with water, drying, filtering, recovering the filtrate, and evaporating the solvent to give an epoxide;

(2) The epoxide and piperazine containing different substituents are dissolved in isopropanol, and refluxed at 0 ° C to 80 ° C for 4-6 hours. The progress of the reaction is detected by TLC. After the reaction is completed, the reaction system is cooled and evaporated. Solvent; purification, recrystallization, to obtain a piperazine derivative against Candida albicans; wherein piperazine containing different substituents is used in an amount of 1:1 in a molar ratio of 4-hydroxycarbazole.

The ratio of the molar amount of the epichlorohydrin described in the step (1) to the molar amount of the 4-hydroxycarbazole is not less than 1, whereby the 4-hydroxycarbazole can be fully utilized; preferably the ring The molar ratio of the oxychloropropane to the molar amount of the 4-hydroxycarbazole is from 1:1 to 2:1; more preferably, the molar amount of the epichlorohydrin is the same as the 4-hydroxyindole The molar ratio of azole was 1.2:1.

The concentration of the NaOH solution described in the step (1) is preferably 40% by mass.

The molar amount of NaOH described in the step (1) is preferably equivalent to twice the molar amount of 4-hydroxycarbazole.

The dropping temperature described in the step (1) is preferably 0 °C.

The specific step of the TLC detection reaction process described in the step (1) is preferably as follows: thin layer chromatography is carried out with silica gel GF254, and the developing solvent is a mixture of petroleum ether: ethyl acetate in a volume ratio of 7:1 to 3:1. The detection of the raw material point disappeared.

The water described in the step (1) is used for suspension dispersion.

The drying described in the step (1) is preferably carried out using anhydrous magnesium sulfate.

The piperazine containing a different substituent described in the step (2) is preferably 1-(4-trifluoromethylphenyl)piperazine, 1-(2-chlorophenyl)piperazine or 1-(2-bromo). Phenyl) piperazine, 1-(4-bromophenyl)piperazine.

The conditions of the reflux reaction described in the step (2) are preferably a reflux reaction at 80 ° C for 6 hours.

The specific step of the TLC detection reaction process described in the step (2) is preferably as follows: silica gel GF254 Thin layer chromatography was carried out, and the developing solvent was a mixture of petroleum ether:ethyl acetate in a volume ratio of 4:1, and the disappearance of the piperazine compound was detected.

The purification described in the step (2) is preferably purified by column chromatography.

The column chromatography packing is preferably 200-300 mesh silica gel.

The column chromatography purification is preferably carried out by using a mixture of petroleum ether and ethyl acetate in a ratio of 6:1 by volume.

The recrystallization described in the step (2) is preferably recrystallization using 95% by mass of ethanol.

The use of the piperazine derivative against Candida albicans for the preparation of a medicament against Candida albicans infection.

The present invention has the following advantages and effects over the prior art:

(1) Previous studies by the inventors of the present application have identified that a diffusible signal factor (DSF) quorum sensing signal and its derivative can strongly interfere with the transformation of the Candida albicans yeast-mycelium state. This study also successfully synthesized and screened compounds with inhibition of Candida albicans adhesion, mycelial morphology transformation and pathogenicity. At the same time, these compounds are less toxic and do not affect the growth of Candida albicans and human cells. These characteristics are expected to promote the development of new antifungal drugs.

(2) The yield of the preparation method provided by the present invention is high, about 75%.

DRAWINGS

Figure 1 is a diagram showing the synthesis process of a piperazine derivative; wherein a is sodium hydroxide.

Figure 2 is a graph showing the results of piperazine derivatives adhering to polystyrene on Candida albicans; wherein, Figure (A) shows 45 synthetic piperazine derivatives against Candida albicans cells at a final concentration of 100 μM. Figure of effect of adhesion; Figure (B) is the result of inhibition of 6 compounds of 22, 24, 25, 26, 27 and 28 at different concentrations from 12.5 μM to 200 μM; fluconazole as a positive control The data shows the average of 8 biological replicates, and the error bars reflect the standard deviation.

Figure 3 is a graph showing the effect of piperazine derivatives on the formation of Candida albicans hyphae; wherein, Figure (A) is the inhibition of the formation of Candida albicans hyphae by 45 synthetic piperazine derivatives at a final concentration of 100 μM. Figure (B) is the inhibition effect of six compounds of 22, 24, 25, 26, 27 and 28 at different concentrations from 50 μM to 200 μM; Figure (C) is DMSO, fluconazole Micrographs of microscopic observations of inhibition of hyphal formation by compounds Nos. 22, 24, 25, 26, 27 and 28 at a final concentration of 100 μM; this data shows the average of three biological experiments, and the error bars reflect the standard difference.

Figure 4 is a diagram showing the results of pathogenicity test of piperazine derivatives against Candida albicans; wherein, Figure A is the end Results of cytotoxicity of 45 synthetic piperazine derivatives at a concentration of 100 μM on A549 cells, and panel B is the effect of 45 synthetic piperazine derivatives at a final concentration of 100 μM on the cytotoxicity of Candida albicans Fig. C is a graph showing the effect of compounds Nos. 25, 26, 27 and 28 on the cytotoxicity of Candida albicans at various concentrations ranging from 3.125 μM to 100 μM; the toxicity of the cells was detected by detecting the release of LDH, and the white rosary was detected. For the cytotoxicity of the bacteria, the LDH release amount of the group to which DMSO was added was taken as 100%, and thereby the ratio of LDH release to other piperazine derivative groups was regulated; the data showed four biological replicates. On average, the error bars reflect the standard deviation.

Figure 5 is a graph showing the effect of piperazine derivatives on the growth rate of Candida albicans; fluconazole as a positive control; the data shows the average of three biological replicates, and the error bars reflect the standard deviation.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

Example 1 Synthesis of Piperazine Derivatives

1. As shown in Figure 1, the synthesis of piperazine derivatives is as follows:

(1) First, 4-hydroxyindole, 4-hydroxycarbazole or 4-hydroxyacetophenone is mixed with epichlorohydrin (molar ratio: 1:1.2), and mass (g) is added dropwise at 0 °C ( The ratio of mL) is 40% sodium hydroxide aqueous solution (the amount of NaOH is 2 times the number of moles of 4-hydroxyindole, 4-hydroxycarbazole or 4-hydroxyacetophenone), and the reaction is completed, and the temperature is raised to 60 ° C. The corresponding epoxy compound was obtained, and the reaction progress was detected by TLC (thin layer chromatography, the material was silica gel GF254) until the starting point disappeared, and the developing solvent was a mixture of petroleum ether: ethyl acetate at a volume ratio of 7:1, and the reaction was completed. After the addition of water, extraction with ethyl acetate, the combined organic phases, washed with water, dried over anhydrous magnesium sulfate, filtered, filtrated

(2) The obtained product is respectively combined with piperazine containing different substituents (the amount is consistent with the number of moles of 4-hydroxyindole, 4-hydroxycarbazole or 4-hydroxyacetophenone, respectively, and the specific compounds are shown in Table 1) The reaction was refluxed at high temperature (80 ° C) for 6 hours in isopropanol, and the progress of the reaction was detected by TLC until the piperazine point disappeared. The developing solvent was a mixture of petroleum ether:ethyl acetate at a volume ratio of 4:1. The reaction system was cooled, and the solvent was evaporated. Purification by column chromatography (petroleum ether: ethyl acetate = 6:1 by volume, the filler is 200-300 mesh silica gel), and recrystallization from 95% by mass of ethanol to give compounds 1 to 45 (see Table 2)). The compounds were identified by nuclear magnetic and mass spectrometry and were consistent with the designed structure. All samples were dissolved in DMSO in the activity experiment and used. The yield of the compound obtained by this method is about 75%.

Table 1

Note: The piperazine structure containing different substituents in Table 1 can be based on "Ge, ZQ; Ji, QG; Chen, CY; Liao, Q.; Wu, HL; Liu, XF; Huang, YR; Yuan, LJ; Liao, F., Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents, Journal of Enzyme Inhibition & Medicinal Chemistry, 2016, 31(2), 219-228".

For example, the synthesis of the compound 25 is specifically as follows: 9.16 g (0.05 mol) of 4-hydroxycarbazole is added to 5.55 g (0.06 mol) of epichlorohydrin, and the concentration is 40% (0/) at 0 °C. v) NaOH solution 10mL, about 1 hour, the temperature is raised to 60 ° C to continue the reaction for several hours, TLC detection reaction process, after the end of the reaction, add appropriate amount of water, extracted with ethyl acetate, the organic phase, washed with water, anhydrous magnesium sulfate dry. Filtration, recovery of the filtrate, and evaporation of the solvent afforded a crude red oil of about 15 g, which is a crude 4-(ethylene oxide-2-yl)methoxy-9H-carbazole, without purification. The above obtained epoxide was dissolved in an appropriate amount of isopropanol with 11.5 g (0.05 mol) of 1-(4-trifluoromethylphenyl)piperazine, and refluxed for 6 hours. The reaction progress was detected by TLC, and the reaction was completed. The reaction system was cooled, and the solvent was evaporated. Purification by column chromatography (petroleum ether: ethyl acetate 6:1), and then recrystallized from 95% ethanol to give compound 25. about 17.50 g, yield 75%.

Table 2

Example 2 Effect Test

1, testing test

(1) Adhesion test

Candida albicans SC5314 strain (ATCC, USA) is a GMM nutrient solution (composed of 6.7 g/L of amino acid-free yeast nitrogen source (YNB) plus 0.2% glucose, ref. "A novel DSF-like signal from Burkholderia cenocepaciainterferes with Candida The culture was carried out overnight in the method of albicans morphological transition, and the OD ₆₀₀ value was adjusted to 0.5, and a certain final concentration of the compound was sequentially added, mixed, and added to a 96-well plate (polystyrene material) at 200 μL/well. The plate was incubated at 37 ° C for 4 hours, and the supernatant was discarded, and 50 μL of crystal violet having a concentration of 0.5% by mass was added to each well for 45 min at room temperature. The crystal violet was discarded and washed 10 times with iced deionized water, and then crystal violet was dissolved with 200 μL of a 75% by volume ethanol, left at room temperature for 30 min, and then the OD ₅₉₀ value was measured with a microplate reader.

(2) Hyphae formation test

Candida albicans SC5314 strain was cultured in GMM nutrient solution at 30 ° C until its OD ₆₀₀ value was 2.0, and diluted 20-fold with GMM nutrient solution. The compound was added to 0.5 mL of the diluted bacterial solution at a certain final concentration, mixed gently, and then placed in a 37 ° C water bath for 4 hours, and fluconazole, BDSF (B. cenocepacia diffusible signal factor, one A freely diffusible signal molecule isolated from B. cepacii, chemically known as cis-2-dodecenoic acid, was purchased and DMSO was used as a control. After the end of the action, centrifugation (5000 rpm, 10 min), the supernatant was discarded, 40 μL of fresh GMM nutrient solution was added for resuspension, and hyphal formation was observed under a Zeiss Axioplan 2 microscope.

(3) Analysis of growth curve of Candida albicans

Candida albicans SC5314 strain was cultured overnight in GMM medium at 30 ° C, its OD ₆₀₀ value was adjusted to 0.05, and the compound was added to give a final concentration of 100 μM. Add 300 μL/well to a 10×10 sterile honeycomb microplate (suitable for use in the Bioscreen-C Automated Growth Curve Analyzer) and place the plate in a Bioscreen-C automatic growth curve analyzer at 30 °C. Shake the culture, the instrument automatically detects the OD ₆₀₀ value of each well every half hour, and continuously monitors for 48 hours.

(4) Cytotoxicity experiment

The cytotoxicity assay was detected by the amount of lactate dehydrogenase LDH released from human lung cancer A549 cells. A549 cells were cultured in 96-well plates at a concentration of 1 × 10 ⁴ cells/well in high-sugar medium DMEM containing 10% (v/v) fetal bovine serum overnight. When the cells were over 80%, the culture solution was discarded, and the cells were washed three times with PBS (0.01 M, pH 7.4). Candida albicans SC5314 strain was cultured in a GMM medium containing 0.2% (w/v) glucose overnight at 30 ° C, and the cells were collected by centrifugation, washed three times with PBS, and dispersed in a concentration of 10 ⁸ CFU/mL. % (v/v) FBS in DMEM cell maintenance solution, adding a certain concentration of compound. After the interaction, it was added to a 96-well cell plate and allowed to act in a cell culture incubator for 8 hours. Both DMSO and no compound were added as control wells.

(5) Mouse infection test

The animal test used BALB/c mice (purchased from the Guangdong Experimental Animal Center). The test was in accordance with the National Animal Health Institute's Health Guidance for the Care and Use of Laboratory Animals (NIH 8023 publication, revised in 1978). Come on. Male mice 6-8 weeks old were randomly assigned to different groups of 8 animals each and weighed. Candida albicans SC5314 was dispersed in PBS at a concentration of 5×10 ⁸ cfu/mL, and Compounds 27 and 28 with a final concentration of 100 μM were added to the PBS containing bacteria, and injected into the tail vein, 100 μL/10 g, simultaneously. 1×PBS (pH 7.4, 0.01 M) and 100 μM fluconazole were used as negative and positive controls, respectively.

2, the experimental results

(1) Piperazine derivatives inhibit adhesion of Candida albicans

Since adhesion is the first step in Candida albicans infection, we first examined whether piperazine derivatives inhibit the adhesion of Candida albicans to polystyrene. As shown in Fig. 2, 11 of the 45 compounds had an adhesion inhibitory ratio of more than 75% (Fig. 2A). Among them, the effects of compounds Nos. 22, 24 and 25-28 are particularly prominent. We continued to test whether this inhibition was concentration-dependent, so we chose to test the inhibitory effects of these compounds at concentrations ranging from 12.5 μM to 200 μM. The results showed that these compounds inhibited more than 65 at a concentration of 12.5 μM. % (Fig. 2B), the results indicate that the piperazine derivative can effectively inhibit the adhesion of Candida albicans.

(2) Piperazine derivatives inhibit the formation of Candida albicans hyphae

The morphological transformation of Candida albicans from the yeast state to the mycelial state is very important for its pathogenicity. We examined the effects of these compounds on the morphological transformation of Candida albicans in vitro. Candida albicans was cultured in a GMM medium at 30 ° C to maintain its yeast morphology. The culture was diluted in a certain ratio and cultured at 37 ° C to promote mycelial formation. The culture medium was added with or without the compound. After 4 hours of incubation, most of the cells in the negative control group added with DMSO formed hyphae, and most of the hyphae formed in the compound group were reduced (Fig. 3). The hyphal inhibition rate of 13 of the compounds exceeded at least 50% at a final concentration of 100 μM (Fig. 3A), and the compounds Nos. 22, 24, 25-28 inhibited mycelial formation in a concentration-dependent manner (Fig. 3B). Further observation of mycelial formation under a microscope, the effect of compounds Nos. 22, 24, 25, 26, 27 and 28 on the formation of Candida albicans hyphae at a concentration of 100 μM can be seen (Fig. 3C). Can inhibit mycelial production.

(3) Piperazine derivatives attenuate the pathogenicity of Candida albicans

To investigate whether piperazine derivatives can affect the pathogenicity of Candida albicans, we tested piperazines. Toxic effects of derivatives on cells and mice with and without Candida albicans. As we expected, the toxicity of Candida albicans on cells was reduced after the addition of some piperazine derivatives (Figure 4). According to the results of the LDH experiment, when the final concentration is 100 μM, the compounds of 8, 23, 24, 35, 37, 38, 39, 40 and 42 have high cytotoxicity, while other compounds have no or very low cells. Toxicity (Figure 4A). When Candida albicans was added to the cells, the piperazine derivatives at the same concentration almost completely inhibited the toxicity of Candida albicans (Fig. 4B). Therefore, we selected compounds 25, 26, 27, and 28 to inhibit the toxicity of Candida albicans at a concentration of 3.125 μM to 100 μM (Fig. 4C). Considering that compounds Nos. 22, 24, and 25-28 have a good effect in inhibiting the formation of biofilm and hyphae, and compounds 22 and 24 have no effect in inhibiting the cytotoxicity of Candida albicans, we mainly analyzed 25- The inhibitory effect of Compound No. 28 on the cytotoxicity of Candida albicans at different concentrations. The results showed that none of the four compounds affected the growth efficiency of Candida albicans (Fig. 5), which reduced the pathogenicity of Candida albicans without killing the fungi, indicating that they are expected to be novel antifungals. Drug use. The results of the mouse infection test also showed that the mice died 100% within 30 days of infection with Candida albicans, but the mortality rates were 29% and 14%, respectively, after the addition of compounds Nos. 27 and 28 (Table 3). We can see that Compound No. 28 is one of the candidates for the new anti-Candida albicans drug.

Table 3 Effect of compounds on the pathogenicity of Candida albicans in a mouse infection model

Compound	Survival rate (%)
DMSO	0
Fluconazole	100
27	71
28	86

In total, we synthesized a series of piperazine derivatives and screened their ability to fight fungal pathogens. Some of these compounds show excellent properties for inhibiting Candida albicans cell adhesion, hyphal formation and pathogenicity, but they are not toxic to Candida albicans cells as well as to human cells. Experimental results show that some of these compounds may be developed as novel drugs against Candida albicans infection.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and combinations thereof may be made without departing from the spirit and scope of the invention. Simplifications should all be equivalent replacements and are included in the scope of the present invention.

Claims (10)

1. A piperazine derivative against Candida albicans characterized by a structural formula as shown in Formula I:

   

   Wherein R is 2-X, 4-X or 4-CX ₃ ; and X is a halogen element.
2. The piperazine derivative of Candida albicans according to claim 1, wherein said R is 4-CF ₃ , 2-Cl, 2-Br or 4-Br.
3. The method for producing a piperazine derivative against Candida albicans according to claim 1 or 2, comprising the steps of:

   (1) Mix 4-hydroxycarbazole and epichlorohydrin, add NaOH solution at -10 °C to 10 °C, heat to 60 °C to continue the reaction, TLC to detect the progress of the reaction, add appropriate amount of water after the reaction, use Extracting with ethyl acetate, combining the organic phases, washing with water, drying, filtering, recovering the filtrate, and evaporating the solvent to give an epoxide;

   (2) The epoxide and piperazine containing different substituents are dissolved in isopropanol and refluxed at 0 °C to 80 °C for 4-6 hours. The progress of the reaction is detected by TLC. After the reaction is completed, the reaction system is cooled and evaporated. Solvent; purification, recrystallization, to obtain a piperazine derivative against Candida albicans; wherein piperazine containing different substituents is used in an amount of 1:1 in a molar ratio of 4-hydroxycarbazole.
4. The method for producing a piperazine derivative against Candida albicans according to claim 3, wherein:

   The ratio of the molar amount of the epichlorohydrin described in the step (1) to the molar amount of the 4-hydroxycarbazole is not less than 1;

   The molar amount of NaOH described in the step (1) is equivalent to twice the molar amount of 4-hydroxycarbazole.
5. The method for producing a piperazine derivative against Candida albicans according to claim 3, wherein:

   The dropping temperature described in the step (1) is 0 ° C;

   The refluxing condition described in the step (2) is a reflux reaction at 80 ° C for 6 hours;

   The drying described in the step (1) is carried out using anhydrous magnesium sulfate.
6. The method for producing a piperazine derivative against Candida albicans according to claim 3, wherein the piperazine containing a different substituent described in the step (2) is 1-(4-trifluoromethyl). Phenyl)piperazine, 1-(2-chlorophenyl)piperazine, 1-(2-bromophenyl)piperazine or 1-(4-bromophenyl)piperazine.
7. The method for producing a piperazine derivative against Candida albicans according to claim 3, wherein:

   The purification described in the step (2) is carried out by column chromatography.
8. The method for producing a piperazine derivative against Candida albicans according to claim 7, wherein:

   The column chromatography packing is 200-300 mesh silica gel;

   The column chromatography was purified by eluting with a mixture of petroleum ether and ethyl acetate in a ratio of 6:1 by volume.
9. The method for producing a piperazine derivative against Candida albicans according to claim 3, wherein the recrystallization described in the step (2) is recrystallization using 95% by mass of ethanol.
10. Use of the piperazine derivative against Candida albicans according to claim 1 or 2 for the preparation of a medicament against Candida albicans infection.

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