Use of compounds in preparing products for preventing and / or treating diseases

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a compound represented by formula (I), a derivative thereof, or a pharmaceutically acceptable salt thereof; wherein X is an oxygen atom, a sulfur atom, an imino group, a hydrocarbon group, or a hydrocarbon group derivative group; and R1 is a polyphenyl group, a phenyl heterocyclic aromatic group, a cycloolefin group, or a derivative group thereof. The compounds of the present invention can effectively inhibit the activity of metallo-β-lactamase.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of compounds in preparing products for preventing and / or treating diseases. Background Art

[0002] In 2017, the World Health Organization (WHO) listed carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter as "extremely hazardous" among the superbugs posing the greatest threat to human health. Carbapenem antibiotics are hailed as "the last line of defense for human health," but infections caused by bacteria resistant to these drugs are clinically untreatable. These bacteria develop resistance to carbapenems through the presence of genes encoding metallo-β-lactamases, which catalyze the hydrolysis of antibiotics. Currently, the most threatening metallo-β-lactamase to humans is New Delhi metallo-β-lactamase 1 (NDM-1). This is because NDM-1 efficiently hydrolyzes β-lactam antibiotics, and the gene encoding NDM is located on a plasmid, allowing for rapid transmission between bacteria and a high probability of mutation.

[0003] At present, the research on inhibitors is all targeting the active center of NDM-1. This is because during the process of NDM-1 hydrolyzing substrates, the substrates react with the active center of NDM-1, Zn 2+ After coordination, the reaction occurs. The active center is the site where the hydrolysis reaction occurs and is crucial in the NDM-1 structure. There are two main routes for the development of inhibitors, either targeted stripping or replacement of the active center Zn 2+ The goal is to disrupt the structure of the NDM-1 active center or mimic the binding of substrate intermediates to the NDM-1 active center, thereby preventing the substrate from entering the active center. Although there are many reports on NDM-1 inhibitors, there are currently no effective inhibitors for clinical application, mainly because these inhibitors are either highly toxic to humans or have poor antibacterial effects. Summary of the Invention

[0004] In view of this, an object of the present invention is to provide use of a compound in preparing a product for preventing and / or treating a disease.

[0005] In a first aspect, the present invention provides a compound represented by formula (I), a derivative thereof, or a pharmaceutically acceptable salt thereof;

[0006]

[0007] Wherein, X is an oxygen atom, a sulfur atom, an imino group, a hydrocarbon group, or a hydrocarbon-derived group. For example, X can be a hydrocarbon-derived group obtained by replacing or modifying a hydrocarbon group with an amino group, a substituted amino group, a carboxyl group, a hydroxyl group, a thiol group, an ester group, an aryl group, a substituted aryl group, an aromatic heterocycle, or a substituted aromatic heterocycle substituent.

[0008] R1 is a polyphenyl group, a phenyl heterocyclic aromatic group, a cycloalkene group or a derivative group thereof. For example, R1 can be a derivative group formed by condensing one or more aromatic or heterocyclic aromatic groups with one or more aromatic or heterocyclic aromatic groups, and groups such as naphthyl, triphenyl, tetraphenyl, benzo polycycloalkenyl, benzo polycycloalkynyl, dibenzo polycycloalkenyl, and benzo polycycloalkynyl can be listed.

[0009] Optionally, R1 is naphthyl, anthracenyl or a benzo five-membered nitrogen-containing heterocyclic ring.

[0010] In a second aspect, the present application provides a method for preparing the above-mentioned compound or its derivative, comprising:

[0011] 1) mixing 1-naphthol, a cesium salt, a solvent and a methyl halogenated acetate to obtain a mixture;

[0012] 2) After the mixture is completely reacted, filtering and drying the filtrate to obtain a first intermediate product;

[0013] 3) mixing the first intermediate product, the alkali hydrate, and the solvent to obtain a reaction system, heating the system to a certain temperature and reacting for 10 to 60 minutes, cooling to room temperature, adjusting the pH value to 0.5 to 2, and then filtering and drying to obtain a second intermediate product;

[0014] 4) mixing the second intermediate product, the solvent, and the organic chloride under nitrogen protection until the reaction system is clear, and then drying to obtain a third intermediate product;

[0015] 5) mixing 6-aminopenicillanic acid, water, a solvent and the third intermediate, reacting at room temperature for 10 to 60 minutes, and then adjusting the pH of the system to 0.5 to 2.0 to obtain the compound or its derivative.

[0016] Optionally, in step 1), the molar ratio of 1-naphthol, cesium salt and methyl halide acetate is 1:1-10:1-10.

[0017] Optionally, in step 2), the reaction temperature is 50-100° C., and the reaction time is 0.5-3 hours.

[0018] Optionally, in step 3), the molar ratio of the first intermediate product to the alkali hydrate is 1:1-10.

[0019] Optionally, in step 3), the reaction temperature is 30-80° C., and the reaction time is 0.5-3 hours.

[0020] Optionally, in step 4), the molar ratio of the second intermediate product to the organic chloride is 1:1-10.

[0021] Optionally, in step 5), the ratio of 6-aminopenicillanic acid to the third intermediate product is 1:1-10.

[0022] Optionally, after step 5), a purification step is also included.

[0023] Optionally, the purification includes extraction, washing, filtration and drying, and recrystallization.

[0024] In another aspect, the present application also provides the use of the above-mentioned compound or its derivative or the compound prepared by the above-mentioned preparation method in the preparation of products for preventing and / or treating diseases caused by super bacteria.

[0025] The beneficial effects of the present invention are:

[0026] The compound of the present invention has high inhibitory activity against metallo-β-lactamase 1, can better inhibit metallo-β-lactamase 1, and provides a new inhibitor for the study of super bacteria-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The NMR of compound 1 1 H spectrum;

[0028] Figure 2 The NMR of compound 1 13 C spectrum;

[0029] Figure 3 is the mass spectrum of compound 1;

[0030] Figure 4 The Michaelis constants for the hydrolysis of five substrates by NDM-1 are shown;

[0031] Figure 5 The catalytic constants for the hydrolysis of five substrates by NDM-1 are shown in Figure 2.

[0032] Figure 6 The results of the antibacterial experiment. DETAILED DESCRIPTION

[0033] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0034] 1. Synthesis of compounds

[0035] 1) 4 g of 1-naphthol and 14 g of cesium carbonate were placed in a two-necked flask, and then 60 mL of acetone and 40 mL of methyl bromoacetate were added to the two-necked flask to obtain a mixture;

[0036] 2) reacting the mixture at 55° C. for 2 h, then filtering and drying the mixture to obtain a first intermediate product;

[0037] 3) Then, 1 g of the first intermediate product and 6 g of lithium hydroxide monohydrate were placed in another two-necked flask, 40 mL of methanol and 10 mL of water were added to the two-necked flask, and the mixture was reacted at 60° C. for 50 min, then cooled to room temperature, and the pH of the system was adjusted to 1.3. The mixture was then filtered and dried to obtain a second intermediate product;

[0038] 4) 2 g of the second intermediate product was placed in another two-necked flask, 30 mL of DCM and 2 mL of DMF were added under nitrogen, and 6 mL of oxalyl chloride was slowly added dropwise to the reaction system until the reaction system was clear, and then the reaction was dried to obtain the third intermediate product;

[0039] 5) 1 g of 6-APA was placed in another two-necked flask, and 15 mL of water and 5 mL of acetone were added to obtain a mixed solution. 2 g of the third intermediate product was dissolved in 10 mL of acetone to obtain an acetone solution. The acetone solution was slowly added dropwise to the mixed solution reaction system under an ice bath. After reacting for 60 min, the pH was adjusted to 1.0. The mixture was then extracted with EA, washed with 20 wt% NaCl solution, and the organic phase was filtered through diatomaceous earth, spin-dried, and recrystallized from PE-acetone to obtain the target product, i.e., compound 1.

[0040] Compound 1 was subjected to NMR 1 H spectroscopy and NMR 13 C spectrum detection, the results are as follows Figure 1-2 shown.

[0041] Depend on Figure 1-2 It can be seen that the synthesized substance is our target compound.

[0042] Compound 1 (the structure of compound 1 is shown below) was subjected to mass spectrometry detection, and the results were as follows: Figure 3 shown.

[0043] Depend on Figure 1-3 It can be seen that the synthesized substance has high purity and is the target compound.

[0044]

[0045] 2. Enzyme kinetics test

[0046] NDM-1 was prepared into a 500 nmol / L solution. NDM-1 and compound 1 were mixed at a molar ratio of 1:20,000. The reaction rate of antibiotic hydrolysis was monitored using a multifunctional microplate reader. The substrate concentration [s] was used as the abscissa and the initial reaction rate v0 of the enzymatic reaction was used as the ordinate. The Michaelis-Menten equation was fitted to obtain the following equation:

[0047]

[0048] The Michaelis constant K is calculated by the Michaelis equation mWith the maximum reaction rate V max , k cat By V max The enzyme concentration [E] is:

[0049] At the same time, single variable experiments were carried out with ampicillin (Amp), penicillin G (PG), penicillin V (PV) and oxacillin (Oxacillin) as controls, that is, except that compound 1 was replaced by ampicillin (Amp), penicillin G (PG), penicillin V (PV) and oxacillin (Oxa), other experimental conditions were the same.

[0050] The results of enzyme kinetic test were as follows Figure 4-5 shown.

[0051] Oxacillin is not a good substrate for metallo-β-lactamases. Figure 4-5 It can be seen that when compound 1 is not considered, penicillin V has the highest affinity for NDM-1 and the smallest catalytic constant. After the R1 group of penicillin V is modified to naphthyl to obtain compound 1, the enzyme kinetic parameter data show that the affinity of compound 1 for NDM-1 is greatly improved, and its catalytic constant is significantly reduced, K m With k cat The decreases were statistically significant. These results suggest that modifying the phenyl group in the antibiotic R1 group to a more aromatic naphthyl group significantly enhances the interaction between the substrate and NDM-1, increasing its affinity and slowing the rate of product dissociation from the active site, resulting in a decrease in the catalytic constant. This suggests that compound 1 is a potent competitive inhibitor of NDM-1, significantly inhibiting its catalytic activity. These results provide new avenues for the design of inhibitors for metallo-β-lactamases, including NDM-1.

[0052] 3. Antibacterial test

[0053] The inhibition zone KB method was used to determine the inhibitory effect of compound 1 on the strain expressing NDM-1 protein, wherein the strain used was Escherichia coli BL21 (DE3) (purchased) containing the NDM-1 expression plasmid (containing the kanamycin resistance gene). Figure 6As shown (the figure shows the total dosage of antibiotics), the size of the inhibition zone of the blank (no substance added), kanamycin, ampicillin, and ampicillin-compound 1 (combination) on the strain was measured respectively. The inhibition zone size reflects the antibacterial effect of different groups. Among them, the working concentrations of antibiotics are as follows: ampicillin 1mg / mL, compound 1 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, kanamycin 1mg / mL. The specific operation steps are as follows:

[0054] (1) Punch the filter paper into 6 mm diameter discs with a hole punch, sterilize and dry them for later use; prepare LB solid medium, sterilize and pour it into a culture dish for later use.

[0055] (2) Ampicillin was prepared into 1 mg / mL solution, and compound 1 was prepared into 1 mg / mL solution.

[0056] 2mg / mL, 3mg / mL and 4mg / mL solutions, and kanamycin was prepared into a 1mg / mL solution.

[0057] (3) Take seven filter paper discs, and soak the No. 1 filter paper disc in 20 μL blank solution as a blank control (A); soak the No. 2 filter paper disc in 20 μL kanamycin solution to determine whether the strain has been successfully transformed with the NDM-1 plasmid (B); soak the No. 3 filter paper disc in 20 μL ampicillin solution to detect the antibacterial effect of ampicillin when used alone (C); After the No. 4-7 filter paper discs were all immersed in 20 μL ampicillin solution, they were immersed in 20 μL of different concentrations of compound 1 solution (1 mg / ml-D, 2 mg / ml-E,

[0058] 3 mg / ml-F and 4 mg / ml-G) to test whether the inhibition of NDM-1 by compound 1 is concentration-dependent. After the filter paper is immersed in the sample, it is air-dried and used for later use.

[0059] (4) Take 200 μL of the bacterial solution and spread it evenly on a Petri dish. Divide the Petri dish into seven sections and place seven pieces of filter paper in each section. Incubate the Petri dish at 37°C for 15 h.

[0060] (5) Take out the culture dish and compare the size of the inhibition zone under different conditions.

[0061] The test results are shown in Table 1 and Figure 6 As shown:

[0062] Table 1 Inhibition zone size

[0063]

[0064]

[0065] From Table 1 and Figure 6As shown, the combination of Compound 1 and ampicillin enhanced the antibacterial effect of ampicillin compared to ampicillin alone, indicating that Compound 1 can effectively inhibit the activity of NDM-1 and restore some of the antibacterial ability of ampicillin. Furthermore, the antibacterial effect of ampicillin significantly increased with increasing dosage of Compound 1, indicating that Compound 1's inhibition of NDM-1 is concentration-dependent and that Compound 1 is an effective competitive inhibitor of NDM-1.

[0066] In summary, the compound represented by formula (I) has a high inhibitory activity against metallo-β-lactamase 1 and can better inhibit metallo-β-lactamase 1.

[0067] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of a compound of formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the activity of metallo-β-lactamase 1; （1）。

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