CN114224893A

CN114224893A - Application of quinazoline derivative in preparation of drug for preventing and treating arsenic-induced liver injury

Info

Publication number: CN114224893A
Application number: CN202210084671.5A
Authority: CN
Inventors: 骆衡; 杨和平; 徐必学; 程莎; 余佳
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Guizhou Natural Products Research Center
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-03-25
Anticipated expiration: 2042-01-25
Also published as: CN114224893B

Abstract

The invention provides application of a quinazoline derivative in preparation of a medicine for preventing and treating arsenic-induced liver injury, wherein the structural formula of the quinazoline derivative is shown as a formula I. The invention belongs to the technical field of medicaments, and provides application of quinazoline derivatives in preparing medicaments for preventing and treating arsenic-induced liver injury by intervening quinazoline derivatives on arsenic poisoning cell models and infected mice from in vivo and in vitro levels and discussing the prevention and protection effects of the quinazoline derivatives on arsenic-induced liver injury.

Description

Application of quinazoline derivative in preparation of drug for preventing and treating arsenic-induced liver injury

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a quinazoline derivative in preparation of a medicine for preventing and treating arsenic-induced liver injury.

Background

Arsenic (As), a naturally occurring environmental poison and carcinogen, readily enters human and animal bodies through food, water and occupational contamination, causing damage to the multiple organ system. At present, more than 2 billion people around the world face the problem of excessive arsenic content in drinking water, which causes a large amount of arsenic poisoning, and particularly, the arsenic poisoning occurs in laggard developing countries. In Guizhou province and Shaanxi province of China, more coal pollution type local arsenic poisoning phenomena exist.

The liver, as the main metabolism and detoxification organ of arsenic, can be high-concentration retained in the metabolic process, thereby causing liver diseases, such as abnormal liver function, hepatomegaly, hepatic fibrosis, cirrhosis and even liver canceration, with different degrees, and becoming one of the main causes of arsenic death. The existing research finds that: oxidative stress and lipid peroxidation are pathogenic factors of liver injury caused by arsenic, and arsenic can generate a large amount of free radicals and non-free radical products in liver metabolism to cause lipid peroxidation of liver cell membranes, so that the structures of the cell membranes and organelles are damaged, and the membrane fluidity is abnormal; a large amount of enzymes (ALT, AST) in liver cells are released into blood, free radical scavenging enzymes (SOD, GPX) are exhausted, liver cell damage is further aggravated, the activity level of Glutathione (GSH) is reduced, and the level of Malondialdehyde (MDA) is increased, so that abnormal liver function and fat metabolism disorder are caused.

Quinazoline compounds (quinazolines) are a class of heterocyclic compounds containing nitrogen, which are ubiquitous in nature. The ring structure of quinazoline is the skeleton of various alkaloids, quinazoline compounds with different structures may have different efficacies, and drugs such as dactinib, gefitinib, doxazosin and the like take quinazoline as pharmacodynamic groups and respectively have excellent efficacies of resisting tumors, resisting hypertension and the like. Quinazoline compounds have the advantages of low toxicity, high efficiency, unique action mode, easy modification and the like, and become one of the research hotspots in the fields of medicinal chemistry and pharmacology.

Quinazoline compounds play an important role in the fields of tumor resistance, hypertension resistance, malaria resistance, bacteria resistance, inflammation diminishing, tuberculosis resistance, insect killing, weeding and the like, and no report on the aspect of preventing and treating liver injury of quinazoline is found.

Disclosure of Invention

In order to enrich the prior art and expand the application range of quinazoline compounds, the invention intervenes quinazoline derivatives on arsenic poisoning cell models and infected mice from in vivo and in vitro levels on the basis of obtaining quinazoline derivatives shown in formula I through early-stage research construction and screening, discusses the prevention and protection effects of the quinazoline derivatives on arsenic-induced liver injury, and provides the application of the quinazoline derivatives in preparing drugs for preventing and treating arsenic-induced liver injury.

The objects of the invention will be further illustrated by the following detailed description.

The invention provides application of a quinazoline derivative in preparing a medicine for preventing and treating arsenic-induced liver injury, wherein the structural formula of the quinazoline derivative is shown as a formula I:

in the invention, the code number of the quinazoline derivative is KZL-047, which is further simplified to KZL in part of the attached drawings of the specification.

Preferably, the arsenic is sodium arsenite.

More preferably, the liver injury comprises: abnormal liver function, hepatomegaly, hepatic fibrosis, liver cirrhosis, and liver canceration.

Compared with the prior art, the invention has the beneficial effects that: the invention proves the efficacy of the quinazoline derivative (KZL-047) shown in the formula I in the aspect of preventing and treating arsenic-induced liver injury from in vivo and in vitro levels, and provides the application of the quinazoline derivative in preparing a medicament for preventing and treating arsenic-induced liver injury. According to the invention, by constructing an arsenic poisoning cell model and intervening KZL-047, the KZL-047 is found to be capable of relieving sodium arsenite-mediated hepatocyte growth inhibition activity and relieving sodium arsenite-induced L-02 cell apoptosis. According to the invention, a mouse model with arsenic poisoning is constructed, and KZL-047 is intervened, so that KZL-047 can be used for preventing and treating mouse liver injury caused by SA.

Drawings

FIG. 1 is a graph showing the effect of KZL-047 at various concentrations on the growth of L-02 cells.

FIG. 2 is a graph showing the effect of varying concentrations of KZL-047 on SA-treated L-02 cell proliferation. Wherein, 2-A is a result graph for detecting that SA has inhibition effect on the growth of L-02 cells in vitro by MTT test and has concentration dependence; 2-B is a morphological map of L-02 cells observed microscopically as the concentration of SA increases; 2-C is a cell proliferation influence result graph of continuously treating the L-02 cells for 48 hours by KZL-047 with different concentrations after SA with 20 mu mol/L is selected as an infection concentration to treat the L-02 cells for 24 hours; 2-D is a morphological examination chart of L-02 cells treated for 24h with SA of 20. mu. mol/L as an infecting concentration, and then cells treated for another 48h with KZL-047 of different concentrations.

FIG. 320. mu. mol/L SA and results of the effect of varying concentrations of KZL-047 on L-02 apoptosis are shown. Wherein, 3-A is a detection result graph of apoptosis level after SA and KZL-047 treatment at different concentrations; 3-B is a flow cytogram of SA-induced apoptosis at 20. mu. mol/L and of different concentrations of KZL-047 to alleviate apoptosis.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

In the present invention, the experimental material is a conventional commercially available product or can be obtained by a conventional technique in the art, the detection method used is performed according to a method commonly used in the art or a kit specification, and a DMEM high-glucose medium supplemented with fetal bovine serum is used as a culture solution for L-02 cells.

The normal human liver cell line (L-02) used in this experiment was purchased from cell banks of Chinese academy of sciences, Shanghai, China; sodium Arsenite (SA) was a standard reagent, purchased from Sigma corporation, usa; tetramethylazodicarbonyl blue (MTT) powder and dimethyl sulfoxide (DMSO), available from Solebao, Beijing; DMEM high-glucose medium, purchased from Hyclone, USA; fetal Bovine Serum (FBS) from the green house company; 96 well plates, available from Nest, usa. 140 male mice of Kunming SPF class, 8-10 weeks old, 25-30g of body weight, provided by Liaoning province experimental animal resource center, and animal license number (license number: SCXK (Liao) 2020-.

Example an MTT assay to examine the Effect of KZL-047 on L-02 cell proliferation

Respectively taking the L-02 cells with good transmission state of three generations, adjusting the cell concentration to 5 cells10³Per well and inoculated in a 96-well plate, 200. mu.L of cell culture medium was added to each well, and after incubation in an incubator at 37 ℃ for 24 hours, KZL-047 was added at different concentrations (DMSO, 1.25, 2.50, 5.0, 10.0, 20.0. mu. mol/L) in 5 wells per concentration. After continuous treatment in an incubator at 37 ℃ for 48 hours, photographs were taken with an inverted fluorescence microscope. Then, 20 μ L of MTT was added to each well, incubation was performed at 37 ℃ for 4h, centrifugation was performed at 3000rpm/min at room temperature for 30min, the liquid in the 96-well plate was aspirated by absorbent paper, 150 μ L of DMSO was added, shaking was performed at room temperature for 15min, absorbance (OD) was measured with a microplate reader under excitation light of 490nm, the measurement results are shown in FIG. 1, and are expressed as the mean. + -. SD of three independent experiments, compared to the control,^*P<0.05，^**P<0.01. the low-concentration KZL-047 can obviously promote the L-02 cell proliferation (P) after acting for 48 hours<0.05) and the high concentration boosting effect is reduced, therefore, 5.0 mu mol/L and 10.0 mu mol/L are selected for intervention in subsequent experiments.

Example II Effect of KZL-047 on SA leads to inhibition of L-02 cell proliferation and apoptosis

After SA was treated continuously at different concentrations (DMSO, 10.0, 20.0, 40.0, 80.0 and 160. mu. mol/L) for 24h according to the method of example one, absorbance (OD) was measured with a microplate reader under excitation light of 490nm, and the measurement results are shown in FIG. 2-A as the mean. + -. SD of three independent experiments, compared with the control group,^*P<0.05，^**P<0.01，^***P<0.001. different concentrations of SA treated L-02 cells for 24h, IC₅₀The value was 25.80. + -. 2.34. mu. mol/L, and the results showed that L-02 cell viability decreased with increasing SA concentration, indicating that SA can inhibit the proliferation of L-02 cells in a concentration-dependent manner, and 20. mu. mol/L was selected as the subsequent contamination and molding concentration. The change of cell morphology was observed by taking a photograph on an inverted fluorescence microscope, and as a result, as shown in FIG. 2-B, the cell morphology was rounded by fusiform and decreased in number. The subsequent experimental design was divided into 4 groups, blank (DMSO), model (20. mu. mol/L SA), and KZL-047 dry run (+ 5. mu. mol/L KZL after 20. mu. mol/L SA treatment, and + 10. mu. mol/L KZL after 20. mu. mol/LSA treatment), the culture was aspirated after 24h SA treatment, the cells were treated for 48h in KZL-047 intervention group by adding cell culture medium containing KZL-047 at the corresponding concentration, and the procedures were carried outThe results of the detection are shown in FIG. 2-C. Compared with the control group, the compound of the formula,^*P<0.05，^**P<0.01; in comparison to the set of models,^aP<0.05，^aaP<0.01. the results show that: when the SA concentration is 20 mu mol/L, the molding is successful, and the cell growth is obviously inhibited compared with a control group (P)<0.05); while the KZL-047 treatment with 5. mu. mol/L and 10. mu. mol/L increases cell growth was still inhibited to some extent compared to the control group (P)<0.05), but the degree of cell growth inhibition was significantly lower than that of the model group (P)<0.05). These results indicate that low concentrations of KZL-047 significantly reduce the inhibitory effect of SA on normal hepatocyte proliferation. Observation of cell morphology As shown in FIG. 2-D, it is clear that after treating hepatocytes with 20. mu. mol/L of SA for 24 hours, the cell morphology began to become irregular from smooth, the boundaries were blurred, and the cell gaps became larger, but after treating with 5. mu. mol/L and 10. mu. mol/L of KZL-047 for 48 hours, the cell morphology gradually recovered, the boundaries became clear, and the rounded cells were reduced. These data indicate that KZL-047 can alleviate growth inhibition of normal hepatocytes after treatment with SA at lower concentrations by 20. mu. mol/L, showing some recovery effect.

The third generation of L-02 cells was performed at 3X 10⁵The culture medium is inoculated into a 6-well plate at the concentration of/mL, 20 mu mol/L SA is added for molding culture for 24h, the original culture medium is completely sucked, KZL-047 with different concentrations is added for culture for 48h, and three parallel experiments are respectively set. The cells were harvested, washed twice with PBS, and the cell concentration was adjusted to 1X 10 with 1 × binding buffer⁶Perml, 5. mu.L FITC and 5. mu.L PI were added to the cells. Gently mixing, incubating for 15min at room temperature in the dark, collecting cells, staining with FITC and PI, and immediately detecting apoptosis by flow cytometry, the results are shown in FIG. 3-B. The potential effect of KZL-047 on relieving SA-induced L-02 apoptosis is detected by a flow cytometer, and the result is shown in figure 3-A. The research result shows that the 20 mu mol/L SA is selected to induce the L-02 apoptosis rate of the human liver cells to be 13.56% (compared with a control group, P is less than 0.01), and the L-02 apoptosis rate is respectively reduced to 8.59% (P is P) by treating the cells with 5 mu mol/L KZL-047 and 10 mu mol/L SA<0.05) and 3.81% (P)<0.01), gradually approaching the condition of untreated normal hepatocytes. In comparison to the blank set, the data is,^*P<0.05，^**P<0.01; in comparison to the set of models,^aP<0.05，^aaP<0.01. expressed as mean ± SD of three independent experiments. The results show that KZL-047 can obviously reduce the normal hepatocyte apoptosis induced by SA.

EXAMPLE three animal Experimental examination of KZL-047

Grouping and administration: adaptively feeding for 1 week (room temperature is 20-24 ℃, humidity is 50-60%, day and night circulation and free water drinking) before formal experiments, randomly and equally dividing 140 SPF male Kunming mice into two large groups for prevention and treatment, wherein each large group is provided with a blank group (physiological saline), a model group (5mg/kg SA), a positive control group (11.375mg/kg bicyclol and 182mg/kg glutathione), a KZL low, medium and high dose group (25, 50 and 100mg/kg), and 7 small groups in total; in the prevention group, a blank group is perfused with physiological saline with the same volume as the stomach, a model group is perfused with SA solution with the dosage of 5mg/kg, and the other 5 groups are separately perfused with drug with the corresponding dosage and SA solution with the dosage of 5mg/kg for 1 time every day and are perfused for 4 weeks continuously; the other groups except the blank group in the treatment group were subjected to molding treatment according to SA solution of 5mg/kg d for 4 consecutive weeks, starting from the fifth week, the model group was perfused with normal saline, and the other 5 groups were administered with the corresponding drugs for 4 weeks.

The measuring and observing method comprises the following steps: after the last administration, all mice were fasted and kept without water for 8 h; collecting blood samples of mice by an eyeball picking method, standing at room temperature for half an hour, centrifuging at 3000 r/min for 15min, collecting supernatant, measuring biochemical indexes such as ALT, AST, TBIL and the like, performing statistical treatment on the obtained data, expressing the data with the average number +/-standard deviation, judging the difference significance by a T test, and respectively showing the results in tables 1 and 2.

No death or clinical abnormalities were observed in all experimental mice. In the prophylactic and therapeutic groups, the amount of food consumed by SA-treated mice was less than in the control and quinazoline-treated mice.

TABLE 1 prevention of ALT, AST, TBIL Activity changes in serum of mice in groups

Note: comparison with blank group，^**P is less than 0.01; in comparison with the set of models,^aaP＜0.01。

TABLE 2 changes in ALT, AST, TBIL Activity in sera of treatment groups of mice

Note: in comparison with the blank set, the results,^**p is less than 0.01; in comparison with the set of models,^aaP＜0.01。

as can be seen from the results of the prevention group (Table 1) and the treatment group (Table 2), after SA treatment is performed in the experimental hour, the ALT, AST and TBIL activities of the model group are all obviously increased, and are significantly different from those of the blank group (P is less than 0.01), which indicates that the molding is successful. In the prevention group, compared with a blank group, the ALT, AST and TBIL activities of the mice in the model group are remarkably increased (P <0.01), the ALT, AST and TBIL activities of the mice in the positive control group and the KZL-047 dose group are not remarkably different (P >0.05), whereas compared with the model group, the ALT, AST and TBIL activities of the mice in the positive control group and the KZL-047 dose group are reduced (P <0.01), and the ALT, AST and TBIL activities of the mice in the KZL-047 dose group and the positive control group are not remarkably different (P >0.05) when the indexes are compared. In the treatment group (see Table 2), the trend of the activity of ALT, AST and TBIL in the serum of the mice is consistent with that of the prevention group, and the activity of the mice is dose-dependent, namely, the activity of the mice is reduced along with the increase of the KZL-047 dose. The quinazoline derivative prepared by the invention has good effects of preventing and treating liver injury, and the effect is equivalent to the activity of a positive medicament.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. The application of the quinazoline derivative in preparing the medicine for preventing and treating the liver injury caused by arsenic is characterized in that: the structural formula of the quinazoline derivative is shown as a formula I:

2. use of a quinazoline derivative according to claim 1 in the manufacture of a medicament for the treatment of liver injury, wherein: the arsenic is sodium arsenite.

3. Use of a quinazoline derivative according to claims 1 and 2 in the manufacture of a medicament for the prevention and treatment of liver damage, characterised in that: the liver injury comprises: abnormal liver function, hepatomegaly, hepatic fibrosis, liver cirrhosis, and liver canceration.