CN110143926B

CN110143926B - Compound with frostbite prevention and treatment effect and application of derivative thereof

Info

Publication number: CN110143926B
Application number: CN201910530639.3A
Authority: CN
Inventors: 赵庆春; 王男; 陆鸿远; 李想; 丁怀伟; 王健
Original assignee: General Hospital of Shenyang Military Region
Current assignee: General Hospital of Shenyang Military Region
Priority date: 2019-02-25
Filing date: 2019-06-19
Publication date: 2020-07-28
Anticipated expiration: 2039-06-19
Also published as: CN110143926A

Abstract

The invention relates to a compound with the application of preventing and treating frostbite and a derivative thereof, and also relates to the application of the compound in preparing a medicament for increasing heat production of an organism and preventing cold injury and the application in preparing a medicament for treating metabolic diseases. The invention provides a potential cold damage resistance medicament for improving the cold resistance of the body for cold region operation and fighters, and can be developed into an effective cold damage resistance medicament.

Description

Compound with frostbite prevention and treatment effect and application of derivative thereof

The technical field is as follows: the invention belongs to the field of biological pharmacy, and relates to a compound with a frostbite prevention effect and application of a derivative thereof.

Background art: china has wide alpine regions, covers northeast, northwest and northeast China, is respectively bordered with Korea, Russia, Mongolia, Pakistan and India in the east, north and west, has long border lines and is an important national defense strategic place. Frostbite occurs in large areas in troops performing military missions in alpine regions, causing a large number of non-combat derepreneurs and seriously weakening the combat capability of troops.

The invention content is as follows:

the purpose of the invention is as follows: the invention aims to provide a compound with the application of preventing and treating frostbite and a derivative thereof, and experiments prove that the compound can be used for preparing medicines for preventing and treating frostbite.

The technical scheme is as follows:

a compound with the function of preventing and treating frostbite has the general formula:

the compound with the effect of preventing and treating frostbite is preferably:

R₁is-OCH₃、-CH₃Any one of-F, -Cl or-Br.

R₂is-OCH₃、-CH₃、-F、-Cl、-Br、-OCH₂COOH、-OCONH₂、-CH2CONH₂、-OCH₂NH₂or-OCH₂CH₂NH₂Any one of them.

R₃is-OCH₂COOH、-CH₃、-F、-Cl、-Br、-OCH₂COOH、-OCONH₂、-CH₂CONH₂、-OCH₂NH₂or-OCH₂CH₂NH₂Any one of them.

The compound with the effect of preventing and treating frostbite R₁is-OCH₃，R₂is-OCH₃，R₃is-OCH₂At the time of COOH, the reaction liquid is,

the compound is:

the compound is used for preparing the medicine for increasing the heat production of the body and preventing cold injury.

The cold injury includes cold injury caused under a low temperature environment and cold injury caused at an ultra-low temperature.

The use of said compounds for the preparation of a medicament for the treatment of metabolic disorders.

The metabolic diseases include diabetes and obesity caused by metabolic problems.

The advantages and effects are as follows:

the invention provides a potential cold damage resistance medicament for improving the cold resistance of the body for cold region operation and fighters, and can be developed into an effective cold damage resistance medicament.

Description of the drawings:

FIG. 1 shows the synthesis of compound ZW290 for preventing and treating cold injury;

FIG. 2 is an amino acid sequence alignment of UCP1 and UCP 2;

FIG. 3 is a three-dimensional structure of UCP 2;

FIG. 4 is a three-dimensional structure of UCP1 modeled from a common source;

FIG. 5 shows the variation of different concentrations of ZW290 on the brown adipocyte lipid droplets;

FIG. 6 is a graph showing the lipid droplet consumption rate of ZW290 on brown adipocytes at different concentrations and different times;

FIG. 7 shows primary pre-brown adipocytes and their differentiated and matured morphology; a: 1d after primary pre-brown adipocytes are attached to the wall; b: cell growth and fusion; c: inducing and differentiating 8d cells to mature; d: oil red O staining after cell maturation;

FIG. 8 is a graph of the effect of different concentrations of ZW290 on mitochondrial membrane potential changes (400 ×);

FIG. 9 is a graph of the effect of different concentrations of ZW290 on ATP content in primary brown adipocytes;

FIG. 10 is a graph of the effect of ZW290 on UCP1 protein expression in brown adipocytes;

FIG. 11 is a graph of the effect of ZW290 on PGC1- α protein expression in brown adipocytes;

FIG. 12 is a graph of the effect of ZW290 on PRDM16 protein expression in brown adipocytes;

FIG. 13 is a graph of the effect of ZW290 on PPAR α protein expression in brown adipocytes;

FIG. 14 is a graph of the effect of ZW290 on HS L protein expression in brown adipocytes;

FIG. 15 is a graph of the effect of ZW290 on the anal temperature of mice at various times after administration at room temperature;

FIG. 16 is a graph showing the effect of ZW290 on the anal temperature of mice when administered for 90min at room temperature;

FIG. 17 is a graph showing the effect of ZW290 on the anal temperature of mice when administered at room temperature for 120 min;

FIG. 18 is the body surface temperature changes of Kunming mice in each group at 120min after 7 days of administration;

FIG. 19 is a core body temperature zone area statistic;

FIG. 20 is a graph of anal temperature changes at different times in various groups of Kunming mice after cold exposure;

FIG. 21 is a graph showing the change in body surface temperature of various groups of Kunming mice after 7 days of administration and after 120min of cold exposure;

FIG. 22 is a core body temperature zone area statistic;

FIG. 23 is a graph of the effect of varying doses of ZW290 compound on the size of fat droplets in brown adipose tissue (400 ×);

FIG. 24 is a graph of the effect of varying doses of ZW290 compound on mitochondrial content and UCP1 expression in brown adipose tissue (400 ×);

FIG. 25 is a graph of the effect of different doses of ZW290 on the expression level of UCP1 protein in brown adipose tissue;

FIG. 26 is a graph of the effect of different doses of ZW290 compound on the size of white adipose tissue droplets (400 ×);

FIG. 27 is a graph of the effect of different doses of ZW290 compound on the amount of UCP1 expression in white adipose tissue (400 ×);

figure 28 is a graph of the effect of different doses of ZW290 compound on survival of cold-exposed mice.

The specific implementation mode is as follows:

the invention provides an effect of ZW-290 in exciting UCP1 to increase body thermogenesis, aiming at developing ZW-290 to obtain a novel medicament for better increasing body thermogenesis and preventing cold injury.

R₁is-OCH₃、-CH₃Any one of-F, -Cl or-Br.

the compound is:

it is called as: (Z) -2- (2-methoxy-4 ((1- (4-methoxyphenyl) -5-oxo-2-thiohydantoin-4-methylene) methyl) phenoxy) acetic acid, abbreviated as ZW290 in the present invention.

The compound ZW290 with the effect of preventing and treating frostbite is synthesized in the following specific mode:

synthesis of Compound 2 in FIG. 1 Aniline (1eq) was added slowly to a stirred ethyl acetate solution at room temperature followed by carbon disulfide (10eq) and sodium bicarbonate (1eq) and stirred at room temperature for 1 hour (until a yellow solid was obtained.) Thiocarbin formation was monitored by T L C to which CoCl was added slowly₂·6H₂O (25 mol%), sodium bicarbonate (1eq) was then added slowly and the reaction mixture was stirred for 1 hour during which time a lumpy color precipitate was observed and precipitated at the bottom of the round bottom flask study the progress of the reaction by T L C after completion of the reaction, the reaction was mixedThe mixture was transferred to a centrifuge tube and the mixture was centrifuged for 10 minutes using a centrifuge. The bulk solids settled at the bottom of the centrifuge tube. The clear solution was concentrated by using a rotary evaporator and the crude mixture was purified by column chromatography on silica gel (60-120 mesh) using 2% ethyl acetate/hexane as eluent. "eq" represents a molar ratio; "mol%" is the percentage content in moles, CoCl₂·6H₂O (25 mol%) represents CoCl₂·6H₂O accounts for 25% of the total material.

Compound 4 in figure 1 was synthesized by adding anhydrous potassium carbonate (1.3eq) to a solution of vanillin (1eq) in anhydrous acetone at room temperature for 10 minutes, then adding ethyl acetate (1.5eq), refluxing the mixture and monitoring by T L C until complete, after cooling, removing the solvent under reduced pressure, adding water to the residue, then extracting with dichloromethane and washing with brine, drying the combined organic extracts with magnesium sulfate, filtering and concentrating in vacuo, adding ethanol or isopropanol and evaporating by azeotropic distillation to remove ethyl bromoacetate in excess, evaporating the solvent to dryness to give a residue which is chromatographed on a silica gel column (dichloromethane/ethyl acetate as eluent).

The synthesis of compound 6 in figure 1: compound 4(1eq) glycine (1eq) vanillin (1eq) was dissolved in glacial acetic acid solution and refluxed for 5 h. Filtering out the precipitate to obtain a compound 5, and hydrolyzing to obtain a compound 6. Obtaining ZW290 compound in the application.

The invention adopts computer-aided drug design technology to carry out homologous modeling on UCP1, as shown in figure 1, and virtual screening is carried out on the basis of the homologous modeling. And then, the screened candidate compound is subjected to cold injury resistance mechanism research through in vivo and in vitro experiments, and a theoretical basis is laid for developing cold injury resistance medicines.

According to the invention, primary pre-brown adipocytes are adopted to pass through JC-1 and ATP content determination kits, WesternBlot and other experiments to prove that ZW290 and the general formula (1) can convert energy into heat by inhibiting ATP synthesis, and the heat production capacity of BAT is improved by improving the expression level of UCP1 and channel protein. Furthermore, ZW290 and general formula (1) can promote BAT to generate heat and improve the cold-resisting capability of the body by increasing the mitochondrial content in BAT and WAT and increasing the expression level of UCP1 protein.

The present invention is further illustrated in detail by the following examples.

Example 1: research on in-vitro promotion of cell energy consumption to generate heat by ZW290

1) Instruments and reagents: DMEM medium (Hyclone); fetal bovine serum (Hyclone corporation); model CKX31 inverted microscope (philippine olympus); constant temperature CO₂Incubator (Thermo corporation, usa); 5810R model desk top high speed cryogenic centrifuge (Eppendorf, Germany).

2) Cell culture, the preadipocytes obtained by primary culture grow in DMEM complete culture medium containing 10% FBS, when the fusion of monolayers is close (the confluence degree is more than 90%), the preadipocytes are changed into induced differentiation solution I (0.5 mmol/L IBMX, 1 mu mol/L dexamethasone and 10 mu g/m L insulin are added into the DMEM complete culture medium), the culture is recorded as induced differentiation day 0, the culture is continued, the culture solution is changed into induced differentiation solution II (10 mu g/m L insulin is added into the DMEM complete culture medium) after induced differentiation day 2, namely 48 hours, the culture is continued, the DMEM culture solution is changed into 1 time every 2 days, the cell morphology change is observed under a microscope, and the culture is carried out in an incubator at 37 ℃ (5% CO₂ Relative humidity 90%), after the cells are induced to differentiate and mature, the next experiment is carried out.

3) Oil red O staining, namely after primary pre-brown adipocytes are induced to differentiate and mature, respectively adding ZW290 with corresponding concentration and t-RA with concentration of 100 mu mol/L, removing original culture solution after 48 hours, adding 1m L10% neutral formaldehyde solution into each hole to fix cells, adding oil red O application solution into the fixed cells for staining at room temperature for about 1 hour, adding 1m L isopropanol into each hole to extract oil red, oscillating for 10 minutes in a micro-oscillator, measuring an OD (recorded as an absorbance A value) at the position of 570nm of an enzyme-linked immunosorbent detector, calculating the lipid droplet consumption rate (the lipid droplet consumption rate is 1-A sample/A control), and repeating the experiment for 3 times.

4) Mitochondrial membrane potential and ATP content determination, namely respectively adding ZW290, t-RA and C L with corresponding concentrations after primary pre-brown adipocytes induce differentiation and maturation _-316243 the experiment was carried out 48h after the action, the specific experimental procedures were carried out according to the instructions of the mitochondrial membrane potential detection kit (JC-1) and the ATP content detection kit of Biyunshi biology Inc. the results show that t-RA positive drug group and C L-316243 positive drug group can significantly reduce the ATP content in primary brown adipocytes and that different administration doses reduce the ATP content in cells in a concentration-dependent manner, compared with the control group, the results show that ZW290 can convert energy into heat by limiting the generation of ATP in cells, and the specific results are shown in FIGS. 7-9.

5) UCP1 and its channel protein content determination by BCA method, determining UCP1, PGC1- α, PRDM16, PPAR α and HS L protein content in mature brown fat cell, and under alkaline condition, determining protein and Cu²⁺Bonding and reducing it to Cu⁺. BCA and Cu²⁺ZW290 can increase the protein expression of UCP1, PGC1- α, PRDM16, PPAR α and HS L in primary brown adipocytes, and has concentration dependence with the best effect at a concentration of 100. mu. mol/L. the results demonstrate that ZW290 can increase the heat production capacity of BAT by increasing the expression levels of UCP1 and pathway proteins in BAT, specific results are shown in FIGS. 10-14.

The experimental phenomena and results are briefly described below with reference to the accompanying drawings:

from fig. 5 and fig. 6, it can be seen that the number of lipid droplets in different concentrations of ZW290 is significantly less than that of the control group, and after 24h, 48h, and 72h of the treatment, the ZW290 is dose-dependent on the lipid droplets in the primary brown fat, and the consumption of the lipid droplets is significantly increased by 50 μmol/L and 100 μmol/L ZW290, and has statistical significance ". P <0.5,". P <0.01, ". P <0.001, which represents significant statistical significance between the two groups.

As shown in FIG. 7, in the process of primary pre-brown adipocytes culture, after 1d, cells adhere to the wall, gradually grow and fuse, and are induced to differentiate into mature cells, which are then stained with oil red to prove the maturation of the cells.

FIG. 8 shows that ZW290 lowers mitochondrial membrane potential in a concentration-dependent manner, and that T-RA and C L as positive drugs are used in cases where ZW 290100. mu. mol/L _-316243 the results are comparable.

As shown in FIG. 9, the t-RA-positive drug group, C L, was found to be present in comparison with the control group _-316243 the medicine group can significantly reduce ATP content in primary brown fat cells, and the ATP content in the cells can be reduced in a concentration-dependent manner by different administration doses. Wherein "ns" represents no statistical difference between the two groups, indicating that the administered group has a comparable result to the positive group.

As shown in FIGS. 10-14, ZW290 can increase the protein expression of UCP1, PGC1- α, PRDM16, PPAR α and HS L in primary brown adipocytes, and has concentration dependence, and the concentration is 100 mu mol/L, which indicates that ZW290 can improve the thermogenic ability of BAT by increasing the expression level of UCP1 and channel proteins in BAT.

Example 2: protective effect of ZW290 on cold injury of mice under cold exposure condition

1) Instruments and reagents: all-trans retinoic acid (Dalian Meiren Biotechnology Ltd., purity)>98％)；CL_-316243 (Sigma, USA, purity)>98%); a compound: ZINC 04660290 (SpeCS, Netherlands, purity)>95%) tissue mitochondrial protein extraction kit (C3606, Shanghai Biyuntian biotechnology, Inc.), mouse anal thermometer (Beijing east west instruments science and technology, Inc.), digital medical thermal infrared imager (MTI-X7 PRO-2013-B, Chongqing Yuanhao medical science and technology, Inc.), paraffin slicer (RM2145, Germany L eica), constant temperature experimental animal refrigerator (RXZ-0250, Haier, Inc.).

2) Experimental animal grouping and administration method comprises dividing Kunming mice 56 into 7 groups according to body weight, wherein the 7 groups comprise blank control group (0.2m L/10 g), model control group (0.2m L/10 g), all-trans retinoic acid group (100mg/kg), and C L _-316243 groups (1mg/kg), ZW 29025 mg/kg, ZW29050mg/kg and ZW 290100 mg/kg, 8 mice in each group were subjected to acclimation for 5 days, and then administered with a gavage of 0.2m L/10 g per day, and the placebo group and the model control group were each administered with an equal volume of physiological saline for 7 days.

3) Acute cold exposure experiment: each experimental group was given the corresponding drug treatment once daily for 7 days. And 8d, respectively and singly putting the mice of each administration group into a single mouse cage, keeping the mouse cage dry, and avoiding the interference of other factors on the body temperature as much as possible without putting padding, mouse grains and water. Placing the mouse cage filled with a single mouse into a constant temperature experiment refrigerator at-20 ℃, taking out the mouse cage after 25min, observing the condition of the mouse, measuring and recording the anal temperature of the mouse taken out at different times (15min, 30min, 45min, 60min, 75min and 90 min).

4) Detecting mitochondria and related protein expression in brown adipose tissues by an HE staining method and an immunohistochemical method: and (5) fixing the specimen for 24h, taking the specimen and trimming the specimen. Mouse brown adipose tissue sections, HE staining and immunohistochemistry were then performed.

5) Examination of survival rates of cold-exposed mice: aiming at more intuitively investigating the protection effect of ZW290 on the cold injury of the mouse, the invention further investigates the influence of different doses of ZW290 on the survival rate of the mouse under the cold condition through a long-term cold exposure experiment and discusses the protection effect of the ZW290 on the cold injury. And respectively placing the model control group and the mice of each group to be administrated in a constant temperature experiment refrigerator (single cage and single mouse without padding) at the temperature of-20 ℃ 24 hours after the last administration, observing whether the mice die every 5 minutes after the mice are placed in the refrigerator for 60 minutes, and recording the survival time of the mice of each group.

As a result: compared with a control group and a model group, ZW290 can obviously improve the temperature of the anus and the body surface of the mouse under the room temperature condition and after cold exposure, and obviously improve the survival rate of the mouse after long-term cold exposure. In addition, UCP1 and mitochondrial content were significantly increased in BAT (brown adipose tissue) and WAT (white adipose tissue). Specific results are shown in fig. 15 to 28.

from fig. 15 to fig. 17, it can be seen that the anal temperature of mice in the dose groups of ZW 29025 mg/kg, ZW29050mg/kg and ZW 290100 mg/kg increased from 90min to 120min, and the anal temperature of mice in the dose group of 100mg/kg increased significantly compared to the blank control group. Normothermia then begins to recover. The results show that: under the condition of room temperature, ZW290 can raise the anal temperature of the mouse.

As can be seen from FIGS. 18 and 19, ZW290 increased the body surface temperature of the mice 120min after administration at room temperature. The results show that: ZW290 can increase the body's ability to withstand cold by increasing the body surface temperature of the mouse at room temperature.

It can be seen from fig. 20 that the mice in the ZW 29025 mg/kg dose group, ZW29050mg/kg dose group, and ZW 290100 mg/kg dose group recovered normal anal temperature levels more rapidly after cold exposure for 25min than in the model group. The results show that: ZW290 can improve the anal temperature level of the mouse in a cold exposure environment, so that the cold resistance of the mouse is improved.

As can be seen from FIGS. 21 and 22, the ZW 29025 mg/kg dose group, the ZW29050mg/kg dose group, and the ZW 290100 mg/kg dose group were able to maintain the body surface temperature of the mice in the cold exposure environment, as compared with the model group. The ZW 290100 mg/kg dose group was most significant. The results show that: ZW290 increases the body's ability to withstand cold by increasing the body surface temperature of the mouse under cold exposure conditions.

As shown in FIG. 23, the volume of lipid droplets was decreased in the model group mice, and the t-RA positive drug group and C L were found to be smaller than those in the blank control group mice _-316243 mice in the positive drug group, ZW 29025 mg/kg dose group, ZW29050mg/kg dose group and ZW 290100 mg/kg dose group all had a different degree of reduction in lipid droplets. And ZW290 group dose-dependently reduced lipid droplets. And (4) prompting by a result: ZW290 decreased lipid droplet content and increased mitochondrial content in brown adipose tissue, indicating that mice may be converted to calories by consuming lipid droplets from brown adipose tissue after administration.

As can be seen from fig. 24 and 25, the expression of UCP1 was significantly increased in the acute cold-exposed mice and the administered group of brown adipose tissues, and was dose-dependent, as compared to the blank group. The results show that: compared with the blank group, the expression of UCP1 in the acute cold-exposed mice and the administered group of brown adipose tissues was significantly increased and dose-dependent. Immunohistochemical results further confirmed the above results, suggesting that ZW290 may increase the protein expression level of UCP1 to improve thermogenic ability of BAT.

As can be seen from fig. 26 and 27, the expression of UCP1 was significantly increased in the acute cold-exposed mice and the administered group of brown adipose tissues, as compared to the blank group. The results show that: ZW290 may reduce lipid droplet size and increase UCP1 protein expression levels in white adipose tissue. It is shown that ZW290 can promote the browning of white fat, thereby increasing the heat production of the mouse body.

As can be seen from FIG. 28, the mice in the model group died from 75min after being placed in the refrigerator to 90min, and the mice in the t-RA group died from 70min to 100min, C L _-316243 and ZW290 can prolong the survival time of mice to different extent, and the mice die at all times from ZW 290100 mg/kg dose to 145 min. The results show that: ZW290 may extend the survival time of cold-exposed mice.

To summarize: ZW290 and the structural general formula thereof can promote cell consumption lipid drop, reduce ATP generation and further play a role in increasing heat production, and ZW290 and the structural general formula thereof can obviously improve the anal temperature and the body surface temperature of the mouse after cold exposure, and obviously improve the survival rate of the mouse after long-term cold exposure. In addition, ZW290 and its general structure can increase the content of mitochondria and UCP1 in BAT and WAT, thereby improving the heat production ability of BAT and enhancing the cold resistance ability of organism.

Claims

1. The application of the compound with the function of preventing and treating frostbite is characterized in that:

the compound is:

the compound is used for preparing medicines for increasing body heat production and preventing cold injury.

2. The use of a compound having a frostbite control effect according to claim 1, wherein: the cold injury includes cold injury caused under a low temperature environment and cold injury caused at an ultra-low temperature.