CN118340765B

CN118340765B - Application of benzamide compound in preparation of wound healing promoting medicine

Info

Publication number: CN118340765B
Application number: CN202410532596.3A
Authority: CN
Inventors: 钱朝南; 陈金东; 黄婕; 周红娟; 刘倚秀
Original assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Current assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Priority date: 2024-04-30
Filing date: 2024-04-30
Publication date: 2024-08-23
Anticipated expiration: 2044-04-30
Also published as: CN118340765A

Abstract

The invention relates to application of benzamide compounds in preparing medicaments for promoting wound healing. The benzamide compound can obviously improve the movement capacity of cells at the concentration lower than 2 mu M, promote the migration and invasion of cells, accelerate the wound healing, particularly reduce the wound healing capacity of diabetic mice by the administration concentration lower than 1mg/kg, reduce the chronic wound healing time, and be widely applicable to the healing of wounds of trauma, burns, ulcers, dermatitis, chronic wounds, childbirth and diabetic patients.

Description

Application of benzamide compound in preparation of wound healing promoting medicine

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of benzamide compounds in preparing medicaments for promoting wound healing.

Background

The skin is the first important defense line of human body lower than external invasion, contains a large amount of nutrients such as protein, fat, moisture and the like, participates in metabolism of human body, has the functions of immunity, resisting invasion of external pathogens, regulating body temperature and the like, has a protective effect, needs to take timely treatment measures when the skin is wounded, promotes wound healing, has slow wound healing process if no intervention is performed, is very likely to produce serious conditions such as inflammation, ischemia or necrosis and the like, and can cause abnormal functions such as scars, pigmentation, ulcers and the like. However, wound repair is complex and typically requires 4 phases: fibrotic clot formation, inflammatory response, revascularization and connective tissue remodeling, of which revascularization is critical, is an abnormally difficult stage of revascularization for healing of chronic wounds, such as very slow revascularization in wounds of diabetics, where the vascular surface area, branch connection number, total vascular length and total branch number are all significantly reduced, and angiogenesis can effectively support wound closure, while vascular lesions are one of the causes of difficult healing of wounds of diabetics. For healing common skin wounds, especially chronic wounds, traditional skin healing methods such as laser, treatment auxiliary materials, negative pressure treatment, skin transplantation and the like are not ideal in effect, and have limitation in clinical application. Therefore, development of new drugs for accelerating wound healing, especially chronic wound healing for diabetics and the like, is a highly-needed problem. At present, for the medicaments for wound healing, including isoquinoline, flavonoid, alkaloid, some Chinese herbal medicines and the like, chinese patent CN104478780A discloses an acyl pyrrole small molecular organic compound, derivatives, application and preparation methods thereof, the compound can be used for promoting angiogenesis, including promoting wound healing, diabetic foot and cardiovascular diseases, but the compound has relatively high dosage in wound healing, even up to 50 mu M, to produce the effect of promoting wound healing, and has poor effect; chinese patent CN109646424a discloses a phenolic compound which achieves a better cell migration promoting ability at a concentration of 20 μm, and which is administered at a relatively high concentration. In summary, delayed or no wound healing and the healing process are persistent clinical problems, and the development difficulty of new compounds for promoting wound healing is high, and the activity of wound healing is still low, so that development of new compounds for promoting wound healing, especially compounds for promoting rapid healing of chronic wounds, is still required.

Disclosure of Invention

Aiming at the prior art problems, the invention provides application of benzamide compounds in preparing medicaments for promoting wound healing, and the benzamide compounds can remarkably improve migration and invasion capacities of cells and can be used for promoting wound healing.

In a first aspect, the invention provides application of benzamide compounds in preparing medicaments for promoting wound healing, wherein the benzamide compounds are compounds with a molecular structural formula shown in (I) or geometric isomers thereof or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof;

（I）；

further, the medicine for promoting wound healing also comprises pharmaceutically acceptable pharmaceutical excipients.

Further, the dosage form of the wound healing promoting medicine includes, but is not limited to, suspension, granule, capsule, powder, tablet, emulsion, solution, drop pill, injection, aerosol, gel, patch, drop or liniment.

Further, the wound healing promoting pharmaceutical indications include, but are not limited to, trauma, burns, ulcers, dermatitis, chronic wounds, childbirth, or diabetic wounds.

In a second aspect, the invention provides a preparation method of the benzamide compound, the molecular structural formula of the benzamide compound is shown as a formula (I),

（I）

The preparation method comprises the following steps: dissolving 1, 4-benzodioxane-6-carboxylic acid in N, N-dimethylformamide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, aniline and N, N-diisopropylethylamine, mixing for reaction, adding ethyl acetate and water for extraction after the reaction is completed, drying an organic phase, filtering, concentrating, adding an organic solvent for pulping, and filtering to obtain a compound shown in a formula (I);

the synthetic route of the benzamide compound structural formula (I) is as follows:

Further, the feeding mole ratio of the 1, 4-benzodioxane-6-carboxylic acid to the aniline is 1:0.8-1.2.

Preferably, the feeding mole ratio of the 1, 4-benzodioxane-6-carboxylic acid to the aniline is 1:1.1.

Further, the reaction temperature is between 0 and 25 ℃.

Further, the organic solvent is selected from any one of petroleum ether, acetone, n-butane, isopropanol, acetonitrile, methanol and ethanol.

Preferably, the organic solvent is selected from petroleum ether.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention provides an application of a benzamide compound in preparing a medicine for promoting wound healing, which can obviously improve the movement capacity of human foreskin fibroblasts HFF-1 and mouse embryo fibroblasts 3T3 under the concentration lower than 2 mu M, improve the migration and invasion penetration capacity of cells, and simultaneously has the administration concentration of 1mg/kg for wound healing of normal mice and the administration concentration of 1mg/kg for wound healing of diabetic mice, so that the benzamide compound can obviously improve the wound healing capacity of the diabetic mice, reduce the chronic wound healing time, and can be widely applied to wound healing of wounds, burns, ulcers, dermatitis, chronic wounds, delivery and diabetic patients.

(2) The invention provides a synthesis way for developing the benzamide compound, which has the beneficial effect that the yield of the benzamide compound reaches more than 39%.

Drawings

FIG. 1 is a ¹ H NMR nuclear magnetic spectrum of a benzamide compound of the present invention.

FIG. 2 is a graph of EH-P002A migration through mouse embryonic fibroblasts 3T 3.

FIG. 3 is a data statistic of EH-P002A on mouse embryonic fibroblast 3T3 migration.

FIG. 4 is a graph of EH-P002A migration through human foreskin fibroblasts HFF-1.

FIG. 5 is a statistical data of EH-P002A migration of human foreskin fibroblasts HFF-1.

FIG. 6 is a graph of EH-P002A invasiveness of mouse embryonic fibroblasts 3T3, wherein A is Control, B is EH-P002A-1. Mu.M, C is EH-P002A-2. Mu.M, and D is EH-P002A-5. Mu.M.

FIG. 7 is a data statistical result of EH-P002A invasion of mouse embryonic fibroblasts 3T 3.

FIG. 8 is a graph of EH-P002A invasiveness of human foreskin fibroblasts HFF-1, wherein A is Control, B is EH-P002A-1. Mu.M, C is EH-P002A-2. Mu.M, and D is EH-P002A-5. Mu.M.

FIG. 9 is a statistical data of EH-P002A invasion of human foreskin fibroblasts HFF-1.

FIG. 10 is a physical graph showing the effect of EH-P002A on wound healing in normal mice.

FIG. 11 is statistical analysis of the effect of EH-P002A on wound healing in normal mice on day 7 and day 9, respectively.

FIG. 12 is a physical graph of the effect of EH-P002A on wound healing in diabetic mice.

FIG. 13 is statistical analysis of the effect of EH-P002A on wound healing in diabetic mice on day 5.

Detailed Description

The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of benzamide Compounds

The synthesis route of the benzamide compound of the invention is as follows:

the specific synthesis steps are as follows:

1.8g of 1, 4-benzodioxane-6-carboxylic acid is dissolved in 20mL of DMF, 2.3g of EDCI, 1mL of aniline and 2mL of DIPEA are added and mixed with stirring, reaction is carried out for 5h at 0 ℃, 50mL of ethyl acetate and 50mL of water are added for extraction after the reaction is finished, the extracted organic phase is added into saturated sodium chloride solution for washing, anhydrous sodium sulfate is dried, then filtration is carried out, 20mL of petroleum ether is added when the liquid is concentrated to 2mL, pulping and then filtration are carried out, thus obtaining benzamide compound (I) which is marked as EH-P002A, and the yield is 39.2%.

（I）

The nuclear magnetic spectrum of the synthesized benzamide compound (I) is shown in the attached figure 1, wherein ¹ H NMR data are as follows:

¹H NMR (400 MHz, Chloroform-d) δ 7.69 (s, 1H), 7.64-7.58 (m, 2H), 7.42 (d, J = 2.2 Hz, 1H), 7.39-7.32 (m, 3H), 7.17-7.10 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.31 (tdd, J = 5.5, 2.7, 1.1 Hz, 4H).

EXAMPLE 2 Effect of benzamide Compounds on 3T3 cell, HFF-1 cell migration

(1) The experimental object: mouse embryonic fibroblast 3T3, human foreskin fibroblast HFF-1;

(2) Experimental drugs: EH-P002A, solvent DMSO (dimethyl sulfoxide);

(3) The experimental method comprises the following steps:

before plating, a marker pen thin head is used to uniformly draw a transverse line behind a 6-hole plate, a straight ruler is used to compare, a transverse line is approximately drawn every 0.5 cm to 1 cm, a through hole is traversed, three lines are generally drawn, the line is sequentially named as a line, b line and c line, the line b crosses the right center, the other two lines are equally spaced and drawn on two sides of the line b, six-hole plate is plated, 2mL complete culture mediums containing 10% FBS (fetal bovine serum) are added into each hole, 2 x10 ⁶ cells are respectively arranged, 2 compound holes are respectively arranged, about 24h cells are cultivated, the number of the cells is suitably regulated so that the cells can be fully covered by over 90% by overnight adherence, the cover of the hole plate is opened the next day, old culture mediums are sucked off, the ruler is perpendicular to a b-line frame and is placed on an orifice plate, a 200 uL gun head is tightly attached to the ruler to uniformly move up and down to manufacture a cell scratch line, two parallel lines are drawn on two sides of the line at equal intervals, the two parallel lines are named line 1, line 2 and line 3 from left to right, cells are washed 3 times by sterile 1 XPBS (phosphate buffer solution diluted to 1 time), after the drawn cells are removed, a complete culture medium containing 20% FBS (fetal bovine serum) is added, a benzamide compound EH-P002A (working concentration is 2 mu M) is added, the culture is carried out in a culture box with the temperature of 37 ℃, and the culture box with the concentration of 5% CO ₂, and the culture is sampled and photographed according to dosing for 0h and 36 h.

The cell scratch test is carried out according to the test method, the obtained results are shown in fig. 2 to 5, and the test results show that: compared with Control (DMSO solution), the migration speed of 2 mu M of EH-P002A treated mouse embryo fibroblast 3T3 and human foreskin fibroblast HFF-1 is obviously accelerated, which proves that the EH-P002A can effectively promote cell migration and movement under very low concentration, and the result has obvious difference (P < 0.0001).

EXAMPLE 3 Effect of benzamide Compounds on 3T3 cell invasion and HFF-1 cell invasion

(2) Experimental drugs: EH-P002A, solvent DMSO (dimethyl sulfoxide);

(3) The experimental method comprises the following steps:

Preparing a cell suspension: digesting cells, centrifuging after stopping digestion, discarding culture solution, washing with PBS (phosphate buffer solution) for 1 time, re-suspending with serum-free culture medium, and adjusting cell density to proper concentration (the plate densities of mouse embryo fibroblast 3T3 and human foreskin fibroblast HFF-1 are 4×10 ⁵/mL);

Inoculating cells: each cell is provided with a negative control group (DMSO with the same dilution ratio as EH-P002A is added), a dosing group (working concentration is respectively 1 mu M, 2 mu M and 5 mu M), each group is provided with 2 compound holes, a proper amount of cell suspension is taken according to cell density, a proper amount of 10% BSA (bovine serum albumin) with the final percentage of 0.1% is added, benzamide compound EH-P002A is added, and finally DMEM (liquid culture medium and high sugar type) is used for supplementing, so that the total volume of each hole is 200 mu L, a Transwell upper chamber paved with glue is gently and evenly added, and 800 mu L of culture medium containing 20% FBS (fetal bovine serum) is generally added in a 24-pore plate lower chamber immediately, and a plate is harvested after 24 hours of dosing;

Cell staining: taking out a Transwell chamber, discarding culture solution in the hole, lightly wiping off cells and gel which are not migrated from the upper chamber by using a cotton swab, placing the cells and the gel into a clean 24-well plate, washing 1 time by using 1 XPBS (phosphate buffer solution diluted to 1 time), fixing the cells for 30 minutes by using methanol, sucking the methanol, placing the cells into a fume hood for air drying, dyeing the cells with 0.1% crystal violet for 20 min, sucking recovered crystal violet, washing 1 time by using PBS, sucking the PBS, and airing the fume hood;

And (3) result statistics: cells were observed under a 5X microscope and randomly in five fields under a 10X microscope, photographed, counted and statistically plotted, and specific results are shown in FIGS. 6 to 9.

The experimental results show that: compared with the control group (solvent DMSO), the number of cells which are treated by EH-P002A with the concentration of 1 mu M,2 mu M and 5 mu M and invaded and migrated through the Transwell is obviously increased, which proves that the EH-P002A can promote the invasion and penetration capacity of the cells at the extremely low concentration, and the experimental results have obvious/extremely obvious difference.

Example 4 test on mice with wound healing promotion

The experiment for promoting the wound healing of the diabetic mice comprises modeling of the diabetic mice, wound construction of the diabetic mice and healing of the wound of the diabetic mice by drug administration, and the specific experimental design is shown in table 1;

1. the specific experimental steps for promoting wound healing of normal mice are as follows:

table 1: design of experiment

(1) Mouse ordering: ordering 13-week-old, male and BALB/c mice from a laboratory animal center, and feeding for 5 days in an SPF feeding room, and performing isolation observation;

(2) Grouping: the mice were randomly divided into Control negative Control groups and EH-P002A dosing groups;

(3) Anesthesia: injecting 5% chloral hydrate into abdominal cavity for anesthesia (100 uL/20 g), placing the fully anesthetized mice on a pad towel in prone position, removing back hair with depilatory cream, sterilizing back skin with 75% alcohol, respectively making 2 circular skin full-layer wounds with diameters of about 15mm on two sides of the highest position of the back midline with a trephine with diameters of 15mm, removing subcutaneous tissues with surgical scissors and forceps, exposing muscle surface fascia, stopping bleeding and sterilizing the wounds, opening the wounds, taking a ruler as a control, photographing and recording the shape and the size of the wounds;

(4) Administration: each mouse of the control group was injected with the same volume (0.1 mL) of saline containing 10% DMSO-40% PEG 400-5% tween 80-45%; EH-P002A (1 mg/kg/d) was administered by intraperitoneal injection; mice were injected with d1, d3, d5, d7, d9, d11 on day of mice trauma;

(5) The anesthetized mice are resuscitated under the temperature control plate and transported back to the EVC cage of the clean rearing room.

The results of the wound healing promotion experiments performed on normal mice in the above manner are shown in fig. 10 and 11.

2. The specific experimental steps of modeling and wound healing promotion of the diabetic mice are as follows:

Table 2: design of experiment

(1) Mouse ordering: ordering 3-week-old male BALB/c mice from the experimental animal center, and feeding for 5 days in an SPF feeding room while performing isolation observation;

(2) Taking the starting of feeding the high-fat feed by the model-building mice as the first day of the experiment, recording the weight weekly, observing the conditions of hair, feeding and urination, removing the feed at night on the 28 th day, and taking off the food without water inhibition after fasting for overnight;

(3) Preparing Streptozotocin (STZ) injection: dissolving 20 mg STZ powder in 4 mL citric acid buffer solution to prepare injection of 5mg/mL, all operations are carried out on ice, taking care of light shielding, and STZ solution is prepared at present;

(4) Injection of STZ: the body weight and fasting blood glucose of the mice were measured the next day, and the model mice were intraperitoneally injected with STZ solution (40 mg/kg/d) once a day for 5 consecutive days, with the first day of injection continuing fasting and the second day of injection beginning to resume high fat feeding; continuously observing the feeding, drinking and urination conditions of the mice;

(5) After 2 weeks of the end of administration, respectively detecting fasting blood sugar of two groups of mice, defining the mice which are stable above 11.1 mmoL/L and are 'polydipsia, polyuria, polyphagia and emaciation' for 3 times in succession as diabetic mice which are successfully molded, taking the mice which do not meet the model standard into a subsequent experiment, taking out the experiment, and counting the molding success rate; during the experiment, the sanitary nursing of the model-making mice is paid attention to, and if the model-making mice show polydipsia and urination, drinking water needs to be supplemented in time, so that the interval for replacing padding is shortened;

(6) Grouping: randomly dividing the model-successfully-molded mice into model Control groups without drug administration and EH-P002A drug administration groups;

(7) Anesthesia: injecting 5% chloral hydrate into abdominal cavity for anesthesia (100 μl/20 g), placing the completely anesthetized mice on a pad towel in prone position, removing back hair with depilatory cream, sterilizing back skin with 75% alcohol, respectively making 2 circular skin full-layer wounds with diameters of about 15 mm on two sides of the highest position of the back midline with trephine with diameters of 15 mm, shaving subcutaneous tissues with surgical scissors and forceps, exposing muscle surface fascia, stopping bleeding and sterilizing the wounds, opening the wounds, taking a ruler as a control, photographing and recording the shape and the size of the wounds;

(8) Administration: each mouse of the model Control group without drug administration was injected with the same volume (0.1 mL) of saline containing 10% DMSO-40% PEG 400-5% tween 80-45%; EH-P002A (1 mg/kg/d) was administered by intraperitoneal injection; mice were injected with d1, d3, d5, d7, d9 on day of mice trauma;

(9) The anesthetized mice are resuscitated under the temperature control plate and transported back to the EVC cage of the clean rearing room.

The results of the wound healing promotion experiments performed on diabetic mice in the above manner are shown in fig. 12 and 13.

3. Experimental result of promoting wound healing of mice

The wound healing promotion experiments were performed on normal mice and diabetic mice, respectively, as described above, and the experimental results of fig. 10 to 13 show that: whether the normal mice are wound healed or the diabetic mice are wound healed, the wound healing speed of the mice with the EH-P002A is faster than that of the mice with the control group without the EH-P002A, wherein the wound healing of the normal mice and the diabetic mice can be obviously promoted when the EH-P002A is administered at a concentration of 1mg/kg, the experimental results have obvious difference (P < 0.01), and compared with the wound healing speed of the normal mice, the diabetic mice are faster, the benzamide compound prepared by the invention can obviously improve the wound healing capacity of the diabetic mice, reduce the chronic wound healing time, and can be widely applied to wound healing of wounds of trauma, burns, ulcers, dermatitis, chronic wounds, delivery and diabetic patients.

It should be noted that, in the present specification, specific features, structures, materials, or characteristics may be arbitrarily combined, and in order to simplify the description, all possible combinations of the features in the foregoing embodiments are not described, and those skilled in the art may combine and combine the features of the different embodiments and the different embodiments described in the present specification without contradiction.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The application of the benzamide compound in preparing the medicine for promoting the wound healing is characterized in that the benzamide compound is a compound with a molecular structural formula shown as (I) or pharmaceutically acceptable salt thereof;（I）。

2. the use of benzamide compounds of claim 1 for the preparation of a medicament for promoting wound healing, wherein the medicament further comprises pharmaceutically acceptable excipients.

3. The use of a benzamide compound according to any one of claims 1 to 2 for the preparation of a medicament for promoting wound healing, wherein the pharmaceutical dosage form comprises granules, capsules, powders, tablets, emulsions, drop pills, injections, aerosols, gels, patches or liniments.

4. The use according to any one of claims 1-2, wherein the pharmaceutical indication comprises burns, ulcers, dermatitis, childbirth or diabetic wounds.

5. The use according to claim 1, wherein the pharmaceutical concentration of the benzamide compound is 1-5 μm.