CN118271276B

CN118271276B - Naphthalenone derivative for promoting wound healing as well as preparation method and application thereof

Info

Publication number: CN118271276B
Application number: CN202410683059.9A
Authority: CN
Inventors: 钱朝南; 陈金东; 黄婕; 刘倚秀; 周红娟
Original assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Current assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Priority date: 2024-05-30
Filing date: 2024-05-30
Publication date: 2024-08-23
Anticipated expiration: 2044-05-30
Also published as: CN118271276A

Abstract

The invention relates to a naphthalenone derivative for promoting wound healing, and a preparation method and application thereof, wherein the naphthalenone derivative comprises a molecular structure shown in a formula (I) or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof; (I) . The naphthalenone derivative is synthesized by using 1, 4-benzodioxane-6-carboxylic acid and amino naphthalenol hydrochloride as raw materials, and the yield can reach more than 60%. The naphthalenone derivative prepared by the invention can obviously improve the movement capability of cells at the concentration lower than 5 mu M, promote the migration and invasion of cells, accelerate the wound healing, particularly reduce the wound healing capability of diabetic mice by the administration concentration lower than 1mg/kg, reduce the chronic wound healing time, and can be widely applied to the healing of wounds of trauma, burns, ulcers, dermatitis, chronic wounds, childbirth and diabetic patients.

Description

Naphthalenone derivative for promoting wound healing as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicine synthesis, and particularly relates to a naphthalenone derivative for promoting wound healing, and a preparation method and application thereof.

Background

The skin is the first important defense line for the human body to resist external attack, contains a large amount of nutrients such as protein, fat, moisture and the like, participates in metabolism of the human body, has the functions of immunity, resisting external pathogen attack, regulating body temperature and the like, has a protective effect, needs to take treatment measures in time when the skin is wounded, promotes wound healing, has slow wound healing process, 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 medicine for wound healing, including isoquinoline, flavonoid, alkaloid, some Chinese herbal medicines and the like, chinese patent CN114380680A discloses that a flavonoid compound can be used for preparing the medicine for promoting wound healing, but the dosage of the compound in wound healing is relatively high, and the administration concentration reaches 30mg/kg in a mouse experiment; 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 long 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 needed.

Disclosure of Invention

Aiming at the prior art problems, the invention provides a naphthalenone derivative, a preparation method and application thereof, wherein the naphthalenone derivative can remarkably improve migration and invasion capacities of cells and can be used for promoting chronic and non-chronic wound healing.

In a first aspect, the present invention provides a naphthalenone derivative, wherein the naphthalenone derivative is a molecular structure shown in formula (I) or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof;

（I）

In a second aspect, the present invention provides a method for preparing a naphthalenone derivative, the method comprising: dissolving 1, 4-benzodioxane-6-carboxylic acid in N, N-Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), amino naphthol hydrochloride and N, N-Diisopropylethylamine (DIPEA), mixing for reaction, adding ethyl acetate and water for extraction after the reaction is finished, drying an organic phase, filtering, concentrating to obtain a crude product, and carrying out column chromatography on the crude product to obtain a naphthalenone derivative, wherein the naphthalenone derivative is a molecular structure shown in a formula (I) or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof.

Further, the synthetic route of the naphthalenone derivative of the molecular structural formula (I) is as follows:

further, the feeding mole ratio of the 1, 4-benzodioxane-6-carboxylic acid, the amino naphthol hydrochloride and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1:0.8-1.5:0.5-1.5.

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

Further, the column chromatography step is as follows: placing 10-20 times of sample amount of silica gel H (Qingdao ocean, 200-300 meshes) in a container, adding a developing agent (petroleum ether: ethyl acetate=10:1 according to the volume ratio) with the mass of 1-3 times of the silica gel, stirring until no bubble exists, uniformly introducing the mixture into a chromatographic column (a small amount of cotton is plugged at the bottom of the column in advance to prevent the silica gel from leaking), settling and compacting the mixture to prepare the chromatographic column, adding a concentrated crude product, eluting the crude product by the developing agent, identifying whether the eluted product is completely discharged by an ultraviolet lamp, concentrating the obtained target product, and weighing the naphthalenone derivative.

In a third aspect, the present invention provides a preparation for promoting wound healing, wherein the preparation comprises a naphthalenone derivative, and the naphthalenone derivative comprises a molecular structure shown as a formula (I) or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof.

（I）

Further, the preparation also comprises pharmaceutically acceptable pharmaceutical excipients.

Further, the dosage forms of the preparation include, but are not limited to, suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, aerosols, gels, patches, drops or liniments.

In a fourth aspect, the invention also provides application of a naphthalenone derivative in preparing a medicament for promoting wound healing, wherein the naphthalenone derivative comprises a molecular structure shown in a formula (I) or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof;

（I）

Further, the pharmaceutical indications include, but are not limited to, chronic or non-chronic wounds;

such non-chronic wounds include, but are not limited to, wounds resulting from trauma, burns, ulcers, dermatitis, or labor.

The chronic wounds include, but are not limited to, diabetic wounds.

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

(1) The invention develops a new synthetic route to prepare a novel naphthalenone derivative, and the synthetic method of the naphthalenone derivative is simple, and the yield is high and can reach more than 60%.

(2) The naphthalenone derivative can obviously improve the movement capability of human foreskin fibroblasts HFF-1 and mouse embryo fibroblasts 3T3 at the concentration lower than 5 mu M, improve the migration and invasion penetration capability of cells, and simultaneously has obvious healing promotion capability on wounds of normal mice and diabetic mice, and improves the healing speed of the wounds, and the drug concentration can be as low as 1mg/kg so as to obviously promote the healing capability of the wounds of the diabetic mice, so that the naphthalenone derivative can obviously improve the chronic wound healing capability, reduce the chronic wound healing time, and can also be widely applied to wounds caused by trauma, burns, ulcers, dermatitis and labor.

Drawings

FIG. 1 is a ¹ H NMR nuclear magnetic spectrum of a naphthalenone derivative of the present invention.

FIG. 2 is a graph of EH-P007U versus mouse embryonic fibroblast 3T3 migration.

FIG. 3 is a data statistic of EH-P007U versus mouse embryonic fibroblast 3T3 migration.

FIG. 4 is a graph of EH-P007U versus human foreskin fibroblast HFF-1 migration.

FIG. 5 is a data statistic of EH-P007U migration of human foreskin fibroblasts HFF-1.

FIG. 6 is a graph of EH-P007U invasiveness against mouse embryonic fibroblasts 3T3, wherein A is Control, B is EH-P007U-1. Mu.M, C is EH-P007U-2. Mu.M, and D is EH-P007U-5. Mu.M.

FIG. 7 is a data statistic of EH-P007U invasion of mouse embryonic fibroblasts 3T 3.

FIG. 8 is a graph of EH-P007U invasiveness to human foreskin fibroblasts HFF-1, wherein A is Control, B is EH-P007U-1. Mu.M, C is EH-P007U-2. Mu.M, and D is EH-P007U-5. Mu.M.

FIG. 9 is a data statistic of EH-P007U invasion of human foreskin fibroblast HFF-1.

FIG. 10 is a physical graph of the effect of EH-P007U on wound healing in normal mice.

FIG. 11 is statistical analysis data of the effect of EH-P007U on wound healing in normal mice.

FIG. 12 is a graphical representation of the effect of EH-P007U on wound healing in diabetic mice.

FIG. 13 is statistical analysis data of the effect of EH-P007U on wound healing in diabetic mice.

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.

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 naphthalenone derivatives

The synthetic route of the naphthalenone derivative of the invention is as follows:

the specific synthesis steps are as follows:

Dissolving 0.3g of 1, 4-benzodioxane-6-carboxylic acid in 10mL of DMF, adding 0.38g of EDCI, 0.358g of amino naphthol hydrochloride and 0.34mL of DIPEA, stirring and mixing, reacting for 5H at 5 ℃, sequentially adding 50mL of ethyl acetate and 50mL of water after the reaction is finished, extracting the obtained organic phase, adding saturated sodium chloride solution for washing, drying by anhydrous sodium sulfate, filtering and concentrating to obtain a crude product, adding the crude product to the top of a column chromatography (the preparation method of the column chromatography is that silica gel H (Qingdao ocean, 200-300 meshes) with 20 times of loading amount is placed in a glass beaker, adding a developing agent (petroleum ether: ethyl acetate=10:1) with one time of the mass of the silica gel, stirring until no bubble is generated, uniformly introducing the mixture into the column chromatography (the bottom of the column is pre-reduced in quantity, preventing the silica gel from leaking), compacting, preparing the column chromatography by using the developing agent (the volume ratio is that petroleum ether: ethyl acetate=10:1), eluting, identifying whether the eluted product is a product is subjected to ultraviolet lamp chromatography, and obtaining the product which is completely recorded as a target molecule of the formula I, namely, the product is the ultraviolet-007, which is the pure-60% of the molecular structure, and the obtained product is completely recorded as the ultraviolet-purifying product is expressed by the U007.

（I）

The nuclear magnetic spectrum of the structural formula (I) of the naphthalenone derivative is shown in the attached figure 1, wherein ¹ H NMR data are as follows:

¹H NMR (400 MHz, Chloroform-d) δ 9.79 (s, 1H), 8.43 (d, J = 8.1 Hz, 1H), 7.98 (s, 1H), 7.84-7.74 (m, 1H), 7.54-7.35 (m, 5H), 7.07 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 4.42- 4.25 (m, 4H).

EXAMPLE 2 Effect of naphthalenone derivatives on migration of 3T3 cells, HFF-1 cells

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

(2) Experimental drugs: EH-P007U, 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, 2 mL complete culture mediums containing 10% FBS (fetal bovine serum) are added into each hole, 2x 10 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 the line b 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 naphthalenone derivative EH-P007U (working concentration is 5 mu M) is added, the culture is carried out in a 37 ℃ incubator with 5% CO ₂, and the culture is carried out according to the sampling of 0h and 24h of medicine addition.

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 rate of mouse embryo fibroblasts 3T3 and human foreskin fibroblasts HFF-1 treated by EH-P007U with low concentration (5 mu M) is obviously accelerated, which shows that the naphthalenone derivative EH-P007U can effectively promote cell migration and movement, and has extremely significant difference (P < 0.0001).

EXAMPLE 3 Effect of naphthalenone derivatives on invasion of 3T3 cells, HFF-1 cells

(2) Experimental drugs: EH-P007U, 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-P007U is added), a dosing group (working concentration is 1 mu M, 2 mu M and 5 mu M), each group is provided with 2 duplicate wells, a proper amount of cell suspension is taken according to the cell density, a proper amount of 10% BSA (bovine serum albumin) with the final percentage of 0.1% is added, then a naphthalenone derivative EH-P007U is added, finally, DMEM (liquid culture medium and high sugar) is used for filling, the total volume of each well is 200 mu L, after uniform mixing, a glue-paved Transwell upper chamber is gently and uniformly added, and 800 mu L of culture medium containing 20% FBS (bovine serum) is generally added into a 24-well plate lower chamber immediately, and a plate is harvested after 24h 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: the significantly increased number of cells invasively migrating through Transwell in EH-P007U treated at a concentration of less than 5. Mu.M compared to the control group indicated that EH-P007U had significantly improved cell invasion and penetration capacity with significant/very significant differences.

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-P007U 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-P007U (1 mg/kg/d, 10 mg/kg/d) 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-P007U 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-P007U (1 mg/kg/d) by intraperitoneal injection; mice were injected with D1, D3, D5, D7, D9, D11 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 or the diabetic mice are wound healed, the mice with EH-P007U have a faster wound healing speed than the control group without the EH-P007U, and have significant differences (P <0.05; P < 0.01), compared with the EH-P007U, the effect of the EH-P007U on the wound healing of the normal mice and the diabetic mice is more obvious, the chronic wounds can be healed rapidly with the administration concentration of only 1mg/kg, and the experimental results have statistical significance.

It should be noted that the above examples merely represent several embodiments of the present invention, which are described in more 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. A naphthalenone derivative, which is characterized in that the naphthalenone derivative is selected from a molecular structure shown in a formula (I) or pharmaceutically acceptable salts thereof;

（I）。

2. A process for preparing a naphthalenone derivative according to claim 1, wherein 1, 4-benzodioxane-6-carboxylic acid is dissolved in N, N-dimethylformamide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, aminonaphthol hydrochloride and N, N-diisopropylethylamine are added and mixed at 0-25 ℃ for reaction, ethyl acetate and water are added for extraction after the reaction is completed, an organic phase is dried, filtered and concentrated to obtain a crude product, and the crude product is subjected to column chromatography to obtain the naphthalenone derivative.

3. The method for preparing a naphthalenone derivative according to claim 2, wherein the 1, 4-benzodioxane-6-carboxylic acid, amino naphthol hydrochloride and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide are added in a molar ratio of 1:0.8-1.5: 0.5 to 1.5.

4. The method for producing a naphthalenone derivative according to claim 2, wherein the column chromatography step is: eluting the crude product with a developing agent, wherein the developing agent is petroleum ether and ethyl acetate=10:1, and identifying and concentrating the eluted product by an ultraviolet lamp to obtain the target product naphthalenone derivative.

5. A formulation for promoting wound healing, comprising a naphthalenone derivative according to claim 1.

6. The wound-healing-promoting formulation of claim 5, further comprising a pharmaceutically acceptable adjuvant.

7. The wound-healing-promoting formulation of claim 5 or 6, wherein the formulation comprises a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an aerosol, a gel, a patch, or a liniment.

8. Use of a naphthalenone derivative according to claim 1 for the preparation of a medicament for promoting wound healing, wherein the wound comprises a chronic wound or a non-chronic wound.

9. The use of a naphthalenone derivative according to claim 8, wherein the non-chronic wounds comprise trauma, burns, ulcers, dermatitis and childbirth wounds.

10. Use of a naphthalenone derivative according to claim 8, wherein the chronic wound comprises a wound of a diabetic patient.