Application of 18 beta-glycyrrhetinic acid derivative in preparation of medicine for treating ulcerative colitis

Abstract

The invention discloses application of an 18 beta-glycyrrhetinic acid derivative in preparation of a medicine for treating ulcerative colitis, and belongs to the technical field of medicines. The structural formula of the 18 beta-glycyrrhetinic acid derivative is shown as the formula 1 in the specification. The 18beta-glycyrrhetinic acid derivative YCY-20 disclosed by the invention is more stable in gastrointestinal tracts, 12.5 mg / Kg of compound is injected into the stomach of a mouse compared with 200mg / Kg of mesalazine, the weight loss and colonic atrophy of the mouse with ulcerative colitis are better relieved, the inflammatory phenotype of the mouse is relieved, and the 18beta-glycyrrhetinic acid derivative YCY-20 has a better effect of treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and more particularly to the application of an 18β-glycyrrhetinic acid derivative in the preparation of a medicament for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD). The inflammation starts from the rectum and extends to the proximal colon, and it is prone to recurrence. Currently, the etiology and pathogenesis of UC are unknown, and its etiology may involve the host immune system, genetic variability, and environmental factors. In recent years, traditional Chinese medicine has been increasingly applied in the treatment of UC. Traditional Chinese medicine treatment has multiple targets and few side effects, and has unique advantages in the treatment of chronic diseases. Gancaoxiexin Decoction can be found in the chapter on "Huo" disease in Zhang Zhongjing's *Synopsis of the Golden Chamber*. The treatment of "Huo" disease with Gancaoxiexin Decoction and "UC" belongs to the treatment of different diseases with the same method, and the prescriptions of the two are relatively similar. Among them, roasted licorice is used as the monarch drug to reinforce the middle-jiao and relieve spasm, so that the deficiency of the stomach can be tonified.

[0003] Glycyrrhizic acid (GL) is a triterpenoid saponin obtained from licorice and is one of the most important components in licorice. It is composed of two molecules of glucuronic acid and one molecule of glycyrrhetinic acid (GA), and it is the main component of the root of *Glycyrrhiza glabra* L. 18β-glycyrrhetinic acid is the aglycone of glycyrrhizic acid and is one of the active metabolites of glycyrrhizic acid under the action of intestinal bacteria. Glycyrrhetinic acid is metabolized in the liver to GA-3-O-monos-β-D-glucuronide (GAMG) and exerts its medicinal effect through blood circulation, so its oral bioavailability is poor.

[0004] Modern research shows that 18β-GA has a wide range of pharmacological effects, such as anti-inflammatory activity, antibacterial activity, antiviral activity, antioxidant activity, and antitumor activity, etc. Currently, there are studies evaluating the effectiveness of glycyrrhizic acid and glycyrrhetinic acid in experimental models of induced colitis. Oral administration or application of enemas containing these drugs, alone or in combination with other substances with anti-inflammatory activity, can reduce the inflammatory process and oxidative tissue damage in the colonic mucosa and improve the epithelial healing of the colonic mucosa. However, 18β-GA has serious side effects, including pseudoaldosteronism and the toxic effect of high concentrations on normal cells, which limits its clinical application to a certain extent.

[0005] Therefore, how to develop the application of an 18β-glycyrrhetinic acid derivative in the preparation of a medicament for treating ulcerative colitis is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides an application of an 18β-glycyrrhetinic acid derivative in the preparation of a drug for treating ulcerative colitis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an application of an 18β-glycyrrhetinic acid derivative in the preparation of a drug for treating ulcerative colitis, and the structural formula of the 18β-glycyrrhetinic acid derivative is shown in Formula 1, denoted as YCY-20;

[0009]

[0010]

[0011] The present invention provides an application of the 18β-glycyrrhetinic acid derivative in the preparation of a drug for improving colon atrophy and relieving colon tissue damage.

[0012] Beneficial effects of the present invention: Compared with the clinically commonly used prescription drug mesalazine (5-aminosalicylic acid), mesalazine belongs to salicylic acid drugs, retains a certain acidity, can directly stimulate the gastric mucosa orally, can cause gastric ulcers and imperceptible gastric bleeding. 5-aminosalicylic acid is not resistant to gastric acid and is quickly absorbed and decomposed in the stomach and small intestine after oral administration, losing its activity and unable to play an anti-inflammatory role. In order to ensure the safety and effectiveness of mesalazine treatment, sustained-release preparations are used for clinical administration. The 18β-glycyrrhetinic acid derivative YCY-20 of the present invention is more stable in the gastrointestinal tract. When mice were intragastrically administered 12.5 mg / Kg of the compound, compared with intragastric administration of 200 mg / Kg of mesalazine, it better alleviated the weight loss and colon atrophy of ulcerative colitis mice, relieved the inflammatory phenotype of mice, and had a good effect on treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Blood compatibility of the compound (where the values are expressed as mean ± standard error, n = 3).

[0014] Figure 2 Graph of body weight changes of mice in each group (where the values are expressed as mean ± standard error, n = 6).

[0015] Figure 3 Graph of DAI changes of mice in each group (where the values are expressed as mean ± standard error, n = 6).

[0016] Figure 4 Graph of colon morphology of mice in each group (where the values are expressed as mean ± standard error, n = 6).

[0017] Figure 5 Graph of colon length of mice in each group (where the values are expressed as mean ± standard error, n = 6).

[0018] Figure 6 H&E staining images of the colon of each group of mice. Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] Example 1

[0021] Preparation method of 18β-glycyrrhetinic acid derivative:

[0022] (1) At room temperature, 18β-GA (2.0039 g, 4.3 mmol), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (1.6486 g, 8.6 mmol), and HOBT (1-hydroxybenzotriazole monohydrate) (0.6586 g, 4.3 mmol) were dissolved by stirring at 800 r / min in 40 mL of DCM (dichloromethane) for 1 min. TEA (triethylamine) (3 mL, 24 mmol) was added for activation for 2 h. After the activation was completed, N-methylpiperazine (3 mL, 26 mmol) was added and the reaction was carried out at 25 °C for 24 h. After the reaction was completed, 40 mL of DCM was added to quench the reaction. The lower organic layer was taken after extraction with 200 mL of distilled water, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation (water bath temperature 40 °C, rotation speed 100 r / min, vacuum degree 100 mbar). The intermediate of GA-piperazine was obtained by column chromatography purification using 200-300 mesh silica gel as the stationary phase and methanol∶petroleum ether∶ethyl acetate = 0.5∶2∶1 as the mobile phase for elution, with a yield of 78.5%.

[0023] (2) At room temperature, the intermediate (2.097 g, 4 mmol), N-Boc-L-methionine (2.3936 g, 9.6 mmol), EDCI (1.8403 g, 9.6 mmol), and DMAP (4-dimethylaminopyridine) (0.1955 g, 1.6 mmol) were dissolved by stirring at 800 r / min in 40 mL of DCM and reacted for 12 h. After the reaction was completed, 40 mL of DCM was added to quench the reaction. The lower organic layer was taken after extraction with 200 mL of distilled water, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation (water bath temperature 40 °C, rotation speed 100 r / min, vacuum degree 100 mbar). The final 18β-GA derivative, denoted as YCY-20, was obtained by column chromatography purification using 200-300 mesh silica gel as the stationary phase and methanol∶petroleum ether∶ethyl acetate = 0.5∶2∶1 as the mobile phase for elution, with a yield of 57.42%;

[0024] 1 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H, C=C-NH), 7.60 (d, J = 7.8 Hz, 1H, H-Ar), 7.34 (d, J = 8.2 Hz, 1H, H-Ar), 7.27 (s, 2H, H-Ar), 7.13 - 7.09 (m, 1H, H-Ar), 6.99 (d, J = 2.3 Hz, 1H, CH=C), 5.68 (s, 1H, CH-12), 5.10 (d, J = 8.2 Hz, 1H, CHNH), 4.66 (q, J = 7.1, 6.5 Hz, 1H, CH-3), 4.46 (dd, J = 10.4, 6.1 Hz, 1H, CHNH), 3.72 (s, 3H, COOCH3), 3.61 (dt, J = 11.2, 4.9 Hz, 4H, N(CH2)2), 3.46 (t, J = 5.1 Hz, 4H, N(CH2)2), 2.32 (s, 1H, CH-9), 2.04 (s, 1H, CH-18), 2.01 - 1.79, 1.59 - 1.46, 1.40, 1.29, 1.26, 1.16, 1.01, 0.78, 0.72 (19H, methylene and methine of triterpenoid structure), 1.69 - 1.59 (m, 3H, CH-25),, 1.43 (s, 9H, Boc-CH3), 1.34 (s, 3H, CH-26), 1.22 (s, 3H, CH-27), 1.11 (d, J = 3.0 Hz, 6H, CH-28 / CH-29), 0.81 (s, 3H, CH-23), 0.77 (s, 3H, CH-24); 1313C NMR (101 MHz, CDCl3) δ 200.0, 174.28 (2C), 172.17, 169.67, 155.85, 128.55, 127.89, 122.81, 122.10, 119.49, 119.06, 111.14, 110.35, 81.96, 79.70, 61.66, 55.00, 52.87, 48.12, 45.30 (2C), 43.94 (2C), 43.89, 43.31, 38.70, 38.02, 37.73, 36.89, 33.11, 32.71, 31.79 (2C), 28.43, 28.36 (Boc-CH3, 3C), 28.14, 27.93, 27.06, 26.72, 26.40, 23.25, 23.11, 18.68, 17.33, 16.58; IR νmax 2974, 2931, 2791, 1712, 1658, 1525, 1452, 1390, 1365, 1290, 1166, 1049, 1001, 983, 875 cm -1 ; HRMS (ESI) m / z calcd for C 45 H 74 N3O6S + (M + H) + 784.52928, found 784.52966.

[0025]

[0026] 1. Hemolysis activity evaluation

[0027] 1.1 Preparation of heparin sodium solution

[0028] Weigh 0.01 - 0.02 g of heparin sodium and dissolve it in 1 mL of physiological saline to obtain the solution.

[0029] 1.2 Blood collection from rat orbital cavity

[0030] Use a 0.5 mm glass capillary pipette filled with heparin sodium solution, insert it into the orbital cavity to collect blood, and store it in a centrifuge tube containing heparin sodium solution.

[0031] 1.3 Preparation of 2% red blood cell suspension

[0032] The blood mixed with heparin sodium solution is centrifuged (2000 rpm, 5 min) to remove the heparin sodium solution, and then washed 2 - 3 times with physiological saline until the supernatant is no longer red but slightly yellow to obtain red blood cells.

[0033] 1.4 Preparation of final concentration

[0034] Weigh the sample precisely with a one-ten-thousandth balance, and then dissolve it in DMSO to a suitable concentration as the stock solution. The final concentration of the sample is prepared according to Table 1.

[0035] Table 1 Reaction system of hemolysis test for SD rat blood cells

[0036]

[0037]

[0038] 1.5 Hemolysis phenomenon and data determination

[0039] Incubate at 37 °C for 1 h, centrifuge (2000 rpm, 5 min), observe the phenomenon in the centrifuge tube visually, and take pictures for record. Take 200 μL of the supernatant and place it in a 96-well plate. Set 3 replicate wells for each compound and repeat 3 times. Measure the absorbance at 540 nm, and calculate the hemolysis rate according to formula (1).

[0040]

[0041] 1.6 Evaluation of hemolytic activity of GA derivatives

[0042] Generally, when the hemolysis rate of a compound is < 5%, it can be considered to have hemolytic safety. The hemolysis test results of 18β-GA derivatives at different concentrations are as Figure 1 shown. The results show that: for the lead compound 18β-GA, the hemolysis rates at 75, 150, and 300 μM concentrations are all greater than 5%, which are 9%, 12%, and 73% respectively, not within the hemolytic safety range. At 300 μM, the hemolysis rate is 73%, showing strong hemolytic toxicity. For the derivatives at the 3 concentrations, the hemolysis rates are all lower than 5%, within the hemolytic safety range.

[0043] 2. Establishment of murine ulcerative colitis and administration method

[0044] 2.1 Grouping

[0045] Male C67BL / 6J mice (7 weeks old, 22 ± 2 g). After one week of adaptive feeding, weigh the mice and randomly divide them into 6 groups with 6 mice in each group. Control group (0.5% CMC-Na), Model group (2.5% DSS + 0.5% CMC-Na), Positive drug group (2.5% DSS + 200 mg / kg SASP), Derivative high-dose group (2.5% DSS + 12.5 mg / kg), Derivative medium-dose group (2.5% DSS + 6.25 mg / kg), Derivative low-dose group (2.5% DSS + 3.125 mg / kg)

[0046] 2.2 Reagent preparation

[0047] 2.5% DSS solution: Weigh 2.5 g of dextran sulfate sodium salt and dissolve it in 300 mL of purified water, stirring until dissolved and clarified.

[0048] 0.5% CMC-Na solution: Weigh 5 g of CMC-Na and dissolve it in 1000 mL of purified water, heat to boiling, and keep stirring until it becomes clear and transparent.

[0049] 2.3 Establishing a DSS-induced murine ulcerative colitis model and drug administration

[0050] Each group was continuously gavaged for ten days. Except for the Control group, the drinking water of the other groups was replaced with 2.5% DSS solution on the third day. The 2.5% DSS solution was freshly prepared daily.

[0051] 2.4 Recording of the conditions of mice and collection and processing of tissues

[0052] From day 0, the body weight of the mice was recorded. From day 1, the health status of the mice was observed at the same time period every day, and the body weight of the mice was recorded. The mice were placed individually in a mouse cage and observed for 15 min. Filter paper was placed below to collect fresh feces, and the fecal traits and fecal occult blood conditions were recorded. The disease activity index (DAI) of the mice was evaluated daily. The DAI scoring criteria were scored according to Table 2.

[0053] Table 2 Disease Activity Index (DAI)

[0054]

[0055]

[0056] 24 h after the intervention ended on the tenth day, the mice were sacrificed by cervical dislocation. The abdominal cavity of the mice was opened, cut along the midline of the abdomen to the anus, the colon of the mice was found in the right posterior abdomen, the mesentery and tissues around the colon were removed, the colon was cleaned from the anus to the cecum, cut at the junction of the cecum and ileum, and the intestinal tissue from the cecum to the anus was taken out, naturally unfolded on graph paper, and the total length of the colon tissue was measured. About 1 cm of colon tissue was intercepted about 1.5 cm from the anus, cut open, rinsed with physiological saline to clean the feces, and fixed in paraformaldehyde at -4°C for 24 h for H&E staining after rinsing clean.

[0057] 3. GA derivatives can improve the weight loss of DSS-induced acute ulcerative colitis in mice

[0058] After induction with 2.5% DSS, the body weight of the mice will change, and the trend of the body weight change of the mice is as Figure 2Shown as follows: During the whole experiment, the body weights of mice in the Control group did not change significantly, while the body weights of the remaining groups decreased on the fourth day after DSS intervention. Compared with the Model group, each treatment group alleviated the decrease in body weight, and the therapeutic effect of the derivative treatment group showed a dose-dependence. The body weight trends of the GA-H group and the SASP group were comparable.

[0059] 4. GA derivatives can improve the disease activity index of DSS-induced acute ulcerative colitis in mice

[0060] The DAI was evaluated at the same time period every day and scored according to the standard. The DAI is a comprehensive evaluation of the clinical manifestations of mice during the inflammatory process and is an important indicator for evaluating the severity of UC mice. As Figure 3 shown, the scoring results indicated that the DAI score of the Model group was the highest. The scores of the GA-H group and the SASP group were similar. It can be concluded from the curve trend that glycyrrhetinic acid derivatives can significantly improve the disease manifestations of UC mice.

[0061] 5. GA derivatives can improve the colon atrophy of DSS-induced acute ulcerative colitis in mice

[0062] After the mice were sacrificed, the intestinal tissues from the cecum to the anus were taken as Figure 4 shown. The gross morphology of the colon tissues of mice in the Control group was normal, the intestinal wall thickness was normal and uniform, and there were no conditions such as congestion and edema. After DSS modeling, due to DSS stimulation, the colon of the experimental mice had a severe intestinal inflammatory reaction, the intestines of the mice would have spasms, the intestinal wall would become thinner, the weight would decrease, and in addition, due to intestinal constriction, the colon length would also become shorter. As Figure 5 shown, under the intervention of 2.5% DSS, compared with the Model group, the colon morphology of the SASA group and the glycyrrhetinic acid derivative treatment group had a certain degree of increase in the colon length of each treatment group, and there was a significant improvement in the occurrence of intestinal congestion, edema and erosion. It shows that glycyrrhetinic acid derivatives have an improving effect on the colon shortening in ulcerative colitis mice.

[0063] 6. GA derivatives can improve the colon tissue pathology of DSS-induced acute ulcerative colitis in mice

[0064] The intestinal epithelial cells of normal mice should be arranged neatly, with normal cell morphology, no goblet cell deficiency, and no inflammatory infiltration. After DSS-induced inflammation, the intestines of mice are damaged, and pathological manifestations of morphological changes and inflammatory manifestations will occur. As Figure 6As shown, in the Control group, the mucosa of the colonic epithelium was smooth and intact, there was no obvious loss of goblet cells, and the intestinal glands were arranged neatly. In the Model group, the mouse intestine showed a loss of goblet cells, disappearance of colonic crypts, severe epithelial loss, partial destruction of the intestinal mucosa, and inflammatory infiltration into the submucosa. The low- and medium-dose GA derivative groups could improve the condition of inflammatory cell infiltration, increase the number of goblet cells between epithelial cells, and the crypt structure was restored to a certain extent; the colonic mucosal tissue structure of the high-dose GA derivative group and the SASP group tended to be close to that of normal intestinal epithelium, the cells were closely arranged, the inflammatory cell infiltration was significantly reduced, and a large number of goblet cells and crypt structures were restored. This indicates that GA derivatives can effectively alleviate the damage of DSS to colonic tissue.

[0065] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of an 18β-glycyrrhetinic acid derivative in the preparation of a medicament for treating ulcerative colitis, characterized in that, The structural formula of the 18β-glycyrrhetinic acid derivative is shown in Formula 1; 2. Use of the 18β-glycyrrhetinic acid derivative according to Claim 1 in the preparation of a drug for improving colonic atrophy and alleviating colonic tissue injury.

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