An indole-azole derivative WM-20, its preparation method and use

Abstract

The application discloses an indole-azole derivative WM-20, a structural formula of which is as shown in the following formula, and a molecular formula of which is C 32 H 32 ClF5N6O2. The application further discloses a preparation method of the indole-azole derivative WM-20 and application of the indole-azole derivative WM-20 to preparation of an anti-inflammatory drug in vitro and in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and specifically relates to an indole-azole derivative WM-20, a preparation method and use thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) with lesions affecting the rectum and colon. Common clinical symptoms include abdominal pain, bloody diarrhea, and rectal bleeding. This disease is usually characterized by a process of repeated attacks and remissions. Most patients with ulcerative colitis suffer from the disease for life after diagnosis, which has a serious impact on their quality of life. According to statistics, the global prevalence of ulcerative colitis was estimated to be 5 million cases in 2023, and the incidence rate is rising sharply, placing a heavy burden on the global healthcare system. Currently, the goal of treating ulcerative colitis is to relieve symptoms, control inflammation, and maintain remission. Commonly used treatments include: aminosalicylates, which primarily inhibit prostaglandin synthesis and reduce intestinal inflammation; glucocorticoids, which are effective for short-term control of acute attacks, but long-term use may cause side effects such as osteoporosis and diabetes; immunosuppressants, which suppress the immune system and reduce inflammation in patients with moderate to severe ulcerative colitis; and biologics and targeted synthetic small molecule drugs, which are often used in patients with moderate to severe ulcerative colitis who have not responded well to other medications. However, these drugs are expensive and may cause numerous adverse reactions. Despite the increasing number of available treatments and treatment options, only a few can maintain and reduce the severity of ulcerative colitis, and real-world remission rates do not exceed 30-60%. More than 10% of patients require proctocolectomy for treatment. Therefore, there is an urgent need to develop new therapeutic agents and effective treatment strategies to improve treatment efficacy, reduce healthcare costs, and meet patients' needs for more effective and personalized treatment options to address this growing global health challenge. Summary of the Invention

[0003] The present invention discloses an indole-azole derivative WM-20 and a preparation method thereof. The indole-azole derivative WM-20 of the present invention has significant in vivo and in vitro anti-inflammatory activity.

[0004] The technical solutions of the present invention are as follows:

[0005] The first aspect of the present invention discloses an indole-azole derivative WM-20, whose structural formula is: The molecular formula is C 32 H 32 ClF5N6O2.

[0006] The second aspect of the present invention discloses a method for preparing the indole-azole derivative WM-20, comprising the following steps:

[0007]

[0008] (1) Indoles containing different substituents, methyl acrylate, and 1,8-diazabicyclo[5.4.0]undec-7-ene were added to acetonitrile and heated at 60°C to obtain compound 1;

[0009] (2) Compound 1 obtained in step (1) and NaBH3CN were added to acetic acid and reacted at 0°C for 12 hours to obtain compound 2;

[0010] (3) Compound 2 obtained in step (2) was refluxed with ethylenediamine in CH3OH to obtain yellow oily substances, namely compounds 3 and 4;

[0011] (4) Compound 3 and compound 4 obtained in step (3) and triethylamine were added to ethanol, heated under reflux for 12 h, concentrated in vacuo, eluted by column chromatography, separated and dried to obtain a light yellow oil, which is compound 5;

[0012] (5) Compound 5 obtained in step (4), 4-trifluoromethylbenzyl bromide and K2CO3 were added to dichloromethane and reacted at room temperature overnight. The mixture was concentrated in vacuo and eluted by column chromatography. The mixture was separated and dried to obtain a yellow viscous substance, which was the indole-azole derivative WM-20.

[0013] Preferably, anhydrous sodium sulfate is used for drying in step (4), and the eluent is a mixture of dichloromethane and triethylamine in a volume ratio of 100:0.5.

[0014] Preferably, the drying in step (5) uses anhydrous sodium sulfate, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of (80-50):1 and 0.5 v / v% Et3N.

[0015] The third aspect of the present invention discloses the use of the indole-azole derivative WM-20 for preparing in vitro and in vivo anti-inflammatory drugs.

[0016] Preferably, the indole-azole derivative WM-20 is used for preparing a drug for treating ulcerative colitis.

[0017] Beneficial effects of the present invention:

[0018] The indole-azole derivative WM-20 of the present invention is a newly synthesized compound used for the preparation of anti-inflammatory drugs, and exhibits significant in vitro and in vivo anti-inflammatory activity. In vitro, it can effectively inhibit the production of NO in LPS-induced RAW264.7 macrophages and reduce the secretion of inflammatory cytokines IL-6 and IL-1β. In vivo, the indole-azole derivative WM-20 can effectively treat ulcerative colitis and downregulate the expression of serum inflammatory factors IL-6, IL-1β, and TNF-α. The indole-azole derivative WM-20 of the present invention provides a new therapeutic strategy for the treatment of ulcerative colitis.

[0019] The present invention relates to a method for preparing an indole-azole derivative, WM-20. The applicants linked an indole or indoline related to ethylenediamine with the active fragment of fluconazole to synthesize a novel compound, WM-20. The resulting novel compound, WM-20, not only exhibits significant anti-inflammatory effects in vitro but is also effective in treating ulcerative colitis in vivo. See the applicants' prior work in ACS Med. Chem. Lett. 2023, 14, 1448-1454 and RSCMed. Chem. 2024, 15, 1236. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Compound WM-20 1 H NMR spectrum.

[0021] Figure 2 Compound WM-20 13 C NMR spectrum.

[0022] Figure 3 is the HRMS spectrum of compound WM-20. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Example 1: Preparation of indole-azole derivative WM-20, according to the steps shown in the figure below:

[0025]

[0026] Step 1: Preparation of Compound 1: 10 mmol of 5-chloroindole, 15 mmol of methyl acrylate, and 3 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were added to a round-bottom flask, and acetonitrile (CH3CN) was used as a solvent. The mixture was heated at 60°C to obtain Compound 1;

[0027] Step 2: Preparation of compound 2: 10 mmol of compound 1 and 20 mmol of NaBH3CN were added to a round-bottom flask, and 20 mL of acetic acid (AcOH) was used as solvent. The mixture was reacted at 0°C for 12 h to obtain compound 2;

[0028] Step 3: Preparation of Compound 3 and Compound 4: 5 mmol of Compound 1 and 25 mmol of ethylenediamine were added to a round-bottom flask and refluxed in methanol (CH3OH) to obtain Compound 3 and Compound 4;

[0029] Step 4: Preparation of Compound 5: To a round-bottom flask were added 5 mmol of compound 3, 5 mmol of compound 4, and 0.3 mmol of triethylamine (Et3N), and the mixture was heated under reflux with ethanol as the solvent for 12 h. After completion of the reaction, the mixture was concentrated in vacuo and then subjected to column chromatography using a mixture of dichloromethane and Et3N (100:0.5) as the eluent. The mixture was separated and dried to obtain a light yellow oily substance, Compound 5.

[0030] Step 5: Preparation of Indole-Azole Derivative WM-20: To a round-bottom flask, add 1 mol of compound 5, 1.2 mmol of 4-trifluoromethylbenzyl bromide, and 2.0 mmol of KCO. The mixture was reacted overnight at room temperature using dichloromethane (DCM). After completion of the reaction, the mixture was concentrated in vacuo and then subjected to column chromatography using a 80-50:1 mixture of dichloromethane and methanol and approximately 0.5 v / v% EtN (0.5 mL of EtN per 100 mL of the solution) as the eluent. The mixture was separated and dried to afford a pale yellow oil, compound WM-20.

[0031] like Figure 1-Figure 3 As shown, the test results are as follows: 1H NMR(400MHz, CDCl3)δ:8.00(s,1H),7.76(s,1H),7.44-7.50(m,3H),7.16(d ,J=8.0Hz,2H),7.00-7.02(m,2H),6.68-6.78(m,2H),6.40(d,J=8.2Hz,1H) ,6.20(s,1H),4.51(q,J=14.2Hz,2H),3.49(s,2H),3.20-3.33(m,5H),3.08 -3.16(m,2H),2.87-2.93(m,3H),2.48-2.52(m,2H),2.39(t,J=6.2Hz,2H); 13 C NMR(100MHz, CDCl3)δ:171.81,151.57,150.67,142.48,132.13,129.98,129.92,129.89,129.83,129.33,127.22,125.47,125.44,125.40,125. 36,124.96,123.20,111.87,111.66,108.14,104.64,104.38,104.11,7 4.63,74.58,59.98,55.68,53.64,46.17,37.76,34.22,28.51; HRMS-ESI calcd.for C 32 H 33 ClF5N6O2(M+H) + 663.2268, found 663.2273.

[0032] Prove that its structural formula is: The molecular formula is C 32 H 32 ClF5N6O2.

[0033] Example 2: In vitro anti-inflammatory activity experiment.

[0034] (1) In vitro anti-inflammatory activity of indole-azole derivative WM-20

[0035] The MTT assay and Griess colorimetric assay were used to evaluate the effects of indole-azole derivative WM-20 on RAW264.7 macrophage activity and LPS-induced NO release in RAW264.7 cells. The results are shown in Table 1. As shown in Table 1, the indole-azole derivative WM-20 had an effect on CC of RAW264.7 cells. 50 The IC value was 27.6±2.94 μM for LPS-induced NO production in RAW264.7 cells. 50The value was: 3.09±0.17μM. To further evaluate the anti-inflammatory activity of the indole-azole derivative WM-20, an LPS-induced RAW 264.7 cell model was used. The results are shown in Table 2. As shown in Table 2, compared with the control group, the expression of inflammatory cytokines IL-6, IL-1β, and TNF-α in LPS-treated RAW 264.7 cells was significantly increased (P<0.05). When the indole-azole derivative WM-20 was co-cultured with LPS-induced RAW264.7 cells, compared with the LPS-stimulated group, 1.25μM of the indole-azole derivative WM-20 significantly inhibited IL-6 production, while 5μM of the indole-azole derivative WM-20 significantly inhibited the secretion of IL-6 and IL-1β, and the differences were statistically significant (P<0.01). In vitro intervention with the indole-azole derivative WM-20 had no significant effect on TNF-α secretion (P>0.05).

[0036] Table 1 Effects of indole-azole derivative WM-20 on RAW264.7 cell viability and LPS-induced NO production in RAW264.7 cells ( n=3)

[0037]

[0038] Table 2 Effects of indole-azole derivative WM-20 on the expression of inflammatory cytokines in LPS-induced RAW264.7 cell model ( n=3)

[0039]

[0040] Note: Compared with the control group, # P<0.05, ## P < 0.01 and ### P < 0.001; *P < 0.05 and **P < 0.01 compared with LPS.

[0041] Therefore, it can be seen from Tables 1 and 2 that the indole-azole derivative WM-20 exhibited strong anti-inflammatory activity in the LPS-induced RAW264.7 macrophage inflammation model.

[0042] Example 3: In vivo anti-inflammatory activity experiment of indole-azole derivative WM-20.

[0043] (1) A DSS (dextrose sulfate sodium salt)-induced ulcerative colitis mouse model was established to investigate the anti-inflammatory effect of the indole-azole derivative WM-20 in vivo. The results are shown in Tables 3 and 4. The body weight of the ulcerative colitis mice began to decrease from the 4th day, and the body weight was significantly reduced on the 7th day compared with the control mice, with a significant difference (P < 0.05). The body weight of the indole-azole derivative WM-20 drug intervention showed an upward trend, but the difference was not significant (P > 0.05). The model group mice suffered more severe diarrhea and rectal bleeding from the 2nd day, and the disease activity index (DAI) score was significantly increased from the 2nd to the 7th day compared with the control group (P < 0.01). Compared with the model group mice, the high-dose group of the indole-azole derivative WM-20 significantly reduced diarrhea, bloody stools, and disease activity index (DAI) scores on the 4th and 5th days. The results in Table 5 show that compared with the control group, the colon of the model group mice was significantly shortened and the H&E staining pathological injury score was significantly increased, with statistically significant differences (P<0.001). Intervention with the indole-azole derivative WM-20 (25 and 50 mg / kg) not only significantly inhibited colon shortening but also significantly reduced the colon pathological injury score, with both differences being statistically significant (P<0.05).

[0044] Table 3 Effects of indole-azole derivative WM-20 on body weight changes in DSS-induced ulcerative colitis model ( n=8)

[0045]

[0046] Note: Compared with the control group, ## P<0.01.

[0047] Table 4 Effects of indole-azole derivative WM-20 on DAI score of DSS-induced ulcerative colitis model ( n=8)

[0048]

[0049] Note: Compared with the control group, ## P < 0.01 and ### P < 0.001; *P < 0.05 compared with LPS.

[0050] Table 5 Effects of indole-azole derivative WM-20 on colon length and pathological damage in DSS-induced ulcerative colitis model ( n=6)

[0051]

[0052] Note: Compared with the control group,### P < 0.001; *P < 0.05 and **P < 0.01 compared with LPS.

[0053] (2) Since IL-6, IL-1β, and TNF-α are important inflammatory markers associated with colitis, and their levels are positively correlated with the severity of colitis, the levels of these markers in colon tissue were detected. The results are shown in Table 6. The levels of IL-6 and IL-1β in the colon tissue of mice in the model group were significantly higher than those in the control group, and the differences were statistically significant (P<0.05). However, compared with the model group, treatment with the indole-azole derivative WM-20 (25 mg / kg and 50 mg / kg) significantly inhibited the levels of IL-6 and TNF-α in colon tissue. The indole-azole derivative WM-20 at a dose of 50 mg / kg significantly reduced the expression of IL-1β, and the difference was statistically significant (P<0.05). This indicates that the indole-azole derivative WM-20 can alleviate intestinal mucosal inflammation in mice with colitis.

[0054] Table 6 Effects of indole-azole derivative WM-20 on the expression of inflammatory cytokines in the colon of DSS-induced ulcerative colitis model ( n=6)

[0055]

[0056] Note: Compared with the control group, # P < 0.05; *P < 0.05 and **P < 0.01 compared with LPS.

[0057] Therefore, it can be seen from Tables 3, 4, 5 and 6 that the indole-azole derivative WM-20 effectively alleviates intestinal inflammation in DSS-induced ulcerative colitis mice and has a good anti-inflammatory effect in vivo.

[0058] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An indole-azole derivative WM-20, characterized in that: Its structural formula is: , molecular formula is C 32 H 32 ClF5N6O2.

2. The method for preparing the indole-azole derivative WM-20 according to claim 1, characterized in that: The steps include: ； (1) 5-chloroindole, methyl acrylate and 1,8-diazabicyclo[5.4.0]undec-7-ene were added to acetonitrile and heated at 60°C to obtain compound 1; (2) Compound 1 obtained in step (1) and NaBH3CN were added to acetic acid and reacted at 0°C for 12 h to obtain compound 2; (3) Compound 2 obtained in step (2) was refluxed with ethylenediamine in CH3OH to obtain a yellow oily substance, which is compound 3; (4) Compound 3 and compound 4 obtained in step (3) and triethylamine were added to ethanol, heated under reflux for 12 h, concentrated under vacuum, and eluted by column chromatography. The mixture was separated and dried to obtain a light yellow oily substance, which was compound 5; (5) Compound 5 obtained in step (4), 4-trifluoromethylbenzyl bromide and K2CO3 were added to dichloromethane and reacted at room temperature overnight. The mixture was concentrated in vacuo and eluted by column chromatography. The mixture was separated and dried to obtain a yellow viscous substance, which was the indole-azole derivative WM-20.

3. The preparation method according to claim 2, characterized in that In step (4), anhydrous sodium sulfate was used for drying, and the eluent was a mixture of dichloromethane and triethylamine in a volume ratio of 100:0.

5.

4. The preparation method according to claim 2, characterized in that In the drying step (5), anhydrous sodium sulfate is used, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of (80-50):1 and 0.5 v / v% Et3N.

5. Use of the indole-azole derivative WM-20 according to claim 1 for preparing in vitro and in vivo anti-inflammatory drugs.

6. The use according to claim 5, characterized in that The indole-azole derivative WM-20 is used for preparing a drug for treating ulcerative colitis in vivo.

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