Indole-azole derivative WM-20 as well as preparation method and application thereof

Abstract

The invention discloses an indole-azole derivative WM-20. The structural formula of the indole-azole derivative WM-20 is # imgabs0 #, and the molecular formula of the indole-azole derivative WM-20 is C32H32ClF5N6O2. The invention also discloses a preparation method of the indole-azole derivative WM-20 and application of the indole-azole derivative WM-20 in preparation of in-vitro and in-vivo anti-inflammatory drugs.

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 involving 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 the patients' quality of life. According to statistics, the global prevalence of ulcerative colitis is 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 therapeutic drugs include: aminosalicylic acid drugs, which mainly inhibit prostaglandin synthesis and reduce intestinal inflammation; glucocorticoids, which are used to control acute attacks in the short term and have significant effects, but long-term use may cause side effects such as osteoporosis and diabetes; immunosuppressants, which are used in patients with moderate to severe ulcerative colitis to suppress the immune system and reduce inflammation; biologics and targeted synthetic small molecule drugs, which are often used in patients with moderate to severe ulcerative colitis who do not respond well to other drugs; but these drugs are relatively expensive and may cause many adverse reactions. Despite the increasing and expanding number of available therapeutic drugs and treatment options, only a few drugs can maintain and reduce ulcerative colitis, and the remission rate in real life does not exceed 30-60%, and more than 10% of patients need to undergo proctocolectomy for treatment. Therefore, there is an urgent need to develop new therapeutic drugs and effective treatment strategies to improve treatment effects, reduce medical costs, and meet patients' needs for more effective and personalized treatment options to meet this increasingly severe global health challenge. Summary of the invention

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

[0004] The technical solution of the present invention is 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) adding compound 1 obtained in step (1) and NaBH3CN to acetic acid and reacting at 0°C for 12h to obtain compound 2;

[0010] (3) reflux the compound 2 obtained in step (2) 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 are added to dichloromethane, and the mixture is reacted at room temperature overnight. The mixture is concentrated in vacuo and eluted by column chromatography. The mixture is separated and dried to obtain a yellow viscous substance, which is 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 new compound, which is used for preparing anti-inflammatory drugs and has significant in vivo and in vitro 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 treatment strategy for treating ulcerative colitis.

[0019] The preparation method of the indole-azole derivative WM-20 of the present invention, the applicant connects the indole or indoline related to ethylenediamine and the active fragment of fluconazole to synthesize a new compound indole-azole derivative WM-20, and the obtained new compound indole-azole derivative WM-20 not only has obvious anti-inflammatory effect in vitro, but also can effectively treat ulcerative colitis in vivo. See the applicant's previous work 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 technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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: Add 10 mmol of 5-chloroindole, 15 mmol of methyl acrylate, and 3 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) into a round-bottom flask, use acetonitrile (CH3CN) as solvent, and heat the reaction at 60°C to obtain compound 1;

[0027] Step 2: Preparation of compound 2: Add 10 mmol of compound 1 and 20 mmol of NaBH3CN into a round-bottom flask, use 20 mL of acetic acid (AcOH) as solvent, and react at 0°C for 12 h to obtain compound 2;

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

[0029] Step 4: Preparation of compound 5: 5 mmol of compound 3, 5 mmol of compound 4 and 0.3 mmol of triethylamine (Et3N) were added to a round-bottom flask, and heated under reflux with ethanol as solvent for 12 h; after the reaction was completed by TLC detection, vacuum concentration was performed and column chromatography was performed with a mixture of dichloromethane and Et3N (100:0.5) as eluent, and light yellow oily substance was separated and dried to obtain compound 5;

[0030] Step 5: Preparation of indole-azole derivative WM-20: 1 mol of compound 5, 1.2 mmol 4-trifluoromethylbenzyl bromide and 2.0 mmol K2CO3 were added to a round-bottom flask, and dichloromethane (DCM) was used as solvent, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed by TLC detection, the mixture was concentrated in vacuo and then subjected to column chromatography, using a mixture of dichloromethane and methanol (80-50): 1 and about 0.5 v / v% Et3N (0.5 mL Et3N was added to 100 mL of the solution) as the eluent, and the mixture was separated and dried to obtain a light yellow oily substance, which was 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 method and Griess colorimetric method 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 can be seen from Table 1, the indole-azole derivative WM-20 has an effect on the CC of RAW264.7 cells. 50 The value was 27.6±2.94μM, and the IC value for LPS-induced NO production in RAW264.7 cells was 50The value is: 3.09±0.17μM. In order to further evaluate the anti-inflammatory activity of the indole-azole derivative WM-20, the LPS-induced RAW 264.7 cell model was used. The results are shown in Table 2. As can be seen from Table 2, compared with the control group, the LPS-treated RAW 264.7 cells significantly increased the expression of inflammatory cytokines IL-6, IL-1β and TNF-α, and the difference was statistically significant (P<0.05); when the indole-azole derivative WM-20 was added and co-cultured with LPS-induced RAW264.7 cells, compared with the LPS stimulation group, 1.25μM of the indole-azole derivative WM-20 significantly inhibited the production of IL-6, while 5μM of the indole-azole derivative WM-20 significantly inhibited the secretion of IL-6 and IL-1β, and the difference was significant (P<0.01). In vitro intervention with the indole-azole derivative WM-20 drug 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 (dextran sulfate sodium salt)-induced ulcerative colitis mouse model was constructed to explore 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. Compared with the control group mice, the body weight was significantly reduced on the 7th day, and the difference was significant (P<0.05). The indole-azole derivative WM-20 drug intervention showed an upward trend in body weight, 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 compared with the control group on the 2nd to 7th day (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, and the difference was statistically significant (P<0.001). The intervention of the indole-azole derivative WM-20 (25 and 50 mg / kg) not only significantly inhibited the shortening of the colon, but also significantly reduced the colon pathological injury score, and the differences were 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 Effect 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 difference was 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 alleviated the intestinal inflammation of DSS-induced ulcerative colitis mice and had a good anti-inflammatory effect in vivo.

[0058] Although 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 the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present 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: The 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) 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; (2) adding compound 1 obtained in step (1) and NaBH3CN to acetic acid and reacting at 0°C for 12h to obtain compound 2; (3) reflux the compound 2 obtained in step (2) with ethylenediamine in CH3OH to obtain yellow oily substances, namely, compounds 3 and 4; (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; (5) Compound 5 obtained in step (4), 4-trifluoromethylbenzyl bromide and K2CO3 are added to dichloromethane, and the mixture is reacted at room temperature overnight. The mixture is concentrated in vacuo and eluted by column chromatography. The mixture is separated and dried to obtain a yellow viscous substance, which is the indole-azole derivative WM-20.

3. The preparation method according to claim 2, characterized in that: In step (4), anhydrous sodium sulfate is used for drying, and the eluent is 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: The drying 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 0.5v / 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.

Images