Glycyrrhetinic acid derivatives, processes for their preparation, and uses thereof

Abstract

The application provides a glycyrrhetinic acid derivative and a preparation method and application thereof. A structural formula of the glycyrrhetinic acid derivative is shown in formula A. The glycyrrhetinic acid derivative has a novel structure, good anticoagulation and anti-inflammatory activities, can be preferably applied to preparation of a medicine with both anticoagulation and anti-inflammatory purposes, has a dual function, and has high application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and particularly to glycyrrhetinic acid derivatives, their preparation methods, and applications. Background Technology

[0002] Thrombotic diseases (local blood coagulation) are among the most morbid and fatal diseases in humans, seriously threatening human health. Currently, anticoagulants are mainly used clinically for the prevention and treatment of venous and arterial thromboembolic diseases. Traditional anticoagulants have many limitations, such as a narrow therapeutic window, slow onset and expiration of action, weakened anticoagulant effect by foods containing vitamin K, interactions with many drugs, and the potential for bleeding complications requiring frequent monitoring. Traditional anticoagulants can no longer meet the clinical needs of related diseases, and there is an urgent need to develop a new generation of anticoagulants with the following characteristics: good anticoagulant effect, high safety, strong specificity, predictable pharmacodynamics and pharmacokinetics, rapid onset and timely elimination of action, a wide effective therapeutic window, no need for clinical monitoring, oral administration, and minimal interactions with food and other drugs. In the cascade reaction of blood coagulation, FXa occupies a central position at the intersection of intrinsic and extrinsic coagulation pathways. FXa is an ideal target for next-generation anticoagulants. The rare case report of rivaroxaban, the first marketed FXa inhibitor, unexpectedly reaching 600 mg (10 mg daily oral dose), also showed no bleeding complications, further confirming the good safety profile of FXa inhibitors as anticoagulants. Glycyrrhetinic acid, derived from the plant kingdom, has been proven to be an ideal lead compound for FXa inhibitors; however, different glycyrrhetinic acid derivatives exhibit significantly different activities, with many even lacking pharmacological activity. Summary of the Invention

[0003] In view of this, the present invention proposes a new glycyrrhetinic acid derivative, its preparation method, and its application. The technical solution of the present invention is achieved as follows: a glycyrrhetinic acid derivative, the structural formula of which is shown in Formula A:

[0004]

[0005] The method for preparing glycyrrhetinic acid derivatives of the present invention includes the following steps:

[0006] (1) Dissolve 1.4-1.6 mmol of compound B, 1.8-2.0 mmol of benzyl bromide and 500-550 mg of potassium carbonate in 4-6 mL of LDMF according to the following proportions, and stir the mixture at room temperature for 8-18 h to obtain the reaction solution;

[0007] (2) Wash the reaction solution from step (1) sequentially with 8-12 ml of water, 8-12 ml of hydrochloric acid, and 8-12 ml of saturated sodium chloride solution. After washing, extract with 8-12 ml of ethyl acetate and collect the organic phase.

[0008] (3) After drying and filtering the organic phase in step (2), the organic phase is concentrated by vacuum distillation to obtain the crude product;

[0009] (4) The crude product in step (3) was purified by silica gel column chromatography to obtain compound A;

[0010] The structural formula of compound B is shown in formula B:

[0011]

[0012] Further, in step (1), the preparation method of compound B includes the following steps: adding zinc amalgam to a solution of 10-11 mmol glycyrrhetinic acid and 200 mL dioxane according to the following proportions, wherein the zinc amalgam is synthesized from 20-25 g zinc powder, 850-950 mg HgBr2 and 150-200 mL 4%-6% v / v HCl in the following mass-volume ratios; adding 8-12 mL concentrated hydrochloric acid dropwise under an ice-salt bath to obtain a mixture; stirring the mixture at room temperature for 5-6 h to obtain a reactant; extracting the reactant with ethyl acetate and collecting the organic phase; drying the organic phase, filtering, concentrating, and obtaining a crude product; passing the crude product through a silica gel column using petroleum ether:ethyl acetate in a volume ratio of 2.8-3.2:0.9-1.1 as the eluent to obtain the target product.

[0013] Further, in step (2), the hydrochloric acid is a 0.8-1.2% v / v HCl solution.

[0014] Further, in step (3), Na2SO4 is used for drying and filtration.

[0015] Further, in step (4), the purification uses an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 0.9–1.1:0.9–1.1.

[0016] The glycyrrhetinic acid derivatives described in this invention are used in the preparation of anticoagulant and / or anti-inflammatory drugs.

[0017] The glycyrrhetinic acid derivative described in this invention is used in the preparation of coagulation factor FXa inhibitors.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) The present invention synthesizes a new glycyrrhetinic acid derivative A with good anticoagulant and anti-inflammatory activities, which can be well applied to the preparation of drugs with both anticoagulant and anti-inflammatory uses, and inhibits IL-1β activity.

[0020] (2) The glycyrrhetinic acid derivative (compound A) prepared by the present invention has dual functions and has higher application value.

[0021] (3) The preparation method of the present invention produces compound A with high purity, and the preparation process is simple and easy to implement. Attached Figure Description

[0022] Figure 1 , 2 : NMR spectrum of compound A.

[0023] Figure 3 The IC50 standard curve shows the inhibitory effect of compound A on human FXa protease.

[0024] Figure 4 The in vitro anti-inflammatory results of compound A were obtained by detecting the expression level of IL-1β protein using immunofluorescence staining.

[0025] Figure 5 The in vitro anti-inflammatory results of compound A were obtained by detecting the expression level of IL-1β gene using RT-qPCR. Detailed Implementation

[0026] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0027] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0029] The compound of this invention has the following Chinese name:

[0030] PE: petroleum ether; EA: ethyl acetate; DMF: dimethylformamide; DMSO: dimethyl sulfoxide.

[0031] In this embodiment of the invention, the concentrated hydrochloric acid used is 37% v / v HCl.

[0032] The structural formula of glycyrrhetinic acid is as follows:

[0033]

[0034] Example 1

[0035] Compound 2: A zinc amalgam (synthesized from 22 g zinc powder, 901 mg HgBr2, and 180 mL 5% v / v HCl) was added to a solution of glycyrrhetinic acid (5 g, 10.62 mmol) and dioxane (200 mL). 10 mL of concentrated hydrochloric acid (37% v / v HCl) was added dropwise under an ice-salt bath to obtain a mixture. The mixture was then stirred at room temperature for 5 h to obtain the reactant. The filtrate after filtration was extracted with water (200 mL) and ethyl acetate (150 mL), and the organic phase was collected and washed with water (200 mL * 2). After washing, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain the crude product. The crude product was subjected to silica gel column chromatography (PE:EA = 3:1, v / v) to obtain purified white solid compound 2 (4.2 g, 87%).

[0036] NMR spectral data of compound 2: 1 H-NMR (400MHz, DMSO-D6) δ5.12(s,1H),4.26(d,J=5.2Hz,1H),2.95(dt,J=9.9,5.6Hz,2H),1.93(dt,J=13.3,6.7Hz,2H),1.81-1.63(m,5H ),1.55-1.38(m,7H),1.27(dq,J=33.8,10.9Hz,5H),1.03(d,J=28.3Hz,7H),0.86(d,J=2.8Hz,8H),0.84-0.74(m,6H),0.74-0.60(m,7H). 13 C-NMR (101MHz, DMSO-D6) δ174.34,145.13,122.17,77.33,55.25,53.97,48.13,47.61,43.66,41.61,41.11,38.93,38.76 ,38.67,36.99,36.66,32.73,32.13,28.73,28.65,27.50,27.00,26.21,23.55,18.54,17.69,17.07,16.56,15.79,0.64.

[0037] The structural formula of compound B is as follows:

[0038]

[0039] Compound A, compound B (700 mg, 1.53 mmol), benzyl bromide (314 mg, 1.836 mmol), and potassium carbonate (529 mg) were dissolved in DMF (5 mL). The mixture was stirred at room temperature for 8–18 h to obtain a reaction solution. The reaction solution was washed successively with water (10 mL), 1% v / v HCl (10 mL), H₂O (10 mL), and saturated NaCl solution (10 mL), and extracted with ethyl acetate (10 mL). The organic phase was collected. The organic phase was dried over Na₂SO₄, filtered, and concentrated by vacuum distillation to obtain a crude product (1.21 g). The crude product was purified by silica gel column chromatography (PE:EA = 1:1, v / v) to obtain compound A (520 mg, 62%).

[0040] The NMR spectrum of compound A is as follows: Figure 1 , 2 As shown.

[0041] NMR spectrum data of compound A: 1 H-NMR (400MHz, DMSO-D6) δ7.38-7.22(m,5H),5.16-5.02(m,2H),5.01(s,1H),2.95(dd,J=9.8,5.8Hz,1H),1.92(d,J=4.2Hz,1H),1.80-1.73(m,3H), 1.65(dd,J=26.4,13.3Hz,4H),1.43(dt,J=14.0,7.8Hz,6H),1.35-0.97(m ,12H),0.90-0.80(m,10H),0.78(d,J=12.3Hz,3H),0.63(d,J=3.7Hz,7H). 13 C-NMR(101MHz,CHLOROFORM-D)δ177.10,144.39,136.51,128.56,128.15,122.63,79.10,66.05,55.24,48.19,47.69,44.36,42.89,41.5 9,39.84,38.86,38.67,38.37,37.01,32.73,32.00,31.38,28.62,28 .18,27.30,27.02,26.22,26.02,23.59,18.44,16.85,15.68,15.59.

[0042] The structural formula of compound A (a glycyrrhetinic acid derivative) is as follows:

[0043]

[0044] Example 2 - Experiment with human FXa protease

[0045] Experimental materials: Human FXa enzyme (Abcam); chromogenic substrate S-2765 (Shanghai Yuanye Biotechnology Co., Ltd.); Tri-HCl buffer (0.05M Tris-HCl, 0.1M NaCl, 0.1% FBS)

[0046] Experimental Method: Add 25 μL of FXa solution diluted in 50 nM buffer, 40 μL of buffer, and 1 μL of sample solution dissolved in DMSO to a 96-well plate. Preheat at 37°C for 10 min. Add 40 μL of chromogenic substrate diluted in 1 mg / mL buffer and measure the absorbance change at 37°C and 405 nM. 1 μL of DMSO serves as a blank control. The inhibition rate is calculated using the following formula:

[0047]

[0048] The IC50 standard curve of the inhibition rate of compound A against human FXa protease is shown below. Figure 3 As shown, the experimental settings (final concentrations) were: 0.009 μM, 0.09 μM, 0.9 μM, 9 μM, and 90 μM.

[0049] The IC50 values ​​of compound A are shown in Table 1 and compared with the reference standard (glycyrrhetinic acid).

[0050] Table 1

[0051] Compound A 7.638±3.41 nM GA (Control) 32.6±1.24uM

[0052] The results showed that, compared with glycyrrhetinic acid, the glycyrrhetinic acid derivative compound A of the present invention has good anticoagulant activity.

[0053] Example 3 Anti-inflammatory experiment

[0054] (1) Experimental method:

[0055] Experimental Overview: RAW264.7 macrophages pre-stimulated with LPS (inducing an inflammatory response) were co-cultured with each group of drugs in vitro for 24 hours, with a blank control included. Subsequently, the expression levels of inflammation-related factors (IL-1β) were detected by Western blotting, RT-qPCR, and immunofluorescence.

[0056] 1. Cell culture and passage

[0057] RAW264.7 cells were cultured at 37°C in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were grown in a 5% CO2 atmosphere at 37°C. When the cell density reached 90%, cells were passaged. Adherent cells were scraped off with a cell scraper, then resuspended in a single suspension by blowing, and transferred to 15 mL centrifuge tubes. The cells were centrifuged at 1000 rpm (8 cm radius) for 5 min, the supernatant was discarded, and the cells were resuspended in fresh culture medium and seeded at a 1:3 ratio.

[0058] 2. Activation of the inflammatory response

[0059] Take RAW264.7 cell suspension (1×10⁻⁶) 6 Adding LPS (1 μg / mL) and culturing for 24 h elicited an inflammatory response.

[0060] 3. Detection of inflammatory factors

[0061] Each group of drugs was added to the above-mentioned RAW264.7 cells with inflammation at a concentration of 300 mg / mL and co-cultured for 24 hours. Then, the expression level of inflammation-related factor (IL-1β) was detected by RT-qPCR and immunofluorescence.

[0062] 1) RT-qPCR

[0063] Total RNA was isolated from RAW264.7 cells using TRIzol reagent (Thermo Scientific, Massachusetts, USA), and its concentration was detected using Nanodrop 2000. cDNA was synthesized using HiScript II reverse transcriptase, and quantitative PCR was performed using SYBR qPCR Master Mix (Vazyme, Nanjing, China) to determine the mRNA levels of IL-1β and TNF-α in the cells. Primer information is as follows:

[0064] Table 2 Primer sequences

[0065]

[0066] 2) Immunofluorescence staining

[0067] 1. Blocking: Completely cover the sample with 5% blank goat serum. The slides need to be placed in a humidified box. For cell culture plates, the well plates can be sealed directly and incubated in a constant temperature and humidity incubator at 37°C for 30 minutes.

[0068] 2. Primary antibody dilution: Dilute the antibody in antibody dilution buffer according to the instructions;

[0069] 3. Primary antibody incubation: Aspirate the blocking solution, add the diluted primary antibody, and incubate overnight at 4°C;

[0070] 4. Warming: Place the sample at room temperature and warm for 15 minutes.

[0071] 5. Washing: Remove antibody working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time;

[0072] 6. Secondary antibody dilution: Dilute the antibody in antibody dilution buffer according to the instructions;

[0073] 7. Secondary antibody incubation: 1 hour at room temperature, protected from light;

[0074] 8. Washing: Remove the secondary antibody working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time;

[0075] 9. Nucleus staining / mounting: Add DAPI working solution to the sample, incubate in the dark at room temperature for 10 minutes; remove the DAPI working solution, wash once with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time; add anti-fluorescence attenuation mounting medium, observe and acquire images under a fluorescence microscope.

[0076] (2) Experimental Results

[0077] In vitro anti-inflammatory drugs such as Figure 4-5 As shown in Table 3-4.

[0078] Table 3 RT-qPCR Detection Results

[0079] IL-1β 1.02 0.53 0.62

[0080] The results showed that, compared with glycyrrhetinic acid, the glycyrrhetinic acid derivative compound A of the present invention has good anti-inflammatory activity.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a glycyrrhetinic acid derivative in the preparation of anticoagulant drugs, wherein the structural formula of the glycyrrhetinic acid derivative is shown in Formula A: 。

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