A resveratrol derivative, its preparation method and application

Abstract

The present invention relates to a resveratrol derivative, a preparation method thereof and an application. The resveratrol derivative of the present invention: 3'-(pyridin-4-yl) resveratrol (BG-7), and its structural formula is shown as follows: It has obvious activity in promoting the enucleation of orthochromatic erythroblasts, can promote the cell cycle detachment of orthochromatic erythroblasts to make them enucleate, and further improve the production efficiency of mature erythrocytes in vitro, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a resveratrol derivative, a preparation method thereof and an application thereof. Background Art

[0002] Blood transfusion is an important means for treating chronic anemia or massive hemorrhage clinically. In recent years, due to the increasing spread of infectious diseases (such as COVID-19), population aging and other blood donation eligibility restrictions, the imbalance between blood supply and demand has occurred, and blood shortage has gradually become a potential global crisis. Based on the dilemma brought about by blood shortage, scientists are eager to find an artificial blood strategy to address this challenge. Currently, the research directions of artificial blood mainly fall into three categories, namely perfluorocarbon emulsions, synthetic heme and hemoglobin, and culturing functional red blood cells using autologous stem cells; as artificial synthetic products, the former two have properties limitations such as poor stability and inability to transport nutrients, and still cannot be compared with natural red blood cells; therefore, culturing functional red blood cells (RBCs) in vitro has become a promising coping strategy; this strategy can minimize the risk of blood-borne pathogen infection, is more easily obtained under strict quality control, and avoids the risk of immune incompatibility.

[0003] In mammals, mature red blood cells are produced through a finely regulated process of erythropoiesis, which begins with the transformation of hematopoietic stem cells (HSCs) into megakaryocyte–erythroid progenitor cells (MEPs). At the end of erythropoiesis, erythroblasts undergo 4 to 5 cell divisions, during which chromatin gradually condenses, histones are released, the cell cycle exits, and the nucleus polarizes. Subsequently, erythroblasts expel the condensed nucleus to form reticulocytes (Retics), and finally, after the degradation and excretion of residual RNA, they further become mature red blood cells (RBCs). The in vitro culture system of erythroid progenitor cells is a dynamic and strictly regulated process. Currently, multiple factors and signaling pathways have been found to affect or participate in the enucleation process of erythroid progenitor cells, but the mechanism remains unclear. To date, hematopoietic stem / progenitor cells, embryonic stem cells, and induced pluripotent stem cells have been used as starting materials for in vitro preparation of RBCs. However, due to the low enucleation rate, large-scale production of mature red blood cells has not been achieved. Therefore, there is an urgent need to study an effective erythroblast enucleation agent to increase the enucleation rate, thereby increasing the yield of RBCs in vitro and serving clinical applications. Summary of the Invention

[0004] The object of the present invention is to provide a resveratrol derivative, its preparation method and application, to solve the problem of low enucleation rate in the process of in vitro culturing functional red blood cells.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In the first aspect of the present invention, a resveratrol derivative is provided, including a compound of formula (I):

[0007]

[0008] In the second aspect of the present invention, a preparation method of the above resveratrol derivative is provided, and the preparation route is as follows:

[0009]

[0010] Wherein,

[0011] Step a: Mix 3,5-dimethoxybenzyl bromide and triethyl phosphite in a round-bottom flask, and then react at 140 °C to obtain Intermediate 1;

[0012] Step b: Dissolve Intermediate 1 in N,N-dimethylformamide under an ice bath, add sodium methoxide and 3-bromo-4-methoxybenzaldehyde successively, and then perform the Horner–Wadsworth–Emmons reaction at room temperature to obtain Intermediate 2;

[0013] Step c: Under argon protection, disperse Intermediate 2, 4-tributylstannylpyridine and Pd(PPh3)4 in anhydrous N,N-dimethylformamide, and perform the Stille reaction at 100 °C to obtain Intermediate 3;

[0014] Step d: Under argon protection, dissolve Intermediate 3 in dichloromethane, and then dropwise add a DCM solution (1 M) of BBr3, and perform demethylation at 20 °C to obtain the target compound BG-7.

[0015] As a preferred technical solution of the present invention, in the said step a, the mass ratio of 3,5-dimethoxybenzyl bromide to triethyl phosphite is (1 - 1.2):3.

[0016] As a preferred technical solution of the present invention, in the said step b, the mass-volume ratio of Intermediate 1 to N,N-dimethylformamide is (1 - 1.2):66, and the unit is g / mL.

[0017] As a preferred technical solution of the present invention, in the said step c, the mass-volume ratio of Intermediate 2 to N,N-dimethylformamide is (1 - 1.2):4, and the unit is g / mL.

[0018] As a preferred technical solution of the present invention, in the said step d, the molar ratio of BBr3 to Intermediate 3 is (5 - 7):1.

[0019] In the third aspect of the present invention, there is provided an application of the above-mentioned resveratrol derivative in the preparation of an erythrocyte enucleation promoter.

[0020] Advantages of the present invention:

[0021] The present invention provides a new compound resveratrol derivative, its preparation method and application. The preparation process is relatively simple, and the raw materials are easily obtained. The present invention for the first time discovers that the resveratrol derivative 3'-(pyridin-4-yl)resveratrol (BG-7) has obvious activity in promoting the enucleation of late erythroblasts, can make them enucleate by promoting the cell cycle detachment of late erythroblasts, and further improve the production efficiency of mature erythrocytes in vitro, and has broad application prospects. Description of the Drawings

[0022] Figure 1 Photographs of the number of TER119 positive cells detected by flow cytometry and the percentage of reticulocytes in the present invention;

[0023] Figure 2 Comparison chart of the effects of compound BG-7 and the blank group on the cell cycle;

[0024] Figure 3 Photograph of the membrane imaged by the ChemiDoc XRS imaging system (Bio-Rad).

[0025] The technical solution of the present invention will be further described in detail below through specific embodiments and examples. Specific Embodiments

[0026] The technical solution of the present invention will be clearly and completely described below through specific examples. Obviously, the described examples are part of the embodiments of the present invention, rather than all of them. All other examples obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0027] Example 1

[0028] Prepare resveratrol derivative (BG-7), namely 3'-(pyridin-4-yl) resveratrol, and its structural formula is shown as follows:

[0029]

[0030] The preparation steps are as follows:

[0031] (1) Mix 3,5-dimethoxybenzyl bromide (6.9 g) and triethyl phosphite (15.5 ml) in a round-bottom flask and react at 140 °C for 3 h to obtain intermediate 1, which is directly used for the next step of feeding without purification;

[0032] (2) Dissolve intermediate 1 (30 mmol) in 20 mL of N,N-dimethylformamide in an ice bath, add sodium methoxide (3.2 g) and 2-bromo-4-methoxybenzaldehyde (6.5 g) successively, and then carry out the Horner–Wadsworth–Emmons reaction at 25 °C for 12 h to obtain intermediate 2 (8.1 g). The total yield of steps 1 and 2 is 77%;

[0033] (3) Under argon protection, intermediate 2 (6.0 g, 17 mmol), 4-tributylstannylpyridine (6.258 g, 17 mmol), and Pd(PPh3)4 (0.98 g, 0.85 mmol) were dispersed in 24 mL of anhydrous N,N-dimethylformamide and subjected to the Stille reaction at 80 °C for 5 h to obtain intermediate 3 (5.4 g) with a yield of 91%;

[0034] (4) Under argon protection, intermediate 3 (0.7 g, 2 mmol) was dissolved in 5 mL of dichloromethane (DCM), and then 10 mL of BBr3 solution (1 M in DCM) was added dropwise. The mixture was stirred at 20 °C for 8 h. After the reaction was complete, 10 mL of water was added to quench the reaction. Finally, the final product BG-7 (0.45 g) was obtained after purification with a yield of 75%.

[0035] The physical and chemical and spectroscopic data of the resveratrol derivative BG-7 prepared in Example 1 are as follows:

[0036] BG-7 = (E)-5-(4-hydroxy-3-(pyridin-4-yl)styryl)benzene-1,3-diol, yellow powder, mp: >250 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.62 (dd, J = 4.4, 1.6 Hz, 2H), 7.67 (dd, J = 4.4, 1.6 Hz, 2H), 7.63 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 8.4, 2.3 Hz, 1H), 7.12 (d, J = 16.3 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 16.4 Hz, 1H), 6.57 (d, J = 2.2 Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H). 13C NMR (100 MHz, Acetone-d6) δ 158.74, 154.29, 149.46, 146.08, 139.82, 129.98, 128.61, 128.08, 127.67, 126.91, 125.71, 123.98, 116.71, 104.87, 101.96.

[0037] Example 2

[0038] Effect of BG-7 on enucleation of late erythroblasts

[0039] Mouse fetal liver TER119-negative cells (FLCs) were seeded at a cell density of 3×103 cells / 200 μl in round-bottom 96-well plates and cultured in Epocontaining medium for 30 h until they exited the cell cycle, and then treated with compound BG-7 (1, 5, 10 μM) dissolved in DMSO for 18 h. TER119 staining and Hoechst staining were used, and flow cytometry (NovoCyte TM ) was used to detect the number of TER119-positive cells and the percentage of reticulocytes.

[0040] As can be seen from Figure 1 , compared with the blank group (Ctrl), compound SY-21 can increase the enucleation efficiency of target cells in a concentration-dependent manner.

[0041] Example 3

[0042] Effect of BG-7 on the exit of orthochromatic erythroblasts from the cell cycle

[0043] 1) Plating and drug addition: A cell suspension of 300,000 / ml was seeded at 1 ml / well in 6-well plates and cultured at 37 °C and 5% CO2 until the TER119-negative cells (FLCs) adhered to the wall and spread to a normal morphology (12 h). A stock solution of compound BG-7 in DMSO was pre-diluted in fresh Epocontaining medium and added to the wells for continued incubation for 24 h.

[0044] 2) Staining and flow cytometry: The cells were digested, washed twice with pre-cooled PBS, centrifuged to obtain the supernatant, added with pre-cooled 70% ethanol solution at -20 °C, fixed at 4 °C for 12 h, centrifuged at 100 g, washed twice with pre-cooled PBS, the supernatant was discarded, 100 μL of PI / RNAse mixture was added in the dark and gently pipetted several times, and incubation was continued in the dark for 30 min, followed by analysis on the machine.

[0045] As can be seen from Figure 2 , compared with the blank group (Ctrl), compound BG-7 can arrest the cell cycle of target cells at the G0 / G1 phase, indicating that BG-7 can promote the exit of target cells from the cell cycle.

[0046] Example 4

[0047] Mechanism of BG-7 promoting enucleation of orthochromatic erythroblasts

[0048] TER119+ erythroblasts were cultured in Epocontaining medium with and without BG-7 (10 μM) for a specific time, and then the cells were lysed in RIPA lysis buffer, and a phosphatase inhibitor and a protease inhibitor mixture were added. Protein samples were separated by SDS-PAGE and wet transferred to a PVDF membrane (Millipore). At room temperature, the membrane was blocked with 5% BSA in TBST for 1 hour, and then probed with appropriate primary antibodies and enzyme-labeled secondary antibodies. Finally, the membrane was imaged using a ChemiDoc XRS imaging system (Bio-Rad).

[0049] It can be seen from Figure 3 that compared with the blank group (Ctrl), 10 μM compound BG-7 can significantly up-regulate the expression levels of p53, p-p53, and p21 proteins. The results indicate that BG-7 may promote terminal enucleation of orthochromatic erythroblasts by activating the p53-p21 signaling pathway.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A resveratrol derivative, characterized in that it is A compound represented by the following formula (I):

2. A method for preparing the resveratrol derivative according to claim 1, characterized in that, The preparation route is as follows:

3. The preparation method of the resveratrol derivative according to claim 2, characterized in that, In step a: 3,5-dimethoxybenzyl bromide and triethyl phosphite are mixed in a round-bottom flask, and then reacted at 140 °C to obtain intermediate 1; In step b: Intermediate 1 is dissolved in N,N-dimethylformamide under an ice bath, sodium methoxide and 3-bromo-4-methoxybenzaldehyde are added successively, and then subjected to the Horner-Wadsworth-Emmons reaction at room temperature to obtain intermediate 2; In step c: Under argon protection, intermediate 2, 4-tributylstannylpyridine and Pd(PPh3)4 are dispersed in anhydrous N,N-dimethylformamide, and subjected to the Stille reaction at 100 °C to obtain intermediate 3; In step d: Under argon protection, intermediate 3 is dissolved in dichloromethane, and then a DCM solution of BBr3 is added dropwise, and the methylation is removed at 20 °C to obtain the target compound BG-7.

4. The preparation method of the resveratrol derivative according to claim 3, characterized in that, In step a, the mass ratio of 3,5-dimethoxybenzyl bromide to triethyl phosphite is (1 to 1.2):

3.

5. The preparation method of the resveratrol derivative according to claim 3, wherein, In step b, the mass-volume ratio of intermediate 1 to N,N-dimethylformamide is (1 to 1.2):66, with the unit of g / mL.

6. The preparation method of the resveratrol derivative according to claim 3, wherein In step c, the mass-volume ratio of intermediate 2 to N,N-dimethylformamide is (1 to 1.2):4, with the unit of g / mL.

7. The preparation method of the resveratrol derivative according to claim 3, characterized in that, In step d, the molar ratio of BBr3 to intermediate 3 is (5 to 7):

1.

8. Use of a resveratrol derivative as described in claim 1 in the preparation of an erythrocyte enucleation promoter.

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