A dendrobium alkaloid small molecule active probe and preparation method

Abstract

The present invention discloses a dendrobium alkaloid small molecule active probe and a preparation method, belonging to the field of biomedicine technology. The chemical structure of the dendrobium alkaloid small molecule active probe is as follows: The dendrobium alkaloid small molecule active probe is used to determine the direct target of dendrobium alkaloid. Experimental results show that the dendrobium alkaloid small molecule probe synthesized for the first time has biological activity similar to that of dendrobium alkaloid and can specifically bind to the target of dendrobium alkaloid. This proves that it can be used as a tool to replace dendrobium alkaloid in binding research on related protein targets, fills the gap in the existing technology, and provides a basis for in-depth research on the mechanism of action of dendrobium alkaloid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a dendrobium alkaloid small molecule active probe and a preparation method thereof. Background Art

[0002] Dendrobine, a sesquiterpene alkaloid compound from the Dendrobium family, is one of the main active ingredients in the plant, exhibiting multiple pharmacological effects, including anti-inflammatory, antiviral, and neuroprotective properties. Previous studies have shown that dendrobine significantly improves motor dysfunction in Parkinson's disease (PD) mice by alleviating dopaminergic neuron damage and death, making it a potential therapeutic agent for the prevention and treatment of Parkinson's disease.

[0003] The identification and related research of drug target proteins have important theoretical guidance and practical value in the field of new drug development. They clearly elucidate the drug's mechanism of action and provide an important theoretical basis for new drug development at the molecular level. Dendrobium is currently the most studied Dendrobium compound, but the direct target of dendrobium has not been reported. The identification and verification of direct targets of dendrobium is of great significance for the development of drugs based on the dendrobium structure and the discovery of drugs based on dendrobium targets. Summary of the Invention

[0004] The present invention synthesizes a dendrobium alkaloid small molecule active probe for the first time. The chemical structure of the dendrobium alkaloid probe is as follows:

[0005]

[0006] Dendrobium alkaloid small molecule activity probe is used to determine the direct target of dendrobium alkaloid.

[0007] The synthesis reaction equation of the dendrobium alkaloid small molecule active probe is as follows:

[0008]

[0009] The preparation steps of the dendrobium alkaloid small molecule active probe are as follows:

[0010] (1) Add 10 mg of dendrobine and 5-15 mg of propargyl bromide to 10 mL of organic solvent;

[0011] (2) Under inert gas conditions, at a temperature of 20-55°C, in the dark, stirring and reacting for 8-36 hours;

[0012] (3) separation and purification to obtain a dendrobium alkaloid small molecule active probe, wherein the dendrobium alkaloid small molecule active probe is a brown solid.

[0013] The technical solutions are further defined as follows:

[0014] In step (1), the organic solvent is at least one of chloroform, dichloromethane, acetonitrile, n-butanol, and methanol.

[0015] In step (2), the inert gas is nitrogen.

[0016] In step (3), the separation and purification is to spin-dry the reaction solution and then separate and purify it on a chromatography plate; the developing agent for purification is a mixture of petroleum ether and acetone in a volume ratio of 7:3.

[0017] The beneficial technical effects of the present invention are embodied in the following aspects:

[0018] 1. This invention synthesizes the dendrobium alkaloid small molecule active probe for the first time.

[0019] 2. The experimental results show that the dendrobium alkaloid small molecule active probe has no toxic effect on cells and significantly improves MPP + The survival rate of induced PD cells was similar to that of dendrobium alkaloids.

[0020] 3. The experimental results show that the dendrobium alkaloid small molecule active probe specifically binds to the target of dendrobium alkaloid, and can be used as a tool to clarify the mechanism of action of dendrobium alkaloid in anti-Parkinson's disease.

[0021] The above results indicate that a biologically active dendrobium alkaloid small molecule probe was synthesized for the first time, which can specifically bind to the protein bound by dendrobium alkaloid in the Parkinson's disease model, and can be used as a research tool for the study of dendrobium alkaloid target fishing and its mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a mass spectrum of the structure of the dendrobium alkaloid small molecule active probe obtained in the present invention;

[0023] Figure 2 This is a hydrogen spectrum of the structure of the dendrobium alkaloid small molecule active probe obtained in the present invention;

[0024] Figure 3 This is the carbon spectrum of the structure of the dendrobium alkaloid small molecule active probe obtained in the present invention;

[0025] Figure 4 This is a graph showing the cytotoxicity of the dendrobium alkaloid small molecule active probe obtained in the present invention;

[0026] Figure 5 Improve the MPP of the dendrobium alkaloid small molecule active probe obtained by the present invention + The results of the survival rate of induced PD cells are shown in the figure;

[0027] Figure 6 The dendrobium alkaloid small molecule active probe obtained by the present invention reduces MPP + Results of lactate dehydrogenase release induced by PD cells;

[0028] Figure 7 The dendrobium alkaloid small molecule active probe obtained by the present invention is in MPP + Figure 2 shows the labeling results of induced PD cells (A: fluorescent labeling image; B: Coomassie brilliant blue image). DETAILED DESCRIPTION

[0029] In order to make the objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the embodiments.

[0030] Example 1: Synthesis and preparation of dendrobium alkaloid small molecule active probe

[0031] The synthesis reaction equation of the dendrobium alkaloid small molecule active probe is as follows:

[0032]

[0033] The specific preparation steps are as follows:

[0034] (1) Accurately weigh 10 mg of dendrobium alkaloids and 9.03 mg of propargyl bromide (the molar ratio of the two is 1:2), place them in an oblique two-way reaction tube, and fully dissolve them in 10 mL of organic solvent. 10 mL of organic solvent is prepared by mixing 5 mL of chloroform and 5 mL of acetonitrile.

[0035] (2) Under nitrogen protection, the mixture was stirred at 25°C in the dark for 12 h to obtain a reaction solution.

[0036] (3) The reaction solution was spin-dried; the sample was redissolved in methanol and spotted on a thin layer plate for chromatography, and purified in a developing solvent, which was a mixture of petroleum ether and acetone in a volume ratio of 7:3. A brown solid was obtained, i.e., 4.6 mg of the dendrobium alkaloid small molecule active probe, with a yield of 46%.

[0037] The characterization data of the obtained product dendrobium alkaloid probe are:

[0038] See also Figure 1 Results: Dendrobium alkaloids probe MS (ES) m / z: calculated value C 19 H 28 NO2 + [M] + 302.2115, mass spectrum value 302.1815.

[0039] See also Figure 2 Results: Dendrobium alkaloid probe 1H NMR (600MHz, CDCl3) δ5.70(d,J=16.5Hz,1H),5.24(dd,J=5.7,3.2Hz,1H),4.77(dd,J=16.4,2.0Hz,1H),4.68(d,J=3. 2Hz,1H),4.50(dd,J=12.4,8.8Hz,1H),3.73(s,3H),3.24(ddd,J=12.3,10.6,1.4Hz,1H),2.90(t,J=2.5Hz,1H),2.80( dd,J=10.4,8.6Hz,1H),2.62(dd,J=5.6,4.3Hz,1H),2.38–2.34(m,1H),2.33–2.29(m,1H),2.25–2.18(m,1H),2.15(d d,J=8.4,6.0Hz,1H),1.74–1.67(m,2H),1.67(s,3H),1.67–1.56(m,1H),1.28(d,J=6.4Hz,3H),0.99(d,J=6.4Hz,3H).

[0040] See also Figure 3 Results: Dendrobium alkaloid probe 13 C NMR (151MHz, CDCl3) δ176.11,82.15,76.40,75.81,72.16,69.41,55.77,52.57 ,51.49,50.55,46.43,45.90,42.88,33.99,32.49,31.33,24.63,21.64,21.10.

[0041] Example 2 Dendrobium alkaloid probe to MPP + Induced protective effect of PD cells

[0042] SH-SY5Y cells were quickly thawed and cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody. The cells were evenly distributed by blowing gently and placed in a cell culture incubator (37° C., 5% CO 2 ) for subsequent experiments.

[0043] 2.1. Dendrobium alkaloid probe has no toxic effect on cells

[0044] SH-SY5Y cells were cultured for 24 h until they were about 90% confluent, and then the cells were evenly seeded in a 96-well plate. After 24 h, the drugs were administered. A blank control group and a dendrobium alkaloid probe group (2.5, 5, 10, 25, 50, 100, and 200 μM) were set up. After 24 h, the cell survival rate was detected by CCK-8.

[0045] See also Figure 4The results showed that the blank control group of dendrobium alkaloid probe with a concentration of 2.5-200 μM had no obvious effect on the cell survival rate, which was 106.34%, 107.68%, 100.20%, 100.81%, 100.00%, 102.55% and 105.21%, respectively, and was not significant compared with that of the control group, indicating that the dendrobium alkaloid probe had no obvious cytotoxicity to the cells.

[0046] 2.2 Dendrobium alkaloid probe significantly improves MPP + Survival rate of induced PD cells

[0047] When SH-SY5Y cells were cultured for 24 h until they were about 90% full, the cells were evenly seeded in a 96-well plate. After 24 h, the drugs were administered and the model was established. The blank group, model group, dendrobium group (10 μM), and dendrobium probe group (10 μM) were set up. MPP was used 2 h after pre-dose. + (2.5 mM) to induce an in vitro Parkinson's disease model, and cell survival rate was detected by CCK-8 after 24 h.

[0048] See also Figure 5 The results showed that compared with the blank control group, the cell survival rate of the 10 μM dendrobium alkaloid probe was 66.77% of the blank control group, and the cell survival rate of the dendrobium alkaloid probe was 65.61% of the blank control group. The results showed that the dendrobium alkaloid probe significantly improved the MPP + The survival rate of PD cells induced by dendrobium was close to that of dendrobium (10 μM), indicating that the dendrobium probe has certain biological activity.

[0049] 2.3. Dendrobium alkaloid probe significantly reduces MPP + Induced lactate dehydrogenase release in PD cells

[0050] The cells were evenly seeded in a 96-well plate, and the drug was administered and the model was established 24 hours later. The maximum enzyme activity control group, blank control group, model group, dendrobium group (10 μM), and dendrobium probe group were set up, and 2.5 μM, 5 μM, and 10 μM pachymic acid were added to the blank control group and the model group, respectively. The same volume of dimethyl sulfoxide (DMSO) was added to the blank control group and the model group. 1-methyl-4-phenylpyridinium ion (MPP) was used 2 hours after pre-dose. + An in vitro Parkinson's disease model was induced with 2.5 mM (100 μL / well) of 1% LDH (2.5 mM). After 24 hours of culture, a lactate dehydrogenase releaser was added to the maximum enzyme activity control well and the cells were repeatedly pipetted. After an additional 1 hour of incubation in a cell culture incubator, the plate was centrifuged at 1000 rpm for 5 minutes. 60 μL of the supernatant was collected and the absorbance was measured at 490 nm on a microplate reader according to the manufacturer's instructions. The lactate dehydrogenase release rate was calculated as follows: (sample absorbance - blank solvent absorbance) / (maximum enzyme activity absorbance - background blank control absorbance) × 100%.

[0051] See also Figure 6 The results showed that the higher the lactate dehydrogenase release rate, the more severe the cell damage. The lactate dehydrogenase release rate of the blank control group was 7.77%, the lactate dehydrogenase release rate of the model group was 46.18%, the lactate dehydrogenase release rate of the dendrobium alkaloid group was 17.55%, and the lactate dehydrogenase release rate of the dendrobium alkaloid probe group with a concentration of 10 μM was 18.40%. The results showed that the dendrobium alkaloid probe significantly reduced MPP + The induced lactate dehydrogenase release of PD cells, and the effect of the dendrobium probe at a concentration of 10 μM was basically the same as that of dendrobium.

[0052] Example 3 Target fishing experiment

[0053] SH-SY5Y cells were cultured in a cell culture dish and tested after 24 hours when the cells were about 90% full. The blank control group (same volume of DMSO), probe group (Dendrobium probe 10μM), and competition group (Dendrobium 10μM + Dendrobium 10μM probe) were set up. MPP was used 2 hours after pre-administration. + (2.5mM) to induce PD cell model.

[0054] After 24 hours, the cell culture medium was discarded, and the cells were washed twice with pre-chilled PBS. The cells were collected by centrifugation (1500 rpm, 4°C, 5 min). The supernatant was discarded, cell lysis buffer was added, and the cells were fully lysed for 30 minutes. The supernatant was collected by centrifugation (15000 rpm, 4°C, 15 min). The protein concentration was determined by BCA kit and adjusted to 1 mg / mL for each group. The rhodamine fluorescent group was attached to the dendrobium alkaloid probe using click chemistry. To each group, 100 μM tetramethylrhodamine-azide (TAMRA-N3), 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), 1 mM tricarboxyethylphosphine (TCEP), and 1 mM anhydrous copper sulfate (CuSO4) were added, mixed thoroughly, and reacted in a rotating mixer at 25°C in the dark for 2 hours. After the reaction, pre-chilled acetone (-20°C) was added to precipitate the protein. The protein was then collected by centrifugation at 15,000 rpm for 10 minutes at 4°C. 200 μL of cell lysis buffer was then added and sonicated to re-dissolve the precipitated protein. Finally, the supernatant was collected by centrifugation (15,000 rpm, 4°C, 15 minutes). 5× Loading Butter was added and the reaction was terminated by boiling. SDS-PAGE electrophoresis was performed, and the gel was observed using a gel imaging analysis system. After completion, the gel was stained with Coomassie Brilliant Blue and photographed.

[0055] See also Figure 7Results A in Figure 1 show that a fluorescent band is clearly visible at 40-55 kDa in the gel, and the band in the competition group is significantly weakened, indicating that the dendrobium alkaloid probe can specifically bind to the protein bound by dendrobium alkaloid and can be used as a research tool for the target and mechanism of action of dendrobium alkaloid.

[0056] See also Figure 7 As shown in result B, the degree of Coomassie brilliant blue staining of the blank control group, probe group, and competition group was basically the same, indicating that the dendrobium alkaloid probe can specifically bind to the protein bound by dendrobium alkaloid and can be used as a research tool for the target of dendrobium alkaloid and its mechanism of action.

Claims

1. A dendrobium alkaloid small molecule active probe, characterized in that: The chemical structure of the dendrobium alkaloid small molecule active probe is as follows: 。 2. The dendrobium alkaloid small molecule active probe according to claim 1 is used for determining the direct target of dendrobium alkaloid for the purpose of non-disease diagnosis and treatment.

3. The method for preparing a dendrobium alkaloid small molecule active probe according to claim 1, characterized in that: The steps are as follows: (1) Add 10 mg of dendrobine and 5-15 mg of propargyl bromide to 10 mL of organic solvent; (2) Under inert gas protection, at a temperature of 20-55°C, in the dark, stir and react for 8-36 hours; (3) Separating and purifying to obtain a dendrobium alkaloid small molecule active probe, wherein the dendrobium alkaloid small molecule active probe is a brown-yellow solid.

4. The preparation method according to claim 3, wherein: In step (1), the organic solvent is at least one of chloroform, dichloromethane, acetonitrile, n-butanol, and methanol.

5. The preparation method according to claim 3, wherein: In step (2), the inert gas is nitrogen.

6. The preparation method according to claim 3, wherein: In step (3), the separation and purification is to spin-dry the reaction solution and then separate and purify it on a chromatography plate; the developing agent for purification is a mixture of petroleum ether and acetone in a volume ratio of 7:3.

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