Sesquiterpene derivative as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpene derivative as well as a preparation method and application thereof. The invention provides a sesquiterpene derivative with a novel structure. The sesquiterpene derivative is obtained by modifying atractylenolide III. An activity research shows that the sesquiterpene derivative has a stronger effect of inhibiting IgE (Immunoglobulin E) or histamine secretion than atractylenolide III. Technicians in the field know that allergy is mediated by IgE, and histamine secretion under IgE stimulation is an important reason for causing various allergy symptoms. Therefore, the sesquiterpene derivative provided by the invention has the prospect of being developed into drugs for preventing and treating diseases, such as food allergy and other allergic diseases, which are prevented and / or treated by inhibiting IgE or histamine secretion.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to the modification, preparation and application of natural products, and in particular to a sesquiterpene derivative and a preparation method and application thereof. Background Art

[0002] Food allergy is a type I hypersensitivity disease mediated by immunoglobulin E (IgE). Its core mechanism is that specific food proteins (such as peanuts, milk, eggs, etc.) bind to the body's IgE antibodies, triggering the degranulation of mast cells and basophils, releasing inflammatory mediators such as histamine and leukotrienes, leading to multi-system symptoms such as skin, respiratory tract, and digestive tract. In severe cases, it can cause anaphylactic shock or even death. With the increasing incidence of allergic diseases worldwide, especially the significant increase in the proportion of food allergies in children, this disease has become a major threat to public health. It not only severely restricts patients' dietary freedom and quality of life, but also comes with long-term psychological burdens and social costs. Current clinical treatment is mainly based on strict avoidance of allergens, supplemented by antihistamines and glucocorticoids to relieve acute symptoms, and epinephrine for emergency rescue. However, the above methods can only temporarily control allergic reactions and cannot block the IgE-mediated immune cascade reaction. In addition, long-term use of drugs may cause side effects such as drug resistance and metabolic disorders. Although desensitization strategies such as oral immunotherapy can induce tolerance through gradual exposure, their efficacy is unstable, treatment cycles are long, and there is a risk of inducing severe allergic reactions. Therefore, the development of new drugs that target the IgE pathway and precisely regulate abnormal immune responses has become a key direction for solving the bottleneck in food allergy treatment.

[0003] Atractylodes lactone III is a natural product. Studies have shown that it may have certain IgE inhibitory activity, but its activity has not reached a significant level, and its application in the treatment of food allergies still faces many challenges. Summary of the Invention

[0004] The present invention aims to solve the technical defect of insufficient IgE inhibitory activity of Atractylodes macrocephala lactone compounds in the prior art, and provides a sesquiterpene derivative, a preparation method of the compound, and use of the compound in preparing anti-allergic drugs.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A compound having a structure as shown in formula (I) or a pharmaceutically acceptable salt or solvate thereof:

[0007]

[0008] A method for preparing the compound represented by the above formula (I) comprises the following steps: dissolving atractyl lactone III and p-formylbenzoic acid in dichloromethane, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine, stirring to react, removing dichloromethane by distillation under reduced pressure after the reaction is completed, adding ethyl acetate to the residue, stirring, washing with a saturated sodium bicarbonate aqueous solution, concentrating the ethyl acetate solution, loading the sample on a normal phase silica gel column chromatography, eluting with an eluent, detecting by TLC, collecting the eluate corresponding to the target compound, and concentrating and drying to obtain the compound.

[0009] Preferably, the molar ratio of the atractyl lactone III to the p-formylbenzoic acid is 1:(1-2).

[0010] Preferably, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to triethylamine is (1-2):2.

[0011] Use of the above-mentioned compound or its pharmaceutically acceptable salt or solvate for preparing a drug for preventing and treating a disease that can be prevented and / or treated by inhibiting IgE or histamine secretion.

[0012] Preferably, the disease to be prevented and / or treated by inhibiting IgE or histamine secretion is an allergic disease.

[0013] More preferably, the allergic disease is food allergy.

[0014] More preferably, the drug uses the above-mentioned compound or its pharmaceutically acceptable salt or solvate as the active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0015] More preferably, the auxiliary material is solid, liquid or semi-solid.

[0016] More preferably, the dosage form is capsule, tablet, injection, granule, pill or powder.

[0017] Beneficial effects:

[0018] The present invention provides a novel sesquiterpene derivative obtained by modifying Atractylodes macrocephala lactone III. Activity studies have shown that the sesquiterpene derivative has a stronger inhibitory effect on IgE or histamine secretion than Atractylodes macrocephala lactone III. Those skilled in the art are aware that allergies are mediated by IgE, and histamine secretion under IgE stimulation is a major cause of various allergic symptoms. Therefore, the sesquiterpene derivative provided by the present invention has the prospect of being developed into a drug for the prevention and treatment of diseases that are prevented and / or treated by inhibiting IgE or histamine secretion, such as allergic diseases such as food allergies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To evaluate the safety of different concentrations of Atractylodes lactone III and the compound of formula (I) on U266 cells;

[0020] Figure 2 The inhibitory effects of different concentrations of Atractylodes lactone III and the compound of formula (I) on the release of IgE from U266 cells;

[0021] Figure 3 To evaluate the safety of different concentrations of Atractylodes lactone III and the compound of formula (I) on RBL-2H3 cells;

[0022] Figure 4 The inhibition rates of different concentrations of Atractylodes lactone III and the compound of formula (I) on histamine release under IgE stimulation. DETAILED DESCRIPTION

[0023] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] Example 1: Preparation of compound of formula (I)

[0025]

[0026] Atractylodes lactone III (1 mmol) and p-formylbenzoic acid (1 mmol) were dissolved in 50 mL of dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.5 mmol) and triethylamine (TEA, 2 mmol) were added. The reaction system was stirred at 25°C for 24 h. After the reaction, dichloromethane was removed by distillation under reduced pressure. 30 mL of ethyl acetate was added to the residue, stirred, and washed three times with 20 mL of saturated sodium bicarbonate aqueous solution. The ethyl acetate solution was concentrated and loaded onto normal phase silica gel column chromatography. The solution was isocratically eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 3:1. The product was detected by TLC. The eluate corresponding to the target compound was collected, concentrated and dried to obtain the compound of formula (I) with an HPLC purity of 98%.

[0027] Spectral data of the compound represented by formula (I): 1 H NMR (500MHz, CDCl3) δ10.10(s,1H),8.14(m,2H),7.96(m,2H),4.87(s,1H),4.60(s,1H),3.70(s,1H),2.92(d,J=14.3Hz,1H) ,2.77(m,1H),2.38(m,2H),2.04(m,1H),1.95(m,1H),1.94(s,3H),1.82(m,1H),1.56(m,1H),1.34–1.25(m,3H),0.98(s,3H). 13C NMR (125MHz, CDCl3) δ191.46,171.16,162.76,157.96,147.97,139.49,134.64,130.37,129.63,124 .35,107.06,104.65,51.29,49.92,40.78,36.67,35.90,24.62,22.19,16.66,8.47.ESI-MS:Calcd.C 23 H 24 O5,[M+Na] + m / z:403.15,found:403.14.

[0028] Example 2: Safety evaluation of Atractylodes lactone III and compound of formula (I) on U266 cells and their inhibition rate on IgE

[0029] 1. Experimental materials and instruments

[0030] Phosphate-buffered saline (PBS), fetal bovine serum (FBS), RPMI1640 medium, trypan blue, and dimethyl sulfoxide (DMSO) were purchased from Beijing Solaibao Technology Co., Ltd. U266 cell lines were obtained from the laboratory of Henan University of Traditional Chinese Medicine, and IgE detection kits were purchased from MabTech Technology Co., Ltd. CO2 incubator (Thermo Fisher Scientific); DK-8D constant temperature water bath (Shanghai Jinghong Laboratory Equipment Co., Ltd.); Centrifuge 5702 centrifuge (Eppendorf, Germany); 1510 microplate reader (Thermo Fisher Scientific); autoclave (Japan); and double-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.). Atractylodes lactone III was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The compound of formula (I) was prepared according to Example 1.

[0031] 2. Experimental methods

[0032] Atractylodes lactone III and the compound of formula (I) were weighed separately and dissolved in DMSO to prepare a 40 mg / mL solution. The U266 frozen cell line was revived in a water bath at 37°C, 10 mL of PBS was added and centrifuged to remove DMSO, and the cells were resuspended in RPMI1640 medium containing 10% FBS and cultured in an incubator at 37°C and 5% CO2. After the cells recovered, the U266 cell density was counted using a cell counting plate and diluted to 1×105 cells / mL using pre-prepared RPMI 1640 complete medium containing 10% fetal bovine serum (without double antibody). The 96-well culture plates were grouped and treated, and blank groups, model groups and experimental groups with different concentrations were set up. Three replicates were set up in each group. 100 μL of complete medium was added to the blank group, and 100 μL of cell suspension with a concentration of 1×105 cells / mL was added to the model group and experimental group. Then, different treatments were performed on them. 100 μL of complete culture medium was added to the blank group and the model group, while 100 μL of complete culture medium containing the tested Atractylodes lactone III or the compound of formula (I) was added to the experimental group, so that the final drug concentrations in the 96-well plate reached 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively. The culture plates were placed in a 37°C incubator and incubated for 72 hours. After the incubation, U266 cells were suspended. After the 96-well plate was mixed evenly, 50 μL was taken out and the survival rate of the cells after treatment with different concentrations of drugs was determined by trypan blue staining. The culture plates were then centrifuged at 1000 rpm for 5 minutes, the supernatant was collected, and the total IgE level was measured using an ELISA kit.

[0033] The IgE experimental data are expressed as inhibition rate (I), where E 实验 represents the detection value of IgE in each experimental group, E 模型 represents the average value of IgE in U266 cells of the model group, E 空白 Represents the blank group test value, the formula is as follows:

[0034] Inhibition rate of each group Ic=100%-(E 实验 -E 空白 ) / (E 模型 -E 空白 )×100%.

[0035] The survival rate and IgE inhibition rate of U266 cells in response to different concentrations of Atractylodes lactone III and the compound of formula (I) were statistically analyzed and compared using bar graphs. ****P<0.0001.

[0036] 3. Results

[0037] The results are as follows Figure 1As shown in Table 1 and Table 2, the compound of formula (I) prepared by the present invention has high safety in the concentration range of 1.25 to 20 μg / mL and has no obvious inhibitory effect on cells. Figure 2 As shown, within the concentration range of 1.25 to 20 μg / mL, the inhibitory effect of the compound of formula (I) on IgE at the same concentration is stronger than that of atractylodes lactone at the same concentration. The above results show that the compound of formula (I) provided by the present invention has a significant inhibitory effect on IgE, and the effect is better than that of atractylodes lactone III. Those skilled in the art know that allergies are mediated by IgE, so the compound of formula (I) has a better anti-allergic effect than atractylodes lactone III.

[0038] Table 1 Inhibition rate of IgE by compounds at different concentrations

[0039]

[0040] Example 3: Inhibition rate of Atractylodes lactone III and compound of formula (I) on histamine release from RBL-2H3 cells stimulated by IgE

[0041] 1. Experimental materials and instruments

[0042] Anti-dinitrophenol (DNP) and IgE monoclonal antibodies (anti-DNP IgE) were purchased from Sigma, USA. Bovine serum albumin (DNP-BSA), PBS buffer, and the CCK8 test kit were purchased from Beijing Solaibao Technology Co., Ltd. RBL-2H3 cell lines were obtained from the laboratory of Henan University of Traditional Chinese Medicine, and the histamine test kit was purchased from MabTech Technology Co., Ltd. A carbon dioxide incubator (Thermo Fisher Scientific); a DK-8D constant temperature water bath (Shanghai Jinghong Laboratory Equipment Co., Ltd.); a Centrifuge 5702 centrifuge (Eppendorf, Germany); a 1510 microplate reader (Thermo Fisher Scientific); an autoclave (Japan); and a double-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.). Atractylodes lactone III was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The compound of formula (I) was prepared according to Example 1.

[0043] 2. Experimental methods

[0044] Atractylodes lactone III and the compound of formula (I) were weighed and dissolved in DMSO to prepare an 80 mg / mL solution. First, RBL-2H3 cells in the logarithmic growth phase were collected and the cell density was adjusted to 1×10 5Cells were seeded into 96-well culture plates at a volume of 100 μL per well. Blank, control, model, and experimental groups at different concentrations were set up, with three replicates per well. The inoculated culture plates were transferred to a 37°C, 5% CO2 constant temperature incubator for incubation. After 24 hours of full adherence and growth, the culture medium was replaced. The 96-well culture plates were divided into groups and treated, with blank, model, and experimental groups at different concentrations, with three replicates per well. 100 μL of complete culture medium was added to the blank group, and 100 μL of a cell suspension at a concentration of 1 × 105 cells / mL was added to the model and experimental groups. Immediately after treatment, the blank, model, and experimental groups were sensitized with 100 μL of complete culture medium containing anti-DNP IgE. After incubation for 24 hours, the cell supernatant was aspirated and discarded. 100 μL of complete culture medium was added to the blank group and the model group, while 100 μL of complete culture medium containing the tested Atractylodes lactone III or the compound of formula (I) was added to the experimental group, respectively, to a final concentration of 10 μg / mL and 20 μg / mL, respectively. After incubation for 30 minutes, the cell supernatant was discarded, and 100 μL of DNP-BSA with a concentration of 1 μg / mL was added to the blank group, the model group, and the experimental group to activate the cells, and the reaction was terminated by ice bath for 10 minutes. The culture plate was centrifuged at 1000 rpm for 5 minutes, the supernatant was collected, and the histamine level was determined using an ELISA kit. In addition, 100 μL of a mixture of culture medium and CCK-8 detection reagent (volume ratio 9:1) was added to each well. After incubation at 37°C in the dark for 4 hours, the absorbance (OD value) of each well at a wavelength of 450 nm was measured using an enzyme reader, and the relative cell viability of each group of cells was calculated using the formula:

[0045] Cell viability (%) = (OD 实验 -OD 空白 ) / (OD 对照 -OD 空白 )×100%.

[0046] Histamine experimental data are expressed as inhibition rate (I), where E 实验 represents the detection value of histamine in each experimental group, E 模型 represents the average value of histamine in the model group, E 空白 Represents the detection value of histamine in the blank group, and the formula is as follows:

[0047] The inhibition rate of each experimental group Ic=100%-(E 实验 -E 空白 ) / (E 模型 -E 空白 )×100%.

[0048] The survival rate and histamine inhibition rate of RBL-2H3 cells at different concentrations of Atractylodes lactone III and the compound of formula (I) were statistically analyzed and compared using bar graphs. ***P<0.001.

[0049] 3. Results

[0050] The results are as follows Figure 3 As shown in Table 2, the compound of formula (I) prepared by the present invention has high safety in the concentration range of 10-20 μg / mL and has no significant effect on cell viability. Figure 4 It is shown that the inhibition rate of histamine release of mast cells stimulated by IgE of the compound of formula (I) at 10 to 20 μg / mL is higher than that of Atractylodes lactone III. The above results show that the compound of formula (I) provided by the present invention can significantly inhibit histamine secretion under IgE stimulation, and the effect is significantly better than that of Atractylodes lactone III. Those skilled in the art know that histamine secretion under IgE stimulation is an important cause of various allergic symptoms. Therefore, the compound of formula (I) has a better anti-allergic effect than Atractylodes lactone III.

[0051] Table 2 Inhibition rate of histamine secretion under IgE stimulation by different concentrations of compounds

[0052]

[0053] The above results indicate that the compound of formula (I) provided by the present invention can inhibit IgE and significantly inhibit the release of histamine under IgE stimulation, and has the prospect of being developed into a drug for anti-allergic diseases such as food allergies.

[0054] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt or solvate thereof:

2. A method for preparing the compound represented by formula (I) according to claim 1, characterized in that: The method comprises the following steps: dissolving atractyl lactone III and p-formylbenzoic acid in dichloromethane, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine, stirring to react, removing dichloromethane by distillation under reduced pressure after the reaction is completed, adding ethyl acetate to the residue, stirring, washing with a saturated sodium bicarbonate aqueous solution, concentrating the ethyl acetate solution, loading the solution on a normal phase silica gel column chromatography, eluting with an eluent, detecting by TLC, collecting the eluate corresponding to the target compound, concentrating and drying to obtain the target compound.

3. The preparation method according to claim 2, wherein: The amount ratio of the atractyl lactone III to the p-formylbenzoic acid is 1:(1-2).

4. The preparation method according to claim 2, wherein: The molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to triethylamine is (1-2):

2.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof for preparing a medicament for preventing and / or treating a disease that can be prevented and / or treated by inhibiting IgE or histamine secretion.

6. The use according to claim 5, characterized in that: The disease to be prevented and / or treated by inhibiting the secretion of IgE or histamine is an allergic disease.

7. The use according to claim 6, characterized in that: The allergic disease is food allergy.

8. The use according to any one of claims 5 to 7, characterized in that: The drug uses the compound according to claim 1 or its pharmaceutically acceptable salt or solvate as the active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that: The auxiliary material is solid, liquid or semi-solid.

10. The use according to claim 8, characterized in that: The dosage form is capsule, tablet, injection, granule, pill or powder.

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