A terpenoid compound, and a preparation method and application thereof

Abstract

The present application relates to Chinese herbal medicine active organic small molecule compound technical field, specifically discloses a kind of terpenoid compound with formula (I) structure;And its preparation method and application are provided.The terpenoid compound with formula (I) structure is found and extracted from the underground part of Salvia przewalskii Maxim, and it is found that the compound can significantly inhibit the activity of PTP1B protein tyrosine phosphatase by pharmacological activity test, and has the possibility to become the lead compound for the development of new drugs for the treatment of type 2 diabetes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of active organic small molecule compounds of Chinese herbal medicine, and particularly relates to a terpenoid compound and a preparation method and application thereof. BACKGROUND

[0002] Type 2 diabetes (T2DM), also known as non-insulin-dependent diabetes mellitus or adult-onset diabetes, is a chronic metabolic disease that usually occurs after the age of 35-40, accounting for more than 90% of all diabetes patients, and one of the obvious features is insulin resistance.

[0003] The etiology of type 2 diabetes is complex, involving genetic, environmental, racial, age, and lifestyle factors. Genetic factors account for a certain proportion of type 2 diabetes, and studies have shown that about 80%-90% of type 2 diabetes patients have other family members with diabetes. Other factors, such as environmental factors, such as obesity, lack of exercise, and poor eating habits, can increase the risk of type 2 diabetes.

[0004] Protein tyrosine phosphatase 1B (PTP1B) belongs to the family of protein tyrosine phosphatases (PTP). PTP1B mainly regulates the phosphorylation level of insulin receptors in cells through dephosphorylation, thereby affecting the downstream insulin signaling pathway. Some early studies have shown that PTP1B inhibitors have potential therapeutic effects in enhancing insulin sensitivity and reducing blood glucose levels, so the development of new drugs targeting PTP1B has always been a hot research field in this field.

[0005] Salvia przewalskii Maxim. is a perennial herb of the Labiatae Salvia genus, distributed in western Gansu, western Sichuan, northwest Yunnan, and Tibet, China. It grows in forest edges, roadsides, ditch edges, and shrubs at an altitude of 2100-4050 meters. The roots are used as medicine, with the same effect as Danshen, and are used as substitutes for Danshen in Sichuan and Lijiang in Yunnan, with the effects of promoting blood circulation, removing blood stasis, and calming the mind. SUMMARY

[0006] The first object of the present application is to provide a terpenoid compound with PTP1B inhibitory activity.

[0007] The second object of the present application is to provide a preparation method of the above-mentioned compound.

[0008] The third object of the present application is to provide the application of the above-mentioned compound.

[0009] The application is realized by the technical scheme below.

[0010] A terpenoid compound has the following formula (I) structure:

[0011]

[0012] The preparation method of the terpenoid compound comprises the following steps:

[0013] S1: dry ground parts of Gynostemma pentaphyllum are sliced and extracted by ethanol to obtain an ethanol extract;

[0014] S2: the ethanol extract is concentrated by vacuum distillation to obtain a total extract, and the total extract is dissolved in water and then extracted by petroleum ether, ethyl acetate and n-butanol to obtain different extract solutions, which are concentrated by vacuum to obtain corresponding extracts;

[0015] S3: the obtained ethyl acetate extract is separated by HP-20 macroporous resin column chromatography to obtain components Fr.1-Fr.5;

[0016] S3-1: component Fr.4 is separated and purified by small-pore resin MCI gel CHP-20P / P120 column chromatography to obtain components Fr.4.1-Fr.4.2;

[0017] S3-2: component Fr.4.1 is separated and purified by Sephadex LH-20 to obtain components Fr.4.1.1-Fr.4.1.4;

[0018] S3-3: component Fr.4.1.2 is separated and purified by normal phase silica gel chromatography to obtain components Fr.4.1.2.1-Fr.4.1.2.4;

[0019] S3-4: component Fr.4.1.2.2 is separated and purified by Sephadex LH-20 to obtain components Fr.4.1.2.2.1-Fr.4.1.2.2.3;

[0020] S3-5: component Fr.4.1.2.2.2 is purified by a semi-preparative liquid chromatography column to obtain a compound having formula (I).

[0021] As preferred, in step S1, the used ethanol is industrial-grade ethanol with a volume fraction of 95%, and the extraction temperature is room temperature; in step S2, the vacuum degree of the vacuum distillation and vacuum concentration is 0.078 MPa, the water bath temperature is 45°C, and the temperature of the water used to dissolve the total extract is 60°C.

[0022] Further preferably, in step S3, the mobile phase used for separation is a methanol / water system, with a volume ratio of 0%, 30%, 50%, 80%, or 100%.

[0023] Further preferably, in step S3-1, the elution system used for separation and purification is a methanol / water system, with a volume fraction of methanol of 30%-100%.

[0024] Further preferably, in step S3-2, the elution system used for separation and purification is a dichloromethane / methanol system with a volume ratio of 1:1.

[0025] Further preferably, in step S3-3, the normal-phase silica gel used has a particle size of 200-300 mesh, and the elution system is a petroleum ether / acetone system with a volume ratio of 1:0-0:1.

[0026] Further preferably, in step S3-4, the elution system used for separation and purification is a methanol system; and in step S3-5, the semi-preparative liquid chromatography column used is a Waters SunFire C18 OBD Prep Column 10μM, 10mm x 250mm, with a mobile phase of a methanol / water system, a volume fraction of methanol of 40%, a flow rate of 2mL / min, a binary infusion pump of Waters 1525, and a PDA detector of Waters 2998.

[0027] The terpenoid compound described above is used for preparing a medicine for inhibiting the activity of PTP1B.

[0028] The terpenoid compound described above is used for preparing a medicine for treating type 2 diabetes.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The diterpenoid compound with the structure of formula (I) is separated from the underground parts of Salvia przewalskii Maxim. It is found through pharmacological activity testing that the compound can significantly inhibit the activity of PTP1B protein tyrosine phosphatase, has the possibility of becoming a new drug for treating type 2 diabetes, and can be used as a new type of small-molecule PTP1B inhibitor lead compound, thereby providing a template molecule for synthetic chemistry and a new idea for developing a new type of small-molecule PTP1B inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the inhibitory activity of compound 1 on PTP1B, IC 50 ;

[0032] Figure 2 is the inhibitory activity of compound 1 on PTP1B, Ki;

[0033] Figure 3 is the docking mode of compound 1 at the allosteric site;

[0034] Figure 4 is the docking mode of compound 1 at the catalytic site;

[0035] Figure 5 is the RMSD of compound 1 at the allosteric site;

[0036] Figure 6 is the RMSD of compound 1 at the catalytic site. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0038] Unless otherwise specified, the materials in the examples are all commercially available products.

[0039] Example 1

[0040] Extraction and preparation of small molecule compound 1 having the structure of formula (I):

[0041] The dried underground part of sage is sliced ​​and extracted with 60L of ethanol at room temperature to obtain an ethanol extract, wherein the ethanol uses industrial-grade ethanol with a volume fraction of 95%. The ethanol extract is concentrated by vacuum distillation to obtain a total extract, wherein the vacuum distillation is performed under conditions of a vacuum degree of 0.078MPa, a water bath temperature of 45°C, and a rotary evaporator speed of 30 rpm. After being dissolved in 60°C warm water, the extract is extracted with petroleum ether, ethyl acetate, and n-butanol, respectively. After the extract is concentrated under vacuum, extracts of a petroleum ether portion, an ethyl acetate portion, and an n-butanol portion are obtained, and the vacuum concentration is performed under conditions of a vacuum degree of 0.078MPa, a water bath temperature of 45°C, and a rotary evaporator speed of 30 rpm. The ethyl acetate extract was separated by HP-20 macroporous resin column chromatography with a methanol / water system as the mobile phase at a volume ratio of (0%, 30%, 50%, 80%, 100%) to obtain components Fr.1–Fr.5. Component Fr.4 was separated and purified by polystyrene small-pore resin MCI gel CHP-20P / P120 column chromatography with a methanol / water system (V MeOH% = 30% - 100%) to obtain components Fr.4.1 - Fr.4.2; component Fr.4.1 was separated and purified by Sephadex LH-20 to obtain components Fr.4.1.1 - Fr.4.1.4, with a dichloromethane / methanol system (volume ratio 1:1) as the elution system; component Fr.4.1.2 was separated and purified by normal-phase silica gel chromatography with a particle size of 200-300 mesh, with a petroleum ether / acetone system (volume ratio 1:0 - 0:1) as the elution system, to obtain components Fr.4.1.2.1 - Fr.4.1.2.4; component Fr.4.1.2.2 was separated and purified by Sephadex LH-20, with a methanol system as the elution system, to obtain components Fr.4.1.2.2.1 - Fr.4.1.2.2.3; component Fr.4.1.2.2.2 was purified by semi-preparative liquid chromatography to obtain compound 1, with a Waters SunFire C18 OBD Prep Column 100A, 10 μM, 10 mm x 250 mm as the liquid chromatography column, a methanol / water system as the mobile phase, a methanol volume fraction of 40%, 2 mL / min, a binary pump Waters 1525 Binary Pump, and a PDA detector Waters 2998.

[0042] Compound 1 was identified to have the following structure (I):

[0043]

[0044] Compound 1: 1 H NMR (600 MHz, CDC13 δ in ppm, J in Hz): δ H 7.63 (d, J = 7.8 Hz, H-7), 7.50 (d, J = 7.8 Hz, H-6), 7.30 (s, H-16), 5.20 (dd, J = 11.5, 5.1 Hz, H-1), 2.38 (dd, J = 8.8, 5.1 Hz, H-2β), 2.15 (s, H3-17), 1.93 (dd, J = 10.5, 3.9 Hz, H-3α), 1.86 (t, J = 12.5, 12.1 Hz, H-3β), 1.60 (ddd, J = 12.5, 11.5, 8.8 Hz, H-2β), 1.42 (s, H3-19), 1.09 (s, H3-18); 13 C NMR (150 MHz, CDC13): δ C168.8 (C-12), 168.7 (C-11), 153.6 (C-14), 148.1 (C-10), 143.8 (C-5), 140.4 (C-16), 131.1 (C-7), 130.1 (C-6), 122.8 (C-15), 122.2 (C-9), 117.1 (C-13), 77.6 (C-1), 36.8 (C-3), 34.6 (C-4), 31.6 (C-18), 30.8 (C-19), 26.1 (C-2), 9.9 (C-17).

[0045] Example 2

[0046] Inhibition activity experiment of compound 1 on PTP1B protein

[0047] 1. Preparation of test solution of compound 1: 1 mg of compound 1 was dissolved in 500 μL of DMSO (dimethyl sulfoxide) and vortexed to mix, and then stored in a refrigerator at -20°C.

[0048] 2. Preparation of positive control drug: 1 mg of ursolic acid was dissolved in 1000 μL of DMSO and vortexed to mix, and then stored in a refrigerator at -20°C.

[0049] 3. Preparation of Time End-Point Assay Buffer: 50 mM Bis-Tris, 100 mM NaCl, 10 mM DTPA and 20 mM substrate pNPP (4-nitrophenyl phosphate disodium salt hexahydrate) were added to deionized water, and the pH value was adjusted to 6.0 with HC1 or NaOH. In this paragraph, mM refers to the molar concentration of the substance in the prepared solution.

[0050] 4. Preparation of Exchange Buffer: 50 mM Bis-Tris, 50 mM Tris, 100 mM sodium acetate, 10 mM DTPA and 5% Tween 80 were added to deionized water, and the pH value was adjusted to 7.0 with HC1 or NaOH. In this paragraph, mM refers to the molar concentration of the substance in the prepared solution.

[0051] 5. Preparation of reaction termination solution: 8.0 g of NaOH was dissolved in 100 mL of deionized water to obtain a 2 mol / L NaOH solution.

[0052] 6. Pretreatment of PTP1B protein: The desalting column used for treatment is Thermo Fisher Zebra series; the protein used in this experiment is purchased from Aikewei Biological Engineering Co., Ltd., 3.16 mg of PTP1B protein is stored in 1 mL of storage solution, which contains 50 mM (here mM refers to the molar concentration of solute in the storage solution) HEPES, 2 mM DTT, 150 mM NaCl and 50% glycerol by volume, the pH value is 7.5 at 4℃, and it is stored in a -20℃ refrigerator. First, centrifuge in a low-temperature ultracentrifuge at 0℃ and 1000g for 2 min, remove the storage solution in the column, then use Exchange Buffer to centrifuge under the same conditions to replace the desalting column buffer four times, then take 20 μL of the protein stock solution and dilute it in 80 μL of Exchange Buffer, finally add the pre-equilibrated desalting column, centrifuge the protein stock solution under the same conditions, and finally obtain 100 μL of desalted PTP1B protein and store it in an ice bath.

[0053] 7. Inhibition rate of test compound 1 on PTP1B activity at 30 μM concentration: First, 120 μL of incubation solution containing 30.03 μM of compound 1 was prepared using Exchange buffer. The specific preparation method of the incubation solution was as follows: first, 10 μL of compound 1 test solution in step 1 was taken and diluted with 90 μL of Exchange buffer, then 6 μL of the diluted compound 1 drug solution was taken and added to 114 μL of Exchange buffer, finally 120 μL of incubation solution containing 30.03 μM of compound 1 was obtained. 58 μL of the incubation solution was taken and added to a 1.5 mL centrifuge tube, then 2 μL of pretreated protein in step 6 was added to prepare an incubation solution with a total volume of 60 μL and a final concentration of 30 μM of compound 1, and incubated at 25°C for 20 min, then 10 μL of the incubation solution (which contained pretreated protein and 30 μM of compound 1) was taken and added to a centrifuge tube containing 490 μL of Time End-Point Assay Buffer, the reaction was terminated after 30 min by adding 2 mol / L NaOH solution, and three sets of parallel experiments were performed; then 200 μL of the reaction solution was taken and placed in a 96-well plate, and the absorbance at 410 nm was detected using an enzyme marker. Another 58 μL of buffer containing 30.03 μM of compound 1 was taken and added to 1 μL of Exchange Buffer and 1 μL of DMSO as a control group, and incubated for 20 min, then 10 μL was added to 490 μL of Time End-Point Assay Buffer, the reaction was terminated after 30 min by adding 2 mol / L NaOH solution, then 200 μL of the reaction solution was taken and placed in a 96-well plate, and the absorbance at 410 nm was detected using an enzyme marker, and the experimental data was analyzed using GraphPad Prism 9 program ANVOA. The experimental results showed that the inhibition rate of compound 1 on PTP1B at 30 μM was 91.61%.

[0054] 8. IC of test compound 1 on PTP1B inhibition activity 50: The incubation solution containing 100 μM final concentration of compound 1 was prepared by using Exchange Buffer, and then serially diluted to obtain the final concentration of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, respectively. 58 μL of different concentrations of incubation solution was taken into a 1.5 mL centrifuge tube, and then 2 μL of pretreated protein in step 6 was added, and incubated at 25 °C for 20 min. Then 10 μL of the incubation solution was taken into a centrifuge tube containing 490 μL of Time end-point Assay buffer, and reacted for 30 min. The reaction was terminated by adding the reaction termination solution, and three sets of parallel experiments were performed. Then 200 μL of the reaction solution was taken into a 96-well plate, and the absorbance at 410 nm was detected by using the microplate reader. Another 58 μL of the incubation solution described in step 7 was taken, and 1 μL of Exchange Buffer and 1 μL of DMSO were added separately, and incubated for 20 min. Then 10 μL was added to 490 μL of Time End-Point Assay Buffer as a control group. After 30 min of reaction, the reaction was quenched by the reaction termination solution, and then 200 μL of the reaction solution was taken into a 96-well plate, and the absorbance at 410 nm was detected by using the microplate reader. The experimental data was analyzed by using the GraphPad Prism 9 program, as shown in Figure 1 . The experimental results show that compound 1 has a significant inhibitory effect on PTP1B, and the IC 50 = 14.80 μM.

[0055] 9. Test the apparent inhibition constant K i : The incubation solution containing different final concentrations of compound 1 was prepared by using Exchange Buffer, and then the same concentration of pretreated protein in step 6 was added. The Time End-Point Assay Buffer was serially diluted to obtain several final concentration reaction solutions. The experiment was that each group of inhibitor concentration corresponded to different concentration of pNPP reaction solution, and after 5 min of reaction, 500 μL of 2 mol / L NaOH was added to terminate the reaction. Finally, the absorbance at 410 nm was measured by using the microplate reader; different concentrations of pNPP solution were prepared, and the absorbance at 410 nm was measured. The standard curve was obtained by using the GraphPad Prism 9 program, and the reaction rate was calculated by combining the standard curve with the absorbance of the kinetic experiment. The experimental data was analyzed by using the Michaelis-Menten equation, Lineweaver-Burk double-reciprocal plot and GraphPad Prism 9 program, as shown in Figure 2 . The experimental results show that compound 1 has a significant inhibitory effect on PTP1B, and the K i = 18.00 μM.

[0056] 10. Molecular docking of compound 1 and target protein PTP1B: All processing and analysis of molecular docking were completed using the package Schrodinger 2021.4, and the crystal structure of human PTP1B protein used for docking was downloaded from the RCSB Protein DataBank database (PDB ID: 7S4F). The ProteinPrep module was used to hydrogenate and charge the PTP1B protein, the Grid Generation module was used to construct the docking pocket, the LigPrep module was used to process the small molecule for docking, the Glide SP algorithm was used for molecular docking, and the PoseViewer was used to visualize the docking results, as shown in FIGS. 10A and 10B. From the docking results, it can be seen that in the allosteric site, compound 1 forms a hydrogen bond with residue GLN262, and forms hydrophobic interactions with residues VAL49, ILE219, and forms a π-π stacking interaction with aromatic amino acid residue TYR46; in the catalytic site, compound 1 forms a hydrogen bond with residues ARG24, ARG254, and forms hydrophobic interactions with amino acid residues ALA27, PHE52, ILE219, MET258, GLN262. Figure 3 、 4

[0057] 11. Molecular dynamics simulation and calculation of binding free energy (MM-PBSA) of compound 1 and PTP1B complex: The molecular dynamics simulation program is the GROMACS2019.6 package, the protein force field is AMBER14SB, the small molecule force field is GAFF2 force field combined with the RESP2 charge model, and the water model is the OPC explicit model. After energy minimization and sufficient pre-equilibration, the simulation system is formally simulated in the NPT system, the total simulation time is 100 ns, the temperature is 268.15 K, and the pressure is 1 atm. After the simulation is completed, the gmx rms module is used to analyze the trend of the ligand-receptor distance over time, as shown in FIGS. 11A and 11B. MM-PBSA is a computational physics method used to calculate the absolute binding free energy of a ligand, which has the advantages of accuracy and computational amount, and is widely used in the field of computer-aided drug design. The gmx_MMPBSA 1.6.2 package is used to calculate the receptor MM-PBSA in the present application, and the calculation parameters all use default parameters, and the Single-Trajectory Method is used for calculation. Among them, the binding free energy of the small molecule in the allosteric site is –14.10 kcal / mol, and the binding free energy in the catalytic site is –21.70 kcal / mol, indicating that compound 1 can bind to both sites, and is more inclined to bind to the catalytic site, which is consistent with the conclusion obtained by experiment, and is a mixed inhibition mode, being both a reversible competitive inhibitor and a reversible non-competitive inhibitor. Figure 5 、 6

[0058] ​​12. The compound 1 having the structure of formula (I) is a chemical substance with specific bioactive function isolated from the underground part of Salvia przewalskii Maxim, which has both chemical and biological dualities, retains the special active molecular structure of the lead compound, and greatly improves the bioactivity and reduces the artificial cost. The compound 1 has a significant inhibitory effect on PTP1B protein, and the IC 50 value is 14.80 μM; the active small molecule compound 1 has the potential to be developed into a new type of drug for treating type 2 diabetes, can be used as a lead compound of a new type of small molecule PTP1B inhibitor, provides a template molecule for synthetic chemistry, and provides a new idea for developing a new type of small molecule PTP1B inhibitor.

[0059] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a terpenoid compound in the preparation of a medicament for inhibiting the activity of PTP1B, characterized in that, The terpenoid compound has the following formula (I) structure:

2. Use of a terpenoid compound for the manufacture of a medicament for the treatment of type 2 diabetes, characterized in that, The terpenoid compound has the following formula (I) structure:

3. Use according to claim 1 or 2, characterized in that, The preparation method of the terpenoid compound comprises the following steps: S1: dry ganci sage underground part slices are extracted by ethanol to obtain an ethanol extract; S2: the ethanol extract is concentrated by reduced pressure distillation to obtain a total extract, and after the total extract is dissolved in water, different extract liquids are extracted by using petroleum ether, ethyl acetate and n-butanol respectively, and the extract liquids are concentrated by reduced pressure to obtain corresponding extracts; S3: the obtained ethyl acetate extract is separated by HP-20 macroporous resin column chromatography to obtain components Fr.1-Fr.5; S3-1: component Fr.4 is separated and purified by small pore resin MCI gel CHP-20P / P120 column chromatography to obtain components Fr.4.1-Fr.4.2; S3-2: component Fr.4.1 is separated and purified by dextran gel Sephadex LH-20 to obtain components Fr.4.1.1-Fr.4.1.4; S3-3: component Fr.4.1.2 is separated and purified by normal phase silica gel chromatography to obtain components Fr.4.1.2.1-Fr.4.1.2.4; S3-4: component Fr.4.1.2.2 is separated and purified by dextran gel Sephadex LH-20 to obtain components Fr.4.1.2.2.1-Fr.4.1.2.2.3; S3-5: component Fr.4.1.2.2.2 is purified by semi-preparative liquid chromatography column to obtain a compound with formula (I).

4. Use according to claim 3, characterized in that, In step S1, the ethanol used is industrial-grade ethanol with a volume fraction of 95%, and the extraction temperature is room temperature; in step S2, the vacuum degree of the reduced pressure distillation and reduced pressure concentration is 0.078 MPa, the water bath temperature is 45°C, and the temperature of the water used to dissolve the total extract is 60°C.

5. Use according to claim 3, characterized in that, In step S3, the mobile phase used for separation is a methanol / water system with a volume ratio of 0%, 30%, 50%, 80% and 100%.

6. Use according to claim 5, characterized in that, In step S3-1, the elution system used for separation and purification is a methanol / water system with a methanol volume fraction of 30%-100%.

7. Use according to claim 6, characterized in that, In step S3-2, the elution system used for separation and purification is a dichloromethane / methanol system with a volume ratio of 1:

1.

8. Use according to claim 7, characterized in that, In step S3-3, the particle size of the normal phase silica gel used is 200-300 mesh, and the elution system is a petroleum ether / acetone system with a volume ratio of 1:0-0:

1.

9. Use according to claim 8, characterized in that, In step S3-4, the elution system used for separation and purification is a methanol system; in step S3-5, the semi-preparative liquid chromatography column used is a Waters SunFire C18 OBD Prep Column 10 μM, 10 mm x 250 mm, the mobile phase is a methanol / water system, the volume fraction of methanol is 40%, the flow rate is 2 mL / min, the binary pump is Waters 1525, and the PDA detector is Waters 2998.

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