Terpenoid as well as preparation method and application thereof

Abstract

The invention relates to the technical field of Chinese herbal medicine active organic small molecule compounds, and particularly discloses a terpenoid with a structure shown in a formula (I). The invention also provides a preparation method and application thereof. The terpenoids with the structure as shown in the formula (I) are found and extracted from the underground part of salvia japonica thunb, and pharmacological activity tests show that the terpenoids can significantly inhibit the activity of PTP1B protein tyrosine phosphatase and have the possibility of becoming lead compounds for research and development of new drugs for treating type II diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of active organic small molecule compounds of Chinese herbal medicines, and specifically relates to a terpene compound and a preparation method and application thereof. Background Art

[0002] Type 2 diabetes mellitus (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 to 40 years and accounts for more than 90% of diabetic patients. One of its obvious characteristics is insulin resistance.

[0003] The causes of type 2 diabetes are complex, involving factors such as genetics, environment, race, age and lifestyle. Genetic factors play a certain role in type 2 diabetes. Studies have shown that about 80%-90% of patients with type 2 diabetes have other family members with diabetes. Other factors, such as environmental factors, such as obesity, lack of exercise, and poor eating habits, will increase the risk of type 2 diabetes.

[0004] Protein Tyrosine Phosphatase 1B (PTP1B) belongs to the protein tyrosine phosphatase (PTP) family. 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 lowering blood sugar levels. Therefore, the development of new drugs targeting PTP1B has always been a hot research area in this field.

[0005] Ganxi sage (Salviaprzewalskii Maxim.) is a perennial herbaceous plant of the genus Salvia in the family Lamiaceae. It is distributed in western Gansu, western Sichuan, northwestern Yunnan, and Tibet in China. It grows on forest edges, roadsides, ditches, and under bushes at an altitude of 2100–4050 meters. The root is used as medicine, and its efficacy is the same as that of Danshen. It is used as a substitute for Gentiana macrophylla in Sichuan and as a substitute for Danshen in Lijiang, Yunnan. It has the effects of promoting blood circulation, removing blood stasis, and calming the nerves. Summary of the invention

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

[0007] The second object of the present invention is to provide a method for preparing the above compound.

[0008] The third object of the present invention is to provide the use of the above compounds.

[0009] The purpose of the present invention is specifically achieved through the following technical solutions:

[0010] A terpene compound having the following structure:

[0011]

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

[0013] S1: slices of the dried underground part of Salvia ganxiensis were extracted with ethanol to obtain an ethanol extract;

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

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

[0016] S3–1: Component Fr.4 was separated and purified using 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 was separated and purified using Sephadex LH–20 to obtain components Fr.4.1.1–Fr.4.1.4;

[0018] S3–3: Component Fr.4.1.2 was separated and purified by normal phase silica gel chromatography into components Fr.4.1.2.1–Fr.4.1.2.4;

[0019] S3–4: Component Fr.4.1.2.2 was separated and purified using 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 using a semi-preparative liquid chromatography column to obtain a compound having the structure of formula (I).

[0021] Preferably, 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.

[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%, 100%.

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

[0026] Further preferably, 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 C18OBD Prep Column 10μM, 10mm x250mm, mobile phase is methanol / water system, volume fraction of methanol is 40%, flow rate is 2mL / min, binary infusion pump is Waters1525, and PDA detector is Waters 2998.

[0027] Application of the above terpenoid compounds in the preparation of drugs for inhibiting PTP1B activity.

[0028] The application of the above terpenoid compounds in the preparation of drugs for treating type 2 diabetes.

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

[0030] The diterpene compound with the structure of formula (I) isolated from the underground part of Salvia glabra was found to significantly inhibit the activity of PTP1B protein tyrosine phosphatase through pharmacological activity tests. It has the potential to become a lead compound for the development of new drugs for the treatment of type 2 diabetes. It can be used as a lead compound for a new small molecule PTP1B inhibitor, providing a template molecule for synthetic chemistry and a new idea for the development of a new small molecule PTP1B inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 is the inhibitory activity of compound 1 against 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 Salvia glabra is sliced ​​and extracted with 60L 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 carried out under the conditions of a vacuum degree of 0.078MPa and a water bath temperature of 45°C, and the speed of a rotary evaporator is 30 rpm. After being dissolved in 60°C warm water, petroleum ether, ethyl acetate and n-butanol are used for extraction respectively. After the extract is concentrated by vacuum distillation, extracts of a petroleum ether part, an ethyl acetate part and a n-butanol part are obtained, and the vacuum distillation is carried out under the conditions of a vacuum degree of 0.078MPa and a water bath temperature of 45°C, and the speed of a rotary evaporator is 30 rpm. The ethyl acetate extract was separated by HP-20 macroporous resin column chromatography, and the mobile phase was a methanol / water system with 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, and the elution system was 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, and the elution system was dichloromethane / methanol system, with a volume ratio of 1:1; component Fr.4.1.2 was separated and purified by normal phase silica gel chromatography with a particle size of 200–300 mesh, and the elution system was petroleum ether / acetone system with a volume ratio of 1:0–0:1, and was divided into 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 LH-20 separation and purification, with a methanol system as the elution system, obtained 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 liquid chromatography column of Waters SunFire C18 OBD Prep Column 100A, 10μM, 10mm x 250mm, a methanol / water system as the mobile phase, a methanol volume fraction of 40%, 2mL / min, a binary infusion pump of Waters 1525BinaryPump, and a PDA detector of Waters2998.

[0042] The structure of compound 1 is as follows:

[0043]

[0044] Compound 1: 1 H NMR(600MHz,CDCl3δinppm,Jin Hz):δ H 7.63(d,J=7.8Hz,H–7),7.50(d,J=7.8Hz,H–6),7.30(s,H–16),5.20(dd,J=11.5,5.1Hz,H–1),2.38(dd,J=8.8,5.1Hz,H–2β),2.15(s,H3–17 ),1.93(dd,J=10.5,3.9Hz,H–3α),1.86(t,J=12.5,12.1Hz,H–3β),1.60(ddd,J=12.5,11.5,8.8Hz,H–2β),1.42(s,H3–19),1.09(s,H3–18); 13 C NMR (150MHz,CDCl3):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] Inhibitory activity of compound 1 on PTP1B protein

[0047] 1. Preparation of the test solution of compound 1: dissolve 1 mg of compound 1 in 500 μL DMSO (dimethyl sulfoxide), vortex to mix, and then freeze in a -20°C refrigerator.

[0048] 2. Preparation of positive control drug: Dissolve 1 mg of ursolic acid in 1000 μL of DMSO, vortex to mix, and then freeze in a -20°C refrigerator.

[0049] 3. Preparation of Time End–Point Assay Buffer: Add 50 mM Bis–Tris, 100 mM NaCl, 10 mM DTPA, and 20 mM substrate pNPP (4-nitrophenylphosphate disodium salt hexahydrate) to deionized water, and adjust the pH to 6.0 with HCl or NaOH. mM in this paragraph refers to the molar concentration of the substance in the prepared solution.

[0050] 4. Exchange Buffer: Add 50 mM Bis-Tris, 50 mM Tris, 100 mM sodium acetate, 10 mM DTPA, and 5% Tween 80 to deionized water, and adjust the pH to 7.0 with HCl or NaOH. mM in this paragraph refers to the molar concentration of the substance in the prepared solution.

[0051] 5. Preparation of reaction termination solution: Take 8.0g NaOH and dissolve it in 100mL deionized water to obtain 2mol / L NaOH solution.

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

[0053] 7. Test the inhibition rate of compound 1 on PTP1B activity at a concentration of 30 μM: First, use Exchange buffer to prepare 120 μL of incubation solution containing 30.03 μM compound 1. Specific preparation method of incubation solution: First, take 10 μL of the compound 1 test solution in step 1, add 90 μL of Exchange buffer to dilute, then take 6 μL of the diluted compound 1 solution and add 114 μL of Exchange buffer, and finally obtain 120 μL of incubation solution with a final concentration of compound 1 of 30.03 μM. Take 58 μL of incubation solution and add it to a 1.5 mL centrifuge tube, then add 2 μL of the pretreated protein in step 6 to prepare an incubation solution with a total volume of 60 μL and a final concentration of 30 μM of compound 1, and keep it warm at 25°C for 20 min. Then take 10 μL of incubation solution (the incubation solution here contains pretreated protein and 30 μM compound 1) and add it to a centrifuge tube containing 490 μL Time End–Point Assay Buffer. After reacting for 30 min, add 2 mol / L NaOH solution to terminate the reaction, and perform three sets of parallel experiments; then take 200 μL of the reaction solution in a 96-well plate, and use an enzyme reader to detect the absorbance at 410 nm. Another 58 μL buffer solution containing 30.03 μM compound 1 was taken, and 1 μL Exchange Buffer and 1 μL DMSO were added separately as the control group. After incubation for 20 minutes, 10 μL was added to 490 μL Time End–Point Assay Buffer. After 30 minutes of reaction, 2 mol / L NaOH solution was added to terminate the reaction. Then 200 μL of the reaction solution was taken into a 96-well plate, and the absorbance at 410 nm was detected by an ELISA instrument. The experimental data were analyzed using the GraphPad Prism 9 program ANVOA. The experimental results showed that the inhibition rate of compound 1 on PTP1B was 91.61% at 30 μM.

[0054] 8. Test the IC of compound 1 against PTP1B inhibition 50: Use Exchange Buffer to prepare an incubation solution containing compound 1 at a final concentration of 100 μM, and dilute it serially to obtain incubation solutions with final concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, respectively. Take 58 μL of incubation solutions of different concentrations and add them to 1.5 mL centrifuge tubes, then add 2 μL of the pretreated protein in step 6, incubate at 25°C for 20 min, then take 10 μL of the incubation solution and add it to a centrifuge tube containing 490 μL of Time end–point Assay buffer, react for 30 min, then add the reaction stop solution to terminate the reaction, perform three sets of parallel experiments, then take 200 μL of the reaction solution in a 96-well plate, and use an ELISA reader to detect the absorbance at 410 nm. Take another 58 μL of the incubation solution described in step 7, add 1 μL of Exchange Buffer and 1 μL of DMSO, incubate for 20 min, take 10 μL and add it to 490 μL of Time End–Point Assay Buffer as the control group. After 30 min of reaction, quench the reaction with the reaction stop solution, then take 200 μL of the reaction solution into a 96-well plate and detect the absorbance at 410 nm with an ELISA reader. The experimental data were fitted and analyzed using the GraphPad Prism 9 program, as shown in Figure 1 The experimental results showed that compound 1 had a significant inhibitory effect on PTP1B, IC 50 =14.80μM.

[0055] 9. Apparent inhibition constant K of the inhibitory activity of test compound 1 on PTP1B i : Use Exchange Buffer to prepare incubation solutions containing different final concentrations of compound 1, and then add the pretreated protein of step 6 at the same concentration. Continuously dilute the Time End–Point Assay Buffer to obtain several final concentration reaction solutions. In the experiment, each group of inhibitor concentrations corresponds to different concentrations of pNPP reaction solutions. After 5 minutes of reaction, 500μL 2mol / LNaOH is added to terminate the reaction, and finally the absorbance at 410nm is measured using an enzyme reader; pNPP solutions of different concentrations are prepared, and the absorbance at 410nm is measured. The standard curve is fitted using the GraphPadPrism 9 program, and the reaction rate is calculated by inverse calculation of the standard curve combined with the absorbance of the kinetic experiment. The experimental data were fitted and analyzed using the Michaelis–Menten equation, the Lineweaver–Burk double reciprocal plot, and the GraphPad Prism 9 program, as shown in the following figure. Figure 2 The experimental results show that compound 1 has a significant inhibitory effect on PTP1B, K i =18.00μM.

[0056] 10. Molecular docking of compound 1 and target protein PTP1B: All molecular docking processing and analysis were completed using the program package Schrodinger 2021.4. The crystal structure of human PTP1B protein used for docking was downloaded from the RCSB ProteinDataBank 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 docking small molecule, the Glide SP algorithm was used for molecular docking, and the PoseViewer was used to visualize the docking results. Figure 3 , 4 As shown in the docking results, it can be seen that in the allosteric site, compound 1 forms hydrogen bonds with residue GLN262, forms hydrophobic interactions with residues VAL49 and ILE219, and forms π-π stacking interactions with aromatic amino acid residue TYR46; in the catalytic site, compound 1 forms hydrogen bonds with residues ARG24 and ARG254, and forms hydrophobic interactions with amino acid residues ALA27, PHE52, ILE219, MET258, and GLN262.

[0057] 11. Molecular dynamics simulation and binding free energy calculation (MM-PBSA) of compound 1 and PTP1B complex: The molecular dynamics simulation program is GROMACS2019.6 program package, the protein force field is AMBER14SB, the small molecule force field is GAFF2 force field combined with RESP2 charge model, and the water model is OPC explicit model. After energy minimization and sufficient pre-equilibrium, the simulation system is formally simulated in the NPT system. The total simulation time is 100ns, the temperature is 268.15K, and the pressure is 1atm. After the simulation is completed, the gmx rms module is used to analyze the trend of the kinetic trajectory ligand receptor distance over time, such as Figure 5 , 6 As shown. MM-PBSA is a computational physics method used to calculate the absolute binding free energy of a ligand. Its advantage is that it takes into account both accuracy and computational complexity, so it is widely used in the field of computer-aided drug design. The present invention uses the gmx_MMPBSA 1.6.2 program package to calculate the receptor MM-PBSA. The calculation parameters all use default parameters and are calculated using the Single-Trajectory Method. The binding free energy of the small molecule at the allosteric site is -14.10 kcal / mol, and the binding free energy at 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 drawn from the experiment. It is a mixed inhibition mode, which is both a reversible competitive inhibitor and a reversible non-competitive inhibitor.

[0058] 12. Compound 1 with the structure of formula (I) is a chemical substance with specific biological activity isolated from the underground part of Salvia serrata. It has both chemical and biological properties. It not only retains the special active molecular structure of the lead compound, but also greatly improves the biological activity and reduces the labor cost. Compound 1 has a significant inhibitory effect on PTP1B protein. IC 50 The value is 14.80μM; the active small molecule compound 1 has the potential to be developed into a new drug for the treatment of type 2 diabetes. It can be used as a lead compound for a new small molecule PTP1B inhibitor, providing a template molecule for synthetic chemistry and a new idea for the development of a new small molecule PTP1B inhibitor.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A terpene compound having the following structure:

2. The method for preparing terpenoid compounds according to claim 1, characterized in that: The steps include: S1: slices of the dried underground part of Salvia ganxiensis were extracted with ethanol to obtain an ethanol extract; S2: The ethanol extract is concentrated by vacuum distillation to obtain a total extract, the total extract is dissolved in water and extracted with petroleum ether, ethyl acetate and n-butanol to obtain different extracts, and the extracts are concentrated by vacuum distillation to obtain the corresponding extracts; S3: Separate the obtained ethyl acetate extract using HP-20 macroporous resin column chromatography to obtain components Fr.1-Fr.5; S3–1: Component Fr.4 was separated and purified using 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 was separated and purified using Sephadex LH–20 to obtain components Fr.4.1.1–Fr.4.1.4; S3–3: Component Fr.4.1.2 was separated and purified by normal phase silica gel chromatography into components Fr.4.1.2.1–Fr.4.1.2.4; S3–4: Component Fr.4.1.2.2 was separated and purified using 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 using a semi-preparative liquid chromatography column to obtain a compound having the structure of formula (I).

3. The method for preparing terpenoid compounds according to claim 2, 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.

4. The method for preparing terpenoid compounds according to claim 2, 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%, 100%.

5. The method for preparing terpenoid compounds according to claim 4, characterized in that: In step S3-1, the elution system used for separation and purification is a methanol / water system, and the volume fraction of methanol is 30%-100%.

6. The method for preparing terpenoid compounds according to claim 5, 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.

7. The method for preparing terpenoid compounds according to claim 6, 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.

8. The method for preparing terpenoid compounds according to claim 7, 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 SunFireC18 OBD Prep Column 10μM, 10mm x 250mm, mobile phase is methanol / water system, volume fraction of methanol is 40%, flow rate is 2mL / min, binary infusion pump is Waters 1525, and PDA detector is Waters 2998.

9. Use of the terpenoid compound according to claim 1 in the preparation of a drug for inhibiting PTP1B activity.

10. Use of the terpenoid compound according to claim 1 in the preparation of a drug for treating type 2 diabetes.

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