Hydroxytetradecene aldehyde and preparation method and application thereof

Abstract

The present invention provides a hydroxytetradecene aldehyde and a preparation method and application thereof, wherein two novel hydroxytetradecene aldehyde compounds 1 and 2 are separated from Amomum villosum, wherein the hydroxytetradecene aldehyde compounds have better safety and tolerance and are more easily accepted by the human body than synthetic compounds. Experimental results show that the hydroxytetradecene aldehyde compounds have obvious PTP1B and Gpa inhibitory activity, indicating that the compounds have a hypoglycemic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug component preparation, and particularly relates to hydroxytetradecenal and a preparation method and application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM), as a complex metabolic disease, has become a major health problem worldwide. Due to long-term hyperglycemia, type 2 diabetes often leads to serious complications such as retinopathy, nephropathy, hypertension, etc. Currently, there are 7 oral hypoglycemic drugs on the market, including biguanides, α-glucosidase inhibitors, insulin secretagogues, insulin sensitizers, glucagon-like peptide-1 (GLP-1) receptor agonists, dipeptidyl peptidase-4 (DPP4) inhibitors, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors. However, various side effects (such as acute hypoglycemia, weight gain, gastrointestinal discomfort, and hepatotoxicity) limit their application. Therefore, new anti-diabetic drugs with high efficacy and low blood sugar risk still need to be studied. At present, drug combination therapy is often used to treat type 2 diabetes. For example, Qtrilmet and TrijardyXR are a combination of SGLT-2 inhibitors, DPP4 inhibitors, and metformin hydrochloride, which have obvious therapeutic advantages compared with single-target drugs. Therefore, compounds with multiple targets are of great value in finding new anti-diabetic drug candidates.

[0003] PTP1B negatively regulates insulin action by dephosphorylating activated insulin receptors and is an important therapeutic target for the development of anti-diabetic drugs. However, PTP1B inhibitors have poor membrane permeability and weak selectivity for T-cell protein tyrosine phosphatase (TCPTP), the protein most similar to PTP1B, which seriously hinders their clinical application. Glycogen phosphorylase a (GPa) is responsible for controlling the rate of glycogen degradation and plays a key role in catalyzing the hydrolysis of glycogen phosphate to form glucose-1-phosphate. Several types of GPa inhibitors have shown hypoglycemic effects in diabetic animals and clinical trials, but have not yet been approved as new drugs.

[0004] Traditional Chinese medicine, which has been used in China for thousands of years, is an important source for exploring new anti-diabetic drugs. Amomum tsao-ko ) is a famous spice in my country, and is often used in traditional Chinese medicine to treat abdominal distension, vomiting, malaria and other diseases. Previous studies have isolated various types of compounds such as diarylheptane, flavonoids, and monoterpenes from Amomum villosum, some of which have antiemetic, antiasthmatic, anti-gastric ulcer, anti-proliferative, antiviral, antioxidant, anti-inflammatory, and antibacterial biological activities; however, no hydroxytetradecene aldehyde compounds with PTP1B and Gpa inhibitory activity have been found in Amomum villosum in existing studies. Summary of the invention

[0005] The object of the present invention is to provide a hydroxytetradecenal and a preparation method and application thereof. The obtained hydroxytetradecenal compound has the activity of inhibiting protein tyrosine phosphatase PTP1B and glycogen phosphorylase a (GPa).

[0006] The present invention first provides a hydroxytetradecene aldehyde, a structural formula of which is

[0007]

[0008] (recorded as Compound 1 in this specification);

[0009] Another structural formula is:

[0010]

[0011] (recorded as Compound 2 in this specification);

[0012] Furthermore, the preparation method of the hydroxytetradecene aldehyde is as follows:

[0013] 1) Crush the dried ripe fruits of Amomum villosum, extract with 90% ethanol by reflux, recover ethanol by decompression to obtain an extract, disperse the extract in water, extract with petroleum ether, and concentrate to obtain a petroleum ether extraction part;

[0014] 2) The petroleum ether extraction portion was subjected to silica gel column chromatography, and a mixed solution of ethyl acetate and petroleum ether was used as the eluent, and eight fractions from Fr.A-1 to Fr.A-8 were obtained by gradient elution;

[0015] 3) The fraction Fr.A-4 was subjected to silica gel column chromatography and eluted with acetone and petroleum ether solution (20:80). The same fractions were combined by TLC detection to obtain fractions Fr.A-4-1 to Fr.A-4-3;

[0016] 4) The Fr.A-4-2 fraction was chromatographed on a Sephadex LH-20 (chloroform-methanol, 50:50) column and further purified by HPLC (Rp-C8 column) and eluted with acetonitrile-water (70:30) to obtain hydroxytetradecene aldehyde compounds 1 and 2.

[0017] The present invention also provides the use of the screened hydroxytetradecene aldehyde compounds 1 and 2 in the preparation of PTP1B or Gpa inhibitors.

[0018] The present invention also provides another use of the screened hydroxytetradecenal compound 1 and compound 2, which is use in the preparation of hypoglycemic drugs.

[0019] In another aspect, the present invention also provides a pharmaceutical composition, comprising any one or both of Compound 1 and Compound 2;

[0020] Furthermore, the medicine also contains a pharmaceutically acceptable carrier or excipient.

[0021] The present invention isolates two novel hydroxytetradecene aldehyde compounds 1 and 2 from Amomum villosum, and the hydroxytetradecene aldehyde compounds have better safety and tolerance, and are more easily accepted by the human body than synthetic compounds. Experimental results show that the hydroxytetradecene aldehyde compounds have obvious PTP1B and Gpa inhibitory activity, indicating that the compounds have a hypoglycemic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Dose-effect diagram of the inhibitory activity of compound 1 of the present invention on Gpa. DETAILED DESCRIPTION

[0023] In order to better understand the essence of the present invention, the test examples and embodiments of the present invention are used below in conjunction with the accompanying drawings to further illustrate the preparation methods, structural identification, pharmacological effects, and preparation methods and drug compositions of compounds 1 and 2, but the present invention is not limited to these test examples and embodiments.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more related listed items.

[0025] Example 1: Preparation and characterization of compounds

[0026] 1. Isolation and preparation of compounds

[0027] 1) Preparation of compounds

[0028] The ripe fruits of Amomum villosum were dried and crushed, and extracted with 90% ethanol under reflux twice, each time for 2 hours. The ethanol extracts were combined, and the ethanol was recovered under reduced pressure to obtain an extract. The extract was dispersed in water and extracted with petroleum ether, and concentrated to obtain the petroleum ether extraction part.

[0029] Then the petroleum ether extraction part was subjected to silica gel column chromatography, and ethyl acetate-petroleum ether (0:100, 2:98, 5:95, 10:90, 20:80 and 100:0, v / v) was used as the eluent, and gradient elution was performed to obtain eight fractions from Fr.A-1 to Fr.A-8; the fraction Fr.A-4 obtained by eluting with ethyl acetate-petroleum ether (10:90) was subjected to silica gel column chromatography, and 10 column volumes were eluted isocratically with acetone-petroleum ether (20:80), and each fraction was 1 / 3 column volume. The same fractions were combined by TLC detection to obtain three fractions Fr.A-4-1 to Fr.A-4-3. Among them, Fr.A-4-2 was subjected to Sephadex LH-20 (chloroform-methanol, 50:50) column chromatography, and further purified by HPLC (Rp-C8 column), eluted with acetonitrile-water (70:30), to obtain compounds 1 and 2.

[0030] 2. Structural data analysis of compounds 1 and 2:

[0031] The optical rotation was measured by a Jascomodel 1020 polarimeter (Horiba, Tokyo, Japan);

[0032] The UV spectra were measured by UV-2401PC UV spectrometer (Shimadzu, Kyoto, Japan);

[0033] Nuclear magnetic resonance spectra (1D and 2D NMR) were measured using an AV800 or AvanceIII-600 superconducting NMR instrument (Bruker, Bremerhaven, Germany), using deuterated methanol as the solvent;

[0034] High-resolution mass spectra (HRMS) were measured using a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan).

[0035] The structural formula of compound 1 is as follows:

[0036]

[0037] (2E,4E,8Z,11Z)-6R-hydroxytetradeca-2,4,8,11-tetraenal (1)

[0038] Molecular formula: C 14 H 20 O2,

[0039] Molecular weight: 220

[0040] Appearance: Light yellow oil

[0041] HRESIMS m / z221.1521 ([M+H] + , calcd. for C 14 H 21 O2, 221.1536, −1.5 mDa);

[0042] [α] D ‒5.4 ( c 0.11, MeOH);

[0043] 1 HNMR and 13 CNMR (DEPT) data are shown in Table 1.

[0044] The structural formula of compound 2 is as follows:

[0045]

[0046] (2E,4E,8Z)-6R-hydroxytetradeca-2,4,8-trienal (2)

[0047] Molecular formula: C 14 H 22 O2

[0048] Molecular weight: 222

[0049] Appearance: Light yellow oil

[0050] HRESIMS m / z 223.1700 ([M+H] + , calcd. for C 14 H 23 O2, 223.1693, +0.7 mDa);

[0051] [α] D ‒3.8 ( c 0.09, MeOH);

[0052] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Table 1.

[0053] Table 1: Compounds 1 and 2 13 C (150 MHz) and 1 HNMR (600 MHz, J in Hz) data table, the test solvent is CDCl3 ( d in ppm).

[0054]

[0055] Example 2: Compounds inhibiting PTP1B and Gpa activity test

[0056] 1 Materials and Methods

[0057] 1.1 Materials

[0058] PTP1B (protein tyrosine phosphatase) was purchased from Sino Biological (Wayne, PA, USA); GPa was purchased from Sigma Aldrich (St. Louis, MO, USA); phosphate buffer (≥99%, Meilun Biotechnology, Dalian); Hepes was purchased from Beijing Xiasi Biotechnology Co., Ltd. (Beijing); glycogen was purchased from Meilun Biotechnology Co., Ltd. (Dalian); α-D-glucose 1-phosphate disodium salt (St. Louis, MO, USA); ammonium molybdate was purchased from Shanghai Jiuding Chemical Co., Ltd. (Shanghai); malachite green was purchased from Beijing Balinwei Technology Co., Ltd. (Beijing), China; bovine serum was purchased from Shanghai Longtian Biotechnology Co., Ltd. (Shanghai).

[0059] 1.2 Instruments

[0060] FlexStation3 desktop multifunctional microplate reader (Bio-RAD680, USA); analytical balance (AG135, Metler Toledo, China); constant temperature box (DHP-9082, Shanghai).

[0061] 1.3 Experimental process

[0062] PTP1B inhibition assay: Working buffer (WB) was prepared by dissolving 3-(N-morpholino)propanesulfonic acid (MOPS, 722.02 mg), dithiothreitol (DTT, 30 mg), EDTA (25.7 mg), bovine serum albumin (BSA, 200 mg) and NaCl (12.1 g) in 100 mL ultrapure water. p-Nitrophenyl phosphate (p-NPP, 31 mg) dissolved in 1 mL ultrapure water was used as a substrate. 70 μL WB, 10 μL PTP1B enzyme (5 mg / L) and 10 μL of the sample to be tested dissolved in DMSO were added to a 96-well plate in sequence. After incubation at 37°C for 15 min, the reaction was initiated by adding 10 μL 100 mM p-NPP and then incubated for 30 min. Add 100 μL of 0.1 M Na2CO3 solution to the reaction mixture to terminate the reaction, measure the absorbance at 405 nm with a microplate reader, and record the results. DMSO was used instead of the test solution for the negative control, and suramin sodium was used for the positive control. The other methods were the same. The PTP1B inhibition rate was calculated as inhibition rate (%) = (△ enzyme - △ sample / △ enzyme - △ negative) × 100%. The experimental results were analyzed using Graphpadprism5 software. The TCPTP measurement method, materials used, and measurement methods are consistent with those of PTP1B.

[0063] GPa inhibition experiment: Preliminary preparation: Dissolve 1.19g Hepes in 100mL pure water to prepare a 50mM buffer solution (pH7.2); then weigh 370mg KCl, 25mg MgCl2•6H2O, 50mg glycogen and 7.5mg 1-phosphoglucose disodium salt and dissolve them in 50mL Hepes buffer to prepare solution I. Add 900mg ammonium molybdate and 34.2mg malachite green to 90mL 1M hydrochloric acid, mix thoroughly, and sonicate until completely dissolved to prepare solution II. First, 10 m L of sample dissolved in DMSO and 50 m 3U / mL GPa enzyme diluted with Hepes buffer was added to the 96-well plate in turn. The blank was incubated in a 25°C incubator for 15 min. m L of solution I initiated the reaction. After incubation for 30 min, 150 m L solution II, incubate in a 25°C incubator for 20 min. Measure the absorbance at 620 nm with a microplate reader and record the results. For the negative control, DMSO was used instead of the test solution, and for the positive control, CP-91149 was used. Other methods were the same. The GPa inhibition rate was calculated as inhibition rate (%) = (△ enzyme - △ sample / △ enzyme - △ negative) × 100%. The experimental results were analyzed using Graphpadprism5 software.

[0064] 2. Results:

[0065] The inhibitory activities of compound 1 and compound 2 on PTP1B and Gpa were evaluated at concentrations of 200 and 100 μmol / L. As shown in Table 2, compound 1 and compound 2 have PTP1B inhibitory activity, with inhibition rates of 40.2±1.0% and 50.3±0.6% at 200 μmol / L and 33.8±1.6% and 41.1±2.6% at 100 μmol / L, respectively. Among them, the IC of compound 2 for inhibiting PTP1B is 50 The activity of compound 1 was 195.5 μmol / L, which was comparable to that of the positive control sodium orthovanadate. Figure 1 ), at 200 μmol / L, the inhibition rate was 94.8±0.2%, and at 100 μmol / L, the inhibition rate was 89.8±2.9%. IC of compound 1 inhibiting Gpa 50 The positive control CP-91149 (IC 50 =194.0μmol / L).

[0066] Table 2: Activity of compounds 1 and 2 in inhibiting PTP1B and GPa

[0067]

[0068] a Values ​​are expressed as mean ± standard deviation (n=3). CP-91149 (IC 50 =194.0μmol / L) is the positive control for GPa inhibition test; sodium orthovanadate (IC 50 =148.0 μmol / L) was the positive control for the PTP1B inhibition experiment.

[0069] The results showed that new hydroxytetradecene aldehyde compounds 1 and 2 were isolated from Amomum villosum, which had PTP1B and Gpa inhibitory activity at concentrations of 200 μmol / L and 100 μmol / L, had a clear hypoglycemic effect, and can be used in hypoglycemic drugs and health foods.

[0070] Example 3 Preparation of medicine

[0071] When preparing the preparation, either Compound 1 or Compound 2 or any combination thereof is used. This example takes Compound 1 with a concentration of 100 μmol / L as an example to list various preparation methods:

[0072] 1. Take compound 1, dissolve it with a small amount of DMSO, add water for injection as usual, filter it, and sterilize it by filling to prepare a 100 μmol / L injection solution.

[0073] 2. Take compound 1, dissolve it with a small amount of DMSO, dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, filter aseptically, dispense into ampoules, freeze-dry at low temperature, and aseptically seal to obtain 100 μmol / L powder injection.

[0074] 3. Take compound 1 and add excipients at a weight ratio of 9:1 to the excipients to prepare a powder.

[0075] 4. Take compound 1, add excipients in a weight ratio of 5:1, and granulate and tablet.

[0076] 5. Take compound 1 and prepare an oral liquid according to a conventional oral liquid preparation method.

[0077] 6. Compound 1 was taken and excipients were added in a weight ratio of 5:1 to the excipients to prepare capsules.

[0078] 7. Take compound 1 and add excipients at a weight ratio of 3:1 to excipients to prepare capsules.

[0079] 8. Take any one of compounds 1 and 2 or any combination thereof, add excipients at a weight ratio of 5:1 to the excipients to prepare granules.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 hydroxytetradecene aldehyde compound, characterized in that The structural formula of the compound is: 。 2. A hydroxytetradecene aldehyde compound, characterized in that The structural formula of the compound is: 。 3. The hydroxytetradecenal compound according to claim 1 or 2, characterized in that The preparation method of the compound is as follows: 1) Crush the dried ripe fruits of Amomum villosum, extract with 90% ethanol by reflux, recover ethanol from the extract under reduced pressure to obtain an extract, disperse the extract in water, extract with petroleum ether, and concentrate to obtain a petroleum ether extraction portion; 2) The petroleum ether extraction portion was subjected to silica gel column chromatography, and a mixed solution of ethyl acetate and petroleum ether was used as the eluent, and eight fractions from Fr.A-1 to Fr.A-8 were obtained by gradient elution; 3) The fraction Fr.A-4 was subjected to silica gel column chromatography and eluted with acetone and petroleum ether solution. The same fractions were combined after TLC detection to obtain fractions Fr.A-4-1 to Fr.A-4-3; 4) The Fr.A-4-2 fraction was chromatographed on a Sephadex LH-20 column and further purified by HPLC using an acetonitrile aqueous solution as eluent to obtain hydroxytetradecene aldehyde compound 1 and compound 2.

4. The hydroxytetradecenal compound according to claim 3, characterized in that The volume ratio of acetone to petroleum ether in the above-mentioned step 3) is 20:

80.

5. The hydroxytetradecenal compound according to claim 3, characterized in that The HPLC purification described in 3) is performed using an Rp-C8 column.

6. The hydroxytetradecenal compound according to claim 3, characterized in that The volume ratio of acetonitrile to water in the above-mentioned step 3) is 70:

30.

7. Use of the hydroxytetradecenal compound according to claim 1 or 2 in the preparation of a PTP1B or Gpa inhibitor.

8. Use of the hydroxytetradecenal compound according to claim 1 or 2 in the preparation of a drug for lowering blood sugar.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any one or two of the hydroxytetradecene aldehyde compounds according to claim 1 or 2 in a pharmacologically effective concentration.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient.

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