Yohimbine alkaloid as well as preparation method and application thereof

Abstract

The invention discloses yohimbine alkaloids as well as a preparation method and application thereof. The yohimbine alkaloid is at least one of a compound 1 to a compound 8. According to the application, novel yohimbine type alkaloid compounds, namely compounds 1 to 8, are found from neriaceae Abrus dichroa plant Codonopsis lanceolata, and research finds that the yohimbine type alkaloid compounds have a remarkable inhibitory effect on protein tyrosine phosphatase, and can be used for preparing a medicine for preventing and treating the diseases caused by the protein tyrosine phosphatase. The invention provides a novel scheme and product for preparing the protein tyrosine phosphatase inhibitor or a therapeutic drug based on the protein tyrosine phosphatase inhibitor.

Description

Technical Field

[0001] This application relates to the technical field of yohimbine alkaloids, and particularly to yohimbine alkaloids, their preparation methods and applications. Background Art

[0002] Alstonia mairei Levl. is a plant of the genus Alstonia in the Apocynaceae family, an erect shrub, also known as Alstonia yunnanensis Diels, Alstonia mairei Levl. var. tomentosa Tsiang, and Alstonia mairei Levl. var. villosa Tsiang. The branches and leaves of Alstonia mairei Levl. can be used as medicine, with the effects of clearing heat, detoxifying, stopping bleeding, relieving pain, and discharging pus and promoting granulation. Apocynaceae plants are rich in monoterpene indole alkaloids (MIAs), which have complex and diverse structures and rich biological activities, such as anti-tumor, anti-inflammatory, and antibacterial. MIAs have a high drug-forming rate, such as vinblastine, reserpine, and ajmaline, which have been clinically used to treat diseases such as cancer, hypertension, and arrhythmia.

[0003] At present, there are few research reports on the alkaloid components in Alstonia mairei Levl. Therefore, discovering new alkaloid structures from Alstonia mairei Levl. has important value and significance for the research of Alstonia mairei Levl. and the development of related drugs. Summary of the Invention

[0004] The purpose of this application is to provide a new class of yohimbine alkaloids, their preparation methods and applications.

[0005] This application adopts the following technical solutions:

[0006] One aspect of this application discloses a class of yohimbine alkaloids, which are at least one of Compound 1 to Compound 8.

[0007] It should be noted that a class of yohimbine-type alkaloid compounds, namely Compounds 1 to 8, have been discovered from the plant Alstonia mairei Levl. of the genus Alstonia in the Apocynaceae family. Compound 1 is yohimbine N4-oxide, Compound 2 is 17-epi-α-yohimbine, Compound 3 is α-yohimbine, Compound 4 is yohimbine, Compound 5 is β-yohimbine, Compound 6 is O-acetylyohimbine, Compound 7 is 19,20-dehydro-β-yohimbine, and Compound 8 is trimethoxybenzoylyohimbine. Through research, eight new yohimbine-type alkaloid compounds have been discovered in this application, and methods for extraction, separation, structure identification, and their new uses have been developed. In particular, Compounds 1, 2, 3, 7, and 8 have good inhibitory effects on protein tyrosine phosphatase (PTP1B).

[0008] Another aspect of this application discloses the use of the yohimbine alkaloids of this application in the preparation of drugs for inhibiting protein tyrosine phosphatase (PTP1B).

[0009] Another aspect of this application discloses a protein tyrosine phosphatase inhibitory drug containing the yohimbine alkaloids of this application.

[0010] It should be noted that through research in this application, it has been found that Compounds 1 to 8, the eight yohimbine-type alkaloid compounds extracted and separated in this application, all have inhibitory effects on protein tyrosine phosphatase. Therefore, these eight yohimbine-type alkaloid compounds can be used to prepare PTP1B inhibitory drugs, providing a new method and product for protein tyrosine phosphatase inhibition.

[0011] Another aspect of this application discloses the use of the yohimbine alkaloids of this application in the preparation of drugs for treating diabetes.

[0012] Another aspect of this application discloses a drug for treating diabetes containing the yohimbine alkaloids of this application.

[0013] It should be noted that those skilled in the art are well aware that PTP1B is a negative regulator of insulin signal transduction. By dephosphorylating tyrosine residues on the insulin receptor (IR) or its substrate (IRS), the insulin receptor is unable to bind to insulin, thereby causing insulin resistance. PTP1B inhibitors can alleviate or even reverse insulin and leptin resistance in obese patients, induce an increase in lipid metabolism levels, and have a therapeutic effect on type 2 diabetes mellitus (T2DM). Therefore, the eight yohimbine-type alkaloid compounds of the present application, as a new class of PTP1B inhibitors, can also be used to treat diabetes, especially type 2 diabetes, providing a new method and product for the treatment of type 2 diabetes.

[0014] Another aspect of the present application discloses a method for preparing the yohimbine alkaloids of the present application, comprising the following steps:

[0015] Step 1: Dry and pulverize the branches and leaves of Alstonia mairei, extract with methanol, and concentrate the extract under reduced pressure to obtain a crude extract paste.

[0016] Step 2: Add warm water to the crude extract paste obtained in Step 1, suspend it, then add an acidic solution to adjust the pH value to acidic, and extract with chloroform to remove non-alkaloid components in the extract, obtaining an acidic aqueous solution.

[0017] Step 3: Add an alkaline solution to the acidic aqueous solution obtained in Step 2 to adjust the pH value to alkaline, obtaining an alkaline aqueous solution.

[0018] Step 4: Extract the alkaline aqueous solution obtained in Step 3 with chloroform and n-butanol respectively to obtain a chloroform fraction paste and an n-butanol fraction paste.

[0019] Step 5: Mix the chloroform fraction paste with silica gel, perform normal-phase silica gel column chromatography, with the mobile phase being dichloromethane-methanol gradient elution, and combine TLC thin-layer chromatographic plate spotting detection to obtain 7 fractions with increasing polarity, namely fractions A to G; perform reverse-phase C-18 silica gel column chromatography on fraction B, with the mobile phase being methanol-water gradient elution, and combine TLC thin-layer chromatographic plate spotting detection to obtain 8 fractions with decreasing polarity, namely fractions B1 to B8; perform Sephadex LH-20 gel column chromatography on fraction B7, with the mobile phase being methanol, to obtain 2 fractions with decreasing molecular weight, namely B7a and B7b; fraction B7b is purified by high-performance liquid chromatography on a C-18 chromatographic column, with the mobile phase being methanol-water-ammonia water, to obtain compound 8.

[0020] Fraction C was subjected to reversed-phase C-18 silica gel column chromatography with a mobile phase of methanol-water gradient elution. Combining with TLC thin-layer chromatographic plate spotting detection, 7 fractions with decreasing polarity were obtained, namely fraction C1 to fraction C7; fraction C4 was subjected to Sephadex LH-20 gel column chromatography with a mobile phase of methanol to obtain 2 fractions with decreasing molecular weight, namely fraction C4a and fraction C4b; fraction C4a was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of methanol-water-ammonia water to obtain compound 6;

[0021] Fraction C4b was purified by high-performance liquid chromatography on a phenyl column with a mobile phase of methanol-water-ammonia water to obtain compound 3;

[0022] Fraction D was subjected to medium-pressure preparative column chromatography on reversed-phase C-18 silica gel with a mobile phase of methanol-water gradient elution. Combining with TLC thin-layer chromatographic plate spotting detection, 5 fractions with decreasing polarity were obtained, namely fraction D1 to fraction D5; fraction D1 was subjected to Sephadex LH-20 gel column chromatography with a mobile phase of methanol to obtain 5 fractions with decreasing molecular weight, namely fraction D1a to fraction D1e; fraction D1c was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of methanol-water-ammonia water to obtain compound 1;

[0023] Fraction D1d was subjected to Sephadex LH-20 gel column chromatography with a mobile phase of methanol to obtain 2 fractions with decreasing molecular weight, namely fraction D1d1 and fraction D1d2; fraction D1d1 was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of methanol-water-ammonia water to obtain compound 4;

[0024] Fraction D1d2 was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of methanol-water-ammonia water to obtain compound 2;

[0025] Fraction D3 was subjected to Sephadex LH-20 gel column chromatography with a mobile phase of methanol to obtain 4 fractions with decreasing molecular weight, namely fraction D3a to fraction D3d; fraction D3b was subjected to reversed-phase phenyl silica gel column chromatography with a mobile phase of methanol-water gradient elution. Combining with TLC thin-layer chromatographic plate spotting detection, 3 fractions with decreasing polarity were obtained, namely fraction D3b1 to fraction D3b3; fraction D3b1 was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of methanol-water-ammonia water to obtain compound 5;

[0026] Fraction D3b2 was purified by high-performance liquid chromatography on a C-18 column with a mobile phase of acetonitrile-water-ammonia water to obtain compound 7.

[0027] Preferably, in step one, the volume concentration of methanol is 80%.

[0028] Preferably, in step one, the number of times of methanol extraction is at least 5 times.

[0029] Preferably, in step two, the temperature of the warm water is 30 - 45 °C.

[0030] Preferably, in step two, the acidic solution is 0.3% dilute hydrochloric acid solution.

[0031] Preferably, in step two, the pH value is adjusted to be acidic, specifically adjusted to a pH value of 2.

[0032] Preferably, in step three, the alkaline solution is an aqueous sodium carbonate solution.

[0033] Preferably, in step three, the pH value is adjusted to be alkaline, specifically adjusted to a pH value of 10.

[0034] Preferably, in step five, the silica gel used for mixing the chloroform part extract with silica gel is 200 - 300 mesh.

[0035] Preferably, in step five, the volume ratio of dichloromethane - methanol is sequentially 100:1 to 0:1.

[0036] Preferably, in step five, the volume ratio of methanol - water is sequentially 35:65 to 100:0.

[0037] Preferably, in step five, in methanol - water - ammonia water, the volume ratio of the total volume of methanol and water to ammonia water is 100:0.2, and the volume ratio of methanol and water is 35:65 to 70:30.

[0038] Preferably, in step five, the volume ratio of acetonitrile - water - ammonia water is sequentially 35:65:0.2.

[0039] Preferably, in step five, the flow rate of the mobile phase in high - performance liquid chromatography is 2.5 mL / min.

[0040] The beneficial effects of this application are as follows:

[0041] A new type of yohimbine - type alkaloid compound was discovered from the plant Alstonia mairei Lévl. of the genus Alstonia in the Apocynaceae family. Research found that this type of yohimbine - type alkaloid compound has significant protein tyrosine phosphatase inhibitory effects, providing a new solution and product for the preparation of protein tyrosine phosphatase inhibitors or therapeutic drugs based on protein tyrosine phosphatase inhibitors. Description of the Drawings

[0042] Figure 1 It is the two - dimensional nuclear magnetic resonance correlation signal diagram of compound 1 in the examples of this application;

[0043] Figure 2 It is the nuclear magnetic resonance of compound 1 in the examples of this application1 1H NMR spectrum;

[0044] Figure 3 1H NMR spectrum of compound 1 in the examples of the present application 13 13C NMR spectrum;

[0045] Figure 4 13C NMR spectrum of compound 2 in the examples of the present application 1 1H NMR spectrum;

[0046] Figure 5 1H NMR spectrum of compound 2 in the examples of the present application 13 13C NMR spectrum. Detailed implementation manners

[0047] When the present application was studying the medicinal plant Alstonia mairei Levl. of the genus Alstonia in the Apocynaceae family, a new type of yohimbine alkaloid compound was discovered, namely compound 1 to compound 8 of the present application. The present application obtained the above-mentioned yohimbine alkaloid compounds, namely compound 1 to compound 8, through repeated column chromatography separation and purification of the chloroform extract of the 80% methanol extract of the branches and leaves of Alstonia mairei Levl. with an alkaline aqueous solution at pH 10.

[0048] In the present application, a variety of modern spectroscopic analysis methods are used, such as high-resolution mass spectrometry, nuclear magnetic resonance 1 1H NMR spectrum, nuclear magnetic resonance 13 13C NMR spectrum, 1 1H– 1 1H COSY spectrum, HSQC spectrum, HMBC spectrum and NOESY spectrum, ultraviolet spectrum and infrared spectrum, etc., to analyze and determine the chemical structures of compound 1 to compound 8.

[0049] Furthermore, the present application's research found that the newly separated yohimbine alkaloid compounds of the present application have significant PTP1B inhibitory effects, and therefore can be used as PTP1B inhibitors. It can be understood that the existing PTP1B inhibitors can play roles such as inhibiting tumor cell proliferation and reducing blood sugar. As an effective PTP1B inhibitor, the newly separated yohimbine alkaloid compounds of the present application also have the same or similar functions and effects, including but not limited to inhibiting tumor cell proliferation and reducing blood sugar.

[0050] The present application will be described in detail below through specific examples in conjunction with the accompanying drawings. The following examples are only used to illustrate the present application and should not be construed as a limitation of the present application. The materials used in the following experiments are all obtained through regular purchases unless otherwise specified.

[0051] Example 1

[0052] 1. Plant material information

[0053] The branches and leaves of Alstonia mairei Levl., a medicinal plant of the genus Alstonia in the Apocynaceae family, were collected from Qiaojia County, Zhaotong City, Yunnan Province in April 2021 and identified by Professor Wang Jianping of the School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology.

[0054] 2. Separation and preparation process

[0055] 20.0 kg of the branches and leaves of Alstonia mairei were dried and pulverized, and extracted 5 times with methanol with a volume concentration of 80%. The extract was concentrated under reduced pressure to obtain a crude extract. The crude extract was suspended in warm water, and 0.3% dilute hydrochloric acid solution was added to adjust the pH to 2. Chloroform was used for extraction to remove non-alkaloid components in the extract, and an acidic aqueous solution was obtained. An aqueous sodium carbonate solution was added to the acidic aqueous solution to adjust the pH to 10 to obtain an alkaline aqueous solution. The alkaline aqueous solution was successively extracted with chloroform and n-butanol to obtain 230.0 g of chloroform extract and 110.5 g of n-butanol extract, respectively. The chloroform extract was mixed with silica gel of 200 - 300 mesh and subjected to normal-phase silica gel column chromatography. The mobile phase was dichloromethane - methanol (100:1, V / V) gradient elution. Combined with TLC thin-layer chromatographic plate spotting detection, 7 fractions with increasing polarity were obtained, namely fraction A to fraction G. Fraction B was subjected to reversed-phase C-18 silica gel medium-pressure preparative column chromatography. The mobile phase was methanol - water (50:50, V / V) gradient elution. Combined with TLC thin-layer chromatographic plate spotting detection, 8 fractions with decreasing polarity were obtained, namely fraction B1 to fraction B8. Fraction B7 was subjected to Sephadex LH-20 gel column chromatography. The mobile phase was methanol to obtain 2 fractions with decreasing molecular weight, namely fraction B7a and fraction B7b. Fraction B7b was purified by high-performance liquid chromatography on a C-18 chromatographic column. The mobile phase was methanol - water - ammonia (70:30:0.2, V / V), and the flow rate was 2.5 mL / min to obtain compound 8 (9.8 mg, t R =34.6 min). Fraction C was subjected to reversed-phase C-18 silica gel medium-pressure preparative column chromatography. The mobile phase was methanol - water (50:50 to 100:0, V / V) gradient elution. Combined with TLC thin-layer chromatographic plate spotting detection, 7 fractions with decreasing polarity were obtained, namely fraction C1 to fraction C7. Fraction C4 was subjected to Sephadex LH-20 gel column chromatography. The mobile phase was methanol to obtain 2 fractions with decreasing molecular weight, namely fraction C4a and fraction C4b. Fraction C4a was purified by high-performance liquid chromatography on a C-18 chromatographic column. The mobile phase was methanol - water - ammonia (67:33:0.2, V / V), and the flow rate was 2.5 mL / min to obtain compound 6 (14.9 mg, t R= 26.3 min). Fraction C4b was purified by high performance liquid chromatography using a phenyl column with a mobile phase of methanol - water - ammonia (65:35:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 3 (21.3 mg, t R = 29.6 min). Fraction D was separated by medium - pressure preparative column chromatography on a reversed - phase C - 18 silica gel column with a gradient elution of methanol - water (35:65, V / V). Detection was carried out by spotting on a TLC thin - layer chromatographic plate, and five fractions with decreasing polarity were combined, namely fraction D1 to fraction D5; fraction D1 was separated by Sephadex LH - 20 gel column chromatography with methanol as the mobile phase to obtain five fractions with decreasing molecular weight, namely fraction D1a to fraction D1e; fraction D1c was purified by high performance liquid chromatography using a C - 18 column with a mobile phase of methanol - water - ammonia (60:40:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 1 (5.2 mg, t R = 43.7 min). Fraction D1d was separated by Sephadex LH - 20 gel column chromatography with methanol as the mobile phase to obtain two fractions with decreasing molecular weight, namely fraction D1d1 and fraction D1d2; fraction D1d1 was purified by high performance liquid chromatography using a C - 18 column with a mobile phase of methanol - water - ammonia (60:40:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 4 (6.6 mg, t R = 27.9 min). Fraction D1d2 was purified by high performance liquid chromatography using a C - 18 column with a mobile phase of methanol - water - ammonia (65:35:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 2 (4.9 mg, t R = 31.8 min). Fraction D3 was separated by Sephadex LH - 20 gel column chromatography with methanol as the mobile phase to obtain four fractions with decreasing molecular weight, namely fraction D3a to fraction D3d; fraction D3b was separated by medium - pressure preparative column chromatography on a reversed - phase phenyl silica gel column with a gradient elution of methanol - water (50:50, V / V). Detection was carried out by spotting on a TLC thin - layer chromatographic plate, and three fractions with decreasing polarity were combined, namely fraction D3b1 to fraction D3b3. Fraction D3b1 was purified by high performance liquid chromatography using a C - 18 column with a mobile phase of methanol - water - ammonia (55:45:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 5 (4.1 mg, t R = 33.1 min). Fraction D3b2 was purified by high performance liquid chromatography using a C - 18 column with a mobile phase of acetonitrile - water - ammonia (35:65:0.2, V / V) at a flow rate of 2.5 mL / min to obtain compound 7 (8.3 mg, t R = 29.4 min).

[0056] 3. Identification of Compounds 1 to 8

[0057] Combined with various spectroscopic analysis methods, including high-resolution mass spectrometry, optical rotation, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance, etc., data analysis was carried out to determine the structures of Compounds 1 to 8.

[0058] Compound 1 (yohimbine N4-oxide): yellow oil; [α]D 25 +36 (c 0.1, CH2Cl2); HR-ESI-MS high-resolution mass spectrometry m / z 371.1965 [M+H] + (C 21 H 27 N2O4 calculated value is 371.1971); 1 H and 13 13C NMR spectral data are shown in Table 1.

[0059] Through HR-ESI-MS high-resolution mass spectrometry data analysis, the molecular formula of Compound 1 has 16 more mass units compared with the known Compound 4 (yohimbine). The NMR data of Compound 1 (Table 1) are similar to those of Compound 4. The main difference is that compared with Compound 4, the carbon-hydrogen signals around N4 in Compound 1 shift significantly to the low field, especially CH-3 (Δδ H 1.13 and Δδ C 9.2), CH2-5 (Δδ Ha 0.80, Δδ Hb 0.45 and Δδ C 12.2) and CH2-21 (Δδ Ha 0.86, Δδ Hb 0.22 and Δδ C 10.5). Combining its molecular formula, it was determined that Compound 1 is the N4 oxidation derivative of Compound 4. After detailed 2D NMMR including 1 H- 1 HCOSY, HMQC and HMBC spectral analysis, the relevant signals are as Figure 2 shown, further supporting the above inference, and thus the planar structure of Compound 1 was determined, as Figure 1 shown. The nuclear magnetic resonance 13 13C spectrum of Compound 1 is as Figure 3 shown.

[0060] The relative configuration of Compound 1 was determined by NOESY correlation signals and coupling constants. First, assume that H-3 is in the α configuration. In its NOESY spectrum, it can be observed that H-3 (δ H 4.62, d, J = 11.9 Hz) and H-15 (δ H2.13, ddd, J = 11.5, 11.5, 3.4 Hz), H-14β and H-20 (δ H 2.34, m), H-20 and H-16 (δ H 2.46, dd, J = 11.5, 2.7 Hz) correlation signals, from which it can be determined that H-15 is in the α configuration, and H-16 and H-20 are in the β configuration. The relatively small coupling constant (J = 2.8 Hz) between H-16 and H-17 indicates that H-17 is in the β configuration. Except for the configuration of N4 + the compound 1 and compound 4 have the same relative configuration. In addition, there are strong NOESY correlation signals between H-3 and H-5α (δ H 3.49, dd, J = 11.7, 5.9 Hz) and H2-21 (δ H 3.18, d, J = 7.8 Hz), indicating that N4 + -O - in compound 1 is β-oriented. Compound 1 has been reported as a synthetic product, but its NMR data has not been reported. Therefore, compound 1 is a new natural product, first isolated from Alstonia mairei, and its NMR signals are assigned for the first time.

[0061] Table 1 1 H and 13 13C NMR data (CDCl3) of compound 1

[0062] No. <![CDATA[δ H (JinHz)]]> <![CDATA[δ C > No. <![CDATA[δ H (JinHz)]]> <![CDATA[δ C > 2 130.5 14β 1.89, ddd(13.2, 12.0, 12.0) 3 4.62,d(11.9) 71.2 15 2.13, ddd(11.5, 11.5, 3.4) 36.4 5α 3.62, ddd(11.7, 11.7, 4.8) 66.4 16 2.46, dd(11.5, 2.7) 53.3 5β 3.49, dd(11.7, 5.9) 17 4.30, ddd(5.6, 2.8, 2.8) 68.6 6α 2.85, dd(15.5, 4.8) 19.2 18α 1.72, dddd(13.7, 13.7, 4.2, 2.5) 33.6 6β 3.38,m 18β 1.96, ddd(13.8, 3.3, 3.3) 7 106.4 19α 1.56, ddd(12.8, 12.8, 3.6) 23.5 8 128.1 19β 1.37, ddd(12.8, 3.5, 3.5) 9 7.44, dd(7.9, 1.2) 119.0 20 2.34,m 36.1 10 7.07, dd(7.9, 7.0, 1.0) 120.4 21α 3.18,d(7.8) 72.7 11 7.09, dd(8.1, 7.0, 1.2) 122.6 21β 3.18,d(7.8) 12 7.34, dd(8.1, 1.0) 112.6 22 175.0 13 138.3 <![CDATA[OCH3]]> 3.79 52.4 14α 2.47, ddd(13.2, 3.1, 3.1) 28.2

[0063] Compound 2 (17-epi-α-yohimbine): white powder; optical rotation value [α] D 25 -21 (c 0.1, CH2Cl2) [α] 25 D -35.0 (c 0.1, methanol); HR-ESI-MS high-resolution mass spectrometry m / z 355.2261 [M + H] + (calculated for C 21 H 27 N2O3, 355.2002); 1 1H and 13 13C NMR spectral data are shown in Table 2.

[0064] Through literature research, it was found that the 13 13C NMR data of compound 2 are consistent with those of the synthetic product 17-epi-α-yohimbine. However, the 1H NMR data of this compound has not been reported in the literature. In this paper, the 1H NMR data are assigned for the first time, as shown in Table 2. The nuclear magnetic resonance 1 1H spectrum of compound 2 is as Figure 4As shown, the nuclear magnetic resonance of Compound 2 13 C spectrum is as Figure 5 shown.

[0065] Table 2 1 H and 13 CNMR data (chloroform-d)

[0066] No. <![CDATA[δ H (JinHz)]]> <![CDATA[δ C > No. <![CDATA[δ H (JinHz)]]> <![CDATA[δ C > 2 135.0 14β 1.67, ddd(11.9, 2.8, 2.6) 3 3.06, dd(11.9, 2.6) 60.7 15 1.76,d(13.0) 37.5 5α 2.51,m 53.4 16 2.61, dd(4.9, 2.5) 49.6 5β 3.00, overlap 17 4.27, ddd(3.2, 3.0, 2.5) 65.6 6α 2.68,m 21.8 18α 1.47, dddd(14.0, 14.0, 4.3, 2.9) 31.7 6β 2.96, overlap 18β 1.98, ddd(14.0, 3.2, 3.2) 7 108.2 19α 2.33, ddd(14.0, 13.8, 3.6) 20.5 8 127.4 19β 1.34, ddd(13.8, 3.6, 3.6) 9 7.45,d(7.7) 118.0 20 2.37, overlap 36.8 10 7.07, dd(7.7, 7.7) 119.3 21α 2.59, dd(11.3, 3.5) 61.5 11 7.11, dd(7.7, 7.7) 121.2 21β 2.89, dd(11.4, 2.0) 12 7.29,d(7.7) 110.7 22 175.7 13 135.9 <![CDATA[OCH3]]> 3.84 52.0 14α 2.43, dd(11.9, 11.9) 29.3

[0067] Compound 3 (α-yohimbine): yellow transparent oil, showing orange-yellow under spraying with Dragendorff's reagent, soluble in chloroform. 1 H NMR (600 MHz, chloroform-d) δ H : 7.96 (s, NH), 7.46 (d, J = 7.7 Hz, H-9), 7.29 (d, J = 7.7 Hz, H-12), 7.12 (dd, J = 7.7, 7.7 Hz, H-11), 7.07 (dd, J = 7.7, 7.7 Hz, H-10), 3.99 (ddd, J = 10.9, 4.5, 4.5 Hz H-17), 3.82 (s, OCH3), 3.12 (dd, J = 11.2, 2.4 Hz, H-3), 2.83 (dd, J = 11.4, 2.0 Hz, H-5α), 2.68 (dd, J = 12.8, 6.3 Hz, H-21α), 2.58 (dd, J = 9.38, 11.3, 4.1 Hz, H-16), 2.42 (m, H-5β): 13 C NMR (150 MHz, chloroform-d) δ C : 174.9 (C-22), 136.1 (C-13), 134.6 (C-2), 127.4 (C-8), 121.5 (C-11), 119.5 (C-10), 118.2 (C-9), 110.9 (C-12), 108.4 (C-7), 66.2 (C-17), 60.6 (C-21), 60.4 (C-3), 54.8 (C-16), 53.4 (C-5), 52.1 (OCH3), 38.1 (C-20), 36.6 (C-15), 33.3 (C-14), 27.8 (C-18), 24.7 (C-19), 21.8 (C-6).

[0068] Compound 4 (yohimbine): colorless transparent oil, showing orange-yellow under spraying with Dragendorff's reagent, soluble in chloroform. 1 H NMR (600 MHz, chloroform-d) δ H: 7.84 (s, NH), 7.47 (d, J = 7.7 Hz, H-9), 7.29 (d, J = 7.7 Hz, H-12), 7.13 (dd, J = 7.7, 7.7 Hz, H-11), 7.08 (dd, J = 7.7, 7.7 Hz, H-10), 4.23 (m, H-17), 3.81 (s, OCH3), 3.31 (dd, J = 11.4, 2.2 Hz, H-3), 3.08 (dd, J = 10.9, 5.5 Hz, H-5α), 2.94 (dd, J = 11.4, 2.8 Hz, H-21β), 2.61 (ddd, J = 11.3, 11.3, 4.3 Hz, H-5β), 2.34 (dd, J = 11.5, 2.2 Hz, H-21α), 2.23 (dd, J = 10.6, 10.6 Hz, H-16); 13 13C NMR (150 MHz, chloroform-d) δ C : 175.8 (C-22), 136.1 (C-13), 134.6 (C-2), 127.5 (C-8), 121.5 (C-11), 119.5 (C-10), 118.2 (C-9), 110.9 (C-12), 108.3 (C-7), 67.1 (C-17), 61.4 (C-21), 60.0 (C-3), 53.0 (C-5), 52.4 (C-16), 52.1 (OCH3), 40.8 (C-20), 36.8 (C-15), 34.4 (C-14), 31.6 (C-18), 23.4 (C-19), 21.8 (C-6).

[0069] Compound 5 (β-yohimbine): yellow transparent oil, showing orange-yellow under spraying with Dragendorff's reagent, soluble in chloroform. 1 1H NMR (600 MHz, chloroform-d) δ H: 7.39 (d, J = 7.8 Hz, H-9), 7.28 (d, J = 8.0 Hz, H-12), 7.05 (dt, J = 8.0 Hz, H-11), 6.95 (dt, J = 8.0 Hz, H-10), 3.81 (s, OCH3), 3.78 (m, H-17), 3.35 (d, J = 11.0 Hz, H-3), 3.12 (dd, J = 11.5, 5.1 Hz, H-5a), 2.97 (dd, J = 11.0, 3.0 Hz, H-21a), 2.90 (m, H-6a), 2.74 (ddd, 3.2 and 1.6 Hz, H-6b), 2.63 (dt, J = 11.5, 4.6 Hz, H-5b), 2.20 (q, J = 11.0 Hz, H-21b), 2.18 (m, H-14a), 2.15 (m, H-16), 2.05 (ddd, J = 12.0, 7.0, 3.0 Hz, H-18a), 1.70 (ddd, J = 12.0, 7.0, 3.0 Hz. H-19a), 1.55 (dt, J = 11.0, 3.0 Hz, H-15), 1.50 (m, H-20), 1.40 (m, H-18b), 1.35 (m, H-14b), 1.20 (m, H-19b). 1 1H NMR (600 MHz, chloroform-d) δ C : 177.0 (C-22), 138.3 (C-13), 135.1 (C-2), 128.4 (C-8), 122.2 (C-11), 120.0 (C-10), 118.8 (C-9), 112.2 (C-12), 108.0 (C-7), 73.1 (C-17), 61.9 (C-21), 61.5 (C-3), 58.9 (C-16), 54.3 (C-5), 52.4 (OCH3), 43.6 (C-15), 40.7 (C-20), 35.2 (C-18), 34.5 (C-14), 29.2 (C-19), 22.4 (C-6).

[0070] Compound 6 (O-acetylyohimbine): Colorless transparent oil, showing orange-yellow color when sprayed with Dragendorff's reagent, soluble in chloroform. 1 1H NMR (600 MHz, chloroform-d) δ H: 7.80 (NH), 7.46 (d, J = 7.8 Hz, H-9), 7.30 (d, J = 7.8 Hz, H-12), 7.13 (dd, J = 7.8, 7.8 Hz, H-11), 7.08 (dd, J = 7.8, 7.8 Hz, H-10), 5.44 (m, H-17), 3.70 (s, OCH3), 3.40 (dd, J = 11.3, 2.3 Hz, H-3), 3.09 (dd, J = 11.3, 6.0 Hz, H-5β), 2.97 (dd, J = 11.2, 3.7 Hz, H-21β), 2.64 (ddd, J = 11.3, 11.3, 4.3 Hz, H-5α), 2.39 (dd, J = 11.7, 2.6 Hz, H-16), 2.23 (dd, J = 10.6, 10.6 Hz, H-21α)), 2.01 (s, COCH3).

[0071] Compound 7 (19,20-dehydro-β-yohimbine): Colorless transparent oil, showing orange-yellow under spraying with Dragendorff's reagent, soluble in chloroform. 1 H NMR (600 MHz, chloroform-d) δ H : 7.47 (d, J = 7.8 Hz, H-9), 7.35 (d, J = 7.8 Hz, H-12), 7.15 (t, J = 7.4 Hz, H-11), 7.06 (t, J = 7.4 Hz, H-10), 5.88 (s, H-19), 4.45 (s, H-17), 4.07 (m, H-21a), 3.98 (m, H-21b), 3.81 (s, OCH3), 3.23 (m, H-6a), 3.16 (m, H-15), 3.07 (m, H-6b), 3.07 (m, H-14a), 2.29 (m, H-18b), 2.52 (m, H-16), 2.52 (m, H-18a), 1.52 (m, H-14b). 13 C NMR (150 MHz, chloroform-d) δ C : 174.7 (C-22), 138.0 (C-13), 127.4 (C-8), 126.8 (C-19), 123.8 (C-11), 120.9 (C-10), 119.3 (C-9), 112.7 (C-12), 106.8 (C-7), 66.7 (C-17), 60.1 (C-21), 52.8 (OCH3), 51.4 (C-16), 34.9 (C-14), 34.6 (C-18), 32.9 (C-15), 20.3 (C-6).

[0072] Compound 8 (trimethoxybenzoylyohimbine): yellow oil, showing orange-yellow under spraying with Dragendorff's reagent, soluble in chloroform. 1 H NMR (600 MHz, chloroform-d) δ H : 7.47 (d, J = 7.6 Hz, H-9), 7.29 (d, J = 7.6 Hz, H-12), 7.13 (dd, J = 7.6, 7.6 Hz, H-11), 7.09 (dd, J = 7.6, 7.6 Hz, H-10), 7.32 (s, H-2’), 7.32 (s, H-6’), 5.57 (d, J = 2.8 Hz, H-17), 3.94 (s, 4’-OCH3), 3.94 (s, 4’-OCH3), 3.91 (s, 5’-OCH3), 3.69 (s, OCH3), 3.33 (d, J = 10.9 Hz, H-3), 3.11 (dd, J = 11.3, 5.8 Hz, H-5β), 3.02 (dd, J = 11.2, 3.7 Hz, H-21β), 2.66 (ddd, J = 11.3, 11.3, 4.3 Hz, H-5α), 2.52 (dd, J = 11.6, 2.8 Hz, H-16), 2.27 (overlap, H-21α), 2.12 (ddd, J = 11.4, 11.4, 3.4 Hz, H-15); 13 C NMR (150 MHz, chloroform-d) δ C : 172.3 (C-22), 165.5 (C-7’), 153.1 (C-3’), 153.1 (C-5’), 142.4 (C-4’), 136.1 (C-13), 134.5 (C-2), 127.5 (C-8), 125.4 (C-1’), 121.5 (C-11), 119.5 (C-10), 118.3 (C-9), 110.9 (C-12), 108.3 (C-7), 107.0 (C-2’), 107.0 (C-6’), 71.3 (C-17), 62.6 (C-21), 61.1 (5’-OCH3), 60.2 (C-3), 56.4 (4’-OCH3), 56.4 (6’-OCH3), 53.2 (C-5), 51.2 (C-16), 52.1 (OCH3), 40.4 (C-20), 37.6 (C-15), 34.5 (C-14), 29.9 (C-18), 24.2 (C-19), 21.9 (C-6).

[0073] The structural formulas of Compounds 1 to 8 are as follows:

[0074]

[0075] Specifically:

[0076]

[0077]

[0078] Example 2

[0079] 1. Experimental method

[0080] Protein tyrosine phosphatase (PTP1B) can hydrolyze the phosphate bond of the substrate p-nitrophenyl phosphate (p-NPP) to generate the dephosphorylated product p-nitrophenol (p-NP), which has a strong absorption at a wavelength of 405 nm. After adding the sample to be tested, the inhibition rate of the compound on PTP1B can be calculated by detecting the change in OD 405 value, and compared with the inhibition rate of the positive drug oleanolic acid to evaluate the inhibitory effect of the compound on PTP1B activity.

[0081] Specifically, this experiment was carried out in a 96-well cell culture plate. The reaction used a citrate buffer system (pH 6.0), and the total reaction system was 100 μL. First, 25 μL of citrate buffer (50 mM) was added to each well, then 25 μL of oleanolic acid or the compound solution to be tested at different concentrations was added, and then 25 μL of PTP1B (30 nM) was added. After gently shaking and mixing, the 96-well cell culture plate was incubated at 37 °C for 10 min. Then 25 μL of p-NPP (2 mM) solution was added, and the incubation was continued at 37 °C for 30 min. 100 μL of NaOH solution (10 M) was added to terminate the reaction, and the absorbance value of its OD 405 was measured, the data was recorded, and the measurement was carried out in parallel 3 times. The PTP1B inhibition rate (100%) = (1 - OD value of the sample group / OD value of the blank group) × 100%. Each group of experiments was set with 3 replicate wells. In this experiment, the blank group was the one where the enzyme solution was replaced with citrate buffer, and other reagents were the same as those in the sample experimental group.

[0082] The initial screening concentration of the compound was 100 μM. For compounds with an inhibition rate greater than 50%, the IC 50 value would be further determined. The compound solution was diluted to concentration gradients of 100, 50, 25, 12.5, 6.25, and 3.125 μM. The PTP1B inhibition rate of the compound at different concentrations was measured by the above method, and the IC 50 value was calculated using GraphPad Prism 8.0 software.

[0083] 2. Test results

[0084] The PTP1B enzyme inhibition activity results of Compounds 1 to 8 are shown in Table 3.

[0085] Table 3 PTP1B Enzyme Inhibitory Activity of Compounds 1 to 8

[0086] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> 1 28.62±1.17 4 >100 7 46.58±2.95 2 80.45±0.21 5 >100 8 21.35±0.51 3 62.93±1.92 6 >100 Oleanolic acid 9.51±0.17

[0087] The results in Table 3 show that compounds 1 and 8 have significant inhibitory effects on the protein tyrosine phosphatase PTP1B, with IC 50 values of 28.62 ± 1.17 μM and 21.35 ± 0.51 μM, respectively. Compounds 2, 3, and 7 all have good PTP1B enzyme inhibitory activity, with IC 50 values of 80.45 ± 0.21 μM, 62.93 ± 1.92 μM, and 46.58 ± 2.95 μM, respectively.

[0088] The above content is a detailed description of the present application in combination with specific embodiments, and it cannot be determined that the implementation mode of the present application is only limited to these descriptions. For those of ordinary skill in the technical field of the present application, without departing from the basic inventive concept of the present application, several simple deductions or substitutions can be made.

Claims

1. Yohimbine alkaloids, characterized in that: is at least one of Compound 1 to Compound 8, 2. The yohimbine alkaloid according to claim 1, wherein: is Compound 3, Compound 5, Compound 6, Compound 7 or Compound 8.

3. Use of the yohimbine alkaloid according to Claim 1 or 2 in the preparation of a protein tyrosine phosphatase inhibitory drug.

4. A protein tyrosine phosphatase inhibitory drug, characterized in that: contains the yohimbine alkaloid according to Claim 1 or 2.

5. Use of the yohimbine alkaloid according to Claim 1 or 2 in the preparation of a drug for treating diabetes.

6. A drug for treating diabetes, characterized in that: contains the yohimbine alkaloid according to Claim 1 or 2.

7. The preparation method of yohimbine alkaloids according to claim 1 or 2, characterized in that: comprises the following steps, Step 1: Dry and pulverize the branches and leaves of Alstonia mairei, extract with methanol, and concentrate the extract under reduced pressure to obtain a crude extract paste; Step 2: Add warm water to the crude extract paste in Step 1, suspend, then add an acidic solution, adjust the pH value to acidic, and extract with chloroform to remove the non-alkaloid components in the extract, obtaining an acidic aqueous solution; Step 3: Add an alkaline solution to the acidic aqueous solution in Step 2, adjust the pH value to alkaline, obtaining an alkaline aqueous solution; Step 4: Extract the alkaline aqueous solution in Step 3 with chloroform and n-butanol respectively to obtain a chloroform fraction paste and an n-butanol fraction paste; Step 5: Mix the chloroform fraction paste with silica gel, perform normal-phase silica gel column chromatography, with the mobile phase being dichloromethane-methanol gradient elution, combine with TLC thin-layer chromatographic plate spotting detection, and merge to obtain 7 fractions with increasing polarity, namely Fraction A to Fraction G; perform reverse-phase C-18 silica gel column chromatography on Fraction B, with the mobile phase being methanol-water gradient elution, combine with TLC thin-layer chromatographic plate spotting detection, and merge to obtain 8 fractions with decreasing polarity, namely Fraction B1 to Fraction B8; perform Sephadex LH-20 gel column chromatography on Fraction B7, with the mobile phase being methanol, to obtain 2 fractions with decreasing molecular weight, namely B7a and B7b; Fraction B7b is purified by high-performance liquid chromatography on a C-18 chromatographic column, with the mobile phase being methanol-water-ammonia water, to obtain Compound 8; Fraction C is subjected to reverse-phase C-18 silica gel column chromatography, with the mobile phase being methanol-water gradient elution, combine with TLC thin-layer chromatographic plate spotting detection, and merge to obtain 7 fractions with decreasing polarity, namely Fraction C1 to Fraction C7; Fraction C4 is subjected to Sephadex LH-20 gel column chromatography, with the mobile phase being methanol to obtain 2 fractions with decreasing molecular weight, namely Fraction C4a and Fraction C4b; Fraction C4a is purified by high-performance liquid chromatography on a C-18 chromatographic column, with the mobile phase being methanol-water-ammonia water, to obtain Compound 6; Fraction C4b is purified by high-performance liquid chromatography on a phenyl chromatographic column, with the mobile phase being methanol-water-ammonia water, to obtain Compound 3; Fraction D was subjected to medium-pressure preparative column chromatography on reverse-phase C-18 silica gel, with the mobile phase being gradient elution of methanol-water. Combining with TLC thin-layer chromatographic plate spotting detection, 5 fractions with decreasing polarity were obtained, namely fraction D1 to fraction D5; fraction D1 was subjected to Sephadex LH-20 gel column chromatography, with the mobile phase being methanol, and 5 fractions with decreasing molecular weight were obtained, namely fraction D1a to fraction D1e; fraction D1c was purified by high-performance liquid chromatography on a C-18 column, with the mobile phase being methanol-water-ammonia water, to obtain compound 1; Fraction D1d was subjected to Sephadex LH-20 gel column chromatography, with the mobile phase being methanol, and 2 fractions with decreasing molecular weight were obtained, namely fraction D1d1 and fraction D1d2; Fraction D1d1 was purified by high-performance liquid chromatography on a C-18 column, with the mobile phase being methanol-water-ammonia water, to obtain compound 4; Fraction D1d2 was purified by high-performance liquid chromatography on a C-18 column, with the mobile phase being methanol-water-ammonia water, to obtain compound 2; Fraction D3 was subjected to Sephadex LH-20 gel column chromatography, with the mobile phase being methanol, and 4 fractions with decreasing molecular weight were obtained, namely fraction D3a to fraction D3d; Fraction D3b was subjected to reverse-phase phenyl silica gel column chromatography, with the mobile phase being gradient elution of methanol-water. Combining with TLC thin-layer chromatographic plate spotting detection, 3 fractions with decreasing polarity were obtained, namely fraction D3b1 to fraction D3b3; Fraction D3b1 was purified by high-performance liquid chromatography on a C-18 column, with the mobile phase being methanol-water-ammonia water, to obtain compound 5; Fraction D3b2 was purified by high-performance liquid chromatography on a C-18 column, with the mobile phase being acetonitrile-water-ammonia water, to obtain compound 7.

8. The preparation method according to claim 7, wherein: In the first step, the volume concentration of methanol is 80%; Preferably, the number of methanol extractions is at least 5 times; Preferably, in the second step, the temperature of the warm water is 30 - 45 °C; Preferably, the acidic solution is 0.3% dilute hydrochloric acid solution; Preferably, the pH value is adjusted to acidic, specifically adjusted to pH 2.

9. The preparation method according to claim 7, characterized in that: In the third step, the alkaline solution is sodium carbonate aqueous solution; Preferably, the pH value is adjusted to alkaline, specifically adjusted to pH 10.

10. The preparation method according to any one of claims 7-9, characterized in that: In the fifth step, the silica gel used for mixing the chloroform part extract with silica gel is 200 - 300 mesh; Preferably, the volume ratio of dichloromethane-methanol is sequentially 100:1 to 0:1; Preferably, the volume ratio of methanol-water is sequentially 35:65 to 100:0; Preferably, in the methanol-water-ammonia water, the total volume ratio of methanol and water to ammonia water is 100:0.2, and the volume ratio of methanol and water is 35:65 to 70:30; Preferably, the volume ratio of acetonitrile-water-ammonia water is sequentially 35:65:0.2; Preferably, the flow rate of the mobile phase of the high-performance liquid phase is 2.5 mL / min.

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