CN101434625A - Anoectochilus roxburghii glycosides, derivatives thereof, preparation and use - Google Patents

Abstract

The invention discloses a protein tyrosine phosphatase 1B (PTP1B) inhibitor auroglutin and its derivatives, and provides its preparation method and application. These compounds have strong hypoglycemic activity and high inhibitory The protein tyrosine phosphatase 1B (PTP1B) activity and lower toxicity can be used to treat diseases such as diabetes and hyperlipidemia.

Description

Aurochinoside and its derivatives, preparation method and use

Technical field:

The invention belongs to the technical field of medicine, and relates to protein tyrosine phosphatase 1B (PTP1B) inhibitors, in particular to auroside and its derivatives and a preparation method thereof, and to auroside and its derivatives in the treatment of diabetes and Application in diseases such as hyperlipidemia.

Background technique:

Diabetes is a systemic chronic secretory metabolic disease associated with genetic factors. It is the metabolic disorder of sugar, fat and protein caused by the absolute or relative insufficiency of insulin in the body. If it is not well controlled, it can lead to chronic complications such as retinopathy, hypertension, kidney disease, coronary heart disease, lower extremity vascular disease, cerebrovascular disease, neuropathy, and eventually blindness, lower extremity gangrene, uremia, stroke or myocardial infarction. Infarction, even life-threatening. According to the statistics of the World Health Organization, in the world, the number of people diagnosed with diabetes in 1985 was 30 million, and it rose to 135 million in 1995. It is predicted that 300 million people will be sick in 2025.

Herba Anoectochili is the dry whole herb of Anoectochilus roxburghii, a perennial rare Chinese herbal medicine. Guangxi, Guangdong, Hainan, Guizhou, Sichuan, Yunnan and other provinces in southern my country have rich medicinal resources, and folk medicines are widely used. Clematis aureus is known among the folks as "Golden Grass", "Miraculous Medicine", "Black Ginseng", etc. In the producing areas of Zhejiang and Fujian, the price of its fresh grass is as high as 360-500 yuan/kg.

Invention content:

Under the funding of the National Natural Science Foundation of China (NSFC30400585) and the Special Fund for Chinese Medicine Research of the State Administration of Traditional Chinese Medicine (04-05ZQ03), under the guidance of hypoglycemic activity, we conducted a systematic study on the hypoglycemic active ingredients of Clematis clematis , to isolate the main active ingredient, kinsenoside (4(R)-β-D-glucopyranosyloxy-butyric acid (γ) lactone, referred to as "auuroside" in this patent application.).

auroglutinin

We studied the hypoglycemic activity of auroside, and discussed the related hypoglycemic mechanism from the aspects of in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. The results showed that the test compound aurochinoside had significant anti-hyperglycemic activity, and the reversal of hyperglycemia was dose-dependent. The results of in vitro PTP1B inhibitory activity screening showed that auroside had a significant inhibitory effect on PTP1B in a significant dose-dependent manner, and the inhibitory effect was parallel to the in vitro anti-hyperglycemic activity. And it has little inhibitory effect on other enzymes of the PTP family, showing highly specific inhibition on PTP1B. As a specific inhibitor of PTP1B, auroside is used for the treatment of type II diabetes and obesity, and it is worthy of further research and development. In addition, kinsenoside can significantly enhance the activity of serum superoxide dismutase and the ability to scavenge hydroxyl free radicals, reduce the peroxidation product malondialdehyde, and significantly reduce the content of active factor NO; pancreas histopathological examination revealed that auroside passes through the islet β The recovery of cell morphology and function plays a hypoglycemic effect; in the oral glucose tolerance test, auroside significantly improved the tolerance to exogenous glucose in hyperglycemic rats and normal rats.

In the acute toxicity test, there was no adverse reaction in the mice in the auroside administration group with a dose of 400 mg/kg, and the toxicity was extremely low. In addition, auroside also has strong hepatoprotective and lipid-lowering effects, while its epimer Goodyeroside A (4(S)-β-D-glucopyranoseoxy-butyric acid (γ) lactone ) does not have anti-hyperglycemic and lipid-lowering effects, indicating that the 4(S) configuration of auroside is the pharmacophore of its anti-hyperglycemic and lipid-lowering effects.

The object of the present invention is to provide protein tyrosine phosphatase 1B (PTP1B) inhibitor aurochinoside and its derivatives and analogs, and simultaneously provide methods for synthesizing these derivatives and analogs. Another object of the present invention is the application of the compound provided by the present invention in treating diseases such as diabetes and hyperlipidemia.

The compound provided by the invention has the following structural formula:

Wherein R is: a hydrogen atom, an alkyl group with 30 carbons or less, -CH ₂ COOR ₁ (wherein R ₁ is a hydrogen atom or an alkyl group with 30 carbons or less), -CH ₂ CH ₂ COOR ₂ (wherein R ₂ is a hydrogen atom or an alkyl group with 30 carbons or less The following alkyl), or -CH ₂ Ar (where Ar is an aromatic or heterocyclic ring such as a benzene ring, pyridine ring, quinoline ring, furan ring, pyrrole ring or thiophene ring with various substituents, where Ar is substituted The group can be a hydrogen atom or an alkyl group with 30 carbons or less, a halogen, a hydroxyl group, an amino group, an ether group, an aliphatic amino group, an aromatic amino group, a carboxyl group, etc.). When said R, R ₁ or R ₂ is an alkyl group with 30 carbons or less, it is preferably methyl, ethyl or propyl.

The structural formula of the derivative of auroglutin provided by the present invention, its pharmaceutically acceptable salt of analog is as shown in (I):

in

The compound when R=H is called auroside in this patent;

The compound when R=-CH ₃ is called compound KD1 in this patent;

The compound when R=-CH ₂ CH ₃ is called compound KD2 in this patent;

The compound when R=-CH ₂ COOH is called compound KD3 in this patent;

The compound when R=-CH ₂ CH ₂ COOH is called compound KD4 in this patent;

The compound when R=-CH ₂ ph is called compound KD5 in this patent;

The compound when R=-CH ₂ -p-hydroxyl-ph is called compound KD6 in this patent;

The compound when R=-CH ₂ -p-carboxyl-ph is called compound KD7 in this patent;

The compound when R=-CH ₂ -p-COOH-CH=CH-ph is called compound KD8 in this patent;

The compound when R=-CH ₂ -2-thiophene is called compound KD9 in this patent;

The compound when R=-CH ₂ -2-[(5'-COOH)-thi ophene] is called compound KD10 in this patent.

The compound of the present invention is prepared by using auroglutinin as the starting material, protecting the hydroxyl group on the glucose group through Ac ₂ O/pyridine, reacting with RX through LDA/HMPA/THF/low temperature conditions, and further reacting in NaOCH ₃ /CH ₃ OH /Low temperature/anhydrous conditions to remove the protective group on the glucose group in a three-step reaction. Its synthetic route is as follows:

Aureoside (compound 1) Fully acetylated auroside (compound 2)

Compound 3-1 R=-CH ₃ Compound KD1 R=-CH ₃

Compound 3-2 R=-CH ₂ Compound KD2 R=-CH ₂

Compound 3-3 R=-CH ₂ COOH Compound KD3 R=-CH ₂ COOH

Compound 3-4 R=-CH ₂ CH ₂ COOH Compound KD4 R=-CH ₂ CH ₂ COOH

Compound 3-5 R=-CH ₂ ph Compound KD5 R=-CH ₂ ph

Compound 3-6 R=-CH ₂ -p-hydroxyl-ph Compound KD6 R=-CH ₂ -p-hydroxyl-ph

Compound 3-7 R=-CH ₂ -p-carboxyl-ph Compound KD7 R=-CH ₂ -p-carboxyl-ph

Compound 3-8 R=-CH ₂ -p-COOH-CH=CH-ph Compound KD8 R=-CH ₂ -p-COOH-CH=CH-ph

Compound 3-9 R=-CH ₂ -2-thiophene Compound KD9 R=-CH ₂ -2-thiophene

Compound 3-10 R=-CH ₂ -2-[(5'-COOH)]-thiophene Compound KD10 R=-CH ₂ -2-[(5'-COOH)]-thiophene

The invention provides novel auroglutin and its derivatives and analogues, as well as a practical preparation method. The experiment of the present invention shows that the acretin and its derivatives have the prospect of inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B) and preparing drugs for treating diabetes and hyperlipidemia.

Description of drawings

Fig. 1 is the structural formula of the compound provided by the present invention.

Detailed ways:

Example 1 Extraction, separation and purification of auroside (compound 1)

1 kg of dried whole herb of Clematis clematis was crushed, then heated and refluxed with methanol to extract 6 times, each time for 2 hours, filtered, and the solvent was recovered. Suspend the crude extract with an appropriate amount of water, extract with petroleum ether, ethyl acetate, and n-butanol in sequence, and concentrate the extract into an extract to obtain 500 g of n-butanol. 10 g of the n-butanol fraction was subjected to repeated silica gel column chromatography, eluted with a chloroform-methanol gradient, and then recrystallized through a Sephadex LH-20 gel column to obtain 1580 mg of auroside.

Auroglutinin: colorless needle crystal, easily soluble in methanol and water, insoluble in chloroform, acetone and ethanol. Melting point 151.0-153°C, Molish reaction positive. IR v _max cm ⁻¹ : 3400 (v _OH ), 2926, 2908 (v _CH ), 1769, 889. Glucose was detected by co-thin layer of acid hydrolysis and standard substance. FAB(-)-MS: m/z 264 ([MH] ⁺ , 355-Gly),

[α] _D ¹⁶ -62.8° (c0.64, H ₂ O). ¹ H-NMR (in Pyr): δ 2.71 (1H, dd, J=18.0Hz, J=2.5Hz, H-3), 2.89 (1H, dd, J=18.0Hz, J=6.3Hz, H-3 ), 3.18(1H, m, H-5'), 3.27(1H, dd, J=8.0Hz, J=6.1Hz, H-2'), 3.29(1H, m, H-4'), 3.34( 1H, m, H-3'), 3.65(1H, dd, J=11.2Hz, J=3.7Hz, H-6'), 3.86(1H, dd, J=10.3Hz, J=4.7Hz, H- 5), 4.36(1H, dd, J=11.7Hz, J=2.3Hz, H-6'), 4.40(1H,dd, J=10.3Hz, J=1.5Hz, H-5), 4.50(1H, m, H-4), 4.77 (1H, d, J = 7.9 Hz, H-1'). ¹³ C-NMR (in MeOD): δ37.0 (C-3), 62.7 (C-6'), 71.4 (C-4'), 74.8 (C-2'), 75.3 (C-5), 76.0 (C-4), 77.9 (C-5'), 78.1 (C-3'), 103.6 (C-1'), 179.0 (C-1). The single crystal of auroside was cultured in methanol, and X-ray diffraction structure analysis was performed to confirm that the 4-position chiral carbon was in the R configuration, and the structure was shown in the figure below.

auroglutinin

Embodiment 2 The synthesis of fully acetylated auroglutinin (compound 2)

Auroglutinin (0.5g, 2mmol) was dissolved in 10ml of anhydrous pyridine, after stirring and dissolving, add 5ml of acetic anhydride, react at room temperature for 7h, TLC tracking detection, the reaction solution was poured into 60ml of saturated _CuSO , Extracted with ethyl acetate (50ml×2), combined the organic layers, washed with 0.5M HCl (20ml), washed with saturated NaCl, and dried over anhydrous Na ₂ SO ₄ overnight. The solvent was distilled off under reduced pressure, and 780 mg of a white solid (that is, fully acetylated auroside) was obtained after purification on a silica gel column.

The synthesis of embodiment 3 compound 3-1～3-10

Fully acetylated auroglutinin (0.5g, 1.2mmol) was dissolved in 10ml of anhydrous THF, the reaction system reached -25°C, 1ml of lithium diisopropylamide (LDA) was added, CH ₃ -Br was added, and the chemical The equivalent ratio is 1:1. After reacting for 1 hour, add 2ml of hexamethyltriamine phosphate (HMPA), evaporate the solvent under reduced pressure, and slowly add 20ml of ice water dropwise. Quickly extracted with ethyl acetate (50ml×2), combined the organic layers and dried over anhydrous Na ₂ SO ₄ overnight. The solvent was distilled off under reduced pressure, and compound 3-1 (white solid, 358 mg) was obtained after silica gel column purification.

Compound 3-2 (white solid, 374mg), compound 3-3 (white solid, 390mg), compound 3-4 (white solid, 402mg), compound 3-5 (white solid, 420mg), compound 3-6 (white solid, 431 mg), compound 3-7 (white solid, 443 mg), compound 3-8 (white solid, 452 mg), compound 3-9 (white solid, 417 mg), compound 3-10 (white solid , 435mg).

The synthesis of embodiment 4 compound KD1～KD10

Dissolve 0.5 g of compound 3-1 in 20 ml of anhydrous methanol, stir to dissolve, add 0.3 g NaOCH ₃ , react at 10°C for 6 h, track and detect by TLC, evaporate the solvent and acetic acid under reduced pressure, and purify on a silica gel column to obtain compound KD1.

Compounds KD2-KD10 were prepared by the same method.

Compound KD1 (R=-CH ₃ , white solid, 358 mg), ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 1.17 (3H, d, J=7.2 Hz, 3-CH ₃ ), 2.76 (1H , m, 3-H), 3.41 (1H, m, 4-H), 4.30 (1H, m, 5-H), 4.54 (1H, m, 5-H), 5.01 (1H, m, 1'- H), 3.40~3.81 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 178.2 (C-2), 42.7 (C-3), 12.8 (3- CH ₃ ), 66.8 (C-4), 72.4 (C-5 ), 102.1 (C-1'), 74.2 (C-2'), 76.9 (C-3'), 71.6 (C-4'), 77.2 (C-5'), 62.5 (C-6'). According to the above data, the compound was identified as 3-methylauroside (ie compound KD1).

Compound KD1

Compound KD2 (R=—CH ₂ CH ₃ , white solid, 269 mg), ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 0.91 (3H, t, J=7.5 Hz, 3-CH ₂ CH ₃ ) , 1.78(2H, m, 3- CH ₂ CH ₃ ), 3.41(1H, m, 4-H), 4.30(1H, m, 5-H), 4.54(1H, m, 5-H), 5.01( 1H, m, 1'-H), 3.40~3.81 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 178.8 (C-2), 49.7 (C-3), 11.5 (3-CH ₂ CH ₃ ), 22.2 (3- CH ₂ CH ₃ ) , 64.8(C-4), 72.8(C-5), 102.1(C-1'), 74.3(C-2'), 76.8(C-3'), 71.7(C-4'), 77.1(C -5'), 62.3 (C-6'). According to the above data, the compound was identified as 3-ethylline glucoside (ie compound KD2).

Compound KD2

Compound KD3 (R=—CH ₂ COOH, white solid, 261 mg), ¹ H NMR (400 MHz, CD ₃ OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 2.93 (1 H, m, 3- CH ₂ COOH), 2.68 (1H, m, 3- CH ₂ COOH), 2.95 (1H, m, 3-H), 3.46 (1H, m, 4-H), 4.31 (1H, m, 5-H), 4.56 (1H, m, 5-H), 5.04 (1H, m, 1'-H), 3.40~3.80 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 177.5 (C-2), 48.4 (C-3), 177.1 (3- CH ₂ COOH), 32.0 (3- CH ₂ COOH), 64.1 (C-4), 72.6(C-5), 102.1(C-1'), 74.3(C-2'), 76.8(C-3'), 71.7(C-4'), 77.1(C-5 '), 62.3 (C-6'). According to the above data, the compound was identified as 3-carboxymethyl auronoside (ie compound KD3).

Compound KD3

Compound KD4 (R=—CH ₂ CH ₂ COOH, white solid, 257 mg), ¹ H NMR (400 MHz, CD ₃ OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 1.92 (2H, m, 3 - CH ₂ CH ₂ COOH), 2.35 (2H, m, 3-CH ₂ CH ₂ COOH), 2.63 (1H, m, 3-H), 3.45 (1H, m, 4-H), 4.30 (1H, m , 5-H), 4.57 (1H, m, 5-H), 5.02 (1H, m, 1'-H), 3.40~3.81 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 178.8 ( C -2), 46.7 (C-3), 178.2 (3-CH ₂ CH ₂ COOH), 8.6 (3- CH ₂ CH ₂ COOH), 31.0(3-CH ₂ CH ₂ COOH), 64.7(C-4), 72.1(C-5), 102.9(C-1'), 74.2(C-2'), 76.7(C-3 '), 71.5 (C-4'), 77.8 (C-5'), 62.9 (C-6'). According to the above data, the compound was identified as 3-carboxyethyl clematin (ie compound KD4).

Compound KD4

Compound KD5 (R=-CH ₂ Ph, white solid, 250 mg), ¹ H NMR (400 MHz, CD ₃ OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 3.20 (1 H, m, 3- CH ₂ ph), 2.96 (1H, m, 3- CH ₂ ph), 7.29-7.41 (5H, m, 3-CH ₂ ph ), 2.98 (1H, m, 3-H), 3.49 (1H, m, 4-H), 4.33(1H, m, 5-H), 4.58(1H, m, 5-H), 5.01(1H, m, 1'-H), 3.40～3.82(6H, m, 2'- H～6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 177.3 (C-2), 55.7 (C-3), 138.6, 128.7, 128.9, 125.4 (3-CH ₂ C ₆ H ₅ ), 32.6 (3 - CH ₂ C ₆ H ₅ ), 64.5(C-4), 72.3(C-5), 102.0(C-1'), 74.6(C-2'), 76.3(C-3'), 71.9( C-4'), 77.0 (C-5'), 62.0 (C-6'). According to the above data, the compound was identified as 3-benzylsiltrin (ie compound KD5).

Compound KD5

Compound KD6 (R=p-hydroxyl-ph-CH ₂ -, white solid, 243 mg), ¹ H NMR (400 MHz, CD ₃ OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 3.21 (1H, m, 3- CH2phOH ), 2.92 (1H, m, 3 _- CH2phOH ), 7.13 (2H _, d, J = 7.8Hz, 3- CH2phOH ), 6.75 (2H, d, J ₌ 7.8 Hz, 3-CH ₂ ph OH), 3.01 (1H, m, 3-H), 3.45 (1H, m, 4-H), 4.34 (1H, m, 5-H), 4.59 (1H, m, 5 -H), 5.02 (1H, m, 1'-H), 3.40~3.81 (6H, m, 2'-H~6'H). ¹³ CNMR (100MHz, CD ₃ OD): δ (ppm) 177.5 (C-2), 55.8 (C-3), 155.7, 115.7, 130.6, 131.2 (3-CH ₂ C ₆ H ₄ OH), 32.8 (3 - CH ₂ C ₆ H ₄ OH), 64.3 (C-4), 72.8 (C-5), 102.9 (C-1'), 74.0 (C-2'), 76.2 (C-3'), 71.5 (C-4'), 77.2 (C-5'), 62.7 (C-6'). According to the above data, the compound was identified as 3-hydroxybenzylsiltrin (ie compound KD6).

Compound KD6

Compound KD7 (R=p-carboxyl-ph—CH ₂ -, white solid, 238 mg), ¹ H NMR (400 MHz, CD ₃ OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 3.28 (1H , m, 3- CH ₂ phCOOH), 2.90 (1H, m, 3- CH ₂ phCOOH), 7.04 (2H, d, J=8.4Hz, 3-CH ₂ ph COOH), 8.19 (2H, d, J= 8.4Hz, 3-CH ₂ ph COOH), 3.05(1H, m, 3-H), 3.48(1H, m, 4-H), 4.32(1H, m, 5-H), 4.52(1H, m, 5-H), 5.05 (1H, m, 1'-H), 3.40~3.80 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 177.2 (C-2), 55.7 (C-3), 143.2, 128.1, 130.2, 127.9 (3-CH ₂ C ₆ H ₄ COOH), 32.6 ( 3 - CH2C6H4COOH), 64.7(C _{- 4} ), 72.5(C-5 ₎ , 102.8(C-1'), 74.3(C-2'), 76.3(C-3'), 71.4 (C-4'), 77.6 (C-5'), 62.3 (C-6'). According to the above data, the compound was identified as 3-carboxybenzylsilaminoside (ie compound KD7).

Compound KD7

Compound KD8 (R=p-COOH-CH=CH-Ph—CH ₂ -, white solid, 232 mg), ¹ H NMR (400 MHz, CD3OD): ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 3.21 (1H, m , 3 _- CH2phCH =CHCOOH), 2.94 (1H, m, 3 _- CH2CH =CHCOOH), 6.75 (2H, d, J=8.1Hz, 3 _- CH2phCH=CHCOOH), 7.68 (2H, d, J ₌ 8.1 Hz, 3- CH2phCH = CHCOOH), 7.61 (1H, d, J = 16.2Hz, 3- _CH2phCH = CHCOOH ), 6.35 (1H, d, J = 16.2Hz, 3-CH ₂ phCH= CH COOH), 3.02(1H, m, 3-H), 3.46(1H, m, 4-H), 4.31(1H, m, 5-H), 4.54(1H , m, 5-H), 5.01 (1H, m, 1'-H), 3.40~3.81 (6H, m, 2'-H~6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 177.1 (C-2), 55.2 (C-3), 137.7, 128.3, 128.9, 132.8 (3-CH ₂ C ₆ H ₄ CH=CHCOOH), 145.1 ( 3 _- CH2C6H4CH ₌ CHCOOH ₎ , 117.3 _{( 3} - CH2C6H4CH= CHCOOH ), 172.1 (3 _{- CH2C6H4CH} =CHCOOH), _{32.8 (} 3 - CH2C6H4CH=CHCOOH), 64.4(C- ₄ ) _, 72.2(C-5 ₎ , 102.3(C-1'), 74.0(C-2'), 76.5(C-3' ), 71.6 (C-4'), 77.2 (C-5'), 62.5 (C-6'). According to the above data, the compound was identified as 3-carboxyvinylbenzylsiltrin (ie compound KD8).

Compound KD8

Compound KD9 (R=-CH ₂ -2'-thiophene, white solid, 241 mg), ¹ H NMR (400 MHz, CD ₃ OD): δ (ppm) 3..01 (1H, m, 3- CH ₂ C ₄ H ₃ S), 2.75 (1H, m, 3- CH ₂ C ₄ H ₃ S), 6.80-7.45 (5H, m, 3-CH ₂ C ₄ H ₃ S), 2.99 (1H, m, 3-H ), 3.45(1H, m, 4-H), 4.32(1H, m, 5-H), 4.55(1H, m, 5-H), 5.02(1H, m, 1'-H), 3.40～3.82 (6H, m, 2'-H ~ 6'H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 177.2 (C-2), 56.2 (C-3), 143.1, 126.6, 126.4, 124.8 (3-CH ₂ CH ₃ S), 23.7 (3 - _{CH2C4H3S )} , 64.2(C- ₄ ), 72.8(C-5), 102.1(C-1'), 74.1(C-2'), 76.5(C-3'), 71.5 (C-4'), 77.5 (C-5'), 62.1 (C-6'). According to the above data, the compound was identified as 3-thienylsilchinoside (ie compound KD9).

Compound KD9

Compound KD10 (R = -CH ₂ -2'-[(5'-COOH)-thiophene], white solid, 228 mg). ¹ H NMR (400MHz, CD ₃ OD): δ (ppm) 3..02 (1H, m, 3- CH ₂ C ₄ H ₃ SCOOH), 2.76 (1H, m, 3- CH ₂ C ₄ H ₃ SCOOH ), 6.92 (1H, d, J=6.4Hz, 3-CH ₂ C ₄ H ₂ S), 7.93 (1H, d, J=6.4Hz, 3-CH ₂ C ₄ H ₂ S), 2.98 (1H, m, 3-H), 3.43 (1H, m, 4-H), 4.30 (1H, m, 5-H), 4.54 (1H, m, 5-H), 5.03 (1H, m, 1'-H ), 3.40~3.82 (6H, m, 2'-H~6'H). ¹³ CNMR (100MHz, CD ₃ OD): δ (ppm) 177.0 (C-2), 56.1 (C-3), 151.0, 124.7, 133.2, 131.5 (3-CH ₂ C ₄ H ₂ SCOOH), 163.4 (3 -CH ₂ C ₄ H ₂ S C OOH), 23.5 (3- CH ₂ C ₄ H ₂ SCOOH), 64.5 (C-4), 72.7 (C-5), 102.3 (C-1'), 74.2 ( C-2'), 76.8 (C-3'), 71.4 (C-4'), 77.4 (C-5'), 62.0 (C-6'). According to the above data, the compound was identified as 3-carboxythiophene silchinoside (ie compound KD10).

Compound KD10

The synthesis of embodiment 5 intermediate compound 8

Compound 5 Compound 6 Compound 7 Compound 8

Reagents and conditions: (a) DMF, POCl ₃ , 80°C, (b) ethanediol, NH _2- NH ₂ /KOH, 180°C (c) NBS, (phCO ₂ ) ₂ , CCl ₄ , 20h, reflux

Compound 6: 10 g (0.12 mol) of thiophene was dissolved in 30 ml of DMF, stirred and dissolved, and 10.8 ml of phosphorus oxychloride was slowly added dropwise, and the addition was completed in one hour. Reaction at 80°C for 10h. After distilling off the solvent under reduced pressure, compound 6 was purified.

Compound 7: Compound 6 (10g, 0.09mol) was dissolved in 30ml of medium ethylene glycol. After stirring and dissolving, an appropriate amount of NH ₂ -NH ₂ and KOH was added, refluxed for 4 hours, followed by TLC detection, and the solvent was evaporated under reduced pressure. Crystallization and silica gel column purification yielded compound 7.

Compound 8: 10 g (0.1 mol) of 2-methylthiophene was dissolved in 40 ml of CCl ₄ , stirred and dissolved, and 17.8 g (0.1 mol) of bromosuccinimide and 1 g of benzoyl peroxide were added. Reflux reaction 20h. TLC tracking detection, the solvent was evaporated under reduced pressure, and compound 8 was obtained after silica gel column purification. ¹ H NMR (400MHz, CD ₃ OD): δ (ppm) 4.97 (2H, s, 2- CH ₂ Br), 6.84 (1H, d, J=6.8Hz, 3-H), 6.95 (1H, t, J=6.8Hz, 4-H), 7.45 (1H, d, J=6.8Hz, 5-H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 139.4 (C-2), 27.1 ( 2- CH2Br ), 126.1 (C-3), 127.3 (C-4), _125.7 (C-5). According to the above data, the compound was identified as 2-bromomethylthiophene (ie compound 8).

Compound 8

The synthesis of embodiment 6 intermediate compound 10

Compound 7 Compound 9 Compound 10

Reagents and conditions: (a) AC ₂ O/AlCl ₃ , CH ₃ CHCl ₂ 40-45°C (b) NaOCl, 70-80°C (c) NBS, (PhCO ₂ ) ₂ , CCl ₄ , 20h, reflux

Compound 9: 10 g (0.1 mol) of 2-methylthiophene was dissolved in 30 ml of CH ₃ CHCl ₂ , stirred and dissolved, and then 11 ml of acetic anhydride and 1 g of aluminum trichloride were added. After reacting at 40-45°C for 3h, add 2.6g of sodium hypochlorite and react at 70-80°C for 5h, TLC tracking detection, and distill off the solvent and acetic acid under reduced pressure. Compound 9 was obtained by silica gel column purification.

Compound 10: Compound 9 (10 g, 0.08 mol) was dissolved in 40 ml of CCl ₄ , stirred and dissolved, then 17.8 g (0.1 mol) of bromosuccinimide and 1 g of benzoyl peroxide were added. Reflux reaction 20h. TLC tracking detection, the solvent was distilled off under reduced pressure, and compound 10 was obtained by silica gel column purification. ¹ H NMR (400MHz, CD ₃ OD): δ (ppm) 4.92 (2H, s, 2- CH ₂ Br), 6.92 (1H, d, J=6.8Hz, 3-H), 7.93 (1H, d, J=6.8Hz, 4-H). ¹³ C NMR (100MHz, CD ₃ OD): δ (ppm) 147.3 (C-2), 27.6 (2- CH ₂ Br), 127.1.1 (C-3) , 134.5 (C-4), 132.7 (C-5), 162.7 (5- COOH ). According to the above data, the compound was identified as 2-bromomethyl-5-carboxythiophene (ie compound 10).

Compound 10

Example 7 STZ-induced anti-hyperglycemia activity in rats

Male wistar rats were taken and fed adaptively for 3 days. After overnight fasting, 50 mg·kg ^-1 streptozotocin (STZ) was injected intraperitoneally to establish a diabetic model. Blood glucose levels were measured after 72 hours ^. They were used as experimental rats with hyperglycemia.

The hyperglycemia rats were randomly divided into model control group, positive control group, drug administration group and normal control group, with 8 rats in each group. On the 3rd day after the establishment of the model, that is, when the blood sugar was significantly increased, the administration was administered by intragastric administration. The monomer auroside and KD1-10 were prepared with distilled water at 5 mg/ml. 15 mg·kg ^-1 body weight, the model control group was given the same volume of normal saline, the positive control group was given 60 mg·kg ^-1 metformin hydrochloride daily, and the normal control group was fed routinely. Diabetic model rats have typical symptoms of "three excesses and one deficiency". For 4 consecutive weeks, daily observation and record of body weight, diet, drinking water, litter, urine sugar level was detected every other day, and blood sugar level was regularly measured every three days.

Experimental results: After intraperitoneal injection of STZ in model rats for 72 hours, blood glucose was significantly higher than that of normal rats. After 4 weeks of administration, there were significant differences in the blood glucose values of each administration group compared with the model control group. During the feeding process, it was also found that the symptoms of "three more and one less" of the rats in this group were significantly improved. Among all the compounds, the compound KD10 (ie, 3-carboxythiophene silchinoside) has the best hypoglycemic activity.

Effects of auroside and KD1-KD10 on blood sugar (n=8, x±s)

Note: ① ^* p<0.05 is significantly different from the normal control group

Δp<0.05 is significantly different from the model control group

Example 8 PTP1B inhibitory activity experiment

Protein tyrosine phosphorylation is an important regulatory step in cell metabolism, signal transduction and cell cycle regulation. Protein tyrosine phosphatase PTP1B is an intracellular protein tyrosine phosphatase widely expressed in vivo and plays an important role in the regulation of insulin sensitivity and energy metabolism. PTP1B is considered to be an important potential target for the treatment of insulin resistance in type II diabetes. By inhibiting PTP1B, the activity of insulin and leptin can be increased, which provides a bright prospect for the treatment of type II diabetes and obesity.

THE BIOMOL GREEN ^TM PTP1B Drug Discovery Kit (Cat#: AK-822 Lot#: T5388) was used to measure the activity of purified PTP1B by colorimetry.

Experimental results: All the tested compounds have a high inhibitory effect on the activity of PTP1B enzyme, and the inhibitory activity of compound KD10 (namely 3-carboxythiophene clematogenin) is the strongest, which is parallel to its higher hypoglycemic activity.

Auroside and KD1-KD10 Inhibit PTP1B Enzyme Activity

Example 9 Determination of the LD ₅₀ of auroside and its compounds KD1～KD10

Determination of LD ₅₀ of auroside: 12 ordinary Kunming mice, 20±2g, half male and half male, fasted for 12 hours after adaptive feeding, and then assigned to three dosage groups to investigate the oral acute toxicity of auroside , half male and half female in each group. According to 10 times the reference dose of 10mg/kg, the low dose of acute toxicity in mice was designed for pre-testing. The three dose groups were 100mg/kg, 200mg/kg, and 400mg/kg, and they were administered orally, and the diet and activities of the animals were observed within a week. , Mao Ze and survival and so on. After intragastric administration, the appearance and behavior of the animals were normal, and there was no toxic reaction in the three dosage groups, and none of them died of poisoning. The results show that there is no toxic reaction when the effective reference dose is 40 times. It is considered that the toxicity level of auroglucoside is very low or non-toxic, and it is safe to take orally, so the dose will not be increased for toxicity test.

Determination of LD ₅₀ of compounds KD1～KD10: Determination was carried out by the same method as determination of auroside LD ₅₀ , the results showed that there was no toxic reaction at 40 times the effective reference dose, and the toxicity levels of compounds KD1～KD10 were considered to be very low or none Toxic, safe to take orally.

Therefore, the toxicity levels of auroside and its compounds KD1-KD10 are very low or non-toxic, and are safe for oral administration.

Example 10 Compound KD10 (i.e. 3-carboxythiophene silchinoside) tablet preparation and method

The main pharmaceutical raw materials include: active substance compound KD10, diluent for pharmaceutical tablet, tablet binder and disintegrant, lubricant. The diluent can be selected from one or more of microcrystalline cellulose, starch, sodium hydroxymethyl starch, and glucosine; the binder can be selected from one of starch slurry, PVP glue, and gelatin slurry; the lubricant can be One of the selected talcum powder and magnesium stearate. The tablet has good stability, and the patient can take the medicine at home according to the doctor's advice, so as to ensure the continuity of treatment.

Get compound KD10 5g, starch 35g, microcrystalline cellulose 35g, hydroxymethyl starch sodium 6g, glycine 20 grind respectively finely, pass 80 mesh sieves, the addition of equal amount mixes evenly, make soft material with starch slurry (5%) , use a 30-mesh sieve to granulate, dry at 70°C, then use a 40-mesh sieve to granulate, add 0.3g of magnesium stearate, mix evenly, compress into tablets, and pack separately to obtain.

Claims (8)

1. the compound that has following structural formula

Wherein R is:

Hydrogen atom;

The following alkyl of 30 carbon;

-CH ₂COOR ₁, R wherein ₁Be hydrogen atom or the following alkyl of 30 carbon;

-CH ₂CH ₂COOR ₂, R wherein ₂Be hydrogen atom or the following alkyl of 30 carbon;

Or

-CH ₂Ar, wherein Ar is for being with aromatic nucleus or fragrant heterocycles such as various substituent phenyl ring, pyridine ring, quinoline ring, furan nucleus, pyrrole ring or thiphene ring, and wherein Ar goes up substituting group and can be hydrogen atom or the following alkyl of 30 carbon, halogen, hydroxyl, amino, ether, fatty amido, aromatic amino, carboxyl etc.

2. compound according to claim 1 is characterized in that, as described R, R ₁Or R ₂When being the following alkyl of 30 carbon, preferable methyl, ethyl or propyl group.

3. the pharmaceutical composition that contains claim 1 or 2 described compounds.

4. claim 1 or 2 described compounds are used for the treatment of application in the diabetes medicament in preparation.

5. claim 1 or 2 described compounds are used for the treatment of application in the high blood cholesterol drug in preparation.

6. claim 1 or the 2 described compounds application in preparation PTP 1B (PTP1B) selective depressant.

7. pharmaceutical composition, it is combined by claim 1 or 2 described compounds and one or more pharmaceutically acceptable carrier.

8. the preparation method of claim 1 or 2 described compounds is characterized in that: pass through Ac ₂Hydroxyl on the O/ pyridine protection glucosyl group is by LDA/HMPA/THF/ cold condition and R-X reaction, further at NaOCH ₃/ CH ₃OH/ low temperature/anhydrous condition is sloughed protecting group on the glucosyl group.

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