LU503166B1 - A compound and its applications - Google Patents

Abstract

The present invention belongs to the technical field of pharmaceutical applications of compounds, The present invention discloses a compound and its application in the preparation of preparations with inhibitory activity against protein tyrosine phosphatase 1B, in drugs for the treatment of type II diabetes in hypoglycemic drugs, in hypolipidemic drugs, in weight loss drugs, and in drugs for the treatment of fatty liver disease.

Description

Specification

A compound and its applications LU503166

Technical field

The present invention belongs to the technical field of pharmaceutical applications of compounds.

Background technology

Microorganisms play a very important role in drug and food development, such as penicillin, streptomycin, statins, etc, and have made a great contribution to human heaith.

The present invention is in the field of pharmaceutical and food technology and specifically relates to a compound prepared by fermentation of eurotium cristatum in different media and isolated by extraction. The compounds and their analogues, derivatives, prodrugs, metabolites and their active phosphate (representative acid addition salts include but are not limited to acetate, adipate, alginate, citrate, aspartate salt, sucrose benzoate, sodium benzenesulfonate, disulphate, butyrate, camphoric acid, sodiumcamphor sulfonate, digluconate, sodium glycerophosphate, hemisulfate, enanthate caproate, fumarate, hydrochloride, hydrobromide, hydroiodic acid salt, 2-

Hydroxyethanesulphonic acid (Isothiosulfate, isothionate), Lactate, maleate, mesylate, nicotinic acid maleate, mesylate, nicotinate, 2-Naphthalenesulfonate, oxalate, palmitate, pectate, persulfate, 3-Phenylpropionic acid salt, picrate, pivalate Similarly, nucleobase can be made from the following substances: naphthalenesulfonate, Oxalate, Palmitate, pectate, persulfate, 3-Phenyipropionic acid salt, picrate, pivalate, propanoate, suceine,

Tartaric Acid Salt, Thiocyanate, phosphorous salts, glutamate, sodium tosylate and

Undecanoic acid. nucleobase can be quaternary ammoniation with the following substances: lower alkyl halides such as chloride, bromide and iodides of the methyl group, ethyl group, propyl group and butyl group; dialkyl sulfate such as Dimethyl sulfate alkyl esters such as Dimethyl sulfate, diethyl, dibutyl and dipentyl esters; long chain halides such as decyl, dodecyl, tetradecyl and octadecyl chlorides, bromides and iodides; aryl alkyl halides such as benzyl bromide and benzyl ethyl group bromide and others. Thus products are obtained that are soluble or dispersible in water or oil.

Examples of acids that can be used to form pharmaceutically acceptable acid addition

Specification salts include inorganic acids such as hydrochloric, hydrobromic, sulphuric and LU503166 phosphoric acids, as well as organic acids such as oxalic, maleic, succinic and citric acids), and their application in the preparation of products for the regulation of blood lipids to improve blood circulation and alleviate fatty liver, as well as in drugs, health foods and functional foods for weight loss.

Summary of the invention

It is an object of the present invention to provide a compound having the following structural formula

ST , A \ / no x

The invention provides compounds for use in the preparation of formulations with inhibitory activity against protein tyrosine phosphatase 1B, in drugs for the treatment of type IT diabetes, in hypoglycaemic drugs, in hypolipidaemic drugs, in weight loss drugs and in drugs for the treatment of fatty liver disease.

Description of attached drawings

Fig 1 shows a reversed-phase high performance liquid chromatogram of the compound.

Fig 2 shows a graph of the results of the compound blood glucose experiment.

Fig 3 is a graph of the results of the compound fatty liver experinient.

Fig 4 is a graph of the results of the compound cholesterol, triglyceride, LDL and body weight experiments,

Specific implementation method

Example 1 1. Isolation of fungi

Eurotium cristatum was isolated from Jingwei Fu tea (Xianyang Jingwei Fu Tea Co,

Ltd.) according to the literature {Yang Ruijuan, Wang Qiaomei, Peng Wenwen, Ji

Aibing, Zhang Wenjie, Yan Liang, Isolation and identification of microorganisms from eight commercially available "Jinhua" teas, Tropical Agricultural Science, 2019, 39(10), 81-88).

Specification 2. Fermentation culture of fungt LU503166

The activated eurotium cristatum was made into a spore suspension and then inoculated onto sterilised tea medium and incubated at 25-32 °C for 10-30 days in a static fermentation. 3. Preparation of active compounds

The fermented product is extracted using, but not limited to, methanol, ethanol, ethyl acetate, acetone, dichloromethane, ether, supercritical extraction and other methods to obtain the extract.

The extracts were dissolved in a mixture of dichloromethane and methanol and the samples were mixed at a weight ratio of crude fermentation extract:column chromatography silica (100-200 mesh) = 1:1.5 and separated by column chromatography under reduced pressure.

The column chromatography was eluted with organic solvent systems of different ratios of n-hexane and dichloromethane in the ratio of 100% n-hexane, 1:99 (n- hexane:dichloromethane to obtain sample A), 2:98 {n-hexane:dichioromethane to obtain sample B), 3:97 (n-hexane:dichloromethane to obtain sample ©), respectively.4:96 (hexane:dichloromethane to obtain sample D), 5:95 (hexane:dichloromethane to obtain sample E), 6:94 (hexane:dichloromethane to obtain sample F), 8:92 (hexane; dichloromethane to obtain sample G) and 10:90 (hexane:dichloromethane to obtain sample H) were eluted to give eight fractions of minor to major polarity.

Sample B was separated using gel Sephadex LH-20 column chromatography, the eluent was hexane:dichlorohexane (2:1 v/v) and 1 fraction was collected per 10 ml and combined to give 5 fractions based on HPLC analysis.

Wherein, fraction 2 is purified by reversed-phase high performance liquid chromatography. The chromatographic column is Agilent reversed-phase C18 column, and the mobile phase is 40% acetonitrile aqueous solution - 100% acetonitrile (20 minutes) to obtain the target compound (as shown in Fig 13. 4 . Structural identification of compounds

HPLC was used to identify the purity of the target compounds, and samples with a

Specification purity greater than 98% were structurally identified using mass spectrometry and NMR LU503166 techniques, NMR was measured with a Bruker AVANCE DRX-500 NMR instrument; mass spectrometry was measured with an An Agilent 6520Q-TOF mass spectrometer was used for the determination.

NMR hydrogen spectrum data of the target compound 6H (Pyridine-d5}: 12 46(s), 10 .94(s), 7 .38(s), 5 42, (tm J = 7,5 Hz), 3 50(d ,J =7.5), 3 25(t,J = 8 0 Hz), ! .77(m), 1 .64(s), 1 .60(s), 1.45(m }. ! .29(m)}, 1 .19(m), 1 .21{(m}, 0.83(t J = 7 0Hz).

NMR carbon spectrum data for this target compounddy (Pyridine-d5 }: 12 .46(s), 94(s), 7.38 (s), 5.42, (tm J=7 .5Hz). 3 .50(d ,J=7.5), 3 25(t j—8 .0Hz), 1.77(m)s 1 .64(s), 1.6068); 1 45(m), 1.29(m). 1.19m), 1.2Um), 0.83(tJ= 7 OHz):

NMR carbon spectrum data for this target compoundôc (Pyridine-dS ): 197 4, 155 2, 148.7, 133.6, 130.4, 128.4, 126.6, 122.8, 118.8, 33.0, 32 4, 30.4, 29.8, 27.8, 26.1, 24.7, 23.2, 18.0, 14.6;

High-resolution EST mass spectrometry of this target compound M+H] mz 305 2116,

C19H2903 “Calculated values305 2111).

The molecular structural formula of the target compound was determined to be as follows

ONG SN go EL ga 7 od Ne FE

Ho \ 5. Inhibitory activity of the compound against protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) is an important target for screening for the treatment of type H diabetes. PTPIB is an important target for the treatment of type IT diabetes. (a) Recombinant mouse PTP1B proteinab42574 (ab42574) was purchased from Abcam. (b) The screening was carried out using a transparent Beckman 96-well plate with a reaction system of 80 pL at 37°C. The substrate concentration, enzyme concentration and reaction time were optimised on this basis,

The final composition of the enzyme reaction system was determined as follows: 10 mmol/L Tris hydrochloric acid, pH 7 6, 10 mmol/L PNPP, 2% DMSO, 50 pg/mL

PTPIB.

The reaction system was mixed and left at 37°C for 30 min. The reaction was terminated by the addition of 1M NaOH and measured on an enzyme calibrator. The absorbance value (A) at 405nm was determined by subtracting the background value and calculating the enzyme activity.

Specification ; (on on 3 1 LU503166 inhibition rate = | 3. bs par t € Opi” EI i +4

The IC50 was calculated to be 9.13 pM. 6 . Hypoglycemic effect of compounds on ob/ob mice

Experimental grouping: ob/ob mice were grouped equally according to blood glucose values and body weight: (1) C57 mice fed normal chow as a blank control group; (2) model control group (ob/ob); (3) metformin 100mg/kg; (4) drug administration group (compound 5 .0 mg/kg) 10 mice in each group were administered continuously for 5 weeks.

Blood glucose levels in the blank, model, and dosing groups, as well as in the positive control group (Fig 2).

Table 1 . Effect of compounds on blood glucose in ob/ob diabetic mice {fasted blood glucose) + FAY TA EEE TN Ta = PEAY EXER de SER USE 3 Ha HESS SEX ai TAN + 188 HY $3e BE RAF 6 . Compound pairs alleviate Hpid markers and reduce body weight in a high-fat feeding model in C57 mice (1) Experimental materials (1)Experimental animals: C57 mice, 20-25g, male, provided by Beijing Huafukang

Biotechnology Co. Ltd. (2)High fat feed: Beijing Huafukang Biotechnology Co. Ltd. (3)Subjects; compound administered at a daily dose of 5 mg/kg body weight. (4) Positive control drug: Simvastatin, 10mg/kg body weight, Hangzhou Merzadone

Pharmaceutical Co. Ltd. (2) Test method.

Forty C57 mice, weighing 20-25g, males were taken. The mice were randomly divided into 2 groups, with 10 in the blank control group and 30 in the high-fat model. After 8 weeks, all animals were fasted for 12 h and then fed a high-fat diet (78.6% basal diet, 10% lard, 10% egg volk powder, 1% cholesterol, 0.4% bile salts} with free access to water. Blood was collected from the venous plexus at the base of the eye and serum TC and TG levels were measured to confirm the moulding status.

The adult model animals were then randomly grouped into groups of 8 animals each and kept as before. (1) C57 mice fed normal chow were used mice as a blank control group; (2) always fed a high-fat diet as a model high-fat control; (3) simvastatin 10 mg/kg; (4) administration of drug group (compound 5 .0 mg/kg).

Specification

Administered by gavage once daily for a total of 8 weeks, LU503166

The same volume of distilled water was given to both the blank control group and the model high-fat control group.

Blood was collected from the fundus venous plexus for 12h after the last dose of the drug at 8 weeks of administration and analysed according to the serum cholesterol and triglyceride kit The blood was analysed using a semi-automatic biochemical analyser according to the test method in the instructions.

The analyzer measures the levels of both indicators in the serum and weighs the weight.

The mice were also dissected, liver sections were taken and the fatty liver development was observed under the microscope.

A significant reduction in fat particles was seen in the administration group (Fig 3).

The results showed a significant reduction in blood cholesterol, triglyceride levels, LDL levels, and body weight in the dosing group compared to the control group (Fig 4).

Table 2 . Effect of compounds on lipid-related indicators in the blood of C57 mice

LIER re 4. free Là a Gamal i 2,58 38% te # SSI ER {0 3 285 AR es + 18s ve tés

During the experiment, the behaviour and activity of C57 mice were observed in real time, and their body weight and death were recorded. Blood samples and liver samples were collected. During the sixteen weeks of the experiment, none of the animals died and their food, water and faeces were normal.

Table 3 . Effect of compounds on body weight of C57 mice

Grouping | Bondy weight {oi 2 ES 25.3 SER FR FR

Claims (7)

Claims

1. A compound of the following structural formula LU503166 ORE Jp HO

2. The compound of claim 1 in the preparation of a formulation with inhibitory activity against protein tyrosine phosphatase 1B .

3. The compound of claim 1 in the preparation of a drug for the treatment of type II diabetes mellitus.

4. The compound of claim 1 in the preparation of a hypoglycemic drug.

5. The compound of claim 1 in the preparation of a hypolipidemic drug.

6. The compound of claim | in the preparation of a weight loss drug.

7. The compound of claim ! in the preparation of a drug for fatty liver disease.