Application of compound in preparation of medicine for treating type 2 diabetes

Abstract

The invention relates to an application of a carboline compound separated from marine organisms, in particular to an application of the compound in preparation of a protein tyrosine phosphatase PTP1B inhibitor and a pharmaceutical preparation for treating type 2 diabetes. The compound can be used as a PTP1B inhibitor for treating type 2 diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry and specifically to the application of a compound in the preparation of drugs for treating diabetes. This compound can be used as a PTP1B inhibitor for the treatment of diabetes. Background Technology

[0002] Clinically, diabetes is classified into type 1 (insulin-dependent, IDDM) and type 2 (non-insulin-dependent, NIDDM), with the latter accounting for approximately 90% of all patients. With lifestyle changes, reduced physical activity, and rising obesity rates, the prevalence of type 2 diabetes in my country continues to rise.

[0003] Insulin resistance is a core element in the development and progression of type 2 diabetes; therefore, intervention strategies that can improve insulin resistance have become an important direction for new drug development.

[0004] Molecular biology studies have confirmed that protein tyrosine phosphatase 1B (PTP1B) negatively regulates the insulin receptor and its substrates through dephosphorylation, thereby inhibiting the insulin signaling cascade. It is widely recognized as a definitive target for the treatment of type 2 diabetes. Elchebly et al. constructed PTP1B knockout mice using homologous recombination, which showed normal growth and reproduction, and significantly increased insulin receptor and IRS-1 phosphorylation levels in their liver and skeletal muscle, resulting in enhanced overall insulin sensitivity (Science, 1999, 283:1544-1548). Klaman et al. further reported that the deletion of this gene can antagonize high-fat diet-induced weight gain and insulin resistance, with the mechanism stemming from a reduction in adipocyte volume rather than a decrease in number (Mol. Cell. Biol., 2000, 20:5479-5489). These studies have established the dual role of PTP1B in regulating insulin sensitivity and lipid storage at the functional level and verified its feasibility as a target for anti-diabetic and anti-obesity drugs.

[0005] In the drug discovery stage, after target identification, it is crucial to obtain lead compounds with good targeted regulatory capabilities. Marine organisms, living in unique environments different from terrestrial environments, often produce structurally unique secondary metabolites to cope with environmental changes. The inventors derived these compounds from the marine organism *Stylosus stenoptera* (…). Styela clava A novel small molecule was isolated (see patent ZL202310785667.6). In vitro activity evaluation showed that it had significant inhibitory activity against PTP1B. Pharmacological experiments also proved that the compound could reduce postprandial blood glucose in mice and had the potential to be developed into a candidate drug for the treatment of type 2 diabetes. Summary of the Invention

[0006] The purpose of this invention is to provide a novel effect of a compound obtained by extraction, isolation, and purification from marine organisms. The inventors have applied for an invention patent for this compound [Patent No.: ZL202310785667.6], and the compound's name is: 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro -β -Carboline, its structural formula is as follows:

[0007] The preparation method of the above compound is as follows: First, the sheath powder of *Stichopus spp.* is alkalized with ammonia water, and then extracted with chloroform at room temperature to remove lipid substances until the chloroform extract is colorless. The sheath residue after chloroform extraction is soaked in 4-10 times its weight of 60-80% ethanol for 12-36 hours, then filtered, and the filtrate is collected. To ensure complete extraction of the compound, it is extracted with 60-90% ethanol 3-6 times, and the filtrates from 3-6 extractions are combined. Then, the ethanol is recovered by vacuum extraction using a rotary evaporator (pressure ≤ 0.1 atm, temperature 45-55 degrees Celsius) to obtain a concentrated extract. Then, an appropriate amount of distilled water was added to the concentrated extract to fully suspend it in water. The extract was then fully extracted with chloroform and ethyl acetate in sequence. The chloroform extract was removed, and the ethyl acetate extract was subjected to multiple chromatographic analyses using a silica gel column (first with 200-300 mesh coarse silica gel, then with silica gel H for thin-layer chromatography). Chromoform-acetone and chloroform-methanol were used as eluents in sequence. The chromatograms containing the same components were combined, and the chromatograms rich in alkaloid components were then separated and purified using a preparative high-performance liquid chromatography column to obtain the brominated β-carboline compounds of the present invention.

[0008] The compounds of this invention can be used as inhibitors of protein tyrosine phosphatase PTP1B, and can be used to prepare drugs that inhibit protein tyrosine phosphatase PTP1B, and can be used to prepare drugs for treating type 2 diabetes.

[0009] The present invention has the following advantages: the compounds of the present invention have a good inhibitory effect on protein tyrosine phosphatase PTP1B, and the compounds of the present invention, as selective PTP1B inhibitors, have important value in the treatment of insulin resistance in diabetes. Detailed Implementation

[0010] The principles and features of the present invention are described in detail below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0011] Example 1: Extraction, purification, and structural identification of the compound described in this invention. Example 1 describes the preparation and structural identification of the compound, which falls under the compound preparation content of the inventor's authorized invention patent [see patent: ZL202310785667.6]. The specific content is as follows: The stalked sea squirt, collected from the seashore of Zhifu Island, Zhifu District, Yantai City, was biologically identified as a stalked sea squirt (a member of the class Cynosquamatidae). Styela clava The collected *Stylosus stenoptera* were rinsed with tap water at room temperature to remove attached impurities and remove any attached substances growing on the surface of the sheath. The sheath and body were then separated. The body was homogenized using a high-speed tissue homogenizer, followed by low-temperature freeze-drying to remove moisture. The dried body tissue was then pulverized using a high-speed universal pulverizer to obtain sheath powder. The *Stylosus stenoptera* sheath powder was alkalized with ammonia water and then extracted with chloroform at room temperature to remove lipids until the final chloroform extract was colorless. The sheath residue after chloroform extraction to remove lipids was then further extracted and purified.

[0012] Take an appropriate amount of the above-mentioned sheath powder and extract it four times at room temperature with 80% ethanol (8 times its weight in volume), each time for 24 hours. Then combine the four ethanol extracts and recover the ethanol at 45°C and 0.1 atm until the final extract has no alcohol odor. Add an appropriate amount of distilled water to fully suspend the extract. Extract it multiple times with chloroform until the final chloroform layer is colorless. Detect the extract with alkaloid detection reagent by thin-layer chromatography. The chloroform extract does not contain alkaloids and should be left unused. The residual sheath powder suspension was extracted five more times with ethyl acetate solvent. The ethyl acetate extracts were collected and combined, and the solvent was recovered by rotary evaporation under reduced pressure (≤0.1 atm) at a temperature ≤45℃, yielding a brown ethyl acetate extract. The extract was weighed, and a silica gel column was prepared using 50 times its weight of silica gel (200-300 mesh). The ethyl acetate extract was loaded onto the silica gel column and eluted with different volume ratios of chloroform:acetone (100:0, 50:1, 20:1, 10:1, 5:1, 2:1, 0:100). The extracts were collected in fractions, and each fraction was analyzed by thin-layer chromatography (using modified bismuth potassium iodide solution as the colorimetric reagent). Fractions with the same components were combined, resulting in six coarse fractions (1-6). Each coarse fraction was further separated and purified using preparative high-performance liquid chromatography (eluting with different volume ratios of methanol and distilled water) to obtain the compound of this invention. Analysis revealed that the compound was named 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro -β -Carboline, with the following structure: .

[0013] This compound has the following physicochemical characteristics: Molecular formula: C 12 H 13 BrN2O; Appearance: White solid; Melting point: mp. 231.4-232.6℃; Ultraviolet (UV) max (MeOH): 218.2, 282.4 nm; Infrared: IR (KBr): 3245, 2973, 1636, 1589, 1458, 1399, 1262, 1151, 967, 927, 803 cm⁻¹ -1 ; Hydrogen spectrum: 1 H NMR and carbon spectrum 13 C NMR (DMSO- d 6) Data is shown in Table 1; Table 1. δ¹¹ NMR spectra of the compounds of this invention H and carbon spectrum δ C data (NMR frequency: 400 MHz for) 1 H and 100 MHz for 13 C, δ, ppm ( J , Hz), Solvent: DMSO- d 6) position <![CDATA[δ C (a lot)]]> <![CDATA[δ H (a lot)]]> 1 53.1 (t) 3.52 (s, 2H) 3 53.0 (t) 2.71 (d,= 4.9Hz, 2H) 4 20.9(t) 2.65 (d,= 4.9Hz, 2H) 4a 106.1 (s) 4b 126.7 (s) 5 103.1 (d) 6.66 (s, 1H) 6 149.6 (s) 7 109.0 (d) 8 113.8 (d) 6.86 (s, 1H) 8a 129.7 (s) 9a 132.5 (s) 9(NH) 10.44 (s, 1H) <![CDATA[N-CH3]]> 2.29 (s, 3H) OH 9.69 (s, 1H) .

[0014] Example 2: Activity test of the PTP1B inhibitor compound of the present invention Test Principle: Human tyrosine phosphatase 1B (hPTP1B) catalytic active domain was expressed in *E. coli* using molecular biology techniques. The purified recombinant hPTP1B protein hydrolyzes the phosphoester bonds of the substrate disodium p-nitrophenyl phosphate (pNPP). The resulting product exhibits strong light absorption at 410 nm. Therefore, changes in enzyme activity can be observed by directly detecting changes in light absorption at 410 nm. Furthermore, the inhibitory effect of the compound of this invention on enzyme activity was observed by adding a specific concentration. The standard activity assay system is as follows: 10 mM Tris·HCl (pH 7.6), 10 mM pNPP, 2% dimethyl sulfoxide (DMSO), 100 nM hPTP1B. Observation Indicators: The light absorption at 410 nm was dynamically measured over 3 minutes. The slope of the first-order reaction of the kinetic curve was used as the enzyme activity indicator.

[0015] The testing principle of this testing system can be expressed by the following formula. .

[0016] Methods for evaluating and interpreting experimental results: The screening results were the percentage inhibition rate of enzyme activity at a compound concentration of 10 mg / mL. When the inhibitory activity of a compound on the enzyme was higher than 50%, the IC50 of the compound was determined according to standard screening methods. 50 Using sodium orthovanadate as a positive control compound, the IC50 of sodium orthovanadate was...50 It is 2.0 mM.

[0017] Experimental results: The IC50 of the compound of this invention against the PTP1B enzyme inhibitory activity 50 It is 3.4 mM.

[0018] Experimental conclusion: Molecular biology experiments show that this compound has a good inhibitory effect on protein tyrosine phosphatase PTP1B.

[0019] Since the compounds of the present invention have the activity of inhibiting protein tyrosine phosphatase PTP1B, they can be prepared into any conventional pharmaceutical preparation according to conventional pharmaceutical techniques, such as tablets, pills, capsules, granules, oral liquids, powder for injection, etc.

[0020] Example 3: Effect of the compounds of the present invention on glucose tolerance in normal mice

[0021] Forty healthy male mice, weighing 22-26g, were used in the experiment. Blood was collected from their tails. Fasting blood glucose levels were measured in each mouse. The mice were then randomly divided into four groups: a saline control group; a low-dose group (20 mg / kg body weight); a high-dose group (50 mg / kg body weight); and acarbose group (20 mg / kg body weight). During the experiment, each group was administered 0.5 mL of the drug solution via intragastric gavage (ig). Ten minutes later, 0.5 mL of starch (10 g / kg) was administered via ig. Blood glucose levels were measured at 0.5, 1, and 2 hours after starch administration.

[0022] As shown in Table 2, the experimental results indicated that blood glucose levels in normal mice significantly increased at 0.5 and 1 hour after starch administration. The compound of this invention significantly reduced blood glucose levels in mice at 0.5 and 1 hour after starch administration.

[0023] Table 2. Effects of the compounds of this invention on glucose tolerance in normal mice (x±s, n=10)

[0024] Compared with the control group, * P<0.05 ** P<0.01.

[0025] Example 4 Preparation of the compound tablets of the present invention: 1000 mg of the compound raw material obtained in Example 1 was accurately weighed, and 29000 mg of pharmaceutical starch and dextrin (weight ratio 3:1) were added as excipients. After mixing evenly, the mixture was granulated, granulated and compressed into tablets to obtain 100 tablets, each weighing 0.30 g, with a compound content of 10 mg / tablet.

[0026] Example 5 Preparation of the lyophilized powder injection of the compound of the present invention: 10 g of the compound raw material obtained in Example 1 and 20 g of pharmaceutical mannitol were accurately weighed and dissolved in 500 mL of water for injection. The solution was stirred thoroughly to dissolve the compound. Water for injection was added to a final volume of 1000 mL. 2.0 g of activated carbon for injection was added. The mixture was heated to 60°C and stirred for 30 minutes. The solution was filtered through a carbon rod and sterilized by filtration through a 0.25 μm microporous membrane. The solution was dispensed into 1000 vials, with a volume of 1.0 mL per vial. The solution was then freeze-dried to obtain the lyophilized powder injection containing 10 mg of the compound per vial.

Claims

1. A compound 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro -β Application of carboline in the preparation of protein tyrosine phosphatase PTP1B inhibitors.

2. The use of the compound of claim 1 in the preparation of a pharmaceutical formulation for treating type 2 diabetes.

Images