CN109912490B

CN109912490B - Indole compound Plannyindole E and preparation method thereof

Info

Publication number: CN109912490B
Application number: CN201811454305.4A
Authority: CN
Inventors: 程永现; 朱宏杰; 涂正超
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-10-16
Anticipated expiration: 2038-11-30
Also published as: CN109912490A

Abstract

The invention is applicable to the field of chemical drugs, and provides an indole compound Plancy indole E with COX-2 and JAK3 inhibitory activity, and a preparation method of the compound. Pharmacological experiments show that the compound Plancyindole E has the activity of obviously inhibiting COX-2 and JAK3, and has practicability in the aspects of inflammatory diseases, immunosuppressant and the like.

Description

Indole compound Plannyindole E and preparation method thereof

Technical Field

The invention belongs to the field of chemical drugs, and particularly relates to an indole compound Plannyindole E and a preparation method thereof.

Background

Inflammatory responses are a complex process involving multiple mediators, with inflammatory factors struggling with the body until a balance is reached. Common anti-inflammatory drugs are classified into steroidal anti-inflammatory drugs (SAIDs) and non-steroidal anti-inflammatory drugs (NSAIDs). SAIDs are glucocorticoid inhibitors such as hydrocortisone and dexamethasone, have excellent anti-inflammatory effect, but cause water salt metabolism and sugar, fat and protein metabolism disorder after long-term use. NSAIDs are mainly used for rheumatic diseases, chronic inflammatory arthritis, respiratory tract infection, sports injury and the like in clinic, and are medicaments which achieve anti-inflammatory action by inhibiting Cyclooxygenase (COX). COX-1 is present in cells of various organs under physiological conditions, and prostaglandin E2(PGE2) synthesized by COX-1 in gastric mucosa has effects of maintaining normal blood flow of gastric tissue and increasing gastric mucosa mucus. COX-2 is expressed in a very small amount in normal tissue cells, and COX-2 is expressed in a large amount only when cells are inflamed by various stimuli. Arachidonic acid is firstly converted into PGG2 under the action of COX-2 and then further converted into PGH2, dehydration reaction or isomerization and the like are carried out under the action of enzyme to form different Prostaglandins (PG), wherein PGE2 can induce inflammatory cells to release chemokines, recruit the inflammatory cells to move, and express a large amount of inflammatory cytokines IL-1, IL-6, IL-23 and the like to play a proinflammatory role. With the increasing demand of NSAIDs, the side effects of the digestive tract caused by NSAIDs are highlighted. After long-term use of traditional non-selective NSAIDs such as aspirin, indomethacin and the like, COX-1 activity can be inhibited, physiological PGs synthesis is reduced, normal physiological functions of the gastrointestinal tract are difficult to maintain, and adverse reactions such as gastric mucosal injury, ulcer, perforation, bleeding and the like are caused. Therefore, for COX-2 target drugs, the first COX-2 inhibitor celecoxib was introduced by the FDA in 1998, which exerts anti-inflammatory and analgesic effects while reducing and eliminating gastrointestinal adverse reactions caused by COX-1 inhibition, and thus is widely used. In recent years, COX-2 has become an important target for drug action, and whether it is a simple COX-2 inhibitor, or a dual inhibitor combined with Lipoxygenase (LOX), or the function of protecting the gastrointestinal tract by using Nitric Oxide (NO), future anti-inflammatory inhibitors targeting COX-2 are always important for the research of NSAIDs.

JAK3 has been studied more and its target is an important approach to the discovery of novel immunosuppressant drugs. Since JAK3 was originally expressed in hematopoietic cells, it is believed that a highly specific JAK3 inhibitor may exert a precise effect on immune cells, making the drug effect discovered superior to other currently targeted immunosuppressive drugs as well.

The ground beetle (Polyphaga plancy i Bolivar) is a whole body of dried female insect of ground beetle of the family turtle, which is recorded in Shen nong Ben Cao Jing and has the functions of removing blood stasis, removing food retention, dredging collaterals, regulating wound and the like, and is a traditional Chinese medicine for insects. The invention is supported by Shenzhen city science initiative project (JCYJ20170412110504956) and the like, the related compounds are from ground beetles, belong to indole novel compounds, and have no related reports on anti-inflammation and prevention and treatment of immune system diseases.

Disclosure of Invention

The indole compound Plannyindole E provided by the invention has the structure shown in the following figure,

the invention also provides a pharmaceutical composition of the novel compound, which comprises Plancy indole E or pharmaceutically acceptable derivatives and salts thereof.

Furthermore, the pharmaceutical composition of the indole compound is a pharmaceutical preparation, and consists of Plancyindole E and Plancyindole C or pharmaceutically acceptable derivatives and salts thereof as active ingredients and pharmaceutically acceptable excipients.

Wherein the solid preparation comprises: tablets, capsules, pills, granules and the like; semisolid preparations include ointments, suppositories, and the like; the liquid formulation comprises: solutions, injections, sprays, and the like; the ophthalmic preparation comprises: eye drops, eye gels, etc.

The invention also discloses a preparation method of the indole compound Planctyindole E, which comprises the following steps:

extracting Eupolyphaga Seu Steleophaga dry insect body (50kg) with 70% ethanol under reflux (300L,4h,3h,3h), and concentrating under reduced pressure to obtain total extract (6.32 kg). After the total extract was suspended with an appropriate amount of water, it was extracted with petroleum ether and ethyl acetate in this order for 3 times to obtain an ethyl acetate-extracted fraction (127 g). The ethyl acetate part extract is subjected to MCI gel CHP 20P column chromatography segmentation (methanol/water, 10% -100%), TLC detection is carried out to combine the same components, and finally 6 groups, Fr.A-Fr.F, are obtained. Fr.D (8.7g) was separated by Sephadex LH-20 column chromatography, eluted with methanol-water (80:20), and combined to give 6 fractions, Fr.D 1-D6. And Fr.D6(1.01g) is separated by Sephadex LH-20 column chromatography, eluted by methanol and combined to obtain 4 groups of Fr.D 6.1-D6.4. Fr.D6.4(200mg) was purified by semi-preparative RP-18HPLC (acetonitrile-water, 21:79, flow rate: 3mL/min) to give Plancyindole E (1.0mg, t:, shown in the figure)_R＝14.0min)。

Furthermore, the indole compound is obtained by extracting from ground beetle or artificially synthesizing.

Furthermore, the indole compound has the application of intervening in inflammation inhibition and immune system diseases.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention discloses a method for extracting and separating the novel compound Plancy indole E from ground beetles for the first time.

2. The novel compound Plancyindole E is a novel compound which is separated and identified from ground beetles for the first time.

3. The novel compound Plancyindole E in the invention has the activity of inhibiting COX-2 and JAK 3.

4. The novel compound Plancy indole E has the application of intervening inflammatory diseases and immune system diseases.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 extraction separation and Structure identification of indole Compound Plancyindole E

The compound structure was identified as follows:

compound Plancyindole E (1), light yellow solid, uv (meoh) max (log)371(4.16),272(4.01), 206(4.35) nm; HRESIMS M/z 282.0768[ M-H ]]–(calcd for C₁₆H₁₂NO₄,282.0772).¹Hand¹³C NMR data, see Table 1.

TABLE 1 preparation of Compound 1¹H and¹³c NMR data

EXAMPLE 2 COX-2 inhibition assay with Compounds

This example uses Cayman's COX Fluorescent inhibition Screening Assay Kit to detect the enzymatic activity of COX-2. The method comprises the following specific steps:

1. compound gradient dilution: compounds were first dissolved in DMSO to 10mM stock and diluted in DMSO 1:3 gradients to 10 concentration gradients of 1000 × Compound series stock before being transferred to 384PP plates adapted to the Compound transfer apparatus for use.

2. Mu.l of enzyme dilution was added to each well, and 10. mu.l of 1xassay buffer was added to the blank well without enzyme.

3. Compounds were transferred to 20nl of 1000 Xgradient diluted compounds and added to the reaction, and DMSO was added to the blank and DMSO control wells. Incubating at room temperature for 10-15 min.

4. The fluorescent substrates ADHP and coenzyme Heme are diluted by 1xassay buffer, the dilution factor of Heme is 100 times, and the dilution factor of ADHP is 40 times. Add 4. mu.l ADHP dilution and 4. mu.l Heme dilution to each well.

5. Preparing a working solution of arachidonic acid and potassium hydroxide as substrates by water, wherein the dilution times are 40 times. Mu.l of substrate was added to each well and the fluorescence signal at 531/595nm was immediately detected dynamically using a microplate reader.

The results show that the novel compounds have COX-2 activity, IC inhibiting activity₅₀As shown in table 2.

EXAMPLE 3 method for detecting Activity of Compound inhibiting protein kinase JAK3

The inhibition of protein kinase activity by the compound is detected by a Z-lyte fluorescence resonance energy transfer method. Protein kinases and their corresponding substrate kits were purchased from Life technologies, Inc. All reactions were carried out in 384 microwell plates, and the enzyme reaction volume was 10. mu.l. The reaction buffer composition was 50mM HEPES pH 7.5,10mM MgCl2,1mM EGTA, 0.01% BRIJ-35. The specific reaction is as follows: firstly, a proper amount of 5 mul of protein kinase and 5 mul of corresponding substrate (the final reaction concentration is 2 mul) mixed solution are added into a micropore plate, and then an Echo520 ultrasonic micro liquid transfer system (America)Labcyte Ltd) Adding a series of compounds diluted in gradient, andthen adding ATP with corresponding concentration, oscillating and mixing uniformly for 5min, and placing in a constant temperature box with 29 ℃ for reaction for 1.5 h; then adding 5 μ l of detection solution (Development Reagent) with corresponding concentration, shaking and mixing for 5min, placing in a constant temperature box of 29 deg.C, and reacting for 1 h; finally, 5 mul of Stop solution (Stop Reagent) is added, after shaking and mixing uniformly, an envision multilabel reader multifunctional enzyme-linked immunosorbent assay instrument of PE company is used for detection, the excitation light wavelength is 400nm, and the emission light wavelength is 445nm and 520nm respectively. The assay was set up with test wells (plus compound, enzyme, substrate and ATP), 0% phosphorylated wells (plus DMSO, substrate and ATP) 100% phosphorylated wells (plus phosphorylated substrate only), 0% inhibitory wells (plus DMSO, enzyme, substrate and ATP), respectively. Calculating the inhibition rate of the compound on enzyme reaction according to the fluorescence ratio, and analyzing and calculating the IC of the compound by GraphPad software₅₀The value is obtained.

The results show that the compound has JAK3 inhibiting activity and IC₅₀As shown in table 2.

TABLE 2 inhibitory Activity of Compounds on COX-2, JAK3

Example 4

The compound in the example 1 is added with injection solvent according to the conventional method, fine filtered, encapsulated and sterilized to prepare injection.

Example 5

The compound in the example 1 is dissolved in sterile water for injection, filtered by a sterile funnel, subpackaged, frozen and dried at low temperature, and then aseptically sealed by melting to obtain the powder injection.

Example 6

The compound in the example 1 can be prepared into tablets or capsules by being matched with various pharmaceutical excipients according to a conventional method. Using the compound of example 1 as a pharmaceutically active ingredient, several conventional excipients as auxiliary ingredients for the preparation of combination pharmaceutical tablets or capsules, samples containing 10-300mg of the pharmaceutical ingredient per tablet or capsule were prepared according to conventional methods.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.

In the above description of the indole compound Plancyindole E and the preparation method thereof provided in the embodiments of the present invention, for those skilled in the art, there may be variations in the specific implementation manners and the application ranges according to the ideas of the embodiments of the present invention, and in summary, the contents of the present specification should not be construed as limiting the present invention.

Claims

1. An indole compound Plancyindole E is characterized in that the structural formula is as follows

2. A method for preparing the compound as claimed in claim 1, wherein 50kg of the dried eupolyphaga sinensis is taken, heated and refluxed with 70% ethanol for extraction for 300L each time for 4h,3h and 3h, and concentrated under reduced pressure to obtain 6.32kg of total extract, after the total extract is suspended with appropriate amount of water, petroleum ether and ethyl acetate are sequentially used for extraction for 3 times to obtain 127g of ethyl acetate extraction part, the ethyl acetate part extract is subjected to MCI gel CHP 20P column chromatography, the solvent system is methanol/water, the elution gradient is 10% -100%, TLC detection is carried out to combine the same components, finally 6 groups are obtained, Fr.A-Fr.F and Fr.D 8.7g are separated by Sephadex LH-20 column chromatography, methanol-water is eluted, the solvent group is 80:20, 6 groups are obtained by combination, Fr.D 1-D6 and Fr.D 61.01g are separated by Sephadex-20 column chromatography, eluting with methanol, mixing to obtain 4 fractions Fr.D6.1-D6.4, Fr.D6.4200 mg, purifying by semi-preparative RP-18HPLC with acetonitrile-water solvent system of 21:79 to obtain Planchyndole E compound with weight of 1.0mg and retention time t_R＝14.0min。

3. Use of a compound according to claim 1 for the preparation of an immunosuppressant medicament.