CN116836165A - A kind of curcumin 2-chloroadenine derivative compound - Google Patents

Abstract

The present invention relates to a curcumin 2-chloroadenine derivative compound or its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal.

Description

A kind of curcumin 2-chloroadenine derivative compound

Technical field

The application of the present invention relates to the field of medical technology, and specifically relates to a curcumin 2-chloroadenine derivative compound.

Background technique

Most of the body's metabolic activities rely on oxidative reactions, but this may lead to aging, disease, and oxidative stress. The body produces antioxidants to regulate these reactions, but sometimes the load of free radicals produced during metabolism is too high, and more antioxidants are needed to slow aging and prevent certain diseases.

Free radicals are a class of highly reactive molecules produced during biological metabolism, including reactive oxidative species containing superoxide anions (O ₂ -) and hydroxyl groups (·OH) and their reactive derivatives. Reactive oxygen species (ROS) can also be produced by phospholipase A2, 5-lipoxygenase (5-LOX), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and the production of reactive oxygen species ( ROS) enzyme-induced production. Free radicals are important in regulating cell growth and signaling, as well as inhibiting bacteria and viruses in the body. However, if free radicals accumulate excessively in the body, reactive oxygen species (ROS) may have toxic effects on cells. The reaction of superoxide and peroxide with metal ions can promote the production of other free radicals, especially hydroxyl radicals, which can react with all components of the cell, including lipid membranes, DNA, and proteins.

Since the 1970s, curcumin has been recognized for its antioxidant effects and its ability to scavenge free radicals has been studied. Curcumin can prevent the oxidation of hemoglobin to methemoglobin or reduce the amount of reactive oxygen species by inhibiting lipopolysaccharide-activated macrophages and reducing nitrate-induced oxidative stress. In 1985, Toda et al. extracted part of curcumin from turmeric roots and found in in vitro experiments that it has strong free radical scavenging ability. Motterlini et al. studied the in vivo antioxidant activity of curcumin and found that it can broadly activate various enzymes in the liver, including glutathione triphosphotransferase, glutathione peroxidase, epoxide hydrolase, and Superoxide dismutase (SOD).

According to the modern understanding of the antioxidant mechanism of curcumin, the main part of its antioxidant activity is the phenolic hydroxyl and β-diketone units. These two structural units can provide proton-blocking antioxidants to combat the effects of free radicals. In addition, the antioxidant activity of curcumin is also closely related to its ability to inhibit lipid peroxidation and maintain the activities of various antioxidant enzymes such as SOD, catalase (CAT), and glutathione peroxidase (GTP). Related. Lipid peroxidation is a free radical-mediated chain reaction that can damage cell membrane structure. Curcumin inhibits lipid peroxidation mainly by removing factors involved in free radical reactions. Since free radicals and reactive oxygen species are the causative factors of many common diseases, it is promising to fully utilize curcumin as an antioxidant and means of scavenging free radicals to develop potential therapeutic drugs.

However, more in-depth research found that curcumin has poor water solubility, less body absorption, rapid metabolism, and low bioavailability, which greatly limits its application.

Contents of the invention

In order to solve or partially solve the problems existing in related technologies, the present invention provides a curcumin 2-chloroadenine derivative compound. The present invention introduces a purine group and derivatizes it with curcumin without affecting the advantages of curcumin itself. Basically, a product with relatively good water solubility and bioavailability was obtained.

The application of the present invention provides a compound or its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal, characterized in that the compound is selected from the group consisting of general formula (I) The compound shown,

The second aspect of the present application provides a pharmaceutical composition, including the above-mentioned compound or its stereoisomer, deuterate, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal, and a pharmaceutical Acceptable carrier or excipient.

The third aspect of the present application provides the use of the above-mentioned compound or its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal in the preparation of anti-cancer drugs.

The fourth aspect of the present application provides a method for preparing the above-mentioned compound.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

"Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" means that the compound of the present invention retains the biological effectiveness and properties of the free acid or free base, and the free acid is combined with a non-toxic inorganic base or Organic base, the salt of the free base obtained by reacting with a non-toxic inorganic acid or organic acid.

"Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention, their pharmaceutically acceptable salts or prodrugs and other chemical components, where "other chemical components" refers to pharmaceutically acceptable Acceptable carriers, excipients and/or one or more other therapeutic agents.

"Carrier" refers to a material that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the compound to which it is administered.

"Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of the compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders agents and disintegrants.

"Prodrug" refers to a compound of the present invention that can be converted into a biologically active compound through metabolism in the body. The prodrugs of the present invention are prepared by modifying the amino group or carboxyl group in the compound of the present invention. The modification can be removed by conventional operations or in vivo to obtain the parent compound. When the prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free amino or carboxyl group.

"Co-crystal" refers to a crystal formed by combining an active pharmaceutical ingredient (API) and a co-crystal form (CCF) under the action of hydrogen bonds or other non-covalent bonds. The pure states of API and CCF are both Solids, and there are fixed stoichiometric ratios between the components. A eutectic is a multicomponent crystal that includes both a binary eutectic formed between two neutral solids and a multicomponent eutectic formed between a neutral solid and a salt or solvate.

"Stereoisomers" refer to isomers produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and do not limit the application of the present invention.

Beneficial technical effects of the present invention:

Improve the bioavailability of curcumin: One of the reasons for suppressing the bioavailability of curcumin is its low water solubility, which limits the absorption and distribution of curcumin in the human body. By improving its water solubility, curcumin can be absorbed more efficiently and work more effectively in the body.

Increase the anti-cancer activity of curcumin: The curcumin derivatives of the present invention have higher anti-cancer effects than curcumin at the same concentration, showing their potential in the anti-cancer field.

Reduced dose and toxicity: The efficacy of curcumin may be dose-limited, and higher doses may cause toxicity. By increasing the bioavailability of curcumin, lower doses can be used to achieve its therapeutic effects, thereby reducing the risk of side effects and reducing toxicity.

Broadening the scope of use: Improving the bioavailability of curcumin could also broaden its scope of use in the treatment of other diseases. For example, improving the limitations of curcumin by targeting new diseases or as a combination of new treatments.

Description of the drawings

Figure 1 is the PDB ligand pattern before treatment in Test Example 4 of the application of the present invention;

Figure 2 is the PDB ligand pattern after treatment in Test Example 4 of the application of the present invention;

Figure 3 is the parameter setting information of the docking GRID in the test example 4 of the application of the present invention;

Figure 4 is a diagram of the docking GRID setup in Test Example 4 of the application of the present invention;

Figure 5 is the conformation information of docking in Test Example 4 of the application of the present invention;

Figure 6 shows the conformation in Test Example 4 of the application of the present invention colored according to van der Waals force;

Figure 7 is the geometric center of the docking conformation in Test Example 4 of the present application;

Figure 8 shows the overall state of the docking conformation and the interaction with the acceptor residues in Test Example 4 of the application of the present invention.

Detailed ways

Alternative embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although alternative embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. As used in this application and the appended claims, the singular forms "a," "the" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will also be understood that the term "and/or" as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

For greater clarity, detailed description is given below through the following examples.

Example 1

Use a balance to weigh 1.69g (0.01mol) of 2-chloroadenine and 3.68g of curcumin (0.005mol) into an Erlenmeyer flask, add 50mL of anhydrous methanol to the Erlenmeyer flask, heat with a magnetic stirrer, and condense React in reflux device for 1.5h. Then use a rotary evaporator to evaporate anhydrous methanol, wash with ether to remove impurities, and dry naturally to obtain 3.91g of curcumin 2-chloroadenine derivative with a yield of 72.73%.

¹ H NMR (DMSO, ppm): 9.73 (br, 2H, Ar-OH); 6.77-7.52 (m, 6H, -C ₆ H ₅ ); 3.85 (s, 6H, -OCH ₃ ); 3.46; (s ,1H,-CH-); 2.51(br,1H,-C-NH-); 8.13(s,1H,-N=CH-). ¹³ C NMR (DMSO, ppm): 44.76 (>CH ₂ ); 188.61 (> C = O); 157.81, 146.11 (> C = NH); 61.12 (-OCH ₃ ); 106.26, 116.75, 121.14, 126.52, 128.53 ,131.77,135.73(C＝C); 154.74(＝CH-NH-).

Test example 1

Experimental purpose: To compare the bioavailability of curcumin and curcumin 2-chloroadenine derivative (referred to as A).

Experimental subjects: The experiment will use Sprague-Dawley rats as model animals.

experimental design:

Prepare reagents and equipment:

Curcumin

Curcumin 2-chloroadenine derivative (A)

Solvent (such as PEG400, DMSO, etc.)

rat feed

Pipettes and syringes

Spectrophotometer or high performance liquid chromatography (HPLC)

centrifuge

Experimental steps:

Step 1: Prepare three groups of Sprague-Dawley rats, 5 rats in each group. Curcumin group and group A were fed respectively.

Step 2: Dissolve curcumin and A in appropriate solvents for oral administration. The dosage was 20 mg/kg per rat.

Step 3: Orally administer curcumin and A solution to rats every day for 7 consecutive days.

Step 4: Collect blood samples from rats at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after dosing on day 7.

Step 5: Centrifuge the blood sample and collect plasma.

Step 6: Determine the concentration of curcumin, A in plasma using a spectrophotometer or HPLC.

Step 7: Draw the plasma drug concentration-time curve and calculate bioavailability parameters such as AUC (area under the curve) and Cmax (maximum plasma concentration).

Experimental data and conclusions:

in conclusion:

According to the experimental data, the following conclusions can be drawn:

The bioavailability of A is significantly better than that of curcumin, with higher AUC and Cmax values.

Through experimental data, it can be seen that A has advantages over curcumin in terms of bioavailability, which will help curcumin derivatives play a greater role in biological and medical applications.

Test example 2

Experimental purpose: To compare curcumin and curcumin 2-chloroadenine derivatives (referred to as A).

experimental design:

Prepare reagents and equipment:

Curcumin

Curcumin 2-chloroadenine derivative (A)

distilled water

test tube

Magnetic stirrers and magnetic stirrers

filter paper and funnel

Spectrophotometer or high performance liquid chromatography (HPLC)

Experimental steps:

Step 1: Weigh 10 mg of curcumin and A respectively.

Step 2: Add the weighed curcumin and A into a test tube containing 10 mL of distilled water.

Step 3: Use a magnetic stirrer and magnetic stir bar to stir the solution in the test tube at a speed of 500 rpm for 30 minutes.

Step 4: After stirring is complete, let the solution in the test tube sit for 5 minutes to allow undissolved solids to settle to the bottom.

Step 5: Filter the clear solution using filter paper and a funnel, and collect the filtrate.

Step 6: Use a spectrophotometer or HPLC to determine the concentration of curcumin and A in the filtrate.

Step 7: Calculate the solubility of the solution in each test tube based on the measured concentration data, expressed in milligrams per milliliter (mg/mL).

Experimental data and conclusions:

Reagents Solubility(mg/mL) Curcumin 0.013 A 5.8

in conclusion:

According to the experimental data, the following conclusions can be drawn:

The water solubility of A is obviously better than that of curcumin, and its solubility is higher.

Through experimental data, it can be seen that A has advantages over curcumin in terms of water solubility, which will help curcumin derivatives play a greater role in biological and medical applications.

Test example 3

Experimental steps:

1. Adjust the density of cancer cells (human lung cancer A549 cells) so that 100 μL is inoculated in each well, which is approximately 1*10^4 cells.

2. Place the 96-well plate in an incubator at 37°C and 5% _CO2 and incubate for 24 hours to allow cells to adhere to the wall.

3. Prepare different concentrations of curcumin and curcumin 2-chloroadenine derivatives and add them to the corresponding wells respectively. Establish a blank group (complete medium) and a negative control group (cells + complete medium).

4. Continue to incubate for 48 hours, and the cell growth trends of each group will begin to differ.

5. After the incubation, add 20μL MTT reagent (5mg/mL) to each well and mix thoroughly. Place the 96-well plate into the incubator again and incubate for 4 hours.

6. Use a pipette to carefully remove excess MTT solution from each well to avoid disturbing the cells. Add 150 μL DMSO to each well to dissolve the generated methylthiazolium blue precipitate.

7. Shake the 96-well plate to fully mix DMSO and the precipitate. After the sample is completely dissolved, use a microplate reader to read the absorbance value (OD value) of each well.

Experimental data and calculations:

The inhibition rates of the three compounds on A549 cells at different concentrations were measured as follows:

Curcumin: 50μM-18.2%, 100μM-34.7%, 200μM-58.5%;

Curcumin 2-chloroadenine derivative: 50μM-41.9%, 100μM-69.8%, 200μM-87.0%;

Calculation method:

Cell inhibition rate = [(negative control group OD - experimental group OD)/negative control group OD] × 100%

in conclusion:

It can be seen from the above data that curcumin derivatives have higher anti-cancer effects than curcumin at the same concentration, showing their potential in the field of anti-cancer. The reasons are:

Increased active sites: Due to the addition of purine groups, curcumin purine derivatives have more active sites than curcumin in terms of anti-tumor effects, allowing them to effectively intervene in the growth, invasion and migration of tumor cells. . In addition, the purine group can further enhance the antioxidant capacity of curcumin, thereby producing better inhibitory effects on tumor cells.

Improved bioavailability: Due to changes in chemical structure, the water solubility of curcumin purine derivatives is significantly improved. This gives the drug advantages in terms of absorption, distribution and metabolism in the body. Compared with curcumin, curcumin purine derivatives can more easily pass through the cell membrane and enter tumor cells to exert their effects. This means that curcumin purine derivatives require lower doses to achieve ideal anti-cancer effects in practical applications.

Cooperate with targeted therapy: Purine groups have important biological functions in organisms, such as the synthesis of DNA and RNA. Curcumin purine derivatives can inhibit tumor growth by interfering with the nucleic acid synthesis process of tumor cells. In addition, they can synergize with other anti-cancer drugs to enhance their anti-cancer effects.

Test example 4

1 Pretreatment of receptors and ligands

Receptor preprocessing: Run the Autodock software and open the downloaded pdb file of the G-quadruplex in the file-readmolecule directory. Remove water molecules, add hydrogen, and calculate point charges. Add atom type: Generally, the rigid docking selection is saved as AD4 type, Edit-Atom-Assign AD4 type. After the preprocessing operation is completed, save the processed receptor molecule as a file with the suffix pdbqt.

Preprocessing of ligands: Open the Autodock program, click on the PDB file of the ligand curcumin 2-chloroadenine derivative, and open the pdb file of the ligand in the Ligand-Input-Open directory. At this time, Autodock will automatically add the file. For hydrogen, point charge calculation and other processing, a prompt will pop up, click OK. After the preprocessing operation is completed, name the processed ligand molecule as a pdbqt file and save it. Figure 1 is the pdb image before ligand treatment, and Figure 2 is the pdbqt image after ligand molecule treatment.

2GRID docking operation

Open the Autodock software and click on the pdbqt files of G-quadruplex and curcumin 2-chloroadenine derivatives.

Click Grid-GridBox-Grid Options, and a prompt will pop up. In it, set the docking grid points. The values set for the X side, Y side, and Z side are 64, 72, and 78. The coordinates set on the X plane, Y plane, and Z plane are 0.056, 1.000, and -1.806. After completing the above steps, find File-Close saving in the Grid Options prompt message and save the above operations. Then save it as a gpf file for later use. The numerical values and graphics input during operation are shown in Figures 3 and 4.

3. Docking and results

After the above operations are completed, perform autogrid operations in Run-Run autogrid. At this time, there will be a prompt message. In the prompt box, add the files generated during the runtime of autogrid, the gpf file generated when the grid points are set, and the glg file generated at the same time when the gpf file is generated. After that, click Lanch and the software will automatically calculate the autogrid.

Open the pdbqt file of the previously processed receptor molecule in Docking-Macromolecule-Set Rigidmacro, then open the pdbqt file of the previously processed ligand molecule in Docking-ligand-open, and click Accept in the pop-up dialog box.

After the above steps are completed, click Docking-search Parameters-Genetic algorithm parameters, select the default in the pop-up prompt message, and click Accept. After entering the parameters, click Docking-Docking Parameters-Set docking run options and then Accept directly.

Finally, after saving the docking, get the dpf file and click the Docking-Output-Lamarckian Ga key to save.

Perform Autodock processing in Run-Run Autodock. A prompt message will pop up. In the pop-up prompt box, add the autodock running program in sequence at the three places to be input, the dpf file generated during the docking operation, and the dlg file generated at the same time when the dpf file is generated. Then click the Launch button, and the program will automatically calculate the autogrid.

Finally, open the dlg file saved during the Docking process in Analyze-Docking-Open, and click Confirm in the pop-up dialog box.

When Analyze-Conformations-Load, the results of the previous docking operation and its conception information are added to the graphics window. After that, a prompt message will pop up. In the prompt, click the number corresponding to it in the list. At this time, you can Observe the docking information of this molecular conformation. Double-click the number in the prompt message to add the conformation information of the molecule to the displayed pop-up window to facilitate observation and analysis.

Analyze-Conformations-Play will pop up a prompt message for playback control. Click the second button from the bottom, and the following prompt message will appear. Select ShowInfo to see the data about the conformation at this time. Select Color by as vdw in the drop-down settings to color the current conformation. .

Load the Receptor rigid molecule in Analyze-Macromolecule-Open, and then you can see the Ligand molecule in the Receptor molecule.

In Analyze-Dockings-ShowasSpheres, the results of molecular docking conformational information obtained in previous operations are expressed in spherical shapes. Each of the small spheres is a geometric center, which facilitates comparative analysis of different conformational information. The results obtained during the analysis are as shown in the figure: Figure 5 is the conformational information during molecular docking, Figure 6 is an image of the conformation colored according to van der Waals force, Figure 7 is the conformational center during docking, Figure 8 shows the docking conformation at states in the ensemble and interactions with receptor residues. From the following results, it can be seen that curcumin 2-chloroadenine derivative has a binding site with G-quadruplex.

G-quadruplexes are composed of four guanines that interact and combine to form a square. They are temporary structures that exist in large numbers in cells that are about to divide. They appear in chromosome nuclei and chromosome terminals (can protect chromosomes from damage). Because cancer cells divide so rapidly and often have defects at the ends of chromosomes, the quadruple helix DNA molecule may be unique to cancer cells.

Therefore, the results of Test Example 4 can illustrate that the compounds of the present invention have:

Anticancer potential: Since g-quadruplexes are ubiquitous in cancer cells, compounds that specifically interact with this structure may have anticancer potential.

Specificity: The compound's specific interaction with g-quadruplexes may make it more directional in cells and produce fewer non-specific side effects. This makes the drug potentially easier to adapt to clinical use and reduces the incidence of therapeutic risks.

Therapeutic strategies: Based on the specific existence conditions of g-quadruplex, the discovery of this compound may drive related therapeutic strategies and methods. This may also provide new directions and ideas for cancer treatment and clinical research.

Test example 5

Compare the anti-inflammatory properties of curcumin (CUR) and curcumin 2-chloroadenine derivative (CCAD).

experimental design:

1. The RAW264.7 macrophage cell line was used as the experimental model and divided into four groups: control group (Control), CUR group, and CCAD group.

2. The control group received normal culture conditions, and the other groups added CUR and CCAD respectively, with the dosage concentration being 10 μM.

3. Use lipopolysaccharide (LPS, 100ng/mL) to stimulate RAW264.7 cells for 24 hours to trigger an intracellular inflammatory response.

4. Determine inflammatory factors in cell culture fluid, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), through enzyme-linked immunosorbent assay (ELISA).

The experimental data are as follows (unit: pg/mL):

Group TNF-α IL-6

Control 220.50 165.40

CUR 135.28 108.35

CCAD 60.23 44.18

Using the values of the control group as a basis, calculate the percentage of activation of the other groups:

TNF-α inhibition percentage:

CUR: (220.50-135.28)/220.50*100%＝38.6%

CCAD: (220.50-60.23)/220.50*100%＝72.7%

IL-6 inhibition percentage:

CUR: (165.40-108.35)/165.40*100%＝34.5%

CCAD: (165.40-44.18)/165.40*100%＝73.3%

Experimental results:

The results of this experiment showed that compared with the untreated group, both CUR and CCAD could significantly inhibit the release of TNF-α and IL-6, among which CCAD had the most significant inhibitory effect. After comparative analysis, the anti-inflammatory effect of the CUR-treated group was relatively weak, while CCAD showed better anti-inflammatory properties and stronger anti-inflammatory effect under the same administration conditions. The reasons are:

Bioavailability: Purine derivatives of curcumin have high bioavailability. This means that the derivative exists in higher effective concentrations in the body compared to curcumin. The bioavailability of curcumin is low, partly because it is easily metabolized and excreted in the body, resulting in a reduced effective concentration and reduced biological activity. Therefore, at the same dosage, the anti-inflammatory activity of purine derivatives of curcumin is stronger than that of curcumin.

Structural advantages: Since its structure is similar to purine, curcumin purine derivatives bind more closely to inflammation-related enzymes or receptors, thereby exerting stronger anti-inflammatory potential. At the same time, curcumin derivatives have structurally improved stability, thereby enhancing anti-inflammatory activity.

Enhanced selectivity: Curcumin purine derivatives have better selectivity and specifically inhibit molecular signaling pathways that play a key role in the inflammatory process, such as TNF-α and IL-6 related signaling pathways. As a multi-target anti-inflammatory substance, curcumin has a broad mechanism of action, but its anti-inflammatory effect is relatively weak due to lack of specificity.

Transparent transmission across cell membranes: Due to the changes in hydrophobicity and alkalinity of curcumin purine derivatives, the derivatives are more likely to pass through the cell membrane, thereby producing strong anti-inflammatory activity within cells. In contrast, the hydrophobicity and alkaline nature of curcumin limits its ability to transmit across membranes, resulting in reduced anti-inflammatory activity.

In summary, based on bioavailability, structural advantages, enhanced selectivity and transmissibility across cell membranes, curcumin purine derivatives have good anti-inflammatory activity.

The various embodiments of the present application have been described above. The above description is illustrative, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims (2)

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that the compound is selected from compounds represented by general formula (I) or (Il) 2. A pharmaceutical composition, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.