Application of natural compound in preparation of medicine for treating or preventing central nervous system disorders and cardiovascular diseases

Abstract

The invention belongs to the technical field of biological medicines, particularly relates to application of natural compounds in preparation of medicines for treating or preventing central nervous system disorders and cardiovascular diseases, and more particularly relates to application of seven phosphodiesterase II activity inhibitors screened out through virtual docking and biological experiment methods and application of the phosphodiesterase II activity inhibitors in preparation of medicines for treating or preventing central nervous system disorders and cardiovascular diseases in preparation of medicines for treating or preventing central nervous system disorders and cardiovascular diseases. And the natural compounds are respectively lysin, welterone, licoflavanone, 6-isopentenyl apigenin, norfarrerol, 6-isoprenyl naringenin and lupinus luteus welterone. The invention further discloses a preparation method of the compound. The compounds can be used for preparing drugs for inhibiting the activity of PDE2, the enzyme level IC50 value of the compounds is obtained through detection, it is determined that the compounds have a good PDE2 enzyme inhibition effect, and the compounds have positive research significance in central nervous system disorder drugs and cardiovascular disease drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to the use of various natural compounds in the preparation of drugs for treating or preventing central nervous system disorders and cardiovascular diseases. Background Art

[0002] Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are crucial second messenger molecules that play an indispensable role in receiving external signal stimuli and regulating the life activities of organisms. They regulate physiological processes through both direct and indirect mechanisms. In cells, the concentration levels of cAMP and cGMP are precisely regulated. This regulatory process depends on the promotion of their synthesis by nucleotide cyclases and the hydrolysis mediated by phosphodiesterases (PDEs). The two work together to maintain their dynamic balance in cells. In particular, PDEs become the only way to degrade these second messenger molecules by catalyzing the hydrolysis and ring-opening reactions of cAMP and cGMP, thereby ensuring the accuracy and timeliness of intracellular signal transmission.

[0003] The PDEs family has 11 subtypes (PDE1-11), among which PDE2 can hydrolyze intracellular cAMP and cGMP to reduce their concentration levels. Overexpression of PDE2 will cause cAMP and cGMP levels to fall below the minimum catalytic activity threshold, resulting in neurological pathological phenomena such as memory loss, depression, and cognitive decline. Inhibiting overexpressed PDE2 and increasing cyclic nucleotide levels are effective targets for the treatment of central nervous system diseases. PDE2 inhibitors exert their physiological effects by inhibiting the activity of PDE2 and restoring c AMP and cGMP concentrations to normal levels. PDE2 inhibitors have therapeutic prospects in the treatment of depression, anxiety, cognitive impairment, and Alzheimer's disease. By inhibiting the activity of PDE2, PDE2 inhibitors can increase the concentration of intracellular cAMP or cGMP, thereby regulating neurotransmitters and signaling pathways related to depression and anxiety and improving patients' symptoms. Phosphodiesterase II activity inhibitors increase the concentration of intracellular cyclic adenosine monophosphate (cAMP), thereby enhancing myocardial contractility and improving cardiac function. They also have certain therapeutic effects on cardiovascular diseases such as heart failure, arrhythmia, and angina pectoris. PDE2 inhibitors may lower blood pressure by dilating blood vessels and reducing peripheral resistance. This mechanism of action makes PDE2 inhibitors one of the potential drugs for the treatment of hypertension.

[0004] Natural products refer to components or metabolites of animal and plant extracts (plant extracts for short) or insects, marine organisms and microorganisms, as well as many endogenous chemical components in humans and animals. Natural products have always been an important source of potential drugs, and natural products and their structural analogs have made significant contributions to disease treatment in history. Discovering, screening and transforming active compounds from natural products has been a hot research direction in drug development in recent years. Summary of the invention

[0005] The present invention screens and provides 7 phosphodiesterase II activity inhibitors from a natural product library. The present invention uses a method of integrating virtual screening and experimental determination to search and discover from the ChemDiv compound library that the 7 compounds shown can inhibit PDE2 enzyme activity, and combines experiments to verify the inhibitory activity of the compounds on PDE2.

[0006] The natural compounds are shown below.

[0007] Prunus quinone (CAS No.: 80453-44-7), its structural formula is:

[0008] Weite ketone (CAS No.: 119240-82-3), its structural formula is:

[0009] Licorice flavanone (CAS No.: 119240-82-3), its structural formula is:

[0010] 6-Isopentenyl apigenin (CAS No.: 68097-13-2), its structural formula is:

[0011] Norrhododendron (CAS No.: 14348-16-4), its structural formula is:

[0012] 6-Isoprenylnaringenin (CAS No.: 68236-13-5), its structural formula is:

[0013] Yellow lupinus weiterone (CAS No.: 104691-86-3), its structural formula is:

[0014] The above compounds can be used as phosphodiesterase II (PDE2) activity inhibitors and can be used to prepare drugs for treating or preventing central nervous system disorders and cardiovascular diseases.

[0015] The compound is prepared into a pharmaceutical composition with one or more pharmaceutically acceptable carriers.

[0016] The pharmaceutical composition enters the human body through the corresponding dosage form via the corresponding carrier, inhibits the activity of phosphodiesterase II (PDE2) to treat PDE2 disorder; and is used through smearing, buccal filling, atomization, enema, oral administration, injection and infusion to achieve the treatment purpose.

[0017] The dosage form of the drug is conventional dosage forms such as sol, water injection, ointment, gel, powder, granule, tablet, capsule, pill, suppository, aerosol, spray, etc.

[0018] The carriers include conventional carriers such as diluents, excipients, fillers, adhesives, etc. in the pharmaceutical field, and novel carriers such as microcapsules, microspheres, microneedles, and nanoparticles.

[0019] Furthermore, central nervous system disorders include sensory disorders, language disorders, movement disorders, cognitive disorders, sleep disorders, higher nervous activity disorders, visual disorders, and swallowing disorders.

[0020] Furthermore, cardiovascular diseases include heart and blood vessel lesions, arrhythmias, cardiomyopathy, endocarditis, and hypertension.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses computer simulation software Discovery Studio to evaluate the effectiveness of the compound, and uses biological experimental methods to detect and verify the activity, and obtains an effective PDE2 enzyme inhibitor, PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The IC value of prunus quinone has a good PDE2 enzyme inhibitory effect. 50 The value was 22.74±1.71μmol / L; Weite ketone IC 50 The value is 10.57±1.07μmol / L, and the IC 50 The IC50 value of 6-isopentenylapigenin was 7.65±0.58μmol / L; the IC50 value of norrhododendron 50 The value was 5.78±0.41μmol / L; 6-isoprenylnaringenin IC 50 The value is 4.22±0.39μmol / L; Yellow Lupinus weiterone IC 50 The value is 0.88±0.12μmol / L. These studies are crucial for the ultimate development of anti-PDE2 drugs and have important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1:2D image of the simulated docking of BAY60-7550 and PDE2 protein molecule 4HTX using Discovery Studio;

[0024] Figure 2 :2D image of the simulated docking of prunus cerevisiae and PDE2 protein molecule 4HTX using Discovery Studio;

[0025] Figure 3 : PDE-Glo TM IC of prunus tinctorius at the PDE2 protein level measured by Phosphodiesterase Assay 50 Value graph;

[0026] Figure 4 :2D image of simulated docking of weiterone and PDE2 protein molecule 4HTX using Discovery Studio;

[0027] Figure 5 : PDE-Glo TM Phosphodiesterase Assay measured the IC of weiterone at the PDE2 protein level 50 Value graph;

[0028] Figure 6 :2D image of simulated docking of licorice flavanone and PDE2 protein molecule 4HTX using Discovery Studio;

[0029] Figure 7 : PDE-Glo TM IC of licorice flavanones at the PDE2 protein level measured by Phosphodiesterase Assay 50 Value graph;

[0030] Figure 8 :2D image of the simulated docking of 6-isopentenyl apigenin and PDE2 protein molecule 4HTX using Discovery Studio;

[0031] Fig. 9 : PDE-Glo TM IC of 6-isopentenylapigenin at PDE2 protein level measured by Phosphodiesterase Assay 50 Value graph;

[0032] Fig.10 :2D image of the simulated docking of nordurin and PDE2 protein molecule 4HTX using Discovery Studio;

[0033] Fig.11: PDE-Glo TM Phosphodiesterase Assay was used to measure the IC of norrhododendronine at the PDE2 protein level 50 Value graph;

[0034] Fig.12 :2D image of the simulated docking of 6-isoprenylnaringenin and PDE2 protein molecule 4HTX using Discovery Studio;

[0035] Fig.13 : PDE-Glo TM Phosphodiesterase Assay measured the IC of 6-isoprenylnaringenin at the PDE2 protein level 50 Value graph;

[0036] Fig.14 :2D image of the simulated docking of yellow lupin weite ketone and PDE2 protein molecule 4HTX using Discovery Studio;

[0037] Fig.15 : PDE-Glo TM IC of yellow lupin weiterone on PDE2 protein level measured by Phosphodiesterase Assay 50 Value graph. DETAILED DESCRIPTION

[0038] 1 Experimental methods

[0039] 1.1 Receptor-based virtual screening

[0040] First, download the PDE2 protein crystal structure 4HTX from the PDB protein database (https: / / www.rcsb.org), and then use Discovery Studio (DS) docking software for molecular docking. The specific steps are as follows:

[0041] (1) Optimization of receptor protein file: Download the 4HTX file from the PDB protein database and open it with DS software. Delete all chains except the third C chain and the water molecules in the protein. Then click Prepare protein to modify the corresponding data and run the program to obtain the optimized protein.

[0042] (2) Prepare the ligand small molecule library: Create a drawing board in the small molecule window of DS, then copy the ligand molecules drawn in Chem Draw to the drawing board, click Prepare Ligans to run, and after the run is completed, adjust the optimized ligands with suitable structures to visible.

[0043] (3) Determine the active site: Select the ligand part after complex coupling with BAY60-7550, then click Attibutes of SBD Sphere to confirm the application and delete other ligands.

[0044] (4) Molecular docking: Click Receptor-Ligand Interactions and select CDocker to run. Adjust the receptor to the optimized protein and the ligand to the visible corresponding structure.

[0045] BAY60-7550 is an important PDE2 inhibitor discovered in recent years. We used molecular docking software to dock it with 4HTX (PDE2 catalytic domain complex crystal) in the PDB database. The interaction force results are as follows: Figure 1 , BAY60-7550 mainly binds to two sub-pockets in the PDE2 receptor protein: the Q pocket containing the glutamine switch and the hydrophobic clamp, and the H pocket containing the hydrophobic amino acid residues. Phe-862 in the PDE2 receptor protein forms a π-π interaction with the imidazotriazine in the BAY60-7550 structure; glutamine (Gln-812, Gln-859) forms a hydrogen bond with the carbonyl group on the imidazotriazine; at the same time, aspartic acid Asp-808 forms a hydrogen bond with the hydroxyl group on the side chain of BAY60-7550; methionine Met-847 has a hydrophobic interaction with the 3,4-dimethoxybenzyl group on the side chain of BAY60-7550.

[0046] 1.2 Experimental steps for in vitro PDE2 enzyme activity detection

[0047] 1) Expression and purification of PDE2 protein: The wild-type and truncated plasmids of pET15b-PDE2A (580-941) used in the invention were donated by Professor Hengming Ke's laboratory at the University of North Carolina, USA. The experimental contents include: (1) Preparation of culture medium, including the preparation of LB culture medium, LB solid culture medium and 2*YT culture medium. (2) Plasmid transformation, specifically adding the plasmid to the LB culture medium prepared in advance and culturing the centrifuged bacterial liquid on the LB solid culture medium after resuscitation on a shaker at 37°C to obtain the PDE2 plasmid. (3) Protein expression and purification, specifically adding the prepared plasmid to the strain and culturing it in the above manner, then adding the cultured bacterial liquid to the YT liquid culture medium until the OD value is measured to be 0.6-0.8, collecting and storing, and purifying the collected solid with a Nice column.

[0048] 2) Experimental steps for in vitro PDE2 enzyme activity detection:

[0049] This experiment was carried out on OptiPlate TMThe reaction was carried out in a microplate constant temperature oscillator at 25°C and 300 r / min. Three parallel experiments were performed for each experiment, and centrifugation was performed for 1 min between each step (speed 1000 r / min).

[0050] (1) Experimental process

[0051] Add 1 μL of the dilution of the test compound and 1.5 μL of the PDE2 protein dilution to the white well plate, shake at room temperature for 30 minutes, add 2.5 μL cNMP, 2.5 μL Termination Buffer and 2.5 μL Detection Solution in turn, centrifuge for 1 minute and shake at room temperature for 20 minutes each time the reagent is added. Finally, add Kinase Reagent buffer and react in the dark for 10 minutes. After the reaction is completed, use a multifunctional microplate reader to read. During the experiment, set up a positive control (no PDE2 protein) and a negative control (no inhibitor), and fill in an equal amount of 1×Reaction Buffer. Each group needs to have 3 parallel experiments.

[0052] (2) BAY 60-7550 positive compound IC 50 Determination

[0053] Dilute BAY 60-7550 to 10 mM using dimethyl sulfoxide (DMSO), and then use 1× Reaction Buffer to further dilute it to a final concentration of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, 1.56 nM, and 0.78 nM in the system; add BAY 60-7550 dilutions of various concentrations and PDE2 protein solution to the wells, and add reagents in sequence according to the above experimental process. After the reaction is completed, read the results with an ELISA reader and calculate the IC using GraphPad Prism. 50 .

[0054] Example 1

[0055]

[0056] The results of the interaction force of linalool are as follows Figure 2 Phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the benzene ring containing two hydroxyl groups of prunus persica and a π-σ force with the tetrahydropyrone ring; glutamine Gln-859 forms a hydrogen bond with the hydroxyl group on the tetrahydropyrone ring of prunus persica; glutamine Gln-812 produces a van der Waals force with prunus persica; aspartic acid Asp-808 produces a hydrogen bond with the hydroxyl group on the benzene ring containing two hydroxyl groups of prunus persica; methionine Met-847 has a hydrophobic effect with the benzene ring of prunus persica on the tetrahydropyrone ring.

[0057] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value was 22.74±1.71μmol / L (such as Figure 3 It has a good PDE2 enzyme inhibitory effect.

[0058] Example 2

[0059]

[0060] The results of the interaction force of ketone are as follows Figure 4 The phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the dihydropyrone ring of ketone and the benzene ring it is connected to; the glutamine Gln-859 forms a hydrogen bond force with the hydroxyl group on the benzene ring of ketone connected to the dihydropyrone ring; the aspartic acid Asp-808 forms a hydrogen bond force with the hydroxyl group on the benzene ring of ketone that contains only one hydroxyl group; the methionine Met-847 produces an alkylation reaction with the methyl group on the isopentene of ketone.

[0061] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value was 10.57±1.07μmol / L (such as Figure 5 It has a good PDE2 enzyme inhibitory effect.

[0062] Example 3

[0063]

[0064] The results of the interaction of licorice flavanones are as follows Figure 6 , phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the benzene ring where the isopentyl of glycyrrhizic flavanone is located and produces an alkyl reaction with the methyl group on the isopentyl; glutamine Gln-859 forms a hydrogen bond force with the hydroxyl group on the benzene ring where the isopentyl of glycyrrhizic flavanone is located; glutamine Gln-812 forms a van der Waals force with glycyrrhizic flavanone; aspartic acid Asp-808 produces a hydrogen bond force with the hydroxyl group close to the carbonyl group of glycyrrhizic flavanone; methionine (Met-847) has a hydrophobic interaction with the benzene ring where the isopentyl of glycyrrhizic flavanone is located and produces an alkyl reaction with the methyl group on the isopentyl of glycyrrhizic flavanone.

[0065] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value was 8.05±0.74μmol / L (such as Figure 7It has a good PDE2 enzyme inhibitory effect.

[0066] Example 4

[0067]

[0068] The interaction results of 6-isopentenyl apigenin are as follows Figure 8 Phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the non-isopentenyl apigenin benzene ring and a π-anion force with the hydroxyl group on it; glutamine (Gln-812, Gln-859) forms a hydrogen bond with the hydroxyl group on the non-isopentenyl apigenin benzene ring; aspartic acid Asp-808 forms a hydrogen bond with the hydroxyl group on the benzene ring where the isopentenyl apigenin is located.

[0069] The IC50 value of the enzyme level detected by PDE-GloTM Phosphodiesterase Assay was 7.65±0.58μmol / L (such as Fig. 9 It has a good PDE2 enzyme inhibitory effect.

[0070] Example 5

[0071]

[0072] The interaction force results of norrhododendron Fig.10 The phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the benzene ring of norcurculin that contains only one hydroxyl group; glutamine (Gln-812, Gln-859) forms a hydrogen bond force with the hydroxyl group on the benzene ring of norcurculin that contains only one hydroxyl group; aspartic acid Asp-808 forms a hydrogen bond force with the hydroxyl group on the benzene ring of norcurculin that is far away from the six-membered ring.

[0073] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value was 5.78±0.41μmol / L (such as Fig.11 It has a good PDE2 enzyme inhibitory effect.

[0074] Example 6

[0075]

[0076] The interaction results of 6-isoprenylnaringenin are as follows Fig.12, phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the benzene ring of 6-isoprenylnaringenin that only contains one hydroxyl group; glutamine Gln-812 forms a hydrogen bond force with the hydroxyl group on the benzene ring of 6-isoprenylnaringenin that only contains one hydroxyl group; glutamine Gln-859 produces a van der Waals force with 6-isoprenylnaringenin; aspartic acid Asp-808 produces a van der Waals force with 6-isoprenylnaringenin; methionine Met-847 produces a van der Waals force with 6-isoprenylnaringenin.

[0077] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value was 4.22±0.39μmol / L (such as Fig.13 It has a good PDE2 enzyme inhibitory effect.

[0078] Example 7

[0079]

[0080] The results of interaction force between yellow lupin and ketone are as follows Fig.14 , phenylalanine Phe-862 in the PDE2 receptor protein forms a π-π force with the three six-membered rings of yellow lupine weite ketone; glutamine Gln-859 forms a hydrogen bond force with the hydroxyl group on the isopentyl benzene ring of yellow lupine weite ketone; glutamine Gln-812 produces a van der Waals force with yellow lupine weite ketone, aspartic acid Asp-808 forms a hydrogen bond force with the hydroxyl group on the isopentyl benzene ring of yellow lupine weite ketone, and methionine Met-847 forms a π-alkyl force with the benzene ring where the isopentyl of yellow lupine weite ketone is located.

[0081] PDE-Glo TM Phosphodiesterase Assay to detect enzyme levels IC 50 The value is 0.88±0.12μmol / L (such as Fig.15 It has a good PDE2 enzyme inhibitory effect.

[0082] In addition, the present invention detected the in vitro enzyme activity of PDE1 and PDE3-11 using the above method, and no obvious inhibitory activity was detected.

[0083] In summary, the 7 compounds described in the present invention are effective phosphodiesterase II (PDE2) inhibitors, and good inhibitory enzyme activity is detected in vitro when the in vitro enzyme activity test is performed, showing a strong inhibitory effect on PDE2, and showing good selectivity for other PDE subtypes. As PDE2 inhibitors, the 7 compounds described in the present invention have potential for treating PDE2 disorders, and are expected to be used as active agents for preparing drugs for treating PDE. Due to the important role of PDE2 in regulating the concentration levels of cAMP and cGMP, they are particularly expected to be used for preparing drugs for treating mental illness or neurosis, cardiovascular diseases, etc., and have medicinal prospects.

[0084] It should be understood and recognized that although the present invention is described in detail above, the above description is intended to illustrate and explain only and should not be used to limit the scope of the present invention, which is defined in the claims of the present application. Other aspects, benefits and modifications are also within the scope of the claims. The contents of all publications cited in this application are all incorporated into this application in the form of reference materials and become a part of this application.

Claims

1. Use of a natural compound in the preparation of a drug for treating or preventing central nervous system disorders or cardiovascular diseases, characterized in that: The natural compound can be used as a phosphodiesterase II activity inhibitor for preparing drugs for treating or preventing central nervous system disorders and cardiovascular diseases. The structural formula of the natural compound is:

2. The use according to claim 1, characterized in that: The compound of claim 1 is prepared into a pharmaceutical composition with one or more pharmaceutically acceptable carriers.

3. The use according to claim 2, characterized in that: The pharmaceutical composition enters the human body through the corresponding dosage form via the corresponding carrier, inhibits the activity of phosphodiesterase II to treat PDE2 disorder; and is used through one or more routes of smearing, buccal administration, packing, atomization, enema, oral administration, injection or infusion to achieve the therapeutic purpose.

4. The use according to claim 3, characterized in that: The dosage form of the medicine is sol, water injection, ointment, gel, powder, granule, tablet, capsule, pill, suppository, aerosol and spray.

5. The use according to claim 2, characterized in that: The carrier includes diluents, excipients, fillers, adhesives, microcapsules, microspheres, microneedles and nanoparticle carriers used in the pharmaceutical field.

6. The use according to claim 1, characterized in that: The central nervous system disorders include sensory disorders, language disorders, movement disorders, cognitive disorders, sleep disorders, higher nervous activity disorders, visual disorders, and swallowing disorders.

7. The use according to claim 1, characterized in that: Cardiovascular diseases include heart and blood vessel diseases, arrhythmias, cardiomyopathy, endocarditis, and hypertension.

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