Application of compound for preparing medicament for treating and / or preventing diseases related to inhibition of PDE2 activity

Abstract

The invention belongs to the technical field of compound medicines, particularly relates to application of a compound to preparation of a medicament for treating and / or preventing diseases related to inhibition of PDE2 activity, and more particularly relates to screening out a compound with a PDE2 activity inhibitor, in particular to comastolol, the compound can be used for preparing a medicament for inhibiting PDE2 activity, and the compound can be used for treating and / or preventing diseases related to inhibition of PDE2 activity. The IC50 value of the enzymology level is obtained through detection, it is determined that the compound has a good PDE2 enzyme inhibition effect, and the compound has positive research significance in drugs for central nervous system degenerative diseases and cardiovascular diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to compounds for preparing medicaments for treating and / or preventing diseases related to inhibition of PDE2 activity. Background Art

[0002] The second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an indispensable role in the intracellular signal transduction process. Phosphodiesterase hydrolysis is the only degradation pathway of cyclic nucleotides. It controls the balance of cAMP and cGMP concentrations, regulates various signal pathways mediated by cyclic nucleotides, and produces physiological effects. Phosphodiesterase II (PDE2) can hydrolyze cGMP and cAMP at the same time and is a dual-substrate enzyme. PDE2 is the most common PDE in the hippocampus and cerebral cortex. It has high expression levels in the frontal lobe, parietal cortex, and amygdala brain regions related to mood changes. Therefore, PDE2 inhibitors have shown an irreplaceable role in the treatment of neurodegenerative diseases and other psychiatric diseases. In animal models, PDE2 inhibitors have been shown to reduce anxiety-like behavior. For example, by constructing PDE2 lentivirus and transfecting it into the hippocampus of mice, the anxiety-like behavior of mice can be reduced and their memory function can be improved. Similar to anxiety disorders, the effects of PDE2 inhibitors in depression are also achieved by regulating the levels of neurotransmitters. By inhibiting the activity of PDE2, the level of cAMP is increased, thereby regulating the excitability of neurons and synaptic plasticity, thereby improving depressive symptoms.

[0003] PDE2 is a member of the phosphodiesterase (PDEs) family and is responsible for the hydrolysis of intracellular cyclic adenosine monophosphate (cAMP) and / or cyclic guanosine monophosphate (cGMP). These two molecules play an important role as secondary messengers in the neurohormonal regulation of the cardiovascular system. The activity of PDE2 is regulated by a variety of factors, including hormones, neurotransmitters, and drugs. PDE2 affects cardiac muscle strength and chronotropic response by regulating the levels of cAMP and cGMP. Studies have shown that PDE2 inhibitors can increase cardiac output and heart rate, thereby improving cardiac function. PDE2 is also expressed in vascular smooth muscle cells, affecting the dilation and contraction of blood vessels by regulating the levels of cAMP and cGMP. PDE2 inhibitors can cause vasodilation and lower blood pressure. Other studies have shown that PDE2 inhibitors can significantly improve the cardiac function of mice with heart failure, reduce cardiac hypertrophy and fibrosis, and reduce the decline in myocardial contractile function. Currently, more and more PDE2 inhibitors have been developed and used to treat cardiovascular diseases.

[0004] The existing PDE2 inhibitors mainly include BAY60-7550, which has high inhibitory activity and selectivity for PDE2, but its poor pharmacokinetic performance, namely low blood-brain barrier permeability, limits its clinical application. Other types of PDE2 inhibitors that have emerged later, such as TAK-915, have not been reported until now due to pharmacokinetic problems, and have not been successfully transformed into clinical therapeutic drugs. Therefore, it is of great significance to develop new PDE2 inhibitors in response to market requirements. Summary of the invention

[0005] The present invention uses drug molecule design computer simulation software to evaluate the effectiveness of known compounds, and uses biological experimental methods to detect and verify the activity. The invention obtains effective PDE2 enzyme inhibitors. Seven phosphodiesterase (PDE2) activity inhibitors are screened out, specifically: coum estrol (CAS: 479-13-0), 7,2',4'-trihydroxy-3-aryl coumarin (CAS: 1401024-37-0), epiberberine (CAS: 6873-09-2), naringenin chalcone (CAS: 73692-50-9), isobavachalcone (CAS: 20784-50-3), 2'-O-methylisoliquiritigenin (CAS: 51828-10-5), Loureirin C (CAS: 1401024-37-0), and 1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1,2-dihydro-2-yl-1 CAS: 116384-24-8).

[0006] The above compounds can be used as phosphodiesterase PDE2 activity inhibitors or as lead compounds of a new series of PDE2 inhibitors to design new PDE2 inhibitors with excellent pharmacokinetics, activity and few side effects.

[0007] Specifically, the diseases or symptoms related to the inhibition of PDE2 activity are at least one of cardiovascular disease and neurodegenerative disease.

[0008] Among them are neurodegenerative diseases including Alzheimer’s, Huntington’s and Parkinson’s diseases, anxiety disorders, and depression;

[0009] Cardiovascular diseases include heart failure, bradycardia, atrioventricular block, hypertension, coronary heart disease, myocardial hypertrophy, and myocardial infarction.

[0010] The drug is administered orally, intravenously or intramuscularly, rectally, parenterally, or by inhalation.

[0011] The compounds shown above can be prepared into preparations with one or more pharmaceutically acceptable excipients, such as diluents, fillers, binders, disintegrants, lubricants, coating agents, emulsifiers, sweeteners, colorants, etc. Dosage forms such as tablets, granules, soft capsules and other sustained-release preparations and targeted preparations can be prepared.

[0012] The pharmaceutical preparation product treats PDE2 disorders by inhibiting the activity of type 2 phosphodiesterase; the therapeutic purpose is achieved by using the drug through oral, oral, sublingual, rectal, parenteral, topical, inhalation and other routes.

[0013] Beneficial Effects

[0014] The enzymatic level IC of the compounds screened by the test 50 The IC value of Coumestrol is 50 The value was 7.71±1.17μmol / L; the IC of 7,2',4'-trihydroxy-3-arylcoumarin 50 The value is 7.27±0.41μmol / L; the IC of Epiberberine 50 The IC50 value of naringeni n chalcone was 39.23±3.05μmol / L; the IC50 value of isobavachalcone was 19.38±2.73μmol / L; the IC50 value of 2'-O-Methylisoliquiritigenin was 50 The value was 7.64±0.57μmol / L; the IC of Loureirin C 50 The value is 22.30±1.35μmol / L. Although it remains at the enzymatic level, it is crucial for the ultimate development of drugs to treat P DE2 disorders and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : PDE-Glo TM Phosphodiesterase Assay measured the IC of Coumestrol at the PDE2 protein level 50 Value graph

[0016] Figure 2 : PDE-Glo TM Phosphodiesterase Assay measured the IC of 7,2',4'-trihydroxy-3-arylcoumarin at the PDE2 protein level 50 Value graph

[0017] Figure 3 : PDE-Glo TM Phosphodiesterase Assay measures the IC of Epiberberine at the PDE2 protein level 50 Value graph

[0018] Figure 4 : PDE-Glo TM IC of Naringenin chalcone at PDE 2 protein level measured by Phosphodiesterase Assay 50 Value graph

[0019] Figure 5 : PDE-Glo TM The IC of Isobavachalcone at the PDE2 protein level was measured by Phosphodiesterase Assay 50 Value graph

[0020] Figure 6 : PDE-Glo TM Phosphodiesterase Assay measured the IC of 2'-O-Methylisoliquiritigenin at the PDE2 protein level 50 Value graph

[0021] Figure 7 : PDE-Glo TM IC of Loureirin C at PDE2 protein level measured by Phosphodiesterase Assay 50 Value graph

[0022] Figure 8 :Docking results of Bay60-7550 and protein 4HTX

[0023] Fig. 9 :Docking results of Coumestrol and protein 4HTX

[0024] Fig.10 :Docking results of 7,2',4'-trihydroxy-3-arylcoumarin and protein 4HTX

[0025] Fig.11 :Docking results of Epiberberine and protein 4HTX

[0026] Fig.12 :Docking results of Naringenin chalcone and protein 4HTX

[0027] Fig.13 :Docking results of Isobavachalcone and protein 4HTX

[0028] Fig.14 :Docking results of 2'-O-Methylisoliquiritigenin and protein 4HTX

[0029] Fig.15 :Docking results of Loureirin C and protein 4HTX DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0031] 1 Experimental methods

[0032] 1.1 Molecular evaluation based on 4HTX protein

[0033] The molecular docking method was used to dock and score the PDE2 protein and the compounds of the present invention using Discovery Studio 2020 Client.

[0034] First, download the macromolecular protein from the PDB library and process the crystal structure of PDE2 (4HTX). Since 4HTX is a tetramer with four identical chains, the three protein chains A, B, and D and their ligands are deleted, and only the C chain is retained for docking. The molecular docking process is: optimize the C chain, mainly by completing the hydrogen on the protein chain; establish the active site with the BAY60-7550 ligand; optimize the small molecule hydrogenation and 3D structure; dock the small molecule with the protein.

[0035] BAY60-7550 is a specific PDE2 inhibitor. In the crystal structure of BAY60-7550 binding to PDE2, BAY60-7550 mainly binds to two pockets of the protein 4HTX: the Q pocket and the H pocket. The most important one that can reflect the PDE2 inhibitory activity is the binding to the Q pocket.

[0036] The docking results of BAY60-7550 and 4HTX protein are as follows: Figure 8

[0037] BAY60-7550 mainly occupies the Q pocket, and the binding to the active site adopts a glutamine switch mechanism, that is, the carbonyl group on the pyrimidinone ring has hydrogen bonds with Gln859 and Gln812, and the pyrimidinone ring forms a π-π conjugation with Phe862 in the hydrophobic clamp. In addition, the imidazole ring forms a hydrogen bond with Tyr655; the pyrimidinone ring has a π-alkyl reaction with P he826 and Leu809; the methyl group on the imidazolone ring has a π-alkyl reaction with Phe862 and Leu809; the propylphenyl group on the side chain of BAY60-7550 forms a π-alkyl conjugation with Leu770; the o-dimethoxyphenyl group has a π-alkyl reaction with Leu770 and Met847; and the alkane on the side chain branch has a π-alkyl reaction with His656 and Tyr655. The key forces for the above docking are the π-π conjugation between the compound and Phe862 and the hydrogen bonding between the compound and Gln859 and Gln812.

[0038] The docking results of the compounds in the present invention are as follows:

[0039] Coumestrol docking results: Fig. 9

[0040] The benzofuran ring and coumarin ring of Coumestrol both have π-π conjugation with Phe862, and the keto carbonyl group of the coumarin ring forms hydrogen bonds with Gln859 and Gln812, proving that Coumestrol has a key interaction with the 4HTX protein. In addition, the hydroxyl group of the benzofuran ring has a hydrogen bond with Asp808 in the M pocket; it has a π-alkyl interaction with Leu809 in the H pocket and Ile826 in the Q pocket; and it also has a π-π conjugation with Tyr655. The coumarin ring has a π-alkyl interaction with Leu809 in the H pocket and Ile826 in the Q pocket; it has a π-π conjugation with Phe830; and it has a π-alkyl interaction with Met847.

[0041] Docking results of 7,2',4'-trihydroxy-3-arylcoumarin: Fig.10

[0042] In the coumarin structure of 7,2',4'-trihydroxy-3-arylcoumarin, the entire coumarin ring has a π-π conjugation with Phe862, and the hydroxyl group on the benzene ring has a hydrogen bond interaction with Gln859 and Gln812. In addition, the coumarin ring has a π-alkyl interaction with Ile826, the benzene ring outside the coumarin ring has a π-π conjugation with Phe830, and the hydroxyl group on the benzene ring has a hydrogen bond interaction with His656.

[0043] Docking results of Epiberberine: Fig.11

[0044] The benzene ring linked to 1,3-dioxolane in Epiberberine has a π-π conjugated interaction with Phe862, and the oxygen on 1,3-dioxolane has a hydrogen bond with Gln812. In addition, the benzene ring linked to 1,3-dioxolane and the methylene group of 1,3-dioxolane have a π-alkyl interaction with Leu809, the benzene ring also has a π-alkyl interaction with Ile826, the methylene group has a π-alkyl interaction with Tyr655, and the other oxygen of 1,3-dioxolane has a hydrogen bond interaction with Tyr655; the nitrogen cation has an electrostatic interaction with Asp808; on the benzene ring with two methoxy groups, the benzene ring has a π-π conjugated interaction with His656, and the methoxy group has a π-alkyl interaction with Phe830.

[0045] Docking results of Naringenin chalcone: Fig.12

[0046] In Naringenin chalcone, there is a π-π conjugation with Phe862 on the benzene ring not connected to the carbonyl carbon, and the phenolic hydroxyl group on this ring has a hydrogen bond interaction with Gln859. In addition, there is a π-π conjugation with Phe830 and a π-alkyl interaction with Met847 on the benzene ring not connected to the carbonyl carbon; there are hydrogen bond interactions between the hydroxyl group and Asp808 and Tyr655 on the phloroglucinol ring.

[0047] Isobavachalcone docking results: Fig.13

[0048] In isobavachalcone, the benzene ring containing the dihydroxyl group has a π-π conjugated interaction with Phe862, and the hydroxyl group on the benzene ring has hydrogen bond interactions with Gln859 and Gln812. In addition, the dimethyl group on the olefin carbon has a π-alkyl interaction with Phe862, Phe830, and Met847, and the benzene ring also has a π-alkyl interaction with Ile826; on other benzene rings, there is a π-alkyl interaction with Leu770 and Leu809.

[0049] Docking results of 2'-O-Methylisoliquiritigenin: Fig.14

[0050] The hydroxyl group at the carbonyl carbon position on the benzene ring of 2'-O-Methylisoliquiritigenin forms hydrogen bonds with key amino acids Gln859 and Gln812, and this ring forms a π-π conjugation with Phe862 and a π-alkylation with Ile826. The methoxy group adjacent to the carbonyl carbon forms a π-alkylation with Met847, Phe830, and Phe862. The benzene ring in the other part forms a π-π conjugation with Asp769, and a π-alkylation with Leu770 and Leu809, and the hydroxyl group on this ring forms a van der Waals force with Thr805.

[0051] Docking results of Loureirin C: Fig.15

[0052] The benzene ring connected to the carbonyl carbon in Loureirin C has a π-π conjugation with Phe862, and the hydroxyl group on the benzene ring has hydrogen bond interactions with Gln859 and Gln812. In addition, the benzene ring connected to the carbonyl carbon also has a π-alkyl interaction with Ile826; on another benzene ring, the phenolic hydroxyl group has a hydrogen bond interaction with Thr768, the methoxy group has a π-alkyl interaction with His656, the benzene ring also has a π-π conjugation with Asp769, and has a π-alkyl interaction with Leu809 and Leu770.

[0053] 1.2 PDE2 in vitro enzyme activity detection

[0054] 1) Expression and purification of PDE2 protein: pET15b-PDE2A (580-941) wild-type and truncated plasmids were donated by Professor Ke Hengming's laboratory at the University of North Carolina, USA. The experimental contents included: transformation of competent cells with plasmid DNA, plasmid extraction, plasmid transfection, protein expression (expression with 2*YT (no NaCl), adding IPTG when OD = 0.6-0.8), purification with Ni column, characterization with 10% SDS-PAGE gel electrophoresis, staining, dialyzing the corresponding protein twice, and finally concentrating and storing with glycerol at -80°C.

[0055] 2) Experimental steps for in vitro PDE2 enzyme activity detection:

[0056] This experiment was carried out on OptiPlate TM The 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 minute between each step (speed 1000 r / min).

[0057] Using PDE-Glo TM Phosphodiesterase Assay measures the inhibitory effect of compounds on PDE2;

[0058] Add 1 μL of the test compound into the well (7 concentrations of each compound were set in sequence, and 3 groups of parallel experiments), and then add 1.5 μL of PDE2 protein, and react at room temperature for 30 minutes;

[0059] Add 2.5 μL of 1 μM cAMP prepared in advance to each well and react at room temperature for 20 minutes;

[0060] Add 2.5 μL PDE-Glo to each well. TM Termination Buffer, 2.5μL PDE-Glo TM Detection Solution, react at room temperature for 20 minutes;

[0061] Finally, add 10 μL Reagents, cover with tin foil to avoid light, react at room temperature for 10 minutes;

[0062] After the reaction was completed, the microplate signal value was read using a multifunctional microplate reader.

[0063] Negative control and positive control were set for each group of experiments. Negative control: When cAMP substrate was added, PDE protein was not added, and PDE protein was not added, the cAMP content was the highest, cAMP bound to protein kinase A, protein kinase A was activated, and the catalytic subunit was released. The catalytic subunit catalyzed the transfer of the terminal phosphate of adenosine triphosphate to protein kinase A, consuming the most ATP in this process, so the luminescence signal was the weakest and the microplate signal value was the smallest. Positive control: When cAMP substrate and PDE protein were added, PDE would hydrolyze most of the c AMP, less protein kinase A would be activated, more ATP could be used for luciferase reaction, luminescence increased, and the microplate signal value was the largest.

[0064] The results of in vitro enzyme activity detection are shown in Table 1:

[0065] Table 1

[0066]

[0067]

[0068] It should be understood and recognized that although the present invention is described above, the above description is only for illustrative purposes 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 as references and become part of this application.

Claims

1. Use of a compound having PDE2 inhibitory activity or a pharmaceutically acceptable salt thereof for preparing a medicament, characterized in that: The compound is used for preparing a medicament for treating and / or preventing diseases related to inhibiting PDE2 activity; the compound is selected from pseudoestrolactone, 7,2',4'-trihydroxy-3-arylcoumarin, epiberberine, naringenin chalcone, isopsoralen chalcone, 2"-O-methyl isoliquiritigenin, and dragon's blood C.

2. The method according to claim 1, characterized in that: The disease or symptom related to the inhibition of PDE2 activity is at least one of cardiovascular disease and neurodegenerative disease.

3. The use according to claim 1, characterized in that: Neurodegenerative diseases include Alzheimer's, Huntington's and Parkinson's diseases, anxiety disorders, and depression.

4. The use according to claim 1, characterized in that: Cardiovascular diseases include heart failure, bradycardia, atrioventricular block, hypertension, coronary heart disease, myocardial hypertrophy, and myocardial infarction.

5. The use according to claim 1, characterized in that: The drug is administered orally, intravenously or intramuscularly, rectally, parenterally, or by inhalation.

6. The use according to claim 1, characterized in that: The drug is formulated into tablets, granules or capsules.

7. A pharmaceutical composition, characterized in that: The composition comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: Excipients include one or more combinations of diluents, fillers, binders, disintegrants, lubricants, coating agents, emulsifiers, sweeteners, and colorants.

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