Phosphodiesterase 2 activity inhibitors

Abstract

This invention belongs to the field of biomedical technology, specifically relating to phosphodiesterase 2 (PDE2) activity inhibitors. Six PDE2 activity inhibitors were screened, providing six compounds: sanguisorbin, Picropodophyllol, aristolochic acid D, Malabaricone A, tetradehydropodophyllotoxin, and Roseoflavin. These compounds can be used to prepare PDE2 activity inhibitor drugs. Biological experimental methods were used to detect and verify the activity, resulting in effective PDE2 enzyme inhibitors. The IC50 level of these inhibitors was measured. 50 It exhibits good PDE2 enzyme inhibition effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving six phosphodiesterase 2 (PDE2) activity inhibitors. Background Technology

[0002] Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers in intracellular signal transduction, acting extensively on intracellular target organs and playing crucial roles in muscle contraction, nerve transmission, and cell growth, thus being closely related to human life activities. Since polymorphonuclear leukocyte inhibitors (PDEs) play a significant role in regulating intracellular cAMP and cGMP concentrations, inhibiting the activity of intracellular PDEs can increase cAMP and cGMP levels, thereby maintaining the balance of various physiological functions.

[0003] Phosphodiesterases (PDEs) are the only enzymes in the human body that catalyze the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Since their discovery, PDEs have attracted considerable attention from researchers and have long been considered important targets in drug development. Currently, several selective inhibitors of different subtypes have been approved for clinical use, including inhibitors of PDE1, PDE3, PDE4, PDE5, and PDE10.

[0004] The single-gene-encoded phosphodiesterase PDE2 is mainly distributed in the brain, heart, and neurons, and exists as a homodimer. It is also found in platelets, lungs, macrophages, endothelial cells, adrenal glands, liver, placenta, skeletal muscle, and pancreas. PDE2 has four substructures: PDE2A1 (from bovine), PDE2A2 (found only in mice), PDE2A3 (from human), and PDE2A4 (from human).

[0005] PDE2 is an attractive target for drug development, playing a crucial role in regulating myocardial contraction, improving cognitive function, and enhancing long-term memory. Existing PDE2 inhibitors include BAY60-7550, EHNA, and dipyridamole, but due to their inability to cross the blood-brain barrier and poor pharmacokinetics, no drugs have yet been marketed. Therefore, developing novel PDE2 inhibitors is of great significance. Summary of the Invention

[0006] This invention provides six phosphodiesterase (PDE2) activity inhibitors, which are the compounds shown below.

[0007] Sangxinsu, its structural formula is: Picropodophyllol, its structural formula is: Aristolochic acid D, its structural formula is: Malabaricone A, its structural formula is: Tetradehydropodophyllotoxin has the following structural formula: Roseoflavin, its structural formula is:

[0008] The compounds shown above can act as inhibitors of phosphodiesterase PDE2 activity.

[0009] The compounds shown above can be formulated into pharmaceutical compositions with one or more pharmaceutically acceptable carriers.

[0010] The carriers include common pharmaceutical materials such as diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

[0011] The dosage form of the drug is tablets, capsules, granules, pills, or other conventionally prepared dosage forms.

[0012] The pharmaceutical composition treats PDE2 disorders by inhibiting phosphodiesterase (PDE2) activity; it can be introduced into the body, such as through injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into muscle, intradermal, subcutaneous, venous, or mucosal tissues; or it can be introduced into the body after being mixed with or encapsulated by other substances.

[0013] The PDE2 disorder is a depressive disorder, which includes: single-episode depressive disorder, recurrent depressive disorder, and bipolar disorder.

[0014] This invention uses computer simulation software to evaluate the effectiveness of compounds and employs biological experimental methods to detect and verify their activity, resulting in an effective PDE2 enzyme inhibitor. The IC50 level of this inhibitor was determined. 50 It exhibits good PDE2 enzyme inhibition effect. Attached Figure Description

[0015] Figure 1 PDE-Glo TM The Phosphodiesterase Assay measured the IC50 of senna at the PDE2 protein level. 50 Value diagram;

[0016] Figure 2 PDE-Glo TM The Phosphodiesterase Assay measured the IC50 of Picropodophyllol at the PDE2 protein level. 50 Value diagram;

[0017] Figure 3 PDE-Glo TMThe Phosphodiesterase Assay measured the IC50 of aristolochic acid D at the PDE2 protein level. 50 Value diagram;

[0018] Figure 4 PDE-Glo TM The Phosphodiesterase Assay measured the IC50 of Malabaricone A at the PDE2 protein level. 50 Value diagram;

[0019] Figure 5 PDE-Glo TM The Phosphodiesterase Assay measured the IC50 of tetradehydropodophyllotoxin at the PDE2 protein level. 50 Value diagram;

[0020] Figure 6 PDE-Glo TM The Phosphodiesterase Assay measured the IC50 of roseoflavin at the PDE2 protein level. 50 Value graph. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0022] 1 Experimental Methods

[0023] 1.1 Receptor-based virtual screening

[0024] Molecular docking was employed using Discovery Studio 2020 Client to dock and score PDE2 protein and sangin. First, the large protein molecule was downloaded from the PDB library. The crystal structure of PDE2 (4HTX) was processed. Since 4HTX is a tetramer with four identical chains, protein chains B, C, and D, along with their ligands, were deleted, leaving only chain A for docking. The molecular docking process included: hydrogenation and dehydration of the protein, hydrogenation of the small molecule and optimization of its 3D structure, establishment of an active site using the BAY60-7550 ligand, and docking of the small molecule with the protein.

[0025] BAY60-7550 is a specific PDE2 inhibitor. In the crystal structure of BAY60-7550 bound to PDE2, BAY60-7550 mainly binds to two sub-pockets of the active site: the Q pocket containing a glutamine switch and a hydrophobic clip, and the H pocket containing hydrophobic amino acid residues. BAY60-7550 mainly occupies the Q pocket, and its binding to the active site also employs a glutamine switch mechanism, namely, hydrogen bonding between the carbonyl group on the pyrimidinone ring and Gln859 and Gln812, and π-π conjugation between the pyrimidinone ring and Phe862 in the hydrophobic clip. Furthermore, the pyrimidinone ring forms a π-π conjugation with Tyr655; the pyrimidinone ring has a π-alkyl interaction with Phe826 and Leu809; the methyl group on the pyrimidinone ring has a π-alkyl interaction with Phe862 and Leu809; the propylphenyl group on the side chain of BAY60-7550 forms a π-π conjugation with Leu809 and Leu770; the o-dimethoxyphenyl group forms a π-π conjugation with Met847; the o-dimethoxyphenyl group has a π-alkyl interaction with Leu770 and Met847; and the alkane on the side chain branch has a π-alkyl interaction with His656, Tyr655, and His660.

[0026] Through docking, the compounds of this invention contain similar interactions to BAY60-7550. Sancinin: The hydroxyl group on the m-dihydroxyphenyl group of benzopyranone forms hydrogen bonds with Gln859 and Gln812; the m-dihydroxyphenyl group forms π-π conjugation with Phe862 and Phe826; benzopyranone forms π-π conjugation with Phe862, Phe830, and Leu770; dimethyl-2H-pyran and His770 and His656 have π-alkyl interactions; the side chain of benzopyranone has π-alkyl interactions with Ile826, Met847, and Leu770. Picropodophyllol: The methoxy group in the middle of the independent benzene ring reacts with Gln859 via hydrocarbon interaction. This ring forms a π-π conjugation with Phe862. The three methoxy groups on the ring react with Phe862, Phe830, Leu809, Tyr655, Met847, Phe826, and Tyr827 via π-alkyl interaction, respectively. The methylene group on the six-membered ring reacts with Thr768 and Asp808 via hydrocarbon interaction. The oxygen group on the five-membered ring reacts with Thr805 via hydrocarbon interaction. The ortho-methylene group reacts with Leu770, Ile870, His773, and Leu809 via π-alkyl interaction. The middle benzene ring reacts with Leu770 and Leu809 via π-alkyl interaction. Aristolochic acid D: The hydroxyl group at the carboxyl terminus has hydrogen bonding with Gln859 and Gln812; the oxygen on the nitro group and the oxygen on the five-membered ring have hydrogen bonding with Tyr827 and Tyr655, respectively; the benzene ring adjacent to the five-membered ring has π-alkyl interactions with Ile826, Phe862, and Leu809; the benzene ring connected to the nitro group has π-π conjugation interactions with Phe862 and Phe830, and π-sulfur interactions with Met847; the lateral benzene ring has π-alkyl interactions with Leu770, and π-sulfur interactions with Met847; the methoxy group on this benzene ring has π-sulfur and π-alkyl interactions with Met847. Malabaricone A: The hydroxyl group on the resorcinol benzene ring has hydrogen bonding with Gln859 and Gln812. This ring forms a π-π conjugation with Tyr655 and has π-alkyl interactions with Ile826 and Leu809. Another benzene ring has a π-alkyl interaction with Leu770. Furthermore, the carbon chain between the two benzene rings has π-alkyl interactions with Met847, Phe862, and Leu858.Tetradehydropodophyllotoxin: The independent benzene ring forms π-π conjugation with Phe862 and Phe830, and has π-sulfur interaction with Met847. The three methoxy groups on this ring have π-alkyl interactions with Phe830, Met847, Phe862, Leu770, and Ile770, respectively. The hydroxyl group on the hydroxy-substituted benzene ring has hydrogen bonding with Asp808. The oxygen on the adjacent five-membered ring and the hydrogen on the methylene group have hydrocarbon interactions with His656. The unsubstituted benzene ring has π-π conjugation with Asp769, and has π-alkyl interactions with Leu770 and Leu809. The methylene group on the adjacent five-membered ring has π-alkyl interactions with Leu809 and His773. Roseoflavin: The two terminal hydroxyl groups on the side chain of the pyrazine have hydrogen bonding with Gln859 and Gln812, and the third hydroxyl group on the side chain has hydrogen bonding with Tyr655; the amino and hydroxyl groups on the piperazine ring have hydrogen bonding with Asp808.

[0027] 1.2 Experimental Procedure for Detecting PDE2 In Vitro Enzyme Activity

[0028] 1) Expression and purification of PDE2 protein: wild-type and truncated pET15b-PDE2A(580-941) plasmids were provided by Professor Hengming Ke's laboratory at the University of North Carolina. The experiment included: plasmid DNA transformation of competent cells, plasmid extraction, plasmid transfection, protein expression (expression using 2*YT (no NaCl), IPTG added when OD=0.6-0.8), purification using a Ni column, characterization by 10% SDS-PAGE gel electrophoresis, staining, dialyzing the corresponding protein twice, and finally concentration and storage at -80 degrees Celsius with glycerol.

[0029] 2) Experimental steps for PDE2 in vitro enzyme activity detection:

[0030] This experiment was conducted on OptiPlate. TM The experiment was conducted in a -384 white well plate and kept at a constant temperature in a microplate shaker at 25 degrees Celsius and 300 rpm. Each experiment was performed in triplicate, and centrifugation was performed for 1 minute between each step (1000 rpm).

[0031] Using PDE-Glo TM The Phosphodiesterase Assay determined the inhibitory effect of the compound on PDE2.

[0032] Add 1 μL of the test compound (7 concentrations for each compound, 3 parallel experiments) to the well, then add 1.5 μL of PDE2 protein and react at room temperature for 30 minutes;

[0033] Add 2.5 μL of pre-prepared 1 μM cAMP to each well and react at room temperature for 20 minutes;

[0034] Add 2.5 μL of PDE-Glo to each well. TM Termination Buffer, 2.5μL PDE-Glo TM Detection Solution, react at room temperature for 20 minutes;

[0035] Finally, add 10 μL of Kinase- The reagent was covered with aluminum foil to protect it from light and reacted at room temperature for 10 minutes.

[0036] After the reaction was completed, the signal values ​​of the microplate were read using a multi-functional microplate reader.

[0037] Each experiment included a negative control and a positive control. Negative control: cAMP substrate was added, but PDE protein was not added. This resulted in the highest cAMP content. cAMP bound to protein kinase A, activating protein kinase A and releasing its catalytic subunit. The catalytic subunit catalyzes the transfer of the terminal phosphate of adenosine triphosphate (ATP) to protein kinase A. This process consumes the most ATP, resulting in the weakest luminescence signal and the lowest microplate signal value. Positive control: cAMP substrate and PDE protein were added. PDE hydrolyzed most of the cAMP, activating less protein kinase A. More ATP was available for the luciferase reaction, increasing luminescence and resulting in the highest microplate signal value.

[0038] Example 1

[0039]

[0040] Sanxinsu via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 2.893 ± 1.34 μmol / L (e.g. Figure 1 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0041]

[0042] Picropodophyllol via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 5.548 ± 0.625 μmol / L (e.g. Figure 2 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0043] Example 3

[0044]

[0045] Aristolochic acid D via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 4.673 ± 0.428 μmol / L (e.g. Figure 3 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0046] Example 4

[0047]

[0048] Malabaricone A via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 37.79 ± 1.25 μmol / L (e.g. Figure 4 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0049] Example 5

[0050]

[0051] Tetradehydropodophyllotoxin via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 9.575 ± 1.698 μmol / L (e.g. Figure 5 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0052] Example 6

[0053]

[0054] Roseoflavin via PDE-Glo TM Phosphodiesterase Assay detects its enzyme level IC 50 The value was 22.99 ± 3.02 μmol / L (e.g. Figure 6 As shown in the figure, it has a good PDE2 enzyme inhibition effect.

[0055] In summary, the six compounds described in this invention are all effective phosphodiesterase (PDE2) inhibitors, effectively inhibiting its enzymatic activity in vitro. Given the crucial role of PDE2 in regulating cAMP and cGMP, these compounds, as PDE2 inhibitors, possess the potential to treat PDE2 disorders and are expected to become drugs for the preparation of treatments for PDE2 disorders.

[0056] It should be understood and acknowledged that although the present invention has been described in detail above, the foregoing description is intended for illustrative purposes only and should not be construed as limiting the scope of the invention, which is defined in the claims of this application. Other aspects, benefits, and modifications are also within the scope of these claims. All publications referenced in this application are incorporated herein by reference and are part of this application.

Claims

1. The use of the compound in the preparation of a drug for treating depressive disorders, characterized in that: The compound is: .

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

3. The application according to claim 2, characterized in that: The carrier includes one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, and lubricant.

4. The application according to claim 1, characterized in that: Injectables, tablets, pills, capsules, suspensions or emulsions containing the compound of claim 1.

Images