PSD-95 PDZ2 peptidomimetic inhibitor and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a PSD-95 PDZ2 peptidomimetic inhibitor and application thereof. A series of compounds are designed and synthesized by taking tetrapeptide ETAV as a template, the structure-function relationship between the compounds and PSD-95 PDZ2 is systematically studied, and an optimal compound 32-2, namely the PSD-95 PDZ2 peptidomimetic inhibitor is screened, has a structural formula shown as a formula (I), has very excellent plasma stability, shows remarkable neuroprotective activity in in-vivo and in-vitro biological activity evaluation, and can be used for preparing the PSD-95 PDZ2 peptidomimetic inhibitor for the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection of the neurological protection. Cerebral infarction caused by cerebral ischemia reperfusion injury is remarkably reduced, and the compound can be used for preparing drugs for treating cerebral arterial thrombosis. (I).

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a PSD-95 PDZ2 peptidomimetic inhibitor and an application thereof in preparing a drug for treating ischemic stroke. Background Art

[0002] Ischemic stroke is a major public health problem, resulting in high mortality and disability in adults and is the second leading cause of death worldwide. During cerebral ischemia, excessive release of glutamate in the synaptic cleft overactivates NMDARs and allows Ca 2+ PSD-95 flows into postsynaptic neurons in large quantities, and binds to the C-terminus of nNOS and NMDAR through the first and second PDZ domains, especially the NMDAR 2B (NMDA Receptor 2B, GluN2B) subunit, activating nNOS to produce excessive NO, thereby inducing neuronal death ( Figure 1 Inhibiting this process has been considered a potential neuroprotective strategy. However, inhibitors that directly inhibit nNOS or NMDAR can cause various side effects (such as movement disorders and memory impairment), and such drugs have not been approved for clinical use. In contrast, PSD-95, as a scaffold protein, contains three PDZ domains, which can bind to NMDAR and nNOS within the cell membrane to form a toxic complex. Inhibitors designed against the first or second PDZ domains of PSD-95 can not only prevent the formation of the NMDAR-PSD-95-nNOS complex, but also do not affect the function of NMDAR and nNOS. Therefore, the development of PSD-95 PDZ2 inhibitors has important clinical value.

[0003] Most of the reported PSD-95 PDZ inhibitors are peptide structures, such as NA-1 ( Figure 2 A 20-decapeptide peptide, consisting of the C-terminal 9 amino acids of the NR2B isoform of the NMDAR and the 11 amino acids of the transmembrane peptide Tat, exerts neuroprotective effects by inhibiting the interactions between the NMDAR and PSD-95, and between PSD-95 and nNOS. Phase I and II clinical trials have been completed, demonstrating for the first time the clinical efficacy of a neuroprotective agent. However, peptide drugs have drawbacks such as a short half-life, poor stability, and inability to be taken orally, which limit their efficacy.

[0004] AVLX-144 is a dimeric peptide drug candidate based on the C-terminal five amino acids of the NR2B isoform of the NMDAR, with polyethylene glycol as a linker. It simultaneously targets both PDZ1 and PDZ2 of PSD-95 to inhibit the formation of the NMDAR-PSD-95-nNOS complex. Compared to NA-1, it has better stability and in vitro protein affinity. Phase I clinical trials have been completed overseas.

[0005] Although reported PSD-95 PDZ peptide inhibitors have shown significant translational potential, their druggability limitations due to poor in vivo stability limit their application. Small molecule drugs could overcome this limitation, making the development of small molecule inhibitors targeting PSD-95 PDZ2 of significant interest. However, the development of small molecules targeting the PDZ domain is extremely challenging, and reported inhibitors are scarce. To date, no small molecule inhibitors of PSD-95 PDZ2 with low micromolar activity have been reported. Peptidomimetics, as intermediates between peptides and small molecules, provide a key approach for the conversion of peptides to small molecules. Summary of the Invention

[0006] The purpose of the present invention is to provide a PSD-95 PDZ2 peptidomimetic inhibitor with significant neuroprotective activity and high plasma stability, which can be used to prepare a drug for treating ischemic stroke.

[0007] The technical solutions of the present invention are as follows: The PSD-95 PDZ2 peptidomimetic inhibitor of the present invention has a structural formula as shown in formula (I), code-named 32-2.

[0008] (I) The present invention provides the use of the PSD-95 PDZ2 peptidomimetic inhibitor in preparing a drug for treating ischemic stroke and in preparing a neuroprotective drug.

[0009] The present invention also provides a drug comprising a therapeutically effective amount of acetate, hydrochloride or other pharmaceutically acceptable salt forms of the PSD-95 PDZ2 peptidomimetic inhibitor.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention used the tetrapeptide ETAV as a design template and standard Fmoc solid-phase synthesis technology to synthesize 31 peptidomimetics. After in vitro fluorescence polarization experiments and cell protection activity experiments, the optimal compound 32-2 was screened out, namely the PSD-95 PDZ2 peptidomimetic inhibitor of the present invention. A series of cell and animal experiments on the optimal compound led to the following conclusions: (1) Fluorescence polarization experiments screened out compound 32-2, which has a higher affinity for PSD-95 PDZ2 protein. Its affinity for PSD-95 PDZ2 protein is twice that of ETAV; (2) Glutamate-induced HT22 cell and primary neuron injury models demonstrated that compound 32-2 exerted a significant protective effect at the cellular level; (3) Compound 32-2 significantly reduced the production of reactive oxygen species and the occurrence of cell apoptosis in the glutamate-induced HT22 cell injury model; (4) Compound 32-2 has excellent plasma stability. The half-life of compound 32-2 is greater than 24 h, and 98% of the peptidomimetic remains after 24 h. (5) Compound 32-2 significantly reduced cerebral infarction caused by cerebral ischemia-reperfusion injury, had significant neuroprotective effects, and could be used to prepare drugs for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 , NMDAR-PSD-95-nNOS complex-mediated cell death and its targeted therapeutic strategies; Figure 2 , representative drugs targeting NMDAR-PSD-95-nNOS; Figure 3 , compound design; Figure 4 , Compound separation and FP affinity determination; A. HPLC spectra of two configurations of compound 31; B. FP detection of two configurations of compound 31; C. HPLC spectra of two configurations of compound 32; D. FP detection of two configurations of compound 32; All FP detection results were repeated 4 times; Figure 5 , circular dichroism spectrum of raw material A11 after splitting and calculated CD spectrum of A11-S; Figure 6 , neuroprotective activity of compounds; A. Evaluation of neuroprotective activity of compounds 31-2 and 32-2 in HT22 cells; B. Evaluation of neuroprotective activity of compound 32-2 in primary cortical neurons; Data are expressed as mean ± SEM, 3 repeated experiments, ns>0.05, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 compared with Glu group, #### P <0.0001 compared with the normal group, using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test; Figure 7 , Effects of compound 32-2 on intracellular ROS; A. Representative images of DCFH-DA staining of HT22 cells; B. Flow cytometry detection of intracellular ROS levels; C. Data analysis of intracellular ROS levels; Data are expressed as mean ± SEM, 3 repeated experiments, **** P<0.0001 compared with Glu group, #### P <0.0001 compared with the Normal group, using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test; Figure 8 Effects of compound 32-2 on the expression of Bcl-2 / Bax / Caspase 3 proteins related to the apoptosis pathway; A. Western blot results; B. Bcl-2; C. Bax; D. Caspase 3; Data are expressed as mean ± SEM, repeated 3 times, * P <0.05, ** P <0.01 compared with the Glu group, using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test; Figure 9 , rat plasma stability of compound 32-2; A. HPLC analysis of compound 32-2, ETAV, and NA-1 at different times of plasma incubation; B. Half-life fitting curve, 3 repeated experiments; Figure 10 , membrane permeability evaluation of compound 32-2; Figure 11 , neuroprotective activity of compounds in vivo; A. Flowchart of rat MCAO model; B. Typical brain slices from sham, model, NA-1, and 32-2 groups; C. Statistical analysis of cerebral infarction volume; 5 experiments were repeated. * P <0.05, *** P <0.001, compared with the model group, using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test; Figure 12 , solid phase synthesis of compounds; Figure 13 , unnatural amino acid synthesis; Figure 14 , chiral separation of compounds; Figure 15 , mass spectrum of compound 32-2; Figure 16 , NMR spectrum of compound 32-2. DETAILED DESCRIPTION

[0012] To further illustrate the present invention, the following will be combined with the accompanying drawings and application examples to provide a more comprehensive and systematic description of the present invention. The technical terms involved in the present invention are all common terms that can be understood by those skilled in the art. In addition, unless otherwise specified, the materials and reagents used in the present invention can be purchased through conventional means.

[0013] Example 1 Compound Design ETAV exhibits poor cell permeability, primarily due to the presence of two carboxyl groups. To address this limitation, the carboxyl group at the P3 position (corresponding to the fourth amino acid from the C-terminus of the peptide ligand) was removed to enhance cell permeability. In addition, a hydrophobic group of appropriate size was added to occupy the cavity formed by Val178 and His225 at the P3 position to improve binding affinity ( Figure 3 Position P2 is modified by replacing the hydroxyl group of the threonine with a hydrophobic group or other hydrogen bond donor, thereby interacting with His225 and improving binding affinity. The alanine (Ala) at position P1 serves solely as a linker, ensuring the correct positioning of the P0 and P2 amino acids. Furthermore, position P1 faces a solvent-exposed region surrounded by hydrophobic residues (Lys168, Lys193, and Ile195), making it a suitable position for introducing hydrophobic groups to enhance binding affinity. The residues at position P0 are crucial for binding affinity because they occupy a hydrophobic pocket formed by the GLGF motif (Gly169, Leu170, Gly171, and Phe172). Therefore, unnatural amino acids of appropriate size were introduced to occupy this hydrophobic pocket.

[0014] Example 2 In vitro affinity evaluation of compounds Fluorescence polarization (FP) assay was performed using 5FAM-labeled NR2B (5FAM-KSGYEKLSSIESDV) and GST-PDZ2 proteins to evaluate the binding affinity of the designed ETAV analogs to PDZ2. K i= 39.02 μM) was used as a positive control (Table 1). In the first round of design, for P3 (Structure (II)), cyclized glutamic acid was used to eliminate the polar carboxyl group to improve cell permeability, resulting in compound 2, which was less potent than ETAV but still maintained weak affinity (22% inhibition at 50 μM). To investigate the effects of ring size and chirality on affinity, compounds 3-6 were prepared. Among them, compound 4 showed moderate affinity at 50 μM, with an inhibition rate of 32% (Table 1). In contrast, compounds containing achiral groups (compound 3), D-amino acids (compound 5), or larger rings (compound 6) showed reduced affinity. These results indicate that P3 can accommodate the size of the five-membered ring. Therefore, a five-membered ring was used as a linker, and a phenyl moiety was introduced (compounds 7-13) to form additional interactions with Val178. Most compounds exhibited improved affinity, with compound 7 (Ki = 53.65 μM) showing comparable affinity to ETAV (Ki = 39.02 μM) (Table 1). Substituting the amide with a thioamide (compound 8, Ki = 21.03 μM) increased binding affinity, a substitution that may improve cell permeability. In addition to the methoxy group, the introduction of acetamido groups at the para (compound 9) and meta (compound 10) positions into the phenyl ring to increase hydrogen bonding interactions slightly improved affinity. However, a comparison of compounds 9, 10, and 11 (all showing Ki ≈ 30 μM) suggests that the increased affinity may be attributed to hydrophobic interactions, as compound 11 lacks a hydrogen bond donor. Introduction of a chlorine atom at the para position (compound 12) resulted in a slight decrease in affinity (Ki = 61.63 μM). Furthermore, replacing the chiral carbon with an unsaturated double bond also reduced binding affinity (compound 13, Ki = 100.78 μM). Based on these results, compounds 8 and 11 were selected to further optimize their affinity and polarity.

[0015] (II) Table 1 P3 position modification and affinity

[0016] Note: The inhibition rate is expressed as the average value. K i The data are expressed as mean ± standard deviation, the experiment was repeated 4 times, and ND means not detected.

[0017] While cyclization of the glutamic acid at P3 reduced binding affinity, this modification significantly reduced polarity and still resulted in partial binding. Therefore, this modification was retained, while P2 (Structure (III)) was substituted with an unnatural amino acid (Table 2). As a class I PDZ domain, PSD-95PDZ2 typically has a threonine or serine at the P2 position, with the hydroxyl groups on these two amino acids forming a key hydrogen bond interaction with the conserved His225. To this end, the hydroxyl group was replaced with an amino group, also a hydrogen bond donor, to yield 14. However, 14 did not bind to PDZ2, likely due to electrostatic repulsion between the protonated amino group and the basic amino acid His225. The introduction of a hydrophobic cyclohexyl group (compound 15) generated a hydrophobic interaction with His225, but FP analysis revealed no binding, demonstrating the strict conformational requirements of the P2 position. These findings highlight the importance of the key hydrogen bond between the β-hydroxyl group of threonine and His225. After this round of structural modifications, the threonine at the P2 position was ultimately retained.

[0018] (III) Table 2 Modification and affinity of P2 position

[0019] Note: NA means no binding, the same below.

[0020] At the P1 (structural formula (IV)) position, alanine is the smallest hydrophobic residue that retains affinity. Structural analysis shows that its side chain is oriented in the solvent-exposed region and has no interaction with surrounding amino acid residues. The P1 position is rich in hydrophobic amino acids such as Lys 193, Ile195, and Lys168, which provides the possibility of introducing larger substituents to interact with these amino acids to enhance binding. Therefore, compounds 16-20 with side chains of different sizes were designed (Table 3). Neither the introduction of aromatic rings (compounds 16 and 17) nor saturated alkanes (compounds 18, 19, and 20) improved the affinity. Subsequently, a long-chain small group was introduced to obtain compound 21, which showed weaker binding (50 μM inhibition rate of approximately 20%). In contrast, long-chain large group compounds (compounds 22-26) showed improved affinity, so this solvent-exposed region may be suitable for long-chain large groups. Among them, compound 22 showed moderate affinity ( K i = 35.05 μM) is comparable to ETAV ( K i= 39 μM). Comparison of compounds 22, 23, and 24 revealed that the hydrogen on the glutamine at P1 may be involved in key interactions, as compound 24 lacks hydrogen and loses its activity. In addition, the introduction of methoxy groups at the para position (compound 25) and meta position (compound 26) of the aniline provides more hydrophobic interactions to improve affinity. However, by comparing the affinities of compounds 22, 25, and 26 (the three compounds K i ≈ 30 μM), and it was found that the methoxy group did not form additional hydrophobic interactions with PDZ2. Based on these results, compound 22 with an appropriate molecular weight and acceptable affinity was selected for further optimization.

[0021] (IV) Table 3 Modification and affinity of P1 position

[0022] The side chain group at position P0 (Formula (V)) determines the compound's selectivity for PDZ domains through steric complementarity with the variable hydrophobic pocket in the PDZ family. Introducing a group of appropriate size into the hydrophobic pocket can enhance protein binding, so compounds 27 and 28 containing large substituents were designed (Table 4). Results showed that compound 27 did not bind to PDZ2, while compound 28 exhibited comparable binding to compound 2 (22% inhibition at 50 μM). Furthermore, compound 29, obtained by introducing o-bromophenylalanine at position P0, showed no detectable binding. These results suggest that the hydrophobic pocket at P0 can only accommodate relatively small hydrophobic groups, and therefore, the valine at P0 was retained in subsequent optimization.

[0023] (V) Table 4 Modification of P0 position and affinity

[0024] Based on the results of these four rounds of optimization, the advantageous groups such as thioamide in compound 8, indole in compound 11 and phenylglutamine in compound 22 were combined to design compounds 30-32, which have better affinity and fewer polar groups. K i = 51.45 μM), the affinities of 31 and 32 were improved, increasing by about 2-fold compared with ETAV, as shown in Table 5.

[0025] Table 5 Affinity of compounds 30, 31 and 32

[0026] Compounds 31 and 32 are racemic products. To determine which absolute configuration is more conducive to binding, two configurations of compounds 31 and 32 were purified by HPLC ( Figure 4 ). Discovery 31-2 ( K i = 14.09 μM) has an affinity ratio of 31-1 ( K i = 70.92 μM) was 4 times higher, which was the same as the result of 31. Compound 32-2 ( K i = 19.45 μM) is also higher than 32-1 ( K i = 84.47 μM) was 4 times higher. In order to confirm the absolute configuration of configuration 2, the intermediate product A11 was chemically resolved to obtain A11-S (corresponding to configuration 2) and A11-R (corresponding to configuration 1). The two configurations were detected by circular dichroism spectroscopy and compared with the calculated CD spectrum. Finally, it was confirmed that the configuration 2 of compound 32-2 was S configuration ( Figure 5 Considering that the affinity of the single-configuration compound is better than that of the racemate, compounds 31-2 and 32-2 were selected for further pharmacological evaluation.

[0027] Example 3 Evaluation of Cellular Neuroprotective Activity To evaluate the neuroprotective efficacy of compounds 31-2 and 32-2, the cytotoxicity and neuroprotective evaluation of the compounds were performed on HT22 neuronal cells using CCK-8 assay. Glutamate (5 mM, 24 h) induced severe neuronal damage, reducing cell viability to 35% of the normal group ( P <0.0001). Compared with the glutamate group, compound 31-2 had significant protective activity at 10 μM (47.22%), which was slightly lower than the protective activity of NA-1 at 10 μM (58.05%) ( Figure 6 In addition, compound 32-2 maintained 51.68% and 58.31% of its activity at 1 μM and 10 μM, respectively ( Figure 6 In contrast, the maximum protective efficacy of compound 32-2 was stronger than that of 31-2, so compound 32-2 was selected to further evaluate its protective activity in primary cortical neuronal plexus. Figure 6 As shown in Figure B, compound 32-2 also exhibited significant neuroprotective activity in primary neurons, where the protective activity of 10 μM 32-2 was comparable to that of 10 μM NA-1, and the protective activity of 20 μM 32-2 was superior to that of 10 μM NA-1.

[0028] Example 4 Effect of Compound 32-2 on Intracellular ROS Blocking the formation of the NMDAR-PSD-95-nNOS complex reduces NO production and reduces oxidative stress, and intracellular ROS is a commonly used indicator for detecting oxidative stress. Therefore, the 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescence assay was used to quantify the intracellular ROS level in HT22 cells. Figure 7 As shown, glutamate (5 mM, 24 h) induced an increase in ROS production ( P <0.0001 vs. normal group). However, ROS level was reduced by 80% after treatment with 10 μM 32-2 ( P <0.0001 vs. glutamate group), with an effect comparable to that of the 10 μM NA-1 group. Flow cytometry revealed similar results: glutamate induced a significant increase in intracellular ROS, while treatment with 10 μM 32-2 reduced ROS levels by 90%. These results confirm that 32-2 alleviates oxidative stress by disrupting the NMDAR-PSD-95-nNOS complex by targeting PSD-95 PDZ2.

[0029] Example 5 Effect of Compound 32-2 on Cell Apoptosis Pathway During ischemic stroke, activation of GluN2B-containing NMDARs triggers excitotoxicity and leads to programmed cell death (especially neuronal apoptosis). To investigate whether 32-2 exerts neuroprotective effects through anti-apoptosis, HT22 cells were treated with 10 μM 32-2 and glutamate for 24 hours, and Western blot analysis of key apoptosis regulators was performed. Figure 8 As shown in the figure, the glutamate group increased the expression levels of the pro-apoptotic protein Bax and the apoptosis marker protein Cleaved-caspase 3, and decreased the expression level of the anti-apoptotic protein Bcl2, showing a pro-apoptotic effect. In the group treated with 32-2, the anti-apoptotic Bcl-2 protein was upregulated by 2 times ( P <0.05 vs. glutamate control) was comparable to the 10 μM NA-1 group. In addition, the apoptosis marker protein Cleave-caspase 3 was upregulated by 55% ( P <0.05 vs. glutamate control), the pro-apoptotic protein Bax was downregulated by approximately 50% ( P <0.05 vs. glutamate control) showed an effect comparable to that of the 10 μM NA-1 group, demonstrating an anti-apoptotic effect. All results were quantified by normalization to tubulin, confirming that 32-2 exerts its neuroprotective effects by inhibiting intracellular apoptosis.

[0030] Example 6 Rat Plasma Stability Test of Compound 32-2 Plasma stability is a key parameter for evaluating the drugability of peptides and peptidomimetics. Due to enzymatic cleavage by proteases and peptidases, peptide-based compounds have low stability in the blood, especially linear peptides, which have a short half-life in plasma. Therefore, in order to evaluate the plasma stability of 32-2, an in vitro stability experiment was conducted in rat plasma at 37°C for 24 hours. The degradation of the test compound over time was monitored by using RP-HPLC analysis. Figure 9 As shown in the results, ETAV had a half-life of 1.17 h and was completely degraded by 8 h. NA-1 had a half-life of 2.02 h and was completely degraded by 8 h. However, compound 32-2 had a half-life of more than 24 h, with 98% of the peptide still remaining after 24 h. These results indicate that compound 32-2 has excellent plasma stability and also confirm that the introduction of unnatural amino acids into peptides can enhance stability.

[0031] Example 7 Evaluation of membrane permeability of compound 32-2 Penetrating the blood-brain barrier is a very important property of drugs for treating neurological diseases. To evaluate the ability of compound 32-2 to penetrate the blood-brain barrier, we used MDCK-MDR1 cells to simulate the blood-brain barrier, detected the amount of compound on both sides of the cell, and calculated the apparent permeability coefficient of the compound. Figure 10 As shown, the apparent permeability coefficient of compound 32-2 is 12.9±1.3×10 -6 cm / s is a highly permeable compound that has the ability to penetrate the blood-brain barrier.

[0032] Example 8 Neuroprotective activity of compounds in vivo The MCAO model is a classic model for simulating ischemic stroke, and the cerebral infarction volume is used as an evaluation indicator. Figure 11 As shown in Figure 2, MCAO injury caused a larger cerebral infarction volume (32.64%, p >0.05 vs. sham operation group). After the rats were injected with 8 mg / kg compound 32-2 through the tail vein, the cerebral infarction area was significantly reduced (25.28%, p >0.05 vs. model group). Considering that ETAV has no neuroprotective activity on cells, animal experiments were not performed, and the positive drug NA-1 was selected for comparison. The cerebral infarction volume of compound 32-2 was slightly lower than that of the NA-1 group (21.71%, p >0.001 vs. model group). These data indicate that compound 32-2 has significant neuroprotective effects in vivo.

[0033] Based on Examples 1-7, the present invention synthesized 31 peptidomimetics using standard Fmoc solid-phase synthesis technology. After in vitro fluorescence polarization experiments and cell protection activity experiments, the optimal compound 32-2 was screened. A series of cell and animal experiments on the optimal compound yielded the following conclusions: (1) Fluorescence polarization experiments screened out compound 32-2, which has a higher affinity for PSD-95 PDZ2 protein. Its affinity for PSD-95 PDZ2 protein is twice that of ETAV. (2) Glutamate-induced HT22 cell and primary neuron injury models demonstrated that compound 32-2 exerted a significant protective effect at the cellular level; (3) Compound 32-2 significantly reduced the production of reactive oxygen species and the occurrence of cell apoptosis in the glutamate-induced HT22 cell injury model; (4) Compound 32-2 significantly reduced cerebral infarction caused by cerebral ischemia-reperfusion injury.

[0034] The experimental part of the embodiment of the present invention is as follows: Compound Synthesis 1.1 Solid Phase Synthesis All peptoids were synthesized via Fmoc-based manual solid-phase synthesis (SPPS) using Wang resin (100-200 mesh, 1 mmol / g, 1% DVB, GL Biochem Ltd.) and Fmoc-protected amino acids purchased from Shanghai Bidex Pharmaceuticals. Some unnatural amino acids were obtained by chemical synthesis. Unless otherwise noted, all reagents and chemicals were of ACS grade or higher and used without further purification. 0.05 g (0.05 mM) Wang resin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 4-dimethylaminopyridine (DMAP), and an Fmoc-protected amino acid (resin / amino acid / EDCI / HOBT / DMAP = 1:3:3:3:0.1) were added and reacted in DCM for 12 h. The solution was then discarded and the resin was blocked with acetic anhydride and pyridine dissolved in DCM for 2 h (resin / acetic anhydride / pyridine = 1:5:5). After loading the first amino acid, the amino acid synthesis steps were repeated, as shown in Figure 2. Figure 12As shown: (1) Fmoc deprotection with 20% piperidine in DMF (2 × 15 min, 10 mL each); (2) washing with DMF (3 × 5 min, 10 mL each) and monitoring by ninhydrin test; (3) Fmoc-amino acid or unnatural amino acid (3 eq.), O-(benzotriazol-1-yl)- N , N , N ′, N ′-Tetramethyluronium hexafluoroammonium phosphate (O-Benzotriazole- N , N , N ', N '-tetramethyl-uronium-hexaf , HBTU) (3 eq.), HOBT (3eq.) and N,N -Diisopropylethylamine ( N , N -Diisopropylethylamine (DIPEA) (6 eq.) was activated in dry DMF for 10 min and then added to the resin and shaken at 25 °C for 1 h; (4) the reaction solution was discarded and the resin was washed with DMF (3 × 5 min, 10 mL each time) and monitored by ninhydrin test. Finally, the resin was treated with cutting solution (5% water: 5% triisopropylsilane: 90% trifluoroacetic acid) for 3 h to cleave the final peptide from the resin. The crude product was analyzed by HPLC and purified by semi-preparative RP-HPLC. After semi-preparative purification, the product peak was collected and lyophilized to obtain the desired peptide.

[0035] In particular, the synthesized compound 32-2 was subjected to mass spectrometry and nuclear magnetic resonance detection, and the results were as follows: Figure 15 and 16 , which are consistent with the theoretical predictions.

[0036] 1.2 Synthesis of intermediates (1) Synthesis of intermediate product A1-5 Add 88 mg (1 mmol, 1 eq.) of itaconic acid and the corresponding amine (1 mmol, 1 eq.) to water and stir at 100 °C for 16 h. After the reaction is complete, add 10 mL of 1 N NaOH to the reaction mixture and filter out the residual amine. The filtrate is acidified with 1 N HCl to pH 2-3 and filtered to obtain solid A1-5, which is then purified without further purification. Figure 13 .

[0037] (2) Synthesis of intermediate product A6-10 Fmoc-L-glutamic acid-1-allyl ester (1 eq.), EDC·HCl (1.3 eq.), HOBt (1.3 eq.) and DIPEA (3 eq.) were added to DMF and stirred for 10 min, followed by the addition of the corresponding amine (1 eq.) and stirring at room temperature overnight. After the reaction, the reaction mixture was poured into water and extracted three times with 10 mL of ethyl acetate (EA). The organic phase was collected and washed with saturated brine, dried over anhydrous MgSO4 and concentrated under reduced pressure. Purification by column chromatography gave the desired compound A6a-10a. A6a-10a (1 eq.), tetrakis(triphenylphosphine)palladium (0.1 eq.) and phenylsilane (3 eq.) were added to DCM and stirred overnight. After the reaction, the mixture was filtered and the filter cake was washed with DCM to give compound A6-10, which was used in the next reaction without further purification, as shown in FIG. Figure 13 .

[0038] (3) Synthesis of intermediates A11 and A12 A1 or A5 (1 eq.) was dissolved in methanol, EDC·HCl (1.3 eq.), DMAP (0.1 eq.), and DIPEA (3 eq.) and stirred at 50°C overnight. The reaction mixture was added with water and extracted three times with 10 mL of EA. The organic phase was collected, washed with saturated brine, dried over anhydrous MgSO₄, and concentrated under reduced pressure. The concentrate was purified by column chromatography to yield the desired compound A11a or A12a. A11a or A12a (1 eq.) and Lawson's reagent (0.6 eq.) were added to dioxane and refluxed overnight under N₂ protection. The reaction mixture was rotary evaporated, and water was added to the residue, which was extracted three times with 10 mL of EA. The organic layer was concentrated and purified by column chromatography using ethyl acetate:petroleum ether (1:4) to yield A11b or A12b. Dissolve A11b or A12b (1 eq.) in MeOH:H2O (1:1), add NaOH (1.2 eq.), and stir at room temperature overnight. Evaporate the reaction mixture under reduced pressure and adjust the pH to 1-2 with 2N HCl. Extract three times with 10 mL of ethyl acetate (EA), combine the organic phases, and concentrate under reduced pressure. The crude product can be used in the next reaction without further purification, as described in the following example. Figure 13 .

[0039] (4) Chiral resolution of intermediate A11 Intermediates A1-A5, A11, and A12 all have one chiral center, and the synthesized product is a racemate. To determine which chiral configuration is more favorable for activity, compound A11 was chemically resolved. Different chiral amines were used as resolving agents to reflux with the carboxyl group to form diastereomeric organic salts. The diastereomeric organic salts were then cooled and crystallized to obtain a single non-commutative isomer organic salt due to the difference in solubility of the diastereomeric organic salts in the solvent. The precipitated solid and the filtrate were alkalized and filtered to remove the chiral amine resolving agent. The filtrate was then acidified to obtain a carboxyl compound with a single configuration, such as Figure 14 .

[0040] Analysis and purification of peptides HPLC analysis conditions for peptides were as follows: the chromatographic column was Positisil OSD-P reversed-phase C18 (analytical type) 5 μm, 4.6 mm × 250 mm; the sample solvents were deionized water and acetonitrile; the aqueous phase was deionized water containing 0.05% TFA, and the organic phase was chromatographic grade ACN containing 0.05% TFA; the elution time was 1-27 min, with a gradient of 5%-40% ACN; the wavelength was 220 nm; the column temperature was 25 ± 2 °C; and the flow rate was set at 1 mL min -1 ; The injection volume was 20 μL.

[0041] HPLC purification conditions for the peptide were as follows: a Positisil reverse-phase C18 (semi-preparative) 5 μm column, 10.0 mm × 250 mm; deionized water and acetonitrile were used as sample solvents; the aqueous phase was deionized water containing 0.05% TFA, and the organic phase was chromatographic-grade ACN containing 0.05% TFA; the elution time was 1–27 min, and the acetonitrile gradient was 5%–40% ACN; the wavelength was 220 nm; the column temperature was 25 ± 2 °C; and the flow rate was set at 3 mL min. -1 ; The injection volume was 2 mL.

[0042] 3. Fluorescence Polarization Assay (FP Assay) Fluorescence polarization assays were performed in TC-treated 384-well black opaque plates. The final volume per well was 50 μL, and the dilution buffer was Tris buffer (150 mM NaCl, 10 mM Tris, pH 7.4). To measure the fluorescence of the fluorescent peptide (5-FAM-NR2B) and GST-PDZ2 155-249 The binding affinity between GST-PDZ2 and PDZ2 was determined by saturation binding experiments. 155-249 Add to a fixed concentration of 5-FAM-NR2B (50 nM), incubate at room temperature for 20-30 minutes, and measure the FP of the sample on an ultrasensitive full-wavelength microplate detector. Adjust the g factor to make sure that the sample does not contain GST-PDZ2. 155-249The FP value of 50 nM probe was 20 mP, and a saturation binding curve was obtained. Competitive binding experiment: Increasing concentrations of compound were added to a fixed concentration of 5-FAM-NR2B (50 nM) and GST-PDZ2 155-249 The samples were incubated with 8 μM at room temperature for 30 min, and the FP of the samples was measured using an ultrasensitive full-wavelength microplate reader. All graphs were generated using GraphPad 7.0 (GraphPad Software, San Diego, CA), and all values ​​reported are the average of at least three separate experiments.

[0043] 4. Neuroprotective activity of compound 32-2 on HT22 cells HT-22 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were grown to 80-90% confluence for passage or seeding and seeded at a density of 5,000 cells / well in 96-well plates for 24 hours. Cells were then incubated with various concentrations of the compound (0.1, 1, and 10 μM) for 2 hours, followed by the addition of glutamate to a final concentration of 5 mM. After 24 hours of incubation with the compound, 10 μL of CCK-8 reagent was added, and absorbance was measured at an optical density (OD) of 450 nm using a microplate reader.

[0044] Compound 32-2 was tested for its neuroprotective activity against primary neurons. Primary cortical neurons were isolated from the cerebral cortex of 18-day-old SD rat embryos. Cerebral hemispheres were dissected, and the pia mater and blood vessels were removed under a microscope. The cells were then digested with 1.25% trypsin at 37°C for 15-30 minutes. Digestion was terminated with 10% fetal bovine serum, and the cells were treated with DNase I for 3 minutes. The cell pellet was resuspended in Neurobasal medium containing 10% B27, 1% anti-antibody, and GlutaMAX, and then filtered through a 40 μm cell strainer. The collected cells were seeded at a density of 106 cells / mL in a 96-well plate coated with 0.01 mg / mL poly-D-lysine. The next day, the medium was completely replaced with Neurobasal medium containing B27, anti-antibody, and GlutaMAX. Half of the medium was replaced every two days. After 9 days of culture, the neurons were incubated with various concentrations of the compound (0.1, 1, and 10 μM) for 2 hours, followed by the addition of glutamate to a final concentration of 25 μM. After 20 h, 10 μL of CCK-8 reagent was added and incubated for 3 h, and the absorbance was measured at the optical density (OD) of 450 nm using a microplate reader.

[0045] Effect of compound 32-2 on intracellular ROS HT-22 cells were cultured at a rate of 2 × 10 5 Cells were seeded into 6-well plates at a density of 10 cells / well. After 24 hours, the culture medium was discarded and the cells were incubated with 10 μM compound diluted in DMEM for 2 hours. Glutamate was then added to a final concentration of 5 mM and incubated for 24 hours. Intracellular ROS levels were measured using the DCFH-DA fluorescent probe. DCFH-DA was diluted in DMEM to a final concentration of 10 μM. After removing the cell culture medium, an appropriately diluted DCFH-DA solution was added and incubated at 37°C in the dark for 20 minutes. The DCFH-DA solution was discarded and the cells were washed three times with PBS to remove any unincorporated DCFH-DA. Intracellular ROS levels were measured using an inverted fluorescence microscope or by flow cytometry using harvested cells.

[0046] Effect of Compound 32-2 on the Expression of Bcl-2 / Bax / Caspase 3, Proteins Related to the Apoptosis Pathway. Cell pretreatment was performed in the same manner as in the ROS quantification experiment. HT22 cells were first pretreated with 10 μM compound diluted in DMEM for 2 h, followed by incubation with 5 mM glutamate (Glu) for 24 h. The culture medium was discarded, and the cells were rinsed three times with ice-cold PBS. 250 μL of RIPA lysis buffer containing 1 mM PMSF was then added to each well of a 6-well plate. The plate was gently shaken at 4°C for 30 min to ensure complete contact of the lysis buffer with the cells. The HT22 cells were scraped off with a cell scraper and transferred to a 1.5 mL microcentrifuge tube. The cells were centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant containing the total protein extract was collected. The extracted protein concentration was determined using a BCA protein assay kit. To 100 μL of protein sample, add 20 μL of 5x SDS-PAGE loading buffer. Denature the sample by boiling it in a 100°C metal bath for 15 minutes. Prepare a 15% separating gel using the SDS-PAGE gel preparation kit and let it solidify at room temperature for 30 minutes. Then prepare a 5% stacking gel and let it solidify before use. Add running buffer and load an equal amount of protein per well, with a total loading volume of <30 μL. Add 5 μL of protein marker to each gel. Run the top stacking gel at 80 V. After 30 minutes, adjust the voltage to 120 V and stop when the protein marker bands are well separated. Remove the gel and transfer to a membrane. Pre-soak the PVDF membrane in methanol for 1 minute. Transfer the membranes in the following order: cathode carbon plate (black) + sponge + filter paper + gel + PVDF membrane + filter paper + sponge + anode carbon plate (red). Clamp the membranes and place them in a tank. Add transfer buffer and transfer the membranes at 300 V for 1 hour. After transfer, remove the PVDF membrane and wash it in TBST buffer for 15 minutes. Block the membrane with 4% BSA in TBST buffer for 2 hours at room temperature. After blocking, wash the membrane three times with TBST buffer for 10 minutes each. Excise the membrane for the target protein and internal control protein based on their molecular weight. Primary antibodies against Cleave-caspase 3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and internal control Tubulin (1:1000) were diluted in TBST buffer and applied to the membrane, followed by overnight incubation at 4°C. Wash the membrane three times with TBST for 10 minutes each. Incubate the membrane with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000) diluted in TBST buffer for 1 hour at room temperature. Wash the membrane three times with TBST for 10 minutes each. Finally, mix equal volumes of Solution A and Solution B of the Ultrasensitive ECL Chemiluminescent Detection Kit, immerse the membrane in the mixture, and then visualize it on an automated chemiluminescent gel imaging analyzer.

[0047] Plasma Stability of Compound 32-2: A 10 mM stock solution of the compound was added to rat plasma (anticoagulated with sodium heparin) to a final concentration of 300 μM and incubated at 37°C. Samples (150 μL) were taken at different time points and quenched with 10% trichloroacetic acid aqueous solution (300 μL), vortexed, incubated at 4°C for 15 minutes, and then centrifuged at 14,000 g for 5 minutes. The supernatant was filtered through a 0.22 μm filter and analyzed by HPLC at a wavelength of 220 nm. Quantification was based on the peak area relative to time 0.

[0048] Membrane permeability evaluation of compound 32-2 was performed by incubating MDCK-MDR1 cells in complete medium (10% FBS, 1% NEAA, 1% double-antibody-containing DMEM) at 37°C and 5% CO2 in culture flasks. After reaching 70.0% to 80.0% confluency, cells were dissociated with 0.25% EDTA-trypsin, counted, and diluted to 1,000,000 cells / mL / well in complete medium. Cells were seeded in Transwell-96 well plates and cultured in a CO2 incubator, with complete medium regularly replaced for 4-8 days. Cells were washed three times with transporter buffer and preincubated in a CO2 incubator for 30 minutes. Transmembrane apparent resistance was measured using a resistance meter. 150 μL of prewarmed drug solution (final concentration 10 μM) was added to each well on side A. 50 μL of the solution was removed from side A as T0. 300 μL of prewarmed transporter buffer was added to each well on side B. Incubate the Transwell-96 plate on a thermostatic shaker (37°C, 40 rpm) for 120 minutes. Remove all liquid from both sides A and B as the T120 sample (100 μL of liquid from side B is used for post-test quality control—Lucifer Yellow permeability). For the remaining samples, a portion of the solution is added to the stop solution (methanol containing the internal standard tolbutamide) in a specific proportion. After the stop, shake the plate on a shaker and centrifuge at 4°C, 4000 rpm for 20 minutes. The supernatant is collected or diluted to a specific proportion for LC-MS / MS analysis.

[0049] Animal activity evaluation of compound 32-2 Focal cerebral ischemia was induced in male SD rats by intraluminal middle cerebral artery occlusion (MCAO). Male SD rats (220−280 g) were randomly divided into 4 groups (n = 5 per group): (1) sham group, (2) model group, (3) NA-1 group, and (4) 32-2 group. Mice were anesthetized with chloral hydrate (400 mg / kg, ip). The left common carotid artery was exposed along the midline of the neck using surgical scissors, and the left internal carotid artery and external carotid artery were isolated. A suture was inserted into the external carotid artery through the opening of the external carotid artery, introduced into the left internal carotid artery, and advanced 20-21 mm past the carotid bifurcation until slight resistance was felt. After 1.5 h of occlusion, the suture was removed (reperfusion), and the drug (8 mg / kg, iv) was administered 2 h after reperfusion. The body temperature of the rats was maintained at 37°C ± 0.5°C throughout the experiment. Rats were killed 24 h after reperfusion, and the brains were stored at -80°C for 15 min. The brains were cut into seven coronal sections with a thickness of 1.0-2.0 mm and stained in 2% TTC solution at 37°C for 30 min.

Claims

1. A PSD-95 PDZ2 peptidomimetic inhibitor characterized by: Its structural formula is shown in formula (I); （Ⅰ）。 2. Use of the PSD-95 PDZ2 peptidomimetic inhibitor according to claim 1 in the preparation of a drug for treating ischemic stroke.

3. Use of the PSD-95 PDZ2 peptidomimetic inhibitor according to claim 1 in the preparation of neuroprotective drugs.

4. A drug, characterized in that: Comprising a therapeutically effective amount of acetate, hydrochloride or other pharmaceutically acceptable salt form of the PSD-95 PDZ2 peptidomimetic inhibitor according to claim 1.

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