PSD-95 PDZ2 peptide-mimicking inhibitors and their applications

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a PSD-95 PDZ2 peptidomimetic inhibitor and application thereof. A series of compounds are designed and synthesized by taking a tetrapeptide ETAV as a template, a structure-activity relationship of the compounds with PSD-95 PDZ2 is systematically studied, and an optimal compound 32-2, i.e., the PSD-95 PDZ2 peptidomimetic inhibitor, is screened, a structural formula of which is shown as formula (I), the PSD-95 PDZ2 peptidomimetic inhibitor has very excellent plasma stability, and exhibits significant neuroprotective activity in in-vivo and in-vitro biological activity evaluation, significantly reduces cerebral infarction caused by cerebral ischemia-reperfusion injury, and can be used for preparing a drug for treating ischemic stroke. (I).

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to PSD-95 PDZ2 peptide inhibitors and their application in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Ischemic stroke is a major public health problem, causing high rates of mortality and disability in adults and is the second leading cause of death worldwide. During cerebral ischemia, excessive release of glutamate from the synaptic cleft overactivates the NMDAR and allows Ca2+ to be released into the brain. 2+ A large influx of PSD-95 into postsynaptic neurons occurs, and PSD-95 binds to the C-terminus of nNOS and NMDAR via the first and second PDZ domains, particularly 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, direct inhibition of nNOS or NMDAR inhibitors can cause various side effects (such as motor and memory impairments), and such drugs are not clinically approved. In contrast, PSD-95, as a scaffold protein containing three PDZ domains, can bind to NMDAR and nNOS within the cell membrane to form toxic complexes. Inhibitors designed targeting the first or second PDZ domain 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, thus the development of PSD-95 PDZ2 inhibitors has significant clinical value.

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

[0004] AVLX-144 is a dimer peptide candidate drug formed using the C-terminal 5 amino acids of the NR2B isoform of NMDAR as a template and polyethylene glycol as a linker. It simultaneously targets PDZ1 and PDZ2 of PSD-95 to inhibit the formation of the NMDAR-PSD-95-nNOS complex. Compared with NA-1, it has better stability and in vitro protein affinity. It has already completed Phase I clinical trials overseas.

[0005] Although reported PSD-95 PDZ peptide inhibitors have shown significant translational potential, their poor in vivo stability and resulting druggability limitations restrict their application. Small molecule drugs can overcome this limitation, making the development of small molecule inhibitors targeting PSD-95 PDZ2 of great significance. However, the development of small molecules targeting the PDZ domain is extremely challenging, and few inhibitors have been reported in the literature. To date, no small molecule inhibitors of PSD-95 PDZ2 with low micromolar activity have been developed. Peptides, as intermediates between peptides and small molecules, provide a key approach for the conversion of peptides into small molecules. Summary of the Invention

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

[0007] The technical solution of the present invention is as follows:

[0008] The PSD-95 PDZ2 peptide inhibitor described in this invention has the structural formula shown in formula (Ⅰ) and is designated as 32-2.

[0009] (I)

[0010] This invention provides the application of the PSD-95 PDZ2 peptide inhibitor in the preparation of drugs for treating ischemic stroke and in the preparation of neuroprotective drugs.

[0011] The present invention also provides a medicament comprising a therapeutically effective amount of the acetate, hydrochloride or other pharmaceutically acceptable salt form of the PSD-95 PDZ2 peptide inhibitor.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] Using the tetrapeptide ETAV as a design template, this invention synthesized 31 peptide-like compounds using standard Fmoc solid-phase synthesis technology. After in vitro fluorescence polarization experiments and cell protection activity experiments, the optimal compound 32-2 was selected, which is the PSD-95 PDZ2 peptide-like inhibitor described in this invention. A series of cell and animal experiments on the optimal compound yielded the following conclusions:

[0014] (1) Fluorescence polarization experiments screened out compound 32-2, which has a higher affinity for PSD-95 PDZ2 protein than ETAV.

[0015] (2) Glutamate-induced HT22 cell and primary neuron injury models demonstrate that compound 32-2 exerts a significant protective effect at the cellular level;

[0016] (3) Compound 32-2 significantly reduced the production of reactive oxygen species and the occurrence of apoptosis in the glutamate-induced HT22 cell injury model;

[0017] (4) Compound 32-2 has excellent plasma stability. The half-life of compound 32-2 is greater than 24 h, and 98% of the peptide remains after 24 h.

[0018] (5) Compound 32-2 significantly reduced cerebral infarction caused by cerebral ischemia-reperfusion injury and has a significant neuroprotective effect. It can be used to prepare drugs for the treatment of ischemic stroke. Attached Figure Description

[0019] Figure 1 NMDAR-PSD-95-nNOS complex-mediated cell death and its targeted therapy strategies;

[0020] Figure 2 Representative drugs targeting NMDAR-PSD-95-nNOS;

[0021] Figure 3 Compound design;

[0022] Figure 4 Compound resolution and FP affinity determination; A. HPLC chromatograms of two configurations of compound 31; B. FP detection of two configurations of compound 31; C. HPLC chromatograms of two configurations of compound 32; D. FP detection of two configurations of compound 32; All FP detection results were repeated 4 times;

[0023] Figure 5 The circular dichroism spectrum of raw material A11 after separation and the calculated CD spectrum of A11-S;

[0024] Figure 6 The neuroprotective activities of the compounds; A. Evaluation of the neuroprotective activities of compounds 31-2 and 32-2 in HT22 cells; B. Evaluation of the neuroprotective activity of compound 32-2 in primary cortical neurons; Data are expressed as mean ± SEM, 3 replicates, ns > 0.05, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 compared to the Glu group, #### P<0.0001 compared with the normal group, one-way ANOVA and Dunnett's multiple comparison test were used;

[0025] Figure 7 The effects of compound 32-2 on intracellular ROS; A. Representative images of HT22 cells stained with DCFH-DA; B. Flow cytometry analysis of intracellular ROS levels; C. Data analysis of intracellular ROS levels; Data are expressed as mean ± SEM, with 3 replicates. **** P <0.0001 compared to the Glu group, #### P <0.0001 compared with the Normal group, one-way ANOVA and Dunnett's multiple comparison test were used;

[0026] Figure 8 The effect of compound 32-2 on the expression levels of apoptosis-related proteins Bcl-2 / Bax / Caspase 3; A. Western blot results; B. Bcl-2; C. Bax; D. Caspase 3; Data are expressed as mean ± SEM, with 3 replicates. * P <0.05, ** P <0.01 compared with the Glu group, one-way ANOVA and Dunnett's multiple comparison test were used;

[0027] Figure 9 A. Rat plasma stability of compound 32-2; B. HPLC analysis of compound 32-2, ETAV, and NA-1 at different incubation times in plasma; C. Half-life fitting curves, with three repeated experiments.

[0028] Figure 10 Evaluation of membrane permeability of compound 32-2;

[0029] Figure 11 A. Neuroprotective activity of the compound in vivo; B. Flowchart of the rat MCAO model; C. Typical brain slices of the sham-operated group, model group, NA-1 group, and 32-2 group; D. Statistical analysis of cerebral infarction volume; 5 experimental replicates. * P <0.05, *** P <0.001, compared with the model group, one-way ANOVA and Dunnett multiple comparison test were used;

[0030] Figure 12 Solid-phase synthesis of compounds;

[0031] Figure 13 Non-natural amino acid synthesis;

[0032] Figure 14 Chiral resolution of compounds;

[0033] Figure 15 Mass spectrum of compound 32-2;

[0034] Figure 16 The nuclear magnetic resonance spectrum of compound 32-2. Detailed Implementation

[0035] To further clarify this invention, the following description, in conjunction with the accompanying drawings and application examples, will provide a more comprehensive and systematic overview. All technical terms used in this invention are common terms that can be understood by those skilled in the art. Furthermore, unless otherwise specified, the materials and reagents used in this invention can be purchased through conventional means.

[0036] Example 1 Compound Design

[0037] ETAV exhibits poor cell permeability, primarily due to the presence of two carboxyl groups. To address this limitation, the carboxyl group at the P3 site (corresponding to the 4th amino acid counted at the C-terminus of the peptide ligand) was removed to enhance cell permeability. Furthermore, appropriately sized hydrophobic groups were used to occupy the cavity formed by Val178 and His225 at the P3 position to improve binding affinity. Figure 3 The binding affinity is enhanced by modifying position P2 with a hydrophobic group or other hydrogen bond donor to interact with His225. Alanine (Ala) at position P1 acts only as a linker, ensuring the correct orientation of amino acids P0 and P2. Furthermore, position P1 faces a solvent-exposed region surrounded by hydrophobic residues (Lys168, Lys193, and Ile195), making it suitable 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, an appropriately sized non-natural amino acid is introduced to occupy this hydrophobic pocket.

[0038] Example 2: Evaluation of the in vitro affinity of the compound

[0039] Fluorescence polarization (FP) assays were performed using 5FAM-labeled NR2B (5FAM-KSGYEKLSSIESDV) and GST-PDZ2 protein to assess the binding affinity of the designed ETAV analog to PDZ2, using ETAV ( K i= 39.02 μM) as a positive control (Table 1). In the first round of design, for P3 (structure (II)), cyclized glutamate eliminated the polar carboxyl group to improve cell permeability, yielding compound 2, which had lower potency 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 some affinity at a concentration of 50 μM, with an inhibition rate of 32% (Table 1). In contrast, compounds containing achiral groups (compound 3), D-type amino acids (compound 5), or larger rings (compound 6) showed reduced affinity. These results indicate that P3 can accommodate the size of a five-membered ring; therefore, a five-membered ring was used as a linker, and phenyl fragments (compounds 7-13) were introduced onto it to form additional interactions with Val178. Most compounds exhibited better 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; this substitution can enhance cell permeability. Introducing para (compound 9) and meta (compound 10) acetamino groups into the benzene ring, in addition to the methoxy group, to increase additional hydrogen bonding interactions, resulted in a slight increase in affinity. However, comparisons of compounds 9, 10, and 11 (all showing Ki ≈ 30 μM) suggest that the increased affinity may be attributed to hydrophobic interactions, as compound 11 lacks a hydrogen bond donor. Introducing a chlorine atom at the para position (compound 12) resulted in a slight decrease in affinity (Ki = 61.63 μM). Simultaneously, substituting the chiral carbon with an unsaturated double bond also decreased 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.

[0040] (II)

[0041] Table 1. P3 Position Modification and Affinity

[0042]

[0043] Note: Inhibition rate is expressed as an average value. K i Data are expressed as mean ± standard deviation, with 4 replicates. ND indicates no detection, and the same applies below.

[0044] Cyclation of glutamate in P3 reduces binding affinity, but this modification significantly reduces polarity while still allowing for partial binding. Therefore, this modification was retained, and non-natural amino acid substitutions were performed on P2 (structure (III)) (Table 2). As a class I PDZ domain, PSD-95PDZ2 typically has threonine or serine at its P2 position, and these amino acids interact with the conserved His225 via a key hydrogen bond through their hydroxyl groups. To this end, the hydroxyl group was replaced with an amino group, also a hydrogen bond donor, to obtain 14. However, 14 did not bind to PDZ2, possibly due to electrostatic repulsion between the protonated amino group and the basic amino acid His225. A hydrophobic interaction was achieved by introducing a hydrophobic cyclohexyl group (compound 15) with His225, but FP results showed no binding, indicating that the P2 position has strict requirements on the chemical conformation. These findings highlight the importance of the key hydrogen bond between the threonine β-hydroxyl group and His225. After this round of structural modifications, threonine at the P2 position was ultimately retained.

[0045] (III)

[0046] Table 2 Modifications and Affinity at P2 Position

[0047]

[0048] Note: NA indicates no binding, the same applies below.

[0049] At site P1 (structural formula (Ⅳ)), alanine is the smallest hydrophobic residue retaining affinity. Structural analysis shows that its side chain is oriented in the solvent-exposed region and does not interact with surrounding amino acid residues. The area around site P1 is rich in hydrophobic amino acids such as Lys 193, Ile195, and Lys168, which provides the possibility of introducing larger substituents to enhance the interaction with these amino acids. Therefore, compounds 16-20 with side chains of different sizes were designed (Table 3). The introduction of aromatic rings (compounds 16 and 17) or saturated alkanes (compounds 18, 19, and 20) did not improve affinity. Subsequently, the introduction of long-chain small groups yielded compound 21, which exhibited weak binding (inhibition rate of approximately 20% at 50 μM). In contrast, long-chain large-group compounds (compounds 22-26) showed improved affinity, suggesting that the 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 atom on glutamine at P1 may be involved in a key interaction, as compound 24 lacks hydrogen and is therefore inactive. Furthermore, introducing methoxy groups at the para (compound 25) and meta (compound 26) positions of aniline provides more hydrophobic interactions to improve affinity. However, comparing the affinity among compounds 22, 25, and 26 (the three compounds...) K i (≈ 30 μM), it was found that the methoxy group did not exhibit additional hydrophobic interactions with PDZ2. Based on these results, compound 22 with a suitable molecular weight and acceptable affinity was selected for further optimization.

[0050] (IV)

[0051] Table 3 Modifications and Affinity at P1 Position

[0052]

[0053] The side chain group at position P0 (structural formula (V)) determines the selectivity of the compound for the PDZ domain through its spatial complementarity with the variable hydrophobic pocket in the PDZ family. Introducing appropriately sized groups into the hydrophobic pocket can make the binding of the compound to the protein more compact; therefore, compounds 27 and 28 (Table 4) containing large substituents were designed. The results showed that compound 27 did not bind to PDZ2, while compound 28 had a comparable binding affinity to compound 2 (inhibition rate of 22% at 50 μM). Furthermore, introducing o-bromophenylalanine at P0 yielded compound 29, which did not show any binding. These results indicate that the hydrophobic pocket at P0 can only accommodate small hydrophobic groups; therefore, valine at P0 was retained in subsequent optimizations.

[0054] (V)

[0055] Table 4 Modifications and Affinity at P0 Position

[0056]

[0057] Based on the results of these four rounds of optimization, compounds 30-32 were designed by combining dominant groups such as thioamide in compound 8, indole in compound 11, and phenylglutamine in compound 22. These compounds exhibit good affinity and have fewer polar groups. Except for compound 30 ( K i = 51.45 μM), and the affinity for 31 and 32 was improved, increasing by about 2 times compared to ETAV, as shown in Table 5.

[0058] Table 5. Affinities of compounds 30, 31, and 32

[0059]

[0060] Compounds 31 and 32 are racemic products. To determine which absolute configuration is more favorable for binding, HPLC was used to purify both configurations of compounds 31 and 32. Figure 4 ). Found 31-2 ( K i = 14.09 μM) has an affinity for 31-1 ( K i = 70.92 μM) is 4 times higher, the same as the result of 31, compound 32-2 ( K i = 19.45 μM) also has a higher affinity than 32-1 ( K i = 84.47 μM) is 4 times higher. 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). Circular dichroism spectroscopy was performed on these two configurations and compared with the calculated CD spectrum. Finally, it was confirmed that configuration 2 of compound 32-2 is the S configuration ( = 84.47 μM). Figure 5 Considering that the affinity of the single-configuration compound is better than that of the racemic version, compounds 31-2 and 32-2 were selected for further pharmacological evaluation.

[0061] Example 3 Evaluation of cellular neuroprotective activity

[0062] To evaluate the neuroprotective efficacy of compounds 31-2 and 32-2, the cytotoxicity and neuroprotective effects of these compounds were assessed in HT22 neurons using a 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 showed significant protective activity at 10 μM (47.22%), slightly lower than the NA-1 protective activity at 10 μM (58.05%). Figure 6 (A). Furthermore, compound 32-2 retained 51.68% and 58.31% of its activity at 1 μM and 10 μM, respectively. Figure 6 (A). In comparison, compound 32-2 exhibited stronger maximum protective potency than 31-2; therefore, compound 32-2 was selected for further evaluation of its protective activity in primary cortical neural clouds. Figure 6 As shown in Figure B, compound 32-2 also exhibited significant neuroprotective activity in primary neurons. The protective activity of 10 μM 32-2 was comparable to that of 10 μM NA-1, while the protective activity of 20 μM 32-2 was superior to that of 10 μM NA-1.

[0063] Example 4: Effects of compound 32-2 on intracellular ROS

[0064] Blocking the formation of the NMDAR-PSD-95-nNOS complex reduces NO production and oxidative stress, and intracellular ROS is a commonly used indicator for detecting oxidative stress. Therefore, the intracellular ROS level in HT22 cells was quantified using a 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescence assay. Figure 7 As shown, glutamate (5 mM, 24 h) induced an increase in ROS production ( P <0.0001 vs. normal group). Treatment with 10 μM 32-2 reduced ROS levels by 80% ( P <0.0001 vs. glutamate group), with effects comparable to the 10 μM NA-1 group. Flow cytometry revealed the same 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 targeting PSD-95 PDZ2 to disrupt the NMDAR-PSD-95-nNOS complex.

[0065] Example 5: Effects of compound 32-2 on the apoptosis pathway

[0066] 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 a neuroprotective effect through anti-apoptosis, HT22 cells were treated with 10 μM 32-2 and glutamate for 24 hours, and Western blot analysis was performed on key apoptosis regulators. Figure 8 As shown, 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 Bcl-2, exhibiting a pro-apoptotic effect. In the group treated with 32-2, the expression level of the anti-apoptotic Bcl-2 protein was upregulated by 2-fold (…). P The effect of <0.05 vs. glutamate control was comparable to that of the 10 μM NA-1 group. Furthermore, 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 The effect of <0.05 vs. glutamate control was comparable to that of the 10 μM NA-1 group, demonstrating an anti-apoptotic effect. All results were quantified by tubulin normalization, confirming that 32-2 exerts its neuroprotective effect by inhibiting intracellular apoptosis.

[0067] Example 6: Rat plasma stability test of compound 32-2

[0068] Plasma stability is a key parameter for evaluating the drug-like properties of peptides and peptide-like compounds. Due to enzymatic cleavage by proteases and peptidases, peptide-based compounds exhibit low stability in blood, especially linear peptides which have short half-lives in plasma. Therefore, to assess the plasma stability of 32-2, an in vitro stability experiment was conducted in rat plasma at 37°C for 24 hours. RP-HPLC analysis was used to monitor the degradation of the test compound over time. Figure 9 As shown, ETAV has a half-life of 1.17 h and is completely degraded after 8 h, NA-1 has a half-life of 2.02 h and is completely degraded after 8 h, while compound 32-2 has a half-life of more than 24 h, and 98% of the peptide remains after 24 h. These results indicate that compound 32-2 has excellent plasma stability and also verify that introducing non-natural amino acids into peptides can enhance stability.

[0069] Example 7 Evaluation of membrane permeability of compound 32-2

[0070] Penetration of the blood-brain barrier is a crucial characteristic of drugs used to treat neurological disorders. 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, measured the amount of the 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. -6 cm / s is a highly permeable compound, capable of penetrating the blood-brain barrier.

[0071] Example 8: Neuroprotective activity of the compound in vivo

[0072] The MCAO model is a classic model for simulating ischemic stroke, using infarct volume as an evaluation indicator. For example... Figure 11 As shown, MCAO injury resulted in a large infarct volume (32.64%). p >0.05 vs. sham-operated group). After intravenous injection of compound 32-2 at 8 mg / kg in the tail vein of rats, the infarct 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 conducted; instead, the positive control drug NA-1 was chosen for comparison. The infarct 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.

[0073] Based on Examples 1-7, this invention synthesized 31 peptide-like compounds using standard Fmoc solid-phase synthesis technology. After in vitro fluorescence polarization experiments and cell protection activity experiments, the optimal compound 32-2 was selected. A series of cell and animal experiments on the optimal compound yielded the following conclusions:

[0074] (1) Fluorescence polarization experiments screened out compound 32-2, which has a higher affinity for PSD-95 PDZ2 protein than ETAV.

[0075] (2) Glutamate-induced HT22 cell and primary neuron injury models demonstrated that compound 32-2 exerted a significant protective effect at the cellular level;

[0076] (3) Compound 32-2 significantly reduced the production of reactive oxygen species and the occurrence of apoptosis in the glutamate-induced HT22 cell injury model;

[0077] (4) Compound 32-2 significantly reduced cerebral infarction caused by cerebral ischemia-reperfusion injury.

[0078] The experimental part of this invention is as follows:

[0079] Compound Synthesis 1.1 Solid-phase Synthesis

[0080] All peptides were synthesized via Fmoc-based manual solid-phase synthesis (SPPS). Wang resin (100-200 mesh, 1 mmol / g, 1% DVB, GL Biochem Ltd.) and Fmoc-protected amino acids purchased from Shanghai Bid Pharmaceutical Co., Ltd. were used for synthesis. Some non-natural amino acids were obtained through chemical synthesis. Unless otherwise specified, all reagents and chemicals were ACS grade or higher and could be used without further purification. 0.05 g (0.05 mM) of Wang resin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 4-dimethylaminopyridine (DMAP), and Fmoc-protected amino acids (in a ratio of 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 for 2 h with acetic anhydride and pyridine dissolved in DCM (resin / acetic anhydride / pyridine = 1∶5∶5). After loading the first amino acid, the amino acid synthesis steps were repeated, as follows: Figure 12 As shown: (1) Fmoc deprotection was performed with 20% piperidine in DMF solution (2 × 15 min, 10 mL each time); (2) washing with DMF (3 × 5 min, 10 mL each time) and monitored by ninhydrin test; (3) Fmoc-amino acid or non-natural amino acid (3 eq.), O-(benzotriazol-1-yl)- N , N , N ′, N ′-Tetramethylurea hexafluoroammonium phosphate (O-Benzotriazole- N , N , N ', N '-tetramethyl-uronium-hexaf , HBTU) (3 eq.), HOBT (3eq.) and N,N -Diisopropylethylamine ( N , N(6 eq.) After activation in dry DMF for 10 min, the product was 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 cleavage 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.

[0081] Specifically, the synthesized compound 32-2 was analyzed by mass spectrometry and nuclear magnetic resonance, and the results are as follows: Figure 15 and 16 All of them are consistent with the theoretical predictions.

[0082] 1.2 Synthesis of intermediate products

[0083] (1) Synthesis of intermediate product A1-5

[0084] Itaconic acid 88 mg (1 mmol, 1 eq.) and the corresponding amine (1 mmol, 1 eq.) were added to water and stirred at 100 °C for 16 h. After the reaction was complete, 10 mL of 1 N NaOH was added to the reaction mixture, and the residual amine was filtered off. The filtrate was acidified to pH 2-3 with 1 N HCl and filtered to obtain solid Al-5, which did not require further purification. Figure 13 .

[0085] (2) Synthesis of intermediate product A6-10

[0086] 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. Then, the corresponding amine (1 eq.) was added and stirred overnight at room temperature. After the reaction was complete, 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. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure. Purification by column chromatography yielded 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 was complete, the mixture was filtered and the filter cake was washed with DCM to obtain compound A6-10, which could be used in the next reaction without further purification. Figure 13 .

[0087] (3) Synthesis of intermediate products A11 and A12

[0088] Dissolve A1 or A5 (1 eq.) in methanol, EDC·HCl (1.3 eq.), DMAP (0.1 eq.), and DIPEA (3 eq.), and stir overnight at 50°C. Add water to the reaction mixture and extract three times with 10 mL EA. Collect the organic phase, wash it with saturated brine, dry it with anhydrous MgSO4, and concentrate it under reduced pressure. Purify the concentrate by column chromatography to obtain the desired compound A11a or A12a. Add A11a or A12a (1 eq.) and Lawson's reagent (0.6 eq.) to dioxane and reflux overnight under N2 protection. Rotary evaporate the reaction mixture, add water to the residue, and extract three times with 10 mL EA. Concentrate the organic layer and purify it by ethyl acetate:petroleum ether (1:4) column chromatography to obtain A11b or A12b. Dissolve A11b or A12b (1 eq.) in MeOH:H2O (1:1), add NaOH (1.2 eq.), and stir overnight at room temperature. 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, such as... Figure 13 .

[0089] (4) Chiral separation of intermediate product A11

[0090] Intermediate products A1-A5, A11, and A12 all possess one chiral center, and the synthesized product is a racemic mixture. To determine which chiral configuration is more favorable for activity, compound A11 was chemically resolved. Different chiral amines were used as resolving agents to react with the carboxyl group under reflux to form diastereomeric organic salts. The differences in solubility of the diastereomeric organic salts in solvents were then used for cooling crystallization to obtain a single non-co-isomeric organic salt. The precipitated solid and filtrate were separately subjected to alkali filtration to remove the chiral amine resolving agent. The filtrate was then acidified to obtain a single-configuration carboxyl compound, such as... Figure 14 .

[0091] Analysis and purification of peptides: HPLC analytical conditions for peptides: Column: Positisil OSD-P reversed-phase C18 (analytical) 5 μm, 4.6 mm × 250 mm; Sample solvent: deionized water and acetonitrile; Aqueous phase: deionized water containing 0.05% TFA; Organic phase: chromatographic grade ACN containing 0.05% TFA; Elution time: 1–27 min; Gradient: 5%–40% ACN; Wavelength: 220 nm; Column temperature: 25 ± 2 ℃; Flow rate: 1 mL·min -1 The injection volume was 20 μL.

[0092] HPLC purification conditions for peptides: 5 μm Positisil reversed-phase C18 (semi-preparative) column, 10.0 mm × 250 mm; sample solvents: deionized water and acetonitrile; aqueous phase: deionized water containing 0.05% TFA; organic phase: chromatographic grade ACN containing 0.05% TFA; elution time: 1–27 min; acetonitrile gradient: 5%–40% ACN; wavelength: 220 nm; column temperature: 25 ± 2 ℃; flow rate: 3 mL·min -1 The injection volume was 2 mL.

[0093] 3. Fluorescence Polarization Assay (FP Assay)

[0094] Fluorescence polarization assays were performed in TC-treated 384-well black opaque plates. The final volume per well was 50 μL, diluted with Tris buffer (150 mM NaCl, 10 mM Tris, pH 7.4). This was done to measure the fluorescent peptide (5-FAM-NR2B) and GST-PDZ2. 155-249 The binding affinity between them was determined through saturation binding experiments. The concentration of GST-PDZ2 was increased in increments. 155-249 Add to a fixed concentration of 5-FAM-NR2B (50 nM), incubate at room temperature for 20-30 minutes, and then measure the sample's FP on an ultrasensitive full-wavelength microplate detector. Adjust the g-factor to ensure the sample is free of GST-PDZ2. 155-249 The FP value of the 50 nM probe was 20 mP, and a saturation binding curve was obtained. Competitive binding experiment: The compound was added at increasing concentrations to fixed concentrations of 5-FAM-NR2B (50 nM) and GST-PDZ2. 155-249 (8 μM) Incubate at room temperature for 30 minutes, then measure the sample FP using an ultrasensitive full-wavelength microplate detector. All graphs were generated using GraphPad 7.0 (GraphPad Software, San Diego, CA), and all reported values ​​are averages of at least three individual experiments.

[0095] 4. Neuroprotective activity experiment of compound 32-2 against HT22 cells

[0096] HT-22 cells were cultured in Dulbecco-modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were passaged or seeded at 80-90% confluence and cultured in 96-well plates at a density of 5000 cells / well for 24 h. Cells were then incubated with different concentrations (0.1, 1, and 10 μM) of the compound for 2 h, followed by the addition of glutamate to a final concentration of 5 mM. After 24 h of co-incubation with the compound and glutamate, 10 μL of CCK-8 reagent was added, and absorbance was measured at 450 nm using a microplate reader.

[0097] Neuroprotective Activity of Compound 32-2 on Primary Neurons: Primary cortical neurons were extracted from the cerebral cortex of 18-day-old SD rat embryos. The cerebral hemispheres were dissected, and the pia mater and blood vessels were removed under a microscope. Cells were then digested with 1.25% trypsin at 37°C for 15–30 minutes. Digestion was terminated with 10% fetal bovine serum, followed by treatment with DNase I for 3 minutes. The cell pellet was resuspended in Neurobasal medium containing 10% B27, 1% penicillin antibiotics, and GlutaMAX, and then filtered through a 40 μm cell filter. The collected cells were seeded at a density of 10⁶ cells / mL into 96-well plates coated with 0.01 mg / mL poly-D-lysine. The next day, the medium was completely replaced with Neurobasal medium containing B27, penicillin antibiotics, and GlutaMAX. Half of the medium was replaced every two days. After culturing neurons for 9 days, they were incubated with different concentrations (0.1, 1, and 10 μM) of the compound for 2 h, 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. The absorbance was then measured using an ELISA reader at an optical density (OD) of 450 nm.

[0098] The effect of compound 32-2 on intracellular ROS was investigated by using HT-22 cells at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well culture plates. After 24 h, the original culture medium was discarded, and the cells were co-incubated with a 10 μM compound diluted in DMEM for 2 h. Glutamate was then added to a final concentration of 5 mM, and incubation continued for 24 h. Intracellular ROS levels were measured using the DCFH-DA fluorescent probe. DCFH-DA was diluted with DMEM to a final concentration of 10 μM. After removing the cell culture medium, an appropriate amount of diluted DCFH-DA solution was added, and the cells were incubated at 37°C in the dark for 20 min. The DCFH-DA solution was discarded, and the cells were washed three times with PBS to remove any untreated DCFH-DA. Intracellular ROS levels were detected using an inverted fluorescence microscope or by flow cytometry after cell collection.

[0099] The effect of compound 32-2 on the expression levels of Bcl-2 / Bax / Caspase 3, a protein related to the apoptosis pathway. Cell pretreatment was the same as in the ROS quantification experiment. HT22 cells were first pretreated with 10 μM of the compound diluted in DMEM for 2 h, followed by co-incubation with 5 mM glutamate (Glu) for 24 h. The culture medium was discarded, and the cells were washed three times with ice-cold PBS. Then, 250 μL of RIPA lysis buffer containing 1 mM PMSF was added to each well of a 6-well plate. The plate was gently shaken at 4 °C for 30 min to ensure complete contact between the lysis buffer and the cells. HT22 cells were scraped off with a cell scraper and transferred to 1.5 mL microcentrifuge tubes. 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 concentration of the extracted protein was determined using a BCA protein quantification kit. Add 20 μL of 5xSDS-PAGE loading buffer to 100 μL of protein sample and boil in a 100 ℃ metal bath for 15 min to denature. Prepare a 15% separating gel according to the SDS-PAGE gel preparation kit, solidify at room temperature for 30 min, and then prepare a 5% stacking gel and solidify it for later use. Add electrophoresis buffer, and then add an equal mass of protein to each well, with a total loading volume <30 μL. Add 5 μL of protein marker to each gel. The electrophoresis voltage of the upper stacking gel is 80 V. After running for 30 min, the voltage is increased to 120 V, and the process is stopped when the protein marker bands are significantly separated. Remove the gel and perform the transfer. The PVDF membrane is soaked in methanol for 1 min beforehand. The transfer sequence is: cathode carbon plate (black) + sponge + filter paper + gel + PVDF membrane + filter paper + sponge - anode carbon plate (red). After clamping, place the membrane in the tank, add transfer buffer, and transfer at 300 V for 1 h. After transfer, the PVDF membrane was removed and washed in TBST buffer for 15 min. Blocked with 4% BSA in TBST buffer at room temperature for 2 h. After blocking, washed three times with TBST buffer for 10 min each time. The membranes containing the target protein and internal control protein were excised according to their molecular weight. Cleave-caspase 3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and internal control Tubulin (1:1000) primary antibodies were diluted with TBST buffer and added to the membrane. The membrane was incubated overnight at 4°C. Afterward, washed three times with TBST for 10 min each time. HRP-labeled goat anti-rabbit secondary antibody (1:10000) was diluted with TBST buffer and incubated at room temperature for 1 h. Washed three times with TBST for 10 min each time. Finally, the membrane was immersed in an equal volume mixture of solution A and solution B from the ultrasensitive ECL chemiluminescence detection kit and then developed in an automated chemiluminescence gel imaging analyzer.

[0100] The plasma stability of compound 32-2 was determined by adding 10 mM of the compound stock solution to rat plasma (containing heparin sodium anticoagulant) to a final concentration of 300 μM and incubating at 37 °C. Samples (150 μL) were taken at different time points and quenched with 300 μL of 10% trichloroacetic acid aqueous solution, vortexed, incubated at 4 °C for 15 min, centrifuged at 14000 g for 5 min, and the supernatant was filtered through a 0.22 μm filter and analyzed by HPLC at 220 nm. Quantification was based on the peak area relative to time 0.

[0101] The membrane permeability of compound 32-2 was evaluated by adding MDCK-MDR1 cells to complete culture medium (10% FBS, 1% NEAA, 1% penicillin-dextrin DMEM solution) and culturing them in culture flasks at 37°C and 5% CO2. When the cells reached 70.0%–80.0% confluence, they were digested with 0.25% EDTA-trypsin and counted. The cells were diluted with complete culture medium to a concentration of 1,000,000 cells / mL / well and seeded into Transwell-96-well plates. The plates were then incubated in a CO2 incubator, with the complete culture medium changed periodically for 4–8 days. The cells were washed three times with transporter buffer and pre-incubated in a CO2 incubator for 30 min. The apparent transmembrane resistance was measured using a resistance meter. 150 μL of pre-warmed drug solution (final concentration 10 μM) was added to each well on side A, and 50 μL of the solution from side A was taken as T0. 300 μL of pre-warmed transporter buffer was added to each well on side B. The Transwell-96-well plate was incubated in a constant-temperature shaker (37°C, 40 rpm) for 120 min. All liquid from both sides A and B was collected as samples for T120 (100 μL of liquid from side B was used for post-experiment quality control – fluorescein permeability). A portion of the solution from each of the other samples was added to a stop solution (methanol containing the internal standard tolbutamide). The plate was then shaken well in a shaker and centrifuged at 4°C, 4000 rpm for 20 minutes. The supernatant was collected or diluted according to a specific ratio for LC-MS / MS analysis.

[0102] Animal activity evaluation of compound 32-2: Focal cerebral ischemia was induced in male SD rats by middle cerebral artery occlusion (MCAO). Male SD rats (220–280 g) were randomly divided into 4 groups (n = 5 per group): (1) sham-operated 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 by making an incision along the midline of the neck with surgical scissors, and the left internal carotid artery and external carotid artery were separated. At the opening of the external carotid artery, a suture was inserted into the external carotid artery, introduced into the left internal carotid artery, and advanced 20–21 mm past the bifurcation of the carotid artery until slight resistance was felt. After occlusion for 1.5 h, the suture was removed (reperfusion), and after reperfusion for 2 h, the drug was administered (8 mg / kg, iv). The rat body temperature was maintained at 37 °C ± 0.5 °C throughout the experiment. Rats were sacrificed 24 h after reperfusion, and their brains were stored at -80℃ for 15 minutes. The brains were then cut into 7 coronal sections of 1.0-2.0 mm thickness and stained in 2% TTC solution at 37℃ for 30 min.

Claims

1. A PSD-95 PDZ2 peptide-like inhibitor, characterized in that: Its structural formula is shown in formula (Ⅰ); （Ⅰ）。 2. The use of the PSD-95 PDZ2 peptide inhibitor according to claim 1 in the preparation of a drug for treating ischemic stroke.

3. A drug, characterized in that: The acetate, hydrochloride, or other pharmaceutically acceptable salt form of the PSD-95 PDZ2 peptide inhibitor of claim 1, comprising a therapeutically effective amount.

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