Use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester in treatment of psychiatric diseases

Abstract

The present invention provides use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester in the preparation of a drug for treating psychiatric diseases. The novel drug for treating psychiatric diseases provided by the present invention may play a role in regulating the imbalance in the excitation / inhibition of a neural circuit by means of mechanisms such as targeted nNOS-PSD95 uncoupling and the agonistic effect of the α2 GABA A receptor, and has a significant inhibitory effect on immune inflammation, which plays an important role in the development and progression of psychiatric diseases. The novel drug provided by the present invention further inhibits central inflammation on the basis of regulating the imbalance in the excitation / inhibition of a central neural circuit.

Description

Application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the treatment of mental illnesses

[0001] This application claims priority to Chinese Patent Application No. 2024108080099, filed on June 21, 2024, entitled "Application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphene ester in the treatment of mental illnesses", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the pharmaceutical field and relates to a novel use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester, more specifically, to the application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the preparation of drugs for treating mental illnesses. These mental illnesses include depression, obsessive-compulsive disorder, and post-traumatic stress disorder. Background Technology

[0003] Mental illnesses are highly prevalent globally, with common ones including depression, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder (PTSD). Depression is a common mental illness with a global prevalence of 4.4% (Lancet 2023 Jan 14; 401(10371):141-153). Its main characteristics are persistent, severe depressive mood and loss of interest or pleasure. Obsessive-compulsive disorder (OCD) is a highly prevalent chronic illness characterized by recurrent obsessive thoughts and compulsive behaviors, with a lifetime prevalence of approximately 2-3% (Nat Rev Dis Primers 2019 Aug 1; 5(1):52). Post-traumatic stress disorder (PTSD) is a severe mental disorder that often develops months or years after experiencing severe trauma. Characteristic symptoms of the disorder include re-experiencing traumatic memories, avoidance of trauma-related stimuli, negative emotions and thoughts, and overreaction, with a prevalence of approximately 6% in the general population (Nat Rev Neurol 2022 May; 18(5):273-288). These diseases not only severely impact patients' daily lives, work, and interpersonal relationships, but also significantly increase the risk of suicide, leading to high social and economic costs.

[0004] Currently, the main clinical treatments for mental illnesses such as depression, OCD, and PTSD include selective serotonin reuptake inhibitors (SSRIs), selective serotonin and norepinephrine reuptake inhibitors (SNRIs), and cognitive behavioral therapy (CBT). Existing drug treatments primarily work by regulating the activity of monoamine neurotransmitter systems such as serotonin (5-HT). Although existing treatments are effective in some cases, their onset of action is often slow, generally requiring several weeks, and many patients experience poor efficacy or intolerable side effects. Therefore, the field of drug development urgently needs innovation, especially in providing new drugs with higher efficacy, faster onset of action, and fewer side effects to meet unmet clinical needs and improve patients' quality of life.

[0005] Mental illnesses such as depression, OCD, and PTSD are closely related to an imbalance between excitation and inhibition in the nervous system. This imbalance is a key molecular pathological feature of major depressive disorder (MDD), as altered GABA and glutamate levels have been found in multiple brain regions of MMD patients (Mol Psychiatry, 2023, 28:3257-3266). Studies have reported structural changes in excitatory glutamatergic and inhibitory GABAergic circuits in OCD patients (Front Psychiatry, 2017, 8:69). This imbalance can lead to repetitive, compulsive behaviors and thoughts. In the brains of PTSD patients, the balance between excitatory and inhibitory neurotransmitters may also be affected (Psychiatry Res, 2014, 224:0). Therefore, drugs aimed at restoring the balance between excitation and inhibition in the nervous system represent a new direction for drug development in the treatment of mental illnesses.

[0006] 2-Hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester is a compound with a multi-target mechanism. It acts on the downstream nNOS-PSD95 target of the NMDAR signaling pathway, acting as an uncoupling agent to reduce the pathological release of NMDAR-mediated NO without affecting the physiological functions of NMDAR and nNOS, and has a significant protective effect against Glu-induced neuronal excitability damage (Patent No. WO2013064031A1). Simultaneously, 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester can selectively activate α2GABA. A Receptors that restore the brain's GABAergic system (Theranostics, 2021, 11:5970-5985). Through these multi-target mechanisms, it is hoped that the excitation / inhibition imbalance in neural circuits of mental illnesses can be effectively regulated.

[0007] Recent studies have shown that central nervous system inflammation plays a crucial role in mental illnesses such as depression, OCD, and PTSD. Compared to healthy controls, patients with depression have elevated levels of pro-inflammatory cytokines and acute-phase proteins (such as IL-6, TNF, and CRP) in their blood (Neuron, 2020, 107:0). Furthermore, research has indicated that neuroinflammation may play an important role in the cortico-striatal-thalamic-cortical circuit of OCD (JAMA Psychiatry, 2017, 74:0). Trauma exposure is associated with pro-inflammatory activity, specifically manifested as elevated levels of circulating pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) and CRP (Neuropsychopharmacology, 2017, 42:254-270), reflecting the potential role of inflammation in PTSD. Elevated levels of pro-inflammatory cytokines and immune system dysregulation may affect brain function through multiple mechanisms, leading to the occurrence and development of these mental illnesses. This provides clues for developing new anti-inflammatory treatment strategies to improve the prognosis of patients with mental illnesses. We recently discovered that 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester is a reversible myeloperoxidase (MPO) inhibitor. MPO is highly expressed in neutrophils, microglia, and macrophages and is a key enzyme mediating immune inflammation and free radical metabolism. Our results indicate that 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester has a significant inhibitory effect on immune inflammation, thus potentially holding great promise for the treatment of depression, OCD, and PTSD. Summary of the Invention

[0008] Technical problem solved: This invention provides the application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the treatment of mental illnesses; 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester is a reported product with nNOS-PSD95 uncoupling and α2GABA... A Compounds with receptor agonist activity can also exert central anti-inflammatory effects by inhibiting myeloperoxidase through a novel mechanism of action independent of the above targets. Through the synergistic effect of the above multi-target mechanisms, they have great therapeutic potential in mental illnesses.

[0009] Technical solution: The present invention provides a drug for treating mental illnesses through a multi-target mechanism as shown in Formula I, wherein the drug is 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof.

[0010] The aforementioned drugs achieve the goal of treating mental illnesses by regulating the imbalance of excitation / inhibition in the central nervous system under pathological conditions and through the central anti-inflammatory mechanism.

[0011] The use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the preparation of drugs for the treatment of depression.

[0012] Application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the preparation of drugs for the treatment of obsessive-compulsive disorder.

[0013] Application of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester in the preparation of drugs for the treatment of post-traumatic stress disorder.

[0014] Use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of heart failure.

[0015] Use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of multiple system atrophy.

[0016] The use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of Parkinson's disease.

[0017] Use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.

[0018] Use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis. Beneficial effects:

[0019] The novel drug provided by this invention for the treatment of mental illnesses may work by targeting nNOS-PSD95 uncoupling and α2GABA. AMechanisms such as receptor agonism regulate the excitation / inhibition imbalance in neural circuits and significantly inhibit immune inflammation, which plays a crucial role in the development of mental illnesses. Considering the complex etiology of mental illnesses, single-target drugs often fail to achieve satisfactory therapeutic effects. The novel drug provided by this invention, while regulating the excitation / inhibition imbalance in central nervous circuits, further inhibits central inflammation, comprehensively targeting various etiological mechanisms of mental illnesses. This approach differs significantly from existing therapeutic drug mechanisms and may offer advantages such as significant therapeutic effect, rapid onset of action, fewer side effects, and overcoming non-responsiveness to existing drugs. Attached Figure Description

[0020] The invention will now be further described with reference to the following non-limiting embodiments and accompanying drawings, wherein:

[0021] Figure 1. Inhibition effect of direct incubation of ZL006-05 with MPO enzyme protein on its activity;

[0022] Figure 2. Effect of ZL006-05 on the MPO activity of BV2 under physiological conditions;

[0023] Figure 3. Effect of ZL006-05 on the MPO activity of BV2 under LPS modeling induction;

[0024] Figure 4. Effect of ZL006-05 on the MPO activity of Ana-1 under physiological conditions;

[0025] Figure 5. Effect of ZL006-05 on the MPO activity of Ana-1 under LPS modeling induction;

[0026] Figure 6. Effect of ZL006-05 on MPO activity in neutrophils under physiological conditions;

[0027] Figure 7. Inhibitory effect of ZL006-05 on LPS-induced expression of inflammation-related proteins in BV2;

[0028] Figure 8. Inhibitory effect of ZL006-05 on LPS-induced expression of inflammation-related proteins in Ana-1;

[0029] Figure 9. Inhibitory effect of ZL006-05 on the expression of inflammation-related proteins in neutrophils induced by LPS and INF-γ.

[0030] Figure 10. Results of environmental rigidity test for each group of animals;

[0031] Figure 11. Results of the elevated cross maze experiment for each group of animals;

[0032] Figure 12. Results of tail suspension test for each group of animals. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] Inhibitory effect of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester (ZL006-05) on myeloperoxidase activity

[0036] 1. Materials and Methods

[0037] 1.1 Cells

[0038] Mouse microglia BV2 were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0039] Mouse macrophages Ana-1 were purchased from Cybex (Shanghai) Cell Technology Co., Ltd.

[0040] 1.2 Isolation of neutrophils

[0041] C57BL / 6J mice were euthanized by cervical dislocation. The femur and tibia were rapidly separated, disinfected with 75% ethanol, and muscle tissue was removed as completely as possible. The femurs and tibias were washed with PBS and collected into pre-cooled 10% FBS RPMI-1640 medium. The femurs and tibias were washed three times with sterile PBS, soaked in physiological saline, and both ends were cut off to expose the medullary cavity. The medullary cavity was washed with serum-free RPMI-1640 medium, and the bone marrow flush was filtered through a 100 μm cell filter. The mixture was centrifuged at 4°C for 1200g for 5 min. The supernatant was discarded, and the mixture was resuspended in 2 mL of FBS-free medium and slowly added to a 15 mL EP tube containing 6 mL of Histopaque 1077. The mixture was centrifuged at 4°C for 2000g for 20 min. The supernatant was discarded, and the mixture was resuspended in 3 mL of FBS-free medium and added to a 15 mL EP tube containing 6 mL of Histopaque 1119. Centrifuge in EP tubes at 4°C, 2000g × 20 min; carefully aspirate the milky white intermediate layer, resuspend and dilute in 5 mL of FBS-free medium, gently mix, centrifuge at 4°C, 1500g × 10 min; discard the supernatant, resuspend cells in 2% FBS medium. The basal medium was RPMI-1640. All the above operations were performed on ice.

[0042] 1.3 Detection of myeloperoxidase (MPO) activity

[0043] Prepare all reagents, samples, positive controls, and standards according to the instructions for use of the MPO Activity Assay Kit. Plot the corresponding standard curve based on the activity of the reactant enzyme (sample to be tested). Add all samples to the test wells (96-well white plate), add the drug / inhibitor / solvent control, add buffer to a volume of 60 μL, mix well, and incubate at room temperature for 10 min. Finally, add 40 μL Reaxtion Mix to a final volume of 100 μL, mix well, and incubate on ice. Measure fluorescence (Ex / Em = 535 / 587 nm) at 37 °C for 5–20 min. Determine myeloperoxidase (MPO) peroxidase activity using the formula.

[0044] 1.3.1 Sample Testing

[0045] 1) The inhibitory effect of ZL006-05 on the activity of MPO enzyme protein solution by direct incubation.

[0046] Add 10 μL of control solvent or ZL006-05 or MPO inhibitor standards at various concentrations to the detection well, then add 45 μL of MPO analysis buffer and 5 μL of MPO enzyme protein, bringing the total volume to 60 μL. Mix well and incubate at room temperature for 10 min. Place on ice and add 40 μL of reaction solution to bring the final volume to 100 μL, then mix well. Measure fluorescence (Ex / Em = 535 / 587 nm) at 37 °C for 5–20 min.

[0047] 2) Inhibitory effect of ZL006-05 on MPO activity in microglia (BV2).

[0048] Mouse microglia BV2 (3.0×10⁻⁶) 5 Cells / well were co-incubated with different concentrations of ZL006-05 for 14 h, the culture medium was discarded, and the cells were washed once with D-Hank's solution. 110 μL / well of MPO analysis buffer was added, and the cells were lysed on ice for 15 min. The procedure was performed on ice, and each sample was divided into two 50 μL parallel wells. 5 μL of MPO analysis buffer was added to well 1, and 5 μL of MPO inhibitor control was added to well 2. 5 μL of MPO analysis buffer was then added to all wells to bring the volume to 60 μL. The mixture was then mixed, and 40 μL of reaction solution was added to bring the final volume to 100 μL. Fluorescence (Ex / Em = 535 / 587 nm) was measured at 37 °C for 5–20 min.

[0049] 3) The inhibitory effect of ZL006-05 on the MPO activity of macrophages (Ana-1).

[0050] Using mouse macrophages Ana-1 (3.0 × 10⁻⁶) 5 (Cells / well), other experiments are the same as for BV2 cells.

[0051] 4) Inhibitory effect of ZL006-05 on MPO activity in neutrophils.

[0052] Mouse neutrophils (4.0 × 10⁻⁶) 4 Cells (per well) were co-incubated with different concentrations of ZL006-05 for 2 h. The culture medium was discarded, and the cells were washed once with D-Hank's solution. 100 μL of MPO analysis buffer was added to each well, and the cells were lysed on ice for 15 min. For the next step, 20 μL of lysis buffer was added to each well. 5 μL of MPO analysis buffer was added to the sample in well 1, and 5 μL of inhibitor control was added to the sample in well 2. 35 μL of MPO analysis buffer was added to all wells to make a volume of 60 μL, and the mixture was stirred. 40 μL of reaction solution was added to make a final volume of 100 μL, and the mixture was stirred. Fluorescence (Ex / Em = 535 / 587 nm) was measured at 37 °C for 5–20 min.

[0053] 1.4 Main Reagents and Drugs

[0054] 1.5 Statistical Analysis

[0055] Quantitative data are expressed as mean ± standard error. One-way ANOVA was performed using SPSS statistical software, and the significance of differences between the two groups was determined post-hocly by LSD test. A p-value < 0.05 was defined as significant.

[0056] 2 Experimental Results

[0057] 2.1 Inhibitory effect of direct incubation of ZL006-05 with MPO enzyme protein on its activity

[0058] The inhibitory effect of ZL006-05 on MPO enzyme protein is shown in Figure 1. All concentrations of ZL006-05 (0.1 μM, 1 μM, 10 μM, 100 μM, 200 μM, 400 μM, and 800 μM) significantly inhibited the catalytic activity of MPO protein, with inhibition rates of 10.9%, 11.5%, 14.5%, 24.0%, 45.2%, 64.7%, and 81.6%, respectively, with an effective concentration of 0.1 μM. The results indicate that ZL006-05 has a certain inhibitory effect on the catalytic activity of MPO enzyme protein.

[0059] For specific results, please refer to Figure 1-ZL006-05 for the inhibitory effect of direct incubation with MPO enzyme protein on its activity. Mean ± standard error. *** P<0.001, compared with the solvent control.

[0060] 2.2 Inhibitory effect of ZL006-05 on MPO in microglia (BV2)

[0061] 2.2.1 Under physiological conditions

[0062] Figure 2 shows the inhibitory effect of ZL006-05 on MPO in microglia (BV2) under physiological conditions. Compared with the solvent control, 0.1 μM, 1 μM, and 10 μM ZL006-05 significantly inhibited MPO activity (0.1 μM, P = 0.001; 1 μM, P < 0.001; 10 μM, P < 0.001). The results indicate that ZL006-05 has a significant dose-dependent inhibitory effect on MPO activity in microglia (BV2).

[0063] For specific results, please refer to Figure 2, which shows the effect of ZL006-05 on the MPO activity of BV2 under physiological conditions. Mean ± standard error. *** P<0.001, compared with the solvent control.

[0064] 2.2.2 LPS-induced conditions

[0065] The inhibitory effect of ZL006-05 on MPO in microglia (BV2) under LPS-induced conditions is shown in Figure 3. Compared with the solvent control, LPS (1 μg / mL) significantly increased MPO activity (P<0.001), indicating that LPS can induce a significant increase in MPO activity in BV2. ZL006-05 (0.01 μM, 0.1 μM, 1 μM, 10 μM) all significantly decreased MPO activity (each concentration of ZL006-05, P<0.001). The results indicate that ZL006-05 has a significant inhibitory effect on LPS-induced MPO activity in microglia (BV2).

[0066] For specific results, please refer to Figure 3, which shows the effect of ZL006-05 on the MPO activity of BV2 under LPS-induced modeling. Mean ± standard error. *** P<0.001, compared with the solvent control, ### P<0.001, compared with the LPS group that was not treated with ZL006-05.

[0067] 2.3 Inhibitory effect of ZL006-05 on macrophage (Ana-1) MPO

[0068] 2.3.1 Under physiological conditions

[0069] The inhibitory effect of ZL006-05 on macrophage (Ana-1) MPO under physiological conditions is shown in Figure 4. Compared with the solvent control, 0.1 μM, 1 μM, and 10 μM ZL006-05 significantly inhibited MPO activity (0.1 μM, P = 0.024; 1 μM, P < 0.001; 10 μM, P < 0.001). The results indicate that ZL006-05 has a significant dose-dependent inhibitory effect on macrophage (Ana-1) MPO activity.

[0070] For specific results, please refer to Figure 4, which shows the effect of ZL006-05 on the MPO activity of Ana-1 under physiological conditions. Mean ± standard error. *** P<0.001, * P<0.05, compared with the solvent control.

[0071] 2.3.2 LPS-induced conditions

[0072] The inhibitory effect of ZL006-05 on MPO in macrophages (Ana-1) under LPS-induced conditions is shown in Figure 5. Compared with the solvent control, LPS (1 μg / mL) significantly increased MPO activity (P = 0.003), indicating that LPS can induce a significant increase in MPO activity in Ana-1 cells. ZL006-05 (0.1 μM, 1 μM, 10 μM) significantly decreased MPO activity (0.1 μM, P = 0.001; 1 μM, P < 0.001; 10 μM, P < 0.001). The results indicate that ZL006-05 has a significant inhibitory effect on LPS-induced MPO activity in macrophages (Ana-1).

[0073] For specific results, please refer to Figure 5, which shows the effect of ZL006-05 on the MPO activity of Ana-1 under LPS-induced modeling. Mean ± standard error. *** P<0.001, compared with the solvent control, ### P<0.001, ## P = 0.001, compared with the LPS group that was not treated with ZL006-05.

[0074] 2.4 Effect of ZL006-05 on MPO activity in neutrophils

[0075] The inhibitory effect of ZL006-05 on neutrophil MPO under physiological conditions is shown in Figure 6. Compared with the solvent control, 0.01 μM, 0.1 μM, 1 μM, and 10 μM ZL006-05 significantly inhibited MPO activity (each concentration of ZL006-05, P < 0.001). The results indicate that ZL006-05 has a significant inhibitory effect on neutrophil MPO activity.

[0076] For specific results, please refer to Figure 6, which shows the effect of ZL006-05 on neutrophil MPO activity under physiological conditions. Mean ± standard error. *** P<0.001, compared with the solvent control.

[0077] Example 2

[0078] Inhibitory effect of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester (ZL006-05) on immune inflammation

[0079] 1. Materials and Methods

[0080] 1.1 Cells

[0081] Mouse microglia BV2 were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0082] Mouse macrophages Ana-1 were purchased from Cybex (Shanghai) Cell Technology Co., Ltd.

[0083] 1.2 Isolation of neutrophils

[0084] C57BL / 6J mice were euthanized by cervical dislocation. The femur and tibia were rapidly separated, disinfected with 75% ethanol, and muscle tissue was removed as completely as possible. The femurs and tibias were washed with PBS and collected into pre-cooled 10% FBS RPMI-1640 medium. The femurs and tibias were washed three times with sterile PBS, soaked in physiological saline, and both ends were cut off to expose the medullary cavity. The medullary cavity was washed with serum-free RPMI-1640 medium, and the bone marrow flush was filtered through a 100 μm cell filter. The mixture was centrifuged at 4°C for 1200g for 5 min. The supernatant was discarded, and the mixture was resuspended in 2 mL of FBS-free medium and slowly added to a 15 mL EP tube containing 6 mL of Histopaque 1077. The mixture was centrifuged at 4°C for 2000g for 20 min. The supernatant was discarded, and the mixture was resuspended in 3 mL of FBS-free medium and added to a 15 mL EP tube containing 6 mL of Histopaque 1119. Centrifuge in EP tubes at 4°C, 2000g × 20 min; carefully aspirate the milky white intermediate layer, resuspend and dilute in 5 mL of FBS-free medium, gently mix, centrifuge at 4°C, 1500g × 10 min; discard the supernatant, resuspend cells in 2% FBS medium. The basal medium was RPMI-1640. All the above operations were performed on ice.

[0085] 1.3 In vitro cell culture induction of inflammatory factor expression and drug administration regimen

[0086] BV2, Ana-1 (3.0×10 5 Cells / well were co-incubated with solvent or different concentrations of ZL006-05 for 2 h, then LPS (1.0 μg / mL) was added for 22 h induction. After 24 h, the culture medium was discarded, D-Hank's solution was added for washing once, and 100 μL of pre-chilled protein lysis buffer RIPA was added to each well. Cells were lysed on ice for 15 min.

[0087] Mouse bone marrow neutrophils (4.0 × 10⁻⁶) 4Cells / well were co-incubated with solvent or different concentrations of ZL006-05 for 2 h, then LPS (1.0 μg / mL) and INF-γ (100 ng / mL) were added for 3 h of induction. After 5 h, the culture medium was discarded, D-Hank's solution was added for washing once, and 50 μL of pre-chilled protein lysis buffer RIPA was added to each well. Cells were lysed on ice for 15 min.

[0088] 1.4 Western Blot (WB) of Proteins

[0089] Western blotting (WB) was used to detect protein expression levels. A chemiluminescence imaging system (manufacturer: Bio-Rad, model: ChemiDoc) was used. TM Protein signals were acquired using MP (Micro-Rad) software, and the relative quantification of proteins was performed using Image Lab software (Manufacturer: Bio-Rad, Model: Image Lab).

[0090] 1.5 Main reagents, consumables and drugs

[0091] 1.6 Statistical Analysis

[0092] Quantitative data are expressed as mean ± standard error. One-way ANOVA was performed using SPSS statistical software, and the significance of differences between the two groups was determined post-hocly by LSD test. A p-value < 0.05 was defined as significant.

[0093] 2 Experimental Results

[0094] 2.1 Inhibitory effect of ZL006-05 on LPS-induced expression of inflammation-related proteins in BV2

[0095] The inhibitory effect of ZL006-05 on the expression of inflammation-related proteins in microglia (BV2) is shown in Figure 7. All concentrations of ZL006-05 significantly reduced iNOS expression (P < 0.001 at 4.0 μM, 2.0 μM, and 1 μM; P = 0.028 at 0.5 μM), in a dose-dependent manner. ZL006-05 (4.0 μM, 2.0 μM, 1 μM, and 0.5 μM) significantly reduced IL-1β expression (P < 0.001 at each concentration), in a dose-dependent manner. ZL006-05 (4.0 μM and 2.0 μM) significantly reduced TNF-α expression (4.0 μM; P < 0.001; 2.0 μM; P = 0.021), in a dose-dependent manner. The results showed that ZL006-05 significantly inhibited the expression of various inflammation-related proteins in BV2 induced by LPS in a dose-dependent manner.

[0096] For specific results, please refer to Figure 7ZL006-05 for the inhibitory effect of LPS on the expression of inflammation-related proteins in BV2. Top: iNOS expression; Middle: IL-1β expression; Bottom: TNF-α expression. Mean ± standard error. *** P<0.001, compared with the solvent control, # P<0.05, ### P<0.001, compared with the LPS group that was not treated with ZL006-05.

[0097] 2.2 Inhibitory effect of ZL006-05 on LPS-induced expression of inflammation-related proteins in Ana-1

[0098] The inhibitory effect of ZL006-05 on the expression of inflammation-related proteins in macrophages (Ana-1) is shown in Figure 8. All ZL006-05 groups (4.0 μM, 2.0 μM, 1 μM, 0.5 μM) significantly reduced the expression of iNOS (each concentration of ZL006-05, P < 0.001) and IL-1β (each concentration of ZL006-05, P < 0.001). ZL006-05 concentrations had no effect on the expression of TNF-α (P = 0.486). The results indicate that ZL006-05 significantly inhibited LPS-induced expression of iNOS and IL-1β in macrophages (Ana-1) in a dose-dependent manner, but had no significant effect on TNF-α expression.

[0099] For specific results, please refer to Figure 8ZL006-05 for the inhibitory effect of LPS on the expression of inflammation-related proteins in Ana-1. Top: iNOS expression; Middle: IL-1β expression; Bottom: TNF-α expression. Mean ± standard error. *** P<0.001, compared with the solvent control, ### P<0.001, compared with the LPS group that was not treated with ZL006-05.

[0100] 2.3 Inhibitory effect of ZL006-05 on neutrophil inflammation

[0101] The inhibitory effect of ZL006-05 on the expression of inflammation-related proteins in neutrophils is shown in Figure 9. Both 4.0 μM and 2.0 μM ZL006-05 significantly reduced IL-1β expression (4.0 μM and 2.0 μM ZL006-05, P < 0.001), in a dose-dependent manner. All concentrations of ZL006-05 significantly reduced TNF-α expression (all concentrations of ZL006-05, P < 0.001). These results indicate that ZL006-05 significantly inhibits the expression of various inflammation-related proteins in neutrophils induced by both LPS and INF-γ in a dose-dependent manner.

[0102] For specific results, please refer to Figure 9 for the inhibitory effect of LPS and INF-γ dual-induced expression of inflammation-related proteins in neutrophils. *** P<0.001, compared with solvent control; ### P<0.001, compared with the LPS and INF-γ groups that were not treated with ZL006-05.

[0103] Example 3

[0104] Pharmacodynamic study of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester (ZL006-05) in an animal model of post-traumatic stress disorder.

[0105] 1. Materials and Methods

[0106] 1.1 Laboratory Animals

[0107] C57BL / 6J mice, male, SPF grade, 8–10 weeks old.

[0108] 1.2 Reagents and Instruments

[0109] 1.3 Animal Groups

[0110] The experimental animals were randomly divided into 8 groups: normal control group, model control group, low-dose ZL006-05 group (2 mg / kg), medium-dose ZL006-05 group (4 mg / kg), high-dose ZL006-05 group (8 mg / kg), ZL006 group (8 mg / kg), dexborneol group (8 mg / kg), and sertraline group (15 mg / kg), with 10 animals in each group. The ZL006, dexborneol, and ZL006-05 groups were administered the corresponding treatment drugs via a single intravenous injection 30 minutes before each behavioral test. Animals in the sertraline group were administered sertraline once daily by gavage before the tail suspension test for 25 days.

[0111] 1.4 Preparation of PTSD Model

[0112] After at least 5 days of acclimatization, a modified single-course long-duration stress-induced PTSD model was used, including 2 hours of confinement, 20 minutes of forced swimming, ether anesthesia until loss of consciousness, and electric shock. First, mice were confined in a 50 mL perforated acrylic tube for 2 hours. Then, the mice were placed in a glass beaker (50 cm high, 24 cm in diameter) filled with water and forced to swim for 20 minutes, with the water temperature maintained at 24°C. After a 15-minute recovery period, the mice were given two intermittent, unavoidable foot shocks (0.8 mA, 10 seconds each, 10-second intervals), and then remained in the shock chamber for another 60 seconds. Finally, the mice were returned to their original cages. Normal control mice were placed in cages or in a shock chamber without electric shocks during the same time.

[0113] 1.5 Behavioral Testing

[0114] On days 9, 11, and 13 after modeling, environmental rigidity test, elevated cross maze test, and tail suspension test were conducted on each group of animals.

[0115] 1.5.1 Environmental Rigidity Test

[0116] The animals were returned to the shock chamber for 5 minutes without being shocked, and the animal's rigidity time, which reflects the situational fear response, was recorded.

[0117] 1.5.2 Elevated Cross Maze Test

[0118] An elevated cross maze, 50 cm off the ground and with arms 30 cm long, was used as the testing tool. The maze consisted of two open arms (30 × 5 cm) and two closed arms (30 × 5 cm, with the closed arms surrounded by a 15 cm high fence around their perimeter and tail ends), connected by a central area (5 × 5 cm). At the start of the experiment, mice were placed in the central area of ​​the maze, facing the same open arm, and allowed to move freely. The video system recorded the animals' activities over 5 minutes.

[0119] 1.5.3 Tail Suspension Test

[0120] The mouse's tail was fixed to the edge of the rack, and it was suspended for 6 minutes on the rack, which was 50 cm above the bottom and 10 cm wide from the back wall. The time the animal remained still during the last 4 minutes was recorded.

[0121] 1.6 Statistical Analysis

[0122] Experimental data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used to analyze differences between groups, and Fisher's LSD test was used for inter-group comparisons. P < 0.05 was defined as statistically significant.

[0123] 2 Results

[0124] 2.1 Environmental rigidity test results

[0125] The results of the environmental rigidity test are shown in Figure 10. Compared with the normal control group, the percentage of rigidity time in the model control group was significantly increased (P<0.001), indicating that the modified single-stage long-term stress induced significant fear memory behavior. Sertraline is a first-line drug for the clinical treatment of PTSD. Chronic administration of sertraline can significantly reduce the percentage of rigidity time (P=0.014); a single dose of ZL006 8 mg / kg can significantly reduce the percentage of rigidity time (P=0.049); a single dose of dextromethorphan 8 mg / kg can significantly reduce the percentage of rigidity time (P=0.015); a single dose of ZL006-05 2, 4, and 8 mg / kg can significantly reduce the percentage of rigidity time (P=0.003, P<0.001, P<0.001), with an onset dose of 2 mg / kg and an optimal dose of 8 mg / kg. The results showed that ZL006-05 had a dose-dependent effect on improving situational fear memory behavior in the PTSD model, and its efficacy was superior to that of ZL006 and dextromethorphan.

[0126] For detailed results, please refer to Figure 10 for the experimental results of environmental rigidity tests in each group of animals. Data are expressed as mean ± standard error. * P<0.05 ** P<0.01 *** P<0.001, compared with the model control group.

[0127] 2.2 Results of the Elevated Cross Maze

[0128] The results of the elevated cross maze experiment are shown in Figure 11. Compared with the normal control group, the percentage of open-arm dwell time in the model control group was significantly reduced (P<0.001), indicating that the modified one-way long-duration stress induced significant anxiety-like behavior. Chronic administration of sertraline significantly increased the percentage of open-arm dwell time (P=0.010); a single dose of ZL006 at 8 mg / kg significantly increased the percentage of open-arm dwell time (P=0.035); a single dose of dextromethorphan at 8 mg / kg showed a trend of increasing the percentage of open-arm dwell time (P=0.071); single doses of ZL006-05 at 2, 4, and 8 mg / kg significantly increased the percentage of open-arm dwell time (P=0.007, P<0.001, P<0.001), with an onset dose of 2 mg / kg and an optimal dose of 8 mg / kg. The results showed that ZL006-05 significantly improved anxiety-like behaviors in the PTSD model in a dose-dependent manner, and its efficacy was superior to that of ZL006 and dextromethorphan.

[0129] For detailed results, please refer to Figure 11 for the elevated cross maze experiment results of each group of animals. Data are expressed as mean ± standard error. * P<0.05 ** P<0.01 *** P<0.001, compared with the model control group.

[0130] 2.3 Tail Suspension Test Results

[0131] The results of the tail suspension test are shown in Figure 12. Compared with the normal control group, the immobility time of the model control group was significantly increased (P<0.001), indicating that the modified single-course long-duration stress induced significant depressive-like behavior. Chronic administration of sertraline significantly reduced the immobility time of the animals (P=0.002); a single dose of ZL006 at 8 mg / kg significantly reduced the immobility time (P=0.003); a single dose of dextromethorphan at 8 mg / kg showed a trend of reducing immobility time (P=0.066); a single dose of ZL006-05 at 2, 4, and 8 mg / kg significantly reduced the immobility time (P<0.001, P<0.001, P<0.001), with an onset dose of 2 mg / kg and an optimal dose of 8 mg / kg. The results showed that ZL006-05 significantly improved depressive-like behaviors in the PTSD model in a dose-dependent manner, and its efficacy was superior to that of ZL006 and dextromethorphan.

[0132] For detailed results, please refer to Figure 12 for the tail suspension test results of each group of animals. Data are expressed as mean ± standard error. * P<0.05 ** P<0.01 *** P<0.001, compared with the model control group.

[0133] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. The use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceutical compositions, in the preparation of a medicament for the treatment of psychotropic disorders, wherein the structure of the 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester is shown below:

2. The application according to claim 1, characterized in that, The use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester in the preparation of a drug for treating depression.

3. The application according to claim 1, characterized in that, The use of the 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester in the preparation of a drug for treating obsessive-compulsive disorder.

4. The application according to claim 1, characterized in that, The use of the 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester in the preparation of a drug for treating post-traumatic stress disorder. 5.2-Hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of heart failure. 6.2-Hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of multiple system atrophy. 7.2-Hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of Parkinson's disease. 8.2-Hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphene ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.

9. The use of 2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl)aminobenzoic acid 2-camphenol ester or a pharmaceutically acceptable salt thereof, and in combination with other pharmaceuticals, in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis.

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