A small molecule activator targeting neurocellular aromatase and its use in cerebral ischemic injury

Abstract

The application discloses a small-molecule activator targeting neural cell aromatase and application thereof in cerebral ischemic injury, and belongs to the technical field of biological medicine. The activator can directly enhance the catalytic activity of aromatase without significantly affecting the gene transcription, so that the local estrogen level in the brain is accurately improved, and a neuroprotective effect is generated. By specifically activating the neuron aromatase, the side effects of systemic estrogen therapy are avoided. In-vivo and in-vitro experiments prove that the activator can effectively increase the estrogen concentration in the target area through local intracerebral administration, and significantly improve the neuron survival rate in an oxygen-glucose deprivation / reoxygenation injury model. The pharmaceutical composition of the application is suitable for local administration or systemic administration of the central nervous system, and can be combined with the existing therapy, so that a brand-new treatment strategy and a drug candidate are provided for the cerebral ischemic injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule activator that targets aromatase in nerve cells and its application in cerebral ischemia-reperfusion injury. Background Technology

[0002] Ischemic brain injury is one of the leading causes of death and long-term disability worldwide. Its core pathological process involves multiple stages, including energy metabolism failure, excitatory amino acid toxicity, oxidative stress, inflammatory response, and ultimately, neuronal apoptosis and necrosis. Currently, the main clinical treatments for acute ischemic stroke are intravenous thrombolysis and endovascular mechanical thrombectomy. However, these methods have extremely narrow treatment windows and can only achieve vascular recanalization, failing to directly intervene in the persistent neuronal damage cascade (Yang SH, Transl Stroke Res, 2021, 12: 937-945), resulting in many patients not benefiting or having poor prognoses. Therefore, developing drugs that can transcend time window limitations and act directly on nerve cells to provide protection is a long-standing key challenge and urgent need in the fields of neuroscience and drug development.

[0003] Activating endogenous neuroprotective mechanisms has become a hot topic in brain injury research in recent years. The brain itself possesses certain self-repair and protective capabilities. Among these, neurosteroids synthesized locally in the brain, especially 17β-estradiol (E2), exhibit powerful neuroprotective effects, including inhibiting apoptosis, reducing neuroinflammation and oxidative stress, promoting synaptic plasticity, and maintaining mitochondrial function (Xu J, Biology (Basel), 2023, 12: 99). However, systemic supplementation with exogenous estrogen can lead to serious systemic side effects such as breast cancer, endometrial hyperplasia, and thrombosis, greatly limiting its clinical translation (Chuffa LG. Steroids, 2017, 118: 93-108). Therefore, strategically and precisely increasing endogenous estrogen levels in affected brain regions, rather than resorting to systemic hormone replacement, has become a more attractive and potentially safer research direction.

[0004] Aromatases are key rate-limiting enzymes in the body that catalyze the conversion of androgens (such as testosterone) into estrogens. In the brain, aromatases are mainly expressed in neurons, particularly in brain regions associated with learning, memory, and mood regulation, such as the hippocampus, hypothalamus, and amygdala. Brain-derived estrogens are primarily synthesized locally by these neurons via aromatases and exert their effects in an autocrine or paracrine manner, which is crucial for maintaining neuronal survival, synaptic function, and coping with stress-induced damage (Faddetta M. Mol Neurobiol, 2025, 63: 286). In pathological states such as ischemic brain injury, endogenous protective mechanisms are activated, including the upregulation of aromatase expression and activity, attempting to counteract damage by increasing local estrogen synthesis. However, this endogenous upregulation is often insufficient or delayed, inadequate to completely counteract the severe damage process (Ma Y. Mol Neurobiol, 2020, 57: 3540-3551).

[0005] The development of existing neuroprotective agents faces numerous bottlenecks. Many drugs targeting single pathological pathways (such as glutamate receptor antagonists and free radical scavengers) have failed in clinical trials, suggesting that a multi-target strategy acting on endogenous pathways may be more effective. Directly targeting aromatase, the "master switch" for endogenous estrogen synthesis, provides a precise entry point for regulating local estrogen levels in the brain. Currently, aromatase inhibitors used clinically (such as letrozole and anastrozole) are mainly used for hormone therapy in breast cancer, inhibiting aromatase activity to lower estrogen levels and treat breast cancer. However, in the field of neurological diseases, particularly for ischemic brain injury, no small molecule compounds that can specifically activate neuronal aromatase have been reported or marketed, leaving related drug development completely undeveloped. Summary of the Invention

[0006] In the treatment of ischemic brain injury, the lack of small molecule compounds that can specifically activate neuronal aromatase (CYP19A1) has created a technological gap that prevents effective neuroprotection through precise enhancement of endogenous estrogen levels in the brain. This invention aims to provide a small molecule activator that targets neuronal aromatase and its application in ischemic brain injury. It provides the first such small molecule activator, laying the foundation for the development of novel and highly targeted neuroprotective drugs.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention provides the use of a small molecule activator targeting aromatase in nerve cells in the preparation of a medicament for the prevention and / or treatment of ischemic brain injury, wherein the small molecule activator is a compound having the structure of formula (I) below, or a pharmaceutically acceptable salt, solvate, prodrug, or polymorph thereof; .

[0008] The ischemic brain injury is selected from ischemic stroke or cerebral ischemia-reperfusion injury.

[0009] Preferably, the ischemic brain injury is ischemic stroke.

[0010] The drug works by activating aromatase in nerve cells, thereby increasing the level of endogenous estrogen in the brain.

[0011] The nerve cells mentioned are neurons.

[0012] The small molecule activator targeting aromatase in nerve cells activates aromatase by directly activating its protein catalytic activity, without significantly upregulating the mRNA expression level of the aromatase-encoding gene Cyp19a1.

[0013] The compound represented by formula (I) is brassinolide gentioside HY-N4177.

[0014] HY-N4177 (Rubrofusarin gentiobioside, CAS No. 50988-92-6), its molecular formula is: C 27 H 32 O 15。

[0015] The present invention provides a pharmaceutical composition for the prevention and / or treatment of ischemic brain injury, comprising a therapeutically effective amount of the above-described small molecule activator targeting neuronal aromatase, and other pharmaceutically acceptable carriers or excipients.

[0016] The concentration of the small molecule activator targeting aromatase in the pharmaceutical composition is from 0.1 μM to 10 μM.

[0017] The pharmaceutical composition is an injectable preparation for local administration to the central nervous system.

[0018] Preferably, the local administration route to the central nervous system is intraventricular injection, intrathecal injection, or direct intracerebral injection.

[0019] The use of the above-mentioned small molecule activator targeting aromatases in nerve cells, or the above-mentioned pharmaceutical composition for the prevention and / or treatment of ischemic brain injury, in the preparation of a medicament for combined administration with at least one other therapeutic agent for ischemic brain injury.

[0020] Other ischemic brain injury treatment agents are selected from thrombolytic drugs, antiplatelet drugs, or neuroprotective agents.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The application provided by this invention is the first to propose and verify the feasibility of using a "small molecule activator targeting aromatase in nerve cells" for the treatment of ischemic brain injury, filling a gap in the research and development of targeted activators in this field. Existing technologies only offer aromatase inhibitors, while this invention provides the first small molecule compound (HY-N4177) that directly activates aromatase activity without significantly affecting its gene transcription, offering a novel drug target and treatment strategy for neuroprotection.

[0022] Furthermore, the small molecule activator precisely enhances local estrogen levels in the brain by specifically activating neuronal aromatase, avoiding side effects such as breast cancer, endometrial hyperplasia, and thrombosis associated with systemic estrogen replacement therapy. In vivo experiments show that local intracerebral administration (e.g., lateral ventricle injection) significantly increases estrogen concentration in the target brain region, while systemic administration has a relatively mild effect on circulating estrogen, demonstrating good tissue selectivity and safety. Through multi-level experiments including high-throughput virtual screening, in vitro enzyme activity detection, and gene overexpression / knockdown models, it was confirmed that HY-N4177 can directly enhance aromatase catalytic activity, and the effect is strictly dependent on the presence of this enzyme. In an oxygen-glucose deprivation / reoxygenation (OGD / R) injury model, HY-N4177 significantly improves neuronal survival and promotes endogenous estrogen synthesis, exhibiting a clear neuroprotective function.

[0023] The pharmaceutical composition provided by this invention can be formulated as an injectable preparation suitable for local administration to the central nervous system (such as intraventricular, intrathecal, or intraparenchymal injection), effectively bypassing the blood-brain barrier to achieve targeted drug delivery within the brain. Simultaneously, this compound also has the potential to take effect through systemic administration (such as intraperitoneal injection), providing diverse options for dosage forms and routes of administration for different clinical scenarios. This activator can be used in combination with existing therapies (such as thrombolytic, thrombectomy, or chemotherapy drugs), potentially producing synergistic effects and reducing toxic side effects by enhancing endogenous protective mechanisms, demonstrating broad prospects for clinical translation and combination therapy. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a high-throughput virtual screening process for targeting Mouse Cyp19a1 based on molecular docking. Figure 2 Figure showing the results of Western blot analysis to assess the basal expression levels of aromatases in major cell types within the central nervous system. Figure 3 To construct and optimize an aromatase activity reaction system based on HT22 cells; where a) is the result of optimized substrate concentration determined by enzyme-linked immunosorbent assay (ELISA); b) is the result of optimized aromatase inhibitor concentration in the negative control group determined by CCK8 assay and ELISA; c) is the result of optimized reaction time determined by ELISA. Figure 4 The figure shows the screening results of 41 candidate compounds in the in vitro aromatase system (HY-4177 in the figure is HY-N4177 of this invention); where a represents the screening results of 27 solution compounds; and b represents the screening results of 14 powder compounds. Figure 5 The figure shows the results of the CCK-8 assay to evaluate the cytotoxicity of four initially screened active compounds; (a) is HY-112683; (b) is HY-N4177 (HY-4177 in the figure is the HY-N4177 of this invention); (c) is HY-W021491; (d) is HY-020106; Figure 6 The figure shows the screening results of gradient concentration experiments for four candidate compounds; (a) is HY-112683; (b) is HY-N4177 (HY-4177 in the figure is the HY-N4177 of this invention); (c) is HY-W021491; (d) is HY-020106; Figure 7 The effect of gradient concentrations of HY-N4177 on the survival rate of HT22 cells is shown in Figure a, where a represents the survival rate of HT22 cells treated with different concentrations of HY-N4177 (maximum 50 μM) and TeS (HY-4177 in the figure is the HY-N4177 of this invention), b represents the survival rate of HT22 cells treated with different concentrations of HY-N4177 (maximum 20 μM) and TeS (HY-4177 in the figure is the HY-N4177 of this invention), and c represents the survival rate of HT22 cells treated with different concentrations of HY-N4177 (maximum 10 μM) and TeS (HY-4177 in the figure is the HY-N4177 of this invention). Figure 8 This study validates the effect of HY-N4177 on aromatase activity in astrocytes overexpressing the Mouse Cyp19a1 gene. Figure a shows the real-time quantitative PCR validation of an astrocyte model specifically overexpressing the Mouse Cyp19a1 gene; figure b shows the effect of HY-N4177 on E2 secretion and Cyp19a1 mRNA transcription levels in astrocytes overexpressing Cyp19a1 (HY-4177 in the figure is the HY-N4177 of this invention). Figure 9 This study validates the aromatase-dependent effect of HY-N4177 on neurons with knocked-down Mouse Cyp19a1 gene. Figure a shows the real-time quantitative PCR validation of a neuronal cell model with specifically knocked-down Mouse Cyp19a1 gene; figure b shows that knocking down Cyp19a1 blocks the promoting effect of HY-N4177 on E2 secretion in neurons (HY-4177 in the figure is the HY-N4177 of this invention). Figure 10 To evaluate the effect of HY-N4177 on systemic estrogen levels using an intraperitoneal injection model (HY-4177 in the figure is the HY-N4177 of this invention), where a is a schematic diagram of the intraperitoneal injection HY-N4177 administration experiment, and b is a comparison of serum E2 levels in mice intraperitoneally injected with HY-N4177 at different time points with the Sham group. Figure 11 To evaluate the effect of HY-N4177 on local intracerebral administration using a lateral ventricle injection model (HY-4177 in the figure is the HY-N4177 of this invention), a is a schematic diagram of the construction of the lateral ventricle injection model, b is that lateral ventricle injection of HY-N4177 can significantly increase the local E2 concentration in the hippocampus of mice. Figure 12 To verify the neuroprotective potential of HY-N4177 (HY-4177 in the figure is the HY-N4177 of this invention) using an oxygen-glucose deprivation / reoxygenation (OGD / R) injury model; wherein, a) HY-N4177 can enhance endogenous estrogen synthesis under ischemic-hypoxic injury stress; b) HY-N4177 can improve neuronal survival under ischemic-hypoxic injury stress.

[0025] Figure 13 To verify the neuroprotective effect of HY-N4177 (HY-4177 in the figure is the HY-N4177 of this invention) in an in vivo middle cerebral artery embolism model (MCAO / R); where a) HY-N4177 can reduce brain injury in mice after cerebral ischemia; b) HY-N4177 can improve sensorimotor function in mice after cerebral ischemia. Detailed Implementation

[0026] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0027] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0028] The hippocampal neuron cell line HT22 used in this invention was purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number CL-0409; the astrocyte cell line was purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number CP-R126; the microglia cell line N9 was purchased from Huatuo Cell Bank, catalog number HTX1877; and the oligodendrocyte cell line OLN-93 was purchased from Shanghai Kanglang Biotechnology Co., Ltd., catalog number KL-C1009R.

[0029] Example 1: Virtual screening of lead compounds targeting Mouse Cyp19a1 protein This embodiment aims to utilize computer-aided drug design technology; see appendix. Figure 1 The specific steps for screening potential aromatase activators from small molecule libraries are as follows: Target protein preparation. A predicted 3D structure model of mouse aromatase (Mouse Cyp19a1) was retrieved and downloaded from the AlphaFold protein structure database (database identifier: AF-P28649-F1). The protein structure was then preprocessed using the Protein Preparation Wizard module in the Schrödinger software suite. Preprocessing steps included adding hydrogen atoms to the protein structure to simulate a physiological pH environment, followed by energy minimization optimization using the OPLS2005 force field to correct potential interatomic conflicts and bond angle anomalies. The convergence criterion for the optimization process was set to a root mean square deviation (RMSD) of atomic positions not exceeding 0.30 Å, resulting in an energy-stable and conformationally sound receptor protein structure for subsequent docking.

[0030] A grid file of receptor binding sites for molecular docking was generated. Using the ReceptorGrid Generation module of the Schrödinger software, the active pocket of the Mouse Cyp19a1 protein, as optimized above, was defined. Based on known substrate binding information, the center coordinates of the grid generation were set near key amino acid residues (e.g., sites 309 and 374 corresponding to the substrate binding pocket). A 20 Å × 20 Å × 20 Å cube was constructed, completely covering the predicted active pocket and its surrounding region, to ensure sufficient space for conformational sampling and binding mode exploration by the ligand molecule during docking.

[0031] Small molecule compound libraries were prepared for screening. Two commercially available small molecule compound libraries were selected: the MCE Bioactive Compound Library (HY-L001V, containing 22,628 compounds with known biological activities) and the MCE Fragment Library (HY-L032V, containing 22,447 molecular fragments). The two-dimensional (2D) structures of all molecules in these libraries were imported into the LigPrep module of the Schrödinger software. In this module, each compound was hydrogenated, its possible ionization states were generated (within the pH range of 7.0 ± 2.0), stereoisomers were generated, and preliminary geometry optimization was performed. Finally, the lowest-energy, normalized three-dimensional (3D) structures were output, forming a virtual screening library containing a total of 45,075 pretreated compounds.

[0032] A hierarchical virtual screening workflow was implemented. Using the Virtual ScreeningWorkflow module of the Schrödinger software, molecular docking calculations were performed one by one with the previously generated Mouse Cyp19a1 acceptor grid. First, the High-Throughput Virtual Screening (HTVS) mode in the Glide module was used for preliminary rapid docking. This mode greatly improves the calculation speed while maintaining a certain level of accuracy, and is used to quickly enrich molecules that may bind to the target from a large number of compounds. After docking, all compounds were ranked according to the Glide score (an approximate estimate of the binding free energy), and the compounds with the highest scores in the top 15% were selected to proceed to the next round of fine screening.

[0033] The approximately 6,761 (top 15%) potential compounds identified in the initial screening underwent more precise molecular docking validation. These compounds were then re-docked using Glide's Standard Precision (SP) mode and High Precision (XP) mode, respectively. The SP mode provides more detailed conformational sampling and scoring functions, while the XP mode employs more stringent van der Waals force screening and more precise electrostatic interaction and desolvation effect models, enabling more accurate prediction of ligand-acceptor binding modes and affinities. By analyzing the binding conformations, key interactions (such as hydrogen bonds, π-π stacking, and hydrophobic interactions) in these high-precision docking results, as well as the final Glide XP score, the binding strength and specificity of each compound to the Mouse Cyp19a1 active pocket were comprehensively evaluated.

[0034] Based on a comprehensive scoring system using high-precision docking, 41 candidate small molecule compounds were selected from 45,075 initial compounds. These compounds had the highest Glide XP scores, the most reasonable predicted binding modes, and the most key interactions with the target pocket. These 41 compounds demonstrated strong potential for high-affinity binding to the Mouse Cyp19a1 protein in the computational model. Subsequently, these 41 virtually screened lead compounds (including 27 in solution and 14 in powder form) were chemically synthesized or procured to obtain physical samples, providing a material basis for subsequent in vitro bioactivity verification experiments.

[0035] Table 1: Detailed information on the 41 candidate compounds obtained from virtual screening

[0036] Through the above implementation scheme, this invention establishes an efficient and accurate computer-aided drug discovery process, successfully virtual screening 41 potential activator candidate molecules targeting Mouse Cyp19a1 from a large-scale compound library, as shown in Table 1. This lays a solid foundation for subsequent experimental verification and the discovery of highly active molecules, and significantly improves the efficiency and targeting of drug discovery.

[0037] Example 2: Construction and optimization of an in vitro aromatase activity detection system based on HT22 cells This embodiment aims to establish a stable and sensitive cellular-level aromatase activity detection platform for the initial screening of activators.

[0038] Detection of basal aromatase expression levels: To establish a cell model that can effectively reflect aromatase activity, it is necessary to screen and determine suitable cell vectors for constructing the in vitro reaction system. Western blotting was used to assess the basal expression levels of aromatase in major cell types within the central nervous system. The steps are as follows: 1) Culture hippocampal neuronal cell lines HT22, astrocytes, microglia N9, and oligodendrocytes OLN-93 separately in high-glucose complete medium containing 10% fetal bovine serum. 2) After the four cell lines have filled the culture flasks, discard the complete medium, wash with PBS, remove sediment, and discard the PBS. 3) Add lysis buffer from RIPA (Beyotime Biotechnology Co., Ltd.), and repeatedly scrape the bottom of the culture flask with a cell scraper. Lyse the cells on ice. Repeat the above steps 6 times. 4) Thoroughly lyse the cells using sonication. Collect the lysis buffer. 5) Pre-chill the centrifuge to 4°C, centrifuge at 12000 RPM for approximately 20 minutes, and discard the precipitate. 6) Add loading buffer and boil the lysis buffer. Store at -80°C. 7) Prepare a 10% separating gel and a 5% stacking gel according to the Beyotime gel preparation kit instructions. 8) Determine protein concentration using the BCA method. 9) Load proteins onto the gel, ensuring a protein concentration of 10 μg / well. Set the electrophoresis voltage to 70V initially, then increase to 100V after 30 minutes. 10) Stop electrophoresis once the bromophenol blue reaches the bottom. Add methanol to prepare transfer buffer and pre-cool. Activate the PVDF membrane using methanol. 11) Carefully cut the electrophoresis gel and the corresponding PVDF membrane, and perform wet transfer on ice. Use constant voltage transfer: maintain the transfer voltage at 100V, with a corresponding transfer current of 180mA-220mA. 12) Stop transfer after 90 minutes. Refer to the marker and carefully cut the membrane according to the approximate location of the target band. Wash with TBST and incubate in skim milk at room temperature for 60 minutes. 13) Dilute the primary antibody with the primary antibody dilution buffer, add the corresponding primary antibody (ARO), and incubate the cut PVDF membrane overnight at 4°C. 14) After washing with PBST, add horseradish peroxidase-labeled secondary antibody diluted 1:5000 and incubate at 37°C for 1 hour. 15) After washing with PBST, add ECL luminescent solution, and measure the optical density value of each group after luminescence.

[0039] The results are as follows Figure 2 As shown, the HT22 hippocampal neuron cell line can stably express high levels of aromatase protein, and therefore was selected as an ideal cell vector for constructing an in vitro drug screening model.

[0040] An aromatase activity reaction system based on HT22 cells was constructed and optimized. The core principle of this system is that aromatase catalyzes the conversion of the substrate testosterone to estradiol (E2). The aromatase activity was indirectly assessed by measuring the E2 level in the reaction system using enzyme-linked immunosorbent assay (ELISA). The procedure for system construction and optimization is as follows: 1) HT22 cells in logarithmic growth phase were stored at a density of 2 × 10⁶ cells per well. 41) Cells were seeded at a density of 1000 mcg / mL in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cells were fully adherent. 2) Substrate concentration optimization: Cells were treated with a series of different concentrations of testosterone (1 μM, 10 μM, 20 μM, 50 μM, 100 μM), and the cell culture supernatant was collected at a fixed time point (6 hours). 3) The procedure was strictly followed according to the instructions of the commercial E2 ELISA kit (Shanghai Xitang Biotechnology, catalog number F10440, specification 96T). The collected cell culture supernatant was added to the wells of the antibody-coated ELISA plate, and the E2 in the supernatant was allowed to bind fully with the antibody at 37°C for 60 minutes. After washing to remove unbound material, horseradish peroxidase-labeled detection antibody was added to form a complex. After washing again, the chromogenic substrate TMB was added and incubated in the dark for 15 minutes. 4) The absorbance of each well was measured at a specific wavelength (450 nm), and the precise concentration of E2 in each sample was calculated using a standard curve to determine the optimal substrate concentration. 5) A control group was established. At the determined optimized substrate concentration, cells were treated with a series of different concentrations of the aromatase inhibitor letrozole (1 nM, 10 nM, 100 nM, 1 μM, 10 μM). Cell culture supernatant was collected at a fixed time point (24 h) and analyzed by ELISA to determine the negative control group. 6) The reaction time was optimized. Cell culture supernatant was collected after a series of different treatment times (6 h, 12 h, 24 h) and analyzed by ELISA to determine the appropriate reaction time.

[0041] The results are as follows Figure 3 As shown, after treating HT22 cells with 10 μM testosterone for 6 hours, the E2 content in the culture supernatant was significantly increased compared with the blank control group, and this increase could be completely or mostly inhibited by 1 μM letrozole.

[0042] Through the above implementation scheme, this invention successfully constructed and optimized a stable, reliable, cell-based in vitro aromatase activity detection platform. This platform utilizes aromatase expressed endogenously in HT22 cells. By detecting the efficiency with which aromatase catalyzes the production of E2 from its substrate testosterone, it can indirectly but effectively reflect the regulatory effect (activation or inhibition) of exogenously added small molecule compounds on aromatase activity, providing a key technical means for rapidly screening potential aromatase activators from a large number of candidate molecules.

[0043] Example 3: In vitro screening and toxicity assessment of candidate compounds This embodiment utilizes the above-described screening system to perform preliminary activity screening and safety assessment on 41 candidate compounds obtained from virtual screening. Based on the established and optimized in vitro aromatase reaction system, the steps for bioactivity testing are as follows: (1) Preliminary screening and identification of active compounds HT22 cells in the logarithmic growth phase were divided into groups of 2 × 10⁶ cells per well. 4 1) Cells were evenly seeded at a density of 1000 mcg in 96-well cell culture plates and cultured at 37°C with 5% CO2 until the cells were fully adherent. 2) The 41 candidate compounds obtained from the first stage of virtual screening (27 of which were in solution and 14 were freshly prepared powder) were prepared to working concentrations using cell culture medium or DMSO. 1 μM of a specific candidate compound and 10 μM of testosterone were added to each well sequentially. A substrate control group (containing only 10 μM of the 41 candidate compounds obtained from the virtual screening, including 27 in solution and 14 in powder form) was set up as a baseline, along with a blank control group (containing no compound) and a negative control group (containing 1 μM letrozole) for calibration. All wells were incubated for 6 hours. After incubation, the cell culture supernatant from each well was carefully aspirated, avoiding cell aspiration, and stored at -80°C or immediately for analysis. 3) The effect of each compound on E2 production was analyzed by ELISA. The results are shown below. Figure 4 As shown, compared with the substrate control group, the four compound treatment groups (HY-112683, HY-N4177, HY-W021491, and HY-W020106) showed a statistically significant increase in E2 content in the cell supernatant. This result indicates that these four candidate molecules can effectively enhance the catalytic activity of aromatase in HT22 cells at a concentration of 1 μM, suggesting their potential as aromatase activators.

[0044] (2) Cytotoxicity assessment To rule out the possibility that the observed E2 elevation was due to cellular stress caused by compound toxicity and to ensure the safety of the candidate compounds, cytotoxicity tests were performed on the four initially screened active compounds. The CCK-8 assay was used for evaluation, and the specific method is as follows: 1) Prepare a single-cell suspension of HT22 cells in the logarithmic growth phase, using 2 × 10⁶ cells per well. 4 Cells were evenly seeded at a density of 100 μg / well in 96-well cell culture plates and incubated at 37°C with 5% CO2 until the cells were fully adherent. A blank control group (containing only culture medium, no cells), a substrate-free control group, a control group, and an experimental group were set up, with 5 replicates for each group. 2) Different concentrations (100 nM, 1 μM, 10 μM, 50 μM) of HY-112683, HY-N4177, HY-W021491, and HY-W020106 were added, and the cells were incubated for another 6 hours. 3) An appropriate amount of CCK8 solution (10% of the culture medium volume) was added to each well, gently shaken to mix, avoiding the formation of air bubbles, and then returned to the incubator for 1 hour in the dark. 4) The absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0045] Experimental results are as follows Figure 5 As shown, at a concentration of 10 μM, the cell viability of the four compound treatment groups did not decrease significantly compared with the substrate control group, indicating that they have no obvious cytotoxicity within the effective concentration range and have good biocompatibility.

[0046] (3) Dosage effect and confirmation of preferred compounds To further confirm the reliability of the agonistic effects of the candidate compounds and screen for molecules with superior performance, a more refined gradient experiment was designed for four molecules: HY-112683, HY-N4177, HY-W021491, and HY-W020106. This screening was conducted in an HT22 cell system, with two variables: first, a fixed compound concentration (1 μM) was used while varying the concentration of the substrate testosterone (10 μM, 20 μM, and 50 μM gradients); second, a fixed testosterone concentration (10 μM) was used while varying the concentration of the compound itself (0.1 μM, 1 μM, 10 μM, and 50 μM gradients). After 6 hours of treatment under each condition, the supernatant was collected for ELISA detection. The results are as follows: Figure 6 As shown, among the four compounds, brassinolide gentioside HY-N4177 exhibited the most prominent and stable performance. At a fixed compound concentration, the amount of E2 produced in the HY-N4177-treated group increased with increasing substrate testosterone concentration, indicating that its agonistic effect was effective under different substrate conditions. At a fixed substrate concentration, the amount of E2 generated also showed a clear concentration-dependent increasing trend with increasing HY-N4177 concentration (from 0.1 μM to 10 μM), strongly demonstrating that the agonistic effect of HY-N4177 on aromatase is an inherent and controllable pharmacological characteristic. Furthermore, as... Figure 7 As shown, at a concentration of 10 μM, HY-N4177 did not cause a significant decrease in the survival rate of HT22 cells, confirming its good cell safety within the effective concentration range.

[0047] Example 4: In vitro verification of the mechanism of action of HY-N4177 This embodiment elucidates the molecular mechanism by which HY-N4177 directly stimulates aromatase proteins using a genetic gain-loss function model. The direct stimulatory mechanism of HY-N4177 on aromatase was verified using an in vitro gene-modified cell model.

[0048] (1) Overexpression model validates direct activation effect The direct effect of HY-N4177 on enzyme activity was verified using an overexpression model. To clarify whether HY-N4177 acts directly on the aromatase itself rather than by upregulating its gene expression, an astrocyte model specifically overexpressing the Mouse Cyp19a1 gene was constructed as follows: 1) Prepare a single-cell suspension of astrocytes in the logarithmic growth phase and evenly seed them into cell culture dishes. Incubate at 37°C with 5% CO2. 2) Once cells have fully adhered, discard the old culture medium and add serum-free medium containing a virus solution specifically overexpressing the Mouse Cyp19a1 gene (MOI=10). Incubate at 37°C for 6-8 hours, gently shaking the culture plate every 2 hours to ensure even virus contact with the cells. 3) After incubation, replace the medium with complete medium containing 10% FBS and continue culturing for 48-72 hours. During this period, observe the proportion of positive cells under a fluorescence microscope. 4) Verify the overexpression efficiency of Cyp19a1 mRNA using real-time quantitative PCR (qPCR). 5) Repeat the above method to construct an astrocyte cell line model specifically overexpressing the MouseCyp19a1 gene. Transfected cells were divided into different treatment groups: empty vector + solvent control group, empty vector + 1 μM HY-N4177 group, Cyp19a1 overexpression + solvent control group, and Cyp19a1 overexpression + 1 μM HY-N4177 group. After treatment with 10 μM testosterone and 1 μM HY-N4177 (or an equal volume of solvent) for 6 hours, cell supernatants were collected for ELISA to detect E2 content. 4) Simultaneously, RNA was extracted from cells for qPCR to detect the transcriptional level of Cyp19a1.

[0049] The results are as follows Figure 8 As shown, in cells overexpressing Cyp19a1, the E2 content in the cell supernatant of the HY-N4177-treated group was significantly higher than that of the untreated overexpression control group; however, qPCR analysis revealed that HY-N4177 treatment did not significantly alter the mRNA transcription level of Cyp19a1. This result suggests for the first time that HY-N4177 increases E2 synthesis by directly enhancing enzyme protein activity, rather than promoting gene transcription.

[0050] (2) Knock down the model validation target dependency The target specificity of its action was verified using a gene knockdown model. To further confirm that the action of HY-N4177 is entirely dependent on the presence of aromatase, a neuronal cell model with specific knockdown of the Mouse Cyp19a1 gene was constructed as follows: 1) Prepare a single-cell suspension of HT22 cells in logarithmic growth phase, evenly seed them in cell culture dishes, and incubate at 37℃ with 5% CO2. 2) After complete cell adhesion, discard the old culture medium and add serum-free medium containing a virus solution specifically knocking down the Mouse Cyp19a1 gene (MOI=10). Incubate at 37℃ for 6-8 h, gently shaking the culture plate every 2 h to ensure uniform virus contact with the cells. 3) After incubation, replace with complete medium containing 10% FBS and continue culturing for 48–72 h. During this period, observe the proportion of positive cells under a fluorescence microscope. 4) Verify the knockdown efficiency of Cyp19a1 mRNA using real-time quantitative PCR (qPCR). 5) Repeat the above method to construct a neuronal cell line model specifically knocking down the Mouse Cyp19a1 gene. Divide the transfected cells into different treatment groups: empty vector control group, empty vector + HY-N4177 group, knockdown control group, and knockdown + HY-N4177 group. After treating each group of cells with 10 μM substrate testosterone and 1 μM HY-N4177 (or an equal volume of solvent) for 6 hours, the cell supernatant was collected for ELISA to detect E2 content. 4) At the same time, RNA was extracted from the cells for qPCR to detect the transcriptional level of Cyp19a1.

[0051] The results are as follows Figure 9 As shown, in the control group, HY-N4177 significantly increased E2 levels; however, in neurons with successfully knocked-down Cyp19a1, regardless of the addition of HY-N4177, the E2 content in the cell supernatant remained at a low level, and the agonistic effect of HY-N4177 completely disappeared. This result corroborates the overexpression experiment, strongly demonstrating that the small molecule compound HY-N4177 functions by directly stimulating the catalytic activity of aromatase (a product of the Cyp19a1 gene), and its effect is strictly dependent on the presence of aromatase protein and does not involve the regulation of Cyp19a1 gene transcription levels.

[0052] Through the above implementation scheme, this invention utilizes in vitro cell models with genetically enhanced function (overexpression) and lost function (knockdown) to conclusively demonstrate the mechanism of action of the candidate molecule HY-N4177 from both positive and negative perspectives: it directly acts on aromatase proteins, enhancing their catalytic activity in converting testosterone to E2 without affecting the enzyme's transcriptional synthesis process. This mechanism elucidation provides crucial experimental evidence for the localization of HY-N4177 as a direct activator of aromatase.

[0053] Example 5: In vivo pharmacodynamic evaluation of HY-N4177 via different routes of administration This embodiment evaluates the in vivo bioactivity of HY-N4177 under different routes of administration. In vivo experiments using different routes of administration were conducted to verify the efficacy of HY-N4177 and determine the optimal administration method.

[0054] (1) Effects of intraperitoneal injection on systemic estrogen levels An intraperitoneal injection model was established to evaluate its effect on systemic estrogen levels. Adult male C57BL / 6J mice aged 6-8 weeks with similar body weights were randomly divided into a sham-operated group (Sham group) and a HY-N4177 intraperitoneal injection group. Mice in the sham-operated group received HY-N4177 (dissolved in a suitable solvent, such as physiological saline containing a small amount of DMSO) twice daily (approximately 12 hours apart) via intraperitoneal injection at a dose of 1 mg / kg body weight. Mice in the Sham group received an equal volume of the solvent. Administration continued for 7 days. The concentration of E2 in orbital serum samples was measured and evaluated using ELISA on days 1, 3, 5, and 7. The ELISA kit steps are as follows: 1) Remove the Mouse E2 ELISA kit from the refrigerator 20 minutes beforehand and allow it to equilibrate to room temperature. 2) Take the serum sample separated from the mouse orbital blood (after blood collection, allow it to stand at room temperature for 30 min, centrifuge at 4℃ and 3000 r / min for 15 min, collect the supernatant, and aliquot at -80℃), thaw it at room temperature, and gently mix. 3) Dilute the concentrated washing buffer with double-distilled water (1:20) and mix well. 4) Take the E2 standard and dilute it to a gradient concentration using sample diluent. 5) Take the 96-well plate pre-coated with E2 antibody, set up standard wells, sample wells, and blank wells (with sample diluent). Add 50 μL of standard or sample to each well, and immediately add 50 μL of HRP enzyme-conjugating antibody working solution. After adding the samples, seal the reaction wells with sealing film and incubate at 37℃ for 60 minutes. 6) Wash 5 times with washing buffer. 7) Add 90 μL of substrate solution to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes. 8) Add 50 μL of stop solution to each well and immediately measure the OD value at 450 nm. 9) Calculate the corrected OD value for the standard and sample wells: Measured OD value - Average OD value of blank wells. Serum E2 levels in the drug-treated group at each time point were compared with those in the Sham group at the same time point. Results are as follows: Figure 10 As shown, compared with the Sham group, mice that received continuous intraperitoneal injections of HY-N4177 exhibited a significant increasing trend in serum E2 levels starting from day 3 of administration, maintaining a high level on days 5 and 7. This indicates that HY-N4177, administered intraperitoneally, can systematically increase estrogen levels in the body, preliminarily confirming its biological activity in vivo.

[0055] (2) Effect of intraventricular injection on local estrogen levels in the brain Considering that the treatment of brain diseases requires effective drug delivery to the central nervous system, and systemic administration may be limited by the blood-brain barrier (BBB), this patent further establishes a lateral ventricle injection model to evaluate the effect of local intracerebral administration. Using 6-8 week old adult male C57BL / 6J mice, a solution containing 2 μM HY-N4177 was precisely injected into the lateral ventricle of the mice using a stereotaxic instrument, once daily for 7 consecutive days. A sham-operated group (injected with an equal volume of artificial cerebrospinal fluid) was established. Mice were sacrificed on days 1, 3, 5, and 7 after administration, and hippocampal tissue was rapidly isolated. The hippocampal tissue was homogenized in pre-cooled buffer, centrifuged, and the supernatant was collected to obtain tissue lysate. The E2 content in the hippocampal tissue lysate was detected by ELISA, an indicator that more directly reflects the estrogen synthesis in the brain (especially the hippocampus, which is closely related to learning and memory). Results are as follows: Figure 11 As shown: Compared with the Sham group, the E2 content in the hippocampus of mice injected with HY-N4177 in the lateral ventricle showed a very significant increase on the 3rd day of administration, and the increase was much greater than that in the intraperitoneal injection group.

[0056] Comparing the results of the two administration routes revealed that while intraperitoneal injection increased E2 levels in systemic circulation, intraventricular injection more rapidly and significantly increased local E2 concentrations in the target brain region (hippocampus). This strongly suggests that systemic administration of HY-N4177 may be somewhat hindered by the blood-brain barrier, while direct intracerebral administration can bypass this barrier, allowing the drug to act more efficiently on aromatases in the central nervous system. Therefore, based on the combined in vitro and in vivo experimental data, intraventricular injection (or a similar local central administration route) is a better administration method for HY-N4177 to treat central nervous system diseases such as ischemic brain injury. This indicates that local intracerebral administration can more efficiently deliver the drug to the target area and significantly increase local estrogen levels in the brain, suggesting it as a superior administration route for treating central nervous system diseases.

[0057] Example 6: Validation of the neuroprotective effect of HY-N4177 in an oxygen-glucose deprivation / reoxygenation (OGD / R) model This embodiment verifies the neuroprotective function of HY-N4177 in a cell model simulating cerebral ischemia. The neuroprotective potential of HY-N4177 is also verified in an oxygen-glucose deprivation / reoxygenation (OGD / R) injury model. The classic OGD / R model was used to simulate the pathological environment of ischemic brain injury to evaluate the role of the candidate activator HY-N4177 in the injury model. The specific methods are as follows: 1) Prepare a single-cell suspension of HT22 cells in the logarithmic growth phase, seed them evenly in cell culture dishes, and incubate them in an incubator at 37°C and 5% CO2. 2) After the cells have fully adhered, remove the normal culture medium and replace it with a sugar-free balanced salt solution. Place the cell culture dishes in a triple-gas incubator filled with 94% N2, 5% CO2, and 1% O2, and incubate at 37°C for 3 hours to induce moderate injury. 3) After OGD treatment, set up an OGD / R model group (complete culture medium replaced), an OGD / R + substrate control group (substrate replaced + complete culture medium), and an OGD / R + HY-N4177 treatment group (substrate replaced + 1 μM HY-N4177 + complete culture medium). Return the cells to normal culture conditions (37°C, 5% CO2) for further culture to establish a reoxygenation injury model. 4) At specific time points (6h, 12h, and 24h) after reoxygenation culture, cell culture supernatant was collected, and the E2 content was detected using ELISA to assess whether HY-N4177 could still effectively promote the secretion of brain-derived estrogen by nerve cells under injury stress. 5) At specific time points (6h, 12h, and 24h) after reoxygenation culture, cell viability was detected using the CCK-8 assay.

[0058] The results are as follows Figure 12 As shown, compared with the normal control group, the E2 content in the cell supernatant of the OGD / R model group changed due to cell damage, and the cell survival rate decreased significantly. However, in the OGD / R+HY-N4177 treatment group, the E2 content in the cell culture supernatant was significantly higher than that of the OGD / R model group. This confirms that even under the stress of ischemic hypoxia, HY-N4177 can effectively activate aromatase in neurons and promote local estrogen synthesis. The cell survival rate in this group was significantly higher than that in the OGD / R model group. This result directly links the aromatase activating activity of HY-N4177 to its neuroprotective function, demonstrating that enhancing endogenous estrogen synthesis through this compound can effectively combat ischemic injury simulated by OGD / R and improve neuronal survival. HY-N4177, by activating aromatase and enhancing endogenous estrogen synthesis, exhibits a clear neuroprotective effect in an in vitro ischemic injury model, providing direct functional evidence for its treatment of ischemic brain injury.

[0059] Example 7: Validation of the neuroprotective effect of HY-N4177 in an in vivo ischemia / reperfusion model (MCAO / R) This embodiment verifies the neuroprotective effect of HY-N4177 in an animal model simulating ischemic stroke. A mouse in vivo middle cerebral artery occlusion (MCAO / R) model, established using the classic suture selection occlusion method, was used to simulate the pathological environment of ischemic brain injury to assess the role of the candidate activator HY-N4177 in the injury model. The specific method is as follows: 1) Six- to eight-week-old SPF-grade male C57BL / 6 mice were selected. After establishing a lateral ventricle injection model, they were randomly divided into a sham-operated group, an MCAO / R model group (WT group), and an MCAO / R+HY-N4177 treatment group. In the model and treatment groups, a middle cerebral artery ischemia / reperfusion (MCAO / R) model was established using the suture occlusion method: a silicone-coated suture was surgically inserted into the origin of the left middle cerebral artery to block blood flow and induce focal cerebral ischemia. After a certain period of ischemia, the suture was removed, restoring blood flow and achieving reperfusion. In the sham-operated group, only the relevant vessels were exposed; no suture was inserted. All other procedures were the same as in the model and treatment groups. 2) At the start of reperfusion, the treatment group received a precise injection of a solution containing 2 μM HY-N4177 into the lateral ventricle of the mice using a stereotaxic instrument, once daily for 7 consecutive days. The model and sham-operated groups received an equal volume of artificial cerebrospinal fluid. 3) A systematic neurobehavioral functional assessment was conducted on day 7 after modeling. This included: a) Neurological deficit scoring: Standard methods such as the Longa scale were used to score the mice's motor function, balance, and forelimb extension. Higher scores indicated more severe deficits. b) Grip strength test: A grip strength meter was used to measure the mice's forelimb grip strength, and the maximum grip strength was recorded in Newtons (N). c) Rotary rod fatigue test: Mice were allowed to walk on an accelerating rotating rotary rod, and the time (in seconds) it took to fall from the rod was recorded to assess their motor coordination and endurance. 4) After the behavioral tests, the mice were euthanized, and a morphological assessment of brain tissue damage was performed. Brain tissue was rapidly removed, and coronal sections were prepared and stained with 2,3,5-triphenyltetrazolium chloride (TTC). Normal brain tissue stained red, while ischemic infarct areas appeared white. The percentage of infarct volume to the ipsilateral cerebral hemisphere volume was calculated using image analysis software.

[0060] The results are as follows Figure 13As shown, the neurological function deficit scores indicated that compared with pre-modeling, the neurological function of MCAO / R modeled mice was significantly impaired, with the treatment group showing less damage than the WT mice. The grip strength test results showed that compared with pre-modeling, the forelimb grip strength of MCAO / R modeled mice was significantly impaired, with the treatment mice showing less damage than the WT mice. The rotundus fatigue test results showed that after 3 days of pre-test training, all mice did not fall off the rotundus within the 5-minute test period before modeling, while the time to fall off the rotundus was significantly shortened after MCAO / R modeling, with the WT mice taking a shorter time than the treatment group. The TTC test results revealed that compared with the WT modeled mice, the infarct volume on the affected side of the brain was significantly reduced in the treatment group. Through the above implementation scheme, this invention comprehensively and powerfully demonstrates, in an animal model that highly simulates clinical ischemic stroke, that HY-N4177 administered via the lateral ventricle can significantly reduce the degree of brain injury after ischemia and promote the recovery of neurological function, providing direct, objective and convincing in vivo functional evidence for its treatment of ischemic brain injury, from multiple dimensions including neurological function, motor ability and brain tissue pathological damage.

[0061] In summary, this invention provides the first experimentally verified small molecule activator (HY-N4177) that can directly activate neuronal aromatase, directly activating enzyme activity without regulating genes; and precisely increasing estrogen levels in the brain through local administration, avoiding the serious side effects of systemic hormone replacement.

[0062] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The use of a small molecule activator targeting aromatase in neural cells in the preparation of a medicament for the prevention and / or treatment of ischemic brain injury, characterized in that, The small molecule activator is a compound having the structure of formula (I) below, or a pharmaceutically acceptable salt, solvate, prodrug, or polymorph thereof; 。 2. The application according to claim 1, characterized in that, The ischemic brain injury is selected from ischemic stroke or cerebral ischemia-reperfusion injury.

3. The application according to claim 1, characterized in that, The drug works by activating aromatase in nerve cells, thereby increasing the level of endogenous estrogen in the brain.

4. The application according to claim 3, characterized in that, The nerve cells mentioned are neurons.

5. The application according to claim 3, characterized in that, The small molecule activator targeting aromatase in nerve cells activates aromatase by directly activating its protein catalytic activity, without significantly upregulating the mRNA expression level of the aromatase-encoding gene Cyp19a1.

6. A pharmaceutical composition for the prevention and / or treatment of ischemic brain injury, characterized in that, It contains a therapeutically effective amount of the small molecule activator that targets aromatases of nerve cells as described in any one of claims 1-6, as well as other pharmaceutically acceptable carriers or excipients.

7. A pharmaceutical composition for the prevention and / or treatment of ischemic brain injury according to claim 6, characterized in that, The concentration of the small molecule activator targeting aromatase in the pharmaceutical composition is from 0.1 μM to 10 μM.

8. A pharmaceutical composition for the prevention and / or treatment of ischemic brain injury according to claim 6, characterized in that, The pharmaceutical composition is an injectable preparation for local administration to the central nervous system.

9. A pharmaceutical composition for the prevention and / or treatment of ischemic brain injury according to claim 8, characterized in that, The local administration routes to the central nervous system are intraventricular injection, intrathecal injection, or direct intracerebral injection.

10. The use of a small molecule activator targeting aromatase of nerve cells in any one of claims 1 to 6, or a pharmaceutical composition for the prevention and / or treatment of ischemic brain injury as described in any one of claims 7 to 9, in the preparation of a medicament for administration in combination with at least one other ischemic brain injury treatment agent; wherein the other ischemic brain injury treatment agent is selected from thrombolytic drugs, antiplatelet drugs, or neuroprotective agents.

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