Application of small molecule activator of targeted nerve cell aromatase in preparation of medicine for treating cerebral ischemia injury

Abstract

The invention discloses a small molecule activator targeting nerve cell aromatase and application of the small molecule activator in cerebral ischemia 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 of aromatase, so that the local estrogen level in the brain is accurately improved, and the neuroprotection effect is achieved. The neuron aromatase is specifically activated, so that the side effect of systemic estrogen treatment is avoided. In-vivo and in-vitro experiments prove that the concentration of estrogen in a target region can be effectively improved through local intracerebral administration, and the survival rate of neurons in an oxygen-glucose deprivation / reoxygenation injury model is remarkably improved. The pharmaceutical composition provided by the invention is suitable for local administration or systemic administration of a central nervous system, can be combined with the existing therapy, and provides a brand new treatment strategy and drug candidate for cerebral ischemia injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of small molecule activators targeting aromatases in nerve cells in the preparation of drugs for treating cerebral ischemia-reperfusion injury. Background Technology

[0002] Ischemic stroke (IS), the most prevalent clinical type of acute brain injury, has a complex pathophysiological mechanism involving a cascade of reactions including energy metabolism failure, excitatory amino acid toxicity, oxidative stress, inflammatory response, and apoptosis. Globally, the incidence of ischemic stroke remains high. In my country, with the accelerating aging of the population and the prevalence of unhealthy lifestyles, the onset of ischemic stroke is showing a trend towards younger ages, making its prevention and control increasingly challenging and a major public health problem urgently needing to be addressed. Therefore, exploring more effective and safer neuroprotective strategies to salvage neurons nearing death in the ischemic penumbra is a core research direction for improving patient prognosis and reducing the social disease burden. Currently, clinical treatment of ischemic stroke mainly focuses on blood flow recanalization. Intravenous thrombolysis (such as recombinant tissue plasminogen activator) and endovascular mechanical thrombectomy are currently the mainstream methods for restoring cerebral perfusion. However, both have strict treatment time windows (usually within 4.5 or 6 hours after onset) and carry serious risks such as hemorrhagic transformation and reperfusion injury, preventing many patients from benefiting due to delayed treatment or contraindications. Even after thrombolysis or thrombectomy, approximately 50% of patients still have poor prognoses after vascular recanalization. Regarding drug development targeting neuroprotection itself, although many promising targets have been identified in basic research, such as N-methyl-D-aspartate (NMDA) receptor antagonists, free radical scavengers, and anti-inflammatory drugs, most have failed to translate into effective clinical therapies in clinical trials. This is mainly attributed to the blood-brain barrier, insufficient drug targeting, systemic side effects, and the difficulty in precisely intervening in complex pathological networks. Therefore, a significant gap exists in the current treatment of ischemic brain injury: the lack of targeted drugs that can efficiently and specifically activate the brain's endogenous protective mechanisms, thereby directly enhancing neuronal survival at the cellular level.

[0003] In recent years, neuroendocrinology research has provided new perspectives on neuroprotection against ischemic brain injury. Estrogen, as a classic sex hormone, has been proven to play a crucial neuroprotective role in the central nervous system. Numerous preclinical studies have shown that both exogenous supplementation and endogenous elevation of estrogen can significantly reduce ischemic brain injury and improve neurological deficits. Its protective mechanisms involve multiple aspects, including anti-apoptosis, anti-inflammation, anti-oxidation, promotion of synaptic plasticity, and maintenance of mitochondrial function. The brain itself has the ability to synthesize estrogen, a process that depends on a key rate-limiting enzyme—aromatase (AROM, encoded by the CYP19A1 gene). Brain-derived estrogens, especially estradiol, which is converted from testosterone by aromatase in neurons, are considered to play an irreplaceable protective role in local, rapid responses to ischemic stress. While direct supplementation with exogenous estrogen has some effect, it also has several drawbacks: First, long-term or systemic use increases the risk of hormone-dependent tumors such as breast cancer and endometrial cancer; second, it may produce adverse endocrine effects such as feminization in male patients, limiting its applicability; and third, it is difficult to simulate the local, timely, and appropriate estrogen secretion patterns in the brain under physiological conditions, easily disrupting systemic hormonal homeostasis. Therefore, how to safely and effectively regulate this endogenous protective pathway and transform it into a practical treatment method remains a key technical bottleneck in this field.

[0004] Aromatase, encoded by the CYP19A1 gene, belongs to the cytochrome P450 superfamily and its function is to convert androstenedione and testosterone into estrone and estradiol, respectively. In the brain, aromatase is mainly expressed in neurons of specific regions. Previous studies have confirmed that aromatase activity in neurons changes after brain ischemia-reperfusion injury, but the specific regulatory mechanisms are complex. Existing research mainly focuses on verifying the importance of the aromatase pathway. The study by Xu et al. (2025) revealed that aromatase expression was significantly increased in penumbra reactive astrocytes after acute brain ischemia-reperfusion injury, and that aromatase exerts a neuroprotective effect by regulating A1 / A2 phenotypic differentiation of astrocytes through estrogen receptor 2 (ESR2). However, the intervention methods in this type of research are mainly gene knockout or downstream receptor agonists (such as the ESR2 agonist DPN), and have not involved the screening or verification of small molecule activators that directly target aromatase itself.

[0005] Currently known aromatase modulators are all inhibitors, such as letrozole and anastrozole, which are mainly used to treat estrogen-dependent breast cancer. There is no systematic research and development targeting aromatase activators. Summary of the Invention

[0006] To address the technical problem that the lack of small molecule compounds in the existing technology that can specifically activate neuronal aromatases, thus preventing the effective neuroprotection of ischemic brain injury by precisely increasing the level of endogenous estrogen in the brain, this invention aims to provide the application of small molecule activators that target neuronal aromatases in the preparation of drugs for treating ischemic brain injury.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof in the preparation of a medicament for the prevention and / or treatment of ischemic brain injury; .

[0008] The ischemic brain injury referred to ischemic stroke, transient ischemic attack, cerebral ischemia-reperfusion injury, vascular dementia, or post-ischemic cerebral inflammation. By activating aromatase and upregulating endogenous estrogen, the above-mentioned multiple pathological cascade reactions can be regulated from upstream, showing broad-spectrum therapeutic potential.

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

[0010] The compound represented by formula (I) is a specific small molecule activator of neuronal aromatase (CYP19A1).

[0011] The compound represented by formula (I) exerts a neuroprotective effect by directly activating the catalytic activity of aromatase, thereby promoting the local synthesis of endogenous brain estrogens.

[0012] Preferably, the endogenous brain estrogen is 17β-estradiol.

[0013] The compound shown in formula (I) activates aromatase by directly activating its protein catalytic activity, without significantly upregulating the mRNA expression level of the aromatase encoding gene Cyp19a1.

[0014] The drug is administered via local intracerebral delivery.

[0015] Preferably, the local intracerebral administration method is selected from lateral ventricle injection, intrathecal injection, or stereotactic injection.

[0016] In a second aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of ischemic brain injury, comprising a therapeutically effective amount of a compound as shown in formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0017] Preferably, the concentration of the compound of formula (I) or its pharmaceutically acceptable salt, solvate, prodrug or polymorph in the pharmaceutical composition is from 0.1 μM to 10 μM.

[0018] The pharmaceutical composition is an injectable preparation for local administration to the central nervous system.

[0019] Preferably, the local administration route to the central nervous system is intraventricular injection, intrathecal injection, or direct intracerebral injection.

[0020] The use of the compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for administration in combination with at least one other ischemic brain injury treatment agent.

[0021] Preferably, the other ischemic brain injury treatment agents are selected from thrombolytic drugs, antiplatelet drugs, or neuroprotective agents.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention, through multi-level experiments including high-throughput virtual screening, in vitro enzyme activity detection, and gene overexpression / knockdown models, has for the first time discovered the novel function of compound (I) (HY-112683) as a specific activator of neuronal aromatase, with its effect strictly dependent on the presence of this enzyme. Its application in the prevention and treatment of ischemic brain injury fills the technological gap in this field, which lacks small-molecule drugs that directly activate aromatase. Pharmaceutically acceptable salts, solvates, prodrugs, or polymorphs of this compound can be applied in pharmaceutical form for the prevention and treatment of ischemic brain injury, expanding the druggability of the compound and providing technological space for drug formulation optimization. It overcomes the technical deficiency of existing technologies in lacking specific neuronal aromatase activators, achieving precise, safe, and multi-target endogenous neuroprotection of the brain, and providing a new direction for drug development in the clinical treatment of ischemic brain injury.

[0023] Furthermore, this compound selectively acts on aromatase in neurons, rather than broadly activating other estrogen synthases or receptors, thereby achieving precise regulation and precisely increasing local estrogen levels in the brain. This avoids the side effects of systemic estrogen replacement therapy, such as breast cancer, endometrial hyperplasia, and thrombosis. The compound's mechanism of action is: direct activation of aromatase catalytic activity → promotion of local synthesis of endogenous brain estrogen → neuroprotective effect, forming a complete causal chain. This differs from indirect mechanisms of action that upregulate gene expression or activate upstream signaling pathways. This compound does not affect the transcriptional level of the Cyp19a1 gene and acts directly on the enzyme protein.

[0024] Furthermore, HY-112683 exhibits excellent tissue selectivity: local intracerebral administration (e.g., lateral ventricle injection) significantly increases estrogen concentration in the target brain region while having a mild effect on systemic circulating estrogen levels. This characteristic allows for precise enhancement of local estrogen levels in the brain, while effectively avoiding serious side effects such as breast cancer, endometrial hyperplasia, and thrombosis associated with systemic estrogen replacement therapy, demonstrating good tissue selectivity and safety. As a known ASCT2 inhibitor, HY-112683 has a well-established physicochemical properties, pharmacokinetic characteristics, and safety data, providing favorable conditions for rapid clinical translation.

[0025] The pharmaceutical composition for the prevention and / or treatment of ischemic brain injury provided by this invention transforms the active ingredient into a specific pharmaceutical composition form, providing a direct technical solution for industrial production and clinical application. By defining "one or more pharmaceutically acceptable carriers or excipients," suitable excipients can be selected for different routes of administration (such as injection or local administration), optimizing the stability, solubility, and bioavailability of the formulation.

[0026] The compounds / compositions of this invention, when used in combination with existing ischemic brain injury treatments, activate endogenous neuroprotective mechanisms. Combined use with thrombolytic drugs (to restore blood flow), antiplatelet drugs (to prevent recurrence), and neuroprotective agents (for multi-target intervention) may produce synergistic effects. Through combined administration, the dosage of single drugs may be reduced, thereby decreasing toxic side effects. This meets the practical needs of multi-drug combination therapy in clinical practice, enhancing the commercial value and clinical acceptance of this invention. Attached Figure Description

[0027] 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; where a represents the screening results of 27 solution compounds and b represents the screening results of 14 powder compounds. Figure 5The 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; (c) is HY-W021491; and (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; (c) is HY-W021491; and (d) is HY-020106. Figure 7 The graph shows the cytotoxicity results of the HY-112683 gradient concentration experiment; Figure 8 To verify the direct agonistic effect of HY-112683 on aromatase using an astrocyte model specifically overexpressing the Mouse Cyp19a1 gene; where a is the real-time quantitative PCR verification image of the astrocyte model specifically overexpressing the Mouse Cyp19a1 gene; b is the effect of HY-112683 promoting E2 secretion in astrocytes overexpressing Cyp19a1 without affecting the Cyp19a1 mRNA transcription level; Figure 9 To verify the aromatase-dependent effect of HY-112683 using a neuronal cell model with specific knockdown of the Mouse Cyp19a1 gene; where a is a real-time quantitative PCR verification image of the neuronal cell model with specific knockdown of the Mouse Cyp19a1 gene; b is the effect of knockdown of Cyp19a1 on blocking the promoting effect of HY-112683 on E2 secretion in neuronal cells. Figure 10 To evaluate the effect of HY-112683 on systemic estrogen levels using an intraperitoneal injection model; Figure 11 To evaluate the effect of HY-112683 on local intracerebral administration using a lateral ventricle injection model, a is a schematic diagram of the lateral ventricle injection model, and b is a diagram showing that lateral ventricle injection of HY-112683 can significantly increase the local E2 concentration in the hippocampus of mice. Figure 12 To verify the neuroprotective potential of HY-112683 using an oxygen-glucose deprivation / reoxygenation (OGD / R) injury model; where a) HY-112683 enhances endogenous estrogen synthesis under ischemic-hypoxic stress; and b) HY-112683 improves neuronal survival under ischemic-hypoxic stress.

[0028] Figure 13To verify the neuroprotective effect of HY-112683 in an in vivo middle cerebral artery embolism (MCAO / R) model; where a) HY-112683 can alleviate brain injury in mice after cerebral ischemia-reperfusion injury; and b) HY-112683 can improve sensorimotor function in mice after cerebral ischemia-reperfusion injury. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Example 1: Virtual screening of lead compounds targeting Mouse Cyp19a1 protein and discovery of HY-112683 This embodiment aims to screen potential aromatase activators from a small molecule library using computer-aided drug design technology. The specific steps 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Table 1: Detailed information on the 41 candidate compounds obtained from virtual screening

[0039] 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.

[0040] Among the 41 candidate compounds obtained from the virtual screening above, compound HY-112683 (V-9302) is one of the specific examples of the compound shown in formula (I), with CAS number 1855871-76-9 and molecular formula C. 34 H 38 N2O4. This compound was further verified in subsequent examples as a specific small-molecule activator of neuronal aromatases.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 3: In vitro screening and activity evaluation of HY-112683 This embodiment utilizes the in vitro aromatase reaction system established and optimized in Example 2 to perform initial activity screening and safety assessment on 41 candidate compounds obtained from virtual screening. The experimental steps 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.

[0048] (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.

[0049] Experimental results are as follows Figure 5 As shown, at a concentration of 10 μM, the cell viability of the HY-112683 treatment group did not decrease significantly compared with the substrate control group, indicating that it has no obvious cytotoxicity within the effective concentration range and has good biocompatibility.

[0050] (3) Dosage effect and confirmation of preferred compounds To further confirm the reliability of the agonist effect of HY-112683, a gradient concentration experiment was designed. This screening was conducted in an HT22 cell system, with two variables: first, a fixed compound concentration (1 μM) was used, while the concentration of the substrate testosterone was varied (three gradients: 10 μM, 20 μM, and 50 μM); second, a fixed substrate testosterone concentration (10 μM) was used, while the concentration of the compound itself was varied (four gradients: 0.1 μM, 1 μM, 10 μM, and 50 μM). 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, at a fixed compound concentration, the amount of E2 produced in the HY-112683-treated group increased with increasing substrate testosterone concentration, indicating that its agonistic effect is effective under different substrate conditions. At a fixed substrate concentration, the amount of E2 produced also showed a clear concentration-dependent increasing trend with increasing HY-112683 concentration (from 0.1 μM to 10 μM), strongly demonstrating that the agonistic effect of HY-112683 on aromatase is an inherent and controllable pharmacological characteristic. Furthermore, as... Figure 7 As shown, at a concentration of 1 μM, HY-112683 did not cause a significant decrease in the survival rate of HT22 cells, confirming its good cell safety within the effective concentration range.

[0051] Example 4: In vitro verification of the mechanism of action of HY-112683 This embodiment elucidates the molecular mechanism by which HY-112683 directly stimulates aromatase proteins using a genetic gain-loss function model. The direct stimulatory mechanism of HY-112683 on aromatase was verified using an in vitro gene-modified cell model.

[0052] (1) Overexpression model validates direct activation effect The direct effect of HY-112683 on enzyme activity was verified using an overexpression model. To clarify whether HY-112683 acts directly on 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-112683 group, Cyp19a1 overexpression + solvent control group, and Cyp19a1 overexpression + 1 μM HY-112683 group. After treatment with 10 μM testosterone and 1 μM HY-112683 (or an equal volume of solvent) for 6 hours, cell supernatants were collected for ELISA to detect E2 levels. 4) Simultaneously, RNA was extracted from cells for qPCR to detect the transcriptional level of Cyp19a1.

[0053] The results are as follows Figure 8 As shown, in cells overexpressing Cyp19a1, the E2 content in the cell supernatant of the HY-112683-treated group was significantly higher than that of the untreated overexpression control group; however, qPCR analysis revealed that HY-112683 treatment did not significantly alter the mRNA transcription level of Cyp19a1. This result suggests for the first time that HY-112683 increases E2 synthesis by directly enhancing enzyme protein activity, rather than promoting gene transcription.

[0054] (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-112683 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-112683 group, knockdown control group, and knockdown + HY-112683 group. After treating each group of cells with 10 μM substrate testosterone and 1 μM HY-112683 (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.

[0055] The results are as follows Figure 9 As shown, in the control group, HY-112683 significantly increased E2 levels; however, in neurons with successfully knocked-down Cyp19a1, regardless of the addition of HY-112683, the E2 content in the cell supernatant remained at a low level, and the agonistic effect of HY-112683 completely disappeared. This result corroborates the overexpression experiment, strongly demonstrating that the small molecule compound HY-112683 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 the aromatase protein and does not involve the regulation of Cyp19a1 gene transcription levels.

[0056] Through the above-described 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-112683 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. This mechanism elucidation provides crucial experimental evidence for the localization of HY-112683 as a direct activator of aromatase.

[0057] (3) Multi-target characteristic analysis of HY-112683 HY-112683 (V-9302) is a known highly selective competitive inhibitor of ASCT2 (SLC1A5) (Schulz et al., J Biol Chem, 2019). ASCT2 is a glutamine transporter that plays an important role in tumor cell metabolism. This invention is the first to discover that this compound also possesses a novel activity of activating aromatase.

[0058] This discovery demonstrates that HY-112683 possesses a dual function of multi-target regulation of cellular metabolism and neuroprotection: on the one hand, it promotes endogenous estrogen synthesis by activating aromatase, exerting anti-inflammatory and anti-apoptotic effects; on the other hand, it regulates glutamine metabolism by inhibiting ASCT2, affecting the energy balance of nerve cells. This multi-target characteristic provides new possibilities for combined treatment strategies for ischemic brain injury and offers new research directions for further in-depth exploration of its neuroprotective mechanisms.

[0059] Example 5: In vivo pharmacodynamic evaluation of HY-112683 via different routes of administration This embodiment evaluates the in vivo bioactivity of HY-112683 under different routes of administration. In vivo experiments using different routes of administration were conducted to verify the efficacy of HY-112683 and determine the optimal administration method.

[0060] (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-112683 intraperitoneal injection group. Mice in the sham-operated group received HY-112683 (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 develop color 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.

[0061] The results are as follows Figure 10 As shown, compared with the Sham group, mice that received consecutive intraperitoneal injections of HY-112683 did not show a statistically significant increase in serum E2 levels at any of the detection time points. This result indicates that HY-112683, when administered systemically, has difficulty effectively entering the central nervous system or has a relatively mild effect on systemic estrogen levels, suggesting that this compound may be blocked by the blood-brain barrier.

[0062] (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-112683 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 local brain regions (especially the hippocampus, which is closely related to learning and memory). Results are as follows: Figure 11As shown: Compared with the Sham group, the E2 content in the hippocampus of mice injected with HY-112683 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.

[0063] (3) Comparison and Advantage Analysis of the Two Routes of Administration Comparison of the two administration routes revealed that HY-112683 is blocked by the blood-brain barrier when administered systemically, while direct intracerebral administration bypasses this barrier, allowing the drug to act more efficiently and significantly on aromatases in the central nervous system. Therefore, based on in vitro and in vivo experimental data, lateral ventricle injection (or a similar local central administration method) is the optimal administration route for HY-112683 in treating central nervous system diseases such as ischemic brain injury. This strategy precisely delivers the drug to the target area, significantly increasing local estrogen levels in the brain while minimizing systemic exposure and related side effects, demonstrating good tissue selectivity and safety.

[0064] Example 6: Verification of the neuroprotective effect of HY-112683 in an oxygen-glucose deprivation / reoxygenation (OGD / R) model This embodiment verifies the neuroprotective function of HY-112683 in a cell model simulating cerebral ischemia. The neuroprotective potential of HY-112683 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-112683 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-112683 treatment group (substrate replaced + 1 μM HY-112683 + 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-112683 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.

[0065] 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-112683 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-112683 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-112683 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-112683, 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.

[0066] Example 7: Validation of the neuroprotective effect of HY-112683 in an in vivo ischemia / reperfusion model (MCAO / R) This embodiment verifies the neuroprotective effect of HY-112683 in an animal model simulating ischemic stroke. A mouse in vivo middle cerebral artery occlusion (MCAO / R) model, prepared using the classic suture occlusion method, was used to simulate the pathological environment of ischemic brain injury to assess the role of the candidate activator HY-112683 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-112683 treatment group. The model and treatment groups used the suture occlusion method to establish a middle cerebral artery ischemia / reperfusion (MCAO / R) model: 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 to restore blood flow and achieve reperfusion. In the sham-operated group, only the relevant vessels were exposed without inserting a suture; the remaining 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-112683 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.

[0067] The results are as follows Figure 13As shown, the neurological function deficit score indicated that compared with pre-modeling, the MCAO / R model mice exhibited significantly impaired neurological function, with the HY-112683 treatment group showing a significantly reduced degree of impairment compared to the model group (P<0.01). Grip strength testing results showed that compared with pre-modeling, the forelimb grip strength of the mice after MCAO / R modeling was significantly impaired, with the HY-112683 treatment group showing stronger grip strength than the model group (P<0.05). The rotarod fatigue test results showed that the time it took for mice to fall from the rotarod was significantly shortened after MCAO / R modeling, while the fall time in the HY-112683 treatment group was significantly prolonged compared to the model group (P<0.01). TTC testing results revealed that compared with WT model mice, the cerebral infarction volume on the affected side was significantly reduced in the HY-112683 treatment group (P<0.01).

[0068] Through the above implementation scheme, this invention comprehensively and powerfully demonstrates, in an animal model that highly simulates clinical ischemic stroke, that HY-112683 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.

[0069] In summary, this invention provides the first experimentally verified small molecule activator (HY-112683) 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.

[0070] 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 compound of formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof in the preparation of a medicament for the prevention and / or treatment of ischemic brain injury; 。 2. The application according to claim 1, characterized in that, The ischemic brain injury is selected from ischemic stroke, transient ischemic attack, cerebral ischemia-reperfusion injury, vascular dementia, or post-ischemic inflammation.

3. The application according to claim 1, characterized in that, The compound represented by formula (I) is a specific small molecule activator of neuronal aromatase.

4. The application according to claim 1, characterized in that, The compound represented by formula (I) exerts a neuroprotective effect by directly activating the catalytic activity of aromatase, thereby promoting the local synthesis of endogenous brain estrogens.

5. The application according to claim 4, characterized in that, The endogenous brain estrogen is 17β-estradiol.

6. The application according to claim 1, characterized in that, The compound shown in formula (I) enhances the catalytic activity of aromatase without significantly affecting the transcription level of the aromatase gene.

7. The application according to claim 1, characterized in that, The drug is administered via local intracerebral administration; preferably, the local intracerebral administration method is selected from lateral ventricle injection, intrathecal injection, or stereotactic injection.

8. A pharmaceutical composition for the prevention and / or treatment of ischemic brain injury, characterized in that, It contains a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof, and one or more pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is an injectable preparation for local administration to the central nervous system.

10. The use of the compound of formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorph thereof, or the pharmaceutical composition of claim 8, in the preparation of a medicament for administration in combination with at least one other ischemic brain injury treatment agent.

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