Vanillic acid derivative compound, therapeutic agent composition for alzheimer's disease comprising same, and therapeutic agent composition for neuroinflammation

Abstract

The present invention relates to a vanillic acid derivative compound, which can be formed by a dehydration condensation reaction between vanillic acid and catecholamine. The vanillic acid derivative compound according to the present invention can be used as a therapeutic agent for Alzheimer's disease and neuroinflammation. In addition, the present invention relates to a composition for treating Alzheimer's disease and a composition for treating neuroinflammation, the compositions comprising the compound.

Description

Vanillic acid derivative compound, composition for treating Alzheimer's disease and composition for treating neuroinflammation comprising the same

[0001] The present invention relates to a compound that can be used in the fields of biotechnology and pharmacology, and relates to a vanillic acid derivative compound that can be used as a therapeutic agent targeting Alzheimer's disease and neuroinflammation.

[0002] Alzheimer's disease is one of the most common neurodegenerative diseases worldwide. One of the key pathological hallmarks of this disease is the excessive accumulation of amyloid beta proteins. Amyloid beta folds abnormally and aggregates, forming plaques between nerve cells and impairing brain function. Furthermore, these plaques promote neuroinflammatory responses, further exacerbating neuronal damage.

[0003] Existing treatments primarily focus on inhibiting or eliminating amyloid-beta accumulation. For example, some drugs attempt to reduce neuronal damage by reducing amyloid-beta production or promoting its clearance from the brain. However, these approaches only target pathways directly related to amyloid-beta, limiting their ability to adequately control other pathological mechanisms, such as neuroinflammation. Neuroinflammation, another potential cause of Alzheimer's disease, is a key factor in its pathological mechanisms. In particular, various inflammatory factors, such as NLRP3, are among the representative protein complexes that induce neuroinflammation during this process.

[0004] Accordingly, there is a growing recognition that, to develop more effective treatments for Alzheimer's disease, an approach that simultaneously inhibits amyloid beta accumulation and targets neuroinflammation is necessary. Based on this background, the present invention proposes a novel vanillic acid derivative (VD) combining dopamine with vanillic acid. This VD not only inhibits amyloid beta aggregation but also simultaneously suppresses key factors that induce neuroinflammation, thereby demonstrating the potential to slow or prevent the progression of Alzheimer's disease.

[0005] Therefore, the present invention aims to overcome the limitations of existing treatments and propose a new paradigm for Alzheimer's disease treatment by developing a novel therapeutic agent that simultaneously suppresses neuroinflammation and amyloid beta accumulation. This new approach, by targeting multiple pathological pathways, including neuroinflammation, could enable more comprehensive and effective Alzheimer's disease treatment.

[0006] One object of the present invention is to provide a vanillic acid derivative compound that can be used as a therapeutic agent for Alzheimer's disease and neuroinflammation.

[0007] Another object of the present invention is to provide an Alzheimer's treatment composition comprising the compound.

[0008] Another object of the present invention is to provide a neuroinflammatory therapeutic composition comprising the compound.

[0009] In one aspect, the present invention provides a compound represented by the following chemical formula 1.

[0010] [Chemical Formula 1]

[0011]

[0012] Here, X is *-(CH2) n -*, n is an arbitrary integer from 0 to 10, each independently selected, and * represents a binding site.

[0013] On the left side of the nitrogen (N) position, there is a structure derived from vanillic acid, and on the right side containing nitrogen (N) there is a structure derived from catecholamine. In the present invention, catecholamine refers to compounds containing a catechol structure and an amine group, and the catechol structure means a structure in which two hydroxyl groups are adjacently bonded to a benzene ring. At this time, the benzene ring may include a chain containing an amine group in addition to the two hydroxyl groups, and the amine group of the amine group may bond to the carboxyl group of vanillic acid through a dehydration condensation reaction to have a structure as shown in the chemical formula 1. Here, the catecholamine may be dopamine, norepinephrine, epinephrine, etc.

[0014] In the present invention, there is a linker, which is a portion connecting the amine group of catechol and the benzene ring, and hereinafter, this portion is referred to as the X site. Here, X can be determined depending on what catecholamine substance binds to vanillic acid. As long as the compound according to an embodiment of the present invention is formed by the bond between vanillic acid and catecholamine as in the above chemical formula 1, bonding or removal of bonding acceptable to those skilled in the art is not excluded from the scope of the present invention.

[0015] In one embodiment, the compound may be represented by Compound 1 below.

[0016]

[0017] The above compound 1 is a substance in which an amide bond is formed by a dehydration condensation reaction between dopamine and vanillic acid by selecting dopamine as the catecholamine substance. Since dopamine has a structure in which two carbon chains are located between the nitrogen atom of the amine group and the benzene ring, n is selected as 2, and X becomes *-(CH2)2-*.

[0018] In one embodiment, the method may be characterized by targeting a protein complex that induces inflammation. By directly targeting a specific protein complex that triggers an inflammatory response, it is expected to have the effect of suppressing or alleviating the inflammatory response. In particular, inflammatory protein complexes are associated with neuronal damage in Alzheimer's disease, and targeting them could be an important therapeutic strategy for inhibiting disease progression.

[0019] In one embodiment, the protein complex may include amyloid beta. Amyloid beta is a key pathological feature of Alzheimer's disease, accumulating in the brain to form plaques and disrupting signal transmission between nerve cells. Targeting the protein complex containing amyloid beta can inhibit amyloid beta accumulation and the resulting inflammatory response, which may play a significant role in delaying or preventing the progression of Alzheimer's disease.

[0020] In one embodiment, the method may feature targeting neuroinflammatory factors. Neuroinflammation is a key pathological mechanism of Alzheimer's disease, leading to neuronal damage and death. Targeting factors that induce neuroinflammation can reduce the inflammatory response and provide neuroprotective effects, representing an important therapeutic strategy applicable not only to Alzheimer's disease but also to other neurodegenerative diseases.

[0021] In one embodiment, the neuroinflammatory factor may include the NLRP3 factor. NLRP3 is a protein complex that plays a key role in neuroinflammatory responses. When activated, it promotes the secretion of inflammatory cytokines, which can cause damage to nerve cells. By suppressing the inflammatory response through the compound of the present invention, damage to nerve cells can be reduced, contributing to slowing the progression of Alzheimer's disease.

[0022] In another aspect, the present invention may be a treatment for Alzheimer's disease comprising the compound. The novel compound of the present invention can be utilized as a treatment that simultaneously targets amyloid beta accumulation and neuroinflammation, the major pathophysiological mechanisms of Alzheimer's disease. This suggests the potential to inhibit disease progression more effectively than existing treatments, potentially providing a new treatment option.

[0023] In another aspect, the present invention may be a composition for treating neuroinflammation comprising the compound. Neuroinflammation plays a key role in the pathological mechanisms of various neurodegenerative diseases. A composition capable of effectively suppressing neuroinflammation may be applicable not only to Alzheimer's disease but also to other neuroinflammation-related diseases. This presents a novel therapeutic strategy targeting neuroinflammation and may play a significant role in the treatment of various neurological diseases.

[0024] The compound according to the present invention can target both amyloid beta involved in the development of Alzheimer's disease and neuroinflammatory factors involved in neuroinflammation, and thus can have the effect of inhibiting both Alzheimer's disease and neuroinflammation.

[0025] The Alzheimer's treatment according to the present invention can target and aggregate amyloid beta, thereby contributing to the effective treatment of Alzheimer's disease.

[0026] The neuroinflammatory therapeutic composition according to the present invention can contribute to exerting a powerful anti-inflammatory effect in the treatment of neuroinflammatory diseases.

[0027] Figure 1 shows the chemical structure of a vanillic acid derivative compound according to the present invention.

[0028] Figure 2 shows a chemical reaction mechanism showing a method for producing a vanillic acid derivative compound according to the present invention.

[0029] Figure 3 is a vanillic acid derivative compound according to the present invention. 1Shows the results of H NMR measurements.

[0030] Figure 4 is a vanillic acid derivative compound according to the present invention. 13 Shows the results of C NMR measurements.

[0031] Figure 5 shows the results of HR-MS measurement of a vanillic acid derivative compound according to the present invention.

[0032] Figure 6 shows the results of analyzing the purity of a vanillic acid derivative compound according to the present invention.

[0033] Figure 7 shows the results of a viability analysis of a vanillic acid derivative compound according to the present invention on BV2 cells.

[0034] Figure 8 shows the results of nitric oxide secretion analysis of a vanillic acid derivative compound according to the present invention.

[0035] Figure 9 shows the Western blot results for inflammation and pro-inflammatory factors of the vanillic acid derivative compound according to the invention.

[0036] Figure 10 shows the experimental results evaluating the protein-ligand interaction (CETSA) of the vanillic acid derivative compound according to the present invention for D1R and D2R.

[0037] Figure 11 shows the Western blot results for the CREB factor of the vanillic acid derivative compound according to the present invention.

[0038] Figure 12 shows the Western blot results for the NF-kB factor of the vanillic acid derivative compound according to the present invention.

[0039] Figure 13 shows a schematic diagram showing the body pathway of a vanillic acid derivative compound according to the present invention.

[0040] Figure 14 shows a Western band of the hippocampus of a dementia rat injected with a vanillic acid derivative compound according to the present invention.

[0041] Figure 15 shows a Western band of the cerebral cortex of a dementia rat injected with a vanillic acid derivative compound according to the present invention.

[0042] Figure 16 shows a graph showing what factors are effective in the hippocampus and cerebral cortex of the vanillic acid derivative compound according to the present invention.

[0043] Figure 17 shows the results of a behavioral experiment on mice with dementia to determine the effect of the compound according to the present invention on dementia.

[0044] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention is susceptible to various modifications and variations, and thus specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. Throughout the description of each drawing, similar reference numerals have been used to designate similar components.

[0045] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, steps, operations, components, parts, or combinations thereof.

[0046] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0047]

[0048] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide, primarily affecting the elderly and a leading cause of dementia. One of the key pathological hallmarks of the disease is the excessive expression and aggregation of amyloid-beta proteins. This excessive expression and aggregation of amyloid-beta proteins leads to neuronal damage and death, ultimately leading to key symptoms of AD, such as memory loss and cognitive decline. Furthermore, Alzheimer's disease is not simply caused by the accumulation of amyloid-beta; neuroinflammation also plays a crucial role in the progression of the disease. In particular, inflammatory factors such as the NLRP3 inflammasome can induce neuroinflammation and further accelerate neuronal damage. This neuroinflammation plays a key role in the pathogenesis of AD, necessitating therapeutic strategies that simultaneously target both factors.

[0049] In order to solve these problems, the present invention developed a vanillic acid derivative compound according to the present invention. The vanillic acid derivative compound according to the present invention is a new substance that can be used as a therapeutic agent for both Alzheimer's disease and neuroinflammation. As an example, a new compound was developed by conjugating dopamine to vanillic acid, which is hereinafter abbreviated as VD (Vanillic-Dopamin). VD aims to inhibit and treat the progression of Alzheimer's disease by simultaneously targeting amyloid beta and neuroinflammatory factors. The purpose of the present invention is to overcome the limitations of existing therapeutic agents and provide a more effective therapeutic agent by simultaneously targeting various pathological factors associated with Alzheimer's disease and neuroinflammation through VD.

[0050] This approach presents a new paradigm for treating Alzheimer's disease, offering the potential to slow or prevent its progression. The development of VD is expected to open new avenues for treating Alzheimer's disease by not only suppressing amyloid beta accumulation but also targeting the root cause of neuroinflammation.

[0051] Figure 1 illustrates the chemical structure of a vanillic acid derivative compound according to one embodiment of the present invention. The present invention is produced by a dehydration-condensation reaction between vanillic acid and a catecholamine. The compound of Figure 1 uses dopamine as the catecholamine substance. The chemical structures of vanillic acid and dopamine are shown in the figure below.

[0052] <Chemical structure of vanillic acid>

[0053]

[0054] <Chemical structure of dopamine>

[0055]

[0056] In the middle of the compound in Figure 1, there is a nitrogen atom (N), which is derived from the amine group of dopamine. On the left side of this nitrogen, the -OH group of the carboxyl group of vanillic acid is detached and the nitrogen atom of dopamine is bonded to form a derivative structure, and on the right side including this nitrogen, one hydrogen of the amine group of dopamine is detached and forms a structure with vanillic acid. This is the result of the -OH group of the carboxyl group of vanillic acid and the -H group of the amine group of dopamine forming an amide bond (-CONH-) between the two molecules through a dehydration condensation reaction, through which a new chemical structure can be formed.

[0057] In Fig. 1, the nitrogen atom and the benzene ring are connected by two carbons, which is a result of dopamine bonding to vanillic acid. If a catecholamine substance other than dopamine is bonded as a catecholamine substance, the structure between the nitrogen atom and the benzene ring may change, and this is hereinafter referred to as the X site. As long as the compound according to the embodiment of the present invention is formed by the bond between vanillic acid and catecholamine as in the above chemical formula 1, bonding or removal of bonding acceptable to those skilled in the art is not excluded from the scope of the present invention.

[0058] Figure 2 illustrates a chemical reaction mechanism illustrating a method for producing a vanillic acid derivative compound according to the present invention. This will be described in detail through the following examples.

[0059] (Example)

[0060] The method for preparing a compound according to the present invention includes a step of dissolving vanillic acid, a catecholamine-based substance, a coupling reagent, an acid catalyst, a base catalyst, and an amide bond formation accelerator in an organic solvent and reacting them to obtain a vanillic acid derivative compound. In one embodiment, the phenylalkylamine-based substance may include dopamine. In one embodiment, the organic solvent may include dimethylformamide (DMF). In the present invention, the coupling reagent is a substance used to couple two molecules, the acid catalyst is a substance that promotes the reaction by interacting with the coupling reagent to provide a proton, the base catalyst is a substance that promotes the reaction by removing a proton in the reaction, and the amide bond formation accelerator is a substance that plays a role in particularly increasing the efficiency of the amide bond and reducing side reactions. In one embodiment, the coupling reagent may include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the acid catalyst may include hydrochloric acid (HCl), the base catalyst may include N,N-diisopropylethylamine, and the amide bond formation promoter may include 1-hydroxybenzotriazole (HOBt).

[0061] A specific example of the manufacturing method is as follows. First, vanillic acid (18.97 mmol) was dissolved in 50 mL of dimethylformamide, and hydroxybenzotriazole (20.87 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20.87 mmol) were added, and the mixture was stirred at 0°C for 30 minutes. Then, dopamine (18.97 mmol) and N,N-diisopropylethylamine (37.94 mmol) were added, and the mixture was stirred at room temperature for 24 hours, and the product was extracted using dichloromethane, water, and brine. The extracted solvent was evaporated to dryness, and the residue was dissolved in methanol and separated by flash chromatography using water / acetonitrile. After removing the solvent from the obtained product and drying it under vacuum, the vanillic acid derivative compound VD according to the present invention could be obtained. The yield at this time was approximately 29%.

[0062] Below, an experimental example to confirm the final synthesized compound, Gd-DO3A-SPA, is described.

[0063] (Experimental Example) Structural Analysis and Purity Analysis of the Final Compound VD

[0064] To confirm whether the vanillic acid derivative compound VD according to the present invention matches the expected structure and to verify whether the synthesis was successful, a structural analysis of the VD of the present invention was performed. For the VD structural analysis, 1 H NMR, 13 The structural properties of the compound were closely investigated by C NMR and HR_MS analyses.

[0065] Figure 3 is a vanillic acid derivative compound according to the present invention. 1This shows the results of H NMR measurements. Hydrogen atoms at specific positions in the compound exhibit different chemical shifts (δ) depending on their chemical environment. The peak at δ = 9.60 (s, 1H) appears to correspond to the -NH- hydrogen atom of the amide bond. This corresponds to a single peak, indicating that there are no other hydrogen atoms surrounding the hydrogen atom. The peaks at δ = 8.76 (d, J = 52.1 Hz, 2H), δ = 8.31 (t, J = 5.6 Hz, 1H), δ = 7.39 (d, J = 2.0 Hz, 1H), δ = 7.31 (dd, J = 8.2, 2.0 Hz, 1H), δ = 6.80 (d, J = 8.2 Hz, 1H), δ = 6.63 (dd, J = 9.5, 5.0 Hz, 2H), and δ = 6.47 (dd, J = 8.0, 2.0 Hz, 1H) indicate that the hydrogens of the aromatic ring are undergoing spin-spin interactions with adjacent hydrogen atoms. These are presumably the hydrogens of the aromatic ring contained in vanillic acid and dopamine. The peak at δ = 3.80 (s, 3H) corresponds to the hydrogen atoms of the methoxy group (-OCH3) that appears as a single peak, and the peak at δ = 3.36 (dd, J = 14.8, 6.0 Hz, 2H) is a doublet (dd) peak that probably corresponds to the methylene group (-CH2-) close to the amide bond. The peak corresponding to δ = 2.66 - 2.59 (m, 2H) is a multiple peak (m) with several overlapping signals that seems to correspond to the methylene group (-CH2-) with complex spin-spin interactions.

[0066] Figure 4 is a vanillic acid derivative compound according to the present invention. 13This shows the results of C NMR measurement. The peak at δ = 166.42 ppm corresponds to the carbonyl carbon of the amide bond and has a high chemical shift value. The peaks corresponding to δ = 149.78 ppm, 147.56 ppm, and 145.49 ppm are likely to correspond to carbon atoms of the catechol structure belonging to the aromatic ring. The peak corresponding to δ = 143.93 ppm appears to correspond to carbon atoms belonging to the aromatic ring of the vanillic acid structure. The peaks corresponding to δ = 130.88 ppm and 126.12 ppm correspond to other carbon atoms of the aromatic ring and may be carbons bonded with different substituents. The peaks corresponding to δ = 121.05 ppm, 119.76 ppm, 116.48 ppm, 115.96 ppm, 115.24 ppm, and 111.66 ppm are carbon atoms of the aromatic ring, which may be carbons bonded to substituents at different positions. The peak corresponding to δ = 56.08 ppm appears to correspond to the carbon atom of the methoxy group (-OCH3). The peaks corresponding to δ = 41.74 ppm, and 35.23 ppm correspond to carbon atoms of the methylene group (-CH2-), which are connected to amide bonds at their respective positions.

[0067] Figure 5 shows the results of HR-MS measurement of a vanillic acid derivative compound according to the present invention. The molecular weight of the manufactured compound can be determined by the HR-MS analysis, and the resulting measured molecular weight [M+H] + The molecular weight of 302.1028 was measured exactly as theoretically calculated. This means that the synthesized compound has the molecular structure C intended for the VD compound. 16 H 17 This indicates that it accurately reflects N1O5.

[0068] The 1H NMR, 13C NMR, and HR-MS analyses of the fourth powder in Figure 2-5 confirmed that the synthesized compound matched the expected chemical structure. The 1H NMR and 13C NMR spectra showed signals expected from a sinapinic acid derivative and a DO3A skeleton, and the HR-MS analysis results indicated that the molecular weight matched very well with the expected structure. These analyses confirmed that the fourth powder was successfully synthesized.

[0069] Figure 6 shows the results of a purity analysis of a vanillic acid derivative compound according to the present invention. The main peak is observed at approximately 3.297 minutes, indicating that the compound was detected as a major component when traveling through the HPLC column. Other peaks may be impurities or reaction byproducts, and these can be considered to assess the purity and purification of the compound. The table records the retention time (RT), area, and height of each peak, providing information on the signal intensity and purity assessment of each. In this analysis, purity can be determined by the ratio of the main peak to the total peak, and the result confirmed the purity to be 96.24%.

[0070] %Theoretical Calculated %Experimentally Measured %CHNOCHNOVD + H2O59.815.964.3629.8759.605.934.4829.61

[0071] Table 1 above shows the results of elemental analysis (EA) that measures the content of major elements in VD. If the error range between the theoretical and experimental values ​​is within ±0.4%, it can be considered an accurate analysis. The error ranges for each element are 0.21% for carbon, 0.03% for hydrogen, 0.12% for nitrogen, and 0.26% for oxygen, which can be considered to be within the allowable error range. In summary, the above analyses confirmed that the VD compound was synthesized with the expected structure and had high purity and an accurate molecular weight. This can be evaluated as proof of the successful synthesis and characterization of the VD compound.

[0072] Below, a method for evaluating the performance of a synthesized material and an evaluation example of the results are described.

[0073] (Evaluation Example 1) Cell viability analysis results

[0074] Figure 7 shows the results of a viability assay for BV2 cells using vanillic acid derivative compounds according to the present invention. The toxicity of the synthesized VD was confirmed through a cell viability assay. BV2 (normal mouse brain) cells were used. They were cultured in a growth medium containing DMEM (DME medium) containing 10% FBS (Fetal Bovine Serum). The cells were cultured in a humidified 5% CO2 atmosphere at 37°C with 1% antibiotic-antimycotic, 1% insulin-transferrin-selenium, and 40 ng / mL dexamethasone. VD dissolved in physiological saline at various concentrations (0.5–400 mM) was administered to the cells. After starvation for 24 hours in serum-free medium, a subsequent 48-hour culture was performed using RPMI 1640 medium containing 10% FBS and drugs (cisplatin and WP1130). Then, fresh complete medium containing Cell Counting Kit-8 (CCK-8) solution was added to the plates and incubated for 3 hours. Cell viability was measured by absorbance at 450 nm using a SpectraMax® i3 (Molecular Devices, CA, US).

[0075] The graph on the left (VD) shows how cell viability changes according to the concentration of VD. At 0 μM (i.e., the control group not treated with VD), cell viability is set to 100%, meaning that all cells survived normally. At high concentrations of VD (over 200 μM), slight cytotoxicity may occur, which may reduce cell viability. However, VD has almost no toxicity at low concentrations (0-100 μM), so it can be confirmed that cell viability is maintained high. The graph on the right (VD + additive) shows the cell viability when propylene glycol (PG), an additive to increase the solubility of VD, was added. It shows a similar pattern to VD treatment alone: ​​92.6% at 0 μM, 84.7% at 100 μM, 60.1% at 200 μM, and 43.1% at 400 μM. This demonstrates that cell viability results when VD and an additive (PG) are used together are virtually identical to those obtained with VD alone. These results provide crucial information for assessing the effect of VD on cell viability and suggest that VD can be used at appropriate concentrations with minimal or no cytotoxicity. This information can be utilized as valuable data in the development of potential VD drugs.

[0076] (Evaluation Example 2) Nitric Oxide Secretion Analysis Results

[0077] Figure 8 shows the results of a nitric oxide (NO) secretion analysis of a vanillic acid derivative compound according to the present invention. This evaluation example was performed to evaluate the effect of VD on nitric oxide (NO) secretion. Nitric oxide, such as NO, is an inflammatory mediator that increases during inflammatory responses or infection and is an important indicator of the severity of inflammation. The anti-inflammatory effect of VD was evaluated by confirming whether VD can inhibit NO secretion. BV2 cells were stabilized, treated with LPS for 2 hours, and then VD was added and cultured for 12 hours. The supernatant was separated from the cultured cells and centrifuged at 5,000 rpm for 2 minutes at 4°C. The analysis was performed using a Griess Reagent kit, and the concentration of NO was calculated by measuring the absorbance at 540 nm.

[0078] Since the drug concentration was confirmed to be non-toxic up to 100 μM in the cytotoxicity test results shown in Figure 7, the next experiment was conducted with a concentration of up to 100 μM. In the group treated with LPS, a large amount of NO was secreted, but in the group treated with LPS and the drug together, less NO was secreted than in the group treated with LPS alone at all concentrations. The above results show that VD can effectively suppress NO secretion in LPS-induced inflammatory responses. This suggests that VD has potential as an anti-inflammatory agent and can regulate inflammatory responses by reducing the production of NO, an inflammatory mediator. This can be an important basis for evaluating the possibility that VD can be used as a therapeutic agent in inflammatory diseases.

[0079] (Evaluation Example 3) In vitro results on VD treatment effect

[0080] This evaluation example is an experiment conducted to determine the fundamental effects of a drug at the cellular level. It aims to verify the potential therapeutic effects of VD at an early stage and to understand the drug's safety, efficacy, and mechanism of action before proceeding to more complex in vivo experiments or clinical trials.

[0081] Figure 9 shows Western blot results for inflammatory and proinflammatory factors of vanillic acid derivative compounds according to the present invention. This evaluation example is an in vitro experiment conducted to evaluate the ability of VD to suppress inflammatory responses. After extracting proteins from treated cells, Western blot analysis was performed for the expression of inflammatory factors such as IL-6, iNOS, NLRP3, and TNF-α. β-Actin was used as an endogenous control to quantify protein expression levels. As a result, the intensity of the inflammatory factor NLRP3 increased in the LPS-only treated group, while the intensity was significantly reduced in the VD drug-treated group. The proinflammatory factors IL-6, iNOS, and TNF-α also showed similar patterns as NLRP3. Therefore, it can be concluded that VD affects both inflammatory and proinflammatory factors. This suggests that VD may be useful in modulating inflammatory responses and treating inflammatory diseases.

[0082] Figure 10 shows the results of an experiment evaluating the protein-ligand interaction (CETSA) of a vanillic acid derivative compound according to the present invention for D1R and D2R. This was performed to identify the receptor targeted by VD. Dopamine receptors 1-5 exist on the cell membranes of neurons, microglia, astrocytes, etc. Among them, we wanted to identify the pathway through which VD exerts its anti-inflammatory effect. Since dopamine receptors are largely divided into group 1 and group 2, representative dopamine receptors 1 (D1R) and 2 (D2R) were identified through the CETSA experiment. The experimental results confirmed that there was no change in D2R, but there was a change in D1R. Through this, it was confirmed that VD affects at least dopamine receptor 1, although it is unclear whether it is an agonist or an antagonist.

[0083] The above experimental results suggest that VD exerts its anti-inflammatory effects by acting on the D1R dopamine receptor. Since it did not affect the D2R receptor, it is likely that VD selectively targets the D1R. This suggests that VD may have a mechanism for modulating or suppressing inflammatory responses through the D1R receptor. This experiment provides important clues to understanding the mechanism of action of VD.

[0084] Figure 11 shows the results of Western blot analysis of vanillic acid derivative compounds according to the present invention for CREB factor. This was performed to evaluate how VD regulates CREB factor, which plays a key role in the inflammatory pathway. CREB (cAMP response element-binding protein) is a transcription factor that regulates various signaling pathways within cells. When CREB is activated, it plays a crucial role in suppressing the NF-κB pathway. Since NF-κB is a key factor in inducing inflammatory responses, CREB activation is closely related to inflammation suppression. After inducing an inflammatory response by treating cells with LPS (1000 ng / mL), VD was treated at concentrations of 10 μM, 50 μM, and 100 μM. A group treated with VD and an additive (propyleneglycol, PG) was also included. Western blot analysis was then performed to analyze the expression of CREB and its activated form, pCREB. β-Actin was used as an endogenous control for protein quantification.

[0085] Looking at the Western blot results and graphs, we can see that the pCREB / CREB ratio significantly increased at 100 μM VD, regardless of the presence or absence of additives. This means that CREB is activated by VD. These experimental results suggest that VD can suppress the NF-κB pathway and regulate inflammatory responses by activating the CREB factor. CREB activation plays an important role in inflammation suppression, and it is possible that VD exerts this effect through CREB. This suggests that VD has the potential to exert a therapeutic effect in inflammatory diseases and that it can exert its inflammation suppression effect through a mechanism that regulates the CREB pathway.

[0086] Figure 12 shows the Western blot results for the NF-κB factor of the vanillic acid derivative compound according to the present invention. This was performed to evaluate how VD regulates the NF-κB pathway, which plays an important role in the inflammatory response. NF-κB is a key transcription factor that regulates the inflammatory response, and when activated in cells, it promotes the expression of inflammatory cytokines. Therefore, inhibition of NF-κB activation may play an important role in suppressing inflammation. CREB is a factor that acts prior to the NF-κB pathway, and the activation of CREB is related to the inhibition of the NF-κB pathway. After activating the NF-κB pathway and inducing an inflammatory response by treating the cells with LPS (1000 ng / mL), VD was treated at a concentration of 100 μM, and a group treated with VD and an additive (propyleneglycol, PG) together was also compared. Western blot was performed to confirm the expression of pNF-κB, the active form of NF-κB, and Lamin B was used as a control for nuclear protein quantification. In the group treated with 100 μM VD, it was confirmed that NF-κB activation was significantly reduced. This suggests that VD can suppress inflammatory responses by inhibiting the NF-κB pathway.

[0087] The above experimental results demonstrate that VD can modulate inflammatory responses by inhibiting the NF-κB pathway. Because NF-κB is a key transcription factor that promotes the expression of inflammatory cytokines, inhibition of the NF-κB pathway by VD could be an important therapeutic strategy for inflammatory diseases. This suggests that VD has potential as a therapeutic agent for suppressing inflammation.

[0088] Figure 13 is a schematic diagram of the mechanism of action of VD, synthesizing the results of previous experiments. It explains how VD regulates the inflammatory response within cells.

[0089] The specific mechanism is as follows. VD transmits a signal by binding to the D1R dopamine receptor. The D1R receptor is a G-protein coupled receptor that activates adenylyl cyclase, promoting the production of cAMP. The increased cAMP activates Protein Kinase A (PKA), and the activated PKA phosphorylates and activates CREB (cAMP response element-binding protein). The activated CREB inhibits the NF-κB pathway, and NF-κB is an important transcription factor that induces the expression of inflammatory cytokines. When CREB is activated by VD, the inflammatory response is reduced through the inhibition of NF-κB. In this way, VD can reduce the expression of inflammatory factors by inhibiting the activation of NF-κB through the CREB pathway. This suggests that VD may be useful in the treatment of inflammatory diseases by regulating factors related to inflammatory cytokines in this pathway.

[0090] (Evaluation Example 4) In vivo results on the VD treatment effect

[0091] This evaluation was conducted to evaluate the effect of administering VD (vanillic acid dopamine derivative) to a 12-month-old dementia mouse model (5xFAD) on the expression of specific proteins.

[0092] An experiment was conducted on 13 12-month-old dementia-affected rats (5xFAD). The rats were divided into three groups: the wild type (normal rats), the saline-injected group (5xFAD + saline), and the vanillic acid dopamine derivative (VD)-injected group (5xFAD + VD). Each group was administered the substance intravenously (IV) six times over a total of 20 days. After completing the experiment, the rats' brains were harvested, and hippocampal tissue was isolated for further analysis. The hippocampal tissue was used to measure the protein expression of specific factors. First, to extract proteins from the tissue, the tissue was homogenized in RIPA (protein extraction solution), and then centrifuged at 13,000 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was collected, and the protein concentration was measured using a BCA protein quantitation kit. The same amount of measured protein was loaded onto an 8.5% SDS-PAGE and transferred to an immobilon-P membrane. The transferred membrane was blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS-T) containing 0.05% Tween 20 (TBS-T) for 3 hours at room temperature. The membrane was then incubated with diluted primary antibodies in 3% BSA overnight at 4°C. The membrane was then washed three times and incubated with secondary antibodies for 1 hour. Western blot bands were detected using PicoEPD Western Reagent and SuperSignal West Femto Maximum Sensitivity Substrate, and images were observed on a chemiluminescence Western imaging system. The obtained images were then analyzed using Image J software.

[0093] Figures 14 and 15 show the results of Western blot experiments analyzing changes in protein expression after VD (vanillic acid-dopamine derivative) treatment in the hippocampus and cerebral cortex, respectively. These experiments were conducted on Alzheimer's disease model mice (5xFAD), and the expression of various inflammatory and neuronal proteins was compared to evaluate the effects of VD. Both Figures 14 and 15 were conducted in two sets, with the results from the first set on the left and the results from the second set on the right.

[0094] The results in Figures 14 and 15 demonstrate that VD can suppress inflammatory and neurodegenerative changes in the hippocampus and cerebral cortex of Alzheimer's disease model mice. This suggests that VD has the potential to inhibit the pathological progression of Alzheimer's disease by reducing the expression of inflammatory cytokines and neurotoxic proteins (e.g., β-amyloid, p-tau).

[0095] Figure 16 is a Western blot result showing the changes in the expression of various inflammatory and neurodegenerative factors in the hippocampus and cerebral cortex following VD (vanillic acid-dopamine derivative) treatment. This figure quantitatively evaluates the anti-inflammatory and neuroprotective effects of VD in Alzheimer's disease model mice (5xFAD). For this purpose, the drug VD was injected into the dementia mice at two concentrations, 2 mg / kg and 4 mg / kg. The inflammatory factors used in the experiment are as follows. NLRP3 is a protein complex that acts as an inflammatory factor, plays an important role in regulating the immune response, and induces an inflammatory response. BACE-1 (Beta-site Amyloid Precursor Protein Cleaving Enzyme 1) is an enzyme that cleaves amyloid precursor protein (APP) to produce beta-amyloid (β-amyloid). Here, beta-amyloid is one of the main causative agents of Alzheimer's disease, and its accumulation is considered one of the pathological hallmarks of Alzheimer's disease. IBA-1 is a microglia activation marker, indicating microglia activation, which plays a crucial role in the brain's immune and inflammatory responses. Finally, GFAP is a marker of astrocyte activation, and increased GFAP expression indicates glial cell activation. When these astrocytes and microglia are activated, they secrete pro-inflammatory cytokines, which can promote inflammatory responses.

[0096] Experimental results showed that VD (vanillic acid dopamine) suppressed the expression of inflammatory and neuroinflammatory factors, such as NLRP3, BACE-1, IBA-1, and GFAP, in both the hippocampus and cerebral cortex. This suggests that it has an effect on Alzheimer's disease, as evidenced by a decrease in beta-amyloid and phytau, factors associated with Alzheimer's disease, as well as a decrease in the levels of inflammatory factors.

[0097] Figure 17 shows the results of a water maze experiment evaluating the cognitive function improvement effect of VD (vanillic acid-dopamine) treatment in a dementia mouse model (5xFAD). Latency to Target is a measurement of the time it takes to reach the target, and the time to reach the target is delayed because the cognitive ability of the dementia mouse declines over time. In the case of dementia mice administered VD, the time to reach the target was shorter compared to dementia mice that were not administered VD, and the improvement was most significant in the VD administration group at a dose of 4 mg / kg. In addition, the time to reach the target was shorter in the drug-administered group compared to dementia mice, and a significant value was shown in the case of administration of 4 mg / kg.

[0098] Occupancy in the Target Quadrant (TQ) refers to the degree to which the target is reached in the fourth quadrant. Compared to the dementia mice, the drug-administered group (particularly the 4 mg / kg group) showed a higher reaching rate. This suggests that VD improved spatial memory in the dementia mice.

[0099] The Number of Target Crossings measures the number of times the target is crossed. The VD-administered group crossed the target area more frequently. This figure was also higher in the drug-administered group (4 mg / kg) compared to the dementia-free group. This indicates that VD improved the learning and memory abilities of the dementia-free mice.

[0100] In conclusion, these experimental results demonstrate that VD has the potential to improve cognitive function in mice with dementia. Dementia mice administered VD showed improved performance on cognitive tests, with the effect being particularly pronounced at a dose of 4 mg / kg.

[0101]

[0102] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. A compound represented by the following chemical formula 1: [Chemical Formula 1] Here, X is *-(CH2) n -*and, n is an arbitrary integer from 0 to 10, each independently selected, * indicates the binding site.

2. In paragraph 1, A compound represented by the following compound 1:

3. In paragraph 1, characterized by targeting protein complexes that cause inflammation; compound.

4. In paragraph 3, The protein complex comprises amyloid beta. compound.

5. In paragraph 1, characterized by targeting neuroinflammatory factors, compound.

6. In paragraph 5, The above neuroinflammatory factors include NLRP3 factors, compound.

7. Comprising a compound according to any one of claims 1 to 6, Alzheimer's disease treatment composition.

8. Comprising a compound according to any one of paragraphs 1 to 6, A composition for treating neuroinflammation.

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