Use of S-9-PAHSA in the preparation of drugs for the treatment of Alzheimer's disease and cognitive impairment

Abstract

This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the use of S-9-PAHSA in the preparation of drugs for treating Alzheimer's disease and cognitive impairment. This invention utilizes the separation and modification of PAHSA isomers to obtain enantiomeric pure S-9-PAHSA. The S-9-PAHSA improves cognitive function in various cognitive impairment models, including 5xFAD, through anti-inflammatory, anti-apoptotic, and oxidative stress-reducing effects, demonstrating good pharmacological activity in improving cognitive impairment. The results of this invention show that S-9-PAHSA reduces neuroinflammation and improves amyloid plaque pathology and cognitive impairment in 5xFAD mice by inhibiting NLRP3 inflammasome formation; S-9-PAHSA can improve working, learning, and memory abilities in diabetic-associated cognitive impairment mice through anti-apoptosis and oxidative stress reduction. This invention provides a novel synthetic route for S-9-PAHSA, which is simple to operate and easy to industrialize.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the pharmaceutical uses of S-9-PAHSA, particularly its use in the preparation of drugs for treating Alzheimer's disease and cognitive impairment. This invention utilizes the resolution and modification of PAHSA isomers to obtain enantiomeric pure S-9-PAHSA, thereby enhancing its pharmacological activities in anti-inflammatory, anti-apoptotic, and oxidative stress-reducing effects, thus treating and improving Alzheimer's disease and other cognitive impairment-related diseases. Background Technology

[0002] Currently, how to prevent and treat cognitive impairment-related diseases such as Alzheimer's disease has become a major issue that urgently needs to be addressed. PAHSA is a novel hydroxystearic acid extracted from natural foods. Our research team was the first in China to successfully synthesize PAHSA in previous studies, and found that it has certain biological activities to improve cognitive impairment in animal experiments.

[0003] Studies have shown that lipids are essential components of life, possessing multiple important functions, including participation in signal transduction, formation of cell barriers, and provision of metabolic energy. Fatty acid esters (FAHFAs) are a novel type of endogenous lipid, particularly abundant in adipose tissue. Research has found a significant correlation between FAHFA levels and age; from childhood to middle age, FAHFA levels in mouse tissues show an increasing trend, while in older mice, FAHFA levels significantly decrease. Therefore, FAHFAs are likely involved in age-related neurodegenerative diseases. Representative components such as PAHSA, especially 9-PAHSA, are expressed at relatively high levels in human serum and tissues; therefore, 9-PAHSA may have a certain ameliorative effect on AD. As is well known, type 2 diabetes is an important independent risk factor for Alzheimer's disease (AD). Studies have shown that elderly patients with type 2 diabetes and cognitive impairment have a large amount of Aβ protein and p-tau protein deposits in functional areas such as the cortex and hippocampus. This suggests that patients with diabetic cognitive impairment have similar pathological features to AD patients, indicating that diabetic cognitive impairment may be a special type of AD and may share common mechanistic pathways with AD. Therefore, 9-PAHSA is expected to become a candidate drug molecule for intervening in cognitive-related diseases such as Alzheimer's disease and diabetes-related cognitive impairment.

[0004] Studies have shown that in the context of Alzheimer's disease (AD), microglia are overactivated, leading to severe chronic neuroinflammation. Therefore, effective anti-inflammatory therapy is an important way to improve AD. Some fatty acids can induce anti-inflammatory effects through GPR120, which is expressed on immune cells such as microglia and bone marrow-derived dendritic cells (BMDCs) that mediate innate immune responses. Saturated fatty acids such as palmitic acid and endotoxin lipopolysaccharide (LPS) can promote the maturation of microglia and BMDCs through the Toll-like receptor (TLR) signaling pathway. It has been confirmed that GPR12O is an important receptor for PAHSA. Cellular studies have shown that 9-PAHSA can activate GPR12O to block LPS activation of BMDCs, mainly by inhibiting LPS-induced expression of CD80, CD86, CD40 and MHCII in BMDCs, while blocking LPS-induced IL-12 secretion in a dose-dependent manner and significantly reducing IL-1β and TNF-α secretion.

[0005] It is well known in the industry that neurological injury is a core event in cognitive impairment-related diseases such as Alzheimer's disease (AD), and reducing neuronal apoptosis is key to improving cognitive impairment. Animal studies have found that bioactive lipids, such as ω-3 fatty acids, can activate neuronal GPR120, promote BCL-2 protein expression, exert anti-apoptotic effects, and alleviate neurological damage caused by cerebral ischemia and other factors. Therefore, this study hypothesizes that PAHSA can...

[0006] The aim is to salvage neuronal loss by activating GPR120 to induce the expression of anti-apoptotic proteins in neurons. Besides GPR120, this study has currently...

[0007] It has been confirmed that PAHSA can promote the expression of anti-apoptotic proteins and reduce neuronal apoptosis by activating carbonic anhydrase III (CA).

[0008] However, studies have also shown that PAHSAs have the following drawbacks in exerting their pharmacological activity to improve cognition: long-term administration of PAHSAs can promote intrahepatic inflammation, hepatocyte edema, and hepatocyte necrosis; long-term low-dose PAHSAs intervention can induce atherosclerosis in diabetic mice, etc. In view of these shortcomings, the research team of this invention aims to provide a new use for S-9-PAHSA in the preparation of drugs for the treatment of Alzheimer's disease and cognitive impairment-related diseases. Specifically, this invention modifies and transforms 9-PAHSA, synthesizing 9-PAHSA and its enantiomers, the pure S-type and R-type, using a new route. The resolved S-9-PAHSA exhibits high biological activity and pharmacological effects in anti-apoptosis and anti-inflammation, which can solve the adverse reactions caused by long-term PAHSA treatment. Summary of the Invention

[0009] The purpose of this invention is to provide a highly bioactive PAHSA isomer, its preparation method, and novel applications, in light of the current state and shortcomings of existing technologies. In particular, it relates to a novel application of S-9-PAHSA in the preparation of therapeutic drugs for Alzheimer's disease and cognitive impairment-related diseases.

[0010] This invention modifies 9-PAHSA and synthesizes its enantiomers, the pure S-type and R-type, via a novel approach. The resolved S-9-PAHSA exhibits high biological activity and pharmacological effects in anti-apoptosis and anti-inflammation, which can address the adverse reactions associated with long-term PAHSA treatment. PAHSA is a novel hydroxystearic acid extracted from natural foods. Previous studies in this invention successfully synthesized PAHSA and demonstrated certain anti-inflammatory and anti-apoptotic pharmacological activities in animal experiments. This invention further investigated the resolution, modification, organic synthesis, and preclinical pharmacodynamic studies of PAHSA, synthesizing a PAHSA type with significant efficacy, good safety, and multi-target anti-DM and arteriosclerosis effects. Ultimately, new drug candidate compounds with high drug activity, few side effects, simple preparation, and low cost were screened.

[0011]

[0012] Specifically,

[0013] This invention employs the following method for the biosynthesis of S-9-PAHSA, modifying and synthesizing 9-PAHSA to obtain its enantiomers, the pure S-type and R-type. The method includes:

[0014] (1) Synthesis of nonadec-1-en-10-ol

[0015]

[0016] In a tetrahydrofuran (THF) aprotic solvent containing magnesium shavings and elemental iodine, 9-bromo-1-nonene was added, nitrogen gas was bubbled in, and the mixture was stirred at 40–80 °C until the solution became colorless. The solution was then refluxed, and 9-bromo-1-nonene was added again, followed by reflux. The mixture was cooled to room temperature, and THF containing decanal was added at 0 °C. The mixture was stirred overnight at room temperature, and the solution was purified by chromatography.

[0017] (2) Synthesis of nonadec-1-en-10-yl palmitate

[0018]

[0019] Weigh out 1-eno-10-ol and nonylmagnesium bromide, dissolve them in dichloromethane, stir magnetically, and then add palmitic acid.

[0020] Anhydride, 4-dimethylaminopyridine, and triethylamine were stirred overnight at room temperature and then subjected to column chromatography to obtain a colorless oily substance, C35H69O2+.

[0021] (3) Synthesis of 9-PAHSA

[0022]

[0023] Weigh out 1-neocene-10-palmitate, dissolve it in dichloromethane, and bubble ozone upwards under a dry ice / acetone bath until the solution turns blue.

[0024] Nitrogen gas was bubbled in until the solution became colorless. Triphenylphosphine was added, and the temperature was raised to room temperature to continue the reaction.

[0025] Add sodium hypochlorite, sodium dihydrogen phosphate, 2-methyl-2-ene, water, and tert-butanol, and react overnight;

[0026] The solution was concentrated, dichloromethane was added, and the solution was washed with hydrochloric acid solution in a separatory funnel. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a waxy substance.

[0027] Dissolved in a small amount of ethyl acetate, followed by column chromatography, the white solid was 9-PAHSA, C34H65O4;

[0028] (4) Differentiation between S-type and R-type PAHSA enantiomers

[0029]

[0030] The above preparation process yielded a mixture of S- and R-enantiomers of 9-PAHSA. To purify the S- and R-enantiomers separately, an auxiliary group was attached to the hydroxyl group of the compound as shown in the figure. The compound was then analyzed on an analytical column for 10–30 min. The S- and R-enantiomers were distinguished by the colorimetric function of the auxiliary group. After deprotection and removal of the auxiliary group, pure S-9-PAHSA and R-9-PAHSA were obtained.

[0031] Clinical trials of this invention have shown that: 1. S-9-PAHSA reduces neuroinflammation and improves amyloid plaque pathology and cognitive impairment in 5xFAD mice by inhibiting NLRP3 inflammasome formation; 2. S-9-PAHSA can improve the working, learning, and memory abilities of diabetic-associated cognitive impairment mice by inhibiting apoptosis and reducing oxidative stress levels; the split S-9-PAHSA has high biological activity and pharmacological effects in anti-apoptosis and anti-inflammation, which can solve the adverse reactions caused by long-term PAHSA treatment.

[0032] This invention demonstrates that the modified PAHSA isomers exhibit good pharmacodynamic activity in improving cognitive-related diseases such as Alzheimer's disease (AD) and diabetes-related cognitive impairment, and also possess favorable pharmacokinetic properties. The preparation method for the PAHSA isomers of this invention is simple to operate and easy to industrialize.

[0033] The S-9-PAHSA of the present invention can be further used to prepare therapeutic drugs for Alzheimer's disease and cognitive impairment-related diseases. Attached Figure Description

[0034] Figure 1 S-9-PAHSA intervention significantly improved the spatial learning and memory abilities of 3-month-old 5xFAD mice.

[0035] Figure 2 S-9-PAHSA can reduce Aβ deposition in the cortex and hippocampus of 3-month-old 5xFAD mouse models.

[0036] Figure 3 S-9-PAHSA can reduce the proliferation of microglia in the cortex and hippocampus of 3-month-old 5xFAD mouse models.

[0037] Figure 4 S-9-PAHSA can reduce neuroinflammation in a 3-month-old 5xFAD mouse model.

[0038] Figure 5 Changes in apoptosis-related proteins in the cerebral cortex of mice after S-9-PAHSA intervention.

[0039] Figure 6 Changes in oxidative stress-related proteins in the cerebral cortex of mice after S-9-PAHSA intervention.

[0040] Figure 7 Y-maze experiment results of diabetic cognitive impairment aged mice after 1 month of S-9-PAHSA intervention. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] Example 1

[0043] The biosynthesis process of S-9-PAHSA:

[0044] (1) Synthesis of nonadec-1-en-10-ol

[0045]

[0046] In 100 mL of tetrahydrofuran (THF) aprotic solvent containing 1.3 g magnesium shavings and 20 g elemental iodine, 1.09 g of 9-bromo-1-nonene was added. Nitrogen gas was bubbled in, and the mixture was stirred at 40–80 °C until the solution became colorless. The solution was refluxed, and then 9–10 g of 9-bromo-1-nonene was slowly added, followed by reflux for 0.5–1 h. The mixture was cooled to room temperature, and 10 mL of THF containing 5.0–5.5 g of decanal was added dropwise using a syringe at 0 °C. The mixture was stirred overnight at room temperature. The solution was then subjected to column chromatography to obtain a pure product (5.58 g, yield 60%).

[0047] (3) Synthesis of nonadec-1-en-10-yl palmitate

[0048]

[0049] Weigh out 1-eno-10-ol and nonylmagnesium bromide, dissolve in 20 ml of dichloromethane, stir magnetically, add palmitic anhydride, 4-dimethylaminopyridine, and triethylamine, and stir overnight (15–20 h) at room temperature. Column chromatography (10% EtOAc / hexanes) yields a colorless oil (7 g, 67% yield), C35H69O2+, molecular weight: 521.5302.

[0050] (3) Synthesis of 9-PAHSA

[0051]

[0052] Weigh out 1-neocene-10-palmitate and dissolve it in 20 mL of dichloromethane. In a dry ice / acetone bath (-78°C), bubble ozone upwards through the solution until it turns blue.

[0053] Nitrogen gas was bubbled in until the solution became colorless. Triphenylphosphine was added, and the mixture was brought to room temperature. The reaction was continued for 2 hours.

[0054] Add sodium hypochlorite, sodium dihydrogen phosphate, 2-methyl-2-ene, water, and tert-butanol, and react overnight.

[0055] The solution was concentrated, dichloromethane was added, and the solution was washed with 10% hydrochloric acid solution in a separatory funnel. The organic phase was washed with anhydrous sodium sulfate.

[0056] The product was dried, filtered, and concentrated by rotary evaporation to obtain a waxy substance.

[0057] Dissolved in a small amount of ethyl acetate, the solution was subjected to column chromatography (20% EtOAc / hexane s) to give a white solid 9-PAHSA, C34H65O4, with a molecular weight of 537.4905.

[0058] (4) Differentiation between S-type and R-type PAHSA enantiomers

[0059]

[0060] The above preparation process yields a mixture of S- and R-enantiomers of 9-PAHSA. To purify the S- and R-enantiomers separately, auxiliary groups are attached to the hydroxyl groups of the compounds shown in the figure. The compounds are then analyzed on an analytical column for 10–30 min, and the S- and R-enantiomers are distinguished by the colorimetric function of the auxiliary groups. Deprotection is then performed to remove the auxiliary groups, yielding pure S-9-PAHSA and R-9-PAHSA.

[0061] Example 2

[0062] Effect Example

[0063] Clinical trials of this invention have shown that the method of resolving and modifying PAHSA isomers to obtain enantiomerically pure S-9-PAHSA has the following technical advantages:

[0064] 1. Improvement of cognitive impairment in 5xFAD model mice: Three-month-old mice in each group were administered S-9-PAHSA 30 mg / kg / day (solvent control and water intervention, respectively) for two months. Immediately after the intervention, the Morris water maze behavioral test was performed. Results showed that S-9-PAHSA intervention significantly improved the spatial learning and memory abilities of 5xFAD mice (e.g., ...). Figure 1 (as shown);

[0065] 2. Reducing Amyloid Deposition in the Brains of 5xFAD Model Mice: Aβ deposition is one of the most important pathological features of AD. Aβ plaque toxicity causes tau protein hyperphosphorylation and neuroinflammation, and the interaction of these three factors can lead to a continuous acceleration of AD progression. Three-month-old 5xFAD mice were treated with S-9-PAHSA (30 mg / kg / day) for two months, and then Thio-S staining analysis was performed on mouse brain slices. The staining revealed a significant reduction in Aβ deposition in the cortex and hippocampus of the S-9-PAHSA intervention group (e.g., ...). Figure 2 (as shown);

[0066] 3. Reducing microglia proliferation in the brains of 5xFAD model mice: Microglia are innate immune cells of the central nervous system and are closely related to the development of neuroinflammation. This study used Iba-1 to label microglia, and the results showed that S-9-PAHSA significantly reduced microglia proliferation and activation in the AD environment (e.g., Figure 3 (as shown);

[0067] 4. Reducing NLRP3 inflammasome formation in the brains of 5xFAD model mice: NLRP3 inflammasomes are important mediators of neuroinflammation. After two months of water intervention, the expression of NLRP3 signaling pathway proteins in the mouse brain was detected. The results showed that S-9-PAHSA significantly reduced the formation of NLRP3 inflammasomes in the mouse brain (e.g., Figure 4 (as shown);

[0068] 5. Neuroprotective effect on diabetic-related cognitive impairment model mice: Mice in each group were treated with solvent control and 9-PAHSA 30 mg / kg in drinking water for one month. The expression of apoptosis-related proteins and oxidative stress proteins in the brain tissue of each group was detected to observe the neuroprotective effect of S-9-PAHSA on diabetic-related cognitive impairment model mice. The results showed that S-9-PAHSA significantly inhibited the level of apoptosis and oxidative stress response (e.g., oxidative stress protein) in the cerebral cortex of diabetic model mice. Figure 5 , Figure 6 (as shown);

[0069] 6. Improvement of Diabetes-Related Cognitive Impairment: Mice in each group were administered solvent-based control and 9-PAHSA 30 mg / kg water intervention for one month. Following this, the spatial memory ability of the mice was assessed using the Y-maze behavioral test. After one month of drug intervention, the percentage of spontaneous alternation in the S-9-PAHSA group was significantly increased. When CAIII was knocked out, the percentage of spontaneous alternation was not significantly different from that in the control group. This indicates that S-9-P has an improving effect on spatial memory in mice, while knockdown of CAIII in the brain...

[0070] Subsequently, this protective effect decreases (e.g.) Figure 7 (As shown).

Claims

1. Use of S-9-PAHSA of the following structure, ###0001### in the preparation of a medicament for the treatment of Alzheimer's disease. .

2. Use according to claim 1, characterized in that: The drug includes S-9-PAHSA as an active ingredient, and a pharmaceutically acceptable carrier.

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