Novel butyric acid lithium proline eutectic salt as well as preparation method and application thereof

Abstract

The invention relates to a novel lithium butyrate proline eutectic salt and a preparation method and application thereof, and belongs to the technical field of medicines. According to the invention, the lithium salt of n-butyric acid and L-proline are co-crystallized for the first time, and a new molecular structure with better stability and stronger drug effect is provided. The novel lithium butyrate proline eutectic salt disclosed by the invention can be used for remarkably inhibiting the activity of GSK3beta in cell and animal models and effectively reducing the total tau protein and the phosphorylation level of the total tau protein, so that the potential value for treating neurodegenerative diseases such as Alzheimer's disease is shown; particularly, the compound has a wide application prospect in the aspects of reducing neuroinflammation and improving the neuroprotection effect. Meanwhile, the compound also has prevention and treatment values in GSK3beta-related diseases such as cancers, mental diseases and diabetes mellitus.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to a novel lithium butyrate proline eutectic salt, its preparation method, and its application. Background Technology

[0002] Glycogen synthase kinase-3β (GSK3β) is an evolutionarily conserved serine / threonine kinase that is widely found in mammalian eukaryotic cells. It can regulate cell differentiation, proliferation, survival, and apoptosis, and serves as a key therapeutic target in a variety of major diseases, including cancer, neurodegenerative diseases, mental illnesses, and type II diabetes.

[0003] In cancer, GSK3β affects tumor progression through multiple pathways. In normal cells, GSK3β, as a key regulator of the Wnt / β-catenin signaling pathway, phosphorylates β-catenin, causing its degradation and inhibiting cell proliferation. However, in some cancers, GSK3β activity is inhibited, leading to β-catenin accumulation and entry into the cell nucleus, activating pro-proliferation genes. Simultaneously, GSK3β participates in cell cycle regulation; abnormal activity can lead to cell cycle dysregulation. Furthermore, it regulates the phosphorylation of apoptosis-related proteins, affecting cell life and death; in some cancers, abnormal activity can hinder apoptosis and promote tumor growth.

[0004] In the context of mental illness, GSK3β is expressed during neurodevelopment and participates in multiple brain signaling pathways; abnormal activity can lead to neurodevelopmental disorders. It also regulates various signal transduction pathways, affecting neuronal function and neural network formation; dysregulation of its activity can result in abnormal signal transduction. Furthermore, GSK3β can regulate gene expression through phosphorylation. These genes are crucial for neurons and neural networks; abnormal activity of GSK3β can cause gene expression disorders, thereby triggering mental illness.

[0005] In type 2 diabetes, GSK3β is a crucial regulator of the insulin signaling pathway. Abnormal activity hinders insulin signaling, leading to insulin resistance, a key characteristic of type 2 diabetes. It also participates in multiple stages of glucose metabolism; abnormalities can cause glucose metabolism disorders and hyperglycemia. Furthermore, GSK3β is involved in the regulation of inflammatory responses; abnormal activity can trigger chronic inflammation, exacerbating insulin resistance and hyperglycemia, and accelerating the progression of type 2 diabetes.

[0006] Alzheimer's disease (AD) is highly representative among neurodegenerative diseases. AD is characterized by cognitive impairment and neuronal degeneration. Its pathological features include β-amyloid (Aβ) deposition and tau protein hyperphosphorylation forming neurofibrillary tangles (NFTs). GSK3β plays a key role in tau protein phosphorylation and the pathological development of AD, therefore GSK3β inhibitors are considered a potential treatment.

[0007] Lithium salts, including lithium carbonate and lithium citrate, are common GSK3β inhibitors and have been used to treat bipolar disorder and other neuropsychiatric disorders due to their neuroprotective effects, showing some potential in the treatment of Alzheimer's disease (AD). However, the current clinical application of lithium salts is limited by toxicity and a narrow therapeutic window.

[0008] To address this issue, researchers have attempted to modify short-chain fatty acids with lithium through co-crystallization, such as lithium isobutyrate-proline. However, current technologies still lack short-chain fatty acid-lithium amino acid co-crystallized salts that offer high activity, low toxicity, strong stability, and good therapeutic effects. Lithium carbonate has also been investigated for its potential use in treating related diseases, but it also has limitations.

[0009] Therefore, developing safer and more effective GSK3β inhibitors is of great significance for the treatment of a variety of related diseases. Summary of the Invention

[0010] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0011] The first aspect of this application provides a lithium butyrate-proline co-crystal salt, which is prepared by co-crystallization of lithium salt of butyrate and proline or proline derivative in an equimolar ratio.

[0012] According to the US Food and Drug Administration's "Regulatory classification of pharmaceutical cocrystals" (2018), a cocrystal is a crystalline substance containing two or more different molecules within the same crystal lattice. In this application, it was unexpectedly discovered that cocrystallizing the lithium salt of the stated butyric acid with the stated proline significantly inhibits GSK3β activity.

[0013] Butyric acid, as a short-chain fatty acid, possesses biological activity and can regulate various physiological processes in the nervous system, thus endowing the cocrystal salt with higher efficacy and stability. In some specific embodiments of this application, lithium butyrate proline cocrystal salt exhibits higher efficacy and lower toxicity compared to lithium isobutyrate proline salt.

[0014] In some embodiments of this application, the proline derivative is proline with chemically modified carboxyl and / or imine sites.

[0015] In some embodiments of this application, the eutectic crystallization is one of solvent evaporation crystallization, cooling crystallization, grinding crystallization, reaction crystallization, hot melt extrusion crystallization, melt crystallization, and acoustic crystallization.

[0016] The second aspect of this application provides a method for preparing lithium butyrate proline eutectic salt as described in any of the first aspects of this application, comprising the following steps: The lithium salt of butyric acid and the proline are dissolved in a solvent in an equimolar ratio. The solution is dissolved under reflux and cooled to precipitate crystals. The crystals are collected and dried to obtain the lithium butyric acid-proline eutectic salt.

[0017] In some embodiments of this application, the solvent is selected from one or more of water, methanol, and ethanol.

[0018] The third aspect of this application provides the use of the lithium butyrate proline cocrystal salt described in any of the first aspects of this application in the preparation of a medicament for at least one of the following: (1) Inhibit GSK3β; (2) Reduce tau protein levels; (3) Reduce the level of phosphorylated tau protein, or reduce the phosphorylation level of tau protein; (4) Reduce nerve inflammation; (5) Enhance neuroprotection; (6) Maintaining neuronal functional stability; (7) Prevention or treatment of neurodegenerative diseases; (8) Prevention or treatment of cancer; (9) Prevention or treatment of mental illnesses; (10) Prevention or treatment of type II diabetes and / or its complications.

[0019] Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved serine / threonine kinase, widely found in mammalian eukaryotic cells. GSK3β acts on numerous signaling proteins, structural proteins, and transcription factors, regulating cell differentiation, proliferation, survival, and apoptosis. GSK3β serves as a therapeutic target for various major diseases, including cancer, neurodegenerative diseases, and mental illnesses.

[0020] In some embodiments of this application, the neurodegenerative disease is selected from one or more of Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, amyotrophic lateral sclerosis, and spinocerebellar ataxia.

[0021] In some embodiments of this application, the cancer is a malignant tumor disease selected from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, etc. One of the following: papillary carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, glioblastoma, and retinoblastoma.

[0022] In some embodiments of this application, the mental illness is selected from one of anxiety disorder, obsessive-compulsive disorder, depression, phobia, and schizophrenia.

[0023] In some embodiments of this application, the type II diabetes complications include acute complications of diabetes and chronic complications of diabetes.

[0024] In some specific embodiments of this application, the type II diabetes complications include acute complications of diabetes and chronic complications of diabetes.

[0025] In some more specific embodiments of this application, the acute diabetic complication is selected from at least one of hypoglycemia, ketoacidosis, nonketotic hyperosmolar coma, and lactic acidosis.

[0026] In some other more specific embodiments of this application, the chronic diabetic comorbidities are selected from at least one of diabetic neuropathy, diabetic gastroparesis, diabetic enteropathy, diabetic neurogenic bladder, erectile dysfunction, diabetic nephropathy, diabetic cardiomyopathy, diabetic foot, diabetic osteoporosis, diabetic comorbidity and diabetic dermatitis.

[0027] The fourth aspect of this application provides a drug comprising the lithium butyrate proline cocrystal salt described in any of the first aspects of this application.

[0028] In some embodiments of this application, the drug further includes at least one pharmaceutically acceptable excipient and / or diluent.

[0029] In some embodiments of this application, the dosage form of the drug is selected from the group consisting of oral preparations, injections, aerosols, suppositories, drops, and transdermal patches.

[0030] In some embodiments of this application, the drug is administered via the respiratory or digestive system; or subcutaneously; or via the nasal mucosa or submucosa; or via the eye or ear; or via the rectum; or via the vagina.

[0031] The fifth aspect of this application provides a method for preventing and / or treating neurodegenerative diseases, comprising the step of administering a therapeutically effective amount of any of the drugs described in the fourth aspect of this application to a subject.

[0032] Compared with the prior art, this application has the following advantages: Butyric acid is an important metabolite of gut microbiota, possessing anti-inflammatory, immunomodulatory, and neuroprotective effects. This application, for the first time, co-crystallizes lithium butyric acid with proline, providing a novel molecular structure with better stability and higher efficacy. The novel lithium butyrate-proline co-crystallized salt of this application significantly inhibits GSK3β activity in cell and animal models and effectively downregulates total tau protein and its phosphorylation levels, demonstrating potential value in treating Alzheimer's disease and other neurodegenerative diseases, especially in reducing neuroinflammation and enhancing neuroprotective effects, showing broad application prospects.

[0033] The method for preparing lithium butyrate-proline cocrystal salt in this application optimizes intermolecular interactions through precise synthetic steps, resulting in a stable cocrystal structure with high bioavailability. The preparation method is simple and efficient, comprising steps such as reflux cocrystallization in anhydrous ethanol and natural cooling crystallization, with mild reaction conditions and high yield.

[0034] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0035] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 The 1H NMR spectrum of lithium butyrate-L-proline eutectic salt (BuLiPro) in Example 2 of this application is shown; Figure 2 The 1H NMR spectrum of lithium butyrate-L-proline eutectic salt (BuLiPro) in Example 2 of this application is shown; Figure 3 The XRD pattern of lithium butyrate-L-proline eutectic salt (BuLiPro) in Example 2 of this application is shown; Figure 4The XRD pattern of L-proline in Example 2 of this application is shown; Figure 5 The DSC diagram of lithium butyrate-L-proline eutectic salt (BuLiPro) in Example 2 of this application is shown; Figure 6 The inhibition curves of lithium butyrate-L-proline eutectic salt (BuLiPro), lithium butyrate (LiB), LiCl, sodium butyrate (NaB), lithium isobutyrate-L-proline eutectic salt (IsoLiPro) and lithium isobutyrate (LiIsobu) on GSK3β are shown in Example 3 of this application. Figure 7 This application demonstrates that lithium butyrate-L-proline cocrystal salt (BuLiPro) significantly downregulated total tau levels in the HEK293 / tau cell line in a dose-dependent manner in Example 4 of this application. Figure 8 This application demonstrates that lithium butyrate-L-proline cocrystal salt (BuLiPro) significantly downregulated total tau levels in the SH-SY5Y / tau stable cell line in a dose-dependent manner in Example 4 of this application. Figure 9 This application demonstrates that lithium butyrate-L-proline cocrystal salt (BuLiPro) in Example 4 of this application downregulated total tau and ptau levels in the HEK293 / tau stable cell line in a dose-dependent manner. Figure 10 The images show the total protein, total tau, and phosphorylated tau levels extracted after HEK293 / tau (P301L) was treated with 20mM lithium concentrations of BuLiPro, LiCl, or IsoLiPro for 1, 2, and 4 hours in Example 4 of this application. Figure 11 The results of total tau immunohistochemical staining of brain tissue from 3xTG mice under different treatments in Example 5 of this application are shown. Figure 12 The results of p-tau181 immunohistochemical staining of brain tissue from 3xTG mice under different treatments in Example 5 of this application are shown. Figure 13 The results of Western blot analysis of tau protein and phosphorylated tau protein in hippocampal tissues of 3xTG mice under different treatments in Example 5 of this application are shown. Figure 14 The results of H&E staining of liver tissue from 3xTG mice under different treatments in Example 5 of this application are shown. Figure 15 The results of H&E staining of kidney tissue from 3xTG mice under different treatments in Example 5 of this application are shown. Figure 16The following are examples of rat survival and vital signs in the subacute toxicity test (28d) of lithium butyrate-L-proline cocrystal salt (BuLiPro) and control compounds in Example 6 of this application. A: Effects of low, medium, and high dose groups of BuLiPro, Li2CO3, and IsoLiPro on the survival rate and body weight of SD rats 28d after oral administration, n=10 (5 females, 5 males). B: Organ coefficients of surviving rats after 28d. Data are expressed as mean ± standard error and analyzed using two-way ANOVA. Compared with the control group, *P<0.05, **P<0.01, ****P<0.0001; Figure 17 and Figure 18 The image shows the H&E staining results of rat kidneys in a subacute toxicity experiment (28 days) using lithium butyrate-L-proline cocrystal salt (BuLiPro) and the control compound, as described in Example 6 of this application. Representative H&E-stained kidney sections from the high (H), medium (M), low (L) dose groups and the satellite group (R) show histopathological changes. Arrows indicate: green—renal tubule dilation, red—renal tubular epithelial cell shedding, black—cast or suspected cast, yellow—epithelial cell edema or vacuolar degeneration, white—luminal narrowing (in the renal tubules or Bowman's capsule), blue—inflammatory exudate. Lithium carbonate shows more extensive and severe pathological changes. Scale bar = 50 μm. Figure 19 The residual lithium content in rat kidney tissue during the subacute toxicity test (28d) of lithium butyrate-L-proline eutectic salt (BuLiPro) and control compound in Example 6 of this application is shown. Detailed Implementation

[0036] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0037] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided that there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed lowest and highest values ​​are considered to be clearly described in this application.

[0038] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0039] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0040] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0042] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0044] Example 1: Preparation of lithium butyrate-L-proline eutectic salt 1. Raw materials butyric acid: 99% purity, 90g; Lithium hydroxide: 35% aqueous solution, 24.47g; L-proline: 99% purity.

[0045] 2. Preparation steps: Under normal pressure and with the temperature controlled at approximately 60°C, 90g of 99% n-butyric acid was added dropwise to 24.47g of an aqueous solution of LiOH (molar ratio of n-butyric acid to LiOH was 1:1), wherein the mass percentage of LiOH in the aqueous solution was 35%. After the addition was complete, the system was kept at 60°C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the resulting solid was dried at 55°C for 15 hours to obtain 83g of lithium n-butyrate. An equimolar amount of lithium n-butyrate and L-proline was added to anhydrous ethanol at a volume ratio of 25, and the mixture was heated under reflux until just dissolved. The mixture was stirred for about 3 hours, allowed to cool naturally to crystallize, and no crystals precipitated. The solid was concentrated under reduced pressure, and then slurried with anhydrous ethanol at 0-5°C for 1 hour. The solid was then obtained by filtration.

[0046] Example 2 Characterization of lithium butyrate-L-proline eutectic salt 1. Nuclear Magnetic Resonance (NMR) Analysis The solid obtained in Example 1 was analyzed by 1H-NMR, and the results are as follows: Figure 1 and Figure 2 As shown.

[0047] Figure 1 In the sample, signals with δ values ​​of 3.93, 3.26–3.20, and 3.17–3.10 were attributed to L-proline, while signals with δ values ​​of 2.20–2.12, 1.37, and 0.70 were attributed to butyric acid.

[0048] Graphical integral: 1H-NMR (400 MHz, D2O) δ 3.93 (dd, J = 8.9, 6.2 Hz, 1H), 3.26-3.20 (m, 1H), 3.17-3.10 (m, 1H), 2.20-2.12 (m, 1H), 1.97 (t, J = 7.5 Hz, 2H), 1.92-1.77 (m, 3H), 1.37 (td, J = 14.9, 7.3 Hz, 2H), 0.70 (t, J = 7.5 Hz, 3H).

[0049] Spectral analysis: δ 21.97 (t, J = 7.5 Hz, 2H), 1.37 (td, J = 14.9, 7.3 Hz, 2H), and 0.70 (t, J = 7.5 Hz, 3H) can be attributed to hydrogen atoms on n-butyric acid; δ 3.93 (dd, J = 8.9, 6.2 Hz, 1H), 3.26–3.20 (m, 1H), 3.17–3.10 (m, 1H), 2.20–2.12 (m, 1H), and 1.92–1.77 (m, 3H) can be attributed to hydrogen atoms on L-proline.

[0050] NMR results showed that the molar ratio of butyric acid to L-proline was 1:1, confirming the composition of the eutectic salt.

[0051] 2. X-ray powder diffraction (XRD): XRD patterns of lithium butyrate and its eutectic salt ( Figure 3 The XRD pattern shows that the 2θ characteristic peaks of the sample are: 7.804°, 12.520°, 13.910°, 14.338°, 17.409°, 20.825°, 21.234°, and 28.274°. The XRD pattern of L-proline (…) Figure 4 The results show that the 2θ characteristic peaks are: 15.110°, 18.001°, 19.518°, 24.770°, 30.528°, and 32.120°.

[0052] As can be seen, compared with lithium butyrate and L-proline alone, the XRD spectrum of the eutectic salt obtained in Example 1 has new characteristic peaks, indicating that a new eutectic structure has been formed.

[0053] 3. Digital Scanning Calorimetry (DSC) DSC chart ( Figure 5 The results show that the eutectic salt has a stable melting point and exhibits enhanced thermal stability compared to the single component.

[0054] 4. Lithium content detection (ICP-MS): 1) Preparation of diluent: Accurately measure 10 mL of nitric acid into a 500 mL volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0055] 2) Preparation of test sample: Stock solution of test sample: Accurately weigh approximately 25 mg of the test sample into a 50 mL polytetrafluoroethylene volumetric flask, dilute to the mark with diluent, and mix well. Test sample solution: Accurately transfer 1 mL of the stock solution of test sample into a 10 mL polytetrafluoroethylene volumetric flask, dilute to the mark with diluent, and mix well.

[0056] 3) Preparation of standard solution: Prepare a 1 ppm Li standard solution according to the concentration of the standard solution.

[0057] The results are shown in Table 1.

[0058] Table 1. ICP-MS Lithium Content Detection Results

[0059] pass 1 H-NMR, 13 Analysis of the sample by C-NMR, XRD, DSC and ICP-MS revealed that the sample is a eutectic salt of butyric acid: lithium: L-proline (1:1:1), named BuLiPro.

[0060] Example 3: Determination of GSK3β Inhibitory Activity Prepare 2×ATP / substrate solution and 2× kinase solution, and add kinase reaction buffer. Using an Echo 655, transfer 100 nmL of the compound and 100 nmL of DMSO dilution to a 384 assay plate. Add 5 μL of 2× kinase solution to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min. Add 5 μL of 2× substrate and ATP solution to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 60 min. Transfer 5 μL of ADP-Glo ​​reagent to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min. Add 10 μL of kinase assay reagent to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min. Use BMG to read the luminescence signal.

[0061] The inhibition rate of the composite well (% inh) = 100 * (high control - cpd well) / (high control - low control).

[0062] IC50 was calculated by fitting the % inhibition value and the logarithm of the compound concentration to a nonlinear regression (dose response-variable slope) using GraphPad 8.0.

[0063] Lithium butyrate-L-proline co-crystal salt (BuLiPro), lithium chloride (LiCl) (CAS: 7447-41-8), lithium butyrate (LiB) (CAS: 21303-03-7), sodium butyrate (NaB), lithium isobutyrate-L-proline co-crystal salt (IsoLiPro) (prepared using the method disclosed in CN114081881A), and lithium isobutyrate (LiIsobu) (CAS: 25179-23-1) were prepared into a 1 mM stock solution, diluted to a 10 mM working solution, and then down-diluted 10 times at a ratio of 1.5 to form a gradient. The inhibition rate was detected according to the GSK3β activity detection method described above, and the IC50 was calculated. The results are as follows: Figure 6 As shown in Table 2.

[0064] Table 2 IC50 of compounds such as BuLiPro against GSK3β

[0065] Depend on Figure 6 As shown in Table 2, the IC50 value of BuLiPro was significantly lower than that of the control compounds (LiB, LiCl, NaB, IsoLiPro and LiIsobu), indicating that it has more efficient GSK3β inhibitory activity.

[0066] Example 4: Cellular Experiment Validation 1. Effects on tau protein in HEK293 / tau cells HEK293 / tau cells were transfected with a vector overexpressing htau441 (human tau 441) protein. After 48 hours, the HEK293 / tau cells were evenly seeded in six-well plates until the confluence reached approximately 70%. (1) After treatment with different concentrations of BuLiPro (0, 1.25, 2.5, 5, 10 mM) for 24 h, total protein was extracted. Sodium butyrate (NaB) was used as a control for butyrate and other metal ion co-crystal salts.

[0067] Total tau protein was analyzed by Western blot (WB) using tau12 antibody. β-actin was used as an internal control for WB protein normalization, and GFP was used as an internal control for exogenous gene expression normalization. Results are as follows: Figure 7 The results showed that the total tau protein level in cells decreased with increasing BuLiPro treatment concentration, indicating that the effect of BuLiPro on tau protein levels was concentration-dependent. The results also showed that BuLiPro significantly reduced tau protein levels in a dose-dependent manner.

[0068] (2) HEK293 / tau (P301L) was treated with 20mM lithium concentrations of BuLiPro, LiCl or IsoLiPro for 1, 2 or 4 hours, and then the total protein, total tau and phosphorylated tau levels were extracted.

[0069] The results are as follows Figure 10 The selective regulation of phosphorylation sites by BuLiPro was analyzed. The effects of BuLiPro (20 mM) on different phosphorylation sites were examined over 1-4 hours. p-S396 phosphorylation significantly decreased to 63.1 ± 7.8% of the control group after 1 hour of treatment, significantly better than the LiCl group (P < 0.01); after 2 hours, it decreased to 56.9 ± 5.6%, also significantly lower than the LiCl group (P < 0.01). p-T181 and AT8 (p-S202 / T205) showed no significant difference compared to LiCl. Short-term treatment with BuLiPro significantly inhibited S396 phosphorylation, significantly better than traditional lithium salts (LiCl), revealing that BuLiPro's regulation of tau phosphorylation differs from that of lithium ions. Furthermore, BuLiPro was significantly superior to IsoLiPro in inhibiting S396 phosphorylation.

[0070] 2. Effects on tau protein in SH-SY5Y / tau cells SH-SY5Y cells were transfected with a lentiviral-packaged tau protein overexpression vector. After 48 hours, SH-SY5Y / tau cells were seeded evenly in six-well plates. When the confluence reached about 70%, the cells were treated with different concentrations of BuLiPro (0, 2.5, 5, 10, 20, 40 mM) for 24 hours, and then the total protein was extracted.

[0071] Total tau protein was analyzed by Western blot (WB) using tau12 antibody, while β-actin was used as an internal control for WB protein standardization.

[0072] The results are as follows Figure 8 As shown, the total tau protein level in cells also showed a decreasing trend with increasing BuLiPro treatment concentration.

[0073] 3. Effects of puromycin on tau protein in HEK293 cells screened. HEK293 cells were transfected with a lentiviral vector overexpressing htau441 protein and screened with puromycin to obtain stable transgenic cells. The HEK293 / tau stable transgenic cells were seeded evenly in six-well plates. After the confluence reached about 70%, the cells were treated with different concentrations of BuLiPro (0, 1.25, 2.5, 5, 10, 20 mM) for 24 h, and then the total protein was extracted.

[0074] Total tau protein was analyzed by Western blot (WB) using tau12 antibody, while β-actin was used as an internal control for WB protein standardization.

[0075] The results are as follows Figure 9 As shown, similarly, the levels of total tau protein and phosphorylation (ptau level) in cells decreased with increasing BuLiPro treatment concentration.

[0076] Example 5: Animal Experiment Verification The experiment was divided into four groups: 3xTg mice / ddH2O group: 3xTg mice / Li2CO3 group (9.24 mg Li / kg); 3xTg mice / BuLiPro low-dose group (9.24 mg Li / kg); 3xTg mice / BuLiPro high-dose group (18.48 mg Li / kg).

[0077] 3xTg mice were administered the drug via gavage at 36 weeks of age, once daily for 12 weeks. After 12 weeks, the mice were anesthetized and perfused, and brain tissue was removed. The hemispheres were sagittal sections, paraffin sections were prepared, and immunohistochemical staining was performed. The hippocampus of the hemispheres was dissected for Western blot analysis.

[0078] See results Figure 11 In the BuLiProHigh and Low groups, tau-positive staining in the CA1 region of the hippocampus was lighter than that in the ddH2O and Li2CO3 groups, and p-tau181-positive staining in the CA3 region was lighter than that in the ddH2O group (e.g., ...). Figure 12 ), and consistent with the WB results of the hippocampus ( Figure 12 It also showed a significant trend of downregulating tau and tau phosphorylation, with the effects of downregulating total tau and p-tau202 / 205, p-tau396 being significantly stronger than those in the Li2CO3 group (see [link to article]). Figure 13 ).

[0079] The results showed that oral administration of BuLiPro significantly downregulated the levels of tau and phosphorylated tau in the brains of 3xTg mice, with better effects than Li2CO3.

[0080] The inventors also prepared paraffin sections of mouse liver and kidney tissue and stained them with H&E. The results are as follows: Figure 14 and Figure 15 As shown.

[0081] The results showed that long-term administration of Li2CO3 caused liver congestion, punctate necrosis, and patchy necrosis in mice, with inflammatory cell infiltration at the interlobular vein walls. The kidneys exhibited pathological changes such as glomerular mesangial proliferation, inflammatory cell infiltration, and fibrosis. These phenomena were not observed in either the high-dose or low-dose BuLiPro groups, suggesting that BuLiPro has strong safety.

[0082] Example 6 Animal safety evaluation SPF-grade SD rats (half male and half female, with females not yet pregnant or having given birth), weighing 180–220 g. All animals were quarantined for 3 days prior to the experiment, with free access to water and food. Test compounds included BuLiPro, Li₂CO₃, and IsoLiPro. Based on acute oral toxicity reactions in rats, control groups (ddH₂O treatment), low (12.82 mg Li / kg), medium (16.67 mg Li / kg), and high (21.67 mg Li / kg) dose groups were established. Additionally, a control satellite group and a high-dose satellite group were established (treated the same as the control and high-dose groups, but continued to be fed for 14 days after cessation of treatment). Each group consisted of 10 animals, half male and half female. All groups were administered the drugs orally via gavage at a dose of 10 mL / kg BW, once daily for 28 consecutive days.

[0083] The results showed that at higher isolithium doses, the lethality of BuLiPro was lower than that of Li2CO3 or IsoLiPro ( Figure 16 Medium dose: In the IsoLiPro group, 4 rats (3 females, 1 male) died in week 4, while no deaths were observed in the BuLiPro and Li2CO3 groups. High dose: In the Li2CO3 group, rat deaths occurred as early as week 2, with a total of 5 deaths (1 female, 4 males), while in the BuLiPro group, the earliest deaths occurred in week 3, with a total of 2 deaths (1 male, 1 female) at the end of the experiment. No obvious abnormalities were observed in the low-dose group during the administration period, while slow weight gain was observed in the medium and high dose groups. Figure 16 , Figure 17 ).

[0084] H&E staining results of rat kidney tissue showed that ( Figure 18In contrast, the high-dose Li2CO3 group exhibited more severe and pronounced renal pathological changes, with severe structural changes in the renal tubules, edema and diffuse shedding of renal tubular epithelial cells, tubular dilation with inflammatory cell infiltration, and granular or protein casts. Extensive inflammatory infiltration was also observed in the renal interstitium around the collecting ducts. No significant recovery was observed after a two-week withdrawal period. Among the high-dose groups, the BuLiPro group showed the mildest lesions, followed by IsoLiPro, with Li2CO3 showing the most severe. The high-dose BuLiPro group only exhibited narrowing of Bowman's capsule and renal tubular lumen, as well as focal glomerular inflammation and tubular epithelial cell shedding. After a two-week withdrawal period, the severity and incidence significantly decreased, showing a trend towards recovery. Figure 18 Similar lesions were still observed in the low-dose Li2CO3 group, but not in the low-dose BuLiPro group. Figure 18 ).

[0085] The inventors further examined the Li accumulation in kidney tissue and found that the Li element accumulation in the high-dose group of Li₂CO₃ was significantly higher than that in BuLiPro ( Figure 19 ).

[0086] In conclusion, BuLiPro demonstrates superior toxicological safety compared to IsoLiPro and Li2CO3 in animal studies.

[0087] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A lithium butyrate proline eutectic salt, characterized in that, It is prepared by co-crystallization of lithium salt of butyric acid and proline or proline derivative in an equimolar ratio.

2. The lithium butyrate proline eutectic salt according to claim 1, characterized in that, The proline derivative is proline with chemically modified carboxyl and / or imine functional groups.

3. The lithium butyrate-proline eutectic salt according to claim 1 or 2, wherein the eutecticization is one of solvent evaporation crystallization, cooling crystallization, grinding crystallization, reaction crystallization, hot melt extrusion crystallization, melt crystallization, and acoustic wave crystallization.

4. The method for preparing lithium butyrate proline eutectic salt according to any one of claims 1 to 3, characterized in that, Includes the following steps: The lithium salt of butyric acid and the proline are dissolved in a solvent in an equimolar ratio. The solution is dissolved under reflux and cooled to precipitate crystals. The crystals are collected and dried to obtain the lithium butyric acid-proline eutectic salt.

5. The method according to claim 4, characterized in that, The solvent is selected from one or more of water, methanol, and ethanol.

6. The use of the lithium butyrate proline cocrystal salt according to any one of claims 1 to 3 in the preparation of a medicament for at least one of the following: (1) Inhibit GSK3β; (2) Reduce tau protein levels; (3) Reduce phosphorylated tau protein levels; (4) Reduce nerve inflammation; (5) Enhance neuroprotection; (6) Maintaining neuronal functional stability; (7) Prevention or treatment of neurodegenerative diseases; (8) Prevention or treatment of cancer; (9) Prevention or treatment of mental illnesses; (10) Prevention or treatment of type II diabetes and / or its complications.

7. A drug, characterized in that, It includes the lithium butyrate proline eutectic salt as described in any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient and / or diluent.

8. The medicament according to claim 7, characterized in that, The dosage form of the drug is selected from one of the group consisting of oral preparations, injections, aerosols, suppositories, drops, and transdermal patches.

9. The drug according to claim 7 or 8, characterized in that, The drug is administered via the respiratory or digestive system; or subcutaneously; or via the nasal mucosa or submucosa; or via the eye or ear; or via the rectum; or via the vagina.

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