Benzimidazole methylenhydrazine thiazole derivatives and their use in the prevention and treatment of neurodegenerative diseases

Abstract

This invention belongs to the field of pharmaceutical technology and relates to benzimidazole methylenehydrazine thiazole compounds of Formula I, pharmaceutically acceptable salts thereof and methods for their preparation, compositions containing one or more compounds, and the use of such compounds in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to benzimidazole methylenehydrazylthiazole compounds and their pharmaceutical uses. Specifically, this invention relates to certain benzimidazole methylenehydrazylthiazole derivatives and their applications in the prevention and treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Background Technology

[0002] Neurodegenerative diseases are progressive functional disorders caused by degenerative changes in the central nervous system. With the accelerating aging process, they are placing an increasingly heavy burden on global healthcare systems. Typical examples include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). These diseases are characterized by irreversible neuronal damage, leading to cognitive decline, motor coordination impairment, and other symptoms. Their high rates of disability and mortality severely impair patients' quality of life, placing a heavy dual burden on patients' families and public health systems.

[0003] However, the clinical treatment of neurodegenerative diseases still faces severe challenges. Existing drugs are mostly limited to symptom relief (such as cholinesterase inhibitors and dopamine replacement therapy), and cannot reverse or delay the degenerative process of nerve cells (Nature, 2016, 539:179). Recent studies have shown that the molecular mechanisms of neurodegenerative diseases involve a multi-dimensional pathological network, including oxidative stress-mediated mitochondrial dysfunction and neuroinflammation caused by excessive microglial activation (Frontiers In Molecular Neuroscience, 2022, 15:844193). By synergistically regulating the above-mentioned pathological cascade reactions, neuroprotective effects are expected to be achieved, providing a new breakthrough for delaying or even blocking disease progression. Compounds with antioxidant properties may have neuroprotective effects, thereby delaying the course of neurodegenerative diseases.

[0004] Among benzimidazole-based methylenehydrazinethiazole compounds, the unique bicyclic structure and modifiability of the benzimidazole fragment make it a common backbone for constructing bioactive molecules. Studies have confirmed that benzimidazole compounds exhibit multi-dimensional application potential in the pharmaceutical field. Currently, several drugs based on this structure have been successfully translated into clinical applications, covering therapeutic areas such as anticancer, antibacterial, anti-inflammatory, analgesic, anti-HIV, anticonvulsant, anti-tuberculosis, antidiabetic, and antihistamine treatments (ChemMedChem, 2021, 16:1861-1877). These drugs, with their low toxicity, high bioavailability, good biocompatibility, and significant efficacy, occupy an important position in the prevention and treatment of various diseases. In recent years, with the deepening of molecular target research, research into revealing new mechanisms and targets of action of benzimidazole compounds continues.

[0005] Thiazoles, as important five-membered sulfur-nitrogen heterocyclic compounds, possess both metabolic stability and target binding ability, making them a superior scaffold for drug design. Several blockbuster drugs contain a thiazole ring, such as the antibacterial sulfathiazole, the anti-HIV ritonavir, the antitumor dasatinib analogue, and vitamin B1 (thiamine). Meanwhile, research on HDAC6 inhibitors and PPARγ agonists continues to drive their application in the treatment of cancer and metabolic diseases (Journal of Medicinal Chemistry, 2016, 59:1545-1555 and Bioorganic & Medicinal Chemistry Letters, 2010, 20:2933-2937). It has been reported that the methylenehydrazylthiazole scaffold has certain inhibitory activity against monoamine oxidase A and monoamine oxidase B (European Journal of Medicinal Chemistry, 2012, 58:405-417). However, current research still faces limitations such as the synthesis of toxic reagents, drug resistance, off-target toxicity, and low oral bioavailability of some compounds. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a class of benzimidazole methylene hydrazylthiazole derivatives, their preparation methods, pharmaceutical compositions, and their use in the preparation of drugs for the prevention or treatment of neurodegenerative diseases.

[0007] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0008] The first aspect of the present invention is to provide a compound or pharmaceutically acceptable salt represented by Formula I:

[0009]

[0010] In the formula:

[0011] R1 is independently selected from hydrogen, C 1-6 Alkyl; R2 can be monosubstituted or polysubstituted in Formula I, independently selected from hydrogen, halogen, amino, carbonyl, cyano, nitro, C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Heteroalkyl, 3-6 membered heterocyclic alkyl, 5-7 membered aryl, 5-7 membered heteroaryl.

[0012] Preferred benzimidazole methylenehydrazinethiazole compounds or their pharmaceutically acceptable salts are characterized in that R1 is independently selected from hydrogen and methyl; and R2 is monosubstituted in Formula I and is independently selected from hydrogen, fluorine, chlorine, bromine, and iodine.

[0013] A more preferred benzimidazole methylenehydrazinethiazole compound or a pharmaceutically acceptable salt thereof, characterized in that R1 is independently selected from hydrogen and methyl; R2 is monosubstituted in Formula I, and R2 is independently selected from hydrogen, fluorine, and chlorine.

[0014] The most preferred benzimidazole methylenehydrazylthiazole compounds or their pharmaceutically acceptable salts are selected from the following group:

[0015] Compound 1

[0016]

[0017] Compound 2

[0018]

[0019] Compound 3

[0020]

[0021] Compound 4

[0022]

[0023] Compound 5

[0024]

[0025] Compound 6

[0026]

[0027] Compound 7

[0028]

[0029] The pharmaceutically acceptable salt described in the first aspect of this invention is selected from salts formed by compounds and inorganic or organic acids. The inorganic acid is selected from sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, and nitric acid, and the organic acid is selected from oxalic acid, citric acid, maleic acid, fumaric acid, malic acid, tartaric acid, methanesulfonic acid, and benzenesulfonic acid.

[0030] The second aspect of the present invention is to provide a method for processing a compound represented by Formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, which mainly includes the following steps:

[0031] Wherein, R1 and R2 are defined independently as described in the first aspect of the present invention; X is bromine or chlorine;

[0032]

[0033] Step 1: In anhydrous ethanol, compounds M1 and M2 react under the catalysis of acetic acid to generate intermediate M3 linked by thiourea.

[0034] Step 2: In anhydrous ethanol, compound M3 and compound M4 react to give the compound shown in Formula I.

[0035] A third aspect of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains an effective amount of the compound of the first aspect of the present invention, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0036] A fourth aspect of the present invention is the use of the compounds described in the first aspect of the invention and their pharmaceutically acceptable salts in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases. The neurodegenerative diseases are selected from Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

[0037] Terminology Explanation

[0038] Pharmaceutically acceptable salts refer to base addition salts formed by the carboxyl group in the molecule and alkali metals such as sodium, potassium, and lithium, alkaline earth metals such as calcium and magnesium, and metals such as aluminum, or ammonia or organic amines; or acid addition salts formed by the amino group in the molecule and inorganic or organic acids. Here, inorganic acids include sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, nitric acid, etc., and organic acids include oxalic acid, citric acid, maleic acid, fumaric acid, malic acid, tartaric acid, sulfonic acid, etc. This invention does not impose any restrictions on pharmaceutically acceptable salts.

[0039] In this invention, alkyl refers to straight-chain and branched saturated aliphatic groups having a specific number of carbon atoms.

[0040] "Halogen" or "halogenated" refers to fluorine, chlorine, bromine, or iodine as substituents. When a halogen atom is used as a substituent, it can replace one, two, or three atoms.

[0041] The compounds of the present invention were prepared as shown in Procedure 1. The compounds of the present invention were obtained from: (a) commercially available raw materials; (b) known raw materials that can be prepared by the methods described in the literature; and (c) intermediates described in the procedures and experiments herein.

[0042] Process 1:

[0043]

[0044] The reaction is carried out in a solvent suitable for the reagents and materials used and for efficient conversion. As is known in the field of organic synthesis, the various functional groups present on the molecule must correspond to the planned chemical transformation. Those skilled in the art can modify the above steps to improve the yield; they can determine the synthetic route based on fundamental knowledge in the art, such as selecting reactants, solvents, and temperatures. Such modifications or variations are within the scope of this invention.

[0045] Beneficial technical effects: The compound has a novel structure and there are no reports of its application in drugs for the prevention and / or treatment of neurodegenerative diseases. Attached Figure Description

[0046] Figure 1. Effects of compound 1 on motor function in PD mice

[0047] Figure 2. Effects of compound 1 on dopaminergic neurons in PD mice

[0048] Figure 3. Changes in body weight in mice after oral administration of compound 1

[0049] Figure 4. Effects of oral administration of compound 1 on organ indices in mice Detailed Implementation

[0050] The following examples further illustrate the present invention. However, the present invention is not limited to these examples. Any modifications made by those skilled in the art to the implementation of the present invention based on the teachings of this specification will fall within the scope of the claims.

[0051] Chemical Experiment Section

[0052] Instrumentation: Melting point was determined using an RY-2 melting point apparatus manufactured by Tianjin Analytical Instrument Factory; the temperature was not calibrated. Proton NMR spectroscopy was performed using a Wuhan Zhongke Spectroscopy Qone series 400MHz superconducting NMR spectrometer with DMSO-d6 as the solvent. ESI-HRMS data were collected using a Thermo Exactive Orbitrap mass spectrometer.

[0053] Example 1: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(4-fluoro)thiazole hydrobromide (Compound 1)

[0054] Step 1: (E)-2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)thiourea

[0055]

[0056] 2-Acetylbenzimidazole (1 g, 6.24 mmol), aminothiourea (569 mg, 6.24 mmol), and 15 mL of anhydrous ethanol were added to a round-bottom flask. Then, 1 mL of glacial acetic acid was added, and the mixture was refluxed at 80 °C for 12 h. The reaction was monitored by TLC until complete. After cooling to room temperature, the solvent was removed by vacuum distillation, and the mixture was filtered through 15 mL of anhydrous methanol to obtain a white solid in 82% yield. 1 H NMR (400MHz, DMSO-d6): δ12.73(s,1H),10.70(s,1H),8.60-8.51(m,2H),7.67(d ,J=8.0Hz,1H),7.49(d,J=8.0Hz,1H),7.22(dt,J=32.0,7.8Hz,2H),2.43(s,3H).

[0057] Step 2: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(4-fluoro)thiazole hydrobromide (Compound 1)

[0058]

[0059] Add (E)-2-(1-(1H-benzo[d]imidazol-2-yl)ethylidene)thiourea (200 mg, 0.86 mmol), 2-bromo-4'-fluoroacetophenone (186 mg, 0.86 mmol), and 10 mL of anhydrous ethanol to a round-bottom flask, and stir at room temperature for 5 h. The reaction was monitored by TLC until complete. Filter to give a yellow solid, 92% yield, melting point: 275.5–276 °C. ESI-HRMS: m / z = 352.1021 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1 H NMR (400MHz, DMSO-d6): δ7.93 (dd, J=8.6, 5.5Hz, 2H), 7.79 (dd, J=6.2, 3.1Hz, 2H), 7.56-7.50 (m, 3H), 7.27 (t, J=8.7Hz, 2H), 2.50 (s, 3H).

[0060] Example 2: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(4-fluoro)thiazole (Compound 2)

[0061]

[0062] Compound 1 from Example 1 was released as follows: 50 mg of compound 1 was weighed and dissolved in 15 mL of ethyl acetate, followed by washing with ammonia (10 mL × 3), and then with saturated brine (10 mL × 3). The organic layer was collected, dried over anhydrous Na₂SO₄, and the solvent was evaporated to obtain a yellow solid with a yield of 95% and a melting point of 244-244.5 °C. ESI-HRMS: m / z = 352.1027 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1 H NMR (400MHz, DMSO-d6): δ12.27(s,1H),11.61(s,1H),7.93(dd,J=8.6,5.8Hz,2H),7.65(d,J=8. 0Hz, 1H), 7.54 (d, J = 7.9Hz, 1H), 7.45 (s, 1H), 7.31-7.21 (m, 3H), 7.21-7.13 (m, 1H), 2.48 (s, 3H).

[0063] Example 3: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(4-fluoro)thiazole hydrochloride (compound 3)

[0064]

[0065] (E)-2-(1-(1H-benzo[d]imidazol-2-yl)ethylidene)thiourea (200 mg, 0.86 mmol), 2-chloro-4'-fluoroacetophenone (148 mg, 0.86 mmol), and 10 mL of anhydrous ethanol were added to a round-bottom flask, and the mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC until complete, and the mixture was filtered to give a yellow solid in 53% yield with a melting point of 247–247.5 °C. ESI-HRMS: m / z = 352.1022 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1H NMR (400MHz, DMSO-d6): δ7.93 (dd, J=8.4, 5.5Hz, 2H), 7.79 (dd, J=6.1, 3.2Hz, 2H), 7.56-7.46 (m, 3H), 7.28 (t, J=8.7Hz, 2H), 2.54 (s, 3H).

[0066] Example 4: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(3-fluoro)thiazole hydrobromide (Compound 4)

[0067]

[0068] The procedure was the same as in Example 1, except that 2-bromo-3'-fluoroacetophenone was used instead of 2-bromo-4'-fluoroacetophenone. The product was a yellow solid with a yield of 89% and a melting point of 268-268.5 °C. ESI-HRMS: m / z = 352.1027 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1 H NMR (400MHz, DMSO-d6): δ7.78 (dd, J=6.1, 3.2Hz, 2H), 7.75 (d, J=8.2Hz, 1H), 7 .72-7.66(m,2H),7.57-7.45(m,3H),7.17(td,J=8.6,2.6Hz,1H),2.50(s,3H).

[0069] Example 5: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(3-fluoro)thiazole (Compound 5)

[0070]

[0071] Compound 4 from Example 4 was released, following the procedure described in Example 2. ESI-HRMS: m / z = 352.1027 [M+H] + calcd for C 18 H 15 FN5S: 352.1027. The product is a yellow solid with a yield of 96% and a melting point of 228-228.5℃. 1HNMR (400MHz, DMSO-d6): δ12.29(s,1H),11.64(s,1H),7.75(d,J=8.2Hz,1H),7 .72-7.51(m,4H),7.47(td,J=8.0,6.2Hz,1H),7.28-7.09(m,3H),2.49(s,3H).

[0072] Example 6: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(2-fluoro)thiazole hydrobromide (Compound 6)

[0073]

[0074] The procedure was the same as in Example 1, except that 2-bromo-2'-fluoroacetophenone was used instead of 2-bromo-4'-fluoroacetophenone. The product was a yellow solid with a yield of 83% and a melting point of 268-268.5 °C. ESI-HRMS: m / z = 352.1021 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1 H NMR (400MHz, DMSO-d6): δ8.05(t,J=7.9Hz,1H),7.79(dd,J=6.1,3.2Hz,2H),7.53(dd,J=6.2 ,3.1Hz,2H),7.48(s,1H),7.44-7.37(m,1H),7.36-7.29(m,2H),3.90(brs,2H),2.51(s,3H).

[0075] Example 7: (E)-2-(2-(1-(1H-benzo[d]imidazol-2-yl)ethylene)hydrazyl)-4-(2-fluoro)thiazole (Compound 7)

[0076]

[0077] Compound 6 from Example 6 was released, following the procedure described in Example 2. The product was a yellow solid, 95% yield, with a melting point of 234-234.5 °C. ESI-HRMS: m / z = 352.1027 [M+H] + calcd for C 18 H 15 FN5S:352.1027. 1HNMR (400MHz, DMSO-d6): δ12.30(s,1H),11.64(s,1H),8.11-8.04(m,1H),7.65(d,J=7.9Hz,1H),7.58-7.51 (m,1H),7.42-7.27(m,4H),7.24(td,J=8.0,7.5,1.2Hz,1H),7.17(ddd,J=8.3,7.2,1.3Hz,1H),2.49(s,3H).

[0078] Pharmacologically active part

[0079] Experimental Example 1: Evaluation of the antioxidant activity of compounds 1-7 in the examples

[0080] 1) Experimental method:

[0081] The test tubes were set up as a control group, model group, positive control group, and compound group, with three replicates for each group. Each tube contained a mixture of 155 μL PBS buffer, 30 μL liver microsomes, and 50 μL cysteine. 2.5 μL DMSO was added to each tube of the control and model groups, and 2.5 μL curcumin (final concentration 10) was added to each tube of the positive control group. -5 M), 2.5 μL of compound was added to each tube (final concentration 10). -5 M). After placing all test tubes in a 37°C water bath and shaking for 15 min, add 13 μL of PBS buffer to the control group, and add 10 μL of PBS buffer to each tube of the model group, positive control group, and compound group. -2 13 μL of M FeSO4 solution was added. All test tubes were placed in a 37°C water bath and shaken for 15 min. Then, 250 μL of trichloroacetic acid (TCA) solution and 500 μL of thiobarbituric acid (TBA) solution were added to each tube. All test tubes were placed in a 100°C water bath for 10 min, shaken to mix, and then 600 μL of the liquid from each tube was transferred to a 1.5 mL EP tube. The tubes were centrifuged at 8000 rpm for 10 min. 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader. The inhibition rate of the compound was calculated using the following formula.

[0082] 2) Experimental results:

[0083] The experimental results are listed in Table 1. The results show that compounds 1, 3, 4, and 6 in the examples have good antioxidant activity.

[0084] Table 1. Antioxidant effects of compounds 1-7

[0085] Sample number MDA generation inhibition rate (%) Compound 1 98.48 Compound 3 96.77 Compound 4 97.21 Compound 6 96.62 Curcumin 98.03

[0086] Experimental Example 2: Evaluation of the protective effect of the compounds in the examples against rotenone-induced PC12 cells.

[0087] 1) Experimental method:

[0088] PC12 cells were cultured in DMEM high-glucose medium containing 10% serum. PC12 cells in the logarithmic growth phase were harvested, digested with trypsin, and then cultured at 8 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of [number] wells in 96-well plates and cultured at 37°C in a 5% CO2 incubator for approximately 24 hours. Cells were then divided into a control group, a model group, and a test compound group. When cells reached approximately 60% confluence, the model group and the test compound group were treated with 15 μM rotenone, while the test compound group was simultaneously treated with 10 μM of different compounds. The control group was treated with an equal volume of DMSO. Cells were cultured for another 24 hours. Cell viability was assessed using the CCK-8 assay. CCK-8 solution was added to each well at a 1:10 ratio, and the plates were incubated. After 4 hours, the absorbance was measured at 450 nm using a microplate reader. The drug protection rate was calculated using the following formula.

[0089]

[0090] 2) Experimental results:

[0091] The experimental results are listed in Table 2. The results show that compound 1 in the examples has a good neuroprotective effect.

[0092] Table 2. Protective effect of compound 1 against rotenone-induced PC12 cells

[0093] Sample number Protection rate (%) Compound 1 40.26

[0094] Experimental Example 3: Evaluation of the inhibitory effect of compounds 1-7 from Examples on monoamine oxidase B.

[0095] 1) Experimental method:

[0096] Prepare a detection buffer (50 mM phosphate buffer, pH 7.4) to dilute the enzyme, substrate, and detection reagents. Then, use an Echo liquid transfer instrument to transfer 200 nL of the test compound (10 μM dissolved in DMSO) to a 384-well black detection plate. Add 10 μL of LMA-B enzyme solution (300 nM), 5 μL of Amplex UltraRed / Horseradish peroxidase (HRP) mixture (200 μM Amplex Red, 1 U / mL HRP), and 5 μL of tyramine hydrochloride (400 μM) sequentially. After each addition, centrifuge at 1000 rpm for 1 minute. After incubation at 25°C in the dark for 60 minutes, use a microplate reader to detect the fluorescence signal. The excitation wavelength is 530 nm, and the emission wavelength is 590 nm. Measure the fluorescence value.

[0097]

[0098] 2) Experimental results:

[0099] The experimental results are listed in Table 3. The results show that compounds 1-7 in the examples did not have a significant MAO-B inhibitory effect.

[0100] Table 3. Inhibitory effects of compounds 1-7 on MAO-B

[0101] Sample number Concentration (μM) Inhibition rate (%) Compound 1 10 20.8 Compound 2 10 1.3 Compound 3 10 22.5 Compound 4 10 18.8 Compound 5 10 9.7 Compound 6 10 19.3 Compound 7 10 9.6 Safenamide 0.3 95.5

[0102] Experiment Example 4: Evaluation of the neuroprotective effect and behavioral improvement of compound 1 in the example using rotenone-induced PD model mice.

[0103] 1) Experimental method:

[0104] I) Establishment and administration of PD mouse model

[0105] After acclimatizing for 3 days, C57BL / 6 mice were randomly divided into four groups according to body weight: control group, rotenone group, compound 1 group, and selegiline group. Mice in the rotenone, compound 1, and selegiline groups were administered 30 mg / kg rotenone by gavage daily for 4 weeks, while the control group received rotenone in 0.5% CMC-Na solution. Two weeks after rotenone gavage, the compound 1 group received 50 mg / kg compound 1 by gavage daily, and the selegiline group received 10 mg / kg selegiline by gavage daily for 4 weeks, followed by behavioral assessments.

[0106] II) Behavioral testing

[0107] a) Rotating Cane: The rotating cane device has a horizontal metal rod (3cm in diameter) and is approximately 50cm long. It is divided into 5 sections by metal plates, and baffles are used to separate the animals so that they do not affect each other. The rotating cane device is set to rotate at a constant speed of 20rpm. The mouse is placed on the rod and the timer is started. The latency period (i.e., the time of the first fall) until the mouse falls off the rod is recorded as the mouse's motor coordination ability.

[0108] b) Grip strength: A nylon rope is suspended horizontally above the mouse cage. Sufficient bedding is placed inside the cage. The mouse's front paws grasp the rope and immediately release it. The time the mouse remains on the rope is recorded and scored. The scoring criteria are as follows: 5 points: Attempts to move horizontally on the rope; 4 points: Limbs and tail wrapped around the rope; 3 points: Attempts to climb the rope or front paws and at least one hind paw are simultaneously attached to the rope; 2 points: Front paws are attached to the rope (greater than 5 seconds); 1 point: Front paws are attached to the rope (less than 5 seconds); 0 points: The mouse falls directly from the rope (no grasping ability).

[0109] c) Balance Beam: The balance beam is a square wooden pole 100cm long and 1cm wide, with a black box lined with sawdust at one end. The experiment was divided into a training period and a testing period. On the first day, the mouse was placed 80cm from the black box end of the balance beam and allowed to walk into the black box. This training was repeated twice. On the second day, the mouse was placed 80cm from one end of the balance beam and allowed to walk to the black box at the other end. The time taken for the mouse to cross the balance beam from the starting position and the number of times it slipped were recorded and scored. The scoring criteria were as follows: 5 points: The mouse will fall off the balance beam; 4 points: The mouse cannot cross the balance beam but can sit on it; 3 points: The mouse can cross the balance beam, but the affected paralyzed hind limb cannot help move forward; 2 points: The mouse crosses the balance beam with a greater than 50% chance of falling; 1 point: The mouse crosses the balance beam with a less than 50% chance of falling; 0 points: The mouse crosses the balance beam without falling.

[0110] III) Immunohistochemical detection

[0111] After the last administration of compound 1, three mice from each group were randomly selected, and their brains were harvested and fixed overnight in 4% paraformaldehyde. After complete fixation and embedding, serial coronal sections of the substantia nigra were prepared using a cryostat, with a thickness of approximately 4 μm. The sections were incubated with 3% H₂O₂ at room temperature for 10 min, washed three times with PBS for 10 min each time. The sections were blocked with sheep serum stock solution at room temperature for 1 h, and then incubated overnight at 4°C with diluted polyclonal rabbit TH antibody (1:500). Biotin-labeled anti-rabbit secondary antibody was then added, and the sections were incubated at room temperature for 2 h. Horseradish peroxidase (HRP)-labeled streptavidin was added, and the sections were incubated at room temperature for 2 h. The sections were developed with DAB working solution for approximately 2 min, and the colorimetric reaction was terminated with tap water. The sections were dehydrated sequentially with alcohol, cleared with xylene, and mounted with neutral resin. Changes in the number of tyrosine hydroxylase (TH)-positive neurons in the brain sections were observed under an optical microscope.

[0112] 2) Experimental results:

[0113] I) Effect of compound 1 in the examples on motor function in rotenone-induced PD mice

[0114] Compared with the control group, rotenone-treated mice exhibited significant motor impairment, indicating successful establishment of the PD model. Administration of compound 1 significantly improved the motor impairment in mice, specifically manifested as a significant increase in time spent on the spinning stick, a significant improvement in grip strength score, a significant decrease in balance beam score, and a significant reduction in the number of falls and the time taken to cross the balance beam (see...). Figure 1) .

[0115] II) Effects of compound 1 in the examples on dopaminergic neurons in rotenone-induced PD mice

[0116] Immunohistochemical results showed that rotenone-treated mice had fewer TH-positive neurons in the substantia nigra, and oral administration of compound 1 increased the number of TH-positive neurons in the substantia nigra and improved the function of dopaminergic neurons in PD mice (see [link to article]). Figure 2) .

[0117] Based on the above results, it can be concluded that compound 1 (50 mg / kg) can significantly improve motor function and reduce dopaminergic neuron damage in PD mice after administration.

[0118] Example 5: Safety evaluation of compound 1 in the examples

[0119] 1) Experimental method:

[0120] I) Experimental animals: Compound 1 was prepared into suspensions of 1 g / kg and 2 g / kg using 0.5% CMC-Na solution. The experimental animals were 18 male ICR mice, weighing 22–24 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0121] II) Administration and Treatment: ICR mice were divided into three groups according to body weight: control group, compound 1 (1 g / kg) group, and compound 1 (2 g / kg) group, with 6 mice in each group. After a single gavage administration, the animals were observed for any abnormalities within 24 hours. The animals were weighed the next day, and on the eighth day, organs including heart, liver, spleen, lung, kidney, and thymus were collected to measure organ indices.

[0122] 2) Experimental results:

[0123] I) Effect of compound 1 on mouse body weight in the examples

[0124] After administration of compound 1, there was no significant change in body weight between the 1 g / kg and 2 g / kg groups and the control group (see [link to compound 1]). Figure 3) .

[0125] II) Effect of Compound 1 on Organ Index in Mice in Examples

[0126] After administration of compound 1, there were no significant differences in the heart, liver, spleen, lung, kidney, and thymus indices of mice in the 1 g / kg and 2 g / kg groups compared with the control group (see [link to article]). Figure 4) The results showed that compound 1 had no significant effect on organ indices.

Claims

1. A benzimidazole methylenehydrazylthiazole compound of formula I or a pharmaceutically acceptable salt thereof: in: R1 is independently selected from hydrogen, C 1-6 Alkyl; R2 can be monosubstituted or polysubstituted in Formula I, independently selected from hydrogen, halogen, amino, carbonyl, cyano, nitro, C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Heteroalkyl, 3-6 membered heterocyclic alkyl, 5-7 membered aryl, 5-7 membered heteroaryl.

2. The benzimidazole methylenehydrazylthiazole compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R1 is independently selected from hydrogen, methyl, ethyl, and propyl; R2 is monosubstituted in Formula I and is independently selected from hydrogen, fluorine, chlorine, bromine, and iodine.

3. The benzimidazole methylenehydrazinethiazole compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: R1 is independently selected from hydrogen and methyl; R2 is monosubstituted in formula I, and R2 is independently selected from hydrogen, fluorine, and chlorine.

4. The benzimidazole methylenehydrazylthiazole compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The compounds are selected from the following group: Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 5. A method for preparing the compound of formula I as described in claim 1, comprising the following main steps: in, R1 and R2 are each defined as described in claim 1, and X is bromine or chlorine; Step 1: In anhydrous ethanol, compounds M1 and M2 react under the catalysis of acetic acid to generate intermediate M3 linked by thiourea. Step 2: In anhydrous ethanol, compound M3 and compound M4 react to give the compound shown in Formula I.

6. A pharmaceutical composition, characterized in that, It includes one or more of the compounds according to any one of claims 1-4, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers or excipients.

7. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound and an inorganic or organic acid, wherein the inorganic acid is selected from sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, and nitric acid, and the organic acid is selected from oxalic acid, citric acid, maleic acid, fumaric acid, malic acid, tartaric acid, methanesulfonic acid, and benzenesulfonic acid.

8. The use of the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 6, in the preparation of a medicament for the prevention and / or treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that, The neurodegenerative diseases mentioned are selected from Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

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