Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of drugs for treating cognitive impairment

Abstract

The present invention relates to the field of medical technology, and in particular to use of 4-guanidino-butyryl-3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propsulfane)-propionyl)-benzoate in the preparation of a drug for treating cognitive impairment. The present invention is expected to provide a new drug option for the clinical treatment of cognitive impairment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of drugs for treating cognitive impairment. Background Art

[0002] The pathogenesis of cognitive impairment is complex, with dysfunctional insulin signaling, glutamate toxicity, and neuroinflammation as key contributors. Insulin activates tyrosine kinases, which aggregate and phosphorylate various substrate-binding proteins, regulating the expression of multiple proteins and influencing neuronal differentiation, synaptic plasticity, and neurotransmitter release. Therefore, dysfunctional insulin signaling in the central nervous system (CNS) impairs neuronal survival and function, ultimately leading to impaired synaptic plasticity. Glutamate is the primary excitatory neurotransmitter in the mammalian CNS. Because glutamate plays a central role in signal transduction, synaptic plasticity, and memory consolidation, extracellular glutamate concentration is a key factor in maintaining CNS homeostasis. Abnormally elevated extracellular glutamate levels can induce excitotoxicity, leading to neuronal dysfunction and even death. Furthermore, neuroinflammation is an immune cascade mediated by glial cells (primarily microglia and astrocytes) in the CNS. Microglial activation is a key pathological hallmark of neuroinflammation. Activated microglia exhibit amebic morphology and phagocytosis. Therefore, changes in microglial morphology can reflect their activation state. Activated microglia can transform into two phenotypes: the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. When microglia transform into a pro-inflammatory phenotype, they release a large amount of pro-inflammatory factors, inducing neuroinflammation, causing synaptic plasticity damage, and ultimately damaging cognitive function. In diabetic patients, dysfunction of the insulin signaling pathway, glutamate toxicity, and the occurrence of neuroinflammation can be observed.

[0003] Currently, the drugs used in clinical practice for cognitive impairment mainly include cholinesterase inhibitors, excitatory amino acid receptor antagonists, ergot alkaloids, neurotrophic agents, etc. Although these drugs have certain therapeutic effects, they cannot achieve satisfactory results, and long-term use can cause gastrointestinal reactions, headaches, fatigue, insomnia, muscle spasms and other side effects.

[0004] Therefore, there is an urgent need to develop a new drug for the treatment of cognitive impairment. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide the use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester (abbreviated as ZYZ451) in the preparation of drugs for treating cognitive impairment.

[0006] To achieve this object, the present invention provides the following technical solutions:

[0007] The invention relates to the use of ZYZ451 in the preparation of a drug for treating cognitive impairment, wherein the active ingredient of the drug is 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester.

[0008] Furthermore, the cognitive impairment includes cognitive impairment caused by diabetes.

[0009] Furthermore, the cognitive impairment includes cognitive impairment caused by islet function deficiency and / or damaged insulin signaling pathway.

[0010] Furthermore, the cognitive impairment includes cognitive impairment caused by glutamate toxicity.

[0011] Furthermore, the cognitive impairment includes cognitive impairment caused by neuroinflammation.

[0012] Furthermore, the 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester has the following structural formula:

[0013] .

[0014] Furthermore, the medicine is a single-ingredient pharmaceutical preparation.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients or carriers.

[0016] Furthermore, the preparation is a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, dripping pill, dry extract, injection or infusion.

[0017] Furthermore, the auxiliary materials include any one or at least two of diluents, flavoring agents, adhesives, fillers, thickeners, disintegrants, solubilizers, suspending agents, lubricants, and buffers.

[0018] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0019] ZYZ451 can reduce fasting blood glucose and improve glucose tolerance in type 2 diabetic mice;

[0020] ZYZ451 can increase the serum insulin content and reduce the glutamate content in the whole brain of type 2 diabetic mice;

[0021] ZYZ451 can significantly alleviate cognitive impairment in type 2 diabetic mice;

[0022] ZYZ451 can increase the density of dendritic spines in the hippocampus of type 2 diabetic mice, increase the slope of fEPSPs in the mPFC-vCA1 pathway, and improve the impairment of long-term protein expression (LTP) in the model mice. ZYZ451 can also increase the number of synapses in the hippocampus and prefrontal cortex, and alleviate damage to the ultrastructure of neurons and synapses in these regions.

[0023] ZYZ451 can increase the expression of synaptic-related proteins PSD95, NMDAR2B, BDNF, and CAMKⅡ in the hippocampus of type 2 diabetic mice;

[0024] ZYZ451 can increase the expression of insulin signaling pathway-related proteins IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the hippocampus of type 2 diabetic mice;

[0025] ZYZ451 can increase the expression of synapse-related proteins and insulin signaling pathway-related proteins PSD95, IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the prefrontal cortex of type 2 diabetic mice;

[0026] ZYZ451 can increase the number of microglial branches in the prefrontal cortex and hippocampus of type 2 diabetic mice and reduce microglial activation;

[0027] ZYZ451 can increase the expression of insulin in pancreatic tissue of type 2 diabetic mice, reduce the loss of pancreatic beta cells, and increase serum insulin levels;

[0028] ZYZ451 can exert neuroprotective effects by activating the insulin signaling pathway, reducing glutamate toxicity and inhibiting neuroinflammation.

[0029] The above experimental results provide a pharmacological basis for the use of ZYZ451 in the preparation of drugs for treating cognitive impairment.

[0030] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the fasting blood glucose monitoring results of Example 1 of the present invention after four weeks of drug administration.

[0032] Figure 2 This is a graph showing the results of glucose changes in a glucose tolerance test in Example 1 of the present invention.

[0033] Figure 3 This is a graph showing the results of glutamate content in whole brain homogenate according to Example 2 of the present invention.

[0034] Figure 4 This is a graph showing the results of insulin content in serum according to Example 2 of the present invention.

[0035] Figure 5 Schematic diagram of immunohistochemical staining of insulin in pancreatic tissue in Example 3 of the present invention.

[0036] Figure 6 Statistical graph of the positive area results of Insulin in pancreatic tissue in Example 3 of the present invention.

[0037] Figure 7 4 is a diagram showing the results of the Y-maze experiment of Example 4 of the present invention.

[0038] Figure 8 This is a graph showing changes in the slope of fEPSPs in the vCA1-mPFC pathway in Example 5 of the present invention.

[0039] Figure 9 This is a quantitative graph of the slope of fEPSPs during the period 40-60 minutes after HFS stimulation in Example 5 of the present invention.

[0040] Figure 10 This is a diagram showing the results of the Golgi staining experiment in the hippocampus of Example 6 of the present invention.

[0041] Figure 11 This is a statistical graph of the results of the Golgi staining experiment in the hippocampus in Example 6 of the present invention.

[0042] Figure 12 This is a diagram showing the results of the Golgi staining experiment in the prefrontal lobe brain region in Example 6 of the present invention.

[0043] Figure 13 This is a statistical graph of the Golgi staining experimental results of the prefrontal lobe brain region in Example 6 of the present invention.

[0044] Figure 14 This is a diagram showing the ultrastructure of neurons in the hippocampus region of Example 7 of the present invention.

[0045] Figure 15 This is a diagram showing the synaptic ultrastructure of the hippocampus in Example 7 of the present invention.

[0046] Figure 16 This is a statistical diagram of the number of synapses in the hippocampus of Example 7 of the present invention.

[0047] Figure 17This is a diagram showing the ultrastructure of neurons in the prefrontal lobe brain region according to Example 7 of the present invention.

[0048] Figure 18 This is a diagram showing the synaptic ultrastructure of the prefrontal brain region according to Example 7 of the present invention.

[0049] Figure 19 This is a statistical diagram of the number of synapses in the prefrontal brain region according to Example 7 of the present invention.

[0050] Figure 20 This is a schematic diagram of the Western blot results of NMDAR2B, PSD95, BDNF, CAMKⅡ, P-mTOR, mTOR, IR, P-AKT, AKT, and β-actin in the hippocampus of Example 8 of the present invention.

[0051] Figure 21 This is a statistical graph of the expression level of NMDAR2B in the hippocampus of Example 8 of the present invention.

[0052] Figure 22 This is a statistical graph of the expression level of PSD95 in the hippocampus of Example 8 of the present invention.

[0053] Figure 23 This is a statistical graph of the expression level of BDNF in the hippocampus of Example 8 of the present invention.

[0054] Figure 24 This is a statistical graph of the expression level of CAMKⅡ in the hippocampus of Example 8 of the present invention.

[0055] Figure 25 This is a statistical graph of the expression levels of P-mTOR / mTOR in the hippocampus of Example 8 of the present invention.

[0056] Figure 26 This is a statistical graph of the expression level of P-mTOR in the hippocampus of Example 8 of the present invention.

[0057] Figure 27 This is a statistical graph of the expression level of mTOR in the hippocampus of Example 8 of the present invention.

[0058] Figure 28 This is a statistical graph of the expression level of IR in the hippocampus of Example 8 of the present invention.

[0059] Figure 29 This is a statistical graph of the expression levels of P-AKT / AKT in the hippocampus of Example 8 of the present invention.

[0060] Figure 30 This is a schematic diagram of the Western blot results of PSD95, P-mTOR, mTOR, IR, P-AKT, AKT, and β-actin in the prefrontal brain region of Example 9 of the present invention.

[0061] Figure 31 Statistical graph of the expression level of PSD95 in the prefrontal brain region in Example 9 of the present invention.

[0062] Figure 32 This is a statistical graph of the expression levels of P-mTOR / mTOR in the prefrontal brain region of Example 9 of the present invention.

[0063] Figure 33 This is a statistical graph of the expression level of P-mTOR in the prefrontal brain region of Example 9 of the present invention.

[0064] Figure 34 This is a statistical graph of the expression level of mTOR in the prefrontal brain region of Example 9 of the present invention.

[0065] Figure 35 This is a statistical graph of the expression level of IR in the prefrontal brain region of Example 9 of the present invention.

[0066] Figure 36 This is a statistical graph of the expression levels of P-AKT / AKT in the prefrontal brain region of Example 9 of the present invention.

[0067] Figure 37 This is a schematic diagram of IBA-1 immunohistochemical staining of microglial cells in the hippocampus of Example 10 of the present invention.

[0068] Figure 38 This is a schematic diagram of the morphology of microglial cells in the hippocampus of Example 10 of the present invention.

[0069] Figure 39 This is a statistical diagram of the number of microglial cell morphologies and branches in the hippocampus of Example 10 of the present invention.

[0070] Figure 40 Schematic diagram of IBA-1 immunohistochemical staining of microglial cells in the prefrontal brain region according to Example 10 of the present invention.

[0071] Figure 41 This is a schematic diagram of the morphology of microglial cells in the prefrontal brain region according to Example 10 of the present invention.

[0072] Figure 42 This is a statistical diagram of the number of microglial cell morphology and branches in the prefrontal brain region according to Example 10 of the present invention.

[0073] Figure 43 This is a schematic diagram of Iba-1 and CD16 / 32 immunofluorescence staining of microglial cells in the hippocampal CA1 brain region in Example 10 of the present invention.

[0074] Figure 44 This is a statistical graph of the expression levels of CD16 / 32 in microglial cells in the hippocampal CA1 brain region in Example 10 of the present invention.

[0075] Figure 45 This is a statistical graph of the expression levels of CD16 / 32 / Iba-1 in microglial cells in the hippocampal CA1 brain region in Example 10 of the present invention.

[0076] Figure 46 Schematic diagram of Iba-1 and CD16 / 32 immunofluorescence staining of microglia in the prefrontal brain region according to Example 10 of the present invention.

[0077] Figure 47 This is a statistical graph of the expression levels of CD16 / 32 in microglia in the prefrontal brain region according to Example 10 of the present invention.

[0078] Figure 48 This is a statistical graph of the expression levels of CD16 / 32 / Iba-1 in microglial cells in the prefrontal brain region according to Example 10 of the present invention. DETAILED DESCRIPTION

[0079] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0080] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0081] Among them, ZYZ451 was prepared using the technical solution disclosed in patent ZL201010100153.5.

[0082] Example 1

[0083] 1. Detecting fasting blood glucose in experimental mice

[0084] Eight-week-old adult male type 2 diabetic mice (db / db mice) were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd. The solvent was double-distilled water, which was prepared on a Milli-Q® IQ 7000 water purifier purchased from Merck.

[0085] Model mice were randomly divided into five groups: model group (double-distilled water group), model group + compound ZYZ451 (10 mg / kg) group, model group + compound ZYZ451 (20 mg / kg) group, and model group + compound ZYZ451 (40 mg / kg) group. Wild-type mice from the same littermate (db / m mice) served as negative controls and were randomly divided into two groups: normal group (double-distilled water group) and normal group + compound ZYZ451 (40 mg / kg) group. Each group consisted of 12 mice. Each experimental group was housed for 4 weeks. Starting from the 12th week, each group received oral administration for 4 weeks. Fasting blood glucose levels were measured weekly during this period.

[0086] The results are as follows Figure 1 As shown, compared with the normal group, the blood glucose level of the mice in the model group was significantly increased ( ## P<0.01); After the intervention of compound ZYZ451 (40 mg / kg), the blood glucose level of diabetic mice decreased significantly in the second week after administration ( * P<0.05).

[0087] Figure 1 n = 12, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001 vs. normal group.

[0088] 2. Glucose tolerance test

[0089] After four weeks of dosing, mice in each group were fasted for 18 hours but not water. 50% glucose (2g / kg) was then injected intraperitoneally, and blood glucose levels were measured by slicing blood from the tail vein. The tails of the mice were disinfected with 75% alcohol. After the alcohol evaporated, the tails were cut, and a drop of blood was squeezed out and placed on a blood glucose test strip. Blood glucose levels were measured at 0 minute before, and 15, 30, 60, and 120 minutes after the intraperitoneal injection. The blood glucose value at 0 minute was the fasting blood glucose (FBG, mmol / L) of the mice.

[0090] The results are as follows Figure 2 As shown, compared with the normal group, the blood glucose changes of the model group mice were significantly increased at 30 minutes, 60 minutes, and 120 minutes ( ##P<0.01, ### P<0.001); after intervention with compound ZYZ451 (10 mg / kg, 20 mg / kg, 40 mg / kg), the blood glucose changes in type 2 diabetic mice were significantly reduced at 30 minutes, 60 minutes, and 120 minutes ( ** P<0.01, *** P<0.001).

[0091] The results showed that compound ZYZ451 could reduce fasting blood sugar and alleviate glucose tolerance in type 2 diabetic mice.

[0092] Figure 2 n = 12, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001 vs. normal group.

[0093] Example 2

[0094] 1. Determination of glutamate content in the whole brain

[0095] The whole brain tissue of the experimental mice was quickly separated on ice and homogenized to extract protein for quantitative analysis. The content of glutamate in the whole brain tissue of the experimental mice was detected using a glutamate kit.

[0096] The results are as follows Figure 3 As shown in Figure 2, the content of glutamate in the whole brain homogenate of the model group mice was significantly increased compared with that of the control group (P # <0.05); after intervention with compound ZYZ451 (40 mg / kg), the glutamate content in the whole brain homogenate of model mice was significantly reduced (P * <0.05).

[0097] 2. Determination of serum insulin content

[0098] The mice were eyeballed and blood was collected in EP tubes. The blood samples were allowed to stand for 30 minutes and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected as serum. The insulin content in the serum of each group of mice was measured using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay kit.

[0099] The results are as follows Figure 4As shown, compared with the control group, the serum insulin content of the model group mice was significantly increased ( # P<0.05); after intervention with compound ZYZ451 (40 mg / kg), the serum insulin content in type 2 diabetic mice was significantly increased ( * P<0.05). Compared with the normal group and the normal + compound ZYZ451 (40 mg / kg) group, the insulin content in the serum of normal mice increased significantly after the intervention of compound ZYZ451 (40 mg / kg) ( ## P<0.01).

[0100] The results showed that compound ZYZ451 could increase the level of insulin in the serum of type 2 diabetic mice and reduce the level of glutamate in the whole brain of type 2 diabetic mice.

[0101] Figure 3 and Figure 4 n = 4, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0102] Example 3

[0103] The experimental mice were perfused with saline and 4% PFA before pancreatic tissue was removed. After 48 hours of fixation with 4% PFA, the tissue was dehydrated and transparentized using a gradient of ethanol solutions and xylene. The tissue was then immersed in wax for 4 hours and embedded. The tissue was cut into 8μm thick slices for insulin immunohistochemical staining. The experimental results are shown in Figure 2. Figure 5 and Figure 6 As shown in Figure 2, compared with the control group, the expression of Insulin in the pancreatic tissue of the model group mice was significantly decreased ( ### P<0.001), indicating that the number of pancreatic beta cells was significantly reduced; after intervention with compound ZYZ451, the expression of Insulin in the pancreatic tissue of model mice was significantly increased ( *** P<0.001), indicating that the number of pancreatic beta cells increased significantly.

[0104] Experimental results showed that compound ZYZ451 could increase the expression of Insulin in the pancreatic tissue of model mice and reduce the damage of pancreatic beta cells.

[0105] Figure 5 and Figure 6 n = 4, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0106] Example 4 Y-maze test

[0107] Before the experiment began, we designated the three arms of the Y-maze as A, B, and C. At the start of the experiment, mice were placed at the end of Arm A, facing the center, and allowed to freely explore the maze for 5 minutes. The order in which the mice passed through all arms during the experiment was recorded. After each mouse was tested, the maze was cleaned with 75% alcohol and allowed to dry completely before testing the next mouse.

[0108] The results are as follows Figure 7 As shown, the number of correct alternations in the model group mice was significantly reduced compared with the normal group ( # P<0.05); after the intervention of compound ZYZ451 (20mg / kg, 40mg / kg), the correct alternation times of the model mice increased significantly ( * P<0.05, ** P<0.01).

[0109] Experimental results showed that compound ZYZ451 can improve the learning and memory abilities of model mice.

[0110] Figure 7 n = 12, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; # P<0.05, ##P<0.01, ### P<0.001 vs. normal group.

[0111] Example 5

[0112] Mice were anesthetized intraperitoneally with 30% urethane and fixed in a stereotaxic apparatus. The skull was exposed, and the CA1 (3.4 mm posterior to bregma, 3.0 mm lateral, 3.0-3.5 mm deep) and mPFC (1.8 mm anterior to bregma, 0.5 mm lateral, 1.5 mm deep) were located with bregma as the origin. After obtaining stable, representative waveforms, single pulses (0.2 ms, 0.03 Hz) were first administered every 30 seconds for 20 minutes, recording as baseline. High-frequency stimulation (HFS) (200 Hz frequency, 12 pulses per train, 200 ms inter-train interval) was then applied, followed by single pulses every 60 seconds for 60 minutes.

[0113] like Figure 8 and Figure 9 As shown, the results showed that compound ZYZ451 could increase the fEPSP slope (fEPSP Slope) in the vCA1-mPFC pathway of model mice and improve the impairment of postsynaptic long-term potentiation.

[0114] in, Figure 8 The arrows indicate HFS stimulation; Figure 8 medium, n = 8; Figure 9 n = 20, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001 vs. normal group.

[0115] Example 6

[0116] After the electrophysiological recording, the mice were decapitated and the brains were quickly isolated and immersed in freshly prepared Golgi buffer for 3 weeks. Coronal brain slices (150 μm) were cut using a vibrating microtome, and the coronal slices were immersed in freshly prepared 6% sodium carbonate solution for 20 minutes and washed with double distilled water for 2 seconds. The slices were dehydrated in 70% ethanol (10 minutes), 90% ethanol (15 minutes) and anhydrous ethanol (20 minutes), respectively. After vitrification in xylene for 20 minutes, the slices were sealed with neutral resin. Pictures were taken using a 100× oil-immersion objective lens under an ordinary optical microscope. The experimental results are shown in Figure 2. Figure 10 and Figure 11 As shown, compared with the control group, the density of dendritic spines in the hippocampus of mice in the model group was significantly reduced ( ### P<0.001); after intervention with compound ZYZ451 (40 mg / kg), the density of dendritic spines in the hippocampus of model mice was significantly increased ( ** P<0.01).

[0117] The experimental results are as follows Figure 12 and Figure 13 As shown, compared with the control group, the density of dendritic spines in the prefrontal cortex of mice in the model group was significantly reduced ( ### P<0.001); after intervention with compound ZYZ451 (40 mg / kg), the density of dendritic spines in the prefrontal cortex of model mice was significantly increased ( ** P<0.01).

[0118] The results showed that compound ZYZ451 could increase the density of dendritic spines in the hippocampus and prefrontal cortex of model mice. Figure 10 、 Figure 11 、 Figure 12 and Figure 13 Each group consisted of 3 mice; n = 21 for each statistical figure. Normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; **P < 0.01, ***P < 0.001 vs. model group; # P<0.05, ### P<0.001 vs. normal group.

[0119] Example 7

[0120] After the electrophysiological experiment, the experimental mice were perfused with 0.01M PBS and then perfused with electron microscopy fixative (4% PFA, 2% glutaraldehyde). After perfusion, the hippocampal CA1 brain region and prefrontal cortex were isolated and then cut into approximately 1mm 3Xiao Ding, fixed with 2.5% glutaraldehyde for 48 hours. The tissue block was placed in 1% osmium acid and fixed for 1 to 2 hours; the tissue block was gradient dehydrated; stained with 70% ethanol uranyl acetate; soaked with propylene oxide and epoxy resin; and embedded in pure epoxy resin. The embedded block was then trimmed and made into ultrathin sections, which were scooped out with a copper mesh and electron stained. Finally, an electron microscope specimen was made, and the ultrastructure of neurons and synapses was observed using a transmission electron microscope. The experimental results are as follows. Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 and Figure 19 As shown in the results, compared with the control group, the chromatin in the nuclei of neurons in the hippocampal CA1 region and prefrontal cortex of the model group mice showed marginalization, expansion and degranulation of the rough endoplasmic reticulum, swelling of the mitochondria and rupture or vacuolation, irregular morphology of the Golgi complex, and lipofuscin deposition. The ultrastructure of the synapses was disordered, the synaptic cleft and the presynaptic and postsynaptic membrane structures were unclear, the number of presynaptic vesicles was reduced, the mitochondria showed vacuolation, the postsynaptic density was reduced in the postsynaptic membrane, and a discontinuous distribution was observed. The number of synapses was significantly reduced ( ## P<0.01, # P<0.05). After treatment with compound ZYZ451 (40 mg / kg), chromatin was evenly distributed in the nuclei of neurons in the hippocampus of model mice, the rough endoplasmic reticulum was slightly expanded, degranulation was reduced, and most mitochondria had regular morphology and neatly arranged mitochondrial cristae, while a small number of mitochondria showed swelling and cristae rupture. The Golgi complex had regular morphology, with no lipofuscin deposition. The synaptic cleft and presynaptic and postsynaptic membrane structures were clear, with a large number of clear and dense synaptic vesicles in the presynaptic component, and the mitochondrial structure in some presynaptic components was irregular. The postsynaptic density was more numerous and thicker than that in the model group, and its arrangement was continuous, and the number of synapses was significantly increased ( *** P<0.001, * P<0.05)).

[0121] The results showed that compound ZYZ451 could improve the ultrastructural damage of neurons and synapses in the hippocampus and prefrontal regions of model mice, and increase the number of synapses in the hippocampus and prefrontal regions of model mice.

[0122] Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 and Figure 19Each group consisted of 3 mice, and n = 3 in each statistical graph. Normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0123] Example 8

[0124] After the electrophysiological recording, the hippocampal tissue of the experimental mice was quickly isolated on ice and homogenized to extract proteins for quantitative analysis. After protein denaturation, protein expression was detected by Western blot. Figures 20 to 29 As shown in the data, in all experimental groups, the expression of PSD95, NMDAR2B, BDNF, CAMKⅡ, IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the hippocampus tissue of model experimental mice was reduced; after intervention with compound ZYZ451 (40 mg / kg), the expression of PSD95, NMDAR2B, BDNF, CAMKⅡ, IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the hippocampus tissue of model mice was significantly increased.

[0125] The results showed that compound ZYZ451 could increase the expression of synaptic-related proteins PSD95, NMDAR2B, BDNF, CAMKII and insulin signaling pathway-related proteins IR, mTOR, P-mTOR, and P-mTOR / mTOR in the hippocampus.

[0126] Figures 20 to 29 n = 5, normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0127] Example 9

[0128] After the electrophysiological recording, the prefrontal cortex tissue of the experimental mice was quickly isolated on ice and homogenized to extract protein for quantitative analysis. After protein denaturation, protein expression was detected by Western blot. Figures 30 to 36 As shown in the results, in all experimental groups, the expression of PSD95, IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the prefrontal tissue of model experimental mice was reduced; after intervention with compound ZYZ451 (40 mg / kg), the expression of PSD95, IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the prefrontal tissue of model mice was significantly increased.

[0129] The results showed that compound ZYZ451 could increase the expression of synaptic-related protein PSD95 and insulin signaling pathway-related proteins IR, mTOR, P-mTOR, P-mTOR / mTOR, and P-AKT / AKT in the prefrontal brain region of model mice.

[0130] Figures 30 to 36 (n=4) Normal group: wild-type mice (db / m mice); Model group: type 2 diabetic mice (db / db mice); Compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; *P<0.05, **P<0.01, ***P<0.001 vs. Model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0131] Example 10

[0132] Iba-1 immunohistochemical staining

[0133] After electrophysiological recordings, the mice were perfused with saline and 4% PFA before brain removal. After postfixation with 4% PFA for 48 hours, the brains were dehydrated and transparentized using a gradient of ethanol and xylene. The brains were then immersed in wax for 4 hours and embedded. The brains were then cut into 8-μm-thick slices for immunohistochemical staining of IBA-1.

[0134] The experimental results are as follows Figure 37 、 Figure 38 and Figure 39As shown, in the hippocampus, compared with the control group, the branching complexity of microglia in the hippocampus of the model group mice was significantly reduced between 9μm and 15μm from the cell nucleus; after intervention with compound ZYZ451 (40mg / kg), the branching complexity of microglia in the hippocampus of the model mice was significantly increased between 9μm and 15μm from the cell nucleus.

[0135] The experimental results are as follows Figure 40 、 Figure 41 and Figure 42 As shown in the figure, in the prefrontal region, the branching complexity of microglia in the prefrontal region of mice in the model group was significantly reduced between 10 μm and 20 μm from the cell nucleus compared with the control group ( ### P<0.001); after intervention with compound ZYZ451 (40 mg / kg), the branching complexity of microglia in the prefrontal cortex of model mice increased significantly between 9 μm and 14 μm from the cell nucleus ( ** P<0.01).

[0136] II. Iba-1 and CD16 / 32 Immunofluorescence Staining

[0137] After the electrophysiological recording, the experimental mice were perfused with saline and 4% PFA before the brain was removed. After post-fixation with 4% PFA for 48 hours, the brain was dehydrated and transparentized with a gradient of ethanol solution and xylene, and then immersed in wax for 4 hours before embedding the tissue block. The tissue block was cut into 8μm thick tissue slices for immunofluorescence staining of Iba-1, M1 type CD16 / 32. The experimental results are shown in Figure 2. Figures 43 to 48 As shown in the figure, in the hippocampus and prefrontal cortex, the expression of CD16 / 32 and CD16 / 32 / Iba-1 in microglia in the prefrontal cortex of mice in the model group was significantly increased compared with that in the control group ( ### P<0.001); after intervention with compound ZYZ451 (40 mg / kg), the expression of CD16 / 32 and CD16 / 32 / Iba-1 in microglia in the prefrontal cortex of model mice was significantly reduced ( *** P<0.001). The experimental results showed that compound ZYZ451 can reduce the activation of microglia in the hippocampus and prefrontal cortex of model mice.

[0138] Figures 37 to 42 There were 3 mice in each group, and n = 3 in each statistical graph; Figures 43 to 48Each group consisted of 4 mice, and n = 4 in each statistical figure; normal group: normal wild-type mice (db / m mice); model group: type 2 diabetic mice (db / db mice); compound ZYZ451: 3,5-dimethoxy-4-(2-amino-3-(2-ynyl-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester; *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group; # P<0.05, ## P<0.01, ### P<0.001vs. normal group.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. -dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment, characterized in that, The active ingredient of the drug is 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester, and its structural formula is ; The cognitive impairment is caused by diabetes, and the drug can lower fasting blood sugar and improve glucose tolerance.

2. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester according to claim 1 in the preparation of a drug for treating cognitive impairment, characterized in that: The drug can protect pancreatic islet cell function and / or the drug can regulate insulin signaling.

3. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester according to claim 1 in the preparation of a drug for treating cognitive impairment, characterized in that: The drug can inhibit diabetes-induced neuroinflammation.

4. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 1, characterized in that: The medicine is a single-ingredient pharmaceutical preparation.

5. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 4, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

6. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 4, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

7. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 4, characterized in that: The preparation is in the form of tablets, capsules, granules, pills, oral liquids, dry suspensions, dripping pills, dry extracts, injections or infusions.

8. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 7, characterized in that: The capsule is a soft capsule.

9. Use of 3,5-dimethoxy-4-(2-amino-3-(2-alkyne-propanesulfane)-propionyl)-benzoic acid 4-guanidino-butyryl ester in the preparation of a drug for treating cognitive impairment according to claim 5, characterized in that: The auxiliary materials include any one or at least two of diluents, flavoring agents, binders, thickeners, disintegrants, solubilizers, suspending agents, lubricants, and buffers.

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