Use of a benzamide derivative for improving cognitive dysfunction

Abstract

Disclosed is a use of a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving cognitive dysfunction, the benzamide derivative having a structural formula of Formula I. The benzamide derivative having the structural formula of Formula I of the present application can improve Aβ 1‑42 induced cognitive dysfunction and APP / PS1 mouse cognitive dysfunction without affecting spontaneous activity of the mice.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to the use of a benzamide derivative to improve cognitive impairment. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized primarily by cognitive impairment, accompanied by behavioral and social dysfunction and a decline in daily living abilities. Unfortunately, the types and efficacy of drugs currently available for treating AD are very limited, and AD cannot be significantly slowed or cured. Clinically, two classes of AD medications—cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists—cannot halt the progression of AD; they only alleviate symptoms and have significant side effects. Summary of the Invention

[0003] The main objective of this invention is to provide a drug that is effective, has few side effects, and is naturally safe for treating and improving diseases and symptoms related to cognitive impairment, in order to solve the problems of common drugs in the prior art that have poor efficacy, cannot stop the progression of AD, can only relieve symptoms, and have large side effects.

[0004] To achieve the above objectives, according to one aspect of the present invention, there is provided the use of a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0005]

[0006] Furthermore, the method for preparing the solution of the benzamide derivative includes: weighing an appropriate amount of the benzamide derivative, adding a reagent to dissolve and dilute it to obtain a solution of the benzamide derivative.

[0007] Furthermore, the reagent is artificial cerebrospinal fluid, such as sterile artificial cerebrospinal fluid.

[0008] Furthermore, the concentration of the benzamide derivative solution is prepared to be 0.01–2 μg / mL, for example, about 0.1 μg / mL, about 0.3 μg / mL, or about 0.9 μg / mL.

[0009] According to another aspect of the invention, there is provided the use of a pharmaceutical composition comprising a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0010]

[0011] Furthermore, the pharmaceutical composition further comprises one or more drugs and / or extracts for improving cognitive dysfunction.

[0012] Furthermore, the drug is selected from one or more of the following: cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, monoclonal antibodies, and gut-brain axis modulators.

[0013] Furthermore, the cholinesterase inhibitor is donepezil, rivastigmine, huperzine A, and / or galantamine.

[0014] Furthermore, the N-methyl-D-aspartate (NMDA) receptor antagonist is memantine.

[0015] Furthermore, the monoclonal antibody is aduretumumab, lencanemab, and / or donepemab.

[0016] Furthermore, the brain-gut axis regulator is mannostatin.

[0017] According to another aspect of the invention, there is a use of a pharmaceutical formulation comprising a benzamide derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0018]

[0019] Furthermore, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

[0020] Furthermore, the drug preparation is in the form of powder, tablet, drop pill, capsule, film, lozenge, granule, injection or oral liquid.

[0021] Furthermore, this cognitive impairment is a neurodegenerative disease, such as Alzheimer's disease.

[0022] The beneficial effects of this invention are:

[0023] The benzamide derivatives of the present invention having the structure shown in Formula I can improve Aβ 1-42 Induced cognitive impairment in mice and APP / PS1 mice without affecting spontaneous activity in mice. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0025] Figure 1 Administration of the benzamide derivative of the present invention having the structural formula shown in Formula I (hereinafter referred to as "Formula I compound") to the lateral ventricle improves Aβ 1-42 A schematic diagram illustrating the induced cognitive impairment in mice. (A)Aβ 1-42 (A) Flowchart of inducing cognitive impairment in mice and administration of compound I to the lateral ventricle; (B) Statistical chart of total distance traveled by mice in the open field experiment; (C) Statistical chart of central dwell time in mice in the open field experiment; (D) Statistical chart of average central velocity in mice in the open field experiment; (E) Swimming trajectory of mice in the Morris water maze experiment; (F) Statistical chart of percentage of time spent in the target quadrant in the Morris water maze experiment; (G) Statistical chart of the number of times mice crossed platforms in the Morris water maze experiment; (H) Statistical chart of spontaneous alternation index in the Y maze experiment; (I) Statistical chart of recognition index in mice in the new object recognition experiment. One-way comparisons of multiple groups of data were performed using one-way ANOVA and Tukey post-hoc analysis. Results are expressed as mean ± standard error. * P<0.05, ** P<0.01, *** P<0.001, ns:P>0.05.

[0026] Figure 2 This diagram illustrates the effect of gavage administration of the benzamide derivative of Formula I (hereinafter referred to as "Compound I") of the present invention on improving cognitive impairment in APP / PS1 transgenic mice. (A) Flowchart of gavage administration to APP / PS1 transgenic mice; (B) Statistical graph of total distance traveled by mice in open field experiments; (C) Statistical graph of central dwell time of mice in open field experiments; (D) Statistical graph of average central velocity of mice in open field experiments; (E) Swimming trajectory of mice in Morris water maze experiments; (F) Statistical graph of percentage of time spent in the target quadrant of mice in Morris water maze experiments; (G) Statistical graph of number of times mice crossed platforms in Morris water maze experiments; (H) Statistical graph of spontaneous alternation index in Y maze experiments; (I) Statistical graph of recognition index of mice in new object recognition experiments. One-way comparison of multiple groups of data was performed using one-way ANOVA and Tukey post-hoc analysis. Results are expressed as mean ± standard error. * P<0.05, ** P<0.01, *** P<0.001, ns:P>0.05. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0029] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0030] Unless otherwise stated, the following definitions will apply in this invention. For the purposes of this invention, chemical elements are defined according to the periodic table, CAS version, and the Handbook of Chemical Physics, 75th Ed., 1994. Furthermore, general principles of organic chemistry are found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007; therefore, all of these references are incorporated herein by reference.

[0031] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0032] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0033] As described in the background section, common drugs in the prior art for treating neurodegenerative diseases with cognitive impairment as the main clinical symptom suffer from poor efficacy, inability to halt the progression of Alzheimer's disease (AD), and only symptom relief with significant side effects. To address these problems, the present invention provides the use of a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0034]

[0035] As used in this invention, the term "salt" refers to a salt of the compound of this invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Suitable salts include all known pharmaceutically acceptable salts, including those formed with organic and inorganic acids. Thus, suitable salts include those formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, lactic acid, valeric acid, acetic acid, succinic acid, fumaric acid, maleic acid, methanesulfonic acid, and benzenesulfonic acid.

[0036] In a preferred embodiment, the method for preparing a solution of the benzamide derivative includes: weighing an appropriate amount of the benzamide derivative, adding a reagent to dissolve and dilute it to obtain a solution of the benzamide derivative.

[0037] In a preferred embodiment, the reagent is artificial cerebrospinal fluid, such as sterile artificial cerebrospinal fluid.

[0038] In a preferred embodiment, the concentration of the benzamide derivative solution is prepared to be 0.01–2 μg / mL, for example, about 0.1 μg / mL, about 0.3 μg / mL, or about 0.9 μg / mL.

[0039] In this invention, when concentration or other parameters are expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.01–2” is disclosed, the described range should be interpreted as including ranges “0.01–2”, “0.01–1.5”, “0.01–1”, “0.01–0.5”, “0.5–2”, “0.50–1.50”, “0.50–1”, “1–2”, “1–1.5”, “1.5–2”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range, and all values ​​within the range achieve the effects of this invention.

[0040] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105; "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315; and "about 0.9" includes ±5% of 0.9, or from 0.855 to 0.945.

[0041] According to another aspect of the invention, there is provided the use of a pharmaceutical composition comprising a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0042]

[0043] In a preferred embodiment, the pharmaceutical composition further comprises one or more drugs and / or extracts for improving cognitive impairment.

[0044] In a preferred embodiment, the drug is selected from one or more of the following: cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, monoclonal antibodies, and gut-brain axis modulators.

[0045] In a preferred embodiment, the cholinesterase inhibitor is donepezil, rivastigmine, huperzine A, and / or galantamine.

[0046] In a preferred embodiment, the N-methyl-D-aspartic acid (NMDA) receptor antagonist is memantine.

[0047] In a preferred embodiment, the monoclonal antibody is aduretumumab, lencanemab, and / or donepemab.

[0048] In a preferred embodiment, the brain-gut axis modulator is mannitol.

[0049] According to another aspect of the invention, there is a use of a pharmaceutical formulation comprising a benzamide derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient in the preparation of a medicament for improving cognitive impairment, the benzamide derivative having the structural formula shown in Formula I:

[0050]

[0051] Pharmaceutical compositions are manufactured using effective doses of the above-described benzamide derivatives of the present invention, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients. The dose of the active compound may vary depending on the route of administration, the patient's age and weight, the nature and severity of the disease to be treated, and similar factors.

[0052] The pharmaceutical compositions of the compounds of the present invention can be administered in any of the following ways: orally, by spray inhalation, rectal administration, nasal administration, buccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or via an external implantation device. Oral, topical, intraperitoneal, or intravenous administration are preferred.

[0053] In a preferred embodiment, the pharmaceutical formulation of the present invention contains at least one pharmaceutically acceptable excipient in an amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.% relative to the weight of the pharmaceutical formulation.

[0054] In this invention, the term "pharmaceutically acceptable" means a substance, such as a carrier or diluent, that does not destroy the biological activity or properties of a compound and is relatively non-toxic, such that, when administered to an individual, it will not cause unwanted biological effects or interact with any of its constituent components in a harmful manner.

[0055] In this invention, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0056] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

[0057] Those skilled in the art will know how to select specific chemical substances within the aforementioned excipient categories. For example, the diluent may be selected from one or more of the following: powdered sugar, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate, and calcium carbonate. The wetting agent may be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbate esters such as polysorbate 80, cetyl alcohol, glyceryl fatty acid esters (such as triacetin, glyceryl monostearate, and the like), polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, sodium lauryl sulfate, sorbitol fatty acid esters, sucrose fatty acid esters, polyoxyethylene ethers, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The binder may be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol, and methylcellulose. The disintegrant may be selected from one or more of the following: carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose. The flavoring agent may be selected from one or more of the following: sorbitol, glucose, mannose, sucrose, and lactose. The dispersant may be selected from one or more of the following: croscarmellose sodium, sodium starch glycolate, and pregelatinized corn starch. The plasticizer may be dibutyl sebacate and / or various citrate esters. The sustained-release agent may be selected from one or more of the following: sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, hydroxyethyl cellulose, gum arabic, gelatin, and shellac. The antioxidant may be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite, and sodium thiosulfate. The lubricant may be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid, and colloidal silica.

[0058] These excipients are preferably drug-inert, or may have synergistic or enhancing effects to enhance the therapeutic activity of the drug composition. The above excipients are merely examples, and the excipients actually used in this invention are not limited to the above excipients. They can be adjusted according to the actual situation to achieve the effects of this invention.

[0059] In a preferred embodiment, the pharmaceutical preparation is a powder, tablet, drop pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0060] The drugs, excipients and dosage forms described above in this invention are merely illustrative examples. In actual use, they are not limited to those listed above. Any drug that can be used in combination with the pharmaceutical composition of this invention is within the scope of protection of this invention.

[0061] In a preferred embodiment, the cognitive impairment is a neurodegenerative disease, such as Alzheimer's disease.

[0062] In this invention, the term "improvement" also includes "prevention," unless specifically stated otherwise. The terms "improved" and "improvedly" should be understood accordingly.

[0063] In this invention, the term "improvement" includes alleviating, suppressing, or preventing symptoms or conditions of a disease; suppressing the development of complications; preventing potential metabolic syndromes; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; mitigating a disease or symptom; reducing complications caused by a disease or symptom; or preventing signs caused by a disease or symptom. As used herein, a pharmaceutical composition or pharmaceutical preparation, after administration, may improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration. Whether the administration is fixed or intermittent, continuous or intermittent, it may be attributable to or related to the administration.

[0064] The present invention also provides the above-described composition or pharmaceutical preparation for improving cognitive impairment, such as Alzheimer's disease, in subjects.

[0065] The present invention also provides a method for improving cognitive impairment, such as Alzheimer's disease, in a subject, comprising administering an effective amount of the above-described composition or the above-described pharmaceutical preparation to the subject.

[0066] In this invention, the term "subject" refers to a mammal. A mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing a model of Alzheimer's disease. Preferably, the subject is a human.

[0067] The “effective amount” of the pharmaceutical composition or formulation used in this invention can achieve the desired therapeutic and / or preventative effects. The effective amount for this purpose will depend on factors such as the pharmaceutical composition, the route of administration, the stage and severity of the disease being treated, the individual’s weight and overall health status, and the judgment of the prescribing physician. The dose can be administered once a week, every two days, once daily, or even several times a day. Dosage units can be administered over a short period (e.g., weeks to months) or a longer period (months to years).

[0068] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0069] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the method of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0070] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0071] Examples

[0072] 1. Experimental materials

[0073] 1.1 Experimental animals

[0074] Male SPF-grade C57BL / 6J mice, weighing 22±2 g, 8-10 weeks old, were purchased from the Comparative Medicine Center of Yangzhou University and quality inspected by the Institute of Hygiene and Environmental Technology of Soochow University. The experimental animal license number is: SCXK (Jiangsu) 2017-0007.

[0075] Male SPF-grade transgenic APP / PS1 mice and wild-type control mice, weighing 25±2 g, 9 months old, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and quality inspected by the Zhejiang Provincial Experimental Animal Quality Supervision and Monitoring Station. The experimental animal license number is: SCXK (Zhejiang) 2019-0004.

[0076] Animal rearing conditions: constant temperature 22±2 °C, humidity 50±5%, light-dark cycle 12 h / 12 h, light time is 07:00-19:00, experimental animals can freely obtain food and water, and the experiment starts after 1 week of environmental adaptation. All operations comply with the regulations of the China Animal Protection Association and the Experimental Animal Management Committee of Zhengzhou University.

[0077] 1.2 Experimental reagents

[0078] The main experimental reagents used in the present invention are shown in Table 1.

[0079] Table 1 Experimental reagents

[0080]

[0081]

[0082] 1.3 Experimental instruments

[0083] The main experimental instruments used in the present invention are shown in Table 2.

[0084] Table 2 Experimental Instruments

[0085] Instrument Name Manufacturer Sartorius analyzes the balance scale Beijing Dolis Balance Co., Ltd. 80-2C Magnetic Stirrer Gongyi Yuhua Instrument Co., Ltd. TS-1 type horizontal shaking table Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd. SHA-C Water Bath Thermostatic Oscillator Jintan Ronghua Instrument Manufacturing Co., Ltd. Ultrapure water system Millipore, USA Mouse respiratory anesthesia machine Shenzhen Ruiwode Life Technology Co., Ltd. ZS-FD / S Digital Display Stereoscopic Brain Array Beijing Zhongshidichuang Technology Development Co., Ltd. Mouse-specific adapter Beijing Zhongshidichuang Technology Development Co., Ltd. Stereo microscope Beijing Zhongshidichuang Technology Development Co., Ltd. Dual-fiber cold light source Beijing Zhongshidichuang Technology Development Co., Ltd. TJ-4A Micro-injection pump controller Baoding Lange Constant Flow Pump Co., Ltd. AH-GSZ High-Speed ​​Skull Drill Huaibei Zhenghua Biological Instrument Equipment Co., Ltd. Open field test instrument Shanghai Jiliang Software Technology Co., Ltd. Morris Water Maze Experiment Shanghai Jiliang Software Technology Co., Ltd. Y-maze experiment apparatus Shanghai Jiliang Software Technology Co., Ltd. New Object Recognition Experimental Instrument Shanghai Jiliang Software Technology Co., Ltd.

[0086] 1.4 Preparation of main reagent solutions

[0087] Sterilized artificial cerebrospinal fluid (ACSF): Weigh 3.66g NaCl, 0.11g KCl, 0.075g Na2HPO4, 0.12g MgSO4, 0.111g CaCl2, 1.05g NaHCO3 and 0.9g D-glucose, dissolve in an appropriate amount of distilled water, bring the volumetric flask to 500mL, and sterilize by autoclaving at 120℃ for 30min.

[0088] Aβ 1-42 Solution: Weigh 1.0 mg Aβ 1-42 Add 2.7 mL of sterile artificial cerebrospinal fluid to fully dissolve the solution, achieving a final concentration of 82 pmol / μL. Incubate at 37°C for 5 days before use.

[0089] The benzamide derivative solution of Formula I of this invention: 1. Weigh 1.0 mg of the benzamide derivative of Formula I of this invention, add 1.0 mL of sterile artificial cerebrospinal fluid and dissolve thoroughly to obtain a 1.0 mg / mL stock solution. Dilute with sterile artificial cerebrospinal fluid to 0.1 μg / μL, 0.3 μg / μL, and 0.9 μg / μL for lateral ventricle administration. 2. Weigh 30 mg of the benzamide derivative of Formula I of this invention, dissolve in 10 mL of 0.5% methylcellulose solution, and administer by gavage.

[0090] Donepezil solution: Weigh 5 mg of donepezil and dissolve it in 10 mL of 0.5% methylcellulose solution for oral administration.

[0091] 2. Animal surgery

[0092] Mice were anesthetized with isoflurane (1.5-5%), and drugs were injected into the target nucleus using stereotactic brain imaging. The specific surgical procedure was as follows: The mice were fixed in a stereotactic brain imaging frame, their heads were shaved, and the skin was incised to expose the skull. The coordinates of the target nucleus were determined based on the mouse brain atlas. Using the anterior fontanelle as a reference point, the mouse skull was three-dimensionally localized, and a high-speed cranial drill was used to drill a hole at the target nucleus coordinates. A microinjector was used to inject the drug into the target nucleus, with the injection volume and speed controlled by a microinjection pump. After injection, the needle was left in place for 5 minutes, the microinjector was slowly withdrawn, and the head skin was sutured. It is important to note that the mouse skull needs to be kept moist throughout the procedure, and analgesics and anti-inflammatory drugs were administered postoperatively. The stereotactic brain imaging injection nucleus of this invention is the lateral ventricle (LV), with coordinates (AP: -0.30, ML: -1.00, DV: -2.50).

[0093] 3. Animal behavioral experiments

[0094] 3.1 Open Field Experiment (OFT)

[0095] Mice were placed in a 50cm×50cm×40cm plastic open field test chamber. A camera recorded the mice's spontaneous activity for 5 minutes. The total distance traveled, central dwell time, and central movement speed of the mice were analyzed using the ANY-MAZE animal behavior video analysis system. After each mouse test, the open field test chamber was wiped with a 75% alcohol solution to remove mouse odor.

[0096] 3.2 Water Maze Test (MWM test)

[0097] The water maze experiment setup consists of a white circular pool (1.2m in diameter and 0.5m in height), a white platform (9cm in diameter), and a video acquisition system. Four markers for mice to identify are placed on the outer wall of the pool, dividing it into four quadrants: I, II, III, and IV. The platform is placed in quadrant IV, 1cm below the water surface, and has a detachable white triangular flag. The water depth is 30cm, the water temperature is maintained at 22-24℃, and a suitable amount of white food additive is added to enable the black mice to be identified by the video acquisition system. The water maze experiment consisted of three phases: Phase 1 (visible platform training period), conducted on days 1-2, involved placing mice into the water sequentially from quadrants I, II, III, and IV, facing the wall. If a mouse could climb onto a platform (carrying a flag, placed in quadrant IV) and remain there for 10 seconds within 90 seconds, the time taken was recorded as the escape latency. If the mouse did not discover the platform within 90 seconds, it was guided to climb onto the platform and remain there for 30 seconds; the latency was recorded as 90 seconds. Phase 2 (hidden platform training period), conducted on days 3-5, involved removing the flags from the platforms, with the training method the same as the visible platform training. Phase 3 (spatial exploration test period), conducted on day 6, involved removing the platforms inside the water tank, and placing mice into the water from quadrant II (diagonally opposite quadrant IV), facing the wall. A camera recorded the mice's swimming activity for 90 seconds. The ANY-MAZE animal behavior video analysis system was used to analyze the mice's movement trajectory, the percentage of swimming time in quadrant IV, and the number of times they crossed platforms.

[0098] 3.3 Y-maze test

[0099] The Y-maze has three uniform arms (40cm × 9cm × 16cm), numbered A, B, and C, with the arms forming a 120° angle, resembling the letter "Y". A mouse is placed face down at the end of the starting arm A, and a camera records the mouse's free movement within the Y-maze for 8 minutes. Each consecutive entry of a mouse into any of the three arms (e.g., ABC, ACB, BCA) constitutes one alternation cycle. The number of alternation cycles is analyzed using the ANY-MAZE animal behavior video analysis system. The percentage of spontaneous alternation responses is calculated as: (Actual number of alternation cycles) / (Total number of arms entered - 2).

[0100] 3.4 Novel Object Recognition Experiment (NOR test)

[0101] This experiment was conducted based on the open field experiment. The experiment consisted of two phases: Phase 1 (training period) involved placing two identical blue cylindrical blocks (of the same size, color, and material) diagonally across the open field box, 5 cm from the side walls. Mice were placed in the box and allowed to explore freely for 5 minutes before being returned to their cages. Phase 2 (testing period) was conducted 24 hours later. One of the cylindrical blocks in the box was randomly replaced with a white cube (different in size, color, and material), which was then presented as a new object. Mice were placed in the box, and a camera recorded their exploration activities for 5 minutes. The ANY-MAZE animal behavior video analysis system was used to analyze the mice's exploration time for the cylinder (Original object A) and the cube (Novel object B). The new object recognition index was calculated as (exploration time of B - exploration time of A) / (total exploration time of A and B).

[0102] 4. Statistical methods

[0103] All experiments and data processing were double-blind. Data analysis was performed using SPSS software (version 22.0), and data plotting was performed using GraphPad Prism software (version 7.00). When comparing multiple groups of data in a one-way manner, we used one-way ANOVA with Tukey's post-hoc analysis. All data are expressed as mean ± standard error (mean ± SEM). * P<0.05 indicates a significant difference. ** P<0.01, *** P<0.001 indicates a highly significant difference.

[0104] 5. Experimental Results

[0105] 5.1. Lateral ventricle administration of the benzamide derivative of Formula I of this invention improves Aβ 1-42 Induced cognitive impairment in mice

[0106] Two-month-old male SPF-grade C57BL / 6J mice were randomly divided into 5 groups, namely, a blank control group (Veh+Veh), a lateral ventricle Aβ group, and a control group. 1-42 Modeling group (Aβ) 1-42 +Veh), lateral ventricle, low-dose administration group of the benzamide derivative of Formula I of the present invention (Aβ) 1-42 +The benzamide derivative of Formula I of the present invention (0.5 μg) and the intermediate dose group of the benzamide derivative of Formula I of the present invention administered to the lateral ventricle (Aβ) 1-42 +The benzamide derivative of Formula I of the present invention (1.5 μg) and the high-dose administration group of the benzamide derivative of Formula I of the present invention in the lateral ventricle (Aβ)1-42 + The benzamide derivative of Formula I of this invention (4.5 μg) was administered to 10 mice in each group. After one week of acclimatization, the mice in the model group and the mice in the benzamide derivative administration group of Formula I of this invention were first injected with Aβ into the lateral ventricle via stereotactic brain injection. 1-42 A mouse model of cognitive impairment was established using 410 pmol / 5 μL of artificial cerebrospinal fluid. Mice in the control group were injected with an equal volume (5 μL) of artificial cerebrospinal fluid into the lateral ventricle via stereotactic brain injection. Three days after surgery, mice in each treatment group were injected into the lateral ventricle via stereotactic brain injection of the benzamide derivative of Formula I (low dose 0.5 μg, medium dose 1.5 μg, high dose 4.5 μg). Three days after recovery, mice in each group underwent open field testing to assess spontaneous activity, Morris water maze testing to assess spatial learning and memory, Y-maze testing to assess spatial working memory, and novel object recognition testing to assess episodic memory. Figure 1 A).

[0107] Open field experiments showed no significant differences among the groups in total distance traveled (F(4,45)=0.036, P=0.997), central dwell time (F(4,45)=0.103, P=0.981), and central movement speed (F(4,45)=0.068, P=0.992). Figure 1 (B-1D). This indicates that intraventricular administration of the benzamide derivative of Formula I of the present invention does not affect spontaneous activity in mice.

[0108] Morris water maze test results showed that, compared with the blank control group, Aβ... 1-42 The percentage of time spent in the target quadrant in the model group mice (F(4,45)=9.848, P<0.001) and the number of times they crossed platforms (F(4,45)=6.751, P<0.01) were significantly decreased, while the groups treated with the benzamide derivative of Formula I of this invention (1.5 and 4.5 μg) in the lateral ventricle significantly increased Aβ. 1-42 The percentage of time spent in the target quadrant and the number of times mice crossed the platform in the model group were significantly different (P<0.05 and P<0.001). Figure 1 E-1G). Results showed that medium to high doses of the benzamide derivative of Formula I of the present invention administered to the lateral ventricle improved Aβ. 1-42 Induced spatial learning and memory impairment.

[0109] The Y-maze test results showed that, compared with the blank control group mice, Aβ... 1-42 The spontaneous alternation index of mice in the model group was significantly decreased (F(4,45)=8.053, P<0.001), while the lateral ventricle of the benzamide derivative group of Formula I of this invention (1.5 and 4.5 μg) showed a significant increase in Aβ.1-42 The spontaneous alternation index of mice in the model group (P<0.05 and P<0.01) was significantly reduced. Figure 1 H). The results showed that medium to high doses of the benzamide derivative of Formula I of the present invention administered to the lateral ventricle could improve Aβ. 1-42 Induced spatial working memory impairment.

[0110] The results of the new object recognition experiment showed that, compared with the blank control group mice, Aβ... 1-42 The recognition index of mice in the model group was significantly decreased (F(4,45)=11.951, P<0.001), while the Aβ of mice in the lateral ventricle administration groups of the benzamide derivative of Formula I of this invention (1.5 and 4.5 μg) was significantly increased. 1-42 The recognition index of mice in the model group (P<0.05 and P<0.001) was significantly lower than that in the model group. Figure 1 I). The results showed that medium to high doses of the benzamide derivative of Formula I of the present invention administered to the lateral ventricle could improve Aβ. 1-42 Induced episodic memory impairment.

[0111] The above results indicate that intraventricular administration of the benzamide derivative of Formula I of the present invention can improve Aβ. 1-42 It induces cognitive impairment in mice without affecting their spontaneous activity.

[0112] 5.2. Gavage administration of the benzamide derivative of Formula I of this invention to improve cognitive impairment in APP / PS1 transgenic mice

[0113] Nine-month-old male SPF-grade transgenic APP / PS1 mice and wild-type control mice were randomly divided into four groups: a blank control group (Veh+Veh), a cognitive impairment model group (APP / PS1+Veh), a group receiving gavage administration of the benzamide derivative shown in Formula I of this invention (APP / PS1+benzamide derivative shown in Formula I of this invention), and a positive control donepezil group (APP / PS1+donepezil), with 10 mice in each group. After one week of acclimatization, APP / PS1 mice were treated by gavage with the benzamide derivative shown in Formula I of this invention (30 mg / kg), while the positive control group APP / PS1 mice were treated by gavage with donepezil (5 mg / kg). The blank control group consisted of wild-type control mice, which, like the cognitive impairment model group mice, were administered the same volume of 0.5% methylcellulose solution by gavage. After administering the drug once daily for four consecutive weeks, mice in each group underwent open field tests to assess spontaneous activity, Morris water maze tests to assess spatial learning and memory, Y-maze tests to assess spatial working memory, and novel object recognition tests to assess episodic memory. Figure 2 A).

[0114] Open field experiments showed no significant differences among the groups in total distance traveled (F(3,36)=0.383, P=0.766), central dwell time (F(3,36)=0.066, P=0.978), and central movement speed (F(3,36)=0.152, P=0.929). Figure 2 (B-2D). This indicates that gavage administration of the benzamide derivative of Formula I of the present invention does not affect the spontaneous activity of mice.

[0115] Morris water maze test results showed that, compared with the blank control group, APP / PS1 mice had significantly decreased percentage of time spent in the target quadrant (F(3,36)=10.240, P<0.01) and number of steps crossed (F(3,36)=6.352, P<0.01). However, gavage administration of the benzamide derivative and donepezil of Formula I of this invention significantly increased the percentage of time spent in the target quadrant (P<0.01 and P<0.01) and number of steps crossed (P<0.05 and P<0.01) in APP / PS1 mice. Figure 2 E-2G). The results showed that gavage administration of the benzamide derivative of Formula I of the present invention improved spatial learning and memory impairment in APP / PS1 mice.

[0116] The Y-maze test results showed that, compared with the blank control group, the spontaneous alternation index of APP / PS1 mice was significantly decreased (F(3,36)=11.325, P<0.01), while gavage administration of the benzamide derivative and donepezil of Formula I of this invention significantly increased the spontaneous alternation index of APP / PS1 mice (P<0.01 and P<0.01, respectively). Figure 2 H). The results showed that gavage administration of the benzamide derivative of Formula I of the present invention improved spatial working memory impairment in APP / PS1 mice.

[0117] The results of the new object recognition experiment showed that compared with the blank control group, the recognition index of APP / PS1 mice was significantly decreased (F(3,36)=14.318, P<0.001), while gavage administration of the benzamide derivative and donepezil shown in Formula I of this invention significantly increased the recognition index of APP / PS1 mice (P<0.01 and P<0.001, respectively). Figure 2 I). The results showed that gavage administration of the benzamide derivative of Formula I of the present invention could improve episodic memory impairment in APP / PS1 mice.

[0118] The above results indicate that gavage administration of the benzamide derivative of Formula I of the present invention can improve cognitive impairment in APP / PS1 mice without affecting spontaneous activity.

[0119] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. Use of a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving Alzheimer's disease, characterized in that, The benzamide derivative has the structural formula shown in Formula I: Formula I.

2. Use of a pharmaceutical composition comprising a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving Alzheimer's disease, characterized in that, The benzamide derivative has the structural formula shown in Formula I: Formula I.

3. The use according to claim 2, characterized in that, The pharmaceutical composition further comprises one or more drugs and / or extracts for improving Alzheimer's disease.

4. The use according to claim 3, characterized in that, The drug is selected from one or more of the following: cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, monoclonal antibodies, and gut-brain axis modulators.

5. The use according to claim 4, characterized in that, The cholinesterase inhibitors are donepezil, rivastigmine, huperzine A, and / or galantamine.

6. The use according to claim 4, characterized in that, The N-methyl-D-aspartate receptor antagonist is memantine.

7. The use according to claim 4, characterized in that, The monoclonal antibody is aduretumumab, lencanemab, and / or donepemab.

8. The use according to claim 4, characterized in that, The brain-gut axis modulator is mannitol.

9. Use of a pharmaceutical formulation comprising a benzamide derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient in the preparation of a medicament for improving Alzheimer's disease, characterized in that, The benzamide derivative has the following structural formula: Formula I.

10. The use according to claim 9, characterized in that, The excipients are selected from one or more of the following: diluents, wetting agents, adhesives, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

11. The use according to claim 9, characterized in that, The pharmaceutical preparations are powders, tablets, pills, capsules, films, lozenges, granules, injections, or oral liquids.

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