A nicotinamide phosphoribosyltransferase agonist, pharmaceutical compositions thereof, and use in reducing muscle fatigue

Abstract

The present application provides a nicotinamide phosphoribosyltransferase agonist, a pharmaceutical composition thereof and an application in relieving muscle fatigue. The agonist is compound YB12 having a structure shown in formula I or compound YB8 having a structure shown in formula II, which improves the intracellular NAD + level, thereby improving the energy metabolism state of skeletal muscle, so as to achieve the purpose of delaying or relieving muscle fatigue. Compound YB8 can further improve the index related to exercise endurance when used in combination with oxidoreductase cofactor pyrroloquinoline quinone (PQQ), showing a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically involving the preparation and application of two drug precursors with anti-exercise fatigue effects, the core feature of which is a strong activating effect on nicotinamide phosphoribosyltransferase. Background Technology

[0002] Muscle fatigue is a physiological phenomenon characterized by a reversible decline in the contractile capacity of skeletal muscles after strenuous or prolonged activity, often accompanied by reduced exercise endurance. It is also commonly seen in pathological conditions such as mitochondrial myopathy and muscle atrophy, severely impacting quality of life. However, existing interventions have significant limitations: natural products have slow onset and limited potency, while central nervous system stimulants, although effective in the short term, have side effects such as addiction and rhythm disturbances. Therefore, developing safe and effective novel anti-fatigue strategies is of great clinical significance.

[0003] NAD + Nicotinamide adenine dinucleotide (NAD) is a key intracellular coenzyme and metabolic regulatory molecule, playing a central role in redox reactions and energy metabolism. In skeletal muscle, NAD... + The level of NAD+ directly determines the mitochondrial ATP production capacity, thus affecting muscle endurance maintenance and fatigue recovery. Under conditions of strenuous exercise or continuous load, intracellular NAD+ levels... + Exhaustion and NAD + A decreased NAD / NADH ratio impairs mitochondrial function and reduces ATP synthesis efficiency, thereby exacerbating muscle fatigue and exercise-related tissue damage. Therefore, maintaining NAD / NADH balance is crucial. + Homeostasis is a potentially effective strategy for improving muscle function.

[0004] NAMPT (nicotinamide phosphoribosyltransferase) acts as NAD+ + Targeting and activating NAMPT, a key rate-limiting enzyme in the salvage synthesis pathway, is crucial for enhancing NAD+ synthesis. + This patent describes an efficient approach to combating exercise-induced fatigue. Currently, there are no reports on the application of NAMPT activators in combating exercise-induced fatigue. This patent prepared and obtained two lead compounds that can effectively activate NAMPT, and verified their preventative and therapeutic effects on exercise-induced fatigue using a mouse model. Summary of the Invention

[0005] This invention aims to address the limitation of existing interventions for muscle fatigue by providing two drug prodrugs capable of regulating NAMPT activity, thereby increasing intracellular NAD+. + This improves the energy metabolism of skeletal muscle, thereby delaying or reducing muscle fatigue.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a nicotinamide phosphoribosyltransferase agonist with anti-exercise fatigue effect, wherein the agonist is compound YB12 or compound YB8.

[0007] The present invention also provides a pharmaceutical composition comprising: (a) the agonist of claim 1; and (b) pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof.

[0008] According to a specific embodiment of the present invention, components (a) and (b) are formulated as independent dosage forms for sequential administration. As a preferred embodiment, the sequential administration refers to administering pyrroquinoline quinone first, followed by the agonist 30 minutes later.

[0009] The present invention further provides the use of the agonist or a pharmaceutical composition comprising the agonist in the preparation of a medicament for relieving muscle fatigue.

[0010] Through a series of in vitro and in vivo experiments, the inventors discovered that compounds YB12 and YB8 can activate NAMPT activity, thereby increasing intracellular NAD+. + In animal exercise models, compound YB8 was found to enhance exercise endurance and reduce muscle fatigue. In particular, compound YB8, when used in combination with the redox cofactor pyrroloquinoline quinone (PQQ), further improved exercise endurance-related indicators, demonstrating a synergistic effect.

[0011] In the first part of this invention, a NAMPT agonist YB12 is provided.

[0012] As a specific embodiment of the present invention, we found that administration of the NAMPT agonist YB12 can effectively increase NAD in muscles. + Content. The NAMPT agonist YB12 has the structure shown in Formula I: Formula I In the second part of this invention, it is clarified that YB12 has the effect of reducing muscle fatigue.

[0013] In one specific embodiment of the present invention, C57BL / 6J mice were used to evaluate motor function and establish an exercise-induced fatigue model. Mice were subjected to weighted swimming, tumbling block, treadmill exercises, and grip strength tests after drug administration. Continuous forced running was used to induce exercise fatigue in the mice. After the final exercise session, serum lactate levels, lactate dehydrogenase activity, and liver and muscle glycogen levels were measured, and NAD+ levels in relevant tissues were determined. +The effects of compound YB12 on exercise fatigue were comprehensively evaluated by measuring ATP levels and observing changes in skeletal muscle tissue structure. Compound YB12 was deemed to have an anti-exercise fatigue effect if the exercise endurance and strength indicators of the tested mice significantly improved, fatigue-related biochemical indicators recovered, and muscle tissue structural damage was reduced.

[0014] Without intervention, exercise-induced fatigue tends to worsen progressively. At the cellular level, its mechanism is mainly related to impaired mitochondrial function; at the tissue level, it often manifests as skeletal muscle ultrastructural damage, accompanied by lactic acid accumulation, glycogen depletion, and insufficient energy supply. This invention mainly studies the exercise capacity of mice and a mouse model of exercise-induced fatigue induced by forced exercise. The main detection indicators are: (1) behavioral indicators such as weighted swimming, tumbling bar, treadmill, and grip strength; (2) serum lactate, lactate dehydrogenase activity, and liver and muscle glycogen levels; (3) tissue structure analysis and NAD in related tissues. + and ATP levels.

[0015] The innovation of this invention lies in the fact that it is the first time that compound YB12 has been shown to increase NAD in the body by regulating NAMPT-related pathways. + This finding improves energy metabolism and exerts an anti-exercise fatigue effect by increasing ATP levels. It provides a new technical strategy for drug intervention in exercise-induced fatigue, with the following advantages: in animal models, YB12 significantly enhances exercise capacity, alleviates exercise fatigue, and reduces skeletal muscle tissue damage. These results provide a basis for developing NAD+-based drug interventions. + This has laid an important foundation for products related to metabolic regulation and resistance to exercise fatigue.

[0016] In a third part of this invention, a NAMPT agonist YB8 is provided.

[0017] As a specific embodiment of the present invention, we found that administering the NAMPT agonist YB8 can effectively increase NAD in muscles. + Content. The NAMPT agonist YB8 has the structure shown in Formula II: Formula II In the fourth part of this invention, it is clarified that YB8 has the effect of reducing muscle fatigue.

[0018] In one specific embodiment of the present invention, C57BL / 6J mice were selected to evaluate exercise capacity. After administration, the mice underwent weighted swimming and treadmill exercise tests to comprehensively evaluate the anti-exercise fatigue effect of compound YB8. When the exercise endurance of the test mice was significantly improved compared to the control group, compound YB8 was determined to have an anti-exercise fatigue effect.

[0019] This invention focuses on the motor ability of mice, selecting behavioral indicators such as weighted swimming and treadmill exercise as core evaluation parameters.

[0020] The innovation of this invention lies in the fact that, through systematic animal experiments, it is the first time that compound YB8 has been demonstrated to significantly improve the body's exercise endurance in an exercise-induced fatigue model, thus clarifying that it has a significant anti-exercise fatigue effect, providing new candidate compounds and experimental evidence for the development of novel anti-fatigue drugs or functional interventions.

[0021] In the fifth part of this invention, it is clarified that the combined use of YB8 and PQQ has a synergistic effect in reducing muscle fatigue.

[0022] In one specific embodiment of the present invention, C57BL / 6J mice were used to evaluate exercise capacity and construct an exercise-induced fatigue model to assess the anti-exercise fatigue effect of compound YB8 combined with pyrroloquinoline quinone (PQQ). Mice underwent treadmill exercise testing, with the combined administration group receiving sequential administration: PQQ was administered first, followed by compound YB8 after a certain interval. Simultaneously, continuous forced running was used to induce exercise fatigue in the mice. After the final exercise, serum lactate levels, liver glycogen, and muscle glycogen levels were measured to evaluate fatigue-related metabolic changes. Furthermore, liver histological observation combined with serum alanine aminotransferase (ALT) level detection was used to assess the safety of the combined administration and whether there were any toxic or abnormal changes. If the exercise endurance and strength indicators of the tested mice significantly improved, and fatigue-related biochemical indicators recovered, then the combined administration of compound YB8 and PQQ was determined to have an anti-exercise fatigue effect.

[0023] Without intervention, exercise-induced fatigue will progressively worsen. This process is related to mitochondrial dysfunction at the microscopic level and manifests as skeletal muscle ultrastructural damage at the macroscopic level, leading to lactic acid accumulation, glycogen depletion, and energy metabolism disorders. This invention mainly studies the exercise capacity of mice and a mouse exercise-induced fatigue model induced by forced exercise. The main detection indicators include: (1) behavioral indicators such as treadmill duration; (2) serum lactic acid, liver glycogen, and muscle glycogen levels; (3) liver tissue H&E staining; and (4) serum ALT levels.

[0024] The innovation of this invention lies in the fact that it is the first time that the combined application of compound YB8 and PQQ can exert a synergistic anti-exercise fatigue effect in vivo. This effect is achieved by improving energy metabolism, enhancing exercise endurance through multiple pathways, and alleviating exercise fatigue. This discovery provides a new technical strategy and experimental basis for the development of anti-fatigue products based on energy metabolism regulation. Attached Figure Description

[0025] Figure 1Effects of YB12 and YB8 on NAMPT enzyme activity and binding activity.

[0026] Figure 2 Effects of YB12 on exercise endurance in mice.

[0027] Figure 3 Effects of YB12 on fatigue-related biochemical indicators in mice.

[0028] Figure 4 Effects of YB12 on muscle tissue morphology and mitochondria in exercise-induced fatigue mice.

[0029] Figure 5 YB12 affects ATP and NAD levels in major organs and muscle tissues of mice. + The effect of content.

[0030] Figure 6 Effects of YB8 on exercise endurance in mice.

[0031] Figure 7 Effects of combined administration of YB8 and PQQ on the time to exhaustion in mice during treadmill exercise and body weight during administration.

[0032] Figure 8 Effects of combined administration of YB8 and PQQ on fatigue-related biochemical indicators in mice.

[0033] Figure 9 Effects of combined administration of YB8 and PQQ on liver tissue morphology in mice.

[0034] Figure 10 Effect of combined administration of YB8 and PQQ on serum ALT levels in mice. Detailed Implementation

[0035] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0036] Example 1 This invention provides a method for preparing compounds YB12 and YB8, comprising the following steps: First, synthesize key intermediate 4 according to route 1.

[0037] Route 1: Reagents and conditions: (a) Triphosgene, DIPEA, DCM, room temperature; (b) H2, Pd / C, DCM, room temperature, overnight.

[0038] Compound 1 and Compound 2 were reacted in dichloromethane (DCM) solvent in the presence of triphosgene and N,N-diisopropylethylamine (DIPEA) at room temperature to give Compound 3 in 37% yield.

[0039] Compound 3 was reacted overnight at room temperature in a hydrogen atmosphere and in DCM solvent under Pd / C catalysis to give key intermediate 4 in 92% yield.

[0040] The final product was synthesized according to route 2.

[0041] Route 2: Reagents and conditions: (a) Triethylamine, DCM, room temperature, 1 h; Intermediate 4 was reacted with 4-methoxybenzenesulfonyl chloride or 4-fluorobenzenesulfonyl chloride at room temperature for 1 hour in the presence of DCM solvent and triethylamine (Et3N) to prepare compounds YB12 and YB8, respectively.

[0042] Example 2 In this invention, C57BL / 6J mice (referred to as C57 mice) were used and purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animal experimental procedures were strictly in accordance with animal ethics requirements to ensure that the mice were treated humanely.

[0043] The specific implementation method for evaluating the motor ability of mice was as follows: experimental mice were randomly divided into experimental and control groups. Mice in the experimental group were administered compound YB12 via intraperitoneal injection daily for 3 consecutive days; mice in the control group were administered an equal volume of the solvent via intraperitoneal injection in the same manner. Thirty minutes after the last administration, mice in each group underwent weighted swimming, rotarod, treadmill exercise, and grip strength tests to comprehensively evaluate the mice's exercise endurance, coordination ability, and muscle strength.

[0044] The specific implementation method of the mouse exercise-induced fatigue model is as follows: A mouse exercise-induced fatigue model is established using a continuous forced exercise method. Specifically, mice are forced to exercise on a treadmill for one hour daily for six consecutive days to induce exercise-induced fatigue. During the last three days of modeling, mice are randomly divided into two groups: a YB12 administration group and a model group. Mice in both groups are intraperitoneally injected with compound YB12 and an equal volume of solvent, respectively, at a dose of 20 mg / kg / day. Normal mice that do not undergo forced exercise serve as a control group, and during the last three days of modeling, they are injected with an equal volume of solvent.

[0045] The specific administration regimen was as follows: a mixed solvent consisting of 9% DMSO (dimethyl sulfoxide), 10% Tween-80 (polysorbate 80), and 81% physiological saline was used as the administration solvent. The dosage of YB12 was 20 mg / kg, and the administration period was 3 days, with the drug administered once a day. The weight changes of mice were recorded daily during the administration period to assess the safety of the drug.

[0046] Sample collection and index detection methods were as follows: Blood and tissue samples were collected from mice 30 minutes after the last exercise following modeling and drug administration. Serum lactate levels, liver glycogen, and muscle glycogen levels were detected using a kit; NAD+ levels in the heart, liver, kidney, brain, and skeletal muscle tissue were also detected. + ATP levels were measured to assess changes in energy metabolism. Furthermore, hematoxylin-eosin (H&E) staining and electron microscopy were used to observe morphological and ultrastructural changes in skeletal muscle tissue. L-lactic acid (LAC) and liver / muscle glycogen assay kits were purchased from Nanjing Jiancheng Bioengineering Institute; enhanced ATP assay kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; NAD+ levels were measured. + The NADH colorimetric assay kit was purchased from Elabscience® in Wuhan. All assays were performed strictly according to the kit instructions.

[0047] like Figure 2 As shown, compared with the control group, the time to exhaustion during weight-bearing swimming, the time spent on the rotarod, the duration of treadmill exercise, and the grip strength of the limbs were significantly improved in the YB12-treated group; Figure 3 As shown, compared with the model group, the YB12-treated mice had significantly lower serum lactate levels, higher LDH activity, and significantly increased liver and muscle glycogen content; Figure 4 As shown, H&E staining revealed no significant histological differences among the groups. Electron microscopy revealed that the skeletal muscle of the model group mice exhibited disordered myofibril arrangement, increased vacuolation, and damaged mitochondrial cristae structure, while the myofibrils of the YB12-treated group mice were relatively well-organized, and the mitochondrial structure remained relatively intact. Figure 5 As shown, YB12 can increase NAD in tissues. + It also increases ATP levels, with a particularly significant effect on skeletal muscle. These experimental results indicate that YB12 effectively improves athletic performance and has a good intervention effect on exercise-induced fatigue by regulating energy metabolism-related pathways.

[0048] Example 3 In this invention, C57BL / 6J mice (referred to as C57 mice) were used and purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animal experimental procedures were strictly in accordance with animal ethics requirements to ensure that the mice were treated humanely.

[0049] The specific implementation method for evaluating the exercise capacity of mice was as follows: Experimental mice were randomly divided into experimental and control groups. Mice in the experimental group were administered compound YB8 via intraperitoneal injection daily for three consecutive days; mice in the control group were administered an equal volume of the solvent via intraperitoneal injection in the same manner. Thirty minutes after the last administration, mice in each group underwent weighted swimming and treadmill exercise tests to comprehensively evaluate their exercise endurance.

[0050] The specific administration regimen was as follows: using 0.5% CMC-Na (sodium carboxymethyl cellulose) as the solvent, the dosage of YB8 was 30 mg / kg, the administration period was 3 days, and the mice were given once a day. During the administration period, the weight and general condition of the mice were recorded daily to assess the safety of the administration.

[0051] like Figure 6 As shown, compared with the control group, the YB8 group mice showed significantly improved time to exhaustion during weight-bearing swimming and duration of treadmill exercise. These experimental results indicate that YB8 can effectively improve athletic performance and demonstrates a good effect in alleviating exercise-induced fatigue.

[0052] Example 4 In this invention, C57BL / 6J mice (referred to as C57 mice) were used and purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animal experimental procedures were strictly in accordance with animal ethics requirements to ensure that the mice were treated humanely.

[0053] The specific implementation method for evaluating the motor ability of mice was as follows: Experimental mice were randomly divided into three groups: a blank control group, a single-drug group, and a combination-drug group. The YB8 and PQQ combination-drug group received sequential administration, first administering pyrrolquinoline quinone (PQQ), followed by compound YB8 30 minutes later. The PQQ and NMN combination-drug group received sequential administration, first administering pyrrolquinoline quinone (PQQ), followed by nicotinamide mononucleotide (NMN) 30 minutes later. The single-drug groups received equivalent doses of PQQ, compound YB8, or NMN, respectively. The blank control group received an equal volume of solvent. All administration methods were oral, once daily for three consecutive days. Thirty minutes after the last administration, mice in each group underwent weighted swimming, rotarod, treadmill, and grip tests to comprehensively evaluate their exercise endurance, coordination, and muscle strength.

[0054] The specific implementation method of the mouse exercise-induced fatigue model is as follows: Mice were induced to develop exercise-induced fatigue using continuous forced exercise. Specifically, for six consecutive days, mice were placed on a treadmill for forced running training, with each session lasting one hour, to gradually induce exercise fatigue. During the last three days of modeling, mice in each group received appropriate treatments. The YB8 and PQQ combined treatment group received PQQ and compound YB8 in the aforementioned sequential manner, while the blank control group received an equal volume of solvent. The grouping and administration methods for mice were the same as those used in the mouse exercise capacity assessment, and all mice in each group underwent continuous forced exercise.

[0055] The specific dosing regimen was as follows: using 0.5% CMC-Na (sodium carboxymethyl cellulose) as the solvent, the dosage of YB8 and PQQ was 30 mg / kg and 15 mg / kg, respectively, and the dosage of NMN was 300 mg / kg. The dosing cycle was 3 days, with the mice administered once daily. The weight and general condition of the mice were recorded daily during the dosing period to assess the safety of the administration.

[0056] Sample collection and index detection methods were as follows: Blood and tissue samples were collected from mice 30 minutes after the last exercise following modeling and drug administration. Serum lactate levels, liver glycogen, and muscle glycogen levels were detected using kits to assess changes in energy metabolism. In addition, mouse liver tissue was subjected to H&E staining to observe histological morphological changes, and serum alanine aminotransferase (ALT) levels were detected to comprehensively evaluate the safety and potential toxicity of the combined drug administration. The LAC, liver / muscle glycogen, and ALT detection kits were purchased from Nanjing Jiancheng Bioengineering Institute, and all tests were performed strictly according to the kit instructions.

[0057] like Figure 7 , 8 As shown, compared with the blank control group and the NMN monotherapy group, the mice in the YB8 monotherapy group showed some improvement in treadmill exercise duration and post-exercise serum lactate levels, while liver glycogen and muscle glycogen levels recovered. Furthermore, the combined administration groups further enhanced the improvement in exercise endurance and metabolic indicators, with the YB8 and PQQ combined administration group showing the most significant improvement, significantly better than the PQQ and NMN combined administration group. Figure 9 , 10 As shown, no significant abnormalities were observed in the liver tissue of mice in each group, and serum ALT levels remained within the normal range. These experimental results indicate that the combined administration of YB8 and PQQ can synergistically improve the body's energy metabolism, enhance exercise endurance through multiple pathways, and alleviate exercise-induced fatigue, demonstrating a stable and significant anti-exercise fatigue effect. Furthermore, this combined administration regimen exhibits good safety and promising application prospects.

[0058] Example 5 This invention evaluates the effects of compounds YB12 and YB8 on NAMPT from two dimensions: "function" and "binding," through biochemical functional experiments (enzyme activity assays) and biophysical interaction (SPR) experiments. In the enzyme activity assay, NAD+ in the reaction system was used as the basis for the evaluation. + The amount of NAMPT produced is used as a detection indicator to reflect the relative catalytic activity of the NAMPT enzyme. Figure 1 Experimental results showed that both could significantly activate NAMPT enzyme in a dose-dependent manner and had micromolar binding capacity to NAMPT, with YB8 exhibiting stronger target binding affinity.

[0059] It should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nicotinamide phosphoribosyltransferase agonist, characterized in that, The agonist is either compound YB12 having the structure shown in Formula I or compound YB8 having the structure shown in Formula II. Formula I Formula II.

2. The agonist according to claim 1, characterized in that, The preparation method of compounds YB12 and YB8 includes the following steps: First, synthesize key intermediate 4 according to route 1: Route 1: Reagents and conditions: (a) Triphosgene, DIPEA, DCM, room temperature; (b) H2, Pd / C, DCM, room temperature, overnight; The final product was synthesized according to route 2: Route 2: Reagents and conditions: (a) Triethylamine, DCM, room temperature, 1 h; Intermediate 4 was reacted with 4-methoxybenzenesulfonyl chloride or 4-fluorobenzenesulfonyl chloride at room temperature for 1 hour in the presence of DCM solvent and triethylamine to prepare compounds YB12 and YB8, respectively.

3. A pharmaceutical composition, characterized in that, It comprises: (a) the agonist of any one of claims 1-2; and (b) pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that, Components (a) and (b) are formulated as separate dosage forms for sequential administration.

5. The pharmaceutical composition according to claim 4, characterized in that, The sequential administration refers to administering pyrroloquinoline quinone first, followed by the agonist 30 minutes later.

6. Use of the agonist of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-5 in the preparation of a medicament for relieving muscle fatigue.

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