Naringenin or derivatives thereof for improving muscle endurance or treating or preventing muscle atrophy or dystrophy

Abstract

The present invention provides compositions, compounds and methods for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need of such treatment. Such compositions include an effective amount of a compound of formula (I), or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The compound is naringenin or a naringenin derivative. Methods of making the compounds or compositions are also provided.

Description

[0001] Priority and related applications

[0002] The present invention claims priority to U.S. Provisional Application No. 62 / 323,146, filed on March 24, 2022, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to compositions having pharmaceutical or functional properties. More specifically, it relates to a composition comprising naringenin or a derivative thereof, a method for preparing the composition, and a method for using the composition, for example as a pharmaceutical composition, a functional composition and / or a dietary supplement. Background Art

[0004] Skeletal muscle is the largest organ in mammals and plays an extremely important role in supporting movement, heat production, and metabolic regulation. However, its function can be impaired by aging, sedentary lifestyles, and muscle-related diseases, resulting in a decrease in endurance or strength. Skeletal muscle atrophy and muscle function decline accompany the aging process (Porter et al., 1995). Studies have shown that elderly people have reduced cross-sectional area of ​​fast muscle fibers, reduced expression of mitochondrial respiratory chain complexes, and decreased aerobic respiratory capacity of skeletal muscle (Carter et al., 2015; Murgia et al., 2017). Correspondingly, from a functional point of view, the absolute grip strength of aged mice is 26% lower than that of adult mice, and the relative grip strength is 15% lower (McArdle et al., 2004).

[0005] Furthermore, mice that did not exercise had greater age-related loss of muscle mass and mitochondrial dysfunction than mice that exercised regularly on a running wheel (Figueiredo et al., 2009). Sedentary older men have a concomitant decrease in type IIa fibers (oxidative muscle fibers) and a decrease in mitochondrial content in all fiber types (St-Jean-Pelletier et al., 2017). Furthermore, various muscle diseases can lead to muscle atrophy and dysfunction. For example, Duchenne muscular dystrophy (DMD), the most common muscular dystrophy in children, causes proximal muscle weakness and hypertrophy of the calves, ultimately leading to degeneration of the skeleton and heart muscle (Falzarano et al., 2015; Kamdar and Garry, 2016). Therefore, healthy skeletal muscle is essential for the organism, and muscle damage can severely affect health and quality of life.

[0006] Strategies are urgently needed to combat muscle loss and functional decline. Exercise has been shown to be an effective strategy that can improve muscle function and reverse muscle aging to a certain extent. However, exercise is not suitable for patients who are bedridden for a long time or have other clinical complications. Therefore, drug therapies are needed to reduce skeletal muscle loss and restore muscle function.

[0007] In recent years, the signaling pathways involved in muscle dysfunction have been elucidated, and different types of molecules, such as proinflammatory cytokines, growth factors, and transforming growth factor-β (TGF-β) family effectors, have been found to be involved in skeletal muscle atrophy (Furrer and Handschin, 2019; Lynch et al., 2007). Some studies have focused on these molecules to investigate therapeutic targets or drugs to improve muscle loss. For example, it has been reported that cyclooxygenase 2 (COX2) inhibitors significantly reduce plasma levels of interleukin 6 (IL-6) and interleukin 1 (IL-1) and increase muscle mass in aged rats (Rieu et al., 2009). Myostatin (MSTN) can significantly reduce muscle mass, and therefore, MSTN has attracted widespread attention as a therapeutic target. LY2495655, a humanized MSTN antibody, increased muscle mass in the elderly in clinical trials; however, grip strength was not affected (Becker et al., 2015). Although some drugs can ameliorate the loss of muscle mass, most trials fail to show significant improvements in functional parameters (Furrer and Handschin, 2019). Skeletal muscle remains one of the most pharmacologically neglected organs.

[0008] In addition to the effectiveness of the drug, the safety of the drug also needs to be considered. Testosterone has been shown to prevent age-related muscle strength loss and improve physical function (Srinivas-Shankar et al., 2010; Storer et al., 2017); however, its clinical use is limited by serious side effects (Grech et al., 2014). Therefore, it is necessary to discover new natural drugs that are safer and more effective than existing drugs to improve the physiological function of skeletal muscle or prevent muscle atrophy.

[0009] Naringenin (NAR), a dihydroflavonoid compound, is commonly found in the form of naringenin in plants of the Rosaceae, Rutaceae, and Citrus families. NAR has been reported to have important biological activities and has potential positive effects on metabolic diseases, cardiovascular diseases, cancer, lung diseases, neurodegenerative diseases, and gastrointestinal pathologies (Rivoira et al., 2021). For example, NAR supplementation can increase energy expenditure, enhance insulin sensitivity, and increase hepatic fatty acid oxidation, thereby reducing fat mass and improving metabolic function (Alam et al., 2013; Goldwasser et al., 2010; Pu et al., 2012; Rebello et al., 2019; Sacks et al., 2018). In terms of mechanism of action, NAR mainly affects energy metabolism through the PPAR family, PGC-1 family, and AMPK signaling pathways (Goldwasser et al., 2011; Mulvihill et al., 2009; Yu et al., 2019). Summary of the invention

[0010] The present disclosure provides a composition and method for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment.

[0011] In the present invention, the inventors found that naringenin (NAR) can improve muscle endurance and alleviate muscle dysfunction in naturally aged mice and mdx mice by increasing the number of oxidative muscle fibers and aerobic metabolism. The transcription factor Sp1 has been identified as a direct target of NAR, which was identified by biotin labeling co-immunoprecipitation mass spectrometry, and its binding site has been further verified to be GLN-110. NAR upregulates the expression of Esrrg by upregulating Sp1 phosphorylation levels and enhancing the binding of Sp1 to the CCCTGCCCTC sequence of the Esrrg promoter. The discovery of the Sp1-ERRγ transcription axis is of great significance in basic skeletal muscle research, and the new function of NAR has potential significance for preventing the decline in aerobic exercise capacity associated with a sedentary lifestyle and age- or disease-related muscle atrophy.

[0012] Based on the study of naringenin, the present invention has obtained various compounds, including derivatives thereof, which are used to improve skeletal muscle endurance, treat muscle atrophy or malnutrition, or prevent muscle atrophy or malnutrition in subjects in need of such treatment. The present invention also has obtained a composition comprising at least one derivative.

[0013] On the one hand, the present disclosure provides a composition for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment. This composition includes an effective amount of a compound of formula (I) as described herein (also having code S1), or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient.

[0014] Formula (I) has the following chemical structure:

[0015]

[0016] In formula (I), R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2 , C1-C6 alkyl, C1-C6 alkoxy and phenyl. 1 , R 2 , R 3 and R4 Each is selected from H, F, Cl, Br, OH and NH 2 Such a compound is naringenin or a naringenin derivative.

[0017] In some embodiments, R 1 , R 2 , R 3 and R 4 One or two of are substituents other than H. For example, in some embodiments, R 1 or R 2 is a substituent other than H, R 3 =H, and R 4 =H.

[0018] The composition may be a pharmaceutical composition, a functional composition and / or a dietary supplement. For example, the composition is a pharmaceutical composition that can be injected or taken orally. The composition is preferably injectable. The concentration of the compound may be in the range of 1 mM to 50 mM, for example, 2 mM to 15 mM, 5 mM to 10 mM, or any other appropriate concentration.

[0019] The excipient may be a solvent, a cosolvent, a colorant, a preservative, an antibacterial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent or any combination thereof. For example, in an injectable composition, the excipient comprises a carrier calculated by weight of 20% DMSO and 80% saline.

[0020] In another aspect, the present disclosure provides a compound having formula (I), as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof. The present disclosure provides any genus or species of compound as described herein. In certain embodiments, R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2 , C1-C6 alkyl, C1-C6 alkoxy and phenyl. At least one R 1 , R 2 , R 3 and R 4 is a substituent other than H. Such a compound is a naringenin derivative. In certain embodiments, R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH and NH 2 Composed of groups.

[0021] In some embodiments, R 1 , R 2 , R 3 and R 4 One or two of are substituents other than H. For example, in some embodiments, R 1 or R 2 is a substituent other than H, R 3 =H, and R 4 =H.

[0022] The compound may be a suitable compound having desired solubility and pharmacological properties.

[0023] In another aspect, the present disclosure provides a method for preparing a composition or compound described herein. The method may include preparing the compound. The method may further include mixing an excipient and the compound.

[0024] In another aspect, the present disclosure provides a method for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment. The method comprises administering an appropriate amount of a composition described herein to a subject in need of such treatment.

[0025] In certain embodiments, in the compound of formula (I), R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2 , C1-C6 alkyl, C1-C6 alkoxy and phenyl. At least one R 1 , R 2 , R 3 and R 4 is a substituent other than H. Such a compound is naringenin or a naringenin derivative. In certain embodiments, each R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH and NH 2 Composed of groups.

[0026] In some embodiments, R 1 , R 2 , R 3 and R 4 One or two of are substituents other than H. For example, in some embodiments, R 1 or R 2 is a substituent other than H, R 3 =H, and R4 =H.

[0027] In some embodiments, the subject is a mammal, preferably a human subject, and can be a healthy human, or an adult with age- or disease-related muscle wasting.

[0028] In some embodiments, the composition is administered by intramuscular injection or oral administration. Preferably, it is administered by injection. In certain embodiments, the composition is administered intramuscularly at a frequency of once a day or once every other day, with an effective dose of the compound in a dose range of 2 mg / Kg to 20 mg / Kg. The dosage can be any suitable dose. For example, in certain embodiments, the dosage range of the effective dose of the compound is 3.6 mg / Kg to 7.6 mg / Kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present disclosure is best understood by the following detailed description and the accompanying illustrations. It is emphasized that, in accordance with common practice, the various features of the illustrations are not necessarily drawn to scale. Instead, the dimensions of the various features may be arbitrarily enlarged or reduced for clarity. In the specifications and illustrations, the same reference numerals represent the same features.

[0030] Figure 1-3 Shows the maximum running distance of middle-aged mice on a treadmill ( Figure 1 ), the ratio of limb grip strength to body weight ( Figure 2 ) and the ratio of gastrocnemius (GAS) muscle weight to body weight ( Figure 3 ), these mice received intragastric injection of vehicle or NAR. n = 6 per group, 10-month-old C57 mice.

[0031] Figure 4-6 The maximum running distance of young mice on a treadmill is shown ( Figure 4 ), the ratio of limb grip strength to body weight ( Figure 5 ) and the ratio of gastrocnemius (GAS) muscle weight to body weight ( Figure 6 ), these mice received intragastric injection of vehicle or NAR. n = 9 / 10, 2-month-old C57 mice per group.

[0032] Figure 7-9 The maximum running distance of C57 or mdx mice on a treadmill is shown. Figure 7 ), the ratio of limb grip strength to body weight ( Figure 8 ) and the ratio of gastrocnemius (GAS) muscle weight to body weight ( Fig. 9 ), these mice received intragastric injection of vehicle or NAR. Each group n = 4-6, 4-month-old C57 mice or 4-month-old mdx mice.

[0033] Fig.10 Shown are the mRNA expression levels of Myh7, Myh2 and Myh4 in gastrocnemius muscle of 10-month-old C57 mice treated with vehicle or NAR. n=6 per group.

[0034] Fig.11 Shown are the quantitative statistical results of MyHC type I myofibers in gastrocnemius muscle of 10-month-old C57 mice treated with vehicle or NAR by immunofluorescence staining.

[0035] Fig.12 Shown are the mRNA expression levels of genes related to aerobic metabolism in the gastrocnemius muscles of 10-month-old C57 mice treated with vehicle or NAR. n=6 per group.

[0036] Fig.13 Shown are the mRNA expression levels of Myh7, Myh2 and Myh4 in gastrocnemius muscle of 2-month-old C57 mice treated with vehicle or NAR. n=9 / 10 per group.

[0037] Fig.14 Shown are the quantitative statistical results of MyHC I myofibers in gastrocnemius muscle of 2-month-old C57 mice treated with vehicle or NAR by immunofluorescence staining. n = 9 / 10 per group.

[0038] Fig.15 Shown are the mRNA expression levels of genes related to aerobic metabolism in the gastrocnemius muscle of 2-month-old C57 mice treated with vehicle or NAR. n=9 / 10 per group.

[0039] Fig.16 Quantitative statistical results of the levels of five oxidative phosphorylation complexes in 2-month-old C57 mice treated with vehicle or NAR by immunoblotting are shown. β-actin was used as an internal control. n = 7 per group.

[0040] Fig.17 Shown are the mRNA expression levels of genes related to aerobic metabolism in the gastrocnemius muscle of 4-month-old C57 mice or mdx mice treated with vehicle or NAR. n = 4-6 per group.

[0041] Fig.18 Shown are the morphologies of C2C12 myotubes after treatment with DMSO or different concentrations (40, 100, 200, 400, 800, 1600 and 2400 μM) of NAR.

[0042] Fig.19 Shown are the mRNA expression levels of Myh7, Myh2, and Myh4 in C2C12 myotubes treated with DMSO or different concentrations of NAR.

[0043] Fig. 20 ATP levels in C2C12 myotubes treated with DMSO or different concentrations of NAR are shown;

[0044] Fig.21 Shown are oxygen consumption rates of C2C12 myotubes treated with DMSO or NAR. Oligomycin was added to block ATP-coupled respiration, FCCP was added to induce maximal respiration, and antimycin A / rotenone was added to block mitochondrial electron transport. Four biological replicates were performed.

[0045] Fig. 22 Statistical results of the levels of five oxidative phosphorylation complexes in C2C12 myotubes treated with DMSO or NAR are shown.

[0046] Fig.23 The mRNA expression levels of genes related to oxidative phosphorylation, tricarboxylic acid cycle, and β-oxidation in C2C12 myotubes treated with DMSO and NAR are shown. Due to limited space, changes without significant differences are not marked.

[0047] Fig.24 Shown are the statistical results of qPCR analysis of ERRγ levels in C2C12 myotubes treated with DMSO or NAR. β-Actin was used as an internal control.

[0048] Fig.25 Shown is qPCR analysis of ERRγ levels in gastrocnemius muscle of middle-aged, young, and mdx mice treated with DMSO or NAR.

[0049] Fig.26 The mRNA expression levels of Esrrg, Myh7, Myh2, Atp5b, and Cpt1b in C2C12 myotubes (NC group and Esrrg knockdown group) treated with DMSO or NAR are shown.

[0050] Fig. 27 Shown are immunoblot analyses of Sp1 levels after treatment of cells with biotin and biotin-labeled NAR.

[0051] Fig.28 Shown are the results of experiments performed in cell lysate (in vitro) using a cellular thermal shift assay (CETSA) to assess the interaction between NAR and Sp1.

[0052] Fig.29 The structural prediction sliding docking model of compound NAR binding to Sp1 is shown. Compound NAR is represented by blue sticks. The yellow dashed lines depict the three hydrogen bonds predicted for NAR binding to the ASN-81, SER-83, and GLN-110 residues of Sp1.

[0053] Figure 30-31 Shown are the maximum treadmill running distances of middle-aged mice treated with and without Mit-A pretreatment ( Fig.30 ) and grip ( Fig.31 ).

[0054] Fig.32 Shown are the quantitative statistical results of immunofluorescence staining of MyHC type I myofibers in the gastrocnemius muscle of middle-aged mice with and without NAR treatment after Mit-A pretreatment.

[0055] Fig.33 The mRNA expression levels of Myh7, Myh2 and Myh4 in the gastrocnemius muscle of middle-aged mice treated with and without NAR after Mit-A pretreatment are shown. n = 6 per group, 10-month-old C57 mice.

[0056] Fig.34 Shown is the qPCR analysis of ERRγ levels in gastrocnemius muscle of middle-aged mice treated with and without NAR after Mit-A pretreatment. β-Actin was used as an internal control. n = 6 per group, 10-month-old C57 mice.

[0057] Fig.35 The mRNA expression levels of energy metabolism-related genes in the gastrocnemius muscle of middle-aged mice treated with and without NAR after Mit-A pretreatment are shown. n = 6 per group, 10-month-old C57 mice.

[0058] Fig.36 Deletion analysis and mutation analysis of potential transcription factor binding sites (Sp1) on the Esrrg promoter in HEK293T cells are shown.

[0059] Fig.37 The figure shows the changes in cellular response to NAR induction after overexpression of Sp1 with a mutated predicted binding site (Sp1-MT-Pocket1-GLN) in HEK293T cells.

[0060] Fig.38 Immunoblot analysis of the effect of NAR on Sp1 phosphorylation levels is shown. Total Sp1 levels were used as a control. DETAILED DESCRIPTION

[0061] In this disclosure, the singular forms "a", "an", and "the" include plural references, and references to a particular value include at least that particular value unless the context clearly indicates otherwise. Thus, for example, reference to "an additive" is a reference to one or more such compounds known to those skilled in the art and their equivalents, and so on. When values ​​are expressed as approximate values, by using the leading word "about", it should be understood that the particular value forms another embodiment. As used herein, "about X" (where X is a value) preferably refers to ±10% of the stated value, inclusive. For example, the phrase "about 8" preferably refers to values ​​of 7.2 to 8.8, inclusive; as another example, the phrase "about 8%" preferably (but not always) refers to values ​​of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is mentioned, the range mentioned should be interpreted to include ranges of "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1-3 and 5", "2-5", etc. Furthermore, when a list of alternatives is positively provided, such a list may be interpreted to mean that any alternative may be excluded, for example, by a negative limitation in the claim. For example, when a range of "1 to 5" is mentioned, the mentioned range should be interpreted to include any case excluding 1, 2, 3, 4, or 5; thus, a reference to "1 to 5" may be interpreted as "1 and 3-5, but not including 2," or simply "where 2 is not included." It is intended that any component, element, attribute, or step positively mentioned herein may be explicitly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or mentioned separately.

[0062] In the present invention, the terms "subject" and "patient" are used interchangeably. As used herein, the term "patient" refers to an animal, preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, mice, etc.) and a primate (e.g., monkeys and humans), and most ideally a human. In certain embodiments, the subject is a non-human animal, such as a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is a human. In another embodiment, the subject is a human adult. In another embodiment, the subject is a human child. In another embodiment, the subject is a human infant.

[0063] In the present invention, the term "drug" refers to any molecule, compound, method and / or substance used to prevent, treat, manage and / or diagnose a disease or condition. The term "effective dose" as used herein refers to a therapeutic dose sufficient to prevent the development, recurrence or onset of a disease or condition, as well as one or more symptoms thereof, enhance or improve the preventive effect of another therapy, reduce the severity and duration of a disease or condition, alleviate one or more symptoms of a disease or condition, prevent the progression of a disease or condition, cause regression of a disease or condition, and / or enhance or improve the therapeutic effect of another therapy.

[0064] As used herein, the phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0065] In the present invention, the term "therapeutic agent" refers to any molecule, compound and / or substance used to treat and / or manage a disease or disorder.

[0066] In the present invention, the terms "therapy" and "treatment" can refer to any method, composition and / or drug used to prevent, treat and / or manage a disease or disorder, or one or more symptoms thereof. In certain embodiments, the terms "treatment" and "therapy" refer to small molecule therapy.

[0067] As used herein, the terms "treat," "treatment," and "therapy" in the context of administering a therapy to a subject refer to reducing or inhibiting the progression and / or duration of a disease or condition, lessening the severity of a disease or condition (such as cancer), and / or ameliorating one or more symptoms thereof as a result of the administration of one or more therapies.

[0068] In the present invention, the term "excipient" refers to an inactive substance that serves as a carrier or medium for a drug or other active substance. Examples of suitable excipients include, but are not limited to, solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0069] In the present invention, the terms "dihydrochromone" and "chromone" are interchangeable and refer to the same chemical structure. Unless otherwise specified, the compounds described herein are racemic or have no chirality. Naringenin (NAR) or NAR derivatives used in this disclosure are racemic.

[0070] The effects of NAR on skeletal muscle are less studied; existing findings are mainly related to diabetes and obesity. In L6 myotube cells, NAR activates AMPK and directly stimulates muscle glucose uptake in an insulin-independent manner, suggesting that NAR may regulate skeletal muscle glucose homeostasis (Zygmunt et al., 2010). NAR can alleviate palmitic acid and fructose-induced insulin resistance by increasing the expression of GLUT4 in mouse skeletal muscle (Mutlur Krishnamoorthy and Carani Venkatraman, 2017). In L6 skeletal muscle cells, NAR increases glucose uptake in myocytes by activating AMPK and thereby increasing GLUT4 translocation (Bhattacharya et al., 2013; Zygmunt et al., 2010). However, the effects of NAR on muscle endurance and muscle atrophy remain unclear.

[0071] In this study, the inventors of the present invention explored whether NAR can improve muscle function and protect muscle from the aging process or muscle disease. Using young adult mice, naturally aged mice and mdx mice (a preclinical model of DMD) as models, the effects of NAR on exercise capacity and aerobic metabolism levels of skeletal muscle were evaluated. The inventors of the present invention found that NAR increased the number of oxidative muscle fibers, enhanced aerobic respiration in vivo and in vitro, and improved muscle dysfunction in naturally aged and mdx mice. Mechanistically, the inventors of the present invention found that Sp1 is a direct binding target of NAR, and NAR affects skeletal muscle by activating the Sp1-estrogen-related receptor γ (ERRγ) transcriptional axis. The results of the present invention will provide new strategies for improving muscle function and treating muscle atrophy.

[0072] Skeletal muscle function may be impaired by aging, sedentary lifestyle, or disease; however, skeletal muscle remains one of the most under-medicated organs to date. This study found that naringenin (NAR) can improve muscle endurance and ameliorate muscle dysfunction in naturally aged mice and mdx mice by increasing the number of oxidative myofibers and aerobic metabolism. By biotinylated immunoprecipitation mass spectrometry, the transcription factor Sp1 was identified as a direct target of NAR, and its binding site was further verified to be GLN-110. NAR upregulated Esrrg expression by upregulating Sp1 phosphorylation levels and enhancing the binding of Sp1 to the CCCTGCCCTC sequence in the Esrrg promoter. The discovery of the Sp1-ERRγ transcriptional axis is of great significance in basic skeletal muscle research, and the new function of NAR has potential significance for preventing the decline in aerobic capacity associated with a sedentary lifestyle and age- or disease-related muscle atrophy.

[0073] The inventors of the present invention have discovered a new function of NAR in improving skeletal muscle endurance and protecting muscle from atrophy in natural aging and mdx mice by binding to the transcription factor Sp1 and promoting Sp1 phosphorylation. Phosphorylated Sp1 directly upregulates the transcription level of estrogen-related receptor γ (ERRγ), thereby regulating energy metabolism and muscle remodeling.

[0074] Naringenin (NAR) improves muscle dysfunction in aging and mdx mice. NAR increases the number of oxidative muscle fibers and enhances aerobic metabolism. NAR binds to Sp1 to increase its phosphorylation and transcription factor activity. Activation of the Sp1-ERRγ axis promotes energy metabolism and muscle remodeling.

[0075] The inventors of the present invention have further modified naringenin to obtain different derivatives thereof which are expected to provide improved properties.

[0076] The present invention also provides a composition comprising naringenin or its derivatives or derivative compounds, a method for preparing the composition, and a method for using the composition, for example, as a pharmaceutical composition, a functional composition and / or a dietary supplement.

[0077] method

[0078] General Chemistry

[0079] NAR (naringenin) (lot number QY00305-180610, purity ≥ 98%) was purchased from Qingyun Bio Co., Ltd., Nanjing, China. The purity of the naringenin probe (NAR-Biotin) was determined by a Waters ACQUITY UPLC system with an ELSD, PDA detector, sample manager, and binary solvent manager. Preparative HPLC was run on a Varian PrepStar system with an Alltech 2424ELSD and 2489PDA, using a Waters Sunfire RP C18 (5 μm, 30×150 mm) column. Electrospray ionization (ESI)-MS spectra were obtained on a Waters 2695 instrument with a 2998PDA detector coupled to a Waters ACQUITY ELSD and a Waters 3100SQDMS detector, using a Waters Sunfire RP C18 column (4.6×150 mm, 5 μm) at a flow rate of 1.0 mL / min. HRESI-MS was performed on a Waters ACQUITY UPLC system (Waters Corporation Milford, MA, USA) equipped with an ESI ion source. MS detection was performed using a Synapt G2-Si Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE III 500 MHz instrument. Chemical shifts are reported in ppm (δ) and coupling constants (J values) are reported in Hertz. Chemical shifts are reported in Me 4 Si is reported in ppm as a reference standard.

[0080] Synthesis of NAR (naringenin) probe (NAR-biotin)

[0081] NAR (272 mg, 1.0 mmol), biotin (244 mg, 1.1 mmol, 1.1 eq.) and DMAP (12.2 mg, 0.1 mmol, 0.1 eq.) were dissolved in 20 ml of anhydrous DMF. EDCI (310 mg, 2.0 mmol, 2.0 eq.) and Et3N (277 μL, 202 mg, 2.0 eq.) were added. The solution was stirred at room temperature overnight. The reaction was quenched with 2M HCl solution, then diluted with 100 ml of water and extracted with ethyl acetate. The combined organic phase was washed with water, brine, and washed with anhydrous MgSO 4Drying. The organic solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (MeCN containing 0.1% HCCOH in water, 40-60%, 0-35 min) to give the product (300 mg, yield 60%). Purity at 288 nm: 99.93%; 1 HNMR (500 MHz, Py-d 5 )δ is 7.64 and 7.38 (d, J = 8.6 Hz, per 1H), 7.60 and 7.47 (br.s, per 1H), 6.50 and 6.43 (d, J = 2.2 Hz, per 1H), 5.55 (dd, J = 13.1, 3.0 Hz, 1H), 4.58 (m, 1H), 4.42 (m, 1H), 3.26 (m, 1H), 3.19 (dd, J = 1 7.0, 13.1Hz, 1H), 2.98 (dd, J=12.5, 5.0Hz, 1H), 2.91 (m, 1H), 2.89 (dd, J=17.0, 3.0Hz , 1H), 2.56 (t, J=7.4Hz, 2H), 1.85-1.94 (m, 2H), 1.70-1.76 (m, 2H), 1.58-1.63 (m, 2H); 13 C NMR (125 MHz, Py-d 5 )δ is 196.31, 172.51, 169.18, 165.64, 164.82, 164.15, 152.07, 137.40, 128.60, 128.60, 123.05, 123.05, 103.27, 97.94, 96.68, 79.48, 62.97, 61.08, 56.71, 43.85, 41.54, 34.60, 29.54, 29.37 and 25.52; ESI-MS m / z 499.24 [M+H] + , 497.24[MH] + ; C 25 H 27 N 2 O 7 S[M+H] + The HRESI-MS (m / z) value of was calculated as 499.1539, found as 499.1544.

[0082] Mice and cell lines

[0083] HEK293T cells (human embryonic kidney cells, female) and C2C12 cells (mouse mesenchymal progenitor cells, sex unknown) were obtained from the American Type Culture Collection (ATCC). These cells were maintained at 37°C and 5% CO 2C2C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco) and penicillin / streptomycin (Gibco 15070063) with 4.5 g / L (25 mM) glucose under conditions of 10% (Gibco). C2C12 cells were induced to differentiate into myotubes using 2% horse serum (Gibco) for three days. Mice used in this experiment included 10-month-old and 2-month-old C57BL / 6N male mice purchased from Vital River and 4-month-old C57BL / 10ScSnJGpt-Dmdem3Cd4 / Gpt(mdx) male mice purchased from GemPharmatech. Before the start of the experiment, these mice were housed in standard cages, 2-6 animals per group, and housed in a temperature-controlled room (21-23°C) under standardized conditions, maintained on a 12:12 h light-dark cycle, and provided with standard mouse chow and water ad libitum. All animal studies were reviewed and approved by the Institutional Animal Care Committee (IACUC) of the Chinese Academy of Sciences.

[0084] Mouse skeletal muscle intramuscular injection

[0085] The gastrocnemius and tibialis anterior muscles of the mouse hind limbs (only in 2-month-old mice) were treated by intramuscular injection every two days for a total of fifteen injections. The volume of intramuscular injection was 40 μL for the gastrocnemius and 20 μL for the tibialis anterior, and the concentration of NAR was 8 mM, containing 20% ​​DMSO and 80% saline. The vehicle was 20% DMSO and 80% saline. For 10-month-old mice, 50 μM Mit-A (Abcam ab142723) was pre-injected to block the transcriptional activity of Sp1.

[0086] Running experiment

[0087] Two days before the experiment, mice were acclimated to the treadmill by running on a LE8710RTS treadmill test system (Panlab / Harvard Apparatus) at a 15-degree incline and a speed of 15 cm / s for 5 min / day. For the exercise experiment, the speed was increased by 5 cm / s every 5 min until it reached 30 cm / s, and the mice were allowed to run until exhaustion. Then, the total distance the mice ran was calculated.

[0088] Grip test

[0089] Muscle grip force was recorded using the GT3 Grip Tester System (Bioseb, Vitrolles, France). Mice were allowed to grasp the metal grid with four paws and then gently pulled back on their tails until they could no longer hold the grid. The peak pull force was recorded on a digital force sensor. The maximum value of 10 measurements was used to represent the grip force of each mouse.

[0090] Immunofluorescence staining of cryosections

[0091] Gastrocnemius muscles were embedded in the embedding medium OCT, then frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80 °C. Frozen sections (8 μm) of the muscle were fixed in cold acetone (precooled to -20 °C) for 20 min, washed three times with PBS, and permeabilized three times with 0.5% Triton X-100 / PBS (PBST) for 10 min each. After washing, slides were blocked by incubation with 5% BSA for 2 h at room temperature and then incubated with the following antibodies: BA-F8 for MHC type 1 (1:100), BF-F3 for MHC2b (1:100), and SC-71 for MHC-2a (1:100) (Developmental Studies Hybridoma Bank, University of Iowa, USA). Sections were incubated with AlexaFluor 350-, 488-, and 594-conjugated secondary antibodies (1:100) (Invitrogen) for 1 h at room temperature in the dark. Then the cells were observed and photographed under a confocal laser scanning microscope (Carl Zeiss LSM710).

[0092] Oxygen consumption measurement

[0093] Cellular oxygen consumption was measured using the XF24 extracellular flux analyzer from Seahorse Bioscience. C2C12 myotubes were treated with DMSO or 400 μM NAR for 24 h on XF24 V28 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA). For detailed experimental procedures, please refer to the experimental guide (Agilent Seahorse XF CellMito Stress Test Kit).

[0094] RNAi interference and inhibitor treatment experiments

[0095] siRNAs (JST) against mouse Sp1 and Esrrg were transfected into C2C12 cells at a final concentration of 50 nM using Lipofectamine 2000 transfection reagent (Invitrogen 11668019) according to the manufacturer's instructions. Cells were differentiated for 3 days and then treated with or without 400 μM NAR for 24 h. For Sp1 and ERRγ inhibition studies, myotubes were pre-treated with DMSO, 250 nM Mit-A, or 10 μM 4-OHT (MCE, HY-16950) for 2 h. Then, they were treated with or without 400 μM NAR for another 24 h.

[0096] Plasmid preparation, cell transfection, and luciferase reporter gene assay

[0097] The total cDNA extracted from the gastrocnemius muscle tissue of mice was amplified by PCR, and after double restriction digestion, it was transformed with the pcDNA3.1 vector digested with the same endonuclease to obtain the Sp1 overexpression vector.

[0098] According to the mouse Esrrg genome sequence provided by the NCBI database, primers suitable for PCR amplification of Esrrg promoter sequences of different lengths were designed using Primer 5 software. The Esrrg promoter sequence obtained by PCR was double-digested with KpnI / HindIII (NEB) restriction endonucleases, recovered by agarose gel electrophoresis, and ligated with the pGL3-basic vector that was also digested with the same endonucleases. PSE-1518, pSE-1518DEL, pSE-1080, and pSE-129 fluorescent reporter plasmids were obtained using T4 ligase (NEB). pSE+3 and pSE+38 fluorescent reporter plasmids were obtained by circular PCR using pSE-129 plasmid as a template. pSE+3MT fluorescent reporter plasmid was obtained by circular PCR using pSE+3 plasmid as a template. In addition, the mutant Sp1 plasmid vectors Sp1-MT-Pocket1-GLN, Sp1-MT-Pocket2-THR, Sp1-MT-Pocket3-Total and Sp1-MT-Pocket4-GLY were obtained by circular PCR using the Sp1 overexpression plasmid as a template.

[0099] HEK293T cells were seeded into 12-well plates 24 h before the experiment. After 24 h, when the cells grew to 85% density, transfection was started. The pGL3-basic plasmid containing Esrrg promoter fragments of different lengths, the internal reference reporter vector plasmid pRL-TK, and the pcDNA-3.1-Sp1 overexpression vector containing these mutant Sp1 vectors were co-transfected into cells in 12-well plates at a ratio of 20:1:20.

[0100] After 24 h, the plates were pretreated with DMSO or NAR for 12 h, washed twice with PBS, and then dual luciferase reporter gene analysis was performed according to the instructions of the Novozymes dual luciferase assay kit. Promoter activity was expressed in relative luciferase units. The ratio of firefly luciferase to marine luciferase was calculated as the promoter efficiency of the corresponding promoter fragment. The pGL3 vector was used as a negative control.

[0101] Gene expression detection

[0102] Total RNA was extracted from mouse skeletal muscle or C2C12 myotubes using TRIzol reagent (Thermo Scientific 15596018). cDNA was synthesized using HiScript II Reverse Transcriptase (Vazyme R201). Real-time quantitative PCR was performed using SYBR Green Fast qPCR Mix (Genstar A304).

[0103] Western immunoblot analysis

[0104] Protein concentration was assessed using a BCA protein assay kit (Beyotime P0012), and 20–30 μg of protein was used in each SDS-PAGE experiment.

[0105] Chromatin immunoprecipitation analysis

[0106] C2C12 cells were grown in 100 mm culture dishes. After differentiation into myotubes, they were treated with 400 μM NAR for 24 h. The specific experimental steps were carried out according to the kit instructions (Millipore #17-408). The nuclei were sonicated in 500 μl of ultrasonic buffer using an ultrasonic disruptor (4417 detector) (15 s on / 30 s off each time, 18 times, power 9 W) to shear DNA to obtain fragments between 300 and 600 bp in length. Immunoprecipitation (IP) was performed overnight at 4 ° C using 5 μg of anti-Sp1 antibody (Abcam ab227383), 5 μg of anti-histone H3 antibody (Millipore) or 5 μg of normal rabbit IgG antibody (Millipore) and 40 μg of chromatin. DNA was analyzed by real-time PCR targeting the Sp1-specific binding region of the Esrrg promoter.

[0107] Acquisition of NAR interacting proteins

[0108] Two 100 mm dishes of C2C12 myotubes were washed three times with PBS and then lysed in 2 mL of hypotonic lysis buffer (20 mM HEPES, 2 mM EDTA, 2 mM MgCl 2, 1% mixture, pH = 7.4). The cells were scraped off using a cell scraper and lysed on ice for 45 min. Then, centrifuged at 12,000 × g for 15 minutes at 4°C. To reduce false positives, 40 μL of streptavidin magnetic beads (Thermo Scientific #65001) were added to the supernatant and incubated at 4°C with reverse rotation for 2 h. The supernatant was divided into two parts: 4 mM NAR-biotin containing 1% DMSO was added to the treatment group, and 4 mM biotin containing 1% DMSO was added to the control group. Each group was incubated upside down at 4°C overnight, and then 20 μL of streptavidin magnetic beads (ThermoScientific #65001) was added to each group. Each group was incubated upside down at 4°C for 2 hours. The supernatant was discarded, and the magnetic beads were washed three times with three buffers (buffer A: TBS, 0.5% Triton X-100, mixture; buffer B: TBS, 0.1% Triton X-100, mixture; buffer C: TBS, mixture). The resulting bound proteins were subjected to SDS-PAGE, Coomassie brilliant blue staining, and liquid chromatography (LC)-tandem mass spectrometry (MS / MS) analysis.

[0109] LC-MS / MS analysis

[0110] After SDS-PAGE and Coomassie Brilliant Blue staining, the gel bands were destained and digested with trypsin overnight. Then, peptides were extracted in multiple steps using different concentrations of acetonitrile.

[0111] LC-MS / MS analysis was performed on a nanoLC-Q Exactive system. Briefly, the flow rate through the column was set at 0.3 μL / min and the applied distal spray voltage was set at 2.0 kV. Data collection was accomplished by performing a full scan (MW300–1,600) followed by a data-dependent MS2 scan of the 20 most abundant ions following the full MS1 scan.

[0112] The peptide mixture obtained by enzymatic digestion was first analyzed by liquid chromatography-tandem mass spectrometry (LC–MS / MS). Then, the protein search was performed in the UniProt-proteome-mouse (update-20171001) database using the SEQUEST HT search engine of Thermo Proteome Discoverer (2.2.0.388). The search parameters were as follows: trypsin digestion, 2 missed cleavage sites, precursor ion mass error less than 10 ppm, fragment ion mass error less than 20 mDa, alkylation of cysteine ​​as a fixed modification, and oxidation of methionine as a variable modification. The filtering parameters of the search results were as follows: Percolator was used for filtering of spectra, Delta Cn less than 0.1, FDR was set to 1%, high peptide confidence was selected, and FDR at the protein level was also set to 1%. Label-free quantification (LFQ) was performed using the Consensus node with the following parameters: unique+razor; intensity-based precursor abundance; normalization mode, none or total peptide amount; ratio calculation, pairwise ratio-based ratio calculation, maximum allowed FC, high (100). Only proteins with an average ratio (treated group / control group) of more than 2 in all three replicates were selected for further GO analysis using the R language.

[0113] RNA-Seq transcriptome analysis

[0114] Total RNA from DMSO- and NAR-treated C2C12 myotubes was extracted by using TRIzol reagent (Thermo Scientific 15596018). The samples were sent to Biomarker Technologies (Beijing, China) for sequencing. The exon model per kilobase per million mapped reads (FPKM) values ​​of mRNA were used for further analysis. Differential expression analysis between the two groups was performed using DESeq2. Genes with adjusted P values ​​< 0.05 found by DESeq2 were considered differentially expressed. FDR < 0.05 and FC ≥ 1.5 were set as the thresholds for significant differential expression. GO enrichment analysis and KEGG pathway enrichment analysis of up-regulated and down-regulated genes in DEGs were performed using GOseq R package and KOBAS software.

[0115] Sp1 phosphorylation detection

[0116] C2C12 myotubes were treated with or without NAR for 24 h and then lysed in RIPA lysis buffer (GenStar E123-01) containing 1% protease inhibitor cocktail (MCE HY-K0010) and 1% phosphatase inhibitor cocktail (MCE HY-K0023). Cells were scraped using a cell scraper and lysed on ice for 45 min. Proteins were pre-cleared with Protein A / G magnetic beads (Thermo Scientific 88803) at 4°C for 1 h. The pre-cleared supernatant was incubated with Sp1 antibody (Abcam ab227383) at 4°C overnight with gentle shaking. After incubation, Protein A / G magnetic beads (Thermo Scientific 88803) were added and incubated at 4°C for 1 h. The protein-antibody-bead complex was washed three times with lysis buffer for 5 min each at 4°C. The complex was resuspended in loading buffer (Thermo Scientific 39000) and boiled for 10 min before Western blot analysis.

[0117] Cell thermal shift assay CETSA

[0118] C2C12 myotubes were cultured in medium containing DMSO or 400 μM NAR for 48 h. Then, the medium was washed with PBS and the cells were collected. RIPA lysis buffer (GenStar #E121-01) containing 1% protease inhibitor (MCE HY-K0010) was added. The cells were repeatedly frozen and thawed in liquid nitrogen three times and then centrifuged at 12,000 × g for 15 min at 4°C. The supernatant of the DMSO group was divided into two parts. One part was treated with 4 mM NAR for 30 min. The other part of the DMSO-treated group and the 400 μM NAR-treated group were treated with DMSO for 30 min. For each group, 50 μL (1 mg / mL) was separated and lysed at S1000 TM The samples were heated at different temperature gradients in a thermal cycler (Bio-Rad) for 5 min. The samples were then centrifuged at 12,000 × g for 15 min. The supernatant was removed to prepare the samples for electrophoresis.

[0119] Prediction of Sp1 protein structure and ligand binding site

[0120] The transcription factor Sp1 is encoded by the Sp1 gene and belongs to the Sp / KLF family (Vellingiri et al., 2020). Several methods have been used to predict the Sp1 protein structure, but the entire structure has not yet been determined.

[0121] The first method is homology modeling. Homology modeling, also known as comparative modeling, is a commonly used method for structure prediction (Muhammed and Aki-Yalcin, 2019). The goal of homology modeling is to construct the three-dimensional structure of a target protein from its primary amino acid sequence based on an alignment with a known sequence (template) (Bordoli et al., 2009). First, the SWISS-MODEL workspace was used to construct the three-dimensional structure of the Sp1 protein. SWISS-MODEL was developed by the Computational Structural Biology Group at the Swiss Institute of Bioinformatics, SIB, and the Biocenter, University of Basel.

[0122] The I-TASSER server, provided by the Yang Zhang research group at the University of Michigan Medical School, is another tool for automated protein structure prediction. I-TASSER (Iterative Threading ASSEMbly Refinement) is a hierarchical protein structure prediction and structure-based functional annotation method. I-TASSER is also a template-based method that uses a multithreaded approach to identify known structures that have structural similarity to the target protein on a limited number of protein folds (Deng et al., 2016; Roy et al., 2010).

[0123] Once the 3D structure of the Sp1 protein was determined, the next step was to predict the ligand binding site for protein-ligand docking. Roll is a new algorithm for predicting binding sites and has been implemented in a program called POCASA (Yu et al., 2010). In this study, the web server of POCASA 1.1 provided by Hokkaido University in Japan was used to predict the ligand binding site of the Sp1 protein. The parameters of POCASA were set to default; for example, the radius of the probe sphere was set to The size of the unit grid is set to

[0124] Molecular docking

[0125] In this study, molecular docking was used to explore the interaction between small molecule NAR and protein Sp1. First, the structure of Sp1 protein was preprocessed using the Protein Preparation Wizard module, including operations such as adding hydrogen, removing water, and minimizing structural constraints. Then, the predicted Sp1 binding site was imported into the program as a docking grid box for the next step of the program. After that, the possible ionization states and stereoisomers of small molecule NAR were generated at the target pH value of 7.0±1.0 (up to 10 for each ligand) by the LigPrep module. Finally, these preprocessed ligands were docked into the predicted binding site of SP1 protein by the Ligand Docking module. All these modules are included in the Maestro 11.2 software (Schrodinger, LLC: New York, NY, 2010).

[0126] Statistical analysis

[0127] All experiments were performed at least three times. The number of replicates (n) for each animal experiment is indicated in the figure legends. When three or more groups were compared, one-way ANOVA was performed with treatment as an independent factor. Two-tailed Student's t test was used for two groups of measurements. Data are presented as mean ± SEM. ns was considered to be non-significant, *P < 0.05 was considered to be statistically significant, and **P < 0.01 was considered to be statistically significant. All statistical analyses were performed using Excel and GraphPad.

[0128] Experimental results:

[0129] 1. NAR improves muscle endurance and protects against age- or disease-related muscle atrophy.

[0130] In order to study the protective effect of NAR against age-related muscle atrophy, 10-month-old male mice were first used as an experimental model. These mice were randomly divided into a control group and a NAR group and injected intramuscularly into the gastrocnemius muscle. The results showed that NAR significantly increased the running distance of mice ( Figure 1 ), suggesting that it enhances muscle endurance. In addition, NAR also increased grip strength ( Figure 2 ) and gastrocnemius relative weight ( Figure 3 ). Thus, in middle-aged mice at 10 months of age, NAR not only prevented muscle degeneration but also enhanced muscle function.

[0131] The inventors of the present invention next sought to determine whether NAR could improve muscle function in young mice under normal physiological conditions. Two-month-old mice were used and it was found that NAR again increased aerobic endurance capacity (running distance) ( Figure 4 ), but for grip ( Figure 5 ) and the relative weight of the gastrocnemius muscle ( Figure 6Thus, NAR also significantly increased running distance in young mice under normal physiological conditions.

[0132] In addition to age-related muscle atrophy, the effect of NAR on disease-related muscle atrophy was further investigated using mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Consistent with literature reports (Ryu et al., 2016), mdx mice showed a typical phenotype of reduced endurance as well as muscle pseudohypertrophy. NAR increased the running distance of mdx mice ( Figure 7 ) and grip ( Figure 8 ), but did not affect body weight or relative gastrocnemius weight ( Fig. 9 ).

[0133] Overall, NAR may improve muscle endurance and protect against age- or disease-related muscle wasting.

[0134] 2. NAR improves muscle endurance by increasing the number of oxidative muscle fibers and enhancing aerobic metabolism.

[0135] Increased muscle endurance and redder muscle phenotypes suggest that NAR may affect muscle fiber type and energy metabolism. Therefore, we further examined the effects of NAR on different types of muscle fibers in the gastrocnemius of 10-month-old mice, the anterior tibialis and gastrocnemius of 2-month-old mice, and the gastrocnemius of 4-month-old mdx mice. The results showed that NAR upregulated the expression of Myh7, the MHC1 gene corresponding to oxidative muscle fibers, and Myh4, the MHC2b gene corresponding to glycolytic muscle fibers, in the gastrocnemius of 10-month-old mice, but did not upregulate Myh2, the MHC2a gene corresponding to oxidative muscle fibers ( Fig.10 ).

[0136] In addition, immunofluorescence (IF) showed that NAR increased the content of type I oxidative muscle fibers in the gastrocnemius muscle ( Fig.11 ). To further explore the effect of NAR on skeletal muscle energy metabolism, the expression levels of several genes related to energy metabolism were examined. Aerobic metabolism-related genes (Mb, Atp5b, Cycs, Cox5b, and Cox2), β-oxidation-related genes (Cpt1b and Ucp3), and glucose metabolism-related genes (Pdk4) were upregulated by NAR ( Fig.12 ), which is consistent with an increase in the content of oxidative myofibers. Therefore, NAR not only increases the content of myofibers to resist muscle atrophy caused by aging, but also improves muscle endurance by increasing the content of oxidative myofibers in mice.

[0137] Similarly, in 2-month-old mice, NAR increased the expression of Myh7 (corresponding to MHC1) and Myh2 (corresponding to MHC2a) in the gastrocnemius muscle without significantly changing the level of Myh4 (corresponding to MHC2b) ( Fig.13 ). These findings are consistent with the results that NAR had no effect on the grip strength of young mice. Therefore, IF staining showed that there were more type I oxidative muscle fibers in the NAR group than in the control group ( Fig.14 In terms of energy metabolism, NAR upregulated the expression of genes related to aerobic metabolism (Mb, Atp5b, Cycs, Cox5b, and Cox2), β-oxidation (Cpt1b and Ucp3), and glucose metabolism (Pdk4) in the gastrocnemius muscle of young mice ( Fig.15 In addition, NAR upregulated the relative levels of five oxidative phosphorylation (OXPHOS) complexes in mitochondria, including complex I subunit NDUFB8, complex II subunit 30 kDa, complex III subunit Core2, complex IV, and ATP synthase α subunit ( Fig.16 Overall, the results suggest that NAR improves muscle endurance in young mice by increasing the number of oxidative muscle fibers and enhancing aerobic metabolism without affecting glycolytic muscle fibers.

[0138] In 4-month-old mdx mice, the expression levels of Mb, Atp5b, Cox2, Ucp3, and Pdk4 were upregulated in the NAR-treated group ( Fig.17 ), suggesting an increase in aerobic metabolism, which may contribute to the improved muscle function in mdx mice after NAR treatment.

[0139] In summary, NAR improves muscular endurance by increasing the number of oxidative muscle fibers and enhancing aerobic metabolism.

[0140] 3. NAR increased the content of oxidative muscle fibers in C2C12 myotubes and enhanced aerobic metabolism.

[0141] To further explore the cellular mechanism by which NAR regulates skeletal muscle function, C2C12 myoblasts were used. C2C12 cells can differentiate and fuse to form myotubes. C2C12 myotubes were treated with different concentrations of NAR (0, 40, 100, 200, 400, 800, 1600, and 2400 μM) for 24 h. When the NAR concentration exceeded 800 μM, many cells died ( Fig.18 ). Therefore, NAR concentrations below 400 μM were selected to treat cells. The expression of oxidative myofiber-related genes Myh7 and Myh2 was upregulated by NAR in a dose-dependent manner, while the expression of glycolytic myofiber-related gene Myh4 was not ( Fig.19 ), which indicates an increase in the content of oxidative muscle fibers.

[0142] The effects of NAR on cellular energy metabolism were then evaluated. The study found that NAR increased total ATP levels ( Fig. 20 ), indicating that NAR promotes energy production. In addition, the cellular bioenergetic characteristics of C2C12 myotubes treated with or without NAR were measured using the Seahorse Cell Energy Metabolometer. Treatment of myotubes with 200 μM NAR increased the oxygen consumption rate (OCR) ( Fig.21 ). These results confirmed that NAR promoted aerobic respiration of C2C12 cells.

[0143] In addition, NAR enhanced the expression of key enzymes in the oxidative phosphorylation (OXPHOS) process, the tricarboxylic acid cycle (TCA) and β-oxidation ( Fig. 22 and 23 ). Therefore, NAR increases the content of oxidative muscle fibers and enhances aerobic respiration by improving mitochondrial activity in skeletal muscle.

[0144] 4. ERRγ mediates NAR-induced enhancement of oxidative muscle fiber number and aerobic metabolism

[0145] The differentially expressed genes (DEGs) between C2C12 myotubes treated with NAR and those not treated with NAR were analyzed. The results showed that the expression of the nuclear transcription factor ERRγ (encoded by Esrrg) was upregulated by NAR to a more significant extent than other nuclear transcription factors. Previous studies have shown that ERRγ is highly expressed in oxidative muscle fibers and that it can regulate many genes related to metabolism (Misra et al., 2017). Therefore, the ERRγ mRNA levels in C2C12 myotubes treated with NAR were examined. The results showed that NAR increased the levels of ERRγ in a dose-dependent manner ( Fig.24 ). In addition, NAR increased ERRγ transcription levels in 10-month-old mice, 2-month-old mice, and mdx mice in vivo ( Fig.25 ). In vitro and in vivo data suggested that ERRγ may be involved in the function of NAR. Subsequently, to test this hypothesis, ERRγ was knocked down at the cellular level using siRNA. The lack of ERRγ inhibited the NAR-mediated upregulation of oxidative myofiber-related Myh7 and Myh2 and aerobic metabolism-related Atp5b and Cpt1b ( Fig.26 ), indicating that ERRγ mediates the NAR-induced increase in oxidative muscle fiber number and aerobic metabolism.

[0146] In summary, ERRγ mediates NAR-induced increases in oxidative muscle fiber number and aerobic metabolism, thereby improving the endurance exercise capacity of mice.

[0147] 5. Sp1 is a direct binding protein of NAR in C2C12 cells

[0148] In order to further clarify the reason why NAR upregulates ERRγ expression, the inventors of the present invention biotinylated NAR. Differentiated C2C12 cells were lysed, and the lysate was immunoprecipitated with biotin and biotin-labeled NAR. Next, affinity magnetic beads were used to enrich nonspecific proteins bound to biotin and specific proteins bound to NAR-biotin. The two groups of proteins obtained were separated by SDS-PAGE and analyzed by mass spectrometry (MS), and the proteins immunoprecipitated with NAR-biotin were compared with the proteins immunoprecipitated with negative control (NC) biotin to identify differential proteins with a change of more than two times. The results showed that a total of 492 proteins were upregulated more than two times in the NAR-biotin group, of which 207 proteins were only present in the NAR-biotin group but not the NC group. Therefore, molecules of transcription-related proteins that are only present in the biotinylated NAR group were enriched ( Fig. 27 ). The transcription factor Sp1 was only present in the biotinylated NAR group. Therefore, SP1 is most likely a binding protein of NAR.

[0149] To verify the direct interaction between NAR and Sp1, we first used immunoblotting to demonstrate that Sp1 was significantly enriched in biotinylated NAR binding proteins ( Fig. 27 ). Then, the binding affinity of NAR to Sp1 was further analyzed using the cellular thermal shift assay (CETSA), which detects the binding affinity of small molecule compounds and target proteins. The results showed that the solubility curve of Sp1 in the C2C12 myotube cell lysate treated with 4mM NAR for 30 minutes was significantly shifted. In the control group, 50% of the Sp1 protein was degraded at 63°C, while in the cell lysate sample treated with 4mM NAR for 30 minutes, the temperature increased to 69°C. The increased stability of the Sp1 protein in the NAR-treated samples indicates that NAR directly binds to the Sp1 protein. Therefore, the above results indicate that NAR may play a regulatory role in skeletal muscle by directly binding to the transcription factor Sp1 ( Fig.28 ).

[0150] In order to better demonstrate the interaction between NAR and Sp1, the 3D structure of Sp1 and the possible interaction sites of NAR / Sp1 were predicted. Small molecule NAR mainly interacts with Sp1 protein through hydrogen (H-) bonds; more specifically, the two hydroxyl groups in the NAR molecule interact with the ASN-81, SER-83, and GLN-110 residues of Sp1 ( Fig.29 ). The NAR conformation with the highest docking score (-6.584) is most likely the NAR / Sp1-bound combination.

[0151] 6. Sp1 is involved in NAR-mediated ERRγ upregulation and improvement of muscle function

[0152] In order to determine whether Sp1 is involved in the function of NAR in skeletal muscle, the effects of NAR on skeletal muscle function and metabolism were first examined at the animal level using Sp1 inhibitors. When mithramycin A (Mit-A), a specific Sp1 inhibitor, was used to inhibit the transcriptional activity of Sp1 in the gastrocnemius muscle of middle-aged mice, the inventors of the present invention found that the enhancing effect of NAR on aerobic exercise distance and grip strength was inhibited ( Fig.30 and 31 The increase in the number of oxidative muscle fibers induced by NAR ( Fig.32 ) and upregulation of oxidative muscle fiber-related genes Myh7 and Myh2 ( Fig.33 ) was also inhibited. In addition, NAR-mediated ERRγ upregulation was inhibited by Mit-A at the RNA level ( Fig.34 In addition, the upregulation of ERRγ downstream genes related to oxidative muscle fiber number or aerobic metabolism, including myoglobin, Atp5b, Cycs, Cox5b, Cpt1b, and Ucp3, was also suppressed ( Fig.35 ). These results suggest that NAR-induced increases in endurance and grip strength in 10-month-old mice are mediated by the transcription factor Sp1 in vivo.

[0153] 7. NAR enhances the interaction between Sp1 and Esrrg promoter by increasing Sp1 phosphorylation

[0154] The above results show that Sp1 mediates NAR-induced ERRγ upregulation, so the next step is to determine how NAR regulates ERRγ expression through Sp1. Through sequence comparison and prediction, the inventors found that this region of the Esrrg promoter contains a sequence (CCCTGCCCTC) that may be bound by Sp1. In order to determine whether the binding of Sp1 to the Esrrg promoter responds to NAR induction, the different effects of NAR on the control vector, the fluorescent reporter vector containing the +3 to +100 region of the Esrrg promoter, and the mutant reporter vector were measured. The results showed that the response of the fluorescent reporter vector pSE+3 to NAR was 4.5 times, which was much higher than the 1.7 times of the control and the 2.5 times of the mutant ( Fig.36 ).

[0155] The inventors of the present invention obtained the possible binding site of NAR and Sp1. Therefore, a fluorescent reporter vector containing the mutated amino acids in the Esrrg promoter +3 to +100 region and the Sp1 vector binding site was used to verify the true binding site of NAR and Sp1. The results showed that the NAR response of the fluorescent reporter vector pSE+3 to the unmutated Sp1 vector was 4.5 times, which was much higher than the 1.7-fold response of the Sp1-MT-Pocket1-GLN vector ( Fig.37). Mutation of GLN-110 in pocket 1 of Sp1 disrupts NAR binding, indicating that NAR interacts with Sp1 protein by forming hydrogen bonds with GLN-110 of Sp1 in pocket 1. The mechanism by which Sp1 directly binds to NAR to increase Sp1 transcriptional activity is still unclear. Recent data indicate that the phosphorylation state of Sp1 plays an important role in regulating multiple genes (Chu, 2012; Tan and Khachigian, 2009). The post-translational phosphorylation level of Sp1 after NAR treatment was determined using immunoprecipitation (IP). The results showed that the phosphorylation of Sp1 increased after NAR binding ( Fig.38 ), which leads to an increase in Sp1 transcriptional activity.

[0156] 8. Reconstruction and synthesis of NAR structure: Improving the water solubility, activity and medicinal properties of NAR through structural modification

[0157] Based on the results of the study of NAR, the structure of NAR was further modified to obtain different derivatives. The goals of these modifications include improving water solubility, enhancing biological or therapeutic activity, and medicinal properties. These derivatives will be used to improve skeletal muscle endurance, treat muscle atrophy or malnutrition, or prevent muscle atrophy or malnutrition in subjects who need such treatment. A composition comprising at least one derivative is obtained. In the present invention, the new compound includes a series of derivatives as described herein.

[0158] Muscle atrophy and dysfunction are major problems in life and health, and finding solutions has always been a research pursuit. In this study, new functions of NAR were discovered. NAR, including its derivatives, improved muscle endurance in the natural aging process and mdx mice and protected them from muscle atrophy by increasing the number of oxidative muscle fibers and enhancing aerobic respiration. Importantly, the transcription factor Sp1 was identified as a direct binding target of NAR. Binding of NAR increased the phosphorylation and transcription factor activity of Sp1. In addition, Sp1 was found to be a new transcription factor of Esrrg, and the Sp1-ERRγ transcriptional axis was found to be involved in NAR-mediated energy metabolism and muscle remodeling. These findings have important implications for preventing the reduction of aerobic exercise capacity and muscle atrophy associated with a sedentary lifestyle and age, and for treating DMD-related muscle weakness.

[0159] New functions and implications of NAR in skeletal muscle in three animal models:

[0160] The reduction of muscle mass and strength with age is a problem that cannot be ignored. Some studies have reported that muscle mass decreases by 3% to 8% every 10 years after the age of 30 (Volpi et al., 2004). Skeletal muscle atrophy and decreased exercise tolerance are the main manifestations of aging, mainly manifested as a reduction in muscle mass and mitochondrial aerobic respiration (Brunner et al., 2007; Carter et al., 2015; Klitgaard et al., 1990; Lanza et al., 2005; Murgia et al., 2017; Short et al., 2005). The inventors used naturally aged 10-month-old mice as a model to demonstrate that NAR can improve endurance and grip and reverse muscle atrophy to a certain extent by increasing the number of oxidative muscle fibers and overall aerobic metabolic capacity. Since muscle atrophy caused by natural aging is the most common type of muscle loss, the inventors first used a naturally aged mouse model to demonstrate the role of NAR in improving muscle function. Against the backdrop of the increasingly serious global aging problem, NAR has great prospects for being widely used to improve or treat muscle atrophy. Even in young adult mouse models, NAR significantly increased running distance, the formation of oxidative muscle fibers, and the expression of key enzymes in the skeletal muscle OXPHOS process. This suggests that NAR can enhance muscle aerobic capacity under normal physiological conditions, suggesting the possibility that it may be applied to endurance athletes such as long-distance runners.

[0161] In addition, this new function of NAR has potential application value for modern youth, in which lack of exercise or sedentary lifestyle leads to reduced skeletal muscle metabolism and increased risk of metabolic diseases such as obesity and diabetes. Notably, NAR also improved endurance and grip strength in mdx muscular dystrophy mice. Although it did not significantly affect the type or content of muscle fibers, NAR's promotion of aerobic metabolism may explain this phenomenon to some extent. In summary, although the mechanism is not fully understood, NAR also expands the potential application of NAR in the treatment of muscle diseases by improving the mdx phenotype. Collectively, these findings suggest that the role of NAR in improving muscle function will benefit multiple populations.

[0162] Sp1, identified as a novel direct target of NARs, mediates NAR function in skeletal muscle:

[0163] Although the various biological activities of NAR in different diseases have been widely studied, there is a lack of research on its direct targets, which greatly limits the application of NAR. To date, only two studies have explored the direct targets of NAR. Collapsin response mediator protein 2 (CRMP2) has been identified as a candidate that directly binds to NAR to explain the protective effect of NAR in Alzheimer's disease (AD) (Yang et al., 2017). Another study showed that NAR directly binds to the PPARα ligand binding domain (LBD) region and activates PPARα through a dual luciferase reporter system, thereby reducing very low density lipoprotein (VLDL) levels and reducing lipid accumulation in the liver (Goldwasser et al., 2011). However, whether Pparα is a direct target of NAR has not been confirmed by additional evidence.

[0164] In the present invention, the inventors of the present invention determined by IP-MS that Sp1 is a direct binding protein of NAR, and verified the binding affinity of NAR to Sp1 by performing CETSA in muscle cells. The activity of Sp1 is regulated by post-translational modifications, such as phosphorylation, glycosylation and acetylation, among which phosphorylation is the most intensively studied. The study found that NAR activates the activity of Sp1 transcription factor by promoting the phosphorylation of Sp1, revealing the mechanism by which NAR regulates Sp1. This is the first time that the direct target of NAR in the regulation of skeletal muscle fiber type and energy metabolism has been determined.

[0165] Sp1 is a novel transcription factor for ERRγ:

[0166] ERRγ is an orphan nuclear estrogen receptor with ligand-independent transcriptional activity. It plays an important role in pathological conditions such as insulin resistance, alcoholic liver damage, and cardiac hypertrophy, and regulates energy metabolism in the heart, skeletal muscle, and pancreatic β cells (Misra et al., 2017). Based on the importance of ERRγ in metabolic homeostasis, ERRγ is a good target for the treatment of metabolic diseases. Various cellular stresses can induce the expression of ERRγ through membrane receptors or intracellular transcription factors.

[0167] In addition, ERRγ expression can be regulated by transcription factors c-Jun, Stat3, CREB, HIF1a, and ATF6a under external stimulation (Misra et al., 2017). In the study, Sp1 was identified as a new transcription factor of ERRγ, providing a new mechanism for the regulation of ERRγ expression. The binding of NAR to Sp1 further promoted the binding of Sp1 to the ERRγ promoter. The establishment of the Sp1-ERRγ axis not only reveals the mechanism of NAR regulating muscle function, but also provides a new signaling pathway for optimizing strategies to improve muscle function.

[0168] In this study, the present invention found that NAR enhances the transcription factor activity of Sp1 by promoting the phosphorylation of Sp1. However, the specific phosphorylation site has not been verified, and how NAR affects the phosphorylation of Sp1 remains unclear. In addition, the relatively low solubility of NAR may limit its effectiveness in animals. Improving the water solubility of NAR will help promote the application of NAR.

[0169] As described herein, the present invention demonstrates for the first time that NAR can improve the endurance and aerobic capacity of skeletal muscle in young mice, protect middle-aged mice from muscle atrophy, and alleviate DMD. In addition, it identifies Sp1 as a new direct target of NAR and establishes a new relationship between Sp1 and the target gene ERR, thereby clarifying the molecular mechanism of how NAR upregulates ERR expression by binding to Sp1 to enhance the interaction between Sp1 and the ERR promoter. Therefore, this study opens up new avenues for the application of NAR and provides a new and safer strategy for improving muscle function and protecting against muscle atrophy caused by aging and disease. NAR derivatives are synthesized with the aim of mimicking NAR or further improving its performance.

[0170] In one aspect, the present disclosure provides a composition for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment. This composition comprises an effective amount of a compound of formula (I) as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient,

[0171] The chemical structure of formula (I) is as follows:

[0172]

[0173] In formula (I), R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2 , C1-C6 alkyl, C1-C6 alkoxy and phenyl. 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH and NH 2 Such a compound is naringenin (NAR) or a naringenin derivative.

[0174] In the naringenin derivatives provided herein, the benzene ring connected to the dihydrochromone is substituted by at least one substituent and at most four substituents R1 , R 2 , R 3 and R 4 In certain embodiments, R 1 , R 2 , R 3 and R 4 One or two of are substituents other than H. For example, in certain embodiments, R 1 or R 2 is a substituent group other than H, R 3 =H, and R 4 =H. When the naringenin derivative contains only one substituent group (R 1 When the compound is represented by (II), it has the chemical structure of formula (II):

[0175]

[0176] The only substituent group (consisting of R 1 The compound may have a chemical structure of formula (III) or (IV):

[0177]

[0178] In the compositions provided herein, examples of suitable compounds include, but are not limited to, the following compounds:

[0179]

[0180]

[0181] In the above compounds, the sample number includes the compound number and the laboratory compound code in parentheses.

[0182] The composition can be a pharmaceutical composition, a functional composition and / or a dietary supplement. For example, the composition is an injectable or oral pharmaceutical composition. The composition is preferably injectable. The concentration of the compound can be in the range of 1-50mM, such as 2-15mM, 5-10mM, or any other appropriate concentration. Examples of appropriate concentrations include, but are not limited to, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM 47mM, 48mM, 49mM, 50mM and any other value between any two of these values.

[0183] The excipient can be a solvent, a cosolvent, a colorant, a preservative, an antibacterial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent or any combination thereof. For example, in an injectable composition, the excipient includes a carrier composed of 20% DMSO and 80% saline by weight. In certain embodiments, these compositions can be administered together with beverages, food or related ingredients.

[0184] In another aspect, the present disclosure provides a compound having formula (I) as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof. Such a compound is a naringenin derivative other than naringenin (NAR).

[0185] The compound may be a suitable compound having desired solubility and pharmacological properties. In certain embodiments, the compound is a compound of formula (I) or a pharmaceutically acceptable solvate thereof.

[0186] The present invention provides any genus or species of compound as described herein, for example having formula (II), (III) or (IV). In certain embodiments, R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2 , C1-C6 alkyl, C1-C6 alkoxy and phenyl. At least one R 1 , R 2 , R 3and R 4 is a substituent other than H. Such a compound is a naringenin derivative. In certain embodiments, R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH and NH 2 Composed of groups.

[0187] In certain embodiments, R 1 , R 2 , R 3 and R 4 One or both of are substituents other than H. For example, in certain embodiments, R 1 or R 2 is a substituent other than H, R 3 =H, and R 4 =H.

[0188] Examples of compounds suitable as naringenin derivatives include, but are not limited to, the examples of compounds described herein, such as Compound Nos. 1-13.

[0189] In another aspect, the present disclosure provides a method for preparing a composition or compound described herein. The method may include preparing a compound. The method may further include mixing an excipient and the compound.

[0190] In another aspect, the present disclosure provides a method for preparing the compound, which is a naringenin derivative as described herein. In certain embodiments, the method comprises the following steps: reacting 1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethanone with substituted p-bis(methoxymethoxy)benzaldehyde, as described below.

[0191] On the other hand, the present disclosure provides a method for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment. The method includes administering an appropriate amount of a composition as described herein to a subject in need of such treatment. The composition comprises an effective amount of a compound of formula (I). The present disclosure also provides a method for preparing a drug for treating any of these medical conditions using a compound of formula (I), such as NAR and NAR derivatives.

[0192] In some embodiments, in the compound of formula (I), R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH, NH 2 、NO 2, C1-C6 alkyl, C1-C6 alkoxy and phenyl. At least one R 1 , R 2 , R 3 and R 4 is a substituent other than H. Such a compound is naringenin or a naringenin derivative. In certain embodiments, each R 1 , R 2 , R 3 and R 4 Each is selected from H, F, Cl, Br, OH and NH 2 Composed of groups.

[0193] In some embodiments, R 1 , R 2 , R 3 and R 4 One or both of are substituents other than H. For example, in certain embodiments, R 1 or R 2 is a substituent other than H, R 3 =H, and R 4 =H.

[0194] In some embodiments, the subject is a mammal, preferably a human subject, and can be a healthy human, or an adult with age- or disease-related muscle wasting.

[0195] In some embodiments, the composition is administered by intramuscular injection or oral administration. Preferably, intramuscular injection is used. In certain embodiments, the composition is injected intramuscularly with an effective dose of the compound, and the dosage range is 2 mg / Kg to 20 mg / Kg, and the frequency is once a day or once every other day. Any appropriate dosage can be used for administration. For example, in certain embodiments, the dosage range of the effective dose of the compound is 3.6 mg / Kg to 7.6 mg / Kg. Examples of suitable doses of the compound of an effective dose include, but are not limited to, 2 mg / Kg, 3 mg / Kg, 3.5 mg / Kg, 4 mg / Kg, 4.5 mg / Kg, 5 mg / Kg, 5.5 mg / Kg, 6 mg / Kg, 6.5 mg / Kg, 7 mg / Kg, 7.5 mg / Kg, 8 mg / Kg, 9 mg / Kg, 10 mg / Kg, 11 mg / Kg, 12 mg / Kg, 13 mg / Kg, 14 mg / Kg, 15 mg / Kg, 16 mg / Kg, 17 mg / Kg, 18 mg / Kg, 19 mg / Kg and 20 mg / Kg. These doses are calculated based on the data obtained so far.

[0196] Examples of compounds:

[0197] The following examples, including synthetic processes, are for illustration only and do not limit the scope of the compounds provided by the present disclosure.

[0198] Compounds 1-13 have been prepared and evaluated. Their structural comparisons with naringenin (abbreviated as NAR, laboratory compound code S12) are summarized in Table 1. The label compound codes of compounds 1-13 are also shown in Table 1.

[0199]

[0200] Table 1

[0201]

[0202] General procedure for the synthesis of 2'-hydroxyacetophenone:

[0203] 2'-Hydroxyacetophenone was synthesized according to Scheme (A) and the steps described below:

[0204]

[0205] 2', 4', 6'-trihydroxyacetophenone (14.74g, 87.68mmol, 1.0eq.) was suspended in dichloromethane (180mL) and stirred under an argon atmosphere. N, N-diisopropylethylamine (DIPEA, 46.05mL, 263.05mmol, 3.0eq.) was added at 0°C, and then bromomethyl methyl ether (MOMBr, 15.75mL, 192.91mmol, 2.2eq.) was added dropwise to the reaction mixture at 0°C. The reaction mixture was stirred for 4 hours at 0°C. The reaction mixture was quenched with 200mL of water, and then the aqueous solution was extracted with dichloromethane (3×120mL). The organic solution was combined and dried (Na 2 SO 4 ), filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE / EtOAc=15 / 1-10 / 1) to give 1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)-1-ethanone (8.4 g, 37.39%). 1 HNMR (400MHz, chloroform-d) δ is 13.71 (s, 1H), 6.26-6.20 (m, 2H), 5.24 (s, 2H), 5.15 (s, 2H), 3.50 (s, 3H), 3.45 (s, 3H) and 2.63 (s, 3H). Other phenolic hydroxyl groups need to be protected by MOM protecting groups and can be synthesized by this method.

[0206] General procedure for the synthesis of 6-hydroxy-[1,1'-biphenyl]-3-carbaldehyde:

[0207] 6-Hydroxy-[1,1'-biphenyl]-3-carbaldehyde was synthesized according to Scheme (B) and the following steps:

[0208]

[0209] 3-Bromo-4-hydroxybenzaldehyde (1.0 g, 5 mmol, 1.0 eq.) was added by conventional MOM protecting group method and purified by silica gel column chromatography (PE / EtOAc=30:1-20:1) to obtain 400 mg of 3-bromo-4-(methoxymethoxy)benzaldehyde with a yield of 32.79%. 3-Bromo-4-(methoxymethoxy)benzaldehyde (400 mg, 1.63 mmol, 1.0 eq.), phenylboronic acid (298.52 mg, 2.45 mmol, 1.5 eq.) and PdCl were added to the stirred solution. 2 (dppf) (59.71 mg, 0.081 mmol, 5 mol%) was reacted in 1,4-dioxane (5.0 mL) at 60°C under argon atmosphere for 24 hours. The solvent was then removed in vacuo. The residue was purified by silica gel column chromatography (PE / EtOAc=40:1-30:1) to give 6-(methoxymethoxy)-[1,1'-biphenyl]-3-carbaldehyde (231 mg, 58.42%). The structure formation was confirmed by LC-ESIMS and found to be m / z 243.2 [M+H] + .

[0210] General steps for synthesizing naringenin derivatives (i.e., the compounds):

[0211] Compounds as naringenin derivatives were synthesized according to Scheme (C), wherein conditions (a) and (b) are alternative:

[0212]

[0213] Taking compound 1 (laboratory compound code: NAR-27) as an example, condition (a) in the synthesis method is 2-(3-fluoro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone.

[0214] Compound 1 (NAR-27) was synthesized by the following scheme (D):

[0215]

[0216] 3-Fluoro-4-hydroxybenzaldehyde (154mg, 1.0mmol, 1.0eq.) was added to a 50mL eggplant-shaped flask equipped with a stirring rod, which contained acetone (10mL), potassium carbonate (276.4mg, 2.0mmol, 2.0eq.) and bromomethyl methyl ether (90μL, 1.1mmol, 1.1eq.). The mixture was heated to reflux for 3 hours. After the reaction was complete, methanol was added to quench the reaction, and a large amount of organic phase was removed by rotary evaporation, and then 20mL of water was added, and then extracted three times with ethyl acetate (20mL). The organic phases were combined, washed with salt water, dried over anhydrous sodium sulfate, and then concentrated in a vacuum to obtain a brown oily liquid. Directly proceed to the next step without treatment.

[0217] In a 50mL eggplant-shaped flask, add KOH (1.0g, 17.8mmol), the unpurified crude product obtained in the previous step and 2-hydroxy-4,6-bis (methoxymethoxy)-1-acetylphenone (256mg, 1.0mmol, 1.0eq.). Then add 20mL of ethanol and 2mL of water, stir at room temperature for 18h. Add 2M HCl solution so that the pH value is adjusted to about 7.5-8 weak alkalinity. Extract three times with ethyl acetate (50mL). Combine organic phases, wash with saturated brine, use anhydrous sodium sulfate drying, then dry and remove solvent under vacuum. The crude product was purified by silica gel column chromatography (PE / EtOAc=15:1-10:1) to obtain 3-(3-fluoro-4-(methoxymethoxy)phenyl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)prop-2-en-1-one as a yellow solid with a mass of 100.0 mg and a total yield of 26.45% for two steps.

[0218] The chalcone obtained in the previous step was placed in a 50 mL eggplant-shaped flask. 3.0 mL of 2 M HCl solution and 10.0 mL of methanol were added, and then heated to reflux for 24 h. The ring-closed product UV had a characteristic absorption peak at 330-360 nm, and the ring-opened product UV had a characteristic absorption peak at 280 nm. After the reaction, an appropriate amount of saturated NaHCO 3 The excess HCl was neutralized and extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography (PE / EtOAc = 5:1) to obtain 2-(3-fluoro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (NAR-27) as a pale yellowish white solid (14.19 mg) with a yield of 21.54%. MF is C 15 H 11 FO 5 , MW is 290.06.1 H NMR (400 MHz, DMSO-d 6 ) δ is 12.13 (s, 1H), 10.81 (s, 1H), 10.06 (s, 1H), 7.33 (dd, J=12.3, 2.1Hz, 1H), 7.13 (dd, J=8.2, 2.1Hz, 1H), 6.97 (t, J=8. 7Hz, 1H), 5.92-5.86 (m, 2H), 5.46 (dd, J=12.8, 3.0Hz, 1H), 3.27 (dd, J=17.1, 12.9Hz, 1H) and 2.70 (dd, J=17.1, 3.0Hz, 1H); 13 C NMR (200 MHz, acetone-d 6 )δ is 196.8, 167.5, 165.0, 164.1, 152.7, 151.5, 146.1, 131.8, 123.9, 118.5, 103.0, 96.8, 95.9, 79.2 and 43.4. 15 H 12 FO 5 + [M+H] + HRMS (ESI) m / z calcd. 291.0663, found 291.0667.

[0219] 2-(3,5-difluoro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 2, NAR-28)

[0220] Compound 2 (NAR-28) was synthesized by the following scheme (E):

[0221]

[0222] The synthesis method is the same as the method of condition (a) above. The product is compound 2 (NAR-28), which is a light yellow solid (38.66 mg, 12.54%); 1 H NMR (500 MHz, acetone-d 6 )δ is 11.71 (s, 2H), 9.23 (s, 1H), 7.34-7.23 (m, 4H), 5.93 (s, 2H), 3.39 (dd, J=8.3, 7.1 Hz, 2H) and 3.01-2.94 (m, 2H). 13 C NMR (150 MHz, acetone-d 6)δ is 196.44, 167.55, 164.93, 163.80, 154.01 (d, J = 7.0 Hz), 152.41 (d, J = 7.0 Hz), 134.83 (t, J = 17.1 Hz), 131.04 (t, J = 6.1 Hz), 110.88 (dd, J = 17.1, 6.1 Hz), 102.97, 96.97, 95.96, 78.63 and 43.29. C 15 H 11 F 2 O 5 + [M+H] + HRMS (ESI) m / z calcd. 293.0575, found 293.0577.

[0223] 2-(3-Chloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 3, NAR-29) Compound 3 (NAR-29) was synthesized by the following scheme (F):

[0224]

[0225] The synthesis method is the same as the method of condition (a) above. The obtained compound 2 (NAR-29) is a light yellow solid (13.98 mg, 4.56%); 1 H NMR (400 MHz, DMSO-d 6 ) δ is 12.13 (s, 1H), 10.83 (s, 1H), 10.39 (s, 1H), 7.50 (d, J=2.1Hz, 1H), 7.29 (dd, J=8.5, 2.2Hz, 1H), 6.99 (d, J= 8.4Hz, 1H), 5.92-5.86 (m, 2H), 5.46 (dd, J=13.0, 3.0Hz, 1H), 3.31-3.23 (m, 1H) and 2.70 (dd, J=17.2, 3.1Hz, 1H); 13 CNMR (200 MHz, acetone-d 6 )δ is 196.8, 167.5, 164.9, 164.1, 154.2, 132.2, 129.2, 127.3, 121.1, 117.4, 103.0, 96.8, 95.8, 79.1 and 43.4. C 15 H 12 C1O 5 + [M+H] + HRMS (ESI) m / z calcd. 307.0368, found 307.0367.

[0226] 2-(3,5-dichloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 4, NAR-30)

[0227] Compound 4 (NAR-30) was synthesized by the following scheme (G):

[0228]

[0229] The synthesis method of NAR-30 is a general procedure using the method of condition (b) above.

[0230] To a solution of 3,5-dichloro-4-hydroxybenzaldehyde (191 mg, 1.0 mmol) and DIPEA (522 μL, 3.0 mmol, 3.0 eq.) in dry ethylene (10 mL), MOMBr (183 μL, 2.2 mmol, 2.2 eq.) was added at 0°C under argon. The reaction mixture was stirred at 0°C for 4 h. The reaction was terminated by adding 100 mL of water and then extracted three times with ethylene (50 mL). The organic phases were combined, washed with brine, and washed with anhydrous MgSO 4 Drying and then evaporation under reduced pressure gave the crude bis-MOM protected intermediate.

[0231] To a solution of 2-hydroxy-4,6-bis(methoxymethoxy)-1-acetophenone (256 mg, 1.0 mmol, 1.0 eq.) in dry THF (10 mL), NaH (60% dispersion in paraffin oil) (80.0 mg, 2.0 mmol, 2.0 eq.) was added portionwise at 0°C under nitrogen atmosphere and vigorous stirring. 2 When the generation stops, the solution of the above-mentioned two MOM protected intermediates in dry THF (5.0 mL) is added dropwise to the reaction mixture for 15 min, and then the reaction mixture is stirred at room temperature for 2 h, unless otherwise stated. After the reaction is completed, the reaction is slowly quenched with ice water, extracted three times with ethyl acetate (20 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and removed in vacuo. The crude product is purified by silica gel column chromatography (PE / EtOAc=15:1-10:1) to obtain 378 mg of yellow solid as 3-(4-trifluoromethylphenyl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)prop-2-ene-1-one, with a yield of 88.30%.

[0232] Put all the chalcone obtained in the previous step into a 50mL eggplant bottle, add 10.0mL methanol and 3.0mL 2M HCl solution, and heat to reflux for 24h. Monitor by TLC and UpLC (the cyclization product has a characteristic absorption peak at 330-360nm UV, and the ring-opening product has a characteristic absorption peak at 280nm UV). After the reaction is completed, add an appropriate amount of saturated NaHCO3 The excess HCl was neutralized and extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo.

[0233] The crude product was purified by silica gel column chromatography (PE / EtOAc=8:1-5:1) to give 2-(3,5-dichloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (NAR-30) as a Type A pale yellow solid (56.78 mg, 16.67%); 1 HNMR (500 MHz, acetone-d 6 )δ is 7.55 (s, 2H), 6.01 (d, J=2.2 Hz, 1H), 5.97 (d, J=2.2 Hz, 1H), 5.50 (dd, J=12.9, 3.1 Hz, 1H), 3.18 (dd, J=17.1, 12.9 Hz, 1H) and 2.82 (dd, J=17.1, 3.1 Hz, 1H). 13 C NMR (125 MHz, acetone-d 6 )δ is 196.4, 167.7, 165.0, 163.8, 150.3, 132.9, 127.7, 122.8, 102.9, 97.0, 96.0, 78.5 and 43.3. 15 H 11 Cl 2 O 5 + [M+H] + HRMS (ESI) m / z calcd. 340.9978, found 340.9981.

[0234] 2-(3-Bromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 5, NAR-31)

[0235] Compound 5 (NAR-31) was synthesized by the following scheme (H):

[0236]

[0237] The synthesis method is the same as the method of condition (b) above. The product is compound 5 (NAR-31), which is a light yellow solid (63.78 mg, 18.16%); 1 H NMR (400 MHz, acetone-d 6) δ is 12.16 (s, 1H), 9.37 (s, 2H), 7.73 (d, J = 2.1Hz, 1H), 7.41 (dd, J = 8.4, 2.2Hz, 1H), 7.07 (d, J = 8.4Hz, 1H), 6. 01-5.94 (m, 2H), 5.49 (dd, J=12.9, 3.0Hz, 1H), 3.20 (dd, J=17.1, 12.9Hz, 1H) and 2.78 (dd, J=17.1, 3.0Hz, 1H); 13 C NMR (125 MHz, acetone-d 6 )δ is 196.9, 167.4, 165.2, 164.1, 155.2, 132.8, 132.4, 128.1, 117.2, 110.3, 103.1, 96.9, 95.9, 79.1 and 43.4. C 15 H 12 B O 5 + [M+H] + HRMS (ESI) m / z calcd. 350.9863, found 350.9861.

[0238] 2-(3,5-dibromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 6, NAR-32)

[0239] Compound 6 (NAR-32) was synthesized by the following scheme (I):

[0240]

[0241] The synthesis method is the same as the method of condition (b) above. The obtained compound 6 (NAR-32) is a light yellow solid (90.21 mg, 20.98%); 1 H NMR (400 MHz, DMSO-d 6 )δ is 12.12 (s, H), 10.87 (s, 1H), 10.17 (s, 1H), 7.73 (s, 2H), 5.96-5.89 (m, 2H), 5.50 ( dd, J=13.0, 2.9Hz, 1H), 3.33 (dd, J=17.1, 13.0Hz, 1H) and 2.75 (dd, J=17.1, 3.0Hz, 1H); 13 C NMR (125 MHz, acetone-d 6 )δ is 196.4, 167.4, 165.2, 164.9, 163.8, 151.8, 134.3, 131.5, 111.5, 103.0, 97.0, 96.9, 78.2 and 43.3. 15 H11 Br 2 O 5 + [M+H] + HRMS (ESI) m / z calcd. 428.8968, found 428.8972.

[0242] 5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-dihydrochromone (Compound 7, NAR-33)

[0243] Compound (NAR-33) was synthesized by the following scheme (J):

[0244]

[0245] The synthesis method is the same as the method of condition (b) above. The obtained compound 7 (NAR-33) is a pale white solid (102.6 mg, 34.47%); 1 H NMR (400 MHz, DMSO-d 6 ) δ is 12.15 (s, 1H), 10.78 (s, 1H), 9.13 (s, 1H), 7.09 (d, J=2.0Hz, 1H), 6.90 (dd, J=8.2, 2.0Hz, 1H), 6.79 (d, J= 8.2Hz, 1H), 5.89 (q, J=2.1Hz, 2H), 5.43 (dd, J=12.8, 2.9Hz, 1H), 3.78 (s, 3H) and 2.68 (dd, J=17.1, 3.0Hz, 1H); 13 CNMR (125 MHz, acetone) δ is 197.2, 167.5, 165.0, 164.3, 148.4, 147.9, 131.2, 120.5, 115.7, 111.2, 103.0, 96.7, 95.8, 80.2, 56.3 and 43.6. 16 H 15 O 6 + [M+H] + HRMS (ESI) m / z calcd. 303.0863, found 303.0873.

[0246] 5,7-Dihydroxy-2-(4-hydroxy-3-nitrophenyl)-4-dihydrochromone (Compound 8, NAR-34)

[0247] Compound 8 (NAR-34) was synthesized by the following scheme (K):

[0248]

[0249] The synthesis method is the same as the method of condition (b) above. The obtained compound 8 (NAR-34) is a pale white solid (8.47 mg, 1.89%); 1 H NMR (400 MHz, DMSO-d 6 ) δ is 12.11 (s, 1H), 8.03 (s, 1H), 7.69 (d, J = 8.9Hz, 1H), 7.17 (d, J = 8.6Hz, 1H), 5. 91 (d, J=7.9Hz, 2H), 5.58 (dd, J=12.9, 2.9Hz, 1H) and 2.76 (dd, J=17.1, 3.1Hz, 1H); 13 C NMR (125 MHz, acetone-d 6 )δ was 195.9, 166.8, 163.5, 162.6, 152.6, 136.6, 133.5, 129.5, 123.7, 119.5, 101.7, 96.1, 95.1, 77.3 and 41.8. LC-ESIMS found m / z 318.2 [M+H] + and m / z 316.2 [MH] - .

[0250] 5,7-Dihydroxy-2-(6-hydroxy-[1,1-biphenyl]-3-yl)-4-dihydrochromone (Compound 9, NAR-35)

[0251] Compound 9 (NAR-35) was synthesized by the following scheme (L):

[0252]

[0253] The synthesis method was the same as the method under the above condition (b). Compound 9 (NAR-35) was obtained as a yellow solid (68 mg, 20.50%). 1 H NMR (400 MHz, DMSO-d 6 ) δ is 12.16 (s, 1H), 10.80 (s, 1H), 9.77 (s, 1H), 7.56 (d, J = 7.5Hz, 2H), 7.44-7.36 (m, 3H), 7.35-7.26 (m, 2H) ), 6.97 (d, J=8.3Hz, 1H), 5.92-5.85 (m, 2H), 5.49 (dd, J=12.8, 3.0Hz, 1H), 2.72 (dd, J=17.1, 3.1Hz, 1H); 13 C NMR (125 MHz, acetone-d 6)δ is 196.5, 166.7, 163.5, 163.0, 154.7, 138.2, 129.5, 129.3, 129.1, 128.0, 127.6, 127.2, 125.7, 116.0, 101.8, 95.8, 95.0, 78.5 and 42.0. C 21 H 17 O 5 + [M+H] + HRMS (ESI) m / z calcd. 349.1071, found 349.1074.

[0254] 5,7-Dihydroxy-2-(2-fluoro-4-hydroxyphenyl)-4-dihydrochromone (Compound 10, NAR-37) Compound 10 (NAR-37) was synthesized by the following scheme (M):

[0255]

[0256] The synthesis method was the same as the method under the above condition (b). Compound 10 (NAR-37) was obtained as a pale white solid (96 mg, 23.09%). 1 H NMR (500 MHz, acetone-d 6 ) δ is 7.46 (t, J=8.6Hz, 1H), 6.76 (dd, J=8.6, 2.4Hz, 1H), 6.66 (dd, J=12.3, 2.4Hz, 1H), 5.95 (t, J=1.7 Hz, 2H), 5.67 (dd, J=13.2, 3.0Hz, 1H), 3.26 (dd, J=17.1, 13.2Hz, 1H), 2.71 (dd, J=17.1, 3.0Hz, 1H); 13 C NMR (125 MHz, acetone-d 6 )δ is 196.9, 167.5, 165.0, 164.2, 162.9, 130.0, 117.2, 112.5, 103.7, 103.5, 102.9, 96.9, 95.8, 74.2 and 42.2. C 15 H 12 FO 5 + [M+H] + HRMS (ESI) m / z calcd. 291.0663, found 291.0680.

[0257] 5,7-Dihydroxy-2-(2,6-difluoro-4-hydroxyphenyl)-4-dihydrochromone (Compound 11, NAR-38)

[0258] Compound 11 (NAR-38) was synthesized by the following scheme (N):

[0259]

[0260] The synthesis method was the same as the method under the above condition (b). The obtained compound 11 (NAR-38) was a pale yellowish white solid (102 mg, 32.11%). 1 H NMR (800 MHz, acetone-d 6 )δ is 6.55 (d, J=10.7 Hz, 2H), 5.96 (d, J=2.2 Hz, 1H), 5.94 (d, J=2.2 Hz, 1H), 5.75 (dd, J=13.9, 3.0 Hz, 1H), 3.48 (dd, J=17.1, 13.8 Hz, 1H) and 2.72 (dd, J=17.1, 3.1 Hz, 1H). 13 C NMR (200 MHz, acetone-d 6 )δ is 196.7, 167.4, 165.1, 164.2, 163.7 (d, J = 11.3 Hz), 162.4 (d, J = 11.3 Hz), 161.3, 105.6, 102.9, 100.5, 100.4, 97.0, 95.7, 71.1 and 41.0. C 15 H 11 F 2 O 5 + [M+H] + HRMS (ESI) m / z calcd. 309.0569, found 309.0573.

[0261] 5,7-Dihydroxy-2-(2-chloro-4-hydroxyphenyl)-4-dihydrochromone (Compound 12, NAR-39)

[0262] Compound 12 (NAR-39) was synthesized by the following scheme (O):

[0263]

[0264] The synthesis method was the same as the method under the above condition (b). Compound 12 (NAR-39) was obtained as a white solid (84 mg, 27.45%). 1 H NMR (500 MHz, acetone-d 6) δ is 7.57 (d, J=8.5Hz, 1H), 6.95 (d, J=2.5Hz, 1H), 6.92 (dd, J=8.5, 2.5Hz, 1H), 5.98 (q, J=2.2Hz, 2H), 5.76 (dd, J=13.2, 2.9Hz, 1H), 3.15 (dd, J=17.1, 13.3Hz, 1H) and 2.75 (dd, J=17.1, 2.9Hz, 1H). 13 C NMR (125 MHz, acetone-d 6 )δ is 196.7, 167.6, 165.0, 164.2, 159.4, 133.6, 129.8, 127.7, 116.9, 115.6, 102.9, 96.9, 95.9, 76.6 and 42.4. 15 H 12 C1O 5 + [M+H] + HRMS (ESI) m / z calcd. 307.0368, found 307.0368.

[0265] 2-(2-Bromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 13, NAR-40)

[0266] Compound 13 (NAR-40) was synthesized by the following scheme (P):

[0267]

[0268] The synthesis method was the same as the method under condition (b) above. The obtained product, compound 13 (NAR-40), was a pale white solid (79 mg, 22.57%). 1 H NMR (800 MHz, acetone-d 6 ) δ is 7.57 (d, J=8.5Hz, 1H), 7.14 (d, J=2.5Hz, 1H), 6.97 (dd, J=8.5, 2.5Hz, 1H), 6.00-5.96 (m, 2 H), 5.71 (dd, J=13.4, 2.9Hz, 1H), 3.12 (dd, J=17.0, 13.3Hz, 1H) and 2.77 (dd, J=17.0, 2.9Hz, 1H). 13 C NMR (200 MHz, acetone-d 6 )δ is 196.6, 167.6, 165.0, 164.2, 159.4, 129.9, 129.3, 123.3, 120.2, 116.2, 103.0, 97.0, 95.9, 78.8 and 42.5. 15 H12 B O 5 + [M+H] + HRMS (ESI) m / z calcd. 350.9863, found 350.9867.

[0269] As described herein, the inventors of the present invention have found that NAR promotes the increase in oxidative muscle fiber number and aerobic metabolism by upregulating relative Esrrg expression. Therefore, upregulation of relative Esrrg expression is a key indicator for evaluating whether NAR analogs have similar functions.

[0270] Compounds 1-13 were used to test relative Esrrg expression. Each compound was dissolved in DSMO and tested after adding water to form an aqueous solution. The term "relative Esrrg expression" used in this article refers to the expression of Esrrg in the compound-treated group relative to the expression of the control group treated with DMSO alone. During the experimental test, C2C12 myotubes were treated with DMSO or NAR (100μM, 200μM and 400μM) or one of the other compounds. Total RNA from C2C12 myotubes was extracted using TRIzol reagent. mRNAj in total RNA was reverse transcribed using oligo-dT primers and HiScript II reverse transcriptase to synthesize cDNA. Quantitative real-time PCR was performed using SYBR Green Fast qPCR Mix. When SYBR Green dye is added to the sample, it immediately binds to the cDNA in the sample. During PCR, DNA polymerase amplifies the target sequence to produce PCR products called "amplicons." Then, SYBR Green dye binds to each newly generated cDNA molecule. As PCR proceeds, more and more amplicons are produced. When SYBR Green dye binds to all cDNAs, the fluorescence intensity increases as the PCR products increase. The Ct value (the number of cycles when the fluorescence signal exceeds a fixed threshold for the no-template control sample) for each compound was obtained. Finally, the relative quantification method was used to analyze the changes in the expression of the Esrrg gene in samples treated with NAR or other compounds relative to those treated with DMSO, using the arithmetic formula 2 -△△Ct The relative expression levels of Esrrg in different samples were obtained.

[0271] The relative Esrrg expression results of compounds 1-13 and NAR are shown in Table 2. When a compound shows a relative Esrrg expression close to or above 2, it is expected that the compound has a desired biological activity, for example, improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need thereof. Relative Esrrg expression measurements are a model for evaluating these desired activities. Naringenin and its derivatives provided in the present disclosure have been shown to have higher relative Esrrg expression and desired biological or pharmaceutical activities.

[0272] Table 2

[0273]

[0274]

[0275] In some embodiments, the only substituent group (consisting of R 1 or R represents) may be located at the ortho position or meta position of the hydroxyl group of the phenyl group connected to the dihydrochromone. The compound has a structure as shown in formula (III) or (IV) respectively.

[0276] For compounds having the chemical structure of formula (III), examples described above and in Table 2 include NAR-27 (R = F), NAR-29 (R = Cl), NAR-31 (R = Br), NAR-33 (R = OMe), NAR-34 (R = NO 2 ) and NAR-35 (R = phenyl). As shown in Table 2, the relative expression results of Esrrg show a trend in the order of substituents: Cl>Br>OMe>F. 2 When the phenyl group or phenyl group was added, the solubility of these compounds in water was low and no results were obtained relative to the expression of Esrrg. Further studies are needed to improve the solubility.

[0277] For compounds having the chemical structure of formula (IIV) and R located at the meta-position of the hydroxyl group, the examples described above and in Table 2 include NAR-37 (R = F), NAR-39 (R = Cl) and NAR-40 (R = Br). As shown in Table 2, the relative Esrrg expression results show a trend in the order of substituents: F and Cl>Br.

[0278] Some of the examples described above and in Table 2 include two substituent groups in addition to the hydroxyl group on the phenyl group attached to the dihydrochromone ring structure. For example, NAR-28 and NAR-38 contain two F substituents, NAR-30 contains two Cl substituents, and NAR-32 contains two Br substituents. Compounds with two halogen substituents may have lower relative Esrrg expression values ​​than their corresponding counterparts with one halogen substituent. However, with the exception of NAR-30, all other compounds with two halogen substituents have relative Esrrg expression values ​​close to or above 2, indicating that these compounds have the desired biological or pharmacological activity.

[0279] Based on these results, when the phenyl group attached to a dihydrochromone is modified with at least one substituent group as described herein (other than the hydroxyl group), compounds having desired biological or pharmaceutical activities can be obtained.

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[0324] Although the subject matter has been described herein in the form of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be interpreted broadly to include other variants and embodiments that may be made by those skilled in the art.

Claims

1. A composition for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment, characterized in that The composition comprises: An effective amount of a compound having the structure of formula (I) or a pharmaceutically acceptable solvate thereof or any combination thereof; and a pharmaceutically acceptable excipient, Wherein, formula (I) has the following chemical structure: in, R1, R2, R3 and R4 are each selected from the group consisting of H, F, Cl, Br, OH, NH2, NO2, C1-C6 alkyl, C1-C6 alkoxy and phenyl.

2. The composition according to claim 1, wherein One or two of R1, R2, R3 and R4 are substituents other than H.

3. The composition according to claim 1, wherein R1, R2, R3 and R4 are each selected from the group consisting of H, F, Cl, Br, OH and NH2.

4. The composition according to claim 1, wherein R1 or R2 is a substituent other than H, R3=H, and R4=H.

5. The composition according to claim 1, wherein The compound is selected from the group consisting of:

6. The composition according to claim 1, wherein The excipient is selected from solvents, co-solvents, colorants, preservatives, antibacterial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents or any combination thereof.

7. The composition according to claim 1, wherein The excipients included a vehicle containing 20% ​​DMSO and 80% saline.

8. The composition according to claim 1, wherein The composition is a pharmaceutical composition, a functional composition and / or a dietary supplement.

9. The composition according to claim 1, wherein The composition is an injectable or oral pharmaceutical composition.

10. The composition according to claim 9, wherein The concentration of the compounds was 1-50 mM.

11. A compound of formula (I), a pharmaceutically acceptable solvate thereof, or any combination thereof, in, Formula (I) has the following chemical structure: in, Each of R1, R2, R3 and R4 is selected from the group consisting of H, F, Cl, Br, OH, NH2, NO2, C1-C6 alkyl, C1-C6 alkoxy and phenyl, and at least one of R1, R2, R3 and R4 is a substituent other than H.

12. The compound according to claim 11, wherein One or two of R1, R2, R3 and R4 are substituents other than H.

13. The compound according to claim 1, wherein R1, R2, R3 and R4 are each selected from the group consisting of H, F, Cl, Br, OH and NH2.

14. The compound according to claim 1, wherein The compound is selected from the group consisting of:

15. A method for preparing the compound according to claim 11, characterized in that: The following steps are involved: 1-(2-Hydroxy-4,6-bis(methoxymethoxy)phenyl)ethanone is reacted with substituted p-bis(methoxymethoxy)benzaldehyde.

16. A method for preparing the composition of claim 1, characterized in that: The method comprises preparing the compound.

17. The method according to claim 16, wherein: The method further comprises mixing the excipient and the compound.

18. A method for improving skeletal muscle endurance, treating muscle atrophy or malnutrition, or preventing muscle atrophy or malnutrition in a subject in need of such treatment, characterized in that The method includes: The composition of claim 1 is administered to a subject in need thereof, wherein the composition contains an effective amount of a compound of formula (I), a pharmaceutically acceptable solvate thereof, or any combination thereof.

19. The method according to claim 18, wherein: The subject is a mammal.

20. The method according to claim 18, wherein: The subject is a human subject.

21. The method according to claim 18, wherein: The composition is administered by intramuscular injection or orally.

22. The method according to claim 18, wherein: The composition is administered once a day or once every other day via intramuscular injection with an effective amount of the compound ranging from 2 to 20 mg / Kg.

23. The method according to claim 22, wherein: The effective dose of the compound is 3.6-7.6 mg / Kg.

24. The method according to claim 18, wherein: One or two of R1, R2, R3 and R4 are substituents other than H, and R1, R2, R3 and R4 are each selected from the group consisting of H, F, Cl, Br, OH and NH2.

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