CN119300816A - Naringenin or its derivatives for improving muscle endurance or treating or preventing muscle atrophy or malnutrition - Google Patents

Abstract

The present invention provides a composition, compound and method 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 comprise an effective amount of a compound having formula (I), or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The compound is naringenin or naringenin derivative. Methods of preparing the compounds or compositions are also provided.

Description

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

PRIORITY AND RELATED APPLICATION

The present invention claims priority from U.S. provisional application 62/323,146 filed 24 at 2022, 03, the entire contents of which are expressly incorporated herein by reference.

Technical Field

The present invention relates to compositions having pharmaceutical or functional properties. And more particularly to a composition comprising naringenin or a derivative thereof, a method of preparing the composition, and a method of using the composition, e.g., as a pharmaceutical composition, a functional composition, and/or a dietary supplement.

Background

Skeletal muscle is the largest organ in mammals and plays an extremely important role in supporting exercise, thermogenesis, and metabolic regulation. However, its function is impaired by aging, sedentary lifestyle and muscle-related diseases, resulting in a decrease in endurance or strength. Skeletal muscle atrophy and muscle dysfunction are accompanied by the aging process (Porter et al, 1995). Studies have shown reduced fast muscle fiber cross-sectional area, reduced mitochondrial respiratory chain complex expression, and reduced skeletal muscle aerobic respiration capacity in elderly people (Carter et al 2015; murgia et al 2017). Accordingly, older mice have 26% lower absolute grip than adult mice and 15% lower relative grip (McArdle et al, 2004).

Furthermore, non-motile mice have more age-related loss of muscle mass and mitochondrial dysfunction than mice that regularly motile on the running wheel (Figueiredo et al, 2009). Sedentary elderly men are accompanied by a decrease in type IIa fibers (oxidized myofibers) and a decrease in mitochondrial content in all fiber types (St-Jean-Pelletier et al, 2017). In addition, various muscle diseases can lead to muscle atrophy and dysfunction. For example, dunaliella Muscular Dystrophy (DMD), the most common muscular dystrophy in children, can lead to proximal muscle weakness and leg hypertrophy, ultimately leading to degeneration of the bones and cardiac muscles (Falzarano et al, 2015; kamdar and Garry, 2016). Healthy skeletal muscles are therefore vital to the organism, and muscle damage can severely impact health and quality of life.

Strategies are urgently needed to combat muscle loss and functional decline. Studies have demonstrated that exercise is an effective strategy that can improve muscle function and reverse muscle aging to some extent. However, exercise is not suitable for patients lying in bed for a long period of time or having other clinical complications. Thus, there is a need for pharmacotherapy to reduce skeletal muscle loss and restore muscle function.

In recent years, signaling pathways involved in muscle dysfunction have been elucidated, and different types of molecules, such as pro-inflammatory cytokines, growth factors, and transforming growth factor-beta (TGF-beta) family effectors, have been found to be involved in skeletal muscle atrophy (Furrer and HANDSCHIN,2019; lynch et al, 2007). There have been some studies focused on these molecules, therapeutic targets or drugs to improve muscle loss. For example, cyclooxygenase 2 (COX 2) inhibitors have been reported to significantly reduce plasma levels of interleukin 6 (IL-6) and interleukin 1 (IL-1) in aged rats and to increase muscle mass (Rieu et al, 2009). Myostatin (MSTN) can significantly reduce muscle mass, and thus MSTN has attracted considerable attention as a therapeutic target. LY2495655, a humanized MSTN antibody, increased muscle mass in the elderly in clinical trials, however, grip was not affected (Becker et al 2015). Although some drugs may improve the reduction in muscle mass, most trials have failed to show significant improvements in functional parameters (Furrer and HANDSCHIN, 2019). Skeletal muscle remains one of the most pharmacologically overlooked organs.

In addition to the effectiveness of the drug, the safety of the drug is also a concern. Testosterone has been shown to prevent age-related muscle strength loss and improve body function (Srinivas-Shankar et al, 2010; storer et al, 2017), however, its clinical use is limited by serious side effects (Grech et al, 2014). Accordingly, there is a need to find new natural drugs that are safer and more effective than existing drugs to improve the physiological function of skeletal muscle or to prevent muscle atrophy.

Naringenin (NAR), a dihydroflavonoid, is commonly found in the form of naringenin in rosaceous, rutaceae and citrus plants. NAR has been reported to have important biological activities and to have a potentially positive impact on metabolic diseases, cardiovascular diseases, cancer, pulmonary diseases, neurodegenerative diseases and gastrointestinal pathology (Rivoira et al, 2021). For example, NAR supplements may increase energy expenditure, enhance insulin sensitivity, and increase liver 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 affects energy metabolism primarily through PPAR family, PGC-1 family and AMPK signaling pathway (Goldwasser et al, 2011; mulvihill et al, 2009; yu et al, 2019).

Disclosure of Invention

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

In the present invention, the inventors found that Naringenin (NAR) can improve muscle endurance and alleviate muscle dysfunction in naturally-aging mice and mdx mice by increasing the number of oxidized muscle fibers and aerobic metabolism. The transcription factor Sp1 has been identified as a direct target of NAR by biotin-labeled co-precipitation mass spectrometry and has been further verified to have its binding site GLN-110.NAR increased Sp1 binding to Esrrg promoter CCCTGCCCTC sequence by up-regulating Sp1 phosphorylation, thereby up-regulating Esrrg expression. The discovery of the Sp 1-ERRgamma transcription axis is of great importance in basic skeletal muscle research, while the novel function of NAR is of potential interest for preventing sedentary lifestyle-related reduced aerobic exercise capacity and age-or disease-related muscle atrophy.

Based on the study of naringin, the present invention has resulted in various compounds, including derivatives thereof, which are useful 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 invention also provides compositions comprising at least one derivative.

In one aspect, the present disclosure provides a composition 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 comprise an effective amount of a compound having formula (I) (also having code S1) as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient.

The formula (I) has the following chemical structure:

in formula (I), R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy and phenyl. In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH and NH ₂. Such compounds are naringenin or naringenin derivatives.

In some embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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.

The composition may be a pharmaceutical composition, a functional composition and/or a dietary supplement. For example, the composition is a pharmaceutical composition, which may be injected or orally administered. The composition is preferably injectable. The concentration of the compound may be in the range of 1mM to 50mM, for example, 2mM to 15mM,5mM to 10mM, or any other suitable concentration.

The excipient may be a solvent, co-solvent, colorant, preservative, antimicrobial agent, filler, binder, disintegrant, lubricant, surfactant, emulsifier, suspending agent, or any combination thereof. For example, in one injectable composition, the excipients include a carrier calculated by weight from 20% dmso and 80% saline.

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 compounds as described herein. In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy, and phenyl. At least one of R ₁、R₂、R₃ and R ₄ is a substituent other than H. Such a compound is a naringin derivative. In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH and NH ₂.

In some embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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.

The compound may be a suitable compound having the desired solubility and pharmacological properties.

In another aspect, the present disclosure provides a method of preparing a composition or compound described herein. Such methods may include preparing the compounds. The method may further comprise mixing the excipient and the compound.

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

In certain embodiments, in the compound having formula (I), R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy, and phenyl. At least one of R ₁、R₂、R₃ and R ₄ is a substituent other than H. Such compounds are naringenin or naringenin derivatives. In certain embodiments, each R ₁、R₂、R₃ and R ₄ is independently selected from the group consisting of H, F, cl, br, OH and NH ₂.

In some embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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.

In some embodiments, the subject is a mammal, preferably a human subject, which may be a healthy human, or an adult with age or disease related muscle atrophy.

In some embodiments, the composition is administered by intramuscular injection or oral administration. Injection administration is preferred. In certain embodiments, the composition is injected intramuscularly at a dose in the range of 2mg/Kg to 20mg/Kg of the compound at an effective dose at a frequency of once daily or once every other day. The dosage may be any suitable dosage. For example, in certain embodiments, the effective dose of the compound ranges from 3.6mg/Kg to 7.6mg/Kg.

Drawings

The disclosure is best understood from the following detailed description and the accompanying drawings. It is emphasized that, in accordance with the conventional practice, the various features of the drawing are not necessarily drawn to scale. On the contrary, the dimensions of the various features may be arbitrarily expanded or reduced for clarity. In the specification and drawings, like reference numerals refer to like features.

Figures 1-3 show the maximum running distance on the treadmill (figure 1), the ratio of limb grip to body weight (figure 2), and the ratio of Gastrocnemius (GAS) muscle weight to body weight (figure 3) of medium-year mice that received an intramuscular injection of solvent carrier or NAR. Each group of n=6, 10 month old C57 mice.

Figures 4-6 show the maximum running distance on the treadmill (figure 4), the ratio of limb grip to body weight (figure 5), and the ratio of Gastrocnemius (GAS) muscle weight to body weight (figure 6) for young mice that received an intramuscular injection of solvent carrier or NAR. Each group of n=9/10, 2 month old C57 mice.

Figures 7-9 show the maximum running distance on the treadmill (figure 7), the ratio of limb grip to body weight (figure 8), and the ratio of Gastrocnemius (GAS) muscle weight to body weight (figure 9) for C57 or mdx mice, which received an intramuscular injection of solvent vehicle or NAR. Each group of n=4-6, C57 mice aged 4 months or mdx mice aged 4 months.

FIG. 10 shows the mRNA expression levels of Myh7, myh2 and Myh4 in gastrocnemius muscle of 10 month old C57 mice treated with solvent vehicle or NAR. Each group n=6.

Figure 11 shows quantitative statistics of type MyHC I muscle fibers in gastrocnemius muscles of 10 month old C57 mice treated with solvent vehicle or NAR by immunofluorescence staining.

FIG. 12 shows mRNA expression levels of genes associated with aerobic metabolism in gastrocnemius muscles of 10 month old C57 mice treated with solvent vehicle or NAR. Each group n=6.

FIG. 13 shows the mRNA expression levels of Myh7, myh2 and Myh4 in gastrocnemius muscle of 2 month old C57 mice treated with solvent vehicle or NAR. Each group n=9/10.

Figure 14 shows quantitative statistics of MyHC I muscle fibers in gastrocnemius muscles of 2 month old C57 mice treated with solvent vehicle or NAR by immunofluorescence staining. Each group n=9/10.

FIG. 15 shows mRNA expression levels of genes associated with aerobic metabolism in gastrocnemius muscles of 2 month old C57 mice treated with solvent vehicle or NAR. Each group n=9/10.

Figure 16 shows quantitative statistics of five oxidative phosphorylation complex levels of 2 month old C57 mice after solvent vehicle or NAR treatment by immunoblotting analysis. Beta-actin was used as an internal control. Each group n=7.

FIG. 17 shows mRNA expression levels of genes associated with aerobic metabolism in gastrocnemius muscle of 4 month old C57 mice or mdx mice treated with solvent vehicle or NAR. Each group n=4-6.

Figure 18 shows the morphology of C2C12 myotubes after treatment with DMSO or NAR at different concentrations (40, 100, 200, 400, 800, 1600 and 2400 μm).

FIG. 19 shows the mRNA expression levels of Myh7, myh2 and Myh4 in C2C12 myotubes treated with DMSO or NAR at different concentrations.

FIG. 20 shows ATP levels in C2C12 myotubes treated with DMSO or NAR at various concentrations;

Figure 21 shows the oxygen consumption rate of C2C12 myotubes treated with DMSO or NAR. Oligomycin was added to prevent ATP-coupled respiration, FCCP was added to induce maximum respiration, and antimycin a/Luo Tengtong was added to prevent mitochondrial electron transport. The experiment was repeated for four biological replicates.

Figure 22 shows statistics of five oxidative phosphorylation complex levels in C2C12 myotubes treated with DMSO or NAR.

FIG. 23 shows mRNA expression levels of genes associated with oxidative phosphorylation, tricarboxylic acid cycle, and β -oxidation in C2C12 myotubes treated with DMSO and NAR. Changes that are not significantly different are not marked due to limited space.

Figure 24 shows qPCR analysis statistics of errγ levels in C2C12 myotubes treated with DMSO or NAR. Beta-actin was used as an internal control.

Figure 25 shows qPCR analysis of errγ levels in gastrocnemius muscle in medium-aged, young and mdx mice treated with DMSO or NAR.

FIG. 26 shows mRNA expression levels of Esrrg, myh7, myh2, atp b and Cpt1b in C2C12 myotubes treated with DMSO or NAR (NC and Esrrg knockdown groups).

FIG. 27 shows immunoblot analysis of Sp1 levels after biotin and biotin-labeled NAR treatment of cells.

FIG. 28 shows the results of an experiment in which a cell thermal transfer assay (CETSA) was performed in cell lysates (in vitro) to assess the interaction between NAR and Sp 1.

Fig. 29 shows a structure-predicted slip docking model for the binding of compound NAR to Sp 1. Compound NAR is represented by a blue bar. The yellow dotted line depicts three hydrogen bonds predicted for NAR to bind to ASN-81, SER-83 and GLN-110 residues of Sp 1.

Figures 30-31 show the maximum treadmill movement distance (figure 30) and grip of nai treated and untreated middle aged mice (figure 31) after Mit-a pretreatment.

FIG. 32 shows quantitative statistics of immunofluorescent staining of type MyHC I muscle fibers in gastrocnemius muscles of naive and untreated medium-aged mice after Mit-A pretreatment.

FIG. 33 shows the mRNA expression levels of Myh7, myh2 and Myh4 in gastrocnemius muscle of naive and untreated medium-aged mice after Mit-A pretreatment. Each group of n=6, 10 month old C57 mice.

Figure 34 shows qPCR analysis of errγ levels in gastrocnemius of nai treated and untreated middle aged mice after Mit-a pretreatment. Beta-actin was used as an internal reference. Each group of n=6, 10 month old C57 mice.

FIG. 35 shows mRNA expression levels of energy metabolism related genes in gastrocnemius muscles of NAR treated and untreated middle aged mice after Mit-A pretreatment. Each group of n=6, 10 month old C57 mice.

FIG. 36 shows deletion and mutation analysis of the potential transcription factor binding site (Sp 1) on Esrrg promoter in HEK293T cells.

FIG. 37 shows the change in response to NAR induction of cells after over-expression of Sp1 (Sp 1-MT-Pocket 1-GLN) mutated at the predicted binding site in HEK293T cells.

FIG. 38 shows an immunoblot analysis of the effect of NAR on Sp1 phosphorylation levels. The total Sp1 level was used as a control.

Detailed Description

In this disclosure, the singular forms "a", "an" and "the" include plural references, and reference to a particular value includes at least that particular value, unless the context clearly dictates otherwise. Thus, for example, reference to "an additive" is a reference to one or more such compounds and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably means + -10% of the stated value, inclusive. For example, the phrase "about 8" preferably refers to a value of 7.2 to 8.8 inclusive, and as another example, the phrase "about 8%" preferably (but not always) refers to a value of 7.2% to 8.8% inclusive. Where present, all ranges are inclusive and combinable. For example, when referring to a range of "1 to 5," the ranges mentioned should be interpreted to include ranges of "1 to 4," "1 to 3," "1-2 and 4-5," "1-3 and 5," "2-5," and the like. Furthermore, when a list of alternatives is provided positively, such list may be interpreted to mean that any alternatives may be excluded, e.g. by a negative limitation in the claims. For example, when referring to a range of "1 to 5," the range referred to should be interpreted as including any case excluding 1,2,3,4, or 5, and thus, the reference to "1 to 5" may be interpreted as "1 and 3-5, but not including 2," or simply "wherein not including 2. It is intended that any component, element, property, or step recited herein in the claims be expressly excluded whether or not such component, element, property, or step is included as a substitute or alone.

In the present invention, the terms "subject" and "patient" are used interchangeably. The term "patient" as used herein refers to animals, preferably mammals, such as non-primates (e.g., cows, pigs, horses, cats, dogs, mice, etc.) and primates (e.g., monkeys and humans), most preferably humans. 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 particular 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.

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 disorder. The term "effective dose" as used herein refers to a dose of a therapy sufficient to prevent the development, recurrence, or onset of a disease or disorder, and one or more symptoms thereof, enhance or improve the prophylactic effect of another therapy, reduce the severity and duration of a disease or disorder, alleviate one or more symptoms of a disease or disorder, prevent the progression of a disease or disorder, cause regression of a disease or disorder, and/or enhance or improve the therapeutic effect of another therapy.

In the present invention, the phrase "pharmaceutically acceptable" means that it has been approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

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.

In the present invention, the terms "therapy" and "treatment" may refer to any method, composition and/or medicament for preventing, treating and/or managing a disease or disorder, or one or more symptoms thereof. In certain embodiments, the terms "treatment" and "therapy" refer to small molecule therapy.

In the context of the present invention, the terms "treat," "therapy" and "treatment" in the context of the management of therapy in a subject refer to the reduction or inhibition of the progression and/or duration of a disease or condition, the reduction of the severity of a disease or condition (e.g., cancer), and/or the amelioration of one or more symptoms thereof due to the management of one or more therapies.

In the present invention, the term "excipient" refers to an inactive substance that is 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, antibacterial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

In the present invention, the terms "dihydrochromone" and "chromone" are interchangeable and all refer to the same chemical structure. Unless otherwise indicated, the compounds described herein are racemic or achiral. Naringenin (NAR) or a NAR derivative used in the present disclosure is racemic.

NAR has less research on skeletal muscle, and the existing findings are mainly related to diabetic obesity. In L6 myotube cells, NAR activates AMPK and stimulates uptake of muscle glucose directly 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 GLUT4 expression in mouse skeletal muscle (Mutlur Krishnamoorthy and CARANI VENKATRAMAN, 2017). In L6 skeletal muscle cells, NAR increases glucose uptake by muscle cells by activating AMPK and thus increasing GLUT4 translocation (Bhattacharya et al, 2013; zygmunt et al, 2010). However, the effect of NAR on muscle endurance and muscle atrophy is still unclear.

In this study, the inventors of the present invention studied whether NAR could improve muscle function and protect muscles from the aging process or muscle diseases. The effect of NAR on motor capacity and skeletal muscle aerobic metabolic levels was assessed using young adult mice, naturally senescent mice, and mdx mice (preclinical model of DMD) as models. The inventors of the present invention found that NAR increased the number of oxidized muscle fibers, enhanced aerobic respiration in vivo and in vitro, and improved natural aging and muscle dysfunction in mdx mice. Mechanistically, the inventors of the present invention found that Sp1 was the direct binding target for NAR, which affects skeletal muscle by activating the Sp 1-estrogen related receptor gamma (ERRgamma) transcription axis. The results of the present invention will provide a new strategy for improving muscle function and treating muscle atrophy.

Skeletal muscle function may be impaired by aging, sedentary lifestyle or disease, however, skeletal muscle has so far been one of the most inadequate organs for drug treatment. This study found that Naringenin (NAR) can improve muscle endurance and improve muscle dysfunction in naturally-aging mice and mdx mice by increasing the number of oxidized muscle fibers and aerobic metabolism. Transcription factor Sp1 was determined to be a direct target of NAR by biotin-labeled co-precipitation mass spectrometry and was further validated as GLN-110 at its binding site. NAR increased Sp1 binding to Esrrg promoter CCCTGCCCTC sequence by up-regulating Sp1 phosphorylation, thereby up-regulating Esrrg expression. The discovery of the Sp 1-ERRgamma transcription axis is of great importance in basic skeletal muscle research, while the novel function of NAR is of potential interest in preventing sedentary lifestyle-related reduced aerobic exercise capacity and age-or disease-related muscle atrophy.

The inventors of the present invention have found a novel function of NAR in promoting Sp1 phosphorylation by binding to the transcription factor Sp1, thereby improving skeletal muscle endurance in naturally aged and mdx mice and protecting the muscle from atrophy. Phosphorylated Sp1 directly upregulates the transcriptional level of estrogen-related receptor gamma (errγ), thereby regulating energy metabolism and muscle remodeling.

Naringenin (NAR) improved natural aging and muscle dysfunction in mdx mice. NAR increases the number of oxidized muscle fibers and enhances aerobic metabolism. NAR binds to Sp1 to increase its phosphorylation and transcription factor activity. Activating the Sp 1-ERRgamma axis promotes energy metabolism and muscle remodeling.

The inventors of the present invention have further modified naringenin to obtain its different derivatives, which are expected to provide improved properties.

The invention also provides a composition comprising naringenin or a derivative, derivative compound thereof, a method of preparing the composition, and a method of using the composition, e.g., as a pharmaceutical composition, a functional composition, and/or a dietary supplement.

Method of

General chemistry

NAR (naringenin) (batch number QY00305-180610, purity. Gtoreq.98%) was purchased from Qingyun Bio Inc. of Nanjing, china. The purity of naringenin probe (NAR-Biotin) was determined by Waters ACQUITY UPLC system with ELSD, PDA detector, sample manager and binary solvent manager. Preparative HPLC was run on VARIAN PREPSTAR systems with Alltech 2424ELSD and 2489PDA using Waters Sunfire RP C18 (5 μm, 30X 150 mm) columns. Electrospray ionization (ESI) -MS spectra were obtained on a Waters 2695 instrument with a 2998PDA detector coupled to Waters ACQUITY ELSD and Waters 3100SQDMS detectors using a Waters Sunfire RP C column (4.6X105 mm,5 μm) at a flow rate of 1.0mL/min. HRESI-MS was carried out on a Waters ACQUITY UPLC system (Waters Corporation Milford, MA, USA) equipped with an ESI ion source. MS detection was performed using Synapt G-Si Q-TOF mass spectrometer (Waters Corporation, milford, mass., USA). ¹ H and ¹³ C NMR spectra were recorded on a Bruker AVANCE III MHz instrument. Chemical shifts are reported in ppm (δ) and coupling constants (J values) are reported in hertz. Chemical shifts are reported in ppm with Me ₄ Si as a reference standard.

Synthesis of NAR (naringenin) probe (NAR-biotin)

NAR (272 mg,1.0 mmol), biotin (244 mg,1.1mmol,1.1 eq.) and DMAP (12.2 mg,0.1mmol,0.1 eq.) were dissolved in 20ml anhydrous DMF. EDCI (310 mg,2.0mmol,2.0 eq.) and Et3N (277. Mu.L, 202mg,2.0 eq.). The solution was stirred at room temperature overnight. The reaction was quenched with 2M HCl solution, then diluted with 100ml of water and extracted with ethyl acetate. The combined organic phases were washed with water, brine and dried over anhydrous MgSO ₄. The organic solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (MeCN with 0.1% hccoh in water, 40-60%,0-35 min) to give the product (300 mg, 60% yield). Purity at 288nm 99.93%; ¹HNMR(500MHz,Py-d₅) delta were calculated as 499.1539 for HRESI-MS (m/z) values of 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＝13.1,3.0Hz,1H)、4.58(m,1H)、4.42(m,1H)、3.26(m,1H)、3.19(dd,J＝17.0,13.1Hz,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);¹³C NMR(125MHz,Py-d₅)δ 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-MSm/z499.24[M+H]⁺,497.24[M-H]⁺;C₂₅H₂₇N₂O₇S[M+H]⁺, found to be 499.1544.

Mice and cell lines

HEK293T cells (human embryonic kidney cells, females) and C2C12 cells (mouse mesenchymal precursor cells, sex unknown) were obtained from the American Type Culture Collection (ATCC). These cells were cultured at 37℃with 5% CO ₂ using 4.5g/L (25 mM) glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (Gibco 15070063). C2C12 cells were induced to differentiate into myotubes using 2% horse serum (Gibco) for three days. The mice used in this experiment included 10 month old and 2 month old C57BL/6N male mice purchased from VITAL RIVER company, and 4 month old C57BL/10ScSnJGpt-Dmdem3Cd4/Gpt (mdx) male mice purchased from GEMPHARMATECH. Prior to the start of the experiment, these mice were housed in standard cages of 2-6 animals each, housed in temperature control chambers (21-23 ℃) under standardized conditions, maintained for a 12:12 hour photoperiod, and provided standard mouse food and water for ad libitum consumption. All animal studies were reviewed and approved by the national academy of sciences animal committee (IACUC).

Skeletal muscle intramuscular injection in mice

The gastrocnemius and tibialis anterior of the hind limb of the mice (in mice of 2 months old only) were treated and given by intramuscular injection once every two days for a total of fifteen injections. The volume of intramuscular injection was 40. Mu.L for gastrocnemius, 20. Mu.L for tibialis anterior, and the concentration of NAR was 8mM, containing 20% DMSO and 80% saline. The vehicle was 20% DMSO and 80% physiological saline. For 10 month old mice, 50. Mu.M of Mit-A (Abcam ab 142723) was pre-injected to block the transcriptional activity of Sp 1.

Running experiment

2 Days prior to the experiment, mice were acclimatized to the treadmill by running exercise at 15cm/s for 5min/day at 15 degree incline on LE8710RTS treadmill detection system (Panlab/Harvard Apparatus). For exercise experiments, the speed was increased by 5cm/s every 5min until 30cm/s was reached and the mice were allowed to run until exhaustion. Then, the total distance the mice run was calculated.

Grip test

Muscle grip was recorded using the GT3 grip test instrument system (Bioseb, france Vitrolles). The mice were allowed to grasp the metal mesh with four paws and then gently pull their tails back until they could no longer grasp the mesh. The peak tension is recorded on the digital force sensor. The maximum of 10 measurements was used to represent the grip of each mouse.

Immunofluorescent staining of frozen sections

The gastrocnemius muscle was embedded in embedding agent OCT, then frozen in liquid nitrogen and stored in a-80 ℃ ultra-low temperature refrigerator. Frozen sections of muscle (8 μm) were fixed in cold acetone (pre-chilled to-20 ℃) for 20min, washed three times with PBS, and infiltrated three times with 0.5% Triton X-100/PBS (PBST) for 10min each. After washing, the slide was blocked by incubation with 5% BSA for 2h at room temperature, followed by incubation with antibodies such as BA-F8 for MHC class 1 (1:100), BF-F3 for MHC2b (1:100), and SC-71 for MHC-2a (1:100) (university of Aihua development study hybridoma pool in U.S.A.). Sections were incubated at room temperature with Alexa Fluor 350-, 488-and 594-bound secondary antibodies (1:100) (Invitrogen) in the dark for 1h. Photographs were then observed under a confocal laser scanning microscope (Carl Zeiss LSM 710).

Oxygen consumption measurement

Cell oxygen consumption was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. C2C12 myotubes were treated with DMSO or 400 μM NAR on XF 24V 28 cell culture microplates (Seahorse Bioscience, north Billerica, mass., USA) for 24h. For detailed experimental procedures, please refer to the experimental guidelines (Agilent Seahorse XF Cell Mito STRESS TEST KIT).

RNAi interference and inhibitor treatment experiments

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

Plasmid preparation, cell transfection and luciferase reporter gene analysis

The total cDNA extracted from the calf muscle tissue of the mice is amplified by PCR to obtain Sp1 cDNA sequence, and the Sp1 cDNA sequence is transformed with pcDNA3.1 vector digested by the same endonuclease after double enzyme digestion to obtain Sp1 over-expression vector.

Primers suitable for PCR amplification of Esrrg promoter sequences of different lengths were designed using Primer 5 software based on the mouse Esrrg genomic sequences provided by NCBI database. The Esrrg promoter sequence obtained by PCR was digested with KpnI/HindIII (NEB) restriction enzymes, recovered by agarose gel electrophoresis, ligated with pGL3-basic vector which was also digested with the enzyme, and the pSE-1518, pSE-1518DEL, pSE-1080 and pSE-129 fluorescent reporter plasmids were obtained with T4 ligase (NEB). pSE+3 and pSE+38 fluorescent reporter plasmids were obtained by circular PCR using the pSE-129 plasmid as a template. pSE+3MT fluorescent reporter plasmid was obtained by circular PCR using pSE+3 plasmid as template. In addition, 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 Sp1 over-expression plasmid as a template.

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

After 24h, plates were pretreated with DMSO or NAR for 12h, washed twice with pbs, and then double luciferase reporter assays were performed as described in Novozymes double luciferase assay kit. Promoter activity is expressed in relative luciferase units. The ratio of firefly luciferase to marine luciferase was calculated as the initiation efficiency of the corresponding promoter fragment. pGL3 vector served as negative control.

Gene expression detection

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 R) and was used. Real-time quantitative PCR was performed using SYBR GREEN FAST qPCRMix (Genstar A) and PCR.

Western immunoblot analysis

Protein concentration was assessed using the BCA protein assay kit (Beyotime P0012), using 20-30 μg of protein per SDS-PAGE experiment.

Chromatin immunoprecipitation analysis

C2C12 cells were grown in 100mm dishes. After differentiation into myotubes, treatment with 400 μmnar was performed for 24h. Specific experimental procedures were performed according to the kit instructions (Millipore # 17-408). The nuclei were sonicated in 500. Mu.l of sonication buffer using a sonicator (4417 probe) (15 s on/30 s off for 18 times, power 9W) to cleave the DNA to obtain fragments between 300 and 600bp in length. Immunoprecipitation (IP) was performed overnight at 4℃using 5. Mu.g of anti-Sp 1 antibody (Abcam ab 227383), 5. Mu.g of anti-histone H3 antibody (Millipore) or 5. Mu.g of normal rabbit IgG antibody (Millipore) and 40. Mu.g of chromatin. DNA was analyzed by real-time PCR for the Esrrg promoter Sp1 specific binding region.

Acquisition of NAR interacting proteins

Two dishes of 100mm C2C12 myotube cells were washed three times with PBS and then lysed in 2mL of hypotonic lysis buffer (20mM HEPES,2mM EDTA,2mM MgCl ₂, 1% mixture, ph=7.4). Cells were scraped off using a cell scraper and lysed on ice for 45min. Then, the mixture was centrifuged at 12000 Xg at 4℃for 15 minutes. To reduce false positives, 40 μl of streptavidin magnetic beads (Thermo Scientific # 65001) were added to the supernatant and incubated for 2h at 4 ℃ with counter-rotation. The supernatant was divided equally into two parts, 4mM NAR-biotin with 1% DMSO was added to the treatment group and 4mM biotin with 1% DMSO was added to the control group. Each group was incubated upside down at 4 ℃ overnight, then 20 μl of streptavidin magnetic beads (Thermo Scientific # 65001) was added to each group. Each group was incubated upside down at 4 ℃ for 2 hours. The supernatant was discarded and the 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 binding proteins were subjected to SDS-PAGE, coomassie blue staining and Liquid Chromatography (LC) -tandem mass spectrometry (MS/MS) analysis.

LC-MS/MS analysis

After SDS-PAGE and Coomassie Brilliant Blue staining, the gel bands were destained and subjected to overnight enzymolysis with trypsin. The peptide fragments were then extracted by multiple steps using different concentrations of acetonitrile.

LC-MS/MS analysis was performed on nanoLC-Q Exactive system. In brief, the flow rate through the column was set at 0.3. Mu.L/min and the applied distal spray voltage was set at 2.0kV. Data collection was accomplished by performing a data dependent MS2 scan of the 20 most abundant ions after a full MS1 scan after a full scan (MW 300-1, 600).

The peptide mixture obtained by enzymatic digestion was first analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A protein search was then performed in the UniProt-protein-mouse (update-20171001) database using the SEQUEST HT search engine of Thermo Proteome Discoverer (2.2.0.388). The search parameters were trypsin digestion with 2 missing cleavage sites, precursor ion mass error less than 10ppm, fragment ion mass error less than 20mDa, alkylation of cysteine as fixed modification, oxidation of methionine as variable modification. The filtering parameters of the search results were Percolator for spectral filtering, delta Cn was less than 0.1, FDR was set to 1%, high peptide confidence was chosen, and FDR at protein level was also set to 1%. Label-free quantification (LFQ) was performed using the Consensus node and parameters of unique + razor, precursor abundance based on intensity, normalized mode, no or total peptide amount, ratio calculation based on the pairwise ratio, maximum allowed FC, high (100). Only proteins with an average ratio (treatment/control) of over 2 in all three replicates were selected for further GO analysis using the R language.

RNA-Seq transcriptome analysis

Total RNA of DMSO and NAR treated C2C12 myotubes was extracted by using TRIzol reagent (Thermo Scientific 15596018). Samples were sent to Biomarker Technologies (Beijing, china) for sequencing. Exon model per kilobase per million mapped reads (FPKM) values of mRNA were used for further analysis. Differential expression analysis was performed on both groups using DESeq 2. Genes with post-regulation P-values <0.05 found by DESeq2 were considered differentially expressed. FDR <0.05 and FC.gtoreq.1.5 were set as thresholds for significant differential expression. GO enrichment analysis and KEGG pathway enrichment analysis were performed on the up-and down-regulated genes in DEGs using GOseq R package and KOBAS software.

Sp1 phosphorylation assay

The C2C12 myotubes were treated with or without NAR for 24h and then lysed in RIPA lysis buffer (GenStar E-123-01) containing 1% protease inhibitor cocktail (MCE HY-K0010) and 1% phosphatase inhibitor cocktail (MCE HY-K0023). Cells were scraped off using a cell scraper and lysed on ice for 45min. Proteins were first pre-cleared with Protein A/G beads (Thermo Scientific 88803) at 4℃for 1h. The supernatant after pre-removal was incubated with Sp1 antibody (Abcam ab 227383) overnight at 4℃with gentle shaking. After incubation, protein A/G beads (Thermo Scientific 88803) were added and incubated for 1h at 4 ℃. The protein-antibody-bead complexes were washed three times with lysis buffer for 5 minutes at a temperature of 4 ℃. The complex was resuspended in loading buffer (Thermo Scientific 39000,390), boiled for 10min and subjected to Westernblot analysis.

CETSA for cell thermal transfer experiments

The C2C12 myotubes were incubated in medium containing DMSO or 400. Mu.M NAR for 48h. 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. Cells were repeatedly freeze-thawed 3 times in liquid nitrogen and then centrifuged at 12000 Xg for 15min at 4 ℃. Supernatants from DMSO groups were split equally into two parts. A portion was treated with 4mM NAR for 30min. Another part of DMSO-treated group and 400 μΜ NAR-treated group were treated with DMSO for 30min. For each group, 50. Mu.L (1 mg/mL) was isolated and heated for 5min under different temperature gradients of a S1000 ^TM thermal cycler (Bio-Rad). The sample was then centrifuged at 12000 Xg for 15min. The supernatant was removed to prepare an electrophoresis sample.

Prediction of Sp1 protein Structure and ligand binding site

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 Sp1 protein structure, but the entire structure has not been determined.

The first method is homology modeling. Homology modeling, also known as comparative modeling, is a commonly used method of structure prediction (Muhammed and Aki-Yalcin, 2019). The goal of homology modeling is to construct its three-dimensional structure from the main amino acid sequence of the target protein based on alignment with a known sequence (template) (Bordoli et al, 2009). First, the SWISS-MODEL workspace was used to construct the three-dimensional structure of Sp1 protein. SWISS-MODEL was developed by the group of computational structure biology of the SIB Swiss bioinformatics institute and the Basel university student center.

The I-TASSER server, offered by the Yang Zhang research group of the university of Michigan medical institute, is another tool for automated protein structure prediction. I-TASSER (ITERATIVE THREADING ASSEMbly Refinement, iterative thread assembly refinement) is a hierarchical protein structure prediction and structure-based functional annotation method. I-TASSER is also a template-based method that employs a multi-threading approach to identify known structures that have structural similarity to a protein of interest over a limited number of protein folds (Deng et al 2016; roy et al 2010).

Once the 3D structure of the Sp1 protein is determined, the next step is 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 procedure named POCASA (Yu et al, 2010). In this study, a network server of POCASA 1.1.1, supplied by the university of Hokkaido, japan, was used to predict the ligand binding site of the Sp1 protein. POCASA parameter settings default, e.g. the radius of the probe sphere is set toThe size of the unit cell is set to

Molecular docking

This study used molecular docking to explore the interaction between small molecule NAR and protein Sp 1. First, the structure of the Sp1 protein is pre-treated using a protein preparation guide module, including operations such as adding hydrogen, removing water, minimizing structural limitations, and the like. The predicted Sp1 binding site was then imported into the program as a docking grid for the next procedure. The possible ionization states and stereoisomers (up to 10 per ligand) of small molecule NARs were then generated by the LigPrep module at the target pH value of 7.0.+ -. 1.0. Finally, these pretreated ligands access the predicted binding site of the SP1 protein by a ligand docking module pair. All of these modules were included in Maestro 11.2 software (Schrodinger, LLC: new York, N.Y., 2010).

Statistical analysis

All experiments were performed at least three times. The number of replicates (n) for each animal experiment is shown in the legend. When three or more groups are compared, one-factor analysis of variance is performed with the process as an independent factor. Two sets of measurements were tested using a two-tailed Student's t. Data are presented as mean ± SEM. ns is considered to be no significant difference, P <0.05 is considered to have a statistical significance, P <0.01 is considered to have a significant statistical significance. Excel and GraphPad were used for all statistical analyses.

Experimental results:

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

To investigate the protective effect of NAR against age-related muscular atrophy, first 10 month old male mice were used as experimental models. These mice were randomized into control and NAR groups and given intramuscular injections of gastrocnemius. The results showed that NAR significantly increased the running distance of the mice (fig. 1), indicating that it enhanced motor endurance of the muscles. In addition, NAR also increases grip (fig. 2) and gastrocnemius relative weight (fig. 3). Thus, in 10 month old middle aged mice, NAR not only prevented muscle degeneration, but also enhanced muscle function.

The inventors of the present invention next attempted to determine whether NAR could improve muscle function in young mice under normal physiological conditions. Using 2 month old mice, NAR was found to again increase aerobic endurance (running distance) (fig. 4), but had no significant effect on the relative weights of grip (fig. 5) and gastrocnemius (fig. 6). Thus, NAR also significantly increases running distance in young mice under normal physiological conditions.

In addition to age-related muscular atrophy, the effect of NAR on disease-related muscular atrophy was further studied using mdx mice, which are animal models of duchenne muscular dystrophy DMD. Consistent with literature reports (Ryu et al, 2016), mdx mice show a typical reduced endurance phenotype as well as muscle pseudohypertrophy. NAR increased running distance (fig. 7) and grip (fig. 8) in mdx mice, but did not affect body weight or gastrocnemius relative weight (fig. 9).

Overall, NAR can improve muscle endurance and protect against age-or disease-related muscle atrophy.

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

Increased muscle endurance and redder muscle phenotype indicate that NAR may affect muscle fiber type and energy metabolism. Thus, the effect of NAR on different types of muscle fibers in the gastrocnemius muscle of 10 month old mice, the anterior tibialis muscle and gastrocnemius muscle of 2 month old mice, and the gastrocnemius muscle of 4 month old mdx mice was further examined. The results showed that NAR upregulated the expression of MHC1 gene Myh7 corresponding to oxidized myofibers and MHC2b gene Myh4 corresponding to glycolytic myofibers in calf muscles of 10 months old mice, but did not upregulate MHC2a gene Myh2 corresponding to oxidized myofibers (FIG. 10).

Furthermore, immunofluorescence (IF) showed that NAR increased the content of type I oxidized muscle fibers in gastrocnemius (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, cox b and Cox 2), beta-oxidation related genes (Cpt 1b and Ucp 3) and glucose metabolism related genes (Pdk 4) were upregulated by NAR (FIG. 12), consistent with an increase in oxidized myofiber content. Thus, NAR not only increases the content of muscle fibers to combat muscle atrophy caused by aging, but also increases muscle endurance by increasing the content of oxidized muscle fibers in mice.

Also, in 2 month old mice, NAR increased expression of Myh7 (corresponding to MHC 1) and Myh2 (corresponding to MHC2 a) in the gastrocnemius muscle without significantly altering the level of Myh4 (corresponding to MHC2 b) (fig. 13). These findings are consistent with the results that NAR has no effect on the grip of young mice. Thus, IF staining showed more type I oxidized myofibers in the NAR group than in the control group (fig. 14). In terms of energy metabolism, NAR upregulated expression of genes related to aerobic metabolism (Mb, atp5b, cycs, cox b and Cox 2), β -oxidation (Cpt 1b and Ucp 3) and glucose metabolism (Pdk 4) in the calf muscle of young mice (FIG. 15). In addition, NAR upregulates the relative levels of five oxidative phosphorylation (OXPHOS) complexes in mitochondria, including complex I subunit NDUFB, complex II subunit 30kDa, complex III subunit Core2, complex IV and ATP synthase alpha subunit (fig. 16). Overall, the results indicate 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.

In mdx mice at 4 months of age, the expression levels of Mb, atp5b, cox2, ucp, and Pdk4 were up-regulated in the NAR treated group (fig. 17), indicating an increase in aerobic metabolism, which may contribute to improvement of muscle function in mdx mice after NAR treatment.

In summary, NAR increases muscle endurance by increasing the number of oxidized muscle fibers and enhancing aerobic metabolism.

3. NAR increases the content of oxidized muscle fibers in the C2C12 myotubes and enhances aerobic metabolism.

To further explore the cellular mechanisms by which NAR regulates skeletal muscle function, C2C12 myoblasts were used. C2C12 cells can differentiate and fuse to form myotubes. The C2C12 myotubes were treated with different concentrations of NAR (0, 40, 100, 200, 400, 800, 1600 and 2400 μm) for 24h. When the NAR concentration exceeded 800. Mu.M, many cells died (FIG. 18). Therefore, NAR concentrations below 400. Mu.M were selected to treat cells. Expression of the oxidized myofiber-related genes Myh7 and Myh2 was upregulated by NAR in a dose-dependent manner, whereas expression of the glycolytic myofiber-related gene Myh4 was absent (fig. 19), indicating an increased oxidized myofiber content.

The effect of NAR on cellular energy metabolism was then assessed. The study found that NAR increased total ATP levels (fig. 20), suggesting that NAR promoted energy production. In addition, the cellular bioenergy characteristics of C2C12 myotubes with or without NAR treatment were measured using a Seahorse cellular energy metabolizer. Treatment of myotubes with 200 μm NAR increased Oxygen Consumption Rate (OCR) (fig. 21). These results confirm that NAR promotes aerobic respiration of C2C12 cells.

In addition, NAR enhanced the expression of key enzymes in oxidative phosphorylation (OXPHOS), tricarboxylic acid cycle (TCA), and β -oxidation processes (fig. 22 and 23). Thus, NAR increases the content of oxidized muscle fibers and enhances aerobic respiration by improving mitochondrial activity in skeletal muscle.

4. ERRgamma mediated enhancement of NAR-induced oxidized myofiber number and aerobic metabolism

The differentially expressed genes between the C2C12 myotubes treated with NAR and untreated with NAR were analyzed (DEGs). As a result, it was found that the expression of the nuclear transcription factor errγ (encoded by Esrrg) was up-regulated by NAR to a more significant extent than other nuclear transcription factors. Previous studies have shown that errγ is highly expressed in oxidized muscle fibers and that it can regulate many genes associated with metabolism (Misra et al, 2017). Thus, errγ mRNA levels in C2C12 myotubes treated with NAR were examined. As a result, NAR was found to increase the level of errγ in a dose-dependent manner (fig. 24). In addition, NAR increased errγ transcript levels in vivo in 10 month old mice, 2 month old mice and mdx mice (fig. 25). In vitro and in vivo data indicate that errγ may be involved in the function of NAR. Subsequently, to verify this hypothesis, errγ was knockdown at the cellular level using siRNA. The deficiency of errγ inhibited the NAR-mediated upregulation of oxidized myofiber-associated Myh7 and Myh2 and aerobic metabolism-associated Atp b and Cpt1b (fig. 26), suggesting that errγ mediates an NAR-induced increase in oxidized myofiber number and aerobic metabolism.

Summarizing, errγ mediates an increase in NAR-induced oxidized myofiber number and aerobic metabolism, thereby improving endurance exercise capacity in mice.

5. Sp1 is a direct binding protein for NAR in C2C12 cells

To further elucidate the reasons why NAR upregulates ERRgamma expression, NAR was biotinylated by the inventors. Differentiated C2C12 cells were lysed and lysates were immunoprecipitated with biotin and biotin-labeled NAR. Next, affinity magnetic beads were used to enrich for non-specific proteins that bind to biotin and specific proteins that bind to NAR-biotin. The two groups of proteins were separated by SDS-PAGE and analyzed by Mass Spectrometry (MS) and the protein immunoprecipitated with NAR-biotin was compared with the Negative Control (NC) biotin immunoprecipitated protein to identify the differential protein which varied more than twice. The results showed that a total of 492 proteins were up-regulated more than twice in the NAR-biotin group, with 207 proteins only present in the NAR-biotin group and not in the NC group. Thus, the molecules of the transcriptional process related proteins present only in the biotinylated NAR group were enriched (FIG. 27). The transcription factor Sp1 is only present in the biotinylated NAR group. Thus, SP1 is most likely the binding protein of NAR.

To verify the direct interaction of NAR with Sp1, western blot was first used to demonstrate that Sp1 was significantly enriched in biotinylated NAR binding protein (fig. 27). The binding affinity of NAR to Sp1 was then further analyzed using a cell thermal transfer assay (CETSA) which detects the binding affinity of the small molecule compound to the protein of interest. The results show a clear shift in the solubility profile of Sp1 in C2C12 myotube cell lysates treated with 4mM NAR for 30 minutes. In the control, 50% of the Sp1 protein was degraded at 63℃whereas in the cell lysate samples treated with 4mM NAR for 30 minutes, the temperature was increased to 69 ℃. An increase in stability of the Sp1 protein in the NAR treated sample indicates that NAR binds directly to the Sp1 protein. Thus, the above results indicate that NAR may exert a regulatory effect in skeletal muscle by directly binding to the transcription factor Sp1 (FIG. 28).

To better demonstrate the interaction of NAR with Sp1, the 3D structure of Sp1 and the possible interaction sites of NAR/Sp1 were predicted. The small NAR molecule interacts with Sp1 protein mainly through hydrogen (H-) bonds, more specifically, two hydroxyl groups in NAR molecule interact with 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 combination of NAR/Sp1 binding.

6. Sp1 is involved in NAR-mediated ERRgamma up-regulation and improvement of muscle function

To determine whether Sp1 is involved in the function of NAR in skeletal muscle, the effect of NAR on skeletal muscle function and metabolism was 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 was inhibited (fig. 30 and 31). The increase in the number of oxidized myofibers induced by NAR (fig. 32) and the upregulation of oxidized myofiber-related genes Myh7 and Myh2 (fig. 33) were also inhibited. Furthermore, NAR-mediated upregulation of ERRgamma was inhibited by Mit-A at the RNA level (FIG. 34). In addition, upregulation of errγ downstream genes associated with oxidized myofiber number or aerobic metabolism, including myoglobin, atp b, cycs, cox b, cpt1b, and Ucp3, was also inhibited (fig. 35). These results indicate that NAR-induced increases in endurance and grip in 10 month old mice are achieved by the in vivo transcription factor Sp 1.

7. NAR enhances Sp1 interaction with Esrrg promoters by increasing Sp1 phosphorylation

The above results show that Sp1 mediates NAR-induced upregulation of errγ, and thus it was next sought to determine how NAR regulates errγ expression via Sp 1. Through sequence comparison and prediction, the inventors found that Esrrg promoter this region contained a sequence that was likely to be bound by Sp1 (CCCTGCCCTC). To determine whether binding of Sp1 to Esrrg promoter was responsive to NAR induction, different effects of NAR on control vectors, fluorescent reporter vectors containing the +3 to +100 region of Esrrg promoter, and mutant reporter vectors were measured. The results showed that the response of the fluorescent reporter vector pSE+3 to NAR was 4.5-fold, much higher than that of the control, 1.7-fold and the mutant, 2.5-fold (FIG. 36).

The inventors of the present invention obtained possible binding sites for NAR to Sp 1. Thus, a fluorescent reporter vector containing the Esrrg promoter +3 to +100 region and mutated amino acids of the Sp1 vector binding site was used to verify the true binding site of NAR to Sp 1. The results showed that the NAR response of the fluorescent reporter vector pSE+3 on the unmutated Sp1 vector was 4.5-fold, much higher than that of the Sp1-MT-Pocket1-GLN vector (FIG. 37). Mutation of GLN-110 in pocket1 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 binds directly to NAR to increase Sp1 transcriptional activity is not yet known. Recent data indicate that the phosphorylation status of Sp1 plays an important role in regulating multiple genes (Chu, 2012;Tan and Khachigian,2009). Post-translational phosphorylation levels of Sp1 after NAR treatment were determined using Immunoprecipitation (IP). The results show that phosphorylation of Sp1 increases after NAR binding (fig. 38), which results in an increase in Sp1 transcriptional activity.

8. Reconstruction and synthesis of NAR structure, improving water solubility, activity and medicinal properties of NAR by structural modification

Based on the results of the study on NAR, the structure of NAR was further modified to obtain different derivatives. These modified targets include increased water solubility, enhanced biological or therapeutic activity, and pharmaceutical properties. These derivatives will be useful for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need of such treatment. A composition comprising at least one derivative is obtained. In the present invention, novel compounds include a range of derivatives as described herein.

Muscle atrophy and dysfunction are major problems for life and health, and finding solutions has been the pursuit of research. In this study, a new function of NAR was discovered. NAR, including its derivatives, improves the natural aging process and muscle endurance of mdx mice by increasing the number of oxidized muscle fibers and enhancing aerobic respiration, and protects them from muscle atrophy. Importantly, the transcription factor Sp1 was identified as a direct binding target for NAR. NAR binding increases Sp1 phosphorylation and transcription factor activity. In addition, sp1 was found to be a novel transcription factor for Esrrg, and the Sp 1-ERRgamma transcription axis was found to be involved in NAR-mediated energy metabolism and muscle remodeling. These findings are of great significance for preventing sedentary lifestyles and age-related reduction of aerobic exercise capacity and muscle atrophy, as well as for treating DMD-related muscle weakness.

New function and meaning of NAR in skeletal muscle in three animal models:

With age, the decrease in muscle mass and strength is a non-negligible problem, and some studies report that muscle mass decreases by 3-8% every 10 years after age 30 (Volpi et al, 2004). Skeletal muscle atrophy and impaired exercise tolerance are the primary manifestations of aging, primarily manifested by a decrease 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 modeled naturally senescent 10 month old mice, demonstrating that NAR can improve endurance and grip and to some extent reverse muscle atrophy by increasing the number of oxidized muscle fibers and overall aerobic capacity. Since muscle atrophy caused by natural aging is the most common type of muscle loss, the inventors first used a mouse model of natural aging to demonstrate the role of NAR in improving muscle function. In the background of the increasing global aging problem, NAR has great promise for widespread use in improving or treating muscle atrophy. Even in young adult mouse models, NAR significantly increases running distance, oxidized muscle fiber formation, and skeletal muscle OXPHOS process key enzyme expression. This suggests that NAR can enhance the aerobic capacity of muscle under normal physiological conditions, suggesting the possibility that it may be applied to endurance athletes such as long-distance runners.

Furthermore, this new function of NAR has potential application to modern young adults, where lack of exercise or sedentary lifestyle leads to decreased skeletal muscle metabolism and increased risk of metabolic diseases such as obesity, diabetes, etc. Notably, NAR also improved endurance and grip strength in mdx muscular dystrophy mice. Although it does not significantly affect the type or content of muscle fibers, the promotion of aerobic metabolism by NAR accounts for this phenomenon to some extent. In summary, although the mechanism is not fully understood, NAR also expands the potential use of NAR in the treatment of muscle diseases by improving the mdx phenotype. Overall, these findings indicate that the role of NAR in improving muscle function would benefit multiple populations.

Sp1, identified as a new direct target of NAR, mediates NAR function in skeletal muscle:

Although NAR has been widely studied for its various biological activities in different diseases, studies on its direct action targets are very lacking, which greatly limits the application of NAR. To date, only two studies have explored the direct effect goal of NAR. Collapsin reaction mediator protein 2 (CRMP 2) has been identified as a candidate for direct binding to NAR to explain the protective effect of NAR in Alzheimer's Disease (AD) (Yang et al, 2017). Another study showed that NAR binds directly to the pparα Ligand Binding Domain (LBD) region and activates pparα via 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 Ppar a is a direct target of NAR has not been confirmed by additional evidence.

In the present invention, the inventors of the present invention determined that Sp1 is a direct binding protein of NAR by IP-MS and verified the binding affinity of NAR to Sp1 by CETSA in muscle cells. Sp1 activity is regulated by post-translational modifications such as phosphorylation, glycosylation, and acetylation, among which phosphorylation is most studied. It was found that NAR activates Sp1 transcription factor activity by promoting phosphorylation of Sp1, revealing the mechanism by which NAR regulates Sp 1. This is the first time that it was determined that NAR is a direct target in skeletal muscle fiber type and energy metabolism regulation.

Sp1 is a novel transcription factor for ERRgamma:

errγ is a parthenous estrogen receptor with ligand independent transcriptional activity, plays an important role in pathological conditions such as insulin resistance, alcoholic liver injury and myocardial hypertrophy, and regulates energy metabolism of heart, skeletal muscle and islet beta cells (Misra et al, 2017). Errγ is a good target for the treatment of metabolic diseases based on its importance in metabolic homeostasis. Various cellular stresses may induce expression of errγ through membrane receptors or intracellular transcription factors.

In addition, errγ expression can be regulated by transcription factors c-Jun, stat3, CREB, HIF1a and ATF6a under external stimuli (Misra et al, 2017). In the research, sp1 is determined as a new transcription factor of ERRgamma, and a new mechanism is provided for the regulation of ERRgamma expression. Binding of NAR to Sp1 further promoted binding of Sp1 to ERRgamma promoter. The establishment of Sp 1-ERRgamma axis not only reveals the mechanism of NAR regulating muscle function, but also provides a new signal path for optimizing the strategy for improving muscle function.

In this study, the present inventors found that NAR enhances the transcription factor activity of Sp1 by promoting phosphorylation of Sp1. However, the specific phosphorylation site has not been verified, and it is still unclear how NAR affects Sp1 phosphorylation. Furthermore, the relatively low solubility of NAR may limit its effectiveness in animals. Increasing the water solubility of NAR will help to facilitate the use of NAR.

As described herein, the present invention demonstrates for the first time that NAR is able to increase skeletal muscle endurance and aerobic metabolic capacity in young mice, protect middle aged mice from muscle atrophy, and alleviate DMD. In addition, it established that Sp1 is a new direct target of NAR and established a new relationship between Sp1 and the gene of interest ERR, thereby elucidating how NAR enhances the interaction between Sp1 and the ERR promoter by binding to Sp1, thereby upregulating the molecular mechanism of ERR expression. Thus, this study opens up a new approach to the application of NAR and provides a new and safer strategy for improving muscle function and protecting muscle atrophy caused by aging and disease. NAR derivatives were synthesized in order to mimic NAR or to further enhance its performance.

In one aspect, the present disclosure provides a composition 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 comprise an effective amount of a compound having formula (I) as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient,

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

In formula (I), R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy and phenyl. In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group of H, F, cl, br, OH and NH ₂. Such compounds are Naringenin (NAR) or naringenin derivatives.

In naringenin derivatives provided herein, the benzene ring linked to the dihydrochromone is modified with at least one substituent and up to four substituents R ₁、R₂、R₃ and R ₄. In certain embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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. When naringenin derivatives contain only one substituent group (represented by R ₁) on the benzene ring of naringenin base structure, such compounds have the chemical structure of formula (II):

The only substituent (represented by R ₁) may be ortho or meta to the hydroxy group. The compound may have a chemical structure of formula (III) or (IV):

Examples of suitable compounds in the compositions provided herein include, but are not limited to, the following:

Among the above compounds, the sample numbers include the compound numbers and laboratory compound codes in brackets.

The composition may 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 may be in the range of 1-50mM, such as 2-15mM,5-10mM, or any other suitable concentration. Examples of suitable 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 and any other value between any two of these values.

The excipient may be a solvent, co-solvent, colorant, preservative, antimicrobial agent, filler, binder, disintegrant, lubricant, surfactant, emulsifier, suspending agent, or any combination thereof. For example, in an injectable composition, the excipients include a carrier consisting of 20% DMSO and 80% saline by weight. In certain embodiments, these compositions may be administered with a beverage, food, or related ingredient.

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 compounds are naringenin derivatives other than Naringenin (NAR).

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

The present invention provides any genus or species of compounds as described herein, e.g., having formula (II), (III) or (IV). In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy, and phenyl. At least one of R ₁、R₂、R₃ and R ₄ is a substituent other than H. Such a compound is a naringin derivative. In certain embodiments, R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH and NH ₂.

In certain embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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.

Examples of compounds suitable as naringenin derivatives include, but are not limited to, examples of compounds described herein, such as compound numbers 1-13.

In another aspect, the present disclosure provides a method of preparing a composition or compound described herein. Such methods may include preparing the compound. The method may further comprise mixing the excipient and the compound.

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

In another aspect, the present disclosure provides a method of improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need of such treatment. The method comprises administering to a subject in need of such treatment an amount of a composition described herein. The composition comprises an effective amount of a compound having formula (I). The present disclosure also provides for the use of compounds having formula (I), such as NAR and NAR derivatives, for the manufacture of a medicament for the treatment of any of these medical conditions.

In some embodiments, in the compound having formula (I), R ₁、R₂、R₃ and R ₄ are each selected from the group consisting of H, F, cl, br, OH, NH ₂、NO₂, C1-C6 alkyl, C1-C6 alkoxy, and phenyl. At least one of R ₁、R₂、R₃ and R ₄ is a substituent other than H. Such compounds are naringenin or naringenin derivatives. In certain embodiments, each R ₁、R₂、R₃ and R ₄ is independently selected from the group consisting of H, F, cl, br, OH and NH ₂.

In some embodiments, one or both of R ₁、R₂、R₃ and R ₄ 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.

In some embodiments, the subject is a mammal, preferably a human subject, which may be a healthy human, or an adult with age or disease related muscle atrophy.

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

Examples of compounds:

the following examples include synthetic procedures for illustration only and do not limit the scope of the compounds provided by the present disclosure.

Compounds 1-13 have been prepared and evaluated. Table 1 summarizes their structural comparison with naringenin (abbreviated NAR, laboratory compound code S12). The tag compound codes for compounds 1-13 are also shown in table 1.

TABLE 1

General procedure for the synthesis of 2' -hydroxyacetophenone:

synthesis of 2' -hydroxyacetophenone according to scheme (A) and the procedure described below:

2',4',6' -trihydroxyacetophenone (14.74 g,87.68mmol,1.0 eq.) was suspended in dichloromethane (180 mL) and stirred under argon atmosphere. N, N-diisopropylethylamine (DIPEA, 46.05mL,263.05mmol,3.0 eq.) was added at 0℃followed by dropwise addition of bromomethyl ether (MOMBr, 15.75mL,192.91mmol,2.2 eq.) to the reaction mixture at 0 ℃. The reaction mixture was stirred at 0 ℃ for 4 hours. The reaction mixture was quenched with 200mL of water, and then the aqueous solution was extracted with dichloromethane (3X 120 mL). The combined organic solutions were dried (Na ₂SO₄), 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 (400 MHz, chloroform-d) delta was 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.

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

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

3-bromo-4-hydroxybenzaldehyde (1.0 g,5mmol,1.0 eq.) was added by the conventional MOM protecting group method and purified by silica gel column chromatography (PE/etoac=30:1-20:1) to give 400mg of 3-bromo-4- (methoxymethoxy) benzaldehyde in 32.79% yield. To the stirred solution were added 3-bromo-4- (methoxymethoxy) benzaldehyde (400 mg,1.63mmol,1.0 eq.) phenylboronic acid (298.52 mg,2.45mmol,1.5 eq.) and PdCl ₂ (dppf) (59.71 mg,0.081mmol,5 mol%) in1, 4-dioxane (5.0 mL) and reacted under argon atmosphere at 60 ℃ 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%). Structure generation was confirmed by LC-ESIMS and found to be M/z243.2 m+h ⁺.

General procedure for the synthesis of naringenin derivatives (i.e. the compounds):

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

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

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

3-fluoro-4-hydroxybenzaldehyde (154 mg,1.0mmol,1.0 eq.) is added to a 50mL eggplant-shaped flask equipped with a stirring bar, which contains acetone (10 mL), potassium carbonate (276.4 mg,2.0mmol,2.0 eq.) and bromomethyl ether (90. Mu.L, 1.1mmol,1.1 eq.). The mixture was heated to reflux for 3 hours. After completion of the reaction, methanol was added to quench the reaction, and a large amount of the organic phase was removed by rotary evaporation, followed by addition of 20mL of water and extraction three times with ethyl acetate (20 mL). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate and then concentrated in vacuo to give a tan oily liquid. The next operation is directly carried out without treatment.

In a 50mL eggplant-shaped flask, KOH (1.0 g,17.8 mmol), the unpurified crude product obtained in the previous step, and 2-hydroxy-4, 6-bis (methoxymethoxy) -1-acetophenone (256 mg,1.0mmol,1.0 eq.) were added. Then 20mL of ethanol and 2mL of water were added and stirred at room temperature for 18h. 2M HCl solution was added to adjust the pH to about 7.5-8 weakly basic. Three extractions were performed with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, then dried under vacuum and the solvent was removed. The crude product was purified by silica gel column chromatography (PE/etoac=15:1-10:1) to give 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 100.0mg and a total yield of 26.45% in two steps.

The chalcone obtained in the previous step was placed in a 50mL eggplant-shaped flask. 3.0mL of 2M HCl solution and 10.0mL of methanol were added, followed by heat refluxing for 24h. Monitored by TLC and UpLC (Ring-closed product UV has a characteristic absorption peak at 330-360nm, ring-open product UV has a characteristic absorption peak at 280 nm). After the reaction, the excess HCl was neutralized with an appropriate amount of saturated NaHCO ₃ and extracted three times with ethyl acetate (20 ml×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 column chromatography on silica gel (PE/etoac=5:1) to give 2- (3-fluoro-4-hydroxyphenyl) -5, 7-dihydroxy-4-dihydrochromone (NAR-27), 14.19mg of pale yellow solid, yield 21.54%. MF is C ₁₅H₁₁FO₅ and MW is 290.06. ¹H NMR(400MHz,DMSO-d₆ ) Delta was 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,1 h), ¹³ C NMR (200 MHz, acetone-d ₆) delta was 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. HRMS (ESI) m/z for C ₁₅H₁₂FO₅ ⁺[M+H]⁺ was calculated to be 291.0663 and found to be 291.0667.

2- (3, 5-Difluoro-4-hydroxyphenyl) -5, 7-dihydroxy-4-dihydrochromone (Compound 2, NAR-28)

Compound 2 (NAR-28) was synthesized by scheme (E) as follows:

The synthesis method is the same as the method of the condition (a) above. The product was compound 2 (NAR-28) as a pale yellow solid (38.66 mg, 12.54%); ¹ H NMR (500 MHz, acetone-d ₆) delta was 11.71 (s, 2H), 9.23 (s, 1H), 7.34-7.23 (m, 4H), 5.93 (s, 2H), 3.39 (dd, J=8.3, 7.1Hz, 2H) and 3.01-2.94 (m, 2H). ¹³ C NMR (150 MHz, acetone-d ₆) delta were 196.44、167.55、164.93、163.80、154.01(d,J＝7.0Hz)、152.41(d,J＝7.0Hz)、134.83(t,J＝17.1Hz)、131.04(t,J＝6.1Hz)、110.88(dd,J＝17.1,6.1Hz)、102.97、96.97、95.96、78.63 and 43.29. HRMS (ESI) m/z for C ₁₅H₁₁F₂O₅ ⁺[M+H]⁺ was calculated to be 293.0575 and found to be 293.0577.

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):

The synthesis method is the same as the method of the condition (a) above. Compound 2 (NAR-29) was obtained as a pale yellow solid (13.98 mg, 4.56%); ¹H NMR(400MHz,DMSO-d₆) delta was 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 C NMR (200 MHz, acetone-d ₆) delta was 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. HRMS (ESI) m/z for C ₁₅H₁₂ClO₅ ⁺[M+H]⁺ was calculated to be 307.0368 and found to be 307.0367.

2- (3, 5-Dichloro-4-hydroxyphenyl) -5, 7-dihydroxy-4-dihydrochromone (Compound 4, NAR-30)

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

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

To a solution of 3, 5-dichloro-4-hydroxybenzaldehyde (191 mg,1.0 mmol) and DIPEA (522 μl,3.0mmol,3.0 eq.) in dry ethylene (10 mL) was added MOMBr (183 μl,2.2mmol,2.2 eq.) under argon at 0 ℃. The reaction mixture was stirred at 0 ℃ for 4h. The reaction was quenched by the addition of 100mL of water, and then extracted three times with ethylene (50 mL). The organic phases were combined, washed with brine, dried over anhydrous MgSO ₄, and then evaporated under reduced pressure to give the crude bis-MOM protected intermediate.

To a solution of 2-hydroxy-4, 6-bis (methoxymethoxy) -1-acetophenone in dry THF (10 mL) (256 mg,1.0mmol,1.0 eq.) was added NaH (60% dispersed in paraffinic oil) (80.0 mg,2.0mmol,2.0 eq.) in portions, under nitrogen and with vigorous stirring at 0 ℃. When H ₂ stopped forming, a solution of the above two MOM protection intermediates in dry THF (5.0 mL) was added dropwise to the reaction mixture for 15min, then the reaction mixture was stirred at room temperature for 2H unless otherwise stated. After the reaction was completed, the reaction was slowly quenched with ice water, extracted three times with ethyl acetate (20 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography (PE/etoac=15:1-10:1) to give 378mg of a yellow solid as 3- (4-trifluoromethylphenyl) -1- (2-hydroxy-4, 6-bis (methoxymethoxy) phenyl) prop-2-en-1-one in 88.30% yield.

All chalcone obtained in the previous step is put into a 50mL eggplant bottle, 10.0mL of methanol and 3.0mL of 2M HCl solution are added, and the mixture is heated and refluxed for 24h. TLC and UpLC monitoring (cyclization product UV has characteristic absorption peak at 330-360nm, ring-opening product UV has characteristic absorption peak at 280 nm). After completion of the reaction, excess HCl was neutralized with an appropriate amount of saturated NaHCO ₃ and extracted three times with ethyl acetate (20 ml×3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and the solvent was removed in vacuo.

The crude product was purified by column chromatography on silica gel (PE/etoac=8:1-5:1) to give 2- (3, 5-dichloro-4-hydroxyphenyl) -5, 7-dihydroxy-4-dihydrochromone (NAR-30) as a pale yellow solid of form a (56.78 mg, 16.67%); ¹ HNMR (500 MHz, acetone-d ₆) δ was 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, 3.1hz, 1H), 3.18 (dd, j=17.1, 12.9hz, 1H) and 2.82 (dd, j=17.1, 3.1hz, 1H). ¹³ C NMR (125 MHz, acetone-d ₆) delta were 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. HRMS (ESI) m/z for C ₁₅H₁₁Cl₂O₅ ⁺[M+H]⁺ was calculated to be 340.9978 and found to be 340.9981.

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

Compound 5 (NAR-31) was synthesized by scheme (H) as follows:

The synthesis method is the same as the method of the condition (b) above. The product was compound 5 (NAR-31) as a pale yellow solid (63.78 mg, 18.16%), ¹ H NMR (400 MHz, acetone-d ₆) delta 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) delta 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. HRMS (ESI) m/z for C ₁₅H₁₂BrO₅ ⁺[M+H]⁺ was calculated to be 350.9863 and found to be 350.9861.

2- (3, 5-Dibromo-4-hydroxyphenyl) -5, 7-dihydroxy-4-dihydrochromone (Compound 6, NAR-32)

Compound 6 (NAR-32) was synthesized by scheme (I) as follows:

The synthesis method is the same as the method of the condition (b) above. Compound 6 (NAR-32) was obtained as a pale yellow solid (90.21 mg, 20.98%); ¹H NMR(400MHz,DMSO-d₆) delta was 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) delta was 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. HRMS (ESI) m/z for C ₁₅H₁₁Br₂O₅ ⁺[M+H]⁺ was calculated to be 428.8968 and found to be 428.8972.

5, 7-Dihydroxy-2- (4-hydroxy-3-methoxyphenyl) -4-dihydro-chromone (Compound 7, NAR-33)

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

The synthesis method is the same as the method of the condition (b) above. Compound 7 (NAR-33) was obtained as a pale white solid (102.6 mg, 34.47%); ¹H NMR(400MHz,DMSO-d₆) delta was 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), and ¹³ C NMR (125 MHz, acetone) delta was 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. HRMS (ESI) m/z for C ₁₆H₁₅O₆ ⁺[M+H]⁺ was calculated to be 303.0863 and found to be 303.0873.

5, 7-Dihydroxy-2- (4-hydroxy-3-nitrophenyl) -4-dihydro-chromone (Compound 8, NAR-34)

Compound 8 (NAR-34) was synthesized by scheme (K) as follows:

The synthesis method is the same as the method of the condition (b) above. Compound 8 (NAR-34) was obtained as a pale white solid (8.47 mg, 1.89%); ¹H NMR(400MHz、DMSO-d₆) delta was 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) delta 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 was found to be M/z318.2[ M+H ] ⁺ and M/z316.2[ M-H ] ^-.

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

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

The synthesis method is the same as the method of the condition (b) above. Compound 9 (NAR-35) was obtained as a yellow solid (68 mg, 20.50%). ¹H NMR(400MHz,DMSO-d₆ ) Delta 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);¹³C NMR(125MHz, acetone-d ₆) delta 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. HRMS (ESI) m/z for C ₂₁H₁₇O₅ ⁺[M+H]⁺ was calculated to be 349.1071 and found to be 349.1074.

5, 7-Dihydroxy-2- (2-fluoro-4-hydroxyphenyl) -4-dihydro-chromone (compound 10, NAR-37) compound 10 (NAR-37) was synthesized by scheme (M) as follows:

The synthesis method is the same as the method of the condition (b) above. Compound 10 (NAR-37) was obtained as a pale white solid (96 mg, 23.09%). ¹ H NMR (500 MHz, acetone-d ₆) delta 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＝1.7Hz,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);¹³C NMR(125MHz, acetone-d ₆) delta 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. HRMS (ESI) m/z for C ₁₅H₁₂FO₅ ⁺[M+H]⁺ was calculated to be 291.0663 and found to be 291.0680.

5, 7-Dihydroxy-2- (2, 6-difluoro-4-hydroxyphenyl) -4-dihydro-chromone (Compound 11, NAR-38)

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

The synthesis method is the same as the method of the condition (b) above. Compound 11 (NAR-38) was obtained as a pale yellow-white solid (102 mg, 32.11%). ¹ H NMR (800 MHz, acetone-d ₆) delta was 6.55(d,J＝10.7Hz,2H)、5.96(d,J＝2.2Hz,1H)、5.94(d,J＝2.2Hz,1H)、5.75(dd,J＝13.9,3.0Hz,1H)、3.48(dd,J＝17.1,13.8Hz,1H) and 2.72 (dd, j=17.1, 3.1hz, 1H). ¹³ C NMR (200 MHz, acetone-d ₆) delta 196.7、167.4、165.1、164.2、163.7(d,J＝11.3Hz)、162.4(d,J＝11.3Hz)、161.3、105.6、102.9、100.5、100.4、97.0、95.7、71.1 and 41.0. HRMS (ESI) m/z for C ₁₅H₁₁F₂O₅ ⁺[M+H]⁺ was calculated to be 309.0569 and found to be 309.0573.

5, 7-Dihydroxy-2- (2-chloro-4-hydroxyphenyl) -4-dihydro-chromone (Compound 12, NAR-39)

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

The synthesis method is the same as the method of the condition (b) above. Compound 12 (NAR-39) was obtained as a white solid (84 mg, 27.45%). ¹ H NMR (500 MHz, acetone-d ₆) delta was 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) delta were 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. HRMS (ESI) m/z for C ₁₅H₁₂ClO₅ ⁺[M+H]⁺ was calculated to be 307.0368 and found to be 307.0368.

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

Compound 13 (NAR-40) was synthesized by scheme (P) as follows:

The synthesis method is the same as the method of the condition (b) above. The resulting product, compound 13 (NAR-40), was a pale white solid (79 mg, 22.57%). ¹ H NMR (800 MHz, acetone-d ₆) delta was 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,2H)、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) delta were 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. HRMS (ESI) m/z for C ₁₅H₁₂BrO₅ ⁺[M+H]⁺ was calculated to be 350.9863 and found to be 350.9867.

As described herein, the inventors of the present invention found that NAR promotes an increase in the number of oxidized muscle fibers and aerobic metabolism by up-regulating the expression of the pair Esrrg. Thus, up-regulation of relative Esrrg expression is a key indicator in assessing whether NAR analogs have similar function.

Compounds 1-13 were used to test relative Esrrg expression. Each compound was dissolved in DSMO and tested after addition of water to form an aqueous solution. The term "relative Esrrg expression" as used herein refers to Esrrg expression in the group treated with the compound relative to the expression in the control group treated with DMSO alone. During experimental testing, C2C12 myotubes were treated with DMSO or NAR (100 μm, 200 μm and 400 μm) or other one compound, respectively. Total RNA from the C2C12 myotubes was extracted using TRIzol reagent. mRNAj in the total RNA was reverse transcribed using oligo-dT primer and HISCRIPT II reverse transcriptase to synthesize cDNA. Quantitative real-time PCR was performed using SYBR GREEN FAST QPCR Mix. The SYBR Green dye will bind to cDNA in the sample immediately after it is added to the sample. During PCR, the DNA polymerase will amplify the target sequence, producing a PCR product called an "amplicon". Then, SYBR Green dye will bind to each nascent cDNA molecule. As PCR proceeds, more and more amplicons are produced. When SYBR Green dye binds to all cDNAs, the fluorescence intensity increases with the increase of PCR products. Ct values for each compound (number of cycles when fluorescence signal exceeds a fixed threshold for no template control samples) were obtained. Finally, the relative quantitative method was used to analyze the change in the amount of gene expression of NAR or other compounds in samples treated with DMSO relative to the amount of gene expression of Esrrg in samples treated with DMSO, and the relative amounts of expression of Esrrg in the different samples were obtained using the arithmetic formula 2 ^-△△Ct.

The relative Esrrg expression results for compounds 1-13 and NAR are shown in Table 2. Where a compound exhibits a relative Esrrg expression approaching or exceeding 2, it is desirable for the compound to have the desired biological activity, e.g., to improve skeletal muscle endurance, treat muscle atrophy or dystrophy, or prevent muscle atrophy or dystrophy in a subject in need thereof. Relative Esrrg expression measurements are models to evaluate these desired activities. Naringenin and its derivatives provided in the present disclosure have been demonstrated to have high relative Esrrg expression and desirable biological or pharmaceutical activity.

TABLE 2

In some embodiments, the only substituent group (represented by R ₁ or R) may be ortho or meta to the hydroxy group of the phenyl group attached to the dihydrochromone. The compound has a structure shown as a formula (III) or (IV) respectively.

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 Esrrg expression results showed a trend of Cl > Br > OMe > F in the order of substituents. When R is NO ₂ or phenyl, the solubility of these compounds in water is low and NO results of relative Esrrg expression are obtained. Further research is required to increase solubility.

For compounds having the chemical structure of formula (IIV) with R in the meta position to the hydroxy group, 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, relative Esrrg expression results showed a trend of F and Cl > Br in order of substituents.

Some examples described above and in table 2 include two substituents, in addition to the hydroxyl group on the phenyl group attached to the azulene 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 having two halogen substituents may have lower relative Esrrg expression values than the corresponding counterpart having one halogen substituent. However, all other compounds with two halogen substituents, except NAR-30, have relative Esrrg expression values near or above 2, indicating that these compounds have the desired biological or pharmacological activity.

Based on these results, when at least one substituent group as described herein (other than hydroxy) is used to modify the phenyl group attached to the dihydrochromone, a compound having the desired biological or pharmaceutical activity can be obtained.

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although the subject matter has been described herein in terms of exemplary embodiments, this is not limited to such. Rather, the appended claims should be construed broadly, to include other variants and embodiments which may be made by those skilled in the art.

Claims (24)

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, R ₁ , R ₂ , R ₃ and R ₄ are each selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxy and phenyl. 2 . The composition according to claim 1 , wherein one or two of R ₁ , R ₂ , R ₃ and R ₄ are substituents other than H. 3. The composition of claim ₁ , wherein _R1 , _R2 , _R3 and _R4 are each selected from the group consisting of H, F, Cl, Br, OH and NH2. The composition according to claim 1 , wherein R ₁ or R ₂ is a substituent other than H, R ₃ ═H, and R ₄ ═H. 5. The composition according to claim 1, wherein the compound is selected from the group consisting of: 6. The composition of claim 1, wherein the excipient is selected from 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. 7. The composition of claim 1, wherein the excipient comprises a vehicle comprising 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 compound is 1-50 mM. 11. A compound of formula (I), a pharmaceutically acceptable solvate thereof, or any combination thereof, Wherein, 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 it comprises the following steps: reacting 1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethanone 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 of claim 16, further comprising 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 comprises: 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 of claim 18, wherein the subject is a mammal. 20. The method of claim 18, wherein the subject is a human subject. 21. The method of 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 of claim 22, wherein the effective dose of the compound is 3.6-7.6 mg/Kg. 24. The method of 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 .