MAKING AND METHOD OF USING THE SAME

PRIORITY CLAIM AND CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/323,146, filed March 24, 2022, which application is expressly incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The disclosure relates to generally a composition having pharmaceutical or functional properties. More particularly, the disclosed subject matter relates to a composition comprising naringenin or a derivative thereof, a method of making the same, and a method of using the same, for example, as a pharmaceutical composition, a functional composition, and/or a dietary supplement.

BACKGROUND

[0003] Skeletal muscle is the largest organ in a body of a mammal and plays an extremely important role in supporting movement, heat production, and metabolic regulation. However, its function is impaired by aging, a sedentary lifestyle, and muscle-related diseases, causing it to show reduced endurance or strength. Skeletal muscle atrophy and reduced muscle function accompany the aging process (Porter et al., 1995). Studies have shown that older individuals have decreased cross-sectional areas of fast muscle fibers, reduced expression of mitochondrial respiratory chain complexes and reduced aerobic respiration in skeletal muscle (Carter et al., 2015; Murgia et al., 2017). Accordingly, in terms of function, older mice have 26% lower absolute grip strength and 15% lower relative grip strength than adult mice (McArdle et al., 2004).

[0004] In addition, mice with lifelong sedentary behavior have more age-related loss of muscle mass and mitochondrial dysfunction than mice that exercise regularly on a running wheel (Figueiredo et al., 2009). Aging in sedentary men is associated with a decline in type Ila fibers (oxidative myofibers) and a decrease in the mitochondrial content of all fiber types (St-Jean- Pelletier et al., 2017). Moreover, various muscular diseases lead to muscle atrophy and dysfunction. For example, Duchenne muscular dystrophy (DMD), the most common muscular dystrophy in children, causes proximal muscle weakness and calf hypertrophy, eventually leading to degeneration of skeletal and cardiac muscle (Falzarano et al., 2015; Kamdar and Garry, 2016). Therefore, healthy skeletal muscle is vital to organisms, and muscle impairment greatly affects health and quality of life.

[0005] Strategies to combat muscle loss and functional decline are urgently needed. Exercise has been demonstrated to be an effective strategy to improve muscle function and reverse muscle aging to some extent. However, exercise is not appropriate for patients who are bedridden for a long time or who have other clinical complications. Therefore, drug therapies to reduce skeletal muscle loss and restore muscle function are need.

[0006] In recent years, the signaling pathways involved in muscle dysfunction have been clarified, and different types of molecular mediators, such as proinflammatory cytokines, growth factors and transforming growth factor-β (TGF-β) family effectors, have been found to participate in skeletal muscle atrophy (Furrer and Handschin, 2019, Lynch et al., 2007).

Focusing on these contributing factors, several studies have identified therapeutic targets or drugs for ameliorating muscle loss. For example, it has been reported that cyclooxygenase 2 (COX2) inhibitors significantly reduce the plasma levels of interleukin 6 (IL-6) and interleukin 1 (IL-1) and increase muscle mass in aged rats (Rieu et al., 2009). Myostatin (MSTN) powerfully reduces muscle mass; therefore, MSTN has drawn widespread attention as a therapeutic target. LY2495655, a humanized MSTN antibody, increased muscle mass in elderly individuals in a clinical trial; however, grip strength was not affected (Becker et al., 2015). Even though decreases in muscle mass can be ameliorated with some drugs, most trials have failed to show significant improvements in functional parameters (Furrer and Handschin, 2019). Skeletal muscle remains one of the most pharmacologically undertreated organs.

[0007] In addition to the effectiveness of a drug, drug safety also needs to be considered. Testosterone has been shown to prevent age-associated loss of muscle strength and improve physical function (Srinivas-Shankar et al., 2010; Storer et al., 2017); however, its clinical use is substantially limited by severe side effects (Grech et al., 2014). Therefore, new natural medicines that are safer and more effective than existing drugs need to be discovered that can improve the physiological function of skeletal muscle or protect against muscle atrophy. [0008] Naringenin (NAR), a dihydroflavonoid, is generally found in the rosaceae, rutaceae, and citrus family plants as naringin. It is reported that NAR has important biological activities and has potential positive effects on metabolic diseases, cardiovascular diseases, cancer, pulmonary disorders, neurodegenerative diseases and gastrointestinal pathologies (Rivoira et al., 2021). For example, NAR supplementation increases energy expenditure, enhances insulin sensitivity, and increases hepatic fatty acid oxidation, thereby reducing fat mass and improving metabolic function (Alam et al., 2013; Goldwasser et al, 2010; Pu et al., 2012; Rebello et al., 2019; Sacks et al., 2018). In terms of the mechanism of action, NAR affects energy metabolism mainly through the PPAR family, PGC-1 family and AMPK signaling pathway (Goldwasser et al., 2011; Mulvihill et al., 2009; Yu et al., 2019).

SUMMARY

[0009] The present disclosure provides a composition and a method for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof.

[0010] In the present disclosure, naringenin (NAR) has been found to improve muscle endurance and ameliorate muscle dysfunction in both naturally aging mice and mdx mice by increasing oxidative myofiber numbers and aerobic metabolism. The transcription factor Spl has been identified as a direct target of NAR by biotin-labeled co-immunoprecipitation mass spectrometry, and the binding site has been further validated to be GLN-1 10 NAR enhances the binding of Spl to the CCCTGCCCTC sequence of the Esrrg promoter by upregulating the level of Spl phosphorylation, thus upregulating Esrrg expression. The discovery of the Spl-ERRγ transcriptional axis is of great significance in basic skeletal muscle research, and the new function of NAR has potential implications for the prevention of sedentary lifestyle-related declines in aerobic exercise capacity and age-or disease-related muscle atrophy.

[0011] Based on the findings with naringenin, different compounds including its derivatives thereof are obtained and are used for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof. Compositions including at least one derivative are also obtained. [0012] 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 thereof. Such a composition comprises an effective amount of a compound having formula (I) (also having a code SI) as described herein or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient

[0013] The formula (I) as a chemical structure below:

[0014] In the formula (I), each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl. In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ . Such a compound is naringenin or a naringenin derivative.

[0015] In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ each is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H.

[0016] The composition can be a pharmaceutical composition, a functional composition, and/or a dietary supplement. For example, the composition is a pharmaceutical composition, which can be injectable or orally administrated. The composition is preferably injectable. The compound may have a concentration in a range of from 1 mM to 50 mM, for example, 2 mM to 15 mM, 5 mM to 10 mM, or any other suitable concentration.

[0017] The excipient may be a solvent, a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrating agent, a lubricant, a surfactant, an emulsifying agent, a suspending agent, or any combination thereof. For example, in one injectable composition, the excipient comprises a vehicle comprising 20% DMSO and 80% saline by weight. [0018] 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 compound genus or species as described herein. In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl. At least one of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. Such a compound is a naringenin derivative. In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ .

[0019] In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H.

[0020] The compound can be a suitable compound having desired solubility and pharmaceutical properties.

[0021] In another aspect, the present disclosure provides a method of making the composition or the compound as described herein. Such a method may include preparing the compound. The method may further comprise mixing the excipient and the compound.

[0022] In another aspect, the present disclosure provides a method of for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof. The method comprises administrating a suitable amount of the composition as described herein into a subject in need thereof.

[0023] In some embodiments, in the compound having formula (I), each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl At least one of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H

Such a compound is naringenin or a naringenin derivative. In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ .

[0024] In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H.

[0025] In some embodiments, the subject is a mammal, preferably a human subject, which can be a healthy human, or an adult having an age-or disease-related muscle atrophy. [0026] In some embodiments, the composition is intramuscularly injected or orally administrated. It is preferably injected. In some embodiments, the composition is intramuscularly injected with a dose of the effective amount of the compound in a range of from 2 mg/Kg to 20 mg/Kg, with a frequency of once daily or once every other day. The administration can be at any suitable dosage. For example, in some embodiments, the dose of the effective amount of the compound is in a range of from 3.6 mg/Kg to 7.6 mg/Kg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.

[0028] FIGS. 1-3 show maximum treadmill running distance (FIG. I), limb grip strength normalized to body weight (FIG. 2), gastrocnemius (GAS) muscle weights normalized to body weight (FIG. 3) of middle-aged mice with gastrocnemius intramuscular injection of vehicle or NAR. n=6/group, 10-month-old C57 mice.

[0029] FIGS. 4-6 show maximum treadmill running distance (FIG. 4), limb grip strength normalized to body weight (FIG. 5), gastrocnemius (GAS) muscle weights normalized to body weight (FIG 6) of young mice with gastrocnemius intramuscular injection of vehicle or NAR n=9-l 0/group, 2-month-old C57 mice.

[0030] FIGS. 7-9 show maximum treadmill running distance (FIG. 7), limb grip strength normalized to body weight (FIG. 8), gastrocnemius (GAS) muscle weights normalized to body weight (FIG. 9) of C57 or mdx mice with gastrocnemius intramuscular injection of vehicle or NAR. n=4-6/group, 4-month-old C57 mice or 4-month-old mdx mice.

[0031] FIG. 10 shows mRNA expression of Myh7, Myh2 and Myh4 in the gastrocnemius muscles of 10-month-old C57 mice treated with vehicle or NAR. n=6/group.

[0032] FIG. 11 shows the quantitative statistical results of MyHC I myofibers by IF staining in the gastrocnemius muscle of 10-month-old C57 mice treated with vehicle or NAR. [0033] FIG. 12 shows mRNA expression of aerobic metabolism-related genes in the gastrocnemius muscles of 10-month-old C57 mice treated with vehicle or NAR. n=6/group.

[0034] FIG. 13 shows mRNA expression of Myh7, Myh2 and Myh4 in the gastrocnemius muscles of 2-month-old C57 mice treated with vehicle or NAR. n=9-10/group.

[0035] FIG. 14 shows the quantitative statistical results of MyHC I myofibers by IF staining in the gastrocnemius muscle of 2-month-old C57 mice treated with vehicle or NAR. n=9-l 0/group.

[0036] FIG. 15 shows mRNA expression of aerobic metabolism -related genes in the gastrocnemius muscles of 2-month-old C57 mice treated with vehicle or NAR. n=9-l 0/group. [0037] FIG. 16 shows the quantitative statistical results of the levels of five OXPHOS complexes by western blot analysis of 2-month-old C57 mice treated with vehicle or NAR. β- Actin was used as the loading control. n=7/group.

[0038] FIG. 17 shows mRNA expression of aerobic metabolism-related genes in the gastrocnemius muscles of 4-month-old C57 mice or mdx mice treated with vehicle or NAR. n=4- 6/group.

[0039] FIG. 18 shows the morphology of C2C 12 myotubes treated with DMSO or different concentrations (40, 100, 200, 400, 800, 1600, and 2400 μM) of NAR.

[0040] FIG. 19 shows mRNA expressioonf M yh7, Myh2 and Myh4 in C2C12 myotubes treated with DMSO or different concentrations of NAR.

[0041] FIG. 20 shows ATP levels in C2C12 myotubes treated with DMSO or different concentrations of NAR.

[0042] FIG. 21 shows OCR ₃ of C2C12 myotubes treated with DMSO or NAR.

Oligomycin was added to block ATP-coupled respiration, FCCP was added to induce maximal respiration, and antimycin A/rotenone was added to block mitochondrial electron transport. n=four biological replicates.

[0043] FIG. 22 shows the statistical results of the levels of five OXPHOS complexes for C2C12 myotubes treated with DMSO or NAR.

[0044] FIG. 23 shows mRNA expression of genes related to OXPHOS, the TCA cycle and β-oxidation in C2C12 myotubes treated with DMSO and NAR. Due to limited space, changes without significant differences are not marked. [0045] FIG. 24 shows qPCR analyses of ERRγ levels statistical results for C2C12 myotubes treated with vehicle (DMSO) or NAR. β- Actin was used as the loading control.

[0046] FIG. 25 shows qPCR analyses of ERRy levels for the gastrocnemius muscles of middle-aged mice, young mice and mdx mice treated with vehicle or NAR.

[0047] FIG. 26 shows mRNA expression of Esrrg, Myh7, Myh2, Atp5b and Cptlb in C2C12 myotubes (NC group and Esrrg knockdown group) treated with DMSO or NAR.

[0048] FIG. 27 shows western blot analyses of Spl levels in two sets of experiments including biotin and NAR-biotin.

[0049] FIG. 28 shows CETSA performed to evaluate the interaction between NAR and Spl in cell lysates (in vitro).

[0050] FIG. 29 shows glide docking model of the compound NAR bound with the Spl predicted structure. The compound NAR is shown as blue sticks. The yellow dashed lines illustrate three predicted H-bonds with the ASN-81, SER-83 and GLN-110 residues of Spl.

[0051] FIGS. 30-31 show maximum treadmill running distance (FIG. 30) and grip strength (FIG. 31) of the middle-aged mice treated with or without NAR after Mit-A pretreatment. n=6/group, 10-month-old C57 mice.

[0052] FIG. 32 shows the quantitative statistical results for MyHC I myofibers in the gastrocnemius muscles by IF staining of middle-aged mice treated with or without NAR after Mit-A pretreatment.

[0053] FIG. 33 shows mRNA expression of My7h, Myh2 and Myh4 in the gastrocnemius muscles of middle-aged mice treated with or without NAR after Mit-A pretreatment. n=6/group, 10-month-old C57 mice.

[0054] FIG. 34 shows qPCR (F) analyses of ERRγ levels in the gastrocnemius muscles of middle-aged mice treated with or without NAR after Mit-A pretreatment. β-Actin was used as the loading control. n=6/group, 10-month-old C57 mice.

[0055] FIG. 35 shows mRNA expression of energy metabolism-related genes in the gastrocnemius from the middle-aged mice treated with or without NAR after Mit-A pretreatment. n=6/group, 10-month-old C57 mice.

[0056] FIG. 36 shows deletion analysis and mutation analysis of the potential transcription factor binding site (Spl) on the Esrrg promoter in HEK293T cells. [0057] FIG. 37 shows analysis of the induction and response of NAR by mutation of the predicted NAR binding site in the Spl overexpression vector (Spl-MT-Pocketl-GLN) in HEK293T cells.

[0058] FIG. 38 shows the western blot analysis of the effect of NAR on the phosphorylation level of Spl. Total Spl level was used as the loading control.

DETAILED DESCRIPTION

[0059] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “an additive” is a reference to one or more of 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 refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to 8.8, inclusive; 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 a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “ 1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e g , by a negative limitation in the claims For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.

[0060] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the term “patient” refers to an animal, preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), and most preferably a human. In some embodiments, the subject is a non-human animal such as a farm animal (e g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is a human. In another embodiment, the subject is a human adult. In another embodiment, the subject is a human child. In yet another embodiment, the subject is a human infant.

[0061] As used herein, the term “agent” refers to any molecule, compound, methodology and/or substance for use in the prevention, treatment, management and/or diagnosis of a disease or condition. As used herein, the term “effective amount” refers to the amount of a therapy that is sufficient to result in the prevention of the development, recurrence, or onset of a disease or condition, and one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity, the duration of a disease or condition, ameliorate one or more symptoms of a disease or condition, prevent the advancement of a disease or condition, cause regression of a disease or condition, and/or enhance or improve the therapeutic effect(s) of another therapy.

[0062] As used herein, the phrase “pharmaceutically acceptable” means 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.

[0063] As used herein, the term “therapeutic agent” refers to any molecule, compound, and/or substance that is used for the purpose of treating and/or managing a disease or disorder. [0064] As used herein, the terms “therapies” and “therapy” can refer to any method(s), composition(s), and/or agent(s) that can be used in the prevention, treatment and/or management of a disease or condition, or one or more symptoms thereof. In certain embodiments, the terms “therapy” and “therapies” refer to small molecule therapy.

[0065] As used herein, the terms “treat,” “treatment,” and “treating” in the context of the administration of a therapy to a subject refer to the reduction or inhibition of the progression and/or duration of a disease or condition, the reduction or amelioration of the severity of a disease or condition, such as cancer, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.

[0066] As used herein, the term “excipient” refers to an inactive substance that serves as the vehicle or medium for a drug or other active substance. Examples of a suitable excipient include, but are not limited to, a solvent, a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrate, a lubricant, a surfactant, an emulsifying agent, a suspending agent, or any combination thereof.

[0067] As used herein, the term “dihydrochromone” and the term “chromanone” are interchangeable and both refer to the same chemical structure. Unless indicated otherwise, the compounds described herein is racemic or has no chirality. Naringenin (NAR) or NAR derivatives used in the present disclosure are racemic.

[0068] The effects of NAR on skeletal muscle are less well studied; the existing findings are mainly related to diabetic obesity. In L6 myotubular cells, NAR activates AMPK and directly stimulates muscle glucose uptake in an insulin-independent manner, which suggests that NAR may regulate skeletal muscle glucose homeostasis (Zygmunt et al., 2010). NAR can alleviate palmitic acid- and fructose-induced insulin resistance by increasing the expression of GLUT4 in mouse skeletal muscle (Mutlur Krishnamoorthy and Carani Venkatraman, 2017). In L6 skeletal muscle cells, NAR increases glucose uptake by myocytes by activating AMPK and thereby increasing GLUT4 translocation (Bhattacharya et al., 2013; Zygmunt et al., 2010). However, the effects of NAR on muscle endurance and muscle atrophy are still unclear.

[0069] In this study, we explored whether NAR can improve muscle function and protect against muscle atrophy in the contexts of aging process or muscle diseases. Using young adult mice, naturally aging mice and mdx mice (a preclinical model of DMD) as models, we assessed the effect of NAR on exercise capacity and aerobic metabolic levels in skeletal muscle. We found that NAR increases oxidative myofiber numbers, enhances aerobic respiration in vivo and in vitro and ameliorates muscle dysfunction in both naturally aging and mdx mice.

Mechanistically, we found that Spl is the direct binding target of NAR and that NAR exerts its effects on skeletal muscle through activation of the Spl -estrogen-related receptor y ( ERRγ) transcriptional axis. Our results will provide new strategies for improving muscle function and treating muscle atrophy.

[0070] Skeletal muscle function can be damaged by aging, a sedentary lifestyle or disease; however, until now, skeletal muscle has remained one of the organs most undertreated with medication. In the present disclosure, naringenin (NAR) was found to improve muscle endurance and ameliorate muscle dysfunction in both naturally aging mice and mdx mice by increasing oxidative myofiber numbers and aerobic metabolism. The transcription factor Spl was identified as a direct target of NAR by biotin-labeled co-immunoprecipitation mass spectrometry, and the binding site was further validated to be GLN-110. NAR enhances the binding of Spl to the CCCTGCCCTC sequence of the Esrrg promoter by upregulating the level of Spl phosphorylation, thus upregulating Esrrg expression. The discovery of the Spl-ERRγ transcriptional axis is of great significance in basic skeletal muscle research, and the new function of NAR has potential implications for the prevention of sedentary lifestyle-related declines in aerobic exercise capacity and age-or disease-related muscle atrophy.

[0071] The inventors have found a new function of NAR in improving skeletal muscle endurance and protecting against muscle atrophy in both naturally aging and mdx mice via binding to transcription factor Spl (Spl) to promote Spl phosphorylation. Phosphorylated Spl directly upregulates the transcript levels of estrogen-related receptor gamma (ERRγ), which in turn regulates energy metabolism and muscle remodeling.

[0072] Naringenin (NAR) ameliorates muscle dysfunction in both natural aging and mdx mice. NAR increases oxidative myofiber numbers and enhances aerobic metabolism. NAR binds to Spl to increase its phosphorylation and transcription factor activity. Activation of the Spl- ERRγ axis promotes energy metabolism and muscle remodeling.

[0073] The inventors further modify naringenin to obtain different derivatives thereof, which are expected to provide improved properties.

[0074] The present disclosure also provides a composition comprising naringenin or a derivative thereof, a derivative compound, a method of making the same, and a method of using the same, for example, as a pharmaceutical composition, a functional composition, and/or a dietary supplement.

[0075] Methods

[0076] General chemistry

[0077] NAR (Naringenin) (Lot# QY00305-180610, purity > 98%) was purchased from

Qing Yun Biology Co., Ltd (Nanjing, China). The purity of the Naringenin probe (NAR-Biotin) was determined on a Waters ACQUITY UPLC system with an ELSD, a PDA detector, a sample manager, and a binary solvent manager. Preparative HPLC was run on a Varian PrepStar system with an Alltech 2424 ELSD and 2489 PDA using a Waters Sunfire RP Cl 8 (5 μM, 30 x 150 mm) column. Electrospray ionization (ESI)-MS spectra were obtained on a Waters 2695 instrument with a 2998 PDA detector coupled with a Waters ACQUITY ELSD and a Waters 3100 SQDMS detector using a Waters Sunfire RP C ₁ 8 column (4.6 x 150 mm, 5 μM) with a 1.0 mL/min flow rate. HRESI-MS was performed on a Waters ACQUITY UPLC System (Waters Corporation, Milford, MA, USA) equipped with an ESI ion source. MS detection was conducted with a Synapt G2-Si Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). 1H and 13C NMR spectra were recorded on a Bruker AVANCE III 500 MHz instrument. Chemical shifts are reported in ppm (8), and coupling constants (J values) are reported in hertz. Chemical shifts are reported in ppm with Me4Si as a reference standard.

[0078] Synthesis of the NAR (Naringenin) probes (NAR-biotin)

[0079] NAR (272 mg, 1 .0 mmol), biotin (244 mg, 1 . 1 mmol, 1.1 eq.) and DMAP (12.2 mg, 0.1 mmol, 0.1 eq.) were dissolved in 20 ml of anhydrous DMF. EDCI (310 mg, 2.0 mmol, 2.0 eq.) and Et3N (277 μL, 202 mg, 2.0 eq.). The solution was stirred at the room temperature overnight. The reaction was quenched with 2 M HC ₁ solution (4 ml), diluted with 100 ml of water, and then extracted with ethyl acetate. The combined organic phase was washed with water, and brine and dried with anhydrous MgSO ₄ . The organic solvents were evaporated under reduced pressure and the residue was purified by preparative HPLC (MeCN in water containing 0. 1% HCCOH, 40-60%, 0-35 min) to give the product (300 mg, 60% yield). Purity at 288 nm: 99.93%; 1H NMR (500 MHz, Py-d5) δ 7.64 and 7.38 (d, J = 8.6 Hz, each 1H), 7.60 and 7.47 (br.s, each 1H), 6.50 and 6.43 (d, J = 2.2 Hz, each 1H), 5.55 (dd, J = 13.1, 3.0 Hz, 1H), 4.58 (m, 1H), 4.42 (m, 1H), 3.26 (m, 1H), 3.19 (dd, J = 17.0, 13.1 Hz, 1H), 2.98 (dd, I = 12.5, 5.0 Hz, 1H), 2.91 (m, 1H), 2.89 (dd, J = 17.0, 3.0 Hz, 1H), 2.56 (t, J = 7.4 Hz, 2H), 1.85-1.94 (m, 2H), 1.70-1.76 (m, 2H), 1.58-1.63 (m, 2H); 13CNMR (125 MHz, 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, 25.52; ESI-MS m/z 499.24 [M+H]+, 497 24 [M-H]+; HRESI-MS (m/z) calc, for C25H27N2O7S [M+H]+: 499.1539; found: 499.1544.

[0080] Mice and cell lines

[0081] HEK293T cells (human embryonic kidney cells, female), and C2C12 cells (mouse mesenchymal precursor cells, sex unknown) obtained from the American Type Culture Collection (ATCC). The cells were cultured at 37 °C, under 5 % CO ₂ in 4.5 g/L (25 mM) glucose Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (Gibco) and penicillin/ streptomycin (Gibico 15070063). C2C12 cell differentiation toward myotubes was induced with 2% horse serum (Gibico) 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 C57BL/10ScSnJGpt-Dmdem3Cd4/Gpt (mdx), male mice aged 4 months purchased from GemPharmatech Until the beginning of the experiments, these mice were housed in standard cages in groups of 2-6 animals under standardized conditions in a temperature-controlled room of 21- 23 °C maintained on a 12:12 h light-dark cycle with standard mouse chow and water available ad libitum. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Standing Committee on Animals at the University of the Chinese Academy of Sciences.

[0082] Mice skeletal muscle intramuscular injection

[0083] The gastrocnemius and tibialis anterior muscles (only in 2-month-old mice) of the hind limbs of mice were treated with vehicle or NAR by intramuscular injection once every two days for a total of fifteen doses. The intramuscular injection volume was 40 pL in the gastrocnemius and 20 pL in the tibialis anterior, and the concentration of NAR was 8 mM in contained 20% DMSO and 80% saline. The vehicle is 20% DMSO and 80% saline. For 10- month-old mice, 50 μM Mit-A (Abeam abl42723) was preinjected to block the transcriptional activity of Sp 1.

[0084] Treadmill test

[0085] The mice were acclimated to the treadmill 2 days prior to the experiments by running for 5 min/day at 15 cm/s and a 15 degrees incline followed by 15 min a day at 25 cm/s and a 15 degrees incline in a LE8710RTS Treadmill Inspection System (Panlab/Harvard Apparatus). For exercise experiments, the speed was increased by 5 cm/s every 5 min until 30 cm/s was reached, and the mice were run until exhaustion. Then, the total distance traveled by the running mice was calculated.

[0086] Grip-strength test

[0087] Muscle strength was recorded using a GT3 grip-test meter system (Bioseb, Vitrolles, France). Mice were allowed to hold on to a metal grid with their four paws and were gently pulled backwards by the tail until they could no longer hold the grid. The peak pull force was recorded on a digital force transducer. The maximum value of 10 measurements was used to represent the grip strength of each mouse.

[0088] IF staining of frozen sections [0089] Gastrocnemius muscles were embedded in optimum cutting temperature (OCT) compound before being frozen in liquid-nitrogen and then stored at 80 °C. The frozen muscle sections (8 μM) were fixed in cold acetone (-20 °C precooling) for 20 min, washed three times with PBS, and permeabilized with 0.5% of Triton X-100/PBS (PBST) three times for 10 min each time. After washing, the slides were blocked through incubation with 5% of BSA at room temperature for 2 h before being incubated with the following antibodies: BA-F8 for MHC type 1 (1 : 100), BF-F3 for MHC 2b (1 :100), and SC-71 for MHC -2a (1 :100) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, USA). The slides were incubated with Alexa Fluor 350-, 488- and 594-conjugated secondary antibodies (1 : 100) (Invitrogen) at room temperature for 1 h in the dark. The slides were then observed under a confocal laser scanning microscope (Carl Zeiss LSM710 and Aperio Versa 200).

[0090] Oxygen consumption measurements

[0091] Oxygen consumption was measured using an XF24 extracellular flux analyzer from Seahorse Bioscience. For this measurement, C2C12 myotubes on an XF24 V28 cell culture microplate (Seahorse Bioscience, North Billerica, MA, USA) were pretreated with DMSO or 400 μM NAR for 24 h. For the detailed experimental procedures, please refer to the experimental guide (Agilent Seahorse XF Cell Mito Stress Test Kit).

[0092] RNAi and inhibition experiments

[0093] siRNAs (JST) targeting mouse Spl and Esrrg were transfected into C2C12 cells at a final concentration of 50 nM using Lipofectamine 2000 transfection reagent (Invitrogen 11668019) according to the manufacturer’s instructions. The cells were then differentiated for 3 days prior to treatment with or without 400 μM NAR for 24 h. For the Spl and ERRγ inhibition study, myotubes were pretreated for 2 h with DMSO, 250 nM Mit-A or 10 μM 4-OHT (MCE, HY-16950). Then, the myotubes were treated with or without 400μM NAR for 24 h.

[0094] Plasmid preparation, cell transfection and luciferase reporter assays

[0095] Spl cDNA sequences were amplified by PCR from total cDNA extracted from mouse gastrocnemius muscle tissue, digested by double digestion and transformed with the pcDNA3.1 vector digested with the same endonuclease to obtain the Spl overexpression vector [0096] Based on the mouse Esrrg genome sequence provided by the NCBI database, Primer 5 software was used to design primers suitable for PCR amplification of Esrrg promoter sequences of different lengths. The Esrrg promoter sequences obtained by PCR were double cleaved with KpnI/Hindlll (NEB) restriction endonucleases, recovered by agarose gel electrophoresis and ligated with the same endonuclease-digested pGL3-basic vector using T4 ligase (NEB) to obtain pSE-1518, pSE-1518DEL, pSE-1080 and pSE-129 fluorescent reporter plasmids. The pSE+3 and pSE+38 fluorescent reporter plasmids were obtained using the pSE- 129 plasmid as a template by circular PCR. The pSE+3MT fluorescent reporter plasmids were obtained using the pSE+3 plasmid as a template by circular PCR. Additionally, the mutated Spl plasmid vectors Spl-MT-Pocketl-GLN, Spl-MT-Pocket2-THR, Spl -MT -Pocket3 -Total and Spl-MT-Pocket4-GLY were obtained using the Spl overexpression plasmid as a template by circular PCR.

[0097] Twenty -four hours before the experiment, HEK293T cells were inoculated into 12-well plates. The transfection was started after 24 h when the cells had grown to 85% density. pGL3-basic plasmids containing Esrrg promoter fragments of different lengths, the internal reference reporter vector plasmid pRL-TK and the pcDNA-3. 1-Spl overexpression vector containing these mutation Spl vectors were cotransfected into the cells in 12-well plates at a ratio of 20: 1 :20.

[0098] After 24 h, the plates were pretreated with DMSO or NAR for 12 h, washed twice with PBS, and then subjected to a dual luciferase reporter gene assay according to the instructions of the Novozymes dual luciferase assay kit. Promoter activity is expressed in relative luciferase units. The ratio of firefly luciferase to renilla luciferase was calculated as the initiation efficiency of the corresponding promoter fragment. The pGL3 vector served as the negative control.

[0099] Gene expression detection

[0100] Total RNA from mouse skeletal muscle or C2C12 myotubes was extracted using TRIzol Reagent (Thermo Scientific 15596018). cDNA was synthesized using HiScript II Reverse Transcriptase (Vazyme R ₂ 01). Quantitative real-time PCR was performed with SYBR Green Fast qPCR Mix (Genstar A304).

[0101] Western blot analysis

[0102] Protein amounts were assessed using a BCA Protein Assay Kit (Beyotime POO 12), and 20-30 pg of protein was used in each SDS-PAGE experiment.

[0103] ChIP assay [0104] C2C12 myoblasts were grown on 100 mm dishes. After differentiation and fusion to form myotubes in differentiation medium, the C2C12 myotubes were treated with 400 μM NAR for 24 h. The specific experimental procedures were carried out according to the kit instructions (Millipore #17-408). Cell nuclei were sonicated to shear DNA in 500 pl of sonication buffer, using a Sonicator (4417 detector) (18 times, 15 s on/30 s off each time, 9 W power) to obtain fragments with lengths between 300 and 600 bp. IP was carried out overnight at 4 °C using 5 pg of an anti-Spl antibody (Abeam ab227383), 5 pg of an anti-histone H3 antibody (Millipore) or 5 pg of normal rabbit IgG antibodies (Millipore) and 40 pg of chromatin. DNA was analyzed by real-time PCR directed to the Spl-specific binding region of the Esrrg promoter.

[0105] Acquisition ofNAR-interacting proteins

[0106] Two 100 mm dishes C2C12 myotubes were washed three times with PBS, and the myotubes were then lysed in 2 mL of Hypotonic Lysis Buffer (20 mM HEPES, 2 mM EDTA, 2 mM MgCl ₂ , 1% cocktail, pH=7.4). The cells were scraped off using a cell scraper and lysed on ice for 45 min. Then, the cells were centrifuged at 12,000 xg for 15 min at 4 °C. To reduce false positives, 40 pL of streptavidin magnetic beads (Thermo Scientific #65001) were added to the supernatant, and the mixture was incubated for 2 h at 4 °C with reverse rotation. The supernatant was divided equally into two parts: 4 mM NAR-biotin containing 1% DMSO was added to the treated group, and 4 mM biotin containing 1% DMSO was added to the control group. The groups were incubated overnight at 4 °C with inversion, and then 20 pL streptavidin magnetic beads (Thermo Scientific #65001) were added to each group. The groups were incubated for 2 h at 4 °C with inversion. The supernatants were discarded, and the magnetic beads were cleaned three times with three kinds of buffer (Buffer A: TBS, 0.5% Triton X-100, cocktail; Buffer B: TBS, 0.1% Triton X-100, cocktail; Buffer C: TBS, cocktail). The obtained binding proteins were subjected to SDS-PAGE, Coomassie Brilliant Blue staining and liquid chromatography (LC)- tandem MS (MS/MS) analysis.

[0107] LC-MS/MS analysis

[0108] After SDS-PAGE and Coomassie Brilliant Blue staining, the gel strips were decolored and enzymatically digested overnight with trypsin. The peptides were then extracted in multiple steps using different concentrations of acetonitrile. [0109] LC-MS/MS analysis was performed on a nanoLC-Q Exactive system. Briefly, the flow rate through the column was set to 0.3 pL/min, and the applied distal spray voltage was set to 2.0 kV. Data collection was performed using one full scan (MW 300-1,600) followed by data- dependent MS2 scans of the 20 most abundant ions after the full MSI scan.

[0110] The peptide mixture obtained by enzymatic digestion was first analyzed by LC- MS/MS. Then, a protein search was performed in the UniProt-proteome-mouse (update- 20171001) database using the SEQUEST HT search engine of Thermo Proteome Discoverer (2.2.0.388). The search parameters were as follows: trypsin digestion with 2 missed cut sites, precursor ion mass error less than 10 ppm, fragment ion mass error less than 20 mDa, alkylation of cysteine as the fixed modification, and oxidation of methionine as the variable modification. The filtering parameters for the search results were as follows: Percolator was used for the filtering of the spectra, the Delta Cn was less than 0.1 , the FDR was set to 1%, high peptide confidence was chosen and the FDR at the protein level was also set to 1%. Label-free quantification (LFQ) was performed using Consensus node and the following parameters for peptide quantification: unique + razor; precursor abundance based on intensity; normalization mode, none or total peptide amount; ratio calculation, pairwise ratio-based, and maximum allowed FC, high (100). Only proteins that were quantified in all three replicates with an average ratio (treated group/control group) above 2 were selected for further GO analysis with the R language.

[0111] Transcriptome analysis by RNA-seq [0112] Total RNA of DMSO- and NAR-treated C2C12 myotubes was extracted by using TRIzol Reagent (Thermo Scientific 15596018). The samples were sent to Biomarker Technologies (Beijing, China) for sequencing The fragments per kilobase of exon model per million mapped reads (FPKM) values of the mRNA were used for further analysis. Differential expression analysis of the two groups was performed using DESeq2. Genes with an adjusted P value < 0 05 found by DESeq2 were considered differentially expressed. An FDR < 0.05 and an FC ≥1.5 were set as the thresholds for significant differential expression. GO enrichment analysis and KEGG pathway enrichment analysis of the upregulated genes and downregulated genes among the DEGs were implemented with the GOseq R packages and KOBAS software. [0113] Spl phosphorylation detection [0114] C2C12 myotubes were treated with or without NAR for 24 h and then lysed in RIPA lysis buffer (GenStar E123-01) containing 1% protease inhibitor cocktail (MCE HY- K0010) and 1% phosphatase inhibitor cocktail (MCE HY-K0023). The cells were scraped off using cell scrapers and lysed on ice for 45 min. The proteins were initially precleared with Protein A/G Magnetic Beads (Thermo Scientific 88803) at 4 °C for 1 h. The precleared supernatant was incubated with the anti-Sp l antibody (Abeam ab227383) at 4 °C overnight with gentle shaking. This incubation was followed by the addition of Protein A/G Magnetic Beads (Thermo Scientific 88803) and incubation at 4 °C for 1 h. The protein-antibody-bead complex was washed three times with lysis buffer for 5 min each time at 4 °C. The complex was resuspended in loading buffer (Thermo Scientific 39000) and boiled for 10 min before being subjected to Western blot analyses.

[01 15] CETSA

[0116] C2C 12 myotubes were cultured for 48 h in medium containing DMSO or 400 μM NAR. Then, the medium was washed away with PBS, and the cells were collected. RIPA lysis buffer (GenStar #E121 -01) containing 1% protease inhibitor cocktail (MCE HY-K0010) was added. The cells were repeatedly frozen and thawed 3 times in liquid nitrogen and then centrifuged at 12,000 xg for 15 min at 4 °C. The supernatant of the DMSO group was divided equally into two parts. One part was treated with 4 mM NAR for 30 min. The other part of the DMSO-treated group and the 400 μM NAR- treated group were treated with DMSO for 30 min. For each group, 50 μL (1 mg/mL) was separated and heated with a S1000™ thermal cycler (Bio-Rad) at different temperature gradients for 5 min. The samples were then centrifuged at 12,000 xg for 15 min. The supernatant was removed to prepare electrophoresis samples.

[0117] Prediction of Spl protein structure and ligand binding site

[0118] The transcription factor protein Spl is encoded by the Spl gene and belongs to the Sp/KLF family (Vellingiri et al., 2020). Several methods have been used to predict the Spl protein structure, but the whole structure has not been determined.

[0119] The first method is homology modeling. Homology modeling, also known as comparative modeling, a commonly used structure prediction method (Muhammed and Aki- Yalcin, 2019). Homology modeling aims to build a 3-dimensional structure of a target protein from its primary amino-acid sequence based on an alignment with a known sequence (template) (Bordoli et al., 2009). Here, the SWISS-MODEL workspace was first employed to build the 3D structure of the Spl protein. The SWISS-MODEL is developed by the Computational Structural Biology Group at the SIB Swiss Institute of Bioinformatics and the Biozentrum of the University of Basel.

[0120] The I-TASSER server, available from Yang Zhang’ s Research Group at University of Michigan Medical School, is another tool for automated protein structure prediction. I-TASSER (Iterative Threading ASSEmbly Refinement) is a hierarchical approach to protein structure prediction and structure-based function annotation. I-TASSER is also atemplate-based method, that employs a multiple-threading approach to identify known structures with structural similarities to the target protein on the basis of a limited number of protein folds (Deng et al., 2016; Roy et al., 2010)

[0121] Once the 3D structure of the Spl protein is derermined, the next setp is to predict ligand binding sites in order to protein-ligand docking. Roll is a new algorithm for predicting bingding sites and has been implemented in a program named POCASA (Yu et al., 2010) In this study, a web server of POCASA 1.1, available from Hokkaido University, Japan, was used to predict the ligand binding sites of the Spl protein The parameter settings of POCASA were set by default; for example, the radius of the probe sphere was set to 2 A, and the size of the unit grid was set to 1 A.

[0122] Molecular docking

[0123] Molecular docking was used to explore the interaction between the small molecule NAR and the protein Spl in this study. First, the structure of the Spl protein was preprocessed, which included addition of hydrogens, removal of waters, structure restraint minimization and other operations, with the Protein Preparation Wizard module. Then, the predicted binding sites of Spl were imported into the program as a docking grid box for the next procedure. After that, the small molecular NAR generated possible ionization states at the target pH of 7.0±l .0 and stereoisomers (at most 10 per ligand) via the LigPrep module. Finally, these preprocessed ligands were docked into the predicted binding sites of the SP1 protein through the ligand docking module. All these modules are included in Maestro 11.2 software (Schrodinger, LLC: New York, NY, 2010).

[0124] Quantification and statistical analysis

[0125] All experiments were performed at least three times. The number of replicates (n) for each animal experiment is shown in the figure legends. When three or more group were compared, one-way ANOVA was conducted with treatment as the independent factor. Two- tailed Student’s t test was used for two groups of measurements. The data are presented as the mean ± SEM. ns was considered to indicate no significant difference, *P <0.05 was considered to indicate statistical significance, and **P <0.01 was considered to indicate extreme statistical significance. Excel and GraphPad were used for all statistical analyses.

[0126] Experimental Results:

[0127] 1 . NAR improves muscle endurance and protects against age- or disease-related muscle atrophy

[0128] To investigate the protective effect of NAR against age-related muscle atrophy, first 10-month-old male mice were used as an experimental model. The mice were randomly divided into vehicle and NAR groups for intramuscular injection of the gastrocnemius. The results showed that NAR significantly increased the running distance of the mice (FIG. 1), suggesting that it enhanced muscle exercise endurance. In addition, NAR increased grip strength (FIG. 2) and the relative weight of the gastrocnemius muscle (FIG. 3). Thus, in 10-month-old aging mice, NAR not only prevented muscle atrophy but also enhanced muscle function.

[0129] The inventors next sought to determine whether NAR improved muscle function in young mice under normal physiological conditions. Using 2-month-old mice, we found that NAR again increased aerobic endurance capacity (running distance) (FIG. 4) but had no significant effect on grip strength (FIG. 5) Congruously, it did not affect the relative weight of the gastrocnemius muscle (FIG. 6). Thus, in young mice under normal physiological conditions, NAR also markedly increased the running distance.

[0130] In addition to age-related muscle atrophy, the effect of NAR on disease-related muscle dystrophy was further investigated using mdx mice, which are a preclinical model of DMD. Consistent with previous publications (Ryu et al., 2016), the mdx mice showed a typical phenotype of reduced endurance capacity, as indicated by running distance (FIG. 7), and muscle pseudohypertrophy (FIG. 9). NAR increased the running distance (FIG. 7) and grip strength (FIG. 8) of the mdx mice but did not influence body weight or the relative content of the gastrocnemius muscle (FIG. 9).

[0131] In summary, NAR can improve muscle endurance and protect against age- or disease-related muscle atrophy [0132] 2. NAR improves muscle endurance by increasing the number of oxidative myofibers and enhancing aerobic metabolism in vivo

[0133] The increased muscle endurance and reddened phenotype suggested that NAR likely influences myofiber types and energy metabolic patterns. Therefore, the effects of NAR on different types of myofibers were further examined in the gastrocnemius muscles of 10- month-old mice, the tibialis anterior and gastrocnemius muscles of 2-month-old mice, and the gastrocnemius muscles of 4-month-old mdx mice. The results showed that NARupregulated the expression of Myh7, the gene corresponding to MHC ₁ in oxidative myofibers, and Myh4, the gene corresponding to MHC2b in glycolytic myofibers, but not Myh2, the gene corresponding to MHC2a in oxidative myofibers (FIG. 10), in the gastrocnemius muscles of 10-month-old mice. [0134] In addition, immunofluorescence (IF) showed that NAR increased the content of type I oxidative myofibers in gastrocnemius muscle (FIG. 1 1). To further explore the effect of NAR on skeletal muscle energy metabolism, the expression levels of several genes related to energy metabolism were examined. Aerobic metabolism-related genes (Mb, Atp5b, Cycs, Cox5b and Cox2), β-oxidation related genes (Cptlb and Ucp3) and glucose metabolism-related gene (Pdk4) were upregulated by NAR (FIG. 12), consistent with the increased content of oxidative myofibers. Therefore, NAR not only increased the content of myofibers to resist the muscle atrophy caused by aging, but also improved muscle endurance by increasing the oxidative myofiber content in aging mice.

[0135] Similarly, in 2-month-old mice, NAR increased the expression of Myh7 (corresponding to MHC ₁ ) and Myh2 (corresponding to MHC2a) in the gastrocnemius muscle, while it did not significantly alter the level ofy Mh4 (corresponding to MHC2b) (FIG. 13). These findings were consistent with the lack of effect of NAR on grip strength in young mice. Accordingly, IF staining showed that there was more type I oxidative myofibers in the NAR group than in the vehicle group (FIG. 14). In terms of energy metabolism, NAR upregulated the expression of aerobic metabolism-related genes (Mb, Atp5b, Cycs, Cox5b and Cox2), β- oxidati on-related genes (Cptlb and t!cp3) and glucose metabolism-related genes (Pdk4) in the gastrocnemius muscles of young mice (FIG. 15). In addition, NAR upregulated the relative levels of the five oxidative phosphorylation (OXPHOS) complexes in mitochondria, including complex I subunit NDUFB8, complex II subunit 30kDa, complex III subunit Core 2, complex IV and ATP synthase subunit alpha (FIG. 16). Taken together, the results indicated that NAR increased muscular endurance in young mice by increasing the number of oxidative myofibers and aerobic metabolism without affecting glycolytic myofibers.

[0136] In 4-month-old mdx mice, the expression levels of Mb, Atp5b, Cox2, Ucp3 and Pdk4 were upregulated in the NAR-treated group (FIG. 17), indicating increased aerobic metabolism, which might have contributed to the improved muscle function in the mdx mice after NAR treatment.

[0137] In summary, NAR improves muscle endurance by increasing the number of oxidative myofibers and enhancing aerobic metabolism in vivo.

[0138] 3. NAR increases the content of oxidative myofibers and enhances aerobic metabolism in C2C12 myotubes

[0139] To further explore the cellular mechanism by which NAR regulates skeletal muscle function, C2C12 myoblasts were employed. C2C 12 myoblasts 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 24 h. Many cells died when the NAR concentration was above 800 μM (FIG. 18). Therefore, NAR at concentrations below 400 μM was chosen to treat cells. The expression of the oxidative myofiber-related genes Myh7 and Myh2 was upregulated by NAR in a dose-dependent manner, while that of the glycolytic myofiber-related gene Myh4 was not (FIG. 19), suggesting an increased content of oxidative myofibers.

[0140] The effects of NAR on cellular energy metabolism were then evaluated. It was found that NAR increased the total ATP level (FIG. 20), which suggested that NAR promoted energy production. Furthermore, the cellular bioenergetic profiles of C2C12 myotubes treated with or without NAR were measured with a Seahorse cellular energy metabolism meter. The myotubes treated with 200 μM NAR had an increased oxygen consumption rate (OCR) (FIG. 21). These results confirmed that NAR promoted aerobic respiration in C2C12 myoblasts

[0141] Also, NAR enhance the expression of key enzymes in the OXPHOS process, the TCA cycle and the β-oxidation process (FIG. 22 and 23). Therefore, NAR increases the oxidative myofiber content and enhances aerobic respiration by improving mitochondrial activity in skeletal muscle.

[0142] 4. ERRγ mediates NAR-induced promotion of the oxidative myofiber numbers and aerobic metabolism [0143] The DEGs between C2C12 myotubes treated with NAR and C2C12 myotubes not treated with NAR were analyzed. It was found that the expression of the nuclear transcription factor ERRγ (encoded by Esrrg) was more significantly upregulated gene by NAR more than other nuclear transcription factors. A previous study has shown that ERRγ is highly expressed in oxidative myofibers and that it can regulate many metabolism-related genes (Misra et al., 2017). Therefore, the mRNA level of ERRγ were examined in C2C12 myotubes treated with NAR. It was found that NAR increased ERRγ levels in a dose-dependent manner (FIG. 24). In addition, the transcript levels of ERRγ were increased by NAR in 10-month-old mice, 2-month-old mice and mdx mice in vivo (FIG. 25). The in vitro and in vivo data suggested that ERRγ might be involved in NAR function. Subsequently, to validate this hypothesis, ERRγ was knocked down with siRNA at the cellular level. Deficiency of ERRγ suppressed NAR-mediated upregulation of oxidative myofiber-related Myh7 and Myh2 and aerobic metabolism-related Atp5b and Cptlb (FIG. 26), indicating that ERRγ mediated the NAR-induced increases in oxidative myofiber numbers and aerobic metabolism.

[0144] In summary, ERRγ mediates NAR-induced promotion of oxidative myofiber numbers and aerobic metabolism, which in turn improves endurance exercise capacity in mice. [0145] 5. Spl is a direct binding protein of NAR in C2C12 cells

[0146] To further clarify the reason for the upregulation of ERRγ expression by NAR, the inventors biotinylated NAR for labeling. The differentiated C2C12 cells were lysed, and the lysates were immunoprecipitated with biotin and NAR-biotin. Next, affinity cleavage magnetic beads were used to enrich the nonspecific proteins bound to biotin as well as specific proteins bound to NAR-biotin. The two sets of proteins obtained were separated by SDS-PAGE and analyzed by mass spectrometry (MS), and the proteins that immunoprecipitated with NAR-biotin were compared with those that immunoprecipitated with the negative control (NO) biotin, to identify the differential proteins with more than twofold change. The results showed that a total of 492 proteins were more than twofold upregulated in the NAR-biotin group, of which 207 proteins were present in the NAR-biotin group but not in the NC group. Therefore, the molecular functions of transcription process-related proteins that were present only in the biotinylated NAR group were enriched (FIG. 27). The transcription factor Spl was present only in the biotinylated NAR group. Therefore, SP1 is most likely the binding protein of NAR. [0147] To validate the direct interaction of NAR with Spl, western blotting was first used to demonstrate that Spl was significantly enriched in biotinylated NAR-bound proteins (FIG. 27). Then, the binding affinity of NAR for Spl was further analyzed using cellular thermal shift assay (CETSA), which detects the binding affinity of small molecule compounds and target proteins. The results showed an obvious shift in the solubility profile of Spl in C2C12 myotube cell lysates treated with 4 mM NAR for 30 min. In the control group, 50% Spl protein degradation occurred at 63 °C, while the temperature increased to 69 °C in the samples treated with 4 mM NAR in cell lysates for 30 min. The increased stability of the Spl protein in the NAR-treated samples indicated that NAR bound directly to the Spl protein. Thus, the above results suggest that NAR may play a regulatory role in skeletal muscle by directly binding to the transcription factor Spl (FIG. 28).

[0148] To better show the interaction of NAR with Spl , we predicted the 3D structure of Spl and the possible sites of NAR/ Spl interaction. The small molecule NAR mainly interacted with the Spl protein via hydrogen (H-)bonds; more specifically, two hydroxyl groups in the NAR molecule interacted with the ASN-81, SER-83 and GLN-110 residues of Spl (FIG. 29). The conformation of NAR with the highest docking score (-6.584) was the most likely combination for NAR/ Spl binding.

[0149] 6. Spl is involved in NAR-mediated upregulation of ERRγ and improvement of muscle function

[0150] To determine whether Spl is involved in the function of NAR in skeletal muscle, we first examined the effects of NAR on skeletal muscle function and metabolism after using Spl inhibitors at the animal level. When we used mithramycin A (Mit-A), a Spl -specific inhibitor, to inhibit the transcriptional activity of Spl in the gastrocnemius muscles of aging mice, we found that the enhancing effects of NAR on aerobic running distance and grip strength were suppressed (FIG. 30 and 31). The increase in the number of oxidative myofibers (FIG. 32) and the upregulation of the oxidative myofiber-related genes Myh7 and Myh2 (FIG. 33) induced by NAR were also suppressed. In addition, NAR-mediated ERRγ upregulation was inhibited by Mit-A at RNA levels (FIG. 34). Furthermore, the upregulation of downstream genes of ERRγ, including myoglobin, Atp5b, Cycs, Cox5b, Cptlb and Ucp3, which are associated with oxidative myofiber numbers or aerobic metabolism was inhibited (FIG. 35). These results suggest that the NAR-induced increases in endurance and grip strength in 10-month-old mice are achieved through the transcription factor Spl in vivo.

[0151] 7. NAR enhances the interaction of Spl with the Esrrg promoter by increasing the phosphorylation of Spl

[0152] The above results showed that Spl mediates NAR-induced ERRγ upregulation, so we next sought to determine how NAR regulates ERRγ expression through Spl. By sequence comparison and prediction, the inventors found that this region of Esrrg promoter contains a sequence (CCCTGCCCTC) that may bind by Spl. To determine whether the binding of Spl with the Esrrg promoter responds to NAR induction, the different effects of NAR on a control reporter vector, a fluorescent reporter vector containing the region from +3 to +100 of the Esrrg promoter, and the mutant reporter vector were measured. The results showed that the response of the fluorescent reporter vector pSE+3 to NAR was 4 5 -fold, much higher than the 1 .7-fold response of the control and the 2.5-fold response of the mutant (FIG. 36).

[0153] The possible binding sites of NAR with Spl were obtained. So a fluorescent reporter vector containing the +3 to +100 region of the Esrrg promoter and a mutant amino acid at the binding site of the Spl vector was used to verify the real binding sites of NAR with Spl. The results showed that the response of the fluorescent reporter vector pSE+3 with the unmutated Spl vector to NAR was 4.5-fold, much higher than the 1.7-fold response of the Spl - MT -Pocket 1-GLN vector (FIG. 37). Mutation of GLN-110 in the pocket 1 of Spl disrupted the binding of NAR, indicating that NAR interacted with the Spl protein via an H-bond with GLN- 110 of Spl in the pocket 1 . The mechanism by which direct binding of Spl to NAR increases Spl transcriptional activity is unclear. Recent data demonstrate an important role for the phosphorylation state of Spl in the regulation of multiple genes (Chu, 2012; Tan and Khachigian, 2009). We assayed the level of posttranslational phosphorylation of Spl following NAR treatment using immunoprecipitation (IP). The results showed that Spl phosphorylation increased upon NAR binding (FIG. 38), which in turn led to an increase in Sp 1 transcriptional activity.

[0154] 8. Reconstruction and synthesis of NAR structure: Improve the water solubility, activity and medicinal properties of NAR through structural modification.

[0155] Based on the findings with naringenin, the structure of narigenin is further modified to obtain different derivatives. The objectives of the modifications include to improve water solubility, enhance biological or therapeutic activities, and medicinal properties. These derivatives thereof are to be used for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof. Compositions including at least one derivative are obtained. In the present disclosure, the new compounds includes a series of derivatives as described herein.

[0156] Muscle loss and dysfunction are major issue in life and health, and finding a solution has long been a research pursuit. In the study of the present disclosure, a new function of NAR has been discovered. NAR, including the derivatives thereof, improves muscle endurance and protects against muscle atrophy in both naturally aging and mdx mice by increasing oxidative myofiber numbers and enhancing aerobic respiration in vivo and in vitro. Importantly, the transcriptional factor Spl was identified as a direct binding target of NAR. Binding of NAR increases Spl phosphorylation and transcription factor activity. Furthermore, Spl was found to be a new transcription factor of Esrrg and the Spl-ERRγ transcriptional axis was discovered to be involved in NAR-mediated energy metabolism and muscle remodeling. These findings have important implications for the prevention of sedentary lifestyle and age- related decreases in aerobic exercise capacity and muscle atrophy, as well as for the treatment of DMD-related muscle weakness.

[0157] The new function and significance of NAR in skeletal muscle in three animal models:

[0158] The loss of muscle mass and strength during aging is an issue that cannot be ignored; some studies have reported that muscle mass decreases by approximately 3-8% per decade after the age of 30 (Volpi et al., 2004). The atrophy of skeletal muscle and the decrease in exercise tolerance due to aging mainly manifest as decreases 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). Using 10-month-old naturally aging mice as a model, the inventors demonstrated that NAR can improve endurance and grip strength and reverse muscle atrophy to a certain extent by increasing the number of slow myofibers and the overall aerobic metabolic capacity. Muscle loss due to natural aging is the most common type of muscle loss; therefore, the inventors first used a mouse model of natural aging to demonstrate the role of NAR in improving muscle function. In the context of increasingly severe aging problems worldwide, NAR shows great promise for widespread application to improve or treat muscle atrophy. Even in the young adult mouse model, NAR significantly increased running distance, oxidative myofiber formation and the expression of key enzymes for the OXPHOS process in skeletal muscle. This indicates that NAR can enhance the aerobic capacity of muscles under normal physiological conditions, suggesting the possibility of its application in endurance athletes such as long-distance runners.

[0159] In addition, this new function of NAR has potential application value for modern youth, among whom a lack of exercise or sedentary lifestyle leads to decreases in skeletal muscle metabolism and increased risks of metabolic diseases such as obesity and diabetes. Notably, NAR also improved endurance and grip strength in mdx muscular dystrophy mice. Although it did not significantly affect the type or content of myofibers, the promoting effect of NAR on aerobic metabolism explains this phenomenon to a certain extent. In conclusion, although the mechanism is not fully understood, the improvement of the mdx phenotype by NAR also expands the potential application of NAR to the treatment of muscular diseases. Together, the findings indicate that the role of NAR in improving muscle function will benefit multiple populations.

[0160] Spl, identified as a new direct target of NAR, mediates NAR function in skeletal muscle:

[0161] Although diverse biological activities of NAR in different diseases have been well studied, there is a lack of research on the direct targets of action, which greatly limits the application of NAR. To date, only two studies have explored the direct targets of NAR. Collapsin response mediator protein 2 (CRMP2) has been identified as a candidate for direct binding with NAR to explain the protective effect of NAR in Alzheimer's disease (AD) (Yang et al., 2017). Another study has demonstrated that NAR binds directly to the PPARa ligand-binding domain (LBD) region and activates PPARa through a dual luciferase reporter system, thereby reducing Very Low Density Lipoprotein (VLDL) levels and decreasing lipid accumulation in the liver (Goldwasser et al., 2011). However, whether Ppara is a direct target of NAR has not yet been proven by additional evidence.

[0162] In the study of the present disclosure, the inventors have identified Spl as a direct binding protein of NAR through IP-MS and validated the binding affinity of NAR for Spl through CETSA in muscle cells. The activity of Spl is regulated by posttranslational modifications, such as phosphorylation, glycosylation and acetylation, among which phosphorylation has been the most studied. It was found that NAR activated Spl transcription factor activity by promoting the phosphorylation of Spl, revealing the mechanism by which NAR regulates Spl . This is the first study to identify the direct target of NAR in regulation of myofiber types and energy metabolism in skeletal muscle.

[0163] Spl is a new transcription factor of ERRγ:

[0164] ERRγ, an orphan nuclear hormone receptor with ligand-independent transcriptional activity, plays an important role in pathological conditions such as insulin resistance, alcoholic liver injury, and cardiac hypertrophy and regulates energy metabolism in cardiac, skeletal muscle and pancreatic beta cells (Misra et al., 2017). Based on the importance of ERRγ in metabolic homeostasis, ERRγ is a good target for the treatment of metabolic diseases. ERRγ expression can be induced by various cellular stresses through membrane receptors or intracellular transcription factors

[0165] In addition, ERRγ expression can be regulated by the transcription factors c-Jun, Stat3, CREB, HIFla, and ATF6a in response to external stimulation (Misra et al., 2017). In our study, we identified Spl as a new transcription factor of ERRγ, providing a new mechanism for the regulation of ERRγ expression. The binding of NAR and Spl further promotes the binding of Spl to the ERRγ promoter. The establishment of the Spl- ERRγ axis not only reveals the mechanism by which NAR regulates muscle function but also provides a new signaling pathway for optimization of strategies to improve muscle function.

[0166] In the present disclosure, it is found that NAR enhances the transcription factor activity of Spl by promoting the phosphorylation of Spl However, the specific phosphorylation site has not yet been verified, and how NAR affects Spl phosphorylation remains unclear. In addition, the relatively low solubility of NAR may limit its effectiveness in animals. Improving the water solubility of NAR will help facilitate the application of NAR.

[0167] As described herein, this work demonstrates for the first time that NAR improves the endurance and aerobic metabolic capacity of skeletal muscle in young mice, protects against muscle atrophy in middle-aged mice and relieves DMD. Furthermore, it identifies Spl as a new direct target of NAR and establishes a new relationship between Spl and the target gene ERR, thereby elucidating the molecular mechanism by which NAR binds Spl to enhance the interaction between Spl and the ERR promoter and thus upregulate ERR expression. Therefore, this study opens new avenues for the application of NAR and provides a new and safer strategy for improving muscle function and protecting against muscle atrophy of aging and disease. NAR derivatives are synthesized with an objective to mimic NAR or further improve the performance. [0168] 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 thereof. Such a composition comprises 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, [0169] The formula (I) has chemical structures below:

[0170] In the formula (I), each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl. In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ . Such a compound is naringenin (NAR) or a naringenin derivative.

[0171] In the narigenin derivatives provided in the present disclosure, the phenyl ring connected with the dihydrochromonone is modified with at least one substitution group and up to four substitution groups R ₁ , R ₂ , R ₃ , and R ₄ . In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ each is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H. When a naringenin derivative contains only one substitution group (represented by R ₁ ) on the phenyl ring of the naringenin base structure, such a compound has a chemical structure having formula (II):

[0172] The only one substitution group (represented by R ₁ ) may be in the ortho- or metaposition relative to the hydroxyl group. The compound may have a chemical structure having formula (III) or (IV):

[0173] In the composition provided in the present disclosure, examples of a suitable compound include, but are not limited to, the following compounds:

[0174] In the compounds above, the sample identification includes the compound number followed by the lab compound code in the parentheses. [0175] The composition can be a pharmaceutical composition, a functional composition, and/or a dietary supplement. For example, the composition is a pharmaceutical composition, which can be injectable or orally administrated. The composition is preferably injectable. The compound may have a concentration in a range of from 1 mM to 50 mM, for example, 2 mM to 15 mM, 5 mM to 10 mM, or any other suitable concentration. Examples of a suitable concentration include, but are not limited to, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, and any other values between any of two of these values

[0176] The excipient may be a solvent, a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrating agent, a lubricant, a surfactant, an emulsifying agent, a suspending agent, or any combination thereof. For example, in one injectable composition, the excipient comprises a vehicle comprising 20% DMSO and 80% saline by weight. In some embodiments, the compositions may be administrated with drink, food, or related ingredients.

[0177] In another aspect, the present disclosure provides a compound having formula (I) as described herein, or a pharmaceutically acceptable solvate thereof, or any combination thereof. Such a compound is a naringenin derivative other than naringenin (NAR).

[0178] The compound can be a suitable compound having desired solubility and pharmaceutical properties. In some embodiments, the compound is a compound having formula (I) or a pharmaceutically acceptable solvate thereof.

[0179] The present disclosure provides any compound genus or species as described herein, for example, having a formula (II), (III), or (IV). In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl. At least one of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. Such a compound is a naringenin derivative In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ . [0180] In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H.

[0181] Examples of a suitable compound as a naringenin derivative include, but are not limited to, the compound examples as described herein, for Compounds No. 1-13.

[0182] In another aspect, the present disclosure provides a method of making the composition or the compound as described herein. Such a method may include preparing the compound. The method may further comprise mixing the excipient and the compound.

[0183] In another aspect, the present disclosure provides a method of preparing the compound, which is a naringenin derivative as described herein. In some embodiments, such a method comprises a step of reacting l-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-l-one with a substituted p-bis(methoxymethoxyl)benzaldehyde as described below.

[0184] In another aspect, the present disclosure provides a method of for improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof. The method comprises administrating a suitable amount of the composition as described herein into a subject in need thereof. The composition comprises an effective amount of a compound having formula (I). The present disclosure also provide the use of the compound having formula (I) such as NAR and NAR derivatives for the manufacture of a medicament for the treatment of any of these medical conditions.

[0185] In some embodiments, in the compound having formula (I), each of R ₁ , R ₂ , Rr, and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, NH ₂ , NO ₂ , C1-C6 alkyl, C1-C6 alkoxyl, and phenyl. At least one of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H.

Such a compound is naringenin or a naringenin derivative In some embodiments, each of R ₁ , R ₂ , R ₃ , and R ₄ is selected from the group consisting of H, F, Cl, Br, OH, and NH ₂ .

[0186] In some embodiments, one or two of R ₁ , R ₂ , R ₃ , and R ₄ is a substitution group other than H. For example, in some embodiments, R ₁ or R ₂ is a substitution group other than H, R ₃ =H, and R ₄ =H.

[0187] In some embodiments, the subject is a mammal, preferably a human subject, which can be a healthy human, or an adult having an age-or disease-related muscle atrophy. [0188] In some embodiments, the composition is intramuscularly injected or orally administrated. It is preferably injected. In some embodiments, the composition is intramuscularly injected with a dose of the effective amount of the compound in a range of from 2 mg/Kg to 20 rng/Kg, with a frequency of once daily or once every other day. The administration can be at any suitable dosage. For example, in some embodiments, the dose of the effective amount of the compound is in a range of from 3.6 mg/Kg to 7.6 mg/Kg. Examples of a suitable dose of the effective amount of the compound include, but are not limited to, 2 mg/Kg, 3 mg/Kg, 3.5 mg/Kg, 4 mg/Kg, 4.5 mg/Kg, 5 mg/Kg, 5.5 mg/Kg, 6 mg/Kg, 6.5 mg/Kg, 7 mg/Kg, 7.5 mg/Kg, 8 mg/Kg, 9 mg/Kg, 10 mg/Kg, 11 mg/Kg, 12 mg/Kg, 13 mg/Kg, 14 mg/Kg, 15 mg/Kg, 16 mg/Kg, 17 mg/Kg, 18 mg/Kg, 19 mg/Kg, and 20 mg/Kg. The doses are calculated based on the data obtained so far. [0189] EXAMPLES OF COMPOUNDS:

[0190] The following examples including the synthesis procedures are described for illustration only, and do not limit the scope of the compound provided in the present disclosure. [0191] Compounds 1-13 were made and evaluated. Table 1 summarizes their structures compared to Naringenin (abbreviated as NAR and having a lab compound code S12). The label compound codes of Compounds 1-13 are also shown in Table 1.

[0192] Table 1

[0193] General Procedure for the Synthesis of 2'-hydroxyacetophenone:

[0194] 2'-hydroxyacetophenone was synthesized based on Scheme (A) and procedures described below:

Scheme (A).

[0195] A suspension of 2',4',6'-Trihydroxyacetophenone (14.74 g, 87.68 mmol, 1.0 equiv) was stirred in dichloromethane (180 mL) under argon atmosphere. N,N- diisopropylethylamine (DIPEA, 46.05 mL, 263.05 mmol, 3.0 equiv) was added at 0 °C, and bromomethyl methyl ether (MOMBr, 15.75 mL, 192.91 mmol, 2.2 equiv) was added dropwise to the reaction mixture at 0 °C. Stir the reaction mixture at 0 °C for 4 h. The reaction mixture was quenched with water (200 mL), and the aqueous solution was extracted with dichloromethane (3x 120 mL). The combined organic solution was dried (Na2SOr), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EtOAc = 15/1-10/1) to give the l-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-l-one (8.4g, 37.39%) . Td NMR (400 MHz, Chloroform-^ d 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), 2.63 (s, 3H). Other phenolic hydroxyl groups need to be protected by MOM protecting group and can be synthesized by this method.

[0196] General Procedure for Synthesis of 6-hydroxy-[l,l'-biphenyl]-3-carbaldehyde: [0197] 6-hydroxy-[l,T-biphenyl]-3-carbaldehyde was synthesized based on Scheme (B) and the following procedures:

Scheme (B).

[0198] 3-bromo-4-hydroxybenzaldehyde (1.0 g, 5 mmol, 1.0 eq.) was added by conventional MOM protecting group method, and purified by silica gel column chromatography (PE/EtOAc = 30: 1 - 20: 1) to obtain 400 mg of 3-bromo-4-(methoxymethoxy)benzaldehyde with a yield of 32.79 %. To a stirred solution of the 3-bromo-4-(methoxymethoxy)benzaldehyde (400 mg, 1.63 mmol, 1.0 equiv), phenylboronic acid (298.52mg, 2.45 mmol, 1.5 equiv ), and PdCh(dppf) (59.71mg, 0.081 mmol, 5 mol %) ) in 1,4-dioxane (5.0 mL) were added at 60 °C for 24 h under an argon atmosphere. Remove the solvent in vacuo. The residue was purified by silica gel column chromatography (PE / EtOAc = 40 : 1 - 30 : 1) to give the 6- (methoxymethoxy)-[l, l'-biphenyl]-3-carbaldehyde (23 Img, 58.42%). Structure generation was confirmed by LC-ESIMS found m/z 243.2 [M+H] ⁺ .

[0199] General Procedure for the Synthesis of Naringenin Derivatives (i.e., the Compounds):

[0200] The compounds as naringenin derivatives were synthesized based on Scheme (C), in which conditions (a) and (b) are alternative:

Scheme (C).

[0201] The synthesis method with condition (a) is described using Compound 1 (lab compound code: NAR-27) as an example, which is 2-(3-Fluoro-4-hydroxyphenyl)-5,7- dihydroxy -4-dihydrochromone. Compound 1 (NAR-27) was synthesized as follows through

Scheme (D):

[0202] 3-Fluoro-4-hydroxybenzaldehyde (154 mg, 1.0 mmol, 1.0 eq.) was added to a 50 mL eggplant-shaped flask with a built-in stirring bar containing acetone (10 mL), and potassium carbonate (276.4 mg, 2.0 mmol , 2.0 eq.) and bromomethyl methyl ether (90 pL, 1.1 mmol, 1.1 eq.). The mixture was heated to reflux for 3 h. After the reaction was completed, methanol was added to quench the reaction, and a large amount of organic phase was removed by rotary evaporation, then 20 mL of water was added, and then extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a tan oily liquid. Proceed directly to the next step without processing.

[0203] In a 50 mL eggplant-shaped flask, KOH (1.0 g, 17.8 mmol), the unpurified crude product from the previous step, and 2-hydroxy-4,6-bis(methoxymethoxy)-l -acetophenone (256 mg, 1.0 mmol, 1 0 eq.) were added. 20 mL of ethanol and 2 mL of water were added, followed by stirring at room temperature for 18 hours. 2 M HC ₁ solution was added to adjust the pH to about 7.5-8 weakly alkaline. Extraction was performed three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and dried under vacuum remove the solvent The crude product was purified by silica gel column chromatography (PE / EtOAc = 15: 1 - 10:1) to give 3-(3-fluoro-4- (methoxymethoxy)phenyl)-l-(2- Hydroxy -4, 6-bis(methoxymethoxy)phenyl)prop-2-en-l -one, yellow solid, mass 100.0 mg, two-step total yield 26.45 %. [0204] The chaicone obtained in the previous step was put into a 50 mL eggplant-shaped flask. 3.0 mL of 2 M HC ₁ solution and 10.0 mL of methanol were added, followed by heating to reflux for 24 h. TLC and UpLC monitoring (the ring-closing product UV has characteristic absorption at 330-360nm, and the ring-opening product UV has characteristic absorption at 280nm). After the reaction, appropriate saturated Nal ICO - was used to neutralize excess HC ₁ , and ethyl acetate (20 mL*3) was used to extract three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and removed the solvent in vacuum. The crude product was purified by silica gel column chromatography (PE / EtOAc = 5 : 1) to give 2-(3-fluoro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (NAR-27), pale yellow-white solid 14.19 mg, yield 21.54 %. The MF is C _{1 5} H _{1 1} FO ₅ , the MW is 290.06. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.13 (s, 1H), 10.81 (s, 1H), 10.06 (s, 1H), 7.33 (dd, J= 12.3, 2.1 Hz, 1H), 7.13 (dd, J= 8.2, 2.1 Hz, 1H), 6.97 (t, J= 8.7 Hz, 1H), 5.92 - 5.86 (m, 2H), 5.46 (dd, J = 12.8, 3.0 Hz, 1H), 3.27 (dd, J= 17.1, 12.9 Hz, 1H), 2.70 (dd, J= 17.1, 3.0 Hz, 1H); ¹³ C NMR (200 MHz, Acetone-d ₆ ) d 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, 43.4. HRMS (ESI) m/z Calcd for C _{1 5} H _{1 2} FO ₅ [M+H] ⁺ 291.0663, found 291.0667.

[0205] 2-(3,5-difluoro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochro mone (Compound 2, NAR-28)

[0206] Compound 2 (NAR-28) was synthesized as follows through Scheme (E):

Scheme (E). [0207] The synthesis method is the same as the method with condition (a) as described above. The product, which is compound 2 (NAR-28), is a pale yellow solid (38.66 mg, 12.54 %); 'l l NMR (500 MHz, Acetone-d ₆ ) 5 11.71 (s, 2H), 9.23 (s, 1H), 7.34 - 7.23 (m, 4H), 5.93 (s, 2H), 3.39 (dd, J= 8.3, 7. 1 Hz, 2H), 3.01 - 2.94 (m, 2H). ¹³ C NMR (150 MHz, Acetone-d ₆ ) δ 196.44, 167.55, 164.93, 163.80, 154.01 (d, J= 7.0 Hz), 152.41 (d, J= 7.0 Hz), 134.83 (t, J = 17.1 Hz), 131.04 (t, J= 6.1 Hz), 110.88 (dd, J= 17.1, 6.1 Hz), 102.97, 96.97, 95.96, 78.63, 43.29. HRMS (ESI) m/z Calcd for C ₁ 5H14ClO ₄ [M+H] ⁺ 293.0575, found 293.0577.

[0208] 2-(3-Chloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 3, NAR-29)

[0209] Compound 3 (NAR-29) was synthesized as follows through Scheme (F):

[0210] The synthesis method is the same as the method with condition (a) as described above. The resulting product, Compound 2 (NAR-29), is a pale yellow white solid (13. 98 mg, 4.56 %); 'H NMR (400 MHz, DMSO-d ₆ ) δ 12.13 (s, 1H), 10.83 (s, 1H), 10.39 (s, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.29 (dd, J= 8.5, 2.2 Hz, 1H), 6.99 (d, J= 8.4 Hz, 1H), 5.92 - 5.86 (m, 2H), 5.46 (dd, J - 13.0, 3.0 Hz, 1H), 3.31 - 3.23 (m, 1H), 2.70 (dd, J= 17.2, 3.1 Hz, 1H); ¹³ C NMR (200 MHz, Acetone-d ₆ ) δ 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, 43.4. HRMS (ESI) m/z Calcd for C _{1 5} H _{1 2} FO ₅ ⁺ [M+H] ⁺ 307 0368, found 307.0367.

[0211] 2-(3,5-Dichloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochro mone(Compound 4, NAR-30) [0212] Compound 4 (NAR-30) was synthesized as follows through Scheme (G):

Scheme (G).

[0213] The synthesis method of NAR-30 is a general procedure using the method with condition (b) as shown above.

[0214] To a solution of 3,5-dichloro-4-hydroxybenzaldehyde (191 mg, 1.0 mmol) and DIPEA (522 pL, 3.0 mmol, 3.0 eq.) in dry CH ₂ CH ₂ ( 10 ml) was added MOM-Br (183 pL, 2.2 mmol, 2.2 eq.) at 0°C under Argon. The reaction mixture was stirred for 4 h at 0°C. The reaction was quenched by adding 100 ml of water and then extracted with CH ₂ CH ₂ (50 ml) for three times. The organic phase was combined, washed with brine, dried with anhydrous MgSO4, and evaporated under reduced pressure to obtained crude di-MOM protected intermediate.

[0215] To a solution of the 2-hydroxy-4,6-bis(methoxymethoxy)-l-acetophenone (256 mg, 1.0 mmol, 1.0 equiv) in dry THF (10 mL), NaH (60% dispersed in paraffin oil) (80.0 mg, 2.0 mmol, 2.0 eq.) was added in portions at 0 °C, under a nitrogen atmosphere and with vigorous stirring. When the evolution of H ₂ ceased, a solution of above di-MOM protected intermediate in dry THF (5.0 mL) was added dropwise over 15 min and the reaction mixture was stirred at room temperature for 2 h, except otherwise stated. After the reaction was completed, quenched the reaction slowly with ice water, extracted three times with ethyl acetate (20 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and removed in vacuo solvent. The crude product was purified by silica gel column chromatography (PE/EtO Ac = 15: 1 - 10: 1) to give 378 mg of a yellow solid as 3-(4-trifluoromethylphenyl)-l-(2-hydroxy-4,6- bis(methoxymethoxy)phenyl)prop-2-en-l-one, the yield was 88.30 %. [0216] All the chaicones obtained in the previous step were put into a 50 mL eggplantshaped flask. 10.0 mL of methanol and 3.0 mL of 2 M HC ₁ solution were added and heated to reflux for 24 hours. TLC and UpLC monitoring (the ring-closing product UV has characteristic absorption at 330-360 nm, and the ring-opening product UV has characteristic absorption at 280nm). After the reaction is completed, neutralize excess HC ₁ with appropriate saturated NaHCO ₃ , and use ethyl acetate (20 mL*3) was extracted three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo.

[0217] The crude product was purified by silica gel column chromatography (PE / EtOAc = 8: 1 - 5: 1) to give 2-(3,5-dichloro-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochro mone (NAR- 30) as A pale yellow white solid (56.78 mg, 16.67 %); ^X H NMR (500 MHz, Acetone-d ₆ ) δ 7.55 (s, 2H), 6.01 (d, .7= 2.2 Hz, 1H), 5.97 (d, J= 2.2 Hz, 1H), 5.50 (dd, J= 12.9, 3.1 Hz, 1H), 3.18 (dd, J= 17.1, 12 9 Hz, 1H), 2.82 (dd, J= 17.1, 3.1 Hz, 1H). ¹³ C NMR (125 MHz, Acetone-d ₆ ) <5 196.4, 167.7, 165.0, 163.8, 150.3, 132.9, 127.7, 122.8, 102.9, 97.0, 96.0, 78.5, 43.3. HRMS (ESI) m/z Calcd for C _{1 5} H _{1 1} ClO ₅ ⁺ [M+H] ⁺ 340.9978, found 340.9981.

[0218] 2-(3-Bromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone (Compound 5, NAR-31)

[0219] Compound 5 (NAR-31) was synthesized as follows through Scheme (H):

Scheme (H).

[0220] The synthesis method is the same as the method with condition (b) as shown above. The product, which is compound 5 (NAR-31), is a pale yellow white solid (63.78 mg, 18. 16 %); ^X H NMR (400 MHz, Acetone-d ₆ ) δ 12. 16 (s, 1H), 9.37 (s, 2H), 7.73 (d, J= 2.1 Hz, 1H), 7.41 (dd, 8.4, 2.2 Hz, 1H), 7.07 (d, J= 8.4 Hz, 1H), 6.01 - 5.94 (m, 2H), 5.49 (dd, J= 12.9, 3.0 Hz, 1H), 3.20 (dd, J= 17.1, 12.9 Hz, 1H), 2.78 (dd, J= 17. 1, 3.0 Hz, 1H); ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 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, 43.4. HRMS (ESI) m/z Calcd for C ₁₅ H ₁₂ BrO ₅ ⁺ [M+H] ⁺ 350.9863, found 350.9861.

[0221] 2-(3,5-Dibromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochrom one(Compound 6, NAR-32)

[0222] Compound 6 (NAR-32) was synthesized as follows through Scheme (I):

Scheme (I).

[0223] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 6 (NAR-32), is a pale yellow white solid (90.21 mg, 20.98 %); ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.12 (s, 1H), 10.87 (s, 1H), 10.17 (s, 1H), 7.73 (s, 2H), 5.96 - 5.89 (m, 2H), 5.50 (dd, J= 13 0, 2.9 Hz, 1H), 3.33 (dd, J= 17 1, 13.0 Hz, 1H), 2.75 (dd, J= 17.1, 3.0 Hz, 1H); ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 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, 43.3. HRMS (ESI) m/z Calcd for C ₁₅ H ₁₂ BrO ₅ ⁺ [M+H] ⁺ 428.8968, found 428.8972

[0224] 5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-dihydrochromon e (Compound 7, NAR-33)

[0225] Compound (NAR-3 ) was synthesized as follows through Scheme (J):

Scheme (J).

[0226] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 7 (NAR-33), is a pale white solid (102.6 mg, 34.47 %); ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.15 (s, 1H), 10.78 (s, 1H), 9.13 (s, 1H), 7.09 (d, .7 = 2.0 Hz, 1H), 6.90 (dd, J= 8.2, 2.0 Hz, 1H), 6.79 (d, J= 8.2 Hz, 1H), 5.89 (q, J= 2.1 Hz, 2H), 5.43 (dd, J= 12.8, 2.9 Hz, 1H), 3.78 (s, 3H), 2.68 (dd, J= 17.1, 3.0 Hz, 1H); ¹³ C NMR (125 MHz, Acetone) δ 97.2, 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, 43.6. HRMS (ESI) m/z Calcd for C ₁₆ H ₁₅ O ₆ ⁺ [M+H] ⁺ 303.0863, found 303.0873.

[0227] 5,7-Dihydroxy-2-(4-hydroxy-3-nitrophenyl)-4-dihydrochromone (Compound 8, NAR-34)

[0228] Compound 8 (NAR-34) was synthesized as follows through Scheme (K):

Scheme (K). [0229] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 8 (NAR-34), is a pale white solid (8.47 mg, 1.89 %); 1H NMR (400 MHz, DMSO-d ₆ ) δ 12.11 (s, 1H), 8.03 (s, 1H), 7.69 (d, J= 8.9 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 5.91 (d, J= 7.9 Hz, 2H), 5.58 (dd, J= 12.9, 2.9 Hz, 1H), 2.76 (dd, J= 17.1, 3.1 Hz, 1H), ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 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, 41.8. LC-ESIMS found m/z 318.2 [M+H] ⁺ and m/z 316.2 [M-H]-.

[0230] 5,7-Dihydroxy-2-(6-hydroxy-[l,l’-biphenyl]-3-yl)-4-dihydro chromone ( Compound 9, NAR-35 )

[0231] Compound 9 (NAR-35) was synthesized as follows through Scheme (L):

Scheme (L).

[0232] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 9 (NAR-35), is a yellow solid (68 mg, 20.50 %) , ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.16 (s, 1H), 10.80 (s, 1H), 9.77 (s, 1H), 7.56 (d, J= 7.5 Hz, 2H), 7.44 - 7.36 (m, 3H), 7.35 - 7.26 (m, 2H), 6.97 (d, J= 8.3 Hz, 1H), 5.92 - 5.85 (m, 2H), 5.49 (dd, J= 12.8, 3.0 Hz, 1H), 2.72 (dd, J= 17.1, 3.1 Hz, 1H); ¹³ C NMR (125 MHz, Acetone- d ₆ ) δ 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, 42.0. HRMS (ESI) m/z Calcd for C ₂₁ H ₁₇ O ₅ ⁺ [M+H] ⁺ 349.1071, found 349.1074.

[0233] 5,7-Dihydroxy-2-(2-fluoro-4-hydroxyphenyl)-4-dihydrochromone ( Compound 10, NAR-37)

[0234] Compound 10 (NAR-37) was synthesized as follows through Scheme (M):

Scheme (M).

[0235] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 10 (NAR-37), is a pale white solid (96 mg, 23.09 %).

³ H NMR (500 MHz, Acetone-d ₆ ) δ 7.46 (t, J = 8.6 Hz, 1H), 6.76 (dd, 8.6, 2.4 Hz, 1H), 6.66 (dd, J= 12.3, 2.4 Hz, 1H), 5.95 (t, J= 1.7 Hz, 2H), 5.67 (dd, J= 13.2, 3.0 Hz, 1H), 3.26 (dd, J = VIA, 13.2 Hz, 1H), 2.71 (dd, J= 17.1, 3.0 Hz, 1H); ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 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, 42.2.

HRMS (ESI) m/z Calcd for C ₁₅ H ₁₂ FO ₅ ⁺ [M+H] ⁺ 291.0663, found 291.0680.

[0236] 5,7-Dihydroxy-2-(2,6-difluoro-4-hydroxyphenyl)-4-dihydrochro mone (

Compound 11, NAR-38)

[0237] Compound 11 (NAR-38) was synthesized as follows through Scheme (N):

Scheme (N). [0238] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 11 (NAR-38), is a pale yellow white solid (102 mg, 32.11 %). 'l l NMR (800 MHz, Acetone-c/r,) 3 6.55 (d, J= 10.7 Hz, 2H), 5.96 (d, J= 2.2 Hz, 1H), 5.94 (d, J= 2.2 Hz, 1H), 5.75 (dd, J= 13.9, 3.0 Hz, 1H), 3.48 (dd, J= 17 1, 13.8 Hz, 1H), 2.72 (dd, J= 17.1, 3.1 Hz, 1H). ¹³ C NMR (200 MHz, Acetone-d ₆ ) δ 196.7, 167.4, 165.1, 164.2, 163. 7 (d, J= 11.3 Hz), 162.4 (d, J= 11.3 Hz), 161.3, 105.6, 102.9, 100.5, 100.4, 97.0, 95.7, 71.1, 41.0. HRMS (ESI) m/z Calcd for C ₁₅ H ₁₁ F ₂ O ₅ ⁺ [M+H] ⁺ 309.0569, found 309.0573.

[0239] 5,7-Dihydroxy-2-(2-chloro-4-hydroxyphenyl)-4-dihydrochromone (Compound 12, NAR-39)

[0240] Compound 12 (NAR-39) was synthesized as follows through Scheme (O):

Scheme (O).

[0241] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 12 (NAR-39), is a white solid (84 mg, 27.45 %). ^L H NMR (500 MHz, Acetone-d ₆ ) δ 7.57 (d, J= 8.5 Hz, 1H), 6.95 (d, J= 2.5 Hz, 1H), 6.92 (dd, J = 8.5, 2.5 Hz, 1H), 5.98 (q, J= 2.2 Hz, 2H), 5.76 (dd, J= 13.2, 2.9 Hz, 1H), 3.15 (dd, J= 17.1, 13.3 Hz, 1H), 2.75 (dd, J= 17. 1, 2.9 Hz, 1H). ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 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, 42.4. HRMS (ESI) m/z Calcd for C ₁₅ H ₁₂ ClO ₅ ⁺ [M+H] ⁺ 307.0368, found 307.0368.

[0242] 2-(2-bromo-4-hydroxyphenyl)-5,7-dihydroxy-4-dihydrochromone ( Compound 13, NAR-40)

[0243] Compound 13 (NAR-40) was synthesized as follows through Scheme (P):

[0244] The synthesis method is the same as the method with condition (b) as shown above. The resulting product, Compound 13 (NAR-40), is a pale white solid (79 mg, 22.57 %). ¹ H NMR (800 MHz, Acetone-d ₆ ) δ 7.57 (d, J= 8.5 Hz, 1H), 7.14 (d, J= 2.5 Hz, 1H), 6.97 (dd, J - 8.5, 2.5 Hz, 1H), 6.00 - 5.96 (m, 2H), 5.71 (dd, .J- 13.4, 2.9 Hz, 1H), 3.12 (dd, J- 17.0, 13.3 Hz, 1H), 2.77 (dd, J= 17.0, 2.9 Hz, 1H). ₁₅ C NMR (200 MHz, Acetone-d ₆ ) δ 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, 42.5. HRMS (ESI) m/z Calcd for C ₁₅ H ₁₂ BrO ₅ ⁺ [M+H] ⁺ 350.9863, found 350.9867.

[0245] As described herein, it was found that the NAR induced promotion of oxidative myofiber numbers and aerobic metabolism by up-regulating the relative Esrrg expression. Therefore, the upregulation of the relative Esrrg expression is the key indicator to evaluate whether NAR analogues have similar functions.

[0246] Compounds 1-13 were tested for relative Esrrg expression. Each compound is dissolved in DSMO (dimethyl sulfoxide) and added to water to form an aqueous solution before testing. The term “relative Esrrg expression” used herein refers to the expression of Esrrg in the treatment group using a compound relative to the expression in the control group with DMSO only. The experimental methods and data analysis methods are shown below: the C2C12 myotubes were treated with DMSO or different concentrations (100, 200 and 400 μM) of NAR or one of the other compounds. Total RNA from C2C12 myotubes was extracted using TRIzol Reagent. The cDNA was synthesized by reverse transcription of mRNA from Total RNA using oligo-dT primers and Hi Script II Reverse Transcriptase. Quantitative real-time PCR was performed with SYBR Green Fast qPCR Mix. When SYBR Green dye is added to a sample, it immediately binds to the cDNA in the sample. During the PCR process, DNA polymerase amplifies the target sequence to produce PCR products, known as 'amplicons'. The SYBR Green dye then binds to each newly generated cDNA molecule. As the PCR progresses, more and more amplicons are generated. As the SYBR Green dye binds to all cDNA, the fluorescence intensity increases as the PCR product increases. The Ct value (the number of cycles when the fluorescent signal exceeds a fixed threshold for no template control sample) of each compound was obtained. Finally, relative quantification was used for the analysis of changes in Esrrg gene expression in NAR or other compounds treated samples relative to DMSO treated sample, and the arithmetic formula 2 ' ^AACt is used to obtain the relative Esrrg expression of different samples. [0247] The results of the relative Esrrg expression of Compounds 1-13 and NAR are shown in Table 2. When a compound shows a relative Esrrg expression close to or higher than 2, it is expected that such a compound has the desired biological activities, for example, improving skeletal muscle endurance, treating muscle atrophy or dystrophy, or preventing muscle atrophy or dystrophy in a subject in need thereof. The relative Esrrg expression measurement is a model to evaluate these desired activities. Narigenin and derivatives provided in the present disclosure have been proved to have a high relative Esrrg expression and desired biological or pharmaceutical activities.

[0248] Table 2

[0249] In some embodiments, the only one substitution group (represented by R ₁ or R) may be in the ortho- or meta- position relative to the hydroxyl group on the phenyl group connected to the dihydrochromone. The compound has a structure as shown in formula (III) or (IV), respectively.

[0250] For the compounds having a chemical structure of formula (III), the examples described above and in Table 2 include NAR-27 (R=F), NAR-29 (R=C ₁ ), 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 follow a trend by the order of the substitution group: Cl > Br > OMe > F. When R is NO ₂ or phenyl, the compounds have low solubility in water, and no results of relative Esrrg expression was obtained. Further studies are to be conducted to improve solubility.

[0251] For the compounds having a chemical structure of formula (IIV) with R in the meta-position relative to the hydroxyl group, the examples described above and in Table 2 include NAR-37 (R=F), NAR-39 (R=C ₁ ), and NAR-40 (R=Br). As shown in Table 2, the relative Esrrg expression results follow a trend by the order of the substitution group: F, and Cl > Br.

[0252] Some examples described above and in Table 2 include two substitution groups additional to the hydroxyl group on the phenyl group connected to the dihydrochromone ring structure. For example, NAR-28 and NAR- 38 contain two F substitutions, NAR-30 contains two Cl substitutions, andNAR-32 contains two Br substitutions. The compounds having two halogen substitutions may have lower relative Esrrg expression values than the corresponding counterparts with one halogen substitution. However, except NAR-30, all other compounds having two halogen substitution have relative Esrrg expression values close to or higher than 2, which demonstrate these compounds have desired biological or pharmaceutical activities.

[0253] Based on these results, when at least one substitution group as described herein additional to the hydroxyl group is used to modify with the phenyl group connected to the dihydrochromone, compounds having desired biological or pharmaceutical activities can be obtained.

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[0298] Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art.