Use of compound (s)-sto021 for the preparation of a medicament against aging

Abstract

The application belongs to the technical field of medicine, and particularly relates to a use of a compound (S)-STO021 in preparation of a medicine for resisting aging and aging-related diseases. The application proves that (S)-STO021 has a better effect of inhibiting cell aging, and particularly relates to improving the morphology of bone marrow mesenchymal stem cells (BMSC) after aging, promoting proliferation and colony formation of the BMSC, inhibiting the positive cell rate of aging heterochromatin foci (SAHF), and significantly inhibiting the functions of aging-related biomarkers, such as the activity of beta-galactosidase (SA-beta-gal), and the expression levels of p16 and p21 related mRNA and protein, and the compound has a good function of preventing cell aging and is safe and reliable. The compound (S)-STO021 is prepared into a medicine for treating aging and aging-related diseases, and has a good commercial value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the use of a compound (S)-STO021 in the preparation of anti-aging drugs. Background Technology

[0002] Aging is a complex biological process, generally defined as the progressive loss of physiological integrity with age, ultimately leading to irreversible functional decline. Aging has become a significant global public health issue, with an aging population causing a continuous increase in the incidence of age-related diseases such as osteoporosis, Alzheimer's disease, and cardiovascular disease. Anti-aging drugs can be categorized by their physicochemical properties into: ① Chemical drugs, including antioxidants, anti-aging hormones, nutrients, monoamine oxidase inhibitors, immunomodulators, biochemical agents, and brain function enhancers; ② Traditional Chinese medicine, including single-herb Chinese medicines such as anti-aging plant-based medicines, anti-aging animal-based medicines, anti-aging mineral-based medicines, and compound preparations. Although a large number of anti-aging drugs exist on the market, with representative drugs being resveratrol, rapamycin, and metformin, some of these are repurposed older drugs, while others are new drugs developed in recent years for other medical purposes such as antihypertensive, lipid-lowering, and anti-tumor treatments. Drugs specifically designed for anti-aging are currently rare. Therefore, the development of specific and effective anti-aging and age-related disease drugs has attracted the attention of scientists in related fields, which can provide important protection for human health during aging.

[0003] Resveratrol is a polyphenolic compound widely found in grape skins, peanuts, Japanese knotweed, veratrum, and cassia seeds. Numerous studies have shown that resveratrol possesses broad biodiversity, involving various pharmacological activities such as anti-aging, anti-tumor, anti-cardiovascular disease, anti-inflammation, anti-oxidation, free radical scavenging, hepatoprotection, neuroprotection, estrogen-like effects, and regulation of bone metabolism. Anti-aging activity is one of the most clearly understood indications. In 2003, K.T. Howitz et al. proposed that resveratrol could act as an activator of the strongest silent information regulation 2 homolog 1 (SIRT1), participating in the regulation of the average lifespan of organic organisms (Nature 2006, 444, 337). SIRT1, a multifunctional transcriptional regulator, can deacetylate various transcription factors (FOXO, PGC1-α, p53, PPAR-γ, and NF-κB) that control metabolic and endocrine signals, thereby regulating their activity. It thus participates extensively in regulating multiple signaling pathways that control mammalian cell lifespan and is closely related to cell survival, metabolic processes, proliferation, aging, and apoptosis (Nature 2011, 477, 482). Resveratrol is a potent inducer of SIRT1, increasing its expression in organs and tissues such as the brain, heart, intestine, kidney, muscle, and adipose tissue. Evidence suggests that resveratrol-induced physiological changes include delayed aging and extended lifespan, with the most significant increase being up to 50% (Trends Endocrinal Metabol, 2009, 20, 325). Furthermore, resveratrol competitively inhibits cAMP phosphodiesterase, leading to inhibited cAMP degradation and increased cAMP expression, thereby activating the cAMP effector protein Epac1 and causing Ca2+ oxidative stress. + The passage is open, Ca2 + Increased influx further activates the CamKK8-AMPK pathway, leading to enhanced NAD+ and SIRT1 activity, ultimately improving age-related metabolic phenotypes (Cell 2012, 148, 421). Therefore, the above research on the mechanism of resveratrol's anti-aging effects not only provides a theoretical basis for its application in anti-aging indications, but also the scientific data that resveratrol intake can extend the lifespan of yeast, nematodes, fruit flies, and lower fish has been widely recognized by scientists (Nature, 2004, 430, 686).

[0004] The resveratrol sesquiomer (±)-Isopaucifloral F, which has phytoestrogens-like anti-osteoporosis activity extracted from the root of the Chinese herb *Gnaphalium affine*, was synthesized as an optical isomer (S)-STO021 by introducing two bisphosphate groups with high bone-targeting properties after drug-like structural optimization.

[0005] The structure of compound (S)-STO021 is shown in formula (I):

[0006]

[0007] The full name of compound (S)-STO021 is: (S)-4-(3-((diethoxyphosphoryl)oxy)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-1H-indene-1-yl)phenyl phosphate diethyl ester, molecular weight: 636.15;

[0008] Compound (S)-STO021, as shown in Formula (I), has demonstrated in vitro effects, exhibiting a dual mechanism of action that inhibits bone resorption and promotes bone formation, particularly excelling in its anti-osteoporosis activity. For example, in a zebrafish model, oral administration at a concentration of 25 μg / mL significantly restored bone mass. In an ovariectomized rat osteoporosis model, administration of compound (S)-STO021 to ovariectomized SD rats at a dose of 4 mg·kg⁻¹ for 24 weeks, as shown by Micro-CT analysis of the bone microstructure of the lumbar vertebrae and femur, revealed that it could restore osteoporosis symptoms in a time- and concentration-dependent manner, superior to or equivalent to the first-line clinical drug alendronate sodium. Especially noteworthy is its good oral bioavailability (46.2%) in rats, making it suitable for oral administration and possessing significant commercial application value in expanding the clinical application of anti-osteoporosis drugs. However, given that compound (S)-STO021 has the dominant resveratrol sesquivalent skeleton, its anti-aging activity has not yet been reported in the literature. Summary of the Invention

[0009] Based on the lack of existing research on the anti-aging activity of compound (S)-STO021, this invention provides the use of compound (S)-STO021 in the preparation of anti-aging drugs.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] This invention provides the use of compound (S)-STO021 in the preparation of medicaments for anti-aging and age-related diseases; the structural formula of compound (S)-STO021 is shown in formula (I):

[0012]

[0013] The full name of compound (S)-STO021 is: (S)-4-(3-((diethoxyphosphoryl)oxy)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-1H-indene-1-yl)phenyl phosphate diethyl ester, molecular weight: 636.15;

[0014] Compound (S)-STO021 is a single-configuration compound obtained by optimizing the structure of resveratrol sesquisome (±)-Isopaucifloral F, which has plant hormone-like activity, from the root of the natural plant *Broom's Root*, through bisphosphonate synthesis and chiral resolution.

[0015] In some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable pharmaceutical salt. The pharmaceutical salt includes inorganic acid salts and organic acid salts, wherein the inorganic acid salts include, but are not limited to: hydrochlorides, sulfates, phosphates, diphosphates, hydrobroms, and sulfates; and the organic acid salts include, but are not limited to: malates, maleates, fumarates, tartrates, succinates, citrates, acetates, lactates, methanesulfonates, p-toluenesulfonates, dihydroxynaphthyl salts, salicylates, and stearates.

[0016] In some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0017] In some embodiments of the present invention, the dosage form of the drug is tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, patches, ophthalmic liquids, or nasal drops. The dosage form of the drug of the present invention is not particularly limited and can be selected according to the therapeutic purpose or administration regimen.

[0018] In some embodiments of the present invention, the anti-aging refers to delaying aging or improving age-related changes in appearance; the age-related diseases include atherosclerosis, skeletal deformities, or type II diabetes, etc.

[0019] In some embodiments of the present invention, the compound (S)-STO021 is used in the preparation of a medicament for restoring the morphology of senescent BMSC cells and promoting the proliferation and colony formation of BMSC cells.

[0020] In some embodiments of the present invention, the compound (S)-STO021 is used in the preparation of a medicament for reducing the rate of heterochromatin foci (SAHF) positive cells in aging.

[0021] In some embodiments of the present invention, the compound (S)-STO021 is used in the preparation of a medicament for reducing the mRNA and protein expression levels of aging markers β-galactosidase (SA-β-gal), p16, and p21.

[0022] Considering that (S)-STO021 has phytoestrogen-like effects, this invention focuses on its systematic functional regulation of related biomarkers involved in human aging. For example, bone marrow mesenchymal stem cells (BMSCs), as long-lived cells, are more prone to senescence under stress conditions. Therefore, this invention induces BMSC cell senescence through stress-stimulating factors, namely ionizing radiation, and intervenes with the drug (S)-STO021. With resveratrol (Res) as a positive control, the effects on senescent BMSCs and their degenerative differentiation potential were observed. This included the positive cell rate of heterochromatin foci (SAHF, which form punctate DNA foci in DAPI-stained senescent cells), a specific marker for identifying senescence-related chromatin changes, as well as the expression of β-galactosidase and p16, biological markers of senescent cells. INK4A and p21 Cip1 The expression of (p16 and p21, which can reflect cellular senescence and biological aging) was investigated, and the effect of (S)-STO021 in preventing cellular senescence was examined.

[0023] This invention has shown in cellular-level studies that (S)-STO021 has a dose-dependent restorative effect on the viability of BMSCs; it can improve the morphology of senescent BMSCs, significantly enhance their proliferation capacity, increase their colony formation rate, and (S)-STO021 significantly reduces the rate of SAHF-positive cells in heterochromatin foci. Staining results for the senescence marker β-galactosidase (SA-β-gal) showed that (S)-STO021 reduces the expression level of SA-β-gal. RT-qRCR and Western Blot results showed that (S)-STO021 can dose-dependently reduce the mRNA and protein expression levels of the senescence markers p16 and p21.

[0024] This invention has found that the compound (S)-STO021 plays a key regulatory role in the function of relevant biomarkers during human aging, demonstrating its significant anti-aging effect and thus achieving a better effect in preventing cell aging.

[0025] This invention found that serum liver and kidney function indicators in the maximum tolerated dose study of compound (S)-STO021 in mice showed that (S)-STO021 did not exhibit significant liver or kidney dysfunction or damage; organ HE staining results also showed no obvious organ inflammation or damage, and its LD50 was [not specified]. 50The therapeutic index (TI) >75, with a dose >300 mg / kg, indicates that (S)-STO021 has a wide therapeutic window and excellent safety profile. In the hERG cardiotoxicity evaluation experiment, (S)-STO021 showed an IC50 of >300 mg / kg on hERG potassium channels. 50 >30μM, low cardiotoxicity.

[0026] Therefore, in this invention, the compound (S)-STO021 exhibits good in vivo safety while possessing anti-aging effects.

[0027] There are no particular restrictions on the timing of administration of the drug of the present invention, and it can be appropriately selected according to the target disease.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] 1) Compound (S)-STO021 has a good effect on improving the senescent morphology of BMSC cells and enhancing their proliferation and colony formation ability. At the same dose, it is superior to or equivalent to the positive control drug resveratrol in vivo.

[0030] 2) Compound (S)-STO021 has the ability to significantly reduce chromatin changes in aging and reduce the rate of heterochromatin foci (SAHF) positive cells. At the same dose, it is superior to the positive control drug resveratrol in vivo.

[0031] 3) Compound (S)-STO021 has a significant ability to reduce the activity of the aging marker β-galactosidase (SA-β-gal) and the gene and protein expression of p16 and p21. At the same dose, it is superior to the positive control drug resveratrol in vivo.

[0032] 4) Compound (S)-STO021 can reduce chromatin changes and the expression of aging biomarkers, and can be used to prepare drugs for anti-aging and aging-related diseases.

[0033] 5) Compound (S)-STO021 has excellent in vivo safety and low cardiotoxicity, and has the potential to be developed into an anti-aging drug. Attached Figure Description

[0034] Figure 1 Effects of compound (S)-STO021 on BMSC cell viability.

[0035] Figure 2 Effects of compound (S)-STO021 on morphological changes in senescent BMSC cells.

[0036] Figure 3 Effects of compound (S)-STO021 on colony formation in senescent BMSC cells.

[0037] Figure 4 Effect of compound (S)-STO021 on the percentage of positive cells for aging-associated heterochromatin foci (SAHF).

[0038] Figure 5 Effect of compound (S)-STO021 on the expression level of aging biomarker β-galactosidase (SA-β-gal).

[0039] Figure 6 Effects of compound (S)-STO021 on the expression levels of aging-related genes p16 and p21 mRNA.

[0040] Figure 7 Effects of compound (S)-STO021 on the protein expression levels of aging-related proteins p16 and p21.

[0041] Figure 8: Maximum tolerated dose assessment and hERG cardiotoxicity evaluation results of compound (S)-STO021 in mice. Figure 8 includes... Figure 8-1 and Figure 8-2 . Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0043] The present invention will be further described below with reference to the embodiments. Due to space limitations, the description of the experimental process cannot be very detailed. All parts not described in detail in the experiment are conventional operations well known to those skilled in the art. However, the embodiments are by no means any limitation on the present invention.

[0044] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents required in the following implementations are all commercially available unless otherwise specified.

[0045] The data analysis in the examples all used Graphpad Prism 8.0 statistical analysis software to perform one-way ANOVA for multiple group comparisons.

[0046] Example 1: Preparation of (S)-4-(3-((diethoxyphosphoryl)oxy)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-1H-indene-1-yl)phenyl phosphate diethyl ester [(S)-STO021]

[0047] The preparation route of (S)-STO021 is shown below:

[0048]

[0049] Reaction conditions: (I) N,O-dimethylhydroxylamine hydrochloride, triethylamine, dichloromethane, 0℃; (II) 3,5-dimethoxybromobenzene, n-butyllithium, anhydrous tetrahydrofuran, nitrogen protection, -78℃; (III) bromotriphenyl(4-((triisopropylsilyl)oxy)benzyl)-λ 5 - Phosphine, bis(trimethylsilyl)aminopotassium, toluene, 60℃; (IV) Tetrabutylammonium fluoride, tetrahydrofuran; (V) Boron trifluoride ether, dichloromethane; (VI) Diethyl chlorophosphate, sodium hydride, anhydrous tetrahydrofuran; (VII) Boron tribromide, dichloromethane, 0℃; (VIII) Chiral column separation conditions: High performance liquid chromatograph: Shimadzu LC-20AT, CP-HPLC-07; Mobile phase: 7:1:2 = n-hexane / dichloromethane / ethanol; Flow rate: 1.0 mL / min; Temperature: 35℃; UV detection wavelength: 254 nm; Run time: 10 min; Injection volume: 5 μL.

[0050] 1) Synthesis of compound 2 (N1,N2-dimethoxy-N1,N2-dimethylamide): Compound 1 (oxalyl chloride) (10.0 mL, 110 mmol) and dimethylhydroxylamine hydrochloride (23.70 g, 237 mmol) were dissolved in 230 mL of anhydrous dichloromethane. Triethylamine (66.6 mL, 480 mmol) was added with stirring under ice bath conditions. The reaction was carried out at 0 °C for 30 min, then heated to room temperature and stirred for 30 min. The reaction was quenched with water, extracted with dichloromethane, and the organic layers were combined and evaporated to dryness to obtain crude compound 2. Recrystallization and drying yielded compound 2. Structural characterization: white crystals; 1 H NMR(CDCl3,400MHz)δ3.16(s,6H),3.66(s,6H)ppm.ESI-MS:m / z 176.1[M+H] + .

[0051] 2) Synthesis of compound 3 (3,3′,5,5′-tetramethoxydiphenylethylenedione): 1-Bromo-3,5-dimethoxybenzene (10.36 g, 47 mmol) was weighed into a 250 mL three-necked flask, and 50 mL of anhydrous tetrahydrofuran was added and stirred until fully dissolved under N2 protection. 18 mL of n-butyllithium (29.00 g, 450 mmol) was added at -78 °C. Compound 2 (2.00 g, 11 mmol) was fully dissolved in 8 mL of anhydrous tetrahydrofuran. The dissolved compound 2 was injected into the three-necked flask using a syringe, and the mixture was stirred at -78 °C for 5 min. The system was then cooled to room temperature, and an appropriate amount of anhydrous ethanol was added, followed by quenching with water. After low-temperature storage, compound 3 precipitated as yellow crystals, which were then filtered to obtain compound 3. Structural characterization: Yellow crystals; 1H NMR (CDCl3, 400MHz) δ6.93 (d, J = 1.7Hz, 4H), 6.73 (s, 2H), 3.97 (s, 12H) ppm. ESI-MS: m / z 330.1 [M+H] + .

[0052] 3) Synthesis of compound 4 (1,2-bis(3,5-dimethoxyphenyl)-3-triphenylsilanedione): Compound 3 (5.00 g, 15 mmol) and Wittig reagent (15.00 g, 25 mmol) were added to a 250 mL round-bottom flask, followed by the addition of 50 mL of anhydrous toluene. The mixture was heated to 60 °C and stirred until homogeneous. Under stirring at 60 °C, 25 mL of potassium bis(trimethylsilyl)amino (21.93 g, 110 mmol) was added dropwise. After the addition was complete, the mixture was stirred for 20 min. A sample was spotted onto a TLC plate, and the reaction was stopped after compound 3 had completely reacted. The mixture was cooled to room temperature, quenched with water, extracted with ethyl acetate, and the organic layers were combined, evaporated to dryness, and purified by silica gel column chromatography [V(petroleum ether):V(ethyl acetate) = 5:1] to obtain compound 4. Structural characterization: Yellow-green oily substance; 1 H NMR(CDCl3,400MHz)δ7.67(m,1H),7.48(d,J=7.6Hz,1H),7.34(d,J=8.6Hz,4H),7.08(d,J=8.6Hz,2H),6.90(s,1H ),6.85(d,J=8.5Hz,2H),6.72(d,J=11.2Hz,1H),3.78(d,J=4.1Hz,1H),1.06(m,33H)ppm.ESI-MS:m / z576.3[M+H] + .

[0053] 4) Synthesis of compound 5 (1,2-bis(3,5-dimethoxyphenyl)-3-hydroxyphenyl ethylenedione): Compound 4 (15.00 g, 26 mmol) was added to a 500 mL round-bottom flask, and 30 mL of anhydrous tetrahydrofuran was added. The mixture was stirred at room temperature for 10 min. Then, 7.2 mL of tetrabutylammonium fluoride (6.80 g, 25 mmol) was added, and the mixture was reacted at room temperature for 1 h. A sample was spotted onto a TLC plate, and the reaction was stopped after compound 4 had fully reacted. The reaction was quenched with water, extracted with ethyl acetate, and the organic layers were combined, evaporated to dryness, and purified by silica gel column chromatography [V(petroleum ether):V(ethyl acetate) = 3:1] to obtain compound 5. Structural characterization: Yellow oily substance; 1H NMR (CDCl3, 400MHz) δ7.26 (s, 1H), 7.16 (d, J = 9.1Hz, 1H), 7.04 (m, 2H), 6.96 (s, 1H), 6.66-6.56 (d d,J=18.8,11.7Hz,3H),6.43(m,2H),5.10(d,1H),3.77(dd,J=17.6,11.3Hz,12H)ppm.ESI-MS:m / z 420.1[M+H] + .

[0054] 5) Synthesis of compound 6 (2-(3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)-4,6-dimethoxyindanone): Compound 5 (9.50 g, 23 mmol) was added to a 500 mL round-bottom flask, and 45 mL of anhydrous dichloromethane was added. The mixture was stirred at room temperature for 10 min. Then, 2.86 mL of boron trifluoride diethyl ether (3.21 g, 23 mmol) was added. The mixture was stirred at room temperature for 30 min. A white solid precipitated, and the reaction was terminated. The resulting white solid was compound 6. Structural characterization: white solid; 1 H NMR (400MHz, CDCl3) δ6.98 (s, 1H), 6.72 (s, 1H), 6.45 (s, 4H), 6.19 (s, 1H), 5.92 (d, J = 2.30Hz, 2H), 4. 90(d,J=2.75Hz,1H),4.27(d,J=2.75Hz,1H),3.90(s,3H),3.72(s,3H),3.68(s,6H)ppm; ESI-MS:m / z 420.1[M+H] + HRMScalcd for C 28 H 28 O6[M+H] + 421.1648, found 421.1651.

[0055] 6) Synthesis of Compound 7 (2-(3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)-4,6-dimethoxyindanone phosphate derivative): Compound 6 (1.20 g, 2.9 mmol) was dissolved in 15 mL of anhydrous tetrahydrofuran in a 100 mL double-necked flask. Sodium hydride (0.47 g, 19 mmol) was added to the flask under nitrogen protection. 0.9 mL of diethyl chlorophosphate (1.0 g, 5.8 mmol) was added dropwise under stirring at room temperature, and the reaction was allowed to proceed for 4 h at room temperature. A sample was spotted onto a TLC plate, and the reaction was stopped after Compound 6 had completely reacted. Compound 7 exhibited characteristic purple-blue fluorescence. The reaction was quenched with water, extracted with ethyl acetate, and the organic layers were combined, evaporated to dryness, and purified by silica gel column chromatography [V(petroleum ether):V(ethyl acetate) = 1:2] to obtain Compound 7. Structural characterization: Pink oily substance;1 H NMR (400MHz, CDCl3) δ6.94(s,1H),6.85(d,J=8.0Hz,2H),6.75(d,J=2.0Hz,2H),6.67(s,1H),6.46(s,2H),6.06(s ,1H),4.66(s,1H),3.76-3.96(m,8H),3.61(s,3H),3.47(d,6H),3.32(d,3H),0.93-1.12(m,12H)ppm.ESI-MS:m / z 692.2[M+H] + .

[0056] Synthesis of (±)-STO021 (2-(3,5-dihydroxyphenyl)-3-(4-hydroxyphenyl)-4,6-dihydroxyindone phosphate derivative): Compound 7 (0.69 g, 1 mmol) was added to a 100 mL round-bottom flask with 20 mL of anhydrous dichloromethane. Under nitrogen protection, the mixture was stirred at 0 °C for 10 min. 7 mL of boron tribromide solution was carefully injected using a syringe. The mixture was then cooled to room temperature and stirred for 3 h. A sample was spotted onto a TLC plate, and the reaction was stopped after compound 5 had fully reacted. The reaction was quenched with ethanol and water, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, evaporated to dryness, and purified by silica gel column chromatography [V(dichloromethane):V(methanol) = 10:1] to obtain product (±)-STO021. Structural characterization: Pinkish-purple solid; 1 H NMR (400MHz, DMSO-d6) δ9.30(s,1H),9.12(d,3H),7.04(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,2H),6.62(d,J=2.0Hz,1H),6.38( d,J=2.0Hz,2H),6.10(d,J=2.0Hz,1H),6.07(t,J=2.0Hz,1H),4.87(s,1H),4.11-3.96(m,8H),1.36-1.06(m,12H)ppm.ESI-MS m / z:637.1[M+H] + .

[0057] 8. Preparation of (S)-STO021: (S)-STO021 was obtained by chiral column separation of synthesized (±)-STO021. The system conditions for chiral separation were as follows: High performance liquid chromatograph: Shimadzu LC-20AT, CP-HPLC-07; Mobile phase: 7:1:2 = n-hexane / dichloromethane / ethanol; Flow rate: 1.0 mL / min; Temperature: 35℃; UV detection wavelength: 254 nm; Run time: 10 min; Injection volume: 5 μL.

[0058] Structural characterization: Pink solid; IR(film):νmax=3422.22,1603.61,1502.88,1245.20,1162.06,1030.22,843.07cm -1 ; 1 H NMR(600MHz,DMSO-d6)δ9.28(s,1H),9.11(s,2H),9.09(s,1H),7.04(d,J=8.3Hz,2H),6.95(d,J=8.3Hz,2H),6.51(s,1H),6.38(s,2H),6 .08(d,J=18.6Hz,2H),4.86(d,J=4.1Hz,1H),4.18–3.94(m,9H),1.25(t,J=7.0Hz,3H),1.20(t,J=7.0Hz,6H),1.15(t,J=7.0Hz,3H)ppm; 13 C NMR(151MHz,DMSO-d6)δ158.05,157.74,152.80,148.30,148.25,144.11,144.05,140.40,135.98,133.86,133.13,133.09,129.1 6,121.03,119.12,106.64,101.68,101.35,98.17,64.27,64.23,64.11,64.05,64.01,55.91,49.33,39.95,18.45,15.69ppm.HRMS calcd forC 29 H 34 O 12 P4[M+H] + 637.1616, found 637.1612.

[0059] Example 2: Compound (S)-STO021 showed a dose-dependent restoration of BMSC cell viability.

[0060] BMSC cells were irradiated according to the irradiation plan for 24 hours, and then the culture medium was replaced with the corresponding group's medium. Cells were cultured for 1-7 days, and cell viability was assessed using a CCK8 assay kit at 3, 5, and 7 days post-irradiation. The culture medium was discarded, and each well was filled with culture medium containing 10% CCK8 reagent. Cells were incubated in the dark, and the absorbance was measured at 450 nm using a microplate reader, indicating cell viability. Results are as follows: Figure 1 As shown, the results indicate that treatment with (S)-STO021 (0.1–10 μM) can restore the viability of irradiated BMSC cells (***p<0.001).

[0061] Example 3: Compound (S)-STO021 restores the morphological damage of BMSC cells to a certain extent.

[0062] The irradiation plan is as follows: 137 Csγ rays, dose 2 Gy. P2-P3 generation mouse BMSCs in good growth condition were inoculated and cultured for 24 h. 137 Csγ-ray irradiation at a dose of 2 Gy was performed. Control group cells (0 Gy) were placed in the irradiator for the same duration but were not exposed to radiation, and subsequent culture conditions were the same as those for the irradiated group cells.

[0063] Treatment with compound (S)-STO021 (0.1–10 μM) 137 BMSCs irradiated with Csγ rays for 24 hours were stained with TRITC-labeled phalloidin for F-actin and DAPI for nucleus staining to observe morphological changes in each group of BMSCs. The method is briefly described below: BMSCs in good growth condition (P2-P3 generation) were collected and cell cultured at 5 × 10⁶ cells / well. 3 Mouse BMSCs were seeded into 48-well plates and cultured for 24 h. After 24 h of irradiation, they were irradiated according to the irradiation plan for each group. Following irradiation, (S)-STO021 at the above concentration was added to each well and cultured for another 24 h. Cells were then fixed with 2.5% glutaraldehyde at room temperature for 10 min, and the fixative was washed with PBS. 200 μL of TRITC-labeled phalloidin working solution was added to each well to cover the cells, and the cells were incubated at room temperature in the dark. After washing with PBS, 200 μL of DAPI solution was added to each well, and the cells were incubated at room temperature in the dark. After washing with PBS, the morphological changes of BMSCs in each group were observed and images were captured using a Leica fluorescence microscope. The results are as follows: Figure 2 As shown, Figure 2 The image shown is a 400X magnified photograph. The results show that the control group (0Gy) cells have a stellate or fibroblast-like morphology with clear boundaries, while the 2Gy dose can cause changes in the morphology of BMSC cells, with fewer cells in the field of view, blurred boundaries, and larger cell bodies. After using the anti-aging positive drug resveratrol and the test drug (S)-STO021, the morphological damage and the reduced number of cells in the field of view were restored to some extent.

[0064] Example 4: Compound (S)-STO021 enhances colony formation in BMSC cells.

[0065] (A) Colony staining using the crystal violet method, briefly described as follows: using... 137After irradiating BMSC cells with Csγ rays for 24 hours, the cells were cultured in a medium containing the corresponding group compounds for 2-3 weeks. When the cells grew into colonies and visible clones appeared in the culture dish, the culture was stopped, staining was performed, and the colony formation of each group of BMSCs was observed under a microscope.

[0066] (B) Quantitative analysis of the colonies was performed to determine the BMSC colony formation rate. Results are as follows: Figure 3 As shown, the results indicate that (S)-STO021 (0.1–10 μM, **p<0.01) can improve the colony formation rate of BMSC cells.

[0067] Example 5: Compound (S)-STO021 significantly reduced the rate of senescence-associated heterochromatin foci (SAHF) positive cells.

[0068] BMSCs were seeded onto cell culture slides and irradiated according to the irradiation plan for each group. The culture medium was replaced 24 hours later for the corresponding group. After one day of culture, DAPI fluorescence staining was used to visualize senescence-related heterochromatin foci in the nuclei of BMSCs from each group. The staining method was briefly described as follows: cells were fixed with 2.5% glutaraldehyde at room temperature for 10 min, washed with PBS, and 200 μL of DAPI solution was added to each well. The cells were incubated at room temperature in the dark. After washing with PBS, observation and photography were performed using a laser confocal microscope to observe senescence-related heterochromatin foci in the nuclei of BMSCs from each group. (Reference) Figure 4 Analysis of the formation of senescence-associated heterochromatin foci (SAHF) at punctate DNA focal points (200X) (A) and the proportion of SAHF-positive cells (B) showed that (S)-STO021 (0.1–10 μM, ***p<0.001) significantly reduced the formation of senescence-associated heterochromatin foci at punctate DNA focal points (SAHF) and significantly reduced the rate of SAHF-positive cells.

[0069] Example 6: Compound (S)-STO021 significantly reduced the expression level of aging-related β-galactosidase (SA-β-gal).

[0070] BMSC cells were irradiated according to the irradiation plan for 24 hours, and then the culture medium was replaced for the corresponding group. When the cells reached approximately 80% confluence, the expression of senescence-associated β-galactosidase (SA-β-gal) was detected using a senescence-associated β-galactosidase chromogenic kit. The staining method was briefly described as follows: fixation with a dedicated fixative at room temperature for 15 minutes, washing with PBS, adding freshly prepared working solution to each well, sealing the culture plate, and incubating overnight at 37°C (CO2-free). After removing the staining solution and washing with PBS, the expression of SA-β-gal in each group was observed under an optical microscope.

[0071] Results Reference Figure 5SA-β-gal staining results (A) and SA-β-gal positive cell proportion analysis (B) showed that both (S)-STO021 (1 μM) and resveratrol (Res, 1 μM) could reduce SA-β-gal expression. Compared with the irradiated group, (S)-STO021 (0.1–10 μM, ***p<0.001) significantly reduced SA-β-gal expression, and the reduction level of SA-β-gal by 1 μM (S)-STO021 was comparable to that of the positive control resveratrol.

[0072] Example 7: Compound (S)-STO021 significantly reduced the mRNA expression levels of aging-related genes p16 and p21.

[0073] BMSC cells were irradiated according to the irradiation plan for 24 hours, and then the culture medium was replaced for the corresponding group. After 4 days of culture, RT-qPCR was used to detect changes in the expression of aging-related genes p21 and p16. The detection method was briefly described as follows: Total RNA was extracted from the collected cells. Using the extracted total RNA as template RNA, reverse transcription was performed to obtain cDNA. The entire reaction was performed using a real-time quantitative PCR system. After the program was completed, the Ct value was saved, and 2... -ΔΔCq The method quantifies the relative mRNA expression level of a specified gene. GAPDH was used as a control for normalization. Results reference: Figure 6 The results showed that (S)-STO021 (0.1–10 μM, ***p<0.001) significantly reduced the mRNA expression levels of p16 and p21 in a dose-dependent manner.

[0074] Example 8: Compound (S)-STO021 significantly reduced the protein expression levels of aging-related proteins p16 and p21.

[0075] Western blot was used to detect changes in the expression of senescence-related proteins p21 and p16. The experimental procedures included total cellular protein extraction, protein concentration determination using the BCA method, protein denaturation, PAGE gel preparation, electrophoresis, membrane transfer, blocking, and antibody incubation. Chemiluminescence was used to obtain developing bands, and ImageJ was used for grayscale analysis of the protein bands. Results are referenced below. Figure 7 The results showed that (S)-STO021 (0.1-10 μM, ***p<0.001) could significantly reduce the expression level of p16 protein, and (S)-STO021 (0.1-10 μM, ***p<0.001) could also reduce the expression level of p21 protein in a dose-dependent manner.

[0076] Example 9: Compound (S)-STO021 exhibits excellent in vivo safety and low cardiotoxicity.

[0077] The maximum tolerated dose of compound (S)-STO021 was investigated in mice. Healthy adult female Balb / c mice weighing 20±2g were randomly divided into 5 groups of 8 mice each: a solvent blank group, a 25mg / kg group, a 75mg / kg group, a 150mg / kg group, and a 300mg / kg group. The mice were administered the drug daily via gavage at a volume of 0.2mL / 10g. The behavioral status and mortality of the mice were continuously observed and recorded for 14 days. Changes in weight and food intake were recorded to preliminarily assess the safety. After the experiment, blood was collected from the mice's eyes, and serum was separated for liver and kidney function tests. Organs were weighed, and the organ-to-body weight ratio was calculated. The organs were then fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE) to examine their histopathological changes.

[0078] The experimental results are shown in Figure 8, which includes... Figure 8-1 and Figure 8-2 , Figure 8-1 There are two pictures, A and B. Figure 8-2 Figures C and D show that (S)-STO021's LD 50 >300mg / kg; Serum liver and kidney function indicators showed that (S)-STO021 did not show obvious liver and kidney dysfunction or damage; HE staining of organs also showed no obvious organ inflammation or damage.

[0079] In (A), a represents the curves showing the change in body weight of mice in the five groups (8 female mice per group) over 14 days. The results show that the weight change trend of (S)-STO021 is the same as that of the blank group (average weight gain of 1.63g), with a slight increase, indicating that oral administration for 14 consecutive days did not have a significant impact on the survival status of the mice in each group. b represents the curves showing the change in food intake of the five groups (8 female mice per group) over 14 days. The results show that (S)-STO021 administration for 14 consecutive days did not have a significant impact on the survival status of the mice in each group. c represents the comparison of the organ-to-body weight ratio of the five groups (8 female mice per group). The results show that compared with the blank solvent group, there was no significant change in the organ-to-body weight ratio in the (S)-STO021 group, indicating that (S)-STO021 has high safety for mice.

[0080] In (B), a, b, and c represent the results of liver function index analysis in mouse serum. Comparing the serum levels of alanine aminotransferase (ALT, a), aspartate aminotransferase (AST, b), and alkaline phosphatase (ALP, c) in the five groups of mice, there was no significant difference between the treated group and the blank solvent group, proving that (S)-STO021 did not cause significant liver damage in mice. d, e, and f represent the results of kidney function index analysis in mouse serum. Comparing the serum levels of creatinine (CREA, d) and urea (UREA, e) in the five groups of mice, there was no significant difference between the treated group and the control group; comparing the uric acid (UA, f) levels (from left to right: 80.0, 48.94, 80.89, 36.19, 36.73 μmol·L⁻¹) showed no significant difference. -1 The study found a significant difference between the 300 mg / kg treatment group and the control group at 300 mg / kg (*P<0.05), indicating that continuous administration of 300 mg / kg (S)-STO021 for 14 days reduced uric acid in mice, but this had no significant effect on renal function.

[0081] (C) indicates the HE staining results of mouse organs after the maximum tolerated dose study. No significant organ toxicity was observed in the heart (Heart) group. In the liver (Liver) group, the lobules and hepatocytes were intact, with blue-stained nuclei. The nuclei were centrally located, round, and large, with clearly visible nucleoli, and no significant abnormalities were observed. In the spleen (Spleen) group, the splenic bodies were normal in morphology, with no significant increase in follicles, and no transformed lymphoblasts were found, showing no significant abnormalities. In the lung (Lung) group, the tissues showed a reticular structure, normal lumen dilation, and no significant abnormalities. In the kidney (Kidney) group, the renal tubules were normally dilated, the glomerular structure was clear, and there was no significant congestion in the intercellular matrix, showing no significant abnormalities. HE staining results confirmed that (S)-STO021 has good in vivo safety.

[0082] The hERG cardiotoxicity evaluation experiment assessed the hERG inhibitory activity of (S)-STO021 using the conventional patch-clamp technique, with the hERG inhibitor quinidine as a positive control and the control group as a blank control.

[0083] (D) represents the hERG cardiotoxicity evaluation results. Typical electrogram results for (S)-STO021 and the positive control (a) show that the negative control showed the highest potassium current, indicating no blockade of the hERG ion channel, while the positive control quinidine almost completely inhibited the potassium current, and (S)-STO021 showed no inhibitory activity. The current inhibition rate of different concentrations of (S)-STO021 in the control group (b) shows that the inhibition rate of (S)-STO021 on the current is close to zero and remains unchanged with varying concentrations. The IC50 of compound (S)-STO021 for its inhibitory activity on the hERG potassium ion channel is shown. 50 The result of >30μM indicates that (S)-STO021 has almost no blocking effect and low cardiotoxicity within the tested concentration range.

[0084] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A compound ( S The use of )-STO021 in the preparation of anti-aging drugs is characterized by, The compound ( S The structure of )-STO021 is shown in equation (I): （Ⅰ）。 2. The use according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable medicinal salts.

3. The use according to claim 2, characterized in that, The pharmaceutical salts include inorganic acid salts and organic acid salts. The inorganic acid salts are selected from one of hydrochloride, phosphate, hydrobromide and sulfate. The organic acid salts are selected from one of malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, dihydroxynaphthylate, salicylate and stearate.

4. The use according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that, The dosage form of the drug is tablet, pill, powder, suspension, emulsion, granule, capsule, syrup, suppository, injection, ointment, patch or nasal drop.

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