Photosensitizer prodrug compound for selectively eliminating senescent cells and preparation method and application thereof

Abstract

The present invention discloses a photosensitizer prodrug compound (structural formula (I)) that selectively clears senescent cells and is specifically activated by the aging-associated β-galactosidase, as well as its preparation method and application. Upon activation by β-galactosidase, the compound rapidly releases its active molecule, anchoring it to proteins with nucleophilic groups. This allows for precise detection and covalent anchoring of senescent cells, thereby achieving selective clearance of senescent cells, reversing senescence-related markers and gene expression, and ameliorating aging-related decline in bodily functions. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a photosensitizer prodrug compound that specifically activates senescence-related beta-galactosidase and selectively eliminates senescent cells, as well as a preparation method and application thereof, belonging to the technical field of medicinal chemistry. Background Art

[0002] Senescence is a major pathogenic factor in numerous diseases, including cancer and fibrosis. Cellular senescence is a stable and arresting end state that emerges after stress-induced cellular damage, and its overdevelopment is considered a fundamental mechanism of aging. The accumulation of senescent cells promotes the secretion of pro-inflammatory and matrix-degrading molecules (termed the senescence-associated secretory phenotype), further driving inflammation-mediated tissue dysfunction and aging. Selective elimination of senescent cells can alleviate these age-related hallmarks of aging and extend healthy lifespan. Surgical resection of senescent tissue is currently used as a treatment for motor dysfunction caused by senescent cell accumulation. However, this treatment is limited to aging muscles and causes difficult-to-repair trauma and pain for patients. Therefore, a better approach is to use drugs that selectively eliminate senescent cells. Initially, such drugs were selected based on their ability to transiently shut down senescence-associated anti-apoptotic pathways. Typical examples include ABT-737 and ABT-263, which target senescent cells by inhibiting anti-apoptotic proteins in the BCL-2 family to induce apoptosis. Natural "selective senescent cell elimination" drugs, such as quercetin and fexitin, have also been shown to be effective against aging-related diseases. However, senescent cells exhibit tremendous heterogeneity and dynamics in vivo, resulting in significant limitations in the accuracy (non-toxicity to non-senescent cells), tractability (controllable dosing), and broad-spectrum activity (effectiveness against different types of senescent cells) of these "selective senescent cell elimination" drugs. Although new strategies have emerged to address these issues, such as glutaminase inhibitors and enzyme-targeted prodrugs, to our knowledge, no single "selective senescent cell elimination" drug strategy has been reported that can achieve all of these goals. Summary of the Invention

[0003] One of the objectives of the present invention is to provide a strategy for constructing a "selective senescent cell elimination" photosensitizer prodrug compound that integrates photodynamic therapy, β-galactosidase-targeted prodrug technology, and protein anchoring technology, thereby providing a "selective senescent cell elimination" photosensitizer prodrug compound with controllability, broad-spectrum activity, and accuracy, and a preparation method thereof.

[0004] A second objective of the present invention is to utilize the aforementioned integrated strategy to develop a photosensitizer prodrug compound for selective senescent cell elimination, specifically recognizing the senescence-associated β-galactosidase, thereby enabling precise detection and covalent binding of senescent cells. Furthermore, utilizing this photosensitizer prodrug compound, which can covalently bind to senescent cells, allows for photodynamic targeted elimination of senescent cells at the single-cell level, reversing senescence-associated markers and gene expression, and ameliorating aging-related decline in bodily functions.

[0005] The implementation process of the present invention is as follows:

[0006] The compound represented by structural formula (I),

[0007]

[0008] Where R is selected from or , X is O or Se.

[0009] The preparation method of the compound of structural formula (I) comprises the following steps:

[0010] (1) Compound A and compound B are catalyzed by an inorganic base to obtain compound C;

[0011]

[0012] (2) Compound C and compound D are catalyzed by an organic base to obtain compound E;

[0013]

[0014] (3) Compound E reacts with a fluorinating agent to obtain Compound F, wherein the fluorinating agent is selected from diethylaminosulfur trifluoride, N-fluorobisbenzenesulfonamide, morpholine sulfur trifluoride, or bis-(2-methoxyethyl)amine sulfur trifluoride;

[0015]

[0016] (4) Compound F undergoes ester exchange reaction to obtain compound G;

[0017] .

[0018] The above preparation methods are all carried out in organic solvents, and in the corresponding steps:

[0019] In step (1), the organic solvent used includes but is not limited to acetonitrile, dichloromethane, 1,4-dioxane, tetrahydrofuran, and toluene, and the inorganic base used includes but is not limited to potassium carbonate, cesium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide;

[0020] In step (2), the organic solvent used includes but is not limited to acetonitrile, methanol, ethanol, isopropanol, toluene, and benzene; the organic base used includes but is not limited to hexahydropyridine, triethylamine, and pyridine;

[0021] In step (3), the organic solvent used includes but is not limited to dichloromethane, tetrahydrofuran, cyclohexane, and 1,4-dioxane;

[0022] In step (4), the organic solvent used includes but is not limited to methanol, ethanol, and isopropanol, and the organic base used includes but is not limited to sodium methoxide, sodium ethoxide, and sodium tert-butoxide.

[0023] The compound represented by structural formula (I) has multiple uses. It can visualize and accurately detect senescent cells enriched in β-galactosidase at the single-cell level. The active molecule generated after reaction with the enzyme can anchor within senescent cells. Upon activation by light of a specific wavelength, it converts intracellular oxygen into reactive oxygen species, selectively eliminating senescent cells and achieving anti-aging effects. It has the potential to reverse the upregulation of aging-related markers and genes, and improve physiological dysfunction caused by aging.

[0024] Specifically, the compound represented by structural formula (I) has one or more of the following applications:

[0025] (1) A composition or preparation for the detection of β-galactosidase;

[0026] (2) Compositions or preparations for detecting β-galactosidase activity in samples at the molecular level, cellular level, tissue section level, and living animal level;

[0027] (3) Compositions or preparations for selectively identifying and anchoring cells with high β-galactosidase expression;

[0028] (4) A composition or preparation for selectively eliminating cells with high β-galactosidase expression;

[0029] (5) Compositions or preparations for preparing aging-related genes for reversing aging in living animals;

[0030] (6) Compositions or preparations for enhancing or improving the behavioral ability or motor ability of aging animals.

[0031] The composition is a pharmaceutical composition, a cosmetic composition, a dietary supplement, a food composition or a health product composition.

[0032] The pharmaceutical composition comprises compound (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier and excipient.

[0033] The pharmaceutical composition is an injection or oral medicine.

[0034] The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, pills, solutions, syrups or injections.

[0035] Advantages of the present invention: (1) For the first time, a new strategy for constructing a "selective senescent cell elimination" photosensitizer prodrug compound that integrates photodynamic therapy, β-galactosidase targeted prodrug technology, and protein anchoring technology is proposed. This integrated strategy provides a new paradigm for the development of "selective senescent cell elimination" drugs, which can overcome the limitations of currently available "selective senescent cell elimination" drugs. (2) A "selective senescent cell elimination" photosensitizer prodrug compound that specifically activates senescence-related β-galactosidase with controllable, broad-spectrum activity, and accuracy is developed. Accurate detection and covalent anchoring of senescent cells are achieved, thereby achieving photodynamic targeted elimination of senescent cells at the single-cell level, reversing senescence-related markers and gene expression, and improving the decline in physical function caused by aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Fluorescence emission spectra and gel electrophoresis experiments after adding β-galactosidase to KSL0608-O / KSL0608-Se;

[0037] Figure 2 Kinetic studies, pH effects, photostability studies, and selectivity studies for different types of nucleophilic small molecules and bovine serum albumin (BSA) after adding β-galactosidase to KSL0608-O / KSL0608-Se;

[0038] Figure 3 To study the photosensitizer activity of KSL0608-O / KSL0608-Se;

[0039] Figure 4 Fluorescence imaging of KSL0608-O / KSL0608-Se in replicative senescent cells and chemically induced senescent cells (including reactive oxygen species, doxorubicin, and mitomycin-induced senescent cells);

[0040] Figure 5 KSL0608-Se-mediated photodynamic therapy targets and eliminates replicative senescent cells and chemically induced senescent cells (including reactive oxygen species-induced, doxorubicin-induced, and mitomycin-induced);

[0041] Figure 6 Single-cell-level visualization of photodynamic therapy experiments using KSL0608-O / KSL0608-Se in a co-incubation system of senescent and young cells;

[0042] Figure 7In vivo and ex vivo organ imaging experiments of KSL0608-O in naturally aged and young mice;

[0043] Figure 8 To investigate the effects of KSL0608-Se-mediated photodynamic therapy on the reversal of aging-related markers and genes in doxorubicin-induced aging mice;

[0044] Figure 9 KSL0608-Se-mediated photodynamic therapy reverses aging-related markers in the liver and kidneys of naturally aged mice;

[0045] Figure 10 KSL0608-Se-mediated photodynamic therapy reverses the expression of aging-related markers in the serum and aging-related genes in the liver of naturally aging mice, thereby improving aging-related physiological dysfunctions. DETAILED DESCRIPTION

[0046] After extensive and in-depth research, the inventors of this application have discovered for the first time a photosensitizer prodrug compound specifically activated by the senescence-associated enzyme β-galactosidase, which selectively eliminates senescent cells. Upon activation by β-galactosidase, the compound rapidly releases its active molecule and further anchors within senescent cells. Upon photoactivation, it releases fluorescence and generates reactive oxygen species, enabling visualization of senescent cells in aging mice at the single-cell level, reversing the expression of senescence-associated markers and genes, and ameliorating aging-related physiological dysfunction. This invention was completed on this basis. It should be understood that, within the scope of the present invention, the aforementioned technical features of the present invention and those described in detail in the Examples below may be combined to form novel or preferred technical solutions.

[0047] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions (such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0048] Example 1

[0049] Preparation of intermediate KSL0608-O1

[0050]

[0051] 4-Hydroxy-3-hydroxymethylbenzaldehyde (0.56 g, 3.65 mmol), Cs2CO3 (5 g, 15.38 mmol), and Na2SO4 (1.39 g, 9.79 mmol) were dissolved in acetonitrile (30 mL). 2,3,4,6-tetraacetyl-α-D-bromogalactose (2.3 g, 2.43 mmol, 1.2 equivalents) was then added to the reaction system. The mixture was stirred at room temperature under nitrogen. After completion of the reaction, the filtrate was collected by filtration, and the solvent was removed to obtain the crude product. After column chromatography, KSL0608-O1 (0.9 g, 51%) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3): δ 9.93 (s, 1H), 7.90 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 5.57–5.52 (m, 1H), 5.50 (s, 1H), 5.20(d, J = 8.0 Hz, 1H), 5.15 (d, J = 11.3 Hz, 1H), 4.68 (s, 2H), 4.27–4.19 (m, 1H), 4.17 (s, 1H), 4.14 (s, 1H), 2.20 (s, 3H), 2.10 (s, 4H), 2.06 (s, 3H), 2.03(s, 3H). 13 C NMR (100 MHz, DMSO -d 6):δ 192.32, 170.43, 170.34, 170.00, 169.79,157.94, 132.64, 131.44, 130.92, 128.28, 114.21, 97.66, 71.11, 70.29, 68.49,67.63, 61.79, 57.53, 20.87.FT-IR (KBr, cm -1 ):3458.47, 3130.24, 1751.69,1688.92, 1605.98, 1496.99, 1400.39, 1239.27, 1126.50,1075.75, 953.02, 907.91,907.91, 739.40, 601.57.ESI MS m / zcalcd. for C 22 H 26 O 12 Na + , 505.1316; found [M+Na] + ,505.1289.

[0052] Preparation of intermediate KSL0608-O2

[0053]

[0054] 2-(2-Methyl-4H-1-benzopyran-4-ylidene)malononitrile (200 mg, 0.96 mmol) and KSL0608-O1 (556 mg, 1.15 mmol) were dissolved in anhydrous acetonitrile (10 mL). Piperidine (150 μL, 1.72 mmol) and acetic acid (75 μL, 1.76 mmol) were added, and the reaction system was heated to reflux. After the reaction was complete, the solvent was removed, and the crude product was separated by column chromatography to obtain KSL0608-O2 (380 mg, 58.8%) as a yellow solid. 1 H NMR (400 MHz, DMSO -d 6):δ 8.74 (d, J = 8.4 Hz, 1H), 7.93 (t, J = 8.4 Hz, 1H), 7.84 (d, J = 9.0 Hz,2H), 7.81 (d, J = 4.4 Hz, 1H), 7.76 (s, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 (t, J =7.7 Hz, 1H), 7.44 (d, J = 16.0 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.09 (s, 1H), 5.52 (d, J = 7.4 Hz, 1H), 5.38 (s, 1H), 5.28 (dd, J = 9.0 Hz, 5.3 Hz, 1H), 5.21(t, J = 5.4 Hz, 1H), 4.50 (dd, J = 12.3 Hz, 5.7 Hz, 1H), 4.41 (d, J= 15.9 Hz, 2H),4.14 (t, J = 6.0 Hz, 2H), 2.16 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H). 13 C NMR (100 MHz, DMSO -d 6): δ 169.35, 169.16, 169.08, 169.02, 156.44,155.04, 151.77, 151.26, 136.91, 133.67, 130.61, 128.83, 127.69, 124.98,124.78, 117.56, 116.86,116.78, 115.75, 114.77, 114.70, 113.92, 105.69, 98.07,70.31, 69.34, 67.58, 65.72, 61.61, 60.32, 59.71, 19.82, 19.67, 19.64,19.55.FT-IR (KBr, cm -1 ):3449.27, 3129.88, 2211.50, 1750.84, 1632.40, 1600.54,1558.07, 1499.50,1481.76, 1400.92, 1238.52, 1130.52, 1076.12, 980.51,596.66.HR-ESI MS m / z calcd. for C 35 H 32 N2O 12 Na + , 695.1847, found [M+Na] + , 695.1860.

[0055] Preparation of compound KSL0608-O3

[0056]

[0057] KSL0608-O2 (100 mg, 0.15 mmol) was dissolved in anhydrous dichloromethane (5 mL). Diethylaminosulfur trifluoride (100 µL, 0.75 mmol) was added dropwise. The reaction was stirred at room temperature under nitrogen until completion. The reaction was quenched by adding saturated ammonium chloride solution. The reaction system was extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was removed to obtain the crude product. Column chromatography afforded KSL0608-O3 (80 mg, 79.7%) as a yellow solid. 1 H NMR (400 MHz, CDCl3): δ 8.86 (d, J = 8.2 Hz, 1H), 7.68 (t, J =7.4 Hz, 1H), 7.56 (d, J = 12.2 Hz, 2H), 7.49 (d, J = 11.1 Hz, 2H), 7.40 (t, J = 7.7Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.70 (d, J = 15.9 Hz, 1H), 5.53–5.46 (m, 1H), 5.44 (s, 1H), 5.38–5.26 (m, 1H), 5.24–5.09(m, 1H), 5.04 (d, J =8.0 Hz, 2H), 4.26–4.15 (m, 1H), 4.16–4.03 (m, 2H), 2.14 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 170.34, 170.16, 170.09,169.50, 157.26,155.82, 152.80, 152.31, 137.47, 134.72, 129.87, 129.63,128.93, 127.03, 126.04, 125.87, 118.58, 118.26, 117.84, 116.72, 115.68,115.07, 106.90, 99.38, 80.12,79.02, 71.34, 70.51, 68.11, 66.73, 62.97, 61.36,29.70, 25.94, 22.66, 20.66.FT-IR (KBr, cm -1 ):3458.83, 3130.14, 2211.89,1752.05, 1634.81, 1559.30, 1499.75, 1400.73, 1239.08, 1076.94, 978.48,601.63.HR-ESI MS m / z calcd. for C 35 H 31 FN2O 11 Na + , 697.1804, found [M+Na] + ,697.1820.

[0058] Preparation of intermediate KSL0608-O

[0059]

[0060] KSL0608-O3 (60 mg, 0.09 mmol) was dissolved in methanol (3 mL), and the reaction system was cooled to 0°C. Sodium methoxide (34 mg, 0.63 mmol) was added and stirred in an ice bath until the reaction was complete. The pH of the reaction system was adjusted to neutral by adding dilute hydrochloric acid, and the solvent was removed. The crude product was separated by column chromatography to obtain KSL0608-O (22 mg, 49%) as a yellow solid. 1 H NMR (400 MHz, DMSO- d 6): δ 8.73 (d, J =8 .3 Hz, 1H),7.93 (t, J = 7.4 Hz, 1H), 7.85 (s, 1H), 7.81–7.78 (m, 1H), 7.75 (t,J = 11.2 Hz,2H), 7.62 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 16.0 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H),7.03 (s, 1H), 5.55 (m, 2H), 5.26 (d, J = 5.1 Hz, 1H), 4.91 (t, J = 7.2 Hz, 2H),4.68 (t, J = 5.1 Hz, 1H), 4.56 (d, J = 4.4 Hz, 1H), 3.72 (s, 1H), 3.61 (d, J = 6.2Hz, 2H), 3.58–3.55 (m, 1H), 3.54–3.51 (m, 1H), 3.46–3.41 (m, 1H). 13 C NMR (150MHz, DMSO- d 6): δ 158.86, 157.09, 153.46, 152.52, 138.60, 135.90, 131.05,129.13, 128.91, 126.63,126.35, 125.12, 119.53, 118.60, 117.74, 117.58,116.38, 115.67, 106.91, 101.74, 80.61, 79.53, 76.18, 73.49, 70.68, 68.57,60.87.FT-IR (KBr, cm -1 ):3445.59, 3173.82, 2819.64, 2352.61, 2209.29, 1631.53,1598.26, 1557.67,1495.77, 1455.57, 1400.51, 1352.64, 1262.78, 1216.50,1123.08, 1085.10, 982.30, 767.40, 548.75.HR-ESI MS m / z calcd. for C 27 H 23 FN2O7Na + ,529.1382, found [M+Na] + , 529.1388。

[0061] Preparation of intermediate KSL0608-Se1

[0062]

[0063] 2-(2-Methyl-4H-1-phenylselenol-4-ylidene)malononitrile (261 mg, 0.96 mmol) and KSL0608-O1 (556 mg, 1.15 mmol) were dissolved in anhydrous acetonitrile (10 mL). Piperidine (150 μL, 1.72 mmol) and acetic acid (75 μL, 1.76 mmol) were added to the reaction system, and the reaction system was heated to reflux. After the reaction was completed, the solvent was removed, and the crude product was separated by column chromatography to obtain KSL0608-Se1 (340 mg, 48%) as a red solid. 1 H NMR (400 MHz, DMSO -d 6):δ 8.61 (d, J = 9.1 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 13.2Hz, 2H), 7.72 (d, J = 6.3 Hz, 2H), 7.68 (s, 3H), 7.33 (d, J = 16.0 Hz, 1H), 7.10(d, J = 8.6 Hz, 1H), 5.51 (d, J = 7.4 Hz, 1H), 5.38 (d, J = 3.1 Hz, 1H), 5.30–5.23(m, 2H), 5.17 (t, J = 5.4 Hz, 1H), 4.51–4.48 (m, 1H), 4.44 (d, J = 5.3 Hz, 1H),4.40 (d, J = 5.4 Hz, 1H), 4.13 (t, J = 4.0 Hz, 1H), 2.16 (s, 3H), 2.06 (d, J = 5.1Hz, 6H), 1.96 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 170.39, 170.19, 170.10,170.06, 158.81, 155.79, 149.15, 137.13, 133.69, 131.81, 131.60, 130.23,129.64, 129.45, 128.60, 128.32, 128.25,126.94, 126.52, 123.59, 116.64,115.46, 115.00, 99.16, 72.83, 71.36, 70.45, 68.61, 66.75, 61.38, 60.84,20.86, 20.74, 20.69, 20.59.FT-IR (KBr, cm -1 ):3415.00, 3129.31, 2208.76,1750.93, 1637.69, 1618.16, 1567.21, 1534.95,1400.55, 1239.36, 1127.53,1075.56, 954.53, 770.31, 601.43.HR-ESI MS m / z calcd. for C 35 H 32 N2O 11 SeNa + ,759.1064, found [M+Na] + , 759.1072.

[0064] Preparation of compound KSL0608-Se2

[0065]

[0066] The synthesis route refers to the synthesis steps of KSL0608-O3, and the obtained KSL0608-Se2 is a red solid. 1 H NMR (400 MHz, DMSO -d 6): δ 8.62 (d, J = 9.4 Hz, 1H), 8.12 (d, J = 9.1 Hz, 1H), 7.93 (s,1H), 7.87 (d, J = 8.8 Hz, 1H), 7.79 (s, 2H), 7.73 (d, J = 12.4 Hz, 1H), 7.68 (s,1H), 7.34 (d, J= 16.0 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 5.57 (d, J = 7.2 Hz, 1H),5.43–5.37 (m, 2H), 5.31–5.25 (m, 3H), 4.54 (t, J = 6.5 Hz, 1H), 4.16–4.13 (t, J =6.5 Hz, 2H), 2.16 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H). 13 C NMR (150 MHz, CDCl3): δ 169.33, 169.15, 169.06, 168.48, 157.76, 154.55, 147.89, 135.71, 132.63, 130.80, 129.20, 128.62, 128.43, 128.27, 127.71, 127.25,125.98,125.91, 125.78, 122.71, 115.54, 114.37, 114.11, 98.42, 79.11, 78.00,70.34, 69.51, 67.12, 65.76, 60.40, 28.68, 19.72, 19.64, 19.56.FT-IR (KBr, cm -1 ):3460.06, 3130.38, 2209.33, 1752.69, 1637.01, 1568.01, 1536.35, 1499.72,1459.43, 1400.49, 1239.10, 1129.06, 1075.89, 953.74, 899.48.HR-ESI MS m / z calcd. for C 35 H 31 FN2O 10 SeNa + , 761.1020, found [M+Na] + , 761.1035.

[0067] Preparation of compound KSL0608-Se

[0068]

[0069] The synthetic route refers to the synthetic steps of KSL0608-O, and the obtained KSL0608-Se is a red solid. 1 H NMR (600 MHz, DMSO -d 6):δ 8.61 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.87 (s,1H), 7.77 (s, 2H), 7.70 (dd, J = 14.6, 7.0 Hz, 2H), 7.65 (t, J = 7.6 Hz, 1H),7.31 (d, J = 16.0 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 5.54 (ddd, J = 59.5, 47.6,11.1 Hz, 2H), 5.27 (d, J = 5.1 Hz, 1H), 4.91 (d, J = 6.2 Hz, 2H), 4.69 (t, J = 5.4Hz, 1H), 4.57 (d, J = 4.4 Hz, 1H), 3.72 (s, 1H), 3.64 (dt, J = 14.4, 7.2 Hz, 2H),3.57 (dt, J = 11.0, 5.5 Hz, 1H), 3.54–3.52 (m, 1H), 3.45–3.42 (m, 1H). 13 C NMR (150 MHz, DMSO -d 6): δ 159.13, 156.92, 151.24, 137.90, 134.15, 132.67, 130.99,130.56,129.67, 129.45, 129.06, 128.68, 127.07, 126.47, 126.29, 123.33,117.52, 116.03, 115.63, 101.78, 80.58, 76.18, 73.53, 71.40, 70.71, 68.59,60.88.FT-IR (KBr, cm -1):3409.47, 3131.23, 2203.05, 1616.37, 1566.74, 1534.65,1505.28, 1478.38, 1455.76, 1400.60, 1348.92, 1303.49, 1257.62,1221.42,1124.95, 1080.32, 949.31, 866.14, 809.31, 768.92, 716.27, 621.22, 575.14.HR-ESI MS m / z calcd. for C 27 H 23 FN2O6SeNa + , 593.0598, found [M+Na] + , 593.0605.

[0070] Example 2

[0071] The "selective elimination of senescent cells" photosensitizer prodrug compound KSL0608-O / KSL0608-Se prepared in Example 1 specifically activated by senescence-related β-galactosidase reacted with Escherichia coli β-galactosidase ( E. coli The changes in fluorescence emission spectra before and after the reaction of β-gal and bovine serum albumin (BSA) were analyzed, as well as protein electrophoresis experiments to verify the ability of the active intermediate to be anchored on proteins with nucleophilic groups.

[0072] Accurately weigh appropriate amounts of KSL0608-O and KSL0608-Se and dissolve them in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. Take 20 μL of the stock solution and add it to a 2 mL centrifuge tube. Then add different volumes of 100 U / mL β-galactosidase standard solution (final concentration varies from 0 to 4 U / mL). Then add 100 μL of 1 mg / mL BSA stock solution to the system. The system is buffered with PBS (10 mmol / L, E. coli For the β-gal assay, the sample was diluted to 2 mL using PBS buffer (pH 7.4) and incubated at 37°C for 30 minutes as the experimental group. For the blank group, 20 μL of the above-mentioned probe stock solution was transferred to a 2 mL centrifuge tube and directly diluted to 2 mL with PBS without the addition of β-galactosidase. The sample was then incubated at 37°C for 30 minutes. Fluorescence spectra of the experimental and blank samples were measured using a fluorescence spectrophotometer. For protein electrophoresis, the photosensitizer stock solution, β-galactosidase, and BSA were added to the centrifuge tube, respectively. After incubation at 37°C for 30 minutes, loading buff solution was added to the tube. The tube was incubated in a 100°C metal bath for 10 minutes before loading into the gel lanes for the experiment.

[0073] like Figure 1 As shown, the photosensitizer KSL0608-O and KSL0608-Se solutions were mixed with different concentrations of E. coli After incubation with β-gal standard solution, the fluorescence spectrum changes significantly. As the enzyme concentration increases, the fluorescence at the maximum emission wavelength of the released active molecules gradually increases. E. coli Within the β-gal concentration range, the fluorescence intensity at the maximum emission wavelength showed a good linear relationship with the enzyme concentration. The maximum emission wavelength of KSL0608-O was 665 nm, and the maximum emission wavelength of KSL0608-Se was 721 nm. In addition, protein electrophoresis experiments showed that the photosensitizer molecules only reacted with BSA or E. coli There was no obvious fluorescent band when incubated with β-gal. E. coli When incubated with β-gal, there is an obvious fluorescent band near 75KDa, indicating that the active intermediate produced can be anchored on the protein.

[0074] Example 3

[0075] Example 1: Enzyme kinetics of the "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se specifically activated by senescence-related β-galactosidase

[0076] The fluorescence intensity of KSL0608-O (10 μM) and KSL0608-Se (10 μM) in PBS buffer solution with pH = 7.4 was investigated over time at a temperature of 37 °C. Figure 2 As shown in Figure a, after adding β-galactosidase and BSA to the KSL0608-O solution, the fluorescence intensity at the maximum emission point of 665 nm increased with the increase of incubation time, and the fluorescence intensity reached saturation when the incubation time was 5 min. Figure 2 As shown in middle b, after adding β-galactosidase and BSA to the KSL0608-Se solution, the fluorescence intensity at the maximum emission point of 721 nm increased with the increase of incubation time, and the fluorescence intensity reached saturation when the incubation time was 10 min.

[0077] Example 4

[0078] Relationship between the fluorescence spectra of the "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se specifically activated by the senescence-associated β-galactosidase prepared in Example 1 and the pH value before and after the reaction with β-galactosidase and BSA.

[0079] The changes in fluorescence intensity of KSL0608-O (10 μM) and KSL0608-Se (10 μM) with β-galactosidase and BSA in PBS buffer solutions of different pH values were investigated at a temperature of 37°C. Figure 2 As shown in Figure c, after adding β-galactosidase and BSA, the KSL0608-O solution has obvious fluorescence response ability in the pH range of 6-8. Figure 2 As shown in middle d, after adding β-galactosidase and BSA, the KSL0608-Se solution has obvious fluorescence response ability in the pH range of 6-8.

[0080] Example 5

[0081] After adding β-galactosidase and BSA to the senescence-associated β-galactosidase-specifically activated "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se prepared in Example 1, the relationship between the fluorescence intensity of the system and the illumination time.

[0082] The photostability of the active molecules KSL0608-O (10 μM) and KSL0608-Se (10 μM) was investigated after they were treated with β-galactosidase and BSA, respectively, at a temperature of 37°C. Figure 2 As shown in Figure f, after adding β-galactosidase and BSA, the fluorescence intensity of the KSL0608-O solution at the maximum emission wavelength of 665 nm did not change significantly with the extension of illumination time, indicating that it has good photostability. Figure 2 As shown in Figure g, after adding β-galactosidase and BSA, the fluorescence intensity of the KSL0608-Se solution at the maximum emission wavelength of 721 nm did not change significantly with the extension of illumination time, indicating that it has good photostability.

[0083] Example 6

[0084] Fluorescence spectra of the "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se specifically activated by the senescence-associated β-galactosidase prepared in Example 1 after addition of β-galactosidase and further incubation with different nucleophilic small molecules and amino acids.

[0085] The fluorescence changes of KSL0608-O and KSL0608-Se (10 μM) after the reaction with β-galactosidase were investigated after the released active molecules reacted with different nucleophilic species at a test temperature of 37 °C. Figure 2As shown in Figure h, after adding β-galactosidase and different nucleophilic species, including BSA, L-cysteine (L-Cys), glutathione (GSH), N-acetylcysteine (NAC) and hydrogen sulfide (H2S), the KSL0608-O solution showed obvious fluorescence only after adding β-galactosidase and BSA at the same time, indicating that the rigidity of the active molecule is enhanced after the intermediate is anchored with BSA, and the fluorescence is enhanced. Figure 2 As shown in Figure (i), after adding β-galactosidase and different nucleophilic species, including BSA, L-cysteine (L-Cys), glutathione (GSH), N-acetylcysteine (NAC), and hydrogen sulfide (H2S), to the KSL0608-Se solution, significant fluorescence was only produced after the simultaneous addition of β-galactosidase and BSA, indicating that the generated intermediate, anchored to BSA, enhances the rigidity of the active molecule and the fluorescence.

[0086] Example 7

[0087] After adding β-galactosidase and BSA to the solution of the senescence-associated β-galactosidase-specifically activated "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se prepared in Example 1, diphenylisobenzofuran (DPBF), a reactive oxygen species (ROS) probe, was added to the system to study the generation of ROS in the system after illumination.

[0088] The ability of the released active molecules to generate reactive oxygen species under light after the reaction of KSL0608-O (10 μM) and KSL0608-Se (10 μM) with β-galactosidase and BSA was investigated at 37°C. Figure 3 As shown, after KSL0608-O (10 μM) reacted with β-galactosidase and BSA, the absorbance intensity of bulk DBPF at 410 nm decreased slightly under 535 nm illumination. In contrast, after KSL0608-Se (10 μM) reacted with β-galactosidase and BSA, the absorbance intensity of bulk DBPF at 410 nm decreased significantly under 535 nm illumination. In the control system containing β-galactosidase and BSA, the UV absorbance intensity of DBPF remained unchanged. Furthermore, after KSL0608-Se reacted with β-galactosidase and BSA and was exposed to light for 90 seconds, the UV absorbance intensity of DPBF in the system decreased significantly. This indicates that the active molecules produced by the reaction of KSL0608-Se with β-galactosidase and BSA can generate a large amount of reactive oxygen species when activated by 535 nm light.

[0089] Example 8

[0090] The application of the "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O / KSL0608-Se prepared in Example 1, which is specifically activated by senescence-associated β-galactosidase, in the detection of replicative senescent cells and chemically induced senescent cells.

[0091] KSL0608-O or KSL0608-Se at a concentration of 10 μM was incubated with A549 cells (non-small cell lung cancer cells), A549 cells induced by mitomycin senescence, HL-7702 cells (normal human liver cells), HL-7702 cells induced by doxorubicin senescence, NRK-52E cells (rat renal tubular epithelial cells), NRK-52E cells induced by mitomycin senescence, MRC-5 cells at passage 28 (human embryonic lung fibroblasts), and MRC-5 cells at passage 40 for 30 minutes at 37°C, and the imaging effects were observed using a fluorescence microscope. Figure 4 As shown, KSL0608-O and KSL0608-Se produced significant fluorescence after incubation with the above senescent cells. X-gal staining results showed that these senescent cells all highly expressed β-galactosidase.

[0092] Example 9

[0093] The "selective elimination of senescent cells" photosensitizer prodrug compound KSL0608-Se, which specifically activates senescence-associated β-galactosidase prepared in Example 1, was verified by an experiment to selectively eliminate senescent cells under light irradiation.

[0094] The CCK-8 method was used to detect the phototoxicity of photosensitizer molecules to the cells in the above four cell models, and to investigate the ability of diagnostic and therapeutic photosensitizer molecules to selectively eliminate senescent cells. KSL0608-Se was incubated with A549 cells, induced A549 cells, HL-7702 cells, induced senescent HL-7702 cells, NRK-52E cells, induced senescent NRK-52E cells, 28th generation MRC-5 cells and 40th generation MRC-5 cells at a concentration of 0-30 μM for 30 minutes, and then irradiated with light (535 nm) for 20 minutes, and the percentage of live cells was tested after 24 hours, 48 hours and 72 hours. Figure 5 As shown in the results, KSL0608-Se can significantly induce apoptosis of senescent cells under light exposure, while showing no significant toxicity to young cells after 24, 48, and 72 hours.

[0095] Example 10

[0096] The "selective elimination of senescent cells" photosensitizer prodrug compound KSL0608-O / KSL0608-Se, which is specifically activated by senescence-associated β-galactosidase prepared in Example 1, was used to verify the visualization of old cells at the single-cell level in a mixed system (young cells and senescent cells).

[0097] After staining young cells with a green fluorescent single-cell dye (CMFDA), they were co-incubated with senescent cells to construct a hybrid system. The hybrid system was incubated with 10 μM photosensitizer molecules KSL0608-O and KSL0608-Se for 30 minutes, and the cell nuclei were stained with Hoechst. The cells were imaged under a confocal microscope. Figure 6 As shown in Figure a, in the mixed system, the 28th generation MRC-5 (green fluorescence) and the 40th generation MRC-5 were specifically labeled by KSL0608-O, indicating that the generated active molecules were targeted and anchored in senescent cells. As the illumination time increased, there was no obvious change in the cells, indicating that KSL0608-O can detect senescent cells at the single cell level. Figure 6 As shown in (b), passage 28 MRC-5 (green fluorescence) and passage 40 MRC-5 were specifically labeled with KSL0608-Se, indicating that the generated active molecules were targeted and anchored within senescent cells. With prolonged illumination, passage 40 MRC-5 cells (red fluorescence) gradually underwent apoptosis, demonstrating that KSL0608-Se can visualize the induction of apoptosis in senescent cells at the single-cell level.

[0098] Example 11

[0099] The ability of the "selective senescent cell elimination" photosensitizer prodrug compound KSL0608-O, which is specifically activated by the senescence-associated β-galactosidase prepared in Example 1, to detect senescent cells in mice.

[0100] The imaging experiment of mice was performed on an in vivo fluorescence imaging system (Perkin Elmer IVIS Spectrum CT imaging system) (Ex: 535 nm, Em: 640-660 nm). Before imaging, the hair on the back and abdomen of the mice was shaved. The aged mice and young mice were intraperitoneally injected with 20 mg / kg of KSL0608-O, and the images were taken 1 hour, 12 hours, 24 hours, 72 hours and 96 hours after the injection. After the imaging was completed, the mice were killed and their major organs (heart, liver, spleen, lungs and kidneys) were dissected for in vitro imaging. Figure 7As shown, compared to young mice, aged mice had a significant fluorescence signal, and the fluorescence intensity gradually increased over time. Furthermore, the liver and kidneys of aged mice showed significant fluorescence signals, indicating that senescent cells in aged mice are primarily concentrated in these two organs. These results also demonstrate that KSL0608-O can detect senescent cells in mice.

[0101] Example 12

[0102] The "selective elimination of senescent cells" photosensitizer prodrug compound KSL0608-Se, which specifically activates senescence-associated β-galactosidase prepared in Example 1, was used to verify the targeted elimination of senescent cells in doxorubicin-induced senescent mice.

[0103] A doxorubicin-induced aging mouse model was constructed, and KSL0608-Se was injected intraperitoneally to investigate its ability to eliminate senescent cells and delay aging in vivo.

[0104] A young control group (only vehicle), a doxorubicin group (only vehicle), and a "KSL0608-Se + light group" (20 mg / kg) were set up. The solvent was 5 vol% DMSO, 70 vol% PBS, and 25 vol% castor oil. Mice in the doxorubicin group received two injections of doxorubicin (3 mg / kg) in advance, 10 days apart. After doxorubicin-induced aging mice were injected intraperitoneally with KSL0608-Se, they were irradiated with a 535 nm laser for 20 minutes 3, 24, and 48 hours later. Figure 8 As shown, the expression of senescence-associated markers γ-H2AX, AST, ALT, p21, and senescence-related genes increased in doxorubicin-induced senescence mice. Treatment with senolytics reduced the expression of these markers. X-gal staining of mouse kidney sections also demonstrated that KSL0608-Se-mediated photodynamic therapy significantly eliminated senescent cells in the kidneys. In summary, KSL0608-Se-mediated photodynamic therapy can eliminate senescent cells in doxorubicin-induced senescence mice and reverse the expression of senescence-associated markers and genes.

[0105] Example 13

[0106] The "selective elimination of senescent cells" photosensitizer prodrug compound KSL0608-Se, which specifically activates senescence-associated β-galactosidase and is prepared in Example 1, was used to verify the targeted elimination of senescent cells in naturally aging mice.

[0107] Naturally aged mice (21 months old) were treated with KSL0608-Se-mediated photodynamic therapy to investigate its ability to eliminate senescent cells in vivo and delay aging.

[0108] A young control group (2 months), an aged control group (21 months), a KSL0608-Se group, and a KSL0608-Se + light group were set up. The mice in the young control group and the aged control group were intraperitoneally injected with lysozyme, and the mice in the KSL0608-Se group were intraperitoneally injected with KSL0608-Se (10 mg / kg). The mice in the "KSL0608-Se + light group" were intraperitoneally injected with KSL0608-Se (10 mg / kg). At 24 hours, 48 hours, 72 hours, and 96 hours after the injection, the mice were irradiated with a 535 nm laser for 20 minutes. The mice were dosed once every two weeks and exposed to light four times for four weeks. The solvent was 5 vol% DMSO, 70 vol% PBS, and 25vol% castor oil. Figure 9 As shown in the results, compared with young control mice, the liver and kidneys of aged control mice showed a significant increase in senescent cells and increased expression of senescence-related markers, including p53, urea, creatinine, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase. After KSL0608-Se-mediated photodynamic therapy, senescent cells in the kidneys and livers of mice were significantly eliminated, and the expression of the above-mentioned senescence-related markers was significantly reduced.

[0109] In addition, if Figure 10 As shown, aging-induced upregulation of senescence-related markers in serum was significantly decreased after KSL0608-Se-mediated photodynamic therapy, including CXC chemokine ligand 1, CXC chemokine ligand 3, interleukin-1β, interleukin-6, matrix metalloproteinase 1, matrix metalloproteinase 7, and tumor necrosis factor α. Behavioral experiments demonstrated that KSL0608-Se-mediated photodynamic therapy ameliorated the aging-induced decline in physiological function in mice. Transcriptome sequencing results demonstrated that KSL0608-Se-mediated photodynamic therapy reversed the expression of aging-related genes in mice. In summary, KSL0608-Se-mediated photodynamic therapy can eliminate senescent cells in naturally aged mice, reverse the expression of senescence-related markers and genes, and improve the decline in physiological function caused by aging.

Claims

1. A compound represented by structural formula (I), Where R is selected from 、 , X is O or Se.

2. The method for preparing the compound according to claim 1, characterized in that The following steps are involved: (1) Compound A and compound B are catalyzed by an inorganic base to obtain compound C; （2） Compound C and compound D are catalyzed by an organic base to obtain compound E; （3） Compound E reacts with a fluorinating agent to obtain compound F, wherein the fluorinating agent is selected from diethylaminosulfur trifluoride, N-fluorobisbenzenesulfonamide, morpholine sulfur trifluoride or bis-(2-methoxyethyl)amine sulfur trifluoride; （4） Compound F undergoes an ester exchange reaction to obtain compound G; 。 3. The preparation method according to claim 2, characterized in that The preparation method is carried out in an organic solvent, and in the corresponding steps: (1) The organic solvent used is selected from acetonitrile, dichloromethane, 1,4-dioxane, tetrahydrofuran, and toluene, and the inorganic base used is selected from potassium carbonate, cesium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide; (2) The organic solvent used is selected from acetonitrile, methanol, ethanol, isopropanol, toluene, and benzene; the organic base used is selected from hexahydropyridine, triethylamine, and pyridine; (3) The organic solvent used is selected from dichloromethane, tetrahydrofuran, cyclohexane, and 1,4-dioxane; (4) The organic solvent used is selected from methanol, ethanol, and isopropanol, and the organic base used is selected from sodium methoxide, sodium ethoxide, and sodium tert-butoxide.

4. The use of the compound according to claim 1, selected from one or more of the following: (1) A composition or preparation for the detection of β-galactosidase; (2) Compositions or preparations for detecting β-galactosidase activity in samples at the molecular level, cellular level, tissue section level, and living animal level; (3) Compositions or preparations for selectively identifying and anchoring cells with high β-galactosidase expression; (4) A composition or preparation for selectively eliminating cells with high β-galactosidase expression; (5) Compositions or preparations for preparing aging-related genes for reversing aging in living animals; (6) Compositions or preparations for enhancing or improving the behavioral ability or motor ability of aging animals.

5. The use according to claim 4, characterized in that The composition is a pharmaceutical composition.

6. The use according to claim 5, characterized in that The pharmaceutical composition comprises compound (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier and excipient.

7. The use according to claim 5, characterized in that The pharmaceutical composition is an injection or oral medicine.

8. The use according to claim 5, characterized in that The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, pills, solutions, syrups or injections.

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