Galactose-masked aminoquinoline compound and application thereof

Abstract

The invention discloses a galactose-masked aminoquinoline compound and application thereof, the structural formula of the galactose-masked aminoquinoline compound is # imgabs0, and R is acetyl, propionyl or butyryl. After being subjected to hydrolysis deacetylation, propionylation or butyrylation by esterase in cells, the compound can be activated by senescence-related beta galactosidase in senescence cell lysosome to release aminoquinoline (NCQ) so as to inhibit the survival of senescence cells in vivo or in vitro.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal compounds, and particularly relates to a galactose-masked aminoquinoline compound and application thereof. Background Art

[0002] Senescent cells are a key factor in promoting aging. Their accumulation in the body can lead to tissue dysfunction and a variety of age-related diseases, including chronic renal failure, cardiovascular disease, and neurodegenerative diseases. Eliminating these senescent cells is expected to reduce chronic low-grade inflammation, improve tissue repair capacity, and thus delay or alleviate many age-related diseases.

[0003] A class of functional molecules that can selectively kill senescent cells, called Senolytics, is expected to alleviate diseases associated with the accumulation of senescent cells. A key feature of senescent cells is that their lysosomes highly express β-galactosidase, namely senescence-associated β-galactosidase (SA-β-Gal), which is also a major marker of senescent cells in the body. Currently, studies have developed galactose derivatives targeting senescence-associated β-galactosidase, which release different cytotoxic molecules under enzyme-catalyzed conditions to inhibit or kill senescent cells. However, most studies in this field use galactose-conjugated broad-spectrum toxic drugs, such as anticancer drugs, which may lead to off-target effects and cause these drugs to have toxic side effects on normal cells, tissues or organs. Summary of the invention

[0004] The present invention aims to overcome the defects of the prior art and provide a galactose-masked aminoquinoline compound.

[0005] Another object of the present invention is to provide the application of the above galactose-masked aminoquinoline compound.

[0006] The technical solution of the present invention is as follows:

[0007] A galactose-masked aminoquinoline compound, the structural formula of which is

[0008]

[0009] Wherein, R is acetyl, propionyl or butyryl.

[0010] In a preferred embodiment of the present invention, R is butyryl or acetyl.

[0011] More preferably, the R is butyryl.

[0012] Use of the galactose-masked aminoquinoline compound in preparing a senescent cell remover.

[0013] Use of the galactose-masked aminoquinoline compound in preparing an anti-aging composition.

[0014] A senescent cell remover, the active ingredient of which includes the galactose-masked aminoquinoline compound.

[0015] In a preferred embodiment of the present invention, the active ingredient is the galactose-masked aminoquinoline compound.

[0016] An anti-aging composition, the effective ingredient of which includes the galactose-masked aminoquinoline compound.

[0017] In a preferred embodiment of the present invention, the active ingredient is the galactose-masked aminoquinoline compound.

[0018] The beneficial effects of the present invention are:

[0019] 1. After being hydrolyzed and deacetylated, propionylated or butyrylated by esterase in the cell, the present invention can be activated by the senescence-associated β-galactosidase in the lysosome of senescent cells to release NCQ, thereby inhibiting the survival of senescent cells in vivo or in vitro.

[0020] 2. When R is a butyryl group, the butyric acid released in the cells of the present invention can synergistically inhibit the survival of senescent cells with NCQ, thereby achieving more efficient and selective removal of senescent cells. It has broad prospects in the future in anti-aging skin health care and anti-aging clinical applications for removing senescent cells in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The schematic diagram of the chemical reaction of the enzyme catalyzing the release of aminoquinoline and butyric acid of the present invention.

[0022] Figure 2 The synthetic route of aminochloroquinoline, O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ in Example 1 of the present invention is shown in FIG.

[0023] Figure 3 The effect of O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ on the survival rate of normal growth and Etoposide-induced senescent mouse MEF cells (mouse embryonic fibroblasts) co-incubated for 12 hours in Example 2 of the present invention is shown.

[0024] Figure 4 The effect of co-incubation of O-Butyl-Gal-NCQ with normal growing and replicatively aged human HFF cells (human foreskin fibroblasts) for 12 hours on cell viability in Example 3 of the present invention is shown.

[0025] Figure 5The effect of O-Butyl-Gal-NCQ in Example 4 of the present invention on the survival rate of normal growth and replicative senescent human HFF cells after incubation for 6 hours is shown. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0027] Example 1

[0028] O-Acetyl-Gal-NCQ was synthesized as Figure 2 As shown in A, it specifically includes the following steps:

[0029] (1) CQ-N3 was synthesized according to the literature method (F. Yu., Y. Wang, Y. Hang, W. Tang, Z. Zhao, D. Oupicky, Journal of Polymer Science Part A: Polymer Chemistry 2019, 57, 2235–2242). Compound CQ-N3 (1.40 g, 3.89 mmol) and triphenylphosphine (6.12 g, 23.34 mmol) were dissolved in a mixed solution of tetrahydrofuran (40 mL) and deionized water (8 mL) and reacted at room temperature for 12 h. The reaction system was rotary evaporated to remove the solvent and then separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 10:1) to obtain the compound aminochloroquinoline (NCQ) (73%, 0.95 g);

[0030] (2) S1 was synthesized according to the literature method (Y. Cai, H. Zhou, Y. Zhu, Q. Sun, Y. Ji, A. Xue, Y. Wang, W. Chen, X. Yu, L. Wang, H. Chen, TLC Li, H. Deng, Cell Research 2020, 7, 574-589). Compound S1 (1000 mg, 1.5 mmol) was dissolved in dichloromethane (15 mL), and aminochloroquinoline (554 mg, 1.66 mmol) and N, N-diisopropylethylamine (585 mg, 4.5 mmol) were added in sequence, and the reaction was stirred at room temperature for 3-5 h. The reaction solution was diluted with dichloromethane (100 mL), and then washed with saturated sodium carbonate aqueous solution (100 mL) and saturated sodium chloride aqueous solution (100 mL). The obtained organic phase was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure and separated and purified by silica gel column chromatography (eluent: ethyl acetate / methanol, 3:1) to obtain O-Acetyl-Gal-NCQ (86%, 1113 mg).

[0031] The structural formula of O-Acetyl-Gal-NCQ is Its characterization data are: 1 H NMR (500MHz, DMSO-d6) δ8.39–8.33(m,2H),7.84(d,J=2.1Hz,1H),7.76(d,J=2.3Hz,1H),7.66(dd,J=8.7,2.2Hz,1H),7.45–7.37(m,2H),7.1 6(t,J=5.8Hz,1H),6.92(d,J=8.0Hz,1H),6.50(d,J=5.6Hz,1H),5.58(d,J=7.6Hz,1H),5.37(d,J=3.5Hz,1H),5.31–5.20(m,2H),5.01(s,2H) ,4.48(t,J=6.4Hz,1H),4.17–4.09(m,2H),3.71(p,J=6.6Hz,1H),3.18(s,2H),3.04(q,J=6.6Hz,2H),2.44(dt,J=20.3,7.2Hz,5H),2.15(s,3 H),2.03(d,J=1.5Hz,6H),1.95(s,3H),1.68(dd,J=11.7,5.4Hz,1H),1.48(dp,J=26.8,7.5Hz,3H),1.25–1.19(m,4H),0.91(t,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ170.42,170.32,170.01,169.34,156.29,152.23,150.05,1 49.58,148.29,140.60,133.87,133.34,127.76,124.85,124.28,118.23,117.93,99 .27,99.04,71.26,70.38,68.15,67.55,64.05,61.70,53.40,52.73,49.06,48.11, 47.57,39.02,33.68,23.90,20.96,20.85,20.80,20.77,20.28,12.02.MALDI-TOFMS calculated for C 40 H 50 ClN5O 14 (M)m / z 859.3043, found 859.931.

[0032] O-Butyl-Gal-NCQ was synthesized as Figure 2 As shown in B, it specifically includes the following steps:

[0033] (1) Add n-butyric anhydride (87.7 g, 555.6 mmol) and pyridine (48.3 g, 611.2 mmol) to β-D-galactose (10 g, 55.6 mmol), stir the reaction at room temperature for 12 h, monitor the complete conversion of β-D-galactose on a TLC plate, and then drain the solvents with an oil pump. The residue was dissolved in ethyl acetate (150 mL). The solution was washed with 1 M aqueous hydrochloric acid solution (150 mL) and saturated aqueous sodium bicarbonate solution (150 mL). The organic phase was separated and dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain compound S2 (100%, 29.44 g);

[0034] (2) Dissolve the above compound S2 in dichloromethane (70 mL), add HBr (30% acetic acid solution; 30 mL) under nitrogen protection and ice bath conditions, and react for 1 hour. Dilute the reaction solution with dichloromethane (200 mL). Wash the resulting solution with ice water (300 mL) and saturated sodium bicarbonate ice water solution (300 mL), respectively. After separation of the organic phase, dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound S3 (95%, 23.40 g);

[0035] (3) Add 4-hydroxy-3-nitrobenzyl alcohol (7.7 g, 46.0 mmol) and anhydrous potassium carbonate (10.6 g, 76.6 mmol) to a solution of the above compound S3 (20.0 g, 38.3 mmol) in N,N-dimethylformamide (200 mL). The reaction solution was stirred at room temperature for 6 h and the solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate (300 mL). The solution was washed with clean water (300 mL) in a saturated sodium bicarbonate aqueous solution (300 mL), and then the organic phase was separated and dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, 1:1) to obtain compound S4 (55%, 12.85 g);

[0036] (4) To a solution of the above compound S4 (1000 mg, 1.6 mmol) in dichloromethane (15 mL) were added p-nitrophenyl chloroformate (660.0 mg, 3.3 mmol) and pyridine (389.0 mg, 4.9 mmol) in sequence. The reaction system was stirred at room temperature for 40 min, and the solvent was removed by rotary evaporation. The residue was separated and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, 3:1) to obtain compound S5 (82%, 1045 mg);

[0037] (5) After dissolving the above compound S5 (1000 mg, 1.29 mmol) in dichloromethane (15 mL), aminochloroquine (NCQ, 475.0 mg, 1.4 mmol) and N,N-diisopropylethylamine (500.0 mg, 3.9 mmol) were added to the reaction system in sequence. The reaction solution was stirred at room temperature for 3-5 h and then diluted with dichloromethane (100 mL). The resulting solution was washed with saturated aqueous sodium carbonate solution (100 mL) and saturated aqueous sodium chloride solution (100 mL) in sequence. After the organic phase was separated, it was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure and separated and purified by silica gel column chromatography (eluent: ethyl acetate / methanol, 5:1) to obtain the compound O-Butyl-Gal-NCQ (86%, 1065.00 mg).

[0038] The structural formula of O-Butyl-Gal-NCQ is Its characterization data are: 1 H NMR(500MHz,DMSO-d6)δ8.70(d,J=9.1Hz,1H),8.45(dd,J=6.4,1.9Hz,1H),8.13 (s,1H),7.97(s,1H),7.86(d,J=2.2Hz,1H),7.66(dd,J=8.7,2.2Hz,1H),7.56(dd ,J=9.1,2.3Hz,2H),7.43(d,J=8.7Hz,1H),6.72(d,J=6.4Hz,1H),5.68(d,J=7.8H z,1H),5.41(d,J=3.7Hz,1H),5.34(dd,J=10.5,3.4Hz,1H),5.28(dd,J=10.4,7.8 Hz,1H),5.03(s,2H),4.56(t,J=6.6Hz,1H),4.17–4.09(m,2H),3.91(q,J=6.9Hz, 1H),2.95(s,4H),2.45–2.38(m,2H),2.32–2.20(m,4H),2.16(td,J=7.4,5.8Hz,2 H),1.88–1.78(m,1H),1.71(s,2H),1.60(p,J=7.2Hz,3H),1.56–1.42(m,7H),1.3 0–1.21(m,4H),1.13(t,J=7.2Hz,3H),0.94(t,J=7.4Hz,3H),0.89–0.80(m,10H). 13C NMR(151MHz,DMSO-d6)δ172.71,172.16,171.70,156.33,152.57,148.36,140.44,135.98 ,133.89,132.93,125.99,125.55,124.42,123.75,118.02,117.04,99.20,98.72,71.23,7 0.36,68.02,67.42,64.34,61.49,52.13,51.17,48.84,47.57,36.42,35.65,35.63,35.59 ,35.52,32.76,20.07,18.60,18.32,18.24,18.09,13.82,13.73,13.71,13.64.MALDI-TOF MS calculated forC 48 H 66 ClN5O 14 (M+H + )m / z972.4295, found 972.063.

[0039] Example 2

[0040] Prepare a 10 mM CQ-OH standard solution: weigh 3.4 mg of CQ-OH and dissolve it in 1 mL of dimethyl sulfoxide to obtain a 10 mM CQ-OH standard solution.

[0041] Prepare a 10 mM O-Acetyl-Gal-NCQ standard solution: weigh 8.6 mg of the O-Acetyl-Gal-NCQ prepared in Example 1 and dissolve it in 1 mL of dimethyl sulfoxide to obtain a 10 mM O-Acetyl-Gal-NCQ standard solution.

[0042] Prepare a 10 mM O-Butyl-Gal-NCQ standard solution: weigh 9.7 mg of O-Butyl-Gal-NCQ prepared in Example 1 and dissolve it in 1 mL of dimethyl sulfoxide to obtain a 10 mM O-Butyl-Gal-NCQ standard solution.

[0043] The three prepared standard solutions (CQ-OH, O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ) were mixed with the cell culture solution at a volume ratio of 1:4000, 1:2000, 1:1000, and 1:500, respectively, to obtain cell culture solutions with 2.5μM, 5μM, 10μM, and 20μM of the corresponding compounds, respectively. The culture solutions of the above different concentrations of CQ-OH, O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ were incubated with normal growth and Etoposide-induced senescent mouse MEF cells (mouse embryonic fibroblasts) in a 96-well culture plate for 12 hours, and then used The cell viability (ATP level) was determined using the Luminescent Cell Viability Assay commercial kit.

[0044] The results are as follows Figure 3 As shown: Under 20μM conditions, CQ-OH caused the survival rate of normal MEF cells to decrease by 10%, and the survival rate of MEF cells in aged mice to decrease by about 30%; O-Acetyl-Gal-NCQ caused the survival rate of normal MEF cells to decrease by about 15%, and the survival rate of MEF cells in aged mice to decrease by about 60%; while O-Butyl-Gal-NCQ caused the survival rate of normal MEF cells to decrease by about 25%, and the survival rate of MEF cells in aged mice to decrease by about 90%.

[0045] The results showed that CQ-OH had a mild inhibitory effect on the survival of MEF cells in aged mice (difference of about 20%). O-Acetyl-Gal-NCQ was more effective than CQ-OH in inhibiting the survival of aged MEF cells, supporting the release of aminoquinoline mediated by senescence-associated β-galactosidase, thereby improving the anti-aging efficiency. Compared with O-Acetyl-Gal-NCQ, O-Butyl-Gal-NCQ further reduced the survival rate of aged MEF cells, which supports the synergistic effect of intracellularly released butyric acid and aminoquinoline to improve the effectiveness and selectivity of inhibiting the survival of senescent cells.

[0046] Example 3

[0047] The two standard solutions (CQ-OH and O-Butyl-Gal-NCQ) prepared in Example 2 were mixed with the cell culture solution at a volume ratio of 1:4000, 1:2000, 1:1000, and 1:500, respectively, to obtain cell culture solutions containing 2.5 μM, 5 μM, 10 μM, and 20 μM CQ-OH and O-Butyl-Gal-NCQ, respectively. The above CQ-OH and O-Butyl-Gal-NCQ culture solutions with different concentrations were incubated with normal growth and replicative senescence human HFF cells (human foreskin fibroblasts) in a 96-well culture plate for 12 hours, and then The cell viability (ATP level) was determined using the Luminescent Cell Viability Assay commercial kit.

[0048] The results are as follows Figure 4 As shown: At a concentration of 20 μM, the clearance rates of O-Butyl-Gal-NCQ in senescent and normal human HFF cells were approximately 50% and 25%, respectively, while the inhibition rates of CQ-OH under the same conditions were approximately 25% and 15%, respectively. This indicates that O-Butyl-Gal-NCQ also effectively inhibits the survival of senescent human HFF cells.

[0049] Example 4

[0050] The prepared O-Butyl-Gal-NCQ standard solution was mixed with the cell culture solution at a volume ratio of 1:2000, 1:1000, 1:500, and 1:250, respectively, to obtain cell culture solutions containing 5μM, 10μM, 20μM, and 40μM O-Butyl-Gal-NCQ, respectively. The culture solutions with different O-Butyl-Gal-NCQ contents were co-cultured with normal growth and replicative senescence human HFF cells in a 96-well culture plate for 6 hours, and then the cells were washed and placed in a new cell culture solution for further incubation for 0, 24, or 48 hours. The cells after the above incubation were stained with Hoechst 33342 and propidium iodide (PI) to distinguish dead cells from surviving cells.

[0051] The results are as follows Figure 5As shown: the subsequent survival rate of senescent HFF cells pretreated with O-Butyl-Gal-NCQ for 6h decreases with time. For example, under 20μM conditions, the survival rate of senescent HFF cells pretreated with O-Butyl-Gal-NCQ in fresh culture medium after 48h incubation is close to zero. Under the same conditions, the survival rate of normal HFF cells pretreated with O-Butyl-Gal-NCQ was not significantly affected. In contrast, in Example 3, incubation of O-Butyl-Gal-NCQ with cells for 12h resulted in a decrease in the survival rate of senescent HFF cells by about 50%, and a decrease in the survival rate of normal HFF by about 25%. In this example, pretreatment with O-Butyl-Gal-NCQ for 6h will result in quantitative clearance of senescent HFF cells 48h later, without affecting the survival of normal HFF. The results show that short-term compound treatment further improves the selectivity of O-Butyl-Gal-NCQ in killing senescent HFF cells, indicating that appropriate administration time and method will improve the selectivity and effectiveness of O-Butyl-Gal-NCQ in inhibiting senescent cells.

[0052] In summary, the O-Butyl-Gal-NCQ in the present invention is hydrolyzed by esterase in cells to remove the butyryl group and generate butyric acid and Gal-NCQ. Gal-NCQ is activated by senescence-related β-galactosidase to release aminoquinoline. Butyric acid and aminoquinoline synergistically promote cell death, achieving more efficient senescent cell clearance (such as Figure 1 A); after the O-Acetyl-Gal-NCQ in the present invention is hydrolyzed and deacetylated by esterase in the cell, the generated Gal-NCQ is activated by senescence-related β-galactosidase to release aminoquinoline, promoting cell death (such as Figure 1 B).

[0053] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A galactose-masked aminoquinoline compound, characterized in that: Its structural formula is Wherein, R is acetyl, propionyl or butyryl.

2. A galactose-masked aminoquinoline compound as claimed in claim 1, characterized in that: The R is a butyryl group or an acetyl group.

3. A galactose-masked aminoquinoline compound as claimed in claim 2, characterized in that: The R is a butyryl group.

4. Use of the galactose-masked aminoquinoline compound according to any one of claims 1 to 3 in the preparation of a senescent cell clearing agent.

5. Use of the galactose-masked aminoquinoline compound according to any one of claims 1 to 3 in the preparation of an anti-aging composition.

6. A senescent cell scavenger, characterized in that: The effective ingredient thereof comprises the galactose-masked aminoquinoline compound according to any one of claims 1 to 3.

7. The senescent cell scavenger according to claim 6, characterized in that: The active ingredient is the galactose-masked aminoquinoline compound.

8. An anti-aging composition, characterized in that: The effective ingredient thereof comprises the galactose-masked aminoquinoline compound according to any one of claims 1 to 3.

9. An anti-aging composition according to claim 8, characterized in that: The active ingredient is the galactose-masked aminoquinoline compound.

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