Galactose masked aminoquinoline compounds and uses thereof

Abstract

The application discloses a galactose-masked aminoquinoline compound and application thereof, and a structural formula of the compound is shown in the following formula: wherein R is acetyl, propionyl or butyryl. After being hydrolyzed by an esterase to remove acetylation, propionylation or butyrylation in cells, the application can be activated by a senescence-associated beta-galactosidase in a lysosome of a senescent cell, so that an aminoquinoline (NCQ) is released to inhibit survival of a senescent cell in vivo or in vitro.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds, and particularly relates to a galactose-masked aminoquinoline compound and application thereof. BACKGROUND

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

[0003] A class of functional molecules capable of selectively killing senescent cells, known as Senolytics, is expected to alleviate diseases related to the accumulation of senescent cells. A key feature of senescent cells is the high expression of lysosomal β-galactosidase, i.e., senescence-associated β-galactosidase (SA-β-Gal), which is also a major marker of senescent cells in the body. Currently, galactose derivatives targeting senescence-associated β-galactosidase have been developed, which release different cytotoxic molecules under enzymatic catalysis to inhibit or kill senescent cells. However, most research in this field uses galactose-conjugated broad-spectrum toxic drugs, such as anticancer drugs, which can cause off-target effects, resulting in toxic side effects of these drugs on normal cells, tissues, or organs. SUMMARY

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

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

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

[0007] A galactose-masked aminoquinoline compound, whose structural formula is

[0008]

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

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

[0011] Further preferably, R is butyryl.

[0012] The use of the above-mentioned galactose-masked aminoquinoline compound in the preparation of a senescent cell clearing agent.

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

[0014] An anti-aging composition, the effective component of which comprises the galactose-masked aminoquinoline compound.

[0015] In a preferred embodiment of the present application, the effective component is the galactose-masked aminoquinoline compound.

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

[0017] In a preferred embodiment of the present application, the effective component is the galactose-masked aminoquinoline compound.

[0018] The present application has the following beneficial effects:

[0019] 1. After being deacetylated, propionylated or butyrylated by esterase in cells, the present application can be activated by senescence-associated beta-galactosidase in lysosomes of senescent cells to release NCQ, so as to inhibit the survival of senescent cells in vivo or in vitro.

[0020] 2. When R is butyryl, the butyric acid released in cells of the present application can synergize with NCQ to inhibit the survival of senescent cells, so as to achieve more efficient and selective elimination of senescent cells, and has a broad prospect in the future anti-aging clinical application of skin anti-aging health care and elimination of senescent cells in the body. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Chemical reaction principle diagram for enzyme-catalyzed release of aminoquinoline and butyric acid of the present application.

[0022] Figure 2 Synthesis route diagram of amino chloroquinoline, O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ in Example 1 of the present application.

[0023] Figure 3 Effect of O-Acetyl-Gal-NCQ and O-Butyl-Gal-NCQ in Example 2 of the present application on the survival rate after being co-incubated with normal growing and Etoposide-induced senescent mouse MEF cells (mouse embryonic fibroblasts) for 12 h.

[0024] Figure 4 Effect of O-Butyl-Gal-NCQ in Example 3 of the present application on the cell viability after being co-incubated with normal growing and replicative senescent human HFF cells (human foreskin fibroblasts; Human Foreskin Fibroblast) for 12 h.

[0025] Figure 5Figure 4 shows the effect of O-Butyl-Gal-NCQ incubation for 6h on the subsequent time survival rate of normal growing and replicative senescent human fibroblast (HFF) cells. DETAILED DESCRIPTION

[0026] The technical solutions of the present application are further described and explained with the specific embodiments in conjunction with the accompanying drawings.

[0027] Example 1

[0028] The synthesis of O-Acetyl-Gal-NCQ is shown in Figure 2, which specifically includes the following steps: Figure 2 A, which specifically includes the following steps:

[0029] (1) Synthesize CQ-N3 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). Dissolve compound CQ-N3 (1.40 g, 3.89 mmol) and triphenylphosphine (6.12 g, 23.34 mmol) in a mixed solution of tetrahydrofuran (40 mL) and deionized water (8 mL), and react at room temperature for 12 h. After the reaction system is rotary evaporated to remove the solvent, it is separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 10:1) to obtain compound amino chloroquinoline (NCQ) (73%, 0.95 g);

[0030] (2) Synthesize S1 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, T. L. C. Li, H. Deng, Cell Research 2020, 7, 574-589). Dissolve compound S1 (1000 mg, 1.5 mmol) in dichloromethane (15 mL), and then add compound amino chloroquinoline (554 mg, 1.66 mmol) and N,N-diisopropylethylamine (585 mg, 4.5 mmol) successively. Stir the reaction at room temperature for 3-5 h. Dilute the reaction solution with dichloromethane (100 mL), and then wash it with saturated sodium carbonate aqueous solution (100 mL) and saturated sodium chloride aqueous solution (100 mL) successively. Dehydrate the obtained organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and separate and purify 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 the O-Acetyl-Gal-NCQ is Its characterization data are: 1 H NMR (500 MHz, DMSO-d6) δ 8.39 - 8.33 (m, 2H), 7.84 (d, J = 2.1 Hz, 1H), 7.76 (d, J = 2.3 Hz, 1H), 7.66 (dd, J = 8.7, 2.2 Hz, 1H), 7.45 - 7.37 (m, 2H), 7.16 (t, J = 5.8 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 5.6 Hz, 1H), 5.58 (d, J = 7.6 Hz, 1H), 5.37 (d, J = 3.5 Hz, 1H), 5.31 - 5.20 (m, 2H), 5.01 (s, 2H), 4.48 (t, J = 6.4 Hz, 1H), 4.17 - 4.09 (m, 2H), 3.71 (p, J = 6.6 Hz, 1H), 3.18 (s, 2H), 3.04 (q, J = 6.6 Hz, 2H), 2.44 (dt, J = 20.3, 7.2 Hz, 5H), 2.15 (s, 3H), 2.03 (d, J = 1.5 Hz, 6H), 1.95 (s, 3H), 1.68 (dd, J = 11.7, 5.4 Hz, 1H), 1.48 (dp, J = 26.8, 7.5 Hz, 3H), 1.25 - 1.19 (m, 4H), 0.91 (t, J = 7.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 170.42, 170.32, 170.01, 169.34, 156.29, 152.23, 150.05, 149.58, 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-TOF MS calculated for C 40 H 50 ClN5O 14 (M) m / z 859.3043, found 859.931.

[0032] O-Butyl-Gal-NCQ synthesis is shown as Figure 2 B, specifically comprising the following steps:

[0033] (1) To β-D-galactose (10 g, 55.6 mmol) was added n-butyric anhydride (87.7 g, 555.6 mmol) and pyridine (48.3 g, 611.2 mmol) and the reaction was stirred at room temperature for 12 h, TLC plate monitoring of the complete conversion of β-D-galactose, then the solvent was removed by oil pump. The resulting residue was dissolved in ethyl acetate (150 mL). The resulting solution was washed with 1 M aqueous hydrochloric acid (150 mL) and saturated aqueous sodium bicarbonate (150 mL) successively. The resulting organic phase was separated and anhydrous sodium sulfate was added to remove the water, then concentrated under reduced pressure to obtain compound S2 (100%, 29.44 g);

[0034] (2) The compound S2 was dissolved in dichloromethane (70 mL), HBr (30% in acetic acid; 30 mL) was added under nitrogen protection and ice bath conditions, and the reaction was carried out for 1 h. The reaction solution was diluted with dichloromethane (200 mL). The resulting solution was washed with ice water (300 mL) and saturated aqueous sodium bicarbonate (300 mL). The organic phase was separated and anhydrous sodium sulfate was added to remove the water, then concentrated under reduced pressure to obtain compound S3 (95%, 23.40 g);

[0035] (3) To a solution of the above compound S3 (20.0 g, 38.3 mmol) in N,N-dimethylformamide (200 mL) was added 4-hydroxy-3-nitrobenzyl alcohol (7.7 g, 46.0 mmol) and anhydrous potassium carbonate (10.6 g, 76.6 mmol). The reaction solution was stirred at room temperature for 6 h, and then the solvent was removed by rotary evaporation. The resulting residue was dissolved in ethyl acetate (300 mL). The resulting solution was washed with water (300 mL) and saturated aqueous sodium bicarbonate (300 mL), then the organic phase was separated and anhydrous sodium sulfate was added to remove the water, and concentrated under reduced pressure. The resulting 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) was added p-nitrophenyl chloroformate (660.0 mg, 3.3 mmol) and pyridine (389.0 mg, 4.9 mmol) successively. The reaction system was stirred at room temperature for 40 min, and then 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) The compound S5 (1000 mg, 1.29 mmol) was dissolved in dichloromethane (15 mL), and then the reaction system was sequentially added with aminochloroquine (NCQ, 475.0 mg, 1.4 mmol) and N,N-diisopropylethylamine (500.0 mg, 3.9 mmol). After the reaction solution was stirred at room temperature for 3-5 h, it was diluted with dichloromethane (100 mL). The obtained solution was sequentially washed with saturated aqueous sodium carbonate solution (100 mL) and saturated aqueous sodium chloride solution (100 mL). 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 the O-Butyl-Gal-NCQ is Its characterization data are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.70 (d, J = 9.1 Hz, 1H), 8.45 (dd, J = 6.4, 1.9 Hz, 1H), 8.13 (s, 1H), 7.97 (s, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.66 (dd, J = 8.7, 2.2 Hz, 1H), 7.56 (dd, J = 9.1, 2.3 Hz, 2H), 7.43 (d, J = 8.7 Hz, 1H), 6.72 (d, J = 6.4 Hz, 1H), 5.68 (d, J = 7.8 Hz, 1H), 5.41 (d, J = 3.7 Hz, 1H), 5.34 (dd, J = 10.5, 3.4 Hz, 1H), 5.28 (dd, J = 10.4, 7.8 Hz, 1H), 5.03 (s, 2H), 4.56 (t, J = 6.6 Hz, 1H), 4.17 - 4.09 (m, 2H), 3.91 (q, J = 6.9 Hz, 1H), 2.95 (s, 4H), 2.45 - 2.38 (m, 2H), 2.32 - 2.20 (m, 4H), 2.16 (td, J = 7.4, 5.8 Hz, 2H), 1.88 - 1.78 (m, 1H), 1.71 (s, 2H), 1.60 (p, J = 7.2 Hz, 3H), 1.56 - 1.42 (m, 7H), 1.30 - 1.21 (m, 4H), 1.13 (t, J = 7.2 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H), 0.89 - 0.80 (m, 10H). 13C NMR (151 MHz, DMSO-d6) δ 172.71, 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, 70.36, 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 for C 48 H 66 ClN5O 14 (M+H + )m / z 972.4295, found 972.063.

[0039] Example 2

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

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

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

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

[0044] The results are shown in Table 1. Figure 3 Under the condition of 20 μM, CQ-OH caused 10% reduction of the normal MEF cell viability, and about 30% reduction of the senescent MEF cell viability; O-Acetyl-Gal-NCQ caused about 15% reduction of the normal MEF cell viability, and about 60% reduction of the senescent MEF cell viability; and O-Butyl-Gal-NCQ caused about 25% reduction of the normal MEF cell viability, and about 90% reduction of the senescent MEF cell viability.

[0045] The results showed that CQ-OH had a mild inhibitory effect on the viability of the senescent MEF cells (about 20% difference). O-Acetyl-Gal-NCQ was more effective than CQ-OH in inhibiting the viability of the senescent MEF cells, which supported the senescence-associated β-galactosidase-mediated release of aminochinol, thus improving the anti-senescence efficiency. Compared with O-Acetyl-Gal-NCQ, O-Butyl-Gal-NCQ further reduced the viability of the senescent MEF cells, which supported the release of butyric acid in the cells to work together with aminochinol, thus improving the effectiveness and selectivity of inhibiting the viability of the senescent cells.

[0046] Example 3

[0047] The two standard solutions (CQ-OH and O-Butyl-Gal-NCQ) prepared in Example 2 were mixed with cell culture solution in the volume ratio of 1 :4000, 1 :2000, 1 :1000, 1 :500, respectively, to obtain cell culture solutions containing CQ-OH and O-Butyl-Gal-NCQ at 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively. The above cell culture solutions with different concentrations of CQ-OH and O-Butyl-Gal-NCQ were incubated with normal growing and replicatively senescent human HFF cells (human foreskin fibroblasts) in 96-well plates for 12 h, and then stained with Hoechst 33342 and propidium iodide (PI) to distinguish dead cells from living cells. The cell viability (ATP level) was determined by using the Luminescent Cell Viability Assay commercial kit.

[0048] The results are shown in Table 1. Figure 4 At a concentration of 20 μM, the clearance rates of O-Butyl-Gal-NCQ for senescent and normal human HFF cells were about 50% and 25%, respectively, while the inhibition rates of CQ-OH under the same conditions were about 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 O-Butyl-Gal-NCQ standard solution prepared was mixed with cell culture solution in the volume ratio of 1 :2000, 1 :1000, 1 :500, and 1 :250, respectively, to obtain cell culture solutions containing O-Butyl-Gal-NCQ at 5 μM, 10 μM, 20 μM, and 40 μM, respectively. The above cell culture solutions with different O-Butyl-Gal-NCQ contents were incubated with normal growing and replicatively senescent human HFF cells in 96-well plates for 6 h, and then the cells were washed and incubated in fresh cell culture solution for 0, 24, or 48 h. The above incubated cells were stained with Hoechst 33342 and propidium iodide (PI) to distinguish dead cells from living cells.

[0051] The results are shown in Table 2. Figure 5As shown: the survival rate of senescent HFF cells pre-treated with O-Butyl-Gal-NCQ for 6h decreased over time. The survival rate of senescent HFF cells pre-treated with O-Butyl-Gal-NCQ for 48h in fresh medium was close to zero, as in the 20μM condition. The survival rate of normal HFF cells pre-treated with O-Butyl-Gal-NCQ was not significantly affected under the same condition. In contrast, incubation of O-Butyl-Gal-NCQ with cells for 12h in Example 3 resulted in a 50% decrease in survival rate of senescent HFF cells and a 25% decrease in survival rate of normal HFF. Pre-treatment of O-Butyl-Gal-NCQ for 6h in this example resulted in a quantitative depletion of senescent HFF cells 48h later, without affecting the survival of normal HFF. This result shows 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 mode will improve the selectivity and effectiveness of O-Butyl-Gal-NCQ in inhibiting senescent cells.

[0052] In summary: O-Butyl-Gal-NCQ in the present application is hydrolyzed by esterase to release butyric acid and Gal-NCQ in cells. Gal-NCQ is activated by senescence-associated β-galactosidase to release aminochinoline. Butyric acid and aminochinoline synergistically promote cell death, achieving more efficient senescent cell depletion (as shown in Figure 1 A); O-Acetyl-Gal-NCQ in the present application is hydrolyzed by esterase to release Gal-NCQ in cells. Gal-NCQ is activated by senescence-associated β-galactosidase to release aminochinoline, promoting cell death (as shown in Figure 1 B).

[0053] The above merely describes preferred embodiments of the present application, and therefore cannot limit the scope of the present application. Any equivalent changes and modifications made according to the scope and content of the present patent should still fall within the scope of the present application.

Claims

1. A galactose masked aminoquinoline compound characterized by: The structural formula is ; wherein R is butyryl.

2. Use of the galactose-masked aminoquinoline compound of claim 1 for the manufacture of a senolitic agent.

3. Use of the galactose-masked aminoquinoline compound of claim 1 for the manufacture of an anti-aging composition.

4. A senescent cell scavenger, characterized in that: an effective ingredient comprising the galactose-masked aminoquinoline compound of claim 1.

5. An agent for eliminating senescent cells according to claim 4, characterized in that: an effective ingredient comprising the galactose-masked aminoquinoline compound of claim 1.

6. An anti-aging composition characterized in that: an effective ingredient comprising the galactose-masked aminoquinoline compound of claim 1.

7. An anti-aging composition according to claim 6, wherein: an effective ingredient comprising the galactose-masked aminoquinoline compound of claim 1.

Images