A class of sugar metabolite molecular probes and preparation method thereof

Abstract

The present application belongs to the field of chemical synthesis and biotechnology, and particularly relates to a kind of sugar metabolism product molecular probes and preparation method thereof. Starting from fructose, a bio-orthogonal group is added to the 4-position hydroxyl group of the compound without affecting the biological activity of the sugar metabolism product. The present application provides a kind of sugar metabolism product probe and preparation method thereof, compared with the prior art, has the following beneficial effects: (1) the present application first synthesizes an endogenous sugar metabolism product molecular probe that can undergo click chemistry reaction, by adding a bio-orthogonal group to the 4-position hydroxyl group, the biological activity of the sugar metabolism product is maintained, which can be used for live cell imaging; (2) the probe of the present application has not been reported, and the synthesis method has less side reaction, mild reaction condition, good selectivity, can reflect the behavior of endogenous sugar metabolism product in cells, including direct reaction of sugar metabolism product in live cells Subcellular localization and tracing, is an important technical support for the study of live cell sugar metabolism.

Description

Technical Field

[0001] The invention belongs to the fields of chemical synthesis and biotechnology, specifically involving a class of molecular probes for sugar metabolites and their preparation methods. Background Technology

[0002] Carbohydrate metabolism is a core component of the body's material and energy metabolism. Cells possess multiple carbohydrate metabolism pathways, such as glycolysis, hexosamine, pentose phosphate, and glycogen synthesis. Among these pathways are several key intermediates, such as fructose-1,6-bisphosphate, fructose-1-phosphate, and fructose-6-phosphate. Fructose-1,6-bisphosphate acts as a signaling molecule in carbohydrate metabolism; its decreased level is sensed by aldolases in the glycolysis pathway, subsequently initiating AMPK activation in the lysosomal pathway. This process does not involve AMP levels at all, representing a novel AMPK activation pathway (Zhang, C. Set et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 2017, 548, 112-116). Fructose-6-phosphate can reverse the increase in lipoprotein lipase protein levels induced by HK-2 inhibitors in microglia (Leng, L., Yuan, Z., Pan, R. et al. Microglial hexokinase 2 deficiency increases ATP generation through lipid metabolism leading to β-amyloid clearance. Nat. Metab. 2022, 4, 1287-1305.).

[0003] To investigate the mechanisms of action of endogenous carbohydrate metabolism signaling molecules, it is crucial to elucidate the spatial distribution of these carbohydrate metabolites within cells. Currently, a commonly used chemical tool for small molecule fluorescence imaging is the copper-catalyzed cycloaddition reaction of azides and alkynes (CuAAC), but the Cu used in this reaction... + Ions are highly toxic to cells, making live-cell imaging difficult. The cycloaddition reaction (SPAAC) of azide and dibenzooctylene (DBCO) does not require the addition of Cu to the reaction system. + Ions, therefore suitable for live-cell imaging. However, there is still a lack of molecular probes of endogenous sugar metabolites that can be used for live-cell labeling and imaging. Summary of the Invention

[0004] To address the shortcomings of existing methods, the first objective of this invention is to provide a class of chemical probes for endogenous sugar metabolites; the second objective of this invention is to provide a method for preparing the aforementioned sugar metabolite probes.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] This invention protects a class of compounds, the structure of which is shown in Formula I:

[0007]

[0008] R1 is selected from H, Specifically, the preferred compounds according to the present invention are as follows:

[0009]

[0010] Adding an azide group to the 4-hydroxyl group of the compound does not affect the biological activity of the sugar compound. The synthetic route for the compound of formula I above is as follows:

[0011]

[0012] (1) Compound A was obtained by reacting fructose with tert-butyldimethylchlorosilane;

[0013] (2) Intermediate 1 was reacted with 2,2-dimethoxypropane and p-toluenesulfonic acid to obtain compound B;

[0014] (3) The intermediate product 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 2-azidoacetic acid were reacted to give compound C;

[0015] (4) Compound C was added to the hydrofluoric acid pyridine complex to obtain compound D;

[0016] (5) The intermediate product 4, N,N-diethylphosphamide ditert-butyl ester, 5-phenyltetrazazole and 3-chloroperoxybenzoic acid were reacted to obtain compound E;

[0017] (6) Reaction of compound E with hydrochloric acid yields the target product of formula I;

[0018]

[0019] The definitions of R1, R2, and R3 are the same as in claim 3.

[0020] The present invention also protects a molecular probe, said probe being a compound of formula I or a pharmaceutically acceptable salt thereof.

[0021] The molecular probe is prepared in the same manner as the aforementioned compound of Formula I or its pharmaceutically acceptable salt.

[0022] Beneficial effects.

[0023] The present invention provides a sugar metabolite probe and its preparation method, which have the following advantages compared with the prior art: (1) The present invention synthesizes a sugar metabolite molecular probe that can undergo click chemistry for the first time. By adding a bioorthogonal group to the 4-position hydroxyl group, the biological activity of endogenous sugar metabolites is maintained, and it can be used for live cell imaging; (2) The probe of this application has not been found in the existing reports of related probes, and the synthesis method has high reaction efficiency, few side reactions, mild reaction conditions, good selectivity, sensitivity and stability, and can reflect the behavior of endogenous sugar metabolites in cells. Attached Figure Description

[0024] Figure 1 The 1H NMR spectrum of compound A is used for characterization.

[0025] Figure 2 The 1H NMR spectrum of compound B is used for characterization.

[0026] Figure 3 The 1H NMR spectrum of compound C is used for characterization.

[0027] Figure 4 The 1H NMR spectrum of compound F is used for characterization.

[0028] Figure 5 The hydrogen nuclear magnetic resonance (HMR) spectrum of compound G is used for characterization.

[0029] Figure 6 The 1H NMR spectrum characterization of the 6-phosphate fructose probe;

[0030] Figure 7 The 1H NMR spectrum was used to characterize compound H.

[0031] Figure 8 The 1H NMR spectrum of compound I is used for characterization.

[0032] Figure 9 The 1H NMR spectrum characterization of the fructose-1,6-bisphosphate molecular probe; Detailed Implementation

[0033] The invention will be further described in detail below with reference to examples. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market. Chemicals and solvents were purchased from Bailinwei Company, Inokai Company, Tongguang Company, and Taosu Company.

[0034] Example 1:

[0035] This embodiment provides a molecular probe for fructose-6-phosphate, with the following structure:

[0036]

[0037] Its preparation method includes the following steps:

[0038]

[0039] (1) Fructose (10 g, 55.51 mmol) and tert-butyldimethylchlorosilane (18 g, 119.4 mmol) were dissolved in pyridine (100 mL) and stirred at room temperature for 12 h. The mixture was then analyzed by thin-layer chromatography. When the starting material disappeared, the mixture was extracted with ethyl acetate and then with saturated sodium chloride aqueous solution. After drying with anhydrous sodium sulfate, the mixture was filtered, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain compound A (9.8 g, yield 43%). 1 H NMR (400MHz, Chloroform-d) δ4.37 (s, 1H), 4.15-4.02 (m, 2H), 3.84-3.71 (m, 4H), 0.92 (d, J = 2.5Hz, 18H), 0.14 (t, J = 1.6Hz, 12H).

[0040]

[0041] (2) Compound A (4 g, 9.7 mmol) was dissolved in 80 mL of ethyl acetate. 2,2-Dimethoxypropane (9.6 mL, 97 mmol) and p-toluenesulfonic acid (168 mg, 0.97 mmol) were added at 0 °C, and the reaction was carried out at room temperature for 5 h. Thin-layer chromatography was used for detection. After the starting material disappeared, triethylamine was added under ice bath conditions to quench the reaction. The mixture was extracted with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Compound B (1.3 g, 30% yield) was purified by silica gel column chromatography. 1 H NMR(400MHz,Chloroform-d)δ4.43(s,1H),4.24-4.19(d,1H),3.92-3.66(m,5H) ),1.51(s,3H),1.30(s,3H),0.90(d,J=11.5Hz,18H),0.09(d,J=27.7Hz,12H).

[0042]

[0043] (3) Compound B (1.5 g, 2.22 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (759.4 mg, 3.68 mmol), and 4-dimethylaminopyridine (40.8 mg, 0.33 mmol) were dissolved in dichloromethane (20 mL), the mixture was evacuated three times, and the mixture was placed in an ice-water bath. 2-Azideacetic acid (676.1 mg, 6.69 mmol) was dissolved in dichloromethane (2 mL) and slowly added to the reaction system. The mixture was stirred for 0.5 h. The reaction was allowed to proceed overnight at room temperature. Thin-layer chromatography was used for detection. After the starting material disappeared, the solvent was removed by rotary evaporation, and compound C (1.524 g, 85% yield) was purified by silica gel column chromatography. 1 H NMR(400MHz,Chloroform-d)δ5.39(d,J=2.6Hz,1H),4.62(s,1H),4.16-4.09(m,1H),3.91-3.82(m,3 H),3.82-3.72(m,3H),1.57(s,3H),1.37(s,3H),0.91-0.89(s,9H),0.89-0.87(s,9H),0.08(m,12H).

[0044]

[0045] (4) Compound C (21 mg, 0.04 mmol) was dissolved in tetrahydrofuran (2 mL) in a plastic tube. The hydrofluoric acid pyridine complex (0.04 mL, 0.02 mmol) was added dropwise under ice bath conditions, and the reaction was allowed to proceed for 1 h. Thin-layer chromatography was performed, and the reaction was quenched with saturated sodium bicarbonate solution. The mixture was extracted with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting product was purified by silica gel column chromatography to obtain compound D (1.9 mg, yield 11.5%). 1 H NMR(400MHz,Chloroform-d)δ5.22(d,J=2.7Hz,1H),4.7(s,1H),4.23-4.16(m,1H),3.90(s,2H),3.87 -3.74(m,4H),2.04(t,J=6.42Hz,1H),1.58(s,3H),1.38(s,3H),0.91(s,9H),0.09(s,6H).

[0046]

[0047] (5) Compound F (72.1 mg, 0.173 mmol) and 5-phenyltetrazolium (24.2 mg, 0.345 mmol) were dissolved in toluene, evaporated to dryness three times, and then dissolved in dichloromethane (1 mL). N,N-diethylphosphamide di-tert-butyl ester (77.5 mg, 0.311 mmol) was added dropwise at 0 °C, and the reaction was carried out at room temperature. Thin-layer chromatography was used for detection. After reacting for 15 h, 85% pure 3-chloroperoxybenzoic acid (74 mg, 0.36 mmol) was added at -78 °C, and the reaction was carried out at 0 °C for 2 h. Thin-layer chromatography was used for detection. When the starting material disappeared, the reaction was quenched by adding triethylamine and saturated sodium thiosulfate solution in sequence under ice bath. The mixture was extracted with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography to obtain compound G (36.8 mg, yield 34.9%). 1 H NMR(600MHz,Chloroform-d)δ5.36(d,J=2.3Hz,1H),4.61(s,1H),4.36-4.26(m,1H),4.22-4.08(m,2H),3.95-3 .83(m,2H),3.82-3.70(m,2H),1.58(s,3H),1.49(s,18H),1.45(s,3H),0.90(s,9H),0.08(s,3H),0.07(s,3H).

[0048]

[0049] (6) Compound F (3.14 mg, 0.006 mmol) and hydrochloric acid (0.5 mL) were added to a round-bottom flask, 0.5 mL of water was added, and the mixture was reacted at room temperature for 12 h. After filtration and evaporation, the target product 6-phosphate fructose probe (1.67 mg, yield 81%) was obtained. 1 H NMR (400MHz, Deuterium Oxide) δ4.25-4.16(m,1H),4.13-4.06(m,2H),4.02-3.84(m,4H),3.56-3.51(m,2H).

[0050] Example 2:

[0051] This embodiment provides a 1,6-bisphosphate fructose probe, with the following structure:

[0052]

[0053] Its preparation method includes the following steps:

[0054]

[0055] (1) Compound C (1.47 g, 2.76 mmol) was dissolved in tetrahydrofuran (30 mL) in a plastic tube. The hydrofluoric acid pyridine complex (2.5 mL, 1.34 mmol) was added dropwise under ice bath conditions, and the reaction was allowed to proceed for 12 h at room temperature. Thin-layer chromatography was used for detection. After the starting material disappeared, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was extracted with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting product was purified by silica gel column chromatography to obtain compound H (688 mg, yield 82%). 1 H NMR(400MHz,Chloroform-d)δ5.22(d,J=2.4Hz,1H),4.67(s,1H),4.23(ddd,J=7.4,5. 2,2.4Hz,1H),3.96-3.70(m,6H),2.10(m,1H),1.95(m,1H),1.59(s,3H),1.39(s,3H).

[0056]

[0057] (2) Compound H (661 mg, 2.18 mmol) and 5-phenyltetrazolium (955 mg, 6.54 mmol) were evaporated twice to toluene, dissolved in dichloromethane (1 mL), and N,N-diethylphosphamide di-tert-butyl ester (1.63 g, 6.54 mmol) was added dropwise. The reaction was carried out at room temperature for 2 h. 75% pure 3-chloroperoxybenzoic acid (1.5 g, 2.18 mmol) was added at -78 °C, and the reaction was carried out at 0 °C for 2 h. Thin-layer chromatography was used for detection. When the starting material disappeared, the reaction was quenched by adding triethylamine and saturated sodium thiosulfate solution in sequence under ice bath. The mixture was extracted with saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography to obtain compound I (360 mg, yield 24%). 1 H NMR (600MHz, CDCl3) δ5.32(m,1H),4.69(s,1H),4.42-4.32(m,1H),4.22-4. 07(m,4H),4.03-3.89(m,2H),1.58(s,3H),1.51-1.45(m,36H),1.38(s,3H).

[0058]

[0059] (3) Compound J (9.5 mg, 0.042 mmol) was transferred to a round-bottom flask, 1 ml of water was added, and 1 ml of concentrated hydrochloric acid was added dropwise under an ice-water bath. The mixture was stirred at room temperature for 24 h. After the reaction was completed, methanol and water were added and evaporated to dryness three times each to obtain the target product, fructose 1,6-bisphosphate probe (5.1 mg, yield 88%). 1H NMR(400MHz,Deuterium Oxide)δ4.40-3.446(m,7H),3.37-3.28(m,1H)。

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof. in R1 is selected from H, 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

3. The method for preparing the compound according to claim 1, characterized in that, (1) Compound A was obtained by reacting fructose with tert-butyldimethylchlorosilane; (2) Compound A was reacted with 2,2-dimethoxypropane and p-toluenesulfonic acid to obtain compound B; (3) Compound B, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 2-azidoacetic acid were reacted to give compound C; (4) Compound C was added to the hydrofluoric acid pyridine complex to obtain compound D; (5) Compound D was reacted with N,N-diethylphosphamide ditert-butyl ester, 5-phenyltetrazazole and 3-chloroperoxybenzoic acid to obtain compound E; (6) Compound E was reacted with type 732 acid resin to obtain the target product of formula (I); R1 is selected from H, R2 is selected from H, TBS R3 is selected from TBS,

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