ADP-ribosylated derivatives as well as preparation method and application thereof

Abstract

The invention discloses an ADP-ribosylated derivative and a preparation method and application thereof, the ADP-ribosylated derivative comprises two types, the I type derivative is an ADPr-fluorescent probe which is synthesized by directly connecting ADPr and a hydroxyl fluorophore through an O-glucosidic bond; the type II derivative is a triazole derivative which is chemically synthesized by ADPr-N and a specific alkynyl compound through click. The invention also specifically discloses a preparation method of the ADP-ribosylated derivative. The prepared I type derivative is a high-performance fluorescent substrate of ARH3; in-vitro enzyme activity detection proves that various II-type derivatives have remarkable inhibitory activity on ARH3, can be used as ARH3 inhibitors for researching life processes related to ADP-ribosylation, and have application potential in ARH3-mediated disease intervention.

Description

A class of ADP-ribosylated derivatives, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a class of ADP-ribosylated derivatives, their preparation methods, and applications. Background Technology

[0002] ADPr-N3 is an inhibitor targeting ADP-ribosylation hydrolases. It binds to alkyne compounds via a click reaction, generating a series of ADPr-triazole compounds. This effectively expands the structural diversity of the molecule, allowing it to interact with new subsites (such as hydrophobic regions and aromatic ring stacking regions) in the enzyme's active pocket, thereby enhancing binding affinity (Ki / IC50 value) and inhibitory efficacy. For different isoforms of ADP-ribosylation hydrolases or related enzyme families, subtype-selective inhibition is achieved by controlling the structure of aromatic ring substituents, reducing off-target effects, by addressing the electronic effects and steric hindrance of the molecule. ADP-ribose molecules contain multiple chiral centers, phosphate ester bonds, and sensitive glycosidic bonds. Organic chemical synthesis of fluorescent probes suffers from long routes, low yields, and purification difficulties. The newly invented method for synthesizing fluorescent probes effectively avoids these problems, obtaining the target product in a single chemical reaction, thus reducing the difficulty of acquiring ADPr-fluorescent probes. Summary of the Invention

[0003] The technical problem solved by this invention is to provide a class of ADP-ribosylated derivatives and their preparation methods. These ADP-ribosylated derivatives include two main types: Type I derivatives are ADPr-fluorescent probes synthesized by directly linking ADPr with a hydroxyl fluorescent group via an O-glycosidic bond, where the hydroxyl fluorescent group is selected from 7-hydroxycoumarins or 9-hydroxy-3-isophenoxazolone; Type II derivatives are triazole derivatives synthesized by click chemistry from ADPr-N3 and specific alkyne compounds. Type I derivatives (such as α-ADPr-9-hydroxy-3-isophenoxazolone) are high-performance fluorescent substrates for ARH3. In vitro enzyme activity assays have demonstrated that various Type II derivatives exhibit significant inhibitory activity against ARH3 and can be used as ARH3 inhibitors to study life processes related to ADP-ribosylation, showing potential application in ARH3-mediated disease intervention.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a class of ADP-ribosylated derivatives, including type I derivatives and type II derivatives, with corresponding structural formulas as follows:

[0005]

[0006] The glycosidic bond between the N / O atoms and the ribose is either α-configuration or β-configuration;

[0007] .

[0008] Furthermore, the type I derivatives include compounds with the following structures:

[0009] , , , , , , , , , , ,

[0010] , .

[0011] Furthermore, the type II derivatives include compounds with the following structures:

[0012] .

[0013] The preparation method of the ADP-ribosylated derivative described in this invention specifically involves: using β-nicotinamide adenine dinucleotide β-NAD... + The synthetic route for preparing type I derivatives is as follows: The reaction substrate is coupled with a 7-hydroxycoumarin compound or a 9-hydroxy-3-isophenoxazolone compound.

[0014] .

[0015] Furthermore, the specific preparation steps of the type I derivative are as follows: under anhydrous and oxygen-free conditions, β-nicotinamide adenine dinucleotide β-NAD... + The target product, type I derivative, is prepared by adding a fluorescent group containing a hydroxyl group, namely 7-hydroxycoumarin or 9-hydroxy-3-isophenoxazolone, to the ionic liquid [TMG][BF4] and then stirring the reaction system at 70-80℃.

[0016] The preparation method of the ADP-ribosylated derivative of the present invention specifically includes the following preparation process: firstly, β-nicotinamide adenine dinucleotide β-NAD... + α- / β-ADPr-N3 was synthesized by reacting with sodium azide, and then the α- / β-ADPr-N3 was coupled with a benzyl ether compound containing an alkyne group via a click reaction to obtain the type II derivative. The synthetic route corresponding to this process is as follows:

[0017] .

[0018] Furthermore, the specific preparation steps of the type II derivative are as follows: β-nicotinamide adenine dinucleotide β-NAD... + Sodium azide was added to saturated brine and stirred at 80-100°C. The reaction product was then purified by high performance liquid chromatography to obtain α- / β-ADPr-N3. The α-ADPr-N3 or β-ADPr-N3 with its single configuration was added to water along with a benzyl ether compound containing an alkyne group, CuSO4·5H2O, sodium ascorbate, and tris(3-hydroxypropyltriazolylmethyl)amine. The reaction system was then subjected to a click reaction at 20-60°C to obtain the target product type II derivative.

[0019] The application of the ADP-ribosylated derivative described in this invention as an ARH3 fluorescent probe, wherein the ADP-ribosylated derivative is the α-configuration of the type I derivative.

[0020] Furthermore, the α-configuration ADP-ribosylated derivative of the type I derivative is used as a hydrolysis fluorescent substrate for ARH3 to detect the hydrolysis rate of ARH3, thereby realizing the detection of ARH3 hydrolysis kinetics.

[0021] The application of the ADP-ribosylated derivative described in this invention as an ARH3 inhibitor, wherein the ADP-ribosylated derivative is an α-configuration ADP-ribosylated derivative of type II derivatives.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a method for using commercially available β-nicotinamide adenine dinucleotide β-NAD. + Using raw materials, this invention provides a simple and rapid one-step chemical reaction to synthesize ADPr-fluorescent probes and a two-step chemical reaction to obtain type II triazole derivatives. The preparation method has the following characteristics: 1. The reaction steps are simple; a fluorescent probe that can be recognized and hydrolyzed by ADPr-hydrolase can be obtained through a single chemical reaction; 2. It allows for convenient and diverse modification of type II triazole derivatives, providing a framework for the synthesis and screening of ARH3 inhibitors. The type I derivatives (such as α-ADPr-9-hydroxy-3-isophenoxazolone) of the ADP-ribosylated derivatives described in this invention are high-performance fluorescent substrates for ARH3 and can be used to detect the hydrolysis rate of ARH3. In vitro enzyme activity assays have demonstrated that various type II derivatives (such as compounds 3bv and 3bx) have significant inhibitory activity against ARH3 and can be used as ARH3 inhibitors to study life processes related to ADP-ribosylation, showing potential application in ARH3-mediated disease intervention. Attached Figure Description

[0023] Figure 1 shows the hydrolysis process of type I derivatives as ARH3 hydrolysis substrates.

[0024] Figure 2 shows the Km / Vmax results of the type I derivative as a substrate for ARH3 hydrolysis.

[0025] Figure 3 shows the inhibitory effect of some type II derivatives on ARH3.

[0026] Figure 4 shows the inhibitory effect of some type II derivatives on ARH3.

[0027] Figure 5 shows the inhibitory effect of some type II derivatives on ARH3.

[0028] Figure 6 shows the inhibitory effect of some type II derivatives on ARH3. Detailed Implementation

[0029] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0030] Example 1

[0031]

[0032] Under anhydrous and oxygen-free conditions, β-NAD + 0.015 mmol (1 equivalent) and 0.75 mmol (50 equivalent) of 7-hydroxycoumarin were added to a system containing 0.75 mmol (50 equivalent) of ionic liquid [TMG][BF4], followed by the addition of 0.75 mmol (50 equivalent) of TMG (tetramethylguanidine). 200 μL of DMSO was added to aid dissolution, and the mixture was stirred and heated at 75 °C for 2 h. Samples were taken during the reaction to monitor the reaction progress. After the reaction was completed, the reaction system was extracted three times with water (adjusted to pH=7) and dichloromethane, and the aqueous phase was collected. The product was separated and purified using semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=40 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm), and the solvent was removed using a freeze dryer to obtain a white powder product.

[0033]

[0034] 1H NMR (600 MHz, D2O) δ 8.45 (s, 1H), 8.11 (s, 1H), 7.96 (d, J = 9.6Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.01 (dd, J = 8.4 Hz, 1.8 Hz, 1H), 6.91(d, J = 2.4 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 6.12 (d, J = 6.0 Hz, 1H), 5.84(d, J = 4.2 Hz, 1H), 4.73 (t, J = 4.8 Hz, 1H), 4.61 – 4.58 (m, 2H), 4.58 -4.56 (m, 1H), 4.55 – 4.53 (m, 1H), 4.45 – 4.43 (m, 1H), 4.42 – 4.39 (m, 2H),4.32 – 4.29 (m, 1H), 4.25 – 4.22 (m, 1H). 31 P NMR (162 MHz, D2O) δ -11.26 (s).MALDI-TOF-MS: m / z C 24 H 27 N5O 16 P2, calculated [M-H] - = 703.45, found 703.45。

[0035]

[0036] 1H NMR (600 MHz, D2O) δ 8.44 (s, 1H), 8.10 (s, 1H), 7.95 (d, J = 9.6Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 5.4 Hz, 1H), 6.91 (s, 1H), 6.43 (d, J = 9.6 Hz, 1H), 6.13 (d, J = 6.0 Hz, 1H), 5.84 (d, J = 4.2 Hz, 1H), 4.72 (t, J = 4.8 Hz, 1H), 4.61 – 4.58 (m, 2H), 4.58 – 4.55 (m, 1H), 4.54 –4.51 (m, 1H), 4.45 – 4.42 (m, 1H), 4.41 – 4.37 (m, 2H), 4.31 – 4.28 (m, 1H), 4.24 – 4.21 (m, 1H). 31 P NMR (162 MHz, D2O) δ -11.32 (s). MALDI-TOF-MS: m / zC 24 H 27 N5O 16 P2, calculated [MH] - = 703.45, found 703.45.

[0037] Example 2

[0038]

[0039] Under anhydrous and oxygen-free conditions, β-NAD + 0.015 mmol (1 equiv) of 4-trifluoromethyl-7-hydroxycoumarin derivative and 0.75 mmol (50 equiv) of 4-trifluoromethyl-7-hydroxycoumarin derivative were added to a system containing 0.75 mmol (50 equiv) of ionic liquid [TMG][BF4]. Then, 0.75 mmol (50 equiv) of TMG was added to the reaction system. 200 μL of DMSO was added to aid dissolution. The resulting mixture was stirred and heated at 75 °C for 2 h. During the reaction, samples were taken to monitor the reaction. After the reaction was completed, the reaction system was extracted three times with water (adjusted to pH=7) and dichloromethane. The aqueous phase was then collected. The product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1MTEAB=40 / 80, within 40 minutes, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0040]

[0041] 1 H NMR (600 MHz, D2O) δ 8.46 (s, 1H), 8.20 (s, 1H), 7.07 (d, J = 9.0Hz, 1H), 6.84 (s, 1H), 6.52 (s, 1H), 6.49 (d, J = 9.0 Hz, 1H), 6.10 (s, 1H),5.51 (s, 1H), 4.65 – 4.60 (m, 1H), 4.52 – 4.47 (m, 1H), 4.42 – 4.28 (m, 2H),4.37 – 4.30 (m, 2H), 4.23 – 4.20 (m, 2H), 4.15 – 4.05 (m, 2H). 31 P NMR (162MHz, D2O) δ -11.3 (d). MALDI-TOF-MS: m / z C 25 H 26 F3N5O 16 P2, calculated [M-H] - =771.08, found 771.08。

[0042]

[0043] 1 H NMR (600 MHz, D2O) δ 8.23 (s, 1H), 8.04 (s, 1H), 7.54 (d, J = 9.0Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.88 (s, 1H), 6.71 (s, 1H), 5.94 (s, 1H),5.67 (s, 1H), 4.61 – 4.57 (m, 1H), 4.51-4.49 (m, 1H), 4.48 – 4.45 (m, 1H),4.42 – 4.39 (m, 1H), 4.37 – 4.30 (m, 2H), 4.25 – 4.18 (m, 2H). 31 P NMR (162MHz, D2O) δ -11.1 (d). MALDI-TOF-MS: m / z C 25 H 26F3N5O 16 P2, calculated [MH] - =771.08, found 771.08.

[0044] Example 3

[0045]

[0046] Under anhydrous and oxygen-free conditions, β-NAD + 0.015 mmol (1 equivalent) and 0.75 mmol (50 equivalent) of 9-hydroxy-3-isophenoxazinone were added to a system containing 0.75 mmol (50 equivalent) of ionic liquid [TMG][BF4], followed by the addition of 0.75 mmol (50 equivalent) of TMG to the reaction system. 200 μL of DMSO was added to aid dissolution, and the resulting mixture was stirred and heated at 75 °C for 2 h. After the reaction was completed, the reaction system was extracted three times with water (adjusted to pH=7) and dichloromethane, and the aqueous phase was collected. The product was separated and purified using semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=40 / 80, within 40 minutes, flow rate=3.0 mL / min, I=254 nm), and the solvent was removed by freeze drying to obtain a dark red powder.

[0047]

[0048] 1 H NMR (600 MHz, D2O) δ 8.03 (s, 1H), 7.93 (s, 1H), 7.61 (d, J = 9.0Hz, 1H), 7.44 (d, J = 9,6 Hz, 1H), 7.10 – 7.06 (m, 1H), 6.88 (d, J = 9.6 Hz,1H), 6.29 (s, 1H), 5.82 (d, J = 5.4 Hz, 1H), 5.79 (s, 1H), 4.55 (t, J =5.4Hz, 1H), 4.50 (t, J = 4.8 Hz, 1H), 4.45 (d, J = 4.8 Hz, 1H), 4.42 – 4.36(m, 2H), 4.26 – 4.21 (m, 1H), 4.18 – 4.12 (m, 2H), 4.09 – 4.03 (m, 1H). 31PNMR (162 MHz, D2O) δ -11.2 (s). MALDI-TOF-MS: m / z C 27 H 28 N6O 16 P2, calculated [MH] - = 754.10, found 754.10.

[0049]

[0050] 1 H NMR (600 MHz, D2O) δ 8.04 (s, 1H), 7.93 (s, 1H), 7.57 (d, J = 8.4Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 9.0 Hz, 1H), 6.95 (s, 1H), 6.86 (d, J = 9.0 Hz, 1H), 6.25 (s, 1H), 5.82 (s, 1H), 5.79 (s, 1H), 4.57 (s,1H), 4.52 – 4.49 (m, 1H), 4.48 – 4.45 (m, 1H), 4.43 – 4.38 (m, 2H), 4.26 –4.22 (m, 2H), 4.19 – 4.14 (m, 2H), 4.08 – 4.05 (m, 1H). 31 P NMR (162 MHz, D2O)δ -11.2 (s). MALDI-TOF-MS: m / z C 27 H 28 N6O 16 P2, calculated [MH] - = 754.10, found754.12.

[0051] Example 4

[0052]

[0053] Add ADPr-N3 (0.01 mmol, 1 equiv), 1-(benzyloxy)-2-((prop-2-yn-1-yloxy)methyl)benzene (0.015 mmol, 1.5 equiv), CuSO4 .5H₂O (0.01 mmol, 1 equiv), NaASC (0.01 mmol, 1 equiv), and THPTA (0.005 mmol, 0.5 equiv) were dissolved in DMSO, and the reaction system was stirred at 40 °C for 3 h. After the reaction was completed, the reaction system was extracted with water / dichloromethane, the aqueous phase was collected, purified by high performance liquid chromatography, and the solvent was removed by freeze drying to obtain a clean product. MALDI-TOF-MS m / z C₃₂H₃₈N₈O₁₅P₂, calculated M=836.19, found 836.19.

[0054] The target products obtained by replacing different 7-hydroxycoumarin compounds using the preparation method of Example 1 are as follows:

[0055]

[0056] 1 H NMR (600 MHz, D2O) δ 8.17 (s, 1H), 8.02 (s, 1H), 7.46 (d, J = 9.0Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 1.8 Hz, 1H), 6.11 (s, 1H), 5.93 (d, J = 4.8 Hz, 1H), 5.64 (s, 1H), 4.59 (t, J = 4.8 Hz, 1H), 4.49 (t, J= 4.8 Hz, 1H), 4.46 (d, J = 4.8 Hz, 1H), 4.39 (d, J = 4.8 Hz, 1H), 4.36 –4.32 (m, 2H), 4.29 – 4.24 (m, 1H), 4.23 – 4.16 (m, 2H), 2.34 (s, 1H). 31 P NMR(162 MHz, D2O) δ -11.24 (s). MALDI-TOF-MS: m / z C 25 H 29 N5O 16 P2, calculated [MH] - = 717.11, found 717.11.

[0057]

[0058] 1H NMR (600 MHz, D2O) δ 8.18 (s, 1H), 8.02 (s, 1H), 7.47 (d, J = 13.8Hz, 1H), 6.88 (d, J = 9.6 Hz, 1H), 6.80 (s, 1H), 6.12 (s, 1H), 5.93 (d, J =7.8 Hz, 1H), 5.64 (s, 1H), 4.59 (t, J = 4.8 Hz, 1H), 4.50 (t, J = 4.8 Hz,1H), 4.46 (d, J = 4.8 Hz, 1H), 4.42 – 4.37 (m, 2H), 4.36 – 4.32 (m, 1H), 4.27– 4.16 (m, 4H), 2.35 (s, 1H). 31 P NMR (162 MHz, D2O) δ -11.31 (s). MALDI-TOF-MS: m / z C 25 H 29 N5O 16 P2, calculated [M-H] - = 717.11, found 717.11。

[0059]

[0060] 1 H NMR (600 MHz, D2O) δ 8.32 (s, 1H), 7.97 (s, 1H), 7.65 – 7.63 (m,3H), 7.58 – 7.56 (m, 2H), 7.32 (d, J = 9.0 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H),6.85 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 6.31 (s, 1H), 5.98 (d, J = 5.4 Hz, 1H),5.79 (d, J = 4.8 Hz, 1H), 4.58 (t, J = 5.4 Hz, 1H), 4.50 – 4.46 (m, 3H), 4.41– 4.38 (m, 1H), 4.33 (dd, J = 6.0 Hz, 2.4 Hz, 1H), 4.29 – 4.26 (m, 2H), 4.23– 4.20 (m, 1H), 4.13 – 4.10 (m, 1H). 31P NMR (162 MHz, D2O) δ -11.2 (d).MALDI-TOF-MS: m / z C 30 H 31 N5O 16 P2, calculated [M-H] - = 779.12, found 779.12。

[0061]

[0062] 1 H NMR (600 MHz, D2O) δ 8.19 (s, 1H), 8.00 (s, 1H), 7.63 – 7.56 (m,3H), 7.51 – 7.46 (m, 2H), 7.32 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H),6.84 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 6.25 (s, 1H), 5.59 (d, J = 4.8 Hz, 1H),5.68 (s, 1H), 4.54 – 4.49 (m, 2H), 4.44 – 4.39 (m, 2H), 4.36 – 4.32 (m, 1H),4.28 – 4.24 (m, 2H), 4.18 – 4.10 (m, 3H). 31 P NMR (162 MHz, D2O) δ -11.2 (d).MALDI-TOF-MS: m / z C 30 H 31 N5O 16 P2, calculated [M-H] - = 779.12, found 779.12。

[0063]

[0064] 1H NMR (600 MHz, D2O) δ 8.27 (s, 1H), 7.95 (s, 1H), 7.51 (d, J = 9.0Hz, 1H), 6.91 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.18(s, 1H), 5.98 (d, J = 5.4 Hz, 1H), 5.77 (d, J = 4.2 Hz, 1H), 4.57 (t, J = 5.4Hz, 1H), 4.50 – 4.46 (m, 3H), 4.43 – 4.39 (m, 1H), 4.34 – 4.31 (m, 1H), 4.30– 4.26 (m, 2H), 4.22 – 4.19 (m, 1H), 4.15 – 4.10 (m, 1H), 2.71 (t, J = 7.2Hz, 2H), 1.68 (q, J = 7.8 Hz, 2H), 1.02 (t, J = 7.8 Hz, 3H). 31 P NMR (162 MHz,D2O) δ -11.2 (d). MALDI-TOF-MS: m / z C 27 H 33 N5O 16 P2, calculated [M-H] - = 745.14,found 745.14。

[0065]

[0066] 1H NMR (600 MHz, D2O) δ 8.14 (s, 1H), 8.02 (s, 1H), 7.48 (d, J = 8.4Hz, 1H), 6.87 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.08(s, 1H), 5.91 (d, J = 5.4 Hz, 1H), 5.65 (s, 1H), 4.57 (t, J = 5.4 Hz, 1H),4.50 (t, J = 6.0 Hz, 1H), 4.46 (t, J = 4.2 Hz, 1H), 4.39 (d, J = 4.8 Hz, 1H),4.36 – 4.31 (m, 2H), 4.26 – 4.23 (m, 1H), 4.22 – 4.16 (m, 3H), 2.62 (td, J =7.2 Hz, 3.6 Hz, 2H), 1.62 (q, J = 7.2 Hz, 2H), 0.99 (t, J = 7.8 Hz, 3H). 31 PNMR (162 MHz, D2O) δ -11.3 (d). MALDI-TOF-MS: m / z C 27 H 33 N5O 16 P2, calculated [M-H] - = 745.14, found 745.14。

[0067]

[0068] 1H NMR (600 MHz, D2O) δ 8.28 (s, 1H), 7.96 (s, 1H), 7.47 (d, J = 9.0Hz, 1H), 6.90 (dd, J = 9.0 Hz, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.20 (s, 1H),5.98 (d, J = 5.4 Hz, 1H), 5.77 (d, J = 4.2 Hz, 1H), 4.59 (t, J = 5.4 Hz, 1H),4.51 – 4.46 (m, 4H), 4.42 – 4.41 (m, 1H), 4.34 – 4.32 (m, 1H), 4.31 – 4.27(m, 2H), 4.22 – 4.18 (m, 2H), 4.13 – 4.10 (m, 1H). 31 P NMR (162 MHz, D2O) δ -11.3 (s). MALDI-TOF-MS: m / z C 25 H 28 ClN5O 16 P2, calculated [M-H] - = 751.07, found751.07。

[0069]

[0070] 1 H NMR (600 MHz, D2O) δ 8.18 (s, 1H), 8.02 (s, 1H), 7.46 (d, J = 9.0Hz, 1H), 6.88 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.12(s, 1H), 5.93 (d, J = 4.8 Hz, 1H), 5.65 (s, 1H), 4.96 (t, J = 4.8 Hz, 1H),4.50 (t, J = 5.4 Hz, 1H), 4.47 (t, J = 4.8 Hz, 1H), 4.40 (d, J = 4.8 Hz, 1H),4.35 – 4.33 (m, 2H), 4.29 – 4.24 (m, 2H), 4.23 – 4.21 (m, 2H), 4.20 – 4.16(m, 2H). 31P NMR (162 MHz, D2O) δ -11.4 (s). MALDI-TOF-MS: m / z C 25 H 28 ClN5O 16 P2, calculated [MH] - = 751.07, found 751.06.

[0071] The target products obtained by replacing different alkynyl-containing benzyl ether compounds according to the preparation method of Example 4 are as follows:

[0072]

[0073] Example 5

[0074] The hydrolysis rate of ARH3 was determined using α-ADP-ribosyl-9-hydroxy-3-isophenoxazinone.

[0075] ARH3 protein concentration determination and preparation: The concentration of ARH3 protein solution was determined using the BCA method. The concentrated ARH3 protein solution was diluted to 22 nM using PBS buffer solution and set aside. Preparation of different concentrations of α-ADPR-9-hydroxy-3-isophenoxazinone: 0.5 μM, 1 μM, 5 μM, 10 μM, and 20 μM aqueous solutions of α-ADPR-9-hydroxy-3-isophenoxazinone were accurately prepared using deionized water, with a volume of 100 μL. The prepared samples were then freeze-dried to remove the solvent and set aside.

[0076] Take 20 μL of 22 nM ARH3 protein solution and dilute it with 80 μL of PBS buffer. Add the diluted protein solution (total 100 μL) to test tubes containing different concentrations of α-ADPR-9-hydroxy-3-isophenoxazinone dry powder. After the dry powder is fully dissolved, quickly transfer it to a cuvette. Place the cuvette in a fluorescence spectrophotometer to monitor the change in fluorescence intensity in real time. The hydrolysis process and Km / Vmax results are shown in Figures 1 and 2. Relevant monitoring parameters: Total monitoring volume: 100 μL; ARH3 concentration in the monitoring system: 4.4 nM; α-ADPR-9-hydroxy-3-isophenoxazinone concentration: 0 μM, 10 μM, 30 μM, 60 μM, 120 μM, 250 μM, and 300 μM; Monitoring mode: Time Scan, 3 scans / second; Total monitoring time: 1000 s; The test system was irradiated with a light source with an excitation wavelength of 550 nm, and the change in fluorescence signal intensity at a wavelength of 588 nm in the monitoring system over time was measured. The relationship between fluorescence change and time was detected in the hydrolysis experiment of ADH3 on ADPr-9-hydroxy-3-isophenoxazinone, and the results were calculated using the Michaelis-Menten equation, yielding Vmax = 1.7 μmol / min / mg and Km = 6.7 μM.

[0077] Example 6

[0078] Determination of the inhibitory effect of ADP-ribosylation inhibitors (type II triazole derivatives)

[0079] ARH3 protein concentration determination and preparation: The concentration of ARH3 protein solution was determined using the BCA method. The concentrated ARH3 protein solution was diluted to 20 nM using PBS buffer solution and set aside. Preparation of different concentrations of α-ADPr-9-hydroxy-3-isophenoxazinone: A 10 μM aqueous solution of α-ADPr-9-hydroxy-3-isophenoxazinone was accurately prepared using deionized water, with a volume of 100 μL. The prepared sample was then freeze-dried to remove the solvent and set aside. Preparation of ADP-ribosylation inhibitors (candidates): The required amount of compound was calculated based on the different molecular weights of compounds from 3bn to 3da. A 10 μM aqueous solution was accurately prepared, with a volume of 100 μL. The prepared sample was then freeze-dried to remove the solvent and set aside.

[0080] Take 20 μL of a 20 nM ARH3 protein solution and dilute it with 75 μL of PBS buffer. Add the diluted protein solution (total 90 μL) to the test tube containing α-ADPr-9-hydroxy-3-isophenoxazinone (a type I ADPr fluorescent probe) and ADP-ribosylation inhibitors (type II triazole derivatives) (3bn~3da). Add 5 μL of DMSO to aid dissolution. After the dry powder is fully dissolved, quickly transfer it to a cuvette and place the cuvette in a fluorescence spectrophotometer to monitor the fluorescence intensity changes in real time. In vitro enzyme activity assays showed that various type II triazole derivatives (such as compounds 3bv and 3bx) have significant inhibitory activity against ARH3 and can be used as ARH3 inhibitors to study life processes related to ADP-ribosylation. They also have potential applications in ARH3-mediated disease intervention, as shown in Figures 3-6. Relevant monitoring parameters: Total monitoring volume: 100 μL; ARH3 concentration in the monitoring system: 4 nM; α-ADPr-9-hydroxy-3-isophenoxazinone concentration: 10 μM; The test system was irradiated with a light source with an excitation wavelength of 550 nm; the change in fluorescence signal intensity at a wavelength of 588 nm in the monitoring system over time was recorded. Monitoring mode: Time Scan, 3 scans / second; Total monitoring time: 400 s.

[0081] K-type inhibitory effect of different ADP-ribosyl inhibitors (candidates) on ARH3 hydrolysis inhibition obs value

[0082] Entry K obs Entry K obs Blank2.94α-ADPr-N30.483bn0.533ch3.023bo0.783ci3.093bp0.223cj3.133bq0.833ck3 .213br1.013cl3.213bs0.493cm3.023bt1.943cn3.243bu0.763co3.043bv0.173cp2.973bw 0.743cq2.973bx0.193cr3.013by0.243cs2.983bz0.773ct2.983ca1.213cu3.213cb1.773 cv3.013cc0.893cw3.023cd2.413cx3.223ce0.623cy2.953cf0.863cz2.963cg2.433da3.22 surface

[0083] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A class of ADP-ribosylated derivatives, characterized in that... Including type I derivatives and type II derivatives, the corresponding structural formulas are as follows: The glycosidic bond between the N / O atoms and the ribose is either α-configuration or β-configuration; 。 2. The ADP-ribosylated derivative according to claim 1, characterized in that... The type I derivatives include compounds with the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The class of ADP-ribosylated derivatives according to claim 1, characterized in that... The type II derivatives include compounds with the following structures: 。 4. A method for preparing a class of ADP-ribosylated derivatives as described in claim 1, characterized in that... The specific preparation process is as follows: using β-nicotinamide adenine dinucleotide (β-NAD) + Using this as a substrate, a type I derivative is prepared by coupling reaction with a 7-hydroxycoumarin compound or a 9-hydroxy-3-isophenoxazolone. The corresponding synthetic route is as follows: 。 5. The method for preparing a class of ADP-ribosylated derivatives according to claim 4, characterized in that... The specific preparation steps of the type I derivative are as follows: under anhydrous and oxygen-free conditions, β-nicotinamide adenine dinucleotide β-NAD... + The target product, type I derivative, is prepared by adding a fluorescent group containing a hydroxyl group, namely 7-hydroxycoumarin or 9-hydroxy-3-isophenoxazolone, to the ionic liquid [TMG][BF4] and then stirring the reaction system at 70-80℃.

6. A method for preparing the ADP-ribosylated derivative according to claim 1, characterized in that... The specific preparation process is as follows: First, β-nicotinamide adenine dinucleotide β-NAD... + α- / β-ADPr-N3 was synthesized by reacting with sodium azide, and then the α- / β-ADPr-N3 was coupled with a benzyl ether compound containing an alkyne group via a click reaction to obtain the type II derivative. The synthetic route corresponding to this process is as follows: 。 7. The method for preparing a class of ADP-ribosylated derivatives according to claim 6, characterized in that... The specific preparation steps for the type II derivative are as follows: β-nicotinamide adenine dinucleotide β-NAD... + Sodium azide was added to saturated brine and stirred at 80-100°C. The reaction product was then purified by high performance liquid chromatography to obtain α- / β-ADPr-N3. The α-ADPr-N3 or β-ADPr-N3 with its single configuration was added to water along with a benzyl ether compound containing an alkyne group, CuSO4·5H2O, sodium ascorbate, and tris(3-hydroxypropyltriazolylmethyl)amine. The reaction system was then subjected to a click reaction at 20-60°C to obtain the target product type II derivative.

8. The application of the ADP-ribosylated derivative according to claim 1 or 2 as an ARH3 fluorescent probe, wherein the ADP-ribosylated derivative is an α-configuration ADP-ribosylated derivative of type I derivative.

9. The application according to claim 8, characterized in that: The α-configuration ADP-ribosylated derivative of the type I derivative is used as a hydrolysis fluorescent substrate for ARH3 to detect the hydrolysis rate of ARH3, thereby realizing the detection of ARH3 hydrolysis kinetics.

10. The use of the ADP-ribosylated derivative of claim 1 or 3 as an ARH3 inhibitor, wherein the ADP-ribosylated derivative is an α-configuration ADP-ribosylated derivative of type II derivatives.

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