Alpha / beta-selectivity adjustable ADPr-phenolic compound synthesis method, compound library and application thereof

Abstract

The invention discloses a synthesis method of an alpha / beta-selectivity adjustable ADPr-phenolic compound, a compound library and application of the compound library, in the presence of an ionic liquid, beta-nicotinamide adenine dinucleotide and a phenolic compound are subjected to a one-step nucleophilic substitution reaction to generate the ADPr-phenolic compound, by accurately regulating and controlling the ionic liquid system, the reaction substrate proportion, the reaction temperature or / and the reaction time, the ADPr-phenolic compound can be efficiently synthesized, the efficient control on the alpha / beta-stereoselectivity of the product is realized, and the high beta-selectivity gt is obtained; according to the present invention, the product with the alpha-selectivity of 99% or 88% can be synthesized, and the synthesized ADPr-phenolic compound can be used for preparing the ADP-ribose hydrolase ARH3 inhibitor, can be used for regulating NAD metabolism, researching protein ADP-ribosylation modification, can be used as the metabolic tracer agent, and can be used as the inhibitor or the tool molecule for other targets.

Description

Technical Field

[0001] This invention belongs to the field of synthesis and application technology of ADPr-phenolic compounds, specifically relating to a method for synthesizing ADPr-phenolic compounds with tunable α / β-selectivity, a compound library, and their applications. Background Technology

[0002] ADPr-phenolic compounds are key structural units in the ADP-ribosylation modification of protein tyrosine, and are also an important class of NAD compounds. + Metabolic tracers and candidate drugs with broad-spectrum antiviral potential. However, their chemical synthesis has long faced significant challenges. Traditional multi-step synthetic methods (Drown, BS, Shirai, T., Rack, JGM, Ahel, I. & Hergenrother, PJ Monitoring Poly(AD Pribosyl) glycohydrolase Activity with a Continuous Fluorescent Substrate. Cell Chem. Biol. 25, 1562-1570 (2018)) typically require 8-11 steps, involving lengthy protection and deprotection steps, harsh reaction conditions, and inefficient phosphorylation / pyrophosphorylation coupling, resulting in relatively low overall yields. Particularly noteworthy is the difficulty in obtaining the α-isomer, which possesses important biological functions, efficiently and selectively using conventional chemical methods.

[0003] In existing technologies, although there are reports of attempts to use NAD... + While thermal dissolution can directly introduce phenolic oxygen groups, it is generally plagued by low yields and poor selectivity (usually yielding only the β-isomer), making it impossible to achieve the controlled synthesis of the α-isomer. Therefore, developing a method with simple steps, mild conditions, precise on-demand control of α / β-stereoselectivity, and applicability to the synthesis of various phenolic substrates is of vital importance for advancing the functional study, tool development, and drug discovery of these molecules. Summary of the Invention

[0004] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing ADPr-phenolic compounds that is simple to operate, has mild conditions, and has a wide range of applicable substrates with tunable α / β-selectivity.

[0005] The second objective of this invention is to provide the application of the ADPr-phenolic compounds synthesized by the above method in the preparation of ADP-ribose hydrolase ARH3 inhibitors.

[0006] A third objective of this invention is to provide ADPr-phenolic compounds synthesized by the above method for use in the preparation of drugs for regulating NAD+. +It has wide applications in metabolism, studying protein ADP-ribosylation modification, as a metabolic tracer, and as an inhibitor or tool molecule targeting other targets (such as PARP, viral meta-domains, etc.).

[0007] The fourth objective of this invention is to provide specific ADPr-phenolic compounds with well-defined structures and biological activities synthesized by the above method.

[0008] To achieve the above objectives, this invention employs the following technical solution: a method for synthesizing α / β-selectivity tunable ADPr-phenolic compounds, the specific synthesis process of which is as follows: in the presence of an ionic liquid, β-nicotinamide adenine dinucleotide (β-NAD...) is synthesized... + ADPr undergoes a one-step nucleophilic substitution reaction with phenolic compounds to generate ADPr-phenolic compounds;

[0009] The phenolic compound is phenol, substituted phenol, naphthol, coumarin, ferulic acid ester or Boc-tyrosine methyl ester, wherein the substituent on the substituted phenol is selected from alkyl, alkoxy, halogen or nitro groups;

[0010] The ionic liquid is selected from one of the following systems:

[0011] Composed of organic base cations and halogen anions, BF4 - PF6 - CF3COO - NTf2 - or CF3SO3 - Composed of ionic liquids;

[0012] An ionic liquid composed of organic base cations and phenoxy anions;

[0013] An ionic liquid system composed of organic base cations, phenoxy anions, and excess free phenol;

[0014] The organic base cation is selected from 1,1,3,3-tetramethylguanidine (TMG), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), N,N-dimethylhexadecylamine (DMA16), N,N-dimethylethylamine (DMEA), N-methylpiperidine, N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), triethanolamine (TEOA), N-methylimidazolium (MIM), or L-arginine (L(+)-Arginine).

[0015] By precisely controlling the ionic liquid system, the ratio of reactants to substrates, the reaction temperature and / or the reaction time, ADPr-phenolic compounds can be synthesized efficiently, and the α / β stereoselectivity of the products can be effectively controlled, yielding products with high β-selectivity >99% or high α-selectivity up to 88%.

[0016] Furthermore, the reaction temperature of the synthesis process is 70–80 °C.

[0017] Furthermore, the substituent on the substituted phenol is C. 1-6 Alkyl or C 1-6 Alkyl group.

[0018] Furthermore, the ionic liquid systems are [DABCO][Cl], [DBU][Cl], [DMA16][Cl], [TMG][Cl], [DABCO][Br], [DBU][Br], [DMA16][Br], [TMG][Br], [DMEA][Br], [DABCO][I], [DBU][I], [TMG][I], [DMA16][I], [N-Methylpiperidine][I], [DABCO][BF4], [DBU][BF4], [DMA16][BF4], [TMG][BF4], [N-Methylpiperidine][BF4], [DMEA][BF4], [DI... [PEA][BF4], [TEA][BF4], [TEOA][BF4], [MIM][BF4], [L(+)-Arginine][BF4], [DABCO][PF6], [DABCO][CF3COO], [DBU][CF3COO], [DABCO][NTf2], [TMG][NTf2], [DBU][NTf2], [N-Methylpiperidine][NTf2], [L(+)-Arginine][NTf2], [N-Methylpiperidine][CF3SO3], or [DABCO][CF3SO3] or one or more of these; or the ionic liquid system is [TMG][ArO] - [DBU][ArO] - ]、[DABCO][ArO - [TMG][p-NO2-PhO-], [DBU][p-NO2-PhO-], or [DABCO][p-NO2-PhO-]; or the ionic liquid system is [TMG][ArO] - ]-ArOH、[DBU][ArO - ]-ArOH or [DABCO][ArO - ]-ArOH.

[0019] Furthermore, the synthetic route for the synthesis process is as follows:

[0020]

[0021] The application of the ADPr-phenolic compounds described in this invention in the preparation of ADP-ribose hydrolase inhibitors.

[0022] Furthermore, the ADP-ribose hydrolase is ADP-ribonuclease 3 (ARH3).

[0023] A pharmaceutical composition comprising a therapeutically effective amount of an ADPr-phenolic compound and a pharmaceutically acceptable salt or carrier.

[0024] The ADPr-phenolic compounds described in this invention are used in the preparation of compounds for regulating NAD. + Applications in products for metabolism, detection of ADP-ribosylase activity, or as metabolic tracers.

[0025] The application of the ADPr-phenolic compounds described in this invention in the preparation of pharmaceutical or tool molecules for inhibiting the activity of poly(ADP-ribose) polymerase (PARP) or viral metadomains.

[0026] The ADPr-phenolic compounds described in this invention specifically include: α-ADPr-p-Cl-phenol, α-ADPr-m-MeO-phenol, α-ADPr-p-Me-phenol, α-ADPr-pI-phenol, α-ADPr-pNP, α-ADPr-coumarin, α-ADPr-tyrosine methyl ester (Boc protected), α-ADPr-phenol, α-ADPr-o-MeO-phenol, α-ADPr-p-MeO-phenol, α-ADPr-p-Br-phenol, α-ADPr-1-naphthol, α-ADPr-2-naphthol, and α-ADPr-ferulic acid. Methyl ester, β-ADPr-p-Cl-phenol, β-ADPr-m-MeO-phenol, β-ADPr-p-Me-phenol, β-ADPr-pI-phenol, β-ADPr-pNP, β-ADPr-coumarin, β-ADPr-tyrosine methyl ester (Boc protected), β-ADPr-phenol, β-ADPr-o-MeO-phenol, β-ADPr-p-MeO-phenol, β-ADPr-p-Br-phenol, β-ADPr-1-naphthol, β-ADPr-2-naphthol, β-ADPr-ferulic acid methyl ester; the structural formulas of ADPr-phenolic compounds are:

[0027]

[0028]

[0029]

[0030] The present invention has the following advantages and beneficial effects:

[0031] (1) Disruptive innovation in synthesis method: For the first time, the synthesis of natural β-NAD was achieved. + Starting with phenolic compounds, ADPr-phenolic compounds can be synthesized in one step with high efficiency and selectivity, shortening the traditional 8-11 step synthetic route to 1 step and significantly improving the overall yield.

[0032] (2) Precise and controllable stereoselectivity: By simply controlling the ionic liquid system, the on-demand preparation of α / β-selectivity of ADPr-phenolic compounds was achieved for the first time under non-enzymatic conditions, overcoming the difficult problem of chemical synthesis of α-isomers.

[0033] (3) Mild and practical reaction conditions: The reaction is not sensitive to moisture and air, does not require strict anhydrous and oxygen-free conditions, and is extremely easy to operate, making it suitable for applications at all levels from conventional laboratories to large-scale production.

[0034] (4) The substrate is extremely versatile: it is applicable to a variety of phenolic substrates with diverse structures, including complex and biologically relevant molecules (such as tyrosine derivatives, coumarin and ferulic acid esters, etc.), and the synthesis of acid-unstable ADPr-Tyr was successfully achieved under acid-free conditions.

[0035] (5) Clear activity and adequate protection: This invention synthesized a series of compounds with clear structures and verified their inhibitory activity against ARH3 through biological experiments. Among them, α-ACP was confirmed as a novel and potent ARH3 inhibitor. Attached Figure Description

[0036] Figure 1 The α-ADPr-pNP synthesized in Example 1 1 H NMR spectrum.

[0037] Figure 2 The α-ADPr-pNP synthesized in Example 1 31 P NMR spectrum.

[0038] Figure 3 The inhibition curves and IC50 values ​​of α-ACP and the positive control α-ADPr-N3 on ARH3 enzyme activity in Example 6 are shown. 50 Comparison chart.

[0039] Figure 4 The equilibrium conformation diagram for the docking simulation of α-ACP and ARH3 molecules.

[0040] Figure 5 This is a comparison of the inhibitory activities of various synthesized α-type ADPr-phenolic compounds on ARH3. Detailed Implementation

[0041] 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.

[0042] Example 1

[0043] Synthesis of α-ADPr-pNP:

[0044] Take 7.5 μmol β-NAD + 375.0 μmol of [TMG][p-NO2-PhO-] ionic liquid was mixed with 1.5 mmol of p-nitrophenol (pNP) and reacted at 75 °C for 6 hours. After the reaction, the mixture was extracted with dichloromethane and water, and the pH of the aqueous phase was adjusted to 6.0. The product was purified by semi-preparative HPLC (C18 column, CH3CN / 0.1M TEAB = 40 / 60), and lyophilized to obtain a white powder product α-ADPr-pNP with a yield of 80% and an α / β selectivity of 83:17. The structure of the product was determined by... 1 H NMR, 31 Confirmed by P NMR and MALDI-TOF-MS.

[0045] Example 2

[0046] Synthesis of α-ADPr-coumarin:

[0047] Take 7.5 μmol β-NAD + 375.0 μmol of [TMG][BF4] ionic liquid, 375.0 μmol of TMG, and 375.0 μmol of 7-hydroxycoumarin were mixed and reacted at 75 °C for 2 hours. The post-treatment and purification methods were the same as in Example 1, yielding α-ADPr-coumarin with a yield of 83% and an α / β selectivity of 71:29.

[0048] Example 3

[0049] Synthesis of α-ADPr-tyrosine methyl ester (Boc protected):

[0050] Take 7.5 μmol β-NAD + 375.0 μmol of [TMG][BF4] ionic liquid, 375.0 μmol of TMG, and 375.0 μmol of Boc-tyrosine methyl ester were mixed and reacted at 75 °C for 2 hours. Post-treatment and purification were performed as in Example 1, yielding the target product in 62% yield with an α / β selectivity of 88:12.

[0051] Example 4

[0052] β-selective synthesis of various ADPr-phenolic compounds:

[0053] Using the [TMG][Br] or [TMG][ArO-] ionic liquid system, reactions with different phenolic substrates (such as phenol, p-methoxyphenol, p-chlorophenol, etc.) can yield the corresponding β-ADPr-phenolic compounds with moderate to excellent yields (66%-96%) and high β-selectivity (β:α up to >99:1), as shown in Table 1.

[0054] Table 1 Synthetic products of different phenolic substrates using the [TMG][Br] or [TMG][ArO-] ionic liquid systems

[0055]

[0056]

[0057] Table 2. Optimization of ionic liquid systems for the synthesis of different products using phenol as a reaction substrate.

[0058]

[0059] Example 5

[0060] Synthesis of α-ACP:

[0061] Referring to a similar [TMG][ArO-]-ArOHα-selective system as in Example 1, α-ADPr-p-Cl-phenol (α-ACP) was synthesized using p-chlorophenol as a substrate. The product was confirmed by NMR and mass spectrometry.

[0062] Example 6

[0063] Assay of the inhibitory activity of α-ACP against ARH3 enzyme:

[0064] ARH3 enzyme solution was added to an assay system containing PBS buffer (pH 7.6), α-ADPr-pNP (200 μM), MgCl2 (10 mM), and different concentrations of α-ACP (0–900 μM gradient), and incubated at 37°C for 30 minutes. The absorbance of p-nitrophenol (pNP) at 400 nm was measured using Nanodrop One. The IC50 was calculated using GraphPad Prism10. 50 Value. The results show that α-ACP affects the IC50 of ARH3. 50 The concentration was 6.87 μM, significantly better than the positive control α-ADPr-N3 (IC50). 50 =22.0μM).

[0065] Example 7

[0066] Verification of the inhibitory activity of ADPr-phenolic compounds against ARH3:

[0067] A series of ADPr-phenolic compounds were synthesized using the method described in this invention, and their inhibitory effects were evaluated according to the ARH3 activity assay method described in Example 6. Key activity data are summarized in Table 3.

[0068] Table 3 shows the activity of a series of ADPr-phenolic compounds.

[0069]

[0070] *Note: A lower relative hydrolysis yield indicates a stronger inhibitory effect of the compound on ARH3. Other compounds not listed in the table but synthesized and characterized by the methods of this invention (such as α / β-ADPr-phenol, α / β-ADPr-o-MeO-phenol, α / β-ADPr-p-MeO-phenol, α / β-ADPr-p-Br-phenol, α / β-ADPr-1-Naphthol, α / β-ADPr-2-Naphthol, α / β-ADPr-Ferulic acid methyl ester, etc.) also constitute a part of this invention.

[0071] Application Example 1

[0072] Application of β-ADPr-pNP as a PARP enzyme substrate:

[0073] The β-ADPr-pNP synthesized by the method in Example 4 of this invention can be used as a chromogenic substrate for poly(ADP-ribosyl) polymerases (such as PARP1) to detect enzyme activity or screen inhibitors. (Drown, BS, Shirai, T., Rack, JGM, Ahel, I. & Hergenrother, PJ Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate. Cell Chem. Biol. 25, 1562-1570 (2018)).

[0074] Application Example 2

[0075] ADPr-phenolic compound library for drug discovery:

[0076] A series of ADPr-phenolic compounds synthesized using the method of this invention (including the α and β configuration products synthesized in Examples 1-5) will be used to form a compound library. This library can be used for high-throughput screening or structure-based drug design targeting specific biological targets (such as SARS-CoV-2 viral metadomains, other ADP-ribonucleases, etc.) to discover new inhibitors or tool molecules. (Anmangandla, A. et al. A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain. ACS Chem. Biol. 18, 1200-1207 (2023). and Peng, K. et al. GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and FluorescencePolarization Tracers for SARS-CoV-2and OtherViral Macrodomains.ACSChem.Biol.19,1093-1105(2024)).

[0077] Comparative Example 1

[0078] Traditional multi-step synthesis method:

[0079] Referring to the background literature (Drown, BS, Shirai, T., Rack, JGM, Ahel, I. & Hergenrother, PJ Monitoring Poly(ADP ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate. Cell Chem. Biol. 25, 1562-1570 (2018)), the synthesis of α-ADPr-pNP requires 11 steps, with an overall yield of only 19%, and the process is cumbersome and time-consuming. Compared with Comparative Example 1, the method of Example 1 of this invention achieves a breakthrough in terms of step, yield, and selectivity control.

[0080] 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 method for synthesizing ADPr-phenolic compounds with tunable α / β selectivity, characterized in that... The specific synthesis process is as follows: in the presence of an ionic liquid, β-nicotinamide adenine dinucleotide undergoes a one-step nucleophilic substitution reaction with a phenolic compound to generate ADPr-phenolic compounds; The phenolic compound is phenol, substituted phenol, naphthol, coumarin, ferulic acid ester or Boc-tyrosine methyl ester, wherein the substituent on the substituted phenol is selected from alkyl, alkoxy, halogen or nitro groups; The ionic liquid is selected from one of the following systems: Composed of organic base cations and halogen anions, BF4 - PF6 - CF3COO - NTf2 - or CF3SO3 - Composed of ionic liquids; An ionic liquid composed of organic base cations and phenoxy anions; An ionic liquid system composed of organic base cations, phenoxy anions, and excess free phenol; By precisely controlling the ionic liquid system, the ratio of reactants to substrates, the reaction temperature and / or the reaction time, ADPr-phenolic compounds can be synthesized efficiently, and the α / β stereoselectivity of the products can be effectively controlled, yielding products with high β-selectivity >99% or high α-selectivity up to 88%.

2. The method for synthesizing α / β-selectively tunable ADPr-phenolic compounds according to claim 1, characterized in that: The reaction temperature during the synthesis process is 70–80℃.

3. The method for synthesizing α / β-selectively tunable ADPr-phenolic compounds according to claim 1, characterized in that: The substituent on the substituted phenol is C 1-6 Alkyl or C 1-6 Alkyl group.

4. The method for synthesizing α / β-selectivity tunable ADPr-phenolic compounds according to claim 1, characterized in that: The ionic liquid systems are [DABCO][Cl], [DBU][Cl], [DMA16][Cl], [TMG][Cl], [DABCO][Br], [DBU][Br], [DMA16][Br], [TMG][Br], [DMEA][Br], [DABCO][I], [DBU][I], [TMG][I], [DMA16][I], [N-Methylpiperidine][I], [DABCO][BF4], [DBU][BF4], [DMA16][BF4], [TMG][BF4], [N-Methylpiperidine][BF4], [DMEA][BF4], [DIPEA] [BF4], [TEA], [TEOA], [MIM], [L(+)-Arginine], [DABCO], [PF6], [DABCO], [CF3COO], [DBU], [CF3COO], [DABCO], [NTf2], [TMG], [DBU], [NTf2], [N-Methylpiperidine], [L(+)-Arginine], [CF3SO3], or [DABCO], [CF3SO3]; or the ionic liquid system is [TMG], [ArO], [PF6], [TEA], [TEOA], [MIM], [L(+)-Arginine], [CF3SO3], [NTf2], [TMG], [NTf2], [N-Methylpiperidine], [NTf2], [L(+)-Arginine], [NTf2], [N-Methylpiperidine], [CF3SO3], or [DABCO], [CF3SO3], or one or more of these; or the ionic liquid system is [TMG], [ArO], [PF6], [TEOA], [MIM], [L(+)-Arginine], [PF6], [DABCO], [CF3COO], [DBU], [NTf2], [NTf2], [N-Methylpiperidine], [CF3SO3], or [DABCO], [CF3SO3]. - [DBU][ArO] - ]、[DABCO][ArO - [TMG][p-NO2-PhO-], [DBU][p-NO2-PhO-], or [DABCO][p-NO2-PhO-]; or the ionic liquid system is [TMG][ArO] - ]-ArOH、[DBU][ArO - ]-ArOH or [DABCO][ArO - ]-ArOH.

5. The use of the ADPr-phenolic compound prepared by the method according to any one of claims 1 to 4 in the preparation of ADP-ribose hydrolase inhibitors.

6. The application according to claim 5, characterized in that: The ADP-ribose hydrolase is ADP-ribonuclease 3.

7. A pharmaceutical composition, characterized in that... The ADPr-phenolic compound prepared by the method of any one of claims 1 to 4, comprising a therapeutically effective amount, and a pharmaceutically acceptable salt or carrier.

8. The ADPr-phenolic compound prepared by the method according to any one of claims 1 to 4, in the preparation of a compound for regulating NAD... + Applications in products for metabolism, detection of ADP-ribosylase activity, or as metabolic tracers.

9. The use of the ADPr-phenolic compound prepared by the method according to any one of claims 1 to 4 in the preparation of a drug or tool molecule for inhibiting the activity of poly(ADP-ribose) polymerase (PARP) or viral metadomains.

10. An ADPr-phenolic compound library, characterized in that... The ADPr-phenolic compounds specifically include: α-ADPr-p-Cl-phenol, α-ADPr-m-MeO-phenol, α-ADPr-p-Me-phenol, α-ADPr-pI-phenol, α-ADPr-pNP, α-ADPr-coumarin, α-ADPr-tyrosine methyl ester (Boc protected), α-ADPr-phenol, α-ADPr-o-MeO-phenol, α-ADPr-p-MeO-phenol, α-ADPr-p-Br-phenol, α-ADPr-1-naphthol, α-ADPr-2-naphthol, and α-ADPr-ferulic acid methyl ester. β-ADPr-p-Cl-phenol, β-ADPr-m-MeO-phenol, β-ADPr-p-Me-phenol, β-ADPr-pI-phenol, β-ADPr-pNP, β-ADPr-coumarin, β-ADPr-tyrosine methyl ester (Boc protected), β-ADPr-phenol, β-ADPr-o-MeO-phenol, β-ADPr-p-MeO-phenol, β-ADPr-p-Br-phenol, β-ADPr-1-naphthol, β-ADPr-2-naphthol, β-ADPr-methyl ferulic acid; the structural formulas of ADPr-phenolic compounds are:

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