Screening method of adenosine LCMT1 inhibitor and preparation method and application of small-molecule inhibitor

Abstract

The invention relates to the technical field of biological medicine, in particular to a screening method of an adenosine LCMT1 inhibitor and a preparation method and application of a small-molecule inhibitor. The derivative comprises an optical isomer, a racemate, a single enantiomer, a possible diastereoisomer of the adenosine LCMT1 inhibitor, or a pharmaceutically acceptable salt, a prodrug, a deuterated derivative, a hydrate and a solvate of the adenosine LCMT1 inhibitor. The LCMT1 inhibitor is the first selective inhibitor aiming at the LCMT1, and plays a very important role in treating various diseases such as liver cancer, diabetes mellitus and glucose metabolism disorder.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a selective LCMT1 inhibitor, its preparation method and application; a fluorescence polarization screening method and system for screening LCMT1 inhibitors, and the synthesis and application of corresponding fluorescent probes. Background Technology

[0002] Among the two families of serine / threonine phosphatases, the protein phosphatase 2A family (PP2A) is known to regulate development, apoptosis, and cell division. LCMT1 (Leucine Carboxyl Methyltransferase-1) is a specific methyltransferase primarily responsible for catalyzing the carboxyl methylation of the conserved leucine residue (Leu309) at the C-terminus of the PP2A catalytic subunit. This modification is crucial for the proper assembly of the PP2A holoenzyme. Currently, there are few reports on LCMT1 inhibitors, and given their increasingly unique advantages in the treatment of various diseases, the development of LCMT1 inhibitors holds great promise.

[0003] Studies have shown that LCMT1 inhibition has advantages in treating and improving liver cancer, diabetes, and glucose metabolism disorders, and is expected to become a new target for next-generation disease treatment. However, in reality, most research on LCMT1 has remained at the level of gene knockout and mechanism, with little data on inhibitor development, and no high-throughput selective screening method for LCMT1 inhibitors. A widely used inhibitor screening method is the enzymatic assay, which uses AdoHcy hydrolase (SAHH) coupled with fluorescence or bioluminescent MTase Glo TM Assay methods directly monitor the production of AdoHcy, but both of these methods require additional coupling enzymes and reagents to generate a detectable signal; high performance liquid chromatography (HPLC) is not suitable for high-throughput screening; isothermal titration calorimetry (ITC) and surface plasmon resonance can directly determine the binding affinity between inhibitors and targets, but the instruments used are expensive and have high requirements for protein stability. Therefore, there is still a lack of an LCMT1 inhibitor and its corresponding screening method to solve the problems of complex detection reagents and expensive instruments in existing technologies. Summary of the Invention

[0004] This invention provides a screening method for adenosine-based LCMT1 inhibitors, as well as a method for preparing small molecule inhibitors and their applications. Its purpose is to fill the gap in existing LCMT1 inhibitor research by providing a screening method for LCMT1 inhibitors and their potential active compounds using the principle of fluorescence polarization. When a fluorescent probe binds to LCMT1, polarization occurs. The inhibitor competitively binds to LCMT1, causing the probe to become detached, thus reducing fluorescence polarization and allowing evaluation of the compound's inhibitory ability against LCMT1. This method can be used for high-throughput screening and can characterize the binding ability of fluorescent ligands to targets or any compounds that interfere with the above interactions. Furthermore, this invention allows for the identification and evaluation of LCMT1 inhibitors that directly or allosterically disrupt the binding to the active site. Compared with other screening methods, this invention is low-cost, convenient, rapid, stable, and reliable, offering significant advantages in high-throughput drug screening.

[0005] To achieve the above objectives, the present invention provides an adenosine LCMT1 inhibitor and its derivatives, wherein the derivatives comprise one or more of the following: an optical isomer, a racemic mixture, a single enantiomer, a possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate of the adenosine LCMT1 inhibitor, and the structure of the adenosine LCMT1 inhibitor is shown in formula (I): ; Where: A is a group having a formula selected from (A-1) or (A-2): ; B 1 and B 2 Selected from chemical bonds or ; C is selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spiral ring, the C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spirocyclic rings can be independently substituted by one or more of the following substituents: hydroxyl, amino, carboxyl, nitro, cyano, one or more halogen atoms, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Any one of oxoalkyl or ester groups; Y is selected from , where n is any natural number among 1, 2, 3, 4, and 5; L is selected from O and NQ. 1 Q 1 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Any one of the alkylamino groups; Q is selected from the middle Any one of them; Z is selected from C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spiral ring, the C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spirocyclic rings can be substituted independently by one or more of the following substituents: hydroxyl, amino, carboxyl, nitro, cyano, one or more halogen atoms, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Any one of oxoalkyl or ester groups; R1 is selected from hydrogen, deuterium, and C. 1-6 Alkyl, C3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Any one of the alkylamino groups; R2 is selected from any one of hydrogen, deuterium, trimethylolpropionyl (TML) groups, medoxomil moiety, tert-butyloxycarbonyl group, p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, fluorenemethoxycarbonyl, p-toluenesulfonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, trifluoroacetyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, benzyl, phthaloyl, nitrobenzenesulfonyl, triphenylmethyl, acetamyl, carbamate, trimethylsilylethoxymethyl, benzyl p-phenylboronic acid, benzyl p-phenylboronic acid, fluoroethyl, and chloroformates (including all chloroformates such as hexyl chloroformate, dodecyl chloroformate, and hexadecyl chloroformate); the trimethylolpropionyl (TML) groups include, but are not limited to: The medoxomil moiety includes, but is not limited to: or ; R3 is selected from C 1-6 Alkyl, benzyl, allyl, medoxomil moiety, C 1-6 Orthoester protecting group, C 1-18 Any of the following silyl ether protecting groups.

[0006] Preferably, the Y in the adenosine LCMT1 inhibitor is selected from... where n is 2; L represents NH; Q is selected from , , Any one of them; R1 is a hydrogen atom; R2 is selected from any one of hydrogen, deuterium, trimethylolpropionate (TML) groups, and medoxomil moiety; the trimethylolpropionate groups include, but are not limited to: The medoxamil moiety is... ; R3 stands for medoxomil moiety. .

[0007] Preferably, the chemical structural formula of the adenosine LCMT1 inhibitor and its derivatives includes any one of the following formulas 1-60: ; ; ; ; ; ; .

[0008] Under the same technical concept, the present invention also provides a method for preparing the aforementioned adenosine LCMT1 inhibitor and its derivatives, wherein the chemical reaction formula of the preparation method includes the following reaction pathway: Path 1: When A is selected from formula (A-1), the aldehyde a1 obtained by modification and oxidation with homoserine as the parent nucleus undergoes a reductive amination reaction with the raw material a2 to obtain intermediate a3, and then undergoes a reductive amination reaction with aldehyde or ketone a4 to obtain intermediate a5. Finally, the protecting group is removed to obtain compound I-(A-1). ; Pathway 2: When A is selected from formula (A-2), the preparation method of intermediate a3 is the same as that of Pathway 1. Intermediate a3 is reduced and aminationd with aldehyde b4 to obtain intermediate b5. After removing the L group protecting group Pg, intermediate b5 undergoes at least one of the following reactions: amide condensation, transesterification or reduced amination, to obtain the product shown in formula I-(A-2). .

[0009] Under the same technical concept, the present invention also provides a fluorescent probe for screening adenosine LCMT1 inhibitors and their derivatives, the structure of which is shown in formula (II): ; Wherein, X is selected from at least one of C, N, O, S, C(O), NHC(O), C(O)NH, S(O), and S(O)2; n is selected from at least one of 2, 3, and 4; The R1 group is a fluorescent group attached to an adenosine analogue and its derivatives, selected from at least one of the following structures or their enantiomers, diastereomers, monomeric isomers, racemates, or pharmaceutically acceptable salts thereof: 5-FAM and its derivatives, NBD and its derivatives, Atto590 and its derivatives, Atto647N and its derivatives, BODIPY and its derivatives, CY3 and its derivatives, and CY5 and its derivatives. The structural formulas of 5-FAM and its derivatives, NBD and its derivatives, Atto590 and its derivatives, Atto647N and its derivatives, BODIPY and its derivatives, CY3 and its derivatives, and CY5 and its derivatives are shown below: ; The R2 and R3 groups are independently selected from hydrogen atoms, halogen atoms, cyano groups, hydroxyl groups, amino groups, carboxyl groups, nitro groups, and C atoms, respectively. 1-4 Alkyl, haloalkyl, C 1-4 Alkoxy, halogenated C l-4 Alkoxy, hydroxy C l-4 Alkyl, carboxyl C l-4 Alkyl, C 6-10 Aryl, C 3-7 Monocyclic or polycyclic cycloalkyl, 5-10-membered heteroaryl, and 4-10-membered heterocycloalkyl groups, wherein each group is optionally substituted by 1, 2, 3, or 4 independently selected from the following substituents: hydrogen atom, halogen atom, cyano, hydroxyl, amino, carboxyl, nitro, C 1-4 Alkyl, haloalkyl, C 1-4 Alkoxy, halogenated C l-4 Alkoxy, hydroxy C l-4 alkyl group, carboxyl group C l-4 Alkyl halide, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 At least one of the haloalkyl groups.

[0010] The uses of the fluorescent probe include: (a) Used to prepare LCMT1 receptor fluorescent probes; (b) Used as a fluorescent probe for the LCMT1 receptor; (c) Used to prepare LCMT1 receptor inhibitors; (d) Used to prepare LCMT1 inhibitor-related drugs.

[0011] Under the same technical concept, the present invention also provides a method for preparing fluorescent probes of the adenosine LCMT1 inhibitor and its derivatives, wherein the chemical reaction formula of the preparation method includes the following reaction pathway: ; Where n = at least one of 2, 3, and 4; Compounds d1 and d2 undergo a reductive amination reaction to generate intermediate d3. Intermediate d3 then reacts with homoserine-derived aldehyde compound d4 via a reductive amination reaction to generate intermediate d5, selectively removing the nitrogen protecting group Pg. 1 Then it is coupled with a fluorescent reagent, and finally all protecting groups Pg are removed.2 and Pg 3 Generate a fluorescent probe as shown in Formula II; the definitions of n, X, R1, R2, and R3 in each formula are as previously shown.

[0012] Under the same technical concept, the present invention also provides a fluorescence polarization screening method for adenosine LCMT1 inhibitors and their derivatives, comprising the following steps: (1) The protein LCMT1 was prepared using the Escherichia coli prokaryotic expression method; (2) Synthesize fluorescent probes; (3) Determine the concentration of the fluorescent probe by measuring its fluorescence intensity and polarization value; (4) Incubate the protein LCMT1 and the fluorescent probe together, and determine the concentration of protein LCMT1 to be used based on the measured fluorescence polarization value; (5) Co-incubate the protein LCMT1, the fluorescent probe, and the target compound to be screened, so that the fluorescent probe and the target compound competitively bind to the LCMT1 protein, and measure the fluorescence polarization value of the system; use the measured fluorescence polarization value as the ordinate and the logarithm of the concentration of the target compound as the abscissa to fit the inhibition curve of the target compound, and calculate the K of the target compound. i The value is used to determine the extent of the inhibitory activity of the target compound to be screened.

[0013] The screening method and system described in this invention are based on the following principle for screening LCMT1 inhibitors: The fluorescent probe's chemical structure consists of two parts: a fluorescent group and an adenosine analog fragment that can bind to the LCMT1 protein. When the fluorescent probe binds to the LCMT1 protein, it generates polarized light, resulting in a higher measured fluorescence polarization value. The LCMT1 inhibitor can competitively bind to the fluorescent probe, causing the LCMT1-bound fluorescent probe to become free, thus reducing the measured fluorescence polarization value. The change in the measured fluorescence polarization value reflects the compound's binding ability to the LCMT1 protein; the stronger the binding ability to the LCMT1 protein, the stronger the competitive inhibition effect.

[0014] Preferably, the preparation of protein LCMT1 by the Escherichia coli prokaryotic expression method in step (1) specifically includes: extracting the expression plasmid from the DH5α strain, introducing the plasmid into the competent cells of the protein expression strain BL21 DE3 for expression, and separating and purifying the LCMT1 protein using a Ni-NTA affinity chromatography column; The method for synthesizing the fluorescent probe in step (2) is as described in the preparation method above; The determination of fluorescence intensity and polarization value of the fluorescent probe in step (3) specifically includes: preparing the fluorescent probe into a DMSO solution of a certain concentration and performing gradient dilution, and determining the concentration of the fluorescent probe to be used based on the measured fluorescence intensity and fluorescence polarization value, wherein the concentration to be used is between 1-1000 nM. In step (4), the co-incubation of protein LCMT1 and fluorescent probe, and the determination of the concentration of protein LCMT1 based on the measured fluorescence polarization value, specifically involves: simultaneously adding fluorescent probe and LCMT1 protein, co-incubating them, and determining the concentration of LCMT1 protein solution based on the measured fluorescence polarization value; the concentration of fluorescent probe used is the concentration of fluorescent probe used in step (3) of the fluorescence polarization screening method, which is 1-1000 nM; LCMT1 protein is serially diluted with buffer solution, and the composition of the buffer solution is 50 mM Tris HCl, pH 8, 150 mM NaCl and buffer solution additives; In step (5), when the protein LCMT1, the fluorescent probe, and the compound to be screened are co-incubated, the protein LCMT1 and the fluorescent probe are used at their respective concentrations, and the compound to be screened is serially diluted. Different gradients of the compound to be screened are then co-incubated with the protein LCMT1 and the fluorescent probe in the dark, and their fluorescence polarization values ​​are detected. The measured fluorescence polarization values ​​are plotted on the ordinate, and the logarithm of the concentration of the compound to be screened is plotted on the abscissa to fit the inhibition curve of the target compound, and the K-value of the compound to be screened is calculated. i The value is used to determine the extent of the inhibitory activity of the target compound to be screened.

[0015] More preferably, in step (1), the expression plasmid is extracted from the DH5α strain using E. coli prokaryotic expression technology and introduced into competent cells of the protein expression strain BL21 DE3. Recombinants are screened using the kanamycin resistance method. The recombinants are inoculated into 1 liter of LB liquid medium (containing 50 μg / mL kanamycin), cultured at 37°C for 4 h, and then 0.6 mM IPTG is added and incubated overnight at 25°C. The bacterial cells are disrupted by sonication, and the lysate supernatant is then purified by Ni-NTA affinity chromatography.

[0016] More preferably, in step (5), the K value of the target compound's inhibitory activity against PARP3 is calculated. i Value (K) i =IC50 / (1+[S] / K m ), K i [S] represents the binding affinity of the inhibitor, IC50 represents the functional strength of the inhibitor; [S] represents the fixed substrate concentration, and K represents the binding affinity of the inhibitor. m This represents the substrate concentration at which the enzyme-catalyzed reaction rate reaches half of its maximum reaction rate. If the substrate concentration is much less than K... m Value, IC50 equals Ki This determines the extent of the inhibitory activity of the target compound.

[0017] More preferably, in step (5), the initial concentration of the compound to be screened is 10 mM, the amount of the compound to be screened added to the black 384-well plate is 1 μL, the volume of the LCMT1 protein buffer added is 99 μL, and the time in the dark is 5-30 min.

[0018] Under the same technical concept, the present invention also provides an application of the fluorescent probe of the adenosine LCMT1 inhibitor and its derivatives, or the fluorescent probe of the adenosine LCMT1 inhibitor and its derivatives prepared by the preparation method, wherein the fluorescent probe and its derivatives used for screening adenosine LCMT1 inhibitors and their derivatives are used in the construction, screening, activity determination, development of related drugs, identification of related active compounds, and identification of related inhibitors in fluorescence polarization screening methods and screening systems for their potential active compounds.

[0019] The above-described solution of the present invention has the following beneficial effects: (1) The LCMT1 inhibitor described in this invention is the first selective LCMT1 inhibitor, which plays a very important role in the treatment of many diseases such as liver cancer, diabetes, and glucose metabolism disorders. (2) The fluorescent probe, screening method and system described in this invention can achieve high-throughput screening of LCMT1 inhibitors and potential active compounds, which is of great significance for the development of LCMT1 inhibitors and related drugs. (3) The screening method and system described in this invention have the advantages of being fast and simple, low cost, easy to operate, stable and reliable, accurate and sensitive, which provides an important foundation for the development of LCMT1 inhibitors. Attached Figure Description

[0020] Figure 1 It is the regression line of fluorescence intensity of fluorescent probe II in buffer solution as a function of probe concentration; Figure 2 This is the fluorescence polarization curve of fluorescent probe II in buffer solution as a function of probe concentration; Figure 3 This is the concentration titration curve of LCMT1 against fluorescent probe II; Figure 4 The curves show the competitive binding of fluorescent probe II to LCMT1 protein in different concentrations of positive compounds. Figure 5 This is the NMR spectrum of compound 23. Detailed Implementation

[0021] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0023] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0024] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Example 1: Prokaryotic expression, isolation, and purification of protein LCMT1 Using E. coli prokaryotic expression technology, the LCMT1 plasmid was transformed into competent cells of the protein expression strain BL21(DE3). The cells were plated on kanamycin-containing plates and incubated overnight at 37°C. Single colonies were picked and inoculated into 100 mL LB medium supplemented with kanamycin, incubated overnight, and then inoculated into 1 L LB medium supplemented with kanamycin for large-scale amplification. The culture was then cooled to 4°C, and 0.5 mM isopropyl thiogalactoside (IPTG) was added to 1 L of medium, followed by initiation at 18°C ​​overnight. The bacterial cells were disrupted by sonication, and the supernatant was purified by Ni column chromatography. The purified LCMT1 protein had an apparent molecular weight of 38 kDa. The protein was then dialyzed overnight at 4°C, digested with enzymes, concentrated, and dispensed.

[0026] Example 2: Preparation of fluorescent probe YGA0-127 ; Synthesis of intermediate S2: Compound 1 (5 g, 16.27 mmol, 1.0 equiv.) was added to a 250 mL three-necked flask and dissolved in dioxane (50 mL, 0.33 M). Nitrogen protection was maintained, and air was purged. DPPA (6.97 mL, 8.9 g, 32.54 mmol, 2.0 equiv.) and DBU (7.29 mL, 7.43 g, 48.81 mmol, 3.0 equiv.) were slowly added to the solution, and the mixture was stirred at room temperature for 4 h. Sodium azide (1.58 g, 24.4 mmol, 1.5 equiv.) and 15-crown-5 (0.322 mL, 358.36 mg, 1.63 mmol, 0.1 equiv.) were added to the reaction mixture, and the temperature was raised from room temperature to 110 °C and refluxed for 4 h. After the reaction was completed as monitored by TLC, the reaction solution was allowed to cool naturally to room temperature. 80 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (30 mL × 5). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-50 / 1) to obtain a colorless oily intermediate S2 (4.86 g, 90% yield).

[0027] ; Synthesis of intermediate S3: Intermediate S2 (4.86 g, 14.63 mmol) was added to a 250 mL inclined two-necked flask, dissolved in methanol (100 mL, 0.15 M), and Pd / C (400 mg, 0.1 equiv.) was added. The air was purged five times with hydrogen, and the reaction mixture was stirred at room temperature under hydrogen atmosphere for 6 h. After the reaction was completed by TLC monitoring, Pd / C was removed by diatomaceous earth filtration, and the crude product was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 - 5 / 1) to give a white solid compound S3 (4 g, yield 89.3%).

[0028] ; Synthesis of intermediate A4-1: Intermediate S3 (2 g, 6.5 mmol) was added to a 100 mL round-bottom flask and dissolved in methanol (30 mL, 0.22 M). Intermediate a (1.8 g, 6.5 mmol, 1.0 equiv.) and sodium cyanoborohydride (490.15 mg, 7.8 mmol, 1.2 equiv.) were then added. The mixture was stirred at room temperature for 5 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-30 / 1) to give a white solid powder product A4-1 (2.12 g, yield 57%). Synthesis of intermediate A5-1: Intermediate A4-1 (600 mg, 1.05 mmol) was added to a 50 mL round-bottom flask and dissolved in methanol (10 mL, 0.1 M). Intermediate b (431.58 mg, 1.58 mmol, 1.5 equiv.) and sodium cyanoborohydride (79.18 mg, 1.26 mmol, 1.2 equiv.) were then added. The mixture was stirred at room temperature for 5 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100 / 1-30 / 1) to give a white solid powder product A5-1 (431 mg, yield 49.6%).

[0029] ; Synthesis of fluorescent probe YGA0-127: Intermediate A5-1 (50 mg, 0.06 mmol) was added to a 25 mL round-bottom flask, followed by dichloromethane (1.0 mL, 0.06 M) and diethylamine (1.0 mL). The mixture was stirred at room temperature for 5 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane / methanol = 30 / 1-10 / 1) to obtain a white powdery solid compound c (33.4 mg, yield 92%). Compound d (20 mg, 0.05 mmol) was added to a 25 mL round-bottom flask, dissolved in DMF (1.0 mL, 0.05 M), followed by HATU (28.5 mg, 0.075 mmol, 1.5 equiv.) and DIPEA (32.3 mg, 0.25 mmol, 5.0 equiv.). After stirring at room temperature for 1 h, compound c (32 mg, 0.05 mmol, 1.0 equiv.) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed as monitored by TLC, DMF was extracted with petroleum ether (20 mL × 3), and then water (5 mL) was added to produce an orange solid, which was the crude compound A6-1. The crude compound A6-1 was separated, dried, and purified by silica gel chromatography (dichloromethane / methanol = 100 / 1-20 / 1) to obtain an orange powdery solid compound 6a (32.3 mg, yield 67%). Compound 6c (32.3 mg, 0.03 mmol) was added to a 10 mL round-bottom flask, dissolved in dichloromethane (1.0 mL, 0.03 M), followed by the addition of trifluoroacetic acid (1.0 mL). The mixture was stirred at room temperature for 0.5 h, then water (0.2 mL) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure, neutralized with saturated sodium bicarbonate, and purified by reversed C18 chromatography (water / methanol = 100 / 1-70 / 30) to obtain the yellow powdery solid target compound probe YGA0-127 (5.9 mg, yield 26%).

[0030] Example 3: Determining the concentration of fluorescent probes used in the screening system The experimental steps are as follows: Taking the fluorescent probe YGA0-127 as an example, the procedure is the same for other fluorescent probes. After freeze-drying the fluorescent probe overnight, prepare a 10 mM DMSO solution. Perform a 3-fold serial dilution of the fluorescent probe in a 96-well plate: add 10 μL of DMSO to wells 2-12 of the 96-well plate, add 5 μL of the initial concentration (10 mM) of the fluorescent probe to the first well, then add another 5 μL of the 10 mM fluorescent probe to the second well, mix thoroughly by pipetting, then add 5 μL to the third well, mix thoroughly by pipetting, and add 5 μL to the fourth well. Repeat the above operation. When reaching the eleventh well, discard 5 μL of the solution, leaving the last well with pure DMSO. This completes the 3-fold serial dilution of the fluorescent probe. Dilute the serially diluted fluorescent probe 100-fold: Take 1 μL of the liquid from each of the 12 wells of a 96-well plate and add it to the odd-numbered wells in a row of a black 384-well plate. Then add 99 μL of buffer solution (in this example, the buffer solution consists of 50 mM pH 8 Tris HCl, 150 mM NaCl, 10 mM MgCl2, and 0.001% Triton X-100), making the final volume of each well 100 μL. The final concentration of the fluorescent probe is 100 μM obtained from the three-fold dilutions. On a multi-plate reader, select Fluorescence Intensity to measure the fluorescence intensity. Select Fluorescence Polarization to measure the fluorescence polarization (plate selected: 384 flat black; emission wavelength: 485 nm; absorption wavelength: 535 nm; Fluorophore: DAPI; Z-position: Calculate from well). Plot the fluorescent probe concentration on the x-axis and the fluorescence intensity and fluorescence polarization on the y-axis, respectively. Figure 1 , 2 Based on the data in section 3, the concentration of the fluorescent probe YGA0-127 was selected to be 70 nM.

[0031] Example 4: Determining the optimal concentration of LCMT1 protein The experimental steps are as follows: Similar to the procedure in Example 3, using fluorescent probe YGA0-127 as an example, the procedure for other fluorescent probes is the same. The LCMT1 protein was serially diluted 3-fold in a 96-well plate: 100 μL of buffer solution (in this example, the buffer solution consisted of 50 mM pH 8 Tris HCl, 150 mM NaCl, 10 mM MgCl2, and 0.001% Triton X-100) was added to well 2-12 of the 96-well plate. 100 μL of the initial concentration of LCMT1 protein (50 μM) was added to well 1. Then, another 50 μL of LCMT1 protein (50 μM) was added to well 2. The mixture was then thoroughly mixed by pipetting up and down, and 50 μL was added to well 3. This process was repeated until well 4. By the eleventh well, 50 μL of solution was removed and discarded, leaving only buffer solution in the last well. This completes the three-fold serial dilution of LCMT1 protein. 1 μL of the fluorescent probe (7 μM) was added to the odd-numbered wells of a black 384-well plate. Then, 99 μL of LCMT1 protein solution was added to the corresponding 96-well plates, ensuring the final concentration of the fluorescent probe was the optimal concentration obtained in Example 3, i.e., 70 nM (1 μL of fluorescent probe solution and 99 μL of buffer solution in the last black 384-well plate). The fluorescence polarization value was measured using a multi-mode microplate reader. Based on the measured data, a nonlinear regression curve was plotted using a one-site-total model on GraphPad to obtain K. d Value, based on Figure 3 The results determined that the optimal concentration of LCMT1 protein was 1.2 μM.

[0032] Example 5: Gold Standard Experiment (Activity Verification of SAH, a Positive Compound for LCMT1 Inhibitors) The experimental steps are as follows: Taking the fluorescent probe YGA0-127 as an example, the procedure is the same for other fluorescent probes. Prepare a 7 μM DMSO solution of the fluorescent probe, a 10 mM DMSO solution of SAH, and a 1.2 μM LCMT1 protein buffer solution (in this example, the buffer solution consists of 50 mM pH 8 Tris HCl, 150 mM NaCl, 10 mM MgCl2, and 0.001% Triton X-100). Perform a three-fold serial dilution of SAH (initial concentration 10 mM): In a row of 12 wells of a 96-well plate, add 5 μL of 10 mM SAH to the first well, 10 μL of DMSO to the 2nd to 12th wells, then add another 5 μL of 10 mM SAH to the second well. Mix thoroughly by pipetting up and down 10 times. Repeat the above steps with 5 μL from the second well to the third well. When operating on the eleventh well, mix by blowing and swishing 10 times, then discard 5 μL of the liquid, ensuring that the 12th well contains pure DMSO. Take 1 μL of the liquid from each of the 12 wells of the 96-well plate and add it to the odd-numbered wells in a row of the black 384-well plate. Then add 1 μL of a 7 μM fluorescent probe (the final concentration of the fluorescent probe in the black 384-well plate is the optimal probe concentration obtained in Example 3, i.e., 70 nM). Next, add 98 μL of 1.2 μM LCMT1 protein buffer solution to each odd-numbered well in the row to dilute the SAH concentration 100-fold. After protecting from light for 5-30 minutes, measure the fluorescence polarization value and calculate the K value based on the obtained fluorescence polarization value. i Value. According to Figure 4 , obtain SAH's K i The value is nM, which is very close to the 70 nM reported in the literature. Therefore, it can be proven that the screening method constructed in this invention is very accurate and reliable in practical applications.

[0033] Example 6: Preparation of Compound 6 ; Step 1: Compound S3 (2 g, 6.5 mmol, 1.0 equiv.) was dissolved in MeOH (20 mL, 0.32 M), compound b (1.78 g, 6.5 mmol, 1.0 equiv.) was added, and NaBH3CN (491.5 mg, 7.8 mmol, 1.2 equiv.) was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-30 / 1) to obtain a white solid powder product S4-1 (2.6 g, yield 72.2%).

[0034] Step 2: Compound S4-1 (100 mg, 0.17 mmol, 1.0 equiv.) was dissolved in MeOH (2.0 mL, 0.08 M), compound 6a (24.5 mg, 0.35 mmol, 2.0 equiv.) was added, and NaBH3CN (12.9 mg, 0.2 mmol, 1.2 equiv.) was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100 / 1-30 / 1) to obtain a white solid powder product S5-6 (73.4 mg, yield 72.0%).

[0035] In the second step, compound S5-6 (70.0 mg, 0.1 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The mixture was stirred at room temperature for 5 h. After the reaction was complete as monitored by TLC, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5.0 mL), concentrated under reduced pressure, and purified by C18 reverse-phase chromatography column (water / methanol = 100 / 1-70 / 30) to obtain product 6 (31.2 mg, yield 77.0%) as a white solid powder.

[0036] 6. Proton NMR spectral data of the white solid powder product: 1 H NMR (500 MHz, DMSO) δ 8.33 (d, J = 5.9 Hz, 1H), 8.15 (d, J = 5.8Hz, 1H), 7.27 (s, 2H), 5.85 (t, J = 4.9 Hz, 1H), 4.65 (dt, J = 17.6, 5.3 Hz,1H), 4.12 (dt, J = 18.6, 4.8 Hz, 1H), 4.04 – 3.93 (m, 1H), 3.59 (s, 1H), 3.42(d, J = 2.4 Hz, 1H), 3.08 (dt, J = 15.1, 2.3 Hz, 1H), 2.77 (dt, J = 12.2, 5.8Hz, 1H), 2.67 (dd, J = 12.7, 6.8 Hz, 1H), 2.64 – 2.61 (m, 1H), 2.60 – 2.53(m, 1H), 1.97 – 1.88 (m, 1H), 1.71 (ddq, J = 21.3, 14.3, 6.1 Hz, 1H). Example 7: Preparation of Compound 19 ; Step 1: Compound S4-1 (2.0 g, 3.5 mmol, 1.0 equiv.) was dissolved in MeOH (20 mL, 0.17 M), compound a (1.3 g, 4.3 mmol, 1.2 equiv.) was added, and NaBH3CN (270.9 mg, 4.3 mmol, 1.2 equiv.) was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-30 / 1) to obtain a white solid powder product A5-1 (2.2 g, yield 76.0%).

[0037] Step 2: Compound A5-1 (2.0 g, 2.4 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and diethylamine (1.0 mL) was added. The mixture was stirred at room temperature for 5 h to remove the amino protecting group. After the reaction was complete as monitored by TLC, water (5 mL) was added and the mixture was extracted three times with dichloromethane (5 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1-10 / 1) to obtain a white solid powder product c (1.3 g, yield 92.0%).

[0038] Step 3: HOBt (16.2 mg, 0.12 mmol, 1.0 equiv.) and EDCI (46.0 mg, 0.24 mmol, 3.0 equiv.) were added to a dichloromethane solution of compound c (50 mg, 0.08 mmol, 1.0 equiv.) and 19a (27.7 mg, 0.1 mmol, 1.2 equiv.) at room temperature. DIPEA (51.6 mg, 0.4 mmol, 5.0 equiv.) was then added, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was then quenched with 5.0 mL of water, and the resulting solution was extracted with 3 × 5 mL of dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1–20 / 1) to give a white powdery solid product 19b (56.8 mg, 82% yield).

[0039] Step 4: Compound 19b (50.0 mg, 0.05 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The mixture was stirred at room temperature for 5 h. After complete reaction by TLC monitoring, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5.0 mL), concentrated under reduced pressure, and purified by C18 reverse-phase chromatography column (water / methanol = 100 / 1-70 / 30) to obtain a white solid powder product 19 (30.0 mg, yield 78.0%).

[0040] 19H NMR spectral data of white solid powder product: 1 H NMR (500 MHz, DMSO) δ 8.79 (d, J = 5.5 Hz, 1H), 8.33 (s, 1H), 8.17 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.76 (dd, J =8.4, 2.1 Hz, 1H), 7.27 (s, 2H), 5.87 (d, J = 5.2 Hz, 1H), 4.61 (t, J = 5.2Hz, 1H), 4.16 – 4.05 (m, 2H), 2.86 (dd, J = 13.8, 5.0 Hz, 1H), 2.79 – 2.72(m, 1H), 2.67 (q, J = 6.3 Hz, 4H), 1.98 (td, J = 17.0, 6.8 Hz, 2H), 1.82 –1.73 (m, 1H), 1.20 (d, J = 6.6 Hz, 6H). Example 8: Preparation of Compound 21 ; Following the synthetic method of compound 19, in the first step, HOBt (16.2 mg, 0.12 mmol, 1.5 equiv.) and EDCI (46.0 mg, 0.24 mmol, 3.0 equiv.) were added to a dichloromethane solution of compound c (50 mg, 0.08 mmol, 1.0 equiv.) and 21a (21.3 mg, 0.1 mmol, 1.2 equiv.) at room temperature. Then, DIPEA (51.6 mg, 0.4 mmol, 5.0 equiv.) was added, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was then quenched with 5.0 mL of water, and the resulting solution was extracted with 3 × 5 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate. The organic layer was concentrated under vacuum and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-20 / 1) to give a white powdery solid product 21b (50.6 mg, yield 79.0%). In the second step, compound 21b (50.0 mg, 0.06 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The mixture was stirred at room temperature for 5 h. After complete reaction by TLC monitoring, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5.0 mL), concentrated under reduced pressure, and purified by C18 reverse-phase chromatography (water / methanol = 100 / 1-70 / 30) to give a white powdery solid product 21 (32.0 mg, yield 85.0%).

[0041] White solid powder product, 19H NMR spectral data 1H NMR (500 MHz, DMSO) δ 8.33 (s,1H), 8.21 – 8.07 (m, 2H), 7.88 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H),7.45 (s, 1H), 7.40 (td, J = 6.1, 2.0 Hz, 2H), 7.34 – 7.26 (m, 3H), 7.23 (q, J= 3.4 Hz, 2H), 5.89 (d, J = 5.1 Hz, 1H), 4.61 (t, J = 5.1 Hz, 1H), 4.09 (dq,J = 10.6, 4.8 Hz, 2H), 3.54 (s, 1H), 3.41 (s, 2H), 3.17 (h, J = 7.1 Hz, 2H), 2.91 (dd, J = 13.8, 4.8 Hz, 1H), 2.83 (dd, J = 13.9, 7.3 Hz, 1H), 2.72 (s,2H), 2.61 (d, J = 7.5 Hz, 2H), 1.94 (s, 1H), 1.75 (s, 1H). Example 9: Preparation of Compound 23 ; Following the synthetic method of compound 19, in the first step, HOBt (16.2 mg, 0.12 mmol, 1.5 equiv.) and EDCI (46.0 mg, 0.24 mmol, 3.0 equiv.) were added to a dichloromethane solution of compound c (50 mg, 0.08 mmol, 1.0 equiv.) and 23a (12.7 mg, 0.1 mmol, 1.2 equiv.) at room temperature. Then, DIPEA (51.6 mg, 0.4 mmol, 5.0 equiv.) was added, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was then quenched with 5.0 mL of water, and the resulting solution was extracted with 3 × 5 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate. The organic layer was concentrated under vacuum and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-20 / 1) to give a white powdery solid product 23b (40.4 mg, yield 70.6%). In the second step, compound 23b (40.0 mg, 0.05 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The mixture was stirred at room temperature for 5 h. After complete reaction by TLC monitoring, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5.0 mL), concentrated under reduced pressure, and purified by C18 reverse-phase chromatography (water / methanol = 100 / 1-70 / 30) to give a white powdery solid product 23 (20.7 mg, yield 72.5%).

[0042] Figure 5 This is the NMR spectrum of compound 23.

[0043] 1H NMR (500 MHz, DMSO) δ 8.85 (s, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 7.88 (d, J = 3.7 Hz, 1H), 7.68 (d, J = 5.0 Hz, 1H), 7.27 (s, 2H), 7.08 (dd, J= 5.0, 3.7 Hz, 1H), 5.85 (d, J = 5.3 Hz, 1H), 4.57 (t, J = 5.4 Hz, 2H), 4.14(t, J = 4.9 Hz, 1H), 4.04 (q, J = 5.6 Hz, 1H), 2.83 (dd, J = 13.7, 5.7 Hz,1H), 2.72 – 2.56 (m, 5 h), 1.88 (s, 1H), 1.68 (d, J = 6.9 Hz, 1H), 1.23 (s, 1H). Example 10: Preparation of Compound 37 In the first step, triethylamine (0.5 mL) was added to a DMF (1.0 mL) solution of compound c (50 mg, 0.08 mmol, 1.0 equiv.) and 37a (30.5 mg, 0.1 mmol, 1.2 equiv.) at room temperature. The mixture was heated to 70 °C and stirred for 5 h. After the reaction was completed by TLC monitoring, 10 mL of water was added and the mixture was extracted with ethyl acetate (10 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1-20 / 1) to obtain a white powdery solid product 37b (44.0 mg, yield 67.4%). In the second step, compound 37b (40.0 mg, 0.05 mmol, 1.0 equiv.) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The mixture was stirred at room temperature for 5 h. After complete reaction by TLC monitoring, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5.0 mL), concentrated under reduced pressure, and purified by C18 reverse-phase chromatography (water / methanol = 100 / 1-70 / 30) to obtain a white solid powder product 37 (24.2 mg, yield 79.6%). The 1H NMR spectrum of the white solid powder product is shown below. 1H NMR (400 MHz, DMSO) δ 8.34 (s, 1H), 8.15 (s, 1H), 7.41 (d, J = 2.1Hz, 1H), 7.38 (dt, J = 6.7, 2.3 Hz, 1H), 7.29 (s, 2H), 7.25 (d, J = 6.5 Hz,2H), 6.89 (t, J = 6.2 Hz, 1H), 6.49 (t, J = 5.4 Hz, 1H), 5.87 (d, J = 5.2 Hz,1H), 5.59 (s, 1H), 4.61 (t, J = 5.2 Hz, 1H), 4.18 (d, J = 6.0 Hz, 2H), 4.10(t, J = 4.8 Hz, 1H), 4.04 (q, J = 5.8 Hz, 1H), 3.10 (dq, J = 12.0, 7.0 Hz,2H), 2.80 (dd, J = 13.7, 5.5 Hz, 1H), 2.70 – 2.62 (m, 2H), 2.56 (d, J = 17.0Hz, 2H), 2.48 – 2.43 (m, 1H), 1.92 (s, 1H), 1.72 – 1.63 (m, 1H), 1.23 (s, 1H).

[0044] Example 11: Determination of the inhibitory activity of compounds on LCMT1 protein using the fluorescence polarization screening method of the present invention. (1) Experimental method:

[0045] Referring to the SAH activity assay method, and using the screening method of this invention, a 7 μM fluorescent probe DMSO solution, a 10 mM compound DMSO solution, and a 1.2 μM LCMT1 protein buffer solution were prepared (in this example, the buffer solution consisted of 50 mM pH 8 Tris HCl, 150 mM NaCl, 10 mM MgCl2, and 0.001% Triton X-100). The compound was serially diluted three times (initial concentration 10 mM): In a row of 12 wells of a 96-well plate, 5 μL of the 10 mM compound solution was added to the first well, 10 μL of DMSO was added to wells 2-12, and then another 5 μL of the 10 mM compound solution was added to the second well. The mixture was then pipetted up and down 10 times to mix. 5 μL from the second well was added to the third well, and the mixture was pipetted up and down 10 times to mix. This process was repeated with 5 μL from the third well. When operating on the eleventh well, mix by blowing and swishing 10 times, then discard 5 μL of the liquid, ensuring that the 12th well contains pure DMSO. Take 1 μL of the liquid from each of the 12 wells of the 96-well plate and add it to the odd-numbered wells in a row of the black 384-well plate. Then add 1 μL of a 7 μM fluorescent probe (the final concentration of the fluorescent probe in the black 384-well plate is the optimal probe concentration obtained in Example 3, i.e., 70 nM). Next, add 98 μL of 1.2 μM LCMT1 protein buffer solution to each odd-numbered well in the row, diluting the compound concentration 100-fold. After incubating in the dark for 5-30 minutes, measure the fluorescence polarization value and calculate the K0 value based on the obtained fluorescence polarization value. i Value. Each experiment was set up with 3 parallel replicates.

[0046] (2) Experimental results:

[0047] The effects of compounds 1-59 on LCMT1 K were determined using the experimental method described above. i The values ​​are shown in Table 1.

[0048] Table 1. Effects of target compounds on PARP1 and PARP2 K i value

[0049] Note: + indicates compound K. i > 10000 nM

[0050] ++ indicates that the compound has 10000 nM > K i > 1000 nM

[0051] +++ indicates that the compound has a density of 1000 nM > K. i > 100 nM

[0052] ++++ indicates compound K i < 100 nM

[0053] Conclusion: The representative compound of this invention can effectively inhibit LCMT1.

Claims

1. An adenosine-based LCMT1 inhibitor and its derivatives, characterized in that: The derivatives comprise one or more of the following: optical isomers, racemates, single enantiomers, possible diastereomers, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, and solvates of adenosine LCMT1 inhibitors, the structures of which are shown in Formula (I): ； Where: A is a group having a formula selected from (A-1) or (A-2): ； B 1 and B 2 Selected from chemical bonds or ; C is selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spiral ring, the C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spirocyclic rings can be independently substituted by one or more of the following substituents: hydroxyl, amino, carboxyl, nitro, cyano, one or more halogen atoms, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Any one of oxoalkyl or ester groups; Y is selected from , where n is any natural number from 1, 2, 3, 4, 5; L is selected from O and NQ. 1 Q 1 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Any one of the alkylamino groups; Q is selected from Any one of them; Z is selected from C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spiral ring, the C 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkylamino, C 4-14 Aryl, 4-14 membered aryl heterocyclic groups and 4-14 membered heterocyclic groups, monocyclic or polycyclic, C 4-10 Spirocyclic rings can be substituted independently by one or more of the following substituents: hydroxyl, amino, carboxyl, nitro, cyano, one or more halogen atoms, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Any one of oxoalkyl or ester groups; R1 is selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 3-7 cycloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Any one of the alkylamino groups; R2 is selected from any one of hydrogen, deuterium, trimethylolpropionate group, medoxamycin group, tert-butyloxycarbonyl group, p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, fluorenemethoxycarbonyl, p-toluenesulfonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, trifluoroacetyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, benzyl, phthaloyl, nitrobenzenesulfonyl, triphenylmethyl, acetamipridyl, carbamate, trimethylsilylethoxymethyl, p-phenylboronic acid benzyl, p-phenylboronic acid benzyloxycarbonyl, fluoroethyl, and chloroformate; the trimethylolpropionate group includes, but is not limited to: The medoxamycin group includes, but is not limited to: or ; R3 is selected from C 1-6 Alkyl, benzyl, allyl, medoxamycin group, C 1-6 Orthoester protecting group, C 1-18 Any of the following silyl ether protecting groups.

2. The adenosine LCMT1 inhibitor according to claim 1, characterized in that: The Y in the adenosine LCMT1 inhibitor is selected from... where n is 2; L represents NH; Q is selected from , , Any one of them; R1 is a hydrogen atom; R2 is selected from any one of hydrogen, deuterium, trimethylolpropionate group, and medoxamycin group; the trimethylolpropionate group includes, but is not limited to: The medoxamic group is ; R3 is a medoxamycin group: .

3. The adenosine LCMT1 inhibitor according to claim 1, characterized in that: The chemical structural formulas of the adenosine LCMT1 inhibitors and their derivatives include any one of the following formulas 1-60: ； ； ； ； ； ； 。 4. A method for preparing an adenosine-based LCMT1 inhibitor and its derivatives, characterized in that: The chemical reaction formula of the preparation method includes the reaction path shown below: Path 1: When A is selected from formula (A-1), the aldehyde a1 obtained by modification and oxidation with homoserine as the parent nucleus undergoes a reductive amination reaction with the raw material a2 to obtain intermediate a3, and then undergoes a reductive amination reaction with aldehyde or ketone a4 to obtain intermediate a5. Finally, the protecting group is removed to obtain compound I-(A-1). ； Pathway 2: When A is selected from formula (A-2), the preparation method of intermediate a3 is the same as that of Pathway 1. Intermediate a3 is reduced and aminationd with aldehyde b4 to obtain intermediate b5. After removing the L group protecting group Pg, intermediate b5 undergoes at least one of the following reactions: amide condensation, transesterification or reduced amination, to obtain the product shown in formula I-(A-2). 。 5. A fluorescent probe for screening adenosine LCMT1 inhibitors and their derivatives, characterized in that, The structure of the fluorescent probe is shown in formula (II): ； Wherein, X is selected from at least one of C, N, O, S, C(O), NHC(O), C(O)NH, S(O), and S(O)2; n is selected from at least one of 2, 3, and 4; R 1 The group is a fluorescent group attached to an adenosine analog and its derivatives, selected from at least one of the following structures or their enantiomers, diastereomers, monomeric isomers, racemates, or pharmaceutically acceptable salts thereof: 5-FAM and its derivatives, NBD and its derivatives, Atto590 and its derivatives, Atto647N and its derivatives, BODIPY and its derivatives, CY3 and its derivatives, and CY5 and its derivatives. The structural formulas of 5-FAM and its derivatives, NBD and its derivatives, Atto590 and its derivatives, Atto647N and its derivatives, BODIPY and its derivatives, CY3 and its derivatives, and CY5 and its derivatives are shown below: ； R 2 R 3 The functional groups are independently selected from hydrogen atoms, halogen atoms, cyano groups, hydroxyl groups, amino groups, carboxyl groups, nitro groups, and C groups. 1-4 Alkyl, haloalkyl, C 1-4 Alkoxy, halogenated C l-4 Alkoxy, hydroxy C l-4 Alkyl, carboxyl C l-4 Alkyl, C 6-10 Aryl, C 3-7 Monocyclic or polycyclic cycloalkyl, 5-10-membered heteroaryl, and 4-10-membered heterocycloalkyl groups, wherein each group is optionally substituted by 1, 2, 3, or 4 independently selected from the following substituents: hydrogen atom, halogen atom, cyano, hydroxyl, amino, carboxyl, nitro, C 1-4 Alkyl, haloalkyl, C 1-4 Alkoxy, halogenated C l-4 Alkoxy, hydroxy C l-4 alkyl group, carboxyl group C l-4 Alkyl halide, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 At least one of the haloalkyl groups.

6. The fluorescent probe according to claim 5, characterized in that, The uses of the fluorescent probe include: (a) Used to prepare LCMT1 receptor fluorescent probes; (b) Used as a fluorescent probe for the LCMT1 receptor; (c) Used to prepare LCMT1 receptor inhibitors; (d) Used to prepare LCMT1 inhibitor-related drugs.

7. A method for preparing a fluorescent probe of an adenosine LCMT1 inhibitor and its derivatives as described in any one of claims 5-6, characterized in that, The chemical reaction formula of the preparation method includes the reaction path shown below: ； Where n = at least one of 2, 3, and 4; Compounds d1 and d2 undergo a reductive amination reaction to generate intermediate d3. Intermediate d3 then undergoes a reductive amination reaction with homoserine-derived aldehyde compound d4 to generate intermediate d5, selectively removing the nitrogen protecting group Pg. 1 Then it is coupled with a fluorescent reagent, and finally all protecting groups Pg are removed. 2 and Pg 3 Generate a fluorescent probe as shown in Formula II; the definitions of n, X, R1, R2, and R3 in each formula are as previously shown.

8. A fluorescence polarization screening method for adenosine LCMT1 inhibitors and their derivatives, characterized in that: Includes the following steps: (1) The protein LCMT1 was prepared using the Escherichia coli prokaryotic expression method; (2) Synthesize fluorescent probes; (3) Determine the concentration of the fluorescent probe by measuring its fluorescence intensity and polarization value; (4) Incubate the protein LCMT1 and the fluorescent probe together, and determine the concentration of protein LCMT1 to be used based on the measured fluorescence polarization value; (5) The protein LCMT1, the fluorescent probe and the compound to be screened are co-incubated to allow the fluorescent probe to competitively bind to the LCMT1 protein and the fluorescence polarization value of the system is measured. Using the measured fluorescence polarization value as the ordinate and the logarithm of the concentration of the target compound as the abscissa, an inhibition curve of the target compound was fitted, and the K of the target compound was calculated. i The value is used to determine the extent of the inhibitory activity of the target compound to be screened.

9. The fluorescence polarization screening method according to claim 8, characterized in that: The preparation of protein LCMT1 using the Escherichia coli prokaryotic expression method in step (1) specifically includes: extracting the expression plasmid from the DH5α strain, introducing the plasmid into the competent cells of the protein expression strain BL21 DE3 for expression, and separating and purifying the LCMT1 protein using a Ni-NTA affinity chromatography column; The method for synthesizing the fluorescent probe in step (2) is as described in the preparation method of claim 7; The determination of fluorescence intensity and polarization value of the fluorescent probe in step (3) specifically includes: preparing the fluorescent probe into a DMSO solution of a certain concentration and performing gradient dilution, and determining the concentration of the fluorescent probe to be used based on the measured fluorescence intensity and fluorescence polarization value, wherein the concentration to be used is between 1-1000 nM. In step (4), the co-incubation of protein LCMT1 and fluorescent probe, and the determination of the concentration of protein LCMT1 based on the measured fluorescence polarization value, specifically involves: simultaneously adding fluorescent probe and LCMT1 protein, co-incubating them, and determining the concentration of LCMT1 protein solution based on the measured fluorescence polarization value; the concentration of fluorescent probe used is the concentration of fluorescent probe determined in step (3) of the fluorescence polarization screening method of claim 8, ranging from 1 to 1000 nM; LCMT1 protein is serially diluted with buffer solution, the composition of which is 50 mM Tris HCl, pH 8, 150 mM NaCl, and buffer solution additives; In step (5), when the protein LCMT1, the fluorescent probe, and the compound to be screened are co-incubated, the protein LCMT1 and the fluorescent probe are used at the concentrations described above, and the compound to be screened is serially diluted. Different concentrations of the compound to be screened are co-incubated with the protein LCMT1 and the fluorescent probe in the dark, and their fluorescence polarization values ​​are detected. The measured fluorescence polarization values ​​are plotted on the ordinate, and the logarithm of the concentration of the compound to be screened is plotted on the abscissa to fit the inhibition curve of the target compound, and the K of the compound to be screened is calculated. i The value is used to determine the extent of the inhibitory activity of the target compound to be screened.

10. The application of a fluorescent probe for screening adenosine LCMT1 inhibitors and their derivatives as described in any one of claims 5-6, or a fluorescent probe for adenosine LCMT1 inhibitors and their derivatives prepared by the method described in claim 7, characterized in that, The fluorescent probes used for screening adenosine LCMT1 inhibitors and their derivatives, and the fluorescent polarization screening methods and systems used for screening potential active compounds of adenosine LCMT1 inhibitors and their derivatives, are used for construction, screening, activity determination, development of related drugs, identification of related active compounds, and identification of related inhibitors.

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