Thioether structure-containing targeted PHGDH active molecular probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a targeted PHGDH active molecular probe containing a thioether structure as well as a preparation method and application of the targeted PHGDH active molecular probe. A series of novel PHGDH small molecular probes containing thioether structures are obtained through screening of a compound library and structural modification, the structural types of PHGDH target probes are enriched, and application of the PHGDH small molecular probes in biological orthogonal reaction is expanded. The invention provides a novel thioether skeleton PHGDH molecular probe, and the probe can efficiently and specifically mark a target protein PHGDH through a Click biological orthogonal reaction technology and identify the interaction between a targeted PHGDH inhibitor and a PHGDH protein. Compared with the prior art, the probe disclosed by the invention has higher selectivity and accuracy, and can be used for effectively marking and identifying target protein in a complex biological system, so that the efficiency of protein interaction analysis is remarkably improved. In addition, through an immunofluorescence co-labeling method, the binding condition of the probe and the target protein can be visually observed, and the targeting property of the inhibitor is further verified.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a class of targeted PHGDH active molecular probes containing a thioether structure, a preparation method thereof, and an application thereof. Background Art

[0002] Amino acid metabolism is one of the main metabolic pathways used by cancer cells to maintain growth and reproduction, and it plays an important role in the regulation of redox, the maintenance of biosynthetic pathways, the regulation of epigenetic modification, and the synthesis of metabolic intermediates. Among the nutrients required for mammalian cell proliferation, serine is the third largest metabolite after glucose and glutamine and ranks second among all amino acids. Serine can promote the generation of energy required for the metabolism of normal cell proliferation and differentiation and the synthesis of many biological macromolecules, and can provide building blocks for molecules involved in various biochemical pathways, including amino acids, lipids, nucleotides, and cofactors.

[0003] Phosphoglycerate dehydrogenase (PHGDH), as the rate-limiting enzyme in the serine synthesis pathway, is closely related to the occurrence of various tumors. In the SSP pathway, PHGDH uses NAD+ as a cofactor to oxidize the glycolytic intermediate 3-PG to 3-PHP, and then through the transamination of PSAT and the dephosphorylation of PSPH, it is converted into serine that provides building blocks for molecules in various biochemical pathways. At the same time, due to the substrate promiscuity of PHGDH, it can also participate in other physiological processes, such as promoting tumorigenesis by accelerating the NADH-dependent reduction of α-ketoglutaric acid (α-KG) to generate the carcinogenic metabolite D-2-hydroxyglutaric acid. In recent years, pharmacological research on the PHGDH target has mainly focused on its high expression, and it has now been found that the expression of this target is upregulated in various tumors such as breast cancer, melanoma, liver cancer, lung cancer, and leukemia. Currently, PHDGH has gradually become a new direction for targeted drug discovery.

[0004] In the development of targeted drugs, the Click bioorthogonal reaction technology has become an important tool for the efficient labeling of targeted proteins and target identification. The Click reaction is a highly efficient and selective chemical reaction that can specifically react with target proteins or other biological macromolecules without interfering with the biological system. The principle of the Click reaction is based on the "click chemistry" reaction, which realizes labeling through the interaction between alkynyl and azide groups, and is widely used in small molecule drug screening, protein interaction research, and drug target identification.

[0005] The research group where the inventor belongs previously designed and synthesized a large number of compounds with a thioether backbone. Through screening and structural modification, a series of new Click reaction probes were obtained. These novel thioether backbone probes can efficiently identify the targeted PHGDH protein through Click technology, enrich the structural types of PHGDH molecular activity probes, have high specificity and activity, and can be used to further screen inhibitors targeting PHGDH and reveal its mechanism of action in cells. Summary of the Invention

[0006] The object of the present invention is to fill the gap in the prior art and provide a class of targeted PHGDH active molecular probes containing a thioether structure, a preparation method thereof, and an application thereof.

[0007] Through screening a compound library and structural modification, the inventor obtained a series of novel PHGDH molecular probes with a thioether backbone that are structurally novel and have strong activity, which can identify the interaction between a targeted PHGDH inhibitor and a protein. The inventor designed and synthesized a large number of thioether backbone compounds. By screening this compound library and structurally modifying some compounds, a class of novel thioether backbone probes capable of identifying the PHGDH target based on the Click bioorthogonal reaction method was obtained, enriching the structural types of PHGDH molecular activity probes.

[0008] The technical solution for the present invention to solve the technical problem is as follows:

[0009] In the first aspect of the present invention, a class of targeted PHGDH active molecular probes containing a thioether structure is provided, which has the structure shown in formula (I):

[0010]

[0011] Wherein:

[0012] R 1 is selected from any one of strong electron-withdrawing groups such as a hydrogen atom, a nitro group, a cyano group, a carboxyl group, or a trifluoromethyl group;

[0013] Both the A fragment and the B fragment are substituted or unsubstituted aromatic rings or heteroaromatic rings. The aromatic ring and the heteroaromatic ring can include any one of five-membered or six-membered rings such as furan, pyridine, pyrazole, thiazole, thiadiazole, oxazole, and tetrazole. The substituent is any one of a mono-substituted or multi-substituted alkyl group, an alkoxy group, a nitro group, a halogen, a trifluoromethyl group, a carbonyl group, a benzoylpiperidine group, a benzamide group, an amide, a phenylmethyl ketone, etc.; the A fragment and the B fragment are the same or different;

[0014] Linker is a link molecule with an adjustable length. The main chain atoms of this connecting chain are selected from carbon, nitrogen, or oxygen atoms, including but not limited to a flexible PEG chain, a saturated carbon chain, or an unsaturated carbon chain, and the chain length can be adjusted;

[0015] C represents a reporter group that can be used to identify protein targets, such as fluorescent groups like FITC, biotin, azides and alkynes that can undergo bioorthogonal Click reactions, and any one of strained alkenes and tetrazines in the inverse electron demand Diels - Alder reaction.

[0016] Preferably, in formula (I),

[0017] R 1 is any one of nitro, cyano, trifluoromethyl;

[0018] A is selected from any one of furan, pyrazole, thiazole, thiadiazole or oxazole;

[0019] B is selected from any one of thiazolyl phenyl ketone, thiazolyl benzoyl piperidine, thiazolyl benzamide, thiadiazolyl amide, thiazolyl amide;

[0020] Linker is selected from saturated carbon chains or unsaturated carbon chains;

[0021] C is selected from any one of azides and alkynes that can undergo bioorthogonal Click reactions, and strained alkenes and tetrazines in the inverse electron demand Diels - Alder reaction.

[0022] More preferably, in formula (I),

[0023] R 1 is nitro;

[0024] A is thiazolyl or oxazolyl;

[0025] B is selected from: any one of thiazolyl phenyl ketone, thiazolyl benzamide, thiadiazolyl amide, thiazolyl amide;

[0026] Linker is selected from saturated carbon chains, such as any one of methyl, ethyl, propyl, isopropyl, butyl, pentyl;

[0027] C is selected from any one of azides and alkynes that can undergo bioorthogonal Click reactions, and strained alkenes in the inverse electron demand Diels - Alder reaction.

[0028] In some most preferred embodiments of the present invention, the molecular probe is selected from any one of the following compounds:

[0029]

[0030]

[0031] In the second aspect of the present invention, a preparation method of a class of targeted PHGDH active molecular probes containing a thioether structure with the structure shown in formula (I) is provided, and can be synthesized through the following route:

[0032]

[0033] Each substituent in the compound of formula (I) is defined as in the first aspect.

[0034] The said method comprises the following steps:

[0035] 1) Compound I-3 is prepared from compound I-1 and compound I-2 by nucleophilic substitution reaction with the elimination of one molecule of hydrogen bromide.

[0036] 2) In the presence of a condensing agent and a base, compound I-3 reacts with compound Linker—C through amide condensation reaction to prepare compound I-4.

[0037] Preferably, in the said step 1), the molar ratio of compound I-1 to compound I-2 is 0.8 - 1:2; in the said step 2), the molar ratio of compound I-3 to compound Linker—C is 0.8 - 1:2.

[0038] Preferably, in the said step 1), the temperature of the substitution reaction is room temperature and the reaction time is 0 - 6 h; in the said step 2), the temperature of the amide condensation reaction is room temperature and the reaction time is 2 - 8 h.

[0039] Preferably, in the said step 2), the condensing agent for the amide condensation reaction is one or more of HOAT, HOBT, HATU, HBTU or BOP; the base for the amide condensation reaction is one or two of N,N - diisopropylethylamine DIPEA or triethylamine TEA; the organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N - dimethylformamide or dimethyl sulfoxide.

[0040] Preferably,

[0041] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is (4 - ethylpiperidin - 1 - yl)(4-(2 - ((5 - nitrothiazol - 2 - yl)thio)thiazol - 4 - yl)phenyl)methanone (LBJ - 1), its preparation method comprises the following steps:

[0042] 1) Prepare 4-(2 - mercaptothiazol - 4 - yl)benzoic acid (probe precursor T - 1), and the reaction route is as follows:

[0043]

[0044] 4-(2-Bromoacetyl)benzoic acid was added to ethanol and stirred until completely dissolved. Ammonium dithiocarbamate was added, and the reaction was carried out at room temperature for 3 h to obtain 4-(2-mercapto-4-thiazolyl)benzoic acid (probe precursor T-1); wherein, the molar ratio of 4-(2-bromoacetyl)benzoic acid to ammonium dithiocarbamate was 1:1.15;

[0045] 2) Preparation of 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzoic acid (probe precursor T-2), the reaction route is as follows:

[0046]

[0047] Probe precursor T-1 and 2-bromo-5-nitrothiazole were added to ethanol and stirred until completely dissolved. Sodium ethoxide was added, and the reaction was carried out at room temperature for 4 h to obtain 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzoic acid (probe precursor T-2); wherein, the molar ratio of probe precursor T-1, 2-bromo-5-nitrothiazole and sodium ethoxide was 8.4:10:1;

[0048] 3) Preparation of (4-ethylpiperidin-1-yl)(4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)phenyl)methanone (LBJ-1), the reaction route is as follows:

[0049]

[0050] Probe precursor T-2 was dissolved in DCM, HATU and 4-ethynylpiperidine hydrochloride were added in sequence. After complete dissolution, DIPEA was added dropwise, and the reaction was carried out at room temperature for 6 h to obtain (4-ethylpiperidin-1-yl)(4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)phenyl)methanone (LBJ-1); wherein, the molar ratio of probe precursor T-2, HATU, 4-ethynylpiperidine hydrochloride and DIPEA was 1:1.4:1:2.3.

[0051] When the targeting PHGDH active molecule probe containing a thioether structure with the structure shown in formula (I) is N-(2-methylbut-3-yn-2-yl)-4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzamide (LBJ-2), the synthesis method is the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride is replaced with the raw material 2-methyl-3-butyn-2-amine.

[0052] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)-N-(prop-2-yn-1-yl)benzamide (LBJ-3), the synthesis method is the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride is replaced with the raw material prop-2-yn-1-amine.

[0053] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)-N-(pent-4-yn-1-yl)benzamide (LBJ-4), the synthesis method is the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride is replaced with the raw material pent-4-yn-1-amine hydrochloride.

[0054] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(2-azidoethyl)-4-(2-(5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzamide (LBJ-5), the synthesis method is the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride is replaced with the raw material 2-azidoethylamine.

[0055] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)undec-10-enamide (LBJ-6), the preparation route is as follows:

[0056]

[0057] Preparation method:

[0058] 1) 2-Amino-5-mercapto-1,3,4-thiadiazole, 2-bromo-5-nitrothiazole and sodium ethoxide are added to ethanol for dissolution, and the reaction is carried out at room temperature for 2 h to obtain the probe precursor T-5; among them, the molar ratio of 2-amino-5-mercapto-1,3,4-thiadiazole, 2-bromo-5-nitrothiazole and sodium ethoxide is 1:0.9:1;

[0059] 2) The probe precursor T-5 obtained in step 1), undec-10-ynoic acid, HATU and DIPEA are dissolved in DMF and heated at 40 °C for reaction for 5 h to obtain N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)undec-10-enamide (LBJ-6). Among them, the molar ratio of the probe precursor T-5, undec-10-ynoic acid, HATU and DIPEA is 1:1:2.5:2.

[0060] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)oct-7-amide (LBJ-7), the synthesis method is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material oct-7-ynoic acid.

[0061] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)hept-6-ylimide (LBJ-8), the synthesis method is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material hept-6-ynoic acid.

[0062] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)hex-5-ylimide (LBJ-9), the synthesis method is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material hex-5-ynoic acid.

[0063] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)pent-4-ylimide (LBJ-10), the synthesis method is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material pent-4-ynoic acid.

[0064] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is 5-azido-N-(2-(5-nitrothiazol-2-yl)thio)thiazol-5-yl)pentamide (LBJ-11), the synthesis method is the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride is replaced with the raw material 5-azidopentanoic acid.

[0065] When the targeted PHGDH active molecular probe containing a thioether structure with the structure shown in formula (I) is 3-(cycloprop-2-en-1-yl)-N-(5-((5-nitrooxazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)propanamide (LBJ-12), the synthesis method is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material 3-(cycloprop-2-en-1-yl)propanoic acid.

[0066] The third aspect of the present invention provides the use of a class of targeted PHGDH active molecular probes containing a thioether structure as described in the first aspect in identifying the interaction between a targeted PHGDH inhibitor and a PHGDH protein. The reporter group of the active molecular probe can be selected from any one of biotin, fluorescent groups such as FITC, azides or alkynes for bioorthogonal Click reactions, and strained olefins and tetrazines in inverse electron demand Diels - Alder reactions.

[0067] The targeted PHGDH active molecular probe labels the PHGDH target protein through a Click bioorthogonal reaction.

[0068] The Click bioorthogonal reaction is an unnatural chemical reaction that can occur in biological systems, and the molecules participating in the reaction do not react with other biomolecules, so it has high selectivity. The Click reaction is widely used in research fields such as probe labeling, target protein identification, and cell imaging. The typical steps of the Click reaction include:

[0069] Selection of reactants: Usually, molecules with "click reaction" properties are selected. These molecules contain functional groups with bioorthogonality (that is, they do not react with other biomolecules in the cell), such as azide and alkyne. The specificity of these groups enables them to carry out precise reactions in a biological environment.

[0070] Setting of reaction conditions: The commonly used type of Click reaction is the copper - catalyzed azide - alkyne cycloaddition (CuAAC). This reaction is carried out in an aqueous solution and has the characteristics of high efficiency and strong selectivity. The reaction usually needs to be carried out under appropriate pH conditions and under the catalysis of copper ions.

[0071] Ligation reaction: Through the Click reaction, a probe molecule containing an alkyne or azide group is efficiently labeled or ligated to a target molecule (such as a protein) or other biological macromolecules. This process can ensure precise labeling and traceability of the target protein in cells or tissues.

[0072] Separation and analysis: After the reaction is completed, the reaction products are separated and detected by techniques such as chromatography, mass spectrometry, and fluorescence imaging. The protein labeled with the probe can be detected by fluorescence or radioactive labeling to identify protein binding and interactions.

[0073] In the Click bioorthogonal reaction of the present invention, the main role of the PHGDH molecular probe is to label and identify the interaction between a targeted PHGDH inhibitor and a PHGDH protein. By synthesizing a thioether - skeleton probe containing appropriate chemical groups, an orthogonal reaction can occur with the PHGDH protein or related targets in cells or tissues, achieving efficient labeling and identification of the target protein.

[0074] For example, in Example 17 of the present invention, the PHGDH molecular probe LBJ-5 verified the target binding property by immunofluorescence co-labeling method. The LBJ-5 probe contains an alkynyl group and can undergo a Click reaction (CuAAC) with an azide-labeled fluorescent molecule. This reaction introduces a fluorescent group onto the target protein bound by the probe, causing the target protein to exhibit a specific fluorescent signal under a fluorescence microscope. By the immunofluorescence co-labeling method, the PHGDH protein was first labeled in the experiment and a fluorescent signal was introduced. Subsequently, after reacting with the LBJ-5 probe, the co-localization of the fluorescent signals was observed. If the two fluorescent signals highly overlap or completely coincide under the microscope, it indicates that LBJ-5 has specifically bound to the target protein PHGDH, and LBJ-5 has good on-target property.

[0075] Unless otherwise specified, the following terms used in the specification and claims have the meanings discussed below:

[0076] The term "aromatic ring" refers to a monocyclic, fused polycyclic or biphenyl group containing 1-12 carbon atoms and having a fully conjugated π-electron system. Non-limiting examples of aromatic rings are phenyl, naphthyl and biphenyl, and the aromatic ring can be substituted or unsubstituted.

[0077] The term "heteroaromatic ring" refers to a monocyclic system containing 1-6 atoms, the system containing one, two, three or four ring heteroatoms which are N, O or S, the remaining ring atoms being C, and having a fully conjugated π-electron system. Non-limiting examples of unsubstituted heteroaromatic rings include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, thiadiazole, pyrazole, pyridine, pyrimidine, tetrazole and triazine. The heteroaromatic ring can be substituted or unsubstituted.

[0078] The term "alkyl" denotes a saturated aliphatic hydrocarbon group of 1-20 carbon atoms, including straight-chain and branched-chain groups. The alkyl can be substituted or unsubstituted. When it is a substituted alkyl, the substituent is preferably one or more.

[0079] The term "alkoxy" denotes -O-(unsubstituted alkyl) and -O-(unsubstituted cycloalkyl). Representative examples include but are not limited to methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, etc.

[0080] The term "trifluoromethyl" denotes -CF 3 group.

[0081] The term "nitro" denotes -NO 2 group.

[0082] The term "cyano" denotes -CN group.

[0083] The term "carbonyl" refers to the -C=O group.

[0084] The term "carboxyl" refers to the -COOH group.

[0085] The term "benzoylpiperidine" refers to group.

[0086] The term "benzamide" refers to group.

[0087] The term "azide" refers to a compound structure with an -N 3 group.

[0088] The term "alkynyl" refers to the -C≡CH group.

[0089] The term "strained alkene" refers to a cyclic alkene group, such as etc.

[0090] The term "tetrazine" refers to group.

[0091] The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine, bromine.

[0092] The term "PEG chain" refers to a polymer chain composed of a series of ethylene glycol units (-O-CH2-CH2-).

[0093] The term "saturated carbon chain" refers to a carbon chain structure in which all carbon atoms are connected by single bonds and there are no double or triple bonds.

[0094] The term "unsaturated carbon chain" refers to a carbon chain structure in which there is one or more unsaturated bonds (such as carbon-carbon double bonds or carbon-carbon triple bonds).

[0095] The present invention has the following technical effects: The present invention provides a novel thioether backbone PHGDH molecular probe, which can efficiently and specifically label the target protein PHGDH through Click bioorthogonal reaction technology and identify the interaction between the PHGDH inhibitor targeting PHGDH and the PHGDH protein. Compared with the prior art, the probe of the present invention has high selectivity and accuracy, can effectively label and identify the target protein in a complex biological system, thus significantly improving the efficiency of protein interaction analysis. In addition, through immunofluorescence co-labeling, the binding situation between the probe and the target protein can be visually observed, further verifying the on-target property of the inhibitor. The present invention also enriches the structural types of PHGDH molecular probes through structural modification, provides a new tool for in-depth research on the PHGDH target, and promotes the screening of PHGDH-targeted inhibitors and their applications in anti-tumor and metabolic diseases. Brief Description of the Drawings

[0096] Figure 1 The result of Coomassie brilliant blue staining based on the ABPP method shows the binding of the PHGDH inhibitor to the target protein.

[0097] Figure 2 It is a validation result graph of the on-target property of the probe molecule LBJ-5 by immunofluorescence co-labeling method. Detailed implementation manners

[0098] The technical solution of the present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the described embodiments.

[0099] For those without specific technologies or conditions indicated in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0100] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available products unless otherwise specified.

[0101] Example 1 Preparation of 4-(2-mercapto-1,3-thiazol-4-yl)benzoic acid (probe precursor T-1)

[0102]

[0103] Add 4-(2-bromoacetyl)benzoic acid (5 g, 0.020 mol) and ethanol (25 mL) to a single-necked flask, stir until completely dissolved, and add ammonium dithiocarbamate (2.49 g, 0.023 mol). React at room temperature for 3 h, stop the reaction when the reaction is completed detected by TLC plate. Add 50 mL of water, filter by suction to obtain a yellow solid, and dry it under vacuum to obtain 3.88 g of probe precursor T-1, with a yield of 79.5%.

[0104] 1 H NMR (300 MHz, DMSO-d 6 ): δ = 8.05 (d, 2H, J = 8.67 Hz, Ar-H), 7.94 (d, 2H, J = 6.77 Hz, Ar-H), 7.89 (s, 1H, Ar-H) ppm. MS (ASAP), [M+H] + calculated for C 10 H 8 NO 2 S 2 + 237.99, found 237.86.

[0105] Example 2 Preparation of 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzoic acid (probe precursor T-2)

[0106]

[0107] Add probe precursor T-1 (2 g, 0.0084 mol), 2-bromo-5-nitrothiazole (2.11 g, 0.010 mol) and ethanol (20 mL) to a single-necked flask, stir until completely dissolved, add sodium ethoxide (0.69 g, 0.0010 mol), and react at room temperature for 4 h. Monitor the reaction by TLC plate until completion, add 50 mL of water, filter by suction, wash the filter cake with DCM by pulping, and dry in vacuo to obtain 2.5 g of a pale yellow solid with a yield of 81.2%. 1 H NMR (300 MHz, DMSO-d 6 ): δ = 8.61 (s, 1H, Ar-H), 8.11 (d, 2H, J = 7.79 Hz, Ar-H), 7.81 (s, 1H, Ar-H), 7.69 (d, 2H, J = 5.87 Hz) ppm. MS (ASAP), [M+H] + calculated forC 13 H 8 N 3 O 4 S 3 + 365.97, found 365.57.

[0108] Example 3 Preparation of (4-ethylpiperidin-1-yl)(4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)phenyl)methanone (LBJ-1)

[0109]

[0110] Add probe precursor T-2 (1 g, 0.0027 mol) to a single-necked flask, dissolve it with DCM (10 mL), sequentially add HATU (1.5 g, 0.0039 mol) and 4-ethynylpiperidine hydrochloride (0.4 g, 0.0027 mol). After complete dissolution, add DIPEA (0.8 g, 0.0062 mol) dropwise and react at room temperature for 6 h. Monitor the reaction by TLC plate until completion. Stop the reaction, extract with water and dichloromethane three times, combine the dichloromethane layers, dry over anhydrous sodium sulfate, distill off the solvent under reduced pressure, and purify by silica gel column chromatography (V 石油醚 : V 乙酸乙酯 = 3:1) to obtain 0.24 g of a pale yellow solid with a yield of 19%. 11H NMR (300 MHz, CDCl3): δ = 8.47 (s, 1H, Ar-H), 8.01 - 7.98 (d, 2H, J = 8.28 Hz, Ar-H), 7.76 (s, 1H, Ar-H), 7.55 - 7.52 (d, 2H, J = 8.31 Hz, Ar-H), 4.16 - 4.02 (m, 2H, CH 2 ), 3.66 (s, 2H, CH 2 ), 3.36 (s, 1H, CH 2 ), 2.77 (s, 1H, CH 2 ), 2.17 (s, 1H, CH), 2.05 (s, 1H, CH 2 ), 1.81 (s, 1H, CH 2 ), 1.29 - 1.24 (t, 1H, J = 8.40 Hz, CH) ppm. MS (ASAP), [M + H] + calculated for C 20 H 17 N 4 O 3 S 3 + 457.04, found 456.96.

[0111] Example 4 Preparation of N-(2-methylbut-3-yn-2-yl)-4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzamide (LBJ-2)

[0112] The synthesis method was the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride was replaced with the raw material 2-methyl-3-butyn-2-amine, with an input amount of 0.4 g, and finally 0.64 g of yellow solid was obtained, with a yield of 30.9%. 1 1H NMR (300 MHz, DMSO-d 6 ): δ = 8.86 (s, 1H, Ar-H), 8.62 (s, 1H, Ar-H), 8.13 - 8.11 (d, 2H, J = 8.40 Hz, Ar-H), 9.00 - 7.97 (d, 2H, J = 8.40 Hz, Ar-H), 3.14 (s, 1H, CH), 1.64 (s, 6H, CH 3 ) ppm. MS (ASAP), [M + H] + calculated for C 18 H 15 N 4 O 3 S 3 + 431.52, found 431.45.

[0113] Example 5 Preparation of 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)-N-(prop-2-yn-1-yl)benzamide (LBJ-3)

[0114] The synthesis method was the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride was replaced with the raw material prop-2-yn-1-amine. The feeding amount was 0.5 g, and finally 2.77 g of yellow solid was obtained with a yield of 76%. 1 H NMR(300MHz,DMSO-d 6 ):δ=8.79(s,1H,Ar-H),8.55(s,1H,Ar-H),8.11-8.09(d,2H,J=8.49Hz,Ar-H),7.99-7.96(d,2H,J=8.50Hz,Ar-H),4.06(s,2H,CH 2 ),3.09(s,1H,CH)ppm.MS(ASAP),[M+H] + calculated forC 16 H 11 N 4 O 3 S 3 + 403.00,found403.21.

[0115] Example 6 Preparation of 4-(2-((5-nitrothiazol-2-yl)thio)thiazol-4-yl)-N-(pent-4-yn-1-yl)benzamide (LBJ-4)

[0116] The synthesis method was the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride was replaced with the raw material pent-4-yn-1-amine hydrochloride. The feeding amount was 0.5 g, and finally 0.72 g of yellow solid was obtained with a yield of 40%. 1 H NMR(300MHz,CDCl 3 ):δ=8.48(s,1H,Ar-H),8.04-8.02(d,2H,J=7.59Hz,Ar-H),7.92-7.89(d,2H,J=8.7Hz,Ar-H),3.65-3.63(d,2H,J=6.18Hz,CH 2 ),2.38-2.34(m,2H,CH 2 ),2.05(s,1H,CH),1.93-1.89(t,2H,J=7.11Hz,CH 2 )ppm.MS(ASAP),[M+H] + calculated for C18 H 15 N 4 O 3 S 3 + 431.52, found 431.12.

[0117] Example 7 Preparation of N-(2-azidoethyl)-4-(2-(5-nitrothiazol-2-yl)thio)thiazol-4-yl)benzamide (LBJ-5)

[0118] The synthesis method was the same as that of LBJ-1, except that the raw material 4-ethynylpiperidine hydrochloride was replaced with the raw material 2-azidoethylamine, and the feeding amount was 0.3 g. Finally, 0.12 g of yellow solid was obtained, and the yield was 9.9%. 1 H NMR(300 MHz, CDCl 3 ): δ = 8.55(s, 1H, Ar-H), 8.12 - 8.10(d, 2H, J = 7.63 Hz, Ar-H), 7.65 - 7.62(d, 2H, J = 8.32 Hz, Ar-H), 3.33 - 3.27(d, 2H, J = 5.87 Hz, CH 2 ), 1.77 - 1.74(t, 2H, J = 6.61 Hz, CH 2 ) ppm. MS(ASAP), [M + H] + calculated for C 15 H 12 N 7 O 3 S 3 + 434.49, found 434.15.

[0119] Example 8 Preparation of N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)undec-10-enamide (LBJ-6)

[0120] The preparation route is as follows:

[0121]

[0122] Add 2-amino-5-mercapto-1,3,4-thiadiazole (2 g, 0.015 mol), 2-bromo-5-nitrothiazole (2.82 g, 0.0135 mol) and sodium ethoxide (1.02 g, 0.015 mol) into a single-necked flask. Add ethanol (15 mL) to dissolve them, and react at room temperature for 2 h. After detecting the end of the reaction by TLC plate, remove the solvent by vacuum distillation, then slurry with water, and obtain 2.64 g of the probe precursor T-5 after vacuum drying, with a yield of 66.6%. Add the probe precursor (1.5 g, 0.0057 mol), undec-10-ynoic acid (1 g, 0.0057 mol), HATU (5.46 g, 0.0143 mol) and DIPEA (1.48 g, 0.0115 mol) into a single-necked flask. After dissolving with DMF (10 mL), heat to 40 °C and react for 5 h. Extract three times with water and dichloromethane, combine the dichloromethane layers, remove the solvent by pressure distillation, and purify by silica gel column chromatography (pure DCM) to obtain 0.64 g of a dark yellow solid, with a yield of 26.4%. 1 H NMR(300MHz,DMSO-d 6 ):δ=8.79(s,1H,Ar-H),2.73-2.72(t,1H,J=2.61Hz,CH),2.17-2.11(m,2H,CH 2 ),1.64-1.59(t,2H,J=7.17Hz,CH 2 ),1.48-1.27(m,12H,CH 2 )ppm.MS(ASAP),[M+H] + calculated for C 16 H 20 N 5 O 3 S 3 + 426.07,found 426.01.

[0123] Example 9 Synthesis method of N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)oct-7-amide (LBJ-7) is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced with the raw material oct-7-ynoic acid, with a feeding amount of 0.5 g, and finally 0.56 g of a light yellow solid is obtained, with a yield of 40.9%. 1 H NMR(300MHz,DMSO-d 6 ):δ=8.79(s,1H,Ar-H),2.75-2.73(t,1H,J=2.73Hz,CH),2.56-2.54(t,2H,J=7.38Hz,CH 2 ),2.19-2.14(m,2H,CH2 ), 1.66 - 1.61 (t, 2H, J = 7.56 Hz, CH 2 ), 1.45 - 1.35 (m, 2H, CH 2 ) ppm. MS (ASAP), [M + H] + calculated for C 13 H 14 N 5 O 3 S 3 + 384.47, found 384.56.

[0124] Example 10 Synthesis method of N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)hept-6-ylimide (LBJ-8) is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced by the raw material hept-6-ynoic acid, the feeding amount is 0.4 g, and finally 0.36 g of yellow solid is obtained, with a yield of 31%. 1 H NMR (300 MHz, DMSO-d 6 ): δ = 8.76 (s, 1H, Ar-H), 2.91 (s, 1H, CH), 2.51 - 2.49 (t, 2H, J = 3.41 Hz, CH 2 ), 2.28 - 2.26 (t, 2H, J = 2.78 Hz, CH 2 ), 1.56 - 1.52 (m, 2H, CH 2 ), 1.43 - 1.41 (m, 2H, CH 2 ) ppm. MS (ASAP), [M + H] + calculated for C 12 H 12 N 5 O 3 S 3 + 370.01, found 370.14.

[0125] Example 11 Synthesis method of N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)hex-5-ylimide (LBJ-9) is the same as that of LBJ-6, except that the raw material undec-10-ynoic acid is replaced by the raw material hex-5-ynoic acid, the feeding amount is 0.6 g, and finally 0.82 g of light yellow solid is obtained, with a yield of 43%. 1 H NMR (300 MHz, DMSO-d 6): δ = 8.79 (s, 1H, Ar-H), 2.82 (s, 1H, CH), 2.66 - 2.61 (t, 2H, J = 7.47 Hz, CH 2 ), 2.26 - 2.21 (m, 2H, CH 2 ), 1.84 - 1.75 (m, 2H, CH 2 ) ppm. MS(ASAP), [M + H] + calculated for C 11 H 10 N 5 O 3 S 3 + 355.99, found 356.02.

[0126] Example 12 Synthesis method of N-(5-((5-nitrothiazol-2-yl)thio)-1,3,4-thiadiazol-2-yl)pent-4-ylimide (LBJ-10) is the same as that of LBJ-6, the difference is that the raw material undec-10-ynoic acid is replaced by the raw material pent-4-ynoic acid, the feeding amount is 0.5 g, and finally 1.13 g of yellow solid is obtained, with a yield of 65%. 1 H NMR(300 MHz, DMSO-d 6 ): δ = 8.79 (s, 1H, Ar-H), 2.85 - 2.83 (t, 1H, J = 2.64 Hz, CH), 2.78 - 2.73 (t, 2H, J = 7.2 Hz, CH 2 ) ppm. MS(ASAP), [M + H] + calculated for C 10 H 8 N 5 O 3 S 3 + 341.98, found 341.86.

[0127] Example 13 Synthesis method of 5-azido-N-(2-(5-nitrothiazol-2-yl)thio)thiazol-5-yl)pentanamide (LBJ-11) is the same as that of LBJ-1, the difference is that the raw material 4-ethynylpiperidine hydrochloride is replaced by the raw material 5-azidopentanoic acid, the feeding amount is 0.1 g, and finally 0.11 g of off-white solid is obtained, with a yield of 42%. 1 H NMR(300 MHz, DMSO-d 6 ): δ = 8.73 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 2.32 - 2.30 (t, 2H, J = 2.48 Hz, CH 2 ), 1.77 - 1.75 (m, 2H, CH2 ), 1.56 - 1.53 (m, 2H, CH 2 ), 1.42 - 1.39 (m, 2H, CH 2 ) ppm. MS(ASAP), [M + H] + calculated for C 11 H 12 N 7 O 3 S 3 + 386.02, found 386.11.

[0128] Example 14 3-(Cycloprop - 2 - en - 1 - yl)-N-(5 - ((5 - nitrooxazol - 2 - yl)thio)-1,3,4 - thiadiazol - 2 - yl)propanamide (LBJ - 12)

[0129] The synthesis method is the same as that of LBJ - 6, except that the raw material undec - 10 - ynoic acid is replaced with the raw material 3-(cycloprop - 2 - en - 1 - yl)propanoic acid, the feeding amount is 0.4 g, and finally 0.46 g of light yellow solid is obtained, with a yield of 38%. 1 H NMR(300 MHz, DMSO - d 6 ): δ = 7.54 (s, 1H, Ar - H), 7.01 - 6.98 (m, 2H, CH), 2.35 - 2.34 (t, 2H, J = 2.67 Hz, CH 2 ), 1.84 - 1.82 (m, 2H, CH 2 ), 1.01 - 1.00 (m, 1H, CH) ppm. MS(ASAP), [M + H] + calculated for C 11 H 10 N 5 O 4 S 2 + 340.02 found 340.05.

[0130] Example 15 Enzymatic Inhibition Activity Test of PHGDH Small - Molecule Probes and Some of Their Precursors

[0131] This example provides the enzymatic inhibition activities of PHGDH small - molecule probes and some of their precursors. By coupling the dehydrogenation reaction of PHGDH with the reaction of myocardial xanthine oxidase catalyzing resazurin, the amount of the coupling reaction product is detected under the conditions of Ex 544 / Em 590 to indirectly determine the PHGDH enzyme inhibition activities of the probe molecules and some of their precursors.

[0132] Experimental method: Detect in an enzyme buffer solution (20 μM NAD +, 56.25 mM N 2 H 4 -H 2 SO 4 pH 9.0, 125 mM Tris-HCl pH 7.5, 2.5 mM EDTA, 0.0125% Tween 20), the compound was diluted with DMSO, then the protein was incubated with different concentrations of the compound in a 384-well plate for 30 min. Finally, substrate buffer (175 μM 3-PG, 25 μM resazurin, 5 μg / mL diaphorase, 56.25 mM N 2 H 4 -H 2 SO 4 pH 9.0, 125 mM Tris-HCl pH 7.5, 2.5 mM EDTA, 0.0125% Tween 20) was added to the reaction system. After incubating at room temperature for 2 h, the fluorescence signal value (Ex 544 / Em 590) was measured using a multi-functional microplate reader and the inhibition rate at different concentrations was calculated. Finally, the PHGDH inhibitory activity IC 50 value of the test compound was obtained by fitting with GraphPad Prism 8.0 software.

[0133] It can be seen from the enzyme inhibition activity data in Table 1 that the probe molecule and its partial precursors all have good affinity for the PHGDH protein.

[0134] Table 1 Enzyme inhibition activity IC of some probe molecules and their partial precursors of the present invention against PHGDH 50 Range

[0135] Number <![CDATA[PHGDHIC 50 (μM)]]> LBJ-1 +++ LBJ-2 +++ LBJ-3 +++ LBJ-4 +++ LBJ-5 +++ LBJ-6 +++ LBJ-7 +++ LBJ-8 +++ LBJ-9 +++ LBJ-10 +++ LBJ-11 ++ LBJ-12 ++ Probe precursor T-2 +++ Probe precursor T-5 +++

[0136] Note: "+++" indicates IC 50 <1 μM, indicating that the compound has extremely strong inhibitory activity against PHGDH and can effectively inhibit the activity of PHGDH at low concentrations. "++" indicates 1 μM ≤ IC 50 <5 μM, indicating that the compound has moderate inhibitory activity against PHGDH and can effectively inhibit PHGDH within a certain concentration range, but its inhibitory effect is slightly weaker than that of strong inhibitors. "+" indicates 5 μM ≤ IC 50 <10 μM, indicating that the compound has weak inhibitory activity against PHGDH and requires a higher concentration to exert a certain inhibitory effect.

[0137] Example 16 Discovery of the target protein of the compound by Gel-based ABPP method

[0138] This example provides the Coomassie brilliant blue staining experimental results of some probe molecules to show the target proteins bound by PHGDH inhibitors. Using the Gel-based Activity-Based Probe Labeling (Gel-based ABPP) method, first, the PHGDH in liver tissue proteins was saturated by pre-incubating the laboratory-synthesized PHGDH probe precursors T-2 and T-5. Then, probe molecules were added for competitive binding. Subsequently, a biotin tag was introduced onto the probe molecules through the click chemical reaction (CuAAC) of azide-biotin. Finally, the protein-probe-biotin complex was incubated with streptavidin beads to enrich the probe-labeled target proteins. The enriched complex was separated by SDS-PAGE gel electrophoresis, and the gel was stained with Coomassie brilliant blue to visualize the target proteins.

[0139] Experimental method: Homogenize the liver tissue sample to extract proteins, adjust to a suitable concentration after quantification, add the PHGDH probe precursors T-2 and T-5 to the protein solution, and incubate at 37 °C for 60 minutes. Add the probe molecules and continue to incubate at 37 °C for 30 minutes to complete the competitive binding. After incubation, add azide-biotin for the click reaction (CuAAC) and react for 30 minutes to bind the biotin label to the probe. Mix the protein-probe-biotin complex solution with pre-washed streptavidin beads, incubate at 4 °C for 60 minutes, and then wash with PBS to remove non-specific proteins. Add lysis buffer and heat to elute the proteins, perform SDS-PAGE gel electrophoresis separation, stain the gel with Coomassie brilliant blue for 30 minutes and decolorize, and observe and record the protein bands on the stained gel.

[0140] From Figure 1 the Coomassie result graph, it can be seen that compared with the experimental group without pre-incubating the PHGDH protein to saturation with the probe precursor, the amounts of some proteins, including PHGDH, in the bands with the pre-added probe precursor are significantly reduced. This indicates that the Gel-based ABPP method can display the target proteins of PHGDH inhibitors.

[0141] Example 17 Verification of the On-Target Activity of PHGDH Inhibitors by Immunofluorescence Co-Labeling

[0142] This embodiment provides a method for verifying the on-target property of an inhibitor using the probe molecule LBJ-5 through immunofluorescence co-labeling. By immunofluorescence method, the target protein is labeled with a specific antibody and fluorescence is shown in a fluorescence channel, and this fluorescence signal is used to indicate the spatial distribution and existence state of the target protein. The derivatized probe molecule LBJ-5 of the inhibitor contains an alkyne group, and the alkyne group can undergo a click chemical reaction (CuAAC) with an azide-labeled fluorescent molecule, and this reaction can introduce a fluorescent group, causing the target protein bound by the probe to emit fluorescence in another fluorescence channel. Observe the co-localization of the two fluorescence signals under a microscope. If the two fluorescence signals overlap or highly coincide at the same position, it indicates that the on-target property of the inhibitor is good, that is, the inhibitor has specifically bound to the target protein.

[0143] Experimental method: Select PHGDH and culture it to an appropriate density by a conventional method. Add different concentrations of the probe LBJ-5 into the cell culture medium and incubate at 37 °C for 1 hour. Fix the cells with 4% paraformaldehyde for 10 - 15 minutes to maintain the cell morphology and lock the protein position. After permeabilizing the cells with a 0.1% Triton X-100 or 0.1% Tween-20 solution for 5 - 10 minutes, incubate the cells with a solution containing a specific anti-PHGDH antibody for 1 - 2 hours to allow the antibody to bind to the PHGDH target protein. After washing, add a secondary antibody (antibody with a fluorescent label). Expose the cells to an azide-labeled fluorescent dye and an appropriate amount of click reaction catalyst (copper ions and stabilizer), and react at 37 °C for 30 minutes to allow the fluorescent molecule to bind to the LBJ-5 probe. Use a fluorescence microscope or a confocal microscope to sequentially capture the fluorescence signals of the two channels. Alexa Fluor 561 channel: Shows the position of the target protein labeled by the PHGDH antibody. Alexa Fluor 647 channel: Shows the distribution of the LBJ-5 probe in the cells.

[0144] Through Figure 2 the immunofluorescence co-labeling result diagram, it can be seen that the two fluorescence signals highly overlap at the same position, that is, the distribution of the probe molecule is basically the same as that of the target protein, and the on-target property of the inhibitor is good.

[0145] The above are only embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent transformations made using the content of the specification of the present invention, directly or indirectly applied in other related technical fields, are equally included in the patent protection scope of the present invention.

Claims

1. A type of PHGDH active molecular probe containing a thioether structure, characterized in that: It has a structure as shown in formula (I): in: R1 is selected from any one of a hydrogen atom, a nitro group, a cyano group, a carboxyl group or a trifluoromethyl group; Both fragment A and fragment B are substituted or unsubstituted aromatic rings or aromatic heterocycles, and the substituents are selected from any one of monosubstituted or polysubstituted alkyl, alkoxy, nitro, halogen, trifluoromethyl, carbonyl, benzoylpiperidine, benzamide group, amide, and phenyl ketone; fragment A and fragment B are the same or different; Linker is a linking molecule with adjustable length, which is a flexible PEG chain, a saturated carbon chain or an unsaturated carbon chain; C represents a reporter group that can be used to identify protein targets, which is selected from any one of a fluorescent group, biotin, an azide and an alkyne group that can undergo a bioorthogonal Click reaction, and a strained olefin and a tetrazine that can undergo a reverse electron demand Diels-Alder reaction.

2. The PHGDH active molecular probe containing a thioether structure according to claim 1, characterized in that: In formula (I), R1 is any one of nitro, cyano, and trifluoromethyl; A is selected from any one of furan, pyrazole, thiazole, thiadiazole or oxazole; B is selected from any one of thiazolylphenyl ketone, thiazolylbenzoylpiperidine, thiazolylbenzamide, thiadiazolylamide, and thiazolylamide; Linker is selected from a saturated carbon chain or an unsaturated carbon chain; C is selected from azides and alkynes that can undergo bioorthogonal Click reactions, and strained olefins and tetrazines that can undergo reverse electron demand Diels-Alder reactions.

3. The PHGDH active molecular probe containing a thioether structure according to claim 1, characterized in that: In formula (I), R1 is nitro; A is thiazolyl or oxazolyl; B is selected from: any one of thiazolylphenyl ketone, thiazolylbenzamide, thiadiazolylamide, and thiazolylamide; Linker is selected from saturated carbon chains; C is selected from any one of azides and alkynes that can undergo bioorthogonal Click reactions, and strained olefins that can undergo reverse electron demand Diels-Alder reactions.

4. The targeted PHGDH active molecular probe containing a thioether structure according to claim 1, characterized in that: The molecular probe is selected from any one of the following compounds: 。 5. The method for preparing a molecular probe targeting PHGDH activity according to any one of claims 1 to 4, characterized in that: The following steps are involved: The method comprises the following steps: 1) Compound I-3 is prepared from Compound I-1 and Compound I-2 by nucleophilic substitution reaction and removal of one molecule of hydrogen bromide; 2) In the presence of a condensing agent and a base, compound I-3 reacts with compound Linker-C via an amide condensation reaction to prepare compound I-4.

6. The preparation method according to claim 5, characterized in that: In the step 1), the molar ratio of compound I-1 to compound I-2 is 0.8 to 1:2; In the step 2), the molar ratio of compound I-3 to compound Linker-C is 0.8 to 1:

2.

7. The preparation method according to claim 5, characterized in that: In the step 1), the temperature of the substitution reaction is room temperature and the reaction time is 0 to 6 hours; In the step 2), the temperature of the amide condensation reaction is room temperature, and the reaction time is 2 to 8 hours.

8. The preparation method according to claim 5, characterized in that: In the step 2), the condensing agent for the amide condensation reaction is one or more of HOAT, HOBT, HATU, HBTU or BOP; the base for the amide condensation reaction is one or both of N,N-diisopropylethylamine DIPEA or triethylamine TEA; the organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide.

9. Use of the PHGDH activity-targeted molecular probe according to any one of claims 1 to 4 in identifying the interaction between a targeted PHGDH inhibitor and a PHGDH protein.

10. The use according to claim 9, characterized in that: The targeted PHGDH active molecular probe labels the PHGDH target protein through a Click bioorthogonal reaction.

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