Dipeptidyl peptidase 4 biosensors, methods of making and using the same

Abstract

The application discloses a kind of formula (I) shown for dipeptidyl peptidase 4 detection bioluminescence probe and its preparation method and application.The probe has good selectivity and sensitivity to dipeptidyl peptidase 4, and can be used as dipeptidyl peptidase 4 bioluminescence probe for marking prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, liver cancer and a variety of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, and specifically discloses a type of bioluminescent probe for the detection of dipeptidyl peptidase 4, its preparation method and application. Background Technology

[0002] Dipeptidyl peptidase 4 (DPP4) is a transmembrane glycoprotein with serine carboxypeptidase function, capable of separating an N-terminal dipeptide from the penultimate proline or alanine residue of a polypeptide. DPP4 can degrade various biological polypeptides, such as glucagon-like 1 and glucose-dependent insulinotropic peptides. Therefore, it plays an important role in glucose metabolism. DPP4 participates in processes such as immune regulation, cell migration, cell adhesion, and apoptosis, and abnormalities in its expression or function are closely related to the occurrence and development of various diseases, including tumors. Studies have shown that DPP4 is overexpressed in various tumor cells, including prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, and liver cancer. Therefore, DPP4 can serve as a biomarker for various cancers and is of great significance for the early diagnosis of cancer.

[0003] Currently, there are many methods for detecting dipeptidyl peptidase 4, such as enzyme-linked immunosorbent assay (ELISA), fluorescence assay, and gel electrophoresis. However, among these analytical methods, ELISA has relatively low detection accuracy; fluorescence assays suffer from significant drawbacks such as photobleaching and autofluorescence due to their dependence on the excitation light source, which limits their further application; and gel electrophoresis has excessively long preparation and processing times, further restricting its application. Therefore, designing and preparing a simpler, faster, more efficient, and more sensitive quantitative detection method for dipeptidyl peptidase 4 remains a hot research topic.

[0004] Bioluminescence is a special type of chemiluminescence that exists within living organisms. It does not rely on the absorption of external light energy by the organism, but rather is a process in which luciferin-like substances within the organism efficiently convert chemical energy into light energy under the catalysis of enzymes. Bioluminescence imaging technology has attracted widespread attention due to its advantages such as non-invasiveness, low background, high sensitivity, visualization, and the ability to achieve real-time dynamic observation, and has gradually developed into an important in vivo imaging method. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a compound represented by formula (I):

[0006]

[0007] Where X is selected from O or NR6; Y is selected from S or NR7; Z is selected from S, Se, or Te; R1 is selected from O or NR8; R6, R7, and R8 may be the same or different, and are independently selected from H or C. 1-12 alkyl;

[0008] R2, R3, R4, and R5 may be the same or different, and are independently selected from H, CN, NH2, NO2, OH, COOH, F, Cl, Br, and I. They may be unsubstituted or optionally substituted by one, two, or more of the following groups: C 1-12 Alkyl, C 1-12 Alkoxy, C 1-12 Alkylthio, -CO-C 1-12 Alkyl, -CONH-C 1-12 Alkyl, -COOC 1-12 Alkyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic groups, C 6-20 Aryl, 5-20 heteroaryl;

[0009] Rs is selected from CN, NO2, OH, COOH, C 1-12 Alkyl, C 3-20 cycloalkyl, C 1-12 Alkoxy, C 1-12 Alkylthio, amino, halogen, halogenated C 1-12 Alkyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic groups, C 6-20 Aryl, 5-20 heteroaryl.

[0010] According to an embodiment of the present invention, X is selected from O or NR6; Y is selected from S or NR7; Z is selected from S, Se, or Te; R1 is selected from O or NR8; R6, R7, and R8 may be the same or different, and are independently selected from H or C. 1-6 alkyl;

[0011] R2, R3, R4, and R5 may be the same or different, and are independently selected from H, CN, NH2, NO2, OH, COOH, F, Cl, Br, I, unsubstituted, or optionally substituted by one, two, or more Rs, of the following groups: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, -CO-C 1-6 Alkyl, -CO-NH-C 1-6 Alkyl, -COOC 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-12 Aryl, 5-12 heteroaryl;

[0012] Rs is selected from F, Cl, Br, I, CN, NH2, NO2, OH, COOH, C 1-6 Alkyl, C 3-12 cycloalkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, halogenated C 1-6Alkyl, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl, 5-12 heteroaryl.

[0013] According to a preferred embodiment of the present invention, R2, R3, R4, and R5 may be the same or different, and are independently selected from H and C. 1-3 Alkyl, C 1-3 Alkyl groups; for example, R2, R3, R4, and R5 are independently selected from the following groups: H, CH3-, CH3O-;

[0014] R1 is selected from NH;

[0015] X is selected from O;

[0016] Y and Z are selected from S.

[0017] As an example, the compound of formula (I) is selected from the following:

[0018]

[0019] The present invention also provides a method for preparing the compound of formula (I) above, comprising the following steps:

[0020]

[0021] Among them, R1, R2, R3, R4, R5, X, Y, and Z have the definitions described above;

[0022] (a) Compound (II) was subjected to a deamination protecting group boc reaction to give compound (I).

[0023] According to an embodiment of the present invention, in step (a), the deamination protecting group is trifluoroacetic acid.

[0024] According to an embodiment of the present invention, in step (a), the reaction is carried out in a solvent environment, wherein the solvent is dichloromethane.

[0025] According to an embodiment of the present invention, the preparation method may be carried out by the following steps: dissolving compound (II) in a solvent (e.g., dichloromethane), adding trifluoroacetic acid, and reacting to obtain compound (I).

[0026] According to an embodiment of the present invention, the compound of formula (I) obtained from the above reaction can be separated and purified by adjusting the acidity or alkalinity. For example, the acidity or alkalinity can be adjusted using an aqueous solution of sodium bicarbonate, and after filtration of the precipitated solid, optional recrystallization (e.g., recrystallization using methanol) can yield a pure compound of formula (I).

[0027] According to an embodiment of the present invention, the preparation method further includes a preparation process for compound (II), which can be prepared by the following method, including:

[0028]

[0029] Among them, R1, R2, R3, R4, R5, X, Y, and Z have the definitions described above;

[0030] (b) Compound (V) and compound (B) are reacted to obtain compound (IV);

[0031] (c) The compound (IV) obtained in step (b) is reacted with p-toluenesulfonyl chloride and compound (A) to give compound (III);

[0032] (d) The compound (III) obtained in step (c) is reacted with D-cysteine ​​hydrochloride to obtain compound (II).

[0033] According to an embodiment of the present invention, in step (b), the reaction is carried out in a solvent, such as dichloromethane.

[0034] According to an embodiment of the present invention, in step (b), the reaction temperature is 10-40°C, for example, room temperature.

[0035] According to an embodiment of the present invention, in step (b), the molar ratio of compound (V) to compound (B) is 1:(1-5), preferably 1:1.1.

[0036] According to an embodiment of the present invention, in step (c), the reaction is carried out in a solvent, for example, the reaction of compound (IV) and p-toluenesulfonyl chloride is carried out in dichloromethane, and the solvent used for the reaction of the resulting intermediate with compound (A) may be acetonitrile.

[0037] According to an embodiment of the present invention, in step (c), the reaction temperature of compound (IV) and p-toluenesulfonyl chloride is 10-40°C, for example, room temperature; the reaction temperature of the resulting intermediate with compound (A) is 60-100°C, for example, 80°C.

[0038] According to an embodiment of the present invention, in step (c), the molar ratio of compound (IV), p-toluenesulfonyl chloride to compound (A) can be 1:(1-5):(1-5), preferably 1:2.2:0.7.

[0039] According to an embodiment of the present invention, in step (d), the reaction is carried out in a solvent, which may be a mixture of methanol, dichloromethane and deionized water.

[0040] According to an embodiment of the present invention, in step (d), the reaction is carried out in the presence of a catalyst, such as potassium carbonate.

[0041] According to an embodiment of the present invention, in step (d), the reaction temperature is 10-40°C, for example, room temperature.

[0042] According to an embodiment of the present invention, in step (d), the molar ratio of compound (III) to D-cysteine ​​hydrochloride is 1:(1-5), preferably 1:1.1.

[0043] The present invention further provides the use of the compound of formula (I) above as a bioluminescent probe for dipeptidyl peptidase 4.

[0044] According to an embodiment of the present invention, the detection of dipeptidyl peptidase 4 by the compound of formula (I) can be performed in a variety of buffer solutions, such as PBS buffer solution, Tris-HCl buffer solution, etc.

[0045] According to an embodiment of the present invention, the detection is performed in the presence of ATP, luciferase, and magnesium ions.

[0046] According to an embodiment of the present invention, the detection is performed in an oxygen-containing environment.

[0047] According to an embodiment of the present invention, the detection can be used for the detection of dipeptidyl peptidase 4 in vitro or in vivo.

[0048] According to an embodiment of the present invention, the detection is for non-disease diagnosis or treatment purposes.

[0049] According to embodiments of the present invention, the dipeptidyl peptidase 4 may be located in prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, and liver cancer.

[0050] The present invention also provides the use of the compound of formula (I) as an intracellular dipeptidyl peptidase 4 imaging agent.

[0051] The present invention also provides the use of the compound of formula (I) for qualitative and quantitative detection of dipeptidyl peptidase 4 in cancer cells and normal somatic cells.

[0052] According to an embodiment of the present invention, the cancer cells are prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, and liver cancer cells.

[0053] The present invention also provides the use of the compound of formula (I) for imaging dipeptidyl peptidase 4 in mice.

[0054] The present invention also provides the use of the compound of formula (I) in the preparation of a kit for detecting dipeptidyl peptidase 4.

[0055] Beneficial effects

[0056] The compound of formula (I) of this invention has fewer synthesis steps, shorter reaction time, convenient purification, and simple process.

[0057] This invention involves chemically modifying D-luciferin-like substances to introduce a glycine-L-proline group with specificity for dipeptidyl peptidase 4 (DPP4) reactions, thereby obtaining a class of bioluminescent probes (compounds of formula (I)) that can be used for dipeptidyl peptidase 4 detection. In D-luciferin-like substances, when the 6' position of compound (I) is hydroxyl / amino and the 4' position is carboxylic acid, it can react with luciferase, ATP, and Mg... 2+ Biological luminescence occurs in the presence of [a specific substance / organism]. The compound of formula (I) of this invention uses a D-luciferin-like substance as its parent material, linking a group capable of recognizing dipeptidyl peptidase 4 to the 6' hydroxyl / amino group of D-luciferin. Therefore, the compound of formula (I) of this invention, as a substrate, cannot be recognized by luciferase and does not possess luminescence ability. When the compound of formula (I) encounters dipeptidyl peptidase 4, the recognition group in the compound reacts with dipeptidyl peptidase 4, subsequently causing the linker to break (the recognition group in formula (I) is a reactive probe, which breaks at the amide site upon encountering dipeptidyl peptidase 4, and then the spontaneously breaking linker detaches due to electron transfer, exposing the hydroxyl / amino group in formula (I)), thereby releasing the D-luciferin-like substance, which then reacts with ATP and Mg [a specific substance / organism]. 2+ Bioluminescence occurs under the action of luciferase (see appendix for details on the luminescence principle). Figure 1 This probe exhibits excellent selectivity and sensitivity for dipeptidyl peptidase 4 and can be used as a bioluminescent probe for dipeptidyl peptidase 4 to label various tumor cells, including prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, and liver cancer.

[0058] The compound of formula (I) of this invention is specific for the detection of dipeptidyl peptidase 4, while other anions, cations, amino acids and in vivo enzymes have no effect on the luminescence of the compound of this invention.

[0059] Terminology Definitions and Explanations

[0060] Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. It should be understood that the foregoing summary and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of this application. In this application, unless otherwise stated, the terms “comprising” and other forms such as “including,” “containing,” and “comprising” are not restrictive.

[0061] Term "C" 1-12 "alkyl" should be understood to refer to a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 12 carbon atoms, preferably C12. 1-6 Alkyl group. "C" 1-6"alkyl" should be understood to preferably represent a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. In particular, the group has 1, 2, 3, 4, 5, or 6 carbon atoms ("C"). 1-6 Alkyl groups, such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, and more particularly, the groups having 1, 2, or 3 carbon atoms (“C”). 1-3 Alkyl), such as methyl, ethyl, n-propyl or isopropyl.

[0062] Term "C" 3-20 "Cycloalkyl" should be understood to refer to a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C". 3-10 cycloalkyl. The term "C" 3-10 "Cycloalkyl" should be understood to refer to a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The C... 3-10 Cycloalkyl groups can be monocyclic hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl, or bicyclic hydrocarbon groups such as decahydronaphthalene ring.

[0063] The term "3-20 membered heterocyclic group" refers to a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5 heteroatoms independently selected from N, O, and S, preferably a "3-10 membered heterocyclic group". The term "3-10 membered heterocyclic group" refers to a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5, preferably 1-3, heteroatoms selected from N, O, and S. The heterocyclic group can be connected to the rest of the molecule via any one of the carbon atoms or a nitrogen atom (if present). Specifically, the heterocyclic group can include, but is not limited to: 4-membered rings, such as azirrobutyl or oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, or trithiaalkyl; or 7-membered rings, such as diazacycloheptyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclic group may be bicyclic, for example, but not limited to, a 5,5-membered ring, such as a hexahydrocyclopentano[c]pyrrole-2(1H)-yl ring, or a 5,6-membered bicyclic ring, such as a hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring. The nitrogen-containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, for example, but not limited to, 2,5-dihydro-1H-pyrrole, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or it may be benzo-fused, for example, but not limited to, dihydroisoquinolinyl. According to the invention, the heterocyclic group is non-aromatic.

[0064] Term "C" 6-20 "Aryl" should be understood as representing a monocyclic, bicyclic, or tricyclic hydrocarbon ring with 6 to 20 carbon atoms that is monovalent and partially aromatic, preferably "C". 6-14 Aryl. The term "C" 6-14 "Aryl" should be understood to preferably represent a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring ("C") having 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. 6-14 Aryl), particularly a ring with 6 carbon atoms (“C6 aryl”), such as phenyl; or biphenyl, or a ring with 9 carbon atoms (“C9 aryl”), such as indenyl or indenyl, or a ring with 10 carbon atoms (“C9 aryl”). 10 Aryl groups, such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl, or rings with 13 carbon atoms (“C”). 13 Aryl groups, such as fluorene groups, or rings with 14 carbon atoms (“C”). 14 Aryl), for example, anthracene.

[0065] The term "5-20-membered heteroaryl" should be understood to include monovalent monocyclic, bicyclic, or tricyclic aromatic ring systems having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O, and S, such as "5-14-membered heteroaryl". The term "5-14-membered heteroaryl" should also be understood to include monovalent monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 5, 6, 9, or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3, heteroatoms independently selected from N, O, and S, and in each case, may be benzofused. Specifically, the heteroaryl group is selected from thienyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazoleyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl, and their benzo[derivatives], such as benzofuranyl, benzothienyl, benzooxazolyl, benzoisooxazolyl, benzoimidazolyl, benzotriazolyl, indazole, indolyl, isindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and their benzo[derivatives], such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or acrylinyl, inazinyl, purinyl, and their benzo[derivatives]; or terpenolyl, phthalazinyl, quinazolinyl, quinoxolinyl, naphridinyl, pteridinyl, carbazolyl, acridineyl, phenazinyl, phenothiazinyl, phenothiazinyl, etc.

[0066] Unless otherwise stated, heterocyclic, heteroaryl, or heteroaryl groups include all their possible isomers, such as their positional isomers. Thus, for some illustrative, non-limiting examples, pyridyl or pyridylene includes pyridin-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-3-yl, pyridin-4-yl, and pyridin-4-yl; thiophene.

[0067] The above refers to the term "C" 1-12 The definition of "alkyl" also applies to compounds containing "C". 1-12 Other terms for "alkyl", such as the term "C 1-12 Alkoxy, halogenated C 1-12 Alkyl group, -CO-C 1-12 Alkyl group, -CONH-C 1-12 Alkyl group, -COOC 1-12 Alkyl groups, etc. Attached Figure Description

[0068] Figure 1 This is a diagram illustrating the reaction mechanism of DPP4-Luc prepared in Example 1 for detecting dipeptidyl peptidase 4.

[0069] Figure 2 Bioluminescence intensity of DPP4-Luc prepared in Example 1 in PBS buffer solution as a function of dipeptidyl peptidase 4 concentration;

[0070] Figure 3 The image shows the bioluminescence intensity of DPP4-Luc prepared in Example 1 for detecting intracellular dipeptidyl peptidase 4. Detailed Implementation

[0071] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0072] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0073] Preparation Example 1

[0074] Preparation of compound (1)

[0075]

[0076] Under nitrogen protection, N-Boc-glycine-L-proline (0.75 mmol, 0.21 g), p-aminobenzyl alcohol (0.82 mmol, 0.1 g), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (0.75 mmol, 0.14 g) were dissolved in 10 mL of dichloromethane and reacted at room temperature for 3–4 h. After the reaction was complete, the organic phase was evaporated to dryness, and the crude product was passed through a silica gel column using ethyl acetate / petroleum ether (v / v = 1:1) as the developing solvent to give a yellow solid (0.18 g, 64%). ESI (C 19 H 27 O5N3):[M+Na + =400.1. 1 H-NMR (300MHz, DMSO-d6), δ: 7.54 (d, 2H), 7.30 (d, 2H), 4.55 (s, 3H), 3.91 (m, 2H), 3.63 (m, 2H), 2.10 (m, 4H), 1.44 (s, 9H).

[0077] Preparation Example 2

[0078] Preparation of compound (2)

[0079]

[0080] Under nitrogen protection, compound (1) (0.45 mmol, 0.17 g) and p-toluenesulfonyl chloride (1 mmol, 0.19 g) were dissolved in 4 mL of dichloromethane. Triethylamine (0.5 mmol, 0.07 mL) was added with stirring, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the organic phase was evaporated to dryness to give a white solid intermediate. Under nitrogen protection, the white solid intermediate (0.3 mmol, 0.12 g), 2-cyano-6-hydroxybenzothiazole (0.3 mmol, 0.06 g), potassium carbonate (1.4 mmol, 0.2 g), and sodium iodide (0.1 mmol, 0.02 g) were dissolved in acetonitrile (5 mL). The reaction system was stirred vigorously at 80 °C for 5 h. After the reaction was complete, the temperature was reduced to room temperature, the reaction solution was filtered, the filtrate was extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The crude product was passed through a silica gel column using ethyl acetate / petroleum ether (v / v = 6:1) as the developing solvent to give a white solid (0.12 g, 62%). ESI (C 27 H 29 O5N5S):[M+H + =536.1. 1 H-NMR(300MHz, CDCl3-d6), δ:9.41(s,1H),8.08(d,1H),7.58(s,1H),7.56(s,1H),7.34(m,5H),5.11(s,2H), 4.78(d,1H),3.97(d,2H),3.56(t,1H),3.42(dd,1H),2.59(m,1H),2.17(m,2H),1.86(ddd,1H),1.45(s,9H).

[0081] Preparation Example 3

[0082] Preparation of compound (3)

[0083]

[0084] Under nitrogen protection, compound (2) (0.37 mmol, 0.19 g) was dissolved in 4 mL of a mixed solvent of methanol and dichloromethane (v / v = 1:1). A solution of D-cysteine ​​hydrochloride (0.4 mmol, 0.07 g) and potassium carbonate (0.4 mmol, 0.06 g) dissolved in 4 mL of a mixed solvent of methanol and deionized water (v / v = 1:1) was added. The reaction was carried out at room temperature for 2 h. After the reaction was complete, the organic solvent was evaporated to dryness, an equal volume of water was added, and the pH was adjusted to approximately 2-3 with 1 M hydrochloric acid solution. The crude product obtained was a yellow solid. Recrystallization of the crude product from methanol yielded a pale yellow product (0.18 g, 80%).

[0085] ESI(C 30 H33 N5O7S2[M+H] + =640.1. 1 H NMR(300MHz,DMSO-d6),δ:10.02(s,1H),8.05(d,1H),7.63(m,2H),7.43(t,2H),7.26(dd,1H),6.82 (t,1H),5.14(s,2H),4.43(d,1H),3.80(d,1H),3.71(m,3H),3.53(m,3H),2.01(m,4H),1.36(d,9H).

[0086] Example 1

[0087] Preparation of compound (4): DPP4-Luc

[0088]

[0089] Under nitrogen protection, compound (3) (1.3 mmol, 0.83 g) was dissolved in 15 mL of dichloromethane, cooled in an ice-water bath, and 9 mL of trifluoroacetic acid was added dropwise. After returning to room temperature, the reaction was carried out at room temperature for 12 h. After the reaction was complete, the organic solvent was evaporated to dryness, an equal volume of water was added, and the pH was adjusted to approximately 7 with sodium bicarbonate solution. The crude product obtained was a yellow solid. Recrystallization of the crude product from methanol yielded a pale yellow product (0.38 g, 55%). ESI (C 25 H 25 N5O5S2):[M+H + =540.1. 1 H NMR(300MHz,DMSO-d6),δ:10.16(s,1H),8.03(d,1H),7.81(d,1H),7.64(d,2H),7.45(d,2H) ,7.22(d,1H),5.12(s,2H),4.47(s,1H),3.79(s,1H),3.68(s,3H),3.65(d,3H),1.97(m,4H).

[0090] Example 2

[0091] Compound (4) (DPP4-Luc) obtained in Example 1 was tested against different concentrations of DPP4 using the following method: DPP4-Luc was prepared into a 120 μM solution using 10 mmol PBS buffer (containing 10 mM MgSO4), and DPP4 was prepared into solutions with concentrations of 2.4 μg / mL, 5.4 μg / mL, 16.2 μg / mL, 27 μg / mL, and 36 μg / mL. Then, 25 μL of the DPP4-Luc solution (compound 4) and 25 μL of the above-mentioned solutions of different concentrations of DPP4 were added to each well of a black 96-well plate. The plate was incubated at 37°C for 60 min. Then, 50 μL of ATP (2 mM) and 50 μL of luciferase solution (60 μg / mL) were added to each well. The bioluminescence intensity of the 96-well plate was measured using a microplate reader. Finally, the bioluminescence spectra of DPP4-Luc at different DPP4 concentrations (0, 0.4 μg / mL, 0.9 μg / mL, 2.7 μg / mL, 4.5 μg / mL, and 6 μg / mL) were measured (see [link to data]). Figure 2 , Figure 2 The three black dots indicate that each data set was measured three times. **** represents the P-value (t-test), ****P<0.0001, and the I-shaped bars represent error bars. Figure 2 As can be seen, when DPP4 is absent in the solution, the luminescence of DPP4-Luc is negligible, while when DPP4 is present, the bioluminescence intensity of the solution gradually increases with the increase of DPP4 concentration. Therefore, the compound DPP4-Luc of this invention can be used for the qualitative and quantitative detection of DPP4.

[0092] Furthermore, DPP4 is overexpressed in various tumor cells, including prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, and liver cancer. Therefore, the compound DPP4-Luc of this invention can be used as a marker for the detection of these cancers.

[0093] Example 3

[0094] The DPP4-Luc obtained in Example 1 was used for cell imaging. The testing method is as follows: This experiment was divided into two groups. One group consisted of liver cancer cells (HepG2) with overexpression of DPP4 enzyme. This group was used as the experimental group and different concentrations of probe solution (DPP4-Luc) were directly added. Figure 3 The leftmost bar chart for each group); the second group, as the control group, used human small cell lung cancer cells (H466) with a small amount of DPP4 enzyme expression, and different concentrations of probe solution (DPP4-Luc) were added. Figure 3 (See the rightmost bar chart for each group). After incubating the cells and probe solution for 10 minutes, the bioluminescence intensity was measured using a microplate reader (see...). Figure 3 ).Depend on Figure 3It is evident that DPP4-Luc in the first group of liver cancer cells with overexpression of the DPP4 enzyme can emit light, and the luminescence intensity is positively correlated with the DPP4 concentration; while in the second group of human small cell lung cancer cells with low expression of the enzyme, DPP4-Luc is almost non-luminescent. Therefore, the DPP4-Luc of this invention exhibits excellent selectivity and sensitivity for detecting DPP4.

[0095] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The compound represented by formula (I): （I） in, X is selected from O or NH; Y is selected from S; Z is selected from S, Se, or Te; R1 is selected from NH; R2, R3, R4, and R5 may be the same or different, and are independently selected from H and C. 1-3 Alkyl or C 1-3 Alkyl group.

2. The compound according to claim 1, wherein, R2, R3, R4, and R5 are independently selected from the following groups: H, CH3-, or CH3O-; Z is selected from S.

3. The compound according to claim 1 or 2, wherein, The compound is selected from the following compounds:

4. A method for preparing the compound according to any one of claims 1-3, wherein, Includes the following steps: Wherein, R1, R2, R3, R4, R5, X, Y and Z have the definitions described in any one of claims 1-3; (a) Compound (II) was subjected to a deamination and protecting group boc reaction to obtain the compound shown in formula (I).

5. Use of the compound according to any one of claims 1-3 as a dipeptidyl peptidase 4 bioluminescent probe for non-disease diagnostic or therapeutic purposes.

6. The use according to claim 5, wherein, The detection of the compound for dipeptidyl peptidase 4 was performed in PBS buffer or Tris-HCl buffer.

7. The use according to claim 6, wherein, The detection was performed in the presence of ATP, luciferase, and magnesium ions.

8. The use according to claim 6, wherein, The test was conducted in an oxygen-containing environment.

9. The use according to claim 5, wherein, The dipeptidyl peptidase 4 is found in prostate cancer, gastric cancer, colorectal cancer, malignant mesothelioma, or liver cancer.

10. Use of the compound according to any one of claims 1-3 as an intracellular dipeptidyl peptidase 4 imaging agent for non-disease diagnostic or therapeutic purposes.

11. Use of the compound according to any one of claims 1-3 for qualitative and quantitative detection of dipeptidyl peptidase 4 content in cancer cells and normal somatic cells for non-disease diagnostic or therapeutic purposes.

12. The use according to claim 11, wherein, The cancer cells are prostate cancer, stomach cancer, colorectal cancer, malignant mesothelioma, or liver cancer cells.

13. Use of the compound of any one of claims 1-3 for dipeptidyl peptidase 4 imaging in mice for non-disease diagnostic or therapeutic purposes.

14. Use of the compound of any one of claims 1-3 in the preparation of a kit for detecting dipeptidyl peptidase 4.

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