Quinone oxidoreductase biological probe and its preparation method and application

Abstract

The present invention discloses a bioluminescent probe for quinone oxidoreductase detection, represented by formula (I), as well as its preparation method and application. This probe exhibits excellent selectivity and sensitivity for quinone oxidoreductase and can be used as a bioluminescent probe for quinone oxidoreductase to label various tumor cells, including lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer, and liver cancer. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically discloses a type of bioluminescent probe for quinone oxidoreductase detection, a preparation method and an application thereof. Background Art

[0002] Quinone oxidoreductase (NQO1) is a flavoenzyme that regulates redox potential. It is widely distributed throughout organs, with highest levels in the liver, kidneys, and gastrointestinal tract. Quinone oxidoreductase catalyzes the reduction of quinone compounds to produce the corresponding hydroquinone compounds, with electrons provided by NADH or NAD(P)H. This process avoids the formation of the metabolic intermediate semiquinone, thereby reducing the harmful effects of quinone compounds, which are carcinogenic and teratogenic. Studies have shown that quinone oxidoreductase is overexpressed in various tumor cells, including lung cancer, colon cancer, breast cancer, bile duct tumors, melanomas, pancreatic cancer, and liver cancer, with levels 5-200 times that of normal cells. Therefore, quinone oxidoreductase can be used 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 quinone oxidoreductase, including colorimetry, fluorescence, high-performance liquid chromatography, and nuclear magnetic resonance. However, among these analytical methods, colorimetry has low accuracy; HPLC requires large amounts of organic solvents, which pollutes the environment, is expensive, and makes trace detection difficult; and fluorescence, due to its dependence on the excitation light source, has significant drawbacks such as photobleaching and autofluorescence, which also limit its further application. Therefore, the design and development of simpler, faster, more efficient, and more sensitive quantitative detection methods for quinone oxidoreductase remains a hot topic.

[0004] Bioluminescence is a special type of chemiluminescence found in living organisms. It is not dependent on the organism's absorption of external light energy, but rather on the efficient conversion of chemical energy into light energy by luciferin-like substances in the body, catalyzed by enzymes. Bioluminescence imaging technology has garnered widespread attention due to its advantages, including non-invasiveness, low background, high sensitivity, visualization, and ability to achieve real-time dynamic observations. It has gradually developed into a key in vivo imaging method. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a compound represented by the following formula (I):

[0006]

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

[0008] R1, R2, R3, R4, R5 are 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: 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 group, C 6-20 Aryl, 5-20 membered 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, halo 1-12 Alkyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered 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; R6, R7 are the same or different and are independently selected from H or C 1-6 alkyl;

[0011] R1, R2, R3, R4, R5 are the same or different and are independently selected from H, CN, NH2, NO2, OH, COOH, F, Cl, Br, I, unsubstituted, or the following groups optionally substituted by one, two or more Rs: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, -CO-C 1-6 Alkyl, -CO-NH-C 1-6 Alkyl, -COOC 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-12 Aryl, 5-12 membered 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 Alkylthio, halo C 1-6 Alkyl, C3-12 Cycloalkyl, 3-12 membered heterocyclic group, C 6-12 Aryl, 5-12 membered heteroaryl.

[0013] According to a preferred embodiment of the present invention, R1, R2, R3, R4, and R5 are the same or different and are independently selected from H, C 1-3 Alkyl, C 1-3 Alkoxy; for example, R1, R2, R3, R4, R5 are independently selected from the following groups: H, CH3-, CH3O-;

[0014] X is selected from O or NH;

[0015] Y and Z are selected from S.

[0016] As an example, the compound of formula (I) is selected from the following:

[0017]

[0018] The present invention also provides a method for preparing the compound of formula (I), comprising the following steps:

[0019]

[0020] wherein R1, R2, R3, R4, R5, X, Y, and Z have the definitions given above;

[0021] (a) Compound (II) is reacted with D-cysteine hydrochloride to obtain compound (I).

[0022] According to an embodiment of the present invention, in step (a), the reaction is carried out in a solvent environment, and the solvent is a mixed solvent of methanol, dichloromethane and deionized water.

[0023] According to an embodiment of the present invention, in step (a), the reaction is carried out in the presence of a catalyst, which may be, for example, potassium carbonate.

[0024] According to an embodiment of the present invention, in step (a), the molar ratio of compound (II) to D-cysteine hydrochloride is 1:(1-5), preferably 1:1.2.

[0025] According to an embodiment of the present invention, the preparation method can adopt the following steps: dissolving compound (II) in a solvent (for example, a mixed solvent of methanol and dichloromethane), adding D-cysteine hydrochloride and potassium carbonate solution dissolved in a mixed solvent of methanol and deionized water, and reacting to obtain compound (I).

[0026] According to an embodiment of the present invention, the compound of formula (I) obtained by the above reaction can be separated and purified by recrystallization. For example, the crude product can be recrystallized using a mixed solvent of dichloromethane and methanol, and then filtered to obtain a pure compound of formula (I).

[0027] According to an embodiment of the present invention, the preparation method further includes the preparation of the compound of formula (II), and the compound of formula (II) can be prepared by the following method, comprising:

[0028]

[0029] wherein R1, R2, R3, R4, R5, X, and Y have the definitions given above;

[0030] (b) reacting compound (V) with 3,3-dimethylacrylic acid to obtain compound (IV);

[0031] (c) reacting the compound (IV) obtained in step (b) with N-bromosuccinimide (NBS) to obtain compound (III);

[0032] (d) reacting the compound (III) obtained in step (c) with the compound (A) to obtain the compound (II).

[0033] According to an embodiment of the present invention, in step (b), the reaction is carried out in a solvent, and the solvent may be, for example, methanesulfonic acid.

[0034] According to an embodiment of the present invention, in step (b), the reaction temperature is 60-100°C, for example 85°C.

[0035] According to an embodiment of the present invention, in step (b), the molar ratio of compound (V) to 3,3-dimethylacrylic acid is 1:(1-5), preferably 1:1.2.

[0036] According to an embodiment of the present invention, in step (c), the reaction is carried out in a solvent, which may be acetonitrile.

[0037] According to an embodiment of the present invention, in step (c), the reaction temperature is 10-40°C, such as room temperature.

[0038] According to an embodiment of the present invention, in step (c), the molar ratio of compound (IV) to N-bromosuccinimide (NBS) may be 1:(1-5), preferably 1:1.5.

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

[0040] According to an embodiment of the present invention, in step (d), the reaction is carried out in the presence of a catalyst, and the catalyst may be, for example, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and 4-lutidine (DMAP).

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

[0042] According to an embodiment of the present invention, in step (d), the molar ratio of compound (III) to compound (A) is 1:(1-5), preferably 1:1.2.

[0043] The present invention further provides the use of the compound of formula (I) as a quinone oxidoreductase bioluminescent probe.

[0044] According to an embodiment of the present invention, the detection of quinone oxidoreductase by the compound of formula (I) can be carried out 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 environment containing oxygen.

[0047] According to an embodiment of the present invention, the assay can be used for the detection of quinone oxidoreductase 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 an embodiment of the present invention, the quinone oxidoreductase may be located in lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer, liver cancer.

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

[0051] The present invention also provides the use of the compound of formula (I) for qualitatively and quantitatively detecting the content of quinone oxidoreductase in cancer cells and normal somatic cells.

[0052] According to an embodiment of the present invention, the cancer cells are lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer or liver cancer cells.

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

[0054] The present invention also provides use of the compound of formula (I) in preparing a kit for detecting quinone oxidoreductase.

[0055] Beneficial effects

[0056] The compound of formula (I) of the present invention has fewer synthesis steps, shorter reaction time, easier purification and simpler process.

[0057] The present invention chemically modifies luciferin-based substances and introduces a quinone propionic acid group with quinone oxidoreductase reaction specificity to obtain a class of bioluminescent probes (compounds of formula (I)) that can be used for quinone oxidoreductase detection. Among luciferin / amino luciferin-based substances, when the 6' position of the compound of formula (I) is a hydroxyl / amino group and the 4 position is a carboxylic acid, it can react with luciferase, ATP and Mg. 2+ Bioluminescence occurs in the presence of. The compound of formula (I) of the present invention uses these luminescent luciferin / aminoluciferin substances as the mother body, and links the group that can recognize quinone oxidoreductase to the 6' hydroxyl / amino group of luciferin / aminoluciferin. Therefore, the compound of formula (I) of the present invention cannot be recognized by luciferase as a substrate and has no luminous ability. When the compound of formula (I) of the present invention encounters quinone oxidoreductase, the recognition group in the compound reacts with quinone oxidoreductase, and then the linking group breaks (the recognition group in the compound of formula (I) falls off when encountering quinone oxidoreductase, exposing the hydroxyl or amino group in the compound of formula (I)), thereby releasing the luciferin / aminoluciferin substance, and in the presence of ATP, Mg 2+ and luciferase to produce bioluminescence (see the attached Figure 1 The probe has good selectivity and sensitivity for quinone oxidoreductase and can be used as a bioluminescent probe of quinone oxidoreductase to label various tumor cells such as lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer, and liver cancer.

[0058] The compound of formula (I) of the present invention has specificity for detecting quinone oxidoreductase, while other anions and cations, amino acids and enzymes in organisms have no effect on the luminescence of the compound of the present invention.

[0059] Finally, the compound of formula (I) of the present invention has simple synthesis steps and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a reaction mechanism diagram of the detection of quinone oxidoreductase by NQO1-Luc prepared in Example 1;

[0061] Figure 2 This is a graph showing the bioluminescence intensity of NQO1-Luc prepared in Example 1 in a PBS buffer solution as the concentration of quinone oxidoreductase changes;

[0062] Figure 3 This is a bioluminescence intensity graph of the NQO1-Luc prepared in Example 1 for detecting intracellular quinone oxidoreductase.

[0063] Definitions and Explanations of Terms

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the subject matter of the claims pertains. It should be understood that the foregoing brief description and the following detailed description are exemplary and illustrative only and do not limit the subject matter of this application in any way. Throughout this application, unless otherwise indicated, the term "include," as well as its alternative forms such as "comprises," "includes," and "comprising" are not intended to be limiting.

[0065] The term "C 1-12 "Alkyl" is understood to mean a straight or branched saturated monovalent hydrocarbon group having 1 to 12 carbon atoms, preferably C 1-6 Alkyl. "C 1-6 “Alkyl” is understood as meaning preferably a linear or branched, saturated, monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl radical 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, etc. or their isomers. In particular, the radical has 1, 2, 3, 4, 5, 6, or more carbon atoms (“C 1-6 alkyl), such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly, said radicals having 1, 2 or 3 carbon atoms ("C 1-3 "alkyl"), for example methyl, ethyl, n-propyl or isopropyl.

[0066] The term "C 3-20 "Cycloalkyl" is understood to mean 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" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. 3-10 The cycloalkyl group may be a monocyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as a decalin ring.

[0067] The term "3-20 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5 heteroatoms independently selected from N, O and S, preferably a "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5, preferably 1-3 heteroatoms selected from N, O and S. The heterocyclyl may be attached to the rest of the molecule through any one of the carbon atoms or the nitrogen atom (if present). In particular, the heterocyclyl may include, but is not limited to, a 4-membered ring such as azetidinyl, oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. 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 hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5,6-membered bicyclic ring such as a hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl ring. The nitrogen-containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. According to the present invention, the heterocyclic group is non-aromatic.

[0068] The term "C 6-20 "Aryl" is understood to mean a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms, preferably "C 6-14 Aryl". The term "C 6-14 "Aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring ("C 6-14 or a ring having 9 carbon atoms ("C9 aryl"), for example indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 aryl) such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C 13 aryl), such as fluorenyl, or a ring having 14 carbon atoms ("C 14 "aryl"), for example anthracenyl.

[0069] The term "5-20 membered heteroaryl" is 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, for example "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" is 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, in particular 5 or 6 or 9 or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S and, in addition, in each case may be benzo-fused. In particular, the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl and the like; or acininyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like.

[0070] Unless otherwise indicated, a heterocyclyl, heteroaryl, or heteroarylene group includes all possible isomeric forms thereof, such as positional isomers thereof. Thus, for some illustrative, non-limiting examples, pyridyl or pyridylene groups include pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-yl, and pyridin-4-ylene; thienyl.

[0071] The above term “C 1-12 The definition of "alkyl" also applies to 1-12 Other terms for "alkyl", such as the term "C 1-12 Alkoxy", "halogenated C 1-12 Alkyl", "-CO-C 1-12 Alkyl", "-CONH-C 1-12 Alkyl", "-COOC 1-12 Alkyl" etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a reaction mechanism diagram of the detection of quinone oxidoreductase by NQO1-Luc prepared in Example 1;

[0074] Figure 2 This is a graph showing the bioluminescence intensity of NQO1-Luc prepared in Example 1 in a PBS buffer solution as the concentration of quinone oxidoreductase changes;

[0075] Figure 3 This is a bioluminescence intensity graph of the NQO1-Luc prepared in Example 1 for detecting intracellular quinone oxidoreductase. DETAILED DESCRIPTION

[0076] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations 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 encompassed within the scope of protection that the present invention is intended to protect.

[0077] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0078] Preparation Example 1

[0079] Preparation of compound (1)

[0080]

[0081] Under nitrogen protection, trimethylhydroquinone (32.9 mmol, 5 g) and 3,3-dimethylacrylic acid (39.5 mmol, 3.9 g) were dissolved in 30 mL of methanesulfonic acid and reacted at 85 ° C for 4-5 hours. After the reaction was completed, it was cooled with ice water, and the reactant was extracted with ethyl acetate. The organic phase was washed three times with saturated sodium bicarbonate and water, and then dried over anhydrous magnesium sulfate. The crude product was recrystallized with n-hexane / ethyl acetate (v / v=2:1) to obtain a white solid (4.7 g, 61%). ESI (C 14 H 18 O3):[MH + ] - =233.1. 1 H-NMR (300MHz, DMSO-d6), δ: 4.69 (s, 1H), 2.56 (s, 2H), 2.37 (s, 3H), 2.23 (s, 3H), 2.9 (s, 3H), 1.46 (s, 6H).

[0082] Preparation Example 2

[0083] Preparation of compound (2)

[0084]

[0085] Under nitrogen protection, compound (1) (4.3 mmol, 1 g) and N-bromosuccinimide (NBS) (6.4 mmol, 1.1 g) were dissolved in 30 mL of acetonitrile and stirred at room temperature for 1 h. After the reaction was completed, the organic phase was dried by rotary evaporation, and the crude product was extracted three times with dichloromethane and then with saturated brine, dried over anhydrous magnesium sulfate, and dried by rotary evaporation to obtain a yellow solid (1 g, 97%). ESI (C 14 H 18 O4):[MH + ] - =249.1. 1 H NMR (300MHz, DMSO-d6), δ: 3.04(s,2H), 2.15(s,3H), 1.96(m,3H), 1.94(m,3H), 1.45(s,6H).

[0086] Preparation Example 3

[0087] Preparation of compound (3)

[0088]

[0089] Under nitrogen protection, compound (2) (4.1 mmol, 1 g), 2-cyano-6-hydroxybenzothiazole (3.4 mmol, 0.6 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (8.4 mmol, 1.3 g) and 4-lutidine (DMAP) (9.1 mmol, 0.8 g) were dissolved in ultra-dry dichloromethane (20 mL) and stirred at room temperature for 12 h. After the reaction was completed, the mixture was extracted three times with dichloromethane. The crude product was purified by silica gel chromatography using a developing solvent, ethyl acetate / petroleum ether (v / v=1:50), to obtain a yellow solid (234 mg, 17%). ESI (C 22 H 20 N2O4S):[MH + ] - =407.1. 1 H NMR (400MHz, DMSO-d6), δ: 8.27 (d, J = 8.5 Hz, 1H), 8.10 (s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 3.26 (s, 2H), 2.12 (s, 3H), 1.86 (s, 6H), 1.49 (s, 6H).

[0090] Example 1

[0091] Preparation of compound (4): NQO1-Luc

[0092]

[0093] Compound (3) (0.6 mmol, 234 mg) was dissolved in 5 mL of a mixed solvent of methanol and dichloromethane (v / v=1:1), and a solution of D-cysteine hydrochloride (0.7 mmol, 112 mg) and potassium carbonate (0.7 mmol, 90 mg) dissolved in 5 mL of a mixed solvent of methanol and deionized water (v / v=1:1) was added. The mixture was reacted at room temperature for 20 min. After the reaction was completed, the organic solvent was dried, an equal volume of water was added, and the pH was adjusted to about 2-3 with a 1 M hydrochloric acid solution. The crude product was obtained as a yellow solid. The crude product was recrystallized from methanol to obtain a slightly yellow product (248 mg, 84%). ESI (C 25 H 24 N2O6S2):[MH + ] - =511.1. 1 H NMR (300MHz, DMSO-d6), δ: 8.16 (d, J = 8.9 Hz, 1H), 7.95 (t, J = 3.6 Hz, 1H), 7.26 (dd, J = 8.9, 2.4 Hz, 1H), 5.40 (dt, J =26.6,13.3Hz,1H),3.87–3.60(m,2H),3.24(d,J=4.1Hz,2H),2.12(s,3H),1.86(d,J=4.3Hz,6H),1.49(s,6H).

[0094] Example 2

[0095] Compound (4) (NQO1-Luc) obtained in Example 1 was tested against different concentrations of NQO1. The test method was as follows: NQO1-Luc was prepared into a 120 μM solution and NQO1 was prepared into 30 μg / mL, 60 μg / mL, 120 μg / mL, 180 μg / mL, and 240 μg / mL solutions using 10 mmol PBS buffer (containing 100 μM NADH and 10 mM MgSO4). 25 μL of NQO1-Luc solution (compound 4) and 25 μL of the above-mentioned different concentrations of NQO1 solution were then added to each well of a black 96-well plate. The plate was placed in a 37°C incubator and incubated for 60 min. Then, 50 μL of ATP (2 mM) and 50 μL of luciferase solution (60 ug / mL) were added to each well. The 96-well plate was assayed for bioluminescence intensity using a microplate reader. Finally, the bioluminescence spectra of NQO1-Luc at different NQO1 concentrations (0, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL) were measured (see Figure 2 ),Depend on Figure 2As can be seen in the results, when NQO1 is absent in the solution, the luminescence of NQO1-Luc is negligible. However, when NQO1 is present, the bioluminescence intensity of the solution gradually increases with increasing NQO1 concentration. Therefore, the compound NQO1-Luc of the present invention can be used for both qualitative and quantitative detection of NQO1.

[0096] In addition, since NQO1 is overexpressed in various tumor cells such as lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer, and liver cancer, the compound NQO1-Luc of the present invention can be used as a detection marker for the above cancers.

[0097] Example 3

[0098] The NQO1-Luc obtained in Example 1 was used for cell imaging. The test method was as follows: This experiment was divided into three groups. One group consisted of lung cancer cells with overexpression of NQO1 enzyme. As the experimental group, different concentrations of probe solution (NQO1-Luc) were directly added ( Figure 3 The second group was used as the control group. Before adding the probe solution, a certain concentration of NQO1 enzyme inhibitor (ie, dihydroxycoumarin) was added, and then different concentrations of probe solution (NQO1-Luc) were added ( Figure 3 The third group was used as a control group, and human embryonic lung cells expressing a small amount of NQO1 enzyme were also added with different concentrations of probe solution (NQO1-Luc) ( Figure 3 After the three groups of cells were incubated with the probe solution for 1 hour, the bioluminescence intensity was measured using a microplate reader (see Figure 3 ).Depend on Figure 3 As can be clearly seen in the figure, the NQO1 inhibitor strongly inhibits NQO1 expression in cells. Consequently, NQO1-Luc in the second group showed virtually no luminescence. However, NQO1-Luc in the first group of lung cancer cells overexpressing NQO1 and in the third group of human embryonic lung cells expressing a low level of the enzyme luminesced, with luminescence intensity positively correlated with NQO1 concentration. This demonstrates that NQO1-Luc of the present invention exhibits excellent selectivity and sensitivity for detecting NQO1.

[0099] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Compound represented by formula (I): （I） in, X is selected from O; Y is selected from S; Z is selected from S; R1, R2, R3, R4, R5 are the same or different and are independently selected from H, CN, NH2, NO2, OH, COOH, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, -CO-C 1-6 Alkyl, -CO-NH-C 1-6 Alkyl, -COOC 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-12 Aryl, 5-12 membered heteroaryl.

2. The compound according to claim 1, characterized in that R1, R2, R3, R4, R5 are the same or different and are independently selected from H, C 1-3 Alkyl, C 1-3 alkoxy; X is selected from O; Y and Z are selected from S.

3. The compound according to claim 1, characterized in that R1, R2, R3, R4, and R5 are independently selected from the following groups: H, CH3-, and CH3O-.

4. The compound according to any one of claims 1 to 3, characterized in that The compound represented by formula (I) is selected from the following: 。 5. The method for preparing the compound represented by formula (I) according to any one of claims 1 to 4, characterized in that: The steps include: wherein R1, R2, R3, R4, R5, X, Y, and Z have the definitions described in any one of claims 1 to 4; (a) Compound (II) is reacted with D-cysteine hydrochloride to obtain a compound represented by formula (I).

6. Use of the compound represented by formula (I) according to any one of claims 1 to 4 as a quinone oxidoreductase bioluminescent probe; When used as a quinone oxidoreductase bioluminescent probe, it can be used for non-disease diagnostic or therapeutic purposes.

7. The use according to claim 6, characterized in that The detection of quinone oxidoreductase by the compound represented by formula (I) is carried out in various buffer solutions.

8. The use according to claim 7, characterized in that The buffer solution is PBS buffer solution or Tris-HCl buffer solution.

9. The use according to claim 7, characterized in that The assay is performed in the presence of ATP, luciferase, and magnesium ions.

10. The use according to claim 7, characterized in that The detection is performed in an environment containing oxygen.

11. The use according to claim 7, characterized in that The assay is used for the detection of quinone oxidoreductase in vitro or in vivo.

12. The use according to claim 7, characterized in that The quinone oxidoreductase is located in lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer, and liver cancer.

13. Use of the compound represented by formula (I) according to any one of claims 1 to 4 as an intracellular quinone oxidoreductase imaging agent; When used as an intracellular quinone oxidoreductase imaging agent, it is used for non-disease diagnostic or therapeutic purposes.

14. Use of the compound represented by formula (I) according to any one of claims 1 to 4 for qualitatively and quantitatively detecting the content of quinone oxidoreductase in cancer cells and normal somatic cells; Used for qualitative and quantitative detection of the content of quinone oxidoreductase in cancer cells and normal somatic cells for non-disease diagnosis or treatment purposes.

15. The use according to claim 14, wherein The cancer cells are lung cancer, colon cancer, breast cancer, bile duct tumor, melanoma, pancreatic cancer or liver cancer cells.

16. Use of the compound of formula (I) according to any one of claims 1 to 4 for imaging quinone oxidoreductase in mice; Used for non-disease diagnostic or therapeutic purposes when imaging quinone oxidoreductase in mice.

17. Use of the compound represented by formula (I) according to any one of claims 1 to 4 in the preparation of a kit for detecting quinone oxidoreductase.

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