WO2019100501A1

WO2019100501A1 - Compound, preparation method therefor and use thereof as fluorescent probe

Info

Publication number: WO2019100501A1
Application number: PCT/CN2017/117565
Authority: WO
Inventors: 吴爱国; 邹瑞芬; 龚秋雨
Original assignee: 中国科学院宁波材料技术与工程研究所
Priority date: 2017-11-27
Filing date: 2017-12-20
Publication date: 2019-05-31
Also published as: CN109836420A; CN109836420B

Abstract

Disclosed are a compound I, a preparation method therefor and the use thereof as a fluorescent probe. The compound I has near infrared excitation/emission, wherein the excitation wavelength ranges from 500 nm-750 nm, and when excited with the maximum absorption wavelength of 670 nm, the emission wavelength thereof is 700 nm-810 nm; in addition, the compound I also has a response of an ultra-high sensitivity to PGP-1 enzyme, with a detection limit of 0.18 ng/mL for the PGP-1 enzyme where λex/em = 670/700 nm. The use of the compound I as a fluorescent probe has realized the detection and fluorescence imaging in vivo of PGP-1 for the first time, and can realize the detection and fluorescence imaging of PGP-1 in vitro, in cells and in vivo, and with the use of the compound I as a fluorescent probe, the detection analysis and imaging for all cells and tissues having inflammatory reactions and suffering from canceration can be performed.

Description

Compound, preparation method thereof and application as fluorescent probe

Technical field

The present application relates to a compound, a process for the preparation thereof and use as a fluorescent probe, and belongs to the field of fluorescent probes.

Background technique

Most naturally occurring or synthetic polypeptide and protein end-capping groups are terminated with N-terminal groups of the three classes of acetyl, formyl and pyroglutamyl (pGlu). Among them, hormones, peptides and proteins which are blocked by pyroglutamyl, up to now, it has been found that enzymatic hydrolysis can only be carried out by pyroglutamate aminopeptidase in the process of degradation in vivo or in vitro.

Since Doolittle and Armentrout first reported pyroglutamyl peptidase 1 (PGP-1) in 1968, researchers have conducted in-depth and meticulous attention to the structure and function of glutamate aminopeptidase. The research found that it plays a very important and fundamental role in the regulation, transmission and regulation of the body's metabolic pathway.

There are currently three types of pyroglutamate aminopeptidases found in living organisms: pyroglutamic acid aminopeptidase 1, pyroglutamic acid aminopeptidase 2, and pyroglutamate aminopeptidase 3 present in serum. Among them, pyroglutamate aminopeptidase 3 present in serum exhibits a very similar property to pyroglutamic acid aminopeptidase 2, that is, a high degree of substrate specificity: only thyrotropin releasing hormone (TRH) And a very small number of tripeptide hormones such as luteinizing hormone releasing hormone (LHRH) which are very similar in structure to enzymatic hydrolysis. Pyroglutamic acid aminopeptidase 1 exhibits broad substrate specificity compared to the harsh substrate specificity of pyroglutaminylaminopeptidase 2 and serum pyroglutamic aminopeptidase 3. In addition to peptides and proteins whose terminal groups are Glu-Pro structures, pyroglutamate aminopeptidase 1 can be subjected to enzymatic hydrolysis to all other dipeptides, peptides and proteins containing Glu-terminated. It is worth mentioning that most of the immunoglobulin proteins are terminated with glutamine, and the blocked glutamine often forms a protein terminated by pyroglutamic acid by cyclization. Therefore, designing a probe with high sensitivity response and excellent selectivity for glutamate aminopeptidase 1 will be very important for the study of biological signaling pathways, metabolism, immune response and pathology. Meaningful work.

At present, although many researchers have conducted extensive research on the distribution, structure and function of three types of pyroglutamate aminopeptidase, they have done a probe design on the response of glutamate aminopeptidase. Little research work. There are currently only two probes available for the detection of pyroglutamate aminopeptidase 1, commercially available L-pyroglutamic acid-7 amino-4-methylcoumarin (CAS: 66642-36-2 , λ _ex / _em = 340/440nm, detection limit 14.6ng / mL); there is a PGP-1 enzyme response probe based on cresyl violet synthesis (λ _ex / _em = 585 / 625nm, detection limit 5.6ng / mL , Chem. Sci., 2016, 7, 4694). It can be seen from the excitation/emission wavelength and detection limit of these two probes that these two probes have great disadvantages in the analysis and detection of enzymes, especially commercial probes whose excitation wavelength is in the ultraviolet region. This is very unfavorable for the detection of cells and organisms, and its high detection limit is also detrimental to the detection of low levels of PGP-1. In addition, the excitation of ultraviolet and visible light has a limited penetration depth to the organism, and it is impossible to detect and image PGP-1 in vivo.

So far, probes that can be used for the detection and imaging of PGP-1 in vivo have not been reported.

Summary of the invention

According to an aspect of the present application, there is provided a compound I having near-infrared excitation/emission wherein an excitation wavelength or absorption wavelength ranges from 500 nm to 750 nm, excitation at a maximum absorption wavelength of 670 nm, and an emission wavelength of 700 to 810 nm; This compound I also has an ultra-high sensitivity response to the PGP-1 enzyme, and the detection limit for the PGP-1 enzyme at λ _ex / _em = 670/700 nm is 0.18 ng/mL.

The compound I includes an organic cation and an inorganic anion;

The organic cation is selected from at least one of the cations having the chemical formula of Formula I;

In the formula I, R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₆ , R ₁₀₇ , R ₁₀₈ , R ₁₀₉ , R ₁₁₀ , R ₁₁₁ , R ₁₁₂ , R ₁₁₃ , R ₁₁₄ , R ₁₁₅ , R ₁₁₆ , R ₁₁₇ , R ₁₁₈ and R _{119 are} independently selected from the group consisting of hydrogen, a C ₁ - C ₂₀ alkane group, and a C ₁ - C ₂₀ alkane group having a substituent; R _{105 is} selected from the group consisting of hydrogen, a C ₁ - C ₂₀ alkane group, and C. ₁ to C ₂₀ an alkane group having a substituent.

The substituent-containing alkane group contains at least one substituent; and the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.

Alternatively, the inorganic anion is selected from at least one of monovalent inorganic anions. Preferably, the inorganic anion is at least one selected from the group consisting of a fluoride ion, a chloride ion, a bromide ion, an iodide ion, and a nitrate ion.

Preferably, in the formula I, R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₆ , R ₁₀₇ , R ₁₀₈ , R ₁₀₉ , R ₁₁₀ , R ₁₁₁ , R ₁₁₂ , R ₁₁₃ , R ₁₁₄ , R ₁₁₅ And R ₁₁₆ and R ₁₁₇ are hydrogen; R ₁₀₅ , R ₁₁₈ and R _{119 are} independently selected from hydrogen, a C ₁ to C ₂₀ alkane group, and a C ₁ to C ₂₀ substituent-containing alkane group; and the substituent-containing group The alkane group contains at least one substituent; the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.

Further preferably, in the formula I, R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₆ , R ₁₀₇ , R ₁₀₈ , R ₁₀₉ , R ₁₁₀ , R ₁₁₁ , R ₁₁₂ , R ₁₁₃ , R ₁₁₄ , R ₁₁₅ , R ₁₁₆ , R ₁₁₇ are hydrogen; R ₁₀₅ is propyl; and R ₁₁₈ and R ₁₁₉ are methyl. That is, the structural formula of the organic cation in the compound I is as shown in the formula V, and its chemical formula is: C ₃₄ H ₃₇ N ₂ O ₃ ⁺ ; the English name is:

3,3-Dimethyl-2-(2-{6-[2-oxo-2-(5-oxo-pyrrolidin-2-yl)-ethyl]-2,3-dihydro-1H-xanthen-s-yl} -vinyl)-1-propyl-3H-indolium;

As an embodiment, the excitation wavelength or absorption wavelength of the compound I is from 500 nm to 750 nm.

As an embodiment, the emission wavelength of the compound I is in the range of 700 nm to 810 nm.

According to still another aspect of the present application, there is provided a method of preparing the compound I, comprising the steps of:

a) adding a basic activator to the solution containing the compound II and the compound III, after washing, washing with water, drying, to obtain a mixture A;

The compound II is selected from at least one of the compounds having the formula of the formula II:

In the formula II, R ₂₀₁ , R ₂₀₂ , R ₂₀₃ , R ₂₀₄ , R ₂₀₆ , R ₂₀₇ , R ₂₀₈ , R ₂₀₉ , R ₂₁₀ , R ₂₁₁ , R ₂₁₂ , R ₂₁₃ , R ₂₁₅ , R ₂₁₆ , R ₂₁₇ , R ₂₁₈ , R ₂₁₉ , R ₂₂₀ , R ₂₂₁ , R ₂₂₂ , R ₂₂₃ , and R _{224 are} independently selected from the group consisting of hydrogen, a C ₁ - C ₂₀ alkane group, and a C ₁ - C ₂₀ alkane group having a substituent; R ₂₀₅ , R _{214 is} independently selected from the group consisting of hydrogen, a C ₁ - C ₂₀ alkane group, and a C ₁ - C ₂₀ alkane group having a substituent; the substituent-containing alkane group having at least one substituent; the substituent being selected from a carboxyl group , at least one of an amino group, a thiol group, an ester group, and an amide group; X ₁ and X _{2 are} independently selected from the group consisting of F, Cl, Br, and I;

The compound III is selected from at least one of the compounds having the formula of the formula III:

In the formula III, R ₃₀₁ , R ₃₀₂ , R ₃₀₃ , and R _{304 are} independently selected from hydrogen, a C ₁ - C ₅ alkane group, and a C ₁ - C ₂₀ substituent-containing alkane group; the substituent-containing alkane group; Containing at least one substituent; the substituent is selected from at least one of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group;

b) adding a catalyst to the mixture A, in an inert atmosphere, after reacting at 60-80 ° C, washed with water, and dried to obtain a mixture B;

c) activating the compound IV by an organic base and/or an inorganic base to obtain the activated compound IV;

The compound IV is selected from at least one of the compounds having the formula of the formula IV:

In Formula IV, R ₄₀₁ , R ₄₀₂ , R ₄₀₃ , R ₄₀₄ , R _{408 are} independently selected from hydrogen, C ₁ -C ₂₀ alkane; R ₄₀₅ , R ₄₀₆ , R _{407 are} independently selected from C ₁ -C ₂₀ An alkane group, a C ₁ - C ₂₀ alkane group having a substituent; the substituent-containing alkane group having at least one substituent; the substituent being selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group At least one

d) after the activated compound IV and the mixture B is mixed, and reacted at -10 ° C to 80 ° C for 0.01 h to 24 h, after the reaction is completed, washed with water and dried to obtain a mixture C;

e) adding an acidic reagent to the mixture C for decarboxylation, and after completion of the reaction, after drying and purification, the compound I is obtained.

Preferably, in the formula II, R ₂₀₁ , R ₂₀₂ , R ₂₀₃ , R ₂₀₄ , R ₂₀₆ , R ₂₀₇ , R ₂₀₈ , R ₂₀₉ , R ₂₁₀ , R ₂₁₁ , R ₂₁₂ , R ₂₁₃ , R ₂₁₅ , R ₂₁₆ , R ₂₁₇ , R ₂₁₈ , R ₂₂₁ and R ₂₂₂ are hydrogen; R ₂₀₅ , R ₂₁₄ , R ₂₁₉ , R ₂₂₀ , R ₂₂₃ , and R _{224 are} independently selected from hydrogen, C ₁ -C ₂₀ alkane, C ₁ ～ A _C20- containing alkane group; the substituent-containing alkane group having at least one substituent; and the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.

Preferably, in the formula III, R ₃₀₁ , R ₃₀₂ , R ₃₀₃ and R ₃₀₄ are hydrogen.

Preferably, in the formula IV, R ₄₀₁ , R ₄₀₂ , R ₄₀₃ , R ₄₀₄ and R ₄₀₈ are hydrogen; R ₄₀₅ , R ₄₀₆ and R _{407 are} independently selected from a C ₁ - C ₂₀ alkane group, C ₁ ～ A _C20- containing alkane group; the substituent-containing alkane group having at least one substituent; and the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.

Alternatively, the solution containing the compound II and the compound III in the step a) is an organic solution in which the compound II and the compound III are dissolved and/or dispersed. The organic solvent is selected from common organic solvents such as at least one of a lower alcohol, a halogenated hydrocarbon, cyclohexane, petroleum ether, ethyl acetate, acetonitrile, tetrahydrofuran, pyridine, dioxane, acetone, methyl ethyl ketone, DMF, DMSO. Kind.

A person skilled in the art can select the ratio of the compound II, the compound III and the basic activator according to actual needs. As an embodiment, the molar ratio of the compound II, the compound III and the basic activator in the step a) is:

Compound II, Compound III and basic activator = 1:1 to 3:1 to 5.

Preferably, the molar ratio of the compound II to the compound III in the step a) is 1:1.

Optionally, the alkaline activator in the step a) is selected from the group consisting of NaH, CaH ₂ , KH, NaOH, KOH, Na ₂ CO ₃ , NaHCO ₃ , K ₂ CO ₃ , KHCO ₃ , MgCO ₃ , Mg (HCO) ₃ ) ₂ , at least one of CaCO ₃ , Ca(HCO ₃ ) ₂ , trimethylamine, triethylamine, DBN, DBU, and sodium alkoxide.

Preferably, the step a) is added with an alkaline activator activated to add an alkaline activator, and then stirred at 10 ° C to 40 ° C (preferably room temperature) for 4 to 6 hours. Further preferably, the alkaline activator is added to the step a) to activate the addition of the basic activator, and then stirred at a preferred room temperature for 4 to 6 hours.

Alternatively, the inert atmosphere in the step b) is at least one selected from the group consisting of nitrogen, helium and argon. Preferably, the inert atmosphere in step b) is nitrogen.

Alternatively, the catalyst in the step b) is at least one selected from the group consisting of FeCl ₂ , SnCl ₂ , CuCl, CoCl, and PbCl ₂ .

One skilled in the art can select the ratio of catalyst to mixture A according to actual needs. Preferably, the molar ratio of the catalyst in step b) to mixture A is:

Catalyst: Mixture A = 1:1 to 5:1.

Optionally, the method of activating Compound IV in step c) comprises activating Compound IV with an organic base and/or an inorganic base activator. Further preferably, the activator is selected from activators for use in the amidation reaction. Still more preferably, the activator is selected from the group consisting of N,N-diisopropylethylamine (abbreviated as DIPEA), 2-(7-benzotriazole)-N,N,N',N'- Tetramethylurea hexafluorophosphate (abbreviated as HATU), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride (abbreviated as EDCI), 1-hydroxybenzotriazole (abbreviated as HOBT), at least one of N-hydroxysuccinimide (abbreviated as NHS).

A person skilled in the art can select the ratio of the compound IV to the mixture B after activation according to actual needs. Preferably, the molar ratio of the activated compound IV to the mixture B in the step d) is:

Compound IV after activation: mixture B = 1:1 to 5:1.

Alternatively, the step e) is carried out by adding an -acid reagent to the mixture C at a temperature not exceeding 0 ° C, and then raising the temperature to 10 ° C to 40 ° C (preferably room temperature).

Optionally, the acidic reagent in the step e) is at least one selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, glacial acetic acid, formic acid, and p-toluenesulfonic acid. Preferably, the acidic reagent in step e) is trifluoroacetic acid.

Optionally, the water washing in the step a), the step b), the step c), the step d), and the step e) is: after mixing the mixture to be washed with water, separating the aqueous phase and the organic phase with a separating funnel, and retaining The organic phase.

Alternatively, the drying in the step a), the step b), the step c), the step d) and the step e) are both rotary evaporation drying, in order to remove water and organic solvent in the system.

As a specific embodiment, the preparation steps of the compound I are as follows:

(1) In a 100 ml round bottom flask, add IR-780 (1 mmol) and m-nitrophenol (1 mmol) in an organic solvent, add an alkaline activator, stir at room temperature for 4-6 hours, wash three times with water, spin Dry solvent; (2) Add appropriate amount of catalyst to (1), react under nitrogen for 70 ° C overnight, wash the mixture three times, spin dry solvent; (3) Add activated BOC-pyroglutamic acid (2) In the reaction at room temperature for 8 h, the mixture is washed 3 times with water, and the solvent is dried; (4) trifluoroacetic acid is slowly added dropwise to (3) at 0 ° C, and the reaction is carried out at room temperature, and the degree of reaction is judged according to the spot plate, and the solvent is dried. Column purification (dichloromethane/methanol: 5/1, volume ratio) gave the compound I.

According to still another aspect of the present application, there is provided a fluorescent probe having a longest excitation wavelength and emission wavelength compared to currently known fluorescent probes for PGP-1 detection and imaging, The long-wavelength excitation/emission enables the first detection and fluorescence imaging of PGP-1 in vivo; the fluorescent probe can detect PGP-1 and fluorescence imaging in vitro, in vivo and in vivo, and can be used for all inflammatory reactions and carcinogenesis. Cells and tissues are assayed and imaged. Furthermore, the fluorescent probe has the lowest detection limit and the highest detection sensitivity for PGP-1 compared to the currently known fluorescent probes for PGP-1 detection and imaging.

The fluorescent probe includes at least one of the compound I and the compound I prepared according to the method.

Alternatively, the fluorescent probe is used to detect pyroglutamate aminopeptidase 1. The fluorescent probe not only specifically recognizes the pyroglutamic acid aminopeptidase PGP-1, but also exhibits fluorescence enhancement.

The lower limit of the detection concentration of the fluorescent probe for the pyroglutamic acid aminopeptidase 1 at the emission wavelength λ _ex 670 nm and the excitation wavelength λ _em 700 nm does not exceed 0.2 ng/mL.

Preferably, the fluorescent probe has a detection concentration lower limit of 0.18 ng/mL for the pyroglutamic acid aminopeptidase 1 at an emission wavelength λ _ex 670 nm and an excitation wavelength λ _em 700 nm.

According to still another aspect of the present application, at least one of the compound I and the compound I prepared according to the method is provided for preparing a detection reagent and/or a coke valley for detecting pyroglutamate aminopeptidase 1 Applied to the aminopeptidase 1 imaging reagent.

According to still another aspect of the present application, there is provided the use of at least one of the compound I, the compound I prepared according to the method, for preparing a detection reagent for detecting inflammatory cells and/or an inflammatory cell imaging reagent.

According to still another aspect of the present application, there is provided the use of at least one of the compound I, the compound I prepared according to the method, for preparing a detection reagent for detecting cancerous cells and/or a cancerous cell imaging reagent.

According to still another aspect of the present application, at least one of the compound I, the compound I prepared according to the method, is provided for preparing a test reagent for evaluating the therapeutic effect of an anti-inflammatory drug and/or a cancer therapeutic drug, and/or Application in imaging reagents.

In the present application, "alkane group" is a group formed by the loss of an arbitrary hydrogen atom of an alkane molecule. The alkane molecules include linear alkanes, branched alkanes, and cycloalkanes.

In the present application, "C ₁ to C ₂₀ " means that the number of carbon atoms is from 1 to 20; for example, "C ₁ - C ₂₀ alkane group" means that the number of carbon atoms is (1 to 20) 1, 2, 3, The alkane molecule of 4 or 5 loses the group formed by any one of the hydrogen atoms.

In the present application, the "alkyl group having a substituent" is a group formed by substituting at least one hydrogen atom of the alkane group with a substituent. The "C ₁ - C ₂₀ alkyl group having a substituent" is a group formed by substituting at least one hydrogen atom on an alkane group having 1 to 20 carbon atoms.

Advantages of the application include, but are not limited to:

(1) The compound I provided by the present application has near-infrared excitation/emission in which an excitation wavelength ranges from 500 nm to 750 nm, is excited at a maximum absorption wavelength of 670 nm, and an emission wavelength is 700 to 810 nm; in addition, the compound I is also a PGP- 1 The enzyme has an ultra-high sensitivity response, and the detection limit of PGP-1 enzyme is 0.18 ng/mL at λ _ex / _em = 670 / 700 nm.

(2) The method for producing the compound I provided by the present invention can give the compound I in high purity and high yield.

(3) The fluorescent probe provided by the present application can not only specifically recognize the pyroglutamic acid aminopeptidase PGP-1, but also exhibits fluorescence enhancement.

(4) The fluorescent probe provided by the present application has the longest excitation wavelength and emission wavelength and long wavelength excitation compared with the currently known fluorescent probe for PGP-1 detection and imaging. /Emission for the first time to achieve PGP-1 in vivo detection and fluorescence imaging; the fluorescent probe can achieve PGP-1 detection and fluorescence imaging in vitro, in vivo and in vivo, and can be used for all cells and tissues with inflammatory reactions and carcinogenesis Perform analysis and imaging.

(5) The fluorescent probe provided by the present application has the lowest detection limit and the highest detection sensitivity for PGP-1 compared with the currently known fluorescent probe for PGP-1 detection and imaging. .

(6) In view of the fact that the expression of PGP-1 in cells/tissues with inflammation and canceration is higher than that of normal cells/tissue, the fluorescent probes provided by the present application can be used for detection analysis and imaging of inflammation and cancer.

DRAWINGS

Figure 1 (a) is a ¹ H NMR spectrum of Sample 1 ^# .

Figure 1 (b) is a ¹³ C NMR spectrum of Sample 1 ^# .

Figure 2 is a high resolution mass spectrum of sample 1 ^# .

Figure 3 is an absorption curve of sample 1 ^# and PGP-1 enzyme before and after reaction in phosphate buffer, pH = 7.4.

FIG 4 is a fluorescent sample ^# 1 at different pH reactive with PGP-1 enzyme enhancing the effect of an excitation wavelength of 670nm; wherein line 1 is 5μM Sample ^# 1 fluorescence result, line 2 is 5μM sample ^# 1 and 100ng / mL Fluorescence results after PGP-1 reaction.

FIG 5 is a fluorescent sample ^# 1 at different temperatures for the reaction with the PGP-1 enzyme enhancing the effect of an excitation wavelength of 670nm, wherein line 1 is 5μM Sample ^# 1 fluorescence result, line 2 is 5μM sample ^# 1 and 100ng / mL Fluorescence results after PGP-1 reaction.

Figure 6 is a result of a selective experiment of sample 1 ^# .

Figure 7 is a graph showing the results of measurement of the detection limit of PGP-1 enzyme by sample 1 ^# .

Figure 8 is a graph showing the results of measurement of the detection limit of PGP-1 enzyme by a commercial probe in a comparative example.

Figure 9 is a result of fluorescence imaging and quantitative analysis of PGP-1 enzyme in sample 1 ^# ; wherein (a) is fluorescence imaging and (b) is quantitative analysis.

Figure 10 is a graph showing the results of fluorescence imaging and quantitative analysis of PGP-1 enzyme in sample 1 ^# ; wherein (a) is fluorescence imaging and (b) is quantitative analysis.

Detailed ways

The present application is described in detail below with reference to the embodiments, but the application is not limited to the embodiments.

Unless otherwise stated, the raw materials and reagents used in this application are all commercially available and used without treatment. The equipment used is the ones recommended by the manufacturer.

In the examples, the commercial probe L-pyroglutamic acid-7-amino-4-methylcoumarin (CAS: 66642-36-2) was purchased from Sigma-Aldrich.

In the examples, IR-780 iodide was purchased from Sigma-Aldrich.

In the examples, the catalyst used was purchased from Sinopharm, Aladdin, Sigma-Aldrich.

In the examples, ¹ H-NMR of nuclear magnetic resonance was measured on a 400 AVANCE III spectrometer (Breker), 400 MHz, CD ₃ OD; carbon spectrum ¹³ C-NMR, 400 MHz, CD ₃ OD.

In the examples, electron bombardment mass spectrometry MS (EI) was performed using a TripleTOF Model 4600 mass spectrometer from AB Sciex.

In the examples, the fluorescence test was performed on a FL3-111 type instrument from Horiba, France.

The yield of Compound I, based on the amount of Compound II, is calculated by the following formula:

Yield % = (the mass actually obtained from the target product, the theoretically expected mass of the target product) × 100%.

Example 1 Preparation of Compound I Sample 1 ^#

In a 100 ml round bottom flask, IR-780 iodine compound (1 mmol) and m-nitrophenol (1 mmol) were dissolved in 10 mL of organic solvent acetonitrile, 2 mol of basic activator Na ₂ CO _{3 was added} , and stirred at room temperature for 4 hours. Thereafter, it was washed three times with water, and dried by rotary evaporation at 40 ° C to obtain a mixture A. To the mixture A, 2 mol of a catalyst SnCl ₂ was added, and the mixture was reacted at 70 ° C for 8 hours under nitrogen atmosphere, the mixture was washed three times with water, and dried by rotary evaporation at 40 ° C to obtain a mixture B. BOC-pyroglutamic acid was activated by DIPEA and HATU to obtain activated BOC-pyroglutamic acid. 2 mol of activated BOC-pyroglutamic acid was added to the mixture B, and the reaction was carried out for 8 hours at room temperature for 3 hours, and dried by rotary evaporation at 40 ° C to obtain a mixture C. 2 ml of trifluoroacetic acid was slowly added dropwise to the mixture C at 0 ° C, and reacted at room temperature for 3 h; after completion of the reaction, it was dried by rotary evaporation at 40 ° C, purified by silica gel column, and mixed with a volume ratio of dichloromethane to methanol of 5/1. Solvent), the compound I was obtained, which was designated as sample ^## , and the yield was 70%.

The ¹ H NMR spectrum and the ¹³ C NMR spectrum of Sample 1 ^# are shown in Fig. 1 (a) and Fig. 1 (b), respectively. The high resolution mass spectrum of sample 1 ^# is shown in Figure 2. It can be seen that the molecular weight of sample 1 ^# is 522.2745.

Example 2 Preparation of Compound I Sample 2 ^# ～样六^#

DETAILED step of preparing a ^# in the same product, except that the preparation conditions (the same sample preparation conditions ^# 1 Example 1 Condition in Table 1 unspecified) Table 1 changes, respectively, to obtain samples No. ² to Sample 6 ^# .

Table 1

The nuclear magnetic characterization results of sample 2 ^# to sample 6 ^# and the high resolution mass spectrum were the same as sample 1 ^# .

Example 3 Preparation of Compound I Sample 7 ^#

(1) In a 100 ml round bottom flask, 2,3,3-trimethylsulfonium, 4-bromobutyric acid, and 1,2 dichlorobenzene were added, and the mixture was heated to 110 ° C for 12 hours, and ethyl acetate was precipitated. Filtration to obtain a pink precipitate; (2) adding dichloromethane, N,N-dimethylformamide, phosphorus oxychloride to cyclohexanone, reacting under reflux for 2 hours, pouring into water for precipitation, Filtering to obtain a yellow precipitate; (3) adding the precipitate obtained in (1) and (2) to acetic anhydride solvent, and adding sodium acetate at 70 ° C for 40 minutes, then pouring into an aqueous solution for precipitation to obtain green Precipitate; (4) Add m-nitrophenol to the (3) and a suitable amount of catalyst, react under nitrogen for 70 ° C overnight, wash the mixture three times, and spin dry the solvent; (5) Activate the BOC-coke valley Add the acid to (4), react at room temperature for 8 hours, wash the mixture three times, and spin dry the solvent; (6) slowly add trifluoroacetic acid to (5) at 0 ° C, react at room temperature for 3 hours, and spin dry the solvent. The column was purified (dichloromethane/methanol: 5/1, volume ratio) to give Compound I with a functional group, which was designated as sample ^# 7.

Sample 7 ^# has the same function as Sample 1 ^# .

The change of absorption spectrum of compound I in Example 4 before and after PGP-1 enzyme reaction

In a graduated small tube, add 5 μL of the probe solution (solvent is DMSO), add 5 μL of PGP-1 enzyme solution (solvent is ultrapure water), and dilute the system to 1 mL with phosphate buffer to obtain the reaction. The final concentrations of the pre-system 1, probe and PGP-1 were 5 μM and 5 μg/mL, respectively; the system before the reaction was reacted at 37 ° C for 30 min to obtain the system 2 after the reaction.

The absorption spectrum was measured on the pre-reaction system 1 and the post-reaction system 2, respectively, and the results are shown in Fig. 3. The line in Figure 3 corresponds to System 1 before the reaction, and the line 2 corresponds to System 2 after the reaction. As can be seen from Fig. 3, the absorption wavelength of the compound I of the present application after the reaction with the enzyme is 500 to 750 nm, so the excitation wavelength at the time of fluorescence detection may be 500 to 750 nm.

Effect of pH of Example 5 on the Reaction of Compound I with PGP-1 Enzyme

The pH of the standard phosphate buffer was adjusted using HCl and NaOH solutions to obtain buffer

solutions having pH

5, 6, 7, 8, and 9, respectively.

In a graduated small test tube, 10 μL of the probe solution was added, and the system was adjusted to 2 mL with the above buffer solutions of different pH values, and reacted at 37 ° C for 30 min to carry out fluorescence spectrometry, and the final concentration of the probe was 5 μM ( The result is for line 1) in Figure 4.

In a graduated test tube, 10 μL of the probe solution and 10 μL of the PGP-1 enzyme solution were added, and the system was adjusted to 2 mL with the above buffer solutions of different pH values, and then reacted at 37 ° C for 30 min to carry out fluorescence spectroscopy. The final concentrations of probe and PGP-1 were determined to be 5 [mu]M and 100 ng/mL, respectively (results for line 2 in Figure 4).

The results are shown in Figure 4. As can be seen from the figure, the compound I provided herein can be used for the detection and imaging of PGP-1 over a wide pH range.

Effect of temperature on the reaction of compound I with PGP-1 enzyme

The fluorescence spectrum was measured using a constant temperature shaker, setting different reaction temperatures, and setting the temperature from 25 ° C to 42 ° C. Specifically,

In a graduated small tube, add 10 μL of the probe solution, dilute the system to 2 mL with phosphate buffer (pH=7.4), react at different temperatures for 30 min, and then measure the fluorescence spectrum. The final concentration of the probe is 5 μM (results for line 1 in Figure 5).

In a graduated small tube, add 10 μL of probe solution and 10 μL of PGP-1 enzyme solution, dilute the system to 2 mL with phosphate buffer (pH=7.4), react at different temperatures for 30 min, and then perform fluorescence. Spectrometry, final concentrations of probe and PGP-1 were 5 [mu]M and 100 ng/mL, respectively (results for line 2 in Figure 5).

The results are shown in Fig. 5. As can be seen from the figure, the compound I provided by the present application can perform detection and imaging of PGP-1 over a wide temperature range.

Specific Recognition of PGP-1 Enzyme by Compound I of Example 7

In a graduated tube, 10 μL of the probe solution was added, and the system was adjusted to a volume of 2 mL with a phosphate buffer (pH = 7.4), and a fluorescence spectrum was measured as a control group at a final probe concentration of 5 μM (results corresponding to Fig. 6). Medium test system 1).

In a graduated test tube, 10 μL of the probe solution and 0.3 mmol of KCl were added, and the system was made up to 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 min to carry out fluorescence spectrometry. The final concentrations of the needle and KCl were 5 μM and 150 mM, respectively (results corresponded to System 2 in Figure 6).

In a graduated small tube, 10 μL of the probe solution and 0.004 mmol of CaCl _{2 were added} , and the system was adjusted to a volume of 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 minutes to carry out fluorescence spectrometry. The final concentrations of probe and CaCl ₂ were 5 μM and 2 mM, respectively (results corresponded to system 3 in Figure 6).

In a graduated small tube, 10 μL of the probe solution and 0.2 μmol of ZnCl _{2 were added} , and the system was adjusted to a volume of 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 minutes for fluorescence spectrometry. The final concentrations of probe and ZnCl ₂ were 5 μM and 100 μM, respectively (results corresponded to system 4 in Figure 6).

In a graduated small tube, 10 μL of the probe solution and 0.004 mmol of MgCl _{2 were added} , and the system was adjusted to a volume of 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 minutes to carry out fluorescence spectrometry. The final concentrations of probe and MgCl ₂ were 5 μM and 2 mM, respectively (results corresponded to system 5 in Figure 6).

In a graduated small tube, 10 μL of the probe solution and 0.2 μmol of CuCl _{2 were added} , and the system was adjusted to a volume of 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 minutes for fluorescence spectrometry. The final concentrations of probe and MgCl ₂ were 5 μM and 100 μM, respectively (results corresponded to system 6 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 0.02 mmol of glucose, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min to conduct fluorescence spectrometry. The final concentrations of needle and glucose were 5 μM and 10 mM, respectively (results corresponded to system 7 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 0.002 mmol of cysteine, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min to perform fluorescence spectroscopy. The final concentrations of the probe and cysteine were determined to be 5 μM and 1 mM, respectively (results corresponded to system 8 in Figure 6).

In a graduated tube, 10 μL of the probe solution and 0.010 mmol of glutathione were added, and the system was adjusted to 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 min to carry out fluorescence spectroscopy. The final concentrations of the probe and glutathione were determined to be 5 μM and 5 mM, respectively (results corresponded to system 9 in Figure 6).

In a graduated tube, 10 μL of the probe solution and 0.02 μmol of ClO ^{− were added} , and the system was adjusted to 2 mL with a phosphate buffer (pH=7.4), and then reacted at 37° C. for 30 minutes to carry out fluorescence spectrometry. probe and ClO ^- the final concentrations of 5μM and 10 M (6 results in the system corresponding to FIG. 10).

In a graduated cuvette, add 10 μL of probe solution and 0.002 mmol of tryptophan, make the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence spectrometry. The final concentrations of probe and tryptophan were 5 μM and 1 mM, respectively (results corresponded to system 11 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 0.002 mmol of alanine, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence spectrometry. The final concentrations of probe and alanine were 5 μM and 1 mM, respectively (results corresponded to system 12 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 0.002 mmol of lysine, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence spectrometry. The final concentrations of probe and lysine were 5 μM and 1 mM, respectively (results corresponded to system 13 in Figure 6).

In a graduated tube, add 10 μL of probe solution and 0.002 mmol of threonine, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence spectrometry. The final concentrations of probe and threonine were 5 μM and 1 mM, respectively (results corresponded to system 14 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 4 μg of esterase, dilute the system to 2 mL with phosphate buffer (pH=7.4), react at 37 ° C for 30 min, and conduct fluorescence spectrometry. The final concentrations of the needle and esterase were 5 μM and 2 μg/ml, respectively (results corresponded to system 15 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 2 μg of proline dipeptidase, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence. The final concentrations of the probe, probe and proline dipeptidase were 5 μM and 1 μg/ml, respectively (results corresponded to system 16 in Figure 6).

In a graduated small tube, add 10 μL of probe solution and 4 μg of trypsin, make the system to 2 mL with phosphate buffer (pH=7.4), react at 37 ° C for 30 min, and conduct fluorescence spectrometry. The final concentrations of needle and trypsin were 5 μM and 2 μg/ml, respectively (results corresponded to system 17 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 600 ng of leucine aminopeptidase, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence. The final concentrations of the probe and leucine aminopeptidase were 5 μM and 300 ng/ml, respectively (results corresponded to system 18 in Figure 6).

In a graduated tube, 10 μL of probe solution and 2 μg of fibroblast activation protein were added, and the system was adjusted to 2 mL with phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 min to carry out fluorescence spectroscopy. The final concentrations of probe and fibroblast activator were determined to be 5 [mu]M and 1 [mu]g/ml, respectively (results correspond to system 19 in Figure 6).

In a graduated cuvette, add 10 μL of probe solution and 600 ng of dipeptidyl peptidase 4, dilute the system to 2 mL with phosphate buffer (pH=7.4), and react at 37 ° C for 30 min for fluorescence. The final concentrations of the probe and dipeptidyl peptidase 4 were 5 μM and 300 ng/ml, respectively (results corresponded to system 20 in Figure 6).

In a graduated test tube, 10 μL of probe solution and 60 ng of PGP-1 enzyme were added, and the system was adjusted to 2 mL with phosphate buffer (pH=7.4), and then reacted at 37 ° C for 30 min for fluorescence spectrometry. The final concentrations of the probe and PGP-1 enzyme were 5 μM and 30 ng/ml, respectively (results corresponded to system 21 in Figure 6).

As can be seen from Figure 6, the compound I provided by the present application has excellent selectivity for PGP-1.

Determination of the detection limit of PGP-1 enzyme by the compound I of Example 8

First, configure a series of different concentrations of PGP-1 enzyme system; secondly, add PGP-1 enzyme solution and probe solution to the graduated test tube; then configure the solution to a final volume of 2 mL with phosphate buffer (pH=7.4). After reacting for 30 min in a shaker at 37 ° C, fluorescence spectroscopy was carried out with an excitation wavelength of 670 nm and an emission wavelength of 700 nm. The final concentration of the probe was 5 μM; the final concentrations of PGP-1 enzyme were 0.01 ng/mL, 0.03 ng/mL, 0.06 ng/mL, 0.10 ng/mL, 0.15 ng/mL, 0.20 ng/mL, and 0.25 ng/mL, respectively.

The results are shown in Fig. 7. According to the calculation formula of the detection limit: 3S/m, S is the standard deviation of the blank reagent, n=11, m is the slope of the linear equation, and finally the compound I to PGP-1 provided by the present application is calculated. The detection limit of the enzyme is 0.18 ng/mL, which is known to be the lowest detection limit currently measured.

Comparative Example 1 Determination of Detection Limit of PGP-1 Enzyme by Commercial Fluorescent Probe

First, configure a series of different concentrations of PGP-1 enzyme system; secondly, add PGP-1 enzyme solution and commercial probe (CAS: 66642-36-2) to the graduated test tube; then use phosphate buffer (pH= 7.4) A solution having a final volume of 2 mL was placed; after being reacted for 30 minutes in a shaker at 37 ° C, fluorescence spectroscopy was performed with an excitation wavelength of 340 nm and an emission wavelength of 440 nm. The final concentration of the probe was 5 μM; the final concentrations of PGP-1 enzyme were 50 ng/mL, 75 ng/mL, 125 ng/mL, 150 ng/mL, 175 ng/mL, 225 ng/mL, and 300 ng/mL, respectively.

The results are shown in Fig. 8. According to the calculation formula of the detection limit: 3S/m, S is the standard deviation of the blank reagent, n=11, m is the slope of the linear equation, and finally the compound I to PGP-1 provided by the present application is calculated. The detection limit of the enzyme was 14.6 ng/mL, which is much higher than the detection limit of Compound I provided by the present application.

Fluorescence imaging and semi-quantitative detection of intracellular PGP-1 enzyme by compound I of Example 9

RAW264.9 cells (from Jiangsu Kaiji Biotechnology Co., Ltd.) were cultured in a 5% carbon dioxide incubator at 37 ° C, and can be co-incubated with different drugs depending on the purpose of the experiment. This example studied drug stimulation. Effect on PGP-1 content.

The blank control cells in Fig. 9 were cultured normally, and the 1# and 2# cells were incubated with lipopolysaccharide for 12 h (concentration: 0.5 μg/mL, 1.0 μg/mL, respectively), and washed with serum-free medium after the incubation. Three times, the same concentration of probe solution (final concentration 5 μM) was added, and incubation was continued for 30 min at 37 ° C. After the incubation was completed, the unreacted probe was washed with the medium, and then the cells were imaged (Fig. 9 left 6 images) ) Semi-quantitative analysis of fluorescence intensity can be performed using analysis software (Figure 9 right panel). It can be seen from Fig. 9 that the expression of PGP-1 is increased in the RAW cells stimulated by the drug, and the fluorescence intensity can be semi-quantitatively analyzed by the analysis software to characterize the expression level of PGP-1 stimulated by different concentrations of the drug. Similarly, studies and semi-quantitative analysis of pharmaceutical inhibitors can also be carried out using Compound I provided herein.

Fluorescence Imaging and Quantitative Detection of PGP-1 Enzyme in Animals by Compound I of Example 10

The compound I provided by the present application has excitation and emission at a near-infrared wavelength, and the advantage of deep penetration in the near-infrared is advantageous for in vivo imaging of fluorescence. Therefore, this example studies the detection of the probe in the body of BABL/c mice. And imaging. In this embodiment, lipopolysaccharide was intradermally injected into the liver, and after 2 hours, 100 μL of a probe having a concentration of 50 μM was injected through the tail vein, and fluorescence imaging was performed using a small animal imager. The results are shown in Fig. 10(a). . 1# mice were used as the control group, and 2# mice were the experimental group for intradermal injection of lipopolysaccharide in the liver. In Fig. 10(b), column 1 corresponds to 1# mouse, and column 2 corresponds to 2# mouse. As can be seen from Fig. 10, the fluorescence signal at the liver of the experimental group injected with lipopolysaccharide in the liver was significantly higher than that of the control group, indicating that the expression of PGP-1 in the liver was increased after drug stimulation, and the compound provided by the present application. I successfully achieved specific recognition and fluorescence imaging of PGP-1 in vivo.

Example 11 Application of Compound I in Cancer Detection

During the process of proliferation and proliferation of non-neuronal cells, the expression of PGP-1 was positively correlated with the rate of cell proliferation. Since the canceration of cells can lead to infinite and rapid division and proliferation of cells, it is possible to predict the cell proliferation of this part by monitoring the change of the content of PGP-1 in a certain part of the body, and then make a judgment reference for the diagnosis of cancer. In this example, BABL/c mice harboring a cervical cancer virus model were subjected to probe injection daily for in vivo imaging to observe changes in the expression level of PGP-1 in the virus-containing site. It was found that the expression of PGP-1 is related to the carcinogenesis of cells. With the deepening of cell carcinogenesis, the expression of PGP-1 is obviously increasing. Therefore, the high sensitivity of this probe and the specific recognition of PGP-1 are expected to be realized. The earliest detection of cancer.

Evaluation of the efficacy of Compound I of Example 12 against cancer drugs in vivo and at the cellular level

Since the canceration of cells is positively correlated with the expression of PGP-1, it can be used as an evaluation of the therapeutic effect of cancer drugs by using the probe prepared by the present invention in combination with a drug for treating cancer. In this example, BABL/c mice inoculated with MCF-7 were injected with a probe after administration of DOX, and then observed changes in the fluorescence signal intensity in the tumor region. The results showed that the degree of canceration in mice was positive with the intensity of the fluorescent signal. Related, so this finding can be used to evaluate the effects of cancer treatment.

Example 13 Compound I was used for imaging glial cells and detection of PGP-1

PGP-1 is specifically expressed in glial cells and can therefore be used to detect lesions in the glial zone. In this embodiment, the compound I prepared by the present invention is structurally modified so that the terminal group at the propane is a carboxyl group-COOH, and then chemically synthesized to be linked to an anticancer drug having an amino group at one end, and then coated with an amphiphilic high on the outside. Molecules form micelles, which are injected into mice with glioma by injection into the tail vein, and the detection of fluorescence is used to determine whether the cancer therapeutic drug has passed the blood-brain barrier system (ie, the BBB system). The therapeutic effect of cancer can be further judged by the change in fluorescence intensity.

Example 14 Compound I for the detection of atherosclerosis

Atherosclerotic plaques often exhibit inflammatory cell infiltration, whereas PGP-1 exhibits high expression in inflammatory regions because it specifically hydrolyzes immunogenic proteins (both Examples 8 and 9 validated this result). This embodiment is based on the high inflammatory expression of the atherosclerotic region, and thus the plaque is realized by the MRI/FL dual-modality imaging technique by combining the probe prepared by the present invention with the clinical steroid-like contrast agent. Accurate positioning. The use of organic small molecule probes for in vivo fluorescence imaging can not only accurately determine the lesion area before surgery, but also play a guiding role in the operation, which plays a very positive role in the accurate resection of the diseased tissue.

Example 15 Compound I for the detection of inflammation at the joint

Since the degree of inflammatoryness of cells is positively correlated with the expression of PGP-1, the probe prepared by the present invention can be used for detection and imaging of inflammatory regions. In the present embodiment, by establishing a model of arthritis, the degree of lesion detection and imaging analysis of inflammation in the joints in the body is achieved. The specific embodiment is that the drug LPS is subcutaneously injected into the sole of the BABL/c mouse, divided into different concentration groups and different time groups, and then the probe is injected subcutaneously at the foot portion for imaging analysis and immunohistochemical analysis. Further verify the analysis. The results show that the fluorescence intensity at the joint is positively correlated with the degree of inflammation, so the probe prepared by the present invention can be used for the analysis and detection of the degree of inflammatory lesions at the joint.

Evaluation of the efficacy of Compound I of Example 16 on anti-inflammatory drugs in vivo and at the cellular level

Since the degree of inflammation of cells and tissues is positively correlated with the expression of PGP-1, it can be used as an evaluation of the therapeutic effect of inflammatory drugs by using the probe prepared by the present invention in combination with a drug for treating inflammation. In the present example, on the basis of Example 14, a mouse having arthritis was treated with an anti-inflammatory drug, and the therapeutic effect of the anti-inflammatory drug was evaluated by analyzing the change in fluorescence intensity. The results show that the intensity of fluorescence is positively correlated with the degree of inflammatoryness, and therefore the probe prepared by the present invention can be used not only for the detection and imaging of inflammatory tissues, but also for the evaluation of the efficacy of anti-inflammatory drugs.

The above description is only a few examples of the present application, and is not intended to limit the scope of the application. However, the present application is disclosed in the preferred embodiments, but is not intended to limit the application, any person skilled in the art, It is within the scope of the technical solution to make a slight change or modification with the technical content disclosed above, which is equivalent to the equivalent embodiment, without departing from the technical scope of the present application.

Claims

A compound I, characterized in that the compound I comprises an organic cation and an inorganic anion;

The organic cation is selected from at least one of the cations having the chemical formula of Formula I;

In the formula I, R 101 , R 102 , R 103 , R 104 , R 106 , R 107 , R 108 , R 109 , R 110 , R 111 , R 112 , R 113 , R 114 , R 115 , R 116 , R 117 , R 118 and R 119 are independently selected from the group consisting of hydrogen, a C 1 - C 20 alkane group, and a C 1 - C 20 alkane group having a substituent; R 105 is selected from the group consisting of hydrogen, a C 1 - C 20 alkane group, and C. 1 to C 20 an alkane group having a substituent;

The substituent-containing alkane group contains at least one substituent; and the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.
The compound I according to claim 1, wherein in the formula I, R 101 , R 102 , R 103 , R 104 , R 106 , R 107 , R 108 , R 109 , R 110 , R 111 , R 112 , R 113 , R 114 , R 115 , R 116 , R 117 are hydrogen;

R 105 , R 118 and R 119 are independently selected from the group consisting of hydrogen, a C 1 - C 20 alkane group, and a C 1 - C 20 alkane group having a substituent; the alkyl group having a substituent containing at least one substituent; The substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.
The compound I according to claim 2, wherein the compound I has an excitation wavelength or an absorption wavelength in the range of 500 nm to 750 nm.
The compound I according to claim 2, wherein the compound I has an emission wavelength in the range of 700 nm to 810 nm.
The method for preparing a compound I according to any one of claims 1 to 4, comprising the steps of:

a) adding a basic activator to the solution containing the compound II and the compound III, after washing, washing with water, drying, to obtain a mixture A;

The compound II is selected from at least one of the compounds having the formula of the formula II:

In the formula II, R 201 , R 202 , R 203 , R 204 , R 206 , R 207 , R 208 , R 209 , R 210 , R 211 , R 212 , R 213 , R 215 , R 216 , R 217 , R 218 , R 219 , R 220 , R 221 , R 222 , R 223 , R 224 are independently selected from hydrogen, C 1 -C 20 alkane; R 205 , R 214 are independently selected from hydrogen, C 1 -C 20 An alkane group, a C 1 - C 20 alkane group having a substituent; the substituent-containing alkane group having at least one substituent; the substituent being selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group At least one; X 1 , X 2 are independently selected from the group consisting of F, Cl, Br, I;

The compound III is selected from at least one of the compounds having the formula of the formula III:

In the formula III, R 301 , R 302 , R 303 , and R 304 are independently selected from hydrogen, a C 1 - C 5 alkane group, and a C 1 - C 5 substituent-containing alkane group; the substituent-containing alkane group; Containing at least one substituent; the substituent is selected from at least one of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group;

b) adding a catalyst to the mixture A, in an inert atmosphere, after reacting at 60-80 ° C, washed with water, and dried to obtain a mixture B;

c) using an organic base and / or an inorganic base activator to activate compound IV, to obtain activated compound IV;

The compound IV is selected from at least one of the compounds having the formula of the formula IV:

In Formula IV, R 401 , R 402 , R 403 , R 404 , R 408 are independently selected from hydrogen, C 1 -C 20 alkane; R 405 , R 406 , R 407 , independently selected from C 1 -C Alkane group of 20 , C 1 - C 20 alkane group having a substituent; the alkyl group containing a substituent having at least one substituent; the substituent being selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group At least one type;

d) after the activated compound IV and the mixture B is mixed, and reacted at -10 ° C to 80 ° C for 0.01 h to 24 h, washed with water and dried to obtain a mixture C;

e) adding an acidic reagent to the mixture C for decarboxylation, and after completion of the reaction, after drying and purification, the compound I is obtained.
The preparation method according to claim 5, wherein in the formula II, R 201 , R 202 , R 203 , R 204 , R 206 , R 207 , R 208 , R 209 , R 210 , R 211 , R 212 , R 213 , R 215 , R 216 , R 217 , R 218 , R 221 and R 222 are hydrogen;

R 205 , R 214 , R 219 , R 220 , R 223 , and R 224 are independently selected from the group consisting of hydrogen, a C 1 - C 20 alkane group, and a C 1 - C 20 alkane group having a substituent; the substituent-containing group The alkane group contains at least one substituent; the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group;

In the formula III, R 301 , R 302 , R 303 , and R 304 are hydrogen;

In the formula IV, R 401 , R 402 , R 403 , R 404 and R 408 are hydrogen; R 405 , R 406 and R 407 are independently selected from C 1 to C 20 alkane groups, and C 1 to C 20 are contained. a substituted alkane group; the substituent-containing alkane group having at least one substituent; and the substituent is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an ester group, and an amide group.
The preparation method according to claim 5, wherein the molar ratio of the compound II, the compound III and the basic activator in the step a) is:

Compound II, Compound III and basic activator = 1:1 to 3:1 to 5.
The preparation method according to claim 5, wherein the alkaline activator in the step a) is selected from the group consisting of NaH, CaH 2 , KH, NaOH, KOH, Na 2 CO 3 , NaHCO 3 , K 2 CO 3 . At least one of KHCO 3 , MgCO 3 , Mg(HCO 3 ) 2 , CaCO 3 , Ca(HCO 3 ) 2 , trimethylamine, triethylamine, DBN, DBU, and sodium alkoxide.
The preparation method according to claim 5, wherein the catalyst in the step b) is at least one selected from the group consisting of FeCl 2 , SnCl 2 , CuCl, and CoCl 2 .
The preparation method according to claim 5, wherein the molar ratio of the catalyst in the step b) to the mixture A is:

Catalyst: Mixture A = 1:1 to 5:1.
The preparation method according to claim 5, wherein the activator for activating the compound IV in the step c) comprises N,N-diisopropylethylamine and 2-(7-benzobenzotriazole). -N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride, 1-hydroxybenzotriene At least one of azole and N-hydroxysuccinimide.
The preparation method according to claim 5, wherein the mass ratio of the activated compound IV to the mixture B in the step d) is:

Compound IV after activation: mixture B = 1:1 to 5:1.
The preparation method according to claim 5, wherein the acidic reagent in the step e) is at least one selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, glacial acetic acid, formic acid, and p-toluenesulfonic acid.
A fluorescent probe comprising at least one of the compound I according to any one of claims 1 to 4 and the compound I obtained by the method according to any one of claims 5 to 13.
The fluorescent probe according to claim 14, wherein the fluorescent probe is for detecting pyroglutamate aminopeptidase 1.
The fluorescent probe according to claim 14, wherein the fluorescent probe has an emission concentration λ ex 670 nm and an excitation wavelength λ em 700 nm. The lower limit of detection concentration of pyroglutamate aminopeptidase 1 does not exceed 0.2 ng/ mL.
The fluorescent probe according to claim 14, wherein the fluorescent probe has a detection concentration lower limit of 0.18 ng/mL for the pyroglutamic acid aminopeptidase 1 at an emission wavelength λ ex 670 nm and an excitation wavelength λ em 700 nm. .
The at least one of the compound I according to any one of claims 1 to 4, and the compound I obtained according to the method of any one of claims 5 to 13, for preparing a detection reagent for detecting pyroglutamic acid aminopeptidase 1 And/or application of pyroglutamate aminopeptidase 1 imaging reagent.
At least one of the compound I according to any one of claims 1 to 4, and the compound I prepared according to the method of any one of claims 5 to 13 for producing a detection reagent for detecting inflammatory cells and/or inflammatory cell imaging Application in reagents.
At least one of the compound I according to any one of claims 1 to 4, and the compound I prepared according to the method of any one of claims 5 to 13, for preparing a detection reagent for detecting cancerous cells and/or cancerous cell imaging Application in reagents.
At least one of the compound I according to any one of claims 1 to 4, and the compound I obtained by the method according to any one of claims 5 to 13, for preparing a therapeutic effect for evaluating an anti-inflammatory drug and/or a cancer therapeutic drug. Application in detection reagents and/or imaging reagents.