Precursor compound for preparing targeted telomerase subunit hTERT (human telomerase reverse transcriptase) molecular probe, molecular probe, preparation method and application

Abstract

The invention relates to the technical field of medical tumor imaging agents, in particular to a precursor compound for preparing a targeted telomerase subunit hTERT molecular probe, the molecular probe, a preparation method and application, the prepared compound NPIP serves as a precursor for preparing the targeted telomerase subunit hTERT molecular probe, and the probe precursor compound comprises an aromatic ester ring part and an onium salt part; the aromatic ester ring part has an active group combined with a telomerase subunit hTERT; the onium salt part is positioned at the para-position of the aromatic ester ring part and is used for carrying out nucleophilic substitution reaction with radionuclide 18F. The compound has a unique structure, a methanol solution of the compound can efficiently leach < 18 > F <-> ions, the defects of a traditional method are avoided, a radioactive molecular probe can be prepared through a one-step method, a foundation is laid for developing a novel tumor imaging agent, the difficulty faced by current cancer diagnosis and treatment is expected to be solved, and the cancer diagnosis and treatment level is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical tumor imaging agents, and particularly relates to a precursor compound, a molecular probe, a preparation method and an application for preparing a molecular probe targeting the telomerase subunit hTERT. Background Art

[0002] The activation of telomerase is an important step in the occurrence and development of cancer. Currently, three subunits of human telomerase have been isolated: human telomerase RNA (hTR, Human telomerase reverse), human telomerase binding protein (TP1, Humantelomerase binding protein1), and human telomerase reverse transcriptase (hTERT, Human telomerase reversetranscriptase). hTR is the template for telomere elongation, and its presence is not necessarily related to telomerase activity. TP1 can specifically bind to hTR to form a template substrate binding site. hTERT is not expressed in most normal cells, but is highly expressed in primary tumors and cancer cell lines, and is proportional to telomerase activity.

[0003] Currently, drugs for treating cancer by inhibiting telomerase activity include the following aspects: nucleotides, oligonucleotides, G-quadruplex stabilizers, immunotherapeutic agents, gene therapeutic agents, and small molecule compounds targeting telomerase subunits hTERT and hTR. Among them, small molecule compounds targeting telomerase subunits hTERT and hTR, such as BIBR1532 ([2-[E]-3-naphthalene-2-yl-but-2-enoylylamino]-benzoi c acid), are aryl vinyl telomerase inhibitors developed and studied by Boehringer Ingelheim GmbH in 2002. Molecular docking simulation studies have found that BIBR1532 can tightly bind in the hydrophobic pocket of the telomerase catalytic subunit hTRTE and interact with multiple amino acid residues. In addition, there are also benzylidene hydrazone compounds, myricetin derivatives, aloe-emodin derivatives, curcumin derivatives, aromatic naphthoquinones, isocoumarin rings, and so on. These small molecule compounds have a characteristic in binding to telomerase: there is a "pocket" in the three-dimensional structure of telomerase that can "accommodate" these compounds.

[0004] Existing telomerase inhibitors are mostly used in the treatment of cancer. Some have entered preclinical trials, but the number of patients who benefit is small, and there are also problems such as slow onset. At the same time, there is little research on telomerase-related proteins, and these proteins are expected to become new anti-cancer targets. In terms of existing technical solutions, in 2016, the Frederik L. Giesel team developed [18F]PSMA-1007 based on 177Lu-PSMA-617, which is similar to 68Ga-PSMA-617 in terms of structure, biodistribution, and tumor uptake; as Figure 1 shown. However, there is currently no research on telomerase receptors in the development of pan-tumor imaging agents, and the structural modification of telomerase inhibitors with radionuclides has great application potential. In addition, the capture methods of radioactive 18F - ion nuclides produced by cyclotrons are basically carried out using QMA carbonate columns for capture, and the reagents for eluting 18F - ions usually include K2.2.2 and physiological saline. These reagents will add extra water molecules, increasing the reaction steps. Although there are many methods for 18F - labeling, the vast majority of radioactive fluorine compounds are prepared by aliphatic and aromatic nucleophilic substitution reactions with 18F - ions. Due to strong hydration, water molecules significantly reduce the nucleophilicity of 18F - . In order to remove most of the water, most of them are removed by inefficient and time-consuming (7-15 minutes) repeated azeotropy with acetonitrile.

[0005] Therefore, there is an urgent need to develop new telomerase inhibitors and related technologies, which is of great significance for solving many problems faced in the current field of cancer diagnosis and treatment. Among them, the preparation of molecular probes targeting the hTERT receptor of telomerase subunits has become one of the key breakthrough points. Considering the decay characteristics of radionuclides, for example, the half-life of 18F - is about 110 min, and its radioactivity will gradually decrease over time. When preparing radioactive molecular probes (tracers, imaging agents), in order to ensure the preparation efficiency and quality, the one-step synthesis method is currently mostly used. This requires that an effective and stable precursor compound must be obtained before preparing the radioactive molecular probe.

[0006] Explanation of terms in the present invention:

[0007] (1) Precursor: It refers to a compound that has no tracer effect itself after chemical structure modification of a drug, but can react quickly with a radionuclide to form a compound with a tracer target in the body.

[0008] (2) Radioactive molecular probe: A compound that can be metabolically bound to a target in the body based on the radioactive characteristics of a radionuclide, and thus realizes PET imaging.

[0009] (3) Structural modification: Appropriate modification of the structure of the active ingredient can improve its performance or increase its functions, which is one of the effective ways to explore and develop new drugs.

[0010] (4) Onium salt: (English: Onium Compound), is a cation obtained by protonating the mononuclear hydrides of nitrogen group elements (Group V / Group VA / Group 15), oxygen group elements (Group VI / Group VIA / Group 16), and halogens (Group VII / Group VIIA / Group 17), as well as some derivative cations formed by replacing hydrogen atoms with other groups (such as: organic radicals, halogen atoms, tetramethylammonium); more complex derivatives contain multiple central atoms, such as iminium ions and arsonium ions. They are also called onium ions, and the compounds formed by these ions are called onium salts.

[0011] (5) PET imaging: PET is short for positron emission tomography (PET), which is an imaging device that reflects the gene, molecular, metabolic, and functional status of lesions. It uses positron radionuclide-labeled glucose and other human metabolites as imaging agents, and reflects their metabolic changes through the uptake of the imaging agent by the lesions, so as to provide biological metabolic information of diseases for clinical use. Summary of the Invention

[0012] In order to solve the deficiencies existing in the above-mentioned prior art, the present invention provides a precursor compound, a molecular probe, a preparation method and an application for preparing a molecular probe targeting the telomerase subunit hTERT. This precursor has the structural characteristics of an onium salt, and its ethanol solution can directly elute 18F - ions from the QMA anion exchange resin. Neither azeotropic evaporation of water, nor an alkali, nor any other additives such as crown ethers are required. Due to its simplicity, this method should be very suitable for automated radiochemical synthesis.

[0013] Specifically, the present invention provides a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT, and this precursor compound includes an aromatic ester ring part A and an onium salt part B;

[0014] The aromatic ester ring part A has an active group that binds to the telomerase subunit hTERT;

[0015] The onium salt part B is located at the para position of the aromatic ester ring part A and is used for nucleophilic substitution reaction with the radioactive nuclide 18 F.

[0016] Furthermore, the compound is NPIP, its molecular formula is C 28 H 23 ClINO8, molecular weight: 663.85, and its molecular structure is as follows:

[0017]

[0018] A preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT, and the synthesis steps of the compound NPIP are as follows:

[0019] S1. Synthesize NPIP-2 from NPIP-1; under nitrogen protection, add TMSOAc (210 mg, 1.591 mmol) to a solution of NPIP-1 (280 mg, 0.612 mmol) in ACN (10 ml), and react at room temperature for 4 h; then add potassium (4-methoxyphenyl)trifluoroborate (123 mg, 0.612 mmol) in ACN (10 ml) to the reaction system and continue to react for 1 h; LCMS shows that the raw materials have reacted completely and the target product NPIP-2 is formed; the reaction solution is directly used for the next step without treatment.

[0020] S2. Synthesize NPIP from NPIP-2; under nitrogen protection, add 1N sodium periodate aqueous solution (10 ml) to the above reaction solution and react at room temperature for 30 min; after the reaction is completed, directly spin-dry the reaction solution, and subject the residue to column chromatography separation and purification after mixing the sample. Eluent: DCM:MeOH (1:0 → 99:1), and 42 mg of off-white solid is obtained with a yield of 12%. The main peak of the liquid chromatography-mass spectrometry combined spectrum of NPIP is LC-MSm / z(ESI): 564.1[M+1]+.

[0021] The reaction formulas in the processes of S1 and S2 are as follows:

[0022]

[0023] A molecular probe targeting the telomerase subunit hTERT, and this molecular probe is 18F-BIBR1532, and its molecular structure is as follows:

[0024]

[0025] Furthermore, the synthesis method is as follows

[0026] Capture 18F - ions with an activated QMA column, and then dissolve 1 mg of the precursor compound NPIP in 2 mL of methanol solution. The molecular formula of the NPIP is C 28 H 23ClINO8, Molecular weight: 663.85; Elute the QMA column, collect the mixed solution with a reaction flask, blow in N2 gas or helium gas, evaporate the methanol solvent to dryness, then add 1 mL of DMSO to the reaction flask to dissolve, heat to 80 - 120 °C, react for 10 min, and take out; add 4 mL of injection-grade H2O to terminate the reaction, purify the reaction solution on a column (C18 column), elute with ethanol, pass through a sterile filter membrane, and dilute with physiological saline to obtain the molecular probe; the reaction formula is as follows:

[0027]

[0028] Among them, LG + includes Me3N + 、PhI + or (4-MeOPh)I + ; X - includes I - 、Br, ClO4 - 、HCO3 - or TfO - ,They are combined pairwise to form an onium salt

[0029] Working principle: (1) Select the telomerase subunit with high expression of telomerase activity in cancer cells as the target to locate cancer cells. Among the above seven types of telomerase inhibitors, only the last type has its action target on the human telomerase subunit, and it binds firmly to telomerase through hydrogen bonds, covalent bonds, and van der Waals forces, forming a "pocket and content" relationship. Comprehensive analysis of the seventh type of inhibitor reveals that their chemical structures have several common structural features (see the following formula): ① There are R-NH-N = C-R1, -NH-CO-peptide bond, and -CH = N-functional groups in the structure. The former is the active group for inhibiting telomerase, while the latter is the necessary group for binding to the human telomerase subunit. ② The entire molecular structure is divided into three parts: polar part - non-polar part - polar part. Such a structure may contribute to the formation of hydrogen bonds. ③ Most of the compounds contain aromatic heterocycles. The planar structure of the aromatic heterocycles may be beneficial for insertion into the "pocket" of telomerase. In addition, taking the 18F - ion as a modifying group, firstly, it cannot affect the pharmacokinetics of this type of inhibitor, and secondly, it does not affect the physicochemical properties and pharmacological characteristics of this type of molecule. Then there are strict requirements for the position where the 18F - ion is introduced. It must be introduced into an unimportant position, neither affecting the binding to the telomerase subunit nor changing the physicochemical and pharmacological properties of this type of molecule.

[0030]

[0031] (2) The structure-activity relationship of this compound;

[0032] The molecular structure of the compound is divided into two parts: the aromatic ester ring part A and the onium salt part B. Part A is the mother of the entire molecule, which has the ability to bind to the telomerase subunit hTERT with an affinity at the nanometer level. Part B is a substituent set at the para position of the molecule. Based on the structural properties of the onium salt (elution QMA and nucleophilic reaction), it can introduce radioactive nuclides at the para position of the mother nucleus (with minimal steric hindrance). 18 The purpose of F is to achieve the radioactivity visualization of the molecule, thus realizing PET imaging.

[0033] (3) The synthetic route of the compound and its application as a precursor compound;

[0034]

[0035] After capturing 18F- ions with an activated QMA column, dissolve 1 mg of the precursor compound with 2 mL of methanol solution, rinse the QMA column, collect the mixed solution with a reaction bottle, blow in N2 gas or helium, evaporate the methanol solvent, then add 1 mL of DMSO to the reaction bottle to dissolve, heat to 80-120°C, react for 10 minutes, and take out. Add 4 ml of H2O for injection to terminate the reaction, and purify the reaction solution on a column (C18 column), elute with ethanol, filter through a sterile filter membrane, and dilute with physiological saline to obtain the final product. .

[0036] Beneficial effects:

[0037] 1. The radioactive molecular probe prepared by the present invention can specifically bind to telomerase and accurately locate malignant cancer cells. Compared with traditional diagnostic methods, it uses radionuclides 18 F labeling technology can achieve efficient tracking of cancer cells in the body. Through PET imaging, the location, size and distribution of cancer cells can be clearly presented, greatly improving the accuracy and efficiency of early cancer diagnosis and buying precious treatment time for patients.

[0038] 2. The NPIP of the present invention has an onium salt structure, and its ethanol solution can be directly eluted from the QMA anion exchange resin to obtain 18F - ions, avoiding the problem of introducing water molecules by using K2.2.2 and saline elution in the traditional method, eliminating the need for the time-consuming (7-15 minutes) acetonitrile azeotropic dehydration step, reducing the number of reaction steps, simplifying the 18F labeling process, and improving the efficiency of the preparation of radioactive molecular probes.

[0039] 3. The present invention first applies telomerase inhibitors to the field of nuclear medicine, providing a new direction for the development of nuclear drugs. As a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT, NPIP is expected to promote the development of novel pan-cancer imaging agents. The emergence of this new type of imaging agent not only enriches the variety of nuclear drugs but also may bring new breakthroughs in the diagnosis and treatment monitoring of various cancers, injecting new vitality into the development of the nuclear medicine field.

[0040] 4. The present invention designs a precursor compound based on in-depth research on the telomerase subunit hTERT, which helps to further explore the mechanism of action of telomerase in the occurrence and development of cancer. Through the study of the interaction between NPIP and the telomerase subunit hTERT, it can provide a theoretical basis for the subsequent development of more therapeutic drugs targeting telomerase-related proteins, explore the therapeutic value of these potential targets, and open up new ways for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagrams of the structures, biodistributions, and tumor uptakes of 177Lu-PSMA-617 and [18F]PSMA-1007 described in the background art.

[0042] Figure 2 Detection data of the liquid phase and mass spectrometry of NPIP;

[0043] Figure 3 Detection data of the liquid phase and mass spectrometry of NPIP;

[0044] Figure 4 Detection data of the liquid phase and mass spectrometry of NPIP;

[0045] Figure 5 Detection data of the liquid phase and mass spectrometry of NPIP. DETAILED DESCRIPTION OF THE INVENTION

[0046] The following will describe the technical solutions in detail with reference to the accompanying drawings in the embodiments of the present invention.

[0047] Example 1

[0048] 1. Design concept and chemical name of the chemical structure of NPIP;

[0049] Chemical name: NPIP

[0050] Use: Precursor labeled with [18F - ion

[0051] CAS No: Unregistered

[0052] Molecular formula: C28H23ClINO8

[0053] Molecular weight: 663.85

[0054]

[0055] In Example 1, the telomerase subunit with high expression of telomerase activity in cancer cells was selected as the target to locate cancer cells. Among the above seven types of telomerase inhibitors, only the last type has its action target on the human telomerase subunit and firmly binds to telomerase through hydrogen bonds, covalent bonds and van der Waals forces, forming a "pocket and content" relationship. Through comprehensive analysis of the seventh type of inhibitors, it is found that their chemical structures have several common structural features (see the following formula): ① There are R-NH-N=C-R1, -NH-CO-peptide bond and -CH=N-functional groups in the structure. The former is the active group that inhibits telomerase, while the latter is the necessary group for binding to the human telomerase subunit. ② The entire molecular structure is divided into three parts: polar part - non-polar part - polar part. Such a structure may contribute to the formation of hydrogen bonds. ③ Most of the compounds contain heteroaromatic rings. The planar structure of the heteroaromatic rings may be beneficial for insertion into the "pocket" of telomerase. In addition, taking the 18F - ion as a modifying group, first, it cannot affect the pharmacokinetics of such inhibitors, and second, it does not affect the physicochemical properties and pharmacological characteristics of this type of molecule. Then, there are strict requirements for the position where the 18F - ion is introduced. It must be introduced into an unimportant position, which neither affects the binding to the telomerase subunit nor changes the physicochemical and pharmacological properties of this type of molecule.

[0056]

[0057] 2. The structure-activity relationship of the compound;

[0058] The molecular structure of the compound is divided into two parts: the aromatic ester ring part A and the onium salt part B. Part A is the parent body of the whole molecule, and it has the ability to bind to the telomerase subunit hTERT, with an affinity at the nanometer level. Part B is a substituent set at the para position of the molecule. According to the structural properties of the onium salt (elution QMA and nucleophilic reaction), the purpose of introducing the radionuclide 18 F at the para position of the parent nucleus (with the smallest steric hindrance) is realized, so as to realize the radioactive visualization of this molecule, that is, PET imaging can be achieved.

[0059] 3. The synthetic route of the compound and its application as a precursor compound;

[0060]

[0061] After capturing 18F- ions with an activated QMA column, dissolve 1 mg of the precursor compound in 2 mL of methanol solution, wash the QMA column, collect the mixed solution with a reaction flask, blow in N2 gas or helium gas, evaporate the methanol solvent to dryness, then add 1 mL of DMSO to the reaction flask to dissolve, heat to 80 - 120 °C, react for 10 min, and take out. Add 4 mL of injectable H2O to terminate the reaction, purify the reaction solution by column chromatography (C18 column), elute with ethanol, pass through a sterile filter membrane, and dilute with physiological saline to obtain the final product.

[0062] The preparation method of the radionuclide structure modification of the telomerase inhibitor and the precursor for preparing the molecular probe in this example is as follows:

[0063] Synthesis of NPIP:

[0064]

[0065] Synthesis of intermediate NPIP-2: Under nitrogen protection, add TMSOAc (210 mg, 1.591 mmol) to a solution of NPIP-1 (280 mg, 0.612 mmol) in ACN (10 ml), and react at room temperature for 4 h. Then add potassium (4-methoxyphenyl)trifluoroborate (123 mg, 0.612 mmol) in ACN (10 ml) to the reaction system and continue to react for 1 h. LCMS shows that the raw materials have reacted completely and the target product is formed. The reaction solution is used directly for the next step without treatment.

[0066] Synthesis of NPIP:

[0067] Under nitrogen protection, add 1 N aqueous sodium periodate solution (10 ml) to the above reaction solution and react at room temperature for 30 min. After the reaction is completed, directly spin-dry the reaction solution, subject the residue to sample mixing for column chromatography separation and purification, eluent: DCM:MeOH (1:0 → 99:1), to obtain 42 mg of an off-white solid, with a yield of 12%. The main peak of the liquid chromatography - mass spectrometry combined spectrum of NPIP is LC - MS m / z (ESI): 564.1 [M + 1]+.

[0068] Full name of NPIP:

[0069] (E)-(6~(4-((2-carboxyphenyl)amino)-4-oxobut-2-en-2-yl)naphthalen-2-yl)(4-methoxyphenyl)iodonium perchlorate; ((E)-(6~(4-((2-carboxyphenyl)amino)-4-oxobut-2-en-2-yl)naphthalen-2-yl)(4-methoxyphenyl)iodonium perchlorate.

[0070] Example 2

[0071] Different from Example 1, systematic experimental tests were conducted on the onium salt substitution in this example. Different substitution positions were selected respectively, and various onium salts formed by pairwise combination of LG + and X - were used to carry out reactions in different solvents such as tetrahydrothiophene sulfone and DMSO. The reaction temperature and time were strictly controlled, and the reaction completion rate (RCC%) was recorded. Through a large number of repeated experiments and data statistical analysis, the results showed that the onium salts could undergo substitution reactions under these different reaction conditions, which provided important experimental basis for the synthesis and application of subsequent related compounds.

[0072] Multiple groups of experiments were carried out on different substitution positions, various combinations of onium salts (LG + X - ) and different solvents (tetrahydrothiophene sulfone, DMSO), and the reaction temperature, time and reaction completion rate were accurately recorded. Based on these reliable data, the feasibility of onium salt substitution was clarified in the present invention. This achievement will be directly applied to the optimization of the synthesis process of the precursor compound for the subsequent preparation of the molecular probe targeting the telomerase subunit hTERT, which is expected to improve the preparation efficiency and product quality. The experimental data are as follows:

[0073]

[0074]

[0075]

[0076] Note: Among them, LG + includes Me3N + , PhI + or (4-MeOPh)I + ; X - includes I - , Br, ClO4 - , HCO3 - or TfO - , and they can be combined pairwise to form onium salts.

Claims

1. A precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT, characterized in that: The precursor compound includes an aromatic ester ring moiety and an onium salt moiety; The aromatic ester ring moiety is the parent of the precursor compound molecule and has an active group for binding to the telomerase subunit hTERT; the onium salt moiety is a para-substituent provided in the precursor compound molecule; The oxonium salt moiety is located at the para position of the aromatic ester ring moiety and is used for nucleophilic substitution reaction with the radionuclide 18 F.

2. The precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT according to claim 1, wherein: The compound is NPIP, and its molecular formula is C 28 H 23 ClINO8, molecular weight: 663.85, and its molecular structure is as follows:

3. A preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT as described in claim 2, characterized in that: The synthesis reaction formula of the compound NPIP is as follows:

4. The preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT as claimed in claim 3, characterized in that: The synthesis steps of the compound NPIP are as follows: S1. Synthesize NPIP-2 from NPIP-1, including: under nitrogen protection, add TMSOAc to the ACN solution of NPIP-1 and react at room temperature; then add the ACN of potassium (4-methoxyphenyl)trifluoroborate to the reaction system and continue to react; LCMS shows that the raw materials have reacted completely and the target product NPIP-2 is formed; S2. Synthesize NPIP from NPIP-2, including: under nitrogen protection, add an aqueous solution of sodium periodate to the above reaction solution and react at room temperature; after the reaction is completed, directly evaporate the reaction solution to dryness, and carry out column chromatography separation and purification on the residue by mixing samples, and elute with an eluent to obtain the target product NPIP as a white solid.

5. The preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT according to claim 4, characterized in that, Step S1 is specifically: Prepare a 10 ml ACN solution of 0.612 mmol of NPIP-1, add 1.591 mmol of TMSOAc to the solution and react at room temperature for 4 h; then prepare 10 ml of ACN of 0.612 mmol of potassium (4-methoxyphenyl)trifluoroborate and add it to the reaction system and continue to react for 1 h; LCMS shows that the raw materials have reacted completely and the target product NPIP-2 is formed; the reaction solution is used directly for the next step without treatment.

6. The preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT as claimed in claim 4, characterized in that, Step S2 is specifically: Add 10 ml of 1N aqueous sodium periodate solution to the above reaction solution and react at room temperature for 30 min; after the reaction is completed, directly evaporate the reaction solution to dryness, and carry out column chromatography separation and purification on the residue by mixing samples. The eluent is DCM:MeOH, 1:0→99:1, and 42 mg of the target product NPIP as a white solid is obtained, with a yield of 12%.

7. The preparation method of a precursor compound for preparing a molecular probe targeting the telomerase subunit hTERT according to claim 6, characterized in that, The main peak of the liquid chromatography-mass spectrometry combined spectrum of NPIP is LC-MS m / z (ESI): 564.1 [M+1]+.

8. A molecular probe targeting the telomerase subunit hTERT, characterized in that: The molecular probe is 18F-BIBR1532, and its molecular structure is as follows:

9. The molecular probe targeting the telomerase subunit hTERT according to claim 8, characterized in that, Its reaction formula is as follows: Wherein: LG + includes Me3N + 、PhI + or (4-MeOPh)I + ; X - includes I - 、Br, ClO4 - 、HCO3 - or TfO - , and they are combined pairwise to form an onium salt.

10. A molecular probe targeting the telomerase subunit hTERT as described in claim 9, characterized in that, Its reaction process is as follows: Capture 18F with an activated QMA column - After the ions, dissolve 1 mg of the precursor compound NPIP in 2 mL of methanol solution. The molecular formula of NPIP is C 28 H 23 ClINO8, molecular weight: 663.85; Elute the QMA column, collect the mixed solution in a reaction flask, blow in N2 gas or helium gas, evaporate the methanol solvent to dryness, then add 1 mL of DMSO to the reaction flask to dissolve, heat to 80 - 120 °C, react for 10 min, take out; Add 4 mL of injectable H2O to terminate the reaction, purify the reaction solution on a column (C18 column), elute with ethanol, pass through a sterile filter membrane, and dilute with physiological saline to obtain the molecular probe.

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