CN113372285A

CN113372285A - Prostate specific membrane antigen inhibitor, radionuclide marker thereof, preparation method and application

Info

Publication number: CN113372285A
Application number: CN202110592501.3A
Authority: CN
Inventors: 周志军; 刘洋; 陈跃; 刘楠; 陈环宇; 冯悦; 孙占良; 赵岩
Original assignee: Affiliated Hospital of Southwest Medical University
Current assignee: Affiliated Hospital of Southwest Medical University
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-09-10

Abstract

The invention discloses a prostate specific membrane antigen inhibitor, a radionuclide marker thereof, a preparation method and application, and belongs to the technical field of biological medicines. The chemical structure of the prostate specific membrane antigen inhibitor is shown as a formula I, and the chemical structure of the radionuclide marker is shown as a formula II, so that the prostate specific membrane antigen inhibitor is used for preparing prostate cancer diagnostic reagents/medicines or/and therapeutic medicines. The compound has novel structure and stable physicochemical property, and can be used for preparing medicaments for diagnosing and treating the prostatic cancer and used in the fields of diagnosis, staging, curative effect evaluation and treatment of the prostatic cancer.

Description

Prostate specific membrane antigen inhibitor, radionuclide marker thereof, preparation method and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a prostate specific membrane antigen inhibitor, a radionuclide marker thereof, a preparation method and application.

Background

Precision-targeted radiopharmaceuticals are typically composed of a radionuclide, a targeting structure, a linking group. With the wide application of Positron Emission Tomography (PET) and single photon emission tomography (SPECT) in medicine, medical isotopes play more and more important roles in medical diagnosis and treatment, especially play irreplaceable roles in diagnosis, location, staging and efficacy evaluation of tumors, and really achieve what you see is what you get. Whereas when the radionuclide is a beta-electron, alpha ion or auger electron, the radiopharmaceutical may be used as a radioligand therapy (RLT) for therapeutic use. Up to now, there have been dozens of radiopharmaceuticals approved for clinical use.

Over the past decades, PET and SPECT radiopharmaceuticals are rapidly developing, while PET drugs are becoming the main imaging technique of nuclear medicine due to their higher sensitivity.¹⁸F has long been the predominant nuclide for PET imaging, of which¹⁸The F-labeled FDG is widely used for diagnosing diseases such as tumor, inflammation and the like in clinic. In recent years, as germanium-gallium generators mature, positive electron species⁶⁸Ga is more stable and convenient in source, and⁶⁸the sensitivity of Ga is higher. In the context of the chemistry of the marking,⁶⁸the labeling chemistry of Ga is generally easier. More importantly, the method comprises the following steps of,⁶⁸metal ligands for Ga, DOTA, DOTAGA, etc. may also be used¹⁷⁷Lu and²²⁵ac and other metal therapeutic nuclides, so that DOTA, DOTAGA and other ligand drugs can be used as diagnosis and treatment integrated nuclear drugs, and NOTA can be used for marking⁶⁸Ga may be labeled¹⁸F, also has wide application.

By the time of the present day, it has been,⁶⁸ga-dotate and¹⁷⁷Lu-DOTATATE has been approved by the U.S. food and drug administration for the diagnosis and treatment of neuroendocrine tumors. While⁶⁸Ga-PSMA-11 also obtains FDA approval for diagnosis, location, staging, retesting and the like of prostate cancer in 12 months of 2020, and becomes the gold standard for diagnosis of metastatic prostate cancer.⁶⁸Ga-PSMA-617，¹⁷⁷Lu-PSMA-617，¹⁷⁷Lu-PSMA-I&T has also completed a multicenter study in phase ii and has been used for the diagnosis and treatment of prostate cancer. In addition, there are a plurality¹⁷⁷Lu or⁶⁸Ga-labeled drugs are in clinical research. Prostate cancer is a common malignancy in men, accounts for the second leading cause of cancer-related death in men, and once prostate cancer has progressed to an advanced stage, the 5-year survival rate is only around 30%. In developed countries, prostate cancer is the first tumor in the incidence of malignant tumors in men, and prostate cancer is prone to biochemical recurrence, bone metastasis, lymph node metastasis, and the like. Once metastasis has occurred, conventional radiation and chemotherapy has had very limited effectiveness (Charalic KLS, Konijnenberg M, Nonnekens J, et al, therapeutics, 2016,6, 104-.

Prostate cancer has characteristic pathological features. On the molecular level, the cancer cell surface often expresses some special receptors, while the prostate cancer is specificThe membrane antigen (PSMA) is one of the important characteristic antigens or glutamate hydrolase. PSMA is a transmembrane protein with about 95% of the amino acids distributed on the outer surface of the cell membrane, with high expression at all stages of prostate cancer and little expression in healthy tissues. There is a clear correlation between the expression level of PSMA and disease progression (Sweat SD, Pacelli A, Murphy GP, et al, Urology,1998,52: 637-40). PSMA is the best target for prostate cancer imaging and radionuclide exposure. The nuclear labeled small molecule inhibitor targeting PSMA mainly focuses on carbamido compounds. Radionuclide ligand therapy with radionuclide-linked targeting modules has shown a high therapeutic potential in recent years. In particular as nuclides¹⁷⁷Represented by Lu¹⁷⁷Lu-PSMA-617(Rahbar K, Schmidt M, Heinzel A, al et., J Nucl Med.2016,57,1334-¹⁷⁷Lu-PSMA-I&T (Okamoto S, Thieme A, Allmann J, et al, J Nucl Med.2017,58, 445-.

The chemical structure of the PSMA targeting drug is closely related to the targeting property. Even if the chemical structures are the same, compounds differing only in chirality may have great differences in drug efficacy. Meanwhile, the effectiveness of the medicine and the absorbed dose of the medicine have close relation with the chemical structure of the medicine. High internalization and uptake rates tend to reduce the dose administered, thereby reducing radiopharmaceutical toxicity. Radiopharmaceuticals that are structurally designed and synthesized by structural analysis to achieve high internalization rates and uptake rates and appropriate metabolic properties are important approaches for the study of PSMA ligand therapeutic drugs.¹⁷⁷Lu-labeled PSMA-617 and PSMA-I&The T medicine still has the problems of insufficient cell internalization rate and cell uptake rate (the uptake rate is about 20 percent, the internalization rate is less than 10 percent), and still has a large promotion space. In addition, none of the prior arts has yet been provided¹⁷⁷The Lu-PSMA radiotherapeutic drugs are approved for clinical use (all in the clinical study phase). And approved diagnostic drugs⁶⁸Ga-PSMA-11 has lower sensitivity under the condition of lower PSA level, and⁶⁸the excessive intake of Ga-PSMA-11 in the kidney often affects the metastasis of the kidneyThe diagnosis of (1).

From a chemical structural point of view, PSMA-based targeted radiopharmaceuticals include several major functional modules: bifunctional chelating groups, connecting groups and targeting groups. Wherein the targeting group has great influence on the targeting property and the affinity of the medicament. The PSMA small molecule inhibitor entering clinical stage adopts dipeptide of Lys-Urea-GLu structural segment, wherein lysine branched amino is directly connected with connecting group by amido bond. Because the targeting group is sterically selective for binding to PSMA enzyme, even minimal structural modifications to the inhibitor may result in significant changes to the inhibitor.

The patent with the application number of 2020107083231 discloses a macrocyclic polyamine carboxylic acid short peptide and a macrocyclic polyamine carboxylic acid short peptide radionuclide marker, which has the advantages of easy preparation, short preparation period, simple radioactive labeling process, mild conditions, stable labeling products in PBS buffer solution and fetal calf serum, clear imaging of the positron marker in a prostate cancer animal model, high target/non-target ratio and can be used for diagnosis, staging, curative effect evaluation and treatment of clinical prostate cancer. The compound has high kidney uptake value and longer metabolism time. Given the proximity of prostate tissue to the kidney, high uptake by the kidney may obscure prostate cancer proximal metastases. On the other hand, the kidney is a dose limiting organ for such radiopharmaceuticals, and when used for therapeutic purposes, higher renal uptake may also lead to renal toxicity. Therefore, there is a need to invent new diagnostic and therapeutic drugs for prostate cancer to make up for the deficiencies of such drugs.

Disclosure of Invention

One of the purposes of the invention is to provide a prostate specific membrane antigen inhibitor shown in formula I, which has novel structure and stable physicochemical properties and can be used in the fields of diagnosis, staging, curative effect evaluation and treatment of prostate cancer.

The invention also aims to provide the radionuclide marker of the prostate specific membrane antigen inhibitor shown in the formula I, which has high marking rate, high cellular uptake and internalization rate and clear prostate cancer development and can be used in the fields of diagnosis, staging and curative effect evaluation of prostate cancer.

The invention also aims to provide a preparation method of the prostate specific membrane antigen inhibitor shown in the formula I.

The fourth object of the present invention is to provide a method for preparing the radionuclide marker.

The fifth purpose of the invention is to provide the application of the prostate specific membrane antigen inhibitor shown in the formula I.

The sixth object of the present invention is to provide the use of the radionuclide marker.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention relates to a prostate specific membrane antigen inhibitor shown as a formula I or pharmaceutically acceptable salt thereof,

wherein n is an integer of 1-5, preferably 3, and X is F, Cl, Br, I, preferably I.

The compound of the formula I is a polyamine carboxylic acid short peptide, and the molecular skeleton of the compound is composed of polyamine polyacid, fatty amine connecting group and monoamide adipic acid. Wherein lysine-monoamide-adipic acid is a PSMA targeting moiety and the polyaminocarboxylic acid is⁶⁸Ga、¹⁷⁷Lu、⁶⁴Cu and²²⁵ac chelating group and fatty amine as connecting group. Has novel structure and stable physicochemical property, and can be used for diagnosis, staging, curative effect evaluation and treatment of prostatic cancer.

The radionuclide marker of the compound of formula I has a structure shown in formula II,

wherein when R is₂When it is DOTA-M, M is⁶⁸Ga，¹⁷⁷Lu，²²⁵Ac or⁶⁴Cu；

When R is₂When NOTA-M, M ═ M⁶⁸Ga or¹⁸F；

When R is₂When it is HBED-CC-M, M is⁶⁸Ga；

n is an integer of 1-5, preferably 3; x ═ F, Cl, Br, I, preferably I.

The compound of formula II is a polyamine carboxylic acid short peptide radionuclide marker, and the molecular skeleton is composed of 1 radionuclide M-labeled polyamine carboxylic acid, a fatty amine connecting group, lysine and monoamide adipic acid. Wherein lysine-monoamide adipic acid is a PSMA targeting moiety, the radionuclide is labeled as an imaging functional module or a treatment functional module of the drug, and the fatty amine is a linking group. When the radionuclide is ═⁶⁸Ga、¹⁸F or⁶⁴When Cu is adopted, the radioactive nuclide is marked as an imaging functional module of the medicine; when the radionuclide is ═¹⁷⁷Lu or²²⁵Ac, the radionuclide is labeled as a therapeutic functional module of the drug. The marker has novel structure, stable physicochemical property, high labeling rate, high uptake of PSMA high-expression cells and clear tumor-bearing mouse imaging, and can be used in imaging and treatment fields of diagnosis, staging, curative effect evaluation and the like of prostatic cancer.

The invention provides a preparation method of a compound of formula I, which comprises the following steps:

step 1, carrying out Mannich reaction on the compound b1 and b2 to generate a compound b 3;

step 2, carrying out nucleophilic substitution reaction on the compound b3 and b4 to generate a compound b 5;

step 3, carrying out azide reduction reaction on the compound b5 to generate a compound b 6;

step 4, performing polypeptide coupling reaction on the compound b6 and b7 to generate a compound b 8;

step 5, removing the protecting group from the compound b8 to obtain a sound field compound shown as a formula I;

the reaction scheme is as follows:

in some embodiments of the invention, the molar ratio of compound b1 to compound b2 is: 1.0 to 3.0;

the molar ratio of compound b3 to compound b4 was: 1.0 to 3.5;

the molar ratio of compound b6 to compound b7 was: 1.0 to 2.0;

in some embodiments of the invention, in step 1, compound b1 is reacted with compound b2 and CH₃Mannich reaction of BNNa in an organic solvent gives compound b 3;

or/and in the step 2, the compound b3 reacts with b4 in a basic organic solvent to generate a compound b 5;

or/and in the step 3, the compound b5 and a reducing agent are subjected to reduction reaction in an organic solvent to generate b 6;

or/and in the step 4, the compound b6 and the compound b7 are subjected to coupling reaction in the presence of a basic solvent and a condensing agent to generate a compound b 8;

or/and in the step 5, compound b8 is subjected to deprotection reaction under the condition of an acidic solvent to generate compound I;

preferably, the base in step 2 and step 4 comprises at least one of triethylamine, diethylamine, pyridine, diisopropylamine, ethylenediamine and cyclohexylamine, and the molar ratio of the base in step 2 to the compound b3 is 1: 1.0 to 5.0; the molar ratio of the base to the compound b6 in step 4 was 1: 1.0 to 5.0;

preferably, the reducing agent in the step 3 is Pd/C and hydrogen or triphenylphosphine, the dosage of Pd/C is 1.25-20.50% of the mole number of the compound b5, the dosage of hydrogen is 1.0-20.0 times of the mole number of b5, and the dosage of triphenylphosphine is 1.0-5.0 times of the mole number of b 5;

preferably, the coupling agent used in the coupling reaction in step 4 is one or more of HATU, HBTU, HOBT, DCC, EDCI, and the molar ratio of the coupling agent to the compound b7 is 1: 0.2 to 1.0;

preferably, the solvent used in step 1 is a polar solvent, and further preferably, at least one of methanol, ethanol, water, formic acid, acetic acid and hydrochloric acid is included;

preferably, the organic solvent in

steps

2 and 4 is an aprotic polar solvent, and further preferably, both comprise at least one of dichloromethane, chloroform, tetrahydrofuran, 1, 4-dioxane and acetonitrile;

preferably, the solvents in steps 3 and 5 are both polar solvents, and further preferably, both include at least one of dichloromethane, chloroform, tetrahydrofuran, 1, 4-dioxane, methanol, water and acetonitrile.

In some embodiments of the invention, the reaction temperature in step 1 is 0-60 ℃, and the reaction time is 4-24 hours;

the reaction temperature in the step 2 is 0-60 ℃, and the reaction time is 4-48 hours;

the reaction temperature in the step 3 is 0-80 ℃, and the reaction time is 1-20 hours;

the reaction temperature in the step 4 is 0-80 ℃, and the reaction time is 2-24 hours;

the reaction temperature in the step 5 is 0-50 ℃, and the reaction time is 0.5-24 hours;

the invention provides a preparation method of a radionuclide marker of a compound shown in formula I, in the method, the compound shown in formula I reacts with a radionuclide salt to generate a radionuclide marker of the compound shown in formula I, and the compound shown in formula II has the following reaction formula:

in some embodiments of the present invention, in the method for preparing the radionuclide label of the compound of formula I, the pH value of the reaction system is 3.5-10.0, the reaction temperature is 25-95 ℃, and the reaction time is 5-60 min;

preferably, the reaction system further comprises a stabilizer, and further preferably, the stabilizer is at least one selected from ethanol, vitamin C, tyrosine, cysteine, serine and gentisic acid.

The pH value is adjusted by adding a buffer solution into a reaction system, wherein the buffer solution is selected from a sodium acetate/acetic acid system, an ammonium acetate/acetic acid system, a sodium acetate/hydrochloric acid system, a HEPES system or a Tris system.

In some embodiments of the present invention, in the method for preparing the radionuclide-labeled compound of the compound of formula I, the reaction solvent is one or a combination of any two or three of a buffer solution, pure water, and 0.85% to 0.9% of physiological saline.

The invention provides application of a compound shown in a formula I in preparation of a prostate cancer diagnostic reagent/medicament or/and a treatment medicament.

The invention provides an application of a radionuclide marker of a compound shown in a formula I in preparation of a prostate cancer diagnostic reagent/medicament or/and a treatment medicament.

English abbreviations for compounds or groups described in the present invention are:

HATU: 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate,

HBTU: benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate,

HOBT: 1-hydroxybenzotriazoles

DCC: n, N' -dicyclohexylcarbodiimide

EDCI: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride

tBu: tert-butyl radical

TEA: triethylamine

DMF: n, N-dimethylformamide

EA: ethyl acetate

EtOH: ethanol

THF: tetrahydrofuran (THF)

Compared with the prior art, the invention has the following beneficial effects:

the synthesized polyamine carboxylic acid short peptide compound of the formula I is easy to prepare, short in preparation period, simple in radioactive labeling process and mild in condition, labeled products are stable in PBS buffer solution and fetal calf serum, a positron marker is clearly imaged in a prostate cancer animal model, the target/non-target ratio is high, and the compound can be used for diagnosis, staging, curative effect evaluation and treatment of clinical prostate cancer.

Drawings

FIG. 1 shows a compound of formula II⁶⁸HPLC plot of the stability of Ga-formula I-DOTA-I3 in fetal calf serum for 120 min.

FIG. 2 HPLC plot of the stability of compound of formula II, formula I-NOTA-I3, in fetal calf serum over 120 min.

FIG. 3 HPLC plot of the stability of compound of formula II formula I-HBED-CC-I3 in fetal calf serum over 120 min.

FIG. 4 Compounds of formula II⁶⁸PET visualization of Ga-formula I-DOTA-I3 in animal models LNCaP tumor-bearing mice.

FIG. 5 PET micrograph of compound of formula II formula I-NOTA-I3 in animal model LNCaP bearing mice.

FIG. 6 PET micrograph of compound of formula II formula I-HBED-CC-I3 in animal model LNCaP bearing mice.

FIG. 7 is a compound of formula II⁶⁸High performance liquid chromatograms of Ga formula I-DOTA-I3;

FIG. 8 is a compound of formula II⁶⁸High performance liquid chromatograms of Ga formula I-NOTA-I3;

FIG. 9 is a compound of formula II⁶⁸High performance liquid chromatogram of Ga formula I-HBED-CC-I3;

FIG. 10 is a nuclear magnetic map of a compound of formula I-DOTA-I3;

FIG. 11 is a nuclear magnetic map of a compound of formula I-NOTA-I3;

FIG. 12 is a nuclear magnetic map of a compound of formula I-HBED-CC-I3;

FIG. 13 is a mass spectrum of a compound of formula I-DOTA-I3;

FIG. 14 is a mass spectrum of a compound of formula I-NOTA-I3;

FIG. 15 is a mass spectrum of a compound of formula I-HBED-CC-I3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

This example discloses the synthesis of compound b3-3 (i.e. compound b3, where n is 3) according to the formula:

the method specifically comprises the following steps: compound b1(1.224g,2.513mmol) and b2-3(335mg,2.639mmol) were dissolved in methanol (40mL) and stirred at 0 ℃ for 30 min. Adding CH into the solution₃BNNa (241mg, 3.77mmol) and stirred at room temperature for 4 hours. The reaction solution was poured into ice water, extracted with ethyl acetate, washed with saturated brine 3 times, and dried over anhydrous sodium sulfate overnight. The organic phase is separated off by filtration and the organic solvent is evaporated off. The product was purified by column chromatography (petroleum ether/ethyl acetate 5/1) to give b3-3 as a yellow oily liquid amounting to 1.15g, yield: and (5) 55.3%.

Example 2

This example discloses the synthesis of compound b5-3 (i.e. compound b5, where n is 3) according to the formula:

b3-3(403mg,0.674mmol) and b4(215mg,0.809mmol) were dissolved in dichloromethane (10mL), and triethylamine (81mg,0.809mmol) was added thereto, followed by stirring at room temperature for 12 hours. The reaction solution was washed 3 times with saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate overnight. The organic phase was rotary evaporated to remove the organic solvent. The product was purified by column chromatography (petroleum ether/ethyl acetate 3/1) to obtain 383 mg in total of b5-3 as a yellow oily liquid product in 68.6% yield.

Example 3

This example discloses the synthesis of compound b6-3 (i.e. compound b6, where n is 3) according to the formula:

reaction b5-3(150mg, 0.181mmol) and triphenylphosphine (71mg, 0.272mmol) were dissolved in tetrahydrofuran (2.5ml) and water (0.5ml), and stirred at room temperature for 5 hours. After completion of the reaction, the reaction solution was poured into ice water and extracted with dichloromethane, washed with saturated brine 3 times, and the organic phase was dried over anhydrous sodium sulfate overnight. The organic phase was collected by filtration and the organic solvent was removed by rotary evaporation. Column chromatography (DCM/MeOH ═ 15/1) afforded the product b6-3 as a white solid, totaling 135 mg, yield: 93.1 percent.

Example 4

This example discloses the synthesis of compound b8-3-DOTA (i.e. compound b8 where n is 3 and R1 is DOTA) having the formula:

b6-3(21mg,0.037mmol) was dissolved in dichloromethane (3mL), HATU (24mg,0.062mmol) was added, and after stirring at room temperature for 30 minutes, b-7-DOTA (25m g,0.031mmol) and diisopropylethylamine (6mg,0.047mmol) were added, and the mixture was stirred at room temperature for 10 hours, the reaction mixture was poured into ice water, extracted with dichloromethane, washed with saturated brine 3 times, the organic phase was collected, and dried over anhydrous sodium sulfate overnight. After the drying agent was removed by filtration, the organic solvent was removed by rotary evaporation. The product was isolated and purified by preparative liquid chromatography to give b8-3-DOTA (7mg) as a white solid in yield: 16.5 percent.

Example 5

This example discloses the synthesis of compound I-DOTA-I3 (i.e., compound I wherein n is 3 and R1 is DOTA) according to the formula:

b8-3-DOTA (7mg, 0.005mmol) was dissolved in a mixed solvent of dichloromethane (0.5ml) and trifluoroacetic acid (0.5ml), and the reaction mixture was stirred at room temperature for 12 hours. And (3) performing rotary evaporation under reduced pressure to remove the organic solvent and trifluoroacetic acid, dissolving the product in methanol again, extracting with diethyl ether to obtain a crude product, and separating and purifying by preparative high performance liquid chromatography to obtain the product I-DOTA-I3.

The compound has a nuclear magnetic spectrum of formula I-DOTA-I3 shown in figure 10 and a mass spectrum shown in figure 13.

Replacing R1 with NOTA, and preparing the compound of formula I-NOTA-I3 by the same method, wherein the nuclear magnetic spectrum is shown in figure 11, and the mass spectrum is shown in figure 14.

Replacing R1 with HBED-CC, and preparing compound of formula I-HBED-CC-I3 by the same method, wherein the nuclear magnetic spectrum is shown in figure 12, and the mass spectrum is shown in figure 15.

Example 6

This example discloses compounds of formula II, i.e.⁶⁸Ga-labelled Compounds of formula I⁶⁸Preparation of Ga-formula I-DOTA-I3, the reaction formula is:

mixing NaAc/HAc buffer solution (pH 4.45, 1mL) with 0.85% physiological saline (1mL) at room temperature, adding formula I-DOTA-I3(10 μ L, 10 μ g), mixing, and adding⁶⁸GaCl₃High purity hydrochloric acid solution (2mCi, 1mL, 0.05mol/L), heated to 85 ℃ for 10 minutes, passed through a C18 lighting reverse phase column, washed with physiological saline and collected as waste. The reverse phase column was washed with 50% medical alcohol (0.4mL), washed with 0.85% physiological saline (8mL), and the washing solution was collected and measured by high performance liquid chromatography (acetonitrile/water, acetonitrile 10% to 90% in 15 minutes, both water and acetonitrile containing 0.1% trifluoroacetic acid), retention time of radioactive peak of product 9.1min, labeling rate: 98 percent.

Obtained in this example⁶⁸The radioactive high performance liquid chromatogram of Ga-formula I-DOTA-I3 is shown in figure 7. The HPLC conditions were as follows: angioent C18 column (250mm x 4.6mm x 3.5 μm), flow rate: 1.0ml/min, column temperature, room temperature. Gradient acetonitrile (0.1% TFA) water (0.1%), where acetonitrile rises from 10% to 90% in 15 minutes and is isocratic at 90%Rinsing for 10 minutes.

Example 7

This example discloses compounds of formula II, i.e.⁶⁸Ga-labelled Compounds of formula I⁶⁸Preparation of Ga-formula I-NOTA-I3, having the formula:

NaAc/HAc buffer (pH 4.2, 1mL) was mixed with 0.85% physiological saline (1mL) at room temperature, followed by addition of formula I-NOTA-I3 (10. mu.L, 10. mu.g), mixing, and addition of the mixture⁶⁸GaCl₃High purity hydrochloric acid solution (2mCi, 1mL, 0.05mol/L), reaction at room temperature for 10 minutes, C18 lighting reverse phase column, normal saline washing and collection as waste liquid. The reverse phase column was washed with 50% medical alcohol (0.4mL), washed with 0.85% physiological saline (8mL), and the washing solution was collected and measured by high performance liquid chromatography (acetonitrile/water, acetonitrile 10% to 90% in 15 minutes, both water and acetonitrile containing 0.1% trifluoroacetic acid), retention time of radioactive peak of product 8.9min, labeling rate: 98 percent.

Obtained in this example⁶⁸The radioactive high performance liquid chromatogram of Ga-type I-NOTA-I3 is shown in figure 8. The HPLC conditions were as follows: angioent C18 column (250mm x 4.6mm x 3.5 μm), flow rate: 1.0ml/min, column temperature, room temperature. The gradient was acetonitrile (0.1% TFA) water (0.1%), with acetonitrile rising from 10% to 90% in 15 minutes and rinsing isocratically at 90% for 10 minutes.

Example 8

This example discloses compounds of formula II, i.e.⁶⁸Ga-labelled Compounds of formula I⁶⁸Preparation of Ga-formula I-HBED-CC-I3, reaction formula is:

mixing NaAc/HAc buffer solution (pH 4.45, 1mL) with 0.85% physiological saline (1mL) at room temperature, adding formula I-HBED-CC-I3(10 μ L, 10 μ g), mixing, and adding⁶⁸GaCl₃High-purity hydrochloric acidThe solution (2mCi, 1mL, 0.05mol/L) was reacted at room temperature for 15 minutes or heated to 85 ℃ for 10 minutes, passed through a C18 lighting reverse phase column, washed with physiological saline and collected as waste. The reverse phase column was washed with 50% medical alcohol (0.4mL), washed with 0.85% physiological saline (8mL), and the washing solution was collected and measured by high performance liquid chromatography (acetonitrile/water, acetonitrile 10% to 90% in 15 minutes, both water and acetonitrile containing 0.1% trifluoroacetic acid), retention time of radioactive peak of product 9.7min, labeling rate: 99 percent.

Obtained in this example⁶⁸The radioactive high performance liquid chromatogram of Ga-type I-HBED-CC-I3 is shown in figure 8. The HPLC conditions were as follows: angioent C18 column (250mm x 4.6mm x 3.5 μm), flow rate: 1.0ml/min, column temperature, room temperature. The gradient was acetonitrile (0.1% TFA) water (0.1%), with acetonitrile rising from 10% to 90% in 15 minutes and rinsing isocratically at 90% for 10 minutes.

Example 9

This embodiment discloses⁶⁸The Ga-formula I-NOTA-I3 is used in a fetal calf serum stability experiment, and specifically comprises the following steps:

taking 900 mul fetal calf serum and 2mL of 3 EP tubes in parallel, adding radiochemical pure>99% of⁶⁸Ga-formula I-NOTA-I3100. mu.L (specific activity: 3. mu. Ci/. mu.L), incubating at 37 ℃ for 30min, 60min,120 min. At each time point, 100 μ L of sample is added into 50 μ L of acetonitrile, and after shaking precipitation and centrifugation, 10 μ L of supernatant is taken, radiochemical purity of the sample is detected by using radioactive high performance liquid chromatography, and the chromatogram is shown in figure 1. And (3) testing results: the radiochemical purity of the sample was 90.9% at 30 minutes, and after standing for 2 hours, 83.7% of radiochemical purity was still maintained. Therefore, the temperature of the molten metal is controlled,⁶⁸ga-labels of formula I⁶⁸Ga-formula I-NOTA-I3 is stable in fetal calf serum.

Example 10

This embodiment discloses⁶⁸Ga-formula I-DOTA-I3 in fetal calf serum stability experiments, the experimental process is the same as example 9, the result is:

the sample is radiochemical pure at 30 minutes94.8%, and still maintain 87.0% radiochemical purity after being placed for 2 hours. Therefore, the temperature of the molten metal is controlled,⁶⁸ga-labels of formula I⁶⁸Ga-formula I-DOTA-I3 has good stability in fetal calf serum. The chromatogram is shown in FIG. 2.

Example 11

This embodiment discloses⁶⁸An in vivo distribution experiment of an LNCAP animal model of Ga-formula I-DOTA-I3 specifically comprises the following steps:

LNCAP cells are planted in the armpit of the right forelimb of a nude mouse, and the nude mouse is divided into two groups, namely a cancer-free cell inoculation group and a cancer-free cell inoculation group, wherein the cancer-free cell group is 3, and the cancer-free cell inoculation group is 9. Feeding mice in clean environment, observing tumor generation and development, dividing the mice into 3 groups (30 min, 60min,120 min) when the tumor grows to about 0.5cm in diameter, and performing intravenous injection⁶⁸Ga-formula I-DOTA-I3 (200. mu.L, 10. mu. Ci), corresponding groups were sacrificed at 30min, 60min,120min after drug injection, dissected, blood, heart, liver, spleen, lung, kidney, muscle, small intestine, salivary gland, tumor, measured for radioactivity counts of each organ tissue, and ID%/g was calculated.

⁶⁸The PET/CT image of the LNCAP tumor-bearing mouse Ga-formula I-DOTA-I3 is shown in figure 4. Wherein A, B is PET/CT image at 10min and 120min respectively.

The test results were as follows:

example 12

This embodiment discloses⁶⁸In vivo distribution experiments in an LNCAP animal model of Ga-formula I-NOTA-I3, the experimental procedure was the same as in example 11,⁶⁸the PET/CT image of the LNCAP tumor-bearing mouse Ga-formula I-NOTA-I3 is shown in FIG. 5. Wherein A, B is PET/CT image at 10min and 120min respectively.

The results are as follows:

％ID/g	30min	60min	120min
				heart and heart	1.34	0.86	0.07
Liver disease	2.15	1.09	0.25
				Spleen	5.82	3.85	0.61
Lung (lung)	4.19	2.72	0.31
				Kidney (A)	46.09	42.80	7.33
Stomach (stomach)	5.50	1.43	1.18
				Small intestine	3.68	1.46	0.28
Muscle	1.20	2.62	0.10
				Bone	1.07	0.25	0.07
Brain	0.24	0.24	0.03
				Blood, blood-enriching agent and method for producing the same	4.40	1.86	0.31
Tumor(s)	12.27	19.86	10.24

Example 13

This embodiment discloses⁶⁸In vivo distribution experiments in LNCAP animal models of Ga-formula I-HBED-CC-I3, the procedure was as in example 11, and animals were sacrificed at 30min, 60min,120min, respectively, in the corresponding groups after drug injection.⁶⁸The PET/CT image of the LNCAP-bearing mouse Ga-formula I-HBED-CC-I3 is shown in figure 6.

Wherein A, B is PET/CT image at 5min and 120min respectively.

The results are as follows:

％ID/g	30min	60min	120min
				heart and heart	2.48	1.46	0.53
Liver disease	3.14	2.29	0.94
				Spleen	23.59	36.83	21.50
Lung (lung)	4.56	5.68	3.10
				Kidney (A)	182.88	230.01	251.98
Stomach (stomach)	2.97	1.81	1.08
				Small intestine	2.81	2.04	0.56
Muscle	1.68	0.77	0.40
				Bone	0.61	0.50	0.22
Brain	0.20	0.12	0.04
				Blood, blood-enriching agent and method for producing the same	5.34	2.93	0.87
Tumor(s)	30.35	36.29	39.57

From the above results, it can be seen that: inhibitors with DOTA and NOTA as chelating groups showed rapid tumor-targeted and normal tissue clearance characteristics in NOD/SCID double-deficient mice. It is particularly noteworthy that the renal clearance rate is much faster than that of compounds with HBED-CC and DOTAGA as chelating groups, exhibiting excellent pharmacokinetic profiles.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A prostate specific membrane antigen inhibitor represented by formula I or its pharmaceutically acceptable salt,

wherein n is an integer of 1-5, preferably 3, X ═ F, Cl, Br, and I, preferably I.

2. The radionuclide marker of the compound of the formula I is characterized in that the structure is shown as the formula II,

When R is₂When NOTA-M, M ═ M⁶⁸Ga or¹⁸F；

When R is₂When it is HBED-CC-M, M is⁶⁸Ga；

n is an integer of 1-5, preferably 3, X ═ F, Cl, Br, and I, preferably I.

3. A process for the preparation of a compound of formula I according to claim 1, comprising the steps of:

step 5, removing a protecting group from the compound b8 to generate a compound shown in the formula I;

the reaction scheme is as follows:

4. a process for the preparation of compounds of formula I according to claim 3,

the molar ratio of compound b1 to compound b2 was: 1.0 to 3.0;

and/or the molar ratio of the compound b3 to the compound b4 is: 1.0 to 3.5;

and/or the molar ratio of the compound b6 to the compound b7 is: 1.0 to 2.0.

5. A process for the preparation of a compound of formula I according to claim 3, characterized in thatIn step 1, compound b1 is reacted with compound b2 and CH₃Mannich reaction of BNNa in a solvent to produce compound b 3;

preferably, the coupling agent used in the coupling reaction in step 4 is at least one of HATU, HBTU, HOBT, DCC, EDCI, and the molar ratio of the coupling agent to compound b7 is 1: 0.2 to 1.0;

preferably, the solvent in step 1 is a polar solvent, and further preferably, the solvent comprises at least one of methanol, ethanol, water, formic acid, acetic acid and hydrochloric acid;

preferably, the organic solvent in steps 2 and 4 is an aprotic polar solvent, and further preferably, both comprise at least one of dichloromethane, chloroform, tetrahydrofuran, 1, 4-dioxane and acetonitrile;

6. The preparation method according to claim 5, wherein the reaction temperature in step 1 is 0-60 ℃ and the reaction time is 4-24 hours;

or/and the reaction temperature in the step 2 is 0-60 ℃, and the reaction time is 4-48 hours;

or/and the reaction temperature in the step 3 is 0-80 ℃, and the reaction time is 1-20 hours;

or/and the reaction temperature in the step 4 is 0-80 ℃, and the reaction time is 2-24 hours;

or/and the reaction temperature in the step 5 is 0-50 ℃, and the reaction time is 0.5-24 hours.

7. A process for the preparation of a radionuclide label for a compound of formula I, characterized in that a compound of formula I is reacted with a radionuclide salt to form a radionuclide label for a compound of formula I a compound of formula ii, which reaction is as follows:

8. the method for preparing the radionuclide-labeled compound of formula I according to claim 7, wherein the reaction system has a pH of 3.5-10.0, a reaction temperature of 25-95 ℃ and a reaction time of 5-60 min;

preferably, the reaction system further comprises a stabilizer, and further preferably, the stabilizer is any one or more selected from ethanol, vitamin C, tyrosine, cysteine, serine and gentisic acid.

9. Application of the compound shown in the formula I in preparation of prostate cancer diagnosis reagents/medicines or/and treatment medicines.

10. The application of the radionuclide marker of the compound shown in the formula I in the preparation of a prostate cancer diagnostic reagent/medicament or/and a treatment medicament.