CN112851637B - PSMA inhibitor, compound, preparation method and application thereof - Google Patents
PSMA inhibitor, compound, preparation method and application thereof Download PDFInfo
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- CN112851637B CN112851637B CN202110089388.7A CN202110089388A CN112851637B CN 112851637 B CN112851637 B CN 112851637B CN 202110089388 A CN202110089388 A CN 202110089388A CN 112851637 B CN112851637 B CN 112851637B
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- psma
- pharmaceutically acceptable
- acceptable salt
- metal ion
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Abstract
The invention discloses a PSMA inhibitor, a compound, a preparation method and application thereof. The structure of the compound is shown as a formula A, wherein R1 is cyclicamethamine or benzylamine, R2 is DOTA (I) or NODA (II) for complexing (radioactive) metal ions, radioactive metal nuclide is used for diagnosis or treatment, and non-radioactive metal nuclide is used for combining radionuclide. The invention also provides a preparation method and application of the compound. The compounds have high affinity with Prostate Specific Membrane Antigen (PSMA), have the characteristic of rapid clearance, and are particularly suitable for diagnosis, staging, treatment and the like of tumor radionuclide targets.
Description
Technical Field
The invention belongs to the technical field of radiopharmaceuticals and preparation thereof, and particularly relates to a PSMA inhibitor, a compound, a preparation method and application thereof.
Background
Prostate cancer is a common malignant tumor of a male reproductive system, and about 130 million prostate cancer patients and 36 million patients die of prostate cancer every year in 2018, which accounts for the second place of the incidence rate of tumors in men; in addition, the advanced prostate cancer mainly comprises endocrine therapy, but the advanced prostate cancer finally progresses to castration-resistant prostate cancer (CRPC), and the total treatment effect of CRPC is poor, the mortality rate is high, and the advanced prostate cancer is always a difficulty of clinical treatment. Due to the extremely complex mechanisms underlying development of CRPC, no consensus therapeutic has been developed to date. In China, the incidence of the prostatic cancer is lower than that of the prostatic cancer in European and American countries, but the prostatic cancer tends to increase year by year, and the prostatic cancer is an important problem affecting the life health of men in China. How to diagnose prostate cancer accurately in early stage is one of the current hotspots for effective treatment of CRPC.
Radical prostatectomy is the most common treatment method for early-stage prostate cancer, the treatment effect of the early-stage prostate cancer is positive, most patients can achieve the aim of curing the prostate cancer, but the prostate cancer is hidden and slow in development, and most patients in the early stage have no obvious clinical symptoms. And the recurrence rate of the patient after radical resection of the prostate cancer is very large, and whether the patient with biochemical recurrence has clinical recurrence or not is judged to have local recurrence if the patient with biochemical recurrence has clinical recurrence, and how to treat clinically, regional lymph node metastasis or distant metastasis and the like have great influence. The existing conventional examination means (serum PSA, B-ultrasound, bone scanning, CT, MRI and the like) of the biochemical relapse patients have certain defects on the diagnosis of early-stage prostatic cancer and the application of the biochemical relapse patients after the prostatic cancer operation, and have limited application value on the biochemical relapse patients with PSA less than 10 ng/ml. How to find biochemical recurrence in early stage and accurately position the recurrence provides great guidance for clinical treatment.
Because the fusion of molecular level functional imaging and fine anatomical imaging is realized by PET/CT and PET/MR, the whole body imaging can be completed by one-time examination, the whole body tomograph in 3 directions of coronal plane, sagittal plane and cross section can be obtained, and whether metastasis exists in other parts of the whole body can be detected simultaneously, therefore, the value of the method in the stage and the re-stage of the prostate cancer is obviously superior to that of the conventional imaging examination such as CT, MRI and the like.
Radiopharmaceutical agents 18 F-FDG is the most commonly used tracer for PET imaging, but well-differentiated prostate cancer imaging often appears to be false negative and is excreted through the urinary system with some impact on prostate imaging, thus its value in prostate cancer diagnosis is limited.
Diagnosis of prostate cancerInjectable drug, U.S. FDA, prepared 11 C-choline, 18 F-sodium fluoride and 18 F-Axumin and the like, but the F-Axumin and the like are not targeted drugs and have low detection rate in the aspects of initial stage and regressions of the prostate cancer and biochemical recurrence diagnosis.
Prostate Specific Membrane Antigen (PSMA) is a type of transmembrane glycoprotein located on the surface of prostate epithelial cells. PSMA is expressed on the surface of normal prostate and prostate proliferating cells and is significantly upregulated in the vast majority of prostate cancer cells. The small glutamic urea molecule and the analogue thereof (Glu-urea-R) are folic acid hydrolase I activity inhibitors, and can competitively inhibit NAALADase enzyme activity of PSMA, so that the small glutamic urea molecule and the analogue thereof can be efficiently and targetedly combined with PSMA on the surface of prostate cancer cells and enter the prostate cancer cells through internalization. The micromolecule PET imaging agent based on the glutamic acid urea structure shows great potential in clinical diagnosis and treatment, and particularly develops 68 Ga-PSMA-11 is approved by FDA in the United states and is sold in the market in 2020, and a great number of therapeutic and diagnostic radioactive medicines based on PSMA are reported. First of all PSMA based on complex radiometal nuclide labeling (patent application No. 201480056250.5 "labeled inhibitors of Prostate Specific Membrane Antigen (PSMA), their use as imaging agents and agents for the treatment of prostate cancer"), followed by further defined binding of the PSMA pocket to a naphthalene group 68 Ga/ 177 Lu-labeled PSMA-617 (patent application No. 201810815832.7 "labeled inhibitor of Prostate Specific Membrane Antigen (PSMA) and uses thereof"), which is currently the major drug based on PSMA nuclide therapy; also having I extending the DOTA linkage after removal of the naphthalene group&T and patent 201580080036857.1 "metal/radiometal-labeled PSMA inhibitors for PSMA-targeted imaging and radiotherapy" and patent 201510067581.5 "inhibitors of Prostate Specific Membrane Antigen (PSMA), biological evaluation and use as imaging agents", etc. The above diagnostic nuclides based on DOTA as link are all 68 Ga, in addition to short half-life and generator production, 68 the high energy beta rays emitted by Ga affect the PET image quality, are limited by the supply of generators and the like, and the research direction is shifted to the most common nuclide 18 F, development of a PSMA-based excretory system 18 F-DCFPyL(201780048128.7)、 18 F(AlF)-PSMA-617(201710368982.3“Al 18 F-labeled PSMA targeted inhibitor, preparation method and application thereof) and urinary system excretion-free PSMA targeted inhibitor 18 F-PSMA-1007 and the like, and 18 a new compound J-7 and the like after adding one methoxyl group to the F-DCFPyL pyridine ring 18 Clinical verification shows that the F-labeled PSMA inhibitor has good effect.
However, the PSMA developer has the problems of low sensitivity, low detectable rate when PSA is less than 1 mu g/L and slow in vivo removal; some of them have high radioactive uptake in the liver, which may affect the diagnosis of liver metastasis. Therefore, it is important to develop imaging agents that clear rapidly in vivo, have high activity, high target/non-target ratio, and low liver uptake.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a compound or a pharmaceutically acceptable salt thereof with high activity, rapid in vivo clearance, high target/non-target ratio and low liver uptake, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
a compound is a compound having a structure represented by formula A or a pharmaceutically acceptable salt thereof,
wherein, R1 is cyclicamide or benzylamine, and R2 is DOTA (I) or NODA (II) for complexing metal ions.
Further, a compound as described above wherein R2 is DOTA or NODA, may complex a radioactive metallic species M or a non-radioactive metallic species for use in diagnosis or therapy, or complex a non-radioactive metallic species for use in combination with other radionuclides.
A PSMA inhibitor, the above compound or a pharmaceutically acceptable salt thereof binding radioactive metal ion, R2 is DOTA, wherein the bound radioactive metal ionM is: 177 Lu、 225 AC、 161 Tb、 133/135 La、 90 Y、 44 Sc、 68 ga or 64 Cu and the like, and the structural formula is as follows:
a PSMA inhibitor, the above compound or a pharmaceutically acceptable salt thereof, which binds a radioactive metal ion, R2 is NODA, wherein the bound radioactive metal ion M is 68 Ga, structural formula as follows:
a PSMA inhibitor is prepared by combining non-radioactive metal ion with the above compound or pharmaceutically acceptable salt, R2 is NODA, wherein metal ion M is aluminum or iron ion, and radionuclide combined with metal ion is 18 F, the structural formula is as follows:
the invention further provides a preparation method of the compounds of the structural formula I-M or the pharmaceutically acceptable salts thereof, which comprises the following steps:
wherein a is the reaction of the compound I-1 and the salt containing the radioactive metal ions under the acidic condition and the heating condition for 5 to 40 minutes.
The invention further provides a preparation method of the compound of the structural formula II-3 or the pharmaceutically acceptable salt thereof, which comprises the following steps:
wherein a is a compound II-1 and a radioactive metal ion under acidic conditions 68 And (3) reacting the salt of Ga under the condition of normal temperature.
The invention further provides a preparation method of the compound of the structural formula II-4 or the pharmaceutically acceptable salt thereof, which comprises the following steps:
wherein a is fluorine-18 ions which firstly form aluminum fluoride with aluminum ions; b is heating under acidic condition, after 5 to 40 minutes, aluminum ions are complexed with NODA in the II-1 compound to form a stable complex; the aluminum ions may be replaced with iron ions.
The radionuclide-labeled compound or pharmaceutically acceptable salt is used for imaging of the target PSMA tumor, and is used for diagnosis, staging or curative effect evaluation of the tumor; the compound or the pharmaceutically acceptable salt is used for treating the PSMA tumor.
The invention has the following beneficial effects:
the invention provides a novel structural inhibitor for targeting prostate specific membrane antigen, which can accelerate the radioactive excretion of non-target area in vivo while keeping high tumor uptake compared with the existing PSMA treatment and imaging agents, and is beneficial to the treatment and imaging of tumors. After the radionuclide is labeled, the mouse in-vivo distribution experiment and clinical imaging show that the tumor obviously takes up radioactivity, and the rest radioactivity is mainly excreted through the kidney and is rapidly metabolized in non-target tissues and organs. 177 Lu labeled compound for treating mice with 22RV1 tumors shows that after 24 hours, radioactivity is mainly concentrated in the tumors, and the survival time of the mice in a treatment group is obviously higher than that in a control group and is also higher than that in the control group 177 Lu-PSMA617 group; 68 Ga/ 18 the micro-PET/CT imaging result of the F-labeled compound shows that the probe is higher in uptake in tumors and has a high target-to-cost ratio of 60 min; can be inhibited by PSMA inhibitor; 18 the F marked compound is shown by the preliminary clinical picture that the tumor takes in obviouslyAnd the background of surrounding tissues is low, so the compound is a novel PSMA tumor imaging agent.
Drawings
FIG. 1 is a mass spectrum of I-112 (2-pyridine-cyclohexylmethylamine-DOTA-PSMA) in example 1 of the present invention;
FIG. 2 is a mass spectrum of I-113 (3 pyridine-cyclohexylmethylamine-DOTA-PSMA) in example 2 of the present invention;
FIG. 3 is a mass spectrum of I-114 (4-pyridine-cyclohexylmethylamine-DOTA-PSMA) in example 3 of the present invention;
FIG. 4 is a mass spectrum of II-114 (4-pyridine-cyclohexylmethylamine-NODA-PSMA) in example 4 of the present invention;
FIG. 5 is a mass spectrum of II-124 (4-pyridine-benzylamine-NODA-PSMA) in example 5 of the present invention;
FIG. 6 is an HPLC radiogram of I-31X in example 9 of the present invention;
FIG. 7 is a PET/CT fusion map of I-31X of tumor-bearing mice in example 9 of the present invention;
FIG. 8 is a PET/CT fusion map of tumor-bearing mice I-31X after 2-PMPA blockade in example 9 of the present invention;
FIG. 9 shows the same patient I-314 and I-314 as in example 10 of the present invention 18 F-7Q-PSMA PET/CT contrast map;
FIG. 10 is a II-414 radiochromatogram obtained in example 11 of the present invention;
FIG. 11 is a PET/CT image of II-414 in a tumor-bearing mouse according to example 11 of the present invention;
FIG. 12 is a PET/CT image of II-414 of example 12 of the present invention on a prostate cancer patient;
FIG. 13 is a PET/CT image of II-424 in example 13 of the present invention on a prostate cancer patient;
FIG. 14 is a radioactive chromatogram of I-214 of example 14 of the present invention;
FIG. 15 is a SPECT image at 24 hours after injection of mouse with tumor I-214 according to example 14 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The following examples do not specify particular techniques or conditions, according to the techniques or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
The invention discloses a preparation method and application of a pyridine PSMA inhibitor for tumor diagnosis and treatment. The pyridine PSMA inhibitor has a structure shown as a formula A, wherein R1 is benzylamine or cyclohexylmethylamine, R2 is DOTA or NODA for complexing (radioactive) metal ions, radioactive metal nuclide is used for diagnosis or treatment, and non-radioactive metal nuclide is used for combining radionuclide.
R1 is:
r2 is:
separately for complexing
(radioactive) metal ions. The radioactive metal ion may be: 177 Lu、 225 AC、 161 Tb、 133/135 La、 90 Y、 44 Sc、 68 ga or 64 Cu; the non-radioactive metal ion may be aluminum or iron ion, and the radionuclide bonded to the metal ion is 18 F。
As an example, compounds may be named according to the following order rule:
B-CDE
b: i is DOTA and II is NODA
C:1,2,3 and 4 are respectively a precursor, 177 Lu、 68 Ga、 18 F
D:1,2 are respectively cyclicaminomethyl and phenylmethylamine
E:2,3,4 denotes the position of the nitrogen on the pyridine ring
For example II-314 denotes NODA complexation 68 Ga-Cyclohexanemethylamine-4-pyridine PSMA
Example 1: synthesis of I-112 (2-pyridine-cyclohexylmethylamine-DOTA-PSMA)
100mg of Fmoc-Glu-urea-Lys-resin (tert-butyl ester protected ureido-lysine resin) (0.22 mmol/g) was placed in a solid phase synthesis tube. DCM (3X 5min X2 mL) and DMF (3X 5min X2 mL). Fmoc was removed and 20% piperidine in DMF (1X 2min X2 mL, 2X 10min X2 mL) was used, followed by washing with DMF (6X 1min X2 mL).
Fmoc-3- (2-pyridine) -D-alanine (3M, 0.06mmol, 26.2mg), HBTU (0.072mmol, 27mg), HOBt (0.072mmol, 10mg), DIPEA (0.15mmol, 25. Mu.L) were taken in 3mL of DMF at room temperature for 15 minutes. The above activated 2-pyridine-alanine was added to the washed resin and reacted under nitrogen for 1 hour. DMF rinse (6X 1min X2 mL). Fmoc was removed and 20% piperidine in DMF (1X 2min X2 mL, 2X 10min X2 mL) was used, followed by washing with DMF (6X 1min X2 mL).
Trans-4- (Fmoc-aminomethyl) cyclohexanecarboxylic acid (3M, 0.06mmol, 23mg), HBTU (0.072mmol, 27mg), HOBt (0.072mmol, 10mg), DIPEA (0.15mmol, 25. Mu.L) were dissolved in 3mL of DMF at room temperature for 15 minutes. The activated cyclohexanecarboxylic acid was added to the washed resin, and reacted under nitrogen for 1 hour. DMF rinse (6X 1min X2 mL). Fmoc was removed and 20% piperidine in DMF (1X 2min X2 mL, 2X 10min X2 mL) was used, followed by washing with DMF (6X 1min X2 mL).
DOTA tri-tert-butyl ester (3M, 0.06mmol, 34.4mg), HBTU (94790-37-1, 0.072mmol, 27mg), HOBt (2592-95-2, 0.072mmol, 10mg), DIPEA (0.15mmol, 25. Mu.L) was taken in 3mL of DMF at room temperature for 15 minutes. The activated DOTA tri-tert-butyl ester was added to the washed resin and reacted under nitrogen for 1 hour.
DMF rinse (6X 1min X2 mL). 4.5mL of TFA, 250. Mu.L of Triisopropylsilane (TIPS) and 250. Mu.L of water were added and reacted at room temperature for 2 hours. The filtrates were collected and washed once with 2mL TFA, and the filtrates combined. Purification by HPLC gave the final product as a white color with the mass spectrum shown in FIG. 1.MS: [ M + H ] (M/z = 993.9)
Example 2: synthesis of I-113 (3-pyridine-cyclohexylmethylamine-DOTA-PSMA)
The Fmoc-3- (2-pyridine) -D-alanine from example 1 was replaced with Fmoc-3- (3-pyridine) -D-alanine, and the remainder was unchanged, and the final synthesis was purified by HPLC to give a white final product: 3 pyridine-cyclicamide-DOTA-PSMA, mass spectrum is shown in figure 2.MS: [ M + H ] (M/z = 993.6)
Example 3: synthesis of I-114 (4-pyridine-cyclohexylmethylamine-DOTA-PSMA)
The Fmoc-3- (2-pyridine) -D-alanine from example 1 was replaced with Fmoc-3- (4-pyridine) -D-alanine, and the remainder was unchanged, and the final synthesis was purified by HPLC to give a white final product: 4 pyridine-cyclicaminomethyl-DOTA-PSMA, mass spectrum is shown in figure 3.MS: [ M + H ] (M/z = 991.99)
Example 4: synthesis of II-114 (4-pyridine-cyclohexylmethylamine-NODA-PSMA)
The DOTA tri-tert-butyl ester in example 3 was replaced by NODA di-tert-butyl ester and the final synthesis was purified by HPLC to give the white final product: 4 pyridine-cyclicamide-NODA-PSMA, mass spectrum is shown in figure 4. And (2) MS: [ M + H ] (M/z = 893.3)
Example 5: synthesis of II-124 (4-pyridine-benzylamine-NODA-PSMA)
Trans-4- (Fmoc-aminomethyl) cyclohexanecarboxylic acid from example 3 was replaced by trans-4- (Fmoc-aminomethyl) benzoic acid, and DOTA tri-tert-butyl ester was replaced by NODA di-tert-butyl ester, and the final synthesis was purified by HPLC to give the white final product: 4 pyridine-benzylamine-NODA-PSMA, mass spectrum is shown in FIG. 5.MS: [ M + H ] (M/z = 887.07)
Since I-112, I-113, I-114, II-114 and II-124 contain carboxyl groups and complexing agents, they can form salts with sodium, calcium, magnesium, etc. in the solvent, which can be used for the next step as precursor labeling.
The above are examples of the precursor preparation method, and examples of the drug preparation method are described below.
Example 6
This example was used to obtain R 2 In the case of DOTA, complexation 177 A PSMA inhibitor of the Lu nuclide having the formula:
wherein R1 is benzylamine or cyclohexylmethylamine;
an exemplary specific preparation method comprises:
(a) Dissolving the I-1 compound in 0.1-0.2mL of ultrapure water, and adding 100mg of Vc or gentisic acid;
(b) According to the following steps: 1 molar ratio (I-1 excess) is added to the desired amount 177 LuCl 3 Adding hydrochloric acid solution (37-14800 MBq/0.1-0.2 mL), adding a certain amount of sodium acetate (1.0 mol/L10-20 μ L), and adjusting the pH of the solution to 4.0;
(c) Heating the mixture at 80-100 deg.C for 30min to obtain I-2;
(d) Analyzing the trace I-2 by HPLC to obtain a product with a purity of more than 99%; without further purification, the pH was adjusted with 0.05mol/L sodium bicarbonate for tumor treatment.
The method is to 177 LuCl 3 For hydrochloric acid solutions 68 GaCl 3 Replacement of hydrochloric acid solution to obtain complex 68 PSMA inhibitors of Ga nuclides. Can also use 225 AC、 161 Tb、 133/135 La、 90 Y、 44 Sc or 64 The salt solution of Cu ions performs the above reaction.
In the final product, metal ions such as sodium, magnesium, calcium and the like exist in the solution, and can be combined with the compound I-2 to form sodium salt and the like.
Example 7
This example was used to obtain R 2 Complexing in the case of NODA 68 A PSMA inhibitor of Ga nuclides having the formula:
wherein R1 is benzylamine or cyclohexylmethylamine;
an exemplary specific preparation method comprises:
(a) Adding a certain amount (100-300 mu L) of 1.0mol/L sodium acetate into 0.1-0.2mL of ultrapure water II-1 to ensure that the pH value of the solution is 4.0 after 2-5mL of 0.05M HCl is added;
(b) Rinsing with 2-5mL of 0.05M HCl 68 GaCl 3
(c) Standing at room temperature for 5min;
(d) Analyzing the radiochemical purity of trace by HPLC, wherein the radiochemical purity is more than 95%; the product does not need further purification, and II-3 can be used for targeting PSMA tumor imaging by adjusting the pH with 0.05mol/L sodium bicarbonate.
Example 8
This example is for obtaining R 2 In the case of NODA, the reaction route of the PSMA inhibitor complexing aluminum fluoride is as follows:
wherein R1 is benzylamine or cyclohexylmethylamine;
an exemplary specific preparation method comprises:
(a) Taking 370-7400 MBq (0.2 mL) 18 Adding F physiological saline solution into 0.1-0.3mL 0.1M pH =4.0 acetic acid buffer solution, and adding 2-10 μ L0.002M AlCl 3 Standing the solution at room temperature for 5-10min;
(b) Adding 150-300 microgram of II-1 compound, and heating at 100-120 deg.C for 10-20min; obtaining a compound II-4;
(c) And purifying the product II-4 by a solid phase extraction method, and can be used for tumor imaging of the target PSMA.
The AlCl is prepared by the method 3 FeCl for solution 3 The PSMA inhibitor of complex ferric fluoride can be prepared by solution replacement. Example 9: i-312, I-313, I-314 (I-312, I-313, I-314) 68 Ga-pyridine-cyclohexylmethylamine-DOTA-PSMA) marking and animal experiment
Opening the heating module to set the temperature at 80 ℃; taking out refrigerated 2 (or 3, or 4) -pyridine-cyclo-hexylmethylamine-DOTA-PSMA (I-112, I-113, I-114) white powder, preparing 4-5mg/mL solution by using ultrapure water, placing 4-6 mu L into a 10mL ampoule, and adding 100-200 mu L of 1.0mol/L high-purity sodium acetate solution into a reaction tube. Taking 2-4mL of 0.05mol/L HCl solution, and leaching 68 Ge/ 68 And (3) discarding the first 1-2mL of the leacheate by using the Ga generator, taking the last 2-3mL of the leacheate (740-1680 MBq), adding into the ampoule, and heating in a heating module at 80 ℃ for 5-10min. Adding appropriate amount of sodium bicarbonate solution to adjust pH to about 6-7, and filtering with sterile filter membrane. Analytical HPLC for the determination of radiochemical purity, respectively, the analytical column being a reverse phase C-18 column, the mobile phase being: 5% acetonitrile (0.1% TFA V/V). After marking, the marking rate reaches 97 percent by HPLC measurement calculation. 68 Ga-2P-C-PSMA(I-312)、 68 Ga-3P-C-PSMA(I-313)、 68 The reference retention times for all three markers of Ga-4P-C-PSMA (I-314) were 5-6min, see FIG. 6.
Establishing a 22RV1 tumor-bearing mouse model with positive right upper limb lotus PSMA expression, taking the solution, diluting tail veins, injecting the diluted solution into a 22RV1 tumor-bearing mouse (100 mu Ci/0.2mL, n = 3), imaging by Micro PET/CT after injection, and calculating ID%/g, wherein the result is as follows:
compound I-31X was distributed in 22RV1 tumor bearing mice (n = 3)
| Compound (I) | Tumor(s) | Kidney (A) | Liver disease | Heart | Lung (lung) | Intestinal tract | Muscle | Tumor/muscle |
| I-312 | 1.6 | 2.98 | 0.29 | 0.37 | 0.17 | 0.28 | 0.08 | 20 |
| I-313 | 2.19 | 3.02 | 0.33 | 0.28 | 0.17 | 0.35 | 0.06 | 36.5 |
| I-314 | 3.42 | 5.08 | 0.48 | 0.6 | 0.27 | 0.27 | 0.08 | 42.7 |
From the distribution of 22RV1 tumor-bearing mice, it was found that there was a higher uptake of all three imaging agent tumors, with a tumor/muscle ratio of 42.7 at 60min for the I-314 compound and radioactivity excreted mainly from the kidneys. The following clinical studies were mainly and I-314 was the main.
The solution is taken, tail vein is diluted and injected into tumor-bearing mice (100-200 mu Ci/0.1-0.2 mL), 1h later, 1.5 percent isoflurane and oxygen mixed gas are continuously inhaled for anesthesia, and the mice are placed in a prone position for flat-bed medical SUPER-NOVA PET/CT scanning to obtain a reconstructed PET/CT MIP image as shown in figure 7.
From figure 7 it is clear that all three tracers are concentrated in the tumor, with a double kidney and bladder image.
To confirm that the tumor is a specific uptake radioactivity, 2-phosphomethylglutaric acid (2-PMPA) is adopted as a blocking agent, and the blocking agent and the imaging agent are injected together (40-60 mu g of 2-PMPA and 0.1-0.2mL of 100-200 mu Ci imaging agent) into a tumor-bearing mouse, so that the uptake of the tumor and the kidney is obviously inhibited, as shown in figure 8.
Example 10: i-314 (DOTA complexation) 68 Ga cyclohexane-4-PSMA) PET/CT imaging in prostate cancer patients
The marking substance I-314 is clinically researched after being ethically examined by clinical scientific research. Injecting the prostate cancer patient by vein according to 148 MBq/person dosage, and carrying out whole body PET/CT static imaging 60min after injection; as a comparison, the subjects were spaced three days apart 18 F-PSMA-7Q examination. The PET image reconstruction parameters were as follows: OSEM iterative algorithm, 3 iterations, 21 subsets, gaussian filtering: full width at half maximum 3.0mm, image matrix: 344, scatter correction. The results are shown in FIG. 9, in which I-314 and 7Q- 18 The imaging results of F-PSMA. Both probes were distributed mainly in the salivary gland, kidney and liver of the subjects, and the prostate cancer foci in situ were clearly visible (arrows in the figure).
Example 11 (DONA complexing Al) 18 Labeling of F-cycliohexylmethylamine-4-pyridine-PSMA) and animal experiment
0.1mol/L of an acetic acid buffer solution having pH =4.0 was prepared, and a 2mM aluminum trichloride solution was prepared from the solution. Will be provided with 18 Transferring F ion to anion exchange column, spraying with 0.2mL physiological saline (18500-29600 MBq), adding 0.1-0.2mL 0.1mol/L acetic acid buffer solution with pH =4.0 and 10-20 μ L2 mM aluminum trichloride solution, standing at room temperature for 5-8min, adding 150-250 μ g II-114, and heating at 100-110 deg.C8-12min. After cooling, purification by solid phase extraction (SEP-PAK C-18 column) gave II-414 aqueous normal saline containing 10% ethanol with labeling efficiency of 60% (uncorrected). The final product was analyzed for radiochemical purity by HPLC using a reverse phase C-18 column as analytical column, 10% acetonitrile as mobile phase (0.1% TFA V/V), a reference retention time of 6min and a radiochemical purity of greater than 95% (FIG. 10).
4-10mg/mL of Vc is added into the final product, the product is placed for 6 hours at room temperature under the radiation concentration of 1850-3700MBq/mL, and the radiochemical purity of the product is still more than 95%.
The injection is diluted into tail vein and injected into 22RV 1-loaded tumor mice (100-200 mu Ci/0.1-0.2mL, n = 3), and after injection, the ID%/g is calculated by Micro PET/CT imaging. The results were as follows:
compound II-414 distributed n =3 in 22RV1 tumor bearing mice
| Compound (I) | Tumor(s) | Kidney (A) | Liver disease | Heart and heart | Lung (lung) | Abdominal cavity | Muscle | Tumor/muscle |
| II-414 | 4.8 | 4.63 | 0.29 | 0.12 | 0.1 | 0.15 | 0.06 | 80 |
From biodistribution, it was found that the tumor has a high uptake, the tumor/muscle ratio reached 80 at 60min, and the radioactivity was mainly excreted from the kidney.
Compound II-414 PET/CT imaging in tumor bearing mouse
Establishing a PSMA positive 22RV1 tumor-bearing mouse model, taking the injection, diluting tail vein, injecting the diluted injection into a tumor-bearing mouse (100-200 mu Ci/0.1-0.2 mL), continuously inhaling 1.5% isoflurane and oxygen mixed gas for anesthesia after 1h, and placing the mouse in a prone position for flat-bed medical SUPER-NOVA PET/CT scanning to obtain a reconstructed PET/CT MIP image. The tumor can obviously take up radioactivity, and the taking amount of the radioactivity is higher than that of the radioactivity in the kidney. To confirm that the tumor was specific for uptake of radioactivity, 2-phosphomethylglutaric acid (2-PMPA) was used as a blocking agent, and co-injection (40-60. Mu.g 2-PMPA and 0.1-0.15mL 100-200. Mu.C imaging agent) with the imaging agent was performed in tumor-bearing mice, which showed significant inhibition of tumor and renal uptake, as shown in FIG. 11.
Example 12 II-414 PET/CT imaging in prostate cancer patients
Labeling substance: II-414 clinical research was conducted after ethical review by clinical scientific research. The prostate cancer patient is injected with 7.4MBq (0.20 mCi)/kg of injection via vein, and the whole body PET/CT static imaging is performed 60min after the injection. The PET image reconstruction parameters were as follows: OSEM iterative algorithm, 3 iterations, 21 subsets, gaussian filtering: full width at half maximum 3.0mm, image matrix: 344, scatter correction. The results are shown in FIG. 12, the radioactivity is mainly distributed in salivary glands and kidney in the subject, and the metastatic tumor focus is clearly visible (arrow in the figure). The imaging agent cleared rapidly and had low peripheral background compared to other PSMA imaging agents.
Example 13 18 Marking of F (Al) -4-pyridine-benzylamine-NODA-PSMA) and PET/CT imaging in prostate cancer patients
The radiochemical purity of the final product II-424, labelled with F-18/aluminium trichloride as in example 11 and with precursor II-124, was greater than 95%.
As in example 12, II-424 was subject to clinical research after ethical review by clinical research, and the injection dose and image processing were consistent with example 12. The results are shown in FIG. 13, where a significant bone metastasis is seen on the MIP plot, and intra-splenic radioactive uptake is higher compared to II-414, but does not affect image quality and clinical diagnosis.
Example 14: i-214 (DOTA complexation) 177 Lu-cyclicaminomethyl-4-pyridine-PSMA) marking and animal imaging experiment and treatment
Opening the heating module to set the temperature at 85 ℃; taking out refrigerated 4-pyridine-cyclohexylmethylamine-DOTA-PSMA (I-114) white powder, preparing 4-6mg/mL solution by using ultrapure water, putting 8-12 mu L into a 10mL ampoule, and adding 100-200 mu L of 1.0mol/L high-purity sodium acetate solution and 80-120mg of Vc into a reaction tube; . Adding 1-3mL of 0.05mol/L hydrochloric acid into an ampoule 177 LuCl 3 Adding a certain amount of sodium acetate into the solution (2GBq, 2: 1mol ratio), and adjusting the pH value of the solution to 4.0; heating in a heating module at 85 deg.C for 30min. Adding appropriate amount of sodium citrate solution to adjust pH to about 6, and filtering with sterile filter membrane. Analytical HPLC for the determination of radiochemical purity, respectively, the analytical column being a reverse phase C-18 column and the mobile phase being: 5% acetonitrile (0.1% TFA V/V). The reference retention time of I-214 was 5.5min, calculated by HPLC measurements after labeling, without further purification, with a radiochemical purity of greater than 98%. The radioactivity chromatogram of I-214 is shown in FIG. 14.
A22 RV1 tumor-bearing mouse model with positive right upper limb lotus PSMA expression is established, the solution is taken, diluted tail veins are injected into a 22RV1 tumor-bearing mouse (15-25 mu Ci/0.2mL, n = 3), the mouse is sacrificed after 24 hours, and the radioactive distribution of all organs is measured as follows.
I-214 distribution in 22RV1 tumor bearing mice 24 hours (n = 3)
| Tumor(s) | Kidney (A) | Liver disease | Heart | Lung (lung) | Spleen | Muscle | Tumor/muscle | |
| ％ID/g | 1.46 | 0.60 | 0.82 | 0.14 | 0.37 | 0.59 | 0.14 | 10 |
Diluting tail vein of the above solution, and injecting into 22RV1 tumor-bearing mice (74 MBq/0.2mL, n = 5) with normal saline as control group; after 24 hours, the treated group was imaged by SPECT equipment, as shown in FIG. 15, and only the tumor was found to have a significantly concentrated radioactivity, and a small amount of radioactivity was found in the renal areas. Tumor to contralateral muscle ratio was calculated using ROI method, 24 hours: 15.9.
two groups of tumor-bearing mice are normally raised until the tumor-bearing mice die naturally, and median survival periods of the two groups are calculated; the median survival in the saline control group was 28 days, while the overall survival in the I-214 treated group was 45 days (P < 0.01), significantly prolonging survival of the mice.
It will be evident to those skilled in the art that the apparatus and method of the present invention are not limited to the details of the foregoing illustrative embodiments, and that the present teachings can be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (8)
1. A compound having a structure represented by formula A or a pharmaceutically acceptable salt thereof,
wherein R1 is cyclicamethamine or benzylamine, R2 is DOTA or NODA for complexing metal ions, the pyridine ring is a 4-position pyridine ring,
r2 may be complexed with radioactive metallic nuclides or non-radioactive metallic nuclides, for diagnostic or therapeutic use, for binding to other radionuclides.
2. A PSMA inhibitor, wherein a radioactive metal ion is bound to the compound or pharmaceutically acceptable salt of claim 1, R2 is DOTA (I), wherein the bound radioactive metal ion M is: 177 Lu、 225 AC、 161 Tb、 133/135 La、 90 Y、 44 Sc、 68 ga or 64 Cu, structural formula as follows:
3. a PSMA inhibitor, wherein a radioactive metal ion is bound to the compound or pharmaceutically acceptable salt of claim 1, R2 is NODA (II), and wherein the bound radioactive metal ion is 68 Ga, structural formula as follows:
4. a PSMA inhibitor, wherein a non-radioactive metal ion is bound to a compound or pharmaceutically acceptable salt of claim 1, R2 is NODA (II), wherein the metal ion M is aluminum or iron, and the radionuclide bound to the metal ion is 18 F, the structural formula is as follows:
5. the PSMA inhibition of claim 2A process for the preparation of a compound of the formula I-M or a pharmaceutically acceptable salt thereof, wherein R1 is benzylamine or cyclohexylmethylamine, M is a radioactive metal ion, is prepared from a compound of the formula I-1 or a pharmaceutically acceptable salt thereof: 177 Lu、 225 AC、 161 Tb、 133/135 La、 90 Y、 44 Sc、 68 ga or 64 Cu，
Wherein a is the reaction of the compound I-1 and the salt containing radioactive metal ions under the acidic condition and under the heating condition.
6. A process for the preparation of a PSMA inhibitor according to claim 3, wherein a compound of formula ii-3 or a pharmaceutically acceptable salt thereof is prepared from a compound of formula ii-1 or a pharmaceutically acceptable salt thereof, wherein R1 is benzylamine or cyclohexylmethylamine and the radioactive metal ion is 68 Ga，
Wherein a is a compound II-1 and a radioactive metal ion under acidic conditions 68 And reacting the salt of Ga under normal temperature conditions.
7. A process for the preparation of a PSMA inhibitor according to claim 4, wherein a compound of formula II-4 or a pharmaceutically acceptable salt thereof is prepared from a compound of formula II-1 or a pharmaceutically acceptable salt thereof, R1 is benzylamine or cyclohexylmethylamine,
wherein a is fluorine-18 ions and aluminum ions firstly form aluminum fluoride; b is heating under acidic condition, complexing aluminum ions with NODA in II-1 compound to form stable complex; the aluminum ions may be replaced with iron ions.
8. Use of the PSMA inhibitor according to any of claims 2 to 4 for the preparation of a targeted PSMA tumor imaging agent; the application of the compound in preparing a medicine for diagnosing, staging or evaluating the curative effect of tumors; the application of the derivative in preparing a PSMA tumor targeted therapeutic drug.
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