A prostate-specific membrane antigen targeting compound and method of making and uses thereof

Abstract

This invention discloses a prodrug compound and its preparation method for prostate-specific membrane antigen (PSMA)-targeted radiodiagnosis and therapy, belonging to the field of PMA-targeted radiopharmaceutical technology development. This invention utilizes amino acid mutation and linker modification strategies to design a series of PSMA-targeting prodrug compounds, expanding the variety of PMA-targeting compounds. Compared with traditional PSMA-617 targeting tracers, the PSMA-targeting tracer provided by this invention exhibits higher tumor uptake rates, more significant tumor therapeutic effects, and greater effectiveness in the clinical diagnosis and treatment of prostate cancer, demonstrating greater clinical application advantages.

Description

Technical Field

[0001] This invention belongs to the field of prostate-specific membrane antigen targeted drug development technology, specifically relating to a compound targeting prostate-specific membrane antigens, its preparation method, and its uses. Background Technology

[0002] Statistics show that the incidence and mortality rates of prostate cancer in my country have been rising rapidly over the past decade. Currently, approximately 10 million men worldwide are diagnosed with prostate cancer. Prostate cancer causes more than 400,000 deaths globally each year, and this number is projected to reach over 800,000 annually by 2040. Between 2015 and 2022, the incidence rate of prostate cancer in my country's cancer registry areas increased from 10.2 per 100,000 to 18.6 per 100,000. According to the Chinese Guidelines for Prostate Cancer Screening, Early Diagnosis and Treatment, most newly diagnosed prostate cancer cases in my country are at an intermediate or late stage, with only about 30% being clinically localized cases. Prostate cancer is seriously threatening men's health and lives.

[0003] Prostate-specific antigen (PSA) is a biomarker used for the diagnosis and screening of prostate cancer patients. It is tissue-specific, existing only in the cytoplasm of epithelial cells of the human prostate gland's acini and ducts, and not expressed in other cells. However, it is not tumor-specific; prostatitis, benign prostatic hyperplasia, and prostate cancer can all lead to elevated total PSA levels. Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein with intracellular and extracellular domains, composed of a 750-amino acid sequence encoded on chromosome 11. It was first discovered in prostate cancer cell lines. PSMA is overexpressed in more than 90% of malignant prostate cancers, including lymph node metastases and bone metastases. Unlike PSA, its expression level increases significantly with increasing malignancy, and its expression level in normal tissues is 100 to 1000 times lower than in prostate cancer, exhibiting good specificity. Therefore, it has become an important molecular target for the clinical diagnosis and treatment of prostate cancer.

[0004] Tumor radionuclide imaging, also known as tumor nuclear medicine molecular imaging, utilizes multimodal imaging equipment and radioactive molecular imaging probes to observe the distribution and metabolic changes of key molecules in body tissues and cells. This allows for insights into the biological characteristics of tumor cells, understanding of tumor metabolic heterogeneity, antigen and receptor distribution heterogeneity, and therapeutic response heterogeneity, thereby providing a basis for accurate tumor diagnosis, staging, treatment selection, and prognosis. The development of sensitive, specific, and efficient radionuclide imaging agents has significant clinical implications.

[0005] One important clinical indication for PSMA imaging is to provide initial staging guidance for specific prostate cancer patients, particularly those whose results are negative or indistinguishable using conventional imaging methods (such as bone scintigraphy and CT). Over the past decade, several PSMA-based drugs have been used in positron emission tomography (PET) imaging and introduced into clinical practice. PSMA-targeted imaging agents [ 18 PSMA-1007 has been successfully used in the clinical diagnosis of prostate cancer patients. Compared with other PSMA-targeted tracers, [ 18 [F]PSMA-1007 uptake is increased in prostate cancer lesions, improving the tumor-to-background ratio and thus enhancing the detection rate of small prostate cancer lesions. However, [ 18 The limitations of PSMA-1007 in clinical application are also evident. Besides the fact that peripheral autonomic ganglia and lymph nodes physiologically express PSMA, thus easily leading to false-positive results, studies have shown that... 18 F]PSMA-1007 imaging exhibits nonspecific bone uptake, as reported in one study, by [ 18 The multiple PSMA hyperuptake lesions diagnosed by F]PSMA-1007, after CT, MRI, and long-term follow-up examinations, did not support the diagnosis of multiple bone metastases. Therefore, these lesions were defined as nonspecific PSMA uptake lesions in bone; furthermore, [ 18 F]PSMA-1007 uptake rate in benign lesions is almost [ 68 Five times that of Ga-PSMA-11; additionally [ 18 [F]PSMA-1007 has a unique metabolic pathway; its metabolism primarily occurs in the liver and gallbladder, rather than the urinary system. Therefore, [ 18 F]PSMA-1007 may cause false-negative diagnostic results for liver metastases. 68 Ga-PSMA-11 is currently the most mature and widely used PSMA-targeted radiotracer for prostate cancer. PSMA-11 uses a glutamic acid-urea-lysine (GUL) structure as its pharmacophore, which can efficiently bind to the chelating agent HBED-CC and enter cells via endocytosis through groups within the PSMA envelope. 68 Ga-PSMA-11 is primarily excreted through the kidneys and cleared rapidly from the bloodstream. Good contrast images can be obtained 1-2 hours after injection. Its physiological uptake is mainly concentrated in the parotid, lacrimal, and submandibular glands, and kidneys. Liver metastases show relatively low uptake. 18 F]PSMA-1007 shows improvement. Clinical studies have shown that compared to traditional diagnostic methods, [ 68Ga-PSMA PET / CT is significant for guiding repeat biopsies in patients with a high suspicion of prostate cancer and negative previous biopsies, as it helps in the localization of primary prostate cancer, provides more accurate assessment of distant metastases and regional lymph node status, and has a high ability to identify intermediate- and high-risk prostate cancer patients. 18 [F]PSMA-1007 is comparable and shows higher value in the detection rate of metastatic lymph nodes. Therefore, as the first FDA-approved PET imaging drug targeting PSMA for prostate cancer, [ 68 The diagnostic advantages of Ga-Ga-PSMA PET / CT in prostate cancer and related diseases are being continuously discovered and reported. With the widespread adoption of PET / CT imaging equipment and radioisotope production equipment such as gallium germanium generators, […] 68 Ga-PSMA may continue to profoundly change the landscape of prostate cancer diagnosis and treatment in my country. Therefore, by employing multiple strategies to modify and transform the structure of PSMA-1007, regulating its pharmacokinetic properties, and combining it with Ga-68 radionuclide labeling, we can improve its metabolic pathways in vivo and increase its uptake in PSMA-positive lesions, thereby enhancing diagnostic accuracy and effectiveness. This is expected to bring more benefits to patients and further enrich the variety of PSMA-targeted radiopharmaceuticals. Summary of the Invention

[0006] The purpose of this invention is to provide a prostate-specific membrane antigen-targeting compound, its preparation method, and its uses.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] One of the technical solutions of the present invention is to provide a precursor compound or an acceptable salt thereof that targets prostate-specific membrane antigen, wherein the general structural formula of the labeled precursor compound that targets prostate-specific membrane antigen is shown in Formula I:

[0009]

[0010] In Formula I, m is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L1 and L2 are independently selected from straight-chain or branched C1-C4 alkyl groups, -NHCO-, -CH2-O-CH2-, natural amino acids, non-natural amino acids, or one of the following structures, where a is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10:

[0011]

[0012] R is a chelating agent, preferably DOTA, DOTAGA, DOTAM, DO3AP, or DO3AP. PrA DO3AP ABn HYNIC, DiAmSar, NOTA, NOTAGA, TRAPH, NOPO, TRAP-Pr, TRAP-OH, TRAPH, AAZTA, H2DEPDPA, TETA, TE2A, CB-TE2A, 177, 178, 179, PCTA, EDTMP, DTPA, PEPA, HEHA, HBED or HBED-CC TFP, the specific structure is shown below:

[0013]

[0014] The structural formula of the precursor compound preceding the radionuclide is any one of 7a to 7m;

[0015]

[0016]

[0017]

[0018] More preferably, the precursor compound preceding the radionuclide has a structural formula selected from:

[0019] The method for synthesizing the aforementioned compound can be summarized as follows:

[0020]

[0021] Add DCM (15 mL / g) to 1 g of 2-chlorotriphenylmethyl resin (0.44 mmol / g), shake and swell at room temperature for 30 min, then filter out the DCM. Weigh 2 mmol of compound 1, dissolve it in a small amount of DCM, add 612 μL of DIPEA to activate it for 5 min, add the mixed solution to the resin, and react under nitrogen protection for 2 h. Filter out the reaction solution, wash the resin 5 times with DMF, add 10 mL of a mixed solution of methanol, DCM, and DIPEA (volume ratio 17:2:1), seal at room temperature for 30 min, filter out the reaction solution, wash 4 times with DMF, add 50% morpholine (DMF) solution (15 g / mL), purge and react for 5 min, continue to add morpholine solution and react for 15 min. Filter out the reaction solution, wash 6 times with DMF, dehydrate with DCM and methanol solution, and dry under vacuum for later use.

[0022] 1 mmol of compound 3 and 0.5 mL of DIPEA were added to 70 mL of dry DCM. 0.34 mmol of triphosgene was added to the solution over 4 hours at 0 °C to obtain compound 4. The reaction was continued with stirring at 25 °C for 1 hour. 0.2 mmol of resin (compound 2) was added to 2 mL of DCM, followed by compound 4. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, the mixture was filtered out and the resin was washed five times with DMF. The mixture was then dried to obtain compound 5. 35 mg of tetrakis(triphenyl)palladium and 200 μL of morpholine (2 mL of DCM) were reacted at room temperature for 3 hours to remove the allyloxycarbonyl protecting group. After the reaction was complete, the mixture was filtered out and the resin was washed five times with DMF to obtain compound 6. Next, 1.4 mL of the corresponding amino acid and HATU were added to compound 6. DIPEA (equivalent ratio 1:3:8) was reacted at room temperature for 2 hours, filtered, and the resin was washed five times with DMF. The mixture was then dried to obtain compound 7. Next, 4-(((((9H-fluorene-9-yl)methoxy)carbonyl)amino)methyl)benzoic acid, DIPEA, and HATU (equivalent ratio 1:1:2) were added to compound 7. After reacting at room temperature for 2 hours, the mixture was filtered, the resin was washed, and the mixture was dried to obtain compound 8. Next, 2 mmol of Fmoc-(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid, DIPEA, and HATU (equivalent ratio 1:1:2) were added to compound 8. After reacting at room temperature for 2 hours, the mixture was filtered, the resin was washed with DMF, and the mixture was dried to obtain compound 9. Next, 2 mmol of Fmoc-(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid, DIPEA, and HATU (equivalent ratio 1:1:2) were added to compound 9. Fmoc-(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid, DIPEA, and HATU (equivalent ratio 1:1:2) were reacted at room temperature for 2 h, filtered, and the resin was washed with DMF and dried to obtain compound 10. Next, 2 mmol of chelate, DIPEA, and HATU (equivalent ratio 1:1:2) were added to compound 10, and the mixture was reacted at room temperature for 12 h. The mixture was then filtered, and the resin was washed with DMF. The mixture was dehydrated, dried, and 4 mL of shear buffer (TFA:TIPS:H2O = 95%:2.5%:2.5%) was added. The mixture was reacted at room temperature for 3 h, filtered, and the liquid was removed under reduced pressure. Ether was added, and the mixture was centrifuged and the liquid was discarded. The ether was removed under reduced pressure, and the product was purified by HPLC to obtain compound 11. The product was verified by LC-MS.

[0023] Furthermore, the present invention also provides a diagnostic radionuclide marker, which is a radionuclide-labeled precursor compound targeting the prostate-specific membrane antigen as described in any one of claims 1-4, wherein the radionuclide is a diagnostic or therapeutic radionuclide selected from... 43 Sc、 44 Sc、 51 Mn, 52 Mn,64 Cu、 67 Ga、 68 Ga、 90 Y、 89 Zr、 99m Tc, 111 ln、 152 Tb, 155 Tb, 201 Tl、 203 Pb, 212 Pb, 213 Bi、 212 Bi、 225 Ac、 211 At、 186 Re、 67 Cu、 188 Re、 177 Lu、 18 F, 76 Br、 77 Br、 123 I, 124 I, 125 I, 89 Zr or 11 At least one of C;

[0024] Preferably, the diagnostic radionuclide labeling compound provided by the present invention has a structural formula selected from:

[0025]

[0026]

[0027] More preferably, the diagnostic radionuclide labeling compound provided by the present invention has a structural formula selected from:

[0028]

[0029] In another aspect, the radionuclide labeling method provided by the present invention involves eluting the radionuclide eluent, mixing it with the labeling precursor compound, adjusting the pH to 4-10 using a buffer solution, wherein the buffer solution is sodium acetate, water, ethanol, phosphate, salt buffer solution or dimethyl sulfoxide; reacting at 20-110°C for 10 minutes to overnight;

[0030] In another aspect, the present invention provides the use of a prostate-specific membrane antigen-targeting precursor compound and its diagnostic radionuclide marker in the preparation of one or more drugs or reagents for the diagnosis and / or treatment of one or more tumors, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic / endocrine disorders or neurological diseases;

[0031] Furthermore, the prostate-specific membrane antigen-targeted radionuclide-labeled compounds provided by this invention are used to diagnose and treat diseases by the following pathogenic cells: prostate tumor cells, metastatic prostate tumor cells, lung tumor cells, kidney tumor cells, liver tumor cells, glioblastoma cells, pancreatic tumor cells, bladder tumor cells, sarcoma cells, melanoma cells, breast tumor cells, colon tumor cells, germ cells, pheochromocytoma, esophageal tumor cells, and gastric tumor cells;

[0032] Furthermore, the diagnostic method provided by the present invention is radionuclide imaging, and more preferably includes PET imaging and SPECT imaging;

[0033] In another aspect, the treatment provided by the present invention is selected from radionuclide therapy, and more preferably includes beta-isotope therapy and alpha-isotope therapy;

[0034] The beneficial technical effects of the present invention are as follows:

[0035] This invention utilizes computer-aided drug design technology combined with experiments to design a series of prostate-specific membrane antigen targeting molecules while retaining the core region "glutamic acid-urea group", thus enriching the types of prostate-specific membrane antigen targeting marker precursor compounds.

[0036] The novel prostate-specific membrane antigen targeting tracer provided by this invention has a higher tumor uptake rate than PSMA-617, and is more advantageous in clinical diagnosis and treatment.

[0037] The reagent kit invention involved in this invention includes the following aspects:

[0038] Aspect 1: A radiolabeling kit for prostate-specific membrane antigen-targeting precursor compounds, comprising...

[0039] A certain amount of acetate or its salt buffer is provided to equilibrate the acidic pH eluent from the Ga-68 generator to a pH value in the range of 3 to 5 when the Ga-68 generator / Ltene-177 accelerator is eluted in the kit.

[0040] The chelate functional group of the prostate-specific membrane antigen-targeting precursor compound is capable of chelating gallium-68 / lutetium-177 under radiolabeling conditions; and a metal inhibitor, which is a co-chelating agent capable of inactivating contaminating metals without interfering with the chelation between gallium-68 / lutetium-177 and the prostate-specific membrane antigen-targeting precursor compound under said radiolabeling conditions, wherein the metal inhibitor is selected from the group consisting of DOTA, DTPA, monosaccharides, and disaccharides, and the prostate-specific membrane antigen-targeting precursor compound and the chelate are from claims 1-5;

[0041] Aspect 2: The radiolabeled kit according to Aspect 1, wherein the acetate, the prostate-specific membrane antigen-targeting precursor compound, and the metal inhibitor are co-lyophilized;

[0042] Aspect 3: The radiolabeling kit according to Aspect 1, wherein the prostate-specific membrane antigen-targeting precursor compound and the metal inhibitor are co-lyophilized, and wherein the acetate or its buffer solution is present alone and may be added subsequently;

[0043] Aspect 4: A radiolabeling kit for prostate-specific membrane antigen-targeting precursor compounds according to any one of Aspects 1 to 3, wherein the acetate or its buffer is present at a specific concentration to equilibrate the acidic eluent from the gallium-68 generator / lutetium-177 accelerator to a pH in the range of 3 to 5 when the generator is eluted in the kit after the addition of hydrochloric acid to the kit.

[0044] Aspect 5: A radiolabeling kit for a prostate-specific membrane antigen-targeting precursor compound according to any one of Aspects 1 to 3, wherein the chelate functional group of the targeting agent is capable of reacting with Ga 3+ / Lu 3+ Formation of stable complexes;

[0045] Aspect 6: A radiolabeling kit for prostate-specific membrane antigen-targeting precursor compounds according to any one of Aspects 1 to 3, wherein the acetate is present in a mass between 0.005 g and 1 g;

[0046] Aspect 7: A radiolabeling kit for prostate-specific membrane antigen-targeting precursor compounds according to any one of Aspects 1 to 3, wherein the metal inhibitor is present in micromolar amounts;

[0047] Aspect 8: The radiolabeling kit according to Aspect 1 is used for operation at 25-120°C;

[0048] Aspect 9: The method according to Aspect 1, wherein the acid is hydrochloric acid;

[0049] Aspect 10: The method according to aspect 1 further includes the step of adding hydrochloric acid to the prostate-specific membrane antigen-targeted precursor compound kit prior to elution;

[0050] Aspect 11: A method for preparing a radiolabeled reagent kit for a prostate-specific membrane antigen-targeting precursor compound according to claim 1, comprising the following steps:

[0051] a) Preparing or providing a solution comprising at least an appropriate amount of the acetate or a buffer thereof equilibrated to a pH value in the range of 3 to 5 with an acidic pH eluent from a gallium-68 generator / lutetium-177; preparing or providing the prostate-specific membrane antigen-targeting prodrug compound; and preparing or providing the metal inhibitor; and

[0052] b) The solution obtained in step a) by freeze-drying;

[0053] Aspect 12: A method for preparing the radiolabeled reagent kit according to claim 1, comprising the following steps:

[0054] a) Prepare or provide a solution comprising the chelate-functionalized target agent and the metal inhibitor;

[0055] b) the solution obtained in step a) and c1) adding the acetate as a powder to the lyophilized product obtained in step b), or c2) adding the acetate buffer to the lyophilized product obtained in step b). Attached Figure Description

[0056] Figure 1 This is the liquid chromatogram of PSMA-HK8.

[0057] Figure 2 This is the liquid chromatogram of PSMA-HK9.

[0058] Figure 3 The image shows the liquid chromatogram of PSMA-HK10.

[0059] Figure 4 The images show mass spectra of PSMA-HK8 to 9, where A is the mass spectrum of PSMA-HK8 and B is the mass spectrum of PSMA-HK9.

[0060] Figure 5 This is the mass spectrum of PSMA-HK10.

[0061] Figure 6 for[ 68 The radioactive HPLC chromatogram of Ga]Ga-PSMA-HK8~10, where A is [ 68 The radioactive HPLC chromatogram of Ga]Ga-PSMA-HK8, B is [ 68 The radioactive HPLC chromatogram of Ga]Ga-PSMA-HK9, where C is [ 68 Radioactive HPLC chromatogram of Ga]Ga-PSMA-HK10.

[0062] Figure 7 for[ 177 The radioactive HPLC chromatogram of Lu-PSMA-HK8~10, where A is [177 The radioactive HPLC chromatogram of Lu-PSMA-HK8, B is [ 177 The radioactive HPLC chromatogram of Lu-PSMA-HK9, where C represents [ 177 Radioactive HPLC chromatogram of Lu]Lu-PSMA-HK9.

[0063] Figure 8 for[ 68 Ga]Ga-PSMA 68 Ga-PSMA-HK8~10 and [ 68 Quantitative analysis of PET images and tumor uptake values ​​of Ga-PSMA-617 in LNCaP-bearing mice, where A represents [ 68 Ga]Ga-PSMA-HK8~10 and [ 68 Ga]Ga-PSMA-617 PET / CT image, B is [ 68 Ga]Ga-PSMA-HK8~10 and [ 68 Quantitative analysis results of tumor uptake values ​​of Ga]Ga-PSMA-617.

[0064] Figure 9 for[ 68 Ga]Ga-PSMA-HK8、[ 68 Ga]Ga-PSMA-HK9、[ 68 Ga]Ga-PSMA-HK10 and [ 68 The biodistribution of Ga-PSMA-617 in blank mice, where A is [ 68 Biological distribution of Ga]Ga-PSMA-HK8, B is [ 68 Biological distribution of Ga]Ga-PSMA-HK9, where C is [ 68 Biological distribution of Ga]Ga-PSMA-HK10.

[0065] Figure 10 For administration [ 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10、[ 177 Lu]Lu-PSMA-617、 177 Tumor volume changes in Lu and the control group.

[0066] Figure 11 For administration [ 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10、[ 177Lu]Lu-PSMA-617、 177 Changes in body weight of Lu and control mice.

[0067] Figure 12 for[ 177 Lu]Lu-PSMA-HK8、[ 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10 and [ 177 The biodistribution of Lu-PSMA-HK617 in blank mice, where A is [ 177 Biological distribution of Lu]Lu-PSMA-HK8, B is [ 177 Biological distribution of Lu]Lu-PSMA-HK9, C is [ 177 Biological distribution of Lu]Lu-PSMA-HK10, D is [ 177 Biological distribution of Lu]Lu-PSMA-617. Detailed Implementation

[0068] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0069] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0070] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0071] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0072] In this invention 177 Lu was purchased through commercial channels, and the radionuclide purity is 98%. 68 Ga from 68 Ge / 68Ga generator (ITM, Germany). Radiometric high-performance liquid chromatography (HPLC) analysis of all radiotracers was performed using an HPLC system (Shimadzu, Japan).

[0073] All animal experiments used in this invention were approved by the Animal Ethics Committee of Nanjing Medical University. All animals were cared for by humans, and experiments were conducted according to the recommendations of the Laboratory Animal Care and Use Committee. The establishment of the tumor-bearing mouse model followed standard procedures. LNCaP (1×10⁻⁶) was administered subcutaneously. 6 A tumor-bearing Nude-Bulb / c mouse model was established using cells / mice. The tumor volume reached approximately 100-300 mm². 3 Mice were used for PET / CT imaging and biodistribution studies. LNCaP cell lines were maintained in a humidified CO2 incubator (37°C, 5% CO2) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Subcultures were performed every two days.

[0074] General formula for synthesis:

[0075]

[0076] General method:

[0077] (1) Resin swelling: Add 2-chlorotriphenylmethyl chloride resin (0.44 mmol / g) to DCM (15 mL / g), shake at room temperature for 30 min to swell, and filter out DCM;

[0078] (2) Amino acid linkage: Weigh 5 equivalents of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((allyloxy)carbonyl)amino)valeric acid or N-[(9H-fluoren-9-methoxy)carbonyl]-N'-[(2-propenoxy)carbonyl]-L-lysine, dissolve in a small amount of DCM, add 5 equivalents of DIPEA to activate for 5 min, add the mixed solution to the resin, and react for 2 h under nitrogen protection;

[0079] (3) Resin blocking: Filter out the reaction solution, wash the resin 5 times with DMF, add blocking solution (methanol:DCM:DIPEA=17:2:1, 10mL / g), shake at room temperature for 30min, filter out the reaction solution, wash with DMF 4 times to obtain compound 2;

[0080] (4) Fmoc removal, amino conversion and resin linkage: Add 50% morpholine (DMF) solution (15 g / mL) to 0.2 equivalents of compound 2, react with gas for 5 min, continue to add morpholine solution and react for 15 min, filter out the reaction solution, and wash with DMF 6 times; add 1 equivalent of compound 3 and 3 equivalents of DIPEA to 70 mL of dry DCM, add 0.3 equivalents of triphosgene to the solution within 4 h at 0 °C to obtain compound 4, continue to stir and react at 25 °C for 1 h, add 0.2 equivalents of resin (Fmoc-removed compound 2) to DCM (4 mL / g), add to compound 4 and react at room temperature for 16 h, filter out after the reaction and wash the resin with DMF 5 times to obtain compound 5;

[0081] (5) Alloc protecting group removal: Take 1 equivalent of compound 5, add 0.15 equivalents of tetra(triphenyl)palladium and 10% morpholine ( / DCM) solution (4 mL / g), react at room temperature for 3 hours to remove the allyloxycarbonyl protecting group, filter out after the reaction and wash the resin 5 times with DMF to obtain compound 6;

[0082] (6) Amino acid linkage: Next, 5 equivalents of Fmoc-(S)-3-amino-4-(2-naphthyl)-butyric acid or Fmoc-3-(1-naphthyl)-L-alanine, 15 equivalents of HATU, and 40 equivalents of DIPEA were added to 1 equivalent of compound 6. After reacting at room temperature for 2 hours, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 7.

[0083] (7) Fmoc removal and amino acid linkage: 50% morpholine (DMF) solution (15 g / mL) was added to 1 equivalent of compound 7, and the reaction was carried out under air for 5 min. Morpholine solution was added and the reaction was carried out for 15 min. The reaction solution was filtered off, washed 6 times with DMF, 5 equivalents of Fmoc-(4-aminomethyl)benzoic acid, 5 equivalents of DIPEA, and 10 equivalents of HATU were added. The reaction was carried out at room temperature for 2 h, filtered off and the resin was washed. The mixture was then dried to obtain compound 8.

[0084] (8) Fmoc removal and amino acid linkage: 50% morpholine (DMF) solution (15 g / mL) was added to 1 equivalent of compound 8, and the reaction was carried out under air for 5 min. Morpholine solution was added and the reaction was carried out for 15 min. The reaction solution was filtered off, washed 6 times with DMF, and 5 equivalents of Fmoc-L-glutamic acid-5-tert-butyl ester, 5 equivalents of DIPEA, and 10 equivalents of HATU were added. The reaction was carried out at room temperature for 2 h, filtered off and the resin was washed. The mixture was then dried to obtain compound 9.

[0085] (9) Fmoc removal and amino acid linkage: 50% morpholine (DMF) solution (15 g / mL) was added to 1 equivalent of compound 9, and the reaction was carried out under air for 5 min. Morpholine solution was added and the reaction was carried out for 15 min. The reaction solution was filtered off, washed 6 times with DMF, and 5 equivalents of Fmoc-L-glutamic acid-5-tert-butyl ester, 5 equivalents of DIPEA, and 10 equivalents of HATU were added. The reaction was carried out at room temperature for 2 h, filtered off and the resin was washed. The mixture was then dried to obtain compound 10.

[0086] (10) DOTA-3OtBu linkage: 10 equivalents of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, 10 equivalents of DIPEA, and 20 equivalents of HATU were added to 1 equivalent of compound 10. After reacting at room temperature for 12 h, the mixture was filtered out, the resin was washed with DMF, dehydrated, and dried to obtain compound 11.

[0087] (11) Shearing and purification: Shearing solution (TFA:TIPS:H2O = 95%:2.5%:2.5%, 24 mL / g) was added to compound 11. After reacting at room temperature for 3 h, the liquid was filtered, the liquid was removed under reduced pressure, ether was added, the liquid was discarded after centrifugation, the ether was removed under reduced pressure, the product was purified by HPLC, and the product was verified by LC-MS.

[0088] Example 1: PSMA-HK8:(((R)-1-carboxy-4-((R)-2-(4-((R)-4-carboxy-2-((R)-4-carboxy- 2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butyramide (methyl)benzamido)-3-(naphth-2-yl)propamido)butyl)carbamoyl)-D-glutamic acid

[0089] (1) Resin swelling: Add 1g of 2-chlorotriphenylmethyl chloride resin to 15mL of DCM, shake at room temperature for 30min to swell, and filter out the DCM;

[0090] (2) Amino acid linkage: Weigh 385.4 mg of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((allyloxy)carbonyl)amino)valeric acid, dissolve it in a small amount of DCM, add 612 μL of DIPEA to activate it for 5 min, add the mixed solution to the resin, and react for 2 h under nitrogen protection;

[0091] (3) Resin blocking: Filter out the reaction solution, wash the resin 5 times with DMF, add 10 mL of blocking solution, shake at room temperature for 30 min, filter out the reaction solution, wash with DMF 4 times to obtain compound 2;

[0092] (4) Fmoc removal, amino conversion and resin connection: Weigh 0.2 mmol of compound 2 according to the resin loading, add 8 mL of 50% morpholine (DMF) solution, react under purging for 5 min, continue to add morpholine solution and react for 15 min, filter out the reaction solution, and wash with DMF 6 times; add 295.8 mg of bis(tert-butyl) L-glutamic acid hydrochloride and 0.5 mL of DIPEA to 70 mL of dry DCM, add 101 mg of triphosgene to the solution within 4 h at 0 °C to obtain compound 4, continue to stir and react at 25 °C for 1 h, add 0.2 mmol of resin (compound 2 with Fmoc removed) to 2 mL of DCM, add to compound 4 and react at room temperature for 16 h, filter out after the reaction and wash the resin with DMF 5 times to obtain compound 5;

[0093] (5) Alloc protecting group removal: 0.2 mmol of compound 5 and 35 mg of tetrakis(triphenyl)palladium were added to 2 mL of 10% morpholine (DCM) and reacted at room temperature for 3 hours to remove the allyloxycarbonyl protecting group. After the reaction was completed, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 6.

[0094] (6) Amino acid linkage: Next, 437 mg Fmoc-3-(1-naphthyl)-L-alanine, 1.1 g HATU, and 1.4 mL DIPEA were added to 0.2 mmol of compound 6. After reacting at room temperature for 2 h, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 7.

[0095] (7) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 7, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 373 mg of Fmoc-(4-aminomethyl)benzoic acid, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 8.

[0096] (8) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 8, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 9.

[0097] (9) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 9, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 10.

[0098] (10) DOTA-3OtBu linkage: 1.1 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, 360 μL of DIPEA, and 2.2 g of HATU were added to 0.2 mmol of compound 10. After reacting at room temperature for 12 h, the mixture was filtered out, the resin was washed with DMF, dehydrated, and dried to obtain compound 11.

[0099] (11) Shearing and purification: 12 mL of shearing solution was added to compound 11, and the reaction was carried out at room temperature for 3 h. The liquid was filtered, the liquid was removed under reduced pressure, ether was added, the liquid was discarded after centrifugation, the ether was removed under reduced pressure, the product was purified by HPLC, and the product was lyophilized to obtain a white solid (36.1 mg, yield 13.9%).

[0100] Product validation was performed by LC-MS, MS(ESI) + m / z = 1280.5[M+H] +

[0101] Example 2: PSMA-HK9:(3R,10R,14R)-1-(4-(R)-4-carboxyl-2-((R)-4-carboxyl-2-(2-(4, 7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butamido)butanaamino)methyl) (phenyl)-3-(naphth-2-ylmethyl)-1,5,12-trioxo-2,6,11,13-tetraazahexadecane-10,14,16-triacetic acid

[0102] (1) Resin swelling: Add 1g of 2-chlorotriphenylmethyl chloride resin to 15mL of DCM, shake at room temperature for 30min to swell, and filter out the DCM;

[0103] (2) Amino acid linkage: Weigh 385.4 mg of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((allyloxy)carbonyl)amino)valeric acid, dissolve it in a small amount of DCM, add 612 μL of DIPEA to activate it for 5 min, add the mixed solution to the resin, and react for 2 h under nitrogen protection;

[0104] (3) Resin blocking: Filter out the reaction solution, wash the resin 5 times with DMF, add 10 mL of blocking solution, shake at room temperature for 30 min, filter out the reaction solution, wash with DMF 4 times to obtain compound 2;

[0105] (4) Fmoc removal, amino conversion and resin connection: Weigh 0.2 mmol of compound 2 according to the resin loading, add 8 mL of 50% morpholine (DMF) solution, react under purging for 5 min, continue to add morpholine solution and react for 15 min, filter out the reaction solution, and wash with DMF 6 times; add 295.8 mg of bis(tert-butyl) L-glutamic acid hydrochloride and 0.5 mL of DIPEA to 70 mL of dry DCM, add 101 mg of triphosgene to the solution within 4 h at 0 °C to obtain compound 4, continue to stir and react at 25 °C for 1 h, add 0.2 mmol of resin (compound 2 with Fmoc removed) to 2 mL of DCM, add to compound 4 and react at room temperature for 16 h, filter out after the reaction and wash the resin with DMF 5 times to obtain compound 5;

[0106] (5) Alloc protecting group removal: 0.2 mmol of compound 5 and 35 mg of tetrakis(triphenyl)palladium were added to 2 mL of 10% morpholine (DCM) and reacted at room temperature for 3 hours to remove the allyloxycarbonyl protecting group. After the reaction was completed, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 6.

[0107] (6) Amino acid linkage: Next, 451 mg Fmoc-(S)-3-amino-4-(2-naphthyl)-butyric acid, 1.1 g HATU, and 1.4 mL DIPEA were added to 0.2 mmol of compound 6. After reacting at room temperature for 2 h, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 7.

[0108] (7) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 7, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 373 mg of Fmoc-(4-aminomethyl)benzoic acid, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 8.

[0109] (8) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 8, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 9.

[0110] (9) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 9, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 10.

[0111] (10) DOTA-3OtBu linkage: 1.1 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, 360 μL of DIPEA, and 2.2 g of HATU were added to 0.2 mmol of compound 10. After reacting at room temperature for 12 h, the mixture was filtered out, the resin was washed with DMF, dehydrated, and dried to obtain compound 11.

[0112] (11) Shearing and purification: 12 mL of shearing solution was added to compound 11, and the reaction was carried out at room temperature for 3 h. The liquid was filtered, the liquid was removed under reduced pressure, ether was added, the liquid was discarded after centrifugation, the ether was removed under reduced pressure, the product was purified by HPLC, and the product was lyophilized to obtain a white solid (26.7 mg, yield 10.3%).

[0113] Product validation was performed by LC-MS, MS(ESI) + m / z = 1294.5 [M+H] +

[0114] Example 3: PSMA-HK10:(((R)-1-carboxy-5-((R)-2-(4-((R)-4-carboxy-2-((R)-4-carboxy- 2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)butamido)butanoyl (amino) methyl) benzamide) 3-(naphth-2-yl)propamido) pentyl) carbamoyl) D-glutamic acid

[0115] (1) Resin swelling: Add 1g of 2-chlorotriphenylmethyl chloride resin to 15mL of DCM, shake at room temperature for 30min to swell, and filter out the DCM;

[0116] (2) Amino acid linkage: Weigh 397 mg of N-[(9H-fluorene-9-methoxy)carbonyl]-N'-[(2-propenoxy)carbonyl]-L-lysine, dissolve it in a small amount of DCM, add 612 μL of DIPEA to activate it for 5 min, add the mixed solution to the resin, and react for 2 h under nitrogen protection;

[0117] (3) Resin blocking: Filter out the reaction solution, wash the resin 5 times with DMF, add 10 mL of blocking solution, shake at room temperature for 30 min, filter out the reaction solution, wash with DMF 4 times to obtain compound 2;

[0118] (4) Fmoc removal, amino conversion and resin connection: Weigh 0.2 mmol of compound 2 according to the resin loading, add 8 mL of 50% morpholine (DMF) solution, react under purging for 5 min, continue to add morpholine solution and react for 15 min, filter out the reaction solution, and wash with DMF 6 times; add 295.8 mg of bis(tert-butyl) L-glutamic acid hydrochloride and 0.5 mL of DIPEA to 70 mL of dry DCM, add 101 mg of triphosgene to the solution within 4 h at 0 °C to obtain compound 4, continue to stir and react at 25 °C for 1 h, add 0.2 mmol of resin (compound 2 with Fmoc removed) to 2 mL of DCM, add to compound 4 and react at room temperature for 16 h, filter out after the reaction and wash the resin with DMF 5 times to obtain compound 5;

[0119] (5) Alloc protecting group removal: 0.2 mmol of compound 5 and 35 mg of tetrakis(triphenyl)palladium were added to 2 mL of 10% morpholine (DCM) and reacted at room temperature for 3 hours to remove the allyloxycarbonyl protecting group. After the reaction was completed, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 6.

[0120] (6) Amino acid linkage: Next, 451 mg Fmoc-3-(1-naphthyl)-L-alanine, 1.1 g HATU, and 1.4 mL DIPEA were added to 0.2 mmol of compound 6. After reacting at room temperature for 2 h, the mixture was filtered out and the resin was washed 5 times with DMF to obtain compound 7.

[0121] (7) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 7, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 373 mg of Fmoc-(4-aminomethyl)benzoic acid, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 8.

[0122] (8) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 8, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 9.

[0123] (9) Fmoc removal and amino acid linkage: 8 mL of 50% morpholine (DMF) solution was added to 0.2 mmol of compound 9, and the mixture was reacted under purging for 5 min. Morpholine solution was added again and the mixture was reacted for 15 min. The reaction solution was filtered off, and the mixture was washed 6 times with DMF. 425 mg of Fmoc-L-glutamic acid-5-tert-butyl ester, 180 μL of DIPEA, and 760 mg of HATU were added. The mixture was reacted at room temperature for 2 h, filtered off, and the resin was washed. The mixture was then dried to obtain compound 10.

[0124] (10) DOTA-3OtBu linkage: 1.1 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, 360 μL of DIPEA, and 2.2 g of HATU were added to 0.2 mmol of compound 10. After reacting at room temperature for 12 h, the mixture was filtered out, the resin was washed with DMF, dehydrated, and dried to obtain compound 11.

[0125] (11) Shearing and purification: 12 mL of shearing solution was added to compound 11. After reacting at room temperature for 3 h, the liquid was filtered, the liquid was removed under reduced pressure, ether was added, the liquid was discarded after centrifugation, the ether was removed under reduced pressure, the product was purified by HPLC, and the product was lyophilized to obtain a white solid (32.7 mg, yield 12.6%).

[0126] Product validation was performed by LC-MS, MS(ESI) + m / z = 648.1 1 / 2[M+H] 2+

[0127] The structural formulas of the synthesized PSMA-HK8~10 are as follows:

[0128]

[0129] Experimental Example 1: Evaluation of the synthesis of labeled precursors targeting prostate-specific membrane antigens

[0130] PSMA-HK8 to PSMA-HK10 prepared in Example 1 were analyzed by MS under the following conditions: ion source: ES; nebulizing gas (NEB): 12.00; nitrogen (CUR): 6.00; ion spray voltage (IS): ±4500; temperature (TEM): 0.00; run time: 0.5–1 minute. The liquid chromatography chromatogram is shown below. Figure 1-3 Mass spectrum (see) Figure 4-5 The mass spectra are shown for PSMA-HK8 to PSMA-HK10, in that order. The results indicate that the synthesized structure is correct.

[0131] Experimental Example 2: Labeling and Quality Control of PET Tracers Targeting Prostate-Specific Membrane Antigens

[0132] PET tracers targeting PSMA 68 Synthesis of Ga]Ga-PSMA-HK8~10. [68 Taking Ga]Ga-PSMA-HK8 as an example, the synthesis methods for other compounds are the same:

[0133] Take 5 mL of 0.1 mol / L HCl solution and slowly inject it into the germanium-gallium generator for rinsing. When about 1.5 mL of rinsing solution has been discharged, start collecting the leaching liquid (HCl system). 68 GaCl3 milk, approximately 1.3 × 10⁻⁶ 9 Bq, 3.5 mL). Weigh 20 μg PSMA-HK1, add HCl (0.6 mol / L, 1.0 mL, pH = 4) solution, and then add all of the above freshly rinsed solution to the system. 68 The GaCl3 milk solution was reacted at 85℃ for 15 min. After the reaction, the liquid in the system was sterilized by passing it through a 0.22 μm needle filter and collected in a receiving bottle to obtain [ 68 [Ga]Ga-PSMA-HK8. The reaction process is as follows:

[0134]

[0135] Detected by RP-HPLC [ 68 The radiochemical purity of Ga[Ga]Ga-PSMA-HK8~10 was determined under the following conditions: column: YMC-tria-C18; mobile phase gradient: mobile phase A, 0.1% aqueous trifluoroacetic acid (TFA), mobile phase B, acetonitrile (0.1% TFA); flow time: 15 min; mobile phase B from 20% to 90%; flow rate: 1.0 mL / min. Results are shown below. Figure 6 , [ 68 The radiochemical yield and radiochemical purity of Ga]Ga-PSMA-HK8~10 are both greater than 99%.

[0136] Experimental Example 3: PET tracer targeting PSMA [ 177 Synthesis of Lu]Lu-PSMA-HK8~10 (by[ 177 Taking Lu]Lu-PSMA-HK8 as an example, the synthesis methods for the other three compounds are the same:

[0137] Take 20 μg of PSMA-HK8 and add it to 500 μl of sodium acetate solution (0.25 M). Add... 177 The LuCl3 solution (HCl) was reacted (2 mL, 0.05 M, 10 GBq) at 90 °C for 20 min. After the reaction, the liquid in the system was sterilized by passing it through a 0.22 μm needle filter and collected in a receiving bottle to obtain... 177 Lu-PSMA-HK8. The reaction process is as follows:

[0138]

[0139] Detected by RP-HPLC [177 Radiochemical purity of Lu-PSMA-HK8-10 was determined under the following conditions: column: YMC-tria-C18; mobile phase gradient: mobile phase A, 0.1% aqueous trifluoroacetic acid (TFA); mobile phase B, acetonitrile (0.1% TFA); flow time: 15 min; mobile phase B from 20% to 90%; flow rate: 1.0 mL / min. Results are shown below. Figure 7 From this, we can calculate [ 177 The radiochemical yield and radiochemical purity of Lu]Lu-PSMA-HK8~10 are both greater than 99%.

[0140] Experiment Example 4 :[ 68 Ga]Ga-PSMA-HK8~10 and [ 68 Ga-PSMA-617 in vivo PET scans of tumor-bearing mice Like research

[0141] PET scans were performed using Inveon Micro-PET / CT (Siemens Medical Solutions, Knoxville, Munich, Germany). Each mouse was injected via tail vein with 3.7 MBq dissolved in 100 μL saline. 68 Ga]Ga-PSMA-HK8~10 and [ 68 Ga]Ga-PSMA-617. Dynamic PET scanning begins immediately after injection and continues (n=3). In [ 68 Static PET images were acquired 1 hour and 2 hours after injection of Ga-PSMA-HK8-10, and tumor uptake values ​​were quantitatively analyzed. Results are shown below. Figure 8 Where A is 68 Ga-PSMA-HK8~10 and 68 PET image of Ga-PSMA-617, B is [ 68 Ga]Ga-PSMA-HK8~10 and [ 68 Quantitative analysis results of tumor uptake values ​​of Ga]Ga-PSMA-617. From Figure 8 It can be seen from this that, 68 Ga]Ga-PSMA-HK9 and [ 68 The tumor uptake value of Ga-PSMA-HK10 is higher than that of [ 68 Ga]Ga-PSMA-617 indicates that the radioactive tracer [ 68 Ga]Ga-PSMA-HK9 and [ 68 Ga]Ga-PSMA-HK10 has higher tumor affinity and uptake value.

[0142] Experimental Example 5: Determination of [ 68 Ga]Ga-PSMA-HK8~10 and [ 68 Ga]Ga-PSMA-617 in vitro biological samples from normal mice Distribution

[0143] Blank ICR mice were administered drugs 68 After Ga-PSMA-HK8-10, its blood, heart, liver, spleen, lungs, kidneys, stomach, large intestine, small intestine, pancreas, muscles, bones, brain, salivary glands, testes, thymus, thyroid gland, bladder, adrenal glands, eyes, gallbladder, esophagus, prostate, and lacrimal glands were collected, and their radioactive uptake values ​​were measured.

[0144] See results Figure 9 , where A is [ 68 In vitro biological distribution of Ga]Ga-PSMA-HK8, B is [ 68 In vitro biodistribution of Ga]Ga-PSMA-HK9, where C is [ 68 In vitro biodistribution of Ga]Ga-PSMA-HK10, where D is [ 68 In vitro biological distribution of Ga]Ga-PSMA-617. Figure 8 show, 68 Ga-PSMA-HK8 to 10 are mainly excreted by the kidneys, with low uptake by other non-target organs, and have good safety profile, making them suitable for further clinical translation studies.

[0145] Experimental Example 6: [ 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10 and [ 177 Treatment with Lu-PSMA-617 and safety evaluation

[0146] 18.5 MBq of [method / treatment] was injected into tumor-bearing mice via the tail vein. 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10 and [ 177 Following Lu-PSMA-617 administration, changes in tumor volume and body weight were observed in tumor-bearing mice (n=3). Figure 10 It can be seen from the medication [ 177 Lu]Lu-PSMA-HK9、[ 177 Lu]Lu-PSMA-HK10 and [ 177 Following Lu-PSMA-617 administration, tumor volume growth was significantly inhibited, among which [ 177 Lu]Lu-PSMA-HK9 and [ 177 The tumor volume in mice in the Lu-PSMA-HK10 administration group was smaller than [ 177 Mice in the Lu-PSMA-617-treated group showed that [Lu]Lu-PSMA-617 administration group mice, indicating that [ 177 Lu]Lu-PSMA-HK9 and [ 177 Lu]Lu-PSMA-HK10 has a stronger tumor-suppressing effect and is more promising in clinical treatment.

[0147] Further determination of drug administration [ 177 Lu]Lu-PSMA-HK9、[177 Lu]Lu-PSMA-HK10、[ 177 The changes in body weight of mice in Lu-PSMA-617 and the control group over 12 days are shown in the following results. Figure 11 . Figure 11 The display shows that the drug was administered [ 177 Lu]Lu-PSMA-HK9 and [ 177 Lu]Lu-PSMA-HK10 after [ 177 Compared to Lu-PSMA-617, the mice showed no significant change in body weight. This indicates that the compound involved in this invention is effective for the indicator of body weight change. 177 Similar to Lu-PSMA-617, it has no obvious toxicity.

[0148] Experimental Example 7: Determination of [ 177 In vitro biodistribution of Lu-PSMA-HK8~10 in normal mice

[0149] Normal mice were given the drug 177 After Lu-PSMA-HK8 to 10, blood, heart, liver, spleen, lungs, kidneys, stomach, large intestine, small intestine, pancreas, muscles, bones, brain, salivary glands, testes, thymus, thyroid gland, bladder, adrenal glands, eyes, gallbladder, esophagus, prostate, and lacrimal glands were collected, and their radioactive uptake values ​​were measured.

[0150] See results Figure 12 , where A is [ 177 In vitro biological distribution of Lu]Lu-PSMA-HK8, B is [ 177 In vitro biodistribution of Lu]Lu-PSMA-HK9, C is [ 177 In vitro biodistribution of Lu-PSMA-HK10, D is [ 177 In vitro biological distribution of Lu]Lu-PSMA-617. Figure 9 show,[ 177 Lu-PSMA-HK8~10 is mainly excreted by the kidneys, with trace amounts of radioactive signal uptake in other non-target organs such as bone marrow and salivary glands. 177 The clinical data results for Lu]Lu-PSMA-617 are consistent, indicating that the labeled prodrug compound described in this invention is suitable for further clinical translational studies.

[0151] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A precursor compound or an acceptable salt thereof that targets a prostate-specific membrane antigen, characterized in that, The general structural formula of the compound is shown in Formula I: Where m is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L1 and L2 are independently selected from straight-chain or branched C1-C4 alkyl groups, -NHCO-, -CH2-O-CH2-, natural amino acids, non-natural amino acids, or one of the following structures, where a is any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10: R is selected from one of the following structures:

2. The precursor compound targeting prostate-specific membrane antigen according to claim 1, or an acceptable salt thereof, characterized in that, m is 0 or 1, n is 2 or 3, L1 is a C atom, and L2 is a C atom.

3. The precursor compound or an acceptable salt thereof targeting prostate-specific membrane antigen according to any one of claims 1 to 2, characterized in that, R is or 4. The prodrug compound targeting prostate-specific membrane antigen according to any one of claims 1-2, or an acceptable salt thereof, wherein the labeled prodrug compound is selected from:

5. The method for preparing the precursor compounds PSMA-HK8, PSMA-HK9, and PSMA-HK10 targeting prostate-specific membrane antigens according to claim 4, comprising the following steps: (1) Using compound 1 as the starting material, compound 1 and DIPEA were added to 2-chloro-triphenylmethyl resin to couple them onto the resin to obtain compound 2. (2) Compound 3 was reacted with DIPEA and triphosgene to obtain compound 4; (3) React compound 2 with compound 4 to obtain compound 5; (4) After the reaction was complete, the resin was filtered out to remove the allyloxycarbonyl protecting group, yielding compound 6. (5) Coupled with Fmoc-3-(1-naphthyl)-L-alanine / Fmoc-(S)-3-amino-4-(2-naphthyl)-butyric acid and Fmoc-(4-aminomethyl)benzoic acid via SPPS method. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((allyloxy)carbonyl)amino)valeric acid or N-[(9H-fluoren-9-methoxy)carbonyl]-N'-[(2-propenoxy)carbonyl]-L-lysine, Fmoc-L-glutamic acid-5-tert-butyl ester, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester was purified by shearing resin to obtain a PET tracer precursor compound targeting prostate-specific membrane antigens.

6. A radiolabeled compound for diagnosis and treatment, comprising a precursor compound of any one of claims 1-4 targeting prostate-specific membrane antigens, or a radiolabeled compound of an acceptable salt thereof, wherein, The radionuclide is a diagnostic or therapeutic radionuclide selected from... 43 Sc、 44 Sc、 51 Mn, 52 Mn, 64 Cu、 67 Ga、 68 Ga、 90 Y、 89 Zr、 99m Tc, 111 ln、 152 Tb, 155 Tb, 201 Tl、 203 Pb, 212 Pb, 213 Bi、 212 Bi、 225 Ac、 211 At、 186 Re、 67 Cu、 188 Re、 177 Lu、 18 F, 76 Br、 77 Br、 123 I, 124 I, 125 I, 89 Zr or 11 At least one of C.

7. A diagnostic radionuclide-labeled compound according to claim 6, wherein, The radionuclide labeling method involves eluting the radionuclide eluent, mixing it with the labeling precursor, and adjusting the pH to 4–10 using a buffer solution, wherein the buffer solution is a sodium acetate, water, ethanol, phosphate, salt buffer solution or dimethyl sulfoxide; and reacting at 20–110°C for 10 minutes to overnight.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one radiolabeled precursor compound targeting prostate-specific membrane antigen according to any one of claims 1 to 4, or at least one labeled precursor compound prepared according to the method of claim 5, or at least one therapeutic radiolabeled compound according to any one of claims 6 to 7, and optionally a pharmaceutically acceptable diluent, carrier, and / or excipient.

9. Use of a radiolabeled precursor compound targeting prostate-specific membrane antigen according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or at least one diagnostic radiolabeled compound according to any one of claims 6-7, or the pharmaceutical composition according to claim 8, in the preparation of a medicament for the prevention, diagnosis and / or treatment of prostate-specific membrane antigen-mediated diseases.

10. The use of the diagnostic radionuclide-labeled compound of claim 6 in the preparation of one or more medicaments or reagents for diagnosing and / or treating one or more tumors, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic / endocrine disorders or neurological diseases.

11. The application according to claim 10, characterized in that, Among them, the radionuclide-labeled compounds used to diagnose and treat diseases include the following pathogenic cells: prostate tumor cells, metastatic prostate tumor cells, lung tumor cells, kidney tumor cells, liver tumor cells, glioblastoma cells, pancreatic tumor cells, bladder tumor cells, sarcoma cells, melanoma cells, breast tumor cells, colon tumor cells, germ cells, pheochromocytoma, esophageal tumor cells, and gastric tumor cells.

12. The application according to claim 10, characterized in that, The diagnostic method is radionuclide imaging, and more preferably includes PET imaging and SPECT imaging. The treatment is selected from radionuclide therapy, and more preferably includes beta radionuclide therapy and alpha radionuclide therapy.

13. A radiolabeling kit for a prostate-specific membrane antigen-targeting precursor compound, comprising: an amount of acetate or a salt buffer thereof, to equilibrate the acidic pH eluent from the gallium-68 generator / lutetium-177 accelerator to a pH value in the range of 3 to 5 when eluted in the kit by the gallium-68 generator / lutetium-177 accelerator; The chelating functional group of the prostate-specific membrane antigen-targeting precursor compound is capable of chelating gallium-68 / lutetium-177 under radiolabeling conditions; and a metal inhibitor, which is a co-chelating agent capable of inactivating contaminating metals without interfering with the chelation between gallium-68 / lutetium-177 and the prostate-specific membrane antigen-targeting precursor compound under said radiolabeling conditions, wherein... The metal inhibitor is selected from the group consisting of DOTA, DTPA, monosaccharides, and disaccharides, and the prostate-specific membrane antigen-targeting precursor compound and chelate are derived from claims 1-5.

14. The radiolabeling kit according to claim 13, wherein, The acetate, the prostate-specific membrane antigen-targeting precursor compound, and the metal inhibitor are co-lyophilized.

15. The radiolabeling kit according to claim 13, wherein, The prostate-specific membrane antigen-targeting precursor compound and the metal inhibitor are co-lyophilized, and the acetate or its buffer solution is present alone and may be added subsequently.

16. The radiolabeling kit according to any one of claims 14 to 15, wherein, The acetate or its buffer solution is present at a specific concentration to equilibrate the acidic eluent from the gallium-68 generator / lutetium-177 accelerator to a pH in the range of 3 to 5 when the generator is eluted in the kit after the addition of hydrochloric acid.

17. The radiolabeling kit according to any one of claims 14 to 16, wherein, The chelate functional group of the target agent can react with Ga 3+ / Lu 3+ Forming stable complexes.

18. The radiolabeling kit according to any one of claims 14 to 15, wherein, The acetate is present in quantities between 0.005 g and 1 g.

19. The radiolabeling kit according to any one of claims 14 to 15, wherein, The metal inhibitor is present in micromolar amounts.

20. The radiolabeling kit according to claim 14 is used for operation at 25-120°C.

21. The radiolabeling kit according to claim 14, wherein, The acid in question is hydrochloric acid.

22. The radiolabeling kit of claim 14, further comprising the step of adding hydrochloric acid to the prostate-specific membrane antigen-targeting precursor compound kit prior to elution.

23. A method for preparing a radiolabeled reagent kit for targeting prostate-specific membrane antigen precursor compounds according to claim 1, comprising the following steps: a) Preparing or providing a solution comprising at least an appropriate amount of the acetate or a buffer thereof equilibrated to a pH value in the range of 3 to 5 with an acidic pH eluent from a gallium-68 generator / lutetium-177; preparing or providing the prostate-specific membrane antigen-targeting prodrug compound; and preparing or providing the metal inhibitor; and b) The solution obtained in step a) by freeze-drying.

24. A method for preparing the radiolabeled reagent kit according to claim 1, comprising the following steps: a) Prepare or provide a solution comprising the chelate-functionalized target agent and the metal inhibitor; b) the solution obtained in step a) and c1) adding the acetate as a powder to the lyophilized product obtained in step b), or c2) adding the acetate buffer to the lyophilized product obtained in step b).

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